Smad on X


EVOLUTIONARY HOMOLOGS

Structure of Smad2/3

The Smad proteins mediate transforming growth factor-beta (TGFbeta) signaling from the transmembrane serine-threonine receptor kinases to the nucleus. To initiate a particular TGFbeta response, a specific TGFbeta ligand binds to a specific pair of transmembrane Ser-Thr receptor kinases, the type I and type II receptors, and this activates the Ser-Thr kinase in the cytoplasmic domain of the type I receptor. The signal is then propagated by type I receptor-mediated phosphorylation of specific R-Smads. The R-Smads, Smad2 and 3, are recruited to the TGFbeta receptors by SARA (Smad anchor for receptor activation). This process appears to involve direct interactions between SARA and Smad2, SARA and TGF receptors, and Smad2 and TGF receptors. SARA does not interact with either Smad1 or Smad5, which share ~80% sequence identity with Smad2. These interactions are important in regulating specific Smad phosphorylation. The phosphorylated R-Smad hetero-oligomerizes with the co-Smad (Smad4), translocates into the nucleus and associates with sequence-specific DNA binding proteins, resulting in the positive or negative regulation of agonist-responsive genes. The crystal structure of a Smad2 MH2 domain in complex with the Smad-binding domain (SBD) of SARA has been determined at 2.2 Å resolution. SARA SBD, in an extended conformation comprising a rigid coil, an alpha helix, and a beta strand, interacts with the beta sheet and the three-helix bundle of Smad2. Recognition between the SARA rigid coil and the Smad2 beta sheet is essential for specificity, whereas interactions between the SARA beta strand and the Smad2 three-helix bundle contribute significantly to binding affinity. Comparison of the structures between Smad2 and a co-mediator Smad suggests a model for how receptor-regulated Smads are recognized by the type I receptors. Despite having generally similar structures, the MH2 domains of Smad2 and Smad4 have different surface features. A direct comparison of the electrostatic potential reveals the presence of a highly positively charged groove next to the L3 loop on Smad2, but not on Smad4. This basic surface contains residues that are conserved in R-Smads but not in co-Smads, suggesting that this region might be important for receptor binding. Analysis of the type I TGFbeta receptor cytoplasmic domain reveals a complementary pattern on its surface. Specifically, the L45 loop, which specifies interactions with Smad2, is located immediately adjacent to the flexible GS region, which is phosphorylated upon ligand binding and becomes very acidic. Thus, the phosphorylated GS region on the type I receptors might interact with a highly basic surface groove on R-Smads to provide binding affinity, whereas the L45 loop on the type I receptors recognizes the L3 loop of specific R-Smads to provide specificity (Wu, 2000).

Ligand-induced phosphorylation of the receptor-regulated Smads (R-Smads) is essential in the receptor Ser/Thr kinase-mediated TGF-ß signaling. The crystal structure of a phosphorylated Smad2, at 1.8 Å resolution, reveals the formation of a homotrimer mediated by the C-terminal phosphoserine (pSer) residues. The pSer binding surface on the MH2 domain, frequently targeted for inactivation in cancers, is highly conserved among the Co- and R-Smads. This finding, together with mutagenesis data, pinpoints a functional interface between Smad2 and Smad4. In addition, the pSer binding surface on the MH2 domain coincides with the surface on R-Smads that is required for docking interactions with the serine-phosphorylated receptor kinases. These observations define a bifunctional role for the MH2 domain as a pSer-X-pSer binding module in receptor Ser/Thr kinase signaling pathways (Wu, 2002).

Interaction of Smad2/3 with cell surface receptors

Smad2 interacts transiently with and is a direct substrate for the transforming growth factor-beta (TGF-beta) type I receptor, TbetaRI. Phosphorylation sites on Smad2 were localized to a carboxyl-terminal fragment containing three serine residues at positions 464, 465, and 467. TbetaRI specifically phosphorylates Smad2 on serines 465 and 467. Serine 464 is not a site of phosphorylation, but is important for efficient phosphorylation of Smad2. Phosphorylation at both sites is required to mediate association of Smad2 with Smad4 in mammalian cells, while in yeast, Smad2 interacts directly with Smad4 and does not require phosphorylation. Mutation of either serine residue 465 or 467 prevents dissociation of Smad2 from activated TbetaRI and blocks TGF-beta-dependent signaling and Smad2 transcriptional activity. These results indicate that receptor-dependent phosphorylation of Smad2 on serines 465 and 467 is required in mammalian cells to permit association with Smad4 and to propagate TGF-beta signals (Abdollah, 1997).

Activin A is a multifunctional protein that is a member of the transforming growth factor-beta (TGF-beta) superfamily. Smad proteins have recently been shown to transduce signals for the TGF-beta superfamily of proteins, and Smad2 was implicated in activin signaling in Xenopus embryos. The receptors and Smad proteins activated by activin A in a human epidermal keratinocyte cell line, HaCaT, have been identified. The major activin receptors expressed on HaCaT cells are activin type II receptor (ActR-II) and activin type IB receptor (ActR-IB). In HaCaT cells, activin A induces the phosphorylation of Smad3 and, to a lesser extent, Smad2, while TGF-beta induces an efficient phosphorylation of both Smad2 and Smad3. Activin A preferentially induces the nuclear translocation of Smad3 in HaCaT cells, whereas TGF-beta strongly induces the nuclear translocation of Smad2, as well as other Smads. Moreover, a constitutively active form of ActR-IB efficiently stimulates the formation of a heteromeric complex between Smad3 and Smad4 in COS cells transfected with Smad cDNAs. These results suggest that activin A binds to a receptor complex of ActR-II and ActR-IB, and preferentially activates Smad3 in HaCaT human keratinocytes (Shimizu, 1998).

SMAD proteins mediate signals from receptor serine-threonine kinases (RSKs) of the TGF-beta superfamily. HGF and EGF, which signal through RTKs, can also mediate SMAD-dependent reporter gene activation and induce rapid phosphorylation of endogenous SMAD proteins by kinase(s) downstream of MEK1. HGF induces phosphorylation and nuclear translocation of epitope-tagged Smad2 and a mutation that blocks TGF-beta signaling also blocks HGF signal transduction. Smad2 may thus act as a common positive effector of TGF-beta- and HGF-induced signals and serve to modulate cross talk between RTK and RSK signaling pathways (de Caestecker, 1998).

Activins, members of the transforming growth factor-ß family, are pleiotropic growth and differentiation factors. Activin A induces B-cell apoptosis. To identify the genes responsible for activin-induced apoptosis, retrovirus-mediated gene trap screening was performed in a mouse B-cell line. The rasGAP-binding protein Dok-1 (p62) was identified as an essential molecule that links activin receptors with Smad proteins. In B cells overexpressing Dok-1, activin A-induced apoptotic responses are augmented. The expression of bcl-XL is down-regulated by inhibition of the ras/Erk pathway. Activin stimulation triggers association of Dok-1 with Smad3, as well as association of Smad3 with Smad4. Dok-1 also associates with both the type I and type II activin receptors. Dok-1 has been characterized previously as a tyrosine-phosphorylated protein acting downstream of the protein tyrosine kinase pathway: intriguingly, activin signaling does not induce tyrosine phosphorylation of Dok-1. These findings indicate that Dok-1 acts as an adaptor protein that links the activin receptors with the Smads, suggesting a novel function for Dok-1 in activin signaling leading to B-cell apoptosis (Yamakawa, 2000).

Vertebral bodies are segmented along the anteroposterior (AP) body axis, and the segmental identity of the vertebrae is determined by the unique expression pattern of multiple Hox genes. Recent studies have demonstrated that a TGF-beta family protein, Gdf11 (growth and differentiation factor 11), and the activin type II receptor, ActRIIB, are involved in controlling the spatiotemporal expression of multiple Hox genes along the AP axis, and that the disruption of each of these genes causes anterior transformation of the vertebrae. However, skeletal defects are more severe in Gdf11-null mice than in ActRIIB-null mice, leaving it uncertain whether Gdf11 signals via ActRIIB. Using genetic and biochemical studies it has been demonstrated that ActRIIB and its subfamily receptor, ActRIIA, cooperatively mediate the Gdf11 signal in patterning the axial vertebrae, and that Gdf11 binds to both ActRIIA and ActRIIB, and induces phosphorylation of Smad2. In addition, these two receptors can functionally compensate for one another to mediate signaling of another TGF-beta ligand, nodal, during left-right patterning and the development of anterior head structure (Oh, 2002).

Tob inhibits bone morphogenetic protein (BMP) signaling by interacting with receptor-regulated Smads in osteoblasts. Evidence is provided that Tob also interacts with the inhibitory Smads 6 and 7. A yeast two-hybrid screen identified Smad6 as a protein interacting with Tob. Tob co-localizes with Smad6 at the plasma membrane and enhances the interaction between Smad6 and activated BMP type I receptors. Furthermore, Xenopus Tob2 has been isolated and has been shown to cooperate with Smad6 in inducing secondary axes when expressed in early Xenopus embryos. Finally, Tob and Tob2 cooperate with Smad6 to inhibit endogenous BMP signaling in Xenopus embryonic explants and in cultured mammalian cells. These results provide both in vitro and in vivo evidence that Tob inhibits endogenous BMP signaling by facilitating inhibitory Smad functions (Yoshida, 2003).

Non-receptor activation of Smad2/3

The intracellular kinase mitogen-activated protein kinase kinase kinase-1 (MEKK-1), an upstream activator of the stress-activated protein kinase/c-Jun N-terminal kinase pathway, can participate in Smad2-dependent transcriptional events in cultured endothelial cells. A constitutively active form of MEKK-1 but not mitogen-activated protein kinase kinase-1 (MEK-1) or TGF-beta-activated kinase-1, two distinct intracellular kinases, can specifically activate a Gal4-Smad2 fusion protein, and this effect correlates with an increase in the phosphorylation state of the Smad2 protein. These effects do not require the presence of the C-terminal SSXS motif of Smad2 that is the site of TGF-beta type 1 receptor-mediated phosphorylation. Activation of Smad2 by active MEKK-1 results in enhanced Smad2-Smad4 interactions, nuclear localization of Smad2 and Smad4, and the stimulation of Smad protein-transcriptional coactivator interactions in endothelial cells. Overexpression of Smad7 can inhibit the MEKK-1-mediated stimulation of Smad2 transcriptional activity. A physiological level of fluid shear stress, a known activator of endogenous MEKK-1 activity in endothelial cells, can stimulate Smad2-mediated transcriptional activity. These data demonstrate a novel mechanism for activation of Smad protein-mediated signaling in endothelial cells and suggest that Smad2 may act as an integrator of diverse stimuli in these cells (Brown, 1999).

Smads transmit signals from transmembrane ser/thr kinase receptors to the nucleus. SARA (for Smad anchor for receptor activation) is a FYVE domain protein that interacts directly with Smad2 and Smad3. [Cysteine-rich RING domains of the FYVE finger subfamily bind specifically to Ptdlns phosphorylated exclusively by phosphoinositide 3-kinases at the D-3 position of the inositol ring, thereby recruiting and activating downstream effectors of Ptdlns(3)P signaling.] SARA functions to recruit Smad2 to the TGFbeta receptor by controlling the subcellular localization of Smad2 and by interacting with the TGFbeta receptor complex. Phosphorylation of Smad2 induces dissociation from SARA with concomitant formation of Smad2/Smad4 complexes and nuclear translocation. Furthermore, mutations in SARA that cause mislocalization of Smad2 inhibit TGFbeta-dependent transcriptional responses, indicating that the regulation of Smad localization is important for TGFbeta signaling. These results thus define SARA as a component of the TGFbeta pathway that brings the Smad substrate to the receptor (Tsukazaki, 1998).

Mesoderm induction and patterning are primarily regulated by the concentration of locally expressed morphogens such as members of the TGFß superfamily. Smad2 functions as a transcription factor to regulate expression of mesodermal genes downstream of such morphogens. Xenopus PIASy (XPIASy: Drosophila homolog - Su(var)2-10), a member of the PIAS (protein inhibitors of activated STAT) family, was identified by yeast two-hybrid screening using Xenopus Smad2 (XSmad2) as a bait. During mesoderm induction, XPIASy is expressed in the animal half of embryos with a ventral high-dorsal low gradient at the marginal zone. XPIASy expression is positively and negatively regulated by activities of the XSmad2 and Wnt pathways, respectively. Interestingly, inhibition of XPIASy by morpholinos induces elongation of animal caps with induction of mesoderm genes even in the absence of their morphogen-mediated activation. In addition, their introduction into the ventral marginal zone results in a secondary axis formation. Gain-of-function analysis has revealed that XPIASy inhibits mesoderm induction by specific and direct downregulation of XSmad2 transcriptional activity. These observations indicate that XPIASy functions as an essential negative regulator of the XSmad2 pathway to ensure proper mesoderm induction at the appropriate time and in the appropriate region, and suggest that both the initial step of morphogen-mediated activation of the XSmad2 pathway and regulation of the final downstream transcription step have crucial roles in mesoderm induction and patterning (Daniels, 2004).

Smad2/3 interaction with Smad4

During early embryogenesis of Xenopus, dorsoventral polarity of the mesoderm is established by dorsalizing and ventralizing agents, which are presumably mediated by the activity of an activin/BVg1-like protein and bone morphogenetic proteins (BMPs). Interestingly, these two TGF-beta subfamilies are found in overlapping regions during mesoderm patterning. This raises the question of how the presumptive mesodermal cells recognize the multiple TGF-beta signals and differentially interpret this information to assign a particular cell fate. The well characterized model of Xenopus mesoderm induction was exploited to determine the intracellular interactions between BMP-2/4 and activin/BVg1 signaling cascades. Using a constitutively active BMP-2/4 receptor that transduces BMP-2/4 signals in a ligand-independent fashion, it has been demonstrated that signals provided by activin/BVg1 and BMP modulate each other's activity; this crosstalk occurs through intracellular mechanisms. In assays using BMP-2/4 and activin/BVg1-specific reporters, it has been determined that the specificity of BMP-2/4 and activin/BVg1 signaling is mediated by Smad1 and Smad2, respectively. These Smads should be considered as the mediators of the intracellular antagonism between BMP-2/4 and activin/BVg1, possibly signaling through sequestration of a limited pool of Smad4. Consistent with such a mechanism, Smad4 interacts functionally with both Smad1 and -2 to potentiate their signaling activities; a dominant negative variant of Smad4 can inhibit both activin/BVg1 and BMP-2/4 mediated signaling. An activin/BVg1-dependent transcriptional complex contains both Smad2 and Smad4 and thereby provides a physical basis for the functional involvement of both Smads in TGF-beta-dependent transcriptional regulation. Thus, Smad4 plays a central role in synergistically activating activin/BVg1 and BMP-dependent transcription, and functions as an intracellular sensor for TGF-beta-related signals (Candia, 1997).

Smad2 and Smad4 are related tumor-suppressor proteins, which, when stimulated by the growth factor TGF-beta, form a complex to inhibit growth. The effector function of Smad2 and Smad4 (Drosophila homolog: Medea) is located in the conserved carboxy-terminal domain (C domain) of these proteins and is inhibited by the presence of the amino-terminal domain (N domain). This inhibitory function of the N domain involves an interaction with the C domain that prevents the association of Smad2 with Smad4. This inhibitory function is increased in tumor-derived forms of Smad2 and 4 that carry a missense mutation in a conserved N domain arginine residue. The mutant N domains have an increased affinity for their respective C domains, inhibit the Smad2-Smad4 interaction, and prevent TGFbeta-induced Smad2-Smad4 association and signaling. Whereas mutations in the C domain disrupt the effector function of the Smad proteins, N-domain arginine mutations inhibit SMAD signaling through a gain of autoinhibitory function. Gain of autoinhibitory function is a new mechanism for inactivating tumor suppressors (Hata, 1997).

Smad2 and Smad3 are structurally highly similar and both mediate TGF-beta signals. Smad4 is distantly related to Smads 2 and 3, and forms a heteromeric complex with Smad2 after TGF-beta or activin stimulation. Smad2 and Smad3 interact with the kinase-deficient TGF-beta type I receptor (TbetaR)-I after it is phosphorylated by TbetaR-II kinase. TGF-beta1 induces phosphorylation of Smad2 and Smad3 in cultured Mv1Lu mink lung epithelial cells. Smad4 is found to be constitutively phosphorylated in Mv1Lu cells: the phosphorylation level remains unchanged upon TGF-beta1 stimulation. Similar results are obtained using HSC4 cells, which are also growth-inhibited by TGF-beta. Smads 2 and 3 interact with Smad4 after TbetaR activation in transfected COS cells. In addition, TbetaR-activation-dependent interaction is observed between Smad2 and Smad3. Smads 2, 3 and 4 accumulate in the nucleus upon TGF-beta1 treatment in Mv1Lu cells, and show a synergistic effect in a transcriptional reporter assay using the TGF-beta-inducible plasminogen activator inhibitor-1 promoter. Dominant-negative Smad3 inhibits the transcriptional synergistic response by Smad2 and Smad4. These data suggest that TGF-beta induces heteromeric complexes of Smads 2, 3 and 4, and their concomitant translocation to the nucleus, which is required for efficient TGF-beta signal transduction (Nakao, 1997).

TGF-beta mediates phosphorylation of Smad2 at two serine residues in the C terminus (namely Ser465 and Ser467), which are then phosphorylated in an obligate order; phosphorylation of Ser465 requires that Ser467 be phosphorylated. Transfection of mutated Smad2 (with the mutation of Ser465 and/or Ser467 to alanine residues) into Mv1Lu cells results in dominant-negative inhibition of TGF-beta signaling. These Smad2 mutants stably interact with an activated TGF-beta receptor complex, in contrast to wild-type Smad2, which interacts only transiently. Mutation of Ser465 and Ser467 in Smad2 abrogate complex formation of this mutant with Smad4 and block the nuclear accumulation not only of Smad2, but also of Smad4. Thus, heteromeric complex formation of Smad2 with Smad4 is required for nuclear translocation of Smad4. Peptides from the C terminus of Smad2 containing phosphorylated Ser465 and Ser467 bind Smad4 in vitro, whereas the corresponding unphosphorylated peptides are less effective. Thus, phosphorylated Ser465 and Ser467 in Smad2 may provide a recognition site for interaction with Smad4, and phosphorylation of these sites is a key event in Smad2 activation (Souchelnytskyi, 1997).

Smad proteins are intracellular mediators of transforming growth factor beta (TGF-beta) and related cytokines and undergo ligand-induced nuclear translocation. The identification of a nuclear localization signal (NLS) in the N-terminal region of Smad 3, the major Smad protein involved in TGF-beta signaling, is described. An NLS-like basic motif [Lys(40)-Lys-Leu-Lys-Lys(44)], conserved among all pathway-specific Smad proteins, not only is responsible for constitutive nuclear localization of the isolated Smad 3 MH1 domain but also is crucial for Smad 3 nuclear import in response to ligand. Mutations in this motif completely abolish TGF-beta-induced nuclear translocation but have no impact on ligand-induced phosphorylation of Smad 3, complex formation with Smad 4, or specific binding to DNA. Hence, Smad 3 proteins with NLS mutations are dominant-negative inhibitors of TGF-beta-induced transcriptional activation. Smad 4, which cannot translocate into the nucleus in the absence of Smad 3 or another pathway-specific Smad, contains a Glu in place of the last Lys in this motif. Smad 3 harboring the same mutation (K44E) does not undergo ligand-induced nuclear import. Conversely, the isolated Smad 4 MH1 domain does not accumulate in the nucleus but becomes nuclear enriched when Glu(49) is replaced with Lys. It is proposed that this highly conserved five-residue NLS motif determines ligand-induced nuclear translocation of all pathway-specific Smads (Xiao, 2000).

Smad interaction with Disabled-2

Using a genetic complementation approach disabled-2 (Dab2: Drosophila homolog Disabled), a structural homolog of the Dab1 adaptor molecule, has been identified as a critical link between the transforming growth factor ß (TGFß) receptors and the Smad family of proteins. Expression of wild-type Dab2 in a TGFß-signaling mutant restores TGFß-mediated Smad2 phosphorylation, Smad translocation to the nucleus and Smad-dependent transcriptional responses. TGFß stimulation triggers a transient increase in association of Dab2 with Smad2 and Smad3, which is mediated by a direct interaction between the N-terminal phosphotyrosine binding domain of Dab2 and the MH2 domain of Smad2. Dab2 associates with both the type I and type II TGFß receptors in vivo, suggesting that Dab2 is part of a multiprotein signaling complex. Together, these data indicate that Dab2 is an essential component of the TGFß signaling pathway, aiding in transmission of TGFß signaling from the TGFß receptors to the Smad family of transcriptional activators (Hocevar, 2001).

The ability of Dab2 to rescue TGFß-induced Smad2 phosphorylation and Smad-dependent transcriptional responses in the mutant 903 cell line points to a critical role for Dab2 in mediating Smad-dependent responses. Additionally, the PTB domain of Dab2 binds directly to the MH2 domain of Smad2 in vitro, and the association of Dab2 with Smad2 and Smad3 occurs in a ligand- and time-dependent manner. Other receptor systems, namely EGF and HGF, have recently been shown to activate Smad2-dependent transcription, while transfection of MEKK1, the upstream activator of the c-Jun N-terminal kinase (JNK) pathway, has been shown to activate Smad2, resulting in its increased association with Smad4 and subsequent nuclear accumulation. These results thus suggest functional crosstalk between signaling pathways, which is also supported by the ability of the Smads to bind to and augment the activity of transcription factors, such as c-Jun and c-Fos, which are known targets of MAPK kinase signaling pathways. The C-terminal region of Dab2 contains proline-rich PXXP sequences that have been shown to bind to SH3-containing signaling proteins. Recently, Dab2 has been shown to interact with Grb2 through this region. Dab2 may also play a role in activation of other signaling pathways through recruitment of SH3-containing signaling molecules to its C-terminal PRD, which may explain why Dab2 can modulate basal as well as TGFß-mediated fibronectin levels in the 903 mutant cell line, a response that is dependent on the JNK pathway and independent of Smad4 expression. The requirement for the C-terminal domain of Dab2 for efficient TGFß signaling is demonstrated by the inability of constructs that lack this domain to complement the mutant cells. It may be that Dab2 serves as a bridge to link the Smad and the JNK signaling pathways, a relationship that has recently been postulated to be required for TGFß-mediated transcriptional responses. Identification of the binding partners for the C-terminal region of Dab2 may thus help to clarify the role of other signaling pathways in TGFß signaling (Hocevar, 2001).

Loss of responsiveness to the growth-inhibitory effects of TGFß is commonly observed in human carcinomas, indicating that inactivation of the TGFß signaling pathway is a common target that may permit cancer initiation and progression. This may occur as a result of mutations in either the type I or type II TGFß receptors or a mutation in a component of the signaling pathway. Dab2 was initially identified as a transcript that was down-regulated in ovarian carcinoma, but was present in normal ovarian epithelial cells. Subsequently, Dab2 expression has been demonstrated to be down-regulated in breast and prostate carcinoma as well. Loss of expression, which is seen early in tumor progression, is not due to loss or gross chromosomal rearrangements of the gene. Re-introduction of Dab2 in ovarian, prostate and choriocarcinoma cell lines results in a decreased growth rate, while Dab2-transfected SKOV3 ovarian carcinoma cells form tumors 50% smaller compared with parental cells when injected into nude mice, demonstrating that Dab2 acts as a tumor suppressor gene. Whether the re-introduction of Dab2 in these cell lines mediates restoration of TGFß signaling is unknown at this time. Dab2 inactivation by mutation or down-regulation, an early event in cancer progression, may thus represent a new mechanism by which cancer cells acquire resistance to the growth-inhibitory effects of TGFß. Further investigation of the role of Dab2 in TGFß signaling may therefore provide important new insight as to how loss of TGFß signaling leads to cancer initiation and progression (Hocevar, 2001).

Nuclear import of Smad2/3 proteins

Smad proteins are cytoplasmic signaling effectors of transforming growth factor-ß (TGF-ß) family cytokines and regulate gene transcription in the nucleus. Receptor-activated Smads (R-Smads) become phosphorylated by the TGF-ß type I receptor. Rapid and precise transport of R-Smads to the nucleus is of crucial importance for signal transduction. Four observations result from focusing on the R-Smad (Smad3). (1) Only activated Smad3 efficiently enters the nucleus of permeabilized cells in an energy- and cytosol-dependent manner. (2) Smad3, via its N-terminal domain, interacts specifically with importin-ß1 and only after activation by receptor. In contrast, the unique insert of exon3 in the N-terminal domain of Smad2 prevents its association with importin-ß1. (3) Nuclear import of Smad3 in vivo requires the action of the Ran GTPase, which mediates release of Smad3 from the complex with importin-ß1. (4) Importin-ß1, Ran, and p10/NTF2 are sufficient to mediate import of activated Smad3. The data describe a pathway whereby Smad3 phosphorylation by the TGF-ß receptor leads to enhanced interaction with importin-ß1 and Ran-dependent import and release into the nucleus. The import mechanism of Smad3 shows distinct features from that of the related Smad2 and the structural basis for this difference maps to the divergent sequences of their N-terminal domains (Furisaki, 2001).

The mechanism of Smad2 import might differ from that of Smad3, despite the conservation of the putative mad homology (MH)1 NLS motif. In agreement with this hypothesis Smad2 can not interact with importin-ß1 due to a unique sequence insert termed TID in its MH1 domain. Interestingly, the same sequence determinant also results in Smad2's inefficient binding to the Smad binding element. Thus, two important functions, nuclear import and DNA binding are coregulated by the same structural motifs in the MH1 domain of Smad2. These results lead to the postulate that in Smad2, importin-mediated nuclear import is deficient and thus the intrinsic import activity of the MH2 domain prevails. In addition, these findings predict that the alternatively spliced form of Smad2 that lacks exon 3 can associate with importin-ß1 and thus be imported by a different mechanism than Smad2 (Furisaki, 2001).

The transcription factor Smad2 is released from cytoplasmic retention by TGFß receptor-mediated phosphorylation, accumulating in the nucleus where it associates with cofactors to regulate transcription. Direct interactions have been uncovered of Smad2 with the nucleoporins CAN/Nup214 and Nup153. These interactions mediate constitutive nucleocytoplasmic shuttling of Smad2. CAN/Nup214 and Nup153 compete with the cytoplasmic retention factor SARA and the nuclear Smad2 partner FAST-1 for binding to a hydrophobic corridor on the MH2 surface of Smad2. TGFß receptor-mediated phosphorylation stimulates nuclear accumulation of Smad2 by modifying its affinity for SARA and Smad4 but not for CAN/Nup214 or Nup153. Thus, by directly contacting the nuclear pore complex, Smad2 undergoes constant shuttling, providing a dynamic pool that is competitively drawn by cytoplasmic and nuclear signal transduction partners (Xu, 2002).

TGFß stimulation leads to phosphorylation and activation of Smad2 and Smad3, which form complexes with Smad4 that accumulate in the nucleus and regulate transcription of target genes. Following TGF-ß stimulation of epithelial cells, receptors remain active for at least 3-4 hr, and continuous receptor activity is required to maintain active Smads in the nucleus and for TGF-ß-induced transcription. Continuous nucleocytoplasmic shuttling of the Smads during active TGF-ß signaling provides the mechanism whereby the intracellular transducers of the signal continuously monitor receptor activity. These data therefore explain how, at all times, the concentration of active Smads in the nucleus is directly dictated by the levels of activated receptors in the cytoplasm (Inman, 2002).

Rather than existing as a static pool of activated Smads in the nucleus, the R-Smads are being constantly dephosphorylated, which results in dissociation of the R-Smad/co-Smad complexes and export of the inactive Smads to the cytoplasm. If the receptors are still active, the Smads will be reactivated and return to the nucleus. If the receptors are no longer active, the Smads accumulate back in the cytoplasm. It is concluded that the complexes dissociate in the nucleus because the export of Smad2 and 3 from the nucleus occurs independently of Smad4 export. Smad4 export depends on the nuclear transport receptor, CRM1, while Smad2 and 3 are actively exported via a CRM1-independent mechanism. Since recent work has demonstrated that the complex formation is mediated by the phosphoserine residues on the R-Smads, it is concluded that dephosphorylation of the R-Smads by an as yet unidentified nuclear phosphatase is responsible for triggering complex dissociation (Inman, 2002).

Loss of mesodermal competence (LMC) during Xenopus development is a well known but little understood phenomenon that prospective ectodermal cells (animal caps) lose their competence for inductive signals, such as activin A, to induce mesodermal genes and tissues after the start of gastrulation. Notch signaling can delay the onset of LMC for activin A in animal caps, although the mechanism by which this modulation occurs remains unknown. Notch signaling also delays the onset of LMC in whole embryos, as it does in animal caps. To better understand this effect and the mechanism of LMC itself, an investiation was carried out to discover at which step of activin signal transduction pathway the Notch signaling acts to affect the timing of the LMC. In this system, ALK4 (activin type I receptor) maintains the ability to phosphorylate the C-terminal region of smad2 upon activin A stimulus after the onset of LMC in both control- and Notch-activated animal caps. However, C-terminal-phosphorylated smad2 can bind to smad4 and accumulate in the nucleus only in Notch-activated animal caps. It is concluded that LMC is induced because C-terminal-phosphorylated smad2 loses its ability to bind to smad4, and consequently can not accumulate in the nucleus. Notch signal activation restores the ability of C-terminal-phosphorylated smad2 to bind to smad4, resulting in a delay in the onset of LMC (Abe, 2005).

During vertebrate development, Activin/Nodal-related ligands signal through Smad2, leading to its activation and accumulation in the nucleus. This study demonstrates that Smad2 constantly shuttles between the cytoplasm and nucleus both in early Xenopus embryo explants and in living zebrafish embryos, providing a mechanism whereby the intracellular components of the pathway constantly monitor receptor activity. An intact microtubule network and kinesin ATPase activity are required for Smad2 phosphorylation and nuclear accumulation in response to Activin/Nodal in early vertebrate embryos and TGF-β in mammalian cells. The kinesin involved is kinesin-1, and Smad2 interacts with the kinesin-1 light chain subunit. Interfering with kinesin activity in Xenopus and zebrafish embryos phenocopies loss of Nodal signaling. These results reveal that kinesin-mediated transport of Smad2 along microtubules to the receptors is an essential step in ligand-induced Smad2 activation (Batut, 2007).

Nuclear export of Smad2 and Smad3 by RanBP3 facilitates termination of TGF-beta signaling

Smad2 and Smad3 (Smad2/3) are key intracellular signal transducers for TGF-beta signaling, and their transcriptional activities are controlled through reversible phosphorylation and nucleocytoplasmic shuttling. However, the precise mechanism underlying nuclear export of Smad2/3 remains elusive. This study reports the essential function of RanBP3 in selective nuclear export of Smad2/3 in the TGF-beta pathway. RanBP3 directly recognizes dephosphorylated Smad2/3, which results from the activity of nuclear Smad phosphatases, and mediates nuclear export of Smad2/3 in a Ran-dependent manner. As a result, increased expression of RanBP3 inhibits TGF-beta signaling in mammalian cells and Xenopus embryos. Conversely, depletion of RanBP3 expression or dominant-negative inhibition of RanBP3 enhances TGFbeta-induced antiproliferative and transcriptional responses. In conclusion, this study supports a definitive role for RanBP3 in mediating Smad2/3 nuclear export and terminating TGF-beta signaling (Dai, 2009).

Smad3-regulated proteasomal degradation events

Atrophin-1-interacting protein 4 (AIP4) is the human homolog of the mouse Itch protein (hItch), an E3 ligase for Notch and JunB. Human enhancer of filamentation 1 (HEF1) has been implicated in signaling pathways such as those mediated by integrin, T cell receptor, and B cell receptor and it functions as a multidomain docking protein. Recent studies suggest that HEF1 is also involved in the transforming growth factor-beta signaling pathways, by interacting with Smad3, a key signal transducer downstream of the TGF-beta type I receptor. The interaction of Smad3 with HEF1 induces HEF1 proteasomal degradation, which was further enhanced by TGF-beta stimulation. The detailed molecular mechanisms of HEF1 degradation regulated by Smad3 were poorly understood. This study reports the function of AIP4 as an ubiquitin E3 ligase for HEF1. AIP4 forms a complex with both Smad3 and HEF1 through its WW domains in a TGF-beta-independent manner and regulates HEF1 ubiquitination and degradation, which can be enhanced by TGF-beta stimulation. These findings reveal a new mechanism for Smad3-regulated proteasomal degradation events and also broaden the network of cross-talk between the TGF-beta signaling pathway and those involving HEF1 and AIP4 (Feng, 2004).

Nuclear interactions of Smad2/3 proteins

Smad3 transduces the signals of TGF-ßs, coupling transmembrane receptor kinase activation to transcriptional control. The membrane-associated molecule SARA (Smad Anchor for Receptor Activation) recruits Smad3 for phosphorylation by the receptor kinase. Upon phosphorylation, Smad3 dissociates from SARA and enters the nucleus, in which its transcriptional activity can be repressed by the Smad co-repressor Ski. Ski is known to interact directly with Smad3 and recruit histone deacetylase to the transcriptional control site, resulting in chromatin remodeling and transcriptional repression. The interaction between Ski and Smad3 is enhanced on TGF-ß stimulation. SARA and Ski recognize specifically the monomeric and trimeric forms of Smad3, respectively. Thus, trimerization of Smad3, induced by phosphorylation, simultaneously activates the TGF-ß signal by driving Smad3 dissociation from SARA and sets up the negative feedback mechanism by Ski. Structural models of the Smad3/SARA/receptor kinase complex and Smad3/Ski complex provide insights into the molecular basis of regulation (Qin, 2002).

Members of the TGF-beta superfamily of signaling molecules work by activating transmembrane receptors with phosphorylating activity (serine-threonine kinase receptors); these in turn phosphorylate and activate SMADs, a class of signal transducers. Activins are growth factors that act primarily through Smad2, possibly in partnership with Smad4, which forms heteromeric complexes with different ligand-specific SMADs after activation. In frog embryos, Smad2 participates in an activin-responsive factor (ARF), which then binds to a promoter element of the Mix.2 gene. The principal DNA-binding component of ARF is FAST-1 (Forkhead activin signal transducer 1), a transcription factor with a novel winged-helix structure. The forkhead domain of FAST-1 is as similar to known members of the forkhead family as these are to one another. Smad4 is present in ARF, and FAST-1, Smad4 and Smad2 co-immunoprecipitate in a ligand-regulated fashion. The site of interaction between FAST-1 and Smad2/Smad4 has been mapped to a novel carboxy-terminal domain of FAST-1, and overexpression of this domain specifically inhibits activin signaling. In a yeast two-hybrid assay, the FAST-1 carboxy terminus interacts with Smad2 but not Smad4. Deletion mutants of the FAST-1 carboxy terminus that still participate in ligand-regulated Smad2 binding no longer associate with Smad4 or ARF. These results indicate that Smad4 stabilizes a ligand-stimulated Smad2-FAST-1 complex as an active DNA-binding factor (Chen, 1997).

In early Xenopus embryos, the prototypical XFast-1/Smad2/Smad4 complex ARF1 is induced at the Mix.2 activin responsive element (ARE) by activin overexpression. ARF2, a related, but much more abundant, complex formed during gastrulation in response to endogenous TGFß family members has been characterized and a novel Fast family member, XFast-3, has been identified as its transcription factor component. Endogenous ARF2 efficiently competes out ARF1 at early gastrulation, due to the ability of XFast-3 to interact with activated Smads (Smad2 and Smad4) with much higher affinity than XFast-1. ARF1 and ARF2 are activated by distinct TGFß family members (activin and Xnr1, and weakly by Xnr2). Using morpholino antisense oligonucleotides to deplete levels of the constituent transcription factors XFast-1 and XFast-3 specifically, an important role has been identified for ARF1 and ARF2 in early Xenopus embryos in controlling the convergent extension movements of gastrulation (Howell, 2002).

Smads regulate the transcription of defined genes in response to TGFbeta receptor activation, although the mechanisms of Smad-mediated transcription are not well understood. The TGFbeta-inducible Smad3 uses Smad4/DPC4 and CBP/p300 as transcriptional coactivators, which both associate with Smad3 in response to TGFbeta. The association of CBP with Smad3 was localized to the carboxyl terminus of Smad3, which is required for transcriptional activation, and a defined segment in CBP. Mad4 shows ligand-inducible interaction with the two CBP segments in two-hybrid assays in Mv1Lu cells. This is in contrast with the lack of Smad4-CBP interaction in coimmunoprecipitation and yeast two-hybrid experiments, suggesting that this interaction is mediated through the ligand-dependent association of Smad4 with endogenous Smad3, which in turn interacts in a ligand-dependent fashion with CBP. In addition, coexpression of Smad4 in SW480.7 cells increases the interaction of Smad3 with the amino- and carboxy-terminal domains of CBP, whereas coexpression of Smad3 promotes the association of Smad4 and CBP in mammalian two-hybrid assays. These results thus suggest a ternary protein complex, whereby the ligand-dependent interaction of Smad3 with CBP (primarily its carboxy-terminal segment) is stabilized by Smad4. This interpretation is consistent with the participation of all three proteins in a nucleoprotein complex at the promoter. The stabilization by Smad4 may be required for the ability of CBP to efficiently coactivate Smad3. CBP/p300 stimulates both TGFbeta- and Smad-induced transcription in a Smad4/DPC4-dependent fashion. Smad3 transactivation and TGFbeta-induced transcription are inhibited by expressing E1A, which interferes with CBP functions (Feng, 1998).

Smad proteins are novel transcriptional regulators mediating the signaling of the transforming growth factor-beta (TGFbeta) superfamily. Coactivators such as p300/CBP promote transactivation by various transcription factors through a direct interaction with them. Adenoviral oncoprotein E1A, which binds p300, inhibits the signaling of TGFbeta. These findings raise the possibility that p300 may be involved in TGFbeta signaling. An investigation was carried out to see whether p300 is involved in transactivation by Smads. p300 enhances the Smad-induced transactivation of p3TP-Lux, a TGFbeta responsive reporter. E1A inhibits this enhancement, and the inhibition requires E1A's ability to bind p300/CBP. p300 and Smad3, as well as Smad2, interact in vivo in a ligand-dependent manner. The binding region in Smad3 is its C-terminal half that possesses an intrinsic transactivation activity. The binding region in p300 maps to its C-terminal 678 amino acids. The minimal Smad2/3-interacting region, as well as the rest of the p300, inhibit the transactivation of p3TP-Lux in a dominant-negative fashion. It is concluded that p300 interacts with Smad2 and Smad3 in a ligand-dependent manner, and enhances the transactivation by Smads. These results present the molecular basis of the transactivation by Smad proteins (Nishihara, 1998).

Human Smad2 and Smad4, two essential Smad proteins involved in mediating TGF-beta transcriptional responses in endothelial and other cell types, can functionally interact with the transcriptional coactivator CREB binding protein (CBP). This interaction is specific in that it requires ligand (TGF-beta) activation and is mediated by the transcriptional activation domains of the Smad proteins. A closely related, but distinct endothelial-expressed Smad protein, Smad7, which does not activate transcription in endothelial cells, does not interact with CBP. Furthermore, Smad2,4-CBP interactions involve the COOH terminus of CBP, a region that interacts with other regulated transcription factors such as certain signal transduction and transcription proteins and nuclear receptors. Smad-CBP interactions are required for Smad-dependent TGF-beta-induced transcriptional responses in endothelial cells, as evidenced by inhibition with overexpressed 12S E1A protein and reversal of this inhibition with exogenous CBP. This report demonstrates a functional interaction between Smad proteins and an essential component of the mammalian transcriptional apparatus (CBP) and extends insight into how Smad proteins may regulate transcriptional responses in many cell types. Thus, functional Smad-coactivator interactions may be an important locus of signal integration in endothelial cells (Topper, 1998).

The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, a yeast two-hybrid screen was performed. The results of the screen and subsequent co-immunoprecipitation studies have identified Smad2 and Smad3, two transcriptional activators that mediate the type beta transforming growth factor (TGF-beta) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulate in the nucleus in the absence of added TGF-beta. DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski represses TGF-beta-induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski represses a TGF-beta-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also represses synergistic activation of promoters by combinations of Smad proteins but fails to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-beta-induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolishes TGF-beta-mediated growth inhibition in a prostate-derived epithelial cell line (W. Xu, 2000).

Smad proteins transduce signals for transforming growth factor-beta (TGF-beta)-related factors. Smad proteins activated by receptors for TGF-beta form complexes with Smad4. These complexes are translocated into the nucleus and regulate ligand-induced gene transcription. 12-O-tetradecanoyl-13-acetate (TPA)-responsive gene promoter elements (TREs) are involved in the transcriptional responses of several genes to TGF-beta. AP-1 transcription factors, composed of c-Jun and c-Fos, bind to and direct transcription from TREs, which are therefore known as AP1-binding sites. Smad3 interacts directly with the TRE and Smad3 and Smad4 can activate TGF-beta-inducible transcription from the TRE in the absence of c-Jun and c-Fos. Smad3 and Smad4 also act together with c-Jun and c-Fos to activate transcription in response to TGF-beta, through a TGF-beta-inducible association of c-Jun with Smad3 and an interaction of Smad3 and c-Fos. These interactions complement interactions between c-Jun and c-Fos, and between Smad3 and Smad4. This mechanism of transcriptional activation by TGF-beta, through functional and physical interactions between Smad3-Smad4 and c-Jun-c-Fos, shows that Smad signaling and MAPK/JNK signaling converge at AP1-binding promoter sites (Zhang, 1998).

Transforming growth factor-beta arrests growth of epithelial cells by inducing the transcription of p15Ink4B, a cyclin-dependent kinase inhibitor. p15Ink4B induction is mediated by a TGF-beta-induced complex of Smad2, Smad3, Smad4 and Sp1. Mutations in the Sp1- or Smad-binding sequences decrease or abolish the TGF-beta responsiveness of the p15Ink4B promoter. Interference with, or deficiency in, Smad2, Smad3 or Smad4 functions also reduces or abolishes the TGF-beta-dependent p15Ink4B induction, whereas the absence of Sp1 reduces the basal and TGF-beta-induced p15Ink4B transcription. In the nucleoprotein complex, Smad2 interacts through its C-domain with Sp1 and enhances the DNA binding and transcriptional activity of Sp1. Smad3 interacts indirectly with Sp1 through its association with Smad2 and/or Smad4, and binds directly to the p15Ink4B promoter. Finally, Smad4 interacts through its N-domain with Sp1. These data demonstrate the physical interactions and functional cooperativity of Sp1 with a complex of Smad2, Smad3 and Smad4 in the induction of the p15Ink4B gene. These findings explain the tumor suppressor roles of Smad2 and Smad4 in growth arrest signaling by TGF-beta (Feng, 2000).

The c-Myc oncogene has been implicated in the genesis of diverse human tumors. Ectopic expression of the c-Myc gene in cultured epithelial cells causes resistance to the antiproliferative effects of TGF-ß. However, little is known about the precise mechanisms of c-Myc-mediated TGF-ß resistance. In this study, it has been revealed that c-Myc physically interacts with Smad2 and Smad3, two specific signal transducers involved in TGF-ß signaling. Through its direct interaction with Smads, c-Myc binds to the Sp1-Smad complex on the promoter of the p15Ink4B gene, thereby inhibiting the TGF-ß-induced transcriptional activity of Sp1 and Smad/Sp1-dependent transcription of the p15Ink4B gene. These results suggest that oncogenic c-Myc promotes cell growth and cancer development partly by inhibiting the growth inhibitory functions of Smads (Feng, 2002).

Cell proliferation and differentiation are regulated by growth regulatory factors such as transforming growth factor-beta and the lipophilic hormone, vitamin D. TGF-beta causes activation of SMAD proteins acting as coactivators or transcription factors in the nucleus. Vitamin D controls transcription of target genes through the vitamin D receptor (VDR). Smad3, one of the SMAD proteins downstream in the TGF-beta signaling pathway, has been found to act in mammalian cells as a coactivator specific for ligand-induced transactivation of VDR by forming a complex with a member of the steroid receptor coactivator-1 protein family in the nucleus. Thus, Smad3 may mediate cross-talk between vitamin D and TGF-beta signaling pathways (Yanagisawa, 2000).

Members of the transforming growth factor-beta superfamily mediate a broad range of biological activities by regulating the expression of target genes. Smad proteins play a critical role in this process by binding directly to the promoter elements and/or associating with other transcription factors. TGF-beta1 up-regulates several genes transcriptionally through Sp1 binding sites; however, the mechanism of TGF-beta induction of gene expression through Sp1 sites is largely unknown. A novel 38-base pair TGF-beta-responsive element has been identified in the human plasminogen activator inhibitor-1 (PAI-1) promoter, which contains two Sp1 binding sites, and is required for TGF-beta-induced Smad-dependent transcriptional activation. Three canonical Sp1 binding sites also support strong transcriptional activation by TGF-beta and Smads from a minimal heterologous promoter. TGF-beta induction of PAI-1 and p21 is blocked by the Sp1 inhibitor mithramycin, implicating Sp1 in the in vivo regulation of these genes by TGF-beta. The association between endogenous Sp1 and Smad3 is induced by TGF-beta in several cell lines; however, Smad4 shows constitutive interaction with Sp1. These data provide novel insights into the mechanism by which TGF-beta up-regulates the expression of several genes by activating Sp1-dependent transcription through the induction of Smad/Sp1 complex formation (Datta, 2000).

Members of the transforming growth factor-ß superfamily play critical roles in controlling cell growth and differentiation. Effects of TGF-ß family ligands are mediated by Smad proteins. To understand the mechanism of Smad function, attempts were made to identify novel interactors of Smads by use of a yeast two-hybrid system. A 396-amino acid nuclear protein termed SNIP1 was cloned and shown to harbor a nuclear localization signal (NLS) and a Forkhead-associated (FHA) domain. The FHA domain has been shown to be a modular phosphothreonine recognition motif expressed on a variety of nuclear proteins, and is suggested to play a docking role analogous to that of the modular phosphotyrosine domain recognition site, SH2. The carboxyl terminus of SNIP1 interacts with Smad1 and Smad2 in yeast two-hybrid as well as in mammalian overexpression systems. However, the amino terminus of SNIP1 harbors binding sites for both Smad4 and the coactivator CBP/p300. Interaction between endogenous levels of SNIP1 and Smad4 or CBP/p300 is detected in NMuMg cells as well as in vitro. Overexpression of full-length SNIP1 or its amino terminus is sufficient to inhibit multiple gene responses to TGF-ß and CBP/p300, as well as the formation of a Smad4/p300 complex. Studies in Xenopus laevis further suggest that SNIP1 plays a role in regulating dorsomedial mesoderm formation by the TGF-ß family member nodal. Thus, SNIP1 is a nuclear inhibitor of CBP/p300 and its level of expression in specific cell types has important physiological consequences by setting a threshold for TGF-ß-induced transcriptional activation involving CBP/p300 (Kim, 2000).

A region of Smad4 called the SAD, located in the middle linker region just amino-terminal to the MH2 domain (amino acids 275-322), has been shown to be both necessary and sufficient for the transcriptional-activating activity of Smad4 through its interaction with CBP/p300. It is noteworthy that Smad4 deletion constructs of the MH2 domain inclusive of the SAD motif are unable to bind SNIP1 either in vitro or in vivo. A recent crystallographic structural analysis of the transcriptionally active domain of Smad4, including the SAD (amino acids 276-552), has shown that inclusion of the SAD alters the previously published structure of the inactive MH2 domain by stabilizing a glutamine-rich helical extension from the core. Thus, it is suggested that inclusion of this domain, in the absence of other constraints imposed by the MH1 domain, may restrict interactions of SNIP1 with the MH2 domain (Kim, 2000 and references therein).

Importantly, SNIP1 also interacts constitutively with CBP/p300 through the same amino-terminal domain that mediates its principal interaction with Smad4. On this basis, a model is proposed in which the relative levels of SNIP1 and nuclear Smad4 contribute to setting limits on both the basal activity as well as the maximum level of transcriptional activation of target genes that can be achieved in a cell following ligand activation. In this model, high levels of SNIP1 relative to nuclear Smad4 will restrict the interaction between the activated Smad complex and CBP/p300 with resultant inhibition of the ligand-dependent transcriptional response. However, high levels of Smad4 relative to that of SNIP1 will favor the formation of transcriptionally active Smad4/coactivator complexes and Smad4/SNIP1 complexes, which may initiate proteasomal degradation of SNIP1. In the case of the homeodomain repressor TGIF, which, like SNIP1, suppresses both basal and TGF-ß-activated transcription, a competitive mechanism has also been proposed whereby the relative levels of TGIF and Smad2 determine formation of mutually exclusive Smad2/inhibitor and Smad2/coactivator complexes. Suppression of signaling responses from TGF-ß family ligands by the adenoviral oncoprotein E1A, which interacts with the MH2 domain R-Smads, has also been shown to involve competition for binding of these Smads to the C/H3 domain of p300. These data suggest that SNIP1 acts in a similar fashion to inhibit interaction of Smad4 with the C/H1 domain of p300 (Kim, 2000 and references therein).

SNIP1 mRNA and protein are broadly expressed, and, in the limited number of cell lines examined, do not change with TGF-ß treatment. However, preliminary observations of highly selective patterns of immunohistochemical staining for SNIP1 in tissues suggest that its expression is under stringent control. Activation of BMP or TGF-ß-signaling pathways can lead to degradation of SNIP1 through a process involving antizyme and the proteasome subunit HsN3. Proteolytic degradation has also been suggested to be important in regulation of the suppressor activity of Ski and SnoN, where it has been proposed that a TGF-ß-signal leads to activation of Smad3, which then mediates degradation of SnoN and Ski. Mechanisms such as these suggest that the balance of activated Smad complexes and repressor proteins in the nucleus is critical to regulation of the signal-transduction pathways from TGF-ß family ligands (Kim, 2000 and references therein).

Although the cloning and characterization of SNIP1 was based on its ability to bind to Smad proteins and to inhibit Smad-dependent signaling, its ability to inhibit the transcriptional activating activity of the NF-kappaB transcriptional activator p65 shows that its action is not limited to Smad-dependent signaling pathways. Because NF-kappaB, like Smad4, interacts with p300 through the C/H1 domain, it is speculated that SNIP1 might also suppress transcription dependent on other factors interacting with this same domain of p300 including Stat2 and Stat3, ets-1, p53 and MDM2. However, the inability of SNIP1 to inhibit the p300-dependent activity of Gal4-p53 now suggests that the specificity of SNIP1 may be more narrowly defined in terms of the region of p300 with which it interacts and may possibly also be restricted by other parameters such as direct binding to the transcription factor itself. As such, SNIP1 could both limit the magnitude of a particular cellular response and serve to mediate an additional level of crosstalk between various transcriptional regulators that interact with it to fine tune cellular proliferation, differentiation, and response to injury and stress (Kim, 2000 and references therein).

Smad transcription factors mediate the actions of TGF-ß cytokines during development and tissue homeostasis. TGF-ß receptor-activated Smad2 regulates gene expression by associating with transcriptional co-activators or co-repressors. The Smad co-repressor TGIF competes with the co-activator p300 for Smad2 association, such that TGIF abundance helps determine the outcome of a TGF-ß response. Small alterations in the physiological levels of TGIF can have profound effects on human development, as shown by the devastating brain and craniofacial developmental defects in heterozygotes carrying a hypomorphic TGIF mutant allele. TGIF levels modulate sensitivity to TGF-ß-mediated growth inhibition, TGIF is a short-lived protein and epidermal growth factor (EGF) signaling via the Ras-Mek pathway causes the phosphorylation of TGIF at two Erk MAP kinase sites, leading to TGIF stabilization and favoring the formation of Smad2-TGIF co-repressor complexes in response to TGF-ß. These results identify the first mechanism for regulating TGIF levels and suggest a potential link for Smad and Ras pathway convergence at the transcriptional level (Lo, 2001).

TGIF acts at the intersection of Ras and Smad pathways. Expression of oncogenic Ha-Ras inhibits G1 cell cycle arrest by saturating concentrations of TGF-ß. In this context activation of the Mek pathway, whether by EGF stimulation, expression of a constitutively active Ras, or expression of an activated Mek, leads to a rapid increase in the level of the TGIF protein, whereas pharmacological inhibition of activated Mek blocks the EGF-induced increase in TGIF level. This enhancement in TGIF level occurs by accumulation of a phosphatase-sensitive, hyperphosphorylated TGIF form, which has a retarded electrophoretic mobility. The increase in phosphorylation of this upper form of TGIF in response to EGF requires a pair of Erk MAP kinase consensus sites near the C-terminus of TGIF. In addition, this upper TGIF form has a longer metabolic half-life than the lower TGIF form, leading to an overall build-up in the steady-state level of TGIF itself and hence its increased assembly with activated Smad and HDAC, forming co-repressor complexes. Thus, the effect of the Ras-Mek pathway on TGIF protein stability described here suggests a novel mechanism for modulating TGF-ß signaling at the transcriptional level (Lo, 2001).

The interplay between the TGF-ß and EGF/Ras signal transduction pathways occurs at other levels as well. These include Ras inhibition of TGF-ß receptor expression and of Smad accumulation in the nucleus. EGF stimulation via Ras activation has been shown to diminish nuclear accumulation of TGF-ß-activated Smad proteins. However, at high levels of TGF-ß signaling, EGF addition or transformation by an oncogenic H-ras allele is unable to prevent Smad entry into the nucleus, even though it can profoundly alter the cellular response to TGF-ß. The subcellular distribution of Smad in the cell is a function of its interactions with protein partners in the cytoplasm and nucleus. Smad proteins have intrinsic nuclear import activity that in the basal state is negated by contacts with SARA (Smad anchor for receptor activation). Likewise, overexpression of a nuclear partner of Smad, namely the Smad DNA binding co-factor FAST1, leads to Smad2 nuclear accumulation in the absence of receptor activation. Receptor-mediated Smad phosphorylation diminishes the affinity of Smad for SARA, which results in Smad movement to the nucleus and association with various protein partners. In light of these insights, attenuation of Smad nuclear accumulation by Ras-Mek signaling could result not only from direct effects on Smad nuclear import and/or export machinery, but also from effects of Ras-Mek signaling on Smad interactions with protein partners (Lo, 2001).

Ras signaling has long been known to act as a modifier of cellular responsiveness to TGF-ß. During embryo development, many processes are cooperatively stimulated by TGF-ß and Ras signaling. In principle, this cooperativity could be achieved by Ras modulating gene activation or repression by Activin, Nodal and other TGF-ß-like signals. Smad complexes activated by these factors can associate with either general co-activators, such as p300/CBP, or co-repressors like TGIF that specifically target nuclear Smad proteins. Regulation of co-activator activity by mitogenic signals, such as EGF, may result in general transcriptional upregulation. Increased TGIF activity in response to the same signals provides a mechanism to repress a specific subset of gene responses. Hence, regulation of TGIF levels by Ras signaling allows an effective and selective way to adjust the level of Smad-activated transcription in vivo. TGIF thus provides a potential link within the nucleus between signals that activate the Ras pathway and TGF-ß morphogens that exert different effects on gene expression at different levels of signal (Lo, 2001).

Likewise, during tumorigenesis, transformation by disregulation of Ras or EGFR and related tyrosine kinases in various types of epithelial cells modifies their responsiveness to TGF-ß by conferring resistance to growth inhibition by TGF-ß, while allowing other responses to TGF-ß, including extracellular matrix production, cellular motility and stimulation of angiogenesis. In fact, TGF-ß collaborates with oncogenic Ras to bring about metastatic and invasive phenotypic alterations in Ras-transformed mammary epithelial cells. Thus, oncogenic Ras signaling can attenuate certain TGF-ß responses while allowing or even enabling others. These results suggest that stabilization of TGIF provides a mechanism for the modification of Smad responses by Ras-Mek signaling. In this context, it is noteworthy that a recently identified form of human TGIF, TGIF2, has been found to be amplified and overexpressed in a third of ovarian cancer cell lines. TGIF and TGIF2 are highly conserved in the C-terminus containing the EGF-inducible phosphorylation sites (Lo, 2001).

Smads are signal mediators for the members of the transforming growth factor-ß superfamily. Upon phosphorylation by the TGF-ß receptors, Smad3 translocates into the nucleus, recruits transcriptional coactivators and corepressors, and regulates transcription of target genes. Smad3 activated by TGF-ß is degraded by the ubiquitin-proteasome pathway. Smad3 interacts with a RING finger protein, ROC1, through its C-terminal MH2 domain in a ligand-dependent manner. An E3 ubiquitin ligase complex ROC1-SCFFbw1a consisting of ROC1, Skp1, Cullin1, and Fbw1a (also termed ßTrCP1) induces ubiquitination of Smad3. Recruitment of a transcriptional coactivator, p300, to nuclear Smad3 facilitates the interaction with the E3 ligase complex and triggers the degradation process of Smad3. Smad3 bound to ROC1-SCFFbw1a is then exported from the nucleus to the cytoplasm for proteasomal degradation. TGF-ß/Smad3 signaling is thus irreversibly terminated by the ubiquitin-proteasome pathway (Fukuchi, 2001).

Among the negative regulators of Smad function, SnoN and Ski are two closely related members of the Ski family of nuclear proto-oncoproteins. When overexpressed, they cause oncogenic transformation of chicken and quail embryo fibroblasts as well as muscle differentiation of quail embryo cells. High levels of Ski or SnoN are detected in many types of human cancer cells. Interestingly, mice lacking one copy of the sno gene were also found to be more susceptible to chemical carcinogens. Thus, the oncogenic potential of SnoN appears to be related to deregulation of its normal expression levels. Human SnoN is a ubiquitously expressed transcriptional corepressor of 684 amino acids that interacts with Smad2, Smad3, and Smad4 to antagonize TGF-ß signaling. SnoN may function to maintain the repressed state of TGF-ß target genes in the absence of TGF-ß and may also participate in negative feedback control of TGF-ß signaling. On TGF-ß stimulation, a rapid degradation of SnoN occurs, most likely mediated by Smad3 and to a lesser extent, Smad2. The removal of the inhibitory SnoN may be crucial for activation of TGF- signaling since this allows the Smads to activate transcription of TGF-ß responsive genes (Stroschein, 2001 and references therein).

On TGF-ß stimulation, Smad3 and Smad2 translocate into the nucleus and induce a rapid degradation of SnoN, allowing activation of TGF-ß target genes. Smad2- or Smad3-induced degradation of SnoN requires the ubiquitin-dependent proteasome and can be mediated by the anaphase-promoting complex (APC) and the UbcH5 family of ubiquitin-conjugating enzymes. Smad3 and to a lesser extent, Smad2, interact with both the APC and SnoN, resulting in the recruitment of the APC to SnoN and subsequent ubiquitination of SnoN in a destruction box (D box)-dependent manner. In addition to the D box, efficient ubiquitination and degradation of SnoN also requires the Smad3 binding site in SnoN as well as key lysine residues necessary for ubiquitin attachment. Mutation of either the Smad3 binding site or lysine residues results in stabilization of SnoN and in enhanced antagonism of TGF-ß signaling. These studies elucidate an important mechanism and pathway for the degradation of SnoN and more importantly, reveal a novel role of the APC in the regulation of TGF-ß signaling (Stroschein, 2001).

TGF-ß/activin-induced Smad2/Smad4 complexes are recruited to different promoter elements by transcription factors, such as Fast-1 or the Mix family proteins Mixer and Milk, through a direct interaction between Smad2 and a common Smad interaction motif (SIM) in the transcription factors. Residues in the SIM critical for Mixer-Smad2 interaction have been identified and their functional importance has been cofirmed by demonstrating that only Xenopus and zebrafish Mix family members containing a SIM with all the correct critical residues can bind Smad2 and mediate TGF-ß-induced transcriptional activation in vivo. Significant sequence similarity has been identified between the SIM and the Smad-binding domain (SBD) of the membrane-associated protein SARA (Smad anchor for receptor activation). Molecular modelling, supported by mutational analyses of Smad2 and the SIM and the demonstration that the SARA SBD competes directly with the SIM for binding to Smad2, indicates that the SIM binds Smad2 in the same hydrophobic pocket as does the proline-rich rigid coil region of the SARA SBD. Thus, different Smad2 partners, whether cytoplasmic or nuclear, interact with the same binding pocket in Smad2 through a common proline-rich motif (Randall, 2002).

Members of the GATA family of zinc finger transcription factors are genetically controlled 'master' regulators of development in the hematopoietic and nervous systems. Whether GATA factors also serve to integrate epigenetic signals on target promoters is, however, unknown. The TGF-ß superfamily is a large group of phylogenetically conserved secreted factors controlling cell proliferation, differentiation, migration, and survival in multiple tissues. GATA-3, a key regulator of T helper cell development, was found to directly interact with Smad3, an intracellular signal transducer of TGF-ß. Complex formation requires a central region in GATA-3 and the N-terminal domain of Smad3. GATA-3 mediates recruitment of Smad3 to GATA binding sites independently of Smad3 binding to DNA, and the two factors cooperate synergistically to regulate transcription from the IL-5 promoter in a TGF-ß-dependent manner. Treatment of T helper cells with TGF-ß promotes the formation of an endogenous Smad3/GATA-3 nuclear complex and stimulates production of the Th2 cytokine IL-10 in a Smad3- and GATA-3-dependent manner. Through its interaction with Smad3, GATA-3 is able to integrate a genetic program of cell differentiation with an extracellular signal, providing a molecular framework for the effects of TGF-ß on the development and function of specific subsets of immune cells and possibly other cell types (Blokzijl, 2002).

Inhibitory Smads (I-Smads), including Smad6 and Smad7, were initially characterized as cytoplasmic antagonists in the transforming growth factor-beta signaling pathway. However, I-Smads are also localized in the nucleus. Smad6 can function as a transcriptional co-repressor. Both Smad6 and Smad7 interact with histone deacetylases (HDACs). The acetylation state of core histones plays a critical role in gene transcription regulation. An HDAC inhibitor, trichostatin A, releases Smad6-mediated transcription repression. Moreover, class I HDACs (HDAC-1 and -3), not class II HDACs (HDAC-4, -5, and -6), co-immunoprecipitate with Smad6. Endogenous HDAC-1 interacts with both Smad6 and Hoxc-8. Mapping of the interaction domain indicates Smad6 MH2 domain is mainly involved in recruiting HDAC-1. Most interestingly, Smad6 also binds to DNA through its MH1 domain, and the MH2 domain of Smad6 masks this binding activity, indicating that Smad6 MH1 and MH2 domains associate reciprocally and inhibit each other's function. Hoxc-8 induces Smad6 binding to DNA as a transcriptional complex. These findings reveal that I-Smads act as antagonists in the nucleus by recruiting HDACs (Bai, 2002).

Goosecoid (Gsc) is a homeodomain-containing transcription factor present in a wide variety of vertebrate species and known to regulate formation and patterning of embryos. In embryonic carcinoma P19 cells, the transcription factor TFII-I forms a complex with Smad2 upon transforming growth factor ß (TGFß)/activin stimulation, is recruited to the distal element (DE) of the Gsc promoter, and activates Gsc transcription. BEN is a member of the TFII-I family of transcription factors. TFII-I and BEN share multiple helix-loop-helix (HLH) domains and a leucine zipper domain. Despite the structural similarity, TFII-I often acts as a transcriptional activator, and BEN often acts as a transcriptional repressor. Downregulation of endogenous TFII-I by small inhibitory RNA in P19 cells abolishes the TGFß-mediated induction of Gsc. Similarly, Xenopus embryos with endogenous TFII-I expression downregulated by injection of TFII-I-specific antisense oligonucleotides exhibit decreased Gsc expression. Unlike TFII-I, the related factor BEN (binding factor for early enhancer) is constitutively recruited to the distal element in the absence of TGFß/activin signaling and is replaced by the TFII-I/Smad2 complex upon TGFß/activin stimulation. Overexpression of BEN in P19 cells represses the TGFß-mediated transcriptional activation of Gsc. These results suggest a model in which TFII-I family proteins have opposing effects in the regulation of the Gsc gene in response to a TGFß/activin signal (Ku, 2005).

Negative regulation of Activin/Nodal signaling by SRF during Xenopus gastrulation; SRF interacts with Smad2 and FAST-1

Activin/Nodal signaling is essential for germ-layer formation and axial patterning during embryogenesis. Recent evidence has demonstrated that the intra- or extracellular inhibition of this signaling is crucial for ectoderm specification and correct positioning of mesoderm and endoderm. This study analyzed the function of Xenopus serum response factor (XSRF) in establishing germ layers during early development. XSRF transcripts are restricted to the animal pole ectoderm in Xenopus early embryos. Ectopic expression of XSRF RNA suppresses mesoderm induction, both in the marginal zone in vivo and caused by Activin/Nodal signals in animal caps. Dominant-negative mutant or antisense morpholino oligonucleotide-mediated inhibition of XSRF function expands the expression of mesendodermal genes toward the ectodermal territory and enhances the inducing activity of the Activin signal. SRF interacts with Smad2 and FAST-1, and inhibits the formation of the Smad2-FAST-1 complex induced by Activin. These results suggest that XSRF might act to ensure proper mesoderm induction in the appropriate region by inhibiting the mesoderm-inducing signals during early embryogenesis (Yun, 2007; full text of article).

SRF acts as a Smad2-binding partner to inhibit Activin/Nodal-dependent transcription. Recent evidence points to several mechanisms by which interference with Smad transcriptional complexes negatively regulates TGF-β signaling. For instance, the oncoprotein Ski, a transcriptional co-repressor, competes with R-Smads for association with Smad4, disrupting the formation of a functional complex between Smad4 and R-Smads. Moreover, Ski can also repress Smads directly by recruiting the transcriptional repressor N-CoR as well as the histone deacetylase complex (HDAC). DRAP1 interacts with FAST-1, thereby preventing FAST-Smad2-Smad4 complex from binding to its cognate DNA targets. In addition, inhibitory Smads (Smad6 and Smad7) compete with R-Smads for binding to activated type-I receptors and thus inhibit the phosphorylation of R-Smads. The data show that SRF precludes the association of Smad2 and FAST-1 induced by Activin signal. This suggests that SRF could function to impede Smad2-FAST complex-mediated transcription in Activin/Nodal signaling. Supporting this, gain-of-function phenotypes of SRF are similar to those of maternal FAST-depleted embryos and can be rescued by coexpression of an activated mutant of FAST-1 (FAST-VP16A). The possibility cannot be excluded that SRF could also affect FAST-independent transcription, since FoxH1 depletion in animal caps has no effect on the induction of Nodal target genes in response to Xnr1 or Activin ligands, whereas overexpression of XSRF significantly inhibits the same response. Given that Smad2 binds to SRF via its MH2 domain, which associates with the general transcriptional co-activators p300 and CAATT-binding factor (CBF), SRF may repress the Smad2-mediated transcription of various genes in a cell context-dependent manner by preventing the interaction of the MH2 domain and these co-activators. By contrast, a recent study shows that SRF associates with Smad3 and activates TGF-β1-dependent transcription during myofibroblast differentiation. In contrast, the general mechanism by which SRF regulates gene transcription is known to involve cooperation with the ternary complex factors (TCF), which are phosphorylated and activated by MAP kinase cascades. In Xenopus, SRF was shown, together with the TCF-type Ets protein Elk-1, to regulate the transcription of Xegr-1, an organizer-specific gene, downstream of the FGF-initiated MAP kinase pathway. Interestingly, TGF-β receptors can activate MAP kinase signaling pathways. These activated MAP kinase cascades inhibit or enhance Smad activity by phosphorylating it, depending on the cell signaling context; but, in some cases, they regulate Smad-independent transcription. It will be interesting to examine whether the TCF- and the Smad-dependent SRF regulation of gene transcription involve distinct signaling cascades or whether both of them could be controlled via MAPK pathways by TGF-β signaling. In addition, it remains to be investigated in more detail how SRF could regulate gene expression in a positive or negative fashion depending on its transcription-factor binding-partners (Yun, 2007).

HEB and E2A function as SMAD/FOXH1 cofactors

Nodal signaling, mediated through SMAD transcription factors, is necessary for pluripotency maintenance and endoderm commitment. A new motif, termed SMAD complex-associated (SCA), was identified that is bound by SMAD2/3/4 and FOXH1 in human embryonic stem cells (hESCs) and derived endoderm. Two basic helix-loop-helix (bHLH) proteins-HEB and E2A-bind the SCA motif at regions overlapping SMAD2/3 and FOXH1. Furthermore, HEB and E2A associate with SMAD2/3 and FOXH1, suggesting they form a complex at critical target regions. This association is biologically important, as E2A is critical for mesendoderm specification, gastrulation, and Nodal signal transduction in Xenopus tropicalis embryos. Taken together, E proteins are novel Nodal signaling cofactors that associate with SMAD2/3 and FOXH1 and are necessary for mesendoderm differentiation (Yoon, 2011).

ChIP-seq was used to generate genome-wide occupancy maps for the Nodal signaling factors SMAD2/3, SMAD3, SMAD4, and FOXH1 in both hESCs and derived endoderm. This study sought to identify novel SMAD complex cofactors by performing de novo motif discovery on the SMAD/FOXH1 genomic targets. Three nonrepetitive motifs were identified that were consistently enriched in all data sets (SMAD2/3, SMAD3, SMAD4, and FOXH1) and in both cell types, hESCs and endoderm. The first and second motifs contain the canonical SMAD- and FOXH1-binding sites, respectively, confirming their genome-wide cooperativity in regulating Nodal signaling and further validating the antibodies used for ChIP. The third motif, CCTGCTG, has not previously been shown to associate with any of the SMAD/FOXH1 complex proteins.This element is referred to as the SCA (SMAD complex-associated) motif (Yoon, 2011).

This study presents strong genomic, biochemical, and functional evidence that E2A and HEB interact with SMAD2/3/4 and FOXH1 to regulate transcription of Nodal target genes. E2A and HEB associate with SMAD2/3 and FOXH1 at the SCA consensus site, which is functionally conserved between frogs and humans. The genomic identification of this site using the power of large sequence reads in multiple data sets provided inroads into testing the interaction of E2A, HEB, and the SMAD/FOXH1 complex. Using biochemical approaches, this study shows that these proteins interact in a DNA-independent manner, but then associate with similar target regions. Based on evidence presented in this study, it is hypothesized that a complex consisting of E2A, HEB, SMAD2/3, and FOXH1 forms within the nucleus in response to Nodal, but that maintenance of this complex is independent of continual Nodal signaling. Overall, it is suggested that E2A and HEB are key regulators of SMAD2/3-mediated transcriptional responses, and thus are fundamental Nodal cofactors that have not previously been implicated in this important developmental pathway (Yoon, 2011).

While genomic and biochemical association is suggestive of a key signaling role, the phenotypic effect of knocking down e2a in X. tropicalis embryos is highly reminiscent of phenotypes resulting from perturbation of other key Nodal signaling factors, such as overexpression of a dominant-negative Nodal receptor or of the Nodal antagonists Cerberus-short and Lefty. Furthermore, it was shown epistatically that e2a knockdown inhibits the ability of both Activin and Xnr1 to induce bottle cell formation, strongly suggesting a key downstream role in the pathway. In the mouse, the roles of HEB and E2A and their family member, E2-2, have been extensively characterized as essential factors in hematopoiesis. The phenotypes of single-gene knockout models for E2A and HEB demonstrated that E2A was the primary E-protein member driving B-cell development, but that both E2A and HEB were required for proper T-cell development. Interestingly, however, there is very strong evidence that these proteins are highly redundant due to their heterodimerizing abilities. Dominant-negative HEB, which can also disrupt E2A function through nonproductive heterodimer formation, causes a stronger phenotype than the heb-null mutation. In B-cell development, HEB, driven by the E2A promoter, can rescue E2A loss of function. These complex genetics and the associated lethality of some compound mutants have made investigation of the roles of these proteins in early embryonic development difficult, and a role for E2A or HEB in early embryogenesis or SMAD/FOXH1 signaling has never been identified. Conditional genetic approaches to ablate several family members during gastrulation will more accurately address the role of E2A and HEB during mammalian germ layer formation. It is noted with interest that loss of e2a function in X. tropcialis achieves an effect on gastrulation not seen in the mouse. It is hypothesized that the expansion of the Nodal pathway in frogs during evolution may have generated less redundancy between the E proteins; this is currently being tested by evaluating compound MOs. Overall, further investigation of the mechanisms used by E2A and HEB to modulate Nodal signal transduction will elucidate new insights into how this important pathway is diversified to induce cell lineages within distinct species (Yoon, 2011).

Axin and GSK3-β control Smad3 protein stability and modulate TGF-β signaling

The broad range of biological responses elicited by transforming growth factor-β (TGF-β) in various types of tissues and cells is mainly determined by the expression level and activity of the effector proteins Smad2 and Smad3. It is not fully understood how the baseline properties of Smad3 are regulated, although this molecule is in complex with many other proteins at the steady state. This stud shows that nonactivated Smad3, but not Smad2, undergoes proteasome-dependent degradation due to the concerted action of the scaffolding protein Axin and its associated kinase, glycogen synthase kinase 3-β (GSK3-β). Smad3 physically interacts with Axin and GSK3-β only in the absence of TGF-β. Reduction in the expression or activity of Axin/GSK3-β leads to increased Smad3 stability and transcriptional activity without affecting TGF-β receptors or Smad2, whereas overexpression of these proteins promotes Smad3 basal degradation and desensitizes cells to TGF-β. Mechanistically, Axin facilitates GSK3-β-mediated phosphorylation of Smad3 at Thr66, which triggers Smad3 ubiquitination and degradation. Thr66 mutants of Smad3 show altered protein stability and hence transcriptional activity. These results indicate that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-β, and suggest a new function of the Axin/GSK3-β complex in modulating critical TGF-β/Smad3-regulated processes during development and tumor progression (Guo, 2008).

Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-β signaling

TGF-beta induces phosphorylation of the transcription factors Smad2 and Smad3 at the C terminus as well as at an interdomain linker region. TGF-beta-induced linker phosphorylation marks the activated Smad proteins for proteasome-mediated destruction. This study identified Nedd4L as the ubiquitin ligase responsible for this step. Through its WW domain, Nedd4L specifically recognizes a TGF-beta-induced phosphoThr-ProTyr motif in the linker region, resulting in Smad2/3 polyubiquitination and degradation. Nedd4L is not interchangeable with Smurf1, a ubiquitin ligase that targets BMP-activated, linker-phosphorylated Smad1. Nedd4L limits the half-life of TGF-beta-activated Smads and restricts the amplitude and duration of TGF-beta gene responses, and in mouse embryonic stem cells, it limits the induction of mesoendodermal fates by Smad2/3-activating factors. Hierarchical regulation is provided by SGK1, which phosphorylates Nedd4L to prevent binding of Smad2/3. Previously identified as a regulator of renal sodium channels, Nedd4L is shown here to play a broader role as a general modulator of Smad turnover during TGF-beta signal transduction (Gao, 2009).

Mutation of Smad2

Smad2 and Smad3 are closely related effectors of TGFbeta/Nodal/Activin-related signaling. Smad3 mutant mice develop normally, whereas Smad2 plays an essential role in patterning the embryonic axis and specification of definitive endoderm. Alternative splicing of Smad2 exon 3 gives rise to two distinct protein isoforms. The short Smad2(Deltaexon3) isoform, unlike full-length Smad2, Smad2(FL), retains DNA-binding activity. Smad2(FL) and Smad2(Deltaexon3) are coexpressed throughout mouse development. Directed expression of either Smad2(Deltaexon3) or Smad3, but not Smad2(FL), restores the ability of Smad2-deficient embryonic stem (ES) cells to contribute descendants to the definitive endoderm in wild-type host embryos. Mice engineered to exclusively express Smad2(Deltaexon3) correctly specify the anterior-posterior axis and definitive endoderm, and are viable and fertile. Moreover, introducing a human Smad3 cDNA into the mouse Smad2 locus similarly rescues anterior-posterior patterning and definitive endoderm formation and results in adult viability. Collectively, these results demonstrate that the short Smad2(Deltaexon3) isoform or Smad3, but not full-length Smad2, activates all essential target genes downstream of TGFbeta-related ligands, including those regulated by Nodal (Dunn, 2005).

Transcriptional targets of Smad2/3 proteins

Transforming growth factor (TGF)-beta family members play a central role in mesoderm induction during early embryogenesis in Xenopus. Although a number of target genes induced as an immediate-early response to activin-like members of the family have been described, little is known about the molecular mechanisms involved. Systematic analysis of the activin induction of the target gene XFKH1 reveals two regions that mediate activin-responsive transcription: one, in the first intron, is targeted directly by the activin-signalling pathway; the other, in the 5' flanking sequences, responds to activin indirectly, possibly being required for the maintenance of gene expression. A 107 bp region of the XFKH1 first intron acts as an enhancer and confers activin inducibility onto a minimal uninducible promoter in the absence of new protein synthesis. It bears little sequence similarity to other activin responsive sequences. Overexpression of a constitutively active derivative of Xenopus Smad2 (XSmad2), which has been implicated as a component of the activin signaling pathway, is sufficient for direct activation of transcription via this enhancer. XSmad2 acts indirectly on the proximal promoter element induced by activin via an indirect mechanism. These results establish the XFKH1 intron enhancer as a direct nuclear target of the activin signaling pathway in Xenopus embryos, and provide strong new evidence that XSmad2 is a transducer of activin signals (Howell, 1997).

Smad7 is an inducible intracellular inhibitor of transforming growth factor-beta (TGF-beta) signaling that is regulated by diverse stimuli, including members of the TGF-beta superfamily. To define the molecular mechanisms of negative control of TGF-beta signaling, the human SMAD7 gene has been isolated and its promoter region has been characterized. A -303 to +672 SMAD7 region contains a palindromic GTCTAGAC Smad binding element (SBE) between nucleotides -179 and -172 that is necessary for the induction of a Smad7 promoter luciferase reporter gene by TGF-beta. Electrophoretic mobility shift assays using oligonucleotide probes have demonstrated that TGF-beta rapidly induces the binding of an endogenous SBE-binding complex (SBC) containing Smad2, Smad3, and Smad4. Transfection assays in mouse embryonic fibroblasts (MEFs), with targeted deletions of either Smad2 or Smad3, and the Smad4-deficient cell line MD-MBA-468 reveal that both Smad3 and Smad4, but not Smad2, are absolutely required for induction of the Smad7 promoter reporter gene by TGF-beta. Furthermore, the TGF-beta-inducible SBE-binding complex is diminished in Smad2-deficient MEFs when compared with wild type MEFs and not detectable in Smad3-deficient MEFs and MD-MBA-468 cells. Taken together, these data demonstrate that TGF-beta induces transcription of the human SMAD7 gene through activation of Smad3 and Smad4 transcription factor binding to its proximal promoter (von Gersdorff, 2000).

The regulation of the activin/nodal-inducible distal element (DE) of the Xenopus goosecoid (gsc) promoter has been investigated. The DE consists of a 29-bp response element that is activated specifically by activin or activin-like signals in the presence of cyclohexamide. The DE responds not only to endogenous activin-like signals in Xenopus, but also to nodals and BVg1. On the basis of its interaction with the DE, a Xenopus homolog of the human Williams-Beuren syndrome critical region 11 (XWBSCR11) has been isolated. This Xenopus protein interacts with pathway-specific Smad2 and Smad3 in a ligand-dependent manner. Interestingly, XWBSCR11 functions cooperatively with FoxH1 (Fast-1) to stimulate DE-dependent transcription. A mechanism is proposed in which FoxH1 functions together with Smads as a cofactor for the recruitment of transcription factors like XWBSCR11 in the process of activin/nodal-mediated gsc-specific induction. This mechanism provides considerable opportunities for modulation of transcription across a variety of activin/nodal-inducible genes, increasing diversity in promoter selection, thus leading to the differential induction of activin/nodal target genes (Ring, 2002).

Smad3 is a direct mediator of transcriptional activation by the TGFß receptor. Its target genes in epithelial cells include cyclin-dependent kinase inhibitors that generate a cytostatic reponse. This study defines how, in the same context, Smad3 can also mediate transcriptional repression of the growth-promoting gene c-myc. A complex containing Smad3, the transcription factors E2F4/5 and DP1, and the corepressor p107 preexists in the cytoplasm. In response to TGFbeta, this complex moves into the nucleus and associates with Smad4, recognizing a composite Smad-E2F site on c-myc for repression. Previously known as the ultimate recipients of cdk regulatory signals, E2F4/5 and p107 act here as transducers of TGFbeta receptor signals upstream of cdk. Smad proteins therefore mediate transcriptional activation or repression depending on their associated partners (Chen, 2002).

Smad2/3 proteins in C. elegans

In C. elegans, a TGFß-related signaling pathway regulates body size. Loss of function of the signaling ligand (dbl-1), receptors (daf-4 and sma-6) or Smads (sma-2, sma-3 and sma-4) results in viable, but smaller animals because of a reduction in postembryonic growth. The tissue specificity of this pathway in body size regulation has been investigated. Different tissues are reduced in size by different proportions, with hypodermal blast cell size most closely proportional to body size. SMA-3 Smad is expressed in pharynx, intestine and hypodermis, as has been previously reported for the type I receptor SMA-6. Furthermore, SMA-3::GFP is nuclear localized in all of these tissues, and nuclear localization is enhanced by SMA-6 activity. Interestingly, SMA-3 protein accumulation is negatively regulated by the level of Sma/Mab pathway activity. Using genetic mosaic analysis and directed expression of SMA-3, it has been found that SMA-3 activity in the hypodermis is necessary and sufficient for normal body size. Since dbl-1 is expressed primarily in the nervous system, these results suggest a model in which postembryonic growth of hypodermal cells is regulated by TGFß-related signaling from the nervous system to the hypodermis (Wang, 2002).

Using different SMA-3::GFP fusion constructs, evidence has been obtained that SMA-3 protein accumulation is negatively regulated by the level of SMA-3 and Sma/Mab pathway activity. Furthermore, a negative-feedback loop is consistent with the lack of effect of overexpressing sma-3. The overexpression of dbl-1 ligand induces a long phenotype and male tail sensory ray defects, while the overexpression of sma-3 does not. In other systems, it has been shown that Smads are degraded by the activity of Smurf E3 ubiquitin ligases. In human or Xenopus, Smurf-1 and Smurf-2 induce R-Smad degradation by the ubiquitin pathway. Through interaction with the anti-Smad Smad7, Smurf-1 can also induce the degradation of type I receptor. Smurf-2 enhances the degradation of type I receptor or SnoN oncogene. The target is selected by the Smad with which it interacts. A Smurf gene, Dsmurf (lack -- FlyBase) is found in Drosophila, where it negatively regulates Dpp signaling in embryonic dorsoventral patterning (Podos, 2001). In C. elegans, several open reading frames are found with homology to human or Xenopus Smurf genes, allowing the possibility that one or more of these genes functions in the Sma/Mab pathway. It will be interesting to determine whether a Smurf gene participates in a negative feedback loop (Wang, 2002).

TGF-ß signaling in the nematode Caenorhabditis elegans plays multiple roles in the development of the animal. The Sma/Mab pathway controls body size, male tail sensory ray identity and spicule formation. Three Smad genes, sma-2, sma-3 and sma-4, are all required for signal transduction, suggesting that the functional complex could be a heterotrimer. Because the C termini of Smads play important roles in receptor-mediated activation and heteromeric complex formation, C-terminal mutations were generated in the C. elegans Smad genes and their activities were tested in vivo in each of their distinct developmental roles. Pseudophosphorylated SMA-3 is dominant negative in body size, but functional in sensory ray and spicule development. Somewhat differently, pseudophosphorylated SMA-2 is active in any tissue. The C-terminal mutants of SMA-4 function like wild type, suggesting that the SMA-4 C terminus is dispensable. Using a combination of different C-terminal mutations in SMA-2 and SMA-3, a complex set of requirements was found for Smad-phosphorylation state that are specific to each outcome. Finally, a physical interaction of SMA-3 was detected with the forkhead transcription factor LIN-31, that is enhanced by SMA-3 pseudophosphorylation and reduced in an unphosphorylatable mutant. It is concluded that the tissue-specific requirements for Smad phosphorylation may result, in part, from the need to interact with tissue-specific transcription co-factors that have different affinities for phosphorylated and unphosphorylated Smad protein (Wang, 2005).

In the heterotrimer, the evidence suggests that two R-Smads will interact with one Co-Smad. The Smad2-Smad4 trimer contains one Smad4 and two Smad2 molecules. The complex of Smad4 and ppSmad3 shows a similar ratio. The pSmad2:Smad4 and pSmad3:Smad4 crystal structures contain a 2:1 R-Smad:Co-Smad ratio. Finally, a trimer formed by Smad2, Smad3 and Smad4 has also been reported. The simplest model of heterotrimer formation would suggest that both R-Smad subunits are phosphorylated. It was found, however, that in C. elegans male tail sensory rays, phosphorylatable SMA-3 is not required for function. Thus, it is possible that a Smad heterotrimer may contain one pR-Smad and one unphosphorylated R-Smad. In the case of male sensory rays, the functional complex must contain both SMA-2 and SMA-3, but only one of them needs to be phosphorylated. This finding has implications for the interpretation of morphogen gradients in other contexts. At high levels of ligand, cells may contain a large proportion of Smad heterotrimers containing two pR-Smads, whereas at lower levels of ligand more of the trimers may contain a single pR-Smad and a single unphosphorylated R-Smad. Thus, the Smad heterotrimer composition may provide a direct measure of ligand concentration that can then be translated into differential gene expression (Wang, 2005).

The DAF-7/TGF-β pathway in C. elegans interprets environmental signals relayed through amphid neurons and actively inhibits dauer formation during reproductive developmental growth. In metazoans, the STAT pathway interprets external stimuli through regulated tyrosine phosphorylation, nuclear translocation, and gene expression, but its importance for developmental commitment, particularly in conjuction with TGF-β, remains largely unknown. This study reports that the nematode STAT ortholog STA-1 accumulates in the nuclei of five head neuron pairs, three of which are amphid neurons involved in dauer formation. Moreover, sta-1 mutants showed a synthetic dauer phenotype with selected TGF-β mutations. sta-1 deficiency is complemented by reconstitution with wild-type protein, but not with a tyrosine mutant. Canonical TGF-β signaling involves the DAF-7/TGF-β ligand activating the DAF-1/DAF-4 receptor pair to regulate the DAF-8/DAF-14 Smads. Interestingly, STA-1 functions in the absence of DAF-7, DAF-4, and DAF-14, but it requires DAF-1 and DAF-8. Additionally, STA-1 expression is induced by TGF-β in a DAF-3-dependent manner, demonstrating a homeostatic negative feedback loop. These results highlight a role for activated STAT proteins in repression of dauer formation. They also raise the possibility of an unexpected function for DAF-1 and DAF-8 that is independent of their normal upstream activator, DAF-7 (Wang, 2006).

A model is proposed for how the STAT pathway might regulate dauer formation. Dauer formation has been shown to be actively inhibited by TGF-β signaling in response to environmental cues, such as the pheromone daumone. The data show that the STAT pathway contributes to this network. The Syn-Daf phenotype of daf-7;sta-1, daf-4;sta-1, and daf-14;sta-1 double mutants shows that STA-1 represses dauer independently of TGF-β (Daf-7), one receptor chain (Daf-4), and a co-Smad (Daf-14). Although it could function in an independent parallel pathway, the absence of Syn-Daf when sta-1 mutations were combined with daf-1 or daf-8 shows that STA-1 is functional only in the presence of these proteins, consistent with STA-1 acting at the DAF-8 step, possibly by direct interaction between the STAT and Smad proteins. This finding also reveals an unexpected noncanonical activity of DAF-8 in the absence of its traditional upstream activators, suggesting that it can respond to additional inputs other than DAF-7. The genetic data suggest that such inputs also function through the DAF-1 type I receptor kinase even in the absence of TGF-β, which performs this function independently of its normal coreceptor, DAF-4. DAF-4-independent activity of DAF-1 has been shown to be capable of regulating dauer formation. These data raise the possibility of a signaling pathway in which DAF-1 and DAF-8 can cooperate with STA-1 to repress dauer formation even in the absence of their traditional dimeric partners (Wang, 2006).

In addition to the cooperative action of TGF-β and STA-1 to inhibit dauer formation, active TGF-β signaling also restricted STA-1 nuclear accumulation to a small subset of posterior ganglion sensory neurons. Additional neurons are permissive for STA-1 activation, but only do so in the absence of TGF-β signals. This seemingly contradictory relationship is indicative of a complex homeostatic interaction of cooperative actions regulated by negative feedback loops. A complementary feedback loop was detected in which loss of STA-1 results in enhanced TGF-β target gene expression, suggesting that STA-1 represses TGF-β while TGF-β represses STA-1. The restricted expression of STA-1 is dependent on DAF-8, but not on DAF-3, raising the possibility that STA-1 expression is directly regulated by DAF-3 when it is derepressed in the absence of DAF-8/14 Smads. The sta-1 promoters from both C. elegans and C. briggsae contain putative Smad binding sequences, similar to sequences described for chemosensory genes regulated by TGF-β, which could be responsible for this regulation. Therefore, it appears that cooperation as well as mutual antagonism combine to maintain homeostatic reproductive growth. It is possible that this mutual antagonism contributes to the partially penetrant phenotypes observed in mutant animals. For instance, sta-1 mutants do not show a daf phenotype at 25°C, although they display a Hid (high temperature-induced dauer formation) phenotype at elevated temperature, suggesting that TGF-β signaling alone is sufficient to suppress dauer formation at ambient, but not elevated, temperature. One reason for the sufficiency of TGF-β signaling in the absence of STA-1 expression, however, may be the upregulation of TGF-β target genes normally held in check by STA-1, resulting in more robust TGF-β signaling in the stat-1 mutants. Conversely, many TGF-β pathway mutants are only partially penetrant, resulting in phenotypes that are not manifested under reduced temperature conditions when wild-type STAT-1 is present. It is possible that increased expression of STA-1 substitutes for TGF-β signaling in maintaining reproductive growth. In this model, upregulation of STA-1 after derepression of DAF-3 during dauer signaling may provide a partial break to dauer entry that contributes to more robust recovery during dauer exit (Wang, 2006).

Smad2/3 proteins and early embryonic development

The normal expression pattern of the Wnt responsive homeobox gene Siamois is restricted to the dorso-vegetal region of the Xenopus embryo. Since the Wnt signaling pathway (via beta-catenin) is active on the entire dorsal side of the early embryo, it seemed curious that Siamois expression is not seen in the dorsal ectoderm. It turns out that only Wnt signaling, via activation of beta-catenin, can directly induce Siamois; induction is not induced by signaling via the SMAD1 (BMP2/4) or SMAD2 (activin/Vg-1) pathways. In normal embryos, the SMAD2 pathway can cooperate with the Wnt pathway to induce expression of Siamois much more strongly than does the Wnt pathway alone. The significance of this cooperation is demonstrated in normal embryos by blocking the SMAD2 signaling pathway with a dominant negative activin receptor. The activin dominant negative receptor blocks this cooperative effect and reduces the expression of Siamois by threefold in early embryos. This cooperative relationship between the SMAD2 and Wnt pathways is reciprocal. Thus, in normal embryos, the Wnt pathway can enhance induction, by the SMAD 2 pathway, of the organizer genes Goosecoid and Chordin but not the pan-mesodermal marker genes Xbra and Eomes. The SMAD 1 pathway, which functions to transduce zygotic BMP2/4 signals, fails to induce Siamois. It is concluded that the Wnt and SMAD2 signaling pathways cooperate to induce the expression of Spemann-organizer specific genes and so help to localize their spatial expression (Crease, 1998).

Smads are central mediators of signal transduction for the TGFbeta superfamily. However, the precise functions of Smad-mediated signaling pathways in early development are unclear. A requirement for Smad2 signaling is demonstrated in dorsoanterior axis formation during Xenopus development. Using two point mutations of Smad2 previously identified in colorectal carcinomas, it has been shown that Smad2 ushers Smad4 to the nucleus to form a transcriptional activation complex with the nuclear DNA-binding protein FAST-1 and that the mutant proteins interact normally with FAST-1 but fail to recruit Smad4 into the nucleus. This mechanism of inhibition specifically restricts the dominant-negative activity of these mutants to the activin/Vg1 signaling pathway without inhibiting BMPs. Furthermore, expression of these mutants in Xenopus animal caps inhibits but does not abolish activin and Vg1 induction of mesoderm and in the embryo results in a truncated dorsoanterior axis. These studies define a mechanism through which mutations in Smad2 may block TGFbeta-dependent signaling and suggests a critical role for inductive signaling mediated by the Smad2 pathway in Xenopus organizer function (Hoodless, 1999).

To examine the role of Smad2 in postgastrulation development, mice with a null mutation in this gene were generated. Smad2-deficient embryos die around day 7.5 of gestation because of failure of gastrulation and failure to establish an anterior-posterior (A-P) axis. Expression of the homeobox gene Hex (the earliest known marker of the A-P polarity and the prospective head organizer) was found to be missing in Smad2-deficient embryos. Homozygous mutant embryos and embryonic stem cells formed mesoderm derivatives revealing that mesoderm induction is SMAD2 independent. In the presence of wild-type extraembryonic tissues, Smad2-deficient embryos develop beyond 7.5 and up to 10.5 days postcoitum, demonstrating a requirement for SMAD2 in extraembryonic tissues for the generation of an A-P axis and gastrulation. The rescued postgastrulation embryos show malformation of head structures, abnormal embryo turning, and cyclopia. These results show that Smad2 expression is required at several stages during embryogenesis (Heyer, 1999).

Knowledge of when and where signaling pathways are activated is crucial for understanding embryonic development. This study systematically analyzes and compares the signaling pattern of four major pathways by localization of the activated key components ß-catenin (Wnt proteins), MAPK (tyrosine kinase receptors/FGF), Smad1 (BMP proteins) and Smad2 (Nodal/activin/Vg1). The distribution of these components has been determined at 18 consecutive stages in Xenopus development, from early blastula to tailbud stages. The image obtained is that of very dynamic and widespread activities, with very few inactive regions. Signaling fields can vary from large gradients to restricted areas with sharp borders. They do not respect tissue boundaries. This direct visualization of active signaling verifies several predictions inferred from previous functional data. It also reveals unexpected signal patterns, pointing to some poorly understood aspects of early development. In several instances, the patterns strikingly overlap, suggesting extensive interplay between the various pathways. To test this possibility, maternal ß-catenin signaling has been manipulated and the effect on the other pathways in the blastula embryo has been determined. The patterns of P-MAPK, P-Smad1 and P-Smad2 are indeed strongly dependent on ß-catenin at this stage: their dorsal accumulation is absent in UV-irradiated embryos. The highest levels are then found symmetrically in the vegetal-equatorial region, similar to ß-catenin. Upon LiCl treatment, P-MAPK and P-Smad2 are strongly activated also in the ventral side. A similar activation is observed at the site of ß-catenin overexpression. Despite extensive colocalization, P-MAPK and P-Smad2 activation appear, nevertheless, spatially more restricted than ß-catenin: in all conditions, high P-MAPK is limited to a broad equatorial ring, while P-Smad2 activation is most prominent in the vegetal hemisphere. These differences obviously reflect the differential distribution of other determinants, which limit activation of P-MAPK to the marginal zone and Smad2 to the vegetal pole. P-Smad1 has an opposite polarity, i.e. weakest in the dorsal animal region. In UV-irradiated embryos, P-Smad1 is also activated on the dorsal side. LiCl treatment or ventral ß-catenin overexpression causes a significant decrease in the ventral side. In conclusion, these data show that maternal ß-catenin signaling is an important factor in controlling intensity and pattern of the other pathways at blastula stages. While other parameters regulate the latitude of the activation fields, ß-catenin can entirely account for the dorsoventral polarity. Mechanistically, ß-catenin probably contributes to Smad2 activation by stimulating Xnrs expression. How ß-catenin controls MAPK and Smad1 remains to be investigated (Schohl, 2002).

TGFß/activin/Nodal receptors activate both Smad2 and Smad3 intracellular effector proteins. The functional activities of these closely related molecules have been extensively studied in cell lines. Both Smad2 and Smad3 are expressed in the early mouse embryo from the blastocyst stage onward and mediate Foxh1-dependent activation of the Nodal autoregulatory enhancer in vitro. Genetic manipulation of their expression ratios reveals that Smad3 contributes essential signals at early post-implantation stages. Thus, loss of Smad3 in the context of one wild-type copy of Smad2 results in impaired production of anterior axial mesendoderm, while selective removal of both Smad2 and Smad3 from the epiblast additionally disrupts specification of axial and paraxial mesodermal derivatives. Smad2;Smad3 double homozygous mutants entirely lack mesoderm and fail to gastrulate. Collectively, these results demonstrate that dose-dependent Smad2 and Smad3 signals cooperatively mediate cell fate decisions in the early mouse embryo (Dunn, 2004).

Both Smad2 and Smad3 associate with the activated Alk4 receptor and can propagate Nodal signaling in P19 embryonal carcinoma cells. However, it remains unknown whether Smad2 and Smad3 both transduce Nodal/Alk4 signals in early mouse embryos. A Foxh1-dependent autoregulatory enhancer, termed the ASE, directs Nodal expression in the early epiblast, visceral endoderm and left lateral plate mesoderm, and interestingly an ASE-lacZ reporter transgene is activated appropriately in the epiblast of Smad2-deficient embryos. This observation suggests that Smad3 functionally compensates for the loss of Smad2 in this genetic context. To test this possibility directly, the abilities were examined of Smad2 and Smad3 to activate a reporter construct containing seven tandem repeats of a Foxh1-responsive 24 bp oligonucleotide from the mouse Nodal ASE. This reporter is activated by Foxh1 in a TGFß-dependent manner in cultured cells. In the presence of a constitutively active Alk4 receptor, both Smad2 and Smad3 give robust amplification of the transcriptional response. These results demonstrate that both Smad2 and Smad3 mediate Foxh1-dependent activation of the Nodal ASE. The present work demonstrates that (1) both Smad2 and Smad3 cooperate with the transcription factor Foxh1 to regulate the Foxh1-dependent autoregulatory enhancer present in the Nodal locus; (2) Smad2 and Smad3 as well as Smad4 transcripts are expressed from the blastocyst stage onwards, with Smad3 expression domains appearing more tightly regulated in comparison to widespread Smad2 expression; (3) Smad2 and Smad3 expression ratios are independently regulated, and (4) combinatorial Smad2 and Smad3 activities indeed regulate mesoderm formation and patterning in the developing mouse embryo (Dunn, 2004).

Human embryonic stem cells (hESCs) self-renew indefinitely and give rise to derivatives of all three primary germ layers, yet little is known about the signaling cascades that govern their pluripotent character. Because it plays a prominent role in the early cell fate decisions of embryonic development, the role of TGFß superfamily signaling was examined in hESCs. In undifferentiated cells, the TGFß/activin/nodal branch is activated (through the signal transducer SMAD2/3) while the BMP/GDF branch (SMAD1/5) is only active in isolated mitotic cells. Upon early differentiation, SMAD2/3 signaling is decreased while SMAD1/5 signaling is activated. The functional role of TGFß/activin/nodal signaling in hESCs was examined and it was found to be required for the maintenance of markers of the undifferentiated state. These findings were extended to show that SMAD2/3 activation is required downstream of WNT signaling, which has been shown to be sufficient to maintain the undifferentiated state of hESCs. Strikingly, in ex vivo mouse blastocyst cultures, SMAD2/3 signaling is also required to maintain the inner cell mass (from which stem cells are derived). These data reveal a crucial role for TGFß signaling in the earliest stages of cell fate determination and demonstrate an interconnection between TGFß and WNT signaling in these contexts (James, 2005).

The function has been investigated of Smicl, a zinc-finger Smad-interacting protein that is expressed maternally in the Xenopus embryo. Inhibition of Smicl function by means of antisense morpholino oligonucleotides causes the specific downregulation of Chordin, a dorsally expressed gene encoding a secreted BMP inhibitor that is involved in mesodermal patterning and neural induction. Chordin is activated by Nodal-related signalling in an indirect manner, and this study shows that Smicl is involved in a two-step process that is necessary for this activation. In the first step, Smad3 (but not Smad2) activates expression of Xlim1 in a direct fashion. In the second, a complex containing Smicl and the newly induced Xlim1 induce expression of Chordin. As well as revealing the function of Smicl in the early embryo, this work yields important new insight in the regulation of Chordin and identifies functional differences between the activities of Smad2 and Smad3 in the Xenopus embryo (Collart, 2005).

Smad2/3 proteins and epidermal development

Members of the TGF-ß superfamily are critical regulators for epithelial growth and can alter the differentiation of keratinocytes. Transduction of TGF-ß signaling depends on the phosphorylation and activation of Smad proteins by heteromeric complexes of ligand-specific type I and II receptors. To understand the function of TGF-ß and activin-specific Smad, transgenic mice were generated that overexpress Smad2 in epidermis under the control of keratin 14 promoter. Overexpression of Smad2 increases endogenous Smad4 and TGF-ß1 expression while heterozygous loss of Smad2 reduces their expression levels, suggesting a concerted action of Smad2 and Smad4 in regulating TGF-ß signaling during skin development. These transgenic mice have delayed hair growth, underdeveloped ears, and shorter tails. In their skin, there is severe thickening of the epidermis with disorganized epidermal architecture, indistinguishable basement membrane, and dermal fibrosis. These abnormal phenotypes are due to increased proliferation of the basal epidermal cells and abnormalities in the program of keratinocyte differentiation. The ectodermally derived enamel structure is also abnormal. Collectively, this study presents the first in vivo evidence that, by providing an auto-feedback in TGF-ß signaling, Smad2 plays a pivotal role in regulating TGF-ß-mediated epidermal homeostasis (Ito, 2001).

The Dead ringer homolog Dril1 as a novel regulator of TGFβ signaling acting through Smad2 in Xenopus

ARID domain proteins are members of a highly conserved family involved in chromatin remodeling and cell-fate determination. Dril1 is the founding member of the ARID family and is involved in developmental processes in both Drosophila and Caenorhabditis elegans. This study describes he first embryological characterization of this gene in chordates. Dril1 mRNA expression is spatiotemporally regulated and is detected in the involuting mesoderm during gastrulation. Inhibition of dril1 by either a morpholino or an engrailed repressor-dril1 DNA binding domain fusion construct inhibits gastrulation and perturbs induction of the zygotic mesodermal marker Xbra and the organizer markers chordin, noggin, and Xlim1. Xenopus tropicalis dril1 morphants also exhibit impaired gastrulation and axial deficiencies, which can be rescued by coinjection of Xenopus laevis dril1 mRNA. Loss of dril1 inhibits the response of animal caps to activin and secondary axis induction by smad2. Dril1 depletion in animal caps prevents both the smad2-mediated induction of dorsal mesodermal and endodermal markers and the induction of ventral mesoderm by smad1. Mesoderm induction by eFGF is uninhibited in dril1 morphant caps, reflecting pathway specificity for dril1. These experiments identify dril1 as a novel regulator of TGFβ signaling and a vital component of mesodermal patterning and embryonic morphogenesis (Callery, 2005).

A DNA binding consensus sequence for murine dril1 has been identified, and the MatInspector program (Genomatix) was used to investigate whether several TGFβ-responsive Xenopus promoters contain this putative dril1 binding site. The activin-inducible gene, Xbra, which is down-regulated in dril morphants, has three putative dril1 binding sites in its promoter and is thus a promising candidate for direct regulation by dril1. The induction of Xlim1 by smad2 is also dril1 dependent, and the intronic region of Xlim1 that mediates activin responsiveness also contains two possible dril1 binding sites. A third activin-inducible gene, HNF1α, contains six sequences matching the dril1 binding consensus, four of which have no overlapping sequence, so it will be interesting to investigate whether dril1 is involved in transcriptional regulation of this gene. Interestingly, putative dril1 binding sites were not identified in several promoters that contain either activin- or BMP-responsive elements (AREs or BREs), including the goosecoid, mix2, and bambi promoters. dril1 is required for induction of goosecoid by smad2, and it was found that both the dril1 morpholino and EnR–dril impair the activation of mix2 by smad2. How might dril1 regulate the expression of these genes if no binding sites are identified in their promoters? If dril1 acts as a regulator of chromatin architecture, it may bind regulatory elements further upstream than the promoter sequences analyzed in this study. Alternatively, the dril1-dependent genes whose promoters lack a dril1 binding site may be indirect targets whose transcription is activated by an intermediary protein. A third possibility is that dril1 can bind to sequences other than the canonical consensus identified by the MatInspector program; however, it is also possible that the consensus sequences identified in Xbra, Xlim1, and HNF1α may not function as dril1 binding sites in vivo. Therefore, it is important to note that silico analysis, while a useful preliminary step, cannot substitute for an empirical investigation of promoter binding (Callery, 2005).

Considering that the regulation of gastrulation by brachyury is conserved among vertebrates, it is likely that dril1 plays a role in gastrulation throughout this group. The involvement of dril1 in gastrulation may be conserved throughout deuterostomes because dril1 has recently been shown to be necessary for gastrulation movements in the echinoderm S. purpuratus. However, there is little similarity between the regulatory networks modulated by dril in the two deuterostome groups: dril depletion has no effect on brachyury, lim1, or bmp4 expression in the echinoderm or on the battery of endomesodermal patterning genes assayed. Even within the echinoderms, the presence of brachyury in the presumptive mesoderm is quite variant -- its roles in endoderm patterning and invagination appear more ancient. A contributory factor in the failure of gastrulation in dril-depleted sea urchin embryos may be the inhibition of goosecoid, which is required for gastrulation and greatly reduced by dril depletion. In vertebrates, smad2 is involved in activation of the goosecoid promoter. The role of smad signaling in echinoderm development is unknown so it is not possible to determine whether dril mediates its effects on this deuterostome group through inhibition of these transcription factors. However, because components of the TGFβ regulatory pathway are known to function in flies and worms, it will be interesting to determine whether dril also modulates this pathway in protostomes (Callery, 2005).

p53 is required for TGF-ß gene responses by cooperating with smads

The p53 tumor suppressor belongs to a family of proteins that sense multiple cellular inputs to regulate cell proliferation, apoptosis, and differentiation. Whether and how these functions of p53 intersect with the activity of extracellular growth factors is not understood. Key cellular responses to TGF-ß signals rely on p53 family members. During Xenopus embryonic development, p53 promotes the activation of multiple TGF-ß target genes. Moreover, mesoderm differentiation is inhibited in p53-depleted embryos. In mammalian cells, the full transcriptional activation of the CDK inhibitor p21WAF1 by TGF-ß requires p53. p53-deficient cells display an impaired cytostatic response to TGF-ß signals. Smad and p53 protein complexes converge on separate cis binding elements on a target promoter and synergistically activate TGF-ß induced transcription. p53 can physically interact in vivo with Smad2 in a TGF-ß-dependent fashion. The results unveil a previously unrecognized link between two primary tumor suppressor pathways in vertebrates (Cordenonsi, 2003).

To identify molecules that modulate TGF-β/Activin/Nodal signaling during development, an unbiased functional screen was performed for genes whose expression promotes the differentiation of embryonic cells into endoderm and mesoderm, as this is the hallmark of TGF-β signaling in early vertebrate embryos. A mouse gastrula (embryonic day [E]6.5) cDNA library was generated, constructed in an RNA expression plasmid. Synthetic mRNA was prepared from pools of 100 bacterial colonies and injected into the animal hemisphere of 2-cell Xenopus embryos. At the blastula stage, the ectoderm was explanted and cultivated until siblings reached the gastrula stage. The injected animal caps were then assayed by RT-PCR to identify pools able to activate the expression of Mixer (endoderm) and Xbra (mesoderm). Of five positive pools, two of the active cDNAs isolated after sib selection corresponded to Smad2 and, unexpectedly, three corresponded to p53AS, a natural variant of p53 generated by alternative splicing at the C terminus. p53AS shares with commonly spliced p53 (p53R) the N-terminal transactivation domain, the central DNA binding and oligomerization domains, but lacks the most C-terminal 26 amino acids of p53R (Cordenonsi, 2003).

A wealth of data indicates that the TGF-β and p53 signaling networks operate independently as powerful tumor suppressors in mammalian cells; yet, the cloning of a p53 isoform in a TGF-β screen unveiled the possibility of a previously unrecognized partnership between these two types of molecules. Evidence is provided that p53 family members are critical determinants for key TGF-β gene responses in different cellular and developmental settings. p53 is shown to associates with Smad2 and Smad3 in vivo in a TGF-β-dependent manner, and p53 family members can strongly cooperate with the activated Smad complex. Several TGF-β target genes in mammalian cells and Xenopus embryos are under such joint control of p53 and Smad (Cordenonsi, 2003).

Using a combination of loss-of-function approaches, evidence of the biological importance of such cooperation is provided. In frog cells, specific depletion of p53 leads to diminished responsiveness to Activin signaling and, in the context of the whole embryo, to severe developmental phenotypes recapitulating aspects of Nodal/Derriere deficiencies. In mammalian cells, the biological relevance of the p53/Smad cooperation was investigated in the context of TGF-β growth arrest program. Transient depletion of p53 or its genetic ablation impairs the antiproliferative response to Activin/TGF-β1 signaling. Finally, in p53 null cancer cells that do not respond to TGF-β signaling, reintroduction of p53 activity leads to the rescue of Smad-dependent growth inhibition (Cordenonsi, 2003).

The combinatorial control of gene expression by p53 and Smad establishes a new tier in the regulation of TGF-β gene responses. These data indicate that p53 neither serves as a DNA binding platform for the Smads, nor can it adjust the general magnitude of gene responses to TGF-β. Depletion of p53 leaves the Smad response fully operational on artificial promoters containing only the Activin/TGF-β responsive element and on some endogenous TGF-β targets, such as goosecoid or TIEG. Instead, p53 appears as an independent input that is integrated on specific target promoters to modulate TGF-β induced transcription. Multiple cellular inputs converge on p53 and it is tempting to speculate that specific posttranslational modifications of p53 may further tune its crosstalk with Smads. A model is proposed in which p53 and the activated Smad complex are recruited at distinct cis-regulatory elements on a common target promoter, leading to synergistic activation of transcription. This model is demonstrated for the Mix.2 promoter, a paradigm of TGF-β-induced transcription. A point mutation in the p53 binding element of the Mix.2 promoter causes a reduced Activin responsiveness in human cells and in the frog embryo, suggesting that p53 activity is required on DNA for full TGF-β transactivation. Of note, a correlation is found between other genes that are under joint control of p53 and Smad, and the presence of a functional p53 binding element in their promoters. This is the case for p21WAF1, PAI-1, and MMP2. In contrast no putative p53 elements were identified in the known regulatory sequences of goosecoid or TIEG, two genes not aided by p53 (Cordenonsi, 2003).

Smad2 and tooth development

The gene for activin ßA is expressed in the early odontogenic mesenchyme of all murine teeth but mutant mice show a patterning defect where incisors and mandibular molars fail to develop but maxillary molars develop normally. In order to understand why maxillary molar tooth development can proceed in the absence of activin, the role of mediators of activin signalling in tooth development was explored. Analysis of tooth development in activin receptor II and Smad2 mutants shows that a similar tooth phenotype to activin ßA mutants can be observed. In addition, a novel downstream target of activin signalling, the Iroquois-related homeobox gene, Irx1, has been identified; its expression in activin ßA mutant embryos is lost in all tooth germs, including the maxillary molars. These results strongly suggest that other TGFß molecules are not stimulating the activin signalling pathway in the absence of activin. This was confirmed by a non-genetic approach using exogenous soluble receptors to inhibit all activin signalling in tooth development. These reproduced the genetic phenotypes. Activin, thus, has an essential role in early development of incisor and mandibular molar teeth but this pathway is not required for development of maxillary molars (Ferguson, 2001).

Smad2 and cleft palate

Transforming growth factor (TGF)-ß3 is an important contributor to the regulation of medial edge epithelium (MEE) disappearance during palatal fusion. SMAD2 phosphorylation in the MEE has been shown to be directly regulated by TGF-ß3. No phospho-SMAD2 was identified in the MEE in Tgf-ß3-null mutant mice (Tgf-ß3-/-); this absence is correlated with the persistence of the MEE and failure of palatal fusion. In the present study, the cleft palate phenotype in Tgf-ß3-/- mice was rescued by overexpression of a Smad2 transgene in Keratin 14-synthesizing MEE cells following mating Tgf-ß3 heterozygous mice with Keratin 14 promoter directed Smad2 transgenic mice (K14-Smad2). Success of the rescue can be attributed to the elevated phospho-SMAD2 level in the MEE, demonstrated by two indirect evidences: (1) the rescued palatal fusion in Tgf-ß3-/-/K14-Smad2 mice, however, never proceed to the junction of primary and secondary palates and the most posterior border of the soft palate, despite phospho-SMAD2 expression in these regions at the same level as in the middle portion of the secondary palate; (2) the K14-Smad2 transgene is unable to restore all the functional outcomes of TGF-ß3. This may indicate an anterior-posterior patterning in the palatal shelves with respect to TGF-ß3 signaling and the mechanism of secondary palatal fusion (Cui, 2005).

The TGFbeta intracellular effector Smad3 regulates neuronal differentiation and cell fate specification in the developing spinal cord

Smad3, a transforming growth factor β/activin signaling effector, is expressed in discrete progenitor domains along the dorsoventral axis of the developing chick spinal cord. Restriction of Smad3 expression to the dP6-p2 and p3 domains together with exclusion from the motoneuron progenitor domain, are the result of the activity of key transcription factors responsible for patterning the neural tube. Smad3-mediated TGFβ activity promotes cell-cycle exit and neurogenesis by inhibiting the expression of Id proteins, and activating the expression of neurogenic factors and the cyclin-dependent-kinase-inhibitor p27kip1. Furthermore, Smad3 activity induces differentiation of selected neuronal subtypes at the expense of other subtypes. Within the intermediate and ventral domains, Smad3 promotes differentiation of ventral interneurons at the expense of motoneuron generation. Consequently, the absence of Smad3 expression from the motoneuron progenitor domain during pattern formation of the neural tube is a prerequisite for the correct generation of spinal motoneurons (Garcia-Campmany, 2007).

SMAD pathway mediation of BDNF and TGFß2 regulation of proliferation and differentiation of hippocampal granule neurons

Hippocampal granule cells self-renew throughout life, whereas their cerebellar counterparts become post-mitotic during early postnatal development, suggesting that locally acting, tissue-specific factors may regulate the proliferative potential of each cell type. Confirming this, conditioned medium from hippocampal cells (CMHippocampus) has been shown to stimulate proliferation in cerebellar cultures and, vice versa, that mitosis in hippocampal cells is inhibited by CMCerebellum. The anti-proliferative effects of CMCerebellum are accompanied by increased expression of the cyclin-dependent kinase inhibitors p21 and p27, as well as markers of neuronal maturity/differentiation. CMCerebellum contains peptide-like factors with distinct anti-proliferative/differentiating and neuroprotective activities with differing chromatographic properties. Preadsorption of CMCerebellum with antisera against candidate cytokines shows that TGFß2 and BDNF can account for the major part of the anti-proliferative and pro-differentiating activities, an interpretation strengthened by studies involving treatment with purified TGFß2 and BDNF. Interference with signaling pathways downstream of TGFß and BDNF using dominant-negative forms of their respective receptors (TGFß2-RII and TRKB) or of dominant-negative forms of SMAD3 and co-SMAD4 negated the anti-proliferative/differentiating actions of CMCerebellum. Treatment with CMCerebellum causes nuclear translocation of SMAD2 and SMAD4, and also transactivates a TGFß2-responsive gene. BDNF actions were shown to depend on activation of ERK1/2 and to converge on the SMAD signaling cascade, possibly after stimulation of TGFß2 synthesis/secretion. In conclusion, these results show that the regulation of hippocampal cell fate in vitro is regulated through an interplay between the actions of BDNF and TGFß (Lu, 2005).

Of the various members of the SMAD system, SMAD2 and SMAD3 mediate TGFß signals. SMAD4 is a requisite partner for transcriptional activity of all SMADs, including SMAD2 and SMAD3; the generation of specific downstream responses is presumed to depend on the formation of specific R-SMAD-SMAD4 complexes that then recruit different sequence-specific DNA-binding factors. CMCerebellum treatment can induce nuclear translocation of EGFP-SMAD2 and EGFP-SMAD4. Essential roles for SMAD3 and SMAD4 have been demonstrated insofar that transient expression of the dominant-negative forms of either of these molecules in hippocampal cells prevents CMCerebellum-induced transactivation of the TGFß reporter gene (3TP-Lux) and abrogates the anti-proliferative and pro-differentiating effects of CMCerebellum. Further support for the view that TGFß2 (at least partially) accounts for the anti-proliferative activity present in CMCerebellum, is provided by the observation that expression of a vector containing a dominant-negative form of TGFßRII in either primary hippocampal cells or a hippocampus-derived cell line (Hib5) abolishes CMCerebellum-induced effects on BrdU incorporation and 3TP-Lux reporter activity (Lu, 2005).

Combinatorial actions of Tgfβ and Activin ligands promote oligodendrocyte development and CNS myelination

In the embryonic CNS, development of myelin-forming oligodendrocytes is limited by bone morphogenetic proteins, which constitute one arm of the transforming growth factor-beta (Tgfβ) family and signal canonically via Smads 1/5/8. Tgfβ ligands and Activins comprise the other arm and signal via Smads 2/3, but their roles in oligodendrocyte development are incompletely characterized. This study reports that Tgfβ ligands and activin B (ActB) act in concert in the mammalian spinal cord to promote oligodendrocyte generation and myelination. In mouse neural tube, newly specified oligodendrocyte progenitors (OLPs) are first exposed to Tgfβ ligands in isolation, then later in combination with ActB during maturation. In primary OLP cultures, Tgfβ1 and ActB differentially activate canonical Smad3 and non-canonical MAP kinase signaling. Both ligands enhance viability, and Tgfβ1 promotes proliferation while ActB supports maturation. Importantly, co-treatment strongly activates both signaling pathways, producing an additive effect on viability and enhancing both proliferation and differentiation such that mature oligodendrocyte numbers are substantially increased. Co-treatment promotes myelination in OLP-neuron co-cultures, and maturing oligodendrocytes in spinal cord white matter display strong Smad3 and MAP kinase activation. In spinal cords of ActB-deficient inhibin Inhbb-/- embryos, apoptosis in the oligodendrocyte lineage is increased and OLP numbers transiently reduced, but numbers, maturation and myelination recover during the first postnatal week. Smad3-/- mice display a more severe phenotype, including diminished viability and proliferation, persistently reduced mature and immature cell numbers, and delayed myelination. Collectively, these findings suggest that, in mammalian spinal cord, Tgfβ ligands and ActB together support oligodendrocyte development and myelin formation (Dutta, 2014).

Smad2/3 proteins and cancer

The Smad2 protein plays an essential role in the transforming growth factor-beta signaling pathway. This pathway mediates growth inhibitory signals from the cell surface to the nucleus. Although Smad2 protein is significantly mutated in human cancers, there is no definitive evidence implicating Smad2 as a tumor-suppressor gene. Overexpression of the tumor-derived missense mutation Smad2.D450E, an unphosphorylable form of Smad2 found in colorectal and lung cancers, does not abolish the TGF-beta-mediated growth arrest, suggesting that resistance to the growth-inhibiting effects of TGF-beta exhibited by human tumors cannot be linked to the inactivation of Smad2 protein. In contrast, overexpression of Smad2.D450E induces cellular invasion, and this effect is enhanced by TGF-beta. A similar invasive phenotype has been obtained in cells expressing another inactivating mutation in Smad2 (Smad2.P445H) found in colorectal cancer. These findings indicate that genetic defects in Smad2 are sufficient to confer the invasion-promoting effect of TGF-beta and reveal that TGF-beta acts through Smad2 to induce cellular invasion by a novel mechanism that is independent of Smad2 phosphorylation by the activated TGF-beta type I receptor (Prunier, 1999).

Smad genes constitute a family of nine members whose products serve as intracellular mediators of transforming growth factor beta signals. SMAD2, which is a tumor suppressor involved in colorectal and lung cancer, has been shown to induce dorsal mesoderm in Xenopus laevis in response to transforming growth factor beta and activins. The smad2 gene is expressed ubiquitously during murine embryogenesis and in many adult mouse tissues. Animals that lack smad2 die before 8.5 days of development (E8.5). E6.5 smad2homozygous mutants are smaller than controls, lack the extraembryonic portion of the egg cylinder, and appear strikingly similar to E6.5 smad4 mutants. This similarity is no longer evident at E7.5, however, because the smad2 mutants contained embryonic ectoderm within their interiors. Molecular analysis has shown that smad2 mutant embryos do not undergo gastrulation or make mesoderm. The results demonstrate that smad2 is required for egg cylinder elongation, gastrulation, and mesoderm induction (Weinstein, 1998).

Smad2 and Smad4 are important for transcriptional and antiproliferative responses to TGF-beta, and their inactivation in human cancers indicates that they are tumor suppressors. A missense mutation at a conserved arginine residue in the amino-terminal MH1 domain of both Smad2 and Smad4 has been identified in tumors from patients with colorectal and pancreatic cancers, respectively. However, the mechanism whereby this mutation interferes with Smad activity is uncertain. These mutations do not disrupt activation of Smads, including receptor-mediated phosphorylation of Smad2, Smad2/Smad4 heteromeric complex formation, and Smad nuclear translocation. In contrast, the mutant Smads are degraded rapidly in comparison with their wild-type counterparts. This decrease in Smad protein stability occurs through induction of Smad ubiquitination by pathways involving the UbcH5 family of ubiquitin ligases. These studies thus reveal a mechanism for tumorigenesis whereby genetic defects in Smads induce their degradation through the ubiquitin-mediated pathway (J. Xu, 2000).


Smad on X : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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