Smads and integration of signaling pathways in the nucleus

A Xenopus TGF-ß responsive immediate-early response gene, Mix.2, encodes a homeobox gene expressed in prospective mesoderm and endoderm just after the mid-blastula transition. An activin-response factor (ARF) binds specifically to a 50-bp Mix.2 promoter element. The ARF complex contains XMAD2, a Xenopus homolog of the Drosophila MAD protein. A second component of ARF has been identified as forkhead activin signal transducer-1 (FAST-1) which contains a domain clearly related to the winged-helix domain of the forkhead/HNF3ß family of transcription factors (see Forkhead). FAST-1 mRNA is present in oocytes and in early embryos until shortly after gastrulation. It is concluded that FAST-1 and XMAD2 are partners in the coactivation of Mix.2 (Chen, 1996).

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 forkhead domains 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 but no longer associate with 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).

Upon ligand binding, the receptors of the TGFbeta family phosphorylate Smad proteins, which then move into the nucleus where they activate transcription. To carry out this function, the receptor-activated Smads 1 and 2 require association with the product of deleted in pancreatic carcinoma, locus 4 (DPC4) also known as Smad4. Smad4 is not required for nuclear translocation of Smads 1 or 2, or for association of Smad2 with a DNA binding partner, the winged helix protein FAST-1. Receptor-activated Smad2 takes Smad4 into the nucleus where, with FAST-1, they form a ternary complex that requires these three components to activate transcription. Smad4 contributes two functions: through its amino-terminal domain, Smad4 promotes binding of the Smad2/Smad4/FAST-1 complex to DNA, and through its carboxy-terminal domain, Smad4 provides an activation function required for Smad1 or Smad2 to stimulate transcription. The dual function of Smad4 in transcriptional activation underscores its central role in TGFbeta signaling (Liu, 1997).

A mammalian forkhead domain protein, FAST2, has been identifed that is required for induction of the goosecoid (gsc) promoter by TGF beta or activin signaling. FAST2 binds to a sequence in the gsc promoter, but efficient transcriptional activation and assembly of a DNA-binding complex of FAST2, Smad2, and Smad4 requires an adjacent Smad4 site. Smad3 is closely related to Smad2 but suppresses activation of the gsc promoter. Inhibitory activity is conferred by the MH1 domain, which unlike that of Smad2, binds to the Smad4 site. Through competition for this shared site, Smad3 may prevent transcription by altering the conformation of the DNA-binding complex. Thus, a mechanism is described whereby Smad2 and Smad3 positively and negatively regulate a TGF beta/activin target gene (Labbe, 1998).

Members of the Smad family of proteins are thought to play important roles in transforming growth factor beta (TGF-beta)-mediated signal transduction. In response to TGF-beta, specific Smads become inducibly phosphorylated, form heteromers with Smad4, and undergo nuclear accumulation. In addition, overexpression of specific Smad combinations can mimic the transcriptional effect of TGF-beta on both the plasminogen activator inhibitor 1 (PAI-1) promoter and the reporter construct p3TP-Lux. Although these data suggest a role for Smads in regulating transcription, the precise nuclear function of these heteromeric Smad complexes remains largely unknown. In Mv1Lu cells , Smad3 and Smad4 form a TGF-beta-induced, phosphorylation-dependent, DNA binding complex that specifically recognizes a bipartite binding site within p3TP-Lux. Smad4 itself is a DNA binding protein that recognizes the same sequence. Interestingly, mutations that eliminate the Smad DNA binding site do not interfere with either TGF-beta-dependent transcriptional activation or activation by Smad3/Smad4 co-overexpression. In contrast, mutation of adjacent AP1 sites within this context eliminates both TGF-beta-dependent transcriptional activation and activation in response to Smad3/Smad4 co-overexpression. Concatemerized AP1 sites, in isolation, are activated by Smad3/Smad4 cooverexpression and, to a certain extent, by TGF-beta. Taken together, these data suggest that the Smad3/Smad4 complex has at least two separable nuclear functions: it forms a rapid, yet transient sequence-specific DNA binding complex, and it potentiates AP1-dependent transcriptional activation. Smads appear to be able to function by two mechanisms: (1) Smad2 can act as a coactivator that inducibly associates with transcription factors but itself does not bind to DNA. (2) Smads such as Drosophila MAD bind to specific DNA sequences, such as the Vestigial promoter. A comparison between the DNA binding site for Smad4 and the DNA binding site for MAD reveals little sequence similarity. This suggests that different Smads will have different DNA binding specificities and thus, different target promoters (Yingling, 1997).

Many of the actions of serine/threonine kinase receptors for the transforming growth factor-beta (TGFbeta) are mediated by DPC4 (Smad4), a human MAD-related protein identified as a tumor suppressor gene in pancreatic carcinoma. Overexpression of DPC4 is sufficient to induce the activation of gene expression and cell cycle arrest, characteristic of the TGFbeta response. The stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is also one of the downstream targets required for TGFbeta-mediated signaling (see Drosophila Basket/JNK). Expression of the dominant-interfering mutant of various components of the SAPK/JNK cascade specifically block both TGFbeta and DPC4-induced gene expression. These dominant-interfering mutants also inhibit TGFbeta-stimulated DPC4 transcriptional activity. Overexpression of DPC4 causes transfected cells to undergo the morphological changes typical of apoptosis. These findings define a mechanism whereby TGFbeta signals mediated by DPC4 and SAPK/JNK cascade are integrated in the nucleus to activate gene expression and identify a new cellular function for DPC4 (Atfi, 1997).

Members of the TGF-beta superfamily influence a broad range of biological activities including stimulation of wound healing and inhibition of cell growth. TGF-beta signals through type I and II receptor serine/ threonine kinases and induces transcription of many genes including plasminogen activator inhibitor-1 (PAI-1). To identify proteins that participate in TGF-beta-induced gene expression, a novel retrovirus-mediated expression cloning strategy has been developed; using this approach, it was established that transcription factor microE3 (TFE3), a basic-helix-loop-helix-zipper (bHLHZIP) domain-containing protein (which binds mu E3 sites in regulatory elements in the immunoglobulin heavy chain gene) is involved in TGF-beta-induced activation of the PAI-1 promoter. TFE3 binds to an E-box sequence in PE2, a 56-bp promoter fragment of the PAI-1 promoter: the mutation of this sequence abolishes TFE3 binding as well as TGF-beta-dependent activation. TFE3 and Smad3 synergistically activate the PE2 promoter. Phosphorylated Smad3 and Smad4 bind to a sequence adjacent to the TFE3-binding site in this promoter. Binding of both TFE3 and the Smad proteins to their cognate sequences is indispensable for TGF-beta-inducible activation of the PE2 promoter. Hence, TFE3 is an important transcription factor in at least one TGF-beta-activated signal transduction pathway (Hua, 1998).

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 superfamily are known to transduce signals via the activation of Smad proteins. Ligand binding to transmembrane cell surface receptors triggers the phosphorylation of pathway-specific Smads. These Smads then complex with Smad 4 and are translocated to the nucleus where they effect gene transcription. Smads 1 and 4 mediate BMP activation of the OPN promoter by inhibiting the interaction of Hoxc-8 protein with a Hox-binding element. While specific DNA sequences are recognized by Smad complexes in several promoters, the role of Smad-binding elements (SBEs) in activation of the OPN promoter by members of the TGFbeta superfamily has not been previously evaluated. In this study the hypothesis was tested that a putative Smad-binding region containing the sequence AGACTGTCTGGAC is involved in the activation of the OPN promoter by members of the TGFbeta superfamily. Functional analyses demonstrate that both the HBE- and Smad-binding regions are involved in BMP-2-induced activation of the promoter, whereas, the HBE appears to be the primary region involved in activation by TGFbeta. Deletion of the first 9 bases in the Smad-binding region substantially reduces BMP-2-mediated activation of the promoter. These results strongly suggest that both the Hox- and the Smad-binding regions play a role in BMP-2-induced activation of the OPN promoter (Hullinger, 2001).

TGF-beta and activin induce the phosphorylation and activation of Smad2 and Smad3, but how these proteins stimulate gene transcription is poorly understood. TGF-beta receptor phosphorylation of Smad3 promotes its interaction with the paralogous coactivators CBP and p300, whereas CBP/p300 binding to nonphosphorylated Smad3 or its oligomerization partner Smad4 is negatively regulated by Smad-intramolecular interactions. Furthermore, p300 and TGF-beta receptor-phosphorylated Smad3 synergistically augment transcriptional activation. Thus, CBP/p300 are important components of activin/TGF-beta signaling and may mediate the antioncogenic functions of Smad2 and Smad4 (Janknecht, 1998).

The transforming growth factor-beta (TGF-beta) superfamily of growth factors and cytokines has been implicated in a variety of physiological and developmental processes within the cardiovascular system. Smad proteins are a recently described family of intracellular signaling proteins that transduce signals in response to TGF-beta superfamily ligands. It has been demonstrated by both a mammalian two-hybrid and a biochemical approach that 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).

Smads regulate the transcription of defined genes in response to TGF-beta receptor activation, although the mechanisms of Smad-mediated transcription are not well understood. The TGF-beta-inducible Smad3 uses the tumor suppressor Smad4/DPC4 and CBP/p300 as transcriptional coactivators, which associate with Smad3 in response to TGF-beta. 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 TGF-beta- and Smad-induced transcription in a Smad4/DPC4-dependent fashion. Smad3 transactivation and TGF-beta-induced transcription are inhibited by expressing E1A, which interferes with CBP functions (Feng, 1998).

Smad4 plays a pivotal role in signal transduction of the transforming growth factor beta superfamily cytokines by mediating transcriptional activation of target genes. Hetero-oligomerization of Smad4 with the pathway-restricted SMAD proteins is essential for Smad4-mediated transcription. Evidence is provided that SMAD hetero-oligomerization is directly required for the Smad4 C-terminal domain [Smad4(C)] to show its transcriptional transactivating activity; this requirement obtains even when Smad4(C) is recruited to promoters by heterologous DNA-binding domains and in the absence of the inhibitory Smad4 N-terminal domain. Defined mutations of GAL4 DNA-binding domain fusion of Smad4(C) disrupts SMAD hetero-oligomerization and consequently suppress transcriptional activation. An orphan transcriptional activator MSG1, a nuclear protein that has strong transactivating activity but apparently lacks DNA-binding activity, functionally interacts with Smad4 and enhances transcription mediated by GAL4 DNA-binding domain-Smad4(C) and full-length Smad4. Transcriptional enhancement by MSG1 depends on transforming growth factor beta signaling and is suppressed by either Smad4(C) mutations that disrupt SMAD hetero-oligomerization or by the presence of the Smad4 N-terminal domain. Furthermore, Smad4(C) does not show any detectable transactivating activity in yeast when fused to heterologous DNA-binding domains. These results demonstrate additional roles for SMAD hetero-oligomerization in Smad4-mediated transcriptional activation. They also suggest that the transcriptional-activating activity observed in the presence of Smad4 in mammalian cells may be derived, at least in part, from endogenously expressed separate transcriptional activators, such as MSG1 (Shioda, 1998).

Smad3 and Smad4 are sequence-specific DNA-binding factors that bind to their consensus DNA-binding sites in response to transforming growth factor beta (TGFbeta) and thereby activate transcription. Recent evidence implicates Smad3 and Smad4 in the transcriptional activation of consensus AP-1 DNA-binding sites, which do not interact with Smads directly. Smad3 and Smad4 are shown to be able to physically interact with AP-1 family members. In vitro binding studies demonstrate that both Smad3 and Smad4 bind all three Jun family members: JunB, cJun, and JunD. The Smad interacting region of JunB maps to a C-terminal 20-amino acid sequence that is partially conserved in cJun and JunD. Smad3 and Smad4 also associate with an endogenous form of cJun that is rapidly phosphorylated in response to TGFbeta. Smad3 is required for the activation of concatamerized AP-1 sites in a reporter construct that has previously been characterized as unable to bind Smad proteins directly. This result provides evidence for the importance of this interaction between Smad and Jun proteins. Together, these data suggest that TGFbeta-mediated transcriptional activation through AP-1 sites may involve a regulated interaction between Smads and AP-1 transcription factors (Liberati, 1999).

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).

Ski was first identified as a viral oncogene (v-ski) from the avian Sloan-Kettering retrovirus (SKV) that transforms chicken embryo fibroblasts. The human cellular homolog c-ski was later cloned based on its homology with v-ski and was found to encode a nuclear protein of 728 amino acids. Compared with c-Ski, v-Ski is truncated mostly at the carboxyl terminus. However, this truncation is not responsible for the activation of ski as an oncogene. Overexpression of wild-type c-Ski also results in oncogenic transformation of chicken and quail embryo fibroblasts. The transforming activity of Ski is likely attributable to overexpression, not truncation, of the c-Ski protein. Consistent with this notion, an elevated level of c-Ski has been detected in several human tumor cell lines derived from neuroblastoma, melanoma, and prostate cancer. c-ski is a unique oncogene; in addition to affecting cell growth, it is also involved in regulation of muscle differentiation. Overexpression of Ski results in muscle differentiation of quail embryo cells and hypertrophy of skeletal muscle in mice. Furthermore, mice lacking c-ski display defective muscle and neuronal differentiation. At the molecular level, Ski can function either as a transcriptional activator or as a repressor depending on the specific promoters involved. It has been shown to bind to DNA, but only in conjunction with other cellular proteins. Ski is a component of the histone deacetylase (HDAC1) complex through binding to the nuclear hormone receptor corepressor (N-CoR) and mSin3A, and mediated transcriptional repression of the thyroid hormone receptor, Mad and pRb. The interaction between Ski and N-CoR is mediated by the amino-terminal part of Ski. This region is also essential for the transforming activity of c-Ski and is conserved among ski family members, including v-Ski and c-SnoN (see Drosophila snoN). This raises an interesting possibility that the transforming activity of Ski may be linked to its function as a transcriptional corepressor (Luo, 1999 and references therein).

Ski can interact directly with Smad2, Smad3, and Smad4 on a TGF beta-responsive promoter element and repress their abilities to activate transcription through recruitment of the nuclear transcriptional corepressor N-CoR, and possibly its associated histone deacetylase complex. Thus Ski is a transcriptional corepressor of Smads. Overexpression of Ski in a TGF beta-responsive cell line renders it resistant to TGF beta-induced growth inhibition and defective in activation of JunB expression. This ability to overcome TGF beta-induced growth arrest may be responsible for the transforming activity of Ski in human and avian cancer cells. These studies suggest a new paradigm for inactivation of the Smad proteins by an oncoprotein through transcriptional repression (Luo, 1999).

Using a nuclear extract from c-ski-transformed cells, a specific DNA-binding site for Ski and its associated proteins was identified (GTCTAGAC) by cyclic amplification and selection of targets (CASTing). The Ski binding site was found to mediate transcriptional repression by Ski, suggesting that Ski may bind to DNA through interaction with the Smads. Ski/Smad3 and Ski/Smad4 complexes can bind to SBE and repress Smad-mediated transcriptional activation. Thus, Smad3 and Smad4 are the DNA-binding partners of Ski in these c-ski-transformed cells. In addition to SBE, Ski has also been found to interact with the nuclear factor I (NFI) binding site through interaction with the NFI protein. However, in this context, Ski functions to potentiate, not repress, NFI-stimulated transcriptional activation. Thus, Ski may interact with different DNA-binding factors and regulate transcription both positively and negatively depending on the proper cellular context or interacting partners (Luo, 1999 and references).

Ski also binds directly to Rb and retinoic acid receptor and to repress transactivation induced by these proteins, probably through similar mechanisms. N-CoR was originally identified as a corepressor that mediates transcriptional repression by the thyroid hormone receptor and Mad. It is a protein of 270 kD and contains three repressor domains in its amino-terminal region. It shows a striking homology to another corepressor, SMRT, and represses transcription by forming complexes with mSin3 and HDAC. Although no specific interactions between the Smads and endogenous mSin3A or HDAC could be detected because of technical difficulties, the recruitment of an N-CoR complex to the Smads suggests that repression of Smad-mediated transcription by Ski may involve deacetylation of nucleosomal histones. Recently, Smad2 has been shown to interact with TGIF, another transcriptional corepressor that recruits HDAC to the Smads. Thus, repression of Smad-mediated transactivation may involve multiple corepressors. Future studies will allow for a determination of whether Ski, Smads, N-CoR, and TGIF are in the same complex or whether Smads interact with different corepressors depending on the expression level of these corepressors in different cell types or at different developmental stages (Luo, 1999 and references therein).

Transforming growth factor-beta family members signal through a unique set of intracellular proteins called Smads. Smad4, previously identified as the tumor suppressor DPC4, is functionally distinct among the Smad family, and is required for the assembly and transcriptional activation of diverse, Smad-DNA complexes. A 48-amino acid proline-rich regulatory element within the middle linker domain of this molecule, the Smad4 activation domain (SAD), is essential for mediating these signaling activities. The functional activity of the SAD is reported in this study. Mutants lacking the SAD are still able to form complexes with other Smad family members and associated transcription factors, but cannot activate transcription in these complexes. Furthermore, the SAD itself is able to activate transcription in heterologous reporter assays, identifying it as a proline-rich transcriptional activation domain, and indicating that the SAD is both necessary and sufficient to activate Smad-dependent transcriptional responses. Transcriptional activation by the SAD is p300-dependent; this activity is associated with a physical interaction of the SAD with the amino terminus of p300. These data identify a novel function of the middle linker region of Smad4, and define the role of the SAD as an important locus determining the transcriptional activation of the Smad complex (de Caestecker, 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).

Signaling by Bmp receptors is mediated mainly by Smad proteins. A targeted null mutation of Ecsit (Drosophila homolog: ECSIT), encoding a signaling intermediate of the Toll pathway, leads to reduced cell proliferation, altered epiblast patterning, impairment of mesoderm formation, and embryonic lethality at embryonic day 7.5 (E7.5) phenotypes that mimic the Bmp receptor type1a (Bmpr1a) null mutant. In addition, specific Bmp target gene expression is abolished in the absence of Ecsit. Biochemical analysis demonstrates that Ecsit associates constitutively with Smad4 and associates with Smad1 in a Bmp-inducible manner. Together with Smad1 and Smad4, Ecsit binds to the promoter of specific Bmp target genes. Finally, knock-down of Ecsit with Ecsit-specific short hairpin RNA inhibits both Bmp and Toll signaling. Therefore, these results show that Ecsit functions as an essential component in two important signal transduction pathways and establishes a novel role for Ecsit as a cofactor for Smad proteins in the Bmp signaling pathway (Xiao, 2003).

The Toll pathway was originally identified in Drosophila through genetic screens for mutants with embryo patterning deficiency. A key component of the pathway is the Toll receptor, whose engagement leads to the activation of transcription factors of the NF-kappaB family. Subsequent studies have shown that the Toll pathway is also essential for host defense in the adult fly. The homologous family of Toll-like receptors (TLRs) in mammals also plays essential roles in innate immunity. The basic signal transduction pathway induced by the Toll receptors is homologous in Drosophila and mammals. Upon activation, TLRs recruit an adapter protein called MyD88, which subsequently recruits a serine-threonine kinase IRAK. IRAK binds to TRAF6, an adaptor protein of the tumor necrosis factor receptor-associated factor (TRAF) family. The assembly of this receptor complex activates IRAK, which undergoes autophosphorylation. Phosphorylated IRAK, together with TRAF6, detaches from the receptor complex and transduces the signal downstream, ultimately leading to activation of the IkappaB kinase (IKK) complex. The IKK complex phosphorylates IkappaB, leading to its ubiquitination and degradation. This process frees NF-kappaB and allows it to translocate into the nucleus, where it helps coordinate immune responses. Two pathways have been proposed to bridge the signal from TRAF6 to the IKK complex. One pathway is through TAK1 and its associated adaptor proteins TAB1 and TAB2, whereas the other one goes through Ecsit and MEKK1 or other MAP3K kinases. However, recent gene targeting results show that TAB2 is not required for NF-kappaB activation in response to signaling through the Toll/IL-1 receptors (Xiao, 2003).

Ecsit is a TRAF6-interacting protein that was discovered in a yeast two-hybrid screen using TRAF6 as bait (Kopp, 1999). The interaction between TRAF6 and Ecsit is conserved in Drosophila. Ecsit also interacts with MEKK1, a MAP3K kinase that can phosphorylate and activate the IKK complex. Expression of a dominant-negative mutant of Ecsit specifically blocks signaling from Toll and IL-1 receptors, but not from the TNF receptor. Therefore, Ecsit may transduce the signal from Toll receptors by bridging TRAF6 to the IKK complex (Kopp, 1999). To determine whether the TAK1/TAB1/TAB2 proteins can substitute for Ecsit in Toll signaling, and to further elucidate the physiological function of Ecsit, the Ecsit gene was deleted in embryonic stem cells and null mutant mice were generated. Ecsit-/- mice died around embryonic day 7.5 (E7.5), and analysis of the mutant embryos revealed a striking similarity to the phenotype of mice lacking Bmpr1a. Further characterization shows that Ecsit is an obligatory intermediate in Bmp signaling that functions as a cofactor for Smad1/Smad4-dependent activation of specific Bmp target genes. In addition, ablation of Ecsit using shRNA results in the block of NF-kappaB activation by LPS, but not TNFalpha, demonstrating the specific involvement of Ecsit in Toll receptor signaling. Therefore, these studies show that Ecsit is an essential component in both Bmp and Toll signaling pathways and is required for early embryogenesis (Xiao, 2003).

DACH1 inhibits TGF-beta signaling through binding Smad4

The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, it is hypothesized that DACH1 might play a similar cellular function. DACH1 has been found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-ß-induced apoptosis. DACH1 represses TGF-ß induction of AP-1 and Smad signaling in gene reporter assays and represses endogenous TGF-ß-responsive genes by microarray analyses. DACH1 binds to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localizes with NCoR in nuclear dotlike structures. NCoR enhances DACH1 repression, and the repression of TGF-ß-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH is sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibits the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-ß signaling by interacting with NCoR and Smad4 (Wu, 2003).

DACH1 functions as a transcriptional repressor of TGF-ß signaling. DACH1 represses TGF-ß-induced activity of both Smad/FAST1 Binding Element (SBE) and AP-1 activity and inhibits TGF-ß-induced apoptosis in MDA-MB-231 cells. NCoR enhances repression of TGF-ß signaling by DACH1. Repression by DACH1 requires Smad4, being abrogated in Smad4-deficient cells and restored by Smad4 coexpression. Repression by DACH1 requires a conserved DS domain that binds the transcriptional co-repressor NCoR. DACH1 and NCoR co-localize in a substantial proportion of subnuclear dotlike structures by confocal microscopy. Together, these findings suggest NCoR may participate in DACH1-mediated repression of gene expression (Wu, 2003).

DACH1 is detectable in MDA-MB-231 cells by Western blotting, and genome-wide analysis of DACH1-responsive genes in these cells indicates that 422 genes of 17,000 are regulated >2-fold by DACH1 expression. Consistent with the reporter gene analysis demonstrating DACH1 inhibition of AP-1 activity, several AP-1-responsive genes are repressed by DACH1 expression, including c-fos, Egr1, cyclin E2, neuregulin, tumor necrosis factor alpha-induced protein 3, cdc25A, FGF5, GRO3, MEF2C, ETR101, and BMP4. A comparison between genes regulated significantly by DACH1 with recent studies of TGF-ß signaling using a similar approach has demonstrated that genes induced by TGF-ß in other cell types are repressed by DACH1 (ATF3, interleukin-11, P2RY2) and several genes repressed by TGF-ß are induced by DACH1 (ID1 and interleukin-1-ß). Comparison between genome wide analysis 'fingerprints' must be considered with caution; however, it is of interest that of 70 genes regulated by TGF-ß, 22 of those genes are also significantly regulated by DACH1 expression; similarly, there is overlap with TGF-ß response genes in recent publications. The functions of these genes are diverse and include cell division, transcriptional regulation, cellular adhesion, extracellular matrix remodeling, and signal transduction. The use of genome-wide expression studies to identify clusters of genes representing a molecular signature of DACH1-regulated activity suggests a normal function for DACH1 in the inhibition of AP-1-regulated genes. The current studies suggest DACH1 may function to regulate aberrant TGF-ß signals that play important roles in human breast cancer progression. TGF-ß itself plays an important role in cancer progression by functioning both as an antiproliferative factor and as a tumor promoter. The numerous components of the signal conduction pathway are tumor suppressors that are functionally mutated in cancer (Wu, 2003).

DACH1 was found within a complex bound to a FAST1/SBE DNA binding site with Smad4. Immunopurified DACH1, however, does not bind DNA directly, suggesting that Smad4 serves as a DNA-bound platform to recruit DACH1. The DACH1 DS domain alone is insufficient for Smad4 binding, which requires the EYAD domain and is defective in SBE and AP-1 repression. DACH1 co-immunoprecipitates with Smad4 from cultured cells, and the association of DACH1 with Smad4 was observed in reciprocal immunoprecipitation. DACH1 associates with Smad4 in vitro using GST pull-down experiments, and, like Ski, multiple domains in DACH1 are required, including both the DS and EYA domains. Using saturating immunoprecipitation, the relative amount of co-precipitated Smad4 was greater for Ski than DACH1. In contrast, the relative abundance of NCoR coprecipitating with DACH1 is relatively greater than that associated with Ski. The finding that the DACH1DeltaDS domain mutant abrogates Ski-mediated repression of SBE activity suggests that DACH1 and Ski may function in a similar pathway (Wu, 2003).

DACH1, like Ski, represses Smad3-regulated transactivation of either SBE or AP-1 activity. These findings with Ski are similar to previous findings but contrast with the effect of Sno-N, which has little effect on Smad3 transactivation. Sno-N is degraded rapidly in response to Smad3 or TGF-ß, whereas Ski expression and DACH1 expression are not affected greatly by TGF-ß. These findings suggest distinct roles for Sno-N versus Ski-N and DACH1 in TGF-ß signaling (Wu, 2003).

DACH1 inhibits TGF-ß- and Smad-induced AP-1 activity. Inhibition of TGF-ß and Smad-induced AP-1 activity requires the DACH1 DS domain. TGF-ß induction of several genes, including PAI-1, clusterin, monocyte chemoattractant protein-1 (JE/MCP-1), type I collagen, and TGF-ß itself depends on AP-1 DNA-binding sites in the promoter region of these genes. Induction of AP-1 activity by TGF-ß involves interactions between Smads and AP-1 transcription factors. Smads bind directly to the Jun family, and both Smad3 and Smad4 can bind JunB, c-Jun, and JunD. Since the regions of DACH1 that bind Smads are required for repression of TGF-ß-induced AP-1 activity, it is likely that DACH1 mediates AP-1 repression through Smad4 association (Wu, 2003).

The identification of DACH1 as a new co-repressor of TGF-ß signaling extends understanding of this key pathway. The role of TGF-ß in cancer includes a complex function as both an antiproliferative activity and as a tumor promoter. DACH1, like Sno-N and v-Ski oncogenes, bind directly to NCoR/SMRT and mSin3. TGF-ß controls a plethora of cellular functions and regulates development and homeostasis. Since DACH1 and SKI have only partially overlapping expression patterns, with DACH1 expressed in neuroblastomas and in cell lines derived from pancreas and breast cancer cell lines, it is possible that DACH1 contributes in a cell type-specific manner to regulate TGF-ß signaling (Wu, 2003).

The family of Smad proteins mediates transforming growth factor-β signaling in cell growth and differentiation. Smad proteins repress or activate TGF-β signaling by interacting with corepressors (e.g., Ski) or coactivators (e.g., CREB binding protein [CBP]), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads, and whether they can interact with Smad3 at the same binding surface on Smad3. This study investigated the interactions among purified constructs of Smad, Ski and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Ski (aa 16-192) interacts directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g., Smad2 or Smad3, to form a hexamer; Ski (aa 16-192) interacts with an R-Smad/Smad4 heterotrimer to form a pentamer. CBP (aa 1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer, and with an R-Smad/Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP compete with each other for binding to Smad3. Mutational studies reveal that domains of Ski and CBP interact with Smad3 at a portion of the Smad anchor for receptor activation (SARA)-binding surface. These results suggest that Ski negatively regulates TGF-β signaling by replacing CBP in R-Smad complexes. A working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-β pathway (Chen, 2007),

Negative control of Smad activity by ectodermin/Tif1gamma patterns the mammalian embryo

The definition of embryonic potency and induction of specific cell fates are intimately linked to the tight control over TGFbeta signaling. Although extracellular regulation of ligand availability has received considerable attention in recent years, surprisingly little is known about the intracellular factors that negatively control Smad activity in mammalian tissues. By means of genetic ablation, the Smad4 inhibitor ectodermin (Ecto, also known as Trim33 or Tif1gamma) was shown to be required to limit Nodal responsiveness in vivo. New phenotypes, which are linked to excessive Nodal activity, emerge from such a modified landscape of Smad responsiveness in both embryonic and extra-embryonic territories. In extra-embryonic endoderm, Ecto is required to confine expression of Nodal antagonists to the anterior visceral endoderm. In trophoblast cells, Ecto precisely doses Nodal activity, balancing stem cell self-renewal and differentiation. Epiblast-specific Ecto deficiency shifts mesoderm fates towards node/organizer fates, revealing the requirement of Smad inhibition for the precise allocation of cells along the primitive streak. This study unveils that intracellular negative control of Smad function by ectodermin/Tif1gamma is a crucial element in the cellular response to TGFbeta signals in mammalian tissues (Morsut, 2010).

Smad targets

Human Mad-3 and Mad-4 target the Plasminogen activator inhibitor-1 promoter. Also hMad-3 and hMad-4 coexpression induces a decrease in cyclin A expression, inducing growth arrest characteristic of the TGF-ß response. Physical interaction of hMAD-3 with receptors can be demonstrated by immunoprecipitation of hMAD-3 with ligand-bound RI-RII complex, but not RII alone, consistent with the higher level of phosphorylation of hMAD-3 on serine and less on threonine. hMAD-3 and hMAD-4 display strong heteromeric and homomeric interactions in a yeast two hybrid assay (Zhang, 1996).

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).

Transcriptional regulation by transforming growth factor beta (TGF-beta) is a complex process which is likely to involve cross talk between different DNA responsive elements and transcription factors to achieve maximal promoter activation and specificity. This work has uncovered a concurrent requirement for two discrete responsive elements in the regulation of the c-Jun promoter: one, a binding site for a Smad3-Smad4 complex and the other an AP-1 binding site. The two elements are located 120 bp apart in the proximal c-Jun promoter, and each is able to independently bind its corresponding transcription factor complex. The effects of independently mutating each of these elements are nonadditive; disruption of either sequence results in complete or severe reductions in TGF-beta responsiveness. This simultaneous requirement for two distinct and independent DNA binding elements suggests that Smad and AP-1 complexes function synergistically to mediate TGF-beta-induced transcriptional activation of the c-Jun promoter (Wong, 1999).

The human type VII collagen gene (COL7A1) recently has been identified as an immediate-early response gene for transforming growth factor beta (TGF-beta)/SMAD signaling pathway. In this study, by using SMAD4-/- breast carcinoma cells, it has been demonstrated that expression of SMAD4 is an absolute requirement for SMAD-mediated promoter activity. The SMAD binding sequence (SBS) representing the TGF-beta response element in the region -496/-444 of the COL7A1 promoter functions as an enhancer in the context of a heterologous promoter. Electrophoretic mobility-shift assays with nuclear extracts from COS-1 cells transfected with expression vectors for SMADs 1-5 indicate that SMAD3 forms a complex with a migration similar to that of the endogenous TGF-beta-specific complex observed in fibroblast extracts. Both SMAD4 and C-terminally truncated SMAD3, but not SMAD2, can bind the COL7A1 SBS. Coexpression of SMAD3 and SMAD4 in COS-1 cells leads to the formation of two complexes: a DNA/protein complex containing SMAD3 alone and another slower-migrating complex containing both SMAD3 and SMAD4, the latter complex not being detected in fibroblasts. Maximal transactivation of COL7A1 SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant overexpression of SMAD3 and SMAD4. These data may represent the first identification of a functional homomeric SMAD3 complex regulating a human gene (Vindevoghel, 1998).

In the forming vertebrate heart, bone morphogenetic protein signaling induces expression of the early cardiac regulatory gene nkx-2.5. A similar regulatory interaction has been defined in Drosophila embryos, where Dpp signaling mediated by the Smad homologs Mad and Medea directly regulates early cardiac expression of tinman. A conserved cluster of Smad consensus binding sequences has been identified in early cardiac regulatory sequences of the mouse nkx-2.5 gene. The importance of the nkx-2.5 Smad consensus region in early cardiac gene expression was examined in transgenic mice and in cultured mouse embryos. In transgenic mice, deletion of the Smad consensus region delays induction of embryonic DeltaSmadnkx-2.5/lacZ gene expression during early heart formation. Induction of DeltaSmadnkx-2.5/lacZ expression is also delayed in the outflow tract myocardium and visceral mesoderm. Targeted mutation of the three Smad consensus sequences inhibits nkx-2.5/lacZ expression in the cardiac crescent, demonstrating a specific requirement for the Smad consensus sites in early cardiac gene induction. Cultured DeltaSmadnkx-2.5/lacZ transgenic mouse embryos also exhibit delayed induction of transgene expression. In the four-chambered heart, deletion of the Smad consensus region results in expanded DeltaSmadnkx-2.5/lacZ transgene expression. Thus, the nkx-2.5 Smad consensus region can have positive or negative regulatory function, depending on the developmental context and cellular environment (Liberatore, 2002).

A target consensus binding sequence (GCCGnCGc) for Drosophila MAD and Medea has been reported based on Dpp-responsive elements in tinman, dmef2, and vestigial genes. In mice, a GCCGnCGC-like motif present in the smad6 promoter is responsive to BMP signals mediated by Smad1/5 and binds Smad5 and Smad4. This 7-bp Smad1/5-induced sequence is present with no mismatches in the mouse nkx-2.5 early cardiac regulatory element. Additional Smad-responsive regulatory elements containing the consensus CAGA are present in human plasminogen activator inhibitor type 1, c-jun, PDGF-B, CARP, and alpha2procollagen genes. Two CAGA consensus sequences are present in addition to the distal GC-rich site between alpha3059 and alpha3012 of the mouse early cardiac regulatory element. The presence of three potential Smad-responsive sequences within a short stretch of DNA is characteristic of genetic elements regulated by Smad-dependent signaling mechanisms. The mouse nkx-2.5 Smad consensus region sequence is highly conserved in human nkx-2.5 genomic DNA with 48/50 identical nucleotides. Therefore, the Smad consensus region represents a potential direct target for BMP-mediated induction of mouse nkx-2.5 gene expression. (Liberatore, 2002).

Heart formation in vertebrates and fruit flies requires signaling by bone morphogenetic proteins (BMPs) to cardiogenic mesodermal precursor cells. The vertebrate homeobox gene Nkx2-5 and its Drosophila ortholog, tinman, are the earliest known markers for the cardiac lineage. Transcriptional activation of tinman expression in the cardiac lineage is dependent on a mesoderm-specific enhancer that binds Smad proteins, which activate transcription in response to BMP signaling, and Tinman, which maintains its own expression through an autoregulatory loop. An evolutionarily conserved, cardiac-specific enhancer of the mouse Nkx2-5 gene contains multiple Smad binding sites, as well as a binding site for Nkx2-5. A single Smad site is required for enhancer activity at early and late stages of heart development in vivo, whereas the Nkx2-5 site is not required for enhancer activity. These findings demonstrate that like tinman, Nkx2-5 is a direct target for transcriptional activation by Smad proteins; however, the independence of this Nkx2-5 enhancer of Nkx2-5 binding suggests a fundamental difference in the transcriptional circuitry for activation of Nkx2-5 and tinman expression during cardiogenesis in vertebrates and fruit flies (Lien, 2002).

The organization of the tinman tin-D and vertebrate Nkx2.5 enhancers was compared. There are four putative Smad4 binding sites, GTCT/AGAC, that are conserved in the AR2 enhancer. It has been shown that the tinman tin-D enhancer contains eight Mad binding sites, three of which can also be bound by Medea. The consensus sequence of the Mad and Medea binding sites is the GC-rich sequence, CGCCGC. However, for the sites that can also be bound by Medea, such as the M2 and M4 sites in the tin-D enhancer, there is an AGAC/GTCT sequence adjacent to the GC-rich sequence. This AGAC/GTCT sequence is identical to the vertebrate Smad4 binding site. Thus, it is likely that Medea actually binds to the AGAC/GTCT sequences instead of the GC-rich sequence (Lien, 2002).

Multiple Mad/Medea binding sites in the tin-D enhancer are required for dorsal mesoderm-specific activity of the enhancer. In the AR2 enhancer, the Smad4 site at -2774 is required for enhancer activity in the cardiac crescent and later in heart development. These findings reveal an evolutionarily conserved role for Smad factors in the activation of cardiac NK-type homeobox genes, and support the notion that Nkx2-5, like tinman, is a direct target of Smad proteins. Interestingly, when the mouse AR1 and AR2 enhancers with the Dpp-responsive tin-D3 enhancer of Drosophila tinman are compared, striking similarities are found among these enhancers. The essential Smad site at -2774 adjacent to the two essential GATA sites and the adjacent 3'-flanking sequences in the AR2 enhancer show high homology to the minimal Dpp response element in the tin-D enhancer. In addition, the core of the mouse AR1 enhancer contains a region with high homology to the region surrounding the essential Smad site at -2774 in the AR2 enhancer. This putative Smad site is also close to the essential GATA site in the AR1 enhancer. However, when this putative Smad site in the AR1 enhancer is mutated, enhancer activity is not abolished, suggesting there might be other redundant Smad sites present in the AR1 enhancer (Lien, 2002).

The Smad sites at the 5' end of the AR2 enhancer are not required for cardiac expression later in development and the mutant enhancer actually shows enhanced activity in the right ventricle, suggesting a negative role for Smad binding to these sites. Thus, it appears that the AR2 enhancer is a target for positive and negative regulation by Smad proteins at different stages of cardiac development. These divergent modes of regulation are likely to reflect differential associations of Smads with positive and negative cofactors that bind nearby sites in the enhancer (Lien, 2002).

Smads typically activate transcription in combination with other cofactors. Since BMPs are expressed in other regions of the embryo in addition to the cardiogenic region, the mechanism for BMP-dependent activation of Nkx2-5 must be coupled to other cell-autonomous regulators expressed prior to Nkx2-5. Understanding how BMP signaling is interpreted in mesodermal cells by cardiogenic cofactors is likely to provide insights into the molecular basis for cardiac specification. In this regard, Smad4 interacts directly with GATA-4, providing a possible molecular basis for transcriptional synergy between these factors and for directly linking cardiac gene regulation with the BMP signaling pathway (Lien, 2002).

While the transcriptional regulation of Nkx2-5 and tinman appear to be similar with respect to the dependence of the AR2 and tin-D enhancers on BMP signaling through Smad proteins, there are also fundamental differences in the regulation of these enhancers. In particular, the tinman tin-D enhancer is controlled through the combined actions of Medea and Tinman, whereas Nkx2-5 does not seem to autoregulate its own expression through the Nkx2-5 binding site in the AR2 enhancer. On the contrary, it has been suggested that Nkx2-5 negatively regulates its own expression, although no evidence was found for enhanced expression of the enhancer with the Nkx2-5 binding site mutation, as might be predicted by such a model (Lien, 2002).

The differences in regulation of tinman and Nkx2-5 transcription reflect the differences in mesoderm specification and patterning of the vertebrate and arthropod body plans. tinman is expressed throughout the nascent mesoderm of Drosophila prior to its subdivision into different sublineages. Expression of tinman in the early mesoderm is mediated by binding of Twist to a separate enhancer. Specification of the dorsal mesoderm occurs in response to Dpp signaling from the dorsal ectoderm. In contrast, Nkx2-5 expression is initiated concomitant with cardiogenic specification in response to BMP signaling from the anterior endoderm. Thus, the mechanism for BMP-dependent activation must be coupled to other cell-autonomous regulators expressed prior to Nkx2-5 itself. Understanding how BMP signaling is interpreted in mesodermal cells by cardiogenic cofactors is likely to provide insights into the molecular basis for cardiac specification (Lien, 2002).

Smad and tumorogenesis

The gene deleted in colorectal cancer ( dcc) was considered to be a candidate tumor suppressor gene until it was cloned and found to be a receptor for the axonal chemoattractant netrin-1. Dcc is a homolog of Drosophila Frazzled. Other genes on chromosome 18q remain as tumor suppressor candidates, including DPC4 (SMAD4) and MADR2 (SMAD2). The DPC4 (SMAD4) gene plays a key role in the TGFbeta signaling pathway. Its mouse homolog Dpc4 (Smad4) has been mutationally inactivated. The homozygous mutants are embryonic lethal, whereas the heterozygotes showed no abnormality. The Dpc4 mutation was introduced into the Apc(delta716) knockout mice, a model for human familial adenomatous polyposis. Because both Apc and Dpc4 are located on chromosome 18, compound heterozygotes were constructed by meiotic recombination to carry both mutations on the same chromosome. In such mice, intestinal polyps develop into more malignant tumors than those in the simple Apc(delta716) heterozygotes, showing an extensive stromal cell proliferation, submucosal invasion, cell type heterogeneity, and in vivo transplantability. These results indicate that mutations in DPC4 (SMAD4) play a significant role in the malignant progression of colorectal tumors. This work establishes that shutting off the TGFbeta signaling pathway by the mutation of Dpc4 in the Apc(delta716) polyp adenomas makes these tumors' growth much more malignant (Takaku, 1998).

Smad4 is a central mediator for TGFß signals, which play important functions in many biological processes. To study the role of Smad4 in mammary gland development and neoplasia, this gene was disrupted in mammary epithelium using a Cre-loxP approach. Smad4 is expressed in the mammary gland throughout development; however, its inactivation did not cause abnormal development of the gland during the first three pregnancies. Instead, lack of Smad4 gradually induced cell proliferation, alveolar hyperplasia and transdifferentiation of mammary epithelial cells into squamous epithelial cells. Consequently, all mutant mice developed squamous cell carcinoma and/or mammary abscesses between 5 and 16 months of age. Absence of Smad4 resulted in ß-catenin accumulation at onset and throughout the process of transdifferentiation, implicating ß-catenin, a key component of the Wnt signaling pathway, in the development of squamous metaplasia in Smad4-null mammary glands. TGFß1 treatment degrades ß-catenin and induces epithelial-mesenchymal transformation in cultured mammary epithelial cells. However, such actions are blocked in the absence of Smad4. These findings indicate that TGFß/Smad4 signals play a role in cell fate maintenance during mammary gland development and neoplasia (Li, 2003).

Ectodermal Smad4 and p38 MAPK are functionally redundant in mediating TGF-beta/BMP signaling during tooth and palate development

Smad4 is a central intracellular effector of TGF-beta signaling. Smad-independent TGF-beta pathways, such as those mediated by p38 MAPK, have been identified in cell culture systems, but their in vivo functional mechanisms remain unclear. This study investigated the role of TGF-beta signaling in tooth and palate development and noted that conditional inactivation of Smad4 in oral epithelium results in much milder phenotypes than those seen with the corresponding receptor mutants, Bmpr1a and Tgfbr2, respectively. Perturbed p38 function in these tissues likewise has no effect by itself; however, when both Smad4 and p38 functions are compromised, dramatic recapitulation of the receptor mutant phenotypes results. Thus, this study demonstrates that p38 and Smad4 are functionally redundant in mediating TGF-beta signaling in diverse contexts during embryonic organogenesis. The ability of epithelium to utilize both pathways illustrates the complicated nature of TGF-beta signaling mechanisms in development and disease (Xu, 2008).

This study shows that p38 MAPK functions redundantly with Smad4 to mediate BMP signaling during tooth development, and that tooth development can be arrested at the bud stage only by blocking both Smad4 and p38 MAPK. This functional redundancy is in sharp contrast to the result of loss of Smad4 in the cranial neural crest-derived dental mesenchyme, in which tooth development is retarded at the dental laminar stage (prior to the bud stage). Thus, there is an absolute requirement for Smad4 in the cranial neural crest-derived dental mesenchyme. In addition, Smad4-mediated TGF-β/BMP signaling is required for the homeobox gene patterning of oral/aboral and proximal/distal domains within the first branchial arch. Therefore, in the CNC-derived mesenchyme, TGF-β/BMP signals rely on Smad4-dependent pathways to mediate epithelial-mesenchymal interactions that control craniofacial organogenesis. Previous studies have shown that BMP4 signaling is critical for mediating cell death in the enamel knot, whereas Msx1-mediated BMP signaling is critical for cell proliferation in the dental mesenchyme. Taken together, the temporal and tissue-specific activation of Smad4-dependent or -independent BMP signaling pathways may regulate different downstream target genes and contribute to the diverse functional outcomes of BMP signaling in regulating the fate of dental epithelial and cranial neural crest-derived mesenchymal cells during tooth development (Xu, 2008).

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Medea: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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