Function and targets Smad2, and SMad3 homologs in mammals

Studies in which the mammalian Smad homologs are transiently overexpressed in cultured cells have implicated Smad2 in TGF-beta signaling, but the physiological relevance of the Smad3 protein in signaling by TGF-beta receptors has not been established. Smad proteins were overexpressed at controlled levels in epithelial cells using a novel approach that combines highly efficient retroviral gene transfer and quantitative cell sorting. Upon TGF-beta treatment Smad3 becomes rapidly phosphorylated at the SSVS motif at its very C terminus. Either attachment of an epitope tag to the C terminus or replacement of these three serine residues with alanine abolishes TGF-beta-induced Smad3 phosphorylation; these proteins act in a dominant-negative fashion to block the antiproliferative effect of TGF-beta in mink lung epithelial cells. A Smad3 protein in which the three C-terminal serines have been replaced by aspartic acids is also a dominant inhibitor of TGF-beta signaling, but can activate plasminogen activator inhibitor 1 (PAI-1) transcription in a ligand-independent fashion when its nuclear localization is forced by transient overexpression. Phosphorylation of the three C-terminal serine residues of Smad3 by an activated TGF-beta receptor complex is an essential step in signal transduction by TGF-beta for both inhibition of cell proliferation and activation of the PAI-1 promoter (X. Liu, 1997).

Signaling by the transforming growth factor-beta (TGF-beta) superfamily of proteins depends on the phosphorylation and activation of SMAD proteins by heteromeric complexes of ligand-specific type I and type II receptors with serine/threonine-kinase activity. The vertebrate SMAD family includes at least nine members, of which Smad2 has been shown to mediate signaling by activin and TGF-beta. The function of Smad2 in mammalian development was investigated by using gene targeting to generate two independent Smad2 mutant alleles in mice. Homozygous mutant embryos fail to form an organized egg cylinder and lack mesoderm, like mutant mice lacking nodal or ActRIB, the gene encoding the activin type-I receptor. About 20 per cent of Smad2 heterozygous embryos have severe gastrulation defects and lack both mandibles and eyes, indicating that the gene dosage of Smad2 is critical for signaling. Mice trans-heterozygous for both Smad2 and nodal mutations display a range of phenotypes, including gastrulation defects, complex craniofacial abnormalities such as cyclopia, and defects in left-right patterning, indicating that Smad2 may mediate nodal signaling in these developmental processes. These results show that Smad2 function is essential for early development and for several patterning processes in mice (Nomura, 1998).

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 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).

The cell cycle inhibitor p21/WAF1/Cip1 (Drosophila homolog: Dacapo) is expressed in many cell types and is regulated by p53-dependent and p53-independent mechanisms. p21 is an important regulator of hepatocyte cell cycle, differentiation, and liver development, but little is known about the regulation of its synthesis in hepatocytes. The p21 gene is shown to be constitutively expressed in human hepatoma HepG2 cells. Deletion analysis of the p21 promoter shows that it contains a distal region (positions -2,300/-210) and a proximal region (positions -124 to -61) that act synergistically to achieve high levels of constitutive expression. The proximal region that consists of multiple Sp1 binding sites is essential for constitutive p21 promoter activity in hepatocytes. This region also mediates the transcriptional activation of the p21 promoter by members of the Smad family of proteins, which play important roles in the transduction of extracellular signals, such as transforming growth factor beta, activin, etc. Constitutive expression of p21 is severely reduced by a C-terminally truncated form of Smad4 that has been shown previously to block signaling through Smads. Smad3/4, and to a much lesser extent Smad2/4, causes high levels of transcriptional activation of the p21 promoter. Transactivation is compromised by N- or C-terminally truncated forms of Smad3. By using Gal4-Sp1 fusion proteins, it has been shown that Smad proteins can activate gene transcription via functional interactions with the ubiquitous factor Sp1. These data demonstrate that Smad proteins and Sp1 participate in the constitutive or inducible expression of the p21 gene in hepatic cells (Moustakas, 1998).

Smad proteins play a key role in the intracellular signaling of transforming growth factor beta (TGF beta), which elicits a large variety of cellular responses. Upon TGF beta receptor activation, Smad2 and Smad3 become phosphorylated and form heteromeric complexes with Smad4. These complexes translocate to the nucleus where they control expression of target genes. However, the mechanism by which Smads mediate transcriptional regulation is largely unknown. Human plasminogen activator inhibitor-1 (PAI-1) is a gene that is potently induced by TGF beta. Smad3/Smad4 binding sequences, termed CAGA boxes, have been identified within the promoter of the human PAI-1 gene. The CAGA boxes confer TGF beta and activin, but not bone morphogenetic protein (BMP) stimulation to a heterologous promoter reporter construct. Importantly, mutation of the three CAGA boxes present in the PAI-1 promoter abolishes TGF beta responsiveness. Thus, CAGA elements are essential and sufficient for the induction by TGF beta. In addition, TGFbeta induces the binding of a Smad3/Smad4-containing nuclear complex to CAGA boxes. Bacterially expressed Smad3 and Smad4 proteins, but neither Smad1 nor Smad2 protein, bind directly to this sequence in vitro. The presence of this box in TGF beta-responsive regions of several other genes suggests that this may be a widely used motif in TGF beta-regulated transcription (Dennler, 1998).

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. By using MDA-MB-468 SMAD4-/- mutant breast carcinoma cells it was 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. Electrophoretic mobility-shift assays using recombinant glutathione S-transferase-SMAD fusion proteins indicate that 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).

Interactions of Smads with one another

Smad2 and Smad4 are related tumour-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 tumour-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 tumour suppressors (Hata, 1997).

Homologs of Drosophila Mad function as downstream mediators of the receptors for transforming growth factor beta (TGFbeta)-related factors. Two homologs, the receptor-associated Smad3 and the tumor suppressor Smad4/DPC4, synergize to induce ligand-independent TGFbeta activities and are essential mediators of the natural TGFbeta response. Smad3 and Smad4 associate in homomeric and heteromeric interactions, as assessed by yeast two-hybrid and coimmunoprecipitation analyses. Heteromeric interactions are mediated through the conserved C-terminal domains of Smad3 and Smad4. Smad4/DPC4 is critical because it is the shared hetero-oligomerization partner for the other SMADs. In Smad3, the homomeric interaction is mediated by the same domain. In contrast, the homomeric association of Smad4 requires both the N-terminal domain and the C-terminal domain, which by itself does not homomerize. Mutations that have been associated with impaired Mad activity in Drosophila or decreased tumor suppressor activity of Smad4/DPC4 in pancreas cancer, including a short C-terminal truncation and two point mutations in the conserved C-terminal domains, impair the ability of Smad3 and Smad4 to undergo homomeric and heteromeric associations. Analyses of the biological activity of Smad3 and Smad4 and their mutants show that full signaling activity correlates with their ability to undergo efficient homomeric and heteromeric interactions. Mutations that interfere with these interactions result in decreased signaling activity. The ability of Smad3 or Smad4 to induce transcriptional activation in yeast was also evaluated. These results correlate the ability of individual Smads to homomerize with transcriptional activation and additionally with their biological activity in mammalian cells (Wu, 1997).

Smads and integration of signaling pathways in the nucleus

TGFbeta initiates a signaling cascade leading to stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) activation. The expression of dominant-interfering forms of various components of the SAPK/JNK signaling pathways [including Rho-like GTPases (see Drosophila Rac1), mitogen-activated protein kinase (MAPK) kinase kinase 1 (MEKK1), MAPK kinase 4 (MKK4), SAPK/JNK, and c-Jun] abolishes TGFbeta-mediated signaling. Therefore, the SAPK/JNK activation most probably contributes to TGFbeta signaling (Atfi, 1997).

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, identified as forkhead activin signal transducer-1 (FAST-1), 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 TGFbeta 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 family members 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).

A mammalian forkhead domain protein, FAST2 is required for induction of the goosecoid (gsc) promoter by TGFbeta 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 TGFbeta/activin target gene (Labbe, 1998).

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 the 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; 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 (F. Liu, 1997).

Members of the Smad family of proteins are thought to play important roles in transforming growth factor beta (TGFbeta)-mediated signal transduction. In response to TGFbeta, 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 TGFbeta 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 TGFbeta-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 TGFbeta-dependent transcriptional activation or activation by Smad3/Smad4 co-overexpression. In contrast, mutation of adjacent AP1 sites within this context eliminates both TGFbeta-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 TGFbeta. 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).

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).

Developmental regulation of Smads

Smad proteins are intracellular signaling molecules and putative transcription factors that transduce signals elicited by members of the transforming growth factor beta (TGFbeta) superfamily. By comparing the expression of Smad1 and Smad2 during embryonic development, it has been shown that mRNAs of both Smad isoforms are present in a variety of tissues. The major sites of expression of both Smads can be correlated with the expression domains of several members of the TGFbeta superfamily. These expression data suggest that Smad proteins are involved in organ development, particularly that of organs arising from mesenchymal-epithelial interactions. A second site of strong expression is the central nervous system. Transcriptional control mediated by Smad1 and Smad2, therefore, may exert an important function in differentiation processes that are controlled by ligands of the TGFbeta superfamily during embryonic development (Dick, 1998).

Smads and cancer

The cloning and targeted disruption of the mouse Smad3 gene has been reported. Smad3 mutant mice are viable and fertile. Between 4 and 6 months of age, the Smad3 mutant mice become moribund with colorectal adenocarcinomas. The neoplasms penetrate through the intestinal wall and metastasize to lymph nodes. These results directly implicate TGFbeta signaling in the pathogenesis of colorectal cancer and provide a compelling animal model for the study of human colorectal cancer (Zhu, 1998).

Evi-1 encodes a zinc-finger protein that may be involved in leukemic transformation of hematopoietic cells. Evi-1 has two zinc-finger domains, one with seven repeats of a zinc-finger motif and one with three repeats, and it has characteristics of a transcriptional regulator. Although Evi-1 is thought to be able to promote growth and to block differentiation in some cell types, its biological functions are poorly understood. The mechanisms that underlie oncogenesis induced by Evi-1 have been studied by investigating whether Evi-1 perturbs signaling through transforming growth factor-beta (TGFbeta), one of the most studied growth-regulatory factors, which inhibits proliferation of a wide range of cell types. Evi-1 is shown to repress TGFbeta signaling and antagonize the growth-inhibitory effects of TGFbeta. Two separate regions of Evi-1 are responsible for this repression; one of these regions is the first zinc-finger domain. Through this domain, Evi-1 interacts with Smad3, an intracellular mediator of TGFbeta signaling, thereby suppressing the transcriptional activity of Smad3. These results define a new function for Evi-1 as a repressor of signaling through TGFbeta (Kurokawa, 1998).

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