Mothers against dpp


Smads and integration of signaling pathways in the nucleus

TGFbeta can override the proliferative effects of EGF and other Ras-activating mitogens in normal epithelial cells. However, epithelial cells harboring oncogenic Ras mutations often show a loss of TGFbeta antimitogenic responses. Oncogenic Ras inhibits TGFbeta signaling in mammary and lung epithelial cells by negatively regulating the TGFbeta mediators Smad2 and Smad3. Oncogenically activated Ras inhibits the TGFbeta-induced nuclear accumulation of Smad2 and Smad3 and Smad-dependent transcription. Ras acting via Erk MAP kinases causes phosphorylation of Smad2 and Smad3 at specific sites in the region linking the DNA-binding domain and the transcriptional activation domain. These sites are separate from the TGFbeta receptor phosphorylation sites that activate Smad nuclear translocation. Mutation of these MAP kinase sites in Smad3 yields a Ras-resistant form that can rescue the growth inhibitory response to TGFbeta in Ras-transformed cells. EGF, which is weaker than oncogenic mutations at activating Ras, induces a less extensive phosphorylation and cytoplasmic retention of Smad2 and Smad3. These results suggest a mechanism for the counterbalanced regulation of Smad2/Smad3 by TGFbeta and Ras signals in normal cells, and for the silencing of antimitogenic TGFbeta functions by hyperactive Ras in cancer cells (Kretzschmar, 1999).

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 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).

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; 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).

The transcription factor FAST-1 has recently been shown to play a key role in the specification of mesoderm by TGFbeta superfamily signals in the early Xenopus embryo. Fast1, a mouse homolog of Xenopus FAST-1, has been cloned and its expression during embryogenesis and function in activin/TGFbeta signal transduction characterized. In vitro, Fast1 associates with Smads in response to an activin/TGFbeta signal to form a complex that recognizes the Xenopus activin responsive element (ARE) targeted by Xenopus FAST-1. In intact cells, introduction of Fast1 confers activin/TGFb regulation of an ARE-luciferase reporter. In embryos, Fast1 is expressed predominantly throughout the epiblast before gastrulation and declines as development progresses. It is proposed that mouse Fast1, like Xenopus FAST-1, mediates TGFbeta superfamily signals specifying developmental fate during early embryogenesis (Weisberg, 1998).

The mechanisms by which transforming growth factor beta (TGF-beta) and related ligands regulate transcription remain poorly understood. The winged-helix (WH) transcription factor fork head activin signal transducer 1 (FAST-1) has been identified as a mediator of activin signaling in Xenopus embryos. From the mouse, a novel WH gene has been cloned that shares many properties with FAST-1. This gene, which has been called FAST-2, is able to mediate transcriptional activation by TGF-beta. FAST-2 also interacts directly with Smad2, a cytoplasmic protein that is translocated to the nucleus in response to TGF-beta, and forms a multimeric complex with Smad2 and Smad4 on the activin response element, a high-affinity binding site for FAST-1. Analysis of the sequences of FAST-1 and FAST-2 reveals substantial protein sequence divergence, as compared to known vertebrate orthologs in the WH family. This suggests that FAST-2 represents a new WH gene related to FAST-1, which functions to mediate TGF-beta signals in mammals. The structure of the FAST-2 gene has been examined and it overlaps with a kinesin motor protein gene. The genes are transcribed in opposite orientations, and their transcripts overlap in the 3' untranslated region (Liu, 1999).

Activation of transforming growth factor beta receptors causes the phosphorylation and nuclear translocation of Smad proteins, which then participate in the regulation of expression of target genes. A novel Smad-interacting protein, SIP1 (Drosophila homolog: Zn finger homeodomain 1), is described that was identified using the yeast two-hybrid system. Although SIP1 interacts with the MH2 domain of receptor-regulated Smads in yeast and in vitro, its interaction with full-length Smads in mammalian cells requires receptor-mediated Smad activation. SIP1 is a new member of the deltaEF1/Zfh-1 family of two-handed zinc finger/homeodomain proteins. Like deltaEF1, SIP1 binds to 5'-CACCT sequences in different promoters, including the Xenopus brachyury promoter. Overexpression of either full-length SIP1 or its C-terminal zinc finger cluster, which bind to the Xbra2 promoter in vitro, prevents expression of the endogenous Xbra gene in early Xenopus embryos. Therefore, SIP1, like deltaEF1, is likely to be a transcriptional repressor, which may be involved in the regulation of at least one immediate response gene for activin-dependent signal transduction pathways. The identification of this Smad-interacting protein opens new routes to investigate the mechanisms by which transforming growth factor beta members exert their effects on expression of target genes in responsive cells and in the vertebrate embryo (Verschueren, 1999).

Binding of TGFß/BMP factors to their receptors leads to translocation of Smad proteins to the nucleus where they activate transcription of target genes. The two-handed zinc finger proteins encoded by Zfhx1a and Zfhx1b, ZEB-1/deltaEF1 and ZEB-2/SIP1, respectively, regulate gene expression and differentiation programs in a number of tissues. ZEB proteins are also crucial regulators of TGFß/BMP signaling with opposing effects on this pathway. Both ZEB proteins bind to Smads, but while ZEB-1/deltaEF1 synergizes with Smad proteins to activate transcription, promote osteoblastic differentiation and induce cell growth arrest, the highly related ZEB-2/SIP1 protein has the opposite effect. Finally, the ability of TGFß to mediate transcription of TGFß-dependent genes and induce growth arrest depends on the presence of endogenous ZEB-1/deltaEF1 protein (Postigo, 2003a).

ZEB proteins are members of a large family of zinc finger proteins known as Zinc finger homeodomain, that was first identified in Drosophila. The genes encoding ZEB-1/deltaEF1 and ZEB-2/SIP1 proteins (Zfhx1 and Zfhx1b, respectively) appear to have evolved from a single Drosophila gene named zfh-1. zfh-1 is crucial for mesodermal (gonadal, skeletal and cardiac muscle) and neural differentiation in flies. The human ortholog of Drosophila zfh-2 seems to be ATBF-1, which has two isoforms: ATBF-1A and ATBF-1B. As in the case of ZEB proteins, a recent report demonstrated that ATBF-1A and ATBF-1B have opposing effects on the regulation of muscle differentiation. These results raise the interesting possibility that in vertebrates, the Drosophila zfh family of zinc finger proteins may have evolved into proteins with opposing activities to balance signaling pathways during tissue differentiation and embryonic development. ZEB-1/deltaEF1 and ZEB-2/SIP1 are structurally quite similar and both repress transcription of a number of genes involved in differentiation and development. However, the results presented here indicate that ZEB proteins function antagonistically in the regulation of TGFß/BMP signaling (Postigo, 2003a).

Balancing signals derived from the TGFbeta family are crucial for regulating cell proliferation and differentiation, and in establishing the embryonic axis during development. TGFbeta/BMP signaling leads to the activation and nuclear translocation of Smad proteins, which activate transcription of specific target genes by recruiting P/CAF and p300. The two members of the ZEB family of zinc finger factors (ZEB-1/deltaEF1 and ZEB-2/SIP1) regulate TGFbeta/BMP signaling in opposite ways: ZEB-1/deltaEF1 synergizes with Smad-mediated transcriptional activation, while ZEB-2/SIP1 represses it. These antagonistic effects by the ZEB proteins arise from the differential recruitment of transcriptional coactivators (p300 and P/CAF) and corepressors (CtBP) to the Smads. Thus, while ZEB-1/deltaEF1 binds to p300 and promotes the formation of a p300-Smad transcriptional complex, ZEB-2/SIP1 acts as a repressor by recruiting CtBP. This model of regulation by ZEB proteins also functions in vivo, where they have opposing effects on the regulation of TGFbeta family-dependent genes during Xenopus development (Postigo, 2003b).

Consistent with a crucial role of activin-like molecules in embryogenesis, activin response elements (AREs) have been reported in several activin-inducible transcription factor genes, such as the homeobox genes goosecoid (gsc), Mix.2, HNF1 alpha, and Xlim-1, a T-box gene Xbrachyury (Xbra), and a forkhead gene XFD-1'. Although the mechanisms regulating transcription of these genes remain poorly understood, identification of activin response factor (ARF) provides an entry point. ARF was first identified as a factor binding to an ARE in the Mix.2 promoter in response to Vg1, TGFbeta and activin. Subsequently, forkhead activin signal transducer-1 (FAST-1), Smad2, Smad3, and Smad4 were identified as components of ARF. The contribution of FAST target sites to the endogenous regulation of mesendodermal genes has not been directly investigated. An element in Xnr1 intron 1 has been identified that is activated by activin and Vg1, autoactivated by Xnrs, and suppressed by ventral inducers like BMP4. Intron 1 contains three FAST binding sites on which FAST/Smad transcriptional complexes can assemble; these sites are differentially involved in intron 1-mediated reporter gene expression. Interference with FAST function abolishes intron 1 activity, and transcriptional activation of Xnrs by activin in embryonic tissue explant assays have identified FAST as an essential mediator of Xnr autoregulation and/or 'signal relay' from activin-like molecules. Furthermore, the mapping of endogenous activators of the Xnr1 intronic enhancer within Xenopus embryos agrees well with the pattern of Xnr1 transcription during embryogenesis. In transgenic mice, Xnr1 intron 1 mimics a similarly located enhancer in the mouse nodal gene, and directs FAST site-dependent expression in the primitive streak during gastrulation, and unilateral expression during early somitogenesis. The FAST cassette is similar in an ascidian nodal-related gene, suggesting an ancient origin for this regulatory module. Thus, an evolutionarily conserved intronic enhancer in Xnr1 is involved in both mesendoderm induction and asymmetric expression during left-right axis formation (Osada, 2000).

The regulation of the activin-inducible distal element (DE) of the Xenopus goosecoid promoter has been characterized. The results show that paired-like homeodomain transcription factors of the Mix family, Mixer and Milk, but not Mix.1, mediate activin/TGF-beta-induced transcription through the DE by interacting with the effector domain of Smad2, thereby recruiting active Smad2/Smad4 complexes to the Mixer/Milk-binding site. A short motif has been identified in the carboxyl termini of Mixer and Milk, that has been demonstrated to be both necessary and sufficient for interaction with the effector domain of Smad2 and is required for mediating activin/TGF-beta-induced transcription. This motif is not confined to these homeodomain proteins, but is also present in the Smad2-interacting winged-helix proteins Xenopus Fast-1, human Fast-1, and mouse Fast-2. Transcription factors of different DNA-binding specificity recruit activated Smads to distinct promoter elements via a common mechanism. These observations, together with the temporal and spatial expression patterns of Mixer and Milk, lead to the proposal of a model for mesoendoderm formation in Xenopus in which these homeodomain transcription factor/Smad complexes play a role in initiating and maintaining transcription of target genes in response to endogenous activin-like signals (Germain, 2000).

Recent studies have already implicated Mixer, Milk, and the Bix proteins in endodermal and mesodermal differentiation, based on experiments in which they were overexpressed in prospective ectoderm (animal caps). However the underlying molecular mechanism was unknown. These data indicate that Mixer/Milk/Bix have little inherent transcriptional activity, but require bound activated Smads to increase their transcriptional potential, and thus in the embryo, it is proposed that Mixer/Milk/Bix would activate mesoendodermal genes by cooperating with Smads activated by an endogenous activin-like signal. A prediction would be that the family member Mix.1, which does not interact with Smads, would have a different activity in vivo. In contrast to Mixer and Milk, overexpression of Mix.1 alone in animal caps does not induce endoderm. The interaction data, together with the expression patterns of these homeodomain proteins, suggest a proposal for how these proteins might function in mesoendodermal formation in the Xenopus embryo. The major activin-like mesoendoderm-inducing activity that would activate Smad2 and Smad4 is zygotic, and requires the maternal transcription factor VegT for its production. This activity is likely to be composed of several different activin-related molecules, including derrière, Xnr1, Xnr2, Xnr4, and activinbetaB. The experiments indicate that Milk and Bix3 are also induced (weakly) in Xenopus embryos by a maternal activator. This could be VegT itself, since the Bix genes have been shown to be VegT targets. Alternatively, the maternal activator could be the signaling pathway activated by the maternal activin-related protein Vg-1, because the Bix genes are also known to be directly induced by activin. Thus, low levels of Milk and Bix3 would be available in the embryo to bind the Smad2/Smad4 complexes activated by the zygotic activin-like ligand, thereby initiating transcription of downstream genes like goosecoid. In addition, there may be low levels of ubiquitously maternally expressed Milk/Bix genes that could account for the cycloheximide-insensitive activin-induced transcription of the DE seen in the animal caps. Milk, Bix3, and also Mixer are themselves induced by the zygotic activin-like signaling pathway. It is proposed that these proteins would be involved in maintaining transcription in response to the zygotic activin-like ligand through their formation of transcriptionally active complexes with activated Smads. Determining precisely which mesoendodermal genes are regulated in this way by which Mixer/Milk family members presents a challenge for the future (Germain, 2000).

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 cooverexpression. In contrast, mutation of adjacent AP1 sites within this context eliminates both TGF-beta-dependent transcriptional activation and activation in response to Smad3/Smad4 cooverexpression. 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).

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).

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 inhibited 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).

The growth factor TGF-beta, bone morphogenetic proteins (BMPs, see Drosophila DPP) and related factors regulate cell proliferation, differentiation and apoptosis, controlling the development and maintenance of most tissues. Their signals are transmitted through the phosphorylation of the tumour-suppressor SMAD proteins by receptor protein serine/threonine kinases (RS/TKs), leading to the nuclear accumulation and transcriptional activity of SMAD proteins. Smad1, which mediates BMP signals, is also a target of mitogenic growth-factor signaling through epidermal growth factor and hepatocyte growth factor receptor protein tyrosine kinases (RTKs). Phosphorylation occurs at specific serines within the region linking the inhibitory and effector domains of Smad1, and is catalyzed by the Erk family of mitogen-activated protein kinases. In contrast to the BMP-stimulated phosphorylation of Smad1, which affects carboxy-terminal serines and induces nuclear accumulation of Smad1, Erk-mediated phosphorylation specifically inhibits the nuclear accumulation of Smad1. Thus, Smadl receives opposing regulatory inputs through RTKs and RS/TKs; it is this balance that determines the level of Smad1 activity in the nucleus, and so possibly the role of Smad1 in the control of cell fate (Kretzschmar, 1997).

Osteoprotegerin (OPG), an osteoblast-secreted decoy receptor, specifically binds to osteoclast differentiation factor and inhibits osteoclast maturation. Members of the TGF-beta superfamily including BMPs stimulate OPG mRNA expression. In this study the transcription mechanism of BMP-induced OPG gene expression has been characterized. Transfection of Smad1 and a constitutively active BMP type IA receptor ALK3 (Q233) stimulates OPG promoter. Deletion analysis of OPG promoter has identified two Hoxc-8 binding sites that respond to BMP stimulation. GST-Hoxc-8 protein binds to these two Hox sites specifically. Consistent with the transfection results of the native promoter, ALK3 or Smad1 linker region (which interacts with Hoxc-8) stimulates the activation of the reporter construct with the two Hox sites. Overexpression of Hoxc-8 inhibits the induced promoter activity. When the two Hox binding sites are mutated, ALK3 or Smad1 linker region no longer activate the transcription. Importantly, Smad1 linker region induces both OPG promoter activity and endogenous OPG protein expression in 2T3 osteoblastic cells. The medium from cells transfected with Smad1 linker region expression plasmid effectively inhibits osteoclastogenesis. Collectively, these data indicate that Hox sites mediate both OPG promoter construct activity and endogenous OPG gene expression in response to BMP stimulation (Wan, 2001).

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).

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).

Following TGFbeta receptor-mediated phosphorylation and association with Smad4, Smad2 moves into the nucleus, binds to target promoters in association with DNA-binding cofactors, and recruits coactivators such as p300/CBP to activate transcription. The homeodomain protein TGIF has been identified as a Smad2-binding protein and a repressor of transcription. A TGFbeta-activated Smad complex can recruit TGIF and histone deacetylases (HDACs) to a Smad target promoter, repressing transcription. Thus, upon entering the nucleus, a Smad2-Smad4 complex may interact with coactivators, forming a transcriptional activation complex, or with TGIF and HDACs, forming a transcriptional repressor complex. Formation of one of these two mutually exclusive complexes is determined by the relative levels of Smad corepressors and coactivators within the cell (Wotton, 1999).

The homeodomain protein TGIF represses transcription in part by recruiting histone deacetylases. TGIF binds directly to DNA to repress transcription or interacts with TGF-beta-activated Smads, thereby repressing genes normally activated by TGF-beta. Loss of function mutations in TGIF result in holoprosencephaly (HPE) in humans. One HPE mutation in TGIF results in a single amino acid substitution in a conserved PLDLS motif within the amino-terminal repression domain. TGIF interacts with the corepressor carboxyl terminus-binding protein (CtBP) via this motif. CtBP, which was first identified by its ability to bind the adenovirus E1A protein, interacts both with gene-specific transcriptional repressors and with a subset of polycomb proteins. Efficient repression of TGF-beta-activated gene responses by TGIF is dependent on interaction with CtBP, and TGIF is able to recruit CtBP to a TGF-beta-activated Smad complex. Disruption of the PLDLS motif in TGIF abolishes the interaction of CtBP with TGIF and compromises the ability of TGIF to repress transcription. Thus, at least one HPE mutation in TGIF appears to prevent CtBP-dependent transcriptional repression by TGIF, suggesting an important developmental role for the recruitment of CtBP by TGIF (Melhuish, 2000).

The cytokines LIF (leukemia inhibitory factor) and BMP2 (bone morphogenetic protein-2) signal through different receptors and transcription factors, namely STATs (signal transducers and activators of transcription) and Smads. LIF and BMP2 act in synergy on primary fetal neural progenitor cells to induce astrocytes. The transcriptional coactivator p300 interacts physically with STAT3 at its amino terminus in a cytokine stimulation-independent manner, and with Smad1 at its carboxyl terminus in a cytokine stimulation-dependent manner. The formation of a complex between STAT3 and Smad1, bridged by p300, is involved in the cooperative signaling of LIF and BMP2 and the subsequent induction of astrocytes from neural progenitors (Nakashima, 1999).

Cell proliferation and differentiation are regulated by growth regulatory factors such as transforming growth factor-beta (TGF-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 in mammalian cells to act 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. Because SMAD proteins are differently expressed in target tissues for TGF-beta, the tissue-specific amounts of endogenous SMAD proteins may contribute to the cooperative actions (Yanagisawa, 1999).

The transforming growth factor-beta (TGFbeta) and Wnt/wingless pathways play pivotal roles in tissue specification during development. Activation of Smads, the effectors of TGFbeta superfamily signals, results in Smad translocation from the cytoplasm into the nucleus where they act as transcriptional comodulators to regulate target gene expression. Wnt/wingless signals are mediated by the DNA-binding HMG box transcription factors lymphoid enhancer binding factor 1/T cell-specific factor (LEF1/TCF) and their coactivator beta-catenin. Smad3 is shown to physically interact with the HMG box domain of LEF1 and TGFbeta and Wnt pathways synergize to activate transcription of the Xenopus homeobox gene twin (Xtwn). Disruption of specific Smad and LEF1/TCF DNA-binding sites in the promoter abrogates synergistic activation of the promoter. Consistent with this observation, introduction of Smad sites into a TGFbeta-insensitive LEF1/TCF target gene confers cooperative TGFbeta and Wnt responsiveness to the promoter. Furthermore, TGFbeta-dependent activation of LEF1/TCF target genes can occur in the absence of beta-catenin binding to LEF1/TCF and requires both Smad and LEF1/TCF DNA-binding sites in the Xtwn promoter. Thus, these results show that TGFbeta and Wnt signaling pathways can independently or cooperatively regulate LEF1/TCF target genes and suggest a model for how these pathways can synergistically activate target genes (Labbe, 2000).

Bone morphogenetic protein (BMP) controls osteoblast proliferation and differentiation through Smad proteins. Tob, a member of the emerging family of antiproliferative proteins, is a negative regulator of BMP/Smad signaling in osteoblasts. The family related to Tob include human Tob2, ANA (BTG3 in mouse), BTG2 (PC3 in rat and TIS21 in mouse), and BTG1. A potential Drosophila homolog, Tob, has been identified. The biological importance of Tob family proteins in vivo has been unclear. Mice carrying a targeted deletion of the tob gene have been shown to have a greater bone mass resulting from increased numbers of osteoblasts. Orthotopic bone formation in response to BMP2 is elevated in tob-deficient mice. Overproduction of Tob represses BMP2-induced, Smad-mediated transcriptional activation. Finally, Tob associates with receptor-regulated Smads (Smad1, 5, and 8) and colocalizes with these Smads in the nuclear bodies upon BMP2 stimulation. The results indicate that Tob negatively regulates osteoblast proliferation and differentiation by suppressing the activity of the receptor-regulated Smad proteins (Yoshida, 2000).

It remains unclear how nuclear localization of Smads is regulated. Smads form a transcriptional activation complex. The proteins in Smad complexes, including p300/CBP, HDACs, Ski, Sin3A, and N-CoR, localize to nuclear bodies. Interestingly, localization of endogenous Smad2 and 3 to nuclear bodies has been observed. Smad1, 5, and 8 localize to nuclear bodies after BMP2 stimulation, when Tob is coexpressed with Smad proteins. Taken together, Tob is suggested to regulate localization of R-Smads in the nucleus in a BMP2-dependent manner and to be involved in Smads-mediated transcriptional regulation (Yoshida, 2000).

On the basis of these results, a model of regulation of BMP signaling by Tob is proposed. BMP induces activation and nuclear translocation of R-Smads, which results in transcriptional activation of various genes, including the genes that stimulate proliferation and differentiation of osteoblasts. The tob gene is also induced in response to BMP2. Consequently, Tob accumulates in the nucleus and recruits R-Smads to nuclear bodies by binding to the MH2 domain of the phosphorylated R-Smads. This initiates a negative feedback mechanism, allowing a precise and timely regulation of BMP signaling and, thus, proper bone formation (Yoshida, 2000).

The balance of positive and negative regulation of BMP signaling may be dependent in part on the relative levels of Smad inhibitors and Smad activators present in the cells. Extracellular signals that regulate the activity of either the Smad inhibitors or Smad activators would also affect this balance. Such signal-dependent modulation of Smad interaction with inhibitors and activators may allow precise regulation of BMP-dependent transcription. It is proposed that Tob, as a Smad-inhibitor, plays a critical role in BMP2/Smad-regulated gene expression in osteoblasts. Recent studies have revealed the involvement of the Tob/BTG family proteins in transcriptional regulation. For example, the homeodomain-containing transcription factor Hoxb9 is identified as a possible functional partner of BTG1 and BTG2. Furthermore, exogenously expressed BTG2 causes significant downregulation of the cyclin D1 transcript. Tob, Tob2, BTG1, and BTG2 are associated with Caf1 (CCR4-associated factor 1). CCR4 (CCR, carbon catabolite repression) consists of a complex of transcription factors required for expression of a number of genes, including the gene encoding alcohol dehydrogenase II. Therefore, the interaction of the Tob/BTG family with Caf1 may affect expression of genes involved in regulation of cell growth and/or differentiation. Thus, in addition to participating in BMP2 signaling through its association with Smad proteins, Tob may affect a variety of transcriptional machineries (Yoshida, 2000).

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).

In response to ligand-dependent signal transduction, Ski/SnoN forms a complex with Smad4 as well as the R-Smads. Ski/SnoN contains discrete and separable sequence elements necessary for binding to Smad4 or R-Smad; thus, it is able to contact both Smad4 and R-Smad simultaneously. The Smad4-Ski interaction plays a central role in the biological activities of Ski as evidenced by the observation that v-Ski, which induces potent oncogenic transformation of chicken embryo cells, interacts only with Smad4 but not Smad2 or Smad3. Furthermore, Ski fails to repress a TGF-ß responsive reporter in cells lacking Smad4, again highlighting the significance of the Smad4-Ski interactions. To reveal the mechanism of Smad4 recognition by Ski/SnoN and to understand Ski-mediated repression of TGF-ß, activin, or BMP signaling, the crystal structure of a complex between a conserved Smad4 binding fragment of Ski and the MH2 domain of Smad4 was determined at 2.85 Å resolution. The structure of the Ski fragment, stabilized by a tightly bound zinc atom, resembles that of the DNA binding SAND domain. Interestingly, the L3 loop region of Smad4 is specifically recognized by a highly conserved interaction loop (I loop) from Ski, whereas the corresponding I loop in the SAND domain is critically involved in DNA binding. The Ski binding surface on Smad4 significantly overlaps with that required for binding of the phosphorylated C terminus of the R-Smad. Consequently, Ski disrupts functional complexes between the Co- and R-Smads both in vitro and in vivo (Wu, 2002).

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).

SIP1, a member of the deltaEF1 family of two-handed zinc finger transcriptional repressors, has been identified as a Smad-binding protein. Mutations in the human SIP1 gene (ZFHX1B) have been implicated in Hirschsprung disease. The structure and transcriptional pattern of the mouse SIP1 gene (Zfhx1b) has been documented and it is compared to homologues from other species. The overall structure of Zfhx1b is highly similar to that of the deltaEF1 gene (Zfhx1a), confirming their close evolutionary relationship. In contrast to Zfhx1a, the 5' untranslated region of the SIP1-encoding mouse gene is very complex and includes several alternative exons. The corresponding 5'-UTR splicing pattern seems to be conserved between species and suggests a role in its transcriptional and/or translational regulation. The gene also codes for an antisense transcript that is highly conserved between human and mouse (Nelles, 2003).

deltaEF1 and SIP1 (or Zfhx1a and Zfhx1b, respectively) are the only known members of the vertebrate Zfh1 family of homeodomain/zinc finger-containing proteins. Similar to other transcription factors, both Smad-interacting protein-1 (SIP1) and deltaEF1 are capable of repressing E-cadherin transcription through binding to the E2 boxes located in its promoter. In the case of deltaEF1, this repression has been proposed to occur via interaction with the corepressor C-terminal binding protein (CtBP). In this study, it is shown by coimmunoprecipitation that SIP1 and CtBP interact in vivo and that an isolated CtBP-binding SIP1 fragment depends on CtBP for transcriptional repression. However, and most importantly, full-length SIP1 and deltaEF1 proteins do not depend on their interaction with CtBP to repress transcription from the E-cadherin promoter. Furthermore, in E-cadherin-positive kidney epithelial cells, the conditional synthesis of mutant SIP1 that cannot bind to CtBP, abrogates endogenous E-cadherin expression in a similar way as wild-type SIP1. These results indicate that full-length SIP1 can repress E-cadherin in a CtBP-independent manner (van Grunsven, 2003).

FoxO Forkhead transcription factors have been shown to act as signal transducers at the confluence of Smad, PI3K, and FoxG1 pathways. Smad proteins activated by TGF-ß form a complex with FoxO proteins to turn on the growth inhibitory gene p21Cip1. This process is negatively controlled by the PI3K pathway, a known inhibitor of FoxO localization in the nucleus, and by the telencephalic development factor FoxG1, which binds to FoxO-Smad complexes and blocks p21Cip1 expression. It is suggested that the activity of this network confers resistance to TGF-ß-mediated cytostasis during the development of the telencephalic neuroepithelium and in glioblastoma brain tumor cells (Seoane, 2004).

BMP signals act in concert with FGF8, WNT11 and WNT antagonists to induce the formation of cardiac tissue in the vertebrate embryo. In an effort to understand how these signaling pathways control the expression of key cardiac regulators, the cis-regulatory elements of the chick tinman homolog chick Nkx2.5 have been characterized. At least three distinct cardiac activating regions (CARs) of chick Nkx2.5 cooperate to regulate early expression in the cardiac crescent and later segmental expression in the developing heart. In this report, attention was focused on a 3' BMP-responsive enhancer, termed CAR3, which directs robust cardiac transgene expression. By systematic mutagenesis and gel shift analysis of this enhancer, it has been demonstrated that GATA4/5/6, YY1 and SMAD1/4 are all necessary for BMP-mediated induction and heart-specific expression of CAR3. Adjacent YY1 and SMAD-binding sites within CAR3 constitute a minimal BMP response element, and interaction of SMAD1/4 with the N terminus of YY1 is required for BMP-mediated induction of CAR3. These data suggest that BMP-mediated activation of this regulatory region reflects both the induction of GATA genes by BMP signals, as well as modulation of the transcriptional activity of YY1 by direct interaction of this transcription factor with BMP-activated SMADs (Lee, 2004).

How might the interaction of SMADs with YY1 modulate the activity of this transcription factor when bound to CAR3? Because YY1 can function as either a transcriptional activator or repressor, SMAD association with YY1 may serve to recruit co-activators that modulate the activity of this transcription factor to become an efficient transcriptional activator. Indeed, recruitment of co-activators such as p300 by TGFß activated SMADs is a well-characterized mechanism for SMAD target gene activation. Similarly, known interacting partners of YY1 also include several members of the histone deacetylase family as well as a histone H4 methylase, which have been implicated in either transcriptional repression or activation of YY1 regulated target genes, respectively. It will be interesting to determine if SMAD association with YY1 alters the interaction of this transcription factor with either of these families of histone modifying enzymes, and to what extent chromatin modification is responsible for appropriate regulation of Nkx2.5 (Lee, 2004).

SMAD-mediated modulation of YY1 activity adds an interesting new facet to the repertoire of functions of YY1 during heart development, which also includes direct recruitment of transcriptional co-activators to promote the expression of cardiac B-type natriuretic peptide, inhibition of the expression of the cardiac {alpha}-actin gene, and both activation and inhibition of the expression of the cardiac-specific Mlc2 gene. Clearly, the context within which YY1 functions is of great importance, and it is likely that transcription factors such as GATA and SMAD proteins, when bound to neighboring cognate binding sites, modulate either the association of co-factors with adjacently bound YY1 or the activity of such co-factors. In addition to the GATA, YY1- and SMAD-binding sites, linker scanning mutational analysis of the chick Nkx2.5 CAR3 BMPRE has revealed other sites yet to be characterized that also have a significant impact on the BMP response of this regulatory element. A complete understanding of complex enhancers such as Nkx2.5 CAR3 will require not only the identification of the transcription factors that regulate their expression but also elucidation of the transcriptional co-factors that are recruited to such regulatory elements in a combinatorial fashion (Lee, 2004).

Members of the T-box gene family play important and diverse roles in development and disease. Functional specificities of the Xenopus T-domain proteins Xbra and VegT, which differ in their abilities to induce gene expression in prospective ectodermal tissue, has been studied. In particular, VegT induces strong expression of goosecoid whereas Xbra cannot. These results indicate that Xbra is unable to induce goosecoid because it directly activates expression of Xom, a repressor of goosecoid that acts downstream of BMP signaling. The inability of Xbra to induce goosecoid is imposed by an N-terminal domain that interacts with the C-terminal MH2 domain of Smad1, a component of the BMP signal transduction pathway. Interference with this interaction causes ectopic activation of goosecoid and anteriorization of the embryo. These findings suggest a mechanism by which individual T-domain proteins may interact with different partners to elicit a specific response (Messenger, 2005).

During spinal cord development, the combination of secreted signaling proteins and transcription factors provides information for each neural type differentiation. Studies using embryonic stem cells show that trimethylation of lysine 27 of histone H3 (H3K27me3) contributes to repression of many genes key for neural development. However, it remains unclear how H3K27me3-mediated mechanisms control neurogenesis in developing spinal cord. This study demonstrates that H3K27me3 controls dorsal interneuron generation by regulation of BMP activity. Expression of Noggin, a BMP extracellular inhibitor, is repressed by H3K27me3. Moreover, Noggin expression is induced by BMP pathway signaling, generating a negative-feedback regulatory loop. In response to BMP pathway activation, JMJD3 histone demethylase interacts with the Smad1/Smad4 complex to demethylate and activate the Noggin promoter. Together, these data reveal how the BMP signaling pathway restricts its own activity in developing spinal cord by modulating H3K27me3 levels at the Noggin promoter (Alizu, 2010).

Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-beta pathways

TGF-beta and BMP receptor kinases activate Smad transcription factors by C-terminal phosphorylation. This study identified a subsequent agonist-induced phosphorylation that plays a central dual role in Smad transcriptional activation and turnover. As receptor-activated Smads form transcriptional complexes, they are phosphorylated at an interdomain linker region by CDK8 and CDK9, which are components of transcriptional mediator and elongation complexes. These phosphorylations promote Smad transcriptional action, which in the case of Smad1 is mediated by the recruitment of YAP (Drosophila homolog: Yorkie) to the phosphorylated linker sites. An effector of the highly conserved Hippo organ size control pathway, YAP supports Smad1-dependent transcription and is required for BMP suppression of neural differentiation of mouse embryonic stem cells. The phosphorylated linker is ultimately recognized by specific ubiquitin ligases, leading to proteasome-mediated turnover of activated Smad proteins. Thus, nuclear CDK8/9 drive a cycle of Smad utilization and disposal that is an integral part of canonical BMP and TGF-beta pathways (Alarcon, 2009).

The present findings reveal a remarkable integration of Smad regulatory functions by agonist-induced, CDK8/9-mediated phosphorylation of the linker region and highlight this event as an integral feature of the transcriptional action and turnover of receptor-activated Smad proteins. Agonist-induced linker phosphorylation of R-Smads is a general feature of BMP and TGF-β pathways, occurring in all the responsive cell types examined, shortly after Smad tail phosphorylation. The evidence identifies CDK9 as the kinases involved and does not support a major role for MAPKs or cell-cycle-regulatory CDKs in this process. CDK8 and cyclinC are components of the Mediator complex that couples enhancer-binding transcriptional activators to RNAP II for transcription initiation. CDK9 and cyclinT1 constitute the P-TEFb complex, which promotes transcriptional elongation. CDK8 and CDK9 phosphorylate overlapping serine clusters in the C-terminal domain of RNAP II, a region which integrates regulatory inputs by binding proteins involved in mRNA biogenesis. Thus, CDK8 and CDK9 may provide coordinated regulation of Smad transcriptional activators and RNAP II (Alarcon, 2009).

Precedent exists for the ability of CDK8 to phosphorylate enhancer-binding transcription factors. The CDK8 ortholog Srb10 in budding yeast phosphorylates Gcn4 marking this transcriptional activator of amino acid biosynthesis for recognition by the SCF(Cdc4) ubiquitin ligase. In mammalian cells, CDK8 phosphorylates the ICD signal transduction component of Notch, targeting it to the Fbw7/Sel10 ubiquitin ligase. However, whereas CDK8-mediated phosphorylation inhibits Gcn4 and Notch activity, this study shows that phosphorylation of agonist-activated Smads by CDK8/9 enables Smad-dependent transcription before triggering Smad turnover (Alarcon, 2009).

Activated Smads undergo proteasome-mediated degradation as well as phosphatase-mediated tail dephosphorylation to keep signal transduction closely tied to receptor activation. This study shows that BMP-induced Smad1-ALP generates binding sites for Smurf1, accomplishing in the nucleus what MAPK-mediated phosphorylation of basal-state Smad1 accomplishes in the cytoplasm. Similarly, TGF-β-induced linker phosphorylation of Smad2/3 provides a binding site for Nedd4L (Alarcon, 2009).

The results also reveal a positive role for ALP in Smad-dependent transcription. Smad proteins with phosphorylation-resistant linker mutations are more stable as receptor-activated signal transducers than their wild-type counterparts, yet they are transcriptionally less active. Indeed, mutation of Smad1 linker phosphorylation sites (in a wild-type Smad5 background) does not result in a straight BMP gain-of-function phenotype but rather in an unforeseen gastric epithelial phenotype. Although the interpretation of this phenotype is confounded by the contribution of MAPK signaling to linker phosphorylation, it is consistent with the present evidence that Smad1 linker phosphorylation plays an active role in BMP signaling (Alarcon, 2009).

Focusing on Smad1 to define this dual role, it was found that the phosphorylated linker sites, together with a neighboring PY motif, are recognized also by the transcriptional coactivator YAP. Smurf1 and YAP present closely related WW domains with a similar selectivity toward linker-phosphorylated Smad1. YAP is recruited with Smad1 to BMP responsive enhancers and knockdown of YAP inhibits BMP-induced Id gene responses in mouse embryonic stem cells. Both BMP and YAP act as suppressors of neural differentiation in specific contexts. This study shows that YAP supports the ability of BMP to block neural lineage commitment through the induction of Id family members, creating a link between YAP-dependent BMP transcriptional output and ES cell fate determination (Alarcon, 2009).

Thus, a common structure fulfills two opposite functions -- Smad1 transcriptional action and turnover -- by recruiting different proteins, YAP and Smurf1, at different stages of the signal transduction cycle. The cyclic recruitment and continuous turnover of transcription factors on target enhancers are required for the proper response of cells to developmental and homeostatic cues. It is proposed that Smad activation by TGF-β family agonists accomplishes this important requirement through linker phosphorylation that triggers transcriptional action and messenger turnover in one stroke (Alarcon, 2009).

Activation of the Hippo pathway by cell density cues triggers a kinase cascade that culminates in the inactivation of YAP (Yorkie in Drosophila), a transcriptional coactivator that acts through interactions with enhancer-binding factors, including TEAD/scalloped, Runx, p73, and others. Yorkie/YAP promotes cell proliferation and survival and organ growth, whereas the upstream components of the inhibitory kinase cascade constrain organ size and act as tumor suppressors. Elucidating the links between the Hippo pathway and other signaling cascades is an important open question. The evidence that YAP is recruited to BMP-activated Smad1 reveals a link between the BMP and the Hippo pathways. Both these signaling cascades have the capacity to control organ size and do so in a manner suggestive of interactions with other patterned signals. An example is the regulation of imaginal disc growth by Dpp via cell competition, a process by which slow proliferating cells are eliminated in favor of their higher-proliferating neighbors. A genetic screen for negative regulators of Dpp signaling that protect cells from being outcompeted identified upstream components of the Hippo pathway. Inactivation of these factors elevated Dpp target gene expression, presumably by failing to inhibit Yorkie, and allowed cells to outcompete their neighbors, suggesting a functional convergence of the Hippo and BMP pathways that foreshadowed the findings of this study (Alarcon, 2009).

Although ALP is a general event in Smad activation, YAP may not be a universal partner of linker-phosphorylated Smad1. Smad ALP likely plays a wider role potentially acting to recruit coactivators other than YAP, depending on the cellular context or the target gene. Also of interest is the identity of factors that may play an analogous role in linker-phosphorylated Smad2/3 in the TGF-β pathway. The linker phosphorylation sites and PY motifs of Smad1 and Smad2/3 are conserved in the otherwise divergent linker regions of the Drosophila orthologs Mad/dSmad1 and dSmad2, respectively. Although the contribution of the MAPK pathway in linker phosphorylation precludes a clearcut genetic investigation of these functions, they are probably conserved across metazoans. A concerted search for Smad phospholinker interacting factors would answer many of these questions and would fully elucidate the role of the Smad linker region as a centerpiece in the function, regulation, and connectivity of Smad transcription factors (Alarcon, 2009).

A Smad action turnover switch operated by WW domain readers of a phosphoserine code

When directed to the nucleus by TGF-β or BMP signals, Smad proteins undergo cyclin-dependent kinase 8/9 (CDK8/9) and glycogen synthase kinase-3 (GSK3) phosphorylations that mediate the binding of YAP and Pin1 for transcriptional action, and of ubiquitin ligases Smurf1 and Nedd4L for Smad destruction. This study demonstrates that there is an order of events-Smad activation first and destruction later-and that it is controlled by a switch in the recognition of Smad phosphoserines by WW domains in their binding partners. In the BMP pathway, Smad1 phosphorylation by CDK8/9 creates binding sites for the WW domains of YAP, and subsequent phosphorylation by GSK3 switches off YAP binding and adds binding sites for Smurf1 WW domains. Similarly, in the TGF-β pathway, Smad3 phosphorylation by CDK8/9 creates binding sites for Pin1 and GSK3, then adds sites to enhance Nedd4L binding. Thus, a Smad phosphoserine code and a set of WW domain code readers (see A Smad action turnover switch operated by WW domain readers of a phosphoserine code) provide an efficient solution to the problem of coupling TGF-β signal delivery to turnover of the Smad signal transducers (Aragón, 2011).

Schnurri-2 controls BMP-dependent adipogenesis via interaction with Smad proteins

Adipocyte differentiation is an important component of obesity, but how hormonal cues mediate adipocyte differentiation remains elusive. BMP stimulates in vitro adipocyte differentiation, but the role of BMP in adipogenesis in vivo is unknown. Drosophila Schnurri (Shn) is required for the signaling of Decapentaplegic, a Drosophila BMP homolog, via interaction with the Mad/Medea transcription factors. Vertebrates have three Shn orthologs, Shn-1, -2, and -3. This study reports that Shn-2-/- mice have reduced white adipose tissue and that Shn-2-/- mouse embryonic fibroblasts cannot efficiently differentiate into adipocytes in vitro. Shn-2 enters the nucleus upon BMP-2 stimulation and, in cooperation with Smad1/4 and C/EBPα, induces the expression of PPARγ2, a key transcription factor for adipocyte differentiation. Shn-2 directly interacts with both Smad1/4 and C/EBPα on the PPARγ2 promoter. These results indicate that Shn-2-mediated BMP signaling has a critical role in adipogenesis (Jin, 2006).

BMP-2 induces PPARγ expression and adipogenesis in C3H10T1/2 cells. The effects of BMP-2 on PPARγ2 promoter activity was analyzed using a PPARγ2 promoter-driven luciferase gene. Wild-type MEFs were transfected with the PPARγ2-Luc reporter, and adipocyte differentiation was induced in the presence or absence of BMP-2. The luciferase levels of wild-type cells increased 73% in the presence of BMP-2, whereas the luciferase levels of Shn-2-/- cells were not affected by BMP-2 treatment. Thus, the PPARγ2 promoter is weakly responsive to BMP-2, and Shn-2 is required for this BMP responsiveness. The low degree of induction by BMP-2 could be due to an imbalance among the transcription factors and the promoter molecule in transfected cells (Jin, 2006).

To further examine the BMP responsiveness of the PPARγ2 promoter and the role of Shn-2, luciferase reporter assays were performed using wild-type MEFs transfected with the PPARγ2-Luc reporter and various combinations of expression plasmids for Smad1/4 and Shn-2. The PPARγ2 promoter contains C/EBP binding sites and its activity is enhanced by C/EBPα and C/EBPδ, and, therefore, the C/EBPα expression plasmid was also used. Without Smad1/4 or C/EBPα, the BMP-2-induced expression of luciferase was not observed, whereas BMP-2 enhanced luciferase expression about 2-fold in the presence of Smad1/4 and C/EBPα. When Smad1/4, C/EBPα, and Shn-2 were coexpressed together, higher BMP-2 responsiveness (3.6-fold) was observed. These results may support the speculation that the appropriate balance of these factors and the PPARγ2 promoter molecule is needed for BMP responsiveness. When Shn-2-/- MEFs were used for similar experiments, BMP-2 enhanced luciferase expression only about 50% in the presence of Smad1/4 and C/EBPα. Exogenous expression of Shn-2 in the mutant cells significantly restored the BMP-2 responsiveness of the PPARγ2 promoter (4.5-fold). These results suggest that Shn-2, Smad1/4, and C/EBPα synergistically mediate the BMP-induced transactivation of the PPARγ2 promoter (Jin, 2006).

Smad3/4 bind to the 5'-AGAC-3' sequence, while Smad1 binds to GC-rich sequences. The mouse and human PPARγ2 promoter regions contain six AGAC sequences but not the GC-rich sequence. The AGAC sequence was also found at ten sites in the 1.2 kb promoter region of the mouse PPARγ1 gene. Among these six putative Smad binding sites in the mouse PPARγ2 promoter, four sites (sites 1, 2, 4, and 6) are conserved in the human PPARγ2 promoter. Mutant mouse PPARγ2-Luc reporters in which the AGAC sites were mutated, and the level of activation of the reporters by Shn-2, Smad1/4, ALK3QD, and C/EBPα was examined. The results indicate that three sites in the upstream region of the promoter (sites 1-3) are required for synergistic activation by these factors. Mutation of any of these three sites significantly reduced activation by Shn-2, Smad1/4, ALK3QD, and C/EBPα. The human PPARγ2 promoter lacks site 3 but has another Smad site further upstream of site 1. The presence of three Smad sites in this region of the mouse and human PPARγ2 promoters may support formation of a Smad1/4-Shn-2-C/EBPα complex to synergistically activate transcription (Jin, 2006).

Vertebrate Shn was originally identified as NF-κB site binding proteins, and the metal finger regions of Drosophila and Xenopus Shn recognize these specific DNA sequences. No NF-κB recognition sequence was found in the PPARγ2 promoter, but one sequence (5'-TCCCACCTCTCCC-3') at -94 to -82 partially resembles the Xenopus Shn binding sequence. However, mutation of this site did not affect the synergistic activation of the PPARγ2 promoter by Shn-2, Smad1/4, ALK3QD, and C/EBPα (Jin, 2006).

To examine whether Shn-2 directly binds to the PPARγ2 promoter, a DNA precipitation assay was performed. FLAG-Shn-2 was expressed in 293T cells, immunoprecipitated by anti-FLAG antibody, and eluted from the immunocomplex using FLAG peptide. The purified Shn-2 protein was mixed with 32P-labeled PPARγ2 promoter fragments and precipitated with anti-Shn-2 antibody. The PPARγ2 promoter fragment was not detected in the immunocomplex. These results suggest that Shn-2 does not directly bind and is recruited by Smad proteins to the PPARγ2 promoter (Jin, 2006).

To investigate the interaction between Shn-2 and Smad1/4, coimmunoprecipitation assays of the exogenously expressed proteins were performed. 293T cells were cotransfected with plasmids to express FLAG-Shn-2, Myc-Smad1, and HA-Smad4, and lysates from transfected cells were immunoprecipitated with an anti-FLAG antibody. Myc-Smad1 and HA-Smad4 were coprecipitated with FLAG-Shn-2. When HA-Smad4 was deleted from this combination, FLAG-Shn-2 coprecipitated lesser amounts of Myc-Smad1. When Myc-Smad1 was deleted, HA-Smad4 was not coprecipitated with FLAG-Shn-2. These results suggest that Shn-2 interacts with the hetero-oligomers of Smad1 and Smad4 (Jin, 2006).

To determine which region of Shn-2 protein is responsible for interaction with Smad1, GST pull-down assays were performed. Two in vitro-translated Shn-2 fragments containing either the N- or C-terminal metal fingers (N1 and C1) bound to a GST-Smad1 resin, whereas the two fragments containing the central region of Shn-2 (HS and CP) exhibited only background and minor binding, respectively. Deletion of the metal finger regions from N1 and C1 abrogated the interaction with Smad1, suggesting that both metal finger regions are important for interactions with Smad1 (Jin, 2006).

The present study demonstrates that Shn-2 enters the nucleus upon BMP stimulation and plays an important role in adipocyte differentiation. The current study strongly suggests that BMP has a critical role in vivo. Upon BMP stimulation, Shn-2 is recruited to the PPARγ2 promoter via an interaction with Smad1. This is the first demonstration that Shn plays a role in vertebrate BMP/TGF-β/activin signaling. Shn-2 is required for efficient transcription of PPARγ2, possibly as a scaffold protein to form a ternary complex with Smad1/4 and C/EBPα. Interestingly, Evi-1, which is also a large protein containing two regions of metal fingers like Shn-2, interacts with and represses TGF-β/BMP-activated transcription through Smad proteins. Following TGF-β stimulation, Evi-1 and the associated corepressor CtBP are recruited to the target promoter. Thus, Shn-2 and Evi-1 interact with Smad proteins via their metal fingers and may stimulate and repress transcription by recruiting coactivator and corepressor, respectively (Jin, 2006).

Although vertebrate Shn proteins were originally identified as the NF-κB site binding proteins, the present study indicates that Shn-2 is recruited to the PPARγ2 promoter via an interaction with Smad1 and C/EBPα. This is similar to the recent report that Drosophila Shn forms a complex with Mad/Medea on the silencer element of the brinker (brk) gene to mediate Dpp-dependent brk gene silencing. The brinker silencer element contains three 5'-AGAC-3' sequences and two GC-rich sequences between them, to which Medea and Mad bind, respectively. However, the GC-rich sequence was not found between three 5'-AGAC-3' sequences in the PPARγ2 promoter. Therefore, more work is required to understand whether Smad1 in the Smad1/4 hetero-oligomers directly recognizes the DNA sequence in the PPARγ2 promoter. Interaction of Shn-2 not only with Smad1/4 but also with C/EBPα may support the idea that Shn-2 serves as a scaffold protein to form a ternary complex with various transcription factors to synergistically activate transcription. In fact, Shn-3 was reported to interact with c-Jun to activate IL-2 gene transcription (Jin, 2006).

Adipogenesis in vitro follows a highly ordered and well-characterized temporal sequence. In cultured cell models, initial growth arrest is induced by the addition of a prodifferentiative hormonal regimen and is followed by one or two additional rounds of cell division (clonal expansion). This process ceases upon induction of PPARγ2 and C/EBPα, which is concomitant with permanent growth arrest followed by expression of the fully differentiated phenotype. E2F1 induces PPARγ2 transcription during clonal expansion, whereas E2F4 represses PPARγ2 expression during terminal adipocyte differentiation. Interaction between Smad and E2F proteins has been shown for the Smad3-E2F4/5 complex mediating TGF-β-induced repression of c-myc. Therefore, Smad1/4-Shn-2 may also participate in E2F-dependent transcriptional regulation of PPARγ2 by directly interacting with E2F1/4. IFNγ decreases the expression of PPARγ2 in preadipocytes, but the mechanism remains to be elucidated. IFNγ induces the expression of Smad7, which prevents TGF-β receptor-mediated Smad3 phosphorylation. IFNγ may suppress PPARγ2 transcription by inducing Smad6, which then prevents BMP receptor-mediated Smad1 phosphorylation. FoxO1 is also known to regulate adipocyte differentiation. FoxO1 is induced in the early stages of adipocyte differentiation, and prevents adipose differentiation by upregulating multiple genes, including cell cycle inhibitors. Insulin leads to nuclear exclusion of FoxO1 and stimulates adipocyte differentiation. Smad proteins activated by TGF-β form a complex with FoxO proteins to turn on the growth-inhibitory gene p21Cip1 , and BMP-7 induces higher p21 expression than TGF-β1. By interacting with FoxO proteins, therefore, Smad1/4-Shn-2 may also regulate transcription not only of PPARγ2 but also of p21 during adipocyte differentiation (Jin, 2006).

Although more work is required to understand the role of Smad1/4-Shn-2 during adipocyte differentiation, identification of BMP signaling as the key regulatory pathway of adipogenesis in vivo may enable the development of drugs to affect this signaling pathway to suppress obesity and obesity-related diseases (Jin, 2006).

Smad targets

TGFbeta-related factors are critical regulators of vertebrate mesoderm development. However, the signaling cascades required for their function during this developmental process are poorly understood. Tlx-2 is a homeobox gene expressed in the primitive streak of mouse embryos. Exogenous BMP-2 rapidly activates Tlx-2 expression in the epiblast of E6.5 embryos. A Tlx-2 promoter element responds to BMP-2 signals in P19 cells; this response is mediated by BMP type I receptors and Smad1. These results suggest that Tlx-2 is a downstream target gene for BMP signaling in the primitive streak where BMP-4 and other TGFbeta-related factors are expressed. Disruption of Tlx-2 function leads to early embryonic lethality. Similar to BMP4 and ALK3 mutants, the mutant embryos display severe defects in primitive streak and mesoderm formation. These experiments identify a BMP/Tlx-2 signaling pathway that is required during early mammalian gastrulation (Tang, 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).

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).

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 (positions -2,300/-210) and a proximal (positions -124 to -61) region 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).

Bone morphogenetic proteins (BMPs) are pleiotropic growth and differentiation factors belonging to the transforming growth factor-beta (TGF-beta) superfamily. Signals of the TGF-beta-like ligands are propagated to the nucleus through specific interaction of transmembrane serine/threonine kinase receptors and Smad proteins. GCCGnCGC has been suggested as a consensus binding sequence for Drosophila Mad regulated by Decapentaplegic. Smad1 is one of the mammalian Smads activated by BMPs. Smad1 is shown to bind to this motif upon BMP stimulation in the presence of the common Smad, Smad4. The binding affinity is likely to be relatively low, because Smad1 binds to three copies of the motif weakly, but more repeats of the motif significantly enhance the binding. Heterologous reporter genes (GCCG-Lux) with multiple repeats of the motif respond to BMP stimulation but not to TGF-beta or activin. Mutational analyses reveal several bases critical for the responsiveness. A natural BMP-responsive reporter, pTlx-Lux, is activated by BMP receptors in P19 cells but not in mink lung cells. In contrast, GCCG-Lux responds to BMP stimulation in both cells, suggesting that it is a universal reporter that directly detects Smad phosphorylation by BMP receptors (Kusanagi, 2000).

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).

The anterior heart field (AHF) mediates formation of the outflow tract (OFT) and right ventricle (RV) during looping morphogenesis of the heart. Foxh1 is a forkhead DNA binding transcription factor in the TGFß-Smad pathway. Foxh1−/− mutant mouse embryos form a primitive heart tube, but fail to form OFT and RV and display loss of outer curvature markers of the future working myocardium, similar to the phenotype of Mef2c−/− mutant hearts. Further, Mef2c is shown to be a direct target of Foxh1, which physically and functionally interacts with Nkx2-5 to mediate strong Smad-dependent activation of a TGFß response element in the Mef2c gene. This element directs transgene expression to the presumptive AHF, as well as the RV and OFT, a pattern that closely parallels endogenous Mef2c expression in the heart. Thus, Foxh1 and Nkx2-5 functionally interact and are essential for development of the AHF and its derivatives, the RV and OFT, in response to TGFß-like signals (von Both, 2004).

Renal dysplasia, the major cause of childhood renal failure in humans, arises from perturbed renal morphogenesis and molecular signaling during embryogenesis. Induction of molecular crosstalk between Smad1 and ß-catenin occurs in the TgAlk3QD mouse model of renal medullary cystic dysplasia. The finding that Myc, a Smad and ß-catenin transcriptional target and effector of renal epithelial dedifferentiation, is misexpressed in dedifferentiated epithelial tubules provides a basis for investigating coordinate transcriptional control by Smad1 and ß-catenin in disease. Enhanced interactions occur between a molecular complex consisting of Smad1, ß-catenin and Tcf4 and adjacent Tcf- and Smad-binding regions located within the Myc promoter in TgAlk3QD dysplastic renal tissue, and Bmp-dependent cooperative control of Myc transcription by Smad1, ß-catenin and Tcf4. Analysis of nuclear extracts derived from TgAlk3QD and wild-type renal tissue revealed increased levels of Smad1/ß-catenin molecular complexes, and de novo formation of chromatin-associated Tcf4/Smad1 molecular complexes in TgAlk3QD tissues. Analysis of a 476 nucleotide segment of the 1490 nucleotide Myc genomic region upstream of the transcription start site demonstrated interactions between Tcf4 and the Smad consensus binding region and associations of Smad1, ß-catenin and Tcf4 with oligo-duplexes that encode the adjacent Tcf- and Smad-binding elements only in TgAlk3QD tissues. In collecting duct cells that express luciferase under the control of the 1490 nucleotide Myc genomic region, Bmp2-dependent stimulation of Myc transcription is dependent on contributions by each of Tcf4, ß-catenin and Smad1. These results provide novel insights into mechanisms by which interacting signaling pathways control transcription during the genesis of renal dysplasia (Hu, 2005)

TBX20 has been shown to be essential for vertebrate heart development. Mutations within the TBX20 coding region are associated with human congenital heart disease, and the loss of Tbx20 in a wide variety of model systems leads to cardiac defects and eventually heart failure. Despite the crucial role of TBX20 in a range of cardiac cellular processes, the signal transduction pathways that act upstream of Tbx20 remain unknown. This study identified and characterized a conserved 334 bp Tbx20 cardiac regulatory element that is directly activated by the BMP/SMAD1 signaling pathway. This element is both necessary and sufficient to drive cardiac-specific expression of Tbx20 in Xenopus, and blocking SMAD1 signaling in vivo specifically abolishes transcription of Tbx20, but not that of other cardiac factors, such as Tbx5 and MHC, in the developing heart. Activation of Tbx20 by SMAD1 is mediated by a set of novel, non-canonical, high-affinity SMAD-binding sites located within this regulatory element, and phospho-SMAD1 directly binds a non-canonical SMAD1 site in vivo. Finally, it was shown that these non-canonical sites are necessary and sufficient for Tbx20 expression in Xenopus, and that reporter constructs containing these sites are expressed in a cardiac-specific manner in zebrafish and mouse. Collectively, these findings define Tbx20 as a direct transcriptional target of the BMP/SMAD1 signaling pathway during cardiac maturation (Mandel 2010).

SMAD proteins control DROSHA-mediated microRNA maturation

MicroRNAs (miRNAs) are small non-coding RNAs that participate in the spatiotemporal regulation of messenger RNA and protein synthesis. Aberrant miRNA expression leads to developmental abnormalities and diseases, such as cardiovascular disorders and cancer; however, the stimuli and processes regulating miRNA biogenesis are largely unknown. The transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) family of growth factors orchestrates fundamental biological processes in development and in the homeostasis of adult tissues, including the vasculature. Induction of a contractile phenotype in human vascular smooth muscle cells by TGF-β and BMPs is mediated by miR-21. miR-21 downregulates PDCD4 (programmed cell death 4), which in turn acts as a negative regulator of smooth muscle contractile genes. Surprisingly, TGF-β and BMP signalling promotes a rapid increase in expression of mature miR-21 through a post-transcriptional step, promoting the processing of primary transcripts of miR-21 (pri-miR-21) into precursor miR-21 (pre-miR-21) by the DROSHA (also known as RNASEN) complex. TGF-β- and BMP-specific SMAD signal transducers are recruited to pri-miR-21 in a complex with the RNA helicase p68 (also known as DDX5), a component of the DROSHA microprocessor complex. The shared cofactor SMAD4 is not required for this process. Thus, regulation of miRNA biogenesis by ligand-specific SMAD proteins is critical for control of the vascular smooth muscle cell phenotype and potentially for SMAD4-independent responses mediated by the TGF-β and BMP signalling pathways (Davis, 2008).

Smad developmental regulation

Smad proteins are intracellular signaling molecules and putative transcription factors that transduce signals elicited by members of the transforming growth factor beta (TGF-beta) 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 TGF-beta 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 TGF-beta superfamily during embryonic development (Dick, 1998).

Gastrulation generates mesoderm and endoderm from embryonic epiblast; soon after, the neural plate is established within the epiblast-both events require FGF signaling. A zinc finger transcriptional activator, Churchill (ChCh), is described that acts as a switch between different roles of FGF. FGF induces ChCh slowly; this activates Smad-interacting-protein-1 (Sip1), which blocks further induction of the mesoderm markers brachyury and Tbx6L by FGF. ChCh is first expressed as cells stop migrating through the primitive streak, and it regulates cell ingression. A simple mechanism is proposed by which FGF sensitizes cells to BMP signals. These results reveal that neural induction requires cessation of mesoderm formation at the midline in addition to the decision between epidermis and neural plate (Sheng, 2003).

Because a temporal arrest in the G(1) phase of the cell cycle is thought to be a prerequisite for cell differentiation, cell cycle factors were investigated that critically influence the differentiation of mouse osteoblastic MC3T3-E1 cells induced by bone morphogenetic protein 2 (BMP-2), a potent inducer of osteoblast differentiation. Of the G(1) cell cycle factors examined, the expression of cyclin-dependent kinase 6 (Cdk6) was found to be strongly down-regulated by BMP-2/Smads signaling, mainly via transcriptional repression. The enforced expression of Cdk6 blocked BMP-2-induced osteoblast differentiation to various degrees, depending on the level of its overexpression. However, neither BMP-2 treatment nor Cdk6 overexpression significantly affected cell proliferation, suggesting that the inhibitory effect of Cdk6 on cell differentiation was exerted by a mechanism that is largely independent of its cell cycle regulation. These results indicate that Cdk6 is a critical regulator of BMP-2-induced osteoblast differentiation and that its Smads-mediated down-regulation is essential for efficient osteoblast differentiation (Ogasawara, 2004).

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. Feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state are upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT are increased, while Smad1 deletion in Nkx2-5 mutants rescue SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies are also rescued by Smad1 deletion. These findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD (Prall, 2007).

Canonical BMP7 activity is required for the generation of discrete neuronal populations in the dorsal spinal cord

BMP activity is essential for many steps of neural development, including the initial role in neural induction and the control of progenitor identities along the dorsal-ventral axis of the neural tube. Taking advantage of chick in ovo electroporation, a novel role was shown for BMP7 at the time of neurogenesis initiation in the spinal cord. Using in vivo loss-of-function experiments, BMP7 activity was shown to be required for the generation of three discrete subpopulations of dorsal interneurons: dI1-dI3-dI5. Analysis of the BMP7 mouse mutant shows the conservation of this activity in mammals. Furthermore, this BMP7 activity appears to be mediated by the canonical Smad pathway; Smad1 and Smad5 activities are similarly required for the generation of dI1-dI3-dI5. Moreover, this role is independent of the patterned expression of progenitor proteins in the dorsal spinal cord, but depends on the BMP/Smad regulation of specific proneural proteins, thus narrowing this BMP7 activity to the time of neurogenesis. Together, these data establish a novel role for BMP7 in primary neurogenesis, the process by which a neural progenitor exits the cell cycle and enters the terminal differentiation pathway (Le Dréau, 2011).

BMP5 and Smads in limb development

In an attempt to identify new genes implicated in the control of programmed cell death during limb development, a cDNA library was generated from the regressing interdigital tissue of chicken embryos. 804 sequences were analzyed from this library and 23 genes were identified involved in apoptosis in different models. One of the genes that came up in the screening was the Bone Morphogenetic Protein family member Bmp5 that had not been previously found to be involved in the control of apoptosis during limb development. In agreement with a possible role in the control of cell death, Bmp5 exhibited a regulated pattern of expression in the interdigital tissue. Transcripts of Bmp5 and BMP5 protein were abundant within the cytoplasm of the fragmenting apoptotic interdigital cells in a way suggesting that delivery of BMPs into the tissue is potentiated during apoptosis. Gain-of-function experiments have demonstrated that BMP5 has the same effect as other interdigital BMPs inducing apoptosis in the undifferentiated mesoderm and growth in the prechondrogenic mesenchyme. Both Smad proteins and MAPK p38 have been characterized as intracellular effectors for the action of BMPs in the developing limb autopod. Activation of Smad signaling involves the receptor-regulated genes Smad1 and -8, and the inhibitory Smad6, and results in both the upregulation of gene transcription and protein phosphorylation with subsequent nuclear translocation. MAPK p38 is also quickly phosphorylated after BMP stimulation in the limb mesoderm. Treatment with the inhibitor of p38, SB203580, revealed that there are interdigital genes induced by BMPs in a p38-dependent manner (DKK, Snail and FGFr3), and genes induced in a p38-independent manner (BAMBI, Msx2 and Smads). Together, these results suggest that Smad and MAPK pathways act synergistically in the BMP pathway controlling limb development (Zuzarte-Luísa, 2004).

BMP/SMAD1 signaling sets a threshold for the left/right pathway in lateral plate mesoderm and limits availability of SMAD4

Bistability in developmental pathways refers to the generation of binary outputs from graded or noisy inputs. Signaling thresholds are critical for bistability. Specification of the left/right (LR) axis in vertebrate embryos involves bistable expression of transforming growth factor beta (TGFbeta) member NODAL in the left lateral plate mesoderm (LPM) controlled by feed-forward and feedback loops. This study provides evidence that bone morphogenetic protein (BMP)/SMAD1 signaling sets a repressive threshold in the LPM essential for the integrity of LR signaling. Conditional deletion of Smad1 in the LPM led to precocious and bilateral pathway activation. NODAL expression from both the left and right sides of the node contributed to bilateral activation, indicating sensitivity of mutant LPM to noisy input from the LR system. In vitro, BMP signaling inhibited NODAL pathway activation and formation of its downstream SMAD2/4-FOXH1 transcriptional complex. Activity was restored by overexpression of SMAD4 and in embryos, elevated SMAD4 in the right LPM robustly activated LR gene expression, an effect reversed by superactivated BMP signaling. It is concluded that BMP/SMAD1 signaling sets a bilateral, repressive threshold for NODAL-dependent Nodal activation in LPM, limiting availability of SMAD4. This repressive threshold is essential for bistable output of the LR system (Furtado, 2008).

Smads and differentiation

Bone morphogenetic protein-2 (BMP-2) inhibits terminal differentiation of C2C12 myoblasts and converts them into osteoblast lineage cells. The possible involvement of Smad proteins, vertebrate homologs of Drosophila Mothers against decapentaplegic, has been examined in the BMP effects on the differentiation of C2C12 myoblasts. C2C12 cells express Smad1, Smad2, Smad4, and Smad5 mRNAs, and expression levels are not altered by treatment with BMP-2 or TGF-beta1. When Smads are transiently transfected into C2C12 cells, both Smad1 and Smad5 induce alkaline phosphatase (ALP) activity and decrease the activity of myogenin promoter/chloramphenicol acetyltransferase (myogenin-CAT) without BMP-2. When C-terminal-truncated Smad1 and Smad5 are transfected into constitutively active BMP receptor type IB (BMPR-IB)-expressing C2C12 cells, BMP signals are blocked, resulting in an increase in myogenin-CAT activity. In contrast, Smad1 and Smad5 decrease myogenin-CAT activity but do not induce ALP activity in MyoD-transfected NIH3T3 fibroblasts. These results suggest that both Smad1 and Smad5 are involved in the intracellular BMP signals that inhibit myogenic differentiation and induce osteoblast differentiation in C2C12 cells, and that the conversion of the two differentiation pathways is regulated independently at a transcriptional level (Yamamoto, 1997).

During development of the cerebellum, sonic hedgehog (Shh) is directly responsible for the proliferation of granule cell precursors in the external germinal layer. Signals able to regulate a switch from the Shh-mediated proliferative response to one that directs differentiation of granule neurons have been sought. Bone morphogenetic proteins (BMPs) are expressed in distinct neuronal populations within the developing cerebellar cortex. Bmp2 and Bmp4 are expressed in the proliferating precursors and subsequently in differentiated granule neurones of the internal granular layer, whereas Bmp7 is expressed by Purkinje neurones. In primary cultures, Bmp2 and Bmp4, but not Bmp7, are able to prevent Shh-induced proliferation, thereby allowing granule neuron differentiation. Furthermore, Bmp2 treatment downregulates components of the Shh pathway in proliferating granule cell precursors. Smad proteins, the only known BMP receptor substrates capable of transducing the signal, are also differentially expressed in the developing cerebellum: Smad1 in the external germinal layer and Smad5 in newly differentiated granule neurons. Among them, only Smad5 is phosphorylated in vivo and in primary cultures treated with Bmp2, and overexpression of Smad5 is sufficient to induce granule cell differentiation in the presence of Shh. A model is proposed in which Bmp2-mediated Smad5 signalling suppresses the proliferative response to Shh by downregulation of the pathway, and allows granule cell precursor to enter their differentiation programme (Rios, 2004).

BMP canonical Smad signaling through Smad1 and Smad5 is required for endochondral bone formation

Bone morphogenetic protein (BMP) signaling is required for endochondral bone formation. However, whether or not the effects of BMPs are mediated via canonical Smad pathways or through noncanonical pathways is unknown. In this study the role was determined of receptor Smads 1, 5 and 8 in chondrogenesis. Deletion of individual Smads results in viable and fertile mice. Combined loss of Smads 1, 5 and 8, however, results in severe chondrodysplasia. Smad1/5CKO (cartilage-specific knockout) mutant mice are nearly identical to Smad1/5CKO;Smad8-/- mutants, indicating that Smads 1 and 5 have overlapping functions and are more important than Smad8 in cartilage. The Smad1/5CKO phenotype is more severe than that of Smad4CKO mice, challenging the dogma, at least in chondrocytes, that Smad4 is required to mediate Smad signaling through BMP pathways. The chondrodysplasia in Smad1/5CKO mice is accompanied by imbalances in cross-talk between the BMP, FGF and Ihh/PTHrP pathways. Ihh is shown to be a direct target of BMP pathways in chondrocytes, and FGF exerts antagonistic effects on Ihh expression. Finally, whether FGF exerts its antagonistic effects directly through Smad linker phosphorylation was tested. The results support the alternative conclusion that the effects of FGFs on BMP signaling are indirect in vivo (Retting, 2009).

Smads and disease

Renal dysplasia, the most frequent cause of childhood renal failure in humans, arises from perturbations in a complex series of morphogenetic events during embryonic renal development. The molecular pathogenesis of renal dysplasia is largely undefined. While investigating the role of a BMP-dependent pathway that inhibits branching morphogenesis in vitro, a novel model of renal dysplasia was generated in a transgenic (Tg) model of ALK3 (activin-like kinase 3; BMPR1A) receptor signaling. This study reports the renal phenotype, and the discovery of molecular interactions between effectors in the BMP and WNT signaling pathways in dysplastic kidney tissue. Expression of the constitutively active ALK3 receptor ALK3QD, in two independent transgenic lines causes renal aplasia/severe dysgenesis in 1.5% and 8.4% of hemizygous and homozygous Tg mice, respectively, and renal medullary cystic dysplasia in 49% and 74% of hemizygous and homozygous Tg mice, respectively. The dysplastic phenotype, which included a decreased number of medullary collecting ducts, increased medullary mesenchyme, collecting duct cysts and decreased cortical thickness, is apparent by E18.5. The pathogenesis of dysplasia in these mice was investigated, and a 30% decrease in branching morphogenesis was demonstrated at E13.5 before the appearance of histopathogical features of dysplasia. The formation of ß-catenin/SMAD1/SMAD4 molecular complexes was also demonstrated in dysplastic renal tissue. Increased transcriptional activity of a ß-catenin reporter gene in ALK3QD;Tcf-gal mice demonstrates functional cooperativity between the ALK3 and ß-catenin-dependent signaling pathways in kidney tissue. Together with the results in the dysplastic mouse kidney, the findings that phospho-SMAD1 and ß-catenin are overexpressed in human fetal dysplastic renal tissue suggest that dysregulation of these signaling effectors is pathogenic in human renal dysplasia. This work provides novel insights into the role that crucial developmental signaling pathways may play during the genesis of malformed renal tissue elements (Hu, 2003).

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Mothers against dpp: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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