- Email: [email protected]
Mads and Smads in TGFβ signalling
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Mads and Smads in TGFI3 signalling Liliana Attisano* and Jeffrey L Wranat The discovery of the Mothers against dpp (Mad) gene in Drosophila has opened a window on an entirely unique signalling pathway that functions to mediate responses to the tumour growth factor ~ (TGFIJ) superfamily. This pathway, which is comprised of a family of proteins related to Mad, acts to convey signals directly from TGFI3 receptors to the nucleus and is implicated in the pathogenesis of human diseases.
Addresses *Department of Anatomy and Cell Biology, University of Toronto, Ontario, Canada, M5S lX8 tProgram in Developmental Biology, The Hospital for Sick Children, 555 UniversityAvenue, and Department of Medical Genetics and Microbiology, University of Toronto, Ontario, Canada, M5X lX8; e-mail: [email protected] Correspondence: Jeff Wrana at The Hospital for Sick Children Current Opinion in Cell Biology 1998, 10:188-194
http://biomednet.com/elecref/0955067401000188 © Current Biology Ltd ISSN 0955-0674 Abbreviations ARE activinresponse element BMP bone morphogenetic protein Dpp decapentaplegic MAD mothersagainst dpp sma small TGF~ transforminggrowth factor
Introduction
Transforming growth factor 13 (TGFIJ) is the prototypic member of a large family of signalling molecules that have critical roles in regulating development and homeostasis in vertebrates and invertebrates. Members of this superfamily signal through heteromeric complexes of type II and type I serine/threonine kinase receptors. Within this complex, receptor II transphosphorylates and activates receptor I which then transmits the signal to a downstream family of signal transduction molecules related to the Drosophila gene Mothers against dpp (Mad). There has been a veritable explosion of information on the role of these proteins in mediating TGFI3 superfamily signalling. As a result, a unique signal transduction pathway has been described that connects the TGFI] receptor with the nuclear transcriptional machinery. T h e elucidation of this pathway has also revealed some insights into the pathogenesis of human disease, as many of the components are found to be mutated in human cancers.
Small a n d M a d A Drosophila screen to pick up maternal-effect enhancers of weak alleles of the TGFIJ-like gene, decapentaplegic (dpp), led to the identification of two genetic loci, Mad and Medea [1]. Mad was the first of these to be cloned
and was shown to possess a sequence unlike anything previously identified in the database [2]. Subsequent work demonstrated that the Mad protein indeed functioned downstream of TKV, the type I receptor for DPP protein, [3,4] and was required for dpp activity in the cell that received DPP signals [5]. With the initial identification of Mad, Sekelsky et al. [2] also identified a Caenorhabditis elegans gene that was closely related to Mad and which they called cem-1. In fact cem-1 actually turned out to be one of the genes essential for male tail-ray development that are mutated in small (sma) mutants [6]. This was particularly intriguing as the phenotype of the sma mutants was similar to that of mutants in the gene for the bone morphogenetic protein (BMP) type II receptor, Daf-4. Furthermore, mosaic analysis suggested that like Mad, the sma genes functioned downstream of daf-4. T h e analysis of sma mutants revealed another intriguing finding--that sma-2, sma-3 and sma-4 all represented Mad-related genes. Moreover, genetic analysis suggested that each of these sma genes was required for all TGF[~ signalling, providing the first evidence that these genes function cooperatively in mediating TGFI3 superfamily signalling. Smads
Numerous Mad-related genes have been identified in vertebrates and have been dubbed Smad genes. Currently, eight members have been reported and comparisons of primary sequences make it immediately evident that the proteins encoded by these genes have two highly conserved domains in the amino- and carboxy-terminal regions (Figure 1). These have been termed the MH1 and MH2 domains, respectively (for Mad Homology 1 and Mad Homology 2). T h e structure of the Smad4 MH2 domain has been solved and reveals that the domain exists in the crystal as a trimer with a symmetrical disk-like arrangement [7"]. T h e non-conserved domains extend from one face of the disk while the other face may provide an interaction region that mediates heteromer formation with other Smads. Interestingly, many tumorigenic and developmental mutations in Mad-related genes map to the protein trimer interfaces or to a region of the disk predicted to mediate heteromeric interactions with other Smads. T h e precise functions of the MH1 and MH2 domains in mediating signalling events are still being unravelled; some significant advances have been made, however. In functional assays, expression of the MH2 domain of Smadl, Smad2 or Smad3 is sufficient to initiate signalling in Xenopus and mammalian cells [8-12]. In addition, the MH1 domain physically interacts with the MH2 domain and a model has been developed in which the MH1 domain autoregulates the activity of the MH2 domain [9]. However, as will be described later, the MH1 domain itself
Mads and Smads in TGFI3 signalling Attisano
Figure 1 (a)
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(b)
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Common
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Smadl 1 Smad5 BMP Vertebrates Smad8 Smad2 -ITG FI~ Smad3 _JActivin
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CurrentOpinionin CellBiology The Smad family of signal transducers. (a) Schematic representation of Smad proteins. The highly conserved MH1 and MH2 domains are separated by a proline-rich non-conserved linker region. The carboxy-terminal SSXS motif present in receptor-regulated Smads and the WW-domain recognition motif (PY) present in some Smads is indicated.(b) Conservation of Smad proteins in vertebrates, Drosophila and C. elegans. Members of the Smad family can be subdivided into three groups, receptor-regulated, common and inhibitory Smads. The placement of the Smads into these functional subgroups has been demonstrated for some, but not all, members. In these cases, placement within a group is based on conservation of sequence or on function deduced from genetic data (for details see text).
may be required for some Smad functions, so the MH1 and MH2 domains may actually be mutually inhibitory in the resting state. Unlike the MH1 and MH2 domains, the so-called linker domain of Smads is less well conserved between species and between different members. T h e function of this domain is much less well defined but in the case of Smad4 may contain an activation domain that is required for signalling [13]. This domain is extremely rich in proline and serine residues and could function as a P E S T domain [5], and a conserved PY motif is found in this region in all of the receptor-regulated Smads. T h e PY motif is a proline-rich sequence of the general form XPPXY and serves as a recognition motif for WW domains [14]. However, WW-domain-containing proteins that interact with Smads have yet to be reported.
Smads transduce signals from the receptor into the nucleus Smads are rapidly phosphorylated in response to TGFI3, activin and BMP signalling. Studies using either epitopetagged Smads or antibodies specific for different Smad isoforms have indicated that these phosphorylation events are highly specific. Thus, Smadl, -5 or -8 are phosphorylated in response to BMP signalling, while Smad2
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and -3 lie in the TGFi3/activin pathway [3,15-21,22°]. Phosphorylation of these Smads correlates with their accumulation in the nucleus [3,8,23,24,25], in Drosophila, elevation of Dpp levels induces the nuclear translocation of Mad in vivo [26]. Interestingly, in the latter case, nuclear levels of Mad could not be identified under endogenous levels of Dpp signalling, suggesting that the amount of translocated Mad required for it to fulfil its nuclear functions is quite low. T h e specific regulation of Smad phosphorylation correlates with functional studies in Xenopus that demonstrate that overexpression of particular receptor-regulated Smads alters cell fates in a pathwayspecific manner. Thus, Smadl, -5 and -8 induce BMP-like effects [15,27°°,28,29] while Smad2 and -3 induce TGF[3 or activin-like responses [22°,27°']. T h e kinase that phosphorylates these Smads is the receptor itself, providing a molecular understanding of why phosphorylation of individual Smads is specific for a particular pathway. In the case of TGFI3, Smad2 and Smad3 can associate with the TGFI3 receptor [22",24"°,25]. This association is mediated by the type I receptor and requires activation of receptor I by the type II receptor [24°°]. Consistent with the role of Smads in transmitting signals into the nucleus, association with type I receptor is extremely transient and kinase-deficient versions of the receptor are required to trap the interaction [24°',25]. Phosphopeptide mapping of the phosphorylation places the receptor-dependent sites on the last two serines in a conserved SSXS motif found at the carboxy-terminal of the protein [30,31]. Work on BMP signalling has shown that a SSXS motif in Smadl is the site for phosphorylation by BMP receptors [17]. Thus, the presence of the SSXS motif at the carboxyl terminus appears to define a class of receptor-regulated Smads, while other classes of Smads, such as Smad4, -6 and -7 which do not possess this motif, do not appear to be substrates of the type I receptors. Phosphorylation of receptor-regulated Smads is mediated by the intracellular domain of the type I receptor and is highly specific. Recent studies suggest that a loop, located between the putative 13sheets 4 and 5 (L45) within the kinase domain, determines intraceltular signalling specificity [32]. This region of the receptor could form part of the Smad recognition site in the kinase domain and thus mediate specificity in signalling. Phosphorylation of receptor-regulated Smads on the SSXS motif is critical for nuclear accumulation and phosphorylation-site mutants prevent translocation of the protein in response to signalling [17,24°°]. In addition to regulating the subcellular localization of Smads, phosphorylation is critical for controlling the assembly of heteromeric complexes of Smad proteins.
Partnership between Smads From the initial genetic data in nematodes it appeared that Smads may function in heteromeric complexes [6] and biochemical and functional assays with mammalian
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Smads support this suggestion. Indeed, phosphorylation of receptor-regulated Smads leads to formation of heteromeric complexes with Smad4. For instance, Smadl, Smad8 and probably Smad5 associate with Smad4 in response to BMP signalling [15,33°°], while Smad2 and $mad3 associate with Smad4 in response to TGFI3 and activin signalling [11,25,33°°]. Moreover, functional studies based on dominant-negative mutants of Smads and rescue of certain TGF[5 transcriptional responses in Smad4-deficient turnout cell lines has led to a model in which Smad4 is considered to be an obligate partner for all TGFI3 superfamily signalling [12,22°,33"°]. However, whether Smad4 is absolutely required for all signalling has yet to be determined. In Drosophila, the genetic screen that led to the isolation of Mad also identified Medea [1] and recent cloning of this gene has revealed that it encodes for a protein closely related to Smad4 (L Raftery, personal communication; see Note added in proof). Thus, partnership between Smads is likely a conserved feature of the pathway. Smad4 has been localized to the cytoplasm of resting cells and requires interaction with a receptor-regulated Smad in order to accumulate in the nucleus [34]. This has important implications because it suggests that the receptor-regulated Smad will have a critical role not only in ushering Smad4 into the nucleus, but also in maintaining signalling specificity in the nucleus by mediating interaction of Smad4 with nuclear targets (Figure 2). Recent studies have also revealed a TGFIS-dependent interaction between Smad2 and Smad3 [25]. Furthermore, Smad2 and -3 appeared to synergistically enhance activation of a particular TGFI~ transcriptional response (the activation of the 3TP promoter). This raises the possibility that complexes of Smad2, -3 and -4 may provide the best signalling. In addition, these data suggest that Smad2 and Smad3, which are highly conserved, may not simply be redundant components of the pathway but may form part of a higher-order heteromeric complex together with Smad4. T h e presence of a common partner in both activin/TGF~ signalling and BMP signalling has led to the interesting observation that these pathways can inhibit each other by competing for this common component. Hence, high levels of BMP signalling can titrate Smad4 and block subsequent signalling by activin [35]. Whether this occurs at endogenous signalling levels, where the amount of Smads translocating to the nucleus can be quite small [26], has yet to be determined. N u c l e a r f u n c t i o n s of S m a d c o m p l e x e s T h e search for effectors in the signal transduction pathways of TGFI] superfamily members has led to the identification of the Xenopus transcription factor, FAST1 [36°°]. FAST1 is a forkhead-containing DNA-binding protein that interacts with an activin-response element (ARE) to mediate activin-dependent induction of the
Figure 2
mad4
Current Opinion in Cell Biology
A model for TGF~ signal transduction. Phosphorylation of Smad2 and Smad3 occurs in response to TGFI3 or activin, whereas Smadl, -5, -8 and Drosophila Mad are phosphorylated in response to BMP-like ligands (see text for details). In C. elegans, regulation by phosphorylation is predicted due to the presence of a carboxy-terminal SSXS and SXT motif in Sma2 and Sma3, respectively. Subsequent to phosphorylation, the receptor-regulated Smad (in this case illustrated by Smad2) physically interacts with Smad4 in the cytoplasm and the complex translocates to the nucleus where it targets resident DNA-binding proteins such as FAST1 or binds directly to DNA. In the case of Drosophila, a similar pathway involving Mad and Medea functions in Dpp signalling (L Attisano, unpublished data). Inhibitory Smads block signalling by associating stably with the type I receptor to prevent access of the receptor-regulated Smads.
Mix2 gene. In the absence of signalling, FAST-1 binds constitutively to the ARE, but upon stimulation with activin, it assembles into a higher-order DNA-binding complex, termed the ARF (activin-responsive factor), which contains both Smad2 and Smad4 [34,36",37]. To form the ARF, Smad2 functions to recruit Smad4 into a complex with FAST1 (Figure 2) and binds to the Smad2 interaction domain, or SID, which is located near the carboxy-terminal region of FAST1, outside of the forkhead DNA-binding domain [34,37]. Once recruited to FAST1, Smad4 appears to be required to stabilize DNA binding of the higher-order complex as well as activate transcription. Interestingly, the functions of Smad4 within the ARF may be separable with the MH1 domain acting to stabilize DNA binding, while the MH2 is required to activate transcription [34]. This observation would be consistent with previous analyses that suggested that both domains in Smad4 were required for the function of the protein [13]. T h e mechanism of ARF formation also serves to illustrate the central role of receptor-activated Smads in each step of the TGF[3 signalling pathway. By functioning as both the substrate of the receptor and the usher that brings Smad4
Mads and Smads in TGF~ signallingAttisanoand Wrana
to its nuclear targets, the receptor-regulated Smads fulfil a critical role in maintaining specificity of the transcriptional response to TGFIB family members. Activin-dependent response elements have also been identified in other target genes. In the goosecoidpromoter, Smad2 appears to regulate transcription through DNA-binding complexes that are distinct from FASTl-containing complexes [35]. Further, an unrelated ARE has been found within the first intron of the Xenopus Forkhead (XFKH)-I gene and appears to represent yet another Smad2 target [38]. So identification of how Smads specifically regulate these diverse response elements will be an interesting area for future research. In addition to targeting resident DNA-binding proteins, recent evidence suggests that Smads can directly bind DNA. In Drosophila, activation of dpp signalling pathways in imaginal disks can induce transcription of the vestigial gene and this is mediated by a specific GC-rich sequence [39°]. Interestingly, a bacterially expressed MH1 domain of MAD can bind to this region, suggesting that MAD itself may mediate Dpp-dependent activation of the element. However, this interaction may not be sufficient, and similar sequences in other promoters do not appear to have a critical role in mediating Dpp-responsiveness [40]. In mammalian cells, TGFI3 induces formation of a phosphorylation-dependent DNA-binding complex composed of Smad3 and Smad4 that recognizes a bipartite site within the promoter of the TGF~-inducible reporter, 3TP-Lux [41]. In this case, DNA binding is mediated by Smad4 and there is no similarity between the bipartite element in 3TP and the Mad site in the vestigial quandrant enhancer. However, the functional significance of the interaction remains unknown, since mutation of the Smad-binding site does not interfere with TGFi3-dependent transcriptional activation of the 3TP promoter [41].
Regulating the S m a d pathway Smads do not appear to possess any enzymatic activity. Thus, the sensitivity and function of the pathway is exquisitely sensitive to the levels of Smad protein. Indeed, earlier studies in Drosophila have shown that reductions in Mad gene dosage are sufficient to suppress phenotypes induced by activation of Dpp signalling [3,4]. Furthermore, in Xenopus, manipulation of Smad protein levels is sufficient to alter cell fate in a linear, pathway-specific manner [27°°,42]. Thus Smadl induces ventral cell fates while Smad2 induces dorsal cell fates consistent with the function of these proteins in BMP and TGFl3/activin signalling, respectively [8,27°°,29]. In mammalian cells, Smad2 expression is strongly upregulated in developing follicular granulosa cells and this correlates with increased sensitivity of the cells to TGFI] [43]. Interestingly, in these cells TGFI3 itself appears to upregulate Smad2 levels, suggesting a positive feedforward mechanism in which TGFIB can autoregulate the sensitivity of its own signalling pathway.
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Apart from regulating Smad protein levels, other mechanisms may exist to control activation of the pathway. Recent evidence has suggested that Smadl may serve as a substrate for mitogen-activated protein kinase (MAPK) [44]. Phosphorylation occurs in the linker region, regulates the subcellular localization of the Smad protein and appears to inhibit BMP signalling. Whether phosphorylation of endogenous Smad protein is similarly regulated is unclear, as is the general biological applicability of this observation, since interactions of TGFI3 and BMP pathways with receptor tyrosine kinase signalling pathways are complex and as often involve synergistic interactions as antagonistic ones. Recently, another class of Smad proteins represented by Smad6 and Smad7 have been identified [45°'-47"',48]. Smad6 and Smad7 are distantly related to receptor-regulated Smads and Smad4 and have a poorly conserved MH1 domain. Interestingly, in the case of Smad6 a short form of the molecule, Smad6(S), comprising only the MH2 domain, is expressed in some cell types [48,49]. Smad6 and Smad7 have a unique function: these Smads act as negative regulators of signalling and potently block both TGFI3 and BMP signalling [45"°-47°',48]. Surprisingly, these proteins can interact stably with the TGFIB and BMP receptors to prevent association and phosphorylation of the receptor-regulated Smads. Thus, these anti-Smads act as intracellular antagonists of type I serine/threonine kinase receptors. In the case of Smad6, additional mechanisms to block signalling may be employed, since Smad6(S) has been shown to associate with numerous Smads, thus raising the possibility that it could block signalling by preventing heteromeric complex formation [48]. Expression of anti-Smads is regulated in a highly specific manner. In the case of Smad6(S) and Smad7, transcription of the genes is strongly induced in endothelial cells in response to fluid laminar shear stress and could function to insulate the endothelium from the effects of circulating TGF~3 family members [48]. In addition, it appears that Smad6 and -7 are early target genes for the Smad signalling pathways and that Smad7 is immediately, but transiently induced by TGF[3 in a variety of cell lines [47°°]. What function this brief pulse of Smad7 expression fulfils is unclear, although it could represent a method for downregulating the sensitivity of cells to TGFI3. In Drosophila a related Smad called Dad (for Daughters against dpp) appears to be regulated by Dpp along the anterior-posterior boundary of the wing imaginal disk and may function to inhibit signalling in a manner analogous to Smad6 and -7 [50]. In C. elegans, Daf-3 may represent another antagonistic Smad [51].
Implications of the S m a d p a t h w a y in d e v e l o p m e n t and h u m a n disease Members of the TGFIB family have critical roles as morphogens during vertebrate and invertebrate development. Intriguingly, cell-fate decisions in response to this family of cytokines is highly dependent on ligand
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concentration, and small changes in activin concentration can induce alternative cell-fate changes in Xenopus [52]. The apparent absence of enzymatic activity thus yields a signalling pathway in which the nuclear concentration of Smad provides a direct and proportional measure of the external ligand concentration. This may be critical for the establishment of TGF[3 signalling gradients that then function to control cell fate precisely, depending on the position of the cell in a morphogenetic field.
in colorectal cancers is similar to observations made on the TGF[3 receptor system. T h e pathway may thus be particularly important in controlling homeostasis of the gastrointestinal epithelium. These studies have served to illustrate that the TGFI3 signal transduction pathway can serve a tumour suppressor function which, when disrupted by mutation, may contribute to tumour progression by disrupting cellular responses to TGFI~.
The role of TGFI] as an antiproliferative factor for a variety of cell types has focused considerable attention on this pathway as a target of tumour suppressor mutations. Thus far we have seen that there are relatively few components linking the cell-surface receptor with the nuclear transcriptional machinery. Consistent with the turnout suppressive role of TGF~, mutations have been identified in numerous components of the pathway. Of particular interest, 90% of colon cancers with microsatellite instability (RER + for 'replication errors') have inactivating mutations in the extracellular domain of TI~RII, the type II receptor for TGF[~ [53]. Turnouts that exhibit microsatellite instability occur either in a hereditary form, termed H N P C C (hereditary nonpolyposis colorectal cancer), or as sporadic cases (reviewed in [54]) and account for approximately 15-17% of all colorectal carcinomas. In contrast, receptor mutations are rare in endometrial cancers with microsatellite instability, emphasizing the importance of TGFI3 signalling in colorectal cancer [55]. In addition to RER+ colorectal cancer, mutations in the kinase domain of T ~ R I I have been identified in squamous head and neck carcinomas [56], implicating the TI3RII gene as a tumour suppressor gene in other turnouts. In addition alterations in the TGF[~ type I receptor, TI3RI, have been found in prostate, colon and gastric cancer cells and a lack of TI3RI protein has been observed in AIDS-related Kaposi's sarcoma (reviewed in [57]). These studies suggest that in addition to the type II receptor, TI3RI is another target for inactivating mutations in cancer.
The isolation of Mad by genetic screens has rapidly led to the elaboration of a pathway that directly connects the TGF[3 receptor with the transcriptional machinery. How this conserved pathway elicits diverse, cell-type specific responses to control developmental and homeostatic processes will undoubtedly become a major area of interest in the future.
Conclusions
Like the receptors, intracellular components of the TGF[~ signalling pathway may also function as tumour suppressors (reviewed in [58]). The genes for both Smad2 and Smad4 map to chromosome 18q21 [16,59°°,60], a region that displays loss of heterozygosity in about 90% of pancreatic tumours and in over 60% of colorectal cancers and evidence now implicates the Smad genes as the candidate tumour suppressor genes at this locus. Somatic missense mutations in Smad4 (DPC4) have been identified in pancreatic carcinomas [59°°], and colorectal cancers [61,62], while two missense mutations and a two base-pair frameshift have also been identified in a sampling of 42 lung cancers [63]. Genetic alterations and inactivating missense mutations in Smad2 (MADR2 or JV-18) have also been identified in sporadic colorectal carcinomas but none in breast cancers and sarcomas [16,60]. Apart from those in Smad2 and Smad4 no other mutations have thus far been reported [49]. The bias for mutations of Smad2
Notes added in proof 1) The identification of medea as a Smad4 homologue, which was referred to in the text as ' L Raftery, personal communication' is now in press [64]. Surprisingly, this paper demonstrates that while Mad is required for all dpp signalling events, medea is only required for a subset of responses, and in the wing disk may function to extend the range of dpp action. 2) Recently, another report on inhibitory Smads has been published that shows that in addition to interacting with type 1 receptors, Smad6 associates with Smadl and may prevent formation of Smadl-Smad4 heteromeric complexes [65]. Acknowledgements
Work in the authors' laboratories is supported by grants from the National Cancer Institute of Canada and the Medical Research Council of Canada. The authors are Medical Research Council of Canada Scholars. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: * of special interest o= of outstanding interest 1.
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Chen X, Rubock MJ, Whitman M: A transcriptional partner for MAD proteins in TGF-~ signalling. Nature 1996, 383:691-696. paper describes the identification of a novel forkhead-containing transcription factor from Xenopus, named FAST1. FAST1 was shown to bind to an activin-response element in the Mix.2 promoter and form a transcriptional activation complex, together with Smad2. Thus, it is the first demonstration of a nuclear target for Smad2.
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activation of the element in vivo, thereby providing evidence that Smads can directly bind DNA to activate transcription. 40. Eresh S, Riese J, Jackson DB, Bohmann D, Bienz M: A CREBbinding site as a target for decapentaplegic signalling during Drosophila endoderm induction. EMBO J 1997, 16:2014-2022.
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Markowitz S, Wang J, Myeroff L, Parsons R, Sun 17, Lutterbaugh J, Fan RS, Zborowska E, Kinzler KW, Vogelstein B et al.: Inactivation of the type II TGF-~ receptor in colon cancer cells with microsatellite instability. Science 1995, 268:13361338.
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Garrigue-Antar L, Muoz-Antonia T, Antonia SJ, Gesmonde J, Vellucci VF, Reiss M: Missense mutations of the transforming growth factor 13type II receptor in human head and neck squamous carcinoma cells. Cancer Res 1995, 55:3982-3987.
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Markowitz SD, Roberts AB: Tumor suppressor activity of the TGF-~ pathway in human cancers. Cytokine Growth Factor Rev 1996, 7:93-102.
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Rustgi AK: MAD about colorectal cancer. Gastroenterology 1996, 111:1387-1389.
Yingling JM, Datto MB, Wong C, Frederick JP, Liberati NT, Wang X-F: Tumour suppressor Smad4 is a transforming growth factor B-inducible DNA binding protein. Mo/Cell Biol 1997, 17:7019-7028.
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Wilson PA, Lagna G, Suzuki A, Hemmati-Brivanlou A: Concentration-dependent patterning of the Xenopus ectoderm by BMP4 and its signal transducer Smadl. Development 1997, 124:3177-3184.
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Li M, Li J, Hoodless PA, Tzukazaki T, Wrana JL, Attisano L, Tsang BK: MADR2 Expression in avian granulosa cells is up-regulated by TGF~ during ovarian follicular development. Endocrinology 1997, 138:3659-3665. Kretzschmar M, Doody J, Massagu~ J: Opposing BMP and EGF signalling pathways converge on the TGF-~ family mediator Smadl. Nature 1997, 389:618-622.
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Hayashi H, Abdollah S, Qiu Y, Cai J, Xu Y-Y, Grinnell BW, Richardson MA, Topper JN, Gimbrone JMA, Wrana JL, Falb D: The MAD-related protein Smad7 associates with the TGFb receptor and functions as an antagonist of TGF~ signaling. Cell 1997,
Imamura T, Takase M, Nishihara A, Oeda E, Hanai J-I, Kawabata M, Miyazono K: Smad6 inhibits signalling by the TGF-~ superfamily. Nature 1997, 389:622-626. See note to Nakano eta/., 1997 [47"].
Hahn SA, Schutte M, Shamsul Hoque ATM, Moskaluk CA, da Costa LT, Rozenblum E, Weinstein CL, Fischer A, Yeo C J, Hruban RH eta/.: DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 1996, 271:350-353. This paper describes the first mammalian Smad gane, Smad4, demonstrates its localization at 18q21 and shows that it is mutated in pancreatic carcinomas. This paper is significant because it defines Smads as a novel class of tumour suppressors that is involved in the development of human cancers.
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Riggins G J, Thiagalingam S, Rozenblum E, Weinstein CL, Kern SE, Hamilton SR, Willson JKV, Markowitz SD, Kinzler KW, Vogelstein B: MAD-related genes in the human. Nat Genet 1996, 13:347-349.
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Thiagalingam S, Langauer C, Leach F, Schutte M, Hahn S, Overhauser J, Willson J, Markowitz S, Hamilton S, Kern S e t al. : Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers. Nat Genet 1996, 13:343346.
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Nakao A, Afrakhte M, Mor~n A, Nakayama T, Christian JL, Heuchel R, Itoh S, Kawabata M, Heldin N-E, Heldin C-H, ten Dijke P: Identification of Smad7, a TGF~-inducible antagonist of TGF-~ signalling. Nature 1997, 389:631-635. These three papers define a novel class of Smad proteins. Unlike the positively acting Smads, the inhibitory Smads identified in these papers function to block TGFI3 and BMP signalling by directly interacting with the receptors to prevent association and phosphorylation of the receptor-regulated Smads. 48.
Topper JN, Cai J, Qiu Y, Anderson KR, Xu Y-Y, Deeds JD, Feeley R, Gimeno C J, Woolf F_A,Tayber O e t a/.: Vascular MADs: two novel MAD-related genes selectively inducible by flow in human vascular endothelium. Proc Nat/Acad Sci USA 1997, 94:9314-9319.
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Riggins G J, Kinzler KW, Vogelstein B, Thiagalingam S: Frequency of Smad gene mutations in human cancers. Cancer Res 1997, 57:2578-2580.
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Tsuneizumi K, Nakayama T, Kamoshida Y, Kornberg TB, Christian JL, Tabata T: Daughters against dpp modulates dpp organizing activity in Drosophila wing development. Nature 1997, 389:627831.
Nagatake M, Takagi Y, Osada H, Uchida K, Mitsudomi T, Saji S, Shimokata K, Takahashi T: Somatic in vivo alteration of the DPC4 gene at 18q21 in human lung cancers. Cancer Res 1996, 56:2718-2720.
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Patterson G, Koweek A, Wong A, Liu Y, Ruvkun G: The DAF-3 Smad protein antagonizes TGF-~-related receptor signalling in the C. elegans dauer pathway. Genes Dev 1997, 11:2679-2690. Green JBA, Smith JC: Graded changes in dose of a Xenopus activin A homologue elicit stepwise transitions in embryonic cell fate. Nature 1990, 347:391-394.
Wisotzkey RG, Mehra A, Sutherland DJ, Dobens LL, Liu X, Dohrmann C, Attisano L, Raftery LA: Medea, a Drosophila Smsd4 homolog that is differentially required to potentiate DPP responses.Development 1998, in press.
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Mads and Smads in TGFI3 signalling Liliana Attisano* and Jeffrey L Wranat The discovery of the Mothers against dpp (Mad) gene in Drosophila has opened a window on an entirely unique signalling pathway that functions to mediate responses to the tumour growth factor ~ (TGFIJ) superfamily. This pathway, which is comprised of a family of proteins related to Mad, acts to convey signals directly from TGFI3 receptors to the nucleus and is implicated in the pathogenesis of human diseases.
Addresses *Department of Anatomy and Cell Biology, University of Toronto, Ontario, Canada, M5S lX8 tProgram in Developmental Biology, The Hospital for Sick Children, 555 UniversityAvenue, and Department of Medical Genetics and Microbiology, University of Toronto, Ontario, Canada, M5X lX8; e-mail: [email protected] Correspondence: Jeff Wrana at The Hospital for Sick Children Current Opinion in Cell Biology 1998, 10:188-194
http://biomednet.com/elecref/0955067401000188 © Current Biology Ltd ISSN 0955-0674 Abbreviations ARE activinresponse element BMP bone morphogenetic protein Dpp decapentaplegic MAD mothersagainst dpp sma small TGF~ transforminggrowth factor
Introduction
Transforming growth factor 13 (TGFIJ) is the prototypic member of a large family of signalling molecules that have critical roles in regulating development and homeostasis in vertebrates and invertebrates. Members of this superfamily signal through heteromeric complexes of type II and type I serine/threonine kinase receptors. Within this complex, receptor II transphosphorylates and activates receptor I which then transmits the signal to a downstream family of signal transduction molecules related to the Drosophila gene Mothers against dpp (Mad). There has been a veritable explosion of information on the role of these proteins in mediating TGFI3 superfamily signalling. As a result, a unique signal transduction pathway has been described that connects the TGFI] receptor with the nuclear transcriptional machinery. T h e elucidation of this pathway has also revealed some insights into the pathogenesis of human disease, as many of the components are found to be mutated in human cancers.
Small a n d M a d A Drosophila screen to pick up maternal-effect enhancers of weak alleles of the TGFIJ-like gene, decapentaplegic (dpp), led to the identification of two genetic loci, Mad and Medea [1]. Mad was the first of these to be cloned
and was shown to possess a sequence unlike anything previously identified in the database [2]. Subsequent work demonstrated that the Mad protein indeed functioned downstream of TKV, the type I receptor for DPP protein, [3,4] and was required for dpp activity in the cell that received DPP signals [5]. With the initial identification of Mad, Sekelsky et al. [2] also identified a Caenorhabditis elegans gene that was closely related to Mad and which they called cem-1. In fact cem-1 actually turned out to be one of the genes essential for male tail-ray development that are mutated in small (sma) mutants [6]. This was particularly intriguing as the phenotype of the sma mutants was similar to that of mutants in the gene for the bone morphogenetic protein (BMP) type II receptor, Daf-4. Furthermore, mosaic analysis suggested that like Mad, the sma genes functioned downstream of daf-4. T h e analysis of sma mutants revealed another intriguing finding--that sma-2, sma-3 and sma-4 all represented Mad-related genes. Moreover, genetic analysis suggested that each of these sma genes was required for all TGF[~ signalling, providing the first evidence that these genes function cooperatively in mediating TGFI3 superfamily signalling. Smads
Numerous Mad-related genes have been identified in vertebrates and have been dubbed Smad genes. Currently, eight members have been reported and comparisons of primary sequences make it immediately evident that the proteins encoded by these genes have two highly conserved domains in the amino- and carboxy-terminal regions (Figure 1). These have been termed the MH1 and MH2 domains, respectively (for Mad Homology 1 and Mad Homology 2). T h e structure of the Smad4 MH2 domain has been solved and reveals that the domain exists in the crystal as a trimer with a symmetrical disk-like arrangement [7"]. T h e non-conserved domains extend from one face of the disk while the other face may provide an interaction region that mediates heteromer formation with other Smads. Interestingly, many tumorigenic and developmental mutations in Mad-related genes map to the protein trimer interfaces or to a region of the disk predicted to mediate heteromeric interactions with other Smads. T h e precise functions of the MH1 and MH2 domains in mediating signalling events are still being unravelled; some significant advances have been made, however. In functional assays, expression of the MH2 domain of Smadl, Smad2 or Smad3 is sufficient to initiate signalling in Xenopus and mammalian cells [8-12]. In addition, the MH1 domain physically interacts with the MH2 domain and a model has been developed in which the MH1 domain autoregulates the activity of the MH2 domain [9]. However, as will be described later, the MH1 domain itself
Mads and Smads in TGFI3 signalling Attisano
Figure 1 (a)
NH2t~
/mmm-"m ~COOH PY
...SSXS*
(b)
Receptor-regulated
Common
Inhibitory
Smadl 1 Smad5 BMP Vertebrates Smad8 Smad2 -ITG FI~ Smad3 _JActivin
Smad4
Smad6 Smad7
Drosophila Mad
Medea
Dad
Sma4
Daf-3
Sma2
C. elegans Sma3
CurrentOpinionin CellBiology The Smad family of signal transducers. (a) Schematic representation of Smad proteins. The highly conserved MH1 and MH2 domains are separated by a proline-rich non-conserved linker region. The carboxy-terminal SSXS motif present in receptor-regulated Smads and the WW-domain recognition motif (PY) present in some Smads is indicated.(b) Conservation of Smad proteins in vertebrates, Drosophila and C. elegans. Members of the Smad family can be subdivided into three groups, receptor-regulated, common and inhibitory Smads. The placement of the Smads into these functional subgroups has been demonstrated for some, but not all, members. In these cases, placement within a group is based on conservation of sequence or on function deduced from genetic data (for details see text).
may be required for some Smad functions, so the MH1 and MH2 domains may actually be mutually inhibitory in the resting state. Unlike the MH1 and MH2 domains, the so-called linker domain of Smads is less well conserved between species and between different members. T h e function of this domain is much less well defined but in the case of Smad4 may contain an activation domain that is required for signalling [13]. This domain is extremely rich in proline and serine residues and could function as a P E S T domain [5], and a conserved PY motif is found in this region in all of the receptor-regulated Smads. T h e PY motif is a proline-rich sequence of the general form XPPXY and serves as a recognition motif for WW domains [14]. However, WW-domain-containing proteins that interact with Smads have yet to be reported.
Smads transduce signals from the receptor into the nucleus Smads are rapidly phosphorylated in response to TGFI3, activin and BMP signalling. Studies using either epitopetagged Smads or antibodies specific for different Smad isoforms have indicated that these phosphorylation events are highly specific. Thus, Smadl, -5 or -8 are phosphorylated in response to BMP signalling, while Smad2
and Wrana
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and -3 lie in the TGFi3/activin pathway [3,15-21,22°]. Phosphorylation of these Smads correlates with their accumulation in the nucleus [3,8,23,24,25], in Drosophila, elevation of Dpp levels induces the nuclear translocation of Mad in vivo [26]. Interestingly, in the latter case, nuclear levels of Mad could not be identified under endogenous levels of Dpp signalling, suggesting that the amount of translocated Mad required for it to fulfil its nuclear functions is quite low. T h e specific regulation of Smad phosphorylation correlates with functional studies in Xenopus that demonstrate that overexpression of particular receptor-regulated Smads alters cell fates in a pathwayspecific manner. Thus, Smadl, -5 and -8 induce BMP-like effects [15,27°°,28,29] while Smad2 and -3 induce TGF[3 or activin-like responses [22°,27°']. T h e kinase that phosphorylates these Smads is the receptor itself, providing a molecular understanding of why phosphorylation of individual Smads is specific for a particular pathway. In the case of TGFI3, Smad2 and Smad3 can associate with the TGFI3 receptor [22",24"°,25]. This association is mediated by the type I receptor and requires activation of receptor I by the type II receptor [24°°]. Consistent with the role of Smads in transmitting signals into the nucleus, association with type I receptor is extremely transient and kinase-deficient versions of the receptor are required to trap the interaction [24°',25]. Phosphopeptide mapping of the phosphorylation places the receptor-dependent sites on the last two serines in a conserved SSXS motif found at the carboxy-terminal of the protein [30,31]. Work on BMP signalling has shown that a SSXS motif in Smadl is the site for phosphorylation by BMP receptors [17]. Thus, the presence of the SSXS motif at the carboxyl terminus appears to define a class of receptor-regulated Smads, while other classes of Smads, such as Smad4, -6 and -7 which do not possess this motif, do not appear to be substrates of the type I receptors. Phosphorylation of receptor-regulated Smads is mediated by the intracellular domain of the type I receptor and is highly specific. Recent studies suggest that a loop, located between the putative 13sheets 4 and 5 (L45) within the kinase domain, determines intraceltular signalling specificity [32]. This region of the receptor could form part of the Smad recognition site in the kinase domain and thus mediate specificity in signalling. Phosphorylation of receptor-regulated Smads on the SSXS motif is critical for nuclear accumulation and phosphorylation-site mutants prevent translocation of the protein in response to signalling [17,24°°]. In addition to regulating the subcellular localization of Smads, phosphorylation is critical for controlling the assembly of heteromeric complexes of Smad proteins.
Partnership between Smads From the initial genetic data in nematodes it appeared that Smads may function in heteromeric complexes [6] and biochemical and functional assays with mammalian
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Smads support this suggestion. Indeed, phosphorylation of receptor-regulated Smads leads to formation of heteromeric complexes with Smad4. For instance, Smadl, Smad8 and probably Smad5 associate with Smad4 in response to BMP signalling [15,33°°], while Smad2 and $mad3 associate with Smad4 in response to TGFI3 and activin signalling [11,25,33°°]. Moreover, functional studies based on dominant-negative mutants of Smads and rescue of certain TGF[5 transcriptional responses in Smad4-deficient turnout cell lines has led to a model in which Smad4 is considered to be an obligate partner for all TGFI3 superfamily signalling [12,22°,33"°]. However, whether Smad4 is absolutely required for all signalling has yet to be determined. In Drosophila, the genetic screen that led to the isolation of Mad also identified Medea [1] and recent cloning of this gene has revealed that it encodes for a protein closely related to Smad4 (L Raftery, personal communication; see Note added in proof). Thus, partnership between Smads is likely a conserved feature of the pathway. Smad4 has been localized to the cytoplasm of resting cells and requires interaction with a receptor-regulated Smad in order to accumulate in the nucleus [34]. This has important implications because it suggests that the receptor-regulated Smad will have a critical role not only in ushering Smad4 into the nucleus, but also in maintaining signalling specificity in the nucleus by mediating interaction of Smad4 with nuclear targets (Figure 2). Recent studies have also revealed a TGFIS-dependent interaction between Smad2 and Smad3 [25]. Furthermore, Smad2 and -3 appeared to synergistically enhance activation of a particular TGFI~ transcriptional response (the activation of the 3TP promoter). This raises the possibility that complexes of Smad2, -3 and -4 may provide the best signalling. In addition, these data suggest that Smad2 and Smad3, which are highly conserved, may not simply be redundant components of the pathway but may form part of a higher-order heteromeric complex together with Smad4. T h e presence of a common partner in both activin/TGF~ signalling and BMP signalling has led to the interesting observation that these pathways can inhibit each other by competing for this common component. Hence, high levels of BMP signalling can titrate Smad4 and block subsequent signalling by activin [35]. Whether this occurs at endogenous signalling levels, where the amount of Smads translocating to the nucleus can be quite small [26], has yet to be determined. N u c l e a r f u n c t i o n s of S m a d c o m p l e x e s T h e search for effectors in the signal transduction pathways of TGFI] superfamily members has led to the identification of the Xenopus transcription factor, FAST1 [36°°]. FAST1 is a forkhead-containing DNA-binding protein that interacts with an activin-response element (ARE) to mediate activin-dependent induction of the
Figure 2
mad4
Current Opinion in Cell Biology
A model for TGF~ signal transduction. Phosphorylation of Smad2 and Smad3 occurs in response to TGFI3 or activin, whereas Smadl, -5, -8 and Drosophila Mad are phosphorylated in response to BMP-like ligands (see text for details). In C. elegans, regulation by phosphorylation is predicted due to the presence of a carboxy-terminal SSXS and SXT motif in Sma2 and Sma3, respectively. Subsequent to phosphorylation, the receptor-regulated Smad (in this case illustrated by Smad2) physically interacts with Smad4 in the cytoplasm and the complex translocates to the nucleus where it targets resident DNA-binding proteins such as FAST1 or binds directly to DNA. In the case of Drosophila, a similar pathway involving Mad and Medea functions in Dpp signalling (L Attisano, unpublished data). Inhibitory Smads block signalling by associating stably with the type I receptor to prevent access of the receptor-regulated Smads.
Mix2 gene. In the absence of signalling, FAST-1 binds constitutively to the ARE, but upon stimulation with activin, it assembles into a higher-order DNA-binding complex, termed the ARF (activin-responsive factor), which contains both Smad2 and Smad4 [34,36",37]. To form the ARF, Smad2 functions to recruit Smad4 into a complex with FAST1 (Figure 2) and binds to the Smad2 interaction domain, or SID, which is located near the carboxy-terminal region of FAST1, outside of the forkhead DNA-binding domain [34,37]. Once recruited to FAST1, Smad4 appears to be required to stabilize DNA binding of the higher-order complex as well as activate transcription. Interestingly, the functions of Smad4 within the ARF may be separable with the MH1 domain acting to stabilize DNA binding, while the MH2 is required to activate transcription [34]. This observation would be consistent with previous analyses that suggested that both domains in Smad4 were required for the function of the protein [13]. T h e mechanism of ARF formation also serves to illustrate the central role of receptor-activated Smads in each step of the TGF[3 signalling pathway. By functioning as both the substrate of the receptor and the usher that brings Smad4
Mads and Smads in TGF~ signallingAttisanoand Wrana
to its nuclear targets, the receptor-regulated Smads fulfil a critical role in maintaining specificity of the transcriptional response to TGFIB family members. Activin-dependent response elements have also been identified in other target genes. In the goosecoidpromoter, Smad2 appears to regulate transcription through DNA-binding complexes that are distinct from FASTl-containing complexes [35]. Further, an unrelated ARE has been found within the first intron of the Xenopus Forkhead (XFKH)-I gene and appears to represent yet another Smad2 target [38]. So identification of how Smads specifically regulate these diverse response elements will be an interesting area for future research. In addition to targeting resident DNA-binding proteins, recent evidence suggests that Smads can directly bind DNA. In Drosophila, activation of dpp signalling pathways in imaginal disks can induce transcription of the vestigial gene and this is mediated by a specific GC-rich sequence [39°]. Interestingly, a bacterially expressed MH1 domain of MAD can bind to this region, suggesting that MAD itself may mediate Dpp-dependent activation of the element. However, this interaction may not be sufficient, and similar sequences in other promoters do not appear to have a critical role in mediating Dpp-responsiveness [40]. In mammalian cells, TGFI3 induces formation of a phosphorylation-dependent DNA-binding complex composed of Smad3 and Smad4 that recognizes a bipartite site within the promoter of the TGF~-inducible reporter, 3TP-Lux [41]. In this case, DNA binding is mediated by Smad4 and there is no similarity between the bipartite element in 3TP and the Mad site in the vestigial quandrant enhancer. However, the functional significance of the interaction remains unknown, since mutation of the Smad-binding site does not interfere with TGFi3-dependent transcriptional activation of the 3TP promoter [41].
Regulating the S m a d pathway Smads do not appear to possess any enzymatic activity. Thus, the sensitivity and function of the pathway is exquisitely sensitive to the levels of Smad protein. Indeed, earlier studies in Drosophila have shown that reductions in Mad gene dosage are sufficient to suppress phenotypes induced by activation of Dpp signalling [3,4]. Furthermore, in Xenopus, manipulation of Smad protein levels is sufficient to alter cell fate in a linear, pathway-specific manner [27°°,42]. Thus Smadl induces ventral cell fates while Smad2 induces dorsal cell fates consistent with the function of these proteins in BMP and TGFl3/activin signalling, respectively [8,27°°,29]. In mammalian cells, Smad2 expression is strongly upregulated in developing follicular granulosa cells and this correlates with increased sensitivity of the cells to TGFI] [43]. Interestingly, in these cells TGFI3 itself appears to upregulate Smad2 levels, suggesting a positive feedforward mechanism in which TGFIB can autoregulate the sensitivity of its own signalling pathway.
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Apart from regulating Smad protein levels, other mechanisms may exist to control activation of the pathway. Recent evidence has suggested that Smadl may serve as a substrate for mitogen-activated protein kinase (MAPK) [44]. Phosphorylation occurs in the linker region, regulates the subcellular localization of the Smad protein and appears to inhibit BMP signalling. Whether phosphorylation of endogenous Smad protein is similarly regulated is unclear, as is the general biological applicability of this observation, since interactions of TGFI3 and BMP pathways with receptor tyrosine kinase signalling pathways are complex and as often involve synergistic interactions as antagonistic ones. Recently, another class of Smad proteins represented by Smad6 and Smad7 have been identified [45°'-47"',48]. Smad6 and Smad7 are distantly related to receptor-regulated Smads and Smad4 and have a poorly conserved MH1 domain. Interestingly, in the case of Smad6 a short form of the molecule, Smad6(S), comprising only the MH2 domain, is expressed in some cell types [48,49]. Smad6 and Smad7 have a unique function: these Smads act as negative regulators of signalling and potently block both TGFI3 and BMP signalling [45"°-47°',48]. Surprisingly, these proteins can interact stably with the TGFIB and BMP receptors to prevent association and phosphorylation of the receptor-regulated Smads. Thus, these anti-Smads act as intracellular antagonists of type I serine/threonine kinase receptors. In the case of Smad6, additional mechanisms to block signalling may be employed, since Smad6(S) has been shown to associate with numerous Smads, thus raising the possibility that it could block signalling by preventing heteromeric complex formation [48]. Expression of anti-Smads is regulated in a highly specific manner. In the case of Smad6(S) and Smad7, transcription of the genes is strongly induced in endothelial cells in response to fluid laminar shear stress and could function to insulate the endothelium from the effects of circulating TGF~3 family members [48]. In addition, it appears that Smad6 and -7 are early target genes for the Smad signalling pathways and that Smad7 is immediately, but transiently induced by TGF[3 in a variety of cell lines [47°°]. What function this brief pulse of Smad7 expression fulfils is unclear, although it could represent a method for downregulating the sensitivity of cells to TGFI3. In Drosophila a related Smad called Dad (for Daughters against dpp) appears to be regulated by Dpp along the anterior-posterior boundary of the wing imaginal disk and may function to inhibit signalling in a manner analogous to Smad6 and -7 [50]. In C. elegans, Daf-3 may represent another antagonistic Smad [51].
Implications of the S m a d p a t h w a y in d e v e l o p m e n t and h u m a n disease Members of the TGFIB family have critical roles as morphogens during vertebrate and invertebrate development. Intriguingly, cell-fate decisions in response to this family of cytokines is highly dependent on ligand
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concentration, and small changes in activin concentration can induce alternative cell-fate changes in Xenopus [52]. The apparent absence of enzymatic activity thus yields a signalling pathway in which the nuclear concentration of Smad provides a direct and proportional measure of the external ligand concentration. This may be critical for the establishment of TGF[3 signalling gradients that then function to control cell fate precisely, depending on the position of the cell in a morphogenetic field.
in colorectal cancers is similar to observations made on the TGF[3 receptor system. T h e pathway may thus be particularly important in controlling homeostasis of the gastrointestinal epithelium. These studies have served to illustrate that the TGFI3 signal transduction pathway can serve a tumour suppressor function which, when disrupted by mutation, may contribute to tumour progression by disrupting cellular responses to TGFI~.
The role of TGFI] as an antiproliferative factor for a variety of cell types has focused considerable attention on this pathway as a target of tumour suppressor mutations. Thus far we have seen that there are relatively few components linking the cell-surface receptor with the nuclear transcriptional machinery. Consistent with the turnout suppressive role of TGF~, mutations have been identified in numerous components of the pathway. Of particular interest, 90% of colon cancers with microsatellite instability (RER + for 'replication errors') have inactivating mutations in the extracellular domain of TI~RII, the type II receptor for TGF[~ [53]. Turnouts that exhibit microsatellite instability occur either in a hereditary form, termed H N P C C (hereditary nonpolyposis colorectal cancer), or as sporadic cases (reviewed in [54]) and account for approximately 15-17% of all colorectal carcinomas. In contrast, receptor mutations are rare in endometrial cancers with microsatellite instability, emphasizing the importance of TGFI3 signalling in colorectal cancer [55]. In addition to RER+ colorectal cancer, mutations in the kinase domain of T ~ R I I have been identified in squamous head and neck carcinomas [56], implicating the TI3RII gene as a tumour suppressor gene in other turnouts. In addition alterations in the TGF[~ type I receptor, TI3RI, have been found in prostate, colon and gastric cancer cells and a lack of TI3RI protein has been observed in AIDS-related Kaposi's sarcoma (reviewed in [57]). These studies suggest that in addition to the type II receptor, TI3RI is another target for inactivating mutations in cancer.
The isolation of Mad by genetic screens has rapidly led to the elaboration of a pathway that directly connects the TGF[3 receptor with the transcriptional machinery. How this conserved pathway elicits diverse, cell-type specific responses to control developmental and homeostatic processes will undoubtedly become a major area of interest in the future.
Conclusions
Like the receptors, intracellular components of the TGF[~ signalling pathway may also function as tumour suppressors (reviewed in [58]). The genes for both Smad2 and Smad4 map to chromosome 18q21 [16,59°°,60], a region that displays loss of heterozygosity in about 90% of pancreatic tumours and in over 60% of colorectal cancers and evidence now implicates the Smad genes as the candidate tumour suppressor genes at this locus. Somatic missense mutations in Smad4 (DPC4) have been identified in pancreatic carcinomas [59°°], and colorectal cancers [61,62], while two missense mutations and a two base-pair frameshift have also been identified in a sampling of 42 lung cancers [63]. Genetic alterations and inactivating missense mutations in Smad2 (MADR2 or JV-18) have also been identified in sporadic colorectal carcinomas but none in breast cancers and sarcomas [16,60]. Apart from those in Smad2 and Smad4 no other mutations have thus far been reported [49]. The bias for mutations of Smad2
Notes added in proof 1) The identification of medea as a Smad4 homologue, which was referred to in the text as ' L Raftery, personal communication' is now in press [64]. Surprisingly, this paper demonstrates that while Mad is required for all dpp signalling events, medea is only required for a subset of responses, and in the wing disk may function to extend the range of dpp action. 2) Recently, another report on inhibitory Smads has been published that shows that in addition to interacting with type 1 receptors, Smad6 associates with Smadl and may prevent formation of Smadl-Smad4 heteromeric complexes [65]. Acknowledgements
Work in the authors' laboratories is supported by grants from the National Cancer Institute of Canada and the Medical Research Council of Canada. The authors are Medical Research Council of Canada Scholars. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: * of special interest o= of outstanding interest 1.
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