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TGF-β signaling from receptors to the nucleus
Microbes and Infection, 1, 1999, 1265−1273 © 1999 Éditions scientifiques et médicales Elsevier. All rights reserved
TGF-β signaling from receptors to the nucleus Anita B. Roberts Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute Building 41, Room C629, 41 Library Drive, MSC 5055, Bethesda, MD 20892-5055, USA
ABSTRACT – In the past three years, a novel signal transduction pathway downstream of the transforming growth factor-beta (TGF-β) superfamily receptor serine-threonine kinases has been shown to be mediated by a family of latent transcription factors called ‘Smads’. These proteins mediate a short-circuited pathway in which a set of receptor-activated Smads are phosphorylated directly by the receptor kinase and then translocate to the nucleus complexed to the common mediator, Smad4, to participate in transcriptional complexes. Smads 2 and 3 mediate signals predominantly from the TGFβ receptors. Of these, specific roles have been ascribed to Smad3 in control of chemotaxis of neutrophils and macrophages and the inhibition of Smad3 activity by the oncogene Evi-1 suggests that it may play a role in leukemogenesis. Other data, such as the induction by the inflammatory cytokine interferon-γ of an inhibitory Smad, Smad7, which blocks the actions of Smad3, suggest that identification of the specific gene targets of Smad proteins in immune cells will provide new insight into the mechanisms of TGF-β action on these cells. © 1999 Éditions scientifiques et médicales Elsevier TGF-β / Smad / signal transduction
1. Introduction Transforming growth factor-beta (TGF-β) has profound effects on the immune system and has been implicated in a broad range of pathogenic mechanisms involving primary effects on immune cells. These include immune defects associated with autoimmune disease, suppression of immune surveillance of tumors, hematopoietic malignancies, susceptibility to parasitic infections, and chronic inflammatory disease leading to fibrotic complications [1]. These effects of TGF-β on immune cell function are mediated by signals transduced by its transmembrane serine-threonine kinase receptors and which ultimately target and regulate the transcription of nuclear genes (reviewed in [2, 3]). While several pathways including those involving ras and mitogen-activated protein kinases (MAP kinases) have been implicated in signaling from TGF-β reviewed in [4]), this brief review will be focused on the biochemical mechanisms of signal transduction by a family of recently identified cytoplasmic proteins called ‘Smad’ proteins, named by contraction of the names of the Drosophila Mad and the Caenorhabditis elegans Sma proteins, shown genetically to function downstream of the TGF-β superfamily ligands Dpp and Daf-7, respectively [5]. This shortcircuited pathway in which Smad proteins are phosphoryMicrobes and Infection 1999, 1265-1273
lated directly by the type I receptor kinase and translocated to the nucleus to participate in transcriptional complexes is conceptually similar to the Jak/Stat pathway which mediates signals from many receptors with important functions in hematopoiesis and in regulation of immune cell activity [6]. Many excellent reviews on the Smad signaling pathway have been published and, when appropriate, will be cited in lieu of the primary references.
2. Receptor serine-threonine kinases of the TGF-β superfamily Cell surface receptors for TGF-β family ligands are distinguished from those of other growth factors and cytokines by their specificity for phosphorylation of serine or threonine, rather than tyrosine residues. Receptor complexes are heterotetrameric, consisting of two ‘type II’ receptors (75–85 kDa), which bind ligand, and two signal transducing ‘type I’ receptors (50–60 kDa) which, in most instances, cannot bind ligand directly and thus are considered to act downstream of the type II receptor (for reviews see [3, 4, 7, 8]). The assembly of the heteromeric complex is initiated by ligand binding and stabilized by interactions between the cytoplasmic domains of the type II and type I receptors. This model for receptor activation involves 1265
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potentiation of the kinase activity of the type I receptor by phosphorylation of several residues in a glycine-serinerich juxtamembrane domain (GS domain) by the type II receptor kinase. Mutation of a single threonine residue to glutamine (T204D) at the interface between the GS domain and the kinase domain of the TGF-β type I receptor results in its constitutive activation, demonstrating that the activated type I kinase alone is sufficient to transduce downstream signals [9]. Mutationally activated type I receptors are often used in studies of signal transduction by members of the TGF-β family, since they eliminate the need for ligand and the type II receptor. Signals from all three isoforms of TGF-β appear to be mediated by a single type II receptor called TβR-II and one type I receptor referred to either as TβR-I or ALK-5 (activin receptor-like kinase). Another type I receptor, ALK-1, expressed on endothelial cells and mutated in the autosomal dominant disorder hereditary hemorrhagic telangiectasia (HHT), can also complex with ligand-bound TβR-II, but its role in signaling is presently not understood [10]. Two other cell surface binding proteins also participate in TGF-β receptor binding in certain cells (reviewed in [3, 4]). Betaglycan, formerly called the ‘type III receptor’, binds all isoforms of TGF-β, but may play a selective role in facilitating interaction of TGF-β2 with TβR-II, since this isoform binds to the type II receptor with significantly lower affinity than that of TGF-β1 and TGF-β3. Sharing some homology with betaglycan is endoglin, which is expressed at highest levels on endothelial cells and which is also inactivated in forms of HHT. Unlike betaglycan, endoglin binds TGF-β1 and TGF-β3 selectively. While the roles of these two proteins are not fully understood, it has been postulated that they may constrain the conformation of the TGF-βs in such a way as to enhance binding to TβR-II.
3. The Smad signal transduction pathway The search for mammalian homologues of the Drosophila Mad and C. elegans Sma proteins identified a family of cytoplasmic mediators termed Smad proteins which share highly conserved N- and C-terminal domains (called Mad homology 1 and 2, MH1 and MH2, respectively) connected by highly divergent proline-rich linker regions (reviewed in [2, 3, 7, 8]). These proteins contain no recognizable protein-protein interaction motifs or enzymatic activities. To date, eight different mammalian Smad genes have been described which fall into three distinct functional sets: receptor-activated Smads, which includes Smads 1, 2, 3, 5, and 8; a single common mediator Smad, Smad4/ DPC4, and inhibitory Smads, Smads 6 and 7 (figure 1A). The present model for downstream signaling via the Smad pathway is that: 1) receptor-activated Smads bind to and are phosphorylated on two C-terminal serine residues in their MH2 domain by the type I receptor kinase; 2) the phosphorylated, pathway-specific Smads then heterooligomerize in the cytoplasm with the common mediator, Smad4; 3) the heteromeric Smad4-containing complex is then translocated to the nucleus where it mediates tran1266
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scriptional activation of the target gene (figure 2). Inhibitory Smads, induced by TGF-β family ligands, function in a negative feedback loop to terminate or reduce the strength of the signal. 3.1. Receptor-activated Smads
Of the five receptor-activated Smads identified thus far, Smad2 and 3 have been shown to mediate signals from TGF-β and activin receptors [11, 12] whereas Smad1, 5, and 8 mediate signals from BMP receptors [13]. There are indications, however, that this delineation may not be so strictly defined. Smad1 is phosphorylated and partners with Smad4 following treatment of human breast cancer cells with either BMP-2 or TGF-β [14] and the Smad5 gene has been implicated in inhibition of the proliferation of primitive human hematopoietic progenitor cells by TGFβ [15]. All of the receptor-activated Smad proteins share a common C-terminal motif, SSXS-COOH, in which the two-carboxyl terminal serine residues are phosphorylated by the activated type I receptor kinase. Mutations in this phosphorylation motif (3S > A) result in dominant negative function, presumably by their stable association with the type I receptor and their subsequent failure to associate with Smad4 [16, 17]. The amino acid sequence at the highly conserved L3 loop locus in the MH2 domain has been shown to be an important determinant of the specificity of the Smad/receptor interaction as has an interacting cluster of four amino acids in the L45 loop of the type I receptor kinase domain [18]. Switching of two amino acids flanking the L3 loop core sequence, Arg-Gln, in Smads 1 and 2 changes their receptor specificity to the TGF-β and BMP-2 receptors, respectively. In the absence of ligand, interactions of the MH1 and MH2 domains are mutually repressive with the MH1 domain interfering with the oligomerizing and transcriptional activating activity of the MH2 domain [19]. Liganddependent relief of this autorepression can be blocked either by mutation of the C-terminal serines (3S > A), by mutation of a critical Gly residue in the L3 loop common to all Smads, or by mutation of a conserved Arg in the MH1 domain which increases the strength of the intramolecular interaction (figure 1B). Although mutations in the MH1 domain are rare, this Arg residue has been in found to be mutated in both Smad2 and Smad4 in a colon carcinoma and pancreatic carcinoma, respectively, suggesting that mutations at this position are selected for in tumors [19]. Recently a novel double zinc-finger lipid-binding, or FYVE-domain protein called SARA (Smad anchor for receptor activation) has been described which provides insight into mechanisms whereby the interaction of specific unphosphorylated Smad proteins with the type I receptor is controlled to facilitate their ligand-dependent phosphorylation [20]. The FYVE domain of SARA is predicted to interact with phosphosphotidyl inositol-3phosphate in the cell membrane while an adjacent Smad binding domain recruits Smad2/3 from the cytoplasmic reservoir; the C-terminal domain anchors the Smad-SARA complex to the receptor complex. Mutation of SARA to induce mislocalization of Smad2 or 3 results in inhibition of the transcriptional activation of target genes. Specific Microbes and Infection 1999, 1265-1273
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Figure 1. Structure-function of Smad proteins. A. Smad proteins share highly conserved MH1 and MH2 domains. Receptor-activated Smad proteins share a C-terminal phosphorylation motif, SSXS, and can be assigned to two classes (those binding BMP or TGF-β / activin receptors, respectively) depending on a conserved sequence in the L3 loop of the MH2 domain. Smad4 lacks the phosphorylation motif and has a unique Smad activation domain (SAD). Inhibitory Smad proteins lack both the MH1 domain and the C-terminal phosphorylation motif. B. The MH1 and MH2 domains of receptor-activated Smads and Smad4 interact in the absence of ligand activation to mutually repress the activities of these two domains. Ligand-induced phosphorylation of receptor-activated Smads or hetero-oligomerization of Smad4 relieves this autorepression and reveals the DNA binding activity of the MH1 domain, and the oligomerization and transcriptional activating activity of the MH2 domain and/or the SAD domain.
roles of SARA in regulating the specificity of the signaling pathway and possibly limiting cross-talk with other pathways have been proposed [20]. 3.2. The common mediator – Smad4/DPC4
Smad4 is unique among all the Smads. It was first identified as a tumor suppressor gene for pancreatic canMicrobes and Infection 1999, 1265-1273
cer, being either mutated or deleted in a significant percentage of pancreatic cancers as well as in a smaller percentage of colon cancers and breast cancers [21]. Based on our present understanding of the singular role of Smad4 in mediating signals from receptor serinethreonine kinases of the TGF-β superfamily, it is clear that functional inactivation of this molecule affects most, if not 1267
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Figure 2. Smad-mediated signaling and cross-talk. Different classes of receptor-activated Smads bind and are phosphorylated by the type I receptor serine-threonine kinase, hetero-oligomerize with Smad4 and are translocated to the nucleus where they participate in transcriptional complexes. Signaling from HGF and EGF receptors via MAP-kinase pathways can also phosphorylate receptor-activated Smads, possibly in the middle linker region, with resultant synergistic (HGF on Smad2) or antagonist (EGF on Smad1) effects on signal transduction, resulting in integrated cross-talk between receptor tyrosine and serine-threonine kinases. Nuclear complexes involve both direct binding of Smad proteins to DNA via their MH1 domains, binding of transcription factors (TF) and/or coactivators such as CBP/p300 through their MH2 domains, and possible involvement of additional activators such as MSG1. Regulation of transcription factor activation and of the coactivators by alternative signaling pathways and by oncoproteins such as ras and E1A represents yet another level of transcriptional cross-talk. all, transcriptional targets of the set of receptor-activated Smads. The recent identification of a colon tumor cell line, Vaco8–2, harboring mutations in both the TGF-β type II receptor and Smad4 accentuates the broader role of Smad4 in pathways other than those specific to TGFβ [22]. Smad4 is not phosphorylated upon receptor activation, nor does it have the critical C-terminal phosphorylation motif common to the receptor-activated Smads (reviewed in [2]). Biochemical evidence for its central role in transduction of signals from the type I receptor serine-threonine kinases comes from the demonstration of its specific association with each of the receptor-activated Smads following their ligand-dependent phosphorylation [23]. Recent data suggest that this funneling of multiple signals through a single node can serve to modulate cross-talk between different receptors of the TGF-β family in that the relative strength of each signal can be controlled by competition for a limited pool of Smad4 [24]. Determination of the tertiary structure of the C-terminal domain of Smad4 shows that it associates as a homotri1268
mer [25], and data now suggest that hetero-oligomeric Smads are also trimeric [26]. Key residues at the trimer protein-protein interfaces, which are targets for mutation of Smad4 in human cancers, interfere with homo- and hetero-oligomerization and nuclear translocation [25]. A subset of individuals with familial juvenile polyposis, an autosomal dominant disease characterized by a predisposition to gastrointestinal cancer, also show truncating frameshift mutations in linker and MH2 domains of Smad4/DPC4 [27]. Similar to the receptor-activated Smads, the MH1 domain of Smad4 is inhibitory to the transcriptional activating activity of its MH2 domain; this inhibition is relieved by association with a phosphorylated receptor-activated Smad. Unique to Smad4, an activation domain (SAD) has been identified near the extreme C-terminal portion of the middle linker region which is required for its transcriptional activating activity [28]. 3.3. Inhibitory Smads
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has been the identification of inhibitory Smads or antiSmads [29–31]. Members of this Smad protein subset, which presently includes the mammalian Smad6, Smad7, share the general features of having an N-terminal domain distinct from that of the MH1 domain shared by other Smads and being more similar to Smad4 in the MH2 domain, in that neither the L3 loop receptor binding motif nor the C-terminal serine phosphorylation motif common to the receptor-activated Smads is present. The conservation of the MH2 domain and the ability of Smads 6 and 7 to interact both with the type I receptors and with other Smads, suggest that this domain may represent a novel interaction domain specific for this pathway, analogous to the SH2/SH3 interaction domains which are hallmarks of tyrosine kinase signaling pathways [32]. Identification in Drosophila of the product of a homologous gene called Daughters against decapentaplegic (Dad), which inhibits patterning by dpp, suggests that this inhibitory pathway evolved early and is basic to the mechanisms of signal transduction by this family of ligands and receptors [33], much as the SOCS/SSI inhibitors serve to regulate signaling in Jak/Stat pathways [34]. The mRNAs for Smad6 and Smad7 are rapidly induced by treatment of cells with TGF-β suggesting that antiSmads are direct effectors of a ligand-induced signal to suppress a response; these anti-Smads are also induced by epidermal growth factor, suggesting that they also serve to mediate cross-talk between signaling pathways from tyrosine kinase and serine-threonine kinase receptors [35]. Vascular endothelial cells subjected to laminar shear stress express an N-terminally truncated Smad6 [29, 30]. Since this truncation changes the interaction pattern, resulting in nonspecific interaction with Smad1, 2, and 4, the possibility exists that expression of the truncated version of this protein by endothelial cells may represent an adaptative response to mechanical stress, altering signaling from multiple TGF-β family ligands. There is presently a lack of consensus regarding the mechanism of action of these anti-Smads, and recent data suggest that both cytoplasmic and nuclear activities might be involved [36].
4. Transcriptional activation of nuclear target genes by Smads The nuclear translocation of receptor-activated Smads in a phosphorylation-dependent manner is consistent with their proposed direct role in transcriptional activation of target genes. However, unlike the consensus cis elements which led to the identification of the Stat transducers of signals from receptor tyrosine kinases [6], the diversity of response elements identified in target genes activated by TGF-β and other superfamily members has proved challenging in terms of understanding the mechanisms of transcriptional activation by Smads. Present models suggest that while Smad proteins can bind DNA directly, they likely also require the cooperation of multiple, diverse, sequence-specific-DNA binding proteins as well as coactivator molecules to mediate their transcriptional effects ([2], see figure 2). An excellent example of this is in the collagen VII promoter where an AP-1 binding site is cenMicrobes and Infection 1999, 1265-1273
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tral to a bipartite Smad binding element, resulting in a 52-bp minimal TGF-β response element [37]. This added level of complexity clearly adds both to the specificity of the response patterns in particular cell types and to the degree of cross-talk between different pathways which may modulate the levels or activity of the transcription factors or the coactivators themselves. 4.1. Direct DNA binding of Smad proteins
The MH1 domain of Smad proteins can bind DNA directly. The only exception to this rule is Smad2 in which an inserted 30 aa sequence encoded by exon 3 precludes DNA binding; a naturally occurring splice variant has been described in which this region is deleted, restoring DNA binding activity comparable to Smad3 [38]. Examination of the Dpp A response elements of the vestigial, labial, and Ultrabithorax genes revealed a consensus GC-rich binding sequence, GCCCnCGc, which binds C-terminally truncated forms of Drosophila MAD (reviewed in [2]). The N-terminal domain of Medea, the Drosophila counterpart of Smad4, has also been shown to bind GC-rich sequences in the tinman promoter. Although these interactions are weak, they are specific and correlate with activation of these enhancer elements in vivo. A pallindromic Smad3/4 binding element, GTCTAGAC, has been defined by a screen with random oligonucleotides [39]. Promoters of TGF-β -responsive genes including plasminogen activator inhibitor-1 [40–42], type VII collagen [37], and JunB [43] as well as the artificial 3TP-Lux reporter [44] contain similar sequences and bind the MH1 domain of Smads 3 and 4 directly. Crystallographic analysis of the MH1 domain of Smad3 with this pallindromic element shows this sequence to be optimal for protein DNA contacts [45]. 4.2. Transcription factor/Smad complexes
The first example of a Smad transcriptional complex was based on characterization of the ‘activin response factor’, ARF, which binds to an activin-response element (ARE) in the promoter of the Xenopus Mix.2 homeobox gene and is rapidly induced by activin independently of new protein synthesis [46]. The essential component of the ARF, a forkhead/winged-helix transcription factor, FAST-1, binds the ARE directly. Both Smad2 and Smad4 are also present in the complex although they do not directly bind the ARE: the MH2 domain of Smad2 interacts with the C-terminal domain (Smad-interaction domain, SID) of FAST-1 directly, while the binding of Smad4 is assumed to be dependent on its association with activated Smad2 and possibly also on its ability to stabilize the DNA complex through its MH1 domain. The identification of both Smad2 and 4 in the active transcriptional complex is consistent with the findings that receptor-activated Smads require Smad4 for transcriptional activation, even though they can translocate to the nucleus in its absence, and that nuclear translocation of Smad4 requires heteromeric association with a receptor-activated Smad. Smad3 can interact directly with c-Jun and c-Fos through its MH1 and MH2 domains, respectively [47] leading to the suggestion that AP-1 DNA binding sites can be activated either by direct binding of Smad3 and Smad4 1269
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to the DNA element itself or by interaction of the heteromeric Smad3/Smad4 complex and the AP-1 transcription factor complex, in such a manner as to provide additional stability to the complex. Besides providing transcriptional diversity, this mechanism also suggests a basis for crosstalk between MAP kinase cascades and JNK kinase and Smad signaling pathways, in addition to direct phosphorylation of Smad proteins by signals from receptor tyrosine kinases which activate MEK1 or a downstream kinase as discussed below [46]. A zinc-finger transcription factor Evi-1 has also been shown to interact with Smad3, but to result in suppression of TGF-β-mediated signaling and antagonism of its growth inhibitory effects [48]. Since this factor is overexpressed in certain hematopoietic malignancies, it may have special significance for regulation of immune cell responses to TGF-β (see below). The ubiquitous transcription factor Sp-1 has also been shown to interact functionally with Smad3/Smad4 complexes in regulation of the p21/WAF1/ Cip1 promoter, although neither a physical association of Smad3 and Sp1 nor binding of the Smad complex to the Sp1 site have been demonstrated [49]. A clear example of an indirect interaction of Smad proteins with transcription factors is provided in a TGF-β -induced element in of the plasminogen activator inhibitor-1 (PAI-1) gene. This element has been shown to require binding of both heteromeric Smad3/ Smad4 and the basic helix-loop-helix transcription factor, TFE3, to adjacent sites, presumably without direct physical interaction of Smads and TFE3 [40]. 4.3. Smad interaction with transcriptional coactivators
Coactivators are a class of proteins which act by bridging transcription factors and components of the basal transcriptional machinery. CREB binding protein (CBP) and its closely related functional homologue p300, are histone acetyltransferases (HATs) previously shown to interact with a variety of transcription factors and nuclear hormone receptors as well as with the basal transcription factor TFIIB and RNA polymerase II. These proteins have now been shown to also interact with Smad proteins in the regulation of transcription. At present there is a lack of consensus as to the particular domain and the particular Smad with which these proteins interact. Depending on the system and the cell type, ligand-activated Smad1, Smad2, and Smad3, as well as Smad4 have all been shown to be capable of binding to CBP/p300 ([50]; reviewed in [2]). The essential nature of this interaction is demonstrated by the fact that disruption of the Smad-CBP/p300 interaction by the adenoviral transforming protein E1A, which binds the coactivators, blocks nearly all Smadstimulated transcriptional responses in cells [51]. Another nuclear protein that strongly activates transcription without binding to DNA, MSG1, has been shown to interact functionally with the SAD domain in the middle linker region of Smad4 and to be required for activation of Smad4-dependent transcription in certain cells [52]. Downregulation of expression of MSG1 by both ras and E1A again provides a framework for cross-talk between different pathways which can modulate the activity of TGF-β signaling. 1270
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In summary, it can be said that the difficulty in defining specific consensus TGF-β-responsive elements results from the diversity of transcription factors that cooperate with Smad signaling protein complexes and the additional requirement for an adjacent cooperating Smad DNA binding site [2].
5. Receptor cross-talk mediated by Smad proteins Depending on the context, TGF-β family ligands can either synergize or antagonize the actions of mitogenic growth factors such as epidermal growth factor (EGF) or hepatocyte growth factor (HGF) which signal through receptor tyrosine kinases. Recent data now implicate Smads in the cross-talk between signal transduction pathways downstream of certain receptor tyrosine kinase and TGF-β family signaling pathways. Specifically, it has been shown that Smad1 can be phosphorylated at several PXPS sites in its middle linker region, which are consensus sites for MAP kinases [53]. Treatment of cells with EGF or hepatocyte growth factor (HGF) results in phosphorylation of Smad1 on these sites by the Erk subfamily of MAP kinases, and in retention of the linker-region phosphorylated Smad1 in the cytoplasm, blocking the nuclear translocation and transcriptional activating activity of Smad1/ Smad4 complexes. It has also been shown that Smads, in certain instances, can mediate synergistic signals from these two receptor families. Examples are the activating phosphorylation and nuclear translocation of Smad2 following treatment of cells with HGF [54] or activation of the SAPK/JNK pathway [55]. Smad7 is also an important focal point of disparate signaling pathways as evidenced by its induction by EGF [35] and interferon-γ [56]. These exciting findings suggest that receptor-activated Smads may be sensors of the competing or synergistic inputs from receptor tyrosine and serine/threonine kinases, and that activation of selected gene targets by Smads may reflect an integrated signal input from these two distinct pathways. There are also examples of downregulation of Jak/Stat signaling from IL-12 and IL-2 by TGF-β but the mechanisms involved have not been identified [57, 58].
6. Implications for Smad regulation of immune cell function While TGF-β is known to regulate many facets of immune cell behavior including the growth of cells and their differentiated function, specific gene targets are still poorly understood [1]. Recent data from targeted deletion of the Smad3 gene suggest that the Smad pathway will be critical to response of these cells to TGF-β [59]. Specifically, symptomatic Smad3 null mice show defects in mucosal immunity associated with thymic involution, enlarged lymph nodes, and formation of bacterial abscesses. Smad3 null T cells, but not B cells, show selective loss of response to the growth suppressive effects of TGF-β following activation, and the chemotactic Microbes and Infection 1999, 1265-1273
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responses of both neutrophils and macrophages to TGF-β are impaired [60]. Since targeted deletion of Smad2 or Smad4 each results in early embryonic lethality and in the inability to form mesoderm [61, 62] or failure to gastrulate [63], respectively, it is not possible at present to investigate effects of their loss on mature lymphoid cells. Certainly, the adult phenotype of the Smad3 null mice suggests that this Smad plays a more restricted role than Smad2 in signaling from TGF-β family ligands including, minimally, TGF-β and activin. Study of selected immune cell populations from these mice now affords a unique opportunity to dissect the roles of Smad2 and Smad3 in pathways of TGF-β signaling and to identify specific gene targets with functional correlates. Again suggestive of an important role of Smad3 in immune cell function is the observation that the t(3q26;21q22) fusion product, AML/Evi-1, interacts with Smad3 and blocks inhibition of myeloid cells by TGFβ [64]. This chromosomal translocation, seen in the blastic crisis of chronic myelogenous leukemia, places the AML1/Evi-1 chimera under control of the AML promoter. AML1 has been implicated in myeloid cell differentiation and shown to bind to enhancer core motifs present in myeloid promoters and lymphoid enhancers. Evi-1, located on chromosome 3q26, is frequently translocated and found to be transcriptionally activated in myeloid leukemias and myelodysplasias. It encodes a nuclear zinc finger protein with transcriptional regulatory activity, although specific target genes have not yet been identified. Following the demonstration that the first zinc finger domain of Evi-1 can bind Smad3 and that it, plus a distinct repressor domain, can block the growth inhibitory effects of TGF-β on epithelial cells [48], it has now been shown that the AML/Evi-1 chimera can similarly bind Smad3, suggesting that the mechanism of leukomogenesis in the AML/Evi-1 translocation may also result from interference with TGF-β signaling mediated by Smad3 [64]. Whereas definitive involvement of Smad signaling in hematological malignancies has not yet been demonstrated, these data strongly suggest that interruption of TGF-β signaling may contribute to progression of leukemias and that Smad3 may play a unique role in this process.
7. Summary An explosion of information in the past three years has resulted in the characterization of a novel signal transduction pathway downstream of the type I receptor serinethreonine kinases of TGF-β superfamily ligands. The Smad protein transducers of signals from these receptors also mediate certain aspects of both cytoplasmic and nuclear signaling cross-talk that contribute to stimulatory or inhibitory effects on TGF-β signaling. While the basic features of this signal transduction mechanism have now been elucidated, many aspects of the regulation of this pathway are still unclear. The genes identified thus far as being regulated directly by Smad-mediated pathways are all immediate-early gene targets, and the mechanisms of involvement of Smad proteins in signals requiring longer duration or greater intensity, such as those resulting in Microbes and Infection 1999, 1265-1273
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control of cell growth, differentiation, or apoptosis are not known. Clearly, there is much still to be learned.
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[17] Souchelnytskyi S., Tamaki K., Engstrom U., Wernstedt C., TenDijke P., Heldin C.H., Phosphorylation of Ser465 and Ser467 in the C terminus of Smad2 mediates interaction with Smad4 and is required for transforming growth factorbeta signaling, J. Biol. Chem. 272 (1997) 28107–28115. [18] Chen Y.G., Hata A., Lo R.S., Wotton D., Shi Y., Pavletich N., Massague J., Determinants of specificity in TGF-beta signal transduction, Genes Dev. 12 (1998) 2144–2152. [19] Hata A., Lo R.S., Wotton D., Lagna G., Massague J., Mutations increasing autoinhibition inactivate tumour suppressors Smad2 and Smad4, Nature 388 (1997) 82–87. [20] Tsukazaki T., Chiang T.A., Davison A.F., Attisano L., Wrana J.L., SARA a FYVE domain protein that recruits Smad2 to the TGF-beta receptor, Cell 95 (1998) 779–791. [21] Hahn S.A., Schutte M., Hoque A.T., Moskaluk C.A., DaCosta L.T., Rozenblum E., Weinstein C.L., Fischer A., Yeo C.J., Hruban R.H., Kern S.E., DPC4 a candidate tumor suppressor gene at human chromosome 18q21, Science 271 (1996) 350–353. [22] Grady W.M., Myerhoff L.L., Swinler S.E., Rajput A., Thiagalingam S., Lutterbaugh J.D., Neumann A., Brattain M.G., Chang J., Kim S.J., Kinzler K.W., Vogelstein B., Willson J.K.V., Markowitz S., Mutational inactivation of transforming growth factor b receptor type II in microsatellite stable colon cancers, Cancer Res. 59 (1999) 320–324. [23] Lagna G., Hata A., Hemmati-Brivanlou A., Massague J., Partnership between DPC4 and SMAD proteins in TGFbeta signalling pathways, Nature 383 (1996) 832–836. [24] Candia A.F., Watabe T., Hawley S.H., Onichtchouk D., Zhang Y., Derynck R., Niehrs C., Cho K.W., Cellular interpretation of multiple TGF-beta signals intracellular antagonism between activin/BVg1 and BMP-2/4 signaling mediated by Smads, Development 124 (1997) 4467–4480. [25] Shi Y., Hata A., Lo R.S., Massague J., Pavletich N.P., A structural basis for mutational inactivation of the tumour suppressor Smad4, Nature 388 (1997) 87–93. [26] Kawabata M., Inoue H., Hanyu A., Imamura T., Miyazono K., Smad proteins exist as monomers in vivo and undergo homo- and hetero- oligomerization upon activation by serine/threonine kinase receptors, EMBO J. 17 (1998) 4056–4065. [27] Howe J.R., Roth S., Ringold J.C., Summers R.W., Jarvinen H.J., Sistonen P., Tomlinson I.P., Houlston R.S., Bevan S., Mitros F.A., Stone E.M., Aaltonen L.A., Mutations in the SMAD4/DPC4 gene in juvenile polyposis, Science 280 (1998) 1086–1088. [28] De Caestecker M.P., Hemmati P., Larisch-Bloch S., Ajmera R., Roberts A.B., Lechleider R.J., Characterization of functional domains within Smad4/DPC4, J. Biol. Chem. 272 (1997) 13690–13696. [29] Topper J.N., Cai J., Qiu Y., Anderson K.R., Xu Y.Y., Deeds J.D., Feeley R., Gimeno C.J., Woolf E.A., Tayber O., Mays G.G., Sampson B.A., Schoen F.J., GimbroneJr M.A., Falb D., Vascular MADs two novel MAD-related genes selectively inducible by flow in human vascular endothelium, Proc. Natl. Acad. Sci. USA 94 (1997) 9314–9319. [30] Imamura T., Takase M., Nishihara A., Oeda E., Hanai J., Kawabata M., Miyazono K., Smad6 inhibits signalling by the TGF-beta superfamily, Nature 389 (1997) 622–626. 1272
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[31] Nakao A., Afrakhte M., Moren A., Nakayama T., Christian J.L., Heuchel R., Itoh S., Kawabata M., Heldin N.E., Heldin C.H., TenDijke P., Identification of Smad7 a TGFbeta-inducible antagonist of TGF-beta signalling, Nature 389 (1997) 631–635. [32] Pawson T., Scott J.D., Signaling through scaffold anchoring and adaptor proteins, Science 278 (1997) 2075–2080. [33] Tsuneizumi K., Nakayama T., Kamoshida Y., Kornberg T.B., Christian J.L., Tabata T., Daughters against dpp modulates dpp organizing activity in Drosophila wing development, Nature 389 (1997) 627–631. [34] Hilton D.J., Richardson R.T., Alexander W.S., Viney E.M., Willson T.A., Sprigg N.S., Starr R., Nicholson S.E., Metcalf D., Nicola N.A., Twenty proteins containing a C-terminal SOCS box form five structural classes, Proc. Natl. Acad. Sci. USA 95 (1998) 114–119. [35] Afrakhte M., Moren A., Jossan S., Itoh S., Sampath K., Westermark B., Heldin C.H., Heldin N.E., TenDijke P., Induction of inhibitory Smad6 and Smad7 mRNA by TGF-beta family members, Biochem. Biophys. Res. Commun. 249 (1998) 505–511. [36] Itoh S., Landstrom M., Hermansson A., Itoh F., Heldin C.H., Heldin N.E., TenDijke P., Transforming growth factor beta 1 induces nuclear export of inhibitory Smad7, J. Biol. Chem. 273 (1998) 29195–29201. [37] Vindevoghel L., Lechleider R.J., Kon A., DeCaestecker M.P., Uitto J., Roberts A.B., Mauviel A., SMAD3/4dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor beta, Proc. Natl. Acad. Sci. USA 95 (1998) 14769–14774. [38] Yagi K., Goto D., Hamamoto T., Takenoshita S., Kato M., Miyazono K., Alternatively spliced variant of Smad2 lacking exon 3, Comparison with wild-type Smad2 and Smad3, J. Biol. Chem. 274 (1999) 703–709. [39] Zawel L., Dai J.L., Buckhaults P., Zhou S., Kinzler K.W., Vogelstein B., Kern S.E., Human Smad3 and Smad4 are sequence-specific transcription activators, Mol. Cell 1 (1998) 611–617. [40] Hua X., Liu X., Ansari D.O., Lodish H.F., Synergistic cooperation of TFE3 and smad proteins in TGF-betainduced transcription of the plasminogen activator inhibitor-1 gene, Genes Dev. 12 (1998) 3084–3095. [41] Dennler S., Itoh S., Vivien D., TenDijke P., Huet S., Gauthier J.M., Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene, EMBO J. 17 (1998) 3091–3100. [42] Song C.Z., Siok T.E., Gelehrter T.D., Smad4/DPC4 and smad3 mediate transforming growth factor-beta (TGFbeta) signaling through direct binding to a novel TGFbeta-responsive element in the human plasminogen activator inhibitor-1 promoter, J. Biol. Chem. 273 (1998) 29287–29290. [43] Jonk L.J., Itoh S., Heldin C.H., TenDijke P., Kruijer W., Identification and functional characterization of a Smad binding element (SBE) in the JunB promoter that acts as a transforming growth factor-beta activin and bone morphogenetic protein-inducible enhancer, J. Biol. Chem. 273 (1998) 21145–21152. Microbes and Infection 1999, 1265-1273
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[44] Yingling J.M., Datto M.B., Wong C., Frederick J.P., Liberati N.T., Wang X.F., Tumor suppressor Smad4 is a transforming growth factor beta-inducible DNA binding protein, Mol. Cell Biol. 17 (1997) 7019–7028. [45] Shi Y., Wang Y.F., Jayaraman L., Yang H., Massague J., Pavletich N.P., Crystal structure of a Smad MH1 domain bound to DNA insights on DNA binding in TGF-beta signaling, Cell 94 (1998) 585–594. [46] Chen X., Weisberg E., Fridmacher V., Watanabe M., Naco G., Whitman M., Smad4 and FAST-1 in the assembly of activin-responsive factor, Nature 389 (1997) 85–89. [47] Zhang Y., Feng X.H., Derynck R., Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-beta-induced transcription, Nature 394 (1998) 909–913. [48] Kurokawa M., Mitani K., Irie K., Matsuyama T., Takahashi T., Chiba S., Yazaki Y., Matsumoto K., Hirai H., The oncoprotein Evi-1 represses TGF-beta signalling by inhibiting Smad3, Nature 394 (1998) 92–96. [49] Moustakas A., Kardassis D., Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members, Proc. Natl. Acad. Sci. USA 95 (1998) 6733–6738. [50] Feng X.H., Zhang Y., Wu R.Y., Derynck R., The tumor suppressor Smad4/DPC4 and transcriptional adaptor CBP/ p300 are coactivators for smad3 in TGF-beta-induced transcriptional activation, Genes Dev. 12 (1998) 2153–2163. [51] Topper J.N., Dichiara M.R., Brown J.D., Williams A.J., Falb D., Collins T., Gimbrone M.A.J., CREB binding protein is a required coactivator for Smad-dependent transforming growth factor beta transcriptional responses in endothelial cells, Proc. Natl. Acad. Sci. USA 95 (1998) 9506–9511. [52] Shioda T., Lechleider R.J., Dunwoodie S.L., Li H., Yahata T., DeCaestecker M.P., Fenner M.H., Roberts A.B., Isselbacher K.J., Transcriptional activating activity of Smad4 roles of SMAD hetero- oligomerization and enhancement by an associating transactivator, Proc. Natl. Acad. Sci. USA 95 (1998) 9785–9790. [53] Kretzschmar M., Doody J., Massague J., Opposing BMP and EGF signalling pathways converge on the TGF- beta family mediator Smad1, Nature 389 (1997) 618–622. [54] DeCaestecker M.P., Parks W.T., Frank C.J., Castagnino P., Bottaro D.P., Roberts A.B., Lechleider R.J., Smad2 transduces common signals from receptor serine-threonine and tyrosine kinases, Genes Dev. 12 (1998) 1587–1592.
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[55] Brown J.D., Dichiara M.R., Anderson K.R., Gimbrone M.A.J., Topper J.N., MEKK-1 a component of the stress (Stress-activated Protein Kinase/c- Jun N-terminal Kinase) pathway can selectively activate Smad2-mediated transcriptional activation in endothelial cells, J. Biol. Chem. (1999) 2748797–8805. [56] Ulloa L., Doody J., Massagué J., Inhibition of transforming growth factor-beta/SMAD signalling by the interferongamma/STAT pathway, Nature (1999) 397710–713. [57] Bright J.J., Kerr L.D., Sriram S., TGF-beta inhibits IL-2induced tyrosine phosphorylation and activation of Jak-1 and Stat-5 in T lymphocytes, J. Immunol. 159 (1997) 175–183. [58] Pardoux C., Ma X., Gobert S., Pellegrini S., Mayeux P., Gay F., Trinchieri G., Chouaib S., Downregulation of Interleukin-12 (IL-12) responsiveness in human T cells by transforming growth factor-b relationship with IL-12 signaling, Blood 93 (1999) 1448–1455. [59] Yang X., Letterio J.J., Chen L., Hayman R., Lechleider R.J., Gu H., Roberts A.B., Deng C., Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-β, EMBO J. (1999) 181280–1291. [60] Ashcroft G.S., Yang X., Glick A., Weinstein M., Letterio J.J., Mizel D.E., Anzano M., Greenwell-Wild T., Wahl S.M., Deng C., Roberts A.B., Mice lacking SMAD3 show accelerated wound healing and an inpaired local inflamatory response, Nature Cell Biol. 1 (1999) 260–266. [61] Waldrip W.R., Bikoff E.K., Hoodless P.A., Wrana J.L., Robertson E.J., Smad2 signaling in extraembryonic tissues determines anterior-posterior polarity of the early mouse embryo, Cell 92 (1998) 797–808. [62] Nomura M., Li E., Smad2 role in mesoderm formation left-right patterning and craniofacial development, Nature 393 (1998) 786–790. [63] Sirard C., De LaPompa J.L., Elia A., Itie A., Mirtsos C., Cheung A., Hahn S., Wakeham A., Schwartz L., Kern S.E., Rossant J., Mak T.W., The tumor suppressor gene Smad4/ Dpc4 is required for gastrulation and later for anterior development of the mouse embryo, Genes Dev. 12 (1998) 107–119. [64] Kurokawa M., Mitani K., Imai Y., Ogawa S., Yazaki Y., Hirai H., The t (3;21) fusion product AML1/Evi-1 interacts with smad3 and blocks transforming growth factorbeta-mediated growth inhibition of myeloid cells, Blood 92 (1998) 4003–4012.
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TGF-β signaling from receptors to the nucleus Anita B. Roberts Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute Building 41, Room C629, 41 Library Drive, MSC 5055, Bethesda, MD 20892-5055, USA
ABSTRACT – In the past three years, a novel signal transduction pathway downstream of the transforming growth factor-beta (TGF-β) superfamily receptor serine-threonine kinases has been shown to be mediated by a family of latent transcription factors called ‘Smads’. These proteins mediate a short-circuited pathway in which a set of receptor-activated Smads are phosphorylated directly by the receptor kinase and then translocate to the nucleus complexed to the common mediator, Smad4, to participate in transcriptional complexes. Smads 2 and 3 mediate signals predominantly from the TGFβ receptors. Of these, specific roles have been ascribed to Smad3 in control of chemotaxis of neutrophils and macrophages and the inhibition of Smad3 activity by the oncogene Evi-1 suggests that it may play a role in leukemogenesis. Other data, such as the induction by the inflammatory cytokine interferon-γ of an inhibitory Smad, Smad7, which blocks the actions of Smad3, suggest that identification of the specific gene targets of Smad proteins in immune cells will provide new insight into the mechanisms of TGF-β action on these cells. © 1999 Éditions scientifiques et médicales Elsevier TGF-β / Smad / signal transduction
1. Introduction Transforming growth factor-beta (TGF-β) has profound effects on the immune system and has been implicated in a broad range of pathogenic mechanisms involving primary effects on immune cells. These include immune defects associated with autoimmune disease, suppression of immune surveillance of tumors, hematopoietic malignancies, susceptibility to parasitic infections, and chronic inflammatory disease leading to fibrotic complications [1]. These effects of TGF-β on immune cell function are mediated by signals transduced by its transmembrane serine-threonine kinase receptors and which ultimately target and regulate the transcription of nuclear genes (reviewed in [2, 3]). While several pathways including those involving ras and mitogen-activated protein kinases (MAP kinases) have been implicated in signaling from TGF-β reviewed in [4]), this brief review will be focused on the biochemical mechanisms of signal transduction by a family of recently identified cytoplasmic proteins called ‘Smad’ proteins, named by contraction of the names of the Drosophila Mad and the Caenorhabditis elegans Sma proteins, shown genetically to function downstream of the TGF-β superfamily ligands Dpp and Daf-7, respectively [5]. This shortcircuited pathway in which Smad proteins are phosphoryMicrobes and Infection 1999, 1265-1273
lated directly by the type I receptor kinase and translocated to the nucleus to participate in transcriptional complexes is conceptually similar to the Jak/Stat pathway which mediates signals from many receptors with important functions in hematopoiesis and in regulation of immune cell activity [6]. Many excellent reviews on the Smad signaling pathway have been published and, when appropriate, will be cited in lieu of the primary references.
2. Receptor serine-threonine kinases of the TGF-β superfamily Cell surface receptors for TGF-β family ligands are distinguished from those of other growth factors and cytokines by their specificity for phosphorylation of serine or threonine, rather than tyrosine residues. Receptor complexes are heterotetrameric, consisting of two ‘type II’ receptors (75–85 kDa), which bind ligand, and two signal transducing ‘type I’ receptors (50–60 kDa) which, in most instances, cannot bind ligand directly and thus are considered to act downstream of the type II receptor (for reviews see [3, 4, 7, 8]). The assembly of the heteromeric complex is initiated by ligand binding and stabilized by interactions between the cytoplasmic domains of the type II and type I receptors. This model for receptor activation involves 1265
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potentiation of the kinase activity of the type I receptor by phosphorylation of several residues in a glycine-serinerich juxtamembrane domain (GS domain) by the type II receptor kinase. Mutation of a single threonine residue to glutamine (T204D) at the interface between the GS domain and the kinase domain of the TGF-β type I receptor results in its constitutive activation, demonstrating that the activated type I kinase alone is sufficient to transduce downstream signals [9]. Mutationally activated type I receptors are often used in studies of signal transduction by members of the TGF-β family, since they eliminate the need for ligand and the type II receptor. Signals from all three isoforms of TGF-β appear to be mediated by a single type II receptor called TβR-II and one type I receptor referred to either as TβR-I or ALK-5 (activin receptor-like kinase). Another type I receptor, ALK-1, expressed on endothelial cells and mutated in the autosomal dominant disorder hereditary hemorrhagic telangiectasia (HHT), can also complex with ligand-bound TβR-II, but its role in signaling is presently not understood [10]. Two other cell surface binding proteins also participate in TGF-β receptor binding in certain cells (reviewed in [3, 4]). Betaglycan, formerly called the ‘type III receptor’, binds all isoforms of TGF-β, but may play a selective role in facilitating interaction of TGF-β2 with TβR-II, since this isoform binds to the type II receptor with significantly lower affinity than that of TGF-β1 and TGF-β3. Sharing some homology with betaglycan is endoglin, which is expressed at highest levels on endothelial cells and which is also inactivated in forms of HHT. Unlike betaglycan, endoglin binds TGF-β1 and TGF-β3 selectively. While the roles of these two proteins are not fully understood, it has been postulated that they may constrain the conformation of the TGF-βs in such a way as to enhance binding to TβR-II.
3. The Smad signal transduction pathway The search for mammalian homologues of the Drosophila Mad and C. elegans Sma proteins identified a family of cytoplasmic mediators termed Smad proteins which share highly conserved N- and C-terminal domains (called Mad homology 1 and 2, MH1 and MH2, respectively) connected by highly divergent proline-rich linker regions (reviewed in [2, 3, 7, 8]). These proteins contain no recognizable protein-protein interaction motifs or enzymatic activities. To date, eight different mammalian Smad genes have been described which fall into three distinct functional sets: receptor-activated Smads, which includes Smads 1, 2, 3, 5, and 8; a single common mediator Smad, Smad4/ DPC4, and inhibitory Smads, Smads 6 and 7 (figure 1A). The present model for downstream signaling via the Smad pathway is that: 1) receptor-activated Smads bind to and are phosphorylated on two C-terminal serine residues in their MH2 domain by the type I receptor kinase; 2) the phosphorylated, pathway-specific Smads then heterooligomerize in the cytoplasm with the common mediator, Smad4; 3) the heteromeric Smad4-containing complex is then translocated to the nucleus where it mediates tran1266
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scriptional activation of the target gene (figure 2). Inhibitory Smads, induced by TGF-β family ligands, function in a negative feedback loop to terminate or reduce the strength of the signal. 3.1. Receptor-activated Smads
Of the five receptor-activated Smads identified thus far, Smad2 and 3 have been shown to mediate signals from TGF-β and activin receptors [11, 12] whereas Smad1, 5, and 8 mediate signals from BMP receptors [13]. There are indications, however, that this delineation may not be so strictly defined. Smad1 is phosphorylated and partners with Smad4 following treatment of human breast cancer cells with either BMP-2 or TGF-β [14] and the Smad5 gene has been implicated in inhibition of the proliferation of primitive human hematopoietic progenitor cells by TGFβ [15]. All of the receptor-activated Smad proteins share a common C-terminal motif, SSXS-COOH, in which the two-carboxyl terminal serine residues are phosphorylated by the activated type I receptor kinase. Mutations in this phosphorylation motif (3S > A) result in dominant negative function, presumably by their stable association with the type I receptor and their subsequent failure to associate with Smad4 [16, 17]. The amino acid sequence at the highly conserved L3 loop locus in the MH2 domain has been shown to be an important determinant of the specificity of the Smad/receptor interaction as has an interacting cluster of four amino acids in the L45 loop of the type I receptor kinase domain [18]. Switching of two amino acids flanking the L3 loop core sequence, Arg-Gln, in Smads 1 and 2 changes their receptor specificity to the TGF-β and BMP-2 receptors, respectively. In the absence of ligand, interactions of the MH1 and MH2 domains are mutually repressive with the MH1 domain interfering with the oligomerizing and transcriptional activating activity of the MH2 domain [19]. Liganddependent relief of this autorepression can be blocked either by mutation of the C-terminal serines (3S > A), by mutation of a critical Gly residue in the L3 loop common to all Smads, or by mutation of a conserved Arg in the MH1 domain which increases the strength of the intramolecular interaction (figure 1B). Although mutations in the MH1 domain are rare, this Arg residue has been in found to be mutated in both Smad2 and Smad4 in a colon carcinoma and pancreatic carcinoma, respectively, suggesting that mutations at this position are selected for in tumors [19]. Recently a novel double zinc-finger lipid-binding, or FYVE-domain protein called SARA (Smad anchor for receptor activation) has been described which provides insight into mechanisms whereby the interaction of specific unphosphorylated Smad proteins with the type I receptor is controlled to facilitate their ligand-dependent phosphorylation [20]. The FYVE domain of SARA is predicted to interact with phosphosphotidyl inositol-3phosphate in the cell membrane while an adjacent Smad binding domain recruits Smad2/3 from the cytoplasmic reservoir; the C-terminal domain anchors the Smad-SARA complex to the receptor complex. Mutation of SARA to induce mislocalization of Smad2 or 3 results in inhibition of the transcriptional activation of target genes. Specific Microbes and Infection 1999, 1265-1273
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Figure 1. Structure-function of Smad proteins. A. Smad proteins share highly conserved MH1 and MH2 domains. Receptor-activated Smad proteins share a C-terminal phosphorylation motif, SSXS, and can be assigned to two classes (those binding BMP or TGF-β / activin receptors, respectively) depending on a conserved sequence in the L3 loop of the MH2 domain. Smad4 lacks the phosphorylation motif and has a unique Smad activation domain (SAD). Inhibitory Smad proteins lack both the MH1 domain and the C-terminal phosphorylation motif. B. The MH1 and MH2 domains of receptor-activated Smads and Smad4 interact in the absence of ligand activation to mutually repress the activities of these two domains. Ligand-induced phosphorylation of receptor-activated Smads or hetero-oligomerization of Smad4 relieves this autorepression and reveals the DNA binding activity of the MH1 domain, and the oligomerization and transcriptional activating activity of the MH2 domain and/or the SAD domain.
roles of SARA in regulating the specificity of the signaling pathway and possibly limiting cross-talk with other pathways have been proposed [20]. 3.2. The common mediator – Smad4/DPC4
Smad4 is unique among all the Smads. It was first identified as a tumor suppressor gene for pancreatic canMicrobes and Infection 1999, 1265-1273
cer, being either mutated or deleted in a significant percentage of pancreatic cancers as well as in a smaller percentage of colon cancers and breast cancers [21]. Based on our present understanding of the singular role of Smad4 in mediating signals from receptor serinethreonine kinases of the TGF-β superfamily, it is clear that functional inactivation of this molecule affects most, if not 1267
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Figure 2. Smad-mediated signaling and cross-talk. Different classes of receptor-activated Smads bind and are phosphorylated by the type I receptor serine-threonine kinase, hetero-oligomerize with Smad4 and are translocated to the nucleus where they participate in transcriptional complexes. Signaling from HGF and EGF receptors via MAP-kinase pathways can also phosphorylate receptor-activated Smads, possibly in the middle linker region, with resultant synergistic (HGF on Smad2) or antagonist (EGF on Smad1) effects on signal transduction, resulting in integrated cross-talk between receptor tyrosine and serine-threonine kinases. Nuclear complexes involve both direct binding of Smad proteins to DNA via their MH1 domains, binding of transcription factors (TF) and/or coactivators such as CBP/p300 through their MH2 domains, and possible involvement of additional activators such as MSG1. Regulation of transcription factor activation and of the coactivators by alternative signaling pathways and by oncoproteins such as ras and E1A represents yet another level of transcriptional cross-talk. all, transcriptional targets of the set of receptor-activated Smads. The recent identification of a colon tumor cell line, Vaco8–2, harboring mutations in both the TGF-β type II receptor and Smad4 accentuates the broader role of Smad4 in pathways other than those specific to TGFβ [22]. Smad4 is not phosphorylated upon receptor activation, nor does it have the critical C-terminal phosphorylation motif common to the receptor-activated Smads (reviewed in [2]). Biochemical evidence for its central role in transduction of signals from the type I receptor serine-threonine kinases comes from the demonstration of its specific association with each of the receptor-activated Smads following their ligand-dependent phosphorylation [23]. Recent data suggest that this funneling of multiple signals through a single node can serve to modulate cross-talk between different receptors of the TGF-β family in that the relative strength of each signal can be controlled by competition for a limited pool of Smad4 [24]. Determination of the tertiary structure of the C-terminal domain of Smad4 shows that it associates as a homotri1268
mer [25], and data now suggest that hetero-oligomeric Smads are also trimeric [26]. Key residues at the trimer protein-protein interfaces, which are targets for mutation of Smad4 in human cancers, interfere with homo- and hetero-oligomerization and nuclear translocation [25]. A subset of individuals with familial juvenile polyposis, an autosomal dominant disease characterized by a predisposition to gastrointestinal cancer, also show truncating frameshift mutations in linker and MH2 domains of Smad4/DPC4 [27]. Similar to the receptor-activated Smads, the MH1 domain of Smad4 is inhibitory to the transcriptional activating activity of its MH2 domain; this inhibition is relieved by association with a phosphorylated receptor-activated Smad. Unique to Smad4, an activation domain (SAD) has been identified near the extreme C-terminal portion of the middle linker region which is required for its transcriptional activating activity [28]. 3.3. Inhibitory Smads
An important new development in our understanding of the mechanism of signal transduction by Smad proteins Microbes and Infection 1999, 1265-1273
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has been the identification of inhibitory Smads or antiSmads [29–31]. Members of this Smad protein subset, which presently includes the mammalian Smad6, Smad7, share the general features of having an N-terminal domain distinct from that of the MH1 domain shared by other Smads and being more similar to Smad4 in the MH2 domain, in that neither the L3 loop receptor binding motif nor the C-terminal serine phosphorylation motif common to the receptor-activated Smads is present. The conservation of the MH2 domain and the ability of Smads 6 and 7 to interact both with the type I receptors and with other Smads, suggest that this domain may represent a novel interaction domain specific for this pathway, analogous to the SH2/SH3 interaction domains which are hallmarks of tyrosine kinase signaling pathways [32]. Identification in Drosophila of the product of a homologous gene called Daughters against decapentaplegic (Dad), which inhibits patterning by dpp, suggests that this inhibitory pathway evolved early and is basic to the mechanisms of signal transduction by this family of ligands and receptors [33], much as the SOCS/SSI inhibitors serve to regulate signaling in Jak/Stat pathways [34]. The mRNAs for Smad6 and Smad7 are rapidly induced by treatment of cells with TGF-β suggesting that antiSmads are direct effectors of a ligand-induced signal to suppress a response; these anti-Smads are also induced by epidermal growth factor, suggesting that they also serve to mediate cross-talk between signaling pathways from tyrosine kinase and serine-threonine kinase receptors [35]. Vascular endothelial cells subjected to laminar shear stress express an N-terminally truncated Smad6 [29, 30]. Since this truncation changes the interaction pattern, resulting in nonspecific interaction with Smad1, 2, and 4, the possibility exists that expression of the truncated version of this protein by endothelial cells may represent an adaptative response to mechanical stress, altering signaling from multiple TGF-β family ligands. There is presently a lack of consensus regarding the mechanism of action of these anti-Smads, and recent data suggest that both cytoplasmic and nuclear activities might be involved [36].
4. Transcriptional activation of nuclear target genes by Smads The nuclear translocation of receptor-activated Smads in a phosphorylation-dependent manner is consistent with their proposed direct role in transcriptional activation of target genes. However, unlike the consensus cis elements which led to the identification of the Stat transducers of signals from receptor tyrosine kinases [6], the diversity of response elements identified in target genes activated by TGF-β and other superfamily members has proved challenging in terms of understanding the mechanisms of transcriptional activation by Smads. Present models suggest that while Smad proteins can bind DNA directly, they likely also require the cooperation of multiple, diverse, sequence-specific-DNA binding proteins as well as coactivator molecules to mediate their transcriptional effects ([2], see figure 2). An excellent example of this is in the collagen VII promoter where an AP-1 binding site is cenMicrobes and Infection 1999, 1265-1273
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tral to a bipartite Smad binding element, resulting in a 52-bp minimal TGF-β response element [37]. This added level of complexity clearly adds both to the specificity of the response patterns in particular cell types and to the degree of cross-talk between different pathways which may modulate the levels or activity of the transcription factors or the coactivators themselves. 4.1. Direct DNA binding of Smad proteins
The MH1 domain of Smad proteins can bind DNA directly. The only exception to this rule is Smad2 in which an inserted 30 aa sequence encoded by exon 3 precludes DNA binding; a naturally occurring splice variant has been described in which this region is deleted, restoring DNA binding activity comparable to Smad3 [38]. Examination of the Dpp A response elements of the vestigial, labial, and Ultrabithorax genes revealed a consensus GC-rich binding sequence, GCCCnCGc, which binds C-terminally truncated forms of Drosophila MAD (reviewed in [2]). The N-terminal domain of Medea, the Drosophila counterpart of Smad4, has also been shown to bind GC-rich sequences in the tinman promoter. Although these interactions are weak, they are specific and correlate with activation of these enhancer elements in vivo. A pallindromic Smad3/4 binding element, GTCTAGAC, has been defined by a screen with random oligonucleotides [39]. Promoters of TGF-β -responsive genes including plasminogen activator inhibitor-1 [40–42], type VII collagen [37], and JunB [43] as well as the artificial 3TP-Lux reporter [44] contain similar sequences and bind the MH1 domain of Smads 3 and 4 directly. Crystallographic analysis of the MH1 domain of Smad3 with this pallindromic element shows this sequence to be optimal for protein DNA contacts [45]. 4.2. Transcription factor/Smad complexes
The first example of a Smad transcriptional complex was based on characterization of the ‘activin response factor’, ARF, which binds to an activin-response element (ARE) in the promoter of the Xenopus Mix.2 homeobox gene and is rapidly induced by activin independently of new protein synthesis [46]. The essential component of the ARF, a forkhead/winged-helix transcription factor, FAST-1, binds the ARE directly. Both Smad2 and Smad4 are also present in the complex although they do not directly bind the ARE: the MH2 domain of Smad2 interacts with the C-terminal domain (Smad-interaction domain, SID) of FAST-1 directly, while the binding of Smad4 is assumed to be dependent on its association with activated Smad2 and possibly also on its ability to stabilize the DNA complex through its MH1 domain. The identification of both Smad2 and 4 in the active transcriptional complex is consistent with the findings that receptor-activated Smads require Smad4 for transcriptional activation, even though they can translocate to the nucleus in its absence, and that nuclear translocation of Smad4 requires heteromeric association with a receptor-activated Smad. Smad3 can interact directly with c-Jun and c-Fos through its MH1 and MH2 domains, respectively [47] leading to the suggestion that AP-1 DNA binding sites can be activated either by direct binding of Smad3 and Smad4 1269
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to the DNA element itself or by interaction of the heteromeric Smad3/Smad4 complex and the AP-1 transcription factor complex, in such a manner as to provide additional stability to the complex. Besides providing transcriptional diversity, this mechanism also suggests a basis for crosstalk between MAP kinase cascades and JNK kinase and Smad signaling pathways, in addition to direct phosphorylation of Smad proteins by signals from receptor tyrosine kinases which activate MEK1 or a downstream kinase as discussed below [46]. A zinc-finger transcription factor Evi-1 has also been shown to interact with Smad3, but to result in suppression of TGF-β-mediated signaling and antagonism of its growth inhibitory effects [48]. Since this factor is overexpressed in certain hematopoietic malignancies, it may have special significance for regulation of immune cell responses to TGF-β (see below). The ubiquitous transcription factor Sp-1 has also been shown to interact functionally with Smad3/Smad4 complexes in regulation of the p21/WAF1/ Cip1 promoter, although neither a physical association of Smad3 and Sp1 nor binding of the Smad complex to the Sp1 site have been demonstrated [49]. A clear example of an indirect interaction of Smad proteins with transcription factors is provided in a TGF-β -induced element in of the plasminogen activator inhibitor-1 (PAI-1) gene. This element has been shown to require binding of both heteromeric Smad3/ Smad4 and the basic helix-loop-helix transcription factor, TFE3, to adjacent sites, presumably without direct physical interaction of Smads and TFE3 [40]. 4.3. Smad interaction with transcriptional coactivators
Coactivators are a class of proteins which act by bridging transcription factors and components of the basal transcriptional machinery. CREB binding protein (CBP) and its closely related functional homologue p300, are histone acetyltransferases (HATs) previously shown to interact with a variety of transcription factors and nuclear hormone receptors as well as with the basal transcription factor TFIIB and RNA polymerase II. These proteins have now been shown to also interact with Smad proteins in the regulation of transcription. At present there is a lack of consensus as to the particular domain and the particular Smad with which these proteins interact. Depending on the system and the cell type, ligand-activated Smad1, Smad2, and Smad3, as well as Smad4 have all been shown to be capable of binding to CBP/p300 ([50]; reviewed in [2]). The essential nature of this interaction is demonstrated by the fact that disruption of the Smad-CBP/p300 interaction by the adenoviral transforming protein E1A, which binds the coactivators, blocks nearly all Smadstimulated transcriptional responses in cells [51]. Another nuclear protein that strongly activates transcription without binding to DNA, MSG1, has been shown to interact functionally with the SAD domain in the middle linker region of Smad4 and to be required for activation of Smad4-dependent transcription in certain cells [52]. Downregulation of expression of MSG1 by both ras and E1A again provides a framework for cross-talk between different pathways which can modulate the activity of TGF-β signaling. 1270
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In summary, it can be said that the difficulty in defining specific consensus TGF-β-responsive elements results from the diversity of transcription factors that cooperate with Smad signaling protein complexes and the additional requirement for an adjacent cooperating Smad DNA binding site [2].
5. Receptor cross-talk mediated by Smad proteins Depending on the context, TGF-β family ligands can either synergize or antagonize the actions of mitogenic growth factors such as epidermal growth factor (EGF) or hepatocyte growth factor (HGF) which signal through receptor tyrosine kinases. Recent data now implicate Smads in the cross-talk between signal transduction pathways downstream of certain receptor tyrosine kinase and TGF-β family signaling pathways. Specifically, it has been shown that Smad1 can be phosphorylated at several PXPS sites in its middle linker region, which are consensus sites for MAP kinases [53]. Treatment of cells with EGF or hepatocyte growth factor (HGF) results in phosphorylation of Smad1 on these sites by the Erk subfamily of MAP kinases, and in retention of the linker-region phosphorylated Smad1 in the cytoplasm, blocking the nuclear translocation and transcriptional activating activity of Smad1/ Smad4 complexes. It has also been shown that Smads, in certain instances, can mediate synergistic signals from these two receptor families. Examples are the activating phosphorylation and nuclear translocation of Smad2 following treatment of cells with HGF [54] or activation of the SAPK/JNK pathway [55]. Smad7 is also an important focal point of disparate signaling pathways as evidenced by its induction by EGF [35] and interferon-γ [56]. These exciting findings suggest that receptor-activated Smads may be sensors of the competing or synergistic inputs from receptor tyrosine and serine/threonine kinases, and that activation of selected gene targets by Smads may reflect an integrated signal input from these two distinct pathways. There are also examples of downregulation of Jak/Stat signaling from IL-12 and IL-2 by TGF-β but the mechanisms involved have not been identified [57, 58].
6. Implications for Smad regulation of immune cell function While TGF-β is known to regulate many facets of immune cell behavior including the growth of cells and their differentiated function, specific gene targets are still poorly understood [1]. Recent data from targeted deletion of the Smad3 gene suggest that the Smad pathway will be critical to response of these cells to TGF-β [59]. Specifically, symptomatic Smad3 null mice show defects in mucosal immunity associated with thymic involution, enlarged lymph nodes, and formation of bacterial abscesses. Smad3 null T cells, but not B cells, show selective loss of response to the growth suppressive effects of TGF-β following activation, and the chemotactic Microbes and Infection 1999, 1265-1273
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responses of both neutrophils and macrophages to TGF-β are impaired [60]. Since targeted deletion of Smad2 or Smad4 each results in early embryonic lethality and in the inability to form mesoderm [61, 62] or failure to gastrulate [63], respectively, it is not possible at present to investigate effects of their loss on mature lymphoid cells. Certainly, the adult phenotype of the Smad3 null mice suggests that this Smad plays a more restricted role than Smad2 in signaling from TGF-β family ligands including, minimally, TGF-β and activin. Study of selected immune cell populations from these mice now affords a unique opportunity to dissect the roles of Smad2 and Smad3 in pathways of TGF-β signaling and to identify specific gene targets with functional correlates. Again suggestive of an important role of Smad3 in immune cell function is the observation that the t(3q26;21q22) fusion product, AML/Evi-1, interacts with Smad3 and blocks inhibition of myeloid cells by TGFβ [64]. This chromosomal translocation, seen in the blastic crisis of chronic myelogenous leukemia, places the AML1/Evi-1 chimera under control of the AML promoter. AML1 has been implicated in myeloid cell differentiation and shown to bind to enhancer core motifs present in myeloid promoters and lymphoid enhancers. Evi-1, located on chromosome 3q26, is frequently translocated and found to be transcriptionally activated in myeloid leukemias and myelodysplasias. It encodes a nuclear zinc finger protein with transcriptional regulatory activity, although specific target genes have not yet been identified. Following the demonstration that the first zinc finger domain of Evi-1 can bind Smad3 and that it, plus a distinct repressor domain, can block the growth inhibitory effects of TGF-β on epithelial cells [48], it has now been shown that the AML/Evi-1 chimera can similarly bind Smad3, suggesting that the mechanism of leukomogenesis in the AML/Evi-1 translocation may also result from interference with TGF-β signaling mediated by Smad3 [64]. Whereas definitive involvement of Smad signaling in hematological malignancies has not yet been demonstrated, these data strongly suggest that interruption of TGF-β signaling may contribute to progression of leukemias and that Smad3 may play a unique role in this process.
7. Summary An explosion of information in the past three years has resulted in the characterization of a novel signal transduction pathway downstream of the type I receptor serinethreonine kinases of TGF-β superfamily ligands. The Smad protein transducers of signals from these receptors also mediate certain aspects of both cytoplasmic and nuclear signaling cross-talk that contribute to stimulatory or inhibitory effects on TGF-β signaling. While the basic features of this signal transduction mechanism have now been elucidated, many aspects of the regulation of this pathway are still unclear. The genes identified thus far as being regulated directly by Smad-mediated pathways are all immediate-early gene targets, and the mechanisms of involvement of Smad proteins in signals requiring longer duration or greater intensity, such as those resulting in Microbes and Infection 1999, 1265-1273
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control of cell growth, differentiation, or apoptosis are not known. Clearly, there is much still to be learned.
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