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Abstract

The transforming growth factor-beta (TGF-β) family of cytokines consists of multi-functional polypeptides that regulate a variety of cell processes, including proliferation, differentiation, apoptosis, extracellular matrix elaboration, angiogenesis, and immune suppression, among others. In so doing, TGF-β plays a key role in the control of cell behavior in both health and disease. In this report, we review what is known about the mechanisms of activation of the peptide, together with details of TGF-β signal transduction pathways. This review summarizes the evidence implicating TGF-β in normal physiological processes of the craniofacial complex—such as palatogenesis, tooth formation, wound healing, and scarring—and then evaluates its role in non-malignant disease processes such as scleroderma, submucous fibrosis, periodontal disease, and lichen planus.

(I) Introduction

Transforming growth factor-β (TGF-β) is a ubiquitous peptide that is expressed by nearly all cell types and is known to regulate an extensive array of cellular processes, including proliferation, differentiation, extracellular matrix (ECM) elaboration, hematopoeisis, angiogenesis, immune responses, and cell death (Massagué et al., 2000; Siegel and Massagué, 2003). In the present review, we summarize what is currently known about the activation of the peptide, together with the biochemical pathways by which the protein elicits a cellular response. The fact that TGF-β is multi-functional has meant that the peptide is increasingly being recognized as being of fundamental importance in a variety of normal physiological processes and in the pathogenesis of certain disease processes. We review the involvement of TGF-β in normal and pathological processes of the oral-facial tissues and include the embryonic development of the palatal shelves and teeth, followed by a consideration of disorders of fibrosis and the immune system. In a second review (also in this issue of Crit Rev Oral Biol Med), the role of TGF-β in epithelial malignancy, particularly with reference to the pathogenesis of oral cancer, is discussed.

(II) TGF-β Ligand

TGF-β is a member of a large family of structurally related growth and differentiation factors that also includes the activins and the bone morphogenic proteins (BMP; Massagué, 1990). There are three TGF-β isoforms in humans—TGF-β1, TGF-β2, and TGF-β3—the amino acid sequences of which are 70–80% homologous and encoded by distinct genes located to 19q13.1-q13.3, 1q41, and 14q24, respectively (Fujii et al., 1986; Barton et al., 1988). The wide distribution of expression of each isoform during embryogenesis suggests that the absence of a single TGF-β gene would not be compatible with normal development. The development of TGF-β null mice, however, not only showed that such mice could survive, albeit with limited post-natal survival, but also the absence of specific TGF-β isoforms resulted in specific non-overlapping phenotypes. These findings, therefore, excluded theories of redundancy among the different TGF-β isoforms (Erickson, 1993). What is now known is that the TGF-β isoforms exhibit overlapping, but distinct, temporal and spatial patterns of expression in vivo. In broad terms, TGF-β1 is expressed in epithelial, hematopoietic, and connective tissue cells, TGF-β2 in epithelial and neuronal cells, and TGF-β3 primarily in mesenchymal cells (Massagué, 1998). In a comprehensive review, Kulkarni et al.(2002) summarized the broad spectrum of findings that have resulted from TGF-β transgenic and gene knockout studies. TGF-β1^−/− null mice display strain-dependent death at mid-gestation, due to defective yolk sac vasculogenesis, progressive T-cell-dependent inflammation and autoimmunity, increased nitric oxide production, decreased bone mass and elasticity, defects of IgG, IgM, and IgE, and colon cancer. TGF-β2^−/− null mice exhibit multiple developmental defects, including cardiac, lung, craniofacial, limb, spinal column, eye, inner ear, and urogenital defects, all of which contribute to perinatal lethality. TGF-β3^−/− null mice die within 24 hours of birth, due to a failure of palate shelf fusion and delayed lung maturation.

TGF-β is secreted as either a small or large latent complex. The small latent complex consists of the carboxy terminal of the TGF-β dimer linked covalently to the amino terminus of the TGF-β precursor (latency-associated peptide; LAP). The large latent complex is composed of the small latent complex linked by disulphide bonds to the latent TGF-β-binding protein (LTBP), an ECM protein that belongs to the fibrillin family of proteins and which has a central role in the processing and secretion of TGF-βs. Several other ECM proteins have also been reported to bind TGF-β1 in vitro, including fibronectin, collagen IV, fibromodulin, decorin, and biglycan. These molecules target latent TGF-β to the ECM and are thought to ‘serve’ activated TGF-β to the signaling TβR-I and TβR-II receptors. The restricted pattern of expression of these binding molecules, therefore, is not only critical for the bio-availability of the peptide, but also is known to modulate individual TGF-β isoform activity (Saharinen et al., 1999).

The small latent complex is released from LTBPs by proteolysis, and then the ligand is activated by disruption of the covalent bonds that bind it to LAP (Khalil, 1999; Annes et al., 2003). This can be achieved by:

physio-chemical processes—acidic cellular micro-environment, extremes of pH, γ-radiation, reactive oxygen species;

enzymatic and protein interactions—proteases (plasmin, cathepsin G, calpain, MMP 2 and MMP 9), cell-cell interactions, glycosidases, thrombospondin, integrins αvβ6 and αvβ8; and

drugs/hormones—anti-estrogens, retinoids, vitamin D3 and derivatives, glucocorticoids.

The αvβ6 integrin activates TGF-β1 and TGF-β3 by inducing a conformational change in the small latent TGF-β complex (Munger et al., 1999; Annes et al., 2003). Similarly, αvβ8 also activates small latent TGF-β complexes and inhibits epithelial cell growth in a TGF-β-dependent way (Cambier et al., 2000; Mu et al., 2002). In 1989, Sato and Rifkin showed that latent TGF-β could be activated by co-culture of pericytes and smooth-muscle cells (Sato and Rifkin, 1989). It is now known that the latent TGF-β complex is bound to the cell surface of the smooth-muscle cells by a mannose-6-phosphate/insulin-like growth factor receptor (M6P/IGFII-R), and, after forming a complex with the urokinase plasminogen activator (uPA) receptor, plasmin is released from its precursor molecule plasminogen to facilitate activation of TGF-β (Godar et al., 1999).

(III) Signaling Pathways

(A) TGF-β receptors

TGF-β initiates signaling by the assembly of receptor complexes (Shi and Massagué, 2003; de Caestecker, 2004). Three major classes of receptor proteins (TβR-I, TβR-II, and TβR-III) have been identified by affinity cross-linking and by cDNA cloning. The genes encoding the receptors are located to chromosomes 9q33-34, 3p22, and 1p32-33 (TβR-I, TβR-II, and TβR-II, respectively; Mathew et al., 1994; Johnson et al., 1995).

TβR-I and TβR-II are protein kinases that contain an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic serine-threonine kinase domain. The type I receptor is distinguished by a highly conserved GS domain containing a repetitive glycine-serine motif between the transmembrane and kinase domains (Rosenzweig et al., 1995). Following binding of the TGF-β molecule to TβR-II, TβR-I is recruited to form a heterotetrameric complex. The constitutively active TβR-II then phosphorylates TβR-I on serine residues within its GS domain (Fig. 1; de Caestecker, 2004). TβR-III (β-glycan), the most abundant of the receptor types, is not directly involved in signaling but is thought to regulate TGF-β access to the signaling receptors TβR-I and TβR-II (Massagué, 1998).

(B) Smad proteins

Activation of TGF-β receptors results in the stimulation of an evolutionarily conserved, signal transduction pathway that is mediated by Smad proteins (Derynck and Zhang, 2003; Shi and Massagué, 2003). The Smad family of receptor substrates was discovered through studies on dpp (decapentaplegic), a Drosophila member of the TGF-β family (Sekelsky et al., 1995). A search for genes involved in dpp function in Drosophila led to the identification of a novel gene named Mad (mothers against dpp). Shortly thereafter, three Mad homologs—called sma-2, sma-3, and sma-4—were identified in the nematode Caenorhabditis elegans (Savage et al., 1996), and up to 8 homologs termed ‘Smads’ (for Sma and Mad homologs) were identified in humans (Smads1-8; Heldin et al., 1997).

(C) Smad structure

Based on structural and functional considerations, Smads fall into 3 subfamilies, namely, the receptor-activated Smads (R-Smads; Smad1, Smad2, Smad3, Smad5, and Smad8), the common-mediator Smad (Co-Smad; Smad4), and the inhibitory Smads (I-Smads; Smad6 and Smad7; Fig. 2). Whereas Smad4 has a critical role in signaling by all the TGF-β superfamily members, R-Smads and I-Smads demonstrate pathway specificity and are selectively activated by different receptors. Smad2, Smad3, and Smad7 are activated primarily in response to TGF-β1 (Fig. 3 ), whereas Smad1, Smad5, and Smad8 are substrates of the BMP type I receptor and mediate BMP signaling. At their N- and C-termini, R-Smads and Co-Smads have highly similar amino-acid sequences termed ‘Mad homology domains’, MH1 and MH2, respectively (Attisano and Wrana, 2000). The MH1 domain predominantly mediates DNA binding and assists in nuclear translocation, whereas the MH2 domain is responsible for receptor interaction, Smad oligomerization, and transcriptional activation (Pierreux et al., 2000; Xiao et al., 2000). The MH1 and MH2 domains are separated by a variable proline-rich linker region which contains multiple consensus phosphorylation sites for mitogen-activated protein kinases (MAPKs) and, in the case of Smad4, a segment adjacent to the MH2 domain known as the Smad4 activation domain (SAD). SAD is essential for the mediation of signaling processes through its ability to confer transcriptional activity by binding to nuclear co-factors (de Caestecker et al., 2000). Although the C-terminal domain of the I-Smads is similar in structure to that of the R-Smads, the N-terminal domain fails to show significant homology (Souchelnytskyi et al., 1998).

(D) Smad function

In the resting state, Smad4 is rapidly and continuously shuttling between the cytoplasm and the nucleus. The distribution of Smad4 is dictated by the relative strengths of the nuclear localization and export signals (NLS and NES) within the MH1 domain of the Smad4 protein (Pierreux et al., 2000; Watanabe et al., 2000; Xiao et al., 2003). Continuous nuclear-cytoplasmic shuttling is not considered to be a property of the R-Smads, because, in the absence of TGF-β, they are retained within the cytoplasm through interaction with other cytoplasmic proteins such as microtubules (Dong et al., 2000) and/or Smad-anchor for receptor activation (SARA) molecules (Tsukazaki et al., 1998). Further, while R-Smads do not contain an NES, they do have an NLS which is capable of mediating ligand-induced nuclear localization; in the absence of a signal, the NLS in Smad2 and Smad3 are masked (Kurisaki et al., 2001).

Upon TGF-β-induced receptor activation, Smad2 and Smad3 are transiently recruited to the receptors via chaperone molecules such as membrane-associated SARA (Tsukazaki et al., 1998). Phosphorylation by TβR-I unmasks the NLS of the R-Smads such that they are released back into the cytoplasm, where they form heterotrimeric complexes with Smad4. Although the exact mechanisms of nuclear import are not completely understood, it is postulated that Smad4 either co-translocates with the R-Smads by utilizing the latter’s nuclear import function (Pierreux et al., 2000) or transports separately using its own nuclear import mechanism (Fink et al., 2003; Xiao et al., 2003). Once inside the nucleus, R-Smads mask the NES of Smad4, and the complex is retained in the nucleus to facilitate transcriptional regulation (Watanabe et al., 2000).

Within the nucleus, Smads function as transcriptional regulators and modulate the expression of target genes. The MH1 domains of Smad3 and Smad4, but not Smad2, bind directly to a specific DNA sequence called the Smad-binding element (SBE; Shi et al., 1998). SBEs have been found in multiple TGF-β-responsive promoter regions in a variety of genes, including PAI-1 (Dennler et al., 1998), JunB (Jonk et al., 1998), and type VII collagen (Vindevoghel et al., 1998). Since Smad protein affinity for this cognate sequence (CAGAC) is very low, additional DNA contacts with two classes of proteins—DNA binding co-factors and transcriptional co-activators and co-repressors—are necessary for specific, high-affinity binding of a Smad complex to a target gene. To date, more than 40 transcription factors, co-activators, and co-repressors are known to interact with Smad proteins (Miyazawa et al., 2002).

It is becoming apparent that Smad2 and Smad3 regulate different subsets of target genes. Smad3/Smad4 complexes activate immediate early genes by binding to SBE core repeats of target promoters, whereas Smad2 mediates the transmodulation of immediate-early and intermediate genes and may antagonize the effects of Smad3; interestingly, Smad3 appears to regulate a different set of genes in keratinocytes and fibroblasts (Yang et al., 2003).

(E) Smad co-factors, co-activators, and co-repressors

Smad co-factors co-operate with activated Smads by binding only to those promoters that fulfill combined sequence specificity. For example, Smad2 and/or Smad3 can associate with c-Jun/c-Fos (AP-1; Zhang et al., 1998), ATF2 (Hanafusa et al., 1999), TFE3 (Hua et al., 1998), PEBP2/CBF, and the vitamin D receptor (Yanagisawa et al., 1999). These interactions may lead to either additive or antagonistic activities regarding gene transcription, depending on the structure of the target promoters.

The binding of the Smad complex to DNA leads to the recruitment of either transcriptional co-activators or co-repressors. p300 and CREB binding proteins (CBP) are transcriptional co-activators and function either by altering chromatin structure so that the underlying DNA sequences are exposed to the transcriptional apparatus (Workman and Kingston, 1998) or by directly recruiting RNA polymerase II to the promoter (Snowden and Perkins, 1998). Smad4 functions to stabilize the R-Smad-CBP/p300 interaction and, therefore, is considered to be an essential co-activator for Smad2/3-dependent transcriptional activation (Feng et al., 1998). In contrast, repression of Smad-activated transcription occurs through the action of co-repressors such as TGIF (Wotton et al., 1999) and the proto-oncogenes Ski and SnoN, which recruit histone deacetylases (Luo et al., 1999).

(F) Regulation of receptor/Smad function

There are several mechanisms by which the function of TGF-β receptors and Smads can be modulated. Receptor activation is modified through the actions of accessory receptors such as Betaglycan, Endoglin, and NMA, and through ligand-dependent endocytic trafficking of activated receptors (Tsang et al., 2000; Blobe et al., 2001a,b; Di Guglielmo et al., 2003). Further, a large variety of proteins has been identified that interact with both the receptor complexes and R-Smads to regulate function. The FYVE domain protein SARA, FKBP12, Hgs, Axin, ELF, Disabled-2, TRAP-1, FTLP, and SANE, together with organelles such as microtubules, can regulate function both positively and negatively (Lutz and Knaus, 2002; ten Dijke et al., 2002; de Caestecker, 2004; Wang and Donahoe, 2004).

In the nucleus, Smad binding to DNA is transient, and deactivation of Smad function occurs through the actions of I-Smads and through R-Smad degradation and post-translational modification. The I-Smad, Smad7, resides within the nucleus in the basal state, is induced in response to TGF-β, and functions via a negative feedback mechanism to control the intensity and duration of Smad signaling (Fig. 3). The direct association of Smad7 with receptor complexes prevents further recruitment and phosphorylation of R-Smads, while its ability to recruit Smurf-1 and Smurf-2 leads to receptor degradation (Ebisawa et al., 2001).

Once receptor signaling is abrogated, Smad2 and Smad3 are rapidly de-phosphorylated, presumably by an as-yet-unidentified R-Smad nuclear phosphatase (Reguly and Wrana, 2003). Evidence is accumulating to indicate that phosphorylated R-Smads are degraded via the ubiquitin proteosome system by the activity of E3 ubiquitin ligases, which function at both the cytoplasmic (Zhang et al., 2001) and nuclear levels (Lo and Massagué, 1999). In contrast, Smad4 is not normally subject to regulation by phosphorylation or ubiquitination, most probably because it functions as a common mediator and is required to be present in significant levels at all times. Analysis of recent data suggests that Smad4 is stabilized by a unique post-translational modification involving a small ubiquitin-like modifier-1 (SUMO-1) which prevents Smad4 ubiquitination-dependent degradation (Lee et al., 2003; Lin et al., 2003). Like the R-Smads, Smad7 also undergoes ligand-dependent ubiquitination and degradation, possibly through its association with the intracellular protein Arkadia (Koinuma et al., 2003).

(G) Alternative signaling pathways

Data are rapidly accumulating to implicate a variety of additional pathways, other than Smad complexes, in TGF-β signaling. These include several small GTPases (Edlund et al., 2002), PKC (Runyan et al., 2003), and phosphatidylinositol 3-kinase (PI3K; Higaki and Shimokado, 1999; Bakin et al., 2000). Of increasing interest is the ability of TGF-β to activate MAPKs, a large family of serine/threonine proteins that function to control gene expression. Three main groups of MAP kinases have been identified, including extracellular-regulated kinases (Erks), which are stimulated by growth factors and are associated with the regulation of cellular proliferation and differentiation, and c-Jun N-terminal kinases (JNK) and p38, both of which are activated by cellular stress and have been implicated in the process of cell death. All three MAP kinase groups are activated by TGF-β, with varying kinetics in different cell types (Hartsough and Mulder, 1995; Atfi et al., 1997; Frey and Mulder, 1997; Reimann et al., 1997; Hannigan et al., 1998; Hanafusa et al., 1999; Sano et al., 1999; Yu et al., 2002; Itoh et al., 2003). In some cases, activation is very rapid (from 10 to 30 min), suggesting that it might occur through a direct, Smad-independent, post-translational modification. In other cases, however, activation is slow (several hours), suggesting an indirect Smad-dependent transcriptional response (Engle et al., 1999; Kim et al., 2002; Yu et al., 2002; Dumont et al., 2003; Itoh et al., 2003). To add further complexity, MAP kinase pathways have been shown to modulate Smad signaling. Activation of Erks, for example, induces phosphorylation in the linker region of R-Smads, thereby inhibiting ligand-induced nuclear accumulation of R-Smads (Kretzschmar et al., 1999), and p38 induces ATF-2 phosphorylation, resulting in synergism with R-Smads and transcriptional activation (Sano et al., 1999).

The mechanisms linking the kinase pathways to TGF-β receptors are unclear although TGF-β-activated kinase 1 (TAK-1) has been shown to be phosphorylated following TGF-β stimulation and leads to the activation of JNK and p38 (Yamaguchi et al., 1995; Shirakabe et al., 1997; Takatsu et al., 2000). TAK-1 binding protein (TAB-1) has been identified as an activator of TAK-1 (Shibuya et al., 1996) and may link with TβR-I through HPK1 and XIAP (Yamaguchi et al., 1999; Zhou et al., 1999).

(H) New modulators of the TGF-β response

Recently, Cordenonsi et al.(2003) demonstrated that the reduction of endogenous p53 levels in HepG2 cells reduced the expression of TGF-β target genes and allowed cells that were normally growth-inhibited by the ligand to become refractory to the peptide. Furthermore, restoring p53 activity in a p53-null cancer cell line, which is normally insensitive to TGF-β signaling, resulted in Smad-dependent inhibition of cell growth. Analysis of the data demonstrates that p53 cooperates with Smads to mediate the TGF-β transcriptional response, but the implications of disrupting p53 activity, as occurs in many tumor cell types, are currently unknown.

The Runt family of transcription factors consists of 3 DNA-binding α subunits (RUNX 1, RUNX 2, RUNX 3), each of which is capable of forming heterodimers with the TGF-β co-activator p300/CBP. All 3 RUNX members bind R-Smads, thymocytes from RUNX 2 transgenic mice are hypersensitive to TGF-β, and loss of RUNX 3, now recognized as a tumor suppressor (particularly in gastric carcinoma), not only induces cell proliferation but also blocks TGF-β-induced apoptosis (Ito and Miyazono, 2003).

(IV) TGF-β Function

TGF-β is a multi-functional cytokine that functions to inhibit growth and induce apoptosis in a variety of cell types, elaborate extracellular matrices, and suppress the immune response. These effects are summarized in Fig. 4 and are discussed in more detail below.

(A) Growth inhibition

TGF-β reversibly inhibits cell proliferation in epithelial, endothelial, hematopoietic, and neural cells. Following ligand binding, Smad complexes co-operate with nuclear co-factors such as E2F4/5 and p107 and transcriptionally repress c-myc (Chen et al., 2002). Concurrently, Smad complexes induce certain cell-cycle inhibitors (cyclin-dependent kinase [CDK] inhibitors), including p21/WAF1 and p15. p15 displaces the kinase inhibitor protein p27 from its complex with cyclinD-cdk4/6, which results in the inhibition of CDK activities associated with early G1 phase progression. Thus, cell-cycle progression is blocked at the G1 restriction point. In addition, elevated p21/WAF1 and the displaced p27 co-operate to inhibit cyclinE-cdk2 activity (Reynisdottir et al., 1995). A major outcome of this nuclear activity is the accumulation of hypo-phosphorylated retinoblastoma protein (pRb), which subsequently represses major gene targets with crucial roles in pushing the cell cycle toward the S phase. These changes have always been thought to be mediated by conventional pathways of TGF-β signal transduction, namely, Smad2/3 complexing with Smad4 after ligand stimulation followed by translocation to the nucleus. A recent study, however, has shown that p21/WAF1 can be up-regulated by TGF-β1 in a Smad4-independent manner through Smad2/3-dependent transcriptional activation of the p21/WAF1 promoter (Ijichi et al., 2004). This is the first study to show that Smad2/3 can regulate gene transcription independently of Smad4, and it raises the possibility that Smad4-independent signaling downstream of TGF-β may be as widespread as Smad4-dependent signaling.

Additional signaling molecules have been reported to be critical for the anti-proliferative response to TGF-β, but their importance has not yet been fully established (Petritsch et al., 2000; Aghdasi et al., 2001).

(B) Apoptosis

TGF-β can induce apoptosis (programmed cell death) in a variety of different cell types. Although suppression of the apoptotic inhibitor Bcl-xL and activation of certain caspases have been implicated, the mechanisms that trigger apoptosis following receptor activation are poorly understood. The septrin-like molecule ARTS, a novel mitochondrial protein, translocates into the nucleus coincident with the induction of apoptosis, and Daxx, a Fas-receptor-associated protein, has been shown to interact with TβR-II and subsequently activate the JNK/Map kinase pathway. Analysis of such data suggests that novel pathways, distinct from Smad signaling, are involved in TGF-β-induced apoptosis (Larisch et al., 2000; Perlman et al., 2001). However, Smad-dependent pathways are also involved in TGF-β-induced apoptosis, because the inhibitory protein, Smad7, can induce apoptosis in epithelial cells, possibly by inhibiting NFκB (Lallemand et al., 2001) or by the activation of JNK, thereby demonstrating the potential for complex interplay between signaling pathways (Mazars et al., 2001). Further, TGF-β-activated Smads have been shown to induce the expression of Gadd45, and this, in turn, leads to p38-dependent apoptosis in murine hepatocytes (Yoo et al., 2003).

(C) Extracellular matrix (ECM) elaboration

TGF-β has a marked effect on ECM composition. The peptide is chemotactic for fibroblasts and causes the synthesis and secretion of ECM molecules such as collagen I, collagen type IV, fibronectin, and tenascin. Further, TGF-β mediates the equilibrium between ECM-degrading proteases and their inhibitors, with the result that ECM proteins are elaborated (Sieweke and Bissell, 1994; Verrecchia and Mauviel, 2002).

(D) Angiogenesis

It is generally accepted that TGF-β plays an important role in vascular remodelling, although its effects appear to be contextual and dependent on the presence of other regulators. Studies have shown that TGF-β can increase the production of vascular endothelial growth factor (VEGF), enhance the effects of basic fibroblast growth factor (bFGF), and inhibit endothelial cell migration. As such, TGF-β has been identified as an important regulator of new vessel growth, vascular network formation, and the establishment and maintenance of vessel wall integrity (Pepper, 1997).

(E) Immunosuppression

TGF-β has significant anti-inflammatory functions. The peptide modulates the proliferation and differentiation of lymphocytes, macrophages, and dendritic cells and, therefore, regulates both antigen- and non-antigen-specific immune processes. The overall response is dependent on the differentiation status of the target cells and the presence of other cytokines; in general, however, TGF-β inhibits B- and T-cell proliferation, inhibits MHC class II expression, and stimulates apoptosis. The net effect is that TGF-β functions as a potent immunosuppressive molecule (Letterio and Roberts, 1998; Dong et al., 2001; Dennler et al., 2002).

(V) Role of TGF-β in Craniofacial Development and Oral Disease

The signaling pathways activated by members of the TGF-β superfamily are known to play critical roles in mammalian development. It is perhaps not unexpected, therefore, that defects in TGF-β signal transduction have been shown to be responsible for several developmental disorders, and that de-regulated TGF-β activity has been implicated in the pathogenesis of a variety of human diseases (Blobe et al., 2000; Massagué et al., 2000). In this article, we will focus specifically on the role of the TGF-β isoforms in craniofacial development and in non-malignant oral disease.

(A) Skeletal development

The abundant and overlapping expression of each TGF-β isoform in bone emphasizes the importance of these molecules in normal hard-tissue development and function. Defects of bone have been reported predominantly in TGF-β2^−/− null mice, leading to numerous defects of the craniofacial, axial, and appendicular skeleton (Sanford et al., 1997). Targeted over-expression of TGF-β2 to osteoblasts in transgenic mice causes an imbalance between osteoblastic and osteoclastic activity and leads to a phenotype that is characterized by bone loss such that it resembles either osteoporosis or hyperparathyroidism. Interestingly, there is also a delay in the ossification of bones and the mineralization of sutures, together with a clavicular malformation that resembles cleidocranial dysplasia (Erlebacher and Derynck, 1996; Kreiborg et al., 1999; Mundlos, 1999; Massagué et al., 2000).

(B) Palatogenesis

In mammalian development, the formation of the palate is required to separate the oropharynx from the nasopharynx. Palatogenesis is a multi-step process involving palatal shelf growth and elevation, followed by fusion of the paired shelves and the disappearance of the medial edge epithelium (MEE) from the midline to allow for mesenchymal confluence (Ferguson, 1988). Defects in any of these processes lead to cleft palate, one of the most common birth defects in humans (Chenevix-Trench et al., 1992). Studies with transgenic mice have implicated the involvement of more than 20 different genes in this process. With regard to the TGF-β isoforms, a cleft palate develops in 100% of TGF-β3^−/− null mice (Proetzel et al., 1995) but occurs in only 23% of TGF-β2-deficient animals (Sanford et al., 1997) and is not seen in TGF-β1 knockouts (Shull et al., 1992; Kulkarni et al., 1993). Further, the inhibition of TGF-β3 results in a failure of plate fusion in vitro (Brunet et al., 1995), and the addition of TGF-β3 to cultures of palatal shelves from TGF-β3-deficient mice is able to reverse the phenotype completely (Kaartinen et al., 1997; Taya et al., 1999). TGF-β3 also induces an epithelial-mesenchymal transdifferentiation of the MEE (Kaartinen et al., 1997; Sun et al., 1998), possibly by regulating the expression of MMPs (Blavier et al., 2001) and proteoglycans (Gato et al., 2002) by Smad2-dependent mechanisms (Cui et al., 2003). These findings have been confirmed recently by Dudas et al.(2004), who showed that TGF-β3-induced palatal fusion was mediated by the Alk-5/Smad pathway.

A role for TGF-β3 in the development of cleft palate in humans is less clear. While some workers have failed to find a link between TGF-β3 and oral clefts (Beaty et al., 2001), others have demonstrated polymorphisms in the TGF-β3 gene that have been linked with the disorder (Lidral et al., 1998); these allelic variants may interact with other risk factors, such as maternal tobacco use, in the development of this syndrome (Romitti et al., 1999).

(C) Tooth morphogenesis

TGF-β isoforms are thought to play a fundamental role in the histomorphogenesis of developing teeth. Dental abnormalities have been described in TGF-β^−/− null mice (D’Souza and Litz, 1995; D’Souza et al., 1998), and inactivation of the TβR-II gene leads to accelerated tooth development (Chai et al., 1999). These findings have subsequently been extended, and it has been postulated that TGF-βs (TGF-β1 and -β3), derived from the enamel organ and immobilized and activated by components of the basement membrane, induce odontoblast differentiation. Odontoblasts, in turn, then express TGF-β, which acts in an autocrine manner to stimulate the secretion of predentin and dentin (Lesot et al., 2001). This proposal is consistent with observations showing the presence of TGF-β isoforms in dentin matrices (Cassidy et al., 1997) and both TGF-β isoforms and TβR-I and TβR-II in odontoblasts (Sloan et al., 1999, 2000). These findings suggest an active TGF-β signaling complex at the primary site of dental hard-tissue formation. Interestingly, members of the TGF-β super-family also seem to be important in hard-tissue formation after pulpal injury and have the potential to induce odontoblast differentiation (Ruch et al., 1995). Further, sequestered TGF-β appears to be released from dentin following tissue injury, leading to the generation of reactionary/reparative dentin in mature teeth (Magloire et al., 2001), a repair process that may have significant implications for the dental profession in future years. However, targeted over-expression of TGF-β1 specifically to tooth germs in transgenic mice results in a phenotype similar to dentin dysplasia, such that excess collagen deposition and defects can be found in tooth mineralization (Thyagarajan et al., 2001).

The role of BMPs and their receptors in tooth morphogenesis is less clear (Cheifetz, 1999). BMP-2, BMP-4, and BMP-7 expression appears to be temporally related, with different expression patterns in the epithelium and mesenchyme associated with the bud, cap, and bell stages of tooth morphogenesis; similarly, BMP receptor expression is also variable. Further work is clearly required in this area, not least because the situation is complicated by species variation, ligand and receptor redundancy, and an unknown contribution of ligand from maternal sources.

(D) Wound healing

The role of TGF-β in the wound-healing process is controversial. Many animal models of impaired wound healing have shown that reduction of endogenous TGF-β1 in the wound bed underlies the defective repair process. This concept, however, has recently been questioned, because TGF-β1^−/− and Smad3^−/− null mice showed accelerated wound healing and a reduction in the area of granulation tissue (Ashcroft et al., 1999; Koch et al., 2000). The findings may be explained in terms of the function of different TGF-β isoforms (O’Kane and Ferguson, 1997). TGF-β1 and TGF-β2 increase significantly within 24 hours of an incisional wound, and later, TGF-β3 is elevated when TGF-β1 levels decrease, suggesting a reciprocal relationship between TGF-β1 and TGF-β3 during wound healing. Both TGF-β3 and a polyclonal neutralizing antibody to TGF-β1,2 significantly reduce scarring in rat incisional wounds (Shah et al., 1992, 1994), possibly because the ratio of TGF-β3 relative to TGF-β1/TGF-β2 has been altered early in the wound-healing cascade. Further, preventing the activation of TGF-β1 by competitive inhibition of binding of the LAP at the M6P/IGFII-R has a marked anti-scarring effect (McCallion and Ferguson, 1996).

The observation that there are decreased levels of TGF-β1 and TGF-β2 in situations of impaired wound healing has prompted the use of exogenous TGF-βs in the management of these problems (Cox et al., 1992; Beck et al., 1993; Robson et al., 1995). Of particular significance to the oral physician in this context is the development of oral mucositis following chemotherapy. Topical application of TGF-β3 prior to chemotherapy with 5-Fluorouracil in hamsters resulted in a significant reduction in oral mucositis (Sonis et al., 1997). Despite these early encouraging results, however, a double-blind, placebo-controlled, multi-center phase II clinical trial has shown that TGF-β3 is not effective in the prevention or alleviation of chemotherapy-induced oral mucositis (Foncuberta et al., 2001). In chronic wounds, high levels of proteases causing cytokine breakdown, the presence of underlying systemic disease, and the lack of vascularity at the wound site, together with the placebo effect of any clinical trial, could contribute to these clinical findings.

The situation is somewhat different in human skin exposed to solar ultraviolet irradiation (Quan et al., 2002). In these circumstances, there is increased expression of TGF-β1 and TGF-β3 but decreased expression of TGF-β2 (the predominant TGF-β isoform in normal skin epithelium); TβR-II is decreased and Smad7 is increased. Taken together, the changes are thought to drive ECM elaboration.

(E) Fibrotic disorders

Scleroderma (systemic sclerosis) is a disease characterized by vascular dysfunction and excessive production of ECM proteins, resulting in fibrosis of the subcutaneous tissues and viscera. Approximately 80% of patients have manifestations in the head and neck region, with dysphagia and reflux esophagitis being common. Other oro-facial problems include progressive trismus, stiffening of the tongue, and, occasionally, a widening of the periodontal membrane space without tooth mobility. The fact that TGF-β stimulates fibroblast proliferation and ECM elaboration suggests the importance of this cytokine in fibrotic disease. Indeed, it has been shown that fibroblasts from scleroderma patients show enhanced sensitivity to TGF-β (Cotton et al., 1998). The mechanisms underlying this hyper-responsiveness to TGF-β in scleroderma have recently been explained in terms of decreased expression of the inhibitory Smad, Smad7, in scleroderma fibroblasts compared with their normal counterparts (Dong et al., 2002).

Another fibrotic disease of relevance to the dental community is oral submucous fibrosis (OSF). This disorder is closely associated with the habit of chewing betel quid, it affects approximately 5 million people in the Indian subcontinent, and it is a potentially malignant condition (Pindborg et al., 1984). OSF is characterized histologically by the deposition of collagen in the oral submucosa, together with epithelial atrophy and dysplasia. The role of TGF-β in OSF is unknown, but it has been reported that the type I plasminogen activator inhibitor (PAI-1), a known Smad-responsive gene, is up-regulated in OSF (Dong et al., 2002), thus giving indirect evidence for enhanced TGF-β activity in OSF. Furthermore, it has been suggested that polymorphisms in the TGF-β gene may affect an individual’s susceptibility to this most debilitating of disorders (Chiu et al., 2002).

(F) Periodontal disease

TGF-β is one of several cytokines known to regulate inflammation and immune responses, and the fact that TGF-β mediates leukocyte recruitment, adhesion, and activation suggests that it has a key role to play in the host’s response to bacterial and immunological challenge. It is somewhat surprising, therefore, that there is not clearer evidence for its involvement in the pathogenesis of periodontal disease (Skaleric et al., 1997; Holla et al., 2002; de Souza et al., 2003; Ejeil et al., 2003). This may be due to several factors, including the multi-factorial nature of the disease, the complex interaction between and among different cytokines at the site of inflammation, and the technical difficulties associated with the measurement of active as opposed to total TGF-β.

(G) Lichen planus

Oral lichen planus is a chronic inflammatory disease that is characterized histologically by a dense sub-epithelial T-cell infiltrate and degeneration of basal keratinocytes; auto-reactive cytotoxic T-cells are known to trigger keratinocyte apoptosis (Sugerman et al., 2000). Recently, the role of TGF-β in the pathogenesis of oral lichen planus was reviewed by Sugerman et al.(2002). These authors suggested that, in view of the fact that TGF-β had potent immunosuppressive activity, possibly mediated by CD4+ TGF-β secreting Th3 regulatory T-cells which suppressed immune responses to self-antigens (Bridoux et al., 1997; Mason and Powrie, 1998), and that TGF-β could interfere with antigen presentation (Letterio and Roberts, 1998), a breakdown of TGF-β activation and/or signal transduction might occur at sites of inflammation in this disease process. This proposal is consistent with the identification of TGF-β+ T-cells in the sub-epithelial lymphocytic infiltrate, but not within the epithelium itself, of oral lichen planus tissues (Khan et al., 2003). Further, over-expression of Smad7 appears to be a characteristic feature of basal keratinocytes in active lichen planus lesions, suggesting that the TGF-β pathway is suppressed (Dr. A. Karatsaidis, personal communication).

Widespread autoimmune-like inflammatory disease is a characteristic of TGF-β1^−/− null mice (Dang et al., 1995; Kulkarni et al., 2002), and in animals where Smad3 function has been disrupted (Yang et al., 1999); replacement of TGF-β in animal models of autoimmune disease significantly ameliorates the disease process (Kuruvilla et al., 1991; Racke et al., 1991; Chen et al., 1998). Importantly, interferon gamma (IFN-γ) is known to inhibit the immunosuppressive activity of TGF-β1 by blocking TGF-β-induced phosphorylation of Smad3 (Ulloa et al., 1999), and it has been suggested that the balance between TGF-β1 and IFN-γ signaling determines the level of immunological activity in mucosal inflammatory disorders (Strober et al., 1997). It is conceivable, therefore, that in active lichen planus, over-production of IFN-γ by Thy1 CD4+ T-cells and/or over-expression of Smad7 down-regulates the immunosuppressive activity of TGF-β1 and induces MHC class II expression and CD8+ cytotoxic T-cell activity. In contrast, in quiescent lesions, the balance between IFN-gamma and TGF-β1 activity would change such that the immunosuppressive function of TGF-β1 would predominate.

(VI) Concluding Remarks

The past 10–15 years have seen significant advances in our understanding of the biochemical pathways associated with TGF-β signal transduction. These discoveries have laid the foundation for an exciting period of further exploration that is likely to be directed toward designing new therapeutic modalities to address some of the disease processes in which TGF-β has been implicated.

A tentative start has been made. Scientists are already examining the prospect of using BMPs to treat anomalies of bone such as fractures, osteoporosis, and periodontal defects and to facilitate osseous-integration of implants (King, 2001; Sykaras and Opperman, 2003). Further, the capacity of TGF-β to suppress immune responses raises the possibility that TGF-β substitutes and/or agonists may be used for the treatment of chronic autoimmune diseases, an approach with some experimental support (Prud’homme and Piccirillo, 2000; Prud’homme et al., 2001). In contrast, an antagonist of TGF-β is also likely to have significant value, primarily in the treatment of fibrotic disorders of the kidney, liver, and lung (Blobe et al., 2000), and also in the prevention of scar formation in the skin and oral mucosa (see above). A variety of different strategies has been used to develop TGF-β antagonists, including recombinant soluble betaglycan (Bandyopadhyay et al., 2002), a soluble type II TGF-β:Fc fusion protein (Muraoka et al., 2002; Yang et al., 2002), the histone deacetylase inhibitor trichostatin A (Rombouts et al., 2002), and retroviral gene therapy with dominant-negative TβR-II constructs (Huang and Lee, 2003). The use of peptides in a therapeutic capacity, however, is limited because of poor bioavailability, poor membrane penetration, and unfavorable pharmacokinetics. One of the challenges for the future, therefore, will be to develop new small molecules that can either mimic or antagonize the effects of TGF-β to produce a therapeutic gain in different disease processes. Further, the key will be to deliver these molecules in an organ-specific way to avoid deleterious side-effects.

The present review goes some way toward describing the importance of TGF-β in oral health and non-malignant disease; a second review (also in this issue of Crit Rev Oral Biol Med) examines the part that the peptide plays in oral malignancy. New information on activation and signaling pathways will provide new targets for therapeutic intervention, and the development of both TGF-β agonists and antagonists will expand the opportunities for intervention in common pathological processes. It is perhaps not an overstatement to say that exciting times are ahead for those who wish to be part of this rapidly expanding field.

Figure 1.
TGF-β receptor activation. TGF-β initiates signaling by the assembly of receptor complexes involving two transmembrane serine-threonine kinase receptors (TβR-I and TβR-II). Following binding of TGF-β to TβR-II, TβR-I is recruited to form a heterotetrameric complex and is phosphorylated at serine residues within its GS domain by the constitutively active TβR-II. The activated TβR-I then activates a cascade of downstream signaling events that ultimately regulates gene transcription.

Figure 2.
Structure of Smad proteins. The R and Co-Smads show considerable homology within both the MH1 and MH2 domains. The MH domains are separated by a variable proline-rich linker region, which, in the case of Smad4, contains a Smad activation domain (SAD). **Smad2 is unable to bind directly to DNA, due to a 30-amino-acid insertion within exon3 of the MH1 domain. I-Smads have C-terminal domains structurally similar to R and Co-Smads. The N-terminus, however, fails to show significant homology.

Figure 3.
Smad signaling. The activated TβR-I kinase phosphorylates the R-Smads, Smad2 and Smad3, which then form heterodimers with the Co-Smad, Smad4. The complex translates to the nucleus and regulates gene transcription by binding directly to DNA via interactions with DNA-binding proteins and/or transcriptional co-activators and co-repressors. The transcription of the I-Smad, Smad7, is induced, and Smad7 exits the nucleus to control the intensity and duration of signaling via a negative feedback mechanism.

Figure 4.
Cellular effects of TGF-β. TGF-β is a multi-functional cytokine that inhibits epithelial cell growth, induces apoptosis in a variety of cell types, stimulates new vessel growth, and is a potent immunosuppressant. In addition, TGF-β functions to elaborate the extracellular matrix.

Footnotes

Acknowledgements

The authors are most grateful to the Shirley Glasstone Hughes Memorial Fund (British Dental Association) for the support of MP. We thank J. Beddoe and L. Jones for help in preparation of the manuscript.

References

Aghdasi B, Ye K, Resnick A, Huang A, Ha HC, Guo X, et al. (2001). FKBP12, the 12 kDA FK506-binding protein, is a physiologic regulator of the cell cycle. Proc Natl Acad Sci USA 98:2425–2430.

Annes JP, Munger JS, Rifkin DB (2003). Making sense of TGF-β activation. J Cell Sci 116:217–224.

Ashcroft GS, Yang X, Glick AB, Weinstein M, Letterio JL, Mizel DE, et al. (1999). Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory immune response. Nat Cell Biol 1:E117–E119.

Atfi A, Djelloul S, Chastre E, Davis R, Gespach C (1997). Evidence for a role of Rho-like GTPases and stress activated protein kinase/c-Jun N-terminal linase (SAPK/JNK) in transforming growth factor β mediated signaling. J Biol Chem 272:1429–1432.

Attisano L, Wrana JL (2000). Smads as transcriptional co-modulators. Cur Opin Cell Biol 12:235–243.

Bakin AV, Tomlinson AK, Bhowmick NA, Moses HL, Arteaga CL (2000). Phosphatidylinositol 3-kinase function is required for transforming growth factor β-mediated epithelial to mesenchymal transition and cell migration. J Biol Chem 275:36803–36810.

Bandyopadhyay A, Lopez-Casillas F, Malik SN, Montiel JL, Mendoza V, Yang J, et al. (2002). Antitumor activity of a recombinant soluble betaglycan in human breast cancer xenograft. Cancer Res 62:4690–4695.

Barton DE, Foellmer BE, Du J, Tamm J, Derynck R, Francke U (1988). Chromosomal mapping of genes for transforming growth factors-β2 and -β3 in man and mouse: dispersion of the TGF-β gene family. Oncogene Res 3:323–331.

Beaty TH, Wang H, Hetmansli JB, Fan YT, Zeiger JS, Liang KY, et al. (2001). A case-control study of nonsyndromic oral clefts in Maryland. Ann Epidemiol 11:434–442.

10.

Beck LS, DeGuzman L, Lee WP, Xu Y, Siegel MW, Amento EP (1993). One systemic administration of transforming growth factor-β1 reverses age- or glucocorticoid-impaired wound healing. J Clin Invest 92:2841–2849.

11.

Blavier L, Lazaryev A, Groffen J, Heisterkamp N, DeClerck YA, Kaartinen V (2001). TGF-β3-induced palatogenesis requires matrix metalloproteinases. Mol Biol 12:1457–1466.

12.

Blobe GC, Schiemann WP, Lodish HF (2000). Role of transforming growth factor β in human disease. N Engl J Med 342:1350–1358.

13.

Blobe GC, Schiemann WP, Pepin MC, Beauchemin M, Moustakas A, Lodish HF, et al. (2001a). Functional roles for the cytoplasmic domain of the type III TGF-β receptor in regulating TGF-β signalling. J Biol Chem 276:24627–24637.

14.

Blobe GC, Liu X, Fang SJ, How T, Lodish HF (2001b). A novel mechanism for regulating transforming growth factor beta (TGF-beta) signaling. Functional modulation of type III TGF-beta receptor expression through interaction with the PDZ domain protein, GIPC. J Biol Chem 276:39608–39617.

15.

Bridoux F, Badou A, Saoudi A, Bernard I, Druet E, Pasquier R, et al. (1997). Transforming growth factor beta (TGF-β)-dependent inhibition of T helper cell 2 (Th2)-induced autoimmunity by self-major histocompatibility complex (MHC) class II specific, regulatory CD4(+) T cell lines. J Exp Med 185:1769–1775.

16.

Brunet CL, Sharpe PM, Ferguson MW (1995). Inhibition of TGF-β3 (but not TGF-β1 or TGF-β2) activity prevents normal mouse embryonic palate fusion. Int J Dev Biol 39:345–355.

17.

Cambier S, Mu DZ, O’Connell D, Boylen K, Travis W, Liu WH, et al. (2000). A role for the integrin αvβ8 in the negative regulation of epithelial cell growth. Cancer Res 60:7084–7093.

18.

Cassidy N, Fahey M, Prime SS, Smith AJ (1997). Comparative analysis of transforming growth factor-beta (TGF-β) isoforms 1–3 in human and rabbit dentine matrices. Arch Oral Biol 42:219–223.

19.

Chai Y, Zhao J, Mogharei A, Xu B, Bringas P Jr, Shuler C, et al. (1999). Inhibition of transforming growth factor-β type II receptor signaling accelerates tooth formation in mouse first branchial arch explants. Mech Dev 86:63–74.

20.

Cheifetz S (1999). BMP receptors in limb and tooth formation. Crit Rev Oral Biol Med 10:182–198.

21.

Chen CR, Kang Y, Siegel PM, Massagué J (2002). E2F4/5 and p107 as Smad cofactors linking the TGF-β receptor to c-myc repression. Cell 110:19–32.

22.

Chen LZ, Hochwald GM, Huang C, Dakin G, Tao H, Cheng C, et al. (1998). Gene therapy in allergic encephalomyelitis using myelin basic protein-specific T cells engineered to express latent transforming growth factor-beta1. Proc Natl Acad Sci USA 95:12516–12521.

23.

Chenevix-Trench G, Jones K, Green AC, Duffey DL, Martin NG (1992). Cleft lip with or without cleft palate: associations with transforming growth factor alpha and retinoic acid receptor. Am J Hum Genet 51:1377–1385.

24.

Chiu CJ, Chang ML, Chiang CP, Hahn LJ, Hsieh LL, Chen CJ (2002). Interaction of collagen-related genes and susceptibility to betel quid-induced oral submucous fibrosis. Cancer Epidemiol Biomarkers Prev 11:646–653.

25.

Cordenonsi M, Dupont S, Maretto S, Insinga A, Imbriano C, Piccolo S (2003). Links between tumor suppressors: p53 is required for TGF-β gene responses by cooperating with Smads. Cell 113:301–314.

26.

Cotton SA, Herrick AL, Jayson MI, Freemont AJ (1998). TGF-β—a role in systemic sclerosis. J Pathol 184:4–6.

27.

Cox DA, Kunz S, Cerletti N, McMaster GK, Burk RR (1992). Wound healing in aged animals: effects of locally applied TGF-β2 in different model systems. In: Angiogenesis—key principles. Steiner R, Weisz PB, Langer R, editors. Basel: Birkhauser, pp. 287–295.

28.

Cui XM, Chai Y, Chen J, Yamamoto T, Ito Y, Bringas P, et al. (2003). TGF-β3-dependent SMAD2 phosphorylation and inhibition of MEE proliferation during palatal fusion. Dev Dyn 227:387–394.

29.

D’Souza RN, Litz M (1995). Analysis of tooth development in mice bearing a TGF-β1 null mutation. Connect Tissue Res 32:41–46.

30.

D’Souza RN, Cavender A, Dickinson D, Roberts A, Letterio J (1998). TGF-β1 is essential for the homeostasis of the dentin-pulp complex. Eur J Oral Sci 106:185–191.

31.

Dang H, Geiser AG, Letterio JJ, Nakabayashi T, Kong L, Fernandes G, et al. (1995). SLE-like autoantibodies and Sjögren’s syndrome-like lymphoproliferation in TGF-β knockout mice. J Immunol 155:3205–3212.

32.

de Caestecker MP (2004). The transforming growth factor-β superfamily of receptors. Cytokine Growth Factor Rev 15:1–11.

33.

de Caestecker MP, Piek E, Roberts AB (2000). Role of transforming growth factor-β signaling in cancer. J Natl Cancer Inst 92:1388–1402.

34.

de Souza AP, Trevilatto PC, Scarel-Caminaga RM, de Brito RB, Line SR (2003). Analysis of the TGF-β-1 promoter polymorphism (C-509T) in patients with chronic periodontitis. J Clin Periodont 30:519–523.

35.

Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, Gauthier JM (1998). Direct binding of Smad3 and Smad4 to critical TGF-β inducible elements in the promoter of human plasminogen activator inhibitor type I gene. EMBO J 17:3091–3100.

36.

Dennler S, Goumans MJ, ten Dijke P (2002). Transforming growth factor β signal transduction. J Leukocyte Biol 71:731–740.

37.

Derynck R, Zhang YE (2003). Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature 425:577–584.

38.

Di Guglielmo GM, Le Roy C, Goodfellow AF, Wrana JL (2003). Distinct endocytic pathways regulate TGF-beta receptor signalling and turnover. Nat Cell Biol 5:410–421.

39.

Dong C, Li Z, Alvarez R, Feng XH, Goldschmidt-Clermont PJ (2000). Microtubule binding to Smads may regulate TGF β activity. Mol Cell 5:27–34.

40.

Dong C, Zhu S, Wang T, Yoon W, Li Z, Alvarez RJ, et al. (2002). Deficient Smad7 expression: a putative molecular defect in scleroderma. Proc Natl Acad Sci USA 99:3908–3913.

41.

Dong Y, Tang L, Letterio JJ, Benveniste EN (2001). The Smad3 protein is involved in TGF-β inhibition of class II transactivator and class II MHC expression. J Immunol 167:311–319.

42.

Dudas M, Nagy A, Laping NJ, Moustakis A, Kaartinen V (2004). TGF-β3-induced palatal fusion is mediated by Alk-5/Smad pathway. Dev Biol 266:96–108.

43.

Dumont N, Bakin AV, Arteaga CL (2003). Autocrine transforming growth factor-β signaling mediates Smad-independent motility in human cancer cells. J Biol Chem 278:3275–3285.

44.

Ebisawa T, Fukuchi M, Murakami G, Chiba T, Tanaka K, Imamura T, et al. (2001). Smurf1 interacts with transforming growth factor-β type I receptor through Smad7 and induces receptor degradation. J Biol Chem 276:12477–12480.

45.

Edlund S, Landstrom M, Heldin CH, Aspenstrom P (2002). Transforming growth factor-β-induced mobilization of the actin cytoskeleton requires signaling by small GTPases Cdc42 and RhoA. Mol Biol Cell 13:902–914.

46.

Ejeil AL, Gaultier F, Igondjo-Tchen S, Senni K, Pellat B, Godeau G, et al. (2003). Are cytokines linked to collagen breakdown during periodontal disease progression? J Periodontol 74:196–201.

47.

Engle SJ, Hoying JB, Boivin GP, Ormsby I, Gartside PS, Doetschman T (1999). Transforming growth factor β1 suppresses nonmetastatic colon cancer at an early stage of tumorigenesis. Cancer Res 59:3379–3386.

48.

Erickson HP (1993). Gene knockouts of c-src and transforming growth factor β1, and tenascin suggest superfluous, non-functional expression of proteins. J Cell Biol 120:1079–1081.

49.

Erlebacher A, Derynck R (1996). Increased expression of TGF-β2 in osteoblasts results in an osteoporosis-like phenotype. J Cell Biol 132:195–210.

50.

Feng XH, Zhang Y, Wu RY, Derynck R (1998). The tumor suppressor Smad4/DPC4 and transcriptional adaptor CBP/p300 are coactivators for Smad3 in TGF-β-induced transcriptional activation. Genes Dev 12:2153–2163.

51.

Ferguson MW (1988). Palate development. Development 103:41–60.

52.

Fink SP, Mikkola D, Willson JK, Markowitz S (2003). TGF-β-induced nuclear localisation of Smad2 and Smad3 in Smad4 null cancer cell lines. Oncogene 22:1317–1323.

53.

Foncuberta MC, Cagnoni PJ, Brandts CH, Mandanas R, Fields K, Derigs HG, et al. (2001). Topical transforming growth factor-β3 in the prevention or alleviation of chemotherapy-induced oral mucositis in patients with lymphomas or solid tumors. J Immunother 24:384–388.

54.

Frey RS, Mulder KM (1997). Involvement of ERK2 and SAPK/JNK activation by TGF-β in the negative growth control of breast cancer cells. Cancer Res 57:628–633.

55.

Fujii D, Brissenden JE, Derynck R, Francke U (1986). Transforming growth factor-β gene maps to human chromosome 19 long arm and to mouse chromosome 7. Somat Cell Mol Genet 12:281–288.

56.

Gato A, Martinez ML, Tudela C, Alonso I, Moro JA, Formosa MA, et al. (2002). TGF-β₃-induced chondroitin sulphate proteoglycan mediates palatal shelf adhesion. Dev Biol 250:393–405.

57.

Godar S, Horejsi V, Weidle UH, Binder BR, Hansmann C, Stockinger H (1999). M6P/IGFII-receptor complexes urokinase receptor and plasminogen for activation of transforming growth factor-β1. Eur J Immunol 29:1004–1013.

58.

Hanafusa H, Ninomiya-Tsuji J, Masuyama N, Nishita M, Fujisawa J, Shibuya H, et al. (1999). Involvement of the p38 mitogen-activated protein kinase pathway in transforming growth factor β induced gene expression. J Biol Chem 274:27161–27167.

59.

Hannigan M, Zhan L, Ai Y, Huang CK (1998). The role of p38 MAP kinase in TGF-β1-induced signal transduction in human neutrophils. Biochem Biophys Res Commun 246:55–58.

60.

Hartsough MT, Mulder KM (1995). Transforming growth factor β activation of p44 mapk in proliferating cultures of epithelial cells. J Biol Chem 270:7117–7124.

61.

Heldin CH, Miyazono K, ten Dijke P (1997). TGF-β signaling from cell membrane to nucleus through SMAD proteins. Nature 390:465–471.

62.

Higaki M, Shimokado K (1999). Phosphatidylinositol 3-kinase is required for growth factor-induced amino acid uptake by vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 19:2127–2132.

63.

Holla LI, Fassmann A, Benes P, Halabala T, Znojil V (2002). 5 polymorphisms in the transforming growth factor-β1 gene (TGF-β1) in adult periodontitis. J Clin Periodontol 29:336–341.

64.

Hua X, Liu X, Ansari DO, Lodish HF (1998). Synergistic cooperation of TFE3 and smad proteins in TGF-β-induced transcription of the plasminogen activator inhibitor-1 gene. Genes Dev 12:3084–3095.

65.

Huang X, Lee C (2003). From TGF-β to cancer therapy. Curr Drug Targets 4:243–250.

66.

Ijichi H, Otsuka M, Tateishi K, Ikenoue T, Kawakami T, Kanai F, et al. (2004). Smad4-independent regulation of p21/WAF1 by transforming growth factor-β. Oncogene 23:1043–1051.

67.

Ito Y, Miyazono K (2003). RUNX transcription factors as key targets of TGF-β superfamily signaling. Curr Opin Genet Dev 13:43–47.

68.

Itoh S, Thorikay M, Kowanetz M, Moustakas A, Itoh F, Heldin CH, et al. (2003). Elucidation of Smad requirement in transforming growth factor-β type I receptor-induced responses. J Biol Chem 278:3751–3761.

69.

Johnson DW, Qumsiyeh M, Benkhalifa M, Marchuk DA (1995). Assignment of human TGF-β type I and type III receptor genes (TGFβR1 and TGFβR3) to 9q33-q34 and 1p32-33, respectively. Genomics 28:356–357.

70.

Jonk LJ, Itoh S, Heldin CH, ten Dijke P, Kruijer W (1998). Identification and functional characterization of a Smad binding element (SBE) in the JunB promoter that acts as a transforming growth factor-β, activin, and bone morphogenetic protein-inducible enhancer. J Biol Chem 273:21145–21152.

71.

Kaartinen V, Cui XM, Heisterkamp N, Groffen J, Shuler CF (1997). Transforming growth factor-β3 regulates transdifferentiation of medial edge epithelium during palate fusion and associated degradation of the basement membrane. Dev Dyn 209:255–260.

72.

Kahn A, Farah CS, Savage NW, Walsh LJ, Harbrow DJ, Sugerman PB (2003). Th1 cytokines in oral lichen planus. J Oral Pathol Med 32:77–83.

73.

Khalil N (1999). TGF-β: from latent to active. Microbes Infect 1:1255–1263.

74.

Kim GY, Mercer SE, Ewton DZ, Yan Z, Jin K, Friedman E (2002). The stress-activated protein kinases p38α and JNK stabilise p21^Cip1 by phosphorylation. J Biol Chem 277:29792–29802.

75.

King GN (2001). The importance of drug delivery to optimize the effects of bone morphogenetic proteins during periodontal regeneration. Curr Pharm Biotechnol 2:131–142.

76.

Koch RM, Roche NS, Parks WT, Ashcroft GS, Letterio JJ, Roberts AB, et al. (2000). Incisional wound healing in transforming growth factor-beta1 null mice. Wound Repair Regen 8:179–191.

77.

Koinuma D, Shinozaki M, Komuro A, Goto K, Saitoh M, Hanyu A, et al. (2003). Arkadia amplifies TGF-β superfamily signalling through degradation of Smad7. EMBO J 22:6458–6470.

78.

Kreiborg S, Jensen BL, Larson P, Schleidt DT, Darvan T (1999). Anomalies of craniofacial skeleton and teeth in cleidocranial dysplasia. J Craniofac Genet Dev Biol 19:75–79.

79.

Kretzschmar M, Doody J, Timokhina I, Massagué J (1999). A mechanism of repression of TGF-β/Smad signaling by oncogenic Ras. Genes Dev 13:804–816.

80.

Kulkarni AB, Huh CG, Becker D, Geiser A, Lyght M, Flanders KC, et al. (1993). Transforming growth factor β1 null mutation in mice causes excessive inflammatory response and early death. Proc Natl Acad Sci USA 90:770–774.

81.

Kulkarni AB, Thyagarajan T, Letterio JJ (2002). Function of cytokines within the TGF-β superfamily as determined from transgenic and gene knockout studies in mice. Curr Mol Med 2:303–327.

82.

Kurisaki A, Kose S, Yoneda Y, Heldin CH, Moustakas A (2001). Transforming growth factor-β induces nuclear import of Smad3 in an importin-β1 and Ran-dependent manner. Mol Biol Cell 12:1079–1091.

83.

Kuruvilla AP, Shah R, Hochwald GM, Liggitt HD, Palladino MA, Thorbecke GJ (1991). Protective effect of transforming growth factor β1 on experimental autoimmune diseases in mice. Proc Natl Acad Sci USA 88:2918–2921.

84.

Lallemand F, Mazars A, Prunier C, Bertrand F, Kornprost M, Gallea S, et al. (2001). Smad7 inhibits the survival nuclear factor kappaB and potentiates apoptosis in epithelial cells. Oncogene 20:879–884.

85.

Larisch S, Yi Y, Lotan R, Kerner H, Eimerl S, Parks WT, et al. (2000). A novel mitochondrial septin-like protein, ARTS, mediates apoptosis dependent on its P-loop motif. Nat Cell Biol 2:915–921.

86.

Lee PS, Chang C, Liu D, Derynck R (2003). Sumoylation of Smad4, the common Smad mediator of transforming growth factor-β family signaling. J Biol Chem 278:27853–27863.

87.

Lesot H, Lisi S, Peterkova M, Mitolo V, Ruch JV (2001). Epigenetic signals during odontoblast differentiation. Adv Dent Res 15:8–13.

88.

Letterio JJ, Roberts AB (1998). Regulation of immune responses by TGF-β. Annu Rev Immunol 16:137–161.

89.

Lidral AC, Romitti PA, Basart AM, Doetschman T, Leysens NJ, Daack-Hirsch S, et al. (1998). Association of MSX1 and TGFβ3 with nonsyndromic clefting in humans. Am J Hum Genet 63:557–568.

90.

Lin X, Liang M, Liang YY, Brunicardi FC, Melchior F, Feng XH (2003). Activation of TGF-β signaling by SUMO-1 modification of tumor suppressor Smad4/DPC4. J Biol Chem 278:18714–18719.

91.

Lo RS, Massagué J (1999). Ubiquitin-dependent degradation of TGF-β-activated Smad2. Nat Cell Biol 8:472–478.

92.

Lutz M, Knaus P (2002). Integration of the TGF-β pathway into the cellular signaling network. Cell Signal 14:977–988.

93.

Luo K, Stroschein SL, Wang W, Chen D, Martens E, Zhou S, et al. (1999). The Ski oncoprotein interacts with the Smad proteins to repress TGF-β signaling. Genes Dev 13:2196–2206.

94.

Magloire H, Romeas A, Melin M, Couble ML, Bleicher F, Farges JC (2001). Molecular regulation of odontoblast activity under dentin injury. Adv Dent Res 15:46–50.

95.

Mason D, Powrie F (1998). Control of immune pathology in regulatory T-cells. Curr Opin Immunol 10:649–655.

96.

Massagué J (1990). The transforming growth factor-beta family. Ann Rev Cell Biol 6:597–641.

97.

Massagué J (1998). TGF-β signal transduction. Ann Rev Biochem 67:753–791.

98.

Massagué J, Blain SW, Roger SL (2000). TGF-β signaling in growth control, cancer, and heritable disorders. Cell 103:295–309.

99.

Mathew S, Murty VV, Cheifetz S, George D, Massagué J, Chaganti RS (1994). Transforming growth factor receptor gene TGFβR2 maps to human chromosome band 3p22. Genomics 20:114–115.

100.

Mazars A, Lallemand F, Prunier C, Marais J, Ferrand N, Pessah M, et al. (2001). Evidence for a role of the JNK cascade in Smad7-mediated apoptosis. J Biol Chem 276:36797–36803.

101.

McCallion RL, Ferguson MWJ (1996). Fetal wound healing and the development of anti-scarring therapies for adult wound healing. In: The molecular and cellular biology of wound repair. Clark RAF, editor. New York: Plenum Press, pp. 561–600.

102.

Miyazawa K, Shinozaka M, Hara T, Furuya T, Miyazono K (2002). Two major Smad pathways in TGF-β superfamily signaling. Genes Cells 7:1191–1204.

103.

Mu D, Cambier S, Fjellbirkeland L, Baron JL, Munger JS, Kawakatsu H, et al. (2002). The integrin alpha(v)beta8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1. J Oral Biol 157:493–507.

104.

Munger JS, Huang X, Kawakatsu H, Griffiths MJ, Dalton SL, Wu J, et al. (1999). The integrin αvβ6 binds and activates latent TGF-β1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96:319–328.

105.

Mundlos S (1999). Cleidocranial dysplasia: clinical and molecular genetics. J Med Genet 36:177–182.

106.

Muraoka RS, Dumont N, Ritter CA, Dugger TC, Brantley DM, Chen J, et al (2002). Blockade of TGF-β inhibits mammary tumor cell viability, migration, and metastases. J Clin Invest 109:1551–1559.

107.

O’Kane S, Ferguson MW (1997). Transforming growth factor βs and wound healing. Int J Biochem Cell Biol 29:63–78.

108.

Pepper MS (1997). Transforming growth factor-beta: vasculogenesis, angiogenesis and vessel wall integrity. Cytokine Growth Factor Rev 8:21–43.

109.

Perlman R, Schiemann WP, Brooks MW, Lodish HF, Weinberg RA (2001). TGF-β-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation. Nat Cell Biol 3:708–714.

110.

Petritsch C, Beug H, Balmain A, Oft M (2000). TGF-β inhibits p70 S6 kinase via protein phosphatase 2A to induce G(1) arrest. Genes Dev 14:3093–3101.

111.

Pierreux CE, Nicolas FJ, Hill CS (2000). Transforming growth factor β-independent shuttling of Smad4 between cytoplasm and nucleus. Mol Cell Biol 20:9041–9054.

112.

Pindborg JJ, Murti PR, Bhonsle RB, Gupta PC, Daftary DK, Mehta FS (1984). Oral submucous fibrosis as a precancerous condition. Scand J Dent Res 92:224–229.

113.

Proetzel G, Pawlowski SA, Wiles MV, Yin M, Boivin GP, Howles PN, et al. (1995). Transforming growth factor-β3 is required for secondary palate fusion. Nat Genet 11:409–414.

114.

Prud’homme GJ, Piccirillo CA (2000). The inhibitory effects of transforming growth factor-β1 (TGF-β1) in autoimmune diseases. J Autoimmun 14:23–42.

115.

Prud’homme GJ, Lawson BR, Theofilopoulos AN (2001). Anticytokine gene therapy of autoimmune diseases. Exp Opin Biol Ther 1:359–373.

116.

Quan T, He TY, Kang S, Voorhees JJ, Fisher GJ (2002). Ultraviolet irradiation alters transforming growth factor β/Smad pathway in human skin in vivo. J Invest Dermatol 119:499–506.

117.

Racke MK, Dhib-Jalbut S, Cannella B, Albert PS, Raine CS, McFarlin DE (1991). Prevention and treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-β1. J Immunol 146:3012–3017.

118.

Reguly T, Wrana JL (2003). In or out? The dynamics of Smad nucleocytoplasmic shuttling. Trends Cell Biol 13:216–220.

119.

Reimann T, Hempel U, Krautwald S, Axmann A, Scheibe R, Seidel D, et al. (1997). Transforming growth factor-β1 induces activation of Ras, Raf-1, MEK and MAPK in rat hepatic stellate cells. FEBS Lett 403:57–60.

120.

Reynisdottir I, Polyak K, Iavarone A, Massagué J (1995). Kip/Cip and Ink4 inhibitors cooperate to induce cell cycle arrest in response to TGF-β. Genes Dev 9:1831–1845.

121.

Robson MC, Phillip LG, Cooper DM, Lyle WG, Robson LE, Odom L, et al. (1995). Safety and effect of transforming growth factor-β2 for treatment of venous stasis ulcers. Wound Rep Reg 3:157–167.

122.

Rombouts K, Niki T, Greenwel P, Vandermonde A, Wielant A, Hellemans K, et al. (2002). Trichstatin A, a histone deacetylase inhibitor, suppresses collagen synthesis and prevents TGF- β1-induced fibrogenesis in skin fibroblasts. Exp Cell Res 278:184–197.

123.

Romitti PA, Lidral AC, Munger RG, Daack-Hirsch S, Burns TL, Murray JC (1999). Candidate genes for nonsyndromic cleft lip and palate and maternal cigarette smoking and alcohol consumption: evaluation of genotype-environment interactions from a population-based case-control study of orofacial clefts. Teratology 59:39–50.

124.

Rosenzweig BL, Imamura T, Okadome T, Cox GN, Yamashita H, ten Dijke P, et al. (1995). Cloning and characterization of a human type II receptor for bone morphogenetic proteins. Proc Natl Acad Sci USA 92:7632–7636.

125.

Ruch JV, Lesot H, Bègue-Kirn C (1995). Odontoblast differentiation. Int J Dev Biol 39:51–68.

126.

Runyan CE, Schnaper HW, Poncelet AC (2003). Smad3 and PKC{delta} mediate TGF-{beta}1-induced collagen I expression in human mesangial cells. Am J Physiol Renal Physiol 285:F413–422.

127.

Saharinen J, Hyytiainen M, Taipale J, Keski-Oja J (1999). Latent transforming growth factor-β binding proteins (LTBPs)—structural extracellular matrix proteins for targeting TGF-β action. Cytokine Growth Factor Rev 10:99–117.

128.

Sanford LP, Ormsby I, Gittenberger-de Groot AC, Sariola H, Friedman R, Boivin GP, et al. (1997). TGF-β2 knockout mice have multiple developmental defects that are non-overlapping with other TGF β knockout phenotypes. Development 124:2659–2670.

129.

Sano Y, Haraada J, Tashiro S, Gotoh-Mandeville R, Maekawa T, Ishii S (1999). ATF-2 is a common nuclear target of Smad and TAK1 pathways in transforming growth factor beta signaling. J Biol Chem 274:8949–8957.

130.

Sato Y, Rifkin DB (1989). Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-β1-like molecule by plasmin during co-culture. J Cell Biol 109:309–315.

131.

Savage C, Das P, Finelli AL, Townsend SR, Sun CY, Baird SE, et al. (1996). Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor β pathway components. Proc Natl Acad Sci USA 93:790–794.

132.

Sekelsky JJ, Newfield SJ, Raftery LA, Chartoff EH, Gelbart WW (1995). Genetic characterization and cloning of mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster. Genetics 139:1347–1358.

133.

Shah M, Foreman DM, Ferguson MW (1992). Control of scarring in adult wounds by neutralising antibody to transforming growth factor β. Lancet 339:213–214.

134.

Shah M, Foreman DM, Ferguson MW (1994). Neutralising antibodies to TGF-β1,2 reduces cutaneous scarring in adult rodents. J Cell Sci 107:1137–1157.

135.

Shi Y, Massagué J (2003). Mechanisms of TGF-β signalling from cell membrane to the nucleus. Cell 113:685–700.

136.

Shi Y, Wang YF, Jayaraman L, Yang H, Massagué J, Pavletich N (1998). Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA-binding in TGF-β signaling. Cell 94:585–594.

137.

Shibuya H, Yamaguchi K, Shirakabe K, Tonegawa A, Gotoh Y, Ueno N, et al. (1996). TAB1: an activator of the TAK1 MAPKKK in TGF-β signal transduction. Science 272:1179–1182.

138.

Shirakabe K, Yamaguchi K, Shibuya H, Irie K, Matsuda S, Moriguchi T, et al. (1997). TAK1 mediates the ceramide signaling to stress-activated protein kinase/c-Jun N-terminal kinase. J Biol Chem 272:8141–8144.

139.

Shull MM, Ormsby L, Kier AB, Pawlowski S, Diebold RJ, Yin M, et al. (1992). Targeted disruption of the mouse transforming growth factor-β1 gene results in multifocal inflammatory disease. Nature 359:693–699.

140.

Siegel PM, Massagué J (2003). Cytostatic and apoptotic actions of TGF-β in homeostasis and cancer. Nature Rev Cancer 3:807–819.

141.

Sieweke MH, Bissell MJ (1994). The tumor-promoting effect of wounding: a possible role for TGF-β-induced stromal alterations. Crit Rev Oncog 5:297–311.

142.

Skaleric U, Kramar B, Petelin M, Pavlica Z, Wahl SM (1997). Changes in TGF-β1 levels in gingiva, crevicular fluid and serum associated with periodontal inflammation in humans and dogs. Eur J Oral Sci 105:136–142.

143.

Sloan AJ, Matthews JB, Smith AJ (1999). TGF-β receptor expression in human odontoblasts and pulpal cells. Histochem J 31:565–569.

144.

Sloan AJ, Perry H, Matthews JB, Smith AJ (2000). Transforming growth factor-β isoform expression in mature human molar teeth. Histochem J 32:247–252.

145.

Snowden AW, Perkins ND (1998). Cell cycle regulation of the transcriptional coactivators p300 and CREB binding protein. Biochem Pharmacol 55:1947–1954.

146.

Sonis ST, Van Vugt AG, Brien JP, Muska AD, Bruskin AM, Rose A, et al. (1997). Transforming growth factor-β3 mediated modulation of cell cycling and attenuation of 5-fluorouracil induced oral mucositis. Oral Oncol 33:47–54.

147.

Souchelnytskyi S, Nakayama T, Nakao A, Moren A, Heldin C, Christian JL, et al. (1998). Physical and functional interaction of murine and Xenopus Smad7 with bone morphogenetic protein receptors and transforming growth factor-β receptors. J Biol Chem 273:25364–25370.

148.

Strober W, Kelsall B, Fuss I, Marth T, Ludviksson B, Ehrhardt R, et al. (1997). Reciprocal IFN-gamma and TGF-β responses regulate the occurrence of mucosal inflammation. Immunol Today 18:61–64.

149.

Sugerman PB, Satterwhite K, Bigby M (2000). Auto-cytotoxic T cell clones in lichen planus. Br J Dermatol 142:449–456.

150.

Sugerman PR, Savage NW, Walsh LJ, Zhao ZZ, Zhou XJ, Khan A, et al. (2002). The pathogenesis of oral lichen planus. Crit Rev Oral Biol Med 13:350–365.

151.

Sun D, Vanderburg CR, Odierna GS, Hay ED (1998). TGFβ3 promotes transformation of chicken palate medial edge epithelium to mesenchyme in vitro. Development 125:95–105.

152.

Sykaras N, Opperman LA (2003). Bone morphogenetic proteins (BMPs): how do they function and what can they offer the clinician? J Oral Sci 45:57–73.

153.

Takatsu Y, Nakamura M, Stapleton M, Danos MC, Matsumoto K, O’Connor MB, et al. (2000). TAK1 participates in c-Jun N-terminal kinase signaling during Drosophila development. Mol Cell Biol 20:3015–3026.

154.

Taya Y, O’Kane S, Ferguson MW (1999). Pathogenesis of cleft palate in TGF-β3 knockout mice. Development 126:3869–3879.

155.

ten Dijke P, Goumans MJ, Itoh F, Itoh S (2002). Regulation of cell proliferation by Smad proteins. J Cell Physiol 191:1–16.

156.

Thyagarajan T, Sreenath T, Cho A, Wright JT, Kulkarni AB (2001). Reduced expression of dentin sialophosphoprotein is associated with dysplastic dentin in mice overexpressing transforming growth factor-β1 in teeth. J Biol Chem 276:11016–11020.

157.

Tsang M, Kim R, de Caestecker MP, Kudoh T, Roberts AB, Dawid IB (2000). Zebrafish NMA is involved in TGFbeta family signaling. Genesis 28:47–57.

158.

Tsukazaki T, Chiang TA, Davison AF, Attisano L, Wrana JL (1998). SARA, a FYVE domain protein that recruits Smad2 to the TGF-β receptor. Cell 95:779–791.

159.

Ulloa L, Doody J, Massagué J (1999). Inhibition of transforming growth factor-β/SMAD signaling by the interferon-γ/STAT pathway. Nature 397:710–713.

160.

Verrecchia F, Mauviel A (2002). Transforming growth factor-beta signaling through the Smad pathway: role in extracellular matrix gene expression and regulation. J Invest Dermatol 118:211–215.

161.

Vindevoghel L, Lechleider RJ, Kon A, de Caestecker MP, Uitto J, Roberts AB, et al. (1998). SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor β. Proc Natl Acad Sci USA 95:14769–14774.

162.

Wang T, Donahoe PK (2004). The immunophilin FKBP12: a molecular guardian of the TGF-beta family type I receptors. Front Biosci 9:619–631.

163.

Watanabe M, Masuyama N, Fukuda M, Nishida E (2000). Regulation of intracellular dynamics of Smad4 by its leucine-rich nuclear export signal. EMBO Rep 1:176–182.

164.

Workman JL, Kingston RE (1998). Alteration of nucleosome structure as a mechanism of transcriptional regulation. Ann Rev Biochem 67:545–579.

165.

Wotton D, Lo RS, Swaby LA, Massagué J (1999). Multiple modes of repression by the Smad transcriptional corepressor TGIF. J Biol Chem 274:37105–37110.

166.

Xiao Z, Liu X, Henis YI, Lodish HF (2000). A distinct nuclear localization signal in the N terminus of Smad3 determines its ligand-induced nuclear translocation. Proc Natl Acad Sci USA 97:7853–7858.

167.

Xiao Z, Latek R, Lodish H (2003). An extended bipartite nuclear localization signal in Smad4 is required for its nuclear import and transcriptional activity. Oncogene 22:1057–1069.

168.

Yamaguchi K, Shirakabe K, Shibuya H, Irie K, Oishi I, Ueno N, et al. (1995). Identification of a member of the MAPKKK family as a potential mediator of TGF-β signal transduction. Science 270:2008–2011.

169.

Yamaguchi K, Nagai S, Ninomiya-Tsuji J, Nishita M, Tamai K, Irie K, et al. (1999). XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB1-TAK1 in the BMP signaling pathway. EMBO J 18:179–187.

170.

Yanagisawa J, Yanagi Y, Masuhiro Y, Suzawa M, Watanabe M, Kashiwagi K, et al. (1999). Convergence of transforming growth factor-beta and vitamin D signaling pathways on SMAD transcriptional coactivators. Science 283:1317–1321.

171.

Yang X, Letterio JJ, Lechleider RJ, Chen L, Hayman R, Gu H, et al. (1999). Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-β. EMBO J 18:1280–1291.

172.

Yang Y, Dukhanina O, Tang B, Mamura M, Letterio J, MacGregor J, et al. (2002). Lifetime exposure to a soluble TGF-β antagonist protects mice against metastases without adverse side effects. J Clin Invest 109:1607–1615.

173.

Yang YC, Piek E, Zavadil J, Liang D, Xie D, Heyer J, et al. (2003). Hierarchical model of gene regulation by transforming growth factor β. Proc Natl Acad Sci USA 100:10269–10274.

174.

Yoo J, Ghiassi M, Jirmanova L, Balliet AG, Hoffman B, Fornace AJ, et al. (2003). TGF-β-induced apoptosis is mediated by Smad-dependent expression of GADD45B through p38 activation. J Biol Chem 278:43001–43007.

175.

Yu L, Hebert MC, Zhang YE (2002) TGF-β receptor-activated p38 MAP kinase mediates Smad-independent TGF-β responses. EMBO J 21:3749–3759.

176.

Zhang Y, Feng XH, Derynck R (1998). Smad3 and Smad4 co-operate with c-Jun/c-Fos to mediate TGF-β-induced transcription. Nature 394:909–913.

177.

Zhang Y, Chang C, Gehling DJ, Hemmati-Brivanlou A, Derynck R (2001). Regulation of Smad degradation and activity by Smurf2, an E3 ubiquitin ligase. Proc Natl Acad Sci USA 98:974–979.

178.

Zhou G, Lee SC, Yao Z, Tan TH (1999). Hematopoietic progenitor kinase 1 is a component of transforming growth factor β-induced c-Jun N-terminal kinase signaling cascade. J Biol Chem 274:13133–13138.