THEME
Fibrogenesis
V. TGF-beta signaling pathways

Rebecca G. Wells

Departments of Internal Medicine and Pathology, Yale School of Medicine, New Haven, Connecticut 06520-8019


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Transforming growth factor (TGF)-beta is a multifunctional peptide growth factor with a wide range of potential effects on growth, differentiation, extracellular matrix deposition, and the immune response. General TGF-beta signaling pathways have been described in detail over the last several years, but factors that determine the nature of the TGF-beta response are poorly understood. In particular, signaling pathways that specifically mediate the matrix effects of TGF-beta have received little attention, although they will be important therapeutic targets in the treatment of pathological fibrosis. This themes article focuses on TGF-beta signaling and highlights potential points for generating matrix-specific responses.

fibrosis; extracellular matrix; transforming growth factor-beta receptors; Smads


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TRANSFORMING GROWTH FACTOR (TGF)-beta is one of the most powerful and widely distributed profibrogenic mediators in the body. It regulates not only deposition of the extracellular matrix (ECM) as part of the normal response to tissue injury but also pathological fibrosis. Alterations in TGF-beta homeostasis are important in fibrotic diseases of multiple tissues. In addition to influencing the quantity and composition of the ECM, TGF-beta has an astonishing range of other potential effects depending on the cellular and environmental context, including control of growth and differentiation and modulation of the immune response.

General TGF-beta signaling pathways from receptors to nucleus have been described in detail over the last several years and are surprisingly simple and direct (18). In contrast, factors that determine the nature of the TGF-beta response are poorly understood, and signaling pathways that specifically mediate the matrix effects of TGF-beta have received little attention. Because TGF-beta has so many potential functions, unraveling the subtleties of its signaling pathways is critical to the understanding of its role in disease and the development of effective therapies.


    TGF-beta IN NORMAL WOUND HEALING AND DISEASE
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TGF-beta enhances fibrogenesis and remodeling in normal healing after tissue injury. It is also an important mediator of multiple fibrotic diseases and syndromes, including pulmonary fibrosis, liver fibrosis, chronic pancreatitis, scleroderma, and renal glomerulosclerosis, and it has a significant role in the fibrotic complications of radiation therapy, chemotherapy, and organ transplantation (2). There are three lines of evidence that TGF-beta has a causal role in pathological fibrosis: 1) TGF-beta levels are increased in diseased organs and are often specifically localized to fibrotic areas; 2) administration of exogenous TGF-beta to laboratory animals leads to the development of fibrosis in some organs; and 3) anti-TGF-beta therapies lessen experimentally induced fibrosis.

The causal link between TGF-beta and fibrosis has been demonstrated particularly clearly for lung, kidney, and liver. In fibrotic diseases of all three tissues, regions of increased matrix show increased expression of TGF-beta , especially the isoform TGF-beta 1 (2, 19), and delivery of exogenous TGF-beta 1 by various means to these tissues results in severe fibrosis in experimental animals (22, 23). Also, there are now multiple reports that the therapeutic administration of TGF-beta binding proteins, including neutralizing antibodies, soluble TGF-beta receptors, and the proteoglycan decorin, ameliorates experimental fibrosis (6, 28, 30, 35). Perhaps most exciting is the report that intratracheal administration of an adenovirus expressing the TGF-beta signaling inhibitor Smad7 prevents bleomycin-induced lung fibrosis in mice (16). These studies definitively establish TGF-beta as a primary mediator in pathological fibrosis, although in transgenic models of TGF-beta overexpression fibrosis occurs in some but not all tissues, and the significance of local (autocrine and paracrine) versus circulating TGF-beta remains unknown.


    THE EFFECTS OF TGF-beta ON THE EXTRACELLULAR MATRIX
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TGF-beta alters the normal balance between ECM synthesis and degradation, inducing an increase in synthesis of matrix components and a parallel decrease in overall ECM proteolytic activity. This results in net fibrogenesis, although it is important to note that the composition as well as quantity of ECM change in response to TGF-beta . Nowhere is this more evident than in the liver, where the increase in fibrillar collagens and electron-dense matrix obliterates the sinusoidal fenestrae, resulting in clinical liver disease. It is also important to note that, although the ECM is generally composed of proteins, proteoglycans, and glycans, its specific composition varies among cell and tissue types, reflecting their specialized functions; the effects of TGF-beta on a given ECM may result from cell-specific signaling pathways. Likewise, the term fibrogenesis is an oversimplification and encompasses a variety of changes that potentially involve multiple signaling pathways, direct and indirect, including the upregulation of other profibrogenic cytokines.

In increasing ECM synthesis, TGF-beta upregulates the fibrillar and, to a lesser extent, nonfibrillar collagens, other matrix components including fibronectin and tenascin, the basement membrane components laminin and entactin, and membrane proteoglycans including perlecan and biglycan (2). In the liver, TGF-beta induces the production of the EIIIA splice variant of fibronectin by sinusoidal endothelial cells, contributing to the activation of fibrogenic hepatic stellate cells (10). TGF-beta also alters the number and variety of integrins expressed on some cells, potentially enhancing their adhesion to the ECM (7).

TGF-beta regulates the expression of many proteins responsible for matrix degradation. Although the effect of TGF-beta on matrix metalloproteinases (MMPs) is mixed, it clearly upregulates protease inhibitors, most notably the tissue inhibitors of metalloproteinases (TIMPs). Additionally, TGF-beta induces expression of plasminogen activator inhibitor (PAI)-1. This results in decreased conversion of plasminogen to plasmin, a protease that directly degrades matrix components and activates MMPs.

One final effect of TGF-beta that warrants mention is its ability to increase its own expression and generate potent autocrine loops. The combination of continued TGF-beta overproduction and highly proliferative cells is characteristic of some fibrotic conditions and may be essential to the development of chronic, progressive fibrosis.


    TGF-beta SIGNALING PATHWAYS
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In the decade since the cloning of the first TGF-beta receptors, a simple and general signaling pathway from ligand to transcription has become clear (Fig. 1). Latent TGF-beta undergoes activation and in turn activates its receptors, the activated receptors phosphorylate and assemble cytoplasmic Smad proteins, and Smad complexes move to the nucleus as transcriptional regulators (18).


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Fig. 1.   General transforming growth factor (TGF)-beta signaling pathway. After TGF-beta undergoes activation, it binds to the type II receptor (II). In some cells, and especially for TGF-beta 2, this interaction is enhanced by the presence of the type III receptor, betaglycan (III). Binding of TGF-beta results in the formation or stabilization of a complex of the type I and II receptors (II/I), and the type II kinase phosphorylates (P) and activates the type I receptor (I). The activated type I receptor kinase phosphorylates receptor-specific Smads, which, for the TGF-beta pathway, include Smad2 and Smad3. This step can be inhibited by Smad7. Phospho-Smad2 and 3 form complexes with the co-Smad (Smad4) and move into the nucleus, where they may interact with other transcription factors or coactivators and corepressors to regulate transcription. The Smads and various nuclear factors may or may not bind directly to DNA. The stoichiometry of the Smad and transcription factor complexes is not known.

TGF-beta is synthesized as a prohormone. Although it undergoes cleavage in the Golgi into an amino terminal latency-associated protein and a mature TGF-beta fragment, the two remain associated, bind to various latent TGF-beta binding proteins, and are secreted as a latent complex. Latent TGF-beta can be activated through multiple mechanisms potentially involving integrin-alpha vbeta 6, mannose-6-phosphate receptors, plasmin, MMPs 2 and 9, and thrombospondin-1 (18).

Once activated, TGF-beta signals through a complex of two related but structurally and functionally distinct serine-threonine kinase receptors, called type I (Tbeta RI) and type II (Tbeta RII). Binding of the homodimeric TGF-beta to Tbeta RII enables the formation and stabilization of type I/type II receptor complexes, most likely heterotetramers. The Tbeta RII kinase then phosphorylates Tbeta RI in a glycine- and serine-rich juxtamembrane region called the GS box. This is the critical event in TGF-beta signaling and serves as the initiation point for downstream events.

A third TGF-beta receptor, betaglycan or Tbeta RIII, is a proteoglycan coreceptor that binds TGF-beta with high affinity through its core. In many cells, it enhances signaling by increasing the affinity of TGF-beta , especially TGF-beta 2, for the kinase receptors. We have recently determined, however, that in LLC-PK1 cells, betaglycan competes with Tbeta RI for Tbeta RII binding, inhibiting signaling (O. Eickelberg, M. Centrella, M. Kashgarian, and R. G. Wells, unpublished observations). Betaglycan is lost in one cell type, hepatic stellate cells, when they adopt a fibrogenic, TGF-beta -responsive phenotype, although the significance of this to fibrogenic signaling is not known (31). In addition to betaglycan, several other receptor-associated proteins with unclear functions have recently been described (18).

The family of Smad proteins, with three distinct subfamilies, comprises the cytoplasmic TGF-beta signaling machinery. The receptor-activated Smads for TGF-beta , Smad2 and Smad3, are phosphorylated by Tbeta RI on carboxy-terminal SSXS sequences. These phosphorylated Smads then form heteromeric complexes with the co-Smad, Smad4, and move into the nucleus. The inhibitory Smads, represented in TGF-beta signaling by Smad7, lack the carboxy-terminal phosphorylation motif of Smad2 and Smad3 but interact with Tbeta RI and the receptor-activated Smads, inhibiting the pathway. Of note, Smad7 expression is induced by TGF-beta , enabling rapid downregulation of the TGF-beta response (15).

Smad complexes, once translocated to the nucleus, can regulate transcription in several ways. Smad3 and Smad4, but notably not Smad2, bind directly to DNA via a GC-rich consensus sequence called a Smad binding element. Several TGF-beta -induced matrix proteins, including collagen VII and PAI-1, as well as the inhibitory Smad7, have copies of this consensus sequence in their promoter regions. Smads (in particular Smad2, which cannot itself bind to DNA) also cooperate with other transcription factors, including activator protein (AP-1), simian virus 40 promoter factor 1 (Sp1), and transcription factor muE3 (TFE3). TFE3 and a Smad3/4 complex, for example, do not physically interact but bind to adjacent sites in the PAI-1 gene promoter, enabling its upregulation in response to TGF-beta (9). Activated Smads in the nucleus can also interact with transcriptional coactivators, including CREB binding protein (CBP) and p300, or corepressors, including TG-interacting factor, c-Ski, and SnoN, for both activation and inhibition of transcription (18).


    SPECIFICITY IN SIGNALING: MATRIX-SPECIFIC PATHWAYS?
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Why do different cells respond to TGF-beta in different ways? Ultimately, the answer is context: the makeup of the surrounding environment and the specific intracellular signaling apparatus. Within this framework, however, the details are poorly understood. There has been no systematic study of TGF-beta signaling in fibrogenesis, and, although TGF-beta induction of PAI-1 expression is well described, signaling pathways for other matrix proteins are still incomplete, particularly in pathological fibrosis. Recent studies addressing TGF-beta pathways to fibrogenesis are highlighted below, as are points in the TGF-beta pathway with the potential to generate response specificity.

TGF-beta levels, isoforms, and activation. The three major TGF-beta isoforms, beta 1, beta 2, and beta 3, have similar biological effects in vitro. They have highly different expression patterns, however, and the phenotypes of their knockout mice are distinct. TGF-beta 1-null mice either die in utero because of defects in vasculogenesis and hematopoiesis or die shortly after birth of overwhelming inflammatory infiltration of multiple organs; TGF-beta 2- and -beta 3-null mice have multiple, although different and nonoverlapping, developmental abnormalities (18). The genes for the three isoforms have differentially regulated promoters, suggesting that expression in time and space rather than biological activity may be responsible for the observed effects of individual isoforms. Additionally, TGF-beta 2 requires betaglycan for high-affinity binding to the receptors, so betaglycan expression is an important determinant of TGF-beta 2 activity. It is notable, however, that TGF-beta 1 is often the major isoform in fibrosis, and it is possible that it has biological effects in vivo that differ from beta 2 and beta 3. The contribution of inflammation to fibrosis is not well understood and is debated; it may be that the dominant role of TGF-beta in inflammation is important to its role in fibrosis.

Activation of latent TGF-beta and the establishment of autocrine loops may be critical in determining whether fibrosis does or does not occur. For example, as mentioned in TGF-beta SIGNALING PATHWAYS, the integrin-alpha vbeta 6 activates TGF-beta in the lung, and its absence is protective in experimental fibrosis (14). The potential upregulation of this integrin in the lung by TGF-beta has not been studied. Although alpha vbeta 6 is not present in the liver, indicating that it cannot regulate fibrosis in all settings, the expression of other cell- and tissue-specific mechanisms of latent TGF-beta activation, and their potential upregulation by TGF-beta itself, may contribute to fibrosis. Additionally, differences in TGF-beta signaling pathways after long- and short-term TGF-beta exposure have not been explored, but lessons from the study of platelet-derived growth factor suggest that there may be a biphasic response with distinct effects at different time points.

TGF-beta receptors. Although fibrosis is associated with increased levels of active TGF-beta , fibrogenic cells are also primed to respond differently to TGF-beta from nonfibrogenic cells. Differences in cellular signaling could occur at several points, beginning with Tbeta RI and Tbeta RII. Mutations in a region of Tbeta RI between the transmembrane domain and the GS box eliminate some, but not all, TGF-beta responses: Ser172 and Thr176 are required for TGF-beta -mediated growth inhibition but not PAI-1 and fibronectin upregulation (20). These residues are not known to be phosphorylated, and the mechanisms by which they modulate TGF-beta signaling are unclear. Phosphorylation of Ser165, which is also proximal to the GS box, has different effects on growth inhibition and matrix induction compared with apoptosis (24), but again the mechanism is unknown. In the type II receptor, Thr315 (which is not autophosphorylated) is required for TGF-beta -mediated growth inhibition but not matrix induction (12). Differential autophosphorylation of the kinase region of Tbeta RII chimeras modulates TGF-beta -mediated growth inhibition (13); it would be intriguing to test these phosphorylation sites for their effects on matrix responses.

Alterations in the Tbeta RI/Tbeta RII ratio may also determine response specificity. There are multiple examples whereby marked shifts in this ratio correlate with phenotypic changes, including the change to a proliferative, profibrogenic state. According to some reports (and my laboratory's unpublished data), Tbeta RII decreases significantly in hepatic stellate cells undergoing activation and becoming fibrogenic, whereas Tbeta RI increases or remains unchanged (19). Tbeta RII in many cell types has a shorter half-life than Tbeta RI and undergoes dramatic ligand-mediated shifts in cell surface expression, suggesting that it is a key regulator of the TGF-beta response (unpublished data). The role of Tbeta RII was tested directly a number of years ago by stably transfecting mink lung epithelial cells with dominant-negative Tbeta RII. The transfected cells demonstrated a dramatic insensitivity to TGF-beta -mediated growth inhibition yet retained normal induction of PAI-1 and fibronectin synthesis (3). The authors of this early report contended that the Tbeta RI and Tbeta RII independently mediate different responses. Although multiple studies have since suggested that this is not the case, the functional consequences (and mechanisms) of variable receptor ratios are still not understood. One possibility is that growth inhibition requires a higher threshold level of signaling than matrix induction and that cells with low levels of Tbeta RII fail to achieve adequate activation for the antiproliferative effects of TGF-beta (33). In this context, it will be interesting to measure Smad phosphorylation in cells with different receptor ratios. Alternative and more speculative explanations include that the type I/type II complex has a variable stoichiometry, that Tbeta RI phosphorylation varies according to Tbeta RII levels, and that there is competition for Tbeta RI binding between Tbeta RII and an as yet unknown binding partner.

Smads. The Smads are likely to play a central role in the generation of response specificity. As noted above, it has been hypothesized that the degree of activation of the cytoplasmic signaling machinery determines the nature of the effects of TGF-beta ; studies in Xenopus demonstrate that the transcriptional response to the TGF-beta superfamily member activin varies with the levels of activated Smads (33). Differences between Smad2 and Smad3 function are also likely to be important. Although the two are grouped together as TGF-beta -specific receptor-activated Smads, Smad2 is unable to bind DNA directly, and Smad2 and Smad3 are antagonistic in some systems, suggesting that their relative levels might determine the biological response (11). In support of this, there is now a report that Smad2 and Smad3 are expressed in distinct patterns in maturing chondrocytes according to their state of differentiation (21). Additionally, we and others have noted that Smad2 activation is decreased compared with Smad3 activation as hepatic stellate cells in culture become fibrogenic (Ref. 5 and unpublished data).

The most definitive evidence that Smad2 and Smad3 mediate different cellular responses comes from the demonstration that a Smad2 knockout is embryonic lethal, whereas Smad3 knockout mice survive to adulthood. The Smad3-null mice, surprisingly, demonstrate enhanced wound healing (1). Fibroblasts from these mice still produce matrix material in response to TGF-beta , leading to the suggestion that Smad2 is the specific mediator of many ECM responses. These animals, and other viable Smad knockout mice, will be useful models for studying the role of Smads in experimental fibrosis.

Another recent report raises the possibility that Smad4 can determine response specificity. Although the generic signaling pathway described in TGF-beta SIGNALING PATHWAYS awards Smad4 a central role, studies in a human fibrosarcoma cell line show that, although Smad4 is required for TGF-beta -mediated PAI-1 induction, it is not required for fibronectin upregulation (8). Instead, fibronectin induction in these cells requires the mitogen-activated protein kinase (MAPK) c-Jun-activated amino terminal kinase (JNK). The role of Smad2 and Smad3 has not been examined in these cells, nor have other cells been examined for Smad4-independent fibronectin upregulation, but the report raises the interesting possibility that there is more variety in TGF-beta signaling pathways than initially supposed. Although it has never been shown that the Smads have non-Smad binding partners after activation, this would clearly be one mechanism for generating different responses.

Smad7 is an inhibitory Smad that may regulate fibrogenesis. Smad7, which binds to the phosphorylated Tbeta RI and prevents the activation of Smad2 and Smad3, is rapidly upregulated in response to TGF-beta , likely via a Smad binding site in its promoter (15). This regulation may be directly relevant to fibrogenesis: it has been reported that hepatic stellate cells lose this response as they activate and become fibrogenic (5). In addition to its regulation by TGF-beta , Smad7 is upregulated by other growth factors, including interferon-gamma and tumor necrosis factor-alpha , suggesting that signaling cross-talk can modulate TGF-beta response specificity through Smad7 (29).

Transcription factors and activators. In the end, signaling specificity comes down to distinct nuclear events. The multiple known and potential interactions between Smads, other transcription factors, and transcriptional coactivators and corepressors provide multiple possible branch points in TGF-beta signaling, and there are already several examples of matrix-specific responses generated by the expression of specific nuclear factors. At an AP-1 site in the human collagenase I (MMP-1) promoter, there is cooperation between c-Jun/c-Fos and Smad3 and Smad4 in inducing TGF-beta -mediated transcription (34). In the PAI-1 gene, Smad3/4 complexes cooperate with the transcription factor TFE3 to regulate transcription (9). Novel regulatory elements may also be important in fibrogenesis; induction of a 30-kDa nuclear protein and its binding to a newly described promoter element during hepatic stellate cell activation is important in TIMP-1 upregulation (26). There are several known corepressors (TGIF, c-Ski, and SnoN) that recruit histone deacetylase; interestingly, histone deacetylase inhibitors blunt matrix production by hepatic stellate cells (17), raising the possibility that the expression of corepressors modulates fibrogenesis.

Cross-talk with other pathways. There are now many examples of interactions between MAPK pathways and TGF-beta signaling, raising the possibility that cross-talk between mitogenic and TGF-beta signaling pathways leads to a coordinate response. As described above, TGF-beta -mediated fibronection but not PAI-1 induction in one cell line is dependent on JNK (8); studies on collagenase I upregulation demonstrate that Smad and JNK signaling pathways converge at AP-1 sites (34). MAPKs, in response to such growth factors as hepatocyte growth factor (HGF) and epidermal growth factor (EGF), phosphorylate internal sites on Smad2 and induce its nuclear translocation (4), although the range of functional effects of this cross-talk is not known. It may be relevant, however, that HGF overexpression led to a reduction in experimental fibrosis in a rat model (27).

Connective tissue growth factor is a mitogenic peptide induced by TGF-beta that stimulates the synthesis of collagen I and fibronectin and may mediate some of the downstream effects of TGF-beta . It is upregulated during activation of hepatic stellate cells, suggesting that its expression is another determinant of a fibrogenic response to TGF-beta (32).

Cross-talk between TGF-beta and integrin signaling pathways is also potentially important, although relatively unexplored. In an osteoblast model, the interaction between integrin-alpha 2beta 1 and collagen resulted in TGF-beta receptor downregulation through a focal adhesion kinase-dependent mechanism, and the promatrix effects of TGF-beta were inhibited by alpha 2beta 1-blocking antibodies (25). In liver as in other tissues, the nature of the surrounding matrix determines whether cells are fibrogenic, so interactions between integrin and TGF-beta signaling mediators could be key determinants of the fibrogenic response.


    SIGNALING SPECIFICITY: THERAPEUTICS AND PERSPECTIVES
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The recent description of general TGF-beta signaling pathways provides an exciting point from which to explore TGF-beta signaling in fibrogenesis. In the future, it will be important to examine proteoglycan and TIMP upregulation by TGF-beta and to determine whether there are broad pathways that regulate the matrix effects of TGF-beta . The multifunctional nature of TGF-beta implies that the design of antifibrosis therapies will require a detailed understanding of specific signaling pathways so that the beneficial effects of TGF-beta , including antimitogenesis, are maintained during long-term treatment.


    ACKNOWLEDGEMENTS

I am grateful to Dr. O. Eickelberg for comments on the manuscript.


    FOOTNOTES

I receive support from the Yale Liver Center (DK-34989) and the National Institute of Diabetes and Digestive and Kidney Diseases (DK-02290 and DK-56016).

Address for reprint requests and other correspondence: R. G. Wells, Depts. of Internal Medicine and Pathology, Yale School of Medicine, P.O. Box 208019, New Haven, CT 06520-8019 (E-mail: rebecca.wells{at}yale.edu).


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Am J Physiol Gastrointest Liver Physiol 279(5):G845-G850
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