Systemic sclerosis (SSc) is a chronic disease of unknown etiology characterized by autoimmunity, vascular damage, and progressive fibrosis of the skin and internal organs. The pathogenesis of fibrosis is not well understood, and there are no effective treatments. Fibroblasts from lesional tissues show evidence of activation, with increased synthesis of collagen, fibronectin, tissue inhibitor of metalloproteinases 1 (TIMP-1), and plasminogen activator inhibitor 1 (PAI-1); secretion of profibrotic cytokines such as transforming growth factor β (TGFβ), interleukin-4 (IL-4), IL-13, and connective tissue growth factor (CTGF); myofibroblast differentiation, with elevated levels of α-smooth muscle actin (α-SMA) and stress fiber formation; enhanced expression of cell surface receptors for TGFβ; and resistance to apoptosis (1). Although the nature of the extracellular signals that initially trigger and subsequently sustain and amplify fibroblast activation and the corresponding intracellular signal transduction pathways are still a subject of controversy, TGFβ is considered to play a fundamental role (2).
TGFβ is the prototype of a superfamily of multifunctional cytokines that includes activins and bone morphogenetic proteins (3). These cytokines regulate cell differentiation, proliferation, function, and survival, and in mesenchymal cells, they stimulate extracellular matrix synthesis and organization. In a variety of in vitro and in vivo experimental models, TGFβ induces the phenotypical features of activation in fibroblasts. Accordingly, TGFβ represents an important potential therapeutic target for preventing or arresting the fibrotic process in SSc.
The mechanisms underlying cellular responses to TGFβ are now understood in some detail. Members of the TGFβ superfamily signal through the sequential activation of 2 distinct serine/threonine kinase cell surface receptors that are expressed on virtually all cell types. Upon ligand binding, a type II receptor (TβRII) recruits and phosphorylates a type I receptor (TβRI), which is also known as activin receptor–like kinase (ALK). Of the 7 known type I (ALK) receptors, ALK-5 is most specific for TGFβ, whereas the closely related ALK-4 and ALK-7 interact with other members of the TGFβ superfamily (4). In addition, ALK-1 was recently shown to function as a type I TGFβ receptor, but it is expressed primarily on endothelial cells or at sites of epithelial–mesenchymal interactions (5).
The Smads have been identified as major signaling molecules downstream of activated TβRI. These highly conserved modular proteins function as signal transducers/transcriptional activators that shuttle between the cytoplasm and the nucleus. In response to TGFβ, ALK-5 phosphorylates Smad2 and Smad3 on serine residues, whereas ALK-1 activates Smad1 and Smad5. Activin signals are also transduced by Smad2 and Smad3, but via the ALK-4 and ALK-7 receptors, whereas Smads 1, 5, and 8 are substrates for the bone morphogenetic protein–activated ALKs. In contrast to these receptor-activated Smads (R-Smads) that are phosphorylated by type I TGFβ receptors, Smad4 serves as a Smad cofactor, and Smad7 functions as an inhibitor of TGFβ-Smad signaling. Upon activation, R-Smads interact with Smad4, and the heteromeric complex is then imported into the nucleus. Within the nucleus, the DNA-bound Smad complex regulates the transcription of target genes directly or in association with transcription factors such as Sp-1 and forkhead activin signal transducer 1 (FAST-1), coactivators such as p300/CREB binding protein, or corepressors such as Ski and SnoN. Inhibitory Smad7 interacts with activated TβRI in competition with R-Smads and enhances receptor ubiquitination and proteasomal degradation in caveolae.
In light of the diversity of cellular responses elicited by TGFβ, it is not surprising that in addition to the canonical Smad pathway, TGFβ also activates alternate signal transduction pathways in a cell type– and context-specific manner. Non-Smad signaling pathways induced by TGFβ in cell lines include protein kinase A, protein kinase C, calmodulin-dependent protein kinase II, the MAP kinases ERK, JNK, and p38, and phosphatidylinositol 3 kinase (PI 3-kinase) (for review, see ref.6). By regulating Smad activation or through intracellular cross-talk with the Smad pathway, these kinases can influence the amplitude and duration of Smad-dependent signaling and may directly induce Smad-independent TGFβ responses. The mechanisms linking non-Smad signaling pathways with activated TGFβ receptors are unknown. Furthermore, because most studies examining the cross-talk between ligand-induced Smad and non-Smad signaling pathways have been performed in transformed or immortalized cell lines, the biologic consequences for non-Smad signaling in normal fibroblasts and their relevance in the physiologic context remain incompletely understood.
Small-molecule inhibitors of individual protein kinases are useful not only for dissecting the mechanisms of signal transduction for specific ligands and for delineating their distinct roles in biologic responses, but they have potential as therapeutic agents as well. A group of competitive ATP binding site inhibitors of ALK-5 has recently been described (7). In immortalized cell lines, the ALK-5 inhibitor SB431542 prevented Smad-dependent transcriptional responses and TGFβ-induced R-Smad phosphorylation in vitro; inhibition of ALK-4 and ALK-7 was also noted (8). To date, however, SB431542 has been studied primarily in the context of in vitro phosphorylation assays with immobilized targets or in transformed epithelial cell lines transfected with constitutively active TβRI kinases and substrates. These artificial experimental models fail to reflect either signal strength and duration or intracellular modulation. The results, therefore, cannot accurately predict the effects on nontransformed primary fibroblasts within the physiologic context of TGFβ signaling.
In the present studies, we examined the effects of SB431542 on newborn foreskin fibroblasts and adult dermal fibroblasts activated by TGFβ in vitro, focusing on cellular responses implicated in the development of fibrosis. The results showed that SB431542 could prevent, as well as reverse, TGFβ-induced stimulation of extracellular matrix synthesis and messenger RNA (mRNA) expression, TGFβ autoinduction, and Smad3- and Smad2-dependent transcriptional responses in vitro, and could prevent fibroblast transdifferentiation into myofibroblasts. Inhibition of profibrotic responses was associated with marked selective suppression of TGFβ-induced phosphorylation and nuclear translocation of endogenous R-Smads. Together, these results indicate that signaling via ALK-5 and downstream Smads plays a fundamental role in mediating TGFβ profibrotic responses in normal fibroblasts. Furthermore, these observations suggest that pharmacologic inhibition of the ALK-5 receptor may represent a novel targeted approach to the control of fibrosis in SSc.
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By inducing fibroblast activation through paracrine mechanisms and by sustaining and amplifying this process via autocrine stimulation, TGFβ plays a fundamental role in normal wound repair and in pathologic fibrosis. Therefore, the expression, activity, and intracellular signaling of TGFβ must be held under tight regulation. The precise molecular mechanisms controlling the responses of the target cell to TGFβ in physiologic tissue remodeling and their deregulation in the pathogenesis of fibrosis, however, remain incompletely understood. In most cells, TGFβ signals are transduced to their transcriptional targets through a dual mechanism involving Smad family proteins and non-Smad pathways. A variety of approaches to the delineation of Smad-dependent versus Smad-independent cellular and transcriptional responses to TGFβ have yielded confusing, and sometimes even contradictory, results. For example, TGFβ stimulation of fibronectin and α-SMA expression has been shown to be Smad-dependent or Smad-independent in different studies and different cell types (21–24). However, global analysis of gene expression has revealed that >95% of transcriptional responses induced by TGFβ in mouse embryonic fibroblasts were Smad3-dependent (25). Furthermore, by using a mutant ALK-5 receptor that retains its kinase activity but is unable to activate downstream Smad signaling, a great majority of TGFβ-induced cellular responses have been shown to be dependent on Smad activation in a variety of cell lines (26).
The present studies revealed that the novel small-molecule ALK-5 inhibitor SB431542 caused a dramatic suppression of TGFβ-induced R-Smad phosphorylation and nuclear accumulation of the R-Smad/Smad4 complex in normal dermal fibroblasts. Blockade of ligand-induced Smad activation was accompanied by inhibition of Smad2- and Smad3-dependent transcriptional responses in transiently transfected dermal fibroblasts. Inhibition by SB431542 appeared to be selective for the Smad pathway, because under the same experimental conditions, TGFβ induction of ERK-1/2 phosphorylation and ERK kinase activity were unaffected by SB431542, as was the more modest stimulation of JNK and p38 phosphorylation. Previous studies have also indicated that ERK-1/2 is activated by TGFβ in mesenchymal cell types (27), but the precise mechanisms underlying this response remain unknown. The results of the present study indicate that TGFβ-induced rapid activation of ERK-1/2 in dermal fibroblasts was Smad-independent. Pretreatment with SB431542 abrogated TGFβ-induced stimulation of extracellular matrix gene products that play important roles in the development of fibrosis. These include type I collagen (the principal structural component of connective tissue in the skin), fibronectin, and TIMP-1 and PAI-1 (which prevent the activities of proteolytic enzymes and thereby contribute to connective tissue accumulation in fibrosis). Significantly, SB431542 not only prevented TGFβ-induced stimulation, but it was also effective in reversing these responses when added to cultures following TGFβ.
Although it has previously been reported that stimulation of fibronectin synthesis by TGFβ is independent of Smads (21), the present results indicate that in dermal fibroblasts, the fibronectin response was abrogated by SB431542, suggesting a Smad-dependent mechanism, which is also consistent with previous reports (28). Northern blot analysis and in situ hybridizations indicated that SB431542 itself had no effect on basal Smad7 mRNA expression levels, but prevented their rapid induction by TGFβ, consistent with the Smad3/4 dependence of this response (29). Therefore, the inhibitory activities of SB431542 on basal and TGFβ-induced extracellular matrix gene expression could not be attributed to the induction of an endogenous inhibitor of TGFβ signaling. Of interest was the finding that by itself, SB431542 reduced the cellular levels of extracellular matrix proteins, suggesting that their basal expression levels in fibroblasts were regulated, at least in part, by endogenous TGFβ through autocrine stimulatory loops involving ALK-5 and Smad activation. Furthermore, the results also demonstrated that ALK-5 blockade reduced the secretion of endogenous TGFβ1 in TGFβ2-stimulated fibroblasts and prevented the increase in TGFβ and CTGF mRNA expression. Autoinduction of TGFβ, a phenomenon important in amplifying the magnitude and duration of cellular responses to TGFβ, has been shown in previous studies with Smad3-null fibroblasts to be Smad3-dependent (24, 30). By inhibiting TGFβ autoinduction, SB431542 could potentially exert a powerful effect, arresting the progression of fibrotic responses.
Importantly, SB431542 prevented TGFβ-induced mRNA expression of α-SMA, its intracellular accumulation, and its incorporation into cytoskeletal stress fibers, the hallmarks of myofibroblast transdifferentiation. Myofibroblasts are terminally differentiated cells with morphologic and functional characteristics that are between those of fibroblasts and those of smooth muscle cells (18). In wound healing, myofibroblasts are thought to arise locally from quiescent fibroblasts under the influence of TGFβ released during tissue injury. In vitro, TGFβ is a potent stimulus of α-SMA expression and organization of filamentous actin into stress fibers, resulting in the transdifferentiation of various types of mesenchymal cells into myofibroblasts. The present findings, together with a recent report demonstrating the requirement for an intact L45 loop of the TβRI (24), suggest that TGFβ-induced fibroblast transdifferentiation into myofibroblasts is an ALK-5/Smad-dependent process. Furthermore, the fact that U0126 partially prevented myofibroblast differentiation induced by TGFβ suggests a contribution of MAP kinase pathways to this response as well. Because myofibroblasts play critical roles in the pathogenesis of tissue fibrosis, these inhibitory effects of SB431542 may be particularly significant in ameliorating the fibrotic process.
Previous studies have indicated that while TGFβ was pivotal in the regulation of myofibroblast differentiation, overexpression of Smad2, but not Smad3, induced myofibroblast features in the absence of TGFβ in lung fibroblasts (19). Although Smad2 and Smad3 are highly homologous, these 2 R-Smads show distinct biologic activities. In particular, Smad2 is considered to play pivotal roles during embryogenesis, whereas Smad3 is critical for cellular responses after birth. In our own previous studies, we have shown that dermal fibroblasts derived from Smad3-null mice retained their ability to induce α-SMA expression and to organize filamentous actin into stress fibers in response to TGFβ, consistent with involvement of Smad2 (31). In contrast to these results, Smad3 was shown to be both necessary and sufficient for myofibroblast transdifferentiation in lung fibroblasts (23). The reasons for these apparently discrepant findings relating to the roles of Smad2 and Smad3 in TGFβ regulation of myofibroblast differentiation in different cell types remain unknown.
Taken together, the present results indicate that the novel small-molecule ALK-5 inhibitor SB431542 is a potent and selective antagonist of TGFβ-induced R-Smad activation in normal dermal fibroblasts. Furthermore, inhibition of ALK-5–mediated Smad signaling was associated with a striking reduction of multiple TGFβ-induced cellular responses involved in fibrogenesis in the absence of detectable cytotoxicity. These results establish a compelling direct link between ligand-induced R-Smad phosphorylation and nuclear accumulation and the transcriptional activation of important target genes. Furthermore, the results indicate that in primary dermal fibroblasts, TGFβ stimulation of extracellular matrix gene expression and myofibroblast transdifferentiation are largely ALK-5/Smad-dependent processes, which is consistent with our own previous findings (32) as well as those of other investigators utilizing mutant ALK-5 receptors or Smad-null embryonic fibroblasts (26). Pharmacologic inhibition of ALK-5 and downstream signaling events may provide a novel approach to the precise delineation of the mechanisms of individual cellular responses elicited by TGFβ.
Selective inhibition of ALK-5 signaling using SB431542 may have important implications for the therapeutic control of pathologic fibrotic responses. In light of the widely recognized pivotal role of Smads and TGFβ in the pathogenesis of fibrosis (33), therapeutic strategies targeting TGFβ are currently under active study. Traditional approaches focus on prereceptor targeting of TGFβ by the use of neutralizing antibodies, naturally occurring inhibitors, or soluble TGFβ receptors in order to reduce the local expression or biologic activity of TGFβ. However, in fibrotic conditions such as SSc, lesional fibroblasts display intrinsic abnormalities in Smad signaling, such as loss of endogenous repressors, that could potentially result in their abnormal responsiveness to TGFβ (for review, see ref.34). Some of the autonomous alterations in the TGFβ pathway described to date in SSc fibroblasts include reduced expression of the inhibitory Smad7 (35), defective activation-induced degradation of cell surface receptors for TGFβ (36), and constitutive nuclear accumulation of activated R-Smads (10). Additional abnormalities of SSc fibroblasts, such as defective regulation of the Smad-associated transcriptional corepressors Ski and SnoN, are currently under investigation.
Together, these intrinsic abnormalities in the regulation of TGFβ/Smad signaling would have the net effect of reducing the target cell's threshold for exogenous TGFβ, markedly enhancing its sensitivity to stimulation. In such a situation, even very small amounts of active TGFβ would be sufficient to elicit full-scale biologic responses. Current therapeutic strategies targeting TGFβ at the prereceptor level are unlikely to be able to reduce TGFβ signaling below the subthreshold levels required for activating hypersensitive SSc fibroblasts. Therefore, strategies for inhibiting TGFβ responses by blocking intracellular signaling via the TGFβ/ALK-5/Smad axis may be more effective in preventing the activation of fibroblasts that are hypersensitive to TGFβ. Whether ALK-5 inhibitors can prevent the constitutive activation of Smad signaling observed in SSc fibroblasts (10) is currently under investigation.
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We are grateful to Drs. N. Laping (GlaxoSmithKline, King of Prussia, PA), R. Derynck (University of California, San Francisco), P. ten Dijke (Ludwig Institute for Cancer Research, Uppsala, Sweden), J. Massague (Howard Hughes Medical Institute, New York, NY), B. Vogelstein (Johns Hopkins University, Baltimore, MD), and K. Miyazono (Cancer Institute, Tokyo, Japan) for providing us with plasmids and reagents. Members of the laboratory staff provided helpful discussions.