Fibrosis, affecting skin, lungs, and other organs, is the most prominent feature of systemic sclerosis (SSc). While many factors are known to contribute to fibrosis, activation of the transforming growth factor β (TGFβ) signaling pathway is considered to play a central role in this process (1). TGFβ is a pleiotropic cytokine with diverse functions in many cell types (2). The Smad pathway is a primary mediator of TGFβ signaling (3). The canonical Smad pathway is activated upon binding of TGFβ to the heteromeric serine/threonine kinase receptors TGFβ receptor type II (TGFβRII) and TGFβRI (activin receptor–like kinase 5 [ALK-5]), which leads to phosphorylation of Smads 2 and 3, oligomerization with Smad4, nuclear translocation, and activation of various transcription programs (2). There is also increasing evidence for the activation of other (noncanonical) signaling pathways downstream of TGFβ, but the specific mechanisms involved in activation of Smad-independent pathways have not been fully elucidated (4). With respect to fibrosis, the nonreceptor tyrosine kinase c-Abl is of special interest, because of the potent antifibrotic effects of the pharmacologic inhibitor of c-Abl, imatinib mesylate. Imatinib mesylate has been shown to inhibit profibrotic effects of TGFβ in cultured cells, including SSc fibroblasts (5), and to prevent lung and kidney fibrosis in experimental mouse models (6, 7). TGFβ activation of c-Abl occurs through phosphatidylinositol 3-kinase and p21-activated kinase 2 and is independent of the Smad2/3 pathway (8).
The role of TGFβ signaling in the process of fibrosis in SSc has been mainly studied using fibroblasts explanted from SSc skin. During early passages in culture, these cells demonstrate an “activated phenotype” characterized by elevated synthesis of extracellular matrix (ECM) proteins (9). Accumulating evidence suggests that alterations of TGFβ signaling may play an essential role in the persistent activation of SSc fibroblasts. The documented changes include up-regulation of αvβ5 and αvβ3 integrins, which were shown to contribute to activation of latent TGFβ and establishment of the autocrine TGFβ loop (10, 11). Additional changes include alterations of the TGFβ receptor ratio (12) and the presence of persistently phosphorylated Smad3 (13). Recent studies from our laboratory using an in vitro model of SSc based on the altered ratio of TGFβ receptors have also established that activation of the Smad1 pathway may represent a novel aspect of profibrotic TGFβ signaling that functions independently of activation of the canonical Smad2/3 pathway (14). Activation of Smad1 downstream of TGFβ was first described in endothelial cells, where TGFβ signals through 2 distinct type I receptors, ALK-5 and ALK-1, and their respective signal transducers (Smads 2 and 3 for ALK-5 and Smads 1 and 5 for ALK-1) (15). Based on our recent study, it appears that this mode of signaling is also operational in fibroblasts, but the contribution of Smad1 signaling to SSc fibrosis has not been examined.
The present study was undertaken to further evaluate the role of Smad1 signaling in SSc fibrosis. Given that the inhibition of the TGFβ-mediated fibrotic response by imatinib mesylate is independent of Smads 2 and 3, we also sought to determine whether Smad1 is a downstream target of imatinib mesylate. Our results show that Smad1 signaling is activated in a subset of SSc fibroblasts. We also demonstrate that imatinib mesylate blocks TGFβ-induced activation of Smad1 and ERK-1/2 pathways in control fibroblasts and induces phosphorylation of Smad1 and ERK-1/2 in SSc fibroblasts. The present study provides novel insights into the mechanism of fibrosis in SSc.
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TGFβ is considered a central mediator of the excessive collagen deposition in SSc, but the molecular mechanisms underlying the chronic fibrotic response are not fully elucidated. This study provides further support for the contribution of a novel fibrogenic pathway mediated by the activation of the ERK-1/2–Smad1–CCN2 cascade to SSc fibrosis. Analyses of SSc skin samples revealed elevated expression of total and phospho-Smad1 protein in a subset of patients with active disease. Fibroblasts cultured from SSc biopsy samples demonstrated elevated levels of Smad1 protein, and in 6 of 7 strains tested also showed the presence of phosphorylated Smad1, indicating that activation of the Smad1 pathway persists in cultured cells. Since several previous studies have shown elevated levels of phospho-Smad3 in SSc fibroblasts (13, 24), these data suggest that distinct Smad pathways may play complementary roles in the activation of cultured SSc fibroblasts. Surprisingly, however, comparison of SSc and healthy skin tissues showed similar levels of phosphorylated Smad3 (24). The reason for the different expression of phosphorylated Smad3 in cells in vivo and in cultured cells remains unknown.
In vitro and in vivo DNA binding assays have demonstrated that Smad1 binds to the GC-rich motif in the proximal CCN2 promoter region. Although this site was previously characterized as an Sp-1 binding site (21), our data strongly suggest that this GC-rich motif represents a Smad1 binding site. This finding is also consistent with the previous studies that showed binding of Smad1 to GC-rich motifs in several other promoters (25). Our recent studies have shown that siRNA-mediated inhibition of Smad1 abrogated TGFβ-induced up-regulation of CCN2 messenger RNA and protein levels as well as AdTGFβRI-induced CCN2 promoter activity (14). In the present study, we extended these analyses to SSc fibroblasts and demonstrated that the Smad1 pathway also contributes to activation of profibrotic genes in these cells. Together with the results of the DNA binding assays, these studies indicate that the CCN2 gene is a direct effector of the Smad1 pathway. CCN2 is required for the profibrotic effects of TGFβ in cultured cells and is considered to be a primary mediator of chronic fibrotic processes in SSc skin (26). Thus, activation of the Smad1 pathway may contribute to maintaining constitutively elevated expression levels of CCN2 in SSc fibroblasts in vivo and in vitro.
TGFβ-dependent activation of the Smad1 pathway was first described in endothelial cells (27). In these cells, TGFβ signals through a heteromeric receptor complex that includes TGFβRII, ALK-5, and ALK-1 (28). While the specific interactions between ALK-5 and ALK-1 are still not fully elucidated, there is evidence that the relative ratio of these 2 receptor subtypes regulates the switch between proliferation and differentiation of endothelial cells (15). However, recent studies have also shown that the ALK-1/Smad1 pathway may play a role in kidney fibrosis (29–31). Furthermore, TGFβ signaling via activation of the ALK-1/Smad1 pathway and subsequent up-regulation of the Id1 gene have been shown to contribute to transdifferentiation of hepatic stellate cells into myofibroblasts (32). These recent studies strongly suggest that activation of the ALK-1/Smad1 pathway may represent a common feature of various fibrotic diseases and that this pathway may play an important role in the development of organ fibrosis, including SSc.
Imatinib mesylate is a tyrosine kinase inhibitor that inhibits a wide range of kinases, including c-Abl (or, Bcr-Abl), c-Kit, and PDGFR. Imatinib mesylate has been effective in preventing the development of organ fibrosis in the kidney, lung, liver, and skin in several animal models (5–7); however, it was significantly less effective in ameliorating an established fibrosis (33, 34). The antifibrotic effects of imatinib mesylate include inhibition of fibroblast proliferation via blockade of PDGFR kinase and inhibition of collagen production in response to TGFβ. While it has been established that the antifibrotic effects of imatinib mesylate are mediated through inhibition of c-Abl, the role of this nonreceptor tyrosine kinase in collagen regulation is currently unknown. Our findings suggest that Smad1 may represent a downstream effector of the c-Abl signaling cascade in dermal fibroblasts. Further studies are needed to clarify the mechanism by which c-Abl exerts its profibrotic effects.
Excessive deposition of collagen and other ECM components is the hallmark of SSc, occurring in all target organs and progressively interrupting circulation. Effective antifibrotic treatments for SSc and other fibrotic diseases are not available at present. Due to its prominent role in matrix regulation, TGFβ signaling is considered an attractive therapeutic target for controlling fibrosis. However, the complexity of the profibrotic effects of TGFβ that involve both Smad3-dependent and -independent mechanisms presents a challenge in selecting appropriate therapeutic targets. For example, pharmacologic inhibitors of TGFβRI kinase that result in blockade of Smad3 signaling (ALK-5 kinase inhibitors) did normalize elevated CCN2 levels in SSc fibroblasts and, in general, ameliorated only selected aspects of SSc fibrosis in vitro (35, 36). Our study suggests that due to the pleiotropic nature of TGFβ signaling, targeting a single component of the TGFβ signaling pathway may not be effective in ameliorating SSc fibrosis. The Smad1 pathway may represent, in addition to Smad3, a potential target for antifibrotic therapy in SSc and other fibrotic diseases.
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Dr. Trojanowska had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Pannu, Smith, Jablonska, Blaszczyk, Trojanowska.
Acquisition of data. Pannu, Asano, Nakerakanti, Smith, Jablonska, Blaszczyk.
Analysis and interpretation of data. Pannu, Asano, Nakerakanti, ten Dijke, Trojanowska.
Manuscript preparation. Pannu, ten Dijke, Trojanowska.
Statistical analysis. Pannu, Asano.
Supply and characterization of key reagents. Ten Dijke.