Contribution of activin receptor–like kinase 5 (transforming growth factor β receptor type I) signaling to the fibrotic phenotype of scleroderma fibroblasts

Abstract

Objective

To use a specific transforming growth factor β receptor type I (TGFβRI; activin receptor–like kinase 5 [ALK-5]) kinase inhibitor (SD208) to determine the role of activation of the TGFβRI kinase (ALK-5) in maintaining the profibrotic phenotype of dermal fibroblasts in systemic sclerosis (SSc).

Methods

The effect of SD208 on the expression of key biochemical markers of the fibrotic phenotype was compared in fibroblasts cultured from clinically involved (lesional) and clinically uninvolved skin of patients with diffuse cutaneous SSc (dcSSc) and in fibroblasts from healthy controls matched for age, sex, and anatomic site. Protein expression was compared together with the ability of fibroblasts to adhere to the extracellular matrix and to remodel and contract a free-floating fibroblast–populated type I collagen lattice.

Results

Inhibiting TGFβRI kinase reduced the expression of a cohort of fibrotic markers by dermal fibroblasts from patients with dcSSc, including type I collagen and β1 integrin. Moreover, inhibition also attenuated the elevated adhesive and contractile abilities of dcSSc fibroblasts.

Conclusion

Our data suggest that some of the key profibrotic features of lesional SSc fibroblasts are dependent upon ALK-5 activity. Thus, TGFβRI kinase–mediated signaling may contribute to dermal fibrosis in dcSSc.

Fibrotic disease, one of the largest groups of diseases for which there is no effective therapy, is characterized by excessive deposition of scar tissue, resulting in tissue destruction, organ dysfunction, or death from systemic organ failure. Examples of such diseases include liver cirrhosis, diabetic nephropathy, idiopathic pulmonary fibrosis, and the connective tissue disease systemic sclerosis (SSc). The pathology of fibrotic diseases is characterized by the excessive production and contraction of extracellular matrix (ECM) by mesenchymal cells, such as fibroblasts, within fibrotic lesions (1, 2).

Fibrosis is believed to result from a failure to terminate the proper wound-healing process. Thus, an appreciation of how normal tissue repair is controlled is likely to be a necessary prerequisite to understand the molecular mechanism underlying fibrotic disease (3, 4). The normal wound-healing process requires the de novo synthesis of connective tissue and matrix that occurs in response to injury. In the case of human skin, the tissue repair program involves the proliferation of fibroblasts, which migrate into the wound and produce and contract the ECM, resulting in wound closure and restoration of proper organ function (3, 4). These events are regulated by a complex set of interactions within a network of profibrotic and antifibrotic secreted proteins, including transforming growth factor β (TGFβ), which has been proposed to be a key mediator of fibrotic disease (5). Upon cutaneous injury, TGFβ is rapidly induced (6). TGFβ induces fibrotic responses in vivo and promotes the ability of fibroblasts to synthesize and contract ECM in vitro (7–9). In animal models of fibrosis, blocking the action of TGFβ prevents the acquisition of fibrosis (for review, see ref. 4). However, whether endogenous TGFβ contributes to chronic pathologic fibrosis in humans is unknown.

Diffuse cutaneous systemic sclerosis (dcSSc) is a disease of unknown etiology characterized by microvascular injury, autoimmune inflammatory responses, and severe and often progressive fibrosis of the skin and internal organs (10, 11). Mortality rates among dcSSc patients are high and are directly related to the extent of scarring (10, 11). Since the fibrosis observed in dcSSc can affect many different organs, elucidating the molecular basis of dcSSc is likely to be beneficial in understanding the nature of fibrotic disease in general (10, 11). Patients with diffuse scleroderma overexpress TGFβ and show dysregulated TGFβ signaling (11–14), including alterations in the ratio of TGFβ receptor type I (TGFβRI) to TGFβRII in a subset of dcSSc patients (14) and overexpression of the TGFβ ancillary receptor endoglin (13).

Small-molecule inhibitors of receptor-regulated kinases have been identified for a number of key intracellular signaling pathways, including kinase moieties in the intracellular domains of TGFβ superfamily receptors. Thus, an inhibitor of activin receptor–like kinase 4 (ALK-4), ALK-5, and ALK-7 has been shown to block downstream Smad-mediated signaling events (15). In common with other small-molecule nonpeptide antagonists, these kinase inhibitors inhibit essential ATP binding to their target enzymes, and structural or conformational differences between each kinase molecule endow specificity. The inhibitor SD208 has been shown to be selective for ALK-5, without activity against other related kinases and has been shown to inhibit experimental fibrosis in vivo in animal models (16). Although inhibiting TGFβ signaling, for example, through the TGFβRI (ALK-5) receptor, has been proposed as a potential antifibrotic strategy, the impact of antagonizing endogenous TGFβ signaling on the phenotype of fibrotic dermal fibroblasts has not yet been investigated. These studies are prerequisite to establishing the likely benefit of such treatments in dcSSc.

In the present study, we used a specific TGFβR inhibitor to compare the impact of antagonizing endogenous TGFβ signaling on the fibrotic phenotype of dcSSc dermal fibroblast with that of normal fibroblasts. Our results yield new insights into the molecular mechanism underlying dcSSc and suggest that TGFβRI kinase (ALK-5) inhibition may be a logical strategy for therapeutic intervention to combat the skin fibrosis observed in dcSSc.

PATIENTS AND METHODS

Cell culture.

Cells were isolated and cultured as previously described (17). Briefly, dermal fibroblasts were isolated from biopsy samples of affected (lesional) areas of patients with early-onset dcSSc (median disease duration 7 months [range 2–9 months from the first non–Raynaud's phenomenon manifestation of SSc]; n = 6). Lesional biopsy samples were from sclerotic areas, but not from the leading edge of an advancing lesion. All patients had physician-documented recent progression of skin thickening, confirming active disease. Age-, sex-, and anatomic site–matched control biopsy tissues were obtained from healthy volunteers. Informed consent was obtained from all study subjects, and ethics approval was obtained from the appropriate committee at University College, London.

All patients fulfilled the American College of Rheumatology criteria for the diagnosis of early diffuse SSc (18), and were in the fibrotic phase of the disease. All study subjects were women. None of the subjects were receiving immunosuppressive medication or corticosteroids at the time of biopsy.

Fibroblasts were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 units/ml of penicillin, and 100 mg/ml of streptomycin, and were cultured in a humidified atmosphere of 5% CO₂ in air. Fibroblasts were subcultured at a cell:medium ratio of 1:4 at confluence. We used a novel, highly specific inhibitor of TGFβRI kinase (SD208; Scios, Fremont, CA) to determine whether the blocking of endogenous TGFβ signaling would modulate the biochemical or functional properties of SSc fibroblasts. SD208 has an 50% inhibition concentration of 49 nmoles/liter, based on direct, cell-free enzymatic assay of TGFβRI kinase (ALK-5) activity with a specificity of >100-fold against TGFβRII and at least 17-fold over members of a panel of related protein kinases, including p38α, p38β, p38δ, JNK1, epidermal growth factor receptor, MAPKAPK-2, MKK-6, ERK-2, protein kinases C, A, and D, Cdc2, and Ca²⁺/calmodulin-regulated kinase II. Based upon data from our earlier studies of the effect of SD208 on murine dermal fibroblast ECM biosynthesis (19), a concentration of 1 μM of SD208 was used for experiments examining control or SSc human dermal fibroblasts.

Western blotting and immunofluorescence analysis.

Cells were cultured until confluence in DMEM/10% FBS and incubated in DMEM/0.5% bovine serum albumin (BSA; Sigma, St. Louis, MO) for 18 hours, and then incubated with 1 μM SD208 (19, 20) for an additional 24 hours. Cells were used at passage 3. Cell layers were harvested using 2% sodium dodecyl sulfate (SDS). Proteins were quantified according to a Bradford kit (Bio-Rad, Richmond, CA), and equal amounts of protein (25 μg) were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) using 4–12% polyacrylamide gels (Invitrogen). Gels were blotted onto nitrocellulose, and proteins were detected using antimoesin, antipaxillin, antivinculin, antiezrin (all from Cell Signaling Technology, Beverly, MA), anti–syndecan 4, anti–α4 integrin, anti–β1 integrin (all from Zymed, Burlingame, CA), or anti-CCN2 (Abcam, Cambridge, UK), appropriate horseradish peroxidase–conjugated secondary antibodies (Zymed), and an enhanced chemiluminescence kit (Amersham, Little Chalfont, UK).

To detect type I collagen, equal amounts of media were precipitated with 30% ammonium sulfate, resuspended in 2% SDS and subjected to SDS-PAGE. Gels were blotted onto nitrocellulose, and type I collagen was detected with an anti–type I collagen antibody (Biodesign International, Kennebunk, ME), as described above. In our experience, ∼70–80% of such SSc cells overexpress type I collagen. Cells from 4 patients displaying elevated levels of type I collagen were used for our studies, and representative blots are shown.

For indirect immunofluorescence analysis, cells were treated with 1 μM SD208 for 24 hours and then fixed in 4% paraformaldehyde (Sigma) for 15 minutes. Localization of α-smooth muscle actin (α-SMA) stress fibers and vinculin-positive focal adhesions were detected using anti–α-SMA and antivinculin antibodies, and appropriate FITC-conjugated secondary antibodies (Jackson ImmunoResearch, Avondale, PA) as previously described (21). Photographs were taken using a Zeiss Axiophot microscope and image analysis software (Zeiss, Welwyn Garden City, UK), and exported into Adobe Photoshop (Adobe Systems, San Jose, CA).

Adhesion assay.

Fibroblasts were isolated and cultured as described above. Fibroblasts isolated from 3 normal individuals and from lesional areas of 3 patients with dcSSc were assayed in triplicate. Fibroblasts were used at passage 3. Adhesion assays were performed as described previously (21) by initially coating the wells of 96-well plates with 6 μg/ml of fibronectin (Sigma) in 0.5% BSA/1× phosphate buffered saline (PBS) overnight at 4°C. Wells were blocked for 1 hour at room temperature in 10% BSA in PBS. Fibroblasts were harvested with 2 mM EDTA in PBS (20 minutes at room temperature), washed twice with serum-free DMEM containing 1% BSA (Sigma), and resuspended in the same medium at 2.5 × 10⁵ cells/ml. To detect cell adhesion, an acid phosphatase assay was used in which adherent cells were quantified by incubation for 2 hours at 37°C with 100 μl of substrate solution (0.1M sodium acetate, pH 5.5, 10 mM p-nitrophenyl phosphate, and 0.1% Triton X-100). The reaction was stopped by the addition of 15 μl of 1N NaOH per well, and absorbance at 450 nm was measured.

Collagen gel contraction.

Experiments were performed essentially as described previously (22). Briefly, 24-well tissue culture plates were precoated with BSA. Cells were used at passage 3. Trypsinized fibroblasts were suspended in MCDB medium (Sigma) and mixed with collagen solution (1 part 0.2M HEPES pH 8.0, 4 parts collagen [Vitrogen-100; 3 mg/ml], and 5 parts 2× MCDB) to yield a final concentration of 80,000 cells per ml and 1.2 mg/ml of collagen. Collagen/cell suspension (1 ml) was added to each well. After polymerization, gels were detached from the wells by adding 1 ml of MCDB medium in the presence or absence of SD208. Contraction of the gel was quantified according to the loss of gel weight and the decrease in gel diameter over a 24-hour period.

RESULTS

Dependency of increased expression of proadhesive and procontractile genes in dcSSc fibroblasts on endogenous TGFβ signaling through TGFβRI kinase (ALK-5).

Dermal dcSSc fibroblasts overexpress an array of proadhesive and procontractile proteins (23), including CCN2, which is also known as connective tissue growth factor (4, 21, 24, 25). To assess whether endogenous TGFβ signaling through the type I TGFβ receptor is involved in the overexpression of profibrotic genes by dcSSc fibroblasts, we incubated dermal fibroblasts derived from healthy controls and from lesional areas of the skin of dcSSc patients for 24 hours with SD208, an ALK-5/TGFβRI kinase inhibitor (19, 20). Cell extracts were then prepared, and subjected to SDS-PAGE and Western blot analysis to detect expression of proadhesive and procontractile proteins.

Inhibition of endogenous TGFβ signaling blocked the overexpression of a cohort of profibrotic proteins (Figure 1), indicating that TGFβ signaling through ALK-5 promoted the expression of these profibrotic proteins in dcSSc fibroblasts. However, overexpression of CCN2 was not blocked with SD208, confirming previously reported data showing that overexpression of CCN2 in lesional dcSSc fibroblasts was independent of the TGFβ response element in the CCN2 promoter (17, 25). Similarly, overexpression of α-SMA and vinculin were not blocked by SD208, suggesting that ALK-5–independent pathways also contribute to fibrosis in scleroderma. Consistent with this notion, application of SD208 has no significant impact on the appearance of α-SMA stress fibers or vinculin-positive focal adhesions (Figure 2), indicating that SD208 did not appreciably affect the formation of the characteristic appearance of a myofibroblast cytoskeleton and “supermature” vinculin-positive focal adhesions (26).

Figure 1. Inhibition of transforming growth factor β receptor type I (TGFβRI) kinase (activin receptor–like kinase 5 [ALK-5]) and blocking of the overexpression of a cohort of profibrotic proteins by dermal fibroblasts from patients with early-onset diffuse cutaneous systemic sclerosis (dcSSc). Cell lysates (25 μg) from dermal fibroblasts derived from normal subjects and from lesional areas of the skin of patients with dcSSc were subjected to Western blot analysis with antimoesin, antivinculin, anti-CCN2, antiezrin, anti–α-smooth muscle actin (anti–α-SMA), anti–α4 integrin, anti–β1 integrin, and anti-GAPDH antibodies. Dermal fibroblasts isolated from 6 normal subjects and from lesional and nonlesional areas of 6 dcSSc patients were assayed. Cells were incubated for 24 hours in the presence or absence of the TGFβRI kinase (ALK-5) inhibitor SD208, and protein extracts were harvested. Shown is a representative blot of the results from 2 subjects of each cohort. Results are representative of a series of independent experiments examining dcSSc (n = 6) or control (n = 6) fibroblast cultures.

Figure 2. Lack of blocking of the appearance of an α-SMA–containing cytoskeleton by TGFβRI kinase (ALK-5) inhibition. Dermal fibroblasts derived from normal subjects and from the lesional skin of dcSSc patients were incubated in the presence and absence of SD208 for 24 hours, fixed in paraformaldehyde, and then stained with anti–α-SMA and antivinculin antibodies and appropriate secondary antibodies. In the context of a monolayer not undergoing active tissue remodeling, the appearance of the α-SMA–containing filament network and intense vinculin-positive “supermature” focal adhesions, which are characteristic of a myofibroblast cytoskeleton (26), was not impaired by SD208 treatment. See Figure 1 for definitions.

Dependency of elevated expression of type I collagen in dcSSc fibroblasts on endogenous TGFβ signaling through TGFβRI kinase (ALK-5).

One of the hallmarks of fibrotic disease, including scleroderma, is the overexpression of type I collagen (1, 2, 7). To determine whether overexpression of type I collagen by SSc fibroblasts was dependent upon TGFβRI kinase (ALK-5) signaling, we assessed whether the overexpression of type I collagen by dcSSc fibroblasts could be blocked with SD208. We found that ALK-5 inhibition potently reduced type I collagen production by dcSSc fibroblasts, suggesting that signaling through ALK-5 contributes significantly to the overexpression of type I collagen by SSc fibroblasts (Figure 3). It is interesting to note that for proteins in which ALK-5 inhibition reduced expression in SSc fibroblasts, ALK-5 inhibition also reduced protein expression in normal fibroblasts (Figures 1 and 3). These results suggest that ALK-5 signaling normally contributes to the expression of these proteins in fibroblasts and that the SSc phenotype results from an exaggeration of these processes.

Figure 3. TGFβRI kinase (ALK-5) inhibition and reduction of the overexpression of type I collagen by dermal fibroblasts from patients with early-onset dcSSc. Equal amounts of conditioned media from dermal fibroblasts derived from normal subjects and from patients with dcSSc were concentrated and subjected to Western blot analysis with an anti–type I collagen antibody, as described in Patients and Methods. Cells were incubated for 24 hours in the presence or absence of TβRI kinase (ALK-5) inhibitor SD208, and protein extracts were harvested. A total of 6 fibroblast lines from each cohort were used. Shown is a representative blot of the results from 2 subjects of each cohort. See Figure 1 for definitions.

Blocking of the elevated adhesive ability of dcSSc fibroblasts by TGFβRI kinase (ALK-5).

Our Western blot analyses revealed that inhibition of TGFβ signaling attenuated a number of proadhesive proteins, including integrin β1. As a component of the α5β1 complex, integrin β1 facilitates binding of normal fibroblast to fibronectin in a TGFβ-dependent manner. To address whether there was a functional consequence of these changes in expression on cell adhesion, we assessed the ability of the ALK-5 receptor inhibitor to alleviate the enhanced adhesive and contractile properties of lesional dcSSc fibroblasts in vitro. To assess the impact of the ALK-5 receptor antagonist on the adhesive abilities of normal and lesional dermal fibroblasts, we coated 96-well plates with fibronectin. Fibroblasts from 3 normal individuals and from lesional areas of 3 patients with dcSSc were cultured. Cells were detached from tissue culture dishes with EDTA and allowed to adhere to fibronectin for 45 minutes in the presence or absence of inhibitors.

We found that fibroblasts cultured from lesional areas of dcSSc patients showed significantly elevated adhesive ability relative to that of fibroblasts cultured from normal donors and from nonlesional areas of dcSSc patients (Figure 4). This enhanced adhesive ability was significantly blocked by the TGFβRI kinase inhibitor and returned to levels in normal fibroblasts (Figure 4). These findings extend our previous data showing that ALK-5 inhibition suppressed the enhanced collagen production by SSc fibroblasts. ALK-5 inhibition also significantly reduced cell adhesion to fibronectin in normal fibroblasts (Figure 4), emphasizing our earlier observation that ALK-5 signaling normally contributes to fibroblast function and that the SSc phenotype results from an exaggeration of these processes.

Figure 4. TGFβRI kinase (ALK-5) inhibition and reduction of the enhanced ability of dermal fibroblasts from lesional areas of patients with dcSSc to adhere to extracellular matrix. Dermal fibroblasts derived from normal subjects and from patients with dcSSc were allowed to adhere to bovine serum albumin or fibronectin for 45 minutes. Unbound cells were removed by washing. The remaining adherent cells were detected as described in Patients and Methods. Cells were then incubated for 24 hours in the presence or absence of the TGFβRI kinase (ALK-5) inhibitor SD208. ALK-5 inhibition reduced the adhesion of both normal and SSc fibroblasts (∗ = P < 0.05). Dermal fibroblasts isolated from 6 normal subjects and from lesional and nonlesional areas of 6 patients with dcSSc were assayed in triplicate. Values are the mean ± SEM. P values less than 0.05 were considered significant. See Figure 1 for definitions.

Blocking of the elevated contractile ability of dcSSc fibroblasts by inhibition of TGFβRI kinase (ALK-5).

Although addition of the ALK-5 inhibitor SD208 did not appreciably affect the formation of the α-SMA cytoskeleton on cells growing on a monolayer (Figure 2), it was possible that the TGFβRI kinase (ALK-5) inhibitor might affect the abilities of normal and lesional dermal dcSSc fibroblasts to contract ECM in a 3-dimensional lattice, a process involving assembly and disassembly of focal adhesion via integrins (27). Thus, we seeded dermal fibroblasts within a collagen matrix and allowed the resultant mixture to polymerize on a tissue culture plate. The solidified gel was then detached from the tissue culture plate and incubated for 24 hours in the presence of 0.5% FBS. We then assessed collagen gel contraction by measuring the size and weight of the contracted gel (22).

We found that compared with nonlesional dcSSc and normal dermal fibroblasts, lesional dcSSc dermal fibroblasts showed a significantly increased ability to contract a collagen matrix (Figure 5). This enhanced activity was significantly blocked by the TGFβRI kinase inhibitor SD208. ALK-5 inhibition also significantly reduced matrix contraction by normal fibroblasts (Figure 5), which again implies that the SSc phenotype results from an exaggeration of these processes.

Figure 5. Enhanced ability of dermal fibroblasts from lesional areas of patients with dcSSc to contract an extracellular matrix, which is dependent on endogenous TGFβRI kinase (ALK-5) signaling. Dermal fibroblasts derived from normal subjects and from patients with dcSSc were seeded into a floating 3-dimensional collagen gel matrix and incubated for 24 hours as described in Patients Methods. Under these conditions, the fibroblasts actively mediate remodeling of the collagen gel matrix (22, 26, 27). The weight of the contracted collagen gel was then measured. Dermal fibroblasts isolated from 6 normal subjects and from lesional and nonlesional areas of 6 patients with dcSSc were assayed in triplicate. Cells were incubated for 24 hours in the presence or absence of the TGFβRI kinase (ALK-5) inhibitor SD208. ALK-5 inhibition reduced matrix contraction by both normal and SSc fibroblasts (∗∗ = P < 0.01). Values are the mean and SEM. P values less than 0.05 were considered significant; P values less than 0.01 were considered highly significant. See Figure 1 for definitions.

Collectively, these results suggest that the phenotypic features of lesional dcSSc fibroblasts that intimately contribute to scar formation (i.e., to the physical appearance of fibrotic lesions in dcSSc skin), namely, enhanced collagen production and the enhanced ability to adhere to and contract ECM, are due to endogenous TGFβ signaling acting through TGFβRI and are apparently further due to an enhancement of the processes that are normally operant in fibroblasts. Thus, ALK-5 inhibition may be of potential benefit in combating fibrosis in early-stage dcSSc.

DISCUSSION

Scleroderma carries high mortality rates and is refractory to treatment. Although the immunologic and vascular components of the disease are amenable to therapeutic intervention, there are currently no effective therapies for the fibrotic component of the disease. Recently, however, several candidate mediators of the scleroderma phenotype have emerged, including TGFβ (4, 11, 28). However, until now, the potential utility of TGFβ receptor antagonists in alleviating symptoms of scleroderma has not been compared or assessed. Fibroblasts from patients with dcSSc overexpress a series of profibrotic proteins. In this study, we investigated the ability of SD208, a TGFβRI kinase (ALK-5) inhibitor (19, 20), to prevent the overexpression of profibrotic proteins by dermal fibroblasts from patients with early-onset dcSSc, as well as the ability of dermal fibroblasts from patients with early-onset dcSSc to excessively adhere to and contract ECM.

Our data include several novel findings. First, we showed that TGFβ signaling, acting through the ALK-5 receptor, leads to the overexpression of a cohort of proadhesive and procontractile genes by dcSSc fibroblasts. The overexpression of type I collagen by dcSSc fibroblasts was dependent upon TGFβ signaling through the ALK-5 receptor. In addition, we demonstrated that dcSSc fibroblasts overexpress β1 and α4 integrins, paxillin, and ezrin, which are involved in cell adhesion and focal adhesion formation and function (29), in a TGFβ/ALK-5–dependent manner. Focal adhesion formation is intimately involved with adhesion and contraction of the ECM. We then showed that dermal fibroblasts from the lesional skin of dcSSc patients excessively adhered to and contracted ECM in a manner that was antagonized by an ALK-5 inhibitor. Scarring, as is observed in dcSSc, is characterized by the excessive production and contraction of matrix. Collectively, these data support the notion that TGFβ signaling promotes the profibrotic phenotype of early-onset dcSSc fibroblasts and that ALK-5 inhibition will be of benefit in combating the excessive scarring observed during cutaneous fibrosis in dcSSc.

However, our studies also showed that ALK-5 inhibition abrogated the production of proadhesive and procontractile elements, type I collagen, cell adhesion, and gel contraction by normal dermal fibroblasts. These results indicated that autocrine ALK-5–dependent signaling contributes to these processes in normal fibroblasts and that the SSc phenotype results from an exaggeration of processes that normally occur in fibroblasts. Since the SSc fibroblasts were more abnormal in these processes, the effect of ALK-5 inhibition was more dramatic in the SSc fibroblasts. This may make the use of an ALK-5 inhibitor problematic clinically, since ALK-5 inhibition could also impair normal baseline fibroblast function, rather than return SSc function to normal.

It has been proposed that alterations in the TGFβ signaling pathway aid in the perpetuation of fibrosis. For example, fibroblasts cultured from some dcSSc patients show alterations in the TGFβRI:TGFβRII ratio, which is important for type I collagen expression in these patients (14). In addition, although it has not been shown that dcSSc fibroblasts produce higher levels of TGFβ protein (30, 31) or enhanced activation of TGFβ, it has been reported that dcSSc fibroblasts express increased levels of type I and type II TGFβ receptors (31) and overexpress the TGFβ ancillary receptor endoglin in a manner that increases with disease severity (13). Adding complexity to this issue, the TGFβ signaling mediator Smad3 has been shown to be activated in leading-edge fibroblasts in a TGFβ ligand–independent manner (30), and although 1 study has indicated that dcSSc fibroblasts possess lower levels of the Smad inhibitor Smad7 (32), other studies have reported either no such elevation or indeed increased Smad7 levels (12, 17, 33). Our data confirm and extend the recently reported data concerning another ALK-5 inhibitor in which blockade of acute response to recombinant TGFβ1 was examined in normal dermal fibroblasts (34).

It is interesting to note that the overexpression of CCN2 and α-SMA, genes that are known to be induced by TGFβ in a Smad-dependent manner (17, 35), were not blocked by ALK-5 inhibition in SSc fibroblasts. Similarly, ALK-5 inhibition, although effective at reducing the contractile phenotype of SSc fibroblasts, did not reverse this aspect of SSc fibroblasts to normal.

Our results are consistent with the notion that hyperactive TGFβ receptor signaling is insufficient to explain the complete dcSSc phenotype (4, 13, 17) and that other pathways also contribute to dcSSc (22). However, these results strongly suggest that TGFβ receptor antagonists are likely to be of benefit in combating particular, specific phenotypic aspects of dermal fibrosis, namely, the excessive production, adhesion, and contraction of ECM, in patients with early-onset dcSSc.