SSc is a rare connective tissue disease of unknown etiology characterized by excessive ECM deposition in the skin and internal organs [2
]. Studies over the past decade aimed at delineating the molecular mechanisms involved in SSc fibrosis have identified TGF-β as a central player and the TGF-β signaling pathway as an important target for therapeutic intervention for SSc [2
]. We have previously identified CD109 as a TGF-β co-receptor that inhibits TGF-β signaling in human keratinocytes [37
]. In the current study, we examine CD109 expression and function in limited SSc and diffuse SSc skin and normal skin in vivo
, and in SSc and normal dermal fibroblasts and epidermal keratinocytes in vitro
. Our results show that CD109 protein levels are elevated in vivo
in both the dermis and epidermis of SSc skin compared with normal skin and in vitro
in SSc dermal fibroblasts and epidermal keratinocytes compared with normal fibroblasts and keratinocytes, respectively. Although cultured SSc skin fibroblasts show higher CD109 protein levels compared with normal skin fibroblasts, they do not appear to display elevated CD109 mRNA levels. Elevated CD109 protein levels were observed in the skin tissue and fibroblasts from patients with both limited SSc and diffuse SSc, suggesting that increased CD109 protein expression might be a general feature of SSc. We also demonstrate that treatment with exogenous TGF-β1
does not alter CD109 protein levels in SSc or normal skin fibroblasts, suggesting that CD109 is not a direct target of TGF-β in these cells. Moreover, we found that CD109 inhibits ECM (fibronectin and collagen type I) and CCN2 protein production in both SSc and normal skin fibroblasts - as evidenced by the findings that CD109 siRNA-transfected SSc and normal skin fibroblasts display elevated fibronectin, collagen type I and CCN2 protein levels, whereas treatment with a recombinant CD109 protein leads to a decrease in levels of these proteins. In addition, we show that CD109 inhibits phosphorylation of Smad2 and Smad3 in SSc and normal fibroblasts. Taken together, this study identifies CD109 as a novel protein overexpressed in limited SSc and diffuse SSc skin fibroblasts and indicates that CD109 inhibits Smad2/3 signaling and ECM/CCN2 production in these cells, as in normal skin fibroblasts.
TGF-β co-receptors including endoglin, betaglycan and CD109 are important regulators of TGF-β signaling, and recent evidence indicates that they may modulate fibrotic gene expression in SSc fibroblasts. Endoglin expression has been reported to be higher in diffuse SSc skin fibroblasts compared with normal fibroblasts, and endoglin expression levels were shown to increase with disease progression [47
]. A more recent study demonstrated that endoglin levels correlate with profibrotic marker (collagen I and CCN2) levels in diffuse SSc skin fibroblasts and that endoglin promotes profibrogenic Smad1 signaling in these cells [48
]. In addition, cell surface endoglin and betaglycan expression levels were shown to be increased in diffuse SSc skin fibroblasts compared with normal fibroblasts and overexpression of betaglycan was sufficient to promote basal and TGF-β-induced CCN2 promoter activity in a mouse fibroblast cell line (NIH3T3) [49
]. Our results showing that CD109 protein levels are elevated in both limited SSc and diffuse SSc skin tissue and cultured fibroblasts compared with controls suggest that upregulation of CD109 in these SSc subtypes may involve a common pathophysiological mechanism. Further studies to determine the expression and function of TGF-β co-receptors in the different clinical subsets of SSc, in particular their relevance to TGF-β-driven profibrogenic Smad1 signaling, is an important avenue for future research.
An important question raised from our results is why CD109 protein levels but not mRNA levels are increased in SSc fibroblasts. Our data showing that the levels of CD109 protein, but not of mRNA, are higher in limited SSc and diffuse SSc skin fibroblasts compared with normal skin fibroblasts suggests that post-transcriptional regulation of CD109 may be altered in limited SSc and diffuse SSc skin. Such alteration may involve enhanced CD109 protein synthesis and/or decreased CD109 protein degradation. Our preliminary results demonstrating that CD109 protein degradation is impaired in SSc fibroblasts (AB and AP, 2012, unpublished results) suggest that impaired CD109 degradation may be responsible for the elevated CD109 protein levels in SSc fibroblasts. It is interesting to note in this regard that several studies have reported increased TGF-β receptor protein levels in SSc fibroblasts [21
], leading to the suggestion that TGF-β receptor stability is increased in SSc due to impairment of TGF-β receptor degradation [44
]. Furthermore, recent microarray data [46
] support our finding that CD109 mRNA levels in SSc and normal fibroblasts are similar. However, quantitative PCR analysis of RNA from a higher number of limited SSc and diffuse SSc dermal fibroblasts with age-matched, sex-matched and gender-matched control samples will be required to confirm these results.
Paradoxically, despite the elevated CD109 protein levels in SSc skin fibroblasts and the demonstrated ability of endogenous CD109 protein to inhibit ECM and CCN2 production, the levels of ECM and CCN2 in SSc fibroblasts remain elevated. One possible explanation for these results is that the upregulation of CD109 in SSc fibroblasts is not sufficient to completely counteract the activation of TGF-β or other profibrotic pathways in SSc. This notion is supported by our finding that addition of a recombinant CD109 protein to SSc fibroblasts, which already express elevated CD109 protein levels, is still able to decrease ECM and CCN2 levels. Whether the upregulation of CD109 is an adaptive response or a consequence of aberrant TGF-β signaling in SSc remains to be determined. Our results showing that CD109 is not a direct target of TGF-β in SSc and normal skin fibroblasts suggests that CD109 upregulation in SSc fibroblasts may involve TGF-β-independent mechanisms.
The most promising finding in the current study is that, despite the already upregulated CD109 protein expression in SSc, the addition of further exogenous CD109 is able to downregulate ECM production. Aberrant activation of autocrine TGF-β signaling is a critical factor involved in the maintenance of the fibrotic phenotype of SSc fibroblasts [16
]. Identifying factors that inhibit TGF-β signaling in fibroblasts may thus lead to the development of novel anti-TGF-β therapies for the treatment of SSc. Our results showing that CD109 siRNA transfection leads to a marked increase, and that addition of recombinant CD109 protein results in a significant decrease, in the production of fibronectin, collagen type I and CCN2 in both SSc and normal skin fibroblasts suggest that CD109 has potent antifibrotic effects in these cells. In addition, our finding that CD109 siRNA knockdown in SSc and normal skin fibroblasts is associated with enhanced Smad2/3 phosphorylation suggests that endogenous CD109 inhibits autocrine TGF-β signaling in these cells. These results suggest that mechanism by which CD109 decreases ECM and CCN2 production in SSc and normal skin fibroblasts may involve inhibition of Smad2/3 phosphorylation. Together, our results suggest that recombinant CD109 protein is a promising candidate for antifibrotic therapy in SSc.
In addition to playing a possible pathophysiological role in SSc, the elevated CD109 protein levels may serve as a biomarker of disease activity in SSc. CD109 is a member of the α2
-macroglobulin/complement family of thioester-containing proteins and has been shown to be expressed in endothelial cells, platelets and activated T cells [28
]. Recent studies have shown that CD109 is differentially expressed in a variety of human tumors [30
], leading to the suggestion that CD109 may represent a novel biomarker for certain cancers. The relatively small number of samples examined in the current study does not allow us to correlate CD109 expression levels with disease severity and/or duration. More work is needed to determine whether CD109 levels are differentially expressed at specific phases of disease activity in limited SSC and diffuse SSc.