TGF-beta/Smad1 signaling defines a subset of scleroderma fibroblasts

Transforming growth factor beta (TGF-beta) is the central fibrotic cytokine of scleroderma (SSc) pathogenesis. Signaling downstream of TGF-beta receptors is propagated through Smad and non-Smad pathways that affect diverse cellular functions including production of extracellular matrix (ECM) proteins. Recent evidence demonstrates that in SSc fibroblasts, TGF-beta signals through phosphorylated Smad1 to upregulate ECM production. Although the TGF-beta pathway is the primary mediator of SSc fibrosis, several additional signaling pathways have been implicated in the activation of SSc fibroblasts. The heterogeneity of the SSc phenotype is illustrated by a gene array of SSc patient skin that identified unique phenotype subsets among patients1. Adding further complexity to the SSc phenotype is the finding that different fibroblast phenotypes with regard to collagen alpha1(I) mRNA production exist within the same SSc skin tissue2. SSc fibroblasts are potentially derived from various cellular origins, which could contribute to phenotype heterogeneity as a result of diverse signaling pathway activation. In this work, the role of TGF-beta/Smad1 signaling in SSc fibrosis was examined in three models of SSc; hTERT clonal cell lines, whole biopsy fibroblast populations, and the col1alpha2-ALK5 mouse model of SSc. hTERT clonal cell lines derived from single SSc fibroblasts demonstrated that in addition to heterogeneous collagen production, signaling pathway activation is heterogeneous in SSc fibroblasts from the same tissue section. Activation of TGF-beta/Smad1 signaling was observed in a subset of the SSc clones with elevated expression of TGF-betaRI(ALK5) and phosphorylated Smad1. Further characterization of this pathway in SSc fibroblasts demonstrated that elevated expression of endoglin plays a role in promoting TGF-beta/Smad1 signaling. Inhibition of endoglin in SSc fibroblasts, in hTERT clonal cell lines, and in normal fibroblasts stimulated with TGF-beta reduced phosphorylated Smad1. Overexpression of constitutively active ALK1 in normal fibroblasts was sufficient to partially induce a fibrotic response by upregulating phosphorylated Smad1 levels and ECM protein production. To determine if activation of TGF-beta/Smad1 signaling in a mouse model of SSc was sufficient to induce fibrosis, TGF-betaRI(ALK5) was overexpressed in fibroblasts of the col1alpha2-ALK5 mouse. Analysis of skin morphology, collagen content, and expression of ECM mRNA and protein levels indicated that the heterozygous col1alpha2-ALK5 mouse does not develop a fibrotic SSc phenotype. This may be a result of weak pCD3-COL1alpha2 promoter activity, ineffective levels of TGF-betaRI(ALK5) receptor overexpression, or the requirement of additional stimuli to induce fibrosis. These studies demonstrate the heterogeneity of SSc fibroblasts and the involvement of multiple signaling pathways in activation of the fibrotic phenotype. Elevated expression of endoglin in a subset of SSc fibroblasts may activate the ALK1 receptor and TGF-beta/Smad1 signaling pathway. Although an attempt to activate this pathway in a mouse model of SSc was unsuccessful, in vitro data support the role of TGF-beta/Smad1 signaling in SSc fibrosis.