Abstract
BackgroundScleroderma (systemic sclerosis; SSc) is a clinically heterogeneous and often lethal acquired disorder of the connective tissue that is characterized by vascular, immune/inflammatory and fibrotic manifestations. Tissue fibrosis is the main cause of morbidity and mortality in SSc and an unmet medical challenge, mostly because of our limited understanding of the molecular factors and signalling events that trigger and sustain disease progression. Recent evidence has correlated skin fibrosis in SSc with stabilization of proto-oncogene Ha-Ras secondary to auto-antibody stimulation of reactive oxygen species production. The goal of the present study was to explore the molecular connection between Ha-Ras stabilization and collagen I production, the main read-out of fibrogenesis, in a primary dermal fibroblast culture system that replicates the early stages of disease progression in SSc.ResultsForced expression of proto-oncogene Ha-Ras in dermal fibroblasts demonstrated the promotion of an immediate collagen I up-regulation, as evidenced by enhanced activity of a collagen I-driven luciferase reporter plasmid and increased accumulation of endogenous collagen I proteins. Moreover, normal levels of Tgfβ transcripts and active transforming growth factor-beta (TGFβ) implied Ha-Ras stimulation of the canonical Smad2/3 signalling pathway independently of TGFβ production or activation. Heightened Smad2/3 signalling was furthermore correlated with greater Smad3 phosphorylation and Smad3 protein accumulation, suggesting that Ha-Ras may target both Smad2/3 activation and turnover. Additional in vitro evidence excluded a contribution of ERK1/2 signalling to improper Smad3 activity and collagen I production in cells that constitutively express Ha-Ras.ConclusionsOur study shows for the first time that constitutively elevated Ha-Ras protein levels can directly stimulate Smad2/3 signalling and collagen I accumulation independently of TGFβ neo-synthesis and activation. This finding therefore implicates the Ha-Ras pathway with the early onset of fibrosis in SSc and implicitly identifies new therapeutic targets in SSc.
Highlights
Scleroderma is a clinically heterogeneous and often lethal acquired disorder of the connective tissue that is characterized by vascular, immune/inflammatory and fibrotic manifestations
Proto-oncogene Ha-Ras increases human pro-α2 collagen gene (COL1A2) expression through R-Smad activation The first set of experiments was designed to test the hypothesis that wild-type proto-oncogene Ha-Ras is directly involved in collagen I stimulation and to investigate the underlying mechanism
Primary fetal Human fetal dermal fibroblasts (hDF) were transiently co-transfected with a vector expressing constitutively active Ha-Ras and a luciferase reporter plasmid driven by the COL1A2 proximal promoter
Summary
Scleroderma (systemic sclerosis; SSc) is a clinically heterogeneous and often lethal acquired disorder of the connective tissue that is characterized by vascular, immune/inflammatory and fibrotic manifestations. Tissue fibrosis is the main cause of morbidity and mortality in SSc and an unmet medical challenge, mostly because of our limited understanding of the molecular factors and signalling events that trigger and sustain disease progression. The goal of the present study was to explore the molecular connection between Ha-Ras stabilization and collagen I production, the main read-out of fibrogenesis, in a primary dermal fibroblast culture system that replicates the early stages of disease progression in SSc. Wound healing is a complex and tightly regulated physiological process that involves several different cell types and a plethora of signalling molecules [1,2,3]. In spite of significant investigative effort, our current knowledge of the molecular and cellular events that promote and sustain myofibroblasts activation is limited and the clinical management of affected patients remains confined to therapies that alleviate secondary symptoms rather than arresting the often fatal consequences of the fibrotic response
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