Monocyte chemoattractant protein 3 as a mediator of fibrosis: Overexpression in systemic sclerosis and the type 1 tight-skin mouse

Abstract

Objective: To determine the gene-expression profile in dermal fibroblasts from type 1 tight-skin (Tsk1) mice, and to examine the expression and potential fibrotic activity of monocyte chemoattractant protein 3 (MCP-3) in Tsk1 mouse and human systemic sclerosis (SSc) skin.

Methods: Complementary DNA microarrays (Atlas 1.2) were used to compare Tsk1 fibroblasts with non-Tsk1 littermate cells at 10 days, 6 weeks, and 12 weeks of age. Expression of MCP-3 protein was assessed by Western blotting of fibroblast culture supernatants, and localized in the mouse and human skin biopsy samples by immunohistochemistry. Activation of collagen reporter genes by MCP-3 was explored in transgenic mouse fibroblasts and by transient transfection assays.

Results: MCP-3 was highly overexpressed by neonatal Tsk1 fibroblasts and by fibroblasts cultured from the lesional skin of patients with early-stage diffuse cutaneous SSc. Immunolocalization confirmed increased expression of MCP-3 in the dermis of 4 of 5 Tsk1 skin samples and 14 of 28 lesional SSc skin samples, compared with that in matched healthy mice (n = 5) and human controls (n = 11). Proalpha2(I) collagen promoter-reporter gene constructs were activated by MCP-3 in transgenic mice and by transient transfection assays. This response was maximal between 16 and 24 hours of culture and mediated via sequences within the proximal promoter. The effects of MCP-3 could be diminished by a neutralizing antibody to transforming growth factor beta.

Conclusion: We demonstrate, for the first time, overexpression of MCP-3 in early-stage SSc and in Tsk1 skin, and suggest a novel role for this protein as a fibrotic mediator activating extracellular matrix gene expression in addition to promoting leukocyte trafficking. This chemokine may be an important early member of the cytokine cascade driving the pathogenesis of SSc.