Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Relevance of Epithelial Phenotypic Switching in the Pathogenesis of Systemic Sclerosis.
Shiwen1, Xu, Sonnylal3, S., Tam2, A., Stratton2, R., Leask2, A., Denton2, C. P., Norman2, J.
Systemic sclerosis (SSc) is a complex disorder of uncertain etiology characterised by progressive vascular and interstitial fibrosis. Recently, we found that epidermal compartment in SSc skin exhibits abnormalities, taking on an activated phenotype reminiscent of that during the wound healing response. Fibroblast-Epithelial cell interactions are believed important during normal tissue repair and aberrant cellular cues may underlie important aspects of scarring and fibrosis. Transgenic mice in which the fibroblast expressed a constitutively active TGFb type I receptor (ALK5) or which expressing CTGF, develop progressive tissue fibrosis most prominent in the skin and lung. Our in vivo data also suggests that activation of TGFb signalling or CTGF over-expression by fibroblasts not only causes stromal activation, but also pathological changes in the phenotype of adjacent epithelium. Here, we focus on the phenotypic profile of the SSc epithelium, the role of fibroblast-derived CTGF in epithelial switching into a mesenchymal-like phenotypic and the contribution this process may play to fibrogenesis.
Whole skin biopsies were obtained from SSc and controls. The epidermis was prepared from some of the skin biopsies, and processed for phospho-kinase profiling (Kinexus: Vancouver, Canada). Human and rodent type II epithelial cells lines (A549 and T2) were grown in grown in DMEM + 10%FBS. Epithelial cells were stimulated with TGFb after serum starvation in the presence and absence of siRNA for specific for CTGF. Markers of epithelial cells and fibroblasts including CTGF, snail, E-cadherin, aSMA, collagen type I and fibronectin were examined using western blot analysis.
Phosphorylation arrays were performed on the epidermal tissue from SSc patients and healthy controls to identify signaling pathways activated in SSc epidermis. A number of EMT-related proteins were found to have elevated phosphorylation states in SSc tissues versus controls, including c-Met, Wee1 protein-tyrosine kinase, STAT3, Integrin-linked protein-serine kinase 1(p<0.05). Further in vitro studies, A549 and T2 epithelial cells exposed to TGF-b develop a mesenchymal-like morphology and molecular markers associated with EMT (snail and CTGF). CTGF-specific siRNA dose-dependently suppressed TGFb-induced snail and CTGF protein expression towards basal levels in A549 cells. Transgenic mouse fibroblasts over-expressing CTGF showed significantly higher expression levels for matrix genes and proteins including collagen type I, fibronectin and increased expression of aSMA.
In this study we show that phosphorylation array analysis revealed induction of EMT-like protein kinase signaling in SSc epidermis. Important growth factor, TGF-b promotes and triggers epithelial cells to undergo a phenotypic switch which is attenuated upon CTGF knock-down by RNA interference. Our data suggests that in SSc, the enhanced expression of TGFb signaling and CTGF expression is likely to indirectly contribute to disease pathogenesis by triggering epidermal cells phenotypic switching towards a mesenchyml cell-like programme.
To cite this abstract, please use the following information:
Shiwen, Xu, Sonnylal, S., Tam, A., Stratton, R., Leask, A., Denton, C. P., et al; Relevance of Epithelial Phenotypic Switching in the Pathogenesis of Systemic Sclerosis. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :613