Proteomic analysis of connective tissue growth factor activation in normal and scleroderma lung fibroblasts

Abstract

Connective tissue growth factor (CTGF, CCN2) is over expressed in lung fibroblasts isolated from patients with interstitial lung disease (ILD) and is considered to be a molecular marker of fibrosis. To understand the significance of elevated CTGF we investigated the changes in lung fibroblast proteome in response to CTGF overexpression. Using two‐dimensional gel electrophoresis (2‐DE) followed by in‐gel proteolytic digestion and mass spectrometric analysis, we identified 13 proteins affected by CTGF. Several of the CTGF‐induced proteins, such as pro‐α (I) collagen and cytoskeletal proteins vinculin, moesin, and ezrin, are known to be elevated in pulmonary fibrosis, whereas nine of 13 proteins have not been studied in pulmonary fibrosis and are, therefore, new ILD targets for CTGF. Our study demonstrates that one of the novel CTGF‐induced proteins, IQ motif containing GTPase activating protein (IQGAP)‐1, is elevated in lung fibroblasts isolated from scleroderma patients with ILD. Scleroderma lung fibroblasts and CTGF‐treated normal lung fibroblasts are characterized by increased collagen synthesis, over‐expression of alpha‐smooth muscle actin, increased collagen gel contraction, and increased rate of migration in a wound healing assay. Depletion of IQGAP‐1 expression by small interfering (si)RNA inhibited CTGF‐induced migration of normal lung fibroblasts and significantly decreased the rate of migration of scleroderma lung fibroblasts. These findings further implicate the importance of CTGF in lung tissue repair and fibrosis and suggest that IQGAP‐1 mediates the migration of lung fibroblasts to the damaged tissue.