Proteomic analysis of CTGF-activated lung fibroblasts: identification of IQGAP1 as a key player in lung fibroblast migration.

Connective tissue growth factor (CTGF, CCN2) is overexpressed in lung fibroblasts isolated from patients with interstitial lung disease (ILD) and systemic sclerosis (SSc, scleroderma) 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 2-dimensional gel electrophoresis 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-alpha (I) collagen and cytoskeletal proteins vinculin, moesin, and ezrin, are known to be elevated in pulmonary fibrosis, whereas 9 of 13 proteins have not been studied in pulmonary fibrosis and are, therefore, novel CTGF-responsive molecules that may have important roles in ILD. Our study demonstrates that 1 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. IQGAP1 is a scaffold protein that plays a pivotal role in regulating migration of endothelial and epithelial cells. Scleroderma lung fibroblasts and normal lung fibroblasts treated with CTGF demonstrated increased rate of migration in a wound healing assay. Depletion of IQGAP1 expression by small interfering RNA inhibited CTGF-induced migration and MAPK ERK1/2 phosphorylation in lung fibroblasts. MAPK inhibitor U0126 decreased CTGF-induced cell migration and did not interfere with CTGF-induced IQGAP1 expression, suggesting that MAPK pathway is downstream of IQGAP1. These findings further implicate the importance of CTGF in lung tissue repair and fibrosis and propose that CTGF-induced migration of lung fibroblasts to the damaged tissue is mediated via IQGAP1 and MAPK signaling pathways.

Down-regulation of collagen and connective tissue growth factor expression with hepatocyte growth factor in lung fibroblasts from white scleroderma patients via two signaling pathways.

OBJECTIVE: To study the mechanisms by which hepatocyte growth factor (HGF) down-regulates collagen and connective tissue growth factor (CTGF) in scleroderma (systemic sclerosis [SSc]) lung fibroblasts. METHODS: CTGF, type I collagen, and IkappaBalpha expression, together with MAPK phosphorylation, were studied by immunoblotting of lung fibroblasts derived from white SSc patients. Matrix metalloproteinase 1 (MMP-1) expression in cell culture medium samples was measured by enzyme-linked immunosorbent assay, MMP-1 activity was studied using an MMP-1 assay, and NF-kappaB DNA binding activity was determined using a transcription factor assay. RESULTS: In lung fibroblasts from white SSc patients, HGF activated MAPK (ERK-1/2) signaling pathways and MMP-1, while it inhibited NF-kappaB and significantly down-regulated CTGF and collagen in a time- and dose-dependent manner. Small interfering RNA (siRNA)-mediated depletion of Grb2 expression disrupted c-Met receptor downstream signaling, which resulted in diminished HGF-induced ERK-1/2 phosphorylation and the recovery of HGF-inhibited expression of MMP-1, NF-kappaB, collagen, and CTGF. The MAPK inhibitor, U0126, blocked MMP-1 activity and restored HGF-inhibited collagen and CTGF accumulation. Inhibition of MMP activity by MMP inhibitor GM1489 and inhibition of MMP-1 expression by siRNA did not prevent HGF-induced ERK-1/2 phosphorylation and NF-kappaB activity, but significantly restored HGF-inhibited collagen and CTGF accumulation. NF-kappaB inhibitor BAY 11-7082 did not interfere with MAPK phosphorylation or MMP-1 expression and activation, but significantly inhibited NF-kappaB DNA binding activity and acted synergistically with HGF to completely diminish the expression of CTGF. CONCLUSION: In lung fibroblasts from white SSc patients, HGF down-regulates the accumulation of CTGF via MAPK/MMP-1 and NF-kappaB signaling pathways, whereas collagen down-regulation is mediated mainly by a MAPK/MMP-1-dependent pathway.

Impairment of the antifibrotic effect of hepatocyte growth factor in lung fibroblasts from African Americans: possible role in systemic sclerosis.

OBJECTIVE: To compare the composition of cytokines in African American and Caucasian patients with systemic sclerosis (SSc; scleroderma) and in healthy individuals, particularly the expression and function of hepatocyte growth factor (HGF). METHODS: Bronchoalveolar lavage (BAL) fluid samples were analyzed using cytokine array techniques. HGF in plasma and cell culture medium samples was measured by enzyme-linked immunosorbent assay. Connective tissue growth factor (CTGF), type I collagen expression, and c-Met receptor phosphorylation were studied by immunoblotting. RESULTS: Overall greater expression of cytokines in BAL fluid from African American patients as compared with Caucasian patients was observed. Significant increases in HGF concentrations were detected in BAL fluid, plasma, and fibroblast culture medium from Caucasian SSc patients. In contrast, African American SSc patients did not demonstrate an increase in HGF. Recombinant HGF readily abolished CTGF expression and collagen accumulation in lung fibroblasts isolated from Caucasian SSc patients. Pretreatment of lung fibroblasts with neutralizing anti-c-Met antibody abolished the effects of HGF on CTGF expression and collagen accumulation, suggesting that the antifibrotic activity of HGF is mediated via c-Met receptor tyrosine kinase. Whereas recombinant HGF rapidly induced c-Met receptor phosphorylation in lung fibroblasts from Caucasian patients, c-Met receptor phosphorylation was significantly reduced in lung fibroblasts from African American subjects. Moreover, recombinant HGF failed to prevent CTGF expression and collagen accumulation in lung fibroblasts derived from African American subjects. CONCLUSION: Ethnic differences exist in terms of antifibrotic HGF expression in lung fibroblasts derived from Caucasian and African American subjects. Reduced levels of HGF as well as a deficiency in c-Met receptor function appear to be present in African American patients with SSc. These findings may explain in part the greater disease severity and worse prognosis observed in African Americans with SSc.