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 I kappa B alpha expression, together with MAPK phosphorylation, were studied by immunoblotting of lung fibroblasts derived from white SSc patients. Matrix metalloproteinase I (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-kappa B 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-kappa B 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-kappa B, 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-kappa B activity, but significantly restored HGF-inhibited collagen and CTGF accumulation. NF-kappa B inhibitor BAY 11-7082 did not interfere with MAPK phosphorylation or MMP-1 expression and activation, but significantly inhibited NF-kappa B 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-kappa B signaling pathways, whereas collagen down-regulation is mediated mainly by a MAPK/MMP-1-dependent pathway.