Platelet derived growth factor-evoked Ca2+ wave and matrix gene expression through phospholipase C in human pulmonary fibroblast.

Abstract

The primary role of fibroblasts is production and degradation of extracellular matrix, and thus it helps in the structural framework of tissues. The close relation between fibroblast malfunction and many diseases such as chronic obstructive pulmonary disease, asthma, and fibrosis is widely accepted. Fibroblasts are known to respond to different growth factors and cytokines including platelet-derived growth factors (PDGF). However, the intracellular signaling mechanisms are not entirely clear. In addition to complex phosphorylation-driven signaling pathways, PDGF is also known to work through Ca(2+) signaling. We hypothesize that in human pulmonary fibroblasts, Ca(2+) waves play an important role in PDGF-mediated changes. To test this hypothesis, we treated human pulmonary fibroblasts, obtained from the lungs of ten donors, with PDGF acutely or overnight plus/minus a variety of blockers under various conditions. Ca(2+) waves were monitored by confocal [Ca(2+)]i fluorimetry, while gene expression of extracellular matrix genes was assessed via RT-PCR method. We found that both acute and overnight PDGF treatment evoked Ca(2+) waves. Removal of external Ca(2+) or depletion of internal Ca(2+) store using Cyclopiazonic acid (CPA) completely occluded PDGF-evoked Ca(2+) waves. Ryanodine, which blocks ryanodine receptor channels, had no effect on PDGF-evoked Ca(2+) wave, whereas the phospholipase C inhibitor U73122 and Xestospongin C, a potent IP3 receptor blocker, reduced the rapid PDGF-response to a relatively slowly-developing rise in [Ca(2+)]i. We also found that PDGF dramatically increased the expression of fibronectin1 and collagen A1 genes, which was reversed by the use of CPA or U73122. Our study indicates that, in human pulmonary fibroblasts, PDGF acts through IP3-induced Ca(2+)-release to trigger Ca(2+) waves, which in turn modulate gene expression of several matrix proteins.