The International Journal of Biochemistry & Cell Biology 45 (2013) 1516–1524 Contents lists available at SciVerse ScienceDirect The International Journal of Biochemistry & Cell Biology journa l h om epa ge: www.elsevier.com/locate/biocel Platelet derived growth factor-evoked Ca 2+ wave and matrix gene expression through phospholipase C in human pulmonary fibroblast Subhendu Mukherjee ∗ , Fuqin Duan, Martin R.J. Kolb, Luke J. Janssen Firestone Institute for Respiratory Health, St. Joseph’s Hospital, Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5 a r t i c l e i n f o Article history: Received 7 January 2013 Received in revised form 2 April 2013 Accepted 11 April 2013 Available online 23 April 2013 Keywords: Calcium signaling Fibrosis Extracellular matrix a b s t r a c t 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 Xestospon- gin C, a potent IP 3 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 col- lagen A1 genes, which was reversed by the use of CPA or U73122. Our study indicates that, in human pulmonary fibroblasts, PDGF acts through IP 3 -induced Ca 2+ -release to trigger Ca 2+ waves, which in turn modulate gene expression of several matrix proteins. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction Fibroblasts are one of the key structural elements in all types of connective tissues of the body. Fibroblasts are metabolically active cells and involved in tissue injury, wound healing, secretion of cytokines, regulation of extracellular matrices, etc. Their primary role is the production of proteins and polysaccharides including collagens, fibronectin, tenascin, proteoglycans, fibronectin, etc., and secretion of these to form the extracellular matrix (ECM) (McAnulty, 2007). On the other hand, fibroblasts also produce matrix metalloproteinases (MMP) and their inhibitors to regulate the degradation of the ECM (McAnulty, 2007). In addition to these, Abbreviations: COPD, chronic obstructive pulmonary disease; CPA, cyclop- iazonic acid; ECM, extracellular matrix; HBSS, Hanks’ buffered saline solution; MMP, matrix metalloproteinases; PDGF, platelet derived growth factor; RyR, ryanodine receptor; U73122, 1-[6-[[(17)-3-methoxyestra-1,3,5(10)-trien-17- yl]amino]hexyl]-1H-pyrrole-2,5-dione. ∗ Corresponding author at: T3338, St. Joseph’s Hospital, 50 Charlton Avenue East, Hamilton, Ontario, Canada L8N 4A6. Tel.: +1 905 522 1155. E-mail address: smukher@mcmaster.ca (S. Mukherjee). fibroblasts play crucial roles in regulating interstitial fluid vol- ume and pressure. Studies suggested that diseases associated with abnormal deposition of ECM are likely to be related to the alteration in fibroblast function (Sivakumar et al., 2012; McAnulty, 2007). This is certainly true for pulmonary fibrosis, but even chronic lung diseases such as chronic obstructive pulmonary disease (COPD) and asthma are accompanied by some degree of fibrotic changes and fibroblast malfunction (Lewis et al., 2005; McAnulty, 2007). Although these diseases are among the most life-threatening dis- eases worldwide, there is no specific treatment targeted to these fibroblast-related pathologies. Recent studies have suggested that fibroblasts play a key role in cancer progression, tumor initiation and development (Marsh et al., 2013). As such, it is very impor- tant to better understand fibroblast biology. Fibroblasts are known to respond to many different growth factors and cytokines; one of them being platelet-derived growth factor (PDGF). The intracellu- lar signaling underlying these responses offer excellent targets for therapeutic intervention, but the exact mechanisms are not entirely clear. PDGF is an important mitogen and chemotactic factor for mes- enchymal cells, such as fibroblasts, vascular smooth muscle cells 1357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2013.04.018