Inducible expression of human phospholipase C-gamma 2 and its activation by platelet-derived growth factor B-chain homodimer and platelet-derived growth factor A-chain homodimer in transfected NIH 3T3 fibroblasts.

Growth factors such as platelet-derived growth factor (PDGF) have been shown to activate phospholipase C-gamma 1 (PLC-gamma 1). We have overexpressed the human PLC-gamma 2 (hPLC-gamma 2) cDNA in murine NIH 3T3 fibroblasts using the interferon-type-I inducible murine Mx promoter. Northern blot analysis revealed an induction of hPLC-gamma 2 mRNA by interferon (IFN) alpha of about 25-fold as compared to the uninduced transcript level. Western blot analysis with anti(bovine PLC-gamma 2) antiserum showed increased hPLC-gamma 2 protein levels in hPLC-gamma 2 transfected cells. Induction with IFN alpha resulted only in a slight further increase. After labelling the cells with [35S]methionine an increase of radioactive label in a protein migrating at 148 kDa could be detected in IFN-alpha-stimulated, hPLC-gamma 2 overexpressing cells. PLC activity in homogenates from hPLC-gamma 2 overexpressing cells was increased as compared to control cells transfected with the vector lacking the hPLC-gamma 2 cDNA insert. There was no difference between in vitro PLC activity in homogenates from PDGF B-chain homodimer (BB) treated and untreated cells. PLC activity was mainly present in the soluble fraction. After incubation of hPLC-gamma 2 overexpressing cells with IFN alpha, the in vitro activity of PLC increased significantly in the soluble fraction. Stimulation with PDGF BB increased inositol phosphate production about 3.5-fold in control cells and about 10-fold in hPLC-gamma 2 overexpressing cells. PDGF A-chain homodimer (AA) showed slightly smaller effects. These results demonstrate that human PLC-gamma 2 can be expressed functionally in murine NIH 3T3 fibroblasts and can be activated by both murine PDGF receptors, alpha and beta type.