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.
Totzke
F
F
University of Freiburg, Institute of Molecular Cell Biology, Federal Republic of Germany.
Marmé
D
D
Hug
H
H
D016475:3T3 Cells; D000818:Animals; D001345:Autoradiography; D015152:Blotting, Northern; D015153:Blotting, Western; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D016898:Interferon-alpha / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor; D014158:Transcription, Genetic; D014162:Transfection; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D007295:Inositol Phosphates; D016898:Interferon-alpha; D007527:Isoenzymes; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D004247:DNA; D008715:Methionine; D017479:Receptors, Platelet-Derived Growth Factor; D010738:Type C Phospholipases