Phospholipase D activation in hepatocyte growth factor-stimulated rat hepatocytes mediates the expressions of c-jun and c-fos: involvement of protein tyrosine kinase, protein kinase C, and Ca2+.

Hepatocyte growth factor (HGF) activated phospholipase D (PLD) in primary-cultured rat hepatocytes, as assessed by the formation of phosphatidylbutanol (PBut), a specific and stable product of PLD activity in the presence of 0.3% butanol. PLD hydrolyzes phosphatidylcholine to choline and phosphatidic acid (PA), which is further metabolized to diacylglycerol (DG) by PA phosphohydrolase (PAP). In HGF-stimulated hepatocytes, butanol prevented the formation of PA and DG. A PAP inhibitor, propranolol, inhibited DG production with a reciprocal increase of PA, implying that PLD played a role in the formation of not only PA but DG. Inhibitors for protein kinase C (PKC), Ro31-8425, H-7, and calphostin C, reduced HGF-induced PLD activation. A protein tyrosine kinase (PTK) inhibitor, genistein but not its inactive analogue daidzein, inhibited PLD activation by HGF. Moreover, depletion of extracellular Ca2+ by omission of Ca2+ or by chelating residual Ca2+ with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) abolished HGF-induced PLD activation. HGF, phorbol myristate acetate (PMA) and a DG analog, oleylacetylglycerol (OAG), activated the expression of c-jun and c-fos messenger RNAs (mRNAs). Ro31-8425, calphostin C, and genistein, which prevented HGF-induced PLD activation, inhibited induction of these immediate early genes. Butanol and propranolol at concentrations which effectively inhibited the formation of DG, suppressed HGF-induced expression of c-jun and c-fos mRNAs. However, HGF-induced mitogen-activated protein kinase (MAPK) activation was not affected by both butanol and propranolol. These results suggest that PTK, PKC, and Ca2+ regulate HGF-induced PLD activation, and that DG produced by PLD pathway may play a role in the induction of immediate early genes, which is activated in MAPK-independent manner, in rat hepatocytes.