Hepatocyte growth factor (HGF) exerts several distinct ef-Hepatocyte growth factor (HGF) activated phospholifects on a variety of cell types: it is the most potent mitogen pase D (PLD) in primary-cultured rat hepatocytes, as for hepatocytes in primary culture as well as other cell assessed by the formation of phosphatidylbutanol types, [1][2][3][4][5] it is a motility and invasion-inducing factor for epi-(PBut), a specific and stable product of PLD activity in thelial and endothelial cells, [6][7][8][9] it has cytotoxic and cytostatic the presence of 0.3% butanol. PLD hydrolyzes phosphatieffects on some cell lines, [10][11][12] and it is an inducer of epithelial dylcholine to choline and phosphatidic acid (PA), which morphogenesis. 13,14 The HGF receptor is the c-met protooncois further metabolized to diacylglycerol (DG) by PA gene product 15,16 and several signaling events have been rephosphohydrolase (PAP). In HGF-stimulated hepatoported following HGF receptor activation. [17][18][19][20][21][22][23] In the previous cytes, butanol prevented the formation of PA and DG.
reports, 19,23 we showed that HGF stimulated inisitol 1,4,5-A PAP inhibitor, propranolol, inhibited DG production trisphosphate formation and increased in intracellular Ca 2/ with a reciprocal increase of PA, implying that PLD concentration. In contrast to rapid and transient change of played a role in the formation of not only PA but DG.
trisphosphate, 1,2-diacylglycerol (DG) formation was bipha-Inhibitors for protein kinase C (PKC), Ro31-8425, H-7, sic and the second sustained phase was mainly caused by and calphostin C, reduced HGF-induced PLD activation.
phosphatidylcholine (PC) hydrolysis, partly by phosphatidyl-A protein tyrosine kinase (PTK) inhibitor, genistein but choline-phospholipase C. 19 In a variety of cell types, 24,25 hownot its inactive analogue daidzein, inhibited PLD activaever, phospholipase D (PLD) is well known to play an imtion by HGF. Moreover, depletion of extracellular Ca 2/ portant role in the generation of second messengers, by omission of Ca 2/ or by chelating residual Ca 2/ with phosphatidic acid (PA), and DG, which are thought to be ethyleneglycol-bis(b-aminoethyl ether)-N,N,N,N-tetrainvolved in induction of early response genes. 26 Hydrolysis of acetic acid (EGTA) abolished HGF-induced PLD activaphospholipid(s), mainly PC, by PLD yields PA and choline. tion. HGF, phorbol myristate acetate (PMA) and a DG PA can be further converted to DG by PA phosphohydrolase analog, oleylacetylglycerol (OAG), activated the expres-(PAP). Moreover, it has recently been reported that vasopression of c-jun and c-fos messenger RNAs (mRNAs). Ro31sin, phorbol myristate acetate (PMA), or A23187 activated 8425, calphostin C, and genistein, which prevented PLD in rat hepatocytes, 27 and that PLD activity in hepato-HGF-induced PLD activation, inhibited induction of cytes appeared to be mediated by protein kinase C (PKC)these immediate early genes. Butanol and propranolol dependent and Ca 2/ -dependent pathways. Furthermore, at concentrations which effectively inhibited the forma-PLD in rat liver plasma membrane is activated by a small G tion of DG, suppressed HGF-induced expression of c-jun protein, RhoA. 28 However, little information is available and c-fos mRNAs. However, HGF-induced mitogen-actiabout PLD in the HGF-mediated signaling pathway in hepavated protein kinase (MAPK) activation was not affected tocytes. by both butanol and propranolol. These results suggest
In the present study, we have examined the PLD activation that PTK, PKC, and Ca 2/ regulate HGF-induced PLD acin HGF-stimulated rat hepatocytes in primary culture. The tivation, and that DG produced by PLD pathway may results suggest that PKC and extracellular Ca 2/ play implay a role in the induction of immediate early genes, portant roles in HGF-stimulated PLD activation, and that which is activated in MAPK-independent manner, in rat the DG production by HGF-mediated PLD activation leads to hepatocytes. (HEPATOLOGY 1996;24:1274-1281.) the expression of c-jun and c-fos messages in rat hepatocytes.
Methods
Abbreviations: HGF, hepatocyte growth factor; DG, 1,2-diacylglycerol; PC, phosphati-Materials. [9, H]Palmitic acid (52.4 Ci/mmol) was obtained dylcholine; PLD, phospholipase D; PA, phosphatidic acid; PAP, phosphatidate phosphofrom Amersham (Buckinghamshire, UK). [Methyl-3 H]thymidine (10 hydrolase; PMA, 4b-phorbol 12-myristate 13-acetate; PKC, protein kinase C; PBut, Ci/mmol) was from American Radiolabeled Chemicals Inc. (St. Louis, phosphatidylbutanol; MAPK, mitogen-activated protein kinase; RT-PCR, reverse tran-MO). LK6D slica gel plates and GF/C (glass microfibre) filters were scriptase-polymerase chain reaction; PTK, protein tyrosine kinase; EGTA, ethyleneglyfrom Whatman (Clifton, NJ). Williams' medium E and reverse trancol-bis(b-aminoethyl ether) N,N,N,N,-tetraacetic acid; mRNA, messenger RNA; TLC, scriptase were from Gibco/BRL (Grand Island, NY). Taq polymerase thin-layer chromatography.