cells. [1][2][3][4] The HGF receptor is the c-met proto-oncogene prod-Treatment of primary cultured rat hepatocytes with hepatouct that has tyrosine kinase activity. Several signaling events cyte growth factor (HGF) gives rise to inositol phosphate forhave been reported to occur after HGF-receptor activation. 5,6 mation, cytosolic calcium oscillation, activation of mitogen-acti-HGF causes the tyrosine phosphorylation of phosphatidylvated protein (MAP) kinase and phospholipase D (PLD), and inositol-specific phospholipase Cg 1 (PI-PLCg 1 ), 7 leading to arachidonic acid release, leading to DNA synthesis. Pretreatinositol trisphosphate (IP 3 ) formation and an increase in ment of cultured hepatocytes with pertussis toxin (PT), which intracellular Ca 2/ concentration ([Ca 2/ ] i ). 8,9 In contrast to is known to adenosine diphosphate-ribosylate Gi and Go guathe rapid and transient change in IP 3 , 1,2-diacylglycerol (1,2nine nucleotide-binding proteins and to inhibit their functions, DG) formation was biphasic, and the second sustained phase partially inhibited HGF-induced [ 3 H]thymidine incorporation was mainly due to phosphatidylcholine hydrolysis. 10 Furin a concentration-dependent manner. These results suggest thermore, it is suggested that phosphatidylinositol 3-kinase that HGF-mediated DNA synthesis of hepatocytes is partly binds to the HGF receptor after stimulation. 11,12 Moreover, regulated via PT-sensitive guanine nucleotide-binding protein.
recent studies have demonstrated that HGF caused activation Therefore, the effects of PT treatment on HGF-induced signalof mitogen-activated protein (MAP) kinase [13][14][15] and arachitransduction pathways were investigated. HGF-induced MAP donic acid (AA) release in primary cultured rat hepatocytes. 14 kinase activation and arachidonic acid release were decreased However, the detailed signaling mechanism remains to be by PT treatment, whereas PLD activation was diminished by clarified.
PT to the level of unstimulated control. PT also interfered
Growth factors such as epidermal growth factor (EGF) and with HGF-induced inositol phosphate formation and cytosolic platelet-derived growth factor (PDGF) exert various cellular calcium oscillation. These results suggest that both PT-sensitive responses after binding to their receptors and activation of and PT-insensitive pathways are involved in HGF-induced sigthe receptor tyrosine kinase activity. Several signal-transducnaling. (HEPATOLOGY 1997;26:295-300.)
ing molecules bind to activated receptors through tyrosine phosphorylation. Tyrosine phosphorylation of PI-PLC-g 1 by Hepatocyte growth factor (HGF) is one of the most potent EGF and PDGF receptors results in formation of IP 3 and 1,2mitogens for hepatocytes in primary culture. This growth DG. 16,17 Usually, growth factor receptors are not assumed to factor also stimulates the growth of a variety of cells, such be coupled with GTP-binding protein (G protein). However, as melanocytes, epidermal keratinocytes, and renal tubular it was demonstrated that pretreatment of hepatocytes in primary culture with pertussis toxin (PT) resulted in inhibition of EGF-mediated increases in inositol phosphates and Ca 2/ Abbreviations: HGF, hepatocyte growth factor; PI-PLCg1, phosphatidylinositol-spemobilization, indicating the possible involvement of PT-sencific phospholipase Cg1; IP, inositol phosphate; IP2, inositol 1,4-bisphosphate; IP3, sitive G protein in EGF signaling. 18-21 These results prompted inositol 1,4,5-trisphosphate; DG, 1,2-diacylglycerol; MAP kinase, mitogen-activated us to investigate the involvement of a PT-sensitive G protein protein kinase; AA, arachidonic acid; EGF, epidermal growth factor; PDGF, plateletderived growth factor; G protein, GTP-binding protein; PT, pertussis toxin; PLD, in the HGF-mediated signal-transduction pathway in primary phospholipase D; ATP, adenosine triphosphate; SDS, sodium dodecyl sulfate; PBut, cultured rat hepatocytes. We report here that the PT pretreatphosphatidylbutanol; EGTA, ethylene glycol-bis(b-aminoethyl ether)-N,N,N,N-tetrament prevented the activation of HGF-stimulated signaling acetic acid; MEM, minimum essential medium; [Ca 2/ ]i, intracellular Ca 2/ concentraevents, such as inositol phosphate formation, Ca 2/ oscillation; PLC, phospholipase C. tion, phospholipase D (PLD) activation, AA release, and MAP