Altered Gq/G11 guanine nucleotide regulatory protein expression in a rat model of hepatocellular carcinoma: Role in mitogenesis

Guanine nucleotide regulatory proteins (G-proteins) represent an important transmembrane pathway whereby extracellular signals are transduced to intra cellular signaling pathways, The mitogen-activated protein kinase (MAPK) ca scade has been identified as a key factor in transducing numerous mitogenic stimuli. MARK activity is regulated via numerous receptor types, including those linked to Gq/G11-proteins, which regulate phospholipase-C activity. We hypothesized that alterations in a Gq/G11-PLC pathway may contribute to the enhanced cellular mitogenesis characteristic of hepatocellular carcinom a (HCC), possibly via a MAPK-dependent pathway. By using an in vivo model o f HCC we investigated changes in Gq/G11-protein expression in tumorigenic t issue versus adjacent, non-neoplastic liver. In addition we addressed the r ole of Gq/G11-proteins in the regulation of MARK-linked mitogenesis by usin g rat hepatic tumorigenic cells (H4IIE) and isolated hepatocytes in culture , Western blot analysis showed significant increases in Gq alpha and G11 al pha expression in tumorigenic liver versus normal liver specimens, an effec t that was augmented in cultured H4IIE cells versus isolated cultured hepat ocytes. Furthermore, phosphoinositol specific phospholipase-C (PLC) activit y was significantly increased in HCC versus normal liver. A specific PLC in hibitor (Et-18-OCH3) caused a dose-dependent decrease in serum stimulated D NA synthesis in both cultured H4IIE cells and isolated rat hepatocytes, the H4IIE cell line showing greater sensitivity to Et-18-OCH3. In addition, se rum-stimulated MAPK activity was significantly enhanced in H4IIE versus cul tured hepatocytes. Moreover, treatment with Et-18-OCH3 significantly attenu ated serum stimulated MAPK activity in both cultured hepatocytes and H4IIE cells. Furthermore, U73122 (Gq alpha-PLC specific uncoupler) and GP2A (Gq a lpha specific inhibitor) mirrored the effects of those observed for Et-18-O CH3 whereas PD98059 (specific MEK inhibitor) completely abolished serum-sti mulated DNA synthesis in tumorigenic H4IIE cells. We conclude that HCC is a ssociated with enhanced Gq/G11-PLC expression/activity as compared with nor mal liver. Furthermore, a PLC-linked MAPK cascade plays a significant role in the progression of the enhanced mitogenesis characteristic of HCC.