Inhibitory guanine nucleotide regulatory protein activation of mitogen-activated protein kinase in experimental hepatocellular carcinoma in vitro

Abstract

Hepatocellular carcinoma (HCC) is associated with altered expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins). This study addresses the interaction between Gi-proteins and the extracellular regulated kinase (ERK) component of the mitogen activated protein kinase (MAPK) cascade in regulating mitogenesis in an experimental model of HCC. Pharmacological agents which selectively interact with specific target proteins involved in signal transduction through a Gi-MAPK pathway have recently become available. These agents in combination with scientific assays allow us to address the role of individual components of this cascade in the regulation of mitogenesis in HCC. These studies were performed using rat hepatic tumorigenic cells (H4IIE) and isolated cultured hepatocytes in vitro in conjunction with pharmacological agents which interact with Gi-protein or MAPK components of intracellular signalling. Direct activation of Gi-proteins with mastoparan M7 (M7) significantly increased nuclear thymidine incorporation in hepatic tumorigenic H4IIE cells in a dose-dependent manner (10-1000 nM, n = 4, P < 0.05), an effect that was abolished by treatment with either pertussis toxin (PTx) or the selective mitogen-activated ERK-regulated kinase (MEK) inhibitor PD098059. In contrast, M7 inhibited nuclear thymidine incorporation in serum-stimulated isolated hepatocytes. ERK2 activity was then determined as the ability of immunoprecipitated ERK2 proteins to phosphorylate the ERK substrate myelin basic protein. These studies demonstrated a time- and dose-dependent increase in ERK2 activity in H4IIE cells following Gi-protein activation with M7, a maximal response being attained at 20 min. In contrast, M7 failed to significantly alter ERK2 activity in isolated cultured hepatocytes at any of the doses or time points assayed (10-5000 nM, 10-120 min). Gi-stimulated ERK activation was completely blocked in tumorigenic cells following treatment with PTx. These data demonstrate for the first time a Gi-linked MAPK cascade in experimental HCC, activation of which stimulates cellular mitogenesis.