Insulin-induced gene 33 mRNA expression in Chinese hamster ovary cells is insulin receptor dependent

Gene 33 (g33) is a non-tissue-specific gene regulated in rat liver and hepa toma cells by insulin and other agents. It is thought to participate in the transition from quiescence to proliferation in mitogen-treated cells. The mechanism(s) by which insulin exerts its action on g33 are not totally unde rstood; it is unclear whether a functional insulin receptor is required for this action. In this study, we evaluate the mechanism for insulin inductio n of g33 mRNA in Chinese hamster ovary (CHO) cells transfected with the neo mycin-resistant plasmid (CHONeoB), human insulin receptor (CHONewIRa), and a kinase-defective insulin receptor mutated at the ATP-binding site (CHOK10 18A). Transfected cells had higher levels of insulin binding than that of C HONeoB cells; insulin-induced phosphorylation of the insulin receptor and i ts intracellular substrates were impaired in CHOK1018A cells. Maximal insul in induction of mRNA(g33) occurred 3 h after hormonal exposure in all cell lines. The degree of insulin stimulation of g33 mRNA levels was four- to si xfold higher in CHONewIRa than in CHONeoB or CHOK1018A cells, which had min imal levels of insulin-stimulated g33 mRNA levels. Half-maximal stimulation of g33 mRNA levels was observed at 0.06 +/- 0.01 nM in CHONewIRa cells, co nsistent with insulin interaction with its own receptor. Wortmannin, an inh ibitor of phosphatidyl inositol 3-kinase (PI3K), had some effects on insuli n stimulation of g33 mRNA in CHO NewIRa cells. PD98059, an inhibitor of mit ogen-activated kinase kinase (MAPKK), and rapamycin, a p70 SG kinase inhibi tor, had minimal effect on insulin stimulation of g33 mRNA in all cells tes ted. By contrast, hydroxy-2-naphthalenylmethyl)phosphonic acid triacetoxyme thyl ester (HNMPA(AM)(3), a selective inhibitor of the insulin receptor tyr osine kinase, caused complete inhibition of insulin stimulation of g33 mRNA levels. These data indicate that the insulin receptor with intact kinase a ctivity is required for insulin stimulation of g33 mRNA levels. They also s uggest that AKT, a PI 3-kinase downstream effector molecule, could mediate insulin stimulation of g33 mRNA. The mechanism(s) of insulin regulation of g33 expression downstream of receptor do not seem to rely entirely on the c lassic insulin receptor transduction pathway, as a minor effect was observe d upon inhibition of MAPKK, suggesting that multiple pathways may be involv ed. J. Cell. Biochem. 77:432-444, 2000. (C) 2000 Wiley-Liss, Inc.