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 hepatoma 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 understood; it is unclear whether a functional insulin receptor is required for this action. In this study, we evaluate the mechanism for insulin induction of g33 mRNA in Chinese hamster ovary (CHO) cells transfected with the neomycin-resistant plasmid (CHONeoB), human insulin receptor (CHONewIRa), and a kinase-defective insulin receptor mutated at the ATP-binding site (CHOK1018A). Transfected cells had higher levels of insulin binding than that of CHONeoB cells; insulin-induced phosphorylation of the insulin receptor and its intracellular substrates were impaired in CHOK1018A cells. Maximal insulin 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 sixfold higher in CHONewIRa than in CHONeoB or CHOK1018A cells, which had minimal 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, consistent with insulin interaction with its own receptor. Wortmannin, an inhibitor of phosphatidyl inositol 3-kinase (PI3K), had some effects on insulin stimulation of g33 mRNA in CHO NewIRa cells. PD98059, an inhibitor of mitogen-activated kinase kinase (MAPKK), and rapamycin, a p70 S6 kinase inhibitor, had minimal effect on insulin stimulation of g33 mRNA in all cells tested. By contrast, hydroxy-2-naphthalenylmethyl)phosphonic acid triacetoxymethyl ester (HNMPA(AM)(3), a selective inhibitor of the insulin receptor tyrosine kinase, caused complete inhibition of insulin stimulation of g33 mRNA levels. These data indicate that the insulin receptor with intact kinase activity is required for insulin stimulation of g33 mRNA levels. They also suggest 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 classic insulin receptor transduction pathway, as a minor effect was observed upon inhibition of MAPKK, suggesting that multiple pathways may be involved.
Meléndez
P A
PA
Department of Biochemistry, University of Puerto Rico, School of Medicine, San Juan, Puerto Rico 00936-5067, USA.
Longo
N
N
Jimenez
B D
BD
Cadilla
C L
CL
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology*; D009281:Naphthalenes / Q000494:pharmacology; D063065:Organophosphonates / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D014176:Protein Biosynthesis; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism*; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D025521:Tumor Suppressor Proteins; D000077191:Wortmannin
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D004791:Enzyme Inhibitors; D005419:Flavonoids; D005947:Glucose; D007328:Insulin; D009281:Naphthalenes; D063065:Organophosphonates; D011505:Protein-Tyrosine Kinases; D011506:Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011972:Receptor, Insulin; D020123:Sirolimus; D025521:Tumor Suppressor Proteins; D000077191:Wortmannin; C484017:ERRFI1 protein, human; C060935:(hydroxy-2-naphthalenylmethyl)phosphonic acid; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one