SIGNAL-TRANSDUCTION PATHWAYS LEADING TO INSULIN-INDUCED EARLY GENE INDUCTION

We examined the signal transduction pathway leading to insulin stimulation of two immediate early genes, c-fos, and the early growth response gene, Egr-1. In Rat 1 fibroblasts overexpressing normal human insulin receptors (HIRc-B), insulin and IGF-I rapidly and transiently induced the expression of both c-fos and Egr-1 mRNA with maximum accumulation at 30 min, declining to basal levels at 120 min. Insulin (100 ng/mL) increased c-fos and Egr-1 mRNA expression 10-fold (EC(50) = 20 ng/mL), whereas IGF-I (100 ng/mL) and serum (20%) led to a 3- and 11.5-fold increase, respectively. Insulin-stimulated c-fos protein expression was maximal at 1 h postinduction and undetectable at 4 h. The effects of insulin and IGF-I on both c-fos mRNA and protein expression were absent in Rat I fibroblasts expressing tyrosine kinase-defective human insulin receptors (A/K1018). In cells expressing insulin receptors in which the two C-terminal tyrosines are mutated to phenylalanine (Y/F2 cells), the insulin stimulated increase in Egr-1 and c-fos mRNA was comparable to that of HIRc cells, whereas, in cells expressing C-terminal truncated receptors (Delta CT cells), the insulin induced increase in Egr-1 mRNA was normal, but the c-fos mRNA response was severely blunted. As expected, the insulin effect to increase ras GTP formation and MAP kinase activity was negligible in A/K1018 cells but normal, or supernormal, in Y/F2 cells. Importantly, stimulation of ras GTP was increased in Delta CT cells, whereas stimulation of MAP kinase activity was almost absent. Thus, the insulin signaling cascade was extinguished downstream of ras GTP formation and upstream of MAP kinase. In conclusion, (1) insulin and IGF-I induce a rapid and transient increase in c-fos and Egr-1 mRNA as detected by an RT/PCR assay in HIRc-B cells, (2) in the absence of a competent receptor tyrosine kinase, insulin does not mediate these biologic effects, and (3) insulin signaling is impaired downstream of p21rasGTP formation in Delta CT cells, and this may account for the differential regulation of Egr-1 vs c-fos mRNA.