Signalling pathways of insulin-like growth factor-I that are augmented by cAMP in FRTL-5 cells

We have reported that pretreatment of rat FRTL-5 thyroid cells with thyrotr opin (TSH) markedly potentiates the mitogenic response to insulin-like grow th factor-I (IGF-I). The present study was undertaken to determine whether the augmentation by cAMP of IGF-I-dependent tyrosine phosphorylation of kno wn IGF-I receptor substrates plays an important role in the cAMP-dependent potentiation of DNA synthesis induced by IGF-I. Pretreatment with TSH or di butyryl cAMP did not affect the IGF-I-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), In contrast, cAMP pretreatment poten tiated the tyrosine phosphorylation of IRS-2 induced by IGF-I, but did not affect the amount of IRS-2, We found that the IGF-I-dependent tyrosine phos phorylation of 66 kDa Shc (Src homology collagen) was markedly increased by cAMP pretreatment, and that this change was mainly due to an increase in t he levels of 66 kDa Shc protein. Under these conditions, cAMP pretreatment significantly increased binding of Grb2 (growth-factor-receptor-bound prote in 2) to Shc in response to IGF-I, and activation of MAP kinase (mitogen-ac tivated protein kinase) induced by IGF-I was also enhanced by cAMP. The pre sence of PD98059, an inhibitor of MEK (MAP-kinase/Erk kinase), during treat ment with IGF-I partially inhibited the cAMP-dependent augmentation of DNA synthesis in response to IGF-I. On the other hand, cAMP pretreatment increa sed binding of the phosphoinositide 3-kinase (PI 3-kinase) p85 subunit to I RS-2, which was reflected in PI 3-kinase activity. LY294002, a PI 3-kinase inhibitor, strongly depressed IGF-I-dependent DNA synthesis after pretreatm ent with and without TSH or dibutyryl cAMP. Our results suggest that the in teraction between cAMP-dependent and IGF-I-dependent pathways leads to an a ugmentation of cell proliferation, which is mediated, at least in part, thr ough the MAP kinase and PI 3-kinase signalling pathways. These effects are mediated by changes in tyrosine phosphorylation of IGF-I receptor substrate s, including IRS-2 and Shc.