The critical role of Shc in insulin-like growth factor-I-mediated mitogenesis and differentiation in 3T3-L1 preadipocytes

Insulin-like growth factor-I (IGF-I) stimulates mitogenesis in proliferatin g preadipocytes, but when cells reach confluence and become growth arrested , IGF-I stimulates differentiation into adipocytes. IGF-I induces signaling pathways that involve IGF-I receptor-mediated tyrosine phosphorylation of Shc and insulin receptor substrate 1 (IRS-1). Either of these adaptor prote ins can lead to activation of the three-kinase cascade ending in activation of the extracellular signal-regulated kinase 1 and -2 (ERK-1 and -2) mitog en-activated protein kinases (MAPKs). Several lines of evidence suggest tha t activation of MAPK inhibits 3T3-L1 preadipocyte differentiation. We have shown that IGF-I stimulation of MAPK activity is lost as 3T3-L1 preadipocyt es begin to differentiate. This change in MAPK signaling coincides with los s of IGF-I-mediated Shc, but not IRS-1, tyrosine phosphorylation. We hypoth esized that downregulation of MARK via loss of proximal signaling through S hc is an early component in the IGF-I switch from mitogenesis to differenti ation in 3T3-L1 preadipocytes. Treatment of subconfluent cells with the MEK inhibitor PD098059 inhibited both IGF-I-activation of MARK as well as H-3- thymidine incorporation. PD098059, in the presence of differentiation-induc ing media, accelerated differentiation in subconfluent cells as measured by expression of adipocyte protein-2 (aP-2), peroxisome proliferator-activate d receptor gamma (PPAR gamma) and lipoprotein lipase (LPL). Transient trans fection of subconfluent cells with Shc-Y317F, a dominant-negative mutant, a ttenuated IGF-I-mediated MAPK activation, inhibited DNA synthesis, and acce lerated expression of differentiation markers aP-2, PPAR gamma, and LPL. We conclude that signaling through Shc to MARK plays a critical role in media ting IGF-I-stimulated 3T3-L1 mitogenesis. Our results suggest that loss of the ability of IGF-I to activate Shc signaling to MARK may be an early comp onent of adipogenesis in 3T3-L1 cells.