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 proliferating 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 proteins 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) mitogen-activated protein kinases (MAPKs). Several lines of evidence suggest that activation of MAPK inhibits 3T3-L1 preadipocyte differentiation. We have shown that IGF-I stimulation of MAPK activity is lost as 3T3-L1 preadipocytes begin to differentiate. This change in MAPK signaling coincides with loss of IGF-I-mediated Shc, but not IRS-1, tyrosine phosphorylation. We hypothesized that down-regulation of MAPK via loss of proximal signaling through Shc is an early component in the IGF-I switch from mitogenesis to differentiation in 3T3-L1 preadipocytes. Treatment of subconfluent cells with the MEK inhibitor PD098059 inhibited both IGF-I-activation of MAPK as well as 3H-thymidine incorporation. PD098059, in the presence of differentiation-inducing media, accelerated differentiation in subconfluent cells as measured by expression of adipocyte protein-2 (aP-2), peroxisome proliferator-activated receptor γ (PPARγ) and lipoprotein lipase (LPL). Transient transfection of subconfluent cells with Shc-Y317F, a dominant-negative mutant, attenuated IGF-I-mediated MAPK activation, inhibited DNA synthesis, and accelerated expression of differentiation markers aP-2, PPARγ, and LPL. We conclude that signaling through Shc to MAPK plays a critical role in mediating IGF-I-stimulated 3T3-L1 mitogenesis. Our results suggest that loss of the ability of IGF-I to activate Shc signaling to MAPK may be an early component of adipogenesis in 3T3-L1 cells. plays an important role in preadipocyte growth and differentiation. IGF-I stimulates mitogenesis in many cell types in culture, including preadipocytes (1), and IGF-I (or pharmacological doses of insulin) is clearly required for preadipocyte differentiation in vitro (2, 3). This dual role of IGF-I, stimulation of both mitogenesis and differentiation, indicates that these responses are not necessarily mutually exclusive. In vitro, IGF-I stimulates differentiation of preadipocytes once density-induced growth arrest has occurred (4). The mechanisms of intracellular signaling used by IGF-I to promote mitogenesis or differentiation of preadipocytes are now beginning to be elucidated. The biological effects of IGF-I are mediated through the IGF-I receptor (IGFR), a member of the tyrosine kinase family of growth factor receptors. The activated IGFR tyrosine kinase phosphorylates specific substrates, such as the adaptor proteins Shc and insulin receptor substrate-1 (IRS-1). Tyrosine phosphorylation of these proteins stimulates specific protein-protein interactions via well characterized domains to mediate diverse signaling pathways (5). Shc, a substrate for many growth factor receptor tyrosine kinases, is a key component of signaling complexes that activate several effector pathways, including the small G- protein Ras (6). Ras then activates the three-kinase cascade terminating in the mitogen-activated protein kinases (MAPKs), extracellular signal regulated kinase 1 (ERK1) and ERK2 (7, 8). These MAPK isoforms mediate the mitogenic effects of IGF-I in a number of cell types (9–11). IRS-1, considered to be the major substrate of the IGFR, can activate multiple downstream targets, including Ras and phosphatidylinositol 3- kinase (PI3K) (5, 12). We have previously shown that IGF-I is a potent stimulator of the MAPKs ERK1 and ERK2 in proliferating 3T3-L1 preadipocytes, and that there is a dramatic decrease in IGF-I-stimulated MAPK activity during early differentiation of 3T3-L1 cells (13). This change in MAPK signaling coincides with the loss of IGF-I-stimulated Shc, but not IRS-1, phosphorylation. This indicates that proximal signaling through Shc to MAPK is down-regulated very early in IGF-I-mediated preadipocyte differentiation. Activation of MAPK in growth-arrested 3T3-L1 cells through transfection of active components of the MAPK cascade (14) or by epidermal growth factor (15, 16) inhibits differentiation. Therefore, down-regulation of MAPK activity may be necessary for preadipocyte differentiation. We hypothesized that down-regulation of MAPK via loss of proximal signaling by Shc is involved in mediating the IGF-I switch from 3T3-L1 mitogenesis to differentiation. We used a synthetic inhibitor of MAPK activation (PD098059) or a dominant-negative form of Shc to inhibit mitogenesis and promote differentiation in 3T3-L1 cells independently of the usual requirement for density-induced growth arrest. PD098059 inhibits high-dose insulin stimulation of MAPK in 3T3-L1 cells (14). The Shc mutant consists of a tyrosine-to-phenylalanine substitution at position 317, rendering it defective in signaling to Ras (17). Our results demonstrate the critical role of Shc in the switch from IGF-I-mediated mitogenesis to IGF-I-mediated differentiation of 3T3-L1 cells.