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The Critical Role of Shc in Insulin-Like Growth Factor-I-Mediated Mitogenesis and Differentiation in 3T3-L1 Preadipocytes

Department of Pediatrics Rhode Island Hospital (C.M.B., P.A.G., R.A.F.) Providence, Rhode Island 02903
Department of Medicine (A.R.F.) Roger Williams Hospital Providence, Rhode Island 02908
Departments of Pediatrics and Pathology and Laboratory Medicine Brown University Providence, Rhode Island 02906

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 (PPAR) 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, PPAR, 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.

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