Mitogen-activated protein kinase activation is not necessary for, but antagonizes, 3T3-L1 adipocytic differentiation.

In 3T3-L1 fibroblasts, Ras proteins mediate both insulin-induced differentiation to adipocytes and its activation of cytosolic serine/threonine kinases, including Raf-1 kinase, mitogen-activated protein kinase (MAPK), and Rsk. Here, we report that insulin- and Ras-induced activation of MAPK is not required for the differentiation process and in fact antagonizes it. The treatment of 3T3-L1 preadipocytes with MEK-specific inhibitor PD98059 blocked insulin- and Ras-induced MAPK activation but had no effect on or slightly enhanced adipocytic differentiation. Tumor necrosis factor alpha (TNF-a), an inhibitor of insulin-stimulated adipogenesis, activated MAPK in 3T3-L1 cells. PD98059 treatment blocked MAPK activation by TNF-a and reversed the blockade of adipogenesis mediated by low (1 ng/ml) TNF-a concentrations. 3T3-L1 transfectants containing hyperactivated MEK1 or overexpressed MAPK displayed impaired adipocytic differentiation. PD98059 treatment also reversed the blockade of differentiation in MEK1 transfectants. These results indicate that MAPK does not promote but can contribute to inhibition of the process of adipocytic differentiation of 3T3-L1 cells. Adipocytic differentiation is a complex process regulated by many hormones, growth factors, and cytokines. Whereas some of those signals stimulate differentiation to adipocytes, others inhibit this event (13, 20), presumably based on the ability to ultimately alter gene expression. Preadipocytic cell lines, such as 3T3-L1, undergo differentiation after continuous exposure to pharmacological doses of insulin or physiologic doses of insulin-like growth factor 1 and are additionally induced by glucocorticoids and fatty acids (27, 37, 41, 43). In contrast, tumor necrosis factor alpha (TNF-a), a cytokine secreted by macrophages and adipose tissue, inhibits adipocytic differentiation (44, 45). However, the mechanisms that mediate the actions of insulin and other ligands on differentiation have yet to be clearly defined. We demonstrated previously that overexpression of Ras induces adipocytic differentiation of 3T3-L1 cells (3, 34), implicating Ras proteins as obligatory signaling intermediates in insulin-stimulated differentiation pathways. Our prior studies also showed that Ras proteins are necessary and sufficient for insulin activation of the cytosolic kinases Raf-1, mitogen-activated protein kinase (MAPK), and Rsk (35, 36); furthermore, Raf-1 participates downstream of Ras in the signaling cascade resulting in adipocytic differentiation. However, in contrast to its role in proliferating cells (39, 51, 54), Raf-1 kinase activation in differentiating 3T3-L1 cells is completely dissociated from activation of MAPK by insulin (35, 36), suggesting that at least two separate signals emerge from Ras after insulin stimulation, regulating either growth or differentiation depending on the state of the cell or the presence of other environmental factors. Dissociation between Raf-1 kinase and MAPK has also previously been described for other cell types (15, 39, 55‐57). To further define the role(s) of divergent signals emanating from Ras in mediating the differentiation process, we examined the consequences of blocking or enhancing specific components of the MEK/MAPK signaling pathway in differentiating 3T3-L1 cells. For this purpose, we used a variety of distinct, separate experimental approaches, including treatment with specific inhibitors of MEK activity and insulin-induced differentiation and transfection with specific forms of MEK and Erk. In sharp contrast to the conclusion of a previous report based on antisense technology (42), our results indicate that MAPK activation is not a component of the signaling cascade which mediates adipogenesis and that MAPK in fact opposes such as differentiation process.