Mechanisms regulating the accumulation of p21(Cip1) during adipocyte hyperplasia

Abstract

Knowledge concerning proliferation and differentiation of preadipocytes is essential to our understanding of adipocyte hyperplasia and its role in the etiology of childhood obesity. As a model of hyperplasia, we investigated mechanisms regulating the cyclin-dependent kinase interacting protein p21(Cip1) during clonal expansion of 3T3-L1 preadipocytes. Treating density-arrested, unstimulated cells with a specific inhibitor of the 26S proteasome resulted in accumulation of p21 protein which localized to the nucleus. These data support the hypothesis that p21 is translocated to the nucleus to be degraded in a proteasome-dependent manner maintaining low protein abundance in density-arrested cells. Stimulating these cells with a differentiation cocktail initiated a series of synchronous cell cycle events including the activation of phosphatidylinositol 3-kinase (PI3K) pathway which temporally preceded transient p21 protein accumulation. We hypothesize that p21 is phosphorylated by Akt near its NLS preventing its degradation, a notion that is supported by the observation that pharmacological inhibition of Akt suppressed p21 accumulation during mid-G1 resulting from decreased protein turnover. We also report that pharmacological inhibition of the nuclear factor kappa B (NFkB) pathway enhanced the accumulation of p21 during mid-G1 and caused cell cycle arrest in a dose dependent manner. Collectively, these data suggest that p21 protein abundance is mediated by mechanisms involving proteasome-dependent degradation by the 26S proteasome, the PI3K and the NFkB signaling pathways. Work supported by grants from American Heart Assoc (0265418U) and NIH (1R21DK072067-01).