Adipocyte hyperplasia is a dynamic process that involves the proliferation and differentiation of preadipocytes. Uncontrolled adipocyte hyperplasia is a contributory factor to the development of obesity, a chronic public health problem increasing to epidemic proportions in developed and developing nations. Critical environmental and nutritional cues drive quiescent preadipocytes to proliferate and differentiate into mature adipocytes. Deciphering the molecular mechanisms that control these processes will enable a better understating of the etiology of obesity. A key player that regulates cell proliferation is the cyclin-dependent kinase inhibitor, p21(Cip1). A previous study showed that ablation of p21 caused dramatic accumulation of adipose tissue mass, increased body weight and increased adipocyte cell number in mice. Using 3T3-L1 preadipocytes as a cell model for adipocyte hyperplasia, this study was undertaken to investigate the mechanisms that regulate p21 during preadipocyte proliferation. Studies presented here demonstrate that p21 protein level is regulated by post-transcriptional mechanisms during preadipocyte proliferation.

The 26S proteasome was determined to play a critical role in regulating the transient protein accumulation of p21 during cell cycle progression. Additionally, low p21 protein abundance in density arrested cells and during G1/S transition was maintained by its rapid, 26S proteasome-dependent, degradation. Moreover, Cdk2 activity and the F-box protein, SKP2, were found to contribute to the ubiquitin and proteasome-dependent degradation of p21 during G1/S transition and S phase progression.

It was determined that the PI3K signaling pathway is essential for the optimal accumulation of p21 at mid-G1 phase of the cell cycle. GSK3 is a well known target of the PI3K signaling pathway that has been shown to regulate the 26S proteasomedependent degradation of p21 and Cyclin D1. However, LiCl, a widely used inhibitor of GSK3, was found to prevent the accumulation of p21 and Cyclin D1 and arrested cells in the G1 phase of cell cycle. Further analyses revealed that LiCl mediated its effects on p21, Cyclin D1 and cell cycle progression, independent of GSK3.

Furthermore, the naturally occurring phytochemical, helenalin, dramatically induced p21 protein level and arrested preadipocytes in the G1 phase of cell cycle. Findings revealed that helenalin enhanced the nuclear abundance of p21, suppressed its UPS-dependent degradation and promoted protein-protein interactions between p21 and Cdk2, Cdk4 and Cyclin D1. Studies presented here strongly suggest that helenalin mediates its anti-proliferative effect through post-transcriptional regulation of p21. Collectively, these novel observations provide valuable insight into molecular mechanisms that regulate p21 protein accumulation during adipocyte hyperplasia.