PEROXISOME PROLIFERATOR ACTIVATED RECEPTOR-GAMMA, CCAAT ENHANCER BINDING-PROTEIN-ALPHA, AND CELL-CYCLE STATUS REGULATE THE COMMITMENT TO ADIPOCYTE DIFFERENTIATION/

Terminal differentiation of stem cells is characterized by cessation o f cell proliferation as well as changes in cell morphology associated with the differentiated state. For adipocyte differentiation, independ ent lines of evidence show that the transcription factors peroxisome p roliferator activated receptor gamma (PPAR gamma) and CCAAT/enhancer-b inding protein alpha (C/EBP alpha) as well as the tumor suppressor ret inoblastoma (Rb) protein are essential. How these proteins promote adi pocyte conversion and how they function cooperatively during the diffe rentiation process remain unclear. We have used retinoic acid (RA) inh ibition of adipogenesis to investigate these issues. RA blocked adipog enesis of 3T3-L1 cells induced to differentiate by ectopic expression of PPAR gamma and C/EBP alpha independently or together. However, unde r these circumstances RA was only effective at preventing adipogenesis when added prior to confluence, suggesting that factors involved in r egulation of the cell cycle might play a role in establishing the comm itment state of adipogenesis that is insensitive to RA. During differe ntiation of wild type 3T3 L1 preadipocytes, we found that Rb protein i s hyperphosphorylated early in adipogenesis, corresponding to previous ly quiescent cells reentering the cell cycle, and later becomes hypoph osphorylated, The data suggest that, together with the coexpression of PPAR gamma and C/EBP alpha, permanent exit from the cell cycle establ ishes the irreversible commitment to adipocyte differentiation.