Changes in the expression of cyclin dependent kinase inhibitors during differentiation of immortalized fibroblasts...

The mechanisms implicated in the differentiation of fibroblastic precursors into adipocytes can be analyzed in vitro using cell models, such as the 3T3-L1 cell line. Since cell differentiation involves an exit from the cell cycle, it is likely that molecules that inhibit proliferation participate in the control of adipogenesis. This study was aimed at determining the role, if any, of several cyclin-dependent kinase (CDK)-inhibitors and the transcription factor C/EBPα in the process of adipocyte differentitation. We analyzed by Western blot the expression of distinct cyclin-dependent kinase (CDK)-inhibitors and C/EBPα during various stages of differentiation of 3T3-L1 cells to adipocytes. We observed specific changes in the expression of CDK inhibitors and C/EBPα, during the various phases of adipogenesis. Levels of p15^INK4B were maximal in confluent cells prior to the induction of differentiation and minimal in differentiated cells. Maximal levels of p16^INK4A were detected following 48 h of differentiation treatment. Highest levels of p18^INK4C were measured during the phase of cell confluence prior to treatment and in differentiated cells. p21^CIP1 was expressed during the exponential growth phase, during exit from clonal expansion, and in differentiated cells, while p27^KIP1 was found above all in confluent and differentiated cells. The present results support the participation of CDK-inhibitors in the process of in vitro adipogenesis. Specifically, the proteins p18INK4C, p21CIP1 and p27KIP1 seem to play an outstanding role in the maintenance of the differentiated state of adipocytes. Understanding the molecular mechanisms involved in adipocyte differentiation will presumably facilitate the design of new drugs aimed at novel therapeutic targets.