A novel gene, fad49, plays a crucial role in the immediate early stage of adipocyte differentiation via involvement in mitotic clonal expansion.

Adipogenesis is accomplished via a complex series of steps, and the events at the earliest stage remain to be elucidated. To clarify the molecular mechanisms of adipocyte differentiation, we previously isolated 102 genes expressed early in mouse 3T3-L1 preadipocyte cells using a PCR subtraction system. About half of the genes isolated appeared to be unknown. After isolating full-length cDNAs of the unknown genes, one of them, named factor for adipocyte differentiation 49 (fad49), appeared to be a novel gene, as the sequence of this clone showed no identity to known genes. FAD49 contains a phox homology (PX) domain and four Src homology 3 (SH3) domains, suggesting that it may be a novel scaffold protein. We found that the PX domain of FAD49 not only has affinity for phosphoinositides, but also binds to its third SH3 domain. Expression of fad49 was transiently elevated 3 h after differentiation was induced, and diminished 24 h after induction. Induction of the fad49 gene was observed in adipocyte differentiable 3T3-L1 cells, but not in non-adipogenic NIH-3T3 cells. RNAi-mediated knockdown of fad49 significantly impaired adipocyte differentiation. Moreover, the knockdown of fad49 by RNAi inhibited mitotic clonal expansion, and reduced the expression of CCAAT/enhancer-binding protein beta (C/EBPbeta) and C/EBPdelta at the immediate early phase. Taken together, these results show that fad49, a novel gene, plays a crucial role in the immediate early stage of adipogenesis.

Selective induction of the tumor marker glutathione S-transferase P1 by proteasome inhibitors.

Exposure of cells to a wide variety of chemoprotective compounds confers resistance to a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of phase 2 detoxification enzymes such as glutathione S-transferases (GSTs). Transcription factor Nrf2, which is sequestered in the cytoplasm by Keap1 (Kelch-like ECH-associated protein-1) under unstimulated conditions, regulates the induction of phase 2 enzymes. In this study, to explore the role of the proteasome in the detoxification response, we tested the effect of proteasome inhibitors such as MG132, clasto-lactacystin beta-lactone, and lactacystin on the induction of GST isozymes and found that these inhibitors selectively induced the class Pi GST isozyme (GST P1). Down-regulation of the proteasome by antisense oligonucleotides or RNA interference indeed resulted in significant up-regulation of GST P1, suggesting that a decline in the proteasome activity could be directly or indirectly linked to the induction of GST P1. From the functional analysis of various deletion constructs of the upstream regulatory region of the GST P1 promoter, GST P1 enhancer I was identified as the response element for proteasome inhibition. Overexpression of the wild-type and dominant-negative forms of Nrf2 and Keap1 had little effect on the induction of GST P1 not only by the proteasome inhibitor, but also by phase 2-inducing isothiocyanate, suggesting that there may be a process of GST P1 induction distinct from other phase 2 gene induction mechanisms. Because GST P1 is highly and specifically induced during early hepatocarcinogenesis as well as in hepatocellular carcinoma cells, these data may provide a potential critical role for the proteasome in the induction of a cellular defense program associated with carcinogenesis.