TRANSFORMING GROWTH-FACTOR-BETA REGULATION OF RETINOBLASTOMA GENE-PRODUCT AND E2F TRANSCRIPTION FACTOR DURING CELL-CYCLE PROGRESSION IN MOUSE FIBROBLASTS

The mechanism by which transforming growth factor beta (TGF beta) exer ts growth stimulatory effects was examined in C3H/10T1/2 mouse fibrobl asts by study of cell cycle regulation of the retinoblastoma gene prod uct (p110(Rb)) and transcriptional regulation of the p110(Rb)-associat ed transcription factor, E2F. Northern blotting analysis shows that TG F beta and/or epidermal growth factor (EGF) stimulate by three to sixf old the level of Rb mRNA which is also reflected by the increased leve ls of p110(Rb). p110(Rb) becomes phosphorylated in mid-G1 and further phosphorylated at the G(1)/S transition. Hyperphosphorylation of p110( Rb) by TGF beta can be observed when cells are in S phase. TGF beta st imulates by three to fourfold the activity of cdk2 kinase consistent w ith the observed phosphorylation of p110(Rb) and also with the possibi lity that the kinase is involved in phosphorylating p110(Rb) close to the G(1)/S transition. Thus, TGF beta as a growth stimulator induces, as does EGF, the phosphorylation of p110(Rb) during cell cycle progres sion. Transient transfection of E2F promoter constructs was used to an alyze the effect of TGF beta on the modulation of E2F-mediated transcr iption. The data revealed that TGF beta can stimulate wild-type adenov iral E2 promoter activity by 12-fold. Taken together, TGF beta-induced phosphorylation of p110(Rb) in mouse fibroblasts appears to exert a p ositive regulatory function upon genes that have a pivotal role in the G(1)/S transition of the cell cycle. (C) 1994 Wiley-Liss, Inc.