p38 mitogen-activated protein kinase contributes to cell cycle regulation by cAMP in FRTL-5 thyroid cells

Objective: Thyrotropin activates the cAMP pathway in thyroid cells, and stimulates cell cycle progression in cooperation with insulin or insulin-like growth factor-I. Because p38 mitogen-activated protein kinases (p38 MAPKs) were stimulated by cAMP in the FRTL-5 rat thyroid cell line, we investigated (I) the effect of the specific inhibition of p38 MAPKs on FRTL-5 cell proliferation and (ii) the mechanism of action of p38 MAPKs on cell cycle control, by studying the expression and/or the activity of several cell cycle regulatory proteins in FRTL-5 cells. Methods: DNA synthesis was monitored by incorporation of [H-3]thymidine into DNA and the cell cycle distribution was assessed by fluorescence-activated cell sorter analysis. Expression of cell cycle regulatory proteins was determined by Western blot analysis. Cyclin-dependent kinase 2 (Cdk2) activity associated to cyclin E was immunoprecipitated and was measured by an in vitro kinase assay. Results: SB203580, an inhibitor of alpha and beta isoforms of p38 MAPKS, but not its inactive analog SB202474, inhibited DNA synthesis and the G1-S transition induced by forskolin plus insulin. SB203580 inhibited specifically p38 MAPK activity but not other kinase activities such as Akt and p70-S6 kinase. Treatment of FRTL-5 cells with SB203580 decreased total and cyclin E-associated Cdk2 kinase activity stimulated with forskolin and insulin. However, inhibition of p38 MAPKs by SB203580 was without effect on total cyclin E and Cdk2 levels. The decrease in Cdk2 kinase activity caused by SB203580 treatment was not due to an increased expression of p21(Cip1) or p27(Kip1) inhibitory proteins. In addition, SB203580 affected neither Cdc25A phosphatase expression nor Cdk2 Tyr-15 phosphorylation. Inhibition of p38 MAPKs decreased Cdk2-cyclin E activation by regulating the subcellular localization of Cdk2 and its phosphorylation on Thr-160. Conclusions: These results indicate that p38 MAPK activity is involved in the regulation of cell cycle progression in FRTL-5 thyroid cells, at least in part by increasing nuclear Cdk2 activity.