Involvement of tyrosine phosphorylation in the regulation of 5'-deiodinases in FRTL-5 rat thyroid cells and rat astrocytes.

Recent studies suggest that protein tyrosine phosphorylation may play a role in the regulation of thyroid growth and function. In the present study, we used genistein, a specific inhibitor of tyrosine phosphorylation, to determine if tyrosine phosphorylation is involved in the regulation of type I 5'-deiodinase (5'D-I) expression in FRTL-5 cells and type II 5'-deiodinase (5'D-II) in rat astrocytes. Incubation of FRTL-5 cells with genistein (100 microM) for 3 days had no effect on cell viability as assessed by trypan blue exclusion. In TSH-deprived cells, incubation of FRTL-5 cells with genistein (100 microM) resulted in a modest, but not significant, decrease in 5'D-I activity. Incubation of FRTL-5 cells with TSH (100 microU/ml), Bu2cAMP (0.5 mM) or forskolin (1 microM) resulted in marked increases in 5'D-I activity. In the presence of genistein (100 microns), however, the TSH, Bu2cAMP and forskolin-induced increases in 5'D-I activity were completely inhibited. In Bu2cAMP-stimulated FRTL-5 cells, incubation with genistein (1, 10, and 100 microM) resulted in a dose-dependent decrease in 5'D-I activity, with 100 microns genistein completely blocking the Bu2cAMP-induced increase in 5'D-I activity. Similarly, we found that in FRTL-5 cells, genistein (100 microns) completely blocked the Bu2cAMP-induced increase in 5'D-I messenger RNA (mRNA) levels, DNA synthesis as assessed by [3H]thymidine incorporation, and the T3-induced increase in 5'D-I activity. To determine if addition of genistein to FRTL-5 cells resulted in a general inhibition of Bu2cAMP-induced responses, we examined its effect on the Bu2cAMP-induced increase in c-fos mRNA levels. Bu2cAMP-induced c-fos mRNA levels were not affected by the treatment of cells with genistein (100 microM). We then examined the effect of genistein on the Bu2cAMP and hydrocortisone-induced 5'D-II activity in cultured rat astrocytes. Genistein (100 microM) had no effect on cell viability as assessed by trypan blue exclusion. In serum deprived astrocytes, addition of Bu2cAMP (1 mM) and hydrocortisone (100 nM) resulted in a 110-fold increase in 5'D-II activity. Addition of genistein (100 microM) to stimulated astrocytes completely blocked the Bu2cAMP and hydrocortisone-induced increase in 5'D-II activity. The present data suggest that tyrosine phosphorylation-dephosphorylation may play an important role in the regulation of thyroid hormone deiodination and action in the thyroid and brain.