Involvement of calcium-dependent mechanisms in T3-induced phosphorylation of vimentin of immature rat testis

Thyroid hormones have been shown to act at extra nuclear sites, inducing target cell responses by several mechanisms, frequently involving intracellular calcium concentration. It has also been reported that cytoskeletal proteins are a target for thyroid and steroid hormones and cytoskeletal rearrangements are observed during hormone-induced differentiation and development of rat testes. However, little is known about the effect of 3,5,3′-triiodo-l-thyronine (T3) on the intermediate filament (IF) vimentin in rat testes. In this study we investigated the immunocontent and in vitro phosphorylation of vimentin in the cytoskeletal fraction of immature rat testes after a short-term in vitro treatment with T3. Gonads were incubated with or without T3 and 32P orthophosphate for 30 min and the intermediate filament-enriched cytoskeletal fraction was extracted in a high salt Triton-containing buffer. Vimentin immunoreactivity was analyzed by immunoblotting and the in vitro 32P incorporation into this protein was measured. Results showed that 1 μM T3 was able to increase the vimentin immunoreactivity and in vitro phosphorylation in the cytoskeletal fraction without altering total vimentin immunocontent in immature rat testes. Besides, these effects were independent of active protein synthesis. The involvement of Ca2+-mediated mechanisms in vimentin phosphorylation was evident when specific channel blockers (verapamil and nifedipine) or chelating agents (EGTA and BAPTA) were added during pre-incubation and incubation of the testes with T3. The effect of T3 was prevented when Ca2+ influx was blocked or intracellular Ca2+ was chelated. These results demonstrate a rapid nongenomic Ca2+-dependent action of T3 in phosphorylating vimentin in immature rat testes.