Thyroid-stimulating hormone and cyclic AMP activate p38 mitogen-activated protein kinase cascade - Involvement of protein kinase A, Rac1, and reactive oxygen species

p38 mitogen-activated protein kinases (p38-MAPKs) are activated by cytokine s, cellular stresses, growth factors, and hormones. We show here that p38-M APKs are activated upon stimulation by thyroid-stimulating hormone (TSH) or cAMP. TSH caused the phosphorylation of p38-MAPK in Chinese hamster ovary cells stably transfected with the human TSH receptor but not in wild-type C hinese hamster ovary cells. The effect of TSH was fully mimicked by the ade nylyl cyclase activator, forskolin, and by a permeant analog of cAMP. The e ffect of forskolin was reproduced in FRTL5 rat thyroid cells. TSH also stim ulated the phosphorylation of MAPK kinase 3 or 6, over the same time scale as that of p38-MAPKs. TSH and forskolin stimulated the activity of the cy-i soform of p38-MAPK assayed by phosphorylation of the transcription factor A TF2. The activity of MAPK-activated protein kinase-2 was stimulated by TSH and forskolin. This stimulation was abolished by SB203580, a specific inhib itor of p38-MAPKs. The protein kinase A inhibitor H89 inhibited the stimula tion of phosphorylation of p38-MAPKs by forskolin, whereas inhibitors of pr otein kinase C, p70(S6k), and phosphatidylinositol 3-kinase were ineffectiv e. Expression of the dominant negative form of Rad, but not that of Ras, bl ocked forskolin-induced p38-MAPK activation. Diphenylene iodonium, a potent inhibitor of NADPH oxidase(s), and ascorbic acid, an effective free radica l scavenger, suppressed TSH- or forskolin-stimulated p38-MAPK phosphorylati on, indicating that the generation of reactive oxygen species plays a key r ole in signaling from cAMP to p38-MAPKs. Inhibition of the p38-MAPK pathway with SB203580 partially but significantly, attenuates cAMP- and TSH-induce d expression of the sodium iodide symporter in FRTL-5 cells. These results point to a new signaling pathway for the G(s)-coupled TSH receptor, involvi ng cAMP, protein kinase A, Rad, and reactive oxygen species and resulting i n the activation of a signaling kinase cascade that includes MAPK kinase 3 or 6, p38-MAPK, and MAPK-activated protein kinase-2.