Abstract
p38 mitogen-activated protein kinases (p38-MAPKs) are activated by cytokines, cellular stresses, growth factors, and hormones. We show here that p38-MAPKs 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 Chinese hamster ovary cells. The effect of TSH was fully mimicked by the adenylyl cyclase activator, forskolin, and by a permeant analog of cAMP. The effect of forskolin was reproduced in FRTL5 rat thyroid cells. TSH also stimulated 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 alpha-isoform of p38-MAPK assayed by phosphorylation of the transcription factor ATF2. The activity of MAPK-activated protein kinase-2 was stimulated by TSH and forskolin. This stimulation was abolished by SB203580, a specific inhibitor of p38-MAPKs. The protein kinase A inhibitor H89 inhibited the stimulation of phosphorylation of p38-MAPKs by forskolin, whereas inhibitors of protein kinase C, p70(S6k), and phosphatidylinositol 3-kinase were ineffective. Expression of the dominant negative form of Rac1, but not that of Ras, blocked forskolin-induced p38-MAPK activation. Diphenylene iodonium, a potent inhibitor of NADPH oxidase(s), and ascorbic acid, an effective free radical scavenger, suppressed TSH- or forskolin-stimulated p38-MAPK phosphorylation, indicating that the generation of reactive oxygen species plays a key role in signaling from cAMP to p38-MAPKs. Inhibition of the p38-MAPK pathway with SB203580 partially but significantly, attenuates cAMP- and TSH-induced 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, involving cAMP, protein kinase A, Rac1, and reactive oxygen species and resulting in the activation of a signaling kinase cascade that includes MAPK kinase 3 or 6, p38-MAPK, and MAPK-activated protein kinase-2.
Highlights
P38 mitogen-activated protein kinases (p38-MAPKs) are activated by cytokines, cellular stresses, growth factors, and hormones
The phosphorylation of p38-MAPK was analyzed as a function of incubation time by immunoblotting using a specific anti-phospho-p38-MAPK antibody. Recombinant human TSH (rhTSH) (1 milliunit/ml) stimulated the phosphorylation of p38-MAPK in a time-dependent manner in hTSHR-CHO cells (Fig. 1A) but not in wild-type CHO cells (Fig. 1B). p38-MAPK phosphorylation occurred after incubation for 20 min and reached a maximum at 60 –90 min
Upstream Activators Involved in Stimulation of p38-MAPK by thyroid-stimulating hormone (TSH) or Forskolin—We examined the possible involvement of phosphatidylinositol 3-kinase (PI3K) or/and p70S6k in TSHinduced p38-MAPK phosphorylation. hTSHR-CHO cells were incubated with 100 nM wortmannin for 30 min and incubated with rhTSH
Summary
P38 mitogen-activated protein kinases (p38-MAPKs) are activated by cytokines, cellular stresses, growth factors, and hormones. HTSHR-CHO cells were incubated with rhTSH, forskolin, or anisomycin, the p38MAPK was immunoprecipitated from cell extracts with an antibody against the ␣-isoform of p38-MAPK, and its activity was assayed using GST-ATF2 as a substrate (Fig. 4). The MAPKAP kinase-2 activity stimulated by TSH was completely inhibited by incubating the cells with hTSHR-CHO cells and FRTL-5 cells were treated (ϩSB) or not (ϪSB) with 20 M SB203580 for 30 min, and cells were treated with 10 M forskolin for the indicated times.
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