Dexamethasone induces the expression of MAPK phosphatase 1. (A) HeLa cells were treated for 2 h with vehicle (methanol) (−) or 100 nM dexamethasone (Dex) (+). Total RNA was prepared and subjected to Northern blotting using probes against MKP-1 (DUSP1), MKP-5 (DUSP10), and MKP-7 and PIR (DUSP11). Exposures of the different Northern blots are not identical, and relative expression levels cannot be inferred. (B) HeLa cells, RAW264.7 (RAW) cells, human skin fibroblasts (HSF), and human peripheral blood T cells were treated for 2 h with vehicle (methanol) or 100 nM dexamethasone. Total RNA was prepared and subjected to Northern blotting using a probe against MKP-1.

Proinflammatory stimuli and dexamethasone cooperatively increase MKP-1 mRNA and protein expression. (A) HeLa-TO cells were stimulated with UV C and simultaneously incubated with dexamethasone (Dex) (100 nM) or vehicle (methanol). After the indicated time period, cells were harvested and MKP-1 mRNA was analyzed by Northern blotting. The 28S rRNA is shown as a loading control. (B) HeLa-TO cells were incubated as described above for panel A, and then MKP-1 protein was immunoprecipitated and detected by Western blotting. (C) HeLa cells were incubated in the absence or presence of IL-1 (20 ng/ml) with or without dexamethasone (100 nM). After the indicated time period, cells were collected, and MKP-1 mRNA was then analyzed by Northern blotting. (D) HeLa cells were incubated as described above for panel C, and MKP-1 protein was immunoprecipitated and detected by Western blotting.

Time courses of p38 activation and MKP-1 expression in the absence or presence of dexamethasone. HeLa cells were left untreated or treated with IL-1 (20 ng/ml) alone or with IL-1 (20 ng/ml) and dexamethasone (Dex) (1 μM), and lysates were prepared after the times indicated (15 min [15′]). (A) MKP-1 was immunoprecipitated from lysate (1.5 mg) and subjected to Western blotting. The band below MKP-1 is the light chain of the immunoprecipitating antibody. (B) Lysate (50 μg) was subjected to Western blotting using an antibody against p38α. (C) MAPK p38 was immunoprecipitated from lysate (500 μg) and used in immune complex kinase assays with recombinant MAPKAPK2 (MK2) as the substrate. (D) Quantitation of the data represented in panel C. The results shown are representative of at least two independent experiments.

Dexamethasone-dependent inhibition of p38 is mediated by a stress-activated protein kinase (SAPK)-specific phosphatase(s). (A) HeLa-TO cells were incubated (+) for 2 h with 100 nM dexamethasone (Dex), and the cells were treated (+) with UV C and sodium orthovanadate was added to the medium at the indicated concentrations. After 30 min, total cell lysates were prepared and analyzed by Western blotting with anti-phospho-p38 antibody or total p38 antiserum. (B) HeLa-TO cells were transiently transfected with 500 ng of either pSR-HA-p38 or pSR-HA-p38^N316. After 24 h, cells were incubated for 2 h with dexamethasone at the indicated concentrations and then treated with UV C (+) or left untreated (−). After 30 min, total cell lysates were prepared and analyzed by Western blotting with anti-phospho-p38 antibody or HA antibody as a control for transfection. (C) HeLa cells were transiently transfected with 500 ng of pSR-HA-p38 and cotransfected with 20 ng (lane 3), 50 ng (lane 4), 75 ng (lane 5), or 150 ng (lane 6) of pFlagCMV-MKP-1 or 20 ng (lanes 8 and 12), 50 ng (lanes 9 and 13), 75 ng (lanes 10 and 14), or 150 ng (lanes 11 and 15) of pFlagCMV-MKP-1^C258S. The empty expression vector pFlagCMV was added as appropriate to bring the total quantity of DNA up to 1 μg. After 24 h, cells were treated as indicated with vehicle (methanol) or 100 nM dexamethasone. After a further 2 h, cells were left untreated or stimulated with UV C, and lysates were prepared 30 min later and subjected to Western blotting using anti-phospho-p38 or anti-HA epitope antibodies. (D) HeLa cells were incubated for 2 h with 100 nM dexamethasone and then stimulated with 10 ng of epidermal growth factor (EGF) per ml for 10 min. Cells were harvested, and total cell lysates were analyzed by Western blotting with anti-phospho-ERK or total ERK antibodies.

Dexamethasone induces MKP-1 mRNA and protein in a time- and dose-dependent manner. (A) HeLa-TO cells were incubated with 100 nM dexamethasone for the times shown, and then MKP-1 mRNA was analyzed by Northern blotting. The 28S rRNA is shown as a loading control. Mean MKP-1/28S rRNA ratios from four experiments are shown. (B) HeLa-TO cells were incubated as described above for panel A, and then MKP-1 protein was immunoprecipitated and detected by Western blotting. (C) HeLa-TO cells were incubated with different doses of dexamethasone (Dex) alone or dexamethasone plus RU486 (RU) (1 μM) for 6 h. MKP-1 mRNA was analyzed by Northern blotting. Mean MKP-1/28S rRNA ratios from three independent experiments are shown. (D) HeLa-TO cells were incubated as described above for panel C, and MKP-1 protein was immunoprecipitated and detected by Western blotting.

MKP-1 mRNA induction by proinflammatory stimuli is dependent upon p38. HeLa cells were incubated for 10 min with different inhibitors as follows: no inhibitor (control) (−), 1 μM SB203580 (SB), 10 μM U0126 (U), 1 μM GF109203X (GF), and 0.5 μM protein kinase A inhibitor (PKI). Some cells were then incubated for 1 h in the presence of 100 nM dexamethasone (Dex lanes). Some cells were then stimulated with 20 ng of IL-1 per ml for 1 h (IL-1 lanes). MKP-1 mRNA was analyzed by Northern blotting. The 28S rRNA is shown as a loading control.

Sustained MAPK p38 activity is necessary for the expression of Cox-2 in response to IL-1. HeLa cells were either not treated (−) or treated by stimulation with 20 ng of IL-1 per ml at time zero. Lysates were prepared 24 h later, and Western blotting was performed to assess the expression of Cox-2 or p38. Dexamethasone (Dex) (1 μM) or SB203580 (SB) (1 μM) was added to the cells 1 h before the IL-1 stimulus (−1), at the same time as the IL-1 stimulus (0), or 2 h after the IL-1 stimulus (+2).