Stimulation of beta(3)-adrenoceptors causes phosphorylation of p38 mitogen-activated protein kinase via a stimulatory G protein-dependent pathway in 3T3-L1 adipocytes

Abstract

1. This study deals with phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) via beta(3)-adrenoceptor (AR) and the signal transduction pathway in 3T3-L1 adipocytes. 2. beta(3)-AR agonist BRL37344A (10 nM) caused phosphorylation and activation of p38 MAPK in 3T3-L1 adipocytes but not in fibroblasts. BRL37344A and also the other beta(3)-AR agonists, CGP12177A and SR58611A, caused p38 MAPK phosphorylation in dose-dependent manners. 3. The p38 MAPK phosphorylations by BRL37344A (10 nM), CGP12177A (100 nM), and SR58611A (10 nM) were not antagonized by beta(1)- and beta(2)-ARs antagonist 1-propranolol (100 nM) but blocked by beta(3)-AR antagonist SR59230A (10 microM), suggesting the phosphorylation was caused via beta(3)-AR. 4. The phosphorylations of p38 MAPK were completely abolished by treatment with cholera toxin (CTX) but not pertussis toxin (100 ng ml(-1), 24 h). Activation of Gs by CTX (100 ng ml(-1)) and adenylyl cyclase by forskolin mimicked p38 MAPK phosphorylation. 5. p38 MAPK phosphorylation by BRL37344A was reduced to almost 50% by cyclic AMP-dependent protein kinase (PKA) inhibitors such as H89 (10 microM) and PKI (10 microM). A src-family tyrosine kinases inhibitor PP2 (1 microM) also halved the p38 MAPK phosphorylation. Combined use of H89 (10 microM) and PP2 (10 microM) did not bring about further inhibition. 6. These results suggest that beta(3)-AR caused phosphorylation of p38 MAPK via Gs protein and partly through a pathway involving PKA and src-family kinase(s), although the contribution of the unidentified pathway remains to be clarified.

Figure 1

Cultivation-dependent occurrence of p38 MAPK phosphorylation and activation by the stimulation with BRL37344A in 3T3-L1 cells. The 3T3-L1 fibroblast cells were grown and treated with differentiation reagents for initiation of adipogenesis. After appropriate cultivation, the cells were serum-starved and stimulated with 10 nM BRL37344A for 30 min at 37°C. Open bars represent the degree of p38 MAPK phosphorylation at each period, expressed as the fold increase in phosphorylation level over respective basal level (a). Values represent the means±s.d. (four independent experiments). The values are significantly different from that obtained at day 0 by one-way ANOVA and Dunnett's multiple comparison (**:P<0.01, ***:P<0.001). The activation of p38 MAPK was confirmed at 8-day cultivation after the initiation by immunoprecipitation kinase assay with ATF-2 as described in the Methods, and representative results are shown (b). A positive control was established using 10 μM anisomycin under the same condition.

Figure 2

Time- (a) and dose-dependency (b) of p38 MAPK phosphorylation by BRL37344A in 3T3-L1 adipocytes. 3T3-L1 adipocytes (35 mm dish, 8-day culture) were treated for the indicated periods with 10 nM BRL37344A (a) or for 30 min at the indicated concentrations with either BRL37344A, CGP12177, or SR58611A (b) at 37°C. The increases of phosphorylation were measured as described in Figure 1. Open circles represent the results obtained at each period or with each agonist (means±s.d. of three independent experiments). The data were compared with the values obtained without agonists as controls by one-way ANOVA and Dunnett's multiple comparison (*:P<0.05, **:P<0.01, ***:P<0.001).

Figure 3

Effects of β-AR antagonists on p38 MAPK phosphorylations by the β₃-AR agonists. 3T3-L1 adipocytes (35 mm dish, 8-day cultivation) were treated with 10 nM BRL37344A, 100 nM CGP12177A and 10 nM SR58611A in the absence or presence of 100 nM l-propranolol (a). The adipocytes were cultured in glass dishes and stimulated by 10 nM BRL37344A for 30 min in the presence of SR59230A at the indicated concentrations (b) or by 100 nM CGP12177A and 10 nM SR58611A for 30 min in the presence of 10 μM SR59230A (c). The increases of p38 MAPK phosphorylations were measured and expressed as described in Figure 1 (means±s.d. of three independent experiments). Open bars in (a,c) represent the results obtained with/without each antagonist. The open circles and closed square in (b) represent the results obtained with 10 nM BRL37344A and SR59230A at the indicated concentrations or that with 10 μM SR59230A alone, respectively. The data in (a) and (c) were compared with the controls (*:P<0.05, ***:P<0.001) or each other (#:P<0.001) by one-way ANOVA and Tukey's multiple comparison. The data in (b) were compared with the values obtained with BRL37344A as the control by one-way ANOVA and Dunnett's multiple comparison (*:P<0.05, ***:P<0.001).

Figure 4

Effects of CTX and PTX on p38 MAPK phosphorylations caused by β₃-AR agonists in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with 100 ng ml⁻¹ CTX for the indicated periods at 37°C. The contents of immuno-reactive Gs, p38 MAPK and β₃-AR proteins are shown in upper, middle, and lower panels of (a), respectively. The hatched and shaded bars express the relative contents of two isoforms of Gs proteins in the membrane of 3T3-L1 adipocytes at indicated periods (b). After treatment with 100 ng ml⁻¹ CTX (c) or PTX (d) for 24 h, 3T3-L1 adipocytes were serum-starved and then stimulated by the β₃-AR agonists as described in the legend for Figure 3. The increases of p38 MAPK phosphorylations were measured as described in Figure 1 and expressed as open bars (means±s.d. of three independent experiments). The data in (b) were compared with values obtained at 0 h as the controls by one-way ANOVA and Dunnett's multiple comparison (*:P<0.001). The data in (c) and (d) were compared with the values obtained without agonists (***:P<0.001) or each other (#:P<0.001) by one-way ANOVA with Tukey's multiple comparison.

Figure 5

p38 MAPK phosphorylations in 3T3-L1 adipocytes by CTX (a and b) and forskolin (c and d). 3T3-L1 adipocytes were treated with 100 ng ml⁻¹ CTX (a) or 1 μM forskolin (c) for the indicated periods and at the indicated concentrations for 4 h with CTX (b) or for 30 min with forskolin (d). The increases of p38 MAPK phosphorylations were measured as described in Figure 1 and expressed as open bars (means±s.d. of three independent experiments). The data were compared with the values obtained at 0 min or 0 h without reagents as the control by one-way ANOVA with Dunnett's multiple comparison (*:P<0.05, **:P<0.01, ***:P<0.001).

Figure 6

Effects of PKA and a src-family kinases inhibitors on p38 MAPK phosphorylation by BRL37344A in 3T3-L1 adipocytes. The adipocytes were treated with H89, PKI-(14 – 22)-amide and/or PP2 at the indicated concentrations for 30 min, and then stimulated with 10 nM BRL37344A for 30 min at 37°C. The degree of p38 MAPK phosphorylation was expressed as open circle and bars as a percentage of control that obtained without inhibitors (means±s.d. of four independent experiments). The open square expressed the basal value obtained without BRL37344A and inhibitors. The data in (a, b and c) were compared with the values obtained without inhibitors as controls by one-way ANOVA with Dunnett's multiple comparison (*:P<0.05, **:P<0.01, ***:P<0.001). The values in (d) are significantly different from that obtained without inhibitors by one-way ANOVA and Tukey's multiple comparison (***:P<0.001).