Article Figures & Data
Figures
- FIG. 1.
CREB is required for the β2AR-induced increase in IgG1 but not for the increase in IgE. Naive B cells were isolated from the spleens of WT and CREB DN mice and then activated by CD40L/IL-4 in the absence (open bars) or presence (filled bars) of terbutaline. After 5 days of culture, cells were collected, total mRNA was isolated, and the level of mature IgG1 (A) or mature IgE (B) mRNA was determined using real-time PCR analysis. The data were normalized to β-actin, and each bar represents the mean fg/ml ± standard error of the mean from three independent experiments, (*, P ≤ 0.05).
- FIG. 2.
β2AR stimulation and PKA activation result in an increase in p38 MAPK phosphorylation in activated B cells. Naive B cells were isolated from the spleens of WT (A and B) and β2AR-deficient mice (C and D) and activated by CD40L/IL-4 in the absence or presence of terbutaline (A and C) or forskolin (B and D). CD40L/IL-4-activated cells (solid squares), terbutaline- or forskolin-exposed cells (triangles), and cells pretreated with H-89 prior to exposure to terbutaline or forskolin (open squares) are analyzed. Total protein was isolated at the indicated time points, and the level of total and phospho-(Thr180/Tyr182)p38 MAPK was analyzed by Western blotting. Band density was determined using densitometry, and each data point represents the mean difference of phospho-(Thr/Tyr)p38 MAPK normalized to total p38 MAPK and compared to resting levels ± standard error of the mean from three independent experiments. Exposure groups were compared to CD40L/IL-4-activated cells for statistical significance, (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).
- FIG. 3.
β2AR stimulation activates the Gs/cAMP/PKA pathway to increase the level of p38 MAPK phosphorylation and activity in an activated B cell. Naive B cells from the spleens of WT mice were activated with CD40L/IL-4 in the absence or presence of terbutaline (Terb), forskolin (Fsk), or cholera toxin (CTx). All data were collected after 10 min of activation, and exposure groups were compared to CD40L/IL-4-activated cells for statistical significance (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (A) B cells were preincubated with IBMX for 10 min and then activated as described above. Cells were lysed, and cAMP accumulation was measured and normalized to total protein isolated. Each bar represents the mean pmol cAMP/mg of protein ± standard error of the mean from three independent experiments. (B) B cells were activated as described and then lysed and assayed for PKA activity. Each bar represents the mean PKA activity in arbitrary units ± standard error of the mean from three independent experiments. (C) Total protein was collected from B cells activated as described and analyzed by Western blotting for total and phospho-(Thr/Tyr)p38 MAPK. Band density was determined using densitometry, and each bar represents the mean difference in phospho-p38 MAPK normalized to total p38 MAPK ± standard error of the mean from three independent experiments. (D) B cells were activated as described above, total protein was collected, and phospho-(Thr/Tyr)p38 MAPK was immunoprecipitated from 80 μg of protein and then assayed for p38 MAPK activity using an in vitro kinase assay and an ATF-2 fusion protein as a target. The level of ATF-2 phosphorylation was determined by Western blot analysis, and the band density was determined using densitometry. Each bar represents the mean difference in phospho-ATF-2 normalized to total p38 MAPK ± standard error of the mean from three independent experiments.
- FIG. 4.
Gi activation is not necessary for the β2AR-induced increase in p38 MAPK phosphorylation in an activated B cell. Naive B cells from the spleens of WT mice were activated with CD40L/IL-4 in the absence or presence of terbutaline or pertussis toxin. All exposure groups were compared to CD40L/IL-4-activated cells for statistical significance (*, P ≤ 0.05; **, P ≤ 0.01). (A) Total protein was collected 10 min after activation and analyzed by Western blotting for total and phospho-(Thr/Tyr)p38 MAPK. Band density was determined using densitometry, and each bar represents the mean difference in phospho-p38 MAPK normalized to total p38 MAPK ± standard error of the mean from three independent experiments. (B) B cells were activated as described, total protein was collected after 10 min of activation, and phospho-(Thr/Tyr)p38 MAPK was immunoprecipitated from 80 μg of protein and assayed for p38 MAPK activity as described for Fig. 3. The level of ATF-2 phosphorylation was determined by Western blot analysis, and band density was determined using densitometry. Each bar represents the mean difference in phospho-ATF-2 normalized to total p38 MAPK ± standard error of the mean from three independent experiments.
- FIG. 5.
β-Arrestin activation is not necessary for a β2AR-induced increase in p38 MAPK phosphorylation in an activated B cell. Naive B cells from the spleens of WT (top panel) or β-arrestin-2-deficient (bottom panel) mice were activated by CD40L/IL-4 in the absence or presence of either terbutaline or ICI118551. Total protein was collected, and total and phospho-(Thr/Tyr)p38 MAPK levels were determined by Western blot analysis. Band density was determined using densitometry, and each bar represents the mean difference in phospho-p38 MAPK normalized to total p38 MAPK ± standard error of the mean from four independent experiments. Exposure groups were compared to CD40L/IL-4-activated cells for statistical significance (*, P ≤ 0.05; **, P ≤ 0.01).
- FIG. 6.
β2AR stimulation alone and in the presence of CD40L/IL-4 activation induces PKA-specific phosphorylation of HePTP in a B cell. (A) Naive B cells were either not activated (Unstim.) or activated with CD40L/IL-4 or terbutaline (Terb) only. Total protein was collected after 10 min of culture and analyzed for total and phospho(Thr/Tyr)-p38 MAPK by Western blotting. One representative blot from three independent experiments is shown. Naive B cells from the spleens of WT mice either were not activated or were activated with CD40L/IL-4 in the presence of terbutaline with or without H-89 pretreatment (B) or CD40L, IL-4, or terbutaline alone (C). Total protein was collected after 10 min of culture and analyzed for total and PKA-specific phospho-(Ser23)HePTP by Western blotting. Band density was determined using densitometry, and each bar represents the mean difference in PKA-specific phospho-(Ser23)HePTP normalized to total HePTP ± standard error of the mean from three independent experiments. Exposure groups were compared to CD40L/IL-4-activated cells (B) or resting cells (C) for statistical significance (*, P ≤ 0.05).
- FIG. 7.
Unphosphorylated forms of HePTP and p38 MAPK interact constitutively, while phosphorylated forms do not interact in a resting or activated B cell. (A) Naive B cells from WT mice were activated with CD40L/IL-4 in the absence or presence of terbutaline or forskolin. Total protein was isolated after 10 min of culture, and coimmunoprecipitation was performed with antibodies to either total p38 MAPKα or phospho-(Thr/Tyr)p38 MAPK. Immunoprecipitates were analyzed by Western blotting for HePTP or total p38 MAPK. One representative blot from three independent experiments is shown. (B) Unactivated CH12.LX B cells were transfected with plasmids containing HA-tagged HePTP-WT, HePTP-S23A, or HePTP-S23D for 48 h, and then the cells were lysed and total protein was collected. Coimmunoprecipitation was performed using an anti-HA antibody, and immunoprecipitates were analyzed by Western blotting for HA-tagged HePTP and total p38 MAPK. One representative blot from three independent experiments is shown.
- FIG. 8.
HePTP regulates the level of p38 MAPK phosphorylation in a B cell. Unactivated CH12.LX B cells were transfected with HePTP shRNA plasmids and cultured for 48 h. (A) The cells were collected and sorted on a BD FACS Aria cell sorter for GFP-positive (HePTP siRNA positive) and GFP negative (HePTP siRNA negative) cells. Total protein was isolated and analyzed for actin and total HePTP (B) or total and phospho-(Thr/Tyr)p38 MAPK (C) levels by Western blotting. Band density was determined using densitometry, and the data represent the percent difference in total HePTP normalized to actin or the mean difference in phospho-p38 MAPK normalized to total p38 MAPK ± standard error of the mean from three independent experiments. (*, P ≤ 0.05).
- FIG. 9.
Model of the mechanism by which β2AR mediates an increase in p38 MAPK phosphorylation to increase the level of IgE. B-cell CD40 engagement by CD40L (CD154) on a T cell induces activation of the MAPK cascade and results in phosphorylation and activation of p38 MAPK. The active p38 MAPK translocates to the nucleus and, along with molecules activated by IL-4 receptor stimulation by IL-4 (not shown), induces class switch recombination and the production of IgE. In the absence of β2AR stimulation (left model), the level of p38 MAPK phosphorylation is regulated by the protein tyrosine phosphatase HePTP. In the presence of β2AR stimulation (right model), HePTP is phosphorylated in a PKA-specific manner, which inactivates the phosphatase and results in the release of p38 MAPK. The free p38 MAPK is phosphorylated by molecules of the MAPK cascade to result in increased levels of phosphorylated p38 MAPK and downstream effects, such as increased production of IgE.
