JAK/STAT5 signaling is regulated by TxA2-TP through cAMP-PKA axis in B cells. (A) BM cells from naive mice were pretreated with the TP agonist U46619 (10 μM) or DMSO for 15 minutes, followed by administration with the indicated pathway inhibitors. Cells were then cultured under conditions to induce B-cell differentiation. The proportions of pre-B cells (IgM−CD43−B220+) were analyzed by flow cytometry after a 6-day culture. The concentration of PKA inhibitor was 5 μM, PKC inhibitor chelerythrine chloride (CC) was 1 μM, and ROCK inhibitor Y-27632 was 5 μM. The JAK3 inhibitor WHI-P131 (15 μM) was used as the positive control. (Left) Representative data from a single experiment; numbers in the plots indicate the percentage of pre-B cells among total living cells. (Right) Mean ± SEM of 3 independent experiments. (B) BM cells from naive mice were pretreated with TP agonist U46619 or DMSO for 15 minutes, followed by administration of PKA inhibitor H89 or vehicle for another 15 minutes; B220+ cells were gated, and the levels of p-STAT5 was determined by flow cytometric analysis. (Upper) Representative data from a single experiment. (Lower) Mean ± SEM of MFI from 3 independent experiments. (C) B220+ cells from BM of naive mice were stimulated with arachidonic acid (1 μM) for 30 minutes, followed by the indicated treatments for 15 minutes. The (upper) intracellular cAMP level and (lower) PKA activity were measured by enzyme-linked immunosorbent assay. TP agonist, U46619 (10 μM); adenylyl cyclase (AC) inhibitor, MDL 12330A (20 μM). The AC activator forskolin (Fsk, 10 μM) was used as the positive control. (D) B220+ cells from COX-1−/− and WT mice BM were stimulated with arachidonic acid for 30 minutes, followed by treatment with U46619 or vehicle for 15 minutes. The cellular cAMP levels and PKA activity were measured by enzyme-linked immunosorbent assay. (C-D) The results are presented as mean ± SEM from 3 independent experiments. PKA activity was normalized against the control group. (E) BM cells were cultured with IL-7 and treated with JAK3 inhibitor WHI-P131 (15 μM) or vehicle. Cells were then administrated with or without PKA activator 6-Bnz-cAMP (200 μM) for 15 minutes. The levels of p-STAT5 in B220+ cells were examined by flow cytometric analysis. Both representative results from (upper) a single experiment and (lower) mean ± SEM of MFI from 3 independent experiments were included. (F) BM cells were stimulated with IL-7 for 15 minutes, following with treatments of PKA activator 6-Bnz-cAMP and/or PKA inhibitor H89 (5 μM) for 15 minutes. The phosphorylation levels of JAK3 and JAK1 were determined by immunoblotting. (G) BM cells from COX-1−/− and WT mice were stimulated with IL-7 for 15 minutes, followed by treatment in the presence or absence of U46619 for 15 minutes. Immunoblotting was used to determine the phosphorylation of JAK3. (F-G) Results are representative of 3 independent experiments. (Right) The relative intensity of WB bands normalized to β-actin. (A-E) *P < .05 and **P < .01, using unpaired Student t tests and 1-way ANOVA.
