Journal: The Journal of Biological Chemistry
Article Title: The proton-sensing OGR1 receptor and hypoxia-inducible factors promote metal ion–induced inflammatory responses in coronary artery smooth muscle cells
doi: 10.1016/j.jbc.2025.110842
Figure Lengend Snippet: NiCl 2 or acidic pH activates CREB through OGR1/G q/11 and PKD-dependent pathways. A and B , the phosphorylation of CREB at Ser133 (p-CREB) is induced by the cAMP pathway or the Ca 2+ signaling pathway in CASMCs. The CREB activation response to NiCl 2 (100–300 μM), ION (1 μM), PGE 2 (1 μM), FSK (10 μM), or the vehicle (Veh) was examined. After serum starvation for 8 h, CASMCs were stimulated at 37 °C for 10 min by the indicated reagents in RPMI-1640-Hepes (pH 7.4)-0.1% BSA. Phospho-CREB and CREB, together with actin contents, were measured in cell lysate by Western blotting as described in the the Experimental procedures section. Gel images are representative results ( A ). The results were also expressed as percentages of each vehicle ( B ). Data are shown as the mean ± SEM of each group (n = 4). Comparisons of each test reagent versus the vehicle were assessed using a one-way ANOVA, followed by the Tukey test. The effects of the test reagents compared with the vehicle were significant for NiCl 2 (100 μM, ∗∗ p = 0.0106; 300 μM, ∗∗ p = 0.0027), ION (∗ p = 0.0336), PGE 2 (∗ p = 0.0237), and FSK (∗∗∗∗ p < 0.0001). C and D , the effect of different inhibitors of the Ca 2+ signaling pathway on the CREB activation induced by NiCl 2 and PGE 2 . After serum starvation for 8 h, CASMCs were pretreated with YM-254890 (100 nM), 2-APB (30 μM), CRT0066101 (1 μM), or DMSO at 37 °C for 30 min in RPMI-1640-0.1% BSA. The cells were stimulated for 10 min by NiCl 2 (300 μM), PGE 2 (1 μM), or the vehicle (Veh) in RPMI-1640-Hepes (pH 7.4)-0.1% BSA containing inhibitors or DMSO. Data are shown as the mean ± SEM of each group (n = 3). Gel images are representative results ( C ). The results were also expressed as percentages of each DMSO ( D ). Comparisons of NiCl 2 versus DMSO were assessed using a two-way ANOVA, followed by the Tukey test. The effect of YM-25489 or CRT0066101 on NiCl 2 action was significant (∗∗ p = 0.0014 or ∗∗ p = 0.0012, respectively); however, that of 2-APB was not significant (N.S. p = 0.8019). E and F , the effect of siRNAs on the CREB activation by induced NiCl 2 . After serum starvation for 8 h, siRNA-transfected CASMCs were incubated at 37 °C for 10 min in RPMI-1640-Hepes (pH 7.4)-0.1% BSA with NiCl 2 (300 μM), pH 6.4 (adjusted with 1 M HCl), PGE 2 (1 μM), or the vehicle (Veh). Error bars represent the mean ± SEM (n = 3). Gel images are representative results ( E ). The results were also expressed as percentages of each vehicle in si-NS cells ( F ). Comparisons of siRNAs specific to OGR1 (si-OGR1) or HIF1A (si-HIF-1α) and EPAS1 (si-HIF-2α) versus control siRNA (si-NS) were assessed using a two-way ANOVA, followed by the Tukey test. The effect of si-OGR1 on the NiCl 2 or pH 6.4 actin was significant (∗∗∗∗ p < 0.0001); however, that of HIF-1α and si-HIF-2α was not significant (N.S. p = 0.1610 and N.S. p = 0.1099, respectively). 2-APB, 2-aminoethoxydiphenyl borate; BSA, bovine serum albumin; CASMC, coronary artery smooth muscle cell; CREB, cAMP-responsive element-binding protein; FSK, forskolin; HIF, hypoxia-inducible factor; ION, ionomycin; PGE 2 , prostaglandin E 2 ; PKD, protein kinase D; NiCl 2 , nickel chloride; OGR1, ovarian cancer G protein–coupled receptor 1.
Article Snippet: Anti-phosphorylated antibodies against CREB (Ser133) antibody (87G3, Cell Signaling Technology), together with anti-CREB antibody (48H2, Cell Signaling Technology), were used to estimate the CREB activation.
Techniques: Phospho-proteomics, Activation Assay, Western Blot, Transfection, Incubation, Control, Binding Assay
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