β2‐adrenergic stimulation induces interleukin‐6 by increasing Arid5a, a stabilizer of mRNA, through cAMP/PKA/CREB pathway in cardiac fibroblasts

Figures

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  Figure 1. β‐adrenergic stimulation remarkably increased IL‐6 mRNA in cardiac fibroblasts (CFs) isolated from adult mice. CFs were treated with ISO (10 μmol/L) for indicated times. The expression of IL‐6, IL‐1β, and TNF‐α mRNA was measured by real‐time RT‐PCR. Results are shown as mean ± SEM (n = 6). **P < .01 vs 0 h by Dunnett test

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  Figure 2. IL‐6 expression was induced predominantly by the stimulation of β2AR, not β3AR. A ‐ C) CFs were treated with the indicated concentrations of ISO (A), SAL (B), or CL‐316243 (C) for 1 h. The expression of IL‐6, IL‐1β, and TNF‐α mRNA was measured by real‐time RT‐PCR. Results are shown as mean ± SEM (A, n = 8, B, C, n = 6). *P < .05, **P < .01 vs vehicle by Dunnett test. D, CFs were treated with the indicated concentrations of SAL for 24 h. IL‐6 protein, secreted by CFs in culture medium, was measured by ELISA. Results are shown as mean ± SEM (n = 6). *P < .05, **P < .01 vs vehicle by Dunnett test

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  Figure 3. The intracellular increase in cAMP‐induced IL‐6, but not IL‐1β or TNF‐α. A, B) CFs were treated with the indicated concentrations of FSK (A) or dBcAMP (B) for 1 h. The expression of IL‐6, IL‐1β, and TNF‐α mRNA was measured by real‐time RT‐PCR. Results are shown as mean ± SEM (n = 6). *P < .05, **P < .01 vs vehicle by Dunnett test

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  Figure 4. β2‐adrenergic stimulation failed to activate NF‐κB signaling pathway in adult murine CFs. A, CFs were treated with vehicle or ISO (1 μmol/L) for 1 h. IL‐1β (10 ng/mL) was used as a positive control. The transcriptional activity of NF‐κB p65 in CFs was measured by ELISA‐based assay. Results are shown as mean ± SEM (n = 6). **P < .01 vs vehicle by Dunnett test. B, C) CFs were treated with ISO (1 μmol/L) B, SAL (1 μmol/L) C, or IL‐1β (10 ng/mL) for indicated times. The phosphorylation of IκBα in CFs was analyzed by Western blotting with antiphospho‐specific IκBα antibody. The experiments were performed 3 times with similar results. The representative images are shown

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  Figure 5. β2AR stimulation upregulated the expression of Aird5a, resulting in IL‐6 mRNA induction. (A) CFs were treated with ISO (10 μmol/L) for indicated times. The expression of Arid5a mRNA was measured by real‐time RT‐PCR. Results are shown as mean ± SEM (n = 6). **P < .01 vs 0 h by Dunnett test. (B‐F) CFs were treated with the indicated concentrations of ISO (B), SAL (C), CL‐316243 (D), FSK (E), or dBcAMP (F) for 1 h. The expression of Arid5a mRNA was measured by real‐time RT‐PCR. Results are shown as mean ± SEM (B: n = 8, C‐F: n = 6). *P < .05, **P < .01 vs vehicle by Dunnett test. G) CFs were isolated from WT mice or Arid5a KO mice and treated with or without SAL (1 μmol/L) for indicated times. The expression of IL‐6 and IL‐1β mRNA in CFs was measured by real‐time RT‐PCR. Results are shown as mean ± SEM (n = 6). **P < .01 vs WT by Student's t test. H) CFs were isolated from WT mice or Arid5a KO mice and treated with or without SAL (1 μmol/L) for 24 h. IL‐6 protein, secreted by CFs in culture medium, was measured by ELISA. Results are shown as mean ± SEM (n = 6). **P < .01 by Tukey‐Kramer test

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  Figure 6. β2‐adrenergic stimulation phosphorylated CREB through cAMP/PKA pathway. A, B, CFs were treated with SAL (1 μmol/L) A, or dBcAMP (3 mmol/L) B, for indicated times. C, CFs were pretreated with the indicated concentrations of H89 for 30 min, followed by the treatment of SAL (1 μmol/L) for 5 min. The phosphorylation of CREB was measured by Western blotting with antiphospho‐CREB antibody. Representative images are shown (left panels). The quantification data are shown as mean ± SEM (A, n = 5, B, n = 6, C, n = 8). *P < .05, **P < .01 vs vehicle, ## P < .01 vs DMSO, SAL by Steel test (A) or Dunnett test (B, C) (right panels)

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  Figure 7. The inhibition of CREB suppressed SAL‐induced mRNA upregulation of Arid5a and IL‐6 expression. CFs were pretreated with the indicated concentrations of 666‐15, an inhibitor of CREB, for 30 min, followed by the treatment with or without SAL (1 μmol/L) for 1 h. The expression of Arid5a and IL‐6 mRNA in CFs was measured by real‐time RT‐PCR. Results are shown as mean ± SEM (n = 6). *P < .05, **P < .01 vs DMSO, vehicle, # P < .05, ## P < .01 vs DMSO, SAL by Dunnett test

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  Figure 8. The stimulation of β2AR induces Arid5a through cAMP/PKA/CREB pathway and upregulates IL‐6 expression in adult murine CFs