ß-Adrenergic receptors stimulate interleukin-6 production through Epac-dependent activation of PKCd/p38 MAPK signalling in neonatal mouse cardiac fibroblasts

BACKGROUND AND PURPOSE IL-6 plays crucial roles in cardiac hypertrophy, cardiac fibrosis and heart failure. Activation of beta-adrenoceptors induced IL-6 production in neonatal mouse cardiac fibroblasts (NMCFs) through a Gs/adenylate cyclase/cAMP/p38 MAPK pathway but independent of PKA. However, how cAMP activates p38 MAPK is still not defined. In this study, we have assessed the role of the exchange protein directly activated by cAMP (Epac) and PKCd in p38 MAPK activation and IL-6 production by stimulated by the beta-adrenoceptor agonist isoprenaline in NMCFs. EXPERIMENTAL APPROACH The IL-6 concentration in cell culture supernatants was measured by ELISA. The levels of phosphorylated and total p38 MAPK and PKCd were determined by Western blot analysis. The translocation of PKCd was determined by immunoblotting the soluble and particulate fractions. Expression of Epac1 or PKCd was knocked down by the corresponding, adenovirus-mediated, small hairpin RNA (shRNA). RESULTS In NMCFs, activation of b-adrenoceptors enhanced PKCd phosphorylation and translocation. Furthermore, knock-down of the PKCd isoform using an adenovirus-mediated shRNA markedly down-regulated IL-6 induction by NMCFs stimulated with isoprenaline. Moreover, knock-down of Epac1 confirmed that Epac1 was upstream of PKCd in IL-6 production. Additionally, both Epac1 and PKCd mediated the p38 MAPK activation induced by isoprenaline. CONCLUSIONS AND IMPLICATIONS b-Adrenoceptor agonists activate a cAMP/ Epac/ PKCd/ p38 MAPK pathway to produce IL-6 in NMCFs. This study identifies Epac as the link between cAMP and p38 MAPK signalling pathways and demonstrates that PKCd can function as a novel downstream effector of this b-adrenoceptor/ cAMP/ Epac pathway.