Differential Roles of PKA and Epac on the Production of Cytokines in the Endotoxin-Stimulated Primary Cultured Microglia

Abstract

To further understand the anti-inflammatory effect of adenosine cyclic 3',5'-monophosphate (cAMP), we examined the effect of protein kinase A (PKA) and cAMP-responsive guanine nucleotide exchange factor (Epac) on the transcription and production of cytokines and on the activity of mitogen-activated protein kinases (MAPK) p38 and glycogen synthase kinase-3β (GSK-3β) in endotoxin-treated rat primary cultured microglia. The PKA specific agonist N6-benzoyladenosine-3,5-cAMP (6-Bnz-cAMP) not only inhibited the transcription and production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) but also enhanced the transcription and expression of IL-10, while the Epac selective analog 8-(4-chlorophenylthio)-2-O-methyladenosine-3,5-cAMP (8-pCPT-2'-O-Me-cAMP) merely repressed the TNF-α expression. Western blots assays indicated that 6-Bnz-cAMP significantly inhibited lipopolysaccharide-induced activation of both p38 and GSK-3β in a dose-dependent manner; in contrast, 8-pCPT-2'-O-Me-cAMP only slightly repressed GSK-3β activity at large doses. Pretreatment with H-89, a specific PKA antagonist, could completely reverse the effect of 6-Bnz-cAMP on cytokines expressions and kinases activities but had no effect on the performance of 8-pCPT-2'-O-Me-cAMP. Our findings indicate that PKA and Epac exert differential effect on the expression of inflammatory cytokines such as TNF-α, IL-1β, and IL-10, possibly owing to the different effects on the downstream effectors, MAPK p38, and GSK-3β.

Histamine H3 receptor in primary mouse microglia inhibits chemotaxis, phagocytosis, and cytokine secretion

“…Although cAMP level controls various cellular functions, the roles of cAMP in microglia remain to be elucidated. For example, the effects of cAMP increase on IL‐1β (Jin et al, ; Liu et al, ; Quan et al, ; Woo et al, ) and IL‐6 (Hashioka et al, ; Si et al, ) were different among studies. In contrast, almost all reports agreed that intracellular Ca 2+ concentration played a key role in cytokine production (Espinosa‐Parrilla et al, ; Sun et al, ).…”

Balancing the immune response in the brain: IL-10 and its regulation

“…Activation of p38 has been shown to induce IL-10 expression in a microglial cell line upon adenosine administration [85]. However, in another study, a down-regulation on the activation of p38 in LPS-stimulated rat primary cultured microglia has been correlated with an enhancement in IL-10 prodution [121]. This controversy may be due to the different models used in both works, and/or to the distinct stimuli provided to microglial cells that may activate different molecular pathways.…”

EP2 Receptor Signaling Pathways Regulate Classical Activation of Microglia

“…Cytokine and chemokine production in bone marrow-derived dendritic cells involves both Epac1 and PKA (58). It has also been reported that high concentrations of PKA and cell-impermeable Epac agonists play differential roles in the production of cytokines in primary cultured microglia stimulated with a high concentration of endotoxin (59). In our study, the actions of selective concentrations of both H89 (5 M) and the cell-permeable Epac agonist (10 M) suggest that cAMP-activated Epac rather than PKA mediates the effect of EP2 receptors on the expression of COX-2, iNOS, and inflammatory cytokines in classically activated rat microglia.…”