ROS/Epac1-mediated Rap1/NF-kappaB activation is required for the expression of BAFF in Raw264.7 murine macrophages

Abstract

B-cell activating factor (BAFF) plays a role for the maturation and the maintenance of B cells. Lipopolysaccharide (LPS) activates toll-like receptor 4 (TLR4)-dependent signal transduction, which resulted in BAFF expression through nuclear factor kappa B (NF-κB) activation. Here, we investigated whether BAFF expression could be regulated by p65 phosphorylation through the production of reactive oxygen species (ROS) or cyclic AMP (cAMP) in Raw264.7 murine macrophages. mBAFF expression was reduced by ROS scavengers and it was increased by dibutyl-cAMP, a cAMP analogue. mBAFF expression and mBAFF promoter activity were increased by co-transfection of p65 but they were reduced by p65-small interference (si) RNA. Serine (Ser) 276 phosphorylation of p65 was increased by LPS-mediated PKA activation or by the treatment with forskolin, adenylate cyclase activator and dibutyl-cAMP. In contrast, p65 phosphorylation at Ser276 was decreased by ROS scavengers. H(2)O(2) increased intracellular cAMP concentration, significantly. While no increase in p65 phosphorylation at Ser276 was detected by the treatment with H(2)O(2), CREB and p65 phosphorylation at Ser133 and Ser536 was observed, respectively. It implicates that p65 phosphorylation at Ser276 is independent of ROS-induced cAMP production. As another cAMP effector protein was cAMP-responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor, NF-κB was activated by the treatment with 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (CPT) that is an activator to Epac. Epac1-mediated Rap1 was activated by the treatment with H(2)O(2) but it was inhibited by ROS scavengers. CPT induced p65 phosphorylation at both Ser276 and Ser536. CPT also increased not only mBAFF expression but mBAFF promoter activity. Data demonstrate that TLR4-mediated mBAFF expression was resulted from the crosstalk of p65 phosphorylation at Ser536 and Ser276 through ROS- and/or cAMP-mediated signal transduction. It suggests for the first time that ROS/Epac1-mediated Rap1/NF-κB pathway could be required for BAFF expression.