Interleukin (IL)-32β-mediated CCAAT/Enhancer-binding Protein α (C/EBPα) Phosphorylation by Protein Kinase Cδ (PKCδ) Abrogates the Inhibitory Effect of C/EBPα on IL-10 Production

We previously reported that IL-32 beta promotes IL-10 production in myeloid cells. However, the underlying mechanism remains elusive. In this study, we demonstrated that IL-32 beta abrogated the inhibitory effect of CCAAT/enhancer-binding protein alpha (C/EBP alpha) on IL-10 expression in U937 cells. We observed that the phosphorylation of C/EBP alpha Ser-21 was inhibited by a PKC delta-specific inhibitor, rottlerin, or IL-32 beta knockdown by siRNA and that IL-32 beta shifted to the membrane from the cytosol upon phorbol 12-myristate 13-acetate treatment. We revealed that IL-32 beta suppressed the binding of C/EBP alpha to IL-10 promoter by using ChIP assay. These data suggest that PKC delta and IL-32 beta may modulate the effect of C/EBP alpha on IL-10 expression. We next demonstrated by immunoprecipitation that IL-32 beta interacted with PKC delta and C/EBP alpha, thereby mediating C/EBP alpha Ser-21 phosphorylation by PKC delta. We showed that IL-32 beta suppressed the inhibitory effect of C/EBP alpha on IL-10 promoter activity. However, the IL-10 promoter activity was reduced to the basal level by rottlerin treatment. When C/EBP alpha serine 21 was mutated to glycine (S21G), the inhibitory effect of C/EBP alpha S21G on IL-10 promoter activity was not modulated by IL-32 beta. Taken together, our results show that IL-32 beta-mediated C/EBP alpha Ser-21 phosphorylation by PKC delta suppressed C/EBP alpha binding to IL-10 promoter, which promoted IL-10 production in U937 cells.