Abstract
We previously reported that IL-32β promotes IL-10 production in myeloid cells. However, the underlying mechanism remains elusive. In this study, we demonstrated that IL-32β abrogated the inhibitory effect of CCAAT/enhancer-binding protein α (C/EBPα) on IL-10 expression in U937 cells. We observed that the phosphorylation of C/EBPα Ser-21 was inhibited by a PKCδ-specific inhibitor, rottlerin, or IL-32β knockdown by siRNA and that IL-32β shifted to the membrane from the cytosol upon phorbol 12-myristate 13-acetate treatment. We revealed that IL-32β suppressed the binding of C/EBPα to IL-10 promoter by using ChIP assay. These data suggest that PKCδ and IL-32β may modulate the effect of C/EBPα on IL-10 expression. We next demonstrated by immunoprecipitation that IL-32β interacted with PKCδ and C/EBPα, thereby mediating C/EBPα Ser-21 phosphorylation by PKCδ. We showed that IL-32β suppressed the inhibitory effect of C/EBPα on IL-10 promoter activity. However, the IL-10 promoter activity was reduced to the basal level by rottlerin treatment. When C/EBPα serine 21 was mutated to glycine (S21G), the inhibitory effect of C/EBPα S21G on IL-10 promoter activity was not modulated by IL-32β. Taken together, our results show that IL-32β-mediated C/EBPα Ser-21 phosphorylation by PKCδ suppressed C/EBPα binding to IL-10 promoter, which promoted IL-10 production in U937 cells.
Highlights
Interleukin 32 (IL-32) promotes IL-10 production in myeloid cells
We observed that the phosphorylation of CCAAT/enhancer-binding protein (C/EBP)␣ Ser-21 was inhibited by a PKC␦-specific inhibitor, rottlerin, or IL-32 knockdown by siRNA and that IL-32 shifted to the membrane from the cytosol upon phorbol 12-myristate 13-acetate treatment
IL-32 Promotes IL-10 Production in U937 Cells upon Phorbol 12-myristate 13-acetate (PMA) Stimulation—We demonstrated previously that IL-32 promoted the production of the anti-inflammatory cytokine IL-10 in myeloid cells [28]
Summary
Results: IL-32-mediated C/EBP␣ serine 21 phosphorylation by PKC␦ induced the dissociation of C/EBP␣ from IL-10 promoter, thereby promoting IL-10 production. Conclusion: IL-32 suppressed the inhibitory effect of C/EBP␣ on IL-10 production by mediating C/EBP␣ serine 21 phosphorylation by PKC␦. We demonstrated that IL-32 abrogated the inhibitory effect of CCAAT/enhancer-binding protein ␣ (C/EBP␣) on IL-10 expression in U937 cells. We revealed that IL-32 suppressed the binding of C/EBP␣ to IL-10 promoter by using ChIP assay These data suggest that PKC␦ and IL-32 may modulate the effect of C/EBP␣ on IL-10 expression. Our results show that IL-32-mediated C/EBP␣ Ser-21 phosphorylation by PKC␦ suppressed C/EBP␣ binding to IL-10 promoter, which promoted IL-10 production in U937 cells. The CCAAT/enhancer-binding protein (C/EBP) family of transcription factors is known to play important roles in cell proliferation and differentiation.
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