Cloning and characterization of the porcine IL-10 promoter

Abstract

Interleukin 10 (IL-10) is an anti-inflammatory and immunosuppressive cytokine that plays an important role in regulating the immune response. Therefore, understanding how IL-10 is regulated is important. The regulatory elements have been well studied in human and mouse promoters and several transcription factors have been showed to be involved in IL-10 transcription. In our study, a 1.5kb fragment of the 5′ flanking region of IL-10 gene was cloned and functionally characterized. Several putative regulatory elements including IRF, AP-1, Sp1, C/EBP, and STAT binding sites were found in the porcine IL-10 (pIL-10) promoter. The pIL-10 promoter deletion mutants were analyzed for their ability to direct luciferase expression in a porcine macrophage cell line (CRL 2843), human gastric carcinoma cell lines with or without Epstein-Barr virus (EBV), AGS–EBV and AGS cell lines. Our data showed that the minimal active pIL-10 promoter region was from −605 to +19, with the inducible activity requiring only one key DNA element, the Sp1 binding site (−398 to −393) upstream of the IL-10 gene starting point in both LPS-stimulated CRL 2843 and AGS–EBV cells. Moreover, our results suggested that the two IRF binding sites (−950 to −942 and −662 to −640) may have a positive role in the activation of the pIL-10 promoter in AGS-EBV cells, but not in LPS-stimulated CRL 2843 cells. These data implicate that the cloned porcine IL-10 promoter could be used to explore the molecular mechanisms underlying the regulation of IL10 production in pigs.