Proinflammatory Interferon Gamma and Nuclear Factor Kappa B Synergestically Activates Inducible Nitric Oxide Synthase Transcription in Human Colon Cancer Cells

Abstract

Introduction: Nitric oxide is an important signaling molecule in numerous physiological and pathological conditions. Compelling experimental data have shown that inducible Nitric Oxide Synthase (iNOS) promotes tumor development in certain cellular context and suppresses tumor development in other cellular conditions. iNOS has been proposed as a key-signaling molecule that promotes inflammationmediated spontaneous cancer development, including human colorectal cancer. However, the underlying molecular mechanisms are relatively unknown. Methods: Tissue microarray slides of normal, ulcerative colitis and colon cancer specimens as well as cell lines of human colon cancer and myeloid cell lines were used to elucidate the molecular mechanisms underlying iNOS expression. Chromatin immunoprecipitation and electrophoretic mobility shift assay were used to determine the IFNγ-activated pSTAT1 and NF-kB association with the chromatin DNA of the nos2 promoter. Results: Analysis of human tissue of ulcerative colitis and colon cancer using immunohistochemistry demonstrated significant upregulation of iNOS protein expression levels in inflammed and colon cancer cells or immune cells within the tumor microenvironment. Similarly, the proinflammatory INFγ and NF-kB signals synergistically induced iNOS expression in human colon cancer cells. RT-PCR analysis demonstrated IFNγ up-regulates IRF8 expression to augment NF-kB induction of iNOS expression. Interestingly, Chip and Gel shift assay suggested p65/p65 and p50/p50 homodimers and not the canonical p65/p50 heterodimer, directly binds to the nos2 promoter to regulate iNOS expression in myeloid cells. Conclusion: The transcriptional regulation of iNOS has been the subject of extensive studies due to its diverse mechanisms of regulation. Our results suggest both IFNγ and NF-kB synergistically induces iNOS expression in tumor cells and myeloid cells. IFNγ-induced IRF8 acts in concert with the NF-kB p65/p65 and p50/p50 homodimers to regulate iNOS expression in both colon carcinoma and myeloid cells.Figure 1Figure 2Figure 3