The NF-κB p65 and p50 homodimer cooperate with IRF8 to activate iNOS transcription.
BACKGROUND
Inducible nitric oxide synthase (iNOS) metabolizes L-arginine to produce nitric oxide (NO) which was originally identified in myeloid cells as a host defense mechanism against pathogens. Recent studies, however, have revealed that iNOS is often induced in tumor cells and myeloid cells in the tumor microenvironment. Compelling experimental data have shown that iNOS promotes tumor development in certain cellular context and suppresses tumor development in other cellular conditions. The molecular mechanisms underlying these contrasting functions of iNOS is unknown. Because iNOS is often induced by inflammatory signals, it is therefore likely that these contrasting functions of iNOS could be controlled by the inflammatory signaling pathways, which remains to be determined.


METHODS
iNOS is expressed in colon carcinoma and myeloid cells in the tumor microenvironment. Colon carcinoma 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-κB association with the chromatin DNA of the nos2 promoter.


RESULTS
We show here that iNOS is dramatically up-regulated in inflammed human colon tissues and in human colon carcinoma as compared to normal colon tissue. iNOS is expressed in either the colon carcinoma cells or immune cells within the tumor microenvironment. On the molecular level, the proinflammatory IFNγ and NF-κB signals induce iNOS expression in human colon cancer cells. We further demonstrate that NF-κB directly binds to the NOS2 promoter to regulate iNOS expression. Although neither the IFNγ signaling pathway nor the NF-κB signaling pathway alone is sufficient to induce iNOS expression in myeloid cells, IFNγ and NF-κB synergistically induce iNOS expression in myeloid cells. Furthermore, we determine that IFNγ up-regulates IRF8 expression to augment NF-κB induction of iNOS expression. More interestingly, we observed that the p65/p65 and p50/p50 homodimers, not the canonical p65/p50 heterodimer, directly binds to the nos2 promoter to regulate iNOS expression in myeloid cells.


CONCLUSIONS
IFNγ-induced IRF8 acts in concert with NF-κB to regulate iNOS expression in both colon carcinoma and myeloid cells. In myeloid cells, the NF-κB complexes that bind to the nos2 promoter are p65/p65 and p50/p50 homodimers.
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Regents University, Augusta, GA, 30912, USA. Predd@gru.edu.|Cancer Center, Georgia Regents University, Augusta, GA, 30912, USA. Predd@gru.edu.|Charlie Norwood VA Medical Center, Augusta, GA, 30904, USA. Predd@gru.edu.
Priscilla S
PS
Simon
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Regents University, Augusta, GA, 30912, USA. Ssharman@gru.edu.|Cancer Center, Georgia Regents University, Augusta, GA, 30912, USA. Ssharman@gru.edu.
Sarah K
SK
Sharman
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Regents University, Augusta, GA, 30912, USA. Clu@gru.edu.|Charlie Norwood VA Medical Center, Augusta, GA, 30904, USA. Clu@gru.edu.
Chunwan
C
Lu
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Regents University, Augusta, GA, 30912, USA. Dyang@gru.edu.|Charlie Norwood VA Medical Center, Augusta, GA, 30904, USA. Dyang@gru.edu.
Dafeng
D
Yang
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Regents University, Augusta, GA, 30912, USA. Apaschall@gru.edu.|Cancer Center, Georgia Regents University, Augusta, GA, 30912, USA. Apaschall@gru.edu.|Charlie Norwood VA Medical Center, Augusta, GA, 30904, USA. Apaschall@gru.edu.
Amy V
AV
Paschall
Medicine, Medical College of Georgia, Georgia Regents University, Augusta, GA, 30912, USA. Stulachan@gru.edu.
Sidhartha S
SS
Tulachan
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Regents University, Augusta, GA, 30912, USA. Kliu@gru.edu.|Cancer Center, Georgia Regents University, Augusta, GA, 30912, USA. Kliu@gru.edu.|Charlie Norwood VA Medical Center, Augusta, GA, 30904, USA. Kliu@gru.edu.
Kebin
K
Liu
D000818:Animals; D045744:Cell Line, Tumor / Q000378:metabolism; D003106:Colon / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism*; D006801:Humans; D050835:Interferon Regulatory Factors / Q000378:metabolism / Q000502:physiology*; D007371:Interferon-gamma / Q000502:physiology; D051379:Mice; D022423:Myeloid Cells / Q000378:metabolism; D052002:NF-kappa B p50 Subunit / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051996:Transcription Factor RelA / Q000502:physiology*; D059016:Tumor Microenvironment / Q000502:physiology; D000099185:Interferon Regulatory Factor-8
D050835:Interferon Regulatory Factors; D007371:Interferon-gamma; D052002:NF-kappa B p50 Subunit; D052247:Nitric Oxide Synthase Type II; D051996:Transcription Factor RelA; D000099185:Interferon Regulatory Factor-8; C496319:NOS2 protein, human