Hypoxia regulates iNOS expression in human normal peritoneal and adhesion fibroblasts through nuclear factor kappa B activation mechanism.
OBJECTIVE
To determine the mechanism by which hypoxia increases expression of iNOS in human normal peritoneal and adhesion fibroblasts.


METHODS
Prospective experimental study.


METHODS
University medical center.


METHODS
Primary cultures of fibroblasts from normal peritoneum and adhesion tissues.


METHODS
Hypoxia-treated cells.


METHODS
We used real-time reverse transcription-polymerase chain reaction to quantify mRNA levels of iNOS and nuclear factor kappa B (NF-kappaB). Western blots were used to determine iNOS, NF-kappaB, IkappaB-alpha, and phospho-IkappaB expression levels in normal peritoneal and adhesion fibroblasts in response to hypoxia.


RESULTS
Hypoxia resulted in a significant increase in iNOS and NF-kappaB expression in normal and adhesion fibroblasts. Furthermore, both cell types manifested lower levels of NF-kappaB, cytoplasmic phospho-IkappaB-alpha, and iNOS proteins. In contrast, they manifested higher levels of cytoplasmic IkappaB-alpha and IkappaB-alpha/NF-kappaB ratios as well as a phosphorylated-IkappaB-alpha/NF-kappaB ratio. Under hypoxic conditions, both cell types exhibited significantly decreased cytoplasmic NF-kappaB, IkappaB-alpha levels, and significantly increased cytoplasmic phospho-IkappaB-alpha, iNOS, and NF-kappaB protein levels.


CONCLUSIONS
Hypoxia increases iNOS expression by a mechanism involving activation of NF-kappaB. The ratio of IkappaB-alpha/NF-kappaB or IkappaB-alpha/p-IkappaB-alpha can be used to monitor activation.
Jiang
Zhong L
ZL
Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Fletcher
Nicole M
NM
Diamond
Michael P
MP
Abu-Soud
Husam M
HM
Saed
Ghassan M
GM
D015153:Blotting, Western; D015687:Cell Hypoxia; D002478:Cells, Cultured; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D010532:Peritoneal Diseases / Q000201:enzymology* / Q000473:pathology; D010537:Peritoneum / Q000201:enzymology* / Q000473:pathology; D010766:Phosphorylation; D011446:Prospective Studies; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D000267:Tissue Adhesions; D015854:Up-Regulation
D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D012333:RNA, Messenger; D000072000:NF-KappaB Inhibitor alpha; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II