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.

Design: Prospective experimental study.

Setting: University medical center.

Patient(s): Primary cultures of fibroblasts from normal peritoneum and adhesion tissues.

Intervention(s): Hypoxia-treated cells.

Main Outcome Measure(s): 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.

Result(s): 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.

Conclusion(s): 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.