A negative feedback mechanism involving nitric oxide and nuclear factor kappa-B modulates endothelial nitric oxide synthase transcription

Nuclear factor kappa B (NF kappa B), commonly a proinflammatory transcription factor, is responsible for increasing transcription of the endothelial cell nitric oxide synthase (eNOS) in response to laminar shear stress. Nitric oxide (NO) production can be stimulated by shear, and NO is known to inhibit NF kappa B activation. We hypothesized that this inhibitory action of NO on NF kappa B activation serves as a negative feedback to inhibit NF kappa B activity and eNOS transcription. Exposure of bovine aortic endothelial cells to laminar shear stimulated steady state eNOS mRNA expression and eNOS promoter activity as measured using an eNOS promoter/CAT construct. These effects of shear were enhanced by the NOS inhibitor L-NAME and decreased by the NO-donor DPTA-NO by 30-50%. The NF kappa B inhibitor panepoxydone prevented the increase in eNOS mRNA caused by shear confirming a role of NF kappa B in this response. Shear stress stimulated a transient (30 min) nuclear translocation of the NF kappa B subunit p50. Treatment with L-NAME increased binding of the NF kappa B subunit p50 to consensus oligonucleotide-coated micro-titer plates, while having only minimal effect on binding of p65, strongly suggesting that nitric oxide mainly inhibits p50 activation. Using the biotin switch method, we found that shear stress stimulates p50 nitrosylation and this was prevented by L-NAME. Moreover, transfection of endothelial cells with a vector encoding the C62S p50, a variant with a point mutation of the nitrosylation site C62, markedly increased nuclear translocation of p50 and doubled eNOS mRNA expression under shear stress compared to that observed in cells transfected with wild-type p50. We conclude that this interaction between shear, NF kappa B activation, NO production and NF kappa B inhibition represents a classical negative feedback loop, which prevents sustained activation of NF kappa B. In the absence of NO, shear stimulation of NF kappa B and eNOS transcription are enhanced. Our findings emphasize the critical role of NO in modulation of the endothelial cell inflammatory state. Several common diseases, including hypercholesteremia, hypertension and diabetes, are associated with eNOS dysfunction. Under these conditions, decreased NO availability may result in sustained activation of NF kappa B in response to shear and unrestrained endothelial inflammation. (c) 2005 Elsevier Ltd. All rights reserved.