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

Nuclear factor kappa B (NFκB), 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 NFκB activation. We hypothesized that this inhibitory action of NO on NFκB activation serves as a negative feedback to inhibit NFκB 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 NFκB inhibitor panepoxydone prevented the increase in eNOS mRNA caused by shear confirming a role of NFκB in this response. Shear stress stimulated a transient (30 min) nuclear translocation of the NFκB subunit p50. Treatment with l-NAME increased binding of the NFκB subunit p50 to consensus oligonucleotide-coated microtiter 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, NFκB activation, NO production and NFκB inhibition represents a classical negative feedback loop, which prevents sustained activation of NFκB. In the absence of NO, shear stimulation of NFκB 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 NFκB in response to shear and unrestrained endothelial inflammation.