TNFα stimulates NO release in EA.hy926 cells by activating the CaMKKβ-AMPK-eNOS pathway

Previously we showed that a mild stimulation of EA.hy926 cells with tumour necrosis factor alpha (TNF alpha) activated mitochondrial biogenesis, probably as a mechanism preventing cell death. This was accompanied by an increased phosphorylation of eNOS and elevation of NO release. The aim of the present study was to explain the biochemical basis of this effect. Our results indicate that eNOS is the only enzyme catalysing NO generation in EA.hy926 cells, and TNF alpha stimulates its activity by activating AMP-activated protein kinase (AMPK). Inhibition of AMPK with Compound C prevents the TNF alpha-induced activatory phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177 and reduces the NO release. AMPK is activated by phosphorylation catalysed by liver kinase B1 (LKB1) and calcium/calmodulin-dependent protein kinase kinase beta (CaMKK beta), which are phosphorylated and thereby activated in the presence of TNFa. Moreover, CaMK beta catalyses an activatory phosphorylation of sirtuin 1, which could deacetylate and activate eNOS both directly and indirectly by an elevating the LKB1 activity. TNF alpha hardly increases the nuclear fraction of sirtuin 1, thus its major activity is probably attributed to the cytosolic pool. This is in line with the elevated activity of eNOS. We conclude that the increased AMPK-dependent phosphorylation of eNOS at least partially explains the stimulation of NO generation by TNF alpha in EA.hy926 cells.