Novel Mechanism for Nicotinamide Phosphoribosyltransferase Inhibition of TNF-α-Mediated Apoptosis in Human Lung Endothelial Cells.
Nicotinamide phosphoribosyltransferase (NAMPT) exists as both intracellular (iNAMPT) and extracellular (eNAMPT) proteins. eNAMPT is secreted into the blood and functions as a cytokine/enzyme (cytozyme) that activates NFκB signaling via ligation of the TLR4 receptor, further serving as a biomarker for inflammatory lung disorders such as the acute respiratory distress syndrome (ARDS). In contrast, iNAMPT is involved in nicotinamide mononucleotide (NMN) synthesis and has been implicated in the regulation of cellular apoptosis, although the exact mechanisms for this regulation are poorly understood. We examined the role of NAMPT in TNF-α-induced human lung endothelial cell (EC) apoptosis and demonstrated that reduced NAMPT expression (siRNA) increases EC susceptibility to TNF-α-induced apoptosis as reflected by PARP-1 cleavage and caspase-3 activation. In contrast, over-expression of NAMPT served to reduce levels of TNF-α-induced EC apoptosis. Inhibition of NMN synthesis by FK866 (a selective NAMPT enzymatic inhibitor) failed to alter TNF-α-induced human lung EC apoptosis suggesting that NAMPT-dependent NAD generation is unlikely to be involved in regulation of TNF-α-induced EC apoptosis. We next confirmed that TNF-α-induced EC apoptosis is attributable to NAMPT secretion into the EC culture media and subsequent eNAMPT ligation of TLR4 on the EC membrane surface. Silencing of NAMPT expression, direct neutralization of secreted eNAMPT by a NAMPT-specific polyclonal antibody (preventing TLR4 ligation), or direct TLR4 antagonism, all served to significantly increase EC susceptibility to TNF-α-induced EC apoptosis. Together, these studies provide novel insights into NAMPT contributions to lung inflammatory events and to novel mechanisms of EC apoptosis regulation.