Novel Mechanism for Nicotinamide Phosphoribosyltransferase Inhibition of TNF-α–mediated Apoptosis in Human Lung Endothelial Cells

Abstract

Nicotinamide phosphoribosyltransferase (NAMPT) exists as both intracellular NAMPT and extracellular NAMPT (eNAMPT) proteins. eNAMPT is secreted into the blood and functions as a cytokine/enzyme (cytozyme) that activates NF-κB signaling via ligation of Toll-like receptor 4 (TLR4), further serving as a biomarker for inflammatory lung disorders such as acute respiratory distress syndrome. In contrast, intracellular NAMPT is involved in nicotinamide mononucleotide 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, overexpression of NAMPT served to reduce degrees of TNF-α-induced EC apoptosis. Inhibition of nicotinamide mononucleotide 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 an 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.

Role of secreted extracellular nicotinamide phosphoribosyltransferase (eNAMPT) in prostate cancer progression: Novel biomarker and therapeutic target

“…Within this cohort of 253 men, seventy-three subjects were subsequently diagnosed with PCa by prostate biopsies and the remainder of the cohort ( n = 180) remained PCa -free for up to a 5 year follow up period [26] . A previously reported non-commercial in-house ELISA assay [11] utilizing a proprietary goat α-NAMPT polyclonal antibody, a rabbit polyclonal antibody (Thermo Scientific cat. PA534858) and a secondary donkey anti-rabbit IgG antibody-HRP (Life Technologies cat.…”

“…PA534858) and a secondary donkey anti-rabbit IgG antibody-HRP (Life Technologies cat. A16035) [11] was used to asses eNAMPT level in the plasma samples of 34 PCa, 130 high risk Not-PCa and 105 healthy control subjects. This ELISA assay was also utilized to assess eNAMPT levels in supernatants from PC3 and DU145 cultures exposed to a variety of stimuli.…”

“…NAMPT-mediated NAD biosynthesis controls the functions of mammalian sirtuin family members, as well as other NAD-consuming enzymes, such as PARPs, in each subcellular compartment, therefore, involving in a variety of critical biological processes, including cell metabolism and stress response [ 8 , 9 ]. In contrast to iNAMPT, we previously have shown that the extracellularly-secreted NAMPT ( eNAMPT) is a key innate immunity regulator and potent damage-associated molecular pattern protein (DAMP) responding to potentially injurious danger signals [10] , [11] , [12] via ligation of Toll-like receptor 4 (TLR4) and activation of this evolutionarily-conserved inflammatory cascade [10] .…”

NAMPT and NAPRT: Two Metabolic Enzymes With Key Roles in Inflammation

“…In the tumor microenvironment, neutralization of eNAMPT using blocking antibodies could be effective to repolarize myeloid cells (TAMs/MDSCs) against tumor. Some groups/companies are working on the production of these antibodies (193), hypothesizing a combination strategy with immunotherapy, or a double inhibition of i/eNAMPT. Blocking eNAPRT in acute inflammatory conditions, such as in septic patients, could be an important strategy to prevent the damaging action of a massive secretion of eNAPRT leading to decreased survival of patients, but this remains, at this moment, only a speculative hypothesis.…”

Immunotherapeutic approaches to curtail COVID-19

“…It was shown that physical stress such as excessive mechanical stress caused by ventilators upregulates the expression of a gene, called nicotinamide phosphoribosyltransferase (NAMPT). As the bioavailability of NAMPT increases, Toll-like receptor 4 (TLR4), which is responsible for lung inflammation, gets activated [75] , [76] . Herein, neutralization of circulating NAMPT by monoclonal antibodies can be another viable approach in preventing the lung injury caused by SARS-CoV-2.…”