Antonio De Flora

Sirtuin 6 (SIRT6) is a member of the mammalian NAD⁺-dependent deac(et)ylase sirtuin family. SIRT6’s anti-inflammatory roles are emerging increasingly often in different diseases and cell types, including endothelial cells. In this study, the role of SIRT6 in pro-inflammatory conditions was investigated by engineering human umbilical vein endothelial cells to overexpress SIRT6 (SIRT6+ HUVECs). Our results showed that SIRT6 overexpression affected the levels of adhesion molecules and sustained megakaryocyte proliferation and proplatelet formation. Interestingly, the pro-inflammatory activation of the ATP/purinergic axis was reduced in SIRT6+ HUVECs. Specifically, the TNFα-induced release of ATP in the extracellular space and the increase in pannexin-1 hemichannel expression, which mediates ATP efflux, were hampered in SIRT6+ cells. Instead, NAD⁺ release and Connexin43 expression were not modified by SIRT6 levels. Moreover, the Ca²⁺ influx in response to ATP and the expression of the purinergic receptor P2X7 were decreased in SIRT6+ HUVECs. Contrary to extracellular ATP, extracellular NAD⁺ did not evoke pro-inflammatory responses in HUVECs. Instead, NAD⁺ administration reduced endothelial cell proliferation and motility and counteracted the TNFα-induced angiogenesis. Altogether, our data reinforce the view of SIRT6 activation as an anti-inflammatory approach in vascular endothelium.

Boosting NAD⁺ levels are considered a promising means to promote healthy aging and ameliorate dysfunctional metabolism. The expression of CD38, the major NAD⁺-consuming enzyme, is downregulated during thermogenesis in both brown and white adipose tissues (BAT and WAT). Moreover, BAT activation and WAT “browning” were enhanced in Cd38^−/− mice. In this study, the role of CD38 in the liver during thermogenesis was investigated, with the liver being the central organ controlling systemic energy metabolism. Wild-type mice and Cd38^−/− mice were exposed to cold temperatures, and levels of metabolites and enzymes were measured in the livers and plasma. During cold exposure, CD38 expression was downregulated in the liver, as in BAT and WAT, with a concomitant increase in NAD(H) and a marked decrease in NADPH levels. Glucose-6-phosphate dehydrogenase and the malic enzyme, along with enzymes in the glycolytic pathway, were downregulated, which is in line with glucose-6-P being re-directed towards glucose release. In Cd38^−/− mice, the cross-regulation between glycolysis and glucose release was lost, although this did not impair the glucose release from glycogen. Glycerol levels were decreased in the liver from Cd38^−/− animals upon cold exposure, suggesting that glyceroneogenesis, as gluconeogenesis, was not properly activated in the absence of CD38. SIRT3 activity, regulating mitochondrial metabolism, was enhanced by cold exposure, whereas its activity was already high at a warm temperature in Cd38^−/− mice and was not further increased by the cold. Notably, FGF21 and bile acid release was enhanced in the liver of Cd38^−/− mice, which might contribute to enhanced BAT activation in Cd38^−/− mice. These results demonstrate that CD38 inhibition can be suggested as a strategy to boost NAD⁺ and would not negatively affect hepatic functions during thermogenesis.

ADP-ribosyl cyclases (ADPRCs) catalyze the synthesis of the Ca²⁺-active second messengers Cyclic ADP-ribose (cADPR) and ADP-ribose (ADPR) from NAD⁺ as well as nicotinic acid adenine dinucleotide phosphate (NAADP⁺) from NADP⁺. The best characterized ADPRC in mammals is CD38, a single-pass transmembrane protein with two opposite membrane orientations. The first identified form, type II CD38, is a glycosylated ectoenzyme, while type III CD38 has its active site in the cytosol. The ectoenzymatic nature of type II CD38 raised long ago the question of a topological paradox concerning the access of the intracellular NAD⁺ substrate to the extracellular active site and of extracellular cADPR product to its intracellular receptors, ryanodine (RyR) channels. Two different transporters, equilibrative connexin 43 (Cx43) hemichannels for NAD⁺ and concentrative nucleoside transporters (CNTs) for cADPR, proved to mediate cell-autonomous trafficking of both nucleotides. Here, we discussed how type II CD38, Cx43 and CNTs also play a role in mediating several paracrine processes where an ADPRC⁺ cell supplies a neighboring CNT-and RyR-expressing cell with cADPR. Recently, type II CD38 was shown to start an ectoenzymatic sequence of reactions from NAD⁺/ADPR to the strong immunosuppressant adenosine; this paracrine effect represents a major mechanism of acquired resistance of several tumors to immune checkpoint therapy.

Nicotinamide adenine dinucleotide (NAD⁺) is a fundamental molecule in the regulation of energy metabolism, representing both a coenzyme and a substrate for different NAD⁺ degrading enzymes. Among these enzymes, CD38 can be seen under two perspectives: as the enzyme synthesizing Ca²⁺-mobilizing second messenger, starting from NAD⁺, and as the major NAD⁺-consumer, to be inhibited to increase NAD⁺ levels. Indeed, the regulation of NAD⁺ availability is a key event during different processes. In this review, we examine the recent studies related to the modulation of CD38 expression and activity, and the consequent changes in NAD(P)(H), in adipose tissue, during inflammation and cold-induced thermogenesis.

In several cell types, a regulated efflux of NAD⁺ across Connexin 43 hemichannels (Cx43 HC) can occur, and extracellular NAD⁺ (NAD⁺_e) affects cell-specific functions. We studied the capability of bone marrow-derived human mesenchymal stem cells (MSC) to release intracellular NAD⁺ through Cx43 HC. NAD⁺ efflux, quantified by a sensitive enzymatic cycling assay, was significantly upregulated by low extracellular Ca²⁺ (5–6-fold), by shear stress (13-fold), and by inflammatory conditions (3.1- and 2.5-fold in cells incubated with lipopolysaccharide (LPS) or at 39°C, respectively), as compared with untreated cells, whereas it was downregulated in Cx43-siRNA-transfected MSC (by 53%) and by cell-to-cell contact (by 45%). Further, we show that NAD⁺_e activates the purinergic receptor P2Y₁₁ and a cyclic adenosin monophosphate (cAMP)/cyclic ADP-ribose/[Ca²⁺]_i signaling cascade, involving the opening, unique to MSC, of L-type Ca²⁺ channels. Extracellular NAD⁺ enhanced nuclear translocation of cAMP/Ca²⁺-dependent transcription factors. Moreover, NAD⁺, either extracellularly added or autocrinally released, resulted in stimulation of MSC functions, including proliferation, migration, release of prostaglandin E₂ and cytokines, and downregulation of T lymphocyte proliferation compared with controls. No detectable modifications of MSC markers and of adipocyte or osteocyte differentiation were induced by NAD⁺_e. Controls included Cx43-siRNA transfected and/or NAD⁺-glycohydrolase-treated MSC (autocrine effects), and NAD⁺-untreated or P2Y₁₁-siRNA-transfected MSC (exogenous NAD⁺). These findings suggest a potential beneficial role of NAD⁺_e in modulating MSC functions relevant to MSC-based cell therapies.

Lipopolysaccharide, the main component of the cell wall of Gram‐negative bacteria, is known to activate microglial cells following its interaction with the CD14/Toll‐like receptor complex (TLR‐4). The activation pathway triggered by lipopolysaccharide in microglia involves enhanced basal levels of intracellular calcium ([Ca²⁺]_i) and terminates with increased generation of cytokines/chemokines and nitric oxide. Here we demonstrate that in lipopolysaccharide‐stimulated murine N9 microglial cells, cyclic ADP‐ribose, a universal and potent Ca²⁺ mobiliser generated from NAD⁺ by ADP‐ribosyl cyclases (ADPRC), behaves as a second messenger in the cell activation pathway. Lipopolysaccharide induced phosphorylation, mediated by multiple protein kinases, of the mammalian ADPRC CD38, which resulted in significantly enhanced ADPRC activity and in a 1.7‐fold increase in the concentration of intracellular cyclic ADP‐ribose. This event was paralleled by doubling of the basal [Ca²⁺]_i levels, which was largely prevented by the cyclic ADP‐ribose antagonists 8‐Br‐cyclic ADP‐ribose and ryanodine (by 75% and 88%, respectively). Both antagonists inhibited, although incompletely, functional events downstream of the lipopolysaccharide‐induced microglia‐activating pathway, i.e. expression of inducible nitric oxide synthase, overproduction and release of nitric oxide and of tumor necrosis factor α. The identification of cyclic ADP‐ribose as a key signal metabolite in the complex cascade of events triggered by lipopolysaccharide and eventually leading to enhanced generation of pro‐inflammatory molecules may suggest a new therapeutic target for treatment of neurodegenerative diseases related to microglia activation.

β-NAD+e (extracellular β-NAD+), present at nanomolar levels in human plasma, has been implicated in the regulation of [Ca2+]i (the intracellular calcium concentration) in various cell types, including blood cells, by means of different mechanisms. Here, we demonstrate that micromolar NAD+e (both the α and the β extracellular NAD+ forms) induces a sustained [Ca2+]i increase in human granulocytes by triggering the following cascade of causally related events: (i) activation of adenylate cyclase and overproduction of cAMP; (ii) activation of protein kinase A; (iii) stimulation of ADP-ribosyl cyclase activity and consequent overproduction of cADP-ribose, a universal Ca2+ mobilizer; and (iv) influx of extracellular Ca2+. The NAD+e-triggered [Ca2+]i elevation translates into granulocyte activation, i.e. superoxide and nitric oxide generation, and enhanced chemotaxis in response to 0.1–10 μM NAD+e. Thus extracellular β-NAD+e behaves as a novel pro-inflammatory cytokine, stimulating human granulocytes and potentially recruiting them at sites of inflammation.

Abstract: CD38, a multifunctional enzyme, generates two potent Ca²⁺‐releasing signal metabolites, cyclic ADP‐ribose (cADPR) and NAADP⁺, thereby upmodulating many important Ca²⁺‐mediated cell functions. A topological paradox has long been recognized, as CD38 is an ectoenzyme, or an intravesicularly located enzyme in subcellular membrane vesicles, therefore apparently shielded from its substrate NAD⁺. Moreover, cADPR generated by CD38 should be unavailable to its target Ca²⁺ stores, the ryanodine receptors (RyR). We have solved this paradox by identifying some NAD+ and cADPR transmembrane transporters, whose interplay mediates a hitherto‐unrecognized subcellular and intercellular trafficking of nucleotides that enhances intracellular Ca²⁺ ([Ca²⁺]_i). Connexin 43 (Cx43) hemichannels mediate an equilibrative transport of NAD+ from the cytosol to the active site of CD38 (either ectocellular or intravesicular). Subsequent translocation of in situ‐generated cADPR to reach the RyR is performed, (i) by CD38 itself (concentrative) or (ii) by nucleoside transporters (NT) (one equilibrative and three concentrative). Besides this autocrine mechanism, the same transporters also mediate intercellular (paracrine) trafficking. Thus, Cx43⁺ and CD38⁺ cells can provide cADPR to neighboring RyR⁺ parenchymal cells and enhance their [Ca²⁺]_i levels and Ca²⁺‐dependent functions accordingly. Examples of cADPR‐responsive cells via paracrine processes include (i) smooth myocytes, (ii) 3T3 murine fibroblasts, (iii) hippocampal neurons, and (iv) human hemopoietic stem cells.

Recently, a new system of astrocyte–neurone glutamatergic signalling has been identified. It is started in astrocytes by ectocellular, CD38‐catalysed conversion of NAD⁺ to the calcium mobilizer cyclic ADP‐ribose (cADPR). This is then pumped by CD38 itself into the cytosol where the resulting free intracellular Ca²⁺ concentration [Ca²⁺]_i transients elicit an increased release of glutamate, which can induce an enhanced Ca²⁺ response in neighbouring neurones. Here, we demonstrate that co‐culture of either cortical or hippocampal astrocytes with neurones results in a significant overexpression of astrocyte CD38 both on the plasma membrane and intracellularly. The causal role of neurone‐released glutamate in inducing overexpression of astrocyte CD38 is demonstrated by two observations: first, in the absence of neurones, induction of CD38 in pure astrocyte cultures can be obtained with glutamate and second, it can be prevented in co‐cultures by glutamate receptor antagonists. The neuronal glutamate‐mediated effect of neurones on astrocyte CD38 expression is paralleled by increased intracellular cADPR and [Ca²⁺]_i levels, both findings indicating functionality of overexpressed CD38. These results reveal a new neurone‐to‐astrocyte glutamatergic signalling based on the CD38/cADPR system, which affects the [Ca²⁺]_i in both cell types, adding further complexity to the bi‐directional patterns of communication between astrocytes and neurones.