Luisa Franco

Connexin 43 hemi channels mediate Ca2+-regulated transmembrane NAD+ fluxes in intact cells

A previously unrecognized passive transport for pyridine dinucleotides has been described recently in the plasmamembrane of several mammalian cells. Despite elucidation of some functional and kinetic properties of this transport system, it is still undefined at the molecular level. Therefore, we have addressed the molecular characterization of the NAD+ transporter and identified it as connexin 43 (Cx43). This is a structural component of hexameric hemichannels that, when juxtaposed on adjacent cells, builds up intercellular gap junctions and mediates exchange of molecules between cells. However, the role of connexin hemichannels as potential pores in individual, noncoupled cells remains elusive. Bidirectional NAD+ transport in isolated Cx43‐expressing murine 3T3 fibroblasts was affected by known modulators of connexin‐mediated intercellular coupling and was completely inhibited by treatment of the cells with a Cx43‐antisense oligonucleotide. NAD+ transport in proteoliposomes reconstituted with 3T3 membrane proteins was inhibited in the presence of a monoclonal anti‐Cx43 antibody. Finally, Cx43 immunopurified to homogeneity was reconstituted in unilamellar proteoliposomes, which displayed full NAD+‐transporting activity. This finding is the first evidence that connexin hemichannels can mediate transmembrane fluxes of a nucleotide in whole cells: The pleiotropy of NAD+‐dependent cellular events, including redox reactions, signaling, and DNA repair, implicates Cx43 hemichannels in intercellular NAD+ trafficking, which suggests new paracrine functions of NAD+.

Autocrine and Paracrine Calcium Signaling by the CD38/NAD+/Cyclic ADP‐Ribose System

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.

The transmembrane glycoprotein CD38 is a catalytically active transporter responsible for generation and influx of the second messenger cyclic ADP-ribose across membranes

CD38 is a type II transmembrane glycoprotein expressed in many vertebrate cells. It is a bifunctional ectoenzyme that catalyzes both the synthesis of Cyclic ADP‐ribose (cADPR) from NAD+ and the degradation of cADPR to ADP‐ribose by means of its ADP‐ribosyl cyclase and cADPR‐hydrolase activities, respectively. The cyclase also converts NGD+ to cyclic GDP‐ribose (cGDPR), which is refractory to cADPR‐hydrolase. cADPR, but not cGDPR, is a potent calcium mobilizer from intracellular stores. It has been demonstrated to be a new second messenger involved in the regulation of calcium homeostasis in many cell types, from plants to mammals. The number of physiological processes shown to be regulated by cADPR is steadily increasing. A topological paradox exists because ectocellularly generated cADPR acts intracellularly. Here we demonstrate that the catalytic functioning of CD38 is accompanied by a cADPR (cGDPR) ‐transporting activity across natural and artificial membranes. In resealed membranes from CD38+ human erythrocytes, transport of catalytically generated cADPR or cGDPR was saturation dependent and occurred against a concentration gradient. Likewise, CD38‐reconstituted proteoliposomes were active in concentrating NAD+ (NGD+) ‐derived cADPR (cGDPR) inside the vesicle compartment. Moreover, the cADPR‐transporting activity in CD38 proteoliposomes prevented the hydrolase‐catalyzed degradation to ADPR that occurs conversely with detergent‐solubilized CD38, resulting in selective influx of cADPR. In the CD38 proteoliposomes, catalytically active CD38 exhibited monomeric, dimeric, and tetrameric structures. In CD38 sense‐ but not in antisense‐transfected HeLa cells, externally added NAD+ resulted in significant, transient increases in cytosolic calcium. These data suggest that transmembrane juxtaposition of two or four CD38 monomers can generate a catalytically active channel for selective formation and influx of cADPR (cGDPR) to reach cADPR‐responsive intracellular calcium stores.—Franco, L., Guida, L., Bruzzone, S., Zocchi, E., Usai, C., De Flora, A. The transmembrane glycoprotein CD38 is a catalytically active transporter responsible for generation and influx of the second messenger cyclic ADP‐ribose across membranes. FASEB J. 12, 1507–1520 (1998)

The temperature-signaling cascade in sponges involves a heat-gated cation channel, abscisic acid, and cyclic ADP-ribose.

Sponges (phylum Porifera) are the phylogenetically oldest metazoan animals, their evolution dating back to 600 million years ago. Here we demonstrate that sponges express ADP-ribosyl cyclase activity, which converts NAD+ into cyclic ADP-ribose, a potent and universal intracellular Ca2+ mobilizer. In Axinella polypoides (Demospongiae, Axinellidae), ADP-ribosyl cyclase was activated by temperature increases by means of an abscisic acid-induced, protein kinase A-dependent mechanism. The thermosensor triggering this signaling cascade was a heat-activated cation channel. Elucidation of the complete thermosensing pathway in sponges highlights a number of features conserved in higher organisms: (i) the cation channel thermoreceptor, sensitive to heat, mechanical stress, phosphorylation, and anesthetics, shares all of the functional characteristics of the mammalian heat-activated background K+ channel responsible for central and peripheral thermosensing; (ii) involvement of the phytohormone abscisic acid and cyclic ADP-ribose as its second messenger is reminiscent of the drought stress signaling pathway in plants. These results suggest an ancient evolutionary origin of this stress-signaling cascade in a common precursor of modern Metazoa and Metaphyta.

Cyclic GMP-dependent and -independent Effects on the Synthesis of the Calcium Messengers Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) have been shown to mobilize intracellular Ca2+ stores by totally independent mechanisms, which are pharmacologically distinct from that activated by inositol trisphosphate. Although cADPR and NAADP are structurally and functionally different, they can be synthesized by a single enzyme having ADP-ribosyl cyclase activity. In this study, three different assays were used to measure the metabolism of cADPR in sea urchin egg homogenates including a radioimmunoassay, a Ca2+release assay, and a thin layer chromatographic assay. Soluble and membrane-bound ADP-ribosyl cyclases were identified and both cyclized NAD to produce cADPR. The soluble cyclase was half-maximally stimulated by 5.3 μm cGMP, but not by cAMP, while the membrane-bound form was independent of cGMP. The two forms of the cyclase were also different in the pH dependence of utilizing nicotinamide guanine dinucleotide (NGD), a guanine analog of NAD, as substrate, indicating they are two separate enzymes. The stimulatory effect of cGMP required ATP or ATPγS (adenosine 5′-O-(3-thiotriphosphate)) and a cGMP-dependent kinase activity was shown to be present in the soluble fraction. The degradation of cADPR to ADP-ribose was catalyzed by cADPR hydrolase, which was found to be predominantly associated with membranes. Similar to the membrane-bound cyclase, the cADPR hydrolase activity was also independent of cGMP. Both the soluble and membrane fractions also catalyzed the synthesis of NAADP through exchanging the nicotinamide group of NADP with nicotinic acid (NA). The base-exchange activity was independent of cGMP and the half-maximal concentrations of NADP and NA needed were about 0.2 mm and 10 mm, respectively. The exchange reaction showed a preference for acidic pH, contrasting with the neutral pH optimum of the cyclase activities. The complex metabolic pathways characterized in this study indicate that there may be a multitude of regulatory mechanisms for controlling the endogenous concentrations of cADPR and NAADP.

Ligand-induced internalization of CD38 results in intracellular Ca2+ mobilization: role of NAD+ transport across cell membranes

CD38, a transmembrane glycoprotein widely expressed in vertebrate cells, is a bifunctional ectoenzyme catalyzing the synthesis and hydrolysis of cyclic ADP‐ribose (cADPR). cADPR is a universal second messenger that releases calcium from intracellular stores. Since cADPR is generated by CD38 at the outer surface of many cells, where it acts intracellularly, increasing attention is paid to addressing this topological paradox. Recently, we demonstrated that CD38 is a catalytically active, unidirectional transmembrane transporter of cADPR, which then reaches its receptor‐operated intracellular calcium stores. Moreover, CD38 was reported to undergo a selective and extensive internalization through non clathrin‐coated endocytotic vesicles upon incubating CD38+ cells with either NAD+ or thiol compounds: these endocytotic vesicles can convert cytosolic NAD into cADPR despite an asymmetric unfavorable orientation that makes the active site of CD38 intravesicular. Here we demonstrate that the cADPR‐generating activity of the endocytotic vesicles results in remarkable and sustained increases of intracellular free calcium concentration in different cells exposed to either NAD+, or GSH, or N‐acetylcysteine. This effect of CD38‐internalizing ligands on intracellular calcium levels was found to involve a two‐step mechanism: 1) influx of cytosolic NAD+ into the endocytotic vesicles, mediated by a hitherto unrecognized dinucleotide transport system that is saturable, bidirectional, inhibitable by 8‐N3‐NAD+, and characterized by poor dinucleotide specificity, low affinity, and high efficiency; 2) intravesicular CD38‐catalyzed conversion of NAD+ to cADPR, followed by out‐pumping of the cyclic nucleotide into the cytosol and subsequent release of calcium from thapsigarginsensitive stores. This unknown intracellular trafficking of NAD+ and cADPR based on two distinctive and specific transmembrane carriers for either nucleotide can affect the intracellular calcium homeostasis in CD38+ cells. —Zocchi, E., Usai, C., Guida, L., Franco, L., Bruzzone, S., Passalacqua, M., De Flora, A. Ligand‐induced internalization of CD38 results in intracellular Ca2+ mobilization: role of NAD+ transport across cell membranes. FASEB J. 13, 273–283 (1999)

A Self-restricted CD38-connexin 43 Cross-talk Affects NAD+ and Cyclic ADP-ribose Metabolism and Regulates Intracellular Calcium in 3T3 Fibroblasts

Connexin 43 (Cx43) hexameric hemichannels, recently demonstrated to mediate NAD+ transport, functionally interact in the plasma membrane of several cells with the ectoenzyme CD38 that converts NAD+ to the universal calcium mobilizer cyclic ADP-ribose (cADPR). Here we demonstrate that functional uncoupling between CD38 and Cx43 in CD38-transfected 3T3 murine fibroblasts is paralleled by decreased [Ca2+] i levels as a result of reduced intracellular conversion of NAD+ to cADPR. A sharp inverse correlation emerged between [Ca2+] i levels and NAD+ transport (measured as influx into cells and as efflux therefrom), both in the CD38+ cells (high [Ca2+] i , low transport) and in the CD38− fibroblasts (low [Ca2+] i , high transport). These differences were correlated with distinctive extents of Cx43 phosphorylation in the two cell populations, a lower phosphorylation with high NAD+ transport (CD38− cells) and vice versa (CD38+ cells). Conversion of NAD+-permeable Cx43 to the phosphorylated, NAD+-impermeable form occurs via Ca2+-stimulated protein kinase C (PKC). Thus, a self-regulatory loop emerged in CD38+ fibroblasts whereby high [Ca2+] i restricts further Ca2+mobilization by cADPR via PKC-mediated disruption of the Cx43-CD38 cross-talk. This mechanism may avoid: (i) leakage of NAD+from cells; (ii) depletion of intracellular NAD+ by CD38; (iii) overproduction of intracellular cADPR resulting in potentially cytotoxic [Ca2+] i .

Extracellular cyclic ADP-ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors

Cyclic ADP‐ribose (cADPR) is a universal second messenger that regulates many calcium‐related cellular events by releasing calcium from intracellular stores. Since these events include enhanced cell proliferation and since the bone marrow harbors both ectoenzymes that generate cADPR from NAD+ (CD38 and BST‐1), we investigated the effects of extracellular cADPR on human hemopoietic progenitors (HP). Exposure of HP to 100 μM cADPR for 24 h induced a significant increase in colony output (P<0.01) and colony size (P<0.003). A horizontal expansion of HP, as demonstrated by a markedly increased replating efficiency in semisolid medium (up to 700 times compared to controls), was also observed, indicating that cADPR priming can affect cell growth for multiple generations over several weeks after exposure. Influx of extracellular cADPR into the cells was demonstrated, and a causal relationship between the functional effects and the increase of intracellular free calcium concentration induced by cADPR on HP was established through the use of specific antagonists. Similar effects on HP were produced by nanomolar concentrations of the nonhydrolyzable cADPR analog 3‐deaza‐cADPR. These data demonstrate that extracellular cADPR behaves as a cytokine enhancing the proliferation of human HP, a finding that may have biomedical applications for the ex vivo expansion of hemopoietic cells.—Podestà, M., Zocchi, E., Pitto, A., Usai, C., Franco, L., Bruzzone, S., Guida, L., Bacigalupo, A., Scadden, D. T., Walseth, T. F., De Flora, A., Daga, A. Extracellular cyclic ADP‐ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors. FASEB J. 14, 680–690 (2000)

CD38 expression and functional activities are up-regulated by IFN-γ on human monocytes and monocytic cell lines

Human CD38, a surface molecule expressed by immature and activated T and B lymphocytes, has been characterized as a molecule transducing activation and proliferation signals, and intervening in adhesion to endothelium via its ligand CD31. CD38 is also a complex ectoenzyme featuring ADP‐ribosyl cyclase/cyclic ADP‐ribose hydrolase activities, leading to the synthesis and degradation of cADPR, a Ca+‐mobilizing agent. We investigated the effects of monocyte‐activating stimuli (IFN‐γ, IL‐2, LPS, TNF‐α, and GM‐CSF) on the expression and function of CD38, starting from the observation that human monocytes and the derived lines U937, THP‐1, and Mono‐Mac‐6 bear the molecule on their surface. Our results indicate that IFN‐γ is a strong up‐modulator of CD38, and IL‐2 increases its expression only modestly. LPS, TNF‐α, and GM‐CSF had no detectable effects. Treatment with IFN‐γ produced a dose‐ and time‐dependent up‐regulation of CD38 in monocytes and monocytic lines, which was paralleled by increased ADP‐ribosyl cyclase/cyclic ADP‐ribose hydrolase activities. Furthermore, CD38 ligation by specific MoAb reduced the IFN‐γ‐dependent enhancement of monocyte‐dynamic adhesion to endothelial monolayers. These findings identify IFN‐γ as a modulator of monocytic CD38 expression and indicate that CD38 plays a specific role in the activation and adhesion processes performed by monocytes.

Structural role of disulfide bridges in the cyclic ADP-ribose related bifunctional ectoenzyme CD38

Human CD38, a type II cell surface glycoprotein, is a bifunctional ectoenzyme catalyzing both ADP‐ribosyl cyclase and cyclic ADP‐ribose (cADPR) hydrolase reactions. It shares a high degree of sequence homology with the cyclase from Aplysia species and studies of site‐directed mutagenesis have recently demonstrated the importance, but not elucidated the role, of several cysteine residues highly conserved between these proteins.N‐Ethylmaleimide, iodoacetamide and thiol‐oxidizing reagents failed to affect either the cyclase or the weaker hydrolase activity of the Aplysia californica protein. Likewise, these reagents did not impair the two activities of CD38 purified from human erythrocytes. β‐mercaptoethanol had no effect on the Aplysia enzyme activities, while it inactivated both the cyclase and the cADPR hydrolase of CD38 by inducing its extensive oligomerization. In intact erythrocytes the β‐mercaptoethanol‐dependent enzyme inactivation was completely prevented by prior cross‐linking of the membrane proteins with glutaraldehyde. These data demonstrate that none of the cysteine residues plays any direct catalytic role in CD38 and Aplysia proteins, and that disulfide bridges are essential for maintaining the monomeric, catalytically active structure of CD38.