Paracrine roles of NAD+ and cyclic ADP-ribose in increasing intracellular calcium and enhancing cell proliferation of 3T3 fibroblasts (original) (raw)
Related papers
Journal of Biological Chemistry, 2001
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 [Ca 2؉ ] i levels as a result of reduced intracellular conversion of NAD ؉ to cADPR. A sharp inverse correlation emerged between [Ca 2؉ ] i levels and NAD ؉ transport (measured as influx into cells and as efflux therefrom), both in the CD38 ؉ cells (high [Ca 2؉ ] i , low transport) and in the CD38 ؊ fibroblasts (low [Ca 2؉ ] 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 Ca 2؉-stimulated protein kinase C (PKC). Thus, a selfregulatory loop emerged in CD38 ؉ fibroblasts whereby high [Ca 2؉ ] i restricts further Ca 2؉ 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 [Ca 2؉ ] i .
Cell Biochemistry and Biophysics, 1998
CD38 is a type-II transmembrane glycoprotein occurring in several hematopoietic and mature blood cells as well as in other cell types, including neurons. Although classified as an orphan receptor, CD38 is also a bifunctional ectoenzyme that catalyzes both the conversion of NAD § to nicotinamide and cyclic ADPribose (cADPR), via an ADP-ribosyl cyclase reaction, and also the hydrolysis of cADPR to ADP-ribose (hydrolase). Major unresolved questions concern the correlation between receptor and catalytic properties of CD38, and also the apparent contradiction between ectocellular generation and intracellular Ca 2+mobilizing activity of cADPR. Results are presented that provide some explanations to this topological paradox in two different cell types. In cultured rat cerebellar granule neurons, extracellular cADPR (either generated by CD38 or directly added) elicited an enhanced intracellular Ca 2+ response to KCl-induced depolarization, a process that can be qualified as a Ca2+-induced Ca 2+ release (CICR) mechanism. On the other hand, in the CD38 § human Namalwa B lymphoid cells, NAD § (and thiol compounds *Author to whom all correspondence and reprint requests should be addressed.
The FASEB Journal
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-N 3 -NAD ؉ , and characterized by poor dinucleotide specificity, low affinity, and high efficiency; 2) intravesicular CD38-catalyzed conversion of NAD ؉ to cADPR, followed by outpumping 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 Ca 2؉ mobilization: role of NAD ؉ transport across cell membranes. FASEB J. 13, 273-283 (1999)
The FASEB Journal, 2002
CD38 is an ectocyclase that converts NAD ؉ to the Ca 2؉-releasing second messenger cyclic ADP-ribose (cADPr). Here we report that in addition to CD38 ecto-catalysis, intracellularly expressed CD38 may catalyze NAD ؉ 3cADPr conversion to cause cytosolic Ca 2؉ release. High levels of CD38 were found in the plasma membranes, endoplasmic reticulum, and nuclear membranes of osteoblastic MC3T3-E1 cells. More important, intracellular CD38 was colocalized with target ryanodine receptors. The cyclase also converted a NAD ؉ surrogate, NGD ؉ , to its fluorescent product, cGDPr (K m ϳ5.13 M). NAD ؉ also triggered a cytosolic Ca 2؉ signal. Similar results were obtained with NIH3T3 cells, which overexpressed a CD38-EGFP fusion protein. The ⌬ ؊49-CD38-EGFP mutant with a deleted amino-terminal tail and transmembrane domain appeared mainly in the mitochondria with an expected loss of its membrane localization, but the NAD ؉-induced cytosolic Ca 2؉ signal was preserved. Likewise, Ca 2؉ release persisted in cells transfected with the Myr-⌬ ؊49-CD38-EGFP or ⌬ ؊49-CD38-EGFP-Fan mutants, both directed to the plasma membrane but in an opposite topology to the full-length CD38-EGFP. Finally, ryanodine inhibited Ca 2؉ signaling, indicating the downstream activation of ryanodine receptors by cADPr. We conclude that intracellularly expressed CD38 might link cellular NAD ؉ production to cytosolic Ca 2؉ signaling.
Journal of Biological Chemistry, 2004
Native human HL-60 cells do not express CD38, a multifunctional ectoenzyme, which generates cyclic ADP-ribose (cADPR), a potent calcium mobilizer. However, when HL-60 cells are induced to differentiate to granulocytes by treatment with retinoic acid (RA), they express CD38 and accumulate cADPR. Both processes play a causal role in RA-induced differentiation. Other granulocyte differentiation-inducers, including dimethyl sulfoxide (Me 2 SO), fail to induce CD38 expression. We investigated whether treatment of HL-60 cells with Me 2 SO involves any changes in the cADPR/intracellular calcium ([Ca 2؉ ] i) signaling system and, specifically, whether Me 2 SO affects those nucleoside transporters (NT) (both equilibrative (ENT) and concentrative (CNT)) that mediate influx of extracellular cADPR. Semiquantitative polymerase chain reaction analysis of transcripts, binding of [ 3 H]nitrobenzylthioinosine (NBMPR) to intact cells, and influx experiments of extracellular cADPR (with selective inhibitors of NT as NBMPR or in specific conditions) were performed in native and Me 2 SO-differentiated HL-60 cells. The native cells showed uptake of cADPR across ENT2, whereas influx of cADPR into the Me 2 SOdifferentiated cells occurred mostly by concentrative processes mediated by CNT3 and by an NBMPR-inhibitable concentrative NT designated cs-csg. Me 2 SO-differentiated, but not native HL-60 cells, accumulated cADPR and showed increased [Ca 2؉ ] i levels when grown in a transwell co-culture setting over CD38-transfected 3T3 fibroblasts where nanomolar cADPR concentrations are present in the medium. NBMPR inhibited both responses of Me 2 SO-induced cells. Thus, concentrative influx of extracellular cADPR across CNT3 and cs-csg NT could substitute in the absence of CD38 in eliciting cADPR-dependent [Ca 2؉ ] i increases in granulocyte-differentiated HL-60 cells, as well as in other CD38 ؊ cells. ADP-ribosyl cyclases play a major role in regulating intracellular calcium levels ([Ca 2ϩ ] i) 1 by converting NAD ϩ to nicotina
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1999
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 t...
Journal of Biological Chemistry, 1997
CD38, a lymphocyte differentiation antigen, is also a bifunctional enzyme catalyzing the synthesis of cyclic ADP-ribose (cADPR) from NAD ؉ and its hydrolysis to ADP-ribose (ADPR). An additional enzymatic activity of CD38 shared by monofunctional ADP-ribosyl cyclase from Aplysia californica is the exchange of the base group of NAD ؉ (nicotinamide) with various nucleophiles. Both human CD38 (either recombinant or purified from erythrocyte membranes) and Aplysia cyclase were found to catalyze the exchange of ADPR with the nicotinamide group of NAD ؉ leading to the formation of a dimeric ADPR ((ADPR) 2). The dimeric structure of the enzymatic product, which was generated by recombinant CD38 and by CD38 ؉ Namalwa cells from as low as 10 M NAD ؉ , was demonstrated using specific enzyme treatments (dinucleotide pyrophosphatase and 5-nucleotidase) and mass spectrometry analyses of the resulting products. The linkage between the two ADPR units of (ADPR) 2 was identified as that between the N 1 of the adenine nucleus of one ADPR unit and the anomeric carbon of the terminal ribose of the second ADPR molecule by enzymatic analyses and by comparison with patterns of cADPR cleavage with Me 2 SO:tert-butoxide. Although (ADPR) 2 itself did not release Ca 2؉ from sea urchin egg microsomal vesicles, it specifically potentiated the Ca 2؉-releasing activity of subthreshold concentrations of cADPR. Therefore, (ADPR) 2 is a new product of CD38 that amplifies the Ca 2؉-mobilizing activity of cADPR.
Journal of Biological Chemistry, 2011
Background: The ADP-ribosyl cyclase CD38 produces cyclic ADP-ribose from NAD ϩ in the extracellular space. Cyclic ADP-ribose induces intracellular Ca 2ϩ mobilization. Results: We demonstrate that connexin 43 hemichannels import cyclic ADP-ribose to the intracellular target ryanodine receptor. Conclusion: Connexin 43 hemichannels mediate the extracellular production and Ca 2ϩ-mobilizing action of cyclic ADP-ribose. Significance: We show that connexin 43 hemichannels resolve the topological hindrance between CD38 and ryanodine receptor. The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD ؉. cADPR increases intracellular Ca 2؉ through the intracellular ryanodine receptor/Ca 2؉ release channel (RyR). It has been known that intracellular NAD ؉ approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. However, it is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR although CD38 itself or nucleoside transporter has been proposed to import cADPR. Moreover, it has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD ؉. Here we demonstrate that Cx43 hemichannels, but not CD38, import cADPR to increase intracellular calcium through RyR. We also demonstrate that physiological stimulation such as Fc␥ receptor (Fc␥R) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD ؉ export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. Protein kinase A (PKA) activation also induced calcium mobilization in the same way as Fc␥R stimulation. Fc␥R stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between Fc␥R stimulation and NAD ؉ /cADPR transport. Cx43 knockdown blocked extracellular cADPR import and extracellular cADPR-induced calcium mobilization in J774 cells. Cx43 overexpression in Cx43-negative cells conferred extracellular cADPR-induced calcium mobilization by the mediation of cADPR import. Our data suggest that Cx43 has a dual function exporting NAD ؉ and importing cADPR into the cell to activate intracellular calcium mobilization. Cyclic ADP-ribose (cADPR) 3 is produced from NAD ϩ by the ADP-ribosyl cyclase CD38, a type II transmembrane glycoprotein first identified as a lymphocyte differentiation antigen. cADPR directly binds to types II and III ryanodine receptors (RyRs) to induce Ca 2ϩ release from RyR containing Ca 2ϩ stores located in the sarcoplasmic and endoplasmic reticulum in a variety of cells (1-3). In a limited number of cell systems, cADPR binds to FKBP12.6 and mediates responsiveness of RyR toward cADPR (4-5). The cADPR-mediated increase in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i) controls various biological processes including egg fertilization (6-7), cell activation, and proliferation (8-9), muscle contraction (10), hormone secretion (11-12), and immune responses (13). The catalytic site of CD38 is located outside of the cell, but the substrate NAD ϩ is inside. Moreover, all cADPR targets are located inside and not in direct contact with extracellular cADPR. Therefore, the CD38/cADPR/Ca 2ϩ signaling system has two topological questions, the way to export NAD ϩ to CD38 and the way to import cADPR to its intracellular targets. These topological problems have been described as the topological paradox (14). Many efforts have been done to solve the topological paradox. It has been reported that approach of intracellular NAD ϩ to ecto-CD38 could be carried out by connexin 43 (Cx43) hemichannels, a component of gap junctions (15), and that approach of cADPR to its intracellular target
The Journal of biological chemistry, 1998
CD38 is a bifunctional ectoenzyme, predominantly expressed on hematopoietic cells during differentiation, that catalyzes the synthesis (cyclase) and the degradation (hydrolase) of cyclic ADP-ribose (cADPR), a powerful calcium mobilizer from intracellular stores. Due to the well established role of calcium levels in the regulation of apoptosis, proliferation, and differentiation, the CD38/cADPR system seems to be a likely candidate involved in the control of these fundamental processes. The ectocellular localization of the cyclase activity, however, contrasts with the intracellular site of action of cADPR. Here we demonstrate that ectocellular expression of human CD38 in CD38 ؊ HeLa and 3T3 cells results in intracellular CD38 substrate (NAD ؉ ؉ NADH) consumption and product (cADPR) accumulation. Furthermore, a causal relationship is established between presence of intracellular cADPR, partial depletion of thapsigargin-sensitive calcium stores, increase in basal free cytoplasmic calcium concentration, and decrease of cell doubling time. The significant shortening of the S phase in CD38 ؉ HeLa cells, as compared with controls, demonstrates an effect of intracellular cADPR on the mammalian cell cycle.