Cyclic ADP-ribose Hydrolase Research Articles

A new antibody for the study of human CD157 expression and function

Abstract CD157 is an ectoenzyme that has both cyclic ADP-ribose hydrolase and ADP-ribosyl cyclase activities. In the human hematopoietic system, CD157 is prevalently expressed by cells of the myelomonocytic lineage, and interacts with extracellular matrix components and regulates leukocyte diapedesis via integrin-mediated signaling. CD157 is implicated in the pathophysiology of several neurological disorders. Although it lacks a cytoplasmatic domain, CD157 is able to transduce intracellular signals by establishing functional and structural crosstalk with β1 (CD29) and β2 (CD18) integrins. CD157 ligation causes integrin– dependent cytoskeletal remodeling and increase in F-actin content. Activation of downstream signaling pathways leads to cell polarization, with CD157 migrating prevalently to the rear of the cells whereas F-actin is localized at the opposite pole. However, the molecular and cellular functions of CD157 still remain poorly understood. Here, we describe the generation of a monoclonal antibody specific for human CD157 (clone W21007F) that can be used in flow cytometry, immunofluorescence and blocking of CD157 mediated adhesion of monocytes to fibronectin. Treatment of the pro-monocytic, myeloid leukemia cell line U937 with clone W21007F induces cell polarization and a marked increase in filamentous actin, with CD157 clustering at the uropod. The majority of F-Actin fibers are located at the opposite end of CD157, and CD157 co-localizes with integrin beta-1 in distinct membrane domains. The reference clone SY/11B5 and isotype control antibody do not show this effect. In conclusion, anti-human CD157 (clone W21007F) is a valuable tool for the study of CD157 function and expression in human cells.

Expanding anti-CD38 immunotherapy for lymphoid malignancies

BackgroundLymphoid neoplasms, including multiple myeloma (MM), non-Hodgkin lymphoma (NHL), and NK/T cell neoplasms, are a major cause of blood cancer morbidity and mortality. CD38 (cyclic ADP ribose hydrolase) is a transmembrane glycoprotein expressed on the surface of plasma cells and MM cells. The high expression of CD38 across MM and other lymphoid malignancies and its restricted expression in normal tissues make CD38 an attractive target for immunotherapy. CD38-targeting antibodies, like daratumumab, have been approved for the treatment of MM and tested against lymphoma and leukemia in multiple clinical trials.MethodsWe generated chimeric antigen receptor (CAR) T cells targeting CD38 and tested its cytotoxicity against multiple CD38high and CD38low lymphoid cancer cells. We evaluated the synergistic effects of all-trans retinoic acid (ATRA) and CAR T cells or daratumumab against cancer cells and xenograft tumors.ResultsCD38-CAR T cells dramatically inhibited the growth of CD38high MM, mantle cell lymphoma (MCL), Waldenstrom’s macroglobulinemia (WM), T-cell acute lymphoblastic leukemia (T-ALL), and NK/T-cell lymphoma (NKTCL) in vitro and in mouse xenografts. ATRA elevated CD38 expression in multiple CD38low cancer cells and enhanced the anti-tumor activity of daratumumab and CD38-CAR T cells in xenograft tumors.ConclusionsThese findings may expand anti-CD38 immunotherapy to a broad spectrum of lymphoid malignancies and call for the incorporation of ATRA into daratumumab or other anti-CD38 immunological agents for cancer therapy.

Maintenance of NAD+ Homeostasis in Skeletal Muscle during Aging and Exercise.

Nicotinamide adenine dinucleotide (NAD) is a versatile chemical compound serving as a coenzyme in metabolic pathways and as a substrate to support the enzymatic functions of sirtuins (SIRTs), poly (ADP-ribose) polymerase-1 (PARP-1), and cyclic ADP ribose hydrolase (CD38). Under normal physiological conditions, NAD+ consumption is matched by its synthesis primarily via the salvage pathway catalyzed by nicotinamide phosphoribosyltransferase (NAMPT). However, aging and muscular contraction enhance NAD+ utilization, whereas NAD+ replenishment is limited by cellular sources of NAD+ precursors and/or enzyme expression. This paper will briefly review NAD+ metabolic functions, its roles in regulating cell signaling, mechanisms of its degradation and biosynthesis, and major challenges to maintaining its cellular level in skeletal muscle. The effects of aging, physical exercise, and dietary supplementation on NAD+ homeostasis will be highlighted based on recent literature.

Perspective: Re-defining "Pheromone" in a Mammalian Context to Encompass Seminal Fluid.

The classical view of “pheromone”—an air-borne chemical signal—is challenged by the camelids in which ovulation is triggered by ß-nerve growth factor carried in seminal plasma, effectively extending the pheromone concept to a new medium. We propose further extension of “pheromone” to include a separate class of seminal fluid molecules that acts on the female reproductive tract to enhance the prospect of pregnancy. These molecules include transforming growth factor-ß, 19-OH prostaglandins, various ligands of Toll-like receptor-4 (TLR4), and cyclic ADP ribose hydrolase (CD38). They modulate the immune response to “foreign” male-derived histocompatibility antigens on both sperm and the conceptus, determine pre-implantation embryo development, and then promote implantation by increasing uterine receptivity to the embryo. The relative abundance of these immunological molecules in seminal plasma determines the strength and quality of the immune tolerance that is generated in the female. This phenomenon has profound implications in reproductive biology because it provides a pathway, independent of the fertilizing sperm, by which paternal factors can influence the likelihood of reproductive success, as well as the phenotype and health status of offspring. Moreover, the female actively participates in this exchange—information in seminal fluid is subject to “cryptic female choice,” a process by which females interrogate the reproductive fitness of prospective mates and invest reproductive resources accordingly. These processes participate in driving the evolution of male accessory glands, ensuring optimal female reproductive investment and maximal progeny fitness. An expanded pheromone concept will avoid a constraint in our understanding of mammalian reproductive biology.

Lack of bone marrow stromal cell antigen-1 (BST-1)/CD157 ameliorated colitis induced by dextran sodium sulfate (DSS)

Abstract Bone marrow stromal cell antigen-1(BST-1)/CD157 is GPI-anchored ectoenzyme having adenosine diphosphate(ADP)-ribosyl cyclase and cyclic ADP-ribose hydrolase activities. BST-1 also functions as a receptor for signal transduction. Recently, various groups reported novel functions of BST-1, such as regulation of intestinal or mesenchymal stem cells. Re-analyses of BST-1 deficient mice (Bst1KO) in C57BL/6 background revealed roles in defense against tuberculosis, and prevention of depressive or anxious behaviors. Thus, BST-1 is now appreciated as an entero-neuro-immune regulator. Since expression of BST-1 in the intestines is common in human and mouse, we examined the roles for BST-1 in DSS-induced colitis using Bst1KO. Drinking water containing 3% DSS was supplied ad libitum to Bst1KO and C57BL/6 (WT) in SPF condition for 5 days, and then water without DSS for 2 days. Decreases of body weights in Bst1KO were similar to WT until day 5, but since then they became significantly slower than WT. Clinical scores based on bleeding and diarrhea were similar in both genotypes. Histological analyses with HE staining revealed the ratio of destroyed epitheliums in the total colon of Bst1KO was 15% whereas that of WT was 50%. Flow cytometrical analyses revealed no difference in the cell numbers of neutrophils and macrophages increased in the colon at day 7. Quantitative real-time PCR revealed that increases of Bst1 expression in the colons of WT started on day 1 and reached to the level more than thousand times at day 5. In the colons of Bst1KO, increases of Il6 expression at day 5 were markedly decreased compared with those of WT. These data indicate that BST-1 has pro-inflammatory roles in DSS-induced colitis through positive regulation of IL-6 production.

Chinese‐German Cooperation Group Tumor Immunology: Another inspiring Meeting in Deidesheim

Chinese‐German Cooperation Group Tumor Immunology: Another inspiring Meeting in Deidesheim

Blockade of CD38 diminishes lipopolysaccharide-induced macrophage classical activation and acute kidney injury involving NF-κB signaling suppression

The CD38, possessing ADP-ribosyl cyclase (ADPR-cyclase) and cyclic ADP-ribose hydrolase (cADPR-hydrolase), is able to regulate a variety of cellular activities. However, the role and mechanisms for CD38 in macrophage activation and sepsis-induced acute kidney injury (AKI) remain to be determined. Here we report that in cultured macrophages, Lipopolysaccharide (LPS) could upregulate CD38 expression in time and dose dependent manner. Knocking down or blockade of CD38 in macrophages could inhibit LPS-induced macrophage M1 polarization accompanied by diminished NF-κB signaling activation. In mouse model with LPS-induced acute kidney injury, blocking CD38 with quercetin could significantly relieve kidney dysfunction, kidney pathological changes as well as inflammatory cell accumulation. Similar to those in the cultured cells, quercetin could inhibit macrophage M1 polarization and NF-κB signaling activation in macrophages from kidneys and spleens in mice after LPS injection. Together, these results demonstrate that CD38 mediates LPS-induced macrophage activation and AKI, which may be treated as a therapeutic target for sepsis-induced AKI in patients.

CD38 Expression in B-Lineage Acute Lymphoblastic Leukemia, a Possible Target for Immunotherapy

Introduction:CD38 is a type II transmembrane glycoprotein expressed on immature T and B lymphocytes (thymocytes and hematogones), NK cells, activated T-cells, plasma-cells, monocytes and red blood cells. This ectoenzyme is a ribonucleosyl cyclase (Cyclic ADP ribose hydrolase) involved in the regulation of calcium fluxes.CD38 is absent from quiescent lymphocytes. The lowest levels are present on erythrocytes while CD38 is brightly expressed by hematogones and plasma-cells. The latter has led to the development of daratumumab, a therapeutic monoclonal antibody used in the treatment of multiple myeloma.Because of the wide distribution of this antigen, other diseases could be considered for such a therapy. Among them, acute lymphoblastic leukemia (ALL) could represent an interesting target.We thus investigated the level of expression of CD38 in a cohort of 128 samples from B-lineage ALL (113 at diagnosis and 15 at relapse). We also compared it to that of the normal counterpart of these blasts, hematogones, because of the potential use of CD38 as a leukemia associated immunophenotype (LAIP). Moreover, in 15 paired samples of diagnosis/relapse, we examined the stability of this expression during disease evolution.Patients, Material and Methods:A total of 62 females and 66 males were included in the cohort, with a median age of 20 years old (range 4 months to 90 years). There were 57 children (below 15 yo), 13 adolescents and young adults (15-25 yo) and 58 adults. CD38 expression was investigated in 45 bone marrow (BM) samples and in 83 peripheral blood (PB) samples. The median level of blasts was 62% in BM and 48% in PB. According to EGIL classification, there were 19 B-I, 66 B-II, 38 B-III, 2 B-IV and intracytoplasmic mu chain was not investigated in 3 cases.In parallel, 26 samples of BM with morphologically evidence hematogones were used to compare the level of expression of CD38 on these cells. Immunophenotyping panels comprised CD38 antibodies conjugated to allophycocyanin and all samples were analyzed on a Canto II flow cytometer (BD biosciences, San Jose, CA).Results:CD38 was always present on hematogones. It was expressed by 122 of the 128 B-ALL samples tested (95,3%). Partial expression, between 20 and 70% of the blasts was noted for 9 patients (7%) while 113 patients (88%) had more than 70% positive blasts. For 102 patients (79,7%), the whole population was stained (100% of the blasts). Among the 15 samples obtained at relapse, CD38 was always expressed, partially only in one case.The mean fluorescence intensity (MFI), expressed using the flow cytometer arbitrary units (AI) ranged between 510 and 1396 AI (median 878 AI) in the group of patients with 20-70% CD38+ blasts (n=9) and between 317 and 26466 AI (median 4704 AI) for the 113 patients with more than 70% CD38+ blasts (n=113).The level of fluorescence was always higher on hematogones compared to blasts, with a median MFI of 14915 AI (range 3834-34501 AI). CD38 expression is also used as a LAIP, to discriminate minimal residual disease (MRD) from regenerating hematogones. In our department, a threshold of 6300 AI (median of hematogones - 2 SD) is used to define the LAIP of CD38low blasts. In the cohort reported here, 82 patients presented this LAIP (64%). Conversely, high expression of CD38 with an MFI >10 000 AI was present for 23 of the 128 patients.For the 15 patients with paired samples of diagnosis and relapse, 9 had low levels of CD38 (LAIP). In none of these cases modulation of CD38 was observed, this LAIP remaining stable over time.Conclusion:CD38 has been reported by several authors as one of the best LAIP marker for the detection of MRD in B-ALL, allowing a good follow-up of the patients because of its stability. Moreover, this study confirms that CD38 could be a valuable therapeutic target in most of B-ALL cases, being expressed in over 95% of the cases. Used together with chemotherapy, daratumumab could thus be useful to treat B-ALL both in first line or at relapse. DisclosuresMoreau:Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Janssen: Honoraria, Speakers Bureau.

All-Trans Retinoic Acid and Interferon-Alpha Increase CD38 Expression on Adult T-Cell Leukemia Cells and Sensitize Them to T Cells Bearing Anti-CD38 Chimeric Antigen Receptors

The survival of patients with adult T-cell leukemia (ATL) has been improved by the introduction of anti-CCR4 monoclonal antibody and the expanding use of allogeneic hematopoietic stem cell transplantation. However, not all patients benefit from these treatment modalities, warranting development of a novel therapeutic strategy. CD38, a cell surface ectoenzyme that functions as cyclic ADP ribose hydrolase, is an attractive target of chimeric antigen receptor (CAR) therapy for lymphoid neoplasms because it is widely expressed on the cells of B- or T-lymphoid malignancies. We have previously demonstrated the prominent cytotoxicity of T cells engineered to express an anti-CD38-CAR against B-lymphoma cells and myeloma cells expressing CD38. To expand the applicability of anti-CD38-CAR against ATL cells that usually express undetectable or low levels of CD38, notably, we were successfully able to induce cell surface CD38 expression in HTLV-1-infected cell lines with all-trans retinoic acid (ATRA) (Yoshida T, et al. 2013 ASH Meeting). In ATL cells freshly isolated obtained from the patients, we were able to induce CD38 with ATRA in 60-80% of the cells; the remaining cells survived under the anti-CD38-CAR treatment. We hereby report our attempts in improving the efficacy of anti-CD38-CAR T cells against ATL cells from the patients through the expression of CD38 enhanced with the entry of agents, which are clinically used. Firstly, we investigated whether ATL cells from patients could be transduced with anti-CD38-CAR and what is the efficiency of transduction into T cells in our settings. ATL cells (CD4+ CD25+ GFP+) transduced with retroviral vector were little detected. CD4- CD25- GFP+ T cells alone were detected in our transduction methods. Transduction efficiency was over 40%. To increase the expression of CD38 on ATL cells, we took notice of the CD38 gene upstream region that contains binding sites for interferon regulatory factor-1 (IRF-1) and peroxisome proliferator-activated receptor (PPAR). We thus investigated whether IFN-α, IFN-γ or troglitazone, which is a PPAR-α and -γ agonist, could enhance CD38 expression in ATL cell lines (MT-4, Su9T, ED, and S1T cells), which are negative for CD38. IFN-α and IFN-γ efficiently enhanced CD38 expression in MT-4 cells in a dose-dependent manner but not in Su9T, ED, and S1T cells. As little as 2.5U/ml of IFN-α induced CD38 expression in MT-4 cells for 18 hours in vitr o (>95% at positivity of CD38). 10-25% increase in CD38 expression was observed in ED cells with 125-250 pM troglitazone after 18 hours of treatment, but not in MT-4, Su9T, and S1T cells. Prolonged exposure to troglitazone was toxic to cells. Combined treatment with 10nM ATRA and IFN-α, which induced higher expression of CD38 than IFN-γ, synergistically enhanced CD38 expression of ATL cells from the patients (>90%at positivity of CD38). We next co-cultured ATL cells form three patients with T cells transduced with mock or anti-CD38-CAR in the presence of both ATRA and IFN-α at effector (E): target (T) ratio of 1: 2 for 3 days. The treatment eradicated more than 95% of these ATL cells, demonstrating that ATL cells can be eliminated by T cells harboring anti-CD38-CAR in the presence of ATRA and IFN-α, which is actively used for ATL patients. CD38 targeting therapy is a feasible method, because an anti-CD38 antibody, daratuzumab, has been used to treat plasma cell myeloma. The safety regarding the clinical use of T cells bearing anti-CD38-CAR still needs to be established. As CAR therapy reportedly causes cytokine storm and can potentially be lethal, we envision an inducible immunotherapy with CAR to be a preferred modality with increased efficacy and safety. Our results provide a rationale for a novel therapeutic strategy involving T cells carrying anti-CD38-CAR in combination with ATRA and IFN-α for patients with ATL. DisclosuresNo relevant conflicts of interest to declare.

Polymorphisms in Genes of Relevance for Oestrogen and Oxytocin Pathways and Risk of Barrett's Oesophagus and Oesophageal Adenocarcinoma: A Pooled Analysis from the BEACON Consortium.

BackgroundThe strong male predominance in oesophageal adenocarcinoma (OAC) and Barrett’s oesophagus (BO) continues to puzzle. Hormonal influence, e.g. oestrogen or oxytocin, might contribute.MethodsThis genetic-epidemiological study pooled 14 studies from three continents, Australia, Europe, and North America. Polymorphisms in 3 key genes coding for the oestrogen pathway (receptor alpha (ESR1), receptor beta (ESR2), and aromatase (CYP19A1)), and 3 key genes of the oxytocin pathway (the oxytocin receptor (OXTR), oxytocin protein (OXT), and cyclic ADP ribose hydrolase glycoprotein (CD38)), were analysed using a gene-based approach, versatile gene-based test association study (VEGAS).ResultsAmong 1508 OAC patients, 2383 BO patients, and 2170 controls, genetic variants within ESR1 were associated with BO in males (p = 0.0058) and an increased risk of OAC and BO combined in males (p = 0.0023). Genetic variants within OXTR were associated with an increased risk of BO in both sexes combined (p = 0.0035) and in males (p = 0.0012). We followed up these suggestive findings in a further smaller data set, but found no replication. There were no significant associations between the other 4 genes studied and risk of OAC, BO, separately on in combination, in males and females combined or in males only.ConclusionGenetic variants in the oestrogen receptor alpha and the oxytocin receptor may be associated with an increased risk of BO or OAC, but replication in other large samples are needed.

Concurrent loss of co-stimulatory molecules and functional cytokine secretion attributes leads to proliferative senescence of CD8+ T cells in HIV/TB co-infection

The role of T-cell immunosenescence and functional CD8+ T-cell responses in HIV/TB co-infection is unclear. We examined and correlated surrogate markers of HIV disease progression with immune activation, immunosenescence and differentiation using T-cell pools of HIV/TB co-infected, HIV-infected and healthy controls. Our investigations showed increased plasma viremia and reduced CD4/CD8 T-cell ratio in HIV/TB co-infected subjects relative to HIV-infected, and also a closer association with changes in the expression of CD38, a cyclic ADP ribose hydrolase and CD57, which were consistently expressed on late-senescent CD8+ T cells. Up-regulation of CD57 and CD38 were directly proportional to lack of co-stimulatory markers on CD8+ T cells, besides diminished expression of CD127 (IL-7Rα) on CD57+CD4+ T cells. Notably, intracellular IFN-γ, perforin and granzyme B levels in HIV-specific CD8+ T cells of HIV/TB co-infected subjects were diminished. Intracellular CD57 levels in HIV gag p24-specific CD8+ T cells were significantly increased in HIV/TB co-infection. We suggest that HIV-TB co-infection contributes to senescence associated with chronic immune activation, which could be due to functional insufficiency of CD8+ T cells.

The PI3K p110δ Regulates Expression of CD38 on Regulatory T Cells

The PI3K pathway has emerged as a key regulator of regulatory T cell (Treg) development and homeostasis and is required for full Treg-mediated suppression. To identify new genes involved in PI3K-dependent suppression, we compared the transcriptome of WT and p110δD910A Tregs. Among the genes that were differentially expressed was the gene for the transmembrane cyclic ADP ribose hydrolase CD38. Here we show that CD38 is expressed mainly by a subset of Foxp3+CD25+CD4+ T cells originating in the thymus and on Tregs in the spleen. CD38high WT Tregs showed superior suppressive activity to CD38low Tregs, which failed to upregulate CD73, a surface protein which is important for suppression. However, Tregs from heterozygous CD38+/− mice were unimpaired despite lower levels of CD38 expression. Therefore, CD38 can be used as a marker for Tregs with high suppressive activity and the impaired Treg function in p110δD910A mice can in part be explained by the failure of CD38high cells to develop.

Special features of quantification of CD8<sup>+</sup>CD38<sup>+ </sup>T-cells by flow cytometry does not represent a good biomarker to monitor the reactivation of cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation

CD38 also known as cyclic ADP ribose hydrolase is a surface glycoprotein expressed by several leukocytes, in particular CD4+ and CD8+ T-cells, B-lymphocytes and natural killer cells.(1) CD38 is part of a large family of nucleotide-metabolizing ectoenzymes that also includes CD26, CD39 and CD73. These molecules are clustered in specialized areas of the cell membrane, playing roles as receptor signaling molecules thus regulating cell activation, defense from pathogens, cell migration, immune synapse formation and also life cycle.(2) CD38 is a powerful disease biomarker for several human disorders, including leukemias and myelomas, and also directly involved in the pathogenesis and prognosis of human virus infections. Several potential clinical applications of CD38 as a putative laboratorial biomarker in the diagnosis, prognosis and therapy of viral infections have been previously reported.(2) Elevated CD38 antigen expression on CD8+ T cells has been pointed out as a stronger marker for the risk of chronic HIV disease progression to AIDS.(3) Moreover, recent studies have demonstrated that a parallel decline of CD8+CD38+ lymphocytes and viremia may occur in treated hepatitis B patients, suggesting its role as a biomarker for active infection.(4) It has been proposed that monitoring of the CD8+CD38+ T-cell subset in peripheral blood represents a powerful approach to detect cytomegalovirus (CMV) infections in kidney transplant recipients.(5) More recently, it has been reported that in fact, the monitoring of CD38 high expression in peripheral blood CD8+ lymphocytes is a useful additional diagnostic marker for CMV infection in patients after kidney transplantation, especially when they are at risk of tissue-invasive disease when CMV DNA copies may not be detectable in peripheral blood.(6) The questions surrounding CD38 are still fascinating and continue to prompt novel investigations. One important matter to be clarified is whether the overexpression of the CD38 molecule by circulating T cells may represent a universal biomarker for CMV infection after all modalities of organ transplantation. In order to address this issue, Lino et al.(7) performed a prospective investigation to clarify the applicability of flow cytometric enumeration of CD8+CD38+ T-cells in peripheral blood as a laboratorial indicator of CVM infection/reactivation in allogeneic hematopoietic stem cell (HSCT) recipients. The data presented in this study revealed that, in contrast to those previously published in kidney transplantation, the overexpression of CD38 in circulating CD8+ T cells does not correlate with the presence on CMV antigenemia. The authors present a plausible explanation for this phenomenon, discussing the particularities of CD38 expression during bone marrow repopulation and immune system reconstitution after HSCT. The cyclic nature of CD38 expression during hematopoietic ontogeny may explain the presence of CD38 overexpression even in those HSCT recipients with undetectable antigenemia. It has been reported that CD38 is expressed early in hematopoietic cells, with downregulation during the cell maturation process and re-expression upon cell activation. This knowledge is relevant to aware flow cytometry readers and medical professionals working in the clinical management of transplant recipients that overexpression of CD38 may not be considered a universal biomarker of CMV infection following organ transplantation. The article is well organized, presenting the technical details necessary to ensure its reproducibility, excellent scientific quality and promises a fruitful reading.

Activation of CD38 by Interleukin-8 Signaling Regulates Intracellular Ca2+ Level and Motility of Lymphokine-activated Killer Cells

CD38 is an ADP-ribosyl cyclase, producing a potent Ca(2+) mobilizer cyclic ADP-ribose (cADPR). In this study, we have investigated a role of CD38 and its regulation through interleukin-8 (IL8) signaling in lymphokine-activated killer (LAK) cells. Incubation of LAK cells with IL8 resulted in an increase of cellular cADPR level and a rapid rise of intracellular Ca(2+) concentration ([Ca(2+)](i)), which was sustained for a long period of time (>10 min). Preincubation of an antagonistic cADPR analog, 8-Br-cADPR (8-bromo-cyclic adenosine diphosphate ribose), abolished the sustained Ca(2+) signal only but not the initial Ca(2+) rise. An inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist blocked both Ca(2+) signals. Interestingly, the sustained Ca(2+) rise was not observed in the absence of extracellular Ca(2+). Functional CD38-null (CD38(-)) LAK cells showed the initial rapid increase of [Ca(2+)](i) but not the sustained Ca(2+) rise in response to IL8 treatment. An increase of cellular cADPR level by cGMP analog, 8-pCPT-cGMP (8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate), but not cAMP analog or phorbol 12-myristate 13-acetate was observed. IL8 treatment resulted in the increase of cGMP level that was inhibited by the IP(3) receptor blocker but not a protein kinase C inhibitor. cGMP-mediated Ca(2+) rise was blocked by 8-Br-cADPR. In addition, IL8-mediated LAK cell migration was inhibited by 8-Br-cADPR and a protein kinase G inhibitor. Consistent with these observations, IL8-induced migration of CD38(-) LAK cells was not observed. However, direct application of cADPR or 8-pCPT-cGMP stimulated migration of CD38(-) cells. These results demonstrate that CD38 is stimulated by sequential activation of IL8 receptor, IP(3)-mediated Ca(2+) rise, and cGMP/protein kinase G and that CD38 plays an essential role in IL8-induced migration of LAK cells.

Estrogen Increases CD38 Gene Expression and Leads to Differential Regulation of Adenosine Diphosphate (ADP)-Ribosyl Cyclase and Cyclic ADP-Ribose Hydrolase Activities in Rat Myometrium1

Hormones influence uterine contractility through their effects on intracellular calcium. The regulation of intracellular calcium in uterine smooth muscle is achieved by several mechanisms and includes mobilization from intracellular stores by inositol 1,4,5-trisphosphate and ryanodine-sensitive channels. Cyclic ADP-ribose (cADPR), a metabolite of NAD(+), is known to mediate calcium release through ryanodine receptor channels. A cell surface glycoprotein, CD38, catalyzes the synthesis and breakdown of cADPR and thus possesses bifunctional enzymatic activity. The regulation of cADPR synthesis by ADP-ribosyl cyclase (cyclase) or degradation by cADP-ribose hydrolase (hydrolase) by hormones in the myometrium is poorly understood. We investigated the effects of estradiol-17 beta on CD38 expression and the synthesis and degradation of cADPR in myometrial smooth muscle obtained from ovariectomized rats. CD38 expression was studied by reverse transcription polymerase chain reaction and Western blot analyses. In uterine microsomal fractions, cyclase and hydrolase activities were measured using nicotinamide guanine dinucleotide and [(32)P]cADPR as substrates, respectively. Microsomal proteins subfractionated by SDS-PAGE and gel filtration were used to determine the fractions containing cyclase and hydrolase activities. The results demonstrate that cyclase and hydrolase activities are associated with a single protein fraction, similar to CD38 in uteri from both ovariectomized and estradiol-treated rats, and estradiol-17 beta causes 1) increased CD38 mRNA and protein expression and 2) significantly enhanced cyclase but not hydrolase activity. The differential regulation of CD38 by estradiol-17 beta, resulting in increased cADPR synthesis, would have profound effects on calcium regulation and myometrial contractility.

Antidiabetic Effect of a Prodrug of Cysteine,l-2-Oxothiazolidine-4-carboxylic Acid, through CD38 Dimerization and Internalization

CD38 is a bifunctional enzyme synthesizing (ADP-ribosyl cyclase) and degrading (cyclic ADP-ribose (cADPR) hydrolase) cADPR, a potent Ca(2+) mobilizer from intracellular pools. CD38 internalization has been proposed as a mechanism by which the ectoenzyme produced intracellular cADPR, and thiol compounds have been shown to induce the internalization of CD38. Here, we show that the disulfide bond between Cys-119 and Cys-201 in CD38 may be involved in CD38 dimerization and internalization. We tested the effect of a reducing agent, l-2-oxothiazolidine-4-carboxylic acid (OTC), a prodrug of cysteine, on CD38 internalization in pancreatic islets. OTC enhanced insulin release from isolated islets as well as CD38 internalization and cytoplasmic Ca(2+) level. Furthermore, islet cells treated with antisense CD38 oligonucleotide showed inhibition of OTC-induced insulin secretion. Intake of OTC in db/db mice ameliorated glucose tolerance, insulin secretion, and morphology of islets when compared with control mice. These data indicate that OTC improves glucose tolerance by enhancing insulin secretion via CD38/cADPR/Ca(2+) signaling machinery. Thus, OTC may represent a novel class of antidiabetic drug.

A Redox Role for Cyclic ADP-Ribose

Hypoxic pulmonary vasoconstriction is a normal physiological response that coordinates pulmonary perfusion to respiratory ventilation. NADH (reduced nicotinamide adenine dinucleotide) is thought to have a role in pulmonary artery contraction. Oxidized NAD (NAD+) is a substrate used by ADP-ribosyl cyclase to make cyclic ADP-ribose (cADPR), which regulates ryanodine receptor-mediated Ca2+ release. Wilson et al. investigated whether the ratio of NAD+:NADH in pulmonary arteries functioned as an oxido-reductive sensing rheostat to control pulmonary vasoconstriction. cADPR production in pulmonary artery smooth muscle homogenates was increased when NADH was added, but concentrations of NAD+ were constant. This result suggests that the enzymatic breakdown of cADPR by cADPR hydrolase is inhibited by the presence of NADH. Pulmonary artery sections incubated in hypoxic conditions produced greatly increased amounts of cADPR, whereas, in other experiments, vasoconstriction was inhibited by pretreatment with a cADPR antagonist, indicating that cADPR, produced in response to lower oxygen tension, can mediate pulmonary artery smooth muscle contraction. The authors suggest that cADPR, cADP hydrolase, and ADP-ribosylase function together to link the redox state of pulmonary smooth cells to Ca2+ release from the ryanodine receptor-regulated sarcoplasmic reticulum and consequent vasoconstriction.H. L. Wilson, M. Dipp, J. M. Thomas, C. Lad, A. Galione, A. M. Evans, ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase act as a redox sensor: A primary role for cyclic ADP-ribose in hypoxic pulmonary vasoconstriction. J. Biol. Chem. 276, 11180-11188 (2001). [Abstract] [Full Text]

A Redox Role for Cyclic ADP-Ribose

Hypoxic pulmonary vasoconstriction is a normal physiological response that coordinates pulmonary perfusion to respiratory ventilation. NADH (reduced nicotinamide adenine dinucleotide) is thought to have a role in pulmonary artery contraction. Oxidized NAD (NAD + ) is a substrate used by ADP-ribosyl cyclase to make cyclic ADP-ribose (cADPR), which regulates ryanodine receptor-mediated Ca 2+ release. Wilson et al. investigated whether the ratio of NAD + :NADH in pulmonary arteries functioned as an oxido-reductive sensing rheostat to control pulmonary vasoconstriction. cADPR production in pulmonary artery smooth muscle homogenates was increased when NADH was added, but concentrations of NAD + were constant. This result suggests that the enzymatic breakdown of cADPR by cADPR hydrolase is inhibited by the presence of NADH. Pulmonary artery sections incubated in hypoxic conditions produced greatly increased amounts of cADPR, whereas, in other experiments, vasoconstriction was inhibited by pretreatment with a cADPR antagonist, indicating that cADPR, produced in response to lower oxygen tension, can mediate pulmonary artery smooth muscle contraction. The authors suggest that cADPR, cADP hydrolase, and ADP-ribosylase function together to link the redox state of pulmonary smooth cells to Ca 2+ release from the ryanodine receptor-regulated sarcoplasmic reticulum and consequent vasoconstriction. H. L. Wilson, M. Dipp, J. M. Thomas, C. Lad, A. Galione, A. M. Evans, ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase act as a redox sensor: A primary role for cyclic ADP-ribose in hypoxic pulmonary vasoconstriction. J. Biol. Chem. 276 , 11180-11188 (2001). [Abstract] [Full Text]

Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle

Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of β-NAD + to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5′-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS–PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K m values of 30.4±1.5 and 695.3±171.2 μM and V max values of 330.4±90 and 102.8±17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca 2+] i in porcine TSM cells.

Crystallization and preliminary X-ray diffraction analysis of the extracellular domain of the cell surface antigen CD38 complexed with ganglioside.

The cell surface antigen CD38 is a multifunctional ectoenzyme that acts as an NAD(+) glycohydrolase, an ADP-ribosyl cyclase, and also a cyclic ADP-ribose hydrolase. The extracellular catalytic domain of CD38 was expressed as a fusion protein with maltose-binding protein, and was crystallized in the complex with a ganglioside, G(T1b), one of the possible physiological inhibitors of this ectoenzyme. Two different crystal forms were obtained using the hanging-drop vapor diffusion method with PEG 10,000 as the precipitant. One form diffracted up to 2.4 A resolution with synchrotron radiation at 100 K, but suffered serious X-ray damage. It belongs to the space group P2(1)2(1)2(1) with unit-cell parameters of a = 47.9, b = 94.9, c = 125.2 A. The other form is a thin plate, but the data sets were successfully collected up to 2.4 A resolution by use of synchrotron radiation at 100 K. The crystals belong to the space group P2(1) with unit-cell parameters of a = 57.4, b = 51.2, c = 101.1 A, and beta = 97.9 degrees, and contain one molecule per asymmetric unit with a VM value of 2.05 A(3)/Da.