Chini, Eduardo N.

In the present study, we have determined the role of the enzyme CD38 upon the synthesis of the Ca2+-releasing nucleotide nicotinic acid—adenine dinucleotide phosphate (NAADP). In rat tissues, we observed that the capacity for NAADP synthesis could be co-immunoprecipitated with CD38 using an anti-CD38 antibody. Furthermore, we observed that several tissues from CD38 knockout mice had no capacity for the synthesis of this nucleotide. In addition, CD38 was also identified as the major enzyme responsible for the synthesis of the second messenger cyclic ADP-ribose. These observations lead to the conclusion that CD38 is the major enzyme responsible for the synthesis of NAADP and cyclic ADP-ribose, and raises the possibility of a new signalling pathway where two different Ca2+-releasing nucleotides are synthesized by the same enzyme.

Nicotinic acid–adenine dinucleotide phosphate (NAADP), a molecule derived from β-NADP, has been shown to promote intracellular calcium release in sea urchin eggs. However, there is little information regarding the role of NAADP in the regulation of intracellular calcium fluxes in mammalian cells. We found recently that several mammalian tissues have a high capacity for NAADP synthesis, as assessed by sea urchin egg bioassay. To determine the functional significance of NAADP production by mammalian tissues, we sought to determine whether NAADP is capable of inducing calcium release from microsomes prepared from cultured cells. We found that NAADP, but not β-NADP, activates a specific microsomal calcium release system in mesangial cells isolated from rat kidney; NAADP was without effect in renal tubular epithelial cells. NAADP-induced calcium release is not affected by inhibitors of the inositol 1,4,5-trisphosphate or ryanodine channels. However, NAADP-elicited calcium release was inhibited by L-type calcium channel blockers and by alkaline phosphatase treatment of NAADP. NAADP also promotes specific microsomal calcium release in rat vascular smooth muscle cells, cardiac myocytes, fibroblasts and a human leukaemia cell line, indicating that the capacity for NAADP-induced calcium release is widespread in mammalian cells. We propose that NAADP may be an important regulator of intracellular calcium in many mammalian tissues.

We investigated the pH dependence and the effects of thimerosal and dithiothreitol (DTT) upon the Ca2+ release induced by cADP-ribose (cADPR) and nicotinate–adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenates. Both Ca2+ release triggered by cADPR and the binding of [3H]cADPR to sea urchin egg homogenates were decreased by alkalization of the assay media from pH 7.2 to 8.9. In contrast, NAADP-triggered Ca2+ release was not influenced by changes in pH. The Ca2+ release induced by cADPR was potentiated by thimerosal and inhibited by DTT, but neither thimerosal nor DTT had any effect upon the Ca2+ release induced by NAADP. We conclude that cADPR-sensitive Ca2+-release mechanisms are dependent on pH of the assay media and are sensitive to thiol group modification. On the other hand, these functional properties are not shared by NAADP-regulated Ca2+ channels.

We investigated the dependence of nicotinate–adenine dinucleotide phosphate (NAADP)-induced Ca2+ release from intracellular stores of sea urchin egg homogenates, upon extravesicular Ca2+. In contrast to the Ca2+ release induced by inositol 1´,4´,5´trisphosphate (IP3) or cyclic ADP-ribose (cADPR), the Ca2+ release induced by NAADP was completely independent of the free extravesicular Ca2+ over a wide range of concentrations (0–0.1 mM). The Ca2+ release triggered by either cADPR or IP3 was biphasically modulated by extravesicular Ca2+, and the Ca2+ release by these agents was abolished when the extravesicular Ca2+ was removed by chelation with 2 mM EGTA. On the other hand, NAADP-triggered Ca2+ release was not influenced by EGTA. These data indicate that while both cADPR and IP3 systems behave as functional Ca2+-induced Ca2+ release mechanisms, NAADP activates a Ca2+ release mechanism which is independent of the presence of extravesicular Ca2+. Therefore, the NAADP-sensitive Ca2+ release mechanisms may have a unique regulatory impact upon intracellular Ca2+ homoeostasis.

Nicotinate adenine dinucleotide phosphate (NAADP) was recently identified [Lee and Aarhus (1995) J. Biol. Chem. 270, 2152-2157; Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] as a potent Ca(2+)-releasing agent in sea urchin egg homogenates. NAADP triggered Ca2+ release by a mechanism that was distinct from inositol 1,4,5-trisphosphate (InsP3)- and cyclic ADP-ribose (cADPR)-induced Ca2+ release. When NAADP was microinjected into intact sea urchin eggs it induced a dose-dependent increase in cytoplasmic free Ca2+ which was independent of the extracellular [Ca2+]. The Ca2+ waves elicited by microinjections of NAADP originated at the site of injection and swept across the cytosol. As previously found in sea urchin egg homogenates, NAADP-induced Ca2+ release in intact eggs was not blocked by heparin or by prior desensitization to InsP3 or cADPR. Thio-NADP, a specific inhibitor of the NAADP-induced Ca2+ release in sea urchin homogenates [Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] blocked NAADP (but not InsP3 or cADPR) injection-induced Ca2+ release in intact sea urchin eggs. Finally, fertilization of sea urchin eggs abrogated subsequent NAADP-induced Ca2+ release, suggesting that the NAADP-sensitive Ca2+ pool may participate in the fertilization response. This study demonstrates that NAADP acts as a selective Ca(2+)-releasing agonist in intact cells.

We have previously shown that amino acid changes in the human Kruppel-Like Factor (KLF) 11 protein is associated with the development of maturity onset diabetes of the young VII, whereas complete inactivation of this pathway by the −331 human insulin mutation causes neonatal diabetes mellitus. Here, we report that Klf11−/− mice have decreased circulating insulin levels, alterations in the control of blood glucose and body weight, as well as serum dyslipidemia, but do not develop diabetes. Functional assays using ex vivo liver tissue sections demonstrate that Klf11−/− mice display increased insulin sensitivity. Genome-wide experiments validated by pathway-specific quantitative PCR arrays reveal that the Klf11−/− phenotype associates to alterations in the regulation of gene networks involved in lipid metabolism, in particular those regulated by peroxisome proliferator-activated receptor-γ. Combined, these results demonstrate that the major phenotype given by the whole-body deletion of Klf11 in mouse is not diabetes but increased insulin sensitivity, likely due to altered transcriptional regulation in target tissues. The absence of diabetes in the Klf11−/− mouse either indicates an interspecies difference for the role of this transcription factor in metabolic homeostasis between mouse and humans, or potentially highlights the fact that other molecular factors can compensate for its absence. Nevertheless, the data of this study, gathered at the whole-organism level, further support a role for KLF11 in metabolic processes like insulin sensitivity, which regulation is critical in several forms of diabetes.

Cellular senescence is a fundamental aging mechanism that has been implicated in many age-related diseases and is a significant cause of tissue dysfunction. Accumulation of senescent cells occurs during aging and is also seen in the context of obesity and diabetes. Senescent cells may play a role in type 2 diabetes pathogenesis through direct impact on pancreatic β-cell function, senescence-associated secretory phenotype (SASP)-mediated tissue damage, and involvement in adipose tissue dysfunction. In turn, metabolic and signaling changes seen in diabetes, such as high circulating glucose, altered lipid metabolism, and growth hormone axis perturbations, can promote senescent cell formation. Thus, senescent cells might be part of a pathogenic loop in diabetes, as both a cause and consequence of metabolic changes and tissue damage. Therapeutic targeting of a basic aging mechanism such as cellular senescence may have a large impact on disease pathogenesis and could be more effective in preventing the progression of diabetes complications than currently available therapies that have limited impact on already existing tissue damage. Therefore, senescent cells and the SASP represent significant opportunities for advancement in the prevention and treatment of type 2 diabetes and its complications.

Abstract We have previously shown that loss of HSulf-1, an endosulfatase that removes 6-O sulfation of HSPGs augmented heparin binding growth factor (HB-GF) signaling resulting in enhanced tumorigenesis, angiogenesis and metastasis in ovarian cancer. However, the role of HSulf-1 in energy metabolism and how the increased growth factor signaling in cells with loss of HSulf-1 altered cellular metabolism in cancer yet to be investigated. Here, we show for the first time that batch clonal lines of HSulf-1 knockdown cells [OV202 (Sh1/2)] and HSulf-1 knock out (KO) MEFs showed a significantly higher glycolytic flux (glucose uptake and lactate production) and reduced oxygen consumption rate (OCR) compared to a non-transduced control (OV202NTC) and wild-type (WT) MEF cells respectively. Conversely, enhanced expression of HSulf-1 in TOV21G cells resulted in lower glycolytic flux and higher OCR. Microarray analysis revealed that several of the genes in the glycolytic pathway such as Glut1, HK2, PFKFB3, ALDOC, PGAM, PKM2 and LDHA were upregulated in Sh1/2, cells compared to NTC cells. Expression of these genes and their respective products were validated both at the mRNA and protein levels in OV202Sh1/2, TOV21G Vec and MEFs KO cells compared to OV202NTC, TOV21G Cl13 and MEF WT respectively. Importantly, we found that loss of HSulf-1 decouples the energy flow from glycolysis to mitochondria by lowering the PDH and up-regulating PDK1 expression. We further demonstrated that loss of HSulf-1 reduced the total cellular ATP levels in Sh1/Sh2, TOV21G VEC, and MEFs KO cells, all consistent with lower mitochondrial activity as evidenced by decreased total number of mitochondria, its cross sectional area and perimeter in Sh1/Sh2, TOV21G Vec and MEF KO cells. This augmented aerobic glycolysis with loss of HSulf-1 was further corroborated by metabolomics study which showed significantly higher glycolytic metabolites such as G-6P, F1,6BP, F2,6BP, 3PG. While most of the TCA intermediates were downregulated in Sh1 and Sh2 cells; glutamate, glutamine, α-ketoglutarate and 2-hydroxyglutarate were higher upon loss of HSulf-1. These results suggests that loss of HSulf-1 plays a pivotal role in reprogramming the energy metabolism towards the Warburg effect which are critical to sustain cancer cells proliferation, transformation and angiogenesis. Citation Format: Susmita Mondal, Debarshi Roy, Juliana Camacho Pereira, Eduardo N. Chini, Vijayalakshmi Shridhar. Loss of sulf-1 reprograms energy metabolism in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3360. doi:10.1158/1538-7445.AM2014-3360

Abstract CD40 and BAFFR signaling play important roles in B cell proliferation and Ig production. In this study, we found that B cells from mice with deletion of Dbc1 gene (Dbc1−/−) show elevated proliferation, and IgG1 and IgA production upon in vitro CD40 and BAFF, but not BCR and LPS stimulation, indicating that DBC1 inhibits CD40/BAFF-mediated B cell activation in a cell-intrinsic manner. Microarray analysis and chromatin immunoprecipitation experiments reveal that DBC1 inhibits B cell function by selectively suppressing the transcriptional activity of alternative NF-κB members RelB and p52 upon CD40 stimulation. As a result, when immunized with nitrophenylated-keyhole limpet hemocyanin, Dbc1−/− mice produce significantly increased levels of germinal center B cells, plasma cells, and Ag-specific Ig. Finally, loss of DBC1 in mice leads to higher susceptibility to experimental autoimmune myasthenia gravis. Our study identifies DBC1 as a novel regulator of B cell activation by suppressing the alternative NF-κB pathway.