SIRT6 deacetylase activity regulates NAMPT activity and NAD(P)(H) pools in cancer cells.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage pathway from nicotinamide. By controlling the biosynthesis of NAD+, NAMPT regulates the activity of NAD+-converting enzymes, such as CD38, poly-ADP-ribose polymerases, and sirtuins (SIRTs). SIRT6 is involved in the regulation of a wide number of metabolic processes. In this study, we investigated the ability of SIRT6 to regulate intracellular NAMPT activity and NAD(P)(H) levels. BxPC-3 cells and MCF-7 cells were engineered to overexpress a catalytically active or a catalytically inactive SIRT6 form or were engineered to silence endogenous SIRT6 expression. In SIRT6-overexpressing cells, NAD(H) levels were up-regulated, as a consequence of NAMPT activation. By immunopurification and incubation with recombinant SIRT6, NAMPT was found to be a direct substrate of SIRT6 deacetylation, with a mechanism that up-regulates NAMPT enzymatic activity. Extracellular NAMPT release was enhanced in SIRT6-silenced cells. Also glucose-6-phosphate dehydrogenase activity and NADPH levels were increased in SIRT6-overexpressing cells. Accordingly, increased SIRT6 levels reduced cancer cell susceptibility to H2O2-induced oxidative stress and to doxorubicin. Our data demonstrate that SIRT6 affects intracellular NAMPT activity, boosts NAD(P)(H) levels, and protects against oxidative stress. The use of SIRT6 inhibitors, together with agents inducing oxidative stress, may represent a promising treatment strategy in cancer.-Sociali, G., Grozio, A., Caffa, I., Schuster, S., Becherini, P., Damonte, P., Sturla, L., Fresia, C., Passalacqua, M., Mazzola, F., Raffaelli, N., Garten, A., Kiess, W., Cea, M., Nencioni, A., Bruzzone, S. SIRT6 deacetylase activity regulates NAMPT activity and NAD(P)(H) pools in cancer cells.

Mesenchymal stem cells from preterm to term newborns undergo a significant switch from anaerobic glycolysis to the oxidative phosphorylation.

We evaluated the energy metabolism of human mesenchymal stem cells (MSC) isolated from umbilical cord (UC) of preterm (< 37 weeks of gestational age) and term (≥ 37 weeks of gestational age) newborns, using MSC from adult bone marrow as control. A metabolic switch has been observed around the 34th week of gestational age from a prevalently anaerobic glycolysis to the oxidative phosphorylation. This metabolic change is associated with the organization of mitochondria reticulum: preterm MSCs presented a scarcely organized mitochondrial reticulum and low expression of proteins involved in the mitochondrial fission/fusion, compared to term MSCs. These changes seem governed by the expression of CLUH, a cytosolic messenger RNA-binding protein involved in the mitochondria biogenesis and distribution inside the cell; in fact, CLUH silencing in term MSC determined a metabolic fingerprint similar to that of preterm MSC. Our study discloses novel information on the production of energy and mitochondrial organization and function, during the passage from fetal to adult life, providing useful information for the management of preterm birth.

The Plant Hormone Abscisic Acid Is a Prosurvival Factor in Human and Murine Megakaryocytes.

Abscisic acid (ABA) is a phytohormone involved in pivotal physiological functions in higher plants. Recently, ABA has been proven to be also secreted and active in mammals, where it stimulates the activity of innate immune cells, mesenchymal and hematopoietic stem cells, and insulin-releasing pancreatic ß cells through a signaling pathway involving the second messenger cyclic ADP-ribose (cADPR). In addition to behaving like an animal hormone, ABA also holds promise as a nutraceutical plant-derived compound in humans. Many biological functions of ABA in mammals are mediated by its binding to the LANCL-2 receptor protein. A putative binding of ABA to GRP78, a key regulator of endoplasmic reticulum stress, has also been proposed. Here we investigated the role of exogenous ABA in modulating thrombopoiesis, the process of platelet generation. Our results demonstrate that expression of both LANCL-2 and GRP78 is up-regulated during hematopoietic stem cell differentiation into mature megakaryocytes (Mks). Functional ABA receptors exist in mature Mks because ABA induces an intracellular Ca2+ increase ([Ca2+] i ) through PKA activation and subsequent cADPR generation. In vitro exposure of human or murine hematopoietic progenitor cells to 10 µm ABA does not increase recombinant thrombopoietin (rTpo)-dependent Mk differentiation or platelet release. However, under conditions of cell stress induced by rTpo and serum deprivation, ABA stimulates, in a PKA- and cADPR-dependent fashion, the mitogen-activated kinase ERK 1/2, resulting in the modulation of lymphoma 2 (Bcl-2) family members, increased Mk survival, and higher rates of platelet production. In conclusion, we demonstrate that ABA is a prosurvival factor for Mks in a Tpo-independent manner.

Abscisic Acid Stimulates Glucagon-Like Peptide-1 Secretion from L-Cells and Its Oral Administration Increases Plasma Glucagon-Like Peptide-1 Levels in Rats.

In recent years, Abscisic Acid (ABA) has been demonstrated to be involved in the regulation of glucose homeostasis in mammals as an endogenous hormone, by stimulating both insulin release and peripheral glucose uptake. In addition, ABA is released by glucose- or GLP-1-stimulated ß-pancreatic cells. Here we investigated whether ABA can stimulate GLP-1 release. The human enteroendocrine L cell line hNCI-H716 was used to explore whether ABA stimulates in vitro GLP-1 secretion and/or transcription. ABA induced GLP-1 release in hNCI-H716 cells, through a cAMP/PKA-dependent mechanism. ABA also enhanced GLP-1 transcription. In addition, oral administration of ABA significantly increased plasma GLP-1 and insulin levels in rats. In conclusion, ABA can stimulate GLP-1 release: this result and the previous observation that GLP-1 stimulates ABA release from ß -cells, suggest a positive feed-back mechanism between ABA and GLP-1, regulating glucose homeostasis. Type 2 diabetes treatments targeting the GLP-1 axis by either inhibiting its rapid clearance by dipeptidyl-peptidase IV or using GLP-1 mimetics are currently used. Moreover, the development of treatments aimed at stimulating GLP-1 release from L cells has been considered as an alternative approach. Accordingly, our finding that ABA increases GLP-1 release in vitro and in vivo may suggest ABA and/or ABA analogs as potential anti-diabetic treatments.

Autocrine abscisic acid mediates the UV-B-induced inflammatory response in human granulocytes and keratinocytes.

UV-B is an abiotic environmental stress in both plants and animals. Abscisic acid (ABA) is a phytohormone regulating fundamental physiological functions in plants, including response to abiotic stress. We previously demonstrated that ABA is an endogenous stress hormone also in animal cells. Here, we investigated whether autocrine ABA regulates the response to UV-B of human granulocytes and keratinocytes, the cells involved in UV-triggered skin inflammation. The intracellular ABA concentration increased in UV-B-exposed granulocytes and keratinocytes and ABA was released into the supernatant. The UV-B-induced production of NO and of reactive oxygen species (ROS), phagocytosis, and cell migration were strongly inhibited in granulocytes irradiated in the presence of a monoclonal antibody against ABA. Moreover, presence of the same antibody strongly inhibited release of NO, prostaglandin E2 (PGE(2)), and tumor necrosis factor-α (TNF-α) by UV-B irradiated keratinocytes. Lanthionine synthetase C-like protein 2 (LANCL2) is required for the activation of the ABA signaling pathway in human granulocytes. Silencing of LANCL2 in human keratinocytes by siRNA was accompanied by abrogation of the UV-B-triggered release of PGE(2), TNF-α, and NO and ROS production. These results indicate that UV-B irradiation induces ABA release from human granulocytes and keratinocytes and that autocrine ABA stimulates cell functions involved in skin inflammation.

The association of human mesenchymal stem cells with BMP-7 improves bone regeneration of critical-size segmental bone defects in athymic rats.

Critical size segmental bone defects are still a major challenge in reconstructive orthopedic surgery. Transplantation of human mesenchymal stem cells (hMSC) has been proposed as an alternative to autogenous bone graft, as MSC can be expanded in vitro and induced to differentiate into bone-regenerating osteoblasts by several bone morphogenetic proteins (BMP). The aim of this study was to investigate whether the association of hMSC and BMP-7, with providing the necessary scaffold to fill the bone loss, improved bone regeneration in a rat model of critical size segmental bone defect, compared to treatment with either hMSC or BMP-7 and the matrix. In addition, we tested whether pre-treatment of hMSC with cyclic ADP-ribose (cADPR), an intracellular Ca2+ mobilizer previously shown to accelerate the in vitro expansion of hMSC (Scarfì S et al, Stem Cells, 2008), affected the osteoinductive capacity of the cells in vivo. X-ray analysis, performed 2, 10 and 16 weeks after transplantation, revealed a significantly higher score in the rats treated with hMSC and BMP-7 compared to controls, receiving either hMSC or BMP-7. Microtomography and histological analysis, performed 16weeks after transplantation, confirmed the improved bone regeneration in the animals treated with the association of hMSC and BMP-7 compared to controls. Pre-treatment with cADPR to stimulate hMSC proliferation in vitro did not affect the bone regenerating capacity of the cells in vivo. These results indicate that the association of in vitro expanded hMSC with BMP-7 provide a better osteoinductive graft compared to either hMSC or BMP-7 alone. Moreover, cADPR may be used to stimulate hMSC proliferation in vitro in order to reduce the time required to obtain a transplantable number of cells, with no adverse effect on the bone regenerating capacity of hMSC.

LANCL2 is necessary for abscisic acid binding and signaling in human granulocytes and in rat insulinoma cells.

Abscisic acid (ABA) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. Recently, ABA was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells, and by human and murine pancreatic beta cells. ABA autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin-sensitive receptor/G-protein complex, cAMP, CD38-produced cADP-ribose and intracellular calcium. Here we show that the lanthionine synthetase C-like protein LANCL2 is required for ABA binding on the membrane of human granulocytes and that LANCL2 is necessary for transduction of the ABA signal into the cell-specific functional responses in granulocytes and in rat insulinoma cells. Co-expression of LANCL2 and CD38 in the human HeLa cell line reproduces the ABA-signaling pathway. Results obtained with granulocytes and CD38(+)/LANCL2(+) HeLa transfected with a chimeric G-protein (G alpha(q/i)) suggest that the pertussis toxin-sensitive G-protein coupled to LANCL2 is a G(i). Identification of LANCL2 as a critical component of the ABA-sensing protein complex will enable the screening of synthetic ABA antagonists as prospective new anti-inflammatory and anti-diabetic agents.

The plant hormone abscisic acid stimulates the proliferation of human hemopoietic progenitors through the second messenger cyclic ADP-ribose.

Abscisic acid (ABA) is a hormone involved in pivotal physiological functions in higher plants, such as response to abiotic stress and control of seed dormancy and germination. Recently, ABA was demonstrated to be autocrinally produced by human granulocytes, beta pancreatic cells, and mesenchymal stem cells (MSC) and to stimulate cell-specific functions through a signaling pathway involving the second messenger cyclic ADP-ribose (cADPR). Here we show that ABA expands human uncommitted hemopoietic progenitors (HP) in vitro, through a cADPR-mediated increase of the intracellular calcium concentration ([Ca(2+)](i)). Incubation of CD34(+) cells with micromolar ABA also induces transcriptional effects, which include NF-kappaB nuclear translocation and transcription of genes encoding for several cytokines. Human MSC stimulated with a lymphocyte-conditioned medium produce and release ABA at concentrations sufficient to exert growth-stimulatory effects on co-cultured CD34(+) cells, as demonstrated by the inhibition of colony growth in the presence of an anti-ABA monoclonal antibody. These results provide a remarkable example of conservation of a stress hormone and of its second messenger from plants to humans and identify ABA as a new hemopoietic growth factor involved in the cross-talk between HP and MSC.

Abscisic acid activates the murine microglial cell line N9 through the second messenger cyclic ADP-ribose.

Abscisic acid (ABA) is a phytohormone regulating important functions in higher plants, notably responses to abiotic stress. Recently, chemical or physical stimulation of human granulocytes was shown to induce production and release of endogenous ABA, which activates specific cell functions. Here we provide evidence that ABA stimulates several functional activities of the murine microglial cell line N9 (NO and tumor necrosis factor-alpha production, cell migration) through the second messenger cyclic ADP-ribose and an increase of intracellular calcium. ABA production and release occur in N9 cells stimulated with bacterial lipopolysaccharide, phorbol myristate acetate, the chemoattractant peptide f-MLP, or beta-amyloid, the primary plaque component in Alzheimer disease. Finally, ABA priming stimulates N9 cell migration toward beta-amyloid. These results indicate that ABA is a pro-inflammatory hormone inducing autocrine microglial activation, potentially representing a new target for anti-inflammatory therapies aimed at limiting microglia-induced tissue damage in the central nervous system.

Cyclic ADP-ribose-mediated expansion and stimulation of human mesenchymal stem cells by the plant hormone abscisic acid.

Abscisic acid (ABA) is a phytohormone involved in fundamental processes in higher plants. Endogenous ABA biosynthesis occurs also in lower Metazoa, in which ABA regulates several physiological functions by activating ADP-ribosyl cyclase (ADPRC) and causing overproduction of the Ca(2+)-mobilizing second messenger cyclic ADP-ribose (cADPR), thereby enhancing intracellular Ca(2+) concentration ([Ca(2+)](i)). Recently, production and release of ABA have been demonstrated to take place also in human granulocytes, where ABA behaves as a proinflammatory hormone through the same cADPR/[Ca(2+)](i) signaling pathway described in plants and in lower Metazoa. On the basis of the fact that human mesenchymal stem cells (MSC) express ADPRC activity, we investigated the effects of ABA and of its second messenger, cADPR, on purified human MSC. Both ABA and cADPR stimulate the in vitro expansion of MSC without affecting differentiation. The underlying mechanism involves a signaling cascade triggered by ABA binding to a plasma membrane receptor and consequent cyclic AMP-mediated activation of ADPRC and of the cADPR/[Ca(2+)](i) system. Moreover, ABA stimulates the following functional activities of MSC: cyclooxygenase 2-catalyzed production of prostaglandin E(2) (PGE(2)), release of several cytokines known to mediate the trophic and immunomodulatory properties of MSC, and chemokinesis. Remarkably, ABA proved to be produced and released by MSC stimulated by specific growth factors (e.g., bone morphogenetic protein-7), by inflammatory cytokines, and by lymphocyte-conditioned medium. These data demonstrate that ABA is an autocrine stimulator of MSC function and suggest that it may participate in the paracrine signaling among MSC, inflammatory/immune cells, and hemopoietic progenitors. Disclosure of potential conflicts of interest is found at the end of this article.