Giovanna Basile

The NAD+-dependent histone deacetylase SIRT6 promotes cytokine production and migration in pancreatic cancer cells by regulating Ca2+ responses

Background: Cytokine secretion has unwanted consequences in malignant and in inflammatory disorders. The deacetylase SIRT6 has pro-inflammatory activity, but the underlying mechanisms and its biological significance remain unclear. Results: SIRT6 enhances cytokine secretion and cell motility in pancreatic cancer cells by activating Ca2+ signaling. Conclusion: SIRT6 promotes Ca2+-dependent responses. Significance: SIRT6 inhibitors may help combat malignant and inflammatory disorders. Cytokine secretion by cancer cells contributes to cancer-induced symptoms and angiogenesis. Studies show that the sirtuin SIRT6 promotes inflammation by enhancing TNF expression. Here, we aimed to determine whether SIRT6 is involved in conferring an inflammatory phenotype to cancer cells and to define the mechanisms linking SIRT6 to inflammation. We show that SIRT6 enhances the expression of pro-inflammatory cyto-/chemokines, such as IL8 and TNF, and promotes cell migration in pancreatic cancer cells by enhancing Ca2+ responses. Via its enzymatic activity, SIRT6 increases the intracellular levels of ADP-ribose, an activator of the Ca2+ channel TRPM2. In turn, TRPM2 and Ca2+ are shown to be involved in SIRT6-induced TNF and IL8 expression. SIRT6 increases the nuclear levels of the Ca2+-dependent transcription factor, nuclear factor of activated T cells (NFAT), and cyclosporin A, a calcineurin inhibitor that reduces NFAT activity, reduces TNF and IL8 expression in SIRT6-overexpressing cells. These results implicate a role for SIRT6 in the synthesis of Ca2+-mobilizing second messengers, in the regulation of Ca2+-dependent transcription factors, and in the expression of pro-inflammatory, pro-angiogenic, and chemotactic cytokines. SIRT6 inhibition may help combat cancer-induced inflammation, angiogenesis, and metastasis.

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 β 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 (Gαq/i) suggest that the pertussis toxin-sensitive G-protein coupled to LANCL2 is a Gi. 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.

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-α 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 β-amyloid, the primary plaque component in Alzheimer disease. Finally, ABA priming stimulates N9 cell migration toward β-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.

ABA- and cADPR-mediated effects on respiration and filtration downstream of the temperature-signaling cascade in sponges

Recently, the thermosensing pathway in sponges (Porifera) was elucidated. The thermosensor triggering this cascade is a heat-activated cation channel, with the phytohormone abscisic acid (ABA), cyclic ADP-ribose (cADPR) and calcium acting as intracellular messengers, similarly to the drought-stress signaling cascade in higher plants. Here, we investigated the functional effects downstream of the temperature-signaling pathway in Axinella polypoides (Porifera, Demonspongiae). Short-term stimulation followed by long-term depression of amino acid incorporation, oxygen consumption and water filtration were observed after exposure of the sponge to a brief heat stress or to micromolar ABA. These effects could be prevented by the targeted interruption of the signaling pathway either at the level of the cation channel thermosensor or at the level of the cADPR-induced intracellular calcium increase. Moreover, release of cyclase activity into the sea water and generation of extracellular cADPR were observed following brief heat stress. Intact sponge cells were sensitive to extracellular cADPR and addition of purified cyclase increased sponge respiration similarly to heat stress. This is the first observation of functional effects exerted on Metazoa by the phytohormone ABA: conservation of the ABA/cADPR stress-signaling cascade points to its early evolution in a common precursor of modern Metazoa and Metaphyta. The functional effects induced by extracellular cyclase/cADPR suggest an evolutionary origin of cADPR as an ancient stress hormone in Porifera.

The plant hormone abscisic acid increases in human plasma after hyperglycemia and stimulates glucose consumption by adipocytes and myoblasts

The plant hormone abscisic acid (ABA) is released from glucose‐challenged human pancreatic β cells and stimulates insulin secretion. We investigated whether plasma ABA increased during oral and intravenous glucose tolerance tests (OGTTs and IVGTTs) in healthy human subjects. In all subjects undergoing OGTTs (n=8), plasma ABA increased over basal values (in a range from 2‐ to 9‐fold). A positive correlation was found between the ABA area under the curve (AUC) and the glucose AUC. In 4 out of 6 IVGTTs, little or no increase of ABA levels was observed. In the remaining subjects, the ABA increase was similar to that recorded during OGTTs. GLP‐1 stimulated ABA release from an insulinoma cell line and from human islets, by ~10‐ and 2‐fold in low and high glucose, respectively. Human adipose tissue also released ABA in response to high glucose. Nanomolar ABA stimulated glucose uptake, similarly to insulin, in rat L6 myoblasts and in murine 3T3‐L1 cells differentiated to adipocytes, by increasing GLUT‐4 translocation to the plasma membrane. Demonstration that a glucose load in humans is followed by a physiological rise of plasma ABA, which can enhance glucose uptake by adipose tissues and muscle cells, identifies ABA as a new mammalian hormone involved in glucose metabolism.—Bruzzone, S., Ameri, P., Briatore, L., Mannino, E., Basile, G., Andraghetti, G., Grozio, A., Magnone, M., Guida, L., Scarfì, S., Salis, A., Damonte, G., Sturla, L., Nencioni, A., Fenoglio, D., Fiory, F., Miele, C., Beguinot, F., Ruvolo, V., Bormioli, M., Colombo, G., Maggi, D., Murialdo, G., Cordera, R., De Flora, A., Zocchi, E. The plant hormone abscisic acid increases in human plasma after hyperglycemia and stimulates glucose consumption by adipocytes and myoblasts. FASEB J. 26, 1251‐1260 (2012). www.fasebj.org

P2X7-mediated Increased Intracellular Calcium Causes Functional Derangement in Schwann Cells from Rats with CMT1A Neuropathy

Charcot-Marie-Tooth (CMT) is the most frequent inherited neuromuscular disorder, affecting 1 person in 2500. CMT1A, the most common form of CMT, is usually caused by a duplication of chromosome 17p11.2, containing the PMP22 (peripheral myelin protein-22) gene; overexpression of PMP22 in Schwann cells (SC) is believed to cause demyelination, although the underlying pathogenetic mechanisms remain unclear. Here we report an abnormally high basal concentration of intracellular calcium ([Ca2+]i) in SC from CMT1A rats. By the use of specific pharmacological inhibitors and through down-regulation of expression by small interfering RNA, we demonstrate that the high [Ca2+]i is caused by a PMP22-related overexpression of the P2X7 purinoceptor/channel leading to influx of extracellular Ca2+ into CMT1A SC. Correction of the altered [Ca2+]i in CMT1A SC by small interfering RNA or with pharmacological inhibitors of P2X7 restores functional parameters of SC (migration and release of ciliary neurotrophic factor), which are typically defective in CMT1A SC. More significantly, stable down-regulation of the expression of P2X7 restores myelination in co-cultures of CMT1A SC with dorsal root ganglion sensory neurons. These results establish a pathogenetic link between high [Ca2+]i and impaired SC function in CMT1A and identify overexpression of P2X7 as the molecular mechanism underlying both abnormalities. The development of P2X7 inhibitors is expected to provide a new therapeutic strategy for treatment of CMT1A neuropathy.

Concentrative Influx of Functionally Active Cyclic ADP-ribose in Dimethyl Sulfoxide-differentiated HL-60 Cells

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 (Me2SO), fail to induce CD38 expression. We investigated whether treatment of HL-60 cells with Me2SO involves any changes in the cADPR/intracellular calcium ([Ca2+]i) signaling system and, specifically, whether Me2SO 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 [3H]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 Me2SO-differentiated HL-60 cells. The native cells showed uptake of cADPR across ENT2, whereas influx of cADPR into the Me2SO-differentiated cells occurred mostly by concentrative processes mediated by CNT3 and by an NBMPR-inhibitable concentrative NT designated cs-csg. Me2SO-differentiated, but not native HL-60 cells, accumulated cADPR and showed increased [Ca2+]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 Me2SO-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 [Ca2+]i increases in granulocyte-differentiated HL-60 cells, as well as in other CD38– cells.

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 (PGE2), 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 PGE2, 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. J. Cell. Physiol. 227: 2502–2510, 2012.

Mitochondrial Dysfunction Induced by a Cytotoxic Adenine Dinucleotide Produced by ADP-ribosyl Cyclases from cADPR

ADP-ribosyl cyclases were previously shown to produce three new adenine dinucleotides, P1,P2 diadenosine 5′-diphosphate (Ap2A) and two isomers thereof (P18 and P24), from cyclic ADP-ribose (cADPR) and adenine (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 14509-14514). The Ap2A isomer P24, containing an unusual C1′-N3 N-glycosidic bond, is shown here to affect mitochondrial function through (i) opening of the permeability transition pore complex (and consequent proton gradient dissipation) and (ii) inhibition of Complex I of the respiratory chain. Whereas proton gradient dissipation is dependent upon the extracellular Ca2+ influx triggered by P24, the effect on oxygen consumption is Ca2+ independent. The proton gradient dissipation induces apoptosis in HeLa cells and thus appears to be responsible for the already described potent cytotoxic effect of P24 on several human cell types. The other products of ADP-ribosyl cyclase activity, Ap2A and cADPR, antagonize P24-induced proton gradient dissipation and cytotoxicity, suggesting that the relative concentration of P24, cADPR, and Ap2A in cyclase-positive cells may affect the balance between cell life and death.

Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-β (TGF-β). Conversely, migration toward ABA, but not toward TGF-β, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.