Francisco J. Bedoya

Oxidative Stress Triggers STAT3 Tyrosine Phosphorylation and Nuclear Translocation in Human Lymphocytes

Oxidizing agents are powerful activators of factors responsible for the transcriptional activation of cytokine-encoding genes involved in tissue injury. In this study we show evidence that STAT3 is a transcription factor whose activity is modulated by H2O2 in human lymphocytes, in which endogenous catalase had previously been inhibited. H2O2-induced nuclear translocation of STAT3 to form sequence-specific DNA-bound complexes was evidenced by immunoblotting of nuclear fractions and electrophoretic mobility shift assays, and vanadate was found to strongly synergize with H2O2. Moreover, anti-STAT3 antibodies specifically precipitated a protein of 92 kDa that becomes phosphorylated on tyrosine upon lymphocyte treatment with H2O2. Phenylarsine oxide, a tyrosine phosphatase inhibitor, and genistein, a tyrosine kinase inhibitor, cooperated and cancelled, respectively, the H2O2-promoted STAT3 nuclear translocation. Evidence is also presented, using Fe2+/Cu2+ions, that ⋅OH generated from H2O2through Fenton reactions could be a candidate oxygen reactive species to directly activate STAT3. Present data suggest that H2O2 and vanadate are likely to inhibit the activity of intracellular tyrosine phosphatase(s), leading to enhanced STAT3 tyrosine phosphorylation and hence its translocation to the nucleus. These results demonstrate that the DNA binding activity of STAT3 can be modulated by oxidizing agents and provide a framework to understand the effects of oxidative stress on the JAK-STAT signaling pathway.

15-Deoxy-Δ12,14-prostaglandin J2 Induces Heme Oxygenase-1 Gene Expression in a Reactive Oxygen Species-dependent Manner in Human Lymphocytes

15-Deoxy-Δ12,14-prostaglandin J2 (15dPGJ2 has been recently proposed as a potent anti-inflammatory agent. However, the mechanisms by which 15dPGJ2 mediates its therapeutic effects in vivo are unclear. We demonstrate that 15dPGJ2 at micromolar (2.5–10 μm) concentrations induces the expression of heme oxygenase-1 (HO-1), an anti-inflammatory enzyme, at both mRNA and protein levels in human lymphocytes. In contrast, troglitazone and ciglitazone, two thiazolidinediones that mimic several effects of 15dPGJ2 through their binding to the peroxisome proliferator-activated receptor (PPAR)-γ, did not affect HO-1 expression, and the positive effect of 15dPGJ2 on this process was mimicked instead by other cyclopentenone prostaglandins (PG), such as PGD2 (the precursor of 15dPGJ2) and PGA1 and PGA2 which do not interact with PPAR-γ. Also, 15dPGJ2 enhanced the intracellular production of reactive oxygen species (ROS) and increased xanthine oxidase activity in vitro. Inhibition of intracellular ROS production by N-acetylcysteine, TEMPO, Me2SO, 1,10-phenanthroline, or allopurinol resulted in a decreased 15dPGJ2-dependent HO-1 expression in the cells. Furthermore, buthionine sulfoximine, an inhibitor of reduced glutathione synthesis, or Fe2+/Cu2+ ions enhanced the positive effect of 15dPGJ2 on HO-1 expression. On the other hand, the inhibition of phosphatidylinositol 3-kinase or p38 mitogen-activated protein kinase, or the blockade of transcription factor NF-κB activation, hindered 15dPGJ2-elicited HO-1 expression. Collectively, the present data suggest that 15dPGJ2 anti-inflammatory actions at pharmacological concentrations involve the induction of HO-1 gene expression through mechanisms independent of PPAR-γ activation and dependent on ROS produced via the xanthine/xanthine oxidase system and/or through Fenton reactions. Both phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase signaling pathways also appear implicated in modulation of HO-1 expression by 15dPGJ2.

Taurine supplementation modulates glucose homeostasis and islet function

Taurine is a conditionally essential amino acid for human that is involved in the control of glucose homeostasis; however, the mechanisms by which the amino acid affects blood glucose levels are unknown. Using an animal model, we have studied these mechanisms. Mice were supplemented with taurine for 30 d. Blood glucose homeostasis was assessed by intraperitoneal glucose tolerance tests (IPGTT). Islet cell function was determined by insulin secretion, cytosolic Ca2+ measurements and glucose metabolism from isolated islets. Islet cell gene expression and translocation was examined via immunohistochemistry and quantitative real-time polymerase chain reaction. Insulin signaling was studied by Western blot. Islets from taurine-supplemented mice had: (i) significantly higher insulin content, (ii) increased insulin secretion at stimulatory glucose concentrations, (iii) significantly displaced the dose-response curve for glucose-induced insulin release to the left, (iv) increased glucose metabolism at 5.6 and 11.1-mmol/L concentrations; (v) slowed cytosolic Ca2+ concentration ([Ca2+]i) oscillations in response to stimulatory glucose concentrations; (vi) increased insulin, sulfonylurea receptor-1, glucokinase, Glut-2, proconvertase and pancreas duodenum homeobox-1 (PDX-1) gene expression and (vii) increased PDX-1 expression in the nucleus. Moreover, taurine supplementation significantly increased both basal and insulin stimulated tyrosine phosphorylation of the insulin receptor in skeletal muscle and liver tissues. Finally, taurine supplemented mice showed an improved IPGTT. These results indicate that taurine controls glucose homeostasis by regulating the expression of genes required for glucose-stimulated insulin secretion. In addition, taurine enhances peripheral insulin sensitivity.

Stimulators of AMP-activated protein kinase inhibit the respiratory burst in human neutrophils

In the present study, we have examined the potential ability of 5′‐AMP‐activated protein kinase (AMPK) to modulate NADPH oxidase activity in human neutrophils. AMPK activated with either 5′‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) or with 5′‐AMP significantly attenuated both phorbol 12‐myristate 13‐acetate (PMA) and formyl methionyl leucyl phenylalanine‐stimulated superoxide anion (O2 −) release by human neutrophils, consistently with a reduced translocation to the cell membrane and phosphorylation of a cytosolic component of NADPH oxidase, namely p47phox. AMPK was found to be present in human neutrophils and to become phosphorylated in response to either AICAR or other stimulators of its enzyme activity. Furthermore, AICAR also strongly reduced PMA‐dependent H2O2 release, and induced the phosphorylation of c‐jun N‐terminal kinase 1 (p46), p38 mitogen‐activated protein kinase and extracellular signal‐regulated kinase. Present data demonstrate for the first time that the activation of AMPK, in states of low cellular energy charge (such as under high levels of 5′‐AMP) or other signals, could be a factor contributing to reduce the host defense mechanisms.

NO induces a cGMP-independent release of cytochrome c from mitochondria which precedes caspase 3 activation in insulin producing RINm5F cells

Exposure of RINm5F cells to interleukin‐1β and to several chemical NO donors such as sodium nitroprusside (SNP), SIN‐1 and SNAP induce apoptotic events such as the release of cytochrome c from mitochondria, caspase 3 activation, Bcl‐2 downregulation and DNA fragmentation. SNP exposure led to transient activation of soluble guanylate cyclase (sGC) and prolonged protein kinase G (PKG) activation but apoptotic events were not attenuated by inhibition of the sGC/PKG pathway. Prolonged activation of the cGMP pathway by exposing cells to the dibutyryl analogue of cGMP for 12 h induced both apoptosis and necrosis, a response that was abolished by the PKG inhibitor KT5823. These results suggest that NO‐induced apoptosis in the pancreatic β‐cell line is independent of acute activation of the cGMP pathway.

Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells

The poly(ADP-ribose) polymerase (PARP) inhibitor, nicotinamide, induces differentiation and maturation of fetal pancreatic cells. In addition, we have previously reported evidence that nicotinamide increases the insulin content of cells differentiated from embryonic stem (ES) cells, but the possibility of nicotinamide acting as a differentiating agent on its own has never been completely explored. Islet cell differentiation was studied by: (i) X-gal staining after neomycin selection; (ii) BrdU studies; (iii) single and double immunohistochemistry for insulin, C-peptide and Glut-2; (iv) insulin and C-peptide content and secretion assays; and (v) transplantation of differentiated cells, under the kidney capsule, into streptozotocin (STZ)-diabetic mice. Here we show that undifferentiated mouse ES cells treated with nicotinamide: (i) showed an 80% decrease in cell proliferation; (ii) co-expressed insulin, C-peptide and Glut-2; (iii) had values of insulin and C-peptide corresponding to 10% of normal mouse islets; (iv) released insulin and C-peptide in response to stimulatory glucose concentrations; and (v) after transplantation into diabetic mice, normalized blood glucose levels over 7 weeks. Our data indicate that nicotinamide decreases ES cell proliferation and induces differentiation into insulin-secreting cells. Both aspects are very important when thinking about cell therapy for the treatment of diabetes based on ES cells.

Nitric oxide repression of Nanog promotes mouse embryonic stem cell differentiation

Exposure of mouse embryonic stem (mES) cells to high concentrations of chemical nitric oxide (NO) donors promotes differentiation, but the mechanisms involved in this process at the gene expression level are poorly defined. In this study we report that culture of mES cells in the presence of 0.25–1.0 mM diethylenetriamine nitric oxide adduct (DETA-NO) leads to downregulation of Nanog and Oct4, the two master genes involved in the control of the pluripotent state. This action of NO was also apparent in the human ES cell line, HS 181. The suppressive action of NO on Nanog gene depends on the activation of p53 repressor protein by covalent modifications, such as pSer15, pSer315, pSer392 and acetyl Lys 379. NO-induced repression of Nanog is also associated with binding of trimethylated histone H3 and pSer315 p53 to its promoter region. In addition, exposure to 0.5 mM DETA-NO induces early differentiation events of cells with acquisition of epithelial morphology and expression of markers of definitive endoderm, such as FoxA2, Gata4, Hfn1-β and Sox 17. This phenotype was increased when cells were treated with valproic acid (VPA) for 10 days.

Low concentrations of nitric oxide delay the differentiation of embryonic stem cells and promote their survival

Nitric oxide (NO) is an intracellular messenger in several cell systems, but its contribution to embryonic stem cell (ESC) biology has not been characterized. Exposure of ESCs to low concentrations (2–20 μM) of the NO donor diethylenetriamine NO adduct confers protection from apoptosis elicited by leukaemia inhibitory factor (LIF) withdrawal. NO blocked caspase 3 activation, PARP degradation, downregulation of the pro-apoptotic genes Casp7, Casp9, Bax and Bak1 and upregulation of the anti-apoptotic genes Bcl-2 111, Bcl-2 and Birc6. These effects were also observed in cells overexpressing eNOS. Exposure of LIF-deprived mESCs to low NO prevented the loss of expression of self-renewal genes (Oct4, Nanog and Sox2) and the SSEA marker. Moreover, NO blocked the differentiation process promoted by the absence of LIF and bFGF in mouse and human ESCs. NO treatment decreased the expression of differentiation markers, such as Brachyury, Gata6 and Gata4. Constitutive overexpression of eNOS in cells exposed to LIF deprivation maintained the expression of self-renewal markers, whereas the differentiation genes were repressed. These effects were reversed by addition of the NOS inhibitor L-NMMA. Altogether, the data suggest that low NO has a role in the regulation of ESC differentiation by delaying the entry into differentiation, arresting the loss of self-renewal markers and promoting cell survival by inhibiting apoptosis.

Synthesis, antiapoptotic biological activity and structure of an oxo-vanadium(IV) complex with an OOO ligand donor set

Abstract The oxo–vanadium(IV) complex VO(oda)(H2O)2 (1) (oda=oxydiacetate, O(CH2COO−)2) was obtained by reaction of aerobic aqueous solutions of VO(acac)2 with oxydiacetic acid, O(CH2COOH)2. The antiapoptotic biological activity of 1 was studied in insulin-producing cells. Chemically generated nitric oxide (NO) triggers apoptotic events, such as the appearance of oligonucleosomes in cytosol, and this response was prevented by the presence of 1 in the culture medium. The molecular structure of 1 has been determined by X-ray diffraction analysis.

Evidence for involvement of c-Src in the anti-apoptotic action of nitric oxide in serum-deprived RINm5F cells.

The mechanism by which nitric oxide (NO) protects from apoptosis is a matter of debate. We have shown previously that phosphorylation of tyrosine residues participates in the protection from apoptosis in insulin-producing RINm5F cells (Inorg. Chem. Commun. 3 (2000) 32). Since NO has been reported to activate the tyrosine kinase c-Src and this kinase is involved in the activation of protein kinase G (PKG) in some cell systems, we aimed at studying the contribution of c-Src and PKG systems in anti-apoptotic actions of NO in serum-deprived RINm5F cells. Here we report that exposure of serum-deprived cells to 10 microM DETA/NO results in protection from degradation of the anti-apoptotic protein Bcl-2, together with a reduction of cytochrome c release from mitochondria and caspase-3 inhibition. Studies with the inhibitors ODQ and KT-5823 revealed that these actions are dependent on both activation of guanylate cyclase and PKG. DETA/NO was also able to induce autophosphorylation and activation c-Src protein both in vivo and in vitro and active c-Src was able to induce tyrosine phosphorylation of Bcl-2 in vitro. The c-Src kinase inhibitor PP1 abrogated the actions of DETA/NO on cGMP formation, PKG activation, caspase activation, cytochrome c release from mitochondria, and Bcl-2 phosphorylation and degradation in serum-deprived cells. We thus propose that activation of c-Src is an early step in the chain of events that signal cGMP-dependent anti-apoptotic actions of NO in mitocohondria.