Tohru Yamamori

SIRT1 promotes endothelium-dependent vascular relaxation by activating endothelial nitric oxide synthase

Reduced caloric intake decreases arterial blood pressure in healthy individuals and improves endothelium-dependent vasodilation in obese and overweight individuals. The SIRT1 protein deacetylase mediates many of the effects of calorie restriction (CR) on organismal lifespan and metabolic pathways. However, the role of SIRT1 in regulating endothelium-dependent vasomotor tone is not known. Here we show that SIRT1 promotes endothelium-dependent vasodilation by targeting endothelial nitric oxide synthase (eNOS) for deacetylation. SIRT1 and eNOS colocalize and coprecipitate in endothelial cells, and SIRT1 deacetylates eNOS, stimulating eNOS activity and increasing endothelial nitric oxide (NO). SIRT1-induced increase in endothelial NO is mediated through lysines 496 and 506 in the calmodulin-binding domain of eNOS. Inhibition of SIRT1 in the endothelium of arteries inhibits endothelium-dependent vasodilation and decreases bioavailable NO. Finally, CR of mice leads to deacetylation of eNOS. Our results demonstrate that SIRT1 plays a fundamental role in regulating endothelial NO and endothelium-dependent vascular tone by deacetylating eNOS. Furthermore, our results provide a possible molecular mechanism connecting the effects of CR on the endothelium and vascular tone to SIRT1-mediated deacetylation of eNOS.

Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint

Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that Ir-induced G2/M arrest contributed to the increase in the mitochondrial ROS level by accumulating cells in the G2/M phase.

Roles of p38 MAPK, PKC and PI3-K in the signaling pathways of NADPH oxidase activation and phagocytosis in bovine polymorphonuclear leukocytes.

Stimulation of bovine polymorphonuclear leukocytes (PMN) with serum‐opsonized zymosan (sOZ) induced the activation of p38 mitogen‐activated protein kinase (MAPK), protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI3‐K) and sOZ‐induced O2 − production was significantly attenuated by their inhibitors (SB203580 for p38 MAPK, GF109203X for PKC and wortmannin for PI3‐K). They caused significant attenuation of sOZ‐induced phosphorylation of p47phox as well. Flow cytometric analysis, however, revealed that SB203580 and wortmannin attenuated phagocytosis, but GF109203X facilitated it. The results suggest that p38 MAPK and PI3‐K participated in both signaling pathways of NADPH oxidase activation (O2 − production) and phagocytosis, and PKC participated in the signaling pathway of NADPH oxidase activation alone.

SIRT1 deacetylates APE1 and regulates cellular base excision repair

Apurinic/apyrimidinic endonuclease-1 (APE1) is an essential enzyme in the base excision repair (BER) pathway. Here, we show that APE1 is a target of the SIRTUIN1 (SIRT1) protein deacetylase. SIRT1 associates with APE1, and this association is increased with genotoxic stress. SIRT1 deacetylates APE1 in vitro and in vivo targeting lysines 6 and 7. Genotoxic insults stimulate lysine acetylation of APE1 which is antagonized by transcriptional upregulation of SIRT1. Knockdown of SIRT1 increases cellular abasic DNA content, sensitizing cells to death induced by genotoxic stress, and this vulnerability is rescued by overexpression of APE1. Activation of SIRT1 with resveratrol promotes binding of APE1 to the BER protein X-ray cross-complementing-1 (XRCC1), while inhibition of SIRT1 with nicotinamide (NAM) decreases this interaction. Genotoxic insult also increases binding of APE1 to XRCC1, and this increase is suppressed by NAM or knockdown of SIRT1. Finally, resveratrol increases APE activity in XRCC1-associated protein complexes, while NAM or knockdown of SIRT1 suppresses this DNA repair activity. These findings identify APE1 as a novel protein target of SIRT1, and suggest that SIRT1 plays a vital role in maintaining genomic integrity through regulation of the BER pathway.

24(S)-Hydroxycholesterol Induces Neuronal Cell Death through Necroptosis, a Form of Programmed Necrosis

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis.

Sos-mediated activation of rac1 by p66shc

The Son of Sevenless 1 protein (sos1) is a guanine nucleotide exchange factor (GEF) for either the ras or rac1 GTPase. We show that p66shc, an adaptor protein that promotes oxidative stress, increases the rac1-specific GEF activity of sos1, resulting in rac1 activation. P66shc decreases sos1 bound to the growth factor receptor bound protein (grb2) and increases the formation of the sos1–eps8–e3b1 tricomplex. The NH2-terminal proline-rich collagen homology 2 (CH2) domain of p66shc associates with full-length grb2 in vitro via the COOH-terminal src homology 3 (C-SH3) domain of grb2. A proline-rich motif (PPLP) in the CH2 domain mediates this association. The CH2 domain competes with the proline-rich COOH-terminal region of sos1 for the C-SH3 domain of grb2. P66shc-induced dissociation of sos1 from grb2, formation of the sos1–eps8–e3b1 complex, rac1-specific GEF activity of sos1, rac1 activation, and oxidative stress are also mediated by the PPLP motif in the CH2 domain. This relationship between p66shc, grb2, and sos1 provides a novel mechanism for the activation of rac1.

P53 Impairs Endothelium-Dependent Vasomotor Function Through Transcriptional Upregulation of P66shc

The transcription factor, p53, and the adaptor protein, p66shc, both play essential roles in promoting oxidative stress in the vascular system. However, the relationship between the two in the context of endothelium-dependent vascular tone is unknown. Here, we report a novel, evolutionarily conserved, p53-mediated transcriptional mechanism that regulates p66shc expression and identify p53 as an important determinant of endothelium-dependent vasomotor function. We provide evidence of a p53 response element in the promoter of p66shc and show that angiotensin II-induced upregulation of p66shc in endothelial cells is dependent on p53. In addition, we demonstrate that downregulation of p66shc expression, as well as inhibition of p53 function in mice, mitigates angiotensin II-induced impairment of endothelium-dependent vasorelaxation, decrease in bioavailable nitric oxide, and hypertension. These findings reveal a novel p53-dependent transcriptional mechanism for the regulation of p66shc expression that is operative in the vascular endothelium and suggest that this mechanism is important in impairing endothelium-dependent vascular relaxation.

Relationship between p38 mitogen-activated protein kinase and small GTPase Rac for the activation of NADPH oxidase in bovine neutrophils

Superoxide production by NADPH oxidase is essential for bactericidal properties of neutrophils. However, molecular mechanisms underlying the activation of this enzyme remain largely unknown. Here, using bovine neutrophils we examined the role of p38 mitogen-activated protein kinase (p38 MAPK) in the signaling pathways of the NADPH oxidase activation. Superoxide production was induced by stimulation with serum-opsonized zymosan (OZ) and attenuated by p38 MAPK inhibitor, SB203580. OZ stimulation induced the translocation of p47(phox) and Rac to the plasma membrane and SB203580 completely blocked the translocation of Rac, but only partially blocked that of p47(phox). Furthermore, SB203580 abolished the OZ-elicited activation of Rac, which was assessed by detecting the GTP-bound form of this protein. Phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002, blocked not only p38 MAPK activation but also Rac activation. However, SB203580 showed no effect on the PI3K activity. These results suggested that PI3K/p38 MAPK/Rac pathway was present in the activation of NADPH oxidase in bovine neutrophils.

Roles of protein kinase C delta in the accumulation of P53 and the induction of apoptosis in H2O2-treated bovine endothelial cells.

To clarify the signaling pathways of oxidative stress-induced apoptosis in bovine aortic endothelial cells (BAEC), we treated cells with 1 mM H 2 O 2 and investigated the roles of protein kinase C i (PKC i ) and Ca 2+ in the accumulation of p53 associated with apoptosis. The treatment of cells with H 2 O 2 caused the accumulation of p53, which was inhibited by rottlerin (a PKC i inhibitor) but not by BAPTA-AM (an intracellular Ca 2+ chelator). PKC i itself was activated through the phosphorylation at tyrosine residues. H 2 O 2 induced the release of cytochrome c and the activation of caspases 3 and 9, and these apoptotic signals were inhibited by rottlerin and BAPTA-AM. These results suggest that PKC i contributes to the accumulation of p53 and that Ca 2+ plays a role in downstream signals of p53 leading to apoptosis in H 2 O 2 -treated BAEC.

Histone Deacetylase-3 Antagonizes Aspirin-Stimulated Endothelial Nitric Oxide Production by Reversing Aspirin-Induced Lysine Acetylation of Endothelial Nitric Oxide Synthase

Rationale: Low-dose acetylsalicylic acid (aspirin) is widely used in the treatment and prevention of vascular atherothrombosis. Cardiovascular doses of aspirin also reduce systemic blood pressure and improve endothelium-dependent vasorelaxation in patients with atherosclerosis or risk factors for atherosclerosis. Aspirin can acetylate proteins, other than its pharmacological target cyclooxygenase, at lysine residues. The role of lysine acetylation in mediating the effects of low-dose aspirin on the endothelium is not known. Objective: To determine the role of lysine acetylation of endothelial nitric oxide synthase (eNOS) in the regulation of endothelial NO production by low-dose aspirin and to examine whether the lysine deacetylase histone deacetylase (HDAC)3 antagonizes the effect of low-dose aspirin on endothelial NO production by reversing acetylation of functionally critical eNOS lysine residues. Methods and Results: Low concentrations of aspirin induce lysine acetylation of eNOS, stimulating eNOS enzymatic activity and endothelial NO production in a cyclooxygenase-1–independent fashion. Low-dose aspirin in vivo also increases bioavailable vascular NO in an eNOS-dependent and cyclooxygenase-1–independent manner. Low-dose aspirin promotes the binding of eNOS to calmodulin. Lysine 609 in the calmodulin autoinhibitory domain of bovine eNOS mediates aspirin-stimulated binding of eNOS to calmodulin and eNOS-derived NO production. HDAC3 inhibits aspirin-stimulated (1) lysine acetylation of eNOS, (2) eNOS enzymatic activity, (3) eNOS-derived NO, and (4) binding of eNOS to calmodulin. Conversely, downregulation of HDAC3 promotes lysine acetylation of eNOS and endothelial NO generation. Conclusions: Lysine acetylation of eNOS is a posttranslational protein modification supporting low-dose aspirin-induced vasoprotection. HDAC3, by deacetylating aspirin-acetylated eNOS, antagonizes aspirin-stimulated endothelial production of NO.