Sir2 (silent information regulator 2) is an NAD ؉ -dependent histone deacetylase that contributes to longevity in yeast. SIRT1, a mammalian Sir2 ortholog, deacetylates histones and various transcription factors, including p53, FOXO proteins, and peroxisome proliferator-activated receptor-␥. We found that its subcellular localization varied in different tissues of the adult mouse. Some subsets of neurons predominantly expressed SIRT1 in the cytoplasm, but ependymal cells expressed it in both the nucleus and cytoplasm. On the other hand, spermatocytes expressed SIRT1 only in the nucleus. Cardiomyocytes in the day 12.5 mouse embryo expressed SIRT1 exclusively in the nucleus, but in the adult heart, they expressed it in both the cytoplasm and nucleus. C2C12 myoblast cells expressed SIRT1 in the nucleus, but it localized to the cytoplasm after differentiation. LY294002, an inhibitor of phosphoinositide 3-hydroxykinase, strongly inhibited the nuclear localization of SIRT1 in undifferentiated C2C12 cells. In a heterokaryon assay, SIRT1 shuttled between the nucleus and cytoplasm, and leptomycin B, an inhibitor of CRM1-mediated nuclear exportation, inhibited this shuttling. Two nuclear localization signals and two nuclear export signals were identified by deletion and site-directed mutation analyses. Overexpressed nuclear (but not cytoplasmic or dominant-negative) SIRT1 enhanced the deacetylation of histone H3 in C2C12 cells. Moreover, only the nuclear form suppressed the apoptosis of C2C12 cells induced by antimycin A, an oxidative stressor. These findings indicate that nucleocytoplasmic shuttling is a novel regulatory mechanism of SIRT1, which may participate in differentiation and in inhibition of cell death.The Sir2 (silent information regulator 2) proteins are a family of class III histone deacetylases found in organisms from bacteria to humans (1). Unlike class I and II histone deacetylases, the Sir2 family requires the cofactor NAD ϩ for catalytic activity (2). In yeast, Sir2 participates in heterochromatic silencing at mating-type loci (3). Sir2 extends the life span of yeast by suppressing recombination in the rDNA region and consequently reducing the formation of extrachromosomal rDNA circles (4), a cause of senescence (5). Caloric restriction extends the life span in organisms ranging from yeast to mammals, and the Sir2 family plays an essential role in this effect (6, 7).In mammals, there are seven members of the Sir2 family, termed sirtuins, of which SIRT1 is the closest homolog of yeast Sir2. SIRT1 regulates metabolic responses in adipocytes and liver. It promotes fat mobilization in white adipocytes by repressing peroxisome proliferator-activated receptor-␥ (8). SIRT1 deacetylates the transcription coactivator PGC1␣, thereby inducing the expression of gluconeogenic genes and the repression of glycolytic genes (9). SIRT1 contributes to cell survival by deacetylating and thereby repressing the activity of the tumor suppressor p53 (10 -12). The transcriptional activation of stress-resistance genes by FOXO pro...

Sir2 is an NAD-dependent histone deacetylase that functions in longevity, gene silencing, heterochromatin formation, DNA repair, and suppression of DNA recombination in yeast. The mammalian homolog Sir2K K (SIRT1) has been shown to inhibit p53-dependent apoptosis, but its physiological roles are still not known. We found that the level of Sir2K K expression during embryogenesis was high. The highest Sir2K K mRNA expression was detected as early as embryonic day (E) 4.5. Although the level was down-regulated during embryogenesis, a high level of expression was still found in the late embryonic stage (E18.5). In embryos, Sir2K K was expressed at high levels in the heart, brain, spinal cord, and dorsal root ganglia. The expression levels in these organs were high on E10.5^E13.5 and low on E16.5. Quantitative reverse transcription polymerase chain reaction showed a 60% reduction in Sir2K K mRNA content in the heart between E12.5 and E14.5. After E14.5, the expression level in the heart remained constant up to 27 months of age. The expression pattern of Sir2K K protein in embryonic hearts was consistent with that of mRNA. These results suggest new roles of Sir2K K not only in early embryogenesis but also in cardiogenesis and neurogenesis with a stage-speci¢c manner. ß

The aim of the present study was to examine the hypothesis that acceleration of gap junction (GJ) closure during ischemia contributes to anti-infarct tolerance afforded by preconditioning (PC). First, the effects of PC on GJ communication during ischemia were assessed. Isolated buffer-perfused rabbit hearts were subjected to 5-min global ischemia with or without PC with two cycles of 5-min ischemia/5-min reperfusion or a GJ blocker (2 mM heptanol), and then the tissue excised from the ischemic region was incubated in anoxic buffer containing lucifer yellow (LY; 2.5 mg/ml), a tracer of GJ permeability, for 20 min at 37 degrees C. PC and heptanol significantly reduced the area to which LY was transported in the ischemic myocardium by 39% and by 54%, respectively. In the second series of experiments, three GJ blockers (heptanol, 18beta-glycyrrhetinic acid, and 2,3-butanedione monoxime) infused after the onset of ischemia reduced infarct size after 30-min ischemia/2-h reperfusion to an extent equivalent to that in the case of PC. In the third series of experiments, Western blotting for connexin43 (Cx43) showed that PC shortened the time to the onset of ischemia-induced Cx43 dephosphorylation but reduced the extent of Cx43 dephosphorylation during a 30-min period of ischemia. Calphostin C, a protein kinase C (PKC) inhibitor, abolished preservation of phosphorylated Cx43 but not the early onset of Cx43 dephosphorylation after ischemia in the preconditioned myocardium. These results suggest that PC-induced reduction of GJ permeability during ischemia, presumably by PKC-mediated Cx43 phosphorylation, contributes to infarct size limitation.

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. Erythropoietin affords additional cardioprotection to preconditioned hearts by enhanced phosphorylation of glycogen synthase kinase-3␤. Am J Physiol Heart Circ Physiol 291: H748 -H755, 2006. First published March 24, 2006 doi:10.1152/ajpheart.00837.2005.-The aim of this study was to determine whether erythropoietin (EPO) affords additional cardioprotection to the preconditioned myocardium by enhanced phosphorylation of Akt, STAT3, or glycogen synthase kinase-3␤ (GSK-3␤). Preconditioning (PC) with 5-min ischemia/5-min reperfusion and EPO (5,000 U/kg iv) reduced infarct size (as % of area at risk, %IS/AR) after 20-min ischemia in rat hearts in situ from 56.5 Ϯ 1.8% to 25.2 Ϯ 2.1% and to 36.2 Ϯ 2.8%, respectively. PC-induced protection was significantly inhibited by a protein kinase C inhibitor, chelerythrine (5 mg/kg), and slightly blunted by a phosphatidylinositol-3-kinase inhibitor, wortmannin (15 g/kg). The opposite pattern of inhibition was observed for EPO-induced protection. The combination of PC and EPO further reduced %IS/AR to 8.9 Ϯ 1.9%, and this protection was inhibited by chelerythrine and wortmannin. The additive effects of PC and EPO on infarct size were mirrored by their effects on the level of phosphorylated GSK-3␤ at 5 min after reperfusion but not their effects on the level of phospho-Akt or phospho-STAT3. To mimic phosphorylation-induced inhibition of GSK-3␤ activity, SB-216763 (SB), a GSK-3␤ inhibitor, was administered before ischemia or 5 min before reperfusion. Infarct size was significantly reduced by preischemic injection (%IS/AR ϭ 40.4 Ϯ 2.2% by 0.6 mg/kg SB and 34.0 Ϯ 1.8% by 1.2 mg/kg SB) and also by prereperfusion injection (%IS/AR ϭ 32.0 Ϯ 2.0% by 1.2 mg/kg SB). These results suggest that EPO and PC afford additive infarct size-limiting effects by additive phosphorylation of GSK-3␤ at the time of reperfusion by Akt-dependent and -independent mechanisms. infarct size; phosphatidylinositol-3-kinase; protein kinase C; Akt HUMAN RECOMBINANT ERYTHROPOIETIN (EPO) has been used for decades as a standard therapy for renal anemia. However, recent studies have shown that EPO has potent cell-protective effects in various tissues, including myocardium, in addition to its effect on erythropoiesis (5-7, 12, 16, 25, 26, 33-35, 43). The presence of the EPO receptor in cardiomyocytes has been indicated in , and administration of EPO before or at the time of ischemia has been shown to reduce necrosis, apoptosis, and ventricular dysfunction after ischemia-reperfusion (6,7,12,16,25,(33)(34)(35)43). These features of EPO-induced cardioprotection are similar to those of ischemic preconditioning (PC). Furthermore, it is interesting to note that there are overlaps in signaling pathways provoked by activated EPO receptors and PC: phosphatidylinositol-3-kinase (PI3K)-Akt pathway, ERK pathway, and STAT-mediated pathway (1, 3, 13, 16 -18, 21, 30, 34 -39, 43). However, it is not clear which signaling molecule primarily determines the level of cardioprotection against infarction. It is also unclear whether sign...