Tsutomu Sasaki

Phosphorylation Regulates SIRT1 Function

Background SIR2 is an NAD+-dependent deacetylase [1]–[3] implicated in the regulation of lifespan in species as diverse as yeast [4], worms [5], and flies [6]. We previously reported that the level of SIRT1, the mammalian homologue of SIR2 [7], [8], is coupled to the level of mitotic activity in cells both in vitro and in vivo [9]. Cells from long-lived mice maintained SIRT1 levels of young mice in tissues that undergo continuous cell replacement by proliferating stem cells. Changes in SIRT1 protein level were not associated with changes in mRNA level, suggesting that SIRT1 could be regulated post-transcriptionally. However, other than a recent report on sumoylation [10] and identification of SIRT1 as a nuclear phospho-protein by mass spectrometry [11], post-translational modifications of this important protein have not been reported. Methodology/Principal Findings We identified 13 residues in SIRT1 that are phosphorylated in vivo using mass spectrometry. Dephosphorylation by phosphatases in vitro resulted in decreased NAD+-dependent deacetylase activity. We identified cyclinB/Cdk1 as a cell cycle-dependent kinase that forms a complex with and phosphorylates SIRT1. Mutation of two residues phosphorylated by Cyclin B/Cdk1 (threonine 530 and serine 540) disturbs normal cell cycle progression and fails to rescue proliferation defects in SIRT1-deficient cells [12], [13]. Conclusions/Significance Pharmacological manipulation of SIRT1 activity is currently being tested as a means of extending lifespan in mammals. Treatment of obese mice with resveratrol, a pharmacological activator of SIRT1, modestly but significantly improved longevity and, perhaps more importantly, offered some protection against the development of type 2 diabetes mellitus and metabolic syndrome [14]–[16]. Understanding the endogenous mechanisms that regulate the level and activity of SIRT1, therefore, has obvious relevance to human health and disease. Our results identify phosphorylation by cell cycle dependent kinases as a major mechanism controlling the level and function of this sirtuin and complement recent reports of factors that inhibit [17], [18] and activate [19] SIRT1 by protein-protein interactions.

Effects of aging and hypertension on endothelium-dependent vascular relaxation in rat carotid artery.

We evaluated the effects of aging and hypertension on endothelium-dependent relaxation of rat common carotid arteries using 14-week-old (young) and 11-month-old (old) Wistar-Kyoto rats (WKY) and age-matched spontaneously hypertensive rats (SHR). Isometric tension of common carotid artery ring segments was measured. With a resting tension of 2.0 g determined from the baseline tension-contraction curves, precontraction was induced by 10(-5) M 5-hydroxytryptamine and endothelium-dependent relaxation was measured by application of either acetylcholine or adenosine 5-triphosphate (ATP). Mean arterial blood pressure was 73.1 +/- 3.0 mm Hg in WKY and 110.0 +/- 3.1 mm Hg in SHR. These baseline values were significantly different. Acetylcholine-induced maximal relaxations were 70.1 +/- 2.6% of the 5-hydroxytryptamine-induced contraction in young WKY, 45.6 +/- 2.1% in old WKY, 35.1 +/- 1.8% in young SHR, and 21.4 +/- 2.5% in old SHR. On the other hand, ATP-induced relaxations were 52.0 +/- 3.2%, 35.7 +/- 3.8%, 21.7 +/- 3.5%, and 17.0 +/- 1.8% in the groups, respectively. Acetylcholine-induced relaxations were significantly different between WKY and SHR, young and old, independently. On the other hand, ATP-induced relaxations were also significantly different between young and old WKY, although no significant difference was observed between young and old SHR. The fact that endothelium-dependent relaxation of a cephalic artery is impaired in old rats and in hypertensive rats suggests that aging and hypertension are risk factors that may augment the disturbance of the cerebral circulation in pathologic conditions.

Effect of carbon dioxide and oxygen on endothelin production by cultured porcine cerebral endothelial cells.

We have previously reported the production of endothelin, a potent vasoconstrictor peptide, by porcine cerebral microvessel endothelia and suggested its important role in the regulation of local blood flow within the brain. In our present study, radioimmunoassay with anti-porcine endothelin antiserum revealed that endothelin, produced by cerebral microvessel endothelia grown on a filter, is released mainly to the basement membrane side, not the vascular lumen side. This finding suggests that endothelin constricts arterioles locally where it is produced by endothelia. We also found that cerebral microvessel endothelia produce less endothelin under low oxygen pressure and more endothelin under low carbon dioxide pressure. Our results suggest that endothelin has a role in the regulation of cerebral blood flow in response to oxygen and carbon dioxide pressure.

Progressive loss of SIRT1 with cell cycle withdrawal

Sir2 is an NAD+‐dependent deacetylase that regulates lifespan in yeast, worms and flies. The mammalian orthologs of Sir2 include SIRT1 in humans and mice. In this study, we analyzed the level of SIRT1 in human lung fibroblasts (IMR90) and mouse embryonic fibroblasts (MEFs) from mice with normal, accelerated, and delayed aging. SIRT1 protein, but not mRNA, decreased significantly with serial cell passage in both human and murine cells. Mouse SIRT1 decreased rapidly in prematurely senescent (p44 Tg) MEFs, remained high in MEFs with delayed senescence (Igf‐1r–/–), and was inversely correlated with senescence‐activated β‐galactosidase (SA‐βGal) activity. Reacquisition of mitotic capability following spontaneous immortalization of serially passaged wild‐type MEFs restored the level of SIRT1 to that of early passage, highly proliferative MEFs. In mouse and human fibroblasts, we found a significant positive correlation between the levels of SIRT1 and proliferating cell nuclear antigen (PCNA), a DNA processing factor expressed during S‐phase. In the animal, we found that SIRT1 decreased with age in tissues in which mitotic activity also declines, such as the thymus and testis, but not in tissues such as the brain in which there is little change in mitotic activity throughout life. Again, the decreases in SIRT1 were highly correlated with decreases in PCNA. Finally, loss of SIRT1 with age was accelerated in mice with accelerated aging but was not observed in long‐lived growth hormone‐receptor knockout mice. Thus, as mitotic activity ceases in mouse and human cells in the normal environment of the animal or in the culture dish, there is a concomitant decline in the level of SIRT1.

Osteogenic potential of five different recombinant human bone morphogenetic protein adenoviral vectors in the rat.

Bone morphogenetic protein (BMP) adenoviral vectors for the induction of osteogenesis are being developed for the treatment of bone pathology. However, it is still unknown which BMP adenoviral vector has the highest potential to stimulate bone formation in vivo. In this study, the osteogenic activities of recombinant human BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 adenoviruses were compared in vitro, in athymic nude rats, and in Sprague–Dawley rats. In vitro osteogenic activity was assessed by measuring the alkaline phosphatase activity in C2C12 cells transduced by the various BMP vectors. The alkaline phosphatase activity induced by 2 × 105u2009PFU/well of BMP viral vector was 4890 × 10−12u2009U/well for ADCMVBMP-9, 302 × 10−12u2009U/well for ADCMVBMP-4, 220 × 10−12u2009U/well for ADCMVBMP-6, 45 × 10−12u2009U/well for ADCMVBMP-2, and 0.43 × 10−12u2009U/well for ADCMVBMP-7. The average volume of new bone induced by 107u2009PFU of BMP vector in athymic nude rats was 0.37±0.03u2009cm3 for ADCMVBMP-2, 0.89±0.07u2009cm3 for ADCMVBMP-4, 1.02±0.07u2009cm3 for ADCMVBMP-6, 0.24±0.05u2009cm3 for ADCMVBMP-7, and 0.63±0.07u2009cm3 for ADCMVBMP-9. In immunocompetent Sprague–Dawley rats, no bone formation was demonstrated in the ADCMVBMP-2, ADCMVBMP-4, and ADCMVBMP-7 groups. ADCMVBMP-6 at a viral dose of 108u2009PFU induced 0.10±0.03u2009cm3 of new bone, whereas ADCMVBMP-9 at a lower viral dose of 107u2009PFU induced more bone, with an average volume of 0.29±0.01u2009cm3.

Expression of survivin, an inhibitor of apoptosis protein, in tumors of the nervous system

Abstract. Survivin is an inhibitor of apoptosis protein that blocks apoptosis by binding to caspases-3 and -7. It is highly expressed in less-differentiated embryonic cells and rapidly dividing tumors, but not in terminally differentiated adult tissues. Elevated survivin levels are found in malignant systemic tumors, and are associated with chemo-resistance, radiation resistance, and poor prognosis. However, expression of survivin in primary nervous system tumors has not been previously characterized. Immunohistochemistry using anti-human survivin antibody (SURV11-A) was performed on formalin-fixed, paraffin-embedded archival tissue from 112 primary central nervous system tumors. Survivin immunoreactivity was seen in most diffuse astrocytomas [WHO II (2/4), III (3/3), IV (9/10), giant-cell glioblastoma (1), and gliosarcoma (1)]. The intensity and degree of survivin expression showed trends with tumor grade, with glioblastomas having the highest positivity. Pilocytic astrocytomas (5) and pleomorphic xanthoastrocytoma (1) were positive to a lesser degree. In oligodendrogliomas (6) and mixed oligo-astrocytomas [grade II (5), II–III (3), and III (7)], oligodendroglial elements appear to be negative compared to positive mini-gemistocytic oligodendrocytes. Ependymomas [grade II (6) and grade III (1)] were positive. Medulloblastomas (5) and retinoblastoma (1/4) showed focal positivity. All meningiomas [grade I (12), II (9), III (4), and grade I (3) and II (5) with frank brain invasion] were intensely positive. All schwannomas (11) and neurofibromas (6) were intensely positive. Thus, survivin is expressed in the majority of the primary nervous system tumors, particularly in glioblastomas, meningiomas, schwannomas and neurofibromas. Overexpression of survivin in meningiomas and benign peripheral nerve sheath tumors contrasts with previous reports relating it to rapid division and poor prognosis.

Indomethacin ameliorates ischemic neuronal damage in the gerbil hippocampal CA1 sector.

The purpose of our experiment was to examine whether the cyclooxygenase inhibitor indomethacin ameliorates neuronal injury in the gerbil hippocampal CA1 sector following 5 minutes of forebrain ischemia. Thirty minutes before bilateral carotid artery occlusion, Mongolian gerbils were injected intraperitoneally with 1 (n = 10), 2 (n = 10), 5 (n = 12), or 10 (n = 7) mg/kg of indomethacin. Seven days after occlusion, the gerbils were perfusion-fixed and neuronal density in the hippocampal CA1 sector was assessed. The mean +/- SEM neuronal density in nine unoperated normal gerbils was 307 +/- 9/mm, in 10 untreated ischemic gerbils 55 +/- 21/mm, and in seven vehicle-treated ischemic gerbils 15 +/- 9/mm. The mean +/- SEM neuronal density in ischemic gerbils treated with 1, 2, 5, or 10 mg/kg indomethacin was 132 +/- 28/mm, 154 +/- 29/mm, 176 +/- 30/mm, and 136 +/- 39/mm, respectively. Indomethacin at any dose significantly ameliorated ischemic neuronal damage in the gerbil hippocampal CA1 sector.

Effect of cyclooxygenase and lipoxygenase inhibitors on delayed neuronal death in the gerbil hippocampus.

The purpose of our study was to examine whether cyclooxygenase and lipoxygenase inhibitors ameliorate delayed neuronal death in the hippocampal CA1 sector in Mongolian gerbils after 5 minutes of forebrain ischemia. Gerbils were injected intraperitoneally with cyclooxygenase inhibitors piroxicam and flurbiprofen or with lipoxygenase inhibitors AA-861 and BW-755C. Seven days after ischemic insult, the animals were perfusion-fixed, and the neuronal density in the hippocampal CA1 sector was estimated. The average neuronal density in unoperated normal gerbils was 247 +/- 9/mm (mean +/- SEM). In ischemic gerbils with vehicle administration, the average neuronal densities were 13 +/- 2, 14 +/- 2, 13 +/- 2, and 13 +/- 1 for piroxicam, flurbiprofen, AA-861, and BW-755C, respectively. The average neuronal densities in ischemic gerbils treated with 1.5 and 10 mg/kg piroxicam and 1.5 and 10 mg/kg flurbiprofen were 13 +/- 2, 194 +/- 9, 19 +/- 5, and 143 +/- 12, respectively. In ischemic gerbils treated with 15 and 100 mg/kg AA-861 and 30 mg/kg BW-755C, the average neuronal densities were 12 +/- 1, 13 +/- 1, and 14 +/- 2, respectively. At their higher doses, both piroxicam and flurbiprofen significantly (p less than 0.01) ameliorated delayed neuronal death in the hippocampal CA1 sector. Our results suggest that cyclooxygenase products play an important role in the development of delayed neuronal injury after cerebral ischemia.

Induction of Hypothalamic Sirt1 Leads to Cessation of Feeding via Agouti-Related Peptide

Silent information regulator (SIR)2 is an nicotinamide adenine dinucleotide dependent deacetylase implicated in the regulation of life span in species as diverse as yeast, worms, and flies. Mammalian Sirt1 is the most closely related homolog of the SIR2 gene. Pharmacological activators of Sirt1 have been reported to increase the life span and improve the health of mice fed a high-fat diet and to reverse diabetes in rodents. Sirt1 links the energy availability status with cellular metabolism in peripheral organs including liver, pancreas, muscle, and white adipose tissue. Insulin and leptin signaling regulate food intake by controlling the expression of orexigenic and anorexigenic neuropeptides in the arcuate nucleus of the hypothalamus via Forkhead box O (Foxo)-1 and signal transducer and activator of transcription-3. Sirt1 has been reported to improve insulin sensitivity in vitro, but the role of hypothalamic Sirt1 in regulating feeding has not been addressed. We found that hypothalamic Sirt1 protein levels increase on feeding, and this induction is abrogated in diet-induced obese mice and db/db mice. We also demonstrate for the first time that Sirt1 protein turnover is regulated by the proteasome and ubiquitination in a hypothalamic cell line and in vivo by feeding, and this regulation is not seen in a pituitary cell line AtT20. Forced expression of wild-type Sirt1 in the mediobasal hypothalamus by adenovirus microinjection suppressed Foxo1-induced hyperphagia, a model for central insulin resistance. Moreover, Sirt1 suppressed Foxo1-dependent expression of the orexigenic neuropeptide Agouti-related peptide in vitro. We propose that on feeding, Sirt1 protein is stabilized in the hypothalamus, leading to decreased Foxo1-dependent expression of orexigenic neuropeptide Agouti-related peptide and cessation of feeding.

Hippocampal unit activity after transient cerebral ischemia in rats.

Single unit activity of CA1 and CA3 neurons in the hippocampus was recorded in rats 1, 2, or 3 days after 10 minutes of transient cerebral ischemia induced by the clamping of both carotid arteries combined with hypotension. In addition, paired pulse inhibition/facilitation of the CA1 population spike was examined on Day 2 using two successive stimuli of the contralateral CA3 region delivered at various intervals. On Day 1, the mean +/- SEM firing rate in the CA1 region was 0.91 +/- 0.42/sec (n = 5), which was not significantly different from the control value of 0.98 +/- 0.26/sec (n = 5). Firing rate increased on Days 2 and 3 to 3.96 +/- 0.69/sec (n = 5), and 6.49 +/- 0.89/sec (n = 5), respectively. In the CA3 region, the mean +/- SEM firing rate of 1.18 +/- 0.27/sec in the five control rats sharply dropped to 0.14 +/- 0.11/sec in the five Day 1 rats and gradually increased to 0.45 +/- 0.11/sec in the five Day 3 rats. Histologic examination of these rats revealed ischemic changes restricted to CA1 neurons on Days 2 and 3. The paired-pulse experiment showed no significant difference between six control and six Day 2 rats in the inhibition of the second population spike with interstimulus intervals of less than 400 msec. At interstimulus intervals of greater than 500 msec there was facilitation of the second spike, which lasted 5 seconds in Day 2 rats. This facilitation was not observed in control rats. Because CA3 neurons constitute the main input to CA1 pyramidal cells, decreased activity of CA3 neurons indicates less excitatory input to CA1 neurons.(ABSTRACT TRUNCATED AT 250 WORDS)