Chih-Chuan Liang

Endothelium-specific overexpression of class III deacetylase SIRT1 decreases atherosclerosis in apolipoprotein E-deficient mice

AIMS Hazardous environmental and genetic factors can damage endothelial cells to induce atherosclerotic vascular disease. Recent studies suggest that class III deacetylase SIRT1 may promote cell survival via novel antioxidative mechanisms. The current study tested the hypothesis that SIRT1, specifically overexpressed in the endothelium, is atheroprotective. METHODS AND RESULTS Human umbilical vein endothelial cells (HUVECs) were used to study the effects of oxidized low-density lipoprotein (LDL) on SIRT1 expression. Endothelial cell-specific SIRT1 transgenic (SIRT1-Tg) mice were used to study the effects of SIRT1 on aortic vascular tone. SIRT1-Tg mice were crossed with apolipoprotein E null (apoE(-/-)) mice to obtain SIRT1-Tg/apoE(-/-) mice for the analysis of atherogenesis in the presence of endothelial overexpression of SIRT1. SIRT1 expression in HUVECs was increased by the treatment with oxidative LDL. Adenoviral-mediated overexpression of SIRT1 was protective of apoptosis of HUVECs. Calorie restriction increased, whereas high-fat diet decreased, the SIRT1 expression in mouse aortas. In SIRT1-Tg mice, high fat-induced impairment in endothelium-dependent vasorelaxation was improved compared with that of wild-type littermates. This was accompanied by an upregualtion of aortic endothelial nitric oxide synthase expression in the SIRT1-Tg mice. The SIRT1-Tg/apoE(-/-) mice had less atherosclerotic lesions compared with apoE(-/-) controls, without affecting blood lipids and glucose levels. CONCLUSION These results suggest that endothelium-specific SIRT1 overexpression likely suppresses atherogenesis via improving endothelial cell survival and function.

Paraoxonase gene polymorphisms, oxidative stress, and diseases

The paraoxonase (PON) gene cluster contains at least three members, including PON1, PON2, and PON3, located on chromosome 7q21.3–22.1. Until now there has been little insight into the role of the respective gene products in human physiology and pathology. However, emerging evidence from biochemical and genetic experiments is providing clues about the role(s) of the products of these genes, which indicates that PON(s) acts as important guardians against cellular damage from toxic agents, such as organophosphates, oxidized lipids in the plasma low-density lipoproteins. In parallel, substantial data have been published on the association between the polymorphisms of PON(s) and coronary heart disease. It has become clear that the polymorphisms significantly affect the prevalence of coronary heart disease. However, the associations between the PON(s) polymorphisms and most of these conditions were found to be inconsistent when additional populations were investigated. This contribution provides an overview of the status of research of each of the three genes and the available association studies and the potential problems in interpreting the data. We also review the current evidence on the association between PON(s) polymorphisms and diseases other than coronary heart disease and some metabolic quantitative phenotypes, such as plasma lipoproteins, plasma glucose, and birthweight. Finally, we suggest directions for the future that might elucidate the role of the PON genetic polymorphisms in this potentially important function of PON(s) and the role in coronary heart disease and other related diseases.

Repression of P66Shc Expression by SIRT1 Contributes to the Prevention of Hyperglycemia-Induced Endothelial Dysfunction

Rationale: Inactivation of the p66Shc adaptor protein confers resistance to oxidative stress and protects mice from aging-associated vascular diseases. However, there is limited information about the negative regulating mechanisms of p66Shc expression in the vascular system. Objective: In this study, we investigated the role of SIRT1, a class III histone deacetylase, in the regulation of p66Shc expression and hyperglycemia-induced endothelial dysfunction. Methods and Results: Expressions of p66Shc gene transcript and protein were significantly increased by different kinds of class III histone deacetylase (sirtuin) inhibitors in human umbilical vein endothelial cells and 293A cells. Adenoviral overexpression of SIRT1 inhibited high-glucose–induced p66Shc upregulation in human umbilical vein endothelial cells. Knockdown of SIRT1 increased p66Shc expression and also increased the expression levels of plasminogen activator inhibitor-1 expression, but decreased manganese superoxide dismutase expression in high-glucose conditions. However, knockdown of p66Shc significantly reversed the effects of SIRT1 knockdown. In addition, p66Shc overexpression significantly decreased manganese superoxide dismutase expression and increased plasminogen activator inhibitor-1 expression in high-glucose conditions, which were recovered by SIRT1 overexpression. Moreover, compared to streptozotocin-induced wild-type diabetic mice, endothelium-specific SIRT1 transgenic diabetic mice had decreased p66Shc expression at both the mRNA and the protein levels, improved endothelial function, and reduced accumulation of nitrotyrosine and 8-OHdG (markers of oxidative stress). We further found that SIRT1 was able to bind to the p66Shc promoter (−508 bp to −250 bp), resulting in a decrease in the acetylation of histone H3 bound to the p66Shc promoter region. Conclusion: Our findings indicate that repression of p66Shc expression by SIRT1 contributes to the protection of hyperglycemia-induced endothelial dysfunction.

SIRT1 Suppresses Activator Protein-1 Transcriptional Activity and Cyclooxygenase-2 Expression in Macrophages

SIRT1 (Sirtuin type 1), a mammalian orthologue of yeast SIR2 (silent information regulator 2), has been shown to mediate a variety of calorie restriction (CR)-induced physiological events, such as cell fate regulation via deacetylation of the substrate proteins. However, whether SIRT1 deacetylates activator protein-1 (AP-1) to influence its transcriptional activity and target gene expression is still unknown. Here we demonstrate that SIRT1 directly interacts with the basic leucine zipper domains of c-Fos and c-Jun, the major components of AP-1, by which SIRT1 suppressed the transcriptional activity of AP-1. This process requires the deacetylase activity of SIRT1. Notably, SIRT1 reduced the expression of COX-2, a typical AP-1 target gene, and decreased prostaglandin E2 (PGE2) production of peritoneal macrophages (pMΦs). pMΦs with SIRT1 overexpression displayed improved phagocytosis and tumoricidal functions, which are associated with depressed PGE2. Furthermore, SIRT1 protein level was up-regulated in CR mouse pMΦs, whereas elevated SIRT1 decreased COX-2 expression and improved PGE2-related macrophage functions that were reversed following inhibition of SIRT1 deacetylase activity. Thus, our results indicate that SIRT1 may be a mediator of CR-induced macrophage regulation, and its deacetylase activity contributes to the inhibition of AP-1 transcriptional activity and COX-2 expression leading to amelioration of macrophage function.

Active Chromatin Hub of the Mouse α-Globin Locus Forms in a Transcription Factory of Clustered Housekeeping Genes

ABSTRACT RNA polymerases can be shared by a particular group of genes in a transcription “factory” in nuclei, where transcription may be coordinated in concert with the distribution of coexpressed genes in higher-eukaryote genomes. Moreover, gene expression can be modulated by regulatory elements working over a long distance. Here, we compared the conformation of a 130-kb chromatin region containing the mouse α-globin cluster and their flanking housekeeping genes in 14.5-day-postcoitum fetal liver and brain cells. The analysis of chromatin conformation showed that the active α1 and α2 globin genes and upstream regulatory elements are in close spatial proximity, indicating that looping may function in the transcriptional regulation of the mouse α-globin cluster. In fetal liver cells, the active α1 and α2 genes, but not the inactive ζ gene, colocalize with neighboring housekeeping genes C16orf33, C16orf8, MPG, and C16orf35. This is in sharp contrast with the mouse α-globin genes in nonexpressing cells, which are separated from the congregated housekeeping genes. A comparison of RNA polymerase II (Pol II) occupancies showed that active α1 and α2 gene promoters have a much higher RNA Pol II enrichment in liver than in brain. The RNA Pol II occupancy at the ζ gene promoter, which is specifically repressed during development, is much lower than that at the α1 and α2 promoters. Thus, the mouse α-globin gene cluster may be regulated through moving in or out active globin gene promoters and regulatory elements of a preexisting transcription factory in the nucleus, which is maintained by the flanking clustered housekeeping genes, to activate or inactivate α-globin gene expression.

SIRT1 Acts as a Modulator of Neointima Formation Following Vascular Injury in Mice

Rationale: Vascular smooth muscle cell (VSMC) proliferation and migration are crucial events involved in the pathophysiology of vascular diseases. Sirtuin 1 (SIRT1), a class III histone deacetylase (HDAC), has been reported to have the function of antiatherosclerosis, but its role in neointima formation remains unknown. Objective: The present study was designed to investigate the role of SIRT1 in the regulation of neointima formation and to elucidate the underlying mechanisms. Methods and Results: A decrease in SIRT1 expression was observed following carotid artery ligation. smooth muscle cell (SMC)–specific human SIRT1 transgenic (Tg) mice were generated. SIRT1 overexpression substantially inhibited neointima formation after carotid artery ligation or carotid artery wire injury. In the intima of injured carotid arteries, VSMC proliferation (proliferating cell nuclear antigen (PCNA)–positive cells) was significantly reduced. SIRT1 overexpression markedly inhibited VSMC proliferation and migration and induced cell cycle arrest at G1/S transition in vitro. Accordingly, SIRT1 overexpression decreased the induction of cyclin D1 and matrix metalloproteinase-9 (MMP-9) expression by treatment with serum and TNF-&agr;, respectively, whereas RNAi knockdown of SIRT1 resulted in the opposite effect. Decreased cyclin D1 and MMP-9 expression/activity were also observed in injured carotid arteries from SMC-SIRT1 Tg mice. Furthermore, 2 targets of SIRT1, c-Fos and c-Jun, were involved in the downregulation of cyclin D1 and MMP-9 expression. Conclusions: Our findings demonstrate the inhibitory effect of SIRT1 on the VSMC proliferation and migration that underlie neointima formation and implicate SIRT1 as a potential target for intervention in vascular diseases.

Sirt1 deacetylates c-Myc and promotes c-Myc/Max association

The c-Myc oncoprotein plays critical roles in multiple biological processes by controlling cell proliferation, apoptosis, differentiation, and metabolism. Especially, c-Myc is frequently overexpressed in many human cancers and widely involved in tumorigenesis. However, how the post-translational modifications, especially acetylation of c-Myc, contribute to its activity in the leukemia cells remains largely unknown. Sirt1, a NAD-dependent class III histone deacetylase, has a paradoxical role in tumorigenesis by deacetylating several transcription factors, including p53, E2F1 and forkhead proteins. In this study, we show that Sirt1 interacts physically with the C-terminus of c-Myc and deacetylates c-Myc both in vitro and in vivo. Moreover, the deacetylation of c-Myc by Sirt1 promotes its association with Max, a partner essential for its activation, thereby facilitating c-Myc transactivation activity on hTERT promoter. Finally, inhibition of endogenous Sirt1 in K562 cells by either RNAi or its inhibitor NAM causes the overall decrease of c-Myc target genes expression, including hTERT, cyclinD2 and LDHA, which further suppress cell proliferation and arrest cell cycle at G1/S phase. Thus, our results demonstrate the positive effect of Sirt1 on c-Myc activity by efficiently enhancing c-Myc/Max association in human leukemia cell line K562, suggesting a potential role of Sirt1 in tumorigenesis.

SIRT1 inhibits angiotensin II-induced vascular smooth muscle cell hypertrophy

Angiotensin II (Ang II) stimulates vascular smooth muscle cell (VSMC) hypertrophy as a critical event in the development of vascular diseases such as atherosclerosis. Sirtuin (SIRT) 1, a nicotinamide adenine dinucleotide dependent deacetylase, has been demonstrated to exert protective effects in atherosclerosis by promoting endothelium-dependent vascular relaxation and reducing macrophage foam cell formation, but its role in VSMC hypertrophy remains unknown. In this study, we tried to investigate the effect of SIRT1 on Ang II-induced VSMC hypertrophy. Results showed that adenoviral-mediated over-expression of SIRT1 significantly inhibited Ang II-induced VSMC hypertrophy, while knockdown of SIRT1 by RNAi resulted in an increased [(3)H]-leucine incorporation of VSMC. Accordingly, nicotinamide adenine dinucleotide phosphate oxidase 1 (Nox1) expression induced by Ang II was inhibited by SIRT1 in VSMCs. SIRT1 activator resveratrol decreased, whereas endogenous SIRT1 inhibitor nicotinamide increased Nox1 expression in A7r5 VSMCs. Furthermore, transcription factor GATA-6 was involved in the down-regulation of Nox1 expression by SIRT1. These results provide new insight into SIRT1s anti-atherogenic properties by suppressing Ang II-induced VSMC hypertrophy.

Modulations of hMOF autoacetylation by SIRT1 regulate hMOF recruitment and activities on the chromatin

A wide variety of nuclear regulators and enzymes are subjected to acetylation of the lysine residue, which regulates different aspects of protein functions. The MYST family histone acetyltransferase, human ortholog of MOF (hMOF), plays critical roles in transcription activation by acetylating nucleosomal H4K16. In this study, we found that hMOF acetylates itself in vitro and in vivo, and the acetylation is restricted to the conserved MYST domain (C2HC zinc finger and HAT), of which the K274 residue is the major autoacetylation site. Furthermore, the class III histone deacetylase SIRT1 was found to interact with the MYST domain of hMOF through the deacetylase catalytic region and deacetylate autoacetylated hMOF. In vitro binding assays showed that non-acetylated hMOF robustly binds to nucleosomes while acetylation decreases the binding ability. In HeLa cells, the recruitment of hMOF to the chromatin increases in response to SIRT1 overexpression and decreases after knockdown of SIRT1. The acetylation mimic mutation K274Q apparently decreases the chromatin recruitment of hMOF as well as the global H4K16Ac level in HeLa cells. Finally, upon SIRT1 knockdown, hMOF recruitment to the gene body region of its target gene HoxA9 decreases, accompanied with decrease of H4K16Ac at the same region and repression of HoxA9 transcription. These results suggest a dynamic interplay between SIRT1 and hMOF in regulating H4K16 acetylation.

Involvement of the p65/RelA subunit of NF-κB in TNF-α-induced SIRT1 expression in vascular smooth muscle cells

The proinflammatory cytokine TNF-alpha plays an important role in stimulating inflammatory responses of vascular smooth muscle cells (VSMCs). The anti-inflammatory function of Sirtuin 1 (SIRT1), a NAD-dependent class III histone/protein deacetylase, has been well documented, but how SIRT1 is regulated under inflammatory conditions is largely unknown. In the present research, we showed that levels of SIRT1 mRNA and protein expression increased in TNF-alpha-treated VSMCs. Overexpression of the p65/RelA subunit of NF-kappaB, a TNF-alpha-activated inflammatory transcription factor, in A7r5 cells, upregulated SIRT1 mRNA and protein expression as well as SIRT1 promoter activity, while knockdown of endogenous p65/RelA expression by RNAi not only led to a decrease in SIRT1s basal protein expression and promoter activity, but almost abolished the TNF-alpha-induced elevation of SIRT1 protein expression and SIRT1 promoter activity. Furthermore, using promoter deletion analysis and chromatin immunoprecipitation assays, we found that p65/RelA bound to the SIRT1 promoter at a consensus NF-kappaB binding site. Our study indicates that p65/RelA mediates the TNF-alpha-induced elevated expression of SIRT1 in VSMCs, shedding new light on the regulation of SIRT1 under inflammatory conditions.