Jing Xu

BACKGROUND: Precise genome editing via homology-directed repair (HDR) after double-stranded DNA (dsDNA) cleavage facilitates functional genomic research and holds promise for gene therapy. However, HDR efficiency remains low in some cell types, including some of great research and clinical interest, such as human induced pluripotent stem cells (iPSCs). RESULTS: Here, we show that a double cut HDR donor, which is flanked by single guide RNA (sgRNA)-PAM sequences and is released after CRISPR/Cas9 cleavage, increases HDR efficiency by twofold to fivefold relative to circular plasmid donors at one genomic locus in 293 T cells and two distinct genomic loci in iPSCs. We find that a 600 bp homology in both arms leads to high-level genome knockin, with 97-100% of the donor insertion events being mediated by HDR. The combined use of CCND1, a cyclin that functions in G1/S transition, and nocodazole, a G2/M phase synchronizer, doubles HDR efficiency to up to 30% in iPSCs. CONCLUSIONS: Taken together, these findings provide guidance for the design of HDR donor vectors and the selection of HDR-enhancing factors for applications in genome research and precision medicine.

Characterization of tumor metabolism with spatial information contributes to our understanding of complex cancer metabolic reprogramming, facilitating the discovery of potential metabolic vulnerabilities that might be targeted for tumor therapy. However, given the metabolic variability and flexibility of tumors, it is still challenging to characterize global metabolic alterations in heterogeneous cancer. Here, we propose a spatially resolved metabolomics approach to discover tumor-associated metabolites and metabolic enzymes directly in their native state. A variety of metabolites localized in different metabolic pathways were mapped by airflow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) in tissues from 256 esophageal cancer patients. In combination with in situ metabolomics analysis, this method provided clues into tumor-associated metabolic pathways, including proline biosynthesis, glutamine metabolism, uridine metabolism, histidine metabolism, fatty acid biosynthesis, and polyamine biosynthesis. Six abnormally expressed metabolic enzymes that are closely associated with the altered metabolic pathways were further discovered in esophageal squamous cell carcinoma (ESCC). Notably, pyrroline-5-carboxylate reductase 2 (PYCR2) and uridine phosphorylase 1 (UPase1) were found to be altered in ESCC. The spatially resolved metabolomics reveal what occurs in cancer at the molecular level, from metabolites to enzymes, and thus provide insights into the understanding of cancer metabolic reprogramming.

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. 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. IntroductionTranscription factor activator protein-1 (AP-1) 3The abbreviations used are: AP-1activator protein-1CRcalorie restrictionbZIPbasic leucine zipperCOX-2cyclooxygenase-2RNAiRNA interferencePGprostaglandinpMΦperitoneal macrophagesPMAphorbol 12-myrisatate 13-acetateALad libitumRSVresveratrolHAhemagglutininGSTglutathione S-transferaseGFPgreen fluorescent proteinIPimmunoprecipitationDMSOdimethyl sulfoxideFITCfluorescein isothiocyanate. represents homodimers or heterodimers of the Jun and Fos families. c-Fos and c-Jun are the subunits predominantly expressed in mammalian cells. In response to growth factors, cytokines, oxidative stress, or pharmacological stimuli, such as phorbol 12-myrisatate 13-acetate (PMA), AP-1 binds to the promoters of target genes to modulate their expression, which is in turn involved in cell proliferation, differentiation, and inflammation (1.Eferl R. Wagner E.F. Nat. Rev. Cancer. 2003; 3: 859-868Crossref PubMed Scopus (1600) Google Scholar). Fos and Jun belong to the basic leucine zipper (bZIP) family, which preferentially binds to 12-O-tetradecanoylphorbol-13-acetate response element sites and to the cAMP response element with slightly lower affinity (2.Angel P. Imagawa M. Chiu R. Stein B. Imbra R.J. Rahmsdorf H.J. Jonat C. Herrlich P. Karin M. Cell. 1987; 49: 729-739Abstract Full Text PDF PubMed Scopus (2149) Google Scholar). The basic fragment of bZIP is responsible for sequence-specific DNA binding, and the leucine zipper is critical for the dimerization of ZIP proteins. In addition to direct binding to cis-acting elements, AP-1 could also interact with other transcriptional regulators, participating in transcriptional regulation, and the bZIP domain contributes to the association with co-activator (3.Miotto B. Struhl K. Mol. Cell Biol. 2006; 26: 5969-5982Crossref PubMed Scopus (36) Google Scholar) or co-repressor (4.Lee S.K. Kim J.H. Lee Y.C. Cheong J. Lee J.W. J. Biol. Chem. 2000; 275: 12470-12474Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The transcriptional activity of AP-1 is regulated by the post-translational modification, including acetylation (5.Vries R.G. Prudenziati M. Zwartjes C. Verlaan M. Kalkhoven E. Zantema A. EMBO J. 2001; 20: 6095-6103Crossref PubMed Scopus (72) Google Scholar). The acetylation modification of transcriptional factors changes the protein-protein or protein-DNA interaction as an important regulatory mechanism of transcription (6.Luo J. Li M. Tang Y. Laszkowska M. Roeder R.G. Gu W. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2259-2264Crossref PubMed Scopus (334) Google Scholar).Cyclooxygenase-2 (COX-2), one of the classic AP-1 targets, is the rate-limiting enzyme for prostaglandin (PG) production. Macrophage-derived PGs regulate functions of both macrophage itself and the neighboring cells as well. Excessive PGE2 inhibits the phagocytosis activity and the bacterial killing function of macrophages (7.Serezani C.H. Chung J. Ballinger M.N. Moore B.B. Aronoff D.M. Peters-Golden M. Am. J. Respir. Cell Mol. Biol. 2007; 37: 562-570Crossref PubMed Scopus (127) Google Scholar, 8.Aronoff D.M. Canetti C. Peters-Golden M. J. Immunol. 2004; 173: 559-565Crossref PubMed Scopus (281) Google Scholar). Increased PGE2 compromises the tumoricidal activity of macrophages and natural killer cells (9.Schultz R.M. Pavlidis N.A. Stylos W.A. Chirigos M.A. Science. 1978; 202: 320-321Crossref PubMed Scopus (156) Google Scholar, 10.Yakar I. Melamed R. Shakhar G. Shakhar K. Rosenne E. Abudarham N. Page G.G. Ben-Eliyahu S. Ann. Surg. Oncol. 2003; 10: 469-479Crossref PubMed Scopus (101) Google Scholar, 11.Yamada H. Kuroda E. Matsumoto S. Matsumoto T. Yamada T. Yamashita U. Clin. Exp. Immunol. 2002; 128: 52-58Crossref PubMed Scopus (21) Google Scholar). The expression and activity of COX-2 are induced by various stimuli such as PMA, inflammatory factors (lipopolysaccharide), growth factors, and cytokines. COX-2 expression is increased in cells from aged animals, whereas calorie restriction (CR), a regimen extending the life span of organisms and maintaining many physiological processes in a youthful state, suppresses the age-related COX-2 increase (12.Kim Y.J. Kim H.J. No J.K. Chung H.Y. Fernandes G. Life Sci. 2006; 78: 2523-2532Crossref PubMed Scopus (36) Google Scholar).The Sir2 gene family regulates transcriptional silencing and extends the lifespan of Saccharomyces cerevisiae and Caenorhabditis elegans. SIRT1, the mammalian orthologue of Sir2, is a NAD+-dependent deacetylase of numerous substrates. SIRT1 has been shown to mediate a variety of physiological events such as cell fate regulation, increased metabolic rate, and higher oxygen consumption by deacetylation of the substrate targets. For example, SIRT1 deacetylates p53 protein at lysine 382 and represses its transcriptional function to protect cells from apoptosis (13.Guarente L. Picard F. Cell. 2005; 120: 473-482Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). Deacetylation of FOXO3 and FOXO4 by SIRT1 switches cells from apoptosis to cell cycle arrest under stress (14.Motta M.C. Divecha N. Lemieux M. Kamel C. Chen D. Gu W. Bultsma Y. McBurney M. Guarente L. Cell. 2004; 116: 551-563Abstract Full Text Full Text PDF PubMed Scopus (1184) Google Scholar, 15.Brunet A. Sweeney L.B. Sturgill J.F. Chua K.F. Greer P.L. Lin Y. Tran H. Ross S.E. Mostoslavsky R. Cohen H.Y. Hu L.S. Cheng H.L. Jedrychowski M.P. Gygi S.P. Sinclair D.A. Alt F.W. Greenberg M.E. Science. 2004; 303: 2011-2015Crossref PubMed Scopus (2597) Google Scholar). SIRT1 deacetylates liver X-activated receptors and positively regulates its activity, promoting cholesterol efflux from cells (16.Li X. Zhang S. Blander G. Tse J.G. Krieger M. Guarente L. Mol. Cell. 2007; 28: 91-106Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). In macrophage cell line MonoMac6, SIRT1 has been reported to inhibit proinflammatory mediator release including interleukin-8 and tumor necrosis factor-α through deacetylating RelA/p65 subunit and inhibiting NF-κB (17.Yang S.R. Wright J. Bauter M. Seweryniak K. Kode A. Rahman I. Am. J. Physiol. Lung Cell Mol. Physiol. 2007; 292: L567-L576Crossref PubMed Scopus (337) Google Scholar).CR extends the life span of organisms such as yeast, nematodes, fruit flies, and mice (13.Guarente L. Picard F. Cell. 2005; 120: 473-482Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). In mammals, CR up-regulates expression of SIRT1 in a variety of tissues including kidney, liver, and fat, and SIRT1 transgenic mice display some phenotypes similar to mice on a calorie-restricted diet (18.Cohen H.Y. Miller C. Bitterman K.J. Wall N.R. Hekking B. Kessler B. Howitz K.T. Gorospe M. de Cabo R. Sinclair D.A. Science. 2004; 305: 390-392Crossref PubMed Scopus (1657) Google Scholar, 19.Bordone L. Cohen D. Robinson A. Motta M.C. van Veen E. Czopik A. Steele A.D. Crowe H. Marmor S. Luo J. Gu W. Guarente L. Aging Cell. 2007; 6: 759-767Crossref PubMed Scopus (584) Google Scholar). SIRT1 is implicated in CR-induced physiological events such as cell survival and senescence effects by regulating its substrates of SIRT1, such as p53 and FOXOs (20.Rogina B. Helfand S.L. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 15998-16003Crossref PubMed Scopus (1054) Google Scholar). CR has also been reported to influence macrophage functions (21.Stapleton P.P. Fujita J. Murphy E.M. Naama H.A. Daly J.M. Nutrition. 2001; 17: 41-45Crossref PubMed Scopus (27) Google Scholar, 22.Sun D. Muthukumar A.R. Lawrence R.A. Fernandes G. Clin. Diagn. Lab. Immunol. 2001; 8: 1003-1011Crossref PubMed Scopus (87) Google Scholar), delay the age-related chronic inflammatory diseases, and decrease AP-1 activity (23.Castello L. Froio T. Cavallini G. Biasi F. Sapino A. Leonarduzzi G. Bergamini E. Poli G. Chiarpotto E. FASEB J. 2005; 19: 1863-1865Crossref PubMed Scopus (48) Google Scholar) and COX-2 expression (12.Kim Y.J. Kim H.J. No J.K. Chung H.Y. Fernandes G. Life Sci. 2006; 78: 2523-2532Crossref PubMed Scopus (36) Google Scholar). Based on these observations, we hypothesized that SIRT1 may decrease AP-1 activity and COX-2 expression to modulate macrophage function and that up-regulation of SIRT1 in macrophages may mimic the beneficial effect of CR on macrophages. In this report, we identified the direct interaction between SIRT1 and the bZIP domain of c-Fos/c-Jun and found that the deacetylase activity of SIRT1 is required for repression of AP-1 transcriptional activity. Furthermore, SIRT1 inhibited COX-2 expression and PGE2 production in macrophages, resulting in improved macrophage phagocytosis and tumoricidal functions. Finally, our results demonstrated that SIRT1 expression is increased in macrophages from CR mice, leading to decreased COX-2 expression and PGE2 production, and improves PGE2-related macrophage functions.DISCUSSIONSIRT1 is a NAD+-dependent histone deacetylase that catalyzes the removal of acetyl groups from a number of histone or non-histone targets, belonging to class III deacetylase (30.Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1290) Google Scholar, 31.Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Crossref PubMed Scopus (2737) Google Scholar). The non-histone substrates of SIRT1 include a variety of important transcription factors and some transcription co-factors, such as p53, FOXOs, and PGC-1α (13.Guarente L. Picard F. Cell. 2005; 120: 473-482Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 32.Gasser 2001; PubMed Scopus Google Scholar). The effects of target deacetylation include changes in cell and The functions of SIRT1 are to be by its deacetylase activity Gu W. Nat. Rev. Cancer. PubMed Scopus Google Scholar). In the we found that SIRT1 decreased c-Fos/c-Jun acetylation induced by and inhibited the transcriptional activity of AP-1. which the effect of SIRT1 J.M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, K.T. Bitterman K.J. Cohen H.Y. S. J.G. Chung P. A. Zhang B. Sinclair D.A. Nature. 2003; PubMed Scopus Google Scholar), decreased AP-1 transcriptional activity with SIRT1. J.M. J. 2005; PubMed Scopus Google Scholar) the effect of SIRT1 on AP-1 Furthermore, we found that SIRT1 inhibited AP-1 DNA binding to the COX-2 in with the that acetylation modification is for the DNA binding activity of transcriptional factors (6.Luo J. Li M. Tang Y. Laszkowska M. Roeder R.G. Gu W. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2259-2264Crossref PubMed Scopus (334) Google Scholar). In our also identified the of SIRT1, the and the The effects on AP-1 transcriptional activity of both domains were similar to SIRT1. The of SIRT1 itself could AP-1, AP-1 activity, that the domain is for the effects on AP-1. these results indicate that SIRT1 deacetylase activity important in AP-1 transcriptional AP-1, the bZIP domain is for transcriptional regulation is responsible for dimerization and DNA binding also for the interaction between the bZIP domain and other transcriptional factors or shown that the basic of c-Fos and c-Jun is involved in the interaction with transcriptional factors, including and T. Mol. Cell. Biol. PubMed Google Scholar). The bZIP domain has also been shown to interact with protein association S.K. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) and the factor (3.Miotto B. Struhl K. Mol. Cell Biol. 2006; 26: 5969-5982Crossref PubMed Scopus (36) Google Scholar). groups reported binding between c-Jun and SIRT1 S. P. R. R. S.K. Chen Y. PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar). In line with these and to we found that SIRT1 directly with the bZIP domain of both c-Fos and c-Jun, resulting in a in the acetylation of c-Fos and c-Jun has been reported to be by at lysine the basic of bZIP domain (5.Vries R.G. Prudenziati M. Zwartjes C. Verlaan M. Kalkhoven E. Zantema A. EMBO J. 2001; 20: 6095-6103Crossref PubMed Scopus (72) Google Scholar), which to the between the acetylation of the basic and transcriptional of the lysine the basic of c-Jun AP-1 transcriptional activity in our that the of SIRT1, which the deacetylase activity, was to the activity of may be the of SIRT1 could of by direct interaction A. M. H. P. L. D. Nature. 2007; PubMed Scopus Google Scholar). SIRT1 a mechanism to protect survival C. S.R. 3: PubMed Scopus (156) Google Scholar). are to whether the binding of SIRT1 to bZIP domain of c-Fos and c-Jun in AP-1 transcriptional regulation in a AP-1 DNA binding in mouse J. E. D. W. A. 2001; PubMed Scopus Google Scholar) and the AP-1 (23.Castello L. Froio T. Cavallini G. Biasi F. Sapino A. Leonarduzzi G. Bergamini E. Poli G. Chiarpotto E. FASEB J. 2005; 19: 1863-1865Crossref PubMed Scopus (48) Google Scholar, H.J. K.J. Chung 2002; Scopus Google Scholar). the beneficial effects of mammalian CR with increased SIRT1 expression (18.Cohen H.Y. Miller C. Bitterman K.J. Wall N.R. Hekking B. Kessler B. Howitz K.T. Gorospe M. de Cabo R. Sinclair D.A. Science. 2004; 305: 390-392Crossref PubMed Scopus (1657) Google Scholar, F. M. Chung N. A. T. R. M. McBurney Guarente L. Nature. 2004; PubMed Scopus Google Scholar). results that overexpression of SIRT1 suppressed AP-1 activity, which to AP-1 inhibition by Furthermore, CR has also been shown to decrease COX-2 expression (12.Kim Y.J. Kim H.J. No J.K. Chung H.Y. Fernandes G. Life Sci. 2006; 78: 2523-2532Crossref PubMed Scopus (36) Google Scholar) and PGE2 R. S. C. M. C. J. Biol. Sci. Sci. 2006; PubMed Scopus Google Scholar). Excessive PGE2 the of tumor cells H. Kuroda E. Matsumoto S. Matsumoto T. Yamada T. Yamashita U. Clin. Exp. Immunol. 2002; 128: 52-58Crossref PubMed Scopus (21) Google Scholar) and the phagocytosis activity of macrophages (7.Serezani C.H. Chung J. Ballinger M.N. Moore B.B. Aronoff D.M. Peters-Golden M. Am. J. Respir. Cell Mol. Biol. 2007; 37: 562-570Crossref PubMed Scopus (127) Google Scholar, 8.Aronoff D.M. Canetti C. Peters-Golden M. J. Immunol. 2004; 173: 559-565Crossref PubMed Scopus (281) Google Scholar). In the macrophages with SIRT1 overexpression displayed similar effects on COX-2 expression, PGE2 production, and macrophage functions, with CR mice macrophages higher SIRT1 was CR has been reported to and chronic inflammatory diseases, such as and (23.Castello L. Froio T. Cavallini G. Biasi F. Sapino A. Leonarduzzi G. Bergamini E. Poli G. Chiarpotto E. FASEB J. 2005; 19: 1863-1865Crossref PubMed Scopus (48) Google Scholar, K. T. Google Scholar, D. R. E. A. 2003; Google Scholar, R. Nat. 2000; PubMed Scopus Google Scholar), the up-regulated SIRT1 in macrophages may a mechanism CR is or of SIRT1 by AP-1 functions with promoting AP-1 transcriptional activity and COX-2 also the CR-induced of PGE2 production and of macrophage functions, which a for deacetylase activity in the of macrophage SIRT1 interacts with the bZIP domain of c-Fos/c-Jun and inhibits AP-1 transcriptional activity. The deacetylase activity of SIRT1 contributes to the decreased AP-1 transcriptional activity, reduced COX-2 expression, decreased PGE2 production, and improved PGE2-related macrophage phagocytosis and tumoricidal functions. Furthermore, SIRT1 may mediate CR-induced amelioration of macrophage functions through its deacetylase activity. that SIRT1 activity in macrophages may be an to inflammatory IntroductionTranscription factor activator protein-1 (AP-1) 3The abbreviations used are: AP-1activator protein-1CRcalorie restrictionbZIPbasic leucine zipperCOX-2cyclooxygenase-2RNAiRNA interferencePGprostaglandinpMΦperitoneal macrophagesPMAphorbol 12-myrisatate 13-acetateALad libitumRSVresveratrolHAhemagglutininGSTglutathione S-transferaseGFPgreen fluorescent proteinIPimmunoprecipitationDMSOdimethyl sulfoxideFITCfluorescein isothiocyanate. represents homodimers or heterodimers of the Jun and Fos families. c-Fos and c-Jun are the subunits predominantly expressed in mammalian cells. In response to growth factors, cytokines, oxidative stress, or pharmacological stimuli, such as phorbol 12-myrisatate 13-acetate (PMA), AP-1 binds to the promoters of target genes to modulate their expression, which is in turn involved in cell proliferation, differentiation, and inflammation (1.Eferl R. Wagner E.F. Nat. Rev. Cancer. 2003; 3: 859-868Crossref PubMed Scopus (1600) Google Scholar). Fos and Jun belong to the basic leucine zipper (bZIP) family, which preferentially binds to 12-O-tetradecanoylphorbol-13-acetate response element sites and to the cAMP response element with slightly lower affinity (2.Angel P. Imagawa M. Chiu R. Stein B. Imbra R.J. Rahmsdorf H.J. Jonat C. Herrlich P. Karin M. Cell. 1987; 49: 729-739Abstract Full Text PDF PubMed Scopus (2149) Google Scholar). The basic fragment of bZIP is responsible for sequence-specific DNA binding, and the leucine zipper is critical for the dimerization of ZIP proteins. In addition to direct binding to cis-acting elements, AP-1 could also interact with other transcriptional regulators, participating in transcriptional regulation, and the bZIP domain contributes to the association with co-activator (3.Miotto B. Struhl K. Mol. Cell Biol. 2006; 26: 5969-5982Crossref PubMed Scopus (36) Google Scholar) or co-repressor (4.Lee S.K. Kim J.H. Lee Y.C. Cheong J. Lee J.W. J. Biol. Chem. 2000; 275: 12470-12474Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The transcriptional activity of AP-1 is regulated by the post-translational modification, including acetylation (5.Vries R.G. Prudenziati M. Zwartjes C. Verlaan M. Kalkhoven E. Zantema A. EMBO J. 2001; 20: 6095-6103Crossref PubMed Scopus (72) Google Scholar). The acetylation modification of transcriptional factors changes the protein-protein or protein-DNA interaction as an important regulatory mechanism of transcription (6.Luo J. Li M. Tang Y. Laszkowska M. Roeder R.G. Gu W. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2259-2264Crossref PubMed Scopus (334) Google Scholar).Cyclooxygenase-2 (COX-2), one of the classic AP-1 targets, is the rate-limiting enzyme for prostaglandin (PG) production. Macrophage-derived PGs regulate functions of both macrophage itself and the neighboring cells as well. Excessive PGE2 inhibits the phagocytosis activity and the bacterial killing function of macrophages (7.Serezani C.H. Chung J. Ballinger M.N. Moore B.B. Aronoff D.M. Peters-Golden M. Am. J. Respir. Cell Mol. Biol. 2007; 37: 562-570Crossref PubMed Scopus (127) Google Scholar, 8.Aronoff D.M. Canetti C. Peters-Golden M. J. Immunol. 2004; 173: 559-565Crossref PubMed Scopus (281) Google Scholar). Increased PGE2 compromises the tumoricidal activity of macrophages and natural killer cells (9.Schultz R.M. Pavlidis N.A. Stylos W.A. Chirigos M.A. Science. 1978; 202: 320-321Crossref PubMed Scopus (156) Google Scholar, 10.Yakar I. Melamed R. Shakhar G. Shakhar K. Rosenne E. Abudarham N. Page G.G. Ben-Eliyahu S. Ann. Surg. Oncol. 2003; 10: 469-479Crossref PubMed Scopus (101) Google Scholar, 11.Yamada H. Kuroda E. Matsumoto S. Matsumoto T. Yamada T. Yamashita U. Clin. Exp. Immunol. 2002; 128: 52-58Crossref PubMed Scopus (21) Google Scholar). The expression and activity of COX-2 are induced by various stimuli such as PMA, inflammatory factors (lipopolysaccharide), growth factors, and cytokines. COX-2 expression is increased in cells from aged animals, whereas calorie restriction (CR), a regimen extending the life span of organisms and maintaining many physiological processes in a youthful state, suppresses the age-related COX-2 increase (12.Kim Y.J. Kim H.J. No J.K. Chung H.Y. Fernandes G. Life Sci. 2006; 78: 2523-2532Crossref PubMed Scopus (36) Google Scholar).The Sir2 gene family regulates transcriptional silencing and extends the lifespan of Saccharomyces cerevisiae and Caenorhabditis elegans. SIRT1, the mammalian orthologue of Sir2, is a NAD+-dependent deacetylase of numerous substrates. SIRT1 has been shown to mediate a variety of physiological events such as cell fate regulation, increased metabolic rate, and higher oxygen consumption by deacetylation of the substrate targets. For example, SIRT1 deacetylates p53 protein at lysine 382 and represses its transcriptional function to protect cells from apoptosis (13.Guarente L. Picard F. Cell. 2005; 120: 473-482Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). Deacetylation of FOXO3 and FOXO4 by SIRT1 switches cells from apoptosis to cell cycle arrest under stress (14.Motta M.C. Divecha N. Lemieux M. Kamel C. Chen D. Gu W. Bultsma Y. McBurney M. Guarente L. Cell. 2004; 116: 551-563Abstract Full Text Full Text PDF PubMed Scopus (1184) Google Scholar, 15.Brunet A. Sweeney L.B. Sturgill J.F. Chua K.F. Greer P.L. Lin Y. Tran H. Ross S.E. Mostoslavsky R. Cohen H.Y. Hu L.S. Cheng H.L. Jedrychowski M.P. Gygi S.P. Sinclair D.A. Alt F.W. Greenberg M.E. Science. 2004; 303: 2011-2015Crossref PubMed Scopus (2597) Google Scholar). SIRT1 deacetylates liver X-activated receptors and positively regulates its activity, promoting cholesterol efflux from cells (16.Li X. Zhang S. Blander G. Tse J.G. Krieger M. Guarente L. Mol. Cell. 2007; 28: 91-106Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). In macrophage cell line MonoMac6, SIRT1 has been reported to inhibit proinflammatory mediator release including interleukin-8 and tumor necrosis factor-α through deacetylating RelA/p65 subunit and inhibiting NF-κB (17.Yang S.R. Wright J. Bauter M. Seweryniak K. Kode A. Rahman I. Am. J. Physiol. Lung Cell Mol. Physiol. 2007; 292: L567-L576Crossref PubMed Scopus (337) Google Scholar).CR extends the life span of organisms such as yeast, nematodes, fruit flies, and mice (13.Guarente L. Picard F. Cell. 2005; 120: 473-482Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). In mammals, CR up-regulates expression of SIRT1 in a variety of tissues including kidney, liver, and fat, and SIRT1 transgenic mice display some phenotypes similar to mice on a calorie-restricted diet (18.Cohen H.Y. Miller C. Bitterman K.J. Wall N.R. Hekking B. Kessler B. Howitz K.T. Gorospe M. de Cabo R. Sinclair D.A. Science. 2004; 305: 390-392Crossref PubMed Scopus (1657) Google Scholar, 19.Bordone L. Cohen D. Robinson A. Motta M.C. van Veen E. Czopik A. Steele A.D. Crowe H. Marmor S. Luo J. Gu W. Guarente L. Aging Cell. 2007; 6: 759-767Crossref PubMed Scopus (584) Google Scholar). SIRT1 is implicated in CR-induced physiological events such as cell survival and senescence effects by regulating its substrates of SIRT1, such as p53 and FOXOs (20.Rogina B. Helfand S.L. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 15998-16003Crossref PubMed Scopus (1054) Google Scholar). CR has also been reported to influence macrophage functions (21.Stapleton P.P. Fujita J. Murphy E.M. Naama H.A. Daly J.M. Nutrition. 2001; 17: 41-45Crossref PubMed Scopus (27) Google Scholar, 22.Sun D. Muthukumar A.R. Lawrence R.A. Fernandes G. Clin. Diagn. Lab. Immunol. 2001; 8: 1003-1011Crossref PubMed Scopus (87) Google Scholar), delay the age-related chronic inflammatory diseases, and decrease AP-1 activity (23.Castello L. Froio T. Cavallini G. Biasi F. Sapino A. Leonarduzzi G. Bergamini E. Poli G. Chiarpotto E. FASEB J. 2005; 19: 1863-1865Crossref PubMed Scopus (48) Google Scholar) and COX-2 expression (12.Kim Y.J. Kim H.J. No J.K. Chung H.Y. Fernandes G. Life Sci. 2006; 78: 2523-2532Crossref PubMed Scopus (36) Google Scholar). Based on these observations, we hypothesized that SIRT1 may decrease AP-1 activity and COX-2 expression to modulate macrophage function and that up-regulation of SIRT1 in macrophages may mimic the beneficial effect of CR on macrophages. In this report, we identified the direct interaction between SIRT1 and the bZIP domain of c-Fos/c-Jun and found that the deacetylase activity of SIRT1 is required for repression of AP-1 transcriptional activity. Furthermore, SIRT1 inhibited COX-2 expression and PGE2 production in macrophages, resulting in improved macrophage phagocytosis and tumoricidal functions. Finally, our results demonstrated that SIRT1 expression is increased in macrophages from CR mice, leading to decreased COX-2 expression and PGE2 production, and improves PGE2-related macrophage functions.

Background The value of native myocardial T1 mapping and extracellular volume (ECV) fraction in patients who have hypertrophic cardiomyopathy (HCM) but no late gadolinium enhancement (LGE) and no hemodynamic obstruction are currently unknown. Purpose To evaluate myocardial fibrosis in patients with nonobstructive HCM and no LGE by using native myocardial T1 mapping and ECV fraction and to study their relationships to left ventricular (LV) function and LV hypertrophy. Materials and Methods Patients with HCM who underwent cardiac MRI between 2012 and 2015 were retrospectively evaluated. Patients were included if they had no LGE at MRI, LV ejection fraction greater than or equal to 45%, and no LV outflow tract obstruction. Healthy participants had similar age and sex distribution. Native myocardial T1 and ECV were measured with MRI. Results A total of 258 patients with HCM (mean age ± standard deviation, 49 years ± 15; 74% men) and 122 healthy participants (mean age, 50 years ± 14; 76% men) were evaluated. Native myocardial T1 was longer and ECV fraction was higher in the patients with HCM relative to the healthy participants (mean native T1, 950 msec ± 48 vs 913 msec ± 46; mean ECV, 24.5% ± 2.8 vs 23.0% ± 2.7; both P < .001). Maximum T1 and ECV values correlated strongly with LV mass index for the entire patient cohort with HCM (both r = 0.86; P < .001) and for the subgroups (r = 0.86 and 0.85 for interventricular septal group and r = 0.88 and 0.86 for apical group; all P < .001). Conclusion Prolonged myocardial T1 and elevated extracellular volume in hypertrophic cardiomyopathy suggests diffuse myocardial fibrosis, even in the absence of regionally apparent late gadolinium enhancement and hemodynamic obstruction, and is associated with left ventricular hypertrophy. © RSNA, 2019 See also the editorial by Bluemke and Lima in this issue.