Search

Supplementary Figure 2 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Materials and Methods, Figure Legends 1-7, Table 1 Legend from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Figure 3 from Functional and Clinical Evidence for NDRG2 as a Candidate Suppressor of Liver Cancer Metastasis

Supplementary Figure 5 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Figure 3 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Table 1 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Figure 1 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Figure 4 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Figure 6 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Figure 7 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Figure 1 from Functional and Clinical Evidence for NDRG2 as a Candidate Suppressor of Liver Cancer Metastasis

We searched for potential suppressors of tumor metastasis by identifying the genes that are frequently down-regulated in hepatocellular carcinomas (HCC) while being negatively correlated with clinical parameters relevant to tumor metastasis, and we report here on the identification of N-myc downstream regulated gene 2 (NDRG2) as a promising candidate. NDRG2 expression was significantly reduced in HCC compared with nontumor or normal liver tissues [87.5% (35 of 40) and 62% (62 of 100) at RNA and protein levels, respectively]. Reduction of NDRG2 expression was intimately associated with promoter hypermethylation because its promoter region was found to carry extensively methylated CpG sites in HCC cell lines and primary tumors. Immunohistochemical analysis of NDRG2 protein in 100 HCC patient tissues indicated that NDRG2 expression loss is significantly correlated with aggressive tumor behaviors such as late tumor-node-metastasis (TNM) stage (P = 0.012), differentiation grade (P = 0.024), portal vein thrombi (P = 0.011), infiltrative growth pattern (P = 0.015), nodal/distant metastasis (P = 0.027), and recurrent tumor (P = 0.021), as well as shorter patient survival rates. Ectopically expressed NDRG2 suppressed invasion and migration of a highly invasive cell line, SK-Hep-1, and experimental tumor metastasis in vivo, whereas small interfering RNA–mediated knockdown resulted in increased invasion and migration of a weakly invasive cell line, PLC/PRF/5. In addition, NDRG2 could antagonize transforming growth factor β1–mediated tumor cell invasion by specifically down-regulating the expression of matrix metalloproteinase 2 and laminin 332 pathway components, with concomitant suppression of Rho GTPase activity. These results suggest that NDRG2 can inhibit extracellular matrix–based, Rho-driven tumor cell invasion and migration and thereby play important roles in suppressing tumor metastasis in HCC. [Cancer Res 2008;68(11):4210–20]

The N-acetyltransferase arrest defective 1 (ARD1) is an important regulator of cell growth and differentiation that has emerged recently as a critical molecule in cancer progression. However, the regulation of the enzymatic and biological activities of human ARD1 (hARD1) in cancer is presently poorly understood. Here, we report that hARD1 undergoes autoacetylation and that this modification is essential for its functional activation. Using liquid chromatography-tandem mass spectrometry and site-directed mutational analyses, we identified K136 residue as an autoacetylation target site. K136R mutation abolished the ability of hARD1 to promote cancer cell growth in vitro and tumor xenograft growth in vivo. Mechanistic investigations revealed that hARD1 autoacetylation stimulated cyclin D1 expression through activation of the transcription factors β-catenin and activator protein-1. Our results show that hARD1 autoacetylation is critical for its activation and its ability to stimulate cancer cell proliferation and tumorigenesis. Cancer Res; 70(11); 4422–32. ©2010 AACR.

Supplementary Methods and Materials, Tables 1-4, Figure Legends 1-3 from Functional and Clinical Evidence for NDRG2 as a Candidate Suppressor of Liver Cancer Metastasis

Background: Macrophages produce many inflammation-associated molecules, released by matrix metalloproteinases, such as adhesion molecules, and cytokines, as well, which play a crucial role in atherosclerosis. In this context, we investigated the relationship between Ninjurin-1 (Ninj1 [nerve injury-induced protein]), a novel matrix metalloproteinase 9 substrate, expression, and atherosclerosis progression. Methods: Ninj1 expression and atherosclerosis progression were assessed in atherosclerotic aortic tissue and serum samples from patients with coronary artery disease and healthy controls, and atheroprone apolipoprotein e–deficient ( Apoe −/− ) and wild-type mice, as well. Apoe −/− mice lacking systemic Ninj1 expression ( Ninj1 −/− Apoe −/− ) were generated to assess the functional effects of Ninj1. Bone marrow transplantation was also used to generate low-density lipoprotein receptor–deficient ( Ldlr −/− ) mice that lack Ninj1 specifically in bone marrow–derived cells. Mice were fed a Western diet for 5 to 23 weeks, and atherosclerotic lesions were investigated. The anti-inflammatory role of Ninj1 was verified by treating macrophages and mice with the peptides Ninj1 1 –56 (ML56) and Ninj1 26 –37 (PN12), which mimic the soluble form of Ninj1 (sNinj1). Results: Our in vivo results conclusively showed a correlation between Ninj1 expression in aortic macrophages and the extent of human and mouse atherosclerotic lesions. Ninj1 -deficient macrophages promoted proinflammatory gene expression by activating mitogen-activated protein kinase and inhibiting the phosphoinositide 3-kinase/Akt signaling pathway. Whole-body and bone marrow–specific Ninj1 deficiencies significantly increased monocyte recruitment and macrophage accumulation in atherosclerotic lesions through elevated macrophage-mediated inflammation. Macrophage Ninj1 was directly cleaved by matrix metalloproteinase 9 to generate a soluble form that exhibited antiatherosclerotic effects, as assessed in vitro and in vivo. Treatment with the sNinj1-mimetic peptides, ML56 and PN12, reduced proinflammatory gene expression in human and mouse classically activated macrophages, thereby attenuating monocyte transendothelial migration. Moreover, continuous administration of mPN12 alleviated atherosclerosis by inhibiting the enhanced monocyte recruitment and inflammation characteristics of this disorder in mice, regardless of the presence of Ninj1. Conclusions: Ninj1 is a novel matrix metalloproteinase 9 substrate in macrophages, and sNinj1 is a secreted atheroprotective protein that regulates macrophage inflammation and monocyte recruitment in atherosclerosis. Moreover, sNinj1-mediated anti-inflammatory effects are conserved in human macrophages and likely contribute to human atherosclerosis.

Summary: CD137, a potent costimulatory receptor for CD8+ T cells, is expressed in various non-T cells, but little is known about its regulatory functions in these cells. In this study, we show that CD137 signaling, specifically in intestinal CD11b−CD103+ dendritic cells (DCs), restricts acute colitis progression. Mechanistically, CD137 engagement activates TAK1 and subsequently stimulates the AMPK-PGC-1α axis to enhance expression of the Aldh1a2 gene encoding the retinoic acid (RA) metabolizing enzyme RALDH2. RA can act on CD11b+CD103− DCs and induce SOCS3 expression, which, in turn, suppresses p38MAPK activation and interleukin-23 (IL-23) production. Administration of RA in DC-specific CD137−/− mice represses IL-23-producing CD11b+CD103− DCs and TH17 cells, indicating that RA is a major inhibitory effector molecule against intestinal CD11b+CD103− DCs. Additionally, the therapeutic effect of the anti-CD137 antibody is abrogated in DC-specific CD137−/− mice. Taken together, our results define a mechanism of paracrine immunoregulation operating between adjacent DC subsets in the intestine. : Jin et al. demonstrate that CD137 signaling functions as an immune checkpoint by controlling the survival and function of regulatory intestinal CD11b−CD103+ dendritic cells to coordinate the balance between regulatory T cells and pathogenic IL-23 during acute colitis. Keywords: CD137, regulatory CD11b−CD103+ DC, Foxp3+ Treg, acute colitis, retinoic acid, RALDH2, IL-23, TH17, immune checkpoint, immune tolerance

Topoisomerase IIIβ (Top3β), the only dual-activity topoisomerase in mammals that can change topology of both DNA and RNA, is known to be associated with neurodevelopment and mental dysfunction in humans. However, there is no report showing clear associations of Top3β with neuropsychiatric phenotypes in mice. Here, we investigated the effect of Top3β on neuro-behavior using newly generated Top3β deficient (Top3β−/−) mice. We found that Top3β−/− mice showed decreased anxiety and depression-like behaviors. The lack of Top3β was also associated with changes in circadian rhythm. In addition, a clear expression of Top3β was demonstrated in the central nervous system of mice. Positron emission tomography/computed tomography (PET/CT) analysis revealed significantly altered connectivity between many brain regions in Top3β−/− mice, including the connectivity between the olfactory bulb and the cerebellum, the connectivity between the amygdala and the olfactory bulb, and the connectivity between the globus pallidus and the optic nerve. These connectivity alterations in brain regions are known to be linked to neurodevelopmental as well as psychiatric and behavioral disorders in humans. Therefore, we conclude that Top3β is essential for normal brain function and behavior in mice and that Top3β could be an interesting target to study neuropsychiatric disorders in humans.

Amino-terminal acetylation is catalyzed by a set of N-terminal acetyltransferases (NATs). The NatA complex (including X-linked Naa10 and Naa15) is the major acetyltransferase, with 40–50% of all mammalian proteins being potential substrates. However, the overall role of amino-terminal acetylation on a whole-organism level is poorly understood, particularly in mammals. Male mice lacking Naa10 show no globally apparent in vivo amino-terminal acetylation impairment and do not exhibit complete embryonic lethality. Rather Naa10 nulls display increased neonatal lethality, and the majority of surviving undersized mutants exhibit a combination of hydrocephaly, cardiac defects, homeotic anterior transformation, piebaldism, and urogenital anomalies. Naa12 is a previously unannotated Naa10-like paralog with NAT activity that genetically compensates for Naa10. Mice deficient for Naa12 have no apparent phenotype, whereas mice deficient for Naa10 and Naa12 display embryonic lethality. The discovery of Naa12 adds to the currently known machinery involved in amino-terminal acetylation in mice.

Amino-terminal acetylation is catalyzed by a set of N-terminal acetyltransferases (NATs). The NatA complex (including X-linked Naa10 and Naa15) is the major acetyltransferase, with 40-50% of all mammalian proteins being potential substrates. However, the overall role of amino-terminal acetylation on a whole-organism level is poorly understood, particularly in mammals. Male mice lacking Naa10 show no globally apparent in vivo amino-terminal acetylation impairment and do not exhibit complete embryonic lethality. Rather Naa10 nulls display increased neonatal lethality, and the majority of surviving undersized mutants exhibit a combination of hydrocephaly, cardiac defects, homeotic anterior transformation, piebaldism and urogenital anomalies. Naa12 is a previously unannotated Naa10-like paralogue with NAT activity that genetically compensates for Naa10. Mice deficient for Naa12 have no apparent phenotype, whereas mice deficient for Naa10 and Naa12 display embryonic lethality. The discovery of Naa12 adds to the currently known machinery involved in amino-terminal acetylation in mice.

Oxidative stress activates macroautophagy/autophagy and contributes to atherogenesis via lipophagic flux, a form of lipid removal by autophagy. However, it is not known exactly how endogenous antioxidant enzymes are involved in lipophagic flux. Here, we demonstrate that the antioxidant PRDX1 (peroxiredoxin 1) has a crucial role in the maintenance of lipophagic flux in macrophages. PRDX1 is more highly expressed than other antioxidant enzymes in monocytes and macrophages. We determined that Prdx1 deficiency induced excessive oxidative stress and impaired maintenance of autophagic flux in macrophages. Prdx1-deficient macrophages had higher intracellular cholesterol mass and lower cholesterol efflux compared with wild type. This perturbation in cholesterol homeostasis was due to impaired lipophagic cholesterol hydrolysis caused by excessive oxidative stress, resulting in the inhibition of free cholesterol formation and the reduction of NR1H3 (nuclear receptor subfamily 1, group H, member 3) activity. Notably, impairment of both lipophagic flux and cholesterol efflux was restored by the 2-Cys PRDX-mimics ebselen and gliotoxin. Consistent with this observation, apoe −/− mice transplanted with bone marrow from prdx1−/−apoe−/− mice had increased plaque formation compared with apoe−/− BM-transplanted recipients. This study reveals that PRDX1 is crucial to regulating lipophagic flux and maintaining macrophage cholesterol homeostasis against oxidative stress. We suggest that PRDX1-dependent control of oxidative stress may provide a strategy for treating atherosclerosis and autophagy-related human diseases.

Cardiac regeneration with adult stem-cell (ASC) therapy is a promising field to address advanced cardiovascular diseases. In addition, extracellular vesicles (EVs) from ASCs have been implicated in acting as paracrine factors to improve cardiac functions in ASC therapy. In our work, we isolated human cardiac mesenchymal stromal cells (h-CMSCs) by means of three-dimensional organ culture (3D culture) during ex vivo expansion of cardiac tissue, to compare the functional efficacy with human bone-marrow derived mesenchymal stem cells (h-BM-MSCs), one of the actively studied ASCs. We characterized the h-CMSCs as CD90low, c-kitnegative, CD105positive phenotype and these cells express NANOG, SOX2, and GATA4. To identify the more effective type of EVs for angiogenesis among the different sources of ASCs, we isolated EVs which were derived from CMSCs with either normoxic or hypoxic condition and BM-MSCs. Our in vitro tube-formation results demonstrated that the angiogenic effects of EVs from hypoxia-treated CMSCs (CMSC-Hpx EVs) were greater than the well-known effects of EVs from BM-MSCs (BM-MSC EVs), and these were even comparable to human vascular endothelial growth factor (hVEGF), a potent angiogenic factor. Therefore, we present here that CD90lowc-kitnegativeCD105positive CMSCs under hypoxic conditions secrete functionally superior EVs for in vitro angiogenesis. Our findings will allow more insights on understanding myocardial repair. [BMB Reports 2020; 53(2): 118-123].

CD137, a potent costimulatory receptor for CD8

Ninjurin1 is a homotypic adhesion molecule that contributes to leukocyte trafficking in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, in vivo gene deficiency animal studies have not yet been done. Here, we constructed Ninjurin1 knock-out (KO) mice and investigated the role of Ninjurin1 on leukocyte trafficking under inflammation conditions such as EAE and endotoxin-induced uveitis. Ninjurin1 KO mice attenuated EAE susceptibility by reducing leukocyte recruitment into the injury regions of the spinal cord and showed less adhesion of leukocytes on inflamed retinal vessels in endotoxin-induced uveitis mice. Moreover, the administration of a custom-made antibody (Ab26-37) targeting the Ninjurin1 binding domain ameliorated the EAE symptoms, showing the contribution of its adhesion activity to leukocyte trafficking. In addition, we addressed the transendothelial migration (TEM) activity of bone marrow-derived macrophages and Raw264.7 cells according to the expression level of Ninjurin1. TEM activity was decreased in Ninjurin1 KO bone marrow-derived macrophages and siNinj1 Raw264.7 cells. Consistent with this, GFP-tagged mNinj1-overexpressing Raw264.7 cells increased their TEM activity. Taken together, we have clarified the contribution of Ninjurin1 to leukocyte trafficking in vivo and delineated its direct functions to TEM, emphasizing Ninjurin1 as a beneficial therapeutic target against inflammatory diseases such as multiple sclerosis.

// Hyesoon Kim 1 , Seung-Hwan Lee 1 , Mi-Ni Lee 2 , Goo Taeg Oh 2 , Kyung-Chul Choi 1,3 and Eun Young Choi 1,3 1 Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul, Republic of Korea, 2 Division of Life Sciences, Ewha Womans University, Seoul, Republic of Korea, 3 Department of Pharmacology, University of Ulsan College of Medicine, Seoul, Republic of Korea Correspondence: Eun Young Choi, email: // Keywords : p53, inflammation, endothelial cell, Del-1 Received : August 23, 2013 Accepted : October 1, 2013 Published : October 3, 2013 Abstract Developmental endothelial locus-1 (Del-1) is an endothelium-derived anti-inflammatory molecule that is downregulated by inflammatory stimuli. Little is known about the molecular mechanisms by which Del-1 transcription is regulated. In the present study, a DNA sequence upstream of the Del-1 gene was analyzed and putative p53 response elements (p53REs) were identified. An approximately 2 kb fragment upstream of the translation start site displayed the highest Del-1 transcriptional activity, and the transcriptional activity of this fragment was enhanced by overexpression of p53. Chemical activation of endogenous p53 elevated the levels of Del-1 mRNA. Site-directed mutagenesis of CATG in the consensus sequences of the 2 kb fragment to TATA significantly reduced the transcription of Del-1. Chromatin immunoprecipitation revealed recruitment of p53 to the p53REs of the Del-1 promoter, resulting in increased Del-1 transcription. Finally, primary endothelial cells isolated from mice with reduced levels of p53 showed a decrease in Del-1 mRNA compared to wild-type endothelial cells. Moreover, Del-1 reciprocally enhanced p53 expression in primary endothelial cells. Thus, these findings suggest that Del-1 is a novel transcriptional target gene of p53.

Objective Blocking agents targeting cell adhesion molecules have been developed to prevent cardiovascular diseases such as atherosclerosis, whereas relatively little attention has been paid to the therapeutic potential of vascular cell adhesion molecule (VCAM)-1 as an inflammatory disease target. Two novel, fully human antibodies, H6 and 7H, against human VCAM-1 (hVCAM-1) were developed and tested to validate the hypothesis that blocking VCAM-1 ameliorates atherosclerosis in apolipoprotein E-deficient (ApoE −/− ) mice. Methods and results Treatment with H6 or 7H effectively inhibited VCAM-1 adhesion to inflammatory cells, and reduced RhoA activation and the production of reactive oxygen species in human umbilical cord vascular endothelial cells. As 7H showed binding affinity to both murine VCAM-1 (mVCAM-1) and hVCAM-1, the therapeutic effects of 7H in ApoE −/− mice were tested. After confirming specific in vivo binding activity of 7H to mVCAM-1, we showed that administering 7H resulted in significantly ameliorated plaque formation compared to administering a control antibody in ApoE −/− mice fed a Western diet for 12 weeks. Also, 7H treatment significantly reduced infiltration of CD45 + cells into plaques and reduced inflammation and improved plaque stability. Conclusion These results indicate that the anti-VCAM-1 antibody attenuates atherosclerosis in ApoE −/− mice, improves plaque inflammation and stability as well as inhibiting the adhesion of inflammatory cell, and suggest that blocking VCAM-1 with a monoclonal antibody may be an effective means of anti-atherosclerotic therapy.

Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. This switch is required for the maintenance of protein homoeostasis and ultimately rescues cells from stress-induced cell death in vitro and in vivo. Therefore, ARD1-mediated Hsp70 acetylation is a regulatory mechanism that temporally balances protein refolding/degradation in response to stress., The chaperone Hsp70 has a dual role, promoting both protein refolding and protein degradation. Seo and Park et al. show that Hsp70 acetylation enhances protein refolding after stress, and that subsequent deacetylation progressively promotes ubiquitin ligase binding and protein degradation.

The N-acetyltransferase arrest defective 1 (ARD1) is an important regulator of cell growth and differentiation that has emerged recently as a critical molecule in cancer progression. However, the regulation of the enzymatic and biological activities of human ARD1 (hARD1) in cancer is presently poorly understood. Here, we report that hARD1 undergoes autoacetylation and that this modification is essential for its functional activation. Using liquid chromatography-tandem mass spectrometry and site-directed mutational analyses, we identified K136 residue as an autoacetylation target site. K136R mutation abolished the ability of hARD1 to promote cancer cell growth in vitro and tumor xenograft growth in vivo. Mechanistic investigations revealed that hARD1 autoacetylation stimulated cyclin D1 expression through activation of the transcription factors β-catenin and activator protein-1. Our results show that hARD1 autoacetylation is critical for its activation and its ability to stimulate cancer cell proliferation and tumorigenesis. Cancer Res; 70(11); 4422–32. ©2010 AACR.

Background— The tumor necrosis factor receptor superfamily, which includes CD40, LIGHT, and OX40, plays important roles in atherosclerosis. CD137 (4-1BB), a member of the tumor necrosis factor receptor superfamily, has been reported to be expressed in human atherosclerotic lesions. However, limited information is available on the precise role of CD137 in atherosclerosis and the effects of blocking CD137/CD137 ligand signaling on lesion formation. Methods and Results— We generated CD137-deficient apolipoprotein E–knockout mice ( ApoE −/− CD137 −/− ) and LDL-receptor–knockout mice ( Ldlr −/− CD137 −/− ) to investigate the role of CD137 in atherogenesis. The deficiency of CD137 induced a reduction in atherosclerotic plaque lesions in both atherosclerosis mouse models, which was attributed to the downregulation of cytokines such as interferon-γ, monocyte chemoattractant protein-1, and tumor necrosis factor-α. CD137 signaling promoted the production of inflammatory molecules, including monocyte chemoattractant protein-1, interleukin-6, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1, in endothelial cells. Stimulation of CD137 ligand signaling activated monocytes/macrophages and augmented the production of proinflammatory cytokines in atherosclerotic vessels. Conclusions— CD137/CD137 ligand signaling plays multiple roles in the progression of atherosclerosis, and thus, blockade of this pathway is a promising therapeutic target for the disease.

We searched for potential suppressors of tumor metastasis by identifying the genes that are frequently down-regulated in hepatocellular carcinomas (HCC) while being negatively correlated with clinical parameters relevant to tumor metastasis, and we report here on the identification of N-myc downstream regulated gene 2 (NDRG2) as a promising candidate. NDRG2 expression was significantly reduced in HCC compared with nontumor or normal liver tissues [87.5% (35 of 40) and 62% (62 of 100) at RNA and protein levels, respectively]. Reduction of NDRG2 expression was intimately associated with promoter hypermethylation because its promoter region was found to carry extensively methylated CpG sites in HCC cell lines and primary tumors. Immunohistochemical analysis of NDRG2 protein in 100 HCC patient tissues indicated that NDRG2 expression loss is significantly correlated with aggressive tumor behaviors such as late tumor-node-metastasis (TNM) stage (P = 0.012), differentiation grade (P = 0.024), portal vein thrombi (P = 0.011), infiltrative growth pattern (P = 0.015), nodal/distant metastasis (P = 0.027), and recurrent tumor (P = 0.021), as well as shorter patient survival rates. Ectopically expressed NDRG2 suppressed invasion and migration of a highly invasive cell line, SK-Hep-1, and experimental tumor metastasis in vivo, whereas small interfering RNA–mediated knockdown resulted in increased invasion and migration of a weakly invasive cell line, PLC/PRF/5. In addition, NDRG2 could antagonize transforming growth factor β1–mediated tumor cell invasion by specifically down-regulating the expression of matrix metalloproteinase 2 and laminin 332 pathway components, with concomitant suppression of Rho GTPase activity. These results suggest that NDRG2 can inhibit extracellular matrix–based, Rho-driven tumor cell invasion and migration and thereby play important roles in suppressing tumor metastasis in HCC. [Cancer Res 2008;68(11):4210–20]

Nature Communications 5: Article number: 4410 (2014); Published: 15 July 2014; Updated: 21 January 2015 In the key for Fig. 4d in this Article, the labels indicating the Ldlr−/− and Ldlr−/−/Retnla-Tg groups were unintentionally placed next to the wrong symbol. The correct version of Fig. 4 appears below.

We investigated the effect of tilianin upon inducible nitric oxide synthesis in the plasma of low-density lipoprotein receptor knock-out (Ldlr-/-) mice fed with high cholesterol diet and in primary peritoneal macrophages of Ldlr-/- mice. High cholesterol diet induced nitric oxide production in the plasma of Ldlr-/- mice. Tilianin reduced the level of nitric oxide (NO) in plasma from Ldlr-/- mice induced by the high cholesterol diet. Tilianin also inhibited the NO production from the primary culture of peritoneal macrophages treated with lipopolysaccharide. The inhibition of NO production was caused by the suppression of inducible nitric oxide synthase (iNOS) gene expression in peritoneal macrophages isolated from Ldlr-/- mice. Moreover, tilianin inhibited the transcriptional activation of iNOS promoter that has NF-kappaB binding element. Thus, these results provide the first evidence that tilianin inhibit iNOS expression and production of NO and may act as a potential anti-inflammatory agent.

Hyperlipidemia is a well-recognized risk factor for atherosclerosis and can be regulated by adipokines. Expression of the adipokine resistin-like molecule alpha (Retnla) is regulated by food intake; whether Retnla has a role in the pathogenesis of hyperlipidemia and atherosclerosis is unknown. Here we report that Retnla has a cholesterol-lowering effect and protects against atherosclerosis in low-density lipoprotein receptor-deficient mice. On a high-fat diet, Retnla deficiency promotes hypercholesterolaemia and atherosclerosis, whereas Retnla overexpression reverses these effects and improves the serum lipoprotein profile, with decreased cholesterol in the very low-density lipoprotein fraction concomitant with reduced serum apolipoprotein B levels. We show that Retnla upregulates cholesterol-7-alpha-hydroxylase, a key hepatic enzyme in the cholesterol catabolic pathway, through induction of its transcriptional activator liver receptor homologue-1, leading to increased excretion of cholesterol in the form of bile acids. These findings define Retnla as a novel therapeutic target for treating hypercholesterolaemia and atherosclerosis.

Runt-related transcription factor 2 (Runx2) transactivates many genes required for osteoblast differentiation. The role of N-alpha-acetyltransferase 10 (NAA10, arrest-defective-1), originally identified in yeast, remains poorly understood in mammals. Here we report a new NAA10 function in Runx2-mediated osteogenesis. Runx2 stabilizes NAA10 in osteoblasts during BMP-2-induced differentiation, and NAA10 in turn controls this differentiation by inhibiting Runx2. NAA10 delays bone healing in a rat calvarial defect model and bone development in neonatal mice. Mechanistically, NAA10 acetylates Runx2 at Lys225, and this acetylation inhibits Runx2-driven transcription by interfering with CBF beta binding to Runx2. Our study suggests that NAA10 acts as a guard ensuring balanced osteogenesis by fine-tuning Runx2 signalling in a feedback manner. NAA10 inhibition could be considered a potential strategy for facilitating bone formation.

Rationale: Peroxiredoxin 2 (Prdx2), a thiol-specific peroxidase, has been reported to regulate proinflammatory responses, vascular remodeling, and global oxidative stress. Objective: Although Prdx2 has been proposed to retard atherosclerosis development, no direct evidence and mechanisms have been reported. Methods and Results: We show that Prdx2 is highly expressed in endothelial and immune cells in atherosclerotic lesions and blocked the increase of endogenous H 2 O 2 by atherogenic stimulation. Deficiency of Prdx2 in apolipoprotein E–deficient (ApoE −/− ) mice accelerated plaque formation with enhanced activation of p65, c-Jun, JNKs, and p38 mitogen-activated protein kinase; and these proatherogenic effects of Prdx2 deficiency were rescued by administration of the antioxidant ebselen. In bone marrow transplantation experiments, we found that Prdx2 has a major role in inhibiting atherogenic responses in both vascular and immune cells. Prdx2 deficiency resulted in increased expression of vascular adhesion molecule-1, intercellular adhesion molecule-1, and monocyte chemotactic protein-1, which led to increased immune cell adhesion and infiltration into the aortic intima. Compared with deficiency of glutathione peroxidase 1 or catalase, Prdx2 deficiency showed a severe predisposition to develop atherosclerosis. Conclusions: Prdx2 is a specific peroxidase that inhibits atherogenic responses in vascular and inflammatory cells, and specific activation of Prdx2 may be an effective means of antiatherogenic therapy.

A variety of benzylidenethiazole analogs have been demonstrated to inhibit 5-lipoxygenase (5-LOX). Here we report the anti-atherogenic potential of 5-(4-hydroxy- 2,3,5-trimethylbenzylidene) thiazolidin-2,4-dione (HMB-TZD), a benzylidenethiazole analog, and its potential mechanism of action in LDL receptor-deficient (Ldlr-/-) mice. HMB-TZD Treatment reduced leukotriene B4 (LTB4) production significantly in RAW264.7 macrophages and SVEC4-10 endothelial cells. Macrophages or endothelial cells pre-incubated with HMB-TZD for 2 h and then stimulated with lipopolysaccharide or tumor necrosis factor-alpha (TNF-α) displayed reduced cytokine production. Also, HMB-TZD reduced cell migration and adhesion in accordance with decreased proinflammatory molecule production in vitro and ex vivo. HMB-TZD treatment of 8-week-old male Ldlr-/- mice resulted in significantly reduced atherosclerotic lesions without a change to plasma lipid profiles. Moreover, aortic expression of pro-atherogenic molecules involved in the recruitment of monocytes to the aortic wall, including TNF-α , MCP-1, and VCAM-1, was downregulated. HMB-TZD also reduced macrophage infiltration into atherosclerotic lesions. In conclusion, HMB-TZD ameliorates atherosclerotic lesion formation possibly by reducing the expression of proinflammatory molecules and monocyte/macrophage recruitment to the lesion. These results suggest that HMB-TZD, and benzylidenethiazole analogs in general, may have therapeutic potential as treatments for atherosclerosis.

Cyclooxygenase (COX) and 5-lipoxygenase (5-LOX), which play pivotal roles in atherogenesis, have been reported to be involved in plaque stability. Licofelone, a dual COX and 5-LOX inhibitor, has been reported to possess anti-atherogenic effect in rabbit atherosclerosis model. We therefore investigated the anti-atherogenic effect of BHB-TZD [5-(3,5-di-tert-butyl-4-hydroxybenzylidene)thiazolidin-2,4-dione], a dual COX and 5-LOX inhibitor, in low density lipoprotein receptor null (LDLR-/-) mice. Fifteen LDLR-/- mice were fed a western diet (control group), whereas 15 were fed a western diet plus 0.1% (w/w) BHB-TZD (BHB-TZD group). After 8 weeks, the BHB-TZD group had markedly lower serum levels of leukotriene B(4) and prostaglandin E(2) than the control group. Interestingly, BHB-TZD treatment also reduced plasma triglyceride level without significant changes in total cholesterol and HDL levels. Compared with control mice, BHB-TZD fed mice had 52% fewer fatty streak lesions in the aortic sinus, as well as fewer initial lesions in the aortic arch. Macrophage infiltration into the lesions was 40% lower, and collagen and smooth muscle cells were increased by 102% and 96%, respectively, in the BHB-TZD group compared with the control group. In addition, aortic expression of proatherogenic molecules including TNF-alpha, IL-1beta, IL-6, MCP-1 and VCAM-1, was lower in the BHB-TZD group than the control group. BHB-TZD treatment also reduced MMP-2 and MMP-9 expressions in aorta. In conclusion, BHB-TZD effectively attenuated atherosclerosis in mouse model, suggesting its therapeutic potential for atherosclerosis.

Purpose: Rho GDP dissociation inhibitor 2 (RhoGDI2) has been identified as a regulator of Rho family GTPase. However, there is currently no direct evidence suggesting whether RhoGDI2 activates or inhibits Rho family GTPase in vivo (and which type), and the role of RhoGDI2 in tumor remains controversial. Here, we assessed the effects of RhoGDI2 expression on gastric tumor growth and metastasis progression. Experimental Design: Proteomic analysis was done to investigate the tumor-specific protein expression in gastric cancer and RhoGDI2 was selected for further study. Immunohistochemistry was used to detect RhoGDI2 expression in clinical samples of primary gastric tumor tissues which have different pathologic stages. Gain-of-function and loss-of-function approaches were done to examine the malignant phenotypes of the RhoGDI2-expressing or RhoGDI2-depleting cells. Results: RhoGDI2 expression was correlated positively with tumor progression and metastasis potential in human gastric tumor tissues, as well as cell lines. The forced expression of RhoGDI2 caused a significant increase in gastric cancer cell invasion in vitro, and tumor growth, angiogenesis, and metastasis in vivo, whereas RhoGDI2 depletion evidenced opposite effects. Conclusion: Our findings indicate that RhoGDI2 is involved in gastric tumor growth and metastasis, and that RhoGDI2 may be a useful marker for tumor progression of human gastric cancer.

Methionine sulfoxide reductase A (MSRA) protects proteins from oxidation, and also helps remove reactive oxygen species (ROS) by recovering antioxidant enzymes inactivated by oxidation. Although its functions have been investigated extensively, little is known about the mechanism by which MSRA is regulated. Arrest defective 1 (ARD1) is an enzyme that catalyzes not only N-terminal acetylation as a cotranslational modification but also lysine acetylation as a posttranslational modification. ARD1, which is expressed in most cell types, is believed to participate in diverse biological processes, but its roles are poorly understood. Given that MSRA was hunted in a yeast two-hybrid screen with ARD1 as the bait, we here investigated whether ARD1 is a novel regulator of MSRA. ARD1 was shown to interact with and acetylate MSRA in both cells and test tubes. It specifically acetylated the K49 residue of MSRA, and by doing so repressed the enzymatic function of MSRA. ARD1 increased cellular levels of ROS, carbonylated proteins and DNA breaks under oxidative stress. Moreover, it promoted cell death induced by pro-oxidants, which was attenuated in MSRA-deficient cells. When mice were exposed to hyperoxic conditions for 2 days, their livers and kidneys were injured and protein carbonylation was increased. The oxidative tissue injury was more severe in ARD1 transgenic mice than in their wild-type littermates. In conclusion, ARD1 has a crucial role in the cellular response to oxidative stress as a bona fide regulator of MSRA. ARD1 is a potential target for ameliorating oxidative injury or for potentiating ROS-producing anticancer agents.