Inducible Depletion of Calpain-2 Mitigates Abdominal Aortic Aneurysm in Mice.

[Figure: see text].

Mechanisms of transcription factor acetylation and consequences in hearts.

Acetylation of proteins as a post-translational modification is gaining rapid acceptance as a cellular control mechanism on par with other protein modification mechanisms such as phosphorylation and ubiquitination. Through genetic manipulations and evolving proteomic technologies, identification and consequences of transcription factor acetylation is beginning to emerge. In this review, we summarize the field and discuss newly unfolding mechanisms and consequences of transcription factor acetylation in normal and stressed hearts. This article is part of a Special Issue entitled: The role of post-translational protein modifications on heart and vascular metabolism edited by Jason R.B. Dyck & Jan F.C. Glatz.

DIAPH1-MFN2 interaction regulates mitochondria-SR/ER contact and modulates ischemic/hypoxic stress.

Inter-organelle contact and communication between mitochondria and sarco/endoplasmic reticulum (SR/ER) maintain cellular homeostasis and are profoundly disturbed during tissue ischemia. We tested the hypothesis that the formin Diaphanous-1 (DIAPH1), which regulates actin dynamics, signal transduction and metabolic functions, contributes to these processes. We demonstrate that DIAPH1 interacts directly with Mitofusin-2 (MFN2) to shorten mitochondria-SR/ER distance, thereby enhancing mitochondria-ER contact in cells including cardiomyocytes, endothelial cells and macrophages. Solution structure studies affirm the interaction between the Diaphanous Inhibitory Domain and the cytosolic GTPase domain of MFN2. In male rodent and human cardiomyocytes, DIAPH1-MFN2 interaction regulates mitochondrial turnover, mitophagy, and oxidative stress. Introduction of synthetic linker construct, which shorten the mitochondria-SR/ER distance, mitigated the molecular and functional benefits of DIAPH1 silencing in ischemia. This work establishes fundamental roles for DIAPH1-MFN2 interaction in the regulation of mitochondria-SR/ER contact networks. We propose that targeting pathways that regulate DIAPH1-MFN2 interactions may facilitate recovery from tissue ischemia.

Aldose reductase promotes diet-induced obesity via induction of senescence in subcutaneous adipose tissue.

OBJECTIVE: Aldose reductase (AKR1B1 in humans; Akr1b3 in mice), a key enzyme of the polyol pathway, mediates lipid accumulation in the murine heart and liver. The study objective was to explore potential roles for AKR1B1/Akr1b3 in the pathogenesis of obesity and its complications. METHODS: The study employed mice treated with an inhibitor of aldose reductase or mice devoid of Akr1b3 were used to determine their response to a high-fat diet. The study used subcutaneous adipose tissue-derived adipocytes to investigate mechanisms by which AKR1B1/Akr1b3 promotes diet-induced obesity. RESULTS: Increased expression of aldose reductase and senescence in the adipose tissue of humans and mice with obesity were demonstrated. Genetic deletion of Akr1b3 or pharmacological blockade of AKRIB3 with zopolrestat reduced high-fat-diet-induced obesity, attenuated markers of adipose tissue senescence, and increased lipolysis. CONCLUSIONS: AKR1B1/Akr1b3 modulation of senescence in subcutaneous adipose tissue contributes to aberrant metabolic responses to high-fat feeding. These data unveil new opportunities to target these pathways to combat obesity.

miR-146a Deficiency Accelerates Hepatic Inflammation Without Influencing Diet-induced Obesity in Mice.

miR-146a, an anti-inflammatory microRNA, is shown to be a negative regulator of adipocyte inflammation. However, the functional contribution of miR-146a in the development of obesity is not defined. In order to determine whether miR-146a influences diet-induced obesity, mice that were either wild type (WT) or miR-146a deficient (KO) were fed with high (60% kcal) fat diet (HFD) for 16 weeks. Deficiency of miR-146a did not influence obesity measured as HFD-induced body weight and fat mass gain, or metabolism of glucose and insulin tolerance. In addition, adipocyte apoptosis, adipose tissue collagen and macrophage accumulation as detected by TUNEL, Picro Sirius and F4/80 immunostaining, respectively, were comparable between the two groups of mice. Although, miR-146a deficiency had no influence on HFD-induced hepatic lipid accumulation, interestingly, it significantly increased obesity-induced inflammatory responses in liver tissue. The present study demonstrates that miR-146a deficiency had no influence on the development of HFD-induced obesity and adipose tissue remodeling, whereas it significantly increased hepatic inflammation in obese mice. This result suggests that miR-146a regulates hepatic inflammation during development of obesity.

Author Correction: Suppression of Transposable Elements in Leukemic Stem Cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Suppression of Transposable Elements in Leukemic Stem Cells.

Genomic transposable elements (TEs) comprise nearly half of the human genome. The expression of TEs is considered potentially hazardous, as it can lead to insertional mutagenesis and genomic instability. However, recent studies have revealed that TEs are involved in immune-mediated cell clearance. Hypomethylating agents can increase the expression of TEs in cancer cells, inducing 'viral mimicry', causing interferon signalling and cancer cell killing. To investigate the role of TEs in the pathogenesis of acute myeloid leukaemia (AML), we studied TE expression in several cell fractions of AML while tracking its development (pre-leukemic haematopoietic stem cells, leukemic stem cells [LSCs], and leukemic blasts). LSCs, which are resistant to chemotherapy and serve as reservoirs for relapse, showed significant suppression of TEs and interferon pathways. Similarly, high-risk cases of myelodysplastic syndrome (MDS) showed far greater suppression of TEs than low-risk cases. We propose TE suppression as a mechanism for immune escape in AML and MDS. Repression of TEs co-occurred with the upregulation of several genes known to modulate TE expression, such as RNA helicases and autophagy genes. Thus, we have identified potential pathways that can be targeted to activate cancer immunogenicity via TEs in AML and MDS.

Aldose reductase modulates acute activation of mesenchymal markers via the ß-catenin pathway during cardiac ischemia-reperfusion.

Aldose reductase (AR: human, AKR1B1; mouse, AKR1B3), the first enzyme in the polyol pathway, plays a key role in mediating myocardial ischemia/reperfusion (I/R) injury. In earlier studies, using transgenic mice broadly expressing human AKR1B1 to human-relevant levels, mice devoid of Akr1b3, and pharmacological inhibitors of AR, we demonstrated that AR is an important component of myocardial I/R injury and that inhibition of this enzyme protects the heart from I/R injury. In this study, our objective was to investigate if AR modulates the ß-catenin pathway and consequent activation of mesenchymal markers during I/R in the heart. To test this premise, we used two different experimental models: in vivo, Akr1b3 null mice and wild type C57BL/6 mice (WT) were exposed to acute occlusion of the left anterior descending coronary artery (LAD) followed by recovery for 48 hours or 28 days, and ex-vivo, WT and Akr1b3 null murine hearts were perfused using the Langendorff technique (LT) and subjected to 30 min of global (zero-flow) ischemia followed by 60 min of reperfusion. Our in vivo results reveal reduced infarct size and improved functional recovery at 48 hours in mice devoid of Akr1b3 compared to WT mice. We demonstrate that the cardioprotection observed in Akr1b3 null mice was linked to acute activation of the ß-catenin pathway and consequent activation of mesenchymal markers and genes linked to fibrotic remodeling. The increased activity of the ß-catenin pathway at 48 hours of recovery post-LAD was not observed at 28 days post-infarction, thus indicating that the observed increase in ß-catenin activity was transient in the mice hearts devoid of Akr1b3. In ex vivo studies, inhibition of ß-catenin blocked the cardioprotection observed in Akr1b3 null mice hearts. Taken together, these data indicate that AR suppresses acute activation of ß-catenin and, thereby, blocks consequent induction of mesenchymal markers during early reperfusion after myocardial ischemia. Inhibition of AR might provide a therapeutic opportunity to optimize cardiac remodeling after I/R injury.

The Formin, DIAPH1, is a Key Modulator of Myocardial Ischemia/Reperfusion Injury.

The biochemical, ionic, and signaling changes that occur within cardiomyocytes subjected to ischemia are exacerbated by reperfusion; however, the precise mechanisms mediating myocardial ischemia/reperfusion (I/R) injury have not been fully elucidated. The receptor for advanced glycation end-products (RAGE) regulates the cellular response to cardiac tissue damage in I/R, an effect potentially mediated by the binding of the RAGE cytoplasmic domain to the diaphanous-related formin, DIAPH1. The aim of this study was to investigate the role of DIAPH1 in the physiological response to experimental myocardial I/R in mice. After subjecting wild-type mice to experimental I/R, myocardial DIAPH1 expression was increased, an effect that was echoed following hypoxia/reoxygenation (H/R) in H9C2 and AC16 cells. Further, compared to wild-type mice, genetic deletion of Diaph1 reduced infarct size and improved contractile function after I/R. Silencing Diaph1 in H9C2 cells subjected to H/R downregulated actin polymerization and serum response factor-regulated gene expression. Importantly, these changes led to increased expression of sarcoplasmic reticulum Ca2+ ATPase and reduced expression of the sodium calcium exchanger. This work demonstrates that DIAPH1 is required for the myocardial response to I/R, and that targeting DIAPH1 may represent an adjunctive approach for myocardial salvage after acute infarction.

SM22α suppresses cytokine-induced inflammation and the transcription of NF-κB inducing kinase (Nik) by modulating SRF transcriptional activity in vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) phenotypic modulation is characterized by the downregulation of SMC actin cytoskeleton proteins. Our published study shows that depletion of SM22α (aka SM22, Transgelin, an actin cytoskeleton binding protein) promotes inflammation in SMCs by activating NF-κB signal pathways both in cultured VSMCs and in response to vascular injury. The goal of this study is to investigate the underlying molecular mechanisms whereby SM22 suppresses NF-κB signaling pathways under inflammatory condition. NF-κB inducing kinase (Nik, aka MAP3K14, activated by the LTßR) is a key upstream regulator of NF-κB signal pathways. Here, we show that SM22 overexpression suppresses the expression of NIK and its downstream NF-κB canonical and noncanonical signal pathways in a VSMC line treated with a LTßR agonist. SM22 regulates NIK expression at both transcriptional and the proteasome-mediated post-translational levels in VSMCs depending on the culture condition. By qPCR, chromatin immunoprecipitation and luciferase assays, we found that Nik is a transcription target of serum response factor (SRF). Although SM22 is known to be expressed in the cytoplasm, we found that SM22 is also expressed in the nucleus where SM22 interacts with SRF to inhibit the transcription of Nik and prototypical SRF regulated genes including c-fos and Egr3. Moreover, carotid injury increases NIK expression in Sm22-/- mice, which is partially relieved by adenovirally transduced SM22. These findings reveal for the first time that SM22 is expressed in the nucleus in addition to the cytoplasm of VSMCs to regulate the transcription of Nik and its downstream proinflammatory NF-kB signal pathways as a modulator of SRF during vascular inflammation.

Aldose Reductase Acts as a Selective Derepressor of PPARγ and the Retinoic Acid Receptor.

Histone deacetylase 3 (HDAC3), a chromatin-modifying enzyme, requires association with the deacetylase-containing domain (DAD) of the nuclear receptor corepressors NCOR1 and SMRT for its stability and activity. Here, we show that aldose reductase (AR), the rate-limiting enzyme of the polyol pathway, competes with HDAC3 to bind the NCOR1/SMRT DAD. Increased AR expression leads to HDAC3 degradation followed by increased PPARγ signaling, resulting in lipid accumulation in the heart. AR also downregulates expression of nuclear corepressor complex cofactors including Gps2 and Tblr1, thus affecting activity of the nuclear corepressor complex itself. Though AR reduces HDAC3-corepressor complex formation, it specifically derepresses the retinoic acid receptor (RAR), but not other nuclear receptors such as the thyroid receptor (TR) and liver X receptor (LXR). In summary, this work defines a distinct role for AR in lipid and retinoid metabolism through HDAC3 regulation and consequent derepression of PPARγ and RAR.

RAGE Suppresses ABCG1-Mediated Macrophage Cholesterol Efflux in Diabetes.

Diabetes exacerbates cardiovascular disease, at least in part through suppression of macrophage cholesterol efflux and levels of the cholesterol transporters ATP binding cassette transporter A1 (ABCA1) and ABCG1. The receptor for advanced glycation end products (RAGE) is highly expressed in human and murine diabetic atherosclerotic plaques, particularly in macrophages. We tested the hypothesis that RAGE suppresses macrophage cholesterol efflux and probed the mechanisms by which RAGE downregulates ABCA1 and ABCG1. Macrophage cholesterol efflux to apolipoprotein A1 and HDL and reverse cholesterol transport to plasma, liver, and feces were reduced in diabetic macrophages through RAGE. In vitro, RAGE ligands suppressed ABCG1 and ABCA1 promoter luciferase activity and transcription of ABCG1 and ABCA1 through peroxisome proliferator-activated receptor-γ (PPARG)-responsive promoter elements but not through liver X receptor elements. Plasma levels of HDL were reduced in diabetic mice in a RAGE-dependent manner. Laser capture microdissected CD68(+) macrophages from atherosclerotic plaques of Ldlr(-/-) mice devoid of Ager (RAGE) displayed higher levels of Abca1, Abcg1, and Pparg mRNA transcripts versus Ager-expressing Ldlr(-/-) mice independently of glycemia or plasma levels of total cholesterol and triglycerides. Antagonism of RAGE may fill an important therapeutic gap in the treatment of diabetic macrovascular complications.

Aldose reductase drives hyperacetylation of Egr-1 in hyperglycemia and consequent upregulation of proinflammatory and prothrombotic signals.

Sustained increases in glucose flux via the aldose reductase (AR) pathway have been linked to diabetic vascular complications. Previous studies revealed that glucose flux via AR mediates endothelial dysfunction and leads to lesional hemorrhage in diabetic human AR (hAR) expressing mice in an apoE(-/-) background. Our studies revealed sustained activation of Egr-1 with subsequent induction of its downstream target genes tissue factor (TF) and vascular cell adhesion molecule-1 (VCAM-1) in diabetic apoE(-/-)hAR mice aortas and in high glucose-treated primary murine aortic endothelial cells expressing hAR. Furthermore, we observed that flux via AR impaired NAD(+) homeostasis and reduced activity of NAD(+)-dependent deacetylase Sirt-1 leading to acetylation and prolonged expression of Egr-1 in hyperglycemic conditions. In conclusion, our data demonstrate a novel mechanism by which glucose flux via AR triggers activation, acetylation, and prolonged expression of Egr-1 leading to proinflammatory and prothrombotic responses in diabetic atherosclerosis.

Early stress evokes age-dependent biphasic changes in hippocampal neurogenesis, BDNF expression, and cognition.

BACKGROUND: Adult-onset stressors exert opposing effects on hippocampal neurogenesis and cognition, with enhancement observed following mild stress and dysfunction following severe chronic stress. While early life stress evokes persistent changes in anxiety, it is unknown whether early stress differentially regulates hippocampal neurogenesis, trophic factor expression, and cognition across the life span. METHODS: Hippocampal-dependent cognitive behavior, neurogenesis, and epigenetic regulation of brain-derived neurotrophic factor (Bdnf) expression was examined at distinct time points across the life span in rats subjected to the early stress of maternal separation (ES) and control groups. We also examined the influence of chronic antidepressant treatment on the neurogenic, neurotrophic, and cognitive changes in middle-aged ES animals. RESULTS: Animals subjected to early stress of maternal separation examined during postnatal life and young adulthood exhibited enhanced hippocampal neurogenesis, decreased repressive histone methylation at the Bdnf IV promoter along with enhanced BDNF levels, and improved performance on the stress-associated Morris water maze. Strikingly, opposing changes in hippocampal neurogenesis and epigenetic regulation of Bdnf IV expression, concomitant with impairments on hippocampal-dependent cognitive tasks, were observed in middle-aged ES animals. Chronic antidepressant treatment with amitriptyline attenuated the maladaptive neurogenic, epigenetic, transcriptional, and cognitive effects in middle-aged ES animals. CONCLUSIONS: Our study provides novel insights into the short- and long-term consequences of ES, demonstrating both biphasic and unique, age-dependent changes at the molecular, epigenetic, neurogenic, and behavioral levels. These results indicate that early stress may transiently endow animals with a potential adaptive advantage in stressful environments but across a life span is associated with long-term deleterious effects.

The DNA methyltranferase Dnmt2 participates in RNA processing during cellular stress.

The strong evolutionary conservation of the DNA methyltransferase, Dnmt2, is at odds with the absence of phenotypic defects in organisms lacking Dnmt2. The cellular processes where Dnmt2 has a role to play also remain largely undiscovered. Here we show that Dnmt2 is a part of RNA processing machinery during cellular stress. In addition to interacting with proteins involved in RNA processing and cellular stress, Dnmt2 exhibits nucleo-cytoplasmic shuttling in response to cellular stress. Normally present in the nucleus, under conditions of stress, Dnmt2 relocalises to the cytoplasmic Stress Granules and RNA processing bodies. Surprisingly, for a DNA methyltransferase, knockout of which showed no phenotypic defects in several species, our results show that transient transfection of Dnmt2 in mammalian cells causes cell lethality. Interestingly, Dnmt2 overexpression altered the expression of several genes involved in viral infection. Taking into consideration its recently identified role in retrotransposon silencing, the role of Dnmt2 in stress granules could represent a primitive cellular defense mechanism against viral infection.

An intronic DNA sequence within the mouse Neuronatin gene exhibits biochemical characteristics of an ICR and acts as a transcriptional activator in Drosophila.

Imprinting control regions (ICRs) are domains within imprinted loci that are essential for their establishment and maintenance. Imprinted loci can extend over several megabases, encompass both maternally and paternally-expressed genes and exhibit multiple and complex epigenetic modifications including large regions of allele-specific DNA methylation. Differential chromatin organisation has also been observed within imprinted loci but is restricted to the ICRs. In this study we report the identification of a novel imprinting control region for the mouse Neuronatin gene. This biochemically defined putative ICR, present within its 250 bp second intron, functions as transcriptional activator in Drosophila. This is unlike other known ICRs which have been shown to function as transcriptional silencers. Furthermore, at the endogenous locus, the activating signal from the ICR extends to the Neuronatin promoter via allele-specific unidirectional nucleosomal positioning. Our results support the proposal that the Neuronatin locus employs the most basic mechanism for establishing allele-specific gene expression and could provide the foundation for the multiplex arrangements reported at more complex loci.