Duk-Hwa Kwon

Dietary protein restriction induces steatohepatitis and alters leptin/signal transducers and activators of transcription 3 signaling in lactating rats.

Dietary protein restriction during lactation affects lipid metabolism and food intake in rats. The goals of this study were to determine the effect of a low-protein diet on a liver damage in lactating rats, to determine whether dietary protein restriction of lactating dams affects the liver health of their offspring and to elucidate the molecular mechanisms underlying the development of hepatic damage. Lactating Sprague-Dawley rats were fed either a control 20% protein diet or an 8% low-protein diet for 11 or 23 days, respectively. After weaning, the offspring were continuously fed either the same control diet or the low-protein diet for an additional 22 days. Feeding a low-protein diet during lactation caused steatohepatitis with severe steatosis, lobular inflammation, ballooning degeneration and fibrosis. Offspring nourished by dams fed a low-protein diet showed simple hepatic steatosis. Combined effects of increased lipogenesis, decreased fatty acid oxidation and impaired very-low-density lipoprotein secretion were responsible for the development of hepatic steatosis. Hepatic up-regulation of genes linked to oxidative stress including nicotinamide adenine dinucleotide phosphate oxidase, inflammation and fibrogenesis supports the development of steatohepatitis in protein-restricted lactating rats. Furthermore, protein-restricted lactating rats showed activation of the leptin/signal transducers and activators of the transcription 3 signaling pathway. Taken together, oxidative stress induced by up-regulation of nicotinamide adenine dinucleotide phosphate oxidase with activation of leptin/signal transducers and activators of the transcription 3 signaling was responsible for development of steatohepatitis in protein-restricted lactating rats. Our findings suggest that protein malnutrition has a potential to induce steatohepatitis/hepatic steatosis in lactating mothers and infants during breast-feeding.

Estrogen-related receptor gamma induces cardiac hypertrophy by activating GATA4

Estrogen-related receptor gamma (ERRγ) is an orphan nuclear receptor that has biological roles mainly in metabolism and that controls metabolic switching in perinatal heart. In adult heart diseases, however, the functional roles of ERRγ have not yet been elucidated. In the present study, we aimed to characterize the role of ERRγ in cardiac hypertrophy. The functional roles of ERRγ in the development of cardiac hypertrophy were examined in primary cultured cardiomyocytes and in animal models. ERRγ expression was increased in hearts from human hypertrophic cardiomyopathy patients and in both cellular and animal models of cardiac hypertrophy. Transgenic overexpression in mouse heart as well as forced expression of ERRγ in cardiomyocytes induced hypertrophic phenotypes. Knock-down of ERRγ blocked agonist-induced hypertrophic phenotypes. ERRγ bound directly to the proximal ERR-responsive element in the GATA4 promoter in a sequence-specific manner and thereby induced transcription. ERRγ-induced hypertrophy was blocked by inhibition of GATA4. GSK-5182, an inverse agonist of ERRγ, completely blocked cardiac hypertrophy in cardiomyocytes. It also prevented aortic banding-induced cardiac hypertrophy and fibrosis in mouse heart. These findings demonstrate a novel ERRγ/GATA4 signal cascade in the development of cardiac hypertrophy and suggest GSK-5182 as a possible therapeutic.

MDM2 E3 ligase-mediated ubiquitination and degradation of HDAC1 in vascular calcification.

Vascular calcification (VC) is often associated with cardiovascular and metabolic diseases. However, the molecular mechanisms linking VC to these diseases have yet to be elucidated. Here we report that MDM2-induced ubiquitination of histone deacetylase 1 (HDAC1) mediates VC. Loss of HDAC1 activity via either chemical inhibitor or genetic ablation enhances VC. HDAC1 protein, but not mRNA, is reduced in cell and animal calcification models and in human calcified coronary artery. Under calcification-inducing conditions, proteasomal degradation of HDAC1 precedes VC and it is mediated by MDM2 E3 ubiquitin ligase that initiates HDAC1 K74 ubiquitination. Overexpression of MDM2 enhances VC, whereas loss of MDM2 blunts it. Decoy peptide spanning HDAC1 K74 and RG 7112, an MDM2 inhibitor, prevent VC in vivo and in vitro. These results uncover a previously unappreciated ubiquitination pathway and suggest MDM2-mediated HDAC1 ubiquitination as a new therapeutic target in VC.

The microRNA miR‐124 inhibits vascular smooth muscle cell proliferation by targeting S100 calcium‐binding protein A4 (S100A4)

S100 calcium‐binding protein A4 (S100A4) induces proliferation and migration of vascular smooth muscle cells (VSMCs). We aimed to find the microRNA regulating S100A4 expression. S100A4 transcripts are abruptly increased in the acute phase of carotid arterial injury 1 day later (at day 1) but gradually decreases at days 7 and 14. Bioinformatics analysis reveals that miR‐124 targets S100A4. VSMC survival is attenuated by miR‐124 mimic but increased by miR‐124 inhibitor. miR‐124 decreases immediately after carotid arterial injury but dramatically increases at days 7 and 14. miR‐124 inhibitor‐induced cell proliferation is blocked by S100A4 siRNA, whereas miR‐124‐induced cell death is recovered by S100A4. Our findings suggest that miR‐124 is a novel regulator of VSMC proliferation and may play a role in the development of neointimal proliferation.

Ret finger protein mediates Pax7-induced ubiquitination of MyoD in skeletal muscle atrophy.

Skeletal muscle atrophy results from the net loss of muscular proteins and organelles and is caused by pathologic conditions such as nerve injury, immobilization, cancer, and other metabolic diseases. Recently, ubiquitination-mediated degradation of skeletal-muscle-specific transcription factors was shown to be involved in muscle atrophy, although the mechanisms have yet to be defined. Here we report that ret finger protein (RFP), also known as TRIM27, works as an E3 ligase in Pax7-induced degradation of MyoD. Muscle injury induced by sciatic nerve transection up-regulated RFP and RFP physically interacted with both Pax7 and MyoD. RFP and Pax7 synergistically reduced the protein amounts of MyoD but not the mRNA. RFP-induced reduction of MyoD protein was blocked by proteasome inhibitors. The Pax7-induced reduction MyoD was attenuated by RFP siRNA and by MG132, a proteasome inhibitor. RFPΔR, an RFP construct that lacks the RING domain, failed to reduce MyoD amounts. RFP ubiquitinated MyoD, but RFPΔR failed to do so. Forced expression of RFP, but not RFPΔR, enhanced Pax7-induced ubiquitination of MyoD, whereas RFP siRNA blocked the ubiquitination. Sciatic nerve injury-induced muscle atrophy as well the reduction in MyoD was attenuated in RFP knockout mice. Taken together, our results show that RFP works as a novel E3 ligase in the Pax7-mediated degradation of MyoD in response to skeletal muscle atrophy.

Small Heterodimer Partner Blocks Cardiac Hypertrophy by Interfering with GATA6 Signaling

Rationale: Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that lacks a conventional DNA-binding domain. Through interactions with other transcription factors, SHP regulates diverse biological events, including glucose metabolism in liver. However, the role of SHP in adult heart diseases has not yet been demonstrated. Objective: We aimed to investigate the role of SHP in adult heart in association with cardiac hypertrophy. Methods and Results: The roles of SHP in cardiac hypertrophy were tested in primary cultured cardiomyocytes and in animal models. SHP-null mice showed a hypertrophic phenotype. Hypertrophic stresses repressed the expression of SHP, whereas forced expression of SHP blocked the development of hypertrophy in cardiomyocytes. SHP reduced the protein amount of Gata6 and, by direct physical interaction with Gata6, interfered with the binding of Gata6 to GATA-binding elements in the promoter regions of natriuretic peptide precursor type A. Metformin, an antidiabetic agent, induced SHP and suppressed cardiac hypertrophy. The metformin-induced antihypertrophic effect was attenuated either by SHP small interfering RNA in cardiomyocytes or in SHP-null mice. Conclusions: These results establish SHP as a novel antihypertrophic regulator that acts by interfering with GATA6 signaling. SHP may participate in the metformin-induced antihypertrophic response.

New Aspects of Vascular Calcification: Histone Deacetylases and Beyond

Vascular calcification is a pathologic phenomenon in which calcium phosphate is ectopically deposited in the arteries. Previously, calcification was considered to be a passive process in response to metabolic diseases, vascular or valvular diseases, or even aging. However, now calcification is recognized as a highly-regulated consequence, like bone formation, and many clinical trials have been carried out to elucidate the correlation between vascular calcification and cardiovascular events and mortality. As a result, vascular calcification has been implicated as an independent risk factor in cardiovascular diseases. Many molecules are now known to be actively associated with this process. Recently, our laboratory found that posttranslational modification of histone deacetylase (HDAC) 1 is actively involved in the development of vascular calcification. In addition, we found that modulation of the activity of HDAC as well as its protein stability by MDM2, an HDAC1-E3 ligase, may be a therapeutic target in vascular calcification. In the present review, we overview the pathomechanism of vascular calcification and the involvement of posttranslational modification of epigenetic regulators.

Identification of long noncoding RNAs involved in muscle differentiation

Long noncoding RNAs (lncRNAs) are a large class of regulatory RNAs with diverse roles in cellular processes. Thousands of lncRNAs have been discovered; however, their roles in the regulation of muscle differentiation are unclear because no comprehensive analysis of lncRNAs during this process has been performed. In the present study, by combining diverse RNA sequencing datasets obtained from public database, we discovered lncRNAs that could behave as regulators in the differentiation of smooth or skeletal muscle cells. These analyses confirmed the roles of previously reported lncRNAs in this process. Moreover, we discovered dozens of novel lncRNAs whose expression patterns suggested their possible involvement in the phenotypic switch of vascular smooth muscle cells. The comparison of lncRNA expression change suggested that many lncRNAs have common roles during the differentiation of smooth and skeletal muscles, while some lncRNAs may have opposite roles in this process. The expression change of lncRNAs was highly correlated with that of their neighboring genes, suggesting that they may function as cis-acting lncRNAs. Furthermore, within the lncRNA sequences, there were binding sites for miRNAs with expression levels inversely correlated with the expression of corresponding lncRNAs during differentiation, suggesting a possible role of these lncRNAs as competing endogenous RNAs. The lncRNAs identified in this study will be a useful resource for future studies of gene regulation during muscle differentiation.

Sumoylation of histone deacetylase 1 regulates MyoD signaling during myogenesis

Sumoylation, the conjugation of a small ubiquitin-like modifier (SUMO) protein to a target, has diverse cellular effects. However, the functional roles of the SUMO modification during myogenesis have not been fully elucidated. Here, we report that basal sumoylation of histone deacetylase 1 (HDAC1) enhances the deacetylation of MyoD in undifferentiated myoblasts, whereas further sumoylation of HDAC1 contributes to switching its binding partners from MyoD to Rb to induce myocyte differentiation. Differentiation in C2C12 skeletal myoblasts induced new immunoblot bands above HDAC1 that were gradually enhanced during differentiation. Using SUMO inhibitors and sumoylation assays, we showed that the upper band was caused by sumoylation of HDAC1 during differentiation. Basal deacetylase activity was not altered in the SUMO modification-resistant mutant HDAC1 K444/476R (HDAC1 2R). Either differentiation or transfection of SUMO1 increased HDAC1 activity that was attenuated in HDAC1 2R. Furthermore, HDAC1 2R failed to deacetylate MyoD. Binding of HDAC1 to MyoD was attenuated by K444/476R. Binding of HDAC1 to MyoD was gradually reduced after 2 days of differentiation. Transfection of SUMO1 induced dissociation of HDAC1 from MyoD but potentiated its binding to Rb. SUMO1 transfection further attenuated HDAC1-induced inhibition of muscle creatine kinase luciferase activity that was reversed in HDAC1 2R. HDAC1 2R failed to inhibit myogenesis and muscle gene expression. In conclusion, HDAC1 sumoylation plays a dual role in MyoD signaling: enhancement of HDAC1 deacetylation of MyoD in the basally sumoylated state of undifferentiated myoblasts and dissociation of HDAC1 from MyoD during myogenesis.

PP2A negatively regulates the hypertrophic response by dephosphorylating HDAC2 S394 in the heart

Cardiac hypertrophy occurs in response to increased hemodynamic demand and can progress to heart failure. Identifying the key regulators of this process is clinically important. Though it is thought that the phosphorylation of histone deacetylase (HDAC) 2 plays a crucial role in the development of pathological cardiac hypertrophy, the detailed mechanism by which this occurs remains unclear. Here, we performed immunoprecipitation and peptide pull-down assays to characterize the functional complex of HDAC2. Protein phosphatase (PP) 2 A was confirmed as a binding partner of HDAC2. PPP2CA, the catalytic subunit of PP2A, bound to HDAC2 and prevented its phosphorylation. Transient overexpression of PPP2CA specifically regulated both the phosphorylation of HDAC2 S394 and hypertrophy-associated HDAC2 activation. HDAC2 S394 phosphorylation was increased in a dose-dependent manner by PP2A inhibitors. Hypertrophic stresses, such as phenylephrine in vitro or pressure overload in vivo, caused PPP2CA to dissociate from HDAC2. Forced expression of PPP2CA negatively regulated the hypertrophic response, but PP2A inhibitors provoked hypertrophy. Adenoviral delivery of a phosphomimic HDAC2 mutant, adenovirus HDAC2 S394E, successfully blocked the anti-hypertrophic effect of adenovirus-PPP2CA, implicating HDAC2 S394 phosphorylation as a critical event for the anti-hypertrophic response. PPP2CA transgenic mice were protected against isoproterenol-induced cardiac hypertrophy and subsequent cardiac fibrosis, whereas simultaneous expression of HDAC2 S394E in the heart did induce hypertrophy. Taken together, our results suggest that PP2A is a critical regulator of HDAC2 activity and pathological cardiac hypertrophy and is a promising target for future therapeutic interventions.Cardiovascular disease: A brake for heart muscle growthA regulatory mechanism that controls how cardiac muscle responds to stress could inform development of new therapies for preventing heart failure. Physiological stimuli ranging from heavy exercise to heart attack can induce hypertrophy, an increase in cardiac muscle mass that is initially beneficial but can lead to organ failure. Researchers led by Gwang Hyeon Eom and Hyun Kook at the Chonnam National University Biomedical Research Center, Hwasungun, South Korea have found that an enzyme called protein phosphatase 2A (PP2A) keeps cardiac hypertrophy in check. PP2A binds to and inhibits a second protein known as HDAC2, which would otherwise stimulate the hypertrophic response to stress. The researchers have identified the biochemical mechanism by which PP2A inactivates HDAC2, and demonstrate that this inhibition effectively protects against hypertrophic cardiac damage in mice, revealing a possible avenue for clinical intervention.