Bradley S. Ferguson

Class I HDACs regulate angiotensin II-dependent cardiac fibrosis via fibroblasts and circulating fibrocytes.

Fibrosis, which is defined as excessive accumulation of fibrous connective tissue, contributes to the pathogenesis of numerous diseases involving diverse organ systems. Cardiac fibrosis predisposes individuals to myocardial ischemia, arrhythmias and sudden death, and is commonly associated with diastolic dysfunction. Histone deacetylase (HDAC) inhibitors block cardiac fibrosis in pre-clinical models of heart failure. However, which HDAC isoforms govern cardiac fibrosis, and the mechanisms by which they do so, remains unclear. Here, we show that selective inhibition of class I HDACs potently suppresses angiotensin II (Ang II)-mediated cardiac fibrosis by targeting two key effector cell populations, cardiac fibroblasts and bone marrow-derived fibrocytes. Class I HDAC inhibition blocks cardiac fibroblast cell cycle progression through derepression of the genes encoding the cyclin-dependent kinase (CDK) inhibitors, p15 and p57. In contrast, class I HDAC inhibitors block agonist-dependent differentiation of fibrocytes through a mechanism involving repression of ERK1/2 signaling. These findings define novel roles for class I HDACs in the control of pathological cardiac fibrosis. Furthermore, since fibrocytes have been implicated in the pathogenesis of a variety of human diseases, including heart, lung and kidney failure, our results suggest broad utility for isoform-selective HDAC inhibitors as anti-fibrotic agents that function, in part, by targeting these circulating mesenchymal cells.

Impact of reference gene selection for target gene normalization on experimental outcome using real-time qRT-PCR in adipocytes

Background With the current rise in obesity-related morbidities, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become a widely used method for assessment of genes expressed and regulated by adipocytes. In order to measure accurate changes in relative gene expression and monitor intersample variability, normalization to endogenous control genes that do not change in relative expression is commonly used with qRT-PCR determinations. However, historical evidence has clearly demonstrated that the expression profiles of traditional control genes (e.g., β-actin, GAPDH, α-tubulin) are differentially regulated across multiple tissue types and experimental conditions. Methodology/Principal Findings Therefore, we validated six commonly used endogenous control genes under diverse experimental conditions of inflammatory stress, oxidative stress, synchronous cell cycle progression and cellular differentiation in 3T3-L1 adipocytes using TaqMan qRT-PCR. Under each study condition, we further evaluated the impact of reference gene selection on experimental outcome using examples of target genes relevant to adipocyte function and differentiation. We demonstrate that multiple reference genes are regulated in a condition-specific manner that is not suitable for use in target gene normalization. Conclusion/Significance Data are presented demonstrating that inappropriate reference gene selection can have profound influence on study conclusions ranging from divergent statistical outcome to inaccurate data interpretation of significant magnitude. This study validated the use of endogenous controls in 3T3-L1 adipocytes and highlights the impact of inappropriate reference gene selection on data interpretation and study conclusions.

Signal-dependent repression of DUSP5 by class I HDACs controls nuclear ERK activity and cardiomyocyte hypertrophy

Cardiac hypertrophy is a strong predictor of morbidity and mortality in patients with heart failure. Small molecule histone deacetylase (HDAC) inhibitors have been shown to suppress cardiac hypertrophy through mechanisms that remain poorly understood. We report that class I HDACs function as signal-dependent repressors of cardiac hypertrophy via inhibition of the gene encoding dual-specificity phosphatase 5 (DUSP5) DUSP5, a nuclear phosphatase that negatively regulates prohypertrophic signaling by ERK1/2. Inhibition of DUSP5 by class I HDACs requires activity of the ERK kinase, mitogen-activated protein kinase kinase (MEK), revealing a self-reinforcing mechanism for promotion of cardiac ERK signaling. In cardiac myocytes treated with highly selective class I HDAC inhibitors, nuclear ERK1/2 signaling is suppressed in a manner that is absolutely dependent on DUSP5. In contrast, cytosolic ERK1/2 activation is maintained under these same conditions. Ectopic expression of DUSP5 in cardiomyocytes results in potent inhibition of agonist-dependent hypertrophy through a mechanism involving suppression of the gene program for hypertrophic growth. These findings define unique roles for class I HDACs and DUSP5 as integral components of a regulatory signaling circuit that controls cardiac hypertrophy.

HDAC6 contributes to pathological responses of heart and skeletal muscle to chronic angiotensin-II signaling

Little is known about the function of the cytoplasmic histone deacetylase HDAC6 in striated muscle. Here, we addressed the role of HDAC6 in cardiac and skeletal muscle remodeling induced by the peptide hormone angiotensin II (ANG II), which plays a central role in blood pressure control, heart failure, and associated skeletal muscle wasting. Comparable with wild-type (WT) mice, HDAC6 null mice developed cardiac hypertrophy and fibrosis in response to ANG II. However, whereas WT mice developed systolic dysfunction upon treatment with ANG II, cardiac function was maintained in HDAC6 null mice treated with ANG II for up to 8 wk. The cardioprotective effect of HDAC6 deletion was mimicked in WT mice treated with the small molecule HDAC6 inhibitor tubastatin A. HDAC6 null mice also exhibited improved left ventricular function in the setting of pressure overload mediated by transverse aortic constriction. HDAC6 inhibition appeared to preserve systolic function, in part, by enhancing cooperativity of myofibrillar force generation. Finally, we show that HDAC6 null mice are resistant to skeletal muscle wasting mediated by chronic ANG-II signaling. These findings define novel roles for HDAC6 in striated muscle and suggest potential for HDAC6-selective inhibitors for the treatment of cardiac dysfunction and muscle wasting in patients with heart failure.

Tubulin hyperacetylation is adaptive in cardiac proteotoxicity by promoting autophagy

Significance Proteotoxicity, or the accumulation of misfolded protein, can cause heart failure and effective therapeutics are needed to reduce protein accumulation in the myocardium. This study shows that inhibiting tubulin deacetylation by histone deacetylase 6 (HDAC6) is protective in a mouse model of proteinopathy-induced heart failure. Inhibiting tubulin deacetylation using the FDA-approved drug suberoylanilide hydroxamic acid (SAHA) reduced protein aggregates in cardiomyocytes and led to substantial improvement in cardiac function. Mechanistically, we show that inhibiting HDAC6 increases autophagy in cardiomyocytes, and that inducing autophagy with voluntary exercise also induces tubulin acetylation. This study shows that tubulin acetylation is important for autophagy stimulation in the heart and, importantly, sheds new light on the mechanism of autophagy induction with HDAC inhibitors. Proteinopathy causes cardiac disease, remodeling, and heart failure but the pathological mechanisms remain obscure. Mutated αB-crystallin (CryABR120G), when expressed only in cardiomyocytes in transgenic (TG) mice, causes desmin-related cardiomyopathy, a protein conformational disorder. The disease is characterized by the accumulation of toxic misfolded protein species that present as perinuclear aggregates known as aggresomes. Previously, we have used the CryABR120G model to determine the underlying processes that result in these pathologic accumulations and to explore potential therapeutic windows that might be used to decrease proteotoxicity. We noted that total ventricular protein is hypoacetylated while hyperacetylation of α-tubulin, a substrate of histone deacetylase 6 (HDAC6) occurs. HDAC6 has critical roles in protein trafficking and autophagy, but its function in the heart is obscure. Here, we test the hypothesis that tubulin acetylation is an adaptive process in cardiomyocytes. By modulating HDAC6 levels and/or activity genetically and pharmacologically, we determined the effects of tubulin acetylation on aggregate formation in CryABR120G cardiomyocytes. Increasing HDAC6 accelerated aggregate formation, whereas siRNA-mediated knockdown or pharmacological inhibition ameliorated the process. HDAC inhibition in vivo induced tubulin hyperacetylation in CryABR120G TG hearts, which prevented aggregate formation and significantly improved cardiac function. HDAC6 inhibition also increased autophagic flux in cardiomyocytes, and increased autophagy in the diseased heart correlated with increased tubulin acetylation, suggesting that autophagy induction might underlie the observed cardioprotection. Taken together, our data suggest a mechanistic link between tubulin hyperacetylation and autophagy induction and points to HDAC6 as a viable therapeutic target in cardiovascular disease.

Inflammatory cytokines epigenetically regulate rheumatoid arthritis fibroblast-like synoviocyte activation by suppressing HDAC5 expression

Objectives Epigenetic modifications play an important role in the regulation of gene transcription and cellular function. Here, we examined if pro-inflammatory factors present in the inflamed joint of patients with rheumatoid arthritis (RA) could regulate histone deacetylase (HDAC) expression and function in fibroblast-like synoviocytes (FLS). Methods Protein acetylation in synovial tissue was assessed by immunohistochemistry. The mRNA levels of HDAC family members and inflammatory mediators in the synovial tissue and the changes in HDAC expression in RA FLS were measured by quantitative (q) PCR. FLS were either transfected with HDAC5 siRNA or transduced with adenoviral vector encoding wild-type HDAC5 and the effects of HDAC5 manipulation were examined by qPCR arrays, ELISA and ELISA-based assays. Results Synovial class I HDAC expression was associated with local expression of tumour necrosis factor (TNF) and matrix metalloproteinase-1, while class IIa HDAC5 expression was inversely associated with parameters of disease activity (erythrocyte sedimentation rate, C-reactive protein, Disease Activity Score in 28 Joints). Interleukin (IL)-1β or TNF stimulation selectively suppressed HDAC5 expression in RA FLS, which was sufficient and required for optimal IFNB, CXCL9, CXCL10 and CXCL11 induction by IL-1β, associated with increased nuclear accumulation of the transcription factor, interferon regulatory factor 1(IRF1). Conclusions Inflammatory cytokines suppress RA FLS HDAC5 expression, promoting nuclear localisation of IRF1 and transcription of a subset of type I interferon response genes. Our results identify HDAC5 as a novel inflammatory mediator in RA, and suggest that strategies rescuing HDAC5 expression in vivo, or the development of HDAC inhibitors not affecting HDAC5 activity, may have therapeutic applications in RA treatment.

Non-sirtuin histone deacetylases in the control of cardiac aging.

Histone deacetylases (HDACs) catalyze the removal of acetyl-groups from lysine residues within nucelosomal histone tails and thousands of non-histone proteins. The 18 mammalian HDACs are grouped into four classes. Classes I, II and IV HDACs employ zinc as a co-factor for catalytic activity, while class III HDACs (also known as sirtuins) require NAD+ for enzymatic function. Small molecule inhibitors of zinc-dependent HDACs are efficacious in multiple pre-clinical models of pressure overload and ischemic cardiomyopathy, reducing pathological hypertrophy and fibrosis, and improving contractile function. Emerging data have revealed numerous mechanisms by which HDAC inhibitors benefit the heart, including suppression of oxidative stress and inflammation, inhibition of MAP kinase signaling, and enhancement of cardiac protein aggregate clearance and autophagic flux. Here, we summarize recent findings with zinc-dependent HDACs and HDAC inhibitors in the heart, focusing on newly described functions for distinct HDAC isoforms (e.g. HDAC2, HDAC3 and HDAC6). Potential for pharmacological HDAC inhibition as a means of treating age-related cardiac dysfunction is also discussed. This article is part of a Special Issue entitled: CV Aging.

Histone deacetylase 3 regulates the inflammatory gene expression programme of rheumatoid arthritis fibroblast-like synoviocytes

Objectives Non-selective histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated anti-inflammatory properties in both in vitro and in vivo models of rheumatoid arthritis (RA). Here, we investigated the potential contribution of specific class I and class IIb HDACs to inflammatory gene expression in RA fibroblast-like synoviocytes (FLS). Methods RA FLS were incubated with pan-HDACi (ITF2357, givinostat) or selective HDAC1/2i, HDAC3/6i, HDAC6i and HDAC8i. Alternatively, FLS were transfected with HDAC3, HDAC6 or interferon (IFN)-α/β receptor alpha chain (IFNAR1) siRNA. mRNA expression of interleukin (IL)-1β-inducible genes was measured by quantitative PCR (qPCR) array and signalling pathway activation by immunoblotting and DNA-binding assays. Results HDAC3/6i, but not HDAC1/2i and HDAC8i, significantly suppressed the majority of IL-1β-inducible genes targeted by pan-HDACi in RA FLS. Silencing of HDAC3 expression reproduced the effects of HDAC3/6i on gene regulation, contrary to HDAC6-specific inhibition and HDAC6 silencing. Screening of the candidate signal transducers and activators of transcription (STAT)1 transcription factor revealed that HDAC3/6i abrogated STAT1 Tyr701 phosphorylation and DNA binding, but did not affect STAT1 acetylation. HDAC3 activity was required for type I IFN production and subsequent STAT1 activation in FLS. Suppression of type I IFN release by HDAC3/6i resulted in reduced expression of a subset of IFN-dependent genes, including the chemokines CXCL9 and CXCL11. Conclusions Inhibition of HDAC3 in RA FLS largely recapitulates the effects of pan-HDACi in suppressing inflammatory gene expression, including type I IFN production in RA FLS. Our results identify HDAC3 as a potential therapeutic target in the treatment of RA and type I IFN-driven autoimmune diseases.

Curcumin inhibits 3T3-L1 preadipocyte proliferation by mechanisms involving post-transcriptional p27 regulation

Previous reports from our lab have shown that Skp2 is necessary for p27 degradation and cell cycle progression during adipocyte differentiation. Data presented here demonstrate that the anti-inflammatory, anti-obesity phytochemical curcumin blocked Skp2 protein accumulation during early adipocyte hyperplasia. In addition, curcumin dose-dependently induced p27 protein accumulation and G1 arrest of synchronously replicating 3T3-L1 preadipocytes. Of note, p27 protein accumulation occurred in the presence of decreased p27 mRNA suggesting a role for post-transcriptional regulation. In support of this hypothesis, curcumin markedly increased p27 protein half-life as well as attenuated ubiquitin proteasome activity suggesting that inhibition of targeted p27 proteolysis occurred through curcumin-mediated attenuation of Skp2 and 26S proteasome activity. While we observed no cytotoxic effects for curcumin at doses less than 20 µM, it is important to note an increase in apoptotic signaling at concentrations greater than 30 µM. Finally, data presented here demonstrate that the anti-proliferative effect of curcumin was critical for the suppression of adipocyte differentiation and the development of the mature adipocyte. Collectively, our data demonstrate that curcumin-mediated post-transcriptional accumulation of p27 accounts in part for the anti-proliferative effect observed in 3T3-L1 preadipocytes.

Impact of Obesity on IL-12 Family Gene Expression in Insulin Responsive Tissues

Mounting evidence has established a role for chronic inflammation in the development of obesity-induced insulin resistance, as genetic ablation of pro-inflammatory cytokines and chemokines elevated in obesity improves insulin signaling in vitro and in vivo. Recent evidence further highlights interleukin (IL)-12 family cytokines as prospective inflammatory mediators linking obesity to insulin resistance. In this study, we present empirical evidence demonstrating that IL-12 family related genes are expressed and regulated in insulin-responsive tissues under conditions of obesity. First, we report that respective mRNAs for each of the known members of this cytokine family are expressed within detectable ranges in WAT, skeletal muscle, liver and heart. Second, we show that these cytokines and their cognate receptors are divergently regulated with genetic obesity in a tissue-specific manner. Third, we demonstrate that select IL-12 family cytokines are regulated in WAT in a manner that is dependent on the developmental stage of obesity as well as the inflammatory progression associated with obesity. Fourth, we report that respective mRNAs for IL-12 cytokines and receptors are also expressed and divergently regulated in cultured adipocytes under conditions of inflammatory stress. To our knowledge, this report is the first study to systemically evaluated mRNA expression of all IL-12 family cytokines and receptors in any tissue under conditions of obesity highlighting select family members as potential mediators linking excess nutrient intake to metabolic diseases such as insulin resistance, diabetes and heart disease.