SNHG16/miR-205/HDAC5 is involved in the progression of renal fibrosis.

Renal interstitial fibrosis (RIF) represents an irreversible and progressive pathological manifestation of chronic renal disease, which ultimately leads to end-stage renal disease. Long noncoding RNAs (lncRNAs) have been suggested to be involved in the progression of RIF. Small nucleolar RNA host gene 16 (SNHG16), a member of lncRNAs, has been found to be involved in the progression of pulmonary fibrosis. This paper first researched the effect of SNHG16 on renal fibrosis. We established a unilateral ureteral obstruction (UUO)-induced mouse RIF model by ligation of the left ureter to evaluate the biological function of SNHG16 in RIF. As a result, SNHG16 was upregulated in UUO-induced renal fibrotic tissues. Knockdown of SNHG16 inhibited RIF and reduced alpha-smooth muscle actin (α-SMA), fibronectin, and college IV expression. miR-205 was a target of SNHG16, and downregulated in UUO-induced renal fibrotic tissues. Inhibition of miR-205 promoted RIF and increased the expression of α-SMA, college IV, and fibronectin. Overexpression of SNHG16 promoted the UUO-induced RIF, but miR-205 abrogated this effect of SNHG16. Histone deacetylase 5 (HDAC5) showed high expression in UUO-induced renal fibrotic tissues. Knockdown of HDAC5 significantly reduced α-SMA, fibronectin, and college IV expression in renal tissues of UUO-induced mice. Inhibition of miR-205 promoted HDAC5 expression, but knockdown of SNHG16 inhibited HDAC5 expression in renal tissues of UUO-induced mice. In conclusion, SHNG16 is highly expressed in renal fibrotic tissues of UUO-induced mice. Knockdown of SHNG16 may prevent UUO-induced RIF by indirectly upregulating HDAC5 via targeting miR-205. SHNG16 may be novel target for treating renal fibrosis.

Epigenetics.

Epigenetics is the study of heritable changes to the genome and gene expression patterns that are not caused by direct changes to the DNA sequence. Examples of these changes include posttranslational modifications to DNA-bound histone proteins, DNA methylation, and remodeling of nuclear architecture. Collectively, epigenetic changes provide a layer of regulation that affects transcriptional activity of genes while leaving DNA sequences unaltered. Sequence variants or mutations affecting enzymes responsible for modifying or sensing epigenetic marks have been identified in patients with congenital heart disease (CHD), and small-molecule inhibitors of epigenetic complexes have shown promise as therapies for adult heart diseases. Additionally, transgenic mice harboring mutations or deletions of genes encoding epigenetic enzymes recapitulate aspects of human cardiac disease. Taken together, these findings suggest that the evolving field of epigenetics will inform our understanding of congenital and adult cardiac disease and offer new therapeutic opportunities.

Histone deacetylase 5-induced deficiency of signal transducer and activator of transcription-3 acetylation contributes to spinal astrocytes degeneration in painful diabetic neuropathy.

Diabetes patients with painful diabetic neuropathy (PDN) show severe spinal atrophy, suggesting pathological changes of the spinal cord contributes to central sensitization. However, the cellular changes and underlying molecular mechanisms within the diabetic spinal cord are less clear. By using a rat model of type 1 diabetes (T1D), we noted an extensive and irreversible spinal astrocyte degeneration at an early stage of T1D, which is highly associated with the chronification of PDN. Molecularly, acetylation of astrocytic signal transducer and activator of transcription-3 (STAT3) that is essential for maintaining the homeostatic astrocytes population was significantly impaired in the T1D model, resulting in a dramatic loss of spinal astrocytes and consequently promoting pain hypersensitivity. Mechanistically, class IIa histone deacetylase, HDAC5 were aberrantly activated in spinal astrocytes of diabetic rats, which promoted STAT3 deacetylation by direct protein-protein interactions, leading to the PDN phenotypes. Restoration of STAT3 signaling or inhibition of HDAC5 rescued astrocyte deficiency and attenuated PDN in the T1D model. Our work identifies the inhibitory axis of HDAC5-STAT3 induced astrocyte deficiency as a key mechanism underlying the pathogenesis of the diabetic spinal cord that paves the way for potential therapy development for PDN.

Synergistic Interaction of the Class IIa HDAC Inhibitor CHDI0039 with Bortezomib in Head and Neck Cancer Cells.

In contrast to class I/IIb/pan histone deacetylase inhibitors (HDACi), the role of class IIa HDACi as anti-cancer chemosensitizing agents is less well understood. Here, we studied the effects of HDAC4 in particular and the class IIa HDACi CHDI0039 on proliferation and chemosensitivity in Cal27 and cisplatin-resistant Cal27CisR head and neck squamous cell cancer (HNSCC). HDAC4 and HDAC5 overexpression clones were generated. HDAC4 overexpression (Cal27_HDAC4) increased proliferation significantly compared to vector control cells (Cal27_VC). Chicken chorioallantoic membrane (CAM) studies confirmed the in vitro results: Cal27_HDAC4 tumors were slightly larger than tumors from Cal27_VC, and treatment with CHDI0039 resulted in a significant decrease in tumor size and weight of Cal27_HDAC4 but not Cal27_VC. Unlike class I/pan-HDACi, treatment with CHDI0039 had only a marginal impact on cisplatin cytotoxicity irrespective of HDAC4 and HDAC5 expression. In contrast, the combination of CHDI0039 with bortezomib was synergistic (Chou-Talalay) in MTT and caspase 3/7 activation experiments. RNAseq indicated that treatment with CHDI0039 alters the expression of genes whose up- or downregulation is associated with increased survival in HNSCC patients according to Kaplan-Meier data. We conclude that the combination of class IIa HDACi with proteasome inhibitors constitutes an effective treatment option for HNSCC, particularly for platinum-resistant cancers.

The Dual Functions of Andrographolide in the Epstein-Barr Virus-Positive Head-and-Neck Cancer Cells: The Inhibition of Lytic Reactivation of the Epstein-Barr Virus and the Induction of Cell Death.

Andrographolide, a medicinal compound, exhibits several pharmacological activities, including antiviral and anticancer properties. Previously, we reported that andrographolide inhibits Epstein-Barr virus (EBV) lytic reactivation, which is associated with viral transmission and oncogenesis in epithelial cancers, including head-and-neck cancer (HNC) cells. However, the underlying mechanism through which andrographolide inhibits EBV lytic reactivation and affects HNC cells is poorly understood. Therefore, we investigated these mechanisms using EBV-positive HNC cells and the molecular modeling and docking simulation of protein. Based on the results, the expression of EBV lytic genes and viral production were significantly inhibited in andrographolide-treated EBV-positive HNC cells. Concurrently, there was a reduction in transcription factors (TFs), myocyte enhancer factor-2D (MEF2D), specificity protein (SP) 1, and SP3, which was significantly associated with a combination of andrographolide and sodium butyrate (NaB) treatment. Surprisingly, andrographolide treatment also significantly induced the expression of DNA Methyltransferase (DNMT) 1, DNMT3B, and histone deacetylase (HDAC) 5 in EBV-positive cells. Molecular modeling and docking simulation suggested that HDAC5 could directly interact with MEF2D, SP1, and SP3. In our in vitro study, andrographolide exhibited a stronger cytotoxic effect on EBV-positive cells than EBV-negative cells by inducing cell death. Interestingly, the proteome analysis revealed that the expression of RIPK1, RIPK3, and MLKL, the key molecules for necroptosis, was significantly greater in andrographolide-treated cells. Taken together, it seems that andrographolide exhibits concurrent activities in HNC cells; it inhibits EBV lytic reactivation by interrupting the expression of TFs and induces cell death, probably via necroptosis.

Histone deacetylase 5 deacetylates the phosphatase PP2A for positively regulating NF-κB signaling.

Histone deacetylase 5 (HDAC5) has been reported to have a strong regulatory function in the proinflammatory response, but the mechanism is still unknown. Here, we identified HDAC5 as a positive regulator of NF-κB signaling in vivo. HDAC5-deficient mice exhibited enhanced survival in response to LPS challenge. Using LPS, TNFα, different kinds of viruses, hydrogen peroxide, or ultraviolet stimulation, we demonstrate that HDAC5-mediated regulation of NF-κB occurs in manners both dependent on and independent of IKK, an upstream kinase in the NF-κB signaling pathway. Deficiency in HDAC5 impaired the phosphorylation of IKKß, subsequent phosphorylation of the NF-κB inhibitor protein IκBα and NF-κB subunit p65. We also show that the phosphatase PP2A repressed transcriptional activation of NF-κB by decreasing phosphorylation of IKKß, p65, and IκBα. In vitro deacetylation experiments and site-directed mutagenesis experiments indicated that HDAC5 directly deacetylated PP2Ac at Lys136, which resulted in the deactivation of PP2A. Our data add mechanistic insight into the cross talk between epigenetic and posttranslational modifications regulating NF-κB signaling and protein phosphatase activation that mediate survival in response to inflammatory challenges.

HDAC5, negatively regulated by miR-148a-3p, promotes colon cancer cell migration.

Histone deacetylases (HDACs) are involved in many processes including tumor cell growth and proliferation and regulation of gene expression. To clarify the role of class IIa HDACs in the metastasis of colon adenocarcinoma, we used the class IIa HDAC inhibitor TMP269 and found that it effectively inhibited the migration ability of colon adenocarcinoma cells. Next, we silenced the member of class IIa HDACs and confirmed that the migratory ability of colon adenocarcinoma cells was significantly inhibited by silencing HDAC5 or HDAC7. HDAC5 plays a variety of roles in human cancers. Here, we examined the role of HDAC5 in colon adenocarcinoma. The results indicated that HDAC5 was highly expressed in tumor tissues and negatively correlated with the expression of miR-148a-3p. Moreover, the expression of HDAC5 was correlated with tumor progression. HDAC5 markedly increased the invasion and migration of cancer cells in vitro, an effect that could be inhibited by overexpression of miR-148a-3p. Following an intraperitoneal injection of colon adenocarcinoma cells in athymic nude mice, HDAC5 promoted tumor implant. Together, these findings showed that HDAC5 overexpression in colon adenocarcinoma is consistent with tumor progression and tumor cell migration and the impact of HDAC5 overexpression is reduced by miR-148a-3p.

HDAC5 inactivates CYR61-regulated CD31/mTOR axis to prevent the occurrence of preeclampsia.

Our study was to pinpoint the significance of histone deacetylase 5 (HDAC5) affecting the pathogenesis of preeclampsia (PE) via CD31/mammalian target of rapamycin (mTOR) axis by regulating cysteine-rich angiogenic inducer 61 (CYR61). Expression of HDAC5, CYR61, and CD31/mTOR in placental tissues of patients with PE and trophoblast cells HTR-8/SVneo cells was determined first followed by their interaction analysis. Following different transfection, the significance of HDAC5 in cell functions was assayed in relation to CYR61 and CD31/mTOR. An in vivo PE mouse model was constructed for further validation. The clinical tissue and in vitro cell experimentations discovered that HDAC5 was downregulated in placental tissues of PE patients and trophoblast cells, while CYR61, CD31, mTOR, and p-mTOR displayed upregulation. After overexpression of HDAC5, trophoblast cell functions were enhanced. HDAC5 reduced the acetylation enrichment of H3K27 to inhibit the expression of CYR61. Furthermore, CYR61 promoted the activation of CD31/mTOR axis, thereby inhibiting HTR-8/SVneo cell functions. The in vivo rat model confirmed the above alterations. Taken together, HDAC5 contributes to downregulation of CYR61 through histone deacetylation, inactivating CD31/mTOR axis, which prevents the occurrence and development of PE.

HDAC5 promotes intestinal sepsis via the Ghrelin/E2F1/NF-κB axis.

In the current study, we sought to determine the roles of histone deacetylase 5 (HDAC5) on the promotion of intestinal sepsis in a mouse model. Dual luciferase reporter gene assay was used to determine the binding relationship between HDAC5 and Ghrelin. Cecal ligation and puncture (CLP) was used as an animal model of intestinal sepsis. The roles of HDAC5 on intestinal sepsis were determined by HDAC5 knockdown, overexpression, and inhibitor (LMK-235) in vivo. Mice intestinal permeability and intestinal epithelial damage were evaluated, and HE staining was used to evaluate the intestinal mucosal injury index. Lipopolysaccharide (LPS)-treated intestinal-derived macrophages served as a cell model of sepsis, followed by the loss-of-function and gain-of-function assays. ELISA was used to determine the levels of inflammatory factors, and TUNEL staining was used to detect intestinal cell apoptosis. HDAC5 was upregulated in the intestine of sepsis patients. This increased HDAC5 expression was positively correlated with the expression of inflammatory factors TNF-α, IL-1ß, IL-6, and HMGB1, as well as the intestinal dysfunction-related factors IFABP. In sepsis mice, the expression of inflammatory factors was reduced by HDAC5 knockdown. HDAC5 knockdown also improved survival, morphology of intestinal tissue, intestinal permeability, and epithelial damage. Ghrelin was bound and inhibited by HDAC5, but E2F1 expression was increased by Ghrelin overexpression, leading to inhibition of the NF-κB pathway. Ghrelin and E2F1 expression were increased by the treatment with HDAC5 inhibitor LMK-235, which inhibited the NF-κB pathway to improve intestinal dysfunction in the sepsis model. In conclusion, HDAC5 inhibits Ghrelin to reduce E2F1 and thus activate the NF-κB pathway, thereby promoting intestinal sepsis.

Peripheral administration of the Class-IIa HDAC inhibitor MC1568 partially protects against nigrostriatal neurodegeneration in the striatal 6-OHDA rat model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterised by nigrostriatal dopaminergic (DA) neurodegeneration. There is a critical need for neuroprotective therapies, particularly those that do not require direct intracranial administration. Small molecule inhibitors of histone deacetylases (HDIs) are neuroprotective in in vitro and in vivo models of PD, however it is unknown whether Class IIa-specific HDIs are neuroprotective when administered peripherally. Here we show that 6-hydroxydopamine (6-OHDA) treatment induces protein kinase C (PKC)-dependent nuclear accumulation of the Class IIa histone deacetylase (HDAC)5 in SH-SY5Y cells and cultured DA neurons in vitro. Treatment of these cultures with the Class IIa-specific HDI, MC1568, partially protected against 6-OHDA-induced cell death. In the intrastriatal 6-OHDA lesion in vivo rat model of PD, MC1568 treatment (0.5 mg/kg i.p.) for 7 days reduced forelimb akinesia and partially protected DA neurons in the substantia nigra and their striatal terminals from 6-OHDA-induced neurodegeneration. MC1568 treatment prevented 6-OHDA-induced increases in microglial activation in the striatum and substantia nigra. Furthermore, MC1568 treatment decreased 6-OHDA-induced increases in nuclear HDAC5 in nigral DA neurons. These data suggest that peripheral administration of Class IIa-specific HDIs may be a potential therapy for neuroprotective in PD.

LMK235, a small molecule inhibitor of HDAC4/5, protects dopaminergic neurons against neurotoxin- and α-synuclein-induced degeneration in cellular models of Parkinson's disease.

Epigenetic modifications in neurodegenerative disease are under investigation for their roles in disease progression. Alterations in acetylation rates of certain Parkinson's disease (PD)-linked genes have been associated with the pathological progression of this disorder. In light of this, and given the lack of disease-modifying therapies for PD, HDAC inhibitors (HDIs) are under consideration as potential pharmacological agents. The neuroprotective effects of pan-HDACs and some class-specific inhibitors have been tested in in vivo and in vitro models of PD, with varying outcomes. Here we used gene co-expression analysis to identify HDACs that are associated with human dopaminergic (DA) neuron development. We identified HDAC3, HDAC5, HDAC6 and HDAC9 as being highly correlated with the DA markers, SLC6A3 and NR4A2. RT-qPCR revealed that mRNA expression of these HDACs exhibited similar temporal profiles during embryonic mouse midbrain DA (mDA) neuron development. We tested the neuroprotective potential of a number of class-specific small molecule HDIs on human SH-SY5Y cells, using neurite growth as a phenotypic readout of neurotrophic action. Neither the class I-specific HDIs, RGFP109 and RGFP966, nor the HDAC6 inhibitor ACY1215, had significant effects on neurite outgrowth. However, the class IIa HDI, LMK235 (a HDAC4/5 inhibitor), significantly increased histone acetylation and neurite outgrowth. We found that LMK235 increased BMP-Smad-dependent transcription in SH-SY5Y cells and that this was required for its neurite growth-promoting effects on SH-SY5Y cells and on DA neurons in primary cultures of embryonic day (E) 14 rat ventral mesencephalon (VM). These effects were also seen in SH-SY5Y cells transfected with HDAC5 siRNA. Furthermore, LMK235 treatment exerted neuroprotective effects against degeneration induced by the DA neurotoxin 1-methyl-4-phenylpyridinium (MPP+), in both SH-SY5Y cells and cultured DA neurons. Treatment with LMK235 was also neuroprotective against axonal degeneration induced by overexpression of wild-type (WT) or A53T mutant α-synuclein in both SH-SY5Y cells and primary cultures of DA neurons. In summary, these data show the neuroprotective potential of the class IIa HDI, LMK235, in cell models of relevance to PD.

Hdac4 Interactions in Huntington's Disease Viewed Through the Prism of Multiomics.

Huntington's disease (HD) is a monogenic disorder, driven by the expansion of a trinucleotide (CAG) repeat within the huntingtin (Htt) gene and culminating in neuronal degeneration in the brain, predominantly in the striatum and cortex. Histone deacetylase 4 (Hdac4) was previously found to contribute to the disease progression, providing a potential therapeutic target. Hdac4 knockdown reduced accumulation of misfolded Htt protein and improved HD phenotypes. However, the underlying mechanism remains unclear, given its independence on deacetylase activity and the predominant cytoplasmic Hdac4 localization in the brain. Here, we undertook a multiomics approach to uncover the function of Hdac4 in the context of HD pathogenesis. We characterized the interactome of endogenous Hdac4 in brains of HD mouse models. Alterations in interactions were investigated in response to Htt polyQ length, comparing mice with normal (Q20) and disease (Q140) Htt, at both pre- and post-symptomatic ages (2 and 10 months, respectively). Parallel analyses for Hdac5, a related class IIa Hdac, highlighted the unique interaction network established by Hdac4. To validate and distinguish interactions specifically enhanced in an HD-vulnerable brain region, we next characterized endogenous Hdac4 interactions in dissected striata from this HD mouse series. Hdac4 associations were polyQ-dependent in the striatum, but not in the whole brain, particularly in symptomatic mice. Hdac5 interactions did not exhibit polyQ dependence. To identify which Hdac4 interactions and functions could participate in HD pathogenesis, we integrated our interactome with proteome and transcriptome data sets generated from the striata. We discovered an overlap in enriched functional classes with the Hdac4 interactome, particularly in vesicular trafficking and synaptic functions, and we further validated the Hdac4 interaction with the Wiskott-Aldrich Syndrome Protein and SCAR Homolog (WASH) complex. This study expands the knowledge of Hdac4 regulation and functions in HD, adding to the understanding of the molecular underpinning of HD phenotypes.

Whole Exome Sequencing Identifies APCDD1 and HDAC5 Genes as Potentially Cancer Predisposing in Familial Colorectal Cancer.

Germline mutations in predisposition genes account for only 20% of all familial colorectal cancers (CRC) and the remaining genetic burden may be due to rare high- to moderate-penetrance germline variants that are not explored. With the aim of identifying such potential cancer-predisposing variants, we performed whole exome sequencing on three CRC cases and three unaffected members of a Polish family and identified two novel heterozygous variants: a coding variant in APC downregulated 1 gene (APCDD1, p.R299H) and a non-coding variant in the 5' untranslated region (UTR) of histone deacetylase 5 gene (HDAC5). Sanger sequencing confirmed the variants segregating with the disease and Taqman assays revealed 8 additional APCDD1 variants in a cohort of 1705 familial CRC patients and no further HDAC5 variants. Proliferation assays indicated an insignificant proliferative impact for the APCDD1 variant. Luciferase reporter assays using the HDAC5 variant resulted in an enhanced promoter activity. Targeting of transcription factor binding sites of SNAI-2 and TCF4 interrupted by the HDAC5 variant showed a significant impact of TCF4 on promoter activity of mutated HDAC5. Our findings contribute not only to the identification of unrecognized genetic causes of familial CRC but also underline the importance of 5'UTR variants affecting transcriptional regulation and the pathogenesis of complex disorders.

Muscle A-kinase-anchoring protein-ß-bound calcineurin toggles active and repressive transcriptional complexes of myocyte enhancer factor 2D.

Myocyte enhancer factor 2 (MEF2) transcription factors are key regulators of the development and adult phenotype of diverse tissues, including skeletal and cardiac muscles. Controlled by multiple post-translational modifications, MEF2D is an effector for the Ca2+/calmodulin-dependent protein phosphatase calcineurin (CaN, PP2B, and PPP3). CaN-catalyzed dephosphorylation promotes the desumoylation and acetylation of MEF2D, increasing its transcriptional activity. Both MEF2D and CaN bind the scaffold protein muscle A-kinase-anchoring protein ß (mAKAPß), which is localized to the nuclear envelope, such that C2C12 skeletal myoblast differentiation and neonatal rat ventricular myocyte hypertrophy are inhibited by mAKAPß signalosome targeting. Using immunoprecipitation and DNA-binding assays, we now show that the formation of mAKAPß signalosomes is required for MEF2D dephosphorylation, desumoylation, and acetylation in C2C12 cells. Reduced MEF2D phosphorylation was coupled to a switch from type IIa histone deacetylase to p300 histone acetylase binding that correlated with increased MEF2D-dependent gene expression and ventricular myocyte hypertrophy. Together, these results highlight the importance of mAKAPß signalosomes for regulating MEF2D activity in striated muscle, affirming mAKAPß as a nodal regulator in the myocyte intracellular signaling network.

Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension.

Here, we report that LMK235, a class I and histone deacetylase (HDAC6)-preferential HDAC inhibitor, reduces hypertension via inhibition of vascular contraction and vessel hypertrophy. Angiotensin II-infusion mice and spontaneously hypertensive rats (SHRs) were used to test the anti-hypertensive effect of LMK235. Daily injection of LMK235 lowered angiotensin II-induced systolic blood pressure (BP). A reduction in systolic BP in SHRs was observed on the second day when SHRs were treated with 3 mg/kg LMK235 every 3 days. However, LMK235 treatment did not affect angiotensin-converting enzyme 1 and angiotensin II receptor mRNA expression in either hypertensive model. LMK235, acting via the nitric oxide pathway, facilitated the relaxing of vascular contractions induced by a thromboxane A2 agonist in the rat aortic and mesenteric artery ring test. In addition, LMK235 increased nitric oxide production in HUVECs and inhibited the increasing of aortic wall thickness in both animal hypertensive models. LMK235 decreased the enhanced cell cycle-related genes cyclin D1 and E2F3 in angiotensin II-infusion mice and restored the decreased p21 expression. In addition, LMK235 suppressed calcium calmodulin-dependent protein kinase II (CaMKII) α, which is related to vascular smooth muscle cell proliferation. Inhibition or knockdown of HDAC5 blocked the CaMKIIα-induced cell cycle gene expression. Immunoprecipitation demonstrated that class I HDACs were involved in the inhibition of CaMKII α-induced HDAC4/5 by LMK235. We suggest that LMK235 should be further investigated for its use in the development of new therapeutic options to treat hypertension via reducing vascular hyperplasia or vasoconstriction.

Central adiponectin induces trabecular bone mass partly through epigenetic downregulation of cannabinoid receptor CB1.

Central adiponectin (APN) in either the globular (gAPN) or full-length forms decreases sympathetic tone and increases trabecular bone mass in mice through the hypothalamus. It is known that cannabinoid type-1 (CB1) receptors are expressed in the hypothalamic ventromedial nucleus and participate in energy metabolism by controlling sympathetic activity. However, whether central APN could influence endocannabinoid signaling through CB1 receptor to regulate bone metabolism has not been characterized. Here we demonstrate that gAPN downregulated CB1 expression in embryonic mouse hypothalamus N1 cells in vitro. gAPN intracerebroventricular (icv) infusions also decreased hypothalamic CB1 expression and bone formation parameters in APN-knockout (APN-KO) and wild-type mice. Most importantly, mice pretreated with icv infusions with the CB1 receptor agonist arachidonyl-2'-chloroethylamine or antagonist rimonabant attenuated or enhanced respectively central APN induction of bone formation. We then investigated whether epigenetic signaling mechanisms were involved in the downregulation of hypothalamic CB1 expression by gAPN. We found gAPN enhanced expression levels of various histone deacetylases (HDACs), especially HDAC5. Furthermore, chromatin immunoprecipitation assays revealed HDAC5 bound to the transcriptional start site transcription start site 2 region of the CB1 promoter. In summary, our study identified a possible novel central APN-HDAC5-CB1 signaling mechanism that promotes peripheral bone formation through epigenetic regulation of hypothalamic CB1 expression.

Protein kinase B/AKT mediates insulin-like growth factor 1-induced phosphorylation and nuclear export of histone deacetylase 5 via NADPH oxidase 4 activation in vascular smooth muscle cells.

Insulin-like growth factor 1 (IGF-1) mediates the generation of reactive oxygen species (ROS) and the activation of growth promoting signaling pathways. Histone deacetylases (HDACs) regulate gene transcription by deacetylating lysine residues in histone and nonhistone proteins and a heightened HDAC activation, notably of HDAC5, is associated with vascular disorders, such as atherosclerosis. Although the contribution of IGF-1 in these pathologies is well documented, its role in HDAC phosphorylation and activation remains unexplored. Here, we examined the effect of IGF-1 on HDAC5 phosphorylation in vascular smooth muscle cells (VSMCs) and identified the signaling pathways involved in controlling HDAC5 phosphorylation and nuclear export. Treatment of A10 VSMCs with IGF-1 enhanced HDAC5 phosphorylation. Blockade of the IGF-1 receptor tyrosine kinase (TK) activity with the specific pharmacological inhibitor, AG1024, significantly inhibited IGF-1-induced HDAC5 phosphorylation, whereas the epidermal growth factor receptor (EGFR) TK antagonist, AG1478, had no effect. Inhibition of the mitogen-activated protein kinase pathway with U0126, SP600125, or SB203580, did not affect HDAC5 phosphorylation, whereas two inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT pathways, wortmannin and SC66, almost completely attenuated IGF-1-induced responses as confirmed by immunoblotting of phospho-HDAC5 and by small interfering RNA (siRNA)-induced AKT silencing. Moreover, the NAD(P)H oxidase (Nox) inhibitor, diphenyleneiodonium (DPI), and Nox4 siRNA, attenuated IGF-1-induced phosphorylation of HDAC5 and AKT. The HDAC5 phosphorylation resulted in its nuclear export, which was reversed by SC66 and DPI. Our results indicate that IGF-1-induced phosphorylation and nuclear export of HDAC5 involve Nox4-dependent ROS generation and PI3K/AKT signaling pathways.

Elevated p70S6K phosphorylation in rat soleus muscle during the early stage of unloading: Causes and consequences.

Currently, there is a lack of investigation into the initial signaling events underlying the development of disuse muscle atrophy. The study was aimed to (i) identify an assumed relationship between AMPK dephosphorylation and p70S6K hyperphosphorylation in the initial period of hindlimb unloading (HS), and (ii) assess the signaling consequences of p70S6K hyperphosphorylation following 24-h HS. For experiment 1, rats were treated with AMPK activator (AICAR) for 6 d before HS as well as during 24-h HS. For experiment 2, rats were treated with mTORC1 inhibitor rapamycin during 24-h HS. The key signaling markers implicated in protein turnover were assessed using WB and RT-PCR. One-day HS resulted in a significant upregulation of MuRF-1 and MAFbx expression, increase in p70S6K (Thr389) and IRS-1 (Ser639) phosphorylation and a significant decrease in phosphorylated AMPK, AKT, FOXO3, total IRS-1 content, and HDAC5 nuclear content. AMPK and p70S6K phosphorylation did not differ from control in AICAR-treated unloaded rats. Rapamycin treatment during unloading abolished p70S6K and E3 ligases upregulation and increased HDAC5 nuclear accumulation. The results of the study suggest that mTORC-1/p70S6K signaling pathway in rat soleus muscle is activated following 24-h mechanical unloading. This activation is facilitated by a decrease in AMPK phosphorylation. Increased p70S6K activity at the initial stage of hindlimb unloading could lead to the upregulation of E3 ligases MAFbx/atrogin-1 and MuRF-1 via nuclear export of HDAC5.

HDAC5 Expression in Urothelial Carcinoma Cell Lines Inhibits Long-Term Proliferation but Can Promote Epithelial-to-Mesenchymal Transition.

Class I histone deacetylases (HDACs) generally promote cell proliferation and tumorigenesis, whereas class IIA HDACs like HDAC4 and HDAC5 may promote or impede cancer development in a tissue-dependent manner. In urothelial carcinoma (UC), HDAC5 is often downregulated. Accordingly, HDAC5 was weakly expressed in UC cell lines suggesting a possible tumor-suppressive function. We therefore characterized the effects of stable HDAC5 expression in four UC cell lines (RT112, VM-Cub-1, SW1710 and UM-UC-3) with different phenotypes reflecting the heterogeneity of UC, by assessing proliferation, clonogenicity and migration ability. Further, we detailed changes in the proteome and transcriptome by immunoblotting, mass spectrometry and RNA sequencing analysis. We observed that HDAC5 overexpression in general decreased cell proliferation, but in one cell line (VM-Cub-1) induced a dramatic change from an epitheloid to a mesenchymal phenotype, i.e., epithelial-mesenchymal transition (EMT). These phenotypical changes were confirmed by comprehensive proteomics and transcriptomics analyses. In contrast to HDAC5, overexpression of HDAC4 exerted only weak effects on cell proliferation and phenotypes. We conclude that overexpression of HDAC5 may generally decrease proliferation in UC, but, intriguingly, may induce EMT on its own in certain circumstances.

Bidirectional regulation of HDAC5 by mAKAPß signalosomes in cardiac myocytes.

Class IIa histone deacetylases (HDACs) are transcriptional repressors whose nuclear export in the cardiac myocyte is associated with the induction of pathological gene expression and cardiac remodeling. Class IIa HDACs are regulated by multiple, functionally opposing post-translational modifications, including phosphorylation by protein kinase D (PKD) that promotes nuclear export and phosphorylation by protein kinase A (PKA) that promotes nuclear import. We have previously shown that the scaffold protein muscle A-kinase anchoring protein ß (mAKAPß) orchestrates signaling in the cardiac myocyte required for pathological cardiac remodeling, including serving as a scaffold for both PKD and PKA. We now show that mAKAPß is a scaffold for HDAC5 in cardiac myocytes, forming signalosomes containing HDAC5, PKD, and PKA. Inhibition of mAKAPß expression attenuated the phosphorylation of HDAC5 by PKD and PKA in response to α- and ß-adrenergic receptor stimulation, respectively. Importantly, disruption of mAKAPß-HDAC5 anchoring prevented the induction of HDAC5 nuclear export by α-adrenergic receptor signaling and PKD phosphorylation. In addition, disruption of mAKAPß-PKA anchoring prevented the inhibition by ß-adrenergic receptor stimulation of α-adrenergic-induced HDAC5 nuclear export. Together, these data establish that mAKAPß signalosomes serve to bidirectionally regulate the nuclear-cytoplasmic localization of class IIa HDACs. Thus, the mAKAPß scaffold serves as a node in the myocyte regulatory network controlling both the repression and activation of pathological gene expression in health and disease, respectively.