Histone Deacetylase 6 Knockout Research Articles

The role of HDAC6 in enhancing macrophage autophagy via the autophagolysosomal pathway to alleviate legionella pneumophila-induced pneumonia

ABSTRACT Legionella pneumophila (L. pneumophila) is a prevalent pathogenic bacterium responsible for significant global health concerns. Nonetheless, the precise pathogenic mechanisms of L. pneumophila have still remained elusive. Autophagy, a direct cellular response to L. pneumophila infection and other pathogens, involves the recognition and degradation of these invaders in lysosomes. Histone deacetylase 6 (HDAC6), a distinctive member of the histone deacetylase family, plays a multifaceted role in autophagy regulation. This study aimed to investigate the role of HDAC6 in macrophage autophagy via the autophagolysosomal pathway, leading to alleviate L. pneumophila-induced pneumonia. The results revealed a substantial upregulation of HDAC6 expression level in murine lung tissues infected by L. pneumophila. Notably, mice lacking HDAC6 exhibited a protective response against L. pneumophila-induced pulmonary tissue inflammation, which was characterized by the reduced bacterial load and diminished release of pro-inflammatory cytokines. Transcriptomic analysis has shed light on the regulatory role of HDAC6 in L. pneumophila infection in mice, particularly through the autophagy pathway of macrophages. Validation using L. pneumophila-induced macrophages from mice with HDAC6 gene knockout demonstrated a decrease in cellular bacterial load, activation of the autophagolysosomal pathway, and enhancement of cellular autophagic flux. In summary, the findings indicated that HDAC6 knockout could lead to the upregulation of p-ULK1 expression level, promoting the autophagy-lysosomal pathway, increasing autophagic flux, and ultimately strengthening the bactericidal capacity of macrophages. This contributes to the alleviation of L. pneumophila-induced pneumonia.

Male histone deacetylase 6 (HDAC6) knockout mice have enhanced ventilatory responses to hypoxic challenge.

Histone deacetylase 6 (HDAC6) is a class II histone deacetylase that is predominantly localized in the cytoplasm of cells. HDAC6 associates with microtubules and regulates acetylation of tubulin and other proteins. The possibility that HDAC6 participates in hypoxic signaling is supported by evidence that 1) hypoxic gas challenges cause microtubule depolymerization, 2) expression of hypoxia inducible factor alpha (HIF-1α) is regulated by microtubule alterations in response to hypoxia, and 3) inhibition of HDAC6 prevents HIF-1α expression and protects tissue from hypoxic/ischemic insults. The aim of this study was to address whether the absence of HDAC6 alters ventilatory responses during and/or after hypoxic gas challenge (10% O2, 90% N2 for 15min) in adult male wildtype (WT) C57BL/6 mice and HDAC6 knock-out (KO) mice. Key findings were that 1) baseline values for frequency of breathing, tidal volume, inspiratory and expiratory times, and end expiratory pause were different between knock-out mice and wildtype mice, 2) ventilatory responses during hypoxic challenge were more robust in KO mice than WT mice for recorded parameters including, frequency of breathing, minute ventilation, inspiratory and expiratory durations, peak inspiratory and expiratory flows, and inspiratory and expiratory drives, and 3) responses upon return to room-air were markedly different in KO compared to WT mice for frequency of breathing, minute ventilation, inspiratory and expiratory durations, end expiratory pause (but not end inspiratory pause), peak inspiratory and expiratory flows, and inspiratory and expiratory drives. These data suggest that HDAC6 may have a fundamentally important role in regulating the hypoxic ventilatory response in mice.

[Inhibition of Histone Deacetylase 6 Ameliorates Podocyte Injury in Diabetic Kidney Disease in Mice].

To investigate the role of histone deacetylase 6 (HDAC6) in podocyte injury in diabetic kidney disease (DKD) in mice. 1) The 8-week-old male CD-1 mice were selected to construct the model of DKD with streptozocin (STZ). After the model was established, the mice were intraperitoneally injected with HDAC6 inhibitor CAY10603 (5mg/kg/daily) or same volume vehicle as control. The mice were divided into four groups, control (CTL)+vehicle (Veh) (n=5), CLT+CAY10603 (n=3), STZ+Veh (n=9), and STZ+CAY10603 (n=7). Mice in STZ+Veh and STZ+CAY10603 groups developed DKD, while mice in the CTL+Veh and CTL+CAY10603 groups were served as normal controls. The therapeutic effect was evaluated through urine albumin-to-creatinine ratio (uACR) and renal pathology after the 2-week treatment with CAY10603. 2) Human podocytes were cultured in vitro and were divided into four groups as follows: CTL, transforming growth factor-β (TGFβ), TGFβ+CAY10603 (250 nmol/L), and TGFβ+CAY10603 (500 nmol/L) groups. The control group did not receive any treatment, the last three groups were given 36-h TGFβ treatment at 5 ng/µL, with or without CAY10603 as indicated for an additional 12 h. Western blot was performed to determine the inhibitory effect of CAY10603 on NLRP3 inflammasome. 3) HDAC6 knockout (KO) mice were generated and used to create STZ-induced model of DKD. The mice were divided into four groups: C57BL/6J wild type (WT) (n=6), HDAC6 KO (n=6), WT+STZ (n=10), and HDAC6 KO+STZ (n=9). Samples were collected 16 weeks after successful modeling and changes in uACR and renal pathology were evaluated accordingly. After 2 weeks of treatment, mice in the STZ+CAY10603 group exhibited reduction in uACR (P<0.05) and inhibition of glomerular mesangium expansion (P<0.05) compared with those of the mice in the STZ+Veh group. There was no statistically significant difference in the indicators between the CTL+Veh group and the CTL+CAY10603 group. In vitro cultured podocytes, compared with the control group, NLRP3 inflammasome activation was seen in the TGFβ group. CAY10603 treatment significantly inhibited the activation of NLRP3 in the dosage-dependent manner (P<0.05). Compared with those of the WT group, the WT+STZ group showed increased uACR (P<0.05), obvious glomerulosclerosis and loss of podocytes numbers. Compared with those of the WT+STZ group, the HDAC6 KO+STZ group showed effectively reduction of uACR (P<0.05) and improvement in the renal pathological changes in mice. There was no significant difference in these aspects between the WT and HDAC6 KO groups. Inhibition of HDAC6 alleviates proteinuria and podocyte injury in the mouse model of DKD by suppressing the activation of NLRP3 inflammasome.

HDAC6 Triggers the ATM-Dependent DNA Damage Response To Promote PRV Replication.

Pseudorabies virus (PRV) infection is modulated by various cellular host factors. In this study, we investigated the role of histone deacetylase 6 (HDAC6) in this process. We determined HDAC6 expression in vitro and performed gene knockout, pharmacological inhibition analyses, immunofluorescence assays, and statistical analyses. We found that the pharmacological and genetic inhibition of HDAC6 significantly decreased PRV replication, whereas its overexpression promoted PRV replication. Additionally, we demonstrated that PRV infection can induce the phosphorylation of histone H2AX and lead to DNA damage response (DDR), and the ataxia telangiectasia mutated (ATM) inhibitor KU55933 inhibits DDR and PRV infection. Mechanistically, the HDAC6 inhibitor tubacin and HDAC6 knockout can decrease DDR. The results of this study suggested that HDAC6 may be a crucial factor in PRV-induced ATM-dependent DDR to promote PRV replication. IMPORTANCE Pseudorabies virus (PRV) is a member of the subfamily Alphaherpesvirinae of the family Herpesviridae. PRV infection in swine can lead to high morbidity and mortality of swine, causing huge economic losses. In particular, PRV variants can cause severe damage to the nervous and respiratory systems of humans, revealing that PRV may be a potential zoonotic pathogen. Vaccines for PRV have been developed that can delay or reduce the epidemic, but they currently cannot eliminate this disease completely. Therefore, studies should investigate new targets for the prevention and control of PRV infection. In this study, we demonstrated that HDAC6 can induce ataxia telangiectasia mutated-dependent DNA damage response to foster PRV replication, indicating that HDAC6 is a therapeutic target for PRV infection.

Upregulation of MDH1 acetylation by HDAC6 inhibition protects against oxidative stress-derived neuronal apoptosis following intracerebral hemorrhage.

Oxidative stress impairs functional recovery after intracerebral hemorrhage (ICH). Histone deacetylase 6 (HDAC6) plays an important role in the initiation of oxidative stress. However, the function of HDAC6 in ICH and the underlying mechanism of action remain elusive. We demonstrated here that HDAC6 knockout mice were resistant to oxidative stress following ICH, as assessed by the MDA and NADPH/NADP+ assays and ROS detection. HDAC6 deficiency also resulted in reduced neuronal apoptosis and lower expression levels of apoptosis-related proteins. Further mechanistic studies showed that HDAC6 bound to malate dehydrogenase 1 (MDH1) and mediated-MDH1 deacetylation on the lysine residues at position 121 and 298. MDH1 acetylation was inhibited in HT22 cells that were challenged with ICH-related damaging agents (Hemin, Hemoglobin, and Thrombin), but increased when HDAC6 was inhibited, suggesting an interplay between HDAC6 and MDH1. The acetylation-mimetic mutant, but not the acetylation-resistant mutant, of MDH1 protected neurons from oxidative injury. Furthermore, HDAC6 inhibition failed to alleviate brain damage after ICH when MDH1 was knockdown. Taken together, our study showed that HDAC6 inhibition protects against brain damage during ICH through MDH1 acetylation.

HDAC6 knockout alleviates pristane-induced lupus

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by excessive inflammation and production of pathogenic antibodies. Histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase. It has been reported that selective HDAC6 inhibition decreases inflammation in lupus mice models. C57BL/6 mice develop SLE-like symptoms following pristane injection. In this study, sex and age-matched wild type and HDAC6−/− mice on the C57BL/6 background were administered 0.5 ml pristane or PBS intraperitoneally (i.p.) at 8–12 weeks-of-age and were euthanized 10 days later. At sacrifice, body weight and spleen weight were measured, sera were collected, splenocytes and peritoneal cells were harvested for flow cytometry. We found pristane administration increased the spleen weight with no difference between WT mice and HDAC6−/− mice. Flow cytometry results showed that there was no difference in T cell or B cell populations in the spleen. Pristane administration promoted the population of CD11b+ Ly6Chigh inflammatory monocytes and CD11b+Ly6G+ neutrophils. Peritoneal recruitment of these inflammatory monocytes and neutrophils in HDAC6−/− mice was significantly decreased compared to the WT mice. Pristane administration also induced the interferon (IFN) signature genes, like Mx1, Oas1a, Irf7, Irf9, Cxcl10, Isg15. qRT-PCR revealed that these IFN signature genes were decreased in HDAC6−/− mice compared to the WT mice. In summary, our results show that HDAC6 knockout inhibits the recruitment of inflammatory monocytes and neutrophils in the peritoneum in early inflammation response to pristane. HDAC6 deletion also inhibited the expression of IFN signature genes with pristine stimulation.

Targeting HDAC6 in the Dorsal Root Ganglia Attenuates Peripheral Nerve Injury‐induced Hypersensitivity

Histone deacetylase 6 (HDAC6) is a Class IIb histone deacetylase, which is primarily localized to the cytoplasm and plays an important role in cell structure and dynamism, transcriptional repression, exocytosis and endocytocis. Preclinical evidence has suggested that HDAC6 inhibitors alleviate signs of chemotherapy‐induced peripheral neuropathy (CIPN), such as mechanical allodynia. However, no group to our knowledge has investigated the mechanism of action of HDAC6 inhibitors in a severe nerve injury model, which has a different pathophysiology than milder models such as CIPN. In this study we use genetically modified mice and the tibial spared nerve injury (SNI) model to determine the impact of dorsal root ganglion (DRG) specific HDAC6 knockout in the induction and maintenance of sensory hypersensitivity. Downregulation of HDAC6 in the DRG was achieved by injection of the left sciatic nerve of adult male HDAC6fl\fl mice with AAV8‐CMV‐Cre‐EGFP or AAV8‐CMV‐EGFP vectors. DRG‐knockdown of HDAC6 prevented the development of mechanical allodynia after SNI. Using in situ hybridization (RNAScope), we also demonstrate that HDAC6 is upregulated at three weeks after the induction of nerve injury in L3‐6 DRG neurons. Furthermore, treatment with the peripherally acting HDAC6 inhibitor ACY1215 (Regenacy Pharmaceuticals, Waltham, MA) after SNI leads to full recovery from mechanical allodynia. We are continuing this work by performing bulk RNA sequencing on L3‐6 DRGs from mice who received 21 days of treatment with ACY1215 or vehicle starting at three weeks after SNI or Sham operation, in order to understand the transcriptomic events and upstream pathways associated with recovery from prolonged neuropathic states.Our findings highlight a promising therapeutic role of HDAC6 inhibitors for the prevention or recovery from sensory hypersensitivity behaviors associated with prolonged peripheral nerve injury. Our future work aims to define the mechanisms underlying HDAC6 action in the DRG and identify new pathways associated with recovery from sensory hypersensitivity to influence novel treatment strategies for pain.

Generation of a homozygous HDAC6 knockout human embryonic stem cell line by CRISPR/Cas9 editing.

Histone deacetylase 6 (HDAC6) is a unique cytoplasmic enzyme in the HDAC family. The HDAC6 has been shown to play important roles in several biological processes. Meanwhile, it is also an attractive therapeutic target for a variety of diseases. However, the mechanism of HDAC6 function is not fully understood yet, and it is still lacking highly specific targeted drugs. Here, we generated a homozygous HDAC6 knockout human embryonic stem cell (hESC) line, WAe009-A-21 by the CRISPR/Cas9-based gene editing method. The WAe009-A-21 cell line does not express HDAC6 protein, while maintaining normal 46, XX karyotype, pluripotency, and trilineage differentiation potential.

Staphylococcus aureus–induced endothelial permeability and inflammation are mediated by microtubule destabilization

Staphylococcus aureus is a major etiological agent of sepsis and induces endothelial cell (EC) barrier dysfunction and inflammation, two major hallmarks of acute lung injury. However, the molecular mechanisms of bacterial pathogen-induced EC barrier disruption are incompletely understood. Here, we investigated the role of microtubules (MT) in the mechanisms of EC barrier compromise caused by heat-killed S. aureus (HKSA). Using a customized monolayer permeability assay in human pulmonary EC and MT fractionation, we observed that HKSA-induced barrier disruption is accompanied by MT destabilization and increased histone deacetylase-6 (HDAC6) activity resulting from elevated reactive oxygen species (ROS) production. Molecular or pharmacological HDAC6 inhibition rescued barrier function in HKSA-challenged vascular endothelium. The HKSA-induced EC permeability was associated with impaired MT-mediated delivery of cytoplasmic linker-associated protein 2 (CLASP2) to the cell periphery, limiting its interaction with adherens junction proteins. HKSA-induced EC barrier dysfunction was also associated with increased Rho GTPase activity via activation of MT-bound Rho-specific guanine nucleotide exchange factor-H1 (GEF-H1) and was abolished by HDAC6 down-regulation. HKSA activated the NF-κB proinflammatory pathway and increased the expression of intercellular and vascular cell adhesion molecules in EC, an effect that was also HDAC6-dependent and mediated, at least in part, by a GEF-H1/Rho-dependent mechanism. Of note, HDAC6 knockout mice or HDAC6 inhibitor-treated WT mice were partially protected from vascular leakage and inflammation caused by both HKSA or methicillin-resistant S. aureus (MRSA). Our results indicate that S. aureus-induced, ROS-dependent up-regulation of HDAC6 activity destabilizes MT and thereby activates the GEF-H1/Rho pathway, increasing both EC permeability and inflammation.

HDAC6 inhibition protects cardiomyocytes against doxorubicin-induced acute damage by improving α-tubulin acetylation

Doxorubicin (Dox) is an efficacious antineoplastic drug but is limited used for its cardiotoxicity. Histone Deacetylase 6 (HDAC6) has been indicated to participate in cardiomyopathies, however, its role in Dox-induced cardiac injury is largely unknown. In this study, we firstly aimed to determine the role of HDAC6 in Dox-induced cardiomyopathy. Immunoblotting revealed that Dox increased HDAC6 protein level and activity and decreased α-tubulin acetylation level in vitro and vivo. HDAC6 knockout (HDAC6−/−) mice showed obvious anti-Dox cardiotoxicity by conserved cardiac function monitored by echocardiography and the protection was reversed by Nocodazole, one drug lowering α-tubulin acetylation. Further mechanism investigation showed that improvement of mitochondria function and autophagy flux was partially inhibited by Nocodazole and Colchicine which lowers α-tubulin acetylation in neonatal rat cardiac myocytes. Aiming at transforming this research to clinical application, we then explored the effect of combined utilization of HDAC6 inhibitor and Dox on tumour and cardiac function. Results showed that Tubastatin A, one HDAC6 selective inhibitor, protected against Dox-induced acute cardiomyopathy without influencing the effect of Dox on inhibiting MDA-MB-231 subcutaneous tumour growth. These findings suggest a new treatment for cancer with Dox by combined utilization with HDAC6 selective inhibitors.

Disruption of lipid-raft localized Gαs/tubulin complexes by antidepressants: a unique feature of HDAC6 inhibitors, SSRI and tricyclic compounds.

Current antidepressant therapies meet with variable therapeutic success and there is increasing interest in therapeutic approaches not based on monoamine signaling. Histone deacetylase 6 (HDAC6), which also deacetylates α-tubulin shows altered expression in mood disorders and HDAC6 knockout mice mimic traditional antidepressant treatments. Nonetheless, a mechanistic understanding for HDAC6 inhibitors in the treatment of depression remains elusive. Previously, we have shown that sustained treatment of rats or glioma cells with several antidepressants translocates Gαs from lipid rafts toward increased association with adenylyl cyclase (AC). Concomitant with this is a sustained increase in cAMP production. While Gαs modifies microtubule dynamics, tubulin also acts as an anchor for Gαs in lipid-rafts. Since HDAC-6 inhibitors potentiate α-tubulin acetylation, we hypothesize that acetylation of α-tubulin disrupts tubulin-Gαs raft-anchoring, rendering Gαs free to activate AC. To test this, C6 Glioma (C6) cells were treated with the HDAC-6 inhibitor, tubastatin-A. Chronic treatment with tubastatin-A not only increased α-tubulin acetylation but also translocated Gαs from lipid-rafts, without changing total Gαs. Reciprocally, depletion of α-tubulin acetyl-transferase-1 ablated this phenomenon. While escitalopram and imipramine also disrupt Gαs/tubulin complexes and translocate Gαs from rafts, they evoke no change in tubulin acetylation. Finally, two indicators of downstream cAMP signaling, cAMP response element binding protein phosphorylation (pCREB) and expression of brain-derived-neurotrophic-factor (BDNF) were both elevated by tubastatin-A. These findings suggest HDAC6 inhibitors show a cellular profile resembling traditional antidepressants, but have a distinct mode of action. They also reinforce the validity of antidepressant-induced Gαs translocation from lipid-rafts as a biosignature for antidepressant response that may be useful in the development of new antidepressant compounds.

HDAC6 regulates sensitivity to cell death in response to stress and post-stress recovery.

Histone deacetylase 6 (HDAC6) plays an important role in stress responses such as misfolded protein-induced aggresomes, autophagy, and stress granules. However, precisely how HDAC6 manages response during and after cellular stress remains largely unknown. This study aimed to investigate the effect of HDAC6 on various stress and post-stress recovery responses. We showed that HIF-1α protein levels were reduced in HDAC6 knockout (KO) MEFs compared to wild-type (WT) MEFs in hypoxia. Furthermore, under hypoxia, HIF-1α levels were also reduced following rescue with either a catalytically inactive or a ubiqiutin-binding mutant HDAC6. HDAC6 deacetylated and upregulated the stability of HIF-1α, leading to activation of HIF-1α function under hypoxia. Notably, both the deacetylase and ubiquitin-binding activities of HDAC6 contributed to HIF-1α stabilization, but only deacetylase activity was required for HIF-1α transcriptional activity. Suppression of HDAC6 enhanced the interaction between HIF-1α and HSP70 under hypoxic conditions. In addition to hypoxia, depletion of HDAC6 caused hypersensitivity to cell death during oxidative stress and post-stress recovery. However, HDAC6 depletion had no effect on cell death in response to heat shock or ionizing radiation. Overall, our data suggest that HDAC6 may serve as a critical stress regulator in response to different cellular stresses.

Histone Deacetylase 6 Regulates BladderArchitecture and Host Susceptibility toUropathogenic Escherichia coli.

Histone deacetylase 6 (HDAC6) is a non-canonical, mostly cytosolic histone deacetylase that has a variety of interacting partners and substrates. Previous work using cell-culture based assays coupled with pharmacological inhibitors and gene-silencing approaches indicated that HDAC6 promotes the actin- and microtubule-dependent invasion of host cells by uropathogenic Escherichia coli (UPEC). These facultative intracellular pathogens are the major cause of urinary tract infections. Here, we examined the involvement of HDAC6 in bladder colonization by UPEC using HDAC6 knockout mice. Though UPEC was unable to invade HDAC6−/− cells in culture, the bacteria had an enhanced ability to colonize the bladders of mice that lacked HDAC6. This effect was transient, and by six hours post-inoculation bacterial titers in the HDAC6−/− mice were reduced to levels seen in wild type control animals. Subsequent analyses revealed that the mutant mice had greater bladder volume capacity and fluid retention, along with much higher levels of acetylated α-tubulin. In addition, infiltrating neutrophils recovered from the HDAC6−/− bladder harbored significantly more viable bacteria than their wild type counterparts. Cumulatively, these changes may negate any inhibitory effects that the lack of HDAC6 has on UPEC entry into individual host cells, and suggest roles for HDAC6 in other urological disorders such as urinary retention.

Abstract B18: Targeting histone deacetylase 6 (HDAC6) depresses proliferation and induces caspase-associated cell death in glioblastoma multiforme (GBM) cells and GBM stem cell-like spheroids

Abstract Glioblastoma multiforme (GBM) comprises the most common and very aggressive form of primary brain tumor with a dismal prognosis and very poor response to the current therapies. HDAC inhibitors (HDACi) are capable of inducing growth arrest and apoptosis in various tumor cell types. HDAC6 is a unique enzyme having two deacetylase domains, and a ubiquitin-binding domain. HDAC6 interacts with a number of proteins in the cytoplasm and is involved in tumorigenesis, cell motility, and metastasis. Significantly, it has been shown that HDAC6 knockout mice are viable. Therefore, in this study, we evaluated HDAC6 as a relevant target for GBM treatment by using a potent HDAC6 inhibitor, CAY 10603. Our data showed that CAY 10603 targets the established GBM cell line U86MG, M-HBT161 early primary cultures, and CD133- and SOX2-positive stem cell-like spheroids. CAY 10603 at 1-5 μM triggered significant inhibition of cell survival and induced apoptotic cell death in GBM cells and GBM spheroids. CAY 10603 reduced cell survival in these cells by 50% (IC50) at 1-3 μM treatment for 48 h. Similarly, CAY 10603 induced significant dose-dependent inhibition of spheroid survival in U87MG and M-HBT161 when the spheroids were grown in a defined GBM stem cell (GSC) growth medium on ultra-low attachment plates for four days. Interestingly, when grown in the same medium but on plates which promote attachment, the spheroids were 3-4 fold more sensitive to CAY 10603, indicating that growth conditions affect the sensitivity of GBM stem cell-like spheroids. Moreover, CAY 10603 triggered cell death in U87MG and M-HBT161 cells as well as CD133/SOX2-expressing spheroids was associated with activation of caspases-3, -6 and -9. Overall, our results show that CAY 10603 robustly inhibits the growth of GBM cells, and is effective in eliminating spheroids that contain GBM cancer stem cells which play a major role in drug resistance and disease recurrence. These results suggest that use of CAY 10603 alone or in combination with other agents may potentially improve the survival of brain tumor patients. Citation Format: Mohammad Reza Saadatzadeh, Khadijeh Bijangi Vishehsaraei, Haiyan Wang, Aaron Cohen-Gadol, Karen E. Pollok, Ahmad R. Safa. Targeting histone deacetylase 6 (HDAC6) depresses proliferation and induces caspase-associated cell death in glioblastoma multiforme (GBM) cells and GBM stem cell-like spheroids. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B18.

New HDAC6-mediated deacetylation sites of tubulin in the mouse brain identified by quantitative mass spectrometry.

The post-translational modifications (PTMs) occurring on microtubules have been implicated in the regulation of microtubule properties and functions. Acetylated K40 of α-tubulin, a hallmark of long-lived stable microtubules, is known to be negatively controlled by histone deacetylase 6 (HDAC6). However, the vital roles of HDAC6 in microtubule-related processes such as cell motility and cell division cannot be fully explained by the only known target site on tubulin. Here, we attempt to comprehensively map lysine acetylation sites on tubulin purified from mouse brain tissues. Furthermore, mass spectrometry-based quantitative comparison of acetylated peptides from wild-type vs HDAC6 knockout mice allowed us to identify six new deacetylation sites possibly mediated by HDAC6. Thus, adding new sites to the repertoire of HDAC6-mediated tubulin deacetylation events would further our understanding of the multi-faceted roles of HDAC6 in regulating microtubule stability and cellular functions.

Histone Deacetylase 6 (HDAC6) Regulates Gene Transcription and Enucleation during Erythroid Differentiation

The deacetylation of histone and non-histone proteins by histone deacetylase (HDACs) plays a critical role in gene transcription and many other cellular processes in eukaryotic cells. Class II deacetylase HDAC6 is mainly localized in the cytoplasm and deacetylates tubulin and other cytoplasmic proteins. Earlier studies from our laboratory showed that HDAC6 can be transported to the nucleus, and can interact and deacetylate nuclear proteins, such as histones. In this study, we investigate the function of HDAC6 during erythroid differentiation. In proerythroblast cells, HDAC6 is detected in the nucleus and gradually migrates to the cytoplasm upon the induction of differentiation, indicating different HDAC6 functions during erythropoiesis. Inhibition of HDAC6 in mouse fetal liver erythroblasts impairs differentiation and enucleation. β-globin gene transcription is reduced by HDAC6 specific inhibitor, Tubastatin A, in cultured fetal erythroblasts. HDAC6 knockout mice also showed impaired globin gene transcription. HDAC6 specifically interacts with serine 2 phosphorylated Pol II and is recruited mainly at the transcribed region of β-globin, correlating with RNA Pol II recruitment. These results suggest that HDAC6 promotes erythroblast differentiation through the regulation of transcription elongation. HDAC6 also plays a role in regulating the enucleation process during the late stage of erythropoiesis. The formation of the contractile actin ring is disrupted and the enucleation process is blocked in cultured mouse fetal erythroblasts treated with the HDAC6 inhibitor. We further investigated the molecular mechanism of HDAC6 regulated enucleation. We found that HDAC6 interacts with and deacetylates mDia2, an effector of Rho GTPases that is required for erythroblast enucleation. Deacetylation of mDia2 is required for the formation of the contractile actin ring and subsequent enucleation. Altogether, our results identify the important role of HDAC6 in regulating transcription and enucleation during the different stages of mammalian erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity

Histone deacetylase 6 (HDAC6), a predominantly cytoplasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates. In an attempt to explore the substrate diversity of HDAC6, we performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. We identified 107 proteins with elevated acetylation in the liver of HDAC6 knockout mice. Three cytoplasmic proteins, including myosin heavy chain 9 (MYH9), heat shock cognate protein 70 (Hsc70), and dnaJ homolog subfamily A member 1 (DNAJA1), were verified to interact with HDAC6. The acetylation levels of these proteins were negatively regulated by HDAC6 both in the mouse liver and in cultured cells. Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1. These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-014-0102-8) contains supplementary material, which is available to authorized users.

Physical Interaction of Histone Deacetylase 6 (HDAC6) with STAT3 Regulates IL-10 Gene Expression and Immune Tolerance Mediated by Antigen-Presenting Cells (APCs)

AbstractAbstract 829APCs are critical in T-cell activation and in the induction of T-cell tolerance. Epigenetic modifications of specific genes in the APC play a key role in this process, and among them, histone deacetylases (HDACs) have emerged as key participants.HDAC6 is a 131 KDa protein with preferential cytoplasmic localization where it regulates the acetylation of proteins involved in cytoskeleton, cell-cell interaction and cell migration. Emerging evidence also implicates HDAC6 in regulation of immune responses, in particular at the level of the APC/T cell immune synapse1 and in the suppressive function of regulatory T-cells2. Expanding upon these immunoregulatory properties, here we show for the first time that HDAC6 physically interacts with STAT3, a transcriptional activator of IL-10 gene expression. By co-immunoprecipitation studies and confocal studies we found that HDAC6 co-localize with STAT3 in the cytoplasm and nuclei of macrophages. Furthermore, by using several HDAC6 and STAT3 mutants we have identified that the aminoacids 503–840 of HDAC6 and the STAT3 domain comprising aminoacids 465–585 are required for this interaction. Functionally, knocking down HDAC6 in a macrophage cell line (RAW264.7-HDAC6KD) resulted in inhibition of STAT3 phosphorylation, decreased recruitment of STAT3 to the IL-10 gene promoter and abrogation of IL-10 production by these cells in response to either LPS or IL-10. Similar results were observed in dendritic cells (DCs) or macrophages isolated from HDAC6 knock-out (KO) mice. Furthermore, HDAC6KD clones or APCs from HDAC6 KO mice displayed an enhanced expression of the co-stimulatory molecule B7.2 and are better activators of antigen-specific CD4+ T-cell responses in vitro. More importantly, these APCs are able of restoring the responsiveness of anergic T-cells from lymphoma-bearing mice. Pharmacologic inhibition of HDAC6 in APCs with Tubastatin A, an isotype-selective HDAC6 inhibitor, yielded similar enhancement of APC and T-cell function in vitro. Further support for HDAC6 as an appealing target in cancer immunotherapy has been recently provided by the significant delay in tumor growth observed in either HDAC6 KO mice or in wild type mice treated with Tubastatin A.In summary, we have shown for the first time that HDAC6 interacts physically with STAT3 and such an interaction is necessary for STAT3 phosphorylation and IL-10 gene expression in APCs. Disrupting the HDAC6/STAT3/IL-10 axis in APCs with selective HDAC6 inhibitors represents a novel approach to overcome tolerogenic pathways in these cells and tip the balance towards effective antitumor immune responses. Disclosures:No relevant conflicts of interest to declare.

Abstract LB-391: HDAC6 is a dual enzyme with deacetylase and E3 ligase activities for MSH2 degradation

Abstract MSH2 is a key DNA mismatch repair (MMR) protein that functions as an MSH2/MSH6 heterodimer (MutSa) in repairing errors after DNA replication as well as sensing certain types of DNA damage. It is believed that reduction of MutSα attenuates recognition of DNA adducts which renders a declined cell cycle arrest and apoptosis upon DNA damage and subsequently leads to chemotolerance. However, the mechanisms of down-regulation of MutSa are largely unknown. Here we show that histone deacetylase 6 (HDAC6) is co-purified with MutSa in HeLa nuclear extracts. Interestingly, we have found that HDAC6 itself serves as a ubiquitin E3 ligase to promote MSH2 polyubiquitination which renders MSH2 degradation by proteasome pathway. Intriguingly, HDAC6 prefers to target MSH2 monomer over MutSa for ubiquitination and degradation. Mechanistically, HDAC6 E3 ligase activity primarily resides in the N-terminal region encompassing the previously identified deacetylase domain 1 (DAC1) whereas its deacetylase activity resides in the deacetylase domain 2 (DAC2). We also showed that HDAC6 E3 ligase activity towards acetylated MSH2 is facilitated by its deacetylase activity in vivo. MSH2 lysine 249 and 892 can be either acetylated or ubiqutinated in vivo. These data suggest that HDAC6 sequentially deacetylates and ubiquitinates MSH2 via its DAC2 and DAC1 domains to target MSH2 degradation in vivo. Moreover, cells lacking or depleted of HDAC6 exhibit elevated levels of MSH2 due to impaired ubiquitin-proteasome-mediated degradation and subsequently prolonged half-life, compared with control cells. In agreement with this, HDAC6 knockout cells display higher MMR activity compared with wild type cells. Importantly, absence or reduction of HDAC6 leads to an increased cellular sensitivity to 6-TG and MNNG, whose DNA lesions were recognized by MutSa, suggesting that HDAC6 governs MSH2-dependent cytotoxicity induced by 6-TG or MNNG. Overall, our results identified a novel ubiquitin E3 ligase activity of HDAC6 and revealed that HDAC6 plays an important role in response to genotoxic stress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-391. doi:1538-7445.AM2012-LB-391

Histone deacetylase 6 gates the synaptic action of acute stress in prefrontal cortex

The prefrontal cortex (PFC), a region responsible for high-order cognitive functions, such as decision-making, attention and working memory, is highly influenced by stress and corticosteroid stress hormones. Recently it has been shown that acute stress affects PFC functions by potentiating glutamatergic transmission via a mechanism dependent on glucocorticoid receptor (GR) and its downstream target, serum and glucocorticoid-inducible kinase (SGK). To identify the key regulators of stress responses, we examined the role of histone deacetylase 6 (HDAC6), a unique member of the HDAC family that could regulate the GR chaperone protein heat shock protein 90 (HSP90), in the synaptic action of acute stress in PFC. We found that HDAC6 inhibition or knockdown blocked the enhancement of glutamatergic transmission and glutamate receptor trafficking by acute stress in vivo or corticosterone treatment in vitro. In addition, HDAC6 inhibition blocked the up-regulation of SGK in animals exposed to acute stress. HSP90 inhibition or knockdown produced a similar blockade of the acute stress-induced enhancement of glutamatergic signalling. These findings have identified HDAC6 as a key molecule gating the effects of acute stress on synaptic functions in the PFC.