Loss of HDAC6, a novel CHIP substrate, alleviates abnormal tau accumulation

The abnormal accumulation of the microtubule-binding protein tau is associated with a number of neurodegenerative conditions, and correlates with cognitive decline in Alzheimer's disease. The ubiquitin ligase carboxy terminus of Hsp70-interacting protein (CHIP) and the molecular chaperone Hsp90 are implicated in protein triage decisions involving tau, and have consequently been targeted for therapeutic approaches aimed at decreasing tau burden. Here, we present evidence that CHIP binds, ubiquitinates and regulates expression of histone deacetylase 6 (HDAC6). As the deacetylase for Hsp90, HDAC6 modulates Hsp90 function and determines the favorability of refolding versus degradation of Hsp90 client proteins. Moreover, we demonstrate that HDAC6 levels positively correlate with tau burden, while a decrease in HDAC6 activity or expression promotes tau clearance. Consistent with previous research on Hsp90 clients in cancer, we provide evidence that a loss of HDAC6 activity augments the efficacy of an Hsp90 inhibitor and drives client degradation, in this case tau. Therefore, our current findings not only identify HDAC6 as a critical factor for the regulation of tau levels, but also indicate that a multi-faceted treatment approach could more effectively arrest tau accumulation in disease.     

HDAC6 deacetylase activity links the tubulin cytoskeleton with immune synapse organization

We investigated the role of acetylated microtubules in the antigen-specific interaction of T helper and antigen-presenting cells (APCs). In T cells, acetylated microtubules concentrated at contact site with APCs, surrounding clusters of CD3 and LFA-1. TcR engagement induced a transient deacetylation of microtubules at early times and an enhanced acetylation at late times. Confocal videomicroscopy studies revealed that the HDAC6 tubulin deacetylase was translocated and concentrated at the contact site of T cells with APCs. Overexpression of HDAC6 but not a dead deacetylase mutant in T cells disorganized CD3 and LFA-1 at the immune synapse. This effect was reverted by treatment with the deacetylase inhibitor trichostatin A. The antigen-specific translocation of the microtubule organizing center (MTOC) and IL-2 production were also severely impaired by overexpression of HDAC6. Our results underscore the key role for HDAC6 in the organization of the immune synapse and the antigen-specific reorientation of the MTOC.     

Histone deacetylase 3 is required for centromeric H3K4 deacetylation and sister chromatid cohesion

We describe here the role of histone deacetylase 3 (HDAC3) in sister chromatid cohesion and the deacetylation of histone H3 Lys 4 (H3K4) at the centromere. HDAC3 knockdown induced spindle assembly checkpoint activation and sister chromatid dissociation. The depletion of Polo-like kinase 1 (Plk1) or Aurora B restored cohesion in HDAC3-depleted cells. HDAC3 was also required for Shugoshin localization at centromeres. Finally, we show that HDAC3 depletion results in the acetylation of centromeric H3K4, correlated with a loss of dimethylation at the same position. These findings provide a functional link between sister chromatid cohesion and the mitotic "histone code".     

Spiruchostatin A inhibits proliferation and differentiation of fibroblasts from patients with pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disorder characterized by the proliferation of interstitial fibroblasts and the deposition of extracellular matrix causing impaired gas exchange. Spiruchostatin A (SpA) is a histone deacetylase inhibitor (HDI) with selectivity toward Class I enzymes, which distinguishes it from other nonspecific HDIs that are reported to inhibit (myo)fibroblast proliferation and differentiation. Because the selectivity of HDIs may be important clinically, we postulated that SpA inhibits the proliferation and differentiation of IPF fibroblasts. Primary fibroblasts were grown from lung biopsy explants obtained from patients with IPF or from normal control subjects, using two-dimensional or three-dimensional culture models. The effect of SpA on fibroproliferation in serum-containing medium ± transforming growth factor (TGF)-β(1) was quantified by methylene blue binding. The acetylation of histone H3, the expression of the cell-cycle inhibitor p21(waf1), and the myofibroblast markers α-smooth muscle actin (α-SMA) and collagens I and III were determined by Western blotting, quantitative RT-PCR, immunofluorescent staining, or colorimetry. SpA inhibited the proliferation of IPF or normal fibroblasts in a time-dependent and concentration-dependent manner (concentration required to achieve 50% inhibition = 3.8 ± 0.4 nM versus 7.8 ± 0.2 nM, respectively; P < 0.05), with little cytotoxicity. Western blot analyses revealed that SpA caused a concentration-dependent increase in histone H3 acetylation, paralleling its antiproliferative effect. SpA also increased p21(waf1) expression, suggesting that direct cell-cycle regulation was the mechanism of inhibiting proliferation. Although treatment with TGF-β(1) induced myofibroblast differentiation associated with increased expression of α-SMA, collagen I and collagen III and soluble collagen release, these responses were potently inhibited by SpA. These data support the concept that bicyclic tetrapeptide HDIs merit further investigation as potential treatments for IPF.     

Prognostic significance of the therapeutic targets histone deacetylase 1, 2, 6 and acetylated histone H4 in cutaneous T-cell lymphoma

Aims:  Aberrant histone acetylation has been associated with malignancy and histone deacetylase (HDAC) inhibitors are currently being investigated in numerous clinical trials. So far, the malignancy most sensitive to HDAC inhibitors has been cutaneous T-cell lymphoma (CTCL). The reason for this sensitivity is unclear and studies on HDAC expression and histone acetylation in CTCL are lacking. The aim of this study was to address this issue.
Methods and results:  The immunohistochemical expression of HDAC1, HDAC2, HDAC6, and acetylated H4 was examined in 73 CTCLs and the results related to histological subtypes and overall survival. HDAC1 was most abundantly expressed ( P  < 0.0001), followed by HDAC2; HDAC6 and H4 acetylation were equally expressed. HDAC2 ( P  = 0.001) and H4 acetylation ( P  = 0.03) were significantly more common in aggressive than indolent CTCL subtypes. In contrast, no differences were observed for HDAC1 and HDAC6. In a Cox analysis, elevated HDAC6 was the only parameter showing significant influence on survival ( P  = 0.04).
Conclusions:  High expression of HDAC2 and acetylated H4 is more common in aggressive than indolent CTCL. HDAC6 expression is associated with a favorable outcome independent of the subtype.     

Histone deacetylase activity is necessary for chromosome condensation during meiotic maturation in Xenopus laevis

Chromosome condensation is thought to be an essential step for the faithful transmission of genetic information during cellular division or gamete formation. The folding of DNA into metaphase chromosomes and its partition during the cell cycle remains a fundamental cellular process that, at the molecular level, is poorly understood. Particularly, the role of histone deacetylase (HDAC) activities in establishing and maintaining meiotic metaphase chromosome condensation has been little documented. In order to better understand how metaphase chromosome condensation is achieved during meiosis, we explored, in vivo, the consequences of HDAC activities inhibition in a Xenopus oocyte model. Our results show that deacetylase activity plays a crucial role in chromosome condensation. This activity is necessary for correct chromosome condensation since the earlier stages of meiosis, but dispensable for meiosis progression, meiosis exit and mitosis entry. We show that HDAC activity correlates with chromosome condensation, being higher when chromosomes are fully condensed and lower during interphase, when chromosomes are decondensed. In addition, we show that, unlike histone H4, Xenopus maternal histone H3 is stored in the oocyte as a hypoacetylated form and is rapidly acetylated when the oocyte exits meiosis.     

HDAC6 controls major cell response pathways to cytotoxic accumulation of protein aggregates

A cellular defense mechanism counteracts the deleterious effects of misfolded protein accumulation by eliciting a stress response. The cytoplasmic deacetylase HDAC6 (histone deacetylase 6) was previously shown to be a key element in this response by coordinating the clearance of protein aggregates through aggresome formation and their autophagic degradation. Here, for the first time, we demonstrate that HDAC6 is involved in another crucial cell response to the accumulation of ubiquitinated protein aggregates, and unravel its molecular basis. Indeed, our data show that HDAC6 senses ubiquitinated cellular aggregates and consequently induces the expression of major cellular chaperones by triggering the dissociation of a repressive HDAC6/HSF1 (heat-shock factor 1)/HSP90 (heat-shock protein 90) complex and a subsequent HSF1 activation. HDAC6 therefore appears as a master regulator of the cell protective response to cytotoxic protein aggregate formation.     

Time Course of Histone Deacetylase 1 and Acetylated H3 and H4 Histones in the Brain of Rats Treated with Ladasten

We studied the effects of single intragastric administration of ladasten in a dose of 50 mg/kg on the time course of histone deacetylase 1 (HDAC1) and levels of acetylated histones H3 (Lys9) and H4 (Lys8) in the striatum, hippocampus, and hypothalamus. Ladasten reduced HDAC1 level in rat striatum and hippocampus and modifi ed H3acK9 and H4acK8 levels in various structures of rat brain.     

Expression of HDAC9 in lung cancer – potential role in lung carcinogenesis

Our previous studies identified important molecules involved in lung carcinogenesis through a comprehensive search for the downstream targets of oncogenic KRAS, and these findings suggested that an investigation into the downstream targets of oncogenic KRAS might represent a useful strategy for elucidating the common molecular bases of lung cancer. Among the downstream targets of oncogenic KRAS, a focus was placed on HDAC9, a member of the histone deacetylase family, in the present study because epigenetic modification of DNA or the histone proteins is known to play an important role in carcinogenesis. The immunohistochemical expression of HDAC9 was examined in surgically resected primary lung cancers (130 adenocarcinoma, 49 squamous cell carcinomas, one large cell carcinoma, and 6 small cell carcinomas) and potential associations between its expression level and pathologic factors were analyzed. The results showed that HDAC9 expression levels were lower in lung cancer cells than in non-tumor epithelial cells, and were also significantly lower in adenocarcinomas among the histological types. Moreover, HDAC9 expression levels were significantly lower in adenocarcinomas with lymphatic canal involvement. The restoration of HDAC9 in lung cancer cells losing its expression severely attenuated their growth activity in vitro. These results suggest that HDAC9 may be a suppressor and its downregulation might promote the progression process, especially in lung adenocarcinomas.     

Normal Shear Stress and Vascular Smooth Muscle Cells Modulate Migration of Endothelial Cells Through Histone Deacetylase 6 Activation and Tubulin Acetylation

Endothelial cells (ECs) line the innermost of the blood vessel wall and are constantly subjected to shear stress imposed by blood flow. ECs were also influenced by the neighboring vascular smooth muscle cells (VSMCs). The bidirectional communication between ECs and VSMCs modulates vascular homeostasis. In this study, the involvement of histone deacetylase 6 (HDAC6) in modulating migration of ECs co-cultured with VSMCs by the normal level of laminar shear stress (NSS) was investigated. ECs was either cultured alone or co-cultured with VSMCs under static conditions or subjected to NSS of 15 dyne/cm² by using a parallel-plate co-culture flow chamber system. It was demonstrated that both NSS and VSMCs could increase EC migration. The migration level of ECs co-cultured with VSMCs under NSS was not higher than that under the static condition. The process of EC migration regulated by VSMCs and NSS was associated with the increased expression of HDAC6 and low level of acetylated tubulin. The increase in HDAC6 expression was accompanied by a time-dependent decrease in the acetylation of tubulin in ECs co-cultured with VSMCs. Inhibition of the HDAC6 by siRNA or tributyrin, an inhibitor of HDACs, induced a parallel alteration in the migration and the acetylated tubulin of ECs co-cultured with VSMCs. It was observed by immunofluorescence staining that the acetylated tubulin was distributed mostly around the cell nucleus in ECs co-cultured with VSMCs. The results suggest that the NSS may display a protective function on the vascular homeostasis by modulating EC migration to a normal level in a VSMC-dependent manner. This modulation process involves the down-regulation of acetylated tubulin which results from increased HDAC6 activity in ECs.     

HIV-1感染引起组蛋白乙酰化和增加p21WAF1表达

目的：DNA甲基化和组蛋白乙酰化是基因表达调控的主要形式。人类免疫缺陷病毒（HIV-1）可引起T淋巴细胞DNA甲基化酶上调。本文旨在明确HIV-1对细胞周期依赖刺激酶抑制剂p21^WAF1表达的影响。方法：建立HIV-1感染的Hut78细胞系；以RT-PCR和Westem blotting 分析p21^WAF1表达情况；以亚硫酸氢钠修饰DNA和基因测序，研究p21^WAF1基因启动子甲基化，以Western blot-ting 和染色体免疫测定探究总组蛋白和与p21^WAF1基因启动子相关的组蛋白乙酰化水平。并以GST pull-down和免疫沉淀分析HIV-1导致乙酰化及乙酰化引起p21^WAF1过表达的可能机理。结果：HIV-1感染后，其反式激活蛋白Tat与辅助转录因子P/CAF、hGCN5结合，共同刺激组蛋白H3乙酰化。尽管p21^WAF1启动子部分区域有甲基化发生，但p21^WAF1表达仍上调。这可能与E2A对p21^WAF1的作用有关。结论：HIV-1感染可引起T淋巴细胞p21^WAF1基因的甲基化和乙酰化紊乱，导致p21^WAF1表达增强。     

Hsp90 inhibitors as promising agents for radiotherapy

The 90-kD heat shock protein (Hsp90) is an abundant molecular chaperone catalyzing maturation and activation of client proteins. A number of the Hsp90 client proteins are components of cancer cell-associated signaling pathways that ensure unlimited growth of tumors and their resistance to chemotherapy and radiotherapy. Upon inhibition of the Hsp90 chaperone function, such client proteins are destabilized and degraded which disrupts multiple pathways essential for tumor cell survival; hence, pharmacological Hsp90 inhibitors could be applied in anticancer therapy. Several Hsp90-inhibiting compounds are currently tested in preclinical or phase I–III clinical trials as single anticancer agents or in combination with conventional drugs and radiation. The present review summarizes the data characterizing Hsp90 inhibitors as agents that sensitize human tumors to irradiation which may improve the outcome of radiotherapy. We also discuss molecular mechanisms of the Hsp90 inhibition-induced radiosensitization and its selectivity toward cancer cells.