Animals ; Embryonic Stem Cells ; metabolism ; Histone Deacetylase 6 ; deficiency ; genetics ; metabolism ; Mice ; Reproduction ; genetics

BACKGROUNDNicotine, a major component of tobacco, is the main cause of smoking addiction. It was found that asthmatic patients who smoke were insensitive to glucocorticoid treatment. In this paper, we investigated whether nicotine could inhibit histone deacetylase 6 activity (HDAC6) and chaperone-dependent activation of the glucocorticoid receptor (GR) in A549 cells. Furthermore, the expression level of heat shock protein 90 (HSP90) was determined.

METHODSQuantitative real-time polymerase chain reaction was used to detect the levels of RNA transcription, and Western blotting was applied to analyze the levels of protein expression of HDAC6, GR, and HSP90 in A549 cells. Moreover, the effects of dexamethasone and trichostatin A were observed in A549 cells.

RESULTSA549 cell proliferation was inhibited in the presence of nicotine, and the level of RNA and protein expression of HDAC6 and GR were down-regulated.

CONCLUSIONSNicotine could inhibit HDAC6 activity and chaperone-dependent activation of GR. This might be the main reason why asthmatic patients who smoke show insensitivity to the glucocorticoid treatment.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; Enzyme Activation ; drug effects ; Histone Deacetylase 6 ; Histone Deacetylases ; genetics ; metabolism ; Humans ; Nicotine ; pharmacology ; Receptors, Glucocorticoid ; genetics ; metabolism

OBJECTIVES: Subarachnoid hemorrhage (SAH) is a serious cerebrovascular disease. Early brain injury (EBI) and cerebral vasospasm are the main reasons for poor prognosis of SAH patients. The specific inhibitor of histone deacetylase 6 (HDAC6), tubastatin A (TubA), has been proved to have a definite neuroprotective effect on a variety of animal models of acute and chronic central nervous system diseases. However, the neuroprotective effect of TubA on SAH remains unclear. This study aims to investigate the expression and localization of HDAC6 in the early stage of SAH, and to evaluate the protective effects of TubA on EBI and cerebral vasospasm after SAH and the underlying mechanisms. METHODS: Adult male SD rats were treated with modified internal carotid artery puncture to establish SAH model. In the first part of the experiment, rats were randomly divided into 6 groups: a sham group, a SAH-3 h group, a SAH-6 h group, a SAH-12 h group, a SAH-24 h group, and a SAH-48 h group. At 3, 6, 12, and 24 h after SAH modeling, the injured cerebral cortex of rats in each group was taken for Western blotting to detect the expression of HDAC6. In addition, the distribution of HDAC6 in the cerebral cortex of the injured side was measured by immunofluorescence double staining in SAH-24 h group rats. In the second part, rats were randomly divided into 4 groups: a sham group, a SAH group, a SAH+TubA_L group (giving 25 mg/kg TubA), and a SAH+TubA_H group (giving 40 mg/kg TubA). At 24 h after modeling, the injured cerebral cortex tissue was taken for Western blotting to detect the expression levels of HDAC6, endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining to detect apoptosis, and hematoxylin and eosin (HE) staining to detect the diameter of middle cerebral artery. RESULTS: The protein expression of HDAC6 began to increase at 6 h after SAH (P<0.05), peaked at 24 h (P<0.001), and decreased at 48 h, but there was still a difference compared with the sham group (P<0.05). HDAC6 is mainly expressed in the cytoplasm of the neurons. Compared with the sham group, the neurological score was decreased significantly and brain water content was increased significantly in the SAH group (both P<0.01). Compared with the SAH group, the neurological score was increased significantly and brain water content was decreased significantly in the SAH+TubA_H group (both P<0.05), while the improvement of the above indexes was not significant in the SAH+TubA_L group (both P>0.05). Compared with the sham group, the expression of eNOS was significantly decreased (P<0.01) and the expressions of iNOS and HDAC6 were significantly increased (P<0.05 and P<0.01, respectively) in the SAH group. Compared with the SAH group, the expression of eNOS was significantly increased, and iNOS and HDAC6 were significantly decreased in the SAH+TubA group (all P<0.05). Compared with the SAH group, the number of TUNEL positive cells was significantly decreased and the diameter of middle cerebral artery was significantly increased in the SAH+TubA group (both P<0.05) . CONCLUSIONS HDAC6 is mainly expressed in neurons and is up-regulated in the cerebral cortex at the early stage of SAH. TubA has protective effects on EBI and cerebral vasospasm in SAH rats by reducing brain edema and cell apoptosis in the early stage of SAH. In addition, its effect of reducing cerebral vasospasm may be related to regulating the expression of eNOS and iNOS.
Rats ; Male ; Animals ; Rats, Sprague-Dawley ; Subarachnoid Hemorrhage/drug therapy* ; Vasospasm, Intracranial/metabolism* ; Histone Deacetylase Inhibitors/therapeutic use* ; Neuroprotective Agents/therapeutic use* ; Histone Deacetylase 6/pharmacology* ; Apoptosis ; Brain Injuries/drug therapy*

Pancreatic cancer is a devastating disease with the worst prognosis among all the major human malignancies. The propensity to rapidly metastasize contributes significantly to the highly aggressive feature of pancreatic cancer. The molecular mechanisms underlying this remain elusive, and proteins involved in the control of pancreatic cancer cell motility are not fully characterized. In this study, we find that histone deacetylase 6 (HDAC6), a member of the class II HDAC family, is highly expressed at both protein and mRNA levels in human pancreatic cancer tissues. HDAC6 does not obviously affect pancreatic cancer cell proliferation or cell cycle progression. Instead, it significantly promotes the motility of pancreatic cancer cells. Further studies reveal that HDAC6 interacts with cytoplasmic linker protein 170 (CLIP-170) and that these two proteins function together to stimulate the migration of pancreatic cancer cells. These findings provide mechanistic insight into the progression of pancreatic cancer and suggest HDAC6 as a potential target for the management of this malignancy.
Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Gene Knockdown Techniques ; Histone Deacetylase 6 ; Histone Deacetylases ; metabolism ; Humans ; Microtubule-Associated Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; Pancreatic Neoplasms ; enzymology ; metabolism ; pathology ; Protein Binding

OBJECTIVES: Neuropathic pain (NP) is a chronic pain caused by somatosensory neuropathy or disease, and genistein (Gen) might be a potential drug for the treatment of NP. Therefore, this study aims to investigate the effect of Gen on lipopolysaccharide (LPS)-induced inflammatory injury of dorsal root ganglion neuron (DRGn) in rats and the possible molecular mechanism. METHODS: The DRGn of 1-day-old juvenile rats were taken for isolation and culture. The DRGn in logarithmic growth phase were divided into a control group, a LPS group, a tubastatin hydrochloride (TSA)+LPS group, a Gen1+LPS group, a Gen2+LPS group, a Gen2+LPS+TSA group, a Gen2+pcDNA-histone deacetylase 6 (HDAC6)+LPS group, and a Gen2+pcDNA3.1+LPS group. The LPS group was treated with 1 μg/mL LPS for 24 h; the TSA+LPS group, the Gen1+LPS group, the Gen2+LPS group were treated with 5 μmol/L TSA, 5 μmol/L Gen, 10 μmol/L Gen respectively for 0.5 h, and then added 1 μg/mL LPS for 24 h; the Gen2+TSA+LPS group was treated with 10 μmol/L Gen and 5 μmol/L TSA for 0.5 h and then added 1 μg/mL LPS for 24 h; the Gen2+pcDNA-HDAC6+LPS group and the Gen2+pcDNA3.1+LPS group received 100 nmol/L pcDNA-HDAC6 and pcDNA3.1 plasmids respectively, and 24 h after transfection, 10 μmol/L Gen was pretreated for 0.5 h, and then added 1 μg/mL LPS for 24 h. Real-time RT-PCR was used to detect the HDAC6 mRNA expression in DRGn; CCK-8 method was used to detect cell viability of DRGn; flow cytometry was used to detect cell apoptosis of DRGn; ELISA was used to detect the levels of IL-1β, IL-6, and TNF-α in DRGn culture supernatant; Western blotting was used to detect the protein expression of HDAC6, Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and NF-κB p65 in DRGn. RESULTS: Compared with the control group, the expression levels of HDAC6 mRNA and protein, the expression levels of TLR4 and MyD88 protein in DRGn of LPS group rats were significantly up-regulated, the ratio of p-NF-κB p65/NF-κB p65 was significantly increased, and the activity of DRGn was significantly decreased, the apoptosis rate was significantly increased, and the levels of IL-1β, IL-6 and TNF-α in the DRGn culture supernatant were significantly increased (all P<0.05). Compared with the LPS group, the expression levels of HDAC6 mRNA and protein, TLR4 and MyD88 protein expression levels in DRGn of the TSA+LPS group, the Gen1+LPS group, the Gen2+LPS group and the Gen2+TSA+LPS group were significantly down-regulated, the ratio of p-NF-κB p65/NF-κB p65 was significantly decreased, the activity of DRGn was significantly increased, the apoptosis rate was significantly decreased, and the levels of IL-1β, IL-6 and TNF-α in the DRGn culture supernatant were significantly decreased (all P<0.05), and the above changes were most obvious in the Gen2+TSA+LPS group. Compared with the Gen2+LPS group, the expression levels of HDAC6 mRNA and protein, TLR4 and MyD88 protein expression levels in DRGn of the Gen2+pcDNA-HDAC6+LPS group were significantly up-regulated, the ratio of p-NF-κB p65/NF-κB p65 was significantly increased, the activity of DRGn was significantly decreased, and the apoptosis rate was significantly increased, and the levels of IL-1β, IL-6 and TNF-α in the DRGn culture supernatant were significantly increased (all P<0.05). CONCLUSIONS Gen can alleviate LPS-induced DRGn inflammatory injury in rats, which might be related to down-regulating the expression of HDAC6 and further inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway.
Animals ; Ganglia, Spinal ; Genistein/pharmacology* ; Histone Deacetylase 6/metabolism* ; Interleukin-6/metabolism* ; Lipopolysaccharides ; Myeloid Differentiation Factor 88 ; NF-kappa B/metabolism* ; Neurons/metabolism* ; RNA, Messenger ; Rats ; Toll-Like Receptor 4/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

Angiogenesis, a process by which the preexisting blood vasculature gives rise to new capillary vessels, is associated with a variety of physiologic and pathologic conditions. However, the molecular mechanism underlying this important process remains poorly understood. Here we show that histone deacetylase 6 (HDAC6), a microtubule-associated enzyme critical for cell motility, contributes to angiogenesis by regulating the polarization and migration of vascular endothelial cells. Inhibition of HDAC6 activity impairs the formation of new blood vessels in chick embryos and in angioreactors implanted in mice. The requirement for HDAC6 in angiogenesis is corroborated in vitro by analysis of endothelial tube formation and capillary sprouting. Our data further show that HDAC6 stimulates membrane ruffling at the leading edge to promote cell polarization. In addition, microtubule end binding protein 1 (EB1) is important for HDAC6 to exert its activity towards the migration of endothelial cells and generation of capillary-like structures. These results thus identify HDAC6 as a novel player in the angiogenic process and offer novel insights into the molecular mechanism governing endothelial cell migration and angiogenesis.
Anilides ; pharmacology ; Animals ; Cell Movement ; Cell Polarity ; Cells, Cultured ; Chick Embryo ; Chickens ; Endothelial Cells ; cytology ; Histone Deacetylase 6 ; Histone Deacetylases ; metabolism ; physiology ; Humans ; Hydroxamic Acids ; pharmacology ; Mice ; Microtubule-Associated Proteins ; metabolism ; physiology ; Neovascularization, Physiologic

OBJECTIVETo investigate the role of histone acetylation in regulating influenza virus replicative intermediate double-stranded RNA (dsRNA)-induced interleukin-6 (IL-6) expression in A549 cells.

METHODSA549 cells were treated with influenza virus replicative intermediate dsRNA, histone deacetylase (HDAC) inhibitor trichostatin A (TSA), or HADC small interfering RNA (siRNA). The changes in the cellular IL-6 promoter activities were detected by dual-luciferase assay, and IL-6 mRNA and protein expressions in the cells were determined using real-time RT-PCR and ELISA, respectively.

RESULTSInfluenza virus replicative intermediate dsRNA obviously up-regulated IL-6 expression in the cells. HDAC inhibitor TSA significantly enhanced the activity of IL-6 promoter and increased IL-6 mRNA expression in A549 cells, and HDAC3 may play an important role in this process. HDAC inhibitor TSA and DNMT inhibitor DAC showed no synergic effect in regulating IL-6 expression.

CONCLUSIONSInfluenza virus replicative intermediate dsRNA-induced IL-6 expression in A549 cells is regulated by histone acetylation.

Acetylation ; Cell Line, Tumor ; Gene Expression Regulation ; Histone Deacetylase Inhibitors ; pharmacology ; Histones ; metabolism ; Humans ; Interleukin-6 ; metabolism ; Orthomyxoviridae ; genetics ; metabolism ; Promoter Regions, Genetic ; RNA, Double-Stranded ; RNA, Messenger ; genetics ; RNA, Viral

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.
Acetylation ; Actins ; chemistry ; metabolism ; Animals ; Cell Line ; Chromatography, High Pressure Liquid ; HSC70 Heat-Shock Proteins ; metabolism ; HSP40 Heat-Shock Proteins ; metabolism ; Histone Deacetylase 6 ; Histone Deacetylases ; metabolism ; Isotope Labeling ; Liver ; metabolism ; Lysine ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Microscopy, Confocal ; Nonmuscle Myosin Type IIA ; metabolism ; Protein Binding ; Proteomics ; Substrate Specificity ; Tandem Mass Spectrometry

OBJECTIVETo study the effect of histone deacetylation 6 (HDAC6) siRNA on the growth of xenografted human laryngeal squamous cell carcinoma cell line Hep-2 in nude mice and underlying mechanism.

METHODSLaryngeal squamous cell carcinoma cell line Hep-2 cells were subcutaneously injected to the back of nude mice and transplanted tumor model was established after one week. Nude mice was divided into three groups including blank control group, empty vector group and HDAC6 siRNA group, and the tumor growth was observed. Ki-67 proliferation index was detected by immunohistochemistry. Western blot, in situ hybridization and immunohistochemistry were used to detect the mRNA and protein expressions of HDAC6 in xenograft. The expressions of Bcl-2 and Bax proteins were examined by Western blotting. Cell apoptosis was detected by TUNEL.

RESULTSThe mean volume of xenograft transfected with HDAC6 siRNA was less than that of xenograft transfected with empty vector or that of xenograft with blank control treatment (P < 0.05). HDAC6 siRNA effectively down-regulated the expressions of HDAC6 mRNA and the expressions of HDAC6 and Bcl-2 proteins, but up-regulated the expression of Bcl-2 protein in xenografts, with significant differences (all P < 0.05). The proliferation index of Ki-67 in HDAC6 siRNA transfection group was significantly lower than that in blank control group or empty vector group (P < 0.05). TUNEL assay demonstrated that HDAC6 evidently evoked cell apoptosis (P < 0.05).

CONCLUSIONHDAC6 siRNA could effectively inhibited the growth of xenografted human laryngeal carcinoma cell line Hep-2 in nude mice, down-regulate the expressions of HDAC6 and bcl-2, and up-regulate the expression of bax.

Animals ; Cell Line, Tumor ; Down-Regulation ; Female ; Histone Deacetylase 6 ; Histone Deacetylases ; genetics ; metabolism ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Transfection ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo detect the expression of histone deacetylase 6 (HDAC6) in laryngeal squamous cell carcinoma, and to analyze the effects of downregulation of HDAC6 expression on cell cycle, proliferation and migration of laryngeal squamous cell carcinoma cell line Hep-2 cells, and to explore their possible molecular mechanisms.

METHODSImmunohistochemistry was used to detect the expression of HDAC6 protein in 55 cases of laryngeal squamous cell carcinoma and 20 cases of normal laryngeal mucosa. HDAC6 siRNA and control siRNA were transfected into Hep-2 cells via lipofectamine 2000, and the interfering effect was analyzed using Western blotting. The effects of downregulation of HDAC6 expression on cell cycle, proliferation and migration were determined by cell counting kit-8 (CCK-8), flow cytometry and Boyden chamber, respectively. Finally, Western blotting was used to detect the expressions of cell cycle, proliferation and migration related proteins.

RESULTSThere was a high level expression of HDAC6 protein in laryngeal squamous cell carcinoma, and its expression was not related to age and sex of the patients (P > 0.05), but closely associated with the degree of histological differentiation, TNM staging and lymph node metastasis (P < 0.05). HDAC6 siRNA effectively down-regulated the expression of HDAC6 protein in laryngeal squamous cell carcinoma cell line Hep-2 cells, and downregulation of its expression obviously inhibited cell proliferation, arrested cell cycle at G(0)/G(1) phase and decreased cell migration ability in Hep-2 cells. Additionally, the downregulation of HDAC6 protein expression markedly decreased the expressions of cyclin D1, cyclin E, cdk2 and MMP-9 proteins, but increased the expressions of p21 and E-cadherin proteins.

CONCLUSIONSHDAC6 may play a pivotal role in the carcinogenesis and development of laryngeal squamous cell carcinoma. The downregulation of HDAC6 expression-mediated cell proliferation inhibition, cell cycle arrest and decreased cell migration ability may be closely associated with the decrease of cyclin D1, cyclin E, cdk2 and MMP-9 proteins and increase of p21 and E-cadherin proteins.

Adult ; Aged ; Cadherins ; metabolism ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; Down-Regulation ; Female ; Histone Deacetylase 6 ; Histone Deacetylases ; genetics ; metabolism ; Humans ; Laryngeal Neoplasms ; genetics ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Middle Aged ; Neoplasm Staging ; Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins p21(ras) ; metabolism ; RNA, Small Interfering ; genetics ; Transfection