trichostatin-a and tubacin

The deleterious effects of glucocorticoids on glucose homeostasis limit their clinical use. There is substantial evidence demonstrating that islet function impaired by long-term glucocorticoids exposure is a core defect in the progression of impaired glucose tolerance to diabetes. The activity of heat-shock protein (Hsp) 90 is required to maintain the hormone-binding activity and stability of glucocorticoid receptor (GR). In the present study, Hsp90 inhibition by 17-DMAG counteracted dexamethasone-mediated inhibition of glucose-stimulated insulin secretion in isolated rat islets as well as expressions of neuropeptide Y (NPY) and somatostatin receptor 3 (SSTR3), two negative regulators of insulin secretion. Like 17-DMAG, both the pan-histone deacetylase (HDAC) inhibitor TSA and HDAC6 inhibitor Tubacin exhibited a similar action in protecting islet function against dexamethasone-induced injury, along with the downregulation of NPY and SSTR3 expressions. The hyperacetylation of Hsp90 by TSA and Tubacin disrupted its binding ability to GR and blocked dexamethasone-elicited nuclear translocation of GR in INS-1 β-cell lines. In addition, Tubacin treatment triggered the GR protein degradation through the ubiquitin-proteasome pathway. These findings suggest that Hsp90 acetylation by inhibiting HDAC6 activity may be a potential strategy to prevent the development of steroid diabetes mellitus via alleviating glucocorticoid-impaired islet function.

Topics: Acetylation; Anilides; Animals; Benzoquinones; Cell Line; Dexamethasone; Glucocorticoids; Histone Deacetylase 6; Histone Deacetylase Inhibitors; HSP90 Heat-Shock Proteins; Hydroxamic Acids; Insulin; Islets of Langerhans; Lactams, Macrocyclic; Male; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Proteolysis; Rats, Sprague-Dawley; Secretory Pathway; Tissue Culture Techniques

Group IVA cytosolic phospholipase A2 (cPLA2 or PLA2G4A) is a key enzyme that contributes to inflammation via the generation of arachidonic acid and eicosanoids. While much is known about regulation of cPLA2 by posttranslational modification such as phosphorylation, little is known about its epigenetic regulation. In this study, treatment with histone deacetylase (HDAC) inhibitors, trichostatin A (TSA), valproic acid, tubacin and the class I HDAC inhibitor, MS-275, were found to increase cPLA2α messenger RNA (mRNA) expression in SH-SY5Y human neuroblastoma cells. Co-treatment of the histone acetyltransferase (HAT) inhibitor, anacardic acid, modulated upregulation of cPLA2α induced by TSA. Specific involvement of class I HDACs and HAT in cPLA2α regulation was further shown, and a Tip60-specific HAT inhibitor, NU9056, modulated the upregulation of cPLA2α induced by MS-275. In addition, co-treatment of with histone methyltransferase (HMT) inhibitor, 5'-deoxy-5'-methylthioadenosine (MTA) suppressed TSA-induced cPLA2α upregulation. The above changes in cPLA2 mRNA expression were reflected at the protein level by Western blots and immunocytochemistry. Chromatin immunoprecipitation (ChIP) showed TSA increased binding of trimethylated H3K4 to the proximal promoter region of the cPLA2α gene. Cell injury after TSA treatment as indicated by lactate dehydrogenase (LDH) release was modulated by anacardic acid, and a role of cPLA2 in mediating TSA-induced injury shown, after co-incubation with the cPLA2 selective inhibitor, arachidonoyl trifluoromethyl ketone (AACOCF3). Together, results indicate epigenetic regulation of cPLA2 and the potential of such regulation for treatment of chronic inflammation.

Topics: Anacardic Acids; Anilides; Benzamides; Cell Line, Tumor; Chromatin Immunoprecipitation; Deoxyadenosines; Epigenesis, Genetic; Fluorescent Antibody Technique; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Group IV Phospholipases A2; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; L-Lactate Dehydrogenase; Lysine; Neuroblastoma; Pyridines; Real-Time Polymerase Chain Reaction; RNA, Messenger; Thiazoles; Thionucleosides; Valproic Acid

Paclitaxel (PTX) is a microtubule-targeting drug widely used for the treatment of a variety of cancers. However, drug resistance can emerge after a series of treatments, and this can seriously affect the patient's prognosis. Here, we analyzed the mechanism of PTX resistance using a human prostate cancer cell line, PC3, and its PTX-resistant subline, PC3-PR. Compared with PC3, PC3-PR exhibited some unique phenotypes that might be associated with PTX resistance, including decreased expression of acetylated α-tubulin and the cell cycle regulator p21, and increased expression of βIII tubulin, histone deacetylase 6 (HDAC6), and the anti-apoptotic protein Bcl2. The drug exporters MDR1 and MRP1 were not involved in PTX resistance. Although cabazitaxel (CTX), a novel taxoid, has been reported to overcome PTX resistance, its mechanism of action is unknown. We found that treatment of PC3-PR cells with CTX induced expression of acetylated α-tubulin and p21, but not the related regulators p27, p15, and p16 or the Bcl2 family proteins. The pan-HDAC inhibitors trichostatin A and suberanilohydroxamic acid and the HDAC6-specific inhibitor tubacin inhibited PC3-PR proliferation and increased expression of p21 and acetylated α-tubulin in a manner similar to CTX. Our data shed light on the cellular response to PTX and CTX.

Topics: Acetylation; Anilides; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Paclitaxel; Prostatic Neoplasms; Protein Stability; Taxoids; Tubulin; Tubulin Modulators; Vorinostat

Tubulin can be acetylated/deacetylated on Lys40 of the α-subunit. Studies of the post-translational acetylation/deacetylation of tubulin using biochemical techniques require tubulin preparations that are enriched in AcTubulin (acetylated tubulin) and (for comparison) preparations lacking AcTubulin. Assembly-disassembly cycling of microtubules gives tubulin preparations that contain little or no AcTubulin. In the present study we demonstrated that this result is owing to the presence of high deacetylating activity in the extracts. This deacetylating activity in rat brain homogenates was inhibited by TSA (Trichostatin A) and tubacin, but not by nicotinamide, indicating that HDAC6 (histone deacetylase 6) is involved. TSA showed no effect on microtubule polymerization or depolymerization. We utilized these properties of TSA to prevent deacetylation during the assembly-disassembly procedure. The effective inhibitory concentration of TSA was 3 μM in the homogenate and 1 μM in the subsequent cycling steps. By comparison with immunopurified AcTubulin, we estimated that ~64% of the tubulin molecules in the three cycled preparations were acetylated. The protein profiles of these tubulin preparations, as assessed by SDS/PAGE and Coomassie Blue staining, were identical to that of a preparation completely lacking AcTubulin obtained by assembly-disassembly cycles in the absence of TSA. The tyrosination state and in vitro assembly-disassembly kinetics were the same regardless of the degree of acetylation.

Topics: Acetylation; Anilides; Animals; Blotting, Western; Brain; Brain Chemistry; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Microtubules; Nerve Tissue Proteins; Polymers; Protein Processing, Post-Translational; Rats; Rats, Wistar; Rosaniline Dyes; Tubulin

We present for the first time that histone deacetylase 6 (HDAC6) regulates EGFR degradation and trafficking along microtubules in Pkd1 mutant renal epithelial cells. HDAC6, the microtubule-associated α-tubulin deacetylase, demonstrates increased expression and activity in Pkd1 mutant mouse embryonic kidney cells. Targeting HDAC6 with a general HDAC inhibitor, trichostatin (TSA), or a specific HDAC6 inhibitor, tubacin, increased the acetylation of α-tubulin and downregulated the expression of EGFR in Pkd1 mutant renal epithelial cells. HDAC6 was co-localized with EGF induced endocytic EGFR and endosomes, respectively. Inhibition of the activity of HDAC6 accelerated the trafficking of EGFR from early endosomes to late endosomes along the microtubules. Without EGF stimulation EGFR was randomly distributed while after stimulation with EGF for 30 min, EGFR was accumulated around α-tubulin labeled microtubule bundles. These data suggested that the Pkd1 mutation induced upregulation of HDAC6 might act to slow the trafficking of EGFR from early endosomes to late endosomes along the microtubules for degradation through deacetylating α-tubulin. In addition, inhibition of HDAC activity decreased the phosphorylation of ERK1/2, the downstream target of EGFR axis, and normalized EGFR localization from apical to basolateral in Pkd1 knockout mouse kidneys. Thus, targeting HDAC6 to downregulate EGFR activity may provide a potential therapeutic approach to treat polycystic kidney disease.

Topics: Anilides; Animals; Endocytosis; Endosomes; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; HEK293 Cells; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Kidney; Lysosomes; Mice; Microtubules; Nocodazole; Phosphorylation; Protein Transport; Proteolysis; RNA, Small Interfering; TRPP Cation Channels; Tubulin; Up-Regulation

Proteolytic systems and the aggresome pathway contribute to preventing accumulation of cytotoxic aggregation-prone proteins. Although polyubiquitylation is usually required for degradation or aggresome formation, several substrates are processed independently of ubiquitin through a poorly understood mechanism. Here, we found that p62/SQSTM1, a multifunctional adaptor protein, was involved in the selective autophagic clearance of a non-ubiquitylated substrate, namely an aggregation-prone isoform of STAT5A (STAT5A_ΔE18). By using a cell line that stably expressed STAT5A_ΔE18, we investigated the properties of its aggregation and degradation. We found that STAT5A_ΔE18 formed non-ubiquitylated aggresomes and/or aggregates by impairment of proteasome functioning or autophagy. Transport of these aggregates to the perinuclear region was inhibited by trichostatin A or tubacin, inhibitors of histone deacetylase (HDAC), indicating that the non-ubiquitylated aggregates of STAT5A_ΔE18 were sequestered into aggresomes in an HDAC6-dependent manner. Moreover, p62 was bound to STAT5A_ΔE18 through its PB1 domain, and the oligomerization of p62 was required for this interaction. In p62-knockdown experiments, we found that p62 was required for autophagic clearance of STAT5A_ΔE18 but not for its aggregate formation, suggesting that the binding of p62 to non-ubiquitylated substrates might trigger their autophagic clearance.

Topics: Adaptor Proteins, Signal Transducing; Anilides; Autophagy; Gene Knockdown Techniques; HeLa Cells; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Mutation; Protein Binding; Protein Multimerization; Protein Transport; Proteolysis; Sequestosome-1 Protein; STAT5 Transcription Factor; Transgenes; Ubiquitination

Polymer-mediated gene delivery is an attractive alternative to viral vectors, but is limited by low efficacies of transgene expression. We report that polymers possess differential efficacies for transfecting closely related human prostate cancer cells, which correlates with dramatically different intracellular fate of nanoscale cargo in these cells. Sequestration of nanoscale cargo (27 nm quantum dots and 150-250 nm polyplexes) at a single location near the microtubule organizing compartment (MTOC) in PC3-PSMA human prostate cancer cells correlated with lower polymer-mediated transgene expression compared to PC3 cells, which showed distributed localization throughout the cytoplasm. We show, for the first time, that treatment with the histone deacetylase 6 (HDAC6) inhibitor tubacin, which acetylates tubulin of microtubules in the cytoplasm, abolished quantum dot and polyplex sequestration at the perinuclear recycling compartment/microtubule organizing center (PNRC/MTOC) and increased polymer-mediated transgene expression by up to forty-fold compared to cells not treated with the HDAC6 inhibitor drug. Treatment with the class I and II HDAC inhibitor trichostatin A (TSA) demonstrated similar levels of transgene expression enhancement. These results indicate that mediators of intracellular trafficking can be employed to modulate nanoparticle fate and enhance the efficacy of nanoscale therapeutics in cells. Simultaneous use of high-efficacy polymers along with mediators of intracellular trafficking is an attractive synergistic strategy for enhancing polymer-mediated transgene expression.

Topics: Anilides; Biological Transport; Cell Line, Tumor; Cytoplasm; DNA; Gene Expression; Gene Transfer Techniques; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Plasmids; Polymers; Prostatic Neoplasms; Quantum Dots; Transgenes

Here we investigated whether the sensitivity of microtubules to severing by katanin is regulated by acetylation of the microtubules. During interphase, fibroblasts display long microtubules with discrete regions rich in acetylated tubulin. Overexpression of katanin for short periods of time produced breaks preferentially in these regions. In fibroblasts with experimentally enhanced or diminished microtubule acetylation, the sensitivity of the microtubules to severing by katanin was increased or decreased, respectively. In neurons, microtubules are notably more acetylated in axons than in dendrites. Experimental manipulation of microtubule acetylation in neurons yielded similar results on dendrites as observed on fibroblasts. However, under these experimental conditions, axonal microtubules were not appreciably altered with regard to their sensitivity to katanin. We hypothesized that this may be attributable to the effects of tau on the axonal microtubules, and this was validated by studies in which overexpression of tau caused microtubules in dendrites and fibroblasts to be more resistant to severing by katanin in a manner that was not dependent on the acetylation state of the microtubules. Interestingly, none of these various findings apply to spastin, because the severing of microtubules by spastin does not appear to be strongly influenced by either the acetylation state of the microtubules or tau. We conclude that sensitivity to microtubule severing by katanin is regulated by a balance of factors, including the acetylation state of the microtubules and the binding of tau to the microtubules. In the neuron, this contributes to regional differences in the microtubule arrays of axons and dendrites.

Topics: Acetylation; Adenosine Triphosphatases; Anilides; Animals; Animals, Newborn; Antibodies, Monoclonal; Cells, Cultured; Fibroblasts; Gene Expression Regulation; Green Fluorescent Proteins; Hippocampus; Histone Deacetylase 6; Histone Deacetylases; Humans; Hydroxamic Acids; Katanin; Microtubules; Neurons; Protein Synthesis Inhibitors; Rats; Transfection; Tubulin

NIMA-related kinases (Neks) belong to a large family of Ser/Thr kinases that have critical roles in coordinating microtubule dynamics during ciliogenesis and mitotic progression. The Nek kinases are also expressed in neurons, whose axonal projections are, similarly to cilia, microtubule-abundant structures that extend from the cell body. We therefore investigated whether Nek kinases have additional, non-mitotic roles in neurons. We found that Nek3 influences neuronal morphogenesis and polarity through effects on microtubules. Nek3 is expressed in the cytoplasm and axons of neurons and is phosphorylated at Thr475 located in the C-terminal PEST domain, which regulates its catalytic activity. Although exogenous expression of wild-type or phosphomimic (T475D) Nek3 in cultured neurons has no discernible impact, expression of a phospho-defective mutant (T475A) or PEST-truncated Nek3 leads to distorted neuronal morphology with disturbed polarity and deacetylation of microtubules via HDAC6 in its kinase-dependent manner. Thus, the phosphorylation at Thr475 serves as a regulatory switch that alters Nek3 function. The deacetylation of microtubules in neurons by unphosphorylated Nek3 raises the possibility that it could have a role in disorders where axonal degeneration is an important component.

Topics: Acetylation; Anilides; Animals; Cell Polarity; Cells, Cultured; Ganglia, Spinal; HeLa Cells; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Mice; Microtubules; Neurons; NIMA-Related Kinases; Protein Isoforms; Protein Serine-Threonine Kinases; Threonine; Tubulin

In this work, the role of HDAC6, a type II histone deacetylase with tubulin deacetylase activity, in lymphocyte polarity, motility, and transmigration was explored. HDAC6 was localized at dynamic subcellular structures as leading lamellipodia and the uropod in migrating T-cells. However, HDAC6 activity did not appear to be involved in the polarity of migrating lymphocytes. Overexpression of HDAC6 in freshly isolated lymphocytes and T-cell lines increased the lymphocyte migration mediated by chemokines and their transendothelial migration under shear flow. Accordingly, the knockdown of HDAC6 expression in T-cells diminished their chemotactic capability. Additional experiments with HDAC6 inhibitors (trichostatin, tubacin), other structural related molecules (niltubacin, MAZ-1391), and HDAC6 dead mutants showed that the deacetylase activity of HDAC6 was not involved in the modulatory effect of this molecule on cell migration. Our results indicate that HDAC6 has an important role in the chemotaxis of T-lymphocytes, which is independent of its tubulin deacetylase activity.

Topics: Acetylation; Anilides; Cell Adhesion; Cell Migration Inhibition; Cell Polarity; Cells, Cultured; Chemotaxis; Gene Expression; Gene Silencing; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Lymphocyte Activation; Mutant Proteins; Protein Transport; T-Lymphocytes; Tubulin