• Journal of Life Science
• /
• v.28 no.8
• /
• pp.885-891
• /
• 2018

Clostridium difficile (C. difficile) toxin A is known to cause acute gut inflammation in humans and animals by triggering cytoskeletal disorganization in gut epithelial cells. In human colonocytes, toxin A blocks microtubule assembly by directly increasing the enzymatic activity of histone deacetylase-6 (HDAC-6), a tubulin-specific deacetylase, thereby markedly decreasing tubulin acetylation, which is essential for microtubule assembly. Microtubule assembly dysfunction-associated alterations (i.e., toxin A-exposed gut epithelial cells) are believed to trigger barrier dysfunction and gut inflammation downstream. We recently showed that potassium acetate blocked toxin A-induced microtubule disassembly by inhibiting HDAC-6. Herein, we tested whether acetic acid (AA), another small acetyl residue-containing agent, could block toxin A-induced tubulin deacetylation and subsequent microtubule assembly. Our results revealed that AA treatment increased tubulin acetylation and enhanced microtubule assembly in an HT29 human colonocyte cell line. AA also clearly increased tubulin acetylation in murine colonic explants. Interestingly, the AA treatment also alleviated toxin A-induced tubulin deacetylation and microtubule disassembly, and MTT assays revealed that AA reduced toxin A-induced cell toxicity. Collectively, these results suggest that AA can block the ability of toxin A to cause microtubule disassembly-triggered cytoskeletal disorganization by blocking toxin A-mediated deacetylation of tubulin.

• BMB Reports
• /
• v.55 no.4
• /
• pp.192-197
• /
• 2022

Cell signals for growth factors depend on the mechanical properties of the extracellular matrix (ECM) surrounding the cells. Microtubule acetylation is involved in the transforming growth factor (TGF)-β-induced myofibroblast differentiation in the soft ECM. However, the mechanism of activation of α-tubulin acetyltransferase 1 (α-TAT1), a major α-tubulin acetyltransferase, in the soft ECM is not well defined. Here, we found that casein kinase 2 (CK2) is required for the TGF-β-induced activation of α-TAT1 that promotes microtubule acetylation in the soft matrix. Genetic mutation and pharmacological inhibition of CK2 catalytic activity specifically reduced microtubule acetylation in the cells cultured on a soft matrix rather than those cultured on a stiff matrix. Immunoprecipitation analysis showed that CK2α, a catalytic subunit of CK2, directly bound to the C-terminal domain of α-TAT1, and this interaction was more prominent in the cells cultured on the soft matrix. Moreover, the substitution of alanine with serine, the 236th amino acid located at the C-terminus, which contains the CK2-binding site of α-TAT1, significantly abrogated the TGF-β-induced microtubule acetylation in the soft matrix, indicating that the successful binding of CK2 and the C-terminus of α-TAT1 led to the phosphorylation of serine at the 236th position of amino acids in α-TAT1 and regulation of its catalytic activity. Taken together, our findings provide novel insights into the molecular mechanisms underlying the TGF-β-induced activation of α-TAT1 in a soft matrix.

• BMB Reports
• /
• v.52 no.10
• /
• pp.619-624
• /
• 2019

The primary cilium is a microtubule-based structure projecting from a cell. Although the primary cilium shows no motility, it can recognize environmental stimuli. Thus, ciliary defects cause severe abnormalities called ciliopathies. Ciliogenesis is a very complex process and involves a myriad of components and regulators. In order to excavate the novel positive regulators of ciliogenesis, we performed mRNA microarray using starved NIH/3T3 cells. We selected 62 murine genes with corresponding human orthologs, with significantly upregulated expression at 24 h after serum withdrawal. Finally, calpain-6 was selected as a positive regulator of ciliogenesis. We found that calpain-6 deficiency reduced the percentage of ciliated cells and impaired sonic hedgehog signaling. It has been speculated that this defect might be associated with decreased levels of ${\alpha}-tubulin$ acetylation at lysine 40. This is the first study to report a novel role of calpain-6 in the formation of primary cilia.

• BMB Reports
• /
• v.56 no.3
• /
• pp.178-183
• /
• 2023

Huntington's disease (HD) is a neurodegenerative disorder, of which pathogenesis is caused by a polyglutamine expansion in the amino-terminus of huntingtin gene that resulted in the aggregation of mutant HTT proteins. HD is characterized by progressive motor dysfunction, cognitive impairment and neuropsychiatric disturbances. Histone deacetylase 6 (HDAC6), a microtubule-associated deacetylase, has been shown to induce transport- and release-defect phenotypes in HD models, whilst treatment with HDAC6 inhibitors ameliorates the phenotypic effects of HD by increasing the levels of α-tubulin acetylation, as well as decreasing the accumulation of mutant huntingtin (mHTT) aggregates, suggesting HDAC6 inhibitor as a HD therapeutics. In this study, we employed in vitro neural stem cell (NSC) model and in vivo YAC128 transgenic (TG) mouse model of HD to test the effect of a novel HDAC6 selective inhibitor, CKD-504, developed by Chong Kun Dang (CKD Pharmaceutical Corp., Korea). We found that treatment of CKD-504 increased tubulin acetylation, microtubule stabilization, axonal transport, and the decrease of mutant huntingtin protein in vitro. From in vivo study, we observed CKD-504 improved the pathology of Huntington's disease: alleviated behavioral deficits, increased axonal transport and number of neurons, restored synaptic function in corticostriatal (CS) circuit, reduced mHTT accumulation, inflammation and tau hyperphosphorylation in YAC128 TG mouse model. These novel results highlight CKD-504 as a potential therapeutic strategy in HD.

• Asian Pacific Journal of Cancer Prevention
• /
• v.13 no.7
• /
• pp.3471-3476
• /
• 2012

Background and Objective: Histone deacetylase (HDAC) inhibitors represent a promising class of potential anticancer agents for treatment of human malignancies. In this study, we investigated the effect of trichostatin A (TSA), one such HDAC inhibitor, in combination with docetaxel (TXT), a cytotoxic chemotherapy agent or erlotinib, a novel molecular target therapy drug, on lung cancer A549 cells. Methods: A549 cells were treated with TXT, erlotinib alone or in combination with TSA, respectively. Cell viability, apoptosis, and cell cycle distribution were evaluated using MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) assay, Hochst33258 staining and flow cytometry. Moreover, immunofluorescent staining and Western blot analysis were employed to examine alterations of ${\alpha}$-tubulin, heat shock protein 90 (hsp90), epidermal growth factor receptor (EGFR), and caspase-3 in response to the different exogenous stimuli. Results: Compared with single-agent treatment, co-treatment of A549 cells with TSA/TXT or TSA/erlotinib synergistically inhibited cell proliferation, induced apoptosis, and caused cell cycle delay at the $G_2/M$ transition. Treatment with TSA/TXT or TSA/erlotinib led to a significant increase of cleaved caspase-3 expression, also resulting in elevated acetylation of ${\alpha}$-tubulin or hsp90 and decreased expression of EGFR, which was negatively associated with the level of acetylated hsp90. Conclusions: Synergistic anti-tumor effects are observed between TXT or erlotinib and TSA on lung cancer cells. Such combinations may provide a more effective strategy for treating human lung cancer.