Histone deacetylase 6 regulates insulin signaling in pancreatic ß cells.

The reduction of pancreatic ß cell mass is one of the key factors for the onset of type 2 diabetes. Many reports have indicated that insulin signaling is important for type 2 diabetes, but the mechanism by which insulin signaling is altered in pancreatic ß cells remains unclear. This study was designed to examine the role of histone deacetylases (HDACs) in the regulation of insulin signaling in pancreatic ß cells. We found that insulin signaling was downregulated by inhibition of HDAC6. HDAC6 expression was specifically observed in pancreatic ß cells and was decreased in the pancreatic islets of a type 2 diabetes mouse model. When a mouse pancreatic ß cell line (MIN6 cells) was treated with palmitic acid to mimic the effect of a high-fat diet on pancreatic ß cells, HDAC6 was imported into the nucleus. These results suggest that HDAC6 plays an important role in the regulation of insulin signaling in pancreatic ß cells. Therefore, clarifying the regulation of insulin signaling by HDAC6 may be a valuable approach for the treatment of type 2 diabetes.

Molecular switching from ubiquitin-proteasome to autophagy pathways in mice stroke model.

The ubiquitin-proteasome system (UPS) and autophagy are two major pathways to degrade misfolded proteins that accumulate under pathological conditions. When UPS is overloaded, the degeneration pathway may switch to autophagy to remove excessive misfolded proteins. However, it is still unclear whether and how this switch occurs during cerebral ischemia. In the present study, transient middle cerebral artery occlusion (tMCAO) resulted in accelerated ubiquitin-positive protein aggregation from 0.5 h of reperfusion in mice brain after 10, 30 or 60 min of tMCAO. In contrast, significant reduction of p62 and induction of LC3-II were observed, peaking at 24 h of reperfusion after 30 and 60 min tMCAO. Western blot analyses showed an increase of BAG3 and HDAC6 at 1 or 24 h of reperfusion that was dependent on the ischemic period. In contract, BAG1 decreased at 24 h of reperfusion after 10, 30 or 60 min of tMCAO after double immunofluorescent colocalization of ubiquitin, HSP70, p62 and BAG3. These data suggest that a switch from UPS to autophagy occurred between 10 and 30 min of cerebral ischemia depending on the BAG1/BAG3 ratio and level of HDAC6.

Interactions between two regulatory proteins of microtubule dynamics, HDAC6, TPPP/p25, and the hub protein, DYNLL/LC8.

Degradation of unwanted proteins is important in protein quality control cooperating with the dynein/dynactin-mediated trafficking along the acetylated microtubule (MT) network. Proteins associated directly/indirectly with tubulin/MTs play crucial roles in both physiological and pathological processes. Our studies focus on the interrelationship of the tubulin deacetylase HDAC6, the MT-associated TPPP/p25 with its deacetylase inhibitory potency and the hub dynein light chain DYNLL/LC8, constituent of dynein and numerous other protein complexes. In this paper, evidence is provided for the direct interaction of DYNLL/LC8 with TPPP/p25 and HDAC6 and their assembly into binary/ternary complexes with functional potency. The in vitro binding data was obtained with recombinant proteins and used for mathematical modelling. These data and visualization of their localizations by bimolecular fluorescence complementation technology and immunofluorescence microscopy in HeLa cells revealed the promoting effect of TPPP/p25 on the interaction of DYNLL/LC8 with both tubulin and HDAC6. Localization of the LC8-2-TPPP/p25 complex was observed on the MT network in contrast to the LC8-2-HDAC6 complex, which was partly translocated to the nucleus. LC8-2 did not influence directly the acetylation of the MT network. However, the binding of TPPP/p25 to a new binding site of DYNLL/LC8, outside the canonical binding groove, counteracted the TPPP/p25-derived hyperacetylation of the MT network. Our data suggest that multiple associations of the regulatory proteins of the MT network could ensure fine tuning in the regulation of the intracellular trafficking process either by the complexation of DYNLL/LC8 with new partners or indirectly by the modulation of the acetylation level of the MT network.

The immunohistochemical expression and potential prognostic value of HDAC6 and AR in invasive breast cancer.

Previous studies have investigated the role of histone deacetylase 6 (HDAC6) in the regulation of androgen receptor (AR) in prostate cancer; however, the role of HDAC6 has not yet been clearly identified in breast cancer. The aim of this study was to examine the expression of HDAC6 and AR, determine the correlation between HDAC6 and AR, and assess the prognostic value of HDAC6 and AR in breast cancer. A total of 228 cases of invasive breast cancer were randomly selected. The expression of HDAC6 and AR was analyzed by immunohistochemistry. χ2 Tests were performed to determine the association between conventional clinicopathological factors and HDAC6, AR, and HDAC6/AR co-expression. Spearman correlation methods were performed to determine the correlation between HDAC6 and AR, and Kaplan-Meier analyses were performed to determine the prognostic impact of HDAC6, AR and HDAC6/AR co-expression; 58.8% (134/228) patients exhibited high expression of HDAC6. High HDAC6 expression was significantly associated with high histologic grade (G3) (P<.001) and p53 overexpression (P=.002). HDAC6 and AR expression levels were significantly associated (r=0.382, P<.01). In estrogen receptor (ER)-negative samples, high expression of HDAC6 was more common in the AR+ groups (P<.001) and correlated with high histologic grade (G3) (P=.009), as well as higher HER2 (P=.006) and p53 levels (P=.012). Higher expression of AR and HDAC6 and HDAC6/AR co-expression had a worse clinical prognosis. The expression levels of HDAC6 and AR are correlated in breast cancer; moreover, HDAC6 and AR have prognostic value in predicting the overall survival (OS) of ER-negative breast cancer patients.

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS.

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography - mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.