Recombinant adenoviruses expressing HPV16/18 E7 upregulate the HDAC6 and DNMT3B genes in C33A cells.

Objective: High-risk human papillomavirus (HPV) is a carcinogenic virus associated with nearly all cases of cervical cancer, as well as an increasing number of anal and oral cancers. The two carcinogenic proteins of HPV, E6 and E7, can immortalize keratinocytes and are essential for HPV-related cellular transformation. Currently, the global regulatory effects of these oncogenic proteins on the host proteome are not fully understood, and further exploration of the functions and carcinogenic mechanisms of E6 and E7 proteins is needed. Methods: We used a previously established platform in our laboratory for constructing recombinant adenoviral plasmids expressing the HPV16 E7 gene to further construct recombinant virus particles expressing HPV16/18 E6, E7, and both E6 and E7 genes. These recombinant viruses were used to infect C33A cells to achieve sustained expression of the HPV16/18 E6/E7 genes. Subsequently, total RNA was extracted and RNA-Seq technology was employed for transcriptome sequencing to identify differentially expressed genes associated with HPV infection in cervical cancer. Results: RNA-Seq analysis revealed that overexpression of the HPV16/18 E6/E7 genes upregulated GP6, CD36, HDAC6, ESPL1, and DNMT3B among the differentially expressed genes (DEGs) associated with cervical cancer. Spearman correlation analysis revealed a statistically significant correlation between the HDAC6 and DNMT3B genes and key pathways, including DNA replication, tumor proliferation signature, G2M checkpoint, p53 pathways, and PI3K/AKT/mTOR signaling pathways. Further, qRT-PCR and Western blot analyses indicated that both HPV16/18 E7 can upregulate the expression of HDAC6 and DNMT3B, genes associated with HPV infection-related cervical cancer. Conclusion: The successful expression of HPV16/18 E6/E7 in cells indicates that the recombinant viruses retain the replication and infection capabilities of Ad4. Furthermore, the recombinant viruses expressing HPV16/18 E7 can upregulate the HDAC6 and DNMT3B genes involved in cervical cancer pathways, thereby influencing the cell cycle. Additionally, HDAC6 and DNMT3B are emerging as important therapeutic targets for cancer. This study lays the foundation for further exploration of the oncogenic mechanisms of HPV E6/E7 and may provide new directions for the treatment of HPV-related cancers.

HDAC6 promotes inflammation in lupus nephritis mice by regulating transcription factors MAFF and KLF5 in renal fibrosis.

AIM: This study explored the effect and mechanism of MAFF and HDAC6 on renal fibrosis and inflammation in lupus nephritis (LN). METHODS: IL-33 treated renal epithelial cells and MRL/lpr mice were respectively used for in vitro and in vivo experiments. The expressions of HDAC6, MAFF, and KLF5 were measured in cells and renal tissues. Before and after cell transfection, the morphological changes in renal tissues were observed using Hematoxylin and eosin (H&E) and Masson staining. The proteinuria, serum creatinine (SCr), blood urea nitrogen (BUN), and double-stranded DNA (dsDNA) levels were detected by biochemical analysis. The expressions of fibrosis and inflammation related proteins (including α-SMA, Vimentin, IL-1ß, IL-6, and TNF-α), p65, and iNOS were also detected. The relationship among MAFF, HDAC6, and KLF5 was determined by chromatin immunoprecipitation and dual luciferase reporter gene assay. RESULTS: Renal tissues and cell models had elevated expressions of HDAC6 and KLF5, and decreased MAFF expression. HDAC6 suppression or MAFF overexpression led to suppression of proteinuria, SCr, BUN, and dsDNA levels, as well as attenuation of inflammatory infiltration and collagen deposition. HDAC6 can suppress MAFF expression via deacetylation to abolish its suppression of KLF5 expression, thus increasing KLF5 expression. In vivo and in vitro experiments showed the suppressive effect of HDAC6 suppression on renal fibrosis and inflammation can be abolished by KLF5 overexpression. CONCLUSION: HDAC6 suppresses MAFF expression via deacetylation to elevate KLF5 expression, which consequently enhances fibrosis and inflammatory response in LN.

Metabolic regulation of cytoskeleton functions by HDAC6-catalyzed α-tubulin lactylation.

Posttranslational modifications (PTMs) of tubulin, termed the "tubulin code", play important roles in regulating microtubule functions within subcellular compartments for specialized cellular activities. While numerous tubulin PTMs have been identified, a comprehensive understanding of the complete repertoire is still underway. In this study, we report that α-tubulin lactylation is catalyzed by HDAC6 by using lactate to increase microtubule dynamics in neurons. We identify lactylation on lysine 40 of α-tubulin in the soluble tubulin dimers. Notably, lactylated α-tubulin enhances microtubule dynamics and facilitates neurite outgrowth and branching in cultured hippocampal neurons. Moreover, we discover an unexpected function of HDAC6, acting as the primary lactyltransferase to catalyze α-tubulin lactylation. HDAC6-catalyzed lactylation is a reversible process, dependent on lactate concentrations. Intracellular lactate concentration triggers HDAC6 to lactylate α-tubulin, a process dependent on its deacetylase activity. Additionally, the lactyltransferase activity may be conserved in HDAC family proteins. Our study reveals the primary role of HDAC6 in regulating α-tubulin lactylation, establishing a link between cell metabolism and cytoskeleton functions.

HDAC6 Deletion Decreases Pristane-induced Inflammation.

Systemic lupus erythematosus is an autoimmune disease characterized by excessive inflammation and production of pathogenic Abs. Histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase. It has been reported that selective HDAC6 inhibition decreases inflammation in lupus mouse models. In this study, sex- and age-matched wild-type (WT) and HDAC6-/- mice on the C57BL/6 background were administered 0.5 ml of pristane or PBS i.p. at 8-12 wk of age and were euthanized 10 d later. At sacrifice, body weight and spleen weight were measured, sera were collected, and splenocytes and peritoneal cells were harvested for flow cytometry. We found pristane administration increased the spleen weight with no difference between WT and HDAC6-/- mice. Pristane administration promoted the population of CD11b+Ly6C++ inflammatory monocytes and CD11b+Ly6G+ neutrophils. Peritoneal recruitment of these inflammatory monocytes and neutrophils was significantly decreased in HDAC6-/- mice compared with the WT mice. Flow cytometry results showed that the number of CD69+ T and B cells was increased in HDAC6-/- mice. Pristane administration also induced the IFN signature genes as determined by RT-qPCR. Furthermore, IFN signature genes were not affected in HDAC6-/- mice compared with the WT mice. In vitro studies in J774A.1 cells revealed that the selective HDAC6 inhibitor (ACY-738) increased acetylation of NF-κB while increasing Stat1 phosphorylation, which resulted in inducible NO synthase production in LPS/IFN-γ-stimulated cells. Taken together, these results demonstrate that although HDAC6 inhibition may inhibit some inflammatory pathways, others remain unaffected.

Histone deacetylase 6 controls cardiac fibrosis and remodelling through the modulation of TGF-ß1/Smad2/3 signalling in post-infarction mice.

Histone deacetylase 6 (HDAC6) belongs to the class IIb group of the histone deacetylase family, which participates in remodelling of various tissues. Herein, we sought to examine the potential regulation of HDAC6 in cardiac remodelling post-infarction. Experimental myocardial infarction (MI) was created in HDAC6-deficient (HDAC6-/-) mice and wild-type (HADC6+/+) by left coronary artery ligation. At days 0 and 14 post-MI, we evaluated cardiac function, morphology and molecular endpoints of repair and remodelling. At day 14 after surgery, the ischemic myocardium had increased levels of HADC6 gene and protein of post-MI mice compared to the non-ischemic myocardium of control mice. As compared with HDAC6-/--MI mice, HADC6 deletion markedly improved infarct size and cardiac fibrosis as well as impaired left ventricular ejection fraction and left ventricular fraction shortening. At the molecular levels, HDAC6-/- resulted in a significant reduction in the levels of the transforming growth factor-beta 1 (TGF-ß1), phosphor-Smad-2/3, collagen I and collagen III proteins and/or in the ischemic cardiac tissues. All of these beneficial effects were reproduced by a pharmacological inhibition of HADC6 in vivo. In vitro, hypoxic stress increased the expressions of HADC6 and collagen I and III gene; these alterations were significantly prevented by the HADC6 silencing and TubA loading. These findings indicated that HADC6 deficiency resists ischemic injury by a reduction of TGF-ß1/Smad2/3 signalling activation, leading to decreased extracellular matrix production, which reduces cardiac fibrosis and dysfunction, providing a potential molecular target in the treatment of patients with MI.

Impact of HDAC6-mediated progesterone receptor expression on the response of breast cancer cells to hormonal therapy.

Modulation of estrogen receptor (ER) and progesterone receptor (PR) expression, as well as their emerging functional crosstalk, remains a potential approach for enhancing the response to hormonal therapy in breast cancer. Aberrant epigenetic alterations induced by histone deacetylases (HDACs) were massively implicated in dysregulating the function of hormone receptors in breast cancer. Although much is known about the regulation of ER signaling by HDAC, the precise role of HDAC in modulating the expression of PR and its impact on the outcomes of hormonal therapy is poorly defined. Here, we demonstrate the involvement of HDAC6 in regulating PR expression in breast cancer cells. The correlation between HDAC6 and hormone receptors was investigated in patients' tissues by immunohistochemistry (n = 80) and publicly available data (n = 3260) from breast cancer patients. We explored the effect of modulating the expression of HDAC6 as well as its catalytic inhibition on the level of hormone receptors by a variety of molecular analyses, including Western blot, immunofluorescence, Real-time PCR, RNA-seq analysis and chromatin immunoprecipitation. Based on our in-silico and immunohistochemistry analyses, HDAC6 levels were negatively correlated with PR status in breast cancer tissues. The downregulation of HDAC6 enhanced the expression of PR-B in hormone receptor-positive and triple-negative breast cancer (TNBC) cells. The selective targeting of HDAC6 by tubacin resulted in the enrichment of the H3K9 acetylation mark at the PGR-B gene promoter region and enhanced the expression of PR-B. Additionally, transcriptomic analysis of tubacin-treated cells revealed enhanced activity of acetyltransferase and growth factor signaling pathways, along with the enrichment of transcription factors involved in the transcriptional activity of ER, underscoring the crucial role of HDAC6 in regulating hormone receptors. Notably, the addition of HDAC6 inhibitor potentiated the effects of anti-ER and anti-PR drugs mainly in TNBC cells. Together, these data highlight the role of HDAC6 in regulating PR expression and provide a promising therapeutic approach for boosting breast cancer sensitivity to hormonal therapy.

Inhibiting histone deacetylase 6 suppresses the proliferation of microvascular endothelial cells by epigenetically activating miR-375-3p, potentially contributing to bone loss during mechanical unloading.

BACKGROUND: Mechanical unloading-induced bone loss threatens prolonged spaceflight and human health. Recent studies have confirmed that osteoporosis is associated with a significant reduction in bone microvessels, but the relationship between them and the underlying mechanism under mechanical unloading are still unclear. METHODS: We established a 2D clinostat and hindlimb-unloaded (HLU) mouse model to simulate unloading in vitro and in vivo. Micro-CT scanning was performed to assess changes in the bone microstructure and mass of the tibia. The levels of CD31, Endomucin (EMCN) and histone deacetylase 6 (HDAC6) in tibial microvessels were detected by immunofluorescence (IF) staining. In addition, we established a coculture system of microvascular endothelial cells (MVECs) and osteoblasts, and qRT‒PCR or western blotting was used to detect RNA and protein expression; cell proliferation was detected by CCK‒8 and EdU assays. ChIP was used to detect whether HDAC6 binds to the miRNA promoter region. RESULTS: Bone mass and bone microvessels were simultaneously significantly reduced in HLU mice. Furthermore, MVECs effectively promoted the proliferation and differentiation of osteoblasts under coculture conditions in vitro. Mechanistically, we found that the HDAC6 content was significantly reduced in the bone microvessels of HLU mice and that HDAC6 inhibited the expression of miR-375-3p by reducing histone acetylation in the miR-375 promoter region in MVECs. miR-375-3p was upregulated under unloading and it could inhibit MVEC proliferation by directly targeting low-density lipoprotein-related receptor 5 (LRP5) expression. In addition, silencing HDAC6 promoted the miR-375-3p/LRP5 pathway to suppress MVEC proliferation under mechanical unloading, and regulation of HDAC6/miR-375-3p axis in MVECs could affect osteoblast proliferation under coculture conditions. CONCLUSION: Our study revealed that disuse-induced bone loss may be closely related to a reduction in the number of bone microvessels and that the modulation of MVEC function could improve bone loss induced by unloading. Mechanistically, the HDAC6/miR-375-3p/LRP5 pathway in MVECs might be a promising strategy for the clinical treatment of unloading-induced bone loss.

SARS-CoV-2 nucleocapsid protein promotes self-deacetylation by inducing HDAC6 to facilitate viral replication.

BACKGROUND: The global outbreak of COVID-19 caused by the SARS-CoV-2 has led to millions of deaths. This unanticipated emergency has prompted virologists across the globe to delve deeper into the intricate dynamicity of the host-virus interface with an aim to identify antiviral targets and elucidate host and viral determinants of severe disease. AIM: The present study was undertaken to analyse the role of histone deacetylase 6 (HDAC6) in regulating SARS-CoV-2 infection. RESULTS: Gradual increase in HDAC6 expression was observed in different SARS-CoV-2-permissive cell lines following SARS-CoV-2 infection. The SARS-CoV-2 nucleocapsid protein (N protein) was identified as the primary viral factor responsible for upregulating HDAC6 expression. Downregulation of HDAC6 using shRNA or a specific inhibitor tubacin resulted in reduced viral replication suggesting proviral role of its deacetylase activity. Further investigations uncovered the interaction of HDAC6 with stress granule protein G3BP1 and N protein during infection. HDAC6-mediated deacetylation of SARS-CoV-2 N protein was found to be crucial for its association with G3BP1. CONCLUSION: This study provides valuable insights into the molecular mechanisms underlying the disruption of cytoplasmic stress granules during SARS-CoV-2 infection and highlights the significance of HDAC6 in the process.

CYLD/HDAC6 signaling regulates the interplay between epithelial-mesenchymal transition and ciliary homeostasis during pulmonary fibrosis.

The primary cilium behaves as a platform for sensing and integrating extracellular cues to control a plethora of cellular activities. However, the functional interaction of this sensory organelle with epithelial-mesenchymal transition (EMT) during pulmonary fibrosis remains unclear. Here, we reveal a critical role for cylindromatosis (CYLD) in reciprocally linking the EMT program and ciliary homeostasis during pulmonary fibrosis. A close correlation between the EMT program and primary cilia is observed in bleomycin-induced pulmonary fibrosis as well as TGF-ß-induced EMT model. Mechanistic study reveals that downregulation of CYLD underlies the crosstalk between EMT and ciliary homeostasis by inactivating histone deacetylase 6 (HDAC6) during pulmonary fibrosis. Moreover, manipulation of primary cilia is an effective means to modulate the EMT program. Collectively, these results identify a pivotal role for the CYLD/HDAC6 signaling in regulating the reciprocal interplay between the EMT program and ciliary homeostasis during pulmonary fibrosis.

HDAC6-mediated deacetylation of FLOT2 maintains stability and tumorigenic function of FLOT2 in nasopharyngeal carcinoma. / HDAC6通过介导FLOT2åŽ»ä¹™é °åŒ–ç»´æŒå ¶åœ¨é¼»å’½ç™Œä¸­çš„ç¨³å®šåŠä¿ƒç˜¤ä½œç”¨.

OBJECTIVES: Flotillin-2 (FLOT2) is a prototypical oncogenic and a potential target for cancer therapy. However, strategies for targeting FLOT2 remain undefined. Post-translational modifications are crucial for regulating protein stability, function, and localization. Understanding the mechanisms and roles of post-translational modifications is key to developing targeted therapies. This study aims to investigate the regulation and function of lysine acetylation of FLOT2 in nasopharyngeal carcinoma, providing new insights for targeting FLOT2 in cancer intervention. METHODS: The PhosphoSitePlus database was used to analyze the lysine acetylation sites of FLOT2, and a lysine acetylation site mutation of FLOT2 [FLOT2 (K211R)] was constructed. Nasopharyngeal carcinoma cells were treated with histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and Sirt family deacetylase inhibitor nicotinamide (NAM). TSA-treated human embryonic kidney (HEK)-293T were transfected with FLOT2 mutant plasmids. Co-immunoprecipitation (Co-IP) was used to detect total acetylation levels of FLOT2 and the effects of specific lysine (K) site mutations on FLOT2 acetylation. Western blotting was used to detect FLOT2/FLAG-FLOT2 protein expression in TSA-treated nasopharyngeal carcinoma cells transfected with FLOT mutant plasmids, and real-time reverse transcription PCR (real-time RT-PCR) was used to detect FLOT2 mRNA expression. Nasopharyngeal carcinoma cells were treated with TSA combined with MG132 or chloroquine (CQ) to analyze FLOT2 protein expression. Cycloheximide (CHX) was used to treat HEK-293T cells transfected with FLAG-FLOT2 (WT) or FLAG-FLOT2(K211R) plasmids to assess protein degradation rates. The BioGrid database was used to identify potential interactions between FLOT2 and HDAC6, which were validated by Co-IP. HEK-293T cells were co-transfected with FLAG-FLOT2 (WT)/FLAG-FLOT2 (K211R) and Vector/HDAC6 plasmids, and grouped into FLAG-FLOT2 (WT)+Vector, FLAG-FLOT2 (WT)+HDAC6, FLAG-FLOT2 (K211R)+Vector, and FLAG-FLOT2 (K211R)+HDAC6 to analyze the impact of K211R mutation on total lysine acetylation levels. In 6-0B cells, overexpression of FLOT2 (WT) and FLOT2 (K211R) was performed, and the biological functions of FLOT2 acetylation site mutants were assessed using cell counting kit-8 (CCK-8), colony formation, and Transwell invasion assays. RESULTS: The PhosphoSitePlus database indicated that FLOT2 has an acetylation modification at the K211 site. Co-IP confirmed significant acetylation of FLOT2, with TSA significantly increasing overall FLOT2 acetylation levels, while NAM had no effect. Mutation at the K211 site significantly reduced overall FLOT2 acetylation, unaffected by TSA. TSA decreased FLOT2 protein expression in nasopharyngeal carcinoma cells without affecting FLOT2 mRNA levels or FLOT2 (K211R) protein expression in transfected cells. The degradation rate of FLOT2 (K211R) protein was significantly slower than that of FLOT2 (WT). The proteasome inhibitor MG132 prevented TSA-induced FLOT2 degradation, while the lysosome inhibitor CQ did not. BioGrid data suggested a potential interaction between FLOT2 and HDAC6, confirmed by Co-IP. Knockdown of HDAC6 in nasopharyngeal carcinoma cells significantly increased FLOT2 acetylation; co-transfection of HDAC6 and FLAG-FLOT2 (WT) significantly decreased total lysine acetylation levels, whereas co-transfection of HDAC6 and FLAG-FLOT2 (K211R) had no effect. Knockdown of HDAC6 significantly reduced FLOT2 protein levels without affecting mRNA levels. MG132 prevented HDAC6-knockdown-induced FLOT2 degradation. Knockdown of HDAC6 significantly accelerated FLOT2 degradation. Nasopharyngeal carcinoma cells transfected with FLOT2 (K211R) showed significantly higher proliferation and invasion than those transfected with FLOT2 (WT). CONCLUSIONS: The K211 site of FLOT2 undergoes acetylation modification, and HDAC6 mediates deacetylation at this site, inhibiting proteasomal degradation of FLOT2 and maintaining its stability and tumor-promoting function in nasopharyngeal carcinoma.

HDAC6 inhibition disrupts HDAC6-P300 interaction reshaping the cancer chromatin landscape.

BACKGROUND: Histone deacetylases (HDACs) are crucial regulators of gene expression, DNA synthesis, and cellular processes, making them essential targets in cancer research. HDAC6, specifically, influences protein stability and chromatin dynamics. Despite HDAC6's potential therapeutic value, its exact role in gene regulation and chromatin remodeling needs further clarification. This study examines how HDAC6 inactivation influences lysine acetyltransferase P300 stabilization and subsequent effects on chromatin structure and function in cancer cells. METHODS AND RESULTS: We employed the HDAC6 inhibitor ITF3756, siRNA, or CRISPR/Cas9 gene editing to inactivate HDAC6 in different epigenomic backgrounds. Constantly, this inactivation led to significant changes in chromatin accessibility, particularly increased acetylation of histone H3 lysines 9, 14, and 27 (ATAC-seq and H3K27Ac ChIP-seq analysis). Transcriptomics, proteomics, and gene ontology analysis revealed gene changes in cell proliferation, adhesion, migration, and apoptosis. Significantly, HDAC6 inactivation altered P300 ubiquitination, stabilizing P300 and leading to downregulating genes critical for cancer cell survival. CONCLUSIONS: Our study highlights the substantial impact of HDAC6 inactivation on the chromatin landscape of cancer cells and suggests a role for P300 in contributing to the anticancer effects. The stabilization of P300 with HDAC6 inhibition proposes a potential shift in therapeutic focus from HDAC6 itself to its interaction with P300. This finding opens new avenues for developing targeted cancer therapies, improving our understanding of epigenetic mechanisms in cancer cells.

Genetic deletion or pharmacologic inhibition of histone deacetylase 6 protects the heart against ischaemia/reperfusion injury by limiting tumour necrosis factor alpha-induced mitochondrial injury in experimental diabetes.

AIMS: The histone deacetylase 6 (HDAC6) inhibitor, tubastatin A (TubA), reduces myocardial ischaemia/reperfusion injury (MIRI) in type 1 diabetic rats. It remains unclear whether HDAC6 regulates MIRI in type 2 diabetic animals. Diabetes augments the activity of HDAC6 and the generation of tumour necrosis factor alpha (TNF-α) and impairs mitochondrial complex I (mCI). Here, we examined how HDAC6 regulates TNF-α production, mCI activity, mitochondria, and cardiac function in type 1 and type 2 diabetic mice undergoing MIRI. METHODS AND RESULTS: HDAC6 knockout, streptozotocin-induced type 1 diabetic, and obese type 2 diabetic db/db mice underwent MIRI in vivo or ex vivo in a Langendorff-perfused system. We found that MIRI and diabetes additively augmented myocardial HDAC6 activity and generation of TNF-α, along with cardiac mitochondrial fission, low bioactivity of mCI, and low production of adenosine triphosphate. Importantly, genetic disruption of HDAC6 or TubA decreased TNF-α levels, mitochondrial fission, and myocardial mitochondrial nicotinamide adenine dinucleotide levels in ischaemic/reperfused diabetic mice, concomitant with augmented mCI activity, decreased infarct size, and improved cardiac function. Moreover, HDAC6 knockout or TubA treatment decreased left ventricular dilation and improved cardiac systolic function 28 days after MIRI. H9c2 cardiomyocytes with and without HDAC6 knockdown were subjected to hypoxia/reoxygenation injury in the presence of high glucose. Hypoxia/reoxygenation augmented HDAC6 activity and TNF-α levels and decreased mCI activity. These negative effects were blocked by HDAC6 knockdown. CONCLUSION: HDAC6 is an essential negative regulator of MIRI in diabetes. Genetic deletion or pharmacologic inhibition of HDAC6 protects the heart from MIRI by limiting TNF-α-induced mitochondrial injury in experimental diabetes.

TonEBP inhibits ciliogenesis by controlling aurora kinase A and regulating centriolar satellite integrity.

BACKGROUND: Primary cilia on the surface of eukaryotic cells serve as sensory antennas for the reception and transmission in various cell signaling pathways. They are dynamic organelles that rapidly form during differentiation and cell cycle exit. Defects in these organelles cause a group of wide-ranging disorders called ciliopathies. Tonicity-responsive enhancer-binding protein (TonEBP) is a pleiotropic stress protein that mediates various physiological and pathological cellular responses. TonEBP is well-known for its role in adaptation to a hypertonic environment, to which primary cilia have been reported to contribute. Furthermore, TonEBP is involved in a wide variety of other signaling pathways, such as Sonic Hedgehog and WNT signaling, that promote primary ciliogenesis, suggesting a possible regulatory role. However, the functional relationship between TonEBP and primary ciliary formation remains unclear. METHODS: TonEBP siRNAs and TonEBP-mCherry plasmids were used to examine their effects on cell ciliation rates, assembly and disassembly processes, and regulators. Serum starvation was used as a condition to induce ciliogenesis. RESULTS: We identified a novel pericentriolar localization for TonEBP. The results showed that TonEBP depletion facilitates the formation of primary cilia, whereas its overexpression results in fewer ciliated cells. Moreover, TonEBP controlled the expression and activity of aurora kinase A, a major negative regulator of ciliogenesis. Additionally, TonEBP overexpression inhibited the loss of CP110 from the mother centrioles during the early stages of primary cilia assembly. Finally, TonEBP regulated the localization of PCM1 and AZI1, which are necessary for primary cilia formation. CONCLUSIONS: This study proposes a novel role for TonEBP as a pericentriolar protein that regulates the integrity of centriolar satellite components. This regulation has shown to have a negative effect on ciliogenesis. Investigations into cilium assembly and disassembly processes suggest that TonEBP acts upstream of the aurora kinase A - histone deacetylase 6 signaling pathway and affects basal body formation to control ciliogenesis. Taken together, our data proposes previously uncharacterized regulation of primary cilia assembly by TonEBP.

Regulation of STAT1 Signaling in Human Pancreatic ß-Cells by the Lysine Deacetylase HDAC6: A New Therapeutic Opportunity in Type 1 Diabetes?

Type 1 diabetes arises from the selective destruction of pancreatic ß-cells by autoimmune mechanisms, and intracellular pathways driven by Janus kinase (JAK)-mediated phosphorylation of STAT isoforms (especially STAT1 and STAT2) are implicated as mediators of ß-cell demise. Despite this, the molecular mechanisms that regulate JAK-STAT signaling in ß-cells during the autoimmune attack remain only partially disclosed, and the factors acting to antagonize proinflammatory STAT1 signaling are uncertain. We have recently implicated signal regulatory protein α (SIRPα) in promoting ß-cell viability in the face of ongoing islet autoimmunity and have now revealed that this protein controls the availability of a cytosolic lysine deacetylase, HDAC6, whose activity regulates the phosphorylation and activation of STAT1. We provide evidence that STAT1 serves as a substrate for HDAC6 in ß-cells and that sequestration of HDAC6 by SIRPα in response to anti-inflammatory cytokines (e.g., IL-13) leads to increased STAT1 acetylation. This then impairs the ability of STAT1 to promote gene transcription in response to proinflammatory cytokines, including interferon-γ. We further found that SIRPα is lost from the ß-cells of subjects with recent-onset type 1 diabetes under conditions when HDAC6 is retained and STAT1 levels are increased. On this basis, we report a previously unrecognized role for cytokine-induced regulation of STAT1 acetylation in the control of ß-cell viability and propose that targeted inhibition of HDAC6 activity may represent a novel therapeutic modality to promote ß-cell viability in the face of active islet autoimmunity.

Insufficient KIF15 during porcine oocyte ageing induces HDAC6-based microtubule instability.

During aging, oocytes display cytoskeleton dynamics defects and aneuploidy, leading to embryonic aneuploidy, which in turn causes miscarriages, implantation failures, and birth defects. KIF15 (also known as Hklp2), a member of the kinesin-12 superfamily, is a cytoplasmic motor protein reported to be involved in Golgi and vesicle-related transport during mitosis in somatic cells. However, the regulatory mechanisms of KIF15 during meiosis in porcine oocytes and the connection with postovulatory aging remain unclear. In present study, we found that KIF15 is expressed during porcine oocyte maturation, and its localization is dependent on microtubule dynamics. Furthermore, the level of KIF15 expression decreased in postovulatory aged oocytes. The decrease in KIF15 blocked polar body extrusion, thereby hindering oocyte maturation. We demonstrated that KIF15 defects contributed to abnormal spindle morphologies and chromosome misalignment, possibly due to microtubule instability, as evidenced by microtubule depolymerization after cold treatment. Additionally, our data indicated that KIF15 modulates HDAC6 to affect tubulin acetylation in oocytes. Taken together, these results suggest that KIF15 regulates HDAC6-related microtubule stability for spindle organization in porcine oocytes during meiosis, which may contribute to the decline in maturation competence in aged porcine oocytes.

Targeting histone deacetylase 6 (HDAC6) to enhance radiation therapy in meningiomas in a 2D and 3D in vitro study.

BACKGROUND: External radiation therapy (RT) is often a primary treatment for inoperable meningiomas in the absence of established chemotherapy. Histone deacetylase 6 (HDAC6) overexpression, commonly found in cancer, is acknowledged as a driver of cellular growth, and inhibiting HDACs holds promise in improving radiotherapeutic efficacy. Downregulation of HDAC6 facilitates the degradation of ß-catenin. This protein is a key element in the Wnt/ß-catenin signalling pathway, contributing to the progression of meningiomas. METHODS: In order to elucidate the associations and therapeutic potential of HDAC6 inhibitors (HDAC6i) in conjunction with RT, we administered Cay10603, HDAC6i, to both immortalised and patient-derived meningioma cells prior to RT in this study. FINDINGS: Our findings reveal an increase in HDAC6 expression following exposure to RT, which is effectively mitigated with pre-treated Cay10603. The combination of Cay10603 with RT resulted in a synergistic augmentation of cytotoxic effects, as demonstrated through a range of functional assays conducted in both 2D as well as 3D settings; the latter containing syngeneic tumour microenvironment (TME). Radiation-induced DNA damage was augmented by pre-treatment with Cay10603, concomitant with the inhibition of ß-catenin and minichromosome maintenance complex component 2 (MCM2) accumulation within the nucleus. This subsequently inhibited c-myc oncogene expression. INTERPRETATION: Our findings demonstrate the therapeutic potential of Cay10603 to improve the radiosensitisation and provide rationale for combining HDAC6i with RT for the treatment of meningioma. FUNDING: This work was funded by Brain Tumour Research Centre of Excellence award to C Oliver Hanemann.

A novel insight into neurological disorders through HDAC6 protein-protein interactions.

Due to its involvement in physiological and pathological processes, histone deacetylase 6 (HDAC6) is considered a promising pharmaceutical target for several neurological manifestations. However, the exact regulatory role of HDAC6 in the central nervous system (CNS) is still not fully understood. Hence, using a semi-automated literature screening technique, we systematically collected HDAC6-protein interactions that are experimentally validated and reported in the CNS. The resulting HDAC6 network encompassed 115 HDAC6-protein interactions divided over five subnetworks: (de)acetylation, phosphorylation, protein complexes, regulatory, and aggresome-autophagy subnetworks. In addition, 132 indirect interactions identified through HDAC6 inhibition were collected and categorized. Finally, to display the application of our HDAC6 network, we mapped transcriptomics data of Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis on the network and highlighted that in the case of Alzheimer's disease, alterations predominantly affect the HDAC6 phosphorylation subnetwork, whereas differential expression within the deacetylation subnetwork is observed across all three neurological disorders. In conclusion, the HDAC6 network created in the present study is a novel and valuable resource for the understanding of the HDAC6 regulatory mechanisms, thereby providing a framework for the integration and interpretation of omics data from neurological disorders and pharmacodynamic assessments.

Endosymbiosis in trypanosomatids: The bacterium division depends on microtubule dynamism.

Microtubules are components of the cytoskeleton that perform essential functions in eukaryotes, such as those related to shape change, motility and cell division. In this context some characteristics of these filaments are essential, such as polarity and dynamic instability. In trypanosomatids, microtubules are integral to ultrastructure organization, intracellular transport and mitotic processes. Some species of trypanosomatids co-evolve with a symbiotic bacterium in a mutualistic association that is marked by extensive metabolic exchanges and a coordinated division of the symbiont with other cellular structures, such as the nucleus and the kinetoplast. It is already established that the bacterium division is microtubule-dependent, so in this work, it was investigated whether the dynamism and remodeling of these filaments is capable of affecting the prokaryote division. To this purpose, Angomonas deanei was treated with Trichostatin A (TSA), a deacetylase inhibitor, and mutant cells for histone deacetylase 6 (HDAC6) were obtained by CRISPR-Cas9. A decrease in proliferation, an enhancement in tubulin acetylation, as well as morphological and ultrastructural changes, were observed in TSA-treated protozoa and mutant cells. In both cases, symbiont filamentation occurred, indicating that prokaryote cell division is dependent on microtubule dynamism.

GDF15 controls primary cilia morphology and function thereby affecting progenitor proliferation.

We recently reported that growth/differentiation factor 15 (GDF15) and its receptor GDNF family receptor alpha-like (GFRAL) are expressed in the periventricular germinal epithelium thereby regulating apical progenitor proliferation. However, the mechanisms are unknown. We now found GFRAL in primary cilia and altered cilia morphology upon GDF15 ablation. Mutant progenitors also displayed increased histone deacetylase 6 (Hdac6) and ciliary adenylate cyclase 3 (Adcy3) transcript levels. Consistently, microtubule acetylation, endogenous sonic hedgehog (SHH) activation and ciliary ADCY3 were all affected in this group. Application of exogenous GDF15 or pharmacological antagonists of either HDAC6 or ADCY3 similarly normalized ciliary morphology, proliferation and SHH signalling. Notably, Gdf15 ablation affected Hdac6 expression and cilia length only in the mutant periventricular niche, in concomitance with ciliary localization of GFRAL. In contrast, in the hippocampus, where GFRAL was not expressed in the cilium, progenitors displayed altered Adcy3 expression and SHH signalling, but Hdac6 expression, cilia morphology and ciliary ADCY3 levels remained unchanged. Thus, ciliary signalling underlies the effect of GDF15 on primary cilia elongation and proliferation in apical progenitors.