Involvement of transcription factor XBP1s in the resistance of HDAC6 inhibitor Tubastatin A to superoxidation via acetylation-mediated proteasomal degradation

doi:10.1016/j.bbrc.2014.05.134

Biochemical and Biophysical Research Communications

Volume 450, Issue 1, 18 July 2014, Pages 433-439

https://doi.org/10.1016/j.bbrc.2014.05.134 Get rights and content

Highlights

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Tubastatin A up-regulated anti-oxidative gene expression related to transcription factor XBP1s.
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XBP1s is involved in the transcriptional regulation and cell growth protection from Tubastatin A.
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Tubastatin A up-regulates XBP1s protein levels that are dependent on HDAC6 inhibition.
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Tubastatin A delays XBP1s protein degradation via acetylation-mediated proteasomal degradation.

Abstract

HDAC6 is a major cytoplasmic deacetylase. XBP1s is a basic-region leucine zipper (bZIP) transcriptional factor. Despite their mutual involvement in the anti-oxidative process, there are no reports about their inter-protein interactions so far. Here we identified a direct link between HDAC6 inhibition and XBP1s transcription activity in anti-oxidative damage. We showed that the specific HDAC6 inhibitor Tubastatin A could up-regulate XBP1s transcriptional activity, thereby increasing anti-oxidative genes expression. Moreover, knock down of XBP1s could significantly abolish the cell growth protection afforded by Tubastatin A. We hypothesize that Tubastatin A acts to increase XBP1s protein levels that are dependent on its HDAC6 deacetylase inhibition via a mechanism involving acetylation-mediated proteasomal degradation, providing novel mechanistic insight into the anti-oxidative effects of HDAC6 inhibition.

Graphical abstract

Introduction

HDAC6 is the principal cytoplasmic deacetylase in mammalian cells [1]. HDAC6-specific substrates are varied, and include α-tubulin [2], cortactin [3], HSP90 [4], IFNαR [5], peroxiredoxin (Prx) I and Prx II [6]. Its involvement in deacetylation gives HDAC6 an important role in the progression of neurodegenerative diseases and lends itself to being a potential therapeutic target [6], [7], [8].

In addition to deacetylation, high levels of reactive oxygen damage cells are also believed to be associated with neurodegenerative disorders [9]. A potential interplay between deacetylation and oxidative stress can be found in data using Tubacin [6] and Tubastatin A [10], both highly selective HDAC6 inhibitors that have also showed good anti-oxidative activity.

Despite this prior work, the mechanism behind the anti-oxidative activity of HDAC6-specific inhibitors has still not been clarified. Two substrates have been found to be directly regulated by HDAC6: the cytoplasmic antioxidants enzymes peroxiredoxin (Prx) I and Prx II both appear to be involved in the anti-oxidative effects of HDAC6 inhibition [6]. Consistent with HDAC6 localization to the cytoplasm and its ability to deacetylate a range of cytoplasmic target proteins, it has been suggested that the effects of HDAC6 inhibition occur through a transcription-independent, local mechanism [6].

To this end, our work provide evidence that XBP1s, a bzip transcription factor that is involved in the mammalian unfolded protein response (UPR), could play an important role in the antioxidative activity of HDAC6 inhibition caused by Tubastatin A. This putative interaction between HDAC6 and nuclear transcription factor XBP1s provides evidence for a transcriptionally-regulated mechanism for HDAC6 function.

Section snippets

Reagents and antibodies

Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Grand Island, New York, USA). Protein A-Agarose, anti-acetyl-tublin, and anti-β-actin antibody were purchased from Sigma–Aldrich (St. Louis, MO, USA). Anti-tubulin antibody was purchased from Epitomics (Burlingame, CA, USA) and Anti-acetyl-histone H3 antibody was from Millipore (Billerica, MA, USA). Anti-acetylated-lysine, anti-histone H3, anti-Flag and anti-HA antibodies were provided by Cell

Tubastatin A up-regulates anti-oxidative gene expression related to transcription factor XBP1s

Hydrogen peroxide (H₂O₂) was used to induce oxidative stress and neuronal damage. Tubastatin A, an HDAC6 inhibitor known to have neuroprotective effects [10], was used to inhibit HDAC6 activity. Prior work has elucidated genes important to human anti-oxidant responses, which include peroxidases, superoxide dismutases and thiol redox regulating genes [15]. Based on this prior characterization, peroxidase genes PRX5 and CAT, superoxide dismutase gene SOD, thiol redox regulating genes TRX-1 and

Discussion

Our understanding of acetylation has long been limited, having been restricted to understanding the processes inside the nucleus (e.g. histones) [20] or to non-histone nuclear transcription factors [21]. It took the discovery of HDAC6 as a microtubule-associated deacetylase for researchers to realize that acetylation is not exclusively located to the nucleus [2]. Now, more and more cytoplasmic proteins have been found to be acetylated and involved in diverse cellular processes [22].

Since HDAC6

Acknowledgments

This work was supported by Grants from the National Natural Science Foundation of China (91029716, 81125023, 81173033, 81270942), National Major Scientific and Technological Special Project for “significant new drugs creation” (2012ZX09301001-004).

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Here, we identified K60 and K77 on XBP1s as two pivotal ubiquitin sites required for its proteasome-dependent degradation. We also constructed a double mutant form of XBP1s (K60/77R) and found that it showed higher capacity in resisting against ubiquitin-mediated protein degradation, increasing nuclear translocation, enhancing transcriptional activity, suppressing ER stress and promoting Foxo1 degradation, compared to that of wild type XBP1s (WT). Consistently, overexpression of the K60/77R XBP1s mutant in DIO mice increased the ability to reduce ER stress and decrease Foxo1 levels, thus contributed to maintaining glucose homeostasis.
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Inhibition of HDAC6 promotes the survival of neurons, conferring neuroprotection. The administration of tubastatin A, which specifically inhibits HDAC6, efficiently promotes the release and transportation of brain-derived neurotrophic factor,13 regulates the growth of axons and promotes the differentiation and migration of neurons,1 remits inflammatory responses, and enhances antioxidation.17,18 Liesz et al.19 reported that tubastatin A significantly promoted the recovery of neural function in experimental mice with cerebral ischemia.
To explore the effect of the inhibitor of histone deacetylase (6HDAC6), tubastatin A, on the functional recovery of injured central branch of dorsal root ganglia (cauda equina).
A total of 30 Sprague–Dawley rats (n = 10 for each group) were divided randomly into sham operation (sham group), cauda equina compression control (CEC control group), and cauda equina compression plus tubastatin A treatment (tubastatin A group). The tail-flick test was performed to detect the sense of pain and warmth as well as motor function. Immunoblotting/immunofluorescence experiments, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and hematoxylin–eosin staining were performed to detect the amount of HDAC6 in dorsal root ganglion (DRG) neurons, degree of apoptosis in DRG neurons, and degree of cauda equina injury, respectively.
The ratio of apoptotic cells in the CEC control group was greater than that in the sham group, whereas it decreased in the tubastatin A group. Hematoxylin–eosin staining revealed that the fibers of cauda equina in the tubastatin A group were more compact compared with those in the CEC control group. The expression of HDAC6 was not different between the sham and CEC control groups, whereas it decreased significantly in the tubastatin A group. Tubastatin A administration shortened tail-flick latency on the seventh day after operation compared with the CEC control group.
Tubastatin A significantly decreased the expression of HDAC6 in DRG neurons with injured cauda equina, inhibited the apoptosis of neural cells and axonal demyelinating changes in cauda equina, and partially promoted the recovery of neural function.
Inhibition of HDAC6 increases acetylation of peroxiredoxin1/2 and ameliorates 6-OHDA induced dopaminergic injury
2017, Neuroscience Letters
Citation Excerpt :
Its involvement in deacetylation gives HDAC6 an important role in the progression of neurodegenerative disorders [8]. Specific substrates of HDAC6 deacetylation are varied, such as peroxiredoxin1 (Prx1), peroxiredoxin2 (Prx2), tubulin, cortactin and HSP90 [12]. But the deacetylation effect of HDAC6 in PD has not been fully illustrated.
Histone deacetylase 6 (HDAC6) has been regarded as an unusual HDAC because of its unique properties. It contains two deacetylase catalytic domains and one ubiquitin-binding domain, thus exerting both enzymatic and non-enzymatic actions on cellular function. To date, the ubiquitin-binding activity of HDAC6 has been implicated in several neurodegenerative disorders including Parkinson’s disease (PD). However, the deacetylation effect of HDAC6 in PD has not been fully illustrated. Therefore, the aim of the present study was to explore the role of deacetyation activity of HDAC6 in PD.
We used an in vivo 6-OHDA induced PD model and a specific HDAC6 inhibitor tubastatin A to investigate the acetylation levels of peroxiredoxin1 (Prx1) and peroxiredoxin2 (Prx2) and to explore the effects of tubastain A on nigrostriatal dopaminergic system.
Our results showed that expression of HDAC6 significantly increased in dopaminergic neurons after 6-OHDA injury. Acetylation levels of Prx1 and Prx2 decreased. Pharmacological inhibition of HDAC6 with specific inhibitor tubastatin A increased acetylation of Prx1 and Prx2, reduced ROS production and ameliorated dopaminergic neurotoxicity.
Our results for the first time provide evidence that HDAC6 medicated deacetylation of Prx1 and Prx2 contributes to oxidative injury in PD, suggesting that the development of specific HDAC6 inhibitor is required to develop more effective therapeutic strategies to treat PD.
Inflammation Improves Glucose Homeostasis through IKKβ-XBP1s Interaction
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Citation Excerpt :
These results indicate that IKKβ increases the amount of XBP1s protein by enhancing its stability. Considering that XBP1s is recycled in a proteasome-dependent manner (Zhang et al., 2014), we also tested whether IKKβ affects the level of ubiquitination of XBP1s. A myc-tagged ubiquitin (Ub-myc)-expressing vector was transfected into cells with a flag-tagged XBP1s (XBP1s-flag), in the presence or absence of an HA-tagged constitutively active IKKβ (IKKβ-HA).
It is widely believed that inflammation associated with obesity has an important role in the development of type 2 diabetes. IκB kinase beta (IKKβ) is a crucial kinase that responds to inflammatory stimuli such as tumor necrosis factor α (TNF-α) by initiating a variety of intracellular signaling cascades and is considered to be a key element in the inflammation-mediated development of insulin resistance. We show here, contrary to expectation, that IKKβ-mediated inflammation is a positive regulator of hepatic glucose homeostasis. IKKβ phosphorylates the spliced form of X-Box Binding Protein 1 (XBP1s) and increases the activity of XBP1s. We have used three experimental approaches to enhance the IKKβ activity in the liver of obese mice and observed increased XBP1s activity, reduced ER stress, and a significant improvement in insulin sensitivity and consequently in glucose homeostasis. Our results reveal a beneficial role of IKKβ-mediated hepatic inflammation in glucose homeostasis.
Lysine acetylation stabilizes SP2 protein in the silkworm Bombyx mori
2016, Journal of Insect Physiology
Lysine acetylation (Kac) is a vital post-translational modification that plays an important role in many cellular processes in organisms. In the present study, the nutrient storage proteins in hemolymph were first found to be highly acetylated—particularly SP2 protein, which contains 20 potential Kac sites. Further results confirmed that lysine acetylation could stabilize and up-regulate the protein level of anti-apoptosis protein SP2, thereby improving the survival of H₂O₂-treated BmN cells and suppressing the apoptosis induced by H₂O₂. The potential mechanism involved in the inhibition of ubiquitin-mediated proteasomal degradation by crosstalk between lysine acetylation and ubiquitination. Our results showed that the increase in the acetylation level by TSA could decrease the ubiquitination and improve the protein level of SP2, indicating that lysine acetylation could influence the SP2 protein level through competition between ubiquitination and the suppression of ubiquitin-mediated proteasomal degradation, thereby stabilizing the protein. SP2 is a major nutrient storage protein from hemolymph for amino acid storage and utilization. The crosstalk between lysine acetylation and ubiquitination of SP2 might imply an important role of lysine acetylation for nutrient storage and utilization in silkworm.
Pharmacological modulation of HDAC1 and HDAC6 in vivo in a zebrafish model: Therapeutic implications for Parkinson's disease
2016, Pharmacological Research
Histone deacetylases (HDACs) are key epigenetic enzymes and emerging drug targets in cancer and neurodegeneration. Pan-HDAC inhibitors provided neuroprotection in Parkinson’s Disease (PD) models, however, the HDAC isoforms with highest neuroprotective potential remain unknown. Zebrafish larvae (powerful pharmacological testing tools bridging cellular and in vivo studies) have thus far been used in PD modelling with limited phenotypic characterization. Here we characterize the behavioural and metabolic phenotypes of a zebrafish PD model induced with MPP⁺, assess the feasibility of targeting zebrafish HDAC1 and HDAC6 isoforms, and test the in vivo effects of their selective inhibitors MS-275 and tubastatin A, respectively. MPP⁺ induced a concentration-dependent decrease in metabolic activity and sensorimotor reflexes, and induced locomotor impairments rescuable by the dopaminergic agonist apomorphine. Zebrafish HDAC1 and HDAC6 isoforms show high sequence identity with mammalian homologues at the deacetylase active sites, and pharmacological inhibition increased acetylation of their respective histone and tubulin targets. MS-275 and tubastatin rescued the MPP⁺-induced decrease in diencephalic tyrosine hydroxylase immunofluorescence and in whole-larvae metabolic activity, without modifying mitochondrial complex activity or biogenesis. MS-275 or tubastatin alone modulated spontaneous locomotion. When combined with MPP⁺, however, neither MS-275 nor tubastatin rescued locomotor impairments, although tubastatin did ameliorate the head-reflex impairment. This study demonstrates the feasibility of pharmacologically targeting the zebrafish HDAC1 and HDAC6 isoforms, and indicates that their inhibition can rescue cellular metabolism in a PD model. Absence of improvement in locomotion, however, suggests that monotherapy with either HDAC1 or HDAC6 inhibitors is unlikely to provide strong benefits in PD. This study highlights parameters dependent on the integrity of zebrafish neuronal circuits as a valuable complement to cell-based studies. Also, the demonstrated feasibility of pharmacologically targeting HDAC1 and HDAC6 in this organism paves the way for future studies investigating HDAC inhibitors in other diseases modelled in zebrafish.

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Involvement of transcription factor XBP1s in the resistance of HDAC6 inhibitor Tubastatin A to superoxidation via acetylation-mediated proteasomal degradation

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Reagents and antibodies

Tubastatin A up-regulates anti-oxidative gene expression related to transcription factor XBP1s

Discussion

Acknowledgments

Mol. Cell

Mol. Cell

Cell

J. Neurol. Sci.

Methods

J. Biol. Chem.

Cell Metab.

Mol. Cell

J. Biol. Chem.

Trends Biochem. Sci.

J. Biol. Chem.

Cell

J. Biol. Chem.

Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally

Mol. Cell. Biol.

HDAC6 is a microtubule-associated deacetylase

Nature

HDAC6 is a specific deacetylase of peroxiredoxins and is involved in redox regulation

Proc. Natl. Acad. Sci. USA