Class I HDACs specifically regulate E‐cadherin expression in human renal epithelial cells

Epithelial‐mesenchymal transition (EMT) and renal fibrosis are closely involved in chronic kidney disease. Inhibition of histone deacetylase (HDAC) has an anti‐fibrotic effect in various diseases. However, the pathophysiological role of isoform‐specific HDACs or class‐selective HDACs in renal fibrosis remains unknown. Here, we investigated EMT markers and extracellular matrix (ECM) proteins in a human proximal tubular cell line (HK‐2) by using HDAC inhibitors or by knockdown of class I HDACs (HDAC1, 2, 3 and 8). Trichostatin A (TSA), MS275, PCI34051 and LMK235 inhibited ECM proteins such as collagen type I or fibronectin in transforming growth factor β1 (TGF‐β1)‐induced HK2 cells. However, restoration of TGF‐β1‐induced E‐cadherin down‐regulation was only seen in HK‐2 cells treated with TSA or MS275, but not with PCI34051, whereas TGF‐β1‐induced N‐cadherin expression was not affected by the inhibitors. ECM protein and EMT marker levels were prevented or restored by small interfering RNA transfection against HDAC8, but not against other class I HDACs (HDAC1, 2 and 3). E‐cadherin regulation is mediated by HDAC8 expression, but not by HDAC8 enzyme activity. Thus, class I HDACs (HDAC1, 2, 3 and 8) play a major role in regulating ECM and EMT, whereas class IIa HDACs (HDAC4 and 5) are less effective.     

Histone Deacetylase Classes I and II Regulate Kaposi's Sarcoma-Associated Herpesvirus Reactivation

In primary effusion lymphoma (PEL) cells infected with latent Kaposi''s sarcoma-associated herpesvirus (KSHV), the promoter of the viral lytic switch gene, Rta, is organized into bivalent chromatin, similar to cellular developmental switch genes. Histone deacetylase (HDAC) inhibitors (HDACis) reactivate latent KSHV and dramatically remodel the viral genome topology and chromatin architecture. However, reactivation is not uniform across a population of infected cells. We sought to identify an HDACi cocktail that would uniformly reactivate KSHV and reveal the regulatory HDACs. Using HDACis with various specificities, we found that class I HDACis were sufficient to reactivate the virus but differed in potency. Valproic acid (VPA) was the most effective HDACi, inducing lytic cycle gene expression in 75% of cells, while trichostatin A (TSA) induced less widespread lytic gene expression and inhibited VPA-stimulated reactivation. VPA was only slightly superior to TSA in inducing histone acetylation of Rta''s promoter, but only VPA induced significant production of infectious virus, suggesting that HDAC regulation after Rta expression has a dramatic effect on reactivation progression. Ectopic HDACs 1, 3, and 6 inhibited TPA-stimulated KSHV reactivation. Surprisingly, ectopic HDACs 1 and 6 stimulated reactivation independently, suggesting that the stoichiometries of HDAC complexes are critical for the switch. Tubacin, a specific inhibitor of the ubiquitin-binding, proautophagic HDAC6, also inhibited VPA-stimulated reactivation. Immunofluorescence indicated that HDAC6 is expressed diffusely throughout latently infected cells, but its expression level and nuclear localization is increased during reactivation. Overall, our data suggest that inhibition of HDAC classes I and IIa and maintenance of HDAC6 (IIb) activity are required for optimal KSHV reactivation.     

Nitration of distinct tyrosine residues causes inactivation of histone deacetylase 2

Histone deacetylases (HDACs) are key molecules involved in epigenetic regulation of gene expression. We have previously demonstrated that oxidative stress caused a reduction in HDAC2, resulting in amplified inflammation and reduced corticosteroid responsiveness. Here we showed nitrative/oxidative stress reduced HDAC2 expression via nitration of distinct tyrosine residues. Peroxynitrite, hydrogen peroxide and cigarette smoke-conditioned medium reduced HDAC2 expression in A549 epithelial cells in vitro. This reduction was due to increased proteasomal degradation following ubiquitination rather than reduction of mRNA expression or stability. HDAC2 was nitrated under nitrative/oxidative stress and in the peripheral lung tissues of smokers and patients with chronic obstructive pulmonary disease. Mutagenesis studies replacing tyrosine (Y) residues with alanine revealed that Y253 is at least partly responsible for the proteasomal degradation of HDAC2 under nitrative stress. Thus, nitration of distinct tyrosine residues modifies both the expression and activity of HDAC2, having an impact on epigenetic regulation.     

Prenatal Exposure to Histone Deacetylase Inhibitors Affects Gene Expression of Autism-Related Molecules and Delays Neuronal Maturation

Valproic acid (VPA) is a multi-target drug and an inhibitor of histone deacetylase (HDAC). We have previously demonstrated that prenatal exposure to VPA at embryonic day 12.5 (E12.5), but not at E14.5, causes autism-like behavioral abnormalities in male mouse offspring. We have also found that prenatal VPA exposure causes transient histone hyperacetylation in the embryonic brain, followed by decreased neuronal cell numbers in the prefrontal and somatosensory cortices after birth. In the present study, we examined whether prenatal HDAC inhibition affects neuronal maturation in primary mouse cortical neurons. Pregnant mice were injected intraperitoneally with VPA (500 mg/kg) and the more selective HDAC inhibitor trichostatin A (TSA; 500 µg/kg) at E12.5 or E14.5, and primary neuronal cultures were prepared from the cerebral cortices of their embryos. Prenatal exposure to VPA at E12.5, but not at E14.5, decreased total number, total length, and complexity of neuronal dendrites at 14 days in vitro (DIV). The effects of VPA weakened at 21 DIV. Exposure to TSA at E12.5, but not at E14.5, also delayed maturation of cortical neurons. In addition, real-time quantitative PCR revealed that the prenatal exposure to TSA decreased neuroligin-1 (Nlgn1), Shank2, and Shank3 mRNA levels and increased contactin-associated protein-like 2 mRNA level. The delay in neuronal maturation was also observed in Nlgn1-knockdown cells, which were transfected with Nlgn1 siRNA. These findings suggest that prenatal HDAC inhibition causes changes in gene expression of autism-related molecules linked to a delay of neuronal maturation.     

The Cdc48-Ufd1-Npl4 complex is central in ubiquitin-proteasome triggered catabolite degradation of fructose-1,6-bisphosphatase

The switch from gluconeogenesis to glycolysis in yeast has been shown to require ubiquitin-proteasome dependent elimination of the key enzyme fructose-1,6-bisphosphatase (FBPase). Prior to proteasomal degradation, polyubiquitination of the enzyme occurs via the ubiquitin-conjugating enzymes Ubc1, Ubc4, Ubc5 and Ubc8 in conjunction with a novel multi-subunit ubiquitin ligase, the Gid complex. As an additional machinery required for the catabolite degradation process, we identified the trimeric Cdc48^Ufd1-Npl4 complex and the ubiquitin receptors Dsk2 and Rad23. We show that this machinery acts between polyubiquitination of FBPase and its degradation by the proteasome.     

Depletion of Histone Deacetylase I Protein: A Common Consequence of Inflammatory Cytokine Signaling?

The dynamics of histone acetylation and deacetylation have long been known to influence gene expression by cellular signaling pathways. However, the mechanisms that regulate histone acetyl transferases (HATs) and histone deacetylases (HDACs) by these pathways have only recently become the focus of scientific investigation, spurred by increasing knowledge that HDACs can promote cancer growth. We recently reported that pro-inflammatory signals such as tumor necrosis factor α (TNFα) induce HDAC1 ubiquitination and proteasomal degradation through the IκB kinase IKKβ. The resulting depletion of cellular HDAC1 levels lead to a consequent depletion of HDAC1 associated with the CDKN1A gene promoter and increased expression of its protein product, p^21WAF1/CIP1. This phenomenon heralds a unique mechanism of HDAC regulation that modulates the pro-inflammatory activity of TNFα and other cytokines at the level of gene expression. Here we discuss the current knowledge of pro-inflammatory cytokine-induced regulation of gene expression, emphasizing the involvement of HDAC1, and its possible implications in inflammation, cancer, and their therapy.     

Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation

In eukaryotes, endoplasmic reticulum-associated degradation (ERAD) functions in cellular quality control and regulation of normal ER-resident proteins. ERAD proceeds by the ubiquitin-proteasome pathway, in which the covalent attachment of ubiquitin to proteins targets them for proteasomal degradation. Ubiquitin-protein ligases (E3s) play a crucial role in this process by recognizing target proteins and initiating their ubiquitination. Here we show that Hrd1p, which is identical to Der3p, is an E3 for ERAD. Hrd1p is required for the degradation and ubiquitination of several ERAD substrates and physically associates with relevant ubiquitin-conjugating enzymes (E2s). A soluble Hrd1 fusion protein shows E3 activity in vitro - catalysing the ubiquitination of itself and test proteins. In this capacity, Hrd1p has an apparent preference for misfolded proteins. We also show that Hrd1p functions as an E3 in vivo, using only Ubc7p or Ubc1p to specifically program the ubiquitination of ERAD substrates.     

Inhibition of histone deacetylase activity on specific embryonic tissues as a new mechanism for teratogenicity

BACKGROUND: the inhibition of histone deacetylase (HDAC) has been reported as an effective mechanism on therapy in neoplastic diseases. Among HDAC inhibitors, Trichostatin A (TSA) and Valproic Acid (VPA) prevent the tumorigenesis in rodent and human models. Malformations as neural tube and axial skeletal defects are well-known VPA side effects. Recent hypotheses suggest the HDAC inhibitor activity as the teratogenic mechanism of VPA. The teratogenic potency of TSA is, at the moment, unknown. The aim of the present work is to investigate the HDAC inhibition on embryos exposed in utero to TSA or VPA and to compare the teratogenic potential of these two molecules on the axial skeleton morphogenesis. METHODS: Pregnant CD mice were i.p. treated on day 8 post coitum (9.00 a.m.) with 400 mg/kg VPA or with 0, 2, 4, 8, 16 mg/kg TSA. Embryos explanted 1 hr after the treatment from some females exposed to 400 mg/kg VPA or to 16 mg/kg TSA were processed for Western blotting and immunohistochemical analysis, in order to evaluate the histone hyperacetylation in the total embryo homogenates and to visualize the hyperacetylated tissues. Foetuses at term were processed for skeletal examination. RESULTS: Both VPA and TSA were able to induce hyperacetylation on embryos, specifically at the level of the caudal neural tube and of somites. At term, TSA showed teratogenic effects at the axial skeleton, quite similar to those observed after VPA exposure. CONCLUSIONS: In conclusion, both VPA and TSA are teratogenic in mice. A direct correlation between somite hyperacetylation and axial abnormalities could suggest the HDAC inhibition as the mechanism of the teratogenic effects.     

Development of the first small molecule histone deacetylase 6 (HDAC6) degraders

Histone deacetylases (HDACs) decrease the acetylation level of histones and other non-histone proteins. Over expression of HDACs have been observed in cancers and other diseases. Targeted protein degradation by “hijacking” the natural ubiquitin-proteasome-system (UPS) recently emerged as a novel technology to “knock-out” endogenous disease-causing proteins. We applied this strategy to the development of the first small molecule degraders for zinc-dependent HDACs by conjugating non-selective HDAC inhibitors with E3 ubiquitin ligase ligands. Through cell-based assays, we discovered novel bifunctional molecules (dHDAC6) that could selectively degrade HDAC6. Further mechanistic studies indicated that HDAC6 was selectively removed by the UPS.     

Distinct Ubiquitination Cascades Act on the Peroxisomal Targeting Signal Type 2 Co‐receptor Pex18p

Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by a peroxisomal targeting sequence (PTS) and ferry them to the peroxisomal membrane. Subsequently, the cargo is translocated into the peroxisomal lumen, whereas the receptor is released to the cytosol for further rounds of protein import. This cycle is controlled by the ubiquitination status of the receptor, which is best understood for the PTS1‐receptor. While polyubiquitination of PTS‐receptors results in their proteasomal degradation, the monoubiquitinated PTS‐receptors are exported to the cytosol and recycled for further rounds of protein import. Here, we describe the identification of two ubiquitination cascades acting on the PTS2 co‐receptor Pex18p. Using in vivo and in vitro approaches, we demonstrate that the polyubiquitination of Pex18p requires the ubiquitin‐conjugating enzyme (E2) Ubc4p, which cooperates with the RING (really interesting new gene)‐type ubiquitin‐protein ligases (E3) Pex2p as well as Pex10p. Monoubiquitination of Pex18p depends on the E2 enzyme Pex4p (Ubc10p), which functions in concert with the E3 enzymes Pex12p and Pex10p. Our findings for the PTS2‐pathway complement the data on PTS1‐receptor ubiquitination and add up to a unified concept of the ubiquitin‐based regulation of peroxisomal import .     

In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation

Trichostatin A (TSA) inhibits all histone deacetylases (HDACs) of both class I and II, whereas trapoxin (TPX) cannot inhibit HDAC6, a cytoplasmic member of class II HDACs. We took advantage of this differential sensitivity of HDAC6 to TSA and TPX to identify its substrates. Using this approach, alpha-tubulin was identified as an HDAC6 substrate. HDAC6 deacetylated alpha-tubulin both in vivo and in vitro. Our investigations suggest that HDAC6 controls the stability of a dynamic pool of microtubules. Indeed, we found that highly acetylated microtubules observed after TSA treatment exhibited delayed drug-induced depolymerization and that HDAC6 overexpression prompted their induced depolymerization. Depolymerized tubulin was rapidly deacetylated in vivo, whereas tubulin acetylation occurred only after polymerization. We therefore suggest that acetylation and deacetylation are coupled to the microtubule turnover and that HDAC6 plays a key regulatory role in the stability of the dynamic microtubules.     

Histone deacetylase 8 safeguards the human ever-shorter telomeres 1B (hEST1B) protein from ubiquitin-mediated degradation

Histone deacetylases (HDACs) are enzymes that regulate the functions of histones as well as nonhistones by catalyzing the removal of acetyl groups from lysine residues. HDACs regulate many biological processes, including the cell division cycle and tumorigenesis. Although recent studies have implicated HDAC8 in tumor cell proliferation, the molecular mechanisms linking HDAC8 to cell growth remain unknown. Here, we report that the human ortholog of the yeast ever-shorter telomeres 1B (EST1B) binds HDAC8. This interaction is regulated by protein kinase A-mediated HDAC8 phosphorylation and protects human EST1B (hEST1B) from ubiquitin-mediated degradation. Phosphorylated HDAC8 preferentially recruits Hsp70 to a complex that inhibits the CHIP (C-terminal heat shock protein interacting protein) E3 ligase-mediated degradation of hEST1B. Importantly, HDAC8 regulation of hEST1B protein stability modulates total telomerase enzymatic activity. Our findings reveal a novel mechanism by which HDAC8 contributes to tumorigenesis by regulating telomerase activity.     

Modulation of pro- and anti-apoptotic factors in human melanoma cells exposed to histone deacetylase inhibitors

Valproic acid (VPA, 2-propylpentanoic acid) is an established drug in the long-term therapy of epilepsy. Recently, VPA was demonstrated to inhibit histone deacetylases (HDACs) class I enzyme at therapeutically relevant concentrations, thereby, mimicking the prototypical histone deacetylase inhibitors, tricostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA). In the present study, we investigated the cellular effects of VPA, TSA and SAHA on four human melanoma cell lines (WM115, WM266, A375, SK-Mel28) with particular reference to the modulation of regulators of apoptosis, including Bcl-2, BclXL, Mcl-1, Apaf-1, BclXs, NOXA, TRAIL-R1, TRAIL-R2, caspase 8, and survivin). Firstly, we found that VPA induced apoptosis in two of the four human melanoma cell lines, while both TSA and SAHA exhibited an antiproliferative and apoptotic effects in all four cell lines, a different expression of Bcl-2 and BclX(L/S) occurred. On the other hand, SAHA and VPA modulated differently pro- and anti-apoptotic factors. In particular, the treatment with VPA enhanced the level of expression of survivin only in VPA-resistant cell lines, whereas down-regulation of survivin was induced by VPA and SAHA in VPA-sensitive cells. In the latter, since activation of caspase 8 was documented, a receptor-mediated apoptosis was suggested. Taken together, our results suggest that HDAC inhibitors may represent a promising therapeutic strategy to treat melanoma.     

Histone deacetylase inhibition decreases preference without affecting aversion for nicotine

Mutations in the parkin gene cause early-onset, autosomal recessive Parkinson's disease. Parkin functions as an E3 ubiquitin ligase to mediate the covalent attachment of ubiquitin monomers or linked chains to protein substrates. Substrate ubiquitination can target proteins for proteasomal degradation or can mediate a number of non-degradative functions. Parkin has been shown to preserve mitochondrial integrity in a number of experimental systems through the regulation of mitochondrial fission. Upon mitochondrial damage, parkin translocates to mitochondria to mediate their selective elimination by autophagic degradation. The mechanism underlying this process remains unclear. Here, we demonstrate that parkin interacts with and selectively mediates the atypical poly-ubiquitination of the mitochondrial fusion factor, mitofusin 1, leading to its enhanced turnover by proteasomal degradation. Our data supports a model whereby the translocation of parkin to damaged mitochondria induces the degradation of mitofusins leading to impaired mitochondrial fusion. This process may serve to selectively isolate damaged mitochondria for their removal by autophagy.     

Ubiquitin-dependent proteasomal degradation of human liver cytochrome P450 2E1: identification of sites targeted for phosphorylation and ubiquitination

Human liver CYP2E1 is a monotopic, endoplasmic reticulum-anchored cytochrome P450 responsible for the biotransformation of clinically relevant drugs, low molecular weight xenobiotics, carcinogens, and endogenous ketones. CYP2E1 substrate complexation converts it into a stable slow-turnover species degraded largely via autophagic lysosomal degradation. Substrate decomplexation/withdrawal results in a fast turnover CYP2E1 species, putatively generated through its futile oxidative cycling, that incurs endoplasmic reticulum-associated ubiquitin-dependent proteasomal degradation (UPD). CYP2E1 thus exhibits biphasic turnover in the mammalian liver. We now show upon heterologous expression of human CYP2E1 in Saccharomyces cerevisiae that its autophagic lysosomal degradation and UPD pathways are evolutionarily conserved, even though its potential for futile catalytic cycling is low due to its sluggish catalytic activity in yeast. This suggested that other factors (i.e. post-translational modifications or "degrons") contribute to its UPD. Indeed, in cultured human hepatocytes, CYP2E1 is detectably ubiquitinated, and this is enhanced on its mechanism-based inactivation. Studies in Ubc7p and Ubc5p genetically deficient yeast strains versus corresponding isogenic wild types identified these ubiquitin-conjugating E2 enzymes as relevant to CYP2E1 UPD. Consistent with this, in vitro functional reconstitution analyses revealed that mammalian UBC7/gp78 and UbcH5a/CHIP E2-E3 ubiquitin ligases were capable of ubiquitinating CYP2E1, a process enhanced by protein kinase (PK) A and/or PKC inclusion. Inhibition of PKA or PKC blocked intracellular CYP2E1 ubiquitination and turnover. Here, through mass spectrometric analyses, we identify some CYP2E1 phosphorylation/ubiquitination sites in spatially associated clusters. We propose that these CYP2E1 phosphorylation clusters may serve to engage each E2-E3 ubiquitination complex in vitro and intracellularly.