Effects of histone acetyltransferase (HAT) and histone deacetylase (HDAC) inhibitors on proliferative, differentiative, and regenerative functions of Toll-like receptor 2 (TLR-2)-stimulated human dental pulp cells (hDPCs).

OBJECTIVES: This in vitro study aimed to modify TLR-2-mediated effects on the paracrine, proliferative, and differentiation potentials of human dental pulp-derived cells using histone acetyltransferase (HAT) and histone deacetylase (HDAC) inhibitors. MATERIALS AND METHODS: Cell viability was assessed using the XTT assay. Cells were either treated with 10 µg/ml Pam3CSK4 only, or pre-treated with valproic acid (VPA) (3 mM), trichostatin A (TSA) (3 µM), and MG-149 (3 µM) for a total of 4 h and 24 h. Control groups included unstimulated cells and cells incubated with inhibitors solvents only. Transcript levels for NANOG, OCT3-4, FGF-1 and 2, NGF, VEGF, COL-1A1, TLR-2, hßD-2 and 3, BMP-2, DSPP, and ALP were assessed through qPCR. RESULTS: After 24 h, TSA pre-treatment significantly upregulated the defensins and maintained the elevated pro-inflammatory cytokines, but significantly reduced healing and differentiation genes. VPA significantly upregulated the pro-inflammatory cytokine levels, while MG-149 significantly downregulated them. Pluripotency genes were not significantly affected by any regimen. CONCLUSIONS: At the attempted concentrations, TSA upregulated the defensins gene expression levels, and MG-149 exerted a remarkable anti-inflammatory effect; therefore, they could favorably impact the immunological profile of hDPCs. CLINICAL RELEVANCE: Targeting hDPC nuclear function could be a promising option in the scope of the biological management of inflammatory pulp diseases.

A Therapeutically Targetable NOTCH1-SIRT1-KAT7 Axis in T-cell Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a NOTCH1-driven disease in need of novel therapies. Here, we identify a NOTCH1-SIRT1-KAT7 link as a therapeutic vulnerability in T-ALL, in which the histone deacetylase SIRT1 is overexpressed downstream of a NOTCH1-bound enhancer. SIRT1 loss impaired leukemia generation, whereas SIRT1 overexpression accelerated leukemia and conferred resistance to NOTCH1 inhibition in a deacetylase-dependent manner. Moreover, pharmacologic or genetic inhibition of SIRT1 resulted in significant antileukemic effects. Global acetyl proteomics upon SIRT1 loss uncovered hyperacetylation of KAT7 and BRD1, subunits of a histone acetyltransferase complex targeting H4K12. Metabolic and gene-expression profiling revealed metabolic changes together with a transcriptional signature resembling KAT7 deletion. Consistently, SIRT1 loss resulted in reduced H4K12ac, and overexpression of a nonacetylatable KAT7-mutant partly rescued SIRT1 loss-induced proliferation defects. Overall, our results uncover therapeutic targets in T-ALL and reveal a circular feedback mechanism balancing deacetylase/acetyltransferase activation with potentially broad relevance in cancer. SIGNIFICANCE: We identify a T-ALL axis whereby NOTCH1 activates SIRT1 through an enhancer region, and SIRT1 deacetylates and activates KAT7. Targeting SIRT1 shows antileukemic effects, partly mediated by KAT7 inactivation. Our results reveal T-ALL therapeutic targets and uncover a rheostat mechanism between deacetylase/acetyltransferase activities with potentially broader cancer relevance. This article is highlighted in the In This Issue feature, p. 1.

Design, synthesis, cytotoxicity, and molecular docking studies of novel thiazolyl-hydrazone derivatives as histone lysine acetyl-transferase inhibitors and apoptosis inducers.

Compounds containing both thiazole and arylsulfone moieties are recognized for their high biological activity and ability to fight a variety of ailments. Thus, in this context, new derivatives of (thiazol-2-yl)hydrazone with an arylsulfone moiety were synthesized as CPTH2 analogs with potent anti-histone lysine acetyl-transferase activity. Compounds 3, 4, 10b, and 11b showed an excellent inhibitory effect on P300 (E1A-associated protein p300), compared to CPTH2. Among all the tested derivatives, compound 10b revealed the highest activity against both P300 and pCAF. In addition, the new hits were tested for anticancer efficacy against two leukemia cell lines. Most of them showed a moderate to potent antitumor effect on the k562 and CCRF-CEM cell lines. Interestingly, the activity of compound 10b against the k562 cell line was found to be higher than that of CPTH2. Furthermore, it showed a good safety profile, better than CPTH2 on normal cells. Molecular docking analysis was carried out to reveal the crucial binding contacts in the inhibition of the P300 and pCAF enzymes.

Minocycline suppresses lipogenesis via inhibition of p300 histone acetyltransferase activity in human SZ95 sebocytes.

BACKGROUND: Minocycline is a second-generation tetracycline drug, which is widely used to treat diverse infectious and inflammatory diseases such as acne vulgaris. The effects of minocycline on acne vulgaris have been mainly attributed to its anti-inflammatory effect; however, its sebum-regulating effect and the relevance to epigenetic regulation in human sebaceous glands remain uninvestigated. OBJECTIVES: To identify the potential underlying epigenetic mechanism of sebum-inhibitory effects of minocycline in human SZ95 sebocytes. METHODS: The quantity of lipid droplets and the expression of key lipogenic genes were analysed in minocycline-treated SZ95 sebocytes. To examine whether the sebum-inhibitory effects of minocycline are relevant to histone acetylation, we analysed the effects of minocycline on p300 HAT and total HDAC activity. To elucidate the functional implication of p300 HAT inhibition by minocycline in sebocytes, we assessed the effect of p300 knockdown, inhibition and overexpression on lipid accumulation in SZ95 sebocytes. RESULTS: Minocycline suppressed the insulin and liver X receptor agonist-induced lipid accumulation and the expression of the key lipogenic transcription factor sterol regulatory element-binding protein 1 (SREBP1) and its downstream genes, fatty acid synthase (FAS) and acetyl-CoA carboxylase α (ACCα). Minocycline inhibited p300 HAT activity in a concentration-dependent manner, but demonstrated no effect on global HDAC activity, resulting in a significant decrease in histone acetylation. Downregulation of p300 by knockdown or inhibition significantly suppressed SREBP1 expression, histone acetylation and lipid accumulation, whereas p300 overexpression enhanced these effects. Moreover, p300 overexpression rescued minocycline-inhibited SREBP1 expression and lipid synthesis. CONCLUSIONS: Our findings revealed a novel sebum-regulating effect of minocycline. Moreover, as p300 HAT is a key epigenetic regulator of sebaceous lipogenesis, its inhibitors could be used for the treatment of acne vulgaris.

Dispersive effects and focused biodistribution of recombinant human hyaluronidase PH20: A locally acting and transiently active permeation enhancer.

BACKGROUND: Recombinant human hyaluronidase PH20 (rHuPH20) facilitates the dispersion and absorption of subcutaneously administered therapeutic agents. This study aimed to characterize the transient, local action of rHuPH20 in the subcutaneous (SC) space using focused biodistribution and dye dispersion studies conducted in mice. MATERIALS AND METHODS: To evaluate the biodistribution of rHuPH20, mice were intradermally administered rHuPH20 (80 U). The enzymatic activity of rHuPH20 was analyzed in the skin, lymph nodes, and plasma. Animal model sensitivity was determined by intravenous administration of rHuPH20 (80 U) to the tail vein. To evaluate local dispersion, mice received an intradermal injection of rHuPH20 followed by an intradermal injection of Trypan Blue dye at a contralateral site 45 minutes later. Dye dispersion was measured using a digital caliper. RESULTS: After intradermal rHuPH20 injection, enzymatic activity was detected within the skin near the injection site with levels decreasing rapidly after 15 minutes. There was no clear evidence of systemic exposure after administration of rHuPH20, and no discernible rHuPH20 activity was observed in lymph or plasma as a function of time after dosing. In the dye dispersion study, delivery of rHuPH20 at one site did not impact dye dispersion at a distal skin site. CONCLUSION: These observations support the classification of rHuPH20 as a transiently active and locally acting permeation enhancer.

Histone acetyltransferase promotes fluoride toxicity in LS8 cells.

Previously we demonstrated that fluoride increased acetylated-p53 (Ac-p53) in LS8 cells that are derived from mouse enamel organ epithelia and in rodent ameloblasts. However, how p53 is acetylated by fluoride and how the p53 upstream molecular pathway responds to fluoride is not well characterized. Here we demonstrate that fluoride activates histone acetyltransferases (HATs) including CBP, p300, PCAF and Tip60 to acetylate p53. HAT activity is regulated by post-translational modifications such as acetylation and phosphorylation. HAT proteins and their post-translational modifications (p300, Acetyl-p300, CBP, Acetyl-CBP, Tip60 and phospho-Tip60) were analyzed by Western blots. p53-HAT binding was detected by co-immunoprecipitation (co-IP). Cell growth inhibition was analyzed by MTT assays. LS8 cells were treated with NaF with/without HAT inhibitors MG149 (Tip60 inhibitor) and Anacardic Acid (AA; inhibits p300/CBP and PCAF). MG149 or AA was added 1 h prior to NaF treatment. Co-IP results showed that NaF increased p53-CBP binding and p53-PCAF binding. NaF increased active Acetyl-p300, Acetyl-CBP and phospho-Tip60 levels, suggesting that fluoride activates these HATs. Fluoride-induced phospho-Tip60 was decreased by MG149. MG149 or AA treatment reversed fluoride-induced cell growth inhibition at 24 h. MG149 or AA treatment decreased fluoride-induced p53 acetylation to inhibit caspase-3 cleavage, DNA damage marker γH2AX expression and cytochrome-c release into the cytosol. These results suggest that acetylation of p53 by HATs contributes, at least in part, to fluoride-induced toxicity in LS8 cells via cell growth inhibition, apoptosis, DNA damage and mitochondrial damage. Modulation of HAT activity may, therefore, be a potential therapeutic target to mitigate fluoride toxicity in ameloblasts.

Recombinant human hyaluronidase PH20 does not stimulate an acute inflammatory response and inhibits lipopolysaccharide-induced neutrophil recruitment in the air pouch model of inflammation.

Hyaluronidase (Hyal) and low m.w. hyaluronan (LMW HA) fragments have been widely reported to stimulate the innate immune response. However, most hyaluronidases used were purified from animal tissues (e.g., bovine testis Hyal [BTH]), and contain endotoxin and other unrelated proteins. We tested a highly purified recombinant human Hyal (rHuPH20) and endotoxin-free HA fragments from M(r) 5,000 to 1,500,000 in the rodent air pouch model of inflammation to determine their potential for stimulation of the innate immune response. Exogenous LMW HA fragments (average M(r) 200,000) failed to induce either cytokine/chemokine production or neutrophil infiltration into the air pouch. Challenging the air pouch with LPS or BTH stimulated production of cytokines and chemokines but rHuPH20 did not, suggesting that neither PH20 nor generation of LMW HA fragments in situ stimulates cytokine and chemokine production. LPS and BTH also induced neutrophil infiltration into the air pouch, which was not observed with rHuPH20 treatment. Endotoxin-depleted BTH had much reduced proinflammatory activity, suggesting that the difference in inflammatory responses between rHuPH20 and BTH is likely due to endotoxin contaminants in BTH. When rHuPH20 was dosed with LPS, the induction of cytokines and chemokines was the same as LPS alone, but neutrophil infiltration was inhibited, likely by interrupting HA-CD44 interaction. Our results indicate that neither rHuPH20 nor its directly generated HA catabolites have inflammatory properties in the air pouch model, and rHuPH20 can instead inhibit some aspects of inflammation, such as neutrophil infiltration into the air pouch.

Epigenetic influence of KAT6B and HDAC4 in the development of skeletal malocclusion.

INTRODUCTION: Genetic influences on the development of malocclusion include heritable effects on both masticatory muscles and jaw skeletal morphology. Beyond genetic variations, however, the characteristics of muscle and bone are also influenced by epigenetic mechanisms that produce differences in gene expression. We studied 2 enzymes known to change gene expressions through histone modifications, chromatin-modifying histone acetyltransferase KAT6B and deacetylase HDAC4, to determine their associations with musculoskeletal variations in jaw deformation malocclusions. METHODS: Samples of masseter muscle were obtained from subjects undergoing orthognathic surgery from 6 malocclusion classes based on skeletal sagittal and vertical dysplasia. The muscles were characterized for fiber type properties by immunohistochemistry, and their total RNA was isolated for gene expression studies by microarray analysis and quantitative real-time polymerase chain reaction. RESULTS: Gene expressions for fast isoforms of myosins and contractile regulatory proteins and for KAT6B and HDAC4 were severalfold greater in masseter muscles from a patient with a deepbite compared with one with an open bite, and genes related to exercise and activity did not differ substantially. In the total population, expressions of HDAC4 (P = 0.03) and KAT6B (P = 0.004) were significantly greater in subjects with sagittal Class III than in Class II malocclusion, whereas HDAC4 tended to correlate negatively with slow myosin type I and positively with fast myosin gene, especially type IIX. CONCLUSIONS: These data support other published reports of epigenetic regulation in the determination of skeletal muscle fiber phenotypes and bone growth. Further investigations are needed to elucidate how this regulatory model might apply to musculoskeletal development and malocclusion.

Accelerating and improving the consistency of rapid-acting analog insulin absorption and action for both subcutaneous injection and continuous subcutaneous infusion using recombinant human hyaluronidase.

Rapid-acting insulin analogs were introduced to the market in the 1990s, and these products have improved treatment of diabetes by shortening the optimum delay time between injections and meals. Compared with regular human insulin, rapid-acting insulin formulations also reduce postprandial glycemic excursions while decreasing risk of hypoglycemia. However, the current prandial products are not fast enough for optimum convenience or control. Recombinant human hyaluronidase (rHuPH20) has been used to increase the dispersion and absorption of other injected drugs, and in the case of prandial insulin analogs, it confers both ultrafast absorption and action profiles. Animal toxicology studies have demonstrated excellent tolerability of rHuPH20, and human studies, involving over 60,000 injections of prandial insulin + rHuPH20 to date, have similarly shown excellent safety and tolerability. Studies using rapid-acting analog insulin with rHuPH20 have included clinic-based pharmacokinetic and glucodynamic euglycemic glucose clamp studies, test meal studies, and take-home treatment studies. Administration methods have included subcutaneous injection of coformulations of rapid-acting insulin + rHuPH20 as well as continuous subcutaneous infusion of coformulations or use of pretreatment of newly inserted infusion sets with rHuPH20 followed by standard continuous subcutaneous insulin infusion therapy. These studies have demonstrated acceleration of insulin absorption and action along with improvement in postprandial glycemic excursions and reduction in hypoglycemia risks. Further, rHuPH20 reduces intrasubject variability of insulin absorption and action and provides greater consistency in absorption and action profiles over wear time of an infusion set. Further studies of rHuPH20 in the take-home treatment setting are underway.

Use of recombinant human hyaluronidase to accelerate rapid insulin analogue absorption: experience with subcutaneous injection and continuous infusion.

OBJECTIVE: To discuss clinical studies in which recombinant human hyaluronidase (rHuPH20) was used to increase insulin dispersion and accelerate its absorption. METHODS: We reviewed 10 pertinent clinical studies, 8 of which had data available. RESULTS: In 4 euglycemic clamp studies, coinjection of rHuPH20 consistently yielded acceleration of insulin absorption, providing twice the insulin exposure during the first hour, greater and earlier peak exposure, and half the exposure beyond 2 hours after injection. Insulin-action profiles were similarly accelerated, with a 15-minute faster onset of insulin action and a 45-minute shorter duration of action for each of the 3 commercial rapid-acting insulin analogues. Infusion aspart insulin formulated with rHuPH20 also accelerated insulin absorption and action over the infusion set life when delivered by insulin pump. Administration of rHuPH20 reduced the inconsistency of insulin absorption and action profiles attributable to 3 factors-lack of reproducibility after identical injections, differences across insulin dose ranges, and changes over infusion site life. The rHuPH20-facilitated ultrafast profile consistently reduced hyperglycemic excursions both in injections immediately preceding liquid test meals and in bolus infusions immediately before solid test meals. rHuPH20-facilitated insulin administration has been well tolerated, with safety and tolerability similar to those with the comparator insulin alone. CONCLUSION: rHuPH20 accelerates insulin-action profiles to an extent comparable to the difference between rapid-acting insulin analogue profiles and those of regular insulin. Studies are currently under way to characterize the effect on diabetes management end points (including hemoglobin A1c, blood glucose, and rates of hyperglycemia) of insulin analogues coformulated with rHuPH20 for treatment of both type 1 and type 2 diabetes.

Histone deacetylase inhibitors: new treatment options for inflammatory joint disease?

Histone deacetylase inhibitors (HDIs) are a new class of compounds that are being developed for the treatment of malignancies such as cutaneous T-cell lymphoma. HDIs inhibit the removal of acetyl groups from histones. The histone acetylation process is dependent on two enzymes, histone acetyl transferase (HAT) and histone deacetylase (HDAC), and regulates the expression of genes, including those encoding cell survival or apoptosis. In addition to regulating cell growth, HDIs exert anti-inflammatory effects by controlling the production of anti-inflammatory cytokines; modulating the function of cells such as T cells, monocytes-macrophages, chondrocytes, and osteoclasts; and modulating angiogenesis. In several animal models of arthritis, HDIs improve the clinical manifestations and prevent damage to the bone and cartilage. In humans, the only relevant data available so far come from studies of HAT and HDAC expression in the synovial membrane of patients with rheumatoid arthritis. HDIs may hold promise for the treatment of inflammatory joint disease.

Síntesis y evaluación de nuevos inhibidores de las HDAC / Synthesis and evaluation of new HDAC inhibitors

La acetilación de residuos de lisina en las histonas está mediada por las enzimas denominadas histona acetiltransferasas (HAT). Los grupos acetilo son eliminados de las e-N-acetil-lisinas por la actividadde las histonas desacetilasas (HDAC). El balance entre las actividades opuestas de las HAT y las HDAC regula el estado de acetilación de las histonas. Este tipo de modificaciones regulan en la célula procesos fundamentales clave en respuesta a señales extracelulares. En general, altos niveles de acetilación (hiperacetilación) se asocian a un incremento de la actividad transcripcional, mientras que bajos niveles de acetilación (hipoacetilación) se asocian a la represión de la expresión genética. Actualmente se conocen diversos tipos de inhibidores de las HDAC que pueden reactivar la expresión genética e inhibir el crecimiento de las células tumorales, por lo que se investiga su uso en el tratamiento frente al cáncer. Sería deseable identificar nuevos inhibidores de las enzimas HDAC para su utilización en el tratamiento o profilaxis de enfermedades en las que la inhibición de dichas enzimas HDAC está implicada. Se han obtenido 10 nuevos inhibidores de las HDAC y se ha evaluado su actividad frente a HDAC aislada. Se discute la importancia de las modificaciones realizadas en el espaciador(AU)

Lysine residues acetylation on histones is mediated by histone acetyltransferase (HAT). The acetyl groups are removed from e-N-acetyl-lysine by the histone deacetylase (HDAC) activity. The balance between the HATs and HDACs activities regulates the histone acetylation status. Such changes regulate key processes in the cell in response to extracellular signals. Mostly, high levels of acetylation(hyperacetylation) are associated with increased transcriptional activity. Low levels of acetylation (hypoacetylation) are associated with repression of gene expression. Currently, different types of HDAC inhibitors are known to reactivate gene expression and inhibit tumor cell growth. We aim at identifying novel HDAC inhibitors for the treatment or prophylaxis of cancer diseases. Ten new HDAC inhibitors have been obtained and their potency as HDAC inhibitors has been evaluated. A structure-activity relationship discussion has been focused on the structural changes made in the spacer(AU)

Lack of effect of RuvB-like proteins on DNA damage signaling activation.

Ataxia telangiectasia mutated (ATM) kinase is a central player in cellular response to DNA damage. Phosphorylation of the histone H2AX by ATM is required for the accumulation of repair proteins at the sites of double-strand breaks. Recently, it was reported that the histone acetyltransferase Tat interactive protein-60 (IPP60) is required to acetylate ATM prior to its activation. The RuvB-like proteins TIP48 and TIP49 are known to be necessary for the assembly and functional activity of the TIP60 acetyltransferase complex. In the present communication, we investigated the requirements of IIP48 and IIP49 for ATM activation by monitoring the cell cycle distribution and H2AX phosphorylation after irradiation of IIP48- and IIP49-depleted cells. We found that neither the cell cycle norgammay-H2AX were affected in IIP48- and IIP49-silenced cells, suggesting that the IIP60 chromatin modification complex is not engaged in DNA damage signaling upstream of ATM.

Amyloid precursor protein regulates brain apolipoprotein E and cholesterol metabolism through lipoprotein receptor LRP1.

Mutations in the amyloid precursor protein (APP) cause early-onset Alzheimer's disease (AD), but the only genetic risk factor for late-onset AD is the varepsilon4 allele of apolipoprotein E (apoE), a major cholesterol carrier. Using Cre-lox conditional knockout mice, we demonstrate that lipoprotein receptor LRP1 expression regulates apoE and cholesterol levels within the CNS. We also found that deletion of APP and its homolog APLP2, or components of the gamma-secretase complex, significantly enhanced the expression and function of LRP1, which was reversed by forced expression of the APP intracellular domain (AICD). We further show that AICD, together with Fe65 and Tip60, interacts with the LRP1 promoter and suppresses its transcription. Together, our findings support that the gamma-secretase cleavage of APP plays a central role in regulating apoE and cholesterol metabolism in the CNS via LRP1 and establish a biological linkage between APP and apoE, the two major genetic determinants of AD.