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Trichostatin A enhances acetylation as well as protein stability of ERα through induction of p300 protein

IntroductionTrichostatin A (TSA) is a well-characterized histone deacetylase (HDAC) inhibitor. TSA modifies the balance between HDAC and histone acetyltransferase activities that is important in chromatin remodeling and gene expression. Although several previous studies have demonstrated the role of TSA in regulation of estrogen receptor alpha (ERα), the precise mechanism by which TSA affects ERα activity remains unclear.MethodsTransient transfection was performed using the Welfect-EX™Plus procedure. The mRNA expression was determined using RT-PCR. Protein expression and interaction were determined by western blotting and immunoprecipitation. The transfection of siRNAs was performed using the Oligofectamine™ reagent procedure.ResultsTSA treatment increased acetylation of ERα in a dose-dependent manner. The TSA-induced acetylation of ERα was accompanied by an increased stability of ERα protein. Interestingly, TSA also increased the acetylation and the stability of p300 protein. Overexpression of p300 induced acetylation and stability of ERα by blocking ubiquitination. Knockdown of p300 by RNA interference decreased acetylation as well as the protein level of ERα, indicating that p300 mediated the TSA-induced stabilization of ERα.ConclusionsWe report that TSA enhanced acetylation as well as the stability of the ERα protein by modulating stability of p300. These results may provide the molecular basis for pharmacological functions of HDAC inhibitors in the treatment of human breast cancer.

Regulation of Nur77 protein turnover through acetylation and deacetylation induced by p300 and HDAC1

Although the roles of Nur77, an orphan member of the nuclear hormone receptor superfamily, in the control of cellular proliferation, apoptosis, inflammation, and glucose metabolism, are well recognized, the molecular mechanism regulating the activity and expression of Nur77 is not fully understood. Acetylation of transcription factors has emerged recently as a major post-translational modification that regulates protein stability and transcriptional activity. Here, we examined whether Nur77 is acetylated, and we characterized potential associated factors. First, Nur77 was found to be an acetylated protein when examined by immunoprecipitation and western blotting using acetyl protein-specific antibodies. Second, expression of p300, which possesses histone acetyltransferase activity, enhanced the acetylation and protein stability of Nur77. Treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A, also increased Nur77 acetylation. Among the several types of HDACs, HDAC1 was found as the major enzyme affecting protein level of Nur77. HDAC1 decreased the acetylation level, protein level, and transcriptional activity of Nur77. Interestingly, overexpression of Nur77 induced expression of both p300 and HDAC1. Finally, the expression of Nur77 increased along with that of p300, but decreased with induction of HDAC1 after treatment with epithelial growth factor, nerve growth factor, or 6-mercaptopurine, suggesting that the self-control of the acetylation status contributes to the transient induction of Nur77 protein. Taken together, these results demonstrate that acetylation of Nur77 is modulated by p300 and HDAC1, and suggest that acetylation is an important post-translational modification for the rapid turnover of Nur77 protein.

Transcriptional activation of hypoxia-inducible factor-1α by HDAC4 and HDAC5 involves differential recruitment of p300 and FIH-1

P300 (uniprotkb:Q09472) physically interacts (MI:0218) with HIF1 alpha (uniprotkb:Q16665) by anti bait coimmunoprecipitation (MI:0006)

Differential Regulation of Estrogen Receptor α Expression in Breast Cancer Cells by Metastasis-Associated Protein 1

Metastasis-associated protein 1 (MTA1) is a component of the nucleosome remodeling and histone deacetylase (HDAC) complex, which plays an important role in progression of breast cancer. Although MTA1 is known as a repressor of the transactivation function of estrogen receptor α (ERα), its involvement in the epigenetic control of transcription of the ERα gene ESR1 has not been studied. Here, we show that silencing of MTA1 reduced the level of expression of ERα in ERα-positive cells but increased it in ERα-negative cells. In both MCF7 and MDA-MB-231, MTA1 was recruited to the region +146 to +461 bp downstream of the transcription start site of ESR1 (ERpro315). Proteomics analysis of the MTA1 complex that was pulled down by an oligonucleotide encoding ERpro315 revealed that the transcription factor AP-2γ (TFAP2C) and the IFN-γ-inducible protein 16 (IFI16) were components of the complex. Interestingly, in MCF7, TFAP2C activated the reporter encoding ERpro315 and the level of ERα mRNA. By contrast, in MDA-MB-231, IFI16 repressed the promoter activity and silencing of MTA1 increased expression of ERα. Importantly, class II HDACs are involved in the MTA1-mediated differential regulation of ERα. Finally, an MDA-MB-231-derived cell line that stably expressed shIFI16 or shMTA1 was more susceptible to tamoxifen-induced growth inhibition in in vitro and in vivo experiments. Taken together, our findings suggest that the MTA1-TFAP2C or the MTA1-IFI16 complex may contribute to the epigenetic regulation of ESR1 expression in breast cancer and may determine the chemosensitivity of tumors to tamoxifen therapy in patients with breast cancer.

Mechanical properties of (Ti,Cr)N coatings deposited by inductively coupled plasma assisted direct current magnetron sputtering

Abstract (Ti,Cr)N coatings were deposited by inductively coupled plasma (ICP) assisted magnetron sputtering and the properties of the coatings were investigated. The (Ti,Cr)N coatings deposited by ICP assisted sputtering showed a much higher micro-hardness (>5000 HK0.01) compared to coatings produced by the conventional DC magnetron sputtering method. The ICP deposited (Ti,Cr)N coatings also showed superior properties in adhesion and wear compared to the DC sputtered (Ti,Cr)N coatings. The superior mechanical properties of ICP deposited (Ti,Cr)N coatings were attributed to the fine and dense micro-structure and high compressive residual stress.

Synthesis and characterization of carbon nitride thin films prepared by rf plasma enhanced chemical vapor deposition

Abstract Amorphous carbon nitride thin films were deposited on Si(100) wafers by radio frequency plasma enhanced chemical vapor deposition (PEVCD) with capacitively coupled plasma using NH 3 and CH 4 as precursors. The influence of the experimental parameters, i.e. the ratio of the NH 3 /CH 4 flow rate, rf power, the deposition temperature and electrode spacing, on the film deposition rate and surface roughness of the films was investigated. The composition, surface morphology and film structure were analyzed by various techniques. The deposition rate was found to be affected by all parameters. In particular, at high deposition temperature or at high NH 3 /CH 4 ratio, the deposition rate was reduced to almost zero. The nitrogen concentration increased rapidly with increasing NH 3 /CH 4 ratio, which then saturated to a certain level ( R D with increasing NH 3 /CH 4 ratio is ascribed to the local desorption of nitrogen in the nitrogen enriched regions or the formation of volatile CN compounds (e.g. C 2 N 2 ), which also explains the limited amount of the maximum nitrogen content. The films consisted mainly of two hybridizations, N–sp 2 C and N–sp 3 C bonds, but the fraction of N–sp 3 C bonds decreased with increasing N content. The size of the sp 2 –C clusters in the films decreased with increasing N addition, suggesting that N incorporation can break the long-range order of the graphitic structure.

The properties of (Ti, Al)N coatings deposited by inductively coupled plasma assisted d.c. magnetron sputtering

(Ti,Al)N coatings were deposited on M2 high speed steel substrates by inductively coupled plasma (ICP) assisted d.c. magnetron sputtering, and the structure and mechanical properties such as hardness, Youngs modulus, wear resistance and adhesion strength were investigated. A TiAl alloy target (Ti/Al — 50:50 at.%) was sputtered in an Ar and N2 atmosphere with 400 W for d.c. magnetron power as well as 400 W for ICP power at 80 mtorr of working pressure. Both the hardness and adhesion strength of the coating were found to increase with increasing substrate bias voltage. The hardness value was higher than 6500 HK0.01 at the bias voltage higher than –50 V. The Youngs modulus of the coating had a maximum value of approximately 450 GPa at −50 V. The wear properties of coatings also improved with the application of the substrate bias voltage. It was found that the grain size decreased (<100 nm), and the columnar structure, which was observed in the absence of bias, disappeared when a bias voltage was applied. The high hardness and good wear property was attributed to the microstructure change from a columnar structure with facet shaped grains to a denser one with small and round shaped grains.

A study on the low temperature coating process by inductively coupled plasma assisted DC magnetron sputtering

Abstract Titanium nitride (TiN x ) was deposited on a plastic Acrylonitrile–butadiene–styrene substrate by inductively coupled plasma (ICP) sputtering. Many PVD coating processes, currently used in industry, are quite inefficient as there is a need for an extensive cooling time. However, the ICP technique is a high-density plasma technique, which allows deposition on substrates like plastics at low temperatures. Through the plasma diagnosis, the plasma density of the ICP sputtering system was superior to that of the other commercial PVD processes.

RORα Induces KLF4-Mediated M2 Polarization in the Liver Macrophages that Protect against Nonalcoholic Steatohepatitis

The regulation of M1/M2 polarization in liver macrophages is closely associated with the progression of nonalcoholic steatohepatitis (NASH); however, the mechanism involved in this process remains unclear. Here, we describe the orphan nuclear receptor retinoic-acid-related orphan receptor α (RORα) as a key regulator of M1/M2 polarization in hepatic residential Kupffer cells (KCs) and infiltrated monocyte-derived macrophages. RORα enhanced M2 polarization in KCs by inducing the kruppel-like factor 4. M2 polarization was defective in KCs and bone-marrow-derived macrophages of the myeloid-specific RORα null mice, and these mice were susceptible to HFD-induced NASH. We found that IL-10 played an important role in connecting the function of M2 KCs to lipid accumulation and apoptosis in hepatocytes. Importantly, M2 polarization was controlled by a RORα activator, JC1-40, which improved symptoms of NASH. Our results suggest that the M2-promoting effects of RORα in liver macrophages may provide better therapeutic strategies against NASH.

NR1D1 Recruitment to Sites of DNA Damage Inhibits Repair and Is Associated with Chemosensitivity of Breast Cancer

DNA repair capacity is critical for survival of cancer cells upon therapeutic DNA damage and thus is an important determinant of susceptibility to chemotherapy in cancer patients. In this study, we identified a novel function of nuclear receptor NR1D1 in DNA repair, which enhanced chemosensitivity in breast cancer cells. NR1D1 inhibited both nonhomologous end joining and homologous recombination double-strand breaks repair, and delayed the clearance of γH2AX DNA repair foci that formed after treatment of doxorubicin. PARylation of NR1D1 by PARP1 drove its recruitment to damaged DNA lesions. Deletion of the ligand binding domain of NR1D1 that interacted with PARP1, or treatment of 6-(5H)-phenanthridinone, an inhibitor of PARP1, suppressed the recruitment of NR1D1 to DNA damaged sites, indicating PARylation as a critical step for the NR1D1 recruitment. NR1D1 inhibited recruitment of the components of DNA damage response complex such as SIRT6, pNBS1, and BRCA1 to DNA lesions. Downregulation of NR1D1 in MCF7 cells resulted in resistance to doxorubicin, both in vitro and in vivo Analysis of four public patient data sets indicated that NR1D1 expression correlates positively with clinical outcome in breast cancer patients who received chemotherapy. Our findings suggest that NR1D1 and its ligands provide therapeutic options that could enhance the outcomes of chemotherapy in breast cancer patients. Cancer Res; 77(9); 2453-63. ©2017 AACR.