BACKGROUND: Matrix metallopeptidases (MMPs) are critical matrix-degrading molecules and they are frequently overexpressed in degenerative discs. This study aimed to investigate the mechanism for MMP upregulation.
METHODS: Immunoblot and RT-qPCR were used for detecting protein and gene expression levels. 4-month-old and 24-month-old C57BL/6 mice were used for evaluating intervertebral disc degeneration (IDD). An ubiquitination assay was used to determine protein modification. Immunoprecipitation and mass spectrometry were used for identifying protein complex members.
RESULTS: We identified the elevation of 14 MMPs among 23 members in aged mice with IDD. Eleven of these 14 MMP gene promoters contained a Runx2 (runt-related transcription factor 2) binding site. Biochemical analyses revealed that Runx2 recruited a histone acetyltransferase p300 and a coactivator NCOA1 (nuclear receptor coactivator 1) to assemble a complex, transactivating MMP expression. The deficiency of an E3 ligase called HERC3 (HECT and RLD domain containing E3 ubiquitin-protein ligase 3) resulted in the accumulation of NCOA1 in the inflammatory microenvironment. High throughput screening of small molecules that specifically target the NCOA1-p300 interaction identified a compound SMTNP-191, which showed an inhibitory effect on suppressing MMP expression and attenuating the IDD process in aged mice.
CONCLUSIONS: Our data support a model in which deficiency of HERC3 fails to ubiquitinate NCOA1, leading to the assembly of NCOA1-p300-Runx2 and causing the transactivation of MMPs. These findings offer new insight into inflammation-mediated MMP accumulation and also provide a new therapeutic strategy to retard the IDD process.

Both epidemiological investigations and animal studies have linked arsenic-contaminated water to cancers, including skin, liver and lung cancers. Besides genotoxicity, arsenic exposure-related pathogenesis of disease is widely considered through epigenetic mechanisms; however, the underlying mechanism remains to be determined. Herein we explore the initial epigenetic changes via acute sodium arsenite (As) exposures of mouse embryonic fibroblast (MEF) cells and histone H3K79 methyltransferase Dot1L knockout (Dot1L-/-) MEF cells. Our RNA-seq and Western blot data demonstrated that, in both cell lines, acute As exposure abolished histone acetyltransferase p300 at the RNA level and subsequent protein level. Consequently, p300-specific main target histone H3K27ac, a marker separating active from poised enhancers, decreased dramatically as validated by both Western blot and ChIP-qPCR/seq analyses. Concomitantly, H3K4me1 as another well-known marker for enhancers also showed significant decreases, suggesting an underappreciated crosstalk between H3K4me1 and H3K27ac involved in As exposure. Significantly, As exposure-reduced H3K27ac and H3K4me1 inhibited the expression of genes including EP300 itself and Kruppel Like Factor 4(Klf4) that both are tumor suppressor genes. Collectively, our investigations identified p300 as an internal bridging factor within cells to sense external environmental As exposure to alter chromatin, thereby changing gene transcription for disease pathogenesis.

OBJECTIVE: To investigate melatonin (MEL) levels in human dental pulp tissue (hDP) in type 2 diabetic (T2D) participants and the underlying molecular mechanisms of its effects in human dental pulp cells (hDPCs) under hyperglycaemia.
METHODS: The study included 16 healthy and 16 T2D participants who underwent vital pulp extirpation for hDP and four healthy participants undergoing third molar extraction for hDPCs analyses. MTT and NRU were used as tests for cytotoxicity. The pulp tissue levels of MEL, inducible NO synthase (iNOS) and superoxide dismutase (SOD) activity, as well as iNOS, histone acetyltransferase p300 (p300) and SOD activity levels in hDPCs incubated with MEL (0.1 and 1.0 mmol L-1 ) under normoglycaemia and hyperglycaemia were measured by enzyme-linked immunosorbent assay. Comparisons between the two groups were made by unpaired t-tests or Mann-Whitney test whilst the chi-square test was used for dichotomous variables. To compare more groups, the Kruskal-Wallis test with Dunn\'s multiple comparison was used, whilst Spearman correlation was used to assess association between two variables.
RESULTS: Melatonin was decreased (124.30 ± 21.6 vs. 240.0 ± 19.1 pg mL-1 , P < 0.01), whilst iNOS levels increased (0.92 ± 0.08 vs. 0.32 ± 0.09 ng mL-1 , P < 0.01) in hDP from T2D compared to nondiabetic participants. In hDPCs, MEL (0.1 and 1.0 mmol L-1 ) had no cytotoxicity. Incubation with 1.0 mmol L-1 of MEL (24 h) decreased hyperglycaemia-induced increases of iNOS (0.34 ± 0.01 ng mL-1 vs. 0.40 ± 0.01 ng mL-1 , P < 0.01) and p300 (11.59 ± 0.58 ng mL-1 vs. 16.12 ± 0.39 ng mL-1 , P < 0.01), and also, increased SOD activity (87.11 ± 3.10% vs. 68.56 ± 3.77%, P < 0.01) to the levels comparable to the normoglycaemic; iNOS and p300 protein expression levels showed strong positive correlation under hyperglycaemia (Spearman r = 0.8242, P < 0.001).
CONCLUSIONS: Type 2 diabetic participants had decreased MEL in hDP. At pharmacological concentrations, MEL is not cytotoxic for hDPCs and normalizes iNOS and SOD activity levels in hyperglyceamic hDPCs suggesting its antioxidant and protective effects in human dental pulp tissue under hyperglycaemia.

Thioredoxin-interacting protein (TXNIP) has been shown to be associated with glucose-induced deterioration of pancreatic beta cell function in diabetes. However, whether epigenetic mechanisms contribute to the regulation of TXNIP gene expression by glucose is not clear. Here we studied how glucose exerts its effect on TXNIP gene expression via modulation of histone acetylation marks. To achieve this, we applied clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) to knock out histone acetyltransferase (HAT) p300 in a rat pancreatic beta cell line INS1 832/13. We also treated the cells and human islets with chemical inhibitors of HAT p300 and histone deacetylase (HDAC). In human islets, diabetes and high glucose resulted in elevated TXNIP and EP300 expression, and glucose-induced TXNIP expression could be reversed by p300 inhibitor C646. In INS1 832/13 cells, Ep300 knock-out by CRISPR/Cas9 elevated glucose-induced insulin secretion and greatly reduced glucose-stimulated Txnip expression and cell apoptosis. This effect could be ascribed to decrease in histone marks H3K9ac and H4ac at the promoter and first coding region of the Txnip gene. Histone marks H3K9ac and H4ac in the Txnip gene in the wild-type cells was inhibited by HDAC inhibitor at high glucose, which most likely was due to enhanced acetylation levels of p300 after HDAC inhibition; and thereby reduced p300 binding to the Txnip gene promoter region. Such inhibition was absent in the Ep300 knock-out cells. Our study provides evidence that histone acetylation serves as a key regulator of glucose-induced increase in TXNIP gene expression and thereby glucotoxicity-induced apoptosis.