1 364 123 AMELIORATION OF UREMIC TOXIN INDOXYL SULFATE-INDUCED OSTEOBLASTIC CALCIFICATION BY SET DOMAIN CONTAINING LYSINE METHYLTRANSFERASE 7/9 PROTEIN. BACKGROUND: VASCULAR CALCIFICATION (VC) IS A VERY COMMON PHENOMENON IN PATIENTS WITH CHRONIC KIDNEY DISEASE (CKD). IT HAS BEEN REPORTED THAT SOME HISTONE METHYLATION PLAY A ROLE IN VC AS AN EPIGENETIC REGULATOR. INDOXYL SULFATE (IS) IS A PROTEIN-BOUND UREMIC TOXIN THAT HAS BEEN PROVEN AS ONE OF THE MAJOR RISK FACTORS OF CARDIOVASCULAR DISEASE IN CKD. SET DOMAIN CONTAINING LYSINE METHYLTRANSFERASE 7/9 (SET7/9) IS ONE OF THE IMPORTANT HISTONE METHYLTRANSFERASES. OBJECTIVES: THIS STUDY AIMED TO DETERMINE THE EFFECT OF IS ON THE EXPRESSION OF SET7/9 AND THE ROLE OF SET7/9 IN IS-INDUCED OSTEOBLASTIC DIFFERENTIATION AND CALCIFICATION OF VASCULAR SMOOTH MUSCLE CELLS (VSMCS). METHODS: VSMCS WERE INCUBATED WITH VARIOUS CONCENTRATIONS OF IS FOR DIFFERENT DURATIONS TO ASSESS OSTEOBLASTIC DIFFERENTIATION AND EXPRESSION OF SET7/9. WESTERN BLOT ANALYSIS AND QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION WERE PERFORMED TO ASSESS THE PROTEIN AND MRNA LEVELS OF SET7/9 RESPECTIVELY. THE CALCIUM CONTENT WAS MEASURED TO EVALUATE CALCIFICATION. RESULTS: OSTEOBLASTIC DIFFERENTIATION AND CALCIFICATION OF VSMCS AND DOWNREGULATION OF THE EXPRESSION OF SET7/9 WERE OBSERVED AFTER IS TREATMENT. THE AUTOPHAGY WAS ACTIVATED AFTER TREATMENT WITH IS, WHEREAS THE INHIBITION OF THE AUTOPHAGY PARTIALLY ATTENUATED THE EFFECT OF IS ON BOTH THE STIMULATION OF THE EXPRESSION OF RUNT-RELATED TRANSCRIPTION FACTOR 2 AND CALCIUM DEPOSITION. CONCLUSIONS: OUR DATA DEMONSTRATED THAT SET7/9 DOWNREGULATION AND AUTOPHAGY ACTIVATION MAY BE THE KEY MECHANISM OF IS-INDUCED VC IN CKD. 2019 2 1663 53 DOWNREGULATION OF DNA METHYLTRANSFERASE-3A AMELIORATES THE OSTEOGENIC DIFFERENTIATION ABILITY OF ADIPOSE-DERIVED STEM CELLS IN DIABETIC OSTEOPOROSIS VIA WNT/BETA-CATENIN SIGNALING PATHWAY. BACKGROUND: DIABETES-RELATED OSTEOPOROSIS (DOP) IS A CHRONIC DISEASE CAUSED BY THE HIGH GLUCOSE ENVIRONMENT THAT INDUCES A METABOLIC DISORDER OF OSTEOCYTES AND OSTEOBLAST-ASSOCIATED MESENCHYMAL STEM CELLS. THE PROCESSES OF BONE DEFECT REPAIR AND REGENERATION BECOME EXTREMELY DIFFICULT WITH DOP. ADIPOSE-DERIVED STEM CELLS (ASCS), AS SEED CELLS IN BONE TISSUE ENGINEERING TECHNOLOGY, PROVIDE A PROMISING THERAPEUTIC APPROACH FOR BONE REGENERATION IN DOP PATIENTS. THE OSTEOGENIC ABILITY OF ASCS IS LOWER IN A DOP MODEL THAN THAT OF CONTROL ASCS. DNA METHYLATION, AS A MECHANISM OF EPIGENETIC REGULATION, MAY BE INVOLVED IN DNA METHYLATION OF VARIOUS GENES, THEREBY PARTICIPATING IN BIOLOGICAL BEHAVIORS OF VARIOUS CELLS. EMERGING EVIDENCE SUGGESTS THAT INCREASED DNA METHYLATION LEVELS ARE ASSOCIATED WITH ACTIVATION OF WNT/BETA-CATENIN SIGNALING PATHWAY. THE PURPOSE OF THIS STUDY WAS TO INVESTIGATE THE INFLUENCE OF THE DIABETIC ENVIRONMENT ON THE OSTEOGENIC POTENTIAL OF ASCS, TO EXPLORE THE ROLE OF DNA METHYLATION ON OSTEOGENIC DIFFERENTIATION OF DOP-ASCS VIA WNT/BETA-CATENIN SIGNALING PATHWAY, AND TO IMPROVE THE OSTEOGENIC DIFFERENTIATION ABILITY OF ASCS WITH DOP. METHODS: DOP-ASCS AND CONTROL ASCS WERE ISOLATED FROM DOP C57BL/6 AND CONTROL MICE, RESPECTIVELY. THE MULTIPOTENCY OF DOP-ASCS WAS CONFIRMED BY ALIZARIN RED-S, OIL RED-O, AND ALCIAN BLUE STAINING. REAL-TIME POLYMERASE CHAIN REACTION (RT-PCR), IMMUNOFLUORESCENCE, AND WESTERN BLOTTING WERE USED TO ANALYZE CHANGES IN MARKERS OF OSTEOGENIC DIFFERENTIATION, DNA METHYLATION, AND WNT/BETA-CATENIN SIGNALING. ALIZARIN RED-S STAINING WAS ALSO USED TO CONFIRM CHANGES IN THE OSTEOGENIC ABILITY. DNMT SMALL INTERFERING RNA (SIRNA), SHRNA-DNMT3A, AND LVRNA-DNMT3A WERE USED TO ASSESS THE ROLE OF DNMT3A IN OSTEOGENIC DIFFERENTIATION OF CONTROL ASCS AND DOP-ASCS. MICRO-COMPUTED TOMOGRAPHY, HEMATOXYLIN AND EOSIN STAINING, AND MASSON STAINING WERE USED TO ANALYZE CHANGES IN THE OSTEOGENIC CAPABILITY WHILE DOWNREGULATING DNMT3A WITH LENTIVIRUS IN DOP MICE IN VIVO. RESULTS: THE PROLIFERATIVE ABILITY OF DOP-ASCS WAS LOWER THAN THAT OF CONTROL ASCS. DOP-ASCS SHOWED A DECREASE IN OSTEOGENIC DIFFERENTIATION CAPACITY, LOWER WNT/BETA-CATENIN SIGNALING PATHWAY ACTIVITY, AND A HIGHER LEVEL OF DNMT3A THAN CONTROL ASCS. WHEN DNMT3A WAS DOWNREGULATED BY SIRNA AND SHRNA, OSTEOGENIC-RELATED FACTORS RUNT-RELATED TRANSCRIPTION FACTOR 2 AND OSTEOPONTIN, AND ACTIVITY OF WNT/BETA-CATENIN SIGNALING PATHWAY WERE INCREASED, WHICH RESCUED THE POOR OSTEOGENIC POTENTIAL OF DOP-ASCS. WHEN DNMT3A WAS UPREGULATED BY LVRNA-DNMT3A, THE OSTEOGENIC ABILITY WAS INHIBITED. THE SAME RESULTS WERE OBTAINED IN VIVO. CONCLUSIONS: DNMT3A SILENCING RESCUES THE NEGATIVE EFFECTS OF DOP ON ASCS AND PROVIDES A POSSIBLE APPROACH FOR BONE TISSUE REGENERATION IN PATIENTS WITH DIABETIC OSTEOPOROSIS. 2022 3 5716 44 SIRT6 PROTECTS VASCULAR SMOOTH MUSCLE CELLS FROM OSTEOGENIC TRANSDIFFERENTIATION VIA RUNX2 IN CHRONIC KIDNEY DISEASE. VASCULAR CALCIFICATION (VC) IS REGARDED AS AN IMPORTANT PATHOLOGICAL CHANGE LACKING EFFECTIVE TREATMENT AND ASSOCIATED WITH HIGH MORTALITY. SIRTUIN 6 (SIRT6) IS A MEMBER OF THE SIRTUIN FAMILY, A CLASS III HISTONE DEACETYLASE AND A KEY EPIGENETIC REGULATOR. SIRT6 HAS A PROTECTIVE ROLE IN PATIENTS WITH CHRONIC KIDNEY DISEASE (CKD). HOWEVER, THE EXACT ROLE AND MOLECULAR MECHANISM OF SIRT6 IN VC IN PATIENTS WITH CKD REMAIN UNCLEAR. HERE, WE DEMONSTRATED THAT SIRT6 WAS MARKEDLY DOWNREGULATED IN PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS) AND IN THE RADIAL ARTERY TISSUE OF PATIENTS WITH CKD WITH VC. SIRT6-TRANSGENIC (SIRT6-TG) MICE SHOWED ALLEVIATED VC, WHILE VASCULAR SMOOTH MUSCLE CELL-SPECIFIC (VSMC-SPECIFIC) SIRT6 KNOCKED-DOWN MICE SHOWED SEVERE VC IN CKD. SIRT6 SUPPRESSED THE OSTEOGENIC TRANSDIFFERENTIATION OF VSMCS VIA REGULATION OF RUNT-RELATED TRANSCRIPTION FACTOR 2 (RUNX2). COIMMUNOPRECIPITATION (CO-IP) AND IMMUNOPRECIPITATION (IP) ASSAYS CONFIRMED THAT SIRT6 BOUND TO RUNX2. MOREOVER, RUNX2 WAS DEACETYLATED BY SIRT6 AND FURTHER PROMOTED NUCLEAR EXPORT VIA EXPORTIN 1 (XPO1), WHICH IN TURN CAUSED DEGRADATION OF RUNX2 THROUGH THE UBIQUITIN-PROTEASOME SYSTEM. THESE RESULTS DEMONSTRATED THAT SIRT6 PREVENTED VC BY SUPPRESSING THE OSTEOGENIC TRANSDIFFERENTIATION OF VSMCS, AND AS SUCH TARGETING SIRT6 MAY BE AN APPEALING THERAPEUTIC TARGET FOR VC IN CKD. 2022 4 2326 45 EPIGENETIC REGULATION OF HOTAIR IN ADVANCED CHRONIC MYELOID LEUKEMIA. PURPOSE: CHRONIC MYELOID LEUKEMIA (CML) ACCOUNTS FOR ~10% OF LEUKEMIA CASES, AND ITS PROGRESSION INVOLVES EPIGENETIC GENE REGULATION. THIS STUDY INVESTIGATED EPIGENETIC REGULATION OF HOTAIR AND ITS TARGET MICRORNA, MIR-143, IN ADVANCED CML. PATIENTS AND METHODS: WE FIRST ISOLATED BONE MARROW MONONUCLEAR CELLS FROM 70 PATIENTS WITH DIFFERENT PHASES OF CML AND FROM HEALTHY DONORS AS NORMAL CONTROL; WE ALSO CULTURED K562 AND KCL22 CELLS, TREATED WITH DEMETHYLATION DRUG; MTT ASSAY, FLOW CYTOMETRY, QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QPCR), METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (MSP), WESTERN BLOT, LUCIFERASE ASSAY, RNA PULL-DOWN ASSAY AND RNA-BINDING PROTEIN IMMUNOPRECIPITATION (RIP) ASSAY WERE PERFORMED. RESULT: AS MEASURED BY QPCR, HOTAIR EXPRESSION IN K562 CELLS, KCL22 CELLS, AND SAMPLES FROM CASES OF ADVANCED-STAGE CML INCREASED WITH LEVELS OF SEVERAL DNA METHYLTRANSFERASES AND HISTONE DEACETYLATES, INCLUDING DNMT1, DNMT3A, HDAC1, EZH2, AND LSD1, AND MIR-143 LEVELS WERE DECREASED AND HOTAIR LEVELS WERE INCREASED. TREATMENT WITH 5-AZACYTIDINE, A DNA METHYLATION INHIBITOR, DECREASED DNMT1, DNMT3A, HDAC1, EZH2, LSD1 MRNA, PROTEIN LEVELS, AND HOTAIR MRNA LEVELS BUT INCREASED MIR-143 LEVELS. HOTAIR KNOCKDOWN AND MIR-143 OVEREXPRESSION BOTH INHIBITED PROLIFERATION AND PROMOTED APOPTOSIS IN KCL22 AND K562 CELLS THROUGH THE PI3K/AKT PATHWAY. RNA PULL-DOWN, MASS SPECTROMETRY, AND RIP ASSAYS SHOWED THAT HOTAIR INTERACTED WITH EZH2 AND LSD1. A DUAL-LUCIFERASE ASSAY DEMONSTRATED THAT HOTAIR INTERACTED WITH MIR-143. CONCLUSION: OUR FINDINGS DEMONSTRATE THE KEY EPIGENETIC INTERACTIONS OF HOTAIR RELATED TO CML PROGRESSION AND SUGGEST HOTAIR AS A POTENTIAL THERAPEUTIC TARGET FOR ADVANCED CML. FURTHERMORE, OUR RESULTS SUPPORT THE USE OF DEMETHYLATION DRUGS AS A CML TREATMENT STRATEGY. 2018 5 4076 31 MATERNAL HIGH-FAT DIET MODIFIES EPIGENETIC MARKS H3K27ME3 AND H3K27AC IN BONE TO REGULATE OFFSPRING OSTEOBLASTOGENESIS IN MICE. STUDIES FROM BOTH HUMANS AND ANIMAL MODELS INDICATED THAT MATERNAL CHRONIC POOR-QUALITY DIET, ESPECIALLY A HIGH FAT DIET (HFD), IS SIGNIFICANTLY ASSOCIATED WITH REDUCED BONE DENSITY AND CHILDHOOD FRACTURES IN OFFSPRING. WHEN PREVIOUSLY STUDIED IN A RAT MODEL, OUR DATA SUGGESTED THAT MATERNAL HFD CHANGES EPIGENETIC MARKS SUCH AS DNA METHYLATION AND HISTONE MODIFICATIONS TO CONTROL OSTEOBLAST METABOLISM. IN MOUSE EMBRYONIC AND POSTNATAL OFFSPRING BONE SAMPLES, A CHIP-SEQUENCING (CHIP-SEQ)-BASED GENOME-WIDE METHOD WAS USED TO LOCATE THE REPRESSIVE HISTONE MARK H3K27ME3 (MEDIATED VIA THE POLYCOMB HISTONE METHYLTRANSFERASE, EZH2) AND EXPRESSIVE HISTONE MARK H3K27AC (P300/CBP MEDIATED) THROUGHOUT THE GENOME. USING ISOLATED MOUSE EMBRYONIC CELLS FROM FOETAL CALVARIA (OSTEOBLAST-LIKE CELLS), H3K27ME3 CHIP-SEQ SHOWED THAT 147 GENE BODIES AND 26 GENE PROMOTERS IN HFD EMBRYOTIC SAMPLES HAD A GREATER THAN TWOFOLD INCREASE IN H3K27ME PEAKS COMPARED TO CONTROLS. AMONG THE HFD SAMPLES, PTHLH AND COL2A1 THAT ARE IMPORTANT GENES PLAYING ROLES DURING CHONDRO- AND OSTEOGENESIS HAD SIGNIFICANTLY ENRICHED LEVELS OF H3K27ME3. THEIR DECREASED MRNA EXPRESSION WAS CONFIRMED BY REAL-TIME PCR AND STANDARD CHIP ANALYSIS, INDICATING A STRONG ASSOCIATION WITH EZH2 MEDIATED H3K27ME3 EPIGENETIC CHANGES. USING EMBRYONIC CALVARIA OSTEOBLASTIC CELLS AND OFFSPRING BONE SAMPLES, H3K27AC CHIP-SEQ ANALYSIS SHOWED THAT OSTEOBLAST INHIBITOR GENES TNFAIP3 AND TWIST1 HAD SIGNIFICANTLY ENRICHED PEAKS OF H3K27AC IN HFD SAMPLES COMPARED TO CONTROLS. THEIR INCREASED GENE EXPRESSION AND ASSOCIATION WITH H3K27AC WERE ALSO CONFIRMED BY REAL-TIME PCR AND STANDARD CHIP ANALYSIS. THESE FINDINGS INDICATE THAT CHRONIC MATERNAL HFD CHANGES HISTONE TRIMETHYLATION AND ACETYLATION EPIGENETIC MARKS TO REGULATE EXPRESSION OF GENES CONTROLLING OSTEOBLASTOGENESIS. 2022 6 2361 22 EPIGENETIC REGULATION OF SKELETAL TISSUE INTEGRITY AND OSTEOPOROSIS DEVELOPMENT. BONE TURNOVER IS SOPHISTICATEDLY BALANCED BY A DYNAMIC COUPLING OF BONE FORMATION AND RESORPTION AT VARIOUS RATES. THE ORCHESTRATION OF THIS CONTINUOUS REMODELING OF THE SKELETON FURTHER AFFECTS OTHER SKELETAL TISSUES THROUGH ORGAN CROSSTALK. CHRONIC EXCESSIVE BONE RESORPTION COMPROMISES BONE MASS AND ITS POROUS MICROSTRUCTURE AS WELL AS PROPER BIOMECHANICS. THIS ACCELERATES THE DEVELOPMENT OF OSTEOPOROTIC DISORDERS, A LEADING CAUSE OF SKELETAL DEGENERATION-ASSOCIATED DISABILITY AND PREMATURE DEATH. BONE-FORMING CELLS PLAY IMPORTANT ROLES IN MAINTAINING BONE DEPOSIT AND OSTEOCLASTIC RESORPTION. A POOR ORGANELLE MACHINERY, SUCH AS MITOCHONDRIAL DYSFUNCTION, ENDOPLASMIC RETICULUM STRESS, AND DEFECTIVE AUTOPHAGY, ETC., DYSREGULATES GROWTH FACTOR SECRETION, MINERALIZATION MATRIX PRODUCTION, OR OSTEOCLAST-REGULATORY CAPACITY IN OSTEOBLASTIC CELLS. A PLETHORA OF EPIGENETIC PATHWAYS REGULATE BONE FORMATION, SKELETAL INTEGRITY, AND THE DEVELOPMENT OF OSTEOPOROSIS. MICRORNAS INHIBIT PROTEIN TRANSLATION BY BINDING THE 3'-UNTRANSLATED REGION OF MRNAS OR PROMOTE TRANSLATION THROUGH POST-TRANSCRIPTIONAL PATHWAYS. DNA METHYLATION AND POST-TRANSLATIONAL MODIFICATION OF HISTONES ALTER THE CHROMATIN STRUCTURE, HINDERING HISTONE ENRICHMENT IN PROMOTER REGIONS. MICRORNA-PROCESSING ENZYMES AND DNA AS WELL AS HISTONE MODIFICATION ENZYMES CATALYZE THESE MODIFYING REACTIONS. GAIN AND LOSS OF THESE EPIGENETIC MODIFIERS IN BONE-FORMING CELLS AFFECT THEIR EPIGENETIC LANDSCAPES, INFLUENCING BONE HOMEOSTASIS, MICROARCHITECTURAL INTEGRITY, AND OSTEOPOROTIC CHANGES. THIS ARTICLE CONVEYS PRODUCTIVE INSIGHTS INTO BIOLOGICAL ROLES OF DNA METHYLATION, MICRORNA, AND HISTONE MODIFICATION AND HIGHLIGHTS THEIR INTERACTIONS DURING SKELETAL DEVELOPMENT AND BONE LOSS UNDER PHYSIOLOGICAL AND PATHOLOGICAL CONDITIONS. 2020 7 5868 36 SUPPRESSIVE EFFECTS OF METFORMIN ON T-HELPER 1-RELATED CHEMOKINES EXPRESSION IN THE HUMAN MONOCYTIC LEUKEMIA CELL LINE THP-1. PURPOSE OF THE STUDY: TYPE 1 AND TYPE 2 DIABETES MELLITUS (DM) ARE CHRONIC T-CELL-MEDIATED INFLAMMATORY DISEASES. METFORMIN IS A WIDELY USED DRUG FOR TYPE 2 DM THAT REDUCES THE NEED FOR INSULIN IN TYPE 1 DM. HOWEVER, WHETHER METFORMIN HAS AN ANTI-INFLAMMATORY EFFECT FOR TREATING DM IS UNKNOWN. WE INVESTIGATED THE ANTI-INFLAMMATORY MECHANISM OF METFORMIN IN THE HUMAN MONOCYTIC LEUKEMIA CELL LINE THP-1. MATERIALS AND METHODS: THE HUMAN MONOCYTIC LEUKEMIA CELL LINE THP-1 WAS PRETREATED WITH METFORMIN AND STIMULATED WITH LIPOPOLYSACCHARIDE (LPS). THE PRODUCTION OF T-HELPER (TH)-1-RELATED CHEMOKINES INCLUDING INTERFERON-GAMMA-INDUCED PROTEIN-10 (IP-10) AND MONOCYTE CHEMOATTRACTANT PROTEIN-1 (MCP-1), TH2-RELATED CHEMOKINE MACROPHAGE-DERIVED CHEMOKINE, AND THE PROINFLAMMATORY CHEMOKINE TUMOR NECROSIS FACTOR-ALPHA WAS MEASURED USING ENZYME-LINKED IMMUNOSORBENT ASSAY. INTRACELLULAR SIGNALING PATHWAYS WERE INVESTIGATED USING WESTERN BLOT ANALYSIS AND CHROMATIN IMMUNOPRECIPITATION ASSAY. RESULTS: METFORMIN SUPPRESSED LPS-INDUCED IP-10 AND MCP-1 PRODUCTION AS WELL AS LPS-INDUCED PHOSPHORYLATION OF C-JUN N-TERMINAL KINASE (JNK), P38, EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK), AND NUCLEAR FACTOR-KAPPA B (NF-KAPPAB). MOREOVER, METFORMIN SUPPRESSED LPS-INDUCED ACETYLATION OF HISTONES H3 AND H4 AT THE IP-10 PROMOTER. CONCLUSIONS: METFORMIN SUPPRESSED THE PRODUCTION OF TH1-RELATED CHEMOKINES IP-10 AND MCP-1 IN THP-1 CELLS. SUPPRESSIVE EFFECTS OF METFORMIN ON IP-10 PRODUCTION MIGHT BE ATTRIBUTED AT LEAST PARTIALLY TO THE JNK, P38, ERK, AND NF-KAPPAB PATHWAYS AS WELL AS TO EPIGENETIC REGULATION THROUGH THE ACETYLATION OF HISTONES H3 AND H4. THESE RESULTS INDICATED THE THERAPEUTIC ANTI-INFLAMMATORY POTENTIAL OF METFORMIN. 2018 8 2784 36 EZH2 PROMOTES EXTRACELLULAR MATRIX DEGRADATION VIA NUCLEAR FACTOR-KAPPAB (NF-KAPPAB) AND P38 SIGNALING PATHWAYS IN PULPITIS. PULPITIS IS A COMPLICATED CHRONIC INFLAMMATORY PROCESS WHICH CAN BE IN A DYNAMIC BALANCE BETWEEN DAMAGE AND REPAIR. THE EXTRACELLULAR MATRIX PLAYS AN IMPORTANT REGULATORY ROLE IN WOUND HEALING AND TISSUE REPAIR. THE AIM OF THIS STUDY WAS TO EXPLORE THE ROLE OF THE EPIGENETIC MARK, ENHANCER OF ZESTE HOMOLOG 2 (EZH2) ON THE DEGRADATION OF EXTRACELLULAR MATRIX DURING PULPITIS. QUANTITATIVE POLYMERASE CHAIN REACTION WAS USED TO ASSESS THE EXPRESSION OF MATRIX METALLOPROTEINASES (MMPS) AND TYPE I COLLAGEN IN HUMAN DENTAL PULP CELLS (HDPCS) UPON EZH2 AND EI1 (EZH2 INHIBITOR) STIMULATION. THE MECHANISM OF EZH2 AFFECTING EXTRACELLULAR MATRIX WAS EXPLORED THROUGH QUANTITATIVE POLYMERASE CHAIN REACTION AND WESTERN BLOT. A RAT MODEL OF DENTAL PULP INFLAMMATION WAS ESTABLISHED, AND THE EXPRESSION OF TYPE I COLLAGEN IN DENTAL PULP UNDER EZH2 STIMULATION WAS DETECTED BY IMMUNOHISTOCHEMICAL STAINING. EZH2 UPREGULATED THE EXPRESSION OF MMP-1, MMP-3, MMP-8, AND MMP-10 AND DECREASED THE PRODUCTION OF TYPE I COLLAGEN IN HDPCS, WHILE EI1 HAD THE OPPOSITE EFFECT. EZH2 ACTIVATED THE NUCLEAR FACTOR-KAPPA B (NF-KAPPAB) AND P38 SIGNALING PATHWAYS IN HDPCS, THE INHIBITION OF WHICH REVERSED THE INDUCTION OF MMPS AND THE SUPPRESSION OF TYPE I COLLAGEN. EZH2 CAN DOWNREGULATE THE TYPE I COLLAGEN LEVELS IN AN EXPERIMENTAL MODEL OF DENTAL PULPITIS IN RATS. EZH2 PROMOTES EXTRACELLULAR MATRIX DEGRADATION VIA NUCLEAR FACTOR-KAPPAB (NF-KAPPAB) AND P38 SIGNALING PATHWAYS IN PULPITIS. EZH2 CAN DECREASE THE TYPE I COLLAGEN LEVELS IN VIVO AND IN VITRO. 2021 9 1906 41 ENHANCER OF ZESTE HOMOLOG 2-CATALYSED H3K27 TRIMETHYLATION PLAYS A KEY ROLE IN ACUTE-ON-CHRONIC LIVER FAILURE VIA TNF-MEDIATED PATHWAY. ACUTE-ON-CHRONIC LIVER FAILURE IS MAINLY DUE TO HOST IMMUNITY SELF-DESTRUCTION. THE HISTONE H3 LYSINE 27 (H3K27) TRIMETHYLATING ENZYME, ENHANCER OF ZESTE HOMOLOG 2 (EZH2) MEDIATES EPIGENETIC SILENCING OF GENE EXPRESSION AND REGULATES IMMUNITY, ALSO INVOLVES PATHOGENESIS OF SEVERAL LIVER DISEASES. THE CURRENT STUDY WAS TO DETERMINE THE ROLE OF METHYLTRANSFERASE EZH2 AND ITS CATALYSED H3K27 TRIMETHYLATION (H3K27ME3) IN LIVER FAILURE, AND TO FURTHER INVESTIGATE THE POTENTIAL TARGET FOR LIVER FAILURE TREATMENT. EZH2 AND ITS CATALYSED H3K27ME3 WERE DETERMINED IN PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) FROM LIVER FAILURE PATIENTS AND KUPFFER CELLS FROM EXPERIMENTAL MICE. FURTHERMORE, GSK126 (AN INHIBITOR FOR EZH2 TRIMETHYLATION FUNCTION) WAS APPLIED IN LIVER FAILURE MICE IN VIVO, AND LIPOPOLYSACCHARIDE-STIMULATED MONONUCLEAR CELLS IN VITRO. EZH2 AND H3K27ME3 WERE SIGNIFICANTLY UPREGULATED IN HUMAN PBMC FROM LIVER FAILURE PATIENTS OR MURINE KUPFFER CELLS FROM THE LIVER FAILURE ANIMALS, RESPECTIVELY. GSK126 AMELIORATED DISEASE SEVERITY IN LIVER FAILURE MICE, WHICH MAYBE ATTRIBUTE TO DOWN-REGULATE CIRCULATING AND HEPATIC PROINFLAMMATORY CYTOKINES, ESPECIALLY TNF VIA REDUCING H3K27ME3. IN-DEPTH CHROMATIN IMMUNOPRECIPITATION ANALYSIS UNRAVELLED THAT DECREASED ENRICHMENT OF H3K27ME3 ON TNF PROMOTOR, RESULTING IN TNF ELEVATION IN KUPFFER CELLS FROM LIVER FAILURE MICE. NUCLEAR FACTOR KAPPA B (NF-KAPPAB) AND PROTEIN KINASE B (AKT) SIGNALLING PATHWAYS WERE ACTIVATED UPON LIPOPOLYSACCHARIDE STIMULATION, BUT ATTENUATED BY USING GSK126, ACCOMPANIED WITH DECREASED TNF IN VITRO. IN CONCLUSION, EZH2 AND H3K27ME3 CONTRIBUTED TO THE PATHOGENESIS OF LIVER FAILURE VIA TRIGGERING TNF AND OTHER INDISPENSABLE PROINFLAMMATORY CYTOKINES. EZH2 WAS TO MODIFY H3K27ME3 ENRICHMENT, AS WELL AS, ACTIVATION OF THE DOWNSTREAM NF-KAPPAB AND AKT SIGNALLING PATHWAYS. 2018 10 5555 27 ROLE OF FLUORIDE INDUCED EPIGENETIC ALTERATIONS IN THE DEVELOPMENT OF SKELETAL FLUOROSIS. FLUORIDE IS AN ESSENTIAL TRACE ELEMENT REQUIRED FOR PROPER BONE AND TOOTH DEVELOPMENT. SYSTEMIC HIGH EXPOSURE TO FLUORIDE THROUGH ENVIRONMENTAL EXPOSURE (DRINKING WATER AND FOOD) MAY RESULT IN TOXICITY CAUSING A DISORDER CALLED FLUOROSIS. IN THE PRESENT STUDY, WE INVESTIGATED THE ALTERATION IN DNA METHYLATION PROFILE WITH CHRONIC EXPOSURE (30 DAYS) TO FLUORIDE (8 MG/L) AND ITS RELEVANCE IN THE DEVELOPMENT OF FLUOROSIS. WHOLE GENOME BISULFITE SEQUENCING (WGBS) WAS CARRIED OUT IN HUMAN OSTEOSARCOMA CELLS (HOS) EXPOSED TO FLUORIDE. WHOLE GENOME BISULFITE SEQUENCING (WGBS) AND FUNCTIONAL ANNOTATION OF DIFFERENTIALLY METHYLATED GENES INDICATE ALTERATIONS IN METHYLATION STATUS OF GENES INVOLVED IN BIOLOGICAL PROCESSES ASSOCIATED WITH BONE DEVELOPMENT PATHWAYS. COMBINED ANALYSIS OF PROMOTER DNA HYPER METHYLATION, STRING: FUNCTIONAL PROTEIN ASSOCIATION NETWORKS AND GENE EXPRESSION ANALYSIS REVEALED EPIGENETIC ALTERATIONS IN BMP1, METAP2, MMP11 AND BACH1 GENES, WHICH PLAYS A ROLE IN THE EXTRACELLULAR MATRIX DISASSEMBLY, COLLAGEN CATABOLIC/ORGANIZATION PROCESS, SKELETAL MORPHOGENESIS/DEVELOPMENT, OSSIFICATION AND OSTEOBLAST DEVELOPMENT. THE PRESENT STUDY SHOWS THAT FLUORIDE CAUSES PROMOTER DNA HYPERMETHYLATION IN BMP1, METAP2, MMP11 AND BACH1 GENES WITH SUBSEQUENT DOWN-REGULATION IN THEIR EXPRESSION LEVEL (RNA LEVEL). THE RESULTS IMPLIES THAT FLUORIDE INDUCED DNA HYPERMETHYLATION OF THESE GENES MAY HAMPER EXTRACELLULAR MATRIX DEPOSITION, CARTILAGE FORMATION, ANGIOGENESIS, VASCULAR SYSTEM DEVELOPMENT AND POROSITY OF BONE, THUS PROMOTE SKELETAL FLUOROSIS. 2019 11 908 30 CHRONIC EXPOSURE TO ENVIRONMENTALLY RELEVANT CONCENTRATION OF FLUORIDE IMPAIRS OSTEOBLAST'S COLLAGEN SYNTHESIS AND MATRIX MINERALIZATION: INVOLVEMENT OF EPIGENETIC REGULATION IN SKELETAL FLUOROSIS. GLOBALLY, 200 MILLION PEOPLE ARE SUFFERING FROM TOXIC MANIFESTATIONS OF FLUORIDE(F), DENTAL AND SKELETAL FLUOROSIS; UNFORTUNATELY, THERE IS NO TREATMENT. TO UNRAVEL THE PATHOGENESIS OF SKELETAL FLUOROSIS, WE ESTABLISHED FLUOROSIS MICE BY TREATING ENVIRONMENTALLY RELEVANT CONCENTRATION OF F (15 PPM NAF) THROUGH DRINKING WATER FOR 4 MONTHS. AS IN SKELETAL FLUOROSIS, LOCOMOTOR DISABILITY, CRIPPLING DEFORMITIES OCCUR AND THUS, OUR HYPOTHESIS WAS F MIGHT ADVERSELY AFFECTS COLLAGEN WHICH GIVES THE BONE TENSILE STRENGTH. THIS WORK INEVITABLY HAD TO BE CARRIED OUT ON OSTEOBLAST CELLS, RESPONSIBLE FOR SYNTHESIS, DEPOSITION, AND MINERALIZATION OF BONE MATRIX. ISOLATED OSTEOBLAST CELLS WERE CONFIRMED BY ALP ACTIVITY AND MINERALIZED NODULES FORMATION. EXPRESSION OF COLLAGEN COL1A1, COL1A2, COL1A1 WAS SIGNIFICANTLY REDUCED IN TREATED MICE. FURTHER, A STUDY REVEALED THE INVOLVEMENT OF EPIGENETIC REGULATION BY PROMOTER HYPERMETHYLATION OF COL1A1; EXPRESSIONAL ALTERATIONS OF TRANSCRIPTION FACTORS, CALCIUM CHANNELS AND OTHER GENES E.G., CBFA-1, TGF-BETA1, BMP1, SP1, SP7, NF-(K)B P65, BMP-2, BGLAP, GPRC6A AND CAV(1.2) ARE ASSOCIATED WITH IMPAIRMENT OF COLLAGEN SYNTHESIS, DEPOSITION AND DECREASED MINERALIZATION THUS, ENFEEBLING BONE HEALTH. THIS STUDY INDICATES THE POSSIBLE ASSOCIATION OF EPIGENETIC REGULATION IN SKELETAL FLUOROSIS. HOWEVER, NO ASSOCIATION WAS FOUND BETWEEN POLYMORPHISMS IN THE COL1A1 (RSAI, HINDIII) AND COL1A2 (RSAI, HINDIII) GENES WITH FLUOROSIS IN MICE. 2023 12 6658 43 UPREGULATED LNCRNA H19 SPONGES MIR-106A-5P AND CONTRIBUTES TO ALDOSTERONE-INDUCED VASCULAR CALCIFICATION VIA ACTIVATING THE RUNX2-DEPENDENT PATHWAY. BACKGROUND: EXCESS ALDOSTERONE IS IMPLICATED IN VASCULAR CALCIFICATION (VC), BUT THE MECHANISM BY WHICH ALDOSTERONE-MR (MINERALOCORTICOID RECEPTOR) COMPLEX PROMOTES VC IS UNCLEAR. EMERGING EVIDENCE INDICATES THAT LONG-NONCODING RNA H19 (H19) PLAYS A CRITICAL ROLE IN VC. WE EXAMINED WHETHER ALDOSTERONE-INDUCED OSTEOGENIC DIFFERENTIATION OF VASCULAR SMOOTH MUSCLE CELLS (VSMCS) THROUGH H19 EPIGENETIC MODIFICATION OF RUNX2 (RUNT-RELATED TRANSCRIPTION FACTOR-2) IN A MR-DEPENDENT MANNER. METHODS: WE INDUCED IN VIVO RAT MODEL OF CHRONIC KIDNEY DISEASE USING A HIGH ADENINE AND PHOSPHATE DIET TO EXPLORE THE RELATIONSHIP AMONG ALDOSTERONE, MR, H19, AND VC. WE ALSO CULTURED HUMAN AORTIC VSMCS TO EXPLORE THE ROLES OF H19 IN ALDOSTERONE-MR COMPLEX-INDUCED OSTEOGENIC DIFFERENTIATION AND CALCIFICATION OF VSMCS. RESULTS: H19 AND RUNX2 WERE SIGNIFICANTLY INCREASED IN ALDOSTERONE-INDUCED VSMC OSTEOGENIC DIFFERENTIATION AND VC, BOTH IN VITRO AND IN VIVO, WHICH WERE SIGNIFICANTLY BLOCKED BY THE MR ANTAGONIST SPIRONOLACTONE. MECHANISTICALLY, OUR FINDINGS REVEAL THAT THE ALDOSTERONE-ACTIVATED MR BOUND TO H19 PROMOTER AND INCREASED ITS TRANSCRIPTIONAL ACTIVITY, AS DETERMINED BY CHROMATIN IMMUNOPRECIPITATION, ELECTROPHORETIC MOBILITY SHIFT ASSAY, AND LUCIFERASE REPORTER ASSAY. SILENCING H19 INCREASED MICRORNA-106A-5P (MIR-106A-5P) EXPRESSION, WHICH SUBSEQUENTLY INHIBITED ALDOSTERONE-INDUCED RUNX2 EXPRESSION AT THE POSTTRANSCRIPTIONAL LEVEL. IMPORTANTLY, WE OBSERVED A DIRECT INTERACTION BETWEEN H19 AND MIR-106A-5P, AND DOWNREGULATION OF MIR-106A-5P EFFICIENTLY REVERSED THE SUPPRESSION OF RUNX2 INDUCED BY H19 SILENCING. CONCLUSIONS: OUR STUDY CLARIFIES A NOVEL MECHANISM BY WHICH UPREGULATION OF H19 CONTRIBUTES TO ALDOSTERONE-MR COMPLEX-PROMOTED RUNX2-DEPENDENT VSMC OSTEOGENIC DIFFERENTIATION AND VC THROUGH SPONGING MIR-106A-5P. THESE FINDINGS HIGHLIGHT A POTENTIAL THERAPEUTIC TARGET FOR ALDOSTERONE-INDUCED VC. 2023 13 5479 37 RESVERATROL ATTENUATES CIGARETTE SMOKE EXTRACT INDUCED CELLULAR SENESCENCE IN HUMAN AIRWAY EPITHELIAL CELLS BY REGULATING THE MIR-34A/SIRT1/NF-KAPPAB PATHWAY. CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) IS CHARACTERIZED BY ACCELERATED LUNG AGING. SMOKING IS THE CRITICAL RISK FACTOR FOR COPD. CELLULAR SENESCENCE OF AIRWAY EPITHELIAL CELLS IS THE CYTOLOGICAL BASIS OF ACCELERATED LUNG AGING IN COPD, AND THE REGULATION OF MICRORNAS (MIRNAS) IS THE CENTRAL EPIGENETIC MECHANISM OF CELLULAR SENESCENCE. RESVERATROL (RES) IS A POLYPHENOL WITH ANTI-AGING PROPERTIES. THIS STUDY INVESTIGATED WHETHER RES ATTENUATES CIGARETTE SMOKE EXTRACT (CSE)-INDUCED CELLULAR SENESCENCE IN HUMAN AIRWAY EPITHELIAL CELLS (BEAS-2B) THROUGH THE MIR-34A/SIRT1/NUCLEAR FACTOR-KAPPAB (NF-KAPPAB) PATHWAY. BEAS-2B CELLS WERE TREATED WITH RES, CSE AND TRANSFECTED WITH MIR-34A-5P MIMICS. CELLULAR SENESCENCE WAS EVALUATED BY SENESCENCE -RELATED BETA-GALACTOSIDASE (SA-BETA-GAL) STAINING AND EXPRESSION OF SENESCENCE-RELATED GENES (P16, P21, AND P53). THE EXPRESSIONS OF MIR-34A-5P, SIRT1, AND NF-KAPPAB P65 WERE EXAMINED USING QUANTITATIVE REAL TIME POLYMERASE CHAIN REACTION AND WESTERN BLOTTING. THE SENESCENCE-ASSOCIATED SECRETORY PHENOTYPE (SASP) CYTOKINES (IL-1BETA, IL-6, IL-8, TNF-ALPHA) WERE ASSESSED BY ENZYME-LINKED IMMUNOSORBENT ASSAY. THE BINDING BETWEEN MIR-34A-5P AND SIRT1 WAS CONFIRMED BY DUAL-LUCIFERASE REPORTER ASSAY. THE RESULTS SHOWED THAT CSE DOSE-DEPENDENTLY DECREASED CELL VIABILITY AND ELEVATED CELLULAR SENESCENCE, CHARACTERIZED BY INCREASED SA-BETA-GAL STAINING AND SENESCENCE-RELATED GENE EXPRESSIONS (P16, P21, AND P53). FURTHER, CSE DOSE-DEPENDENTLY INCREASED THE EXPRESSION OF MIR-34A-5P AND SASP CYTOKINES (IL-1BETA, IL-6, IL-8, TNF-ALPHA) IN BEAS-2B CELLS. PRETREATMENT WITH RES INHIBITED CSE-INDUCED CELLULAR SENESCENCE AND SECRETION OF SASP CYTOKINES (IL-1BETA, IL-6, IL-8, TNF-ALPHA) IN A DOSE-DEPENDENT MANNER. MOREOVER, RES REVERSED THE CSE-INDUCED DOWN-REGULATION OF SIRT1 AND UP-REGULATION OF MIR-34A-5P AND NF-KAPPAB P65. SIRT1 IS A TARGET OF MIR-34A-5P. OVEREXPRESSION OF MIR-34A-5P VIA TRANSFECTION WITH MIR-34A-5P MIMIC IN BEAS-2B CELLS ATTENUATED THE INHIBITORY EFFECT OF RES ON CELLULAR SENESCENCE, ACCOMPANIED BY REVERSING THE EXPRESSION OF SIRT1 AND NF-KAPPAB P65. IN CONCLUSION, RES ATTENUATED CSE-INDUCED CELLULAR SENESCENCE IN BEAS-2B CELLS BY REGULATING THE MIR-34A/SIRT1/NF-KAPPAB PATHWAY, WHICH MAY PROVIDE A NEW APPROACH FOR COPD TREATMENT. 2022 14 1826 46 EFFECTS OF HISTONE DEACETYLASE INHIBITOR ON EXTRACELLULAR MATRIX PRODUCTION IN HUMAN NASAL POLYP ORGAN CULTURES. BACKGROUND: NASAL POLYPOSIS IS ASSOCIATED WITH A CHRONIC INFLAMMATORY CONDITION OF THE SINONASAL MUCOSA AND INVOLVES MYOFIBROBLAST DIFFERENTIATION AND EXTRACELLULAR MATRIX (ECM) ACCUMULATION. EPIGENETIC MODULATION BY HISTONE DEACETYLASE (HDAC) INHIBITORS INCLUDING TRICHOSTATIN A (TSA) HAS BEEN REPORTED TO HAVE INHIBITORY EFFECTS ON MYOFIBROBLAST DIFFERENTIATION IN LUNG AND RENAL FIBROBLASTS. THE PURPOSE OF THIS STUDY WAS TO INVESTIGATE THE INHIBITORY EFFECT OF TSA ON MYOFIBROBLAST DIFFERENTIATION AND ECM PRODUCTION IN NASAL POLYP ORGAN CULTURES. METHODS: NASAL POLYP TISSUES FROM 18 PATIENTS WERE ACQUIRED DURING ENDOSCOPIC SINUS SURGERY. AFTER ORGAN CULTURE, NASAL POLYPS WERE STIMULATED WITH TGF-BETA1 AND THEN TREATED WITH TSA. ALPHA-SMOOTH MUSCLE ACTIN (ALPHA-SMA), FIBRONECTIN, AND COLLAGEN TYPE I EXPRESSION LEVELS WERE EXAMINED BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION (PCR), REAL-TIME PCR, WESTERN BLOT, AND IMMUNOFLUORESCENT STAINING. HDAC2, HDAC4, AND ACETYLATED H4 EXPRESSION LEVELS WERE ASSAYED BY WESTERN BLOT. CYTOTOXICITY WAS ANALYZED BY THE TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE BIOTIN-DUTP NICK END LABELING ASSAY. RESULTS: THE EXPRESSION LEVELS OF ALPHA-SMA, FIBRONECTIN, AND COLLAGEN TYPE 1 WERE INCREASED IN NASAL POLYP AFTER TRANSFORMING GROWTH FACTOR (TGF) BETA1 TREATMENT. TSA-INHIBITED TGF-BETA1 INDUCED THESE GENE AND PROTEIN EXPRESSION LEVELS. FURTHERMORE, TSA SUPPRESSED PROTEIN EXPRESSION LEVELS OF HDAC2 AND HDAC4. HOWEVER, TSA INDUCED HYPERACETYLATION OF HISTONES H4. TREATMENT WITH TGF-BETA1 WITH OR WITHOUT TSA DID NOT HAVE CYTOTOXIC EFFECT. CONCLUSION: THESE FINDINGS PROVIDE NOVEL INSIGHTS INTO THE EPIGENETIC REGULATION IN MYOFIBROBLAST DIFFERENTIATION AND ECM PRODUCTION OF NASAL POLYP. TSA COULD BE A CANDIDATE OF A THERAPEUTIC AGENT FOR REVERSING THE TGF-BETA1-INDUCED ECM SYNTHESIS THAT LEADS TO NASAL POLYP DEVELOPMENT. 2013 15 4298 27 MICRORNA-146A GOVERNS FIBROBLAST ACTIVATION AND JOINT PATHOLOGY IN ARTHRITIS. SYNOVIAL FIBROBLASTS ARE KEY CELLS ORCHESTRATING THE INFLAMMATORY RESPONSE IN ARTHRITIS. HERE WE DEMONSTRATE THAT LOSS OF MIR-146A, A KEY EPIGENETIC REGULATOR OF THE INNATE IMMUNE RESPONSE, LEADS TO INCREASED JOINT DESTRUCTION IN A TNF-DRIVEN MODEL OF ARTHRITIS BY SPECIFICALLY REGULATING THE BEHAVIOR OF SYNOVIAL FIBROBLASTS. ABSENCE OF MIR-146A IN SYNOVIAL FIBROBLASTS DISPLAY A HIGHLY DEREGULATED GENE EXPRESSION PATTERN AND ENHANCED PROLIFERATION IN VITRO AND IN VIVO. DEFICIENCY OF MIR-146A INDUCES DEREGULATION OF TUMOR NECROSIS FACTOR (TNF) RECEPTOR ASSOCIATED FACTOR 6 (TRAF6) IN SYNOVIAL FIBROBLASTS, LEADING TO INCREASED PROLIFERATION. IN ADDITION, LOSS OF MIR-146A SHIFTS THE METABOLIC STATE OF FIBROBLASTS TOWARDS GLYCOLYSIS AND AUGMENTS THE ABILITY OF SYNOVIAL FIBROBLASTS TO SUPPORT THE GENERATION OF OSTEOCLASTS BY CONTROLLING THE BALANCE OF OSTEOCLASTOGENIC REGULATORY FACTORS RECEPTOR ACTIVATOR OF NF-KAPPAB LIGAND (RANKL) AND OSTEOPROTEGERIN (OPG). BONE MARROW TRANSPLANTATION EXPERIMENTS CONFIRMED THE IMPORTANCE OF MIR-146A IN THE RADIORESISTANT MESENCHYMAL COMPARTMENT FOR THE CONTROL OF ARTHRITIS SEVERITY, IN PARTICULAR FOR INFLAMMATORY JOINT DESTRUCTION. THIS STUDY THEREFORE IDENTIFIES MICRORNA-146A AS AN IMPORTANT LOCAL EPIGENETIC REGULATOR OF THE INFLAMMATORY RESPONSE IN ARTHRITIS. IT IS A CENTRAL ELEMENT OF AN ANTI-INFLAMMATORY FEEDBACK LOOP IN RESIDENT SYNOVIAL FIBROBLASTS, WHO ARE ORCHESTRATING THE INFLAMMATORY RESPONSE IN CHRONIC ARTHRITIS. MIR-146A RESTRICTS THEIR ACTIVATION, THEREBY PREVENTING EXCESSIVE TISSUE DAMAGE DURING ARTHRITIS. 2017 16 1667 38 DOWNREGULATION OF PCAF BY MIR-181A/B PROVIDES FEEDBACK REGULATION TO TNF-ALPHA-INDUCED TRANSCRIPTION OF PROINFLAMMATORY GENES IN LIVER EPITHELIAL CELLS. ABERRANT CELLULAR RESPONSES TO PROINFLAMMATORY CYTOKINES, SUCH AS TNF-ALPHA, ARE PATHOGENIC FEATURES IN MOST CHRONIC INFLAMMATORY DISEASES. A VARIETY OF EXTRACELLULAR AND INTRACELLULAR FEEDBACK PATHWAYS HAS EVOLVED TO PREVENT AN INAPPROPRIATE CELLULAR REACTION TO THESE PROINFLAMMATORY CYTOKINES. IN THIS STUDY, WE REPORT THAT TNF-ALPHA TREATMENT OF HUMAN AND MOUSE CHOLANGIOCYTES AND HEPATOCYTES DOWNREGULATED EXPRESSION OF P300/CBP-ASSOCIATED FACTOR (PCAF), A COACTIVATOR AND AN ACETYLTRANSFERASE THAT PROMOTES HISTONE ACETYLATION AND GENE TRANSCRIPTION. OF THESE UPREGULATED MICRORNAS IN TNF-ALPHA-TREATED CELLS, MIR-181A/B (MIR-181A AND MIR-181B) SUPPRESSED TRANSLATION OF PCAF MRNA. FUNCTIONAL MANIPULATION OF MIR-181A/B CAUSED RECIPROCAL ALTERATIONS IN PCAF PROTEIN EXPRESSION IN CULTURED CHOLANGIOCYTES AND HEPATOCYTES. INHIBITION OF MIR-181A/B FUNCTION WITH ANTI-MIRS BLOCKED TNF-ALPHA-INDUCED SUPPRESSION OF PCAF EXPRESSION. PROMOTER RECRUITMENT OF PCAF WAS SHOWN TO BE ASSOCIATED WITH TNF-ALPHA-INDUCED TRANSCRIPTION OF INFLAMMATORY GENES. INTRIGUINGLY, PRETREATMENT OF CELLS WITH TNF-ALPHA INHIBITED TRANSCRIPTION OF INFLAMMATORY GENES IN RESPONSE TO SUBSEQUENT TNF-ALPHA STIMULATION. OVEREXPRESSION OF PCAF OR INHIBITION OF MIR-181A/B FUNCTION WITH ANTI-MIRS ATTENUATED THE INHIBITORY EFFECTS OF TNF-ALPHA PRETREATMENT ON EPITHELIAL INFLAMMATORY RESPONSE TO SUBSEQUENT TNF-ALPHA STIMULATION. DOWNREGULATION OF PCAF AND THE INHIBITORY EFFECTS OF TNF-ALPHA PRETREATMENT ON LIVER EPITHELIAL INFLAMMATORY RESPONSE WERE FURTHER CONFIRMED IN A MOUSE MODEL OF TNF-ALPHA I.P. INJECTION. THESE DATA SUGGEST THAT PCAF IS A TARGET FOR MIR-181A/B, AND DOWNREGULATION OF PCAF BY TNF-ALPHA PROVIDES NEGATIVE FEEDBACK REGULATION TO INFLAMMATORY REACTIONS IN LIVER EPITHELIAL CELLS, A PROCESS THAT MAY BE RELEVANT TO THE EPIGENETIC FINE-TUNING OF EPITHELIAL INFLAMMATORY PROCESSES IN GENERAL. 2012 17 2748 40 EXPRESSION AND FUNCTION OF EZH2 IN SYNOVIAL FIBROBLASTS: EPIGENETIC REPRESSION OF THE WNT INHIBITOR SFRP1 IN RHEUMATOID ARTHRITIS. OBJECTIVES: TO STUDY THE EXPRESSION, REGULATION AND FUNCTION OF THE HISTONE METHYLTRANSFERASE ENHANCER OF ZESTE HOMOLOGUE 2 (EZH2) IN SYNOVIAL FIBROBLASTS (SF) FROM PATIENTS WITH RHEUMATOID ARTHRITIS (RA) AND OSTEOARTHRITIS (OA). METHODS: SF WERE OBTAINED FROM RA AND OA PATIENTS UNDERGOING JOINT SURGERY. EXPRESSION LEVELS WERE ASSESSED BY QUANTITATIVE REAL-TIME PCR AND WESTERN BLOT. KINASE INHIBITORS AND REPORTER GENE ASSAYS WERE EMPLOYED TO STUDY SIGNALLING PATHWAYS. FUNCTIONAL ANALYSES INCLUDED EZH2 OVEREXPRESSION BY PLASMID TRANSFECTION AND GENE SILENCING BY SMALL INTERFERING RNA. CHROMATIN IMMUNOPRECIPITATION ASSAY WAS USED TO ANALYSE HISTONE METHYLATION WITHIN DISTINCT PROMOTER REGIONS. RESULTS: BY STUDYING THE EXPRESSION AND FUNCTION OF EZH2 IN SF THE AUTHORS FOUND THAT EZH2 IS OVEREXPRESSED IN RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS (RASF) AND FURTHER INDUCED BY TUMOUR NECROSIS FACTOR ALPHA THROUGH THE NUCLEAR FACTOR KAPPA B AND JUN KINASE PATHWAYS. AS A TARGET GENE OF EZH2 THE AUTHORS IDENTIFIED SECRETED FRIZZLED-RELATED PROTEIN 1 (SFRP1), AN INHIBITOR OF WNT SIGNALLING, WHICH IS ASSOCIATED WITH THE ACTIVATION OF RASF, AND SHOW THAT SFRP1 EXPRESSION CORRELATES WITH THE OCCUPATION OF ITS PROMOTER WITH ACTIVATING AND SILENCING HISTONE MARKS. CONCLUSIONS: THESE DATA STRONGLY SUGGEST THAT THE CHRONIC INFLAMMATORY ENVIRONMENT OF THE RA JOINT INDUCES EZH2 AND THUS MIGHT CAUSE CHANGES IN THE EPIGENETIC PROGRAMMES OF SF. 2011 18 4640 37 NEURONAL NUCLEAR CALCIUM SIGNALING SUPPRESSION OF MICROGLIAL REACTIVITY IS MEDIATED BY OSTEOPROTEGERIN AFTER TRAUMATIC BRAIN INJURY. BACKGROUND: TRAUMATIC BRAIN INJURY (TBI) IS CHARACTERIZED BY MASSIVE CHANGES IN NEURONAL EXCITATION, FROM ACUTE EXCITOTOXICITY TO CHRONIC HYPER- OR HYPOEXCITABILITY. NUCLEAR CALCIUM SIGNALING PATHWAYS ARE INVOLVED IN TRANSLATING CHANGES IN SYNAPTIC INPUTS AND NEURONAL ACTIVITY INTO DISCRETE TRANSCRIPTIONAL PROGRAMS WHICH NOT ONLY AFFECT NEURONAL SURVIVAL AND SYNAPTIC INTEGRITY, BUT ALSO THE CROSSTALK BETWEEN NEURONS AND GLIAL CELLS. HERE, WE REPORT THE EFFECTS OF BLUNTING NEURONAL NUCLEAR CALCIUM SIGNALS IN THE CONTEXT OF TBI. METHODS: WE USED AAV VECTORS TO EXPRESS THE GENETICALLY ENCODED AND NUCLEAR-TARGETED CALCIUM BUFFER PARVALBUMIN (PV.NLS.MCHERRY) OR THE CALCIUM/CALMODULIN BUFFER CAMBP4.MCHERRY IN NEURONS ONLY. UPON TBI, THE EXTENT OF NEUROINFLAMMATION, NEURONAL DEATH AND SYNAPTIC LOSS WERE ASSESSED BY IMMUNOHISTOCHEMISTRY AND TARGETED TRANSCRIPTOME ANALYSIS. MODULATION OF THE OVERALL LEVEL OF NEURONAL ACTIVITY WAS ACHIEVED BY PSAM/PSEM CHEMOGENETICS TARGETED TO PARVALBUMIN INTERNEURONS. THE FUNCTIONAL IMPACT OF NEURONAL NUCLEAR CALCIUM BUFFERING IN TBI WAS ASSESSED BY QUANTIFICATION OF SPONTANEOUS WHISKING. RESULTS: BUFFERING NEURONAL NUCLEAR CALCIUM UNEXPECTEDLY RESULTED IN A MASSIVE AND LONG-LASTING INCREASE IN THE RECRUITMENT OF REACTIVE MICROGLIA TO THE INJURY SITE, WHICH WAS CHARACTERIZED BY A DISEASE-ASSOCIATED AND PHAGOCYTIC PHENOTYPE. THIS EFFECT WAS ACCOMPANIED BY A SUBSTANTIAL SURGE IN SYNAPTIC LOSS AND SIGNIFICANTLY REDUCED WHISKING ACTIVITY. TRANSCRIPTOME ANALYSIS REVEALED A COMPLEX EFFECT OF TBI IN THE CONTEXT OF NEURONAL NUCLEAR CALCIUM BUFFERING, WITH UPREGULATION OF COMPLEMENT FACTORS, CHEMOKINES AND INTERFERON-RESPONSE GENES, AS WELL AS THE DOWNREGULATION OF SYNAPTIC GENES AND EPIGENETIC REGULATORS COMPARED TO CONTROL CONDITIONS. NOTABLY, NUCLEAR CALCIUM BUFFERING LED TO A SUBSTANTIAL LOSS IN NEURONAL OSTEOPROTEGERIN (OPG), WHEREAS STIMULATION OF NEURONAL FIRING INDUCED OPG EXPRESSION. VIRAL RE-EXPRESSION OF OPG RESULTED IN DECREASED MICROGLIAL RECRUITMENT AND SYNAPTIC LOSS. OPG UPREGULATION WAS ALSO OBSERVED IN THE CSF OF HUMAN TBI PATIENTS, UNDERSCORING ITS TRANSLATIONAL VALUE. CONCLUSION: NEURONAL NUCLEAR CALCIUM SIGNALS REGULATE THE DEGREE OF MICROGLIAL RECRUITMENT AND REACTIVITY UPON TBI VIA, AMONG OTHERS, OSTEOPROTEGERIN SIGNALS. OUR FINDINGS SUPPORT A MODEL WHEREBY NEURONAL ACTIVITY ALTERED AFTER TBI EXERTS A POWERFUL IMPACT ON THE NEUROINFLAMMATORY CASCADE, WHICH IN TURN CONTRIBUTES TO THE OVERALL LOSS OF SYNAPSES AND FUNCTIONAL IMPAIRMENT. 2022 19 1632 38 DNMTS ARE INVOLVED IN TGF-BETA1-INDUCED EPITHELIAL-MESENCHYMAL TRANSITIONS IN AIRWAY EPITHELIAL CELLS. CHRONIC RHINOSINUSITIS (CRS) PATHOGENESIS IS CLOSELY RELATED TO TISSUE REMODELING, INCLUDING EPITHELIAL-MESENCHYMAL TRANSITION (EMT). EPIGENETIC MECHANISMS PLAY KEY ROLES IN EMT. DNA METHYLATION, MEDIATED BY DNA METHYLTRANSFERASES (DNMTS), IS AN EPIGENETIC MARKER THAT IS CRITICAL TO EMT. THE GOAL OF THIS STUDY WAS TO DETERMINE WHETHER DNMTS WERE INVOLVED IN TGF-BETA1-INDUCED EMT AND ELUCIDATE THE UNDERLYING MECHANISMS IN NASAL EPITHELIAL CELLS AND AIR-LIQUID INTERFACE CULTURES. GLOBAL DNA METHYLATION AND DNMT ACTIVITY WERE QUANTIFIED. DNMT EXPRESSION WAS MEASURED USING REAL-TIME PCR (QRT-PCR) IN HUMAN CRS TISSUES. MRNA AND PROTEIN LEVELS OF DNMTS, E-CADHERIN, VIMENTIN, ALPHA-SMA, AND FIBRONECTIN WERE DETERMINED USING RT-PCR AND WESTERN BLOTTING, RESPECTIVELY. DNMT1, DNMT3A, AND DNMT3B GENE EXPRESSION WERE KNOCKED DOWN USING SIRNA TRANSFECTION. MAPK PHOSPHORYLATION AND EMT-RELATED TRANSCRIPTION FACTOR LEVELS WERE DETERMINED USING WESTERN BLOTTING. SIGNALING PATHWAYS WERE ANALYZED USING SPECIFIC INHIBITORS OF MAPK. WE DEMONSTRATED THESE DATA IN PRIMARY NASAL EPITHELIAL CELLS AND AIR-LIQUID INTERFACE CULTURES. GLOBAL DNA METHYLATION, DNMT ACTIVITY, AND DNMT EXPRESSION INCREASED IN CRS TISSUES. DNMT EXPRESSION WAS POSITIVELY CORRELATED WITH LUND-MCKAY CT SCORES. TGF-BETA1 DOSE-DEPENDENTLY INDUCED DNMT EXPRESSION. FURTHER, 5-AZA INHIBITED TGF-BETA1-INDUCED DNMT, SNAIL, AND SLUG EXPRESSION RELATED TO EMT, AS WELL AS P38 AND JNK PHOSPHORYLATION IN A549 CELLS AND TGF-BETA1-INDUCED DNMT EXPRESSION AND EMT IN PRIMARY NASAL EPITHELIAL CELLS AND AIR-LIQUID INTERFACE CULTURES. TGF-BETA1-INDUCED DNMT EXPRESSION LEADS TO DNA METHYLATION AND EMT VIA P38, JNK, SNAIL, AND SLUG SIGNALING PATHWAYS. INHIBITION OF DNMT SUPPRESSED THE EMT PROCESS AND THEREFORE IS POTENTIALLY A CRS THERAPEUTIC STRATEGY. 2022 20 4303 48 MICRORNA-223 INHIBITS TISSUE FACTOR EXPRESSION IN VASCULAR ENDOTHELIAL CELLS. OBJECTIVE: ATHEROSCLEROSIS IS A CHRONIC INFLAMMATORY PROCESS, IN WHICH VASCULAR ENDOTHELIAL CELLS (ECS) BECOME DYSFUNCTIONAL OWING TO THE EFFECTS OF CHEMICAL SUBSTANCES, SUCH AS INFLAMMATORY FACTOR AND GROWTH FACTORS. TISSUE FACTOR (TF) EXPRESSION IS INDUCED BY THE ABOVE CHEMICAL SUBSTANCES IN ACTIVATED ECS. TF INITIATES THROMBOSIS ON DISRUPTED ATHEROSCLEROTIC PLAQUES WHICH PLAYS AN ESSENTIAL ROLE DURING THE ONSET OF ACUTE CORONARY SYNDROMES (ACS). INCREASING EVIDENCES SUGGEST THE IMPORTANT ROLE OF MICRORNAS AS EPIGENETIC REGULATORS OF ATHEROSCLEROTIC DISEASE. THE AIM OF OUR STUDY IS TO IDENTIFY IF MICRORNA-223 (MIR-223) TARGETS TF IN ECS. METHODS AND RESULTS: BIOINFORMATIC ANALYSIS SHOWED THAT TF IS A TARGET CANDIDATE OF MIR-223. WESTERN BLOTTING ANALYSIS REVEALED THAT TUMOR NECROSIS FACTOR ALPHA (TNF-ALPHA) INCREASED TF EXPRESSION IN AORTA OF C57BL/6J MICE AND CULTURED ECS (EA.HY926 CELLS AND HUVEC) AFTER 4 H TREATMENT. IN TNF-ALPHA TREATED ECS, TF MRNA WAS ALSO INCREASED MEASURED BY REAL-TIME PCR. REAL-TIME PCR RESULTS SHOWED THAT MIR-223 LEVELS WERE DOWNREGULATED IN TNF-ALPHA-TREATED AORTA OF C57BL/6J MICE AND CULTURED ECS. TRANSFECTION OF ECS WITH MIR-223 MIMIC OR MIR-223 INHIBITOR MODIFIED TF EXPRESSION BOTH IN MRNA AND PROTEIN LEVELS. LUCIFERASE ASSAYS CONFIRMED THAT MIR-223 SUPPRESSED TF EXPRESSION BY BINDING TO THE SEQUENCE OF TF 3'-UNTRANSLATED REGIONS (3'UTR). TF PROCOAGULANT ACTIVITY WAS INHIBITED BY OVEREXPRESSING MIR-223 WITH OR WITHOUT TNF-ALPHA STIMULATION. CONCLUSIONS: MIR-223-MEDIATED SUPPRESSION OF TF EXPRESSION PROVIDES A NOVEL MOLECULAR MECHANISM FOR THE REGULATION OF COAGULATION CASCADE, AND SUGGESTS A CLUE AGAINST THROMBOGENESIS DURING THE PROCESS OF ATHEROSCLEROTIC PLAQUE RUPTURE. 2014