1 4356 120 MIR-30A-5P PROMOTES GLOMERULAR PODOCYTE APOPTOSIS VIA DNMT1-MEDIATED HYPERMETHYLATION UNDER HYPERHOMOCYSTEINEMIA. ABNORMAL ELEVATION OF HOMOCYSTEINE (HCY) LEVEL IS CLOSELY RELATED TO THE DEVELOPMENT AND PROGRESSION OF CHRONIC KIDNEY DISEASE (CKD), WITH THE MOLECULAR MECHANISMS THAT ARE NOT FULLY ELUCIDATED. GIVEN THE DEMONSTRATION THAT MIR-30A-5P IS SPECIFICALLY EXPRESSED IN GLOMERULAR PODOCYTES, IN THE PRESENT STUDY WE AIMED TO INVESTIGATE THE ROLE AND POTENTIAL UNDERLYING MECHANISM OF MIR-30A-5P IN GLOMERULAR PODOCYTE APOPTOSIS INDUCED BY HCY. WE FOUND THAT ELEVATED HCY DOWNREGULATES MIR-30A-5P EXPRESSION IN THE MICE AND HCY-TREATED PODOCYTES, AND MIR-30A-5P DIRECTLY TARGETS THE 3'-UNTRANSLATED REGION (3'-UTR) OF THE FORKHEAD BOX A1 (FOXA1) AND OVEREXPRESSION OF MIR-30A-5P INHIBITS FOXA1 EXPRESSION. BY NMS-PCR AND MASSARRAY QUANTITATIVE METHYLATION ANALYSIS, WE SHOWED THE INCREASED DNA METHYLATION LEVEL OF MIR-30A-5P PROMOTER BOTH AND . MEANWHILE, DUAL-LUCIFERASE REPORTER ASSAY SHOWED THAT THE REGION BETWEEN --1400 AND --921 BP OF MIR-30A-5P PROMOTER IS A POSSIBLE REGULATORY ELEMENT FOR ITS TRANSCRIPTION. MECHANISTIC STUDIES INDICATED THAT DNA METHYLTRANSFERASE ENZYME 1 (DNMT1) IS THE KEY REGULATOR OF MIR-30A-5P, WHICH IN TURN ENHANCES MIR-30A-5P PROMOTER METHYLATION LEVEL AND THEREBY INHIBITS ITS EXPRESSION. TAKEN TOGETHER, OUR RESULTS REVEALED THAT EPIGENETIC MODIFICATION OF MIR-30A-5P IS INVOLVED IN GLOMERULAR PODOCYTE INJURY INDUCED BY HCY, PROVIDING A DIAGNOSTIC MARKER CANDIDATE AND NOVEL THERAPEUTIC TARGET IN CKD INDUCED BY HCY. 2022 2 3388 47 HOMOCYSTEINE INDUCES PODOCYTE APOPTOSIS BY REGULATING MIR-1929-5P EXPRESSION THROUGH C-MYC, DNMT1 AND EZH2. CHRONIC KIDNEY DISEASE (CKD) IS A COMMON AND COMPLEX DISEASE IN KIDNEYS WHICH HAS BEEN ASSOCIATED WITH AN INCREASED RISK OF RENAL CELL CARCINOMA. ELEVATED HOMOCYSTEINE (HCY) LEVELS ARE KNOWN TO INFLUENCE THE DEVELOPMENT AND PROGRESSION OF CKD BY REGULATING PODOCYTE INJURY AND APOPTOSIS. TO INVESTIGATE THE MOLECULAR MECHANISMS TRIGGERED IN PODOCYTES BY HCY, WE USED CBS(+/-) MICE AND OBSERVED THAT HIGHER HCY LEVELS INCREASED THE APOPTOSIS RATE OF PODOCYTES WITH ACCOMPANYING GLOMERULAR DAMAGE. HCY-INDUCED PODOCYTE INJURY AND APOPTOSIS IN CBS(+/-) MICE WAS REGULATED BY INHIBITION OF MICRORNA (MIR)-1929-5P EXPRESSION. OVEREXPRESSION OF MIR-1929-5P IN PODOCYTES INHIBITED APOPTOSIS BY UPREGULATING BCL-2. FURTHERMORE, THE EXPRESSION OF MIR-1929-5P WAS REGULATED BY EPIGENETIC MODIFICATIONS OF ITS PROMOTER. HCY UPREGULATED DNA METHYLTRANSFERASE 1 (DNMT1) AND ENHANCER OF ZESTE HOMOLOG 2 (EZH2) LEVELS, RESULTING IN INCREASED DNA METHYLATION AND H3K27ME3 LEVELS ON THE MIR-1929-5P PROMOTER. ADDITIONALLY, WE OBSERVED THAT C-MYC RECRUITED DNMT1 AND EZH2 TO THE MIR-1929-5P PROMOTER AND SUPPRESSED THE EXPRESSION OF MIR-1929-5P. IN SUMMARY, WE DEMONSTRATED THAT HCY PROMOTES PODOCYTE APOPTOSIS THROUGH THE REGULATION OF THE EPIGENETIC MODIFIERS DNMT1 AND EZH2, WHICH ARE RECRUITED BY C-MYC TO THE PROMOTER OF MIR-1929-5P TO SILENCE MIR-1929-5P EXPRESSION. 2021 3 2197 36 EPIGENETIC MODIFICATION OF BDNF MEDIATES NEUROPATHIC PAIN VIA MIR-30A-3P/EP300 AXIS IN CCI RATS. RECENT INVESTIGATION OF MICRORNAS ON CHRONIC PAIN HAS DEVELOPED A BREAKTHROUGH IN NEUROPATHIC PAIN MANAGEMENT. IN THE PRESENT STUDY, DECREASED EXPRESSION OF MIR-30A-3P WAS REPORTED USING QRT-PCR ANALYSIS AND LOSS OF MIR-30A-3P PROMOTED NEUROPATHIC PAIN PROGRESSION IN SCIATIC NERVE CHRONIC CONSTRICTIVE INJURY RATS THROUGH DETERMINING THE PAIN THRESHOLD. WE PREDICTED MIR-30A-3P COULD TARGET E-CADHERIN TRANSCRIPTIONAL ACTIVATOR (EP300) VIA BIOINFORMATICS ANALYSIS. MEANWHILE, WE FOUND THAT BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) IS INVOLVED IN NEUROPATHIC PAIN. HERE, WE EXHIBITED THAT EP300 EPIGENETICALLY UP-REGULATED BDNF VIA ENHANCING ACETYLATED HISTONE H3 AND H4 ON THE PROMOTER. FOR ANOTHER, MIR-30A-3P WAS ABLE TO MODIFY THE LEVEL OF BDNF AND ACETYLATED HISTONE H3 AND H4. LOSS OF MIR-30A-3P ENHANCED EP300 AND BDNF COLOCALIZATION IN CCI RATS. SUBSEQUENTLY, IT WAS SHOWN THAT INCREASED EP300 INDUCED NEUROPATHIC PAIN BY AN ENHANCEMENT OF NEURONAL BDNF LEVEL IN VIVO. TO SUM UP, IT WAS REVEALED THAT EPIGENETIC MODIFICATION OF BDNF PROMOTED NEUROPATHIC PAIN VIA EP300 INDUCED BY MIR-30A-3P IN CCI RATS. 2020 4 273 35 AGE-INDUCED SUPPRESSION OF EZH2 MEDIATES INJURY OF PODOCYTES BY REDUCING H3K27ME3. BACKGROUND: CHRONIC HYPERGLYCEMIA, A PIVOTAL FEATURE OF DIABETES MELLITUS (DM), INITIATES THE FORMATION OF ADVANCED GLYCATION END PRODUCTS (AGES) AND THE DYSREGULATION OF EPIGENETIC MECHANISMS, WHICH MAY CAUSE INJURY TO RENAL PODOCYTES, A CENTRAL FEATURE OF DIABETIC KIDNEY DISEASE (DKD). PREVIOUS DATA OF OUR GROUP SHOWED THAT AGES SIGNIFICANTLY REDUCE THE EXPRESSION OF NIPP1 (NUCLEAR INHIBITOR OF PROTEIN PHOSPHATASE 1) IN PODOCYTES IN VITRO AS WELL AS IN HUMAN AND MURINE DKD. NIPP1 WAS SHOWN BY OTHERS TO INTERACT WITH ENHANCER OF ZESTE HOMOLOG 2 (EZH2), WHICH CATALYZES THE REPRESSIVE METHYLATION OF H3K27ME3 ON HISTONE 3. THEREFORE, WE HYPOTHESIZED THAT AGES CAN DIRECTLY INDUCE EPIGENETIC CHANGES IN PODOCYTES. METHODS: WE ANALYZED THE RELEVANCE OF AGES ON EZH2 EXPRESSION AND ACTIVITY IN A MURINE PODOCYTE CELL LINE. CELLS WERE TREATED WITH 5 MG/ML GLYCATED BSA FOR 24 H. TO DETERMINE THE MEANING OF EZH2 SUPPRESSION, EZH2 ACTIVITY WAS INHIBITED BY INCUBATING THE CELLS WITH THE PHARMACOLOGICAL METHYLTRANSFERASE INHIBITOR 3-DEAZANEPLANOCIN A; EZH2 EXPRESSION WAS REPRESSED WITH SIRNA. MRNA EXPRESSION WAS ANALYZED WITH REAL-TIME PCR, AND PROTEIN EXPRESSION WITH WESTERN BLOT. EZH2 EXPRESSION AND LEVEL OF H3K27 TRIMETHYLATION IN PODOCYTES OF DIABETIC DB/DB MICE, A MOUSE MODEL FOR TYPE 2 DM, WERE ANALYZED USING IMMUNOFLUORESCENCE. RESULTS: OUR DATA DEMONSTRATED THAT AGES DECREASE EZH2 EXPRESSION IN PODOCYTES AND CONSEQUENTLY REDUCE H3K27ME3. THIS SUPPRESSION OF EZH2 MIMICKED THE AGE EFFECTS AND CAUSED AN UPREGULATED EXPRESSION OF PATHOLOGICAL FACTORS THAT CONTRIBUTE TO PODOCYTE INJURY IN DKD. IN ADDITION, ANALYSES OF DB/DB MICE SHOWED SIGNIFICANTLY REDUCED H3K27ME3 AND EZH2 EXPRESSION IN PODOCYTES. MOREOVER, THE SUPPRESSION OF NIPP1 AND EZH2 SHOWED SIMILAR EFFECTS REGARDING PODOCYTE INJURY. CONCLUSIONS: OUR STUDIES PROVIDE A NOVEL PATHWAY HOW AGES CONTRIBUTE TO PODOCYTE INJURY AND THE FORMATION OF THE SO-CALLED METABOLIC MEMORY IN DKD. 2020 5 4159 30 MECP2 CONTROLS AN EPIGENETIC PATHWAY THAT PROMOTES MYOFIBROBLAST TRANSDIFFERENTIATION AND FIBROSIS. BACKGROUND & AIMS: MYOFIBROBLAST TRANSDIFFERENTIATION GENERATES HEPATIC MYOFIBROBLASTS, WHICH PROMOTE LIVER FIBROGENESIS. THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA (PPARGAMMA) IS A NEGATIVE REGULATOR OF THIS PROCESS. WE INVESTIGATED EPIGENETIC REGULATION OF PPARGAMMA AND MYOFIBROBLAST TRANSDIFFERENTIATION. METHODS: CHROMATIN IMMUNOPRECIPITATION (CHIP) ASSAYS ASSESSED THE BINDING OF METHYL-CPG BINDING PROTEIN 2 (MECP2) TO PPARGAMMA AND CHROMATIN MODIFICATIONS THAT SILENCE THIS GENE. MECP2(-/Y) MICE AND AN INHIBITOR (DZNEP) OF THE EPIGENETIC REGULATORY PROTEIN EZH2 WERE USED IN THE CARBON TETRACHLORIDE MODEL OF LIVER FIBROSIS. LIVER TISSUES FROM MICE WERE ASSESSED BY HISTOLOGIC ANALYSIS; MARKERS OF FIBROSIS WERE MEASURED BY QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR). REVERSE TRANSCRIPTION PCR DETECTED CHANGES IN EXPRESSION OF THE MICRORNA MIR132 AND ITS TARGET, ELONGATED TRANSCRIPTS OF MECP2. MYOFIBROBLASTS WERE TRANSFECTED WITH MIR132; PPARGAMMA AND MECP2 EXPRESSIONS WERE ANALYZED BY QPCR OR IMMUNOBLOTTING. RESULTS: MYOFIBROBLAST TRANSDIFFERENTIATION OF HEPATIC STELLATE CELLS IS CONTROLLED BY A COMBINATION OF MECP2, EZH2, AND MIR132 IN A RELAY PATHWAY. THE PATHWAY IS ACTIVATED BY DOWN-REGULATION OF MIR132, RELEASING THE TRANSLATIONAL BLOCK ON MECP2. MECP2 IS RECRUITED TO THE 5' END OF PPARGAMMA, WHERE IT PROMOTES METHYLATION BY H3K9 AND RECRUITS THE TRANSCRIPTION REPRESSOR HP1ALPHA. MECP2 ALSO STIMULATES EXPRESSION OF EZH2 AND METHYLATION OF H3K27 TO FORM A REPRESSIVE CHROMATIN STRUCTURE IN THE 3' EXONS OF PPARGAMMA. GENETIC AND PHARMACOLOGIC DISRUPTIONS OF MECP2 OR EZH2 REDUCED THE FIBROGENIC CHARACTERISTICS OF MYOFIBROBLASTS AND ATTENUATED FIBROGENESIS. CONCLUSIONS: LIVER FIBROSIS IS REGULATED BY AN EPIGENETIC RELAY PATHWAY THAT INCLUDES MECP2, EZH2, AND MIR132. REAGENTS THAT INTERFERE WITH THIS PATHWAY MIGHT BE DEVELOPED TO REDUCE FIBROGENESIS IN CHRONIC LIVER DISEASE. 2010 6 5972 27 TET REPRESSION AND INCREASED DNMT ACTIVITY SYNERGISTICALLY INDUCE ABERRANT DNA METHYLATION. CHRONIC INFLAMMATION IS DEEPLY INVOLVED IN VARIOUS HUMAN DISORDERS, SUCH AS CANCER, NEURODEGENERATIVE DISORDERS, AND METABOLIC DISORDERS. INDUCTION OF EPIGENETIC ALTERATIONS, ESPECIALLY ABERRANT DNA METHYLATION, IS ONE OF THE MAJOR MECHANISMS, BUT HOW IT IS INDUCED IS STILL UNCLEAR. HERE, WE FOUND THAT EXPRESSION OF TET GENES, METHYLATION ERASERS, WAS DOWNREGULATED IN INFLAMED MOUSE AND HUMAN TISSUES, AND THAT THIS WAS CAUSED BY UPREGULATION OF TET-TARGETING MIRNAS SUCH AS MIR20A, MIR26B, AND MIR29C, LIKELY DUE TO ACTIVATION OF NF-KAPPAB SIGNALING DOWNSTREAM OF IL-1BETA AND TNF-ALPHA. HOWEVER, TET KNOCKDOWN INDUCED ONLY MILD ABERRANT METHYLATION. NITRIC OXIDE (NO), PRODUCED BY NOS2, ENHANCED ENZYMATIC ACTIVITY OF DNA METHYLTRANSFERASES (DNMTS), METHYLATION WRITERS, AND NO EXPOSURE INDUCED MINIMAL ABERRANT METHYLATION. IN CONTRAST, A COMBINATION OF TET KNOCKDOWN AND NO EXPOSURE SYNERGISTICALLY INDUCED ABERRANT METHYLATION, INVOLVING GENOMIC REGIONS NOT METHYLATED BY EITHER ALONE. THE RESULTS SHOWED THAT A VICIOUS COMBINATION OF TET REPRESSION, DUE TO NF-KAPPAB ACTIVATION, AND DNMT ACTIVATION, DUE TO NO PRODUCTION, IS RESPONSIBLE FOR ABERRANT METHYLATION INDUCTION IN HUMAN TISSUES. 2020 7 164 31 ABNORMAL HISTONE METHYLATION IS RESPONSIBLE FOR INCREASED VASCULAR ENDOTHELIAL GROWTH FACTOR 165A SECRETION FROM AIRWAY SMOOTH MUSCLE CELLS IN ASTHMA. VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF), A KEY ANGIOGENIC MOLECULE, IS ABERRANTLY EXPRESSED IN SEVERAL DISEASES INCLUDING ASTHMA WHERE IT CONTRIBUTES TO BRONCHIAL VASCULAR REMODELING AND CHRONIC INFLAMMATION. ASTHMATIC HUMAN AIRWAY SMOOTH MUSCLE CELLS HYPERSECRETE VEGF, BUT THE MECHANISM IS UNCLEAR. IN THIS STUDY, WE DEFINED THE MECHANISM IN HUMAN AIRWAY SMOOTH MUSCLE CELLS FROM NONASTHMATIC AND ASTHMATIC PATIENTS. WE FOUND THAT ASTHMATIC CELLS LACKED A REPRESSION COMPLEX AT THE VEGF PROMOTER, WHICH WAS PRESENT IN NONASTHMATIC CELLS. RECRUITMENT OF G9A, TRIMETHYLATION OF HISTONE H3 AT LYSINE 9 (H3K9ME3), AND A RESULTANT DECREASE IN RNA POLYMERASE II AT THE VEGF PROMOTER WAS CRITICAL TO REPRESSION OF VEGF SECRETION IN NONASTHMATIC CELLS. AT THE ASTHMATIC PROMOTER, H3K9ME3 WAS ABSENT BECAUSE OF FAILED RECRUITMENT OF G9A; RNA POLYMERASE II BINDING, IN ASSOCIATION WITH TATA-BINDING PROTEIN-ASSOCIATED FACTOR 1, WAS INCREASED; H3K4ME3 WAS PRESENT; AND SP1 BINDING WAS EXAGGERATED AND SUSTAINED. IN CONTRAST, DNA METHYLATION AND HISTONE ACETYLATION WERE SIMILAR IN ASTHMATIC AND NONASTHMATIC CELLS. THIS IS THE FIRST STUDY, TO OUR KNOWLEDGE, TO SHOW THAT AIRWAY CELLS IN ASTHMA HAVE ALTERED EPIGENETIC REGULATION OF REMODELING GENE(S). HISTONE METHYLATION AT GENES SUCH AS VEGF MAY BE AN IMPORTANT NEW THERAPEUTIC TARGET. 2012 8 5442 28 RENIN-ANGIOTENSIN BLOCKADE RESETS PODOCYTE EPIGENOME THROUGH KRUPPEL-LIKE FACTOR 4 AND ATTENUATES PROTEINURIA. PROTEINURIA IS A CENTRAL COMPONENT OF CHRONIC KIDNEY DISEASE AND AN INDEPENDENT RISK FACTOR FOR CARDIOVASCULAR DISEASE. KIDNEY PODOCYTES HAVE AN ESSENTIAL ROLE AS A FILTRATION BARRIER AGAINST PROTEINURIA. KRUPPEL-LIKE FACTOR 4 (KLF4) IS EXPRESSED IN PODOCYTES AND DECREASED IN GLOMERULAR DISEASES LEADING TO METHYLATION OF THE NEPHRIN PROMOTER, DECREASED NEPHRIN EXPRESSION AND PROTEINURIA. TREATMENT WITH AN ANGIOTENSIN RECEPTOR BLOCKER (ARB) REDUCED METHYLATION OF THE NEPHRIN PROMOTER IN MURINE GLOMERULI OF AN ADRIAMYCIN NEPHROPATHY MODEL WITH RECOVERY OF KLF4 EXPRESSION AND A DECREASE IN ALBUMINURIA. IN PODOCYTE-SPECIFIC KLF4 KNOCKOUT MICE, THE EFFECT OF ARB ON ALBUMINURIA AND THE NEPHRIN PROMOTER METHYLATION WAS ATTENUATED. IN CULTURED HUMAN PODOCYTES, ANGIOTENSIN II REDUCED KLF4 EXPRESSION AND CAUSED METHYLATION OF THE NEPHRIN PROMOTER WITH DECREASED NEPHRIN EXPRESSION. IN PATIENTS, NEPHRIN PROMOTER METHYLATION WAS INCREASED IN PROTEINURIC KIDNEY DISEASES WITH DECREASED KLF4 AND NEPHRIN EXPRESSION. KLF4 EXPRESSION IN ARB-TREATED PATIENTS WAS HIGHER IN PATIENTS WITH THAN WITHOUT ARB TREATMENT. THUS, ANGIOTENSIN II CAN MODULATE EPIGENETIC REGULATION IN PODOCYTES AND ARB INHIBITS THESE ACTIONS IN PART VIA KLF4 IN PROTEINURIC KIDNEY DISEASES. THIS STUDY PROVIDES A NEW CONCEPT THAT RENIN-ANGIOTENSIN SYSTEM BLOCKADE CAN EXERT THERAPEUTIC EFFECTS THROUGH EPIGENETIC MODULATION OF THE KIDNEY GENE EXPRESSION. 2015 9 1494 40 DNA HYPERMETHYLATION IN HYPERHOMOCYSTEINEMIA CONTRIBUTES TO ABNORMAL EXTRACELLULAR MATRIX METABOLISM IN THE KIDNEY. HYPERHOMOCYSTEINEMIA (HHCY) IS PREVALENT IN PATIENTS WITH CHRONIC KIDNEY DISEASE (CKD) AND END-STAGE RENAL DISEASE (ESRD). EMERGING STUDIES SUGGEST THAT EPIGENETIC MECHANISMS CONTRIBUTE TO THE DEVELOPMENT AND PROGRESSION OF FIBROSIS IN CKD. HHCY AND ITS INTERMEDIATES ARE KNOWN TO ALTER THE DNA METHYLATION PATTERN, WHICH IS A CRITICAL REGULATOR OF EPIGENETIC INFORMATION. IN THIS STUDY, WE HYPOTHESIZED THAT HHCY CAUSES RENOVASCULAR REMODELING BY DNA HYPERMETHYLATION, LEADING TO GLOMERULOSCLEROSIS. WE ALSO EVALUATED WHETHER THE DNA METHYLATION INHIBITOR, 5-AZA-2'-DEOXYCYTIDINE (5-AZA) COULD MODULATE EXTRACELLULAR MATRIX (ECM) METABOLISM AND REDUCE RENOVASCULAR FIBROSIS. C57BL/6J (WILD-TYPE) AND CYSTATHIONINE-BETA-SYNTHASE (CBS(+/-)) MICE, TREATED WITHOUT OR WITH 5-AZA (0.5 MG/KG BODY WEIGHT, I.P.), WERE USED. CBS(+/-) MICE SHOWED HIGH PLASMA HCY LEVELS, HYPERTENSION, AND SIGNIFICANT GLOMERULAR AND ARTERIOLAR INJURY. 5-AZA TREATMENT NORMALIZED BLOOD PRESSURE AND REVERSED RENAL INJURY. CBS(+/-) MICE SHOWED GLOBAL HYPERMETHYLATION AND UP-REGULATION OF DNA METHYLTRANSFERASE-1 AND -3A. METHYLATION-SPECIFIC PCR SHOWED AN IMBALANCE BETWEEN MATRIX METALLOPROTEINASE (MMP)-9 AND TISSUE INHIBITOR OF METALLOPROTEINASE (TIMP)-1 AND -2 AND ALSO INCREASED COLLAGEN AND GALECTIN-3 EXPRESSION. 5-AZA REDUCED ABNORMAL DNA METHYLATION AND RESTORED THE MMP-9/TIMP-1, -2 BALANCE. IN CONCLUSION, OUR DATA SUGGEST THAT DURING HHCY, ABNORMAL DNA METHYLATION AND AN IMBALANCE BETWEEN MMP-9 AND TIMP-1 AND -2 LEAD TO ECM REMODELING AND RENAL FIBROSIS. 2015 10 3306 40 HIGH-PHOSPHATE-INDUCED CALCIFICATION IS RELATED TO SM22ALPHA PROMOTER METHYLATION IN VASCULAR SMOOTH MUSCLE CELLS. HYPERPHOSPHATEMIA IS CLOSELY RELATED TO VASCULAR CALCIFICATION IN PATIENTS WITH CHRONIC KIDNEY DISEASE. VASCULAR SMOOTH MUSCLE CELLS (VSMCS) EXPOSED TO HIGH PHOSPHATE CONCENTRATIONS IN VITRO UNDERGO PHENOTYPIC TRANSITION TO OSTEOBLAST-LIKE CELLS. MECHANISMS UNDERLYING THIS TRANSDIFFERENTIATION ARE NOT CLEAR. IN THIS STUDY WE USED TWO IN VITRO MODELS, HUMAN AORTIC SMOOTH MUSCLE CELLS AND RAT AORTIC RINGS, TO INVESTIGATE THE PHENOTYPIC TRANSITION OF VSMCS INDUCED BY HIGH PHOSPHATE. WE FOUND THAT HIGH PHOSPHATE CONCENTRATION (3.3 MMOL/L) IN THE MEDIUM WAS ASSOCIATED WITH INCREASED DNA METHYLTRANSFERASE ACTIVITY AND METHYLATION OF THE PROMOTER REGION OF SM22ALPHA. THIS WAS ACCOMPANIED BY LOSS OF THE SMOOTH MUSCLE CELL-SPECIFIC PROTEIN SM22ALPHA, GAIN OF THE OSTEOBLAST TRANSCRIPTION FACTOR CBFA1, AND INCREASED ALKALINE PHOSPHATASE ACTIVITY WITH THE SUBSEQUENT IN VITRO CALCIFICATION. THE ADDITION OF A DEMETHYLATING AGENT (PROCAINE) TO THE HIGH-PHOSPHATE MEDIUM REDUCED DNA METHYLTRANSFERASE ACTIVITY AND PREVENTED METHYLATION OF THE SM22ALPHA PROMOTER, WHICH WAS ACCOMPANIED BY AN INCREASE IN SM22ALPHA EXPRESSION AND LESS CALCIFICATION. ADDITIONALLY, DOWNREGULATION OF SM22ALPHA, EITHER BY SIRNA OR BY A METHYL GROUP DONOR (S-ADENOSYL METHIONINE), RESULTED IN OVEREXPRESSION OF CBFA1. IN CONCLUSION, WE DEMONSTRATE THAT METHYLATION OF SM22ALPHA PROMOTER IS AN IMPORTANT EVENT IN VASCULAR SMOOTH MUSCLE CELL CALCIFICATION AND THAT HIGH PHOSPHATE INDUCES THIS EPIGENETIC MODIFICATION. THESE FINDINGS UNCOVER A NEW INSIGHT INTO MECHANISMS BY WHICH HIGH PHOSPHATE CONCENTRATION PROMOTES VASCULAR CALCIFICATION. 2010 11 4362 40 MIR?152 REGULATES TGF?BETA1?INDUCED EPITHELIAL?MESENCHYMAL TRANSITION BY TARGETING HPIP IN TUBULAR EPITHELIAL CELLS. RENAL FIBROSIS IS A COMMON PATHOLOGICAL FEATURE OF CHRONIC KIDNEY DISEASES, AND THEIR DEVELOPMENT AND PROGRESSION ARE INFLUENCED BY EPIGENETIC MODIFICATIONS INCLUDING ABERRANT MICRORNA (MIRNA OR MIR) EXPRESSION. MIRNAS HAVE BEEN DEMONSTRATED TO MODULATE THE AGGRESSIVENESS OF VARIOUS CANCERS AND HAVE EMERGED AS POSSIBLE THERAPEUTIC AGENTS FOR THE MANAGEMENT OF RENAL FIBROSIS. TRANSFORMING GROWTH FACTOR BETA1 (TGF?BETA1)?INDUCED EPITHELIAL?MESENCHYMAL TRANSITION (EMT) OF TUBULAR EPITHELIAL CELLS SERVES A ROLE IN THE INITIATION AND PROGRESSION OF RENAL FIBROSIS. FURTHERMORE, RECENT RESULTS INDICATED THAT THE PROGRESSION OF EMT IS REVERSIBLE. THE PRESENT STUDY AIMED TO CLARIFY THE ROLE OF MIR?152 IN EMT OF THE TUBULAR EPITHELIAL CELL LINE HK?2, STIMULATED BY TGF?BETA1, USING IN VITRO TRANSFECTION WITH A MIR?152 MIMIC AND TO FURTHER INVESTIGATE THE UNDERLYING MECHANISM OF MIR?152 ACTIVITY. IN THE PRESENT STUDY, MIR?152 EXPRESSION WAS SIGNIFICANTLY REDUCED IN TGF?BETA1?TREATED HK?2 CELLS, ACCOMPANIED BY AN INCREASED EXPRESSION OF HEMATOPOIETIC PRE?B?CELL LEUKEMIA TRANSCRIPTION FACTOR (PBX)?INTERACTING PROTEIN (HPIP). ADDITIONALLY, MIR?152 OVEREXPRESSION INHIBITED TGF?BETA1?INDUCED EMT AND SUPPRESSED HPIP EXPRESSION BY DIRECTLY TARGETING THE 3' UNTRANSLATED REGION OF HPIP IN HK?2 CELLS. FURTHERMORE, UPREGULATION OF HPIP REVERSED MIR?152?MEDIATED INHIBITORY EFFECTS ON THE EMT. COLLECTIVELY, THE RESULTS SUGGEST THAT DOWNREGULATION OF MIR?152 INITIATES THE DEDIFFERENTIATION OF RENAL TUBULES AND PROGRESSION OF RENAL FIBROSIS, WHICH MAY PROVIDE IMPORTANT TARGETS FOR PREVENTION STRATEGIES OF RENAL FIBROSIS. 2018 12 1615 36 DNA METHYLTRANSFERASE 3B PLAYS A PROTECTIVE ROLE AGAINST HEPATOCARCINOGENESIS CAUSED BY CHRONIC INFLAMMATION VIA MAINTAINING MITOCHONDRIAL HOMEOSTASIS. MOST HEPATOCELLULAR CARCINOMAS (HCCS) DEVELOP ON THE BASIS OF CHRONIC HEPATITIS, BUT THE MECHANISM OF EPIGENETIC REGULATION IN INFLAMMATORY HEPATOCARCINOGENESIS HAS YET TO BE ELUCIDATED. AMONG DE NOVO DNA METHYLTRANSFERASES (DNMTS), DNMT3B HAS LATELY BEEN REPORTED TO ACT SPECIFICALLY ON ACTIVELY TRANSCRIBED GENES, SUGGESTING THE POSSIBILITY THAT IT PLAYS A ROLE IN THE PATHOGENESIS OF CANCER. WE CONFIRMED THAT DNMT3B ISOFORMS LACKING ITS CATALYTIC DOMAIN WERE HIGHLY EXPRESSED IN HCCS COMPARED WITH NON-TUMOROUS LIVER TISSUE. TO ELUCIDATE THE ROLE OF DNMT3B IN HEPATOCARCINOGENESIS, WE GENERATED A GENETICALLY ENGINEERED MOUSE MODEL WITH HEPATOCYTE-SPECIFIC DNMT3B DELETION. THE LIVER OF THE DNMT3B-DEFICIENT MICE EXHIBITED AN EXACERBATION OF THIOACETAMIDE-INDUCED HEPATITIS, PROGRESSION OF LIVER FIBROSIS AND A HIGHER INCIDENCE OF HCC COMPARED WITH THE LIVER OF THE CONTROL MICE. WHOLE-GENOME BISULFITE SEQUENCING VERIFIED A LOWER CG METHYLATION LEVEL IN THE DNMT3B-DEFICIENT LIVER, DEMONSTRATING DIFFERENTIALLY METHYLATED REGIONS THROUGHOUT THE GENOME. TRANSCRIPTOME ANALYSIS REVEALED DECREASED EXPRESSION OF GENES RELATED TO OXIDATIVE PHOSPHORYLATION IN THE DNMT3B-DEFICIENT LIVER. MOREOVER, PRIMARY HEPATOCYTES ISOLATED FROM THE DNMT3B-DEFICIENT MICE SHOWED REDUCED MITOCHONDRIAL RESPIRATORY CAPACITY, LEADING TO THE ENHANCEMENT OF OXIDATIVE STRESS IN THE LIVER TISSUE. OUR FINDINGS SUGGEST THE PROTECTIVE ROLE OF DNMT3B AGAINST CHRONIC INFLAMMATION AND HCC DEVELOPMENT VIA MAINTAINING MITOCHONDRIAL HOMEOSTASIS. 2020 13 3468 36 HYPOXIA-INDUCED DNA HYPERMETHYLATION IN HUMAN PULMONARY FIBROBLASTS IS ASSOCIATED WITH THY-1 PROMOTER METHYLATION AND THE DEVELOPMENT OF A PRO-FIBROTIC PHENOTYPE. BACKGROUND: PULMONARY FIBROSIS IS A DEBILITATING AND LETHAL DISEASE WITH NO EFFECTIVE TREATMENT OPTIONS. UNDERSTANDING THE PATHOLOGICAL PROCESSES AT PLAY WILL DIRECT THE APPLICATION OF NOVEL THERAPEUTIC AVENUES. HYPOXIA HAS BEEN IMPLICATED IN THE PATHOGENESIS OF PULMONARY FIBROSIS YET THE PRECISE MECHANISM BY WHICH IT CONTRIBUTES TO DISEASE PROGRESSION REMAINS TO BE FULLY ELUCIDATED. IT HAS BEEN SHOWN THAT CHRONIC HYPOXIA CAN ALTER DNA METHYLATION PATTERNS IN TUMOUR-DERIVED CELL LINES. THIS EPIGENETIC ALTERATION CAN INDUCE CHANGES IN CELLULAR PHENOTYPE WITH PROMOTER METHYLATION BEING ASSOCIATED WITH GENE SILENCING. OF PARTICULAR RELEVANCE TO IDIOPATHIC PULMONARY FIBROSIS (IPF) IS THE OBSERVATION THAT THY-1 PROMOTER METHYLATION IS ASSOCIATED WITH A MYOFIBROBLAST PHENOTYPE WHERE LOSS OF THY-1 OCCURS ALONGSIDE INCREASED ALPHA SMOOTH MUSCLE ACTIN (ALPHA-SMA) EXPRESSION. THE INITIAL AIM OF THIS STUDY WAS TO DETERMINE WHETHER HYPOXIA REGULATES DNA METHYLATION IN NORMAL HUMAN LUNG FIBROBLASTS (CCD19LU). AS IT HAS BEEN REPORTED THAT HYPOXIA SUPPRESSES THY-1 EXPRESSION DURING LUNG DEVELOPMENT WE ALSO STUDIED THE EFFECT OF HYPOXIA ON THY-1 PROMOTER METHYLATION AND GENE EXPRESSION. METHODS: CCD19LU WERE GROWN FOR UP TO 8 DAYS IN HYPOXIA AND ASSESSED FOR GLOBAL CHANGES IN DNA METHYLATION USING FLOW CYTOMETRY. REAL-TIME PCR WAS USED TO QUANTIFY EXPRESSION OF THY-1, ALPHA-SMA, COLLAGEN I AND III. GENOMIC DNA WAS BISULPHITE TREATED AND METHYLATION SPECIFIC PCR (MSPCR) WAS USED TO EXAMINE THE METHYLATION STATUS OF THE THY-1 PROMOTER. RESULTS: SIGNIFICANT GLOBAL HYPERMETHYLATION WAS DETECTED IN HYPOXIC FIBROBLASTS RELATIVE TO NORMOXIC CONTROLS AND WAS ACCOMPANIED BY INCREASED EXPRESSION OF MYOFIBROBLAST MARKERS. THY-1 MRNA EXPRESSION WAS SUPPRESSED IN HYPOXIC CELLS, WHICH WAS RESTORED WITH THE DEMETHYLATING AGENT 5-AZA-2'-DEOXYCYTIDINE. MSPCR REVEALED THAT THY-1 BECAME METHYLATED FOLLOWING FIBROBLAST EXPOSURE TO 1% O2. CONCLUSION: THESE DATA SUGGEST THAT GLOBAL AND GENE-SPECIFIC CHANGES IN DNA METHYLATION MAY PLAY AN IMPORTANT ROLE IN FIBROBLAST FUNCTION IN HYPOXIA. 2012 14 4302 38 MICRORNA-223 CONTROLS THE EXPRESSION OF HISTONE DEACETYLASE 2: A NOVEL AXIS IN COPD. REDUCED ACTIVITY OF HISTONE DEACETYLASE 2 (HDAC2) HAS BEEN DESCRIBED IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD), BUT THE MECHANISMS RESULTING IN DECREASED EXPRESSION OF THIS IMPORTANT EPIGENETIC MODIFIER REMAIN UNKNOWN. HERE, WE EMPLOYED SEVERAL IN VITRO EXPERIMENTS TO ADDRESS THE ROLE OF MICRORNAS (MIRNAS) ON THE REGULATION OF HDAC2 IN ENDOTHELIAL CELLS. MANIPULATION OF MIRNA LEVELS IN HUMAN PULMONARY ARTERY ENDOTHELIAL CELLS (HPAEC) WAS ACHIEVED BY USING ELECTROPORATION WITH ANTI-MIRNAS AND MIRNA MIMICS. TARGET PREDICTION SOFTWARE IDENTIFIED MIR-223 AS A POTENTIAL REPRESSOR OF HDAC2. IN SUBSEQUENT STIMULATION EXPERIMENTS USING INFLAMMATORY CYTOKINES KNOWN TO BE INCREASED IN PATIENTS WITH COPD, MIR-223 WAS FOUND TO BE SIGNIFICANTLY INDUCED. FUNCTIONAL ANALYSIS DEMONSTRATED THAT OVEREXPRESSION OF MIR-223 DECREASED HDAC2 EXPRESSION AND ACTIVITY IN HPAEC. CONVERSELY, HDAC2 EXPRESSION AND ACTIVITY WAS PRESERVED IN ANTI-MIR-223-TREATED CELLS. DIRECT MIRNA-TARGET INTERACTION WAS CONFIRMED BY REPORTER GENE ASSAY. IN A NEXT STEP, REDUCED EXPRESSION OF HDAC2 WAS FOUND TO INCREASE THE LEVELS OF THE CHEMOKINE FRACTALKINE (CX3CL1). IN VIVO STUDIES CONFIRMED ELEVATED EXPRESSION LEVELS OF MIR-223 IN MICE EXPOSED TO CIGARETTE SMOKE AND IN EMPHYSEMATOUS LUNG TISSUE FROM LPS-TREATED MICE. MOREOVER, A SIGNIFICANT INVERSE CORRELATION OF MIR-223 AND HDAC2 EXPRESSION WAS FOUND IN TWO INDEPENDENT COHORTS OF COPD PATIENTS. THESE DATA EMPHASIZE THAT MIR-223, THE MOST PREVALENT MIRNA IN COPD, CONTROLS EXPRESSION AND ACTIVITY OF HDAC2 IN PULMONARY CELLS, WHICH, IN TURN, MIGHT ALTER THE EXPRESSION PROFILE OF CHEMOKINES. THIS PATHWAY PROVIDES A NOVEL PATHOGENIC LINK BETWEEN DYSREGULATED MIRNA EXPRESSION AND EPIGENETIC ACTIVITY IN COPD. KEY MESSAGES: HISTONE DEACETYLASE 2 IS DIRECTLY TARGETED BY MIR-223. LEVELS OF MIR-223 ARE INDUCED BY INTERLEUKIN-1BETA AND TUMOR NECROSIS FACTOR-ALPHA. MIR-223 CONTROLS THE EXPRESSION OF FRACTALKINE BY TARGETING HISTONE DEACETYLASE 2. MIR-223 LEVELS ARE INCREASED IN COPD MOUSE MODELS. MIR-223 LEVELS INVERSELY CORRELATE WITH HDAC2 EXPRESSION IN COPD PATIENTS. 2016 15 6411 37 THE SITE SPECIFIC DEMETHYLATION IN THE 5'-REGULATORY AREA OF NMDA RECEPTOR 2B SUBUNIT GENE ASSOCIATED WITH CIE-INDUCED UP-REGULATION OF TRANSCRIPTION. BACKGROUND: THE NMDA RECEPTOR REPRESENTS A PARTICULARLY IMPORTANT SITE OF ETHANOL ACTION IN THE CNS. WE RECENTLY REPORTED THAT NMDA RECEPTOR 2B (NR2B) GENE EXPRESSION WAS PERSISTENTLY UP-REGULATED FOLLOWING CHRONIC INTERMITTENT ETHANOL (CIE) TREATMENT. INCREASING EVIDENCE THAT EPIGENETIC MECHANISMS ARE INVOLVED IN DYNAMIC AND LONG-LASTING REGULATION OF GENE EXPRESSION IN MULTIPLE NEUROADAPTIVE PROCESSES PROMPTED US TO INVESTIGATE THE ROLE OF DNA METHYLATION IN MEDIATING CIE-INDUCED UP-REGULATION OF NR2B GENE TRANSCRIPTION. TO DISSECT THE CHANGES OF DNA METHYLATION IN THE NR2B GENE, WE HAVE SCREENED A LARGE NUMBER OF CPG SITES WITHIN ITS 5'-REGULATORY AREA FOLLOWING CIE TREATMENT. METHODS: PRIMARY CORTICAL CULTURED NEURONS WERE SUBJECTED TO ETHANOL TREATMENT IN A CIE PARADIGM. BISULFITE CONVERSION FOLLOWED BY PYROSEQUENCING WAS USED FOR QUANTITATIVE MEASUREMENT AND ANALYSIS OF CPG METHYLATION STATUS WITHIN THE 5'-REGULATORY AREA OF THE NR2B GENE; CHROMATIN IMMUNOPRECIPITATION (CHIP) ASSAY WAS USED TO EXAMINE DNA LEVELS ASSOCIATED WITH METHYLATION AND TRANSCRIPTION FACTOR BINDING. ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA) AND IN VITRO DNA METHYLATION ASSAYS WERE PERFORMED TO DETERMINE THE DIRECT IMPACT OF DNA METHYLATION ON THE INTERACTION BETWEEN DNA AND TRANSCRIPTION FACTOR AND PROMOTER ACTIVITY. RESULTS: ANALYSIS OF INDIVIDUAL CPG METHYLATION SITES WITHIN THE NR2B 5'REGULATORY AREA REVEALED THREE REGIONS WITH CLUSTERS OF SITE-SPECIFIC CPG DEMETHYLATION FOLLOWING CIE TREATMENT AND WITHDRAWAL. THIS WAS CONFIRMED BY CHIP SHOWING SIMILAR DECREASES OF METHYLATED DNA IN THE SAME REGIONS. THE CIE-INDUCED DEMETHYLATION IS CHARACTERIZED BY BEING LOCATED NEAR CERTAIN TRANSCRIPTION FACTOR BINDING SEQUENCES, AP-1 AND CRE, AND OCCURRED DURING TREATMENT AS WELL AS AFTER ETHANOL WITHDRAWAL. FURTHERMORE, THE INCREASE IN VITRO OF METHYLATED DNA DECREASED TRANSCRIPTION FACTOR BINDING ACTIVITY AND PROMOTER ACTIVITY. AN ADDITIONAL CHIP ASSAY INDICATED THAT THE CIE-INDUCED DNA DEMETHYLATION IS ACCOMPANIED BY INCREASED OCCUPATION BY TRANSCRIPTION FACTORS. CONCLUSIONS: THESE RESULTS SUGGEST AN IMPORTANT ROLE OF DNA DEMETHYLATION IN MEDIATING CIE-INDUCED NR2B GENE UP-REGULATION, THUS IMPLICATING A NOVEL MOLECULAR SITE OF ALCOHOL ACTION. 2010 16 3944 36 LNCRNA H19-EZH2 INTERACTION PROMOTES LIVER FIBROSIS VIA REPROGRAMMING H3K27ME3 PROFILES. LIVER FIBROSIS IS A WOUND-HEALING PROCESS CHARACTERIZED BY EXCESS FORMATION OF EXTRACELLULAR MATRIX (ECM) FROM ACTIVATED HEPATIC STELLATE CELLS (HSCS). PREVIOUS STUDIES SHOW THAT BOTH EZH2, AN EPIGENETIC REGULATOR THAT CATALYZES LYSINE 27 TRIMETHYLATION ON HISTONE 3 (H3K27ME3), AND LONG NON-CODING RNA H19 ARE HIGHLY CORRELATED WITH FIBROGENESIS. IN THE CURRENT STUDY, WE INVESTIGATED THE UNDERLYING MECHANISMS. VARIOUS MODELS OF LIVER FIBROSIS INCLUDING MDR2(-/-), BILE DUCT LIGATION (BDL) AND CCL(4) MICE WERE ADAPTED. WE FOUND THAT EZH2 WAS MARKEDLY UPREGULATED AND CORRELATED WITH H19 AND FIBROTIC MARKERS EXPRESSION IN THESE MODELS. ADMINISTRATION OF EZH2 INHIBITOR 3-DZNEP CAUSED SIGNIFICANT PROTECTIVE EFFECTS IN THESE MODELS. FURTHERMORE, TREATMENT WITH 3-DZNEP OR GSK126 SIGNIFICANTLY INHIBITED PRIMARY HSC ACTIVATION AND PROLIFERATION IN TGF-BETA-TREATED HSCS AND H19-OVEREXPREESING LX2 CELLS IN VIVO. USING RNA-PULL DOWN ASSAY COMBINED WITH RNA IMMUNOPRECIPITATION, WE DEMONSTRATED THAT H19 COULD DIRECTLY BIND TO EZH2. INTEGRATED ANALYSIS OF RNA-SEQUENCING (RNA-SEQ) AND CHROMATIN IMMUNOPRECIPITATION SEQUENCING (CHIP-SEQ) FURTHER REVEALED THAT H19 REGULATED THE REPROGRAMMING OF EZH2-MEDIATED H3K27ME3 PROFILES, WHICH EPIGENETICALLY PROMOTED SEVERAL PATHWAYS FAVORING HSCS ACTIVATION AND PROLIFERATION, INCLUDING EPITHELIAL-MESENCHYMAL TRANSITION AND WNT/BETA-CATENIN SIGNALING. IN CONCLUSION, HIGHLY EXPRESSED H19 IN CHRONIC LIVER DISEASES PROMOTES FIBROGENESIS BY REPROGRAMMING EZH2-MEDIATED EPIGENETIC REGULATION OF HSCS ACTIVATION. TARGETING THE H19-EZH2 INTERACTION MAY SERVE AS A NOVEL THERAPEUTIC APPROACH FOR LIVER FIBROSIS. 2023 17 3295 34 HIGH PHOSPHATE-INDUCED DOWNREGULATION OF PPARGAMMA CONTRIBUTES TO CKD-ASSOCIATED VASCULAR CALCIFICATION. MEDIAL ARTERIAL CALCIFICATION ASSOCIATED WITH HYPERPHOSPHATEMIA IS A MAIN CAUSE OF CARDIOVASCULAR MORTALITY IN PATIENTS WITH CHRONIC KIDNEY DISEASE (CKD), BUT THE MECHANISMS UNDERLYING HIGH PHOSPHATE-INDUCED VASCULAR CALCIFICATION REMAIN LARGELY UNKNOWN. HERE, WE OBSERVED A SIGNIFICANT DECREASE IN THE EXPRESSION OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-GAMMA (PPARGAMMA) IN CALCIFIED ARTERIES BOTH IN CKD PATIENTS AND IN A MOUSE MODEL OF CKD WITH HYPERPHOSPHATEMIA. IN VITRO, HIGH PHOSPHATE TREATMENT LED TO A DECREASED EXPRESSION OF PPARGAMMA IN MOUSE VASCULAR SMOOTH MUSCLE CELLS (VMSCS), ACCOMPANIED BY APPARENT OSTEOGENIC DIFFERENTIATION AND CALCIFICATION. PRETREATMENT WITH PPARGAMMA AGONIST ROSIGLITAZONE SIGNIFICANTLY REVERSED HIGH PHOSPHATE-INDUCED VSMCS CALCIFICATION. FURTHER INVESTIGATION SHOWED THAT METHYL-CPG BINDING PROTEIN 2 (MECP2)-MEDIATED EPIGENETIC REPRESSION WAS INVOLVED IN HIGH PHOSPHATE-INDUCED PPARGAMMA DOWNREGULATION. MOREOVER, THE EXPRESSION OF KLOTHO THAT HAS THE ABILITY TO INHIBIT VASCULAR CALCIFICATION BY REGULATING PHOSPHATE UPTAKE DECREASED WITH THE PPARGAMMA REDUCTION IN VSMCS AFTER HIGH PHOSPHATE TREATMENT, AND ROSIGLITAZONE FAILED TO INHIBIT HIGH PHOSPHATE-INDUCED CALCIFICATION IN VSMCS WITH KNOCKDOWN OF KLOTHO OR IN AORTIC RINGS FROM KLOTHO-DEFICIENT (KL/KL) MICE. FINALLY, AN IN VIVO STUDY DEMONSTRATED THAT ORAL ADMINISTRATION OF ROSIGLITAZONE COULD INCREASE KLOTHO EXPRESSION AND PROTECT AGAINST HIGH PHOSPHATE-INDUCED VASCULAR CALCIFICATION IN CKD MICE. THESE FINDINGS SUGGEST THAT THE INHIBITION OF PPARGAMMA EXPRESSION MAY CONTRIBUTE TO THE PATHOGENESIS OF HIGH PHOSPHATE-INDUCED VASCULAR CALCIFICATION, WHICH MAY PROVIDE A NEW THERAPEUTIC TARGET FOR VASCULAR CALCIFICATION IN CKD PATIENTS. 2018 18 6419 38 THE TET2-UPF1 COMPLEX MODULATES MRNA STABILITY UNDER STRESS CONDITIONS. INTRODUCTION: ENVIRONMENTAL STRESS PROMOTES EPIGENETIC ALTERATIONS THAT IMPACT GENE EXPRESSION AND SUBSEQUENTLY PARTICIPATE IN THE PATHOLOGICAL PROCESSES OF THE DISORDER. AMONG EPIGENETIC REGULATIONS, TEN-ELEVEN TRANSLOCATION (TET) ENZYMES OXIDIZE 5-METHYLCYTOSINE (5MC) TO 5-HYDROXYMETHYLCYTOSINE (5HMC) IN DNA AND RNA AND FUNCTION AS CRITICAL PLAYERS IN THE PATHOGENESIS OF DISEASES. OUR PREVIOUS RESULTS SHOWED THAT CHRONIC STRESS INCREASES THE EXPRESSION OF CYTOPLASMIC TET2 IN THE HIPPOCAMPUS OF MICE EXPOSED TO CHRONIC MILD STRESS (CMS). WHETHER THE CYTOPLASMIC TET2 ALTERS RNA 5HMC MODIFICATION IN CHRONIC STRESS-RELATED PROCESSES REMAINS LARGELY UNKNOWN. METHODS: TO EXPLORE THE ROLE OF CYTOPLASMIC TET2 UNDER CMS CONDITIONS, WE ESTABLISHED CMS MICE MODEL AND DETECTED THE EXPRESSION OF RNA 5HMC BY DOT BLOT. WE VERIFIED THE INTERACTION OF TET2 AND ITS INTERACTING PROTEIN BY CO-IMMUNOPRECIPITATION COMBINED WITH MASS SPECTROMETRY AND SCREENED DOWNSTREAM TARGET GENES BY CLUSTER ANALYSIS OF TET2 AND UPSTREAM FRAMESHIFT 1 (UPF1) INTERACTING RNA. THE EXPRESSION OF PROTEIN WAS DETECTED BY WESTERN BLOT AND THE EXPRESSION OF THE SCREENED TARGET GENES WAS DETECTED BY QRT-PCR. RESULTS: IN THIS STUDY, WE FOUND THAT INCREASED CYTOPLASMIC TET2 EXPRESSION UNDER CMS CONDITIONS LEADS TO INCREASE IN TOTAL RNA 5HMC MODIFICATION. TET2 INTERACTED WITH THE KEY NON-SENSE-MEDIATED MRNA DECAY (NMD) FACTOR UPF1, REGULATED THE STABILITY OF STRESS-RELATED GENES SUCH AS UNC5B MRNA, AND MIGHT THEREBY AFFECT NEURODEVELOPMENT. DISCUSSION: IN SUMMARY, THIS STUDY REVEALED THAT TET2-MEDIATED RNA 5HMC MODIFICATION IS INVOLVED IN STRESS-RELATED MRNA STABILITY REGULATION AND MAY SERVE AS A POTENTIAL THERAPEUTIC TARGET FOR CHRONIC STRESS-RELATED DISEASES SUCH AS DEPRESSION. 2023 19 6431 38 THE USE OF TARGETED NEXT GENERATION SEQUENCING TO EXPLORE CANDIDATE REGULATORS OF TGF-BETA1'S IMPACT ON KIDNEY CELLS. AIMS/HYPOTHESIS: TRANSFORMING GROWTH FACTOR-BETA (TGF-BETA1) PLAYS AN IMPORTANT REGULATORY ROLE IN THE PROGRESSION OF CHRONIC KIDNEY FAILURE. FURTHER, DAMAGE TO KIDNEY GLOMERULAR MESANGIAL CELLS IS CENTRAL TO THE PROGRESSION OF DIABETIC NEPHROPATHY. THE AIM OF THIS STUDY WAS TO EXPLORE THE GENETIC ASSOCIATIONS BETWEEN MRNA, MICRORNA, AND EPIGENETICS IN MESANGIAL CELLS IN RESPONSE TO TGF-BETA1. METHODS: THE REGULATORY EFFECTS OF TGF-BETA1 ON MESANGIAL CELLS WERE INVESTIGATED AT DIFFERENT MOLECULAR LEVELS BY TREATING MESANGIAL CELLS WITH TGF-BETA1 FOR 3 DAYS FOLLOWED BY GENOME-WIDE MIRNA, RNA, DNA METHYLATION, AND H3K27ME3 EXPRESSION PROFILING USING NEXT GENERATION SEQUENCING (NGS). RESULTS: OUR RESULTS PROVIDE THE FIRST COMPREHENSIVE, COMPUTATIONALLY INTEGRATED REPORT OF RNA-SEQ, MIRNA-SEQ, AND EPIGENOMIC ANALYSES ACROSS ALL GENETIC VARIATIONS, CONFIRMING THE OCCURRENCE OF DNA METHYLATION AND H3K27ME3 IN RESPONSE TO TGF-BETA1. OUR FINDINGS SHOW THAT THE EXPRESSION OF KLF7 AND GJA4 ARE INVOLVED IN TGF-BETA1 REGULATED DNA METHYLATION. OUR DATA ALSO PROVIDE EVIDENCE OF THE ASSOCIATION BETWEEN EPIGENETIC CHANGES AND THE EXPRESSION OF GENES CLOSELY RELATED TO TGF-BETA1 REGULATION. CONCLUSION: THIS STUDY HAS ADVANCED OUR CURRENT KNOWLEDGE OF MECHANISMS THAT CONTRIBUTE TO THE EXPRESSION OF TGF-BETA1-REGULATED GENES INVOLVED IN THE PATHOGENESIS OF KIDNEY DISEASE. THE MOLECULAR UNDERPINNINGS OF TGF-BETA1 STIMULATION OF KIDNEY CELLS WAS DETERMINED, THEREBY PROVIDING A ROBUST PLATFORM FOR FURTHER TARGET EXPLORATION. 2018 20 3431 43 HYDROGEN SULFIDE ALLEVIATES HYPERTENSIVE KIDNEY DYSFUNCTION THROUGH AN EPIGENETIC MECHANISM. HYPERTENSION IS A MAJOR RISK FACTOR FOR CHRONIC KIDNEY DISEASE (CKD), AND RENAL INFLAMMATION IS AN INTEGRAL PART IN THIS PATHOLOGY. HYDROGEN SULFIDE (H(2)S) HAS BEEN SHOWN TO MITIGATE RENAL DAMAGE THROUGH REDUCTION IN BLOOD PRESSURE AND ROS; HOWEVER, THE EXACT MECHANISMS ARE NOT CLEAR. WHILE SEVERAL STUDIES HAVE UNDERLINED THE ROLE OF EPIGENETICS IN RENAL INFLAMMATION AND DYSFUNCTION, THE MECHANISMS THROUGH WHICH EPIGENETIC REGULATORS PLAY A ROLE IN HYPERTENSION ARE NOT WELL DEFINED. IN THIS STUDY, WE SOUGHT TO IDENTIFY WHETHER MICRORNAS ARE DYSREGULATED IN RESPONSE TO ANGIOTENSIN II (ANG II)-INDUCED HYPERTENSION IN THE KIDNEY AND WHETHER A H(2)S DONOR, GYY4137, COULD REVERSE THE MICRORNA ALTERATION AND KIDNEY FUNCTION. WILD-TYPE (C57BL/6J) MICE WERE TREATED WITHOUT OR WITH ANG II AND GYY4137 FOR 4 WK. BLOOD PRESSURE, RENAL BLOOD FLOW, AND RESISTIVE INDEX (RI) WERE MEASURED. MICRORNA MICROARRAYS WERE CONDUCTED AND SUBSEQUENT TARGET PREDICTION REVEALED GENES ASSOCIATED WITH A PROINFLAMMATORY RESPONSE. ANG II TREATMENT SIGNIFICANTLY INCREASED BLOOD PRESSURE, DECREASED BLOOD FLOW IN THE RENAL CORTEX, INCREASED RI, AND REDUCED RENAL FUNCTION. THESE EFFECTS WERE AMELIORATED IN MICE TREATED WITH GYY4137. MICROARRAY ANALYSIS REVEALED DOWNREGULATION OF MIR-129 IN ANG II-TREATED MICE AND UPREGULATION AFTER GYY4137 TREATMENT. QUANTITATION OF PROTEINS INVOLVED IN THE INFLAMMATORY RESPONSE AND DNA METHYLATION REVEALED UPREGULATION OF IL-17A AND DNA METHYLTRANSFERASE 3A, WHEREAS H(2)S PRODUCTION ENZYMES AND ANTI-INFLAMMATORY IL-10 WERE REDUCED. TAKEN TOGETHER, OUR DATA SUGGEST THAT DOWNREGULATION OF MIR-129 PLAYS A SIGNIFICANT ROLE IN ANG II-INDUCED RENAL INFLAMMATION AND FUNCTIONAL OUTCOMES AND THAT GYY4137 IMPROVES RENAL FUNCTION BY REVERSING MIR-129 EXPRESSION.NEW & NOTEWORTHY WE INVESTIGATED EPIGENETIC CHANGES THAT OCCUR IN THE HYPERTENSIVE KIDNEY AND HOW H(2)S SUPPLEMENTATION REVERSES ADVERSE EFFECTS. INFLAMMATION, ABERRANT METHYLATION, AND DYSFUNCTION WERE OBSERVED IN THE HYPERTENSIVE KIDNEY, AND THESE EFFECTS WERE ALLEVIATED WITH H(2)S SUPPLEMENTATION. WE IDENTIFY MIR-129 AS A POTENTIAL REGULATOR OF BLOOD PRESSURE AND H(2)S REGULATION. 2017