1 85 115 A NOVEL INDOLE COMPOUND MA-35 ATTENUATES RENAL FIBROSIS BY INHIBITING BOTH TNF-ALPHA AND TGF-BETA(1) PATHWAYS. RENAL FIBROSIS IS CLOSELY RELATED TO CHRONIC INFLAMMATION AND IS UNDER THE CONTROL OF EPIGENETIC REGULATIONS. BECAUSE THE SIGNALING OF TRANSFORMING GROWTH FACTOR-BETA(1) (TGF-BETA(1)) AND TUMOR NECROSIS FACTOR-ALPHA (TNF-ALPHA) PLAY KEY ROLES IN PROGRESSION OF RENAL FIBROSIS, DUAL BLOCKADE OF TGF-BETA(1) AND TNF-ALPHA IS DESIRED AS ITS THERAPEUTIC APPROACH. HERE WE SCREENED SMALL MOLECULES SHOWING ANTI-TNF-ALPHA ACTIVITY IN THE COMPOUND LIBRARY OF INDOLE DERIVATIVES. 11 OUT OF 41 INDOLE DERIVATIVES INHIBITED THE TNF-ALPHA EFFECT. AMONG THEM, MITOCHONIC ACID 35 (MA-35), 5-(3, 5-DIMETHOXYBENZYLOXY)-3-INDOLEACETIC ACID, SHOWED THE POTENT EFFECT. THE ANTI-TNF-ALPHA ACTIVITY WAS MEDIATED BY INHIBITING IKAPPAB KINASE PHOSPHORYLATION, WHICH ATTENUATED THE LPS/GAIN-INDUCED HEPATIC INFLAMMATION IN THE MICE. ADDITIONALLY, MA-35 CONCURRENTLY SHOWED AN ANTI-TGF-BETA(1) EFFECT BY INHIBITING SMAD3 PHOSPHORYLATION, RESULTING IN THE DOWNREGULATION OF TGF-BETA(1)-INDUCED FIBROTIC GENE EXPRESSION. IN UNILATERAL URETER OBSTRUCTED MOUSE KIDNEY, WHICH IS A RENAL FIBROSIS MODEL, MA-35 ATTENUATED RENAL INFLAMMATION AND FIBROSIS WITH THE DOWNREGULATION OF INFLAMMATORY CYTOKINES AND FIBROTIC GENE EXPRESSIONS. FURTHERMORE, MA-35 INHIBITED TGF-BETA(1)-INDUCED H3K4ME1 HISTONE MODIFICATION OF THE FIBROTIC GENE PROMOTER, LEADING TO A DECREASE IN THE FIBROTIC GENE EXPRESSION. MA-35 AFFECTS MULTIPLE SIGNALING PATHWAYS INVOLVED IN THE FIBROSIS AND MAY RECOVER EPIGENETIC MODIFICATION; THEREFORE, IT COULD POSSIBLY BE A NOVEL THERAPEUTIC DRUG FOR FIBROSIS. 2017 2 4362 34 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 3 1461 42 DISRUPTION OF RCAN1.4 EXPRESSION MEDIATED BY YY1/HDAC2 MODULATES CHRONIC RENAL ALLOGRAFT INTERSTITIAL FIBROSIS. CHRONIC ALLOGRAFT DYSFUNCTION (CAD) IS A MAJOR FACTOR THAT HINDERS KIDNEY TRANSPLANT SURVIVAL IN THE LONG RUN. EPITHELIAL-MESENCHYMAL TRANSITION (EMT) HAS BEEN CONFIRMED TO SIGNIFICANTLY CONTRIBUTE TO INTERSTITIAL FIBROSIS/TUBULAR ATROPHY (IF/TA), WHICH IS THE MAIN HISTOPATHOLOGICAL FEATURE OF CAD. ABERRANT EXPRESSION OF THE REGULATOR OF CALCINEURIN 1 (RCAN1), RECOGNIZED AS AN ENDOGENOUS INHIBITOR OF THE CALCINEURIN PHOSPHATASE, HAS BEEN SHOWN TO BE EXTENSIVELY INVOLVED IN VARIOUS KIDNEY DISEASES. HOWEVER, IT REMAINS UNCLEAR HOW RCAN1.4 REGULATES IF/TA FORMATION IN CAD PATIENTS. HEREIN, AN IN VIVO MOUSE RENAL TRANSPLANTATION MODEL AND AN IN VITRO MODEL OF HUMAN RENAL TUBULAR EPITHELIAL CELLS (HK-2) TREATED WITH TUMOR NECROSIS FACTOR-ALPHA (TNF-ALPHA) WERE EMPLOYED. OUR RESULTS PROVED THAT RCAN1.4 EXPRESSION WAS DECREASED IN VIVO AND IN VITRO, IN ADDITION TO THE UP-REGULATION OF YIN YANG 1 (YY1), A TRANSCRIPTION FACTOR THAT HAS BEEN REPORTED TO CONVEY MULTIPLE FUNCTIONS IN CHRONIC KIDNEY DISEASE (CKD). KNOCKING IN OF RCAN1.4 EFFICIENTLY ATTENUATED CHRONIC RENAL ALLOGRAFT INTERSTITIAL FIBROSIS IN VIVO AND INHIBITED TNF-ALPHA-INDUCED EMT IN VITRO THROUGH REGULATING ANTI-OXIDATIVE STRESS AND THE CALCINEURIN/NUCLEAR FACTOR OF ACTIVATED T CELLS CYTOPLASMIC 1 (NFATC1) SIGNALING PATHWAY. IN ADDITION, SUPPRESSION OF YY1 MEDIATED BY SHRNA OR SIRNA ALLEVIATED TNF-ALPHA-INDUCED EMT THROUGH ABOLISHING REACTIVE SPECIES PARTLY IN AN RCAN1.4-DEPENDENT MANNER. NOTABLY, WE CONFIRMED THAT YY1 NEGATIVELY REGULATED RCAN1.4 TRANSCRIPTION BY DIRECTLY INTERACTING WITH THE RCAN1.4 PROMOTER. IN ADDITION, HISTONE DEACETYLASE 2 (HDAC2) INTERACTED WITH YY1 TO FORM A MULTI-MOLECULAR COMPLEX, WHICH WAS INVOLVED IN TNF-ALPHA-INDUCED RCAN1.4 TRANSCRIPTIONAL REPRESSION. THEREFORE, RCAN1.4 IS SUGGESTED TO BE MODULATED BY THE YY1/HDAC2 TRANSCRIPTION REPRESSOR COMPLEX IN AN EPIGENETIC MANNER, WHICH IS A MEDIATED NEPHROPROTECTIVE EFFECT PARTLY THROUGH MODULATING O2?- GENERATION AND THE CALCINEURIN/NFATC1 SIGNALING PATHWAY. THUS, THE YY1-RCAN1.4 AXIS CONSTITUTES AN INNOVATIVE TARGET FOR IF/TA TREATMENT IN CAD PATIENTS. 2023 4 3330 32 HISTONE DEACETYLASE INHIBITOR GIVINOSTAT ALLEVIATES LIVER FIBROSIS BY REGULATING HEPATIC STELLATE CELL ACTIVATION. HEPATIC FIBROSIS, A COMMON PATHOLOGICAL MANIFESTATION OF CHRONIC LIVER INJURY, IS GENERALLY CONSIDERED TO BE THE END RESULT OF AN INCREASE IN EXTRACELLULAR MATRIX PRODUCED BY ACTIVATED HEPATIC STELLATE CELLS (HSCS). THE AIM OF THE PRESENT STUDY WAS TO TARGET THE MECHANISMS UNDERLYING HSC ACTIVATION IN ORDER TO PROVIDE A POWERFUL THERAPEUTIC STRATEGY FOR THE PREVENTION AND TREATMENT OF LIVER FIBROSIS. IN THE PRESENT STUDY, A HIGH?THROUGHPUT SCREENING ASSAY WAS ESTABLISHED, AND THE HISTONE DEACETYLASE INHIBITOR GIVINOSTAT WAS IDENTIFIED AS A POTENT INHIBITOR OF HSC ACTIVATION IN VITRO. GIVINOSTAT SIGNIFICANTLY INHIBITED HSC ACTIVATION IN VIVO, AMELIORATED CARBON TETRACHLORIDE?INDUCED MOUSE LIVER FIBROSIS AND LOWERED PLASMA AMINOTRANSFERASES. TRANSCRIPTOMIC ANALYSIS REVEALED THE MOST SIGNIFICANTLY REGULATED GENES IN THE GIVINOSTAT TREATMENT GROUP IN COMPARISON WITH THOSE IN THE SOLVENT GROUP, AMONG WHICH, DERMOKINE (DMKN), MESOTHELIN (MSLN) AND UROPLAKIN?3B (UPK3B) WERE IDENTIFIED AS POTENTIAL REGULATORS OF HSC ACTIVATION. GIVINOSTAT SIGNIFICANTLY REDUCED THE MRNA EXPRESSION OF DMKN, MSLN AND UPK3B IN BOTH A MOUSE LIVER FIBROSIS MODEL AND IN HSC?LX2 CELLS. KNOCKDOWN OF ANY OF THE AFOREMENTIONED GENES INHIBITED THE TGF?BETA1?INDUCED EXPRESSION OF ALPHA?SMOOTH MUSCLE ACTIN AND COLLAGEN TYPE I, INDICATING THAT THEY ARE CRUCIAL FOR HSC ACTIVATION. IN SUMMARY, USING A NOVEL STRATEGY TARGETING HSC ACTIVATION, THE PRESENT STUDY IDENTIFIED A POTENTIAL EPIGENETIC DRUG FOR THE TREATMENT OF HEPATIC FIBROSIS AND REVEALED NOVEL REGULATORS OF HSC ACTIVATION. 2021 5 1966 31 EPIGENETIC ALTERATION OF PRKCDBP IN COLORECTAL CANCERS AND ITS IMPLICATION IN TUMOR CELL RESISTANCE TO TNFALPHA-INDUCED APOPTOSIS. PURPOSE: PRKCDBP IS A PUTATIVE TUMOR SUPPRESSOR IN WHICH ALTERATION HAS BEEN OBSERVED IN SEVERAL HUMAN CANCERS. WE INVESTIGATED EXPRESSION AND FUNCTION OF PRKCDBP IN COLORECTAL CELLS AND TISSUES TO EXPLORE ITS CANDIDACY AS A SUPPRESSOR IN COLORECTAL TUMORIGENESIS. EXPERIMENTAL DESIGN: EXPRESSION AND METHYLATION STATUS OF PRKCDBP AND ITS EFFECT ON TUMOR GROWTH WERE EVALUATED. TRANSCRIPTIONAL REGULATION BY NF-KAPPAB SIGNALING WAS DEFINED BY LUCIFERASE REPORTER AND CHROMATIN IMMUNOPRECIPITATION ASSAYS. RESULTS: PRKCDBP EXPRESSION WAS HARDLY DETECTABLE IN 29 OF 80 (36%) PRIMARY TUMORS AND 11 OF 19 (58%) CELL LINES, AND ITS ALTERATION CORRELATED WITH TUMOR STAGE AND GRADE. PROMOTER HYPERMETHYLATION WAS COMMONLY FOUND IN CANCERS. PRKCDBP EXPRESSION INDUCED THE G(1) CELL-CYCLE ARREST AND INCREASED CELLULAR SENSITIVITY TO VARIOUS APOPTOTIC STRESSES. PRKCDBP WAS INDUCED BY TNFALPHA, AND ITS LEVEL CORRELATED WITH TUMOR CELL SENSITIVITY TO TNFALPHA-INDUCED APOPTOSIS. PRKCDBP INDUCTION BY TNFALPHA WAS DISRUPTED BY BLOCKING NF-KAPPAB SIGNALING WHILE IT WAS ENHANCED BY RELA TRANSFECTION. THE PRKCDBP PROMOTER ACTIVITY WAS INCREASED IN RESPONSE TO TNFALPHA, AND THIS RESPONSE WAS ABOLISHED BY DISRUPTION OF A KAPPAB SITE IN THE PROMOTER. PRKCDBP DELAYED THE FORMATION AND GROWTH OF XENOGRAFT TUMORS AND IMPROVED TUMOR RESPONSE TO TNFALPHA-INDUCED APOPTOSIS. CONCLUSIONS: PRKCDBP IS A PROAPOPTOTIC TUMOR SUPPRESSOR WHICH IS COMMONLY ALTERED IN COLORECTAL CANCER BY PROMOTER HYPERMETHYLATION, AND ITS GENE TRANSCRIPTION IS DIRECTLY ACTIVATED BY NF-KAPPAB IN RESPONSE TO TNFALPHA. THIS SUGGESTS THAT PRKCDBP INACTIVATION MAY CONTRIBUTE TO TUMOR PROGRESSION BY REDUCING CELLULAR SENSITIVITY TO TNFALPHA AND OTHER STRESSES, PARTICULARLY UNDER CHRONIC INFLAMMATORY MICROENVIRONMENT. 2011 6 5088 35 PIPERLONGUMINE REGULATES EPIGENETIC MODULATION AND ALLEVIATES PSORIASIS-LIKE SKIN INFLAMMATION VIA INHIBITION OF HYPERPROLIFERATION AND INFLAMMATION. PSORIASIS IS AN AUTOIMMUNE SKIN DISEASE, WHERE CHRONIC IMMUNE RESPONSES DUE TO EXAGGERATED CYTOKINE SIGNALING, ABNORMAL DIFFERENTIATION, AND EVASION OF KERATINOCYTES APOPTOSIS PLAYS A CRUCIAL ROLE IN MEDIATING ABNORMAL KERATINOCYTES HYPERPROLIFERATION. FROM THE THERAPEUTIC PERSPECTIVE, THE MOLECULES WITH STRONG ANTI-PROLIFERATIVE AND ANTI-INFLAMMATORY PROPERTIES COULD HAVE TREMENDOUS RELEVANCE. IN THIS STUDY, WE DEMONSTRATED THAT PIPERLONGUMINE (PPL) TREATMENT EFFECTIVELY ABROGATED THE HYPERPROLIFERATION AND DIFFERENTIATION OF KERATINOCYTES BY INDUCING ROS-MEDIATED LATE APOPTOSIS WITH LOSS OF MITOCHONDRIAL MEMBRANE POTENTIAL. BESIDES, THE ARREST OF CELL CYCLE WAS FOUND AT SUB-G1 PHASE AS A RESULT OF DNA FRAGMENTATION. MOLECULARLY, INHIBITION OF STAT3 AND AKT SIGNALING WAS OBSERVED WITH A DECREASE IN PROLIFERATIVE MARKERS SUCH AS PCNA, KI67, AND CYCLIN D1 ALONG WITH ANTI-APOPTOTIC BCL-2 PROTEIN EXPRESSION. KERATIN 17 IS A CRITICAL REGULATOR OF KERATINOCYTE DIFFERENTIATION, AND IT WAS FOUND TO BE DOWNREGULATED WITH PPL SIGNIFICANTLY. FURTHERMORE, PROMINENT ANTI-INFLAMMATORY EFFECTS WERE OBSERVED BY INHIBITION OF LIPOPOLYSACCHARIDE (LPS)/IMIQUIMOD (IMQ)-INDUCED P65 NF-KAPPAB SIGNALING CASCADE AND STRONGLY INHIBITED THE PRODUCTION OF CYTOKINE STORM INVOLVED IN PSORIASIS-LIKE SKIN INFLAMMATION, THUS LED TO THE RESTORATION OF NORMAL EPIDERMAL ARCHITECTURE WITH REDUCTION OF EPIDERMAL HYPERPLASIA AND SPLENOMEGALY. IN ADDITION, PPL EPIGENETICALLY INHIBITED HISTONE-MODIFYING ENZYMES, WHICH INCLUDE HISTONE DEACETYLASES (HDACS) OF CLASS I (HDAC1-4) AND CLASS II (HDAC6) EVALUATED BY IMMUNOBLOTTING AND HDAC ENZYME ASSAY KIT. IN ADDITION, OUR RESULTS SHOW THAT PPL EFFECTIVELY INHIBITS THE NUCLEAR TRANSLOCATION OF P65 AND A HISTONE MODULATOR HDAC3, THUS SEQUESTERED IN THE CYTOPLASM OF MACROPHAGES. FURTHERMORE, PPL EFFECTIVELY ENHANCED THE PROTEIN-PROTEIN INTERACTIONS OF HDAC3 AND P65 WITH IKAPPABALPHA, WHICH WAS DISRUPTED BY LPS STIMULATION AND WERE EVALUATED BY CO-IP AND MOLECULAR MODELING. COLLECTIVELY, OUR FINDINGS INDICATE THAT PIPERLONGUMINE MAY SERVE AS AN ANTI-PROLIFERATIVE AND ANTI-INFLAMMATORY AGENT AND COULD SERVE AS A POTENTIAL THERAPEUTIC OPTION IN TREATING PSORIASIS. 2020 7 3468 30 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 8 6294 26 THE PROINFLAMMATORY CYTOKINE TNFALPHA INDUCES DNA DEMETHYLATION-DEPENDENT AND -INDEPENDENT ACTIVATION OF INTERLEUKIN-32 EXPRESSION. IL-32 IS A CYTOKINE INVOLVED IN PROINFLAMMATORY IMMUNE RESPONSES TO BACTERIAL AND VIRAL INFECTIONS. HOWEVER, THE ROLE OF EPIGENETIC EVENTS IN THE REGULATION OF IL-32 GENE EXPRESSION IS UNDERSTUDIED. HERE WE SHOW THAT IL-32 IS REPRESSED BY DNA METHYLATION IN HEK293 CELLS. USING CHIP SEQUENCING, LOCUS-SPECIFIC METHYLATION ANALYSIS, CRISPR/CAS9-MEDIATED GENOME EDITING, AND RT-QPCR (QUANTITATIVE RT-PCR) AND IMMUNOBLOT ASSAYS, WE FOUND THAT SHORT-TERM TREATMENT (A FEW HOURS) WITH THE PROINFLAMMATORY CYTOKINE TUMOR NECROSIS FACTOR ALPHA (TNFALPHA) ACTIVATES IL-32 IN A DNA DEMETHYLATION-INDEPENDENT MANNER. IN CONTRAST, PROLONGED TNFALPHA TREATMENT (SEVERAL DAYS) INDUCED DNA DEMETHYLATION AT THE PROMOTER AND A CPG ISLAND IN THE IL-32 GENE IN A TET (TEN-ELEVEN TRANSLOCATION) FAMILY ENZYME- AND NF-KAPPAB-DEPENDENT MANNER. NOTABLY, THE HYPOMETHYLATION STATUS OF TRANSCRIPTIONAL REGULATORY ELEMENTS IN IL-32 WAS MAINTAINED FOR A LONG TIME (SEVERAL WEEKS), CAUSING ELEVATED IL-32 EXPRESSION EVEN IN THE ABSENCE OF TNFALPHA. CONSIDERING THAT IL-32 CAN, IN TURN, INDUCE TNFALPHA EXPRESSION, WE SPECULATE THAT SUCH FEEDFORWARD EVENTS MAY CONTRIBUTE TO THE TRANSITION FROM AN ACUTE INFLAMMATORY RESPONSE TO CHRONIC INFLAMMATION. 2019 9 3049 30 GENOME-WIDE ANALYSIS REVEALS ZINC TRANSPORTER ZIP9 REGULATED BY DNA METHYLATION PROMOTES RADIATION-INDUCED SKIN FIBROSIS VIA THE TGF-BETA SIGNALING PATHWAY. RADIATION-INDUCED SKIN FIBROSIS IS A DETRIMENTAL AND CHRONIC DISORDER THAT OCCURS AFTER RADIATION EXPOSURE. DNA METHYLATION HAS BEEN CHARACTERIZED AS AN IMPORTANT REGULATORY MECHANISM OF MULTIPLE PATHOLOGICAL PROCESSES. IN THIS STUDY, WE COMPARED THE GENOME-WIDE DNA METHYLATION STATUS IN RADIATION-INDUCED FIBROTIC SKIN AND ADJACENT NORMAL TISSUES OF RATS BY METHYLATED DNA IMMUNOPRECIPITATION SEQUENCING. RADIATION-INDUCED FIBROTIC SKIN SHOWED DIFFERENTIALLY METHYLATED REGIONS ASSOCIATED WITH 3,650 PROTEIN-CODING GENES, 72 MICRORNAS, 5,836 LONG NONCODING RNAS AND 3 PIWI-INTERACTING RNAS. BY INTEGRATING THE MRNA AND METHYLATION PROFILES, THE ZINC TRANSPORTER SLC39A9/ZIP9 WAS INVESTIGATED IN GREATER DETAIL. THE PROTEIN LEVEL OF ZIP9 WAS INCREASED IN IRRADIATED SKIN TISSUES OF HUMANS, MONKEYS, AND RATS, ESPECIALLY IN RADIOGENIC FIBROTIC SKIN TISSUES. RADIATION INDUCED THE DEMETHYLATION OF A CPG DINUCLEOTIDE IN EXON 1 OF ZIP9 THAT RESULTED IN RECRUITMENT OF THE TRANSCRIPTIONAL FACTOR SP1 AND INCREASED ZIP9 EXPRESSION. OVEREXPRESSION OF ZIP9 RESULTED IN ACTIVATION OF THE PROFIBROTIC TRANSFORMING GROWTH FACTOR-BETA SIGNALING PATHWAY THROUGH PROTEIN KINASE B IN HUMAN FIBROBLASTS. IN ADDITION, RADIATION-INDUCED SKIN FIBROSIS WAS ASSOCIATED WITH INCREASED ZINC ACCUMULATION. THE ZINC CHELATOR N,N,N',N'-TETRAKIS(2-PYRIDYLMETHYL)-1,2-ETHYLENEDIAMINE ABROGATED ZIP9-INDUCED ACTIVATION OF THE TRANSFORMING GROWTH FACTOR-BETA SIGNALING PATHWAY AND ATTENUATED RADIATION-INDUCED SKIN FIBROSIS IN A RAT MODEL. IN SUMMARY, OUR FINDINGS ILLUSTRATE EPIGENETIC REGULATION OF ZIP9 AND ITS CRITICAL ROLE IN PROMOTING RADIATION-INDUCED SKIN FIBROSIS. 2020 10 6549 34 TRANSFORMING GROWTH FACTOR BETA INHIBITS MUC5AC EXPRESSION BY SMAD3/HDAC2 COMPLEX FORMATION AND NF-KAPPAB DEACETYLATION AT K310 IN NCI-H292 CELLS. AIRWAY MUCUS SECRETION IS AN ESSENTIAL INNATE IMMUNE RESPONSE FOR HOST PROTECTION. HOWEVER, OVERPRODUCTION AND HYPERSECRETION OF MUCUS, MAINLY COMPOSED OF THE GEL- FORMING MUC5AC PROTEIN, ARE SIGNIFICANT RISK FACTORS FOR PATIENTS WITH ASTHMA AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD). THE TRANSFORMING GROWTH FACTOR BETA (TGFBETA) SIGNALING PATHWAY NEGATIVELY REGULATES MUC5AC EXPRESSION; HOWEVER, THE UNDERLYING MOLECULAR MECHANISM IS NOT FULLY UNDERSTOOD. HERE, WE SHOWED THAT TGFBETA SIGNIFICANTLY REDUCES THE EXPRESSION OF MUC5AC MRNA AND ITS PROTEIN IN NCI-H292 CELLS, A HUMAN MUCOEPIDERMOID CARCINOMA CELL LINE. THIS REDUCED MUC5AC EXPRESSION WAS RESTORED BY A TGFBETA RECEPTOR INHIBITOR (SB431542), BUT NOT BY THE INHIBITION OF NF-KAPPAB (BAY11-7082 OR TRIPTOLIDE) OR PI3K (LY294002) ACTIVITIES. TGFBETA-ACTIVATED SMAD3 DOSE-DEPENDENTLY BOUND TO MUC5AC PROMOTER. NOTABLY, TGFBETA-ACTIVATED SMAD3 RECRUITED HDAC2 AND FACILITATED NUCLEAR TRANSLOCATION OF HDAC2, THEREBY INDUCING THE DEACETYLATION OF NF-KAPPAB AT K310, WHICH IS ESSENTIAL FOR A REDUCTION IN NF-KAPPAB TRANSCRIPTIONAL ACTIVITY. BOTH TGFBETA-INDUCED NUCLEAR TRANSLOCATION OF SMAD3/HDAC2 AND DEACETYLATION OF NF-KAPPAB AT K310 WERE SUPPRESSED BY A SMAD3 INHIBITOR (SIS3). THESE RESULTS SUGGEST THAT THE TGFBETA-ACTIVATED SMAD3/HDAC2 COMPLEX IS AN ESSENTIAL NEGATIVE REGULATOR FOR MUC5AC EXPRESSION AND AN EPIGENETIC REGULATOR FOR NF-KAPPAB ACETYLATION. THEREFORE, THESE RESULTS COLLECTIVELY SUGGEST THAT MODULATION OF THE TGFBETA1/SMAD3/HDAC2/NF-KAPPAB PATHWAY AXIS CAN BE A PROMISING WAY TO IMPROVE LUNG FUNCTION AS A TREATMENT STRATEGY FOR ASTHMA AND COPD. 2021 11 26 33 A 6-ALKYLSALICYLATE HISTONE ACETYLTRANSFERASE INHIBITOR INHIBITS HISTONE ACETYLATION AND PRO-INFLAMMATORY GENE EXPRESSION IN MURINE PRECISION-CUT LUNG SLICES. LYSINE ACETYLATIONS ARE POST-TRANSLATIONAL MODIFICATIONS OF CELLULAR PROTEINS, THAT ARE CRUCIAL IN THE REGULATION OF MANY CELLULAR PROCESSES. LYSINE ACETYLATIONS ON HISTONE PROTEINS ARE PART OF THE EPIGENETIC CODE REGULATING GENE EXPRESSION AND ARE INSTALLED BY HISTONE ACETYLTRANSFERASES. OBSERVATIONS THAT INFLAMMATORY LUNG DISEASES, SUCH AS ASTHMA AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE, ARE CHARACTERIZED BY INCREASED HISTONE ACETYLTRANSFERASE ACTIVITY INDICATE THAT DEVELOPMENT OF SMALL MOLECULE INHIBITORS FOR THESE ENZYMES MIGHT BE A VALUABLE APPROACH TOWARDS NEW THERAPIES FOR THESE DISEASES. THE 6-ALKYLSALICYLATE MG149 IS A CANDIDATE TO EXPLORE THIS HYPOTHESIS BECAUSE IT HAS BEEN DEMONSTRATED TO INHIBIT THE MYST TYPE HISTONE ACETYLTRANSFERASES. IN THIS STUDY, WE DETERMINED THE K(I) VALUE FOR INHIBITION OF THE MYST TYPE HISTONE ACETYLTRANSFERASE KAT8 BY MG149 TO BE 39 +/- 7.7 MUM. UPON INVESTIGATING WHETHER THE INHIBITION OF HISTONE ACETYLTRANSFERASES BY MG149 CORRELATES WITH INHIBITION OF HISTONE ACETYLATION IN MURINE PRECISION-CUT LUNG SLICES, INHIBITION OF ACETYLATION WAS OBSERVED USING AN LC-MS/MS BASED ASSAY ON HISTONE H4 RES 4-17, WHICH CONTAINS THE TARGET LYSINE OF KAT8. FOLLOWING UP ON THIS, UPON TREATMENT WITH MG149, REDUCED PRO-INFLAMMATORY GENE EXPRESSION WAS OBSERVED IN LIPOPOLYSACCHARIDE AND INTERFERON GAMMA STIMULATED MURINE PRECISION-CUT LUNG SLICES. BASED ON THIS, WE PROPOSE THAT 6-ALKYLSALICYLATES SUCH AS MG149 HAVE POTENTIAL FOR DEVELOPMENT TOWARDS APPLICATIONS IN THE TREATMENT OF INFLAMMATORY LUNG DISEASES. 2017 12 4020 35 LOW-MOLECULAR-WEIGHT FIBROBLAST GROWTH FACTOR 2 ATTENUATES HEPATIC FIBROSIS BY EPIGENETIC DOWN-REGULATION OF DELTA-LIKE1. LIVER FIBROSIS, A MAJOR CAUSE OF END-STAGE LIVER DISEASES, IS CLOSELY REGULATED BY MULTIPLE GROWTH FACTORS AND CYTOKINES. THE CORRELATION OF FIBROBLAST GROWTH FACTOR 2 (FGF2) WITH CHRONIC LIVER INJURY HAS BEEN REPORTED, BUT THE EXACT FUNCTIONS OF DIFFERENT FGF2 ISOFORMS IN LIVER FIBROGENESIS REMAIN UNCLEAR. HERE, WE REPORT ON THE DIFFERENTIAL EXPRESSION PATTERNS AND FUNCTIONS OF LOW- AND HIGH-MOLECULAR-WEIGHT FGF2 (NAMELY, FGF2(LMW) AND FGF2(HMW) , RESPECTIVELY) IN HEPATIC FIBROGENESIS USING A CCL4 -INDUCED MOUSE LIVER FIBROSIS MODEL. FGF2(HMW) DISPLAYED A ROBUST INCREASE IN CCL4 -INDUCED HEPATIC FIBROSIS AND PROMOTED FIBROGENESIS. IN CONTRAST, ENDOGENOUS FGF2(LMW) EXHIBITED A SLIGHT INCREASE IN HEPATIC FIBROSIS AND SUPPRESSED THIS PATHOLOGICAL PROGRESSION. MOREOVER, EXOGENOUS ADMINISTRATION OF RECOMBINANT FGF2(LMW) POTENTLY AMELIORATED CCL4 -INDUCED LIVER FIBROSIS. MECHANISTICALLY, WE SHOWED THAT FGF2(LMW) TREATMENT ATTENUATED HEPATIC STELLATE CELL ACTIVATION AND FIBROSIS BY EPIGENETIC DOWN-REGULATION OF DELTA-LIKE 1 EXPRESSION THROUGH THE P38 MITOGEN-ACTIVATED PROTEIN KINASE PATHWAY. CONCLUSION: FGF2(LMW) AND FGF2(HMW) HAVE DISTINCT ROLES IN LIVER FIBROGENESIS. THESE FINDINGS DEMONSTRATE A POTENT ANTIFIBROTIC EFFECT OF FGF2(LMW) ADMINISTRATION, WHICH MAY PROVIDE A NOVEL APPROACH TO TREAT CHRONIC LIVER DISEASES. 2015 13 4233 31 METHYLATION OF SEPTIN9 MEDIATED BY DNMT3A ENHANCES HEPATIC STELLATE CELLS ACTIVATION AND LIVER FIBROGENESIS. LIVER FIBROSIS, RESULTING FROM CHRONIC AND PERSISTENT INJURY TO THE LIVER, IS A WORLDWIDE HEALTH PROBLEM. ADVANCED LIVER FIBROSIS RESULTS IN CIRRHOSIS, LIVER FAILURE AND EVEN HEPATOCELLULAR CANCER (HCC), OFTEN EVENTUALLY REQUIRING LIVER TRANSPLANTATION, POSES A HUGE HEALTH BURDEN ON THE GLOBAL COMMUNITY. HOWEVER, THE SPECIFIC PATHOGENESIS OF LIVER FIBROSIS REMAINS NOT FULLY UNDERSTOOD. NUMEROUS BASIC AND CLINICAL STUDIES HAVE PROVIDED EVIDENCE THAT EPIGENETIC MODIFICATIONS, ESPECIALLY DNA METHYLATION, MIGHT CONTRIBUTE TO THE ACTIVATION OF HEPATIC STELLATE CELLS (HSCS), THE PIVOTAL CELL TYPE RESPONSIBLE FOR THE FIBROUS SCAR IN LIVER. HERE, REDUCED REPRESENTATION BISULFITE SEQUENCING (RRBS) AND BISULFITE PYROSEQUENCING PCR (BSP) ANALYSIS IDENTIFIED HYPERMETHYLATION STATUS OF SEPTIN9 (SEPT9) GENE IN LIVER FIBROGENESIS. SEPT9 PROTEIN WAS DRAMATICALLY DECREASED IN LIVERS OF CCL4-TREATED MICE AND IMMORTALIZED HSC-T6 CELLS EXPOSED TO TGF-BETA1. NEVERTHELESS, THE SUPPRESSION OF SEPT9 COULD BE BLOCKED BY DNMT3A-SIRNA AND DNA METHYLTRANSFERASE INHIBITOR, 5-AZA-2'-DEOXYCYTIDINE (5-AZADC). OVEREXPRESSED SEPT9 ATTENUATED TGF-BETA1-INDUCED EXPRESSION OF MYOFIBROBLAST MARKERS ALPHA-SMA AND COL1A1, ACCOMPANIED BY UP-REGULATION OF CELL APOPTOSIS-RELATED PROTEINS. CONVERSELY, RNAI-MEDIATED SILENCING OF SEPT9 ENHANCED ACCUMULATION OF EXTRACELLULAR MATRIX. THESE OBSERVATIONS SUGGESTED THAT SEPT9 CONTRIBUTED TO ALLEVIATE LIVER FIBROSIS MIGHT PARTIALLY THROUGH PROMOTING ACTIVATED HSCS APOPTOSIS AND THIS ANTI-FIBROGENESIS EFFECT MIGHT BE BLOCKED BY DNMT-3A MEDIATED METHYLATION OF SEPT9. THEREFORE, PHARMACOLOGICAL AGENTS THAT INHIBIT SEPT9 METHYLATION AND INCREASE ITS EXPRESSION COULD BE CONSIDERED AS VALUABLE TREATMENTS FOR LIVER FIBROSIS. 2017 14 3128 39 GIPC-REGULATED IGFBP-3 PROMOTES HSC MIGRATION IN VITRO AND PORTAL HYPERTENSION IN VIVO THROUGH A BETA1-INTEGRIN PATHWAY. BACKGROUND & AIMS: TRANSFORMING GROWTH FACTOR (TGF-BETA)-INDUCED ACTIVATION OF QUIESCENT HEPATIC STELLATE CELLS (HSCS) AND THEIR TRANSFORMATION TO MYOFIBROBLASTS IS A KEY EVENT IN LIVER FIBROSIS AND PORTAL HYPERTENSION. GIPC (ALSO REFERRED TO AS SYNECTIN) IS A DOWNSTREAM SIGNAL ACTIVATION MOLECULE OF TGF-BETA AND OTHER RECEPTORS. IN THIS STUDY, WE SOUGHT TO IDENTIFY NOVEL GENES TARGETED BY TGF-BETA AND GIPC AND ELUCIDATE IF AND HOW THEY MAY CONTRIBUTE TO LIVER FIBROSIS. METHODS: WE PERFORMED SEQUENTIAL MESSENGER RNA SEQUENCING ANALYSIS ON TGF-BETA-STIMULATED HSCS AND THEN ON TGF-BETA-STIMULATED HSCS IN THE PRESENCE AND ABSENCE OF GIPC ALSO REFERRED TO AS SYNECTIN (GIPC) KNOCKDOWN. INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 (IGFBP-3) TRANSPORT PROTEIN EMERGED AS A TOP ACTIVATION TARGET OF BOTH TGF-BETA AND GIPC. QUANTITATIVE POLYMERASE CHAIN REACTION, ENZYME-LINKED IMMUNOSORBENT ASSAY, TARGETED CHROMATIN IMMUNOPRECIPITATION, AND WESTERN BLOT ANALYSIS WERE DONE FOR FURTHER CONFIRMATION. RESULTS: IGFBP-3, AN INSULIN GROWTH FACTOR TRANSPORT PROTEIN, EMERGED AS A TOP ACTIVATION TARGET OF BOTH TGF-BETA AND GIPC, WHICH WAS CONFIRMED BY QUANTITATIVE POLYMERASE CHAIN REACTION, ENZYME-LINKED IMMUNOSORBENT ASSAY, AND WESTERN BLOT ANALYSIS. TARGETED CHROMATIN IMMUNOPRECIPITATION SHOWED THAT GIPC INCREASES THE HISTONE 3 LYSINE 27 (H3K27) ACETYLATION ACTIVATING MARK AND CONCURRENTLY DECREASES THE H3K27 INHIBITORY TRIMETHYLATION (H3K27M3) MARK, PROVIDING AN EPIGENETIC CORRELATE TO THE GENE REGULATION CHANGES. IN VIVO, GLOBAL KNOCKOUT OF IGFBP-3 MICE RESULTED IN ATTENUATION OF HSC ACTIVATION MARKERS AND ATTENUATION OF PORTAL PRESSURE IN RESPONSE TO CHRONIC LIVER INJURY MODELS. ANALYSIS OF SERUM LEVELS FROM CIRRHOTIC PATIENTS ALSO SHOWED AN IGFBP-3 INCREASE OF MORE THAN 2-FOLD COMPARED WITH HEALTHY CONTROLS. FINALLY, IN VITRO MECHANISM STUDIES SHOWED THAT IGFBP-3 PROMOTES HSC MIGRATION THROUGH INTEGRIN-DEPENDENT PHOSPHORYLATION OF PROTEIN KINASE B. CONCLUSIONS: TGF-BETA UP-REGULATES IGFBP-3 THROUGH GIPC, LEADING TO INCREASED HSC MIGRATION IN VITRO AND PROMOTES PORTAL HYPERTENSION IN VIVO. THESE STUDIES SUPPORT THE ROLE OF IGFBP-3 AS A POTENTIAL PATHOPHYSIOLOGIC TARGET OR BIOMARKER IN CHRONIC LIVER DISEASE. 2020 15 222 24 ACUTE LIVER STEATOSIS TRANSLATIONALLY CONTROLS THE EPIGENETIC REGULATOR MIER1 TO PROMOTE LIVER REGENERATION IN A STUDY WITH MALE MICE. THE EARLY PHASE LIPID ACCUMULATION IS ESSENTIAL FOR LIVER REGENERATION. HOWEVER, WHETHER THIS ACUTE LIPID ACCUMULATION CAN SERVE AS SIGNALS TO DIRECT LIVER REGENERATION RATHER THAN SIMPLY PROVIDING BUILDING BLOCKS FOR CELL PROLIFERATION REMAINS UNCLEAR. THROUGH IN VIVO CRISPR SCREENING, WE IDENTIFY MIER1 (MESODERM INDUCTION EARLY RESPONSE 1) AS A KEY EPIGENETIC REGULATOR THAT BRIDGES THE ACUTE LIPID ACCUMULATION AND CELL CYCLE GENE EXPRESSION DURING LIVER REGENERATION IN MALE ANIMALS. PHYSIOLOGICALLY, LIVER ACUTE LIPID ACCUMULATION INDUCES THE PHOSPHORYLATION OF EIF2S1(EUKARYOTIC TRANSLATION INITIATION FACTOR 2), WHICH CONSEQUENTLY ATTENUATED MIER1 TRANSLATION. MIER1 DOWNREGULATION IN TURN PROMOTES CELL CYCLE GENE EXPRESSION AND REGENERATION THROUGH CHROMATIN REMODELING. IMPORTANTLY, THE LIPIDS-EIF2S1-MIER1 PATHWAY IS IMPAIRED IN ANIMALS WITH CHRONIC LIVER STEATOSIS; WHEREAS MIER1 DEPLETION SIGNIFICANTLY IMPROVES REGENERATION IN THESE ANIMALS. TAKEN TOGETHER, OUR STUDIES IDENTIFY AN EPIGENETIC MECHANISM BY WHICH THE EARLY PHASE LIPID REDISTRIBUTION FROM ADIPOSE TISSUE TO LIVER DURING REGENERATION IMPACTS HEPATOCYTE PROLIFERATION, AND SUGGEST A POTENTIAL STRATEGY TO BOOST LIVER REGENERATION. 2023 16 3327 32 HISTONE DEACETYLASE 4 PROMOTES CHOLESTATIC LIVER INJURY IN THE ABSENCE OF PROHIBITIN-1. PROHIBITIN-1 (PHB1) IS AN EVOLUTIONARILY CONSERVED PLEIOTROPIC PROTEIN THAT PARTICIPATES IN DIVERSE PROCESSES DEPENDING ON ITS SUBCELLULAR LOCALIZATION AND INTERACTOME. RECENT DATA HAVE INDICATED A DIVERSE ROLE FOR PHB1 IN THE PATHOGENESIS OF OBESITY, CANCER, AND INFLAMMATORY BOWEL DISEASE, AMONG OTHERS. DATA PRESENTED HERE SUGGEST THAT PHB1 IS ALSO LINKED TO CHOLESTATIC LIVER DISEASE. EXPRESSION OF PHB1 IS MARKEDLY REDUCED IN PATIENTS WITH PRIMARY BILIARY CIRRHOSIS AND BILIARY ATRESIA OR WITH ALAGILLE SYNDROME, TWO MAJOR PEDIATRIC CHOLESTATIC CONDITIONS. IN THE EXPERIMENTAL MODEL OF BILE DUCT LIGATION, SILENCING OF PHB1 INDUCED LIVER FIBROSIS, REDUCED ANIMAL SURVIVAL, AND INDUCED BILE DUCT PROLIFERATION. IMPORTANTLY, THE MODULATORY EFFECT OF PHB1 IS NOT DEPENDENT ON ITS KNOWN MITOCHONDRIAL FUNCTION. ALSO, PHB1 INTERACTS WITH HISTONE DEACETYLASE 4 (HDAC4) IN THE PRESENCE OF BILE ACIDS. HENCE, PHB1 DEPLETION LEADS TO INCREASED NUCLEAR HDAC4 CONTENT AND ITS ASSOCIATED EPIGENETIC CHANGES. REMARKABLY, HDAC4 SILENCING AND THE ADMINISTRATION OF THE HDAC INHIBITOR PARTHENOLIDE DURING OBSTRUCTIVE CHOLESTASIS IN VIVO PROMOTE GENOMIC REPROGRAMMING, LEADING TO REGRESSION OF THE FIBROTIC PHENOTYPE IN LIVER-SPECIFIC PHB1 KNOCKOUT MICE. CONCLUSION: PHB1 IS AN IMPORTANT MEDIATOR OF CHOLESTATIC LIVER INJURY THAT REGULATES THE ACTIVITY OF HDAC4, WHICH CONTROLS SPECIFIC EPIGENETIC MARKERS; THESE RESULTS IDENTIFY POTENTIAL NOVEL STRATEGIES TO TREAT LIVER INJURY AND FIBROSIS, PARTICULARLY AS A CONSEQUENCE OF CHRONIC CHOLESTASIS. 2015 17 1210 32 COX2 ENHANCES NEOVASCULARIZATION OF INFLAMMATORY TENOCYTES THROUGH THE HIF-1ALPHA/VEGFA/PDGFB PATHWAY. TENDON INJURIES ARE AMONG THE MOST CHALLENGING IN ORTHOPEDICS. DURING THE EARLY TENDON REPAIR, NEW BLOOD VESSEL FORMATION IS NECESSARY. HOWEVER, EXCESSIVE ANGIOGENESIS ALSO EXACERBATES SCAR FORMATION, LEADING TO PAIN AND DYSFUNCTION. A SIGNIFICANTLY WORSE OUTCOME WAS ASSOCIATED WITH HIGHER EXPRESSION LEVELS OF HYPOXIA-INDUCIBLE FACTOR-1 ALPHA (HIF-1ALPHA), AND ITS TRANSCRIPTIONAL TARGETS VASCULAR ENDOTHELIAL GROWTH FACTOR A (VEGFA) AND PLATELET-DERIVED GROWTH FACTOR B (PDGFB), BUT THE UNDERLYING MOLECULAR MECHANISMS REMAIN UNCLEAR. IN THIS STUDY, LIPOPOLYSACCHARIDE (LPS) WAS USED TO INDUCE AN INFLAMMATORY RESPONSE IN TENOCYTES. LPS INCREASED THE TENOCYTES' INFLAMMATORY FACTOR COX2 EXPRESSION AND ACTIVATED THE HIF-1ALPHA/VEGFA/PDGFB PATHWAY. MOREOVER, THE CONDITIONED MEDIUM FROM THE TENOCYTES BOOSTED RAT AORTIC VASCULAR ENDOTHELIAL CELL (RAOEC) ANGIOGENESIS. FURTHERMORE, TRICHOSTATIN A (TSA), AN INHIBITOR OF HISTONE DEACETYLASE, WAS USED TO TREAT INFLAMMATORY TENOCYTES. THE EXPRESSION LEVELS OF HIF-1ALPHA AND ITS TRANSCRIPTIONAL TARGETS VEGFA AND PDGFB DECREASED, RESULTING IN RAOEC ANGIOGENESIS INHIBITION. FINALLY, THE DUAL-LUCIFERASE REPORTER GENE ASSAY AND CHROMATIN IMMUNOPRECIPITATION (CHIP) ASSAY PROVED THAT THE HIF-1ALPHA/PDGFB PATHWAY PLAYED A MORE CRITICAL ROLE IN TENOCYTE ANGIOGENESIS THAN THE HIF-1ALPHA/VEGFA PATHWAY. TSA COULD ALLEVIATE ANGIOGENESIS MAINLY THROUGH EPIGENETIC REGULATION OF THE HIF-1ALPHA/PDGFB PATHWAY. TAKEN TOGETHER, TSA MIGHT BE A PROMISING ANTI-ANGIOGENESIS DRUG FOR ABNORMAL ANGIOGENESIS, WHICH IS INDUCED BY TENDON INJURIES. 2021 18 2244 30 EPIGENETIC MODULATION OF COLLAGEN 1A1: THERAPEUTIC IMPLICATIONS IN FIBROSIS AND ENDOMETRIOSIS. PROGRESSIVE FIBROSIS IS RECALCITRANT TO CONVENTIONAL THERAPY AND COMMONLY COMPLICATES CHRONIC DISEASES AND SURGICAL HEALING. WE EVALUATE HERE A NOVEL MECHANISM THAT REGULATES SCAR-TISSUE COLLAGEN (COL1A1/COL1A1) EXPRESSION AND CHARACTERIZES ITS TRANSLATIONAL RELEVANCE AS A TARGETED THERAPY FOR FIBROSIS IN AN ENDOMETRIOSIS DISEASE MODEL. ENDOMETRIOSIS IS CAUSED BY DISPLACEMENT AND IMPLANTATION OF UTERINE ENDOMETRIUM ONTO ABDOMINAL ORGANS AND SPREADS WITH PROGRESSIVE SCARRING. TRANSCRIPTION FACTOR KLF11 IS SPECIFICALLY DIMINISHED IN ENDOMETRIOSIS LESIONS. LOSS OF KLF11-MEDIATED REPRESSION OF COL1A1/COL1A1 EXPRESSION RESULTED IN INCREASED FIBROSIS. TO DETERMINE THE BIOLOGICAL SIGNIFICANCE OF COL1A1/COL1A1 EXPRESSION ON FIBROSIS, WE MODULATED ITS EXPRESSION. IN HUMAN ENDOMETRIAL-STROMAL FIBROBLASTS, KLF11 RECRUITED SIN3A/HDAC (HISTONE DEACETYLASE), RESULTING IN COL1A1-PROMOTER DEACETYLATION AND REPRESSION. THIS ROLE OF KLF11 WAS PHARMACOLOGICALLY REPLICATED BY A HISTONE ACETYL TRANSFERASE INHIBITOR (GARCINOL). IN CONTRAST, OPPOSITE EFFECTS WERE OBTAINED WITH A HDAC INHIBITOR (SUBEROYL ANILIDE HYDROXAMIC ACID), CONFIRMING REGULATORY SPECIFICITY FOR THESE RECIPROCALLY ACTIVE EPIGENETIC MECHANISMS. FIBROSIS WAS CONCORDANTLY REVERSED IN KLF11(-/-)ANIMALS BY HISTONE ACETYL TRANSFERASE INHIBITOR AND IN WILD-TYPE ANIMALS BY HDAC INHIBITOR TREATMENTS. ABERRANT LESIONAL COL1A1 REGULATION IS SIGNIFICANT BECAUSE FIBROSIS DEPENDED ON LESION RATHER THAN HOST GENOTYPE. THIS IS THE FIRST REPORT DEMONSTRATING FEASIBILITY FOR TARGETED PHARMACOLOGICAL REVERSAL OF FIBROSIS, AN INTRACTABLE PHENOTYPE OF DIVERSE CHRONIC DISEASES. 2016 19 984 27 CHRONIC PSYCHOLOGICAL STRESS ALTERS GENE EXPRESSION IN RAT COLON EPITHELIAL CELLS PROMOTING CHROMATIN REMODELING, BARRIER DYSFUNCTION AND INFLAMMATION. CHRONIC STRESS IS COMMONLY ASSOCIATED WITH ENHANCED ABDOMINAL PAIN (VISCERAL HYPERSENSITIVITY), BUT THE CELLULAR MECHANISMS UNDERLYING HOW CHRONIC STRESS INDUCES VISCERAL HYPERSENSITIVITY ARE POORLY UNDERSTOOD. IN THIS STUDY, WE EXAMINED CHANGES IN GENE EXPRESSION IN COLON EPITHELIAL CELLS FROM A RAT MODEL USING RNA-SEQUENCING TO EXAMINE STRESS-INDUCED CHANGES TO THE TRANSCRIPTOME. FOLLOWING CHRONIC STRESS, THE MOST SIGNIFICANTLY UP-REGULATED GENES INCLUDED ATG16L1, COQ10B, DCAF13, NAT2, PTBP2, RRAS2, SPINK4 AND DOWN-REGULATED GENES INCLUDING ABAT, CITED2, CNNM2, DAB2IP, PLEKHM1, SCD2, AND TAB2. THE PRIMARY ALTERED BIOLOGICAL PROCESSES REVEALED BY NETWORK ENRICHMENT ANALYSIS WERE INFLAMMATION/IMMUNE RESPONSE, TISSUE MORPHOGENESIS AND DEVELOPMENT, AND NUCLEOSOME/CHROMATIN ASSEMBLY. THE MOST SIGNIFICANTLY DOWN-REGULATED PROCESS WAS THE DIGESTIVE SYSTEM DEVELOPMENT/FUNCTION, WHEREAS THE MOST SIGNIFICANTLY UP-REGULATED PROCESSES WERE INFLAMMATORY RESPONSE, ORGANISMAL INJURY, AND CHROMATIN REMODELING MEDIATED BY H3K9 METHYLATION. FURTHERMORE, A SUBPOPULATION OF STRESSED RATS DEMONSTRATED VERY SIGNIFICANTLY ALTERED GENE EXPRESSION AND TRANSCRIPT ISOFORMS, ENRICHED FOR THE DIFFERENTIAL EXPRESSION OF GENES INVOLVED IN THE INFLAMMATORY RESPONSE, INCLUDING UPREGULATION OF CYTOKINE AND CHEMOKINE RECEPTOR GENE EXPRESSION COUPLED WITH DOWNREGULATION OF EPITHELIAL ADHERENS AND TIGHT JUNCTION MRNAS. IN SUMMARY, THESE FINDINGS SUPPORT THAT CHRONIC STRESS IS ASSOCIATED WITH INCREASED LEVELS OF CYTOKINES AND CHEMOKINES, THEIR DOWNSTREAM SIGNALING PATHWAYS COUPLED TO DYSREGULATION OF INTESTINAL CELL DEVELOPMENT AND FUNCTION. EPIGENETIC REGULATION OF CHROMATIN REMODELING LIKELY PLAYS A PROMINENT ROLE IN THIS PROCESS. RESULTS ALSO SUGGEST THAT SUPER ENHANCERS PLAY A PRIMARY ROLE IN CHRONIC STRESS-ASSOCIATED INTESTINAL BARRIER DYSFUNCTION. 2022 20 4001 39 LOSS OF MEN1 LEADS TO RENAL FIBROSIS AND DECREASES HGF-ADAMTS5 PATHWAY ACTIVITY VIA AN EPIGENETIC MECHANISM. BACKGROUND: RENAL FIBROSIS IS A SERIOUS CONDITION THAT RESULTS IN THE DEVELOPMENT OF CHRONIC KIDNEY DISEASES. THE MEN1 GENE IS AN EPIGENETIC REGULATOR THAT ENCODES THE MENIN PROTEIN AND ITS ROLE IN KIDNEY TISSUE REMAINS UNCLEAR. METHODS: KIDNEY HISTOLOGY WAS EXAMINED ON PARAFFIN SECTIONS STAINED WITH HEMATOXYLIN-EOSIN STAINING. MASSON'S TRICHROME STAINING AND SIRIUS RED STAINING WERE USED TO ANALYZE RENAL FIBROSIS. GENE AND PROTEIN EXPRESSION WERE DETERMINED BY QUANTITATIVE REAL-TIME PCR (QPCR) AND WESTERN BLOT, RESPECTIVELY. IMMUNOHISTOCHEMISTRY STAINING IN THE KIDNEY TISSUES FROM MICE OR PATIENTS WAS USED TO EVALUATE PROTEIN LEVELS. FLOW CYTOMETRY WAS USED TO ANALYZE THE CELL CYCLE DISTRIBUTIONS AND APOPTOSIS. RNA-SEQUENCING WAS PERFORMED FOR DIFFERENTIAL EXPRESSION GENES IN THE KIDNEY TISSUES OF THE MEN1F/F AND MEN1?/? MICE. CHROMATIN IMMUNOPRECIPITATION SEQUENCING (CHIP-SEQ) WAS CARRIED OUT FOR IDENTIFICATION OF MENIN- AND H3K4ME3-ENRICHED REGIONS WITHIN THE WHOLE GENOME IN THE MOUSE KIDNEY TISSUE. CHIP-QPCR ASSAYS WERE PERFORMED FOR OCCUPANCY OF MENIN AND H3K4ME3 AT THE GENE PROMOTER REGIONS. LUCIFERASE REPORTER ASSAY WAS USED TO DETECT THE PROMOTER ACTIVITY. THE EXACERBATED UNILATERAL URETERAL OBSTRUCTION (UUO) MODELS IN THE MEN1F/F AND MEN1?/? MICE WERE USED TO ASSESS THE PHARMACOLOGICAL EFFECTS OF RH-HGF ON RENAL FIBROSIS. RESULTS: THE EXPRESSION OF MEN1 IS REDUCE IN KIDNEY TISSUES OF FIBROTIC MOUSE AND HUMAN DIABETIC PATIENTS AND TREATMENT WITH FIBROTIC FACTOR RESULTS IN THE DOWNREGULATION OF MEN1 EXPRESSION IN RENAL TUBULAR EPITHELIAL CELLS (RTECS). DISRUPTION OF MEN1 IN RTECS LEADS TO HIGH EXPRESSION OF ALPHA-SMA AND COLLAGEN 1, WHEREAS MEN1 OVEREXPRESSION RESTRAINS EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT) INDUCED BY TGF-BETA TREATMENT. CONDITIONAL KNOCKOUT OF MEN1 RESULTED IN CHRONIC RENAL FIBROSIS AND UUO-INDUCED TUBULOINTERSTITIAL FIBROSIS (TIF), WHICH IS ASSOCIATED WITH AN INCREASED INDUCTION OF EMT, G2/M ARREST AND JNK SIGNALING. MECHANISTICALLY, MENIN RECRUITS AND INCREASES H3K4ME3 AT THE PROMOTER REGIONS OF HEPATOCYTE GROWTH FACTOR (HGF) AND A DISINTEGRIN AND METALLOPROTEINASE WITH THROMBOSPONDIN MOTIFS 5 (ADAMTS5) GENES AND ENHANCES THEIR TRANSCRIPTIONAL ACTIVATION. IN THE UUO MICE MODEL, EXOGENOUS HGF RESTORED THE EXPRESSION OF ADAMTS5 AND AMELIORATED RENAL FIBROSIS INDUCED BY MEN1 DEFICIENCY. CONCLUSIONS: THESE FINDINGS DEMONSTRATE THAT MEN1 IS AN ESSENTIAL ANTIFIBROTIC FACTOR IN RENAL FIBROGENESIS AND COULD BE A POTENTIAL TARGET FOR ANTIFIBROTIC THERAPY. 2022