Dataset Information

Methylation of RCAN1.4 mediated by DNMT1 and DNMT3b enhances hepatic stellate cell activation and liver fibrogenesis through Calcineurin/NFAT3 signaling.

ABSTRACT: Background: Liver fibrosis is characterized by extensive deposition of extracellular matrix (ECM) components in the liver. RCAN1 (regulator of calcineurin 1), an endogenous inhibitor of calcineurin (CaN), is required for ECM synthesis during hypertrophy of various organs. However, the functional role of RCAN1 in liver fibrogenesis has not yet been addressed. Methods: We induced experimental liver fibrosis in mice by intraperitoneal injection of 10 % CCl4 twice a week. To investigate the functional role of RCAN1.4 in the progression of liver fibrosis, we specifically over-expressed RCAN1.4 in mice liver using rAAV8-packaged RCAN1.4 over-expression plasmid. Following the establishment of the fibrotic mouse model, primary hepatic stellate cells were isolated. Subsequently, we evaluated the effect of RCAN1.4 on hepatic fibrogenesis, hepatic stellate cell activation, and cell survival. The biological role and signaling events for RCAN1 were analyzed by protein-protein interaction (PPI) network. Bisulfite sequencing PCR (BSP) was used to predict the methylated CpG islands in the RCAN1.4 gene promoter. We used the chromatin immunoprecipitation (ChIP assay) to investigate DNA methyltransferases which induced decreased expression of RCAN1.4 in liver fibrosis. Results: Two isoforms of RCAN1 protein were expressed in CCl4-induced liver fibrosis mouse model and HSC-T6 cells cultured with transforming growth factor-beta 1 (TGF-?1). RCAN1 isoform 4 (RCAN1.4) was selectively down-regulated in vivo and in vitro. The BSP analysis indicated the presence of two methylated sites in RCAN1.4 promoter and the downregulated RCAN1.4 expression levels could be restored by 5-aza-2'-deoxycytidine (5-azadC) and DNMTs-RNAi transfection in vitro. ChIP assay was used to demonstrate that the decreased RCAN1.4 expression was associated with DNMT1 and DNMT3b. Furthermore, we established a CCl4-induced liver fibrosis mouse model by injecting the recombinant adeno-associated virus-packaged RCAN1.4 (rAAV8-RCAN1.4) over-expression plasmid through the tail vein. Liver- specific-over-expression of RAN1.4 led to liver function recovery and alleviated ECM deposition. The key protein (a member of the NFAT family of proteins) identified on PPI network data was analyzed in vivo and in vitro. Our results demonstrated that RCAN1.4 over-expression alleviates, whereas its knockdown exacerbates, TGF-?1-induced liver fibrosis in vitro in a CaN/NFAT3 signaling-dependent manner. Conclusions: RCAN1.4 could alleviate liver fibrosis through inhibition of CaN/NFAT3 signaling, and the anti-fibrosis function of RCAN1.4 could be blocked by DNA methylation mediated by DNMT1 and DNMT3b. Thus, RCAN1.4 may serve as a potential therapeutic target in the treatment of liver fibrosis.

SUBMITTER: Pan XY

PROVIDER: S-EPMC6599664 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Methylation of RCAN1.4 mediated by DNMT1 and DNMT3b enhances hepatic stellate cell activation and liver fibrogenesis through Calcineurin/NFAT3 signaling.

Pan Xue-Yin XY You Hong-Mei HM Wang Ling L Bi Yi-Hui YH Yang Yang Y Meng Hong-Wu HW Meng Xiao-Ming XM Ma Tao-Tao TT Huang Cheng C Li Jun J

Theranostics 20190609 15

Background: Liver fibrosis is characterized by extensive deposition of extracellular matrix (ECM) components in the liver. RCAN1 (regulator of calcineurin 1), an endogenous inhibitor of calcineurin (CaN), is required for ECM synthesis during hypertrophy of various organs. However, the functional role of RCAN1 in liver fibrogenesis has not yet been addressed. Methods: We induced experimental liver fibrosis in mice by intraperitoneal injection of 10 % CCl4 twice a week. To ...[more]

PMID: 31285763

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Project description:Background & aimsInterleukin-15 (IL-15) and its high affinity receptor interleukin-15 receptor alpha (IL-15Rα) are widely expressed in immune cells and hepatic resident cells. IL-15 signaling has important functions in homeostasis of natural killer (NK), natural killer T (NKT) and cytotoxic T (CD8(+) T) cells, and in liver regeneration. We hypothesized that IL-15 has a protective role in liver fibrosis progression by maintaining NK cell homeostasis.MethodsFibrosis was induced using two mechanistically distinct models. Congenic bone marrow transplantation was used to evaluate the contribution of IL-15 signaling from various compartments to NK, CD8(+) T and NKT cell homeostasis and fibrogenesis. The gene expression profile of hepatic stellate cell (HSC) from IL-15Rα knockout (IL-15RαKO) mice and wild-type mice were captured using microarray analysis and validated in isolated HSC. Quantitative real-time PCR was used to assess repressors of collagen transcription.ResultsIL-15RαKO mice exhibited more fibrosis in both models. IL-15 signaling from specific types of hepatic cells had divergent roles in maintaining liver NK, CD8(+) T and NKT cells, with a direct and protective role on radio-resistant non-parenchymal cells beyond the control of NK homeostasis. HSCs isolated from IL-15RαKO mice demonstrated upregulation of collagen production. Finally, IL-15RαKO HSC with or without transforming growth factor beta (TGF-β) stimulation exhibited increased expression of fibrosis markers and decreased collagen transcription repressors expression.ConclusionsIL-15Rα signaling has a direct anti-fibrotic effect independent of preserving NK homeostasis. These findings establish a rationale to further explore the anti-fibrotic potential of enhancing IL-15 signaling in HSCs.Lay summaryWe investigated how a cellular protein, Interleukin-15 (IL-15), decreases the amount of scar tissue that is formed upon liver injury. We found that IL-15 and its receptor decrease the amount of scar tissue that is created by specialized liver cells (called stellate cells) and increase the number of a specific subgroup of immune cells (natural killer cells) that are known to eliminate stellate cells.Transcript profiling accession numberGSE45612, GSE 68001 and GSE 25097.

Project description:Exposure to alcohol and its metabolites can initiate hepatic injury and fibrogenesis. Fibrosis is mediated through hepatic stellate cell (HSC) activation, leading to global changes in mRNA and microRNA (miR) expression. miRs are expressed in cells or shuttled to exosomes which can be detected in tissue culture media (TCM) and biological fluids. The mechanisms and function underlying the differential expression and processing of miRs and their downstream effects during hepatic injury remain poorly understood.Expression of primary (pri)-miR17-92. and individual members of this cluster, miR17a, 18a, 19a, 20a, 19b, and 92, were examined in primary HSCs and human LX2 cells exposed to alcohol-conditioned media (CM), liver tissue from a rodent model of alcoholic injury, and in exosomes from TCM and plasma of rodent models and patients with alcoholic liver disease (ALD). miR expression was examined in HSCs transduced with an AAV2 vector carrying GFP-miR19b or GFP-control transgene under the collagen promoter.Profibrotic markers were enhanced in primary HSCs and LX2 cells exposed to alcohol-CM, concomitant with decreased miR19b expression and a significant increase in pri-miR17-92. Increased pri-miR17-92 was confirmed in a rodent model of alcohol-induced liver injury. Individual members of the cluster were inversely proportionate in cells and exosomes. AAV2-mediated miR19b overexpression inhibited miR17-92 and altered expression of individual cluster members in cells and exosomes. Expression of individual miR17-92 cluster members in plasma exosomes isolated from patients with ALD was similar to that seen in a rodent model of alcoholic injury and in vitro.Reintroduction of miR19b inhibits HSC activation and modulates expression of pri-miR17-92 and the inverse expression of individual cluster members in cells and exosomes. Better understanding of miR17-92 processing may provide mechanistic insights into the role of individual miRs and exosomes during hepatic injury, revealing new therapeutic targets.

Dataset Information

Methylation of RCAN1.4 mediated by DNMT1 and DNMT3b enhances hepatic stellate cell activation and liver fibrogenesis through Calcineurin/NFAT3 signaling.

Publications

Methylation of RCAN1.4 mediated by DNMT1 and DNMT3b enhances hepatic stellate cell activation and liver fibrogenesis through Calcineurin/NFAT3 signaling.

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