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PRMT5 Prevents Cardiomyocyte Hypertrophy via Symmetric Dimethylating HoxA9 and Repressing HoxA9 Expression

ABSTRACT: The present study reveals a link between protein arginine methyltransferase 5 (PRMT5) and Homebox A9 (HoxA9) in the regulation of cardiomyocyte hypertrophy. In cardiomyocyte hypertrophy induced by ?-adrenergic receptor agonist isoprenaline (ISO), PRMT5 expression was decreased while HoxA9 was upregulated. Silencing of PRMT5 or inhibition of PRMT5 by its pharmacological inhibitor EPZ augmented the expressions of cardiomyocyte hypertrophic genes brain natriuretic peptide (BNP) and ?-Myosin Heavy Chain (?-MHC), whereas overexpression of PRMT5 inhibited ISO-induced cardiomyocyte hypertrophy, suggesting that PRMT5 ameliorates cardiomyocyte hypertrophy. On the contrary, HoxA9 promoted cardiomyocyte hypertrophy, as implied by the gain-of-function and loss-of-function experiments. HoxA9 was involved in the regulation of PRMT5 in cardiomyocyte hypertrophy, since HoxA9 knockdown prevented si-RPMT5-induced cardiomyocyte hypertrophy, and HoxA9 expression impaired the anti-hypertrophic effect of PRMT5. Co-immunoprecipitation experiments revealed that there were physical interactions between PRMT5 and HoxA9. The symmetric dimethylation level of HoxA9 was decreased by ISO or EPZ treatment, suggesting that HoxA9 is methylated by PRMT5. Additionally, PRMT5 repressed the expression of HoxA9. Chromatin immunoprecipitation (ChIP) assay demonstrated that HoxA9 could bind to the promoter of BNP, and that this binding affinity was further enhanced by ISO or EPZ. In conclusion, this study suggests that PRMT5 symmetric dimethylates HoxA9 and represses HoxA9 expression, thus impairing its binding to BNP promoter and ultimately protecting against cardiomyocyte hypertrophy. These findings provide a novel insight of the mechanism underlying the cardiac protective effect of PRMT5, and suggest potential therapeutic strategies of PRMT5 activation or HoxA9 inhibition in treatment of cardiac hypertrophy.

SUBMITTER: Cai S

PROVIDER: S-EPMC7793800 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Project description:Chronic catecholamine stress (CCS) induces the occurrence of cardiomyopathy-pathological cardiac hypertrophy (PCH), which is characterized by left ventricular systolic dysfunction (LVSD). Recently, mounting evidence has implicated myocardial inflammation in the exacerbation of pathological cardiac remodeling. However, there are currently no well-defined treatment interventions or regimes targeted at both the attenuation of maladaptive myocardial hypertrophy and inflammation during CCS to prevent PCH. G protein-coupled receptor kinase 5 (GRK5) and adenylyl cyclases (ACs)-cAMP mediates both cardiac and inflammatory responses. Also, GRK5 and ACs are implicated in stress-induced LVSD. Herein, we aimed at preventing PCH during CCS via modulating adaptive cardiac and inflammatory responses by inhibiting GRK5 and/or stimulating ACs. Isoproterenol-induced cardiomyopathy (ICM) was modeled using 0.5 mg/100 g/day isoproterenol injections for 40 days. Alterations in cardiac and inflammatory responses were assessed from the myocardia. Similarities in the immunogenicity of cardiac troponin I (cTnI) and lipopolysaccharide under CCS were assessed, and Amlexanox (35 μM/ml) and/or Forskolin (10 μM/ml) were then employed in vitro to modulate adaptive inflammatory responses by inhibiting GRK5 or activating ACs-cAMP, respectively. Subsequently, Amlexanox (2.5 mg/100 g/day) and/or Forskolin (0.5 mg/100 g/day) were then translated into in vivo during CCS to modulate adaptive cardiac and inflammatory responses. The effects of Amlexanox and Forskolin on regulating myocardial systolic functions and inflammatory responses during CCS were ascertained afterward. PCH mice had excessive myocardial hypertrophy, fibrosis, and aggravated LVSD, which were accompanied by massive CD68+ inflammatory cell infiltrations. In vitro, Forskolin-AC/cAMP was effective than Amlexanox-GRK5 at downregulating proinflammatory responses during stress; nonetheless, Amlexanox and Forskolin combination demonstrated the most efficacy in modulating adaptive inflammatory responses. Individually, the translated Amlexanox and Forskolin treatment interventions were ineffective at subduing the pathological remodeling and sustaining cardiac function during CCS. However, their combination was potent at preventing LVSD during CCS by attenuating maladaptive myocardial hypertrophy, fibrosis, and inflammatory responses. The treatment intervention attained its potency mainly via Forskolin-ACs/cAMP-mediated modulation of cardiac and inflammatory responses, coupled with Amlexanox inhibition of GRK5 mediated maladaptive cascades. Taken together, our findings highlight the Amlexanox and Forskolin combination as a potential therapeutic intervention for preventing the occurrence of pathological cardiac hypertrophy during chronic stress.

Project description:Background: Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that symmetrically di-methylates arginine residues on both histone and non-histone protein substrates. Accumulating evidence suggests that PRMT5 exerts its oncogenic properties in a wide spectrum of human malignancies. However, the underlying mechanisms by which PRMT5 contributes to the progression of colorectal cancer (CRC) remain to be defined. Methods: Western blot and real-time PCR were used to analyze the expression of CDKN2B. Co-immunoprecipitation (Co-IP), immunofluorescence and GST pulldown assays were employed to investigate the interaction between PRMT5 and EZH2. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were performed to validate CDKN2B as a direct target of PRMT5/EZH2. DNA methylation status at the CpG islands of promoter region of CDKN2B gene was analyzed by bisulfite sequencing. The effect of PRMT5/EZH2 on malignant phenotypes was examined through in vitro and in vivo assays. PRMT5 and EZH2 protein expression levels in CRC tissues were analyzed by immunohistochemistry (IHC) staining. Results: We observed that PRMT5-deficient CRC cells exhibit proliferation defects in vitro. PRMT5 was identified as a major transcriptional repressor of CDKN2B (p15INK4b) for determining CRC progression. Mechanistically, PRMT5-mediated histone marks H4R3me2s and H3R8me2s were predominantly deposited at the promoter region of CDKN2B gene in CRC cells. Knockdown of PRMT5 in CRC cells decreased the accumulation of H4R3me2s and H3R8me2s marks and reduced the CpG methylation level of CDKN2B promoter, then re-activated CDKN2B expression. Strikingly, silencing of CDKN2B partially abrogated the proliferation defects caused by PRMT5 depletion in vitro and in vivo. Furthermore, we proved that PRMT5 interacted with Enhancer of zeste homolog 2 (EZH2), leading to enhanced EZH2 binding and H3K27me3 deposition together with decreased transcriptional output of CDKN2B gene. Importantly, we found that the combined interventions exerted a synergistic inhibitory effect of combined treatment with PRMT5i (GSK591) and EZH2i (GSK126) on the growth of CRC cells/xenografts in vitro and in vivo. Moreover, PRMT5 and EZH2 were found to be significantly elevated and associated with poor prognosis in CRC patients. Conclusion: PRMT5 functionally associates with EZH2 to promote CRC progression through epigenetically repressing CDKN2B expression. Thus, our findings raise the possibility that combinational intervention of PRMT5 and EZH2 may be a promising strategy for CRC therapy.

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PRMT5 Prevents Cardiomyocyte Hypertrophy via Symmetric Dimethylating HoxA9 and Repressing HoxA9 Expression

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