The histone demethylase JMJD2B regulates endothelial-to-mesenchymal transition - PubMed (original) (raw)

. 2020 Feb 25;117(8):4180-4187.

doi: 10.1073/pnas.1913481117. Epub 2020 Feb 7.

Simone F Glaser 1 2 3, Andreas W Heumüller 1 2 3, Patrick Hofmann 1 2, Marion Muhly-Reinholz 1, Ariane Fischer 1, Stefan Günther 4 5, Karoline E Kokot 6, David Hassel 7 8, Sandeep Kumar 9, Hanjoong Jo 9 10, Reinier A Boon 1 2, Wesley Abplanalp 1 2, David John 1 2, Jes-Niels Boeckel 1 6, Stefanie Dimmeler 11 2

Affiliations

The histone demethylase JMJD2B regulates endothelial-to-mesenchymal transition

Simone F Glaser et al. Proc Natl Acad Sci U S A. 2020.

Erratum in

Abstract

Endothelial cells play an important role in maintenance of the vascular system and the repair after injury. Under proinflammatory conditions, endothelial cells can acquire a mesenchymal phenotype by a process named endothelial-to-mesenchymal transition (EndMT), which affects the functional properties of endothelial cells. Here, we investigated the epigenetic control of EndMT. We show that the histone demethylase JMJD2B is induced by EndMT-promoting, proinflammatory, and hypoxic conditions. Silencing of JMJD2B reduced TGF-β2-induced expression of mesenchymal genes, prevented the alterations in endothelial morphology and impaired endothelial barrier function. Endothelial-specific deletion of JMJD2B in vivo confirmed a reduction of EndMT after myocardial infarction. EndMT did not affect global H3K9me3 levels but induced a site-specific reduction of repressive H3K9me3 marks at promoters of mesenchymal genes, such as Calponin (CNN1), and genes involved in TGF-β signaling, such as AKT Serine/Threonine Kinase 3 (AKT3) and Sulfatase 1 (SULF1). Silencing of JMJD2B prevented the EndMT-induced reduction of H3K9me3 marks at these promotors and further repressed these EndMT-related genes. Our study reveals that endothelial identity and function is critically controlled by the histone demethylase JMJD2B, which is induced by EndMT-promoting, proinflammatory, and hypoxic conditions, and supports the acquirement of a mesenchymal phenotype.

Keywords: EndMT; H3K9me3; JMJD2B; SULF1; epigenetics.

Copyright © 2020 the Author(s). Published by PNAS.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.

Fig. 1.

Regulation of JMJD2B by EndMT-promoting stimuli in vitro and in vivo. (A) JMJD2B mRNA was measured in untreated (Ctrl) or with differentiation medium (DM) + TGF-β2- or IL-1β-treated HUVECS by RT-qPCR and depicted as fold to Ctrl (n = 3). (B) HUVECs were cultivated for 24 h under 0.2% O2 (hypoxia) or 21% O2 (normoxia), and mRNA level were determined by RT-qPCR and normalized to RPLP0 (2-ΔCt) (n = 3). (C) Representative immunoblot (IB) and densiometric quantification of JMJD2B protein level in hypoxia (1% O2) or normoxia, β-ACTIN served as a loading control (n = 5). (D) RNA expression levels of JMJD2B in HUVECs incubated under static and laminar flow conditions determined by RNA sequencing (n = 2). (E_–_G) Partial carotid artery mice model (nonligated, right carotid artery; ligated, left carotid artery) after 12 h and 48 h of ligation. (E) Jmjd2b mRNA levels were determined by RT-qPCR; values were normalized to 18s rRNA level (n = 4). (F_–_G) Jmjd2b mRNA levels were determined by RNA sequencing; values are depicted as fold to nonligated (F) or Fragments Per Kilobase Million (FPKM) values as Pearson’s correlation (G). Data are depicted as mean ± SEM. Statistical significance was determined using Student’s t test or ANOVA Bonferroni post hox test (A_–_F), and Pearson’s correlation (G); *P < 0.05.

Fig. 2.

Fig. 2.

Function of JMJD2B in EndMT. (A) SM22 and JMJD2B protein after siRNA-mediated knockdown of JMJD2B or scrambled siRNA followed by EndMT induction. Topoisomerase I served as loading control. Quantification is shown below, n = 4. (B) SM22 and (C) CNN1 mRNA levels after siJMJD2B or siScr in HUVECs, followed by EndMT induction, determined by RT-qPCR, normalized to RPLP0 mRNA (2-ΔCt) (n = 4). (D) Knockdown of JMJD2B in HUVECS using the CRISPR-Cas9 technology. mRNA of SM22 was quantified via qRT-PCR (n = 7). (E) SM22 immunofluorescence (IF) staining after siJMJD2B or siScr followed by EndMT. Endothelial marker VE-cadherin (CDH5) appears in green, nuclei in white (DAPI), and SM22 in red. (Scale bar, 50 μm.) (F) Percentage of SM22pos. cells per population after siJMJD2B or siScr followed by EndMT, depicted as SM22 negative (white), intermediate (pink), and positive (red) cells (n = 3). (G) CDH5 cell gaps per square micrometer measured using IF staining after siJMJD2B and EndMT (n = 4). (H) Permeability of HUVECs treated with siRNAs and EndMT (n = 4). Data are depicted as mean ± SEM. Statistical significance was determined using Student’s t test or ANOVA Bonferroni post hoc test; *P < 0.05.

Fig. 3.

Fig. 3.

Jmjd2b is a positive regulator of EndMT in vivo. (A) Knockout strategy and DNA gel of the floxed Jmjd2b and the Cdh5-CreERT2 allele. Recombination after TAM treatment led to the deletion of exon 5 in the Jmjd2b ORF. Jmjd2b DNA in total heart of Ctrl (3.1 kbp) or Jmjd2biEC-KO (1.4 kbp) mice, with or w/o TAM. (B) Schema of TAM-induced EC deletion of Jmjd2b with a subsequent AMI induction. (C) Jmjd2b mRNA of isolated EC of control (Ctrl) or Jmjd2biEC-KO mice, determined by RT-qPCR, depicted as percentage of Ctrl, n = 7–10. (D) Single-cell RNA sequencing showing percentage of cells positive [≥1 unique molecular identifiers (UMI) counts] for EC marker (Pecam1 and Cdh5) only (gray) and EC plus mesenchymal cell marker (MSC) gene expression (red) 3 d post-AMI. (E) Representative images of IHC stainings of Ctrl and Jmjd2biEC-KO mice hearts 14 d post-AMI. Hoechst appears in blue, lectin in white, and S100A4 in red, scale bar = 20 μM. (F) S100A4+ and lectin+ cells were counted in the sections of Ctrl or Jmjd2biEC-KO mice, normalized to area of lectin, depicted as fold Ctrl, n = 7–10. Data are depicted ±SEM. Student’s t test (C and F), χ2 test with Yates correction (D); *P < 0.05.

Fig. 4.

Fig. 4.

JMJD2B is a positive regulator of EndMT-related genes in endothelial cells. (A) Genomewide CircosPlot showing H3K9me3 ChIP-Seq peaks in control or EndMT-treated cells. Blue, control; red, EndMT. (B) H3K9me3 level in EndMT fold control of selected EndMT-associated genes determined by ChIP-Seq (P < 0.05, n = 3). (C) Gene ontology (

http://pantherdb.org

) analysis of H3K9me3 ChIP-Seq gene-specific reduced peaks in EndMT. (D_–_F) Differential gene expression of (D) CNN1, (E) AKT3, (F) SULF1 between Ctrl (n = 2227 cells), and EndMT (n = 1038 cells) single-cell RNA sequencing libraries. Data display individual gene UMI counts normalized to the total UMI count of all genes per cell. (G) Microarray RNA sequencing data after silencing of JMJD2B in TGF-β2-stimulated ECs (n = 3). Heatmap, depicting high expressed genes. High expression is given in red and low expression in blue. (H and I) ChIP-RT-qPCR showing level of H3K9me3 at (H) CNN1 and (I) AKT3 promoter, normalized to IgG (n = 5). (J) AKT3 mRNA expression after silencing of JMJD2B in TGF-β2-stimulated ECs using microarray RNA sequencing (n = 3). (K) Representative IB and densitometric quantification of protein levels of AKT3 after siRNA-mediated knockdown of JMJD2B. GAPDH served as loading control (n = 4). Data are depicted as mean ± SEM. Statistical significance was determined using Student’s t test, or likelihood-ratio test (B and D–F); *P < 0.05.

Fig. 5.

Fig. 5.

Analysis of JMJD2B target genes in EndMT. (A) SULF1 mRNA expression after silencing of JMJD2B in TGF-β2-stimulated ECs using microarray RNA sequencing (n = 3). (B) mRNA levels of SULF1 after siRNA-mediated knockdown of JMJD2B using RT-qPCR, normalized to RPLP0 (n = 4). (C) ChIP-Seq RT-qPCR showing H3K9me3 levels at promoter of SULF1, normalized to IgG (n = 5). (D_–_F) mRNA levels after siRNA-mediated knockdown of SULF1 or siScr siRNA in HUVECs followed by EndMT. Total RNA was isolated and mRNA levels were determined by RT-qPCR, normalized to GAPDH (2-ΔCt). (D) SULF1, (E) SM22, (F) TGF-β2 expression levels are depicted as fold siScr Ctrl (n = 3). (G) TGF-β2 mRNA level after siRNA-mediated knockdown of JMJD2B in HUVECs. mRNA levels were determined by RT-qPCR, normalized to RPLP0 (2-ΔCt), depicted as fold to siScr Ctrl (n = 9). (H) Cartoon summarizing our findings. Data are depicted as mean ± SEM. Statistical significance was determined using Student’s t test; *P < 0.05.

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