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EZH2 Modulates the DNA Methylome and Controls T Cell Adhesion Through Junctional Adhesion Molecule A in Lupus Patients.

ABSTRACT: OBJECTIVE:EZH2 is an epigenetic regulator that mediates H3K27 trimethylation (H3K27me3) and modulates DNA methylation. The aim of this study was to characterize the role of EZH2 in CD4+ T cells in the pathogenesis of systemic lupus erythematosus. METHODS:EZH2 expression levels were determined in CD4+ T cells isolated from lupus patients and healthy controls. The epigenetic effects of EZH2 overexpression in CD4+ T cells were evaluated using a genome-wide DNA methylation approach. Gene expression profiles and microRNAs (miRNAs) were assessed by quantitative polymerase chain reaction, while protein expression was examined by Western blotting. A cell adhesion assay was used to assess adhesion of CD4+ T cells to human microvascular endothelial cells. RESULTS:EZH2 and H3K27me3 levels were increased in CD4+ T cells from lupus patients compared to healthy controls. T cell production of EZH2 was down-regulated in the presence of miR-26a and miR-101, and levels of both miRNAs were reduced in lupus CD4+ T cells. Overexpression of EZH2 induced in CD4+ T cells resulted in significant DNA methylation changes. Genes involved in leukocyte adhesion and migration, including F11R (which encodes junctional adhesion molecule A [JAM-A]), became hypomethylated in CD4+ T cells when EZH2 was overexpressed. Overexpression of EZH2 resulted in increases in JAM-A expression and CD4+ T cell adhesion. Preincubation of EZH2-transfected CD4+ T cells with neutralizing antibodies against JAM-A significantly blunted cell adhesion. Similarly, CD4+ T cells from lupus patients overexpressed JAM-A and adhered significantly more to endothelial cells than to T cells from healthy controls. Blocking JAM-A or EZH2 significantly reduced the capacity of lupus CD4+ T cells to adhere to endothelial cells. CONCLUSION:The results of this study identify a novel role of EZH2 in T cell adhesion mediated by epigenetic remodeling and up-regulation of JAM-A. Blockade of EZH2 or JAM-A might have therapeutic potential by acting to reduce T cell adhesion, migration, and extravasation in patients with lupus.

SUBMITTER: Tsou PS

PROVIDER: S-EPMC5745285 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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EZH2 Modulates the DNA Methylome and Controls T Cell Adhesion Through Junctional Adhesion Molecule A in Lupus Patients.

Tsou Pei-Suen PS Coit Patrick P Kilian Nathan C NC Sawalha Amr H AH

Arthritis & rheumatology (Hoboken, N.J.) 20171215 1

<h4>Objective</h4>EZH2 is an epigenetic regulator that mediates H3K27 trimethylation (H3K27me3) and modulates DNA methylation. The aim of this study was to characterize the role of EZH2 in CD4+ T cells in the pathogenesis of systemic lupus erythematosus.<h4>Methods</h4>EZH2 expression levels were determined in CD4+ T cells isolated from lupus patients and healthy controls. The epigenetic effects of EZH2 overexpression in CD4+ T cells were evaluated using a genome-wide DNA methylation approach. G ...[more]

PMID: 28973837

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Project description:INTRODUCTION:The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. METHODS:MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and ?1-integrin, we examined activation of the ?1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and ?1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. RESULTS:JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the ?1-integrin substrate fibronectin. This was accompanied by reduced protein expression of ?1-integrin and its binding partners ?V- and ?5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and ?1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF-6 and the Rap1 activator PDZ-GEF2 in MCF7 cells and in primary cultures from breast cancer patients. CONCLUSIONS:Our findings provide compelling evidence of a novel role for JAM-A in driving breast cancer cell migration via activation of Rap1 GTPase and ?1-integrin. We speculate that JAM-A over-expression in some breast cancer patients may represent a novel therapeutic target to reduce the likelihood of metastasis.

Dataset Information

EZH2 Modulates the DNA Methylome and Controls T Cell Adhesion Through Junctional Adhesion Molecule A in Lupus Patients.

Publications

EZH2 Modulates the DNA Methylome and Controls T Cell Adhesion Through Junctional Adhesion Molecule A in Lupus Patients.

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