Session Title: T-cell Biology and Targets in Autoimmune Disease
Session Type: Abstract Submissions (ACR)
Although many reports have focused on the impaired MEK-ERK signaling pathway in T cells from systemic lupus erythematosus (SLE) patients which results in suppression of DNA methyltransferase (DNMTs) expression and induction of gene transcription of methylation-sensitive genes, the involved mechanisms are still unclear. Here we investigated whether the catalytic subunit of protein phosphatase 2A (PP2Ac) which is overexpressed in SLE T cells contributes to inhibition of MEK-ERK signaling and DNA methylation.
Human peripheral CD3 positive T cells from normal subjects were treated with the selective chemical inhibitor, okadaic acid (OA) or transfected with siPP2Ac to achieve sufficient suppression of the PP2Ac enzymatic activity. After stimulation with PMA and ionomycin, DNA, RNA and protein were extracted. The ratio of phosphorylated over total MEK and ERK protein were determined by western blotting. The enzyme activity of DNMTs and the level of global DNA methylation status were calculated by ELISA. The transcription level of two methylation sensitive genes, CD70 and CD11a was quantified by real time RT-PCR.
Chemical suppression or siRNA silencing of PP2Ac in T cells resulted in sustained phosphorylation of MEK and ERK following stimulation with PMA and ionomycin compared to T cells in which PP2Ac was not manipulated. PP2Ac suppression resulted in increased DNMT enzymatic activity of DNMT, DNA hypermethylation and decreased expression of methylation-sensitive genes.
Our results demonstrate that PP2A regulates DNA methylation levels by influencing the phosphorylation levels of the MEK/ERK pathway. We propose that enhanced PP2Ac in SLE T cells may dephosphorylate and activate the upstream signaling pathway of DNMT and disturb the tight control of methylation-sensitive genes such as CD70 and CD11a which are involved in SLE pathogenesis. In addition, based on our previous report that PP2Ac itself is regulated though DNA methylation around a cAMP response element (CRE) binding site located in the proximal promoter, and therefore PP2A may represent a potent accelerator of DNA demethylation through an additional positive feedback mechanism.
K. S. Watanabe,
G. C. Tsokos,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/suppression-of-pp2ac-causes-dna-hypermethylation-through-enhanced-pmekperk-activity-in-t-cells/