Tumor Necrosis Factor (TNF)-α Induction of CXCL10 in Endothelial Cells Requires Protein Arginine Methyltransferase 5 (PRMT5)-mediated Nuclear Factor (NF)-κB p65 Methylation

Daniel P Harris,Smarajit Bandyopadhyay,Tyler J Maxwell,Belinda Willard,Paul E Dicorleto

doi:10.1074/jbc.m114.547349

Abstract

The chemokine CXCL10/IP-10 facilitates recruitment of Th1-type leukocytes to inflammatory sites. In this study, we show that the arginine methyltransferase PRMT5 is critical for CXCL10 transcription in TNF-α-activated human endothelial cells (EC). We found that depletion of PRMT5 results in significantly reduced levels of CXCL10 mRNA, demonstrating a positive role for PRMT5 in CXCL10 induction. Chromatin immunoprecipitation experiments revealed the presence of the symmetrical dimethylarginine modification catalyzed by PRMT5 associated with the CXCL10 promoter in response to TNF-α. However, symmetrical dimethylarginine-modified proteins were not detected at the promoter in the absence of PRMT5, indicating that PRMT5 is essential for methylation to occur. Furthermore, NF-κB p65, a critical driver of TNF-α-mediated CXCL10 induction, was determined to be methylated at arginine residues. Crucially, RNAi-mediated PRMT5 depletion abrogated p65 methylation and CXCL10 promoter binding. Mass spectrometric analysis in EC identified five dimethylated arginine residues in p65, four of which are uncharacterized in the literature. Expression of Arg-to-Lys point mutants of p65 demonstrated that both Arg-30 and Arg-35 must be dimethylated to achieve full CXCL10 expression. In conclusion, we have identified previously uncharacterized p65 post-translational modifications critical for CXCL10 induction.

Highlights

NF-␬B post-translational modifications contribute to gene-specific transcriptional activation
We previously demonstrated the requirement of Protein arginine methyltransferase 5 (PRMT5) for the induction of the endothelial cells (EC)-leukocyte adhesion molecules E-selectin and VCAM-1 [2]
The mechanism we report here differs from our previous report of E-selectin and VCAM-1 induction in that HOXA9 is not involved and that PRMT5 is not detected by ChIP on the promoter itself

Summary

Background

NF-␬B post-translational modifications contribute to gene-specific transcriptional activation. Significance: Methylation of specific NF-␬B arginine residues contributes to promoter targeting and transcriptional activation. We show that the arginine methyltransferase PRMT5 is critical for CXCL10 transcription in TNF-␣-activated human endothelial cells (EC). PRMT5 Methylation of p65 Activates CXCL10 Expression transcription factor NF-␬B is activated by kinases that phosphorylate p65 and trigger the degradation of inhibitory I␬B subunits These events free NF-␬B to translocate into the nucleus where it associates with ␬B sites in target promoters [16]. A major unresolved question in the study of NF-␬B remains how the various post-translational modifications of NF-␬B enable specific gene activation, differential kinetics, transcription magnitude, and inducer-specific responses [18]. We report that the arginine methyltransferase PRMT5 post-translationally modifies the p65 subunit of the NF-␬B Rel-homology domain, a step imperative to the CXCL10 induction by TNF-␣

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

DiCorleto