Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Microbiology and Immunology

Supervisor

Dr. Rodney DeKoter

Abstract

High titers of anti-citrullinated protein antibodies have been detected in sera of rheumatoid arthritis (RA) patients, implicating citrullinating enzymes in the pathogenesis of RA. Peptidylarginine deiminase type IV (PAD4) is a member of the PAD family of enzymes that catalyze the post- translational modification of arginine to citrulline and has been linked with RA. However, little is known about its transcriptional regulation. Therefore, our aim was to determine how transcription of PAD4 is activated in the myeloid lineage. Using bioinformatics, a potential nuclear factor kappa B (NF-kB) binding site was identified on the PAD4 promoter. Luciferase assays were used to test promoter activity in human and murine myeloid cells. Interestingly, mutation of the NF-κB binding site significantly lowered promoter activity in WEHI-3B cells but significantly increased it in both HL-60 and THP-1 cell lines. In addition, PAD4 mRNA was significantly lowered in response to TNF-α treatment in HL-60 cells but increased in WEHI-3B cells. Finally, chromatin immunoprecipitation (ChIP) using anti-p50 and anti-p65 antibodies revealed that there was a significant increase in p50 enrichment at the PAD4 promoter, but not p65 in cells treated with TNF-α. Our results suggest that NF-κB may play an important role in the transcriptional regulation of PAD4 in human and murine immune systems.

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