Functional evaluation of TNFAIP3 (A20) in rheumatoid arthritis.
OBJECTIVE
To determine the protein expression of TNFAIP3 in synovium and to show the capability of 6q23 intergenic SNPs, associated with rheumatoid arthritis (RA) susceptibility, to influence TNFAIP3 gene transcription.


METHODS
Immunohistochemistry for TNFAIP3, NF-kB p65 and phosphorylated NF-kB p65 protein expression was performed in 6 RA knee joint synovium samples compared to 9 osteoarthritis (OA) samples. Luciferase reporter gene assays were used to examine the regulatory ability of RA associated SNP variants on TNFAIP3 promoter activity. Sense and antisense constructs were prepared for rs6920220 alleles, together with each of the 4 SNPs in r2=1 with it (rs6933404, rs2327832, rs6927172 and rs17264332), coupled to the TNFAIP3 promoter. Transient transfections were performed in a human T lymphoblastoid (CEMC7A) cell line. Bioinformatic software was utilised to prioritise SNPs for further investigation. Electrophoretic mobility shift assays (EMSA), using CEMC7A nuclear extracts, were conducted for the rs6927172 SNP alleles.


RESULTS
TNFAIP3 protein expression was seen in the synovium samples and differential TNFAIP3 protein expression between RA vs. OA synoviocytes observed. Within RA synoviocytes TNFAIP3 expression is predominately cytoplasmic, whereas in OA its expression is strongly nuclear and cytoplasmic. For 3 of the 5 SNPs investigated (rs6920220, rs6933404, rs6927172) evidence of repressor activity of TNFAIP3 transcription was seen and EMSA data showed evidence of differential transcription factor binding to rs6927172 alleles.


CONCLUSIONS
This is the first observation of TNFAIP3 protein expression in RA and OA synovium. In vitro analysis of 6q23 intergenic SNPs supports the possibility of the functional regulation of TNFAIP3.
Elsby
L M
LM
Arthritis Research Campaign's Epidemiology Unit (arc/EU), Manchester Academic Health Sciences Centre, University of Manchester, United Kingdom. laura_elsby@hotmail.com
Orozco
G
G
Denton
J
J
Worthington
J
J
Ray
D W
DW
Donn
R P
RP
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001172:Arthritis, Rheumatoid / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D015415:Biomarkers / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006801:Humans; D007124:Immunoenzyme Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008875:Middle Aged; D010003:Osteoarthritis / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D020641:Polymorphism, Single Nucleotide; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D013583:Synovial Membrane / Q000378:metabolism / Q000473:pathology; D014158:Transcription, Genetic; D014162:Transfection; D055815:Young Adult
D015415:Biomarkers; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; C426747:TNIP3 protein, human
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