Wnt1 inducible signaling pathway protein-3 regulation and microsatellite structure in arthritis.
OBJECTIVE
Rheumatoid arthritis (RA) synovial tissue expresses several embryonic gene families, including wingless (wnt) and their receptors, frizzled (fz). The Wnt proteins, including Wnt-1, activate the Wnt inducible signaling pathway proteins (WISP), which are members of the CCN family that regulate cell growth and differentiation. WISP3 is of particular interest because it contains a microsatellite region in its coding region that is susceptible to frameshift mutations and leads to a truncated protein. To investigate the contribution of WISP3 to synovial inflammation, we evaluated its expression and regulation in arthritis.


METHODS
mRNA and protein expression of WISP3 were determined by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. For mutation analysis, PCR product amplified from genomic DNA of synovial tissue and cultured fibroblast-like synoviocytes (FLS) was subcloned and sequenced.


RESULTS
WISP3 mRNA is expressed in synovial tissue, but is 11-fold higher in RA than osteoarthritis (OA) or normal samples. Surprisingly, WISP3 protein levels are similar in RA, OA, and normal synovium samples. Immunohistochemistry of synovial tissue reveals that WISP3 protein is located primarily in the synovial intimal lining. WISP3 mRNA expression is also 6-fold higher in RA FLS compared with OA FLS and 50-fold higher in RA than in normal FLS. When RA FLS are stimulated with interleukin 1 or tumor necrosis factor-a, WISP3 mRNA is significantly increased. The cytokines also increase WISP3 mRNA in OA FLS, but the maximal level in stimulated OA FLS is still less than medium-treated RA FLS. Mutation analysis in the coding region microsatellite of the WISP3 gene in RA and OA synovium and FLS shows a limited number of insertion and deletion mutations.


CONCLUSIONS
WISP3 gene expression is higher in RA synovium and FLS compared with OA and normal synovial tissue and is further induced by proinflammatory cytokines in vitro. Protein levels are not increased, indicating discoordinate regulation of WISP3 protein and mRNA. Although functionally relevant mutations were observed in genomic DNA, they were noted in both OA and RA samples.
Cheon
Hyeonjoo
H
Division of Rheumatology, Allergy, and Immunology, University of California at San Diego School of Medicine, La Jolla, California 92093-0656, USA.
Boyle
David L
DL
Firestein
Gary S
GS
D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D014408:Biomarkers, Tumor / Q000378:metabolism; D015153:Blotting, Western; D055511:CCN Intercellular Signaling Proteins; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D016368:Frameshift Mutation; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins / Q000235:genetics / Q000378:metabolism*; D007375:Interleukin-1 / Q000494:pharmacology; D018895:Microsatellite Repeats / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010003:Osteoarthritis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000378:metabolism / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D014408:Biomarkers, Tumor; D055511:CCN Intercellular Signaling Proteins; C400081:CCN6 protein, human; D018969:Insulin-Like Growth Factor Binding Proteins; D007375:Interleukin-1; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha