Localization and expression of cartilage oligomeric matrix protein by human rheumatoid and osteoarthritic synovium and cartilage

Abstract

Synovium and cartilage from patients with osteoarthritis or rheumatoid arthritis were analyzed for expression of cartilage oligomeric matrix protein. Immunostaining of synovium with antiserum to cartilage oligomeric matrix protein demonstrated positive staining in both diseases. In osteoarthritis, there was positive staining within the synovial cells and immediately subjacent connective tissue, with less intense staining in the deeper connective tissue. In rheumatoid arthritis, there was less intense staining within the synovial cells and marked intense staining in the deeper connective tissue. In situ hybridization performed with an antisense digoxigenin-labeled riboprobe to human cartilage oligomeric matrix protein confirmed the presence of cartilage oligomeric matrix protein mRNA in the cells of the synovial lining in both types of synovium. Quantitative polymerase chain reaction with a cartilage oligomeric matrix protein MIMIC demonstrated increased cartilage oligomeric matrix protein mRNA in rheumatoid cartilage and synovium as compared with osteoarthritic cartilage and synovium, respectively; mRNA levels in rheumatoid synovium were similar to those from osteoarthritic chondrocytes. As a result of the high expression of cartilage oligomeric matrix protein from rheumatoid synovium, inflammatory synovium should be considered as a potential tissue source of cartilage oligomeric matrix protein in any investigation of biological markers of cartilage metabolism. The upregulated expression of cartilage oligomeric matrix protein in inflammatory tissues suggests its in vivo regulation by cytokines.

Cartilage Oligomeric Matrix Protein Associates with Granulin-Epithelin Precursor (GEP) and Potentiates GEP-stimulated Chondrocyte Proliferation

“…5B). In line with the previous findings, COMP was also primarily intracellular in the early hypertrophic zone but was in the pericellular matrix in the calcifying zone chondrocytes (3,53,64). Distinct localization of GEP and COMP in various chondrocytes suggests that they may play important roles at various stages in skeletogenesis.…”

“…To identify protein interaction partners of COMP, an extracellular matrix protein that has been implicated in the regulation of chondrogenesis and cartilage development (3,6,53), we screened the yeast expression cDNA library using the EGF repeat domain of COMP as bait and identified the GEP, a growth factor that has not been previously described in cartilage, as a direct binding protein of COMP.…”

“…After washing five times with immunoprecipitation buffer, bound proteins were released by boiling in 20 l of 2ϫ SDS loading buffer for 3 min (52). Released proteins were examined by Western blotting with anti-COMP antibodies (3,(53)(54)(55), and the ECL chemiluminescent system (Amersham Biosciences).…”

“…In the case of the antibody-blocking experiment, we first generated affinity-purified anti-COMP antibodies in order to study the dosage response in a controlled condition. Briefly, the anti-COMP serum (3,(53)(54)(55) was incubated with Affi-Gel-10 beads (BioRad) to which purified COMP was covalently linked. The bound antibodies were eluted from the beads with 0.15 M glycine buffer (pH 2.5) and immediately neutralized with 1.5 M Tris-HCl buffer (pH 8.0) (56).…”

ADAMTS-12 Associates with and Degrades Cartilage Oligomeric Matrix Protein

“…Briefly, a COMP stable line was transfected with either control or expression constructs encoding wild-type ADAMTS-12 (pcDNA3-ADAMTS12-HA-FLAG) or ADAMTS-12 with mutant catalytic domain (pcDNA3-ADAMTS12-MUT) (39). Seventy-two hours after transfection, the media were collected and subjected to 8% nonreduced SDS-PAGE and intact COMP, and fragments were detected by Western blotting with polyclonal rabbit anti-COMP antiserum, as previously described (13,37,38). An in vitro digestion assay with purified COMP and the cell lysates carrying ADAMTS-12 was then performed.…”