The Research For Carbonic Anhydrase I Stimulates Abnormal Calcification In Arthritis

Ankylosing spondylitis (AS) is a chronic inflammatory joint disease and is the majorsubtype of spondyloarthritides, chiefly affecting the axial skeleton including the sacroiliacjoints and the spine and also the peripheral joints and the enthuses, Coupling ofinflammation and subsequent fibrosis and calcification, In advanced stages of the diseasethe fusion typically ascends the spine, forming a long bony column referred toas“bamboo spine”. Abnormal bone formation is main feature of ankylosing spondylitis.Histopathological experiments demonstrated that severe forms of AS significantlycorrelate with villous chronic synovitis, including obliterating vascularitis, fibrosclerosis,necrosis and calcification of disintegrated synovial structures. The global disease activityof AS significantly correlates with hyperplasia of the synovial membrane as well as withinflammatory infiltration of macrophages, especially the CD163+subset, andpolymorphonuclear leukocytes. In order to research the role of the synovial membranesin AS, we previously used a proteomic approach, screened novel AS-specific proteins bysimultaneously comparing the expression profiles of synovial membranes from patientswith AS, rheumatoid arthritis (RA) and osteoarthritis (OA). The proteomic study revealeda significantly increased expression of carbonic anhydrase I (CA1) in the synovialmembrane of patients with AS compared with those of patients with RA and OA.Immunohistochemistry and western blotting analysis confirmed the above findings.Carbonic anhydrases are enzymes that catalyze the hydration of carbon dioxide andthe dehydration of bicarbonate, others found that CA1stimulates CaCO3precipitation invitro. Precipitation of calcium salt is an essential step for new bone formation. However, no direct data support that CA1is involved in bio-mineralization and bone formation incells and tissues, and it is not determined if CA1is AS-diseased gene and how CA1isinvolved in the pathogenic process of the disease.OBJECTIVE: This study investigated function of CA1for bio-mineralization incultured cells and determined the effect on arthritis and bone formation in transgenicmice with over-expression of the enzyme.METHODS: Calcification was induced in Saos-2cells, a human osteosarcoma cellline, using osteogenic medium (OM) supplemented with ascorbic acid andβ-glycerophosphate. Calcification in the cells was determined by Alizarin Red-S staining.Expressions of CA1, alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin(OCN), osterix (OSX) and runt-related transcription factor-2(Runx2), protein markers ofossification, were determined by real-time PCR and western blotting. The cultured cellswere also treated with acetazolamide, an anti-carbonic anhydrase drug, to determine theeffect of CA1on bio-mineralization. Transgenic mice with over-expression of CA1(CA1-Tg) were prepared and arthritis was induced with bovine collagen II (CII) in theseanimals. The symptom of CIA was evaluated by examining clinical score, incidence andfootpad swelling of CA1-Tg with wide type mice and transgenic mice withover-expression of PADI4(PADI4-Tg), a known gene susceptible to rheumatoid arthritis，as controls. X-ray assay and histochemistry were used to examine joint destruction ofthese mice. ELISA was used to measure the levels of interleukin-1α (IL-1α), IL-1β, IL-17and tumor necrosis factor-α (TNF-α) in sera of the animals.RESULTS: Following the induction of OM, Saos-2cells produced a great amountof calcium-rich deposits. Increased transcriptions of ALP, BSP, OCN, OSX and Runx2were detected in the cultured cells, indicating that OM initiated the process of thebio-mineralization and ossification. CA1also had a significantly increased expressionduring the process. Following the treatment of acetazolamide, the expression of CA1wasobviously declined, and the mineralized nodule formation was also decreased. Followingintroduction of collagen II, the incidence of CIA was observed in60%(6/10) of CA1-Tgmice,20%(2/10) of PADI4-Tg mice,20%(2/10) of wide-type mice and0%(0/10)CA1-Tg mice with injection of BSA by the day of84th. The arthritis severity score was up to5.5±0.84for CA1-Tg mice on day98th after the first injection, but the score waslow than2for the immunized wild type mice and PADI4–Tg mice under the samecondition. No clinical sign of arthritis was found for CA1-Tg mice with treatment BSA.Hind paw thickness in CA1-Tg mice was3.46±0.107mm and thicker than that ofPADI4-Tg mice (2.225±0.08mm), wild type mice (2.075±0.06mm) and BSA-treatedCA1-Tg mice (2.04±0.07mm). X-ray assay showed new bone formation with possiblebone fusion in spinal of CA1-Tg mice. Histochemistry showed inflammation, synovialhyperplasia and bone erosion in joints of CA1-Tg mice, but not in PADI4-Tg andwide-type mice. Levels of IL-1α、IL-1β、IL-17and TNF-α were significantly increased insera of CA1-Tg mice with CIA than the controls.CONCLUSION: Over-expression of CA1stimulates bio-mineralization andcontributes to abnormal bone formation of arthritis.