Expression and function of NG2/CSPG4 in human chondrocytes

Abstract

Introduction: NG2/CSPG4 is a unique transmembrane chondroitin sulphate proteoglycan molecule expressed as a core protein and a chondroitin sulphate proteoglycan (CSPG) up to 400kD. NG2/CSPG4 mediates the communication between the extracellular and intracellular compartments through interactions with collagen VI, growth factors and the actin cytoskeleton. NG2/CSPG4 affects cell migration, spreading, apoptosis and proliferation processes. NG2/CSPG4 has been shown to be expressed in developing and adult cartilage where less is known of its function. I tested the hypothesis: NG2/CSPG4 is an important regulator of chondrocytes function and has the potential to be a therapeutic target for treatment of diseases of cartilage such as osteoarthritis and chondrosarcoma. To do this, I had the following aims: 1) investigate whether different types of chondrocytes show variation in the form or distribution of NG2/CSPG4 expression and 2) through a knockdown approach develop a model to study the functional roles of NG2/CSPG4 in human chondrocytes. Materials and Methods: JJ012, a chondrosarcoma cell line, chondrocytes derived from human articular cartilage and C20/A4 an immortalised chondrocyte cell line were used. NG2/CSPG4 expression was investigated by RT-PCR western blotting, flow-cytometry and immunocytochemistry. NG2/CSPG4 interaction with Golgi complex and endoplasmic reticulum (ER) was assessed by double immunofluorescence. Biochemical interactions were assessed by immunoprecipitation and mass spectroscopy. For NG2/CSPG knockdown, a viral transduction method was carried out using 5 different constructs. Different functional roles of NG2/CSPG4 were investigated. The role of NG2/CSPG4 in gene regulation was studied by shRNA knockdown of NG2/CSPG4 in JJ012 cells and RTPCR. Results: NG2/CSPG4 mRNA was detectable in all cells tested. Western blotting showed expression of only a 270kD core protein in JJ012 and C20A4 cells. Using two different anti NG2/CSPG4 antibodies human OA chondrocytes were seen to express multiple molecular weight forms differentially recognised with and without chondroitinase ABC pre-treatment. Expression of NG2/CSPG4 in JJ012 cells was predominantly membrane associated whilst in OA chondrocytes and C20A4 cells, additional, predominant punctuate cytoplasmic distribution was evident. In OA chondrocytes NG2/CSPG4 co-localised with the Golgi complex and ER. Immunoprecipitation and mass spectrometry data demonstrated associations between NG2/CSPG4 and both collagen VI and thrombopoietin in OA chondrocytes. A model of NG2/CSPG4 gene knockdown was achieved in JJ012 chondrosarcoma cell line, known as B3. B3 cells spread more and migrate less than JJ012 cells; with a significant difference observed in migration (after 10hours: the closed area was 81.4% for JJ012 and 54.6% for B3). There was no difference in cell adhesion to collagen I, II, VI and fibronectin. EGTA inhibited cell adhesion to fibronectin in dose dependent manner with no significant difference observed between both JJ012 and B3 cells. EDTA reduced adhesion of B3 cells but not JJ012 to fibronectin. A significant difference in cell proliferation was detected with no change in apoptosis. Following NG2/CSPG4 knockdown in JJ012 cells there was no difference in expression of aggrecan, collagen II and SOX-9. In contrast, B3 cells showed a decreased expression of MPP3 and ADAMTS-4, a complete loss of ADAMTS-5 and increased expression of MMP13. Conclusions: I have identified altered expression and multiple forms of NG2/CSPG4 in different types of chondrocytes and shown association of this molecule with type VI collagen and thrombopoietin. Creation of a chondrocyte cell line that has stable knockdown of NG2/CSPG4 allowed further investigation of NG2/CSPG function in chondrocytes. NG2/CSPG4 knockdown reduced the cellular migration and proliferation and increased the chondrocyte spreading. The adhesion mechanism in JJ012 appears to be calcium dependent. Loss of NG2/CSPG4 induced changes in expression of aggrecanases and MMPs. Altered expression or associations of NG2/CSPG4 with extracellular ligands or intracellular signalling cascades may be important in the pathogenesis of OA by regulating proteolytic activity or apoptosis related pathways. NG2/CSPG4 is a potential therapeutic target in degenerative and neoplastic diseases of cartilage.