|dc.contributor.author||Jamil, Nuor Sabah Mohammed||en
|dc.description.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.||en
|dc.contributor.sponsor||Medical Research Council (MRC)||en
|dc.publisher||The University of Edinburgh||en
|dc.relation.hasversion||"Therapeutic Molecular Targets in Human Chondrosarcoma". NSM Jamil, SEM Howie and DM Salter. Review paper published in the International Journal of Experimental Pathology, 2010, Volume 91, 387-393.||en
|dc.relation.hasversion||"The expression and function of NG2/CSPG4 in human chondrocytes". NSM Jamil, SEM Howie and DM Salter. Abstract published in the International Journal Of Experimental Pathology, Volume 93, Issue 4, Pages 243–304, A1–A31||en
|dc.relation.hasversion||British Society for Matrix Biology- Autumn 2011 Meeting Report. Nuor Jamil, Sally Dreger and Christos Gavriilidis, recipients of BSMB Reporter Bursaries. The meeting report was published in the International Journal of Experimental Pathology, Volume 93, Issue 4, Pages 243–304, and A1–A31.||en
|dc.relation.hasversion||"The development of a model to study NG2/CSPG4 function in chondrocytes". NSM Jamil, SEM Howie and DM Salter. Abstract published in the International Journal of Experimental Pathology, 2011, Volume92, A1-A38||en
|dc.relation.hasversion||British Society for Matrix Biology- Autumn 2010 Meeting Report. Matthew Gardiner, Stephanie Dakin, Douglas Dyer, Maan Al-Abbasi and Nuor Jamil, recipients of BSMB Reporter Bursaries. The meeting report was published in the International Journal of Experimental Pathology, 2011, Volume92, A1-A38.||en
|dc.relation.hasversion||"Differential Expression of NG2/CSPG4 in articular Chondrocytes transformed and Chondrosarcoma cell lines". NSM Jamil, SEM Howie and DM Salter. Abstract published in the International Journal of Experimental Pathology, 2010, Volume 91, A1-A42.||en
|dc.title||Expression and function of NG2/CSPG4 in human chondrocytes||en
|dc.type||Thesis or Dissertation||en
|dc.type.qualificationname||PhD Doctor of Philosophy||en