Chondrocyte cluster formation in agarose cultures as a functional assay to identify genes expressed in osteoarthritis.
Understanding altered gene expression in osteoarthritic cartilage can lead to new targets for drug intervention. We established a functional assay based on chondrocyte cluster formation, a phenotype associated with osteoarthritis (OA), to screen an OA cartilage gene library. Previous reports have demonstrated that normal chondrocytes grown in suspension culture maintain their chondrocytic phenotype, however, certain growth factors such as basic fibroblast growth factor (bFGF) will induce the cells to proliferate in tight clusters similar to those seen in osteoarthritic cartilage. In this study we validate that overexpression of bFGF by retrovirally transduced normal chondrocytes would similarly induce the proliferation of tight cell clusters. We then used this approach as a basis to set up a functional screen where an entire OA cartilage cDNA library was tranduced into normal chondrocytes to search for other genes that would also induce cluster formation. Seven potential genes were isolated from the OA gene library, including BPOZ, IL-17 receptor C, NADH ubiquinone oxidoreductase, COMP, Soluble carrier 16 (MCT 3), C1r, and bFGF itself. None of the identified genes were upregulated by bFGF, however, all of them upregulated the expression of bFGF suggesting a common pathway. Although cluster formation is not considered to be destructive in OA cartilage, it is consistent with the disease and could yield answers to the altered phenotype. Further studies are needed to elucidate how these genes are linked to the disease state.
Quintavalla
Joseph
J
Department of Bone, Muscle and Gastrointestinal, Novartis Institutes for BioMedical Research, East Hanover, New Jersey, USA. joseph.quintavalla@pharma.novartis.com
Kumar
Chandrika
C
Daouti
Sherif
S
Slosberg
Eric
E
Uziel-Fusi
Susan
S
D002358:Cartilage, Articular; D002449:Cell Aggregation; D002455:Cell Division; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism* / Q000502:physiology; D003584:Cytological Techniques; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D010003:Osteoarthritis / Q000235:genetics / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D012685:Sepharose; D014161:Transduction, Genetic; D015854:Up-Regulation
D016222:Fibroblast Growth Factor 2; D012685:Sepharose