SOX9 transduction of a human chondrocytic cell line identifies novel genes regulated in primary human chondrocytes and in osteoarthritis

Abstract

The transcription factor SOX9 is important in maintaining the chondrocyte phenotype. To identify novel genes regulated by SOX9 we investigated changes in gene expression by microarray analysis following retroviral transduction with SOX9 of a human chondrocytic cell line (SW1353). From the results the expression of a group of genes (SRPX, S100A1, APOD, RGC32, CRTL1, MYBPH, CRLF1 and SPINT1) was evaluated further in human articular chondrocytes (HACs). First, the same genes were investigated in primary cultures of HACs following SOX9 transduction, and four were found to be similarly regulated (SRPX, APOD, CRTL1 and S100A1). Second, during dedifferentiation of HACs by passage in monolayer cell culture, during which the expression of SOX9 progressively decreased, four of the genes (S100A1, RGC32, CRTL1 and SPINT1) also decreased in their expression. Third, in samples of osteoarthritic (OA) cartilage, which had decreased SOX9 expression compared with age-matched controls, there was decreased expression of SRPX, APOD, RGC32, CRTL1 and SPINT1. The results showed that a group of genes identified as being upregulated by SOX9 in the initial SW1353 screen were also regulated in expression in healthy and OA cartilage. Other genes initially identified were differently expressed in isolated OA chondrocytes and their expression was unrelated to changes in SOX9. The results thus identified some genes whose expression appeared to be linked to SOX9 expression in isolated chondrocytes and were also altered during cartilage degeneration in osteoarthritis.

Figure 1

Retroviral expression of SOX9 in SW1353 chondrosarcoma cells. (a) Phase contrast micrograph demonstrating the morphology of SW1353 chondrosarcoma cells transduced with a retrovirus containing GFP or bicistronic SOX9-GFP. Scale Bar = 50 μm. (b) Cell lysates from GFP- or SOX9-SW1353 cells were analysed by western blotting using an anti-SOX9 antibody. (c) Real time PCR analysis of cDNA derived from green-fluorescent protein (GFP; black bars) or bicistronic SOX9-GFP (grey bars) transduced SW1353 chondrosarcoma cells.

Figure 2

Regulation of candidate genes during chondrocyte dedifferentiation. Real time PCR analysis of candidate gene expression in cDNA from human articular chondrocytes at passage (P) 0, 1 or 2. Mean fold-change values (where P0 = 1) with standard errors are presented from chondrocytes cultures obtained from 3 donors. * indicates significant difference in expression compared with passage 0 levels P < 0.05 by paired students t-test.

Figure 3

Comparison of the expression levels of candidate genes in normal and osteoarthritic cartilage. Real time PCR analysis of candidate gene expression in globally amplified cDNA representative of mRNA levels from normal (n = 8) or osteoarthritic (n = 15) human articular cartilage samples. Cartilage for the analysis was derived from either the medial or lateral femoral condyles. NM = normal medial, NL = normal lateral, OM = osteoarthritic medial and OL = osteoarthritic lateral. Symbols above bars indicate statistically significant regulation of that gene caused by:* disease (P < 0.05 mixed effects regression model) or ◆ joint location (P < 0.05 mixed effects regression model).