Involvement of fibroblast growth factor 18 in dedifferentiation of cultured human chondrocytes

Abstract

Objective: Chondrocytes inevitably decrease production of cartilaginous matrices during long-term cultures with repeated passaging; this is termed dedifferentiation. To learn more concerning prevention of dedifferentiation, we have focused here on the fibroblast growth factor (FGF) family that influences chondrocyte proliferation or differentiation.

Materials and methods: We have compared gene expression between differentiated cells in passage 3 (P3) and dedifferentiated ones in P8 of human cultured chondrocytes. We also performed ligand administration of the responsive factor or its gene silencing, using small interfering RNA (siRNA).

Results: FGFs 1, 5, 10, 13 and 18 were higher at P8 compared to P3, while FGFs 9 and 14 were lower. Especially, FGF18 showed a 10-fold increase by P8. Ligand administration of FGF18 in the P3 cells, or its gene silencing using siRNA in the P8 cells, revealed dose-dependent increase and decrease respectively in type II collagen/type I collagen ratio. Exogenous FGF18 also upregulated expression of transforming growth factor beta (TGF-beta), the anabolic factor of chondrocytes, in P3 chondrocytes, but P8 cells maintained a low level of TGF-beta expression, suggesting a decrease in responsiveness of TGF-beta to FGF18 stimulation in the dedifferentiated chondrocytes.

Conclusion: FGF18 seems to play a role in maintenance of chondrocyte properties, although its expression was rather high in dedifferentiated chondrocytes. Upregulation of FGF18 in dedifferentiated chondrocytes implied that it may be a marker of dedifferentiation.

Figure 1

Gene expression of COL1A1, COL2A1 and the COL2A1/COL1A1 expression ratio in chondrocytes cultured to passages 3 (P3) and P8. (a) Agarose gels after migration of PCR amplification products from P3 chondrocytes (left) and P8 ones (right). GAPDH was used as internal positive control. (b) Gene expression of chondrocytes cultured to P3 and P8 were examined using real time RT‐PCR. COL1A1 expression in the P8 chondrocytes showed approximately 7‐fold increase compared to that in P3 cells, while COL2A1 expression in P8 cells diminished to nearly two‐thirds of P3. Consequently, the COL2A1/COL1A1 expression ratio in P8 cells was ten times less than that in P3 cells. **P < 0.01 versus P3.

Figure 2

Expression profile of FGF family genes in chondrocytes cultured in passage 3 (P3) and P8, by microarray analysis. Gene expression in chondrocytes originating from two donors (samples #1 and #2) duplicated and showed downregulation of FGFs 1, 5, 10, 13, and 18, and upregulation of FGFs 9 and 14. Results from microarray analysis were expressed as log‐transformed fold changes in P3 and P8 cells.

Figure 3

Gene expression of FGF18 in cultured chondrocytes originating from eight donors (Samples #3–10). Normalized copy units of FGF18 in the chondrocyte culture in passage 8 (P8) were compared to those in P3 cells, using real time RT‐PCR, showing that 6 out of 8 samples significantly increased in FGF18 expression. **P < 0.01 versus P3.

Figure 4

Gene expressions of COL1A1, COL2A1 and COL2A1/COL1A1 expression ratio in chondrocytes cultured to passage 3 with administration of FGF18 ligand to the culture medium. Gene expression was evaluated using real time RT‐PCR. COL1A1 expression significantly diminished due to doses of FGF18, while decrease in COL2A1 expression was significant, but partial. Consequently, COL2A1/COL1A1 expression ratio increased with doses of FGF18. *P < 0.05, **P < 0.01 versus control.

Figure 5

Gene expression of COL1A1, COL2A1 and COL2A1/COL1A1 expression ratio in chondrocytes cultured to passage 8, with expression level of FGF18 gene suppressed using siRNA. (a) Cy3‐labelled negative control of siRNA (Cy3‐siRNA) was transfected well into chondrocytes at passage 8. Untransfected cells (Untransfected) at the same density and magnification were the control in this experiment. Left: fluorescence image, Right: phase contrast image. Bar = 100 μm. (b) Two siRNA constructs (#1 and #2) effectively suppressed expression of FGF18, compared to negative control of siRNA (cont). Gene expressions were evaluated using real time RT‐PCR. COL1A1 expression increased parallel to FGF18 expression levels, although COL2A1 expression diminished in contrast. Consequently, COL2A1/COL1A1 expression ratio decreased according to FGF18 expression level. **P < 0.01 versus control.

Figure 6

Gene expressionsof FGFR1, FGFR2, FGFR3 and FGFrl1 in chondrocytes cultured to passage 3 (P3) and P8. No significant differences in gene expressions were found using real time RT‐PCR.

Figure 7

Gene expression of TGF‐β1 in chondrocytes cultured to passage (P3) and P8, and in which FGF18 ligands were administered to the culture medium in P4. Gene expressions were shown to be evaluated using real time RT‐PCR. TGF‐β1 expression in chondrocytes at P8 significantly diminished when compared to those of P3. In contrast, it significantly increased as a result of doses of FGF18 in P4. **P < 0.01 versus P4 without FGF18 administration, *P < 0.05 versus P3.