A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation

Abstract

Activating mutations in FGF receptor 3 (FGFR3) cause several human dwarfism syndromes by affecting both chondrocyte proliferation and differentiation. Using microarray and biochemical analyses of FGF-treated rat chondrosarcoma chondrocytes, we show that FGF inhibits chondrocyte proliferation by initiating multiple pathways that result in the induction of antiproliferative functions and the down-regulation of growth-promoting molecules. The initiation of growth arrest is characterized by the rapid dephosphorylation of the retinoblastoma protein (pRb) p107 and repression of a subset of E2F target genes by a mechanism that is independent of cyclin E-Cdk inhibition. In contrast, hypophosphorylation of pRb and p130 occur after growth arrest is first detected, and may contribute to its maintenance. Importantly, we also find a number of gene expression changes indicating that FGF promotes many aspects of hypertrophic differentiation, a notion supported by in situ analysis of developing growth plates from mice expressing an activated form of FGFR3. Thus, FGF may coordinate the onset of differentiation with chondrocyte growth arrest in the developing growth plate.

Figure 1.

FACScan™ analysis of RCS and ROS cells in response to FGF treatment. Growing cultures were treated with 5 ng/ml FGF1 and 10 mg/ml heparin for the indicated times and subjected to flow cytometry. UN indicates RCS (A) or ROS (B) cells treated with heparin only. Numbers on the y axis indicate percentage of total cells analyzed per sample.

Figure 2.

Gene expression profiles after FGF treatment of RCS cells. RNA samples prepared from RCS cells after 0, 1, 3, 6, 10, or 24 h of FGF treatment were converted into biotinylated cRNAs and hybridized to rat microarrays (Affymetrix). Genes up-regulated (A) or down-regulated (B) by greater than threefold with respect to untreated control samples were subjected to hierarchical clustering as detailed in Materials and methods. Average expression is defined by the GeneSpring^® software (Silicon Genetics) and assigned a value of 1, whereas values greater or less than this value are visualized as more intense shades of red or green, respectively. Individual genes are represented in rows from left to right, and their relative expression level is depicted according to the colorimetric scale at each time point indicated above the dendrograms. Major patterns within the dendrograms were determined for selected nodes and are displayed to the right of each dendrogram. By relating each of these expression profiles to changes in the cell cycle as defined by the FACScan™ analysis, genes within these subgroups were defined as early (E), mid-(M), or late (L) response genes.

Figure 3.

Confirmation of the microarray results using Northern analysis. RNA was isolated from RCS cells treated with FGF for the indicated times and subjected to Northern analysis using radiolabeled probes for PC3, osteopontin (OPN), or osteoprotegerin (OPG) as described in Materials and methods.

Figure 4.

E2F target genes are down-regulated in RCS cells in response to FGF. The expression profiles of rat homologues of previously identified human E2F target genes (Ren et al., 2002) were subjected to k-means clustering as detailed in Materials and methods. Each line within the graphs represents the relative expression profile for one of the genes listed.

Figure 5.

Hypophosphorylation of the pRbs occurs with different kinetics and by distinct mechanisms in response to FGF. (A) Kinetics of p107, p130, and pRb dephosphorylation. Total protein lysates were prepared from RCS cells at the times of FGF treatment indicated and subjected to Western analysis using the antibodies indicated. Bars to the left of the image depict the position of the hyperphosphorylated (top) or hypophosphorylated (bottom) forms of each protein. (B) RCS cells were treated with FGF in the presence or absence of actinomycin D, and whole-cell extracts were subjected to Western analysis using p107 or pRb antibodies. Samples derived from cells treated with FGF (fgf), acinomycin D (act), or both (fgf+act) are indicated above the lanes. (C) Western analysis of p21 expression. Protein extracts from FGF-treated RCS cells were subjected to Western analysis using anti-p21 antibody. (D) Kinetics of cyclin E–Cdk inhibition and association with p21. Cyclin E–Cdk2 complexes were immunoprecipitated from protein extracts of RCS cells after treatment with FGF for the times indicated. Kinase activity was assessed in vitro in the presence of γ[³²P]ATP and histone H1 substrate as detailed in Materials and methods. The remainder of the immunoprecipitate was used for Western analysis using antibodies against Cdk2 or p21.

Figure 6.

In situ hybridization. Tibia sections from P15 wild-type (WT; A–D and I–L) or FGFR3 mutant (369/369; E–H and M–P) mice were stained with Alcian blue, hemotoxylin, and eosin (A and E). R, P, and H depict the regions of the reserve, proliferative, and hypertrophic zones, respectively; B indicates trabecular bone and sco the secondary center of ossification. Sections were hybridized with ³⁵S-labeled RNA probes for collagen X (ColX; B and F), Indian hedgehog (Ihh; C and G), Id-3 (D and H), Id-1 (J and N), or osteopontin (Opn; L and P) and visualized using darkfield microscopy. After hybridization, sections in J, L, N, and P were stained with hemotoxylin to more precisely localize the regions expressing Id-1 and Opn (I, K, M, and O). Arrows indicate chondrocytes expressing OPN.

Figure 7.

Model depicting the multiple pathways induced in response to FGF treatment of RCS cells leading to cell cycle arrest and initiation of differentiation. The initiation of growth arrest encompasses gene expression changes and signaling events observed during the early and mid-response stages. The major pathways serve to down-regulate growth-promoting functions and activate antiproliferative functions as discussed in the text. These events converge to establish the maintenance of growth arrest, which is characterized by cyclin E–Cdk inhibition, the hypophosphorylation of all three pRbs, further down-regulation of E2F target genes and genes encoding other cell cycle components, and the induction of genes associated with hypertrophic differentiation during the mid- and late response stages.