Fibroblast growth factor signaling uses multiple mechanisms to inhibit Wnt-induced transcription in osteoblasts

Abstract

Fibroblast growth factor (FGF) and Wnt signals are both critical for proper bone development. We previously reported that the expression of activating FGF receptor mutations in osteoblasts downregulated the expression of many genes reported as targets of Wnt signaling, suggesting an antagonistic effect between Wnt signaling, which promotes osteoblast differentiation and function, and FGF signaling, which inhibits these processes. To analyze the effect of FGF on Wnt signaling in osteoblasts, we created reporter cell lines where a Wnt-responsive promoter drives luciferase expression and showed that Wnt3a-induced luciferase expression was specifically inhibited by FGF treatment. FGF specifically prevented the formation of a Wnt-induced transcriptional complex of TCF1 and -4 with beta-catenin on DNA. FGF did not significantly affect the activation of beta-catenin, although it reduced both the expression of TCF/LEF factors and their induction by Wnt. Microarray analysis using osteoblasts treated with Wnt3a and FGF alone or in combination showed that about 70% of the genes induced by Wnt3a were downregulated by combined FGF treatment. These included novel and previously identified Wnt target genes and genes involved in osteoblast differentiation. Furthermore, FGF alone could downregulate the expression of four Fzd Wnt receptor genes. Our results show that FGF antagonizes Wnt signaling by inhibiting Wnt-induced transcription and suggest that multiple mechanisms, including downregulation of TCFs and Wnt receptors, contribute to this effect.

FIG. 1.

Wnt3a induces luciferase in reporter osteoblast lines. (A) Alkaline phosphatase assay. Parental OB1 cells were plated and grown in differentiation medium for 5 days in the presence of PBS buffer as a control or 100 ng/ml Wnt3a or 10 ng/ml FGF1 or both. (B) Luciferase assay of OB cell reporter clones stably transfected with the TOP-luciferase or the FOP-luciferase cassette. Each cassette, schematically represented above the histograms, contains the luciferase reporter gene (white box) and the minimal herpesvirus thymidine kinase viral promoter (gray) cloned downstream of eight copies of wild-type (TOP; green box) or mutated (FOP; red box) TCF/LEF binding sites. The cell clones were treated for 18 h with 100 ng/ml Wnt3a or 20 mM LiCl. The resulting luciferase activity, expressed as induction compared to that for untreated cells, is the mean for duplicate samples. The experiment was repeated three times with similar results. (C) Wnt3a dose-response luciferase assay. OB-TOP#1 cells were treated for 20 h with increasing concentrations of Wnt3a. (D) Wnt3a and LiCl time course luciferase assay. OB-TOP#1 cells were treated with 150 ng/ml of Wnt3a or 20 mM LiCl for the indicated times. (E and F) Western blot analysis, with monoclonal antibodies raised against active β-catenin, of total lysates from OB-TOP cells treated with increasing concentrations of Wnt3a (E) or with 150 ng/ml Wnt3a (F) for the indicated times. (G) Dose-response luciferase assay of OB-TOP#1 cells to LiCl or Wnt3a conditioned medium (cm).

FIG. 2.

Downregulation of Wnt/β-catenin signaling by FGF. (A) Luciferase assay of OB-TOP cells incubated with 1, 5, or 25 ng/ml of FGF1 and harvested after 12 or 24 h. One hundred percent Wnt3a conditioned medium (Wnt3a CM) and control CM were used as positive and negative controls, respectively. (B) Luciferase assay of four independent OB-TOP clones treated for 12 h with increasing amounts of Wnt3a CM in the absence or in the presence of 10 ng/ml of FGF1. (C) OB-TOP reporter cells were pretreated for 1 h with the indicated growth factor. Wnt3a at 100 ng/ml (+) or PBS buffer (−) was added and the cells were further incubated for 15 h before being harvested. Growth factor concentrations were 10 ng/ml FGF1, 50 ng/ml BMP2, 50 ng/ml IGF1, ng/ml TGFβ, and 100 ng/ml parathyroid hormone-related peptide (PTHrP). The resulting luciferase activity is from one representative experiment and is the mean of triplicates ± standard deviation. (D) Luciferase response to Wnt or to Wnt and FGF of OB reporter cells pretreated with dimethyl sulfoxide (−) or 20 uM of the indicated inhibitors: SB203580 (SB), LY294002 (LY), and U0126 (U). For each pretreatment, the luciferase activity obtained for Wnt3a-treated cells was arbitrarily set as 100%. The resulting luciferase activity is from one representative experiment and is the mean of duplicates.

FIG. 3.

Time course analysis of the inhibition of Wnt3a-induced luciferase activity by FGF. (A) (Top) Schematic representation of the experiment. The horizontal line represents time in h. The arrows indicate the time of treatment. (Bottom) OB-TOP#1 reporter cells were treated for 10 h with 100 ng/ml of Wnt3a alone (black bar) or together with FGF1 (white bars) at the indicated time. The resulting luciferase activity, expressed as luciferase units, is the mean of triplicates ± standard deviation. (B) (Top) Schematic representation of the time course experiment; (bottom) cells were treated for 6, 15, or 24 h with Wnt3a alone or together with FGF added at time +3, 0, −3, or −10.

FIG. 4.

FGF does not affect β-catenin stabilization by Wnt but blocks the formation of a β-catenin/TCF nuclear complex. (A) Western blot analysis of nuclear (lanes 1 to 4) and cytoplasmic (lanes 5 to 8) protein extracts from OB-TOP#1 cells untreated or treated with Wnt, with FGF, or with FGF and Wnt added together for 6 h. Antibodies raised against active β-catenin (ABC), total β-catenin, TCF3/4, TCF4, and Sox2 were used. Antibodies raised against tubulin (cytoplasmic) and TFII-I (nuclear) were used as controls to verify the purity and equal loading of the samples. (B) Western blot analysis of nuclear extracts from OB-TOP#1 cells that had been pretreated with FGF for 10 h before Wnt addition. The length of Wnt treatment was 5 h (lanes 2 and 4) or 10 h (lanes 3 and 5). Lane 6, FGF only, 20 h total. (C) EMSA comparing nuclear protein complex assemblies on the TCF/LEF consensus DNA probe. Nuclear extracts are from cells treated as described for panel B. ns, a nonspecific DNA/protein complex. Lanes 9 to 13, competition experiment with 10× and 100× molar excesses of unlabeled probes containing the wild-type (TOP) or mutated (FOP) TCF/LEF consensus. (D) EMSA of nuclear extract from OB-TOP#1 cells that are untreated or treated with Wnt, with FGF and Wnt together, or with FGF only (lanes 1 to 4) for 6 h. Supershift experiment: nuclear extracts from OB-TOP#1 cells untreated (lanes 5 to 8) or treated with Wnt3a for 6 h (lanes 9 to 12) were preincubated with 500 ng of the indicated antibodies (Ab) before EMSA. Asterisks indicate the positions of supershifted DNA complexes. (E) EMSA of nuclear extracts from the same experiment shown in panel D preincubated with 500 ng of the indicated antibodies before EMSA.

FIG. 5.

Regulation of mRNA levels by Wnt3a and FGF1. (A) The graph shows representative expression patterns of selected genes at the 6-h and 12-h time points assayed by microarray analysis and classified by the criteria used to compile Table 1. The log values of the normalized intensity values are shown rather than absolute changes. Four Wnt3a-upregulated genes are shown, including two unchanged (Ahr, green; Rhou, red) and two downregulated (Gadd45g, light blue; Timp3, dark blue) by FGF1 alone. (B) The effects of Wnt and FGF treatment on various Wnt target and Fzd receptor genes were validated in an independent experiment by RT-PCR. Relative mRNA levels are expressed compared to the levels in untreated cultures. Bars indicate Wnt only (black), Wnt plus FGF (gray), and FGF only (white). OB-TOP#1 cells were pretreated with FGF for 10 h and with Wnt3a for an additional 10 h.

FIG. 6.

Wnt induction of several target genes is attenuated in Sox2-expressing cells. Real-time RT-PCR analysis of six Wnt target genes in OB1 clones treated with Wnt3a for 16 h. Relative mRNA levels are expressed compared to the level for untreated cells for each clone. Lanes: 1, control OB1 cells transfected with vector alone (open bars); 2, OB1 clone expressing basal levels of Sox2 (gray bars); 3, OB1#16 expressing high levels of Sox2 (black bars). (Inset) Levels of Sox2 protein. β-Catenin was used as a loading control.

FIG. 7.

Schematic representation of the multiple mechanisms by which FGF inhibits canonical Wnt signaling in osteoblasts. FGF signals (i) upregulate factors such as Sox2 and Mitf that can interfere with Wnt signaling by binding β-catenin (β cat); (ii) lower the transcript levels of Fzd receptors Fzd1, -2, -7, and -8; and (iii) decrease the formation of Wnt-induced transcriptional complexes containing TCFs and β-catenin on TCF/LEF binding sites by inhibiting TCF1 and -4 expression, thus preventing the transcription of Wnt target genes. Some Wnt target genes may also be directly downregulated by FGF signaling through FGF response elements (FRE) in independent regulatory regions or by destabilizing transcripts.