Runx2 is required for the proliferation of osteoblast progenitors and induces proliferation by regulating Fgfr2 and Fgfr3

Abstract

Runx2 and Sp7 are essential transcription factors for osteoblast differentiation. However, the molecular mechanisms responsible for the proliferation of osteoblast progenitors remain unclear. The early onset of Runx2 expression caused limb defects through the Fgfr1-3 regulation by Runx2. To investigate the physiological role of Runx2 in the regulation of Fgfr1-3, we compared osteoblast progenitors in Sp7^-/- and Runx2^-/- mice. Osteoblast progenitors accumulated and actively proliferated in calvariae and mandibles of Sp7^-/- but not of Runx2^-/- mice, and the number of osteoblast progenitors and their proliferation were dependent on the gene dosage of Runx2 in Sp7^-/- background. The expression of Fgfr2 and Fgfr3, which were responsible for the proliferation of osteoblast progenitors, was severely reduced in Runx2^-/- but not in Sp7^-/- calvariae. Runx2 directly regulated Fgfr2 and Fgfr3, increased the proliferation of osteoblast progenitors, and augmented the FGF2-induced proliferation. The proliferation of Sp7^-/- osteoblast progenitors was enhanced and strongly augmented by FGF2, and Runx2 knockdown reduced the FGF2-induced proliferation. Fgfr inhibitor AZD4547 abrogated all of the enhanced proliferation. These results indicate that Runx2 is required for the proliferation of osteoblast progenitors and induces proliferation, at least partly, by regulating Fgfr2 and Fgfr3 expression.

Figure 1

Limb development in Tg(Prrx1-EGFP-Runx2) mice (A–H) Whole mount in situ hybridization. Whole mount in situ hybridization of the forelimb buds of wild-type mice (wt) (A,C,E,G) and Tg(Prrx1-EGFP-Runx2) mice with strong expression (Tg) (B,D,F,H) at E10.0 (A,B) and E10.5 (C–H) using Fgf10 (A,B), Fgf8 (C,D), Fgf4 (E,F), and Shh (G,H) probes. (I–P) Histological analysis. A histological analysis of the forelimb buds of a wild-type mouse (wt) (I,K,M,O) and Tg(Prrx1-EGFP-Runx2) (Tg) mouse with strong expression (Tg) (J,L,N,P) at E10.5. (I,J) H-E staining. The AER is observed in the wild-type mouse (I, arrowhead), but not in the Tg(Prrx1-EGFP-Runx2) mouse (J). D, dorsal; V, ventral. (K,L) Immunohistochemical analysis of Runx2 protein expression. The boxed region in L is amplified in the window. (M,N) In situ hybridization using the Fgf8 probe. (O,P) TUNEL staining. F₀ littermates of wild-type and Tg(Prrx1-EGFP-Runx2) mice were compared in each whole mount in situ hybridization and histological analysis. Scale bars: 20 μm (A,B); 200 μm (C–H); 200 μm (I–P); 20 μm (inset in L).

Figure 2

Real-time RT-PCR analyses of the expression of Fgfr1, Fgfr2, and Fgfr3 The expression of Fgfr1, Fgfr2, and Fgfr3 and their respective IIIb and IIIc isoform mRNA in Tg(Prrx1-EGFP) and Tg(Prrx1-EGFP-Runx2) mice was measured by real-time RT-PCR in triplicate. EGFP-positive cells were collected from limb buds of more than 70 F₀ EGFP-positive embryos each in Tg(Prrx1-EGFP-Runx2) mice and Tg(Prrx1-EGFP) mice at E10.5 by sorting EGFP-positive cells using FACS, the EGFP-positive cells obtained in each sorting were pooled, and mRNA was extracted from the pooled cells. We normalized values to that of Gapdh. Values in wild-type mice were defined as 1, and the relative levels are shown. Data are the mean ± SD. *p < 0.05, **p < 0.01.

Figure 3

(A) Induction of Fgfr1, Fgfr2, and Fgfr3 by Runx2 in vitro. RNA was extracted from wild-type osteoblast progenitors that had been infected with an adenovirus expressing type II Runx2 and EGFP or EGFP alone. Samples were harvested 12 and 24 hrs after infection. Values in cells infected with the EGFP-expressing adenovirus were defined as 1, and the relative levels are shown. Data are the mean ± SD of 4 wells. **p < 0.01. (B) Suppression of Fgfr1, Fgfr2, and Fgfr3 expression by Runx2 siRNA. Osteoblast progenitors were prepared from the calvariae of wild-type newborn mice, and transfected with Runx2 siRNA. RNA was extracted 48 hours after transfection, and real-time RT-PCR was performed. Values in siRNA for the control were defined as 1, and the relative levels are shown. Data are the mean ± SE of 3 wells. **p < 0.01. Similar results were obtained in three independent experiments and representative data are shown.

Figure 4

Reporter and ChIP assays of Fgfr1, Fgfr2, and Fgfr3 promoters. (A–C) Reporter assays of the Fgfr1 promoter. (A) Schematic diagrams of the reporter vectors of the Fgfr1 promoter and their luciferase (Luc) activities. (B) The nucleotide sequence containing putative Runx2-binding sites in the 0.2-kb fragment. Ets1-binding sites are underlined and putative Runx2-binding sites (R1 and R2) are boxed. The mutated sequences are shown above the boxes and lines. (C) Reporter activities of the 0.2-kb construct (p0.2k r1) and the 0.2-kb constructs carrying a mutated R1, R2, or Ets1 site. (D–F) Reporter assays of the Fgfr2 promoter. (D) Schematic diagrams of the reporter vectors of the Fgfr2 promoter and their luciferase activities. (E) The nucleotide sequence containing putative Runx2-binding sites in the 0.39-kb fragment. The putative Runx2-binding sites (R1–4) are boxed, and the mutated sequences are shown above or below the boxes. (F) Reporter activities of the 0.39-kb construct (p0.39k r2) and the 0.39-kb constructs carrying mutated R1, R2, R3, or R4. (G–I) Reporter assays of the Fgfr3 promoter. (G) Schematic diagrams of the reporter vectors of the Fgfr3 promoter and their luciferase activities. (H) The nucleotide sequence containing putative Runx2-binding sites in the 0.23-kb fragment. The putative Runx2-binding sites (R1, R2) are boxed, and the mutated sequences are shown above the boxes. (I) Reporter activities of the 0.23-kb construct (p0.23k r3) and the 0.23-kb constructs carrying mutated R1, R2, or R1 and R2. Vertical lines in the diagrams represent the positions of consensus Runx2-binding motifs (A,D,G), and arrows indicate reported transcription start sites (B,E,H). In all reporter assays, C3H10T1/2 cells were transfected with an empty (open column) or Runx2-expressing (closed column) vector. Data are the mean ± SD of 4 wells. *p < 0.01 versus the control. Three independent experiments were performed and representative data are shown. (J) ChIP assays. DNA before (input) and after immunoprecipitation with a monoclonal anti-Runx2 antibody (Runx2) or mouse IgG (IgG) was amplified by PCR using primers that amplify the sequences of Fgfr1 (−88 ~+199), Fgfr2 (−83 ~+216), and Fgfr3 (−188 ~+178). Similar results were obtained in three independent experiments and representative data are shown.

Figure 5

Histological analysis of Sp7^−/− and Runx2^−/− mice Frontal sections of wild-type (wt) (A,D,G,J,M,P), Sp7^−/− (B,E,H,K,N,Q), and Runx2^−/− (C,F,I,L,O,R) mice at E18.5 were stained with H-E (A–C,G–I,M–O), or subjected to in situ hybridization using the Col1a1 probe (D–F,J–L,P–R). The boxed regions in A, B, and C are magnified in G and M, H and N, and I and O, respectively. The boxed regions in D, E, and F are magnified in J and P, K and Q, and L and R, respectively. Brackets in (G–L) indicate the layers of osteoblastic cells or osteoblast progenitors in the calvarial region. The widths of calvariae (wt: n = 8, Sp7^−/−: n = 6, Runx2^−/−: n = 5), and Col1a1-positive area in mandibles (wt: n = 4, Sp7^−/−: n = 3, Runx2^−/−: n = 3) were measured and shown in S and T, respectively. The values in wild-type mice were set as 1, and the relative levels are shown in T. Bars: 500 μm (A–F), 100 μm (G–L), 200 μm (M–R).

Figure 6

Runx2 and Fgfr2 expression and BrdU labeling in the calvaria and mandible of wild-type, Sp7^−/−, and Runx2^−/− mice (A–U) Frontal sections of wild-type (A,D,G,J,M,P,S) and Sp7^−/− (B,E,H,K,N,Q,T), and Runx2^−/− (C,F,I,L,O,R,U) mice at E18.5 were reacted with the anti-Runx2 antibody (A–F,M–O) and anti-Fgfr2 antibody (G–I,P–R) or subjected to BrdU labeling (J–L,S–U). The boxed regions in A are magnified in D and M, the boxed regions in B are magnified in E and N, and the boxed regions in C are magnified in F and O. The boxed regions in (D–F) and (M–O) are magnified in (D’–F’) and (M’–O’), respectively. The similar regions of (D’–F’) are shown in (G–I) and (J–L), and those of (M’–O’) are shown in (P–R) and (S–U). Brackets in (D’–L) indicate the layers of osteoblastic cells or osteoblast progenitors in the calvarial region, short arrows in (G–I) indicate muscle fibers, arrowheads in (G–I) and (P–R) indicate chondrocytes, and long arrows in R indicate neurons. Bars: 500 μm (A–C), 100 μm (D–F,M–O), 20 μm (D’-L, M’–U). (V) Real-time RT-PCR analysis. RNA was extracted from the calvariae of wild-type, Sp7^−/−, and Runx2^−/− mice at E18.5. The values in wild-type mice were set as 1, and relative levels are shown. Data are the mean ± SE of 4–5 mice. *vs. wild-type mice. *p < 0.05, **^,††p < 0.01. (W) Western blot analysis. Protein was extracted from the calvariae of wild-type, Sp7^+/−, and Sp7^−/− mice at E18.5. β-actin was used as an internal control. (X) Quantification of Western blot bands. The normalized values of Runx2 protein bands in wild-type mice were set as 1, and the relative levels in Sp7^−/− embryos are shown. Data are the mean ± SE of 3 bands. (Y and Z) BrdU-positive osteoblastic cells and osteoblast progenitors in calvariae (Y) and mandibles (Z) were counted and shown as a percentage of the number of osteoblastic cells and osteoblast progenitors. n = 6. **^,††p < 0.01.

Figure 7

Analyses of the proliferation of osteoblast progenitors in vitro (A) Reductions in Fgfr1, Fgfr2, and Fgfr3 mRNA by the introduction of respective siRNA into wild-type osteoblast progenitors. Each value of Fgfr1–3 in the introduction of control siRNA was set as 1 and the relative levels are shown. n = 3. **p < 0.01. (B) Effects of FGF2 and each siRNA for Fgfr1, Fgfr2, and Fgfr3 on the proliferation of wild-type osteoblast progenitors. The values of the vehicle in the control siRNA were set as 1, and the relative levels are shown. n = 4. * vs. the respective vehicle. ^†vs. the respective experiment in the control siRNA. **^,††p < 0.01. (C) Effects of Runx2 on proliferation and the FGF2-induced proliferation of wild-type osteoblast progenitors and the inhibition by AZD4547 (50 nM). The values of the vehicle in the GFP group were set as 1, and the relative levels are shown. n = 4. * vs. the respective vehicle. †vs. the respective experiment in the GFP group. **^,††,##p < 0.01. (D) Effects of inhibitors on the FGF2-induced proliferation of wild-type osteoblast progenitors. AZD: AZD4547 (50 nM), U: U0126 (50 μM), LY: LY294002 (1 μM), Akt-I: Akt inhibitor (2.5 μM). The values in the vehicle were set as 1, and the relative levels are shown. n = 4. * vs. the respective vehicle. ^†vs. the respective experiment in the vehicle group. ^†p < 0.05, ^**,††p < 0.01. (E,F) Western blots of activated p42/44 MAP kinase (E) and Akt (F). IGF-1 was used as a positive control for Akt activation (F). (G) Effects of FGF2 and AZD4547 (50 nM) on the proliferation of Sp7^−/− osteoblast progenitors. The values in the vehicle of wild-type osteoblast progenitors were set as 1, and relative levels are shown. n = 4. * vs. the respective vehicle. ^†vs. the respective experiment in the wild-type group. *^,†,#p < 0.05, **^,††,##p < 0.01. (H) Real time RT-PCR analysis using RNA from Sp7^−/− osteoblast progenitors. The values for control siRNA were set as 1 and the relative levels are shown. n = 3. **p < 0.01. (I) The effects of siRNA for Runx2 on the FGF2-induced proliferation of Sp7^−/− osteoblast progenitors. The values for the vehicle were set as 1 and the relative levels are shown. n = 4. * vs. the respective vehicle. †vs. the respective experiment in the control siRNA. **^,††p < 0.01. Similar results were obtained in two to four independent experiments and representative data are shown in A–I.

Figure 8

Droplet digital RT-PCR, ChIP, and cell proliferation analyses (A) Droplet digital RT-PCR analysis. The expression levels of Fgfr1–3 were compared among wild-type, Sp7^−/−, and Runx2^−/−calvariae. n = 4. *vs. wild-type mice. ^†vs. the respective mouse in Fgfr1. ^#vs. the respective mouse in Fgfr2. **^,††,##p < 0.01. (B) ChIP assay. DNA was extracted from Sp7^−/− calvariae, and DNA before (input) and after immunoprecipitation with the monoclonal anti-Runx2 antibody (Runx2) or mouse IgG (IgG) was amplified by PCR using the same primers as those in Fig. 4J. (C) The effects of FGF2, Wnt3a, Ihh, Shh, and PTHrP (1–34) in the proliferation of GFP- or Runx2-transfected wild-type osteoblast progenitors. n = 4. * vs. the vehicle in GFP-transfected cells. ^†vs. the vehicle in Runx2-transfected cells. *p < 0.05, **^,††p < 0.01. (D) Proliferation of wild-type and Runx2^−/− osteoblast progenitors. n = 4. **p < 0.01. Similar results were obtained in three independent experiments and representative data are shown in B-D.

Figure 9

Comparison of the proliferation of osteoblast progenitors in Sp7^−/−Runx2^+/+ and Sp7^−/−Runx2^+/– mice (A–J) Frontal sections of Sp7^−/−Runx2^+/+ (A,C,E,G,I) and Sp7^−/−Runx2^+/– (B,D,F,H,J) mice at E18.5 were stained with H-E (A–D,G,H), or subjected to BrdU labeling (E,F,I,J). The upper boxed regions in A and B are magnified in C and D, and the lower boxed regions in A and B are magnified in G and H, respectively. Serial sections were used for BrdU labeling (E,F: calvarial region; I,J: mandibles). The brackets in (C–F) indicate the layers of osteoblast progenitors in the calvarial region. (K–M) The width of calvariae (K) and the percentage of BrdU-positive osteoblast progenitors in calvariae (L) and mandibles (M). The data are the mean ± SE of 3 mice. Bars: 500 μm (A,B), 200 μm (C–J).