FGF2 regulation of Ank and Tnap expression in primary pre-osteoblastic cells. Primary mouse calvarial cells and BMSCs were treated with FGF2 as indicated, before mRNA isolation. (A–D) FGF2 increases Ank and inhibits Tnap mRNA expression in (A and C) primary calvarial cells and (B and D) BMSCs. RNA was isolated and used for quantitative real-time PCR. mRNA levels are presented as normalized to GAPDH. *p < 0.05 vs. no treatment. (E) The regulation of Ank and Tnap mRNA expression by FGF2 is not cell type specific. RNA was isolated and used for quantitative real-time PCR. mRNA was normalized to GAPDH and is reported as fold induction (Ank) or inhibition (Tnap) by FGF2.

FGF2 specifically induces activity of a PC-1 gene promoter/firefly luciferase reporter construct in pre-osteoblastic cells. (A) PC-1 gene promoter/firefly luciferase construct. A 2.8-kb 5′-untranslated region of the PC-1 gene was cloned by PCR and ligated into the PGL3 firefly luciferase reporter vector. Four potential Runx2 binding sites are present within the promoter sequence: two within a distal region of the promoter and two within a more proximal region. Regions of high homology between mouse and human gene sequences are highlighted in gray. Also indicated are primer sets designed to amplify Runx2 binding site regions for ChIP analysis. (B) FGF2 specifically drives the PC-1 promoter in pre-osteoblastic cells. MC3T3(E1(C4) cells were transfected with PGL3/PC1 firefly luciferase and a control renilla luciferase construct, treated with FGF2 or BMP2, and analyzed for luciferase activity. *p < 0.05 vs. no treatment.

Runx2 associates with the PC-1 gene promoter. (A) Nuclear extract specifically binds to Runx2 binding sites within PC-1 promoter oligonucleotides. MC3T3E1(C4) cell nuclear extract was incubated with radiolabeled Runx2 binding site oligonucleotides or mutant Runx2 binding site oligonucleotides (Table 1), and increasing amounts of nonradiolabeled competitor wildtype or competitor mutant oligonucleotide. Samples were analyzed by PAGE. (B) Runx2 from nuclear extract binds to Runx2 binding sites within PC-1 promoter oligonucleotides. Radiolabeled oligonucleotides were incubated with MC3T3E1(C4) nuclear extract and Runx2 or control IgG antibody. Samples were analyzed by polyacrylamide gel electrophoresis. (C) FGF2 promotes Runx2 recruitment to the endogenous PC-1 gene promoter. Cross-linked chromatin isolated from untreated or FGF2 treated MC3T3E1(C4) cells was immunoprecipitated with Runx2 or control IgG antibody. PCR was performed using primers generated to detect Runx2 binding site regions of the distal or proximal PC-1 gene promoter (Figure 3; Table 1). Arrow indicates PC-1 promoter-specific amplification product. (D) ChIP PCR amplification is specific to the PC-1 gene promoter. PCR of cross-linked chromatin was performed using primers specific to exon 18 of the PC-1 gene.

FGF2 induction of PC-1 expression in primary pre-osteoblastic cells. Primary mouse calvarial cells and BMSCs were treated with FGF2 as indicated, before mRNA isolation or cell lysis. (A and B) FGF2 increases PC-1 mRNA expression in (A) primary calvarial cells and (B) BMSCs. RNA was isolated and used for semiquantitative RT-PCR. PC-1 mRNA levels are presented as normalized to GAPDH. (C and D) FGF2 increases PC-1 enzyme activity in (C) primary calvarial cells and (D) BMSCs. Equivalent amounts of cellular extract were incubated with colorimetric substrate and absorbance was read at 405 nm. *p < 0.05 vs. no treatment. (E) The induction of PC-1 mRNA by FGF2 is cell type specific. RNA was isolated and used for semiquantitative RT-PCR. PC-1 mRNA was normalized to GAPDH and is reported as fold induction by FGF2.

Rapid and direct induction of PC-1 mRNA by FGF2. MC3T3E1(C4) cells were treated with FGF2 as indicated. RNA was isolated and used for semiquantitative RT-PCR. PC-1 mRNA levels are presented as normalized to GAPDH. (A) Dose-dependent induction of PC-1 mRNA by FGF2. Cells were incubated with increasing doses of FGF2 for 12 h before RNA isolation. (B) Time-dependent induction of PC-1 mRNA by FGF2. Cells were incubated with FGF2 for increasing lengths of time, as indicated, before RNA isolation. (C) FGF2 induces transcription of PC-1. Cells were pretreated with 1 μM actinomycin D for 1 h before treatment with FGF2. (D) FGF2 induction of PC-1 does not require a protein intermediate. Cells were pretreated with 10 μM cycloheximide for 1 h before treatment with FGF2. *p < 0.05 vs. no treatment.

Runx2 is required for the induction of PC-1 expression by FGF2. (A and B) Runx2 increases FGF2-induced PC-1 promoter activity in MC3T3E1(C4) (A) and COS7 (B) cells. Cells were transfected with PGL3/PC1 firefly luciferase, control renilla luciferase constructs, and expression vectors for LacZ or Runx2. Cells were treated with FGF2 and analyzed for luciferase activity. *p < 0.05 vs. no treatment or between indicated groups. (C and E) Runx2 enhances PC-1 mRNA expression in response to FGF2. C3H10T1/2 cells (C) or Runx2^−/− calvarial osteoblastic cells (E). Cells were transduced with adenoviral vectors expressing LacZ or Runx2 and treated with FGF2 as indicated. PC-1 and GAPDH mRNA levels were measured by real-time PCR, and PC-1 mRNA levels are presented as normalized to GAPDH. *p < 0.05 vs. no treatment or between indicated groups. (D and F) FGF2 treatment does not enhance Runx2 expression. C3H10T1/2 cells (D) or Runx2^−/− calvarial osteoblastic cells (F) were transduced with adenoviral vectors expressing LacZ or Runx2, and treated with FGF2. Runx2 and GAPDH mRNA levels were measured by real-time PCR, and Runx2 mRNA levels are presented as normalized to GAPDH. *p < 0.05 vs. LacZ. (G) FGF2 treatment does not increase Runx2 nuclear expression in calvarial pre-osteoblastic cells. MC3T3E1(C4) nuclear extracts were analyzed for Runx2 protein and control β-actin protein expression levels by immunoblot analysis. (H) Runx2 binding site mutations in the PC-1 promoter significantly diminish promoter responsiveness to FGF2. MC3T3E1(C4) cells were transfected with wildtype PGL3/PC1 or Runx2 binding site mutant PGL3/PC1 firefly luciferase, and a control renilla luciferase construct. Cells were treated with FGF2 and analyzed for luciferase activity. *p < 0.05 vs. wildtype PGL3/PC1.