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. 2001 Nov;21(21):7416-28.
doi: 10.1128/MCB.21.21.7416-7428.2001.

Common regulation of growth arrest and differentiation of osteoblasts by helix-loop-helix factors

N Funato  1 , K Ohtani, K Ohyama, T Kuroda, M Nakamura
Affiliations

Common regulation of growth arrest and differentiation of osteoblasts by helix-loop-helix factors

N Funato et al. Mol Cell Biol. 2001 Nov.

Abstract

Cellular differentiation entails the coordination of cell cycle arrest and tissue-specific gene expression. We investigated the involvement of basic helix-loop-helix (bHLH) factors in differentiation of osteoblasts using the human osteoblastic cell line MG63. Serum starvation induced growth arrest at G1 phase, accompanied by expression of cyclin-dependent kinase inhibitor p21(WAF1/Cip1). Reporter assays with the p21 gene promoter demonstrated that the combination of E2A (E12 or E47) and coactivator CBP was responsible for p21 induction independent of p53. Twist inhibited E2A-CBP-dependent activation of the exogenous and endogenous p21 promoters. Ids similarly inhibited the exogenously transfected p21 promoter; however less antagonistic effect on the endogenous p21 promoter was observed. Twist was predominantly present in nuclei in MG63 cells growing in complete medium, while it localized mainly in the cytoplasm after serum starvation. The fibroblast growth factor receptor 3 gene (FGFR3), which generates signals leading to differentiation of osteoblasts, was found to be controlled by the same transcriptional regulation as the p21 gene. E2A and Twist influenced alkaline phosphatase expression, a consensus marker of osteoblast differentiation. Expression of E2A and FGFR3 was seen at the location of osteoblast differentiation in the calvaria of mouse embryos, implicating bHLH molecules in physiological osteoblast differentiation. These results demonstrate that a common regulatory system is involved in at least two distinct steps in osteoblastic differentiation. Our results also provide the molecular basis of Saethre-Chotzen syndrome, caused by mutations of the TWIST and FGFR3 genes.

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Figures

FIG. 1
FIG. 1
Effects of serum starvation on cell cycle arrest and p21 expression in MG63 cells. (A) G1 arrest by serum starvation. MG63 cells were cultured in DMEM containing 10% FBS (a) or 0.1% FBS (b) for 48 h and analyzed for their DNA content by flow cytometry. (B) Activation of p21 promoter by serum starvation. MG63 cells were transfected with 2.5 μg of p21-luc, cultured in DMEM containing either 10, 2, or 0.1% FBS for 48 h, and collected for luciferase assay. Luciferase activity is expressed as fold increase over that for cells cultured in DMEM containing 10% FBS. (C) Inhibition of cell growth by p21 as measured by BrdU incorporation. MG63 cells were transfected with pCMV-p21 and pCMV-β-gal plasmids and maintained in DMEM containing 10% FBS for 48 h. BrdU was added 1 h before fixation. Arrowheads, positions corresponding to cells. The cells were stained for β-Gal (a), BrdU (b), and DNA (c). DAPI was used for counterstaining to visualize the nucleus. Scale bar, 20 μm.
FIG. 2
FIG. 2
Effects of serum starvation on expression of p21, E2A, Twist, and Id1 in MG63 cells. (A) Proliferating MG63 cells in DMEM containing 10% FBS (a, c, e, and g) and G1-arrested MG63 cells in DMEM containing 0.1% FBS (b, d, f, and h) were stained with antibodies for p21 (fluorescein; a and b), E2A (rhodamine; c and d), Twist (rhodamine; e and f), and Id1 (rhodamine; g and h) and observed under a fluorescence microscope. Scale bar, 20 μm. (B) MG63 cells with (+) or without (−) the E12-expressing plasmid were cultured in indicated concentrations of FBS. Equal amounts of whole-cell extracts were analyzed by Western blotting with antibodies against E2A and α-tubulin, Lane +, expression of E12 in cells exogenously transfected.
FIG. 3
FIG. 3
Activation of p21 promoter by E12 and E47 independent of p53. (A) Transcriptional activation of p21 promoter by E12 and E47. MG63 cells were cotransfected with 2.0 μg of the p21-luc reporter plasmid in combination with pCMV-E12, pCMV-E47, or pCMV-del E12 (Δ1-507). The cells were cultured in DMEM containing 2% FBS for 48 h and collected for luciferase assay. Luciferase activity mediated by pcDNA3 is arbitrarily set at 1. (B) Schematic diagram of luc reporter constructs. The 2.4-kb promoter sequence of the p21 gene (solid line) was cloned at the site upstream of the luciferase gene in p21-luc. Possible binding sites for E2A (inverted triangles) and p53 (boxes) in the promoter are shown. Dashed lines, internal deletions. (C) Two micrograms of p21 promoter deletion mutant reporter plasmids with or without the E12 or E47 expression plasmid (2 μg) along with the pCMV-β-gal plasmid was transfected into MG63 cells. The cells were cultured in DMEM containing 2% FBS for 48 h and collected for luciferase assay. Luciferase activities were normalized against β-Gal activities. Activities relative to that mediated by the empty pcDNA3 are shown.
FIG. 4
FIG. 4
Effects of E12 on endogenous p21 expression and BrdU incorporation in MG63 cells. (A) Activation of endogenous p21 by E12 overexpression. MG63 cells were transfected with pCMV-E12 and maintained in DMEM containing 10% FBS for 72 h. The cells were fixed and stained for E12 (rhodamine; a), p21 (fluorescein; b), and DNA (DAPI; c). DAPI was used for counterstaining to visualize the nucleus. Arrowheads, cells transfected with E12. (B) Effects of E12 on cell proliferation. MG63 cells were treated by the same procedure as for panel A. BrdU was added 1 h before fixation. Arrowheads, positions corresponding to cells. The cells were stained for E12 (rhodamine; a), BrdU (fluorescein; b), and DNA (DAPI; c). (C) Impairment of E12-dependent p21 induction by the p21 antisense plasmid. MG63 cells were transfected with pCMV-E12 and an antisense p21 construct (pCMV-p21as) and maintained in DMEM containing 10% FBS for 72 h. The cells were fixed and stained for E12 (rhodamine; a), p21 (fluorescein; b), and DNA (DAPI; c). Arrowheads, cells expressing E12. (D) Impairment of E12-mediated cell cycle attenuation by the p21 antisense plasmid. MG63 cells were treated by the same procedure as for panel C. BrdU was added 1 h before fixation. Arrowheads, positions corresponding to cells. The cells were stained for E12 (rhodamine; a), BrdU (fluorescein; b), and DNA (DAPI; c). Scale bar, 20 μm.
FIG. 5
FIG. 5
Effects of Twist, Id1, Id2, and CBP on E2A-dependent transactivation of p21. (A) Inhibition of E12-dependent transactivation of p21 by Twist, Id1, and Id2. MG63 cells were cotransfected with 2.5 μg of p21-luc in combination with 2.0 μg of either pCMV-E12, pCMV-Twist, pCMV-Twist (Y103X), pCMV-Id1, or pCMV-Id2. The cells were cultured in DMEM containing 2% FBS for 48 h and collected for luciferase assay. Activation mediated by pcDNA3 is arbitrarily set at 1. (B) Inhibition of E47-dependent transactivation of p21 by Twist, Id1, and Id2. MG63 cells were cotransfected as for panel A except for pCMV-E12, which was replaced with 2.0 μg of pCMV-E47. (C) CBP-mediated suppression of inhibition of E12-dependent p21 transcription by Twist. MG63 cells were cotransfected with 1.0 μg of p21-luc and 0.25 μg of either pcDNA3 or pCMV-E12. The cells were cultured in DMEM containing 2% FBS for 48 h and collected for luciferase assay. Activation mediated by pcDNA3 is arbitrarily set at 1. (D) Twist-mediated inhibition of p21 gene expression induced by serum starvation. MG63 cells were transfected with 2.5 μg of p21-luc and 2.5 μg of pCMV-Twist or pCMV-Twist (Y103X). The cells were cultured in DMEM containing either 10, 2, or 0.1% FBS for 48 h and collected for luciferase assay. Luciferase activity is expressed as fold increase over that for vehicle-transfected cells cultured in DMEM containing 10% FBS. Empty vector pcDNA3 was included to adjust DNA amounts.
FIG. 6
FIG. 6
Inhibition of E12-dependent p21 induction by Twist and Ids overexpression. MG63 cells were transfected with pCMV-E12 and either pCMV-Twist (A), pCMV-Id1 (B), or pCMV-Id2 (C). DNA amounts used for transfection were adjusted by empty vectors. The cells were maintained in DMEM containing 10% FBS for 72 h. The cells were fixed and stained for E12 (rhodamine; a), p21 (fluorescein; b), and DNA (DAPI; c). Arrowheads, cells expressing E12. Scale bar, 20 μm.
FIG. 7
FIG. 7
Effects of E2A, Twist, Id1, Id2, and CBP on FGFR3 transcription in MG63 cells. (A) Transcriptional activation of the FGFR3 promoter by E12 and E47. MG63 cells were cotransfected with 2.5 μg of the FGFR3-luc reporter plasmid along with pCMV-E12 or pCMV-E47. Cells were harvested 48 h after transfection and then assayed for their luciferase activities. (B) Schematic diagram of luc reporter constructs. The 1.5-kb promoter sequence of the FGFR3 gene (solid line) was cloned at the site upstream of the luciferase gene in FGFR3-luc. Possible binding sites for E2A (inverted triangles) in the promoter are shown. (C) Two micrograms of FGFR3 promoter deletion mutant reporter plasmids with or without the E47 expression plasmid (2 μg) along with the pCMV-β-gal plasmid was transfected into MG63 cells, and luciferase assays were performed. Luciferase activities were normalized against β-Gal activities. Activities relative to that mediated by the empty pcDNA3 are shown. (D) Inhibition of E2A-dependent transactivation of FGFR3 by Twist, Id1, and Id2. MG63 cells were cotransfected with 2.5 μg of p21-luc in combination with 2.0 μg of pCMV-E12, pCMV-E47, pCMV-Twist, pCMV-Twist (Y103X), pCMV-Id1, and pCMV-Id2. The cells were harvested 48 h after transfection and then assayed for reporter gene expression. (E) CBP-mediated suppression of inhibition of E12-dependent FGFR3 transcription by Twist. MG63 cells were cotransfected with 1.0 μg of FGFR3-luc and 0.25 μg of either pcDNA3 or the pCMV-E12 expression vector. Activation mediated by pcDNA3 is arbitrarily set at 1. (F) Additive effect of E2A and 1,25-(OH)2D3 on FGFR3 expression. MG63 cells were transfected with 2.5 μg of FGFR3-luc and 2.5 μg of pCMV-E12. The cells were cultured in DMEM containing either 10, 2, or 0.1% FBS with or without 1,25-(OH)2D3 for 48 h and collected for luciferase assay. Luciferase activity is expressed as fold increase over that for vehicle-transfected cells cultured in DMEM containing 10% FBS. DNA amounts were adjusted by empty vectors.
FIG. 8
FIG. 8
Osteoblast differentiation in vivo and in vitro. (A) E2A and FGFR3 expression in the developing calvarial bones. Frontal sections were stained with antibodies for E2A (a and b) and FGFR3 (c). Solid arrowheads (a to c) and open arrowhead (a), cortical plate and the ventricular zone, respectively. The region between arrows (b and c) indicate parietal bones with osteoblast differentiation. Box (a), magnified area shown in panels b and c. cc, cerebral cortex; pb, parietal bone; tc, temporal cartilages; ep, epithelium. Scale bar, 200 μm. (B) Activation of endogenous FGFR3 by E47 overexpression. MG63 cells were transfected with pCMV-E47 and pnGFP and maintained in DMEM containing 2% FBS for 72 h. The cells were fixed and stained for FGFR3 (rhodamine; b) and DNA (DAPI; c). pnGFP (fluorescein; a) was used as a marker to visualize the transfected cells. DAPI was used for counterstaining to visualize the nucleus. Arrowheads, cells transfected with E47. Scale bar, 20 μm. (C) Effects of 1,25-(OH)2D3 with serum starvation on expression of endogenous FGFR3 in MG63 cells. Proliferating MG63 cells in DMEM containing 10% FBS (a) and G1-arrested MG63 cells in DMEM containing 0.1% FBS with 1,25-(OH)2D3 (b) were stained with antibodies for FGFR3 and observed under a confocal laser microscope. Scale bar, 20 μm. (D) ALP activity in MG63 cells. Cells were transfected with either pCMV-E12, pCMV-Twist, or empty vector and cultured with or without 1,25-(OH)2D3 for 96 h. The cells were harvested, and cell extracts were prepared for determining ALP activities. Values are means with standard errors of three independent experiments.
FIG. 9
FIG. 9
Regulation of p21 and FGFR3 expression by E2A, Twist, Ids, and CBP during osteoblast differentiation. Based on our results, we propose a regulatory mechanism by which expression of p21 and FGFR3 is controlled by E2A proteins, inhibitory HLH proteins, and coactivator CBP. E2A proteins are activators for both the p21 and FGFR3 genes. Twist inhibits the function of the E2A-CBP complex. The model provides the integrated regulation of the expression of p21 necessary for G1 arrest of the cell cycle and the expression of FGFR3 as a signal molecule for maturation of osteoblasts. Dysfunction of Twist and overactivation of FGFR3 signaling result in craniosynostosis because of the acceleration of bone differentiation. Twist alters the expression of FGFR3, thereby providing a direct link between the Twist and FGFR3 pathways.
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