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The role of FGF-2 and BMP-2 in regulation of gene induction, cell proliferation and mineralization.

Hughes-Fulford M, Li CF - J Orthop Surg Res (2011)

Bottom Line: Photomicroscopy was used to identify newly mineralized tissue and fluorescence was used to quantify mineralization.We found that FGF-2 significantly reduced gene expression associated with mineralization, e.g. collagen type-1 (col1a1), fibronectin (fn), osteocalcin (oc), IGF-1, noggin, bone morphogenic protein (bmp-2) and alkaline phosphatase (alp).In contrast, BMP-2 significantly stimulated expression of the mineralization associated genes but had little or no effect on gene expression associated with growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Research, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121, USA. millie.hughes-fulford@ucsf.edu

ABSTRACT

Introduction: The difficulty in re-growing and mineralizing new bone after severe fracture can result in loss of ambulation or limb. Here we describe the sequential roles of FGF-2 in inducing gene expression, cell growth and BMP-2 in gene expression and mineralization of bone.

Materials and methods: The regulation of gene expression was determined using real-time RTPCR (qRTPCR) and cell proliferation was measured by thymidine incorporation or fluorescent analysis of DNA content in MC3T3E1 osteoblast-like cells. Photomicroscopy was used to identify newly mineralized tissue and fluorescence was used to quantify mineralization.

Results: Fibroblast growth factor-2 (FGF-2) had the greatest ability to induce proliferation after 24 hours of treatment when compared to transforming growth factor beta (TGFβ, insulin-like growth factor-1 (IGF-1), bone morphogenic protein (BMP-2), platelet derived growth factor (PDGF) or prostaglandin E₂ (PGE₂). We found that FGF-2 caused the most significant induction of expression of early growth response-1 (egr-1), fgf-2, cyclo-oxygenase-2 (cox-2), tgfβ and matrix metalloproteinase-3 (mmp-3) associated with proliferation and expression of angiogenic genes like vascular endothelial growth factor A (vegfA) and its receptor vegfr1. We found that FGF-2 significantly reduced gene expression associated with mineralization, e.g. collagen type-1 (col1a1), fibronectin (fn), osteocalcin (oc), IGF-1, noggin, bone morphogenic protein (bmp-2) and alkaline phosphatase (alp). In contrast, BMP-2 significantly stimulated expression of the mineralization associated genes but had little or no effect on gene expression associated with growth.

Conclusions: The ability of FGF-2 to re-program a mineralizing gene expression profile to one of proliferation suggests that FGF-2 plays a critical role of osteoblast growth in early fracture repair while BMP-2 is instrumental in stimulating mineralization.

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FGF-2 and BMP-2, the yin and yang of mineralization: Contrast of effect of 24 hours of treatment with FGF-2 or BMP-2 on fold increase in abundance of mineralization-related gene expression. Mineralizing MC3T3-E1 cells were prepared as described in Materials and Methods. They were then treated with either FGF-2 or BMP-2 for 24 hours at which time RNA was collected and analyzed for relative abundance using qRTPCR. Each bar represents mean ± SD triplicate independent biological samples each time point corrected to cyclophilin. (*p < 0.05; **p < 0.01 with two-tail student t-test compare to 0 hour of each gene.) *<0.05; **<0.01; ***<0.0001
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Figure 3: FGF-2 and BMP-2, the yin and yang of mineralization: Contrast of effect of 24 hours of treatment with FGF-2 or BMP-2 on fold increase in abundance of mineralization-related gene expression. Mineralizing MC3T3-E1 cells were prepared as described in Materials and Methods. They were then treated with either FGF-2 or BMP-2 for 24 hours at which time RNA was collected and analyzed for relative abundance using qRTPCR. Each bar represents mean ± SD triplicate independent biological samples each time point corrected to cyclophilin. (*p < 0.05; **p < 0.01 with two-tail student t-test compare to 0 hour of each gene.) *<0.05; **<0.01; ***<0.0001

Mentions: Since FGF-2 increased growth associated genes, we used BMP-2, a known promoter of mineralization, to study relative abundance of gene expression in mineralizing cells after 24 hours of treatment. As seen in Table 2, we found that BMP-2 treatment caused significant increases in genes associated with mineralization including cola1, fn, noggin and oc. Moreover, BMP-2 treatment caused little or no changes in expression of genes associated with angiogenesis and migration e.g. VEGF and MMP3. When compared with relative gene abundance of FGF-2 treated cells (Figure 3) we found that in general, BMP-2 maintained the mineralizing RNA profile of igf-1, alp, and bmp-2 and significantly increased expression of other genes associated with mineralization like col1a1, fn, ilgf-1, noggin and oc. Fgf-2, on the other hand, significantly suppressed expression of mineralizing genes.


The role of FGF-2 and BMP-2 in regulation of gene induction, cell proliferation and mineralization.

Hughes-Fulford M, Li CF - J Orthop Surg Res (2011)

FGF-2 and BMP-2, the yin and yang of mineralization: Contrast of effect of 24 hours of treatment with FGF-2 or BMP-2 on fold increase in abundance of mineralization-related gene expression. Mineralizing MC3T3-E1 cells were prepared as described in Materials and Methods. They were then treated with either FGF-2 or BMP-2 for 24 hours at which time RNA was collected and analyzed for relative abundance using qRTPCR. Each bar represents mean ± SD triplicate independent biological samples each time point corrected to cyclophilin. (*p < 0.05; **p < 0.01 with two-tail student t-test compare to 0 hour of each gene.) *<0.05; **<0.01; ***<0.0001
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3044105&req=5

Figure 3: FGF-2 and BMP-2, the yin and yang of mineralization: Contrast of effect of 24 hours of treatment with FGF-2 or BMP-2 on fold increase in abundance of mineralization-related gene expression. Mineralizing MC3T3-E1 cells were prepared as described in Materials and Methods. They were then treated with either FGF-2 or BMP-2 for 24 hours at which time RNA was collected and analyzed for relative abundance using qRTPCR. Each bar represents mean ± SD triplicate independent biological samples each time point corrected to cyclophilin. (*p < 0.05; **p < 0.01 with two-tail student t-test compare to 0 hour of each gene.) *<0.05; **<0.01; ***<0.0001
Mentions: Since FGF-2 increased growth associated genes, we used BMP-2, a known promoter of mineralization, to study relative abundance of gene expression in mineralizing cells after 24 hours of treatment. As seen in Table 2, we found that BMP-2 treatment caused significant increases in genes associated with mineralization including cola1, fn, noggin and oc. Moreover, BMP-2 treatment caused little or no changes in expression of genes associated with angiogenesis and migration e.g. VEGF and MMP3. When compared with relative gene abundance of FGF-2 treated cells (Figure 3) we found that in general, BMP-2 maintained the mineralizing RNA profile of igf-1, alp, and bmp-2 and significantly increased expression of other genes associated with mineralization like col1a1, fn, ilgf-1, noggin and oc. Fgf-2, on the other hand, significantly suppressed expression of mineralizing genes.

Bottom Line: Photomicroscopy was used to identify newly mineralized tissue and fluorescence was used to quantify mineralization.We found that FGF-2 significantly reduced gene expression associated with mineralization, e.g. collagen type-1 (col1a1), fibronectin (fn), osteocalcin (oc), IGF-1, noggin, bone morphogenic protein (bmp-2) and alkaline phosphatase (alp).In contrast, BMP-2 significantly stimulated expression of the mineralization associated genes but had little or no effect on gene expression associated with growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Research, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121, USA. millie.hughes-fulford@ucsf.edu

ABSTRACT

Introduction: The difficulty in re-growing and mineralizing new bone after severe fracture can result in loss of ambulation or limb. Here we describe the sequential roles of FGF-2 in inducing gene expression, cell growth and BMP-2 in gene expression and mineralization of bone.

Materials and methods: The regulation of gene expression was determined using real-time RTPCR (qRTPCR) and cell proliferation was measured by thymidine incorporation or fluorescent analysis of DNA content in MC3T3E1 osteoblast-like cells. Photomicroscopy was used to identify newly mineralized tissue and fluorescence was used to quantify mineralization.

Results: Fibroblast growth factor-2 (FGF-2) had the greatest ability to induce proliferation after 24 hours of treatment when compared to transforming growth factor beta (TGFβ, insulin-like growth factor-1 (IGF-1), bone morphogenic protein (BMP-2), platelet derived growth factor (PDGF) or prostaglandin E₂ (PGE₂). We found that FGF-2 caused the most significant induction of expression of early growth response-1 (egr-1), fgf-2, cyclo-oxygenase-2 (cox-2), tgfβ and matrix metalloproteinase-3 (mmp-3) associated with proliferation and expression of angiogenic genes like vascular endothelial growth factor A (vegfA) and its receptor vegfr1. We found that FGF-2 significantly reduced gene expression associated with mineralization, e.g. collagen type-1 (col1a1), fibronectin (fn), osteocalcin (oc), IGF-1, noggin, bone morphogenic protein (bmp-2) and alkaline phosphatase (alp). In contrast, BMP-2 significantly stimulated expression of the mineralization associated genes but had little or no effect on gene expression associated with growth.

Conclusions: The ability of FGF-2 to re-program a mineralizing gene expression profile to one of proliferation suggests that FGF-2 plays a critical role of osteoblast growth in early fracture repair while BMP-2 is instrumental in stimulating mineralization.

Show MeSH
Related in: MedlinePlus