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. 2014 Dec 26;289(52):36303-14.
doi: 10.1074/jbc.M114.619569. Epub 2014 Nov 11.

Knockout of nuclear high molecular weight FGF2 isoforms in mice modulates bone and phosphate homeostasis

Collin Homer-Bouthiette  1 , Thomas Doetschman  2 , Liping Xiao  3 , Marja M Hurley  4
Affiliations

Knockout of nuclear high molecular weight FGF2 isoforms in mice modulates bone and phosphate homeostasis

Collin Homer-Bouthiette et al. J Biol Chem. .

Abstract

We previously reported that targeted overexpression of the fibroblast growth factor 2 (FGF2) high molecular weight (HMW) isoforms in osteoblastic lineage cells in mice resulted in phenotypic changes, including dwarfism, rickets, osteomalacia, hypophosphatemia, increased serum parathyroid hormone, and increased levels of the phosphatonin FGF23 in serum and bone. This study examined the effects of genetically knocking out the FGF2HMW isoforms (HMWKO) on bone and phosphate homeostasis. HMWKO mice were not dwarfed and had significantly increased bone mineral density and bone mineral content in femurs and lumbar vertebrae when compared with the wild-type (WT) littermates. Micro-computed tomography analysis of femurs revealed increased trabecular bone volume, thickness, number, and connective tissue density with decreased trabecular spacing compared with WT. In addition, there was significantly decreased cortical porosity and increased cortical thickness and sub-periosteal area in femurs of HMWKO. Histomorphometric analysis demonstrated increased osteoblast activity and diminished osteoclast activity in the HMWKO. In vitro bone marrow stromal cell cultures showed there was a significant increase in alkaline phosphatase-positive colony number at 1 week in HMWKO. At 3 weeks of culture, the mineralized area was also significantly increased. There was increased expression of osteoblast differentiation marker genes and reduced expression of genes associated with impaired mineralization, including a significant reduction in Fgf23 and Sost mRNA. Normal serum phosphate and parathyroid hormone were observed in HMWKO mice. This study demonstrates a significant negative impact of HMWFGF2 on biological functions in bone and phosphate homeostasis in mice.

Keywords: Bone; Fibroblast Growth Factor (FGF); RNA; Transcription Regulation; Wnt Signaling.

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Figures

FIGURE 1.
FIGURE 1.
X-ray images show no differences in HMWKO versus WT mice and dual beam x-ray absorptiometry analysis of excised femurs and vertebrae display increased BMD/BMC in HMWKO. A, x-ray images of WT and HMWKO mice. Dual beam x-ray measurements are as follows: B, femur BMD; C, femur BMC; D, L1–5 BMD; E, L1–5 BMC. Values are the mean ± S.E. (n = 15–27) * indicates significantly different from WT group (p < 0.05).
FIGURE 2.
FIGURE 2.
Micro-CT analysis revealed increased trabecular bone measurements in HMWKO mice. A, representative micro-CT three-dimensional trabecular structure of femurs of 2-month-old male mice. Three-dimensional trabecular microstructural parameters were calculated using two-dimensional data obtained from micro-CT, with calculated morphometric indices included. B, bone volume density; C, trabecular thickness; D, trabecular number; E, trabecular spacing; and F, connective (Conn.) tissue density. (n = 6) * indicates significantly different from WT group (p < 0.05).
FIGURE 3.
FIGURE 3.
HMWKO mice have increased cortical bone measurements when compared with WT mice by micro-CT. A, representative micro-CT images of cortical bone. Cortical bone measurements are as follows: B, sub-periosteal area; C, cortical thickness; D, cortical mask; E, cortical porosity (n = 6). * indicates significantly different from WT (p < 0.05).
FIGURE 4.
FIGURE 4.
Static bone histomorphometric analysis of trabecular bone in femurs showed HMWKO mice osteoblast/osteoclast activity favor bone mineralization. A, ×10 representative images of tartrate-resistant acid phosphatase. B, BV/TV. C, percent osteoblast surface (%ObS). D, percent osteoclast surface (%OcS); E, osteoclast number/bone surface (OcNo/BS) (n = 7–10). * indicates significantly different from WT (p < 0.05).
FIGURE 5.
FIGURE 5.
Dynamic histomorphometric analysis of trabecular bone in femurs demonstrates increased bone mineralization rate in HMWKO mice. A, fluorescent imaging of calcein and xylenol orange from representative WT and HMWKO mice utilizing a ×5 lens. Smaller image uses a ×20 lens. Dynamic histomorphometry measurements are as follows: B, inter-label thickness. C, mineral apposition rate (MAR). D, mineralizing surface/bone surface (MS/BS), and E, bone formation rate/bone surface (BFR/BS) (n = 9–10). * indicates significantly different from WT (p < 0.05).
FIGURE 6.
FIGURE 6.
Dynamic histomorphometric analysis of cortical bone of femurs from WT and HMWKO mice. Dynamic histomorphometry measurements are as follows: A, mineral apposition rate (MAR); B, bone formation rate/bone surface (BFR/BS); and C, mineralizing surface/bone surface (MS/BS) in periosteal layer. * indicates significantly different from WT (p < 0.05). n = 9. D, fluorescent imaging of calcein and xylenol orange labeling in periosteum from representative WT and HMWKO mice.
FIGURE 7.
FIGURE 7.
Increased alkaline phosphatase-positive colonies and mineralized nodules in bone marrow cultures from HMWKO mice versus WT cultures. A, representative images of ALP-positive colonies at days 7, 14, and 21 of culture. B, representative images of von Kossa staining at days 7, 14, and 21 of culture. C, quantitative analysis of ALP-positive colony number; D, ALP-positive colony area measured by ImageJ software (National Institutes of Health). Number of wells measured at day 7 (n = 15 for WT and KO), day 14 (WT, n = 15; KO, n = 21), and day 21 (WT, n = 17; KO, n = 21). E, quantitative analysis of von Kossa-positive colony area utilizing ImageJ software. Number of wells measured at day 7 (n = 15 for WT and KO), day 14 (WT, n = 14; KO, n = 20), and day 21 (WT, n = 16; KO, n = 21). * indicates significantly different from WT (p < 0.05).
FIGURE 8.
FIGURE 8.
Transcriptional regulation in HMWKO mice favors expression of genes that promote osteoblast differentiation and matrix mineralization in bone. Shown is real time RT-PCR analysis of Col1a1 (A), Runx2 (B), osterix (C), OC (D), Op (E), Dmp1 (F), Phex (G), Mgp (H), Dkk1 (I), and Mepe (J) mRNA expression in flushed tibial bones (n = 7–13). * indicates significantly different from WT (p < 0.05).
FIGURE 9.
FIGURE 9.
Modulation of Fgf2 and Fgf23 mRNA in HMWKO bones. Shown is real time RT-PCR analysis of Fgf2 (A) and Fgf23 (B) mRNA expression in flushed tibial bones (n = 7–11). * indicates significantly different from WT (p < 0.05).
FIGURE 10.
FIGURE 10.
Serum FGF23, calcium, PTH, and phosphate levels were not significantly different in WT and HMWKO. A, serum FGF23. B, serum calcium. C, serum PTH. D, serum phosphate. Serum FGF23 and PTH (n = 8–10). Serum calcium and Pi (n = 16–19).
FIGURE 11.
FIGURE 11.
Decreased serum sclerostin and decreased Sost mRNA and protein in bones of HMWKO mice compared with WT. A, serum sclerostin. B, RT-PCR Sost mRNA. C, representative immunofluorescent images for sclerostin expression in femurs. Decreased sclerostin expression (red) in osteocytes of cortical bone in HMWKO compared with WT. Nuclei were counterstained with DAPI (blue) (serum n = 10–11). * indicates significantly different from WT (p < 0.05).
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