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. 2014 Apr;29(4):820-9.
doi: 10.1002/jbmr.2087.

Inactivation of Vhl in osteochondral progenitor cells causes high bone mass phenotype and protects against age-related bone loss in adult mice

Tujun Weng  1 , Yangli Xie, Junlan Huang, Fengtao Luo, Lingxian Yi, Qifen He, Di Chen, Lin Chen
Affiliations

"VSports注册入口" Inactivation of Vhl in osteochondral progenitor cells causes high bone mass phenotype and protects against age-related bone loss in adult mice

VSports注册入口 - Tujun Weng et al. J Bone Miner Res. 2014 Apr.

Abstract (V体育官网)

Previous studies have shown that disruption of von Hippel-Lindau gene (Vhl) coincides with activation of hypoxia-inducible factor α (HIFα) signaling in bone cells and plays an important role in bone development, homeostasis, and regeneration. It is known that activation of HIF1α signaling in mature osteoblasts is central to the coupling between angiogenesis and bone formation. However, the precise mechanisms responsible for the coupling between skeletal angiogenesis and osteogenesis during bone remodeling are only partially elucidated. To evaluate the role of Vhl in bone homeostasis and the coupling between vascular physiology and bone, we generated mice lacking Vhl in osteochondral progenitor cells (referred to as Vhl cKO mice) at postnatal and adult stages in a tamoxifen-inducible manner and changes in skeletal morphology were assessed by micro-computed tomography (µCT), histology, and bone histomorphometry. We found that mice with inactivation of Vhl in osteochondral progenitor cells at the postnatal stage largely phenocopied that of mice lacking Vhl in mature osteoblasts, developing striking and progressive accumulation of cancellous bone with increased microvascular density and bone formation. These were accompanied with a significant increase in osteoblast proliferation, upregulation of differentiation marker Runx2 and osteocalcin, and elevated expression of vascular endothelial growth factor (VEGF) and phosphorylation of Smad1/5/8 VSports手机版. In addition, we found that Vhl deletion in osteochondral progenitor cells in adult bone protects mice from aging-induced bone loss. Our data suggest that the VHL-mediated signaling in osteochondral progenitor cells plays a critical role in bone remodeling at postnatal/adult stages through coupling osteogenesis and angiogenesis. © 2014 American Society for Bone and Mineral Research. .

Keywords: AGING OSTEOPOROSIS; BONE MASS; HYPOXIA-INDUCIBLE FACTOR α (HIFα); MUTANT MICE; VON HIPPEL-LINDAU GENE (VHL). V体育安卓版.

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Conflict of interest statement (VSports手机版)

Disclosures

All authors state that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1
Inactivation of Vhl in osteochondroprogenitors in adult mice causes large amounts of cancellous bone in tibias and spine. (A) Representative 3D µCT images of tibias from Cre-negative controls (Vhl-floxed mice) and Vhl cKO at 2 months old. Cre-negative control and Vhl cKO mice were treated with same amounts of TM. Mice were injected with TM daily for 5 continuous days at the age of 1 month and analyzed 1 month later. (B) Representative image of spine from 4-month-old Cre-negative control and Vhl cKO mice. Mice at the age of 2 months were injected with TM and analyzed 2 months later. X-ray (top) and µCT (bottom) images showed increased bone mass in Vhl cKO mice. (C) Representative images stained with H&E in tibias and spine showed excessive trabecular bone in Vhl cKO mice 2 months after TM treatment. Original magnification: (top) ×40; (bottom) ×20. (D) Quantification of BV/TV and Tb.N in spine was determined by bone histomorphometry analysis. Boxed areas showed in C were analyzed with OsteoMeasure software. ** p < 0.001. BV/TV = trabecular bone volume; Tb.N = trabecular number; TM = tamoxifen.
Fig. 2
Fig. 2
Induced disruption of Vhl in osteochondral progenitor cells does not alter calvarial bone and cortical bone in femurs. Mice were injected daily with TM for 5 days at the age of 2 months and analyzed 2 months later. (A) Representative µCT images of calvaria from 4-month-old Cre-negative control and Vhl cKO mice. The top is 3D reconstructed µCT images of calvaria and the bottom is 2D µCT images of calvaria. (B) 2D cortical bone images at the femoral mid-shaft from 4-month-old Cre-negative control and Vhl cKO mice. (C) Comparable cortical thickness in mid-femoral between Cre-negative control and Vhl cKO mice quantified by µCT. (D) No changes in Ct.Ar/Tt.Ar in the mid-shaft of femurs from Cre-negative control and Vhl cKO mice were observed assessed by µCT. Ct.Ar/Tt.Ar=cortical bone fraction; TM=tamoxifen.
Fig. 3
Fig. 3
Histological and histomorphometric analysis showed that enhanced bone formation and bone resorption in Cre-negative control and Vhl cKO mice at age of 4 months. Mice were treated with TM for 5 days at 2 months of age. (A) Von Kossa/toluidine blue-stained undecalcified sections in the distal femurs from Cre-negative control and Vhl cKO mice. Calcified bone was stained in black, and blue osteoblasts was surrounding the black trabeculae. Original magnification: (top) ×40; (bottom) ×200. (B) Disorganized double-calcein labeling was observed in the tibias from 4-month-old Vhl cKO mice. Original magnification: ×400. (C) Representative images of TRAP staining in the proximal tibia from Cre-negative control and Vhl cKO mice. Original magnification: ×400. (D) Quantification of structural and cellular parameters in the proximal tibias in Cre-negative control and Vhl cKO mice was determined by OsteoMeasure image-analysis software. (E) Real-time RT-PCR analysis showed that expression of osteoclast-related genes Trap and Mmp-9 in long bones from Vhl cKO mice was higher than that in Cre-negative control mice at age of 4 months. BV/TV = bone volume/tissue volume; Tb.Sp = trabecular separation; Tb.N = trabecular number; Tb.Th = trabecular thickness; N.Ob/T.Ar = osteoblast number per tissue volume; N.Oc/T. Ar = osteoclast number per tissue volume; TM = tamoxifen.
Fig. 4
Fig. 4
Mice with Vhl deletion in osteochondroprogenitors in adult protected from aging-induced bone loss. (A) Representative µCT 3D images in distal femoral trabeculae from Cre-negative control and Vhl cKO mice at age of 4 months and 12 months. Mice were injected with TM for 5 days at 2 months old. (B) Changes of trabecular morphology in the distal femora from Cre-negative control and Vhl cKO mice measured by µCT at the age of 4 months and 12 months. *p < 0.05; **p < 0.001. BV/TV = bone volume/tissue volume; Tb.Th = trabecular thickness; Tb.Sp = trabecular separation; Th.N = trabecular number; TM = tamoxifen.
Fig. 5
Fig. 5
Inactivation of Vhl in osteochondroprogenitors accelerated proliferation and differentiation on osteoblast lineage. (A) IHC for HIF-1α (left) and HIF-2α (right) in the distal femora from 4-month-old Cre-negative control and Vhl cKO mice 2 months after TM treatment. Original magnification, ×200. (B) IHC detecting incorporated BrdU in tibiae from Cre-negative control and Vhl cKO mice, which was at age of 1 month and after TM injection for 7 days. Original magnification, ×200. Quantification of BrdU showed higher proliferation of osteoblast lineage in the Vhl cKO mice compared with that in Cre-negative controls. *p < 0.05. (C) IHC for osteoblast differentiation markers Runx2 (left) and osteocalcin (right) in the tibias from Cre-negative control and Vhl cKO mice at age of 12 months. Original magnification, ×400. (D) H&E staining exhibited increased osteoblast lineage cell around trabecular bone in Vhl cKO mice at 4 months of age after deletion of Vhl for 2 months. IHC = immunohistochemistry; TM = tamoxifen.
Fig. 6
Fig. 6
Deletion of VHL in osteochondral progenitor cells enhances VEGF expression and activates BMP signaling. (A) Immunohistochemical analysis of VEGF level in tibial sections from 4-month-old Vhl cKO mice and Cre-negative controls 8 weeks after TM injection. Original magnification: ×400. (B) IHC for CD31-positive endothelium in tibial sections from Cre-negative control and Vhl cKO mice at 4 months old. Original magnification: ×200. (C) IHC analysis showed increased expression of p-smad1/5/8 in tibiae of Vhl cKO mice compared with that in Cre-negative control mice 7 days after TM induction at 1 month old. Magnification: (left) ×40 and (right) ×400. (D) Real-time RT-PCR mRNA expression analyses were performed for evaluating expression of Vhl, Vegf, Bmp-2, Bmp-4, and Bmp-7 in chondrocytes isolated from knee joint of Cre-negative control and Vhl cKO mice at 5 days old after 4-OH-TM treatment for 48 hours and results were expressed as fold changes compared to Cre-negative controls. The real-time RT-PCR analysis was repeated three times. *p < 0.05. TM = tamoxifen; VEGF = vascular endothelial growth factor; BMP = bone morphogenic protein; IHC = immunohistochemistry.
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