Fig 1.
The role of HIF-α pathyway in osteocyte morphology.
(A) Immunohistochemical staining indicated that there were more osteoblasts and osteocytes expressing HIF-1α and HIF-2α in ΔVHL mice than control group. (B) Representative 3D-reconstituted images of the confocal z-series slices from CON and conditional ΔVHL mice, visualized by Texas red-X-conjugated phalloidin. Bar, 10 μm. (C) Surface renderings of osteocyte cell bodies of CON and ΔVHL from the 3D-reconstituted images by IMRIS enable morphometric analyses; Bar, 10 μm. (D) Morphology of ex vivo osteocytes from the bones of CON andΔVHL mice and Immunofluorescence staining of ex vivo osteocytes with sclerostin (SOST) (E) Osteocytes from CON and conditional ΔVHL mice show stellate morphology with extensive processes in the CON. The number of dendrites is significantly decreased and less interconnected in the ΔVHL mice. (F) Osteocytes (Oc) within the lacuna (Lac) and the well-defined cell processes within the canaliculi (Can) are observed in the cross section (arrows).and section of an osteocyte intersecting three well-defined cell processes (arrows) in the CON. Osteocytes from conditional ΔVHL mice clearly display less abundant cell processes within the canaliculi (Can).
Table 1.
Morphometric data of the osteocytes in cortical bone at the femoral diaphysis of ΔVhl.
Fig 2.
The role of HIFα pathway in the differentiation of osteocytes.
(A) Quantitative PCR analysis of the differentiation markers of osteocytes. (B) Western blot analysis of DMP-1 and sclerostin proteins in the tibia of 2-month-old CON and ΔVHL mice. (C) Immunohistochemical analysis of DMP-1. Compared to the findings from CON, DMP-1 expression in the conditional ΔVHL osteocytes is dramatically reduced (arrows). (D) Immunolocalization of sclerostin in transverse sections of the mid-femoral diaphyses of 6-week-old mice. (E) The percentage of sclerostin-positive osteocytes in the mid-femoral diaphyses of the CON and ΔVHL mice (n = 6 mice for each genotype *** p < 0.001). (F) Serum levels of sclerostin in 3-month-old CON and ΔVHL mice (n = 9 mice for each genotype *** p < 0.001).
Fig 3.
Deletion of Vhl in osteocytes and mature osteoblasts results in increased cortical osteocyte apoptosis.
(A) H&E-stained sections of the cortical bone from femurs of 6-week-old and 8-month-old CON and ΔVHL mice. Empty osteocyte lacunae (arrows) can be observed in the sections obtained from the ΔVHL mice; all the osteocyte lacunae are filled with cells, as seen in the sections obtained from the CON mice. (B) Quantification of the empty osteocyte lacunae in the cortical bones of femurs from 6-week-old to 8-month-old CON and ΔVHL mice.(C) TUNEL staining of cortical bone at the diaphyses from femurs of CON and ΔVHL mice at 6 weeks and 8 months of age. (D) Frequency of TUNEL-positive lacunae. The number of TUNEL-positive lacunae was counted in mice at 6 weeks to 8 months of age, and was presented as a percentage of the total number of lacunae in the cortical bone of femurs. (E) Representative TEM images of osteocytes in the femoral midshaft. (F) Polarized microscopy of cortical bone at diaphyses of the femurs obtained from 8-month-old CON and ΔVHL mice. (G) High-power TEM images of Collagen fibrils (n = 6–9 mice for each genotype *** p < 0.001).
Fig 4.
Morphological changes in the lacunocanalicular system (LCS) of ΔVHL.
(A, B) The conditional ΔVHL mouse showed disorganized LCS. (arrows indicate perpendicular to the longitudinal axis of the cortical bone). (C) Basic fuchsin staining of bone tissues from CON and ΔVHL mice. (D) SEM images of the cortex of humeri in 6-week-old mice.
Fig 5.
ΔVHL mice shown promoted the proliferation and differentiation of osteoblasts/osteoprogenitors and increased levels of β-catenin protein in osteoblasts/osteoprogenitors.
(A) H&E staining showed diaphyseal regions of the murine femurs at 3 weeks of age. Abundant bone marrow stromal cells surround the numerous trabeculae in the ΔVHL mouse. (B) Representative images of EdU-labeled proliferating bone marrow stromal cells (red) merged with Hoechst-stained-nuclei (blue). (C) Representative histological sections of diaphyseal regions of femurs from 3-week-old ΔVHL and CON mice, after staining with anti-PCNA antibodies. (D) Immunocytochemical analysis reveals that the osterix protein is strongly detected in the abundant stromal cells in the diaphyseal regions of the femur. (E) Sections from 1-month-old CON and ΔVHL mice were IHC-stained using an antibody against β-catenin. β-Catenin-positive osteoblasts/osteoprogenitors in the diaphyseal regions of the femurs are stained brown in the ΔVHL mouse. (F) Total protein extracts were prepared from the tibiae of 1-month-old CON and ΔVHL mice and used for western blot analyses for β-catenin.
Fig 6.
Diagram showing changes in the osteocytes after activation of the HIFα pathway and modulation of WNT/β-catenin signaling.
(A) WNT/β-catenin signaling induces the proliferation of mesenchymal/osteoblastic progenitors and enhances their differentiation into the osteoblastic lineage. This process is inhibited by sclerostin secreted through the canaliculi under normal conditions. (B) After activation, the HIFα pathway reduces the sclerostin from calcified bone to the soft bone marrow, further activating WNT/β-catenin signaling.