Activation Of HIFα Pathway From Osteoblasts And Osteocytes Influence Bone Growth And Homeostasis

Hypoxia inducible factor is the most direct and may be the only regulatory factor under hypoxic condition, it also plays a key role in cell differentiation and survival.During the intracellular metabolic processes of HIFα, the E3 ubiquitin ligase Von Hippel-Lindau(VHL) binds to the hydroxylated HIFα and targets to the proteasome for degradation, thereby suppressing the transduction of hypoxia/HIFs signal pathway. Osteocytes are matured osteoblast, which is the last state of MSC ’s differentiation. Osteocytes can be found throughout the bone matrix, through cell membrane connections can form net-like structure. During bone tissue’s development and metabolize osteocytes served an important role such like regulater and inter-cellular bio-reaction activator.Objective: 1. Investigate the role of HIF pathway in the maturation of osteocytes. 2. Determine the role of HIF pathway during bone modeling and remodeling. 3. Investigate the causes of high bone mass after activation HIF pathway.Methods: 1. Osteocyte were stained with Texas red-X-conjugated phalloidin and observed using a confocal laser scanning microscope in vivo and ex-vivo.Morphometry of osteocytes in the long bone was analyzed using extensive three-dimensional reconstructing software IMARIS. For Visualization of the Lacuno-Canalicular Network, bone segments were placed in basic fuchsin after observed using confocal laser scanning microscopy. For resin-casted SEM, polished resin-casted bone samples were exposed to acid etching.Detect the apoptosis and ultrastructure of osteocytes by TUNEL and transmission electron microscopy. 2.Analyse the bone mineral density and rate of formation by X-ray,Micro-CT and double tetracycline labeling. Investigate the process of endochondral ossification using HE, safranin fast green and immunohistochemistry.Analyse the mineralization conditions of bone by backscattered electron microscopy. Detect bone mechanical properties by biomechanical testing. 3.Assessment the proliferation and differentiation of mesenchymal stem cells by EDU labeling and immunohistochemistry.Detect the bone homeostasis by double tetracycline labeling,TRAP staining and ELISA.Results: 1.In the activation of HIFα mouse, deformed osteocyte phenotype, the loss of formation of osteocyte processes,consequently the overall density of the dendrite network in the ΔVhl cortex was much reduce.Accordingly, the lacuno-canalicular network which represent the negative imprint of the osteocyte network within the bone matrix, exhibit a massive loss of canalicular network.Empty lacunae were distributed throughout cortical bone up to 70% in ΔVhl mice, empty lacunae appear to result from osteocyte apoptosis, since staining of osteocytes with TUNEL is increased in ΔVhl relative to wild-type bones. 2. The long bone of ΔVhl mouse showed striking and progressive increases in bone volume as early as 21 d of age, exhibited severe osteopetrosis, and these differences became even more significant as the animals aged.Whereas mice lacking Vhl had no detectable changes in calvarial bone morphology. Activation of HIFα in osteocyte leads to massive accumulation of calcified cartilage due to the lack of osteoclasts, there was an almost complete absence of bone marrow cells in contrast to the presence of bone marrow cells in the same area under the growth plate of control long bone. 3.Activation HIFα pathway reduced expression and distribution of the osteocyte-derived Wnt antagonist sclerostin, increasedβ-Catenin Expression in endocortical mesenchymal stem cells(MSCs), thereby promoting it proliferation and osteogenic differentiation.At the same time, the formation of osteoclast,serum TRAP, double tetracycline labeling and new bone formation by Masson staining increased.Conclusion:1.Mice(ΔVhl) conditionally inactivated vhl in mature osteoblasts and osteocyte using Cre recombinase driven by the osteocalcin promoter(ocn-cre) and,as a consequence, activation of hypoxia-inducible factor α(HIFα) signaling from mature osteoblasts and osteocytes,exhibit the disturb of osteocyte and Lacuno-Canalicular Network and also induce osteocyte apoptosis. 2.Mice activation HIFα pathway have disrupted endochondral ossification and osteopetrosis,but the structure of over produced bone in ΔVhl mice is abnormal, and the mechanical properties of ΔVhl mice bone has changed. 3.Activation HIFα pathway in osteocyte modulation of Wnt signaling in cells of the osteoblastic lineage and increased the bone remodeling...