The Role Of HIF-1αand Wnt/β-catenin In The Osteogenic Differentiation Of ST2 Cells Subjected To Cyclical Stretch

Background:The blood vessels in the periodontal tissue are compressed in the process of orthodontal tooth movement, resulting in the low oxygen pressure,which in turn reduced the oxygen tension. Then the modeling and remodeling processes of the alveolar bone were promoted, stimulating the formation and resorption of bone. The appearance and activity of osteoblasts was core of the modeling and remodeling of bone, which were derived from MSCs--the multipotent stem cells that can differentiate into a variety of cell types, including osteoblasts. In this study, we used the ST2 cell, which were derived from the BC8 mice and can differentiate into a variety of cell types, including osteoblasts. Hypoxia occurs when the blood supply to tissues is reduced or disrupted. The transcription factor hypoxia-inducible factor-1α(HIF-1α) is activated under hypoxia and promotes expression of genes required for adaptation and cell survival, and also regulates both bone remodeling (HIF-1) regulates multiple downstream genes that modulate cell metabolism, survival, death, angiogenesis, hematopoiesis, and other functions. HIF-1αhas been proved an essential role in the adaption of hypoxia for organisms. However, the regulation of HIF-1αin the process of the osteogenic differentiation has little been known. Wnts are secreted, lipid-modified glycoproteins that activate cell surface receptor-mediated signal transduction pathways to regulate a variety of cellular activities, including cell fate determination, proliferation, migration, polarity, and gene expression, inhibiting adipogenesis and promoting osteoblast maturation, which is the hot spot in the previous signal conduction research area.Objective:This study firstly constructs ST2 cell in vitro model of mechanical stimulation, then the model was subjected the adequate Cyclical Stretch. The application of MTT assay can permit observing ST2 cell proliferation and a certain size and frequency cylical streth. Immunohistochemistry, MTT assay, RT-PCR were used to observe the change of expression and activity of Wnt/β-catenin, HIF-1αand the downstream gene such as the RUNX2, as well as the activity of ALP in the early osteogenic differentiation. By doing this, we hope identifying the Role of Wnt/β-catenin and HIF-1αUnderlying the Remodeling of Bone Marrow Stromal cells Subjected to Cyclical Stretch.Materials and Methods:1. Culture the ST2 cell in vitro and constuct the model of mechanical stimulation2. Under the condition of normoxia and hypoxia, the ST2 cell were subjected to the adequate the size and frequency cylical stretch, then Immunohistochemistry, MTT assay, RT-PCR were used to observe the change of expression and activity of Wnt/β-catenin, HIF-1αand the downsteam gene such as the RUNX2, as well as the activity of ALP in the early osteogenic differentiation.3. The experimental data were analyzed by SPSS 11.0 statistical package and GraphPad Prism version 5.0.Results:1. The results of MTT assay showed that the cyclical stretch of 15% magnitude and the frequency of 0.25Hz improve the ODs, and the ODs has a relationship with the time. the results was also the same when the BMSCs were cultured in the medicum that had been chemical hypoxia by DFO.2. With and without DFO, the expression of HIF-1αmRNA with cyclical stress was higher than that without the stress.3. Without the DFO, the expression of ALP with cyclical stress were higher that the group without the stress. To 48 hours the activity of the expression of ALP mRNA reached the peak 4. The results of RE-PCR showed that GSK-3β,β-catenin, LRP-5 and RUNX2 expressed in every group. For the GSK-3β, it has a negative correlation between the HIF-1αand ALP; for theβ-catenin, LRP-5 and RUNX2, it has positive correlation between the HIF-1αand ALP (P<0.05)5. With and without DFO, the results of Immunohistochemistry showed that the HIF-1αprotein expressed in each group.Conclusion:1. The cyclical stretch(15%,0.25 Hz) and the chemical hypoxia (130μmol/L)using DFO may promote the proliferation and the osteogenic differentiation of the ST2 cells.2. The cyclical stretch may promote the expression of HIF-la.3. The Wnt/β-catenin signal pathway may regulate the osteogenic differentiation of the ST2 cells under the cyclical stretch and chemical hypoxia.