Study On The Mechanism Of Hypoxia-Inducible Factor-1α Regulating Osteoclast Production

Background: Osteoporosis is a Systemic bone destruction disease that main cause of osteoblast and osteoclast dysfunction,manifested as decreased bone mass and bone density,prone to fractures.Osteoclasts are unique multi-nucleated giant cells formed from bone marrow hematopoietic stem cells through the synergistic action of multiple systemic hormones and cytokines.The hypoxic condition of bone microenvironment which osteoclasts survived in and abnormal osteoclasts’ function are common feature of bone loss diseases such as osteoporosis,rheumatoid arthritis and pathological fractures.Hypoxia-inducible factor-1(HIF-1)is the main cytokine that regulates cellular response to hypoxic environment.Hypoxia-inducible factor-1α(HIF-1α),a subunit of HIF-1 that regulated by the oxygen content is essential for osteoclast differentiation and bone resorption,and can be used as a potential target for the prevention and treatment of osteoporosis.However,the molecular mechanism of HIF-1α regulation of osteoclasts has not been elucidated.Objective: To induce the differentiation of RAW264.7 cells into osteoclasts by nuclear factor κB receptor activating factor ligand(RANKL).The osteoclast model was treated with hypoxia analogues to increase the intracellular HIF-1α concentration,simulate the hypoxic microenvironment,and explore the possible role and specific mechanism of HIF-1α expression on osteoclast differentiation in vitro,which provides new ideas for further research on bone loss diseases caused by abnormal differentiation of osteoclasts such as osteoporosis.Methods: L-mimosine,as a prolyl hydroxylase inhibitor,can protect HIF-1α from being decomposed by prolyl hydroxylase under normoxic conditions,thereby increasing the content of intracellular HIF-1α.The effect of different concentrations(0,50,100,150,200 μM)of L-mimosine on RAW264.7 cell viability was verified by CCK-8 cytotoxicity experiments.In the presence of RANKL,RAW264.7 cell is cultured and stimulated by Lmimosine at different concentrations(0,50,100,150 μM)to maintain different levels of intracellular HIF-1α.The regulation of osteoclast differentiation by different levels of HIF-1α was compared by tartrate-resistant acid phosphatase(TRAP)staining.The regulatory effect of different levels of HIF-1α on the production of F-actin rings in osteoclasts was evaluated by rhodamine-phalloidin staining experiments.Real-time PCR was used to analyze different levels of HIF-1α for key transcription factor NFATc1,c-Fos,c-Jun,and osteoclast-specific genes Cathepsin K,β3-integrin,TRAP,and matrix metallo-proteinase-9(MMP-9).The regulation of different levels of HIF-1α on the key transcription factors NFATc1,c-Fos and c-Jun of osteoclasts was analyzed by western blot.The regulation of protein expression by different levels of HIF-1α on the key signaling pathways MAPK(JNK,ERK,p38)of osteoclast differentiation was analyzed by western blot.BAY-87-2243 is a specific inhibitor of HIF-1α,the effect of different concentrations of BAY-87-2243(0,5,10,20,40 n M)on RAW264.7 cell viability was verified by CCK-8 cytotoxicity experiments and experimental concentrations of BAY-87-2243(10n M)were added to Lmimosine-containing experiments with RANKL to inhibit the regulatory effect of HIF-1αon osteoclasts,and TRAP staining,rhodamine-phalloidin staining,Real-time PCR and western blot experiments were repeated.All experiments were repeated three times,and all data were expressed as mean ± standard deviation(SD).Statistical analysis was performed using SPSS package version 18.0 and Graph Pad Prism 8.0.One-way ANOVA using Tukey’s test was used to calculate and analyze statistical differences between groups with statistical significance set to *P<0.05.Results: L-mimosine did not affect cell activity in the experimental concentration(0,50,100,150μM),and increased the content of intracellular HIF-1α in a dose-dependent manner,as well as the differentiation of osteoclasts and the production of F-actin loops also increased sequentially with the increase of intracellular HIF-1α levels.The key transcription factors and specific genes of osteoclasts also showed an increasing trend.These are consistent with the upregulation trend of the MAPK(JNK,ERK,p38)signaling pathway and NFATc1,c-Fos,and c-Jun protein expression.In the experimental group after adding BAY-87-2243,the level of HIF-1α did not increase with the increase of Lmimosine concentration,osteoclast differentiation and F-actin ring did not increase significantly,and the level of related genes and proteins did not change significantly.Conclusion: Our results suggest that the elevated level of intracellular HIF-1αpromotes the expression of osteoclast-related genes and directly increases RANKLmediated osteoclast differentiation of RAW264.7 cells in vitro by upregulating the MAPK pathway.This provides new ideas and directions for the prevention and treatment of osteoporosis and other bone loss diseases.