Tumor Necrosis Factor ? Regulation Of GLUT1 Expression During Osteoblast Differentiation

Bone diseases are listed by the World Health Organization(WHO)as the most harmful to the human body in addition to cardiovascular disease,cancer and diabetes.Bone mass reduction,bone sclerosis and osteoporosis are related to bone differentiation disorders,and the occurrence and treatment of bone diseases are closely related to nutritional metabolism.Therefore,it is very important to explore bone differentiation and metabolic regulation of bone diseases.Bone differentiation is a form of bone formation,and the balance between bone formation and bone resorption determines the strength and integrity of the bone.Bone formation and absorption imbalance will lead to a variety of bone diseases,such as necrosis of femoral head traumatic bone disease,osteoporosis,and metabolic bone disease,in patients with rheumatoid arthritis,the level and activity of tumor necrosis factor(TNF)–? are enhanced,which destroys the patient's joints and bones.Similarly,in patients with diabetes and obesity,there is a high level of TNF?,which is lower in bone density and bone mass than in healthy people,and is more likely to be osteoporosis.Anti-TNF? therapy is an effective method to improve bone mineral density.Previous studies have focused on the regulation of differentiation,activation and apoptosis of osteoclasts by binding TNF? to its cell surface receptors and activating several signaling pathways,including the NF-?B pathway.Therefore,studying the regulation mechanism of TNF? on osteoblast differentiation may provide a new target for the treatment of inflammatory bone disease.Studies have shown that glucose is the main nutrients,glucose in insulin dependent way using Glut1 shuttle in osteoblast,Glut1 before Runx2 expressing in the process of bone formation,the accumulation of Runx2 and osteogenetic differentiation must have enough glucose to provide energy.Studies have shown that TNF? affects the expression of Glut1 in cancer cells,but it is not clear whether it can be used to regulate Glut1 in osteoblast differentiation.Therefore,based on the above,the effects of TNF? on the differentiation of osteoblasts were detected by using the pre-osteoblast MC3T3-E1.The effects of TNF? on the gene and protein of osteogenic differentiation were detected by Western blot(WB),real-time fluorescence quantitative PCR and ALP enzyme activity detection.In order to further explore the molecular mechanism of TNF? in osteoblast differentiation,the role of NF-?B signaling pathway was investigated by knockdown p65.In addition,we observed the regulation effect of TNF? on Glut1,the role of Glut1 in osteogenic differentiation was investigated by knockdown or overexpress Glut1.Main research results in MC3T3-E1 cells:(1)TNF? can activate MAPK,AKT and NF-?B signaling pathways.(2)TNF? can inhibit the osteogenic differentiation of MC3T3-E1 cells.Real-time fluorescent quantitative PCR and WB results indicate that TNF? inhibits the expression of osteogenic differentiation marker genes and proteins,and the ALP activity test demonstrates that TNF? has a concentration dependence on the inhibition of alkaline phosphatase activity.(3)Knockdown p65 increase the expression of protein Runx2.But it does not save the inhibitory effect of TNF? on Runx2.(4)TNF? was treated with a short time to increase the protein expression of Glut1,and the protein expression of Glut1 was inhibited for a long time.(5)The protein expression of Runx2 decreased with the knockdown of Glut1,and the ALP staining and ALP enzyme activity test showed that the expression and enzyme activity of the ALP were reduced by knockdown Glut1.Overexpression of Glut1 increases the protein expression of Runx2,but does not save the inhibitory effect of TNF? on Runx2.Conclusion: In MC3T3-E1 cells,TNF? can activate MAPK,AKT and NF-?B signaling pathways.TNF? inhibited osteogenesis differentiation of MC3T3-E1 cells;Inhibition the NF-?B signaling pathway can promote the expression of Runx2,but it cannot save the inhibitory effect of TNF? on Runx2,which proved that TNF? does not affect osteoblast differentiation only by NF-?B signal pathway.In addition,TNF? can affect the expression of Glut1,and Glut1 can regulate the protein expression level of Runx2,however,the overexpression of Glut1 cannot prevent TNF? from inhibiting Runx2.These results show that the effects of TNF? on Runx2 and ossification are performed by multiple signaling pathway.Our results further explore the molecular mechanisms of TNF? affecting osteogenesis differentiation and may provide new targets for the treatment of inflammatory bone disease.