Study On Molecular Mechanism For HIF-1α Increased Expression Of IL-6 And IL-8 In Osteoblast Cells

Objectives: In the process of bone formation and regeneration,the blood supply of the bone tissue plays a vital role.Hypoxia is the main driving force to promote angiogenesis,and stabilizes HIF-1α expression in the cells by inhibiting the degradation of HIF-1α,which accumulates and transfers to the nucleus combining with HIF-1β into HIF-1,and activates the downstream target genes transcription of VEGF by combining with hypoxia reaction element as a transcription factor,and therefore promotes the formation of blood vessels.In physiological and pathological conditions,the reduced blood supply of bone tissue can make the bone cells in hypoxia condition,and osteoblasts in the surface of the trabecular bone feel the change of blood flow and oxygen pressure reduced in the bone tissue.Our previous study find HIF-1α and downstream target genes expression of VEGF levels increase significantly in human osteogenesis MG-63 cells under hypoxia and simulated hypoxia.Both IL-6 and IL-8 play an important regulating role in development of angiogenesis as a former angiogenic factor,and osteoblasts can secrete two cytokines.Therefore,we hypothesize that HIF-1α may regulate the expression of IL-6 and IL-8 and play an important role in the process of in bone formation and angiogenesis.On the basis of previous work,we continue to take MG-63 cell as experimental models,and explore the effect of HIF-1α on expression of IL-6 and IL-8 in osteoblasts as well as related mechanism.Methods:1 The mRNA expression levels of IL-6,IL-8 and VEGF were detected by reverse transcription polymerase chain reaction(RT-PCR).2 Secretion capacity of IL-6,IL-8 and VEGF were examined by enzyme-linked immnosorbent assay(ELISA).3 The ss HIF-1α cDNA expression vector is imported into MG-63 cells by liposome method,and G418 screens positive cell clones,and Western Blot identifys HIF-1α high expression cell clone after expanded culture,and observation the effect of HIF-1α on IL-6,IL-8 and VEGF expression in the osteoblasts.4 The effect of HIF-1α overexpression on regulating IL-6,IL-8 gene promoter transcriptional activity in MG-63 cells under the condition of low oxygen by luciferase reporter gene experiment technology.5 The influence of Low oxygen regulating the ability about endogenous HIF-1α combining with IL-6,IL-8 gene promoters in MG-63 is detected by using the chromatin immune coprecipitation technology(ChIP).Results:1 The mRNA expression of IL-6,IL-8 and VEGF in MG-63 cells has improved significantly compared with the control group under the condition of hypoxia and overexpressing of endogenous HIF-1α.2 The protein secretion capacity of IL-6,IL-8 and VEGF in MG-63 cells has improved significantly compared with the control group under the condition of hypoxia and overexpressing of endogenous HIF-1α.3 Overexpressing HIF-1α in MG-63 cells can improve IL-6 and IL-8 promoters transcription activity under the condition of low oxygen.4 IL-6 and IL-8 promoters region have HIF-1α binding sites in MG-63 cells,suggesting that both are HIF-1α downstream target genes,and hypoxia can promote the ability of HIF-1α combining with IL-6,IL-8 gene promoters.Conclusions:1 Exogenous(hypoxia)and endogenous overexpression of HIF-1α can increase the expression of IL-6,IL-8 and VEGF in the osteoblasts.2 HIF-1α can promote their transcription and raise both expression by combining with IL-6,IL-8 gene promoters.