Hypoxia regulates angeogenic-osteogenic coupling process via up-regulating IL-6 and IL-8 in human osteoblastic cells through hypoxia-inducible factor-1α pathway.

Inappropriate angiogenesis and osteogenesis are considered as the crucial factors of osteoporotic fracture. Hypoxia is a primary driving force for regulating the angiogenic-osteogenic coupling process. Our recent results indicated that hypoxia could improve angiogenesis as well as differentiation and activity of osteoblastic cells via up-regulating VEGF through HIF-1α pathway. Here we demonstrated that in human osteoblastic MG-63, U2-OS and Saos-2 cells, besides VEGF, the other two pro-angiogenic factors IL-6 and IL-8 were also up-regulated by hypoxia and CoCl (a mimic of hypoxia). Mechanism studies indicated overexpression of HIF-1α (generated from transfection with a plasmid encoding sense HIF-1α) markedly increased the levels of IL-6 and IL-8 in osteoblastic cells. Furthermore, a luciferase reporter assay was performed using the reporter vector containing the IL-6 or IL-8 promoter sequence to illustrate observably increased activity of hypoxia-induced IL-6 and IL-8 promoter caused by overexpression of HIF-1α. Additionally, chromatin immune-precipitation analysis showed hypoxia increased the DNA binding ability of HIF-1α to IL-6 or IL-8 promoter. Analysis in vitro by MTT test and Boyden chamber assay showed exogenous IL-6 and IL-8 (a relatively short period of treatment with recombinant IL-6 or IL-8 equivalent to the autocrine levels) could significantly promote the proliferation of human osteoblastic, endothelial and monocytic cells, as well as the migration of human endothelial cells. Taken together, these results indicate that IL-6 and IL-8 in osteoblastic cells may also contribute to the angiogenic-osteogenic coupling process via HIF-1α pathway. Besides VEGF, IL-6- or IL-8-targeted adjunctive therapy maybe a new strategy to improve the treatment of osteoporosis.