Abstract:

Objective To investigate the regulation of interaction of osteoblasts and osteoclasts by hypoxia/hypoxia-inducible factor-1α (HIF-1α) pathway. Methods Osteoblasts were obtained from newborn (2 to 3 d) conditional gene knockout mice, osteoclast precursor cells were harvested from C57BL/6 mice aged 4 to 8 weeks, and co-culture system of osteoblasts and osteoclast precursor cells was established (wild-type, HIF-1α^-/-, Vhl^-/-, HIF-1α^-/-/Vhl^-/- co-culture). RT-PCR was employed to detect the expression of receptor activator of nuclear factor-κB ligand (RANKL) mRNA and osteoprotegerin (OPG) mRNA in osteoblasts and that of marker enzyme gene TRAP mRNA in osteoclasts, and resorption pits produced by osteoclasts were observed with toluidine blue staining. Results RT-PCR revealed that compared with wild-type co-culture, the expression of RANKL mRNA increased and that of OPG mRNA decreased in osteoblasts of HIF-1α^-/- co-culture (P<0.05 for both), the expression of TRAP mRNA increased in osteoclasts of HIF-1α^-/- co-culture, the expression of RANKL mRNA decreased and that of OPG mRNA increased in osteoblasts of Vhl^-/-co-culture and HIF-1α^-/-/Vhl^-/- co-culture (P<0.05 for all), and the expression of TRAP mRNA decreased in osteoclasts of Vhl^-/-co-culture and HIF-1α^-/-/Vhl^-/- co-culture. With the time increase of co-culture, the expression of RANKL mRNA and OPG mRNA in osteoblasts gradually decreased, and that of TRAP mRNA in osteoclasts gradually increased. Observation under inverted microscope with toluidine blue staining indicated that resorption pits of osteoclasts began to appear on the ninth day of co-culture, the area and depth of resorption pits gradually increased with the time increase of co-culture, the area and depth of resorption pits increased after HIF-1α^-/- co-culture, and those decreased after Vhl^-/- co-culture and HIF-1α^-/-/Vhl^-/- co-culture. Conclusion Osteoblasts may inhibit the differentiation of osteoclasts after the activation of hypoxia/HIF-1α pathway, while osteoblasts may promote the differentiation of osteoclasts after interruption of hypoxia/HIF-1α pathway.

Key words: osteoblast, osteoclast, co-culture, hypoxia-inducible factor-1α, Vhl, gene knockout