Fig. 5. HIF-1α indirectly promotes RANKL-mediated osteoclastogenesis via HIF-2α.

a Primary cells were infected with the indicated MOI of Ad-Hif1a or Ad-Hif2a on the 3rd day of differentiation and cultured until day 6. Expression pattern of Hif1a, Hif2a, and Rankl was determined using RT-PCR, and representative data were shown (n = 3). b ChIP was performed with anti-HIF-1α antibody and primers spanning the HRE motif of the promoter region of the Hif2a gene (n = 3). c, d qRT-PCR analyses of Hif1a, Hif2a, and Rankl in pre-osteoblasts transfected with Hif2a siRNA in Hif1a-overexpressing cells (c) and transfected with Hif1a siRNA in Hif2a-overexpressing cells (d) (n ≥ 3). e Calvarial pre-osteoblasts infected with Ad-C, Hif1a, or Hif2a adenovirus were cultured with BMMs in a medium containing L-AA (50 μg/ml), β-Gp (5 mM), and VitD₃ (10 nM) for 5 days. TRAP staining and quantitative analysis of multinucleated cells are shown (n = 6). Scale bar, 100 μm. f, g qRT-PCR analyses of Ocn and Runx2 in pre-osteoblasts transfected with Hif2a siRNA in HIF-1α-overexpressing cells (f) and transfected with Hif1a siRNA in HIF-2α-overexpressing cells (g) (n = 4). Values are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.