Fig. 2
From: Controlling hypoxia-inducible factor-2α is critical for maintaining bone homeostasis in mice
HIF-2α blocks osteoblast differentiation by inhibiting osteocalcin expression. a Primary calvarial preosteoblasts from WT mice were cultured in osteogenic differentiation medium containing 50 μg·mL−1 L-AA and 5 μmol·L−1 β-GP for 24 days. The transcript and protein levels of HIF-2α on the indicated culture days were determined by qRT-PCR and western blotting, respectively. The expression levels of Hif-2α, Ocn, Runx2, Rankl, and Hif-1α were analyzed by qRT-PCR (n = 3). b Western blotting and quantification of protein levels of HIF-1α and HIF-2α in undifferentiated or differentiated osteoblasts under normoxia or hypoxia (n = 4). DM, differentiation media. c Alkaline phosphatase (ALP) and alizarin red S (ARS) staining in primary calvarial preosteoblasts cultured in control media (CM) or differentiation media (DM). Calvarial preosteoblasts were obtained from WT or Hif-2α+/− mice (n = 3). d Transcript levels of Hif-2α, Ocn, and Runx2 were detected by qRT-PCR in primary cultured calvarial preosteoblasts infected with 400 multiplicity of infection (MOI) of Ad-C or the indicated MOI of Ad-Hif-2α (n > 3). e Detection of the indicated mRNAs by qRT-PCR in osteoblasts transfected with control siRNA (si-C) or the indicated amounts (nM) of Hif-2α-siRNA (n = 6). f RUNX2-responsive luciferase reporters (6xOSE-luc or OG2-luc) were transfected into primary calvarial preosteoblasts infected with Ad-Hif-2α or Ad-C. Luciferase assays were performed, and the data are presented as fold changes relative to each CM group (n = 3). g qRT-PCR analysis of Twist1 and Twist2 (n = 4). h TWIST2 immunostaining in osteoblasts of bone tissue from WT and Hif-2α+/− mice. Dotted lines indicate osteoblasts (scale bar: 10 μm). Quantification of TWIST2-positive osteoblasts is shown (n = 7). i Detection of the mRNA levels of Hif-2α, Twist2, Runx2, and Ocn following the siRNA-mediated silencing of Twist2 (Twist2-siR) in HIF-2α-overexpressing cells (n = 3). j ChIP assays were performed using primer pairs (1 and 2) designed to span the putative HIF-2α binding sites within the Twist2 promoter, along with an anti-HIF-2α antibody. k Representative µCT images and measurements of bone volume of calvarial defect models infected with Ad-C or Ad-Hif-2α and coinjected with adenovirus encoding Twist2 shRNA (Ad-shTwist2) (n = 3). Values are presented as the mean ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.005)
