Fig. 2: HIF-1α inhibits osteoblast differentiation via the TWIST2-RUNX2-OCN axis.

a Osteogenic differentiation of calvarial pre-osteoblasts was induced by DM, and 200 or 400 MOI of Ad-Hif1a was infected at differentiation-inducing culture day 3. Subsequently, cells were harvested on the 6th day of differentiation. Relative mRNA levels of Hif1a, Ocn, and Runx2 were quantitated by qRT-PCR (n ≥ 3). b Osteogenic phenotypes were determined by ALP and ARS staining. The representative captured images of 24-well plates were displayed (n = 3). c 5-mm diameter critical-sized defects were created in 6-week-old male mice and covered with collagen sponges without (Veh) or with 300 ng BMP-2. For each group, collagen sponges containing Ad-C or Ad-Hif1a were applied. After 2 weeks, the size and bone volume of calvarial defects were measured. The representative μCT images were shown (n = 5). d Cells were transfected with siRNA against Hif1a or control-siRNA (si-C) on differentiation day 3 and cultured for 3 days. Transcript levels of Hif1a, Ocn, and Runx2 were determined by qRT-PCR (n = 3). e The representative images of ALP and ARS staining (n = 3). f The pre-osteoblasts were co-transfected with RUNX2-responsive luciferase reporter constructs (6XOSE-Luc or OG2-Luc) and infected together with indicated MOI of Ad-Hif1a. g The relative RNA levels of Twist1 and Twist2 isotypes in Hif1a-overexpressing cells (n = 4). h ChIP analysis showing the HIF-1α binding to the Twist2 promoter region. i mRNA level of Twist2 in pre-osteoblasts transfected with Hif1a siRNA (n = 4). j Cells were transfected with siRNA against Twist2 or si-C following infection of Ad-Hif1a. Transcript levels of Hif1a, Twist2, Ocn, and Runx2 were analyzed (n = 5). Values are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.