HIF-1α inhibits Wnt pathway in vitro.

<p>(A) HIF-1α inhibited Topflash reporter activity in a dose-dependent manner. HEK293 cells were transfected with a Topflash reporter along with 50 ng β-catenin without or with increasing amounts of an HIF-1α-expression plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values were presented as the mean ±S.D. (B) HIF-1α cooperated with Osx to inhibit Wnt pathway activity. HEK293 cells were transfected with a Topflash reporter along with 25 ng β-catenin without or with different groups of HIF-1α expression plasmid and Osx plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values were presented as the mean ±S.D.</p

Inhibition of HIF-1α by siRNA results in downregulation of Sost expression in osteoblasts.

<p>MC3T3 osteoblasts were transfected with siRNA control or siRNA against HIF-1α. RNA was isolated 24 hr post-transfection and quantitated by quantitative real-time RT-PCR for HIF-1α and Sost, and HSP90 was used as a negative control. The RNA level from the control siRNA group was normalized to a value of 1. Values were presented as the mean ±S.D. si-control: si-RNA control; si-HIF-1α: si-RNA against HIF-1α. A paired <i>t</i>-test was performed comparing si-control group and si-HIF-1α group. *: A star indicates statistical significance compared to control group.</p

Hypoxia inhibits osteoblast proliferation.

<p>(A) Osteoblast number counts in the growth medium. MC3T3 osteoblastic cells were cultured in Alpha Minimum Essential Medium, and maintained for different time points as indicated from 4 hr to 72 hr in normoxic (20%O₂) or hypoxia (1%O₂) condition. (B) Inhibition of HIF-1α expression by siRNA resulted in an increase of osteoblast growth. MC3T3 osteoblastic cells were transfected by siRNA, and cultured under hypoxia for 48 hr. si-control: si-RNA control; si-HIF-1α: si-RNA against HIF-1α. A paired <i>t</i>-test was performed comparing si-control group and si-HIF-1α group. *: A star indicates statistical significance compared to control group.</p

Inhibition of HIF-1α by siRNA results in upregulations of Cyclin D1 and c-Myc expressions in osteoblasts.

<p>MC3T3 osteoblasts were transfected with siRNA control or siRNA against HIF-1α. RNA was isolated 24 hr post-transfection and quantitated by quantitative real-time RT-PCR. The RNA level from the control siRNA group was normalized to a value of 1. Values were presented as the mean ±S.D. si-control: si-RNA control; si-HIF-1α: si-RNA against HIF-1α. A paired <i>t</i>-test was performed comparing si-control group and si-HIF-1α group. *: A star indicates statistical significance compared to control group.</p

Binding of the HIF-1 complex to the HRE of Sost promoter under hypoxia.

<p>Nuclear extracts were isolated from MC3T3 cells in either normoxia or hypoxia conditions from 16 h and used as the HIF-1 protein resource. DNA oligonucleotides of <i>Sost</i> were labeled by Biotin. Nuclear extracts and biotin-labeled DNA probe were incubated. Protein-DNA complexes were separated on 4% polyacrylamide gels, and visualized by a Chemiluminescent Nucleic Acid detection Module. Complexes observed in extracts under normoxia (N, lane 1) or hypoxia conditions (Hyp, lane 2) are indicated by the arrows. Two hundred-fold molar excess of unlabeled <i>Sost</i> promoter oligos were used under hypoxia condition (lane 3).</p

Hypoxia leads to downregulation of Wnt targets.

<p>(A) Increase of HIF-1α expression in RNA level in osteoblasts under hypoxia. RNA levels were normalized to heat shock protein 90 (HSP90). A paired <i>t</i>-test was performed comparing control group (20% O₂) and hypoxia group (1% O₂). *: A star indicates statistical significance compared to control group. (B) Western blotting analysis of HIF-1α expression in protein level in osteoblasts under hypoxia. Heat shock protein 90 (HSP90) was used as a loading control. (C) RNA expression levels of cyclin D1 and c-Myc as determined by quantitative real-time RT-PCR. MC3T3 osteoblasts were cultured for 48 hr under hypoxia (1%O₂). RNA was isolated and quantitated by real-time RT-PCR. The RNA level from normoxic condition (20%O₂) group was normalized to a value of 1. Values were presented as the mean ±S.D. A paired <i>t</i>-test was performed comparing control group (20% O₂) and hypoxia group (1% O₂). *: A star indicates statistical significance compared to control group. (D) RNA expression levels of cyclin D1 and c-Myc treated with DFO as determined by quantitative real-time RT-PCR. MC3T3 osteoblasts were cultured for 48 hr under hypoxia (1%O₂), and treated with desferrioxamine (DFO). +:100 uM; ++:200 uM. The RNA level from normoxic condition (20%O₂) group was normalized to a value of 1. Values were presented as the mean ±S.D. A paired <i>t</i>-test was performed comparing control group (20% O₂) and hypoxia group (1% O₂). *: A star indicates statistical significance compared to control group. A paired <i>t</i>-test was also performed comparing 1% O₂ group and DFO group (+ and ++). **: Two stars indicate statistical significance compared to 1% O₂ group.</p

Effect of HIF-1α on Sost promoter activity.

<p>(A) HIF-1α activates the <i>Sost</i> promoter in a dose-dependent manner. HEK293 cells were transfected with a 1 kb <i>Sost</i> promoter-luciferase reporter gene without or with increasing amounts of an HIF-1α-expression plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values are presented as the mean ±S.D. (B) Jab1 does not activate <i>Sost</i> promoter activity. HEK293 cells were transfected with a 1 kb <i>Sost</i> promoter-luciferase reporter gene without or with increasing amounts of a Jab1-expression plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values are presented as the mean ±S.D.</p

Identification of the minimal region in the promoter of Sost gene for HIF-1α activation.

<p>(A) Schematic representation of the <i>Sost</i> deletion mutants. HRE: hypoxia response element. Sost-1 kb, Sost-540 bp, Sost-260 bp and Sost-106 bp <i>Sost</i> promoter reporter plasmids were constructed, using luciferase (LUC) as a reporter. (B) Deletion analysis of the <i>Sost</i> promoter-reporter constructs. Sost-1 kb, Sost-540 bp, Sost-260 bp and Sost-106 bp promoter-reporter plasmids (300 ng each) were cotransfected with 200 ng of the HIF-1α expression plasmid in HEK293 cells. Twenty-four hours post-transfection, cell extracts were prepared and analyzed for luciferase activity. Luciferase activity was normalized by β-galactosidase activity. Values are presented as the mean ±S.D.</p