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

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

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