Journal: Bone research

Article Title: Hypoxia-inducible factor-1α restricts the anabolic actions of parathyroid hormone

doi: 10.1038/boneres.2014.5

Figure Lengend Snippet: Parathyroid hormone increases the expression of Hif-1α both in vitro and in vivo . ( a and b ) Levels of Hif-1α, Hif-2α and Hif-1β protein were measured in wild-type osteoblasts at the indicated time points after treatment with 10 nmol·L −1 PTH ( a ) and after 6 h of treatment with 200 nmol·L −1 DFO ( b ). ( c ) Immunohistochemical analysis of Hif-1α expression in the trabecular bone of 10-week-old female wild-type mice treated with 40 μg·kg −1 PTH or vehicle for 4 h. Representative tissue sections show increased Hif-1α expression in the osteoblasts (arrows) of PTH-treated mice relative to untreated controls. ( d and e ) mRNA levels of Hif-1α and Hif-2α were measured in vitro ( d ) and in vivo ( e ) after treatment with PTH or vehicle. Spp1 (Osteopontin) and Ptgs2 (Cox2) were used as controls. * P ≤0.05. DFO, desferoxamine.

Article Snippet: Antibodies for Hif-1α (NB100–105), Hif-2α (NB100–122) and Hif-1β (NB100–124) were obtained from Novus Biological.

Techniques: Expressing, In Vitro, In Vivo, Immunohistochemical staining

Journal: Bone research

Article Title: Hypoxia-inducible factor-1α restricts the anabolic actions of parathyroid hormone

doi: 10.1038/boneres.2014.5

Figure Lengend Snippet: cAMP/PKA signaling induces Hif-1α expression. ( a ) Levels of Hif-1α protein were measured in wild-type osteoblasts following 4 h of treatment with 10 nmol·L −1 PTH or vehicle (NT), preceded by 30 min of treatment with 100 μg·mL −1 cycloheximide (Chx). ( b ) Levels of Akt (S473) and p70 S6 kinase protein phosphorylation were measured in wild-type osteoblasts following 1 h of treatment with 10 nmol·L −1 PTH. ( c ) Hif-1α protein expression was measured in control and mTOR-deficient osteoblasts following treatment with 10 nmol·L −1 PTH for 4 h. ( d ) Densitometric quantitation of Hif-1α protein as shown in a and c . ( e and g ) Akt and p70 S6 kinase phosphorylation ( e ) and Hif-1α protein expression ( g ) were measured in wild-type osteoblasts following 30 min ( e ) or 6 hours ( g ) of treatment with Forskolin at the indicated concentrations. ( f ) Quanitification of Akt phosphorylation shown in e in Forskolin-treated osteoblasts relative to untreated. ( h ) Relative expression of Hif-1α protein as shown in g . ( i and l ) Levels of Akt and p70 S6 kinase phosphorylation ( i ) and Hif-1α protein expression ( l ) were measured following 30 min ( i ) or 6 h ( l ) of treatment with 10 nmol·L −1 PTH, preceded by 30 min of treatment with 10 μmol·L −1 H-89. ( j and k ) Quantification of Akt ( j ) and p70 S6 kinase ( k ) phosphorylation shown in i in PTH- and/or H-89-treated osteoblasts relative to untreated. ( m ) Relative expression of Hif-1α protein as shown in l . * P ≤0.05.

Article Snippet: Antibodies for Hif-1α (NB100–105), Hif-2α (NB100–122) and Hif-1β (NB100–124) were obtained from Novus Biological.

Techniques: Expressing, Phospho-proteomics, Control, Quantitation Assay

Journal: Bone research

Article Title: Hypoxia-inducible factor-1α restricts the anabolic actions of parathyroid hormone

doi: 10.1038/boneres.2014.5

Figure Lengend Snippet: Bone formation after PTH administration is increased in mice lacking Hif-1α in osteoblasts and osteocytes. Ten-week-old female control and Hif-1α mutant (ΔHif-1α) mice were treated with vehicle or PTH for 6 weeks by daily s.c. injection. ( a ) Representative microCT images illustrate trabecular bone structure in the distal femur of control and ΔHif-1α mice after treatment with PTH at the indicated doses. ( b – d ) Bone volume per tissue volume (BV/TV) ( b ), trabecular number (Tb. N) ( c ) and trabecular thickness (Tb. Th) ( d ) were quantified by microCT. ( e – g ) The mineralizing surface per bone surface (MS/BS) ( e ), mineral apposition rate (MAR) ( f ) and bone formation rate per bone surface (BFR/BS) ( g ) were assessed by dynamic histomorphometry. ( h and i ) Levels of P1NP ( h ) and CTx ( i ) were measured in serum collected from control and ΔHif-1α mice. Results from e through i are presented as % change from baseline levels in saline-treated animals, which were equivalent in control and ΔHif-1α mice. * P ≤0.05 vs. control mice with matched treatment. P1NP, amino pro-peptide of type 1 collagen; CTx, C-terminal telopeptide; s.c., subcutaneous.

Article Snippet: Antibodies for Hif-1α (NB100–105), Hif-2α (NB100–122) and Hif-1β (NB100–124) were obtained from Novus Biological.

Techniques: Control, Mutagenesis, Injection, Saline

Journal: Bone research

Article Title: Hypoxia-inducible factor-1α restricts the anabolic actions of parathyroid hormone

doi: 10.1038/boneres.2014.5

Figure Lengend Snippet: Hif-1α inhibits β-catenin activity following PTH administration.( a ) Relative expression of Hif-1α mRNA in osteoblasts following adenoviral Cre-mediated deletion. ( b ) Levels of cAMP production were measured by immunoassay after control and Hif-1α-deficient (ΔHif-1α) osteoblasts were treated with 10 nmol·L −1 PTH for 15 min. ( c ) Phosphorylation levels of Creb and Erk in control and ΔHif-1α osteoblasts were measured following 0–30 min of treatment with 10 nmol·L −1 PTH. ( d and e ) Relative expression of Axin2 ( d ) and Nkd2 ( e ) mRNA in control and ΔHif-1α osteoblasts treated with 10 nmol·L −1 PTH for 8 h or left untreated (NT). ( f ) Immunoblot analysis of Hif-1α levels in control and Vhl-deficient (ΔVhl) osteoblasts 48 h after adenoviral Cre infection. ( g ) Relative expression of Axin2 mRNA in control and ΔVhl osteoblasts treated with 10 nmol·L −1 PTH for 4 h or left untreated (NT). ( h ) Protein levels of β-catenin in nuclear extracts isolated from control and ΔHif-1α osteoblasts treated with 10 nmol·L −1 PTH for 4 h or left untreated (NT). Tbp was used as a nuclear control. ( i ) IP analysis of Hif-1α and β-catenin interaction following 4 h of 10 nmol·L −1 PTH treatment in wild-type cells. ( j ) Quantitative chromatin IP analysis of β-catenin occupancy at the Axin2 promoter in control and ΔHif-1α osteoblasts after 8 h of treatment with 10 nmol·L −1 PTH. ( k ) Relative expression of Axin2 mRNA in samples collected from the femur of control and ΔHif-1α mice after treatment with saline (NT) or 40 µg·kg −1 PTH for 16 h. * P ≤0.05. IP, immunoprecipitation; Tbp, TATA-binding protein.

Article Snippet: Antibodies for Hif-1α (NB100–105), Hif-2α (NB100–122) and Hif-1β (NB100–124) were obtained from Novus Biological.

Techniques: Activity Assay, Expressing, Control, Phospho-proteomics, Western Blot, Infection, Isolation, Chromatin Immunoprecipitation, Saline, Immunoprecipitation, Binding Assay