ABSTRACT The hypoxia inducible factors (Hifs) are evolutionarily conserved transcriptional factors that control homeostatic responses to low oxygen. In developing bone, Hif-1 generated

signals induce angiogenesis necessary for osteoblast specification, but in mature bone, loss of Hif-1 in osteoblasts resulted in a more rapid accumulation of bone. These findings suggested

that Hif-1 exerts distinct developmental functions and acts as a negative regulator of bone formation. To investigate the function of Hif-1α in osteoanabolic signaling, we assessed the

effect of Hif-1α loss-of-function on bone formation in response to intermittent parathyroid hormone (PTH). Mice lacking Hif-1α in osteoblasts and osteocytes form more bone in response to

β-catenin transcriptional activity. Together, these data indicate that Hif-1α functions in the mature skeleton to restrict osteoanabolic signaling. The availability of pharmacological agents

that reduce Hif-1α function suggests the value in further exploration of this pathway to optimize the therapeutic benefits of PTH. SIMILAR CONTENT BEING VIEWED BY OTHERS OSTEOCYTE

_EGLN1_/PHD2 LINKS OXYGEN SENSING AND BIOMINERALIZATION VIA FGF23 Article Open access 18 January 2023 DIFFERENTIAL BUT COMPLEMENTARY ROLES OF HIF-1Α AND HIF-2Α IN THE REGULATION OF BONE

HOMEOSTASIS Article Open access 23 July 2024 KINDLIN-2 REGULATES SKELETAL HOMEOSTASIS BY MODULATING PTH1R IN MICE Article Open access 26 December 2020 INTRODUCTION Parathyroid hormone (PTH),

an 84-amino-acid polypeptide, is an essential regulator of mineral homeostasis and bone remodeling. Released by the chief cells of the parathyroid gland in response to deviations in serum

calcium levels, PTH primarily acts on kidney and bone to increase calcium reabsorption and liberate calcium from bone matrix, respectively.1 Additionally, intermittent PTH administration is

recognized for its anabolic effects in bone, and the first 34 amino acids of the hormone form the basis for the only Food and Drug Administration-approved anabolic agent to treat

osteoporosis.2–4 The anabolic actions of PTH have been extensively studied in laboratory rodent models.5 Through its actions on the PTH receptor,6 expressed by osteoblasts and osteocytes,

histological analyses suggest that intermittent PTH increases bone acquisition by increasing the number of bone-forming osteoblasts.7–9 More recent molecular analyses have attempted to

identify signaling mechanisms and components that allow PTH to reduce bone cell apoptosis,9 stimulate progenitor cell recruitment10 and activate formerly quiescent bone lining cells.11 In

addition to the activation of cyclic AMP (cAMP) and protein kinase A signaling,12 components of the insulin-like growth factor pathway,13–15 the transforming growth factor-β pathway10,16 and

Wnt/β-catenin signaling17,18 have all been demonstrated to be required for the full osteo-anabolic response to PTH. The identification of factors or signaling mechanisms that inhibit bone

formation after PTH administration has been less common,19,20 but examining such mechanisms could facilitate the development of strategies to increase the therapeutic efficacy of

intermittent PTH. Hypoxia inducible factor-1 (Hif-1) is most widely recognized for its role in the cellular response to molecular oxygen levels.21,22 A basic helix–loop–helix transcription

factor, the activity and cellular abundance of Hif-1 is regulated by an oxygen-dependent proteolysis mechanism. At normal oxygen tensions, the α-subunit of the protein (Hif-1α) undergoes

prolyl hydroxylation, which initiates recognition by the von Hippel–Lindau (Vhl) tumor suppressor protein, a component of the E3 ubiquitin ligase that targets Hif-1α for proteasomal

degradation. When oxygen tensions fall below 5%, prolyl hydroxylation is inhibited; Hif-1α accumulates and translocates to the nucleus where it forms a dimer with the Hif-1β subunit. _In

vitro_ studies suggest that Hif-1 regulates the expression of several hundred genes involved in angiogenic and metabolic responses,21 and utilizes both direct promoter binding23 as well as

indirect mechanisms to alter gene expression.24 Within bone, Hif-1 exerts distinct developmental functions. In developing bone, Hif-1 generated signals are required for angiogenesis, which

appears to be necessary for initial specification of bone-forming osteoblasts. Consistent with this idea, mice lacking Hif-1α in osteoblasts and osteocytes develop poorly vascularized bones

with reduced cortical and trabecular bone volume, while Hif overexpression results in highly vascularized and dense bone.25,26 As Hif-1α mutant mice mature, a second inhibitory function

emerges such that Hif-1 acts as a negative regulator of bone formation. In this regard, cortical and trabecular bone volume normalize with age and Hif-1α mutants are more sensitive to

mechanical stimuli.27 In this study, we investigated the function of Hif-1α in osteo-anabolic signaling by assessing the effect of Hif-1α loss-of-function on bone formation in response to

parathyroid hormone. In addition to hypoxia, Hif-1α expression is induced by a number of stimuli and signaling pathways critical for normal osteoblast function, including some that are used

by PTH to increase bone formation.27–29 Moreover, PTH stimulates vascular remodeling in bone,30 which suggests two potential mechanisms by which Hif-1 might impact PTH-induced anabolism.

Here, we demonstrate that PTH administration results in Hif-1α expression by osteoblasts both _in vivo_ and _in vitro_, and that by interacting with β-catenin, Hif-1α suppresses the anabolic

response. As a result, the removal of Hif-1α from osteoblasts and osteocytes sensitizes bone to PTH treatment by enhancing the activity of osteoblasts. These findings indicate that Hif-1α

is a more general suppressor of osteo-anabolic signaling and acts to inhibit signals beyond those associated with mechanical loading. MATERIALS AND METHODS GENERATION OF TRANSGENIC MICE The

generation of mice lacking Hif-1α in osteoblasts and osteocytes (ΔHif-1α) was described previously.26,27 Briefly, OC-Cre mice31 were crossed with mice in which the second exon of Hif-1α is

floxed.32 Mice containing Hif-2α-floxed alleles,33 Vhl-floxed alleles34 and mTOR-floxed (mTOR: mammalian target of rapamycin) alleles35 have been described previously. All mice were

maintained on a C57BL/6 background. PCR analysis from ear or tail biopsies was used to confirm genotypes. The Institutional Animal Care and Use Committee of the Johns Hopkins University

School of Medicine approved all animal procedures. ADMINISTRATION OF HUMAN PTH _IN VIVO_ Female ΔHif-1α and control mice were grown until 10 weeks of age at which point daily (7 day per

week) subcutaneous injections of 100 µL vehicle or human PTH 1–34 (Bachem Inc., Torrance, CA, USA) were initiated. PTH concentrations (20 µg·kg−1 or 40 µg·kg−1) were adjusted weekly based on

body mass measurements. All mice were sacrificed at 16 weeks of age. Blood samples were collected at sacrifice for analysis of serum markers of bone resorption and formation. Serum was

collected and immediately stored at −80 °C. Serum concentrations of C-terminal telopeptide (RatLaps; IDS Inc., Scottsdale, AZ, USA) and N-terminal propeptide of type 1 procollagen (P1NP; IDS

Inc.) were determined via commercially available ELISA. Two additional groups of female ΔHif-1α and control mice were treated with PTH (40 μg·kg−1 subcutaneous) for 4 or 16 h and then

sacrificed. Femurs were dissected and prepared for immunohistochemical analysis of Hif-1α expression (sc-10790; Santa Cruz, Dallas, TX, USA) according to standard techniques or homogenized

in TRIzol (Invitrogen, Grand Island, NY, USA) for RNA analysis after flushing the bone of marrow. SKELETAL ANALYSIS To examine bone architecture, the mouse femur was scanned using a desktop

microtomographic imaging system (Skyscan 1172; Skyscan, Kontich, Belgium) in accordance with the recommendations of the American Society for Bone and Mineral Research.36 The femur was

scanned at 50 keV and 200 mA using a 0.5 mm aluminum filter with an isotropic voxel size of 10 µm. The resulting two-dimensional images are shown in gray scale. Trabecular bone parameters

were assessed in the distal femur 500 μm proximal to the growth plate and extending for 2 mm (200 CT slices). Cortical bone parameters were assessed at the femoral midshaft and represent an

average of 50 CT slices (500 µm). Dynamic measures of bone formation were assessed by injection of two sequential 0.2 mL doses of calcein (0.8 mg·mL−1) delivered 3 and 10 days prior to

sacrifice. The femur was fixed in ethanol, dehydrated and embedded in methylmethacrylate. Three micron sections were cut with a Microm microtome and stained with Mason-Goldner trichrome

stain. The number of osteoblasts and osteoclasts per bone perimeter were measured at standardized sites under the growth plate at a magnification of ×200 using a semi-automatic method

(Osteoplan II; Kontron, Munich, Germany). These parameters comply with the guidelines of the nomenclature committee of the American Society for Bone and Mineral Research.37,38 OSTEOBLAST

ISOLATION AND CULTURE Osteoblasts were isolated from the calvaria of newborn Hif-1α-floxed, Hif-2α-floxed, mTOR-floxed and Vhl-floxed mice by serial digestion in 1.8 mg·mL−1 collagenase type

I and maintained in α-MEM (minimum essential medium, alpha modification) supplemented with 10% FBS (Fetal bovine serum) and 1% penicillin/streptomycin. To disrupt Hif-1α, Hif-2α, mTOR or

Vhl expression, osteoblasts were infected with control adenovirus expressing green fluorescent protein or adenovirus expressing _Cre_ recombinase (Vector Biolabs, Philadelphia, PA, USA) at

an MOI (multiplicity of infection) of 100. Osteoblasts were harvested 48 h after adenoviral infection and deletion efficiency was assessed in a portion of the cell population by real-time

PCR. The remaining cells were replated for stimulation with PTH. Pharmacological agents were obtained from Sigma Aldrich, dissolved in DMSO and added to cell cultures with appropriate

vehicle controls 30–60 min before PTH treatments. QUANTITATIVE REAL-TIME PCR AND CHROMATIN IMMUNOPRECIPITATION Total RNA was extracted from osteoblasts or homogenized femurs using TRIzol

(Invitrogen) and 1 µg was reverse transcribed using the iScript cDNA synthesis system (Bio-Rad, Hercules, CA, USA). Two microliters of cDNA was subjected to PCR amplification using the iQ

SYBR Green Supermix (Bio-Rad). Primer sequences were obtained from PrimerBank (http://pga.mgh.harvard.edu/primerbank/index.html). Reactions were normalized to endogenous β-actin reference

transcript. Chromatin immunoprecipitation assays were performed using an Agarose ChIP Kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions and a ChIP-qualified

antibody specific for β -catenin (PAS-16192; Thermo Scientific, Waltham, MA, USA). Precipitated DNA (2 µL) was subjected to PCR amplification by qPCR and normalized to reference reactions

utilizing input DNA. Primer sequences for the Axin2 promoter are available upon request. PROTEIN ISOLATION AND ASSAYS Protein was extracted from cultured osteoblasts in 0.1% Triton X-100

containing protease and phosphatase inhibitors. The extracts were separated on 10% SDS/polyacrylamide gels and transferred to PVDF (polyvinyl difluoride) membranes. Antibodies for Hif-1α

(NB100–105), Hif-2α (NB100–122) and Hif-1β (NB100–124) were obtained from Novus Biological. Antibodies for phospho-Akt (S473, 9721), Akt (9272), phospho-p70 S6 kinase (9206), p70 S6 kinase

(9202), phospho-Creb (9198), Creb (9197), phospho-Erk (9101), Erk (9107) and β -catenin (2698) were obtained from Cell Signaling Technologies, Danvers, MA, USA. Bound antibodies were

visualized using either the Supersignal West Femto or West Pico Substrates (Pierce). Co-immunoprecipitation was performed overnight at 4 °C in a reaction containing 2 µg of antibody specific

for Hif-1α (Novus) or β-catenin (Cell Signaling Technologies, San Diego, CA, USA). STATISTICAL ANALYSIS Results are expressed as mean±s.e.m. All statistical tests were two-sided. A

_P_-value less than 0.05 was considered significant. Comparability of two groups of data was assessed using a Student’s _t_-test. RESULTS PTH STIMULATES HIF-1Α EXPRESSION IN OSTEOBLASTS Mice

that lack Hif-1α in osteoblasts and osteocytes (Hif-1αflox/flox; Oc-CreTG/+, hereafter referred to as ΔHif-1α) exhibit early deficits in both cortical and trabecular bone architecture that

are at least partially attributable to impairments in skeletal vascularization.25,26 As the mutant mice mature, bone architecture normalizes,27 indicating that Hif-1α exerts distinct

developmental functions and likely acts to suppress osteo-anabolic signaling. To assess this inhibitory function, we examined the influence of Hif-1α on the response of osteoblasts to PTH.

To establish that Hif-1α regulates osteo-anabolic signaling in response to PTH, we first examined the ability of the hormone to induce the expression of Hif-1α in osteoblasts. In cultures of

normoxic calvarial osteoblasts, PTH (10 nmol·L−1) rapidly increased Hif-1α protein levels, with expression levels peaking between 2 and 4 h after stimulation and remaining elevated through

8 h of treatment (Figure 1a). Protein levels of Hif-2α and Hif-1β were not affected. However, desferoxamine, an iron chelator that inhibits the activity of the prolyl-hydroxylase enzymes

that initiate the targeting of Hif-α subunits for proteasomal degradation, induced the expression of both Hif-1α and Hif-2α (Figure 1b), indicating that the effects of PTH are specific for

Hif-1α. PTH administration also increased Hif-1α expression _in vivo_ as PTH treated mice (40 µg·kg−1) exhibited robust expression of Hif-1α in osteoblasts lining trabecular bone surfaces, a

subset of osteocytes, and marrow components (Figure 1c), while saline treated animals exhibited only weak expression in these cell populations. Both _in vitro_ (Figure 1d) and _in vivo_

(Figure 1e), the levels of Hif-1α mRNA were unaffected by PTH stimulation, suggesting that the induction of Hif-1α protein occurs via a post-transcriptional mechanism. CAMP/PROTEIN KINASE A

(PKA) SIGNALING INDUCES HIF-1Α EXPRESSION IN RESPONSE TO PTH PTH could increase Hif-1α protein levels without affecting Hif-1α transcription by specifically enhancing Hif-1α translation or

by inhibiting proteasomal degradation. Since PTH retained the capacity to increase Hif-1α protein levels in osteoblasts deficient for Vhl (data not shown), we focused on the induction of new

Hif-1α synthesis. As expected, pre-treatment of osteoblast cultures with cycloheximide, to inhibit new protein synthesis, abolished the effect of PTH on Hif-1α protein levels (Figure 2a and

2d). Moreover, increases in the phosphorylation of Akt (S473) and p70 S6 kinase (Figure 2b) indicated that PTH activates mTOR, a key regulator of Hif expression in response to anabolic

signals.39,40 Adenoviral Cre-mediated disruption of mTOR expression in osteoblasts containing mTORflox/flox alleles, via an 83% reduction in mTOR mRNA levels, completely inhibited the effect

of PTH on Hif-1α protein (Figure 2c and 2d). We next explored the signaling mechanisms by which PTH activates mTOR and ultimately increases Hif-1α protein. Because an increase in cellular

cAMP signaling is a primary response to PTH binding to its receptor,6,12 we initially assessed the effect of pharmacologically raising cAMP levels on mTOR activity and Hif-1α expression.

Forskolin, which activates adenylyl cyclase, dose-dependently stimulated the phosphorylation of Akt and p70 S6 kinase (Figure 2e and 2f), indicating that mTOR was activated, and increased

the levels of Hif-1α protein (Figure 2g and 2h). To confirm these results, we pre-treated osteoblast cultures with H-89 to antagonize the activity of PKA, the downstream mediator of cAMP

signaling. This approach greatly impaired the ability of PTH to stimulate the phosphorylation of Akt and p70 S6 kinase (Figure 2i–2k), even though baseline levels of p70 phosphorylation were

increased by H-89, and abolished the increase in Hif-1α protein (Figure 2l and 2m). Together, these data suggest a mechanism whereby PTH activates cAMP/PKA signaling which in turn activates

the mTOR pathway to regulate Hif-1α expression. ΔHIF-1Α MICE ARE MORE SENSITIVE TO ANABOLIC PTH TREATMENT To directly assess the effect of Hif-1α expression on the anabolic response of bone

to PTH, we generated cohorts of 10-week-old female control and ΔHif-1α mice and administered daily injections of PTH or saline, as a control, for 6 weeks. MicroCT analysis revealed

equivalent trabecular bone architecture in the distal femur of saline treated control and ΔHif-1α mice, which is consistent with our previous study27 that demonstrated a normalization of

bone volume in Hif-1α mutants (Figure 3a–d). PTH injections (40 µg·kg−1 BW, Body weight) produced the predicted anabolic response and increased trabecular bone volume by 155.41% in control

mice (Figure 3a and 3b) by increasing trabecular number (Figure 3c) and thickness (Figure 3d). In Hif-1α mutants, PTH increased bone volume an additional 22.86% relative to the treated

control mice (182.19% increase _versus_ ΔHif-1α, saline-treated controls), due to significantly larger increases in trabecular thickness and slightly larger increases in trabecular number.

PTH also significantly increased cortical bone thickness, but the effect was indistinguishable between control and Hif-1α mutant mice (data not shown). Likewise, increases in body weight

were equivalent among control and Hif-1α mutant mice (data not shown). When we reduced the daily dose of PTH to 20 µg·kg−1, the increase in trabecular bone volume in the mutant mice (182.79%

increase _versus_ ΔHif-1α, saline-treated controls) was identical to that of the 40 µg·kg−1 treatment group, while the percent increase in the control mice was reduced by 29.91% (Figure 3a

and 3b). Taken together, these data suggest that disrupting the expression of Hif-1α increases the sensitivity of bone to intermittent PTH. To understand the cellular basis for the enhanced

anabolic response in ΔHif-1α mice, we performed dynamic histomorphometric and serological analyses of bone formation. Since all measures were equivalent in the saline-treated control and

ΔHif-1α mice, results are presented as the relative increase above this baseline level (Figure 3e–3i). PTH increased the mineralizing surface per bone surface to a similar extent in control

and ΔHif-1α mice (Figure 3e), but the mutant mice exhibited a 23.2% increase in mineral apposition rate while no effect of PTH on this parameter was observed in the control mice (Figure 3f).

This led to a greater overall increase in bone formation rate in the mutant mice relative to the control mice (Figure 3g). Similarly, serum levels of P1NP (Figure 3h), a marker of bone

formation, were increased to a greater extent in the mutant mice relative to controls, while the levels of C-terminal telopeptide (Figure 3i), a marker of bone resorption, were increased to

a similar degree. Thus, the amplified sensitivity of Hif-1α mutant mice to PTH and augmented bone formation response appear to be due to an increase in the functional activity of individual

osteoblasts. HIF-1Α ANTAGONIZES THE ACTIONS OF Β-CATENIN AFTER PTH STIMULATION Finally, we assessed cellular signaling mechanisms that might account for the increased responsiveness of

Hif-1α mutant mice to PTH. Calvarial osteoblasts were isolated from Hif-1αflox/flox mice and infected with adenoviral constructs expressing _Cre_ to eliminate Hif-1α expression (Figure 4a)

or green fluorescent protein as a control. We next examined the effect of eliminating Hif-1α expression on the activation of the primary PTH-responsive pathways, but the increase in cellular

cAMP levels (Figure 4b) and the phosphorylation of Creb and Erk (Figure 4c) were similar in control and Hif-1α deficient osteoblasts. By contrast, the increase in Axin2 and Nkd2 mRNA levels

was enhanced after PTH treatment in ΔHif-1α osteoblasts relative to those of controls (Figure 4d and 4e), suggesting that the activation of β-catenin was increased.41 To test the

specificity of this apparent inhibitory effect of Hif-1α on β-catenin activity, we overexpressed Hif-1α by eliminating the expression of Vhl (Figure 4f), and as expected this genetic

manipulation impaired the ability of PTH to increase the expression of Axin2 (Figure 4g). The expression of Hif-1α in response to PTH did not alter the accumulation or nuclear localization

of β-catenin (Figure 4h), but rather Hif-1α directly interacted with β-catenin (Figure 4i) and acted to inhibit the binding of β-catenin to the promoter of target genes (Figure 4j). We

observed a similar effect _in vivo_ as PTH produced a significant increase in Axin2 mRNA levels in the femurs of ΔHif-1α mice, but not those of control mice. These data imply that the

enhanced bone formation response evident in Hif-1α mutant mice stems from the elimination of Hif-1α-mediated suppression of β-catenin signaling. DISCUSSION In this study, we demonstrate that

the transcription factor Hif-1α acts to suppress the anabolic actions of parathyroid hormone. Hif-1α protein levels were rapidly upregulated both _in vitro_ and _in vivo_ by PTH stimulation

and mice rendered deficient for Hif-1α in osteoblasts and osteocytes were more responsive to intermittent administration of the hormone. The more dramatic increase in bone formation evident

in Hif-1α mutant mice appears to result from an increase in the performance of individual osteoblasts secondary to an enhancement of β-catenin target gene expression. In addition to

hypoxia, Hif-1α is induced by a number of anabolic signals relevant to bone metabolism. Mechanical loading,42 growth factors and paracrine factors like prostaglandins43–45 all increase

Hif-1α protein levels. Some of these factors have also been implicated in the anabolic response of bone to PTH. Insulin-like growth factor-1, for instance, induces Hif-1α expression,46 and

removal of its receptor diminishes bone formation in response to the hormone.13 While insulin-like growth factor-1 might partially contribute to the induction of Hif-1α after PTH

stimulation, the rapid effect we observed suggests a more direct effect on Hif-1α synthesis. Indeed, our data suggest that PTH activates cAMP/PKA signaling to increase the activity of mTOR,

which can directly regulate Hif-1α translation.39,40,47 While not directly examined here, studies in other tissues indicate that PKA can activate mTOR signaling via a number of mechanisms,

including the engagement of PI3K/Akt signaling and the phosphorylation of TOR complex components.48,49 As alluded to above, mTOR signaling has also been demonstrated to be an important

component of the anabolic response of bone to PTH, as rapamycin treatment inhibits the increase in trabecular bone volume resulting from high-dose PTH administration by reducing osteoblastic

activity.29 In light of our data, it would appear that mTOR plays a dual role in the response of bone to intermittent PTH, facilitating anabolism while also establishing a mechanism to

suppress anabolic signaling via induction of Hif-1α expression. We focused our analysis on the effects of Hif-1α on β-catenin signaling because of its well-documented anabolic role in the

skeleton, but we cannot rule out the possibility that Hif-1α also regulates the expression of factors that inhibit mTOR signaling.50 Even though angiogenesis is a well-explored response to

increased Hif-1α signaling and PTH has been shown to induce skeletal vascular remodeling,30 it does not appear that this effect factors into the anabolic response we observed. If vascular

remodeling and the relocalization of blood vessels to sites of new bone formation were regulated by Hif-1α, we would have expected the disruption of Hif-1α expression to impair the

osteo-anabolic response. However, we cannot completely rule out a compensatory effect of the closely related Hif-2α. Rather, as we have suggested previously,27 it is likely that Hif-1α

assumes an inhibitory role in the mature skeleton to prevent unchecked anabolic signaling and the generation of signals that impinge on cellular function. Hif-1α is induced by reactive

oxygen species, and Hif-1-generated signals in turn reduce new oxidant production.51–54 Therefore, induction of Hif-1α may ensure cellular longevity and facilitate cellular repair after the

generation of cellular stressors during an anabolic response. The results presented here are consistent with our previous finding that Hif-1α acts to inhibit the anabolic response to a

tibia-loading regime and potentially does so by suppressing β-catenin activity.27 However, several important differences exist when the cellular basis of each response is considered. Our

mechanical loading study demonstrated that Hif-1α inhibited the anabolic response in the cortical bone envelope, but in these studies, PTH produced similar increases in cortical bone

thickness in control and mutant mice. Likewise, the number of osteoblasts activated by mechanical loading was greatly increased in Hif-1α mutant mice, but the effect of PTH on bone formation

in these mice appears to be related to a larger increase in the functional output of individual osteoblasts. Here, the mineralizing surface was similarly enhanced in control and mutant

mice, but the mineral apposition rate and P1NP levels were enhanced by the genetic removal of Hif-1α. While we cannot exclude the possibility that the differential effects are simply the

result of different bone compartments, these data suggest that the suppressive actions of Hif-1α may be dependent on the cellular context and the stimulus. Nonetheless, the significant

increase in the anabolic effect of each of these stimuli suggests that Hif-1α activity could be targeted in therapeutic paradigms. Previous studies have identified pharmacological molecules

that impair Hif-1α transcriptional activity or interaction with binding partners.55–57 While these studies have primarily focused on the prevention of tumor growth and tumor-induced

angiogenesis, these agents or molecules with similar functions could be adapted to enhance anabolic therapies in bone. After prolonged treatment with intermittent PTH, markers of bone

formation begin to decline, suggestive of the development of a resistance to the anabolic effects of the therapy.58,59 Whether increased expression of Hif-1α contributes to this effect will

require additional studies. However, our studies suggest that an agent that inhibits the expression of Hif-1α or impairs the interaction of Hif-1α and β-catenin could be used to lower the

therapeutic dose of PTH necessary to decrease fracture risk or increase the anabolic response. In summary, our studies support a role for Hif-1α as a negative regulator of osteo-anabolic

signaling. In early development, Hif-1α functions in bone cells to facilitate the vascularization of long bones, a process that is required for normal bone acquisition. As bone matures,

Hif-1α assumes a new function that likely acts to restrain osteoblast and osteocyte activity and does so by interfering with a key component of the Wnt signaling pathway. Our studies provide

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ACKNOWLEDGEMENTS We thank Dr C Lynch for providing the mTORflox/flox mice. Support was provided by a Career Development Award (RCR, BX001284) from the Veterans Administration. AUTHOR

INFORMATION AUTHORS AND AFFILIATIONS * Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA Julie L Frey, David P Stonko & Ryan C Riddle *

Division of Nephrology, Bone & Mineral Metabolism, University of Kentucky, Lexington, KY, USA Marie-Claude Faugere * Veterans Administration Medical Center, Baltimore, MD, USA Ryan C

Riddle Authors * Julie L Frey View author publications You can also search for this author inPubMed Google Scholar * David P Stonko View author publications You can also search for this

author inPubMed Google Scholar * Marie-Claude Faugere View author publications You can also search for this author inPubMed Google Scholar * Ryan C Riddle View author publications You can

also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Ryan C Riddle. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest.

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THIS ARTICLE CITE THIS ARTICLE Frey, J., Stonko, D., Faugere, MC. _et al._ Hypoxia-inducible factor-1α restricts the anabolic actions of parathyroid hormone. _Bone Res_ 2, 14005 (2014).

https://doi.org/10.1038/boneres.2014.5 Download citation * Received: 30 November 2013 * Revised: 24 December 2013 * Accepted: 01 January 2014 * Published: 13 May 2014 * DOI:

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