Hypoxia-inducible Factor-1α Protein Negatively Regulates Load-induced Bone Formation
2011; Elsevier BV; Volume: 286; Issue: 52 Linguagem: Inglês
10.1074/jbc.m111.276683
ISSN1083-351X
AutoresRyan C. Riddle, Julie M. Leslie, Ted S. Gross, Thomas L. Clemens,
Tópico(s)Mitochondrial Function and Pathology
ResumoMechanical loads induce profound anabolic effects in the skeleton, but the molecular mechanisms that transduce such signals are still poorly understood. In this study, we demonstrate that the hypoxia-inducible factor-1α (Hif-1α) is acutely up-regulated in response to exogenous mechanical stimuli secondary to prostanoid signaling and Akt/mTOR (mammalian target of rapamycin) activation. In this context, Hif-1α associates with β-catenin to inhibit Wnt target genes associated with bone anabolic activity. Mice lacking Hif-1α in osteoblasts and osteocytes form more bone when subjected to tibia loading as a result of increased osteoblast activity. Taken together, these studies indicate that Hif-1α serves as a negative regulator of skeletal mechanotransduction to suppress load-induced bone formation by altering the sensitivity of osteoblasts and osteocytes to mechanical signals. Mechanical loads induce profound anabolic effects in the skeleton, but the molecular mechanisms that transduce such signals are still poorly understood. In this study, we demonstrate that the hypoxia-inducible factor-1α (Hif-1α) is acutely up-regulated in response to exogenous mechanical stimuli secondary to prostanoid signaling and Akt/mTOR (mammalian target of rapamycin) activation. In this context, Hif-1α associates with β-catenin to inhibit Wnt target genes associated with bone anabolic activity. Mice lacking Hif-1α in osteoblasts and osteocytes form more bone when subjected to tibia loading as a result of increased osteoblast activity. Taken together, these studies indicate that Hif-1α serves as a negative regulator of skeletal mechanotransduction to suppress load-induced bone formation by altering the sensitivity of osteoblasts and osteocytes to mechanical signals. IntroductionThe ability of bone to serve as an effective weight-bearing structure depends upon its capacity to adapt to its functional environment. As such, skeletal mass is continually added and removed according to physical demands. When mechanical loads exceed those associated with habitual use, new bone is formed via an osteogenic response that is characterized by increased osteoblast proliferation, enhanced osteogenic gene expression, and decreased osteoclast activity (1Hillam R.A. Skerry T.M. J. Bone Miner. Res. 1995; 10: 683-689Crossref PubMed Scopus (153) Google Scholar, 2Pead M.J. Skerry T.M. Lanyon L.E. J. Bone Miner. Res. 1988; 3: 647-656Crossref PubMed Scopus (194) Google Scholar). Conversely, when routine loads are removed or reduced, osteoblastic activity is suppressed and bone loss ensues (3Morey E.R. Baylink D.J. Science. 1978; 201: 1138-1141Crossref PubMed Scopus (417) Google Scholar). In this way, skeletal architecture can be optimized to prevent fracture and minimized to limit the energy expenditure necessary for maintenance.Previous studies using both in vivo and in vitro models of mechanical loading have begun to identify anabolic signaling mechanisms associated with load-induced bone formation. These mechanisms include the rapid release of autocrine/paracrine factors such as ATP, prostaglandin, and nitric oxide (4Bakker A.D. Soejima K. Klein-Nulend J. Burger E.H. J. Biomech. 2001; 34: 671-677Crossref PubMed Scopus (251) Google Scholar, 5Genetos D.C. Geist D.J. Liu D. Donahue H.J. Duncan R.L. J. Bone Miner. Res. 2005; 20: 41-49Crossref PubMed Google Scholar, 6Pead M.J. Lanyon L.E. Calcif. Tissue Int. 1989; 45: 34-40Crossref PubMed Scopus (124) Google Scholar) and the activation of intracellular calcium and kinase signaling pathways (7Riddle R.C. Taylor A.F. Genetos D.C. Donahue H.J. Am. J. Physiol. Cell Physiol. 2006; 290: C776-C784Crossref PubMed Scopus (171) Google Scholar, 8You J. Reilly G.C. Zhen X. Yellowley C.E. Chen Q. Donahue H.J. Jacobs C.R. J. Biol. Chem. 2001; 276: 13365-13371Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). A growing body of work indicates that Wnt signaling is an important regulator of skeletal response to mechanical loading (9Sawakami K. Robling A.G. Ai M. Pitner N.D. Liu D. Warden S.J. Li J. Maye P. Rowe D.W. Duncan R.L. Warman M.L. Turner C.H. J. Biol. Chem. 2006; 281: 23698-23711Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 10Case N. Ma M. Sen B. Xie Z. Gross T.S. Rubin J. J. Biol. Chem. 2008; 283: 29196-29205Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Experimental loading regulates the expression of several Wnt ligands, receptors, and antagonists, which in turn increases β-catenin nuclear translocation (10Case N. Ma M. Sen B. Xie Z. Gross T.S. Rubin J. J. Biol. Chem. 2008; 283: 29196-29205Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 11Santos A. Bakker A.D. Zandieh-Doulabi B. de Blieck-Hogervorst J.M. Klein-Nulend J. Biochem. Biophys. Res. Commun. 2010; 391: 364-369Crossref PubMed Scopus (88) Google Scholar, 12Yang Z. Bidwell J.P. Young S.R. Gerard-O'Riley R. Wang H. Pavalko F.M. J. Cell Physiol. 2010; 223: 435-441PubMed Google Scholar) and the expression of its target genes (13Xia X. Batra N. Shi Q. Bonewald L.F. Sprague E. Jiang J.X. Mol. Cell. Biol. 2010; 30: 206-219Crossref PubMed Scopus (111) Google Scholar, 14Armstrong V.J. Muzylak M. Sunters A. Zaman G. Saxon L.K. Price J.S. Lanyon L.E. J. Biol. Chem. 2007; 282: 20715-20727Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 15Robling A.G. Niziolek P.J. Baldridge L.A. Condon K.W. Allen M.R. Alam I. Mantila S.M. Gluhak-Heinrich J. Bellido T.M. Harris S.E. Turner C.H. J. Biol. Chem. 2008; 283: 5866-5875Abstract Full Text Full Text PDF PubMed Scopus (993) Google Scholar).Other studies suggest the existence of negative feedback mechanisms that operate to modulate skeletal mechanotransduction. For example, during prolonged exposure to fluid flow, osteoblasts exhibit refractory periods in which the activation of anabolic signaling mechanisms is impaired (16Donahue S.W. Donahue H.J. Jacobs C.R. J. Biomech. 2003; 36: 35-43Crossref PubMed Scopus (90) Google Scholar, 17Hung C.T. Pollack S.R. Reilly T.M. Brighton C.T. Clin. Orthop. Relat. Res. 1995; : 256-269PubMed Google Scholar). Additionally, intermittent intervals of loading interspersed with periods of rest produce more bone than sustained loading, suggesting the engagement of mechanisms that desensitize bone cells to mechanical signals (18Srinivasan S. Ausk B.J. Poliachik S.L. Warner S.E. Richardson T.S. Gross T.S. J. Appl. Physiol. 2007; 102: 1945-1952Crossref PubMed Scopus (77) Google Scholar, 19Robling A.G. Burr D.B. Turner C.H. J. Exp. Biol. 2001; 204: 3389-3399Crossref PubMed Google Scholar, 20Srinivasan S. Agans S.C. King K.A. Moy N.Y. Poliachik S.L. Gross T.S. Bone. 2003; 33: 946-955Crossref PubMed Scopus (97) Google Scholar). As described below, previous studies from a number of laboratories suggested that the transcription factor, Hif-1α, 3The abbreviations used are: Hifhypoxia-inducible factorVhlVon Hippel-LindaumTORmammalian target of rapamycinCTcomputer tomographymicroCTmicro-computer tomographyPGE2prostaglandin E2. might function as a regulator of bone cell sensitivity to mechanical stimuli.Hif-1, a transcription factor originally identified as a regulator of the cellular response to molecular oxygen levels (21Semenza G.L. Genes Dev. 2000; 14: 1983-1991PubMed Google Scholar, 22Semenza G.L. Cell. 2001; 107: 1-3Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar), activates angiogenic and glycolytic gene programs required for cells to adapt to hypoxia. The cellular levels of Hif-1 and its nuclear translocation are governed by regulated proteolysis. The α-subunit of the molecule contains an oxygen-dependent degradation domain that is subject to prolyl hydroxylation and subsequent E3 ubiquitin ligation by the tumor suppressor protein von Hippel-Lindau (Vhl) and proteasomal degradation. Inhibition of prolyl hydroxylation under hypoxic conditions allows Hif-1α to accumulate and translocate to the nucleus, where it forms a dimer with the Hif-1β subunit (21Semenza G.L. Genes Dev. 2000; 14: 1983-1991PubMed Google Scholar, 22Semenza G.L. Cell. 2001; 107: 1-3Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar).It is increasingly recognized that the Hif-1 pathway also regulates cellular functions independent of those related to hypoxia. A number of growth factors (21Semenza G.L. Genes Dev. 2000; 14: 1983-1991PubMed Google Scholar, 22Semenza G.L. Cell. 2001; 107: 1-3Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar) and paracrine factors (23Treins C. Giorgetti-Peraldi S. Murdaca J. Semenza G.L. Van Obberghen E. J. Biol. Chem. 2002; 277: 27975-27981Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 24Shi Y.H. Wang Y.X. Bingle L. Gong L.H. Heng W.J. Li Y. Fang W.G. J. Pathol. 2005; 205: 530-536Crossref PubMed Scopus (86) Google Scholar, 25Phillips R.J. Mestas J. Gharaee-Kermani M. Burdick M.D. Sica A. Belperio J.A. Keane M.P. Strieter R.M. J. Biol. Chem. 2005; 280: 22473-22481Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 26Liu X.H. Kirschenbaum A. Lu M. Yao S. Dosoretz A. Holland J.F. Levine A.C. J. Biol. Chem. 2002; 277: 50081-50086Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) activate the Hif-1 pathway in several cell types under normoxia. In osteoblasts, insulin-like growth factor-1 stabilizes Hifα via a mechanism that involves PI3-kinase/Akt signaling (27Akeno N. Robins J. Zhang M. Czyzyk-Krzeska M.F. Clemens T.L. Endocrinology. 2002; 143: 420-425Crossref PubMed Scopus (99) Google Scholar), and the expression of Hif-1α is necessary for normal osteoblast proliferation (28Shomento S.H. Wan C. Cao X. Faugere M.C. Bouxsein M.L. Clemens T.L. Riddle R.C. J. Cell Biochem. 2010; 109: 196-204Crossref PubMed Scopus (82) Google Scholar). Additionally, mechanical signals prevent the degradation of Hif α-subunits in both skeletal and cardiac muscle (29Milkiewicz M. Doyle J.L. Fudalewski T. Ispanovic E. Aghasi M. Haas T.L. J. Physiol. 2007; 583: 753-766Crossref PubMed Scopus (82) Google Scholar, 30Kim C.H. Cho Y.S. Chun Y.S. Park J.W. Kim M.S. Circ. Res. 2002; 90: E25-E33Crossref PubMed Google Scholar). In response to these stimuli, Hif-1 activates angiogenic and metabolic responses that are required for the anabolic response.In the course of analyzing the role of Hif-1α in skeletal development, we created mice that lacked this transcription factor in osteoblasts and osteocytes (31Wang Y. Wan C. Deng L. Liu X. Cao X. Gilbert S.R. Bouxsein M.L. Faugere M.C. Guldberg R.E. Gerstenfeld L.C. Haase V.H. Johnson R.S. Schipani E. Clemens T.L. J. Clin. Invest. 2007; 117: 1616-1626Crossref PubMed Scopus (547) Google Scholar). The immature Hif-1α mutants had deficits in cortical and trabecular bone, which we attributed to impaired development of the skeletal vasculature (31Wang Y. Wan C. Deng L. Liu X. Cao X. Gilbert S.R. Bouxsein M.L. Faugere M.C. Guldberg R.E. Gerstenfeld L.C. Haase V.H. Johnson R.S. Schipani E. Clemens T.L. J. Clin. Invest. 2007; 117: 1616-1626Crossref PubMed Scopus (547) Google Scholar). Surprisingly, as these mice matured, they acquired more cortical bone when compared with controls, suggesting that the loss of Hif-1α caused an enhanced adaptive response in cortical bone architecture. In this study, we show that in vitro fluid flow markedly up-regulates Hif-1α, which partners with β-catenin to inhibit Wnt target genes associated with bone anabolic activity. Consequently, removal of Hif-1α from osteoblasts sensitizes bone to load-induced bone formation in vivo by enhancing the response of osteoblasts to mechanical stimuli. Our findings indicate that Hif-1α functions as a negative regulator of skeletal mechanotransduction to suppress load-induced bone formation.DISCUSSIONIn this study, we show that activation of the transcription factor Hif-1α is a primary response to mechanical loading of bone and that it appears to function in the osteoblast as a negative regulator of load-induced bone formation. Our studies were prompted by observations in mice lacking Hif-1α specifically in osteoblasts and osteocytes, which exhibited dramatic shifts in cortical bone architecture during postnatal development. Thus, deficits in cortical bone seen in immature ΔHif-1α mice, likely due in part to impaired development of the skeletal vasculature (31Wang Y. Wan C. Deng L. Liu X. Cao X. Gilbert S.R. Bouxsein M.L. Faugere M.C. Guldberg R.E. Gerstenfeld L.C. Haase V.H. Johnson R.S. Schipani E. Clemens T.L. J. Clin. Invest. 2007; 117: 1616-1626Crossref PubMed Scopus (547) Google Scholar), were reversed such that the mature mutants had more cortical bone when compared with controls. We hypothesized that this developmental switch in cortical bone architecture might represent enhanced adaptation to mechanical loading associated with increased ambulation during postnatal life. The studies described here were designed to explore the possible role of Hif-1α in this phenomenon.The contrasting skeletal phenotypes observed as the ΔHif-1α mice mature suggest that Hif-1 exerts distinct functions at different times during skeletal development. During early development, the accumulation of both trabecular and cortical bone in the axial skeleton of ΔHif-1α mice is reduced, likely due to a decrease in vascularization critical for the initial specification and differentiation of bone forming osteoblasts. As the mouse matures and increases its ambulatory activity, it is possible that Hif-1α-generated signals impinge on other cellular functions including those involved in adaptation of bone to mechanical loads. In this regard, Hif-1α is known to be regulated by mechanical events in other tissues. For example, loading of the rat extensor digitorum longus muscle induces the accumulation of Hif α-subunits (29Milkiewicz M. Doyle J.L. Fudalewski T. Ispanovic E. Aghasi M. Haas T.L. J. Physiol. 2007; 583: 753-766Crossref PubMed Scopus (82) Google Scholar). Likewise, hemodynamic loading or stretching of cardiac tissue by expanding an intraventricular balloon stimulated Hif-1α accumulation in the cardiac muscle (30Kim C.H. Cho Y.S. Chun Y.S. Park J.W. Kim M.S. Circ. Res. 2002; 90: E25-E33Crossref PubMed Google Scholar). In this context, Hif-1α-generated signals likely participate in mechanisms that enable muscle to adapt to increased mechanical forces as well as the heightened demand for oxygen delivery evidenced by up-regulation of angiogenic gene expression in the stretched myocardium. This situation differs from the exogenous mechanical stimuli implemented in the current study, which are not associated with significant hypoxia or increased angiogenesis (45Piekarski K. Munro M. Nature. 1977; 269: 80-82Crossref PubMed Scopus (382) Google Scholar), but rather, are considered anabolic. On the other hand, application of much more intensive loads that induce bone fatigue with significant tissue damage is associated with increased skeletal vascularity likely due to tissue hypoxia (46McKenzie J.A. Silva M.J. Bone. 2011; 48: 250-258Crossref PubMed Scopus (53) Google Scholar, 47Matsuzaki H. Wohl G.R. Novack D.V. Lynch J.A. Silva M.J. Calcif. Tissue Int. 2007; 80: 391-399Crossref PubMed Scopus (37) Google Scholar).An important conceptual conclusion from our studies is that Hif-1α normally functions to attenuate anabolic responses to exogenous loads. As mentioned above, intermittent application of mechanical loading with interspersed periods of rest is a more effective regimen for increasing bone formation than uninterrupted sustained loading (18Srinivasan S. Ausk B.J. Poliachik S.L. Warner S.E. Richardson T.S. Gross T.S. J. Appl. Physiol. 2007; 102: 1945-1952Crossref PubMed Scopus (77) Google Scholar, 19Robling A.G. Burr D.B. Turner C.H. J. Exp. Biol. 2001; 204: 3389-3399Crossref PubMed Google Scholar, 20Srinivasan S. Agans S.C. King K.A. Moy N.Y. Poliachik S.L. Gross T.S. Bone. 2003; 33: 946-955Crossref PubMed Scopus (97) Google Scholar). Unregulated anabolic signaling in loaded osteoblasts would increase oxidative, metabolic, and genetic stress and ultimately limit their performance. Interestingly, reactive oxygen species, which are generated by mechanical loading in other tissues (48Powers S.K. Jackson M.J. Physiol. Rev. 2008; 88: 1243-1276Crossref PubMed Scopus (1577) Google Scholar), stabilize Hif α-subunits (49Simon M.C. Adv. Exp. Med. Biol. 2006; 588: 165-170Crossref PubMed Scopus (81) Google Scholar, 50Brunelle J.K. Bell E.L. Quesada N.M. Vercauteren K. Tiranti V. Zeviani M. Scarpulla R.C. Chandel N.S. Cell Metab. 2005; 1: 409-414Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar), and in turn, Hif-1-generated signals inhibit reactive oxygen species production (51Kim J.W. Tchernyshyov I. Semenza G.L. Dang C.V. Cell Metab. 2006; 3: 177-185Abstract Full Text Full Text PDF PubMed Scopus (2515) Google Scholar, 52Papandreou I. Cairns R.A. Fontana L. Lim A.L. Denko N.C. Cell Metab. 2006; 3: 187-197Abstract Full Text Full Text PDF PubMed Scopus (1576) Google Scholar). These observations suggest that Hif-1α may function transiently as a negative regulator of load-induced bone formation to ensure periods of quiescence during which time cells can recover from the potentially damaging effects of cellular stressors.Several lines of evidence support the conclusion that Hif-1α attenuates load-induced bone formation by interfering with β-catenin, a critical regulator of osteoblast specification and function (53Holmen S.L. Zylstra C.R. Mukherjee A. Sigler R.E. Faugere M.C. Bouxsein M.L. Deng L. Clemens T.L. Williams B.O. J. Biol. Chem. 2005; 280: 21162-21168Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 54Hill T.P. Später D. Taketo M.M. Birchmeier W. Hartmann C. Dev. Cell. 2005; 8: 727-738Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar). First, Hif-1α directly interacted with β-catenin. Second, osteoblasts deficient in Hif-1α exhibited enhanced activation of a β-catenin reporter gene, enhanced target gene expression, and increased association with Tcf4 after exposure to fluid flow. Conversely, osteoblasts overexpressing Hif-1α had diminished measures of β-catenin activity. Moreover, previous work in other cell types support our findings. For example, inhibition of Wnt/β-catenin signaling has been reported in colon cancer and non-small lung cancer cell lines exposed to hypoxia (55Kaidi A. Williams A.C. Paraskeva C. Nat. Cell Biol. 2007; 9: 210-217Crossref PubMed Scopus (375) Google Scholar, 56Lim J.H. Chun Y.S. Park J.W. Cancer Res. 2008; 68: 5177-5184Crossref PubMed Scopus (78) Google Scholar). In these studies, Hif-1α interacted with β-catenin via its NH2 terminus and thereby inhibited the interaction of β-catenin with TCF4. However, the regulation of Wnt/β-catenin signaling may be cell- and differentiation stage-specific as Hif-1 was recently reported to enhance canonical Wnt signaling in embryonic stem cells but to have no effect on the pathway in neuroprogenitor cells (57Mazumdar J. O'Brien W.T. Johnson R.S. LaManna J.C. Chavez J.C. Klein P.S. Simon M.C. Nat. Cell Biol. 2010; 12: 1007-1013Crossref PubMed Scopus (366) Google Scholar). Finally, it should be noted that other factors including FoxO1 (58Almeida M. Han L. Martin-Millan M. O'Brien C.A. Manolagas S.C. J. Biol. Chem. 2007; 282: 27298-27305Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar) and NMP4-CIZ (12Yang Z. Bidwell J.P. Young S.R. Gerard-O'Riley R. Wang H. Pavalko F.M. J. Cell Physiol. 2010; 223: 435-441PubMed Google Scholar) have also been shown to modulate β-catenin activity in osteoblasts, with the actions of the latter being responsive to experimental loading. Whether or to what extent these factors cooperate with Hif-1α to regulate mechanical responses to loading in bone remains to be determined but fully supports the importance of controlling β-catenin activity.In summary, we have identified Hif-1α as a negative regulator of load-induced bone formation. Our results suggest a model in which loading activates the canonical Wnt/β-catenin pathway, which induces bone anabolic pathways in osteoblasts. Concomitant up-regulation of Hif-1α attenuates the magnitude of Wnt/β-catenin activity by sequestration of β-catenin, thereby enabling periods of quiescence during which time cells can recover from the potentially damaging effects of cellular stressors. Our findings advance the understanding of the mechanisms by which bone cells perceive and respond to changes in their mechanical environment and may lead to the development of strategies designed to increase bone mass via an anabolic response. IntroductionThe ability of bone to serve as an effective weight-bearing structure depends upon its capacity to adapt to its functional environment. As such, skeletal mass is continually added and removed according to physical demands. When mechanical loads exceed those associated with habitual use, new bone is formed via an osteogenic response that is characterized by increased osteoblast proliferation, enhanced osteogenic gene expression, and decreased osteoclast activity (1Hillam R.A. Skerry T.M. J. Bone Miner. Res. 1995; 10: 683-689Crossref PubMed Scopus (153) Google Scholar, 2Pead M.J. Skerry T.M. Lanyon L.E. J. Bone Miner. Res. 1988; 3: 647-656Crossref PubMed Scopus (194) Google Scholar). Conversely, when routine loads are removed or reduced, osteoblastic activity is suppressed and bone loss ensues (3Morey E.R. Baylink D.J. Science. 1978; 201: 1138-1141Crossref PubMed Scopus (417) Google Scholar). In this way, skeletal architecture can be optimized to prevent fracture and minimized to limit the energy expenditure necessary for maintenance.Previous studies using both in vivo and in vitro models of mechanical loading have begun to identify anabolic signaling mechanisms associated with load-induced bone formation. These mechanisms include the rapid release of autocrine/paracrine factors such as ATP, prostaglandin, and nitric oxide (4Bakker A.D. Soejima K. Klein-Nulend J. Burger E.H. J. Biomech. 2001; 34: 671-677Crossref PubMed Scopus (251) Google Scholar, 5Genetos D.C. Geist D.J. Liu D. Donahue H.J. Duncan R.L. J. Bone Miner. Res. 2005; 20: 41-49Crossref PubMed Google Scholar, 6Pead M.J. Lanyon L.E. Calcif. Tissue Int. 1989; 45: 34-40Crossref PubMed Scopus (124) Google Scholar) and the activation of intracellular calcium and kinase signaling pathways (7Riddle R.C. Taylor A.F. Genetos D.C. Donahue H.J. Am. J. Physiol. Cell Physiol. 2006; 290: C776-C784Crossref PubMed Scopus (171) Google Scholar, 8You J. Reilly G.C. Zhen X. Yellowley C.E. Chen Q. Donahue H.J. Jacobs C.R. J. Biol. Chem. 2001; 276: 13365-13371Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). A growing body of work indicates that Wnt signaling is an important regulator of skeletal response to mechanical loading (9Sawakami K. Robling A.G. Ai M. Pitner N.D. Liu D. Warden S.J. Li J. Maye P. Rowe D.W. Duncan R.L. Warman M.L. Turner C.H. J. Biol. Chem. 2006; 281: 23698-23711Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 10Case N. Ma M. Sen B. Xie Z. Gross T.S. Rubin J. J. Biol. Chem. 2008; 283: 29196-29205Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Experimental loading regulates the expression of several Wnt ligands, receptors, and antagonists, which in turn increases β-catenin nuclear translocation (10Case N. Ma M. Sen B. Xie Z. Gross T.S. Rubin J. J. Biol. Chem. 2008; 283: 29196-29205Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 11Santos A. Bakker A.D. Zandieh-Doulabi B. de Blieck-Hogervorst J.M. Klein-Nulend J. Biochem. Biophys. Res. Commun. 2010; 391: 364-369Crossref PubMed Scopus (88) Google Scholar, 12Yang Z. Bidwell J.P. Young S.R. Gerard-O'Riley R. Wang H. Pavalko F.M. J. Cell Physiol. 2010; 223: 435-441PubMed Google Scholar) and the expression of its target genes (13Xia X. Batra N. Shi Q. Bonewald L.F. Sprague E. Jiang J.X. Mol. Cell. Biol. 2010; 30: 206-219Crossref PubMed Scopus (111) Google Scholar, 14Armstrong V.J. Muzylak M. Sunters A. Zaman G. Saxon L.K. Price J.S. Lanyon L.E. J. Biol. Chem. 2007; 282: 20715-20727Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 15Robling A.G. Niziolek P.J. Baldridge L.A. Condon K.W. Allen M.R. Alam I. Mantila S.M. Gluhak-Heinrich J. Bellido T.M. Harris S.E. Turner C.H. J. Biol. Chem. 2008; 283: 5866-5875Abstract Full Text Full Text PDF PubMed Scopus (993) Google Scholar).Other studies suggest the existence of negative feedback mechanisms that operate to modulate skeletal mechanotransduction. For example, during prolonged exposure to fluid flow, osteoblasts exhibit refractory periods in which the activation of anabolic signaling mechanisms is impaired (16Donahue S.W. Donahue H.J. Jacobs C.R. J. Biomech. 2003; 36: 35-43Crossref PubMed Scopus (90) Google Scholar, 17Hung C.T. Pollack S.R. Reilly T.M. Brighton C.T. Clin. Orthop. Relat. Res. 1995; : 256-269PubMed Google Scholar). Additionally, intermittent intervals of loading interspersed with periods of rest produce more bone than sustained loading, suggesting the engagement of mechanisms that desensitize bone cells to mechanical signals (18Srinivasan S. Ausk B.J. Poliachik S.L. Warner S.E. Richardson T.S. Gross T.S. J. Appl. Physiol. 2007; 102: 1945-1952Crossref PubMed Scopus (77) Google Scholar, 19Robling A.G. Burr D.B. Turner C.H. J. Exp. Biol. 2001; 204: 3389-3399Crossref PubMed Google Scholar, 20Srinivasan S. Agans S.C. King K.A. Moy N.Y. Poliachik S.L. Gross T.S. Bone. 2003; 33: 946-955Crossref PubMed Scopus (97) Google Scholar). As described below, previous studies from a number of laboratories suggested that the transcription factor, Hif-1α, 3The abbreviations used are: Hifhypoxia-inducible factorVhlVon Hippel-LindaumTORmammalian target of rapamycinCTcomputer tomographymicroCTmicro-computer tomographyPGE2prostaglandin E2. might function as a regulator of bone cell sensitivity to mechanical stimuli.Hif-1, a transcription factor originally identified as a regulator of the cellular response to molecular oxygen levels (21Semenza G.L. Genes Dev. 2000; 14: 1983-1991PubMed Google Scholar, 22Semenza G.L. Cell. 2001; 107: 1-3Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar), activates angiogenic and glycolytic gene programs required for cells to adapt to hypoxia. The cellular levels of Hif-1 and its nuclear translocation are governed by regulated proteolysis. The α-subunit of the molecule contains an oxygen-dependent degradation domain that is subject to prolyl hydroxylation and subsequent E3 ubiquitin ligation by the tumor suppressor protein von Hippel-Lindau (Vhl) and proteasomal degradation. Inhibition of prolyl hydroxylation under hypoxic conditions allows Hif-1α to accumulate and translocate to the nucleus, where it forms a dimer with the Hif-1β subunit (21Semenza G.L. Genes Dev. 2000; 14: 1983-1991PubMed Google Scholar, 22Semenza G.L. Cell. 2001; 107: 1-3Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar).It is increasingly recognized that the Hif-1 pathway also regulates cellular functions independent of those related to hypoxia. A number of growth factors (21Semenza G.L. Genes Dev. 2000; 14: 1983-1991PubMed Google Scholar, 22Semenza G.L. Cell. 2001; 107: 1-3Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar) and paracrine factors (23Treins C. Giorgetti-Peraldi S. Murdaca J. Semenza G.L. Van Obberghen E. J. Biol. Chem. 2002; 277: 27975-27981Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 24Shi Y.H. Wang Y.X. Bingle L. Gong L.H. Heng W.J. Li Y. Fang W.G. J. Pathol. 2005; 205: 530-536Crossref PubMed Scopus (86) Google Scholar, 25Phillips R.J. Mestas J. Gharaee-Kermani M. Burdick M.D. Sica A. Belperio J.A. Keane M.P. Strieter R.M. J. Biol. Chem. 2005; 280: 22473-22481Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 26Liu X.H. Kirschenbaum A. Lu M. Yao S. Dosoretz A. Holland J.F. Levine A.C. J. Biol. Chem. 2002; 277: 50081-50086Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) activate the Hif-1 pathway in several cell types under normoxia. In osteoblasts, insulin-like growth factor-1 stabilizes Hifα via a mechanism that involves PI3-kinase/Akt signaling (27Akeno N. Robins J. Zhang M. Czyzyk-Krzeska M.F. Clemens T.L. Endocrinology. 2002; 143: 420-425Crossref PubMed Scopus (99) Google Scholar), and the expression of Hif-1α is necessary for normal osteoblast proliferation (28Shomento S.H. Wan C. Cao X. Faugere M.C. Bouxsein M.L. Clemens T.L. Riddle R.C. J. Cell Biochem. 2010; 109: 196-204Crossref PubMed Scopus (82) Google Scholar). Additionally, mechanical signals prevent the degradation of Hif α-subunits in both skeletal and cardiac muscle (29Milkiewicz M. Doyle J.L. Fudalewski T. Ispanovic E. Aghasi M. Haas T.L. J. Physiol. 2007; 583: 753-766Crossref PubMed Scopus (82) Google Scholar, 30Kim C.H. Cho Y.S. Chun Y.S. Park J.W. Kim M.S. Circ. Res. 2002; 90: E25-E33Crossref PubMed Google Scholar). In response to these stimuli, Hif-1 activates angiogenic and metabolic responses that are required for the anabolic response.In the course of analyzing the role of Hif-1α in skeletal development, we created mice that lacked this transcription factor in osteoblasts and osteocytes (31Wang Y. Wan C. Deng L. Liu X. Cao X. Gilbert S.R. Bouxsein M.L. Faugere M.C. Guldberg R.E. Gerstenfeld L.C. Haase V.H. Johnson R.S. Schipani E. Clemens T.L. J. Clin. Invest. 2007; 117: 1616-1626Crossref PubMed Scopus (547) Google Scholar). The immature Hif-1α mutants had deficits in cortical and trabecular bone, which we attributed to impaired development of the skeletal vasculature (31Wang Y. Wan C. Deng L. Liu X. Cao X. Gilbert S.R. Bouxsein M.L. Faugere M.C. Guldberg R.E. Gerstenfeld L.C. Haase V.H. Johnson R.S. Schipani E. Clemens T.L. J. Clin. Invest. 2007; 117: 1616-1626Crossref PubMed Scopus (547) Google Scholar). Surprisingly, as these mice matured, they acquired more cortical bone when compared with controls, suggesting that the loss of Hif-1α caused an enhanced adaptive response in cortical bone architecture. In this study, we show that in vitro fluid flow markedly up-regulates Hif-1α, which partners with β-catenin to inhibit Wnt target genes associated with bone anabolic activity. Consequently, removal of Hif-1α from osteoblasts sensitizes bone to load-induced bone formation in vivo by enhancing the response of osteoblasts to mechanical stimuli. Our findings indicate that Hif-1α functions as a negative regulator of skeletal mechanotransduction to suppress load-induced bone formation.
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