Transient Changes in Oxygen Tension Inhibit Osteogenic Differentiation and Runx2 Expression in Osteoblasts

Abstract

Vascular disruption following bony injury results in a hypoxic gradient within the wound microenvironment. Nevertheless, the effects of low oxygen tension on osteogenic precursors remain to be fully elucidated. In the present study, we investigated in vitro osteoblast and mesenchymal stem cell differentiation following exposure to 21% O2 (ambient oxygen), 2% O2 (hypoxia), and <0.02% O2 (anoxia). Hypoxia had little effect on osteogenic differentiation. In contrast, short-term anoxic treatment of primary osteoblasts and mesenchymal precursors inhibited in vitro bone nodule formation and extracellular calcium deposition. Cell viability assays revealed that this effect was not caused by immediate or delayed cell death. Microarray profiling implicated down-regulation of the key osteogenic transcription factor Runx2 as a potential mechanism for the anoxic inhibition of differentiation. Subsequent analysis revealed not only a short-term differential regulation of Runx2 and its targets by anoxia and hypoxia, but a long-term inhibition of Runx2 transcriptional and protein levels after only 12-24 h of anoxic insult. Furthermore, we present evidence that Runx2 inhibition may, at least in part, be because of anoxic repression of BMP2, and that restoring Runx2 levels during anoxia by pretreatment with recombinant BMP2 rescued the anoxic inhibition of differentiation. Taken together, our findings indicate that brief exposure to anoxia (but not 2% hypoxia) down-regulated BMP2 and Runx2 expression, thus inhibiting critical steps in the osteogenic differentiation of pluripotent mesenchymal precursors and committed osteoblasts. Vascular disruption following bony injury results in a hypoxic gradient within the wound microenvironment. Nevertheless, the effects of low oxygen tension on osteogenic precursors remain to be fully elucidated. In the present study, we investigated in vitro osteoblast and mesenchymal stem cell differentiation following exposure to 21% O2 (ambient oxygen), 2% O2 (hypoxia), and <0.02% O2 (anoxia). Hypoxia had little effect on osteogenic differentiation. In contrast, short-term anoxic treatment of primary osteoblasts and mesenchymal precursors inhibited in vitro bone nodule formation and extracellular calcium deposition. Cell viability assays revealed that this effect was not caused by immediate or delayed cell death. Microarray profiling implicated down-regulation of the key osteogenic transcription factor Runx2 as a potential mechanism for the anoxic inhibition of differentiation. Subsequent analysis revealed not only a short-term differential regulation of Runx2 and its targets by anoxia and hypoxia, but a long-term inhibition of Runx2 transcriptional and protein levels after only 12-24 h of anoxic insult. Furthermore, we present evidence that Runx2 inhibition may, at least in part, be because of anoxic repression of BMP2, and that restoring Runx2 levels during anoxia by pretreatment with recombinant BMP2 rescued the anoxic inhibition of differentiation. Taken together, our findings indicate that brief exposure to anoxia (but not 2% hypoxia) down-regulated BMP2 and Runx2 expression, thus inhibiting critical steps in the osteogenic differentiation of pluripotent mesenchymal precursors and committed osteoblasts. It has long been known that hypoxia is a prominent component of the microenvironment in both bony and soft tissue injury (1Heppenstall R.B. Grislis G. Hunt T.K. Clin. Orthop. 1975; 106: 357-365Crossref PubMed Scopus (85) Google Scholar, 2Knighton D.R. Silver I.A. Hunt T.K. Surgery. 1981; 90: 262-270PubMed Google Scholar, 3Brighton C.T. Ray R.D. Soble L.W. Kuettner K.E. J. Bone Joint Surg. Am. 1969; 51: 1383-1396Crossref PubMed Scopus (64) Google Scholar, 4Brighton C.T. Krebs A.G. J. Bone Joint Surg. Am. 1972; 54: 323-332Crossref PubMed Scopus (145) Google Scholar). Interruption of vascular flow with fracture or surgical osteotomy results in a transient hypoxic gradient within the wound, with oxygen tension falling to 0-2% in the central wound region (1Heppenstall R.B. Grislis G. Hunt T.K. Clin. Orthop. 1975; 106: 357-365Crossref PubMed Scopus (85) Google Scholar, 2Knighton D.R. Silver I.A. Hunt T.K. Surgery. 1981; 90: 262-270PubMed Google Scholar, 3Brighton C.T. Ray R.D. Soble L.W. Kuettner K.E. J. Bone Joint Surg. Am. 1969; 51: 1383-1396Crossref PubMed Scopus (64) Google Scholar, 4Brighton C.T. Krebs A.G. J. Bone Joint Surg. Am. 1972; 54: 323-332Crossref PubMed Scopus (145) Google Scholar, 5Grundnes O. Reikeras O. Acta Orthop. Scand. 1992; 63: 487-491Crossref PubMed Scopus (72) Google Scholar, 6Gordillo G.M. Sen C.K. Am. J. Surg. 2003; 186: 259-263Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Thus, the complex osteogenic regenerative response that ensues following injury begins in the setting of reduced oxygen (7Heppenstall R.B. Goodwin C.W. Brighton C.T. J. Bone Joint Surg. Am. 1976; 58: 1153-1156Crossref PubMed Scopus (40) Google Scholar). In addition to inflammatory cell recruitment, matrix processing, and angiogenesis, the activity of mesenchymal osteoblast precursors is a key component of this cascade (6Gordillo G.M. Sen C.K. Am. J. Surg. 2003; 186: 259-263Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 8Knighton D.R. Hunt T.K. Scheuenstuhl H. Halliday B.J. Werb Z. Banda M.J. Science. 1983; 221: 1283-1285Crossref PubMed Scopus (533) Google Scholar). Both committed resident osteoblasts and recruited precursor cells are exposed to the hypoxic post-injury milieu. Given that normal osteoblast function is critical to the deposition of mineralized matrix, the hallmark of successful fracture repair, it is important to understand the response of osteoprogenitors to a hypoxic microenvironment. Recent investigations have yielded significant insights into the transcriptional regulation of osteogenic differentiation (9Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3668) Google Scholar, 10Ducy P. Starbuck M. Priemel M. Shen J. Pinero G. Geoffroy V. Amling M. Karsenty G. Genes Dev. 1999; 13: 1025-1036Crossref PubMed Scopus (711) Google Scholar, 11Karsenty G. Ducy P. Starbuck M. Priemel M. Shen J. Geoffroy V. Amling M. Bone. 1999; 25: 107-108Crossref PubMed Scopus (119) Google Scholar). Primary cell culture models and established cell lines that recapitulate osteoblast maturation in vitro have allowed the identification of closely regulated transcription factors (e.g. Runx2, Osx, and members of the Ets family) and osteogenic markers (e.g. collagens, alkaline phosphatase, and osteocalcin) specific to each stage of the osteoblast life cycle (12Karsenty G. Endocrinology. 2001; 142: 2731-2733Crossref PubMed Scopus (169) Google Scholar). Runx2, a member of the runt family of transcription factors, is both necessary and sufficient for osteoblast differentiation (9Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3668) Google Scholar, 13Ducy P. Dev. Dyn. 2000; 219: 461-471Crossref PubMed Scopus (398) Google Scholar). Runx2 activity promotes the expression of a number of osteogenic markers including collagen I, osteopontin, and osteocalcin by binding to responsive elements within the promoters of these genes (11Karsenty G. Ducy P. Starbuck M. Priemel M. Shen J. Geoffroy V. Amling M. Bone. 1999; 25: 107-108Crossref PubMed Scopus (119) Google Scholar, 12Karsenty G. Endocrinology. 2001; 142: 2731-2733Crossref PubMed Scopus (169) Google Scholar). Its clinical significance is highlighted by the observation that humans or mice heterozygous for Runx2 mutations develop cleidocranial dysplasia, whereas postnatal impairment of Runx2 results in osteopenia (10Ducy P. Starbuck M. Priemel M. Shen J. Pinero G. Geoffroy V. Amling M. Karsenty G. Genes Dev. 1999; 13: 1025-1036Crossref PubMed Scopus (711) Google Scholar). The effect of reduced oxygen on genes involved in osteoblast proliferation and differentiation in because of the results for primary established cell lines and for oxygen of the cell exposed to O2 a in both cell proliferation and alkaline expression, to of osteoblast to cells at 21% O2 M. PubMed Scopus Google Scholar). investigations in primary and that hypoxic inhibition of alkaline was not to the but was to primary cells as Am. J. Orthop. Full Text Full Text PDF PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar). in to the effect in in proliferation was in both primary cell to a hypoxic effect on proliferation and differentiation in primary osteoblasts. a cell exposed to 2% O2 for to h was to levels of the transcription factor and the bone osteocalcin Cell. PubMed Scopus (85) Google Scholar). this on cells important into the hypoxic regulation of osteogenic expression, a of the hypoxic osteoblast transcriptional response to be elucidated. Furthermore, it be to cell to primary osteoblasts that Runx2 expression be osteogenic potential in cell lines as H. J. Cell 1983; PubMed Scopus Google Scholar, G. J. Bone 1999; PubMed Scopus Google Scholar). Nevertheless, evidence is that hypoxia factors implicated in the post-injury microenvironment. involved in are osteoblasts expression of vascular vascular mesenchymal stem the and specific of the B.J. Am. J. 2000; PubMed Google Scholar). to osteoblast function by with and during bone formation J. Werb Z. 1999; PubMed Scopus Google Scholar). of osteoblast function that as members of the and factor J. PubMed Scopus (169) Google Scholar, B.J. Surg. 2000; PubMed Scopus Google Scholar, B.J. H. Endocrinology. 2000; PubMed Scopus Google Scholar, M. J. Cell PubMed Scopus Google Scholar). by osteoblasts important for this cell in the response that is critical to fracture J. Clin. Orthop. PubMed Scopus Google Scholar). Given that reduced oxygen is a component of the fracture we to a of its effects on osteoblast expression and short-term hypoxic and anoxic to in the hypoxic in vitro osteogenic differentiation of primary osteoblasts and mesenchymal that a to <0.02% O2 long-term osteogenic differentiation cell In we present evidence differential regulation of Runx2 expression by anoxia as a potential mechanism for this and Bone Cell osteoblast established on J. J. Cell PubMed Google Scholar). and mice of and to and with by with with of by and in allowed to and for for The cell was in in bone mesenchymal stem cells and in in Cell that was cells for cells at 21% O2 and in at to hypoxia or anoxia Cell viability was by and as H. M.J. J. Full Text Full Text PDF PubMed Scopus Google Scholar). osteoblasts and for with each of number and of The number of cells was by for cells of The of to cells was each cells in a and by and cell of and osteoblasts. In by with in with the for flow of to the to and osteoblasts and cells with h the effects of cells exposed to 21% (ambient oxygen), 2% (hypoxia), or <0.02% O2 for and was by of a in hypoxia and to of with the as as and hypoxic or anoxic cells or to 21% O2 for long-term differentiation. cells at or <0.02% within the hypoxia to effects on and Bone exposure to or <0.02% cells in vitro osteogenic differentiation in 21% O2 with and was after treatment with reduced or and in osteoblasts we a of BMP2 or to anoxic exposure for h for for expression analysis or of mineralized matrix deposition by bone nodule or the for calcium and at low and to the of bone nodule The for and <0.02% with at 21% O2 that to a of In the we of the cell after and <0.02% O2 to that had not been anoxia a of calcium a following the a of calcium on the of calcium to with in to a complex in that at osteoblasts exposed to or <0.02% O2 for h was and for analysis on the for and are of was for was with 21% O2 or or <0.02% O2 in and <0.02% O2 each with 21% O2 for h at and a and to the Microarray or G. G. M. H. 2001; PubMed Google Scholar). was for each and in Microarray that the following for and and are to with or in expression by or 2% O2 to 21% O2 as the of by as H. J. R. G. R. R. 2000; PubMed Scopus Google Scholar). Both the and the of for a have been to expression are G. G. M. H. 2001; PubMed Google Scholar, H. J. R. G. R. R. 2000; PubMed Scopus Google Scholar). the number of genes the of the that are not with or In contrast, of the of at 2% or or genes within the at or <0.02% The in this was to the to a of genes with or and to of to the at and 2% O2 for specific genes of The effect of oxygen on transcriptional response was by the in at and 2% O2 for of in genes analysis of or to the and to expression during the differentiation we by transcription of of each in to the with the in each was at least in was for to or and to and osteoblasts osteogenic differentiation following exposure to or <0.02% O2 as in and a brief to and of protein on gradient and to Runx2 and and the Bone by and Bone Both resident and recruited precursor involved in post-injury are exposed to wound oxygen levels as low as 0-2% O2 (1Heppenstall R.B. Grislis G. Hunt T.K. Clin. Orthop. 1975; 106: 357-365Crossref PubMed Scopus (85) Google Scholar, 2Knighton D.R. Silver I.A. Hunt T.K. Surgery. 1981; 90: 262-270PubMed Google Scholar, 3Brighton C.T. Ray R.D. Soble L.W. Kuettner K.E. J. Bone Joint Surg. Am. 1969; 51: 1383-1396Crossref PubMed Scopus (64) Google Scholar, 4Brighton C.T. Krebs A.G. J. Bone Joint Surg. Am. 1972; 54: 323-332Crossref PubMed Scopus (145) Google Scholar, 5Grundnes O. Reikeras O. Acta Orthop. Scand. 1992; 63: 487-491Crossref PubMed Scopus (72) Google Scholar, 6Gordillo G.M. Sen C.K. Am. J. Surg. 2003; 186: 259-263Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, R.B. Goodwin C.W. Brighton C.T. J. Bone Joint Surg. Am. 1976; 58: 1153-1156Crossref PubMed Scopus (40) Google Scholar). the of and O2 tissue is not and the effect of oxygen on we exposed primary osteoblasts to h of or <0.02% The cells in osteogenic differentiation for at 21% O2 and for bone nodule in a and and for the <0.02% O2 to the and 2% O2 revealed and mineralized in the <0.02% O2 the and primary of osteoblasts osteoprogenitors at of Cell 1997; PubMed Scopus Google we to reduced O2 the long-term differentiation of cells with osteogenic Primary bone have been to be of and differentiation R. D.R. Science. 1999; PubMed Scopus Google Scholar). exposed to h of or <0.02% O2 by osteogenic differentiation in 21% O2 for to the results for primary bone nodule formation as by and was for the <0.02% O2 to the and 2% O2 a and Given that in response to hypoxia have been for primary we exposed primary and the cell to h of and <0.02% by of differentiation at 21% each cell we the of for extracellular matrix deposition at analysis of cells was by the bone nodule by that of that had not anoxic In to the anoxic inhibition of osteogenic differentiation for primary osteoblasts and the cell not a significant in bone nodule formation following anoxic treatment as h of the of oxygen is a function of both and oxygen we a brief of anoxia in vitro osteogenic differentiation. and exposed to and h of <0.02% O2 and for at 21% the bone nodule as to that had not been anoxia and exposed to h of <0.02% O2 in bone nodule formation with at 21% whereas h of anoxia in a anoxic exposure to not bone nodule formation for osteoblasts and with the results bone nodule formation by cells was not at of the In a we the calcium in for h with or <0.02% O2 by to of differentiation. with the bone nodule calcium was in at to 21% O2 of by Cell a to in long-term differentiation and <0.02% O2 a of in cell viability at each oxygen we and of to the of significant in viability after or h of anoxic or after of following h of anoxia was a in viability in long-term we exposed to h of <0.02% to 21% O2 for to of differentiation. in cell viability by of as or by analysis of cells with and the by and cell these for primary osteoblasts not that the in bone nodule formation not be for by in cell viability following anoxic in the of or during long-term and Hypoxia in in that brief exposure to but not hypoxia, inhibited in vitro osteogenic we to a of the transcriptional this Given that h of anoxic treatment the inhibition of bone nodule we to the expression of osteoblasts for h at or 2% O2 to cells at 21% of expression revealed that exposure to <0.02% O2 a of expression as with 2% O2 down-regulation to for <0.02% to for 2% was by a in in the <0.02% 2% O2 for <0.02% for 2% that the of expression for 2% O2 with the of oxygen with anoxic the effects of anoxia hypoxia on the expression of known genes in a of genes with a in expression the the as was with a to a of in cell with oxygen J. Full Text PDF PubMed Google Scholar). a of genes at 2% O2 effect was for and of have been implicated in or cell activity R. J. 2003; PubMed Scopus Google Scholar, M.J. J. 2003; PubMed Google Scholar, M. J. Full Text Full Text PDF PubMed Scopus Google Scholar, P. O. M. PubMed Scopus Google Scholar). with V. J. M. V. 1997; PubMed Scopus Google and not in our not at both and 2% to 21% factor protein member factors and binding factor binding protein binding factor in of complex protein binding protein binding protein factor protein regulated protein in a Runx2 by but 2% the mechanism the anoxic inhibition of osteogenic we the for in osteogenic of genes with of osteoblast and differentiation revealed that inhibited with anoxia to down-regulation to 21% with little or at 2% O2 down-regulation to but not 2% O2 down-regulated Runx2, and and markers of osteogenic differentiation and findings with our observation that anoxia (but not 2% hypoxia) inhibited osteogenic differentiation Given that the transcription factor Runx2 is for normal osteoblast that anoxic was with down-regulation of Runx2 and and that anoxia but not hypoxia inhibited bone nodule we that anoxic down-regulation of Runx2 for the inhibition of differentiation. we to oxygen Runx2 expression in osteoblasts. our we osteoblasts in or differentiation with h of <0.02% O2 and Runx2 expression by after treatment as as after and h of and Runx2 expression was down-regulated by and only during the In contrast, osteoblasts exposed to 2% O2 for h not Runx2 inhibition during this our down-regulation of Runx2 following h of <0.02% O2 The down-regulation by is with that the of in expression with R. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Bone PubMed Scopus Google Scholar). Runx2 and of and osteoblast differentiation is a that to in we transient hypoxia or anoxia in long-term transcriptional that osteogenic differentiation. Furthermore, we to Runx2 protein levels by brief anoxic of Runx2, osteoblasts collagen into the extracellular in of differentiation matrix and the osteogenic osteocalcin (12Karsenty G. Endocrinology. 2001; 142: 2731-2733Crossref PubMed Scopus (169) Google Scholar, 13Ducy P. Dev. Dyn. 2000; 219: 461-471Crossref PubMed Scopus (398) Google Scholar). to osteoblast expression for Runx2, and during long-term differentiation. exposed for h to or <0.02% O2 in or differentiation for as and expression was following treatment and at and in addition to inhibition of Runx2 expression following h of <0.02% we a of Runx2 in osteoblasts that had anoxic treatment In contrast, Runx2 transcription was in osteoblasts that in 21% O2 the Runx2 expression low in with the transcriptional Runx2 protein levels following anoxic and low the differentiation Given that Runx2 elements that and we to anoxic exposure the expression of these genes by osteoblasts (12Karsenty G. Endocrinology. 2001; 142: 2731-2733Crossref PubMed Scopus (169) Google Scholar, 13Ducy P. Dev. Dyn. 2000; 219: 461-471Crossref PubMed Scopus (398) Google Scholar). of expression revealed immediate anoxic effect at h Furthermore, to the results for Runx2, osteoblasts exposed to h of anoxia expression at and to cells that had not been collagen expression in cells with long-term culture of osteoblasts L.W. R. J. Bone 1992; PubMed Scopus Google Scholar). of the osteogenic osteocalcin was inhibited in osteoblasts the following anoxic exposure osteocalcin in osteoblasts and was with the (but not bone nodule after anoxic treatment in and of Runx2 by BMP2 of Runx2 is to osteogenic factors that osteoblast differentiation been implicated in its expression, including and factor family members (12Karsenty G. Endocrinology. 2001; 142: 2731-2733Crossref PubMed Scopus (169) Google Scholar, G. J. Cell. 2003; PubMed Scopus Google Scholar). our for of to the expression of potential of we differential regulation of members of the factor including bone we a anoxic down-regulation of BMP2, of Runx2 transcription with a established in bone and J. Bone PubMed Scopus Google Scholar, J. J. 2000; PubMed Google Scholar, 2003; PubMed Scopus Google Scholar). was by anoxia In to BMP2, has been to Runx2 transcription in primary osteoblasts Ducy P. Karsenty G. R. J. 2001; PubMed Scopus Google Scholar). and factors implicated in osteoblast differentiation and and and not in our and <0.02% not that inhibition of BMP2 expression to the anoxic down-regulation of Runx2 expression in we to restoring BMP2 levels during the of brief anoxic exposure Runx2 expression in primary osteoblasts (9Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3668) Google Scholar, PubMed Scopus Google Scholar). recombinant BMP2 or to osteoblasts and exposed to h of or <0.02% analysis revealed that anoxic osteoblasts with a significant in Runx2 with our findings In contrast, anoxic osteoblasts with BMP2 Runx2 levels to that BMP2 repression of Runx2 levels Runx2 expression in anoxic osteoblasts not levels in osteoblasts Given that our analysis revealed a significant inhibition of with we both and treatment not inhibition of Runx2 by anoxia in in Runx2 during as with evidence repression of Runx2 transcription Ducy P. Karsenty G. R. J. 2001; PubMed Scopus Google Scholar). in anoxic inhibition of Runx2 to the findings that of Runx2 levels was specific to Given that BMP2 Runx2 levels anoxic we BMP2 the of osteoblasts to long-term differentiation. Thus, we osteoblasts with BMP2 to h of anoxic in osteogenic differentiation for at 21% O2 with the of Runx2 levels by BMP2 the setting of we normal bone nodule formation in BMP2 osteoblasts of or had not the anoxic down-regulation of not the anoxic inhibition of differentiation Runx2 with that of its BMP2, as critical to the regulation of osteoblast differentiation. a to in the setting of a reduced oxygen we have investigated the effects of oxygen levels and <0.02% on cell and primary cell involved in bone that primary osteoblasts and bone mesenchymal but not the cell to (but not 2% was with in vitro or during of differentiation. in the long-term response to h of <0.02% O2 to the transcriptional of osteoblasts following this brief treatment osteoblasts not only had a transcriptional response (e.g. but hypoxic specific elements in anoxic that <0.02% but not 2% a cell the only and by has been to be reduced has not been implicated in hypoxic in in that is the protein for its not to the transcriptional by genes 1999; PubMed Scopus Google Scholar). the of by anoxia in our a for in cells as this the of our analysis was a down-regulation of Runx2, collagens, and markers of osteoblast differentiation following h of the of osteoprogenitors in a low oxygen microenvironment has not been and in and differentiation have been for primary osteogenic was in our as whereas primary osteoblasts and mesenchymal stem cells to anoxic the cell was the mechanism for this differential response to anoxia is this to the primary and cells in response to cell to the regulation of Runx2 expression, as H. J. Cell 1983; PubMed Scopus Google Scholar, G. J. Bone 1999; PubMed Scopus Google Scholar). present evidence that down-regulation of Runx2, of osteoblast the inhibition of osteogenic differentiation by normal Runx2 expression, osteoblasts (9Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3668) Google Scholar, 13Ducy P. Dev. Dyn. 2000; 219: 461-471Crossref PubMed Scopus (398) Google for Runx2 as a that the expression of a number of osteogenic markers including collagen I, osteopontin, and osteocalcin (11Karsenty G. Ducy P. Starbuck M. Priemel M. Shen J. Geoffroy V. Amling M. Bone. 1999; 25: 107-108Crossref PubMed Scopus (119) Google Scholar, 12Karsenty G. Endocrinology. 2001; 142: 2731-2733Crossref PubMed Scopus (169) Google Scholar). The critical of Runx2 is established in as both are in and mineralized bone M.J. Cell. 1997; 89: Full Text Full Text PDF PubMed Scopus Google Scholar). in the Runx2 in humans results in cleidocranial dysplasia, by the of and osteogenic M.J. R. Cell. 1997; 89: Full Text Full Text PDF PubMed Scopus Google Scholar). In our in vitro osteoblast and exposure to anoxia was with inhibition of Runx2, evidence of significant during h of we a long-term down-regulation of Runx2 and protein as transcriptional levels of of its and In contrast, exposure to 2% O2 inhibited bone nodule formation Runx2 Furthermore, in primary osteoblasts and anoxia not the differentiation of It has been in the cell that Runx2 expression not with differentiation potential or of osteogenic cells that levels of Runx2 not bone or osteocalcin G. J. Bone 1999; PubMed Scopus Google Scholar). The Runx2 expression and osteogenic differentiation in the cell was not inhibited by the of Runx2 as a potential of anoxic inhibition of differentiation in primary osteoblasts. Runx2 down-regulation is a critical in the anoxic inhibition of we the in Runx2 expression that with anoxia the of osteoblasts to Runx2 be responsive to a of of Runx2 transcription to be (12Karsenty G. Endocrinology. 2001; 142: 2731-2733Crossref PubMed Scopus (169) Google Scholar, G. J. Cell. 2003; PubMed Scopus Google Scholar). to our analysis on known osteogenic and revealed differential regulation of members of the factor we down-regulation of and BMP2, with significant in and family of are by osteoblasts and have key in bone and the effect of Runx2 expression has been to Runx2 transcription in primary osteoblasts the effect in Ducy P. Karsenty G. R. J. 2001; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). was not by hypoxia or we that anoxia inhibited with not the anoxic inhibition of Runx2 or osteogenic differentiation. In in Runx2 levels in both and anoxic and thus was to the anoxic inhibition of differentiation. that anoxic regulation of is not a of anoxic inhibition of Runx2 expression and osteoblast differentiation in our In to BMP2 is a established of osteogenic differentiation in cell (12Karsenty G. Endocrinology. 2001; 142: 2731-2733Crossref PubMed Scopus (169) Google Scholar, V. M.J. Science. PubMed Scopus Google Scholar). Given our of significant anoxic down-regulation of BMP2, we its potential in anoxic regulation of Runx2 we that treatment with BMP2 (but not or to anoxic exposure Runx2 expression to the levels with osteoblasts. exposure of osteoblasts to anoxia in Runx2 expression levels with for the in Runx2 BMP2 is that anoxia Runx2 transcription and by inhibiting the Runx2 a transcriptional and by important of its expression, as anoxic of BMP2 transcription for the in Runx2 with anoxia is a but Nevertheless, Runx2 levels by BMP2 was sufficient to the long-term inhibition of differentiation. Thus, we that anoxic exposure has a on osteogenic differentiation by inhibiting Runx2 transcription a that BMP2 repression In anoxic in osteogenic differentiation in inhibition of Runx2 and a transcriptional response with 2% the hypoxic response that of anoxia to the of or in In has been to be to oxygen 21% in and in and injury (1Heppenstall R.B. Grislis G. Hunt T.K. Clin. Orthop. 1975; 106: 357-365Crossref PubMed Scopus (85) Google Scholar, 2Knighton D.R. Silver I.A. Hunt T.K. Surgery. 1981; 90: 262-270PubMed Google Scholar, 3Brighton C.T. Ray R.D. Soble L.W. Kuettner K.E. J. Bone Joint Surg. Am. 1969; 51: 1383-1396Crossref PubMed Scopus (64) Google Scholar, 4Brighton C.T. Krebs A.G. J. Bone Joint Surg. Am. 1972; 54: 323-332Crossref PubMed Scopus (145) Google Scholar, 5Grundnes O. Reikeras O. Acta Orthop. Scand. 1992; 63: 487-491Crossref PubMed Scopus (72) Google Scholar, 6Gordillo G.M. Sen C.K. Am. J. Surg. 2003; 186: 259-263Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, R.B. Goodwin C.W. Brighton C.T. J. Bone Joint Surg. Am. 1976; 58: 1153-1156Crossref PubMed Scopus (40) Google Scholar, 8Knighton D.R. Hunt T.K. Scheuenstuhl H. Halliday B.J. Werb Z. Banda M.J. Science. 1983; 221: 1283-1285Crossref PubMed Scopus (533) Google Scholar). Thus, 2% O2 a hypoxic as that with a injury or tissue be both and In contrast, cells the of oxygen to vascular or injury a by cell elements and differentiation as by BMP2 and the osteoblast transcriptional response during transient of reduced oxygen a osteoblast in the of following are to J. for with