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Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 38, Issue of September 17, pp. 40007–40016, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Transient Changes in Oxygen Tension Inhibit Osteogenic Differentiation and Runx2 Expression in Osteoblasts* Received for publication, April 2, 2004, and in revised form, July 7, 2004 Published, JBC Papers in Press, July 19, 2004, DOI 10.1074/jbc.M403715200 Ali Salim‡§, Randall P. Nacamuli‡, Elise F. Morgan‡, Amato J. Giaccia§, and Michael T. Longaker‡ From the Departments of ‡Surgery and §Radiation Oncology, Stanford University School of Medicine, Stanford, California 94305-5148 of mesenchymal osteoblast precursors is a key component of Vascular disruption following bony injury results in a hypoxic gradient within the wound microenvironment. this cascade (6, 8). Both committed resident osteoblasts and Nevertheless, the effects of low oxygen tension on osteo- recruited precursor cells are exposed to the hypoxic post-injury genic precursors remain to be fully elucidated. In the milieu. Given that normal osteoblast function is critical to the present study, we investigated in vitro osteoblast and deposition of mineralized matrix, the hallmark of successful mesenchymal stem cell differentiation following expo- fracture repair, it is important to understand the response of sure to 21% O (ambient oxygen), 2% O (hypoxia), and 2 2 osteoprogenitors to a hypoxic microenvironment. <0.02% O (anoxia). Hypoxia had little effect on osteo- Recent investigations have yielded significant insights into genic differentiation. In contrast, short-term anoxic the transcriptional regulation of osteogenic differentiation (9 – treatment of primary osteoblasts and mesenchymal pre- 11). Primary cell culture models and established cell lines that cursors inhibited in vitro bone nodule formation and recapitulate osteoblast maturation in vitro have allowed the extracellular calcium deposition. Cell viability assays identification of closely regulated transcription factors (e.g. revealed that this effect was not caused by immediate or Runx2, Osx, and members of the Ets family) and osteogenic delayed cell death. Microarray profiling implicated markers (e.g. collagens, alkaline phosphatase, and osteocalcin) down-regulation of the key osteogenic transcription fac- specific to each stage of the osteoblast life cycle (12). Runx2, a tor Runx2 as a potential mechanism for the anoxic inhi- bition of differentiation. Subsequent analysis revealed member of the runt family of transcription factors, is both not only a short-term differential regulation of Runx2 necessary and sufficient for osteoblast differentiation (9, 13). and its targets by anoxia and hypoxia, but a long-term Runx2 activity promotes the expression of a number of osteo- inhibition of Runx2 transcriptional and protein levels genic markers including collagen I, osteopontin, and osteocal- after only 12–24 h of anoxic insult. Furthermore, we cin by binding to responsive elements within the promoters of present evidence that Runx2 inhibition may, at least in these genes (11, 12). Its clinical significance is highlighted by part, be because of anoxic repression of BMP2, and that the observation that humans or mice heterozygous for Runx2 restoring Runx2 levels during anoxia by pretreatment mutations develop cleidocranial dysplasia, whereas postnatal with recombinant BMP2 rescued the anoxic inhibition impairment of Runx2 results in osteopenia (10). of differentiation. Taken together, our findings indicate The effect of reduced oxygen on genes involved in osteoblast that brief exposure to anoxia (but not 2% hypoxia) down- proliferation and differentiation remains unclear, in part be- regulated BMP2 and Runx2 expression, thus inhibiting cause of the sometimes conflicting results observed for primary critical steps in the osteogenic differentiation of plu- cultures versus established cell lines and for different oxygen ripotent mesenchymal precursors and committed tensions. An early study of the immortalized osteoblast-like cell osteoblasts. line MC3T3-E1 exposed to chronic 10% O demonstrated a decrease in both cell proliferation and alkaline phosphatase It has long been known that hypoxia is a prominent compo- expression, an early to middle marker of osteoblast differenti- nent of the microenvironment in both bony and soft tissue ation, relative to cells at 21% O (14). Additional investigations injury (1– 4). Interruption of vascular flow with fracture or in primary osteoblast-enriched cultures from rat calvaria and surgical osteotomy results in a transient hypoxic gradient human periodontal ligament confirmed that hypoxic (5–10%) within the wound, with oxygen tension falling to 0 –2% in the inhibition of alkaline phosphatase was not limited to the central wound region (1– 6). Thus, the complex osteogenic re- MC3T3-E1 line but was applicable to primary cells as well (15, generative response that ensues following injury begins in the 16). However, in contrast to the effect seen in MC3T3-E1, an setting of reduced oxygen (7). In addition to inflammatory cell increase in proliferation was observed in both primary cell recruitment, matrix processing, and angiogenesis, the activity types, perhaps alluding to a reciprocal hypoxic effect on prolif- eration and differentiation in primary osteoblasts. Recently, a human osteosarcoma cell line continuously exposed to 2% O * This work was supported by National Institutes of Health Grants R01 DE13028 and R01 DE14526 and The Oak Foundation (to M. T. L.) for up to 96 h was shown to express diminished levels of the and National Institutes of Health Grant R01 CA88480 (to A. J. G.). The transcription factor RUNX2 and the late bone marker osteocal- costs of publication of this article were defrayed in part by the payment cin (17). Whereas this study on transformed cells provided of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate important initial insight into the hypoxic regulation of osteo- this fact. genic gene expression, a broad understanding of the hypoxic ¶ To whom correspondence should be addressed: Dept. of Surgery, osteoblast transcriptional response remains to be elucidated. Stanford University School of Medicine, 257 Campus Dr., Stanford, Furthermore, it may be difficult to extrapolate tumor cell line CA 94305-5148. Tel.: 650-736-1707; Fax: 650-736-1705; E-mail: [email protected]. derived conclusions to primary osteoblasts given that Runx2 This paper is available on line at http://www.jbc.org 40007 This is an Open Access article under the CC BY license. 40008 Anoxia Inhibits Osteogenic Differentiation TM with the TACS Annexin V-FITC Apoptosis Detection kit (R&D Sys- expression may be dissociated from osteogenic potential in tems, Minneapolis, MN) for flow cytometry-based detection of apoptotis immortalized cell lines such as MC3T3-E1 (18, 19). according to the manufacturer’s directions. Nevertheless, evidence is accumulating that hypoxia regu- Exposure to Ambient, Hypoxic, and Anoxic Conditions—At 95% con- lates osteoblast-secreted growth factors implicated in the post- fluence, calvarial osteoblasts and MC3T3-E1 cells underwent media injury microenvironment. For example, cytokines potentially change with fresh -minimal essential medium, 10% fetal bovine se- TM involved in osteoblast-endothelial interactions are oxygen-sen- rum. MSCs received fresh MSCGM . After 6 h (to minimize the effects of medium replacement upon gene expression), cells were exposed to sitive. Hypoxic osteoblasts exhibit increased expression of vas- 21% (ambient oxygen), 2% (hypoxia), or 0.02% O (anoxia) for 0, 3, 6, cular endothelial growth factor-A (VEGF-A), perhaps the most 12, and 24 h. Oxygen was supplanted by infusion of a 5% CO , 95% potent and specific of the angiogenic cytokines (20). VEGF-A nitrogen gas in 37 °C humidified hypoxia work stations (Bactron An- appears to regulate osteoblast function by coupling ossification aerobic/Environmental Chamber, Sheldon Corporation, Cornelius, OR, with cartilage resorption and angiogenesis during endochon- and Ruskinn Microaerophilic Work station, Ruskinn Technology, dral bone formation (21). Additional hypoxia-regulated modu- Leeds, United Kingdom). These chambers employ an intermediate “pass-box” to minimize mixing of ambient air with the internal chamber lators of osteoblast function that also act as endothelial mito- environment as well as real-time temperature and oxygenation level gens include members of the transforming growth factor- monitoring. (TGF-), insulin-like growth factor, and fibroblast growth fac- After hypoxic or anoxic treatments, cells underwent total RNA iso- tor families (22–25). Their elaboration by osteoblasts further lation using TRIzol reagent (Invitrogen) or were returned to 21% O for suggests an important role for this cell type in regulating the long-term differentiation. For samples derived from cells at 2 or 0.02% angiogenic response that is critical to fracture repair (26). O , RNA collections were done within the hypoxia chamber to limit reoxygenation effects on gene expression. Given that reduced oxygen is a major component of the Long-term Differentiation Experiments and Bone Nodule Staining— fracture microenvironment, we sought to gain a better under- Following exposure to 21, 2, or 0.02% O , cells underwent in vitro standing of its effects on osteoblast gene expression and func- osteogenic differentiation in 21% O via basal medium supplementation tion. We first examined whether short-term hypoxic and anoxic with 1 M dexamethasone, 5 mM -glycerophosphate, and 100 g/ml insults, analogous to those observed in the post-fracture hy- ascorbic acid. Medium was replaced immediately after initial treatment poxic gradient, would affect in vitro osteogenic differentiation with reduced or ambient oxygen, and every 2 days thereafter. For experiments in which osteoblasts were cytokine stimulated, we used a of primary osteoblasts and multipotent mesenchymal precur- single dose of BMP2 (100 ng/ml), TGF-1 (5 ng/ml), TGF-2 (5 ng/ml), sors. We demonstrate that a 12–24-h reduction from 21 to insulin-like growth factor-1 (20 ng/ml) or vehicle immediately prior to 0.02% O suppressed long-term osteogenic differentiation anoxic exposure for 24 h (all cytokines from R&D Systems). without altering cell viability. In addition, we present evidence Cultures were maintained for 28 days for gene expression analysis or implicating differential regulation of Runx2 expression by an- assessment of mineralized matrix deposition by bone nodule staining oxia as a potential mechanism for this effect. via 0.5% alizarin red or the von Kossa method for calcium phosphates, and digitally photographed at low and high magnifications. Low mag- MATERIALS AND METHODS nification (whole plate) von Kossa images were analyzed using Scion Image Software version 4.0.2 (Scion Corp., Frederick, MD) to determine Calvarial Osteoblast, MC3T3-E1, and Bone Marrow-derived Mesen- the area of bone nodule staining. The values reported for 2 and 0.02% chymal Cell Cultures—Primary mouse calvarial osteoblast cultures O -treated plates represent relative staining compared with parallel were established based on described techniques (27). Briefly, frontal cultures at 21% O that were normalized to a value of “one.” In the same and parietal bones from 6-day-old CD-1 mice were stripped of their manner, we quantified staining of the three cell types after 3, 6, 12, and pericranium and dura mater. Calvaria from five to six pups were 24hof 0.02% O relative to parallel cultures that had not been anoxia pooled, minced, and washed with sterile phosphate-buffered saline. treated. We also performed a colorimetric determination of calcium Osteoblasts were released by five sequential 20-min digestions with concentration using a Calcium Reagent Set (Biotron Diagnostics, 0.1% collagenase (Invitrogen). Digestions were stopped with 5 volumes Hemet, CA) following the manufacturer’s instructions. This kit provides of -minimal essential medium, 10% fetal bovine serum. Fractions 2–5 a quantitative assessment of calcium concentration based on the ability were collected, pelleted by centrifugation, and resuspended in -mini- of calcium to react with cresolphthalein complexone in 8-hydroxyquin- mal essential medium, 10% fetal bovine serum plus penicillin/strepto- olone to form a complex purple in color that absorbs at 570 nm. mycin. Cells were allowed to attach and expand for 48 h, then replated Gene Profiling Using Microarrays—RNA from mouse calvarial osteo- for experimentation without further passaging. blasts exposed to 21, 2, or 0.02% O for 24 h was purified and reverse The osteoblast-like immortalized cell line MC3T3-E1 (American transcribed for microarray analysis on cDNA chips from the Stanford Type Culture Collection, Manassas, VA) was maintained in subconflu- Functional Genomics Facility. Each array contains 42,000 mouse ent cultures in -minimal essential medium, 10% fetal bovine serum elements, representing over 30,000 unique genes. Detailed protocols for plus pencillin/streptomycin. Human bone marrow-derived mesenchy- probe synthesis and hybridization are available on-line. Briefly, 30 g mal stem cells (MSCs) were purchased from Cambrex BioScience of total RNA was used for single-stranded cDNA probe synthesis incor- (Walkersville, MD) and maintained in an undifferentiated state in TM TM porating aminoallyl-dUTP, which was coupled with either Cy3 (for 21% manufacturer recommended medium (Poietics MSCGM Mesen- O samples) or Cy5 (for 2 or 0.02% O samples). Type I experiments chymal Stem Cell Medium) that was replaced every 2 days. Early 2 2 were performed in which 2 and 0.02% O samples were each hybrid- passage (3) cells were utilized for experimentation. All cells were ized with pooled 21% O reference samples for 16 h at 65 °C. Arrays maintained at 21% O and 5% CO in humidified incubators at 37 °C 2 2 were washed, scanned, and analyzed using a GenePix Scanner and prior to hypoxia or anoxia experiments. software (Axon Instruments, Foster City, CA). After gridding, array Cell viability was assessed by Hoescht and propidium iodide staining data were uploaded to the Stanford Microarray Data base or SMD (29). as previously described (28). Briefly, osteoblasts and MSCs were incu- Total RNA from three independent experiments was pooled for each bated for 15 min with 2 g each of bis-benzamide (Hoescht stain array probe, and array hybridizations were performed in duplicate. number 33342, Sigma) and propidium iodide (Sigma) per 1 ml of me- Microarray Data Analysis—Using on-line software from SMD, data dium. The number of non-viable cells was determined by scoring three points that met the following spot quality criteria were selected for randomly selected low-magnification fields for double-stained cells (in- analysis: spot regression correlation 0.7, mean channel intensity 2.5 dicating loss of membrane integrity). The ratio of viable to total cells median background, and “spot flag” and “failed” filters 0. These was then determined. For each sample, 500 cells were scored in a user-selectable criteria are intended to exclude spots with non-uniform, blinded fashion, and experiments were repeated three times. Viability dim, or otherwise unreliable signals. Relative changes in gene expres- data were confirmed by trypan blue exclusion and cell counts of sion were evaluated by -fold change (i.e. 0.02 or 2% O relative to 21% trypsinized MSCs and osteoblasts. In addition, MSCs released by O reference) as determined from the log of red/green normalized ratio trypsinization were incubated with propidium iodide in conjunction 2 2 reported by SMD, as previously described (30). Both the -fold change values and the standard deviation (S.D.) of -fold changes for a particu- lar array have been used to determine whether expression changes are The abbreviations used are: VEGF-A, vascular endothelial growth significantly different from array background (29, 30). Describing the factor-A; TGF-, transforming growth factor-; MSC, mesenchymal stem cell; QRT, quantitative real-time. number of genes whose -fold changes fall outside the range of the Anoxia Inhibits Osteogenic Differentiation 40009 mean 2 S.D. allows comparisons between populations that are not identical, e.g. those with different mean -fold changes or distributions. In contrast, reporting absolute -fold change allows comparison of the same gene across two populations (i.e. relative -fold change of gene A at 0.02 versus 2% O ) or two or more genes within the same populations (i.e. gene A versus geneBat2or 0.02% O ). The approach undertaken in this study was to use the 2 S.D. criterion to identify a subset of genes with “significant” up- or down- regulation, and then to use comparisons of -fold change values to directly compare the responses at 0.02 and 2% O for specific genes and/or functional groupings of genes. The overall effect of oxygen level on transcriptional response was assessed by comparing the variances in -fold change at 0.02 and 2% O using Levine’s test for homogeneity of variances. For PCR data, -fold change values in individual genes were compared via analysis of variance or paired t tests (with Bonferroni adjustments to control the Type I error rate). All statistical analyses were performed using JMP 5.0.1 (SAS Institute, Cary, NC). Quantitative Real-time PCR—For selective microarray confirmation and to investigate gene expression during the 28-day differentiation period, we performed quantitative real-time polymerase chain reaction (QRT-PCR). We obtained cDNA by reverse transcription of 1 gof DNase-treated total RNA from each sample using random hexamer priming in 50-l reactions according to the manufacturer’s recommen- dations (Taqman® Reverse Transcription Reagent Kit, Applied Biosys- tems, Foster City, CA). We proceeded with QRT-PCR using the Applied Biosystems Prism® 7900HT Sequence Detection System. A non-multi- plexed SYBR® Green assay in which each cDNA sample was evaluated at least in triplicate 20-l reactions was used for all target transcripts. Expression values were normalized to 18S or -actin. QRT-PCR prim- ers were designed using Primer Express version 2.0.0 (Applied Biosys- tems) and tested to confirm appropriate product size and optimal concentrations. Western Blot Analysis—Mouse osteoblasts underwent osteogenic dif- ferentiation following exposure to 21 or 0.02% O as described above. Cells were lysed in 9 M urea, 75 mM Tris-HCl, pH 7.5, and 0.15 M FIG.1. Mouse osteoblast differentiation following a 24-h expo- -mercaptoethanol. After a brief sonication, lysates were centrifuged to sure to ambient air (21% O ), hypoxia (2% O ), or anoxia (<0.02% 2 2 remove debris and quantitated. Forty micrograms of protein were elec- O ). Osteoblasts were stained for bone nodules after 28 days of differ- trophoresed on 3– 8% gradient SDS-polyacrylamide gels and trans- entiation (basal medium supplemented with 1 M dexamethasone, 5 ferred to nitrocellulose membranes. Membranes were probed using mM -glycerophosphate, and 100 g/ml ascorbic acid) at 21% O . Rep- commercial Runx2 and -actin antibodies (Santa Cruz Biotechnology) resentative low magnification views of von Kossa (a) and alizarin red (b) and the ECL detection kit (Amersham Biosciences). staining revealed diminished bone nodule formation following 0.02% O exposure compared with 21 and 2% O . Representative high magni- 2 2 RESULTS fication fields of von Kossa (c) and alizarin red (d) staining confirmed reduced mineralized nodule formation by the anoxia-treated group. Anoxia Inhibits Bone Nodule Formation by Calvarial Osteo- blasts and Bone Marrow-derived Mesenchymal Stem Cells— Both resident and recruited precursor populations involved in post-injury repair are transiently exposed to wound oxygen levels as low as 0 –2% O (1–7). Without the re-establishment of nutrient and O delivery, tissue regeneration is not possible and necrosis ensues. To investigate the effect of oxygen depri- vation on osteoprogenitor cells, we exposed primary mouse calvarial osteoblasts to 24 h of 21, 2, or 0.02% O . The cells were subsequently maintained in osteogenic differentiation media for 28 days at 21% O and assayed for bone nodule formation. As demonstrated in Fig. 1, a and b, von Kossa and alizarin red staining for the 0.02% O group were diminished relative to the 21 and 2% O groups. High magnification views FIG.2. Long-term osteogenic differentiation of human bone revealed fewer and smaller mineralized nodules in the 0.02% marrow-derived mesenchymal stem cells following 24 h of 21, 2, O group than the two other groups (Fig. 1, c and d). or <0.02% O . Representative high magnification views of von Kossa Because primary cultures of calvarial osteoblasts likely in- (a) and alizarin red (b) staining at 28 days demonstrated diminished clude osteoprogenitors at different stages of commitment (31), bone nodule deposition by cells treated for 24 h with 0.02% O . Bone nodule formation did not appear to be affected by 24 h of 2% O . we next sought to determine whether reduced O altered the 2 long-term differentiation of uncommitted cells with osteogenic potential. Primary human bone marrow-derived MSCs have observed for primary versus immortalized cells, we exposed previously been shown to be capable of osteogenic, adipogenic, primary osteoblasts, MSCs, and the osteoblast-like cell line chondrogenic, and myogenic differentiation (32). We exposed MC3T3-E1 to 24 h of 21, 2, and 0.02% O , followed by 28 days MSCs to 24 h of 21, 2, or 0.02% O followed by osteogenic of differentiation at 21% O . For each cell type, we estimated 2 2 differentiation in 21% O for 28 days. Similar to the results the degree of von Kossa staining for extracellular matrix dep- obtained for primary osteoblasts, bone nodule formation as osition at 28 days using image analysis software. Relative assayed by von Kossa, and alizarin red staining was dimin- staining of anoxia-treated cells was determined by dividing the ished for the 0.02% O group relative to the 21 and 2% O calculated bone nodule area by that of parallel cultures that 2 2 groups (Fig. 2, a and b). had not received anoxic treatment. In marked contrast to the Given that differences in response to hypoxia have been anoxic inhibition of osteogenic differentiation observed for pri- 40010 Anoxia Inhibits Osteogenic Differentiation tion in bone nodule formation compared with cultures at 21% O , whereas 12 h of anoxia resulted in a 60% reduction (Fig. 3b). Shorter anoxic exposure periods (3 to 6 h) did not inhibit bone nodule formation for osteoblasts and MSCs. Consistent with the results obtained above, bone nodule formation by MC3T3-E1 cells was not affected at any of the durations tested (Fig. 3b). In addition, using a cresolphthalein complexone- based colorimetric assay we quantitatively determined the cal- cium concentration in MSC cultures treated for 24 h with 21 or 0.02% O followed by up to 28 days of differentiation. Con- sistent with the bone nodule quantitation data, calcium con- centration was 5-fold lower in anoxia-treated MSC cultures at 28 days relative to 21% O cultures (Fig. 3c). Anoxic Inhibition of Osteogenic Differentiation Is Not Caused by Increased Cell Death—As a first step to determine if ob- served differences in long-term differentiation between 21 and 0.02% O groups were a result of alterations in cell viability at each oxygen concentration, we performed Hoescht and pro- pidium iodide staining of MSCs to determine the proportion of nonviable (double-stained) cells. No significant differences in viability were observed immediately after 3, 6, 12, or 24 h of anoxic exposure, or after 2 days of reoxygenation following 24 h of anoxia (Fig. 4a). To determine whether there was a change in viability in long-term culture, we exposed MSCs to 24 h of 0.02% O , then returned them to 21% O for up to 28 days of 2 2 differentiation. We observed no alterations in cell viability by either visual inspection of cultures stained as above, or by FACS analysis of cells incubated with propidium iodide and the early apoptosis marker annexin V (Fig. 4b). Viability assess- ment by trypan blue exclusion and cell count confirmed these results, which were similar for primary calvarial osteoblasts (data not shown). These data suggested that the differences in bone nodule formation described above could not be accounted for by alterations in cell viability immediately following anoxic treatment, in the first 2 days of reoxygenation, or during long- FIG.3. Quantitative assessment of bone nodule staining and term culture. culture calcium concentration. a, relative bone nodule staining at Anoxia and Hypoxia Result in Distinct Transcriptional Pro- 28 days for primary mouse osteoblasts (Ob), human MSC (MSC), and an files in Osteoblasts—Having determined that brief exposure to osteoblast-like cell line (MC3T3) following 24 h of 21, 2, or 0.02% O . anoxia, but not hypoxia, inhibited in vitro osteogenic differen- For each cell type, the degree of von Kossa staining was estimated using tiation, we sought to gain a better understanding of the tran- image analysis software and expressed relative to parallel cultures kept at 21% O (as described under “Materials and Methods”). Osteoblasts 2 scriptional changes underlying this effect. Given that 24 h of and MSC cultures, but not MC3T3, demonstrated diminished bone anoxic treatment exhibited the most marked inhibition of bone nodule formation. b, relative bone nodule staining of each cell type after nodule formation, we used cDNA microarrays to compare the exposure to 0, 3, 6, 12, or 24 h of 0.02% O . For osteoblast and MSC cultures, anoxia for 12 or 24 h significantly reduced bone nodule stain- gene expression profiles of mouse osteoblasts cultured for 24 h ing, whereas 3 or 6 h exposure did not. c, relative calcium concentration at 0.02 or 2% O relative to identical cells maintained at 21% in MSC cultures exposed to 24 h of 21% O (squares)or 0.02% O 2 2 O . Examination of overall gene expression patterns revealed (circles), then differentiated in osteogenic medium for the indicated that exposure to 0.02% O induced a larger range of relative days (d). Values represent the calcium concentration as compared with 2 day1(i.e. immediately following the initial 24-h treatment period) expression as compared with 2% O (31.8-fold down-regulation measured by a colorimetric assay as described under “Materials and to 55.4-fold up-regulation for 0.02% O , versus 5.0 to 13.1 for Methods.” Each bar or point represents the mean S.D. for three 2% O ). This was reflected by a greater variance in -fold separate experiments. *, p 0.05 for the indicated anoxic groups changes in the 0.02% than 2% O groups (variance 3.62 for compared with the same cell type maintained at 21% O (analysis of 2 variance or paired t tests). 0.02% O versus 0.17 for 2% O , p 0.001). 2 2 We hypothesized that the markedly larger range of relative expression for 0.02 versus 2% O corresponded with the more mary osteoblasts and MSCs, the MC3T3-E1 cell line did not 2 severe deprivation of oxygen with anoxic exposure. Directly reveal a significant decrease in bone nodule formation follow- comparing the effects of anoxia versus hypoxia on the relative ing anoxic treatment (Fig. 3a). expression of known oxygen-sensitive genes confirmed this. For As Little as 12 h of Anoxia Inhibits Long-term Osteogenic example, in a subset of genes with a change in relative expres- Differentiation—Because the severity of oxygen deprivation is sion greater than 2 S.D. from the array sample mean (Table a function of both duration and oxygen tension, we next exam- I), the largest functional grouping comprised 10 glycolytic en- ined whether a more brief period of anoxia would inhibit in zymes (represented as 12 isoforms). This finding was consistent vitro osteogenic differentiation. Osteoblasts, MSCs, and MC3T3-E1s were exposed to 0, 3, 6, 12, and 24 h of 0.02% O with a shift to a primarily anaerobic glycolytic mode of metab- olism observed in many cell types with oxygen deprivation (33). and subsequently differentiated for 28 days at 21% O . We then estimated the bone nodule area as above (relative to cultures We noted a more marked activation of all glycolytic pathway genes at 0.02 than 2% O (Table I). This effect was also true that had not been anoxia treated). Osteoblasts and MSCs ex- posed to 24 h of 0.02% O demonstrated an 70 – 80% reduc- for VEGF-A, adrenomedullin, and -integrin, all of which have 2 1 Anoxia Inhibits Osteogenic Differentiation 40011 FIG.4. Short- and long-term cell vi- ability assessment of MSCs. a, by Hoe- scht and propidium iodide staining, the proportion of viable to total MSCs did not change immediately after 3, 6, 12, and 24 h of the indicated oxygen levels, or 2 days following 24 h exposure (to assess any early reoxygenation effect). Each bar represents the mean S.D. for three sep- arate experiments. b, representative pho- tomicrographs of MSCs stained by Hoe- scht and propidium iodide at 14 and 28 days of differentiation following the ini- tial 24-h exposure to 0.02 or 21% O .A few red (nonviable) condensed nuclei can be seen among blue (viable) nuclei. No significant annexin V-fluorescein isothio- cyanate signal was detected by fluores- cence-activated cell sorter analysis of par- allel cultures at 14 and 28 days following exposure to 24 h of 0.02 or 21% O . been implicated in angiogenesis or modulating endothelial cell Given that the transcription factor Runx2 is required for activity (Table I) (34 –37). Consistent with previous reports normal osteoblast differentiation, that anoxic insult was asso- (38), VEGF-B and -C isoforms were not induced in our microar- ciated with down-regulation of Runx2 and several downstream rays (data not shown). targets, and that anoxia but not hypoxia inhibited bone nodule Runx2 Transcriptional Levels Are Inhibited by Anoxia but formation, we hypothesized that anoxic down-regulation of Not 2% O (Hypoxia)—To investigate the mechanism underly- Runx2 could account for the observed inhibition of differentia- ing the anoxic inhibition of osteogenic differentiation, we inter- tion. Therefore, we sought to further define how oxygen regu- rogated the array data sets for changes in osteogenic marker lates Runx2 expression in osteoblasts. To confirm our microar- expression. Analysis of seven genes associated with various ray findings, we treated calvarial osteoblasts in basal or stages of osteoblast growth and differentiation revealed that differentiation media with 24 h of 0.02% O and assayed they were all inhibited with anoxia (38 to 86% down-regulation Runx2 expression by QRT-PCR immediately after treatment relative to 21% O ), with little or no overall change at 2% O (33 (Fig. 5b, 24 h) as well as after 6 and 12 h of reoxygenation (Fig. 2 2 down-regulation to 30% up-regulation) (Fig. 5a). Anoxia but 5b, 30 and 36 h). Runx2 expression was markedly down-regu- not 2% O down-regulated Runx2, three bone-associated colla- lated by anoxia, and only minimally recovered during the gens (ColI, ColIII, and ColVI), and two additional markers of reoxygenation period examined. In contrast, osteoblasts ex- osteogenic differentiation (Osteopontin and Osteoglycin) (Fig. posed to 2% O for 24 h did not demonstrate Runx2 inhibition 5a). These findings were consistent with our observation that during this period (Fig. 5c). Of note, our QRT-PCR data dem- anoxia (but not 2% hypoxia) inhibited osteogenic differentia- onstrated an 80% down-regulation of Runx2 following 24 h of tion (Figs. 1–3). 0.02% O (Fig. 5b). The more conservative down-regulation 2 40012 Anoxia Inhibits Osteogenic Differentiation TABLE I Genes expressed 2 S.D. from array mean at both 0.02 and 2% oxygen % Change relative to 21% oxygen Class Gene symbol Accession no. 0.02 2 Angiogenesis Adrenomedullin Adm BG063461 350 110 Integrin 1 (fibronectin receptor ) Itgb1 AW544628 470 130 Vascular endothelial growth factor A Vegfa AW913188 420 190 Cellular stress response Heat shock 70-kDa protein 5 (glucose-regulated protein, 78 kDa) Hspa5 AV171683 1720 50 Homocysteine-inducible, endoplasmic reticulum stress-inducible, Herpud1 AV086303 1920 60 ubiquitin-like domain member 1 Glycolytic Aldolase 1, A isoform Aldo1 AI323970 510 220 Enolase 1, non-neuron Eno1 AI839722 490 330 Enolase 3, muscle Eno3 AA162439 310 130 Glucose phosphate isomerase 1 Gpi1 AV051832 320 290 Glyceraldehyde-3-phosphate dehydrogenase Gapd AV005835 610 220 Inducible 6-phosphofructo-2-kinase Pfkfb3 AA107038 580 150 Lactate dehydrogenase 1, A chain Ldh1 AV094945 360 230 Phosphofructokinase, liver, B-type Pfkl AV050313 580 450 Phosphofructokinase, platelet Pfkp AV025773 400 230 Phosphoglycerate kinase 1 Pgk1 AI841020 1480 590 Phosphoglycerate mutase 1 Pgam1 AV074144 450 230 Triose-phosphate isomerase Tpi AV021585 370 300 Growth factors and inhibitors Heparin binding epidermal growth factor-like growth factor Hegfl AV032861 360 170 Insulin-like growth factor binding protein 4 Igfbp4 BG245853 350 180 Transforming growth factor-2 Tgfb2 AI117710 80 90 Hif1- hydroxylation EGL nine homolog 1 (C. elegans) Egln1 BG066218 860 150 Proline 4-hydroxylase, 1 polypeptide P4ha1 AW548258 690 130 Proline 4-hydroxylase, II polypeptide P4ha2 BG063134 610 130 Prolyl 4-hydroxylase, beta polypeptide P4hb BG073729 710 100 Immune signaling Chemokine (C-X-C motif) ligand 12 Cxcl12 AA162879 70 60 Lymphoid enhancer binding factor 1 Lef1 AA119479 290 190 Macrophage migration inhibitory factor Mif AA163695 470 180 Poliovirus receptor-related 2 Pvrl2 AV084691 390 50 Pre-B-cell colony-enhancing factor Pbef AV108470 660 130 Signaling intermediate in Toll pathway-evolutionarily conserved Sitpec AV061837 980 180 Matrix Basigin Bsg AI836069 640 140 Lamin A Lmna AV006486 70 90 Tissue inhibitor of metalloproteinase 3 Timp3 AV022861 70 90 Vinculin Vcl AV011931 80 110 Metabolic (non-glycolytic) ATP synthase, H transporting mitochondrial F1 complex Atp5b AV006330 610 310 Carbonic anhydrase 2 Car2 AW544570 540 290 Flavin containing monooxygenase 5 Fmo5 AV009454 410 250 Glycogen synthase 3, brain Gys3 AV012422 570 120 Nucleotide binding Interferon-stimulated protein (20 kDa) Isg20 AV064198 2130 320 Nuclease sensitive element binding protein 1 Nsep1 AV297118 810 50 Polymerase (DNA directed), 1 Pola1 AV095001 280 50 RNA binding motif protein 6 Rbm6 BG076295 70 50 Splicing factor 3a, subunit 2, 66 kDa Sf3a2 AV166430 970 50 Miscellaneous Apolipoprotein E Apoe AV030294 2200 250 BCL2/adenovirus interacting protein 1 Bnip3 BG074532 4780 460 Dickkopf homolog 1 (Xenopus laevis) Dkk1 BG064961 620 340 Hypoxia induced gene 1 Hig1 AV093606 2290 310 Kidney androgen regulated protein Kap AV002536 530 160 Microfibrillar associated protein 5 Mfap5 AV024041 270 390 Plasma membrane associated protein, S3–12 S3-12 AV006013 680 250 Tubulin 5 Tubb5 AV109614 410 320 noted by microarray (Fig. 5a) is consistent with prior analyses tional changes that could affect osteogenic differentiation. Fur- demonstrating that cDNA array data may underestimate the thermore, we sought to determine whether Runx2 protein lev- degree of change in expression when compared with QRT-PCR els were affected by brief anoxic exposure. Following induction (39, 40). of Runx2, proliferating osteoblasts deposit collagen I into the Anoxic Treatment Down-regulates Long-term Runx2 Expres- extracellular space. Osteoblasts in later stages of differentia- sion and Alters Patterns of Collagen I and Osteocalcin Expres- tion orchestrate matrix calcification and produce the late os- sion—Because osteoblast differentiation is a multistage proc- teogenic marker osteocalcin (12, 13). We therefore used QRT- ess that lasts up to one month in vitro, we next determined if PCR to assay osteoblast gene expression for Runx2, ColI, and transient hypoxia or anoxia resulted in long-term transcrip- Osteocalcin during long-term differentiation. Osteoblasts were Anoxia Inhibits Osteogenic Differentiation 40013 FIG.5. Effect of anoxia versus hypoxia on expression of Runx2 in calvarial osteoblasts. a, calvarial osteoblast microarray analysis revealed uniform down-regulation of Runx2 and other markers of os- teogenesis by anoxia but not 2% hypoxia (including alkaline phospha- tase and bone-associated collagens, which are downstream effectors of the Runx2 pathway). y axes represent the percentage change in gene expression from 21% for 2% O (gray bars) and 0.02% O (black bars). 2 2 In a, OP, osteopontin; and OG, osteoglycin. To confirm our microarray FIG.6. Relative expression in calvarial osteoblasts of Runx2 findings, we performed QRT-PCR using mouse osteoblasts following by QRT-PCR and Western blot (a) and collagen I (col I)(b) and 24 h of anoxic (b) or hypoxic (c) treatment, and after 6 –12 h of reoxy- osteocalcin (c) by QRT-PCR. Mouse osteoblasts were exposed to 24 h genation. In b, N,U indicates normoxic (21% O ) undifferentiated cells, 2 of normoxic/ambient air (21% O ) or anoxia (0.02% O ) and cultured 2 2 whereas A,U indicates anoxia-treated undifferentiated cells. In con- for 28 days in the presence or absence of osteogenic differentiation trast, N,D and A,D cells were placed in osteogenic differentiation me- media. N,U denotes normoxic (21% O ) undifferentiated cells, whereas dium from the beginning of the experiment. In c, H,U denotes 2% A,U indicates anoxia-treated undifferentiated cells. In contrast, N,D hypoxia-treated undifferentiated osteoblasts, whereas H,D reflects hy- and A,D cells underwent 24 h of normoxic or anoxic exposure followed poxia-treated cells in differentiation medium (described under “Mate- by osteogenic differentiation. †, p 0.05 for A,U compared with N,U at rials and Methods”). N,U and N,D are as described above. *, p 0.05 for 24 h (immediately following treatment). *, p 0.05 for A,D compared A,D compared with N,D groups at the same time point (paired t tests). with N,D groups at 7, 10, 14, and 28 days (paired t tests). Each bar Each bar represents the mean S.D. of three separate experiments. All represents the mean S.D. for three separate experiments. All QRT- values were normalized to 18 S expression. PCR values were normalized to 18 S expression. For Runx2 Western blot images, the normoxic band is shown on the left and the anoxic band on the right for each indicated time point pair (with the corresponding exposed for 24 h to 21 or 0.02% O then placed in fresh -actin control). maintenance or differentiation media for 28 days as described under “Materials and Methods.” Gene expression was analyzed immediately following treatment (24 h), and at 7, 10, 14, and 28 undergone initial anoxic treatment (Fig. 6a). In contrast, days (Fig. 6). Runx2 transcription was induced strongly in differentiating Interestingly, in addition to inhibition of Runx2 expression osteoblasts that were maintained in 21% O throughout the immediately following 24 h of 0.02% O , we noted a persistent experiment. As expected, Runx2 expression remained rela- attenuation of Runx2 in differentiating osteoblasts that had tively low in undifferentiated groups. Consistent with the tran- 40014 Anoxia Inhibits Osteogenic Differentiation scriptional data, Runx2 protein levels were decreased following anoxic treatment, and remained low throughout the 28-day differentiation period (Fig. 6a). Given that Runx2 binds cis-acting elements that activate ColI and Osteocalcin promoters, we sought to determine whether an- oxic exposure altered the expression profiles of these well char- acterized genes expressed by differentiating osteoblasts (12, 13). Analysis of ColI expression revealed an immediate anoxic inhib- itory effect at 24 h (Fig. 6b). Furthermore, similar to the results obtained for Runx2, differentiating osteoblasts exposed to 24 h of anoxia demonstrated attenuated ColI expression at 10, 14, and 28 days relative to cells that had not been anoxia-treated (Fig. 6b). Type I collagen expression in undifferentiated cells gradually decreased over 28 days, consistent with long-term culture of post-confluent osteoblasts (41). Transcription of the late osteogenic marker osteocalcin was also inhibited in differentiating osteoblasts throughout the 28 days following anoxic exposure (Fig. 6c, A, D). Whereas lower than their normoxic counterparts, osteocalcin levels, neverthe- less, increased in anoxia-treated osteoblasts between 14 and 28 days. This was consistent with the diminished (but not absent) bone nodule staining after anoxic treatment observed in Figs. 1 and 2. Short-term Restoration of Runx2 Expression by BMP2 Ad- ministration Rescues Anoxic Inhibition of Differentiation—Be- cause Runx2 is essential to osteogenic differentiation, several growth factors that influence osteoblast differentiation have, accordingly, been implicated in regulating its expression, in- cluding TGF-, insulin-like growth factor, and fibroblast growth factor family members (12, 42). We therefore examined our microarray data set for mediators of osteogenesis to define the expression profile of potential upstream regulators of Runx2. Interestingly, we noted differential regulation of mem- bers of the transforming growth factor superfamily, including several bone morphogenic proteins (Fig. 7a). Specifically, we observed a 72% anoxic down-regulation of BMP2, an activator of Runx2 transcription (via Smad-mediated signaling) with a well established role in bone development and repair (40, 43, 44). TGF-2 was also significantly repressed by anoxia (by 80%) (Fig. 7a). In contrast to BMP2, TGF- signaling has been FIG.7. Exogenous administration of recombinant Bmp2 res- shown to repress Runx2 transcription in differentiating pri- cues the anoxic down-regulation of runx2 transcriptional levels mary calvarial osteoblasts (45). Additional paracrine and au- and long-term inhibition of differentiation. a, microarray profiling of transforming growth factor- and bone-morphogenic protein mem- tocrine growth factors implicated in osteoblast differentiation bers at 2 and 0.02% O . Relative to 21% O , TGF-1 levels were not 2 2 (including insulin-like growth factor-1 and -2, and fibroblast significantly altered, whereas TGF-2 was repressed under anoxia. Of growth factor-1 and -2 genes) were not significantly changed in the prosteogenic bmps, only BMP2 and (to a lesser degree) BMP4 were our 2 and 0.02% microarray data sets (data not shown). significantly down-regulated by anoxia. b, QRT-PCR of Runx2 following 24 h of calvarial osteoblast stimulation with the indicated cytokines or We hypothesized that anoxia-mediated inhibition of BMP2 vehicle, in the presence or absence of anoxia. Only BMP2 resulted in expression may contribute to the anoxic down-regulation of sustained Runx2 mRNA levels despite anoxic exposure. All QRT-PCR Runx2. Because Bmp2 can up-regulate Runx2 expression in values were normalized to 18 S expression. c, representative photomi- certain systems, we sought to determine whether restoring crographs of von Kossa-stained osteoblasts that were pre-treated with the indicated cytokines during anoxic exposure, followed by 28 days of BMP2 levels during the period of brief anoxic exposure could differentiation. Normoxic and anoxic vehicle-treated osteoblasts are “rescue” Runx2 expression in primary osteoblasts (9, 46). We also shown. Relative staining was quantitated as described under “Ma- therefore administered recombinant BMP2 or vehicle to calvar- terials and Methods” using low-magnification (whole plate) images. ial osteoblasts and exposed them immediately to 24 h of 21 or 0.02% O . QRT-PCR analysis revealed that anoxic osteo- 2 kine stimulation experiments using both TGF-1 and TGF-2. blasts treated with vehicle exhibited a significant decrease in TGF-1 treatment did not prevent inhibition of Runx2 by an- Runx2 mRNA level, consistent with our findings above (Fig. oxia and, in fact, resulted in decreased Runx2 during normoxia 7b). In contrast, anoxic osteoblasts pretreated with BMP2 ex- as well, consistent with prior evidence implicating TGF--me- hibited similar Runx2 mRNA levels to normoxic vehicle-treated diated repression of Runx2 transcription (45). Also, TGF-2 cells, indicating that BMP2 administration can “rescue” anox- administration resulted in anoxic inhibition of Runx2 similar to ia-mediated repression of Runx2 levels (Fig. 7b). Interestingly, the vehicle-treated group (Fig. 7b). These findings confirmed Runx2 expression in anoxic BMP2-treated osteoblasts did not that restoration of Runx2 mRNA levels was specific to Bmp2 achieve levels observed in normoxic BMP-2-stimulated osteo- pre-treatment. blasts (Fig. 7b). Given that BMP2 administration “rescued” Runx2 mRNA lev- Given that our microarray analysis revealed a significant els despite anoxic exposure, we asked whether BMP2 could re- inhibition of TGF-2 with anoxia, we performed parallel cyto- store the ability of anoxia-treated osteoblasts to undergo long- Anoxia Inhibits Osteogenic Differentiation 40015 term differentiation. Thus, we pre-treated osteoblasts with Runx2 are deficient in functional osteoblasts, hypertrophic car- BMP2 prior to 24 h of anoxic exposure, then maintained them in tilage, and mineralized bone (48). Mutations in the Runx2 locus osteogenic differentiation media for 28 days at 21% O (without in humans results in cleidocranial dysplasia, characterized by additional BMP2). Consistent with the aforementioned restora- the absence of clavicles, open fontanelles, and short stature, tion of Runx2 mRNA levels by BMP2 (in the setting of anoxia), among other osteogenic defects (49). we observed normal bone nodule formation in BMP2 pre-treated In our in vitro system, calvarial osteoblast and MSC expo- osteoblasts (Fig. 7c). Administration of TGF-1 or TGF-2 (which sure to anoxia was associated with profound inhibition of had not prevented the anoxic down-regulation of Runx2) did not Runx2, without evidence of significant recovery during 12 h of negate the anoxic inhibition of differentiation (Fig. 7c). These reoxygenation. Interestingly, we also observed a long-term data further implicate Runx2 modulation, along with that of its down-regulation of Runx2 mRNA and protein (as well as tran- upstream activator BMP2, as critical to the oxygen-dependent scriptional levels of two of its targets, ColI and Osteocalcin). In regulation of osteoblast differentiation. contrast, exposure to 2% O neither inhibited bone nodule formation nor altered Runx2 expression. Furthermore, unlike DISCUSSION in primary calvarial osteoblasts and MSCs, anoxia did not As a first step to understanding osteoprogenitor behavior in inhibit the differentiation of MC3T3-E1 cells. It has been the setting of a reduced oxygen microenvironment, we have shown in the MC3T3-E1 cell line that Runx2 expression may investigated the effects of three oxygen levels (21, 2, and not correlate with differentiation potential or induction of os- 0.02% O ) on an immortalized cell line and two primary cell teogenic markers, i.e. MC3T3-E1 cells that express relatively types involved in bone repair. We demonstrated that mouse high levels of Runx2 may not go onto form bone nodules or primary osteoblasts and human bone marrow-derived mesen- express osteocalcin (19). The dissociation between Runx2 ex- chymal cells, but not the cell line MC3T3-E1, were sensitive to pression and osteogenic differentiation in the MC3T3-E1 cell anoxia. Anoxic (but not 2% hypoxic) insult was associated with line (which was not inhibited by anoxia) underscores the im- diminished in vitro osteogenesis without inducing necrosis or portance of Runx2 as a potential mediator of anoxic inhibition apoptosis during 28 days of differentiation. of differentiation in primary osteoblasts. Differences in the long-term phenotypic response to 24 h of 2 To further investigate if Runx2 down-regulation is a critical versus 0.02% O prompted us to explore the transcriptional step in the anoxic inhibition of differentiation, we examined profiles of osteoblasts following this brief treatment period. whether abrogating the decrease in Runx2 expression that Anoxic osteoblasts not only had a more drastic transcriptional occurs with anoxia could restore the ability of osteoblasts to response (e.g. more marked glycolytic induction) but also acti- differentiate. Whereas Runx2 may be responsive to multiple vated pathways distinct from their hypoxic counterparts. For signal transduction pathways, a complete understanding of example, specific heat-shock elements were activated in anoxic up-stream regulators of Runx2 transcription remains to be cells, suggesting that 0.02% O , but not 2% O , induced a 2 2 determined (12, 42). This prompted us to further focus our selective cell stress response. Of the numerous heat shock microarray analysis on known osteogenic mediators, and re- proteins examined, only Hspa5 and Hsp30 were up-regulated vealed differential regulation of members of the transforming by anoxia. Whereas Hspa5 has been shown to be up-regulated growth factor superfamily. Specifically, we noted marked under reduced oxygen, Hsp30 has not previously been impli- down-regulation of TGF-2 and BMP2, with no significant cated in hypoxic stress in mammalian cells. Interestingly, stud- alterations in TGF-1 and several BMP family members. ies in Saccharomyces cerevisiae suggest that Hsp30 is unique Whereas several isoforms of TGF- are expressed by osteo- among the heat shock protein family, for its induction does not blasts and have key roles in bone development and remodeling, appear to require the same transcriptional apparatus shared the effect of TGF- signaling upon Runx2 expression remains by other heat shock genes (47). Perhaps the selective activation unclear. TGF- has been shown to repress Runx2 transcription of Hsp30 by anoxia in our system may suggest a separate in differentiating primary calvarial osteoblasts while having pathway for activation in mammalian cells as well, although the opposite effect in myoblasts (45, 46). Whereas TGF-1 was this would require subsequent investigation. not significantly altered by hypoxia or anoxia, we found that Perhaps the most interesting finding of our microarray anal- anoxia inhibited TGF-2 mRNA levels. Pre-treatment with ysis was a uniform down-regulation of Runx2, bone-associated either TGF- isoform did not ameliorate the anoxic inhibition collagens, and additional markers of osteoblast differentiation of Runx2 or osteogenic differentiation. In fact, TGF-1 admin- following 24 h of anoxia. As mentioned earlier, the behavior of istration resulted in decreased Runx2 levels in both normoxic osteoprogenitors in a low oxygen microenvironment has not and anoxic osteoblasts, and thus neither isoform was able to been well characterized, and differences in proliferative and rescue the anoxic inhibition of differentiation. These data sug- differentiation responses have been observed for primary ver- gest that anoxic regulation of TGF- is not a likely mediator of sus immortalized osteogenic cells. This was true in our study as anoxic inhibition of Runx2 expression and osteoblast differen- well; whereas primary calvarial osteoblasts and mesenchymal tiation in our system. stem cells were quite sensitive to anoxic insult, the MC3T3-E1 In contrast to TGF-, BMP2 is a well established inducer of cell line was not. Whereas the mechanism for this differential osteogenic differentiation in multiple cell types (12, 50). Given response to anoxia is unclear, this finding may allude to the our finding of significant anoxic down-regulation of BMP2,we inherent differences between primary and immortalized cells further evaluated its potential role in anoxic regulation of in their response to cell stress, and/or to the regulation of Runx2 expression. Indeed, we found that treatment with BMP2 Runx2 expression, as discussed below (18, 19). (but not TGF-1or-2) just prior to anoxic exposure restored We present evidence that down-regulation of Runx2,anes- Runx2 expression to the levels observed with normoxic un- sential mediator of osteoblast differentiation, may explain the stimulated osteoblasts. However, exposure of BMP2-treated inhibition of osteogenic differentiation by anoxia. Without nor- mal Runx2 expression, osteoblasts cannot differentiate (9, 13), osteoblasts to anoxia resulted in lower Runx2 expression levels compared with normoxic BMP2-stimulated cells. A possible for Runx2 acts as a master gene that regulates the expression of a number of osteogenic markers including collagen I, os- explanation for the persistent anoxia-mediated decrease in Runx2 mRNA despite BMP2 administration is that anoxia may teopontin, and osteocalcin (11, 12). The critical role of Runx2 is well established in vivo as well. 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Journal
Journal of Biological Chemistry – Unpaywall
Published: Sep 1, 2004