1Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta; 2Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; 3Amersham Biosciences, Piscataway, New Jersey; and 4Dutch Experiment Support Center, Department of Oral Biology, Academic Center for Dentistry Amsterdam, Free University, Amsterdam, The Netherlands
Submitted 5 May 2004 ; accepted in final form 29 January 2005
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ABSTRACT |
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microarray; bone loss; alkaline phosphatase; runx2; osteomodulin
Unfortunately, it has been difficult and impractical to conduct well-controlled in vitro studies in sufficient numbers in real microgravity conditions because of the limited and expensive nature of spaceflight missions. Thus, to investigate pathophysiology during spaceflight, several ground-based systems, including the two-dimensional (2-D) and three-dimensional (3-D) clinostats and the rotating wall vessel, have been developed to simulate microgravity using cultured cells and tissues (1, 19, 24, 29). Simulated microgravity is based on the hypothesis that sensing no weight would have effects similar to those of weightlessness (13). The 3-D clinostat simulates microgravity by continuously moving the gravity vector in three dimensions before the cell has enough time to sense it, which is a method called gravity-vector averaging.
Previous studies have indicated that spaceflight-induced bone loss may be due in part to decreased osteoblastic function with or without enhancing osteoclastic bone resorption (8). Using 2-D clinostats and rotating wall vessels, simulated microgravity has been shown to inhibit markers of bone mass formation such as alkaline phosphatase (alp) activity and runt-related transcription factor 2 (runx2) activity (24, 42). While these studies examined only a few candidate genes that are likely to be involved in bone mass regulation, systematic and unbiased characterization of gene expression profiles scanning the majority of genes has not been performed. In the present study, we hypothesized that bone loss due to simulated microgravity is regulated by preosteoblastic gene expression, which inhibits differentiation of the preosteoblasts into mature osteoblasts.
To test this hypothesis, we 1) developed and characterized an in vitro cell culture system using preosteoblast cells (2T3) exposed to simulated microgravity conditions produced by a 3-D clinostat called the Random Positioning Machine (RPM), 2) examined the cell proliferation and the alkaline phosphatase activities of 2T3 cells, 3) performed DNA microarray studies, and 4) validated the microarray data using quantitative real-time PCR and immunoblotting.
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METHODS |
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Cell seeding into OptiCells and simulated microgravity studies. Confluent 2T3 cells grown in T-75 flasks were trypsinized using 0.05% trypsin-EDTA (Sigma), and 2 million cells were seeded into a gas-permeable cell culture disk (OptiCell) according to the manufacturer's instructions. As shown in Fig. 1A, an OptiCell disk is a sealed cell culture disk encapsulated by two optically clear, gas-permeable polystyrene membranes containing two ports that allow access to the contents of the OptiCell. The internal disk dimensions are 74.8 x 65 x 2.06 mm, and they can be filled with 1014 ml of medium. To seed cells on both membranes on each side of the OptiCell, the disks were turned over every 5 min for 1 h. Cells were then grown for 3 days to confluence in 14 ml of growth medium before exposure to the stimulus. The day on which the OptiCells were mounted on the RPM was referred to as day 0. On day 0, the medium was replaced with 14 ml of fresh growth medium, and all of the air bubbles were removed. We found removing the air bubbles to be a critical step in preventing potentially uncharacterized mechanical perturbation during RPM exposure.
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Cell proliferation assay. To determine cell proliferation, attached cells were collected by trypsinization after experimental treatments, and cell numbers were determined using an aliquot of cell suspension and counted with a Coulter counter.
Alkaline phosphatase assay. After collecting culture medium following exposure to the simulated gravity, cells were scraped in 500 µl of lysis buffer containing 0.2% Nonidet P-40 in 1 mM MgCl2 and stored at 80°C until needed. Alkaline phosphatase (ALP) activity was determined using a Diagnostics ALP assay kit (Sigma) according to the manufacturer's instructions (26). Aliquots of lysate (20 µl) and p-nitrophenol standard (Sigma) were used for the assay.
RNA isolation, reverse transcription, and quantitative real-time PCR. Total RNA was prepared by using the RNeasy Mini kit (Qiagen) and reverse transcribed by using random primers and a SuperScript II kit (Life Technologies) (31). The synthesized and purified cDNA was amplified using a LightCycler (Roche Applied Science), and the size of each PCR product was verified by performing agarose gel electrophoresis as we described previously (31). The mRNA copy numbers were determined on the basis of standard curves generated with the genes of interest and 18S templates. The 18S primers (50 nM at 61°C annealing temperature; Ambion) were used as an internal control for real-time PCR using capillaries (Roche Applied Science), recombinant Taq polymerase (Invitrogen), and Taq start antibody (Clontech). The primer pairs for the quantitative real-time PCR are listed in Table 1 along with their annealing temperatures, extension times, and base pair yields. Real-time PCR for the listed genes was performed in PCR buffer (20 mM Tris-Cl, pH 8.4, 25°C, and 4 mM MgCl2, to which was added 250 µg/ml bovine serum albumin and 200 µM deoxynucleotides) containing SYBR Green (1:84,000 dilution), 0.05 U/µl Taq DNA polymerase, and Taq Start antibody (1:100 dilution) as we described previously (31).
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Stress and strain analyses.
The attached cells grown on the OptiCell membranes could be exposed to mechanical forces such as fluid shear stress and strain in addition to simulated microgravity during the RPM rotation. To visualize the dynamics of the fluid within the OptiCell, we marked the OptiCell disks with calibrated grids and filled them with water containing colored bead markers (1.018 g/ml density; Amersham Biosciences) with a density close to that of water. Short movies were then recorded using a digital camera mounted on the RPM to track the bead movements over precalibrated grids to estimate flow velocities. The shear stress () was calculated using Newton's law of viscosity as follows:
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Another potential force that cells in the OptiCell disks may experience during the random rotations of the RPM is strain caused by the momentum force of the fluid exerted on the membranes due to sudden directional changes. The strain () was calculated using the 1-D wave equation with fixed boundary conditions at both ends of the OptiCell frame. In addition, we assumed that the maximum height of the stretched membrane (h) was located at half the membrane length (L). The static solution to this wave equation for the first harmonic with the above constraints can be described as follows:
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We calculated the arc for the filled OptiCell when RPM was not rotating, where the height is equal to h, and then the arc at h + h, where
h is the change in gap height due to additional membrane stretch occurring during sudden directional changes by the RPM rotation. The strain (
) of the membrane along the short and long sides was calculated as follows:
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Statistical analysis. Statistical analysis was performed using Student's t-test for all experiments. A significance level of P < 0.05 from three or more independent experiments was considered statistically significant.
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RESULTS |
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To examine the effects of simulated microgravity on osteoblasts, we developed an in vitro system to expose 2T3 cells grown in gas-permeable culture disks (OptiCell) to the RPM. The morphologies of 2T3 cells grown in standard tissue culture dishes and OptiCells were indistinguishable (data not shown). When cells grown in OptiCells were exposed to the simulated microgravity or static 1-g conditions, the pH of the medium remained neutral at or near pH 7.4 (data not shown). As shown in Fig. 2A, the morphologies of 2T3 cells exposed to static 1-g controls and the RPM were not significantly different from each other.
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Exposure of 2T3 cells to simulated microgravity inhibits alkaline phosphatase activity. Because alkaline phosphatase is an established marker for osteoblast differentiation and bone mass formation (39), we chose to determine whether exposing 2T3 cells to simulated microgravity using the RPM would inhibit enzyme activity. As shown in Fig. 3, alkaline phosphatase activity of 2T3 cells increased during culture as expected. The alkaline phosphatase activity of the static-cultured cells dramatically increased more than eightfold within 2 days between days 1 and 3. By day 5, the activity in control cells reached a maximum (24 ± 1 µmol·min1·mg of protein1), which remained at the maximum at day 7. In contrast, exposure of 2T3 cells to the RPM significantly blunted the culture time-dependent increase in alkaline phosphatase activity (Fig. 3). Unlike the static control group, the enzyme activity of the RPM group at day 3 did not increase significantly above the day 1 level. By day 9, the alkaline phosphatase activity was fourfold that of the day 1 level. As shown in Fig. 3, the enzyme activity of the static 1-g group was 2.7 times higher than that of the RPM group at day 9. This finding that simulated microgravity significantly decreased alkaline phosphatase activity is consistent with the inhibitory effects of microgravity on osteoblast differentiation and bone formation.
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These results are consistent with the notion that simulated microgravity decreases the expression of genes necessary for differentiation, matrix formation, and subsequent mineralization while increasing the expression of genes that trigger osteoclast activity.
CodeLink bioarray was verified using quantitative reverse transcriptase real-time PCR. To verify the results of the microarray studies using real-time PCR, we decided to choose genes that have been shown to be involved in osteoblast differentiation and bone mass regulation. The same samples used for the CodeLink bioarray assays were used for real-time PCR. All real-time PCR data shown in Fig. 5, AE, were normalized to the internal control, 18S, and the relative changes were determined by dividing the amount of a gene exposed to simulated microgravity by the static 1-g control (Fig. 5F). 2T3 cells exposed to simulated microgravity had decreases in alp, runx2, pthr1, and osteomodulin (omd) gene expression of 0.2, 0.5, 0.2, and 0.2-fold, respectively, as evaluated using the CodeLink bioarray. In addition, 2T3 cells showed an increase in ctsk of 1.66-fold. To verify these results, quantitative RT-PCR was performed. While the primers used for omd and ctsk were designed by us, the other primers used for quantitative real-time PCR were as described in the publications listed in Table 1. The gene expression changes revealed using real-time PCR for alp, runx2, pthr1, and omd were 0.2, 0.7, 0.3, and 0.2-fold, respectively. The change in ctsk revealed using RT-PCR was 1.67-fold. In addition, we confirmed the expression of nongravisensitive genes such as bone morphogenic protein 4 (BMP4) and cystatin C (cys C) using immunoblotting. We show that the changes for BMP4 and cys C determined using Western blot analysis were 1.12- and 1.13-fold, respectively, and that those found using Codelink were 1.03- and 0.71-fold, respectively (Fig. 5G). The different methodologies used (real-time PCR, immunoblotting, and microarray assay) produced highly consistent results, providing a level of assurance regarding the validity of the microarray data.
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DISCUSSION |
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Microgravity-induced bone loss in humans and animals has been shown to be mediated at least in part by osteoblast differentiation, and alkaline phosphatase is a well-known marker for it (3, 6, 20). Our finding that 2T3 cell exposure to the RPM decreased alp and runx2 expression is consistent with the spaceflight data obtained with osteoblasts as well as with other simulated microgravity data using cultured calvaria, human mesenchymal stem cells, and MC3T3-E1 cells exposed to a rotating wall vessel (5, 7, 25, 42). The decrease in alkaline phosphatase activity could be used as an indicator of spaceflight-induced inhibition of preosteoblast differentiation to osteoblasts, leading to bone loss (10). The runx2 gene, a member of the runt homology domain transcription factor family, which regulates osteocalcin, is an essential transcription factor for osteoblast differentiation and bone formation. We found that runx2 was downregulated almost twofold below the static 1-g control. Osteocalcin protein levels in conditioned medium, however, were too low to be detected in our studies because of a relatively short experimental duration of up to 9 days, and this finding is consistent with the findings described in a previous report (11). On the other hand, osteocalcin level was shown to be decreased by exposure to rotating wall vessel in mouse calvaria and a different osteoblast cell line, MC3T3-E1 (25, 42). Osteomodulin belongs to a small, leucine-rich proteoglycan family and is involved in bone matrix formation (4). Our present study showing the downregulation of osteomodulin by simulated microgravity supports our hypothesis. The decrease in parathyroid hormone-related protein, which plays a role in Ca2+ mobilization, has been linked to the decrease in bone density and bone loss in rats during spaceflight (32). In this light, our result demonstrating that PTH receptor 1 mRNA levels decrease by simulated microgravity is also consistent with the spaceflight data. In addition to the downregulated genes, several genes and proteins were upregulated. Cathepsin K, which is a member of the papain family of cysteine proteases, is expressed mostly in osteoclasts and plays a critical role in bone resorption (22, 41). More recently, however, cathepsin K has been found in nonosteoclastic cells such as thyroid epithelial cells (41). As far as we know, our present study represents the first time that cathepsin K expression has been found in osteoblasts. At present, the biological and pathophysiological implications of cathepsin K expression in osteoblasts are not clear. Cathepsin K induced by simulated microgravity could be responsible for bone loss either by directly increasing osteoclastic activity or through an indirect osteoblast-dependent mechanism.
We also examined some genes that were not shown to change in response to simulated microgravity according to the microarray data. For example, BMP-4 and cys C mRNA levels in the RPM-exposed group showed 1.0- and 0.7-fold changes, respectively, over the controls. We performed Western blot analysis to examine their protein expression levels with specific antibodies to BMP-4, cys C, and actin (as a control) using lysates obtained from 2T3 cells exposed to 3 days of simulated microgravity or static 1-g control conditions. As expected, we did not find any significant difference in their protein expression levels (Fig. 5).
In addition to simulated microgravity, cells in our in vitro system showed a low level of shear stress and strain on the basis of our computational modeling studies. Our model predicts a minor portion of the cells close to the long frames of the OptiCell disk showing significantly less than 1 dyn/cm2 of shear stress for a brief moment (<4 s/min) and <200 microstrains of mechanical strain for a fraction of the duration of the RPM's rotation. However, the levels of these forces are significantly lower than the reported force magnitudes (as low as 2 dyn/cm2 shear stress and 500 microstrains) needed to stimulate signaling in osteoblasts (16, 17, 21, 28). However, it also was shown that as low as 0.14 dyn/cm2 of continuous shear exposure for 4 h increased cyclooxygenase 2 expression (35). Therefore, our results need to be interpreted with the caution that at least some of the observed RPM effects may be due to mechanical forces other than simulated microgravity.
In summary, we have developed a novel in vitro system using RPM and OptiCell disks with 2T3 cells, which seemed to recapitulate the bone loss-like response under microgravity conditions during spaceflight. Our data show that exposure to simulated microgravity changed gene expression profiles and the inhibition of differentiation of preosteoblasts to osteoblasts, eventually leading to reduced bone formation. At this point, whether these two events, differentiation and gene expression change, occur in sequence or concurrently is not clear. It is likely, however, these two events are closely interrelated. In addition to tabulating known and expected genes (e.g., alp, runx2, omd, pthr1, and ctsk), we have developed a list of unknown and uncharacterized genes that dramatically changed after exposure of 2T3 cells to simulated microgravity. The functional characterization of these expected and unexpected genes will provide critical insight into understanding the mechanisms of microgravity-induced bone loss. Moreover, these studies may lead to the identification of the novel targets of therapeutic interventions to prevent or to cure bone loss in astronauts as well as in the general patient population with metabolic bone diseases.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* S. J. Pardo and M. J. Patel contributed equally to this work.
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