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Keywords:

  • glucocorticoids;
  • glucocorticoid receptor antagonist (RU486);
  • mesenchymal stem/stromal cells;
  • proliferation;
  • differentiation;
  • ovariectomy

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Glucocorticoids (GCs) potentially regulate the proliferation, differentiation, and premature senescence of bone marrow mesenchymal stem/stromal cells (MSCs). In the present study we investigated the effects mediated by endogenous GCs and the effects of an antagonist of the glucocorticoid receptor, RU486, on the proliferative and differentiation capabilities of MSCs using an ovariectomized (OVX) animal model. Following ovariectomy and a decrease in systemic estradiol levels, the serum concentration of corticosterone is significantly increased in OVX rats. Compared to sham-operated controls, the total superoxide dismutase (SOD) activity in serum of OVX rats and the proliferation of their MSCs are significantly reduced. Furthermore, the osteogenic differentiation capabilities of OVX rat MSCs are significantly decreased, while adipogenic capabilities tend to increase. Subcutaneous administration of RU486 effectively increases the population and proliferative capacity of the MSCs in OVX rats. RU486 treatment also improves osteogenic capabilities and down-regulates adipogenic capabilities of MSCs. These results strongly indicate that the elevated levels of endogenous GCs induced by estrogen depletion might accelerate the premature senescence of MSCs and reduce their proliferative and osteogenic differentiation capabilities, while the blockage of the effects of endogenous GCs may restore their capabilities. These responses could potentially be developed to protect the capabilities of MSCs from oxidative stress-induced premature senescence and extend their lifespan in patients with advancing age and estrogen depletion. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 760–767, 2013

Glucocorticoids (GCs) have been shown to participate in the regulation of multiple differentiations of bone marrow mesenchymal stem/stromal MSCs and inhibit their proliferation.1, 2 These adrenal steroids potentially reduce telomerase activity during differentiation and shorten telomere length.3–5 In addition, the effects mediated by GCs may accelerate cell aging by increasing oxidative stress and down-regulating the levels of antioxidants.6–8 Our previous publications clearly demonstrated that the blockage of the effects mediated by the endogenous GCs contained in fetal bovine serum using RU486, which is an antagonist of the glucocorticoid receptor (GR), or the small interfering RNA of GR, significantly improve the proliferative and osteochondral differentiation capabilities of human MSCs in vitro.9, 10 In contrast, dexamethasone (synthetic GC) treatment reduces MSC proliferation and down-regulates the expression of fibroblast growth factor (FGF)-2 which is a stemness gene that inhibits senescence.11 These data suggest that GCs may accelerate the premature senescence of MSCs and the blockage of endogenous GCs thus potentially improves MSC proliferation and osteochondral differentiation capabilities by postponing the onset of senescence in these stem cells.

Ovariectomized (OVX) rats are a well-established animal model characterized by a deficiency of endogenous estrogens and reduced activities of antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S transferase (GST).12, 13 Enhanced responses mediated by elevated levels of endogenous GCs have been also reported in these OVX rats.14 Bone marrow MSCs in OVX rats are characterized by decreased proliferation and osteogenic capabilities and increased adipogenic differentiation, which are similar properties to those detected in MSCs from patients with advanced age and estrogen depletion.15, 16 These observations indicate that OVX rats can be used to investigate the effects of endogenous GCs and their blockage on the regulation of bone marrow MSCs. In this study, we investigated the effects of endogenous GCs and their blockage with RU486 treatment on MSC proliferative and differentiation capabilities using an OVX rat model. We observed that RU486 treatment effectively restores the proliferative and differentiation capabilities of MSCs of OVX rats and blocks the reduction in antioxidant levels in OVX rats.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

OVX Rats, RU486 Treatment, and Bone Marrow MSC Preparation

Female Fisher-344 rats (6 months old) that were ovariectomized or sham operated were purchased from the Charles River Laboratory (Wilmington, MA). RU486 (Sigma, St Louis, MO) was suspended in olive oil at 10 mg/ml and injected subcutaneously at a dose of 10 mg/kg body weight twice a week. This dosage has been demonstrated to effectively block the effects mediated by endogenous GCs in OVX rats.14 Rats injected with vehicle alone served as controls in all experiments. Blood samples from rats that had received different treatments were collected to measure the levels of antioxidant activity and steroid hormone after 6 weeks. In order to avoid the diurnal variations in plasma corticosterone level that occur during the course of a day, all blood samples were collected at the same time (9–10 a.m.) on experimental days. Rats were subsequently euthanized by CO2 inhalation. Bone marrow plugs from tibias and femurs were flushed out using PBS as described in our previous studies.17 Cells were placed in a 100-mm petri dish and incubated with Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen, Madison, WI) and 1% antibiotic-antimycotic (Sigma) at 37°C in 5% CO2. After 48 h nonadhesive cells were removed using medium exchange and the MSCs were cultured in vitro. The sample size is six for all experimental groups and each measurement was triplet.

Quantitation of Serum Steroid and SOD Levels

Serum estradiol and corticosterone levels in OVX rats, the dominant estrogens and GCs in rodents, were measured using commercially available EIA kits according to the manufacturer's instructions (ENZO Life Sciences, Farmingdale, NY). The activity of SOD in rat serum, which is a key antioxidant, was evaluated by measuring the dismutation of superoxide radicals generated by xanthine oxidase and hypoxanthine using a commercial kit according to the manufacturer's protocol (Cayman Chemical, Ann Arbor, MI).

Measurement of the Population Size and Proliferative Capacity of MSCs

MSCs were collected 1 week after in vitro culture and counted using a cytometer to determine their population size in rats receiving different treatments. The MSC isolated from one tibia and one femur from each animal represent the population size of the cells. The MSCs were subsequently plated in 96-well plates at 1,000 cells/well. Total DNA was quantitated every other day using a DNA quantification kit (FluoReporter® Blue Fluorometric dsDNA Quantitation Kit, Invitrogen) according to the manufacturer's protocols. The difference in total DNA content as measured every 2 days was used to determine the proliferation rate of MSCs from OVX rats that had received different treatments as described in our previous studies.9, 10

Measurement of Differentiation Capabilities of MSCs

The differentiation capabilities of rat MSCs were analyzed after the cells were cultured in 6-well (104 cells/well) plates and exposed to osteogenic or adipogenic differentiation medium for up to 3 weeks.17 The differentiation medium for osteogenesis consisted of the basic medium supplemented with 10 nM dexamethasone, 10 mM β-glycerophosphate, and 0.05 mM ascorbic acid-2-phosphate. For adipogenesis, the medium was supplemented with 50 nM dexamethasone, 10 µM insulin, and 0.5 mM isobutyl-methylxanthine. We used real-time polymerase chain reaction (PCR) to quantify the expression of genes for specific markers of MSC differentiations. Expression of alkaline phosphatase (ALP), core-binding factor alpha (Cbfa)-1, and osteocalcin (OCN) were used to indicate osteogenic differentiation of MSCs. Expression of peroxisome proliferator-activated receptors (PPAR)-γ2 and lipoprotein lipase (LPL) were used to indicate adipogenic differentiation. The real-time PCR primers and probes for multiple differentiation markers of were described in our previous study.17 The Syber Green Real-time PCR primers and probe for human GAPDH were purchased commercially (ABI, CA). In addition, the ALP, calcium content, and lipid accumulation of MSCs following osteogenic or adipogenic differentiation were also measured in triplicate as described in our previous studies.18

Statistics

All quantitative data are expressed as mean ± standard deviations. One-way ANOVA was used to analyze the differences, including steroid levels, SOD and MSC characteristics, capabilities and senescence, between sham operated controls, OVX rats and OVX rats receiving RU486 treatments with commercially available statistical software (SPSS, Inc., Chicago, IL). p values less than 0.05 was considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Body weight, Steroid Levels and SOD Activity of OVX Rats Treated With RU486

Six weeks after ovariectomy rats have significantly increased body weights and lower serum estradiol levels compared to sham-operated controls (Fig. 1A and B). The total SOD activity, a key antioxidant in OVX rats is also significantly reduced (Fig. 1C). However, serum corticosterone levels, a dominant endogenous GC in rodents, in OVX rats are almost threefold higher than those in control rats (Fig. 1D). While treatment with RU486 had no detectable effects on weight, the total SOD activity in serum of OVX rats treated with RU486 are significantly higher than OVX rats without RU486 treatment (Fig. 1C). RU486 treatment slightly increases levels of endogenous estradiol as well as corticosterone. However, these increases were not statistically significant due to sample variations.

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Figure 1. The characteristics of rats after received different treatments. (A) Body weight; (B) serum estradiol; (C) serum total SOD activity; (D) serum corticosterone. N = 6; mean ± SD; *p < 0.05.

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The Population Size and Proliferative Capabilities of MSCs From OVX Rats Treated With RU486

Figure 2 shows the number and proliferation rate of bone marrow MSCs from OVX rats that received different treatments. While no statistical significance was observed between the number of MSC isolated from OVX rats compared to sham operated controls, the number of MSCs isolated from one femur and one tibia of each OVX rat treated with RU486 were significantly higher than that of OVX rats treated with vehicle (Fig. 2A). The proliferation rates of MSCs from OVX rats indicated by the difference in total DNA content were significantly lower than those detected in sham-operated controls (Fig. 2B). However, RU486 treatment significantly improved the proliferation rate of MSCs from OVX rats (Fig. 2B).

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Figure 2. The population size and proliferative capabilities of MSCs from rats 6 weeks after received different treatments. (A) The MSC number isolated from a pair of femur and tibia of rats; (B) Percentage of increased DNA amount of MSCs after 48 h. N = 6; mean ± SD; *p < 0.05.

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Differentiation Capabilities of MSCs From OVX Rats Treated With RU486

After exposure to osteogenic differentiation medium, mineral deposition indicated by black dots in von-Kossa staining detected in MSCs from sham-operated control (Fig. 3A and D) and OVX rats treated with RU486 (Fig. 3C and F) were increased, while no detectable change was observed in MSC from OVX rats (Fig. 3B and E). Quantitatively, the mRNA expression for osteogenic markers, including ALP and OCN, of MSCs isolated from OVX rats were significantly lower than those of sham-operated controls (Fig. 4A–C). Expression of the mRNA for Cbfa-1 also tended to be lower in MSCs of OVX rats, while no statistical significance was observed because of a small sample size (Fig. 4C). However, the expression of mRNA for these osteogenic markers in MSCs from OVX rat treated with RU486 were comparable to those detected in sham operated controls (Fig. 4A–C). Treatment with RU486 significantly increased the expression of mRNA for OCN (Fig. 4B). Similarly, the ALP and calcium content of MSCs from OVX rats were significantly decreased compared to that of sham operated controls (Fig. 4D and E). RU486 treatment restored these osteogenic markers of MSCs from OVX rats (Fig. 4D and E) and significantly increased calcium content (Fig. 4E).

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Figure 3. Images of von-Kossa stained MSCs from different treatment groups after in vitro osteogenic differentiation. MSCs from sham operated controls (A and D), OVX rats (B and E), and OVX rats with RU486 treatment (C and F) 3 weeks after exposure to control and osteogenic differentiation medium, respectively. Bar = 100 µm.

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Figure 4. Osteogenic capabilities of MSCs from rats that received different treatments. (A–C) The mRNA expressions for ALP (A), OCN (B) and Cbfa-1 (E) in MSCs of sham-operated controls, OVX rats and OVX rats with RU486 treatment 1 week after osteogenic differentiation in vitro. (D and E) ALP activity (D) and calcium content (E) in MSCs from different treatment groups 3 weeks after osteogenic differentiation in vitro. N = 6; mean ± SD; *p < 0.05.

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After exposure to adipogenic differentiation medium, lipid accumulations, as detected by Oil-red-O staining, were observed in MSCs from all treatment groups (Fig. 5). The lipid in MSCs from OVX rats (Fig. 5E) were higher than those detected in sham operated controls (Fig. 5D). RU486 treatment reduced the lipid accumulation in MSCs of OVX rats (Fig. 5F) to levels similar to sham-operated controls. Quantitatively, the expression of mRNA for the adipogenic markers of MSCs, including LPL and PPAR, were increased in OVX rats compared to that of sham-operated controls while statistical difference of fold change is not significant because of individual variations. However, the expressions of these markers of MSCs of OVX rat treated with RU486 are reduced to the comparable levels of sham controls (Fig. 6A and B). Similarly, the extraction of Oil-red-O staining from MSCs of OVX rats tend to be higher than that of sham controls, while no statistically significance was observed. RU486 treatment reduced the lipid accumulation of MSCs of OVX rats to the comparable levels of sham controls (Fig. 6C).

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Figure 5. Images of Oil-red-O stained MSCs from different treatment groups after in vitro adipogenic differentiation. MSCs from sham operated controls (A and D), OVX rats (B and E), and OVX rats with RU486 treatment (C and F) 3 weeks after exposure to control and adipogenic differentiation medium, respectively. Bar = 100 µm.

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Figure 6. Quantitative measurements of adipogenic capabilities of MSCs from rats that received different treatments. (A and B) the mRNA expressions for PPAR-γ (A) and LPL (B) in MSCs from sham-operated control, OVX rats, and OVX rats with RU486 treatment 1 week after exposure to adipogenic differentiation medium. (C) Extraction of Oil-red-O dye in MSCs from rats with different treatments 3 weeks after exposure to adipogenic differentiation medium. N = 6; mean ± SD.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Steroid hormones, including estrogens and GCs, have been demonstrated to extensively regulate the levels of antioxidants and oxidative stress in many tissue and cell types, although they may exert opposite effects on these parameters.19 Physiologically estrogens function as antioxidants and promote MSC proliferation and osteogenic differentiation,20–22 while GCs inhibit MSC proliferation and accelerate senescence by increasing oxidative stress.2–6, 23 GCs have been demonstrated to play a critical role in bone metabolism by actively regulating proliferation, differentiation, and senescence of bone marrow mesenchymal stem/stromal cells (MSCs).6–8 Thus, the variations of these steroid hormones in patients with advancing age and postmenopausal play important roles in causing oxidative stress-induced premature senescence of MSCs. By effectively regulating or blocking these GC-mediated effects we may be able to reduce oxidative stress and increase the levels of antioxidants and by so doing to potentially prevent MSC senescence and rejuvenate their physiological capabilities. The present study demonstrated, for the first time, that the levels of corticosterone, the dominant endogenous GC in rodent models, are increased as estrogen levels decrease. These changes in hormone levels significantly reduce the activity of SOD, a key antioxidant in host animals as well as the proliferative and osteogenic capabilities of MSCs. These reductions in MSC capabilities and antioxidant levels can be restored by treating the experimental animals with the GR antagonist, RU486. Since human with advanced age and those who are postmenopausal have elevated endogenous GC levels and greatly reduced estrogen levels, the results presented here clearly indicate that the blockage of endogenous GCs may effectively prevent the premature senescence of MSCs and extend their lifespan.

MSCs from OVX rats have also been used as a model for MSCs in humans with advancing age and menopause, since both are also characterized with oxidative stress and significantly reduced estrogen levels. In these patients, the MSC population size is dramatically reduced and these cells exhibit accelerated senescence, including short telomere length and rapid loss of proliferative capabilities. These MSCs lose their capacity to undergo osteochondral differentiation and tend to differentiate into adipocytes. In the present study we demonstrated that the proliferative and osteogenic differentiation capabilities of rat MSCs are significantly reduced after ovariectomy, while adipogenic differentiation capabilities are increased. These characteristics of rat MSCs, which are consistent with other published studies, are similar to those of MSCs in humans who are of advanced age and post-menopausal.24, 25 The MSCs from these patients are characterized with premature senescence.26 Collectively, these data strongly suggest that MSCs from OVX rats display accelerated senescence as compared to control sham-operated animals and this conclusion is consistent with what is seen in human MSCs during aging-associated estrogen depletion. Although we did not observe a statistically significant reduction in the MSC population size in the OVX rats utilized in the present study, this is probably caused by relatively young OVX rats used and a short observation time. We assume that an aged OVX rat model and an extended period of estrogen depletion may be helpful the in future to determine the variations in MSC population size and effects of the blockage of endogenous GCs in OVX rat models.

It has been demonstrated that oxidative stress in OVX rats is increased by estrogen and progesterone depletion. However, effects mediated by elevated levels of endogenous GC in OVX rats have also been reported.14 In this study we quantitated a significant increase in endogenous corticosterone levels in OVX rats along with decreased levels of endogenous estradiol. Since GCs and estrogens appear to mediate opposite effects on the regulation of oxidative stress and antioxidant levels, this suggests that the oxidative stress and reduced levels of antioxidants detected in rats following ovariectomy are caused by both estrogen depletion and an increase in endogenous GC levels. This is consistent with clinical observations that patients who have lower endogenous estrogen levels have enhanced GC-mediated response during aging and after the menopause.27 The acceleration of senescence of MSCs in these patients is assumed to be caused by oxidative stress, which in turn is increased by both estrogen depletion and elevated endogenous GC levels. In the present study we confirmed this assumption by finding that RU486 treatment effectively increases antioxidant levels as well as the proliferation and osteogenic differentiation capabilities of MSCs of OVX rats. While RU486 acts as an antagonist for both GR and progesterone receptors (PR), progesterone-mediated effects on MSCs are minor compared to the effects of GC. The effects mediated by RU486 in OVX rats in present study are most likely accomplished by blocking the effects mediated by intracellular GRs. Also supporting this conclusion is the fact that progesterone levels are extremely low in OVX rats.28 Therefore, these data indicate that the reduction in MSC capabilities and antioxidants in estrogen-depleted states are caused, at least partially, by the elevation of endogenous GCs. By blocking these endogenous GCs we potentially protect the capabilities of MSCs from the oxidative stress induced by these adrenal steroids.

Although GCs regulate adipogenesis and fat storage, RU486 treatment has limited effects on the body weight of OVX rats as seen in the present study. One potential explanation is that the variations in body weight of OVX rats are caused primarily by estrogen insufficiency. Estrogens have been demonstrated to actively regulate the balance of MSC differentiation between osteogenesis and adipogenesis and have inhibitory effects on adipogenesis.29 Estrogens influence adipose metabolism and the insufficiency of these steroids directly enhances adipogenesis and increases body weight. As estrogens exert positive regulation on antioxidants and MSC capabilities, combinational usage of estrogen supplements with the blockage of endogenous GCs may synergistically prevent MSC senescence and senescence-associated disorders in advancing age and in estrogen depleted patients. Future studies to optimize the effects of estrogens, combined with blockage of endogenous GCs, could potentially improve MSC capabilities by reducing oxidative stress and increasing antioxidant levels.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

This research was partially supported by NIH/NIDCR (1R03DE017715) and the Start-up funding from the Dows Institute for Dental Research, College of Dentistry, University of Iowa.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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