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Endocrinology Vol. 140, No. 7 3210-3218
Copyright © 1999 by The Endocrine Society

ARTICLES

Progesterone- and Dexamethasone-Dependent Osteoprogenitors in Bone Cell Populations Derived from Rat Vertebrae Are Different and Distinct1

Yoichiro Ishida and Johan N. M. Heersche

Faculty of Dentistry, University of Toronto (Y.I., J.N.M.H.), Toronto, Ontario, Canada M5G 1G6; and the Department of Orthopedic Surgery, Yamaguchi University School of Medicine (Y.I.), Yamaguchi 755-8505, Japan

Address all correspondence and requests for reprints to: Dr. Yoichiro Ishida, M.D., Ph.D., Department of Orthopedic Surgery, Yamaguchi University School of Medicine, 1144 Kogushi, Ube-City, Yamaguchi 755-8505, Japan. E-mail: myishida{at}ymg.urban.ne.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous experiments have demonstrated that bone cell populations derived from explants of lumbar vertebral bone of adult female rats contain osteoprogenitors that require dexamethasone (Dex) or progesterone (Prog) to proliferate and differentiate into fully differentiated bone-forming osteoblasts. We now show that the Prog-dependent population cannot be detected in male rats after sexual maturation, but is present in prepubertal rats of both sexes and can be induced in adult male-derived populations by culturing the explants in medium containing 17ß-estradiol (10-9–10-8 M). This suggested that the Prog- and Dex-dependent osteoprogenitors in adult female-derived populations were probably distinct populations and that the survival of the Prog-dependent osteoprogenitors and/or their ability to proliferate are dependent on the presence of estrogen. We then proceeded to prove this by using replica plating. When one of the paired colonies duplicated was cultured in medium containing Dex (10-8 M) and the other in medium containing Prog (10-5 M), 5.0% of duplicates formed bone in Prog only, 11.1% formed bone in Dex only, and 3.4% formed bone in both Prog and Dex. In all cases the size of the bone-forming colonies in Dex-treated cultures was larger than that in Prog-treated cultures, indicating that the effects of Dex on osteoprogenitor proliferation are greater than those of Prog. The results demonstrate the existence of three classes of osteoprogenitors in adult female rat-derived bone cell populations: a class responding to Dex only, a class responding to Prog only, and a class responding to both Dex and Prog. The results also indicate that the effects of Prog are not mediated by Prog binding to the glucocorticoid receptor and imply that Prog plays an important role in maintaining bone mass through regulating the class of osteoprogenitors responsive to Prog.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CONTINUOUS increase in life expectancy in most developed countries has meant that osteoporosis, characterized by decreased bone mineral density and increased susceptibility to fractures, is also becoming an increasing public health problem. In women, the cessation of ovarian follicle development that characterizes the menopause leads to a marked reduction in serum levels of estrogen and progesterone (Prog) (1). Prog and estrogen deficiency are considered major contributors to the development of postmenopausal osteoporosis (2, 3, 4), and recent data suggest that estrogen-progestin combination therapy is one of the most effective therapies to increase bone mass in postmenopausal women (5, 6, 7, 8).

We have demonstrated recently that cell populations derived from vertebral bone of adult female rats contain osteoprogenitors that are stimulated by Prog and dexamethasone (Dex) to proliferate and differentiate into fully differentiated osteoblast colonies forming bone nodules, whereas estrogen by itself has no effect (9, 10). We also found that in adult male rats, the Prog-dependent osteoprogenitors were undetectable, whereas Dex-dependent osteoprogenitors were found in similar numbers in adult male and female rats (11), suggesting that Prog- and Dex-dependent osteoprogenitors might be two distinct classes of progenitors. Further experiments demonstrated that the number of Prog-dependent osteoprogenitors in female rats increased after treatment of the cultures with 17ß-estradiol (E2) (10-9–10-6 M) (11). Therefore, we hypothesize that an important contribution to postmenopausal bone loss in humans could be the disappearance of a class of Prog-responsive osteoprogenitors. In the present study, we have investigated whether changes in the number of Prog- and Dex-dependent osteoprogenitors occur when sexual maturity is reached, whether E2 regulates responsiveness to Prog and Dex in these osteoprogenitors, and whether Prog-dependent osteoprogenitors and Dex-dependent osteoprogenitors are different and distinct populations. To do this, we first investigated the effects of Prog and Dex on bone nodule formation by osteoprogenitors present in adult and prepubertal male and female rats. We next examined the effect of E2 on Prog- and Dex-induced bone nodule formation in these osteoprogenitors. We then used replica plating techniques to analyze the progeny of one osteoprogenitor under the same or different culture conditions and evaluated whether, when one of the duplicates was cultured in Prog-containing medium and the other in Dex-containing medium, bone formation would occur in the same colonies. The results clearly indicated the existence of a class of Prog-dependent osteoprogenitors that is different and distinct from the class of Dex-dependent osteoprogenitors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of materials
Glass beads (Fisher Scientific, Ottawa, Canada) and polyester membranes (Swiss Silk, Zurich, Switzerland) used for replica plating were prepared as described by Collins (12). Circles were cut from 17-µm pore size polyester membrane to fit the culture dishes (Becton Dickinson and Co., Lincoln Park, NJ), and notches were cut for orientation. The glass beads and polyester membranes were soaked for 2 h in bleach (sodium hypochlorite solution, 6% available chlorine) diluted 1:25 in water. After washing in running water, glass beads and polyester membranes were boiled in detergent for 5 min (Alconox, Inc., New York, NY), washed in running tap water for several hours, then washed with several changes of distilled water followed by three washes with 70% ethanol, and sterilized by autoclaving.

Culture techniques, replica plating techniques, staining procedures, and colony quantitation
The methods for cell culture of bone cell populations derived from lumbar vertebrae of young adult (250 g) or prepubertal (75 g) rats were reported previously (9). In brief, cancellous bone fragments (~1 mm3) of lumbar vertebral bodies (L1–L6) of male or female White Wistar rats (Charles River Canada, Inc., Quebec, Canada) were placed in 10-µl plasma clots in 35-mm culture dishes, which were allowed to clot (~4 h) before adding medium ({alpha}MEM, Flow Laboratories, Inc., Mclean, VA) supplemented with 10% FBS [BioWhittaker, Inc. (Walkersville, MD), and Life Technologies (Grand Island, NY); standard medium]. After culturing the explants in standard medium for 11 days, the outgrowth cells were released with a 1:1 (vol/vol) mixture of 0.05% trypsin and 0.3% collagenase (Sigma Chemical Co., St. Louis, MO), suspended in standard medium supplemented with 50 µg/ml ascorbic acid (control medium), plated in 35-mm culture dishes at a colony density (replica plating experiments) or at a density of 5 x 104 cells/35-mm culture dish (bone nodule assay), and cultured in control medium as described for individual experiments. For bone nodule assays, culture medium was changed after 24 h to control medium with or without Dex (Sigma Chemical Co.), Prog (Calbiochem, San Diego, CA), or E2 (Sigma Chemical Co.) as described for individual experiments. To investigate the effect of E2, phenol red-free {alpha}MEM (Flow Laboratories, Inc., Rockville, MD) was used throughout the culture period (explant culture and subculture). For replica plating experiments, on days 1–11 of culture, a replica polyester membrane was placed on top of the cells in the culture dish and weighted in place with 3-mm glass beads. The replica polyester membrane was taken out of the dish on days 3–9 of culture as described for individual experiments and placed in a fresh dish such that the colonies were on the upper surface of the membrane. The master with the original colonies and the replica with the duplicated colonies were maintained in medium containing optimal concentrations of Prog (10-5 M) or Dex (10-8 M) for inducing bone nodules (9, 11) as described for individual experiments.

In all cultures, media were changed every second day. To induce mineralization of osteoid nodules, 5 mM ß-glycerophosphate was added for the last 4–5 days of culture (13). On days 10–29 of culture after separating the membranes from the culture dishes, the cultures were evaluated for the presence of colonies (by staining with hematoxylin or Coomassie brilliant blue), for alkaline phosphatase (AP)-positive colonies with a cytochemical stain for AP, and for mineralized bone nodules by staining with the von Kossa technique as described previously (9, 11). Further processing is described in individual experiments.

The research protocols were approved by the University of Toronto’s animal care committee and biohazards committee.

Statistics
Results are expressed as the mean ± SEM. Comparisons of two groups were performed using unpaired two-tailed Student’s t test. Three or more groups were compared using ANOVA, followed by Scheffe’s post-hoc test. The colony matching rate was calculated as follows: colony matching rate (%) = [a/(a + b + c)] x 100, where a is the number of paired colonies, b is the number of unpaired colonies with a colony present only in the master dish, and c is the number of unpaired colonies with a colony present only in the replica membrane.

The difference between the number of colonies in masters and replicas was analyzed by {chi}2 test. We also compared the agreement between duplicate colonies forming bone nodules in medium containing either Dex or Prog with the chance agreement using {chi}2 test. When the smallest expected frequency was less than 5, the difference was analyzed by Fisher’s exact test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Prog- and Dex-dependent osteoprogenitors in bone cell populations derived from vertebrae of adult and prepubertal male and female rats
To investigate whether the previously reported absence of Prog-dependent osteoprogenitors in mature male rats (11) was related to sexual maturation, we investigated the presence of Prog-dependent osteoprogenitors in cell populations derived from vertebral bone of prepubertal and adult rats of both sexes. The results shown in Fig. 1AGo indicate that Dex (10-8 M) and Prog (10-7–10-5 M) both induced an increase in the number of bone-forming osteoblast colonies (bone nodules) in cell populations derived from prepubertal rats of both sexes. In bone cell populations derived from adult rat vertebrae, Prog-induced bone nodule formation was seen only in the female-derived cell populations (Fig. 1BGo). The Dex-induced increase in the number of bone nodules in populations derived from either prepubertal or adult rats was independent of the sex of the animal (Fig. 1Go, A and B; 10-8 M Dex, the concentration previously shown to be optimal in this system; concentrations tested, 10-10–10-6 M) (9, 11, 14). Under control conditions very few bone nodules could be detected (A: prepubertal male, 1.75 ± 0.75; female, 0.75 ± 0.48; B: adult male, 0 ± 0; female, 0.33 ± 0.24; mean ± SEM bone nodules/35-mm dish).



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  		Figure 1. Sex-specific effects of Prog and Dex on bone nodule formation in cell populations derived from prepubertal (A) and adult (B) rat vertebrae. Explants were cultured in standard medium for 11 days. Outgrowth cells were plated at a density of 5.0 x 104 cells/35-mm dish and cultured in control medium with or without Prog (10-7–10-5 M) or Dex (10-8 M). At day 20 of culture, cultures were fixed and stained with the von Kossa technique. Each point is the mean of six cultures. SEM is shown by vertical lines. Significant differences from control values are indicated as *, P < 0.05; **, P < 0.001 by the Scheffé’s posthoc test; {ddagger}, P < 0.001 by Student’s t test.

 
Effect of E2 on Prog- and Dex-dependent osteoprogenitors
As the number of Prog-dependent osteoprogenitors in bone cell cultures derived from female rats increased after treatment of the cultures with E2 (11), we investigated whether E2 treatment could induce a Prog response in adult male-derived bone cell populations (Fig. 2Go). The results show that treatment of the cultures with E2 (10-9–10-8 M) throughout the culture period (explant culture and subculture) resulted in the induction of a significant effect of Prog on bone nodule formation in adult male-derived cell populations (by three-way ANOVA, P < 0.001). E2 had no significant effect on Dex-induced bone nodule formation (by three-way ANOVA, P = 0.658).



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  		Figure 2. E2 effect on Prog (A) or Dex (B)-induced bone nodule formation in adult male-derived cell populations. The explants and corresponding subcultures were cultured throughout the culture period under three different conditions: i) no E2; ii) 10-9 M E2; and iii) 10-8 M E2. In all groups, explants were cultured for 11 days. Outgrowth cells were plated at a density of 5.0 x 104 cells/35-mm dish and then cultured in the original culture medium with or without Prog (10-6–10-5 M) or Dex (10-8 M). At day 20 of culture, cultures were fixed and stained. Each point is the mean of five culture dishes. All culture media were phenol red-free. SEM is shown by vertical lines. Significant differences from corresponding controls are indicated as *, P < 0.01 by the Scheffé’s posthoc test; **, P < 0.001 by Student’s t test.

 
We next investigated the effect of E2 on Prog- and Dex-induced bone nodule formation in immature and mature male and female rats. As shown in Fig. 3Go, E2 (10-9–10-8 M) treatment enhanced the Prog (10-5 M)-induced increase in the number of bone nodules in populations derived from adult female rats and prepubertal rats in both sexes and induced a Prog response in adult male-derived populations. Again, E2 had no significant effect on Dex-induced bone nodule formation in any of the groups. Figure 4Go shows the effect of E2 on the dose dependency of Prog-induced bone nodule formation in adult female-derived cell populations. The Prog dose-response curve in E2 (10-8 M)-treated populations was sifted to the left, with 3 x 10-7 M Prog being the minimally effective dose in E2-treated populations and 3 x 10-6 M being the minimally effective dose in non-E2-treated populations.



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  		Figure 3. Effects of E2 in combination with Prog or Dex on bone nodule formation in prepubertal- and adult-derived cell populations. Explants were cultured in standard medium (phenol red-free) for 11 days. Outgrowth cells were plated at a density of 5.0 x 104 cells/35-mm dish and cultured in control medium (phenol red-free) containing E2 (10-9–10-8 M) alone (A) or in combination with 10-5 M Prog (B) or 10-8 M Dex (C). At day 21 of culture, cultures were fixed and stained. Each point is the mean of four culture dishes. SEM is shown by vertical lines. Significant differences (compared with the corresponding cultures without E2) are indicated as *, P < 0.05; **, P < 0.01 by the Scheffé’s posthoc test.

 


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  		Figure 4. Effects of E2 on the dose dependency of Prog-induced bone nodule formation in adult female rat-derived cell populations. Explants were cultured in standard medium (phenol red-free) for 11 days. Outgrowth cells were plated at a density of 5.0 x 104 cells/35-mm dish and cultured in control medium (phenol red-free) with or without Prog (10-8–10-5 M) and in the presence or absence of E2 (10-8 M). At day 21 of culture, cultures were fixed and stained. Each point is the mean of six culture dishes. SEM is shown by vertical lines. Significant differences are indicated as *, P < 0.05; **, P < 0.001 by the Scheffé’s posthoc test (compared with the control value).

 
Replica plating experiments
The results reported here and those reported previously strongly suggest that the Prog- and Dex-dependent osteoprogenitors are different and distinct. We now report replica-plating experiments that strongly support this view. Figure 5Go shows photographs of a representative master (Fig. 5AGo) and replica (Fig. 5BGo) culture for cell populations derived from adult female rat vertebral bone plated at colony density. Three transfer patterns can be seen: 1) paired colonies seen in both master and replica cultures, 2) colonies seen only in master cultures, and 3) colonies seen only in replica cultures. As pointed out previously, the time point at which the replica membrane is placed in the culture and the point at which the replica and master cultures are separated affect colony transfer (15). This was also the case in our culture system; as shown in Fig. 6Go, the colony matching rate was maximal when the membranes were added on day 1 of culture. With regard to time of membrane separation, the maximal colony matching rate was seen when membranes were removed on day 9 of culture after adding the membrane. Thus, for our replica plating experiments, we chose day 1 of culture for membrane addition and day 9 of culture for membrane separation.



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  		Figure 5. Photomicrographs of representative master and replica cultures. Explants from adult female vertebral bone were cultured in standard medium for 11 days. Outgrowth cells were plated at a density of 1,000 cells/35-mm dish on a culture dish (master). A polyester membrane (replica) was placed on the master dish on day 5 of culture and separated after another 5 days. At day 10 of separated culture in control media, the master (A) and replica (B) cultures were fixed and stained with Coomassie brilliant blue staining. (a) Paired colonies, (b) an unpaired colony on the master dish, and (c) an unpaired colony on the replica membrane.

 


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  		Figure 6. The effects of time of adding and separating membranes on the transfer efficiency. Explants of adult female vertebrae were cultured in standard medium for 11 days. Outgrowth cells were plated at a density of 1,500 cells/35-mm dish on master dishes. Replica membranes were placed on the master dishes at days 1, 4, 8, and 11 of culture and separated after another 3, 6, and 9 days. The master and replica cultures were incubated further for 20 days and stained with hematoxylin. Each point is the mean of six paired cultures. SEM is shown by vertical lines.

 
Bone nodule formation in duplicated colonies when both are cultured in either Dex- or Prog-containing medium
When both master and replica colonies were cultured in 10-8 M Dex-containing medium, the percentage of paired colonies forming a bone nodule in the master culture only was 2.8%, the percentage of colonies with a bone nodule in the replica culture only was 9.3%, and the percentage with a bone nodule in both master and replica cultures was 50.5% (Fig. 7AGo). Osteoprogenitor proliferation and differentiation (bone nodule formation) in duplicated colonies on either master or replica when cultured under similar conditions was not significantly different (by {chi}2 test, P = 0.174). Figure 7BGo shows the effect of 10-5 M Prog (optimal concentration for inducing bone nodules) (9, 11, 16) on bone nodule formation in duplicated colonies in masters and replicas. The percentage of paired colonies mineralized in master cultures only was 4.7%, that in replica cultures only 2.7%, and that in both master and replica cultures was 10.9%. Again, no significant difference existed between master and replica colonies (by {chi}2 test, P = 0.532).



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  		Figure 7. The effect of Dex (A) and Prog (B) on bone nodule formation in colonies on masters and replicas. Explants of adult female vertebral bone were cultured in standard medium for 11 days. Outgrowth cells were plated at a density of 1,500 cells/35-mm dish on master dishes and cultured in control medium. Replica membranes were placed on the master dishes on day 1 and separated after another 9 days. The paired cultures (master and replica) were both cultured in control medium with either Prog (10-5 M) or Dex (10-8 M) for a further 29 days, fixed, and stained with Coomassie brilliant blue and the von Kossa technique. Only paired colonies were evaluated for bone nodule formation. +/+, bone nodule in both master and replica; +/-, bone nodule in master only; -/+, bone nodule in replica only; -/-, no bone nodule in either master or replica. The total number of paired colonies was 216 for A, 258 for B, present in 22 paired cultures (A), and 31 paired cultures (B).

 
Bone nodule formation in duplicated colonies with one member of the pair exposed to Prog and the other to Dex
When masters and replicas were cultured under different conditions (one in 10-5 M Prog, the other in 10-8 M Dex), a significant difference was found with regard to bone nodule formation (Fig. 8Go). The percentage of duplicated colonies exhibiting a bone nodule in Prog-containing medium only was 5.0%, that exhibiting a bone nodule in Dex-containing medium only was 11.1%, and that forming a bone nodule in both Prog-containing medium and Dex-containing medium was 3.4%. In this experiment, 80.5% of duplicated colonies did not form a bone nodule under any of the conditions. Table 1Go summarizes the effects of Dex and Prog on bone nodule formation in four pooled experiments. When both the master and replica plates were cultured in the same medium (both cultured in either Dex-containing or Prog-containing medium), it was only a chance occurrence that bone formation did not occur in both colonies (i.e. the percentage of agreement with regard to bone formation was <100% only by chance). However, when different media (Dex-containing and Prog-containing) were used for the duplicated colonies, the agreement was less than what was expected by chance occurrence if the ligands affected different classes of osteoprogenitors. We compared the agreement between bone nodule formation in Dex- and Prog-treated duplicate colonies with the chance agreement and found that it was significantly less (by {chi}2 test). Thus, the results strongly indicate the existence of a class of osteoprogenitors responding to Dex only and a class responding to Prog only. The results also indicate the presence of a class of osteoprogenitors that responds to both Dex and Prog.



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  		Figure 8. Bone nodule formation in Prog- and Dex-treated paired colonies. Explants from adult female rat vertebrae were cultured in standard medium for 11 days. Outgrowth cells were plated at a density of 1,500 cells/35-mm dish on master dishes. Replica membranes were placed onto the dishes at day 1 of culture and separated after another 9 days. Masters and replicas were cultured in different medium [one in the presence of Prog (10-5 M), the other containing Dex (10-8 M)]. At day 28 of separated culture, the master and replica cultures were fixed and stained with Coomassie brilliant blue and the von Kossa technique. The total number of paired cultures in this experiment was 46. The total number of paired colonies was 261, +/+, colonies forming bone in both Dex and Prog; +/-, colonies forming bone in Dex only; -/+, colonies forming bone in Prog only; -/-, colonies not forming bone in either Dex or Prog.

 

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  		Table 1. Bone nodule formation in paired colonies of osteoprogenitors cultured in either Dex or Prog

 
AP expression in paired colonies with one of the paired colonies exposed to Prog and the other to Dex
We previously reported that both Prog and Dex increased the number of AP-positive colonies in adult female rat-derived bone cell populations (9). We, therefore, investigated both AP expression and bone nodule formation in paired colonies, with one cultured in medium containing Prog (10-5 M) and the other in medium containing Dex (10-8 M). As expected (Fig. 9Go), colonies that formed bone nodules were all AP positive. In addition, AP-positive colonies not expressing bone were present in the cultures. The percentage of paired colonies expressing AP in Dex was 45.0% (6.5% expressed AP in Dex only), whereas 48.7% of paired colonies expressed AP in Prog (10.2% expressed AP in Prog only). There was no significant difference between Dex- and Prog-treated cultures in terms of the total number of paired colonies expressing AP (by {chi}2 test, P = 0.389). Table 2Go summarizes the effects of Dex and Prog on AP expression in 503 paired colonies. The results indicate the existence of a class of progenitors for cells expressing AP in Dex only, a class of progenitors for cells expressing AP in Prog only and a large population of a class of progenitors for cells that expresses AP in both Dex and Prog.



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  		Figure 9. The relationship between bone nodule formation and staining for AP. Explants from adult female rat vertebrae were cultured in standard medium for 11 days. Outgrowth cells were plated at a density of 1,500 cells/35-mm dish on master dishes. Replica membranes were placed onto the dishes at day 1 of culture and separated after another 9 days. Masters and replicas were cultured in different medium [one in the presence of Prog (10-5 M), the other containing Dex (10-8 M)]. At day 28 of separated culture, the master and replica cultures were fixed and stained for AP, with Coomassie brilliant blue and with the von Kossa technique. Total number of paired cultures was 46. +/+, AP-positive in both Dex and Prog; +/-, AP-positive in Dex only; -/+, AP-positive in Prog only; -/-, no AP staining in either Dex or Prog.

 

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  		Table 2. AP expression in paired colonies cultured in either Dex or Prog

 
The size of AP-positive paired colonies formed in either Prog- or Dex-containing medium was analyzed in four types of colonies: those forming bone in both Dex and Prog, those forming bone in Dex only, those forming bone in Prog only, and those not forming bone in either Dex or Prog (Fig. 10Go). The AP-positive colonies were classified into three groups according to their colony sizes: 1) large (>=3.5 mm), 2) medium (>=1.5 and <3.5 mm), and 3) small (<1.5 mm). In colonies forming bone in Prog only (Fig. 10CGo) and in those not forming bone in either Dex or Prog (Fig. 10DGo), no significant differences in the size distribution of AP-positive colonies were found in Dex- and Prog-treated cultures (by {chi}2 test, P = 0.667 and P = 0.703, respectively). In the AP-positive colonies forming bone in both Dex and Prog (Fig. 10AGo) and in those forming bone in Dex only (Fig. 10BGo), the size distribution of AP-positive colonies was significantly different in Dex- and Prog-treated cultures (by {chi}2 test, P = 0.005 and P = 0.008, respectively), i.e. the AP-positive colonies maintained in Dex-containing medium were larger than the paired colonies maintained in Prog-containing medium. The size distribution of the AP-positive colonies in Prog-containing medium was the same in all four categories (by {chi}2 test, P = 0.826), whereas the size distribution of colonies in Dex-containing medium was significantly different in the four categories (by {chi}2 test, P < 0.0001), i.e. the majority of the duplicate colonies cultured in Dex-containing medium of the category responding to both Dex and Prog and those responding to Dex only were large colonies (78% and 42%, respectively), whereas only a small fraction of the colonies in the category responding to Prog only and those not responding to either Prog or Dex formed large colonies (7.7% and 7.4%, respectively).



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  		Figure 10. The size distribution of AP-positive colonies in Prog- and/or Dex- dependent bone-forming paired colonies. Explants from adult female rat vertebrae were cultured in standard medium for 11 days. Outgrowth cells were plated at a density of 1,500 cells/35-mm dish on master dishes. Replica membranes were placed onto the dishes at day 1 of culture and separated after another 9 days. Masters and replicas were cultured in different medium [one in Prog (10-5 M), the other in Dex (10-8 M)]. At day 28 of separated culture, the master and replica cultures were fixed and stained for AP, with Coomassie brilliant blue and with the von Kossa technique. (A) colonies forming bone in both Dex and Prog (n = 20), (B) colonies forming bone in Dex only (n = 58), (C) colonies forming bone in Prog only (n = 28), and (D) colonies not forming bone in either Dex or Prog (n = 108). Data derived from 46 paired cultures.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown here that in bone cell populations derived from vertebral bone of adult female rats there exists a population of osteoprogenitors that is responsive to Prog, but not to Dex, and one that is responsive to Dex, but not to Prog. The data also indicate the presence of a small fraction of osteoprogenitors that is responsive to both Prog and Dex.

Whether the action of Prog on osteoblast-like cells is mediated through Prog receptors or through glucocorticoid receptors has been frequently debated (for review, see Ref. 17), as both Prog and glucocorticoid receptors are expressed in most osteoblast-like cells (18, 19, 20, 21, 22), and Prog can compete with glucocorticoids for the glucocorticoid receptor (23, 24, 25). Recent data, however, suggested a direct effect of Prog on osteoblasts (26, 27, 28, 29, 30, 31, 32, 33, 34). The present demonstration of the presence of a class of osteoprogenitors that is responsive to Prog, but not to Dex, provides direct evidence that in bone cell populations derived from adult female rat vertebrae, the stimulatory effect of Prog on bone nodule formation in osteoprogenitors is not solely mediated by Prog binding to the glucocorticoid receptors. As there is no statistically significant difference in the capability of osteoprogenitors of forming bone between the master colony and the replica colony when they are cultured on different substrata but both in either Prog- or Dex-containing medium, this finding is not due to technical aspects of the methods used or to the difference between master and replica surfaces.

We have previously shown that Prog responsiveness of bone-forming osteoprogenitors depends strongly on the serum component of the culture medium, with 10-7 M Prog being the minimally effective dose in some sera and 10-5 M being the minimally effective dose in others (16), whereas the maximum effective concentration of Prog was 10-5 M. We have also shown that the maximum effective concentration of Dex for stimulating osteoprogenitor proliferation and differentiation (bone nodule formation) was 10-8 M and that this response did not depend on the serum component of the culture medium (11, 16). As the main aim of the present study was to investigate whether Prog-dependent osteoprogenitors and Dex-dependent osteoprogenitors were different and distinct, we decided to use the most effective doses of Prog (10-5 M) and Dex (10-8 M) (16). In the normal 4-day estrous cycle of the female rat, serum Prog levels are between 1.6 x 10-8 and 2 x 10-7 M (35, 36, 37, 38, 39). Prog levels during pregnancy increase up to about 5 x 10-7 M (39, 40, 41). Thus, the concentrations of Prog effective in our system are close to or within the range of physiological concentrations. In addition, the concentrations of Prog present throughout the 48- to 72-h culture period may be lower than those at the start of the culture (just after medium change) as a result of hormone conversion by the cells cultured, as culture media are routinely changed only every 48–72 h. Indeed, in our culture systems, active progestin Prog is converted to 20{alpha}-hydroxyprogesterone (a biologically inactive steroid) in adult rat-derived bone cell populations during culture, resulting in a lower effective concentration (42).

In the present study, Prog stimulated bone nodule formation by osteoprogenitors in cell populations derived from both male and female prepubertal rats to the same degree, whereas in adult male rats this Prog-dependent population disappeared. Importantly, in cell populations derived from both adult and prepubertal rats, Dex enhanced bone nodule formation of osteoprogenitors derived from both sexes to the same degree. This suggests that the development and/or maintenance of the Prog-dependent osteoprogenitors in rat skeleton is sex (hormone) dependent. With regard to this, it has been reported that in rat brain, estrogen regulates sex development by up-regulating Prog receptors (43, 44, 45). The experiments presented here indeed indicated that estrogen treatment enhances the Prog-induced increase in the number of bone nodules using bone cell populations derived from rats of both sexes in both age categories. Estrogen had no significant effect on Dex-induced bone nodule formation in these experiments, indicating that the effect is specific for Prog-induced bone nodule formation. Importantly, when the cultures obtained from adult male rats were treated with estrogen, Prog-dependent osteoprogenitors could be induced. These data are compatible with the recent findings that men with either estrogen resistance (by a disruptive mutation in the estrogen receptor gene) (46) or aromatase deficiency (by a disruptive mutation in the aromatase gene) (47) have decreased bone mass, suggesting that estrogen plays an important role in determining and maintaining bone mass not only in female skeleton but also in male skeleton. Taken together, these data suggest that the one of the mechanisms by which estrogen affects bone mass may be that estrogen up-regulates or induces Prog responsiveness of osteoprogenitors.

In preliminary experiments, we measured Prog receptor content with a sensitive radioligand ([3H]R5020) binding method (43) and found detectable levels of Prog receptors in adult female rat-derived bone cells, but not in adult male rat-derived bone cells (unpublished data, in collaboration with Dr. N. J. MacLusky, Division of Reproductive Science, Toronto Hospital Research Institute, Toronto, Canada). However, levels were insufficient to perform Scatchard analysis and thus evaluate changes in receptor levels in our cultures under different conditions. This observation is in agreement with the general observation that the concentrations of Prog receptors in osteoblast-like cells are extremely low, especially in the rat osteoblast-like cell line UMR-106 (19, 29).

In the present experiments, all colonies of osteoprogenitors that formed a bone nodule in either Dex- or Prog-containing medium were AP positive. Interestingly, in addition to the colonies of AP-positive osteoprogenitors capable of forming a bone nodule, colonies of AP-positive cells that could not form bone after either Dex or Prog treatment were also present in adult female rat-derived cell populations. These observations are compatible with the view that nonosteogenic cells also express AP, e.g. reticular cells (48), endothelial cells (49), growth cartilage cells (50), and preadipocytes and adipocytes (51, 52). However, we cannot exclude the possibility that these colonies represent late stage osteoprogenitors that could not sufficiently proliferate to form a colony capable of forming bone. The majority of AP-positive colonies expressed AP in both Dex-containing and Prog-containing media (86% of AP-positive colonies in Dex; 80% of AP-positive colonies in Prog), but the data also suggest the existence of a population expressing AP in Dex-containing medium only and a population expressing AP in Prog-containing medium only. Importantly, all paired colonies of osteoprogenitors forming bone in Dex only, Prog only, or both Prog and Dex were present in the population expressing AP in both Dex and Prog.

With regard to the size distribution of AP-positive paired colonies in Prog- and Dex-treated cultures forming bone in both Prog and Dex, the size of the colonies in Dex was clearly larger than that in Prog. However, the number of AP-positive colonies in Prog-treated cultures and that in Dex-treated cultures were the same. If we consider AP to be an early marker of osteogenic differentiation (53), and colony size to reflect cell proliferation (54), the results suggest that the effect of Prog may predominantly be a stimulation of progenitor commitment to the osteoblast lineage, whereas the effect of Dex may represent both increased progenitor commitment to osteoblasts and stimulation of osteoprogenitor proliferation. This view is compatible with the observation that in human osteoblast cultures, messenger RNA encoding the Prog receptor is maximally expressed in preosteoblasts and decreases as the osteoblast matures (30).

In conclusion, in the present study we provide the first direct evidence that in bone cell populations derived from adult female rat vertebrae there exist two independent classes of osteoprogenitors: a class responding to Dex only and a class responding to Prog only. The data also indicate the presence of a population responding to both Dex and Prog. These results are compatible with the view that in the female skeleton one of the mechanisms maintaining bone mass is related to effects of both estrogen and Prog on a class of osteoprogenitors responsive to Prog. As considerable evidence has accumulated to indicate that the rat is a good model to study the processes associated with age-related or postmenopausal bone loss in humans (39, 55, 56, 57), these findings may provide new insight into the significance of a specific class of Prog-dependent osteoprogenitors and their regulation by estrogen not only in the rat skeleton, but also in the human skeleton.


   Acknowledgments
 
We thank Dr. A. Scima for assistance with data analysis, and Mrs. I. Tertinegg, Dr. D. Jia, and Dr. C. G. Bellows for assistance with these experiments and for critical discussions throughout these studies.


   Footnotes
 
1 This work was supported by the Medical Research Council of Canada, Grant MT-12395. Back

Received October 13, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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