Attenuation of the self-renewal of transit-amplifying osteoblast progenitors in the murine bone marrow by 17β-estradiol (original) (raw)
Proliferative and self-renewal properties of murine CFU-Fs and CFU-OBs. The proliferative status of CFU-Fs and CFU-OBs was assessed in vivo by administering 5-fluorouracil (5-FU; 150 μg/g body weight) to 4-month-old Swiss-Webster mice. Femoral marrow cells were obtained for determination of CFU-F and CFU-OB number 5 days later. The number of CFU-Fs and CFU-OBs in the femurs of animals receiving 5-FU was significantly reduced compared with that of animals injected with vehicle (Table 1). However, when expressed per 106 marrow cells, there was no appreciable change due to the coincident reduction in total number of marrow cells in the isolate. In three separate experiments we found that mice injected with 5-FU had only 29% ± 8% (SEM) of CFU-F progenitors and 15% ± 4% of CFU-OBs, as compared with mice receiving vehicle, indicating that the majority of CFU-Fs and CFU-OBs in adult mice are undergoing cell division.
Inhibition of CFU-F and CFU-OB production in vivo by 5-FU
We next performed in vitro studies to establish whether CFU-Fs and CFU-OBs are capable of self-renewal — i.e., whether they produce identical daughter CFU-F and CFU-OB progenitors. Bone marrow cells were cultured for 7 days to allow formation of colonies of fibroblastic cells from the progenitors present in the marrow isolate. Then, cells from each of 12 randomly selected colonies were enzymatically dispersed using cloning cylinders, and secondary cultures were established to determine the number of CFU-F and CFU-OB progenitors present within each colony. Each colony contained numerous CFU-F progenitors, ranging in number from 12 to 296 (Figure 1a); however, only 5 of the 12 colonies contained CFU-OBs, ranging in number between 24 to 144 (Figure 1b). Interestingly, the ratio of CFU-OBs to CFU-Fs within each colony was highly variable (0.1–0.8), and CFU-OBs were never greater than CFU-Fs. Additional experiments (n = 4) to determine the prevalence of progenitors among all adherent cells in 5- to 6-day marrow cell cultures indicated that 1.6 ± 0.3% were CFU-Fs and 0.8 ± 0.2 % were CFU-OBs.
Content of (a) CFU-Fs and (b) CFU-OBs in fibroblastic colonies present in cultures of murine bone marrow cells. Cells were enzymatically dispersed from each of 12 randomly selected fibroblastic colonies (containing greater than 50 cells) that developed in 7-day cultures of femoral marrow cells and assayed for CFU-Fs and CFU-OBs as described in Methods. Bars represent the number of CFU-F or CFU-OB colonies obtained in the secondary culture divided by the fraction of cells used to establish the secondary culture. “0” indicates no colonies detected.
The quantification of CFU-F and CFU-OB production in whole bone marrow cultures was optimized by culturing freshly isolated bone marrow cells within three-dimensional type I collagen gels instead of on tissue culture plasticware. Culture of marrow cells in this kind of environment has been shown to facilitate long-term viability and hematopoietic activity (32, 33). Moreover, cells precultured within collagen gels were easily dispersed with bacterial collagenase to establish secondary cultures for subsequent assay of CFU-Fs and CFU-OBs. As shown in Figure 2a, CFU-F and CFU-OB number increased exponentially during the first 7 days of culture of isolated marrow cells within the collagen gel. This increase was inhibited by addition of 5-FU (Figure 2b), indicating that proliferation was required for self-renewal to occur. After 16 days of culture, CFU-OB number was reduced fourfold from its value at 7 or 11 days, but CFU-Fs were unchanged.
Time kinetics of CFU-F and CFU-OB production by murine bone marrow cells cultured in type I collagen gels. (a) Marrow cell cultures were established in type I collagen gels (10 × 106 cells per gel), and CFU-F and CFU-OB content was assessed either immediately (day 0) or after culture for 2, 7, 11, or 16 days. The data shown represent the mean (± SEM) number of CFU-Fs and CFU-OBs per gel. Preliminary experiments established that the number of CFU-Fs and CFU-OBs in the initial marrow isolate not put into the gel was indistinguishable from the 0 time values (not shown). Error bars are not visible because the symbols are larger than the error bars. Data were analyzed using mixed-effect ANOVA as described in Methods. Significant (P < 0.01) increases in progenitor number versus day 0 were detected at all time points, except for CFU-OBs at day 2. CFU-OB number at day 16 was significantly less than at day 11 (P < 0.05). (b) Marrow cell cultures were established in type I collagen gels as in a without (vehicle) or with 5 μg/ml 5-FU. Progenitor number was then determined after 2 or 5 days of culture. The number of progenitors in the initial isolate (day 0) is expressed per 10 × 106 marrow cells. A_P_ < 0.05 treatment versus vehicle.
If the increase in CFU-Fs and CFU-OBs during culture truly is due to self-renewal, the behavior of the newly generated daughter cells should be identical to that of the parental cell obtained in the bone marrow isolate. To determine whether this is indeed the case, we compared the characteristics of colonies formed by marrow-derived and in vitro–generated progenitors. Figure 3 shows that alkaline phosphatase staining of CFU-F colonies and von Kossa staining of CFU-OB colonies were indistinguishable, regardless of whether they were formed from freshly isolated marrow progenitors or from in vitro–generated progenitors. Estimates of colony size using a calibrated eyepiece reticle on a dissecting microscope indicated that the majority of colonies (both CFU-F and CFU-OB) ranged from 200 and 400 μm in diameter, regardless of whether the progenitors were obtained from the bone marrow or generated in vitro (data not shown).
Colonies formed from freshly isolated bone marrow progenitors and from progenitors generated in vitro are morphologically indistinguishable. (a) Colonies formed from freshly isolated bone marrow cells. (b) Colonies formed from marrow cells cultured for 5 days in a collagen gel. The left panel of each section shows a 10-cm2 well containing CFU-Fs stained for alkaline phosphatase (top portion) or CFU-OBs stained with von Kossa to detect mineral (bottom portion). The right panel of each section shows a photomicrograph (×2) of a typical alkaline phosphatase–positive CFU-F colony (top portion) and a von Kossa-stained CFU-OB colony (bottom portion).
Next, the rate of osteoblast differentiation within the colonies was examined using marrow cells from OG2-lacZ mice (Figure 4). These mice harbor a transgene expressing β-galactosidase under the control of the OG2 osteocalcin promoter, which is activated only at the later stages of osteoblast differentiation (25, 34, 35). Staining of colonies with X-gal showed that von Kossa-positive CFU-OB colonies contained blue X-gal–stained cells (Figure 4a), demonstrating the presence of osteoblastic cells with active OG2 promoter. As shown in Figure 4b, the development of fibroblastic colonies occurred over the same period from marrow-derived or in vitro–generated progenitors. More important, the time at which blue cells — representing osteoblastic cells with active OG2 promoter — appeared within these colonies was practically identical for marrow-derived and in vitro–derived progenitors. Thus, in both sets of cultures, no β-galactosidase–positive cells were observed at 2, 5, and 10 days of culture, but by 15 days a few such cells appeared. Thereafter, the number of colonies containing β-galactosidase–positive cells increased. This was followed by the mineral deposition within these colonies, as evidenced by von Kossa staining at 25 days of culture.
The time kinetics of colony development and osteoblast differentiation from freshly isolated bone marrow progenitors and from progenitors generated in vitro are identical. (a) Colony histology. Photomicrographs show colonies with a mineralized von Kossa–stained matrix and X-gal–stained blue cells (left panel, ×2; right panel, ×10). Arrows indicate selected X-gal–stained blue cells that are osteoblastic as evidenced by the active OG2 promoter. (b) Kinetics of colony development. Femoral marrow cells were isolated from OG2-lacZ mice and either maintained in culture for 2, 5, 10, 15, 20, or 25 days (left panel) or cultured in type I collagen gels for 6 days and then enzymatically released and cultured for the same period as the freshly isolated cells (right panel). At each time point, cultures were stained with X-gal to detect β-galactosidase–positive cells and according to von Kossa to detect mineral. Colonies comprising at least 50 cells were scored as fibroblastic, β-galactosidase–positive (at least 10 blue cells), or von Kossa–positive. The data shown represent the mean number (± SD) of each type of colony per 106 cells seeded (n = 3 per group). Error bars are not visible days 2 and 5, because the symbol is larger than the error bar. Essentially identical results were obtained in a second experiment.
These findings indicate that the new CFU-OBs generated in vitro are at a similar if not identical stage of differentiation as the parental cell obtained from the bone marrow and that CFU-OBs are capable of both limited self-renewal and differentiation. These are the characteristics of early transit-amplifying cells (16). Because we did not analyze differentiation markers of other cell types, however, we do not know whether the same is true for the subpopulation of CFU-Fs that do not differentiate into osteoblasts. Therefore, subsequent studies were restricted to the investigation of CFU-OB behavior.
17β-estradiol suppresses CFU-OB self-renewal. In view of the finding that CFU-OBs are proliferating self-renewing progenitors, the increase in their number after ovariectomy that we detected in our earlier studies (6) could be due to removal of a suppressive effect of estrogens on self-renewal. To investigate whether this is the case, in the present report we added 17β-estradiol to bone marrow cells cultured in collagen gels and determined the number of CFU-OBs after 5 days of treatment. Whereas CFU-OBs increased 19-fold in cultures incubated with vehicle, cultures incubated with as little as 1 nM 17β-estradiol exhibited only 10- to 12-fold increase (Figure 5). In six separate experiments, 10 nM 17β-estradiol inhibited the self-renewal of CFU-OBs by an average of 53% ± 7% (SEM).
17β-estradiol attenuates the self-renewal of CFU-OBs. Duplicate cultures of marrow cells in collagen gels (5 × 106 per gel) were maintained in the absence (Veh) or presence of 10–11 to 10–8 M 17β-estradiol (E2) for 6 days and then assayed for CFU-OB content. Assay of freshly isolated marrow cells indicated that there were 90 ± 14 CFU-OBs per 5 × 106 cells used to establish each culture. Thus, there was a 19.2-fold increase in CFU-OBs in cultures maintained in vehicle. The data shown represent the mean (± SEM) of CFU-OBs. A_P_ < 0.05 treatment versus vehicle using mixed-effects ANOVA model. Linear contrast testing indicated a significant (P < 0.05) dose-dependent effect.
We also examined the effect of 17β-estradiol on CFU-OB number in vivo. In this experiment, mice were ovariectomized and given 17β-estradiol (20 ng/g body weight) or vehicle at 16 and 18 days after the operation. This time was chosen for analysis because of evidence that CFU-OBs are increased in ovariectomized mice as early as 14 days after the operation (6). On the 20th day after the operation (4 days after initiation of 17β-estradiol treatment), femoral marrow cells were obtained and pooled (six mice per group), and the number of CFU-OBs was determined in triplicate cultures. The number of CFU-OBs in marrow of mice receiving 17β-estradiol was approximately 50% of that seen in mice receiving vehicle (41 ± 9 SD versus 80 ± 15 per 106 marrow cells, respectively; P < 0.01 by Student’s t test).
The role of the ER in the inhibitory effect of 17β-estradiol on CFU-OB self-renewal was examined next. Consistent with a receptor-mediated action, the pure ER antagonist ICI 182,780 (50 nM) blunted the suppressive effect of 1 nM 17β-estradiol on CFU-OB self-renewal (Figure 6a). Moreover, whereas 10 nM 17β-estradiol inhibited CFU-OB production in cultures from ERα+/+ mice by 61 ± 18%, it failed to suppress CFU-OB production when added to marrow cultures established from ERα–/– mice (Figure 6b). CFU-OBs from ERα–/– mice were indistinguishable from that of ERα+/+ mice with respect to colony size and von Kossa staining (not shown). These findings indicate that the α isoform of the ER mediates the suppressive effect of estrogen.
The role of the ER in the suppressive effect of 17β-estradiol on CFU-OB self-renewal. (a) ICI 182,780 blocks the effect of 17β-estradiol. Marrow cell cultures were established in collagen gels (7.5 × 106 per gel) in the absence or presence of 50 nM ICI 182,780. The cultures were maintained for 6 days without (Veh) or with 1 nM 17β-estradiol and then assayed for CFU-OB number. Assay of freshly isolated cells indicated that there were 281 ± 17 CFU-OBs per 7.5 × 106 cells used to establish each culture. Thus, there was a 5.4-fold increase in CFU-OBs in cultures maintained in vehicle in the absence of ICI 182,780. Bars represent the mean number (± SEM) of CFU-OBs per gel. A_P_ < 0.05 treatment versus vehicle as determined by mixed-effects ANOVA. (b) Lack of effect of 17β-estradiol on CFU-OBs from ERα–/– mice. Marrow cells were obtained from ERα+/+ or ERα–/– mice, and collagen gel cultures were established using 8 × 106 cells per gel. Cultures were maintained in the absence or presence of 10 nM 17β-estradiol for 5 days and then assayed for CFU-OBs. Assay of freshly isolated cells from ERα+/+ or ERα–/– mice indicated that there were 248 ± 56 or 384 ± 56 CFU-OBs, respectively, per 8 × 106 cells used to establish each culture. Thus, there was a 7.3-fold (ERα+/+) or 6.5-fold (ERα–/–) increase in CFU-OBs in cultures maintained in vehicle. Bars represent the mean number (± SEM) of progenitors. A_P_ < 0.05 treatment versus vehicle as determined by mixed-effects ANOVA.
Expression of ERα in murine bone marrow cells. In view of the finding that 17β-estradiol suppressed CFU-OB self-renewal in an ERα-dependent fashion, immunocytochemical studies were performed to demonstrate the ERα protein in cells present in colonies of fibroblastic cells. Two Ab’s, MC-20 and ERnt, which recognize epitopes in the COOH-terminal (36) and NH2-terminal (24) portions of murine ERα, respectively, were used. The colonies that form in 5- to 6-day cultures comprise large cells with fibroblast-like morphology, as well as small (5–10 μm) round cells with a high nucleus/cytoplasm ratio (Figure 7a). Occasionally the latter were on top of fibroblastic cells. Both anti-ERα Ab’s predominantly stained these small cells, and the staining was seen in both the nucleus and the cytoplasm (Figure 7b). In three separate bone marrow preparations, an average of 2–3% of the cells exhibited staining for ERα with either Ab. Large fibroblastic cells stained weakly if at all, and no staining was seen with the nonimmune control IgG. Cytoplasmic and nuclear ERα immunoreactivity was also observed in MCF-7 breast cancer cells, used here as a positive control (Figure 7c, top panels), whereas neither Ab stained HeLa cells (Figure 7c, bottom panels), which lack ER (30). The majority of the cells staining positively with the MC-20 Ab did not express alkaline phosphatase, a marker of mesenchymal/osteoblastic cells (Figure 7d). Instead, this enzyme was observed almost exclusively in large fibroblastic cells, most of which lacked ERα immunostaining. MC-20 staining was also absent from cells of the monocyte/macrophage lineage, detected with anti-CD11b Ab (Figure 7e).
Immunocytochemical detection of ERα in cultured murine bone marrow cells. (a) Hematoxylin and eosin (H&E) staining of a fibroblastic colony. The top panel shows a low-power view; bar, 120 μm. The box in the top panel indicates the area shown at high power in the bottom panel; bar, 15 μm. The colonies comprise fibroblast-like cells (arrow) as well as small round cells with high nucleus/cytoplasm ratio (arrowheads). (b) Immunoperoxidase staining (reddish brown) of bone marrow cells with MC-20 or ERnt anti-ERα Ab. Cells incubated with nonimmune rabbit (rIgG) or mouse IgG (mIgG) instead of anti-ERα Ab exhibited no staining. (c) Immunoperoxidase staining of MCF-7 cells (top panels) or HeLa cells (bottom panels) with MC-20 or ERnt Ab. (d) Photomicrographs of the same field taken with fluorescence illumination to visualize ERα-positive cells stained with MC-20 and FITC-labeled anti-rabbit Ab (left panel) and bright-field illumination to visualize alkaline phosphatase–positive (AP-positive) cells (right panel). Arrows indicate position of ERα-positive cells. (e) Photomicrographs of the same field taken with bright-field illumination to visualize ERα-positive cells after immunoperoxidase staining with MC-20 as in b (reddish brown, left panel) and fluorescence illumination to visualize CD11b-positive macrophages detected with FITC (right panel). Arrows indicate position of CD11b-positive cells. b–e: bars, 15 μm.