Estrogen receptor-α signaling in osteoblast progenitors stimulates cortical bone accrual (original) (raw)
Generation of ERαf/f;Prx1-cre mice. To elucidate the biologic role of the ERα in cells of the osteoblast lineage, we crossed mice harboring a floxed _ER_α allele (ER_α_f/f mice) (7) with mice expressing the Cre recombinase under the control of regulatory elements of the Prx1 gene, which is expressed in pluripotent mesenchymal progenitors of the appendicular, but not the axial, skeleton and their progeny (9). The Prx1-cre transgene activates a reporter gene (R26R) in all the osteoblasts and osteocytes present in cortical and cancellous bone as well as growth plate chondrocytes, at least up to 5 weeks of age (10). Here, we established that _Prx1-cre_–mediated recombination was maintained in all these cell types up to at least 28 weeks of age (Figure 1A), which is the oldest age of mice examined in the studies reported herein. The effectiveness of _ER_α gene deletion was demonstrated by a 60%–80% decrease in _ER_α mRNA levels from cultured osteoblastic cells (Figure 1B) and in _ER_α genomic DNA from femoral shafts (Figure 1C) of ER_α_f/f;Prx1-cre mice. _ER_α mRNA expression in osteoclasts, livers, and spleens was unaffected (Figure 1B). Total body weight, femoral length (Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI65910DS1), and the morphology of the growth plate (Figure 1D) were also unaffected by _ER_α deletion in _Prx1-cre_–expressing cells. Likewise, ER_α_f/f;Prx1-cre mice had normal uterine weight, indicating that estrogen levels were not affected (Supplemental Table 1).
Deletion of _ER_α in _Prx1-cre_–expressing cells decreases cortical bone mass. (A) X-gal–stained histological frozen sections of the distal femurs of 24-week-old R26R control and Prx1-cre;R26R mice. The left panels show a low-magnification image of the distal femur. Scale bar: 500 μm. The right panels show a high-magnification image of the cancellous (top) and cortical bone (bottom). Scale bar: 100 μm. (B) _ER_α mRNA levels in cultured osteoblasts (Ob) and osteoclasts (Oc) (6 wells) and livers and spleens (n = 7–9/group). (C) Quantitative PCR of loxP-flanked genomic DNA (gDNA), normalized to a control locus, isolated from collagenase-digested femurs and tibia cortical bone (n = 5–7/group). (D) Safranin-O–stained histological sections of the distal femurs of 12-week-old mice (cartilage stains red). Scale bar: 500 μm. (E) BMDs determined by DEXA in female mice at 8 (n = 9–11/group) and 22 (n = 10/group) weeks of age. (F) Cortical thickness determined at the midshaft and cancellous bone volume measured at the distal end by micro-CT in femurs from 8-week-old female (n = 9–11/group) and male mice (n = 6–12/group). BV/TV, bone volume per tissue volume. Bars represent mean and SD. *P < 0.05 by Student’s t test; #P < 0.05 versus wild-type, ER_α_f/f, and Prx1-cre mice by 2-way ANOVA.
ERαf/f;Prx1-cre mice have low femoral bone mass. Female ER_α_f/f;Prx1-cre mice exhibited low bone mineral density (BMD) in the femur up to at least 22 weeks of age, as determined by dual-energy x-ray absorptiometry (DEXA), compared with ER_α_f/f littermates (Figure 1E). In line with the fact that the Prx1-cre transgene is not expressed in the axial skeleton, spinal BMD was unaltered, providing additional evidence for the specificity of the _ER_α deletion. Micro-CT analysis revealed that the low femoral BMD resulted from decreased cortical thickness but no change in cancellous bone mass (Figure 1F). Albeit, trabecular number was reduced and trabecular spacing was increased in the ER_α_f/f;Prx1-cre mice at 12 weeks of age (Supplemental Table 1). As with the femoral BMD, the decrease in cortical thickness was present up to 28 weeks of age. Similar to that of the females, male ER_α_f/f;Prx1-cre mice had decreased cortical thickness but normal cancellous bone mass, as determined at 6 weeks of age (Supplemental Figure 1A) or 8 weeks of age (Figure 1F). Nonetheless, in contrast to that in the females, the cortical decrement was no longer present in 18-week-old ER_α_f/f;Prx1-cre male mice (Supplemental Figure 1B).
In agreement with the imaging studies, dynamic histomorphometric analysis of femoral bone sections from 8-week-old ER_α_f/f;Prx1-cre females revealed a 40% decrease in mineral apposition rate (MAR) with no change in mineralizing surface (MS), resulting in an overall 50% decrease in bone formation rate (BFR) at the periosteal surface, as compared with the control littermates (Figure 2). However, there was no change in any of the parameters at the endocortical surface. Moreover, consistent with the lack of an effect of _ER_α deletion on cancellous bone mass, the number of osteoblasts and osteoclasts as well as dynamic measures of bone formation were unaffected in the cancellous bone of ER_α_f/f;Prx1-cre mice (Supplemental Figure 2, A and B). The number as well as the size of the adipocytes in the bone marrow was unaffected in the ER_α_f/f;Prx1-cre mice at 12 weeks of age (Supplemental Figure 2C).
ER_α_f/f;Prx1-cre mice have decreased periosteal bone formation. MAR, MS, and BFR, as determined by tetracycline labels, shown in the photomicrographs (scale bar: 20 μm), in longitudinal undecalcified sections of femurs from 8-week-old female mice (n = 6–7/group). Bars represent mean and SD. *P < 0.05 by Student’s t test.
Deletion of ERα from Osterix1-cre–expressing cells recapitulates the skeletal phenotype of the ERαf/f;Prx1-cre mice. The skeletal phenotype of the ER_α_f/f;Prx1-cre mice could be the result of the loss of receptor function in a pluripotent uncommitted mesenchymal progenitor, a descendent osteoblast progenitor, or terminally differentiated osteoblasts and osteocytes. To distinguish between the first and the second of these possibilities, we next generated mice in which the _ER_α was deleted from cells expressing Osterix1 (Osx1) using an Osx1-GFP::cre deleter strain (11). The Osx1-GFP::cre transgene is expressed in osteoblast progenitors residing in the bone-forming regions of the perichondrium and primary spongiosa as well as in hypertrophic chondrocytes. In addition, Osx1/GFP-expressing cells are present in the thin periosteal layer overlaying the cortical bone surface (12).
_ER_α genomic DNA in femoral shafts of ERαf/f;Osx1-cre mice was decreased by 70% as compared with that in Osx1-cre control mice (Figure 3A). _ER_α mRNA expression in cultured Osx1-GFP–positive calvaria cells isolated by flow cytometry was similarly decreased by 70% (Figure 3B). In contrast, _ER_α mRNA expression in bone marrow–derived osteoclasts was indistinguishable between ER_α_f/f;Osx1-cre and the control mice. Body weight in female Osx1-cre or ER_α_f/f;Osx1-cre mice was lower as compared with that in wild-type or ER_α_f/f littermate control mice (Supplemental Figure 3A), as was femoral length (Supplemental Figure 3B). These findings are in line with a previous report showing that the Osx1-cre transgene decreases body size (13). The smaller body size and the reduced femoral length in both Osx1-cre and ER_α_f/f;Osx1-cre mice notwithstanding, these 2 measures were indistinguishable between Osx1-cre and ER_α_f/f;Osx1-cre mice. This result indicates that, whereas Osx1-cre expression in and of itself had an effect, _ER_α deletion per se did not affect body size or femoral length. Nonetheless, spine and femoral BMD was significantly lower in ER_α_f/f;Osx1-cre mice when compared with that of Osx1-cre mice at 12 or 24 weeks of age (Figure 3C).
Deletion of ERα in _Osx1-cre_–expressing cells decreases cortical bone mass. (A) Quantitative PCR of loxP-flanked genomic DNA, normalized to a control locus, isolated from collagenase-digested femur and tibia cortical bone (n = 4–6/group). (B) ER_α mRNA levels by quantitative PCR in cultured Osx1-GFP–positive cells and osteoclasts (triplicate cultures). (C) Longitudinal BMD determined by DEXA in female mice (n = 6–11/group). (D) Cortical bone measurements determined by micro-CT in the midshaft region of femurs and (E) in the fifth lumbar vertebra of 24-week-old mice described in C. (F) Cancellous bone mass determined by micro-CT in the distal end of the femurs and the fifth lumbar vertebrae of mice described in D. Bars represent mean and SD. †_P < 0.05 by Student’s t test; *P < 0.05 versus Osx1-cre; #P < 0.05 versus wild-type or ER_α_f/f by 2-way ANOVA.
In line with the smaller femur size, mice expressing Osx1-cre had decreased cortical thickness as well as smaller outer and inner femoral midshaft bone perimeters when compared with wild-type or ER_α_f/f littermate controls (Figure 3D). Once again, in spite of the effects of the Osx1-cre transgene by itself, cortical thickness was further decreased by _ER_α deletion in ER_α_f/f;Osx1-cre mice when compared with Osx1-cre control mice at 24 weeks of age. The decrease in cortical thickness in the ER_α_f/f;Osx1-cre mice was due to deficient periosteal apposition, as evidenced by a decrease in the outer perimeter of the midshaft, while the inner perimeter was unaffected. Notably, ER_α_f/f;Osx1-cre mice also exhibited decreased cortical thickness in the vertebrae (Figure 3E), demonstrating that the cortical bone phenotype caused by the _ER_α deletion was not restricted to long bones. The cancellous bone volume in the femurs or vertebrae of ER_α_f/f;Osx1-cre mice, on the other hand, was indistinguishable among wild-type, ER_α_f/f, and Osx1-cre littermate controls (Figure 3F). Moreover, trabecular number and spacing were unaffected by _ER_α deletion when compared with Osx1-cre mice, but trabecular thickness was decreased (Supplemental Figure 3C). Thus, deletion of _ER_α in osteoblast progenitors led to a phenotype similar to that caused by _ER_α deletion from uncommitted mesenchymal progenitors.
Deletion of ERα in osteoblasts and osteocytes does not alter bone mass. Next, we investigated whether the effect of the _ER_α deletion from _Prx1-cre_– or _Osx1-cre_–expressing cells on bone was the result of loss of function in osteoblast precursors as opposed to their differentiated descendants, i.e., mature osteoblasts and osteocytes. To do this, we generated mice in which the _ER_α was deleted from osteoblasts and osteocytes expressing α1(I)-collagen (Col1a1) using a Col1a1-cre deleter strain (14). The Col1a1-cre transgene activated the reporter gene R26R in all the osteoblasts and osteocytes present in cortical and cancellous bone, demonstrating extremely high efficiency of cre-mediated recombination in these cell types (Supplemental Figure 4). Bone marrow–derived osteoblasts from ER_α_f/f;Col1a1-cre mice exhibited a 70% reduction in _ER_α expression levels (Figure 4A). _ER_α mRNA expression in livers and spleens was unaffected. Deletion of _ER_α from _Col1a1_-expressing cells had no effect on bone mass, as measured by serial DEXA BMD measurements between 4 and 12 weeks of age in the spines and femurs of female and male mice (Supplemental Figure 5). The lack of an effect of the _ER_α deletion in _Col1a1_-expressing cells on cortical thickness (Figure 4B), cancellous bone mass (Figure 4C), and microarchitecture (Supplemental Table 2) was confirmed by micro-CT analysis in 12- and 26-week-old mice. In spite of the absence of an effect on bone mass, ER_α_f/f;Col1a1-cre mice did exhibit the anticipated increase in cancellous osteoblast apoptosis (Figure 4D).
Deletion of ERα in _Col1a1-cre_–expressing cells does not alter bone mass. (A) _ER_α mRNA levels in cultured osteoblasts (triplicate wells), liver, and spleen (n = 6/group). (B) Cortical thickness measured by micro-CT at the midshaft of the femurs of 12-week-old (n = 11–14/group) and 26-week-old (n = 8–11/group) female mice. (C) Cancellous bone mass determined by micro-CT in the fifth lumbar vertebra of mice described in B. (D) Osteoblast apoptosis in undecalcified sections of L1–L4 vertebra, stained by ISEL, from 26-week-old female mice (n = 4/group). Bars represent mean and SD. *P < 0.05 by Student’s t test.
Osteoblastogenesis is attenuated in ERαf/f;Prx1-cre and ERαf/f;Osx1-cre mice. Having established that the _ER_α expressed in osteoblast progenitors, but not mature osteoblasts or osteocytes, was responsible for optimal periosteal bone formation, we went on to investigate the mechanism(s) responsible. To do this, we examined osteoprogenitor proliferation, differentiation, and life span using cultures of periosteal- or bone marrow–derived osteoblastic cells isolated from femurs. In agreement with the decreased periosteal BFR, periosteal cells from ER_α_f/f;Prx1-cre mice exhibited a markedly decreased capacity to form mineralized nodules (Figure 5A) and to secrete osteocalcin (Figure 5B), both under basal conditions or in response to BMP-2. These changes were accompanied by a reduction in the number of cells due to a decrease in their proliferation (Figure 5C) and a small increase in apoptosis (Figure 5D). Moreover, the expression of genes involved in osteoblastogenesis, such as Osx1, Col1a1, and Bglap but not Runx2, was also decreased in cells from ER_α_f/f;Prx1-cre mice as compared with cells from littermate controls (Figure 5E), suggesting that osteoblast differentiation was also affected by the _ER_α deletion in progenitor cells. Similar results were obtained with bone marrow–derived cells (Supplemental Figure 6, A–C).
Deletion of ERα decreases proliferation and differentiation of osteoblast progenitors from the periosteum. (A) Mineralized matrix visualized and quantified following Alizarin Red staining and (B) osteocalcin levels in the medium of periosteal cell cultures pooled from 3 mice treated with vehicle (veh) or rhBMP-2 (25 ng/ml) for 21 days (triplicate cultures). Original magnification, ×63 (bottom row). (C) BrdU incorporation and (D) caspase-3 activity in periosteal cells cultured for 3 day (6 wells). AFU, arbitrary fluorescence units. (E) mRNA levels of the indicated genes determined by quantitative PCR in periosteal cells cultured with ascorbic acid for 14 days (triplicate cultures). (F) Mineralized matrix quantified following Alizarin Red staining in periosteal cells cultured with vehicle or E2 (10–8 M) in the presence of ascorbic acid for 21 days (triplicate cultures). Bars represent mean and SD. *P < 0.05 by 2-way ANOVA; #P < 0.05 by Student’s t test.
To establish whether the number of mesenchymal progenitors was affected by the _ER_α deletion, we quantified the number of progenitors able to form CFU-fibroblasts (CFU-F), CFU-osteoblasts (CFU-OB), and CFU-adipocytes (CFU-AD) in ex vivo bone marrow cultures from ER_α_f/f;Prx1-cre mice, ER_α_f/f;Osx1-cre mice, and their respective control mice. The number of CFU-F, CFU-AD, and CFU-OB was increased in ER_α_f/f;Prx1-cre mice compared with that in ER_α_f/f control mice (Supplemental Figure 7A). In contrast, the number of CFU-F and CFU-OB in ER_α_f/f;Osx1-cre mice was indistinguishable from that in the Osx1-cre control mice (Supplemental Figure 7B). As in the case of the ER_α_f/f;Prx1-cre mice, the number of CFU-AD was elevated in ER_α_f/f;Osx1-cre mice. Interestingly, CFU-OB colonies from ER_α_f/f;Prx1-cre and ER_α_f/f;Osx1-cre mice were smaller and displayed irregular shapes, consistent with defective osteoblast differentiation.
We have shown earlier that estrogens restrain osteoblastogenesis (15, 16). In agreement with those earlier results, addition of 17β-estradiol to periosteal- (Figure 5F) or bone marrow–derived cell cultures (Supplemental Figure 6D) from control mice had an attenuating effect on osteoblastogenesis, strongly suggesting that the actions of the estrogen-activated ERα are opposite to the ones of the unliganded ERα.
The unliganded ERα potentiates the Wnt/β-catenin signaling pathway. Wnt/β-catenin signaling is essential for osteoblastogenesis, and its effects may be potentiated by ERα (17, 18). In agreement with this evidence, the pro-proliferative and pro-osteoblastogenic actions of Wnt3 were blunted in periosteal cells from ER_α_f/f;Prx1-cre (Figure 6A) and ER_α_f/f;Osx1-cre mice (Figure 6B). Albeit, 17β-estradiol had no effect on basal or Wnt3-stimulated proliferation and attenuated Wnt3-induced alkaline phosphatase (AP) activity, demonstrating that the effects of the receptor are independent or even opposite to those of its ligand. Practically identical results were obtained with bone marrow–derived cells (Supplemental Figure 8, A and B). To confirm that the effects of ERα on Wnt signaling were independent of estrogens, we silenced _ER_α in C2C12 cells using shRNA directed against _ER_α (sh-_ER_α). Knockdown of _ER_α levels in sh-_ER_α cells was confirmed by quantitative PCR (Figure 6C). Similar to the periosteal cells, Wnt3-induced AP activity was greatly attenuated in sh-_ER_α cells (Figure 6D). In addition, the increase in TCF transcription induced by Wnt3 in nontarget shRNA cells was greatly attenuated in sh-_ER_α cells (Figure 6E). In contrast, 17β-estradiol attenuated Wnt3-induced AP activity in C2C12 cells and had no effect on TCF transcriptional activity. Dkk1, Sost, Fzd2, and Fzd4 were not affected by _ER_α deletion (Supplemental Figure 8C), excluding the possibility that the attenuation of Wnt signaling was secondary to upregulation of Wnt inhibitors.
The unliganded _ER_α potentiates the Wnt/β-catenin signaling pathway. BrdU incorporation and AP activity in periosteal cell cultures pooled from 3 mice from each of the (A) ER_α_f/f;Prx1-cre and ER_α_f/f littermate groups and (B) the ER_α_f/f;Osx1-cre and Osx1-cre littermate groups, preincubated for 1 hour with vehicle or E2 (10–8 M), followed by incubation without or with Wnt3 (25 ng/ml) for 3 days. (C) _ER_α mRNA levels determined by quantitative PCR in C2C12 cells transduced with lentiviruses encoding a nontarget shRNA (sh-control) or sh-_ER_α. (D) AP activity in cells treated as in A. (E) Luciferase activity in C2C12 cells transfected with a TCF-luc reporter construct and pretreated as in A, followed by treatment without or with Wnt3 (12.5 ng/ml) for 24 hours. Bars represent mean and SD. *P < 0.05 by 1-way ANOVA with Bonferroni’s test; #P < 0.05 by Student’s t test.
ERαf/f;Prx1-cre mice do not lose cortical bone after ovariectomy. In our previous work, _ER_α deletion from osteoclasts prevented ovariectomy-induced (OVX-induced) loss of cancellous, but not cortical, bone (7). We therefore investigated whether the ERα in cells of the osteoblast lineage could indirectly influence endocortical resorption. Acquisition of peak bone mass in both the Prx1-cre and Col1a1-cre strains occurred at approximately 16 weeks of age. Control and ER_α_f/f;Prx1-cre mice were ovariectomized at 8 or 24 weeks of age, and the effects of the loss of estrogens were examined 3 and 6 weeks later, respectively. Mice ovariectomized at 8 weeks of age showed the expected loss of uterine weight (Supplemental Figure 9A). Littermate control mice exhibited decreased bone accrual at the spine and femur, as determined by DEXA BMD (Figure 7A). ER_α_f/f;Prx1-cre mice also exhibited decreased bone accrual at the spine. In contrast, femoral DEXA BMD was unaltered in the ovariectomized ER_α_f/f;Prx1-cre mice. Nonetheless, micro-CT analysis revealed that OVX did cause loss of cancellous bone accrual (Figure 7B), a decrease in trabecular number, and an increase in trabecular separation (Supplemental Figure 10A) in both the littermate controls and ER_α_f/f;Prx1-cre mice. On the other hand, OVX caused a decrease in the accrual of cortical BMD in control but not in ER_α_f/f;Prx1-cre mice (Figure 7C). In agreement with these findings, histomorphometric analysis of the endocortical surface of femoral bone sections revealed an increase in the number of osteoclasts in the ER_α_f/f control mice following OVX (Figure 7D). This increase was abrogated in the ER_α_f/f;Prx1-cre mice. Similar findings to those from the OVX experiment with 8-week-old (growing) mice were obtained in mice ovariectomized at 24 weeks of age (Figure 7E and Supplemental Figure 9B).
Cortical bone is preserved in ER_α_f/f;Prx1-cre mice following OVX. (A–D) Eight-week-old female mice were sham operated or ovariectomized and euthanized 3 weeks later (n = 10/group). (A) The percentage of change from the initial BMD was determined by DEXA measurements 1 day before surgery and before death. (B) Cancellous bone mass and (C) BMD of cortical bone at the distal femur determined by micro-CT. (D) Osteoclast number per mm of endocortical bone surface in longitudinal decalcified sections of femurs (n = 10/group). In the photomicrographs, osteoclasts (stained red by TRAP; scale bar: 20 μm) are indicated by the arrows. (E) Twenty-two-week-old mice were sham operated or ovariectomized and euthanized 6 weeks later (n = 4–6/group). The percentage of change in BMD was determined as in B. (F) Twenty-week-old mice were sham operated or ovariectomized and euthanized 6 weeks later (n = 11/group). The percentage of change in BMD was determined as in A. Bars represent mean and SD. *P < 0.05 by 2-way ANOVA.
Consistent with the critical role of estrogens in epiphyseal closure at the end of puberty in humans as well as the fact that Prx1-cre is expressed in growth plate chondrocytes (Figure 1A and ref. 10), OVX of 8-week-old mice with deletion of _ER_α from _Prx1-cre_–expressing cells did not cause an increase in bone length, which was readily seen in the ERα intact mice (Supplemental Figure 10B).
Last, in contrast to the ER_α_f/f;Prx1-cre mice, ER_α_f/f;Col1a1-cre mice ovariectomized at 20 week of age exhibited a similar loss of spinal and femoral BMD as that of their littermate controls 6 weeks following OVX (Figure 7F).