Estrogen receptor-α signaling in osteoblast progenitors stimulates cortical bone accrual

Abstract

The detection of estrogen receptor-α (ERα) in osteoblasts and osteoclasts over 20 years ago suggested that direct effects of estrogens on both of these cell types are responsible for their beneficial effects on the skeleton, but the role of ERα in osteoblast lineage cells has remained elusive. In addition, estrogen activation of ERα in osteoclasts can only account for the protective effect of estrogens on the cancellous, but not the cortical, bone compartment that represents 80% of the entire skeleton. Here, we deleted ERα at different stages of differentiation in murine osteoblast lineage cells. We found that ERα in osteoblast progenitors expressing Osterix1 (Osx1) potentiates Wnt/β-catenin signaling, thereby increasing proliferation and differentiation of periosteal cells. Further, this signaling pathway was required for optimal cortical bone accrual at the periosteum in mice. Notably, this function did not require estrogens. The osteoblast progenitor ERα mediated a protective effect of estrogens against endocortical, but not cancellous, bone resorption. ERα in mature osteoblasts or osteocytes did not influence cancellous or cortical bone mass. Hence, the ERα in both osteoblast progenitors and osteoclasts functions to optimize bone mass but at distinct bone compartments and in response to different cues.

Figure 1. Deletion of ERα 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) ERα 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, ERα^f/f, and Prx1-cre mice by 2-way ANOVA.

Figure 2. ERα^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.

Figure 3. 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) ERα 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 ERα^f/f by 2-way ANOVA.

Figure 4. Deletion of ERα in Col1a1-cre–expressing cells does not alter bone mass.

(A) ERα 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.

Figure 5. 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.

Figure 6. The unliganded ERα 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) ERα^f/f;Prx1-cre and ERα^f/f littermate groups and (B) the ERα^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) ERα mRNA levels determined by quantitative PCR in C2C12 cells transduced with lentiviruses encoding a nontarget shRNA (sh-control) or sh-ERα. (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.

Figure 7. Cortical bone is preserved in ERα^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.

Figure 8. Site-specific effects of ERα on different bone compartments.

(Box i) The cortical and (Box ii) the cancellous bone compartments as well as (Box iii) the growth plate of a long bone are depicted in the respective blue boxes. The cells responsible for the particular action along with their dependency or lack thereof on estrogens (E₂) in each compartment are deduced from the respective cell-specific ERα deletion murine models of the present work and two earlier papers in which ERα was selectively deleted in osteoclasts (7) and chondrocytes (48). Red arrows point to the site at which the effect summarized in the blue boxes occurs.