The estrogen receptor-alpha in osteoclasts mediates the protective effects of estrogens on cancellous but not cortical bone

Abstract

Estrogens attenuate osteoclastogenesis and stimulate osteoclast apoptosis, but the molecular mechanism and contribution of these effects to the overall antiosteoporotic efficacy of estrogens remain controversial. We selectively deleted the estrogen receptor (ER)alpha from the monocyte/macrophage cell lineage in mice (ERalpha(LysM)(-/-)) and found a 2-fold increase in osteoclast progenitors in the marrow and the number of osteoclasts in cancellous bone, along with a decrease in cancellous bone mass. After loss of estrogens these mice failed to exhibit the expected increase in osteoclast progenitors, the number of osteoclasts in bone, and further loss of cancellous bone. However, they lost cortical bone indistinguishably from their littermate controls. Mature osteoclasts from ERalpha(LysM)(-/-) were resistant to the proapoptotic effect of 17beta-estradiol. Nonetheless, the effects of estrogens on osteoclasts were unhindered in mice bearing an ERalpha knock-in mutation that prevented binding to DNA. Moreover, a polymeric form of estrogen that is not capable of stimulating the nuclear-initiated actions of ERalpha was as effective as 17beta-estradiol in inducing osteoclast apoptosis in cells with the wild-type ERalpha. We conclude that estrogens attenuate osteoclast generation and life span via cell autonomous effects mediated by DNA-binding-independent actions of ERalpha. Elimination of these effects is sufficient for loss of bone in the cancellous compartment in which complete perforation of trabeculae by osteoclastic resorption precludes subsequent refilling of the cavities by the bone-forming osteoblasts. However, additional effects of estrogens on osteoblasts, osteocytes, and perhaps other cell types are required for their protective effects on the cortical compartment, which constitutes 80% of the skeleton.

Figure 1

Generation of conditional ERα mice. A targeting vector was constructed in which ERα exon 3 was flanked by loxP sites. The targeting vector was used to electroporate embryonic stem (ES) cells, and a correctly targeted clone was injected into blastocysts to generate chimeric mice. After germline transmission of the targeted allele, the frt-flanked neomycin cassette was removed by crossing with mice expressing the FLPe recombinase in germ cells.

Figure 2

ERα_LysM ^−/− mice express lower levels of ERα in macrophages and osteoclasts. A–D, Quantitative RT-PCR analysis of mRNA from: A, macrophages formed from nonadherent bone marrow cells cultured for 4 d in the present of M-CSF. B, Mature osteoclasts generated from bone marrow cells cultured with M-CSF and RANKL for 5 d. C, Osteoblastic cells derived from calvaria of 2- to 3-month-old mice. D, Soft tissues harvested from 12-wk-old mice (n = 5). Bars, Mean ± sd, *, P < 0.05. In panels A and B, the mean was calculated using cells from three individual animals and triplicate determinations per animal. In panel C, the mean was calculated from triplicate determinations obtained from cells pooled from three animals.

Figure 3

ERα_LysM^−/− mice exhibit no changes in total body or uterine weight. A, Total body weight of two cohorts of female ERα_LysM^−/− and ERα^f/f littermate mice. No differences were observed between the two genotypes (n = 9–23 mice per group). B, Wet uterine weight of 28-wk-old female mice which were sham-operated or ovariectomized (OVX) 6 wk earlier (n =10–13 mice per group). Bars, Mean ± sd; *, P < 0.05 vs. respective sham-operated mice.

Figure 4

ERα_LysM^−/− mice exhibit increased number of osteoclast progenitors and osteoclasts. A, Number of TRAP-positive cells developed from bone marrow cells, extracted from femurs of 12-wk-old mice, and cultured in the presence of M-CSF and RANKL for 5 d. Triplicate cultures were performed from each of seven individual animals. B and C, Histomorphometric analysis of longitudinal undecalcified sections of L1–L3 vertebrae from 12-wk-old female mice (n = 8 mice per group). D, BMDs determined by DEXA in two cohorts (4–12 and 18–28 wk of age) of female ERα_LysM^−/− and ERα^f/f littermate mice (n = 7–23 mice per group). Oc, Osteoclasts; Ob, osteoblasts; BFR, bone formation rate; BA/TA, bone area per tissue area. *, P < 0.05 vs. ERα^f/f mice.

Figure 5

ERα_LysM^−/− mice lose bone mass after OVX. Female mice (22 wk of age) were sham operated or ovariectomized and killed 6 wk later. A, Number of TRAP-positive cells generated from bone marrow cells cultured with M-CSF and RANKL for 5 d. B and C, Histomorphometric analysis of longitudinal undecalcified sections of L1–L3 vertebrae from 28-wk-old female mice (n = 8 mice per group). D, First and third panels depict BMD determinations by DEXA before mice were killed. Second and fourth panels depict the percent change from the initial BMD, which was determined 1 d before surgery (n = 10–13 mice per group). BA/TA, Bone area per tissue area; Tb, trabecular; ns, not significant. *, P < 0.05.

Figure 6

Cancellous but not cortical bone is preserved in OVX ERα_LysM^−/−mice. A and B, Micro-CT measurements were made in bones of the 28-wk-old mice described in Fig. 2 (n = 10–13 mice per group). A, Representative images of vertebral cancellous bone (top) and analysis of vertebral and femoral cancellous bone (bottom). ^†, P < 0.05 vs. respective sham-operated mice. μ-CT, μ-computed tomography. B, Cortical thickness determined in femurs. BV/TV, bone volume per tissue volume; Tb, trabecular; ns, not significant; *, P < 0.05.

Figure 7

Estrogens induce osteoclast apoptosis via a nonclassical ERα action. A, Caspase 3 activity (left panel) or TUNEL assay (middle panel) in mature osteoclasts generated from bone marrow cells from C57BL/6 mice cultured with M-CSF and RANKL for 5 d and treated with vehicle (veh), E₂, or DHT (10⁻⁸ m) for 12 h. The photomicrographs (×40) show representative areas of the osteoclast culture stained with TUNEL (right panel); the arrow indicates an apoptotic osteoclast. AFU, Arbitrary fluorescent units. B, Caspase 3 activity (right panel) in mature osteoclasts, developed from nonadherent bone marrow cells from FasL^gld/gld or wild-type mice, and treated with veh or 10⁻⁸ m of each of the indicated compounds for 12 h. C, Number of TRAP-positive cells generated from bone marrow cells from the same mice as in panel C, cultured with M-CSF and RANKL for 5 d in the presence of 10⁻⁸ m of the indicated compounds. D, C2C12 cells were transfected with an ERE-luc reporter plasmid and treated with 10⁻⁸ m of each of the indicated compounds for 24 h. Luciferase activity is depicted as relative luciferase units (RLU) normalized for Renilla activity. Bars represent the mean ± sd of triplicate determinations, *, P < 0.05 vs. vehicle. E, Caspase 3 activity in mature osteoclasts developed from nonadherent bone marrow cells from wild-type or FasL^gld/gld mice and treated with vehicle (veh) or 50 μm etoposide for 12 h. Bars, Mean ± sd of triplicate determinations, *, P < 0.05 vs. respective vehicle. F, mRNA levels of FasL by quantitative RT-PCR in mature osteoclasts generated as in panel A and treated with veh, E₂, DHT, dendrimer conjugate, and EDC (10⁻⁸ m) for 6 h. G, Western blot analysis of FasL in mature osteoclasts treated with 10⁻⁸ m E₂ for the indicated times. H, Western blot analysis of FasL in mature osteoclasts treated with 10⁻⁸ m concentration of the indicated compounds for 6 h. I, Caspase 3 activity (left panel) or TUNEL assay (right panel) in mature osteoclasts [generated in bone marrow cultures from ERα^NERKI/− or wild-type littermates as described in panel A treated for 12 h with vehicle (veh) or E₂ (10⁻⁸ m]. J, Number of osteoclasts in cancellous bone of undecalcified vertebral sections (L1–L3) from 26-wk-old female ERα^+/+ littermate control and ERα^NERKI/− mice ovariectomized 6 wk earlier (n = 6 mice per group). Means were calculated from triplicate determination unless indicated otherwise. *, P < 0.05 vs. respective veh or sham-operated controls. DC, dendrimer conjugate; Etop, etoposide; Oc, osteoclast; WT, wild type.