Estrogen Regulates Bone Turnover by Targeting RANKL Expression in Bone Lining Cells

Abstract

Estrogen is critical for skeletal homeostasis and regulates bone remodeling, in part, by modulating the expression of receptor activator of NF-κB ligand (RANKL), an essential cytokine for bone resorption by osteoclasts. RANKL can be produced by a variety of hematopoietic (e.g. T and B-cell) and mesenchymal (osteoblast lineage, chondrocyte) cell types. The cellular mechanisms by which estrogen acts on bone are still a matter of controversy. By using murine reconstitution models that allow for selective deletion of estrogen receptor-alpha (ERα) or selective inhibition of RANKL in hematopoietic vs. mesenchymal cells, in conjunction with in situ expression profiling in bone cells, we identified bone lining cells as important gatekeepers of estrogen-controlled bone resorption. Our data indicate that the increase in bone resorption observed in states of estrogen deficiency in mice is mainly caused by lack of ERα-mediated suppression of RANKL expression in bone lining cells.

Figure 1

Selective deletion of estrogen receptor α in the mesenchymal or hematopoietic compartment. (A) Relative to baseline (BL) controls, uterine weight decreased in non-irradiated (No BMT) WT mice 4 weeks after OVX, similar to the decrease seen 4 weeks after irradiation (BMT) of vehicle-treated ovary-intact WT mice (Veh); 17ß-estradiol (E2) treatment of irradiated WT mice significantly increased uterine weight. (B) Experimental design. All irradiated mice were supplemented with physiological doses of E2 (10 µg/kg s.c. in B/R 5 times per week) during the 4-week recovery phase post-transplantation. (C) µCT images of the femoral distal metaphysis of non-irradiated (upper panels) SHAM and vehicle- or 17ß-estradiol (E2)-treated OVX WT and αERKO mice, and vehicle- or estradiol-treated WT and αERKO mice transplanted (BMT, lower panels) with unfractionated bone marrow cells from αERKO or WT mice, 4 weeks post-OVX. (D) Total and trabecular BMD measured by pQCT and bone volume (BV/TV) measured by µCT in the distal femoral metaphysis, bone formation rate (BFR/BS) and osteoclast number (N.Oc/B.Pm) in cancellous bone of the distal femoral metaphysis measured by histomorphometry, total BMD of the femoral shaft measured by pQCT, and (E) bone volume (BV/TV) as well as trabecular BMD of the L4 vertebrae in non-irradiated and reconstituted WT and αERKO mice treated with vehicle or physiological doses of E2, 4 weeks post-OVX. All irradiated mice were supplemented with physiological doses of E2 during the 4-week recovery phase post-transplantation. Data represent mean ± SD of 8–10 animals each. *p < 0.05 by one-way ANOVA followed by SNK test.

Figure 2

Selective inhibition of RANKL in the hematopoietic or mesenchymal compartment. (A) µCT images of the distal femoral metaphysis of non-irradiated vehicle-treated OVX WT and huRANKL-KI mice (upper panels), and vehicle- or AMG161 (10 mg/kg twice weekly)-treated WT and huRANKL-KI mice transplanted (BMT) with unfractionated bone marrow cells from huRANKL-KI or WT mice, 4 weeks post-OVX. (B) Total and trabecular BMD measured by pQCT, bone volume (BV/TV) measured by µCT, and bone formation rate (BFR/BS) and osteoclast number (N.Oc/B.Pm) measured by histomorphometry in cancellous bone of the distal femoral metaphysis, urinary deoxypyridinoline/creatinine (DPD/Crea) excretion measured by ELISA, and (C) bone volume (BV/TV) as well as trabecular BMD of the L4 vertebrae in non-irradiated and reconstituted WT and huRANKL-KI mice treated with vehicle or AMG161, 4 weeks post-OVX. All irradiated mice were supplemented with physiological doses of E2 during the 4-week recovery phase post-transplantation. Data represent mean ± SD of 8–10 animals each. *Denotes p < 0.05 by one-way ANOVA followed by SNK test.

Figure 3

Estradiol targets RANKL expression in bone lining cells. (A) Anti-RANKL immunohistochemistry on cryosections of undecalcified distal femurs in vehicle-treated SHAM, and vehicle- or 17ß-estradiol (10 µg/kg in B/R 5 times per week)-treated OVX WT mice, 2 weeks post-OVX. (B) Percent RANKL-positive osteoblasts per bone surface (BS), percent RANKL-positive bone lining cells (LC) per bone surface, and percent RANKL-positive osteocytes in vehicle-treated SHAM and vehicle- or E2-treated OVX WT mice, 2 weeks post-OVX. (C) Anti-RANKL immunohistochemistry on cryosections of undecalcified proximal tibias in vehicle-treated SHAM and vehicle- or E2 (2.5 µg/kg in B/R 5 times per week)–treated OVX Fischer 344 rats, 2 weeks post-OVX. (D) Percent RANKL-positive osteoblasts per bone surface (BS) and percent RANKL-positive bone lining cells per bone surface in vehicle-treated SHAM and vehicle- or E2-treated OVX rats, 2 weeks post-OVX. Arrows in A and C mark RANKL-positive osteoblasts, arrowheads mark RANKL-positive lining cells. *In B and D denotes P < 0.05 vs. Sham, ^#denotes P < 0.05 vs. OVX + vehicle. Data in B and D are mean ± SD of 4–5 animals each.

Figure 4

In situ mRNA profiling of bone cells in cryosections by laser capture microdissection (LCM). (A) Harvesting of osteoblasts (OB), bone lining cells (LC), and osteocytes (OC) by LCM in 4-µm-thick cryosections of mouse femurs. Left panels show sections before LCM with cells to be harvested marked by arrows, right panels show the same section after LCM. Note that the retraction of the bone marrow facilitates harvesting of especially bone lining cells which remain attached to the bone surface. Cryosections were briefly stained with Histostain (Arcturus). Asterisks mark bone tissue. (B) mRNA expression profiling of collagen 1a1 (Col1a1), Runx2, alkaline phosphatase (Alpl), Podoplanin (Pdpl), sclerostin (Sost), dentin matrix protein-1 (Dmp1), RANKL, and OPG by qRT-PCR on RNA isolated from distal femoral cancellous bone osteoblasts (OB), osteocytes (OC), and bone lining cells (LC) harvested by LCM in cryosections of undecalcified bones from WT mice. (C) mRNA expression of ERα and ß in distal femoral cancellous bone osteoblasts, osteocytes, and bone lining cells harvested by LCM in cryosections of undecalcified bones from WT mice. mRNA expression of ERß was expressed relative to ERα expression in osteoblasts. (D) mRNA expression of RANKL in distal femoral cancellous bone osteoblasts, osteocytes, and bone lining cells harvested by LCM in cryosections of undecalcified bones from vehicle-treated SHAM and vehicle- or E2-treated OVX WT mice. Data in B-D represent mean ± SD of 3–5 animals each. In B and C, *denotes P < 0.05 vs. osteoblasts, ^#denotes P < 0.05 vs. lining cells by one-way ANOVA followed by SNK test. In D, *denotes P < 0.05 vs. OVX + vehicle, ^†denotes P < 0.05 vs. the same treatment in osteoblasts and lining cells by one-way ANOVA followed by SNK test.

Figure 5

Model of how estrogen regulates osteoclastic bone resorption. Estradiol (E2) primarily suppresses RANKL expression in bone lining cells. Membrane-bound or soluble RANKL interacts with RANK expressed on mature osteoclasts and osteoclast precursors to stimulate bone resorption. The decoy receptor osteoprotegerin (OPG) binds and, thus, biologically inactivates membrane-bound and soluble RANKL. Estrogen deficiency reduces the suppression of RANKL expression by bone lining cells, leading to increased osteoclastic bone resorption. Estradiol may also regulate OPG expression in bone lining cells.