CoChat
Sign Up
Cited by 251Bone Metabolism and DiseasesEstrogen and related hormone effectsBone health and osteoporosis research

The DOI matches its Crossref record; 68 of the 69 references it cites were found in a registry or an index.

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

Open access
Streicher, Carmen, Heyny, Alexandra, Andrukhova, Olena, Haigl, Barbara, Slavic, Svetlana, Schüler, Christiane, Kollmann, Karoline, Kantner, Ingrid, Sexl, Veronika, Kleiter, Miriam, Hofbauer, Lorenz C., Kostenuik, Paul J., Erben, Reinhold G.·2017·Scientific Reports
DOI 10.1038/s41598-017-06614-0PMC PMC5527119
Open this paper in CoChat — free accountOpen PDF

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.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1038/s41598-017-06614-0

Cites

68 of 68 references verified0 citation errors · 0 not checkable

Statistics

Figures

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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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.
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.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 69 references

Cited by

Sorted by citations

Items 201–251 of 251

Related

See all

Metadata

Authors
Streicher, C., Heyny, A., Andrukhova, O., Haigl, B., Slavic, S., Schüler, C., Kollmann, K., Kantner, I., Sexl, V., Kleiter, M., Hofbauer, L. C., Kostenuik, P. J., & Erben, R. G.
Affiliations
  • University of Veterinary Medicine Vienna (AT)
  • Technische Universität Dresden (DE)
  • Amgen (United States) (US)
Corresponding author
Erben, Reinhold G.
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2017-07-25
Volume
7
Issue
1
Pages
6460-6460
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-017-06614-0
PMC
PMC5527119
OpenAlex
W2738919786
Cited by
251
References
69
Citations by year
2026: 30 · 2025: 36 · 2024: 32 · 2023: 29 · 2022: 40 · 2021: 26 · 2020: 25 · 2019: 18 · 2018: 13 · 2017: 1
FWCI
6.37
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Austrian Science Fund (FWF P 21904-B11)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bone Metabolism and Diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Endocrinology › Estrogen and related hormone effects
  • Health Sciences › Medicine › Orthopedics and Sports Medicine › Bone health and osteoporosis research
Keywords
estrogen, estrogen receptor alpha, RANKL, bone lining cells, bone resorption, bone turnover, bone remodeling, estrogen deficiency, osteoclasts, skeletal homeostasis, hematopoietic cells, mesenchymal cells
MeSH terms
Alkaline Phosphatase, Animals, Bone and Bones, Estrogens, Female, Gene Expression Regulation, Humans, Isoenzymes, Mice, Transgenic, Rats, Inbred F344, Bone Density, Bone Marrow Transplantation, Bone Remodeling, Mice, Knockout, Estrogen Receptor alpha, RANK Ligand, GPI-Linked Proteins, Mesenchymal Stem Cells

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review