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. 2000 Feb 15;97(4):1566-71.
doi: 10.1073/pnas.97.4.1566.

RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism

J Li  1 , I Sarosi, X Q Yan, S Morony, C Capparelli, H L Tan, S McCabe, R Elliott, S Scully, G Van, S Kaufman, S C Juan, Y Sun, J Tarpley, L Martin, K Christensen, J McCabe, P Kostenuik, H Hsu, F Fletcher, C R Dunstan, D L Lacey, W J Boyle
Affiliations

RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism

J Li et al. Proc Natl Acad Sci U S A. .

Abstract

We have generated RANK (receptor activator of NF-kappaB) nullizygous mice to determine the molecular genetic interactions between osteoprotegerin, osteoprotegerin ligand, and RANK during bone resorption and remodeling processes. RANK(-/-) mice lack osteoclasts and have a profound defect in bone resorption and remodeling and in the development of the cartilaginous growth plates of endochondral bone. The osteopetrosis observed in these mice can be reversed by transplantation of bone marrow from rag1(-/-) (recombinase activating gene 1) mice, indicating that RANK(-/-) mice have an intrinsic defect in osteoclast function. Calciotropic hormones and proresorptive cytokines that are known to induce bone resorption in mice and human were administered to RANK(-/-) mice without inducing hypercalcemia, although tumor necrosis factor alpha treatment leads to the rare appearance of osteoclast-like cells near the site of injection. Osteoclastogenesis can be initiated in RANK(-/-) mice by transfer of the RANK cDNA back into hematopoietic precursors, suggesting a means to critically evaluate RANK structural features required for bone resorption. Together these data indicate that RANK is the intrinsic cell surface determinant that mediates osteoprotegerin ligand effects on bone resorption and remodeling as well as the physiological and pathological effects of calciotropic hormones and proresorptive cytokines.

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Figures

Figure 1
Figure 1
Gene targeting at the RANK locus. (A) Restriction maps of part of RANK locus being targeted (Top) and the targeting construct (Middle). In the RANK targeting construct, the PGK-neo cassette was placed in reverse orientation to replace coding exons 2 and 3A. Exons are shown as boxes. (Bottom) The small open box and arrowheads represent the 5′ flanking probe and PCR primers used for genotyping. SA, short arm; LA, long arm. Restriction sites: S, SpeI; H, HincII; N, NotI. (B) Southern blot of SpeI-digested genomic DNA using the 5′ flanking probe as indicated in A. Positions of wild-type allele (WT, 8 kb) and targeted allele (KO, 1 kb) also are indicated. (C) Northern blot of intestine poly(A)+ RNA using cDNA probe encompassing the deleted RANK exons. Positions of the RNA size markers are indicated, and the same blot was reprobed with internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (Bottom). (D) Immunoprecipitation and Western blot analysis of RANK protein. RANK was immunoprecipitated from total intestine lysate by using polyclonal antibody raised against the extracellular region of RANK protein. Cell lysate from 1 × 107 RAW264.7 cells also was immunoprecipitated as positive control.
Figure 2
Figure 2
Severe osteopetrosis in RANK−/− mice. (A and B) Radiographs of the leg, pelvis, and vertebrae regions of RANK+/+ (A) and RANK−/− (B) littermates. Arrowheads indicate the growth plate area. (C and D) Hematoxylin/eosin staining of tibia from RANK+/+ (C) and RANK−/− (D) littermates demonstrates severe osteopetrosis in RANK knockout mice. Note in RANK−/− mice the significantly widened growth plate (*) and the highly vascularized invasive growth (arrowheads) within the growth plate. (E and F) TRAP staining of bone sections from RANK+/+ (E) and RANK−/− (F) littermates shows the absence of TRAP+ osteoclasts (arrowheads in E) in RANK−/− mice. (Original magnifications: A–D, ×4; E and F, ×60.)
Figure 3
Figure 3
RANK−/− mice have an intrinsic defect in osteoclast development. (A) Osteoclast differentiation from RANK+/+, RANK+/−, and RANK−/− splenic hematopoietic progenitors in the presence of CSF-1 (30 ng/ml) and various concentrations of recombinant murine OPGL as measured by TRAP solution assay. (Right) Representative photographs (×20) of TRAP-stained culture treated with CSF-1 (30 ng/ml) and OPGL (500 ng/ml). (B, C, and D) Radiographs of femur and tibia/fibula 4 weeks after bone marrow adoptive transfer from rag1−/− mice. Note the massive decrease of bone radiodensity in rag1−/−RANK−/− mice (D) compared to nontransferred RANK−/− littermate (C), to a level similar to that of rag1−/−RANK+/+ mice (B). (E–G) Hematoxylin/eosin and TRAP (Insets) staining of tibia 4 weeks after bone marrow adoptive transfer from rag1−/− mice. Overall bone morphology of rag1−/−RANK−/− (H) mice is much closer to that of the rag1−/−RANK+/+ littermate (F) than to the nontransplanted RANK−/− littermate (G). Note the obvious increase of marrow space and the appearance of many TRAP+ multinucleated osteoclasts (arrowheads). (Original magnifications: ×4; Insets, ×60.)
Figure 4
Figure 4
Retroviral reconstitution of osteoclastogenic potential in RANK−/− mice. RANK−/− splenic hematopoietic cells were transduced with control vector MSCV (A–C), or MSCV-RANK retroviruses (D–F). After viral infection, cytocentrifuge preparations of cells transduced with MSCV (A) or MSCV-RANK retrovirus (D) were stained with anti-RANK antibodies. Virus-transduced hematopoietic cells were cultured for an additional 7 days in the presence of CSF-1 and OPGL. Multinucleated TRAP+ osteoclasts are demonstrated only in cultures transduced with MSCV-RANK retrovirus (E), not in cultures transduced with MSCV (B). Scanning electron microscopic images of bone slices exposed to MSCV- (C) or MSCV-RANK- (F) transduced hematopoietic cells show resorption lacunae only in MSCV-RANK-transduced cultures (F). Hematoxylin/eosin staining of femurs from RANK−/− mice 6 weeks after they received transplantation of RANK−/− splenic hematopoietic cells transduced with either MSCV (G) or MSCV-RANK (H) retroviruses. Note the significantly increased marrow space in the metaphysis region of H. Osteoclasts that are positively stained with TRAP (red in I) or cathepsin K (brown in J) can be detected only in adjacent sections of H. (Original magnifications: A, B, D, and E, ×40; G and H, ×4; C and F, ×350, I and J, ×60.)
Figure 5
Figure 5
Effects of calciotropic hormones and cytokines challenge in RANK−/− mice. WT, wild type; KO, knockout. RANK+/+ and RANK−/− mice were challenged with (A) murine OPGL, (B) human PTHrP, (C) 1α,25-(OH)2D3, (D) human IL-1β, and (E) murine TNF-α, using PBS (A, B, D, and E) or corn oil (C) as control. (Left) Whole blood ionized calcium levels are shown. (Right) TRAP staining and cathepsin K fluorescent immuno-histochemistry on sections of calvaria from challenged RANK−/− mice are shown. Note the complete absence of any TRAP and cathepsin K-positive osteoclast in OPGL (A), PTHrP (B), 1α,25-(OH)2D3 (C), or IL-1β (D) challenged RANK−/− mice, and the appearance of TRAP and cathepsin K-positive osteoclasts (Inset in E) in TNF-α-challenged RANK−/− mice. *, Statistically different from control group, P < 0.001 (group n = 5, mean ± SEM). (Original magnification: ×10; Inset in E, ×20.)
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