Osteoblast Lineage Cells I

0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society

Vol. 141, No. 12 Printed in U.S.A.

The Expression of Osteoprotegerin and RANK Ligand and the Support of Osteoclast Formation by StromalOsteoblast Lineage Cells Is Developmentally Regulated* FRANCESCA GORI, LORENZ C. HOFBAUER†, COLIN R. DUNSTAN, THOMAS C. SPELSBERG, SUNDEEP KHOSLA, AND B. LAWRENCE RIGGS Endocrine Research Unit and Department of Biochemistry (F.G., L.C.H., S.K., B.L.R.) and Molecular Biology (T.C.S.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905; and Amgen, Inc. (C.R.D.), Thousand Oaks, California ABSTRACT The one or more molecular mechanisms that determine the obligatory sequence of resorption followed by formation during bone remodeling is unclear. RANK ligand (RANK-L) is an essential requirement for osteoclastogenesis, and its activity is neutralized by binding to the soluble decoy receptor, osteoprotegerin (OPG). Because both molecules are produced by osteoblast lineage cells, we studied their developmental regulation in a conditionally immortalized human marrow stromal (hMS[2–15]) cell line. These cells can simulate the complete developmental sequence from undifferentiated precursor(s) to cells with the complete osteoblast phenotype that are capable of forming mineralized nodules. During osteoblast differentiation, RANK-L messenger RNA levels decreased by 5-fold, whereas OPG

T

HE TWO components of bone remodeling—resorption and formation—are closely coordinated. They always occur in the same location, resorption always precedes formation, and the amount of old bone that is removed by the resorption phase normally is replaced by the ensuing formation phase (1). This coordination requires the coordinated action of osteoblasts and osteoclasts across their entire developmental sequence (2). Osteoblasts arise from multipotential stromal cells in bone marrow. As they differentiate, they commit to the osteoblast pathway, progressively acquire the characteristics of the osteoblast phenotype, and are able to secrete mineralizable matrix when fully mature (3). Osteoclasts are multinucleated giant cells that originate from hematopoietic cells of monocyte/macrophage lineage in bone marrow. Having once migrated to the bone surface, preosteoclasts, under the influence of the bone marrow microenviroment, differentiate into mature osteoclasts that are capable of resorbing bone (4, 5). The molecular mechanism(s) responsible for the coordinated sequence (coupling) of osteoclastogenesis and osteoblastogenesis during the bone remodeling sequence remains unclear. Traditionally, this linkage between bone formation Received April 7, 2000. Address all correspondence and requests for reprints to: B. Lawrence Riggs, M.D., Mayo Clinic, 200 First Street SW, Plummer North 6, Rochester, Minnesota 55905. E-mail: [email protected]. * This work was supported by Grant AG-04875 from the National Institute on Aging. † Recipient of a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (Ho 1875/2–1).

messenger RNA levels increased by 7-fold, resulting in a 35-fold change in the RANK-L/OPG ratio. OPG protein also increased by 6-fold. Mouse bone marrow cells generated osteoclast-like cells in coculture with undifferentiated hMS(2–15) cells, but did not when cocultured with hMS(2–15) cells in varying stages of differentiation, unless an excess of RANK-L was added. Thus, undifferentiated marrow stromal cells with a high RANK-L/OPG ratio can initiate and support osteoclastogenesis, but after differentiation to the mature osteoblast phenotype, they cannot. We speculate that the developmental regulation of OPG and RANK-L production by stromal/osteoblast cells contributes to the coordinated sequence of osteoclast and osteoblast differentiation during the bone remodeling cycle. (Endocrinology 141: 4768 – 4776, 2000)

and bone resorption has been attributed to osteoclast-toosteoblast signaling, possibly mediated by the release of growth factors from bone matrix during bone resorption (6, 7). However, clearly osteoblast-to-osteoclast signaling also occurs. Indeed, there are several reasons for believing that the differentiation of osteoclast precursors is linked to the differentiation of the osteoblast precursors. First, cell-to-cell contact of osteoblast lineage cells with preosteoclasts may be required for differentiation of osteoclast precursors into multinucleated osteoclasts (4, 8). Second, when osteoblastogenesis is impaired, as in the SAMP6 mouse, osteoclastogenesis also is impaired (9), and when osteoblastogenesis is absent, as in the Osf2/Cbfa1 knock-out mouse, osteoclastogenesis is absent (10 –13). Third, orchiectomy fails to stimulate osteoclastogenesis in the SAMP6 mouse (14). Finally, the two factors that are essential for regulating osteoclastogenesis—M-CSF and RANK (receptor activator of NF␬B) ligand (RANK-L)1 (15, 16)—as well as the soluble decoy receptor for RANK-L, osteoprotegerin (OPG)2 (17, 18), which regulates the biological availability of RANK-L in the bone microenvironment, are produced by osteoblasts. Recently, OPG was identified independently by three groups (17, 18, 19) as a novel member of the tumor necrosis factor (TNF) receptor family that is produced by osteoblast lineage cells. OPG inhibits both the formation and matura1 Also known as osteoprotegerin ligand (OPG-L) (15) and TNFrelated activation-induced cytokine, (TRANCE) (16). 2 Also known as OPG are osteoclastogenesis inhibitory factor (13) and TNF receptor-like molecule 1 (TRI) (14).

4768

RANK-L/OPG EXPRESSION BY OSTEOBLASTS

tion of osteoclasts (17, 18, 19). Mice with targeted deletion of the OPG gene manifest severe osteoporosis (20, 21), whereas transgenic mice overexpressing OPG have suppressed bone resorption and increased bone mass (17). Even more recently, RANK-L ligand (RANK-L), the natural ligand of OPG and a member of the TNF family, was identified as a key effector of osteoclast differentiation (15, 16). RANK-L induces osteoclast formation and differentiation (15, 16) and also directly enhances activity of mature osteoclast by 7-fold (22). RANK-L exerts its biological effects by binding to its receptor, RANK3 (23–25), on osteoclast lineage cells in either a soluble (15) or the membrane-bound (15, 16) form, the latter requiring cell-to-cell contact. Binding of RANK-L to OPG neutralizes its activity (15, 16). Provided permissive levels of M-CSF are present, RANK-L is both necessary and sufficient for osteoclastogenesis (15, 16, 26). Thus, the biological availability of RANK-L is critically dependent on the RANK-L/ OPG production ratio in the bone microenvironment. We recently developed and characterized conditionally immortalized human marrow stromal (hMS) cell lines that can simulate the complete developmental sequence of osteoblast differentiation from uncommitted precursor cells to mature osteoblastic cells that are capable of forming mineralized nodules (27, 28). Here, we report the use of one of these cell lines, hMS (2–15), to test the hypothesis that developmental regulation of the RANK-L/OPG production ratio is associated with reciprocal changes in the ability to support osteoclastogenesis. If so, this mechanism could contribute to the coordinated sequence of osteoclast and osteoblast differentiation during the bone remodeling cycle. Materials and Methods Reagents Tissue culture media FBS, trypsin-EDTA and penicillin-streptomycin were obtained from Life Technologies, Inc. (Grand Island, NY). Unless otherwise indicated, reagents were purchased from Sigma (St. Louis, MO). Tissue culture plastic wares were purchased from Corning, Inc. (Corning, NY). Molecular biology reagents and enzymes were purchased from Roche Molecular Biochemicals (Indianapolis, IN). The RNA STAT-60 kit for the RNA isolation was obtained from Tel-Test, Inc. (Friendwoods, TX). The random primer labeling kit (Decaprime II) was from Ambion, Inc. (Austin, TX). l-Ascorbate phosphate (Asc-P) was obtained from Wako Pure Chemical Industries Ltd. (Richmond, VA). 1␣,25-dihydroxyvitamin D3 (1␣,25(OH)2D3), and radioisotopes were purchased from NEN Life Science Products (Boston, MA). Polytract messenger RNA (mRNA) isolation system and the Wizard PCR Preps DNA purification system were purchased from Promega Corp. (Norwalk, CT). The human ␤-actin insert and ExpressHyb solution were obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). Kits for the measurement of human interleukin (IL)-1␤, human IL-6, IL-11, recombinant human TNF-␣ and recombinant human macrophages-colony stimulating factor (M-CSF) were purchased from R&D System (Minneapolis, MN). Kits for the measurement of procollagen protein were gifts from Metra Biosystems (Mountain View, CA). CH3/HeN 4- to 6-weekold, male mice were obtained from Charles River Laboratories, Inc. Wilmington, MA). Acid Phosphatase Activity Assay and Leukocyte Acid Phosphatase Assay were purchased from Sigma (St. Louis, MO).

Cell culture Conditionally immortalized human marrow stromal (hMS) cell lines were established in our laboratory by transfecting hMS cells with a gene 3 Also known as the osteoclast differentiation and activation receptor (ODAR) (25).

4769

coding for a temperature-sensitive mutant TsA58 of SV40 large Tantigen (SV40 LTA) (27). Incubation of the cells at the permissive temperature (34 C), when the SV40 LTA is active, results in an increased rate of cell proliferation but little or no cell differentiation. Incubation at the restrictive temperature (39.5 C), when the SV40 LTA is inactive, results in little or no cell division but rapid induction of cell differentiation. The undifferentiated cells are bipotential, and, under appropriate conditions can be induced to differentiate along the osteoblastic or adipocytic pathways (27, 28). All six hMS cell lines that we had previously characterized in our laboratories have similar phenotypes and are stable until at least passage 12 (27). For these present studies, we used the hMS (2–15) cell line from passage 9 to passage 12. The hMS (2–15) cells were cultured in an humidified atmosphere 5% CO2/95%, and the cell population was expanded at 34 C in a growth medium of ␣-MEM containing 10% (vol/vol) heat inactivated (HI)-FBS, 0.2 ␮g/ml geneticin (G418), and 1% (vol/vol) of penicillin 10,000 U/ml-streptomycin 10,000 ␮g/ml. Medium was changed twice a week. To induce hMS (2–15) cell differentiation along the osteoblast pathway, both an increase in the temperature to 39.5 C and exposure to differentiation medium are required. The osteoblast differentiation medium contained dexamethasone (Dex) 10⫺8 m, ␤-glycerol-phosphate (␤-GP) 10 mm and l-ascorbate-phosphate (Asc-P) 100 ␮m.

Semiquantitative RT-PCR Cells were plated at density of 1.8 ⫻ 105 cells in six-well microtiter plates in growth medium and cultured for 2 days at 34 C. The cells were then cultured at 39.5 C for 2 days in growth medium (undifferentiated) or in osteoblast differentiation medium for 2, 7, 14, or 21 days. Total cellular RNA was isolated using the RNA-STAT kit. Complementary DNA (cDNA) was synthesized from 1 ␮g total RNA in a 20 ␮l reaction mix containing 1⫻ incubation buffer for AMV reverse transcriptase (RT) 5⫻, 2.5 ␮m of poly䡠dT, 1 mm each of dATP, dCTP, dGTP and dTTP, 20 U of RNase inhibitor and 20 U of AMVRT (avian Moloney virus reverse transcriptase) for 2 h at 42 C. Aliquots of cDNA were amplified in a 25 ␮l PCR mixture which contained 0.2 ␮m of 5⬘ and 3⬘ oligo-primers, 1⫻ of expanded high fidelity PCR buffer 10⫻ with 15 mm MgCl2, 0.1 nm each of dATP, dCTP, dGTP, and dTTP, 0.25 ␮l of [␣-32P] dCTP (10 ␮Ci/␮l) and 0.35 U of expanded high fidelity Taq DNA polymerase. For each assay performed, each cDNA sample was run in duplicate. Amplification reactions specific for the following cDNAs were carried out: alkaline phosphatase (AP), type I collagen (Col I) OPG, RANK-L, and the housekeeping gene GAPDH. Amplifications were performed in a GeneAmp 9600 thermal cycler (Perkin-Elmer Corp., Norwalk, CT). Primer sequences and amplification profiles used for AP, Col I, and RANK-L were reported previously (27, 29) except for OPG PCR products. OPG PCR product identity was confirmed by sequence analysis in an automated DNA sequencer (Perkin-Elmer Corp.). The PCR primer sequences for OPG used in the studies were: OPG sense 5⬘-GTGTCTTTGGTCTCCTGCTAA-3⬘ OPG antisense 5⬘-GGGCTTTGTTTTGATGTTTC-3⬘. The PCR product size for OPG was 271 bp. PCR products were analyzed by electrophoresis of 9-␮l samples in 1.5% (wt/vol) agarose gels. The amplified DNA fragments were visualized by ethidium bromide staining and quantified by counting the radioactivity in gel slices. The quantitative differences between cDNA samples were normalized to the radioactivity present in the GAPDH PCR products.

Bone protein assays hMS (2–15) cells were plated in 48-well microtiter plates at a density of 2 ⫻ 104 cells in standard growth medium. After 2 days at 34 C, the cells were incubated in standard growth medium at 39.5 C for 2 days (undifferentiated) or in osteoblast differentiation medium for 2, 7, 14, or 21 days. The AP activity in cell lysates was quantified at 37 C in assay buffer containing 0.75 m 2-amino-2-methyl-1-propanol, pH 10.3 for 1 h using p-nitrophenylphosphate as a substrate. The release of p-nitrophenol was monitored by measuring absorbance at 410 nm (30). The media were collected, centrifuged, to remove cell debris, and then used for carboxypeptide of type I procollagen (PICP) measurement by ELISA. Results of Col I secretion and AP activity were normalized to total cell protein, as measured by the Bradford protein assay method.

4770


Endo • 2000 Vol. 141 • No. 12

Measurement of mineralized matrix formation Cells were plated at 5 ⫻ 104 cells/well in 12 -ell microtiter plates in standard growth medium. After 2 days at 34 C, the cells were incubated in standard growth medium at 39.5 C for 2 days (undifferentiated) or in osteoblast differentiation medium for 2, 7, 14, or 21 days. The formation of mineralized matrix nodules was determined by alizarin red-S staining (28, 31). Briefly, the cells were fixed in 70% ethanol for 1 h at room temperature. The fixed cells were washed with PBS and stained with 40 mm alizarin red-S pH 4.2, for 10 min at room temperature. The cells were washed with distilled water five times and rinsed with PBS for 15 min. For quantitative assessment, the alizarin red-S dye was eluted and measured spectrophotometrically as previously described (28, 32).

Northern blot hybridization Poly (A)⫹ RNA were isolated from hMS (2–15) cells cultured at 39.5 C for 2 days in growth medium (undifferentiated) or in osteoblast differentiation medium for 2 or 21 days, using the RNAase kit and the Polytract mRNA isolation system. Total RNA and Poly (A)⫹ RNA were resolved on a 1% (wt/vol) agarose/formaldehyde gel using continuos buffer circulation (33) and then transferred to a nylon membrane (Hybond N⫹, Amersham Pharmacia Biotech, Arlington Heights, IL) by capillary blotting (34). Fifty nanograms of the probe of full-length OPG, RANK-L and ␤-actin cDNA probes, which hybridized to mRNA species of 6.0, 2.9, 2.4, and 2.0 kb respectively, were radiolabeled with 5 ␮l [␣-32P] dCTP to a specific activity of ⬎ 109 cpm/␮g DNA using a random primer DNA labeling kit (35). Hybridization and stringent washing were carried out as previously reported (36). Band intensity was quantified by densitometry. Control hybridization with human ␤-actin cDNA verified that equal amounts of RNA were loaded.

OPG, IL-1␤, IL-6, IL-11, TNF-␣, and M-CSF secretion Media from hMS (2–15) cells treated at 39.5 C for 2 days in growth medium (undifferentiated) or in osteoblast differentiation medium for 2, 7, 14, or 21 days, were collected and then centrifuged to remove cell debris. IL-1␤, IL-6, IL-11, M-CSF, and TNF-␣ secretion was measured by a sandwich ELISA. OPG was measured by ELISA as previously reported (17).

In vitro osteoclastogenesis assay Nonadherent bone marrow stromal cells were obtained from 4- to 6-week-old male CH3/HeN mice and prepared as previously reported (18). For coculture experiments, hMS (2–15) cells, at density of 5 ⫻ 104/well in 24-well microtiter plates, were cultured at 39.5 C either with undifferentiated cells for 2 days in growth medium at 39.5 C (Day 0) or for 2, 7, 14, or 21 days at 39.5 C in osteoblast differentiation medium. At each of these time points, 1 ⫻ 106 nonadherent mouse marrow stromal cells (as osteoclast progenitor cells) were added to the hMS (2–15) cells. The cocultured cells were treated at 37 C for 12 days in the presence of Dex 10⫺7 m and 1␣,25 (OH)2D3 10⫺9 m and in the presence or in the absence of 30 ng/ml of M-CSF. In parallel experiments, RANK-L at dose of 25 ng/ml was added to cocultures of hMS (2–15) treated for 21 days in osteoblast differentiation medium and nonadherent mouse marrow stromal cells. New medium containing fresh reagents was added every 3– 4 days. Osteoclast formation was measured by quantitating the presence of TRAP multinucleated positive cells (more than three nuclei) using cytochemical staining.

Experimental protocol Figure 1 schematically outlines the standard experimental protocol for evaluating changes in RANK-L and OPG and osteoblast to osteoclast signaling during osteoblast differentiation. Undifferentiated hMS cells are plated and allowed to attach at 34 C in growth medium for 2 days. The temperature is then increased to 39.5 C and the cells are maintained in the growth medium for an additional 2 days. Under these culture conditions, as we have previously demonstrated (27), the hMS cells will maintain their undifferentiated state short-term, and, thus, this initial 2-day culture period serves as a baseline for assessing subsequent changes occurring during differentiation. To initiate differentiation, the temperature is maintained at 39.5 C, and the growth medium is replaced

FIG. 1. Experimental design. The undifferentiated hMS cells are plated in plastic microtiter wells and allowed to attach at 34 C in growth medium (GM). The temperature is then increased to 39.5 C, and the hMS cell culture is continued in GM for another 2 days. During this interval, the hMS cells maintain in their undifferentiated state and, thus, can serve as a baseline (Day 0) for subsequent differentiation to the osteoblast phenotype. The GM is then replaced with osteoblast differentiation medium (DM) to induce differentiation along the osteoblast pathway. Changes in osteoblast phenotype markers, and in RANK-L, OPG, and select cytokines are assessed after 0, 2, 7, 14, and 21 days of exposure to the DM. Other hMS(2–15) cells that had been exposed to osteoblast differentiation medium for 0, 2, 7, 14, and 21 days were then cocultured with mouse marrow cells for an additional 12 days to assess their ability to support osteoclast formation. See text for composition of GM and DM and for other details. with differentiation medium. Experimental measurements are made at baseline (0 days exposure) and after 2, 7, 14, and 21 days of exposure to the differentiation medium. Other hMS (2–15) cells that had been exposed to differentiation medium for each of these intervals were then cocultured for 12 days with mouse marrow cells to assess osteoclast formation as described in the previous section.

Statistical analysis All values are expressed as mean ⫾ sem. Multiple measures ANOVA were used to evaluate differences between the sample of interest and its respective control. A P-value ⬍ 0.05 was considered statistically significant.

Results Time course of osteoblast differentiation markers

We previously reported that hMS (2–15) cells express mRNA levels for specific osteoblastic markers (27). As shown in Fig. 2A, the mRNA levels for Col I and AP measured by semiquantitative RT-PCR, demonstrate that, hMS (2–15) cells differentiate along the osteoblast pathway when cultured in osteoblastic differentiation medium. The mRNA expression for Col I was maximal (60% increase over values observed in undifferentiated cells) during the first 2 days of treatment with the osteoblastic differentiation medium (P ⬍ 0.005 by multiple measures ANOVA). Maximal AP mRNA levels (320% over values of undifferentiated cells) occurred at 14 days (P ⬍ 0.0001 by multiple measures ANOVA). Consistent with the increased Col I and AP mRNA levels, culture in osteoblast differentiation medium for 2, 7, 14, or 21 days induced increases in type I procollagen and AP activity of up to 8-fold and 28-fold, respectively, over baseline values in undifferentiated cells, at 14 days and 21 days, respectively (P ⬍ 0.001 for COL-1 secretion and P ⬍ 0.0001 for AP activity by multiple measures ANOVA). (Fig. 2B) The discrepancy observed in the fold increase between mRNA and protein


4771

FIG. 2. Time course of relative changes in markers of osteoblast differentiation and in OPG, and RANK-L mRNA levels in undifferentiated hMS(2–15) cells and during osteoblast differentiation. Data are shown for undifferentiated cells before exposure to osteoblast differentiation medium (Day 0) and at 2, 7, 14, and 21 days of culture in osteoblast differentiation medium. A, Semiquantitative RT-PCR. The levels of Col I (䡺) or AP (F) mRNAs were expressed as ratio to GAPDH levels. B, Bone protein secretion. Col I secretion (䡺) was expressed in nmol of type I procollagen for mg of total protein and AP activity (F) was expressed as nmol PNPP/h/mg of total protein. Results are expressed as the percent of the mean of the control values. C, Mineralized nodule formation assessed by alizarin red-S histochemical staining. The alizarin red-S dye was eluted and measured spectrophotometrically. Results are expressed as mmol of alizarin red S per mg of total cellular protein. D, Changes in mRNA levels for OPG (f), and RANK-L over 21 days of differentiation as assessed by semiquantitative RT-PCR (F). Results were expressed as ratio to GAPDH levels and values at day 0 were set at 100%. The data are representative of at least three separate experiments. The results are the means ⫾ SEM of quadruplicates. P ⬍ 0.0001 for AP mRNA and activity and P ⬍ 0.005 for Col I mRNA and P ⬍ 0.001 for Col I secretion and P ⬍ 0.0001 for alizarin red S compared with the corresponding control values as assessed by multiple measures ANOVA.

levels for both AP and Col I may be due to a posttranscriptional increase of gene translation. However, our studies did not directly address this issue. Undifferentiated hMS (2–15) cells cultured for 2 days in growth medium or partially differentiated hMS (2–15) cells cultured for 2, or 7 days with osteoblast differentiation medium did not form mineralizable matrix. In contrast, the matrix secreted by hMS (2–15) cells cultured in osteoblast differentiation medium, began to mineralize after 14 days and showed intense alizarin red-S staining nodules after 21 days (data not shown). Quantification of mineralization by elution of the dye from stained mineral deposits also showed large increases in mineralized matrix (0.7 ␮mol of dye/mg protein) at 21 days (P ⬍ 0.0001 compared with the corresponding central values by multiple measures ANOVA) (Fig. 2C). OPG mRNA levels increase and RANK-L mRNA levels decrease during osteoblast differentiation

OPG and RANK-L mRNA levels were first assessed by semiquantitative RT-PCR (Fig. 2D). During differentiation along the osteoblast pathway, OPG mRNA levels were increased with a maximal effect of 2.4-fold over values of

undifferentiated cells at 21 days. In contrast, over the same time interval the RANK-L mRNA levels were decreased by 2-fold over values observed in undifferentiated cell. When mRNA levels were assessed by Northern blot analysis, the relative changes in steady-state mRNA were even greater. As assessed by densitometry, gene expression of RANK-L was decreased by 5-fold and that for OPG was increased by 7-fold at 21 days, representing an apparent change in the RANKL/OPG ratio of approximately 35-fold with the acquisition of the osteoblast phenotype (P ⬍ 0.0001 for changes from baseline for RANK-L, OPG or RANK-L/OPG ratio) (Fig. 3). OPG protein secretion increases with osteoblast differentiation

To confirm the increase of constitutive OPG mRNA levels at the protein levels, OPG protein secretion was measured in the conditioned media harvested from hMS (2–15) cells at baseline and at different time intervals. Consistent with the increased OPG mRNA levels, OPG protein secretion increased by 6-fold at 21 days compared with values in undifferentiated cells (P ⬍ 0.001 for changes from baseline by multiple measures ANOVA) (Fig. 4).

4772

Endo • 2000 Vol. 141 • No. 12


entiation was associated with the decreased capability to support osteoclastogenesis, hMS (2–15) were cocultured with mouse marrow cells, as a source for osteoclast precursors. As shown in Fig. 5A, coculture with undifferentiated (Day 0) hMS (2–15) cells supported the formation of TRAP positive multinucleated cells. When hMS (2–15) cells were exposed to the osteoblast differentiation medium for 2, 7, 14, or 21 days, before being cocultured for an additional 12 days with mouse marrow stromal cells, TRAP positive, multinucleated osteoclasts were not detected (Figs. 5 and 6, B–E). However, when the cells where cultured in osteoblast differentiation medium for 21 days in the presence of 25 ng/ml of RANK-L, the fully differentiated hMS (2–15) cells were able to support osteoclastogenesis as shown by the presence of TRAP multinucleated positive cells (Figs. 5 and 6F). Similar results from experiments performed in the absence of M-CSF (data not shown) demonstrated that this cytokine is not required for osteoclast formation in our coculture system. Presumably, the permissive concentration of M-CSF required for osteoclastogenesis was supplied by the hMS (2–15) cells. The development of multinucleated, TRAP⫹ cells in cultured bone marrow was associated with large increases in mRNA for calcitonin receptor as assessed by RT-PCR (data not shown). Discussion FIG. 3. OPG, and RANK-L mRNA levels as assessed by Northern analysis. Data are shown for undifferentiated cells before exposure to osteoblast differentiation medium (Day 0) and at 0, 2, and 21 days of culture in osteoblast differentiation medium. A, Northern Blot. Poly (A)⫹ RNA isolated from hMS(2–15) cells was transferred to nylon membranes and hybridized to 32P-labeled cDNA probe for OPG (2.9 kb) and RANK-L (2.4 kb). The experiments were carried out four times, and a representative blot is shown. Control hybridization with ␤-actin cDNA probe (2.0 kb) verified the amounts of mRNA loaded. B, Densitometric analysis. The bars represent the ratio of the optical density of the band, normalized to the optical density of ␤-actin. The relative changes from that of the undifferentiated cells (Day 0) are given with these values set at an arbitrary value of 1.0. The results are the means ⫾ SEM of four different experiments. Note that the apparent RANK-L/OPG mRNA ratio decreases progressively as the hMS(2–15) cells differentiate.

Cytokines secretion

Over the same interval, we assessed protein secretion for IL-1␤, TNF-␣, IL-6, IL-11, and M-CSF, cytokines known to increase bone resorption (3–5) (Fig. 4). Cytokine levels in conditioned media were relatively high for IL-6, but were 28-fold and 170-fold lower for IL-11 and M-CSF, respectively. Neither IL-1␤ nor TNF-␣ were detectable in the conditioned media. Acquisition of a mature osteoblast phenotype was associated with a decrease in IL-6 and IL-11 secretion with a nadir compared with values of undifferentiated cells at 21 days of 25- and 45-fold, respectively. Interestingly, M-CSF secretion was increased by 8-fold over values in undifferentiated cells by 7 days and then decreased. All changes were P ⬍ 0.001 by multiple measures ANOVA. Undifferentiated, but not differentiating or fully differentiated, hMS (2–15) cells can support osteoclastogenesis

To assess whether the decrease in the apparent RANKL/OPG production ratio observed during osteoblast differ-

We designed our studies to answer two questions. Can osteoclastogenesis be related to the stage of osteoblast differentiation? If so, what are the molecular signaling mechanisms? The study was made possible by the availability of the hMS (2–15) cell line that we created by conditionally immortalizing stromal cells from normal human bone marrow (27). These cells have a phenotype of uncommitted bone stromal cell precursors, but under appropriate culture conditions will acquire the phenotype of the mature osteoblast (27, 28). Because cellular and molecular events observed during the differentiation of the hMS (2–15) cells parallel those of osteoblast differentiation in vivo, they are an excellent in vitro model for addressing the role of bone marrow stromal cells/osteoblasts in regulating osteoclastogenesis. When cultured in a differentiation medium, the hMS (2– 15) cells differentiate over a 21-day interval and acquire the mature osteoblast phenotype characterized by increased mRNA and protein for two characteristic osteoblast genes, type I collagen and alkaline phosphatase, and the ability to form mineralized nodules. As have others, we find that glucocorticoids, ␤-glycerol-phosphate, and l-ascorbate-phosphate are required for differentiation of bone marrow stromal cells into mature osteoblasts in vitro (27, 37– 41). The other characteristic osteoblast phenotype gene is osteocalcin. However, osteocalcin expression requires 1␣,25(OH)2D3 treatment, which we could not include because its presence would permit some of the uncommitted precursor cells to differentiate along the adipocytic pathway (28). When the early undifferentiated hMS (2–15) cells are cocultured with mouse bone marrow cells, the murine preosteoclasts differentiated to TRAP-positive multinucleated osteoclast-like cells, but when they were cocultured with cells cultured in osteoblast differentiation medium for 2, 7, 14, or


4773

FIG. 4. OPG, IL-6, IL-11 and M-CSF secretion. Data are shown for undifferentiated cells before exposure to osteoblast differentiation medium (Day 0) and at 2, 7, 14, and 21 days of culture in osteoblast differentiation medium. OPG, IL-6 (both expressed in ng/ml), IL-11 and M-CSF (expressed in pg/ml) secretion was measured by a sandwich ELISA. The data shown are representative of three separate experiments carried out in triplicate ⫾ SEM P ⬍ 0.0001 for OPG, IL-6, IL-11 and M-CSF as measured by multiple measures ANOVA.

FIG. 5. Osteoclastogenesis assay. Data are shown for undifferentiated cells before exposure to osteoblast differentiation medium (Day 0) and at 2, 7, 14, and 21 days of culture in osteoblast differentiation medium. At each time point, hMS(2–15) cells were cocultured at 37 C for 12 days with 1 ⫻ 106 nonadherent mouse marrow stromal cells in the presence of Dex 10⫺7 M, 1␣,25(OH)2D3 10⫺9 M and M-CSF 30 ng/ml. hMS(2–15) cells differentiated for 21 d were also cocultured with RANK-L 25 ng/ml. TRAP multinucleated positive cells (more than 3 nuclei) were measured by cytochemical staining and positive cells counted for 30 fields. Values for days 2, 7, 14, and 21 are zero. The data shown are representative of three separate experiments carried out in triplicate ⫾ SEM P ⬍ 0.05 as measured by multiple measures ANOVA.

21 days they were unable to do so. Thus, we answered the first question affirmatively: in an in vitro model, early undifferentiated stromal-osteoblast cells support osteoclastogenesis, but, as sequential osteoblastic differentiation occurs, the ability to support osteoclast formation is lost. The ability to support osteoclastogenesis was restored by the addition of only RANK-L. We next investigated the molecular mechanism(s) mediating these changes. Because RANK-L is an obligate require-

ment for osteoclastogenesis and is produced by osteoblasts, we hypothesized that signaling is dependent on its biological availability; this, in turn, is inversely proportional to the local concentration of the soluble neutralizing receptor, OPG, that competes with RANK for binding to RANK-L. Over 21 days of exposure to osteoblast differentiation medium, there was a steep decreasing gradient in the apparent RANK-L/OPG mRNA ratio: steady-state levels of RANK-L mRNA were high in the undifferentiated cells and during osteoblast differentiation decreased by 5-fold, whereas OPG mRNA and protein increased by 7- and 6-fold, respectively. Unfortunately, because sensitive antibodies were not available, we could not measure RANK-L protein and, thus, could not determine the RANK-L/OPG protein ratio directly. However, the decrease at the mRNA level suggests that, RANK-L protein probably was also decreased. In addition, the developmental changes in the apparent RANK-L/OPG gradient correlate with the results of the osteoclastogenesis assay during the coculture experiments; the early undifferentiated cells (that are associated with a high RANK-L/OPG expression ratio) can support osteoclastogenesis, whereas the partially or completely differentiated cells (with a progressively lower ratio) cannot. Moreover, treatment of the late differentiated cells with RANK-L restores their ability to support osteoclastogenesis. In the osteoclastogenesis assays, similar results occurred in the absence or in the presence of M-CSF indicating that the hMS cells make sufficient M-CSF to allow osteoclast to form in coculture. Thus, our data also answer the second question: a high RANK-L/OPG concentration ratio in the bone microenvironment is the main molecular signaling mechanism determining osteoclastogenesis. This mechanism is also supported by the recent report of Nagai and Sato (42). These workers assessed the steady-state levels of RANK-L and OPG mRNA by RT-PCR in several stromal or osteoblastic cell lines that either supported or failed to support osteoclasto-

4774


Endo • 2000 Vol. 141 • No. 12

FIG. 6. Representative fields for TRAP multinucleated positive cells from osteoclastogenesis assay. Data are shown for undifferentiated cells before exposure to osteoblast differentiation medium (Day 0) (A) and at 2 (B), 7 (C), 14 (D), and 21 (E) days of culture in osteoblast differentiation medium. At each time point, hMS(2–15) cells were cocultured at 37 C for 12 days with 1 ⫻ 106 nonadherent mouse marrow stromal cells in the presence of Dex 10⫺7 M, 1␣,25(OH)2D3 10⫺9 M and M-CSF 30 ng/ml. hMS(2–15) cells differentiated for 21 days were also cocultured with RANK-L 25 ng/ml (F). TRAP multinucleated positive cells (more than three nuclei) were measured by cytochemical staining. Note that only undifferentiated cells (A), and the cell receiving RANK-L treatment were capable of osteoclast formation.

genesis, both with and without treatment with 1␣,25(OH)2D3 and prostaglandin E2. They found that the ability of the human leukemic HL60 cell line to support osteoclast formation was critically dependent on the apparent RANK-L/OPG ratio. However, the HL60 cell line does not require the presence of M-CSF to differentiate into osteoclast cells, suggesting that it already is partially differentiated toward osteoclasts. Hofbauer et al. (29) found that 48 h exposure of the hMS cells to dexamethasone directly increased RANK-L mRNA and decreased OPG mRNA expression and protein production in hMS cells. However, in these studies, early, immature hMS cells were treated acutely with dexamethasone in a culture medium without either FCS or other differentiating compounds. This direct effect is consistent with the presence of a glucocorticoid response element in the RANK-L promoter (43). However, glucocorticoids are also required for hMS differentiation, and 21- day exposure to a differentiation medium including dexamethasone, l-ascorbate-phosphate, and FCS resulted in a progressive acquisition of the mature osteoblast phenotype osteoblastic features by the hMS (2–15) cells. Under these conditions, the ratio of RANK-L/OPG decreased progressively as the cells differentiated, a change that was in the opposite direction from the direct effect observed in the early cells. During the 21 days of progressive osteoblastic differentiation, the concentration of dexamethasone in the culture medium remained constant. Thus, the effect of dexamethasone on bone differentiation markers is likely indirect and is due to the differentiation process itself. A number of proinflammatory cytokines, including IL-1␤, TNF-␣, IL-6, and IL-11, stimulate bone resorption. Therefore, we evaluated the secretion of several of these during the differentiation of the hMS (2–15) cells along the osteoblast pathway. The IL-6 secretion was relatively high whereas secretion of IL-11 and, especially, M-CSF was much lower. IL-1␤ and TNF-␣ were undetectable, an observation that is

consistent with their production occurring mainly in cells of the monocyte-macrophage lineage (44). As differentiation progressed along the osteoblast pathway, IL-6 and IL-11 secretion were decreased significantly by 25- and by 45-fold, respectively. Although IL-6 may not affect gene expression of RANK-L directly, the high production of this cytokine by early undifferentiated marrow stromal cells could still enhance bone resorption by increasing the number of preosteoclasts upon which RANK-L could act and by enhancing the activity of mature osteoclasts (45). Unexpectedly, M-CSF production increased significantly during middle phase of osteoblast differentiation of hMS (2–15) cells, although its concentrations were much lower than IL-6. In contrast to RANK-L, only low levels of M-CSF appear to be required for acquisition of osteoclast phenotype (15, 16, 46) and in our studies, exogenous M-CSF was not required for osteoclast formation. Moreover, concentrations of M-CSF were low and varied between 0.1 ng/ml and 0.8 ng/ml during hMS (2–15) cell differentiation along the osteoblast lineage, whereas the concentration of OPG protein was much higher at all time-points and increased from 2 to 15 ng/ml in a time-dependent manner during osteoblast differentiation. Thus, it appears that small changes in M-CSF production that we observed during hMS (2–15) cell differentiation probably are insufficient to override the concurrent progressive decrease in the RANK-L/OPG ratio. Although observations in vitro cannot necessarily be extrapolated to physiological mechanisms in vivo, we speculate the developmental regulation of the RANKL/OPG ratio that we have observed in vitro may have an in vivo counterpart that contributes to coordination of osteoclast and osteoblast differentiation. Using a specific monoclonal antibody, Weng et al. (47) have recently established that marrow stromal cells are anatomically located close to endosteal and trabecular surfaces and rarely are found elsewhere in bone marrow. In such an anatomical location, they would be ideally situated


to attract and activate osteoclast precursors and, thus, to initiate the resorption phase of bones remodeling. As osteoclastogenesis proceeds, the stromal cells differentiate toward the mature osteoblast phenotype and lose their ability to support osteoclastogenesis. Following termination of the resorption phase by apoptosis of osteoclasts (48), the now fully mature osteoblasts would be positioned to respond to chemotactic signals during the brief reversal phase by migrating to the now empty surfaces of the resorption lacunae. They could then initiate the ensuing formation phase by refilling the resorption space with newly formed bone. Further studies in vivo are needed to confirm or refute this hypothesis. In summary, we have shown that osteoblast differentiation in vitro is associated with a steep gradient ranging from a high RANK-L/OPG production ratio in the early undifferentiated hMS (2–15) cells that are able to support osteoclastogenesis to a low ratio in the late differentiated hMS (2–15) cells that cannot. We speculate that the developmental regulation of this ratio may contribute to the coordinated sequence of osteoclast and osteoblast differentiation during the bone remodeling cycle.

16.

17.

18.

19.

20.

21.

Acknowledgment We would like to thank Bethany Ngo, Marcy J. Schroeder, and Kevin C. Hicok for their technical help.

22.

References 1. Frost HM 1963 Remodeling dynamics. In: Frost HM (ed) Bone Remodeling Biodynamics. Little Brown, Boston, pp 65–78 2. Parfitt AM 1994 Osteonal and hemi-osteonal remodeling: the spiral and temporal framework for signal traffic in adult human bone. J Cell Biochem 55:273–286 3. Aubin JE, Turkesen K, Herersche JN 1993 Osteoblastic cell lineage. In: Noda M (ed) Cellular and Molecular Biology of Bone. Academic Press, London, pp 1– 45 4. Suda T, Nakamura I, Jimi E, Takahaschi N 1992 Modulation of osteoclast differentiation. Endocr Rev 13:66 – 80 5. Roodman GD 1994 Advances in bone biology: the osteoclast. Endocr Rev 17:308 –332 6. Howard GA, Bottemiller BL, Turner RT, Rader JI, Baylink DJ 1981 Parathyroid hormone stimulates bone formation and resorption in organ culture: evidence for a coupling mechanism. Proc Natl Acad Sci USA 78:3204 –3203 7. Bonewald LF, Mundy GR 1990 Role of transforming growth factor-␤ in bone remodeling. Clin Orthop 250:261–276 8. Takahashi N, Akatsu T, Udagawa N, Sasaki T, Yamaguchi A, Mosely JM, Martin TJ, Suda T 1988 Osteoblastic cells are involved in osteoclast formation. Endocrinology 123:2600 –2602 9. Jilka RL, Weinstein RS, Takahashi K, Parfitt AM, Manolagas SC 1996 Linkage of decreased bone mass with impaired osteoblastogenesis in a murine model of accelerated senescence. J Clin Invest 97:1732–1740 10. Ducy P, Starbuck M, Priemel M, Shen J, Pinero G 1999 A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development. Genes Dev 13:1025–1036 11. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G 1997 Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89:747–754 12. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Jishimoto T 1997 Targeted disruption of cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755–764 13. Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ 1997 Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89:765–771 14. Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC 1997 The effect of androgen deficiency on murine bone remodeling and bone mineral density are mediated via cells of the osteoblastic lineage. Endocrinology 138:4013– 4021 15. Lacey DL, Timms E, Tan HL, Kelley MJ. Dunstan CR, Burgess T, Elliot R, Colombero A, Elliot G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy

23.

24.

25.

26.

27.

28.

29.

30. 31. 32. 33. 34.

4775

E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ 1998 Osteoprotegerin (OPG) ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165–176 Yasuda H, Shima N, Nagkagawa N, Yamaguchi K, Kinosaki M, Mochizuchi SI, Tomayasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N, Suda T 1998 Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastgenesis-inhibitory factor and is identical to TRANCE/RANKL Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennet L, Boone T, Shimamoto G, DeRose M, Eliott R, Colombero A, Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L, Huges TM, Hill D, Pattosion W, Campbell P, Sander S, Van G, Tarpley J, Derby P, Lee R, Amgen EST Program and Jboyle WJ 1997 Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89:309 –319 Yasuda H, Shima N, Nakagawa N, Mochizuchi SI, Yano K, Fujise N, Sato Y, Goto M, Yamaguchi K. Kuriyama M, Kanno T, Murakami A, Tsuda E, Morinaga T, Higashio K 1998 Identity of osteoclastogenesis factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 39:1329 –1337 Tan KB, Harrop J, Reddy M, Young P, Terrett J, Emery J, Moore G, Truneh A 1997 Characterization of a novel TNF-like ligand and recently described TNF ligand and TNF receptor superfamily genes and their constitutive and inducible expression in hematopoietic cells. Gene 204:35– 46 Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Hxu W, Lacey DL, Boyle WK, Simonet WS 1998 Osteoprotegerindeficient mice develop early onset osteoporosis and arterial calcification. Gene Dev 12:1260 –1268 Mizuno A, Amizuka S, Irie K, Murakami A, Fujise N, Kanno T, Sato Y, Nakagawa N, Yasuda H, Mochizuki S, Gomibuchi T, Yano K, Shima N, Washida N, Tsuda E, Morinaga T, Higashio K, Ozawa H 1998 Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin. Biochem Biophys Res Commun 247:610 – 615 Burgess TL, Quian Y-X, Kaufman S, Ring BD, Van G, Capparelli C, Kelley M, Hsu H, Boyle WJ, Dunstan CR, Hu S, Lacey DL 1999 The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. J Cell Biol 145:527–538 Nakagawa N, Kinosaki M, Yamaguchi K, Shima N, Yasuda H, Yano K, Morinaga T, Higashio K 1998 RANK is the essential signaling receptor of osteoclast differentiation factor in osteoclastogenesis. Biochem Biophys Res Commun 253:395– 400 Anderson MA, Maraskovsky E, Billingsley WL, Dougall WC, Tometsko ME, Roux ER, Teepe MC, DuBose RF, Cosman D, Galibert L 1997 A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390:175–179 Hsu H, Lacey DL, Dunstan CR, Solovyev I, Colombero A, Timms E, Tan H-L, Elliot G, Kelley MJ, Sarosi I, Wang L, Xia X-Z, Elliot R, Chiu L, Black T, Scully S, Capparelli C, Morony S, Shimamoto G, Bass MB, Boyle WJ 1999 Tumor necrosis receptor family member RANK mediated osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl Acad Sci USA 96:3540 –3545 Quinn JMW, Elliott J, Gilliespie MT, Martin TJ 1998 A combination of osteoclast differentiation factor and macrophages-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro. Endocrinology 139:4424 – 4427 Hicok KC, Thomas T, Gori F, Rickard DJ, Spelsberg TC, Riggs BL 1998 Development and characterization of conditionally immortalized osteoblast precursor cell lines from human bone marrow stroma. J Bone Miner Res 13:205–217 Gori F, Thomas T, Hicok KC, Spelsberg TC, Riggs BL 1999 Differentiation of human marrow stromal cells: bone morphogenetic protein-2 increases OSF2/CBFA1, enhances osteoblast commitment and inhibits late adipocyte maturation. J Bone Miner Res 14:1522–1535 Hofbauer LC, Gori F, Riggs BL, Lacey DL, Dunstan CR, Spelsberg TC, Khosla S 1999 Stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin production by glucocorticoids in human osteoblastic lineage cells: potential paracrine mechanism of glucocorticoid-induced osteoporosis. Endocrinology 140:4382– 4389 Puzas JE, Brand JS 1985 Bone cell phosphotyrosine phosphate: characterization and regulation by calcitropic hormones. Endocrinology 116:2463–2468 Vacca LL 1985 Calcification In: Laboratory Manual of Histochemistry. Raven Press, New York, pp 334 –336 Bodine PVN, Trailsmith M, Komm BS 1996 Development and characterization of a conditionally transformed adult human osteoblastic cell line. J Bone Miner Res 11:806 – 819 Lehrach H, Diamond D, Wozney JM, Boedtker H 1997 RNA molecular weight determination by gel electrophoresis under denaturating conditions, a critical reexamination. Biochem Cell 89:747–754 Thomas PS 1980 Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulosa. Proc Natl Acad Sci USA 77:5201–5201

4776


35. Feinberg AP, Vogelstein B 1983 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6 –13 36. Hofbauer LC, Dunstan CR, Spelsberg TC, Riggs BL, Khosla S 1998 Osteoprotegerin production by human osteoblast lineage cells is stimulated by vitamin D, bone morphogenetic protein-2 and cytokines. Biochem Biophys Res Commun 250:776 –781 37. Cheng S, Yang YW, Rifas L, Zhang S, Avioli LV 1994 Differentiation of human bone marrow osteogenic stromal cells in vitro. Induction of the osteoblast phenotype by dexamethasone. Endocrinology 134:277–286 38. Grigoriadis AE, Heersche JN, Aubin JE 1988 Differentiation of muscle, fat, cartilage and bone from progenitor cells present in a bone-derived clonal cell population: effect of dexamethasone. J Cell Biol 106:2139 –2151 39. Locklin RM, Williamson MC, Bereford JN, Triffit JT, Owen ME 1995 In vitro effects of growth factors and dexamethasone on rat marrow stromal cells. Clin Orthop 313:27–35 40. Rickard DJ, Sullivan TA, Shenker BJ, Leboy PS, Kazhdan I 1994 Induction of rapid osteoblast differentiation in rat bone marrow stromal cell culture by dexamethasone and BMP-2. Dev Biol 12:871– 880 41. Franceschi RT 1992 The role of ascorbic acid in mesenchymal differentiation. Nutr Rev 50:65–70 42. Nagai M, Sato N 1999 Reciprocal gene expression of osteoclastogenesis in-

43. 44. 45.

46.

47.

48.

Endo • 2000 Vol. 141 • No. 12

hibitory factor and osteoclast differentiation factor regulates osteoclast formation. Biochem Biophys Res Commun 257:719 –723 Kitazawa R, Kitazawa S, Maeda S 1999 Promotor structure of mouse RANKL/ TRANCE/OPGL/ODF gene. Biochim Biophys Acta 1445:134 –1341 Romas E, Martin TJ 1997 Cytokines in the pathogenesis of osteoporosis. Osteoporos Int 7:S47–S53 Adebanjo OA, Moonga BS, Yamate T, Sun L, Minkin C, Abe E, Zaidi M 1998 Mode of action of interleukin-6 on mature osteoclasts. Novel interactions with extracellular Ca2⫹ sensing in the regulation of osteoclastic bone resorption. J Cell Biol 142:1347–1356 Fuller K, Wong B, Choi Y, Chambers TJ 1998 TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts. J Exp Med 188:997–1001 Weng L, Falla N, Van Den Heuvel R, Raymackers J, Karperien M, Van Bezooijen R, Van Vlasselaer P, Lowik C, Merregaert J 2000 The monoclonal antibodies 18d7/91f2 recognize a receptor regulatory protein on mouse bone marrow stromal cells J Bone Miner Res 15:1286 –1300 Hughes DE, Dai A, Tiefee JC, Li HH, Mundy GR, Boyce BF 1996 Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-␤. Nat Med 2:1132–1136