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RESEARCH COMMUNICATION |
a Department of Microbiology and Immunology, and Walther Oncology Center, Indiana University School of Medicine and the Walther Cancer Institute, Indianapolis, Indiana 46202–5120, USA
b Department of Biochemistry, School of Dentistry, Showa University, Shinagawa-ku, Tokyo 142, Japan
c Cytokine Research Laboratory, Department of Molecular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA
d Department of Microbiology, Chosun University School of Dentistry, Kwang Ju 501–759, Korea
e Department of Oral Microbiology, Seoul National University, School of Dentistry, Chongro-Ku, Seoul, Korea
f Human Genome Sciences, Inc., Rockville, Maryland 20850, USA
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: TNFR • anti-TR1 mAb • osteoclast • immunization • monoclonal antibody
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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By searching an expressed sequence tag (EST) database, we identified a new member of the TNFR superfamily that, unlike other members, is secretory. Abundant mRNA expression of this gene was found in primary fibroblasts and primary osteoblasts, as well as in osteogenic sarcoma cell lines that had characteristics of osteoblasts. Because of its unique pattern of mRNA expression, we examined whether it was associated with fibroblast growth and bone cell differentiation. We found that the molecule exhibited broad biological activities, including fibroblast proliferation, inhibition of osteoclastogenesis, and inhibition of bone resorption.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
Male Balb/c mice 6 to 8 wk old were purchased from Taconic (Germantown, N.Y.). A DdY strain of 16-day pregnant, 6- to 9-wk-old males, or 1- to 2-day-old newborn mice were obtained from Shizuoka Laboratories Animal Center (Shizuoka, Japan).
Chemicals
1
-Dihydroxyvitamin D3 [1, 25(OH)2 D3] was purchased from either Wako Pure Chemical Industries Ltd. (Osaka, Japan) or Biomolecule, Inc. (Plymouth Meeting, Pa.). Eel calcitonin was kindly provided by Asahi Chemical Industry Co. (Shizuoka, Japan). [125I]-labeled human calcitonin and 45Ca were obtained from Amersham, Buckinghamshire, England.
Cells
Human primary osteoblast, isolated and cultured from the rib of a 9-month-old human male, was provided by Dr. Joseph Bidwell, Indiana University School of Dentistry, Indianapolis. Osteoblasts were stimulated with parathyroid hormone (PTH) in some experiments. Osteogenic sarcoma cell lines MG63 and 143B were obtained from the American Type Culture Collection (ATCC; Rockville, Md.). T cell lines (Jurkat and CEM), B cell lines (SKW6.4, DAKAKI), the hematopoietic progenitor cell line (KG1a), a kidney cell line (HEK 293), and a fibroblast cell line IMR 90 were also obtained from the ATCC. Peripheral blood mononuclear cells were prepared by Ficoll-Hypaque gradient centrifugation.
RNA and DNA blot hybridization
Total RNA was extracted from primary cells and cell lines with Trireagent (Molecular Research Center, Cincinnati, Ohio). The RNA was used for Northern blot and reverse transcriptase polymerase chain reaction (RT-PCR) analysis. A human multiple-tissue Northern blot purchased from Clontech (Palo Alto, Calif.) and blots containing multiple cell lines prepared at Human Genome Sciences, Inc. (Rockville, Md.) were used in some experiments.
Production of recombinant TR1, TR1-Fc, and GST-TR1 fusion proteins
The TR1 NH2-terminal region including 208 amino acids was expressed as an Fc fusion protein in NIH 3T3 cells, as described previously (18). The TR1-Fc fusion protein was purified by protein G chromatography, and the amino acid sequence of the amino terminus was determined by automatic peptide sequencer. The purified TR1-Fc was also used for other biological assays.
To produce TR1-GST fusion protein, the entire open reading frame excluding signal peptide was fused in frame with the glutathione-S-transferase (GST) gene, using the PGEX vector (Pharmacia, Piscataway, N.J.). The TR1-GST protein was expressed in Escherichia coli strain top 1 (Stratagene, La Jolla, Calif.) and purified by affinity chromatography over GST beads. Recombinant full-length TR1 protein was also produced by a baculovirus expression system and purified by a BioCAD system from PerSeptive Biosystems (Framingham, Mass.) and heparin-agarose columns.
Immunization and monoclonal antibody production
Balb/c mice (8 wk old) were immunized with 50 µg of TR1-GST emulsified in complete Freund's adjuvant. Three intraperitoneal injections were administered 2 wk apart. Three days after the last injection, the mice were killed and their spleens were removed. Spleen cells were fused with SP2/0 myeloma cells and the hybridoma supernatants were screened by enzyme-linked immunosorbent assay (ELISA) for TR1 using purified TR1-Fc fusion protein. Monoclonal antibody (mAb) isotyping was performed by using the Immunopure Monoclonal Antibody Isotyping Kit (Pierce, Rockford, Ill.). mAb was purified over a protein G Sepharose column (Zymed Lab, South San Francisco, Calif.).
Immunoprecipitation
MG63 cells were cultured in Met/Cys-free RPMI medium (ICN, Pharmaceutical Inc., Irvine, Calif.) for 1 h and then labeled with trans-[35S] (100 µCi/ml, ICN) for 4 h. The culture supernatant was harvested with the addition of a protein inhibitor tablet (GIBCO, BRL, Gaithersburg, Md.) and concentrated by Centricon-5000 (Millipore, Bedford, Mass.). The supernatant was used to immunoprecipitate the TR1 with anti-TR1 mAb. Immunoprecipitates were run on a sodium dodecyl sulfate 10% polyacrylamide gel, transferred to nylon membrane, and exposed to X-ray film.
Radiolabeling of antibodies
Anti-human Fc antibodies were purchased from Jackson Laboratories (Bar Harbor, Maine) and radiolabeled to high specific activity by using Iodobeads according to directions given by the manufacturer (Pierce). Mouse anti-human Fc (10 µg) was incubated with 1.0 mCi of Na 125I (2000 Ci/mmol) (Amersham Corp.) in iodination buffer at room temperature for 15 min. Protein-bound 125I was separated from free 125I by a Sephadex G-25 column. The 125I-labeled antibody yielded specific activities in the range of 1.5 to 2.5 x 1016 cpm/mmol protein.
Receptor binding assays
Receptor binding assays on THP-1 cells were performed in 96-well culture plates. Cells (5x106 cells per well) were incubated with an excess amount of a mixture of goat and rabbit immunoglobulin G (IgG) in a binding medium (RPMI 1640, 1% bovine serum albumin (BSA), 0.2% sodium azide, and 20 mM Hepes, pH 7.2) for 1 h at room temperature to mask Fc receptors on the cells. Cells were washed twice with phosphate-buffered saline (PBS) and incubated in the binding medium containing various concentrations of TR1-Fc for 2 h at room temperature. Cells were then washed once with PBS and incubated with 125I-labeled mouse anti-human IgG (20 ng/ml) in the binding medium for 1 h at room temperature. Cells and unbound 125I-labeled antibody were separated by the phthalate oil separation method (19). In all assays, nonspecific bindings were determined by inclusion of a 500-fold molar excess of unlabeled anti-human IgG in the reaction mixture. Specific bindings were calculated by subtracting the nonspecific binding from each data point.
Flow cytometric studies
Cells were stained and analyzed on a FACScan (Beckton Dickinson, San Jose, Calif.). THP-1 or human fibroblasts were washed three times in staining medium consisting of PBS, 1% BSA, and 0.1% glucose before staining. 5.0 x 105 cells were resuspended in 100 µl of staining medium containing a saturating concentration of biotinylated TR1 (0.5 µg/sample) for THP-1 cells or TR1-Fc (1.0 µg/sample) for fibroblasts, and incubated at 4°C for 30 min. After washing, cells were stained with phosphatidylethanolamine-streptavidin (0.5 µg/sample) for THP-1 cells or with goat anti-human IgG-FITC (1 µg/sample) for fibroblasts diluted in staining medium at 4°C for 20 min. Cells were either immediately analyzed or fixed with 1% paraformaldehyde for later analysis. Human IgG1 was used as a negative control of TR1-Fc and murine IgG1-biotin was used as negative control of TR1-biotin. Gates were set on live cells only, based on forward vs. side scatter profiles.
Fibroblast proliferation assays
Fibroblast growth-stimulatory assays were carried out essentially according to the procedure described in ref 20. Briefly, confluent human diploid foreskin fibroblasts (at passage level 12–15; 8x103/well) were plated in 0.1 ml of the medium [RPMI-1640 plus 10% fetal bovine serum (FBS)] in 96-well Falcon plates. After overnight incubation in a CO2 incubator at 37°C, the medium was removed and a serial dilution of the TNF or recombinant TR1 was layered in 0.1 ml of RPMI-1640 medium. During the last 24 h of a 72 h incubation, tritiated thymidine (6.7 Ci/mmol) was added to each well (0.5 mCi/well). In some experiments, anti-TR1 mAb was added to block the TR1 effects. Relative cell viability was calculated as the amount of thymidine incorporated in treated cells divided by that in the untreated cells, and expressed as a percentage.
Coculture assays for osteoclastogenesis
To prepare primary osteoblastic cells, a total of 20 to 30 calvaria collected from newborn mice were subjected to five sequential digestions using 0.1% collagenase (Wako Pure Chemical Industries Ltd.) and 0.2% dispase (Godo Shusei, Tokyo, Japan). Bone marrow cells were obtained from adult mice. Calvarial cells were cocultured with bone marrow cells as described (21, 22). In short, primary calvarial cells (2x104 per well) and nucleated marrow cells (5x105 per well) were cocultured in 48-well plates (Corning Glass, Corning, N.Y.) with 0.3 ml of -MEM (GIBCO BRL) containing 10% fetal bovine serum (JRH Biosciences, Lenexa, Kans.), in the presence or absence of TR1 or other chemicals. Cultures were incubated in quadruplicate and cells were replenished on day 3 with fresh medium. Osteoclast-like multinucleated cell (OCL) formation was evaluated after culturing for 6 to 7 days. Adherent cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP), and the number of TRAP-positive osteoclasts was scored as described (23). For TRAP staining, adherent cells were fixed with 10% formaldehyde in PBS for 3 min. After treatment with ethanol/acetone (50/50 vol/vol) for 1 min, the well surface was air-dried and incubated for 10 min at room temperature in an acetate buffer (0.1 M sodium acetate, pH 5.0) containing 0.01% naphthol AS-MS phosphate (Sigma, St. Louis, Mo.) as a substrate and 0.03% red violet LB salt (Sigma) as a stain for the reaction product, in the presence of 50 mM sodium tartrate. TRAP-positive cells appeared dark red. The expression of calcitonin receptors was also assessed by autoradiography using [125I]-labeled human calcitonin, as described (21).
To test the effect of anti-TR1 mAb in osteoclast formation, murine bone marrow cells were cultured (24) in the presence or absence of anti-TR1 mAb or other chemicals. Briefly, bone marrow cells were flushed from femurs of 6- to 8-wk-old male Balb/c mice (Taconic, Germantown, N.Y.). The bone marrow cells were plated at 2 x 106 cells/well in 24-well plates (Costar, Cambridge, Mass.) with 10-8 M 1,25(OH)2 D3 (Biomolecule, Plymouth Meeting, Pa.) in the presence or absence of anti-TR1 mAb. Cultures were performed in triplicate; half of the medium was replaced with fresh medium every other day. TRAP staining was performed on day 7. In certain experiments, the assays were performed on dentine slices and the pit formation was examined (25).
Dentine resorption assay
Osteoclasts were obtained by coculture of bone marrow cells and osteoblastic cells on collagen gels for 6–7 days in the presence of 1,25(OH)2D3, as described (23). Collagen gels were digested with 0.2% bacterial collagenase. Osteoclast preparations were allowed to settle on dentine slices in the wells of a 96-well plate (Corning). After a settling period of 1 h, the slices were removed and put into 48-well plates and incubated in -MEM containing 10% FBS for 72 h. Resorption pits were visualized by staining with Mayers hematoxylin and the stained areas were identified by light microscopy (26). We used an image analysis system (ImageHyper II, InterQuest Co., Osaka, Japan), and measured the total pit areas in four randomly selected areas of dentine slices.
Fetal long-bone organ culture system
Bone-resorbing activity was assessed by using a modification of Raisz's method (see ref 27), as reported previously. Pregnant mice were injected subcutaneously with 25 µCi of 45Ca (Amersham) on day 15 of gestation. On day 16, shafts of the radii and ulnae were excised and cultured in BGJb medium (GIBCO BRL). After preincubation for 24 h, the bones were transferred to 0.5 ml BGJb containing 0.2% BSA and incubated for 72 h in the presence or absence of test materials. Bone-resorbing activity was expressed as the percent release of 45Ca from prelabeled bones, as described previously (27).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1A
shows the amino acid sequence of TR1 deduced from the longest open reading frame of one of the isolated cDNAs. The open reading frame encodes 401 amino acids. There are five potential N-linked glycosylation sites. Because other members of the TNFR family are receptors, we initially sought both signal and transmembrane sequences. The first 21 amino acids were readily recognizable and confirmed later by NH2-terminal sequencing of recombinant protein as a signal sequence. This protein, however, contained no transmembrane-spanning domain (TM). Because a cDNA sequence can contain artifact or can represent an alternatively spliced form or faulty transcripts from tumor cell lines, we examined its transcript in primary cell culture. Because TR1 mRNA was produced abundantly in osteoblasts (see below), TR1 mRNA from a primary culture of osteoblasts was examined for the presence of TM by RT-PCR. RT-PCR primers are designed to trap a potential TM of TR1 cDNA that spans from the third cysteine-rich motif to several regions toward the carboxyl terminus. As shown in Fig. 1B, all the mRNA from different cells produced identical PCR products whose sizes are identical to that of cDNA template, indicating that TR1 does not contain TM in primary osteoblasts and may be a novel secretory protein among the TNFR family. T cell lines such as Jurkat and CEM, B cell lines such as Skw6.4 and DAKAKI, and progenitor cell line KG1a did not produce an RT-PCR product, indicating that TR1 mRNA was not expressed in these cells (data not shown). Along with other members of the TNFR family, TR1 contains the characteristic cysteine-rich motifs that have been shown to represent a repetitive structural unit. Figure 1C shows the potential cysteine-rich motif aligned among TNFR-I, TNFR-II, CD40, 4–1BB, TR2, and TR1. TR1 contained two perfect and two imperfect cysteine-rich motifs. The carboxyl-terminal half did not show any recognizable protein motif.
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A single TR1 transcript of 2.4 kb was detected at high levels in the heart, placenta, lung, liver, and kidney and at lower levels in the thymus, prostate, testis, ovary, and small intestine (
Fig. 1D). Expression was also detected in venous endothelial cells, foreskin fibroblast, lung emphysema cell line, and ovarian cancer cell line OVCAR3; relatively higher levels were found in the osteosarcoma MG63 cell line and human smooth muscle cell line AOSMIC ( Fig. 1D). FISH mapping indicated that the TR1 gene was located within bands 8q23–24.1 (data not shown).
The NH2-terminal 208 amino acids excluding signal sequence were expressed in NIH 3T3 cells as a Fc fusion protein, and the NH2-terminal amino acid sequence was determined. The processed mature TR1 protein starts with amino acid 22 (glutamic acid) and therefore consists of 380 amino acids. To determine the molecular size of natural TR1, 35S-labeled MG63 culture medium was used to immunoprecipitate with two different anti-TR1 mAb's: TR1BK1 and TR1BK2. The molecular size of natural TR1 was determined to be 62 kDa ( Fig. 1E).
Receptor for TR1 (TR1-R) is expressed on monocyte cell line and human fibroblast
Because of TR1's unique pattern of mRNA expression, we examined whether monocytic cell lines (potential osteoclast precursors) and fibroblasts express TR1-R. As shown in Fig. 2A,
a monocyte cell line THP-1 and human fibroblasts (such as lung fibroblast cell line IMR-90 and primary cultures of foreskin fibroblast) carried TR1-R. Scatchard analysis (28) indicated two classes of high- and medium-high affinity receptors with a kDa of 45 and 320 pM, respectively. The numbers of binding sites were 65 per THP-1 cell for the high-affinity and 158 per THP-1 cells for the medium-high affinity receptors ( Fig. 2B).
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TR1 induces the proliferation of normal human fibroblasts
TR1 induced the proliferation of normal human foreskin fibroblasts in a dose-dependent manner and potentiated TNF-induced proliferation of these cells ( Fig. 3A).
Proliferation induction was completely inhibited by a neutralizing mAb ( Fig. 3B). Since the TR1 gene was highly expressed in these cells and induced by TNF- and interleukin 1 (IL-1), TR1 may serve as an autocrine growth factor for the human fibroblast.
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TR1 inhibits osteoclastogenesis
Osteoblastic stromal cells play an important role in modulating the development of osteoclast progenitors by mechanisms requiring cell-to-cell interaction (29, 30). Because TR1 was expressed abundantly in osteoblastic cells, we examined a potential role for the TR1 in osteoclastogenesis. We examined TRAP-positive OCL formation in cocultures of osteoblasts and bone marrow cells in the presence of 1,25(OH)2D3. OCLs were formed in coculture where 1,25(OH)2D3 was added; without the bone-resorbing factor, no OCLs were formed ( Fig. 4A).
An autoradiographic study using labeled calcitonin revealed that TRAP-positive multinucleated and mononuclear cells formed in these cocultures possessed calcitonin receptors (data not shown). Recombinant human TR1-Fc fusion protein (TR1-Fc) dose-dependently inhibited OCL formation, yielding maximal inhibition at 30 ng/ml ( Fig. 4A). OCL formation can be enhanced by a number of agents that act through different second messenger systems, such as vitamin D receptor, cAMP, and gp 130 (29). TR1-Fc (30 ng/ml) completely inhibited OCL differentiation in cocultures induced by the osteoclastogenic agents 1,25(OH)2D3, prostaglandin E2, parathyroid hormone, or IL-I and IL-11 (data not shown). We next addressed the action of TR1 on the process of OCL formation. Treatment of cocultures revealed that the inhibitory actions of TR1-Fc (30 ng/ml) on OCL formation occurred during the last 2 days of coculture whereas TR1-Fc had no effect during the first 2 days of coculture ( Fig. 4B). These results suggest that TR1 inhibits the terminal differentiation process but not the proliferation stage of osteoclast progenitors.
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TR1 inhibits mature osteoclastic bone resorption
We examined the role of TR1 in bone resorption by mature osteoclasts. Bone resorption was measured by transferring mature osteoclasts, formed in cocultures on collagen gels, onto dentine slices. No osteoclasts were newly formed from their progenitors in the culture on dentine slices for a period of 72 h in the absence of bone-resorbing factors (26). Pit formation over a period of 72 h by these mature osteoclasts was strongly reduced by TR1-Fc at a concentration of 100–1000 ng/ml ( Fig. 5A, B).
It has been reported that osteoclasts in a bone-resorbing state exhibited ringed structures of F-actin dots (actin ring) (31). Disruption of actin rings by various inhibitors always caused inhibition of the pit-forming activity of osteoclasts (32). TR1-Fc (100 ng/ml) induced disrupted actin rings of osteoclasts on culture wells within 24 h (data not shown). After 48 h, about 80% of TRAP-positive OCLs were detached from the dish in the presence of TR1-Fc (100 ng/ml) ( Fig. 5C). No morphological changes were induced by TR1 in osteoblastic cells contaminating the OCL preparation (data not shown). Finally, we tested the effects of TR1-Fc in the bone organ culture system. Similarly, TR1-Fc (100–1000 ng/ml) completely inhibited calcium release induced by 1,25(OH)2D3 in organ cultures of mouse fetal long bone ( Fig. 6).
Eel calcitonin (10-10 M) as a positive control also inhibited calcium release in the presence of 1,25(OH)2D3. Our findings suggest that TR1 inhibits osteoclast terminal differentiation from its progenitors and inhibits the function of mature osteoclasts.
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Monoclonal antibody to TR1 enhances osteoclast formation
When anti-TR1 mAb (TR1BK1) was added to the osteoclastogenesis assay containing murine bone marrow cells plus 1,25(OH)2D3, numerous TRAP-positive pit-forming giant cells appeared, indicating that anti-TR1 mAb neutralized an osteoclastogenesis inhibitor activity ( Fig. 7A).
In fact, TR1BK1 neutralized the osteoclastogenesis inhibitory activity of exogenous TR1-Fc ( Fig. 7B). This result indicates that, in the mixed culture of bone marrow cells with 1,25(OH)2D3, osteoclastogenesis factors and osteoclastogenesis inhibitors are secreted and mAb blocking of TR1 activity with anti-TR1 mAb led to the enhanced osteoclastogenesis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As do other members of this family, TR1 appears to exert a broad spectrum of bioactivities in different cell types, ranging from fibroblast to osteoclast precursors. Fibroblasts and potential osteoclast precursors both express receptors for TR1. The biochemical basis of the differential effects needs to be examined. It is intriguing that THP-1 cell carries two different classes of TR1 receptors. To rule out the possibility that one of the receptors is in fact an Fc receptor (because we used TR-1-Fc fusion protein), we performed two other experiments: 1) human IgG1 binding on THP-1 cells, and 2) blocking of TR1-Fc binding by anti-TR1 mAb. Both experiments indicated that the binding was specific to TR1: 1) human IgG1 binding was comparable to background binding, which is TR1-Fc binding with an excess amount of cold anti-human IgG and 125I-anti-human IgG1; and 2) treatment of TR1-Fc with anti-TR1 mAb (TR1BK1) produced only a background level of binding. Because most of the ligands for TNFRs belong to the TNF family, the TR1 receptor may be a type 2 membrane protein, as has been shown with other members of the family. Whether both classes of TR1 receptors are functional and mediate signals from TR1 remains to be determined. The second class (medium-high affinity) of TR1 receptor may include other members of the TNF ligand superfamily, representing a cross-reaction. In ELISA assays, in fact, TR1 showed binding to other TNF ligands such as lymphotoxin-
, a newly identified member (unpublished results). Cloning and characterization of the TR1 receptor gene will aid in understanding the nature of the two classes of TR1 receptors and the biochemical signals for the diverse biological functions of TR1.
Two papers have been published on the cytokine that is structurally identical to TR1 and shows biological activities of osteoclast inhibition; it has been named osteoprotegerin (35) and osteoclastogenesis inhibitory factor (36). Osteoclastogenesis inhibition of TR1 is consistent with that of these two reports. Our studies with anti-TR1 mAb are noteworthy in that anti-TR1 mAb blocks the TR1 activities in mixed bone marrow culture and promotes osteoclast formation by almost 600-fold. This mAb is cross-reacted with murine TR1. Ostoblastic stromal cells are essential for osteoclast differentiation from their progenitors (29, 30). We have shown that TR1 mRNA is expressed abundantly in primary osteoblastic cells, which suggests that constitutive production of TR1 by osteoblastic cells suppresses osteoclast differentiation in the absence of bone-resorbing factors in our cocultures. This mAb will be useful when studying the mode of TR1 action in osteoclast precursors and the in vivo effect of blocking TR1 in normal physiological conditions.
In a fibroblast, however, TR1 induced proliferation in a dose-dependent manner and potentiated TNF-induced proliferation. These results are reminiscent of TNF and the Fas ligand effect on fibroblasts. Since the TR1 gene was highly expressed in human fibroblasts, which carried the TR1 receptor, TR1 may function as an autocrine growth factor for a certain population of human fibroblasts.
This novel cytokine is a strong inhibitor of osteoclast differentiation in cocultures of osteoblastic and hemopoietic cells. Ovariectomy-induced bone loss was prevented by administering osteoprotegerin to rats (35). Tansgenic mice expressing high levels of osteoprotegerin were born with osteopetrosis due to a marked reduction in the number of osteoclasts (35). We have shown here that TR1 strongly inhibited not only osteoclastogenesis in a coculture system, but also pit-forming activity by mature osteoclasts and bone-resorbing activity in an organ culture system. It is reported that osteoprotegerin treatment rapidly reduced ionized calcium in hypercalcemic mice injected with tumor cells (37), which confirms our results. These results clearly indicate that TR1 inhibits osteoclast terminal differentiation from its progenitors and inhibits the function of mature osteoclasts.
It has been reported that the survival of mature osteoclasts is dependent on osteoblastic cells or several cytokines (38). Our results suggest that TR1 treatment in mature osteoclasts inhibits the survival of multinucleated osteoclasts and pit-forming activity on dentine slices. It is not yet certain what the mechanism of inhibitory action is in bone resorption by TR1. In our studies, an ~10-fold higher concentration of TR1 was necessary for complete inhibition in pit formation by mature osteoclasts and bone resorption in organ cultures compared to osteoclast formation in cocultures. To elucidate these different dose-responses of TR1, identification of the target cells of TR1 in inhibitory action in these different culture systems needs to be addressed. This novel protein can be used as a therapeutic agent in certain metabolic bone diseases.
We have shown that TR1 is a soluble member of the TNFR family and is produced primarily from fibroblasts and immature osteoblasts. It promotes fibroblast proliferation and inhibits osteoclast formation and its function. TR1BK1, an mAb to the TR1, inhibits the TR1 effect on fibroblast growth and osteoclastogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication December 17, 1997. Accepted for publication January 23, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,25-dihydroxyvitamin D3 have ruffled borders and resorb dentin. Anat. Rec. 224, 379–391[Medline]
maintain the survival of osteoclast–like cells. Endocrinology 136, 806–811
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A. Lipton, S. M. Ali, K. Leitzel, V. Chinchilli, L. Witters, L. Engle, D. Holloway, P. Bekker, and C. R. Dunstan Serum Osteoprotegerin Levels in Healthy Controls and Cancer Patients Clin. Cancer Res., July 1, 2002; 8(7): 2306 - 2310. [Abstract] [Full Text] [PDF]
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 M. Schoppet, K. T. Preissner, and L. C. Hofbauer RANK Ligand and Osteoprotegerin: Paracrine Regulators of Bone Metabolism and Vascular Function Arterioscler. Thromb. Vasc. Biol., April 1, 2002; 22(4): 549 - 553. [Abstract] [Full Text] [PDF]
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K. Itoh, N. Udagawa, T. Katagiri, S. Iemura, N. Ueno, H. Yasuda, K. Higashio, J. M. W. Quinn, M. T. Gillespie, T. J. Martin, et al. Bone Morphogenetic Protein 2 Stimulates Osteoclast Differentiation and Survival Supported by Receptor Activator of Nuclear Factor-{kappa}B Ligand Endocrinology, August 1, 2001; 142(8): 3656 - 3662. [Abstract] [Full Text] [PDF]
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 D. Furuya, R. Kaneko, A. Yagihashi, T. Endoh, T. Yajima, D. Kobayashi, K. Yano, E. Tsuda, and N. Watanabe Immuno-PCR Assay for Homodimeric Osteoprotegerin Clin. Chem., August 1, 2001; 47(8): 1475 - 1477. [Full Text] [PDF]
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M.A. Taubman and T. Kawai Involvement of T-Lymphocytes in Periodontal Disease and in Direct and Indirect Induction of Bone Resorption Critical Reviews in Oral Biology & Medicine, January 1, 2001; 12(2): 125 - 135. [Abstract] [Full Text] [PDF]
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N. Udagawa, N. Takahashi, H. Yasuda, A. Mizuno, K. Itoh, Y. Ueno, T. Shinki, M. T. Gillespie, T. J. Martin, K. Higashio, et al. Osteoprotegerin Produced by Osteoblasts Is an Important Regulator in Osteoclast Development and Function Endocrinology, September 1, 2000; 141(9): 3478 - 3484. [Abstract] [Full Text] [PDF]
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 E. A. Gonzalez The role of cytokines in skeletal remodelling: possible consequences for renal osteodystrophy Nephrol. Dial. Transplant., July 1, 2000; 15(7): 945 - 950. [Full Text] [PDF]
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L. C. Hofbauer and A. E. Heufelder The Role of Receptor Activator of Nuclear Factor-{kappa}B Ligand and Osteoprotegerin in the Pathogenesis and Treatment of Metabolic Bone Diseases J. Clin. Endocrinol. Metab., July 1, 2000; 85(7): 2355 - 2363. [Full Text]
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 R. Yu, S. Mandlekar, S. Ruben, J. Ni, and A-N. T. Kong Tumor Necrosis Factor-related Apoptosis-inducing Ligand-mediated Apoptosis in Androgen-independent Prostate Cancer Cells Cancer Res., May 1, 2000; 60(9): 2384 - 2389. [Abstract] [Full Text]
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 K. Kobayashi, N. Takahashi, E. Jimi, N. Udagawa, M. Takami, S. Kotake, N. Nakagawa, M. Kinosaki, K. Yamaguchi, N. Shima, et al. Tumor Necrosis Factor {alpha} Stimulates Osteoclast Differentiation by a Mechanism Independent of the ODF/RANKL-RANK Interaction J. Exp. Med., January 17, 2000; 191(2): 275 - 286. [Abstract] [Full Text] [PDF]
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L. C. Hofbauer, F. Gori, B. L. Riggs, D. L. Lacey, C. R. Dunstan, T. C. Spelsberg, and S. Khosla Stimulation of Osteoprotegerin Ligand and Inhibition of Osteoprotegerin Production by Glucocorticoids in Human Osteoblastic Lineage Cells: Potential Paracrine Mechanisms of Glucocorticoid-Induced Osteoporosis Endocrinology, October 1, 1999; 140(10): 4382 - 4389. [Abstract] [Full Text]
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 D. W. Powell, R. C. Mifflin, J. D. Valentich, S. E. Crowe, J. I. Saada, and A. B. West Myofibroblasts. I. Paracrine cells important in health and disease Am J Physiol Cell Physiol, July 1, 1999; 277(1): C1 - C19. [Abstract] [Full Text] [PDF]
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 T. Suda, N. Takahashi, N. Udagawa, E. Jimi, M. T. Gillespie, and T. J. Martin Modulation of Osteoclast Differentiation and Function by the New Members of the Tumor Necrosis Factor Receptor and Ligand Families Endocr. Rev., June 1, 1999; 20(3): 345 - 357. [Abstract] [Full Text]
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K. Thirunavukkarasu, D. L. Halladay, R. R. Miles, X. Yang, R. J. S. Galvin, S. Chandrasekhar, T. J. Martin, and J. E. Onyia The Osteoblast-specific Transcription Factor Cbfa1 Contributes to the Expression of Osteoprotegerin, a Potent Inhibitor of Osteoclast Differentiation and Function J. Biol. Chem., August 11, 2000; 275(33): 25163 - 25172. [Abstract] [Full Text] [PDF]
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M. Wan, X. Shi, X. Feng, and X. Cao Transcriptional Mechanisms of Bone Morphogenetic Protein-induced Osteoprotegrin Gene Expression J. Biol. Chem., March 23, 2001; 276(13): 10119 - 10125. [Abstract] [Full Text] [PDF]
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H. Zhou, V. Kartsogiannis, Y. S. Hu, J. Elliott, J. M. W. Quinn, W. J. McKinstry, M. T. Gillespie, and K. W. Ng A Novel Osteoblast-derived C-type Lectin That Inhibits Osteoclast Formation J. Biol. Chem., April 27, 2001; 276(18): 14916 - 14923. [Abstract] [Full Text] [PDF]
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K. Thirunavukkarasu, R. R. Miles, D. L. Halladay, X. Yang, R. J. S. Galvin, S. Chandrasekhar, T. J. Martin, and J. E. Onyia Stimulation of Osteoprotegerin (OPG) Gene Expression by Transforming Growth Factor-beta (TGF-beta ). MAPPING OF THE OPG PROMOTER REGION THAT MEDIATES TGF-beta EFFECTS J. Biol. Chem., September 21, 2001; 276(39): 36241 - 36250. [Abstract] [Full Text] [PDF]
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M. Schoppet, K. T. Preissner, and L. C. Hofbauer RANK Ligand and Osteoprotegerin: Paracrine Regulators of Bone Metabolism and Vascular Function Arterioscler. Thromb. Vasc. Biol., April 1, 2002; 22(4): 549 - 553. [Abstract] [Full Text] [PDF]
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