Osteostat/Tumor Necrosis Factor Superfamily 18 Inhibits Osteoclastogenesis and Is Selectively Expressed by Vascular Endothelial Ce
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《内分泌学杂志》
Human Genome Sciences, Inc., Rockville, Maryland 20850
Abstract
Vascular endothelial cells (EC) participate in the process of bone formation through the production of factors regulating osteoclast differentiation and function. In this study, we report the selective expression in primary human microvascular EC of Osteostat/TNF superfamily 18, a ligand of the TNF superfamily. Osteostat protein is detectable in human microvascular EC and is highly up-regulated by IFN- and IFN-. Moreover, an anti-Osteostat antibody strongly binds to the vascular endothelium in human tissues, demonstrating that the protein is present in the EC layers surrounding blood vessels. Functional in vitro assays were used to define Osteostat involvement in osteoclastogenesis. Both recombinant and membrane-bound Osteostat inhibit differentiation of osteoclasts from monocytic precursor cells. Osteostat suppresses the early stage of osteoclastogenesis via inhibition of macrophage colony-stimulating factor-induced receptor activator of NF-B (RANK) expression in the osteoclast precursor cells. This effect appears to be specific for the differentiation pathway of the osteoclast lineage, because Osteostat does not inhibit lipopolysaccharide-induced RANK expression in monocytes and dendritic cells, or activation-induced RANK expression in T cells. These findings demonstrate that Osteostat is a novel regulator of osteoclast generation and substantiate the major role played by the endothelium in bone physiology.
Introduction
THE MAINTENANCE OF skeletal mass is controlled by the opposing actions of two cell types: the bone-forming osteoblasts, derived from stromal cells, and the bone-resorbing osteoclasts, belonging to the myeloid lineage. However, other cellular types play a crucial role in bone physiology. In particular, vascular endothelial cells (EC) are of central importance in the process of bone formation and remodeling. Vascularization precedes and supports osteogenesis, and, in the bone tissue, there is a close physical association among vascular EC, osteoblasts and osteoclasts. EC produce several factors regulating osteoclast activity (1, 2, 3, 4). Stimulated EC release TNF-, which induces receptor activator of NF-B ligand (RANKL) expression in osteoblasts and has multiple actions on osteoclasts (5, 6, 7, 8, 9). In addition, human microvascular EC (HMVEC) express two TNF-related proteins crucial for bone metabolism: RANKL and osteoprotegerin (OPG) (10). RANKL is an integral modulator of bone formation inducing the differentiation of progenitor cells into fully mature osteoclasts and enhancing the survival and activation state of differentiated osteoclasts (11, 12, 13, 14, 15, 16). Administration of RANKL caused bone loss and development of osteoporosis in normal mice. In contrast, RANKL null mice had a reduced number of osteoclasts and consequent osteopetrosis (17). Although RANKL is expressed predominantly in osteoblasts/stroma cells and activated T cells, it is produced also by other cell types (10, 18, 19, 20). OPG lacks a transmembrane domain and functions as a soluble receptor (21, 22, 23). The physiological role of this protein was discovered via the generation of OPG transgenic mice (21), which display a decrease in osteoclasts that is associated with a generalized increase in bone density and profound osteopetrosis. OPG is a receptor for RANKL, and thus blocks the differentiation of osteoclasts through inhibition of the binding of RANKL to its receptor, receptor activator of NF-B (RANK). As RANKL, OPG is produced by several cell types.
TNF superfamily 18 (TNFSF18) was initially identified as a member of the human TNF ligand superfamily through high throughput sequencing of human cDNAs (24, 25). Hydrophilicity analysis of the full-length cDNA clone predicts for a 177-amino acid type II transmembrane protein. The predicted protein comprises of a short intracellular domain, a hydrophobic transmembrane domain, an extracellular domain with two potential glycosylation sites near the extracellular C-terminal region. The protein is a ligand for a TNF-like receptor [TNFRSF18/glucocorticoid-induced TNF-related (GITR)/activation-inducible TNF receptor (AITR)] and has been named the ligand for activation-inducible TNF receptor (AITRL) (24) and human ligand for glucocorticoid-induced TNF-related (GITRL) (25). However, no clear functional activity of the human protein has been reported so far. In this study, we show that TNFSF18 is a strong negative regulator of osteoclastogenesis, and hence, we have called the protein Osteostat to reflect its biological role. It is constitutively expressed in HMVEC and suppresses the differentiation of myeloid precursor cells into osteoclasts via inhibition of RANK expression in the monocytic cells.
Materials and Methods
Proteins and antibodies
RANKL, macrophage colony-stimulating factor (M-CSF), IFN-, IL-4, granulocyte macrophage colony-stimulating factor (GM-CSF), IL-2, CD40 ligand, RANK, AITRL, TNFRSF18, and vascular endothelial growth factor were purchased from PeproTech (Rocky Hill, NJ); IFN-2a and IFN- were purchased from PBL Biomedical Laboratories (New Brunswick, NJ); and lipopolysaccharide (LPS), phorbol-12-myristate-13-acetate and ionomycin were purchased from Sigma-Aldrich (St. Louis, MO). Rabbit polyclonal antibodies were affinity purified from antisera generated by immunizing rabbits with recombinant Osteostat. Goat polyclonal anti-GITRL and GITRL were from R&D Systems (Minneapolis, MN). BLyS, OPG-Fc, LIGHT, and APRIL were produced by Human Genome Sciences, Inc. (Rockville, MD).
Cells
Monocytes were purified from human peripheral blood mononuclear cells (PBMC) by centrifugation of leukapheresis preparations (BRT Inc., Baltimore, MD) through Histopaque (Sigma-Aldrich) gradients followed by counter-flow centrifugal elutriation. Monocyte-derived dendritic cells were obtained by culturing monocytes for 7–10 d in RPMI containing 10% fetal bovine serum, 2 mM L-glutamine, and 50 μg/ml gentamicyn, supplemented with 50 ng/ml GM-CSF and 20 ng/ml IL-4. T cells were purified from blood mononuclear cells using magnetic beads separation (Miltenyi Biotec, Auburn, CA). Primary HMVEC were purchased from Clonetics (Gaithersburg, MD) and grown following the manufacturer’s instructions.
TaqMan real-time RT-PCR analysis
Osteostat mRNA levels were assessed using a 7700 TaqMan Sequence Detector [Applied Biosystems Inc. (ABI), Foster City, CA]. Total RNA was isolated from homogenized cell lysates using a RNeasy mini kit (Qiagen, Valencia, CA) following the manufacturer’s protocol, and 50 ng used in a one-step 25-μl quantitative reverse RT-PCR. As a control for genomic contamination, parallel reactions were set up without RT. Osteostat mRNA quantitation was conducted with the comparative cycle threshold method using an 18S rRNA as the endogenous reference, as described in Gibson et al. (26) and in the ABI user bulletin no. 2. Osteostat cDNA amplification primers and probe were generated using Primer Express Software (ABI) anticipated to give a theoretical PCR efficiency of one. The 18S rRNA primers and probes were obtained from ABI. Probes were labeled at the 5' end with the reporter dye 6-carboxyfluorescein and on the 3' end with the quencher dye 5-(and 6)-carboxytetramethylrhodamine (BioSource International, Camarillo, CA). Reactions were conducted at 48 C for 30 min, 95 C for 10 min, followed by 40 cycles of denaturation and annealing/extension at 95 C for 15 sec and 60 C for 1 min, respectively. The sequences for primers and probe were as follows: 5'-GGCTCCCAATGCAAACTACAAT-3'; 5'-TGTTAGAGTTTGTATCATGTCTTTGTTTTT-3'; 5'-TACAGCCGCACCTCAAAAGGAGCTACT-3'.
Expression of membrane-bound Osteostat
The full-length Osteostat open reading frame (TNFSF18; GenBank accession no. AF125303) was PCR amplified using the following oligonucleotide primers: 5'-CAGACTGGATCCGCCACCATGTGTTTGAG-CCACTTGG-3' and 5'-CAGACTGGTACCGTATCTCTCTGCAGATCC-AACC-3'. The upper primer introduced a Kozak consensus sequence before the initiating methionine and tailed the amplicon with a 5' BamHI site. The lower primer annealed to the 3' untranslated region of the Osteostat cDNA and introduced a 3' Asp718 site. The PCR amplicon was digested with BamHI and Asp718 and ligated to like digested pC4, a mammalian expression vector (27). The pC4:Osteostat vector was transiently transfected into HEK293T cells using lipofectamine (Invitrogen, Carlsbad, CA), and 48 h after transfection cells were collected for further analysis. As a control, pC4 vector alone was transfected in parallel.
Osteostat gene expression array analysis
cDNA array filters were constructed comprising PCR amplicons from approximately 6000 independent genes selected from the Human Genome Sciences database (28). The genes selected (including Osteostat) were arrayed in duplicate. The array filters were hybridized to over 300 human normal/patient RNA samples isolated from a wide range of cell, tissue, and organ types procured from the Cooperative Human Tissue Network, the National Disease Research Interchange, and from a single commercial site (Clontech, Palo Alto, CA). Hybridization probes from individual RNA samples were generated using 33P-labeled first strand cDNA synthesized from 3 μg total RNA. After overnight hybridization, the filters were washed under stringent conditions and hybridization signal detected after 4 d using a Fuji BAS 2500 phosphorimaging system (FujiFilm, Burbank, CA). Signals from duplicate spots including Osteostat were captured using ImaGene 4.0 software (BioDiscovery, El Segundo, CA). After linear signal normalization, the average and coefficient of variation for each duplicate pair of Osteostat DNA spots hybridized to each RNA sample was stored for further analysis.
Western blot analysis
Soluble fractions of cell lysates (equivalent of 2 x 106 cells per lane) were loaded on a 4–20% Tris-glycine gel (Invitrogen). Resolved proteins were transferred onto polyvinylidene difluoride membranes, and membranes immunoblotted with 0.4 μg/ml of goat polyclonal antihuman GITRL or antihuman RANK antibody. A rabbit antigoat IgG-HRP conjugated antibody (Chemicon International, Temecula, CA) was used for detection. The results were developed by ECL+plus immunoblotting detection system (Amersham Biosciences, Piscataway, NJ). For loading controls, either nonspecific bands were used or membranes were stripped and then reprobed with a monoclonal anti-actin antibody (Sigma-Aldrich).
Immunohistochemistry
Five-micrometer sections of formalin-fixed paraffin-embedded tissues were deparaffinized and hydrated, blocked for endogenous peroxidase, and processed for heat-induced antigen retrieval in 0.01 M citrate buffer with 0.01% Nonidet P-40 (Sigma-Aldrich), pH 6. In addition, sections were serially incubated with goat serum, avidin, and biotin (Vector Laboratories, Burlingame, CA) to block nonspecific epitopes and avidin or biotin binding sites. The primary rabbit-anti-Osteostat polyclonal antibody at 3 μg/ml was incubated in the absence or presence of 15 μg/ml Osteostat before application to tissue sections. The ABC/peroxidase method of detection (Vector), with a diaminobenzidine chromogen (DAB kit; Sigma-Aldrich) were used to visualize specific binding.
Expression and purification of recombinant Osteostat
Osteostat nucleotide sequence corresponding to the predicted extracellular amino acids of Osteostat (T53-S177) based on hydrophobicity profiling were PCR amplified from the full-length Osteostat cDNA (24) using a 5' primer (GGATATCATATGACCGCTAAAGAACCGTGTATGGCTAAGT) and a 3' primer (GACAGTGGTACCTTACTAGGAGATGAATTGGGGAT) that tailed the amplicon with NdeI and Asp718 restriction sites and introduced an initiating methionine. The resulting amplicon was digested with NdeI/Asp718 and subcloned into like digested pHE4 and expressed in the Escherichia coli W3110 strain. Inclusion bodies were solubilized in 1.5 M guanidine hydrochloride, followed by refolding. The protein was purified using exchange columns (Poros QS-50 and CM-20) and eluted out from the last column using a salt and a pH gradient. The protein had a trimeric configuration in solution similarly to other TNF-like ligands.
Osteoclast formation
Human monocytes were cultured for 5–7 d in phenol red-free -MEM containing 10% fetal bovine serum in 24-well tissue culture plates (Corning, Corning, NY) at 1 x 106 cells/ml in the presence of 50–100 ng/ml human RANKL and 25 ng/ml human M-CSF. In several experiments, monocytes were cocultured with Osteostat-transfected cells following the conditions specified above.
Tartrate-resistant acid phosphatase (TRAP) staining and resorption assay
Cells were fixed in 3% formaldehyde for 10 min at room temperature, rinsed with water, and stained with Fast Garnet in the presence of 100 nM sodium tartrate using TRAP assay kit from Sigma-Aldrich. TRAP-positive cells containing three or more nuclei were counted as osteoclasts by light microscopy. To determine the resorption activity of generated osteoclasts, human monocytes were cultured in 24-well plates on Osteologic disks (BD BioCoat Osteologic Bone Cell Culture System; Becton Dickinson, Bedford, MA) in -MEM, as described above. After osteoclast formation was observed, the disks were recovered, cells were lysed in a solution of 3% bleach and 2.5% sodium hydroxide, and disks were rinsed in water and air dried. The resorption areas were screened and analyzed by Microst Automated Image Analyzer (Millenium Biologix Inc., Kingston, Ontario, Canada).
Viability assay
The assay was conducted as in Magan et al. (29). Briefly, monocytes were cultured in polypropylene tubes in DMEM containing 25 mM glucose, 20 mM L-glutamine, 25 mM HEPES buffer (pH 7.3), and 50 μg/ml gentamicin. After 48 h of incubation in the absence or presence of stimulants, apoptotic cells were detected by using an Annexin-FITC apoptosis kit according to the manufacturer’s instructions (BD Pharmingen, Mountain View, CA). Flow cytometric data (FACScan; Becton Dickinson) were collected from 10,000 cells and reported as the percentage of positive apoptotic cells. Three independent experiments were done.
Surface plasmon resonance studies
Binding analysis was performed using a BIAcore 3000 biosensor (BIAcore AB, Uppsala, Sweden), following the manufacturer’s recommendations, along with BIAevaluation software version 3.2. TNF-like receptors RANK, DR4, or TNFRSF18 were immobilized onto a CM5 sensor chip via amine groups, using N-ethyl-N'-(dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide chemistry, to a density of approximately 400 relative response units. Unoccupied sites were blocked with ethanolamine. The chip was thoroughly primed with HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20). Serial dilutions of Osteostat or RANKL (range 0.156–10 μg/ml) in HBS-EP buffer were injected at a flow rate of 15 μl/min at 25 C for a total time of 1.6 min. The off rate was determined by washing unbound RANKL or Osteostat in the presence of HBS-EP buffer and measuring the net relative response units remaining during a period of 3.0 min. After each cycle, the surface was regenerated using 10 mM glycine-HCl, pH 1.5. The resonance signal measured on the control flow cells was subtracted from the signal measured on the experimental flow cell. Each sample set was repeated three times for a total of 24 sample injections. All values are reported as the mean ± SD.
Results
Genes displaying EC-enriched expression were identified by hybridizing approximately 300 individual RNA samples isolated from a range of human cell and tissue types, including normal or diseased, resting or activated on a cDNA array comprising approximately 6000 individual human genes encoding predominantly secreted and membrane-bound proteins. The gene expression profiles of the 20 genes of the 6000 analyzed that demonstrated the most enriched expression in EC relative to the other genes analyzed are presented in Fig. 1. Within this group are genes previously demonstrated to be expressed in EC or EC-enriched tissues, including SERPINE (30), the seven transmembrane receptor ETL (31), and the cysteine-rich angiogenesis inducers, CYR61 and CTGF (32). Genes of complete unknown function (e.g. NM_025107) were also identified. One of the genes that demonstrated a clear enriched expression in EC types was the TNF-like molecule TNFSF18, also called AITRL/TL6 or GITRL (24, 25), which we have named in this study as Osteostat. To determine the levels of Osteostat in EC or other cell types, quantitative PCR analysis was used. Substantial levels of Osteostat mRNA were found only in EC types, such as primary HMVEC and human umbilical vein endothelial cell. Examining cells from seven donors, Osteostat mRNA displayed a mean expression ratio relative to 18S rRNA of 1.2 x 10–3 (±0.23 x 10–3) in HMVEC, whereas the transcript was close to the background level (2 x 10–9) in PBMC. Furthermore, the presence of Osteostat protein in primary HMVEC was detectable by Western blot using goat polyclonal antibodies anti-Osteostat (Fig. 2A). A specific band of approximately 22 kDa corresponding to the full-length protein, as found in cells transfected with the Osteostat gene, was visible in the blots from lysates of normal HMVEC from the majority of the donors tested (five of seven). In contrast, the protein level was usually too low to be detectable by Western blot in the EC obtained from large blood vessels, such as human umbilical vein endothelial cell and aortic EC (data not shown). No protein expression was detectable by Western blot in other cell types, such as monocytes (data not shown). These results are in agreement with Kwon et al. (24), reporting that no Osteostat message was detectable in T, B, and monocytic cell lines and PBMC by RT-PCR.
To test whether Osteostat expression could be modulated, HMVEC cells were treated with several factors or cytokines. Although proinflammatory cytokines, LPS, or hormones did not significantly modulate the protein expression (data not shown), type I IFNs (IFN- and IFN-) strongly increased the protein level in all the donors tested (n = 5). A representative donor is shown in Fig. 2A. To examine whether the increased protein levels were caused by increased levels of steady-state mRNA, real-time PCR analyses were performed with mRNA obtained from HMVEC of dermal (HMVEC-d) or lung origins cultured in absence or in presence of type I IFN or other factors. A total of five donors were tested, and the results obtained with representative donors are shown in Fig. 2B. Treatment with type II IFN, CD40 ligand, or vascular endothelial growth factor did not change significantly Osteostat mRNA level in HMVEC-d; in contrast, as shown in a different donor, the transcript basal expression level was increased 2-fold by IFN- and 3-fold by IFN-. HMVEC obtained from lungs of an additional donor showed a similar response to IFN treatment. In these cells, IFN- and IFN- produced, respectively, more than 4- and 7-fold increase of Osteostat mRNA level. Collectively, these experiments demonstrated that Osteostat is constitutively expressed in HMVEC, and its expression is significantly up-regulated by type I IFN.
Subsequent experiments demonstrated that Osteostat is present in vivo in the EC layers surrounding human blood vessels (Fig. 3). Staining of sections of human skin with Osteostat-specific rabbit polyclonal antibodies clearly demonstrated the presence of the protein on the vascular EC (Fig. 3A). A dense circumferential band of positive EC was observed in the tunica intima of the vessels with variable staining of the smooth muscle layer (tunica media). In the tunica adventitia, prominent staining of the microvessels supplying larger vessels was often observed. Incubation with rabbit IgG did not result in vessel staining (Fig. 3B). The reactivity was specific because it was completely abrogated by competition with recombinant Osteostat (Fig. 3C), whereas competition with an unrelated protein, BLyS, was ineffective (Fig. 3D). The same pattern of reactivity was observed in vascular EC of human lungs (data not shown).
Considering the key role the TNF superfamily plays in modulating a host of cellular responses, we were intrigued to characterize the biological activity of Osteostat. We conducted studies to determine whether the restricted expression of Osteostat in HMVEC was related to osteoclastogenesis, in light of the known contribution of the vascular endothelium in bone remodeling. To this end, 293T cells were transiently transfected with the full-length Osteostat gene and the expression of the protein was assessed by Western blot (Fig. 2A). The cells were then used in a standard in vitro system of osteoclast generation, based on the effect of RANKL and M-CSF to induce the differentiation of monocytes into osteoclasts (11, 33, 34). In this experimental system, human monocytes in culture for 5–7 d with the two osteotropic cytokines fuse and differentiate into osteoclasts, which are multinucleated cells characterized by high TRAP activity. Addition of the 293T-Osteostat cells to the monocytes caused a strong suppression of osteoclastogenesis (Fig. 4A). Fifty thousand Osteostat-transfectants per well completely inhibited the formation of TRAP+ multinucleated cells, whereas vector-transfectants were ineffective. Interestingly, in contrast to the Osteostat-transfected cells, conditioned medium from 293T-Osteostat did not block the differentiation of monocytes to osteoclasts (data not shown). These data, together with the observation that the amino acid sequence of Osteostat lacks putative signal peptide and known protease-sensitive motifs (24, 35, 36), suggest that the protein might be produced mainly in a membrane-bound form. To further assess the effect of Osteostat on osteoclastogenesis, a recombinant soluble protein, comprising the extracellular domain of the full-length protein, was expressed and purified in E. coli. Addition of the recombinant protein to the osteoclastogenesis assay suppressed the formation of multinucleated TRAP+ cells (Fig. 4B). At concentrations above 100 ng/ml, Osteostat caused complete inhibition of osteoclast formation. Identical concentrations of BLyS recombinant protein were used as control and did not inhibit the generation of TRAP+ cells. The protein showed a level of potency comparable to a recombinant OPG-Fc (data not shown).
In the next series of experiments (Fig. 5), we used the resorption assay to further validate the functional effect of Osteostat. Monocytes were differentiated to osteoclasts on disks coated with a synthetic bone-matrix of calcium phosphate. In presence of osteotropic factors, monocytes fused to form the multinucleated TRAP+ cells, which degraded the bone-matrix creating lacunae on the surface of the disks. Addition of Osteostat completely suppressed the formation of osteoclasts and, consequently, of the lacunae. In the resorption assay, concentrations above 100 ng/ml of recombinant protein caused a complete inhibition of degradation of the calcium phosphate matrix, whereas other ligands of the TNF superfamily (BLyS, APRIL, or LIGHT) were completely ineffective (data not shown).
In kinetics studies, addition of Osteostat at the beginning of the culture (d 0 or +1) was highly effective in inhibiting the generation of osteoclasts and, consequently, the degradation of the bone-matrix. In contrast, a delayed addition of Osteostat had no significant effect (Fig. 6). These findings demonstrated that the suppressive effect of the protein occurred during the first 2 d of culture, suggesting that Osteostat inhibits the early phase of the osteoclast differentiation from their precursors.
To evaluate whether Osteostat has a direct effect on osteoclast progenitor cell survival, monocytes cultured in the presence of Osteostat were evaluated for apoptosis. As shown in Table 1, the level of monocyte apoptosis decreased in the presence of Osteostat, demonstrating that Osteostat mediates a survival signal to monocytes under the experimental conditions used. This direct effect mediated by Osteostat on monocytes is supported with our observation that Osteostat can bind directly to monocytes (data not shown). As the known cognate receptor for Osteostat is not believed to be expressed on monocytes as evidenced by its absence of expression in PBMC (24), we were prompted to determine whether Osteostat can associate with RANK, a TNF receptor superfamily member expressed in monocytes and a mediator of osteclastogenesis. However, BIAcore analysis failed to demonstrate any specific binding between Osteostat and RANK (Table 2). In contrast and in agreement with earlier reports (24, 25), Osteostat demonstrates binding to TNFRSF18 with an affinity of approximately 20 nM, and RANKL demonstrates an affinity of approximately 1 nM to RANK.
The induction of RANK expression is the crucial step regulating cell differentiation in the early stage of osteoclastogenesis (15). Therefore, we tested the effect of Osteostat on RANK expression in the myeloid precursor cells. Human monocytes were treated with M-CSF in absence or presence of Osteostat for 2 d, and the cell lysates were analyzed by Western blots using a polyclonal Ab specific for RANK. As shown in Fig. 7A, Osteostat treatment suppressed RANK expression, endogenous or M-CSF induced. The inhibition was detectable also at the level of RANK mRNA (Fig. 7B), as measured by quantitative PCR analysis. Osteostat did not cause a general suppression of M-CSF activation of monocytes, because the induction of other genes by M-CSF, such as TNFR1, was not altered following Osteostat treatment as measured by quantitative PCR (data not shown). Osteostat effect seems to be specific for the signaling pathway regulating osteoclast differentiation because the protein did not affect LPS-induced increase of RANK protein levels in monocytes or in dendritic cells. In addition, it did not inhibit RANK expression in T cells after activation (Fig. 7C). In summary, these results indicate that Osteostat specifically inhibits M-CSF-induced RANK expression in monocytes.
Discussion
In this study, we have identified Osteostat as a novel potent inhibitor of osteoclastogenesis that is expressed selectively in EC. Although the human Osteostat gene was cloned several years ago, its biological activity has remained ill-defined. It was reported that Osteostat induced NF-B activation in cells cotransfected with the ligand and the receptor AITR (24), and that it rescued Jurkat cells transfected with human GITR from activation-induced cell death (25). Using a well-defined in vitro system, we have demonstrated that Osteostat blocks the differentiation of myeloid precursor cells into osteoclasts via the inhibition of RANK expression in the precursor cells.
Osteoclast precursors are derived from the pluripotential hematopoietic stem cell and are present in circulation in the monocytic fraction (37, 38). Although several cytokines influence the differentiation process, it was demonstrated that M-CSF and RANKL are the two essential factors controlling it (11, 33, 39). Arai et al. (15) have identified an early and a late stage in osteoclast development. In the early stage, the presence of M-CSF in the microenvironment is critical because it induces RANK expression in the precursor cells. Mice lacking functional M-CSF have, indeed, very few osteoclasts and are osteopetropic (40). Subsequently in the late stage, RANKL becomes the indispensable factor driving the final differentiation into osteoclasts. Without RANKL-RANK interaction and in presence of M-CSF, the precursor monocytic cells differentiated into tissue macrophages by default. In this model, Osteostat, by inhibiting the M-CSF-induced up-regulation of RANK in the early stage of osteoclastogenesis, blocks RANKL signaling and, consequently, osteoclast formation. It is possible that other molecular events are initiated by Osteostat in the precursor cells and they warrant further research. However, it is evident from our experiments that the effect of Osteostat is restricted to the early stage of osteoclast development, because delayed addition of the protein did not affect differentiation. The described mechanism of activity differentiates Osteostat from other cytokines known to suppress osteoclast generation, such as IFN- that inhibits RANK signaling through degradation of TRAF-6 (41), or IL-4 that blocks RANKL-dependent activation of NF-B and MAP kinase signaling (42).
cDNA array analysis of approximately 6000 cDNAs encoding membrane-bound or secreted proteins identified approximately 15–20 genes including Osteostat that demonstrated selective transcriptional up-regulation in EC. Clearly understanding the biological roles of these proteins in the vascular system will be of great interest. More detailed analysis of Osteostat expression revealed that its mRNA is expressed in normal EC, and Osteostat protein is readily detectable in the tissue microvascular endothelium. Both Osteostat mRNA and protein expression are strongly up-regulated by class I IFNs, although not significantly changed by other stimuli. It is of interest that IFN- was reported to be a potent inhibitor of osteoclast formation by interfering with RANKL-induced expression of c-Fos (43). The restricted expression in the microvascular endothelium could suggest a potential physiological role for the protein. Osteoclast precursor cells belonging to the monocytic lineage reach through the circulation the areas of the peripheral skeleton where active bone remodeling or formation takes place, called basic multicellular unit (BMU) (44). The cells cross by diapedesis the endothelial layer of the blood vessel situated at the center of each BMU, and migrate toward the edge of the unit where they differentiate into osteoclasts after the interaction with osteoblasts and stroma. Although the total production of monocytes in humans is approximately 5 x 108 cells per hour, only a very small fraction of the cells becomes osteoclasts. Osteoclastogenesis takes place exclusively in bones despite the fact that M-CSF and RANKL proteins are found in soft tissues (20, 45). It has been proposed that the preosteoclasts leave the circulation only when they reach a BMU in response to a unique combination of chemotactic signals and adhesion molecules. It is intriguing to speculate that the presence of Osteostat in the microvascular endothelium might constitute part of a negative signaling system that does not allow the monocytic cells extravasating outside the BMU to become osteoclasts. Specifically inhibiting the up-regulation of RANK, Osteostat could contribute to drive the monocytes toward the macrophage differentiation pathway.
At present, it is unclear which receptor is used by Osteostat to deliver the anti-osteoclastogenic signal to the monocytic cells. A receptor, TNFRSF18, also called AITR or human GITR, was identified in humans (23, 24). A murine GITR was recently cloned (46). The murine receptor has two major differences compared with human GITR, being characteristically induced by dexamethasone and presenting a mismatch in the first cysteine-rich pseudorepeat; so that it was proposed that the two receptors might serve distinct functions (24). In the murine system, blocking of GITR signaling caused an enhancement of T cell regulatory function (47, 48). It is possible that Osteostat uses an unknown high-affinity receptor on the preosteoclasts. This hypothesis is supported by the findings that TNFRSF18 mRNA is not detectable or is expressed at very low levels in human monocytes, although it is highly expressed on activated T cells (Refs.24 and 25 and our unpublished data). It is also unclear whether Osteostat is the functional human counterpart of the recently cloned murine GITRL (49, 50). This membrane protein is 51% identical to Osteostat and is highly expressed in mouse dendritic cells (50) and in macrophages (51). From the published reports it is evident that murine GITRL is the ligand for murine GITR, and this pair may be involved in the regulation of immune responses presumably by interaction of dendritic cells and CD25+CD4+ suppressor/regulatory T cells.
In conclusion, we have characterized a new powerful and specific effect of a member of the TNF superfamily. This study adds an additional player in the complex network that regulates bone physiology and potentially opens the way to the generation of a new drug for the treatment of bone diseases.
Acknowledgments
We greatly appreciate the technical support and advice provided by Drs. Reiner Gentz, Shashi Kaithamana, Yanggu Shi, Henrik Olsen, and David LaFleur.
Footnotes
First Published Online September 22, 2005
Abbreviations: AITR, Activation-inducible TNF receptor; AITRL, AITR ligand; BMU, basic multicellular unit; EC, endothelial cell; GITR, glucocorticoid-induced TNF-related; GITRL, GITR ligand; GM-CSF, granulocyte M-CSF; HMVEC, human microvascular endothelial cell; HMVEC-d, HMVEC of dermal origin; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; OPG, osteoprotegerin; PBMC, peripheral blood mononuclear cell; RANK, receptor activator of NF-B; RANKL, RANK ligand; TNFSF18, TNF superfamily 18; TRAP, tartrate-resistant acid phosphatase.
Accepted for publication September 13, 2005.
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Abstract
Vascular endothelial cells (EC) participate in the process of bone formation through the production of factors regulating osteoclast differentiation and function. In this study, we report the selective expression in primary human microvascular EC of Osteostat/TNF superfamily 18, a ligand of the TNF superfamily. Osteostat protein is detectable in human microvascular EC and is highly up-regulated by IFN- and IFN-. Moreover, an anti-Osteostat antibody strongly binds to the vascular endothelium in human tissues, demonstrating that the protein is present in the EC layers surrounding blood vessels. Functional in vitro assays were used to define Osteostat involvement in osteoclastogenesis. Both recombinant and membrane-bound Osteostat inhibit differentiation of osteoclasts from monocytic precursor cells. Osteostat suppresses the early stage of osteoclastogenesis via inhibition of macrophage colony-stimulating factor-induced receptor activator of NF-B (RANK) expression in the osteoclast precursor cells. This effect appears to be specific for the differentiation pathway of the osteoclast lineage, because Osteostat does not inhibit lipopolysaccharide-induced RANK expression in monocytes and dendritic cells, or activation-induced RANK expression in T cells. These findings demonstrate that Osteostat is a novel regulator of osteoclast generation and substantiate the major role played by the endothelium in bone physiology.
Introduction
THE MAINTENANCE OF skeletal mass is controlled by the opposing actions of two cell types: the bone-forming osteoblasts, derived from stromal cells, and the bone-resorbing osteoclasts, belonging to the myeloid lineage. However, other cellular types play a crucial role in bone physiology. In particular, vascular endothelial cells (EC) are of central importance in the process of bone formation and remodeling. Vascularization precedes and supports osteogenesis, and, in the bone tissue, there is a close physical association among vascular EC, osteoblasts and osteoclasts. EC produce several factors regulating osteoclast activity (1, 2, 3, 4). Stimulated EC release TNF-, which induces receptor activator of NF-B ligand (RANKL) expression in osteoblasts and has multiple actions on osteoclasts (5, 6, 7, 8, 9). In addition, human microvascular EC (HMVEC) express two TNF-related proteins crucial for bone metabolism: RANKL and osteoprotegerin (OPG) (10). RANKL is an integral modulator of bone formation inducing the differentiation of progenitor cells into fully mature osteoclasts and enhancing the survival and activation state of differentiated osteoclasts (11, 12, 13, 14, 15, 16). Administration of RANKL caused bone loss and development of osteoporosis in normal mice. In contrast, RANKL null mice had a reduced number of osteoclasts and consequent osteopetrosis (17). Although RANKL is expressed predominantly in osteoblasts/stroma cells and activated T cells, it is produced also by other cell types (10, 18, 19, 20). OPG lacks a transmembrane domain and functions as a soluble receptor (21, 22, 23). The physiological role of this protein was discovered via the generation of OPG transgenic mice (21), which display a decrease in osteoclasts that is associated with a generalized increase in bone density and profound osteopetrosis. OPG is a receptor for RANKL, and thus blocks the differentiation of osteoclasts through inhibition of the binding of RANKL to its receptor, receptor activator of NF-B (RANK). As RANKL, OPG is produced by several cell types.
TNF superfamily 18 (TNFSF18) was initially identified as a member of the human TNF ligand superfamily through high throughput sequencing of human cDNAs (24, 25). Hydrophilicity analysis of the full-length cDNA clone predicts for a 177-amino acid type II transmembrane protein. The predicted protein comprises of a short intracellular domain, a hydrophobic transmembrane domain, an extracellular domain with two potential glycosylation sites near the extracellular C-terminal region. The protein is a ligand for a TNF-like receptor [TNFRSF18/glucocorticoid-induced TNF-related (GITR)/activation-inducible TNF receptor (AITR)] and has been named the ligand for activation-inducible TNF receptor (AITRL) (24) and human ligand for glucocorticoid-induced TNF-related (GITRL) (25). However, no clear functional activity of the human protein has been reported so far. In this study, we show that TNFSF18 is a strong negative regulator of osteoclastogenesis, and hence, we have called the protein Osteostat to reflect its biological role. It is constitutively expressed in HMVEC and suppresses the differentiation of myeloid precursor cells into osteoclasts via inhibition of RANK expression in the monocytic cells.
Materials and Methods
Proteins and antibodies
RANKL, macrophage colony-stimulating factor (M-CSF), IFN-, IL-4, granulocyte macrophage colony-stimulating factor (GM-CSF), IL-2, CD40 ligand, RANK, AITRL, TNFRSF18, and vascular endothelial growth factor were purchased from PeproTech (Rocky Hill, NJ); IFN-2a and IFN- were purchased from PBL Biomedical Laboratories (New Brunswick, NJ); and lipopolysaccharide (LPS), phorbol-12-myristate-13-acetate and ionomycin were purchased from Sigma-Aldrich (St. Louis, MO). Rabbit polyclonal antibodies were affinity purified from antisera generated by immunizing rabbits with recombinant Osteostat. Goat polyclonal anti-GITRL and GITRL were from R&D Systems (Minneapolis, MN). BLyS, OPG-Fc, LIGHT, and APRIL were produced by Human Genome Sciences, Inc. (Rockville, MD).
Cells
Monocytes were purified from human peripheral blood mononuclear cells (PBMC) by centrifugation of leukapheresis preparations (BRT Inc., Baltimore, MD) through Histopaque (Sigma-Aldrich) gradients followed by counter-flow centrifugal elutriation. Monocyte-derived dendritic cells were obtained by culturing monocytes for 7–10 d in RPMI containing 10% fetal bovine serum, 2 mM L-glutamine, and 50 μg/ml gentamicyn, supplemented with 50 ng/ml GM-CSF and 20 ng/ml IL-4. T cells were purified from blood mononuclear cells using magnetic beads separation (Miltenyi Biotec, Auburn, CA). Primary HMVEC were purchased from Clonetics (Gaithersburg, MD) and grown following the manufacturer’s instructions.
TaqMan real-time RT-PCR analysis
Osteostat mRNA levels were assessed using a 7700 TaqMan Sequence Detector [Applied Biosystems Inc. (ABI), Foster City, CA]. Total RNA was isolated from homogenized cell lysates using a RNeasy mini kit (Qiagen, Valencia, CA) following the manufacturer’s protocol, and 50 ng used in a one-step 25-μl quantitative reverse RT-PCR. As a control for genomic contamination, parallel reactions were set up without RT. Osteostat mRNA quantitation was conducted with the comparative cycle threshold method using an 18S rRNA as the endogenous reference, as described in Gibson et al. (26) and in the ABI user bulletin no. 2. Osteostat cDNA amplification primers and probe were generated using Primer Express Software (ABI) anticipated to give a theoretical PCR efficiency of one. The 18S rRNA primers and probes were obtained from ABI. Probes were labeled at the 5' end with the reporter dye 6-carboxyfluorescein and on the 3' end with the quencher dye 5-(and 6)-carboxytetramethylrhodamine (BioSource International, Camarillo, CA). Reactions were conducted at 48 C for 30 min, 95 C for 10 min, followed by 40 cycles of denaturation and annealing/extension at 95 C for 15 sec and 60 C for 1 min, respectively. The sequences for primers and probe were as follows: 5'-GGCTCCCAATGCAAACTACAAT-3'; 5'-TGTTAGAGTTTGTATCATGTCTTTGTTTTT-3'; 5'-TACAGCCGCACCTCAAAAGGAGCTACT-3'.
Expression of membrane-bound Osteostat
The full-length Osteostat open reading frame (TNFSF18; GenBank accession no. AF125303) was PCR amplified using the following oligonucleotide primers: 5'-CAGACTGGATCCGCCACCATGTGTTTGAG-CCACTTGG-3' and 5'-CAGACTGGTACCGTATCTCTCTGCAGATCC-AACC-3'. The upper primer introduced a Kozak consensus sequence before the initiating methionine and tailed the amplicon with a 5' BamHI site. The lower primer annealed to the 3' untranslated region of the Osteostat cDNA and introduced a 3' Asp718 site. The PCR amplicon was digested with BamHI and Asp718 and ligated to like digested pC4, a mammalian expression vector (27). The pC4:Osteostat vector was transiently transfected into HEK293T cells using lipofectamine (Invitrogen, Carlsbad, CA), and 48 h after transfection cells were collected for further analysis. As a control, pC4 vector alone was transfected in parallel.
Osteostat gene expression array analysis
cDNA array filters were constructed comprising PCR amplicons from approximately 6000 independent genes selected from the Human Genome Sciences database (28). The genes selected (including Osteostat) were arrayed in duplicate. The array filters were hybridized to over 300 human normal/patient RNA samples isolated from a wide range of cell, tissue, and organ types procured from the Cooperative Human Tissue Network, the National Disease Research Interchange, and from a single commercial site (Clontech, Palo Alto, CA). Hybridization probes from individual RNA samples were generated using 33P-labeled first strand cDNA synthesized from 3 μg total RNA. After overnight hybridization, the filters were washed under stringent conditions and hybridization signal detected after 4 d using a Fuji BAS 2500 phosphorimaging system (FujiFilm, Burbank, CA). Signals from duplicate spots including Osteostat were captured using ImaGene 4.0 software (BioDiscovery, El Segundo, CA). After linear signal normalization, the average and coefficient of variation for each duplicate pair of Osteostat DNA spots hybridized to each RNA sample was stored for further analysis.
Western blot analysis
Soluble fractions of cell lysates (equivalent of 2 x 106 cells per lane) were loaded on a 4–20% Tris-glycine gel (Invitrogen). Resolved proteins were transferred onto polyvinylidene difluoride membranes, and membranes immunoblotted with 0.4 μg/ml of goat polyclonal antihuman GITRL or antihuman RANK antibody. A rabbit antigoat IgG-HRP conjugated antibody (Chemicon International, Temecula, CA) was used for detection. The results were developed by ECL+plus immunoblotting detection system (Amersham Biosciences, Piscataway, NJ). For loading controls, either nonspecific bands were used or membranes were stripped and then reprobed with a monoclonal anti-actin antibody (Sigma-Aldrich).
Immunohistochemistry
Five-micrometer sections of formalin-fixed paraffin-embedded tissues were deparaffinized and hydrated, blocked for endogenous peroxidase, and processed for heat-induced antigen retrieval in 0.01 M citrate buffer with 0.01% Nonidet P-40 (Sigma-Aldrich), pH 6. In addition, sections were serially incubated with goat serum, avidin, and biotin (Vector Laboratories, Burlingame, CA) to block nonspecific epitopes and avidin or biotin binding sites. The primary rabbit-anti-Osteostat polyclonal antibody at 3 μg/ml was incubated in the absence or presence of 15 μg/ml Osteostat before application to tissue sections. The ABC/peroxidase method of detection (Vector), with a diaminobenzidine chromogen (DAB kit; Sigma-Aldrich) were used to visualize specific binding.
Expression and purification of recombinant Osteostat
Osteostat nucleotide sequence corresponding to the predicted extracellular amino acids of Osteostat (T53-S177) based on hydrophobicity profiling were PCR amplified from the full-length Osteostat cDNA (24) using a 5' primer (GGATATCATATGACCGCTAAAGAACCGTGTATGGCTAAGT) and a 3' primer (GACAGTGGTACCTTACTAGGAGATGAATTGGGGAT) that tailed the amplicon with NdeI and Asp718 restriction sites and introduced an initiating methionine. The resulting amplicon was digested with NdeI/Asp718 and subcloned into like digested pHE4 and expressed in the Escherichia coli W3110 strain. Inclusion bodies were solubilized in 1.5 M guanidine hydrochloride, followed by refolding. The protein was purified using exchange columns (Poros QS-50 and CM-20) and eluted out from the last column using a salt and a pH gradient. The protein had a trimeric configuration in solution similarly to other TNF-like ligands.
Osteoclast formation
Human monocytes were cultured for 5–7 d in phenol red-free -MEM containing 10% fetal bovine serum in 24-well tissue culture plates (Corning, Corning, NY) at 1 x 106 cells/ml in the presence of 50–100 ng/ml human RANKL and 25 ng/ml human M-CSF. In several experiments, monocytes were cocultured with Osteostat-transfected cells following the conditions specified above.
Tartrate-resistant acid phosphatase (TRAP) staining and resorption assay
Cells were fixed in 3% formaldehyde for 10 min at room temperature, rinsed with water, and stained with Fast Garnet in the presence of 100 nM sodium tartrate using TRAP assay kit from Sigma-Aldrich. TRAP-positive cells containing three or more nuclei were counted as osteoclasts by light microscopy. To determine the resorption activity of generated osteoclasts, human monocytes were cultured in 24-well plates on Osteologic disks (BD BioCoat Osteologic Bone Cell Culture System; Becton Dickinson, Bedford, MA) in -MEM, as described above. After osteoclast formation was observed, the disks were recovered, cells were lysed in a solution of 3% bleach and 2.5% sodium hydroxide, and disks were rinsed in water and air dried. The resorption areas were screened and analyzed by Microst Automated Image Analyzer (Millenium Biologix Inc., Kingston, Ontario, Canada).
Viability assay
The assay was conducted as in Magan et al. (29). Briefly, monocytes were cultured in polypropylene tubes in DMEM containing 25 mM glucose, 20 mM L-glutamine, 25 mM HEPES buffer (pH 7.3), and 50 μg/ml gentamicin. After 48 h of incubation in the absence or presence of stimulants, apoptotic cells were detected by using an Annexin-FITC apoptosis kit according to the manufacturer’s instructions (BD Pharmingen, Mountain View, CA). Flow cytometric data (FACScan; Becton Dickinson) were collected from 10,000 cells and reported as the percentage of positive apoptotic cells. Three independent experiments were done.
Surface plasmon resonance studies
Binding analysis was performed using a BIAcore 3000 biosensor (BIAcore AB, Uppsala, Sweden), following the manufacturer’s recommendations, along with BIAevaluation software version 3.2. TNF-like receptors RANK, DR4, or TNFRSF18 were immobilized onto a CM5 sensor chip via amine groups, using N-ethyl-N'-(dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide chemistry, to a density of approximately 400 relative response units. Unoccupied sites were blocked with ethanolamine. The chip was thoroughly primed with HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20). Serial dilutions of Osteostat or RANKL (range 0.156–10 μg/ml) in HBS-EP buffer were injected at a flow rate of 15 μl/min at 25 C for a total time of 1.6 min. The off rate was determined by washing unbound RANKL or Osteostat in the presence of HBS-EP buffer and measuring the net relative response units remaining during a period of 3.0 min. After each cycle, the surface was regenerated using 10 mM glycine-HCl, pH 1.5. The resonance signal measured on the control flow cells was subtracted from the signal measured on the experimental flow cell. Each sample set was repeated three times for a total of 24 sample injections. All values are reported as the mean ± SD.
Results
Genes displaying EC-enriched expression were identified by hybridizing approximately 300 individual RNA samples isolated from a range of human cell and tissue types, including normal or diseased, resting or activated on a cDNA array comprising approximately 6000 individual human genes encoding predominantly secreted and membrane-bound proteins. The gene expression profiles of the 20 genes of the 6000 analyzed that demonstrated the most enriched expression in EC relative to the other genes analyzed are presented in Fig. 1. Within this group are genes previously demonstrated to be expressed in EC or EC-enriched tissues, including SERPINE (30), the seven transmembrane receptor ETL (31), and the cysteine-rich angiogenesis inducers, CYR61 and CTGF (32). Genes of complete unknown function (e.g. NM_025107) were also identified. One of the genes that demonstrated a clear enriched expression in EC types was the TNF-like molecule TNFSF18, also called AITRL/TL6 or GITRL (24, 25), which we have named in this study as Osteostat. To determine the levels of Osteostat in EC or other cell types, quantitative PCR analysis was used. Substantial levels of Osteostat mRNA were found only in EC types, such as primary HMVEC and human umbilical vein endothelial cell. Examining cells from seven donors, Osteostat mRNA displayed a mean expression ratio relative to 18S rRNA of 1.2 x 10–3 (±0.23 x 10–3) in HMVEC, whereas the transcript was close to the background level (2 x 10–9) in PBMC. Furthermore, the presence of Osteostat protein in primary HMVEC was detectable by Western blot using goat polyclonal antibodies anti-Osteostat (Fig. 2A). A specific band of approximately 22 kDa corresponding to the full-length protein, as found in cells transfected with the Osteostat gene, was visible in the blots from lysates of normal HMVEC from the majority of the donors tested (five of seven). In contrast, the protein level was usually too low to be detectable by Western blot in the EC obtained from large blood vessels, such as human umbilical vein endothelial cell and aortic EC (data not shown). No protein expression was detectable by Western blot in other cell types, such as monocytes (data not shown). These results are in agreement with Kwon et al. (24), reporting that no Osteostat message was detectable in T, B, and monocytic cell lines and PBMC by RT-PCR.
To test whether Osteostat expression could be modulated, HMVEC cells were treated with several factors or cytokines. Although proinflammatory cytokines, LPS, or hormones did not significantly modulate the protein expression (data not shown), type I IFNs (IFN- and IFN-) strongly increased the protein level in all the donors tested (n = 5). A representative donor is shown in Fig. 2A. To examine whether the increased protein levels were caused by increased levels of steady-state mRNA, real-time PCR analyses were performed with mRNA obtained from HMVEC of dermal (HMVEC-d) or lung origins cultured in absence or in presence of type I IFN or other factors. A total of five donors were tested, and the results obtained with representative donors are shown in Fig. 2B. Treatment with type II IFN, CD40 ligand, or vascular endothelial growth factor did not change significantly Osteostat mRNA level in HMVEC-d; in contrast, as shown in a different donor, the transcript basal expression level was increased 2-fold by IFN- and 3-fold by IFN-. HMVEC obtained from lungs of an additional donor showed a similar response to IFN treatment. In these cells, IFN- and IFN- produced, respectively, more than 4- and 7-fold increase of Osteostat mRNA level. Collectively, these experiments demonstrated that Osteostat is constitutively expressed in HMVEC, and its expression is significantly up-regulated by type I IFN.
Subsequent experiments demonstrated that Osteostat is present in vivo in the EC layers surrounding human blood vessels (Fig. 3). Staining of sections of human skin with Osteostat-specific rabbit polyclonal antibodies clearly demonstrated the presence of the protein on the vascular EC (Fig. 3A). A dense circumferential band of positive EC was observed in the tunica intima of the vessels with variable staining of the smooth muscle layer (tunica media). In the tunica adventitia, prominent staining of the microvessels supplying larger vessels was often observed. Incubation with rabbit IgG did not result in vessel staining (Fig. 3B). The reactivity was specific because it was completely abrogated by competition with recombinant Osteostat (Fig. 3C), whereas competition with an unrelated protein, BLyS, was ineffective (Fig. 3D). The same pattern of reactivity was observed in vascular EC of human lungs (data not shown).
Considering the key role the TNF superfamily plays in modulating a host of cellular responses, we were intrigued to characterize the biological activity of Osteostat. We conducted studies to determine whether the restricted expression of Osteostat in HMVEC was related to osteoclastogenesis, in light of the known contribution of the vascular endothelium in bone remodeling. To this end, 293T cells were transiently transfected with the full-length Osteostat gene and the expression of the protein was assessed by Western blot (Fig. 2A). The cells were then used in a standard in vitro system of osteoclast generation, based on the effect of RANKL and M-CSF to induce the differentiation of monocytes into osteoclasts (11, 33, 34). In this experimental system, human monocytes in culture for 5–7 d with the two osteotropic cytokines fuse and differentiate into osteoclasts, which are multinucleated cells characterized by high TRAP activity. Addition of the 293T-Osteostat cells to the monocytes caused a strong suppression of osteoclastogenesis (Fig. 4A). Fifty thousand Osteostat-transfectants per well completely inhibited the formation of TRAP+ multinucleated cells, whereas vector-transfectants were ineffective. Interestingly, in contrast to the Osteostat-transfected cells, conditioned medium from 293T-Osteostat did not block the differentiation of monocytes to osteoclasts (data not shown). These data, together with the observation that the amino acid sequence of Osteostat lacks putative signal peptide and known protease-sensitive motifs (24, 35, 36), suggest that the protein might be produced mainly in a membrane-bound form. To further assess the effect of Osteostat on osteoclastogenesis, a recombinant soluble protein, comprising the extracellular domain of the full-length protein, was expressed and purified in E. coli. Addition of the recombinant protein to the osteoclastogenesis assay suppressed the formation of multinucleated TRAP+ cells (Fig. 4B). At concentrations above 100 ng/ml, Osteostat caused complete inhibition of osteoclast formation. Identical concentrations of BLyS recombinant protein were used as control and did not inhibit the generation of TRAP+ cells. The protein showed a level of potency comparable to a recombinant OPG-Fc (data not shown).
In the next series of experiments (Fig. 5), we used the resorption assay to further validate the functional effect of Osteostat. Monocytes were differentiated to osteoclasts on disks coated with a synthetic bone-matrix of calcium phosphate. In presence of osteotropic factors, monocytes fused to form the multinucleated TRAP+ cells, which degraded the bone-matrix creating lacunae on the surface of the disks. Addition of Osteostat completely suppressed the formation of osteoclasts and, consequently, of the lacunae. In the resorption assay, concentrations above 100 ng/ml of recombinant protein caused a complete inhibition of degradation of the calcium phosphate matrix, whereas other ligands of the TNF superfamily (BLyS, APRIL, or LIGHT) were completely ineffective (data not shown).
In kinetics studies, addition of Osteostat at the beginning of the culture (d 0 or +1) was highly effective in inhibiting the generation of osteoclasts and, consequently, the degradation of the bone-matrix. In contrast, a delayed addition of Osteostat had no significant effect (Fig. 6). These findings demonstrated that the suppressive effect of the protein occurred during the first 2 d of culture, suggesting that Osteostat inhibits the early phase of the osteoclast differentiation from their precursors.
To evaluate whether Osteostat has a direct effect on osteoclast progenitor cell survival, monocytes cultured in the presence of Osteostat were evaluated for apoptosis. As shown in Table 1, the level of monocyte apoptosis decreased in the presence of Osteostat, demonstrating that Osteostat mediates a survival signal to monocytes under the experimental conditions used. This direct effect mediated by Osteostat on monocytes is supported with our observation that Osteostat can bind directly to monocytes (data not shown). As the known cognate receptor for Osteostat is not believed to be expressed on monocytes as evidenced by its absence of expression in PBMC (24), we were prompted to determine whether Osteostat can associate with RANK, a TNF receptor superfamily member expressed in monocytes and a mediator of osteclastogenesis. However, BIAcore analysis failed to demonstrate any specific binding between Osteostat and RANK (Table 2). In contrast and in agreement with earlier reports (24, 25), Osteostat demonstrates binding to TNFRSF18 with an affinity of approximately 20 nM, and RANKL demonstrates an affinity of approximately 1 nM to RANK.
The induction of RANK expression is the crucial step regulating cell differentiation in the early stage of osteoclastogenesis (15). Therefore, we tested the effect of Osteostat on RANK expression in the myeloid precursor cells. Human monocytes were treated with M-CSF in absence or presence of Osteostat for 2 d, and the cell lysates were analyzed by Western blots using a polyclonal Ab specific for RANK. As shown in Fig. 7A, Osteostat treatment suppressed RANK expression, endogenous or M-CSF induced. The inhibition was detectable also at the level of RANK mRNA (Fig. 7B), as measured by quantitative PCR analysis. Osteostat did not cause a general suppression of M-CSF activation of monocytes, because the induction of other genes by M-CSF, such as TNFR1, was not altered following Osteostat treatment as measured by quantitative PCR (data not shown). Osteostat effect seems to be specific for the signaling pathway regulating osteoclast differentiation because the protein did not affect LPS-induced increase of RANK protein levels in monocytes or in dendritic cells. In addition, it did not inhibit RANK expression in T cells after activation (Fig. 7C). In summary, these results indicate that Osteostat specifically inhibits M-CSF-induced RANK expression in monocytes.
Discussion
In this study, we have identified Osteostat as a novel potent inhibitor of osteoclastogenesis that is expressed selectively in EC. Although the human Osteostat gene was cloned several years ago, its biological activity has remained ill-defined. It was reported that Osteostat induced NF-B activation in cells cotransfected with the ligand and the receptor AITR (24), and that it rescued Jurkat cells transfected with human GITR from activation-induced cell death (25). Using a well-defined in vitro system, we have demonstrated that Osteostat blocks the differentiation of myeloid precursor cells into osteoclasts via the inhibition of RANK expression in the precursor cells.
Osteoclast precursors are derived from the pluripotential hematopoietic stem cell and are present in circulation in the monocytic fraction (37, 38). Although several cytokines influence the differentiation process, it was demonstrated that M-CSF and RANKL are the two essential factors controlling it (11, 33, 39). Arai et al. (15) have identified an early and a late stage in osteoclast development. In the early stage, the presence of M-CSF in the microenvironment is critical because it induces RANK expression in the precursor cells. Mice lacking functional M-CSF have, indeed, very few osteoclasts and are osteopetropic (40). Subsequently in the late stage, RANKL becomes the indispensable factor driving the final differentiation into osteoclasts. Without RANKL-RANK interaction and in presence of M-CSF, the precursor monocytic cells differentiated into tissue macrophages by default. In this model, Osteostat, by inhibiting the M-CSF-induced up-regulation of RANK in the early stage of osteoclastogenesis, blocks RANKL signaling and, consequently, osteoclast formation. It is possible that other molecular events are initiated by Osteostat in the precursor cells and they warrant further research. However, it is evident from our experiments that the effect of Osteostat is restricted to the early stage of osteoclast development, because delayed addition of the protein did not affect differentiation. The described mechanism of activity differentiates Osteostat from other cytokines known to suppress osteoclast generation, such as IFN- that inhibits RANK signaling through degradation of TRAF-6 (41), or IL-4 that blocks RANKL-dependent activation of NF-B and MAP kinase signaling (42).
cDNA array analysis of approximately 6000 cDNAs encoding membrane-bound or secreted proteins identified approximately 15–20 genes including Osteostat that demonstrated selective transcriptional up-regulation in EC. Clearly understanding the biological roles of these proteins in the vascular system will be of great interest. More detailed analysis of Osteostat expression revealed that its mRNA is expressed in normal EC, and Osteostat protein is readily detectable in the tissue microvascular endothelium. Both Osteostat mRNA and protein expression are strongly up-regulated by class I IFNs, although not significantly changed by other stimuli. It is of interest that IFN- was reported to be a potent inhibitor of osteoclast formation by interfering with RANKL-induced expression of c-Fos (43). The restricted expression in the microvascular endothelium could suggest a potential physiological role for the protein. Osteoclast precursor cells belonging to the monocytic lineage reach through the circulation the areas of the peripheral skeleton where active bone remodeling or formation takes place, called basic multicellular unit (BMU) (44). The cells cross by diapedesis the endothelial layer of the blood vessel situated at the center of each BMU, and migrate toward the edge of the unit where they differentiate into osteoclasts after the interaction with osteoblasts and stroma. Although the total production of monocytes in humans is approximately 5 x 108 cells per hour, only a very small fraction of the cells becomes osteoclasts. Osteoclastogenesis takes place exclusively in bones despite the fact that M-CSF and RANKL proteins are found in soft tissues (20, 45). It has been proposed that the preosteoclasts leave the circulation only when they reach a BMU in response to a unique combination of chemotactic signals and adhesion molecules. It is intriguing to speculate that the presence of Osteostat in the microvascular endothelium might constitute part of a negative signaling system that does not allow the monocytic cells extravasating outside the BMU to become osteoclasts. Specifically inhibiting the up-regulation of RANK, Osteostat could contribute to drive the monocytes toward the macrophage differentiation pathway.
At present, it is unclear which receptor is used by Osteostat to deliver the anti-osteoclastogenic signal to the monocytic cells. A receptor, TNFRSF18, also called AITR or human GITR, was identified in humans (23, 24). A murine GITR was recently cloned (46). The murine receptor has two major differences compared with human GITR, being characteristically induced by dexamethasone and presenting a mismatch in the first cysteine-rich pseudorepeat; so that it was proposed that the two receptors might serve distinct functions (24). In the murine system, blocking of GITR signaling caused an enhancement of T cell regulatory function (47, 48). It is possible that Osteostat uses an unknown high-affinity receptor on the preosteoclasts. This hypothesis is supported by the findings that TNFRSF18 mRNA is not detectable or is expressed at very low levels in human monocytes, although it is highly expressed on activated T cells (Refs.24 and 25 and our unpublished data). It is also unclear whether Osteostat is the functional human counterpart of the recently cloned murine GITRL (49, 50). This membrane protein is 51% identical to Osteostat and is highly expressed in mouse dendritic cells (50) and in macrophages (51). From the published reports it is evident that murine GITRL is the ligand for murine GITR, and this pair may be involved in the regulation of immune responses presumably by interaction of dendritic cells and CD25+CD4+ suppressor/regulatory T cells.
In conclusion, we have characterized a new powerful and specific effect of a member of the TNF superfamily. This study adds an additional player in the complex network that regulates bone physiology and potentially opens the way to the generation of a new drug for the treatment of bone diseases.
Acknowledgments
We greatly appreciate the technical support and advice provided by Drs. Reiner Gentz, Shashi Kaithamana, Yanggu Shi, Henrik Olsen, and David LaFleur.
Footnotes
First Published Online September 22, 2005
Abbreviations: AITR, Activation-inducible TNF receptor; AITRL, AITR ligand; BMU, basic multicellular unit; EC, endothelial cell; GITR, glucocorticoid-induced TNF-related; GITRL, GITR ligand; GM-CSF, granulocyte M-CSF; HMVEC, human microvascular endothelial cell; HMVEC-d, HMVEC of dermal origin; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; OPG, osteoprotegerin; PBMC, peripheral blood mononuclear cell; RANK, receptor activator of NF-B; RANKL, RANK ligand; TNFSF18, TNF superfamily 18; TRAP, tartrate-resistant acid phosphatase.
Accepted for publication September 13, 2005.
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