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Endocrinology, doi:10.1210/en.2005-0065
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Endocrinology Vol. 146, No. 5 2324-2335
Copyright © 2005 by The Endocrine Society

Functional Expression of ß-Chemokine Receptors in Osteoblasts: Role of Regulated upon Activation, Normal T Cell Expressed and Secreted (RANTES) in Osteoblasts and Regulation of Its Secretion by Osteoblasts and Osteoclasts

Shozo Yano, Romuald Mentaverri, Deepthi Kanuparthi, Sanghamitra Bandyopadhyay, Alicia Rivera, Edward M. Brown and Naibedya Chattopadhyay

Division of Endocrinology, Diabetes, and Hypertension (S.Y., R.M., D.K., E.M.B., N.C.), Department of Medicine and Membrane Biology Program, Brigham and Women’s Hospital and Department of Laboratory Medicine (A.R.), Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115; and Genetics and Aging Unit (S.B.), Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129

Address all correspondence and requests for reprints to: Naibedya Chattopadhyay, Ph.D., Division of Endocrinology, Diabetes, and Hypertension, Brigham and Women’s Hospital and Harvard Medical School, 221 Longwood Avenue, Boston, Massachusetts 02115. E-mail: naibedya{at}rics.bwh.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The expression and functions of receptors for the ß-chemokine, regulated upon activation, normal T cell expressed, and secreted (RANTES)/CCL5, were investigated in osteoblasts. Both primary osteoblasts and the MC3T3-E1 osteoblast cell line express the RANTES receptors, CCR1, 3, 4, and 5 (by RT-PCR), which encode functional receptors in osteoblasts as shown by [125I]-RANTES binding followed by Scatchard analysis. Expression of all four RANTES receptor mRNAs in osteoblast is in contrast to the reports of expression of CCR1 being the only RANTES receptor expressed by osteoclasts. Exogenous RANTES elicits chemotaxis of osteoblasts and promotes cell survival via phosphatidylinositol 3-kinase with attendant phosphorylation of Akt. Osteoclastic RANTES, obtained from the conditioned medium of receptor activator of nuclear factor-{kappa}B ligand-differentiated RAW264.7 cells also induces chemotaxis of MC3T3-E1 cells. Incubating the conditioned medium with an anti-RANTES neutralizing antibody attenuated this effect. RANTES secretion from osteoblast is inhibited by differentiation promoting hormones, e.g. 1,25 (OH)2D3 and dexamethasone, whereas macrophage inflammatory protein-1{alpha} (but not macrophage inflammatory protein-1ß) and elevated calcium induce it. Elevated calcium also stimulated RANTES secretion by osteoclasts. Therefore, RANTES is an osteoblast chemoattractant and a survival-promoting molecule whose regulation in osteoblast is varied. Furthermore, RANTES secreted from osteoclasts induces osteoblast chemotaxis. Therefore, expression of RANTES and its receptors in both osteoblasts and osteoclasts could enable this chemokine to act in autocrine/paracrine modes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN OSTEOBLASTS, WHICH ARE derived from the mesenchymal stromal cell lineage, cellular proliferation or differentiation is regulated by numerous factors, including mechanical stress, mineral intake, and age, through systemic/local hormones and/or cytokines. Previous studies have shown that stromal cells and osteoblasts also have chemotactic potential in response to fragments of collagen or osteocalcin (1); growth factors such as platelet-derived growth factor, vascular endothelial growth factor, IGFs, TGFß, and bone morphogenetic proteins (2, 3, 4, 5, 6, 7, 8); cytokines such as IL-4 or -13 (9); and extracellular divalent cations, including calcium (Ca2+) and zinc, possibly mediated by a calcium-sensing receptor (10, 11, 12). The capacity to undergo chemotaxis is an important attribute of osteoblasts, which contributes importantly to their migration to sites of bone resorption. Traditionally, substances/factors released from bone matrix during bone resorption are thought to influence osteoblast chemotaxis, with scant reference to the possible production of chemoattractants by osteoclasts per se.

Recently some chemokine receptors and their ligands have been identified in osteoblasts or osteoblast-like cells, especially under inflammatory conditions (13, 14, 15, 16, 17, 18, 19). Emerging evidence suggests pivotal roles for chemokines in various cellular functions and human diseases beyond the realm of immune function (20, 21), such as their roles in angiogenesis or the development of the central nervous system (22, 23). Therefore, we hypothesized that chemokines might participate in normal bone turnover.

Chemokines are a family of chemotactic cytokines that regulate trafficking and homing of leukocytes and other cells at various differentiation stages (24). Receptors for chemokines are seven membrane-spanning, G protein-coupled receptors, which are classified into four major groups, CC, CXC, CXXXC, and XC, according to the number and spacing of conserved N-terminal cysteine residues. Because most of these receptors share their ligands, targeted deletion of a single chemokine or chemokine receptor has not induced obvious phenotypes under normal circumstances, with the exception of CXCR4 and CXCR2 (25). In this study, we focused on the functions of regulated upon activation, normal T cell expressed and secreted (RANTES) among the CC chemokine (also called ß-chemokine) receptors (CCR), such as those for monocyte chemoattractant proteins and macrophage inflammatory proteins (MIP)-1{alpha} and -ß.

RANTES (also called CCL5) is a small protein of 68 amino acids and is known as a proinflammatory chemokine. Although RANTES was originally identified as a T cell-specific gene, its expression has been detected in a variety of tissues or cell lines, such as spleen, thymic cortex, tonsil, kidney, Wilm’s tumor, the rhabdomyosarcoma cell line RD, and the osteosarcoma cell line MG63 (26, 27, 28). RANTES is a chemoattractant for monocytes, memory T lymphocytes, and eosinophils but not for B cells or killer T cells (28, 29). Previous work in bone suggests that RANTES is involved in the pathological progression of rheumatoid arthritis, osteoarthritis, osteomyelitis, and posttraumatic responses (15, 16). RANTES, along with other ß-chemokines, has been shown to induce osteoclast chemotaxis (30). Here for the first time, we demonstrate that RANTES receptors are expressed in primary calvarial osteoblasts and MC3T3-E1 cells, in which RANTES promotes cell migration and survival. We also show that cell migration can be induced by the RANTES secreted from osteoclasts in a paracrine mode of action, although the components for an autocrine mode of action, e.g. expression of both the receptors and their ligands on the same cell, are present in both osteoblasts and osteoclasts. Collectively, these data strongly point toward RANTES being an important molecule for communication between osteoclasts and osteoblasts and shed new light on the functions of this chemokines in osteoblast biology.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA). Recombinant mouse RANTES, receptor activator of nuclear factor-{kappa}B ligand (RANKL), MIP-1{alpha}, MIP-1ß, an anti-RANTES neutralizing antibody, and a murine RANTES ELISA kit were purchased from R&D Systems (Minneapolis, MN). 1,25 (OH)2D3 was from Biomol (Plymouth Meeting, PA) and dexamethasone from Sigma-Aldrich (St. Louis, MO). The phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, and antibodies against total and phosphorylated (Ser473) Akt were obtained from Cell Signaling Technology (Beverly, MA). A terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) kit was purchased from Roche Diagnostics Corp. (Indianapolis, IN). The enhanced chemiluminescence kit, Supersignal, was purchased from Pierce (Rockford, IL). Protease inhibitors were from Boehringer-Ingelheim (Mannheim, Germany). All other reagents were from Sigma Chemical Co. (St. Louis, MO).

Cell culture
The mouse calvarial osteoblast cell line, MC3T3-E1, was purchased from American Type Culture Collection (Manassas, VA) and cultured in {alpha}MEM containing 10% fetal bovine serum and 1% penicillin-streptomycin. Rat calvarial osteoblasts were cultured from 21-d fetal rats (Sprague Dawley). Twenty-five to 30 calvariae were harvested at room temperature. Humane handling of rats was carried out according to the guidelines of The Center for Animal Resources and Comparative Medicine at Harvard Medical School. Primary culture of calvarial osteoblasts was performed by a previously described method (31). Briefly, cells were released by repeated digestion of the calvariae with 0.05% trypsin and 0.1% collagenase P. After cells from the first two digestions were discarded, cells from the next three digestions were pooled and cultured in DMEM containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin in 5% CO2 at 37 C. After 3 d of culture, cells were serum-starved in DMEM (0.5 mM Ca2+, 4 mM L-glutamine, 1% penicillin-streptomycin, and 0.2% BSA) for 4 h before experimentation. All experiments were done within 7 d after beginning the culture.

The RAW 264.7 monocyte/macrophage mouse cell line was obtained from American Type Culture Collection and maintained in culture according to their instructions. Cells were cultured routinely in {alpha}MEM containing 10% heat-inactivated fetal calf serum. To differentiate the cells to osteoclast-like cells, RAW cells were gently scraped and seeded in either 100-mm2 plates or 24-well plates at a density of 250,000/plate or 3 x 103 cells/well, respectively, and cultured for 5–6 d in DMEM supplemented with 10% fetal calf serum and 50 ng/ml recombinant murine RANKL. Medium was replaced on the third day, and cells were cultured for 2 more days, at which point large numbers of multinucleated cells were observed. To purify these multinucleated cells from contaminating undifferentiated cells, which adhere less tightly to the substratum, we followed a previously published protocol (32). The purity of the differentiated osteoclasts was confirmed by comparing their expression of tartrate-resistant acid phosphatase (TRAP) mRNA with that of undifferentiated RAW cells using quantitative real-time PCR. Purity was also assessed in each experiment, using TRAP staining with a leukocyte acid phosphatase kit (387-A, Sigma). Only preparations in which the purity was close to 90% were used for further experiments.

[125I]-RANTES binding assay
The protocol used was a minor modification from the one previously used by our laboratory (33). MC3T3-E1 cells and primary calvarial osteoblasts were seeded at a density of 20,000 cells/well and were allowed to grow to 85–90% confluency. The monolayers were then washed with HANK-isotonic solution (H1387, Sigma-Aldrich) containing (grams per liter): 8 NaCl, 0.35 NaCO2, 0.4 KCl, 0.06 KHPO4, 0.05 NaH2PO4, 0.09815 MgSO4, 0.14 CaCl2, 10 Tris-3[N-morpholino]propanesulfonic acid (pH 7.4), and 0.02 mg/ml BSA at 4 C. Cells were then incubated with various concentrations of radiolabeled RANTES (20–110 pM, or 44 nCi to 242 nCi/ml) in the presence or absence of 400 nM unlabeled RANTES for 1 h at 4 C to avoid internalization of the radioligand. A 20-µl aliquot was removed to measure the total radioactivity in each well. Then cells were washed three times with six volumes of Hanks’ solution at 4 C to remove unbound, radiolabeled RANTES. Labeled cells were incubated with 0.5 ml phosphate buffer solution containing 5 mM EGTA buffer and 0.01% Triton X-100 for 15 min at 4 C to detach the cell monolayer. Cells were scraped and transferred into 4-ml plastic tubes to measure cell-bound radioactivity in a {gamma}-counter. A 30-µl aliquot was also removed to determine the protein content of each well using the BCA method (Pierce). The specific binding was calculated from the total binding in the presence or absence of unlabeled RANTES. To determine the maximal binding capacity and dissociation constant (Kd) for the binding of RANTES, a Scatchard plot was performed as described previously (34, 35). Similarly, an analysis of the displacement of radiolabeled RANTES by unlabeled RANTES was performed using different concentrations of unlabeled RANTES in the presence of a constant concentration of 90 pM [125I]-RANTES as described above. All linear or nonlinear curve fittings were performed as described in the figure legends using Sigmaplot (version 5, SPSS Science, Chicago, IL), unless otherwise stated.

Measurement of RANTES in cell culture supernatants
Cells prepared in 24-well plates were starved in serum-free medium containing 0.2% BSA for 4 h and then incubated with this medium in the presence of various mediators or vehicles. Supernatants collected after 18 h were used for measurement of RANTES. A Quantikine colorimetric sandwich ELISA kit (R&D Systems) was used according to the manufacturer’s instructions. To compare RANTES secretion from osteoblasts and osteoclasts, each sample was corrected using total protein levels.

RT-PCR
Total RNA was prepared using the TRIzol reagent (Invitrogen). One or 2 µg total RNA were employed for the synthesis of single-stranded cDNA (cDNA synthesis kit; Invitrogen). Primer pairs used for PCR are described in Table 1Go. Hot start PCR protocol was used as described before (31). The PCR products were fractionated on 1.5% agarose gels and gel purified. Bidirectional sequencing of the PCR fragments were performed employing the same primer pairs used for PCR by means of an automated sequencer (AB377; Applied Biosystems, Foster City, CA) and dideoxy terminator Taq technology in the DNA Sequence Faculty of the University of Maine (Orono, ME).


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  		TABLE 1. Primer pairs used for studying various genes

 
Quantitative real-time PCR
SYBR green chemistry was used to perform quantitative determinations of the mRNAs for CCRs, RANTES, and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), following an optimized protocol (31). The design of sense and antisense oligonucleotide primers was based on published cDNA sequences using the Primer Express (version 2.0.0, Applied Biosystems). Primer sequences are listed in Table 1Go, and cDNA was synthesized with the Omniscript reverse transcription kit (QIAGEN, Valencia, CA). For real-time PCR, the cDNA was amplified using an ABI PRISM 7000 sequence detection system (PE Applied Biosystems). The double-stranded DNA-specific dye SYBR Green I was incorporated into the PCR buffer provided in the QuantiTech SYBR PCR kit (QIAGEN) to allow for quantitative detection of the PCR product in a 30-µl reaction volume. The temperature profile of the reaction was 95 C for 15 min, 40 cycles of denaturation at 94 C for 15 sec, and annealing and extension at 60 C for 1 min. GAPDH was used to normalize differences in RNA isolation, RNA degradation, and the efficiencies of the reverse transcription. The size of the PCR product was first verified on a 2.0% agarose gel, and then it was subjected to melting-curve analysis.

Determination of chemotactic activity using a chemotaxis bioassay
Chemotactic activity was assessed using a previously described protocol from our laboratory (10). Briefly, cells incubated overnight in 3 ml of 0.2% BSA-containing medium were scraped, isolated by pipetting, and resuspended with 0.5 ml of serum-free medium. Preincubation of cells with LY294002 (10 µM) was performed for 30 min before scraping. Chemotactic activity was determined in a Blind Well Boyden chamber system (Neuroprobe Inc., Gaithersburg, MD) with a polyvinylpyrrolidone-free polycarbonate membrane (pore size 8 µm; Neuroprobe). Serum-free medium alone or that supplemented with RANTES was placed in the lower chamber, and cells were loaded in the upper chamber at a concentration of 1–5 x 104 cells in 100 µl. Cell viability was assessed by Trypan blue exclusion. The chamber was incubated at 37 C for 5 h, and the membrane was removed. After the cells on the upper side were scraped off, the membrane was fixed with 96% methanol and stained with Giemsa. Cells that had migrated to the lower side of the membrane were counted under a microscope in each of eight high-power fields.

To determine whether RANTES secreted from osteoclasts induced chemotaxis of osteoblasts, RAW264.7 cells were differentiated and purified by the method described earlier in 100-mm2 plates. After purification, the cells were cultured in 5 ml medium for 24 h in the presence of 50 ng/ml RANKL. The conditioned medium was then concentrated in a Speed Vac concentrator to a 1 ml volume. Conditioned medium prepared in this way generally contained approximately 15–20 ng/ml RANTES as determined by ELISA. Two hundred microliters of the concentrated conditioned medium was incubated with 2.5 µg/ml of either mouse IgG or anti-RANTES neutralizing antibody (monoclonal) at room temperature for 2 h. One hundred microliters of these media were then used to study chemotaxis of MC3T3-E1 cells as described above.

Western blot analysis
Calvarial osteoblasts were incubated with 50 ng/ml RANTES for various durations after 4 h of serum deprivation. Cells were rinsed with ice-cold PBS and scraped on ice into lysis buffer that contained 10 mM Tris-HCl (pH 7.4), 1 mM EGTA, 1 mM EDTA, 0.25 M sucrose, 1% Triton X-100, 1 mM dithiothreitol, and a cocktail of protease inhibitors (10 µg/ml each of aprotinin, leupeptin, and calpain inhibitor as well as 100 µg/ml Pefabloc). The cell lysates were then sonicated for 30 sec. Nuclei and cell debris were removed by centrifugation (6000 x g for 10 min), and the resultant total cellular lysate in the supernatant was used for SDS-PAGE. Immunoblot analysis was performed as described previously (31). The blots were incubated overnight at 4 C with gentle shaking with a phospho-Akt antibody at a 1:1000 dilution. The blots were extensively washed with PBS containing 1% Triton X-100 and 0.15% dry milk (washing solution) at room temperature and were further incubated with a 1:2000 dilution of horseradish peroxidase-coupled goat antimouse IgG (Sigma) in PBS containing 1% Triton X-100 for 1 h at room temperature. The blots were then washed, and the signal was visualized by chemiluminescence according to the manufacturer’s protocol (Supersignal, Pierce). After stripping of the blots, total Akt immunoreactivity was determined in the same membrane. National Institutes of Health (NIH) image software (version 1.62) was used to quantify the signal intensity.

TUNEL assay
The TUNEL reaction was performed to detect apoptosis using a previously published protocol from our laboratory (36). Rat primary calvarial osteoblasts were cultured on coverslips to no more than 50–60% confluence. Cells were then washed twice with PBS and incubated with serum-free medium containing vehicle, 50 ng/ml RANTES, or 50 ng/ml RANTES + 10 µM LY294002, for 24 h. This step was repeated after the first 24-h period for another 24 h. After a total of 48 h treatment in this manner, apoptosis was assessed using the in situ cell death detection kit (Roche Diagnostics), following the manufacturer’s recommendations. Briefly, cells were washed with PBS once, fixed with 3.8% formalin for 5 min, washed again with PBS, and permeabilized on ice with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min. Slides were rinsed twice with PBS and then incubated for 60 min at 37 C with terminal deoxynucleotidyl transferase enzyme in reaction buffer. The slides were rinsed three times with PBS and mounted with Vectashield (Vector Laboratories, Burlingame, CA). Samples were analyzed by fluorescence microscopy. TUNEL-positive nuclei were detected by the bright color in condensed or ruptured nuclei. The rate of apoptosis was calculated as the ratio of the number of apoptotic osteoblasts to the total number of osteoblasts (both apoptotic and nonapoptotic cells).

Statistics
All data are presented as the mean ± SE of the indicated number of experiments. Data were analyzed by one-way ANOVA followed by Fisher’s protected least significant difference post hoc test or Student’s t test when appropriate. P < 0.05 was taken to indicate a statistically significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of CC chemokine receptors in the MC3T3-E1, mouse calvarial cell line, and rat primary calvarial osteoblasts
The 10 human CCRs identified to date have been classified into CCR1–10. Previous studies have demonstrated that RANTES binds to CCR1, -3, -4, and -5. Therefore, we initially evaluated the expression of CCR1, -3, -4, and -5 in the MC3T3-E1 cell line by RT-PCR using the primer pairs described in Table 1Go. Total RNA extracted from the cells was subjected to DNase digestion to remove any contaminating DNA before RT-PCR. Figure 1AGo shows amplification of the expected mRNA fragments encompassed by the primer sets, which have the expected molecular sizes for the respective CCRs. No products were detected when the reverse transcription was omitted during synthesis of cDNA (data not shown). Bidirectional sequencing of these amplified products using the primers employed for the respective PCR amplification revealed more than 99% homology with the corresponding region of the cloned mRNAs. Therefore, the MC3T3-E1 mouse calvarial osteoblast cell line expresses the mRNAs for CCR1, -3, -4, and -5. We next studied the relative abundance of the four receptors by real-time quantitative PCR using the mouse-specific primers described in Table 1Go. Figure 1BGo shows that in MC3T3-E1 cells, the mRNAs for CCRs 4 and -5 are expressed at modestly lower levels than those for CCR-1 and -3. Quantitative PCR data are expressed in arbitrary units after normalization with mouse GAPDH mRNA.



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  		FIG. 1. Identification and determination of relative levels of expression of CCRs in osteoblasts. A, One microgram of total RNA from MC3T3-E1 cells was used for RT-PCR amplification of CCR mRNAs using mouse gene-specific primers that are described in Table 1Go. The RT-PCR yielded amplified cDNA products derived from bona fide transcripts of CCR1, -3, -4, and -5 in MC3T3-E1 cells, as determined from the size of the products and their sequences. B, Quantitative real-time PCR was used to determine the relative levels of expression of the CCRs in MC3T3-E1 cells. C, One microgram of total RNA from rat calvarial osteoblasts was used for RT-PCR amplification of CCR mRNAs using the rat gene-specific primers shown in Table 1Go. RT-PCR yielded amplified cDNA products derived from transcripts of CCR1, -3, -4, and -5 in primary calvarial osteoblasts as determined from the sizes of the products and their sequences. D, Quantitative real-time PCR was used to determine the relative levels of expression of CCRs in rat primary calvarial osteoblasts. Data are expressed in arbitrary units and were calculated from three independent experiments. *, P < 0.05.

 
We then extended our study to primary osteoblastic cells. Rat calvarial osteoblasts were prepared following an optimized protocol (31), which yields more than 90% pure cultures as assessed by RUNX-2/cbfa-1 immunostaining. Figure 1CGo shows that, similar to MC3T3-E1 cells, calvarial osteoblasts express mRNAs for CCR1, -3, -4, and -5. The levels of expression of the mRNAs for CCR1, -4, and -5 in these cells were similar, whereas that for CCR3 mRNA was 30–50% less than the others (Fig. 1DGo). Quantitative PCR data are expressed in arbitrary units after normalization with rat GAPDH mRNA.

We next studied whether the expression of the mRNAs for these four receptors known to bind RANTES is associated with functional binding sites using a radioreceptor assay. To test whether RANTES specifically binds to a receptor, we measured [125I]-RANTES binding to cultured MC3T3-E1 osteoblasts in the presence or absence of various concentrations of unlabeled RANTES. The total [125I]-RANTES bound was a saturable process in an hour at 4 C and was blocked more than 60% with unlabeled RANTES, consistent with a specific receptor interaction (data not shown). Analysis of the displacement data by unlabeled RANTES indicated an IC50 for RANTES receptor of 456 ± 22 pM (Rosenthal plot) (35) with a maximal binding of 141.28 ± 38 fmol/µg protein. To directly measure the binding of RANTES to its receptor(s) in MC3T3-E1 cells, we measured the specific binding of [125I]-RANTES as a function of the added concentration of [125I]-RANTES at 4 C (to avoid internalization of the radioligand) (Fig. 2AGo). Scatchard analysis of the experimental points revealed the presence of a binding site with an intrinsic substrate Kd of 107.55 pM. We also found that the estimated maximal binding capacity of 161.89 ± 32 fmol/µg protein (Fig. 2BGo) was not significantly different from that obtained using the ligand displacement protocol (e.g. a constant concentration of [125I]-RANTES and increasing concentrations of unlabeled RANTES; see above). [125I]-RANTES binding to rat primary calvarial osteoblasts revealed a similar binding kinetics and Scatchard analysis of the specific binding showed a Kd of 88.3 ± 13 pM with a binding capacity of 132.7 ± 24 fmol/µg protein (data not shown).



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  		FIG. 2. Binding of RANTES to MC3T3-E1 cells. Total and nonspecific binding of [125I]-RANTES as a function of different concentrations of radiolabeled RANTES was performed as described in Materials and Methods. A, Specific binding of RANTES was estimated from the difference between the total and nonspecific binding in the presence of 500 nM RANTES. B, Scatchard plot analysis yield a Kd of 107.55 pM with a maximal binding site of 161.89 ± 32 fmol/µg protein. Values are the mean of mean ± SE of three experiments in duplicate determinations.

 
Taken together, the data from the MC3T3-E1 cell line and the primary calvarial osteoblast cultures show that osteoblastic cells express all four RANTES receptor mRNAs that translate to functional receptor protein(s) exhibiting similar binding characteristics.

RANTES promotes osteoblast migration via PI3K pathway
Having shown functional expression of RANTES receptors in osteoblast cells by radioreceptor assay, we then studied the biological actions of RANTES. Figure 3AGo shows that RANTES induced chemotaxis of mouse MC3T3-E1 cells in a concentration-dependent manner using the Boyden chamber chemotaxis assay. The maximum effect was observed at 50 ng/ml (~600 nmol) RANTES. A similar stimulation of cell migration by RANTES was observed in primary rat calvarial osteoblasts (Fig. 3BGo). Because chemotaxis of preosteoblasts to sites of recent bone resorption is an important physiological function, our data show that RANTES is capable of inducing this function.



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  		FIG. 3. RANTES promotes migration of osteoblastic cells. A, Concentration-dependent increase in the chemotaxis of MC3T3-E1 cells in a Boyden’s chamber assay as described in Materials and Methods. B, Concentration-dependent increase in the chemotaxis of rat primary calvarial osteoblasts in a Boyden’s chamber assay as described in Materials and Methods. Three independent experiments were carried out in both cases. *, **, Significant increase over the vehicle control (V); P < 0.05; ** > *.

 
We then studied the mechanism underlying RANTES-mediated chemotaxis. Activation of the PI3K pathway is a key mediator of the chemoattractant function of diverse molecules, including RANTES (37, 38, 39, 40). Using 10 µM of the specific PI3K inhibitor, LY294002, we observed significant inhibition of RANTES-induced chemotaxis in primary osteoblasts (Fig. 4AGo). We then used Western blotting to study whether RANTES promotes phosphorylation of Akt kinase (also known as protein kinase B), which is immediately downstream of PI3K. Figure 4BGo shows that 50 ng/ml RANTES phosphorylates Akt within 2 min in calvarial osteoblasts.



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  		FIG. 4. RANTES-induced osteoblast chemotaxis involves activation of the PI3K-Akt pathway. A, Rat calvarial osteoblasts treated with 10 µM of a specific inhibitor of PI3K (LY294002) as described in Materials and Methods exhibited significant inhibition of RANTES-induced chemotaxis. *, Significantly more than the other treatments; P < 0.05. V, Vehicle. B, Rat osteoblasts were starved in serum-free medium for 2 h and then stimulated by addition of medium with 50 ng/ml of RANTES. Cell lysates were collected at the indicated time points for Western blotting. Note that RANTES increases Akt phosphorylation within 2 min in rat primary osteoblasts. The lower panel shows the intensity of phosphorylated Akt and was measured using the NIH Image Program and was normalized using the total Akt signal. Data are the mean of three independent experiments (P < 0.05). pAkt, Phospho-Akt; tAkt, total Akt.

 
RANTES prevents apoptosis of osteoblasts
Because Akt kinase, when activated, inhibits apoptosis by phosphorylating a variety of substrates, such as glycogen synthase kinase-3ß, Bcl-2 antagonist of cell death, and caspase-9 (41), we examined whether RANTES prevents apoptosis in osteoblasts. Because 50 ng/ml RANTES induced maximal chemotaxis and produced half-maximal binding in the radioreceptor assay, we used this concentration of RANTES to study its survival-promoting role in osteoblasts. Apoptosis was assessed 48 h after serum deprivation with or without 50 ng/ml of RANTES by TUNEL staining. Figure 5Go shows that RANTES treatment reduced serum deprivation-induced apoptosis by 66%. In addition, LY294002 largely abrogated this effect, indicating that the inhibitory effect of RANTES on osteoblast apoptosis is mediated, in large part, by the PI3K pathway.



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  		FIG. 5. RANTES protects rat calvarial osteoblasts from apoptosis induced by serum deprivation. Cells were cultured on coverslips to 50–60% confluency and then washed twice with PBS and incubated in serum-free medium containing vehicle (V), RANTES alone, or RANTES + LY294002 for 24 h. This step was repeated once more, and the cells were cultured for another 24 h before fixing in 3.5% formalin. The cells were then processed for the TUNEL assay, which revealed that RANTES treatment significantly protects cells from apoptosis, whereas LY294002 reduces this protective effect. Apoptotic cell number was divided by total cell number (apoptotic + viable) to determine the ratio of apoptotic cells. Data were pooled from four independent experiments; *, P < 0.05.

 
RAW264.7-differentiated osteoclasts secrete RANTES, which induces chemotaxis of osteoblasts
Because osteoblasts express abundant receptors for all four CCRs and exogenous RANTES induces biological responses such as chemotaxis and cell survival, we hypothesized that osteoclasts could be one of the sources of RANTES in the bone microenvironment. To address this question, we used murine-derived RAW264.7 cells differentiated to osteoclast-like cells by RANKL (50 ng/ml). We purified multinucleated cells as described in Materials and Methods after treatment for 5 d with RANKL. Figure 6Go, A–C, shows that our method of purification yielded a very pure population of osteoclasts because the levels of expression of the mRNAs for TRAP and CCR1, as assessed by quantitative PCR, were 30- and 20-fold higher than in the undifferentiated RAW264.7 cells, respectively. In parallel, TRAP histochemistry revealed approximately 90% TRAP-positive cells following the method of osteoclast differentiation that we employed (data not shown). Multinucleated osteoclasts purified in this manner were then used for measuring RANTES secretion by ELISA. We observed that elevated Ca2+ increased RANTES secretion from these cells in a concentration-dependent fashion (Fig. 6DGo). Therefore, RANTES secretion in osteoclast is regulated by an important physiological ion, Ca2+.



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  		FIG. 6. Purified multinucleated osteoclasts (Diff. OC) secrete RANTES, which is up-regulated by high Ca2+; secreted RANTES induces MC3T3-E1 cell chemotaxis. A, Purity of differentiated osteoclasts (OC) was assessed by TRAP mRNA expression using quantitative PCR and comparing its expression level with that of undifferentiated RAW264.7 (RAW) cells. *, Significantly more than bone marrow cells (BM); P < 0.05 (n = 3). B, Detection of CCR1 mRNA in RAW and Diff. OC by RT-PCR using mouse-specific primers from 1.0 µg total RNA from each cell type. The amplified product was derived from bona fide CCR1 mRNA as revealed by its molecular size and sequencing. C, Greater expression of CCR1 (positive control) in differentiated RAW264.7 cells, compared with undifferentiated cells, further confirms the purity of the osteoclast preparation after its differentiation. *, Significantly more than BM, P < 0.05 (n = 3). D, Purified, multinucleated osteoclasts obtained after differentiation of RAW264.7 cells (Diff. OC) secrete RANTES, which is up-regulated by increasing levels of extracellular Ca2+. ** > * > 0.5 and 2.5 mM Ca2+; P < 0.05 (n = 3). E, RANTES secreted from RAW264.7 differentiated osteoclasts induces osteoblast chemotaxis. Conditioned medium obtained from the differentiated RAW264.7 osteoclasts was processed as described in Materials and Methods and preincubated with 2.5 µg of either mouse IgG or anti-RANTES neutralizing antibody (Ab). These conditioned media were then used to study chemotaxis of MC3T3-E1 cells in a Boyden’s chamber assay. Data are pooled from four independent experiments each performed in triplicate. *, P < 0.05.

 
After showing that osteoclasts express and secrete RANTES, and that high Ca2+ could stimulate its secretion, we next evaluated whether the immunoreactive RANTES that is secreted by osteoclasts is biologically relevant. We took conditioned medium from osteoclasts derived from RAW264.7 cells and studied its effect on the chemotaxis of MC3T3-E1 cells in the Boyden chamber assay after incubating the medium with either mouse IgG or neutralizing anti-RANTES monoclonal antibody. Figure 6EGo shows that, whereas conditioned medium in the presence of mouse IgG-induced chemotaxis of MC3T3-E1 cells, neutralizing antibody against RANTES inhibited chemotaxis by almost 50%. Therefore, we conclude that RANTES secreted by the osteoclasts is capable of inducing osteoblast chemotaxis.

Regulation of RANTES secretion from osteoblasts
In addition to showing expression of CCRs capable of binding RANTES in osteoblasts, we further showed that osteoblasts, whose secretion of RANTES is differentially regulated by various physiological factors of importance that are relevant to osteoblasts. Figure 7Go shows that increasing concentrations of Ca2+ robustly up-regulate RANTES secretion from both MC3T3-E1 and rat primary osteoblastic cells. A similar stimulatory effect of Ca2+ on RANTES secretion was observed in RAW264.7 differentiated osteoclasts (Fig. 6DGo).



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  		FIG. 7. Elevated levels of Ca2+ up-regulate RANTES secretion from osteoblastic cells. A, Ca2+ dose-dependently increases RANTES secretion from MC3T3-E1 cell line. ** > * > 0.5 and 2.5 mM Ca2+; P < 0.05 (n = 3). B, Increased RANTES secretion by elevated levels of Ca2+ from rat primary calvarial osteoblasts. ** > * > 0.5 mM Ca2+; P < 0.05 (n = 3).

 
It has recently been shown that osteoblasts produce much less MIP-1{alpha} than do osteoclast-like RAW264.7 cells (30). Because MIP-1{alpha} largely binds to CCR1 and CCR5 (42), whose mRNAs are abundantly expressed in the osteoblasts studied here, we tested its effect on RANTES secretion from these cells. Figure 8Go shows a concentration-dependent stimulation of RANTES secretion from both MC3T3-E1 and calvarial osteoblast cells by MIP-1{alpha}. MIP-1ß, on the other hand, was without effect (data not shown). Interestingly, primary osteoblasts responded more vigorously than MC3T3-E1 cells because the maximum increases in RANTES secretion at 100 ng/ml MIP-1{alpha} were 96 and 43%, respectively.



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  		FIG. 8. MIP-1{alpha} up-regulate RANTES secretion from MC3T3-E1 cell line (A) and rat primary calvarial osteoblasts (B) (n = 3). @ > # > * > vehicle-treated control (V) (P < 0.05).

 
In contrast to the effects of elevated Ca2+ and MIP-1{alpha} on RANTES secretion, hormones that are known to promote osteoblast differentiation, e.g. 1,25 (OH)2 vitamin D3 and dexamethasone (43), down-regulated RANTES secretion from calvarial osteoblasts in a concentration-dependent fashion (Fig. 9Go). Dexamethasone seems to be more potent because it significantly inhibited RANTES secretion at a concentration as low as 10–10 M, whereas the lowest concentration at which 1,25 (OH)2 vitamin D3 exerted its inhibitory action was 10–9 M.



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  		FIG. 9. Hormones inducing differentiation of osteoblasts inhibit RANTES secretion. A, 1,25 (OH)2 vitamin D3. B, Dexamethasone (n = 3); @ > # > * > vehicle-treated control (V) (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Chemokines are group of molecules that induce chemotaxis of a variety of cell types, although additional functions of chemokines have also been described (for review, see Refs. 20 and 44). Chemotaxis is an important biological process for osteoclasts and osteoblasts, in view of the need for orderly recruitment of these cells (and/or their precursors) to sites of bone resorption and formation, respectively. RANTES has been shown to promote osteoclast migration, but it had no effect on bone resorption (30, 45). It is thought that osteoblasts, although generally studied in pathological conditions, are the source of chemokines, including RANTES (15, 16, 19, 46) and that osteoclasts express the receptors for this chemokine, constitutes a paracrine mode of communication.

Our current experiments demonstrated that rat primary calvarial osteoblasts express receptors for RANTES (CCRs), although we could not formally rule out the possibility that a small population of contaminating cells expressed the receptor transcripts. Therefore, we also showed that an established osteoblast cell line, MC3T3-E1 cells, also express CCRs 1, -3, -4, and -5. However, there were modest differences in the relative levels of expression of these CCRs between primary osteoblasts and MC3T3-E1 cells, e.g. a lesser expression of CCR3 in rat calvarial osteoblasts and of CCR4 in the MC3T3-E1 cell line. These differences in the expression profiles of the CCRs might be the result of species variation and/or the immortalization process used to generate MC3T3-E1 cells. It would be a matter of significant importance to determine whether expression of all four receptors is biologically redundant or each receptor performs distinct functions in osteoblasts.

As the first step toward documenting that expression of the CCR mRNAs in osteoblasts is associated with expression of functional receptor protein(s), we performed a radioreceptor assay and showed that these receptor mRNAs indeed translate into RANTES binding. We could not, however, distinguish the binding of the ligand to the various CCRs, which would require the availability of antagonists specific for the individual CCRs. Because MIP-1{alpha} and MIP-1ß bind to CCR1 and CCR5, it is possible that these chemokines might also have functional effects on osteoblasts, a possibility that could be pursued in future studies and might provide some insights into which receptors mediate which actions in osteoblasts. In addition, because osteoblasts secrete RANTES, our data showing expression of functional RANTES receptor(s) indicate a potential autocrine mode of action of this chemokine.

Because osteoblasts express functional CCR protein(s), we reasoned that RANTES could exert a paracrine action on them and that osteoclasts could represent a physiologically relevant source of this chemokine. We showed that RAW264.7 cells differentiated to osteoclasts express and secrete RANTES, which is up-regulated by elevated Ca2+. Therefore, in osteoclasts the situation is similar to that in osteoblasts in that RANTES has the potential to act in an autocrine mode of communication as these cells exhibit robust CCR1 expression. In addition, paracrine modes of action whereby RANTES secreted by osteoclasts acts on osteoblasts, and vice versa, also remains a possibility. Therefore, delineating the relative importance of the autocrine and/or paracrine actions of RANTES and other ß-chemokines in osteoblasts and osteoclasts is a much-needed subject of future investigation. As a first approach to addressing this issue, we show in this report that exogenous RANTES elicits several biological actions in osteoblasts.

Because RANTES is a chemokine, we first studied its effect on the chemotaxis of osteoblasts. Physiologically, preosteoblast migration to the site of bone resorption during the reversal phase of the bone remodeling cycle is thought to be evoked by various factors released from the resorbed bone matrix (e.g. TGFß and bone morphogenetic proteins) (2, 3). However, there has been little work investigating the possibility that factors released from osteoclasts per se also participate in promoting chemotaxis of preosteoblasts. Our data show that both exogenous RANTES and conditioned medium derived from RAW264.7 cells differentiated to osteoclasts elicit osteoblast chemotaxis. This action of RANTES is mediated by its cognate receptor(s), acting via the PI3K pathway. Moreover, treating osteoblasts with RANTES resulted in phosphorylation of Akt, the kinase that is downstream of PI3K, which would be expected to exert a prosurvival effect. Indeed, we observed that RANTES prevents osteoblasts from undergoing apoptosis induced by serum starvation. It is conceivable that, during the course of their migration to sites of recent bone resorption, there is a need for protection from apoptotic cell death induced by the plethora of cytokines and Ca2+ released from the bone matrix. Involvement of the PI3K-Akt pathway has also been observed in platelet-derived growth factor-induced chemotaxis in an osteoblast cell line (4, 40). Therefore, migration of osteoblasts in response to RANTES, along with the antiapoptotic action of the latter, are functional evidence of the expression of functional CCR(s) in osteoblasts, which could be activated in a paracrine fashion, plausibly by RANTES released by osteoclasts.

The paracrine mode of RANTES action in the bone microenvironment has been hypothesized due to recent reports including this report that osteoclast secretes RANTES, which could act on osteoblast. A report based on mRNA profiling demonstrated that RANKL increases RANTES mRNA more than 10-fold in both mouse and human osteoclast precursors (47). In addition, RANTES secretion was found to increase in a time-dependent fashion in RAW264.7 cells treated with RANKL, indicating that the process of differentiation could up-regulate RANTES secretion in RAW264.7 cells (32). We observed expression of RANTES in addition to CCR1 mRNA in RAW264.7 differentiated osteoclasts, which suggests existence of an autocrine mode of RANTES action in this cells. Osteoblast in contrast expresses all four RANTES receptors and is a rich source of RANTES, suggesting a greater possibility of autocrine mode of action. However, given the expression of RANTES and its receptors in both cell types also raises the curious possibility of paracrine mode of communication between these two cell types. Therefore, we speculate that RANTES secreted by the osteoclast could promote osteoblast migration. We indeed have shown that RANTES secreted from differentiated RAW264.7 cells induces chemotaxis of MC3T3-E1 cells. Definitive evidence that the RANTES secreted by osteoclasts was the cause of the osteoblastic chemotaxis was obtained by showing that the migration of MC3T3-E1 cells by the conditioned medium was attenuated by incubating it with neutralizing anti-RANTES antibody.

RANTES secretion was up-regulated in both osteoblasts and osteoclasts by elevated extracellular Ca2+, which would be encountered in the bone microenvironment during bone resorption. High Ca2+ exerts a mitogenic effect on osteoblasts (31), and, therefore, it is conceivable that RANTES up-regulation takes place as a function of the proliferation in these cells. Induction of RANTES secretion by high Ca2+ could be physiologically important because the process of bone resorption by osteoclasts sets in motion changes in osteoblast function, such as chemotaxis, that culminate in bone formation by differentiated osteoblasts at the site at which bone had been resorbed. Therefore, it is possible that osteoclasts, in response to high local Ca2+ generated as a result of their resorptive activity, secrete more RANTES, which, in turn, induces osteoblast migration.

Furthermore, we observed that another ß-chemokine, MIP-1{alpha}, which acts by binding to CCR1 and CCR5, induced RANTES secretion from osteoblasts. MIP-1{alpha} secretion is high in osteoclasts and their precursor cells, compared with osteoblasts, and we have shown expression of CCR1 and CCR5 mRNAs in osteoblasts. Therefore, a concentration-dependent increase in RANTES by MIP-1{alpha} indicates that these receptors are functional in osteoblasts. Also, because RANTES regulates several important osteoclast functions and MIP-1{alpha} is secreted predominantly by osteoclasts, it is conceivable that communication between osteoclasts and osteoblasts could be regulated by these two chemokines.

Differentiation-promoting factors, such as 1,25 (OH)2 vitamin D3 and dexamethasone, inhibit RANTES secretion from osteoblasts. The ability of dexamethasone to inhibit the up-regulation of RANTES mRNA as a result of infection with Mycobacterium tuberculosis in the osteoblast-like MG63 osteosarcoma cell line suggested an inhibitory effect of glucocorticoid on RANTES expression (48). However, 10-fold higher concentration of the hormone was required to maximally inhibit RANTES secretion (e.g. 10–6 M vs. the 10–7 M observed in our study with normal osteoblasts). It is possible that M. tuberculosis infection up-regulates RANTES expression in osteosarcoma cells to such a high level that a supraphysiological concentration of dexamethasone is required to inhibit it.

Although we have shown several functions of RANTES in osteoblasts that would be expected to favor bone formation, we need to understand in greater detail the importance of this chemokine, if any, for bone formation and resorption in vivo. CCRs and their ligands are known for their redundancy of binding, and a similar situation may exist in osteoblasts because we observed the expression of multiple types of CCRs. Therefore, it will be important to identify which, if any, of these receptors participate in normal bone turnover. Gene targeting techniques, receptor-selective inhibitors, and neutralizing antibodies will be of use in addressing these issues. Other classes of chemokines and chemokine receptors may also be involved in normal bone physiology. Further studies are needed to clarify these issues.

Taken together, our data show for the first time that osteoblasts respond to RANTES by virtue of their functional expression of CCRs. We also show that RANTES produced by osteoclasts can induce osteoblast chemotaxis and that exogenous addition of RANTES protects these cells against apoptosis. Osteoblasts and osteoclasts possess the capacity to respond to both autocrine and paracrine modes of action of RANTES owing to their expression of both RANTES and its receptors. As illustrated in Fig. 10Go, inflammatory/osteoclastogenic cytokines, such as TNF{alpha} and IL-1ß (30), elevated Ca2+ (owing to increased osteoclastic activity), and MIP-1{alpha} from osteoclast precursor cells, will induce RANTES secretion by osteoblasts, which consequently will promote migration of preosteoclasts to the site of future bone resorption (e.g. mediated by the osteoblast via RANKL). Elevated Ca2+ in the resorptive microenvironment will produce increased RANTES production from osteoclasts, which, in turn, will induce migration of preosteoblasts to sites of bone resorption. It is also conceivable that increased RANTES secretion by the osteoblasts nearest the resorbing osteoclast as a result of their exposure to higher concentrations of Ca2+ and/or MIP-1{alpha} could then pass along the chemotactic signal (RANTES) to preosteoblasts farther away, a sort of an amplification mechanism. This creates a possible scenario in which RANTES secreted by bone cells could participate both in osteoclastogenesis and recruitment of osteoblasts and their protection against apoptosis during the resorptive and formative phases of bone turnover (49), respectively.



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  		FIG. 10. Schematic illustration of RANTES function and regulation in bone. Cytokines from bone marrow cells or osteoclast (OCs) precursor cells, such as TNF{alpha}, IL-1ß, and MIP-1{alpha}, induce RANTES secretion from osteoblasts (OBs). Increased Ca2+, released from the bone matrix during bone resorption, could induce RANTES secretion from both osteoblasts and osteoclasts. RANTES produced by osteoclasts, in turn, induces osteoblastic chemotaxis by virtue of the latter’s expression of CCRs, thereby promoting cellular migration to the site of resorption and pari pasu increased cellular viability. Up-regulation of RANTES secretion from osteoblasts in response to the various factors shown here would promote OC migration but could also induce migration of additional preosteoblasts toward the resorption site by a paracrine mechanism.

 


   Footnotes
 
This work was supported by National Institutes of Health Grants AR02115 (to N.C.); HL67699 and DK069388 (to AR); and DK48330, DK52005, DK67111, and DK67155 (to E.M.B.).

First Published Online February 17, 2005

Abbreviations: Ca2+, Calcium; CC, ß-chemokine; CCR, CC chemokine receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Kd, dissociation constant; MIP, macrophage inflammatory protein; PI3K, phosphatidylinositol 3-kinase; RANKL, receptor activator of nuclear factor-{kappa}B ligand; RANTES, regulated upon activation normal T cell expressed and secreted; TRAP, tartrate-resistant acid phosphatase; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

Received January 18, 2005.

Accepted for publication February 9, 2005.


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 Results
 Discussion
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FASEB J, December 1, 2006; 20(14): 2562 - 2564.
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