Parathyroid Hormone Stimulates Osteoblastic Expression of MCP-1 to Recruit and Increase the Fusion of Pre/Osteoclasts

The clinical findings that alendronate blunted the anabolic effect of human parathyroid hormone (PTH) on bone formation suggest that active resorption is involved and enhances the anabolic effect. PTH signals via its receptor on the osteoblast membrane, and osteoclasts are impacted indirectly via the products of osteoblasts. Microarray with RNA from rats injected with human PTH or vehicle showed a strong association between the stimulation of monocyte chemoattractant protein-1 (MCP-1) and the anabolic effects of PTH. PTH rapidly and dramatically stimulated MCP-1 mRNA in the femora of rats receiving daily injections of PTH or in primary osteoblastic and UMR 106-01 cells. The stimulation of MCP-1 mRNA was dose-dependent and a primary response to PTH signaling via the cAMP-dependent protein kinase pathway in vitro. Studies with the mouse monocyte cell line RAW 264.7 and mouse bone marrow proved that osteoblastic MCP-1 can potently recruit osteoclast monocyte precursors and facilitate receptor activator of NF-κB ligand-induced osteoclastogenesis and, in particular, enhanced fusion. Our model suggests that PTH-induced osteoblastic expression of MCP-1 is involved in recruitment and differentiation at the stage of multinucleation of osteoclast precursors. This information provides a rationale for increased osteoclast activity in the anabolic effects of PTH in addition to receptor activator of NF-κB ligand stimulation to initiate greater bone remodeling. The clinical findings that alendronate blunted the anabolic effect of human parathyroid hormone (PTH) on bone formation suggest that active resorption is involved and enhances the anabolic effect. PTH signals via its receptor on the osteoblast membrane, and osteoclasts are impacted indirectly via the products of osteoblasts. Microarray with RNA from rats injected with human PTH or vehicle showed a strong association between the stimulation of monocyte chemoattractant protein-1 (MCP-1) and the anabolic effects of PTH. PTH rapidly and dramatically stimulated MCP-1 mRNA in the femora of rats receiving daily injections of PTH or in primary osteoblastic and UMR 106-01 cells. The stimulation of MCP-1 mRNA was dose-dependent and a primary response to PTH signaling via the cAMP-dependent protein kinase pathway in vitro. Studies with the mouse monocyte cell line RAW 264.7 and mouse bone marrow proved that osteoblastic MCP-1 can potently recruit osteoclast monocyte precursors and facilitate receptor activator of NF-κB ligand-induced osteoclastogenesis and, in particular, enhanced fusion. Our model suggests that PTH-induced osteoblastic expression of MCP-1 is involved in recruitment and differentiation at the stage of multinucleation of osteoclast precursors. This information provides a rationale for increased osteoclast activity in the anabolic effects of PTH in addition to receptor activator of NF-κB ligand stimulation to initiate greater bone remodeling. Parathyroid hormone (PTH) 2The abbreviations used are: PTH, parathyroid hormone; h, human; r, rat; RT, reverse transcription; ELISA, enzyme-linked immunosorbent assay; RANKL, receptor activator of NF-κB ligand; M-CSF, macrophage colony-stimulating factor; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; TRAP, tartrate-resistant acid phosphatase. 2The abbreviations used are: PTH, parathyroid hormone; h, human; r, rat; RT, reverse transcription; ELISA, enzyme-linked immunosorbent assay; RANKL, receptor activator of NF-κB ligand; M-CSF, macrophage colony-stimulating factor; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; TRAP, tartrate-resistant acid phosphatase. is a principal hormone regulating bone remodeling via its actions on both bone formation and bone resorption. The finding that the anabolic effect of human PTH-(1-34) on bone formation was blunted when bone resorption was inhibited indicated that active resorption is involved and enhances the in vivo anabolic effect of PTH (1Delmas P.D. Vergnaud P. Arlot M.E. Pastoureau P. Meunier P.J. Nilssen M.H. Bone (Elmsford). 1995; 16: 603-610Crossref PubMed Scopus (146) Google Scholar, 2Black D.M. Greenspan S.L. Ensrud K.E. Palermo L. McGowan J.A. Lang T.F. Garnero P. Bouxsein M.L. Bilezikian J.P. Rosen C.J. N. Engl. J. Med. 2003; 349: 1207-1215Crossref PubMed Scopus (1007) Google Scholar, 3Finkelstein J.S. Hayes A. Hunzelman J.L. Wyland J.J. Lee H. Neer R.M. N. Engl. J. Med. 2003; 349: 1216-1226Crossref PubMed Scopus (714) Google Scholar). However, the molecular basis of the osteoclast activation in this process has not been fully studied. In order to identify the key mediators for the anabolic effects of PTH, we performed microarray using RNA from the femora of 3-month-old Sprague-Dawley female rats injected daily with hPTH-(1-34) to increase bone formation or vehicle. This was compared with rats receiving hPTH-(1-34) in a catabolic protocol by continuous infusion. PTH-(1-34) regulated numerous genes (∼1,000), but differentially, in both regimes (see accompanying article, Ref. 4Li X. Liu H. Qin L. Tamasi J. Bergenstock M. Shapses S. Feyen J.H.M. Notterman D.A. Partridge N.C. J. Biol. Chem. 2007; 282: 33086-33097Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Notably, in the anabolic protocol, a number of cytokines and chemokines, RANKL, interleukin-6, CXCL1, and CCL2 (MCP-1), were highly induced. In contrast, none of these were significantly increased, by microarray, in the catabolic protocol. The chemokine, monocyte chemoattractant protein-1 (MCP-1, CCL2), was found to be the most highly stimulated gene from 14-day intermittent hPTH-(1-34) or hPTH-(1-31) administration (both cause over 100-fold changes). With continuous infusion of PTH, the MCP-1 mRNA was elevated less than 2-fold, and this difference indicates a potentially important role for MCP-1 in the anabolic effect of PTH. Chemokines are bioactive peptides that regulate leukocyte activation and migration. They are essential mediators of inflammation and are crucial for the control of viral infections. MCP-1 is one of the CC chemokines, one of the four main subfamilies of chemokines, consisting of C, CC, CXC, and CX3C, according to the location of the N-terminal four cysteine residues (5Sodhi A. Montaner S. Gutkind J.S. Nat. Rev. Mol. Cell Biol. 2004; 5: 998-1012Crossref PubMed Scopus (136) Google Scholar). As a potent chemokine for monocytes and macrophages, MCP-1 is implicated in the pathogenesis of diseases characterized by monocytic infiltrates (6Wise G.E. Frazier-Bowers S. D'Souza R.N. Crit. Rev. Oral. Biol. Med. 2002; 13: 323-334Crossref PubMed Scopus (226) Google Scholar). It can be commonly detected at the site of tooth eruption, rheumatoid arthritic bone degradation, and bacterially induced bone loss (6Wise G.E. Frazier-Bowers S. D'Souza R.N. Crit. Rev. Oral. Biol. Med. 2002; 13: 323-334Crossref PubMed Scopus (226) Google Scholar) due to stimulation by inflammatory mediators. MCP-1 is also expressed by mature osteoclasts, and its expression is stimulated by receptor activator of NF-κB ligand (RANKL) and regulated by nuclear factor κB (6Wise G.E. Frazier-Bowers S. D'Souza R.N. Crit. Rev. Oral. Biol. Med. 2002; 13: 323-334Crossref PubMed Scopus (226) Google Scholar, 7Ishida N. Hayashi K. Hoshijima M. Ogawa T. Koga S. Miyatake Y. Kumegawa M. Kimura T. Takeya T. J. Biol. Chem. 2002; 277: 41147-41156Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 8Cappellen D. Luong-Nguyen N.H. Bongiovanni S. Grenet O. Wanke C. Susa M. J. Biol. Chem. 2002; 277: 21971-21982Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). It is known that expression of MCP-1 is temporally and spatially associated with the recruitment of monocytes in both osseous inflammation and during developmentally regulated bone remodeling in the process of tooth eruption (9Graves D.T. Jiang Y. Valente A.J. Histol. Histopathol. 1999; 14: 1347-1354PubMed Google Scholar). In vitro studies have shown that osteoclasts in both mice and humans may form directly from precursor cell populations of monocytes and macrophages (10Udagawa N. Takahashi N. Akatsu T. Tanaka H. Sasaki T. Nishihara T. Koga T. Martin T.J. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7260-7264Crossref PubMed Scopus (818) Google Scholar). It is very likely that MCP-1 may play a role in hormone-stimulated bone remodeling through action on osteoclasts. However, the relationship between MCP-1 and PTH, one of the most effective hormones regulating bone remodeling, has never been investigated. Here we propose a model suggesting that PTH-induced osteoblastic expression of MCP-1 is involved in osteoclast recruitment and differentiation at the stage of fusion and multinucleation of osteoclast precursors. This hypothesis provides a rationale for increased osteoclast activity in the anabolic effect of PTH, apart from RANKL stimulation, to initiate greater bone remodeling in a transient time-dependent fashion. Collectively, our data strongly point toward MCP-1 being an important molecule for communication between osteoblasts and osteoclasts in the anabolic effect of PTH. Chemicals—Synthetic PTH-(1-34) (human), PTH-(3-34) (bovine), and PTH-(1-31) (human), were purchased from Bachem (Torrance, CA). Inhibitors H-89 and GF109203X were purchased from Calbiochem. Rat PTH-(1-34), 8-bromo-cAMP, PMA, and cycloheximide were obtained from Sigma. Recombinant rat/murine RANKL, M-CSF, MCP-1, and neutralizing anti-MCP-1 antibody were purchased from Chemicon (Temecula, CA). Penicillin/streptomycin was obtained from Invitrogen. In Vivo Treatment of PTH in Rats—Sprague-Dawley rats (3-month-old female, about 250 g) were purchased from Taconic Farms, Inc. (Germantown, NY). For the anabolic protocol, rats were injected subcutaneously with vehicle (0.9% saline solution) or hPTH-(1-34) (8 μg/100 g) and euthanized using CO2 at the indicated time after injection. For the catabolic protocol, vehicle (0.9% saline solution) or hPTH-(1-34) in a final volume of 200 μl was continuously infused into animals at a nominal pumping rate of 1 μl/h by Alzet osmotic pumps (DURECT Corp.) implanted subcutaneously; continuous infusion of PTH-(1-34) was at 4 μg/100 g/day for the indicated time to 3-month-old female Sprague-Dawley rats (at least four animals per group). The animal protocols were approved by the Robert Wood Johnson Medical School Institutional Animal Care and Use Committee. The primary spongiosa samples from the distal femur were harvested immediately after sacrifice as described previously (11Onyia J.E. Bidwell J. Herring J. Hulman J. Hock J.M. Bone (Elmsford). 1995; 17: 479-484Crossref PubMed Scopus (150) Google Scholar). Analysis of mRNA Abundance by Real Time RT-PCR—Cells or tissues were harvested at the indicated time points after PTH treatment. As described earlier (12Qin L. Li X. Ko J.K. Partridge N.C. J. Biol. Chem. 2005; 280: 3104-3111Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), total RNA was isolated using TRI Reagent® (Sigma) followed by purification with the RNeasy kit (Qiagen). A TaqMan® reverse transcription kit (Applied Biosystems) was used to reverse-transcribe mRNA into cDNA. Following this, PCR was performed on Opticon (MJ Research) using a SYBR® Green PCR core kit (Applied Biosystems). The primers used for the RT-PCR are summarized in Table 1. For cell culture samples, glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. For femoral samples, β-actin was used as an internal control.TABLE 1Sequences of primers for real time RT-PCRGene5′-Primer3′-PrimerMouse β-actinTCCTCCTGAGCGCAAGTACTCTCGGACTCATCGTACTCCTGCTTMouse TRAPCACTCCCACCCTGAGATTTGTGACGGTTCTGGCGATCTCTTTGCMouse CCR2ATTCTCCACACCCTGTTTCGGATTCCTGGAAGGTGGTCAARat MCP-1TAGCATCCACGTGCTGTCTATGCTGCTGGTGATTCTCTTGRat β-actinTCCTGAGCGCAAGTACTCTGTGCGGACTCATCGTACTCCTGCTTRat RANKLACCAGCATCAAAATCCCAAGGGACGCTAATTTCCTCACCARat OPGATACAGACAGCTGGCACACGTGCTTTCGATGACGTCTCACRat glyceraldehyde-3-phosphate dehydrogenaseAACCCATCACCATCTTCCAGGGCCTTCTCCATGGTGGTGAA Open table in a new tab Cell Culture—UMR 106-01 cells were maintained in Eagle's minimal essential medium supplemented with 5% fetal bovine serum, nonessential amino acids, 25 mm HEPES (pH 7.4), 1% penicillin/streptomycin (5,000 units/ml), and 100 μg/ml streptomycin. For serum starvation, osteoblastic cells were allowed to grow to 70-80% confluence and then switched to serum-free minimal essential medium for 1 day before the addition of PTH. Rat primary calvarial osteoblastic cells were obtained and cultured as described previously (13Shalhoub V. Gerstenfeld L.C. Collart D. Lian J.B. Stein G.S. Biochemistry. 1989; 28: 5318-5322Crossref PubMed Scopus (60) Google Scholar). Before PTH treatment, these cells were serum-starved for 1 day. RAW 264.7 cells were plated at 106 cells/cm2 concentration in Dulbecco's minimal essential medium (supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 5,000 units/ml). The cells were cultured in 5% CO2 in a humidified environment. Mouse osteoclasts were cultured from bone marrow isolated from femora and tibiae of 4-6-week-old wild-type B6/129 mice. Briefly, bone marrow was flushed from long bones with serum-free α-minimum Eagle's medium, and the marrow clumps were broken down by passing media through a syringe with an 18-gauge needle; after centrifugation, the cell pellets were resuspended in 10% α-minimum Eagle's medium and incubated in tissue culture dishes at 37 °C in 5% CO2. Cells were stimulated with the indicated concentration of cytokines (M-CSF, 30 ng/ml; RANKL, 50 ng/ml) from the time of culture; fresh cytokines were added every other day until day 6 or day 10 (for bone resorption assay only) (14Kitaura H. Sands M.S. Aya K. Zhou P. Hirayama T. Uthgenannt B. Wei S. Takeshita S. Novack D.V. Silva M.J. Abu-Amer Y. Ross F.P. Teitelbaum S.L. J. Immunol. 2004; 173: 4838-4846Crossref PubMed Scopus (153) Google Scholar, 15Abu-Amer Y. Erdmann J. Alexopoulou L. Kollias G. Ross F.P. Teitelbaum S.L. J. Biol. Chem. 2000; 275: 27307-27310Abstract Full Text Full Text PDF PubMed Google Scholar). MCP-1 ELISA—A rat MCP-1-specific ELISA kit was purchased from BIOSOURCE to measure the protein level of MCP-1 in cell culture media. Assays were performed as recommended by the manufacturer to determine levels of secreted MCP-1 in UMR 106-01 cell culture media that had been concentrated in Amicon Ultra spin columns (Millipore, Billerica, MA) from 4 ml to a 500-μl volume. Values were calculated from standard curves set up for each assay. Supernatants were always run in duplicate, and the variance between the two replicates averaged less than 10%. Chemotaxis Assay—This method was performed as described previously (16Wuyts A. Proost P. Froyen G. Haelens A. Billiau A. Opdenakker G. Van Damme J. Methods Enzymol. 1997; 287: 13-33Crossref PubMed Scopus (15) Google Scholar, 17Votta B.J. White J.R. Dodds R.A. James I.E. Connor J.R. Lee-Rykaczewski E. Eichman C.F. Kumar S. Lark M.W. Gowen M. J. Cell. Physiol. 2000; 183: 196-207Crossref PubMed Scopus (55) Google Scholar, 18Chen X.D. Qian H.Y. Neff L. Satomura K. Horowitz M.C. J. Bone Miner. Res. 1999; 14: 362-375Crossref PubMed Scopus (67) Google Scholar). Briefly, serial dilutions of MCP-1 or conditioned media from UMR 106-01 cell culture media treated with PTH-(1-34) for 2 h were added to the bottom wells of a 96-well chemotaxis chamber (Neuro Probe, Inc.) with osteoclasts or the precursors scraped off from the culture dishes with a rubber-tipped cell scraper (19Collin-Osdoby P. Yu X. Zheng H. Osdoby P. Methods Mol. Med. 2003; 80: 153-166PubMed Google Scholar) and placed on the top of the membrane (8-μm pores). After incubating 6 h at 37 °C in a humidified incubator, chambers were disassembled, and the excess cells were mechanically removed from the applied side of the membranes. Membranes were fixed with methanol and processed for TRAP staining followed by counterstaining with hematoxylin. Cells positive for TRAP were identified as osteoclasts. Migration was quantitated by cell counts in six fields (×200 magnification) of each well on the semi-membrane by light microscopy using image analysis software (SPOT Advanced; Diagnostic Instruments, Inc.). The conditioned media were concentrated in Amicon Ultra spin columns (Millipore, Billerica, MA) from 10 ml to a 100-μl volume before inclusion in the chemotaxis assay. Conditioned media prepared in this way generally contained ∼25 ng/ml MCP-1 (in the concentrated media) as determined by ELISA. In experiments to test the specificity of the chemotactic effect of MCP-1, the medium containing the recombinant MCP-1 or concentrated conditioned media was incubated with 2.5 μg/ml of either rabbit IgG or anti-MCP-1 neutralizing antibody (polyclonal) at room temperature for 2 h before being used to study chemotaxis of pre/osteoclast cells as described above. Osteoclastogenesis—A serial dilution of recombinant rat MCP-1 (10-200 ng/ml) with or without RANKL and M-CSF (in primary osteoclast culture only) was added to cell culture media for the indicated time, and osteoclast differentiation was then assessed by measuring the activity of TRAP, a marker enzyme of osteoclasts, using acid phosphatase kit 387-A (Sigma) and by the direct enumeration of TRAP-positive, morphologically distinct, multinucleated osteoclasts (20Igarashi K. Woo J.T. Stern P.H. Biochem. Pharmacol. 2002; 63: 523-532Crossref PubMed Scopus (40) Google Scholar, 21Woo J.T. Kasai S. Stern P.H. Nagai K. J. Bone Miner. Res. 2000; 15: 650-662Crossref PubMed Scopus (73) Google Scholar). Briefly, the specimens were fixed for 30 s and then stained with naphthol AS-BI-phosphate and a tartrate solution for 30 min at 37 °C, followed by counterstaining with a hematoxylin solution. Osteoclasts were determined to be TRAP-positive staining multinuclear (>3 nuclei) cells using light microscopy. The total number of TRAP-positive cells and the number of nuclei per TRAP-positive cell in each well were counted. The morphological features of osteoclasts were also photographed. Bone Resorption Assay—Mice bone marrow cells were plated onto BioCoat™ Osteologic™ disks (BD Biosciences) under the same culture conditions as described above. The disk incorporates an artificial bone analog in the form of sub-micron calcium phosphate films that can be resorbed on transparent quartz substrates. After a 10-day culture, 1 ml of bleach solution (∼6% NaOCl, ∼5.2% NaCl) was added to each well. Five min later, the disks were washed with distilled water to remove adherent cells. The disks were then air dried and examined by light microscopy. Statistics—All data are based on a minimum of three replicate experiments performed independently on different occasions, unless otherwise stated. Data are shown as mean ± S.E. in the figures. Student's t test or one-way analysis of variance and Tukey's test was used for statistical analyses. PTH Regulates MCP-1 mRNA Abundance in Vivo and in Vitro—Recent microarray studies from our laboratory identified MCP-1 as the most highly regulated target gene in an anabolic PTH protocol in rats, which was not significantly stimulated in a catabolic protocol (4Li X. Liu H. Qin L. Tamasi J. Bergenstock M. Shapses S. Feyen J.H.M. Notterman D.A. Partridge N.C. J. Biol. Chem. 2007; 282: 33086-33097Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). We confirmed this result by performing real time RT-PCR using RNA isolated from the metaphyseal region of the femora of rats that had received intermittent injections of PTH. As shown in Fig. 1A, daily injections of hPTH-(1-34) for 1, 3, 7, 10, and 14 days greatly stimulated MCP-1 mRNA expression in this bone tissue in rats 1 h after the injection. This induction occurs very quickly, since there is a 32-fold increase in the mRNA expression of MCP-1 1 h after even one PTH injection. Interestingly, the fold simulation of MCP-1 mRNA level kept increasing with longer PTH treatment resulting in an increase of more than 200-fold with 14 daily intermittent injections of PTH. In the PTH catabolic treatment model where hPTH-(1-34) was continuously infused into rats subcutaneously, the MCP-1 mRNA level was significantly increased in this region of bone at 6 h and 6 and 14 days but was never greater than 2.5-fold of the vehicle controls (Fig. 1B). To confirm that the stimulation of MCP-1 is an osteoblastic response to PTH, we examined the MCP-1 mRNA level after PTH-(1-34) (10-8 m) administration in osteoblastic cells, the UMR 106-01 cell line, and primary osteoblastic cells. The results indicate that PTH treatment stimulates MCP-1 mRNA levels by 20-fold in the UMR cells with the peak level around 90 min after PTH treatment (Fig. 1C). Correspondingly, ELISA showed that secreted MCP-1 protein in UMR cell culture media reached a peak level 2 h after PTH-(1-34) (10-8 m) treatment (Fig. 2). Furthermore, MCP-1 stimulation by PTH is also observed in rat primary calvarial osteoblastic cells (Fig. 1D). These cells mimic the development of osteoblasts in vivo by transitioning sequentially through three stages (proliferation, differentiation, and mineralization) when cultured in vitro. At all developmental stages, MCP-1 levels were stimulated at 1 h and then decreased to basal levels thereafter. The highest stimulation (6.4-fold) by PTH is observed in the differentiation stage, and the lowest (2.7-fold) is seen in the mineralization stage. It is possible that the osteocyte is also able to produce MCP-1, but these cells would make a minor contribution in the region of the femora (primary spongiosa) analyzed. PTH Stimulation of MCP-1 Is Dose-dependent via the PKA Pathway and Is a Primary Response—We observed significant increases in MCP-1 mRNA in UMR cells at low doses of PTH (10-10 m) when we performed a dose-response experiment with PTH-(1-34). Although 10-10 m PTH was sufficient to increase MCP-1 mRNA significantly (p < 0.05), maximal effects were observed with 10-8 m PTH, and half-maximal stimulation was calculated to occur with 5 × 10-9 m PTH (Fig. 3A). PTH signals through both PKA and PKC pathways after binding with its receptor, PTH1R, on the osteoblast membrane (22Civitelli R. Reid I.R. Westbrook S. Avioli L.V. Hruska K.A. Am. J. Physiol. 1988; 225: E660-E667Google Scholar, 23Partridge N.C. Kemp B. Veroni M.C. Martin T.J. Endocrinology. 1981; 108: 220-225Crossref PubMed Scopus (143) Google Scholar). To study which pathway PTH uses to regulate MCP-1, we took advantage of different peptide fragments of PTH that activate different pathways as well as inhibitors and activators of these pathways. As shown in Fig. 3C, hPTH-(1-31) (which activates PKA but not PKC) (24Chakravarthy B.R. Durkin J. Rixon R.H. Whitfield J.F. Biochem. Biophys. Res. Commun. 1990; 171: 1105-1110Crossref PubMed Scopus (50) Google Scholar, 25Sabatini M. Lesur C. Pacherie M. Pastoureau P. Kucharczyk N. Fauchere J.L. Bonnet J. Bone (Elmsford). 1996; 18: 59-65Crossref PubMed Scopus (37) Google Scholar) retained the ability to stimulate MCP-1 mRNA levels after a 1-h treatment of UMR 106-01 cells (5.7-fold increase compared with 6-fold increase by rPTH-(1-34)). However, bovine PTH-(3-34) (which activates PKC but not PKA) (25Sabatini M. Lesur C. Pacherie M. Pastoureau P. Kucharczyk N. Fauchere J.L. Bonnet J. Bone (Elmsford). 1996; 18: 59-65Crossref PubMed Scopus (37) Google Scholar) failed to regulate the expression of MCP-1. In comparison, 8-bromo-cyclic AMP, a cell-permeable cAMP analog, stimulated the expression of MCP-1 about 5-fold, whereas PMA, an activator of the PKC pathway, had no effect. Consistent with these data, in the presence of the PKA inhibitor H-89, but not in the presence of the PKC inhibitor GF109203X, rPTH-(1-34) lost its ability to induce MCP-1, suggesting that this regulation is PKA-dependent but not PKC-dependent in osteoblastic cells. To determine whether PTH induction of MCP-1 is a primary response, UMR 106-01 cells were treated with rPTH-(1-34) (10-8 m) in the presence or the absence of cycloheximide. Fig. 3B shows that cycloheximide had no effect on the PTH induction of MCP-1, suggesting that this process does not require de novo protein synthesis (i.e. this effect is a primary response). Both MCP-1- and PTH-treated UMR Cell Line Conditioned Media Have Chemotactic Effects on Pre/Osteoclasts—Because the expression of the C-C motif chemokine receptor-2 and -4 (CCR2 and CCR4, the receptors for MCP-1) could not be detected by real time RT-PCR in osteoblastic cells (data not shown), we studied the effect of MCP-1 on osteoclasts and their precursors. First, murine macrophage RAW 264.7 cells were used, which can differentiate into osteoclastic cells. RAW cells respond to RANKL stimulation in vitro to generate multinucleated osteoclasts (RAW osteoclasts), a hallmark characteristic of fully differentiated osteoclasts (26Hsu H. Lacey D. Dunstan C.R. Solovyev I. Colombero A. Timms E. Tan H.L. Elliott G. Sarosi I. Wang L. Xia X.Z. Elliott R. Chiu L. Black T. Scully S. Capparelli C. Morony S. Shimamoto G. Bass M.B. Boyle W.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3540-3545Crossref PubMed Scopus (1398) Google Scholar). RAW cells do not require M-CSF as a permissive factor in RANKL-induced formation of osteoclast differentiation and function (19Collin-Osdoby P. Yu X. Zheng H. Osdoby P. Methods Mol. Med. 2003; 80: 153-166PubMed Google Scholar, 26Hsu H. Lacey D. Dunstan C.R. Solovyev I. Colombero A. Timms E. Tan H.L. Elliott G. Sarosi I. Wang L. Xia X.Z. Elliott R. Chiu L. Black T. Scully S. Capparelli C. Morony S. Shimamoto G. Bass M.B. Boyle W.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3540-3545Crossref PubMed Scopus (1398) Google Scholar, 27Yamamoto A. Miyazaki T. Kadono Y. Takayanagi H. Miura T. Nishina H. Katada T. Wakabayashi K. Oda H. Nakamura K. Tanaka S. J. Bone Miner. Res. 2002; 17: 612-621Crossref PubMed Scopus (76) Google Scholar), so the cells are only treated with RANKL to differentiate them into mature osteoclasts. In Fig. 4A, both recombinant MCP-1- and PTH-treated UMR cell conditioned media have strong chemotactic effects on RAW cells without RANKL treatment. From Fig. 4D we can see that the chemotactic effects of MCP-1 are blocked by the presence of a neutralizing antibody. More importantly, the MCP-1 antibody also significantly decreased the chemotactic potency of PTH-treated UMR cell culture media. Furthermore, we extended our study using osteoclast precursor cells isolated from mouse long bones to confirm the chemotactic effect on primary osteoclastic cultures. Recombinant MCP-1- and PTH-treated UMR cell culture media similarly showed strong chemotactic effects on primary osteoclast precursors (Fig. 4B). Taken together, the results indicate that MCP-1 has a strong chemotactic effect on pre-osteoclast cells, and MCP1 is likely a major chemotactic factor in PTH-treated UMR media, which causes pre-osteoclast migration. RAW cells and the pre-osteoclasts from mouse long bone were induced to differentiate by treatment with RANKL for 6 days. The osteoclasts were used in chemotactic assays. As shown in Fig. 4C, MCP-1- and PTH-treated UMR culture media also demonstrated a chemotactic effect on these differentiated multinucleated cells. Overall, the results indicate that MCP-1 has chemotactic effects on both the mature osteoclast and its precursor cells. MCP-1 Enhances RANKL-induced Osteoclastogenesis and Fusion—We found that exogenous MCP-1 positively stimulated osteoclast differentiation. Adding MCP-1 to the standard M-CSF and RANKL regime of primary osteoclast cultures for 6 days (with fresh cytokines fed every other day) significantly increased TRAP mRNA expression dose-dependently (50 and 100% increases with MCP-1 of 50 and 100 ng/ml specifically) (Fig. 5A). The effect of MCP-1 to facilitate pre-osteoclast differentiation was quite remarkable, with significantly increased number of nuclei in the TRAP-positive cells as well as fusion resulting in morphologically larger multinucleated osteoclasts (Fig. 5, B and D) when compared with cultures without addition of MCP-1. MCP-1 Increases Osteoclastic Mineral Dissolution in Vitro—More importantly, by using BD Biocoat osteologic disks with cultured mouse bone macrophages, we found MCP-1 significantly increased the dissolved areas at concentrations of both 50 and 100 ng/ml (Fig. 5, C and E). The mineral dissolution activity was increased by 2- and 3.5-fold with addition of 50 and 100 ng/ml of MCP-1. Parathyroid hormone is now accepted to have anabolic effects when injected intermittently; in fact, it is a highly effective treatment for osteoporosis. When given this way, PTH not only increases bone formation but also promotes bone remodeling (28Neer R.M. Arnaud C.D. Zanchetta J.R. Prince R. Gaich G.A. Reginster J.Y. Hodsman A.B. Eriksen E.F. Ish-Shalom S. Genant H.K. Wang O. Mitlak B.H. N. Engl. J. Med. 2001; 344: 1434-1441Crossref PubMed Scopus (3781) Google Scholar). Both clinical and animal studies have shown that bisphosphonates, inhibitors of bone resorption, can blunt the anabolic response to PTH, suggesting that active bone resorption enhances the anabolic actions of PTH (1Delmas P.D. Vergnaud P. Arlot M.E. Pastoureau P. Meunier P.J. Nilssen M.H. Bone (Elmsford). 1995; 16: 603-610Crossref PubMed Scopus (146) Google Scholar, 2Black D.M. Greenspan S.L. Ensrud K.E. Palermo L. McGowan J.A. Lang T.F. Garnero P. Bouxsein M.L. Bilezikian J.P. Rosen C.J. N. Engl. J. Med. 2003; 349: 1207-1215Crossref PubMed Scopus (1007) Google Scholar, 3Finkelstein J.S. Hayes A. Hunzelman J.L. Wyland J.J. Lee H. Neer R.M. N. Engl. J. Med. 2003; 349: 1216-1226Crossref PubMed Scopus (714) Google Scholar). Bone resorption requires the activation of the pre-osteoclast to form mature osteoclasts. This involves migration, expression of certain molecular markers, and the increas