Transforming Growth Factor-β Controls Human Osteoclastogenesis through the p38 MAPK and Regulation of RANK Expression
2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês
10.1074/jbc.m303905200
ISSN1083-351X
AutoresM.A. Karsdal, Pernille Hjorth, Kim Henriksen, Tove Kirkegaard, Karina L. Nielsen, Henriette Lou, Jean‐Marie Delaissé, Niels T. Foged,
Tópico(s)Bone health and treatments
ResumoAlthough RANK-L is essential for osteoclast formation, factors such as transforming growth factor-β (TGF-β) are potent modulators of osteoclastogenic stimuli. To systematically investigate the role of TGF-β in human osteoclastogenesis, monocytes were isolated from peripheral blood by three distinct approaches, resulting in either a lymphocyte-rich, a lymphocyte-poor, or a pure osteoclast precursor (CD14-positive) cell population. In each of these osteoclast precursor populations, the effect of TGF-β on proliferation, TRAP activity, and bone resorption was investigated with respect to time and length of exposure. When using the highly pure CD14 osteoclast precursor cell population, the effect of TGF-β was strongly dependent on the stage of osteoclast maturation. When monocytes were exposed to TGF-β during the initial culture period (days 1–7), TRAP activity and bone resorption were increased by 40%, whereas the cell number was reduced by 25%. A similar decrease in cell number was observed when TGF-β was present during the entire culture period (days 1–21), but in direct contrast, TRAP activity, cell fusion, cathepsin K, and matrix metalloproteinase (MMP)-9 expression as well as bone resorption were almost completely abrogated. Moreover, we found that latent TGF-β was strongly activated by incubation with MMP-9 and suggest this to be a highly relevant mechanism for regulating osteoclast activity. To further investigate the molecular mechanism responsible for the divergent effects of continuous versus discontinuous exposure to TGF-β, we examined RANK expression and p38 MAPK activation. We found the TGF-β strongly induced p38 MAPK in monocytes, but not in mature osteoclasts, and that continuous exposure of TGF-β to monocytes down-regulated RANK expression. The current results suggest that TGF-β promotes human osteoclastogenesis in monocytes through stimulation of the p38 MAPK, whereas continuous exposure to TGF-β abrogates osteoclastogenesis through down-regulation of RANK expression and therefore attenuation of RANK-RANK-L signaling. Although RANK-L is essential for osteoclast formation, factors such as transforming growth factor-β (TGF-β) are potent modulators of osteoclastogenic stimuli. To systematically investigate the role of TGF-β in human osteoclastogenesis, monocytes were isolated from peripheral blood by three distinct approaches, resulting in either a lymphocyte-rich, a lymphocyte-poor, or a pure osteoclast precursor (CD14-positive) cell population. In each of these osteoclast precursor populations, the effect of TGF-β on proliferation, TRAP activity, and bone resorption was investigated with respect to time and length of exposure. When using the highly pure CD14 osteoclast precursor cell population, the effect of TGF-β was strongly dependent on the stage of osteoclast maturation. When monocytes were exposed to TGF-β during the initial culture period (days 1–7), TRAP activity and bone resorption were increased by 40%, whereas the cell number was reduced by 25%. A similar decrease in cell number was observed when TGF-β was present during the entire culture period (days 1–21), but in direct contrast, TRAP activity, cell fusion, cathepsin K, and matrix metalloproteinase (MMP)-9 expression as well as bone resorption were almost completely abrogated. Moreover, we found that latent TGF-β was strongly activated by incubation with MMP-9 and suggest this to be a highly relevant mechanism for regulating osteoclast activity. To further investigate the molecular mechanism responsible for the divergent effects of continuous versus discontinuous exposure to TGF-β, we examined RANK expression and p38 MAPK activation. We found the TGF-β strongly induced p38 MAPK in monocytes, but not in mature osteoclasts, and that continuous exposure of TGF-β to monocytes down-regulated RANK expression. The current results suggest that TGF-β promotes human osteoclastogenesis in monocytes through stimulation of the p38 MAPK, whereas continuous exposure to TGF-β abrogates osteoclastogenesis through down-regulation of RANK expression and therefore attenuation of RANK-RANK-L signaling. The skeleton is a dynamic tissue that is undergoing continuous remodeling to sustain calcium homeostasis, repair microfractures, and react to strain and stress of the skeleton. The remodeling process is a complex process and relies on the coupling between bone resorption and formation that involves osteoclasts, osteoblasts, and osteocytes. The constant regeneration of bone emphasizes the delicate balance between bone resorption and bone formation, which, if altered, may lead to pathological conditions such as osteoporosis or osteopetrosis. The investigation of the cellular actions of the major players of bone remodeling may therefore contribute significantly to the discovery of new and better drugs for the treatment of osteoporosis (1Karsdal M.A. Larsen L. Engsig M.T. Lou H. Ferreras M. Lochter A. Delaisse J.M. Foged N.T. J. Biol. Chem. 2002; 277: 44061-44067Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Of particular interest for the regulation of bone turnover is the growth factors and cytokines produced in the bone microenvironment and the signal events involved in their regulation of resorption. One of many important cellular events in bone remodeling is osteoclastic bone resorption, which is preceded by osteoclastogenesis and followed by apoptosis. The life, function, and death of osteoclasts are known to be influenced at many different levels by a variety of growth factors, including but not restricted to M-CSF, 1The abbreviations used are: M-CSF, macrophage colony-stimulating factor; R-M-CSF, M-CSF receptor; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; PBS, phosphate-buffered saline; CTX, C-terminal type I collagen fragment(s); ELISA, enzyme-linked immunosorbent assay; MMP, matrix metalloproteinase; MAPK, mitogen-activated protein kinase.1The abbreviations used are: M-CSF, macrophage colony-stimulating factor; R-M-CSF, M-CSF receptor; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; PBS, phosphate-buffered saline; CTX, C-terminal type I collagen fragment(s); ELISA, enzyme-linked immunosorbent assay; MMP, matrix metalloproteinase; MAPK, mitogen-activated protein kinase. vascular endothelial growth factor, parathyroid hormone, TGF-β, and tumor necrosis factor-α (2Lee S.E. Chung W.J. 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Most importantly, complex and heterogeneous cell culture systems make accurate assessment of the direct effect of TGF-β virtually impossible, since TGF-β stimulates, for example, osteoprotegerin and other factors in stromal cells that indirectly inhibit osteoclastogenesis (31Thirunavukkarasu K. Miles R.R. Halladay D.L. Yang X. Galvin R.J. Chandrasekhar S. Martin T.J. Onyia J.E. J. Biol. Chem. 2001; 276: 36241-36250Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 32Takai H. Kanematsu M. Yano K. Tsuda E. Higashio K. Ikeda K. Watanabe K. Yamada Y. J. Biol. Chem. 1998; 273: 27091-27096Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Thus, since TGF-β exerts pleiotropic effects on a large range of bone cells, this has hampered the molecular understanding of the exact role of this growth factor. The study of the direct effect of TGF-β on human osteoclastogenesis has been extremely limited because of technical difficulties with obtaining human osteoclasts in culture. However, with the recently established technique of peripheral blood monocyte purification followed by in vitro differentiation into mature osteoclasts, the possibility of investigating the direct effects of TGF-β on human osteoclastogenesis has been enabled. However, this approach results in a highly heterogeneous cell population consisting of, among other cells, T-cells, B-cells, and osteoclast precursors. This procedure has been further refined by the finding that the osteoclasts precursors reside in the CD14-positive population (33Shalhoub V. Elliott G. Chiu L. Manoukian R. Kelley M. Hawkins N. Davy E. Shimamoto G. Beck J. Kaufman S.A. Van G. Scully S. Qi M. Grisanti M. Dunstan C. Boyle W.J. Lacey D.L. Br. J. Haematol. 2000; 111: 501-512Crossref PubMed Scopus (105) Google Scholar, 34Nicholson G.C. 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We find that TGF-β modulates all of the osteoclastic phenotypes investigated (i.e. proliferation, fusion, TRAP activity, cathepsin K, and MMP-9 expression) as well as the resorptive activity itself. By further investigation of TGF-β-controlled osteoclastogenesis, we found that TGF-β potently induced p38 MAPK and regulated RANK expression, and we therefore present data suggesting how TGF-β can modulate human osteoclastogenesis. Cell Culture—Isolation of CD-14-positive monocytes with primary coated Dynabeads M-450 (111.11 Dynal Biotech) was as follows. Human monocytes were isolated from peripheral blood, obtained from healthy volunteers both male and female in the age range of 18–67 years. In the present studies, we did not observe differences between age and sex; however, the design was not optimized to investigate such effects. The blood was diluted 1:1 with PBS (catalog no. BE17-512F; Bio-Whittaker). The blood/PBS was carefully layered on the on Ficoll-Paque (catalog no. 17-1440-03; Amersham Biosciences). This gradient was centrifuged at 2000 rpm for 20 min. The lymphocytes were collected from the interface between the plasma and the Ficoll-Paque and washed with ice-cold PBS followed by centrifugation at 2000 rpm for 12 min. The wash was repeated twice, after which the cells were resuspended with cold PBS containing 2% serum (catalog no. S0415; Biochrom). The cells were kept on ice while the preparation of beads was done. For 300 million cells, 125 μl of beads (10 million beads) were used (Dynal Biotech). The tube was placed with the beads and ice-cold PBS in the magnetic device (Dynal Biotech) for 2 min, after which the supernatant was discarded. This wash was repeated three times. 300 million cells were added to the beads and incubated at 4 °C with end-over-end homogenization for 20 min. After the incubation at 4 °C, the tube was placed in the magnetic device for 2 min, after which the supernatant was discarded. Thereafter, 5 ml of PBS containing 2% serum was added, following which the beads were gently resuspended. The tube was placed in the magnetic device for 2 min. This wash was repeated at least five times. Finally, the cells were resuspended in α-minimal essential medium containing 10% serum, 100 units/ml penicillin, 100 μg/ml streptomycin. The cells were seeded in 75-cm2 bottles and cultured in α-minimal essential medium containing 10% serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 25 ng/ml M-CSF (catalog no. 216-MC; R&D Systems), 25 ng/ml Rank Ligand (catalog no. 310-01; Peprotech), or TRANCE (catalog no. 390-TN; R&D Systems). After 3 days, the cells were washed with PBS twice. Hereafter trypsin was added, and the cells and trypsin were incubated at 37 °C for approximately 20 min. The cells were scraped off and reseeded as indicated. The cells were cultured as described above in α-minimal essential medium containing 10% serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 25 ng/ml M-CSF (catalog no. 216-MC; R&D Systems), and 25 ng/ml Rank Ligand (catalog no. 310-01; Peprotech) or TRANCE (catalog no. 390-TN; R&D Systems). TGF-β1 (catalog no. 100-B-010; R&D Systems) was used when indicated at 10 ng/ml. To quantify the cell number, the AlamarBlue assay (Trek Diagnostics) was used according to the manufacturer's instructions. TRAP Assay—At the end of each culture period, TRAP activity was measured in the conditioned media by adding a colorimetric substrate, 6 mm p-nitrophenyl phosphate, in the presence of 25 mm sodium tartrate at pH 5.5. The reaction products were quantified by measuring optical absorbance at 405 nm. Enzyme Assays—Human pro-MMP-2 was a kind gift from Drs. F. Frankenne and J. M. Foidart (University of Liege, Belgium). Mouse pro-MMP-9 was purified from cell culture media of transformed baby hamster kidney cells, by means of two consecutive chromatographic steps on gelatin-Sepharose and concanavalin A-Sepharose columns (Amersham Biosciences) (47Morodomi T. Ogata Y. Sasaguri Y. Morimatsu M. Nagase H. Biochem. J. 1992; 285: 603-611Crossref PubMed Scopus (178) Google Scholar). Pro-MMPs were activated with 4-aminophenyl mercuric acetate. The molar concentration of active MMPs (48Knight C.G. Methods Enzymol. 1995; 248: 85-101Crossref PubMed Scopus (74) Google Scholar) was determined by using the MMP inhibitor BB94 (kindly provided by Dr. H. Van Wart; Roche Applied Science). Latent TGF was incubated with 4-aminophenyl mercuric acetate-activated MMP for 5 or 24 h unless otherwise indicated in a buffer containing 50 mm Tris-Cl, 0.15 m NaCl, 10 mm CaCl2, 50 μm ZnSO4, 0.05% Brij, pH 7.5). Activation of latent TGF-β was followed by Western blotting. Bone Resorption—Bone resorption was measured by formation of resorption pits. Peripheral blood mononuclear cells and CD14-positive cells were cultured for the indicated time periods before adherent cells were scraped gently off with a cotton swab and bone slices were washed in distilled water. Resorption pits were stained with Mayer's hematoxylin and subsequently washed. Resorbed bone area was measured using CAST-GRID software (Microsoft Corp., Olympus, WA), and results are expressed as resorbed bone area in percentage of total bone. The measurement of C-terminal type I collagen fragments (CTX) release from bone slices was performed by the CrossLaps for culture ELISA kit (Nordic Bioscience Diagnostics), which was used according to the manufacture's instructions. Cell Fusion—Cell fusion was determined of peripheral blood mononuclear cells and differentiated CD14-positive cells cultured for 7 days in the presence of factors as indicated. After culture, the cells were fixed in 3.7% formaldehyde and methanol, each for 5 min, and incubated in Wright-Giemsa solution (1% of Wright solution and 10% of Giemsa solution diluted in water) for 10 min at room temperature. Cell fusion, as measured by cells with three or more nuclei, was determined under a light microscope using CAST-GRID software (Microsoft). Western Blotting—Western blotting was performed on total cell lysates in radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.4, 30 mm NaCl, 5 mm EDTA, 1% Nonidet P40, 1% deoxycholic acid, 0.1% SDS) containing 10 mm NaF and 50 mm Na3VO4. Equal protein levels were determined and adjusted by the Bio-Rad DC protein assay according to the manufacturer's descriptions. Samples were resolved on 10% SDS-polyacrylamide gels and electroblotted onto nitrocellulose membranes (Bio-Rad). After blotting, equal and sufficient loading was investigated by Ponceau Red solution (catalog no. P-7170; Sigma) stainings of the membranes, after which the membranes were blocked overnight at 4 °C with TBS-T (50 mm Tris-HCl, pH 7.5, 0.1% Tween 20, 100 mm NaCl) containing 5% milk powder. Membranes were then incubated for 1 h at ambient temperature with antibodies against either MMP-9 (catalog no. MAB13418; Chemicon), cathepsin K (catalog no. MAB3324; Chemicon), p38 MAPK total form (catalog no. 9212; Cell signaling), p38 MAPK active form (catalog no. 9216; Cell Signaling), RANK (catalog no. AB1861; Chemicon), R-M-CSF (catalog no. AF329; R&D Systems), or TGF-β (catalog no. MAB240; R&D Systems). After washing vigorously with TBS-T for 1 h, membranes were incubated for 1 h at ambient temperature with horseradish peroxidase-conjugated antibodies (DAKO) and developed with an enhanced chemiluminescence kit (ECL™; Amersham Biosciences), according to the manufacturer's instructions. Statistics—All graphs show one representative experiment of at least three, each with four individual replications. All graphs show the mean of four replications and S.D. values. All statistical calculations have been performed by Student's two-tailed unpaired t test assuming normal distribution with equal variance. Statistical significance is given by the number of asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.01. The Effect of TGF-β in a Lymphocyte-rich Environment—To systematically investigate the role of TGF-β on human osteoclastogenesis, we isolated monocytes from peripheral blood by three distinct approaches, resulting in (i) lymphocyte rich osteoclast precursor population, (ii) a lymphocyte-poor osteoclast precursor population consisting only of adherent cells, and (iii) a pure osteoclast precursor population obtained by CD14-positive magnetic cell sorting technique. In each of these populations, we investigated the effects of TGF-β on proliferation, TRAP activity, fusion, and resorption. The lymphocyte-rich culture system is a highly heterogeneous population that contains, among other cell types, CD19 B-cells (9%), CD3 T-cells (70%), CD14 monocytes (14%), and CD56 natural killer cells (6%) (33Shalhoub V. Elliott G. Chiu L. Manoukian R. Kelley M. Hawkins N. Davy E. Shimamoto G. Beck J. Kaufman S.A. Van G. Scully S. Qi M. Grisanti M. Dunstan C. Boyle W.J. Lacey D.L. Br. J. Haematol. 2000; 111: 501-512Crossref PubMed Scopus (105) Google Scholar). This heterogeneous population has previously been used to investigate the effects of TGF-β on human osteoclastogenesis (25Massey H.M. Scopes J. Horton M.A. Flanagan A.M. Bone. 2001; 28: 577-582Crossref PubMed Scopus (56) Google Scholar). However, since approximately 85% of the monocyte fraction is not osteoclast precursors, the direct and indirect effects of TGF-β are impossible to distinguish. 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Therefore, to reproduce previous published results and to expand these with the time-dependent effects of TGF-β on osteoclastogenesis, we used the described lymphocyte-rich cell population and cultured these cells in the presence of RANK-L and M-CSF (both at 25 ng/ml) and exposed the cells to TGF-β (10 ng/ml) for the indicated time periods (Fig. 1). We found, as previously described (25Massey H.M. Scopes J. Horton M.A. Flanagan A.M. Bone. 2001; 28: 577-582Crossref PubMed Scopus (56) Google Scholar), that TGF-β stimulated bone resorption, namely by ∼400% measured by CTX analysis (Fig. 1C) and pit area measurements (data not shown). However, resorption was only stimulated when TGF-β was present in the initial part of the culture period (Fig. 1C). When TGF-β was present for a longer time or throughout the culture period, resorption was restored to control levels (Fig. 1C). Supporting this observation, TRAP activity followed the same pattern of modulation by TGF-β (Fig. 1B). Most interestingly, proliferation was just slightly and nonsignificantly increased when TGF-β was present at the initial culture period and just slightly and nonsignificantly decreased when present through the culture period (Fig. 1A).
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