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Interleukin-1β and tumor necrosis factor-α, but not interleukin-6, stimulate osteoprotegerin ligand gene expression in human osteoblastic cells
Bone Vol. 25, No. 3 September 1999:255–259
RAPID COMMUNICATION
Interleukin-1 and Tumor Necrosis Factor-␣, But Not Interleukin-6, Stimulate Osteoprotegerin Ligand Gene Expression in Human Osteoblastic Cells L. C. HOFBAUER,1 D. L. LACEY,2 C. R. DUNSTAN,2 T. C. SPELSBERG,3 B. L. RIGGS,1 and S. KHOSLA1 1
Endocrine Research Unit and 3Department of Biochemistry and Molecular Biology, Mayo Clinic and Mayo Foundation, Rochester, MN, USA Amgen Inc., Thousand Oaks, CA, USA
2
factor (TNF) ligand family.1,20,36,37 Independent in vitro studies have demonstrated that OPG-L, in the presence of macrophagestimulating factor (M-CSF), is both necessary and sufficient for complete osteoclastogenesis from multipotential osteoclast precursor cells to fully committed, mature osteoclasts.20,26,37 Moreover, OPG-L administration to normal mice resulted in enhanced osteoclastogenesis, severe osteoporosis, and malignant hypercalcemia in vivo.20 The soluble receptor, osteoprotegerin (OPG), a member of the TNF receptor (TNF-R) superfamily,19,28,30,38 neutralizes the effects of OPG-L.8,20,26,37 Overexpression of OPG in transgenic mice resulted in osteopetrosis (generalized increased bone mass), and the administration of OPG to normal animals prevented ovariectomy-induced bone loss.28 In contrast, targeted ablation of the OPG gene in knockout mice resulted in unopposed actions of OPG-L, and to early-onset, severe osteoporosis.5,23 Because OPG-L, in the presence of M-CSF, is the only cytokine required for osteoclastogenesis in vitro, and genetic manipulation of this system resulted in both extremes of skeletal phenotype (osteopetrosis, osteoporosis) in vivo, it has been suggested that other calcitropic hormones and cytokines may regulate osteoclastogenesis and bone resorption by modulating the expression of OPG-L and OPG by osteoblastic cells.11 Interleukin-1 (IL-1), tumor necrosis factor-␣ (TNF-␣), and IL-6 are key bone-resorbing cytokines that have also been implicated in the pathogenesis of postmenopausal bone loss.15,22,25 Although these cytokines clearly increase osteoclastogenesis,15,22,25 it is unclear whether these effects are mediated by increased OPG-L production by cells in the bone microenvironment or are independent of them. To test this, we assessed the effects of these cytokines on OPG-L mRNA steady-state levels by northern analysis in human osteoblastic and bone marrow stromal cells.
Recent studies have identified osteoprotegerin ligand (OPG-L) as the essential factor required for osteoclastogenesis, and that the effects are prevented by its soluble receptor, osteoprotegerin (OPG). However, there are limited data at present on the regulation of OPG-L expression in human osteoblastic cells by other cytokines. Because interleukin (IL)-1, tumor necrosis factor (TNF)-␣, and IL-6 all increase osteoclastogenesis, we assessed whether OPG-L mRNA steady-state levels were regulated by these cytokines in human osteoblastic cells. By northern analysis, IL-1 (5 nmol/L) and TNF-␣ (9 nmol/L) increased OPG-L mRNA steady-state levels by up to two- to three-fold in normal marrow stromal cells (MS), an immortalized marrow stromal cell line (hMS), and the osteosarcoma cell line, MG-63, whereas IL-6 (2 nmol/L, with or without its soluble receptor) had no effect on OPG-L mRNA levels in any of these cells. IL-1 and TNF-␣ increased OPG-L mRNA steady-state levels in the normal MS cells and the hMS cell line in a timeand dose-dependent fashion by up to 4.1-fold and up to 2.6-fold, respectively. Our data are thus consistent with the hypothesis that the proinflammatory and bone-resorbing cytokines, IL-1 and TNF-␣, but not IL-6, may stimulate osteoclastogenesis by inducing the expression of OPG-L. (Bone 25:255–259; 1999) © 1999 by Elsevier Science Inc. All rights reserved. Key Words: Interleukin (IL)-1; IL-6; Osteoblast; Osteoprotegerin; Osteoprotegerin ligand; Tumor necrosis factor-␣ (TNF-␣). Introduction Enhanced osteoclastogenesis is a hallmark of various forms of metabolic bone diseases, including postmenopausal and glucocorticoid-induced osteoporosis, and leads to increased bone resorption and bone loss. In the bone microenvironment, osteoclast differentiation is regulated by the coordinated synthesis and action of cytokines produced by bone marrow stromal cells and osteoblasts.22,25 Recently, osteoprotegerin ligand (OPG-L) has been identified as a novel member of the tumor necrosis
Materials and Methods Materials Culture flasks and dishes were obtained from Corning (Corning, NY). Cell culture medium and supplements were purchased from Sigma (St. Louis, MO). The random primer labeling kit (Decaprime II) was from Ambion (Austin, TX) and [␣-32P]-dCTP was from DuPont-NEN (Boston, MA). The human -actin cDNA insert and ExpressHyb solution were obtained from Clontech (Palo Alto, CA). Recombinant human TNF-␣, IL-1, IL-6, and
Address for correspondence and reprints: Sundeep Khosla, M.D., Mayo Clinic and Mayo Foundation, Joseph 5-194, 200 First Street SW, Rochester, MN 55905. E-mail: [email protected] © 1999 by Elsevier Science Inc. All rights reserved.
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IL-6 soluble receptor (IL-6sR) were from R&D Systems (Minneapolis, MN). Cell Cultures The following human osteoblastic cells were used: (i) a conditionally immortalized bipotential marrow stromal cell line (hMS)9; (ii) normal marrow stromal cells (MS) obtained by bone marrow aspiration in healthy subjects16; and (iii) the human osteosarcoma cell line, MG-63, obtained from the American Tissue Culture Collection. The MS cells were obtained following approval by our institutional review board. All cells were maintained in phenol-free medium supplemented with 10% fetal calf serum (FCS), and were grown in serum-free medium supplemented with 0.125% (w/v) bovine serum albumin (BSA) for 2 days prior to RNA isolation. The conditionally immortalized cell line, hMS, proliferates at 33.5°C (the permissive temperature, when the temperature-sensitive mutant SV-40 large T antigen is active) and differentiates at 39.5°C (the restrictive temperature, when the SV-40 large T antigen is inactive).9 The hMS cell line was studied at 39.5°C, when these cells are essentially a clonal population of normal marrow stromal cells, and the MS cells and the MG-63 osteosarcoma cells were grown at 37°C. RNA Isolation and Northern Blot Analysis Total RNA was isolated from cell cultures using the RNeasy kit and the QiaShredder from Qiagen (Hilden, Germany). Poly-A⫹ RNA was isolated using the PolyATract mRNA kit from Promega (Madison, WI). Poly-A⫹ RNA (0.3–2.0 g) was separated on a 1.5% (w/v) agarose/formaldehyde gel21 and then transferred to a nylon membrane (Hybond N⫹, Amersham, Arlington Heights, IL) by capillary blotting.31 The human cDNA inserts, an OPG-L cDNA that hybridized to a 2.4 kb mRNA,20 a full-length OPG cDNA that hybridized to a 2.9 kb mRNA,12 and a -actin cDNA that hybridized to a 2.0 kb mRNA were radiolabeled by random primer labeling.7 Hybridization and stringent washing were carried out as reported elsewhere.12 Control hybridization with human -actin cDNA verified that equal amounts of RNA were loaded. All experiments were carried out at least three times, band intensity was quantified by densitometry, and the data of representative blots are shown as the gene:-actin ratio.
Figure 1. Northern blot analysis of the regulation of OPG-L and OPG mRNA steady-state levels by cytokines in the osteosarcoma cell line, MG-63. One microgram of poly-A⫹ RNA was isolated from MG-63 cells (grown at 37°C in serum-free medium ⫹ 0.125% [w/v] BSA) with various agents for 12 h and analyzed by northern blot hybridization (C, control; IL-1, interleukin-1 [5 nmol/L]; TNF, tumor necrosis factor-␣ [9 nmol/L]; or IL-6, interleukin-6 [2 nmol/L] in the absence (⫺) or presence (⫹) of IL-6 soluble receptor [2 nmol/L]. Upper panel: OPG-L mRNA (2.4 kb); middle panel: OPG mRNA (2.9 kb); lower panel: control hybridization with the housekeeping gene -actin (2.0 kb). The number underneath the OPG-L and OPG bands indicates the gene:-actin ratio as compared with control (normalized to 1.0).
treatment of MS cells with IL-1 occurred in a dose-dependent fashion with a maximum effect (4.1-fold increase) at a concentration of 5 nmol/L (Figure 3A). The time course experiment indicated an increase of OPG-L mRNA steady-state levels as early as 6 h following the exposure of the cells to IL-1 (5 nmol/L) with a maximum after 12 h (Figure 3B). In addition, TNF-␣ treatment for 12 h also stimulated OPG-L mRNA steadystate levels by the hMS cell line in a dose- and time-dependent fashion, by 2.6-fold (Figure 4A,B). Various doses of IL-6 with or without IL-6sR had no effect on OPG-1 mRNA steady-state levels in any osteoblastic cell system.
Results IL-1 (5 nmol/L) and TNF-␣ (9 nmol/L) increased OPG-L mRNA steady-state levels (a single OPG-L mRNA species of 2.4 kb) by 2.2-fold and 1.9-fold, respectively, in the differentiated osteosarcoma cell line, MG-63 (Figure 1, lanes 2 and 3). In contrast, IL-6 (nmol/L) had no effect on OPG-L mRNA steadystate levels either without or with IL-6sR, which may be required for appropriate IL-6 action (Figure 1, lanes 4 and 5).24 Rehybridization of the membrane with the radiolabeled OPG cDNA probe demonstrated upregulation of OPG mRNA steady-state levels by IL-1 and TNF-␣, but not by IL-6, indicating that the proinflammatory cytokines (IL-1, TNF-␣) induce both OPG-L and OPG mRNA steady-state levels. In the hMS cell line, IL-1 (5 nmol/L) and TNF-␣ (9 nmol/L) also increased OPG-L mRNA steady-state levels, by 2.5-fold and 2.1-fold, respectively, whereas IL-6 (2 nmol/L) had no effect (Figure 2). Addition of IL-6sR to IL-6 did not result in any detectable effects on OPG-L mRNA in the hMS cell line. A similar increase of OPG-L mRNA steady-state levels was also observed in the primary bone marrow stromal cells following treatment with IL-1 and TNF-␣, but not with IL-6 (data not shown). The increase of OPG-L mRNA steady-state levels following
Figure 2. Northern blot analysis of the regulation of OPG-L mRNA steady-state levels by cytokines in the human immortalized marrow stromal cell line (hMS). Poly-A⫹ RNA (0.5 g) was isolated from the human immortalized marrow stromal cell line (hMS) grown at 39.5°C in serum-free medium ⫹ 0.125% (w/v) BSA with various agents for 12 h and analyzed by northern blot hybridization (C, control; IL-1, interleukin-1 [5 nmol/L]; TNF, tumor necrosis factor-␣ [9 nmol/L]; IL-6, interleukin-6 [2 nmol/L]). Upper panel: OPG-L mRNA (2.4 kb); lower panel: control hybridization with the housekeeping gene -actin (2.0 kb). The number underneath the -actin bands indicates the OPG-L:-actin ratio as compared with control (normalized to 1.0).
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Figure 3. Northern blot analysis of the regulation of OPG-L mRNA by IL-1 in primary marrow stromal cells. (A) Dose response. (B) Time course. Poly-A⫹ RNA (2 g) was isolated from primary marrow stromal cells, obtained by bone marrow aspiration, that were grown at 37°C in serum-free medium ⫹ 0.125% (w/v) BSA. (A) Dose response: cells were treated with IL-1 (various concentrations: 0 pmol/L, 50 pmol/L, 500 pmol/L, 5 nmol/L) for 12 h. (B) Time course: cells were treated with IL-1 (5 nmol/L) for the time (in hours) indicated. Upper panel: OPG-L mRNA (2.4 kb); lower panel: control hybridization with the housekeeping gene -actin (2.0 kb). The number underneath the -actin bands indicates the OPG-L:-actin ratio as compared with their respective controls (normalized to 1.0).
Discussion The balance between bone resorption and bone formation is regulated by an integrated network of cytokines that includes macrophage colony-stimulating factor (M-CSF), IL-1, IL-4, IL-6, IL-11, prostaglandin estradiol (PGE2), TNF-␣, and transforming growth factor- (TGF-), which are synthesized by bone marrow stromal cells and osteoblasts and which modulate osteoclast differentiation and activity.15,22,25 Although there is consensus about the role of this cytokine network, the importance of one of these cytokines relative to the others on osteoclastogenesis has remained unclear.15,22,25 We report in the present study that the proinflammatory and bone-resorbing cytokines,
Figure 4. Northern blot analysis of the regulation of OPG-L mRNA by TNF-␣ in a human immortalized stromal cell line (hMS). (A) Dose response. (B) Time course. One (A) or 0.3 (B) micrograms of poly-A⫹ RNA was isolated from the human immortalized stromal cell line (hMS) grown at 39.5°C in serum-free medium ⫹ 0.125% (w/v) BSA. (A) Dose response: cells were treated with TNF-␣ (various concentrations: 0 pmol/L, 900 pmol/L, 9 nmol/L) for 12 h. (B) Time course: cells were treated with TNF-␣ (9 nmol/L) for the time (in hours) indicated. Upper panel: OPG-L mRNA (2.4 kb); lower panel: control hybridization with the housekeeping gene -actin (2.0 kb). The number underneath the -actin bands indicates the OPG-L:-actin ratio as compared with their respective controls (normalized to 1.0).
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IL-1 and TNF-␣, but not IL-6, upregulate OPG-L mRNA steady-state levels in various human osteoblastic cell systems. The stimulation by IL-1 and TNF-␣ of OPG-L mRNA steadystate levels was detected in all human osteoblastic cell systems, including the immortalized adult bone marrow stromal cell line (hMS), primary bone marrow stromal cells (MS), and the osteosarcoma cells line, MG-63, indicating a consistent regulation regardless of their stage of differentiation, osteoblastic phenotype, absolute constitutive OPG-L mRNA steady-state levels, and the presence of the SV-40 large T antigen. The stimulation was substantial in magnitude (three- to four-fold) and was cytokine dose- and time-dependent, suggesting that these effects are physiologically relevant. In this study, we did not assess OPG-L regulation at the protein level, because thus far no antibodies are available for ELISA or western analysis. OPG-L1,20,36,37 and OPG19,28,30,38 have recently been identified as the pivotal paracrine system regulating osteoclastogenesis, bone resorption, and bone remodeling. The OPG-L/OPG system is likely to be more important for osteoblast-osteoclast interactions than other cytokines for several reasons: First, OPG-L is produced by cells of the osteoblastic lineage, and, in the presence of the permissive factor M-CSF, obviates the need for other cytokines to initiate and complete osteoclastogenesis in vitro.20,26,37 Second, in contrast to other calcitropic cytokine systems, the OPG-L/OPG system is unique because both of the two extremes of skeletal phenotypes (osteopetrosis and osteoporosis) could be generated by overexpression vs. targeted ablation of the soluble receptor, OPG.5,23,28 Third, OPG has been shown to be upregulated by bone morphogenetic protein-2,12 1,25dihydroxyvitamin D3,12 and the antiresorptive factors TGF-29 and estrogen,14 and downregulated by the bone-resorbing agents, glucocorticoids13,34 and PGE2.3 In addition, more recent studies have also demonstrated the upregulation of OPG-L mRNA steady-state levels in osteoblastic lineage cells by 1,25-dihydroxyvitamin D3,37 IL-11,37 parathyroid hormone,37 PGE2,37 and dexamethasone,13 and the downregulation by TGF-.29 Finally, the bone-resorbing effects of some of the factors that stimulate OPG-L mRNA steady-state levels (1,25-dihydroxyvitamin D3, parathyroid hormone, PGE2) could be prevented by cotreatment with OPG.32 In further support of this are the findings of a recent report that mice with a targeted ablation of the OPG-L gene have osteopetrosis and lack mature osteoclasts.18 Thus, our data, along with these lines of evidence, indicate that the OPG-L/OPG system may be the final and common pathway for mediating the effects of other proinflammatory and bone-resorbing cytokines on osteoclastogenesis and bone resorption. Of note, IL-6 had no effect on OPG-L and OPG mRNA steady-state levels. A recent study indicated that IL-6 may regulate osteoclastogenesis independent of OPG-L.10 In mice with targeted ablation of NF-B1 and NF-B2, transcription factors required for OPG-L signaling, no osteoclastogenesis occurred in cultured spleen cells following treatment with OPG-L, M-CSF, IL-1, and TNF-␣. However, IL-6 (in the presence of IL-6sR) induced osteoclast formation in NF-B double knockout mice (6% of that induced by OPG-L in wildtype mice), suggesting an OPG-L-independent signaling pathway.10 IL-1 and TNF-␣ also stimulate other bone-resorbing cytokines, including IL-622,25 and IL-11.27 Moreover, the production of IL-1 and TNF-␣ by marrow stromal cells is also regulated by other hormones and cytokines,25 and is increased in estrogen deficiency, and suppressed by estrogen treatment.17 Of note, as demonstrated in the present study and in previous studies,4,12,35 TNF-␣ and - and IL-1␣ and - have been shown to upregulate both gene expression4,12,35 and protein production of OPG12 (which neutralizes the proresorptive activity of OPG-L) by hu-
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man osteoblastic lineage cells. Thus, the effect of these cytokines in stimulating osteoclastogenesis may depend on the ratio of OPG-L:OPG generated in the bone microenvironment. A similar situation appears to exist in the case of other cytokines and their endogenous antagonists, including IL-1, soluble IL-1 receptor, IL-1 receptor antagonist,6,33 and matrix metalloproteinases and tissue inhibitor of metalloproteinases.2 Regulation of the ratio agonist:antagonist may represent another level of regulation in these complex systems. In the case of the OPG-L/OPG system, it appears that TNF-␣ and IL-1 induce parallel, rather than reciprocal, changes of both components. It should be noted, however, that while mRNA levels of both OPG-L and OPG increased following stimulation by TNF-␣ and IL-1, the net effect of these changes on osteoclastogenesis clearly depends on the biological dose responses to these agents as well as on other issues such as relative changes in the respective protein concentrations. Clearly, further studies are needed to address these issues. In conclusion, we find that IL-1 and TNF-␣, but not IL-6, stimulate steady-state mRNA levels of OPG-L, which is a critical factor for osteoclastogenesis in various human osteoblastic lineage cells. These data suggest that an increase in OPG-L production following exposure of osteoblastic lineage cells to IL-1 and TNF-␣ may provide an important paracrine mechanism whereby these proinflammatory cytokines stimulate osteoclastogensesis, thus promoting bone resorption and bone loss in vivo.
Acknowledgments: The authors acknowledge the technical assistance of M. J. Schroeder, B. Ngo, and R. A. Soderberg. This work was supported by Grant AG-04875 from the National Institutes of Health. Dr. Hofbauer is a recipient of a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (Ho 1875/1-1).
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¨ ., and Ohlsson, C. 35. Vidal, O. N. A., Sjo¨gren, K., Eriksson, B. I., Ljunggren, O Osteoprotegerin mRNA is increased by interleukin-␣ in the human osteosarcoma cell line MG-63 and in human osteoblast-like cells. Biochem Biophys Res Commun 248:696 –700; 1998. 36. Wong, B. R., Rho, J., Arron, J., Robinson, E., Orlinick, J., Chao, M., Kalachikov, S., Cayani, E., Bartlett, F. S., Frankel, W. N., Young Lee, S., and Choi, Y. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-jun N-terminal kinase in T cells. J Biol Chem 272: 25190 –25194; 1997. 37. Yasuda, H., Shima, N., Nakagawa, N., Yamaguchi, K., Kinosaki, M., Mochizuki, S.-I., Tomoyasu, A., Yano, K., Goto, M., Murakami, A., Tsuda, E., Morinaga, T., Higashio, K., Udagawa, N., Takahashi, N., and Suda, T. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95:3597–3602; 1998. 38. Yasuda, H., Shima, N., Nakagawa, N., Mochizuki, S.-I., Yano, K., Fujise, N., Sato, Y., Goto, M., Yamaguchi, K., Kuriyama, M., Kanno, T., Murakami, A., Tsuda, E., Morinaga, T., and Higashio, K. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): A mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 139:1329 – 1337; 1998.
Date Received: January 29, 1999 Date Revised: April 6, 1999 Date Accepted: May 5, 1999
RAPID COMMUNICATION
Interleukin-1 and Tumor Necrosis Factor-␣, But Not Interleukin-6, Stimulate Osteoprotegerin Ligand Gene Expression in Human Osteoblastic Cells L. C. HOFBAUER,1 D. L. LACEY,2 C. R. DUNSTAN,2 T. C. SPELSBERG,3 B. L. RIGGS,1 and S. KHOSLA1 1
Endocrine Research Unit and 3Department of Biochemistry and Molecular Biology, Mayo Clinic and Mayo Foundation, Rochester, MN, USA Amgen Inc., Thousand Oaks, CA, USA
2
factor (TNF) ligand family.1,20,36,37 Independent in vitro studies have demonstrated that OPG-L, in the presence of macrophagestimulating factor (M-CSF), is both necessary and sufficient for complete osteoclastogenesis from multipotential osteoclast precursor cells to fully committed, mature osteoclasts.20,26,37 Moreover, OPG-L administration to normal mice resulted in enhanced osteoclastogenesis, severe osteoporosis, and malignant hypercalcemia in vivo.20 The soluble receptor, osteoprotegerin (OPG), a member of the TNF receptor (TNF-R) superfamily,19,28,30,38 neutralizes the effects of OPG-L.8,20,26,37 Overexpression of OPG in transgenic mice resulted in osteopetrosis (generalized increased bone mass), and the administration of OPG to normal animals prevented ovariectomy-induced bone loss.28 In contrast, targeted ablation of the OPG gene in knockout mice resulted in unopposed actions of OPG-L, and to early-onset, severe osteoporosis.5,23 Because OPG-L, in the presence of M-CSF, is the only cytokine required for osteoclastogenesis in vitro, and genetic manipulation of this system resulted in both extremes of skeletal phenotype (osteopetrosis, osteoporosis) in vivo, it has been suggested that other calcitropic hormones and cytokines may regulate osteoclastogenesis and bone resorption by modulating the expression of OPG-L and OPG by osteoblastic cells.11 Interleukin-1 (IL-1), tumor necrosis factor-␣ (TNF-␣), and IL-6 are key bone-resorbing cytokines that have also been implicated in the pathogenesis of postmenopausal bone loss.15,22,25 Although these cytokines clearly increase osteoclastogenesis,15,22,25 it is unclear whether these effects are mediated by increased OPG-L production by cells in the bone microenvironment or are independent of them. To test this, we assessed the effects of these cytokines on OPG-L mRNA steady-state levels by northern analysis in human osteoblastic and bone marrow stromal cells.
Recent studies have identified osteoprotegerin ligand (OPG-L) as the essential factor required for osteoclastogenesis, and that the effects are prevented by its soluble receptor, osteoprotegerin (OPG). However, there are limited data at present on the regulation of OPG-L expression in human osteoblastic cells by other cytokines. Because interleukin (IL)-1, tumor necrosis factor (TNF)-␣, and IL-6 all increase osteoclastogenesis, we assessed whether OPG-L mRNA steady-state levels were regulated by these cytokines in human osteoblastic cells. By northern analysis, IL-1 (5 nmol/L) and TNF-␣ (9 nmol/L) increased OPG-L mRNA steady-state levels by up to two- to three-fold in normal marrow stromal cells (MS), an immortalized marrow stromal cell line (hMS), and the osteosarcoma cell line, MG-63, whereas IL-6 (2 nmol/L, with or without its soluble receptor) had no effect on OPG-L mRNA levels in any of these cells. IL-1 and TNF-␣ increased OPG-L mRNA steady-state levels in the normal MS cells and the hMS cell line in a timeand dose-dependent fashion by up to 4.1-fold and up to 2.6-fold, respectively. Our data are thus consistent with the hypothesis that the proinflammatory and bone-resorbing cytokines, IL-1 and TNF-␣, but not IL-6, may stimulate osteoclastogenesis by inducing the expression of OPG-L. (Bone 25:255–259; 1999) © 1999 by Elsevier Science Inc. All rights reserved. Key Words: Interleukin (IL)-1; IL-6; Osteoblast; Osteoprotegerin; Osteoprotegerin ligand; Tumor necrosis factor-␣ (TNF-␣). Introduction Enhanced osteoclastogenesis is a hallmark of various forms of metabolic bone diseases, including postmenopausal and glucocorticoid-induced osteoporosis, and leads to increased bone resorption and bone loss. In the bone microenvironment, osteoclast differentiation is regulated by the coordinated synthesis and action of cytokines produced by bone marrow stromal cells and osteoblasts.22,25 Recently, osteoprotegerin ligand (OPG-L) has been identified as a novel member of the tumor necrosis
Materials and Methods Materials Culture flasks and dishes were obtained from Corning (Corning, NY). Cell culture medium and supplements were purchased from Sigma (St. Louis, MO). The random primer labeling kit (Decaprime II) was from Ambion (Austin, TX) and [␣-32P]-dCTP was from DuPont-NEN (Boston, MA). The human -actin cDNA insert and ExpressHyb solution were obtained from Clontech (Palo Alto, CA). Recombinant human TNF-␣, IL-1, IL-6, and
Address for correspondence and reprints: Sundeep Khosla, M.D., Mayo Clinic and Mayo Foundation, Joseph 5-194, 200 First Street SW, Rochester, MN 55905. E-mail: [email protected] © 1999 by Elsevier Science Inc. All rights reserved.
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IL-6 soluble receptor (IL-6sR) were from R&D Systems (Minneapolis, MN). Cell Cultures The following human osteoblastic cells were used: (i) a conditionally immortalized bipotential marrow stromal cell line (hMS)9; (ii) normal marrow stromal cells (MS) obtained by bone marrow aspiration in healthy subjects16; and (iii) the human osteosarcoma cell line, MG-63, obtained from the American Tissue Culture Collection. The MS cells were obtained following approval by our institutional review board. All cells were maintained in phenol-free medium supplemented with 10% fetal calf serum (FCS), and were grown in serum-free medium supplemented with 0.125% (w/v) bovine serum albumin (BSA) for 2 days prior to RNA isolation. The conditionally immortalized cell line, hMS, proliferates at 33.5°C (the permissive temperature, when the temperature-sensitive mutant SV-40 large T antigen is active) and differentiates at 39.5°C (the restrictive temperature, when the SV-40 large T antigen is inactive).9 The hMS cell line was studied at 39.5°C, when these cells are essentially a clonal population of normal marrow stromal cells, and the MS cells and the MG-63 osteosarcoma cells were grown at 37°C. RNA Isolation and Northern Blot Analysis Total RNA was isolated from cell cultures using the RNeasy kit and the QiaShredder from Qiagen (Hilden, Germany). Poly-A⫹ RNA was isolated using the PolyATract mRNA kit from Promega (Madison, WI). Poly-A⫹ RNA (0.3–2.0 g) was separated on a 1.5% (w/v) agarose/formaldehyde gel21 and then transferred to a nylon membrane (Hybond N⫹, Amersham, Arlington Heights, IL) by capillary blotting.31 The human cDNA inserts, an OPG-L cDNA that hybridized to a 2.4 kb mRNA,20 a full-length OPG cDNA that hybridized to a 2.9 kb mRNA,12 and a -actin cDNA that hybridized to a 2.0 kb mRNA were radiolabeled by random primer labeling.7 Hybridization and stringent washing were carried out as reported elsewhere.12 Control hybridization with human -actin cDNA verified that equal amounts of RNA were loaded. All experiments were carried out at least three times, band intensity was quantified by densitometry, and the data of representative blots are shown as the gene:-actin ratio.
Figure 1. Northern blot analysis of the regulation of OPG-L and OPG mRNA steady-state levels by cytokines in the osteosarcoma cell line, MG-63. One microgram of poly-A⫹ RNA was isolated from MG-63 cells (grown at 37°C in serum-free medium ⫹ 0.125% [w/v] BSA) with various agents for 12 h and analyzed by northern blot hybridization (C, control; IL-1, interleukin-1 [5 nmol/L]; TNF, tumor necrosis factor-␣ [9 nmol/L]; or IL-6, interleukin-6 [2 nmol/L] in the absence (⫺) or presence (⫹) of IL-6 soluble receptor [2 nmol/L]. Upper panel: OPG-L mRNA (2.4 kb); middle panel: OPG mRNA (2.9 kb); lower panel: control hybridization with the housekeeping gene -actin (2.0 kb). The number underneath the OPG-L and OPG bands indicates the gene:-actin ratio as compared with control (normalized to 1.0).
treatment of MS cells with IL-1 occurred in a dose-dependent fashion with a maximum effect (4.1-fold increase) at a concentration of 5 nmol/L (Figure 3A). The time course experiment indicated an increase of OPG-L mRNA steady-state levels as early as 6 h following the exposure of the cells to IL-1 (5 nmol/L) with a maximum after 12 h (Figure 3B). In addition, TNF-␣ treatment for 12 h also stimulated OPG-L mRNA steadystate levels by the hMS cell line in a dose- and time-dependent fashion, by 2.6-fold (Figure 4A,B). Various doses of IL-6 with or without IL-6sR had no effect on OPG-1 mRNA steady-state levels in any osteoblastic cell system.
Results IL-1 (5 nmol/L) and TNF-␣ (9 nmol/L) increased OPG-L mRNA steady-state levels (a single OPG-L mRNA species of 2.4 kb) by 2.2-fold and 1.9-fold, respectively, in the differentiated osteosarcoma cell line, MG-63 (Figure 1, lanes 2 and 3). In contrast, IL-6 (nmol/L) had no effect on OPG-L mRNA steadystate levels either without or with IL-6sR, which may be required for appropriate IL-6 action (Figure 1, lanes 4 and 5).24 Rehybridization of the membrane with the radiolabeled OPG cDNA probe demonstrated upregulation of OPG mRNA steady-state levels by IL-1 and TNF-␣, but not by IL-6, indicating that the proinflammatory cytokines (IL-1, TNF-␣) induce both OPG-L and OPG mRNA steady-state levels. In the hMS cell line, IL-1 (5 nmol/L) and TNF-␣ (9 nmol/L) also increased OPG-L mRNA steady-state levels, by 2.5-fold and 2.1-fold, respectively, whereas IL-6 (2 nmol/L) had no effect (Figure 2). Addition of IL-6sR to IL-6 did not result in any detectable effects on OPG-L mRNA in the hMS cell line. A similar increase of OPG-L mRNA steady-state levels was also observed in the primary bone marrow stromal cells following treatment with IL-1 and TNF-␣, but not with IL-6 (data not shown). The increase of OPG-L mRNA steady-state levels following
Figure 2. Northern blot analysis of the regulation of OPG-L mRNA steady-state levels by cytokines in the human immortalized marrow stromal cell line (hMS). Poly-A⫹ RNA (0.5 g) was isolated from the human immortalized marrow stromal cell line (hMS) grown at 39.5°C in serum-free medium ⫹ 0.125% (w/v) BSA with various agents for 12 h and analyzed by northern blot hybridization (C, control; IL-1, interleukin-1 [5 nmol/L]; TNF, tumor necrosis factor-␣ [9 nmol/L]; IL-6, interleukin-6 [2 nmol/L]). Upper panel: OPG-L mRNA (2.4 kb); lower panel: control hybridization with the housekeeping gene -actin (2.0 kb). The number underneath the -actin bands indicates the OPG-L:-actin ratio as compared with control (normalized to 1.0).
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Figure 3. Northern blot analysis of the regulation of OPG-L mRNA by IL-1 in primary marrow stromal cells. (A) Dose response. (B) Time course. Poly-A⫹ RNA (2 g) was isolated from primary marrow stromal cells, obtained by bone marrow aspiration, that were grown at 37°C in serum-free medium ⫹ 0.125% (w/v) BSA. (A) Dose response: cells were treated with IL-1 (various concentrations: 0 pmol/L, 50 pmol/L, 500 pmol/L, 5 nmol/L) for 12 h. (B) Time course: cells were treated with IL-1 (5 nmol/L) for the time (in hours) indicated. Upper panel: OPG-L mRNA (2.4 kb); lower panel: control hybridization with the housekeeping gene -actin (2.0 kb). The number underneath the -actin bands indicates the OPG-L:-actin ratio as compared with their respective controls (normalized to 1.0).
Discussion The balance between bone resorption and bone formation is regulated by an integrated network of cytokines that includes macrophage colony-stimulating factor (M-CSF), IL-1, IL-4, IL-6, IL-11, prostaglandin estradiol (PGE2), TNF-␣, and transforming growth factor- (TGF-), which are synthesized by bone marrow stromal cells and osteoblasts and which modulate osteoclast differentiation and activity.15,22,25 Although there is consensus about the role of this cytokine network, the importance of one of these cytokines relative to the others on osteoclastogenesis has remained unclear.15,22,25 We report in the present study that the proinflammatory and bone-resorbing cytokines,
Figure 4. Northern blot analysis of the regulation of OPG-L mRNA by TNF-␣ in a human immortalized stromal cell line (hMS). (A) Dose response. (B) Time course. One (A) or 0.3 (B) micrograms of poly-A⫹ RNA was isolated from the human immortalized stromal cell line (hMS) grown at 39.5°C in serum-free medium ⫹ 0.125% (w/v) BSA. (A) Dose response: cells were treated with TNF-␣ (various concentrations: 0 pmol/L, 900 pmol/L, 9 nmol/L) for 12 h. (B) Time course: cells were treated with TNF-␣ (9 nmol/L) for the time (in hours) indicated. Upper panel: OPG-L mRNA (2.4 kb); lower panel: control hybridization with the housekeeping gene -actin (2.0 kb). The number underneath the -actin bands indicates the OPG-L:-actin ratio as compared with their respective controls (normalized to 1.0).
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IL-1 and TNF-␣, but not IL-6, upregulate OPG-L mRNA steady-state levels in various human osteoblastic cell systems. The stimulation by IL-1 and TNF-␣ of OPG-L mRNA steadystate levels was detected in all human osteoblastic cell systems, including the immortalized adult bone marrow stromal cell line (hMS), primary bone marrow stromal cells (MS), and the osteosarcoma cells line, MG-63, indicating a consistent regulation regardless of their stage of differentiation, osteoblastic phenotype, absolute constitutive OPG-L mRNA steady-state levels, and the presence of the SV-40 large T antigen. The stimulation was substantial in magnitude (three- to four-fold) and was cytokine dose- and time-dependent, suggesting that these effects are physiologically relevant. In this study, we did not assess OPG-L regulation at the protein level, because thus far no antibodies are available for ELISA or western analysis. OPG-L1,20,36,37 and OPG19,28,30,38 have recently been identified as the pivotal paracrine system regulating osteoclastogenesis, bone resorption, and bone remodeling. The OPG-L/OPG system is likely to be more important for osteoblast-osteoclast interactions than other cytokines for several reasons: First, OPG-L is produced by cells of the osteoblastic lineage, and, in the presence of the permissive factor M-CSF, obviates the need for other cytokines to initiate and complete osteoclastogenesis in vitro.20,26,37 Second, in contrast to other calcitropic cytokine systems, the OPG-L/OPG system is unique because both of the two extremes of skeletal phenotypes (osteopetrosis and osteoporosis) could be generated by overexpression vs. targeted ablation of the soluble receptor, OPG.5,23,28 Third, OPG has been shown to be upregulated by bone morphogenetic protein-2,12 1,25dihydroxyvitamin D3,12 and the antiresorptive factors TGF-29 and estrogen,14 and downregulated by the bone-resorbing agents, glucocorticoids13,34 and PGE2.3 In addition, more recent studies have also demonstrated the upregulation of OPG-L mRNA steady-state levels in osteoblastic lineage cells by 1,25-dihydroxyvitamin D3,37 IL-11,37 parathyroid hormone,37 PGE2,37 and dexamethasone,13 and the downregulation by TGF-.29 Finally, the bone-resorbing effects of some of the factors that stimulate OPG-L mRNA steady-state levels (1,25-dihydroxyvitamin D3, parathyroid hormone, PGE2) could be prevented by cotreatment with OPG.32 In further support of this are the findings of a recent report that mice with a targeted ablation of the OPG-L gene have osteopetrosis and lack mature osteoclasts.18 Thus, our data, along with these lines of evidence, indicate that the OPG-L/OPG system may be the final and common pathway for mediating the effects of other proinflammatory and bone-resorbing cytokines on osteoclastogenesis and bone resorption. Of note, IL-6 had no effect on OPG-L and OPG mRNA steady-state levels. A recent study indicated that IL-6 may regulate osteoclastogenesis independent of OPG-L.10 In mice with targeted ablation of NF-B1 and NF-B2, transcription factors required for OPG-L signaling, no osteoclastogenesis occurred in cultured spleen cells following treatment with OPG-L, M-CSF, IL-1, and TNF-␣. However, IL-6 (in the presence of IL-6sR) induced osteoclast formation in NF-B double knockout mice (6% of that induced by OPG-L in wildtype mice), suggesting an OPG-L-independent signaling pathway.10 IL-1 and TNF-␣ also stimulate other bone-resorbing cytokines, including IL-622,25 and IL-11.27 Moreover, the production of IL-1 and TNF-␣ by marrow stromal cells is also regulated by other hormones and cytokines,25 and is increased in estrogen deficiency, and suppressed by estrogen treatment.17 Of note, as demonstrated in the present study and in previous studies,4,12,35 TNF-␣ and - and IL-1␣ and - have been shown to upregulate both gene expression4,12,35 and protein production of OPG12 (which neutralizes the proresorptive activity of OPG-L) by hu-
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man osteoblastic lineage cells. Thus, the effect of these cytokines in stimulating osteoclastogenesis may depend on the ratio of OPG-L:OPG generated in the bone microenvironment. A similar situation appears to exist in the case of other cytokines and their endogenous antagonists, including IL-1, soluble IL-1 receptor, IL-1 receptor antagonist,6,33 and matrix metalloproteinases and tissue inhibitor of metalloproteinases.2 Regulation of the ratio agonist:antagonist may represent another level of regulation in these complex systems. In the case of the OPG-L/OPG system, it appears that TNF-␣ and IL-1 induce parallel, rather than reciprocal, changes of both components. It should be noted, however, that while mRNA levels of both OPG-L and OPG increased following stimulation by TNF-␣ and IL-1, the net effect of these changes on osteoclastogenesis clearly depends on the biological dose responses to these agents as well as on other issues such as relative changes in the respective protein concentrations. Clearly, further studies are needed to address these issues. In conclusion, we find that IL-1 and TNF-␣, but not IL-6, stimulate steady-state mRNA levels of OPG-L, which is a critical factor for osteoclastogenesis in various human osteoblastic lineage cells. These data suggest that an increase in OPG-L production following exposure of osteoblastic lineage cells to IL-1 and TNF-␣ may provide an important paracrine mechanism whereby these proinflammatory cytokines stimulate osteoclastogensesis, thus promoting bone resorption and bone loss in vivo.
Acknowledgments: The authors acknowledge the technical assistance of M. J. Schroeder, B. Ngo, and R. A. Soderberg. This work was supported by Grant AG-04875 from the National Institutes of Health. Dr. Hofbauer is a recipient of a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (Ho 1875/1-1).
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Date Received: January 29, 1999 Date Revised: April 6, 1999 Date Accepted: May 5, 1999