IL-4 Inhibits Bone-Resorbing Activity of Mature Osteoclasts by Affecting NF-B and Ca2+ Signaling1
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免疫学杂志 2005年第14期
Abstract
IL-4 is an important immune cytokine that regulates bone homeostasis. We investigated the molecular mechanism of IL-4 action on bone-resorbing mature osteoclasts. Using a highly purified population of mature osteoclasts, we show that IL-4 dose-dependently inhibits receptor activator of NF-B ligand (RANKL)-induced bone resorption by mature osteoclasts. We detected the existence of IL-4R mRNA in mature osteoclasts. IL-4 decreases TRAP expression without affecting multinuclearity of osteoclasts, and inhibits actin ring formation and migration of osteoclasts. Interestingly, IL-4 inhibition of bone resorption occurs through prevention of RANKL-induced nuclear translocation of p65 NF-B subunit, and intracellular Ca2+ changes. Moreover, IL-4 rapidly decreases RANKL-stimulated ionized Ca2+ levels in the blood, and mature osteoclasts in IL-4 knockout mice are sensitive to RANKL action to induce bone resorption and hypercalcemia. Furthermore, IL-4 inhibits bone resorption and actin ring formation by human mature osteoclasts. Thus, we reveal that IL-4 acts directly on mature osteoclasts and inhibits bone resorption by inhibiting NF-B and Ca2+ signaling.
Introduction
Osteoclasts, the multinuclear cells (MNCs)3 responsible for bone resorption, play a crucial role in bone remodeling. The bone loss in many important skeletal disorders such as osteoporosis, rheumatoid arthritis, hypercalcemia of malignancy, and bone metastases occurs mainly because of increased osteoclast activity (1). A major breakthrough in understanding the regulation of osteoclastogenesis occurred after the discovery of novel molecules such as receptor activator of NF-B (RANK), RANK ligand (RANKL), and osteoprotegerin (2, 3, 4, 5, 6, 7). RANKL, in the presence of M-CSF, mediates osteoclastogenesis through binding to its receptor RANK on osteoclast precursors (5, 7). Transgenic and gene knockout studies in mice established the absolute dependency of osteoclast differentiation and activation on the expression of RANKL and RANK (7, 8). The distinct signaling pathways such as NF-B, JNK, p38, ERK, and Src pathways mediated by protein kinases are activated by RANKL during osteoclastogenesis and bone resorption (9).
RANKL also plays an important role in survival and activation of mature osteoclasts and rapidly induces actin ring formation (10, 11, 12). In vivo studies have shown that RANKL increases blood ionized Ca2+ levels within 1 h suggesting its direct effect on preexisting mature osteoclasts (11). Stimulation of RANK on mature osteoclasts by RANKL results in activation of transcriptional factor NF-B and Ca2+ signaling (13, 14). The study of molecular mechanisms by which cytokines secreted by T cells or other immune cells regulate bone-resorbing activity of mature osteoclasts had been limited because of difficulties in obtaining a sufficient number of mature osteoclasts. Induction of mature osteoclasts by RANKL in mouse and human osteoclast precursors is an excellent in vitro model to investigate the novel mechanisms of cytokines that regulate bone resorption.
IL-4 is a 19-kDa pleiotropic type I cytokine secreted by activated TH2 lymphocytes, mast cells, eosinophils, and basophils (15). IL-4, an important immune cytokine that regulates function of lymphocytes and macrophages, also regulates osteoclastogenesis and bone resorption (16, 17). Recent work has clarified the role and molecular mechanisms by which IL-4 inhibits RANKL-induced osteoclast differentiation in osteoclast precursors (18, 19, 20, 21). However, the mechanism of IL-4 action on mature osteoclasts and its function is not fully delineated. Moreno et al. (21) have reported the inhibitory effect of IL-4 on mouse mature osteoclast function and showed that the effect requires STAT6. In this study, we prepared a large number of highly purified mature osteoclasts induced by RANKL using both mice and human osteoclast precursors, and provide further advances that clarify in detail the mechanisms of IL-4 action on bone-resorbing mature osteoclasts. We show here that IL-4 acts directly on mature osteoclasts and significantly inhibits bone resorption and tartrate-resistant acid phosphatase (TRAP) expression. IL-4 inhibits bone resorption by disruption of RANKL-induced actin ring formation in mouse and as well as human mature osteoclasts. Furthermore, IL-4 prevents RANKL-induced nuclear translocation of p65 NF-B subunit, and intracellular Ca2+ changes in mature osteoclasts. In addition, RANKL-induced hypercalcemia in vivo is attenuated by IL-4 and accentuated by IL-4 deficiency. In conclusion, IL-4 acts directly on mature osteoclasts and inhibits bone resorption by inhibiting NF-B and Ca2+ signaling.
Results
IL-4 inhibits bone resorption by mature osteoclasts
Mature osteoclasts were prepared from osteoclast precursors as described in Materials and Methods and purified (>95% pure) by removing mononuclear cells using EDTA treatment. To examine the effect of IL-4 on bone resorption, purified mature osteoclasts on bone slices were further incubated for 48 h with M-CSF (30 ng/ml) and RANKL (30 ng/ml) with or without different concentrations of IL-4. As shown in Fig. 1A, IL-4 inhibited the RANKL-induced bone resorption in a dose-dependent manner. IL-4 (20 ng/ml) also significantly decreased the size of an individual resorption pit (Fig. 1, B and C). The inhibitory effect of IL-4 on bone resorption was confirmed by anti-mouse IL-4 Ab. As shown in Fig. 1D, simultaneous addition of anti-IL-4 Ab neutralized the inhibitory effect of IL-4 in a dose-dependent manner. These results suggest that IL-4 inhibits bone resorption by direct action on activated mature osteoclasts.
IL-4 effects depend upon binding to and signaling through a receptor complex consisting of the IL-4R chain and the common -chain (25). Receptors of IL-4 are expressed on a wide range of cells including hemopoietic cells (15). To examine whether mature osteoclasts express IL-4R, we incubated mature osteoclasts with M-CSF and RANKL and assessed for IL-4R mRNA expression. As shown in Fig. 1E, mature osteoclasts induced by RANKL on day 4 (0 h) expressed IL-4R, and cells further incubated for 24 and 48 h also showed strong expression of IL-4R.
IL-4 inhibits TRAP expression in mature osteoclasts
To investigate the mechanism of IL-4 action on activated mature osteoclasts, we first examined whether IL-4 inhibits bone resorption by inducing the apoptosis in mature osteoclasts. Osteoclasts were incubated for 48 h with M-CSF and RANKL in the absence or presence of various concentrations of IL-4. No apoptotic changes such as chromosome condensation, nuclear fragmentation were seen in the presence of IL-4 (data not shown). We then examined the effect of IL-4 on TRAP expression. As shown in Fig. 2A, IL-4 dose-dependently decreased TRAP expression, and the majority of MNCs were TRAP-negative and increased with increasing concentrations of IL-4 (Fig. 2B). These TRAP-negative MNCs did not express CTR (data not shown). Fig. 2C shows the effect of IL-4 on TRAP expression in mature osteoclasts. The TRAP-negative MNCs in the presence of IL-4 were fused to form giant cells with accumulation of large vacuoles. These results suggest that IL-4 inhibits expression of TRAP in mature osteoclasts without affecting the multinuclearity of cells.
Effect of IL-4 on TRAP, CTR, and RANK mRNA expression by mature osteoclasts
To examine the effect of IL-4 on expression of osteoclast-specific genes TRAP and CTR, mature osteoclasts were incubated with M-CSF and RANKL in the absence or the presence of IL-4 (20 ng/ml), and the mRNA expression was analyzed. RANKL-activated mature osteoclasts showed strong expression of TRAP and CTR genes at 24 and 48 h, and it was down-regulated by IL-4 (Fig. 3). RANKL-RANK interaction is required for activation of mature osteoclasts (10, 11, 13); therefore, effect of IL-4 on RANK mRNA expression was examined. IL-4 showed no effect on RANK expression, suggesting the inhibitory effect of IL-4 on bone resorption is not mediated by blockade in RANK expression in mature osteoclasts.
IL-4 inhibits actin ring formation and osteoclast migration induced by RANKL
Activated status of the mature osteoclasts is indicated by formation of distinct polymerized actin rings (26, 27). RANKL induces actin ring formation and motility of mature osteoclasts (10, 11). Therefore, we determined whether IL-4 inhibits actin ring formation and osteoclast migration induced by RANKL. Mature osteoclasts were incubated for 6 h with M-CSF and RANKL with or without IL-4 (20 ng/ml). As shown in Fig. 4, A and C, RANKL rapidly induced the formation of complete and well-defined actin rings in mature osteoclasts. In the presence of IL-4, two changes were seen in the structure of actin rings. IL-4 disrupted the formation of RANKL-induced actin rings (Fig. 4B), and also significantly decreased the intensity of actin rings (Fig. 4D). Fig. 4, E and F, is the graphical representation of the intensity of actin rings shown in Fig. 4, C and D, respectively. IL-4 significantly decreased the number of well-defined actin rings, and the majority of actin rings were either disrupted or less intense (Fig. 4G). Using Transwell migration assay, we found that IL-4 (20 ng/ml) significantly inhibited RANKL-induced migration of majority of osteoclasts (Fig. 4H). These results suggest that IL-4 inhibits bone resorption by disruption of actin rings and prevention of osteoclast migration.
IL-4 prevents RANKL-induced nuclear translocation of p65 NF-B subunit and intracellular Ca2+ changes
To further address the molecular mechanism by which IL-4 inhibits bone resorption, we examined the effect of IL-4 on NF-B. RANKL is a strong activator of NF-B that plays a functional role in bone resorption, and NF-B knockout mice are osteopetrotic because of defective osteoclast formation (28, 29, 30). In our studies using NF-B inhibitors, we also confirmed the functional role of NF-B in bone resorption. When mature osteoclasts were preincubated for 4 h with curcumin, a strong inhibitor of NF-B (31), RANKL does not induce bone resorption (data not shown). This effect of curcumin at low concentration was without inducing the apoptosis of mature osteoclasts (data not shown). To examine the effect of IL-4 on NF-B, mature osteoclasts were incubated with M-CSF with or without IL-4, and stimulated with RANKL. RANKL stimulated nuclear translocation of the p65 NF-B subunit within 15 min and decreased its cytoplasmic level (Fig. 5A, upper panel). Interestingly, IL-4 totally prevents the nuclear translocation of p65 with accumulation of this protein in the cytoplasm (Fig. 5A, lower panel). The number of MNCs showing nuclear translocation of p65 was decreased significantly in the presence of IL-4 (Fig. 5B). Activation of NF-B and its nuclear translocation in mature osteoclasts by RANKL is associated with increase in intracellular Ca2+ (13, 14). Therefore, we examined whether IL-4 also prevents RANKL-induced intracellular Ca2+ changes. We found that IL-4, in a time-dependent manner, prevented the transient increase in both cytoplasmic and nuclear Ca2+ induced by RANKL (Fig. 5C). These results suggest that decrease in intracellular Ca2+ by IL-4 is associated with decrease in NF-B activation.
Effect of IL-4 on RANKL-induced hypercalcemia in mice
RANKL has been shown to activate preexisting mature osteoclasts and stimulate hypercalcemia in mice (11). Also, hypercalcemia in many bone metastases and adult T cell leukemia occur due to increase in RANKL secretion (32). Because IL-4 has previously been shown to inhibit parathyroid hormone-related protein-induced hypercalcemia in mice (33, 34), we examined the in vivo effect of IL-4 on RANKL-induced hypercalcemia. Adult male mice were injected with RANKL in the absence or the presence of different concentrations of IL-4, and levels of ionized Ca2+ were examined. As shown in Fig. 6A, RANKL rapidly stimulated hypercalcemia in dose-dependent manner by increasing blood ionized Ca2+ levels. IL-4 significantly decreased RANKL-stimulated ionized Ca2+ level in 1 h (Fig. 6B). These results suggest that IL-4 acts on preexisting mature osteoclasts and decreases ionized Ca2+ levels in blood.
Discussion
In previous studies using complex in vitro and in vivo models, IL-4 has been shown to inhibit osteoclastogenesis and bone resorption (16, 17). These studies suggested that IL-4 may target both osteoclast precursors and mature osteoclasts. Recently, IL-4 has been shown to act directly on osteoclast precursors and inhibit osteoclastogenesis through inhibition of RANKL signaling pathways (18, 19, 20, 21). In this study, we investigated the mechanism by which IL-4 inhibits bone-resorbing activity of mature osteoclasts. Purification of mature osteoclasts induced by RANKL has enabled us to fully delineate the mechanism of IL-4 action.
Although RANKL alone is sufficient for activation of isolated rat mature osteoclasts (11), we found that mature osteoclasts require both M-CSF and RANKL for survival and full activation. IL-4 inhibited bone resorption, and anti-IL-4 Ab neutralized its effect, suggesting the direct action of IL-4 on mature osteoclasts. IL-4 also decreased the individual pit size, suggesting that each osteoclast is defective in terms of resorptive activity. Receptors for IL-4 have been found on a various cell types of both hemopoietic and nonhemopoietic lineages (15). In the present study, we provide the evidence of presence of IL-4R on mature osteoclasts by RT-PCR. The expression of IL-4R has previously been noted on human mature osteoclasts from giant cell tumor of bone (35). IL-13, another T cell-derived cytokine, which shares numerous biologic properties with IL-4 (15), showed no effect on bone resorption by mature osteoclasts (data not shown). These divergent results may be partially due to the sharing and differential expression of IL-4 and IL-13 receptor components on various cell types (36, 37).
No apoptotic changes were seen in mature osteoclasts in the presence of IL-4. We found that IL-4 markedly inhibited TRAP expression in mature osteoclasts without affecting its multinuclearity. The enzyme TRAP is strongly expressed in actively bone-resorbing mature osteoclasts, and TRAP-deficient mice have been shown to exhibit osteopetrotic phenotype with normal differentiation of osteoclasts that are dysfunctional in vitro, suggesting a role of TRAP in the bone resorption process (38, 39, 40). Transgenic mice overexpressing the TRAP gene showed mild osteoporosis, with decreased trabecular bone density (41). Overexpression of IL-4 in transgenic mice showed normal numbers of osteoclasts; however, their function was altered by the decrease in TRAP expression (42). Thus, our results suggest that IL-4 inhibits bone resorption predominantly by decrease in TRAP expression and not by apoptosis of mature osteoclasts. Increased number of vacuoles in the presence of IL-4 is consistent with those of Suter et al. (39) in which TRAP-deficient osteoclasts showed the accumulation of vacuoles. IL-4 has been known to act directly on macrophages and induces their fusion to form foreign body giant cells (43). In our studies also IL-4 induced the fusion of TRAP-negative MNCs.
There is an excellent correlation between actin ring formation and bone resorption (26, 27). Our results demonstrate that IL-4-treated mouse and human mature osteoclasts showed reduced intensity and disruption of actin ring structures. The disrupted actin rings and diffuse cytoplasmic staining observed in the presence of IL-4 reflects the improper assembly of osteoclast cytoskeleton. The disassembly of actin rings would not allow the formation of the tight-sealing zone, resulting in the formation of the leaky zone. IL-4 also inhibited the migration of osteoclasts induced by RANKL. Thus, in our study, decreased TRAP expression, structural disturbances in actin rings, and inhibition of osteoclast migration by IL-4 contributed largely to the reduced bone resorption and pit size by mature osteoclasts. We also observed that IL-4 inhibits the formation of actin rings in 2 h in isolated osteoclasts of 2- to 5-day-old mice (data not shown).
Expression of RANK on mature osteoclasts provides evidence that it is required for signaling in activated osteoclasts (13). Also RANK-dependent signaling is essential for osteoclast cytoskeleton organization and resorption (44). Because IL-4 does not inhibit RANK expression we investigated the molecular mechanisms of IL-4 action by examining its effect on NF-B activation. NF-B activation is essential for the osteoclast differentiation, and its role has been implicated in bone resorption (29, 30). In our study, IL-4 inhibited the nuclear translocation of NF-B induced by RANKL. Also, increase of intracellular Ca2+ in mature osteoclasts in response to RANKL was prevented by IL-4. RANKL-induced Ca2+ signaling is more prominent in mature osteoclasts than in precursors, and elevation of intracellular Ca2+ regulates NF-B nuclear translocation in mature osteoclasts (13, 14). Bizzari et al. (45) have reported increase in intracellular Ca2+ in mature osteoclasts by IL-4. In their study, effect of IL-4 was examined on cytoplasmic calcium level only for 10 min. However, in our study, we studied both cytoplasmic and nuclear calcium levels up to 30 min. Furthermore, IL-4 inhibits RANKL-induced hypercalcemia in vivo in 1 h suggesting its direct inhibitory action on preexisting active mature osteoclasts. Hypercalcemia is one of the most frequent and serious complications experienced by patients with adult T cell leukemia leading to accumulation of osteoclasts and marked increase in bone resorption (32). As reported previously (33, 34), IL-4 is a strong inhibitor of hypercalcemia, and in our studies we show that IL-4 knockout mice are more sensitive to RANKL action in inducing hypercalcemia. Also IL-4 knockout mice showed more sensitivity to RANKL for in vitro osteoclast formation and bone resorption.
Binding of IL-4 to its receptor recruits the members of the Janus tyrosine kinase family that activates STAT6 (15). Recently, Moreno et al. (21) has shown that IL-4 inhibits bone resorption through STAT6-dependent mechanism. However, we provide further advances that IL-4 inhibition of NF-B activation and Ca2+ signaling may be central to the mechanism of action of IL-4. It is possible that IL-4 inhibits activity of mature osteoclasts through inhibition of NF-B pathway in STAT6-dependent manner. Our observation of IL-4 inhibition of RANKL induced intracellular Ca2+ changes besides inhibition of NF-B is novel; however, the mechanism by which IL-4 acts on intracellular Ca2+ is yet to be determined. Our results suggest that IL-4-induced disruption of actin ring is associated with the decrease of intracellular Ca2+. This is consistent with the recent report that depletion of intracellular Ca2+ leads to the disruption of the F-actin in smooth muscle cells (46). We also provide in vivo validation of in vitro data that RANKL-induced hypercalcemia is attenuated by IL-4 and accentuated by IL-4 deficiency. In conclusion, our results suggest that IL-4 acts directly on mature osteoclasts and inhibits bone resorption through inhibition of NF-B activation and Ca2+ signaling probably by IL-4R-mediated mechanism.
In vivo, we found a normal number of osteoclasts and an unaltered level of basal ionized calcium levels in IL-4 knockout mice, suggesting that endogenous IL-4 has no physiological significance in bone metabolism. Increased evidence has revealed that IL-4 inhibits bone resorption not only through inhibition of osteoclast formation, but also through suppression of bone resorption by mature osteoclasts (Ref. 21 and our results). It is also reported that IL-4 prevents bone and cartilage destruction in collagen-induced arthritis (47). Our results suggest the pathological significance of IL-4 in the bone. IL-4 inhibition of NF-B activation in mature osteoclasts and its in vivo rapid action to decrease acute hypercalcemia induced by RANKL increases its therapeutic potential in skeletal disorders such as osteoporosis, rheumatoid arthritis, and hypercalcemia of malignancy cases, when delivered as a recombinant cytokine or in combination with other drugs in gene therapy. Our results also strengthened the potent inhibitory nature of IL-4 by showing its inhibitory effects on bone resorption by human mature osteoclasts.
Acknowledgments
We extend our sincere thanks to Dr. G. C. Mishra, Director, National Center for Cell Science, for encouragement and support. We thank S. D. Yogesha for critically reading the manuscript, Satish Pote for technical assistance, and Ashwini Atre for confocal microscopy. We also thank Dr. Cecilia Dayaraj from National Institute of Virology (Pune, India) for help in confocal microscopy.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Department of Biotechnology, Government of India. L.S.M. is the recipient of Senior Research Fellowship from Department of Biotechnology (India). S.M.K. is the recipient of Senior Research Fellowship from the Council for Scientific and Industrial Research (New Delhi, India).
2 Address correspondence and reprint requests to Dr. Mohan R. Wani, National Center for Cell Science, University of Pune Campus, Pune-411 007, India. E-mail address: mohanwani@nccs.res.in
3 Abbreviations used in this paper: MNC, multinuclear cell; CTR, calcitonin receptor; RANK, receptor activator of NF-B; RANKL, RANK ligand; TRAP, tartrate-resistant acid phosphatase; S, sense; AS, antisense.
Received for publication February 18, 2005. Accepted for publication May 10, 2005.
References
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IL-4 is an important immune cytokine that regulates bone homeostasis. We investigated the molecular mechanism of IL-4 action on bone-resorbing mature osteoclasts. Using a highly purified population of mature osteoclasts, we show that IL-4 dose-dependently inhibits receptor activator of NF-B ligand (RANKL)-induced bone resorption by mature osteoclasts. We detected the existence of IL-4R mRNA in mature osteoclasts. IL-4 decreases TRAP expression without affecting multinuclearity of osteoclasts, and inhibits actin ring formation and migration of osteoclasts. Interestingly, IL-4 inhibition of bone resorption occurs through prevention of RANKL-induced nuclear translocation of p65 NF-B subunit, and intracellular Ca2+ changes. Moreover, IL-4 rapidly decreases RANKL-stimulated ionized Ca2+ levels in the blood, and mature osteoclasts in IL-4 knockout mice are sensitive to RANKL action to induce bone resorption and hypercalcemia. Furthermore, IL-4 inhibits bone resorption and actin ring formation by human mature osteoclasts. Thus, we reveal that IL-4 acts directly on mature osteoclasts and inhibits bone resorption by inhibiting NF-B and Ca2+ signaling.
Introduction
Osteoclasts, the multinuclear cells (MNCs)3 responsible for bone resorption, play a crucial role in bone remodeling. The bone loss in many important skeletal disorders such as osteoporosis, rheumatoid arthritis, hypercalcemia of malignancy, and bone metastases occurs mainly because of increased osteoclast activity (1). A major breakthrough in understanding the regulation of osteoclastogenesis occurred after the discovery of novel molecules such as receptor activator of NF-B (RANK), RANK ligand (RANKL), and osteoprotegerin (2, 3, 4, 5, 6, 7). RANKL, in the presence of M-CSF, mediates osteoclastogenesis through binding to its receptor RANK on osteoclast precursors (5, 7). Transgenic and gene knockout studies in mice established the absolute dependency of osteoclast differentiation and activation on the expression of RANKL and RANK (7, 8). The distinct signaling pathways such as NF-B, JNK, p38, ERK, and Src pathways mediated by protein kinases are activated by RANKL during osteoclastogenesis and bone resorption (9).
RANKL also plays an important role in survival and activation of mature osteoclasts and rapidly induces actin ring formation (10, 11, 12). In vivo studies have shown that RANKL increases blood ionized Ca2+ levels within 1 h suggesting its direct effect on preexisting mature osteoclasts (11). Stimulation of RANK on mature osteoclasts by RANKL results in activation of transcriptional factor NF-B and Ca2+ signaling (13, 14). The study of molecular mechanisms by which cytokines secreted by T cells or other immune cells regulate bone-resorbing activity of mature osteoclasts had been limited because of difficulties in obtaining a sufficient number of mature osteoclasts. Induction of mature osteoclasts by RANKL in mouse and human osteoclast precursors is an excellent in vitro model to investigate the novel mechanisms of cytokines that regulate bone resorption.
IL-4 is a 19-kDa pleiotropic type I cytokine secreted by activated TH2 lymphocytes, mast cells, eosinophils, and basophils (15). IL-4, an important immune cytokine that regulates function of lymphocytes and macrophages, also regulates osteoclastogenesis and bone resorption (16, 17). Recent work has clarified the role and molecular mechanisms by which IL-4 inhibits RANKL-induced osteoclast differentiation in osteoclast precursors (18, 19, 20, 21). However, the mechanism of IL-4 action on mature osteoclasts and its function is not fully delineated. Moreno et al. (21) have reported the inhibitory effect of IL-4 on mouse mature osteoclast function and showed that the effect requires STAT6. In this study, we prepared a large number of highly purified mature osteoclasts induced by RANKL using both mice and human osteoclast precursors, and provide further advances that clarify in detail the mechanisms of IL-4 action on bone-resorbing mature osteoclasts. We show here that IL-4 acts directly on mature osteoclasts and significantly inhibits bone resorption and tartrate-resistant acid phosphatase (TRAP) expression. IL-4 inhibits bone resorption by disruption of RANKL-induced actin ring formation in mouse and as well as human mature osteoclasts. Furthermore, IL-4 prevents RANKL-induced nuclear translocation of p65 NF-B subunit, and intracellular Ca2+ changes in mature osteoclasts. In addition, RANKL-induced hypercalcemia in vivo is attenuated by IL-4 and accentuated by IL-4 deficiency. In conclusion, IL-4 acts directly on mature osteoclasts and inhibits bone resorption by inhibiting NF-B and Ca2+ signaling.
Results
IL-4 inhibits bone resorption by mature osteoclasts
Mature osteoclasts were prepared from osteoclast precursors as described in Materials and Methods and purified (>95% pure) by removing mononuclear cells using EDTA treatment. To examine the effect of IL-4 on bone resorption, purified mature osteoclasts on bone slices were further incubated for 48 h with M-CSF (30 ng/ml) and RANKL (30 ng/ml) with or without different concentrations of IL-4. As shown in Fig. 1A, IL-4 inhibited the RANKL-induced bone resorption in a dose-dependent manner. IL-4 (20 ng/ml) also significantly decreased the size of an individual resorption pit (Fig. 1, B and C). The inhibitory effect of IL-4 on bone resorption was confirmed by anti-mouse IL-4 Ab. As shown in Fig. 1D, simultaneous addition of anti-IL-4 Ab neutralized the inhibitory effect of IL-4 in a dose-dependent manner. These results suggest that IL-4 inhibits bone resorption by direct action on activated mature osteoclasts.
IL-4 effects depend upon binding to and signaling through a receptor complex consisting of the IL-4R chain and the common -chain (25). Receptors of IL-4 are expressed on a wide range of cells including hemopoietic cells (15). To examine whether mature osteoclasts express IL-4R, we incubated mature osteoclasts with M-CSF and RANKL and assessed for IL-4R mRNA expression. As shown in Fig. 1E, mature osteoclasts induced by RANKL on day 4 (0 h) expressed IL-4R, and cells further incubated for 24 and 48 h also showed strong expression of IL-4R.
IL-4 inhibits TRAP expression in mature osteoclasts
To investigate the mechanism of IL-4 action on activated mature osteoclasts, we first examined whether IL-4 inhibits bone resorption by inducing the apoptosis in mature osteoclasts. Osteoclasts were incubated for 48 h with M-CSF and RANKL in the absence or presence of various concentrations of IL-4. No apoptotic changes such as chromosome condensation, nuclear fragmentation were seen in the presence of IL-4 (data not shown). We then examined the effect of IL-4 on TRAP expression. As shown in Fig. 2A, IL-4 dose-dependently decreased TRAP expression, and the majority of MNCs were TRAP-negative and increased with increasing concentrations of IL-4 (Fig. 2B). These TRAP-negative MNCs did not express CTR (data not shown). Fig. 2C shows the effect of IL-4 on TRAP expression in mature osteoclasts. The TRAP-negative MNCs in the presence of IL-4 were fused to form giant cells with accumulation of large vacuoles. These results suggest that IL-4 inhibits expression of TRAP in mature osteoclasts without affecting the multinuclearity of cells.
Effect of IL-4 on TRAP, CTR, and RANK mRNA expression by mature osteoclasts
To examine the effect of IL-4 on expression of osteoclast-specific genes TRAP and CTR, mature osteoclasts were incubated with M-CSF and RANKL in the absence or the presence of IL-4 (20 ng/ml), and the mRNA expression was analyzed. RANKL-activated mature osteoclasts showed strong expression of TRAP and CTR genes at 24 and 48 h, and it was down-regulated by IL-4 (Fig. 3). RANKL-RANK interaction is required for activation of mature osteoclasts (10, 11, 13); therefore, effect of IL-4 on RANK mRNA expression was examined. IL-4 showed no effect on RANK expression, suggesting the inhibitory effect of IL-4 on bone resorption is not mediated by blockade in RANK expression in mature osteoclasts.
IL-4 inhibits actin ring formation and osteoclast migration induced by RANKL
Activated status of the mature osteoclasts is indicated by formation of distinct polymerized actin rings (26, 27). RANKL induces actin ring formation and motility of mature osteoclasts (10, 11). Therefore, we determined whether IL-4 inhibits actin ring formation and osteoclast migration induced by RANKL. Mature osteoclasts were incubated for 6 h with M-CSF and RANKL with or without IL-4 (20 ng/ml). As shown in Fig. 4, A and C, RANKL rapidly induced the formation of complete and well-defined actin rings in mature osteoclasts. In the presence of IL-4, two changes were seen in the structure of actin rings. IL-4 disrupted the formation of RANKL-induced actin rings (Fig. 4B), and also significantly decreased the intensity of actin rings (Fig. 4D). Fig. 4, E and F, is the graphical representation of the intensity of actin rings shown in Fig. 4, C and D, respectively. IL-4 significantly decreased the number of well-defined actin rings, and the majority of actin rings were either disrupted or less intense (Fig. 4G). Using Transwell migration assay, we found that IL-4 (20 ng/ml) significantly inhibited RANKL-induced migration of majority of osteoclasts (Fig. 4H). These results suggest that IL-4 inhibits bone resorption by disruption of actin rings and prevention of osteoclast migration.
IL-4 prevents RANKL-induced nuclear translocation of p65 NF-B subunit and intracellular Ca2+ changes
To further address the molecular mechanism by which IL-4 inhibits bone resorption, we examined the effect of IL-4 on NF-B. RANKL is a strong activator of NF-B that plays a functional role in bone resorption, and NF-B knockout mice are osteopetrotic because of defective osteoclast formation (28, 29, 30). In our studies using NF-B inhibitors, we also confirmed the functional role of NF-B in bone resorption. When mature osteoclasts were preincubated for 4 h with curcumin, a strong inhibitor of NF-B (31), RANKL does not induce bone resorption (data not shown). This effect of curcumin at low concentration was without inducing the apoptosis of mature osteoclasts (data not shown). To examine the effect of IL-4 on NF-B, mature osteoclasts were incubated with M-CSF with or without IL-4, and stimulated with RANKL. RANKL stimulated nuclear translocation of the p65 NF-B subunit within 15 min and decreased its cytoplasmic level (Fig. 5A, upper panel). Interestingly, IL-4 totally prevents the nuclear translocation of p65 with accumulation of this protein in the cytoplasm (Fig. 5A, lower panel). The number of MNCs showing nuclear translocation of p65 was decreased significantly in the presence of IL-4 (Fig. 5B). Activation of NF-B and its nuclear translocation in mature osteoclasts by RANKL is associated with increase in intracellular Ca2+ (13, 14). Therefore, we examined whether IL-4 also prevents RANKL-induced intracellular Ca2+ changes. We found that IL-4, in a time-dependent manner, prevented the transient increase in both cytoplasmic and nuclear Ca2+ induced by RANKL (Fig. 5C). These results suggest that decrease in intracellular Ca2+ by IL-4 is associated with decrease in NF-B activation.
Effect of IL-4 on RANKL-induced hypercalcemia in mice
RANKL has been shown to activate preexisting mature osteoclasts and stimulate hypercalcemia in mice (11). Also, hypercalcemia in many bone metastases and adult T cell leukemia occur due to increase in RANKL secretion (32). Because IL-4 has previously been shown to inhibit parathyroid hormone-related protein-induced hypercalcemia in mice (33, 34), we examined the in vivo effect of IL-4 on RANKL-induced hypercalcemia. Adult male mice were injected with RANKL in the absence or the presence of different concentrations of IL-4, and levels of ionized Ca2+ were examined. As shown in Fig. 6A, RANKL rapidly stimulated hypercalcemia in dose-dependent manner by increasing blood ionized Ca2+ levels. IL-4 significantly decreased RANKL-stimulated ionized Ca2+ level in 1 h (Fig. 6B). These results suggest that IL-4 acts on preexisting mature osteoclasts and decreases ionized Ca2+ levels in blood.
Discussion
In previous studies using complex in vitro and in vivo models, IL-4 has been shown to inhibit osteoclastogenesis and bone resorption (16, 17). These studies suggested that IL-4 may target both osteoclast precursors and mature osteoclasts. Recently, IL-4 has been shown to act directly on osteoclast precursors and inhibit osteoclastogenesis through inhibition of RANKL signaling pathways (18, 19, 20, 21). In this study, we investigated the mechanism by which IL-4 inhibits bone-resorbing activity of mature osteoclasts. Purification of mature osteoclasts induced by RANKL has enabled us to fully delineate the mechanism of IL-4 action.
Although RANKL alone is sufficient for activation of isolated rat mature osteoclasts (11), we found that mature osteoclasts require both M-CSF and RANKL for survival and full activation. IL-4 inhibited bone resorption, and anti-IL-4 Ab neutralized its effect, suggesting the direct action of IL-4 on mature osteoclasts. IL-4 also decreased the individual pit size, suggesting that each osteoclast is defective in terms of resorptive activity. Receptors for IL-4 have been found on a various cell types of both hemopoietic and nonhemopoietic lineages (15). In the present study, we provide the evidence of presence of IL-4R on mature osteoclasts by RT-PCR. The expression of IL-4R has previously been noted on human mature osteoclasts from giant cell tumor of bone (35). IL-13, another T cell-derived cytokine, which shares numerous biologic properties with IL-4 (15), showed no effect on bone resorption by mature osteoclasts (data not shown). These divergent results may be partially due to the sharing and differential expression of IL-4 and IL-13 receptor components on various cell types (36, 37).
No apoptotic changes were seen in mature osteoclasts in the presence of IL-4. We found that IL-4 markedly inhibited TRAP expression in mature osteoclasts without affecting its multinuclearity. The enzyme TRAP is strongly expressed in actively bone-resorbing mature osteoclasts, and TRAP-deficient mice have been shown to exhibit osteopetrotic phenotype with normal differentiation of osteoclasts that are dysfunctional in vitro, suggesting a role of TRAP in the bone resorption process (38, 39, 40). Transgenic mice overexpressing the TRAP gene showed mild osteoporosis, with decreased trabecular bone density (41). Overexpression of IL-4 in transgenic mice showed normal numbers of osteoclasts; however, their function was altered by the decrease in TRAP expression (42). Thus, our results suggest that IL-4 inhibits bone resorption predominantly by decrease in TRAP expression and not by apoptosis of mature osteoclasts. Increased number of vacuoles in the presence of IL-4 is consistent with those of Suter et al. (39) in which TRAP-deficient osteoclasts showed the accumulation of vacuoles. IL-4 has been known to act directly on macrophages and induces their fusion to form foreign body giant cells (43). In our studies also IL-4 induced the fusion of TRAP-negative MNCs.
There is an excellent correlation between actin ring formation and bone resorption (26, 27). Our results demonstrate that IL-4-treated mouse and human mature osteoclasts showed reduced intensity and disruption of actin ring structures. The disrupted actin rings and diffuse cytoplasmic staining observed in the presence of IL-4 reflects the improper assembly of osteoclast cytoskeleton. The disassembly of actin rings would not allow the formation of the tight-sealing zone, resulting in the formation of the leaky zone. IL-4 also inhibited the migration of osteoclasts induced by RANKL. Thus, in our study, decreased TRAP expression, structural disturbances in actin rings, and inhibition of osteoclast migration by IL-4 contributed largely to the reduced bone resorption and pit size by mature osteoclasts. We also observed that IL-4 inhibits the formation of actin rings in 2 h in isolated osteoclasts of 2- to 5-day-old mice (data not shown).
Expression of RANK on mature osteoclasts provides evidence that it is required for signaling in activated osteoclasts (13). Also RANK-dependent signaling is essential for osteoclast cytoskeleton organization and resorption (44). Because IL-4 does not inhibit RANK expression we investigated the molecular mechanisms of IL-4 action by examining its effect on NF-B activation. NF-B activation is essential for the osteoclast differentiation, and its role has been implicated in bone resorption (29, 30). In our study, IL-4 inhibited the nuclear translocation of NF-B induced by RANKL. Also, increase of intracellular Ca2+ in mature osteoclasts in response to RANKL was prevented by IL-4. RANKL-induced Ca2+ signaling is more prominent in mature osteoclasts than in precursors, and elevation of intracellular Ca2+ regulates NF-B nuclear translocation in mature osteoclasts (13, 14). Bizzari et al. (45) have reported increase in intracellular Ca2+ in mature osteoclasts by IL-4. In their study, effect of IL-4 was examined on cytoplasmic calcium level only for 10 min. However, in our study, we studied both cytoplasmic and nuclear calcium levels up to 30 min. Furthermore, IL-4 inhibits RANKL-induced hypercalcemia in vivo in 1 h suggesting its direct inhibitory action on preexisting active mature osteoclasts. Hypercalcemia is one of the most frequent and serious complications experienced by patients with adult T cell leukemia leading to accumulation of osteoclasts and marked increase in bone resorption (32). As reported previously (33, 34), IL-4 is a strong inhibitor of hypercalcemia, and in our studies we show that IL-4 knockout mice are more sensitive to RANKL action in inducing hypercalcemia. Also IL-4 knockout mice showed more sensitivity to RANKL for in vitro osteoclast formation and bone resorption.
Binding of IL-4 to its receptor recruits the members of the Janus tyrosine kinase family that activates STAT6 (15). Recently, Moreno et al. (21) has shown that IL-4 inhibits bone resorption through STAT6-dependent mechanism. However, we provide further advances that IL-4 inhibition of NF-B activation and Ca2+ signaling may be central to the mechanism of action of IL-4. It is possible that IL-4 inhibits activity of mature osteoclasts through inhibition of NF-B pathway in STAT6-dependent manner. Our observation of IL-4 inhibition of RANKL induced intracellular Ca2+ changes besides inhibition of NF-B is novel; however, the mechanism by which IL-4 acts on intracellular Ca2+ is yet to be determined. Our results suggest that IL-4-induced disruption of actin ring is associated with the decrease of intracellular Ca2+. This is consistent with the recent report that depletion of intracellular Ca2+ leads to the disruption of the F-actin in smooth muscle cells (46). We also provide in vivo validation of in vitro data that RANKL-induced hypercalcemia is attenuated by IL-4 and accentuated by IL-4 deficiency. In conclusion, our results suggest that IL-4 acts directly on mature osteoclasts and inhibits bone resorption through inhibition of NF-B activation and Ca2+ signaling probably by IL-4R-mediated mechanism.
In vivo, we found a normal number of osteoclasts and an unaltered level of basal ionized calcium levels in IL-4 knockout mice, suggesting that endogenous IL-4 has no physiological significance in bone metabolism. Increased evidence has revealed that IL-4 inhibits bone resorption not only through inhibition of osteoclast formation, but also through suppression of bone resorption by mature osteoclasts (Ref. 21 and our results). It is also reported that IL-4 prevents bone and cartilage destruction in collagen-induced arthritis (47). Our results suggest the pathological significance of IL-4 in the bone. IL-4 inhibition of NF-B activation in mature osteoclasts and its in vivo rapid action to decrease acute hypercalcemia induced by RANKL increases its therapeutic potential in skeletal disorders such as osteoporosis, rheumatoid arthritis, and hypercalcemia of malignancy cases, when delivered as a recombinant cytokine or in combination with other drugs in gene therapy. Our results also strengthened the potent inhibitory nature of IL-4 by showing its inhibitory effects on bone resorption by human mature osteoclasts.
Acknowledgments
We extend our sincere thanks to Dr. G. C. Mishra, Director, National Center for Cell Science, for encouragement and support. We thank S. D. Yogesha for critically reading the manuscript, Satish Pote for technical assistance, and Ashwini Atre for confocal microscopy. We also thank Dr. Cecilia Dayaraj from National Institute of Virology (Pune, India) for help in confocal microscopy.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Department of Biotechnology, Government of India. L.S.M. is the recipient of Senior Research Fellowship from Department of Biotechnology (India). S.M.K. is the recipient of Senior Research Fellowship from the Council for Scientific and Industrial Research (New Delhi, India).
2 Address correspondence and reprint requests to Dr. Mohan R. Wani, National Center for Cell Science, University of Pune Campus, Pune-411 007, India. E-mail address: mohanwani@nccs.res.in
3 Abbreviations used in this paper: MNC, multinuclear cell; CTR, calcitonin receptor; RANK, receptor activator of NF-B; RANKL, RANK ligand; TRAP, tartrate-resistant acid phosphatase; S, sense; AS, antisense.
Received for publication February 18, 2005. Accepted for publication May 10, 2005.
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