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gp130 Signaling Pathway in Bone Metabolism
14 The Role of the Interleukin‐6/gp130 Signaling Pathway in Bone Metabolism Xin‐Hua Liu,* Alexander Kirschenbaum,{ Shen Yao,* and Alice C. Levine* *Department of Medicine, Division of Endocrinology, Diabetes and Bone Diseases Mount Sinai School of Medicine, New York, New York 10029 { Department of Urology, Mount Sinai School of Medicine New York, New York 10029
I. II. III. IV.
Introduction EVect of IL‐6 on Bone Resorption EVect of IL‐6 on Bone Formation Interactions Between IL‐6/gp130 Signaling and the OPG/RANKL/RANK System V. Cross Talk Between IL‐6 and Steroid Hormones, PTH, PGE2, and Cytokines A. IL‐6 and Sex Steroids B. IL‐6 and PTH C. IL‐6 and PGE2 D. IL‐6 and Other Inflammatory Cytokines VI. gp130 Signaling and Bone Remodeling VII. Summary References
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0083-6729/06 $35.00 DOI: 10.1016/S0083-6729(06)74014-6
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I. INTRODUCTION The interleukin‐6 (IL‐6) family of cytokines includes IL‐6, IL‐11, leukemia inhibitory factor (LIF), oncostain M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin‐1 (CT‐1) (Kishimoto et al., 1995). Increasing evidence suggests that IL‐6‐type cytokines regulate immune and inflammatory responses, hepatic acute‐phase protein synthesis, hematopoiesis, and bone metabolism (Kishimoto, 1989). All of the members of the IL‐6 family have overlapping functions and utilize the same receptor subunit, glycoprotein 130 (gp130), a critical signal‐transducing component previously identified as IL‐6 receptor‐b (IL‐6Rb) (Kishimoto et al., 1995). In target cells, IL‐6 can simultaneously induce functionally distinct and even contradictory signals through its receptor complex, IL‐6Ra (or soluble receptor, sIL‐6R) and gp130. The binding of IL‐6 to IL‐6Ra induces dimerization of gp130 (Taga and Kishimoto, 1997), and initiates a cascade of intracellular signaling that leads to activation of signal transducers and activators of transcription (STAT)1/3 and/or SHP2/ras/MAPK, a process that culminates in modulation of gene transcription (Heymann and Rousselle, 2000; Kamimura et al., 2003; O’Brien et al., 2000). There is ample evidence that IL‐6‐type cytokines play an important role in skeletal homeostasis (Manolagas and Jilka, 1995; Martin et al., 1998; Suda et al., 1999). IL‐6 influences both osteoblast and osteoclast activities through a variety of complex mechanisms. For example, IL‐6 has been shown to promote the diVerentiation of macrophages and bone marrow stromal cells into osteoclasts and osteoblasts, respectively (Erices et al., 2002; Manolagas, 1998; Sims et al., 2004). In addition, IL‐6 interacts with other factors that are critically involved in bone remodeling, including the OPG/RANKL/RANK system, sex steroids (estrogens and androgens), prostaglandin E2 (PGE2), tumor necrosis factor (TNF)‐a, parathyroid hormone (PTH), IL‐11, and IL‐1, to regulate bone turnover (Grey et al., 1999; Liu et al., 2005; Steeve et al., 2004). In this chapter, we will highlight the biological eVects and clinical relevance of IL‐6 and its associated receptors in bone development and remodeling. Recent data on the regulation of IL‐6/gp130 signaling pathway in bone homeostasis will also be discussed with an emphasis on how one cytokine can initiate diverse eVects in bone in a cell‐specific fashion.
II. EFFECT OF IL‐6 ON BONE RESORPTION Bone tissue remodeling results from the coordinate activities of osteoblasts and osteoclasts. Osteoclasts are derived from hematopoietic precursors of the bone marrow (Roodman, 1996) and are highly specialized, multinucleated cells that are uniquely capable of lacunar bone resorption. Imbalances in the bone remodeling process result in metabolic bone diseases characterized
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by either enhanced osteoclast activity and increased bone resorption (i.e., osteoporosis and osteolytic bone lesions in cancer) or increased osteoblastic bone formation (i.e., osteopetrosis and prostate cancer‐induced osteoblastic metastases) (Fohr et al., 2003; Ohlsson et al., 1998). IL‐6 increases osteoclast recruitment by acting on early hematopoietic cells from the granulocyte‐macrophage lineage that contain the progenitors of the osteoclastic lineage (Otsuka et al., 1991). IL‐6 has also been shown to play a specific role in postmenopausal osteoporosis (Jilka et al., 1992; Poli et al., 1994; Tarura et al., 1993). A possible mechanism for this eVect was decreased osteoclast formation due to the lack of IL‐6 (Tarura et al., 1993). A cross‐ sectional study in humans compared bone turnover and bone mineral density (BMD) in healthy postmenopausal women with diVerent G‐C polymorphisms at position –174 in the IL‐6 promoter. Patients with either CG or GG genotypes were associated with higher bone resorption and lower BMD at the hip and forearm compared with those in patients with the CC genotype (Ferrari, 2001). The study suggests that allelic variations in the IL‐6 promoter influence IL‐6 production and contribute to increased bone resorption and decreased BMD. High IL‐6 levels also correlate with high bone resorption indices in patients with rheumatoid arthritis. Clinical studies reveal a negative correlation between BMD and the combination of intracellular and cell‐ surface‐bound levels of IL‐6 in peripheral blood monocytes from rheumatoid arthritis patients (Verbruggen et al., 1999). In multiple myeloma, IL‐6 is one of the cytokines that promote bone destruction (Kuehl and Bergsagel, 2002). Multiple myeloma is a plasma cell dyscrasia characterized by proliferation of malignant B cells in the bone marrow and severe bone loss. Higher IL‐6 serum levels in multiple myeloma are associated with more advanced stages of the disease (II and III). Furthermore, the serum level of IL‐6 has been identified as a significant prognostic marker in multiple myeloma. Ludwig et al. (1991) found that survival times diVered significantly between patients whose IL‐6 levels at the time of diagnosis fell below 7 pg/ml and those with higher levels. The 50% of patients in the former category exhibited a medium survival of 53.7 months as compared with only 2.7 months in the latter category (Ludwig et al., 1991). Although the mechanisms underlying these observed eVects of IL‐6 are not well understood, one report demonstrated that IL‐6 modulates trabecular and endochondral bone turnover in vivo by stimulating osteoclast diVerentiation (Rozen et al., 2000). Bone remodeling is the result of the coordinate and interactive eVects of both types of osteogenic cells (osteoblasts and osteoclasts). Although high levels of IL‐6 stimulate bone resorption directely, IL‐6, like many other regulatory factors (i.e., growth factors, hormones, cytokines), predominantly modulates osteoclast activity and bone resorption indirectly by influencing osteoblast diVerentiation and secretion. In bone cells, IL‐6 production is limited to cells of the osteoblastic lineage (Holt et al., 1996; Legrand‐Poels et al., 2000). Moreover, although both osteoblasts and osteoclasts express the
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IL‐6 receptor, functional studies demonstrate that IL‐6‐induced osteoclast diVerentiation is dependent upon IL‐6 receptor expression by the osteoblast, but not the osteoclast (Udagawa et al., 1995). In agreement with these observations, our in vitro studies demonstrate that IL‐6 significantly stimulates osteoclast diVerentiation only when they are grown in coculture with osteoblasts (Liu et al., 2005). However, a direct eVect of IL‐6 on inducing osteoclast formation has also been reported in several osteolytic bone disorders (Kudo et al., 2003).
III. EFFECT OF IL‐6 ON BONE FORMATION Mature osteoblasts are derived from multipotent mesenchymal stem cells of the bone marrow that also give rise to the fibroblast cells of the marrow stroma, chondrocytes, adipocytes, and muscle cells (Aubin, 1998). Stromal cells of the bone marrow and osteoblasts exhibit an extensive overlap of phenotypic properties (Manolagas, 1998). The initial evidence that the IL‐6 family of cytokines influences bone formation was provided by the observation that overexpression of either LIF or OSM gene (two IL‐6 family members) in mice induces excessive bone formation (Lowe et al., 1991; Malik et al., 1995; Metcalf and Gearing, 1989). In addition, IL‐6, LIF, and OSM have been reported to confer an antiapoptotic phenotype on osteoblastic cells via enhanced transcriptional activation of the p21 gene (Bellido et al., 1998; Jilka et al., 1998; Steeve et al., 2004). IL‐6 also promotes osteogenic lineage commitment. IL‐6 plus soluble IL‐6 receptor or LIF stimulates the commitment of embryonic fibroblasts (12th–14th day of gestation) toward the osteoblast phenotype without any promotion of diVerentiation toward adipocytes, chondrocytes, or muscle cells (Tagochi et al., 1998). It has also been reported that the diVerentiation of mesenchymal progenitors of osteoblasts in the murine bone marrow can be stimulated by IL‐6 plus soluble IL‐6 receptor or LIF (Tagochi et al., 1998). IL‐6 as well as LIF, IL‐11, CNTF, and OSM promote the diVerentiation of committed osteoblast cells toward a more mature phenotype, and this action of the IL‐6 family of cytokines is mediated by the activation of the Janus Kinase (JAK)/STAT pathway (Bellido et al., 1997). However, Hughes and Howells (1993) have shown an inhibitory eVect of IL‐6 on bone formation in vitro. These apparently contradictory observations may be due to the diVerences in the experimental systems and methods utilized in the two studies. Both osteoblasts and osteoclasts express the IL‐6 receptor subunits, that is, gp130 and soluble IL‐6 receptor. However, the intracellular signaling cascades induced by IL‐6 activation of gp130 are complex and may be cell specific. It is possible that the observed dual eVect of IL‐6 on bone is due to cell‐ specific activation of diVerent intracellular signaling pathways in osteoblasts versus osteoclasts (Sims et al., 2004).
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IV. INTERACTIONS BETWEEN IL‐6/GP130 SIGNALING AND THE OPG/RANKL/RANK SYSTEM Osteoblast function is intimately tied to osteoclast activity. The interactions between osteoblasts and osteoclasts can be established through cell–cell contact (Jimi et al., 1996) and are mediated by receptor activator of nuclear factor‐kappaB (NF‐kB) ligand (RANKL) produced by osteoblasts and receptor activator of NF‐kB (RANK) on the osteoclast surface (Nakagawa et al., 1998; Yasuda et al., 1998). Bone marrow stromal cells (osteoblast precursor cells) also produce a soluble glycoprotein called osteoprotegerin (OPG), a decoy receptor for RANKL that prevents osteoclast activation (Simonet and Luthy, 1997; Yasuda et al., 1998). OPG and RANKL, synthesized by stromal cells/osteoblasts, have been identified as the two principal cytokines that regulate osteoclast diVerentiation and activation (Lacey et al., 1998; Simonet and Luthy, 1997). Hormones, growth factors, cytokines, and prostaglandins regulate these processes (Brandstrom et al., 2001; Ohlsson et al., 1998) mainly via eVects on osteoblasts. In general, the osteoblasts receive input directly from a variety of regulatory factors and then transmit these signals to the neighboring osteoclasts via the OPG/RANKL/RANK system. RANKL and IL‐6 production by osteoblasts/stromal cells are coupled in the process of osteoclastogenesis and in diseases where there is excessive osteolysis (Steeve et al., 2004). IL‐6 modulates this vital communication network between the osteoblasts and osteoclasts. The first action of IL‐6 is to stimulate osteoblastic production and secretion of factors that regulate osteoclastic activity, like RANKL (Steeve et al., 2004). Although IL‐6 was reported to increase RANKL secretion by osteoblasts in one study (Nakashima et al., 2000), we observed only a minor eVect of IL‐6 on this parameter (Liu et al., 2005). Our data are in agreement with that reported by Steeve et al. (2004) and O’Brien et al. (2000) that neither IL‐6 nor soluble IL‐6 receptor stimulates bone resorption when added separately. In contrast, significant stimulation of RANKL secretion and osteoclastogenesis was observed when IL‐6 and sIL‐6R were added in combination (O’Brien et al., 2000; Steeve et al., 2004). In addition, these studies showed that combination treatment with IL‐6 and sIL‐6R receptor stimulates OPG secretion by osteoblasts and inhibits RANK expression by osteoclasts (O’Brien et al., 2000; Steeve et al., 2004). However, we reported somewhat divergent eVects of IL‐6 on the OPG/RANKL/RANK system. Our in vitro studies demonstrate that IL‐6 alone has no significant eVect on RANKL secretion by osteoblasts. IL‐6 addition, however, increases RANK expression by osteoclasts. Finally, although exogenous IL‐6 had no detectable eVect on osteoblastic OPG production, IL‐6 neutralizing antibodies increased OPG secretion and reversed the observed suppression of OPG secretion by PGE2, suggesting a role for endogenous IL‐6 in mediating basal and inducible OPG production by osteoblasts (Liu et al., 2005). The net
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FIGURE 1. Interaction between IL‐6 and the OPG/RANKL/RANK system in osteoclastogenesis. The binding of RANKL to RANK activates osteoclasts, whereas RANKL binding to OPG, a decoy receptor, suppresses osteoclast activation. IL‐6 primarily modulates osteoclast functions via eVects on osteoblasts and the OPG/RANKL/RANK system. Overall eVect is inhibition of OPG, activation of RANKL/RANK, and promotion of osteoclast activation.
result of these eVects of IL‐6 (suppression of osteoblast OPG production and increased RANK expression in osteoclasts) is increased osteoclastic activity (Fig. 1). However, the precise mechanisms underlying the interaction between IL‐6 and the OPG/RANKL/RANK system in the regulation of osteoclastogenesis are unclear. It has been proposed that the activation of STAT3 induced by IL‐6/sIL‐6R/gp130 signaling not only increases RANKL expression but also enhances the sensitivity of osteoclast precursors to RANKL stimulation (Steeve et al., 2004), presumably by increasing RANK expression on these cells (Liu et al., 2005). Moreover, both RANKL and IL‐6 have been shown to activate protein kinase C (PKC) and the members of the MAPK signaling pathway (Sims et al., 2004; Steeve et al., 2004), such as ERK1/2, JNK, and p38, suggesting that IL‐6/sIL‐6/gp130 signaling and the OPG/RANKL/RANK system may share similar downstream signaling pathways and have synergistic eVects on osteoclastogenesis.
V. CROSS TALK BETWEEN IL‐6 AND STEROID HORMONES, PTH, PGE2, AND CYTOKINES The process of bone remodeling is the result of an elaborate network composed of growth factors, sex steroids, cytokines, and prostaglandins. IL‐6 has been shown to specifically interact with these other factors to promote osteoclastogenesis.
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A. IL‐6 AND SEX STEROIDS
Androgens and estrogens are key regulators of bone metabolism. Loss of gonadal function in either sex increases osteoclast precursor formation and inhibits osteoblast activity in the bone marrow, resulting in increased bone resorption and bone loss. Replacement of sex steroids in hypogonadal individuals of either sex prevents these changes (Bellido et al., 1995; Jilka et al., 1992). Estrogens specifically inhibit osteoclast activation with little or no direct eVect on osteoblasts. This eVect of estrogen is due, at least in part, to an interaction with IL‐6 (Jilka et al., 1992; Manolagas, 1998). It has been shown that loss of estrogens increases IL‐6 production in response to 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) or PTH by in vitro bone marrow cell cultures. IL‐6‐deficient mice do not exhibit an increase in osteoclastogenesis after ovariectomy and are protected from the bone loss caused by disrupted ovarian function (Poli et al., 1994). Increased osteoclastogenic activity resulting from loss of ovarian function in vivo is accompanied by elevated levels of IL‐6 in the microenvironment of the bone marrow (Jilka et al., 1992). Notably, these changes were prevented by the administration of 17b‐estradiol or neutralizing antibodies to IL‐6 (Jilka et al., 1992; Poli et al., 1994). Taken together, these data indicate that estrogen signaling in osteoclasts is mediated by IL‐6. This eVect of estrogens is regulated by protein–protein interactions between the estrogen receptor and other transcriptional factors such as NF‐kB or NF‐IL‐6 (Galien et al., 1996; Hughes and Howells, 1993). Similarly, in a separate study, orchiectomy in male mice resulted in an increase in osteoclast progenitors in the bone marrow, and this eVect was prevented by either androgen replacement or administration of IL‐6 neutralizing antibodies (Bellido et al., 1995). Bone histomorphometric analysis of trabecular bone reveals that IL‐6‐deficient mice exhibit no evidence of bone loss or elevated osteoclastic activity (Bellido et al., 1995; Manolagas, 1998). These data indicate that sex steroids, both estrogens and androgens, inhibit IL‐6 gene expression, and that IL‐6 mediates their eVects on bone metabolism.
B. IL‐6 AND PTH
PTH stimulates bone resorption indirectly by promoting the release of paracrine agents produced by osteoblasts, which recruit and activate osteoclasts. Among those factors, IL‐6 has been shown to be one of the critical cytokine mediators of PTH action. IL‐6 is produced by osteoblasts in response to PTH (Lowik et al., 1989). PTH‐induced bone resorption by osteoclast‐like cells is inhibited by neutralizing antibodies to the IL‐6 receptor (Greenfield et al., 1995). In vivo studies confirm that IL‐6 levels are increased by PTH in experimental animals (Grey et al., 1999). In addition,
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circulating levels of IL‐6 are elevated in patients with primary hyperparathyroidism and correlate with increased biochemical markers of bone resorption, suggesting that IL‐6 plays a permissive role in PTH‐induced bone eVects (Grey et al., 1999). C. IL‐6 AND PGE2
The roles of cyclooxygenase (COX)‐2 and PGE2, a major eicosanoid product of the COX‐2‐catalyzed reaction, in bone remodeling have been described in studies with mice that are genetically deficient in COX‐2. Mice lacking COX‐2 expression display reduced bone resorption in response to PTH or 1,25(OH)2D3 (Okada et al., 2000). In an in vitro study, COX‐2 and PGE2 have been shown to play important roles in osteoclast formation (Ono et al., 2002) and are required for debris‐induced osteoclastogenesis and osteolysis in an in vivo mouse calvaria model (Zhang et al., 2001). COX‐2/ PGE2 also participate in bone formation. Systemic or local injection of PGE2 stimulates bone formation in response to mechanical strain (Suponitzky and Weinreb, 1998), and this eVect is mediated by the COX‐2‐catalyzed pathway (Forwood, 1996). In addition, COX‐2 has been demonstrated to be a critical regulator of mesenchymal cell diVerentiation into osteoblasts and an essential element in bone repair (Zhang et al., 2002). These data indicate that COX‐2 and PGE2 are involved in both cytokine‐mediated osteoclast activation and osteoblastic bone formation. Moreover, it has been demonstrated that the actions of PGE2 in bone homeostasis are mediated through the cAMP signaling pathway, activated by the binding of PGE2 to subtypes of E‐series of prostaglandin (EP) receptor, particularly the EP2 and EP4 receptor subtypes (Li et al., 2000; Miyaura et al., 2000). Several lines of evidence support the notion that IL‐6 interacts with PGE2 in diVerent systems. EP4 receptor knockout mice have reduced circulating levels of IL‐6 and significantly less IL‐6 production by liver and macrophages (McCoy et al., 2002). COX‐2‐promoted human oropharyngeal carcinoma growth is mediated by IL‐6 (Hong et al., 2000). We observed that PGE2 stimulation of human prostate intraepithelial neoplasia cell growth is through activation of the IL‐6/gp130/STAT3 signaling pathway (Liu et al., 2002). Consistent with this data, it has been shown that stromal osteoblast cells support osteoclast diVerentiation by their ability to secrete IL‐6 and RANKL in response to PTH, 1,25(OH)2D3, and PGE2 (Gruber et al., 2000; Kozawa et al., 1998). These data provide mechanistic evidence of cross‐talk between the COX‐2/PGE2 and IL‐6 signaling systems. As previously mentioned, we demonstrated that IL‐6 stimulation of osteoclast diVerentiation in cocultures (with osteoblasts) was due to both an increase in RANK expression by osteoclasts and reciprocal interactions with the COX‐2/PGE2 system (Liu et al., 2005). In that same report, neutralizing antibodies to IL‐6 increased basal OPG secretion by osteoblasts and reversed the inhibitory
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eVects of PGE2 on this parameter, indicating that endogenous IL‐6 mediates PGE2 eVects on osteoblastic OPG secretion. IL‐6 also increased COX-2 expression and PGE2 production in osteoblasts. Finally, there is evidence that IL‐6 interacts with the EP receptor system in inflammatory carcinogenesis and rheumatoid arthritis (Hong et al., 2000; Sugiyama, 2001) through activation of the gp130/STAT3 signaling pathway (Gruber et al., 2000; Ni et al., 2000). Our data indicate that IL‐6 increases the expression of the EP4 receptor subtype in both osteoblasts and osteoclast precursors. IL‐6 also induces the EP2 receptor subtype expression in osteoblasts (Liu et al., 2005). The induction of EP4 and EP2 by IL‐6 in osteoblasts, in turn, amplifies the eVects of PGE2 on the inhibition of OPG production and stimulation of membrane‐bound RANKL expression and soluble RANKL release. All of these events, coordinately, tip the balance of the OPG/ RANKL/RANK system in favor of increased osteoclastogenesis and enhanced bone resorption. These results demonstrate significant cross talk between the IL‐6/gp130 and COX‐2/PGE2 signaling systems in the regulation of osteoclastogenesis and indicate that these eVects are mediated by the OPG/RANKL/RANK system.
D. IL‐6 AND OTHER INFLAMMATORY CYTOKINES
TNF‐a and IL‐1 are important cytokines that are involved in the regulation of bone metabolism. Experimental data indicate that TNF‐a, IL‐1, and IL‐6 stimulate osteoclast diVerentiation in a synergistic fashion (Ragab et al., 2002), primarily via eVects on the OPG/RANKL/RANK system (Steeve et al., 2004). These factors increase the production of both OPG and RANKL, but tip the balance in favor of increased RANKL/OPG production. TNF‐a has also been shown to stimulate both the proliferation and diVerentiation of cells in the osteoclast lineage (Kobayashi et al., 2000; Kudo et al., 2002). TNF‐a can act in combination with RANKL produced in the marrow microenvironment to enhance the actions of macrophage colony‐stimulating factor (MCSF) on osteoclast formation (Kudo et al., 2002), and this eVect can be significantly increased by combination treatment with TNF‐a and IL‐6 (Gorny et al., 2004). In addition, TNF‐a stimulates a significant increase in IL‐6 production by osteoblasts. However, it is not clear whether the eVect of TNF‐a is directly mediated by its eVect on IL‐6 secretion in bone cells. IL‐1 increases RANKL expression by osteoblasts and this eVect is significantly enhanced by IL‐6 (Gorny et al., 2004; Kudo et al., 2002). The interactive eVects of IL‐6, TNF‐a, and IL‐1 have been proposed to involve activation/ inactivation of a cascade of intracellular signaling events including the activation of two members of the MAPK family (i.e., ERK and p38) and JNK (Steeve et al., 2004).The ERKpathway negatively regulates osteoclastogenesis whereas the p38 pathway promotes this process (Steeve et al., 2004).
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VI. GP130 SIGNALING AND BONE REMODELING IL‐6‐type cytokines regulate bone remodeling by activating the gp130 receptor subunit, which, in turn, stimulates the intracellular cascade of signal transduction and induces target gene expression. The expression levels of gp130 in bone marrow stromal cells have been shown to determine the magnitude of the cascade signals generated by IL‐6‐type cytokines (O’Brien, et al., 2000). Recent studies demonstrate that gp130 signaling proceeds via at least two intracellular pathways, that is, the STAT1/3 (Stahl et al., 1995) and SHP2/ras/MAPK (Fukuta, 1996) pathways. The activation of these pathways is ligand‐ and tissue‐specific and leads to induction of distinct sets of target genes and, thus, distinct biological consequences (Kamimura et al., 2003; Sims et al., 2004). The eVects of the activation of these two gp130‐ associated pathways on bone remodeling have been investigated. Sims et al. (2004) analyzed mice in which gp130 signaling via either the STAT1/3 or MAPK pathway was attenuated by conditional mutation and demonstrated a dual role for gp130 in osteoclastogenesis based on its simultaneous expression in osteoblast and osteoclast precursors (Gao et al., 1998; Sims et al., 2004). These studies also revealed that there are three key pathways by which gp130 signaling contributes to bone cell interactions. First, the activation of STAT1/3 pathway induced by gp130 cytokines plays an essential role in stimulating chondrocyte proliferation and disruption of this pathway results in premature growth plate closure and reduced bone size. Second, the SHP2/ ras/MAPK pathway of the gp130 signaling plays an equally key role in the inhibition of osteoclastogenesis. In the absence of this latter pathway, a high level of osteoclastogenesis is observed that leads to trabecular and cortical bone loss. Finally, IL‐6 stimulates osteoblasts, and this eVect is mediated by the gp130‐STAT1/3 pathway. The IL‐6/gp130‐STAT1/3 activation of osteoblast function is exaggerated in the absence of the gp130‐SHP2/ras/MAPK pathway, producing increased osteoblastic bone formation (Sims et al., 2004). The exact pathway whereby IL‐6 type cytokines stimulate osteoclastic activity has not been elucidated. These available data indicate that IL‐6‐type cytokines have diverse eVects on bone metabolism depending upon cell‐ specific intracellular signaling cascades (either STAT1/3 or SHP2/ras/ MAPK pathway, or both) in response to gp130 activation.
VII. SUMMARY There is increasing evidence that the IL‐6 family of cytokines and signaling pathways downstream of the common gp130 receptor subunit play critical roles in the processes of bone homeostasis and remodeling. The binding of IL‐6 to sIL‐6R is a prerequisite step for the activation of
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gp130/STAT3 signaling. Once gp130 is activated, there are at least two intracellular signaling cascades that can transmit divergent signals to bone cells. Although there is some conflicting data regarding the eVects of IL‐6 on bone, it is generally accepted that the primary eVect of this cytokine group is the stimulation of osteoclastic bone resorption. In addition, IL‐6 eVects on osteoclasts appear to be predominantly indirect via modulation of osteoblasts and the OPG/RANKL/RANK system that coordinates the functions and activities of osteoblasts and osteoclasts. IL‐6 alone has minimal eVects on osteoclast diVerentiation and activation. However, IL‐6 acts synergistically with growth factors, steroid hormones, inflammatory cytokines, and prostaglandins in bone cells. Reciprocal interactions between IL‐6 and PGE2 are particularly relevant to bone metabolism. IL‐6 enhances the expression of two subtypes of EP receptor and mediates PGE2‐induced suppression of OPG secretion. A better understanding of the complex, cell‐ specific, interactive eVects of IL‐6 and various bone modulators may uncover new paradigms for the treatment of bone diseases.
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I. II. III. IV.
Introduction EVect of IL‐6 on Bone Resorption EVect of IL‐6 on Bone Formation Interactions Between IL‐6/gp130 Signaling and the OPG/RANKL/RANK System V. Cross Talk Between IL‐6 and Steroid Hormones, PTH, PGE2, and Cytokines A. IL‐6 and Sex Steroids B. IL‐6 and PTH C. IL‐6 and PGE2 D. IL‐6 and Other Inflammatory Cytokines VI. gp130 Signaling and Bone Remodeling VII. Summary References
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I. INTRODUCTION The interleukin‐6 (IL‐6) family of cytokines includes IL‐6, IL‐11, leukemia inhibitory factor (LIF), oncostain M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin‐1 (CT‐1) (Kishimoto et al., 1995). Increasing evidence suggests that IL‐6‐type cytokines regulate immune and inflammatory responses, hepatic acute‐phase protein synthesis, hematopoiesis, and bone metabolism (Kishimoto, 1989). All of the members of the IL‐6 family have overlapping functions and utilize the same receptor subunit, glycoprotein 130 (gp130), a critical signal‐transducing component previously identified as IL‐6 receptor‐b (IL‐6Rb) (Kishimoto et al., 1995). In target cells, IL‐6 can simultaneously induce functionally distinct and even contradictory signals through its receptor complex, IL‐6Ra (or soluble receptor, sIL‐6R) and gp130. The binding of IL‐6 to IL‐6Ra induces dimerization of gp130 (Taga and Kishimoto, 1997), and initiates a cascade of intracellular signaling that leads to activation of signal transducers and activators of transcription (STAT)1/3 and/or SHP2/ras/MAPK, a process that culminates in modulation of gene transcription (Heymann and Rousselle, 2000; Kamimura et al., 2003; O’Brien et al., 2000). There is ample evidence that IL‐6‐type cytokines play an important role in skeletal homeostasis (Manolagas and Jilka, 1995; Martin et al., 1998; Suda et al., 1999). IL‐6 influences both osteoblast and osteoclast activities through a variety of complex mechanisms. For example, IL‐6 has been shown to promote the diVerentiation of macrophages and bone marrow stromal cells into osteoclasts and osteoblasts, respectively (Erices et al., 2002; Manolagas, 1998; Sims et al., 2004). In addition, IL‐6 interacts with other factors that are critically involved in bone remodeling, including the OPG/RANKL/RANK system, sex steroids (estrogens and androgens), prostaglandin E2 (PGE2), tumor necrosis factor (TNF)‐a, parathyroid hormone (PTH), IL‐11, and IL‐1, to regulate bone turnover (Grey et al., 1999; Liu et al., 2005; Steeve et al., 2004). In this chapter, we will highlight the biological eVects and clinical relevance of IL‐6 and its associated receptors in bone development and remodeling. Recent data on the regulation of IL‐6/gp130 signaling pathway in bone homeostasis will also be discussed with an emphasis on how one cytokine can initiate diverse eVects in bone in a cell‐specific fashion.
II. EFFECT OF IL‐6 ON BONE RESORPTION Bone tissue remodeling results from the coordinate activities of osteoblasts and osteoclasts. Osteoclasts are derived from hematopoietic precursors of the bone marrow (Roodman, 1996) and are highly specialized, multinucleated cells that are uniquely capable of lacunar bone resorption. Imbalances in the bone remodeling process result in metabolic bone diseases characterized
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by either enhanced osteoclast activity and increased bone resorption (i.e., osteoporosis and osteolytic bone lesions in cancer) or increased osteoblastic bone formation (i.e., osteopetrosis and prostate cancer‐induced osteoblastic metastases) (Fohr et al., 2003; Ohlsson et al., 1998). IL‐6 increases osteoclast recruitment by acting on early hematopoietic cells from the granulocyte‐macrophage lineage that contain the progenitors of the osteoclastic lineage (Otsuka et al., 1991). IL‐6 has also been shown to play a specific role in postmenopausal osteoporosis (Jilka et al., 1992; Poli et al., 1994; Tarura et al., 1993). A possible mechanism for this eVect was decreased osteoclast formation due to the lack of IL‐6 (Tarura et al., 1993). A cross‐ sectional study in humans compared bone turnover and bone mineral density (BMD) in healthy postmenopausal women with diVerent G‐C polymorphisms at position –174 in the IL‐6 promoter. Patients with either CG or GG genotypes were associated with higher bone resorption and lower BMD at the hip and forearm compared with those in patients with the CC genotype (Ferrari, 2001). The study suggests that allelic variations in the IL‐6 promoter influence IL‐6 production and contribute to increased bone resorption and decreased BMD. High IL‐6 levels also correlate with high bone resorption indices in patients with rheumatoid arthritis. Clinical studies reveal a negative correlation between BMD and the combination of intracellular and cell‐ surface‐bound levels of IL‐6 in peripheral blood monocytes from rheumatoid arthritis patients (Verbruggen et al., 1999). In multiple myeloma, IL‐6 is one of the cytokines that promote bone destruction (Kuehl and Bergsagel, 2002). Multiple myeloma is a plasma cell dyscrasia characterized by proliferation of malignant B cells in the bone marrow and severe bone loss. Higher IL‐6 serum levels in multiple myeloma are associated with more advanced stages of the disease (II and III). Furthermore, the serum level of IL‐6 has been identified as a significant prognostic marker in multiple myeloma. Ludwig et al. (1991) found that survival times diVered significantly between patients whose IL‐6 levels at the time of diagnosis fell below 7 pg/ml and those with higher levels. The 50% of patients in the former category exhibited a medium survival of 53.7 months as compared with only 2.7 months in the latter category (Ludwig et al., 1991). Although the mechanisms underlying these observed eVects of IL‐6 are not well understood, one report demonstrated that IL‐6 modulates trabecular and endochondral bone turnover in vivo by stimulating osteoclast diVerentiation (Rozen et al., 2000). Bone remodeling is the result of the coordinate and interactive eVects of both types of osteogenic cells (osteoblasts and osteoclasts). Although high levels of IL‐6 stimulate bone resorption directely, IL‐6, like many other regulatory factors (i.e., growth factors, hormones, cytokines), predominantly modulates osteoclast activity and bone resorption indirectly by influencing osteoblast diVerentiation and secretion. In bone cells, IL‐6 production is limited to cells of the osteoblastic lineage (Holt et al., 1996; Legrand‐Poels et al., 2000). Moreover, although both osteoblasts and osteoclasts express the
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IL‐6 receptor, functional studies demonstrate that IL‐6‐induced osteoclast diVerentiation is dependent upon IL‐6 receptor expression by the osteoblast, but not the osteoclast (Udagawa et al., 1995). In agreement with these observations, our in vitro studies demonstrate that IL‐6 significantly stimulates osteoclast diVerentiation only when they are grown in coculture with osteoblasts (Liu et al., 2005). However, a direct eVect of IL‐6 on inducing osteoclast formation has also been reported in several osteolytic bone disorders (Kudo et al., 2003).
III. EFFECT OF IL‐6 ON BONE FORMATION Mature osteoblasts are derived from multipotent mesenchymal stem cells of the bone marrow that also give rise to the fibroblast cells of the marrow stroma, chondrocytes, adipocytes, and muscle cells (Aubin, 1998). Stromal cells of the bone marrow and osteoblasts exhibit an extensive overlap of phenotypic properties (Manolagas, 1998). The initial evidence that the IL‐6 family of cytokines influences bone formation was provided by the observation that overexpression of either LIF or OSM gene (two IL‐6 family members) in mice induces excessive bone formation (Lowe et al., 1991; Malik et al., 1995; Metcalf and Gearing, 1989). In addition, IL‐6, LIF, and OSM have been reported to confer an antiapoptotic phenotype on osteoblastic cells via enhanced transcriptional activation of the p21 gene (Bellido et al., 1998; Jilka et al., 1998; Steeve et al., 2004). IL‐6 also promotes osteogenic lineage commitment. IL‐6 plus soluble IL‐6 receptor or LIF stimulates the commitment of embryonic fibroblasts (12th–14th day of gestation) toward the osteoblast phenotype without any promotion of diVerentiation toward adipocytes, chondrocytes, or muscle cells (Tagochi et al., 1998). It has also been reported that the diVerentiation of mesenchymal progenitors of osteoblasts in the murine bone marrow can be stimulated by IL‐6 plus soluble IL‐6 receptor or LIF (Tagochi et al., 1998). IL‐6 as well as LIF, IL‐11, CNTF, and OSM promote the diVerentiation of committed osteoblast cells toward a more mature phenotype, and this action of the IL‐6 family of cytokines is mediated by the activation of the Janus Kinase (JAK)/STAT pathway (Bellido et al., 1997). However, Hughes and Howells (1993) have shown an inhibitory eVect of IL‐6 on bone formation in vitro. These apparently contradictory observations may be due to the diVerences in the experimental systems and methods utilized in the two studies. Both osteoblasts and osteoclasts express the IL‐6 receptor subunits, that is, gp130 and soluble IL‐6 receptor. However, the intracellular signaling cascades induced by IL‐6 activation of gp130 are complex and may be cell specific. It is possible that the observed dual eVect of IL‐6 on bone is due to cell‐ specific activation of diVerent intracellular signaling pathways in osteoblasts versus osteoclasts (Sims et al., 2004).
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IV. INTERACTIONS BETWEEN IL‐6/GP130 SIGNALING AND THE OPG/RANKL/RANK SYSTEM Osteoblast function is intimately tied to osteoclast activity. The interactions between osteoblasts and osteoclasts can be established through cell–cell contact (Jimi et al., 1996) and are mediated by receptor activator of nuclear factor‐kappaB (NF‐kB) ligand (RANKL) produced by osteoblasts and receptor activator of NF‐kB (RANK) on the osteoclast surface (Nakagawa et al., 1998; Yasuda et al., 1998). Bone marrow stromal cells (osteoblast precursor cells) also produce a soluble glycoprotein called osteoprotegerin (OPG), a decoy receptor for RANKL that prevents osteoclast activation (Simonet and Luthy, 1997; Yasuda et al., 1998). OPG and RANKL, synthesized by stromal cells/osteoblasts, have been identified as the two principal cytokines that regulate osteoclast diVerentiation and activation (Lacey et al., 1998; Simonet and Luthy, 1997). Hormones, growth factors, cytokines, and prostaglandins regulate these processes (Brandstrom et al., 2001; Ohlsson et al., 1998) mainly via eVects on osteoblasts. In general, the osteoblasts receive input directly from a variety of regulatory factors and then transmit these signals to the neighboring osteoclasts via the OPG/RANKL/RANK system. RANKL and IL‐6 production by osteoblasts/stromal cells are coupled in the process of osteoclastogenesis and in diseases where there is excessive osteolysis (Steeve et al., 2004). IL‐6 modulates this vital communication network between the osteoblasts and osteoclasts. The first action of IL‐6 is to stimulate osteoblastic production and secretion of factors that regulate osteoclastic activity, like RANKL (Steeve et al., 2004). Although IL‐6 was reported to increase RANKL secretion by osteoblasts in one study (Nakashima et al., 2000), we observed only a minor eVect of IL‐6 on this parameter (Liu et al., 2005). Our data are in agreement with that reported by Steeve et al. (2004) and O’Brien et al. (2000) that neither IL‐6 nor soluble IL‐6 receptor stimulates bone resorption when added separately. In contrast, significant stimulation of RANKL secretion and osteoclastogenesis was observed when IL‐6 and sIL‐6R were added in combination (O’Brien et al., 2000; Steeve et al., 2004). In addition, these studies showed that combination treatment with IL‐6 and sIL‐6R receptor stimulates OPG secretion by osteoblasts and inhibits RANK expression by osteoclasts (O’Brien et al., 2000; Steeve et al., 2004). However, we reported somewhat divergent eVects of IL‐6 on the OPG/RANKL/RANK system. Our in vitro studies demonstrate that IL‐6 alone has no significant eVect on RANKL secretion by osteoblasts. IL‐6 addition, however, increases RANK expression by osteoclasts. Finally, although exogenous IL‐6 had no detectable eVect on osteoblastic OPG production, IL‐6 neutralizing antibodies increased OPG secretion and reversed the observed suppression of OPG secretion by PGE2, suggesting a role for endogenous IL‐6 in mediating basal and inducible OPG production by osteoblasts (Liu et al., 2005). The net
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FIGURE 1. Interaction between IL‐6 and the OPG/RANKL/RANK system in osteoclastogenesis. The binding of RANKL to RANK activates osteoclasts, whereas RANKL binding to OPG, a decoy receptor, suppresses osteoclast activation. IL‐6 primarily modulates osteoclast functions via eVects on osteoblasts and the OPG/RANKL/RANK system. Overall eVect is inhibition of OPG, activation of RANKL/RANK, and promotion of osteoclast activation.
result of these eVects of IL‐6 (suppression of osteoblast OPG production and increased RANK expression in osteoclasts) is increased osteoclastic activity (Fig. 1). However, the precise mechanisms underlying the interaction between IL‐6 and the OPG/RANKL/RANK system in the regulation of osteoclastogenesis are unclear. It has been proposed that the activation of STAT3 induced by IL‐6/sIL‐6R/gp130 signaling not only increases RANKL expression but also enhances the sensitivity of osteoclast precursors to RANKL stimulation (Steeve et al., 2004), presumably by increasing RANK expression on these cells (Liu et al., 2005). Moreover, both RANKL and IL‐6 have been shown to activate protein kinase C (PKC) and the members of the MAPK signaling pathway (Sims et al., 2004; Steeve et al., 2004), such as ERK1/2, JNK, and p38, suggesting that IL‐6/sIL‐6/gp130 signaling and the OPG/RANKL/RANK system may share similar downstream signaling pathways and have synergistic eVects on osteoclastogenesis.
V. CROSS TALK BETWEEN IL‐6 AND STEROID HORMONES, PTH, PGE2, AND CYTOKINES The process of bone remodeling is the result of an elaborate network composed of growth factors, sex steroids, cytokines, and prostaglandins. IL‐6 has been shown to specifically interact with these other factors to promote osteoclastogenesis.
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A. IL‐6 AND SEX STEROIDS
Androgens and estrogens are key regulators of bone metabolism. Loss of gonadal function in either sex increases osteoclast precursor formation and inhibits osteoblast activity in the bone marrow, resulting in increased bone resorption and bone loss. Replacement of sex steroids in hypogonadal individuals of either sex prevents these changes (Bellido et al., 1995; Jilka et al., 1992). Estrogens specifically inhibit osteoclast activation with little or no direct eVect on osteoblasts. This eVect of estrogen is due, at least in part, to an interaction with IL‐6 (Jilka et al., 1992; Manolagas, 1998). It has been shown that loss of estrogens increases IL‐6 production in response to 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) or PTH by in vitro bone marrow cell cultures. IL‐6‐deficient mice do not exhibit an increase in osteoclastogenesis after ovariectomy and are protected from the bone loss caused by disrupted ovarian function (Poli et al., 1994). Increased osteoclastogenic activity resulting from loss of ovarian function in vivo is accompanied by elevated levels of IL‐6 in the microenvironment of the bone marrow (Jilka et al., 1992). Notably, these changes were prevented by the administration of 17b‐estradiol or neutralizing antibodies to IL‐6 (Jilka et al., 1992; Poli et al., 1994). Taken together, these data indicate that estrogen signaling in osteoclasts is mediated by IL‐6. This eVect of estrogens is regulated by protein–protein interactions between the estrogen receptor and other transcriptional factors such as NF‐kB or NF‐IL‐6 (Galien et al., 1996; Hughes and Howells, 1993). Similarly, in a separate study, orchiectomy in male mice resulted in an increase in osteoclast progenitors in the bone marrow, and this eVect was prevented by either androgen replacement or administration of IL‐6 neutralizing antibodies (Bellido et al., 1995). Bone histomorphometric analysis of trabecular bone reveals that IL‐6‐deficient mice exhibit no evidence of bone loss or elevated osteoclastic activity (Bellido et al., 1995; Manolagas, 1998). These data indicate that sex steroids, both estrogens and androgens, inhibit IL‐6 gene expression, and that IL‐6 mediates their eVects on bone metabolism.
B. IL‐6 AND PTH
PTH stimulates bone resorption indirectly by promoting the release of paracrine agents produced by osteoblasts, which recruit and activate osteoclasts. Among those factors, IL‐6 has been shown to be one of the critical cytokine mediators of PTH action. IL‐6 is produced by osteoblasts in response to PTH (Lowik et al., 1989). PTH‐induced bone resorption by osteoclast‐like cells is inhibited by neutralizing antibodies to the IL‐6 receptor (Greenfield et al., 1995). In vivo studies confirm that IL‐6 levels are increased by PTH in experimental animals (Grey et al., 1999). In addition,
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circulating levels of IL‐6 are elevated in patients with primary hyperparathyroidism and correlate with increased biochemical markers of bone resorption, suggesting that IL‐6 plays a permissive role in PTH‐induced bone eVects (Grey et al., 1999). C. IL‐6 AND PGE2
The roles of cyclooxygenase (COX)‐2 and PGE2, a major eicosanoid product of the COX‐2‐catalyzed reaction, in bone remodeling have been described in studies with mice that are genetically deficient in COX‐2. Mice lacking COX‐2 expression display reduced bone resorption in response to PTH or 1,25(OH)2D3 (Okada et al., 2000). In an in vitro study, COX‐2 and PGE2 have been shown to play important roles in osteoclast formation (Ono et al., 2002) and are required for debris‐induced osteoclastogenesis and osteolysis in an in vivo mouse calvaria model (Zhang et al., 2001). COX‐2/ PGE2 also participate in bone formation. Systemic or local injection of PGE2 stimulates bone formation in response to mechanical strain (Suponitzky and Weinreb, 1998), and this eVect is mediated by the COX‐2‐catalyzed pathway (Forwood, 1996). In addition, COX‐2 has been demonstrated to be a critical regulator of mesenchymal cell diVerentiation into osteoblasts and an essential element in bone repair (Zhang et al., 2002). These data indicate that COX‐2 and PGE2 are involved in both cytokine‐mediated osteoclast activation and osteoblastic bone formation. Moreover, it has been demonstrated that the actions of PGE2 in bone homeostasis are mediated through the cAMP signaling pathway, activated by the binding of PGE2 to subtypes of E‐series of prostaglandin (EP) receptor, particularly the EP2 and EP4 receptor subtypes (Li et al., 2000; Miyaura et al., 2000). Several lines of evidence support the notion that IL‐6 interacts with PGE2 in diVerent systems. EP4 receptor knockout mice have reduced circulating levels of IL‐6 and significantly less IL‐6 production by liver and macrophages (McCoy et al., 2002). COX‐2‐promoted human oropharyngeal carcinoma growth is mediated by IL‐6 (Hong et al., 2000). We observed that PGE2 stimulation of human prostate intraepithelial neoplasia cell growth is through activation of the IL‐6/gp130/STAT3 signaling pathway (Liu et al., 2002). Consistent with this data, it has been shown that stromal osteoblast cells support osteoclast diVerentiation by their ability to secrete IL‐6 and RANKL in response to PTH, 1,25(OH)2D3, and PGE2 (Gruber et al., 2000; Kozawa et al., 1998). These data provide mechanistic evidence of cross‐talk between the COX‐2/PGE2 and IL‐6 signaling systems. As previously mentioned, we demonstrated that IL‐6 stimulation of osteoclast diVerentiation in cocultures (with osteoblasts) was due to both an increase in RANK expression by osteoclasts and reciprocal interactions with the COX‐2/PGE2 system (Liu et al., 2005). In that same report, neutralizing antibodies to IL‐6 increased basal OPG secretion by osteoblasts and reversed the inhibitory
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eVects of PGE2 on this parameter, indicating that endogenous IL‐6 mediates PGE2 eVects on osteoblastic OPG secretion. IL‐6 also increased COX-2 expression and PGE2 production in osteoblasts. Finally, there is evidence that IL‐6 interacts with the EP receptor system in inflammatory carcinogenesis and rheumatoid arthritis (Hong et al., 2000; Sugiyama, 2001) through activation of the gp130/STAT3 signaling pathway (Gruber et al., 2000; Ni et al., 2000). Our data indicate that IL‐6 increases the expression of the EP4 receptor subtype in both osteoblasts and osteoclast precursors. IL‐6 also induces the EP2 receptor subtype expression in osteoblasts (Liu et al., 2005). The induction of EP4 and EP2 by IL‐6 in osteoblasts, in turn, amplifies the eVects of PGE2 on the inhibition of OPG production and stimulation of membrane‐bound RANKL expression and soluble RANKL release. All of these events, coordinately, tip the balance of the OPG/ RANKL/RANK system in favor of increased osteoclastogenesis and enhanced bone resorption. These results demonstrate significant cross talk between the IL‐6/gp130 and COX‐2/PGE2 signaling systems in the regulation of osteoclastogenesis and indicate that these eVects are mediated by the OPG/RANKL/RANK system.
D. IL‐6 AND OTHER INFLAMMATORY CYTOKINES
TNF‐a and IL‐1 are important cytokines that are involved in the regulation of bone metabolism. Experimental data indicate that TNF‐a, IL‐1, and IL‐6 stimulate osteoclast diVerentiation in a synergistic fashion (Ragab et al., 2002), primarily via eVects on the OPG/RANKL/RANK system (Steeve et al., 2004). These factors increase the production of both OPG and RANKL, but tip the balance in favor of increased RANKL/OPG production. TNF‐a has also been shown to stimulate both the proliferation and diVerentiation of cells in the osteoclast lineage (Kobayashi et al., 2000; Kudo et al., 2002). TNF‐a can act in combination with RANKL produced in the marrow microenvironment to enhance the actions of macrophage colony‐stimulating factor (MCSF) on osteoclast formation (Kudo et al., 2002), and this eVect can be significantly increased by combination treatment with TNF‐a and IL‐6 (Gorny et al., 2004). In addition, TNF‐a stimulates a significant increase in IL‐6 production by osteoblasts. However, it is not clear whether the eVect of TNF‐a is directly mediated by its eVect on IL‐6 secretion in bone cells. IL‐1 increases RANKL expression by osteoblasts and this eVect is significantly enhanced by IL‐6 (Gorny et al., 2004; Kudo et al., 2002). The interactive eVects of IL‐6, TNF‐a, and IL‐1 have been proposed to involve activation/ inactivation of a cascade of intracellular signaling events including the activation of two members of the MAPK family (i.e., ERK and p38) and JNK (Steeve et al., 2004).The ERKpathway negatively regulates osteoclastogenesis whereas the p38 pathway promotes this process (Steeve et al., 2004).
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VI. GP130 SIGNALING AND BONE REMODELING IL‐6‐type cytokines regulate bone remodeling by activating the gp130 receptor subunit, which, in turn, stimulates the intracellular cascade of signal transduction and induces target gene expression. The expression levels of gp130 in bone marrow stromal cells have been shown to determine the magnitude of the cascade signals generated by IL‐6‐type cytokines (O’Brien, et al., 2000). Recent studies demonstrate that gp130 signaling proceeds via at least two intracellular pathways, that is, the STAT1/3 (Stahl et al., 1995) and SHP2/ras/MAPK (Fukuta, 1996) pathways. The activation of these pathways is ligand‐ and tissue‐specific and leads to induction of distinct sets of target genes and, thus, distinct biological consequences (Kamimura et al., 2003; Sims et al., 2004). The eVects of the activation of these two gp130‐ associated pathways on bone remodeling have been investigated. Sims et al. (2004) analyzed mice in which gp130 signaling via either the STAT1/3 or MAPK pathway was attenuated by conditional mutation and demonstrated a dual role for gp130 in osteoclastogenesis based on its simultaneous expression in osteoblast and osteoclast precursors (Gao et al., 1998; Sims et al., 2004). These studies also revealed that there are three key pathways by which gp130 signaling contributes to bone cell interactions. First, the activation of STAT1/3 pathway induced by gp130 cytokines plays an essential role in stimulating chondrocyte proliferation and disruption of this pathway results in premature growth plate closure and reduced bone size. Second, the SHP2/ ras/MAPK pathway of the gp130 signaling plays an equally key role in the inhibition of osteoclastogenesis. In the absence of this latter pathway, a high level of osteoclastogenesis is observed that leads to trabecular and cortical bone loss. Finally, IL‐6 stimulates osteoblasts, and this eVect is mediated by the gp130‐STAT1/3 pathway. The IL‐6/gp130‐STAT1/3 activation of osteoblast function is exaggerated in the absence of the gp130‐SHP2/ras/MAPK pathway, producing increased osteoblastic bone formation (Sims et al., 2004). The exact pathway whereby IL‐6 type cytokines stimulate osteoclastic activity has not been elucidated. These available data indicate that IL‐6‐type cytokines have diverse eVects on bone metabolism depending upon cell‐ specific intracellular signaling cascades (either STAT1/3 or SHP2/ras/ MAPK pathway, or both) in response to gp130 activation.
VII. SUMMARY There is increasing evidence that the IL‐6 family of cytokines and signaling pathways downstream of the common gp130 receptor subunit play critical roles in the processes of bone homeostasis and remodeling. The binding of IL‐6 to sIL‐6R is a prerequisite step for the activation of
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gp130/STAT3 signaling. Once gp130 is activated, there are at least two intracellular signaling cascades that can transmit divergent signals to bone cells. Although there is some conflicting data regarding the eVects of IL‐6 on bone, it is generally accepted that the primary eVect of this cytokine group is the stimulation of osteoclastic bone resorption. In addition, IL‐6 eVects on osteoclasts appear to be predominantly indirect via modulation of osteoblasts and the OPG/RANKL/RANK system that coordinates the functions and activities of osteoblasts and osteoclasts. IL‐6 alone has minimal eVects on osteoclast diVerentiation and activation. However, IL‐6 acts synergistically with growth factors, steroid hormones, inflammatory cytokines, and prostaglandins in bone cells. Reciprocal interactions between IL‐6 and PGE2 are particularly relevant to bone metabolism. IL‐6 enhances the expression of two subtypes of EP receptor and mediates PGE2‐induced suppression of OPG secretion. A better understanding of the complex, cell‐ specific, interactive eVects of IL‐6 and various bone modulators may uncover new paradigms for the treatment of bone diseases.
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