Selective Estrogen Receptor Modulators. Antonio Cano

Chapter 7. Physiological Regulation of Bone Metabolism and Estrogen Agonism

• Miguel Angel Garci'a-Pérez

The adult skeleton is periodically remodeled by transitory anatomic structures that contain juxtaposed osteoclast and osteoblast teams and that replace old bone with new bone. The purpose of this remodeling is both to prevent bone aging and repair the damage that occurs as well as to guarantee a contribution of minerals, especially calcium, to body cells for their correct function. In the last few years, due mainly to the research in molecular biology and cellular differentiation and to studies of genetically manipulated mice, it has been possible to discover many aspects both of the cellular and molecular bases of this bone remodeling as well as of the differentiation and function of the two main implied cell types: osteoblasts and osteoclasts.

This chapter will focus mainly on the effects that the modulation of the estrogen receptor (ER) determines on bone metabolism. This information will contribute to a better understanding of the data presented in the next chapter, which is dedicated to demonstrated SERM actions on bone. Much of our knowledge on the role of ER agonism in the field derives from the observation of the action of estrogens. Particular attention will be paid in this chapter to the role that estrogens have in normal bone remodeling and the one that is established when the protection of sex steroids ceases during menopause. Certainly, estrogens and androgens slow the rate of bone remodeling and protect against bone loss. Conversely, loss of estrogen leads to an increased rate of remodeling and inclines the balance between bone resorption and formation in favor of the former.

The regulation of this process is very complex because there are many cytokines and growth factors implicated and because systemic hormones control production of numerous local mediators in the bone microenvironment. Nevertheless, it has recently been possible to expand our knowledge of the factors that govern this bone remodeling with the discovery of decisive molecules for the differentiation and function of osteoclasts. These molecules are proteins belonging to the tumor necrosis factor (TNF) superfamily: osteoprotegerin (OPG), the receptor activator of nuclear factor-кB ligand (RANKL), and their receptor RANK. Nevertheless, other molecules such as TNF-a, IL-1, and IL-6 are important mediators during bone remodeling, in particular after estrogen deficiency.

7.1

Normal Bone Remodeling

The skeleton is a specialized and dynamic organ subjected to continuous regeneration. In adult skeleton, this process, known as bone remodeling, consists of the renovation of old bone by new bone in the same anatomical place (Frost 1973). In adult vertebrates, 10% of the skeletal bone mass is replaced every year, amounting to a complete structural overhaul every decade. Bone resorption and formation are closely linked within spatiotemporal anatomic structures called the basic multicellular unit (BMU) (Parfitt 1994). A working and mature BMU consists of an osteoclast team in the front degrading bone followed by an osteoblast team forming new bone (Fig. 7.1). Although the role of this bone remodeling in the mature skeleton is not completely clarified, it is believed that it serves not only to repair damage, to prevent bone aging and the underlying consequences, but also to assure appropriate blood levels of calcium, which is needed for cell function (Manolagas 2000).

The two main arguments in favor of bone remodeling being principally an autocrine-paracrine function are that bone remodeling occurs simultaneously in multiple locations and that cells of osteoblast lineage participate in osteoclast differentation (Rodan et al. 1981; Lacey et al. 1998; Simonet et al. 1997; Yasuda et al. 1998). Early progenitors of hematopoietic lineage differentiate into osteoclasts when they receive appropriate signals from stromal/osteoblastic (stromal/OB) support cells. These support cells express M-CSF and RANKL to promote differentiation of osteoclast progenitors. In addition, the process is subject to both negative and positive control by acomplex network of transcriptional regulators, of circulating hormones, and of locally produced cytokines acting on RANKL and OPG synthesis such as parathyroid hormone (PTH), 1,25-vitamin D3 (vitamin D3), TNF-α, IL-1, and IL-6 (Manolagas et al. 1995).

The factors responsible for the initiation of a BMU are unknowns, although there is evidence to suggest that osteocytes are implicated (Verborgt et al. 2000). The osteocytes are the most abundant cells in bone, and they derive from osteoblasts that have been absorbed into the bone matrix as a consequence of the bone-forming function of osteoblasts. Osteocytes communicate with each other and with the cells that line the bone surface via an extensive canalicular network (Jilka 2003), so that they can detect areas of bone that should be repaired and transmit the appropriate signals to osteoprogenitors in BM to begin a new BMU (Jilka 2003). The mechanism by which bone cells reach BMUs await full clarification, especially in those parts of the skeleton where hematopoietic marrow is sparse or absent. In these parts, the circulatory route is the only route by which osteoclasts can reach bone, although this is not valid for osteoblasts since preosteoblasts are not known to circulate (Parfitt 2000). Recruitment of osteoblasts can be due to the release of growth factors from the bone matrix during bone resorption, to derived signals from endothelial cells that participate in the BMU, to differentiation of the near stromal cells, or even to differentiation of cells that initially displayed a vascular phenotype (Parfitt 2000, 2001).

Fig. 7.1. Bone remodeling cycle in basic multicellular unit (BMU). After microdamage to bone, or following mechanical stress or chemical or cellular signaling, a BMU will originate. The preosteoclasts, which are related to macrophages, appear and, by means of different stages, in which the activation of several genes intervenes, and after the action of soluble cytokines, they transform into multinucleated osteoclasts. The mature osteoclasts resorb bone (A-C). While advancing the BMU, new osteoclasts are continuously activated and start resorption. After resorption, osteoclasts disappear (probably by apoptosis), and this or other poorly known signals, such as bone-derived growth factors that are released by resorption, probably attract osteoblasts (D). Osteoblasts start the formation of new bone by secreting osteoid that later mineralizes (E). The final osteoblasts turn into lining, while some of the osteoblasts turn into osteocytes that remain in the bone, connected by long cell processes that can sense mechanical stresses to the bones called canaliculi

7.2

Executive Cells: Osteoblasts and Osteoclasts

The most important cells implied in bone remodeling are the osteoclasts and osteoblast, although in this process different cellular types such as endothelial cells, stromal cells, lining cells, osteocytes, and T-cells participate. Bone formation is a complex process involving the commitment of osteoprogenitor cells, the proliferation of preosteoblasts, and their differentiation into mature and functional osteoblasts. Osteoblasts come from multipotential undifferentiated mesenchymal stem cells that are able to form cartilage, bone, muscle cells, or adipocytes after induction by hormonal or local factors. Several experiments have demonstrated that adipocytes and osteoblasts share a common precursor cell (Pittenger et al. 1999; Triffitt 1996). This cell differentiates into one or the other cell depending on the expression of specific transcription factors. Thus, the expression of peroxisome proliferator activated receptor у2 (PPARy2) is requested for commitment to the adipocyte lineage, whereas mesenchymal cells expressing Cbfa1/Runx2 are committed to osteoblast lineage (Rodan et al. 1997).

Many transcription factors and proteins are involved not only in the formation and differentiation of osteoblasts but also in their inhibition. Among them, bone morphogenetic proteins (BMPs) and core binding factor a1 (Cbfa1) are crucial molecules in bone biology because they induce differentiation of mesenchymal cells toward cells of osteoblastic lineage (Canalis et al. 2003). The BMPs are members of the TGF-β superfamily of proteins that includes TGF-β, activins, and inhibins. BMPs are the only factors able to initiate osteoblastoge- nesis from noncommitted progenitors (Abe et al. 2000). An essential function of BMPs is to induce the differentiation of mesenchymal cells toward cells of osteoblastic lineage and promote osteoblast maturation and function, which requires interactions between BMPs and other factors like Smad and Cbfa1 (Yamaguchi et al. 1996). BMPs are modulated by numerous secreted factors such as Noggin, Chordin, SOST, and Gremlin, which inhibit BMP action by binding BMPs (Lee et al. 2000; Balemans et al. 2002). Cbfa1 is a specific transcription factor of osteoblasts whose deficiency causes an arrest of osteoblast development, absence of osteoblasts, and lack of bone formation (Ducy et al. 1997). Several studies have demonstrated that Cbfa1 is crucial also for postnatal differentiation and maintenance of osteoblasts and for the function of mature osteoblasts (Ducy et al. 1998, 1999). Regulation of osteoblastogenesis is much more complex and involves a large number of genes, but it is not the topic of this chapter (Canalis et al. 2003; Balemans et al. 2002; Harada et al, 2003; Ducy et al. 2000).

Once stem cells are committed to the osteoblast lineage, proliferating osteoprogenitors become preosteoblasts, cell growth declines, and there is a progressive expression of differentiation markers by osteoblasts (Stein et al. 1996). Osteoblastic differentiation is characterized by the sequential expression of alkaline phosphatase (ALP), an early marker of osteoblastic phenotype, followed by the synthesis and deposition of collagen type I, bone matrix proteins, and glycosaminoglycans and an increased expression of os teocalcin and bone sialoprotein at the onset of mineralization. When bone matrix has been deposited and calcified, most of the osteoblasts reduce their activity of matrix synthesis and become flattened lining cells. Around 10% of the osteoblasts are absorbed into the matrix synthesized by themselves and thus becoming osteocytes, which remain connected to each other by cytoplasmic extensions located in canaliculi. This allows for the transfer of molecules and nutrients from the older bone to the bone surface. Due to the proximity of the bone matrix, the osteocytes and lining cells sense external mechanical signals and transfer this information to other cells by changes in integrins and the cytoskeletal network. More than half of the osteoblasts undergo apoptosis when bone formation concludes (Jilka et al. 1998).

Osteoclasts are large, highly specialized, and polarized multinucleated cells with a characteristic trait. Their cell membrane has folds and invaginations at the interface with the bone surface called “ruffled border". Resorption occurs under this membrane in a microcompartment localized between the ruffled border and thebone matrix. Osteoclasts resorb bone bymeans of producing hydrogen ions to solubilize the mineral phase and to secrete proteolitic enzymes to degrade the organic matrix. To function, osteoclasts should be attached to the bone surface and secrete several enzymes such as tartrate-resistant acid phosphatase (TRAP; a phenotypic marker of these cells), cathepsins, and matrix metalloproteases (Blair 1998; Boyle et al. 2003). The acidification of the mineralized matrix depends on proton production by carbonic anhydrase II, whose deficiency induces osteopetrosis due to a lack of bone resorption (Sly et al. 1983). In addition, indicating the importance of cathepsin K, knockout mice of this protein exhibit an inhibition of bone resorption and display an osteopetrotic phenotype (Gowen et al. 1999). The most important integrin responsible for osteoclast attachment to bone is the vitronectin receptor (aVβ3). If this integrin is inhibited, bone resorption is impaired, thus showing the importance of attachment to the bone for osteoclast function (Helfrich et al. 1992).

Osteoclasts are derived from hematopoietic stem cells of the monocyte- macrophage lineage, which also produces monocytes and macrophages (Kuri- hara et al. 1989; Roodman 1996). The point at which the committed osteoclast progenitor separates from the macrophage lineage is not clear, but when it receives the appropriate signal, this progenitor abandons the BM and goes to bone either by means of circulation or by direct migration. Deletion of gene encoding for molecules that regulate osteoclastogenesis (OCS) results in osteopetrosis due to a failure in osteoclast formation, and, in occasions, an absence of macrophages also occurs. The transcription factor PU.1 is critical for both the initial commitment of both cellular types, since its deficiency results in osteopetrosis with neither osteoclasts nor macrophages (Tondravi et al. 1997). Macrophage-colony stimulating factor (M-CSF) is needed for both early as well as for committed progenitors, promoting proliferation and survival (Kodama et al. 1991). The absence of c-Fos, however, results in osteopetrosis in the absence of osteoclasts, but with normal macrophage numbers, showing a step that allows for the differentiation of osteoclast and macrophage lineages (Grigoriadis et al. 1994). Other crucial proteins for the formation and differentiation of osteoclasts, whose discovery has been one of the most remarkable contributions to osteoclast biology in recent years and to which an entire section of this chapter is dedicated, are RANKL, RANK, and OPG (Lacey et al. 1998; Simonet et al. 1997; Anderson et al. 1997).

7.3

Role of Proinflammatory Cytokines in Bone Resorption

Early stages of hematopoiesis and OCS progress along similar pathways; therefore it is normal that cytokines and growth factors implied in hematopoiesis also affect the development of osteoclasts. The first evidence of this implication came from the discovery that supernatants of activated human monocytes stimulated bone resorption (Horton et al. 1972). This activity was called osteoclast-activating factor (OAF) and later identified as interleukin 1 (IL-1) (Dewhirst et al. 1985). Afterwards, IL-6 and TNF-a were identified, which, like IL-1, are essential mediators in inflammatory responses. Many other cytokines that stimulate bone resorption like IL-3, IL-11, IL-15, IL-17, and GM-CSF and others that inhibit it such as IL-4, IL-10, IL-18, and IFN-y were also identified (Manolagas 2000; Manolagas et al. 1995; Jilka 1998). All these agents directly affect OCS or indirectly act as local regulators of the action of systemic hormones like PTH, vitamin D3, and estrogens. This chapter will focus on the involvement of the cytokines most implicated in bone resorption such as IL-1, TNF-a, and IL-6.

Interleukin 1 (IL-1) is produced mainly by activated monocytes-macrophages, and its principal action is to stimulate thymocytes. A pleiotropic cytokine, IL-1 induces the expression of a large diversity of cytokines such as IL-6, leukaemia inhibitory factor (LIF), and other proinflammatory molecules (Di- marello 1994). IL-1 and TNF-a carry out as part of their function increasing the expression of NF-kB and JNK (c-Jun N-terminal kinase). The importance of IL-1 in OCS is demonstrated because the IL-1-receptor-deficient mouse is resistant to ovariectomy (OVX)-induced bone loss (Lorenzo et al. 1998). The importance in pathological bone loss is also illustrated by the fact that treatment with IL-1 receptor antagonist slows down bone erosion for patients affected with rheumatoid arthritis (Kwan et al. 2004). IL-1 increases osteoclast differentiation rather than mature osteoclast activity, and infusion of IL-1 into mice induces hypercalcemia and bone resorption. Finally, IL-1 and TNF-a stimulate OCS by inducing the expression of RANKL, and it has been demonstrated that IL-1 mediates the osteoclastogenic effect of TNF-a by enhancing stromal cell expression of RANKL (Wei et al. 2005).

Tumor necrosis factor a (TNF-a) is a multifunctional cytokine produced by activated monocytes-macrophages. TNF-a is one of the most potent os- teoclastogenic cytokines produced in inflammation, and, in addition, TNF-a induces IL-1 synthesis. Like the other known stimulators of bone resorption, it acts through osteoblastic cells; however, it has been demonstrated that TNF-a is able to induce osteoclast formation from stromal-depleted macrophages, with potency similar to that of RANKL (Kobayashi et al. 2000). TNF-a is able to induce bone resorption in vitro (Thomson et al. 1987) as well as in vivo (Koning et al. 1988). Osteoclasts induced by TNF-a have the capacity to form resorption pits on dentine slices only in the presence of IL-1a. TNF-a, together with IL-1, plays an important role in bone resorption in inflammatory diseases (Kobayashi et al. 2000). Inhibition of TNF by TNF binding protein (TNFbp) completely prevents bone loss and osteoclast formation (Kimble et al. 1997).

Interleukin-6 (IL-6) is a member of the gp130 cytokine family and is constitutively produced by several cells of bone microenvironment, particularly by osteoblasts and their precursors (Heymann et al. 2000). The main function in bone is on OCS and bone resorption, and its effects are connected to those of IL-1, TNF-a, and PTHrP. IL-6 induces osteoclastlike formation by inducing IL-1 synthesis, and the addition of anti-IL-1 inhibits osteoclast formation by IL-6 (Kurihara et al. 1990). Moreover, IL-6 mediates the effects of TNF-a and enhances PTHrP-induced hypercalcemia and bone resorption by increasing the osteoclast progenitor pool and differentiation into mature osteoclasts (Devlin et al. 1998).

Independently, if these cytokines can exert their bone resorption functions without RANKL, they all stimulate the production of RANKL for stromal/OB cells, and conversely RANKL is able to increase IL-1 and TNF-a synthesis in vitro. To complicate this scenario, these systems of cytokines connect with the network of systemic hormones, such as PTH, PTH-related protein (PTHrP), vitamin D3, estrogens, androgens, glucocorticoids, and T4, since the hormones regulate the production of many of these cytokines by stromal/OB cells (Manolagas et al. 1995; Bellido et al. 1995; Lakatos et al. 1997).

7.4

The RANKL/RANK/OPG System

During the 1970s data on the expression of receptors for known stimulators of bone resorption, like PTH and vitamin D3, demonstrated that these receptors were not present on osteoclasts or their precursor cells, but were on osteoblasts (Rodan et al. 1981). Moreover, cellular interactions between stromal/OB cells and hematopoietic cells of BM are critical for osteoclast development, and this requirement of interaction became a common denominator for all known OCS stimulators (Kelly et al. 1998). These precedents served to formulate the hypothesis that in the surface of these cells exists an “osteoclast differentiating factor” (ODF) (Suda et al. 1992). The molecular mechanism of dependence that OCS has with stromal/OB cells has been explained recently with the discovery of a new bone system of cytokines belonging to the TNF superfamily of receptors and ligands. These crucial proteins for the differentiation and function of osteoclasts are RANKL, its receptor RANK, and OPG.

RANKL, also known as TRANCE, OPGL, or ODF, was cloned almost simultaneously by 4 groups (Lacey et al. 1998; Yasuda et al. 1998; Anderson et al. 1997; Wong et al. 1997). RANKL is expressed in stromal/OB cells, and its expression is increased for factors that induce bone resorption as glucocorticoids, IL-1, IL-6, IL-11, IL-17, TNF-a, PGE2, PTH, or vitamin D3 (Lacey et al. 1998; Yasuda et al. 1998). RANKL stimulates the differentiation and survival of osteoclast precursors, activates to mature osteoclast, and prolongs its lifespan by inhibiting its apoptosis (Lacey et al. 1998; Yasuda et al. 1998). RANKL, together with M-CSF, is necessary and sufficient to carry out all the steps of OCS, even in the absence of stromal cells. The administration of RANKL to the mouse induces a severe osteoporosis, hypercalcemia, and rapid bone loss (Lacey et al. 1998). Conversely, RANKL-deficient mice have a severe osteopetrosis phenotype with the absence of mature osteoclasts, defects in the dental eruption, and several defects in the maturation of T- and B-cells and in the formation of the lymphatic node (Kong et al. 1999a; Kong et al. 1999b). Several agents regulate RANKL expression (Table 7.1). Thus, IL-1, IL-6, TNF-a, vitamin D3, and PTH are compounds that stimulate the production of RANKL, whereas TGF-β is the main factor that inhibits RANKL expression (Khosla 2001; Hofbauer et al. 2000; Suda et al. 1999). Estrogens do not seem to modulate the in vitro expression of RANKL, although the OVX accompanies an increase in the expression of RANKL (Suda et al. 1999) and OPG (Hofbauer et al. 1999).

The receptor for RANKL is RANK, also known as ODAR (Anderson et al. 1997; Hsu et al. 1999). RANK is expressed in osteoclast precursors, mature osteoclasts, condrocytes, fibroblasts, and immune system cells (Anderson et al. 1997; Hsu et al. 1999). The binding of RANKL with RANK on preosteoclasts initiates the OCS and the activation of osteoclasts (Anderson et al. 1997; Hsu et al. 1999; Nakagawa et al. 1998). RANK-deficient mice display a phenotype characterized by osteopetrosis and several defects in the immune system similar to that observed in RANKL-deficient mice (Dougall et al. 1999). Consistent with this hypothesis, RANK-deficient mice are resistant to bone resorption induced by TNF-a, IL-1β, or vitamin D3 (Li et al. 2000). In agreement with this, mice deficient in molecules implied in the transduction pathway from RANK like TRAF-6 or NF-kB1/NK-kB2 also show an osteopetrotic phenotype, demonstrating that signals through RANK are necessary for the differentiation and activation of osteoclasts (Lomaga et al. 1999; Iotsova et al. 1997). Unlike RANKL and OPG, the expression of RANK on osteoclastic cells is stable, with few variations for osteopetrotic agents (Hofbauer et al. 2000). In the immune system, however, the expression of RANKL in T-cells is activated for IL-4 and TGF-β, while the expression of RANK on dendritic cells is upregulated by CD40-L (Table 7.1) (Anderson et al. 1997).

The osteoprotegerin (OPG), also known as OCIF, TR1, or FDCR-1, is the first soluble protein that belongs to the TNF superfamily (Simonet et al. 1997; Kwon et al. 1998; Yun et al. 1998). Unlike RANK and RANKL, OPG is expressed in high concentrations in a variety of tissues and cellular types such as skin, bones, large arteries, and the gastrointestinal tract (Simonet et al. 1997). In bone, OPG is produced by stromal/OB cells (Hofbauer et al. 1999) and works as a “decoy receptor” for RANKL, competing with RANK for binding RANKL. Therefore, OPG is a potent inhibitor of the OCS. In vitro, OPG inhibits the differentiation and survival of osteoclast precursors, blocks their activation, and induces their apoptosis (Lacey et al. 1998; Yasuda et al. 1998; Hofbauer et al. 1999). OPG in vivo overexpression induces severe osteopetrosis similar to that in RANKL- and RANK-deficient mice, although without showing the effects on the immune system of these (Simonet et al. 1997). OPG-deficient mice show a severe osteoporosis, an arterial calcification that suggests a role in the vascular system, and an altered B-cell maturation and antibody response (Yun et al. 2001). OPG is also produced by osteoblasts in response to anabolic agents such as estrogens and BMPs, and administration of recombinant OPG to the mouse results in an increase ofbone mass and prevents the bone loss induced by OVX (Simonet et al. 1997; Yasuda et al. 1998; Udagawa et al. 2000). The production of OPG is stimulated by IL-1, TNF-a, TGF-в, BMP-2, BMP-7, vitamin D3,17β- estradiol, and calcium, while it is diminished by PGE2, glucocorticoids, PTH, and cyclosporine A (Hofbauer et al. 2000; Suda et al. 1999).

The proposed model to explain OCS is schematized in Fig. 7.2. Several agents, induced or not for estrogen deficiency, stimulate the expression of RANKL on stromal/OB cells. The binding of RANKL with its receptor RANK on osteoclastic precursors, together with M-CSF, is a necessary and sufficient condition to carry out all the steps in the formation and differentiation of the osteoclasts. Undoubtedly all this is much more complex than what is described here since at least 24 genes that positively and negatively regulate OCS have been described (Boyle et al. 2003).

Inflammation and autoimmunity often are associated with the destruction of bone, but the molecular link between these two processes had long been unclear. The role of bone in the generation of immune system cells is evident since they are formed in the marrow housed within the bone; however, the role of these cells on bone is not so clear. RANKL and RANK were described initially in activated T-cells and in dendritic cells, respectively, where they have functions in the regulation of cellular lifespan and immunomodulation (Anderson et al. 1997; Wong et al. 1997). Recently the term osteoimmunology has been coined to describe the link between the immune system and bone (Arron et a. 2000). Several data support this idea:

1. T-cell-deficient mice do not lose bone after OVX (Cenci et al. 2000).

2. Activated monocytes or T-cells can induce OCS through the secretion of proresorptive cytokines IL-1, TNF-a, and IL-11, which upregulate RANKL in osteoblasts (Hofbauer et al. 1999).

3. Activated T-cells also express RANKL (Anderson et al. 1997).

4. The systemic activation of T-cells leads to a RANKL-dependent increase of OCS (Kong et al. 1999b).

5. Mice lacking CTLA4, in which T-cells are systemically activated, exhibit osteoporosis (Kong et al. 1999b).

Nevertheless, T-cells also secrete cytokines, including IFN-y, IL-12, IL-18, TGF-в, and IFN-в, which inhibit the pro-osteoclastogenic effects of RANKL.

Fig. 7.2. Osteoclastogenesis. Several bone resorbing factors such as IL-1, TNF-a, vitamin D3, and dexamethasone stimulate the expression of RANKL on the membranes of stromal/OB cells, although it can be secreted into circulation. The binding of RANKL with RANK on osteoclast precursors, together with M-CSF, is the signal required to initiate and maintain all steps of the OCS. The OPG is another member of the TNF superfamily and acts as a decoy receptor for RANKL. OPG is secreted by stromal/OB cells and inhibits osteoclast formation by blocking the RANKL/RANK signal pathway

Therefore, T-cells participate in bone loss during inflammation or when T- cells are chronically activated (rheumatoid arthritis, periodontitis, infections, bone prothesis) and recently have been implicated in postmenopausal bone loss (Cenci et al. 2000,2003; Roggia et al. 2001).

7.5

Bone Remodeling After Estrogen Deficiency

Osteoporosis is a consequence of the reduction of skeletal mass caused by an imbalance between bone resorption and bone formation. The loss of gonadal function and aging are the two main factors that contribute to the development of osteoporosis. Around the fourth or fifth decade of life, men and women lose bone at a rate of 0.3-0.5% per year. After menopause, the rate of bone loss increases to 10% a year (Nordin et al. 1990; Riggs et al. 1986, 1998). The bone loss due to estrogen withdrawal is associated with increments in both bone resorption as well as in bone formation, with the former exceeding the latter. This indicates the birth of new BMUs or an increase in the lifespan of current BMUs (Manolagas 2000). Estrogen deficiency increases the activation frequency (birth rate) of BMUs, which leads to higher bone turnover and induces a remodeling imbalance by prolonging the resorption phase (osteoclast apoptosis is reduced (Hughes et al. 1996)) and a shortening of the formation phase (osteoblast apoptosis is increased (Manolagas 2000)). Unlike postmenopausal bone loss, in which there is a net increase in the number of osteoclasts, bone loss associated with aging is related to a reduced offer of osteoblasts in relation to the demand of the newly created BMUs (Erikson et al. 1990). Both types of bone loss affect different bone types; while postmenopausal bone loss occurs mainly in trabecular bone, the age-associated one occurs primarily in cortical bone.

For a long time it was suspected that estrogens exerted a direct action on bone cells since these cells have active receptors for estrogens (Eriksen et al. 1988; Oursler et al. 1991). The abrupt increase in bone remodeling as a consequence of estrogen deprivation is accompanied by an increase in the production of several cytokines and growth factors (Manolagas 2000; Manolagas et al. 1995; Jilka 1998; Pacifici et al. 1998). Many studies on bone estrogen action have focused on the role of cytokines and molecules such as IL-1, IL-6, TNF-a, GM-CSF, M-CSF, and PGE2 (Fig. 7.3). These factors induce bone resorption, and their expression increases with estrogen deficiency and decreases with estrogen administration (Manolagas 2000; Manolagas et al. 1995; Hofbauer et al. 2000; Riggs et al. 1998; Pacifici et al. 1998). In 1987 it was demonstrated that cultures of monocytes from osteoporotic women have higher levels of IL-1 than those in women with normal bone turnover (Pacifici et al. 1987). These authors also demonstrated an increase in the production of IL-1 and TNF for cultures of monocytes from women subjected to OVX but not if these women took estrogens (Pacifici et al. 1991). In mice, treatment with inhibitors of IL-1 and TNF prevents bone loss induced by OVX (Yamamoto et al. 1996), and OVX is not followed by bone loss in IL-1 type I receptor (IL-1RI)-deficient mice or in mice that overexpress a soluble form of the TNF receptor, that which makes them unable to respond to TNF-a (Ammann et al. 1997). Moreover, treatment of ovariectomized mice with an inhibitor of TNF, such as TNF-binding protein (TNFbp), prevents bone loss induced by OVX (Kimble et al. 1997).

While studies have repeatedly demonstrated that IL-1 and TNF-a are potent inductors of bone loss after OVX, the role of IL-6 is more uncertain. The IL-6- deficient mouse does not lose bone mass after OVX, although it has increased bone turnover (Poli et al. 1994), while the mouse overexpressing IL-6 does not present osteoporosis, which seems to be a contradiction (Kitamura et al. 1995). Moreover, neutralizing antibodies against IL-6 prevents an increase in osteoclast number after OVX (Kimble et al. 1997; Jilka et al. 1992), although it does not prevent bone loss after OVX, nor does it diminish in vivo bone resorption (Kimble et al. 1997). All this suggests that IL-6 contributes to the expansion of osteoclasts from hematopoietic precursors, although it does not seem to be a dominant factor in estrogen-deficiency-induced bone loss (Manolagas 2000; Hofbauer et al. 2000).

Fig. 7.3. Osteoclastogenesis after estrogen deficiency. Estrogen deprivation leads to an increase in the synthesis of RANKL for stromal/OB cells of the BM. This increase in the expression of RANKL leads to an increase in OCS. Estrogen deficiency also induces the synthesis and secretion of cytokines, such as IL-6 and M-CSF, that increase the number of preosteoclasts in the BM, and thus increases OCS. Nonetheless, certain cells of the immune system, such as monocytes and T-cells, intervene in the process when the supply of estrogens fails. These cells secrete IL-1 and TNF-a that are powerful inductors of OCS. When estrogens or agonists of estrogen receptors like raloxifene are administered, the synthesis and secretion of many of the mentioned cytokines diminish and the synthesis and liberation of OPG and TGF-β are stimulated. These molecules inhibit OCS by inhibiting the RANKL/RANK signal pathway and by promoting osteoclast apoptosis

In summary, IL-1 and TNF-a activate mature osteoclasts indirectly via a primary effect on osteoblasts and by inhibiting osteoclast apoptosis. In addition, they increase osteoclast formation either by directly stimulating the proliferation of osteoclast precursors or by increasing the pro-osteoclastogenic capacity ofbone stromal cells. Although in vitro TNF-a and IL-1 can apparently induce the development of TRAP+ osteoclasts in the absence of RANKL/RANK, all data seem to indicate that TNF-a and IL-1 potentiate osteoclast development via the activation of common second messenger systems, such as NF-kB activation, and that the effects on OCS require the RANKL/RANK system (Jones et al. 2002).

If the RANKL/OPG system is a final effector on the biology of osteoclasts, then this system should be the basis for the antiresorptive effects of estrogen. Indeed, estrogen stimulates OPG synthesis for osteoblastic cells (Hofbauer et al. 1999), estrogen deficiency induced by OVX results in a decrease in OPG and increased RANKL production, an action that is prevented by estradiol administration, and OPG administration prevents bone loss induced by OVX (Simonet et al. 1997; Hofbauer et al. 2000; Hofbauer 1999). In addition, estrogen can suppress RANKL and M-CSF-induced differentiation of myelomonocytic precursors into multinucleated TRAP+ osteoclasts through an ER-dependent mechanism that does not require mediation by stromal cells (Shevde et al. 2000). Finally, treatment with estradiol inhibits the response of osteoclast precursors to the action of RANKL (Srivastava et al. 2001).

As previously stated, T-cells intervene in bone loss that is established in states of inflammation. There are, however, more data that implicate T-cells in bone loss associated with estrogen deficiency (Fig. 7.3). Bone loss after OVX is prevented by estrogen administration, by the administration of TNFbp, and by a neutralizing antibody to TNF-a. There is no bone loss after OVX in T- cell-deficient mice (Cenci et al. 2000). In addition, it has been shown that enhanced T-cell production of TNF is a key mechanism by which estrogen deficiency induces bone loss in vivo (Roggia et al. 2001). Activated T-cells also produce IFN-y, which strongly suppresses OCS by interfering with the RANKL/RANK signaling pathway via the induction of TRAF6 degradation that results in an inhibition of the RANKL-induced activation of NF-kB and JNK (Takayanagi et al. 2000). OVX increases TNF levels in BM by increasing the production of TNF by T-cells, which is induced by a complex mechanism driven by IFN-y (Cenci et al. 2003; Gao et al. 2004). This cytokine augments antigen presentation by enhancing MHCII expression on BM macrophages through induction of class II transactivator (CIITA) expression (Cenci et al. 2003). Upregulation of antigen presentation results, in turn, in increased T- cell activation, proliferation, and lifespan. Therefore, TNF produced by T-cells plays a pivotal role in the mechanism of estrogen-deficiency-induced bone loss. The mechanism by which OVX upregulates the production of IFN-y remains undetermined, but TFG-β could be involved since it has been reported that TFG-β represses the production of IFN-y by directly targeting T-cells and inhibiting their proliferation and differentiation (Kehrl et al. 1986; Gorelik et al. 2002).

7.6

Effects of Estrogen and Agonist of Estrogen Receptor on Bone Cells

Bone cells contain estrogen receptors (Eriksen et al. 1988; Oursler et al. 1991; Vidal et al. 1999). Estrogens act directly on osteoblasts and affect cell proliferation and the expression of many genes coding for enzymes, bone matrix proteins, transcription factors, and hormone receptors, as well as growth factors and cytokines (Spelsberg et al. 1999). It has been shown that estrogen inhibits the synthesis of IL-1, IL-6, TNF-a, and IL-11, as well as IL-6 synthesis in response to IL-1 (Manolagas 2000; Jilka 1998; Jilka et al. 1992; Kimble et al. 1996). Estrogen has also been shown to induce the synthesis of BMP-6, OPG, TGF-β, NF-kB, and c-Fos (Stein et al. 1995; Tau et al. 1998; Rickard et al. 1998). The main in vivo action of estrogens on the skeleton is to inhibit bone resorption. This action is indirect since it implies the regulation of cytokines and growth factor production by osteoblasts. Estrogens should have a direct action on osteoclasts, however, since active ERs have been described on osteoclasts. The most important action is probably that estrogens induce osteoclast apoptosis (Hughes et al. 1996; Kameda et al. 1997). This estrogen-mediated induction of apoptosis may be enhanced in vivo by TGF-β since this molecule produces osteoclast apoptosis and its production is increased by estrogens (Hughes et al. 1996). Estrogens have also shown the capacity to inhibit the expression of TRAP, to increase the induction in the expression of an IL-1 decoy receptor gene (Sunyer et al.1999), and to inhibit certain steps in the RANK-JNK signal transduction pathway by suppressing activation of MKK4 and JNK, and c-Jun expression and its subsequent AP-1 transactivation of transcription (Srivastava et al. 2001, 1999). Some evidence suggests that estrogens increase osteoblast formation, differentiation, proliferation, and function, although results vary among the different model systems (Manolagas 2000; Chow et al. 1992; Gohel et al. 1999).

It has been proposed that estrogen’s effects may be mediated by different cell signaling pathways. It has been described that the antiapoptotic effect of estradiol on osteoblasts and osteocytes can be mediated for rapid, nongenomic, and sex-nonspecific signaling through the ligand binding domain of the ER that is localized exclusively in the cell membrane (Kousteni et al. 2001). In addition, investigators have identified a synthetic ligand called estren that reverses bone loss in ovariectomized females (Kousteni et al. 2002), which activates only a subset of these pathways, suggesting that bypassing the traditional estrogen pathways can prevent bone loss without the associated side effects on reproductive organs. This compound exhibits no classical sex steroid hormone activity, and it is a potent activator of the rapid cell-membrane-mediated Src- MAPK pathways in cell culture models that induce a rapid activation ofMAPK (Kousteni et al. 2003). This extranuclear mode of action of estren has led to the definition of a new class of pharmacotherapeutic agents called ANGELS (Activators of Nongenotropic Estrogen-like Signaling) (Manolagas et al. 2002).

The beneficial role of estrogen replacement therapy (ERT) to prevent bone loss has been largely demonstrated (Nelson et al. 2002; Wells et al. 2002). Recently, however, the results of the great clinical study WHI (Women’s Health Initiative) was published in which several side effects, such as an increase in breast cancer incidence and several vascular problems of ERT, were reported (Kobayashi et al. 2000; Rossouw et al. 2002). All of this has led to the large present effort to find new alternatives to ERT. Some of these alternatives are phytoestrogens and SERMs (Selective Estrogen Receptor Modulators).

The ideal SERM would have the beneficial effects of estrogen in bone without the undesirable effects in the breast and uterus, the current gold standard being raloxifene. SERMs are compounds that bind to estrogen receptors and exhibit estrogen agonistic effects on bone and lipid metabolism and estrogen antagonistic effects on uterine endometrium and breast tissue. Because of its tissue selectivity, raloxifene may have fewer side effects than is typically observed with ERT. The beneficial role of raloxifene in bone loss, in the decrease in bone fractures (Delmas et al. 1997; Ettinger et al. 1999), in the decrease in the incidence of breast cancer (Cummings et al. 1999), and in cardiovascular problems (Barrett-Connor et al. 2002) is well established. It has been demonstrated in osteoporotic postmenopausal women that raloxifene decreases levels of the cytokines involved in bone resorption such as IL-6 and TNF-a. This suggests that modulation of soluble factors could play a pivotal role in the mechanisms of the osteoprotective effect of raloxifene (Gianni et al. 2004).

Raloxifene, like 17β-estradiol, significantly reduces the number of osteoclasts in culture, inhibits bone resorption in a pit assay, increases osteoblast proliferation, increases Cbfa1 transcription factor mRNA, prevents the TNF-a- induced IL-1β increase, and stimulates TGF-в expression in rat bone (Taranta et al. 2002; Tou et al. 2001; Yang et al. 1996). Moreover, it has been shown that raloxifene can suppress RANKL and M-CSF-induced differentiation of myelomonocytic precursors into multinucleated TRAP+ osteoclasts through an ER-dependent mechanism that does not require mediation by stromal cells (Shevde et al. 2000). Raloxifene decreases levels of RANKL (Cheung et al. 2003) and stimulates OPG production and inhibits IL-6 production by human osteoblasts, and therefore, since OPG production increases with osteoblastic maturation, enhancement of OPG production by raloxifene could be related to the stimulatory effects on osteoblastic differentiation (Viereck et al. 2003). It seems, however, that the stimulation of bone formation by raloxifene differs from that of estradiol (Qu et al. 1999). Finally, raloxifene, like estradiol, directly decreases the expression of beta3-integrin mRNA and protein, which suggests that the inhibitory action of raloxifene and estradiol on bone resorption may affect adhesion and, like estradiol, prevent the increase in B-cells induced by OVX (Saintier et al. 2004; Onoe et al. 2000).

7.7

Conclusions

Bone metabolism has quickly become a topic of fascinating research. The bone, far from being a metabolically inactive tissue, is a tissue where different cell types and different molecules carry out numerous and varied functions. This has been due largely to the discovery of the RANKL/RANK/OPG system of cytokines. These new molecules are decisive in OCS, bone metabolism, and bone loss, but they are also important for other tissues and cells. Indeed, these proteins are critical in several systems: the immune system, where they have functions that affect cell survival and the immunomodulation of T-, B-, and dendritic cells; the vascular system; and the endocrine system.

This chapter has focused on normal bone remodeling and that which is established after estrogen deficiency. Bone remodeling is regulated not only by this new system of cytokines, but also by other molecules, especially when gonadal function ceases. This constitutes a complex scenario in which transcription factors, systemic hormones, growth factors, and cytokines, together with a variety of cells like osteoclasts, osteoblasts, osteocytes, endothelial cells, lining cells, T-cells, B-cells, and dendritic cells, cohabit and interrelate.

All these discoveries have generated new therapeutic possibilities based on the use of OPG and on inhibitors of the RANKL/RANK signaling pathway, not only for the treatment of postmenopausal bone loss, but also for other pathologies. Special mention should be made of the new therapeutic possibilities constituted by ANGELS, since everything seems to indicate that research is at the threshold of a new way of inhibiting bone loss without the side effects of classic ERT.

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