Bone Resorption Is Regulated by Circadian Clock in Osteoblasts.

Bacterial components and bone resorption

Receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) produced by osteoblasts/stromal cells are involved as positive and negative regulators in osteoclast formation. Three independent signals have been proposed to induce RANKL expression in osteoblasts/stromal cells: vitamin D receptor-, cAMP-, and gp130-mediated signals. We previously reported that intracellular calcium-elevating compounds such as ionomycin, cyclopiazonic acid, and thapsigargin induced osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts. Increases in calcium concentration in culture medium also induced osteoclast formation in cocultures. Treatment of primary osteoblasts with these compounds or with high calcium medium stimulated the expression of both RANKL and OPG messenger RNAs (mRNAs). 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-tetra(acetoxymethyl)ester, an intracellular calcium chelator, suppressed both ionomycin-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, also stimulated osteoclast formation in these cocultures and the expression of RANKL and OPG mRNAs in primary osteoblasts. Protein kinase C inhibitors such as calphostin and staurosporin suppressed ionomycin- and PMA-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Ionomycin stimulated RANKL mRNA expression in ST2 and MC3T3-G2/PA6 cells, but not in MC3T3-E1 or NIH-3T3 cells. These effects were closely correlated with osteoclast formation in response to ionomycin in cocultures with these stromal cell lines. OPG strongly inhibited osteoclast formation induced by calcium-elevating compounds and PMA in cocultures, suggesting that RANKL expression in osteoblasts is a rate-limiting step for osteoclast induction. Forskolin, an activator of cAMP signals, also stimulated osteoclast formation in cocultures. Forskolin enhanced RANKL mRNA expression but suppressed OPG mRNA expression in primary osteoblasts. These results suggest that the calcium/protein kinase C signal in osteoblasts/stromal cells is the fourth signal for inducing RANKL mRNA expression, which, in turn, stimulates osteoclast formation.

Reduced mechanical stress is a major cause of osteoporosis in the elderly, and the osteocyte network, which comprises a communication system through processes and canaliculi throughout bone, is thought to be a mechanosensor and mechanotransduction system; however, the functions of osteocytes are still controversial and remain to be clarified. Unexpectedly, we found that overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteoblast and osteoclast differentiation were unaffected by BCL2 transgene in vitro. However, the cortical bone mass increased due to enhanced osteoblast function and suppressed osteoclastogenesis at 4 months of age, when the frequency of TUNEL-positive lacunae reached 75%. In the unloaded condition, the trabecular bone mass decreased in both wild-type and BCL2 transgenic mice at 6 weeks of age, while it decreased due to impaired osteoblast function and enhanced osteoclastogenesis in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Rankl and Opg were highly expressed in osteocytes, but Rankl expression in osteoblasts but not in osteocytes was increased at unloading in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Sost was locally induced at unloading in wild-type mice but not in BCL2 transgenic mice, and the dissemination of Sost was severely interrupted in BCL2 transgenic mice, showing the severely impaired osteocyte network. These findings indicate that the osteocyte network is required for the upregulation of Rankl in osteoblasts and Sost in osteocytes in the unloaded condition. These findings suggest that the osteocyte network negatively regulate bone mass by inhibiting osteoblast function and activating osteoclastogenesis, and these functions are augmented in the unloaded condition at least partly through the upregulation of Rankl expression in osteoblasts and that of Sost in osteocytes, although it cannot be excluded that low BCL2 transgene expression in osteoblasts contributed to the enhanced osteoblast function.

Hypertension is a major risk factor for cardiovascular disease (CVD) and is associated with increased bone loss due to excessive activity of the local renin-angiotensin system (RAS). Angiotensinogen/Angiotensin (ANG) II/Angiotensin II type 1 receptor (AT1R) axis is considered as the core axis regulating RAS activity. Azilsartan is an FDA-approved selective AT1R antagonist that is used to treat hypertension. This study aimed to determine whether azilsartan affects formation of osteoclast, resorption of bone, and the expression of cytokines linked with osteoclastogenesis during lipopolysaccharide (LPS)-triggered inflammation in vivo. In vivo, following a 5-day supracalvarial injection of LPS or tumor necrosis factor-alpha (TNF-α) with or without azilsartan, the proportion of bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, which are identified as osteoclasts on mice calvariae were counted. The mRNA expression levels of TRAP, cathepsin K, receptor activator of NF-κB ligand (RANKL), and TNF-α were also evaluated. In vitro, the effect of azilsartan (0, 0.01, 0.1, 1, and 10 μM) on RANKL and TNF-α-triggered osteoclastogenesis were investigated. Also, whether azilsartan restrains LPS-triggered TNF-α mRNA and protein expression in macrophages and RANKL expression in osteoblasts were assessed. Furthermore, western blotting for analysis of mitogen-activated protein kinases (MAPKs) signaling was conducted. Azilsartan-treated calvariae exhibited significantly lower bone resorption and osteoclastogenesis than those treated with LPS alone. In vivo, LPS with azilsartan administration resulted in lower levels of receptor activator of RANKL and TNF-α mRNA expression than LPS administration alone. Nevertheless, azilsartan did not show inhibitory effect on RANKL- and TNF-α-triggered osteoclastogenesis in vitro. Compared to macrophages treated with LPS, TNF-α mRNA and protein levels were lower in macrophages treated by LPS with azilsartan. In contrast, RANKL mRNA and protein expression levels in osteoblasts were the same in cells co-treated with azilsartan and LPS and those exposed to LPS only. Furthermore, azilsartan suppressed LPS-triggered MAPKs signaling pathway in macrophages. After 5-day supracalvarial injection, there is no difference between TNF-α injection group and TNF-α with azilsartan injection group. These findings imply that azilsartan prevents LPS-triggered TNF-α production in macrophages, which in turn prevents LPS-Triggered osteoclast formation and bone resorption in vivo.

Vitamin D was discovered as an anti-rachitic agent preventing a failure in bone mineralization, but it is now established that the active form of vitamin D3 (1α,25(OH)2D3) induces bone resorption. Discovery of the receptor activator of nuclear factor -κB ligand (RANKL) uncovered the molecular mechanism by which 1α,25(OH)2D3 stimulates bone resorption. Treating osteoblastic cells with 1α,25(OH)2D3 stimulates RANKL expression, which in turn induces osteoclastogenesis. Nevertheless, active vitamin D compounds such as calcitriol (1α,25(OH)2D3), alfacalcidol (1α(OH)D3) and eldecalcitol (1α,25-dihydroxy-2β-(3-hydroxypropoxy) vitamin D3) have been used as therapeutic drugs for osteoporosis, as they increase bone mineral density (BMD) in osteoporotic patients. Paradoxically, the increase in BMD is caused by the suppression of bone resorption. Several studies have been performed to elucidate the mechanism by which active vitamin D compounds suppress bone resorption in vivo. Our study showed that daily administration of eldecalcitol to mice suppressed neither the number of osteoclast precursors in the bone marrow nor the number of osteoclasts formed in ex vivo cultures. Eldecalcitol administration suppressed RANKL expression in osteoblasts. This review discusses how the difference between in vitro and in vivo effects of active vitamin D compounds on bone resorption is induced.

Magnolol, a compound from the traditional Korean herb Magnolia sp., has been exhaustively investigated as a therapeutic agent against several diseases including systemic and local inflammation. We examined the effects of magnolol on osteoclastic differentiation associated with inflammation. Magnolol markedly reduced interleukin (IL)-1-induced osteoclast formation in co-cultures of murine osteoblasts and bone marrow cells, whereas it had no effect on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation in bone marrow macrophage cultures. In osteoblasts, magnolol markedly inhibited both the up-regulation of RANKL expression and the production of prostaglandin E2 (PGE2) in response to IL-1 treatment. Addition of exogenous PGE2 reversed the inhibitory effects of magnolol on IL-1-induced RANKL expression in osteoblasts and osteoclast formation in co-cultures. Magnolol inhibited IL-1-induced PGE2 production, at least in part by suppressing cyclooxygenase-2 (COX-2) expression. Taken together, these results demonstrate that magnolol inhibits IL-1-induced RANKL expression in osteoblasts through suppression of COX-2 expression and PGE2 production, resulting in inhibition of osteoclast differentiation in co-cultures.

Dlx5, a Positive Regulator of Osteoblastogenesis, is Essential for Osteoblast-Osteoclast Coupling

Prostaglandin E2 (PGE2) stimulates osteoclast formation by increasing receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) mRNA expression via cAMP-protein kinase A (PKA) pathways in osteoblasts. Since phosphodiesterases (PDEs) balance the concentration of intracellular cAMP stimulated by PGE2, we investigated the role of PDEs in PGE2-mediated osteoclast formation using various cAMP-specific PDEs inhibitors. In the presence of PGE(2), PDE3 and 4 inhibitors were shown to dose-dependently increase the osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. In agreement with this finding, they stimulated PGE2-induced cAMP production followed by increased RANKL mRNA expression in osteoblasts, suggesting that PDE3 and 4 negatively regulate PGE2-mediated RANKL expression in osteoblasts. RT-PCR analysis revealed that PDE3A, 3B, 4A, 4B and 4D are expressed in osteoblasts. The PDE8 inhibitor did not increase osteoclast formation, although it stimulated PGE2-induced RANKL mRNA expression in osteoblasts. The four subtypes of PGE receptors are designated EP1, EP2, EP3, and EP4. PDE3 and 4 inhibitors were found to increase EP1/3, EP4 and/or EP2 agonist-stimulated RANKL expression, indicating that PDE3 and PDE4 negatively regulate PGE2-induced RANKL mRNA expression through four EPs. Taken together, these data suggest that PDE3 and PDE4 could have important pharmacological and clinical implications in bone-related diseases.

More than a decade has passed since the publication of the first report implicating T cells in the bone loss induced by ovariectomy (ovx).1 Since then there has been extraordinary progress in the understanding of the regulatory network that links the hemopoietic and the mesenchymal compartments of the bone marrow (BM), the interactions between the immune system and bone, and the role of lymphocytes as mediators of the effects of calciotrophic hormones in bone. Collectively this body of knowledge has led to the firm establishment of "osteoimmunology" as a novel discipline and a promising area of investigation. The objective of this perspective is to revisit the exciting hypothesis that T cells play a pivotal role in the mechanism of ovx-induced bone loss. The bone-sparing activity of estrogens is due to a repression of bone remodeling coupled with a balancing effect on bone formation and resorption.2 The dominant acute effect of estrogen is the blockade of new osteoclast formation, cells which arise by cytokine-driven proliferation and differentiation of monocyte precursors that circulate within the hematopoietic cell.3 This process is facilitated by BM stromal cells (SCs), which provide physical support for nascent osteoclasts and produce soluble and membrane-associated factors essential for the proliferation and differentiation of osteoclast precursors. This inhibitory effect on osteoclastogenesis is associated with a repressive effect on osteoblastogenesis.4 Another critical effect of estrogen is that of increasing osteoclast apoptosis5-7 while blocking the apoptosis of osteoblasts and osteocytes.2, 8 It has been proposed that the effects of estrogens on the generation of osteoblasts and the lifespan of osteoblasts and osteoclasts result from extranuclear actions of the endoplasmic reticulum (ER) and activation of cytoplasmic kinases.2, 7 The decline of ovarian function at menopause results in decreased production of estrogen and a parallel increase in pituitary follicle-stimulating hormone (FSH) levels. The combined effects of estrogen deprivation and elevated FSH production cause a marked stimulation of bone resorption9 and a period of rapid bone loss that is central to the pathogenesis of postmenopausal osteoporosis. In mice the acute effects of menopause are modeled by ovx, a procedure that stimulates bone resorption by increasing osteoclast formation3 and lifespan.5-7 This initial phase of bone loss is followed by a slower but more prolonged loss of mainly cortical bone due to incomplete refilling of the resorption cavities due to insufficient osteoblast (OB) activity and lifespan.10 An expansion of the osteoclastic pool is therefore the key mechanism responsible for the bone loss that occurs early after ovx. The net bone loss caused by ovx is limited, in part, by an increase in bone formation resulting from stimulated osteoblastogenesis.11 This compensation is fueled by an expansion of the pool of BM–SCs, increased commitment of such pluripotent precursors toward the osteoblastic lineage,11 and enhanced proliferation of early OB precursors.4 Subsequent escalations in OB apoptosis12, 13 extensions of osteoclast lifespan5, 6 and increased secretion of cytokines that suppress bone formation, such as interleukin 7 (IL-7) and tumor necrosis factor (TNF), are the likely reasons for why bone formation does not increase as much as resorption after ovx. The stimulatory effect of ovx on SCs is equally relevant for osteoclastogenesis as one of the consequences of estrogen deprivation is the formation of osteoblastic cells with an increased osteoclastogenic activity14; ie, the capacity to support osteoclast formation. Evidence now suggests that T cells play a pivotal role in the mechanism of ovx-induced bone loss. Core observations include reports showing that ovx fails to induce trabecular and cortical bone loss in: T-cell–deficient nude mice,1, 15-17, wild-type (WT) mice deleted of T cells by injection of anti–T cell antibodies,18 mice treated with Abatacept19 (an agent that blocks T cell costimulation and induces T cell anergy and apoptosis)20, 21 and mice lacking the T cell costimulatory molecule CD40 ligand (CD40L).18 The fact that nude mice are protected against ovx-induced bone loss has been confirmed independently.22 By contrast, Lee and colleagues23 showed that nude mice are protected against the loss of cortical, but not trabecular bone induced by ovx. In another study, T-cell–deficient and B-cell–deficient mice were found to lose bone after ovx.24 The discrepancy between our reports1, 15-19, 25 and those of others23, 24 is likely explained by differences in the experimental design, the lack of B cells in some models, and compensatory mechanisms such as an increase in natural killer (NK) cells producing the osteoclastogenic factor IL-17.26 Both CD4+ and CD8+ cells have been found to play a role in ovx-induced bone loss. CD4+ cells include the TH1, Th2, and Th17 subsets. Th17 are regarded as the most osteoclastogenic subset of T CD4+ cells because they produce high levels of IL-17, receptor activator of NF-κB ligand (RANKL), and TNF, and low levels of interferon gamma (IFNγ).27, 28 The differentiation of Th17 is inhibited by estrogen via a direct effect mediated by estrogen receptor alpha (ERα).29 The role of Th17 in ovx-induced bone loss remains to be determined because one study reported that IL-17R–null mice are more susceptible to ovx-induced bone loss than controls,30 while another group found these mice to be protected from ovx-induced bone loss.26 More abundant information is available about regulatory T cells (Tregs), a population capable of suppressing the effector function of TH1, Th2, and Th17 T cells. Tregs are defined by the expression of the transcription factor FoxP3. Tregs inhibit monocyte differentiation into osteoclasts in vitro and in vivo, and blunt bone resorption31, 32 through the secretion of IL-4, IL-10, and transforming growth factor beta 1 (TGFβ1).33 Attesting to the relevance of Tregs, studies have shown that estrogen increases the relative number of Tregs.27, 34 Moreover, transgenic mice overexpressing Tregs develop progressive high bone mass due to inhibition of bone resorption, and are protected against ovx-induced bone loss.35 Moreover, adoptive transfer of Tregs into T cell–deficient mice increases bone mass, indicating that Tregs directly affect bone homeostasis without the need to engage other T cell lineages.35 Two mechanisms have been described to explain how T cells contribute to ovx-induced bone loss (Fig. 1). The first involves an increase in T cell activation, leading to enhanced production of TNF by BM T cells. The second is a regulatory crosstalk between T cells and SCs, resulting in enhanced production of osteoclastogenic cytokines by SCs. Schematic representation of the role of T cells in the mechanism by which ovx promotes osteoclastogenesis, osteoblastogenesis, and hemopoiesis. Estrogen deficiency promotes T cell activation by increasing the interaction of antigen (Ag)-loaded MHC molecules with bone marrow macrophages (BMM) and dendritic cells (DC) with the T cell receptor (TCR). The Ags are likely to be non-self peptides derived from the intestinal macrobiota. T cell activation also requires at least two costimulatory signals provided by the binding of BMM and DC-expressed CD40 and CD80 to the T cell surface molecules CD40L and CD28, respectively. A critical upstream event is the increased production of reactive oxygen species (ROS) that activate DCs by increasing their expression of CD80. The expansion of T cells in the BM is partially driven by an ovx-induced increase in the thymic output of naïve T cells. Activated T cells secrete TNF that stimulates osteoclast formation primarily by potentiating the response to RANKL. In addition, T cell–expressed CD40L and DLK1/FA-1 increase the osteoclastogenic activity of SC by blunting their secretion of OPG and augmenting their production of RANKL, M-CSF, and other proinflammatory factors. The survival of naïve T cells and some memory T cells requires the low-affinity engagement of the T cell receptor (TCR) by a diverse repertoire of self-antigens (Ags) bound to major histocompatibility complex (MHC) molecules expressed on Ag-presenting cells.36, 37 By contrast, binding of foreign Ags such as bacterial Ags with MHC molecules is followed by high-affinity interactions with TCRs that drive T cell activation. Both low-affinity interactions with self-Ags and high-affinity foreign Ag/MHC/TCR interactions are referred to as "Ag presentation." Ovx induces T cell expression of activation markers and promotes T cell proliferation, expansion, and acquisition of effector functions. These are all features of T cells exposed to foreign Ags.36 The gastrointestinal tract is colonized for life with 100 trillion indigenous bacteria, creating a diverse ecosystem known as the microbiota, whose contributions to human health are profound.38 The macrobiota is likely to represent the source of foreign Ag that drives the expansion of T cells induced by ovx. Although direct verification of a role for macrobiota in T cell expansion is still lacking, compelling supportive data are available. For example, adoptive transfer of T cells into T cell–deficient mice is followed by rapid engraftment and expansion of donor T cells into the host. This process is driven by foreign Ags.36 Attesting to a role of the macrobiota, the expansion of transferred T cells into T cell–deficient mice is greatly reduced in host mice raised in a germ-free environment.36 Moreover, mice maintained in germ-free conditions display increased bone mass due to the lack of immune cell activation.39 T cells are key inducers of bone-wasting because ovx increases T cell TNF production to a level sufficient to augment RANKL-induced osteoclastogenesis.1 This effect is due to an increased number of TNF-producing T cells15 and enhanced production of TNF per cell.18, 40 Ovx also increases the population of premature senescent CD4 + CD28–T cells,40 a lineage that produces high levels of TNF. The presence of increased levels of T cell–produced TNF in the BM of ovx animals is well documented.15, 19, 40, 41 Studies have also shown that menopause increases T cell activation and T cell production of TNF and RANKL in humans.42, 43 The role of TNF in ovx-induced bone loss has been demonstrated in multiple models. For example, ovx fails to induce bone loss in TNF-null mice and in animals lacking the p55 TNF receptor.15 Likewise, transgenic mice insensitive to TNF due to the overexpression of a soluble TNF receptor,44 and mice treated with the TNF inhibitor TNF binding protein45 are protected from ovx-induced bone loss. The specific relevance of T cell TNF production in vivo was demonstrated by the finding that although reconstitution of nude recipient mice with T cells from WT mice restores the capacity of ovx to induce bone loss, reconstitution with T cells from TNF-deficient mice does not.15 The mechanism by which estrogen deficiency expands the pool of TNF-producing T cells is summarized in Figure 1 and involves reactivation of thymic function and induction of T cell activation in the BM. T cell activation is driven by enhanced Ag presentation by macrophages and dendritic cells (DCs).46, 47 The thymus undergoes progressive structural and functional decline with age, coinciding with increased circulating sex-steroid levels at puberty.48 By middle age most parenchymal tissue is replaced by fat, and in both mice and humans fewer T cells are produced and exported to secondary lymphoid organs. However, the thymus continues to generate new T cells even into old age.49, 50 In fact, active lymphocytic thymic tissue has been documented in adults up to 107 years of age.51 Under severe T cell depletion secondary to human immunodeficiency virus (HIV) infection, chemotherapy, or bone marrow transplant, an increase in thymic output (known as thymic rebound) becomes critical for long-term restoration of T cell homeostasis. For example, middle-aged women treated with autologous bone marrow transplants develop thymic hypertrophy and a resurgence of thymic T cell output, which contributes to the restoration of a wide T cell repertoire,52 although the intensity of thymic rebound declines with age. The mechanism driving thymic rebound is not completely understood, but one factor involved is IL-7.53 Both androgens and estrogen suppress thymic function.54, 55 Accordingly, castration reverses thymic atrophy and increases export of recent thymic emigrants to the periphery,56 whereas sex steroids inhibit thymic regeneration by promoting thymocyte apoptosis and arresting thymocyte/prelymphocyte differentiation.57 Restoration of thymic function after castration occurs in young58 as well as in very old rodents.59, 60 In accordance with the notion that estrogen deficiency induces a rebound in thymic function, ovx increases the thymic export of naïve T cells.61 Indeed, stimulated thymic T cell output accounts for ∼50% of the increase in the number of T cells in the periphery. Moreover, thymectomy decreases the bone loss induced by ovx by ∼50%, thus demonstrating that the thymus plays a previously unrecognized role in the pathogenesis of ovx-induced bone loss in mice.61 The remaining bone loss is a consequence of the peripheral expansion of naïve and memory T cells. This finding suggests the tantalizing hypothesis that estrogen deficiency–induced thymic rebound may be responsible for the exaggerated bone loss in young women undergoing surgical menopause62 or for the rapid bone loss characteristic of women in their first 5 to 7 years after natural menopause.63 Indeed, an age-related decrease in estrogen deficiency–induced thymic rebound could mitigate the stimulatory effects of sex steroid deprivation and explain why the rate of bone loss in postmenopausal women diminishes as aging progresses.63 The most upstream effects of ovx in the BM are to stimulate the production of reactive oxygen species (ROS) and to impair the generation of antioxidants.10, 19, 64, 65 In response to ovx, ROS are produced by most BM cells, including T cells.41 ROS play an important role in postmenopausal bone loss by generating a more oxidized bone microenvironment.66, 67 Multiple enzymatic pathways regulate the intracellular redox state through modulation of ROS levels.68 Ovx blunts the BM levels of glutathione (GSH), a critical ROS scavenger, and reduces expression of APE1/Ref-1 and Prx-1 proteins, which collectively limit the production of intracellular ROS.69 ROS have important direct effects on osteoblasts and osteoclasts; these effects have been addressed elsewhere.7, 70, 71 However, additional pivotal effects of ROS include expanding the pool of mature DCs that express the costimulatory molecule CD80, and increasing DC-mediated Ag presentation.19 Antioxidants potently inhibit DC differentiation and their ability to activate T cells,72, 73 in part by suppressing expression of MHC class II and costimulatory molecules in response to Ag.74 N-acetyl-cysteine (NAC), which acts as an intracellular scavenger of ROS by restoring intracellular concentrations of GSM, can block DC maturation75 and DC-mediated T cell activation.76 In vivo support for a role of ROS is provided by experiments demonstrating that administration of antioxidants prevents ovx-induced bone loss,19, 64, 70 while depletion of glutathione by buthionine sulfoximine (BSO), which inhibits glutathione synthesis, enhances bone loss.64 Bone loss caused by BSO has significant similarities to bone loss induced by estrogen deficiency, as both processes are TNF-dependent.77 A second, direct upstream effect of estrogen deficiency is to blunt BM levels of TGFβ,78 a powerful repressor of T cell activation. TGFβ acts as an immunosuppressant by inhibiting T cell activation and T cell production of inflammatory cytokines, including IFNγ. Demonstrating the relevance of the repressive effects of TGFβ on T cell function, mice with T cell–specific blockade of TGFβ signaling were found to be completely resistant to the bone-sparing effects of estrogen.16 Gain of function experiments confirmed that elevation of the systemic levels of TGFβ prevents ovx-induced bone loss and bone turnover.16 The key downstream mechanism by which ovx increases Ag presentation by macrophages is a stimulatory effect on the expression of the gene encoding Class II Transactivator (CIITA). The product of CIITA is a non-DNA binding factor induced by IFNγ that functions as a transcriptional coactivator at the MHC II promoter.79 Increased CIITA expression in macrophages results from ovx-mediated increases in IFNγ production by CD4+ T cells and the responsiveness of CIITA to IFNγ.46 This cytokine was initially described as an anti-osteoclastogenic cytokine because it is a potent inhibitor of osteoclastogenesis in vitro.80 The notion that IFNγ is an inhibitor of bone resorption was reinforced by the finding that silencing of IFNγR−/− signaling leads to a more rapid onset of collagen-induced arthritis and bone resorption81 as compared to WT controls, and by the report that IFNγ decreases serum calcium and osteoclastic bone resorption in nude mice.82, 83 Several mechanisms have been proposed to explain the anti-osteoclastogenic activity of IFNγ, including inhibition of RANKL signaling through the degradation of TNF receptor–associated factor 6 (TRAF6),80 stimulation of apoptosis mediated by Fas/FasL signals,84 and inhibition of RANK and c-Fms gene expression.85 However, the finding that IFNγ is an effective treatment for osteopetrosis both in humans86 and rodents87 demonstrates that the net effect of IFNγ in vivo is to stimulate osteoclastic bone resorption. In keeping with a net pro-resorptive effect of IFNγ in vivo are reports demonstrating that IFNγ−/− and IFNγR−/− mice are protected against ovx-induced bone loss.17, 46 Mice lacking IFNγ production are also protected against infection-induced alveolar bone loss,88 whereas in erosive tuberculoid leprosy and psoriatic arthritis IFNγ production correlates positively with tissue destruction.89 In addition, randomized controlled trials have shown that IFNγ does not prevent bone loss in patients with rheumatoid arthritis (RA),90 nor the bone-wasting effect of cyclosporine A.91 Finally, disruption of IFNγ signaling in vivo results in a strong and sustained inhibition of markers of osteoclastic activity.92 These opposing in vitro and in vivo effects of IFNγ are explained by the fact that IFNγ influences osteoclast formation via both direct and indirect effects.17 IFNγ directly blocks osteoclast formation through targeting of maturing osteoclast.93 However, IFNγ is also a potent inducer of antigen presentation and thus of T cell activation. Therefore, when IFNγ levels are increased in vivo, activated T cells secrete pro-osteoclastogenic factors and this activity offsets the anti-osteoclastogenic effects of IFNγ.17 It should also be mentioned that it is now recognized that IFNγ affects bone turnover by promoting the commitment of BM–SCs into the osteoblastic lineage and their differentiation into mature osteoblasts.94 Accordingly, treatment with IFNγ reverses ovx-induced bone loss by promoting bone formation.92 Another mechanism by which estrogen regulates T cell TNF production is by repressing the production of IL-7, a potent lymphopoietic cytokine and an inducer of bone destruction in vivo.95 Attesting to the relevance of this factor, IL-7 levels are significantly elevated following ovx,61, 96 and in vivo IL-7 blockade is effective in preventing ovx-induced bone destruction.96 The elevated BM levels of IL-7 contribute to the expansion of the T cell population in peripheral lymphoid organs through several mechanisms. First, IL-7 directly stimulates T cell proliferation.61 Second, IL-7 increases antigen presentation by upregulating the production of IFNγ. Third, IL-7 and TGFβ regulate The in TGFβ signaling characteristic of estrogen deficiency may to stimulate IL-7 thus driving the of osteoclastogenic cytokine production and bone In estrogen deficiency, IL-7 bone loss by suppressing bone formation and thus bone formation from resorption. A that the crosstalk between T cells and SCs is relevant for ovx-induced bone loss was provided by the finding that activated T cells induce SC apoptosis via the ligand a which blunts the compensatory increase in bone formation that bone loss in ovx More abundant information is available about the T crosstalk driven by the also known as is a key surface ligand expressed on T CD40L to and several CD40 is expressed on cells, hemopoietic and cells of the osteoblastic CD40L has been to because T cells, through the production of the anti-osteoclastogenic factor by B mice a reduced bone due to exaggerated bone bone has also been found in by a in which CD40L production is due to a in the CD40L However, mice lacking T cell–expressed CD40L are protected against hormone bone the that CD40L may in while promoting bone resorption conditions of bone Studies with mice and with WT mice treated with the CD40L have that silencing of CD40L completely prevents ovx-induced bone A mechanism was shown to be First, silencing of CD40L blocks the activation of T cells and the resulting production of TNF. Second, CD40L is for ovx to increase the proliferation and the differentiation of SCs and their capacity to support osteoclast formation through enhanced production of factor and RANKL, and secretion of a critical additional mechanism by which T cells bone homeostasis in ovx mice is through a crosstalk between T cells and SCs that results in enhanced osteoclastogenesis to a enhanced In ovx increases the number of activated T cells that the expression of and RANKL by SCs, and ovx the SC production of The net result is a significant increase in the rate of The interaction of CD40L with CD40 on SCs in the of estrogen deficiency to the effects of costimulation on B cell OPG the of osteoclast formation in of bone loss. An additional mechanism of T crosstalk the novel of bone mass antigen has been for a known as This factor can be to generate a soluble known as In conditions DLK1/FA-1 are produced by SCs, B cells, and T cells. These factors stimulate osteoclastogenesis and block by the production of TNF, IL-7, and other inflammatory cytokines by SCs. Ovx increases the production of DLK1/FA-1 by activated CD4+ and CD8+ T cells, resulting in a stimulation of the production of osteoclastogenic cytokines by SCs. Attesting to the relevance of mice are significantly protected against ovx-induced bone The hypothesis that the immune system is pivotal for the bone loss about by menopause remains to be demonstrated in it remains to the of why the immune system in and is involved in the mechanism of ovx-induced bone loss. may by postmenopausal bone loss as an of an event critical for the need to stimulate bone resorption in the This process is essential to the increased for calcium about by The for this event is the acute in estrogen levels A second to the period is in associated with is by proliferation of the and formation of These are by and is now that regulates and to by the production of RANKL and a system that is for bone homeostasis is also a key of The in levels in the period results in reduced tissue levels of RANK and RANKL, with the effect of the for The increase in the and TNF levels in the BM induced by estrogen levels the of calcium from the to the and then to the a state of to the is induced by an increase in the number of A to the of is the loss of such immune and the restoration of a immune This is through a decrease in the resulting from the acute in steroid levels. However, a between immune to the and bone is provided by the that OPG is expressed by human it is to that of ovarian function induces bone loss through an immune response because natural has these key within the immune system to (Fig. Schematic representation of in the period and of the involved cells and The that estrogen and are followed by a stimulation of bone resorption, to and of immune to the Activated T cells drive bone resorption by TNF. A decrease in the number of Tregs induces the of the immune The loss of OPG may contribute to the and the increased bone resorption. A decrease in levels of RANKL and RANK induce the of the for with increased of calcium from the the in the contribute to an event critical for The data the hypothesis that of ovarian function induces bone loss through an immune response because natural has key within the immune system to The data support the hypothesis that the bone loss induced by estrogen deficiency is due to a complex of hormones and cytokines that to the process of bone Ovx T cell TNF production by increasing T cell activity in the and the peripheral lymphoid organs. T cell precursors the BM and to the T cell and expansion in the of from the these new T cells to peripheral lymphoid including the BM Ovx induces T cell activation in the BM in part by directly promoting antigen and in part via stimulation of IL-7 and IFNγ production and of TGFβ The net result of these actions is an increase in the number of TNF-producing T cells. The elevated levels of TNF increase RANKL-induced osteoclast formation. Estrogen deficiency also T cell activation and osteoclastogenesis by leading to an in The combined effect of IFNγ and ROS enhances Ag T cell activation. T cells stimulate RANKL and production by SCs, through CD40L and The that has of

Disuse osteoporosis, which occurs commonly in prolonged bed rest and immobilization, is becoming a major problem in modern societies; however, the molecular mechanisms underlying unloading-driven bone loss have not been fully elucidated. Bone adjusts its shape and strength against mechanical stress. Osteocytes are the most abundant cells in bone and comprise the communication system through the processes and canaliculi throughout bone. The osteocyte network is considered to be an ideal mechanosensor and mechanotransduction system. We found that overexpression of BCL2 in osteoblasts reduces the number of osteocyte processes, probably due to the function of Bcl2 that modulates cytoskeletal reorganization, and induces the apoptosis of osteocytes, in which the transgene expression was reduced, presumably caused by an insufficient supply of oxygen, nutrients, and survival factors due to the reduced osteocyte processes. Our BCL2 transgenic mouse with accumulated dead osteocytes is a useful model to analyze the function of osteocytes, because a repair process, which replaces dead osteocytes with new osteocytes by bone resorption and formation, was not evident in the mice irrespective of the massive accumulation of dead osteocytes We searched for the molecules responsible for disuse osteoporosis using BCL2 transgenic mice. Pyruvate dehydrogenase kinase isozymes (Pdk1, Pdk2, Pdk3, and Pdk4) are negative regulators of pyruvate dehydrogenase complex (PDC), which converts pyruvate to acetyl-CoA in the mitochondria, linking glycolysis to the energetic and anabolic functions of the tricarboxylic acid (TCA) cycle. Pdk4 was upregulated in femurs and tibiae of wild-type mice but not of BCL2 transgenic mice after tail suspension. Bone in Pdk4-/- mice developed normally and was maintained. At unloading, however, bone mass was reduced due to enhanced osteoclastogenesis and Rankl expression in wild-type mice but not in Pdk4-/- mice. Osteoclast differentiation of Pdk4-/- bone marrow-derived monocyte/macrophage lineage cells (BMMs) in the presence of M-CSF and RANKL was suppressed, and osteoclastogenesis was impaired in the coculture of wild-type BMMs and Pdk4-/- osteoblasts, in which Rankl expression and promoter activity were reduced. Further, introduction of Pdk4 into Pdk4-/- BMMs and osteoblasts enhanced osteoclastogenesis and Rankl expression and activated Rankl promoter. These findings indicate that upregulation of Pdk4 expression in osteoblasts and bone marrow cells after unloading is, at least in part, responsible for the enhancement of osteoclastogenesis and bone resorption after unloading [1].

Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment

Parathyroid hormone (PTH) and the sympathetic tone promote Rankl expression in osteoblasts and osteoclast differentiation by enhancing cyclic adenosine monophosphate production through an unidentified transcription factor for PTH and through ATF4 for the sympathetic tone. How two extracellular cues using the same second messenger in the same cell elicit different transcriptional events is unknown. In this paper, we show that PTH favors Rankl expression by triggering the ubiquitination of HDAC4, a class II histone deacetylase, via Smurf2. HDAC4 degradation releases MEF2c, which transactivates the Rankl promoter. Conversely, sympathetic signaling in osteoblasts favors the accumulation of HDAC4 in the nucleus and its association with ATF4. In this context, HDAC4 increases Rankl expression. Because of its ability to differentially connect two extracellular cues to the genome of osteoblasts, HDAC4 is a critical regulator of osteoclast differentiation.

The discoveries that sex steroid receptors for estrogen [estrogen receptor (ER)], androgen [androgen receptor (AR)], and progesterone [progesterone receptor (PR)] are present in the bone-forming osteoblasts (OB) and bone-resorbing osteoclasts (OCL), and that these cells are targets for sex steroid action, have generated much excitement in the field of bone biology. The subsequent discoveries that the sex steroids regulate the production of growth factors and cytokines in these OB and OCL cells and that these factors mediate much of the steroid action on the skeleton have added to the excitement and complexity of the system. Because of the required brevity of this minireview, we will discuss only selected aspects of this field using a limited number of references and review articles. For more information in this field, readers are referred to several recent books and chapters that review bone biology, bone diseases, and the role of sex steroids in bone biology and disease (1–8). As a brief background to this unique system, the skeletal maintenance involves a continuous remodeling at discrete sites termed bone-remodeling units (BRU). As outlined in Fig. 1, the multinucleated boneresorbing OCLs first dissolve bone at these BRU sites, resulting in resorption cavities. While the mechanism(s)/signal(s) that initiates resorption at a given site is unknown, there appears to be a marked regulation of overall bone resorption via the regulation of the OCL differentiation and activity (5–8). These processes are discussed in more detail below. The bone-forming OBs are then recruited by unknown signals, to replace the previously resorbed bone. The identities of the primary factors that couple these processes of resorption to formation to maintain bone mass are also addressed later in this review and are still the subject of much debate. During periods of skeletal growth in children, the increase in bone formation largely involves bone modeling (accretion of bone at surfaces in the absence of bone resorption). The response to mechanical stresses also utilizes mostly modeling. During early adulthood (20–40 yr), bone resorption and formation are in balance and bone mass is maintained. The increased serum estrogen (E) level, which occurs at the onset of puberty in girls, is accompanied by an increase in growth velocity and, ultimately, in the closure of the epiphyseal growth plate and the cessation of linear growth (9). E is the primary hormone responsible for maintaining bone mass in adult women and may serve a similar role along with androgen, in adult men. Finally, during periods of bone loss, such as in postmenopause in women and aging in both genders, bone resorption outpaces bone formation and, in some cases, the bone loss is sufficient to cause osteoporosis. It is currently accepted that in postmenopausal females, a deficiency of E results in osteoporosis, whereas in aging males, the deficiency of E and androgens (A) may lead to bone loss (1–3). Ovariectomized (OVX) animals and postmenopausal women experience increased bone turnover, increased bone resorption, and an overall decrease in bone mass. On the other hand, E replacement therapy inhibits this bone loss by inhibiting bone turnover and bone resorption, which prevents the development of osteoporosis (1, 3). Although, the complete mechanisms of action of E in this protection against bone loss are still unclear, new studies are beginning to elucidate the molecular processes involved. The following review summarizes some of the important molecular events in the actions of sex steroids, primarily estrogens, which 0888-8809/99/$3.00/0 Molecular Endocrinology Copyright © 1999 by The Endocrine Society

It is well known that GH is important in the regulation of longitudinal bone growth. Its role in the regulation of bone metabolism in man has not been understood until recently. Several in vivo and in vitro studies have demonstrated that GH is important in the regulation of both bone formation and bone resorption. In Figure 9 a simplified model for the cellular effects of GH in the regulation of bone remodeling is presented (Fig. 9). GH increases bone formation in two ways: via a direct interaction with GHRs on osteoblasts and via an induction of endocrine and autocrine/paracrine IGF-I. It is difficult to say how much of the GH effect is mediated by IGFs and how much is IGF-independent. GH treatment also results in increased bone resorption. It is still unknown whether osteoclasts express functional GHRs, but recent in vitro studies indicate that GH regulates osteoclast formation in bone marrow cultures. Possible modulations of the GH/IGF axis by glucocorticoids and estrogens are also included in Fig. 9. GH deficiency results in a decreased bone mass in both man and experimental animals. Long-term treatment (> 18 months) of GHD patients with GH results in an increased bone mass. GH treatment also increases bone mass and the total mechanical strength of bones in rats with a normal GH secretion. Recent clinical studies demonstrate that GH treatment of patients with normal GH secretion increases biochemical markers for both bone formation and bone resorption. Because of the short duration of GH treatment in man with normal GH secretion, the effect on bone mass is still inconclusive. Interestingly, GH treatment to GHD adults initially results in increased bone resorption with an increased number of bone-remodeling units and more newly produced unmineralized bone, resulting in an apparent low or unchanged bone mass. However, GH treatment for more than 18 months gives increased bone formation and bone mineralization of newly produced bone and a concomitant increase in bone mass as determined with DEXA. Thus, the action of GH on bone metabolism in GHD adults is 2-fold: it stimulates both bone resorption and bone formation. We therefore propose "the biphasic model" of GH action in bone remodeling (Fig. 10). According to this model, GH initially increases bone resorption with a concomitant bone loss that is followed by a phase of increased bone formation. After the moment when bone formation is stimulated more than bone resorption (transition point), bone mass is increased. However, a net gain of bone mass caused by GH may take some time as the initial decrease in bone mass must first be replaced (Fig. 10). When all clinical studies of GH treatment of GHD adults are taken into account, it appears that the "transition point" occurs after approximately 6 months and that a net increase of bone mass will be seen after 12-18 months of GH treatment. It should be emphasized that the biphasic model of GH action in bone remodeling is based on findings in GHD adults. It remains to be clarified whether or not it is valid for subjects with normal GH secretion. A treatment intended to increase the effects of GH/IGF-I axis on bone metabolism might include: 1) GH, 2) IGF, 3) other hormones/factors increasing the local IGF-I production in bone, and 4) GH-releasing factors. Other hormones/growth factors increasing local IGF may be important but are not discussed in this article. IGF-I has been shown to increase bone mass in animal models and biochemical markers in humans. However, no effect on bone mass has yet been presented in humans. Because the financial cost for GH treatment is high it has been suggested that GH-releasing factors might be used to stimulate the GH/IGF-I axis. The advantage of GH-releasing factors over GH is that some of them can be administered orally and that they may induce a more physiological GH secretion. (ABSTRACT TRUNCATED)

CYR61/CCN1 is a matricellular protein that resides in the extracellular matrix, but serves regulatory rather than structural roles. CYR61/CCN1 is found in mineralized tissues and has been shown to influence bone healing in vivo and osteogenic differentiation in vitro. In this study we generated Cyr61 bone-specific knockout mice to examine the physiological role of CYR61/CCN1 in bone development and maintenance in vivo. Extensive analysis of Cyr61 conditional knockout mice showed a significant decrease in both trabecular and cortical bone mass as compared to WT littermates. Our data suggest that CYR61/CCN1 exerts its effects on mature osteoblast/osteocyte function to modulate bone mass. Specifically, changes were observed in osteocyte/osteoblast expression of RankL, VegfA, and Sost. The increase in RankL expression was correlated with a significant increase in osteoclast number; decreased VegfA expression was correlated with a significant decrease in bone vasculature; increased Sost expression was associated with decreased Wnt signaling, as revealed by decreased Axin2 expression and increased adiposity in the bone marrow. Although the decreased number of vascular elements in bone likely contributes to the low bone mass phenotype in Cyr61 conditional knockout mice, this cannot explain the observed increase in osteoclasts and the decrease in Wnt signaling. We conducted in vitro assays using UMR-106 osteosarcoma cells to explore the role CYR61/CCN1 plays in modulating Sost mRNA and protein expression in osteocytes and osteoblasts. Overexpression of CYR61/CCN1 can suppress Sost expression in both control and Cyr61 knockout cells, and blocking Sost with siRNA can rescue Wnt responsiveness in Cyr61 knockout cells in vitro. Overall, our data suggest that CYR61/CCN1 modulates mature osteoblast and osteocyte function to regulate bone mass through angiogenic effects as well as by modulating Wnt signaling, at least in part through the Wnt antagonist Sost. © 2018 American Society for Bone and Mineral Research.