Mediators of the biphasic responses of bone to intermittent and continuously administered parathyroid hormone

Parathyroid hormone (PTH) has biphasic effects on bone: continuous treatment is catabolic whereas intermittent treatment is anabolic. The mechanism(s) responsible for these differing effects are still unclear, partly because of the previous non-availability of a model system in which effects on both formation and resorption indices could be studied concomitantly. In cultured marrow cells from 6-week old C57BL/6 mice, we demonstrated that 4 days of intermittent PTH treatment increased mRNA for osteoblast differentiation markers (Runx2, alkaline phosphatase (AP), and type I procollagen (COL1A1) whereas continuous treatment resulted in production of large numbers of TRAP-positive multinucleated osteoclasts. Although IGF-I mRNA did not increase after intermittent treatment, it was consistently higher than after continuous treatment, and the addition of an anti-IGF-I neutralizing antibody prevented the increase in bone formation indices observed with intermittent treatment. By contrast, after continuous treatment, gene expression of RANK ligand (RANKL) was increased and that of osteoprotegerin (OPG) was decreased, resulting in a 25-fold increase in the RANKL/OPG ratio. In this model system, the data suggest that intermittent PTH treatment enhances osteoblast differentiation through an IGF-I dependent mechanism and continuous PTH treatment enhances osteoclastogenesis through reciprocal increases in RANKL and decreases in OPG.     

Impaired osteoclast formation in bone marrow cultures of Fgf2 null mice in response to parathyroid hormone

Fibroblast growth factor (FGF)-2 and parathyroid hormone (PTH) are potent inducers of osteoclast (OCL) formation, and PTH increases FGF-2 mRNA and protein expression in osteoblasts. To elucidate the role of endogenous FGF-2 in PTH responses, we examined PTH-induced OCL formation in bone marrow cultures from wild type and mice with a disruption of the Fgf2 gene. FGF-2-induced OCL formation was similar in marrow culture from both genotypes. In contrast, PTH-stimulated OCL formation in bone marrow cultures or co-cultures of osteoblast-spleen cells from Fgf2-/mice was significantly impaired. PTH increased RANKL mRNA expression in osteoblasts cultures from both genotypes. After 6 days of treatment, osteoprotegerin protein in cell supernatants was 40-fold higher in vehicle-treated and 30-fold higher in PTH-treated co-cultures of osteoblast and spleen cells from Fgf2-/mice compared with Fgf2+/+ mice. However, a neutralizing antibody to osteoprotegerin did not rescue reduced OCL formation in response to PTH. Injection of PTH caused hypercalcemia in Fgf2+/+ but not Fgf2-/mice. We conclude that PTH stimulates OCL formation and bone resorption in mice in part by endogenous FGF-2 synthesis by osteoblasts. Because RANKL- and interleukin-11-induced OCL formation was also reduced in bone marrow cultures from Fgf2-/mice, we further conclude that endogenous FGF-2 is necessary for maximal OCL formation by multiple bone resorbing factors.     

The Proteasome Inhibitor Carfilzomib Suppresses Parathyroid Hormone-induced Osteoclastogenesis through a RANKL-mediated Signaling Pathway

Parathyroid hormone (PTH) induces osteoclast formation and activity by increasing the ratio of RANKL/OPG in osteoblasts. The proteasome inhibitor carfilzomib (CFZ) has been used as an effective therapy for multiple myeloma via the inhibition of pathologic bone destruction. However, the effect of combination of PTH and CFZ on osteoclastogenesis is unknown. We now report that CFZ inhibits PTH-induced RANKL expression and secretion without affecting PTH inhibition of OPG expression, and it does so by blocking HDAC4 proteasomal degradation in osteoblasts. Furthermore, we used different types of culture systems, including co-culture, indirect co-culture, and transactivation, to assess the effect of CFZ on PTH action to induce osteoclastogenesis. Our results demonstrated that CFZ blocks PTH-induced osteoclast formation and bone resorption by its additional effect to inhibit RANKL-mediated IκB degradation and NF-κB activation in osteoclasts. This study showed for the first time that CFZ targets both osteoblasts and osteoclasts to suppress PTH-induced osteoclast differentiation and bone resorption. These findings warrant further investigation of this novel combination in animal models of osteoporosis and in patients.     

Suppression of osteoprotegerin expression by prostaglandin E2 is crucially involved in lipopolysaccharide-induced osteoclast formation

LPS is a potent stimulator of bone resorption in inflammatory diseases. The mechanism by which LPS induces osteoclastogenesis was studied in cocultures of mouse osteoblasts and bone marrow cells. LPS stimulated osteoclast formation and PGE(2) production in cocultures of mouse osteoblasts and bone marrow cells, and the stimulation was completely inhibited by NS398, a cyclooxygenase-2 inhibitor. Osteoblasts, but not bone marrow cells, produced PGE(2) in response to LPS. LPS-induced osteoclast formation was also inhibited by osteoprotegerin (OPG), a decoy receptor of receptor activator of NF-kappaB ligand (RANKL), but not by anti-mouse TNFR1 Ab or IL-1 receptor antagonist. LPS induced both stimulation of RANKL mRNA expression and inhibition of OPG mRNA expression in osteoblasts. NS398 blocked LPS-induced down-regulation of OPG mRNA expression, but not LPS-induced up-regulation of RANKL mRNA expression, suggesting that down-regulation of OPG expression by PGE(2) is involved in LPS-induced osteoclast formation in the cocultures. NS398 failed to inhibit LPS-induced osteoclastogenesis in cocultures containing OPG knockout mouse-derived osteoblasts. IL-1 also stimulated PGE(2) production in osteoblasts and osteoclast formation in the cocultures, and the stimulation was inhibited by NS398. As seen with LPS, NS398 failed to inhibit IL-1-induced osteoclast formation in cocultures with OPG-deficient osteoblasts. These results suggest that IL-1 as well as LPS stimulates osteoclastogenesis through two parallel events: direct enhancement of RANKL expression and suppression of OPG expression, which is mediated by PGE(2) production.     

Constitutively active PTH/PTHrP receptor specifically expressed in osteoblasts enhances bone formation induced by bone marrow ablation

Bone is maintained by continuous bone formation by osteoblasts provided by proliferation and differentiation of osteoprogenitors. Parathyroid hormone (PTH) activates bone formation, but because of the complexity of cells in the osteoblast lineage, how these osteoprogenitors are regulated by PTH in vivo is incompletely understood. To elucidate how signals by PTH in differentiated osteoblasts regulate osteoprogenitors in vivo, we conducted bone marrow ablation using Col1a1‐constitutively active PTH/PTHrP receptor (caPPR) transgenic mice. These mice express caPPR specifically in osteoblasts by using 2.3 kb Col1a1 promoter and showed higher trabecular bone volume under steady‐state conditions. In contrast, after bone marrow ablation, stromal cells recruited from bone surface extensively proliferated in the marrow cavity in transgenic mice, compared to limited proliferation in wild‐type mice. Whereas de novo bone formation was restricted to the ablated area in wild‐type mice, the entire marrow cavity, including not only ablated area but also outside the ablated area, was filled with newly formed bone in transgenic mice. Bone mineral density was significantly increased after ablation in transgenic mice. Bone marrow cell culture in osteogenic medium revealed that alkaline phosphatase‐positive area was markedly increased in the cells obtained from transgenic mice. Furthermore, mRNA expression of Wnt‐signaling molecules such as LRP5, Wnt7b, and Wnt10b were upregulated after marrow ablation in bone marrow cells of transgenic mice. These results indicate that constitutive activation of PTH/PTHrP receptor in differentiated osteoblasts enhances bone marrow ablation‐induced recruitment, proliferation, and differentiation of osteoprogenitors. J. Cell. Physiol. 227: 408–415, 2012. © 2011 Wiley Periodicals, Inc.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

Effects of arachidonic acid, docosahexaenoic acid, prostaglandin E(2) and parathyroid hormone on osteoprotegerin and RANKL secretion by MC3T3-E1 osteoblast-like cells

Bone is continuously remodeled through resorption by osteoclasts and the subsequent synthesis of the bone matrix by osteoblasts. Cell-to-cell contact between osteoblasts and osteoclast precursors is required for osteoclast formation. RANKL (receptor activator of nuclear factor-kappaB ligand) expressed on osteoblastic cell membranes stimulates osteoclastogenesis, while osteoprotegerin (OPG) secreted by osteoblasts inhibits osteoclastogenesis. Although polyunsaturated fatty acids (PUFAs) have been implicated in bone homeostasis, the effects thereof on OPG and RANKL secretion have not been investigated. MC3T3-E1 osteoblasts were exposed to the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA); furthermore, the bone-active hormone parathyroid hormone (PTH) and the effects thereof were tested on OPG and RANKL secretion. Prostaglandin E(2) (PGE(2)), a product of AA metabolism that was previously implicated in bone homeostasis, was included in the study. AA (5.0-20 microg/ml) inhibited OPG secretion by 25-30%, which was attenuated by pretreatment with the cyclooxygenase blocker indomethacin, suggesting that the inhibitory effect of AA on OPG could possibly be PGE(2)-mediated. MC3T3-E1 cells secreted very low basal levels of RANKL, but AA stimulated RANKL secretion, thereby decreasing the OPG/RANKL ratio. DHA suppressed OPG secretion to a smaller extent than AA. This could, however, be due to endogenous PGE(2) production. No RANKL could be detected after exposing the MC3T3-E1 cells to DHA. PTH did not affect OPG secretion, but stimulated RANKL secretion. This study demonstrates that AA and PTH reduce the OPG/RANKL ratio and may increase osteoclastogenesis. DHA, however, had no significant effect on OPG or RANKL in this model.     

RANKL与骨重建

骨是一种动态更新的组织,它不断进行骨吸收（bone resorption）与骨形成（bone formation）的平衡,这个过程称之为骨重建（bone remodeling）．核因子κB受体活化因子配体（receptor activator of nuclear factor κB ligand,RANKL）是骨吸收和骨形成耦联的关键,具有诱导破骨细胞（osteoclast, OC）生成、活化,抑制破骨细胞凋亡的作用．RANKL最初发现于活化的T细胞,但骨重建过程中RANKL主要来源于骨细胞、成骨细胞和骨髓基质细胞．RANKL/核因子κB受体活化因子（receptor activator of nuclear factor κB,RANK）/骨保护素（osteoprotegerin, OPG）信号通路在成骨细胞调控破骨细胞生成的过程中起着重要的调节作用,是维持骨重建平衡的关键．本文就RANKL及其在骨中的分子作用机制作一综述.     

Normal human osteoclasts formed from peripheral blood monocytes express PTH type 1 receptors and are stimulated by PTH in the absence of osteoblasts

The prevailing view for many years has been that osteoclasts do not express parathyroid hormone (PTH) receptors and that PTH's effects on osteoclasts are mediated indirectly via osteoblasts. However, several recent reports suggest that osteoclasts express PTH receptors. In this study, we tested the hypothesis that human osteoclasts formed in vitro express functional PTH type 1 receptors (PTH1R). Peripheral blood monocytes (PBMC) were cultured on bone slices or plastic culture dishes with human recombinant RANK ligand (RANKL) and recombinant human macrophage colony-stimulating factor (M-CSF) for 16-21 days. This resulted in a mixed population of mono- and multi-nucleated cells, all of which stained positively for the human calcitonin receptor. The cells actively resorbed bone, as assessed by release of C-terminal telopeptide of type I collagen and the formation of abundant resorption pits. We obtained evidence for the presence of PTH1R in these cells by four independent techniques. First, using immunocytochemistry, positive staining for PTH1R was observed in both mono- and multi-nucleated cells intimately associated with resorption cavities. Second, PTH1R protein expression was demonstrated by Western blot analysis. Third, the cells expressed PTH1R mRNA at 21 days and treatment with 10(-7) M hPTH (1-34) reduced PTH1R mRNA expression by 35%. Finally, bone resorption was reproducibly increased by two to threefold when PTH (1-34) was added to the cultures. These findings provide strong support for a direct stimulatory action of PTH on human osteoclasts mediated by PTH1R. This suggests a dual regulatory mechanism, whereby PTH acts both directly on osteoclasts and also, indirectly, via osteoblasts.     

Osteoclastogenic activity and RANKL expression are inhibited in osteoblastic cells expressing constitutively active Gα12 or constitutively active RhoA

Gα₁₂–RhoA signaling is a parathyroid hormone (PTH)‐stimulated pathway that mediates effects in bone and may influence genetic susceptibility to osteoporosis. To further elucidate effects of the pathway in osteoblasts, UMR‐106 osteoblastic cells were stably transfected with constitutively active (ca) Gα₁₂ or caRhoA or dominant negative (dn) RhoA and co‐cultured with RAW 264.7 cells to determine effects on hormone‐stimulated osteoclastogenesis. Whereas PTH and calcitriol‐stimulated osteoclastogenesis in co‐cultures with UMR‐106 cells expressing pcDNA or dominant negative RhoA, the osteoclastogenic effects of PTH and calcitriol were significantly attenuated when the UMR‐106 cells expressed either caRhoA or caGα₁₂. These inhibitory effects were partially reversed by the Rho kinase inhibitor Y27632. None of the constructs affected osteoclastogenesis in untreated co‐cultures, and the constructs did not inhibit the osteoclastogenic responses to receptor activator of NFκB ligand (RANKL). To investigate the mechanism of the inhibitory effects of caGα₁₂ and caRhoA, expression of RANKL, osteoprotegerin (OPG), osteopontin (OPN), and intercellular adhesion molecule‐1 (ICAM) in response to PTH or calcitriol was examined in the UMR‐106 cells. In the cells expressing pcDNA or dnRhoA, PTH and calcitriol increased RANKL mRNA and decreased OPG mRNA, whereas these effects were absent in the cells expressing caGα₁₂ or caRhoA. Basal expression of RANKL and OPG was unaffected by the constructs. The results suggest that Gα₁₂–RhoA signaling can inhibit hormone‐stimulated osteoclastogenesis by effects on expression of RANKL and OPG. Since PTH can stimulate the Gα₁₂–RhoA pathway, the current findings could represent a homeostatic mechanism for regulating osteoclastogenic action. J. Cell. Biochem. 111: 1531–1536, 2010. © 2010 Wiley‐Liss, Inc.     

PTH/cAMP/PKA signaling facilitates canonical Wnt signaling via inactivation of glycogen synthase kinase-3beta in osteoblastic Saos-2 cells

Although the intermittent administration of PTH is known to stimulate the bone formation, the underlying mechanisms are not fully understood. Here we investigated the crosstalk between PTH/cAMP signaling and canonical Wnt signaling using the human osteoblastic cell line Saos-2. Treatment with PTH or forskolin, an activator of adenylate cyclase, facilitated T-cell factor (TCF)-dependent transactivation in a dose-dependent manner, which was abolished by pre-treatment with a PKA inhibitor, H89. Wnt3a and forskolin synergistically increased the TCF-dependent transactivation. Interestingly, intermittent treatment with PTH enhanced the TCF-dependent transactivation more profoundly than continuous treatment. In addition to the effects on TCF-dependent reporter activity, treatment with PTH or forskolin resulted in the increased expression of endogenous targets of Wnts, Wnt-induced secreted protein 2 (WISP2) and naked cuticle 2 (NKD2). We then investigated the convergence point of PTH/cAMP signaling and the canonical Wnt pathway. Western blotting demonstrated that GSK-3beta was rapidly phosphorylated at Ser(9) on treatment with PTH or forskolin, leading to its inactivation. Moreover, overexpression of a constitutively active mutant of GSK-3beta abolished the TCF-dependent transactivation induced by forskolin. On the other hand, overexpression of the Wnt antagonist Dickkopf-1 (DKK1) failed to cancel the effects of forskolin on the canonical Wnt pathway. Interestingly, treatment with Wnt3a markedly reduced the forskolin-induced expression of receptor activator of NF-kappaB ligand (RANKL), a target gene of PTH/cAMP/PKA. These results suggest that cAMP/PKA signaling activates the canonical Wnt pathway through the inactivation of GSK-3beta, whereas Wnt signaling might inhibit bone resorption through a negative impact on RANKL expression in osteoblasts.     

Parathyroid hormone temporal effects on bone formation and resorption

Parathyroid hormone (PTH) paradoxically causes net bone loss (resorption) when administered in a continuous fashion, and net bone formation (deposition) when administered intermittently. Currently no pharmacological formulations are available to promote bone formation, as needed for the treatment of osteoporosis. The paradoxical behavior of PTH confuses endocrinologists, thus, a model bone resorption or deposition dependent on the timing of PTH administration would de-mystify this behavior and provide the basis for logical drug formulation. We developed a mathematical model that accounts for net bone loss with continuous PTH administration and net bone formation with intermittent PTH administration, based on the differential effects of PTH on the osteoblastic and osteoclastic populations of cells. Bone, being a major reservoir of body calcium, is under the hormonal control of PTH. The overall effect of PTH is to raise plasma levels of calcium, partly through bone resorption. Osteoclasts resorb bone and liberate calcium, but they lack receptors for PTH. The preosteoblastic precursors and preosteoblasts possess receptors for PTH, upon which the hormone induces differentiation from the precursor to preosteoblast and from the preosteoblast to the osteoblast. The osteoblasts generate IL-6; IL-6 stimulates preosteoclasts to differentiate into osteoclasts. We developed a mathematical model for the differentiation of osteoblastic and osteoclastic populations in bone, using a delay time of 1 hour for differentiation of preosteoblastic precursors into preosteoblasts and 2 hours for the differentiation of preosteoblasts into osteoblasts. The ratio of the number of osteoblasts to osteoclasts indicates the net effect of PTH on bone resorption and deposition; the timing of events producing the maximum ratio would induce net bone deposition. When PTH is pulsed with a frequency of every hour, the preosteoblastic population rises and decreases in nearly a symmetric pattern, with 3.9 peaks every 24 hours, and 4.0 peaks every 24 hours when PTH is administered every 6 hours. Thus, the preosteoblast and osteoblast frequency depends more on the nearly constant value of the PTH, rather than on the frequency of the PTH pulsations. Increasing the time delay gradually increases the mean value for the number of osteoblasts. The osteoblastic population oscillates for all intermittent administrations of PTH and even when the PTH infusion is constant. The maximum ratio of osteoblasts to osteoclasts occurs when PTH is administered in pulses of every 6 hours. The delay features in the model bear most of the responsibility for the occurrence of these oscillations, because without the delay and in the presence of constant PTH infusions, no oscillations occur. However, with a delay, under constant PTH infusions, the model generates oscillations. The osteoblast oscillations express limit cycle behavior. Phase plane analysis show simple and complex attractors. Subsequent to a disturbance in the number of osteoblasts, the osteoblasts quickly regain their oscillatory behavior and cycle back to the original attractor, typical of limit cycle behavior. Further, because the model was constructed with dissipative and nonlinear features, one would expect ensuing oscillations to show limit cycle behavior. The results from our model, increased bone deposition with intermittent PTH administration and increased bone resorption with constant PTH administration, conforms with experimental observations and with an accepted explanation for osteoporosis.     

甲状旁腺素和胰岛素样生长因子促成骨样细胞ROS 17/2.8增殖分化中的协同作用

 间歇性小剂量地给予甲状旁腺素 (parathyroid hormone,PTH)可促进成骨 .胰岛素样生长因子 - I(insulin- like growth factor- I,IGF- I)由成骨细胞所产生并贮存于骨基质中 ,可促进成骨细胞的增殖分化 .为进一步了解向钙性激素和骨源性生长因子对骨生长的影响 ,利用成骨样细胞 ROS1 7/ 2 .8进行体外实验 ,观察了 PTH和 IGF- I这两种在骨生长和代谢中有重要作用的激素和因子相互作用的效果 ,并对其相互作用机制作出初步探讨 .结果显示 :联合使用 IGF- I及 PTH(间歇性给药 )时 ,(1 ) SRB(sodium rhodamine B,SRB)染色显示经 PTH(1 0 -9mol/ L,间歇给药 )和 IGF- I(1 0 -9mol/ L)联合处理的细胞 ,其数目明显增加 ,且明显高于单独处理组 ;(2 ) 3H- Td R参入增加 ,也明显高于单独处理组 ;(3)与增殖相关的原癌基因 (c- fos,c- jun,c- ki- ras)的表达增强 ,明显高于单独处理组 ;(4)骨钙素 (osteocalcin)基因 m RNA表达增强 ,明显高于单独处理组 ;(5) IGF- I(1 0 -8mol/L,1 0 -9mol/ L)可使 PTH受体基因 m RNA表达增强 .这些结果提示 PTH和 IGF- I在成骨样细胞ROS 1 7/ 2 .8增殖分化中具有协同作用 ,原癌基因的表达增强可能是其作用的一个环节 .此外 ,IGF- I可能通过增强 PTH受体表达 ,使细胞对 PTH的反应性增强     

Insulin-like growth factor I supports differentiation of cultured osteoblast-like cells

Rat calvaria cells grown in culture for one week had properties of osteoblasts: a high content in alkaline phosphatase and a marked cyclic AMP response to parathyroid hormone (PTH). In short-term experiments, insulin-like growth factor I (IGF I) stimulated the incorporation of [14C] glucose into glycogen. When IGF I was present in the medium during 6 days the cell number increased slightly and there was a substantial, disproportionate rise in alkaline phosphatase activity of the cultures. Thus, IGF I stimulates growth, and in addition, and in contrast to other growth factors, mainly enhances differentiation of osteoblasts.     

Expression of RANKL/OPG during bone remodeling in vivo

The interaction between receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) plays a dominant role in osteoclastogenesis. As both proteins are produced by osteoblast lineage cells, they are considered to represent a key link between bone formation and resorption. In this study, we investigated the expression of RANKL and OPG during bone remodeling in vivo to determine the relationship between osteoclastogenic stimulation and osteoblastic differentiation.Total RNA was prepared from rat femurs after marrow ablation on days 0, 3, 6, and 9. The temporal activation patterns of osteoblast-related genes (procollagen α1 (I), alkaline phosphatase, osteopontin, and osteocalcin) were examined by Northern blot analysis. An appreciable increase in the expression of these osteoblast markers was observed on day 3. The peak increase in gene expression was observed on day 6 followed by a slight reduction by day 9. Real-time PCR analysis showed that the OPG mRNA expression was markedly upregulated on day 6 and slightly decreased on day 9. In contrast, RANKL mRNA expression was increased by more than 20-fold on day 9. The RANKL/OPG ratio, an index of osteoclastogenic stimulation, peaked on day 9. Histological analysis showed that RANKL and OPG immunoreactivity were predominantly associated with bone marrow cells. The expression of bone formation markers was activated in the bone formation phase, followed by the stimulation of RANKL/OPG expression in the bone resorption phase, which confirmed that these molecules are key factors linking bone formation to resorption during bone remodeling.     

Green tea polyphenol (−)‐epigallocatechin gallate suppressed the differentiation of murine osteoblastic MC3T3‐E1 cells

Recently, various physiological effects of the tea polyphenol catechin for alleviating diseases such as cancer, arteriosclerosis, hyperlipidaemia and osteoporosis have been reported. However, the physiological effect of catechin on bone metabolism remains unclear. We examined the physiological effect of EGCG [(?)‐epigallocatechin‐3‐gallate], which is the main component of green tea catechin, on osteoblast development using the precursor cell line of osteoblasts, MC3T3‐E1, and co‐culture of the osteoblasts from mouse newborn calvaria and mouse bone marrow cells. Although EGCG did not affect the viability and proliferation of MC3T3‐E1 cells, EGCG inhibited the osteoblast differentiation. Furthermore, EGCG did not affect the mineralization of differentiated MC3T3‐E1 cells, and reduced osteoclast formation in co‐culture. These results suggest that EGCG can effectively suppress bone resorption, and can be used as an effective medicine in the treatment of the symptoms of osteoporosis.     

Artesunate inhibits RANKL‐induced osteoclastogenesis and bone resorption in vitro and prevents LPS‐induced bone loss in vivo

Osteoclasts are multinuclear giant cells responsible for bone resorption in lytic bone diseases such as osteoporosis, arthritis, periodontitis, and bone tumors. Due to the severe side‐effects caused by the currently available drugs, a continuous search for novel bone‐protective therapies is essential. Artesunate (Art), the water‐soluble derivative of artemisinin has been investigated owing to its anti‐malarial properties. However, its effects in osteoclastogenesis have not yet been reported. In this study, Art was shown to inhibit the nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis, the mRNA expression of osteoclastic‐specific genes, and resorption pit formation in a dose‐dependent manner in primary bone marrow‐derived macrophages cells (BMMs). Furthermore, Art markedly blocked the RANKL‐induced osteoclastogenesis by attenuating the degradation of IκB and phosphorylation of NF‐κB p65. Consistent with the in vitro results, Art inhibited lipopolysaccharide (LPS)‐induced bone resorption by suppressing the osteoclastogenesis. Together our data demonstrated that Art inhibits RANKL‐induced osteoclastogenesis by suppressing the NF‐κB signaling pathway and that it is a promising agent for the treatment of osteolytic diseases.     

Role of gap junction, hemichannels, and connexin 43 in mineralizing in response to intermittent and continuous application of parathyroid hormone

Intermittent administration stimulates bone formation, whereas sustained elevation of parathyroid hormone (PTH) as in hyperparathyroidism stimulates bone resorption. Even though PTH(1-34) is the only anabolic agent clinically approved for the treatment of osteoporosis, the molecular mechanism whereby PTH mediates these opposing effects depending on timing of administration is not well understood. In this study, we sought to determine the involvement of gap junctions and hemichannels, and the protein that forms them, connexin 43 (Cx43), in the effect of PTH(1-34) on osteoblast mineralization. The osteoblast-like cell line MLO-A5 that rapidly mineralizes in culture was used. Intermittent PTH enhances mineralization, whereas continuous PTH inhibits this process. The mineralization was significantly inhibited by 18 beta-glycyrrhetinic acid, an inhibitor known to block gap junctions and hemichannels. When the cells were treated with PTH(1-34), gap junctional coupling was increased; however, the degree of stimulation was similar between intermittent and continuous treatment. The permeabilization to dye was not detected under various intermittent or continuous PTH treatments. On the other hand, the overall level of Cx43 protein increased in response to continuous PTH treatment. In contrast, when the cells were subjected to intermittent treatment overall level of Cx43 was unchanged, but there was an increase of connexons associated with an increase in Cx43 expression on the cell surface. Our results suggest that Cx43 overall expression, connexon formation and cell surface expression are differentially regulated by intermittent and continuous PTH(1-34), implying the involvement of Cx43 and Cx43-forming channels in mediating the effects of PTH on bone formation.