The stiffness and collagen control differentiation of osteoclasts with an altered expression of c-Src in podosome.

Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the stiffness of bones: regular calcified bone (bone that is resorbable by osteoclasts) and un-calcified osteoid bone (bone that is un-resorbable by osteoclasts). The osteolytic cytokine RANKL promotes osteoclast differentiation; however, the roles of the physical interactions of osteoclasts with calcified and un-calcified bone at the sealing zones and the subsequent cellular signaling remain unclear. In this study, we investigated podosomes, actin-rich adhesion structures (actin-ring) in the sealing zone that participates in sensing hard stiffness with collagen in the physical environment during osteoclast differentiation. RANKL-induced osteoclast differentiation induction was promoted when Raw264.7 cells were cultured on collagen-coated plastic dishes but not on non-coated plastic dishes, which was associated with the increased expression of podosome-related genes and Src. In contrast, when cells were cultured on collagen gel, expression of podosome-related genes and Src were not upregulated. The induction of podosome-related genes and Src requires hard stiffness with RGD-containing substratum and integrin-mediated F-actin polymerization. These results indicate that osteoclasts sense both the RGD sequence and stiffness of calcified collagen through their podosome components regulating osteoclast differentiation via the c-Src pathway.

Effects of 4'-Demethylnobiletin and 4'-Demethyltangeretin on Osteoclast Differentiation In Vitro and in a Mouse Model of Estrogen-Deficient Bone Resorption.

Citrus nobiletin (NOB) and tangeretin (TAN) show protective effects against disease-related bone destruction. We achieved demethylation of NOB and TAN into 4'-demethylnobiletin (4'-DN) and 4'-demethyltangeretin (4'-DT) using enzyme-manufacturing methods. In this study, we examined the effects of 4'-DN and 4'-DT on in vitro osteoclast differentiation, and on in vivo osteoporotic bone loss in ovariectomized (OVX) mice. 4'-DN and 4'-DT clearly suppressed the osteoclast differentiation induced by interleukin IL-1 or RANKL treatment. 4'-DN and 4'-DT treatments resulted in higher inhibitory activity in osteoclasts in comparison to NOB or TAN treatments. RANKL induced the increased expression of its marker genes and the degradation of IκBα in osteoclasts, while these were perfectly attenuated by the treatment with 4'-MIX: a mixture of 4'-DN and 4'-DT. In an in silico docking analysis, 4'-DN and 4'-DT directly bound to the ATP-binding pocket of IKKß for functional inhibition. Finally, the intraperitoneal administration of 4'-MIX significantly protected against bone loss in OVX mice. In conclusion, 4'-DN, 4'-DT and 4'-MIX inhibited the differentiation and function of bone-resorbing osteoclasts via suppression of the NF-κB pathway. Novel 4'-DN, 4'-DT and 4'-MIX are candidates for maintaining bone health, which may be applied in the prevention of metabolic bone diseases, such as osteoporosis.

Prostate cancer expressing membrane-bound TGF-α induces bone formation mediated by the autocrine effect of prostaglandin E2 in osteoblasts.

Prostate cancer highly metastasizes to bone, and such cancer is associated with severe bone resorption and bone formation at the site of metastasis. Prostaglandin E2 (PGE2) promotes bone resorption in inflammatory diseases; however, the roles in prostate cancer-induced bone formation are still unclear. In the present study, we investigated the effects of membrane-bound TGF-α on prostate cancer-induced bone formation through autocrine PGE2 signaling in osteoblasts. In the prostate cancer explant experiment into tibiae, injected prostate cancer cells induced bone formation with the increased expression of osteogenic genes, such as Runx2 and Wnt5a, and prostaglandin synthase Ptgs2. In osteoblasts, PGE2 increased the number of calcified bone nodules with enhanced expression of Runx2 and Wnt5a. We also screened the factors involved in cancer progression, and 11 EGF family members were found to be expressed in the human prostate cancer cell line PC3. PC3 highly expressed amphiregulin, HB-EGF, and especially TGF-α. Treatment with recombinant TGF-α increased the Ptgs2 expression and PGE2 production in osteoblasts, which promoted the formation of calcified bone nodules, suggesting that the interaction between PC3 and osteoblasts promoted PGE2 production. In co-culture of osteoblasts and fixed PC3 cells, the phosphorylation of EGFR and ERK and subsequent Ptgs2 expression and PGE2 production were increased, an effect that was attenuated by treatment with inhibitors of EGFR and ERK. These results indicate that membrane-bound TGF-α enhances ERK signaling while also inducing PGE2-mediated bone formation in osteoblasts, thus suggesting that prostate cancer regulates both PGE2-mediated bone resorption and bone formation at the site of bone metastasis of prostate cancer.

A mouse model of lung cancer induced via intranasal injection for anticancer drug screening and evaluation of pathology.

The pathologies and lethality of lung cancers are associated with smoking, lifestyle, and genomic factors. Several experimental mouse models of lung cancer, including those induced via intrapulmonary injection and intratracheal injection, have been reported for evaluating the pharmacological effect of drugs. However, these models are not sufficient for evaluating the efficacy of drugs during screening, as these direct injection models ignore the native processes of cancer progression in vivo, resulting in the inadequate pathological formation of lung cancer. In the present study, we developed a novel intranasal injection model of lung cancer simulating the native lung cancer pathology for anticancer drug screening. A mouse lung cancer cell line (Lewis lung carcinoma; LCC) was intranasally injected into mouse lungs, and injected cell number-dependent cancer proliferation was apparent in both the left and right lungs. Human non-small-cell lung cancer (NCI-H460) cells were also intranasally injected into nude mice and similarly showed injected cell number-dependent cancer growth. For the pharmacological evaluation of cisplatin, two different treatment frequencies were tested four times per month and twice a month. The intranasal injection model confirmed that cisplatin suppressed lung cancer progression to a greater extent under the more frequent treatment condition. In conclusion, these results indicated that our intranasal injection model is a powerful tool for investigating lung cancer pathology and may aid in the development of new anti-lung cancer agents.

RNA-binding protein LIN28A upregulates transcription factor HIF1α by posttranscriptional regulation via direct binding to UGAU motifs.

Hypoxia-inducible factor 1α (HIF1α) is a transcription factor that regulates angiogenesis under hypoxic conditions. To investigate the posttranscriptional regulatory mechanism of HIF1α, we performed a cell-based screening to reveal potential cis-elements and the regulatory RNA-binding proteins that act as trans-factors. We found that LIN28A promoted HIF1α protein expression independently of the downregulation of microRNA let-7, which is also directly mediated by LIN28A. Transcriptome analysis and evaluation of RNA stability using RNA-seq and SLAM-seq analyses, respectively, revealed that LIN28A upregulates HIF1A expression via mRNA stabilization. To investigate the physical association of LIN28A with HIF1A mRNA, we performed enhanced crosslinking immunoprecipitation in 293FT cells and integrally analyzed the transcriptome. We observed that LIN28A associates with HIF1A mRNA via its cis-element motif "UGAU". The "UGAU" motifs are recognized by the cold shock domain of LIN28A, and the introduction of a loss-of-function mutation to the cold shock domain diminished the upregulatory activities performed by LIN28A. Finally, the microvessel density assay showed that the expression of LIN28A promoted angiogenesis in vivo. In conclusion, our study elucidated the role of LIN28A in enhancing the HIF1α axis at the posttranscription layer.

Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle.

Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the DMD gene encoding dystrophin protein. To quantitively assess human dystrophin protein in muscle biopsy samples, it is imperative to consistently detect as low as 0.003% of the dystrophin protein relative to the total muscle protein content. The quantitation of dystrophin protein has traditionally been conducted using semiquantitative immunoblotting or immunohistochemistry; however, there is a growing need to establish a more precise quantitative method by employing liquid chromatography-mass spectrometry (LC-MS) to measure dystrophin protein. In this study, a novel quantification method was established using a mouse experiment platform applied to the clinical quantification of human dystrophin protein. The method using a spike-in approach with a triple quadrupole LC-MS quantitated the amount of dystrophin in wild-type and human DMD transgenic mice but not in DMD-null mice. In conclusion, we established a quantitating method of dystrophin using HPLC-LC-MS with a novel spike-in approach. These results indicate that our methodology could be applied to several LC-MS devices to enable the accurate measurement of dystrophin protein in patients with DMD.

Caveolin-3 regulates the activity of Ca2+/calmodulin-dependent protein kinase II in C2C12 cells.

Caveolins, encoded by the Cav gene family, are the main components of caveolae. Caveolin-3 (Cav3) is specifically expressed in muscle cells. Mutations in Cav3 are responsible for a group of muscle diseases called caveolinopathies, and Cav3 deficiency is associated with sarcolemmal membrane alterations, disorganization of T-tubules, and disruption of specific cell-signaling pathways. However, Cav3 overexpression increases the number of sarcolemmal caveolae and muscular dystrophy-like regenerating muscle fibers with central nuclei, suggesting that the alteration of Cav3 expression levels or localization influences muscle cell functions. Here, we used mouse C2C12 myoblasts in which Cav3 expression was suppressed with short hairpin RNA and found that Cav3 suppression impaired myotube differentiation without affecting the expression of MyoD and Myog. We also observed an increase of intracellular Ca2+ levels, total calpain activity, and Ca2+-dependent calmodulin kinase II (CaMKII) levels in Cav3-depleted myoblasts. Importantly, those phenotypes due to Cav3 suppression were caused by the ryanodine receptor activation. Furthermore, pharmacological inhibition of CaMKII rescued the impairment of myoblast differentiation due to Cav3 knockdown. Our results suggest that Cav3 regulates intracellular Ca2+ concentrations by modulating ryanodine receptor activity in muscle cells and that CaMKII suppression in muscle could be a novel therapy for caveolinopathies.

Endosomal TLR3 signaling in stromal osteoblasts induces prostaglandin E2-mediated inflammatory periodontal bone resorption.

Toll-like receptors (TLRs) are pattern recognition receptors that play a critical role in innate immune diseases. TLR3, which is localized in the endosomal compartments of hematopoietic immune cells, is able to recognize double-stranded RNA (dsRNA) derived from viruses and bacteria and thereby induce innate immune responses. Inflammatory periodontal bone resorption is caused by bacterial infections, which initially is regulated by innate immunity; however, the roles of TLR3 signaling in bone resorption are still not known. We examined the roles of TLR3 signaling in bone resorption using poly(I:C), a synthetic dsRNA analog. In cocultures of mouse bone marrow cells and stromal osteoblasts, poly(I:C) clearly induced osteoclast differentiation. In osteoblasts, poly(I:C) increased PGE2 production and upregulated the mRNA expression of PGE2-related genes, Ptgs2 and Ptges, as well as that of a gene related to osteoclast differentiation, Tnfsf11. In addition, we found that indomethacin (a COX-2 inhibitor) or an antagonist of the PGE2 receptor EP4 attenuated the poly(I:C)-induced PGE2 production and subsequent Tnfsf11 expression. Poly(I:C) also prolonged the survival of the mature osteoclasts associated with the increased mRNA expression of osteoclast marker genes, Nfatc1 and Ctsk. In ex vivo organ cultures of periodontal alveolar bone, poly(I:C) induced bone-resorbing activity in a dose-dependent manner, which was attenuated by the simultaneous administration of either indomethacin or an EP4 antagonist. These data suggest that TLR3 signaling in osteoblasts controls PGE2 production and induces the subsequent differentiation and survival of mature osteoclasts. Endogenous TLR3 in stromal osteoblasts and osteoclasts synergistically induces inflammatory alveolar bone resorption in periodontitis.

Gram-positive bacteria cell wall-derived lipoteichoic acid induces inflammatory alveolar bone loss through prostaglandin E production in osteoblasts.

Periodontitis is an inflammatory disease associated with severe alveolar bone loss and is dominantly induced by lipopolysaccharide from Gram-negative bacteria; however, the role of Gram-positive bacteria in periodontal bone resorption remains unclear. In this study, we examined the effects of lipoteichoic acid (LTA), a major cell-wall factor of Gram-positive bacteria, on the progression of inflammatory alveolar bone loss in a model of periodontitis. In coculture of mouse primary osteoblasts and bone marrow cells, LTA induced osteoclast differentiation in a dose-dependent manner. LTA enhanced the production of PGE2 accompanying the upregulation of the mRNA expression of mPGES-1, COX-2 and RANKL in osteoblasts. The addition of indomethacin effectively blocked the LTA-induced osteoclast differentiation by suppressing the production of PGE2. Using ex vivo organ cultures of mouse alveolar bone, we found that LTA induced alveolar bone resorption and that this was suppressed by indomethacin. In an experimental model of periodontitis, LTA was locally injected into the mouse lower gingiva, and we clearly detected alveolar bone destruction using 3D-µCT. We herein demonstrate a new concept indicating that Gram-positive bacteria in addition to Gram-negative bacteria are associated with the progression of periodontal bone loss.

A common SNP risk variant MT1-MMP causative for Dupuytren's disease has a specific defect in collagenolytic activity.

Dupuytren's Disease (DD) is a common fibroproliferative disease of the palmar fascia. We previously identified a causal association with a non-synonymous variant (rs1042704, p.D273N) in MMP14 (encoding MT1-MMP). In this study, we investigated the functional consequences of this variant, and demonstrated that the variant MT1-MMP (MT1-N273) exhibits only 17% of cell surface collagenolytic activity compared to the ancestral enzyme (MT1-D273). Cells expressing both MT1-D273 and MT1-N273 in a 1:1 ratio, mimicking the heterozygous state, possess 38% of the collagenolytic activity compared to the cells expressing MT1-D273, suggesting that MT1-N273 acts in a dominant negative manner. Consistent with the above observation, patient-derived DD myofibroblasts with the alternate allele demonstrated around 30% of full collagenolytic activity detected in ancestral G/G genotype cells, regardless of the heterozygous (G/A) or homozygous (A/A) state. Small angle X-ray scattering analysis of purified soluble Fc-fusion enzymes allowed us to construct a 3D-molecular envelope of MT1-D273 and MT1-N273, and demonstrate altered flexibility and conformation of the ectodomains due to D273 to N substitution. Taking together, rs1042704 significantly reduces collagen catabolism in tissue, which tips the balance of homeostasis of collagen in tissue, contributing to the fibrotic phenotype of DD. Since around 30% of the worldwide population have at least one copy of the low collagenolytic alternate allele, further investigation of rs1042704 across multiple pathologies is needed.

Molecular evidence of IGFBP-3 dependent and independent VD3 action and its nonlinear response on IGFBP-3 induction in prostate cancer cells.

BACKGROUND: Clinical trials have been conducted to clarify the beneficial effects of VD3 (1α,25-dihydroxy vitamin D3, also known as calcitriol) treatment in prostate cancer. However, the molecular mechanisms underlying these effects are not fully understood. Recent studies on IGFBP-3 have indicated its intracellular functions in cell growth and apoptosis. The aim of this study was to confirm the benefits of low-dose VD3 treatment and clarify the molecular mechanisms underlying these beneficial effects in prostate cancer cells. METHODS: The molecular effects of simultaneous treatment of LNCaP cells and their genetically modified cell lines with low concentration of docetaxel and VD3 were biologically and biochemically analyzed. To further determine the effects of VD3 treatment on IGFBP-3 induction system, cells were temporarily treated with VD3 in combination with a transcriptional inhibitor or protein synthesis inhibitor. Bcl-2 protein and its mRNA behavior were also observed in Igfbp-3 expression-modified LNCaP cells to determine the involvement of IGFBP-3 in the suppression of Bcl-2 by VD3 treatment. RESULTS: Changes in IGFBP-3 expression levels in LNCaP cells indicated that it mediated the inhibition of cell growth induced by VD3 treatment. IGFBP-3 was also found to be a mediator of the enhanced cytotoxicity of prostate cancer cells to VD3 in combination with the anti-cancer drug. We further identified the distinct property of the IGFBP-3 induction system, wherein temporal VD3 stimulation-induced prolonged IGFBP-3 expression and VD3 treatment-induced increase in IGFBP-3 expression were optimized based on the protein concentration rather than the mRNA concentration. Meanwhile, Bcl-2 expression was down-regulated by VD3 treatment in an IGFBP-3-independent manner. CONCLUSION: These findings indicate the molecular mechanisms of IGFBP-3 induction stimulated by VD3 and IGFBP-3 independent Bcl-2 suppression by VD3 treatment in prostate cancer cells. The results could prompt a re-evaluation of VD3 usage in therapy for patients with prostate cancer.

The Combination of Soy Isoflavones and Resveratrol Preserve Bone Mineral Density in Hindlimb-Unloaded Mice.

It is well known that physical inactivity during space flight or prolonged bed rest causes rapid bone loss. Soy isoflavones (ISOs) and resveratrol (RES) have been reported to be useful to maintain a positive balance for bone turnover. Therefore, we examined the combined effects of ISO and RES on bone loss that was induced by hindlimb-unloading in mice. Female eight-week-old ddY mice were divided into the following six groups (n = 6-8 each): normally housed mice, loading mice, hindlimb-unloading (UL) mice fed a control diet, UL mice fed a 0.16% ISO conjugates, UL mice fed a 0.15% RES diet, and UL mice fed a 0.16% ISO and 0.15% RES diet. After three weeks, femoral bone mineral density was markedly decreased in unloading mice. The combination of ISO and RES prevented bone loss and especially maintained the trabecular bone mineral density more effectively compared with cortical bones. ISO and/or RES inhibited the increase in the RANKL/OPG expression ratio in bone marrow cells in UL mice. These results suggest that the combination of ISO and RES had a preventive effect against bone loss induced by hindlimb-unloading in mice. These osteoprotective effects of ISO and RES may result from the inhibition of bone resorption.

Sustained anti-osteoporotic action of risedronate compared to anti-RANKL antibody following discontinuation in ovariectomized mice.

Bisphosphonates and the anti-receptor activator of nuclear factor-kappa B ligand (RANKL) antibody denosumab are effective anti-resorptive drugs commonly prescribed for osteoporosis. Both drugs may, however, have intolerable side effects; so, it is critical to examine their residual efficacy such as maintenance of bone mass following cessation. Therefore, we compared the changes in bone histology following discontinuation of the aminobisphosphonate risedronate and anti-RANKL antibody in ovariectomized (OVX) mice. Twelve-week-old female C57BL/6 N mice were OVX or sham operated. Four weeks after surgery, mice were treated with vehicle, a single injection of anti-RANKL antibody (5 mg/kg), or risedronate (5 µg/kg/day, s.c.) for 4 weeks (the treatment period), followed by vehicle treatment for an additional 4 weeks (discontinuation period). The lumbar spine and proximal tibia were evaluated by micro-computed tomography. In addition, the lumbar spine, proximal tibia, and the femoral shaft were examined by bone histomorphometry. After 4 weeks of discontinuation, OVX mice initially treated with the anti-RANKL antibody exhibited a trend of bone loss associated with increased turnover in both trabecular and cortical bones, although the difference was not significant. By contrast, OVX mice treated with risedronate exhibited maintained or even increased bone mass and suppressed bone turnover. Patients discontinuing denosumab should be carefully monitored for recurrent osteoporosis symptoms, and a replacement drug should be considered.

Quantitative Measurements of Intercellular Adhesion Strengths between Cancer Cells with Different Malignancies Using Atomic Force Microscopy.

Intercellular adhesion strengths between two kinds of murine breast cancer cells with different malignancies were measured quantitatively using a metal cup-attached chip with atomic force microscopy (AFM). The cup-attached chip was used to approach a cell, pick it up, and then approach another cell, and the adhesion strengths were measured according to the contact time of the cells between 0 to 60 s. Separation work was used as a parameter for quantitative comparisons of the strengths. As a result, the work of a highly metastatic cancer cell (FP10SC2) was greater than a low metastatic cancer cell (4T1-LM) throughout all contact times examined. Adhesion was analyzed from a point of a view of binding kinetics of receptors on cells, and two possibilities were found: one was the number of cell adhesive receptors increased, and the other was the work to separate single molecular binding increased with increasing cancer cell malignancy. These results indicated quantitative measurements of intercellular adhesion strengths using AFM yielded information to understand the mechanism of the cancer progression from a new perspective.

Hypergravity and microgravity exhibited reversal effects on the bone and muscle mass in mice.

Spaceflight is known to induce severe systemic bone loss and muscle atrophy of astronauts due to the circumstances of microgravity. We examined the influence of artificially produced 2G hypergravity on mice for bone and muscle mass with newly developed centrifuge device. We also analyzed the effects of microgravity (mostly 0G) and artificial produced 1G in ISS (international space station) on mouse bone mass. Experiment on the ground, the bone mass of humerus, femur and tibia was measured using micro-computed tomography (µCT), and the all bone mass was significantly increased in 2G compared with 1G control. In tibial bone, the mRNA expression of bone formation related genes such as Osx and Bmp2 was elevated. The volume of triceps surae muscle was also increased in 2G compared with 1G control, and the mRNA expression of myogenic factors such as Myod and Myh1 was elevated by 2G. On the other hand, microgravity in ISS significantly induced the loss of bone mass on humerus and tibia, compared with artificial 1G induced by centrifugation. Here, we firstly report that bone and muscle mass are regulated by the gravity with loaded force in both of positive and negative on the ground and in the space.

Beta-Cryptoxanthin Inhibits Lipopolysaccharide-Induced Osteoclast Differentiation and Bone Resorption via the Suppression of Inhibitor of NF-κB Kinase Activity.

Beta-cryptoxanthin (ß-cry) is a typical carotenoid found abundantly in fruit and vegetables such as the Japanese mandarin orange, persimmon, papaya, paprika, and carrot, and exerts various biological activities (e.g., antioxidant effects). We previously reported that ß-cry suppressed lipopolysaccharide (LPS)-induced osteoclast differentiation via the inhibition of prostaglandin (PG) E2 production in gingival fibroblasts and restored the alveolar bone loss in a mouse model for periodontitis in vivo. In this study, we investigated the molecular mechanism underlying the inhibitory effects of ß-cry on osteoclast differentiation. In mouse calvarial organ cultures, LPS-induced bone resorption was suppressed by ß-cry. In osteoblasts, ß-cry inhibited PGE2 production via the downregulation of the LPS-induced mRNA expression of cyclooxygenase (COX)-2 and membrane-bound PGE synthase (mPGES)-1, which are PGE synthesis-related enzymes, leading to the suppression of receptor activator of NF-κB ligand (RANKL) mRNA transcriptional activation. In an in vitro assay, ß-cry directly suppressed the activity of the inhibitor of NF-κB kinase (IKK) ß, and adding ATP canceled this IKKß inhibition. Molecular docking simulation further suggested that ß-cry binds to the ATP-binding pocket of IKKß. In Raw264.7 cells, ß-cry suppressed RANKL-mediated osteoclastogenesis. The molecular mechanism underlying the involvement of ß-cry in LPS-induced bone resorption may involve the ATP-competing inhibition of IKK activity, resulting in the suppression of NF-κB signaling.

Low Molecular-Weight Curdlan, (1→3)-ß-Glucan Suppresses TLR2-Induced RANKL-Dependent Bone Resorption.

Fungal ß-glucan is a potent immunological stimulator, and that it activates both the innate immune system and adaptive immunity. Curdlan is (1→3)-ß-glucan, a linear form of ß-glucan with a high molecular weight; it modulates the immune response. However, its role in bone tissue is controversial, and the effects of curdlan on bone tissues are unknown. Toll-like receptors (TLRs) play critical roles in innate immunity, and various ligands for TLRs are thought to regulate the host defense mechanisms against pathogens. TLR2 is known to form heterodimers with TLR6, and the TLR2-TLR6 heterodimer (TLR2/6) recognizes diacylated lipopeptides from Gram-positive bacteria. In the present study, we prepared low molecular-weight curdlan, (1→3)-ß-D-glucan, and examined its effects on bone resorption induced by TLR2/6 signaling. In co-cultures of bone marrow cells and osteoblasts, low molecular-weight curdlan suppressed the osteoclast formation induced by TLR2/6 ligand, and attenuated bone resorption in mouse calvarial organ cultures. Curdlan acted on mouse osteoblasts and suppressed the expression of receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL), a key molecule for osteoclastogenesis. Curdlan also acted on mouse bone marrow macrophages and suppressed RANKL-dependent osteoclast differentiation from osteoclast precursor cells. The present study indicates that low molecular-weight curdlan attenuated TLR2-induced inflammatory bone resorption. Curdlan, (1→3)-ß-glucan may be a natural agent with beneficial effects on bone health in humans.

Effects of Polymethoxyflavonoids on Bone Loss Induced by Estrogen Deficiency and by LPS-Dependent Inflammation in Mice.

Polymethoxyflavonoids (PMFs) are a family of the natural compounds that mainly compise nobiletin, tangeretin, heptamethoxyflavone (HMF), and tetramethoxyflavone (TMF) in citrus fruits. PMFs have shown various biological functions, including anti-oxidative effects. We previously showed that nobiletin, tangeretin, and HMF all inhibited interleukin (IL)-1-mediated osteoclast differentiation via the inhibition of prostaglandin E2 synthesis. In this study, we created an original mixture of PMFs (nobiletin, tangeretin, HMF, and TMF) and examined whether or not PMFs exhibit co-operative inhibitory effects on osteoclastogenesis and bone resorption. In a coculture of bone marrow cells and osteoblasts, PMFs dose-dependently inhibited IL-1-induced osteoclast differentiation and bone resorption. The optimum concentration of PMFs was lower than that of nobiletin alone in the suppression of osteoclast differentiation, suggesting that the potency of PMFs was stronger than that of nobiletin in vitro. The oral administration of PMFs recovered the femoral bone loss induced by estrogen deficiency in ovariectomized mice. We further tested the effects of PMFs on lipopolysaccharide-induced bone resorption in mouse alveolar bone. In an ex vivo experimental model for periodontitis, PMFs significantly suppressed the bone-resorbing activity in organ cultures of mouse alveolar bone. These results indicate that a mixture of purified nobiletin, tangeretin, HMF, and TMF exhibits a co-operative inhibitory effect for the protection against bone loss in a mouse model of bone disease, suggesting that PMFs may be potential candidates for the prevention of bone resorption diseases, such as osteoporosis and periodontitis.

Raloxifene reduces the risk of local alveolar bone destruction in a mouse model of periodontitis combined with systemic postmenopausal osteoporosis.

OBJECTIVE: Periodontitis is characterized by local inflammation leading to tooth loss and severe destruction of alveolar bone. Raloxifene is a selective estrogen receptor modulator (SERM) that halts estrogen deficiency-induced systemic bone loss in postmenopausal osteoporosis without the side effects of cancer in breast and uterus. In this study, we examined the effects of raloxifene on alveolar bone mass in a mouse model with estrogen deficiency-induced periodontitis. METHODS: Periodontitis was induced by the injection of lipopolysaccharide (LPS) into the lower gingiva in ovariectomized (OVX) mice, and the alveolar bone and femur bone mineral density (BMD) were analyzed by dual-energy X-ray absorptiometry. To explore the direct osteoclast inhibitory effect of raloxifene, a co-culture system for osteoclast formation and organ culture of alveolar bone was established. RESULTS: When OVX mice were treated with raloxifene, the bone loss in both alveolar bone and femur were abrogated. Interleukin 1 and/or LPS stimulated the osteoclast formation and bone-resorbing activity; however, raloxifene did not show any inhibitory effect on the osteoclast formation or function. In vivo local injection of raloxifene also did not prevent bone resorption in a mouse model of periodontitis. However, the systemic treatment of raloxifene using a mini-osmotic pump did prevent the loss of BMD of alveolar bone induced by LPS. CONCLUSION: These results suggest that the SERM raloxifene systemically maintain alveolar bone mass in a mouse model of periodontitis with osteoporosis. Increasing the alveolar bone mass by SERMs treatment in patients with postmenopausal osteoporosis may be a useful approach to preventing the destruction of alveolar bone in late-onset periodontitis.

Effects of O-methylated (-)-epigallocatechin gallate (EGCG) on LPS-induced osteoclastogenesis, bone resorption, and alveolar bone loss in mice.

(-)-Epigallocatechin-3-O-gallate (EGCG), present in green tea, exhibits antioxidant and antiallergy effects. EGCG3″Me, a 3-O-methylated derivative of EGCG, has been reported to show similar biological functions; the inhibitory activity of EGCG3″Me in a mouse allergy model was more potent than that of EGCG, probably due to the efficiency of absorption from the intestine. However, the functional potency of these EGCGs is controversial in each disease model. We previously observed that EGCG suppressed inflammatory bone resorption and prevented alveolar bone loss in a mouse model of periodontosis. In this study, we examined the role of EGCG3″Me in bone resorption using a mouse model of periodontitis. Lipopolysaccharide (LPS)-induced osteoclast formation was suppressed by adding EGCG3″Me to cocultures of osteoblasts and bone marrow cells, and LPS-induced bone resorption was also inhibited by EGCG3″Me in calvarial organ cultures. EGCG3″Me acted on osteoblasts and suppressed prostaglandin E (PGE) production, which is critical for inflammatory bone resorption, by inhibiting the expression of COX-2 and mPGES-1, key enzymes for PGE synthesis. In osteoclast precursor macrophages, EGCG3″Me suppressed RANKL-dependent differentiation into mature osteoclasts. In a mouse model of periodontitis, LPS-induced bone resorption was suppressed by EGCG3″Me in organ culture of mouse alveolar bone, and the alveolar bone loss was further attenuated by the treatment of EGCG3″Me in the lower gingiva in vivo. EGCG3″Me may be a potential natural compound for the protection of inflammatory bone loss in periodontitis.