Tucatinib alleviates postmenopausal osteoporosis by suppressing osteoclast differentiation via regulating the DRP1/NFATc1/CTSK signaling pathway.

BackgroundAlthough extracellular signal-regulated kinase 5 (ERK5) is known to be critical for osteoclast differentiation, there are few studies on how fluid shear stress (FSS) regulates osteoclast differentiation through the ERK5 signaling pathway. We examined the expression of nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells and its downstream factors, including cathepsin K (CTSK), tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinases-9 (MMP-9) and their relationship with ERK5.Material/MethodsRAW264.7 cells were treated with RANKL, XMD8-92 (ERK5 inhibitor), and then loaded onto 12 dyn/cm2 FSS for 4 days. Endpoints measured were osteoclast differentiation, bone resorption, and TRAP activity. Cell viability was detected by using the Cell Counting Kit-8 (CCK-8) assay. Western blot was used to analyze protein expression of phosphorylated-ERK5 (p-ERK5), NFATc1, CTSK, TRAP, and MMP-9.ResultsFSS inhibited osteoclast differentiation and expression of NFATc1, CTSK, TRAP, and MMP-9; cell viability was not affected. ERK5 expression increased by FSS but not by RANKL, and it was blocked by XMD8-92. Furthermore, FSS suppressed osteoclast differentiation in RAW264.7 cells through ERK5 pathway.ConclusionsOur findings demonstrated that FSS inhibited osteoclast differentiation in RAW264.7 cells via the ERK5 pathway through reduced NFATc1 expression and its downstream factors MMP-9, CTSK, and TRAP.

BackgroundSodium pentosan polysulfate (NaPPS) was testified as a chondroprotective drug in with a detailed rationale of the disease-modifying activity. This study was undertaken to determine whether anti-osteoarthritis drug, NaPPS inhibited osteoclasts (OC) differentiation and function. Canine bone marrow mononuclear cells (n = 6) were differentiated to OC by maintaining with receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) for up to 7 days with the treatment of NaPPS at concentration of 0, 0.2, 1 and 5 μg/mL. Differentiation and function of OC were accessed using tartrate-resistant acid phosphate (TRAP) staining and bone resorption assay, while monitoring actin ring formation. Invasion and colocalization patterns of fluorescence-labeled NaPPS with transcribed gene in OC were monitored. Gene expression of OC for cathepsin K (CTK), matrix metallopeptidase-9 (MMP-9), nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), c-Fos, activator protein-1(AP-1) and carbonic anhydrase II was examined using real-time PCR.ResultsSignificant inhibition of OC differentiation was evident at NaPPS concentration of 1 and 5 μg/mL (p < 0.05). In the presence of 0.2 to 5 μg/mL NaPPS, bone resorption was attenuated (p < 0.05), while 1 and 5 μg/mL NaPPS achieved significant reduction of actin ring formation. Intriguingly, fluorescence-labeled NaPPS invaded in to cytoplasm and nucleus while colocalizing with actively transcribed gene. Gene expression of CTK, MMP-9 and NFATc1 were significantly inhibited at 1 and 5 μg/mL (p < 0.05) of NaPPS whereas inhibition of c-Fos and AP-1 was identified only at concentration of 5 μg/mL (p < 0.05).ConclusionsTaken together, all the results suggest that NaPPS is a novel inhibitor of RANKL and M-CSF-induced CTK, MMP-9, NFATc1, c-Fos, AP-1 upregulation, OC differentiation and bone resorption which might be a beneficial for treatment of inflammatory joint diseases and other bone diseases associated with excessive bone resorption.

Zoledronate and high glucose levels influence osteoclast differentiation and bone absorption via the AMPK pathway

MCP-1 (monocyte chemotactic protein-1) is a CC chemokine that is induced by receptor activator of NFkappaB ligand (RANKL) in human osteoclasts. In the absence of RANKL, treatment of human peripheral blood mononuclear cells with macrophage colony-stimulating factor and MCP-1 resulted in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells that are positive for calcitonin receptor (CTR) and a number of other osteoclast markers, including nuclear factor of activated t cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). Although NFATc1 was strongly induced by MCP-1 and was observed in the nucleus, MCP-1 did not permit the formation of bone-resorbing osteoclasts, although these cells had the typical TRAP(+)/CTR(+) multinuclear phenotype of osteoclasts. Despite a similar appearance to osteoclasts, RANKL treatment was required in order for TRAP(+)/CTR(+) multinuclear cells to develop bone resorption activity. The lack of bone resorption was correlated with a deficiency in expression of certain genes related to bone resorption, such as cathepsin K and MMP9. Furthermore, calcitonin blocked the MCP-1-induced formation of TRAP(+)/CTR(+) multinuclear cells as well as blocking osteoclast bone resorption activity, indicating that calcitonin acts at two stages of osteoclast differentiation. Ablation of NFATc1 in mature osteoclasts did not prevent bone resorption activity, suggesting NFATc1 is involved in cell fusion events and not bone resorption. We propose that the MCP-1-induced TRAP(+)/CTR(+) multinuclear cells represent an arrested stage in osteoclast differentiation, after NFATc1 induction and cellular fusion but prior to the development of bone resorption activity.

Osteoclasts, which are specialized bone multinuclear cells, are responsible for bone lytic diseases such as osteoporosis. 3',4',7,8-tetrahydroxyflavone is a flavonoid from Acacia confusa. In the present study, we found that 3',4',7,8-tetrahydroxyflavone markedly inhibited receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastic differentiation from mouse bone marrow-derived macrophages (BMMs). 3',4',7,8-tetrahydroxyflavone also reduced the mRNA expression levels of osteoclastic marker genes including the calcitonin receptor (CTR) and cathepsin K. In addition, 3',4',7,8-tetrahydroxyflavone decreased the bone resorption activity of osteoclasts on dentin slices. We found that 3',4',7,8-tetrahydroxyflavone inhibited RANKL-induced expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), a key transcription factor of osteoclast differentiation. Furthermore, ectopic overexpression of a constitutively active form of NFATc1 completely rescued the anti-osteoclastogenic effect of 3',4',7,8-tetrahydroxyflavone, suggesting that the anti-osteoclastogenic effect was mainly attributed to the reduction in NFATc1 expression. Taken together, our data suggest that 3',4',7,8-tetrahydroxyflavone inhibits osteoclast differentiation and bone loss and may therefore be considered a promising drug candidate for treating or preventing bone-lytic diseases.

BackgroundVitamin D is an important steroid that can regulate bone metabolism including osteoclast (OC) differentiation. Transient receptor potential cation channel subfamily V member 5 (TRPV5), is a calcium channel protein involved in OC differentiation. However, the impact of vitamin D on TRPV5 expression during OC differentiation is not clear.ObjectivesTo determine if 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates the expression of TRPV5 during OC differentiation.MethodsBone marrow mononuclear macrophage (BMMs) were induced to differentiate into OC with or without treatment with 10 nM 1,25(OH)2D3. The expression levels of vitamin D receptor (VDR) and TRPV5 were examined. The expression of several OC markers, including tartrate resistant acid phosphatase (TRAP), carbonic anhydrase II (Ca II), cathepsin K (CTSK), and vacuolar-type H+-ATPase (V-ATPase) were also detected.ResultsWe found that the VDR was expressed in murine bone marrow-derived macrophages at the early stage of OC differentiation. TRPV5 expression was increased during OC differentiation, which was down-regulated by 1,25(OH)2D3 after a prolonged exposure. The 1,25(OH)2D3 and TRPV5 inhibitors inhibited OC differentiation.Conclusions1,25(OH)2D3 can inhibit TRPV5 expression as well as TRPV5 inhibitors during OC differentiation. This suggests that 1,25(OH)2D3 may suppress OC differentiation by inhibiting TRPV5 expression.

Potential Role of Pancreatic and Enteric Hormones in Regulating Bone Turnover

Bone Health in Chronic Kidney Disease–Mineral and Bone Disease

To explore the effects of ferric ion on the differentiation from both RAW264.7 and bone marrow macrophages to osteoclast in vitro and bone resorption in vivo. In the presence of 50 ng/ml receptor activator of nuclear factor kappa-B ligand (RANKL), RAW264.7 was treated with ferric ammonium citrate (FAC). The formation of osteoclast was observed by staining of tartrate-resistant acid phosphatase (TRAP) and the TRAP positive cell counted. The expression levels of TRAP, cathepsin-K, nuclear factor of activated T cells c1 (NFATc1) and (receptor activator of NF-κB) RANK were measured by reverse transcription-polymerase chain reaction (RT-PCR).The control and iron overload groups were established by the intraperitoneal injection of normal saline and FAC. In vivo imaging system was employed to determine the bone density of femoral midportion and the fourth lumbar vertebra. After that, the bone marrow cells of femurs were used for osteoclast culture. The serum levels of ferritin, TRAP-5b, RANKL, osteoprotegerin (OPG) and C-terminal cross-linking telopeptide (CTX) were measured by enzyme-linked immunosorbent assay (ELISA). Ferric ion could stimulate the formation of TRAP positive cells in a dose-dependent manner. The expression levels of TRAP, cathepsin-K and NFATc1 in the FAC treated group were significantly higher those of the control group (P < 0.05) while the expression of RANK showed no statistical difference among these groups (P = 0.967). The bone marrow density of femoral midportion and the fourth lumbar vertebra of the iron-overload group decreased significantly versus the control group. The concentrations of ferritin, TRAP-5b, RANKL and CTX of the iron overload group were markedly higher than those of the control group (P < 0.05). Moreover, the concentration of ferritin showed a positive correlation with TRAP-5b and CTX respectively in the iron-overload group (r = 0.65, r = 0.76, P < 0.05). But no significant differences existed in the concentration of OPG for two groups (P > 0.05). Ferric ion may enhance the differentiation of osteoclast in vitro as well as bone resorption in vivo.

Genes Involved in Osteoclastogenesis

To investigate the effect of zoledronate (ZOL) on osteoclast differentiation and bone resorption under high glucose, and the regulation mechanism of p38 mitogen activated kinase (p38 MAPK) signaling pathway in this process. RAW264.7 cells were divided into four groups: low group, high group, low+ZOL group and high+ZOL group after induced into osteoclasts. Cell proliferation activity was determined by MTT assay. The migration of RAW264.7 cells were examined Optical microscopy. Immunofluorescence microscopy was used to observe the cytoskeleton and sealing zones of osteoclasts. After adding group 5: high + ZOL + SB203580 group, trap staining was used to identify the number of positive osteoclasts in each group. The number and area of resorption lacunae were observed by SEM. The mRNA and protein expression of osteoclast related factors were detected by real-time PCR and Western blotting. The cells in the 5 groups showed similar proliferative activity. High glucose promoted the migration of RAW264.7 cells (P < 0.05), inhibited the clarity of cytoskeleton and the formation of sealing zones in the osteoclasts. Exposure to high glucose significantly lowered the expressions of p38 MAPK, p-p38 MAPK, NFATc1, CTSK and TRAP, and inhibited osteoclast differentiation and bone absorption (P < 0.05). Treatment with ZOL obviously suppressed the migration ability of RAW264.7 cells, further reduced the clarity of the cytoskeleton, inhibited the formation of sealing zones of the osteoclasts, lowered the expressions of p38 MAPK, p-p38 MAPK, NFATc1, CTSK, and TRAP (P < 0.05), and inhibited osteoclast differentiation and bone absorption. Treatment with SB203580 obviously inhibited osteoclast differentiation and bone resorption and the expressions of P38 MAPK, p-p38 MAPK, NFATc1, CTSK and TRAP (P < 0.05). High glucose inhibits osteoclast differentiation and bone resorption. ZOL inhibits osteoclast differentiation and bone resorption in high-glucose conditions by regulating p38 MAPK pathway, which can be a new pathway for ZOL to regulate diabetic osteoporosis.

DNA damage-inducible transcript 3 restrains osteoclast differentiation and function.

5,7-Dihydroxycoumarin counteracts osteoporosis by inhibition of PI3K/Akt signaling to suppress osteoclastogenesis: A study-based network pharmacology and experimental validation.

Event Abstract Back to Event Ethyl-3, 4-dihydroxybenzoate as a novel dual-action therapeutic agent by regulating both bone formation and resorption Byeong-Ju Kwon1, 2, Min-Ah Koo1, 2, Gyeung Mi Seon1, 2, Min Sung Kim1, 2, Mi Hee Lee1, Jae-Jin Han3 and Jong-Chul Park1, 2 1 Yonsei University College of Medicine, Cellbiocontrol Laboratory,Department of Medical Engineering, Korea 2 Yonsei University College of Medicine, Brain Korea 21 PLUS Project for Medical Science, Korea 3 Cellsafe, Korea Introduction: Bone tissue engineering aims to induce new functional bone regeneration via the synergistic combination of biomaterials, cells, and factor therapy. The interaction between biomaterials and the surrounding tissue at the implant interface is essential for success or failure of implants. Biomaterial interacts with both osteoblasts and osteoclasts. Bone remodeling is controlled by two equal equal, but opposing, forces: bone formation by osteoblasts and bone destruction or resorption by osteoclasts. Successful bone remodeling should be balanced between bone formation and bone resorption. Ethyl-3, 4-dihydroxybenzoate (E-3, 4-DHB) is a component of the Rubus coreanus extract, and the extract has recently been shown to have a bone protecting-effect on postmenopausal osteoporosis in ovariectomized rats. However, the dual function of E-3, 4-DHB of inducing osteoblastic differentiation and inhibiting osteoclast differentiation has not been previously evaluated and warrants further evaluation. Materials and methods: The osteoinductive ability of E-3, 4-DHB in preosteoblats and human mesenchymal stem cells (hMSCs) was examined. E-3, 4-DHB for future use in bone tissue engineering was evaluated by examination of early markers of differentiation (such as alkaline phosphatase [ALP], activity runt-related transcription factor 2 [Runx-2], and collagen type I expression) and late markers of osteoblast differentiation (bone nodule formation). Also, to evaluate the osteoinductive activity in a three-dimensional (3D) dynamic culture system, hMSCs were cultured in a HARV bioreactor for 21 days. In addition, differentiation of osteoclasts in response to E-3, 4-DHB was observed with TRAP activity and TRAP staining. Finally, in vivo study, alginate gel comprised of E-3, 4-DHB and cells was transplanted into the back subcutis of mice. Results and discussion: Our results have shown that the ALP activity, Runx2 expression, collagen type 1 expression, and bone nodule formation of E-3, 4-DHB treated group have been significantly increased in comparison with the untreated group. hMSCs cultured in a bioreactor with osteogenic media containing E-3, 4-DHB showed more red or black color using ARS and von Kossa staining, compared with the hMSCs cultured in osteogenic media (control). In our in vitro study using RAW264.7 cells, we found that E-3, 4-DHB dose-dependently inhibited RANKL-induced osteoclast differentiation. These results suggest that E-3, 4-DHB has an inhibitory effect of osteoclast formation. Also, we verified the osteoinductive activity through animal testing. Conclusion: Our results show that E-3, 4-DHB might have beneficial effects through regulation of both osteoblast and osteoclast differentiation. Therefore, we suggest that E-3, 4-DHB could be a strong candidate for dual regulation to increase osteoblast differentiation and decrease osteoclast differentiation. Keywords: Bone Regeneration, Regenerative Medicine, biomaterial, in vivo tissue engineering Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials in constructing tissue substitutes Citation: Kwon B, Koo M, Seon G, Kim M, Lee M, Han J and Park J (2016). Ethyl-3, 4-dihydroxybenzoate as a novel dual-action therapeutic agent by regulating both bone formation and resorption. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02296 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers' terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Byeong-Ju Kwon Min-Ah Koo Gyeung Mi Seon Min Sung Kim Mi Hee Lee Jae-Jin Han Jong-Chul Park Google Byeong-Ju Kwon Min-Ah Koo Gyeung Mi Seon Min Sung Kim Mi Hee Lee Jae-Jin Han Jong-Chul Park Google Scholar Byeong-Ju Kwon Min-Ah Koo Gyeung Mi Seon Min Sung Kim Mi Hee Lee Jae-Jin Han Jong-Chul Park PubMed Byeong-Ju Kwon Min-Ah Koo Gyeung Mi Seon Min Sung Kim Mi Hee Lee Jae-Jin Han Jong-Chul Park Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Glycyrrhizic acid alleviates liver fibrosis in vitro and in vivo via activating CUGBP1-mediated IFN-γ/STAT1/Smad7 pathway