PSTP-3,5-Me Inhibits Osteoclast Differentiation and Bone Resorption

Osteogenesis is an orchestrated process regulated by osteoclastogenesis and osteoblastogenesis. Excessive osteoclastogenesis causes bone diseases, such as osteoporosis. Although a few drugs are effective in osteoporosis treatment, these drugs lead to side effects, including cellulitis, flatulence, and hypocalcemia. In this study, we reported a 2-(N-Phenylmethylsulfonamido)-N-(2-(phenylthio)phenyl)propanamide (PSTP) compound, PSTP-3,5-Me, as a potential therapeutic agent for osteoporosis. Mouse bone marrow-derived macrophages (BMMs) were differentiated into osteoclasts by receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in the presence of PSTP-3,5-Me. PSTP-3,5-Me inhibited osteoclast differentiation by reduced tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and suppressed the expression of osteoclast marker genes, such as cathepsin K (Ctsk) and TRAP (Acp5). We investigated signaling pathways mediated by RANKL and its receptor, RANK, and found that PSTP-3,5-Me inhibits nucleus translocation of nuclear factor of activated T cell cytoplasmic-1 (NFATc1). Moreover, PSTP-3,5-Me inhibited F-actin ring formation and mineral resorption. Overall, our data suggests that PSTP-3,5-Me attenuates osteoclast differentiation by blocking the activation of NFATc1.

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Kalkitoxin Reduces Osteoclast Formation and Resorption and Protects against Inflammatory Bone Loss

International Journal of Molecular Sciences ◽

10.3390/ijms22052303 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2303

Author(s):

Liang Li ◽

Ming Yang ◽

Saroj Kumar Shrestha ◽

Hyoungsu Kim ◽

William H. Gerwick ◽

...

Keyword(s):

Bone Loss ◽

Transmembrane Protein ◽

Osteoclast Differentiation ◽

Mapk Signaling ◽

Osteoclast Formation ◽

Bone Diseases ◽

Tartrate Resistant Acid Phosphatase ◽

Nuclear Factor ◽

Mineral Density ◽

Multinucleated Cells

Osteoclasts, bone-specified multinucleated cells produced by monocyte/macrophage, are involved in numerous bone destructive diseases such as arthritis, osteoporosis, and inflammation-induced bone loss. The osteoclast differentiation mechanism suggests a possible strategy to treat bone diseases. In this regard, we recently examined the in vivo impact of kalkitoxin (KT), a marine product obtained from the marine cyanobacterium Moorena producens (previously Lyngbya majuscula), on the macrophage colony-stimulating factor (M-CSF) and on the receptor activator of nuclear factor κB ligand (RANKL)-stimulated in vitro osteoclastogenesis and inflammation-mediated bone loss. We have now examined the molecular mechanism of KT in greater detail. KT decreased RANKL-induced bone marrow-derived macrophages (BMMs) tartrate-resistant acid phosphatase (TRAP)-multinucleated cells at a late stage. Likewise, KT suppressed RANKL-induced pit area and actin ring formation in BMM cells. Additionally, KT inhibited several RANKL-induced genes such as cathepsin K, matrix metalloproteinase (MMP-9), TRAP, and dendritic cell-specific transmembrane protein (DC-STAMP). In line with these results, RANKL stimulated both genes and protein expression of c-Fos and nuclear factor of activated T cells (NFATc1), and this was also suppressed by KT. Moreover, KT markedly decreased RANKL-induced p-ERK1/2 and p-JNK pathways at different time points. As a result, KT prevented inflammatory bone loss in mice, such as bone mineral density (BMD) and osteoclast differentiation markers. These experiments demonstrated that KT markedly inhibited osteoclast formation and inflammatory bone loss through NFATc1 and mitogen-activated protein kinase (MAPK) signaling pathways. Therefore, KT may have potential as a treatment for destructive bone diseases.

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Inhibition of Osteoclastogenesis by Thioredoxin-Interacting Protein-Derived Peptide (TN13)

Journal of Clinical Medicine ◽

10.3390/jcm8040431 ◽

2019 ◽

Vol 8 (4) ◽

pp. 431 ◽

Cited By ~ 2

Author(s):

Mi Kim ◽

Won Kim ◽

Jae-Eun Byun ◽

Jung Choi ◽

Suk Yoon ◽

...

Keyword(s):

Bone Loss ◽

Osteoclast Differentiation ◽

Bone Diseases ◽

Raw 264.7 Cells ◽

Tartrate Resistant Acid Phosphatase ◽

Nuclear Factor ◽

Activated T Cells ◽

Interacting Protein ◽

Hematopoietic Stem ◽

Thioredoxin Interacting Protein

Overactivated osteoclasts lead to many bone diseases, including osteoporosis and rheumatoid arthritis. The p38 MAPK (p38) is an essential regulator of the receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis and bone loss. We previously reported TAT conjugated thioredoxin-interacting protein-derived peptide (TAT-TN13) as an inhibitor of p38 in hematopoietic stem cells (HSCs). Here, we examined the role of TAT-TN13 in the differentiation and function of osteoclasts. TAT-TN13 significantly suppressed RANKL-mediated differentiation of RAW 264.7 cells and bone marrow macrophages (BMMs) into osteoclasts. TAT-TN13 also inhibited the RANKL-induced activation of NF-κB and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), leading to the decreased expression of osteoclast-specific genes, including tartrate-resistant acid phosphatase (TRAP) and Cathepsin K. Additionally, TAT-TN13 treatment protected bone loss in ovariectomized (OVX) mice. Taken together, these results suggest that TAT-TN13 inhibits osteoclast differentiation by regulating the p38 and NF-κB signaling pathway; thus, it may be a useful agent for preventing or treating osteoporosis.

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Ostericum koreanum Reduces LPS-Induced Bone Loss Through Inhibition of Osteoclastogenesis

The American Journal of Chinese Medicine ◽

10.1142/s0192415x15500317 ◽

2015 ◽

Vol 43 (03) ◽

pp. 495-512 ◽

Cited By ~ 2

Author(s):

Ju-Young Kim ◽

Sung-Jun Ahn ◽

Jong Min Baek ◽

Kwon-Ha Yoon ◽

Myeung Su Lee ◽

...

Keyword(s):

Bone Marrow ◽

Bone Loss ◽

Bone Structure ◽

Osteoclast Differentiation ◽

Bone Diseases ◽

Therapeutic Drug ◽

Tartrate Resistant Acid Phosphatase ◽

Mouse Bone Marrow ◽

Actin Ring ◽

Ostericum Koreanum

The roots of Ostericum koreanum (OK) Maximowicz have traditionally been used to produce an herbal medicine reported to possess anti-inflammatory, anti-oxidant, antimicrobial, and antitumor activities; however, its effect on bone metabolism has not yet been reported. The present study examined the effects of OK extract on lipopolysaccharide (LPS)-induced bone loss in mice by investigating bone structure and the levels of the receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) in serum and bone marrow fluid (BMF). The effects of OK extract on osteoclastogenesis were also investigated in mouse bone marrow macrophages by examining the formation of tartrate-resistant acid phosphatase (TRAP)-positive cells, the actin ring, and bone resorption activity. OK reduced LPS-induced bone destruction in vivo via a decrease in the RANKL/OPG ratio. Furthermore, it suppressed the formation of TRAP-positive cells and the actin ring, and reduced the bone-resorbing activity of mature osteoclasts. OK also significantly down-regulated the expression of various osteoclast-specific genes. However, it did not affect osteoblast differentiation, or the expression of genes involved in this process. These results demonstrated that OK prevented LPS-induced bone loss by decreasing the RANKL/OPG ratio in serum and BMF, and inhibited osteoclast differentiation and function, suggesting that OK represents a potential therapeutic drug for the treatment of osteoclast-associated bone diseases.

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Inhibitory Effects of 2N1HIA (2-(3-(2-Fluoro-4-Methoxyphenyl)-6-Oxo-1(6H)-Pyridazinyl)-N-1H-Indol-5-Ylacetamide) on Osteoclast Differentiation via Suppressing Cathepsin K Expression

Molecules ◽

10.3390/molecules23123139 ◽

2018 ◽

Vol 23 (12) ◽

pp. 3139 ◽

Cited By ~ 6

Author(s):

Sun-Hee Ahn ◽

Zhihao Chen ◽

Jinkyung Lee ◽

Seok-Woo Lee ◽

Sang Min ◽

...

Keyword(s):

Bone Resorption ◽

Osteoclast Differentiation ◽

Cell Formation ◽

Cathepsin K ◽

Bone Diseases ◽

Specific Gene ◽

Tartrate Resistant Acid Phosphatase ◽

Potential Candidate ◽

Dependent Manner ◽

Gene Markers

Osteoclasts are large multinucleated cells which are induced by the regulation of the receptor activator of nuclear factor kappa-Β ligand (RANKL), which is important in bone resorption. Excessive osteoclast differentiation can cause pathologic bone loss and destruction. Numerous studies have targeted molecules inhibiting RANKL signaling or bone resorption activity. In this study, 11 compounds from commercial libraries were examined for their effect on RANKL-induced osteoclast differentiation. Of these compounds, only 2-(3-(2-fluoro-4-methoxyphenyl)-6-oxo-1(6H)-pyridazinyl)-N-1H-indol-5-ylacetamide (2N1HIA) caused a significant decrease in multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cell formation in a dose-dependent manner, without inducing cytotoxicity. The 2N1HIA compound neither affected the expression of osteoclast-specific gene markers such as TRAF6, NFATc1, RANK, OC-STAMP, and DC-STAMP, nor the RANKL signaling pathways, including p38, ERK, JNK, and NF-κB. However, 2N1HIA exhibited a significant impact on the expression levels of CD47 and cathepsin K, the early fusion marker and critical protease for bone resorption, respectively. The activity of matrix metalloprotease-9 (MMP-9) decreased due to 2N1HIA treatment. Accordingly, bone resorption activity and actin ring formation decreased in the presence of 2N1HIA. Taken together, 2N1HIA acts as an inhibitor of osteoclast differentiation by attenuating bone resorption activity and may serve as a potential candidate in preventing and/or treating osteoporosis, or other bone diseases associated with excessive bone resorption.

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Optimized Extract from Corylopsis coreana Uyeki (Hamamelidaceae) Flos Inhibits Osteoclast Differentiation

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2018/6302748 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9

Author(s):

Yongjin Lee ◽

Jung-Eun Kim ◽

Kwang-Jin Kim ◽

Seung-Sik Cho ◽

Young-Jin Son

Keyword(s):

Osteoclast Differentiation ◽

Cathepsin K ◽

Inhibitory Effect ◽

Osteoclast Formation ◽

Tartrate Resistant Acid Phosphatase ◽

Alcoholic Extract ◽

Nuclear Factor ◽

Activated T Cells ◽

Fractures Of The Spine ◽

The Stability

Osteoporosis is a metabolic disorder that decreases the stability against fractures of the spine, femur, and radius by weakening the strength and integrity of bones. Receptor activator of nuclear factor-kappa B ligand signaling ultimately activated nuclear factor-activated T cells c1, a major transcription factor for osteoclast formation. This study researched the effects of Corylopsis coreana (C. coreana) Uyeki flos extracts on the antiosteoclastic potential of macrophages and the phytochemicals contained therein. The alcoholic extract of C. coreana Uyeki flos inhibited the differentiation of osteoclast. We carried out the experiments of the pattern of differentiation of osteoclasts based on the alcoholic percentage of extracts. Among them, 80% alcoholic extract showed the highest inhibitory effect. The alcoholic extract was composed of phytochemicals such as bergenin, quercetin, and quercitrin. This extract inhibited not only mRNA expression levels of NFATc1, osteoclast-associated receptor (OSCAR), cathepsin K, and tartrate-resistant acid phosphatase (TRAP), but also the translational expression of NFATc1. The inhibitory effect for osteoclast differentiation of the alcoholic extract was confirmed using the resorption pit assay. This is the first scientific report of the antiosteoclastic effects of C. coreana Uyeki flos extract, which can be applied therapeutically for the treatment of osteoporosis.

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Calcitonin inhibits osteoclast formation in mouse haematopoetic cells independently of transcriptional regulation by receptor activator of NF-κB and c-Fms

Journal of Endocrinology ◽

10.1677/joe-07-0338 ◽

2007 ◽

Vol 195 (3) ◽

pp. 415-427 ◽

Cited By ~ 45

Author(s):

Susanne Granholm ◽

Pernilla Lundberg ◽

Ulf H Lerner

Keyword(s):

Transmembrane Protein ◽

Osteoclast Differentiation ◽

Inhibitory Effect ◽

Osteoclast Formation ◽

Tartrate Resistant Acid Phosphatase ◽

Mouse Bone Marrow ◽

Osteoclast Progenitor ◽

Receptor Activator ◽

Macrophage Colony ◽

A Disintegrin And Metalloproteinase

The effects of calcitonin (CT) on osteoclast formation and gene expression have been studied in cultured mouse spleen cells and mouse bone marrow macrophages (BMMs). CT inhibited the formation of multinucleated osteoclasts and resorption pits in spleen cell cultures and BMM as well as in CD115+ CD3− CD45R−sorted BMM cultures, incubated in the presence of macrophage colony-stimulating factor and receptor activator of NF-κB ligand (RANKL). No effect on apoptosis by CT was observed. CT did not affect the mRNA expressions of RANK and c-Fms, or the mRNA expressions of a wide variety of transcription factors and genes important for osteoclast differentiation and activity. CT induced inhibition of tartrate-resistant acid phosphatase (TRAP), positive multinucleated osteoclast formation was not associated with any decrease of total TRAP activity, resulting in a large number of TRAP+ mononucleated cells in CT-treated cultures. CT did not affect the mRNA expression of dendritic cell-specific transmembrane protein, d2 isoform of vacuolar (H+) ATPase vo domain, a disintegrin and metalloproteinase domain 8 (ADAM8), ADAM12, DNAX-activating protein or Fc receptor common γ chain suggested to be involved in fusion of mononucleated osteoclast progenitor cells. The inhibitory effect by CT was mimicked not only by compounds activating cAMP and protein kinase A (PKA) but also by a cAMP analogue activating the exchange protein directly activated by cAMP (Epac) pathway. It is concluded that CT, through cAMP/PKA/Epac cascades, inhibits osteoclast formation and that this effect is not associated with decreased transcription of genes known to be important for osteoclast progenitor cell differentiation, fusion or function.

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miRNA-340 inhibits osteoclast differentiation via repression of MITF

Bioscience Reports ◽

10.1042/bsr20170302 ◽

2017 ◽

Vol 37 (4) ◽

Cited By ~ 25

Author(s):

Hongying Zhao ◽

Jun Zhang ◽

Haiyu Shao ◽

Jianwen Liu ◽

Mengran Jin ◽

...

Keyword(s):

Transcription Factor ◽

Osteoclast Differentiation ◽

Cathepsin K ◽

Luciferase Reporter ◽

Pcr Analysis ◽

Tartrate Resistant Acid Phosphatase ◽

Qrt Pcr ◽

Reverse Transcription Pcr ◽

Macrophage Colony ◽

And Function

Many miRNAs play critical roles in modulating various biological processes of osteoclast differentiation and function. Microphthalmia-associated transcription factor (MITF), a target of miR-340, served as pivotal transcription factor involved in osteoclast differentiation. However, the role of miR-340 and MITF during osteoclast differentiation has not yet been clearly established. Tartrate-resistant acid phosphatase (TRAP) staining assay was performed to identify osteoclasts differentiated from bone marrow-derived macrophages (BMMs). Quantitative reverse transcription PCR (qRT-PCR) or Western blotting was undertaken to examine the mRNA or protein expression respectively. Luciferase reporter assay was performed to investigate the interaction between miR-340 and MITF. MITF was knocked down and miR-340 was overexpressed and transfected into BMMs to detect their effects on osteoclast differentiation. Firstly, qRT-PCR analysis showed that miR-340 was down-regulated during osteoclast differentiation stimulated by macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB (RANK) ligand (RANKL). Besides, we found that overexpression of miRNA-340 inhibited osteoclast differentiation and suppressed both the mRNA and protein level of MITF. Finally, Western blot and qRT-PCR analysis revealed that silencing MITF inhibited TRAP, calcitonin receptor, V-ATPase d2, and cathepsin K. miR-340 suppresses osteoclast differentiation by inhibiting MITF. Our findings may provide promising therapeutic targets for osteoclast-associated diseases.

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N-[2-(4-Acetyl-1-Piperazinyl)Phenyl]-2-(3-Methylphenoxy)Acetamide (NAPMA) Inhibits Osteoclast Differentiation and Protects against Ovariectomy-Induced Osteoporosis

Molecules ◽

10.3390/molecules25204855 ◽

2020 ◽

Vol 25 (20) ◽

pp. 4855

Author(s):

Jinkyung Lee ◽

Sun-Hee Ahn ◽

Zhihao Chen ◽

Sohi Kang ◽

Dong Kyu Choi ◽

...

Keyword(s):

Bone Resorption ◽

Bone Loss ◽

Osteoclast Differentiation ◽

Cathepsin K ◽

Bone Diseases ◽

Drug Candidate ◽

Tartrate Resistant Acid Phosphatase ◽

Dependent Manner ◽

Osteoclast Activity ◽

Protein Levels

Osteoclasts are large, multinucleated cells responsible for bone resorption and are induced in response to the regulatory activity of receptor activator of nuclear factor-kappa B ligand (RANKL). Excessive osteoclast activity causes pathological bone loss and destruction. Many studies have investigated molecules that specifically inhibit osteoclast activity by blocking RANKL signaling or bone resorption. In recent years, we screened compounds from commercial libraries to identify molecules capable of inhibiting RANKL-induced osteoclast differentiation. Consequently, we reported some compounds that are effective at attenuating osteoclast activity. In this study, we found that N-[2-(4-acetyl-1-piperazinyl)phenyl]-2-(3-methylphenoxy)acetamide (NAPMA) significantly inhibited the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells from bone marrow-derived macrophages in a dose-dependent manner, without cytotoxic effects. NAPMA downregulated the expression of osteoclast-specific markers, such as c-Fos, NFATc1, DC-STAMP, cathepsin K, and MMP-9, at the transcript and protein levels. Accordingly, bone resorption and actin ring formation were decreased in response to NAPMA treatment. Furthermore, we demonstrated the protective effect of NAPMA against ovariectomy-induced bone loss using micro-CT and histological analysis. Collectively, the results showed that NAPMA inhibited osteoclast differentiation and attenuated bone resorption. It is thus a potential drug candidate for the treatment of osteoporosis and other bone diseases associated with excessive bone resorption.

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Inhibitory effects of ChondroT and its constituent herbs on RANKL-induced osteoclastogenesis

BMC Complementary and Alternative Medicine ◽

10.1186/s12906-019-2737-8 ◽

2019 ◽

Vol 19 (1) ◽

Author(s):

Rui Hong Guo ◽

Seon-Jong Kim ◽

Chan-hun Choi ◽

Chang-su Na ◽

Bok Yun Kang ◽

...

Keyword(s):

Molecular Mechanisms ◽

Osteoclast Differentiation ◽

Cathepsin K ◽

Mapk Signaling ◽

Bone Diseases ◽

Ring Formation ◽

Tartrate Resistant Acid Phosphatase ◽

Actin Ring ◽

Specific Proteins ◽

Underlying Mechanisms

Abstract Background ChondroT is a complex herbal medicine consisting of water extracts of Ostericum koreanum (Maxim.) Kitag., Lonicera japonica Thunb., Angelica gigas Nakai, Clematis manshurica Rupr., and Phellodendron amurense Rupr. (6:4:4:4:3). Previous studies have reported that ChondroT possesses chondroprotective and anti-inflammatory, anti-osteoarthritic, and anti-hyperuricemic activities. The study is aim to demonstrate the effects of ChondroT and its five constituent herbs on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and the underlying mechanisms. Methods Osteoclastogenesis was identified in bone marrow-derived macrophages (BMDMs) by tartrate-resistant acid phosphatase (TRAP) staining assay, actin ring formation assay and the bone resorption assay. For the molecular mechanisms, activation of RANKL-induced NF-κB and MAPK signaling pathways and the expression levels of osteoclast-specific proteins were investigated by Western blotting. Cell viability was assessed by MTT assay. Actin ring formation and NF-κB translocation were evaluated by immunostaining. Results ChondroT and each of its constituent herbs significantly suppressed osteoclast differentiation dose dependently, and decreased actin ring formation as well as bone-resorbing capacity. Mechanistically, ChondroT and its constituent herbs downregulated the expressional levels of osteoclast-specific proteins such as NFATc1, c-Fos, Cathepsin K, and matrix metalloproteinase 9 (MMP9) by suppressing NF-κB translocation to nucleus and MAPKs phosphorylation at different levels. Compared to its five constituent herbs, ChondroT exhibited the best inhibitory efficiency against osteoclastogenesis. Conclusions Taken together, ChondroT has anti-osteoclastogenesis properties by inhibiting NF-κB and MAPKs pathways. It could be considered as a potential therapeutic candidate for the treatment of osteoclast-related bone diseases.

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Effect of graded calcium supplementation in low-nutrient density feed on tibia composition and bone turnover in meat ducks

British Journal Of Nutrition ◽

10.1017/s0007114518002556 ◽

2018 ◽

Vol 120 (11) ◽

pp. 1217-1229 ◽

Cited By ~ 3

Author(s):

Huaiyong Zhang ◽

Qiufeng Zeng ◽

Shiping Bai ◽

Jianping Wang ◽

Xuemei Ding ◽

...

Keyword(s):

Mechanical Properties ◽

Bone Turnover ◽

Calcium Supplementation ◽

Osteoclast Differentiation ◽

Cathepsin K ◽

Positive Control ◽

Marker Genes ◽

Specific Gene ◽

Tartrate Resistant Acid Phosphatase ◽

Nutrient Density

AbstractBoth genetic selection and increasing nutrient density for improving growth performance had inadvertently increased leg problems of meat ducks, which adversely affects animal welfare. We hypothesised that slowing weight gain with improving tibia quality probably enhanced tibial mechanical properties and alleviated leg deformities. Therefore, the present study aimed to evaluate the effect of graded Ca supplementation in a low-nutrient density (LND) diet on tibia composition and bone turnover in meat ducks. A total of 720 15-d-old male meat ducks were randomly assigned and fed a standard nutrient density positive control (PC) diet containing 0·9 % Ca, and four LND diets with 0·5, 0·7, 0·9 and 1·1 % Ca, respectively. Ducks fed the 0·5 % Ca LND diet and the PC diet had higher incidence of tibial dyschondroplasia (TD). When compared with the 0·5 % Ca LND diet, LND diets with ≥0·7 % Ca significantly improved tibia composition, microarchitecture and mechanical properties, and consequently decreased the incidence of TD. Furthermore, LND diets with ≥0·7 % Ca increased osteocyte-specific gene mRNA expression, blocked the expression of osteoblast differentiation marker genes including osteocalcin, collagenase-1 and alkaline phosphatase (ALP), and also decreased the expression of osteoclast differentiation genes, such as vacuolar-type H+-ATPase, cathepsin K and receptor activator of NF-κB. Meanwhile bone markers such as serum ALP, osteocalcin (both osteoblast markers) and tartrate-resistant acid phosphatase (an osteoclast marker) were significantly decreased in at least 0·7 % Ca treated groups. These findings indicated that LND diets with ≥0·7 % Ca decreased bone turnover, which subsequently increased tibia quality for 35-d-old meat ducks.

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