A Comparison between the Effects of Hydrophobic and Hydrophilic Statins on Osteoclast Function In Vitro and Ovariectomy-Induced Bone Loss In Vivo

AbstractOsteal macrophages (osteomacs) support osteoblast function and promote bone anabolism, but their contribution to osteoporosis has not been explored. While mouse ovariectomy models have been repeatedly used, variation in strain, experimental design and assessment modalities, have contributed to no single model being confirmed as comprehensively replicating the full gamut of osteoporosis pathological manifestations. We validated an ovariectomy model in adult C3H/HeJ mice and demonstrated that it presents with human post-menopausal osteoporosis features, including reduced bone volume in axial and appendicular bone and bone loss in both trabecular and cortical bone including increased cortical porosity. Bone loss was associated with increased osteoclasts on trabecular and endocortical bone and decreased osteoblasts on trabecular bone. Importantly, this OVX model was characterised by delayed fracture healing. Using this validated model, we demonstrated that osteomacs are increased post-ovariectomy on both trabecular and endocortical bone. Dual F4/80 (pan-macrophage marker) and TRAP staining revealed osteomacs frequently located near TRAP+ osteoclasts and containing TRAP+ intracellular vesicles. Using an in vivo inducible macrophage depletion model that does not simultaneously deplete osteoclasts, we observed that osteomac loss was associated with elevated extracellular TRAP in bone marrow interstitium and increased serum TRAP. Using in vitro high-resolution confocal imaging of mixed osteoclast-macrophage cultures on bone substrate, we observed macrophages juxtaposed to osteoclast basolateral functional secretory domains scavenging degraded bone by-products. These data demonstrate a role for osteomacs in supporting osteoclastic bone resorption through phagocytosis and sequestration of resorption by-products. Finally, using Siglec1 knockout mice, we demonstrated that loss of the macrophage-restricted molecule Siglec-1/CD169 is sufficient to cause age-associated low bone mass, emphasizing the macrophages, independent of osteoclasts, contribute to optimal skeletal health. Overall, our data expose a novel role for osteomacs in supporting osteoclast function and provide the first evidence of their involvement in osteoporosis pathogenesis.

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Engineering of L-Plastin Peptide-Loaded Biodegradable Nanoparticles for Sustained Delivery and Suppression of Osteoclast Function In Vitro

International Journal of Cell Biology ◽

10.1155/2019/6943986 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13 ◽

Cited By ~ 4

Author(s):

Sunipa Majumdar ◽

Aniket S. Wadajkar ◽

Hanan Aljohani ◽

Mark A. Reynolds ◽

Anthony J. Kim ◽

...

Keyword(s):

Bone Resorption ◽

Bone Loss ◽

Sustained Release ◽

Double Emulsion ◽

Small Molecular Weight ◽

Rhodamine Phalloidin ◽

Amino Terminal ◽

Osteoclast Function

We have recently demonstrated that a small molecular weight amino-terminal peptide of L-plastin (10 amino acids; “MARGSVSDEE”) suppressed the phosphorylation of endogenous L-plastin. Therefore, the formation of nascent sealing zones (NSZs) and bone resorption are reduced. The aim of this study was to develop a biodegradable and biocompatible PLGA nanocarrier that could be loaded with the L-plastin peptide of interest and determine the efficacy in vitro in osteoclast cultures. L-plastin MARGSVSDEE (P1) and scrambled control (P3) peptide-loaded PLGA-PEG nanoparticles (NP1 and NP3, respectively) were synthesized by double emulsion technique. The biological effect of nanoparticles on osteoclasts was evaluated by immunoprecipitation, immunoblotting, rhodamine-phalloidin staining of actin filaments, and pit forming assays. Physical characterization of well-dispersed NP1 and NP3 demonstrated ~130-150 nm size, < 0.07 polydispersity index, ~-3 mV ζ-potential, and a sustained release of the peptide for three weeks. Biological characterization in osteoclast cultures demonstrated the following: NP1 significantly reduced (a) endogenous L-plastin phosphorylation; (b) formation of NSZs and sealing rings; (c) resorption. However, the assembly of podosomes which are critical for cell adhesion was not affected. L-plastin peptide-loaded PLGA-PEG nanocarriers have promising potential for the treatment of diseases associated with bone loss. Future studies will use this sustained release of peptide strategy to systematically suppress osteoclast bone resorption activity in vivo in mouse models demonstrating bone loss.

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A novel BRD4 inhibitor suppresses osteoclastogenesis and ovariectomized osteoporosis by blocking RANKL-mediated MAPK and NF-κB pathways

Cell Death and Disease ◽

10.1038/s41419-021-03939-7 ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Ying Liu ◽

Wenjie Liu ◽

Ziqiang Yu ◽

Yan Zhang ◽

Yinghua Li ◽

...

Keyword(s):

Bone Loss ◽

Osteoporosis Treatment ◽

Inhibitory Effect ◽

Specific Gene ◽

Actin Ring ◽

Treatment Target ◽

Significant Inhibitory Effect ◽

Promising Treatment

AbstractBromodomain-containing protein 4 (BRD4) has emerged as a promising treatment target for bone-related disorders. (+)-JQ1, a thienotriazolodiazepine compound, has been shown to inhibit pro-osteoclastic activity in a BRD4-dependent approach and impede bone loss caused by ovariectomy (OVX) in vivo. However, clinical trials of (+)-JQ1 are limited because of its poor druggability. In this study, we synthesized a new (+)-JQ1 derivative differing in structure and chirality. One such derivative, (+)-ND, exhibited higher solubility and excellent inhibitory activity against BRD4 compared with its analogue (+)-JQ1. Interestingly, (-)-JQ1 and (-)-ND exhibited low anti-proliferative activity and had no significant inhibitory effect on RANKL-induced osteoclastogenesis as compared with (+)-JQ1 and (+)-ND, suggesting the importance of chirality in the biological activity of compounds. Among these compounds, (+)-ND displayed the most prominent inhibitory effect on RANKL-induced osteoclastogenesis. Moreover, (+)-ND could inhibit osteoclast-specific gene expression, F‐actin ring generation, and bone resorption in vitro and prevent bone loss in OVX mice. Collectively, these findings indicated that (+)-ND represses RANKL‐stimulated osteoclastogenesis and averts OVX-triggered osteoporosis by suppressing MAPK and NF-κB signalling cascades, suggesting that it may be a prospective candidate for osteoporosis treatment.

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Estrogen Decreases Cytoskeletal Organization by Forming an ERα/SHP2/c-Src Complex in Osteoclasts to Protect against Ovariectomy-Induced Bone Loss in Mice

Antioxidants ◽

10.3390/antiox10040619 ◽

2021 ◽

Vol 10 (4) ◽

pp. 619

Author(s):

Hyun-Jung Park ◽

Malihatosadat Gholam-Zadeh ◽

Sun-Young Yoon ◽

Jae-Hee Suh ◽

Hye-Seon Choi

Keyword(s):

Bone Resorption ◽

Bone Loss ◽

Ring Formation ◽

Tartrate Resistant Acid Phosphatase ◽

Actin Ring ◽

Collagen Crosslinks ◽

Trap Staining ◽

Src Activation

Loss of ovarian function is closely related to estrogen (E2) deficiency, which is responsible for increased osteoclast (OC) differentiation and activity. We aimed to investigate the action mechanism of E2 to decrease bone resorption in OCs to protect from ovariectomy (OVX)-induced bone loss in mice. In vivo, tartrate-resistant acid phosphatase (TRAP) staining in femur and serum carboxy-terminal collagen crosslinks-1 (CTX-1) were analyzed upon E2 injection after OVX in mice. In vitro, OCs were analyzed by TRAP staining, actin ring formation, carboxymethylation, determination of reactive oxygen species (ROS) level, and immunoprecipitation coupled with Western blot. In vivo and in vitro, E2 decreased OC size more dramatically than OC number and Methyl-piperidino-pyrazole hydrate dihydrochloride (MPPD), an estrogen receptor alpha (ERα) antagonist, augmented the OC size. ERα was found in plasma membranes and E2/ERα signaling affected receptor activator of nuclear factor κB ligand (RANKL)-induced actin ring formation by rapidly decreasing a proto-oncogene tyrosine-protein kinase, cellular sarcoma (c-Src) (Y416) phosphorylation in OCs. E2 exposure decreased physical interactions between NADPH oxidase 1 (NOX1) and the oxidized form of c-Src homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), leading to higher levels of reduced SHP2. ERα formed a complex with the reduced form of SHP2 and c-Src to decrease c-Src activation upon E2 exposure, which blocked a signal for actin ring formation by decreased Vav guanine nucleotide exchange factor 3 (Vav3) (p–Y) and Ras-related C3 botulinum toxin substrate 1 (Rac1) (GTP) activation in OCs. E2/ERα signals consistently inhibited bone resorption in vitro. In conclusion, our study suggests that E2-binding to ERα forms a complex with SHP2/c-Src to attenuate c-Src activation that was induced upon RANKL stimulation in a non-genomic manner, resulting in an impaired actin ring formation and reducing bone resorption.

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Scutellarein inhibits RANKL‐induced osteoclast formation in vitro and prevents LPS‐induced bone loss in vivo

Journal of Cellular Physiology ◽

10.1002/jcp.27888 ◽

2018 ◽

Vol 234 (7) ◽

pp. 11951-11959 ◽

Cited By ~ 3

Author(s):

Fangsheng Fu ◽

Siyuan Shao ◽

Ziyi Wang ◽

Fangming Song ◽

Xixi Lin ◽

...

Keyword(s):

Bone Loss ◽

Osteoclast Formation

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Arctigenin inhibits RANKL‐induced osteoclastogenesis and hydroxyapatite resorption in vitro and prevents titanium particle–induced bone loss in vivo

Journal of Cellular Biochemistry ◽

10.1002/jcb.27815 ◽

2018 ◽

Vol 120 (4) ◽

pp. 5367-5376 ◽

Cited By ~ 2

Author(s):

Gejin Wei ◽

Tihong Liang ◽

Chengming Wei ◽

Xiaolian Nong ◽

Qiteng Lu ◽

...

Keyword(s):

Bone Loss ◽

Titanium Particle

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

Journal of Cellular Physiology ◽

10.1002/jcp.25907 ◽

2017 ◽

Vol 233 (1) ◽

pp. 476-485 ◽

Cited By ~ 21

Author(s):

Cheng-Ming Wei ◽

Qian Liu ◽

Fang-Ming Song ◽

Xi-Xi Lin ◽

Yi-Ji Su ◽

...

Keyword(s):

Bone Resorption ◽

Bone Loss

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Crataegus pinnatifida Bunge Inhibits RANKL-Induced Osteoclast Differentiation in RAW 264.7 Cells and Prevents Bone Loss in an Ovariectomized Rat Model

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2021/5521562 ◽

2021 ◽

Vol 2021 ◽

pp. 1-12

Author(s):

Minsun Kim ◽

MinBeom Kim ◽

Jae-Hyun Kim ◽

SooYeon Hong ◽

Dong Hee Kim ◽

...

Keyword(s):

Bone Loss ◽

Rat Model ◽

Osteoclast Differentiation ◽

Ovariectomized Rat ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Mineral Density ◽

Crataegus Pinnatifida

Osteoporosis is characterized by a decrease in bone microarchitecture with an increased risk of fracture. Long-term use of primary treatments, such as bisphosphonates and selective estrogen receptor modulators, results in various side effects. Therefore, it is necessary to develop alternative therapeutics derived from natural products. Crataegus pinnatifida Bunge (CPB) is a dried fruit used to treat diet-induced indigestion, loss of appetite, and diarrhea. However, research into the effects of CPB on osteoclast differentiation and osteoporosis is still limited. In vitro experiments were conducted to examine the effects of CPB on RANKL-induced osteoclast differentiation in RAW 264.7 cells. Moreover, we investigated the effects of CPB on bone loss in the femoral head in an ovariectomized rat model using microcomputed tomography. In vitro, tartrate-resistant acid phosphatase (TRAP) staining results showed the number of TRAP-positive cells, and TRAP activity significantly decreased following CPB treatment. CPB also significantly decreased pit formation. Furthermore, CPB inhibited osteoclast differentiation by suppressing NFATc1, and c-Fos expression. Moreover, CPB treatment inhibited osteoclast-related genes, such as Nfatc1, Ca2, Acp5, mmp9, CtsK, Oscar, and Atp6v0d2. In vivo, bone mineral density and structure model index were improved by administration of CPB. In conclusion, CPB prevented osteoclast differentiation in vitro and prevented bone loss in vivo. Therefore, CPB could be a potential alternative medicine for bone diseases, such as osteoporosis.

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