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

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.

Download Full-text

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.

Download Full-text

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

Download Full-text

Pristimerin Inhibits Osteoclast Differentiation and Bone Resorption in vitro and Prevents Ovariectomy-Induced Bone Loss in vivo

Drug Design Development and Therapy ◽

10.2147/dddt.s275128 ◽

2020 ◽

Vol Volume 14 ◽

pp. 4189-4203

Author(s):

Peng Sun ◽

Qichang Yang ◽

Yanben Wang ◽

Jiaxuan Peng ◽

Kangxian Zhao ◽

...

Keyword(s):

Bone Resorption ◽

Bone Loss ◽

Osteoclast Differentiation

Download Full-text

The Role of IL-1βin the Bone Loss during Rheumatic Diseases

Mediators of Inflammation ◽

10.1155/2015/782382 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 63

Author(s):

Piero Ruscitti ◽

Paola Cipriani ◽

Francesco Carubbi ◽

Vasiliki Liakouli ◽

Francesca Zazzeroni ◽

...

Keyword(s):

Bone Resorption ◽

Bone Loss ◽

Rheumatic Diseases ◽

Nuclear Factor Kappa B ◽

Inflammatory Diseases ◽

Receptor Activator ◽

Main Factor

Several inflammatory diseases have been associated with increased bone resorption and fracture rates and different studies supported the relation between inflammatory cytokines and osteoclast activity. The main factor required for osteoclast activation is the stimulation by receptor activator of nuclear factor kappa-B ligand (RANKL) expressed on osteoblasts. In this context, interleukin- (IL-) 1β, one of the most powerful proinflammatory cytokines, is a strong stimulator of in vitro and in vivo bone resorption via upregulation of RANKL that stimulates the osteoclastogenesis. The resulting effects lead to an imbalance in bone metabolism favouring bone resorption and osteoporosis. In this paper, we review the available literature on the role of IL-1βin the pathogenesis of bone loss. Furthermore, we analysed the role of IL-1βin bone resorption during rheumatic diseases and, when available, we reported the efficacy of anti-IL-1βtherapy in this field.

Download Full-text

Inhibition of bone resorption in vitro and prevention of ovariectomy-induced bone loss in vivo by flurbiprofen nitroxybutylester (HCT1026)

Arthritis & Rheumatism ◽

10.1002/1529-0131(200109)44:9<2185::aid-art372>3.0.co;2-3 ◽

2001 ◽

Vol 44 (9) ◽

pp. 2185-2192 ◽

Cited By ~ 18

Author(s):

Kenneth J. Armour ◽

Robert J. Van 't Hof ◽

Katharine E. Armour ◽

Anne C. Torbergsen ◽

Piero Del Soldato ◽

...

Keyword(s):

Bone Resorption ◽

Bone Loss

Download Full-text

Osteomacs support osteoclast-mediated resorption and contribute to bone pathology in a postmenopausal osteoporosis mouse model

10.1101/2021.02.05.429872 ◽

2021 ◽

Author(s):

Lena Batoon ◽

Susan M. Millard ◽

Liza J. Raggatt ◽

Andy C. Wu ◽

Simranpreet Kaur ◽

...

Keyword(s):

Bone Loss ◽

Confocal Imaging ◽

Skeletal Health ◽

Post Menopausal Osteoporosis ◽

Delayed Fracture Healing ◽

Osteoclast Function ◽

Post Menopausal ◽

By Products

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.

Download Full-text

Tyrosine Phosphatase Epsilon Is a Positive Regulator of Osteoclast Function in Vitro and In Vivo

Molecular Biology of the Cell ◽

10.1091/mbc.e03-04-0207 ◽

2004 ◽

Vol 15 (1) ◽

pp. 234-244 ◽

Cited By ~ 62

Author(s):

Riccardo Chiusaroli ◽

Hilla Knobler ◽

Chen Luxenburg ◽

Archana Sanjay ◽

Shira Granot-Attas ◽

...

Keyword(s):

Bone Resorption ◽

Bone Metabolism ◽

Tyrosine Phosphatase ◽

Serum Levels ◽

Normal Serum ◽

Bone Degradation ◽

Trabecular Bone Mass ◽

Osteoclast Function

Protein tyrosine phosphorylation is a major regulator of bone metabolism. Tyrosine phosphatases participate in regulating phosphorylation, but roles of specific phosphatases in bone metabolism are largely unknown. We demonstrate that young (<12 weeks) female mice lacking tyrosine phosphatase epsilon (PTPϵ) exhibit increased trabecular bone mass due to cell-specific defects in osteoclast function. These defects are manifested in vivo as reduced association of osteoclasts with bone and as reduced serum concentration of C-terminal collagen telopeptides, specific products of osteoclast-mediated bone degradation. Osteoclast-like cells are generated readily from PTPϵ-deficient bone-marrow precursors. However, cultures of these cells contain few mature, polarized cells and perform poorly in bone resorption assays in vitro. Podosomes, structures by which osteoclasts adhere to matrix, are disorganized and tend to form large clusters in these cells, suggesting that lack of PTPϵ adversely affects podosomal arrangement in the final stages of osteoclast polarization. The gender and age specificities of the bone phenotype suggest that it is modulated by hormonal status, despite normal serum levels of estrogen and progesterone in affected mice. Stimulation of bone resorption by RANKL and, surprisingly, Src activity and Pyk2 phosphorylation are normal in PTPϵ-deficient osteoclasts, indicating that loss of PTPϵ does not cause widespread disruption of these signaling pathways. These results establish PTPϵ as a phosphatase required for optimal structure, subcellular organization, and function of osteoclasts in vivo and in vitro.

Download Full-text

Structural Dissection andIn VivoEffectiveness of a Peptide Inhibitor ofPorphyromonas gingivalisAdherence toStreptococcus gordonii

Infection and Immunity ◽

10.1128/iai.00361-10 ◽

2010 ◽

Vol 79 (1) ◽

pp. 67-74 ◽

Cited By ~ 73

Author(s):

Carlo Amorin Daep ◽

Elizabeth A. Novak ◽

Richard J. Lamont ◽

Donald R. Demuth

Keyword(s):

Bone Resorption ◽

Bone Loss ◽

Alveolar Bone ◽

Alveolar Bone Loss ◽

Peptide Inhibitor ◽

Dental Biofilm ◽

Streptococcal Antigen ◽

Α Helix

ABSTRACTThe interaction of the minor fimbrial antigen (Mfa) with streptococcal antigen I/II (e.g., SspB) facilitates colonization of the dental biofilm byPorphyromonas gingivalis.We previously showed that a 27-mer peptide derived from SspB (designated BAR) resembles the nuclear receptor (NR) box protein-protein interacting domain and potently inhibits this interactionin vitro. Here, we show that the EXXP motif upstream of the NR core α-helix contributes to the Mfa-SspB interaction and that BAR reducesP. gingivaliscolonization and alveolar bone lossin vivoin a murine model of periodontitis. Substitution of Gln for Pro1171or Glu1168increased the α-helicity of BAR and reduced its inhibitory activityin vitroby 10-fold and 2-fold, respectively. To determine if BAR preventsP. gingivalisinfectionin vivo, mice were first infected withStreptococcus gordoniiand then challenged withP. gingivalisin the absence and presence of BAR. Animals that were infected with either 109CFU ofS. gordoniiDL-1 or 107CFU ofP. gingivalis33277 did not show a statistically significant increase in alveolar bone resorption over sham-infected controls. However, infection with 109CFU ofS. gordoniifollowed by 107CFU ofP. gingivalisinduced significantly greater bone loss (P< 0.01) than sham infection or infection of mice with either organism alone.S. gordonii-infected mice that were subsequently challenged with 107CFU ofP. gingivalisin the presence of BAR exhibited levels of bone resorption similar to those of sham-infected animals. Together, these results indicate that both EXXP and the NR box are important for the Mfa-SspB interaction and that BAR peptide represents a potential therapeutic that may limit colonization of the oral cavity byP. gingivalis.

Download Full-text