Osteoclast Resorptive Activity Research Articles

Abstract 2886: Osteoclast and neuronal cross-talk as a mechanism for chest-wall pain caused by thoracic radiosurgery for lung cancer

Abstract Radiation therapy (RT)-induced chest wall pain (CWP) is a severe complication after thoracic stereotactic body radiation therapy (SBRT) for lung tumors; with increased survivorship, ~30% of patients develop severe CWP. The causes of CWP are unknown, but could involve: 1] damage to intercostal peripheral nerves; 2] bone loss from affected osteoclast activity, or 3] both (secreted factors from activated osteoclasts can affect nerves). This study tested the potential for radiation to affect osteoclasts and/or sensory neurons to induce pain. Mature osteoclasts differentiated from RAW264.7 cells plated on Osteoassay surfaces were exposed to an acute 10 Gy Cs137 γ-rays and bone resorption was assessed. We collected conditioned media (CM) from these cells at 48h and applied to cultured primary mouse dorsal root ganglia neurons (irradiated and non-irradiated neuron), assessing neurite growth and pain-associated marker (CGRP, SP) expression through immunohistochemistry and RT-qPCR. Risedronate (50µM), an anti-resorptive agent, was administered pre-radiation to inhibit osteoclast activity. Unpaired t-tests were utilized for statistical analyses (α=0.05). Osteoclast resorption activity was increased by 48h after irradiation. These irradiated, activated osteoclasts exhibited increased mRNA expression of differentiation (RANK) and activity (CTSK, MMP9) markers. Neurons treated with CM from irradiated osteoclasts exhibited elevated levels of CGRP (+38.5%) and SP (+29.5%) compared to cells treated with CM from non-irradiated cells. Consistently, in co-cultures of osteoclasts and neurons exposed to radiation, irradiated neurons exhibited significantly increased neuropeptide levels. Risedronate not only reduced osteoclast activity but also inhibited neuropeptide expression in irradiated neurons within these co-cultures. Moreover, neuropeptide levels were similar between directly irradiated neurons and non-irradiated neurons. However, irradiated neurons exposed to CM from either irradiated or non-irradiated osteoclasts showed a marked 4-fold increase in neuropeptide levels vs neurons that were non-irradiated and then received osteoclast CM. Our findings indicate that radiation activates osteoclasts and leads to direct bone loss in vitro and signaling between irradiated osteoclasts and non-irradiated neurons increases pain responses. Importantly, this response is enhanced when neurons were also previously irradiated. During SBRT, intercostal nerves may be influenced by adjacent irradiated osteoclasts, increasing expression of pain-related neuropeptides as the osteoclasts break down bone, thus enhancing the risk of CWP as treatment progresses across time. Understanding these interactions could reveal potential targets for preventing CWP, allowing higher tumor radiation doses with less toxicity. Support: The Milton Raben Foundation. Citation Format: Sun Park, Megan Peters, Joseph Moore, Kaitlyn Reno, Michael Farris, Christopher Peters, Jeffery Willey. Osteoclast and neuronal cross-talk as a mechanism for chest-wall pain caused by thoracic radiosurgery for lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2886.

The PDE4 Inhibitors Roflumilast and Rolipram Rescue ADO2 Osteoclast Resorption Dysfunction.

Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption caused by heterozygous missense mutations in the chloride channel 7 (CLCN7). Adenylate cyclase, which catalyzes the formation of cAMP, is critical for lysosomal acidification in osteoclasts. We found reduced cAMP levels in ADO2 osteoclasts compared to wild-type (WT) osteoclasts, leading us to examine whether regulating cAMP would improve ADO2 osteoclast activity. Although forskolin, a known activator of adenylate cyclase and cAMP levels, negatively affected osteoclast number, it led to an overall increase in ADO2 and WT osteoclast resorption activity in vitro. Next, we examined cAMP hydrolysis by the phosphodiesterase 4 (PDE4) proteins in ADO2 versus WT osteoclasts. QPCR analysis revealed higher expression of the three major PDE4 subtypes (4a, 4b, 4d) in ADO2 osteoclasts compared in WT, consistent with reduced cAMP levels in ADO2 osteoclasts. In addition, we found that the PDE4 antagonists, rolipram and roflumilast, stimulated ADO2 and WT osteoclast formation in a dose-dependent manner. Importantly, roflumilast and rolipram displayed a concentration-dependent increase in osteoclast resorption activity which was greater in ADO2 than WT osteoclasts. Moreover, treatment with roflumilast rescued cAMP levels in ADO2 OCLs. The key findings from our studies demonstrate that osteoclasts from ADO2 mice exhibit reduced cAMP levels and PDE4 inhibition rescues cAMP levels and ADO2 osteoclast activity dysfunction in vitro. The mechanism of action of PDE4 inhibitors and their ability to reduce the high bone mass of ADO2 mice in vivo are currently under investigation. Importantly, these studies advance the understanding of the mechanisms underlying the ADO2 osteoclast dysfunction which is critical for the development of therapeutic approaches to treat clinically affected ADO2 patients.

Murine IRF8 Mutation Offers New Insight into Osteoclast and Root Resorption.

Interferon regulatory factor 8 (IRF8), a transcription factor expressed in immune cells, functions as a negative regulator of osteoclasts and helps maintain dental and skeletal homeostasis. Previously, we reported that a novel mutation in the IRF8 gene increases susceptibility to multiple idiopathic cervical root resorption (MICRR), a form of tooth root resorption mediated by increased osteoclast activity. The IRF8 G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. To investigate the molecular basis of MICRR and IRF8 function in osteoclastogenesis, we generated Irf8 knock-in (KI) mice using CRISPR/Cas9 technique modeling the human IRF8G388S mutation. The heterozygous (Het) and homozygous (Homo) Irf8 KI mice showed no gross morphological defects, and the development of hematopoietic cells was unaffected and similar to wild-type (WT) mice. The Irf8 KI Het and Homo mice showed no difference in macrophage gene signatures important for antimicrobial defenses and inflammatory cytokine production. Consistent with the phenotype observed in MICRR patients, Irf8 KI Het and Homo mice demonstrated significantly increased osteoclast formation and resorption activity in vivo and in vitro when compared to WT mice. The oral ligature-inserted Het and Homo mice displayed significantly increased root resorption and osteoclast-mediated alveolar bone loss compared to WT mice. The increased osteoclastogenesis noted in KI mice is due to the inability of IRF8G388S mutation to inhibit NFATc1-dependent transcriptional activation and downstream osteoclast specific transcripts, as well as its impact on autophagy-related pathways of osteoclast differentiation. This translational study delineates the IRF8 domain important for osteoclast function and provides novel insights into the IRF8 mutation associated with MICRR. IRF8G388S mutation mainly affects osteoclastogenesis while sparing immune cell development and function. These insights extend beyond oral health and significantly advance our understanding of skeletal disorders mediated by increased osteoclast activity and IRF8's role in osteoclastogenesis.

Inhibition of receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation and bone resorption by Gryllus bimaculatus extract: An in vitro study.

Excessive bone-resorbing osteoclast activity during bone remodeling is a major feature of bone diseases, such as osteoporosis. Therefore, the inhibition of osteoclast formation and bone resorption can be an effective therapeutic target for various bone diseases. Gryllus biomaculatus (GB) has recently been approved as an alternative food source because of its high nutritional value and environmental sustainability. Traditionally, GB has been known to have various pharmacological properties, including antipyretic and blood pressure-lowering activity, and it has recently been reported to have various biological activities, including protective effects against inflammation, oxidative stress, insulin resistance, and alcohol-induced liver injury. However, the effect of GB on osteoclast differentiation and bone metabolism has not yet been demonstrated. In this study, we confirmed the inhibitory effect of GB extract (GBE) on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. To determine the effect of GBE on RANKL-induced osteoclast differentiation and function, we performed TRAP and F-actin staining, as well as a bone-resorbing assay. The intracellular mechanisms of GBE responsible for the regulation of osteoclastogenesis were revealed by Western blot analysis and quantitative real-time polymerase chain reaction. We investigated the relationship between GBE and expression of osteoclast-specific molecules to further elucidate the underlying mechanisms. It was found that GBE significantly suppressed osteoclastogenesis by decreasing the phosphorylation of Akt, p38, JNK, and ERK, as well as Btk-PLCγ2 signaling, in pathways involved in early osteoclastogenesis as well as through the subsequent suppression of c-Fos, NFATc1, and osteoclastogenesis-specific marker genes. Additionally, GBE inhibited the formation of F-actin ring-positive osteoclasts and bone resorption activity of mature osteoclasts. Our findings suggest that GBE is a potential functional food and therapeutic candidate for bone diseases involving osteoclasts.

Osteoclast-specific Plastin 3 knockout in mice fail to develop osteoporosis despite dramatic increased osteoclast resorption activity.

PLS3 loss-of-function mutations in humans and mice cause X-linked primary osteoporosis. However, it remains largely unknown how PLS3 mutations cause osteoporosis and which function PLS3 plays in bone homeostasis. A recent study showed that ubiquitous Pls3 KO in mice results in osteoporosis. Mainly osteoclasts were impacted in their function However, it has not been proven if osteoclasts are the major cell type affected and responsible for osteoporosis development in ubiquitous Pls3 KO mice. Here, we generated osteoclast-specific Pls3 KO mice. Additionally, we developed a novel polyclonal PLS3 antibody that showed specific PLS3 loss in immunofluorescence staining of osteoclasts in contrast to previously available antibodies against PLS3, which failed to show PLS3 specificity in mouse cells. Moreover, we demonstrate that osteoclast-specific Pls3 KO causes dramatic increase in resorptive activity of osteoclasts in vitro. Despite these findings, osteoclast-specific Pls3 KO in vivo failed to cause any osteoporotic phenotype in mice as proven by micro-CT and three-point bending test. This demonstrates that the pathomechanism of PLS3-associated osteoporosis is highly complex and cannot be reproduced in a system singularly focused on one cell type. Thus, the loss of PLS3 in alternative bone cell types might contributes to the osteoporosis phenotype in ubiquitous Pls3 KO mice.

Evidence that pyrophosphate acts as an extracellular signalling molecule to exert direct functional effects in primary cultures of osteoblasts and osteoclasts.

Extracellular pyrophosphate (PPi) is well known for its fundamental role as a physiochemical mineralisation inhibitor. However, information about its direct actions on bone cells remains limited. This study shows that PPi decreased osteoclast formation and resorptive activity by ≤50%. These inhibitory actions were associated with reduced expression of genes involved in osteoclastogenesis (Tnfrsf11a, Dcstamp) and bone resorption (Ctsk, Car2, Acp5). In osteoblasts, PPi present for the entire (0-21days) or latter stages of culture (7-21/14-21days) decreased bone mineralisation by ≤95%. However, PPi present for the differentiation phase only (0-7/0-14days) increased bone formation (≤70%). Prolonged treatment with PPi resulted in earlier matrix deposition and increased soluble collagen levels (≤2.3-fold). Expression of osteoblast (RUNX2, Bglap) and early osteocyte (E11, Dmp1) genes along with mineralisation inhibitors (Spp1, Mgp) was increased by PPi (≤3-fold). PPi levels are regulated by tissue non-specific alkaline phosphatase (TNAP) and ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). PPi reduced NPP1 expression in both cell types whereas TNAP expression (≤2.5-fold) and activity (≤35%) were increased in osteoblasts. Breakdown of extracellular ATP by NPP1 represents a key source of PPi. ATP release from osteoclasts and osteoblasts was decreased ≤60% by PPi and by a selective TNAP inhibitor (CAS496014-12-2). Pertussis toxin, which prevents Gαi subunit activation, was used to investigate whether G-protein coupled receptor (GPCR) signalling mediates the effects of PPi. The actions of PPi on bone mineralisation, collagen production, ATP release, gene/protein expression and osteoclast formation were abolished or attenuated by pertussis toxin. Together these findings show that PPi, modulates differentiation, function and gene expression in osteoblasts and osteoclasts. The ability of PPi to alter ATP release and NPP1/TNAP expression and activity indicates that cells can detect PPi levels and respond accordingly. Our data also raise the possibility that some actions of PPi on bone cells could be mediated by a Gαi-linked GPCR.

2,5-Dihydroxyacetophenone attenuates RANKL-mediated osteoclastogenesis by affecting the NFATc1 signaling pathway in vitro

Excessive bone resorption activity of osteoclasts is a common characteristic of osteolytic conditions such as osteoporosis and inflammatory bone diseases. Natural compounds with antiosteoclastogenic function seem to be beneficial for the treatment of osteolytic diseases. In this study, we evaluated the effects of 2,5-dihydroxyacetophenone (DHAP), a phenolic compound in Ganoderma bambusicola, on osteoclastogenesis induced in vitro by the receptor activator of nuclear factor-κB ligand (RANKL). DHAP inhibited the differentiation, actin ring formation, and bone resorption activity of osteoclasts. In particular, DHAP inhibited the transcriptional activity of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) during osteoclastogenesis. This inhibition resulted in reduced expression levels of cathepsin k (Ctsk), tartrate-resistant acid phosphatase (Trap), and NFATc1 (Nfatc1), thereby reducing the differentiation of osteoclasts. However, DHAP did not affect reactive oxygen species production or activator protein 1 (AP-1) and nuclear factor kappa B (NF-κB) signaling. Our findings suggest that DHAP inhibits RANKL-induced osteoclastogenesis by inhibiting the NFATc1 signaling pathway.

FRI673 REMS Technology For The Evaluation Of BMD In Hypothyroidism

Abstract Disclosure: F.A. Lombardi: None. P. Pisani*: None. A.P. Quezada*: None. M. Muratore*: None. M. Di Paola*: None. E. Casciaro*: None. F. Conversano*: None. S. Casciaro: None. *Equal contributors in alphabetical order Thyroid hormones play a crucial role in skeletal development, adult bone turnover, and bone maintenance, but they can also have detrimental effects on bone formation when there is thyroid dysfunction. Thyrotoxicosis (hyperthyroidism), including subclinical illness, can affect bone metabolism, resulting in decreased Bone Mineral Density (BMD) and increased fractures risk, and is a significant contributor to secondary osteoporosis, especially in postmenopausal women. On the other hand, little is known about the impact of overt or subclinical hypothyroidism on bone metabolism. However, hypothyroidism lessens bone turnover, reducing both osteoclastic bone resorption and osteoblastic activity. Such alterations in bone metabolism could potentially result in an increase in bone mineralization, although data on BMD and the risk of fracture in hypothyroidism are lacking and conflicting. Radiofrequency Echographic Multi-spectrometry (REMS) is a novel radiation-free technology for assessing BMD and fracture risk. In this context, the goal of this study was to assess BMD using REMS in patients with and without hypothyroidism, in order to look a possible correlation between the disease, BMD loss and fracture risk. Methods: A sample of 51 Caucasian females with hypothyroidism was compared to a reference group of healthy women who were matched for gender, ethnicity, age, and body mass index (BMI). Using REMS technology, the BMD was measurement at the lumbar spine in all participants at the study. To determine whether the two groups’ lumbar spine BMD value varied, a t-test was run. Results: Patients with and without hypothyroidism were matched in order to get similar anthropometric parameters. In patient with and without hypothyroidism mean ± standard deviation (SD) for age and BMI are (64.8 ± 10.2, 65.2 ± 9.8) and (25.1 ± 3.6, 25.0 ± 3.7), respectively. Patients with hypothyroidism were shown to have a significative lumbar spine BMD difference (0.801 g/cm2 ± 0.063 vs 0.831 g/cm2 ± 0.071, p=0.03) respect to the healthy subjects. Conclusions: Although just a small number of hypothyroidism patients are included in this investigation, without any indication of the drug therapy and its effect, it has been shown that, as compared to controls, women with hypothyroidism have a net decline in REMS-measured BMD values. Based on this thought, REMS technology, thanks to its precision and diagnostic accuracy, might improve the efficient care and follow-up programmes of these patients. Presentation: Friday, June 16, 2023

Cilengitide inhibits osteoclast adhesion through blocking the αvβ3-mediated FAK/Src signaling pathway

The remodeling of actin cytoskeleton of osteoclasts on the bone matrix is essential for osteoclastic resorption activity. A specific regulator of the osteoclast cytoskeleton, integrin αvβ3, is known to provide a key role in the degradation of mineralized bone matrixes. Cilengitide is a potent inhibitor of integrins and is capable of affecting αvβ3 receptors, and has anti-tumor and anti-angiogenic and apoptosis-inducing effects. However, its function on osteoclasts is not fully understood. Here, the cilengitide role on nuclear factor κB ligand-receptor activator (RANKL)-induced osteoclasts was explored. Cells were cultured with varying concentrations of cilengitide (0,0.002,0.2 and 20 μM) for 7 days, followed by detected via Cell Counting Kit-8, staining for tartrate resistant acid phosphatase (TRAP), F-actin ring formation, bone resorption assays, adhesion assays, immunoblotting assays, and real-time fluorescent quantitative PCR. Results demonstrated that cilengitide effectively restrained the functionality and formation of osteoclasts in a concentration-dependent manner, without causing any cytotoxic effects. Mechanistically, cilengitide inhibited osteoclast-relevant genes expression; meanwhile, cilengitide downregulated the expression of key signaling molecules associated with the osteoclast cytoskeleton, including focal adhesion kinase (FAK), integrin αvβ3 and c-Src. Therefore, this results have confirmed that cilengitide regulates osteoclast activity by blocking the integrin αvβ3 signal pathway resulting in diminished adhesion and bone resorption of osteoclasts.

DNA damage-inducible transcript 3 deficiency promotes bone resorption in murine periodontitis models.

Periodontitis is a multifactorial inflammatory disease that leads to the destruction of supporting structures of the teeth. DNA damage-inducible transcript 3 (DDIT3) plays crucial roles in cell survival and differentiation. DDIT3 regulates bone mass and osteoclastogenesis in femur. However, the role of DDIT3 in periodontitis has not been elucidated. This research aimed to explore the role and mechanisms of DDIT3 in periodontitis. DDIT3 gene knockout (KO) mice were generated using a CRISPR/Cas9 system. Experimental periodontitis models were established to explore the role of DDIT3 in periodontitis. The expression of DDIT3 in periodontal tissues was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). The alveolar bone phenotypes were observed by micro-CT and stereomicroscopy. The inflammation levels and osteoclast activity were examined by histological staining, immunostaining, and qRT-PCR. Bone marrow-derived macrophages (BMMs) were isolated to confirm the effects of DDIT3 on osteoclast formation and function in vitro. The increased expression of DDIT3 in murine inflamed periodontal tissues was detected. DDIT3 knockout aggravated alveolar bone loss and enhanced expression levels of inflammatory cytokines in murine periodontitis models. Increased osteoclast formation and higher expression levels of osteoclast-specific markers were observed in the inflamed periodontal tissues of KO mice. In vitro, DDIT3 deficiency promoted the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts and the bone resorption activity of mature osteoclasts. Our results demonstrate that DDIT3 deletion aggravated alveolar bone loss in experimental periodontitis through enhanced inflammatory reactions and osteoclastogenesis. The anti-inflammation and the inhibition of bone loss by DDIT3 in murine periodontitis provides a potential novel therapeutic strategy for periodontitis.

Casticin suppresses RANKL‑induced osteoclastogenesis and prevents ovariectomy‑induced bone loss by regulating the AKT/ERK and NF‑κB signaling pathways.

Postmenopausal osteoporosis is a systemic metabolic disease that chronically endangers public health and is typically characterized by low bone mineral density and marked bone fragility. The excessive bone resorption activity of osteoclasts is a major factor in the pathogenesis of osteoporosis; therefore, strategies aimed at inhibiting osteoclast activity may prevent bone decline and attenuate the process of osteoporosis. Casticin (Cas), a natural compound, has anti‑inflammatory and antitumor properties. However, the role of Cas in bone metabolism remains largely unclear. The present study found that the receptor activator of nuclear factor‑κΒ (NF‑κB) ligand‑induced osteoclast activation and differentiation were inhibited by Cas. Tartrate‑resistant acid phosphatase staining revealed that Cas inhibited osteoclast differentiation, and bone resorption pit assays demonstrated that Cas affected the function of osteoclasts. Cas significantly reduced the expression of osteoclast‑specific genes and related proteins, such as nuclear factor of activated T cells, cytoplasmic 1 and c‑Fos at the mRNA and protein level in a concentration‑dependent manner. Cas inhibited osteoclast formation by blocking the AKT/ERK and NF‑κB signaling pathways, according to the intracellular signaling analysis. The microcomputed tomography and tissue staining of tibiae from ovariectomized mice revealed that Cas prevented the bone loss induced by estrogen deficiency and reduced osteoclast activity invivo. Collectively, these findings indicated that Cas may be used to prevent osteoporosis.

Abstract 2481: Human osteoclast assays for studying the effects of bone metastasis treatments with different mechanisms of action

Abstract Metastasis to bone is one of the common complications associated with different types of advanced cancer. Zoledronic acid, a third generation nitrogen containing bisphosphonate, accumulates in bones where it binds to hydroxyapatite crystals. For years, zoledronic acid has been regarded as the standard of care for patients with advanced cancer and bone metastasis to prevent skeletal complications. As an alternative therapeutic option, denosumab has also been found to be effective in delaying skeletal-related events in advanced cancer. Denosumab, a human monoclonal antibody against receptor activator of nuclear factor kappa-B ligand (RANKL), blocks the RANKL/RANK signalling pathway. In addition to the skeletal effects, both zoledronic acid and denosumab have shown direct effect on cancer cells. In this study, effects of zoledronic acid and denosumab on osteoclast differentiation and bone resorption activity were studied using human CD34+ osteoclast precursor cells that were cultured on bovine bone slices in 96-well plates for 7 days in the presence of RANKL and M-CSF, allowing them to differentiate into mature osteoclasts. In the osteoclast differentiation assay, zoledronic acid (0.0001 - 1 000 µM) and denosumab (0.01 - 10 µg/ml) were added into the culture medium at day 0. Tartrate-resistant acid phosphatase 5b (TRACP 5b) activity was measured in the culture medium at day 7 as an index of osteoclast differentiation. In the osteoclast activity assay, the test compounds were added into the culture medium at day 7, and the formed osteoclasts were allowed to resorb bone for 3 days. C-terminal crosslinked telopeptides of type I collagen (CTX-I) was measured in the culture medium at day 10 as an index of bone resorption. Denosumab and zoledronic acid showed strong concentration dependent inhibition of osteoclast differentiation and resorption activity. We conclude that this osteoclast culture system can be used for identifying new potential compounds affecting the disease process of bone metastases with different mechanisms of action. The manifold effects of zoledronic acid and denosumab on cancer cells remain to be studied in more detail. Citation Format: Katja M. Fagerlund, Mervi Ristola, Mari I. Suominen, Jenni H. Mäki-Jouppila, Jukka P. Rissanen. Human osteoclast assays for studying the effects of bone metastasis treatments with different mechanisms of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2481.

The Secretome of Parental and Bone Metastatic Breast Cancer Elicits Distinct Effects in Human Osteoclast Activity after Activation of β2 Adrenergic Signaling.

The sympathetic nervous system (SNS), particularly through the β2 adrenergic receptor (β2-AR), has been linked with breast cancer (BC) and the development of metastatic BC, specifically in the bone. Nevertheless, the potential clinical benefits of exploiting β2-AR antagonists as a treatment for BC and bone loss-associated symptoms remain controversial. In this work, we show that, when compared to control individuals, the epinephrine levels in a cohort of BC patients are augmented in both earlier and late stages of the disease. Furthermore, through a combination of proteomic profiling and functional in vitro studies with human osteoclasts and osteoblasts, we demonstrate that paracrine signaling from parental BC under β2-AR activation causes a robust decrease in human osteoclast differentiation and resorption activity, which is rescued in the presence of human osteoblasts. Conversely, metastatic bone tropic BC does not display this anti-osteoclastogenic effect. In conclusion, the observed changes in the proteomic profile of BC cells under β-AR activation that take place after metastatic dissemination, together with clinical data on epinephrine levels in BC patients, provided new insights on the sympathetic control of breast cancer and its implications on osteoclastic bone resorption.

GIP reduces osteoclast activity and improves osteoblast survival in primary human bone cells.

Drugs targeting the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) are emerging as treatments for type-2 diabetes and obesity. GIP acutely decreases serum markers of bone resorption and transiently increases bone formation markers in short-term clinical investigations. However, it is unknown whether GIP acts directly on bone cells to mediate these effects. Using a GIPR-specific antagonist, we aimed to assess whether GIP acts directly on primary human osteoclasts and osteoblasts. Osteoclasts were differentiated from human CD14+ monocytes and osteoblasts from human bone. GIPR expression was determined using RNA-seq in primary human osteoclasts and in situ hybridization in human femoral bone. Osteoclastic resorptive activity was assessed using microscopy. GIPR signaling pathways in osteoclasts and osteoblasts were assessed using LANCE cAMP and AlphaLISA phosphorylation assays, intracellular calcium imaging and confocal microscopy. The bioenergetic profile of osteoclasts was evaluated using Seahorse XF-96. GIPR is robustly expressed in mature human osteoclasts. GIP inhibits osteoclastogenesis, delays bone resorption, and increases osteoclast apoptosis by acting upon multiple signaling pathways (Src, cAMP, Akt, p38, Akt, NFκB) to impair nuclear translocation of nuclear factor of activated T cells-1 (NFATc1) and nuclear factor-κB (NFκB). Osteoblasts also expressed GIPR, and GIP improved osteoblast survival. Decreased bone resorption and improved osteoblast survival were also observed after GIP treatment of osteoclast-osteoblast co-cultures. Antagonizing GIPR with GIP(3-30)NH2 abolished the effects of GIP on osteoclasts and osteoblasts. GIP inhibits bone resorption and improves survival of human osteoblasts, indicating that drugs targeting GIPR may impair bone resorption, whilst preserving bone formation.

Compressed Prostate Cancer Cells Decrease Osteoclast Activity While Enhancing Osteoblast Activity In Vitro.

Once prostate cancer cells metastasize to bone, they perceive approximately 2 kPa compression. We hypothesize that 2 kPa compression stimulates the epithelial-to-mesenchymal transition (EMT) of prostate cancer cells and alters their production of paracrine signals to affect osteoclast and osteoblast behavior. Human DU145 prostate cancer cells were subjected to 2 kPa compression for 2 days. Compression decreased expression of 2 epithelial genes, 5 out of 13 mesenchymal genes, and increased 2 mesenchymal genes by DU145 cells, as quantified by qPCR. Conditioned medium (CM) of DU145 cells was added to human monocytes that were stimulated to differentiate into osteoclasts for 21 days. CM from compressed DU145 cells decreased osteoclast resorptive activity by 38% but did not affect osteoclast size and number compared to CM from non-compressed cells. CM was also added to human adipose stromal cells, grown in osteogenic medium. CM of compressed DU145 cells increased bone nodule production (Alizarin Red) by osteoblasts from four out of six donors. Compression did not affect IL6 or TNF-α production by PC DU145 cells. Our data suggest that compression affects EMT-related gene expression in DU145 cells, and alters their production of paracrine signals to decrease osteoclast resorptive activity while increasing mineralization by osteoblasts is donor dependent. This observation gives further insight in the altered behavior of PC cells upon mechanical stimuli, which could provide novel leads for therapies, preventing bone metastases.

Research Progress on Mechanism and Application of Zinc in Osteogenesis

As the second most abundant trace metal element in the human body, zinc plays an important role in bone homeostasis and bone disease treatment. Adequate amounts of zinc not only mediates osteogenesis-related pathways through zinc dependent enzymes and zinc finger proteins to induce the differentiation of mesenchymal stem cells into osteoblasts and promote osteogenesis, but also inhibits osteoclast resorption activity, reducing bone resorption and thus promoting osteogenesis. In recent years, the importance of zinc in bone development, regeneration and homeostasis has been gradually discovered. The mechanism and application of zinc's osteogenic effect have been extensively studied. The aim of this paper is to summarize the research on zinc osteogenic mechanism and review its application research in bone tissue engineering in recent years.

Osteoblast-Mediated Resorption of Porous Bioactive SCPC Granules Enhances Bone Regeneration in Human Extraction Sockets

Bone graft materials are widely used in orthopedic and maxillofacial surgeries. The controlled resorbability of the graft material is essential for bone regeneration. Hydroxyapatite and biphasic calcium phosphate bone grafts have poor resorption and limited bone conductive effects. Histology analyses of bone biopsy from SCPC grafted human extraction sockets showed complete bone regeneration and graft resorption in absence of osteoclasts and macrophages. The hypothesis of the present study is that bioactive SCPC inhibits osteoclast’s activity due to the presence of resorbable silica phase in the material. Our objective is to analyze the effect of SCPC dissolution products on the resorption activity of osteoclasts. The conditioned medium was prepared by immersion of SCPC resorbable bioactive SCPC porous granules (Shefabone, Inc, USA) in cell culture medium at various ratios at 37°C for 3 days. The concentration of Si ions released from the SCPC granules into cell culture medium was measured using ICP-OES. Osteoclast precursors derived from human bone marrow were seeded on bone slices and cultured in the conditioned medium containing 10% FBS and osteoclast induction factors. Osteoclast differentiation and resorption were evaluated by TRAP staining and measurement of the volume of resorption pits on the bone slices. Mature multinuclear giant TRAP-positive osteoclasts were observed on the bone substrates after 14 days incubation in control medium containing osteoclast induction factors. In conditioned medium, the number of multinuclear TRAP-positive cells was significantly decreased as the concentration of SCPC dissolved silica increased. The dissolution of silica from SCPC into the culture medium correlates well with down regulation of osteoclast differentiation and the rapid bone regeneration in human bone defects.

Avicularin Alleviates Osteoporosis in Ovariectomized Mice by Inhibiting Osteoclastogenesis through NF-κB Pathway Inhibition.

Osteoporosis (OP) is mainly manifested by bone loss and bone degeneration. OP is considered a risk factor for pathological fractures, as well as impacts the health of middle-aged and elderly individuals. Drug therapy remains the main treatment scheme for OP; however, its efficacy is limited and has been associated with serious side effects. Therefore, it is important to develop new, effective, and safe treatment methods for OP. Avicularin (AL) is a flavonoid and quercetin derivative from various plants. Our study showed that AL disrupts osteoclast activation and resorptive function via inhibition of the RANKL-induced osteoclast differentiation together with the resorption capacity of bone marrow-derived macrophages (BMMs). Hence, AL prevents the activation and resorptive activity of osteoclasts. The results of qPCR showed that genes related to osteoclasts exhibited downregulated expression after AL treatment. Furthermore, AL inhibited RANKL-induced phosphorylation as well as degradation of the inhibitor IκBα of the NF-κB pathway, together with P65 phosphorylation in BMMs. We used an OP mouse model that was established by ovariectomy (OVX). Relative to untreated OP mice, mice that received AL treatment showed a significant increase in bone mineral density; however, the expression of TRAP, NFATC1, mmp9, and CTX-1 was significantly reduced. These results indicate that AL disrupts osteoclastogenesis via inhibition of the NF-κB pathway, which in turn improves OVX-induced OP.

The effects of seeding density and osteoclastic supplement concentration on osteoclastic differentiation and resorption

The bone resorbing osteoclasts are a complex type of cell essential for in vivo bone remodeling. There is no consensus on medium composition and seeding density for in vitro osteoclastogenesis, despite the importance thereof on osteoclastic differentiation and activity. The aim of this study was to investigate the relative effect of monocyte or peripheral blood mononuclear cell (PBMC) seeding density, osteoclastic supplement concentration and priming on the in vitro generation of functional osteoclasts, and to explore and evaluate the usefulness of commonly used markers for osteoclast cultures. Morphology and osteoclast formation were analyzed with fluorescence imaging for tartrate resistant acid phosphatase (TRAP) and integrin β3 (Iβ3). TRAP release was analyzed from supernatant samples, and resorption was analyzed from culture on Corning® Osteo Assay plates. In this study, we have shown that common non-standardized culturing conditions of monocyte or PBMCs had a significant effect on the in vitro generation of functional osteoclasts. We showed how increased osteoclastic supplement concentrations supported osteoclastic differentiation and resorption but not TRAP release, while priming resulted in increased TRAP release as well. Increased monocyte seeding densities resulted in more and large TRAP positive bi-nuclear cells, but not directly in more multinucleated osteoclasts, resorption or TRAP release. Increasing PBMC seeding densities resulted in more and larger osteoclasts and more resorption, although resorption was disproportionally low compared to the monocyte seeding density experiment. Exploration of commonly used markers for osteoclast cultures demonstrated that Iβ3 staining was an excellent and specific osteoclast marker in addition to TRAP staining, while supernatant TRAP measurements could not accurately predict osteoclastic resorptive activity. With improved understanding of the effect of seeding density and osteoclastic supplement concentration on osteoclasts, experiments yielding higher numbers of functional osteoclasts can ultimately improve our knowledge of osteoclasts, osteoclastogenesis, bone remodeling and bone diseases.

Astragalin mitigates inflammatory osteolysis by negatively modulating osteoclastogenesis via ROS and MAPK signaling pathway

Inflammatory bone destruction has gradually attracted attention worldwide and has been observed in several kinds of pathological bone diseases, such as osteoarthritis, osteomyelitis, rheumatic arthritis, and other infectious clinical trials in the skeletal system. In this regard, excessive osteoclasts and bone resorption activity participate in osteolytic processes. Thus, negatively modulating osteoclast differentiation and bone erosion has been considered an effective therapeutic strategy to limit the poor progression of inflammatory osteolysis. Astragalin (AST) is a bioactive component of traditional Chinese drugs, such as Rosa agrestis, which presents anti-inflammatory and antioxidant effects. However, it is unclear how AST may play an essential role in regulating the dynamic balance of the bone matrix by affecting osteoclastogenesis. This study found that AST could inhibit osteoclastic formation and bone resorption activity in a dose‐dependent manner without cytotoxicity. Administration of AST also inhibited the expression of cathepsin K, c-Fos, NFATc1, and TRAP at different stages of mRNA and protein levels during osteoclastogenesis. Reactive oxygen species (ROS) signalling could also be modulated by treatment with AST during RANKL-induced osteoclast differentiation through the Nrf2-HO1 signalling pathway. Additionally, AST could negatively regulate mitogen-activated protein kinase (MAPK) signalling in this process. In vivo, AST significantly reduced lipopolysaccharide (LPS)-induced bone loss in an osteolytic mouse model. AST might be a promising therapeutic candidate for treating osteolytic bone diseases in the future.