Osteoclast Proliferation Research Articles

Cell migration within porous electrospun nanofibrous scaffolds in a mouse subcuticular implantation model.

Cellular infiltration into electrospun nanofibers (NFs) is limited due to the dense structure and small pore sizes. We developed a programmed NF collector that can fabricate porous NFs with desired pore sizes and thickness. Previously we demonstrated improved cellular proliferation and differentiation of osteoblasts, osteoclasts, and fibroblasts with increased pore sizes of polycaprolactone (PCL) NF in-vitro. This study investigated in-vivo host cell migration and vascular ingrowth within porous NF sheets implanted subcutaneously in a mouse model. Two types of PCL NFs with well-defined pore sizes were created using varying speeds of the NF collector: NF-zero (no movement, pore size 14.4 ± 8.9 µm2) and NF-high (0.232 mm/min, pore size 286.7 ± 381.9 µm2). The NF obtained by using classical flat NF collector (2D NF, pore size 1.09 ± 1.7 µm2) was a control. The three formulae of NFs were implanted subcutaneously in 18 BALB/cJ mice. Animals were killed 7 and 28-days after implantation (n = 3 per group per time point). The tissue with implanted NFs were collected for histologic analysis. Overall, 7-day samples showed little inflammatory response. At 28-days, the degree of tissue penetration of PCL NF sheet matrices was linked to pore size and area. NFs with the largest pore area had more efficient tissue migration and new blood vessel formation compared to those with smaller pore sizes. No newly formed blood vessels were observed in the 2D NF group. A porous NF scaffold with controllable pore size has potential for tissue repair/regeneration in situ with potential for many applications in orthopaedics.

Role of oxytocin in bone.

Oxytocin (OT) is a posterior pituitary hormone that, in addition to its role in regulating childbirth and lactation, also exerts direct regulatory effects on the skeleton through peripheral OT and oxytocin receptor (OTR). Bone marrow mesenchymal stem cells (BMSCs), osteoblasts (OB), osteoclasts (OC), chondrocytes, and adipocytes all express OT and OTR. OT upregulates RUNX2, BMP2, ALP, and OCN, thereby enhancing the activity of BMSCs and promoting their differentiation towards OB rather than adipocytes. OT also directly regulates OPG/RANKL to inhibit adipocyte generation, increase the expression of SOX9 and COMP, and enhance chondrocyte differentiation. OB can secrete OT, exerting influence on the surrounding environment through autocrine and paracrine mechanisms. OT directly increases OC formation through the NκB/MAP kinase signaling pathway, inhibits osteoclast proliferation by triggering cytoplasmic Ca2+ release and nitric oxide synthesis, and has a dual regulatory effect on OCs. Under the stimulation of estrogen, OB synthesizes OT, amplifying the biological effects of estrogen and OT. Mediated by estrogen, the OT/OTR forms a feedforward loop with OB. Apart from estrogen, OT also interacts with arginine vasopressin (AVP), prostaglandins (PGE2), leptin, and adiponectin to regulate bone metabolism. This review summarizes recent research on the regulation of bone metabolism by OT and OTR, aiming to provide insights into their clinical applications and further research.

Kurarinone, a flavonoid from Radix Sophorae Flavescentis, inhibits RANKL-induced osteoclastogenesis in mouse bone marrow-derived monocyte/macrophages.

Inflammation-induced osteoclast proliferation is a crucial contributor to impaired bone metabolism. Kurarinone (KR), a flavonoid extracted from the Radix Sophorae Flavescentis, exhibits notable anti-inflammatory properties. Nevertheless, the precise influence of KR on osteoclast formation remains unclear. This study's objective was to assess the impact of KR on osteoclast activity in vitro and unravel its underlying mechanism. Initially, a target network for KR-osteoclastogenesis-osteoporosis was constructed using network pharmacology. Subsequently, the intersecting targets were identified through the Venny platform and a PPI network was created using Cytoscape 3.9.1. Key targets within the network were identified employing topological algorithms. GO enrichment and KEGG pathway analysis were then performed on these targets to explore their specific functions and pathways. Additionally, molecular docking of potential core targets of KR was conducted, and the results were validated through cell experiments. A total of 83 target genes overlapped between KR and osteoclastogenesis-osteoporosis targets. Enrichment analysis revealed their role in inflammatory response, protein tyrosine kinase activity, osteoclast differentiation, and MAPK and NF-κB signaling pathways. PPI analysis and molecular docking demonstrate that key targets MAPK14 and MAPK8 exhibit more stable binding with KR compared to other proteins. In vitro experiments demonstrate that KR effectively inhibits osteoclast differentiation and bone resorption without cellular toxicity. It suppresses key osteoclast genes (NFATc1, c-Fos, TRAP, MMP9, Ctsk, Atp6v2), hinders IκB-α degradation, and inhibits ERK and JNK phosphorylation, while not affecting p38 phosphorylation. The results indicate that KR may inhibit osteoclast maturation and bone resorption by blocking NF-κB and MAPK signaling pathways, suggesting its potential as a natural therapeutic agent for osteoporosis.

A targeted and core-shell structured gambogic acid nanomedicine for ameliorating breast cancer-associated osteolysis

The treatment of breast cancer-associated bone tumors is of critical importance and highly challenging, primarily owing to the malignant interplay between tumor proliferation and osteolysis within the bone tumor microenvironment. Although many efforts have been devoted, it is still urgent and desirable to find a robust therapeutic agent that can simultaneously and efficiently inhibit the proliferation of bone tumor and osteoclast differentiation. In this work, we have fabricated a bone-targeting nanoplatform based on bioinspired polydopamine-coated gambogic acid nanoparticles (GA@PDA NPs) integrated with mild temperature photothermal therapy (PTT) and chemotherapy towards the treatment of bone tumors. In particular, GA not only inhibits osteoclast differentiation and proliferation of bone tumor cells, but also downregulates heat shock protein 90 (Hsp90) in the treated tumor cells to sensitize the PTT effect, and the resulting core-shell structured nanomedicine could allow the spatiotemporal control of chemotherapy and mild temperature PTT. Those bioinspired nanomedicine could exhibit excellent tumor suppression and osteolysis inhibition against a xenograft tumor bone metastasis model, and this novel nanoformulation provides a creative paradigm for the treatment of breast cancer-associated osteolysis.

Effect of primary osteoblast-derived extracellular vesicles on osteoclast differentiation.

To investigate the effect of osteoblast-derived extracellular vesicles (OB-EVs) on the proliferation and differentiation of osteoclasts, and to explore the possible molecular mechanism of extracellular vesicles involved in the communication between osteoblasts and osteoclasts. Primary osteoblasts were isolated from newborn mouse calvarial bone and induced by β-glycero phosphate, ascorbic acid and dexamethasone. Osteogenic feature was tested by alkaline phosphatase (ALP) and alizarin red S staining. Extracellular vesicles were isolated by ultracentrifugation from the cell culture supernatant. Vesicle morphology was observed by transmission electron microscopy, and the characteristic markers of tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (Alix) and cluster of differentiation 9 (CD9) on the surface of extracellular vesicles were identified by Western blotting. Cell counting kit 8 (CCK-8) assay was used to determine the proliferation effect of OB-EVs on mouse mononuclear macrophage RAW264.7 cells. Furthermore, the expression level of specific markers of osteoclast differentiation in RAW264.7 cells was detected by Western blotting after the combined effect of OB-EVs and receptor activator for nuclear factor κB ligand (RANKL). The number of osteoclasts was observed and compared with OB-EVs-treated mouse bone marrow-derived macrophages (BMMs) by tartrate-resistant acid phosphatase (TRAP) staining, and the effect of OB-EVs on osteoclast differentiation was determined. The extracted OB-EVs showed a double-layer cup-like structure with a diameter of 30-150 nm, and TSG101, Alix and CD9 were expressed. RAW264.7 cells were stimulated with OB-EVs, and the results of CCK-8 assay showed that high concentration of OB-EVs (more than 20 μg/mL) inhibited cell proliferation (P<0.05). Western blotting analysis showed that the expression of osteoclast differentiation marker proteins such as c-Fos, activated T cell nuclear factor (NFATc1) and c-Jun N-terminal kinase (JNK) in RAW264.7 cells were significantly increased, and the promoting effect was enhanced with increasing of OB-EVs concentration (P<0.05). In addition, the combination of OB-EVs and RANKL on BMMs showed that the number of TRAP-positive cells was significantly higher than that of the RANKL induction group alone (P<0.05). OB-EVs can promote the differentiation of osteoclast precursor cells into osteoclasts, but high concentration of OB-EVs can inhibit proliferation of RAW264.7 cells.

Characterization of a spontaneous osteopetrosis model using RANKL-dysfunctional mice

Tumor necrosis factor superfamily member 11 (TNFSF11), or receptor activator of nuclear factor-κB ligand (RANKL), is a crucial osteoclast-stimulating factor binding to RANK on osteoclast membranes. Mouse models are powerful tools for understanding the genetic mechanisms of related diseases. Here, we examined the utility of Tnfsf11 mutation in mice for understanding the mechanisms of bone remodeling and dysmorphology. The Tnfsf11gum mouse, discovered in 2011 at Jackson Laboratory, was used to study the genetic landscape associated with TNFSF11 inactivation in bone marrow tissues. Tnfsf11gum/+ and Tnfsf11+/+ mice were subjected to Micro-CT observation, ELISA analysis, histological evaluation, and massively-parallel mRNA sequencing (RNA-Seq) analysis. Tnfsf11gum/+ mice exhibited severe osteopetrotic changes in the bone marrow cavity, along with significantly lower serum RANKL levels and a reduced number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the bone marrow compared to those in Tnfsf11+/+ mice. However, tooth eruption between Tnfsf11gum/+ and Tnfsf11+/+ mice did not differ. Furthermore, genes involved in osteoblast proliferation and differentiation, including Gli1, Slc35b2, Lrrc17, and Junb were differentially expressed. Heterozygous mutation of TNFSF11 was also associated with a slightly increased expression of genes involved in osteoclast proliferation and differentiation, including Tcirg1, Junb, Anxa2, and Atp6ap1. Overall, we demonstrate that single gene mutations in Tnfsf11 cause bone resorption instability without significantly altering the genes related to osteoblast and osteoclast activity in the bone marrow cavity, thus establishing an optimal resource as an experimental animal model for bone resorption in bone biology research.

Iron and bones: effects of iron overload, deficiency and anemia treatments on bone.

Iron is a vital trace element and exerts opposing effects on bone in both iron overload and iron deficiency situations. Remarkably, iron supplementation through intravenous infusion in patients with iron deficiency can also have detrimental effects on bone in special cases. The diverse mechanisms underlying these effects and their manifestations contribute to the complexity of this relationship. Iron overload impacts both bone resorption and formation, accelerating bone resorption while reducing bone formation. These effects primarily result from the direct action of reactive oxygen species (ROS), which influence the proliferation, differentiation, and activity of both osteoclasts and osteoblasts differently. This imbalance favors osteoclasts and inhibits the osteoblasts. Simultaneously, multiple pathways, including bone morphogenic proteins, RANK ligand, and others, contribute to these actions, leading to a reduction in bone mass and an increased susceptibility to fractures. In contrast, iron deficiency induces low bone turnover due to energy and co-factor deficiency, both of which require iron. Anemia increases the risk of fractures in both men and women. This effect occurs at various levels, reducing muscular performance and, on the bone-specific level, decreasing bone mineral density. Crucially, anemia increases the synthesis of the phosphaturic hormone iFGF23, which is subsequently inactivated by cleavage under physiological conditions. Thus, iFGF23 levels and phosphate excretion are not increased. However, in specific cases where anemia has to be managed with intravenous iron treatment, constituents-particularly maltoses-of the iron infusion suppress the cleavage of iFGF23. As a result, patients can experience severe phosphate wasting and, consequently, hypophosphatemic osteomalacia. This condition is often overlooked in clinical practice and is often caused by ferric carboxymaltose. Ending iron infusions or changing the agent, along with phosphate and vitamin D supplementation, can be effective in addressing this issue.

Transcriptome Analysis Elucidates the Potential Key Genes Involved in Rib Development in bmp6-Deficient Silver Carp (Hypophthalmichthys molitrix).

Bone morphogenetic protein 6 (BMP-6) is a constituent of the TGF-β superfamily, known for its ability to stimulate bone and cartilage formation. The investigation of bmp6's involvement in the formation of intermuscular bones in fish has garnered significant attention in recent years. The rib cage is an important skeletal structure that plays a protective function for internal organs in fish. However, there has been limited research conducted on the effects of the bmp6 gene on rib development. Silver carp is one of four major fish in China, favoured for its affordability and tender muscle. Nevertheless, the presence of numerous intermuscular bones in silver carp significantly hinders the advancement of its palatability and suitability for processing. This study showcases the effective utilisation of CRISPR/Cas9 technology for the purpose of disrupting the bmp6 gene in silver carp, leading to the creation of chimeras in the P0 generation, marking the first instance of such an achievement. The chimeras exhibited complete viability, normal appearance, and partial intermuscular bones loss, with approximately 30% of them displaying rib bifurcation or bending. Subsequently, a transcriptome analysis on ribs of P0 chimeras and wild-type silver carp was conducted, leading to the identification of 934 genes exhibiting differential expression, of which 483 were found to be up-regulated and 451 were found to be down-regulated. The results of the KEGG analysis revealed that the "NF-kappa B signalling pathway", "Hippo signalling pathway", "osteoclast differentiation", and "haematopoietic cell lineage" exhibited enrichment and displayed a significant correlation with bone development. The up-regulated genes such as tnfα, fos, and ctgf in pathways may facilitate the proliferation and differentiation of osteoclasts, whereas the down-regulation of genes such as tgfb2 and tgfbr1 in pathways may hinder the formation and specialisation of osteoblasts, ultimately resulting in rib abnormalities. This study presents novel findings on the impact of bmp6 gene deletion on the rib development of silver carp, while simultaneously investigating the previously unexplored molecular mechanisms underlying rib defects in fish.

Effect of antidepressants on bone mineral density and bone metabolism in ovariectomized depressed rats

Purpose: To investigate the effect of antidepressants on bone mineral density and bone metabolism in ovariectomized rats with depression. Methods: 48 female rats were randomly and equally assigned to six groups, namely, sham (Sn), ovariectomized depression (OD), ovariectomized depression rats treated with sertraline (ODs), citalopram (ODx), reboxetine (ODr), and venlafaxine groups (ODw). Behavioral alterations and bone-related parameters were evaluated before and after treatment. Results: After treatment, ODs, ODx, ODw, and ODr groups had higher levels of horizontal and vertical exercise scores, sugar water consumption, osteoclasts, and serum CTX-1 (p &lt; 0.05) compared to OD group. Osteocytes, serum serotonin (5-HT), osteocalcin (BGP), and type I procollagen N-terminal propeptide (PINP) were significantly decreased (p &lt; 0.05) after treatment in ODs, ODx, ODw, and ODr groups. A positive correlation was observed between 5-HT, BGP, PINP, and whole-body bone mineral density (r = 0.931, 0.907, and 0.843, p &lt; 0.05) respectively, while a negative correlation was observed between collagen type I C-terminal propeptide (CTX-I) and bone mineral density (r = -0.855, p &lt; 0.05). Conclusion: Antidepressants reduce bone mineral density and osteocyte proliferation, while increasing osteoclast proliferation. These effects are associated with reductions in 5-HT, PINP and BGP levels, and increase in CTX-I.

Abstract PO1-12-02: Impact of Discontinuation of Denosumab on Bone Health in Breast Cancer Patients

Abstract Denosumab (Dmab), an inhibitor of osteoclast activity and proliferation is approved for treatment of women on aromatase inhibitor (AI) therapy for early breast cancer (60 mg Q 6-months) and metastatic disease (120 mg Q 4-weeks) to protect against bone loss and prevention of skeletal-related events (SREs). 60 mg given twice a year has shown fracture reduction, especially in women on aromatase inhibitor therapy, and recent data supports benefits in disease free survival, bone-metastasis free-survival and overall survival with Dmab in early breast cancer. It appears superior in reduction of SREs versus zoledronic acid (ZA) in established skeletal metastatic disease; thus, a preferred choice in the oncology setting. In the osteoporosis setting, withdrawal of Dmab therapy leads to a “rebound” increase of bone resorption, rapid bone loss and potential vertebral fractures. Increased risk of spontaneous and multiple vertebral fractures has been seen in this patient population. To prevent this, after discontinuation of Dmab, the addition of another anti-resorptive such as ZA is initiated. Dmab withdrawal events are uncommonly reported in patients with metastatic skeletal disease due to generally poor prognosis. Dmab given as 120 mg/month in this setting may need to be curtailed for a variety of indications including hypocalcemia, major dental procedures, osteonecrosis of the jaw or atypical femoral fracture. In the oncologic setting, it is unclear if discontinuation of Dmab will lead to rebound bone resorption or increased incidence of fractures. We describe the case is of a 32-year-old woman with a history of T2N3M1, oligometastatic breast cancer (solitary bone metastases at L1, biopsy-proven) that was ER/PR/HER2 positive. She was treated with neoadjuvant trastuzumab, pertuzumab, taxotere and carboplatin followed by bilateral mastectomy, and radiation to chest wall and L1. She received total estrogen blockade initially with GnRH-agonist and exemestane and ultimately bilateral salpingo-oophorectomy plus exemestane. She continues pertuzumab and trastuzumab (80 treatments to date) for the past 5 years and denosumab 120 mg/month for 14 treatments and then every 3 months, 20 total treatments. Development of hip pain (regular runner) and concern for atypical fracture vs metastatic disease led to bone scan and PET/CT. Metastatic disease was ruled out and stress reaction was diagnosed. Dmab therapy was discontinued with monitoring of her metabolic bone turnover. 1 year after her last Dmab injection, bone turnover markers began to increase and ultimately demonstrated a very significant “rebound phenomenon.” Subsequently, a single dose of 120 mg denosumab decreased bone markers to previously low levels which remain low on routine monitoring, table below. In future, ZA will be initiated and continued on a yearly basis. Dmab has replaced ZA in managing bone health in breast cancer patients. Many oncologists use this without the assistance of bone health endocrinologists, who are an integral part of our cancer center. Bone modifying agents are often discontinued without consideration of the negative impact this may have. As this is a major survivorship issue, we document this case with literature review, to raise awareness for oncologists prescribing these medications. Citation Format: Susan Tannenbaum, Alvaro Alvarez Soto, Pamela Taxel. Impact of Discontinuation of Denosumab on Bone Health in Breast Cancer Patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-12-02.

P-Smad3 differentially regulates the cytological behavior of osteoclasts before and after osteoblasts maturation.

A series of previous investigations have revealed that p-Smad3 plays a facilitative role in the differentiation and maturation of osteoblasts, while also regulating the expression of certain intercellular communication factors. However, the effects of p-Smad3 in osteoblasts before and after maturation on the proliferation, migration, differentiation, apoptosis and other cellular behaviors of osteoclasts have not been reported. MC3T3-E1 cells were cultured in osteogenic induction medium for varying durations, After that, the corresponding conditioned medium was collected and the osteoclast lineage cells were treated. To elucidate the regulatory role of p-Smad3 within osteoblasts, we applied the activator TGF-β1 and inhibitor SIS3 to immature and mature osteoblasts and collected corresponding conditioned media for osteoclast intervention. We observed an elevation of p-Smad3 and Smad3 during the early stage of osteoblast differentiation, followed by a decline in the later stage. we discovered that as osteoblasts mature, their conditioned media inhibit osteoclasts differentiation and the osteoclast-coupled osteogenic effect. However, it promotes apoptosis in osteoclasts and the angiogenesis coupled with osteoclasts. p-Smad3 in immature osteoblasts, through paracrine effects, promotes the migration, differentiation, and osteoclast-coupled osteogenic effects of osteoclast lineage cells. For mature osteoblasts, p-Smad3 facilitates osteoclast apoptosis and the angiogenesis coupled with osteoclasts. As pre-osteoblasts undergo maturation, p-Smad3 mediated a paracrine effect that transitions osteoclast cellular behaviors from inducing differentiation and stimulating bone formation to promoting apoptosis and coupling angiogenesis.

Nodal negatively regulates osteoclast differentiation by inducing STAT1 phosphorylation.

Several members of the transforming growth factor beta (TGF-β) superfamily regulate the proliferation, differentiation, and function of bone-forming osteoblasts and bone-resorbing osteoclasts. However, it is still unknown whether Nodal, a member of the TGF-β superfamily, serves a function in bone cells. In this study, we found that Nodal did not have any function in osteoblasts but instead negatively regulated osteoclast differentiation. Nodal inhibited RANKL-induced osteoclast differentiation by downregulating the expression of pro-osteoclastogenic genes, including c-fos, Nfatc1, and Blimp1, and upregulating the expression of antiosteoclastogenic genes, including Bcl6 and Irf8. Nodal activated STAT1 in osteoclast precursor cells, and STAT1 downregulation significantly reduced the inhibitory effect of Nodal on osteoclast differentiation. These findings indicate that Nodal activates STAT1 to downregulate or upregulate the expression of pro-osteoclastogenic or antiosteoclastogenic genes, respectively, leading to the inhibition of osteoclast differentiation. Moreover, the inhibitory effect of Nodal on osteoclast differentiation contributed to the reduction of RANKL-induced bone loss in vivo.

Relative contributions of osteal macrophages and osteoclasts to postnatal bone development in CSF1R-deficient rats and phenotype rescue following wild-type bone marrow cell transfer.

Macrophage and osteoclast proliferation, differentiation and survival are regulated by colony-stimulating factor-1 receptor (CSF1R) signaling. Osteopetrosis associated with Csf1 and Csf1r mutations has been attributed to the loss of osteoclasts and deficiency in bone resorption. Here we demonstrate that homozygous Csf1r mutation in rat leads to delayed postnatal skeletal ossification associated with substantial loss of osteal macrophages (osteomacs) in addition to osteoclasts. Osteosclerosis and site-specific skeletal abnormalities were reversed by intraperitoneal transfer of wild-type bone marrow cells (BMT) at weaning. Following BMT, IBA1+ macrophages were detected before TRAP+ osteoclasts at sites of ossification restoration. These observations extend evidence that osteomacs independently contribute to bone anabolism and are required for normal postnatal bone growth and morphogenesis.

Abstract 6866: Investigating the role of Epha2 in breast cancer-mediated myeloid cell expansion and function

Abstract Bone metastases are present in over 70% of metastatic breast cancer patients, and current therapy reduces osteolysis in tumor-induced bone disease (TIBD) but does not improve overall survival. Frequently, TIBD is characterized by an increase in osteoclast proliferation and bone-resorbing activity, and patients often experience pain and pathologic fractures. Increased bone resorption releases growth factors that aid in the progression of tumor growth causing the ‘vicious cycle’. Additionally, bone metastatic patients are often resistant to first- and second-line therapies highlighting an importance for research in this area. The receptor tyrosine kinase, Ephrin-type-A 2 receptor (EphA2), participates in both forward and reverse signaling during receptor/ligand engagement and participates in many physiological and pathological processes, including bone homeostasis, immunity, and cancer. Interestingly, EphA2 is highly expressed in bone metastases, and previous evidence suggests that EphA2 can promote TIBD and reduce anti-tumor immunity. We therefore hypothesize that increased expression of EphA2 in breast cancer cells at the site of bone metastasis promotes expansion of myeloid cells, leading to TIBD. Previous data shows a reduction in osteoclast number and bone lesions following EphA2 knockout. However, the mechanism by which EphA2 mediates the expansion of osteoclasts is not well understood. In agreement with previous work, our data shows that EphA2 deletion in murine breast cancer (BC) cell line, 4T1, reduces bone lesion area and number and the presence of bone metastasis-associated macrophages. Additionally, we observed a significant reduction in Ly6Chi cells. To assess the effects of EphA2 deletion on the behavior of 4T1 cells, we conducted a wound healing assay and observed a decrease in wound healing capacity in 4T1 cells following EphA2 deletion. This suggests that EphA2 expression in 4T1 cells may increase their metastatic potential. We will repeat these studies and incorporate factors from the bone marrow. Our future studies will investigate the role of EphA2 signaling and reverse signaling in BC cell lines, myeloid progenitor cells, osteoclasts, and bone metastasis-associated macrophages. We will utilize murine and human BC cell lines to elucidate the effects of EphA2 deletion in BC on immune cell populations, bone metastasis, and tumor progression. Additionally, we will investigate how EphA2 reverse signaling impacts osteoclasts’ ability to modulate immune cells. We aim to reduce TIBD and tumor burden by reducing the expansion of osteoclasts and shifting the immune cells to an anti-tumorigenic phenotype. Our studies will provide additional insight into the underlying mechanisms of the vicious cycle which will offer new treatment strategies for patients with osteolytic breast cancer. Citation Format: Dominique Parker, Verra Ngwa, Jin Chen, Julie Rhoades. Investigating the role of Epha2 in breast cancer-mediated myeloid cell expansion and function [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 6866.

Regulation of Glycosylation in Bone Metabolism.

Glycosylation plays a crucial role in the maintenance of homeostasis in the body and at the onset of diseases such as inflammation, neurodegeneration, infection, diabetes, and cancer. It is also involved in bone metabolism. N- and O-glycans have been shown to regulate osteoblast and osteoclast differentiation. We recently demonstrated that ganglio-series and globo-series glycosphingolipids were essential for regulating the proliferation and differentiation of osteoblasts and osteoclasts in glycosyltransferase-knockout mice. Herein, we reviewed the importance of the regulation of bone metabolism by glycoconjugates, such as glycolipids and glycoproteins, including our recent results.

D-alanine suppressed osteoclastogenesis derived from bone marrow macrophages and downregulated ERK/p38 signalling pathways

ObjectivesD-alanine is a residue of the backbone structure of Type Ⅰ Lipoteichoic acid (LTA), which is a virulence factor in inflammation caused by gram-positive bacteria. However, the role of D-alanine in infectious bone destruction has not been investigated. We aimed to explore the role of D-alanine in the proliferation, apoptosis, and differentiation of osteoclasts. DesignMouse bone marrow-derived macrophages (BMMs) were isolated as osteoclast precursors and stimulated with D-alanine. Cell proliferation and apoptosis were detected using CCK-8 and flow cytometry, respectively. The formation of osteoclasts morphologically observed by tartrate-resistant acid phosphatase staining (TRAP) and immunofluorescence staining. The expressions of osteoclastogenic genes were measured by real-time RT-PCR. The protein expressions of osteoclastogenic markers, p38, and ERK1/2 MAPK signalling were measured by western blot. The expression level of soluble Sema4D was detected via enzyme-linked immunosorbent assay (ELISA). ResultsThe cell proliferation of BMMs was significantly inhibited by D-alanine in a dose-dependent manner. Apoptosis of BMMs was markedly activated with the stimulation of D-alanine. The differentiation of BMMs into osteoclasts was significantly inhibited by D-alanine, and the gene and protein expressions of NFATc1, c-Fos, and Blimp decreased. Western blot showed that D-alanine inhibited the phosphorylated p38 and ERK1/2 signalling pathways of BMMs. Moreover, the expression level of soluble Sema4D significantly decreased in the supernatant of BMMs due to the D-alanine intervention. ConclusionD-alanine plays a pivotal role in the inhibition of RANKL-induced osteoclastogenesis and might become a potential therapeutic drug for bone-resorptive diseases.

The hagfish genome and the evolution of vertebrates

As the only surviving lineages of jawless fishes, hagfishes and lampreys provide a crucial window into early vertebrate evolution1–3. Here we investigate the complex history, timing and functional role of genome-wide duplications4–7 and programmed DNA elimination8,9 in vertebrates in the light of a chromosome-scale genome sequence for the brown hagfish Eptatretus atami. Combining evidence from syntenic and phylogenetic analyses, we establish a comprehensive picture of vertebrate genome evolution, including an auto-tetraploidization (1RV) that predates the early Cambrian cyclostome–gnathostome split, followed by a mid–late Cambrian allo-tetraploidization (2RJV) in gnathostomes and a prolonged Cambrian–Ordovician hexaploidization (2RCY) in cyclostomes. Subsequently, hagfishes underwent extensive genomic changes, with chromosomal fusions accompanied by the loss of genes that are essential for organ systems (for example, genes involved in the development of eyes and in the proliferation of osteoclasts); these changes account, in part, for the simplification of the hagfish body plan1,2. Finally, we characterize programmed DNA elimination in hagfish, identifying protein-coding genes and repetitive elements that are deleted from somatic cell lineages during early development. The elimination of these germline-specific genes provides a mechanism for resolving genetic conflict between soma and germline by repressing germline and pluripotency functions, paralleling findings in lampreys10,11. Reconstruction of the early genomic history of vertebrates provides a framework for further investigations of the evolution of cyclostomes and jawed vertebrates.

Research Progress in the Molecular Regulatory Mechanisms of Alveolar Bone Restoration

Alveolar bone, the protruding portion of the maxilla and the mandible that surrounds the roots of teeth, plays an important role in tooth development, eruption, and masticatory performance. In oral inflammatory diseases, including apical periodontitis, periodontitis, and peri-implantitis, alveolar bone defects cause the loosening or loss of teeth, impair the masticatory function, and endanger the physical and mental health of patients. However, alveolar bone restoration is confronted with great clinical challenges due to the the complicated effect of the biological, mechanical, and chemical factors in the oral microenvironment. An in-depth understanding of the underlying molecular regulatory mechanisms will contribute to the exploration of new targets for alveolar bone restoration. Recent studies have shown that Notch, Wnt, Toll-like receptor (TLR), and nuclear factor-κB (NF-κB) signaling pathways regulate the proliferation, differentiation, apoptosis, and autophagy of osteoclasts, osteoblasts, osteocytes, periodontal ligament cells, macrophages, and adaptive immune cells, modulate the expression of inflammatory mediators, affect the balance of the receptor activator for nuclear factor-κB ligand/receptor activator for nuclear factor-κB/osteoprotegerin (RANKL/RANK/OPG) system, and ultimately participate in alveolar bone restoration. Additionally, alveolar bone restoration involves AMP-activated protein kinase (AMPK), phosphatidyl inositol 3-kinase/protein kinase B (PI3K/AKT), Hippo/YAP, Janus kinase/signal transducer and activator of transcription (JAK/STAT), and transforming growth factor β (TGF-β) signaling pathways. However, current studies have failed to construct mature molecular regulatory networks for alveolar bone restoration. There is an urgent need for further research on the molecular regulatory mechanisms of alveolar bone restoration by using new technologies such as single-cell transcriptome sequencing and spatial transcriptome sequencing.

The autocrine action of salidroside on osteoclast during osteoclastogenesis via hypoxia-inducible factor-1α pathway.

The objective of this study was to investigate the potential of salidroside (SAL) (a major active compound in Rhodiola rosea L.) in regulating osteoclast differentiation and function by modulating the HIF-1α pathway and its downstream target genes. The expression of HIF-1α and its downstream target genes was examined at both mRNA and protein levels in osteoclasts treated with SAL. Immunofluorescence analysis was performed to assess the nuclear translocation and transcriptional activity of HIF-1α in response to SAL. MTT, flow cytometry, qPCR, TRAP staining and bone resorption assays were used to evaluate the potential effect of salidroside on osteoclasts. SAL enhanced the expression of HIF-1α and its downstream target genes in osteoclasts. Immunofluorescence analysis confirmed the facilitation of HIF-1α nuclear translocation and transcriptional activity by SAL. In addition, SAL enhanced osteoclast viability, differentiation and bone resorption activity in an autocrine manner through HIF-1α/VEGF, IL-6 and ANGPTL4 pathways. SAL promotes osteoclast proliferation, differentiation and bone resorption through HIF-1α/VEGF, IL-6 and ANGPTL4 pathways.

Type H vessels: functions in bone development and diseases.

Type H vessels are specialized blood vessels found in the bone marrow that are closely associated with osteogenic activity. They are characterized by high expression of endomucin and CD31. Type H vessels form in the cancellous bone area during long bone development to provide adequate nutritional support for cells near the growth plate. They also influence the proliferation and differentiation of osteoprogenitors and osteoclasts in a paracrine manner, thereby creating a suitable microenvironment to facilitate new bone formation. Because of the close relationship between type H vessels and osteogenic activity, it has been found that type H vessels play a role in the physiological and pathological processes of bone diseases such as fracture healing, osteoporosis, osteoarthritis, osteonecrosis, and tumor bone metastasis. Moreover, experimental treatments targeting type H vessels can improve the outcomes of these diseases. Here, we reviewed the molecular mechanisms related to type H vessels and their associated osteogenic activities, which are helpful in further understanding the role of type H vessels in bone metabolism and will provide a theoretical basis and ideas for comprehending bone diseases from the vascular perspective.