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Cell cytoskeleton and proliferation study for the RANKL-induced RAW264.7 differentiation.

ABSTRACT: Although document studies (including ours) have been reported the achieved in vitro osteoclastic cellular model establishment from the RAW264.7 cell lineage, there was no study directly reported that American Type Culture Collection (ATCC) cell bank has various RAW264.7 cell lineages. Besides that, for our knowledge there was only one study compared the two different RAW264.7^TIB-71 and RAW264.7^CRL-2278 cell lineages for their osteoclastic differentiation, and they concluded that the RAW264.7^CRL-2278 demonstrated to generate much osteoclast than RAW264.7^TIB-71 . However, on the contrary to their results we noticed the fusion of RAW264.7^TIB-71 in our previous studies was much compromising. Therefore, we try to explore the two cell lineages for their properties in osteoclastic differentiation with an in-depth cellular cytoskeletal study. Our current study has showed that comparing to the RAW264.7^CRL-2278 , RAW264.7^TIB-71 demonstrated a much higher efficacies for RANKL-stimulated osteoclastic differentiation. Besides that, in our depth cytoskeletal studies, we found that the RANKL-induced RAW264.7^TIB-71 cells could finally differentiate into mature osteoclasts. However, regardless the various pre-treatment conditions, there was no mature osteoclast formed in RANKL-induced RAW264.7^CRL-2278 cell lineage.

SUBMITTER: Kong L

PROVIDER: S-EPMC8107080 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Effect of l-caldesmon on osteoclastogenesis in RANKL-induced RAW264.7 cells.

Project description:Non-muscle caldesmon (l-CaD) is involved in the regulation of actin cytoskeletal remodeling in the podosome formation, but its function in osteoclastogenesis remains to be determined. In this study, RANKL-induced differentiation of RAW264.7 murine macrophages to osteoclast-like cells (OCs) was used as a model to determine the physiological role of l-CaD and its phosphorylation in osteoclastogenesis. Upon RANKL treatment, RAW264.7 cells undergo cell-cell fusion into multinucleate, and TRAP-positive large OCs with a concomitant increase of l-CaD expression. Using gain- and loss-of-function in OC precursor cells followed by RANKL induction, we showed that the expression of l-CaD in response to RANKL activation is an important event for osteoclastogenesis, and bone resorption. To determine the effect of l-CaD phosphorylation in osteoclastogenesis, three decoy peptides of l-CaD were used with, respectively, Ser-to-Ala mutations at the Erk- and Pak1-mediated phosphorylation sites, and Ser-to-Asp mutation at the Erk-mediated phosphorylation sites. Both the former two peptides competed with the C-terminal segment of l-CaD for F-actin binding and accelerated formation of podosome-like structures in RANKL-induced OCs, while the third peptide did not significantly affect the F-actin binding of l-CaD, and decreased the formation of podosome-like structures in OCs. With the experiments using dephosphorylated and phosphorylated l-CaD mutants, we further showed that dephosphorylated l-CaD mutant facilitated RANKL-induced TRAP activity with an increased cell fusion index, whereas phosphorylated l-CaD decreased the TRAP activity and cell fusion. Our findings suggested that both the level of l-CaD expression and the extent of l-CaD phosphorylation play a role in RANKL-induced osteoclast differentiation.

| S-EPMC5993584 | biostudies-literature

3BP2 adapter protein is required for receptor activator of NF?B ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells.

Project description:The adapter protein 3BP2 (also known as SH3BP2 and Abl SH3-binding protein 2) has been involved in leukocyte signaling and activation downstream immunoreceptors. Genetic studies have further associated 3BP2 mutations to the human disease cherubism and to inflammation and bone dysfunction in mouse. However, how wild type 3BP2 functions in macrophage differentiation remains poorly understood. In this study, using small interfering RNA-mediated silencing of 3BP2 in the RAW264.7 monocytic cell line, we show that 3BP2 was required for receptor activator of NFkappaB ligand (RANKL)-induced differentiation of RAW264.7 cells into multinucleated mature osteoclasts but not for granulocyte macrophage-colony stimulating factor/interleukin-4-induced differentiation into dendritic cells. 3BP2 silencing was associated with impaired activation of multiple signaling events downstream of RANK, including actin reorganization; Src, ERK, and JNK phosphorylation; and up-regulation of osteoclastogenic factors. In addition, 3BP2 knockdown cells induced to osteoclast by RANKL displayed a reduced increase of Src and nuclear factor of activated T cells (NFATc1) mRNA and protein expression. Importantly, 3BP2 interacted with Src, Syk, Vav, and Cbl in monocytic cells, and the introduction of constitutively active mutants of Src and NFATc1 in 3BP2-deficient cells restored osteoclast differentiation. Finally, the expression of a 3BP2 cherubism mutant was found to promote increased Src activity and NFAT-dependent osteoclast formation. Together, this study demonstrates that wild type 3BP2 is a key regulator of RANK-mediated macrophage differentiation into osteoclast through Src and NFATc1 activation.

| S-EPMC2898328 | biostudies-literature

Effects of Rambutan Peel (Nepheliumlappaceum) PhenolicExtract on RANKL-Induced Differentiation of RAW264.7 Cells into Osteoclasts and Retinoic Acid-Induced Osteoporosis in Rats.

Project description:Previous studies have shown that rambutan peel phenolic (RPP) extract has excellent biological activities due to its abundant phenolic content and profile. In this study, the potential anti-osteoporosis (OP) effects of RPP were evaluated by suppressing receptor activator nuclear factor-kappa B ligand (RANKL)-induced differentiation of RAW264.7 cells into osteoclasts and amelioratingretinoic acid-induced OP in rats. Our results showed that RPP efficiently decreased the formation of tartrate-resistant acid phosphatase (TRAP)-positive cells and reduced total TRAP activity in RAW264.7 cells under RANKL stimulation. RPP treatment significantlyameliorated retinoid acid-induced calcium loss in rats (p < 0.05). The serum phosphorus level of osteoporotic rats was increased by RPP treatment, and the serum levels of total alkaline phosphatase and osteocalcin in osteoporotic rats were further reduced. RPP treatment improved the qualities of the femur and tibia, such asbone mineral density, bone length, bone maximum load, cortical bone area ratio, and trabecularelative bone density in osteoporotic rats to some extent. Furthermore, histological analysis showed that RPP effectively improved the bone microstructure of osteoporotic rats by regulating the cortical bone thickness and trabecular bone separation. These results indicate that RPP could have potential applications as a newnutraceutical and functional food in the prevention of OP.

| S-EPMC7230481 | biostudies-literature

Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-κB and NFATc1 activation and DC-STAMP expression.

Project description:Aconiti Lateralis Radix Preparata is a traditional Chinese medicine used to treat chronic arthritis and is highly effective against rheumatoid arthritis. However, the effects of aconine, a derivative of aconitum alkaloids, on osteoclasts, which can absorb bone, remain unknown. Here, we investigated the effects of aconine on osteoclast differentiation and bone resorption in vitro.The viability of mouse leukemic monocyte/macrophage cell line RAW264.7 was measured using CCK-8 assays. Osteoclast differentiation was induced by incubation of RAW264.7 cells in the presence of RANKL, and assessed with TRAP staining assay. Bone resorption was examined with bone resorption pits assay. The expression of relevant genes and proteins was analyzed using RT-PCR and Western blots. The activation of NF-κB and nuclear factor of activated T-cells (NFAT) was examined using stable NF-κB and NFATc1 luciferase reporter gene systems, RT-PCR and Western blot analysis.Aconine (0.125, 0.25 μmol/L) did not affect the viability of RAW264.7 cells, but dose-dependently inhibited RANKL-induced osteoclast formation and bone resorptive activity. Furthermore, aconine dose-dependently inhibited the RANKL-induced activation of NF-κB and NFATc1 in RAW264.7 cells, and subsequently reduced the expression of osteoclast-specific genes (c-Src, β3-Integrin, cathepsin K and MMP-9) and the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which played an important role in cell-cell fusion.These findings suggest that aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing the activation of NF-κB and NFATc1 and the expression of the cell-cell fusion molecule DC-STAMP.

| S-EPMC4753374 | biostudies-other

Melatonin-Induced Cytoskeleton Reorganization Leads to Inhibition of Melanoma Cancer Cell Proliferation.

Project description:Neuroindole melatonin, a hormone synthesized during the night mainly-but not exclusively-by the pineal gland of all vertebrates, functions as an adapting signal to the light-dark cycle. Its antioxidant, neuroprotective, anti-inflammatory, and antitumor properties are all well-known and widely reported. Melanoma is one of the most common carcinomas among developed countries and a type of tumor particularly difficult to fight back in medium/advanced stages. In contrast to other types of cancer, influence of melatonin on melanoma has been scarcely investigated. Thus, we have chosen the murine melanoma model B16-F10 cell line to study antiproliferative and antitumoral actions of melatonin. For this purpose, we combined both, cell culture and in vivo models. Melatonin reduced either, growth rate or migration of B16-F10 cells. Furthermore, melanin synthesis was altered by melatonin, promoting its synthesis. Melatonin also induced a G2/M cell cycle arrest and altered the cytoskeletal organization. To corroborate these results, we tested the effect of melatonin in the in vivo model of B16-F10 cell injection in the tail vein, which causes numerous lung metastases. Two different strategies of melatonin administration were used, namely, in drinking water, or daily intraperitoneal injection. However, contrary to what occurred in cell culture, no differences were observed between control and melatonin treated groups. Results obtained led us to conclude that melatonin exerts an antiproliferative and anti-migrating effect on this melanoma model by interfering with the cytoskeleton organization, but this pharmacological effect cannot be translated in vivo as the indole did not prevent metastasis in the murine model, suggesting that further insights into the effects of the indole in melanoma cells should be approached to understand this apparent paradox.

| S-EPMC7014391 | biostudies-literature

<i>Artocarpus tonkinensis</i> Extract Inhibits LPS-Triggered Inflammation Markers and Suppresses RANKL-Induced Osteoclastogenesis in RAW264.7.

Project description:Artocarpus tonkinensis (At) leaf decoction, a traditional remedy prepared in North Vietnam by the Hmong ethnic group, is a tea extract rich in bioactive compounds that may have therapeutic effects in arthritis and backache. Indeed, it has been demonstrated that At is able to inhibit Th17 lymphocytes development and to protect mice in an experimental model of collagen-induced arthritis. By resorting to macrophage in vitro models of inflammation and osteoclastogenesis, we showed that At extract significantly reduced nitric oxide synthase 2 (NOS2) activity and IL-6 production by RAW 264.7 murine cells. Moreover, At demonstrated an anti-osteoclastogenic effect, as revealed by complete inhibition of TRAP-positive osteoclast formation and decreased expression of key osteoclast-related genes. This At activity likely relies on the inhibition of RANK downstream signaling pathway, as the activation of non-receptor tyrosine kinase Src is reduced upon RANKL-At exposure. Protective effect of At against bone loss was also enlightened in vivo by collagen-induced arthritis (CIA) experiment demonstrating that, although paw edema was only weakly opposed by drinking At decoction, bone and cartilage were well preserved in CIA+At mice and joint tissue expressed decreased levels of osteoclast marker genes respect to CIA control group. Maesopsin 4-O-β-D-glucoside (i.e., TAT-2, one of the main decoction bioactive components) was capable to contrast NOS2 activity, IL-6 expression and osteoclast formation, too, albeit to a lesser extent when compared to At decoction. Overall, this study enlightens another At cell target, macrophages, beside Th17 lymphocytes, and suggests that the anti-arthritic beneficial effects of At decoction largely derives from its ability to counteract not only inflammation, but also osteoclastogenesis.

| S-EPMC7862131 | biostudies-literature

Vav1 inhibits RANKL-induced osteoclast differentiation and bone resorption.

Project description:Vav1 is a Rho/Rac guanine nucleotide exchange factor primarily expressed in hematopoietic cells. In this study, we investigated the potential role of Vav1 in osteoclast (OC) differentiation by comparing the ability of bone marrow mononuclear cells (BMMCs) obtained from Vav1-deficient (Vav1-/-) and wild-type (WT) mice to differentiate into mature OCs upon stimulation with macrophage colony stimulating factor and receptor activator of nuclear kappa B ligand in vitro. Our results suggested that Vav1 deficiency promoted the differentiation of BMMCs into OCs, as indicated by the increased expression of tartrate-resistant acid phosphatase, cathepsin K, and calcitonin receptor. Therefore, Vav1 may play a negative role in OC differentiation. This hypothesis was supported by the observation of more OCs in the femurs of Vav1-/- mice than in WT mice. Furthermore, the bone status of Vav1-/- mice was analyzed in situ and the femurs of Vav1-/- mice appeared abnormal, with poor bone density and fewer number of trabeculae. In addition, Vav1-deficient OCs showed stronger adhesion to vitronectin, an ?v?3 integrin ligand important in bone resorption. Thus, Vav1 may inhibit OC differentiation and protect against bone resorption. [BMB Reports 2019; 52(11): 659-664].

| S-EPMC6889896 | biostudies-literature

RAW264.7 cells: RANKL vs Bone Metastasis Tumor Cell Condidtioned Media

Project description:Transcriptional profiling of differential miRNA expression in mouse RAW264.7 preosteoclast cells comparing control untreated cells with RAW264.7 cells treated for 6 days. Treatment conditiones tested included 50ng/mL RANKL or indicated bone metastasis tumor cell conditioned media from 4T1, 4T1.2, TSU-PR1, and TSU-PR1-B2 cell lines. Two-condition experiment, Control cells vs. treated cells.

2013-10-14 | E-GEOD-44936 | biostudies-arrayexpress

MicroRNA-455-3p regulates proliferation and osteoclast differentiation of RAW264.7 cells by targeting PTEN.

Project description:BackgroundMacrophages are one of the important cells in immune system. In this article, we aim to explore the regulatory role of miR-455-3p on proliferation and osteoblast differentiation of RAW264.7 cells.MethodsExpression levels of genes and proteins in cells were tested via qRT-PCR and western blot. The targeted correlation between miR-455-3p and PTEN was identified by luciferase analysis. MTT assay and flow cytometry were applied to detect the proliferation and apoptosis of cells. Osteoclastogenesis was completed by stimulating RAW 264.7 cells with RANKL. Tartrate-resistant acid phosphatase (TRAP) activity in different groups of cells were assessed.ResultsFirstly, we determined that up-regulation of miR-455-3p promoted the proliferation and inhibited apoptosis of RAW 264.7 cells. MiR-455-3p deficiency played opposite effect in RAW 264.7 cells. Additionally, osteoclastogenesis-related factors (TRAP, CTSK and NFATc1) expression levels were remarkably up-regulated in miR-455-3p-mimic group of RAW264.7 cells treated with RANKL, but decreased in inhibitor group. Luciferase assay proved that miR-455-3p targeted PTEN. We took a further step and found overexpression of PTEN significantly inhibited the increased proliferation and osteoblast differentiation of RAW264.7 cells induced by miR-455-3p.ConclusionsOur findings supported basic to explore the molecular mechanism of proliferation and osteoblast differentiation of RAW264.7 cells.

| S-EPMC8994399 | biostudies-literature

RANKL-Induced Btn2a2 - A T Cell Immunomodulatory Molecule - During Osteoclast Differentiation Fine-Tunes Bone Resorption.

Project description:Butyrophilins, which are members of the extended B7 family of immunoregulators structurally related to the B7 family, have diverse functions on immune cells as co-stimulatory and co-inhibitory molecules. Despite recent advances in the understanding on butyrophilins' role on adaptive immune cells during infectious or autoimmune diseases, nothing is known about their role in bone homeostasis. Here, we analyzed the role of one specific butyrophilin, namely Btn2a2, as we have recently shown that Btn2a2 is expressed on the monocyte/macrophage lineage that also gives rise to bone degrading osteoclasts. We found that expression of Btn2a2 on monocytes and pre-osteoclasts is upregulated by the receptor activator of nuclear factor κ-B ligand (RANKL), an essential protein required for osteoclast formation. Interestingly, in Btn2a2-deficient osteoclasts, typical osteoclast marker genes (Nfatc1, cathepsin K, TRAP, and RANK) were downregulated following RANKL stimulation. In vitro osteoclast assays resulted in decreased TRAP positive osteoclast numbers in Btn2a2-deficient cells. However, Btn2a2-deficient osteoclasts revealed abnormal fusion processes shown by their increased size. In vivo steady state µCT and histological analysis of bone architecture in complete Btn2a2-deficient mice showed differences in bone parameters further highlighting the fine-tuning effect of BTN2a2. Moreover, in rheumatoid arthritis patients and experimental arthritis, we detected significantly decreased serum levels of the secreted soluble Btn2a2 protein. Taken together, we identified the involvement of the immunomodulatory molecule Btn2a2 in osteoclast differentiation with potential future implications in basic and translational osteoimmunology.

| S-EPMC8371446 | biostudies-literature

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