ULK1 Suppresses Osteoclast Differentiation and Bone Resorption via Inhibiting Syk-JNK through DOK3
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ABSTRACT:
SUBMITTER: Zhang Y
PROVIDER: S-EPMC8608530 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We have recently demonstrated NFAT activating protein with ITAM motif 1 (NFAM1) signaling increases osteoclast (OCL) formation/bone resorption associated with the Paget's disease of bone, however, the underlying molecular mechanisms of the NFAM1 regulation of OCL differentiation and bone resorption remains unclear. Here, we showed that RANK ligand stimulation enhances NFAM1 expression in preosteoclast cells. Conditioned media collected from RANKL stimulated RAW264.7 NFAM1 knockdown (KD) stable cells showed inhibition of interleukin-6 (2.5-fold), tumour necrosis factor-α (2.2-fold) and CXCL-5 (3-fold) levels compared to wild-type (WT) cells. Further, RANKL stimulation significantly increased p-STAT6 expression (5.5-fold) in WT cells, but no significant effect was observed in NFAM1-KD cells. However, no changes were detected in signal transducer and activator of transcription 3 levels in either of cell groups. Interestingly, NFAM1-KD suppressed the RANKL stimulated c-fos, p-c-Jun and c-Jun N-terminal kinase (JNK) activity in preosteoclasts. We further showed that the suppression of JNK activity is through inhibition of p-SAPK/JNK in these cells. In addition, NFATc1 expression, a critical transcription factor associated with osteoclastogenesis is significantly inhibited in NFAM1-KD preosteoclast cells. Interestingly, NFAM1 inhibition suppressed the OCL differentiation and bone resorption capacity in mouse bone marrow cell cultures. We also demonstrated inhibition of tartrate-resistant acid phosphatase expression in RANKL stimulated NFAM1-KD preosteoclast cells. Thus, our results suggest that NFAM1 control SAPK/JNK signaling to modulate osteoclast differentiation and bone resorption.
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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].
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Project description:Osteogenesis is an orchestrated process regulated by osteoclastogenesis and osteoblastogenesis. Excessive osteoclastogenesis causes bone diseases, such as osteoporosis. Although a few drugs are effective in osteoporosis treatment, these drugs lead to side effects, including cellulitis, flatulence, and hypocalcemia. In this study, we reported a 2-(N-Phenylmethylsulfonamido)-N-(2-(phenylthio)phenyl)propanamide (PSTP) compound, PSTP-3,5-Me, as a potential therapeutic agent for osteoporosis. Mouse bone marrow-derived macrophages (BMMs) were differentiated into osteoclasts by receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in the presence of PSTP-3,5-Me. PSTP-3,5-Me inhibited osteoclast differentiation by reduced tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and suppressed the expression of osteoclast marker genes, such as cathepsin K (Ctsk) and TRAP (Acp5). We investigated signaling pathways mediated by RANKL and its receptor, RANK, and found that PSTP-3,5-Me inhibits nucleus translocation of nuclear factor of activated T cell cytoplasmic-1 (NFATc1). Moreover, PSTP-3,5-Me inhibited F-actin ring formation and mineral resorption. Overall, our data suggests that PSTP-3,5-Me attenuates osteoclast differentiation by blocking the activation of NFATc1.
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Project description:Osteoclasts (OC) are critical cells responsible for many bone diseases such as osteoporosis. It is of great interest to identify agents that can regulate the activity of OC to treat osteolytic bone diseases. In this study, we found that baicalin exerted a two-way regulatory effect on OC in a concentration-dependent manner in vitro and in vivo. In detail, baicalin at a low concentration (below 1 μmol/L) enhanced OC differentiation and bone resorption, but baicalin at a high concentration (above 2 μmol/L) exhibited inhibitory effects on OC. We demonstrated that baicalin at low concentrations enhanced the mitogen-activated protein kinase (MAPK) (ERK) signalling pathway and activated c-Fos and NFATc1 expression, and thus enhanced gene expression, OC differentiation and bone resorption. However, baicalin at higher levels not only suppressed ERK phosphorylation and c-fos and NFATc1 expression, but also altered the expression of apoptosis-related proteins, and therefore inhibiting OC function. This dual effect was further verified in an LPS-induced mouse calvarial osteolysis model, evidenced by enhanced osteolysis at a lower concentration but reduced bone loss at a higher concentration. Overall, our findings indicate that baicalin exerts dose-dependent effects on OC formation and function. Therefore, caution should be applied when using baicalin to treating OC-related bone diseases.
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Project description:Key osteoclast (OCL) regulatory gene promoters in bone marrow-derived monocytes harbor bivalent histone modifications that combine activating Histone 3 lysine 4 tri-methyl (H3K4me3) and repressive H3K27me3 marks, which upon RANKL stimulation resolve into repressive or activating architecture. Enhancer of zeste homologue 2 (EZH2) is the histone methyltransferase component of the polycomb repressive complex 2, which catalyzes H3K27me3 modifications. Immunofluorescence microscopy reveals that EZH2 localization during murine osteoclastogenesis is dynamically regulated. Using EZH2 knockdown and small molecule EZH2 inhibitor GSK126, we show that EZH2 plays a critical epigenetic role in OCL precursors (OCLp) during the first 24?hours of RANKL activation. RANKL triggers EZH2 translocation into the nucleus where it represses OCL-negative regulators MafB, Irf8, and Arg1. Consistent with its cytoplasmic localization in OCLp, EZH2 methyltransferase activity is required during early RANKL signaling for phosphorylation of AKT, resulting in downstream activation of the mTOR complex, which is essential for induction of OCL differentiation. Inhibition of RANKL-induced pmTOR-pS6RP signaling by GSK126 altered the translation ratio of the C/EBP?-LAP and C/EBP?-LIP isoforms and reduced nuclear translocation of the inhibitory C/EBP?-LIP, which is necessary for transcriptional repression of the OCL negative-regulatory transcription factor MafB. EZH2 in multinucleated OCL is primarily cytoplasmic and mature OCL cultured on bone segments in the presence of GSK126 exhibit defective cytoskeletal architecture and reduced resorptive activity. Here we present new evidence that EZH2 plays epigenetic and cytoplasmic roles during OCL differentiation by suppressing MafB transcription and regulating early phases of PI3K-AKT-mTOR-mediated RANKL signaling, respectively. Consistent with its cytoplasmic localization, EZH2 is required for cytoskeletal dynamics during resorption by mature OCL. Thus, EZH2 exhibits complex roles in supporting osteoclast differentiation and function. © 2019 American Society for Bone and Mineral Research.
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Project description:Rosiglitazone has the potential to activate peroxisome proliferator-activated receptor-? (PPAR?), which in turn can affect bone formation and resorption. However, the mechanisms by which rosiglitazone regulates osteoclastic orosteoblastic differentiation are not fully understood. This study examines how rosiglitazone affects osteoclast formation, bone resorption and osteoblast differentiation from mouse bone marrow. Rosiglitazone treatment not only inhibited the formation of tartrate-resistant acid phosphatase-positive cells, but also prevented pit formation by bone marrow cells in a dose- and time-dependent manner. Rosiglitazone also suppressed the receptor activator of nuclear factor (NF)-?B ligand (RANKL) receptor(RANK) expression but increased PPAR?2 expression in the cells. In addition, rosiglitazone diminished RANKL induced activation of NF-?B-DNA binding by blocking I?B?phosphorylation. Furthermore, it reduced collagen and osteocalcin levels to nearly zero and prevented mRNA expression of osteoblast-specific proteins including runtrelated transcription factor-2, osteocalcin, and type I collagen.However, mRNA levels of adipocyte-specific marker, aP2, were markedly increased in the cells co-incubated with rosiglitazone. These results suggest that PPAR? activation by rosiglitazone inhibits osteoblast differentiation with increased adipogenesis in bone marrow cells and also may prevent osteoclast formation and bone resorptionin the cells.
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Project description:Monotropein (Mon) is a kind of iridoid glycoside plant secondary metabolite primarily present in some edible and medicinal plants. The aim of this study was to investigate the effect of Mon on lipopolysaccharide (LPS)-induced inflammatory bone loss in mice and osteoclasts (OCs) derived from bone marrow-derived macrophages (BMMs), and explore the mechanisms underlying the effect of Mon on LPS-induced osteoclastogenesis. It was found that Mon markedly attenuated deterioration of the bone micro-architecture, enhanced tissue mineral content (TMC) and bone volume/total volume (BV/TV), reduced structure model index (SMI) and trabecular separation/spacing (Tb.Sp) in the bone tissue and decreased the activities of tartrate resistant acid phosphatase-5b (TRACP-5b), receptor activator NF-κB (RANK), and receptor activator NF-κB ligand (RANKL) as well as the serum levels of interleukin 6 (IL-6) and interleukin 1β (IL-1β) in LPS-treated mice. In addition, Mon treatment reduced the number of TRAP positive OCs in the bone tissue of LPS-treated mice and also exerted a stronger inhibitory effect on formation, differentiation, and F-actin ring construction of OCs derived from BMMs. Mon significantly inhibited the expression of the nuclear factor of activated T-cells c1 (NFATc1) and the immediate early gene (C-Fos) and nuclear translocation of NFATc1 in LPS-treated OCs, thereby inhibiting the expression of matrix metalloproteinase-9 (MMP-9), cathepsin K (CtsK), and TRAP. Mon significantly inhibited the expression of TRAF6, phosphorylation of P65, and degradation of IKBα, thus inhibiting the activation of NF-κB pathway in LPS-induced inflammatory mice and OCs derived from BMMs, and also inhibited LPS-induced phosphorylation of protein kinase B (Akt) and Glycogen synthase kinase 3β (GSK-3β) in OCs derived from BMMs. In conclusion, these results suggested that Mon could effectively inhibit osteoclastogenesis both in vitro and in vivo and therefore may prove to be potential option for prevention and treatment of osteoclastic bone resorption-related diseases.
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Project description:Claudin 18 (Cldn-18) belongs to a large family of transmembrane proteins that are important components of tight junction strands. Although several claudin members are expressed in bone, the functional role for any claudin member in bone is unknown. Here we demonstrate that disruption of Cldn-18 in mice markedly decreased total body bone mineral density, trabecular bone volume, and cortical thickness in Cldn-18(-/-) mice. Histomorphometric studies revealed that bone resorption parameters were increased significantly in Cldn-18(-/-) mice without changes in bone formation. Serum levels of tartrate-resistant acid phosphatase 5b (TRAP5b) and mRNA expression levels of osteoclast specific markers and signaling molecules were also increased. Loss of Cldn-18 further exacerbated calcium deficiency induced bone loss by influencing bone resorption, thereby resulting in mechanically weaker bone. In vitro studies with bone marrow macrophages revealed Cldn-18 disruption markedly enhanced receptor activator of NF-?B ligand (RANKL)-induced osteoclast differentiation but not macrophage colony-stimulating factor (MCSF)-induced bone marrow macrophage (BMM) proliferation. Consistent with a direct role for Cldn-18 in regulating osteoclast differentiation, overexpression of wild type but not PDZ binding motif deleted Cldn-18 inhibited RANKL-induced osteoclast differentiation. Furthermore, our findings indicate that Cldn-18 interacts with Zonula occludens 2 (ZO-2) to modulate RANKL signaling in osteoclasts. In conclusion, we demonstrate that Cldn-18 is a novel negative regulator of bone resorption and osteoclast differentiation.
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