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Overexpression of miR?146a blocks the effect of LPS on RANKL?induced osteoclast differentiation.

ABSTRACT: The concept that inflammation serves a leading role in osteoclast?induced bone loss under pathological circumstances is now widely accepted. In the present study, it was observed that lipopolysaccharides (LPSs) demonstrated a synergic effect on receptor activator of nuclear factor ??B ligand (RANKL)?induced osteoclast differentiation in Raw264.7 cells, with increasing levels of multiple pro?inflammatory cytokines including interleukin (IL)?6, tumor necrosis factor?? and IL?1?. Furthermore, microRNA (miR)?146a was highly induced by LPS and RANKL co?stimulation during the process of osteoclast differentiation. Overexpression of miR?146a inhibited osteoclast transformation by targeting the key regulators of nuclear factor (NF)??? signaling, TNF receptor?associated factor 6 and interleukin?1 receptor?associated kinase 1. The downstream activation of NF??? signaling was also inhibited by transfection with a miR?146a mimic. Altogether, the results of the present study demonstrated that miR?146a prevents osteoclast differentiation induced by LPS and RANKL co?stimulation, suggesting that miR?146a may be a promising therapeutic target for treatment of inflammation mediated bone loss.

SUBMITTER: Gao Y

PROVIDER: S-EPMC6236290 | biostudies-other | 2018 Dec

REPOSITORIES: biostudies-other

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Overexpression of miR‑146a blocks the effect of LPS on RANKL‑induced osteoclast differentiation.

Gao Yingjian Y Wang Bo B Shen Conghuan C Xin Weiwei W

Molecular medicine reports 20181030 6

The concept that inflammation serves a leading role in osteoclast‑induced bone loss under pathological circumstances is now widely accepted. In the present study, it was observed that lipopolysaccharides (LPSs) demonstrated a synergic effect on receptor activator of nuclear factor κ‑B ligand (RANKL)‑induced osteoclast differentiation in Raw264.7 cells, with increasing levels of multiple pro‑inflammatory cytokines including interleukin (IL)‑6, tumor necrosis factor‑α and IL‑1β. Furthermore, micro ...[more]

PMID: 30387844

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Project description:BackgroundResolvin E1 (RvE1) derived from the ω-3 polyunsaturated fatty acid eicosapentaenoic acid is known to be a potent pro-resolving lipid mediator that prevents chronic inflammation and osteoclastogenesis. We investigated the inhibitory effects of RvE1 on osteoclastogenesis and bone resorption to clarify its therapeutic potential for rheumatoid arthritis (RA).MethodsReceptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation was assessed with tartrate-resistant acid phosphatase staining. RANKL-induced bone resorption was assessed by the measurement of pit formation using calcium phosphate-labeled fluorescent polyanionic molecules in RAW264.7 cells as osteoclast precursors. The effects of RvE1 on the RANKL-induced mRNA expression of osteoclast-specific genes and transcriptional factors such as c-fos and nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells were measured by quantitative real-time PCR. The distribution of NFATc1 induced by RANKL was evaluated by immunofluorescence staining in RAW264.7 cells. To analyze the mechanism of the inhibitory effect of RvE1 on osteoclastogenesis, we measured IL-17-induced RANKL mRNA expression in MC3T3-E1 osteoblast cells treated with RvE1 using quantitative real-time PCR and determined the level of prostaglandin E2 (PGE2) production by enzyme-linked immunosorbent assay.ResultsRvE1 significantly suppressed RANKL-induced osteoclast differentiation and bone resorption. RvE1 inhibited the RANKL-induced mRNA expression of osteoclast-specific genes along with the transcription factors NFATc1 and c-fos. Moreover, NFATc1 translocation from the cytoplasm to the nucleus of RAW264.7 cells was suppressed following RvE1 treatment. RvE1 also inhibited IL-17-induced RANKL mRNA expression and PGE2 production in MC3T3-E1 cells.ConclusionRvE1 inhibited osteoclastogenesis and bone resorption by suppressing RANKL-induced NFATc1 and c-fos expression in osteoclasts and IL-17-induced RANKL expression through the autocrine action of PGE2 in osteoblasts. Our data suggest RvE1 as a new therapeutic target of RA.

Project description:Mitochondrial biogenesis, the generation of new mitochondrial DNA and proteins, has been linked to osteoclast (OC) differentiation and function. In this study we used mice with mutations in key alternative NF-?B pathway proteins, RelB and NF-?B-inducing kinase (NIK), to dissect the complex relationship between mitochondrial biogenesis and osteoclastogenesis. In OC precursors lacking either NIK or RelB, receptor activator of NF-?B ligand (RANKL) was unable to increase mitochondrial DNA or oxidative phosphorylation (OxPhos) protein expression, which was associated with lower oxygen consumption rates. Transgenic OC precursors expressing constitutively active NIK showed normal RANKL-induced mitochondrial biogenesis (OxPhos expression and mitochondria copy number) compared to controls, but larger mitochondrial dimensions and increased oxygen consumption rates, suggesting increased mitochondrial function. To deduce the mechanism for mitochondrial biogenesis defects in NIK-deficient and RelB-deficient precursors, we examined expression of genes known to control this process. PGC-1? (Ppargc1b) expression, but not PGC-1?, PPRC1, or ERR?, was significantly reduced in RelB(-/-) and NIK(-/-) OCs. Because PGC-1? has been reported to positively regulate both mitochondrial biogenesis and differentiation in OCs, we retrovirally overexpressed PGC-1? in RelB(-/-) cells, but surprisingly found that it did not affect differentiation, nor did it restore RANKL-induced mitochondrial biogenesis. To determine whether the blockade in osteoclastogenesis in RelB-deficient cells precludes mitochondrial biogenesis, we rescued RelB(-/-) differentiation via overexpression of NFATc1. Mitochondrial parameters in neither WT nor RelB-deficient cultures were affected by NFATc1 overexpression, and bone resorption in RelB(-/-) was not restored. Furthermore, NFATc1 co-overexpression with PGC-1?, although allowing OC differentiation, did not rescue mitochondrial biogenesis or bone resorption in RelB(-/-) OCs, by CTX-I levels. Thus, our results indicate that the alternative NF-?B pathway plays dual, but distinct, roles in controlling the independent processes of OC differentiation and OC mitochondrial biogenesis. Furthermore, the inability of PGC-1? to drive mitochondrial biogenesis in OCs without RelB indicates a cell-type specificity in mitochondria regulation.

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Overexpression of miR?146a blocks the effect of LPS on RANKL?induced osteoclast differentiation.

Publications

Overexpression of miR‑146a blocks the effect of LPS on RANKL‑induced osteoclast differentiation.

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