Dataset Information

Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations.

ABSTRACT: Osteoclasts (OCs) are bone-resorptive cells critical for maintaining skeletal integrity through coupled bone turnover. OC differentiation and activation requires receptor activator of NF-kB ligand (RANKL) signaling through the p38 MAPK pathway. However the role of the p38 MAPK substrate, MAPK-activated protein kinase 2 (MK2), is not clearly delineated. Within the bone marrow exists a specific subpopulation of defined osteoclast progenitor cells (dOCPs) with surface expression of B220(-)Gr1(-)CD11b(lo/-)CD115(+) (dOCP(lo/-)). In this study, we isolated dOCPs from male and female mice to determine sex-specific effects of MK2 signaling in osteoclastogenesis (OCgen). Male Mk2(-/-) mice display an increase in the dOCP(lo) cell population when compared to Mk2(+/+) mice, while female Mk2(-/-) and Mk2(+/+ )mice exhibit no difference. Defined OCPs from male and female Mk2(+/+) and Mk2(-/-) bone marrow were treated with macrophage colony stimulation factor (M-CSF) and RANKL cytokines to promote OCgen. RANKL treatment of dOCP(lo) cells stimulated p38 and MK2 phosphorylation. Tartrate-resistant acid phosphatase (TRAP) assays were used to quantify OC number, size, and TRAP enzyme activity post-RANKL stimulation. MK2 signaling was critical for male dOCP(lo) OCgen, yet MK2 signaling regulated OCgen from female dOCP- and CD11b(hi) subpopulations as well. The functional gene, Ctsk, was attenuated in both male and female Mk2(-/-) dOCP(lo)-derived OCs. Conversely, MK2 signaling was only critical for gene expression of pre-OC fusion genes, Oc-stamp andTm7sf4, in male OCgen. Therefore, these data suggest there is a sexual dimorphism in MK2 signaling of OCP subpopulations.

SUBMITTER: Herbert BA

PROVIDER: S-EPMC4422514 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations.

Herbert Bethany A BA Valerio Michael S MS Gaestel Matthias M Kirkwood Keith L KL

PloS one 20150506 5

Osteoclasts (OCs) are bone-resorptive cells critical for maintaining skeletal integrity through coupled bone turnover. OC differentiation and activation requires receptor activator of NF-kB ligand (RANKL) signaling through the p38 MAPK pathway. However the role of the p38 MAPK substrate, MAPK-activated protein kinase 2 (MK2), is not clearly delineated. Within the bone marrow exists a specific subpopulation of defined osteoclast progenitor cells (dOCPs) with surface expression of B220(-)Gr1(-)CD1 ...[more]

PMID: 25946081

Similar Datasets

Project description:Mechanical forces are essential to maintain skeletal integrity, and microgravity exposure leads to bone loss. The underlying molecular mechanisms leading to the changes in osteoblasts and osteoclast differentiation and function remain to be fully elucidated. Because of the infrequency of spaceflights and payload constraints, establishing in vitro and in vivo systems that mimic microgravity conditions becomes necessary. We have established a simulated microgravity (modeled microgravity, MMG) system to study the changes induced in osteoclast precursors. We observed that MMG, on its own, was unable to induce osteoclastogenesis of osteoclast precursors; however, 24 h of MMG activates osteoclastogenesis-related signaling molecules ERK, p38, PLC?2, and NFATc1. Receptor activator of NFkB ligand (RANKL) (with or without M-CSF) stimulation for 3-4 days in gravity of cells that had been exposed to MMG for 24 h enhanced the formation of very large tartrate-resistant acid phosphatase (TRAP)-positive multinucleated (>30 nuclei) osteoclasts accompanied by an upregulation of the osteoclast marker genes TRAP and cathepsin K. To validate the in vitro system, we studied the hindlimb unloading (HLU) system using BALB/c mice and observed a decrease in BMD of femurs and a loss of 3D microstructure of both cortical and trabecular bone as determined by micro-CT. There was a marked stimulation of osteoclastogenesis as determined by the total number of TRAP-positive multinucleated osteoclasts formed and also an increase in RANKL-stimulated osteoclastogenesis from precursors removed from the tibias of mice after 28 days of HLU. In contrast to earlier reported findings, we did not observe any histomorphometric changes in the bone formation parameters. Thus, the foregoing observations indicate that microgravity sensitizes osteoclast precursors for increased differentiation. The in vitro model system described here is potentially a valid system for testing drugs for preventing microgravity-induced bone loss by targeting the molecular events occurring in microgravity-induced enhanced osteoclastogenesis.

Project description:BackgroundPhosphorylation modification of BCL2 is involved in receptor activator of nuclear factor-κB ligand (RANKL)-induced autophagy of osteoclast precursors (OCPs) and osteoclastogenesis. As an antiapoptotic molecule, the role of BCL2 phosphorylation in osteoclastogenesis is unknown. This study aimed to explore how BCL2 phosphorylation at specific sites regulates osteoclastogenesis.MethodsWe first examined the effects of RANKL on BCL2 phosphorylation at different sites (Ser70 and Ser87) in OCPs. In vivo, transgenic mice overexpressing RANKL (Tg-hRANKL mice) were used to observe the effects of RANKL on phosphorylated BCL2 at different sites in OCPs of trabecular bone. Subsequently, using site-directed mutagenesis, we observed the respective effect of BCL2 mutations at different phosphorylation sites in OCPs on osteoclastogenesis, apoptosis, autophagy and the affinity between BCL2 and Beclin1/BAX under RANKL intervention.ResultsRANKL promoted BCL2 phosphorylation at the Ser70 (S70) site, but not the Ser87 (S87) site, in OCPs. Moreover, Tg-hRANKL mice had stronger BCL2 phosphorylation capacity at S70, not S87, in the OCPs of trabecular bone than wild-type mice in the same nest. Furthermore, BCL2 mutation at S70, not S87, inhibited RANKL-induced osteoclast differentiation and bone resorption activity. In addition, BCL2 mutation at S70 promoted OCP apoptosis, while BCL2 mutation at S87 showed the opposite effect. Remarkably, the BCL2 mutation at S70, not S87, inhibited OCP autophagic activity. Furthermore, BCL2 mutation at S70 enhanced the coimmunoprecipitation of BCL2 and Beclin1, whereas BCL2 mutation at S87 enhanced the coimmunoprecipitation of BCL2 and BAX in OCPs. More importantly, OCP autophagy, osteoclast differentiation and resorption pits inhibited by BCL2 mutation at S70 could be reversed by Beclin1 upregulation with TAT-Beclin1.ConclusionRANKL activates OCP autophagy through BCL2 phosphorylation at S70, thereby promoting osteoclastogenesis, which indicates that the inactivation of BCL2 at S70 in OCPs may be a therapeutic strategy for pathological bone loss.

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.

Dataset Information

Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations.

Publications

Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets