scholarly journals METTL3 Modulates Osteoclast Differentiation and Function by Controlling RNA Stability and Nuclear Export

2020 ◽  
Vol 21 (5) ◽  
pp. 1660 ◽  
Author(s):  
Di Li ◽  
Luhui Cai ◽  
Runsha Meng ◽  
Zhihui Feng ◽  
Qiong Xu
Keyword(s):  
Nuclear Export ◽  
Osteoclast Differentiation ◽  
Rna Stability ◽  
Cell Lineage ◽  
Specific Gene ◽  
Bone Homeostasis ◽  
Protein Levels ◽  
Lineage Differentiation ◽  
And Function ◽  
Skeletal Integrity

Osteoclast differentiation and function are crucial for maintaining bone homeostasis and preserving skeletal integrity. N6-methyladenosine (m6A) is an abundant mRNA modification that has recently been shown to be important in regulating cell lineage differentiation. Nevertheless, the effect of m6A on osteoclast differentiation remains unknown. In the present study, we observed that the m6A level and methyltransferase METTL3 expression increased during osteoclast differentiation. Mettl3 knockdown resulted in an increased size but a decreased bone-resorbing ability of osteoclasts. The expression of osteoclast-specific genes (Nfatc1, c-Fos, Ctsk, Acp5 and Dcstamp) was inhibited by Mettl3 depletion, while the expression of the cellular fusion-specific gene Atp6v0d2 was upregulated. Mechanistically, Mettl3 knockdown elevated the mRNA stability of Atp6v0d2 and the same result was obtained when the m6A-binding protein YTHDF2 was silenced. Moreover, the phosphorylation levels of key molecules in the MAPK, NF-κB and PI3K-AKT signaling pathways were reduced upon Mettl3 deficiency. Depletion of Mettl3 maintained the retention of Traf6 mRNA in the nucleus and reduced the protein levels of TRAF6. Taken together, our data suggest that METTL3 regulates osteoclast differentiation and function through different mechanisms involving Atp6v0d2 mRNA degradation mediated by YTHDF2 and Traf6 mRNA nuclear export. These findings elucidate the molecular basis of RNA epigenetic regulation in osteoclast development.

scholarly journals Steroidogenic Factor-1 (SF-1)-Driven Differentiation of Murine Embryonic Stem (ES) Cells into a Gonadal Lineage

Endocrinology ◽  
2011 ◽  
Vol 152 (7) ◽  
pp. 2870-2882 ◽  
Author(s):  
Unmesh Jadhav ◽  
J. Larry Jameson
Keyword(s):  
Target Genes ◽  
De Novo ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Culture Conditions ◽  
Steroidogenic Factor 1 ◽  
Lineage Differentiation ◽  
And Function

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells (SF-1-ES cells) has been shown to prime the cells for steroidogenesis. When provided with exogenous cholesterol substrate, and after treatment with retinoic acid and cAMP, SF-1-ES cells produce progesterone but do not produce other steroids such as cortisol, estradiol, or testosterone. In this study, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic lineages. When embryoid body formation was used to facilitate cell lineage differentiation, SF-1-ES cells were found to be restricted in their differentiation, with fewer cells entering neuronal pathways and a larger fraction entering the steroidogenic lineage. Among the differentiation protocols tested, leukemia inhibitory factor (LIF) removal, followed by prolonged cAMP treatment was most efficacious for inducing steroidogenesis in SF-1-ES cells. In this protocol, a subset of SF-1-ES cells survives after LIF withdrawal, undergoes morphologic differentiation, and recovers proliferative capacity. These cells are characterized by induction of steroidogenic enzyme genes, use of de novo cholesterol, and production of multiple steroids including estradiol and testosterone. Microarray studies identified additional pathways associated with SF-1 mediated differentiation. Using biotinylated SF-1 in chromatin immunoprecipitation assays, SF-1 was shown to bind directly to multiple target genes, with induction of binding to some targets after steroidogenic treatment. These studies indicate that SF-1 expression, followed by LIF removal and treatment with cAMP drives ES cells into a steroidogenic pathway characteristic of gonadal steroid-producing cells.

scholarly journals Plasma membrane calcium ATPase regulates bone mass by fine-tuning osteoclast differentiation and survival

2012 ◽  
Vol 199 (7) ◽  
pp. 1145-1158 ◽  
Author(s):  
Hyung Joon Kim ◽  
Vikram Prasad ◽  
Seok-Won Hyung ◽  
Zang Hee Lee ◽  
Sang-Won Lee ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Bone Mass ◽  
Osteoclast Differentiation ◽  
Premenopausal Women ◽  
Fine Tuning ◽  
Bone Homeostasis ◽  
Regulatory Loop ◽  
And Function

The precise regulation of Ca2+ dynamics is crucial for proper differentiation and function of osteoclasts. Here we show the involvement of plasma membrane Ca2+ ATPase (PMCA) isoforms 1 and 4 in osteoclastogenesis. In immature/undifferentiated cells, PMCAs inhibited receptor activator of NF-κB ligand–induced Ca2+ oscillations and osteoclast differentiation in vitro. Interestingly, nuclear factor of activated T cell c1 (NFATc1) directly stimulated PMCA transcription, whereas the PMCA-mediated Ca2+ efflux prevented NFATc1 activation, forming a negative regulatory loop. PMCA4 also had an anti-osteoclastogenic effect by reducing NO, which facilitates preosteoclast fusion. In addition to their role in immature cells, increased expression of PMCAs in mature osteoclasts prevented osteoclast apoptosis both in vitro and in vivo. Mice heterozygous for PMCA1 or null for PMCA4 showed an osteopenic phenotype with more osteoclasts on bone surface. Furthermore, PMCA4 expression levels correlated with peak bone mass in premenopausal women. Thus, our results suggest that PMCAs play important roles for the regulation of bone homeostasis in both mice and humans by modulating Ca2+ signaling in osteoclasts.

scholarly journals PIP5k1β controls bone homeostasis through modulating both osteoclast and osteoblast differentiation

2019 ◽  
Vol 12 (1) ◽  
pp. 55-70 ◽  
Author(s):  
Xiaoying Zhao ◽  
Penglei Cui ◽  
Guoli Hu ◽  
Chuandong Wang ◽  
Lei Jiang ◽  
...  
Keyword(s):  
Osteoblast Differentiation ◽  
Nuclear Translocation ◽  
Osteoclast Differentiation ◽  
Bone Homeostasis ◽  
Osteoporosis Prevention ◽  
Cytoskeletal Organization ◽  
Fos Expression ◽  
And Migration ◽  
And Function ◽  
Proliferation And Migration

Abstract PIP5k1β is crucial to the generation of phosphotidylinosotol (4, 5)P2. PIP5k1β participates in numerous cellular activities, such as B cell and platelet activation, cell phagocytosis and endocytosis, cell apoptosis, and cytoskeletal organization. In the present work, we aimed to examine the function of PIP5k1β in osteoclastogenesis and osteogenesis to provide promising strategies for osteoporosis prevention and treatment. We discovered that PIP5k1β deletion in mice resulted in obvious bone loss and that PIP5k1β was highly expressed during both osteoclast and osteoblast differentiation. Deletion of the gene was found to enhance the proliferation and migration of bone marrow-derived macrophage-like cells to promote osteoclast differentiation. PIP5k1β−/− osteoclasts exhibited normal cytoskeleton architecture but stronger resorption activity. PIP5k1β deficiency also promoted activation of mitogen-activated kinase and Akt signaling, enhanced TRAF6 and c-Fos expression, facilitated the expression and nuclear translocation of NFATC1, and upregulated Grb2 expression, thereby accelerating osteoclast differentiation and function. Finally, PIP5k1β enhanced osteoblast differentiation by upregulating master gene expression through triggering smad1/5/8 signaling. Therefore, PIP5k1β modulates bone homeostasis and remodeling.

scholarly journals PIP5k1 β controls bone homeostasis through modulating both osteoclast and osteoblast differentiation

2018 ◽  
Author(s):  
Xiaoying Zhao ◽  
Guoli Hu ◽  
Chuandong Wang ◽  
Lei Jiang ◽  
Jingyu Zhao ◽  
...  
Keyword(s):  
Osteoblast Differentiation ◽  
Nuclear Translocation ◽  
Osteoclast Differentiation ◽  
Bone Homeostasis ◽  
Osteoporosis Prevention ◽  
Genes Expression ◽  
And Migration ◽  
And Function ◽  
Insight Into ◽  
Proliferation And Migration

AbstractPIP5K1β is crucial to generation of phosphotidylinosotol (4, 5) P2. PIP5K1β participates in numerous cellular activities, such as B cell and platelet activation, cell phagocytosis and endocytosis, cell apoptosis, and cytoskeletal organization. In the present work, we aimed to make insight into the function of PIP5K1β in osteoclastogenesis and osteogenesis to provide promising strategies for osteoporosis prevention and treatment. We discovered that PIP5k1β deletion in mice resulted in obvious bone loss and PIP5K1β was highly expressed both during osteoclast and osteoblast differentiation, besides, PIP5K1β deletion enhanced the proliferation and migration of BMMs to promote osteoclast differentiation. PIP5k1β−/− osteoclast exhibited normal cytoskeleton architecture but stronger resorption activity. PIP5k1β deficiency also promoted activation of MAPK and Akt signaling, enhanced TRAF6 and c-Fos expression, facilitated the expression and nuclear translocation of NFATC1 and upregulated Grb2 expression, thereby accelerating osteoclast differentiation and function. Finally, PIP5K1β enhanced osteoblast differentiation by upregulating master genes expression through triggering smad1/5/8 signaling. Thereby, PIP5K1β modulate bone homeostasis and remodeling.

IMMU-38. DEEP IMMUNOPROFILING OF HUMAN GLIOBLASTOMA (GBM) REVEALS DIFFERENCES IN THE TUMOR IMMUNE CELL INFILTRATE IN PATIENTS WITH HIGH VS. LOW PLASMA CELL-FREE DNA (CFDNA)

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi100-vi101
Author(s):  
Stephen J Bagley ◽  
Cecile Alanio ◽  
Jacob Till ◽  
Aseel Abdalla ◽  
Zev Binder ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Cell ◽  
Cell Lineage ◽  
Predictive Biomarker ◽  
High Plasma ◽  
Lineage Differentiation ◽  
Tumor Infiltrate ◽  
Poor Outcomes ◽  
And Function ◽  
Immune Cell Infiltrates

Abstract BACKGROUND We have previously demonstrated that high baseline plasma cfDNA concentration is associated with poor survival in patients with newly diagnosed GBM. The mechanism of this association remains unknown. To explore whether differences in the immune landscape between high- vs. low-cfDNA patients may play a role in their divergent clinical outcomes, we phenotyped tumors from patients with high vs. low cfDNA using mass cytometry by time of flight (CyTOF). METHODS We performed CyTOF on frozen tumor infiltrate suspension from a pilot cohort of patients with previously untreated GBM with known baseline plasma cfDNA concentration (Bagley, Clin Cancer Res 2020). CyTOF was used to simultaneously measure expression of 39 molecules related to immune cell lineage, differentiation state, and function. Differences in immune cell infiltrates between high- and low-cfDNA patients were assessed using Mann-Whitney U tests. RESULTS Four patients with high cfDNA (median 57, range 33-90 ng/mL) were compared to six patients with low cfDNA (median 12, range 7-16 ng/mL). Immune cell infiltrates with increased adaptive cells (high monocytes and T cells, p=0.05) were present in high-cfDNA compared to low-cfDNA patients. While > 70% of the infiltrating T cells were exhausted in both groups, the pattern of exhaustion was significantly different in high- vs. low-cfDNA patients, with less CXCR5+CD69+ and more CXCR5-CD69- (p=0.008) progenitor exhausted T cells in cfDNA-high patients. CONCLUSIONS In this GBM pilot study, we demonstrated differences in the tumor immune infiltrate in patients with high vs. low baseline plasma cfDNA concentration. Preclinical studies will be needed to determine if this explains the association between high plasma cfDNA and poor outcomes previously observed in patients. Our results may have implications for the use of cfDNA concentration as a predictive biomarker for immunotherapy, as tumors with more intermediate progenitor (CXCR5-CD69-) exhausted T cells may respond better to PD-1 checkpoint blockade.

scholarly journals Sestrin2 Regulates Osteoclastogenesis via the p62-TRAF6 Interaction

2021 ◽  
Vol 9 ◽  
Author(s):  
Sue Young Oh ◽  
Namju Kang ◽  
Jung Yun Kang ◽  
Ki Woo Kim ◽  
Jong-Hoon Choi ◽  
...  
Keyword(s):  
Osteoclast Differentiation ◽  
Ros Generation ◽  
Nuclear Factor ◽  
Bone Homeostasis ◽  
Activated T Cells ◽  
Stress Injury ◽  
Mitogen Activated Protein ◽  
Intracellular Ca ◽  
And Function

The receptor activator of nuclear factor-kappa B ligand (RANKL) mediates osteoclast differentiation and functions by inducing Ca2+ oscillations, activating mitogen-activated protein kinases (MAPKs), and activating nuclear factor of activated T-cells type c1 (NFATc1) via the RANK and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) interaction. Reactive oxygen species (ROS) also plays an important role during osteoclastogenesis and Sestrin2, an antioxidant, maintains cellular homeostasis upon stress injury via regulation of ROS, autophagy, and inflammation. However, the role of Sestrin2 in osteoclastogenesis remains unknown. In this study, we investigated the role of Sestrin2 in the RANKL-RANK-TRAF6 signaling pathway during osteoclast differentiation. Deletion of Sestrin2 (Sesn2) increased bone mass and reduced the number of multinucleated osteoclasts on bone surfaces. RANKL-induced osteoclast differentiation and function decreased in Sesn2 knockout (KO) bone marrow-derived monocytes/macrophages (BMMs) due to inhibition of NFATc1 expression, but osteoblastogenesis was not affected. mRNA expression of RANKL-induced specific osteoclastogenic genes and MAPK protein expression were lower in Sesn2 KO BMMs than wild-type (WT) BMMs after RANKL treatment. However, the Sesn2 deletion did not affect ROS generation or intracellular Ca2+ oscillations during osteoclastogenesis. In contrast, the interaction between TRAF6 and p62 was reduced during osteoclasts differentiation in Sesn2 KO BMMs. The reduction in the TRAF6/p62 interaction and TRAP activity in osteoclastogenesis in Sesn2 KO BMMs was recovered to the WT level upon expression of Flag-Sesn2 in Sesn2 KO BMMs. These results suggest that Sestrin2 has a novel role in bone homeostasis and osteoclasts differentiation through regulation of NFATc1 and the TRAF6/p62 interaction.

scholarly journals HDAC2 regulates FoxO1 during RANKL-induced osteoclastogenesis

2016 ◽  
Vol 310 (10) ◽  
pp. C780-C787 ◽  
Author(s):  
Ce Dou ◽  
Nan Li ◽  
Ning Ding ◽  
Chuan Liu ◽  
Xiaochao Yang ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Osteoclast Differentiation ◽  
Nuclear Factor Κb ◽  
Negative Regulator ◽  
Bone Homeostasis ◽  
Akt Activation ◽  
Histone Deacetylase 2 ◽  
Forkhead Box ◽  
Receptor Activator ◽  
And Function

The bone-resorbing osteoclast (OC) is essential for bone homeostasis, yet deregulation of OCs contributes to diseases such as osteoporosis, osteopetrosis, and rheumatoid arthritis. Here we show that histone deacetylase 2 (HDAC2) is a key positive regulator during receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption. Bone marrow macrophages (BMMs) showed increased HDAC2 expression during osteoclastogenesis. HDAC2 overexpression enhanced, whereas HDAC2 deletion suppressed osteoclastogenesis and bone resorption using lentivirus infection. Mechanistically, upon RANKL activation, HDAC2 activated Akt; Akt directly phosphorylates and abrogates Forkhead box protein O1 (FoxO1), which is a negative regulator during osteoclastogenesis through reducing reactive oxygen species. HDAC2 deletion in BMMs resulted in decreased Akt activation and increased FoxO1 activity during osteoclastogenesis. In conclusion, HDAC2 activates Akt thus suppresses FoxO1 transcription results in enhanced osteoclastogenesis. Our data imply the potential value of HDAC2 as a new target in regulating osteoclast differentiation and function.

scholarly journals Distinct roles for the hypoxia-inducible transcription factors HIF-1α and HIF-2α in human osteoclast formation and function

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Helen J. Knowles
Keyword(s):  
Bone Resorption ◽  
Osteoclast Differentiation ◽  
Hypoxia Inducible Factor ◽  
Osteoclast Formation ◽  
Bone Homeostasis ◽  
Hypoxic Conditions ◽  
Human Osteoclast ◽  
Inhibition Of Glycolysis ◽  
And Function ◽  
Hif Pathway

AbstractBone homeostasis is maintained by a balance between osteoblast-mediated bone formation and osteoclast-driven bone resorption. Hypoxia modulates this relationship partially via direct and indirect effects of the hypoxia-inducible factor-1 alpha (HIF-1α) transcription factor on osteoclast formation and bone resorption. Little data is available on the role(s) of the HIF-2α isoform of HIF in osteoclast biology. Here we describe induction of HIF-1α and HIF-2α during the differentiation of human CD14+ monocytes into osteoclasts. Knockdown of HIF-1α did not affect osteoclast differentiation but prevented the increase in bone resorption that occurs under hypoxic conditions. HIF-2α knockdown did not affect bone resorption but moderately inhibited osteoclast formation. Growth of osteoclasts in 3D gels reversed the effect of HIF-2α knockdown; HIF-2α siRNA increasing osteoclast formation in 3D. Glycolysis is the main HIF-regulated pathway that drives bone resorption. HIF knockdown only affected glucose uptake and bone resorption in hypoxic conditions. Inhibition of glycolysis with 2-deoxy-d-glucose (2-DG) reduced osteoclast formation and activity under both basal and hypoxic conditions, emphasising the importance of glycolytic metabolism in osteoclast biology. In summary, HIF-1α and HIF-2α play different but overlapping roles in osteoclast biology, highlighting the importance of the HIF pathway as a potential therapeutic target in osteolytic disease.

scholarly journals Intercellular Communication between Keratinocytes and Fibroblasts Induces Local Osteoclast Differentiation: a Mechanism Underlying Cholesteatoma-Induced Bone Destruction

2016 ◽  
Vol 36 (11) ◽  
pp. 1610-1620 ◽  
Author(s):  
Yoriko Iwamoto ◽  
Keizo Nishikawa ◽  
Ryusuke Imai ◽  
Masayuki Furuya ◽  
Maki Uenaka ◽  
...  
Keyword(s):  
Intercellular Communication ◽  
Osteoclast Differentiation ◽  
Bone Destruction ◽  
Nuclear Factor Κb ◽  
Bone Diseases ◽  
Epidermal Cyst ◽  
Bone Homeostasis ◽  
Lethal Complications ◽  
And Function

Bone homeostasis is maintained by a balance in activity between bone-resorbing osteoclasts and bone-forming osteoblasts. Shifting the balance toward bone resorption causes osteolytic bone diseases such as rheumatoid arthritis and periodontitis. Osteoclast differentiation is regulated by receptor activator of nuclear factor κB ligand (RANKL), which, under some pathological conditions, is produced by T and B lymphocytes and synoviocytes. However, the mechanism underlying bone destruction in other diseases is little understood. Bone destruction caused by cholesteatoma, an epidermal cyst in the middle ear resulting from hyperproliferation of keratinizing squamous epithelium, can lead to lethal complications. In this study, we succeeded in generating a model for cholesteatoma, epidermal cyst-like tissue, which has the potential for inducing osteoclastogenesis in mice. Furthermore, anin vitrococulture system composed of keratinocytes, fibroblasts, and osteoclast precursors was used to demonstrate that keratinocytes stimulate osteoclast differentiation through the induction of RANKL in fibroblasts. Thus, this study demonstrates that intercellular communication between keratinocytes and fibroblasts is involved in the differentiation and function of osteoclasts, which may provide the molecular basis of a new therapeutic strategy for cholesteatoma-induced bone destruction.

scholarly journals PKC-δ deficiency in B cells displays osteopenia accompanied with upregulation of RANKL expression and osteoclast–osteoblast uncoupling

2020 ◽  
Vol 11 (9) ◽  
Author(s):  
Shangfu Li ◽  
Qiuli Liu ◽  
Depeng Wu ◽  
Tianwei He ◽  
Jinbo Yuan ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Bone Structure ◽  
Bone Cells ◽  
Osteoclast Differentiation ◽  
Bone Homeostasis ◽  
Osteoblast Proliferation ◽  
And Function

Abstract PKC-δ is an important molecule for B-cell proliferation and tolerance. B cells have long been recognized to play a part in osteoimmunology and pathological bone loss. However, the role of B cells with PKC-δ deficiency in bone homeostasis and the underlying mechanisms are unknown. We generated mice with PKC-δ deletion selectively in B cells by crossing PKC-δ-loxP mice with CD19-Cre mice. We studied their bone phenotype using micro-CT and histology. Next, immune organs were obtained and analyzed. Western blotting was used to determine the RANKL/OPG ratio in vitro in B-cell cultures, ELISA assay and immunohistochemistry were used to analyze in vivo RANKL/OPG balance in serum and bone sections respectively. Finally, we utilized osteoclastogenesis to study osteoclast function via hydroxyapatite resorption assay, and isolated primary calvaria osteoblasts to investigate osteoblast proliferation and differentiation. We also investigated osteoclast and osteoblast biology in co-culture with B-cell supernatants. We found that mice with PKC-δ deficiency in B cells displayed an osteopenia phenotype in the trabecular and cortical compartment of long bones. In addition, PKC-δ deletion resulted in changes of trabecular bone structure in association with activation of osteoclast bone resorption and decrease in osteoblast parameters. As expected, inactivation of PKC-δ in B cells resulted in changes in spleen B-cell number, function, and distribution. Consistently, the RANKL/OPG ratio was elevated remarkably in B-cell culture, in the serum and in bone specimens after loss of PKC-δ in B cells. Finally, in vitro analysis revealed that PKC-δ ablation suppressed osteoclast differentiation and function but co-culture with B-cell supernatant reversed the suppression effect, as well as impaired osteoblast proliferation and function, indicative of osteoclast–osteoblast uncoupling. In conclusion, PKC-δ plays an important role in the interplay between B cells in the immune system and bone cells in the pathogenesis of bone lytic diseases.

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