scholarly journals MiRNAs Expression Profiling in Raw264.7 Macrophages after Nfatc1-Knockdown Elucidates Potential Pathways Involved in Osteoclasts Differentiation

Biology ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1080
Author(s):  
Roberta Russo ◽  
Francesca Zito ◽  
Nadia Lampiasi
Keyword(s):  
Potential Role ◽  
Osteoclast Differentiation ◽  
Negative Control ◽  
Differentially Expressed ◽  
Bone Homeostasis ◽  
Disease States ◽  
Raw264.7 Macrophages ◽  
Mirnas Expression ◽  
Rankl Stimulation

Differentiation of macrophages toward osteoclasts is crucial for bone homeostasis but can be detrimental in disease states, including osteoporosis and cancer. Therefore, understanding the osteoclast differentiation process and the underlying regulatory mechanisms may facilitate the identification of new therapeutic targets. Hereby, we tried to reveal new miRNAs potentially involved in the regulation of early steps of osteoclastogenesis, with a particular focus on those possibly correlated with NFATc1 expression, by studying miRNAs profiling. During the first 24 h of osteoclastogenesis, 38 miRNAs were differentially expressed between undifferentiated and RANKL-stimulated RAW264.7 cells, while 10 miRNAs were differentially expressed between RANKL-stimulated cells transfected with negative control or NFATc1-siRNAs. Among others, the expression levels of miR-411, miR-144 and members of miR-29, miR-30, and miR-23 families changed after RANKL stimulation. Moreover, the potential role of miR-124 during osteoclastogenesis was explored by transient cell transfection with anti-miR-124 or miR-124-mimic. Two relatively unknown miRNAs, miR-880-3p and miR-295-3p, were differentially expressed between RANKL-stimulated/wild-type and RANKL-stimulated/NFATc1-silenced cells, suggesting their possible correlation with NFATc1. KEGG enrichment analyses showed that kinase and phosphatase enzymes were among the predicted targets for many of the studied miRNAs. In conclusion, our study provides new data on the potential role and possible targets of new miRNAs during osteoclastogenesis.

scholarly journals FRI0376 EFFECT OF CARBAMYLATED LOW-DENSITY LIPOPROTEINS ON BONE CELLS HOMEOSTASIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 784.2-785
Author(s):  
B. Lucchino ◽  
M. Leopizzi ◽  
T. Colasanti ◽  
V. DI Maio ◽  
C. Alessandri ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Bone Cells ◽  
Osteoclast Differentiation ◽  
Low Density Lipoproteins ◽  
Tartrate Resistant Acid Phosphatase ◽  
Low Density ◽  
Bone Homeostasis ◽  
Research Support ◽  
Erosive Disease

Background:Carbamylation is a post-translational modification occurring under several conditions such as uremia, smoking and chronic inflammation as in rheumatoid arthritis (RA). Low-density lipoproteins (LDL) represent a target of carbamylation. Carbamylated-LDL (cLDL) have an increased inflammatory and atherogenic potential. Growing evidence supports an influence of modified lipids on bone cells homeostasis. However, the role of cLDL on bone cells physiology is still unknown.Objectives:Considering the rate of carbamylation and the role of anti-carbamylated proteins antibodies as markers of erosive disease in RA, the purpose of this study is to investigate the effect of cLDL on bone homeostasis.Methods:In-vitrocarbamylation of LDL was performed as previously described by Ok et al. (Kidney Int. 2005). Briefly, native LDL (nLDL) were treated with potassium cyanate (KOCN) for 4 hours, followed by excessive dialysis for 36 hours to remove KOCN. Both osteoclasts (OCs) and osteoblasts (OBLs) were treated at baseline with 20 μg/ml, 100 μg/ml and 200 μg/ml of cLDL or nLDL. To induce osteoclast differentiation, CD14+ monocytes were isolated from peripheral blood of healthy donors by magnetic microbeads separation and then cultured on a 96-wells plate in DMEM media supplemented with RANKL and M-CSF. After 10 days cells were fixed, stained for tartrate-resistant acid phosphatase (TRAP), a marker of OC differentiation, and counted. OBLs were isolated from bone specimens of 3 patients who had undergone to knee or hip arthroplasty for osteoarthritis and treated for 5 days with different concentrations of cLDL and nLDL. OBLs were fixed and stained for alkaline phosphatase positive activity (ALP), a marker of osteogenic differentiation. Total RNA was extracted from cell lysates. Copies of single-stranded complementary DNA (cDNA) were synthesized and analyzed by real-time PCR to evaluate RANKL and Osteoprotegerin (OPG) mRNA expression levels.Results:In OCLs culture, cLDL significantly decreased the number of OC compared to untreated cells (200 μg/ml p=0,0015) and nLDL treated cells (200 μg/ml p= 0,011; 20 μg/ml p= 0,0014) (Fig 1). Moreover, treatment with cLDL induced an increase of not terminally differentiated OCs, reduced dimensions of OCs, less intense TRAP staining and vacuolization (Fig 2). In OBLs culture, cLDL (20, 100 μg/ml) significantly reduced the ALP activity of OBLs compared with untreated cells (p<0.05) (Fig 3). nLDL did not affect the ALP expression. Treatment with cLDL stimulated RANKL mRNA expression in osteoblasts increasing the RANKL/OPG ratio (Fig 4).Fig 1.Fig 2.Fig 3.Fig 4.Conclusion:cLDL induce a significant depression of OC and OBL differentiation. Moreover, cLDL increase RANKL expression in OBL, unbalancing bone tissue turnover towards bone resorption. Accordingly, cLDL could be implicated in the bone loss characterizing several conditions associated to an increased carbamylation, such as RADisclosure of Interests:Bruno Lucchino: None declared, Martina Leopizzi: None declared, Tania Colasanti: None declared, Valeria Di Maio: None declared, cristiano alessandri Grant/research support from: Pfizer, Guido Valesini: None declared, fabrizio conti Speakers bureau: BMS, Lilly, Abbvie, Pfizer, Sanofi, Manuela Di Franco: None declared, Francesca Romana Spinelli Grant/research support from: Pfizer, Consultant of: Novartis, Gilead, Lilly, Sanofi, Celgene, Speakers bureau: Lilly

scholarly journals Sorting Mechanisms for MicroRNAs into Extracellular Vesicles and Their Associated Diseases

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1044 ◽  
Author(s):  
Michael Groot ◽  
Heedoo Lee
Keyword(s):  
Extracellular Vesicles ◽  
Potential Role ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Transport Proteins ◽  
Caveolin 1 ◽  
Argonaute 2 ◽  
Disease States ◽  
Mirna Content

Extracellular vesicles (EV) are secretory membranous elements used by cells to transport proteins, lipids, mRNAs, and microRNAs (miRNAs). While their existence has been known for many years, only recently has research begun to identify their function in intercellular communication and gene regulation. Importantly, cells have the ability to selectively sort miRNA into EVs for secretion to nearby or distant targets. These mechanisms broadly include RNA-binding proteins such as hnRNPA2B1 and Argonaute-2, but also membranous proteins involved in EV biogenesis such as Caveolin-1 and Neural Sphingomyelinase 2. Moreover, certain disease states have also identified dysregulated EV-miRNA content, shedding light on the potential role of selective sorting in pathogenesis. These pathologies include chronic lung disease, immune response, neuroinflammation, diabetes mellitus, cancer, and heart disease. In this review, we will overview the mechanisms whereby cells selectively sort miRNA into EVs and also outline disease states where EV-miRNAs become dysregulated.

The regulation of E3 ubiquitin ligases Cbl and its cross talking in bone homeostasis

2020 ◽  
Vol 15 ◽  
Author(s):  
Xiaobin Yang ◽  
Dingjun Hao ◽  
Baorong He
Keyword(s):  
Cellular Proliferation ◽  
Bone Matrix ◽  
Osteoclast Differentiation ◽  
Ubiquitin Ligases ◽  
E3 Ubiquitin Ligases ◽  
Bone Homeostasis ◽  
And Migration ◽  
First Time ◽  
Definite Role

: The E3 ubiquitin ligases Cbl has been found play an important role in regulating cellular proliferation and migration. Whereas the excessive differentiation of osteoclast and/or its over expressing of resorptive functions could lead the pathological bone homeostasis by overly bone matrix degradation. Since the first time of the important role of Cbl in the regulating osteoclast differentiation (also named osteoclastogenesis) has been reported in decades ago. The extensively studies have been conducted for in-depth exploring the Cbl’s definite role during osteoclastogenesis, as well as its cross talking with other signaling pathways (such as: Src and PI3K signaling) in bone homeostasis. Herein, our current study aim to briefly conclude the current studies of osteoclastogenesis and the regulatory role of Cbl, as well as its cross-talking in bone homeostasis.

Long Noncoding RNA Expression Profiling By Microarray in Diffuse Large B-Cell Lymphoma and Preliminary Bioinformatics Study

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5286-5286
Author(s):  
Honghui Huang ◽  
Fei Xiao ◽  
Lijing Shen ◽  
Xiaofeng Han ◽  
Fangyuan Chen ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Expression Profiling ◽  
Kegg Pathway ◽  
Expression Profiles ◽  
B Cell Lymphoma ◽  
Differentially Expressed ◽  
Newly Diagnosed ◽  
Disease States ◽  
Mrna Expression Profiling

Abstract Objective: Application of high-throughput genomics technology to analysis the differences in lncRNA/mRNA expression profiles between the patients with diffuse large B cell lymphoma (DLBCL) and reactive hyperplasia of lymph node (RLN), and between different disease states of DLBCL, in order to explore the role of the aberrantly expressed lncRNAs in the pathogenesis of DLBCL. Materials and methods: Tumor tissue samples from five newly diagnosed DLBCL, 3 relapsed DLBCL patients and 4 samples of RLN were collected. Agilent Human lncRNA/mRNA Microarray (4×180K) was used to profile differentially expression of lncRNA/mRNAs. Bioinformatics methods were performed for data analysis, including screening of differentially expressed lncRNA/mRNAs, gene ontology (GO) and KEGG pathway enrichment analysis, and lncRNA/mRNA co-expression network construction. Portion of dysregulated lncRNAs from the microarray data analysis was selected for validation using quantitative real-time polymerase chain reaction (qRT-PCR). Results: (1) In patients with newly diagnosed DLBCL, compared to RLN, a total of 5,806 dysregulated lncRNAs were identified (P<0.05, FC≥2-fold). Among these, 2,650 lncRNAs were significantly upregulated, while 3,156 lncRNAs were significantly downregulated. From the mRNA expression profiling data, 5,628 differently expressed mRNAs were found (P<0.05, FC≥2-fold). Among them, 2,045 were significantly upregulated and 3,583 downregulated. The most enriched GO term and KEGG pathway annotation associated with DLBCL was cell cycle regulation. The coding and non-coding gene expression network construction showed the significantly correlation between lnc-FAM27B-22:1 and mRNAs, including CDCA7L, CEBPB, CHRDL1 and SMIM9. We speculated that lnc-FAM27B-22:1 might have a critical function in pathogenesis of DLBCL. (2) In patients with primary refractory DLBCL, compared with chemo-sensitive patients, a total of 425 dysregulated lncRNAs were identified(P<0.05, FC≥2-fold). Among these, 124 lncRNAs were significantly upregulated, while 301 lncRNAs were significantly downregulated. From the mRNA expression profiling data, 251 differently expressed mRNAs were found(P<0.05, FC≥2-fold). Among them, 90 were significantly upregulated and 106 downregulated. The most enriched GO terms targeted by dysregulated transcripts were involved in a variety of functions. KEGG pathway analysis showed that 25 pathways corresponded to dysregulated transcripts and the most enriched networks correlated with tumor were Wnt and P53 signaling pathway. (3) In patients with relapsed DLBCL, compared with newly diagnosed patients, a total of 605 dysregulated lncRNAs were identified(P<0.05, FC≥2-fold). Among these, 169 lncRNAs were significantly upregulated, while 436 lncRNAs were significantly downregulated. From the mRNA expression profiling data, 365 differently expressed mRNAs were found(P<0.05, FC≥2-fold). Among them, 201 were significantly upregulated and 164 downregulated. The most enriched GO terms targeted by dysregulated transcripts were involved in a variety of functions. KEGG pathway analysis showed that 24 pathways corresponded to dysregulated transcripts and the most enriched network correlated with tumor was JAK-STAT signaling pathway. (4) Fourteen dysregulated lncRNAs expressions were analyzed in the same sample series using qRT-PCR to validate the microarray analysis results. In all but one lncRNA, consistent trend of expression changes was observed in two methods. Conclusions: In patients with newly diagnosed DLBCL versus RLN, and between different disease states of DLBCL, the lncRNA/mRNA expression profiles were significantly altered. Differentially expressed mRNAs are involved in a number of biological functions and signaling pathways. It prompt lncRNA may play an important role in the mechanism of occurrence, drug resistance and relapse of DLBCL. The above results provide the theory basis for the further study on the role of differentially expressed lncRNA in the development of DLBCL and its potential application as the possible biomarkers of early diagnosis and prognosis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Autoamplification of NFATc1 expression determines its essential role in bone homeostasis

2005 ◽  
Vol 202 (9) ◽  
pp. 1261-1269 ◽  
Author(s):  
Masataka Asagiri ◽  
Kojiro Sato ◽  
Takako Usami ◽  
Sae Ochi ◽  
Hiroshi Nishina ◽  
...  
Keyword(s):  
Essential Role ◽  
Osteoclast Differentiation ◽  
Ectopic Expression ◽  
Epigenetic Mechanism ◽  
Bone Homeostasis ◽  
Hematopoietic Stem ◽  
Vitro Effect

NFATc1 and NFATc2 are functionally redundant in the immune system, but it was suggested that NFATc1 is required exclusively for differentiation of osteoclasts in the skeletal system. Here we provide genetic evidence that NFATc1 is essential for osteoclast differentiation in vivo by adoptive transfer of NFATc1−/− hematopoietic stem cells to osteoclast-deficient Fos−/− mice, and by Fos−/− blastocyst complementation, thus avoiding the embryonic lethality of NFATc1−/− mice. However, in vitro osteoclastogenesis in NFATc1-deficient cells was rescued by ectopic expression of NFATc2. The discrepancy between the in vivo essential role of NFATc1 and the in vitro effect of NFATc2 was attributed to selective autoregulation of the NFATc1 gene by NFAT through its promoter region. This suggested that an epigenetic mechanism contributes to the essential function of NFATc1 in cell lineage commitment. Thus, this study establishes that NFATc1 represents a potential therapeutic target for bone disease and reveals a mechanism that underlies the essential role of NFATc1 in bone homeostasis.

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.

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