Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation

Russo, Roberta; Mallia, Selene; Zito, Francesca; Lampiasi, Nadia

doi:10.3390/cells8020131

Cited by 15 publications

(21 citation statements)

References 42 publications

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“…The remarkable upregulation of NFATc1 mRNA observed in RAW 264.7 cells since the 1st day after RANKL stimulation is a hallmark aspect of osteoclastogenesis. By our previous qPCR and bioinformatic analyses, we found that NFATc1 significantly influences the expression of 31 genes, which are differently involved in osteoclastogenesis [ 9 ]. A variety of data in literature support the idea that the initial induction of NFATc1 protein depends upon the cooperation of NFATc2 and NF-κB (in particular, components p50 and p65, named RelA ), both recruited to the NFATc1 promoter at the very early phase of OC differentiation, i.e., 1 h after RANKL stimulation.…”

Section: Discussionmentioning

confidence: 99%

“…Given that the expression of DC-STAMP mRNA is upregulated from the 1st day of RANKL stimulation, we can hypothesize that RANKL-induced DC-STAMP expression is necessary to restore it on the surface of differentiating OCs as a response to its previous internalization, which is in agreement with data reported in literature [ 30 ]. The mRNA expression levels of DC-STAMP and NFATc1 peaked at the same time points after RANKL stimulation and NFATc1-knockdown significantly inhibited DC-STAMP expression [ 9 ]. Several results suggest that there is a mutual regulation between DC-STAMP and NFATc1 at both gene and protein expression levels [ 30 ].…”

Section: Discussionmentioning

confidence: 99%

“…NFATc1 has been shown to play a role as a master regulator of osteoclastogenesis, regulating the expression of numerous osteoclast-specific molecules involved in fusion, in differentiation, and maturation of multi-nucleated OCs, and in bone remodeling [ 8 ]. Our previous results showed that the expression of osteoclast hallmarks depends on the induction of NFATc1 and ingenuity pathway analysis (IPA) correlated NFATc1 with the activation of ERK and p-38 MAPKs [ 9 ].…”

Section: Introductionmentioning

confidence: 99%

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Osteoclasts Differentiation from Murine RAW 264.7 Cells Stimulated by RANKL: Timing and Behavior

Lampiasi

Russo

Kireev

et al. 2021

Biology

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The development of multi-nucleated cells is critical for osteoclasts (OCs) maturation and function. Our objective was to extend knowledge on osteoclastogenesis, focusing on pre-OC fusion timing and behavior. RAW 264.7 cells, which is a murine monocyte-macrophage cell line, provide a valuable and widely used tool for in vitro studies on osteoclastogenesis mechanisms. Cells were treated with the receptor activator of nuclear factor κ-B ligand (RANKL) for 1–4 days and effects on cell morphology, cytoskeletal organization, protein distribution, and OC-specific gene expression examined by TEM, immunofluorescence, and qPCR. Multinucleated cells began to appear at two days of Receptor Activator of Nuclear factor κ-B Ligand (RANKL) stimulation, increasing in number and size in the following days, associated with morphological and cytoskeletal organization changes. Interesting cellular extensions were observed in three days within cells labeled with wheat germ agglutinin (WGA)-Fluorescein isothiocyanate (FITC). The membrane, cytoplasmic, or nuclear distribution of RANK, TRAF6, p-p38, pERK1/2, and NFATc1, respectively, was related to OCs maturation timing. The gene expression for transcription factors regulating osteoclastogenesis (NFATc1, c-fos, RelA, MITF), molecules involved in RANKL-signaling transduction (TRAF6), cytoskeleton regulation (RhoA), fusion (DC-STAMP), migration (MMP9), and OC-specific enzymes (TRAP, CtsK), showed different trends related to OC differentiation timing. Our findings provide an integrated view on the morphological and molecular changes occurring during RANKL stimulation of RAW 264.7 cells, which are important to better understand the OCs’ maturation processes.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Osteoclasts Differentiation from Murine RAW 264.7 Cells Stimulated by RANKL: Timing and Behavior

Lampiasi

Russo

Kireev

et al. 2021

Biology

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“…M-CSF mainly promotes cellular proliferation, acting through ERK1/2-activation, whereas RANKL induces a switch versus differentiation [ 16 ]. It has been reported that RANKL, but not M-CSF, also induced a delayed (24 h) activation of p38 MAPKs that coincided with the initiation of OCs differentiation [ 16 , 17 ]. In particular, the axis RANKL-RANK-TRAF6-p38 MAP kinase promotes the expression of osteoclastogenic transcription factors, including NFATc1 [ 16 ].…”

Section: Introductionmentioning

confidence: 99%

Long-Lasting Activity of ERK Kinase Depends on NFATc1 Induction and Is Involved in Cell Migration-Fusion in Murine Macrophages RAW264.7

Russo

Mallia

Zito

et al. 2020

IJMS

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Macrophages are mononuclear cells that become osteoclasts (OCs) in the presence of two cytokines, macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL). RANKL binding to its specific receptor RANK leads to OCs differentiation mainly by nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). In our previous study, the analysis of the protein network in NFATc1-knockdown cells, using the Ingenuity Pathway Analysis (IPA), showed a link between NFATc1 and Mitogen-activated protein kinase kinase (MEK)-extracellular receptor kinase (ERK) signaling pathway. Therefore, this study aimed to extend our knowledge of the relationship between NFATc1 and the ERK. Here, we demonstrate that delayed ERK1/2 phosphorylation in pre-OC RANKL-induced depends on NFATc1. Indeed, the knockdown of NFATc1 reduced the phosphorylation of ERK1/2 (60%) and the pharmacological inhibition of the ERK1/2 kinase activity impairs the expression of NFATc1 without preventing its translocation into the nucleus. Furthermore, silencing of NFATc1 significantly reduced RANKL-induced migration (p < 0.01), and most pre-OCs are still mononuclear after 48 h (80 ± 5%), despite the presence of actin rings. On the other hand, the inhibitors FR180204 and PD98059 significantly reduced RANKL-induced cell migration (p < 0.01), leading to a reduction in the number of multinucleated cells. Finally, we suggest that long-lasting ERK activity depends on NFATc1 induction and is likely linked to cell migration, fusion, and OC differentiation.

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“…We further investigated the molecular mechanisms by which RANKL-induced osteoclastogenesis is increased by isorhamnetin 3-O-neohesperidoside. NFATc1 is a master transcription factor that regulates osteoclastogenesis 21,22 ; NFATc1-deficient osteoclast precursor cells failed to differentiate into osteoclasts in response to RANKL stimulation, while NFATc1 caused precursor cells to undergo efficient osteoclast differentiation without RANKL signalling 21,23,24 . Western blotting showed that the expression of NFATc1 increased gradually from day 1 to day 5 after RANKL induction.…”

Section: Discussionmentioning

confidence: 99%

Isorhamnetin 3-O-neohesperidoside promotes the resorption of crown-covered bone during tooth eruption by osteoclastogenesis

Zheng

Shang

et al. 2020

Sci Rep

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Delayed resorption of crown-covered bone is a critical cause of delayed tooth eruption. Traditional herbal medicines may be good auxiliary treatments to promote the resorption of crown-covered bone. This study was carried out to analyse the effect of isorhamnetin 3-O-neohesperidoside on receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis in vitro and resorption of the crown-covered bone of the lower first molars in mice in vivo. Isorhamnetin 3-O-neohesperidoside promoted osteoclastogenesis and the bone resorption of mouse bone marrow macrophages (BMMs) and upregulated mRNA expression of the osteoclast-specific genes cathepsin K (CTSK), vacuolartype H +-ATPase d2(V-ATPase d2), tartrate resistant acid phosphatase (TRAP) and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). NFATc1, p38 and AKT signalling was obviously activated by isorhamnetin 3-O-neohesperidoside in osteoclastogenesis. Isorhamnetin 3-O-neohesperidoside aggravated resorption of crown-covered bone in vivo. In brief, isorhamnetin 3-O-neohesperidoside might be a candidate adjuvant therapy for delayed intraosseous eruption. The development of osseous eruption is an indispensable stage in the tooth eruption process 1,2. Osteoclast differentiation is stimulated, causing the resorption of crown-covered bone of an erupting tooth, which forms an intraosseous eruption canal 3. Osteoclastogenesis in the crown-covered bone is essential. Impaired osseous eruption, in which osteoclastogenesis is disturbed, is common in clinical practice 4 and can manifest as either delayed or the complete absence of eruption 5,6. Although unerupted teeth are usually asymptomatic, they may cause cosmetic and pathologic complications 4 Treatments include orthodontic uprighting, surgical-orthodontic uprighting, surgical uprighting, surgical repositioning, surgical exposure or the removal of pathologic conditions 7. However, these treatments are very complex and invasive. Research shows that osteoclastogenesis is regulated by a key factor termed receptor activator of NF-κB ligand (RANKL). RANKL agonists or osteoclastogenesis-related drugs can be used to treat delayed tooth eruption 8,9. Traditional Chinese medicine, which is without toxic side effects may also be a good auxiliary treatment for delayed tooth eruption. Isorhamnetin 3-O-neohesperidin, known as Pu huang in Chinese 10 , is the main active substance of T. angustifolia, can also be isolated from the leaves of Acacia salicina 11. Because of its antioxidant, antiatherogenic and anti-inflammatory activities, Pu Huang has been widely used for the treatment of haematuria, dysmenorrhea, uterine bleeding and trauma in China for a long time 12. Isorhamnetin 3-O-neohesperidin has been reported to protect cells against oxidative stress by inhibiting H 2 O 2-induced genotoxicity and DNA damage induced by hydroxyl radicals 13. Intestinal flora including Escherichia sp. 23 and sp. 30, can convert isorhamnetin 3-O-neohesperidin to three main metabolites, isorhamnetin-3-O-glucoside(I3OG), isorhamnet...

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Gene Expression Profiling of NFATc1-Knockdown in RAW 264.7 Cells: An Alternative Pathway for Macrophage Differentiation

Cited by 15 publications

References 42 publications

Osteoclasts Differentiation from Murine RAW 264.7 Cells Stimulated by RANKL: Timing and Behavior

Osteoclasts Differentiation from Murine RAW 264.7 Cells Stimulated by RANKL: Timing and Behavior

Long-Lasting Activity of ERK Kinase Depends on NFATc1 Induction and Is Involved in Cell Migration-Fusion in Murine Macrophages RAW264.7

Isorhamnetin 3-O-neohesperidoside promotes the resorption of crown-covered bone during tooth eruption by osteoclastogenesis

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