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1.
Nat Commun ; 15(1): 7324, 2024 Aug 25.
Article in English | MEDLINE | ID: mdl-39183203

ABSTRACT

During the progression of proliferative vitreoretinopathy (PVR) following ocular trauma, previously quiescent retinal pigment epithelial (RPE) cells transition into a state of rapid proliferation, migration, and secretion. The elusive molecular mechanisms behind these changes have hindered the development of effective pharmacological treatments, presenting a pressing clinical challenge. In this study, by monitoring the dynamic changes in chromatin accessibility and various histone modifications, we chart the comprehensive epigenetic landscape of RPE cells in male mice subjected to traumatic PVR. Coupled with transcriptomic analysis, we reveal a robust correlation between enhancer activation and the upregulation of the PVR-associated gene programs. Furthermore, by constructing transcription factor regulatory networks, we identify the aberrant activation of enhancer-driven RANK-NFATc1 pathway as PVR advanced. Importantly, we demonstrate that intraocular interventions, including nanomedicines inhibiting enhancer activity, gene therapies targeting NFATc1 and antibody therapeutics against RANK pathway, effectively mitigate PVR progression. Together, our findings elucidate the epigenetic basis underlying the activation of PVR-associated genes during RPE cell fate transitions and offer promising therapeutic avenues targeting epigenetic modulation and the RANK-NFATc1 axis for PVR management.


Subject(s)
NFATC Transcription Factors , Retinal Pigment Epithelium , Signal Transduction , Vitreoretinopathy, Proliferative , Animals , Vitreoretinopathy, Proliferative/metabolism , Vitreoretinopathy, Proliferative/genetics , Vitreoretinopathy, Proliferative/pathology , Retinal Pigment Epithelium/metabolism , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Mice , Male , Mice, Inbred C57BL , Humans , Enhancer Elements, Genetic/genetics , Epigenesis, Genetic , Disease Models, Animal , Eye Injuries/metabolism , Eye Injuries/genetics , Eye Injuries/pathology , Gene Expression Profiling , Multiomics
2.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 49(5): 655-666, 2024 May 28.
Article in English, Chinese | MEDLINE | ID: mdl-39174879

ABSTRACT

OBJECTIVES: Progressive bone resorption and destruction is one of the most critical clinical features of middle ear cholesteatoma, potentially leading to various intracranial and extracranial complications. However, the mechanisms underlying bone destruction in middle ear cholesteatoma remain unclear. This study aims to explore the role of parathyroid hormone-related protein (PTHrP) in bone destruction associated with middle ear cholesteatoma. METHODS: A total of 25 cholesteatoma specimens and 13 normal external auditory canal skin specimens were collected from patients with acquired middle ear cholesteatoma. Immunohistochemical staining was used to detect the expressions of PTHrP, receptor activator for nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) in cholesteatoma and normal tissues. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the presence of TRAP positive multi-nucleated macrophages in cholesteatoma and normal tissues. Mono-nuclear macrophage RAW264.7 cells were subjected to interventions, divided into a RANKL intervention group and a PTHrP+ RANKL co-intervention group. TRAP staining was used to detect osteoclast formation in the 2 groups. The mRNA expression levels of osteoclast-related genes, including TRAP, cathepsin K (CTSK), and nuclear factor of activated T cell cytoplasmic 1 (NFATc1), were measured using real-time polymerase chain reaction (real-time PCR) after the interventions. Bone resorption function of osteoclasts was assessed using a bone resorption pit analysis. RESULTS: Immunohistochemical staining showed significantly increased expression of PTHrP and RANKL and decreased expression of OPG in cholesteatoma tissues (all P<0.05). PTHrP expression was significantly positively correlated with RANKL, the RANKL/OPG ratio, and negatively correlated with OPG expression (r=0.385, r=0.417, r=-0.316, all P<0.05). Additionally, the expression levels of PTHrP and RANKL were significantly positively correlated with the degree of bone destruction in cholesteatoma (r=0.413, r=0.505, both P<0.05). TRAP staining revealed a large number of TRAP-positive cells, including multi-nucleated osteoclasts with three or more nuclei, in the stroma surrounding the cholesteatoma epithelium. After 5 days of RANKL or PTHrP+RANKL co-intervention, the number of osteoclasts was significantly greater in the PTHrP+RANKL co-intervention group than that in the RANKL group (P<0.05), with increased mRNA expression levels of TRAP, CTSK, and NFATc1 (all P<0.05). Scanning electron microscopy of bone resorption pits showed that the number (P<0.05) and size of bone resorption pits on bone slices were significantly greater in the PTHrP+RANKL co-intervention group compared with the RANKL group. CONCLUSIONS: PTHrP may promote the differentiation of macrophages in the surrounding stroma of cholesteatoma into osteoclasts through RANKL induction, contributing to bone destruction in middle ear cholesteatoma.


Subject(s)
Bone Resorption , Cell Differentiation , Cholesteatoma, Middle Ear , Macrophages , Osteoclasts , Osteoprotegerin , Parathyroid Hormone-Related Protein , RANK Ligand , Animals , Humans , Male , Mice , Bone Resorption/metabolism , Cholesteatoma, Middle Ear/metabolism , Cholesteatoma, Middle Ear/pathology , Macrophages/metabolism , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Osteoclasts/metabolism , Osteoprotegerin/metabolism , Parathyroid Hormone-Related Protein/metabolism , RANK Ligand/metabolism , RANK Ligand/genetics , RAW 264.7 Cells
3.
Nat Commun ; 15(1): 5994, 2024 Jul 17.
Article in English | MEDLINE | ID: mdl-39013863

ABSTRACT

Chromatin remodeler ARID1A regulates gene transcription by modulating nucleosome positioning and chromatin accessibility. While ARID1A-mediated stage and lineage-restricted gene regulation during cell fate canalization remains unresolved. Using osteoclastogenesis as a model, we show that ARID1A transcriptionally safeguards the osteoclast (OC) fate canalization during proliferation-differentiation switching at single-cell resolution. Notably, ARID1A is indispensable for the transcriptional apparatus condensates formation with coactivator BRD4/lineage-specifying transcription factor (TF) PU.1 at Nfatc1 super-enhancer during safeguarding the OC fate canalization. Besides, the antagonist function between ARID1A-cBAF and BRD9-ncBAF complex during osteoclastogenesis has been validated with in vitro assay and compound mutant mouse model. Furthermore, the antagonistic function of ARID1A-"accelerator" and BRD9-"brake" both depend on coactivator BRD4-"clutch" during osteoclastogenesis. Overall, these results uncover sophisticated cooperation between chromatin remodeler ARID1A, coactivator, and lineage-specifying TF at super-enhancer of lineage master TF in a condensate manner, and antagonist between distinct BAF complexes in the proper and balanced cell fate canalization.


Subject(s)
Cell Differentiation , Cell Lineage , DNA-Binding Proteins , Osteoclasts , Osteogenesis , Transcription Factors , Animals , Transcription Factors/metabolism , Transcription Factors/genetics , Osteoclasts/metabolism , Osteoclasts/cytology , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Mice , Osteogenesis/genetics , Osteogenesis/physiology , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Chromatin Assembly and Disassembly , Gene Expression Regulation , Mice, Inbred C57BL , Cell Proliferation , Single-Cell Analysis , Bromodomain Containing Proteins , Nuclear Proteins
4.
Am J Physiol Cell Physiol ; 327(3): C545-C556, 2024 Sep 01.
Article in English | MEDLINE | ID: mdl-38946247

ABSTRACT

Euryhaline fish experience variable osmotic environments requiring physiological adjustments to tolerate elevated salinity. Mozambique tilapia (Oreochromis mossambicus) possess one of the highest salinity tolerance limits of any fish. In tilapia and other euryhaline fish species, the myo-inositol biosynthesis (MIB) pathway enzymes, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1.1), are among the most upregulated mRNAs and proteins indicating the high importance of this pathway for hyperosmotic (HO) stress tolerance. These abundance changes must be precluded by HO perception and signaling mechanism activation to regulate the expression of MIPS and IMPA1.1 genes. In previous work using a O. mossambicus cell line (OmB), a reoccurring osmosensitive enhancer element (OSRE1) in both MIPS and IMPA1.1 was shown to transcriptionally upregulate these enzymes in response to HO stress. The OSRE1 core consensus (5'-GGAAA-3') matches the core binding sequence of the predominant mammalian HO response transcription factor, nuclear factor of activated T-cells (NFAT5). HO-challenged OmB cells showed an increase in NFAT5 mRNA suggesting NFAT5 may contribute to MIB pathway regulation in euryhaline fish. Ectopic expression of wild-type NFAT5 induced an IMPA1.1 promoter-driven reporter by 5.1-fold (P < 0.01). Moreover, expression of dominant negative NFAT5 in HO media resulted in a 47% suppression of the reporter signal (P < 0.005). Furthermore, reductions of IMPA1.1 (37-49%) and MIPS (6-37%) mRNA abundance were observed in HO-challenged NFAT5 knockout cells relative to control cells. Collectively, these multiple lines of experimental evidence establish NFAT5 as a tilapia transcription factor contributing to HO-induced activation of the MIB pathway.NEW & NOTEWORTHY In our study, we use a multi-pronged synthetic biology approach to demonstrate that the fish homolog of the predominant mammalian osmotic stress transcription factor nuclear factor of activated T-cells (NFAT5) also contributes to the activation of hyperosmolality inducible genes in cells of extremely euryhaline fish. However, in addition to NFAT5 the presence of other strong osmotically inducible signaling mechanisms is required for full activation of osmoregulated tilapia genes.


Subject(s)
Inositol , Myo-Inositol-1-Phosphate Synthase , Osmotic Pressure , Tilapia , Up-Regulation , Animals , Tilapia/genetics , Tilapia/metabolism , Inositol/metabolism , Myo-Inositol-1-Phosphate Synthase/genetics , Myo-Inositol-1-Phosphate Synthase/metabolism , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Fish Proteins/genetics , Fish Proteins/metabolism , Cell Line , Signal Transduction , Transcription, Genetic , Osmoregulation/genetics , Transcriptional Activation
5.
Sci Rep ; 14(1): 15678, 2024 07 08.
Article in English | MEDLINE | ID: mdl-38977785

ABSTRACT

Aging and lack of exercise are the most important etiological factors for muscle loss. We hypothesized that new factors that contribute to muscle loss could be identified from ones commonly altered in expression in aged and exercise-limited skeletal muscles. Mouse gastrocnemius muscles were subjected to mass spectrometry-based proteomic analysis. The muscle proteomes of hindlimb-unloaded and aged mice were compared to those of exercised and young mice, respectively. C1qbp expression was significantly upregulated in the muscles of both hindlimb-unloaded and aged mice. In vitro myogenic differentiation was not affected by altering intracellular C1qbp expression but was significantly suppressed upon recombinant C1qbp treatment. Additionally, recombinant C1qbp repressed the protein level but not the mRNA level of NFATc1. NFATc1 recruited the transcriptional coactivator p300, leading to the upregulation of acetylated histone H3 levels. Furthermore, NFATc1 silencing inhibited p300 recruitment, downregulated acetylated histone H3 levels, and consequently suppressed myogenic differentiation. The expression of C1qbp was inversely correlated with that of NFATc1 in the gastrocnemius muscles of exercised or hindlimb-unloaded, and young or aged mice. These findings demonstrate a novel role of extracellular C1qbp in suppressing myogenesis by inhibiting the NFATc1/p300 complex. Thus, C1qbp can serve as a novel therapeutic target for muscle loss.


Subject(s)
Muscle Development , Muscle, Skeletal , NFATC Transcription Factors , Animals , Male , Mice , Acetylation , Cell Differentiation , Histones/metabolism , Mice, Inbred C57BL , Muscle Development/genetics , Muscle, Skeletal/metabolism , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics
6.
Endocrinology ; 165(8)2024 Jul 01.
Article in English | MEDLINE | ID: mdl-39024412

ABSTRACT

Osteocytes are embedded in lacunae and connected by canaliculi (lacuno-canalicular network, LCN). Bones from mice with X-linked hypophosphatemia (Hyp), which have impaired production of 1,25 dihydroxyvitamin D (1,25D) and hypophosphatemia, have abnormal LCN structure that is improved by treatment with 1,25D or an anti-FGF23 targeting antibody, supporting roles for 1,25D and phosphate in regulating LCN remodeling. Bones from mice lacking the vitamin D receptor (VDR) in osteocytes (Vdrf/f;Dmp1Cre+) and mice lacking the sodium phosphate transporter 2a (Npt2aKO), which have low serum phosphate with high serum 1,25D, have impaired LCN organization, demonstrating that osteocyte-specific actions of 1,25D and hypophosphatemia regulate LCN remodeling. In osteoclasts, nuclear factor of activated T cells cytoplasmic 1 (NFATc1) is critical for stimulating bone resorption. Since osteocytes also resorb matrix, we hypothesize that NFATc1 plays a role in 1,25D and phosphate-mediated LCN remodeling. Consistent with this, 1,25D and phosphate suppress Nfatc1 mRNA expression in IDG-SW3 osteocytes, and knockdown of Nfatc1 expression in IDG-SW3 cells blocks 1,25D- and phosphate-mediated suppression of matrix resorption gene expression and 1,25D- and phosphate-mediated suppression of RANKL-induced acidification of the osteocyte microenvironment. To determine the role of NFATc1 in 1,25D- and phosphate-mediated LCN remodeling in vivo, histomorphometric analyses of tibiae from mice lacking osteocyte-specific Nfatc1 in Vdrf/f;Dmp1Cre+ and Npt2aKO mice were performed, demonstrating that bones from these mice have decreased lacunar size and expression of matrix resorption genes, and improved canalicular structure compared to Vdrf/f;Dmp1Cre+ and Npt2aKO control. This study demonstrates that NFATc1 is necessary for 1,25D- and phosphate-mediated regulation of LCN remodeling.


Subject(s)
Bone Remodeling , Fibroblast Growth Factor-23 , NFATC Transcription Factors , Osteocytes , Phosphates , Vitamin D , Animals , Male , Mice , Bone Remodeling/drug effects , Familial Hypophosphatemic Rickets/metabolism , Familial Hypophosphatemic Rickets/genetics , Mice, Inbred C57BL , Mice, Knockout , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Osteocytes/metabolism , Osteocytes/drug effects , Phosphates/metabolism , Receptors, Calcitriol/metabolism , Receptors, Calcitriol/genetics , Sodium-Phosphate Cotransporter Proteins, Type IIa/genetics , Sodium-Phosphate Cotransporter Proteins, Type IIa/metabolism , Vitamin D/pharmacology , Vitamin D/analogs & derivatives , Female
7.
FASEB J ; 38(13): e23779, 2024 Jul 15.
Article in English | MEDLINE | ID: mdl-38967255

ABSTRACT

Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre-expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl (Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), the number of TRAP-positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild-type (WT) G9a-expressing adenovirus in G9a KO cells restored the number of TRAP-positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For in vivo experiments, we created G9a conditional knock-out (cKO) mice by crossing G9afl/fl mice with Rank Cre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C-terminal telopeptide of type I collagen (CTX), a bone-resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone-resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function.


Subject(s)
Bone Resorption , Cell Differentiation , Histone-Lysine N-Methyltransferase , NFATC Transcription Factors , Osteoclasts , Osteogenesis , Animals , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Mice , Osteoclasts/metabolism , Bone Resorption/metabolism , Osteogenesis/physiology , Mice, Knockout , RANK Ligand/metabolism , Mice, Inbred C57BL , Cells, Cultured
8.
Sci Signal ; 17(845): eadg4124, 2024 Jul 16.
Article in English | MEDLINE | ID: mdl-39012937

ABSTRACT

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus that is linked directly to the development of Kaposi's sarcoma. KSHV establishes a latent infection in B cells, which can be reactivated to initiate lytic replication, producing infectious virions. Using pharmacological and genetic silencing approaches, we showed that the voltage-gated K+ channel Kv1.3 in B cells enhanced KSHV lytic replication. The KSHV replication and transcription activator (RTA) protein increased the abundance of Kv1.3 and led to enhanced K+ channel activity and hyperpolarization of the B cell membrane. Enhanced Kv1.3 activity promoted intracellular Ca2+ influx, leading to the Ca2+-driven nuclear localization of KSHV RTA and host nuclear factor of activated T cells (NFAT) proteins and subsequently increased the expression of NFAT1 target genes. KSHV lytic replication and infectious virion production were inhibited by Kv1.3 blockers or silencing. These findings highlight Kv1.3 as a druggable host factor that is key to the successful completion of KSHV lytic replication.


Subject(s)
Herpesvirus 8, Human , Kv1.3 Potassium Channel , NFATC Transcription Factors , Virus Replication , Herpesvirus 8, Human/physiology , Herpesvirus 8, Human/genetics , Herpesvirus 8, Human/metabolism , Humans , Kv1.3 Potassium Channel/metabolism , Kv1.3 Potassium Channel/genetics , Kv1.3 Potassium Channel/antagonists & inhibitors , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Immediate-Early Proteins/metabolism , Immediate-Early Proteins/genetics , Trans-Activators/metabolism , Trans-Activators/genetics , B-Lymphocytes/virology , B-Lymphocytes/metabolism , Calcium/metabolism , Sarcoma, Kaposi/virology , Sarcoma, Kaposi/metabolism , Sarcoma, Kaposi/genetics
9.
Inflamm Res ; 73(9): 1581-1599, 2024 Sep.
Article in English | MEDLINE | ID: mdl-39052064

ABSTRACT

OBJECTIVE AND DESIGN: The exact immunological mechanism of widespread chronic inflammatory skin disorder psoriasis has not been fully established. CD11b+Gr.1+ myeloid-derived cells are immature heterogeneous cells with T-cell suppressive property in neoplasia; however, influence of these cells on adaptive immunity is highly contextual; therefore, we dubbed these cells as myeloid-derived adjuster cells (MDAC). We studied imiquimod induced psoriasis in mouse model and evaluated for the first time the RORγt-NFAT1 axis in MDACs and the function, differentiation and interaction of these cells with T cells. MATERIALS AND METHODS: The status of T cells and MDACs; their functionality and differentiation properties, and the roles of RORγt and NFAT1 in MDACs were evaluated using flow cytometry, qRT-PCR and confocal imaging. RESULTS: We found gradual increase in T cells and MDACs and an increase in the number of IL17 -secreting MDACs and T cells in the skin of psoriatic animals. We also noted that MDAC differentiation is biased toward M1 macrophages and DCs which perpetuate inflammation. We found that psoriatic MDACs were unable to suppress T-cell proliferation or activation but seemingly helped these T cells produce more IL17. Inhibition of the RORγt/NFAT1 axis in MDACs increased the suppressive nature of MDACs, allowing these cells to suppress the activity of psoriatic T-cells. CONCLUSION: Our results indicate that altered MDAC properties in psoriatic condition sustains pathological inflammation and RORγt and NFAT1 as promising intervention target for psoriasis management.


Subject(s)
CD11b Antigen , Cell Differentiation , Imiquimod , Interleukin-17 , NFATC Transcription Factors , Nuclear Receptor Subfamily 1, Group F, Member 3 , Psoriasis , Animals , Mice , Antigens, Ly , CD11b Antigen/metabolism , Cell Differentiation/drug effects , Inflammation/chemically induced , Interleukin-17/metabolism , Mice, Inbred BALB C , Myeloid Cells/drug effects , Myeloid Cells/metabolism , Myeloid Cells/immunology , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Phenotype , Psoriasis/chemically induced , Psoriasis/immunology , Skin/pathology , Skin/immunology , Skin/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/drug effects , Male
11.
Exp Mol Med ; 56(8): 1791-1806, 2024 Aug.
Article in English | MEDLINE | ID: mdl-39085359

ABSTRACT

Bone homeostasis is maintained by an intricate balance between osteoclasts and osteoblasts, which becomes disturbed in osteoporosis. Metallothioneins (MTs) are major contributors in cellular zinc regulation. However, the role of MTs in bone cell regulation has remained unexplored. Single-cell RNA sequencing analysis discovered that, unlike the expression of other MT members, the expression of MT3 was unique to osteoclasts among various macrophage populations and was highly upregulated during osteoclast differentiation. This unique MT3 upregulation was validated experimentally and supported by ATAC sequencing data analyses. Downregulation of MT3 by gene knockdown or knockout resulted in excessive osteoclastogenesis and exacerbated bone loss in ovariectomy-induced osteoporosis. Transcriptome sequencing of MT3 knockdown osteoclasts and gene set enrichment analysis indicated that the oxidative stress and redox pathways were enriched, which was verified by MT3-dependent regulation of reactive oxygen species (ROS). In addition, MT3 deficiency increased the transcriptional activity of SP1 in a manner dependent on intracellular zinc levels. This MT3-zinc-SP1 axis was crucial for the control of osteoclasts, as zinc chelation and SP1 knockdown abrogated the promotion of SP1 activity and osteoclastogenesis by MT3 deletion. Moreover, SP1 bound to the NFATc1 promoter, and overexpression of an inactive SP1 mutant negated the effects of MT3 deletion on NFATc1 and osteoclastogenesis. In conclusion, MT3 plays a pivotal role in controlling osteoclastogenesis and bone metabolism via dual axes involving ROS and SP1. The present study demonstrated that MT3 elevation is a potential therapeutic strategy for osteolytic bone disorders, and it established for the first time that MT3 is a crucial bone mass regulator.


Subject(s)
Metallothionein 3 , Osteoclasts , Osteogenesis , Osteoporosis , Animals , Osteoporosis/metabolism , Osteoporosis/genetics , Osteoporosis/pathology , Osteoporosis/etiology , Osteogenesis/genetics , Mice , Osteoclasts/metabolism , Metallothionein 3/metabolism , Cell Differentiation/genetics , Female , Gene Expression Regulation , Reactive Oxygen Species/metabolism , Sp1 Transcription Factor/metabolism , Sp1 Transcription Factor/genetics , Zinc/metabolism , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Metallothionein/metabolism , Metallothionein/genetics , Mice, Knockout
12.
Nutr Metab Cardiovasc Dis ; 34(11): 2455-2463, 2024 Nov.
Article in English | MEDLINE | ID: mdl-39069466

ABSTRACT

BACKGROUND AND AIMS: Adipose tissue (AT) serves as a vital energy storage site and plays a pivotal role in metabolic regulation, exhibiting a high response to insulin. Impairment in this response may closely associate with obesity, and NFAT (nuclear factor of activated T cells) family genes may be involved in the process. However, human data linking NFAT and AT remains elusive. The aim of this study was to assess the expression of NFAT family genes and markers of adipogenesis in subcutaneous adipose tissue (SAT) among normal-weight and overweight/obese individuals before and after weight loss, in relation to insulin sensitivity. METHODS AND RESULTS: The study included 45 participants, 15 normal-weight (control group) and 30 overweight or obese, who underwent a 12-week dietary intervention (DI) program. Before and after the program hyperinsulinemic-euglycemic clamp and SAT biopsy were conducted. Before DI, a positive correlations was observed in the expression of NFATc1, NFATc4, and NFAT5 with insulin sensitivity. The expression of NFAT family genes and markers of adipogenesis in SAT was lower in individuals with overweight or obesity compared to normal-weight. Additionally, a positive correlation was noted between NFAT family genes and adipogenesis markers both before and after weight loss. Following the DI program, there was an increase in the expression of NFATc3, NFATc4, and NFAT5 in SAT. CONCLUSION: Decreased SAT expression of NFAT genes in obesity is partly reversed in response to weight loss. NFAT genes in SAT are associated with insulin sensitivity and adipogenesis. Registration number for clinical trial: NCT01393210.


Subject(s)
Adipogenesis , Insulin Resistance , NFATC Transcription Factors , Obesity , Subcutaneous Fat , Weight Loss , Humans , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Weight Loss/genetics , Obesity/genetics , Obesity/metabolism , Subcutaneous Fat/metabolism , Male , Adult , Female , Adipogenesis/genetics , Insulin Resistance/genetics , Middle Aged , Treatment Outcome , Case-Control Studies , Time Factors
13.
Adv Exp Med Biol ; 1441: 885-900, 2024.
Article in English | MEDLINE | ID: mdl-38884757

ABSTRACT

The process of valve formation is a complex process that involves intricate interplay between various pathways at precise times. Although we have not completely elucidated the molecular pathways that lead to normal valve formation, we have identified a few major players in this process. We are now able to implicate TGF-ß, BMP, and NOTCH as suspects in tricuspid atresia (TA), as well as their downstream targets: NKX2-5, TBX5, NFATC1, GATA4, and SOX9. We know that the TGF-ß and the BMP pathways converge on the SMAD4 molecule, and we believe that this molecule plays a very important role to tie both pathways to TA. Similarly, we look at the NOTCH pathway and identify the HEY2 as a potential link between this pathway and TA. Another transcription factor that has been implicated in TA is NFATC1. While several mouse models exist that include part of the TA abnormality as their phenotype, no true mouse model can be said to represent TA. Bridging this gap will surely shed light on this complex molecular pathway and allow for better understanding of the disease process.


Subject(s)
Disease Models, Animal , Signal Transduction , Tricuspid Atresia , Animals , Tricuspid Atresia/genetics , Tricuspid Atresia/metabolism , Tricuspid Atresia/pathology , Humans , Mice , Univentricular Heart/genetics , Univentricular Heart/metabolism , Univentricular Heart/physiopathology , Univentricular Heart/pathology , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/genetics , Receptors, Notch/metabolism , Receptors, Notch/genetics
14.
Peptides ; 179: 171267, 2024 Sep.
Article in English | MEDLINE | ID: mdl-38908517

ABSTRACT

Signs and symptoms of hypernatremia largely indicate central nervous system dysfunction. Acute hypernatremia can cause demyelinating lesions similar to that observed in osmotic demyelination syndrome (ODS). We have previously demonstrated that microglia accumulate in ODS lesions and minocycline protects against ODS by inhibiting microglial activation. However, the direct effect of rapid rise in the sodium concentrations on microglia is largely unknown. In addition, the effect of chronic hypernatremia on microglia also remains elusive. Here, we investigated the effects of acute (6 or 24 h) and chronic (the extracellular sodium concentration was increased gradually for at least 7 days) high sodium concentrations on microglia using the microglial cell line, BV-2. We found that both acute and chronic high sodium concentrations increase NOS2 expression and nitric oxide (NO) production. We also demonstrated that the expression of nuclear factor of activated T-cells-5 (NFAT5) is increased by high sodium concentrations. Furthermore, NFAT5 knockdown suppressed NOS2 expression and NO production. We also demonstrated that high sodium concentrations decreased intracellular Ca2+ concentration and an inhibitor of Na+/Ca2+ exchanger, NCX, suppressed a decrease in intracellular Ca2+ concentrations and NOS2 expression and NO production induced by high sodium concentrations. Furthermore, minocycline inhibited NOS2 expression and NO production induced by high sodium concentrations. These in vitro data suggest that microglial activity in response to high sodium concentrations is regulated by NFAT5 and Ca2+ efflux through NCX and is suppressed by minocycline.


Subject(s)
Hypernatremia , Microglia , Minocycline , Nitric Oxide Synthase Type II , Nitric Oxide , Microglia/metabolism , Microglia/drug effects , Animals , Nitric Oxide/metabolism , Hypernatremia/metabolism , Hypernatremia/pathology , Hypernatremia/genetics , Minocycline/pharmacology , Mice , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide Synthase Type II/genetics , Sodium/metabolism , Cell Line , Calcium/metabolism , Sodium-Calcium Exchanger/metabolism , Sodium-Calcium Exchanger/genetics , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics
15.
J Cell Mol Med ; 28(12): e18413, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38894694

ABSTRACT

Cardiac hypertrophy, worldwide known as an adaptive functional compensatory state of myocardial stress, is mainly believed to proceed to severe heart diseases, even to sudden death. Emerging studies have explored the microRNA alteration during hypertrophy. However, the mechanisms of microRNAs involved in cardiac hypertrophy are still uncertain. We studied young rats to establish abdominal aorta coarctation (AAC) for 4 weeks. With the significant downregulated cardiac function and upregulated hypertrophic biomarkers, AAC-induced rats showed enlarged myocardiocytes and alterations in microRNAs, especially downregulated miR-31-5p. miR-31-5p targets the 3'UTR of Nfatc2ip and inhibits myocardial hypertrophy in vitro and in vivo. Furthermore, we verified that Nfatc2ip is necessary and sufficient for cardiac hypertrophy in neonatal rat cardiomyocytes. Moreover, we found miR-31-5p inhibited the colocalization of Nfatc2ip and hypertrophic gene ß-Mhc. Luciferase assay and ChiP-qPCR test demonstrated that Nfatc2ip binded to the core-promoter of ß-Mhc and enhanced its transcriptional activity. Above all, our study found a new pathway, mir-31-5p/Nfatc2ip/ß-Mhc, which is involved in cardiac hypertrophy, suggesting a potential target for intervention of cardiac hypertrophy.


Subject(s)
Cardiomegaly , MicroRNAs , Myocytes, Cardiac , NFATC Transcription Factors , MicroRNAs/genetics , MicroRNAs/metabolism , Animals , Cardiomegaly/genetics , Cardiomegaly/metabolism , Cardiomegaly/pathology , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Rats , Male , Rats, Sprague-Dawley , Gene Expression Regulation , 3' Untranslated Regions , Disease Models, Animal
16.
Aging (Albany NY) ; 16(11): 9569-9583, 2024 06 11.
Article in English | MEDLINE | ID: mdl-38862240

ABSTRACT

The global prevalence of osteoporosis is being exacerbated by the increasing number of aging societies and longer life expectancies. In response, numerous drugs have been developed in recent years to mitigate bone resorption and enhance bone density. Nonetheless, the efficacy and safety of these pharmaceutical interventions remain constrained. Corylin (CL), a naturally occurring compound derived from the anti-osteoporosis plant Psoralea corylifolia L., has exhibited promising potential in impeding osteoclast differentiation. This study aims to evaluate the effect and molecular mechanisms of CL regulating osteoclast differentiation in vitro and its potential as a therapeutic agent for osteoporosis treatment in vivo. Our investigation revealed that CL effectively inhibits osteoclast formation and their bone resorption capacity by downregulating the transcription factors NFATc1 and c-fos, consequently resulting in the downregulation of genes associated with bone resorption. Furthermore, it has been observed that CL can effectively mitigate the migration and fusion of pre-osteoclast, while also attenuating the activation of mitochondrial mass and function. The results obtained from an in vivo study have demonstrated that CL is capable of attenuating the bone loss induced by ovariectomy (OVX). Based on these significant findings, it is proposed that CL exhibits considerable potential as a novel drug strategy for inhibiting osteoclast differentiation, thereby offering a promising approach for the treatment of osteoporosis.


Subject(s)
Bone Resorption , Cell Differentiation , Osteoclasts , Osteoporosis , Animals , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteoporosis/drug therapy , Cell Differentiation/drug effects , Mice , Bone Resorption/drug therapy , Female , Ovariectomy/adverse effects , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , RAW 264.7 Cells , Osteogenesis/drug effects , Flavonoids
17.
Cell Biol Toxicol ; 40(1): 44, 2024 Jun 11.
Article in English | MEDLINE | ID: mdl-38862832

ABSTRACT

BACKGROUND: Vasculogenic mimicry (VM) is an enigmatic physiological feature that influences blood supply within glioblastoma (GBM) tumors for their sustained growth. Previous studies identify NFATC3, FOSL1 and HNRNPA2B1 as significant mediators of VEGFR2, a key player in vasculogenesis, and their molecular relationships may be crucial for VM in GBM. AIMS: The aim of this study was to understand how NFATC3, FOSL1 and HNRNPA2B1 collectively influence VM in GBM. METHODS: We have investigated the underlying gene regulatory mechanisms for VM in GBM cell lines U251 and U373 in vitro and in vivo. In vitro cell-based assays were performed to explore the role of NFATC3, FOSL1 and HNRNPA2B1 in GBM cell proliferation, VM and migration, in the context of RNA interference (RNAi)-mediated knockdown alongside corresponding controls. Western blotting and qRT-PCR assays were used to examine VEGFR2 expression levels. CO-IP was employed to detect protein-protein interactions, ChIP was used to detect DNA-protein complexes, and RIP was used to detect RNA-protein complexes. Histochemical staining was used to detect VM tube formation in vivo. RESULTS: Focusing on NFATC3, FOSL1 and HNRNPA2B1, we found each was significantly upregulated in GBM and positively correlated with VM-like cellular behaviors in U251 and U373 cell lines. Knockdown of NFATC3, FOSL1 or HNRNPA2B1 each resulted in decreased levels of VEGFR2, a key growth factor gene that drives VM, as well as the inhibition of proliferation, cell migration and extracorporeal VM activity. Chromatin immunoprecipitation (ChIP) studies and luciferase reporter gene assays revealed that NFATC3 binds to the promoter region of VEGFR2 to enhance VEGFR2 gene expression. Notably, FOSL1 interacts with NFATC3 as a co-factor to potentiate the DNA-binding capacity of NFATC3, resulting in enhanced VM-like cellular behaviors. Also, level of NFATC3 protein in cells was enhanced through HNRNPA2B1 binding of NFATC3 mRNA. Furthermore, RNAi-mediated silencing of NFATC3, FOSL1 and HNRNPA2B1 in GBM cells reduced their capacity for tumor formation and VM-like behaviors in vivo. CONCLUSION: Taken together, our findings identify NFATC3 as an important mediator of GBM tumor growth through its molecular and epistatic interactions with HNRNPA2B1 and FOSL1 to influence VEGFR2 expression and VM-like cellular behaviors.


Subject(s)
Cell Movement , Cell Proliferation , Glioblastoma , Heterogeneous-Nuclear Ribonucleoprotein Group A-B , NFATC Transcription Factors , Neovascularization, Pathologic , Proto-Oncogene Proteins c-fos , Humans , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-fos/genetics , Glioblastoma/metabolism , Glioblastoma/pathology , Glioblastoma/genetics , Glioblastoma/blood supply , Cell Line, Tumor , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Animals , Cell Proliferation/genetics , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/pathology , Cell Movement/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism , Vascular Endothelial Growth Factor Receptor-2/genetics , Gene Expression Regulation, Neoplastic , Mice , Brain Neoplasms/metabolism , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Brain Neoplasms/blood supply , Mice, Nude
18.
Sci Rep ; 14(1): 14732, 2024 06 26.
Article in English | MEDLINE | ID: mdl-38926604

ABSTRACT

Excess amounts of histones in the cell induce mitotic chromosome loss and genomic instability, and are therefore detrimental to cell survival. In yeast, excess histones are degraded by the proteasome mediated via the DNA damage response factor Rad53. Histone expression, therefore, is tightly regulated at the protein level. Our understanding of the transcriptional regulation of histone genes is far from complete. In this study, we found that calcineurin inhibitor treatment increased histone protein levels, and that the transcription factor NFATc1 (nuclear factor of activated T cells 1) repressed histone transcription and acts downstream of the calcineurin. We further revealed that NFATc1 binds to the promoter regions of many histone genes and that histone transcription is downregulated in a manner dependent on intracellular calcium levels. Indeed, overexpression of histone H3 markedly inhibited cell proliferation. Taken together, these findings suggest that NFATc1 prevents the detrimental effects of histone H3 accumulation by inhibiting expression of histone at the transcriptional level.


Subject(s)
Calcineurin , Histones , NFATC Transcription Factors , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Histones/metabolism , Calcineurin/metabolism , Humans , Cell Proliferation , Gene Expression Regulation , Promoter Regions, Genetic , Signal Transduction , Transcription, Genetic , Calcium/metabolism
19.
Int Immunopharmacol ; 137: 112500, 2024 Aug 20.
Article in English | MEDLINE | ID: mdl-38889511

ABSTRACT

Toll-like receptor 4 (TLR4) acts as a double-edged sword in the occurrence and development of periodontitis. While the activation of TLR4 in macrophages aids in clearing local pathogens, it can also disrupt innate immune responses, upsetting microecological balance and accelerating the destruction of periodontal bone tissues. To date, the effects of TLR4 on osteogenesis and osteoclastogenesis in periodontitis have not been comprehensively studied. In this study, we investigated the development of periodontitis in the Tlr4-/- mice by ligating their second molars with silk threads. Compared to wild-type (WT) mice, Tlr4-/- mice demonstrated increased resistance to periodontitis-associated bone destruction, as evidenced by decreased bone resorption and enhanced bone regeneration. Mechanistically, the deletion of Tlr4 not only inhibited osteoclast formation by reducing the expression of NFATc1, CTSK and TRAP, but also enhanced osteogenic abilities through increased expression of OCN, OPN and RUNX2. In conclusion, TLR4 tips the balance of osteoclastogenesis and osteogenesis, thereby promoting periodontal bone destruction in periodontitis.


Subject(s)
Mice, Knockout , Osteoblasts , Osteoclasts , Osteogenesis , Periodontitis , Toll-Like Receptor 4 , Animals , Toll-Like Receptor 4/metabolism , Toll-Like Receptor 4/genetics , Periodontitis/immunology , Periodontitis/genetics , Periodontitis/pathology , Osteoclasts/physiology , Osteoclasts/immunology , Mice , Osteoblasts/metabolism , Osteoblasts/immunology , Mice, Inbred C57BL , Male , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Humans , Alveolar Bone Loss/immunology , Alveolar Bone Loss/pathology
20.
Biochem Biophys Res Commun ; 719: 150063, 2024 Jul 30.
Article in English | MEDLINE | ID: mdl-38749090

ABSTRACT

Osteoclasts are multinucleated cells with bone resorption activity. Excessive osteoclast activity has been implicated in osteoporosis, rheumatoid arthritis, and bone destruction due to bone metastases from cancer, making osteoclasts essential target cells in bone and joint diseases. C-terminal domain nuclear envelope phosphatase 1 (Ctdnep1, formerly Dullard) is a negative regulator of transforming growth factor (TGF)-ß superfamily signaling and regulates endochondral ossification in mesenchymal cells during skeletal development. In this study, we investigated the role of Ctdnep1 in the Receptor activator of nuclear factor-kappa B ligand (RANKL)-induced RAW264.7 osteoclast differentiation. Expression of Ctdnep1 did not change during osteoclast differentiation; Ctdnep1 protein localized to the cytoplasm before and after osteoclast differentiation. Small interfering RNA-mediated knockdown of Ctdnep1 increased tartrate-resistant acid phosphatase-positive multinucleated osteoclasts and the expression of osteoclast marker genes, including Acp5, Ctsk, and Nfatc1. Interestingly, the knockdown of Ctdnep1 increased the protein level of Nfatc1 in cells unstimulated with RANKL. Knockdown of Ctdnep1 also enhanced calcium-resorbing activity. Mechanistically, the knockdown of Ctdnep1 increased the phosphorylation of RANKL signaling components. These results suggest that Ctdnep1 negatively regulates osteoclast differentiation by suppressing the RANKL signaling pathway.


Subject(s)
Cell Differentiation , Osteoclasts , RANK Ligand , Animals , Mice , Gene Knockdown Techniques , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/genetics , Osteoclasts/metabolism , Osteoclasts/cytology , RANK Ligand/metabolism , RAW 264.7 Cells , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/metabolism
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