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1.
Front Immunol ; 13: 850287, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35401555

RESUMO

The ocular surface is continuously exposed to various environmental factors, and innate and adaptive immunity play crucial roles in ocular surface diseases (OSDs). Previously, we have reported that the topical application of RCI001 affords excellent anti-inflammatory and antioxidant effects in dry eye disease and ocular chemical burn models. In this study, we examined the inhibitory effects of RCI001 on the Rac1 and NLRP3 inflammasomes in vitro and in vivo. Following RCI001 application to RAW264.7 and Swiss 3T3 cells, we measured Rac1 activity using a glutathione-S-transferase (GST) pull-down assay and G-protein activation assay kit. In addition, we quantified the expression of inflammatory cytokines (interleukin [IL]-1ß, IL-6, and tumor necrosis factor [TNF]-α) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells using ELISA and real-time PCR. In the mouse ocular alkali burn model, RCI001 was administered via eye drops (10 mg/mL, twice daily) for 5 days, and 1% prednisolone acetate (PDE) ophthalmic suspension was used as a positive control. Corneal epithelial integrity (on days 0-5) and histological examinations were performed, and transcript and protein levels of Rac1, NLRP3, caspase-1, and IL-1ß were quantified using real-time PCR and western blotting in corneal tissues collected on days 3 and 5. We observed that RCI001 dose-dependently inhibited Rac1 activity and various inflammatory cytokines in LPS-stimulated murine macrophages. Furthermore, RCI001 restored corneal epithelial integrity more rapidly than corticosteroid treatment in chemically injured corneas. Compared to the saline group, activation of Rac1 and the NLRP3 inflammasome/IL-1ß axis was suppressed in the RCI001 group, especially during the early phase of the ocular alkali burn model. Topical RCI001 suppressed the expression of activated Rac1 and inflammatory cytokines in vitro and rapidly restored the injured cornea by inhibiting activation of Rac1 and the NLRP inflammasome/IL-1ß axis in vivo. Accordingly, RCI001 could be a promising therapeutic agent for treating OSDs.


Assuntos
Anti-Inflamatórios , Queimaduras Químicas , Inflamassomos , Células 3T3 , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Queimaduras Químicas/tratamento farmacológico , Citocinas/metabolismo , Queimaduras Oculares/tratamento farmacológico , Inflamassomos/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células RAW 264.7
2.
Cells ; 11(6)2022 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-35326460

RESUMO

G-protein-coupled receptors (GPCRs) represent a family with over 800 members in humans, and one-third of these are targets for approved drugs. A large number of GPCRs have unknown physiologic roles. Here, we investigated GPR27, an orphan GPCR belonging to the family of super conserved receptor expressed in the brain, with unknown functions. Cytosolic levels of L-lactate ([lactate]i), the end product of aerobic glycolysis, were measured with the Laconic fluorescence resonance energy transfer nanosensor. In single 3T3 wild-type (WT) embryonic cells, the application of 8535 (1 µM), a surrogate agonist known to activate GPR27, resulted in an increase in [lactate]i. Similarly, an increase was recorded in primary rat astrocytes, a type of neuroglial cell abundant in the brain, which contain glycogen and express enzymes of aerobic glycolysis. In CRISPR-Cas9 GPR27 knocked out 3T3 cells, the 8535-induced increase in [lactate]i was reduced compared with WT controls. Transfection of the GPR27-carrying plasmid into the 3T3KOGPR27 cells rescued the 8535-induced increase in [lactate]i. These results indicate that stimulation of GPR27 enhances aerobic glycolysis and L-lactate production in 3T3 cells and astrocytes. Interestingly, in the absence of GPR27 in 3T3 cells, resting [lactate]i was increased in comparison with controls, further supporting the view that GPR27 regulates L-lactate homeostasis.


Assuntos
Astrócitos , Ácido Láctico , Células 3T3 , Animais , Astrócitos/metabolismo , Glicogênio/metabolismo , Ácido Láctico/metabolismo , Camundongos , Ratos , Receptores Acoplados a Proteínas G/metabolismo
3.
FASEB J ; 36(3): e22147, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35104016

RESUMO

Diabetes mellitus (DM) and osteoporosis are two common diseases that may develop as a cause-and-effect relationship since the incidence of osteoporotic fractures is significantly increased in DM patients. However, the pathophysiology of diabetic osteoporosis is yet to be clearly understood. Iron overload has been reported to lead to bone loss and closely related to osteoporosis. In this study, we hypothesized that high glucose and high fat (HGHF) may induce osteoblastic ferroptosis for the pathogenesis of diabetic osteoporosis and explored the possible molecular mechanisms behind. Using the diabetic rat model established by HGHF feeding with a subsequent intraperitoneal injection of a single low dose of streptozocin, we found that the serum ferritin level (a biomarker for body iron store) was significantly elevated in HGHF-fed rats and the expression of SLC7A11 and GPX4 (inhibitory marker proteins for ferroptosis) was markedly attenuated in the bone tissue of the rats with diabetic bone loss as compared to the normal rats. In an osteoblast cell model, treatment of pre-osteoblastic MC3T3-E1 cells with high glucose and palmitic acid (HGPA) not only suppressed osteoblast differentiation and mineralization but also triggered ferroptosis-related osteoblastic cell death. m6 A-seq revealed that m6 A methylation on ASK1 was 80.9-fold higher in HGPA-treated cells. The expression of p-ASK1 and p-p38 was also significantly elevated in the HGPA-treated cells. Knockout of METTL3 (methyltransferase-like 3), one of the major m6 A methyltransferases, in MC3T3-E1 cells not only abrogated HGPA-induced activation of ASK1-p38 signaling pathway but also attenuated the level of ferroptosis. Therefore, HGHF-induced ferroptosis in osteoblasts may be the main cause of osteoporosis in DM via activation of METTL3/ASK1-p38 signaling pathway, and inhibition of ferroptosis in osteoblasts may provide a potential therapeutic strategy for diabetic osteoporosis.


Assuntos
Diabetes Mellitus/metabolismo , Ferroptose/fisiologia , Glucose/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Metiltransferases/metabolismo , Osteoblastos/metabolismo , Osteoporose/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células 3T3 , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Dieta Hiperlipídica/efeitos adversos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Osteogênese/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia
4.
PLoS One ; 17(2): e0263141, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35120168

RESUMO

Corneal grafts are the imperative clinical treatment for canine corneal blindness. To serve the growing demand, this study aimed to generate tissue-engineered canine cornea in part of the corneal epithelium and underlying stroma based on canine limbal epithelial stem cells (cLESCs) seeded silk fibroin/gelatin (SF/G) film and canine corneal stromal stem cells (cCSSCs) seeded SF/G scaffold, respectively. Both cell types were successfully isolated by collagenase I. SF/G corneal films and stromal scaffolds served as the prospective substrates for cLESCs and cCSSCs by promoting cell adhesion, cell viability, and cell proliferation. The results revealed the upregulation of tumor protein P63 (P63) and ATP-binding cassette super-family G member 2 (Abcg2) of cLESCs as well as Keratocan (Kera), Lumican (Lum), aldehyde dehydrogenase 3 family member A1 (Aldh3a1) and Aquaporin 1 (Aqp1) of differentiated keratocytes. Moreover, immunohistochemistry illustrated the positive staining of tumor protein P63 (P63), aldehyde dehydrogenase 3 family member A1 (Aldh3a1), lumican (Lum) and collagen I (Col-I), which are considerable for native cornea. This study manifested a feasible platform to construct tissue-engineered canine cornea for functional grafts and positively contributed to the body of knowledge related to canine corneal stem cells.


Assuntos
Materiais Biocompatíveis/química , Epitélio Corneano/patologia , Regeneração , Células-Tronco/citologia , Células Estromais/citologia , Células 3T3 , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Aquaporina 1/metabolismo , Proliferação de Células , Colágeno Tipo I/metabolismo , Transplante de Córnea , Cães , Proteínas do Olho/metabolismo , Fibroblastos/citologia , Fibroínas/química , Gelatina/química , Genes Supressores de Tumor , Imuno-Histoquímica , Técnicas In Vitro , Lumicana/metabolismo , Camundongos , Resistência à Tração , Engenharia Tecidual , Tecidos Suporte
5.
Int J Mol Sci ; 23(4)2022 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-35216266

RESUMO

BRIL (bone restricted ifitm-like; also known as IFITM5) is a transmembrane protein expressed in osteoblasts. Although its role in skeletal development and homeostasis is unknown, mutations in BRIL result in rare dominant forms of osteogenesis imperfecta. The pathogenic mechanism has been proposed to be a gain-of or neomorphic function. To understand the function of BRIL and its OI type V mutant (MALEP BRIL) and whether they could activate signaling pathways in osteoblasts, we performed a luciferase reporter assay screen based on the activity of 26 transcription factors. When overexpressed in MC3T3-E1 and MLO-A5 cells, the MALEP BRIL activated the reporters dependent on MEF2, NFATc, and NR4A significantly more. Additional co-transfection experiments with MEF2C and NFATc1 and a number of their modulators (HDAC4, calcineurin, RCAN, FK506) confirmed the additive or synergistic activation of the pathways by MALEP, and suggested a coordinated regulation involving calcineurin. Endogenous levels of Nr4a members, as well as Ptgs2, were upregulated by MALEP BRIL. Y2H and co-immunoprecipitation indicated that BRIL interacted with CAML, but its contribution as the most upstream stimulator of the Ca2+-calcineurin-MEF2/NFATc cascade was not confirmed convincingly. Altogether the data presented provide the first ever readout to monitor for BRIL activity and suggest a potential gain-of-function causative effect for MALEP BRIL in OI type V, leading to perturbed signaling events and gene expression.


Assuntos
Proteínas de Membrana/genética , Mutação/genética , Fatores de Transcrição NFATC/genética , Osteoblastos/metabolismo , Osteogênese Imperfeita/genética , Ativação Transcricional/genética , Células 3T3 , Animais , Calcineurina/genética , Cálcio/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Osteogênese Imperfeita/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética
6.
Int J Mol Sci ; 23(4)2022 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-35216233

RESUMO

The primary cilium is a hair-like immotile organelle with specific membrane receptors, including the receptor of Hedgehog signaling, smoothened. The cilium organized in preosteoblasts promotes differentiation of the cells into osteoblasts (osteoblast differentiation) by mediating Hedgehog signaling to achieve bone formation. Notably, 4.1G is a plasma membrane-associated cytoskeletal protein that plays essential roles in various tissues, including the peripheral nervous system, testis, and retina. However, its function in the bone remains unexplored. In this study, we identified 4.1G expression in the bone. We found that, in the 4.1G-knockout mice, calcium deposits and primary cilium formation were suppressed in the trabecular bone, which is preosteoblast-rich region of the newborn tibia, indicating that 4.1G is a prerequisite for osteoblast differentiation by organizing the primary cilia in preosteoblasts. Next, we found that the primary cilium was elongated in the differentiating mouse preosteoblast cell line MC3T3-E1, whereas the knockdown of 4.1G suppressed its elongation. Moreover, 4.1G-knockdown suppressed the induction of the cilia-mediated Hedgehog signaling and subsequent osteoblast differentiation. These results demonstrate a new regulatory mechanism of 4.1G in bone formation that promotes the primary ciliogenesis in the differentiating preosteoblasts and induction of cilia-mediated osteoblast differentiation, resulting in bone formation at the newborn stage.


Assuntos
Diferenciação Celular/fisiologia , Cílios/metabolismo , Cílios/fisiologia , Proteínas dos Microfilamentos/metabolismo , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Osteogênese/fisiologia , Células 3T3 , Animais , Osso e Ossos/metabolismo , Osso e Ossos/fisiologia , Calcificação Fisiológica/fisiologia , Linhagem Celular , Camundongos , Camundongos Knockout , Transdução de Sinais/fisiologia
7.
Molecules ; 27(4)2022 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-35209035

RESUMO

Three-dimensional cell culture has become a reliable method for reproducing in vitro cellular growth in more realistic physiological conditions. The surface hydrophobicity strongly influences the promotion of cell aggregate formation. In particular, for spheroid formation, highly water-repellent coatings seem to be required for the significant effects of the process. In this work, surfaces at different wettability have been compared to observe their influence on the growth and promotion of aggregates of representative mammalian cell lines, both tumoral and non-tumoral (3T3, HaCat and MCF-7 cell lines). The effect of increased hydrophobicity from TCPS to agarose hydrogel to mixed organic-inorganic superhydrophobic (SH) coating has been investigated by optical and fluorescence microscopy, and by 3D confocal profilometry, in a time scale of 24 h. The results show the role of less wettable substrates in inducing the formation of spheroid-like cell aggregates at a higher degree of sphericity for the studied cell lines.


Assuntos
Técnicas de Cultura de Células , Proliferação de Células , Hidrogéis/química , Esferoides Celulares/metabolismo , Células 3T3 , Animais , Humanos , Interações Hidrofóbicas e Hidrofílicas , Células MCF-7 , Camundongos , Esferoides Celulares/citologia
8.
Int J Mol Sci ; 23(1)2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35008918

RESUMO

Over the years, natural-based scaffolds have presented impressive results for bone tissue engineering (BTE) application. Further, outstanding interactions have been observed during the interaction of graphene oxide (GO)-reinforced biomaterials with both specific cell cultures and injured bone during in vivo experimental conditions. This research hereby addresses the potential of fish gelatin/chitosan (GCs) hybrids reinforced with GO to support in vitro osteogenic differentiation and, further, to investigate its behavior when implanted ectopically. Standard GCs formulation was referenced against genipin (Gp) crosslinked blend and 0.5 wt.% additivated GO composite (GCsGp/GO 0.5 wt.%). Pre-osteoblasts were put in contact with these composites and induced to differentiate in vitro towards mature osteoblasts for 28 days. Specific bone makers were investigated by qPCR and immunolabeling. Next, CD1 mice models were used to assess de novo osteogenic potential by ectopic implantation in the subcutaneous dorsum pocket of the animals. After 4 weeks, alkaline phosphate (ALP) and calcium deposits together with collagen synthesis were investigated by biochemical analysis and histology, respectively. Further, ex vivo materials were studied after surgery regarding biomineralization and morphological changes by means of qualitative and quantitative methods. Furthermore, X-ray diffraction and Fourier-transform infrared spectroscopy underlined the newly fashioned material structuration by virtue of mineralized extracellular matrix. Specific bone markers determination stressed the osteogenic phenotype of the cells populating the material in vitro and successfully differentiated towards mature bone cells. In vivo results of specific histological staining assays highlighted collagen formation and calcium deposits, which were further validated by micro-CT. It was observed that the addition of 0.5 wt.% GO had an overall significant positive effect on both in vitro differentiation and in vivo bone cell recruitment in the subcutaneous region. These data support the GO bioactivity in osteogenesis mechanisms as being self-sufficient to elevate osteoblast differentiation and bone formation in ectopic sites while lacking the most common osteoinductive agents.


Assuntos
Biopolímeros/farmacologia , Diferenciação Celular , Grafite/farmacologia , Osteogênese , Células 3T3 , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Osteogênese/efeitos dos fármacos , Porosidade , Espectroscopia de Infravermelho com Transformada de Fourier , Tela Subcutânea/efeitos dos fármacos , Tecidos Suporte/química , Difração de Raios X , Microtomografia por Raio-X
9.
Aging (Albany NY) ; 14(1): 253-271, 2022 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-34982732

RESUMO

Osteopontin (OPN) has been proved to be closely related to the pathogenesis of osteoarthritis (OA), but the role of OPN in the pathogenesis of OA has not been fully clarified. Current studies on OPN in OA mostly focus on articular cartilage, synovial membrane and articular fluid, while ignoring its role in OA subchondral bone turnover and remodeling. In this study, we used a destabilization OA mouse model to investigate the role of OPN in OA subchondral bone changes. Our results indicate that increased expression of OPN accelerates the turnover and remodeling of OA subchondral bone, promotes the formation of h-type vessels in subchondral bone, and mediates articular cartilage degeneration induced by subchondral bone metabolism. In addition, our results confirmed that inhibition of PI3K/AKT signaling pathway inhibits OPN-mediated OA subchondral bone remodeling and cartilage degeneration. This study revealed the role and mechanism of OPN in OA subchondral bone, which is of great significance for exploring specific biological indicators for early diagnosis of OA and monitoring disease progression, as well as for developing drugs to regulate the metabolism and turnover of subchondral bone and alleviate the subchondral bone sclerosis of OA.


Assuntos
Remodelação Óssea/fisiologia , Osso e Ossos/metabolismo , Osteoartrite/metabolismo , Osteopontina/metabolismo , Células 3T3 , Animais , Osso e Ossos/irrigação sanguínea , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Cromonas/farmacologia , Regulação da Expressão Gênica/fisiologia , Camundongos , Morfolinas/farmacologia , Osteopontina/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
10.
Cell Rep ; 38(3): 110277, 2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-35045290

RESUMO

Exosomes/small extracellular vesicles (sEVs) can serve as multifactorial mediators of cell-to-cell communication through their miRNA and protein cargo. Quantitative proteomic analysis of five cell lines representing metabolically important tissues reveals that each cell type has a unique sEV proteome. While classical sEV markers such as CD9/CD63/CD81 vary markedly in abundance, we identify six sEV markers (ENO1, GPI, HSPA5, YWHAB, CSF1R, and CNTN1) that are similarly abundant in sEVs of all cell types. In addition, each cell type has specific sEV markers. Using fat-specific Dicer-knockout mice with decreased white adipose tissue and increased brown adipose tissue, we show that these cell-type-specific markers can predict the changing origin of the serum sEVs. These results provide a valuable resource for understanding the sEV proteome of the cells and tissues important in metabolic homeostasis, identify unique sEV markers, and demonstrate how these markers can help in predicting the tissue of origin of serum sEVs.


Assuntos
Biomarcadores/sangue , Exossomos/metabolismo , Proteoma/metabolismo , Células 3T3 , Adiponectina/sangue , Tecido Adiposo/metabolismo , Animais , Camundongos
11.
Fitoterapia ; 157: 105130, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35051554

RESUMO

Petasites japonicus is one of the most popular edible wild plants in Japan. Many biological effects of P. japonicus have been reported, including anti-allergy, anti-inflammation, and anticancer effects. Although its anti-obesity effect has been reported in several studies, the most important component responsible for this activity has not been fully elucidated. On screening the components that suppress adipocyte differentiation in 3T3-F442A cells, we found that the extract of the flower buds of P. japonicus has anti-adipogenic effect. Among the known major components of P. japonicus, petasin exhibited a potent anti-adipogenic effect at an IC50 value of 0.95 µM. Quantitative analysis revealed that the active component responsible for most of the anti-adipogenic effects of P. japonicus extract is petasin. Petasin suppressed the expression of markers of mature adipocytes (PPARγ, C/EBPα, and aP2). However, as isopetasin and petasol, analogs of petasin, did not exhibit these effects, it indicates that a double bond at the C11-C12 position and an angeloyl ester moiety were essential for the activity. Petasin affected the late stage of adipocyte differentiation and inhibited the expression of lipid synthesis factors (ACC1, FAS, and SCD1). Additionally, it was revealed that petasin could be efficiently extracted using hexane with minimal amount of pyrrolizidine alkaloids, the toxic components. These findings indicate that P. japonicus extract containing petasin could be a promising food material for the prevention of obesity.


Assuntos
Adiposidade/efeitos dos fármacos , Obesidade/prevenção & controle , Petasites/química , Sesquiterpenos/farmacologia , Células 3T3/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Compostos Azo , Western Blotting , Corantes , Flores/química , Concentração Inibidora 50 , Japão , Camundongos , Polifenóis/análise , Alcaloides de Pirrolizidina/química , Reação em Cadeia da Polimerase em Tempo Real , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação , Relação Estrutura-Atividade
12.
FASEB J ; 36(2): e22153, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34997955

RESUMO

DNA methylation is an epigenetic modification critical for the regulation of chromatin structure and gene expression during development and disease. The ten-eleven translocation (TET) enzyme family catalyzes the hydroxymethylation and subsequent demethylation of DNA by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Little is known about TET protein function due to a lack of pharmacological tools to manipulate DNA hydroxymethylation levels. In this study, we examined the role of TET-mediated DNA hydroxymethylation during BMP-induced C2C12 osteoblast differentiation using a novel cytosine-based selective TET enzyme inhibitor, Bobcat339 (BC339). Treatment of C2C12 cells with BC339 increased global 5mC and decreased global 5hmC without adversely affecting cell viability, proliferation, or apoptosis. Furthermore, BC339 treatment inhibited osteoblast marker gene expression and decreased alkaline phosphatase activity during differentiation. Methylated DNA immunoprecipitation and bisulfite sequencing showed that inhibition of TET with BC339 led to increased 5mC at specific CpG-rich regions at the promoter of Sp7, a key osteoblast transcription factor. Consistent with promoter 5mC marks being associated with transcriptional repression, luciferase activity of an Sp7-promoter-reporter construct was repressed by in vitro DNA methylation or BC339. Chromatin immunoprecipitation analysis confirmed that TET2 does indeed occupy the promoter region of Sp7. Accordingly, forced overexpression of SP7 rescued the inhibition of osteogenic differentiation by BC339. In conclusion, our data suggest that TET-mediated DNA demethylation of genomic regions, including the Sp7 promoter, plays a role in the initiation of osteoblast differentiation. Furthermore, BC339 is a novel pharmacological tool for the modulation of DNA methylation dynamics for research and therapeutic applications.


Assuntos
Diferenciação Celular/fisiologia , DNA/metabolismo , Osteoblastos/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Células 3T3 , Animais , Apoptose/fisiologia , Biomarcadores/metabolismo , Linhagem Celular , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Desmetilação do DNA , Metilação de DNA/fisiologia , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Regiões Promotoras Genéticas/genética
13.
Cell Rep ; 38(2): 110240, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35021086

RESUMO

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.


Assuntos
Linhagem da Célula/genética , Células-Tronco/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Células 3T3 , Animais , Proteína Axina/metabolismo , Diferenciação Celular , Proliferação de Células , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição , Via de Sinalização Wnt/fisiologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética
14.
FASEB J ; 36(2): e22115, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35032415

RESUMO

Bone loss is a hallmark of inflammatory bone diseases caused by aberrantly activated osteoclasts (OCLs). Studies have shown that OCLs exhibit various phenotypes and functions due to variations in the source(s) of precursor cells, cytokine expressions, and microenvironment-dependent factors. During these conditions, inflammatory osteoclasts (iOCLs) lose their immune-suppressive effect relative to OCLs under physiological conditions. This induces TNF α-producing CD4+ T cells in an antigen-dependent manner and finally leads to cascade amplification of iOCLs. OCL-derived exosomes have been reported to regulate OCL formation and inhibit the osteoblast activity. However, the specific function and mechanism of iOCL-derived exosomes on osteoblast have not been studied yet. In the present study, we compare the osteoblast promoting activities of iOCL-derived exosomes and OCL-derived exosomes. We found that iOCLs exosomes specifically target osteoblasts through ephrinA2/EphA2. Mechanistically, the lncRNA LIOCE is enriched in iOCL exosomes and promotes the osteoblast activity after being incorporated into osteoblasts. Furthermore, our results revealed that exosomal lncRNA LIOCE stabilizes osteogenic transcription factor Osterix by interacting and reducing the ubiquitination level of Osterix. This study demonstrated that the bone loss is alleviated in the inflammatory osteolysis mice model after injection of iOCL exosomes encapsulating lncRNA LIOCE. The role of exosomes encapsulating lncRNA LIOCE in promoting bone formation was well established in the rat bone repair model. Our results indicate that iOCL-derived exosomal lncRNA LIOCE promotes bone formation by upregulating Osx expression, and thus, the exosomes encapsulating lncRNA LIOCE may be an effective strategy to increase bone formation in osteoporosis and other bone metabolic disorders.


Assuntos
Exossomos/genética , Inflamação/genética , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Osteogênese/genética , RNA Longo não Codificante/genética , Fator de Transcrição Sp7/genética , Células 3T3 , Animais , Diferenciação Celular/genética , Linhagem Celular , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteólise/genética , Osteoporose/genética , Ratos , Fatores de Transcrição/genética , Ubiquitinação/genética , Regulação para Cima/genética
15.
Cancer Lett ; 530: 170-180, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35077804

RESUMO

C/EBPß has recently emerged as a pro-leukemogenic transcription factor that cooperates with oncoprotein MYB to maintain proliferation and differentiation block of AML cells, making C/EBPß an interesting drug target for AML. Here we have studied the inhibitory potential and biological effects of a synthetic analog of the natural product helenalin, a known inhibitor of C/EBPß. The synthetic compound inhibits C/EBPß by covalent binding to cysteine residues in the transactivation domain, thereby causing up-regulation of differentiation-associated genes, cell death and reduced self-renewal potential of AML cells. Suppression of these effects by ectopic expression of C/EBPß or MYB and gene expression profiling validate C/EBPß as a relevant target of the helenalin-mimic and highlight its role as a pro-leukemogenic factor. Overall, our work demonstrates that the synthetic helenalin mimic acts as a covalent inhibitor of C/EBPß and identifies the cysteine residues in the transactivation domain of C/EBPß as ligandable sites. The helenalin mimic can be considered a potential "lead molecule" but needs further development towards more effective C/EBPß inhibitors before being used as a therapeutic agent.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Sesquiterpenos de Guaiano/farmacologia , Ativação Transcricional/efeitos dos fármacos , Células 3T3 , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HEK293 , Células HL-60 , Humanos , Leucemia Mieloide Aguda/genética , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Células THP-1
16.
J Mol Histol ; 53(1): 75-83, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34676487

RESUMO

Although endoplasmic reticulum (ER) stress is thought to be involved in various diseases such as cancer, metabolic, and inflammatory disorders, the relationship between ER stress and bone diseases, are remains unclear. Tunicamycin-treated MC3T3-E1 osteoblasts were used as the ER stress model in this study. 635 nm light-emitting diode irradiation (635 nm-IR) was carried out for 1 h before and after inducing ER stress. To investigate the effects of 635 nm-IR on ER stress-induced MC3T3-E1 osteoblasts and the underlying mechanism, western blot, reverse transcription polymerase chain reaction, alkaline phosphatase and Alizarin red staining, 2',7'-dichlorodyhydrofluorescein diacetate assay, Fluo-3AM and immunocytochemistry were performed. Pretreatment with 635 nm-IR effectively prevented intracellular reactive oxygen species production and alleviated ER stress through the pancreatic ER kinase (PERK)-eukaryotic initiation factor 2 (eIF2)-activating transcription factor 4 (ATF4)-nuclear factor-like 2 (Nrf2) signaling pathway. Hence, 635 nm-IR may serve a protective role in the treatment of ER stress-related bone diseases.


Assuntos
Estresse do Retículo Endoplasmático/efeitos da radiação , Lasers Semicondutores , Osteoblastos/efeitos da radiação , Células 3T3 , Fator 4 Ativador da Transcrição/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Western Blotting , Sobrevivência Celular , Células Cultivadas , Fator de Iniciação 2 em Eucariotos/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Osteoblastos/metabolismo , Osteogênese/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais
17.
Biomed Pharmacother ; 146: 112497, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34891117

RESUMO

Rhubarb as an herbal medicine has been shown to exhibit antiadipogenic activity. This study evaluated and compared the lipid-lowering activity of five rhubarb hydroxyanthraquinones (HAQs), including chrysophanol, aloe emodin, emodin, physcion, and rhein, aiming to identify candidate compounds for obesity treatment. Examination of the antiobesity effects of HAQs in 3T3-L1 adipocytes and high-fat diet (HFD)-induced obese rats showed that these anthraquinone compounds inhibited lipid accumulation in 3T3-L1 cells before and after differentiation. Emodin and rhein showed greater inhibition than the other compounds; dosage at 50 µM reduced intracellular triglyceride (TG) by about 30% in the differentiated adipocytes. Both compounds also revealed lipolytic effects to increase glycerol release from adipocytes. Adipokine overexpression induced by differentiation was downregulated by emodin and rhein through mitogen-activated protein kinase (MAPK) signaling. Despite their structural similarity, emodin and rhein exhibited different mechanisms on adipogenesis and lipid metabolism. Rhein restrained lipid deposition by controlling adipogenic transcriptional factors and lipolytic lipases during differentiation. The lipid-lowering effects of emodin did not use these pathways but reduced levels of lipogenic enzymes. HFD consumption in rats significantly increased body weight, visceral fat mass and adipocyte size, which were attenuated by intraperitoneal delivery of emodin or rhein. Rhein showed greater amelioration of obesity than emodin, decreasing plasma cholesterol by 29% and 14%, respectively. HAQs also suppressed cytokine upregulation in the liver and adipose tissues of obese rats. Rhein is a potential antiobesity agent through its ability to regulate obesity-associated adipogenesis, lipolysis and inflammation.


Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Antraquinonas/farmacologia , Fármacos Antiobesidade/farmacologia , Rheum/química , Células 3T3 , Animais , Sobrevivência Celular/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Emodina/farmacologia , Glicerol/metabolismo , Fígado/efeitos dos fármacos , Camundongos , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Regulação para Cima
18.
Biol Trace Elem Res ; 200(2): 740-748, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34031801

RESUMO

Little attention has been paid to the tolerance of osteoblasts to fluoride in distinct differentiation stages, and the role of TGF-ß1 in fluoride-treated osteoblast differentiation of progenitors and precursors was rarely mentioned in previous studies. The present study aimed to clarify how fluoride affected different differentiation stages of osteoblasts, and to elucidate the role of TGF-ß1 in this process. We assessed cell migration, proliferation, DNA damage, and apoptosis of early-differentiated osteoblasts derived from bone marrow stem cells (BMSCs) exposed to fluoride with or without TGF-ß1. Subsequently, MC3T3-E1 cells cultured with mineral induction medium were treated with fluoride to test fluoride's effect on late-differentiated osteoblasts. The specific fluoride concentrations and treatment times were chosen to evaluate the role of TGF-ß1 in fluoride-induced osteoblastic differentiation and function. Results showed early-differentiated osteoblasts treated with a low dose of fluoride grew and moved more rapidly. TGF-ß1 promoted cell proliferation and inhibited cell apoptosis in early-differentiated osteoblasts exposed to a low fluoride dose, but enhanced apoptosis at higher fluoride conditions. In the late-differentiated osteoblasts, the fluorine dose range with anabolic effects was narrowed, and the fluoride range with catabolic effects was widened. Treatment with a low fluoride dose stimulated the alkaline phosphatase (ALP) expression. TGF-ß1 treatment inhibited Runx2 expression but increased RANKL expression in late-differentiated osteoblasts exposed to fluoride. Meanwhile, TGF-ß1 treatments activated Smad3 phosphorylation but blocked Wnt10b expression in osteoblasts. We conclude that TGF-ß1 plays an essential role in fluoride-induced differentiation and osteoblast function via activation of Smad3 instead of Wnt10 signaling.


Assuntos
Fluoretos , Osteoblastos/efeitos dos fármacos , Fator de Crescimento Transformador beta1 , Células 3T3 , Animais , Diferenciação Celular , Fluoretos/farmacologia , Camundongos , Osteogênese , Transdução de Sinais
19.
Acta Pharmacol Sin ; 43(2): 367-375, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33875797

RESUMO

The excess deposition of underlying extracellular matrix (ECM) in adipose tissue is defined as adipose tissue fibrosis that is a major contributor to metabolic disorder such as obesity and type 2 diabetes. Anti-fibrosis therapy has received much attention in the treatment of metabolic disorders. Orosomucoid (ORM) is an acute-phase protein mainly produced by liver, which is also an adipokine. In this study, we investigated the effects of ORM on adipose tissue fibrosis and the potential mechanisms. We showed that ORM1-deficient mice exhibited an obese phenotype, manifested by excessive collagen deposition in adipose tissues and elevated expression of ECM regulators such as metalloproteinases (MMP-2, MMP-13, MMP-14) and tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, TIMP-3). Administration of exogenous ORM (50 mg· kg-1· d-1, ip) for 7 consecutive days in high-fat diet (HFD)-fed mice and leptin receptor (LepR)-deficient db/db mice attenuated these abnormal expressions. Meanwhile, ORM administration stimulated AMP-activated protein kinase (AMPK) phosphorylation and decreased transforming growth factor-ß1 (TGF-ß1) level in adipose tissues of the mice. In TGF-ß1-treated 3T3-L1 fibroblasts, ORM (10 µg/mL) improved the impaired expression profiles of fibrosis-related genes, whereas a selective AMPK inhibitor dorsomorphin (1 µmol/mL) abolished these effects. Together, our results suggest that ORM exerts a direct anti-fibrosis effect in adipose tissue via AMPK activation. ORM is expected to become a novel target for the treatment of adipose tissue fibrosis.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Adipocinas/farmacologia , Tecido Adiposo/efeitos dos fármacos , Orosomucoide/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células 3T3 , Tecido Adiposo/diagnóstico por imagem , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Western Blotting , Dieta Hiperlipídica/efeitos adversos , Fibrose , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Orosomucoide/deficiência
20.
J Cell Sci ; 135(1)2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34859814

RESUMO

Adipocytes are key to metabolic regulation, exhibiting insulin-stimulated glucose transport that is underpinned by the insulin-stimulated delivery of glucose transporter type 4 (SLC2A4, also known and hereafter referred to as GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, and increase cell surface GLUT4 levels. Adipocytokines, such as adiponectin, are secreted via a similar mechanism. We used genome editing to knock out syntaxin-4, a protein reported to mediate fusion between GLUT4-containing vesicles and the plasma membrane in 3T3-L1 adipocytes. Syntaxin-4 knockout reduced insulin-stimulated glucose transport and adiponectin secretion by ∼50% and reduced GLUT4 levels. Ectopic expression of haemagglutinin (HA)-tagged GLUT4 conjugated to GFP showed that syntaxin-4-knockout cells retain significant GLUT4 translocation capacity, demonstrating that syntaxin-4 is dispensable for insulin-stimulated GLUT4 translocation. Analysis of recycling kinetics revealed only a modest reduction in the exocytic rate of GLUT4 in knockout cells, and little effect on endocytosis. These analyses demonstrate that syntaxin-4 is not always rate limiting for GLUT4 delivery to the cell surface. In sum, we show that syntaxin-4 knockout results in reduced insulin-stimulated glucose transport, depletion of cellular GLUT4 levels and inhibition of adiponectin secretion but has only modest effects on the translocation capacity of the cells. This article has an associated First Person interview with Hannah L. Black and Rachel Livingstone, joint first authors of the paper.


Assuntos
Adipócitos , Adiponectina , Células 3T3 , Células 3T3-L1 , Adipócitos/metabolismo , Adiponectina/genética , Animais , Membrana Celular/metabolismo , Transportador de Glucose Tipo 4/genética , Humanos , Insulina/metabolismo , Camundongos , Proteínas Qa-SNARE/genética
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