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Objective To investigate the effects and mechanisms of Zhenxin Anshen Prescription(composed of Os Draconis,Ostreae Concha,Lophatheri Herba,Drynariae Rhizoma,Poria)on mice with atopic dermatitis(AD)based on the calcium channel regulator 1(ORAI1)/nuclear factor of T-cells(NFAT)signalling axis.Methods Thirty-six BALB/c mice were randomly divided into a blank control group,a model group,a Cetirizine group(1.3 mg·kg-1)and a Zhenxin Anshen Prescription group(36.36 g·kg-1),with nine mice in each group.AD mouse model was established using 1-chloro-2,4-dinitrobenzene(DNCB)induction.The drug was administered by gavage once a day for 2 weeks.At the end of drug administration,the area of skin lesions was measured and the severity of skin lesions was scored;spleen mass was measured and spleen index was calculated;pathological changes of skin lesion tissues were observed by HE staining;interleukin 4(IL-4),IL-13 and thymic stromal lymphopoietin(TSLP)in serum were detected by ELISA;and the protein expression levels of ORAI1,calmodulin phosphatase A(CaN)and nuclear factor of T cells 2(NFAT2)were detected by Western Blot.Results Compared with the blank control group,the skin lesion score of mice in the model group was significantly increased(P<0.01),the skin lesion area was significantly enlarged(P<0.01);the thickness of the epidermis and dermis were significantly increased(P<0.01),hyperkeratosis of the epidermis,hypertrophy of the stratum spinosum,and infiltration of inflammatory cells such as eosinophils and lymphocytes can be seen in the dermis;the splenic index and serum IL-4,IL-13,TSLP levels were significantly increased(P<0.01);protein expression levels of CaN,NFAT2,ORAI1 were significantly increased in the skin lesion tissues(P<0.01).Compared with the model group,the dermatitis score of mice in the Zhenxin Anshen Prescription group was significantly decreased(P<0.01),the lesion area was significantly reduced(P<0.01),the epidermal and dermal thicknesses(P<0.01),the hyperkeratosis of epidermis was alleviated,the spinous layer was slightly hypertrophic,and there was a small amount of inflammatory cell infiltration in the dermis;the splenic index and the levels of serum IL-4,IL-13,and TSLP were significantly decreased(P<0.01);the protein expressions levels of CaN,NFAT2,and ORAI1 in the skin lesion tissues were significantly decreased(P<0.01).Conclusion Zhenxin Anshen Prescription can ameliorate dermatopathological injury in DNCB-induced AD mice,and the mechanism may be related to its ability to inhibit the protein expressions of ORAI1,CaN and NFAT2,reduce the levels of serum type 2 inflammatory factors TSLP,IL-4 and IL-13,and ameliorate cutaneous inflammation and itching through immunomodulation.
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ObjectiveTo explore the underlying mechanism of Tripterygium wilfordii polyglycoside tablets (TWPT) in the prevention and treatment of kidney injury in diabetic nephropathy (DN) through the nuclear factor of activated T-cells 2(NFAT2)/cyclooxygenase-2(COX-2) pathway. MethodForty-two male SD rats of SPF grade were selected and randomly divided into a normal group (n=8) and an experimental group (n=34) after one week of adaptive feeding. The rats in the normal group were fed conventionally. The DN model was established in rats of the experimental group by intraperitoneal injection of streptozotocin (STZ) following one week of feeding on a high-fat and high-glucose diet. After the death and failure cases during modeling were eliminated, the remaining 24 model rats were randomly divided into model group, valsartan (8.33 mg·kg-1·d-1) group, and TWPT (5 mg·kg-1·d-1) group. Rats in normal group and model group were given equal amounts of normal saline by gavage. After six weeks, body weight was measured and urine samples were collected. Blood samples were collected from the abdominal aorta, and then the rats were sacrificed for sampling. Biochemical indicators, such as serum blood urea nitrogen (BUN), serum creatinine (SCr), alanine aminotransferase (ALT), blood lipid, blood glucose, and 24-hour urine total protein (24 h UTP), were determined. Hematoxylin-eosin (HE) staining and Masson staining were used to observe the pathology of the kidney. Enzyme-linked immunosorbent assay (ELISA) was used to detect NFAT2 and COX-2 expression levels in the serum. Western blot and Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)were adopted to detect NFAT2, COX-2 protein and mRNA expression in kidney tissues, respectively. ResultCompared with the normal group, the model group showed elevated 24 h UTP, BUN, SCr, CHO, TG, and FBG, increased serum NFAT2 and COX-2 production and expression (P<0.01), and elevated protein and mRNA expression of NFAT2 and COX-2 in kidney tissues (P<0.01). In addition, the pathology of the kidney showed enlarged glomeruli, mild proliferation of mesangial cells, and widened mesangial stroma. Compared with the model group, the TWPT group showed decreased 24 h UTP, BUN, SCr, CHO, TG, and FBG (P<0.05,P<0.01), basically normal glomerular morphology, decreased expression of serum NFAT2 and COX-2 (P<0.01), and down-regulated protein and mRNA expression of NFAT2 and COX-2 in kidney tissues (P<0.01). ConclusionTWPT can alleviate 24 h UTP in DN model rats, protect renal function, and improve renal pathology, and its mechanism of action may be related to the down-regulation of NFAT2/COX-2 expression in the serum and kidney tissues.
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Increasing evidences suggest the important role of calcium homeostasis in hallmarks of cancer, but its function and regulatory network in metastasis remain unclear. A comprehensive investigation of key regulators in cancer metastasis is urgently needed. Transcriptome sequencing (RNA-seq) of primary esophageal squamous cell carcinoma (ESCC) and matched metastatic tissues and a series of gain/loss-of-function experiments identified potassium channel tetramerization domain containing 4 (KCTD4) as a driver of cancer metastasis. KCTD4 expression was found upregulated in metastatic ESCC. High KCTD4 expression is associated with poor prognosis in patients with ESCC and contributes to cancer metastasis in vitro and in vivo. Mechanistically, KCTD4 binds to CLIC1 and disrupts its dimerization, thus increasing intracellular Ca2+ level to enhance NFATc1-dependent fibronectin transcription. KCTD4-induced fibronectin secretion activates fibroblasts in a paracrine manner, which in turn promotes cancer cell invasion via MMP24 signaling as positive feedback. Furthermore, a lead compound K279-0738 significantly suppresses cancer metastasis by targeting the KCTD4‒CLIC1 interaction, providing a potential therapeutic strategy. Taken together, our study not only uncovers KCTD4 as a regulator of calcium homeostasis, but also reveals KCTD4/CLIC1-Ca2+-NFATc1-fibronectin signaling as a novel mechanism of cancer metastasis. These findings validate KCTD4 as a potential prognostic biomarker and therapeutic target for ESCC.
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ObjectiveTo explore the effect of Xiao Xianxiongtang (XXXT) on the transforming growth factor (TGF)-β1-induced invasion, metastasis, and epithelial-mesenchymal transition (EMT) of gastric cancer MGC-803 cells and the underlying mechanism. MethodThe molecular docking between XXXT and nuclear factor of activated T cells (NFAT) was performed by CB-DOCK (http://clab.labshare.cn/cb-dock/). The invasion and metastasis model of MGC-803 cells was established with 10 μg·L-1 TGF-β1. MGC-803 cells were classified into blank group, model group, 0.1 g·L-1 XXXT group, 0.2 g·L-1 XXXT group, and 0.4 g·L-1 XXXT group. For further clarifying the key role of Wnt5a/Ca2+/NFAT signaling pathway in the inhibition of XXXT on gastric cancer, MGC-803 cells were transfected with Wnt5a overexpression plasmid, and then the cells were classified into blank plasmid group, Wnt5a-OE group, blank plasmid + XXXT (0.4 g·L-1) group, and Wnt5a-OE + XXXT (0.4 g·L-1) group. Cell viability was determined by cell counting kit-8 (CCK-8) assay, cell invasion and migration ability by Transwell invasion assay and wound healing assay, expression of EMT-related proteins (E-cadherin, N-cadherin, Vimentin, Snail) and Wnt5a/Ca2+/NFAT signaling pathway-related key proteins [Wnt5a, calcineurin (CaN), NFAT1, and p-NFAT1] by Western blot, and changes in intracellular Ca2+ concentration by immunofluorescence assay. ResultMolecular docking suggested that XXXT acted on Wnt5a/Ca2+/NFAT signaling pathway. XXXT (0.1, 0.2, 0.4 g·L-1) significantly promoted the loss of MGC-803 cell viability (P<0.05,P<0.01). It inhibited cells from invading the transwell lower chamber and slowed down the healing of cell wounds in a dose-dependent manner (P<0.05, P<0.01). Moreover, it promoted the expression of E-cadherin while suppressed the expression of N-cadherin, Vimentin, and Snail (P<0.05, P<0.01). Further experiments showed that XXXT could inhibit the expression of Wnt5a, CaN, NFAT1, and p-NFAT1, and reduce the nuclear expression of NFAT1 and the transcription activity mediated by NFAT1, so as to reduce the cellular Ca2+ concentration (P<0.05, P<0.01). XXXT can reverse the effect of Wnt5a (P<0.05, P<0.01). ConclusionXXXT can attenuate the invasion, metastasis, and EMT of MGC-803 cells via the Wnt5a/Ca2+/NFAT pathway, thereby weakening the tumor-promoting effect of TGF-β1. In summary, XXXT may exert therapeutic effect on gastric cancer by regulating the invasion, metastasis, and EMT of gastric cancer cells.
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Aim To study the protective effect of cyclosporine A (CsA) on daunorubicin (DNR) injured H9c2 cardiomyoblasts. Methods H9c2 cells were pre-treated with CsA for 2 hours, then co-cultured with 1 μmol · L
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Objective:This studu aims to investigate the effect of aqueous extract of modified Xiao Xianxiongtang on the epithelial mesenchymal transition(EMT) and the change of its invasion and migration ability of human gastric cancer MGC-803 cells mediated by transforming growth factor-<italic>β</italic><sub>1</sub>(TGF-<italic>β</italic><sub>1</sub>),and to explore the mechanism of regulating Wnt5a/Ca<sup>2+</sup>/ activated T-cell nuclear factor(NFAT) signaling pathway to inhibit EMT and invasion and metastasis of MGC-803 cells. Method:TGF-<italic>β</italic><sub>1</sub>(10 μg·L<sup>-1</sup>)was used to induce EMT and the invasion and metastasis model of human gastric cancer MGC-803 cells. Transwell chamber experiment, scratchhealing experiment, Western blot and immunofluorescence assay were used to detect cell invasion and migration ability, expression of EMT marker protein and key protein expression of Wnt5a/Ca<sup>2+</sup>/NFAT signaling pathway, and intracellular Ca<sup>2+</sup> concentration. Result:Compared with the blank group, TGF-<italic>β</italic><sub>1</sub> could significantly enhance the invasion and migration ability of MGC-803 cells(<italic>P</italic><0.01), down-regulate the level of E-cadherin(<italic>P</italic><0.01), up-regulate protein expressions of N-cadherin, Snail and Vimentin(<italic>P</italic><0.01), and induce cell Wnt5a, calcineurin (CaN), total protein of activated T-cell nuclear factor 1(NFAT1), up-regulation of phosphorylated proteins p-NFAT1 and NFAT1 nucleoprotein and intracellular accumulation of Ca<sup>2+</sup>(<italic>P</italic><0.01). Compared with the TGF-<italic>β</italic><sub>1</sub> group, modified Xiao Xianxiongtang (10, 20, 40 mg·L<sup>-1</sup>) could significantly inhibit this phenomenon,and 40 mg·L<sup>-1</sup> had the best effect(<italic>P</italic><0.05,<italic>P</italic><0.01).The specific inhibitors of Wnt5a/Ca<sup>2+</sup>/NFAT signaling pathway (<italic>R</italic>)-(+)-Bay-K-8644 and modified Xiao Xianxiongtang (40 mg·L<sup>-1</sup>) could significantly inhibit theinvasion and migration of MGC-803 cells mediated by TGF-<italic>β</italic><sub>1</sub>, up-regulate the level of E-cadherin, and down-regulate expressions of N-cadherin, Snail, Vimentin, Wnt5a, CaN and NFAT1 proteins and reduce the intracellular accumulation of Ca<sup>2+</sup>(<italic>P</italic><0.05,<italic>P</italic><0.01).Moreover, (<italic>R</italic>)-(+)-Bay-K-8644 combined with modified Xiao Xianxiongtang (40 mg·L<sup>-1</sup>) had stronger inhibitory effect(<italic>P</italic><0.05,<italic>P</italic><0.01). Conclusion:These results suggest that modified Xiao Xianxiongtang can inhibit the EMT mediated by TGF-<italic>β</italic><sub>1</sub> via Wnt5a/Ca<sup>2+</sup>/NFAT signaling pathway,thereby reducing the invasion and migration ability of MGC-803 cells.
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BACKGROUND: Regular exercise training possesses health promotion effect. Low-to-moderate intensity continuous aerobic exercise has been an important strategy for primary and secondary prevention of chronic diseases such as hypertension; however, the effect of high-intensity interval training is still debated. OBJECTIVE: To explore the effects of high-intensity interval training on pathological cardiac hypertrophy and investigate the possible mechanism in spontaneously hypertensive rats. METHODS: Thirty male spontaneously hypertensive rats were randomly assigned into a control group and a training group. Fifteen Wistar-Kyoto rats were used as normotensive group. Rats in the normotensive and control group were housed at rest, while those in the training group were subjected to a high-intensity interval training lasting for 8 weeks. After experiment, blood pressure was detected using a non-invasive blood pressure tester, and cardiac structure and function were measured by echocardiogram. Histopathological detection was performed by hematoxylin-eosin and Masson staining to determine myocardial cross-sectional area. mRNA expression of fetal genes including atrial natriuretic peptide and brain natriuretic peptide were detected by RT-PCR. Protein expression of PI3-K, Akt, CnAβ and NFATc3 was detected using western blot assay. RESULTS AND CONCLUSION: Compared with the normotensive group, the blood pressure level was significantly elevated (P 0.05) in the control group. Compared with the control group, the blood systolic pressure was lowered (P 0.05). Therefore, the 8-week high-intensity interval training can induce the transfer from pathological hypertrophy to physiological hypertrophy and enhance heart function in spontaneously hypertensive rats via the activation of PI3-K/Akt signal transduction pathway; however, the Cn/NFAT pathway cannot be inhibited.
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OBJECTIVE: To establish a stably-transfected cell line with adr adrenergic receptorα2B-AR) and enhanced green fluorescent protein (EGFP) labeled nucleus factor of activated T cells 2 (NFAT2) (EGFP-NFAT2) in U20S. METHODS U20S cells that stably expressed EGFP-NFAT2(U20S-EGFP-NFAT2 cells) were transfected with pcDNA3.1 expressing hygromycin B (Hygro) resistance gene and α2B-AR gene (pcDNA3.1-Hygro-α2B-AR) recombinant plasmid. The transfected cells were selected by Hygro (200 mg · L-1) for 10 d before being screened on the nucleus translocation assay of EGFP-NFAT2. Then, the selected cells were evaluated by Z' factor for functional stability and azrAR expression in the selected cells was analyzed by quantitative real-time PCR (qRT-PCR) and Western blotting. Finally, the activity of α2B-AR on NFAT2 nucleus translocation was evaluated by α2-AR agonist dexme-detomidine hydrochloride (DMED) or antagonist atipamezole hydrochloride (ATI) treatment. RESULTS Recombinant plasmid pcDNA3.1-Hygro-α2B-AR was established and transfected to U20S cells stably expressing EGFP-NFAT2. In the stably-transfected No.12 cell strain, the relative nuclear translocation index (0.445) was the highest among the selected cell lines, and Z' factors on three independent experiments were 0.664, 0.533 and 0.634, respectively. The mRNA expression of α2B-AR of No. 12 cell strain was detected by qRT-PCR and was about 140 times that of U20S-EGFP-NFAT2 cells (P<0.01) in 20 generations. The protein band of α2B-AR was also detected in No. 12 cell strain whereas no band of α2B-AR was detected in U20S-EGFP-NFAT2 cell by Western blotting. DMED concentration-depend-ently increased the relative translocation nuclear index in U20S-EGFP-NFAT2-α2B-AR cell [EC50= (2.616±0.121) nmol·L-1]. ATI concentration-dependency decreased the relative nuclear translocation index in U20S-EGFP-NFAT2-α2B-AR cell [IC50 =(89.05±0.22) nmol-L-1]. CONCLUSION The stably-transfected U20S-EGFP-NFAT2-α2B-AR cell line is established and can be used for high throughout screening of biased chemicals and the study on the mechanism of α2B-AR.
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<p><b>OBJECTIVE</b>To determine whether hyperglycemia activates NFAT2 in cultured podocytes to cause podocyte apoptosis and explore the role of NFAT2 in high glucose-induced podocyte apoptosis.</p><p><b>METHODS</b>Immortalized mouse podocytes were cultured in the presence of normal (5.3 mmol/L) or high glucose (10, 20, 30, and 40 mmol/L) or pretreated with 11R-vivit (100 nmol/L) or cyclosporine A (500 nmol/L) before exposure to 20 mmol/L glucose for different durations (0.5-48 h). The activation of NFAT2 in the podocytes was detected using Western blotting and immunofluorescence assay. The role of NFAT2 in hyperglycemia-induced podocyte apoptosis was explored by observing the inhibition of NFAT2 activation by 11R-vivit using flow cytometry. Intracellular Ca was monitored in high glucose-treated podocytes using Fluo-3/AM. The mRNA and protein expressions of the apoptosis gene Bax were detected using real time-qPCR and Western blotting.</p><p><b>RESULTS</b>Exposure to high glucose in the medium time- and dose-dependently activated NFAT2 in cultured podocytes. Pretreatment with cyclosporine A or 11R- VIVIT completely blocked nuclear accumulation of NFAT2. Treatment with 11R- vivit also inhibited high glucoseinduced apoptosis in cultured podocytes. Exposure to high glucose obviously increased [Ca]I in the podocytes to cause activation of calcineurin and the subsequent increment of nuclear accumulation of NFAT2 and Bax expression.</p><p><b>CONCLUSIONS</b>High glucose-induced apoptosis in podocytes is mediated by calcineurin/NFAT2/Bax signaling pathway, which may serve as a potential target for therapeutic intervention.</p>
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Objective To investigate the expressions of apoptosis associated protein 3 (APR3) and nuclear factor 3 of activated T-cell (NFAT3) in the tissue of epithelial ovarian tumors and its correlation with the clinicopathological features.Methods 92 patients with epithelial ovarian tumor were collected,23 cases with malignant tumor,24 cases with borderline tumor,45 cases with benign tumor.The expressions of APR3 and NFAT3 were detected by immunohistochemical methods,and the differences of different types of epithelial ovarian tumor were compared.The correlation of the expressions of APR3 and NFAT3 with the clinicopathological features of epithelial ovarian tumor was analyzed.The correlation of the expressions of APR3 with the expressions of NFAT3 in epithelial ovarian tumor was analyzed.Results The positive expression rate of APR3 in patients with malignant epithelial ovarian tumors (78.26%) was significantly higher than borderline tumors (41.67 %) and benign tumors (22.22 %),the differences were statistically significant (x2 =5.864,7.632,all P < 0.05).The expression of APR3 in patients with malignant epithelial ovarian tumors was significantly correlated with differentiation,clinical stage,lymph node and abdominal organs metastasis and ascites (x2 =7.425,7.262,8.421,5.031,all P < 0.05).The positive expression rate of NFAT3 in patients with malignant epithelial ovarian tumors (56.52%) was significantly higher than borderline tumors (29.17%) and benign tumors(17.78%),the differences were statistically significant (x2 =6.829,7.547,all P <0.05).The expression of NFAT3 in patients with malignant epithelial ovarian tumors was significantly correlated with differentiation,clinical stage,lymph node and abdominal organs metastasis (x2 =5.253,6.367,8.021,all P < 0.05).The expressions of APR3 and NFAT3 in patients with malignant epithelial ovarian tumors were positively correlated (r =0.032,P < 0.05).Conclusion The expressions of APR3 and NFAT3 in the tissue of malignant epithelial ovarian tumor obviously increase,are significantly correlated with differentiation,clinical stage,lymph node and abdominal organs metastasis and are positively correlated,and it may be correlated with the development and progression of malignant epithelial ovarian tumor.
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Objective To explore the effects of various radiation doses on the NFAT3/c4 signaling pathway and the improvement effect of exogenous brain-derived neurotrophic factor(BDNF)on this pathway. Methods Four groups of one-month-old Sprague-Dawley rats received radiation doses of 0,2,10,and 20 Gy, respectively, in a single radiation. At three days after radiation, exogenous BDNF was injected stereotaxically into the bilateral hippocampus. Western blotting and RT-PCR were used to assess the levels of NFAT3/c4-related proteins in the hippocampus. Results The results of Western blotting and RT-PCR showed that the level of NFAT3/c4 was reduced in a dose-and time-dependent manner after ionizing radiation. Compared with the radiation alone group,the ionizing radiation plus BDNF group had significantly increased levels of NFAT3/c4 and CaN with increases in radiation dose and time. Conclusions Whole brain radiotherapy inhibits the CaN/NFAT3/c4 signaling pathway. Exogenous BDNF can promote the NFAT-dependent transcription and then improve the cognitive function.
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Objective To investigate the effects on IL-6 and PGE2 expression in wear-particles-induced osteoblast cells by blocking calcium phosphatase (Cn)/ activated T nuclear factor (NFAT) pathway. Methods Fetal Sprague-Dawley rats were used in this study. Osteoblast were prepared from the calvariae of rats . Osteoblast cells were incubated in four group according to different supplementation:(1) neither Ti particles nor 11R-VIVIT (Control group), (2) only Ti particles (Ti group), (3) both Ti particles and 11R-VIVIT (Ti/VIVIT group), and (4) only 11R-VIVIT (VIVIT group). Cells were incubated for 96 hours and the expression of NFATc1 protein was detected by western blot. The expression of IL-6 and PGE2 in liquid supernatant of osteoblast were detected at 6, 24 and 96 hours by ELISA. Results The expression of NFATc1 in the Ti group was higher than that in the Control group (P < 0.01), but in Ti/VIVIT group that was significantly lower than in the titanium particle group (P < 0.01). The IL-6 and PGE2 expression in the supernatant of the Ti group were significantly increased than those in the control group (P < 0.05). The IL-6 and PGE2 in the Ti/VIVIT group were significantly lower than that in the Ti group (P < 0.05). Conclusions 11R-VIVIT peptide specific blockade of Cn/NFAT signaling pathway significantly inhibited IL-6 and PGE2 of osteoblast cells induced by titanium particles.
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Understanding the cellular and molecular mechanisms involved in the development and progression of pulmonary hypertension (PH) remains imperative if we are to successfully improve the quality of life and life span of patients with the disease. A whole plethora of mechanisms are associated with the development and progression of PH. Such complexity makes it difficult to isolate one particular pathway to target clinically. Changes in intracellular free calcium concentration, the most common intracellular second messenger, can have significant impact in defining the pathogenic mechanisms leading to its development and persistence. Signaling pathways leading to the elevation of [Ca2+]cyt contribute to pulmonary vasoconstriction, excessive proliferation of smooth muscle cells and ultimately pulmonary vascular remodeling. This current review serves to summarize the some of the most recent advances in the regulation of calcium during pulmonary hypertension.
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Humanos , Calcio , Concentración de Iones de Hidrógeno , Hipertensión Pulmonar , Miocitos del Músculo Liso , Calidad de Vida , Sistemas de Mensajero Secundario , VasoconstricciónRESUMEN
The activation of nuclear factor of activated T cells 5 (NFAT5), a well-known osmoprotective factor, can be induced by isotonic stimuli, such as activated Toll-like receptors (TLRs). It is unclear, however, how NFAT5 discriminates between isotonic and hypertonic stimuli. In this study we identified a novel context-dependent suppression of NFAT5 target gene expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) or a high salt (NaCl) concentration. Although LPS and NaCl both used NFAT5 as a core transcription factor, these stimuli mutually inhibited distinct sets of NFAT5 targets within the cells. Although reactive oxygen species (ROS) are essential for this inhibition, the source of ROS differed depending on the context: mitochondria for high salt and xanthine oxidase for TLRs. Specifically, the high salt-induced suppression of interleukin-6 (IL-6) production was mediated through the ROS-induced inhibition of NFAT5 binding to the IL-6 promoter. The context-dependent inhibition of NFAT5 target gene expression was also confirmed in mouse spleen and kidney tissues that were cotreated with LPS and high salt. Taken together, our data suggest that ROS function as molecular sensors to discriminate between TLR ligation and osmotic stimuli in RAW 264.7 macrophages, directing NFAT5 activity toward proinflammatory or hypertonic responses in a context-dependent manner.
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Animales , Masculino , Ratones , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-6/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Manitol/farmacología , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Rotenona/farmacología , Cloruro de Sodio/farmacología , Receptores Toll-Like , Factores de Transcripción/genéticaRESUMEN
Durante a transcrição gênica em eucariotos, a produção de níveis significativos de mRNA é conferida pela presença de diversos sítios de ligação para fatores de transcrição específicos, localizados dentro e fora do promotor basal. Dentre os fatores de transcrição que se ligam a estes sítios estão as proteínas da família NFAT (nuclear factor of activated T cells), composta pelos NFAT1-5, sendo o NFAT1 o alvo desse estudo. O NFAT pode regular seus genes alvo direta ou indiretamente e, neste processo, a ativação ou a repressão da transcrição pode ser alterada pela interação com diferentes parceiros proteicos. Esses dois pontos da regulação transcricional mediados pelo NFAT foram avaliados em duas etapas distintas. Na primeira, avaliamos se a regulação do proto-oncogene c-Myc pelo NFAT1 ocorria de forma direta. Tanto NFAT quanto c-Myc estão envolvidos na regulação de genes de ciclo celular, apoptose e angiogênese. Já tinha sido mostrado que as vias de sinalização que ativam NFAT induzem a expressão de c-Myc e que o NFAT1 se liga a um elemento localizado no promotor mínimo de c-Myc, apesar da importância desse elemento não ter sido avaliada no contexto do promotor completo de c-Myc. Nós mostramos que a regulação desse promotor pelo NFAT1 é mais complexa do que se acreditava. Além desse sítio proximal, o NFAT1 se liga a sítios distais com diferentes afinidades. Nossos resultados sugerem que alguns elementos NFAT são negativos e dominantes, enquanto outros são positivos e recessivos. Além disso, demonstramos que a cooperação com p300 aumenta a transativação do promotor de c-Myc mediada pelo NFAT1. Por último, mostramos que os novos sítios identificados também são responsivos ao NFAT2, outro membro da família, que pode tanto ter funções opostas quanto redundantes com o NFAT1. Assim, sugerimos que a contribuição do NFAT para a regulação da expressão de c-Myc é direta e depende do balanço entre a ligação do NFAT aos sítios positivos e negativos e da cooperação com cofatores transcricionais. Na segunda parte do trabalho, focamos nas implicações da interação recém-identificada entre NFAT1 e a proteína IRF-2BP2. A IRF-2BP2 foi pescada num ensaio de duplo híbrido com a região TAD-C do NFAT1, região esta que é menos conservada entre as proteínas NFAT. Mostramos que a IRF- 2BP2 reprime a expressão de genes de citocina induzida pelo NFAT1 e que ela age somente sobre a função do NFAT1 dentro da família NFAT. Nossos dados também sugerem que a IRF-2BP2 não se ligue ao DNA e, portanto, funcione como um correpressor transcricional. Ainda mostramos que a superexpressão de IRF-2BP2 leva ao atraso da progressão das fases G0/G1 para a fase S do ciclo celular e à alteração da expressão de genes de ciclo celular, como de c-Myc, que também é regulado por NFAT1. Esses dados sugerem que a IRF-2BP2 possa ter um papel bem mais amplo na regulação do NFAT1, não se restringindo apenas à modulação de genes de citocinas. Se isso se confirmar, a interação entre NFAT1 e IRF-2BP2 poderá contribuir para explicar as diferenças fenotípicas exercidas pelos diferentes membros da família NFAT.
During gene transcription in eukaryotes, the production of significant mRNA levels is conferred by the presence of several binding sites for specific transcription factors, located inside and outside of the basal promoter. Among the transcription factors that bind to these sites are proteins of the NFAT family (nuclear factor of activated T cells), comprising the NFAT1-5, being the NFAT1 the target of this study. The NFAT can regulate their target genes directly or indirectly and in this process, the activation or repression of transcription can be altered by interaction with different partner proteins. These two points of NFAT-mediated transcriptional regulation were assessed here in two stages. In the first one, we evaluated whether the regulation of the proto-oncogene c-Myc by NFAT1 occurred directly. Both c-Myc as NFAT regulate genes involved in cell cycle, apoptosis and angiogenesis. It has already been shown that the signaling pathways that activate NFAT induce the c-Myc expression and that NFAT1 binds to an element located at the minimal c-Myc promoter, although the importance of this element has not been assessed in the full c-Myc promoter context. We demonstrated that the regulation of this promoter by NFAT1 is more complex than previously conceived. In addition to this proximal site, NFAT1 binds to distal sites with different affinities. Our results suggest that some NFAT elements are negative and dominant, while others are positive and recessive. Furthermore, we demonstrated that cooperation with p300 enhances NFAT1-mediated transactivation of the c-Myc promoter. Finally, we show that the newly identified sites are also responsive to NFAT2, another member of NFAT family, which can both have opposite as well as redundant functions with NFAT1. We suggest that NFAT1 the contribution of NFAT to the regulation of c-Myc expression is direct and may depend on a balance between the binding to positive and negative NFAT-responsive elements, and cooperation with transcriptional cofactors. In the second part of this work, we focused on the implications of the recently identified interaction between NFAT1 and the protein IRF-2BP2. The IRF-2BP2 was identified in a two-hybrid assay with the TAD-C region of the NFAT1, a region which is less conserved among the NFAT proteins. We showed that IRF-2BP2 represses the expression of cytokine genes induced by NFAT1 and regulates specifically the function of NFAT1 among the NFAT family members. Our data also suggest that the IRF- 2BP2 does not bind to DNA and therefore functions as a transcriptional correpressor. Therefore, the overexpression of IRF-2BP2 leads to a delayed transition from G0/G1 to S phase of cell cycle and also alters the expression of many cell cycle genes, such as c-Myc, which is also regulated by NFAT1. These data suggest that the IRF-2BP2 may have a much greater role regulating NFAT1, not being restricted to the modulation of cytokine genes. If this is confirmed, the interaction between NFAT1 and IRF-2BP2 may help to explain the phenotypic differences exerted by different NFAT family members.
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Regulación de la Expresión Génica , Factores de Transcripción NFATCRESUMEN
Tonicity-responsive enhancer binding protein (TonEBP) is a signal transcription factor of transporters such as sodium-myo-inositol cotransporter (SMIT), aldose reductase. TonEBP has a variety of functions such as control of intracellular osmolytes and immunomodulating. It is known that TonEBP is abundant in the placenta, but location and function aren't known. The aim of this study is to describe the localization of TonEBP in the placenta. We assayed the immunohistochemistry of TonEBP and performed in situ hybridization of SMIT in normal human full term placenta. In normal human full term placenta, TonEBP was in villous trophoblasts, extravillous trophoblasts and some endothelial cells. The result of the in situ hybridization of SMIT was similar to that of immunohistochemistry of TonEBP. Neither TonEBP nor SMIT was present in TonEBP knockout mouse placenta. This shows TonEBP is a key factor in SMIT transcription. TonEBP may play an important role in transporting of inositol to fetus in placenta.
Asunto(s)
Animales , Humanos , Ratones , Aldehído Reductasa , Proteínas Portadoras , Células Endoteliales , Feto , Inmunohistoquímica , Hibridación in Situ , Inositol , Ratones Noqueados , Placenta , Factores de Transcripción , TrofoblastosRESUMEN
Platinum nanoparticles (PtNP) exhibit remarkable antioxidant activity. There is growing evidence concerning a positive relationship between oxidative stress and bone loss, suggesting that PtNP could protect against bone loss by modulating oxidative stress. Intragastric administration of PtNP reduced ovariectomy (OVX)-induced bone loss with a decreased level of activity and number of osteoclast (OC) in vivo. PtNP inhibited OC formation by impairing the receptor activator of nuclear factor-kappaB ligand (RANKL) signaling. This impairment was due to a decreased activation of nuclear factor-kappaB and a reduced level of nuclear factor in activated T-cells, cytoplasmic 1 (NFAT2). PtNP lowered RANKL-induced long lasting reactive oxygen species as well as intracellular concentrations of Ca2+ oscillation. Our data clearly highlight the potential of PtNP for the amelioration of bone loss after estrogen deficiency by attenuated OC formation.
Asunto(s)
Animales , Ratones , Nanopartículas del Metal/administración & dosificación , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/metabolismo , Osteoclastos/efectos de los fármacos , Osteoporosis/tratamiento farmacológico , Ovariectomía/efectos adversos , Estrés Oxidativo/efectos de los fármacos , Platino (Metal)/administración & dosificación , Ligando RANK/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Os fatores de transcrição da família NFAT (nuclear factor of activated T cells) foram inicialmente descritos por desempenhar um papel central na resposta imune. Esta família de fatores de transcrição é composta por cinco diferentes genes, NFAT1-5, que codificam diferentes isoformas protéicas. Os membros NFAT1-4 possuem dois importantes domínios de ativação da transcrição (TAD transcription activation domain) localizados nas regiões N- e C-terminais (TAD-N; TAD-C) da proteína. Estes domínios possuem relativamente pouca conservação de sequência e são, possivelmente, as principais regiões responsáveis pelas diferenças na regulação gênica entre os diferentes membros da família NFAT. Diversos estudos já demonstraram que as proteínas NFAT desempenham papéis não redundantes no processo de tumorigênese. Recentemente, nosso grupo demonstrou que o NFAT1/C reverte a transformação celular induzida pelo oncogene H-RasV12 em fibroblastos NIH3T3 pela ativação do mecanismo de morte celular. O NFAT1 já foi descrito por regular genes envolvidos na apoptose, tais como FasL, TNF-alfa e Nur77. O NFAT1 regula forma direta a expressão de FasL e TNF-alfa e de forma indireta o Nur77 agindo como um coativador de MEF2D. Nesta dissertação, nós demonstramos que as isoformas predominantes do NFAT1, B e C (CANFAT1/ B; CA-NFAT1/C), são capazes de induzir apoptose em fibroblastos NIH3T3. Além disso, demonstramos que a retirada dos resíduos 699 a 888 do TAD-C abole por completo a capacidade de induzir apoptose do NFAT1/C e induz a hiperproliferação de fibroblastos NIH3T3. No entanto, apesar da apoptose induzida pelo NFAT1/C ser dependente de resíduos de aminoácidos presentes no seu TAD-C, demonstramos que o TAD-C não é capaz de induzir apoptose em fibroblastos NIH3T3 por si só. O TAD-C também não age como um dominante negativo do NFAT1/C indicando que esta região da proteína não interage com nenhuma proteína parceira para induzir apoptose. Além disso, demonstramos que a apoptose induzida pelo NFAT1/C é dependente da sua ligação ao DNA. Em conjunto, estes dados sugerem que o gene induzido para ativar a apoptose no nosso modelo é diretamente regulado pelo NFAT1/C e depende do seu TAD-C para ser ativado.
Transcription factors of the NFAT family (nuclear factor of activated T cells) were initially described to play a central role in immune response. This family of transcription factors consists of five different genes, NFAT1-5, which encode different protein isoforms. Members NFAT1-4 possess two significant transcription activation domains (TAD) located in the N- and C-terminal protein regions (TAD-N and TAD-C). These domains have relatively little conservation of sequence and are, possibly, the main regions responsible for differences in gene regulation between different NFAT family members. Several studies have shown that NFAT proteins play non-redundant roles in the process of tumorigenesis. Recently, our group has shown that the NFAT1/C reverses HRasV12- induced cell transformation in NIH3T3 fibroblasts by the activation of cell death mechanism. The NFAT1 has been described to regulate genes involved in apoptosis such as FasL, TNF-alfa and Nur77. The NFAT1 directly regulates the expression of FasL and TNF-alfa and indirectly the expression of Nur77 acting as a coactivator of MEF2D. In this dissertation, we demonstrated that the predominant isoforms of NFAT1, B and C (CA-NFAT1/B, CANFAT1/ C), are able to induce apoptosis in NIH3T3 fibroblasts. Furthermore, we demonstrated that the removal of residues 699 to 888 of TAD-C completely abolishes the ability to induce apoptosis of NFAT1/C and induces the proliferation of NIH3T3 fibroblasts. However, despite the apoptosis induced by NFAT1/C to be dependent on amino acid residues present in their TAD-C, we demonstrated that the TAD-C is not able to induce apoptosis in NIH3T3 fibroblasts alone. The TAD-C also does not act as a dominant negative NFAT1/C indicating that this region of the protein does not interact with any protein partner to induce apoptosis. Furthermore, we demonstrated that apoptosis induced by NFAT1/C is dependent on its binding to DNA. Together, these results suggest that the gene induced to activate apoptosis in our model is directly regulated by NFAT1/C and depends on your TAD-C to be activated.
Asunto(s)
Masculino , Femenino , Humanos , Apoptosis , Muerte Celular/genética , Genes Supresores de Tumor , Factores de Transcripción NFATCRESUMEN
PURPOSE: Dendritic cells (DCs) are considered the most professional antigen-presenting cells (APCs) because of their unique role in initiating immunity against threatening antigens. Recently, hypertonicity has been suggested to be involved in the activation and development of immune cells such as T cells. And tonicity enhancer binding protein (TonEBP) has been thought to play a pivotal role in this process. Here, we studied the maturation status of DCs and expression of TonEBP in DCs exposed to hypertonic condition. METHODS: Murine bone marrow-derived DCs were generated in the presence of GM-CSF for 6 days, and then exposed to hypertonic media. We evaluated the functional capacities and maturation of DCs using flow cytometry, mixed lymphocyte reaction, and cytokine analysis. Also we investigated the expression of TonEBP in DCs cultured in variable hypertonic media. RESULTS: Mild hypertonicity made CD11c+ DCs to have up-regulation of CD40, 80, and 86. DCs exposed to 320 mOsm/kg media stimulated allogeneic T cell proliferation most effectively compared to DCs exposed to other tonic conditions. However, DCs exposed to 400 mOsm/kg media showed similar stimulatory capacities to isotonic control. Consistent with the phenotype changes, IL-1, 6, and TNF-alpha secretion increased in CD11c+ DCs exposed to mild hypertonic condition. Though we confirmed that TonEBP was expressed in CD11c+ DCs, the amount of upregulation was not dependent on the degree of hypertonicity. CONCLUSION: Our results suggest that hypertonicity enhances the maturation and activation of DCs.
Asunto(s)
Células Presentadoras de Antígenos , Proteínas Portadoras , Proliferación Celular , Células Dendríticas , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Interleucina-1 , Prueba de Cultivo Mixto de Linfocitos , Concentración Osmolar , Fenotipo , Linfocitos T , Factor de Necrosis Tumoral alfa , Regulación hacia ArribaRESUMEN
Nuclear factor of activated T-cells (NFAT) proteins are, calcium-regulated transcription factors, key regulator of stimulation-dependent gene activation. In our microarray analysis for the genes expressed in human black and white hairs, NFAT2 was significantly upregulated in the white hair, compared to the black hair. The aim of this study was to investigate functional role of NFAT2 in melanogenesis. Western blot analysis was performed to investigate the expression of NFAT2 protein in B16 melanoma cells. Our data showed that NFAT2 expression was increased in the hypopigmented B16 cells, while tyrosinase and MITF expression was decreased. To investigate the potential role of NFAT2, the recombinant adenovirus expressing microRNA specific for NFAT2 was transduced into the cultured B16 melanoma cells. Consistently, inhibition of NFAT2 enhanced tyrosinase activity and melanin content. Moreover, cyclosporine A, which is known as a calcineurin inhibitor blocking NFAT activation, enhanced tyrosinase activity and melanin content. These data suggest that NFAT2 may play an important role in regulation of melanogenesis in melanocyte.