scholarly journals Canonical, Non-Canonical and Atypical Pathways of Nuclear Factor кb Activation in Preeclampsia

2020 ◽  
Vol 21 (15) ◽  
pp. 5574
Author(s):  
Agata Sakowicz ◽  
Michalina Bralewska ◽  
Tadeusz Pietrucha ◽  
Dominika E Habrowska-Górczyńska ◽  
Agnieszka W Piastowska-Ciesielska ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Protein Level ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Rt Pcr ◽  
Protein Levels ◽  
Activation Pathways ◽  
Expression And Activity

Although higher nuclear factor κB (NFκB) expression and activity is observed in preeclamptic placentas, its mechanism of activation is unknown. This is the first study to investigate whether the canonical, non-canonical, or atypical NFκB activation pathways may be responsible for the higher activation of NFκB observed in preeclamptic placentas. The study included 268 cases (130 preeclamptic women and 138 controls). We studied the expression of the genes coding for NFκB activators (NIK, IKKα, IKKβ, and CK2α) and inhibitors (IκBα and IκBβ) using RT-PCR in real time. The RT-PCR results were verified on the protein level using ELISA and Western blot. To determine the efficiency of the pathways, the ratios of activator(s) to one of the inhibitors (IκBα or IκBβ) were calculated for each studied pathway. The preeclamptic placentas demonstrated significantly lower IKKα and CK2α but higher IκBα and IκBβ protein levels. In addition, the calculated activator(s) to inhibitor (IκBα or IκBβ) ratios suggested that all studied pathways might be downregulated in preeclamptic placentas. Our results indicate that preeclamptic placentas may demonstrate mechanisms of NFκB activation other than the canonical, non-canonical, and atypical forms. In these mechanisms, inhibitors of NFκB may play a key role. These observations broaden the existing knowledge regarding the molecular background of preeclampsia development.

scholarly journals Cardiac deletion of α-E-catenin leads to redused expression of the main component of desmosomes – γ-catenin

1970 ◽  
Vol 21 ◽  
pp. 293-296
Author(s):  
V. V. Balatskyi ◽  
L. L. Matsevych ◽  
O. O. Piven
Keyword(s):  
Heart Failure ◽  
Transgenic Mice ◽  
Western Blot ◽  
Real Time ◽  
Knockout Mice ◽  
Protein Level ◽  
Conditional Knockout ◽  
Protein Levels ◽  
Main Component ◽  
Real Time Qpcr

Aim. In our present work, we have addressed to the γ-catenin, known main component of desmosomes, expression in hearts with heterozygous and homozygous knockout of α-E-catenin gene. Methods. Alpha-E-catenin conditional knockout mice were bred with α-MHC-Cre transgenic mice. We analyze expression of γ-catenin with real time qPCR and Western blot. Results. Cardiac α-E-catenin deletion leads to downregulation of γ-catenin mRNA and protein levels only in homozygous mice, while we not observed any perturbation of γ-catenin expression in heterozygous mice. Conclusions. We have shown that homozygous knockout of α-E-catenin gene in embryonic heart occur reduction of the main component of desmosomes – γ-catenin mRNA and protein level of expression, which can lead to disruption of the desmosomes structure in adult myocardium. Keywords: α-E-catenin, heart failure, γ-catenin.

scholarly journals The PAAD/PYRIN-Family Protein ASC Is a Dual Regulator of a Conserved Step in Nuclear Factor κB Activation Pathways

2002 ◽  
Vol 196 (12) ◽  
pp. 1605-1615 ◽  
Author(s):  
Christian Stehlik ◽  
Loredana Fiorentino ◽  
Andrea Dorfleutner ◽  
Jean-Marie Bruey ◽  
Eugenia M. Ariza ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Adaptor Protein ◽  
Large Family ◽  
Nuclear Factor Κb ◽  
Inhibitory Influence ◽  
Nuclear Factor ◽  
Death Domain ◽  
Protein Levels ◽  
Inflammatory Stimuli ◽  
Activation Pathways

Apoptosis-associated speck-like protein containing a Caspase recruitment domain (ASC) belongs to a large family of proteins that contain a Pyrin, AIM, ASC, and death domain-like (PAAD) domain (also known as PYRIN, DAPIN, Pyk). Recent data have suggested that ASC functions as an adaptor protein linking various PAAD-family proteins to pathways involved in nuclear factor (NF)-κB and pro-Caspase-1 activation. We present evidence here that the role of ASC in modulating NF-κB activation pathways is much broader than previously suspected, as it can either inhibit or activate NF-κB, depending on cellular context. While coexpression of ASC with certain PAAD-family proteins such as Pyrin and Cryopyrin increases NF-κB activity, ASC has an inhibitory influence on NF-κB activation by various proinflammatory stimuli, including tumor necrosis factor (TNF)α, interleukin 1β, and lipopolysaccharide (LPS). Elevations in ASC protein levels or of the PAAD domain of ASC suppressed activation of IκB kinases in cells exposed to pro-inflammatory stimuli. Conversely, reducing endogenous levels of ASC using siRNA enhanced TNF- and LPS-induced degradation of the IKK substrate, IκBα. Our findings suggest that ASC modulates diverse NF-κB induction pathways by acting upon the IKK complex, implying a broad role for this and similar proteins containing PAAD domains in regulation of inflammatory responses.

scholarly journals Interleukin-37 contributes to the pathogenesis of gout by affecting PDZ domain-containing 1 protein through the nuclear factor-kappa B pathway

2020 ◽  
Vol 48 (9) ◽  
pp. 030006052094871
Author(s):  
Wei Wan ◽  
Yeqing Shi ◽  
Lianmei Ji ◽  
Xiaofang Li ◽  
Xia Xu ◽  
...  
Keyword(s):  
Western Blot ◽  
Western Blotting ◽  
Protein Level ◽  
Nuclear Factor Kappa B ◽  
Mrna Level ◽  
Pdz Domain ◽  
Nuclear Factor Κb ◽  
Pyrrolidine Dithiocarbamate ◽  
Nuclear Factor ◽  
Translational Level

Objective Our objective was to explore the molecular pathogenesis of the onset of gout and the mechanism underlying the effect of interleukin (IL)-37 on PDZ domain-containing 1 (PDZK1) protein through the nuclear factor-κB signaling pathway. Methods Real-time PCR and western blotting were used to detect expression of PDZK1 mRNA and protein, respectively, in the HK-2 cell line. The inhibitors pyrrolidine dithiocarbamate (PDTC) and wortmannin were added to HK-2 cells stimulated by IL-37, and changes in PDZK1 protein were detected by western blotting. Results Based on our previous research, we used 10 µmol/L PDTC. We detected no significant change in PDZK1 at the mRNA level among the IL-37, PDTC+IL-37, and wortmannin+IL-37 groups. With increasing IL-37 concentration, the protein level of PDZK1 increased. After adding wortmannin, the protein level of PDZK1 increased with increasing concentration of IL-37, albeit not significantly, and the level of PDZK1 remained lower than that with IL-37 alone. After adding PDTC, the protein level of PDZK1 showed a trend to decrease with increasing concentrations of IL-37 up to 40 ng/mL. The immunofluorescence results supported the western blot results. Conclusions IL-37 can affect protein expression of PDZK1, but not at the translational level, in the pathogenesis of gout.

scholarly journals Expression pattern of Wif1 and β-catenin during development of anorectum in fetal rats with anorectal malformations

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4445 ◽  
Author(s):  
Xiao Bing Tang ◽  
Huan Li ◽  
Jin Zhang ◽  
Wei Lin Wang ◽  
Zheng Wei Yuan ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Expression Pattern ◽  
Immunohistochemical Staining ◽  
Anorectal Malformations ◽  
Rt Pcr ◽  
Rat Embryos ◽  
Fetal Rats ◽  
Normal Rat ◽  
Wnt Inhibitory Factor 1

Purpose This study was performed to investigate the expression pattern of Wnt inhibitory factor 1 (Wif1) and β-catenin during anorectal development in normal and anorectal malformation (ARM) embryos and the possible role of Wif1 and β-catenin in the pathogenesis of ARM. Methods ARM was induced with ethylenethiourea on the 10th gestational day in rat embryos. Cesarean deliveries were performed to harvest the embryos. The expression pattern of Wif1 and β-catenin protein and mRNA was evaluated in normal rat embryos (n = 288) and ARM rat embryos (n = 306) from GD13 to GD16 using immunohistochemical staining, Western blot, and real time RT-PCR. Results Immunohistochemical staining revealed that in normal embryos Wif1 was constantly expressed in the cloaca from GD13 to GD16. On GD13 and GD14, Wif1-immunopositive cells were extensively expressed in the cloaca. On GD15, the expression of Wif1 were mainly detected on the very thin anal membrane. In ARM embryos, the epithelium of the hindgut and urorectal septum demonstrated faint immunostaining for Wif1 from GD14 to GD16. Western blot and real time RT-PCR revealed that Wif1 and β-catenin protein and mRNA expression level was significantly decreased in the ARM groups compared with the normal group on GD14 and GD15 (p < 0.05). Conclusions This study demonstrated that the expression pattern of Wif1 and β-catenin was disrupted in ARM embryos during anorectal morphogenesis, which demonstrated that downregulation of Wif1 and β-catenin at the time of cloacal separation into the primitive rectum and urogenital septum might related to the development of ARM.

scholarly journals p62 acts as an oncogene and is targeted by miR-124-3p in glioma

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Danni Deng ◽  
Kaiming Luo ◽  
Hongmei Liu ◽  
Xichen Nie ◽  
Lian Xue ◽  
...  
Keyword(s):  
Glioma Cells ◽  
Nuclear Factor Κb ◽  
Autophagic Flux ◽  
Nuclear Factor ◽  
Oxygen Species ◽  
Human Glioma ◽  
Protein Levels ◽  
Novel Therapeutic

Abstract Background Glioma is the most common central nervous system (CNS) tumour. p62, an important autophagy adaptor, plays a crucial role in cancer. However, the role of p62 in the progression of glioma is poorly characterized. Methods We examined the expression of p62 in glioma tissues and cell lines. Then we investigated the function of p62 in vitro, and clarified the mechanism underlying the regulation of p62 expression. Results We revealed that p62 was upregulated at both the mRNA and protein levels in human glioma tissues irrelevant to isocitrate dehydrogenase (IDH) status. Then, we found that overexpression of p62 promoted glioma progression by promoting proliferation, migration, glycolysis, temozolomide (TMZ) resistance and nuclear factor κB (NF-κB) signalling pathway, and repressing autophagic flux and reactive oxygen species (ROS) in vitro. In accordance with p62 overexpression, knockdown of p62 exerted anti-tumour effects in glioma cells. Subsequently, we demonstrated that miR-124-3p directly targeted the 3′-UTR of p62 mRNA, leading to the downregulation of p62. Finally, we found that p62 function could be partially reversed by miR-124-3p overexpression. Conclusions Our results demonstrate that p62 can be targeted by miR-124-3p and acts as an oncogene in glioma, suggesting the potential value of p62 as a novel therapeutic target for glioma.

REAL TIME RT-PCR VEGF mRNA EXPRESSION AND ERK1 EXPRESSION AND ACTIVITY IN RENAL TUMORS

1999 ◽  
pp. 148
Author(s):  
Donata Villari ◽  
Giulio Nicita ◽  
Carmela Tricarico ◽  
Astrid Parenti ◽  
Alessandro Della Melina ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Renal Tumors ◽  
Rt Pcr ◽  
Vegf Mrna ◽  
Vegf Mrna Expression ◽  
Expression And Activity

scholarly journals Expression and activity of Apaf1 and caspase-9 in granulosa cells during follicular atresia in pig ovaries

Reproduction ◽  
2003 ◽  
pp. 113-120 ◽  
Author(s):  
T Matsui ◽  
N Manabe ◽  
Y Goto ◽  
N Inoue ◽  
S Nishihara ◽  
...  
Keyword(s):  
Western Blot ◽  
Granulosa Cells ◽  
Blot Analysis ◽  
Crucial Role ◽  
Mitochondrial Pathway ◽  
Signalling Pathway ◽  
Follicular Atresia ◽  
Rt Pcr ◽  
Caspase 9 ◽  
Expression And Activity

Apoptosis in granulosa cells plays a crucial role in ovarian follicular atresia, but the intracellular regulating mechanism, especially the mitochondrion-dependent apoptosis signalling pathway, is still largely unknown. This study examined whether the mitochondrial pathway is associated with granulosa cell apoptosis during atresia in pig ovaries. Both mRNAs of caspase-9 and apoptotic protease-activating factor 1 (Apaf1), which are major signal transducing components in the mitochondrial pathway, were detected in granulosa cells in healthy, early atretic and progressed atretic follicles by RT-PCR. No changes in the expression of Apaf1 mRNA were seen during follicular atresia, but the expression of caspase-9 mRNA increased during atresia. Apaf1 protein was steadily detected in granulosa cells prepared from healthy, early atretic and progressed atretic follicles by western blot analysis, but high expression of the precursor of caspase-9 (procaspase-9) was detected only in granulosa cells of healthy follicles. Decreased procaspase-9 protein was demonstrated during follicular atresia. Proteolytic activity of caspase-9 increased during atresia, in agreement with the diminution of procaspase-9 protein. Intensive expression of caspase-9 mRNA was demonstrated in the granulosa cells of early atretic and progressed atretic follicles but not in those of healthy follicles. These results indicate that the mitochondrial signalling pathway, which is mediated by Apaf1 and caspase-9, plays a crucial role in determining the fate of granulosa cells during atresia in pig ovaries.

scholarly journals Torque Teno Virus (SANBAN Isolate) ORF2 Protein Suppresses NF-κB Pathways via Interaction with IκB Kinases

2007 ◽  
Vol 81 (21) ◽  
pp. 11917-11924 ◽  
Author(s):  
Hong Zheng ◽  
Linbai Ye ◽  
Xiaonan Fang ◽  
Baozong Li ◽  
Yuhua Wang ◽  
...  
Keyword(s):  
Western Blot ◽  
Nuclear Factor Κb ◽  
Torque Teno Virus ◽  
Dependent Manner ◽  
Nuclear Factor ◽  
Innate And Adaptive Immunity ◽  
Reading Frame ◽  
Orf2 Protein ◽  
First Time ◽  
Dose Dependent

ABSTRACT Since the first discovery of Torque teno virus (TTV) in 1997, many researchers focused on its epidemiology and transcriptional regulation, but the function of TTV-encoded proteins remained unknown. The function of the TTV open reading frame (ORF) in the nuclear factor κB (NF-κB) pathway has not yet been established. In this study, we found for the first time that the TTV ORF2 protein could suppress NF-κB activity in a dose-dependent manner in the canonical NF-κB pathway. By Western blot analysis, we proved that the TTV ORF2 protein did not alter the level of NF-κB expression but prevented the p50 and p65 subunits from entering the nucleus due to the inhibition of IκBα protein degradation. Further immunoprecipitation assays showed that the TTV ORF2 protein could physically interact with IKKβ as well as IKKα, but not IKKγ. Luciferase assays and Western blot experiments showed that the TTV ORF2 protein could also suppress NF-κB activity in the noncanonical NF-κB pathway and block the activation and translocation of p52. Finally, we found that the TTV ORF2 protein inhibited the transcription of NF-κB-mediated downstream genes (interleukin 6 [IL-6], IL-8, and COX-2) through down-regulation of NF-κB. Together, these data indicate that the TTV ORF2 protein suppresses the canonical and noncanonical NF-κB pathways, suggesting that the TTV ORF2 protein may be involved in regulating the innate and adaptive immunity of organisms, contributing to TTV pathogenesis, and even be related to some diseases.

scholarly journals Liraglutide Exerts Antidiabetic Effect via PTP1B and PI3K/Akt2 Signaling Pathway in Skeletal Muscle of KKAy Mice

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Wenjun Ji ◽  
Xinlin Chen ◽  
Juan Lv ◽  
Meng Wang ◽  
Shuting Ren ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Western Blot ◽  
Real Time ◽  
Signaling Pathway ◽  
Real Time Pcr ◽  
Glucose Transporter ◽  
Tyrosine Phosphatase ◽  
Gene Expressions ◽  
Protein Levels ◽  
Kkay Mice

Background. Liraglutide (a glucagon-like peptide 1 analog) was used for the treatment of type 2 diabetes (T2DM) which could produce glucose-dependent insulin secretion.Aim. The aim was to investigate whether liraglutide could improve myofibril and mitochondria injury in skeletal muscle and the mechanisms in diabetic KKAy mice.Method. We divided the male KKAy mice into 2 groups: liraglutide group (250 μg/kg/day liraglutide subcutaneous injection) and model group; meanwhile, the male C57BL/6J mice were considered as the control. After 6 weeks, the ultrastructure of skeletal muscle was observed by electron microscope. The gene expressions of protein tyrosine phosphatase 1B (PTP1B), phosphatidylinositol 3-kinase (PI3K), and glucose transporter type 4 (GLUT4) were determined by real-time PCR. The protein levels of the above molecules and phospho-Akt2 (p-Akt2) were measured by Western blot.Results. Liraglutide significantly ameliorated the injury of mitochondria by increasing the number (+441%) and the area (+113%) of mitochondria and mitochondrial area/100 µm2(+396%) in skeletal muscle of KKAy mice. The results of real-time PCR and Western blot showed that liraglutide downregulated PTP1B while it upregulated PI3K and GLUT4 (P<0.01). The protein level of p-Akt2/Akt2 was also increased (P<0.01).Conclusion. These results revealed that liraglutide could improve myofibril and mitochondria injury in skeletal muscle against T2DM via PTP1B and PI3K/Akt2 signaling pathway.

scholarly journals NF-κB and pSTAT3 synergistically drive G6PD overexpression and facilitate sensitivity to G6PD inhibition in ccRCC

2020 ◽  
Author(s):  
Qiao Zhang ◽  
Zhe Yang ◽  
Yueli Ni ◽  
Honggang Bai ◽  
Qiaoqiao Han ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Signaling Pathway ◽  
Protein Interaction ◽  
Xenograft Model ◽  
Metabolic Reprogramming ◽  
Regulatory Function ◽  
Rt Pcr ◽  
Repressive Effect ◽  
The Impact

Abstract Background: Glucose 6-phosphate dehydrogenase (G6PD) serves key roles in cancer cell metabolic reprogramming, and has been reported to be involved in certain carcinogenesis. Previous results from our laboratory demonstrated that overexpressed G6PD was a potential prognostic biomarker in clear cell renal cell carcinoma (ccRCC), the most common subtype of kidney cancer. G6PD could stimulate ccRCC growth and invasion through facilitating reactive oxygen species (ROS)-phosphorylated signal transducer and activator of transcription 3 (pSTAT3) activation and ROS-MAPK-MMP2 axis pathway, respectively. However, the reasons for ectopic G6PD overexpression and the proliferation repressive effect of G6PD inhibition in ccRCC are still unclear. Methods: The impact of ROS accumulation on NF-κB signaling pathway and G6PD expression was determined by real-time RT-PCR and Western blot in ccRCC cells following treatment with ROS stimulator or scavenger. The regulatory function of NF-κB signaling pathway in G6PD transcription was analyzed by real-time RT-PCR, Western blot, luciferase and ChIP assay in ccRCC cells following treatment with NF-κB signaling activator/inhibitor or lentivirus infection. ChIP and Co-IP assay was performed to demonstrate protein-DNA and protein-protein interaction of NF-κB and pSTAT3, respectively. MTS assay, human tissue detection and xenograft model were conducted to characterize the association between NF-κB, pSTAT3, G6PD expression level and proliferation functions. Results: ROS-stimulated NF-κB and pSTAT3 signaling over-activation could activate each other, and exhibit cross-talks in G6PD aberrant transcriptional regulation. The underlying mechanism was that NF-κB signaling pathway facilitated G6PD transcription via direct DNA–protein interaction with p65 instead of p50. p65 and pSTAT3 formed a p65/pSTAT3 complex, occupied the pSTAT3-binding site on G6PD promoter, and contributed to ccRCC proliferation following facilitated G6PD overexpression. G6PD, pSTAT3, and p65 were highly expressed and positively correlated with each other in ccRCC tissues, confirming that NF-κB and pSTAT3 synergistically promote G6PD overexpression. Moreover, G6PD inhibitor exhibited tumor-suppressor activities in ccRCC and attenuated the growth of ccRCC cells both in vitro and in vivo . Conclusion: ROS-stimulated aberrations of NF-κB and pSTAT3 signaling pathway synergistically drive G6PD transcription through forming a p65/pSTAT3 complex. Moreover, G6PD activity inhibition may be a promising therapeutic strategy for ccRCC treatment.

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