scholarly journals Pyroptosis in pterygium pathogenesis

2018 ◽  
Vol 38 (3) ◽  
Author(s):  
Naiyu Sun ◽  
Hong Zhang
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Pathological Process ◽  
Qrt Pcr ◽  
Caspase 1 ◽  
Human Pterygium Fibroblasts ◽  
Programed Cell Death ◽  
E Cadherin

Pterygium is a common ocular disease characterized by proliferating fibrovascular tissue. Pyroptosis, a recently discovered programed cell death, is known to be associated with oxidative stress, one of the main causes of pterygia. Here, we aimed to study the role of pyroptosis in pterygium pathogenesis. The expression of nod-like receptor pyrins-3 (NLRP3), caspase-1, IL-18, and IL-1β was analyzed in 60 human pterygium tissues and 60 human conjunctival epithelium tissues using real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot analysis. Human conjunctival epithelial cells (HConECs) and human pterygium fibroblasts (HPFs) were primary cultured and the level of pyroptosis-associated factors was detected. Both cells were treated with H2O2, and cell lysis was detected by lactate dehydrogenase (LDH) release assay, the expression of the factors by qRT-PCR, Western blot analysis, and immunostaining. The downstream factors IL-18 and IL-1β were measured after inhibition of caspase-1 to confirm the caspase-1-dependent pyroptosis. α-SMA and E-cadherin were detected as indicators of pyroptosis-induced myofibroblast activation in HPFs. We discovered that the expression of the factors was significantly increased in pterygium and that caspase-1-dependent pyroptosis presents in both H2O2-treated HPFs and HConECs during which the expression of these factors was significantly elevated and the elevation of downstream factors IL-18 and IL-1β was restrained after caspase-1 inhibition. α-SMA increase and E-cadherin down-regulation were detected in H2O2-treated HPFs and the changes were reversed by caspase-1 inhibition. Pyroptosis displays a role in the pathological process of pterygium formation and progression. Pyroptosis appears to be an intriguing target to prevent pterygium pathogenesis.

scholarly journals Quercetin Protects RIMVECs From Inflammatory Damage, Pyroptosis and Cell Migration by Inhibiting the TLR4/NF-κB/NLRP3 Pathway

2021 ◽  
Author(s):  
Huixin Zhang ◽  
Yeye Li ◽  
Zhongjie Liu
Keyword(s):  
Cell Migration ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Inflammatory Factors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Caspase 1 ◽  
Inflammatory Damage

Abstract Background: Intestinal mucosal microvascular endothelial cells (MEC) have multiple functions and play an important role in intestinal bowel diseases (IBD). Quercetin is a flavonoid found in many plants and fruits. It was reported that quercetin can treat several gastrointestinal cancers, but its effect on bacterial enteritis and pyroptosis-related diseases has been rarely studied. This article aims to explore the effect and mechanism of quercetin on inflammatory injury and pyroptosis of RIMVECs.Methods: The inflammatory damage and pyroptosis in RIMVECs were induced by LPS and ATP. Real-time quantitative polymerase chain reaction (RT-qPCR), western blot analysis, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence methods were used to detect TLR4/NF-κB/NLRP3 pathways, inflammatory factors (IL-1β and IL-18) and pyroptosis-related proteins (Caspase-1 and GSDMD). The expression and distribution of ZO-1 were detected by western blot analysis and immunofluorescence method. The late apoptosis and necrosis of cells were measured by cell flow cytometry. Results: The results showed that different concentrations (5, 10, 20μM) of quercetin not only significantly reduced the protein and mRNA levels of TLR4, NLRP3, Caspase-1 and GSDMD, but also down-regulated the protein expression, mRNA and secretion of IL-1β and IL-18. Quercetin also inhibited the phosphorylation of NF-κB p65 and the degradation of IκB. At the same time, quercetin increased the cell migration rate and the expression level of ZO-1, and reduced the number of late apoptotic cells (P<0.05). Conclusions: Our data indicated that Quercetin reduced the inflammatory response and pyroptosis induced by LPS/ATP through the TLR4/NF-κB/NLRP3 pathway, and protected the migration and tight junctions of RIMVECs.

Abstract 663: Identification of 2 Novel Candidate Genes of Atherosclerosis Susceptibility on Mouse Chromosome 3: Pla2g12a and Elovl6

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Alexandros Nicolaou ◽  
Kristina Sass ◽  
Bernd H Northoff ◽  
Daniel Teupser ◽  
Lesca M Holdt
Keyword(s):  
Western Blot ◽  
Candidate Genes ◽  
Mouse Chromosome ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ldl Receptor ◽  
Atherosclerotic Lesion ◽  
Lesion Size ◽  
Specific Expression ◽  
Qrt Pcr

Quantitative trait locus (QTL) mapping in an F2 intercross (n=452) of atherosclerosis-susceptible C57BL/6 (B6) and atherosclerosis-resistant FVB mice on the LDL-receptor deficient background revealed a novel atherosclerosis susceptibility locus on mouse chromosome (Chr) 3. In previous work the susceptible genetic region on Chr3 was narrowed to 80 - 160 MB and validated by congenic FVB.Chr3 B6/B6 mice. We hypothesized that underlying genetic variation in this region leads to differential expression of causal genes, thereby affecting atherosclerosis susceptibility. We performed transcriptome-wide expression analyses in livers of congenic FVB.Chr3 B6/B6 and FVB mice (n=4/4) using Illumina Ref-8 arrays followed by validation in livers of congenic FVB.Chr3 B6/B6 and FVB mice (n=8/9) as well as in livers of B6 and FVB mice (n=5/5) by quantitative real-time PCR (qRT-PCR). C is -regulation was investigated in F2 livers (n=47) by correlating the expression to the genotype. Tissue-specific expression of genes was examined by qRT-PCR in parental B6 and FVB mice. Western blot analysis and immunohistochemical staining (IHC) were performed. Mechanisms of atherogenesis were investigated by RNAi. Pla2g12a and Elovl6 were identified as candidate genes co-segregating with the atherosclerosis QTL at marker rs13464244. Pla2g12a mRNA expression was inversely correlated (r 2 =0.2, p=0.002) with atherosclerotic lesion size in F2 mice while Elovl6 expression was positively correlated (r 2 =0.18, p=0.002). qRT-PCR revealed a strong expression of Pla2g12a in muscle and fat tissues whereas Elovl6 was highly expressed in liver and fat tissues. Western blot analysis revealed significantly decreased protein expression of Pla2g12a in livers of B6 compared to FVB and an increased expression of Elovl6 in B6 mice. IHC staining of Pla2g12a and Elovl6 in aortic roots indicated high expression in macrophages and predominantly in endothelial cells. siRNA knockdown of Elovl6 was associated with reduced adhesion and increased apoptosis. In conclusion, we identified Elovl6 and Pla2g12a as promising candidate genes of atherosclerosis susceptibility on mouse Chr3. Further work is necessary to better understand the influence of these two genes on atherosclerosis development.

The N-Terminally Truncated p53 Isoform Δ40p53 Influences Prognosis in Mucinous Ovarian Cancer

2012 ◽  
Vol 22 (3) ◽  
pp. 372-379 ◽  
Author(s):  
Gerda Hofstetter ◽  
Astrid Berger ◽  
Regina Berger ◽  
Arijana Zorić ◽  
Elena I. Braicu ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Confidence Interval ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Hazard Ratio ◽  
Histological Subtypes ◽  
Mutational Status

ObjectiveThe tumor suppressor p53 generates the N-terminally truncated isoforms Δ40p53 and Δ133p53 that possess the ability to modulate p53 function in vitro. The aim of the present study was to evaluate the clinical relevance of p53 isoforms in the main histological subtypes of ovarian cancer.MethodsΔ40p53, Δ133p53, and full-length p53 (FLp53) expression was determined in 45 mucinous, 30 endometrioid, and 91 serous ovarian cancer specimens as well as 42 normal ovarian tissues using reverse transcriptase–quantitative polymerase chain reaction. In a subgroup of mucinous ovarian cancer cases, Δ40p53 expression was examined using Western blot analysis. A functional yeast-based assay and subsequent sequencing were performed to analyze the p53 mutational status.ResultsIn endometrioid cancer specimens, Δ133p53 expression was significantly lower than in mucinous and serous cases (P = 0.016) or in normal tissues (P = 0.004). Mucinous cancer samples showed elevated Δ40p53 expression as compared with normal ovarian tissues (P = 0.003). In addition, high Δ40p53 expression constituted an independent prognostic marker for recurrence-free but not for overall survival in patients with mucinous ovarian cancer (hazard ratio, 0.267; 95% confidence interval, 0.094–0.756 [P = 0.013]; hazard ratio, 0.453, 95% confidence interval, 0.193–1.064 [P = 0.069]). Western blot analysis confirmed the presence of p53β and Δ40p53α in a subset of patients with mucinous ovarian cancer. Expression of p53 isoforms was not associated with p53 mutational status or clinicopathologic parameters.ConclusionsWe show that expression of p53 isoforms differs in histological subtypes, thus supporting the hypothesis that histological subtypes represent distinct disease entities. In addition, we provide first evidence for a favorable role of Δ40p53 in patients with mucinous ovarian cancer.

scholarly journals Inhibition of NHE1 Induced Apoptosis in Cytarabine Resistant Leukemia Cell Lines and Primary Leukemia Cells from AML Patients, Which Showed Increased Intracellular pH and NHE1 Activity

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3614-3614 ◽  
Author(s):  
Shin Young Hyun ◽  
Young Kyung Kim ◽  
Ji Eun Jang ◽  
Yundeok Kim ◽  
Yu Ri Kim ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Qrt Pcr ◽  
Nhe1 Activity ◽  
Induced Apoptosis ◽  
Primary Leukemia

Abstract Background: Na/H exchanger 1 (NHE1), an important participant in the precise regulation system of intracellular pH (pHi), is known to be involved in pathological processes such as cell transformation, maintenance and active progression of the neoplastic process. Some studies have showed that leukemic cells showed higher pHi than normal cells, and NHE1 inhibitor could induce acidification and apoptosis of the leukemic cells. In this study, we tried to elucidate the role of NHE1 in leukemic cells according to cytarabine (AraC) resistance. Materials and Methods: Two human AML cell lines, AraC sensitive (AS)-OCI-AML2 cells and AraC resistant (AR)-OCI-AML2 cells, primary leukemic cells from AML patients, and normal bone marrow mononuclear cells (BMMNC) from healthy donor were analyzed. The pH-sensitive fluorescent dye, 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) was used to measure pHi and NHE1 activity. The fluorescent ratio of the 490/440 nm was calibrated intracellularly. The expression of NHE1 was measured by qRT-PCR and western blot analysis. To inhibit the NHE1, the amiloride analogue, 5-(N,N-hexamethylene) amiloride (HMA) (10 µM, 20 µM, 30 µM) was used. Results: To confirmed AraC sensitivity, cell lines were treated with 10 µM AraC for 24 hours, and apoptosis fraction in AS-OCI-AML2 cells and AR-OCI-AML2 cells were 53.1±7.2 % and 4.0±0.8 %, respectively. The pHi of AR-OCI-AML2 cells was significantly higher than AS-OCI-AML2 cells (7.839±0.033 vs. 7.589±0.129, P=0.045) and BMMNC (7.839±0.033 vs. 7.578±0.035, P=0.083), and these differences were associated with higher NHE1 activity. Compared AS-OCI-AML2 cells, AR-OCI-AML2 cells showed significantly higher NHE1 expression by western blot analysis (Figure 1), and NHE1 mRNA levels (0.039±0.014 vs. 1.565±0.070, P<.001) by qRT-PCR. Treatment with HMA (20 µM) could induce apoptosis both on AS-OCI-AML2 cells (26.9±2.8%) and AR-OCI-AML2 cells (37.4±18.8%). Interestingly, induction of apoptosis by HMA was dose-dependent both in AS-OCI-AML2 cells and AR-OCI-AML2 cells, and higher concentration of HMA (30 µM) could induce apoptosis on most of AR-OCI-AML2 cells (68.7±20.2%). Co-treatment experiment with 10 µM AraC and 20 µM HMA in AS-OCI-AML2 cells showed additive effect on inducing apoptosis (AraC vs. HMA vs. HMA+AraC = 53.1±12.4 vs. 53.1±12.4 vs. 67.20±4.3%, Figure 2), but in AR-OCI-AML2 cells, co-treatment did not show additional or synergistic effect on inducing apoptosis (AraC vs. HMA vs. HMA+AraC = 4.0±0.1 vs. 27.1±2.2 vs. 28.1±2.0%, Figure 2). As in the cell lines, primary leukemia cells from patients with AraC resistance showing higher pHi and NHE activity than those from patients without. HMA could induce apoptosis on primary cell lines regardless AraC sensitivity. Conclusions: In this study, we first showed that NHE1 inhibition could induce apoptosis in leukemia cells regardless AraC sensitivity. Apoptotic activity was related with higher pHi and NHE activity in AraC resistant cell lines and primary leukemic cells. NHE inhibition induced apoptosis may be independent with AraC induced apoptosis. The heterogeneity in pHi and NHE activity within leukemic cells may be related to alteration in drug delivery machinery or dormant status of leukemia cells. Further experimental and clinical studies are needed to elucidate the therapeutic application of NHE1 inhibitor to AraC resistant AML. Figure 1. Western blot analysis showed higher level of expression of Na/H exchanger I in AR-AML-OCI2 cells than AS-AML-OCI2 cells. Figure 1. Western blot analysis showed higher level of expression of Na/H exchanger I in AR-AML-OCI2 cells than AS-AML-OCI2 cells. Figure 2. Percentage of apoptotic cells after treatment with 20 µM HMA and/or 10 µM AraC. Figure 2. Percentage of apoptotic cells after treatment with 20 µM HMA and/or 10 µM AraC. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effect of Sox9 on TGF-β1-mediated atrial fibrosis

2021 ◽  
Author(s):  
Hechuan Wang ◽  
Yiqi Chen ◽  
Shuting Zhao ◽  
Xiaowen Wang ◽  
Kai Lu ◽  
...  
Keyword(s):  
Cell Migration ◽  
Western Blot ◽  
Blot Analysis ◽  
Protein Level ◽  
Western Blot Analysis ◽  
Transforming Growth Factor ◽  
Quantitative Polymerase Chain Reaction ◽  
Right Atrial ◽  
Atrial Fibrosis ◽  
Tgf Β1

Abstract Atrial fibrosis is a crucial mechanism responsible for atrial fibrillation (AF). Sex-determining region Y-box containing gene 9 (Sox9) plays a pivotal role in fibrosis of many organs such as the skin, kidney, and liver. However, there are few studies about the occurrence and maintenance of Sox9 in atrial fibrosis. In this study, we investigated the role of Sox9 in the fibrotic phenotype of human atrial tissues and rat atrial fibroblasts in vitro. In the human right atrial tissue, Masson’s trichrome staining, immunofluorescence, real-time quantitative polymerase chain reaction, and western blot analysis were carried out to explore the relationship between Sox9 and atrial fibrosis at the morphological, functional, and molecular levels. In cultured atrial fibroblasts, Sox9 was overexpressed by adenovirus or depleted by siRNA, and then, recombinant human transforming growth factor (TGF)-β1 was added. Immunofluorescence analysis, western blot analysis, Transwell assay, and scratch assay were used to analyze the cells. In patient atrial tissues, Sox9 was increased with worsened atrial fibrosis, and this increase was related to AF severity. In rat atrial fibroblasts, Sox9 was promoted by TGF-β1, and the α-smooth muscle actin (α-SMA) protein level and the ability of cell migration were increased after Sox9 overexpression by adenovirus, while the α-SMA protein level and the cell migration ability were decreased after Sox9 depletion by siRNA. In conclusion, Sox9 is involved in the regulation of fibrosis in the atria and may be located downstream of TGF-β1. Our findings may provide a new perspective to treat atrial fibrosis during AF.

scholarly journals HYAL1 Is Downregulated in Idiopathic Pulmonary Fibrosis and Inhibits HFL-1 Fibroblast Proliferation When Upregulated

2020 ◽  
Vol 2020 ◽  
pp. 1-15 ◽  
Author(s):  
Dong Leng ◽  
Xiaoxi Huang ◽  
Jiawen Yi ◽  
Hongying Zhao ◽  
Yuhui Zhang
Keyword(s):  
Data Mining ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Western Blot ◽  
Microarray Data ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Lung Fibroblast ◽  
Fibroblast Proliferation ◽  
Qrt Pcr

Background. Idiopathic pulmonary fibrosis (IPF), the most common interstitial lung disease, arises from transforming growth factor beta 1- (TGFβ1-) induced aberrant fibroproliferation in response to epithelial injury. The TGFβ1 antagonists—hyaluronidases (HYALs)—have been used to clinically treat pulmonary fibrosis. This study focused on characterizing the effect of HYAL1, the main enzyme in hyaluronan degradation, on human lung fibroblast proliferation and apoptosis, and elucidating its potential underlying mechanism of action. Methods. We first performed microarray data mining of previously published gene expression datasets to identify key gene signatures in IPF lung tissues. HYAL1 expression levels in IPF and normal lung tissues were then characterized using immunohistochemistry followed by real-time quantitative reverse transcription-PCR (qRT-PCR) and western blot analysis on isolated fibroblasts from fresh lung tissues of IPF and healthy donors. A human fetal lung fibroblast HFL-1 cell line, which was used in place of primary lung fibroblasts, was used to assess the proliferative or apoptotic effects associated with lentiviral-induced HYAL1 overexpression using CCK-8 cell proliferation assay and Annexin V-APC staining. The identification of potentially associated molecular pathways was performed using microarray analysis followed by qRT-PCR and western blot analysis. Results. Lung tissue microarray data mining and immunohistochemistry revealed significantly downregulation of HYAL1 in IPF lung tissue. However, HYAL1 expression level in IPF fibroblasts was significantly upregulated at the mRNA level, but not altered at the protein level. HYAL1 overexpression in HFL-1 fibroblasts reduced fibroproliferation modestly but did not promote apoptosis. In addition, HYAL1 overexpression led to concomitant transcription factor downregulation, bone morphogenetic protein receptor 2 (BMPR2) signaling activation, but had no effect on TGFβ receptor 2 (TGFβR2) signaling. Conclusions. We showed that HYAL1 overexpression could prevent HFL-1 fibroproliferation. Furthermore, our findings suggest that transcriptional regulators and BMP receptor signaling may be involved in HYAL1 modulation in IPF therapy.

PIP4KIIα Gene Silencing by Lentivirus-Mediated Delivery of shRNA in Human K562 Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3200-3200
Author(s):  
Tânia Regina Zaccariotto ◽  
Daniela Maria Ribeiro ◽  
Joao Machado-Neto ◽  
Magnun N N Santos ◽  
Carolina Lanaro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Globin Gene ◽  
K562 Cells ◽  
Globin Genes ◽  
Qrt Pcr ◽  
Globin Gene Expression

Abstract Abstract 3200 Background: Phosphatidylinositol-phosphate-kinase type II alpha (PIP4KIIα) belongs to a family of lipid kinases responsible for the production of a variety of lipid second messengers, such as PI4,5P2 (phosphatidylinositol 4,5-biphosphate), and appears to be implicated in the regulation of gene expression, pre-mRNA processing and mRNA export. In a previous study, two transcripts, PIP4KIIα and β-globin, were found to be overexpressed in reticulocytes from two siblings with Hb H disease, suggesting a possible relationship between this enzyme and the production of globins, particularly β-globin. Recently, we established a gene expression pattern for PIP4KIIα in healthy individuals during in vitro erythropoiesis and observed a gradual increase in the expression of this gene during erythroid differentiation similar to that observed for globin genes, reinforcing the hypothesis of a relationship between PIP4KIIα and globin expression. Aim: To investigate the effects of PIP4KIIα gene silencing on the expression of α- and γ-globin genes in human K562 cells. Methods: Two different human K562 cells cultures were transduced with a lentiviral vector encoding PIP4KIIα-specific shRNA or non-relevant control shRNA. After transduction the positive cells were selected by adding puromycin to the culture and collected 2, 6, 8 and 10 days later to analyze gene and protein expression. PIP4KIIα and α- and γ-globin gene expression was assessed by qRT-PCR and quantified using the equation RQ=2−ΔΔCt. Western blot analysis was performed to determine PIP4KIIα protein expression. β-actin and GAPDH were used as endogenous controls in the qRT-PCR, and β-actin in the Western blot. Results: Analysis of the results showed that there was a statistically significant reduction in PIP4KIIα mRNA levels in knockdown cells (79%) (0.208 ± 0.048; p<0.0001) compared with the control culture. Western blot analysis corroborated these findings. PIP4KIIα silencing resulted in an 18% (0.927 ± 0.244; p=0.09) and 44% (0.625 ± 0.124; p=0.03) reduction in the expression of α- and γ-globin genes, respectively, compared with the control. Conclusion: Although the reduction in α-globin gene expression did not achieve statistical significance, our results revealed alterations in α- and γ-globin gene expression in PIP4KIIα knockdown cells, suggesting a parallelism between the expression of PIP4KIIα and globin genes and reinforcing the hypothesis that the former may be involved in regulation of the latter. This work was supported by FAPESP, CNPq and INCTS. Disclosures: No relevant conflicts of interest to declare.

FGF-7 facilitates the process of psoriasis by inducing TNF-α expression in HaCaT cells

2019 ◽  
Vol 51 (10) ◽  
pp. 1056-1063 ◽  
Author(s):  
Jiaojiao Pu ◽  
Rui Wang ◽  
Guanglin Zhang ◽  
Ju Wang
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Specific Antibody ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Hacat Cells ◽  
Tnf Α

Abstract The purpose of this study was to uncover the mechanism of tumor necrosis factor (TNF)-α induction by fibroblast growth factor-7 (FGF-7) in human HaCaT cells and the potential role of FGF-7-specific antibody F-9 in psoriatic therapy. TNF-α expression in HaCaT cells induced by FGF-7 was analyzed by quantitative polymerase chain reaction, western blot analysis, and enzyme-linked immunosorbent assays. In vivo, the BALB/c mouse psoriasis model established by topical application of imiquimod (IMQ) was used to determine the role of FGF-7-specific antibody (F-9) in skin inflammation. We found that induction of TNF-α expression by FGF-7 in HaCaT cells was suppressed by FGF-7-specific antibody F-9. Western blot analysis results showed that FGF-7 induced TNF-α expression in HaCaT cells via the FGF receptor 2 (FGFR2)/AKT/NF-κB signaling pathway. In vivo, F-9 could significantly ameliorate the inflammations in a mouse psoriatic model evaluated by Psoriasis Area and Severity Index scores and ear thickness, which was consistent with the results of hematoxylin–eosin staining, immunohistochemistry assay, and western blot analysis. These results indicate that FGF-7 induces TNF-α expression in HaCaT cells and FGF-7 antibody F-9 alleviates IMQ-induced psoriasiform in mice. Therefore, FGF-7/FGFR2 signaling pathway is a potential target for psoriasis treatment.

Abstract 447: Activation of IL-1β-Producing Inflammasomes Triggered by RNA Receptor RIG-I in Mouse Endothelial Cells

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Yang Zhang ◽  
Xiang Li ◽  
Xiao-Xue Li ◽  
Ashley L Pitzer ◽  
Pin-Lan Li
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammasome Activation ◽  
Type I ◽  
Mrna Levels ◽  
Cholesterol Crystals ◽  
Double Stranded Rna ◽  
Caspase 1

Retinoic acid-inducible gene-I (RIG-I) is a putative RNA helicase and recently identified as a cytosolic RNA receptor in mammalian cells. The role of RIG-I in the regulation of vascular function under physiological and pathological conditions is unknown. The present study tested whether RIG-I activation triggers inflammasome formation, turning on inflammation in mouse endothelial cells (EOMA cell line). By real time RT-PCR and Western blot analysis, transfection of mouse ECs with RIG-I specific agonist, 5’-triphosphate double-stranded RNA (3pRNA, 0.5 mg/L) increased RIG-I mRNA level by 106% and protein level by 81% compared to those in control double-stranded RNA (dsRNA) transfected ECs. ELISA analyses showed that 3pRNA significantly increased release of type I IFN alpha by 31 folds and IL-1 beta (a prototype cytokine from inflammasome activation) by 8 folds in these ECs. Proatherogenic stimulation of mouse ECs with cholesterol crystals or 7-ketocholesterol also markedly increased protein expression of RIG-I, but had no effect on RIG-I mRNA levels. Measurements of active caspase-1, an inflammasome activation marker using FLICA fluorescent probe that specifically binds to cleaved caspase-1, demonstrated that 3pRNA doubled FLICA positive cells compared to that in control dsRNA transfected ECs. Interestingly, cholesterol crystals significantly increased FLICA positive cells by 3 folds. This activation of caspase-1 in ECs by cholesterol crystals was further confirmed by increase in cleaved caspase-1 (p10) using Western blot analysis and by enhanced IL-1 beta release as detected by ELISA. In the presence of 3pRNA, cholesterol crystal-induced inflammasome activation was not further augmented. These data indicate that increased expression and activity of RIG-I activate IL-1 beta producing inflammasomes in ECs, which may represent an early molecular mechanism mediating vascular inflammation or injury upon atherogenic stimulations.

Functional Analysis of Cyclin D1 in Mantle Cell Lymphoma (MCL) by Specific Lentiviral shRNA Mediated Knockdown.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1584-1584
Author(s):  
Margit Klier ◽  
Natasa Anastasov ◽  
Daniela Angermeier ◽  
Mark Raffeld ◽  
Falko Fend ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Infection Rates ◽  
Facs Analysis ◽  
Cyclin D2 ◽  
Qrt Pcr

Abstract Introduction: Cyclin D1 overexpression is the hallmark of MCL. However, the importance of cyclin D1 for the maintenance of MCL still remains to be defined. Therefore, the aim of this study is to elucidate the role of cyclin D1 overexpression using the siRNA technology in well-characterized MCL cell lines, as a model system. Material and Methods: A highly efficient cyclin D1-shRNA (96% knockdown) was identified using a lacZ-cyclin D1 fusion gene reporter system in HEK-293T cells. This shRNA was cloned into a lentiviral transfer vector carrying GFP as a reporter gene, which enables the detection of infected cells by FACS analysis. Seven MCL cell lines were analyzed (Granta 519, Jeko-1, Rec-1, Z-138, UPN-1, Hbl-2 and JVM-2), using appropriate controls. Western Blot analysis and qRT-PCR were performed to quantitate the knockdown effect. The effect of cyclin D1 knockdown on proliferation, cell cycle, and viability was analyzed by MTT assay and FACS analysis. Results: The infection rates varied among the different MCL cell lines. Rec-1 and Hbl-2 showed low infection rates (50%) even at high MOI’s (multiplicity of infection), whereas UPN-1 and JVM-2 had moderate infection rates (80%). Jeko-1, Granta 519 and Z-138 showed high infection rates (almost 100% of the cells). Despite the good tranfection rate, the downregulation of cyclin D1, as measured by Western Blot and qRT-PCR, was about 80% in Granta 519, and 65% in Jeko-1 and Z-138. No IFN response, as secondary effect was identified. Interestingly, no apoptosis was observed, and there was only a moderate retardation of growth (60% of control cells) with 10% shift from the S phase to G1 phase of the cell cycle when compared to the controls, suggesting that other cell cycle proteins might compensate, at least partially, for the loss of cyclin D1. Accordingly, cyclin D2 showed upregulation in Western blot analysis and qRT-PCR, whereas the phosphorylation status of retinoblastoma protein on Ser780 was reduced and the expression of the CDK inhibitor p27Kip1 increased. No changes were observed in the expression of cyclin D3, Cyclin E, CDK4 and CDK2. Conclusions: In this study, a system that enables the specific downregulation of cyclin D1 in MCL cell lines was established. Surprisingly, the downregulation of cyclin D1 in MCL cell lines resulted in only a moderate inhibition on cell growth with no apoptosis. The reasons for this might be 1) that the upregulation of cyclin D2 compensates for cyclin D1 downregulation, and/or 2) that the chromosomal translocation leading to cyclin D1 overexpression is an initiating event in MCL lymphomagenesis followed by secondary genetic events at later stages of the disease, which make cyclin D1 dispensable. This finding has important implications for MCL therapy, as strategies targeting only cyclin D1 might be hampered by the redundancy of the system, resulting in a low probability of treatment response.

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