scholarly journals Involvement of TLR7 and TLR8 in conceptus development and establishment of pregnancy in sheep

Reproduction ◽  
2015 ◽  
Vol 149 (4) ◽  
pp. 305-316 ◽  
Author(s):  
Irene Ruiz-González ◽  
Megan Minten ◽  
Xiaoqiu Wang ◽  
Kathrin A Dunlap ◽  
Fuller W Bazer
Keyword(s):  
Estrous Cycle ◽  
Antisense Oligonucleotides ◽  
Endogenous Retrovirus ◽  
Innate Immune ◽  
Reproductive Status ◽  
Loss Of Function ◽  
Interferon Tau ◽  
Trophectoderm Cells ◽  
Conceptus Development

Toll-like receptors (TLRs) belong to the innate immune system and regulate inflammatory events that affect mammalian reproduction. In Study 1, we demonstrated that abundance of ovine TLR1–TLR9 mRNAs in the uterus differs due to reproductive status (TLR2, TLR3, TLR7, and TLR8) and the day of the estrous cycle and pregnancy (TLR1–TLR3, TLR5–TLR7, and TLR9). Expression of TLR7 and TLR8 proteins was localized primarily to uterine epithelia and stroma and regulated in a temporal manner. In Study 2, we determined that ovine conceptuses express TLR7 and TLR8 on all days studied and that expression of the envelope protein of ovine endogenous retrovirus (enJSRV-Env) declined in conceptus trophectoderm from Day 13 to Day 16 of pregnancy. In Study 3, loss-of-function experiments were conducted in vivo using morpholino antisense oligonucleotides (MAOs) injected into the uterine lumen to block synthesis of TLR7 and TLR8 proteins, individually and jointly. Conceptuses were recovered on Day 16 to assess their morphology. MAO-treated conceptuses were developmentally retarded, produced less interferon tau (IFNT), and had fewer binucleate cells (BNCs) compared with MAO-Controls. Moreover, expression of enJSRV-Env mRNA in MAO-TLR7 conceptuses was greater than that for MAO-Control and MAO-TLR8 conceptuses, but similar to MAO-TLR7/TLR8 conceptuses. Results of this study indicated differences in TLR1–TLR9 expression due to reproductive status and the day of the estrous cycle and pregnancy. TLR7 and TLR8 also influence development, enJSRV-Env abundance, secretion of IFNT, and formation of BNCs by conceptuses. These findings corroborate our hypothesis that TLR7 and TLR8 mediate pathways whereby enJSRV-Env regulates key peri-implantation events in conceptus development and differentiated functions of trophectoderm cells.

scholarly journals Several fibroblast growth factors are expressed during pre-attachment bovine conceptus development and regulate interferon-tau expression from trophectoderm

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 259-269 ◽  
Author(s):  
Flavia N T Cooke ◽  
Kathleen A Pennington ◽  
Qien Yang ◽  
Alan D Ealy
Keyword(s):  
Growth Factors ◽  
Fibroblast Growth Factors ◽  
Mrna Levels ◽  
Fibroblast Growth ◽  
Blastocyst Development ◽  
Interferon Tau ◽  
Trophectoderm Cells ◽  
Conceptus Development

The trophectoderm-derived factor interferon tau (IFNT) maintains the uterus in a pregnancy-receptive state in cattle and sheep. Fibroblast growth factors (FGFs) are implicated in regulatingIFNTexpression and potentially other critical events associated with early conceptus development in cattle. The overall objectives of this work were to identify the variousFGFsand FGF receptors (FGFRs) expressed in elongating pre-attachment bovine conceptuses and determine if these FGFs regulate conceptus development and/or mediate IFNT production.In vitro-derived bovine blastocysts andin vivo-derived elongated conceptuses collected at day 17 of pregnancy express at least fourFGFRsubtypes (R1c,R2b,R3c,R4). In addition, transcripts forFGF1,2, and10but notFGF7are present in elongated bovine conceptuses. The expression pattern ofFGF10most closely resembled that ofIFNT, with both transcripts remaining low in day 8 and day 11 conceptuses and increasing substantially in day 14 and day 17 conceptuses. Supplementation with recombinant FGF1, 2 or 10 increasedIFNTmRNA levels in bovine trophectoderm cells and bovine blastocysts and increased IFNT protein concentrations in trophectoderm-conditioned medium. Blastocyst development was not affected by any of the FGFs. In summary, at least four FGFRs reside in pre- and peri-attachment bovine conceptuses. Moreover, conceptuses express at least three candidate FGFs during elongation, the time of peakIFNTexpression. These findings provide new insight for how conceptus-derived factors such as FGF1, 2, and 10 may controlIFNTexpression during early pregnancy in cattle.

PSXVI-12 Revolutionizing in vitro endometrial cell studies: A novel methodology to obtain bovine endometrial cells

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 325-326
Author(s):  
Cecilia Constantino Rocha ◽  
Felipe Alves Correa Carvalho da Silva ◽  
Thiago Martins ◽  
Marcela Marrero ◽  
John Driver ◽  
...  
Keyword(s):  
Estrous Cycle ◽  
Bos Indicus ◽  
Transcript Abundance ◽  
Specific Method ◽  
Interferon Tau ◽  
Endometrial Cells ◽  
Physiological Relevance ◽  
Estrous Cycle Stage

Abstract Cultured primary endometrial cells are used extensively to study uterine function in cattle. However, most protocols harvest endometrial cells from slaughtered animals at estimated stages of the estrous cycle. The goal of this study was to establish and validate an in vivo, minimally invasive, and estrous cycle stage-specific method to obtain endometrial cells for culture. In Experiment 1, the uterine body of Bos indicus-influenced cows was sampled using a cytology brush (cytobrush) 4 days post estrus (D4; n = 13). Brushes were transported in medium (DMEM/F12, 3% Penicillin/Streptomycin and 2% of Fungizone) to the laboratory at ambient temperature. Cells were cultured in medium containing 10% FBS at 5% of CO2 (38°C). Confluent cells (~7 days of culture) were sub-cultured for two subsequent passages. Pools (n = 4) of cells from 2–3 animals, were frozen, thawed, and re-plated (passage 3). The relative transcript abundance of PPIA, ACTB, KRT18, VIM, OXTR, PGR, ESR1 and IFNAR1 were analyzed by qPCR and compared among fresh cells and cells from each passage. Abundance of KRT18 and VIM transcripts was similar across passages, while PGR, ESR1, OXTR and IFNAR1 transcripts decreased by 90, 96, 84, and 82 %; respectively in cultured compared to fresh cells (P < 0.05). In Experiment 2, passage 3 cells were cultured for 24 hours with 0 or 1ng/mL of recombinant bovine interferon-tau (rbIFNT; n = 3 replicates/treatment). The relative expression of a classical interferon stimulated gene, ISG15, was evaluated by qPCR. Expression of ISG15 was 6-fold greater (P < 0.05) in the rbIFNT treated cells compared to controls. In conclusion, the culture of endometrial cells collected by cytobrush is feasible, generates a monolayer enriched in epithelial cells and may be used as a model for physiological studies involving IFNT signaling. Further experiments to ascertain the physiological relevance of this model are underway.

scholarly journals Progesterone and interferon tau-regulated genes in the ovine uterine endometrium: identification of periostin as a potential mediator of conceptus elongation

Reproduction ◽  
2009 ◽  
Vol 138 (5) ◽  
pp. 813-825 ◽  
Author(s):  
Hyo Won Ahn ◽  
Jennifer L Farmer ◽  
Fuller W Bazer ◽  
Thomas E Spencer
Keyword(s):  
Early Pregnancy ◽  
Calcium Binding ◽  
Collagen Type ◽  
Cdna Array ◽  
Interferon Tau ◽  
Uterine Endometrium ◽  
S100 Calcium Binding Protein ◽  
Trophectoderm Cells ◽  
Conceptus Development

During early pregnancy in ruminants, progesterone (P4) and interferon tau (IFNT) act on the endometrium to regulate genes hypothesized to be important for conceptus development and implantation. The present study was conducted to verify several candidate genes (actin α-2, smooth muscle, aorta (ACTA2), collagen, type III, α-1 (COL3A1), periostin (POSTN), secreted protein acidic cysteine-rich (SPARC), S100 calcium-binding protein A2 (S100A2),STAT5Aand transgelin (TAGLN)) regulated by pregnancy, P4, and/or IFNT in the endometrium determined using a custom ovine cDNA array.S100A2mRNA was detected primarily in endometrial epithelia and conceptuses.S100A2mRNA increased in endometrial epithelia from days 10 to 16 in cyclic ewes and from days 10 to 14 in pregnant ewes and declined thereafter. The abundance ofS100A2mRNA was less in endometrial luminal epithelium of IFNT-infused ewes receiving P4. Expression ofCOL3A1, SPARC, ACTA2, andTAGLNwas independent of pregnancy, P4, or IFNT.POSTNmRNA was detected primarily in compact stroma of intercaruncular and caruncular endometria, but not in the conceptus. EndometrialPOSTNmRNA increased between days 12 and 14 in pregnant but not cyclic ewes, andPOSTNmRNA was more abundant in uterine stroma of ewes treated with P4. POSTN protein was detected in uterine flushings of pregnant ewes and found to mediate attachment and stimulate migration of ovine trophectoderm cellsin vitro. These results support the ideas that POSTN and S100A2 are regulated by P4and IFNT respectively, and that POSTN is involved in conceptus elongation during early pregnancy.

Loss of Function of the VHL Tumor Suppressor Gene Resurrects An Endogenous Retrovirus in Kidney Cancer That Encodes a Tumor Antigen Targeted by Allogeneic T-Cells Following Hematopoietic Cell Transplantation (HCT).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 467-467
Author(s):  
Elena Cherkasova ◽  
Elizabeth Malinzak ◽  
Sheila Rao ◽  
Vera N. Senchenko ◽  
Anna V. Kudryavtseva ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Suppressor ◽  
Kidney Cancer ◽  
Hematopoietic Cell Transplantation ◽  
Clear Cell ◽  
Endogenous Retrovirus ◽  
Loss Of Function ◽  
Rt Pcr ◽  
Normal Tissues

Abstract Abstract 467 Renal cell carcinoma (RCC) has recently been identified as a target for a graft-vs-tumor (GVT) effect following allogeneic hematopoietic cell transplantation (HCT). At the NHLBI, 30 of 76 patients (39%) with metastatic RCC were observed to have delayed tumor regression following a reduced intensity allogeneic HCT consistent with a GVT effect. A better understanding of the tumor antigens targeted by donor immune cells could lead to the development of transplant approaches that enhance GVT effects while avoiding GVHD. We detected RCC-reactive CD8+ T-cells by ELISPOT analysis in the blood of several responding patients with metastatic RCC following HCT that were absent before transplantation. Using PBMC obtained from a patient who had a graft-vs-RCC effect associated with prolonged disease free survival, we isolated CD8+ T-cell clones with RCC-specific tumor cytotoxicity. Utilizing cDNA expression cloning, we identified a 10 amino acid peptide to be the target antigen of RCC-specific T-cells. The genes encoding this antigen were found to be derived from a human endogenous retrovirus type E (named CT-RCC HERV-E) previously unknown to be expressed in any human tissues. Remarkably, RT-PCR showed this endogenous RV was expressed in the majority (13/17) of clear cell tumors but was not expressed in normal kidney tissues, or the non-clear cell histological subtypes of kidney cancer (0/16). A number of different endogenous retroviruses have recently been shown to be expressed in both solid tumors and hematological malignancies and can induce cytotoxic T-cell responses in vivo. However, the factors regulating transcriptional activity of most endogenous retrovirus that lead to tumor-restricted expression are poorly understood. Because the clear-cell histological subtype of RCC is characterized by inactivation of the VHL tumor-suppressor, we hypothesized VHL might regulate expression of CT-RCC HERV-E. RT-PCR showed VHL expression was absent in all clear cell tumors expressing the CT-RCC HERV-E. The HERV-E 5' LTR has a promoter with HIF response elements (HRE) that were found by bisulfite PCR sequencing to be extensively methylated in all normal tissues and non-clear cell RCC tumors but de-methylated in all HERV-E expressing clear cell kidney tumors. Transfection of wild type VHL into VHL mutated clear cell carcinomas dramatically reduced expression of HERV-E derived transcripts. In VHL mutant RCC cells, HIF-2α over-expression was required but alone was insufficient to induce HERV-E expression; SiRNA inhibition of HIF-2α silenced HERV-E expression, although the provirus was only expressed in clear cell tumors that expressed HIF-2α and had de-methylated proviral HREs. Conclusion: These data suggest loss of function of the VHL tumor suppressor leads to selective expression of an endogenous retrovirus in kidney cancer that serves as an antigenic target of T-cells in vivo. Because CT-RCC HERV-E is not expressed in normal tissues, antigens derived from this provirus could serve as excellent targets for cellular immunity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals microRNA-186 in extracellular vesicles from bone marrow mesenchymal stem cells alleviates idiopathic pulmonary fibrosis via interaction with SOX4 and DKK1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jing Zhou ◽  
Yang Lin ◽  
Xiuhua Kang ◽  
Zhicheng Liu ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Extracellular Vesicles ◽  
Luciferase Assay ◽  
Therapeutic Effects ◽  
Loss Of Function ◽  
Fibroblast Activation ◽  
Promising Therapeutic Approach

Abstract Background Previous reports have identified that human bone marrow mesenchymal stem cell-derived extracellular vesicles (BMSC-EVs) with their cargo microRNAs (miRNAs) are a promising therapeutic approach for the treatment of idiopathic pulmonary fibrosis (IPF). Therefore, we explored whether delivery of microRNA-186 (miR-186), a downregulated miRNA in IPF, by BMSC EVs could interfere with the progression of IPF in a murine model. Methods In a co-culture system, we assessed whether BMSC-EVs modulated the activation of fibroblasts. We established a mouse model of PF to evaluate the in vivo therapeutic effects of BMSC-EVs and determined miR-186 expression in BMSC-EVs by polymerase chain reaction. Using a loss-of-function approach, we examined how miR-186 delivered by BMSC-EVs affected fibroblasts. The putative relationship between miR-186 and SRY-related HMG box transcription factor 4 (SOX4) was tested using luciferase assay. Next, we investigated whether EV-miR-186 affected fibroblast activation and PF by targeting SOX4 and its downstream gene, Dickkopf-1 (DKK1). Results BMSC-EVs suppressed lung fibroblast activation and delayed IPF progression in mice. miR-186 was downregulated in IPF but enriched in the BMSC-EVs. miR-186 delivered by BMSC-EVs could suppress fibroblast activation. Furthermore, miR-186 reduced the expression of SOX4, a target gene of miR-186, and hence suppressed the expression of DKK1. Finally, EV-delivered miR-186 impaired fibroblast activation and alleviated PF via downregulation of SOX4 and DKK1. Conclusion In conclusion, miR-186 delivered by BMSC-EVs suppressed SOX4 and DKK1 expression, thereby blocking fibroblast activation and ameliorating IPF, thus presenting a novel therapeutic target for IPF.

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

scholarly journals Roles for RNA export factor, Nxt1, in ensuring muscle integrity and normal RNA expression in Drosophila

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kevin van der Graaf ◽  
Katia Jindrich ◽  
Robert Mitchell ◽  
Helen White-Cooper
Keyword(s):  
Mrna Export ◽  
Rna Stability ◽  
Muscle Degeneration ◽  
Loss Of Function ◽  
Cellular Functions ◽  
Partial Loss ◽  
Export Pathway ◽  
Pathway Gene ◽  
Rna Export

Abstract The mRNA export pathway is responsible for the transport of mRNAs from the nucleus to the cytoplasm, and thus is essential for protein production and normal cellular functions. A partial loss of function allele of the mRNA export factor Nxt1 in Drosophila shows reduced viability and sterility. A previous study has shown that the male fertility defect is due to a defect in transcription and RNA stability, indicating the potential for this pathway to be implicated in processes beyond the known mRNA transport function. Here we investigate the reduced viability of Nxt1 partial loss of function mutants, and describe a defect in growth and maintenance of the larval muscles, leading to muscle degeneration. RNA-seq revealed reduced expression of a set of mRNAs, particularly from genes with long introns in Nxt1 mutant carcass. We detected differential expression of circRNA, and significantly fewer distinct circRNAs expressed in the mutants. Despite the widespread defects in gene expression, muscle degeneration was rescued by increased expression of the costamere component tn (abba) in muscles. This is the first report of a role for the RNA export pathway gene Nxt1 in the maintenance of muscle integrity. Our data also links the mRNA export pathway to a specific role in the expression of mRNA and circRNA from common precursor genes, in vivo.

Flagellin induces innate immune genes in bronchial epithelial cells in vivo: Role of TET2

2021 ◽  
Author(s):  
Wanhai Qin ◽  
Xanthe Brands ◽  
Cornelis Veer ◽  
Alex F. Vos ◽  
Brendon P. Scicluna ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Bronchial Epithelial Cells ◽  
Innate Immune ◽  
Bronchial Epithelial ◽  
Immune Genes ◽  
Innate Immune Genes

scholarly journals β-elemene alleviates airway stenosis via the ILK/Akt pathway modulated by MIR143HG sponging miR-1275

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Guoying Zhang ◽  
Cheng Xue ◽  
Yiming Zeng
Keyword(s):  
Cell Cycle ◽  
Cell Viability ◽  
Cell Model ◽  
Protective Efficacy ◽  
Rabbit Model ◽  
Akt Pathway ◽  
Loss Of Function ◽  
Airway Stenosis

Abstract Background We have previously found that β-elemene could inhibit the viability of airway granulation fibroblasts and prevent airway hyperplastic stenosis. This study aimed to elucidate the underlying mechanism and protective efficacy of β-elemene in vitro and in vivo. Methods Microarray and bioinformatic analysis were used to identify altered pathways related to cell viability in a β-elemene-treated primary cell model and to construct a β-elemene-altered ceRNA network modulating the target pathway. Loss of function and gain of function approaches were performed to examine the role of the ceRNA axis in β-elemene's regulation of the target pathway and cell viability. Additionally, in a β-elemene-treated rabbit model of airway stenosis, endoscopic and histological examinations were used to evaluate its therapeutic efficacy and further verify its mechanism of action. Results The hyperactive ILK/Akt pathway and dysregulated LncRNA-MIR143HG, which acted as a miR-1275 ceRNA to modulate ILK expression, were suppressed in β-elemene-treated airway granulation fibroblasts; β-elemene suppressed the ILK/Akt pathway via the MIR143HG/miR-1275/ILK axis. Additionally, the cell cycle and apoptotic phenotypes of granulation fibroblasts were altered, consistent with ILK/Akt pathway activity. In vivo application of β-elemene attenuated airway granulation hyperplasia and alleviated scar stricture, and histological detections suggested that β-elemene's effects on the MIR143HG/miR-1275/ILK axis and ILK/Akt pathway were in line with in vitro findings. Conclusions MIR143HG and ILK may act as ceRNA to sponge miR-1275. The MIR143HG/miR-1275/ILK axis mediates β-elemene-induced cell cycle arrest and apoptosis of airway granulation fibroblasts by modulating the ILK/Akt pathway, thereby inhibiting airway granulation proliferation and ultimately alleviating airway stenosis.

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