mike across america 2020

10 Dec mike across america 2020

Through overexpression experiments, we show that miR-200s repress endogenous ZEB1 and ZEB2 expression in human myometrial cells. Near term, a decrease in circulating P4 and/or a decrease in PR function results in down-regulation of ZEB1 expression, and, in turn, up-regulation of the miR-200 family, further suppressing ZEB1 and ZEB2. As can be seen, P4 had a pronounced effect on the up-regulation of ZEB1 promoter activity in cells cotransfected with WT PR-B; however, this was largely prevented in cells cotransfected with mutPR-BDBD (Fig. Furthermore, few studies have … Researchers are still trying to understand what causes this strong correlation between neural and social networks. Expression of PR in responsive tissues is driven by estrogen-bound ER and, … 2008 Dec;118(12):3829-32. doi: 10.1172/JCI37785. HEK293 cells were transfected with various PR expression plasmids and ZEB1-Luciferase reporter constructs using Fugene 6 transfection reagent (Roche) according to the manufacturer’s protocol. Data are the mean ± SD values from three replicate experiments (Student’s t test, *P < 0.05, **P < 0.01). 2000). 2007 Jun 1;7 Suppl 1(Suppl 1):S10. doi: 10.1152/ajprenal.00638.2016. Online ISSN 1091-6490. Relaxin (RLX), a polypeptide hormone produced in the corpus luteum of pregnancy as well as in the placenta and decidua inhibits PIP 2 turnover and subsequent signaling in human myometrium. -Corticotrophin-releasing hormone (CRH): promotes uterine quiescence during pregnancy . Work in the laboratory of C.R.M. Figure 8. Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. S1C). At present, the exact factors initiating human parturition remain unknown, and labour may occur due to a loss of uterine quiescence, an increase in uterine contractility, or a combination of both. For example, P4 and PR inhibit activation of COX-2 expression in myometrial cells through direct interaction of PR with NF-κB p65 (5) and by P4-induced expression of the NF-κB inhibitor, IκB-α (6). Dysregulation of the myome-trial contractility can lead to either preterm or slow-to- Using a ChIP Assay Kit (catalog no. These collective findings suggest that P4 can inhibit miR-200b/429 expression and that this may be mediated, in part, via P4 induction of ZEB1. (G) Overexpression of ZEB1 and ZEB2 in hTERT-HM cells inhibits oxytocin-mediated contraction. In an effort to identify miRNAs that mediate myometrial transition to a contractile phenotype in preparation for labor, we performed microarray analysis to compare the miRNA expression profile in RNA from isolated myometrium of three pools (six uteri each) from eighteen 15.5 d post-coitum (dpc) vs. three pools (six uteri each) from eighteen 18.5 dpc (with term labor being 19.0 dpc) pregnant mice. Treatment of the cells with P4 (10−7 M) for 12 or 24 h caused a pronounced induction of ZEB1 mRNA, whereas no effect of P4 on ZEB2 expression was evident (Fig. Among the family members, miR-200b and miR-429 showed the most dramatic increases in expression between 15.5 dpc and term (Fig. Genomic fragments containing ZEB1 and ZEB2 were each released from vectors (kindly provided by Yujiro Higashi, Osaka University, Osaka, Japan) and subcloned into pACCMVpLpA(−)loxP-SSP adenoviral vectors as described in SI Materials and Methods. There is near-complete homology of the miR-200 family between the mouse and human; the genes cluster identically and differ in their mature sequences by only two nucleotides in the 3′-region of miR-429 (Fig. Because both ZEB1 and ZEB2 are up-regulated in the uterus throughout most of pregnancy, this raises the question as to what factor(s) cause the pregnancy-associated induction of ZEB2. As mentioned, ZEBs act as transcriptional repressors by recruiting corepressors to the promoters of target genes (20). COVID-19 is an emerging, rapidly evolving situation. We also wanted to determine whether similar changes in miR-200b/429 and ZEB1 and ZEB2 expression were associated with preterm labor. β-Actin (Abcam) was detected as a loading control. National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error. Throughout most of pregnancy, uterine quiescence is maintained by increased progesterone receptor (PR) transcriptional activity, whereas spontaneous labor is initiated/facilitated by a concerted series of biochemical events that activate inflammatory pathways and have a negative impact on PR function. The action of oxytocin as an uterotonic agent is widely accepted. Would you like email updates of new search results? Excitingly, we observed that ZEB1 and ZEB2 inhibit expression of the contraction-associated genes, oxytocin receptor and connexin-43, and block oxytocin-induced contractility in human myometrial cells. Mice found to have vaginal plugs at 0600 hours the following day were considered to be 0.5 dpc. Conversely, parturition is associated with a decline in maternal circulating P4 and/or a decrease in the function of the PR, termed “functional P4 withdrawal,” (2, 3) and an increased inflammatory response within the uterus and cervix (4). Through overexpression studies of ZEB1 and ZEB2 in cultured human myometrial cells, we have shown that ZEB1 and ZEB2 down-regulate the expression of CXN-43 and OXTR and that ZEB overexpression inhibits oxytocin-mediated contraction of myometrial cells embedded in a collagen matrix. Thus, it is the balance between uterine stress-mediated activation of non-apoptotic CASP3 and augmented adaptive UPR signaling that facilitates quiescence. To this end, we conducted temporal expression analyses of CXN-43 and OXTR in the same myometrial samples used for analyses of miR-200b/429, ZEB1, and ZEB2. P4 injection had a modest effect to decrease myometrial expression of miR-200b/429 (Fig. 3G); this presumably occurs via ZEB1 suppression of the miR-200 family. GSE25017). CXN-43 and OXTR mRNA showed temporal expression profiles that were coordinately up-regulated with down-regulation of ZEB1 and ZEB2 in the pregnant mouse uterus during late gestation (Fig. (D) Contraction-associated genes CXN-43 and OXTR were significantly up-regulated in the same samples as in A and B, beginning at 18.5 dpc. A representative gel is shown below the data for each group. 1B). Cells starved of endogenous steroids (see MATERIALS AND METHODS) were grown to 100% confluence (t 0h)and treated with 10 … This negative feedback loop, which is supported by the present findings in the pregnant mouse and human myometrium and in cultured human myometrial cells as well as by published reports by others (17, 18), results in further induction of contraction-associated gene expression and labor. Parturition: activation of stimulatory pathways or loss of uterine quiescence? miR-200b/429 are significantly up-regulated in RU486-induced (C) and LPS-induced (D) preterm labor. HHS RNA was DNase-treated (Invitrogen) and reverse-transcribed using the SuperScript III-RT kit (Invitrogen) or TaqMan miRNA reverse transcription kit (Applied Biosystems). 4F), a decline in ZEB1 results in a reciprocal up-regulation of the miR-200 family. 3B). 4B). It is accepted that whilst hormones such as oxytocin, vasopressin and prostaglandin F2α induce myometrial contractions, essentially via an elevation of intracellular calcium, other ligands, such as β-adrenoceptor agonists, calcitonin gene-related peptide, and prostaglandin E2, promote uterine quiescence via their ability to increase intracellular cyclic AMP levels. Adequate uterine contractility is involved in the transport of the semen and gametes and in successful embryo implantation, whereas inadequate uterine contractility may lead to ectopic pregnancies, miscarriages, retrograde bleeding and endometriosis (Leyendecker et al., 2004; Bulletti and de Ziegler, 2005; Kissler et al., 2005). The levels of each mRNA/miRNA were normalized to h36B4/U6 and expressed as the fold change over control. (A) Transfection of hTERT-HM cells with miRmimics of miR-200b/429 causes a significant reduction in endogenous ZEB1 and ZEB2 within 24 h. (B) Primary murine myometrial cells were infected with recombinant adenoviruses expressing ZEB1, ZEB2, or β-gal (control); levels of miR-200b and miR-429 were analyzed by qRT-PCR 96 h posttransduction. Therefore, we hypothesize that normal transient stress insults may precondition the uterus and prevent contractility by increasing the capacity of the myometrium to experience ER stress in the absence of precocious increases in adaptive UPR … Paradoxically, during uterine quiescence CRH may act as a myometrial relaxant rather than as a promoter of parturition. Timed-pregnant ICR/CD1 female mice were injected s.c. daily with P4 (1 mg in 0.5 mL of sterile sesame oil) or with sesame oil (0.5 mL) in the flank region from 15.5–18.5 dpc. The miRNA microarray was performed (LC Sciences) on 18 biological replicates of murine myometrium at 15.5 dpc and on an equal number of replicates at 18.5 dpc. In addition, mice with a smooth muscle-specific deletion of the CXN-43 gene manifest a significant delay in the timing of parturition (28). ↵*This Direct Submission article had a prearranged editor. Notably, the contraction-associated genes CXN-43 and OXTR were found to be colocalized in the murine myometrium with ZEB1 and ZEB2 during late gestation (Fig. Myometrial contraction is regulated by a number of factors, such as female sexual hormones, the adrenergic receptor (AR) system, ion channels and transmitters. TREK-1 channel, a stretch-activated tetraethyl ammonium-insensitive potassium (K+) channel, has been described in smooth muscle of human myometrium. The miR-200 family was found to be up-regulated (Fig. Our findings suggest that elevated circulating P4 throughout most of pregnancy directly up-regulates expression of ZEB1 in the myometrium via binding of PR to the ZEB1 promoter. Diagrammed above each graph are the locations of E-boxes within the gene promoter. We also observed up-regulation of the miR-200 family and down-regulation of ZEB1 and ZEB2 in two different mouse models of preterm labor. Therefore, these relaxatory hormones, together with progesterone, control contractile pathways within the gravid uterus and collectively ensure myometrial quiescence during most of pregnancy. However, the exact cellular and molecular events are still in question. The cells were cultured with or without P4 (10−7 M) for 24 h and assayed for luciferase activity. ... the placental hormone equine chorionic gonadotropin released by endometrial cup acts like LH and FSH and stimulates additional ovulations, … Clipboard, Search History, and several other advanced features are temporarily unavailable. Collectively, our findings suggest that ZEB1 is a key PR target gene in the myometrium that inhibits expression of contraction-associated genes and the miR-200 family throughout most of pregnancy (Fig. Progesterone and Estrogen Contributions Both estrogen and progesterone are components of a broader-based molecular system that implements and maintains uterine quiescence. The decline in PR function near term causes a down-regulation of ZEB1 gene expression, which, in turn, results in derepression and up-regulation of miR-200b/429 expression. In mammalian pregnancy, uterine quiescence is maintained by elevated circulating progesterone (P4) acting via the progesterone receptor (PR). (C) ZEB1 but not ZEB2 mRNA is significantly increased by P4 treatment (same samples as in B).  |  Primary murine myometrial cells infected with recombinant adenoviruses expressing CMV/ZEB1 manifested induction of endogenous ZEB2 mRNA after 72 and 96 h. Expression of ZEB2 was determined by qRT-PCR, normalized to GAPDH, and expressed as the fold increase over ZEB2 expression in cells transduced with β-gal–expressing adenoviruses. To explore the reciprocal repression of the miR-200 family by ZEB1 and ZEB2, primary cultures of mouse myometrial cells were infected with recombinant adenoviruses overexpressing ZEB1, ZEB2, or β-gal (control). miR-200b/429 are up-regulated and ZEB1 and ZEB2 are down-regulated in two models of preterm labor. In this study, we uncovered a previously undescribed regulatory pathway whereby micro-RNAs (miRNAs) serve as hormonally modulated and conserved mediators of contraction-associated genes in the pregnant uterus in the mouse and human. The steroid hormones progesterone (P4) and estradiol-17β (E2), produced by the placenta in humans and the ovaries in rodents, serve crucial roles in the maintenance of pregnancy, and the initiation of parturition. The low progesterone concentration could be the possible cause of abortion in dexamethasone treated sheep in this study. 3D) were significantly increased in the myometrium of the P4-injected mice, whereas ZEB2 mRNA was unaffected (Fig. S3). For RU486-induced preterm labor, 15.5-dpc timed-pregnant ICR/CD1 female mice were injected s.c. in the right flank with RU486 [200 μg of RU486 in 1 mL of sterile 5% (vol/vol) ethyl alcohol (EtOH); Sigma] or 5% EtOH (1 mL). hCG is a glycoprotein αβ-dimer. 3G). The identification of miRNAs as hormonally regulated modulators of gene expression prompted us to investigate their roles in P4 and PR regulation of contraction-associated genes during pregnancy and labor. β-actin was used as a loading control. We do not capture any email address. To compare their temporal expression in the mouse myometrium during late gestation, we next examined miR-200b/429 and ZEB1 and ZEB2 by quantitative RT-PCR (qRT-PCR) from 15.5 dpc to labor. (C) hTERT-HM cells were infected with recombinant adenoviruses expressing ZEB1, ZEB2, or β-gal (control); CXN-43 and OXTR mRNA levels were analyzed by qRT-PCR 48 h posttransduction. The mice were killed on the birth of one pup, and time-matched controls were killed directly afterward. We postulated that P4 induction of ZEB1 causes suppression of the miR-200 family, which, in turn, relieves suppression of ZEB2, resulting in its subsequent induction. Experimental strategy for hormone additions to human uterine smooth muscle cells (hUtSMCs). We thank Dr. Y. Higashi (Osaka University, Osaka, Japan) for murine ZEB1 and ZEB2 expression plasmids, as well as Dr. A. Pertsemlidis for array analysis, Dr. W. Renthal and Dr. P. Kumar for expertise with ChIP, A. Click for help with immunohistochemistry, and Dr. F. Grinnell and Dr. C. Ho for instruction in collagen matrix assays (University of Texas Southwestern Medical Center). FSH Actions and Pregnancy: Looking Beyond Ovarian FSH Receptors. (G) Overexpression of ZEB1 causes induction of ZEB2 mRNA expression. Enter multiple addresses on separate lines or separate them with commas. Following transduction of myometrial cells with recombinant adenoviruses expressing either ZEB1 or ZEB2, CXN-43 and OXTR mRNA levels were found to be significantly suppressed at 48 h (Fig. designed research; N.E.R., C.-C.C., and J.P.-K. performed research; N.E.R., K.C.W., and R.D.G. Together, these data suggest that ZEB1 is a direct PR target gene. Using miRNA and gene expression microarray analyses of uterine tissues, we identified a conserved family of miRNAs, the miR-200 family, that is highly induced at term in both mice and humans as well as two coordinately down-regulated targets, zinc finger E-box binding homeobox proteins ZEB1 and ZEB2, which act as transcriptional repressors. 1G). Steroid hormone regulation of myometrial cell to cell communication ; progeterone causes … (Multiple comparison test compared with 15.5 dpc: *P < 0.05, **P < 0.01; n = 10 mice each for 15.5 dpc and laboring groups, 5 mice each for 16.5–18.5 dpc.) 3E). Expression of each miRNA/mRNA was determined by qRT-PCR, normalized to U6/GAPDH, and expressed as the fold change over vehicle-treated controls. The purpose of this study was to evaluate a possible effect of RLX on OTR … Oxytocin, a key hormone that enhances uterine contractility at term (24), significantly induced contraction of collagen gel matrices embedded with untransduced and β-gal–transduced hTERT-HM cells (Fig. The physiology of uterine quiescence and contractility is very complex. 1E) and ZEB1 and ZEB2 down-regulated in laboring human myometrium as compared with the myometrium of nonlaboring women at term (Fig. Although the evidence is still limited, a growing body of research suggests music may have beneficial effects for diseases such as Parkinson’s. Epub 2008 Nov 20. Mean ± SEM values are shown (Student’s t test, *P < 0.05, **P < 0.01; n = 7 per group). The appropriate timing of the onset of labor is critical to a successful pregnancy, with potentially devastating consequences resulting to both the mother and child with the onset of preterm labor. For example, at 20.5 dpc in the rat (which corresponds to 17.5 dpc in the mouse), a “synthetic to contractile switch” occurs during which myometrial proliferation declines, whereas myocyte attachment to the basement membrane and reorganization of myometrial cells increase (26). Mature miR-200b and miR-429 are significantly up-regulated (A) and ZEB1 and ZEB2 mRNA are significantly down-regulated (B) across late gestation in the mouse myometrium, beginning at 17.5 dpc. Experimental Physiology (2001) 86.2, 265-272. Study design: A multicenter, double-blind, placebo-controlled trial was designed for patients in preterm labor who responded to early intravenous treatment with atosiban. By contrast, ZEB1 mRNA (Fig. These findings suggest that alterations in myometrial expression of miR-200b/429, ZEB1, and ZEB2 may play a role in the induction of preterm labor. CXN-43 is a protein responsible for the formation of gap junctions in the myometrium, which mediate intercellular coupling to coordinate the synchronous myometrial contractions necessary for successful labor. In ChIP studies, we observed that endogenous ZEB1 binds to the promoters of the OXTR, CXN-43, and miR200b-a-429 cluster and that ZEB1 binding significantly declines between 15.5 and 18.5 dpc, in concert with the gestational up-regulation of expression of these genes. Cyclic AMP signalling pathways in the regulation of uterine relaxation. 2018 Dec 1;159(12):4033-4042. doi: 10.1210/en.2018-00497. (E) ZEB1 but not ZEB2 mRNA levels are induced in T74D cells treated with 10−7 M P4 for 12 or 24 h. Expression of each gene was determined by qRT-PCR, normalized to h36B4, and expressed as the fold increase over vehicle-treated control cells. wrote the paper. As shown in Fig. J Physiol. USA.gov. Experiments were repeated twice with similar results. 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