Prokineticin 1 Induces Inflammatory Response in Human Myometrium
2011; Elsevier BV; Volume: 179; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2011.08.029
ISSN1525-2191
AutoresMarta R. Gorowiec, Rob D. Catalano, Jane E. Norman, Fiona C. Denison, Henry N. Jabbour,
Tópico(s)Pregnancy-related medical research
ResumoThe infiltration of human myometrium and cervix with leukocytes and the formation of a pro-inflammatory environment within the uterus have been associated with the initiation of both term and preterm parturition. The mechanism regulating the onset of this pro-inflammatory cascade is not fully elucidated. We demonstrate that prokineticin 1 (PROK1) is up-regulated in human myometrium and placenta during labor. The expression of PROK1 receptor remains unchanged during labor and is abundantly expressed in the myometrium. Gene array analysis identified 65 genes up-regulated by PROK1 in human myometrium, mainly cytokines and chemokines, including IL-1β, chemokine C-C motif ligand 3, and colony-stimulating factor 3. In addition, we demonstrate that PROK1 increases the expression of chemokine C-C motif ligand 20, IL-6, IL-8, prostaglandin synthase 2, and prostaglandin E2 and F2α secretion. The treatment of myometrial explants with 100 ng/mL of lipopolysaccharide up-regulates the expression of PROK1, PROK1 receptor, and inflammatory mediators. The infection of myometrial explants with lentiviral microRNA targeting PROK1, preceding treatment with lipopolysaccharide, reduces the expression of inflammatory genes. We propose that PROK1 is a novel inflammatory mediator that can contribute to the onset of human parturition at term and partially mediate premature onset of inflammatory pathways during bacterial infection. The infiltration of human myometrium and cervix with leukocytes and the formation of a pro-inflammatory environment within the uterus have been associated with the initiation of both term and preterm parturition. The mechanism regulating the onset of this pro-inflammatory cascade is not fully elucidated. We demonstrate that prokineticin 1 (PROK1) is up-regulated in human myometrium and placenta during labor. The expression of PROK1 receptor remains unchanged during labor and is abundantly expressed in the myometrium. Gene array analysis identified 65 genes up-regulated by PROK1 in human myometrium, mainly cytokines and chemokines, including IL-1β, chemokine C-C motif ligand 3, and colony-stimulating factor 3. In addition, we demonstrate that PROK1 increases the expression of chemokine C-C motif ligand 20, IL-6, IL-8, prostaglandin synthase 2, and prostaglandin E2 and F2α secretion. The treatment of myometrial explants with 100 ng/mL of lipopolysaccharide up-regulates the expression of PROK1, PROK1 receptor, and inflammatory mediators. The infection of myometrial explants with lentiviral microRNA targeting PROK1, preceding treatment with lipopolysaccharide, reduces the expression of inflammatory genes. We propose that PROK1 is a novel inflammatory mediator that can contribute to the onset of human parturition at term and partially mediate premature onset of inflammatory pathways during bacterial infection. Human parturition is a pro-inflammatory event associated with the influx of leukocytes, such as neutrophils and macrophages, to the uterus.1Thomson A.J. Telfer J.F. Young A. Campbell S. Stewart C.J. Cameron I.T. Greer I.A. Norman J.E. Leukocytes infiltrate the myometrium during human parturition: further evidence that labor is an inflammatory process.Hum Reprod. 1999; 14: 229-236Crossref PubMed Scopus (436) Google Scholar, 2Osman I. Young A. Ledingham M.A. Thomson A.J. Jordan F. Greer I.A. Norman J.E. Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labor at term.Mol Hum Reprod. 2003; 9: 41-45Crossref PubMed Scopus (543) Google Scholar Indeed, 40% to 60% of genes up-regulated in the myometrium and cervix at labor are involved in inflammation and chemotaxis.3Bollapragada S. Youssef R. Jordan F. Greer I. Norman J. Nelson S. Term labor is associated with a core inflammatory response in human fetal membranes, myometrium, and cervix.Am J Obstet Gynecol. 2009; 200: 104e101-104e111Google Scholar Recruited leukocytes secrete cytokines, including IL-1β, IL-6, and IL-8,4Young A. Thomson A.J. Ledingham M. Jordan F. Greer I.A. Norman J.E. Immunolocalization of proinflammatory cytokines in myometrium, cervix, and fetal membranes during human parturition at term.Biol Reprod. 2002; 66: 445-449Crossref PubMed Scopus (276) Google Scholar and prostaglandins E2 (PGE2) and F2α (PGF2α). These cytokines and prostaglandins can stimulate production of matrix metalloproteinases that are involved in membrane rupture5Vega-Sanchez R. Gomez-Lopez N. Flores-Pliego A. Clemente-Galvan S. Estrada-Gutierrez G. Zentella-Dehesa A. Maida-Claros R. Beltran-Montoya J. Vadillo-Ortega F. Placental blood leukocytes are functional and phenotypically different than peripheral leukocytes during human labor.J Reprod Immunol. 2010; 84: 100-110Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar and can also promote cervical ripening.6Stygar D. Wang H. Vladic Y.S. Ekman G. Eriksson H. Sahlin L. Increased level of matrix metalloproteinases 2 and 9 in the ripening process of the human cervix.Biol Reprod. 2002; 67: 889-894Crossref PubMed Scopus (115) Google Scholar However, during labor, the greatest influx of leukocytes is observed in the myometrium.1Thomson A.J. Telfer J.F. Young A. Campbell S. Stewart C.J. Cameron I.T. Greer I.A. Norman J.E. Leukocytes infiltrate the myometrium during human parturition: further evidence that labor is an inflammatory process.Hum Reprod. 1999; 14: 229-236Crossref PubMed Scopus (436) Google Scholar Cytokines, such as IL-1β, released by leukocytes, promote the synthesis of prostaglandin synthase 2 (PTGS2)–dependent prostaglandins (PGE2 and PGF2α), which are important regulators of myometrial contractility.7Rauk P.N. Chiao J.P. Interleukin-1 stimulates human uterine prostaglandin production through induction of cyclooxygenase-2 expression.Am J Reprod Immunol. 2000; 43: 152-159Crossref PubMed Scopus (108) Google Scholar, 8Zaragoza D.B. Wilson R.R. Mitchell B.F. Olson D.M. The interleukin 1beta-induced expression of human prostaglandin F2alpha receptor messenger RNA in human myometrial-derived ULTR cells requires the transcription factor, NFkappaB.Biol Reprod. 2006; 75: 697-704Crossref PubMed Scopus (37) Google Scholar More important, independently of prostaglandins, IL-1β also regulates basal and store-operated calcium entry in myometrial smooth muscle cells, which is important for contractions of individual smooth muscle cells.9Tribe R.M. Moriarty P. Dalrymple A. Hassoni A.A. Poston L. Interleukin-1beta induces calcium transients and enhances basal and store operated calcium entry in human myometrial smooth muscle.Biol Reprod. 2003; 68: 1842-1849Crossref PubMed Scopus (81) Google ScholarThe same pro-inflammatory events can be initiated before 37 weeks of gestation and lead to premature onset of parturition and preterm birth. Nearly 11% of all singleton deliveries are preterm, and this constitutes the biggest cause of neonatal morbidity and mortality.10Norman J.E. Morris C. Chalmers J. The effect of changing patterns of obstetric care in Scotland (1980–2004) on rates of preterm birth and its neonatal consequences: perinatal database study.PLoS Med. 2009; 6: e1000153Crossref PubMed Scopus (85) Google Scholar, 11Heron M. Hoyert D.L. Murphy S.L. Xu J. Kochanek K.D. Tejada-Vera B. Deaths: final data for 2006.Natl Vital Stat Rep. 2009; 57: 1-134PubMed Google Scholar Although several factors, such as smoking, alcohol, advanced maternal age, and structural abnormalities of the cervix,12Goldenberg R.L. Culhane J.F. Iams J.D. Romero R. Epidemiology and causes of preterm birth.Lancet. 2008; 371: 75-84Abstract Full Text Full Text PDF PubMed Scopus (4722) Google Scholar, 13Norman J.E. Preterm labor: cervical function and prematurity.Best Pract Res Clin Obstet Gynaecol. 2007; 21: 791-806Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar can contribute to preterm initiation of labor, bacterial infection in the uteroplacental unit is believed to be a major cause. Such infections are detected in 79% of births at 23 weeks and in 11% of births at 31 to 34 weeks.14Onderdonk A.B. Hecht J.L. McElrath T.F. Delaney M.L. Allred E.N. Leviton A. Colonization of second-trimester placenta parenchyma.Am J Obstet Gynecol. 2008; , 15Muglia L.J. Katz M. The enigma of spontaneous preterm birth.N Engl J Med. 2010; 362: 529-535Crossref PubMed Scopus (498) Google Scholar In most cases of preterm birth, even in the absence of infection, histological evidence of inflammation is apparent in the uteroplacental unit.16Goldenberg R.L. Hauth J.C. Andrews W.W. Intrauterine infection and preterm delivery.N Engl J Med. 2000; 342: 1500-1507Crossref PubMed Scopus (1955) Google Scholar, 17Smith R. Parturition.N Engl J Med. 2007; 356: 271-283Crossref PubMed Scopus (378) Google Scholar Intrauterine inflammation is associated with adverse perinatal outcome.18Lee S.E. Romero R. Jung H. Park C.W. Park J.S. Yoon B.H. The intensity of the fetal inflammatory response in intraamniotic inflammation with and without microbial invasion of the amniotic cavity.Am J Obstet Gynecol. 2007; 197: 294.e1-294.e6Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar However, therapies for preterm birth have predominantly focused on inhibition of the onset of labor. Therefore, there is increasing recognition that effective therapies should target myometrial contractility and inhibit/reduce intrauterine inflammation.Prokineticins 1 and 2 (PROK1 and PROK2, respectively) are multifunctional secreted proteins that signal via two G protein–coupled receptors termed PROK receptors 1 and 2 (PROKR1 and PROKR2, respectively). PROKs induce smooth muscle contractility of the gut,19Schweitz H. Pacaud P. Diochot S. Moinier D. Lazdunski M. MIT(1), a black mamba toxin with a new and highly potent activity on intestinal contraction.FEBS Lett. 1999; 461: 183-188Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 20Li M. Bullock C.M. Knauer D.J. Ehlert F.J. Zhou Q.Y. Identification of two prokineticin cDNAs: recombinant proteins potently contract gastrointestinal smooth muscle.Mol Pharmacol. 2001; 59: 692-698Crossref PubMed Scopus (238) Google Scholar which is mediated via increased calcium influx into smooth muscle cells.19Schweitz H. Pacaud P. Diochot S. Moinier D. Lazdunski M. MIT(1), a black mamba toxin with a new and highly potent activity on intestinal contraction.FEBS Lett. 1999; 461: 183-188Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 20Li M. Bullock C.M. Knauer D.J. Ehlert F.J. Zhou Q.Y. Identification of two prokineticin cDNAs: recombinant proteins potently contract gastrointestinal smooth muscle.Mol Pharmacol. 2001; 59: 692-698Crossref PubMed Scopus (238) Google Scholar PROKs also have potent effects in multiple other processes, including neurogenesis, angiogenesis, hematopoiesis, and nociception.21Zhou Q.Y. Meidan R. Biological function of prokineticins.Results Probl Cell Differ. 2008; 46: 181-199Crossref PubMed Scopus (12) Google Scholar There is increasing evidence that PROK1, via PROKR1, can modulate immune responses. PROK1 can promote differentiation of bone marrow progenitors into macrophage-like adherent cells.22Dorsch M. Qiu Y. Soler D. Frank N. Duong T. Goodearl A. O'Neil S. Lora J. Fraser C.C. PK1/EG-VEGF induces monocyte differentiation and activation.J Leukoc Biol. 2005; 78: 426-434Crossref PubMed Scopus (79) Google Scholar In addition, PROK1 induces the expression of chemokines, including chemokine C-C motif ligand 4 (CCL4), CXCL1, and IL-8 (CXCL8) in human monocytes, which constitutes an important regulatory mechanism for leukocyte recruitment to the site of inflammation.23Monnier J. Quillien V. Piquet-Pellorce C. Leberre C. Preisser L. Gascan H. Samson M. Prokineticin 1 induces CCL4, CXCL1 and CXCL8 in human monocytes but not in macrophages and dendritic cells.Eur Cytokine Netw. 2008; 19: 166-175PubMed Google ScholarPROKs may be important regulators of female reproductive functions, including regulation of uterine receptivity24Evans J. Catalano R.D. Morgan K. Critchley H.O. Millar R.P. Jabbour H.N. Prokineticin 1 signaling and gene regulation in early human pregnancy.Endocrinology. 2008; 149: 2877-2887Crossref PubMed Scopus (84) Google Scholar and placental function.25Denison F.C. Battersby S. King A.E. Szuber M. Jabbour H.N. Prokineticin-1: a novel mediator of the inflammatory response in third-trimester human placenta.Endocrinology. 2008; 149: 3470-3477Crossref PubMed Scopus (34) Google Scholar, 26Hoffmann P. Feige J.J. Alfaidy N. Expression and oxygen regulation of endocrine gland-derived vascular endothelial growth factor/prokineticin-1 and its receptors in human placenta during early pregnancy.Endocrinology. 2006; 147: 1675-1684Crossref PubMed Scopus (90) Google Scholar, 27Hoffmann P. Saoudi Y. Benharouga M. Graham C.H. Schaal J.P. Mazouni C. Feige J.J. Alfaidy N. Role of EG-VEGF in human placentation: physiological and pathological implications.J Cell Mol Med. 2009; 13: 2224-2235Crossref PubMed Scopus (79) Google Scholar Considering their well-described roles in mediating inflammatory pathways and smooth muscle contractility, we hypothesized that PROK1, via PROKR1, may be important in regulating events leading to the onset of parturition. This study was designed to determine the expression and potential role of PROK1 and PROKR1 in the human myometrium during labor.Materials and MethodsPatients and Tissue CollectionFull-thickness lower-segment biopsy specimens of human myometrium, placenta, and fetal membranes were collected from women undergoing elective caesarean section at term (>37 weeks) before the onset of labor (NL) and women in spontaneous labor at term (>37 weeks) who required emergency lower-segment caesarean section (L). Women with medical complications, such as diabetes or hypertension, or with symptoms of infection (determined by temperature >38°C) were excluded from the study. Patients were also excluded if the pregnancy was multiple or if they received either prostaglandin for induction or oxytocin for augmentation of labor. Written consent was obtained from each patient before recruitment, in accordance with approval from the Lothian Local Research Ethics Committee (REC reference no. 07/S1103/25). Immediately after collection, tissue was placed in RNAlater (Applied Biosystems, Warrington, UK) for RNA extraction, fixed in 4% neutral-buffered formalin, and wax embedded for immunohistochemistry (IHC).Tissue Culture and TreatmentsMyometrial tissue explants from women at term and not in labor were collected for in vitro studies. Tissue was routinely maintained in RPMI 1640 medium (GIBCO, Invitrogen, Paisley, UK) supplemented with 100 IU penicillin and 100 μg/mL streptomycin (Sigma, Poole, UK) at 37°C and 5% CO2, as recommended. After collection, explants were finely chopped into equal portions with scissors and incubated overnight. Tissue was treated with vehicle (sterile double-distilled water), 100 ng/mL of lipopolysaccharide (LPS) from Salmonella abortus equi (Alexis Biochemicals, Axxora, Nottingham, UK), or 40 nmol/L PROK1 (PeproTech, London, UK) for 2, 4, 6, 8, and 24 hours. In previous studies, the PROK1 and LPS doses used in this study have given optimal responses.28Evans J. Catalano R.D. Brown P. Sherwin R. Critchley H.O. Fazleabas A.T. Jabbour H.N. Prokineticin 1 mediates fetal-maternal dialogue regulating endometrial leukemia inhibitory factor.FASEB J. 2009; 23: 2165-2175Crossref PubMed Scopus (94) Google Scholar, 29Maldonado-Pérez D. Golightly E. Denison F.C. Jabbour H.N. Norman J.E. A role for lipoxin A4 as anti-inflammatory and proresolution mediator in human parturition.FASEB J. 2011; 25: 569-575Crossref PubMed Scopus (38) Google Scholar Tissue and supernatants were collected at all time points. All experiments were performed in duplicate.Gene Array and Data AnalysisTotal RNA was extracted from myometrial explants treated with either vehicle or 40 nmol/L PROK1 for 6 and 24 hours (n = 6 for each time point). Sample preparation, array hybridization, and data analysis were performed by the Finnish Microarray and Sequencing Centre and the Turku Centre for Biotechnology (Turku, Finland). Briefly, total RNA was biotin labeled by a single in vitro transcription amplification step. Samples were hybridized to Illumina Human HT-12 v.3 Expression BeadChips (Illumina, San Diego, CA) and stained with streptavadin-Cy3. Signal intensities within data sets were normalized by applying quantile normalization. R package Limma30Gentleman R. Bioinformatics and Computational Biology Solutions Using R and Bioconductor. Springer, New York, Cambridge2005Crossref Google Scholar (Bioconductor, Open Source Software for Bioinformatics) was used for performing the statistical testing between the groups. Functional grouping of the gene list into statistically significant overrepresented ontologies was performed using the protein analysis through evolutionary relationships database analysis tools (PANTHER).31Thomas P.D. Kejariwal A. Guo N. Mi H. Campbell M.J. Muruganujan A. Lazareva-Ulitsky B. Applications for protein sequence-function evolution data: mRNA/protein expression analysis and coding SNP scoring tools.Nucleic Acids Res. 2006; 34: W645-W650Crossref PubMed Scopus (386) Google ScholarTaqMan Quantitative Real-Time PCRTissue was homogenized in QIAzol (Qiagen, Crawley, UK), followed by RNA extraction with on-column DNase digestion using the RNeasy Mini Kit, according to the manufacturer's instructions (Qiagen). RNA was reverse transcribed using the VILO SuperScript VILO cDNA Synthesis Kit (Invitrogen). TaqMan quantitative real-time PCR was performed using an ABI Prism 7500 System (Applied Biosystems). The sequences of all primers and FAM-labeled probes are listed in Table 1. The expression of target genes was normalized to RNA loading, measured with primers and VIC-labeled probe for ribosomal 18S RNA. Relative gene expression was calculated by using the comparative CT method. Results of in vitro experiments are presented relative to expression in vehicle-treated samples. A comparison of gene expression in tissues collected from women in labor versus not in labor is presented as relative to a calibrator endometrial sample included in all reactions.28Evans J. Catalano R.D. Brown P. Sherwin R. Critchley H.O. Fazleabas A.T. Jabbour H.N. Prokineticin 1 mediates fetal-maternal dialogue regulating endometrial leukemia inhibitory factor.FASEB J. 2009; 23: 2165-2175Crossref PubMed Scopus (94) Google ScholarTable 1Sequences of Primers and Probes Used for Analysis of Gene Expression by TaqMan Quantitative Real-Time PCRGeneSequence or category numberPROK1 Forward5′-GTGCCACCCGGGCAG-3′ Reverse5′-AGCAAGGACAGGTGTGGTGC-3′ FAM5′-ACAAGGTCCCCTTGTTCAGGAAACGCA-3′CCL20 Forward5′-TCCTGGCTGCTTTGATGTCA-3′ Reverse5′-CCAAGACAGCAGTCAAAGTTGCT-3′ FAM5′-TGCTGCTACTCCACCTCTGCGGC-3′PROKR1 Forward5′-TCTTACAATGGCGGTAAGTCCA-3′ Reverse5′-CTCTTCGGTGGCAGGCAT-3′ FAM5′-TGCAGACCTGGACCTCAAGACAATTGG-3′IL-1β Forward5′-CGCATCCAGCTACGAAT-3′ Reverse5′-CATGGCACAACAACTGA-3′ FAM5′-CGACCACCACTACAGC-3′IL-6 Forward5′-GCCGCCCCACACAGACA-3′ Reverse5′-CCGTCGAGGATGTACGGAAT-3′ FAM5′-CCACTCACCTCTTCAGAACGAATTGACAAAC-3′IL-8 Forward5′-CTGGCCGTGGCTCTCTT-3′ Reverse5′-TTAGCACTCCTTGGCAAAACTG-3′ FAM5′-CCTTCCTGATTTCTGCAGCTCTGTGTGAA-3′COX-2 Forward5′-CCTTCCTCCTGTGCCTGATG-3′ Reverse5′-ACAATCTCATTTGAATCAGGAAGCT-3′ FAM5′-TGCCCGACTCCCTTGGGTGTCA-3′18S Forward5′-CGGCTACCACATCCAAGGAA-3′ Reverse5′-GCTGGAATTACCGCGGCT-3′ VIC5′-TGCTGGCACCAGACTTGCCCTC-3′CCL3Hs00234142-m1CSF3Hs99999083-m1VCAM1Hs01003372-m1TACC2Hs00610617-m1CCL2Hs00234140-m1CCL8Hs00271615-m1 Open table in a new tab IHC DataParaffin-embedded sections were dewaxed in xylene and dehydrated in graded ethanol. Antigen retrieval was performed by boiling for 5 minutes in 0.01 mol/L citrate buffer (pH 6.0), followed by quenching the activity of endogenous peroxidase with 30% H2O2 in methanol. Sections were blocked in a solution of 20% normal goat serum and 5% bovine serum albumin at room temperature, followed by incubation with rabbit anti-human PROK1 at 1:500 dilution (Phoenix Pharmaceuticals, Belmont, CA) or PROKR1 at 1:250 dilution (Life Span Biosciences, Atlanta, GA) overnight at 4°C; negative controls were incubated with rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Subsequently, sections were incubated with biotinylated goat anti-rabbit antibody (Vector Laboratories, Peterborough, UK) (1:500), followed by incubation with streptavidin–horseradish peroxidase (GE Healthcare UK, Little Chalfont) (1:1000). Staining was detected with 3,3′-diaminobenzidine (Vector Laboratories, Peterborough, UK) as the chromogen.Lentivirus miRNA Gene SilencingLentivirus microRNA (miRNA) targeting PROK1 mRNA was used to knock down PROK1 expression, as previously described.28Evans J. Catalano R.D. Brown P. Sherwin R. Critchley H.O. Fazleabas A.T. Jabbour H.N. Prokineticin 1 mediates fetal-maternal dialogue regulating endometrial leukemia inhibitory factor.FASEB J. 2009; 23: 2165-2175Crossref PubMed Scopus (94) Google Scholar, 32Cook I.H. Evans J. Maldonado-Pérez D. Critchley H.O. Sales K.J. Jabbour H.N. Prokineticin-1 (PROK1) modulates interleukin (IL)-11 expression via prokineticin receptor 1 (PROKR1) and the calcineurin/NFAT signalling pathway.Mol Hum Reprod. 2010; 16: 158-169Crossref PubMed Scopus (26) Google Scholar Briefly, myometrial explants (n = 8) were finely chopped and subsequently infected with lentivirus containing scrambled sequence (plenti6/V5-EmGFP-miR-neg) as a negative control or PROK1 miRNA (pLenti6/V5-EmGFP-hum-PROK1-72-287) chained constructs for 72 hours. Subsequently, explants were incubated in the presence or absence of 100 ng/mL of LPS for 24 hours. Tissue and supernatants were then harvested, and RNA was extracted from tissues for PCR analysis. Results are presented relative to expression in vehicle-treated samples.Measurement of Prostaglandins PGE2 and PGF2αMyometrium explants (n = 5) were incubated in the presence or absence of 40 nmol/L PROK1 for 24 hours. Medium was collected and assayed by enzyme-linked immunosorbent assay for PGE2 and PGF2α, as previously described.33Denison F.C. Grant V.E. Calder A.A. Kelly R.W. Seminal plasma components stimulate interleukin-8 and interleukin-10 release.Mol Hum Reprod. 1999; 5: 220-226Crossref PubMed Scopus (78) Google Scholar Results are presented as pg/mg tissue.Measurement of IL-1β, CCL3, and CCL20Myometrial explants were infected with lentiviral constructs containing scrambled or PROK1 miRNA and subsequently treated with 100 ng/mL of LPS for 24 hours, as previously described. Conditioned media were collected and assayed for IL-1β, CCL3, and CCL20 by using appropriate DuoSet ELISA Development Systems, according to the manufacturer's protocols (R&D Systems, Abingdon, UK). Briefly, 96-well microplates were coated overnight with 100 μL of working concentration of appropriate Capture Antibody diluted in PBS (1:180). The following day, the plates were blocked with 300 μL of 1% bovine serum albumin in PBS for 1 hour at room temperature. Subsequently, 100 μL of samples and appropriate standards were added for 2 hours, followed by incubation with Detection Antibody diluted (1:180) for 2 hours and then incubation with streptavidin–horseradish peroxidase (1:200) for 20 minutes. After the addition of TMB Substrate Solution, optical density was determined at 450 nm using a microplate reader. Results are presented relative to expression in vehicle-treated samples.Statistical AnalysisData were analyzed using an unpaired t-test with Welch's correction or a paired t-test (GraphPad Prism 5.0; GraphPad Software, La Jolla, CA). The n values represent the number of independent experiments. Unless otherwise indicated, data are given as mean ± SEM.ResultsExpression of PROK1 Increases in Uteroplacental Tissues at LaborPROK1 mRNA expression is significantly elevated during labor (L) versus not labor (NL) in the myometrium (16.78 ± 7.0-fold versus 1.15 ± 0.37-fold; P < 0.05) and placenta (1.97 ± 0.56-fold versus 0.9 ± 0.17-fold; P < 0.05) but not in fetal membranes (Figure 1A). PROKR1 expression does not change at term with the onset of labor in any of the uteroplacental tissues. However, expression is highest in the myometrium compared with other uteroplacental tissues (P < 0.0001), suggesting that myometrium may be the main target tissue for PROK1 signaling during labor (Figure 1B).We subsequently focused on the role of PROK1 and PROKR1 in human myometrium. During IHC, PROK1 was localized in the smooth muscle bundles in the myometrium of women at term before the onset of labor (Figure 2A) and during labor (Figure 2B). Similarly, PROKR1 was immunolocalized to the smooth muscle bundles in myometrial tissue collected from women at term before the onset of labor (Figure 2C) and during labor (Figure 2D). During labor, PROKR1 expression is additionally localized to the vasculature (Figure 2D).Figure 2Immunolocalization of PROK1 and PROKR1 in myometrium collected from women at term before the onset of labor or during labor. PROK1 is localized to the smooth muscle bundles (A, not in labor; B, in labor). PROKR1 was also immunolocalized to the smooth muscle bundles in myometrial tissue collected from women not in labor (C) and in labor (D). During labor, PROKR1 expression is also localized to vasculature. Control sections were negative for immunoreactivity (insets). E, endothelium; SMB, smooth muscle bundles (n = 16: n = 8 for myometrium collected from women not in labor, and n = 8 for myometrium collected from women in labor).View Large Image Figure ViewerDownload Hi-res image Download (PPT)PROK1/PROKR1 Regulates Inflammatory Mediators in Human MyometriumAfter having established that PROK1 expression is elevated in the myometrium during labor and that PROKR1 transcript levels are highest in myometrial tissue, we sought to identify the downstream targets of PROK1/PROKR1 signaling in the myometrium. Gene array analysis was used to identify PROK1-regulated transcripts in myometrial explants at term (in six independent samples), treated with 40 nmol/L PROK1 for 6 or 24 hours and compared with vehicle treatment for each time point. The array analysis identified both up- and down-regulated transcripts at both time points (see Supplemental Figure S1, A and B, at http://ajp.amjpathol.org; http://www.ncbi.nlm.nih.gov/geo, Accession no. GSE28272), with an increase in the number of regulated transcripts from 6 to 24 hours (see Supplemental Figure S1C at http://ajp.amjpathol.org). The analysis shown there was a distinct response at 6 hours after PROK1 treatment compared with the response at 24 hours, although approximately 30% of the genes at 6 hours were common to 24-hour treatment, indicating a sustained regulation of a subset of genes. Genes were selected to verify the array analysis by real-time PCR. These genes were selected to represent high- and low-intensity signals and genes that were up- or down-regulated at either time point (Figure 3A). PCR verification confirmed the increased expression of CCL3 and CCL8 at 6 hours (Figure 3C) and IL-1β, CCL3, colony-stimulating factor 3 (CSF3), and vascular cell adhesion molecule 1 (VCAM1) at the 24-hour time point (Figure 3B). However, PCR did not validate regulation of transforming acidic coiled-coil–containing protein 2 and CCL2. The PCR validation permitted cutoff values to be established to generate a reliable list of 65 PROK1 regulated genes (Table 2). The gene list indicated that the response to PROK1 at 6 hours was mainly constituted of chemokine genes (7 of the 11 genes). Also, chemokines (ie, CCL3, CCL3L1, and CCL3L3) represented the genes that were regulated at both 6 and 24 hours after PROK1 treatment. The response to PROK1 treatment after 24 hours consisted mainly of chemokines and cytokines (ie, 22 of the 57 genes). A functional grouping of the gene list into statistically significant overrepresented ontologies revealed the immune response and related pathways to predominate, including macrophage activation and response to interferon-γ (Table 3).Figure 3Validation of gene array analysis by quantitative real-time PCR. A: List of genes selected to verify the array analysis, including genes representing high- and low-intensity signals and genes that were up- or down-regulated at either the 6- or 24-hour time point. Adj, adjusted; FC, fold change. B: Treatment of myometrial explants with PROK1 for 24 hours up-regulates expression of IL-1β, CCL3, CSF3, and VCAM1, whereas levels of transforming acidic coiled-coil–containing protein 2 remained unaffected. C: Treatment of myometrial explants with 40 nmol/L PROK1 for 6 hours elevated expression of CCL3 and CCL8, whereas there was no difference in the expression of CCL2. *P < 0.05, **P < 0.005, and ***P < 0.0001. n/s, not significant (n = 6). Results are presented as mean ± SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 2Differential Gene Expression of 65 Genes after PROK1 Treatment of Term Myometrial ExplantsGeneMean fold changeAdjusted P valueTime (hours)ADAMDEC11.60.074124AMPD31.60.086124BDKRB11.90.050624C15orf483.20.032724C8orf41.60.050624CCL203.90.070924CCL35.50.00016CCL32.60.029624CCL3L13.60.00176CCL3L12.50.032724CCL3L34.90.00116CCL3L33.20.024924CCL4L12.60.01186CCL4L26.90.00006CCL83.60.00716CCR72.00.096924CD821.80.042024CSF34.50.024924CX3CL13.00.01006CXCL12.60.024924CXCL23.10.047424CXCL55.00.000024CXCL65.30.006224CYP27B13.00.070924ELL21.60.008524FBP11.80.070924FCGR2A1.60.090024G0S21.90.084124HCK1.80.079
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