Epithelial Membrane Protein Research Articles

FAK activation and the role of epithelial membrane protein 2 (EMP2) in collagen gel contraction.

Proliferative vitreoretinopathy (PVR) occurs in approximately 10% of patients after retinal detachment. PVR results from a multiphase process that leads to an aberrant wound-healing strategy with contractile cellular forces and tractional retinal detachment (TRD). Epithelial membrane protein (EMP) 2 controls cell surface expression and function of integrin isoforms associated with cellular contraction in many cell types. Since EMP2 is highly expressed in retinal pigment epithelium, this study investigates the role of EMP2 in collagen gel contraction. EMP2 expression was recombinantly modified in the ARPE-19 cell line. Cell surface integrin expression was assessed by flow cytometry. Collagen gel contraction was assessed by using an in vitro assay and the percentage of contraction was quantified. Proliferation and migration were measured by BrdU incorporation and a wound-healing assay, respectively. Cellular invasion was investigated with polycarbonate membranes coated with collagen. EMP2 expression levels correlated positively with the ability to contract collagen gels. Compared with wild-type ARPE-19 cells, the cells with increased EMP2 expression exhibited enhanced contraction (P = 0.02), and decreased EMP2 expression concomitantly resulted in decreased contraction (P = 0.002). EMP2 overexpression resulted in reduced proliferation, migration, and integrin alpha1 and alpha2 integrin expression. EMP2 overexpression was associated with a 70% increase in FAK activation (P = 0.0003) and relative resistance of gel contraction to inhibitors of FAK/Src activation. ARPE-19-mediated collagen gel contraction is a multistep process that requires integrin ligation and activation of the FAK/Src complex. EMP2 positively modulates collagen gel contraction by ARPE-19 cells through increased FAK activation.

Steroid hormone regulation of EMP2 expression and localization in the endometrium

BackgroundThe tetraspan protein epithelial membrane protein-2 (EMP2), which mediates surface display of diverse proteins, is required for endometrial competence in blastocyst implantation, and is uniquely correlated with poor survival from endometrial adenocarcinoma tumors. Because EMP2 is differentially expressed in the various stages of the murine and human estrous cycle, we tested the hypothesis that the steroid hormones progesterone and estrogen influence EMP2 expression and localization.MethodsFrozen human proliferative and secretory endometrium were collected and analyzed for EMP2 expression using SDS-PAGE/Western blot analysis. The response of EMP2 to progesterone and estradiol was determined using a combination of real-time PCR, SDS-PAGE/Western blot analysis, and confocal immunofluorescence in the human endometrial carcinoma cell line RL95-2. To confirm the in vitro results, ovariectomized mice were treated with progesterone or estradiol, and EMP2 expression was analyzed using immunohistochemistry.ResultsWithin normal human endometrium, EMP2 expression is upregulated in the secretory phase relative to the proliferative phase. To understand the role of steroid hormones on EMP2 expression, we utilized RL95-2 cells, which express both estrogen and progesterone receptors. In RL95-2 cells, both estradiol and progesterone induced EMP2 mRNA expression, but only progesterone induced EMP2 protein expression. To compare steroid hormone regulation of EMP2 between humans and mice, we analyzed EMP2 expression in ovarectomized mice. Similar to results observed in humans, progesterone upregulated endometrial EMP2 expression and induced EMP2 translocation to the plasma membrane. Estradiol did not promote translocation to the cell surface, but moderately induced EMP2 expression in cytoplasmic compartments in vivo.ConclusionThese findings suggest that targeting of EMP2 to specific locations under the influence of these steroid hormones may be important for integrating the molecular responses required for implantation competence.

The Epithelial Membrane Protein 1 is a Novel Tight Junction Protein of the Blood—Brain Barrier

In the central nervous system, a constant microenvironment required for neuronal cell activity is maintained by the blood-brain barrier (BBB). The BBB is formed by the brain microvascular endothelial cells (BMEC), which are sealed by tight junctions (TJ). To identify genes that are differentially expressed in BMEC compared with peripheral endothelial cells, we constructed a subtractive cDNA library from porcine BMEC (pBMEC) and aortic endothelial cells (AOEC). Screening the library for differentially expressed genes yielded 26 BMEC-specific transcripts, such as solute carrier family 35 member F2 (SLC35F2), ADP-ribosylation factor-like 5B (ARL5B), TSC22 domain family member 1 (TSC22D1), integral membrane protein 2A (ITM2A), and epithelial membrane protein 1 (EMP1). In this study, we show that EMP1 transcript is enriched in pBMEC compared with brain tissue and that EMP1 protein colocalizes with the TJ protein occludin in mouse BMEC by coimmunoprecipitation and in rat brain vessels by immunohistochemistry. Epithelial membrane protein 1 expression was transiently induced in laser-capture microdissected rat brain vessels after a 20-min global cerebral ischemia, in parallel with the loss of occludin immunoreactivity. The study identifies EMP1 as a novel TJ-associated protein of the BBB and suggests its potential role in the regulation of the BBB function in cerebral ischemia.

Differential gene expression with co‐engagement of FcεRI and CC chemokine receptor 1

The classical pathway for mast cell degranulation is mediated through the high affinity IgE receptor FcεRI but the optimal mast cell degranulation in the conjunctiva also requires CC chemokine ligand 3 (CCL3). To examine the transcriptional changes underlying the rat basophilic leukemia mast cell line's response to costimulation of FceRI and CCL3 receptor CC chemokine receptor 1 (CCR1), we screened more than 31,000 probe sets using Affymetrix Rat Expression Array 230 2.0. Genes whose expression changed only with activation of both receptors were designated as synergy‐related genes. Among the 212 synergy‐related genes, 93 genes are functionally annotated, fourteen genes are involved in signaling pathways, 112 genes are up‐regulated while 100 genes are down‐regulated. The expression changes of the most up‐regulated synergy‐related genes including Epithelial Membrane Protein 1 (Emp1), Regulator of G protein Signaling 1 (Rgs1), and RT1 class Ib, locus S3 (RT1‐S3) were validated at both mRNA and protein levels. Their function was also analyzed by siRNA inhibition followed by degranulation and chemotaxis assays. This project is funded by MCCID EU 504926 EST program, European Commission, Brussels, Belgium.

Selective inhibition of binding of Bacillus thuringiensis Cry1Ab toxin to cadherin-like and aminopeptidase proteins in brush-border membranes and dissociated epithelial cells from Bombyx mori

Binding analyses with denatured epithelial membrane proteins from Bt (Bacillus thuringiensis) demonstrated at least two kinds of proteins, APNs (aminopeptidases N) and cadherin-like proteins, as possible receptors for the Cry1A class of Bt toxins. Two alternative models have been proposed, both based on initial toxin binding to a cadherin-like protein, but one involving APN and the other not. We have used two Bombyx mori strains (J65 and Kin), which are highly susceptible to Cry1Ab, to study the role of these two types of receptors on Cry1Ab toxin binding and cytotoxicity by means of the inhibitory effect of antibodies. BBMVs (brush-border membrane vesicles) of strain J65 incubated with labelled 125I-Cry1Ab revealed a marked reduction in reversible and irreversible binding when anti-BtR175 (a cadherin-like protein) was used for BBMV pre-treatment. By contrast, the anti-APN1 antibody specifically affected the irreversible binding, while the reversible binding component was not affected. This is the first time that binding of Cry1Ab to APN1 and to a cadherin-like protein from BBMVs in solution has been shown. Dissociated epithelial cells from the Kin strain were used to test the inhibitory effect of the antibodies on the cytotoxicity of Cry1Ab. Pre-incubation of the cells with the anti-BtR175 antibody conferred protection against Cry1Ab, but not the anti-APN1 antibody. Therefore our results seem to support the two models of the mode of action of Cry1Ab in Lepidoptera, depending on whether BBMVs or intact dissociated cells are used, suggesting that both pathways may co-operate for the toxicity of Cry1A toxins in vivo.

The SNMP/CD36 gene family in Diptera, Hymenoptera and Coleoptera: Drosophila melanogaster, D. pseudoobscura, Anopheles gambiae, Aedes aegypti, Apis mellifera, and Tribolium castaneum

Sensory neuron membrane proteins (SNMPs) are membrane bound proteins initially identified in olfactory receptor neurons of Lepidoptera and are thought to play a role in odor detection; SNMPs belong to a larger gene family characterized by the human protein CD36. We have identified 12–14 candidate SNMP/CD36 homologs from each of the genomes of Drosophila melanogaster, D. pseudoobscura, Anopheles gambiae and Aedes aegypti (Diptera), eight candidate homologs from Apis mellifera (Hymenoptera), and 15 from Tribolium castaneum (Coleoptera). Analysis (sequence similarity and intron locations) suggests that the insect SNMP/CD36 genes fall into three major groups. Group 1 includes the previously characterized D. melanogaster emp (epithelial membrane protein). Group 2 includes the previously characterized D. melanogaster croquemort, ninaD, santa maria, and peste. Group 3 genes include the SNMPs, which fall into two subgroups referred to as SNMP1 and SNMP2. D. melanogaster SNMP1 (CG7000) shares both significant sequence similarity and five of its six intron insertion sites with the lepidopteran Bombyx mori SNMP1. The topological conservation of this gene family within the three major holometabolous lineages indicates that it predates the coleopteran and hymenoptera/dipera/lepidoptera split 300+ million years ago. The current state of knowledge of the characterized insect members of this gene family is discussed.

The Tetraspan Protein EMP2 Regulates Expression of Caveolin-1

Caveolin-1 is the primary component of caveolae and functions in a variety of intracellular activities, including membrane trafficking and signal transduction. EMP2 (epithelial membrane protein 2) is a tetraspan protein recently identified as a novel regulator of caveolin-1 expression. In this study, we analyzed the mechanism of EMP2-mediated caveolin-1 regulation. In NIH 3T3 cells and in the human retinal pigment epithelium cell line (ARPE-19), EMP2 regulates caveolin-1 transcription and more substantially its protein levels. EMP2-mediated down-regulation of caveolin-1 does not affect caveolin-1 translational efficiency, phosphorylation, or proteasome-mediated degradation. Analysis of caveolin-1 protein half-life indicates the EMP2-mediated loss of caveolin-1 occurs rapidly. Protease inhibition and laser confocal microscopy associates this fate with specific intracellular compartmentalization, including early lysosomal delivery. These findings elucidate a new mechanism of caveolin-1 regulation and define an additional role for EMP2 as a key regulator of cell membrane composition.

Epithelial membrane protein 2 modulates infectivity of Chlamydia muridarum (MoPn)

Chlamydiae are bacterial pathogens which have evolved efficient strategies to enter, replicate, and survive inside host epithelial cells, resulting in acute and chronic diseases in humans and other animals. Several candidate molecules in the host receptor complex have been identified, but the precise mechanisms of infection have not been elucidated. Epithelial membrane protein-2 (EMP2), a 4-transmembrane protein, is highly expressed in epithelial cells in sites of chlamydial infections. Here we show that infectivity of the Chlamydia muridarum (MoPn) is associated with host cellular expression of EMP2 in multiple cell lines. Recombinant knockdown of EMP2 impairs infectivity, whereas infectivity is augmented in cells recombinantly modified to over-express EMP2. An epithelial cell line without native expression of EMP2 is relatively resistant to MoPn infection, whereas infectivity is markedly increased by recombinant expression of EMP2 in that cell line. Blockade of surface EMP2 using a specific anti-EMP2 antibody significantly reduces chlamydial infection efficiency. In addition, MoPn infectivity as measured in the EMP2 overexpressing cell line is not heparin-dependent, suggesting a possible role for EMP2 in the non-reversible phase of early infection. These findings identify EMP2 as a candidate host protein involved in infection of C. muridarum (MoPn).

156: Variants in the Kallikrein (KLK) Gene Cluster and Epithelial Membrane Protein 3 (EMP3) on 19Q13 are Associated with Prostate Cancer in Men of Caucasian Ancestry

You have accessJournal of UrologyModerated Poster 6, Sunday, May 20, 2007, 8:00 - 10:00 am1 Apr 2007156: Variants in the Kallikrein (KLK) Gene Cluster and Epithelial Membrane Protein 3 (EMP3) on 19Q13 are Associated with Prostate Cancer in Men of Caucasian Ancestry Ranjan Deka, Prodipto Pal, Huifeng Xi, Guangyun Sun, Ritesh Kaushal, Joshua J. Meeks, C. Shad Thaxton, Brian K. Suarez, and William J. Catalona Ranjan DekaRanjan Deka More articles by this author , Prodipto PalProdipto Pal More articles by this author , Huifeng XiHuifeng Xi More articles by this author , Guangyun SunGuangyun Sun More articles by this author , Ritesh KaushalRitesh Kaushal More articles by this author , Joshua J. MeeksJoshua J. Meeks More articles by this author , C. Shad ThaxtonC. Shad Thaxton More articles by this author , Brian K. SuarezBrian K. Suarez More articles by this author , and William J. CatalonaWilliam J. Catalona More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)30421-XAboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "156: Variants in the Kallikrein (KLK) Gene Cluster and Epithelial Membrane Protein 3 (EMP3) on 19Q13 are Associated with Prostate Cancer in Men of Caucasian Ancestry." The Journal of Urology, 177(4S), p. 53 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 177Issue 4SApril 2007Page: 53 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Ranjan Deka More articles by this author Prodipto Pal More articles by this author Huifeng Xi More articles by this author Guangyun Sun More articles by this author Ritesh Kaushal More articles by this author Joshua J. Meeks More articles by this author C. Shad Thaxton More articles by this author Brian K. Suarez More articles by this author William J. Catalona More articles by this author Expand All Advertisement Loading ...

Chemokine receptor signaling in mast cells (89.27)

Abstract Mast cells play an important role in IgE-associated allergic disorders and immune response to parasites. In a former study, we demonstrated that chemokine CCL3 (MIP-1α) acted as a co-stimulator for FcεRI-mediated activation on mast cells. To advance our understanding on this finding, we studied gene profiles in Rat Basophilic Leukaemia mast cells after co-activation of CCR1 (a receptor of CCL3) and FcεRI using the Affymetrix GeneChip Arrays. Emp1 (Epithelial Membrane protein 1), RT1-S3 (non-classical type 1 MHC gene), and Slc35e4 (solute carrier family 35, member E4) were found to be the top possible synergy-related genes. Validations of this data are being carried out using real time PCR, western-blotting and siRNA technologies. Further to our in vivo findings of the inhibition of the immediate hypersensitivity reactions in the conjunctiva and the attenuation of mast cell degranulation in MIP-1α deficient mice, we would like to check if MIP-1α and CCR1 play a critical role to produce these effects. We reconstituted the bone marrow-derived mice mast cells (BMMC) from naïve mice into mast cell-deficient mice and confirmed that the reconstituted BMMC home to their eye. The next step is to examine whether CCR1 and CCL3 are critical for mast cell activation and allergic conjunctivitis-like symptoms following induction of murine allergic conjunctivitis using CCR1- and CCL3- deficient mice.

Promoter hypermethylation and aberrant expression of EMP3at 19q13.3 in oligodendrogliomas and astrocytomas

Allelic losses on the long arm of chromosome 19 are found in the majority of oligodendroglial tumors and approximately 20–50% of astrocytic tumors. Losses on 19q are one of the earliest events in oligodendrogliomas, whereas such losses are linked to progression in astrocytomas. Due to a t(1;19) (q10;p10) translocations oligodendrogliomas show a loss of the entire long arm of chromosome 19, whereas astrocytomas exhibit interstitial and telomeric deletions without translocations. However, altered tumor suppressor genes (TSG) on 19q are still elusive in gliomas. We used cDNA microarray expression profiling to identify differentially expressed TSG candidates between gliomas with and without losses on 19q. Thereby, we found that 19q-deleted gliomas, when compared to gliomas without 19q deletion, showed lower expression of the epithelial membrane protein 3 (EMP3) gene at 19q13.3. Recently, EMP3 has been postulated as a hypermethylated TSG candidate in neuroblastomas and astrocytomas.

222 SPECIES SPECIFICITY OF PORCINE SPERM MOTILITY REDUCTION BY A HIGH MOLECULAR WEIGHT FRACTION OF OVIDUCTAL FLUID

The significance of sperm motility with respect to fertilization is widely recognized and used as a criterion to assess the quality of ejaculates. Observations of sperm behavior in the oviductal isthmus of several species have shown that their motility is suppressed in this physiological environment because the spermatozoa bind to the oviductal epithelial cells, forming a sperm reservoir prior to ovulation (Hunter 1981 J. Reprod. Fertil. 63, 109–117; Hunter and Wilmut 1984 Reprod. Nutr. Dev. 24, 597–608). Once the spermatozoa are released from the reservoir, they progress toward the ampullar region to reach the oocyte, and an increase in motility at this point could, potentially, be crucial. It has been demonstrated that a soluble fraction of oviductal epithelial cell apical plasma membrane proteins (sAPM) suppresses sperm motility and enhances sperm survival (Holt et al. 2005 Reprod. Fertil. Dev. 17, 683–692; Satake et al. 2006 J. Exp. Biol. 209, 1560–1572). However, few studies to date have investigated the influence of oviductal fluid (OF), the natural medium into which spermatozoa are released from the reservoir, on sperm motility. Consequently, this study aimed to determine the effects of different soluble fractions of OF on sperm motility and the species specificity of such effects. OF from pigs and cows was collected and selectively filtered to obtain two different fractions with molecular weights higher or lower than 100 kD. Diluted semen samples from 14 different boars were exposed to bicarbonate/CO2 (to stimulate maximum motility) in the presence or absence of OF fractions. Sperm trajectories were measured using a Hobson Sperm Tracker (Hobson Tracker, Ltd., Sheffield, UK) and analyzed by PATN analysis as described previously to identify subpopulations of high and low motility spermatozoa (Abaigar et al. 1999 Biol. Reprod. 60, 32–41; Satake et al. 2006). The results showed that neither of the bovine OF fractions affected the proportions of the fast linear boar sperm subpopulation in the samples, which was similar to that of the control. However, when the high molecular weight fraction of porcine OF was used, a significant suppression of the fast linear sperm subpopulation was observed (P ≤ 0.05). These data support the hypothesis that species-specific, high molecular weight components in OF are involved in the suppression of sperm motility. Further studies are required to confirm the significance of this finding, although it may not be unreasonable to speculate that the OF, in addition to other sperm selection mechanisms, acts to protect oocytes against fertilization by poor quality spermatozoa (Okada et al. 1986 J. Submicrosc. Cytol. 18, 233–247). In fact, similar results demonstrating that oviductal fluid decreases sperm motility have been obtained in cow (Grippo et al. 1995 J. Reprod. Fertil. 105, 57–64) and rabbit (Overstreet and Cooper 1979 J. Reprod. Fertil. 55, 53–59). This work was supported by MEC and FEDER (PR2006-0506 and AGL2006-03495).

EMP3 overexpression is associated with oligodendroglial tumors retaining chromosome arms 1p and 19q

The epithelial membrane protein 3 (EMP3) gene located on chromosome 19q13 has been implicated as a candidate tumor suppressor gene (TSG) in neuroblastomas and gliomas. The aim of this study was to investigate whether EMP3 is involved in oligodendroglial tumors (OTs), which frequently carry combined chromosomes 1p and 19q deletion. We first investigated the transcript level of EMP3 in a cohort of 57 OTs by quantitative real-time RT-PCR. Our results showed that 10 (18%) tumors had reduced EMP3 expression level compared to normal brains. Six of these tumors carried chromosome 19q13 deletion but no statistical correlation was found between the 2 parameters. Intriguingly, a similar proportion (11 of 57, 19%) of tumors displayed EMP3 overexpression, with 8 of them having transcript level >10-fold higher than normal brain. All 11 OTs retained chromosomes 1p36 and 19q13, and a significant association was found between EMP3 overexpression and balanced chromosomes 1p36 and 19q13 (p = 0.004). The methylation status of EMP3 was evaluated by bisulfite sequencing in 29 OTs with diverse expression levels. All tumors except 3 showed aberrant methylation of EMP3 and no correlation was observed between transcript level and methylation status, suggesting that methylation alone does not mediate transcriptional down-regulation of EMP3 in OTs. In conclusion, our study demonstrates that EMP3 overexpression is involved in OTs retaining chromosomes 1p and 19q and does not support EMP3 as the target TSG on chromosome 19q13 in OTs.

CKAP4/p63 Is a Receptor for the Frizzled-8 Protein-related Antiproliferative Factor from Interstitial Cystitis Patients

Antiproliferative factor (APF) is a low molecular weight sialoglycopeptide that is secreted by bladder cells from interstitial cystitis patients and is a potent inhibitor of both normal bladder epithelial and bladder carcinoma cell proliferation. We hypothesized that APF may produce its antiproliferative effects by binding to a transmembrane receptor. This study demonstrates that cytoskeleton-associated protein 4/p63 (CKAP4/p63), a type II transmembrane receptor, binds with high affinity to APF. The antiproliferative activity of APF is effectively inhibited by preincubation with anti-CKAP4/p63-specific antibodies, as well as by short interfering RNA knockdown of CKAP4/p63. Immunofluorescent confocal microscopy showed co-localization of anti-CKAP4/p63 and rhodamine-labeled synthetic APF binding in both cell membrane and perinuclear areas. APF also inhibits the proliferation of HeLa cervical carcinoma cells that are known to express CKAP4/p63. These data indicate that CKAP4/p63 is an important epithelial cell receptor for APF.

In Vivo Transcript Profiling and Phylogenetic Analysis Identifies Suppressor of Cytokine Signaling 2 as a Direct Signal Transducer and Activator of Transcription 5b Target in Liver

The GH-activated signal transducer and activator of transcription 5b (STAT5b) is an essential regulator of somatic growth. The transcriptional response to STAT5b in liver is poorly understood. We have combined microarray-based expression profiling and phylogenetic analysis of gene regulatory regions to study the interplay between STAT5b and GH in the regulation of hepatic gene expression. The acute transcriptional response to GH in vivo after a single pulse of GH was studied in the liver of hypophysectomized rats in the presence of either constitutively active or a dominant-negative STAT5b delivered by adenoviral gene transfer. Genes showing differential expression in these two situations were analyzed for the presence of STAT5b binding sites in promoter and intronic regions that are phylogenetically conserved between rats and humans. Using this approach, we showed that most rapid transcriptional effects of GH in the liver are not results of direct actions of STAT5b. In addition, we identified novel STAT5b cis regulatory elements in genes such as Frizzled-4, epithelial membrane protein-1, and the suppressor of cytokine signaling 2 (SOCS2). Detailed analysis of SOCS2 promoter demonstrated its direct transcriptional regulation by STAT5b upon GH stimulation. A novel response element was identified within the first intron of the human SOCS2 gene composed of an E-box followed by tandem STAT5b binding sites, both of which are required for full GH responsiveness. In summary, we demonstrate the power of combining transcript profiling with phylogenetic sequence analysis to define novel regulatory paradigms.

Expression of epithelial membrane protein‐2 is associated with endometrial adenocarcinoma of unfavorable outcome

Epithelial membrane protein 2 (EMP2) is an estrus-regulated tetraspan protein that is required for endometrial competence in blastocyst implantation. EMP2 controls surface levels of several classes of integrin and other cell-interaction molecules, and their trafficking to glycolipid-enriched lipid raft domains is important in receptor signaling. These features suggest that EMP2 may contribute to neoplastic traits of endometrial cancer. The objective of this study was to determine the prevalence of EMP2 expression in endometrial neoplasms and its clinical significance. EMP2 immunophenotype, histologic diagnosis, grade, the presence of lymphovascular invasion, disease stage, and clinical follow-up were determined for 99 endometrial cancers. Significant EMP2 expression (EMP2 positive) was observed in 12 of 99 cancers (9 endometrioid [6 International Federation of Gynecology and Obstetrics Grade 3], 1 serous, 1 mixed endometrioid and serous, and 1 mixed endometrioid and clear cell), and weak EMP2 expression was observed in 11 cancers. EMP2-positive tumors were more likely than others to be myometrium invasive, high stage, and recurrent, persistent, or fatal. The overall median survival for patients with EMP2-positive tumor was only 23 months, whereas the medial survival was not reached for patients with EMP2-weak and EMP2-negative tumors. The median disease-free interval was only 11 months for patients with EMP2-positive tumors and was not reached for patients with EMP2-weak and EMP2-negative tumors. A multivariate analysis of disease-free survival demonstrated independent, negative prognostic significance for EMP2 expression, high stage, and high-risk histologic subtypes. EMP2 expression is a feature of some prognostically unfavorable endometrial cancers. Its utility for clinical decision making and its biologic role in endometrial cancer deserves further study in a larger series of patients.

Knockdown of the tetraspan protein epithelial membrane protein-2 inhibits implantation in the mouse

The establishment of pregnancy requires a successful molecular interaction between the trophectoderm cells of the blastocyst stage embryo and the endometrial cells of the uterus. These interactions are complex and require synchronous development and coordinated endocrine, paracrine, and autocrine communication. In this study, we demonstrate that the tetraspan protein epithelial membrane protein-2 (EMP2) is involved in these molecular interactions during implantation. EMP2, which is highly expressed in the uterus, translocates from an intracellular location to the apical surface of the endometrial epithelium during the window of implantation and is expressed in decidualized stromal cells. We developed plasmid constructs that utilized either ribozyme-mediated or short hairpin RNA-mediated mechanisms to target endometrial EMP2 mRNA for destruction. These constructs were transfected into the mouse uterus on day 1 of pregnancy using the technique of in vivo reproductive tract gene transfer. Reduction in EMP2 expression by either method resulted in a significant decrease in the number of implantation sites in the treated uterine horns as compared to control horns. These studies indicate a previously unknown function of tetraspan proteins in implantation and could provide a molecular framework for the development of therapeutic modalities for both contraception and fertility.

312 EXPOSURE OF SPERMATOZOA TO SOLUBILIZED EXTRACTS OF THE OVIDUCTAL EPITHELIUM APICAL PLASMA MEMBRANE ENHANCES FERTILIZATION IN PORCINE IN VITRO FERTILIZATION

In vitro production (IVP) of porcine embryos is currently suboptimal compared with IVP in species such as mice and cattle. In vitro fertilization (IVF) usually involves the co-culture of oocytes and spermatozoa in a medium droplet. Oocyte quality is the focus of many studies. In vivo, the quality of spermatozoa is as important as the oocyte, and females have many mechanisms to select the highest quality spermatozoa for their oocytes. Oviductal proteins have been shown to affect sperm motility of subpopulations within an ejaculate. The present study was carried out to investigate normal and polyspermic fertilization rates of spermatozoa exposed to oviductal epithelial apical plasma membrane (APM) proteins, a mixture of peripheral proteins extracted by 1 M NaCl from isolated oviductal apical plasma membranes, prior to co-culture with oocytes in IVF. Porcine oocytes were aspirated from ovaries and grade I quality oocytes (cumulus–oocyte complexes with a spherical shape, visible nucleus, even-density cytoplasm, and multiple layers of cumulus cells) were selected and matured for 48 h in TCM-199 supplemented with LH (0.5 �g mL-1), FSH (0.5 �g mL-1), and EGF (10 ng mL-1). Ejaculates were washed through a Percoll gradient to obtain a concentrated pellet. Spermatozoa were diluted in capacitation–fertilization medium in the presence or absence of APM proteins (100 �g mL-1), incubated for 10 min, and then co-cultured with oocytes for 6 h in modified Tween medium B with milk powder medium (Abeydeera and Day 1997 Theriogenology 48, 537–544) supplemented with BSA (0.4%) and sodium bicarbonate (5 mM). Presumptive zygotes were cultured in NCSU23 medium for a further 48 h. The oocytes/zygotes were then fixed and stained with propidium iodide for evaluation by confocal microscopy for fertilization and cleavage (n = 1235 oocytes). Fertilization rates were compared between treatments in a chi-squared test using the Mantel-Haenszel approach. The overall fertilization rate was significantly higher (78 vs. 86%) when spermatozoa were incubated in the presence of APM proteins (P < 0.05), and in the group of fertilized oocytes, polyspermic fertilization (47 vs. 21%) was significantly reduced when spermatozoa were exposed to APM proteins (P < 0.01). However, cleavage rates were not different. These results suggest that exposure of spermatozoa to APM proteins prior to IVF increases the fertilization rate and decreases the incidence of polyspermic penetration.

Epithelial membrane protein-2 regulates surface expression of αvβ3 integrin in the endometrium

The four-transmembrane protein epithelial membrane protein-2 (EMP2) was recently identified as an endometrial protein necessary for blastocyst implantation, but the mechanism of this role is uncertain. In other cell types, EMP2 controls delivery of certain classes of proteins to the cell surface, including various integrin isoforms (a class of receptors implicated in endometrial–blastocyst interaction). Since αvβ3 integrin is an important endometrial molecule involved in blastocyst interaction, we evaluated the role of EMP2 in modulating integrin expression in the HEC1A endometrial cell line and endometrial epithelium in vivo. Elevation of EMP2 expression in HEC1A cells selectively increased the expression of αvβ3 integrin on the plasma membrane and was functional as judged by increased cell binding to an αvβ3 ligand, fibronectin. Conversely, reduction in EMP2 expression using an EMP2 specific ribozyme decreased the cell αvβ3 surface expression. The influence of EMP2 on αvβ3 integrin was also observed in vivo as reduction of EMP2 using ribozymes or short hairpin RNA diminished αvβ3 integrin expression in glandular and luminal uterine epithelium. Colocalization and coimmunoprecipitation studies suggested that EMP2 and αvβ3 integrin predominantly exist in a physically associated state. This study demonstrates for the first time the influence of EMP2 on αvβ3 surface expression and suggests that surface trafficking of integrin αvβ3 by EMP2 during the window of implantation may be a mechanism for its requirement in endometrial–blastocyst interaction.

Epithelial membrane protein-1 is a biomarker of gefitinib resistance.

We describe a molecular resistance biomarker to gefitinib, epithelial membrane protein-1 (EMP-1). Gefitinib is a small-molecule inhibitor that competes for the ATP-binding site on EGF receptor (EGFR) and has been approved for patients with advanced lung cancers. Treatment with gefitinib has resulted in clinical benefit in patients, and, recently, heterozygous somatic mutations within the EGFR catalytic domain have been identified as a clinical correlate to objective response to gefitinib. However, clinical resistance to gefitinib limits the utility of this therapeutic to a fraction of patients, and objective clinical responses are rare. We aimed to assess the molecular phenotype and mechanism of in vivo gefitinib resistance in xenograft models and in patient samples. We generated in vivo gefitinib-resistance models in an adenocarcinoma xenograft model by serially passaging tumors in nude mice in presence of gefitinib until resistance was acquired. EMP-1 was identified as a surface biomarker whose expression correlated with acquisition of gefitinib resistance. EMP-1 expression was further correlated with lack of complete or partial response to gefitinib in lung cancer patient samples as well as clinical progression to secondary gefitinib resistance. EMP-1 expression and acquisition of gefitinib clinical resistance was independent of gefitinib-sensitizing EGFR somatic mutations. This report suggests the role of the adhesion molecule, EMP-1, as a biomarker of gefitinib clinical resistance, and further suggests a probable cross-talk between this molecule and the EGFR signaling pathway.