Biomedical subjects

J Julian

Publications and source records attributed to J Julian.

At least 37 records · Page 2Linked to original sources

PubMed1998

Regional specialization of the cell membrane-associated, polymorphic mucin (MUC1) in human uterine epithelia.

The cell membrane-associated, polymorphic mucin, MUC1, has been proposed to hinder implantation by virtue of its anti-adhesive properties. Consistent with this proposal is the observation of a dramatic decrease in MUC1 protein and mRNA expression in the uterine epithelium of several species at the time of implantation. In contrast, little change in glandular epithelial expression of MUC1 protein or its mRNA during the peri-implantation period has been detected in humans. However, expression in the luminal epithelium, i.e. the epithelium involved in embryo attachment, has not been reported. Using tissue samples with a clearly defined luminal epithelium and antibodies directed against the cytoplasmic domain found in all cell-associated MUC1 species (CT-1) and against two MUC1 ectodomain epitopes, HMFG-1 and HMFG-2, we demonstrate that MUC1 expression in the luminal epithelium is maintained throughout the menstrual cycle. The staining observed with CT-1 correlates with that seen with HMFG-2, but not HMFG-1. HMFG-1 reactivity was high in all regions except basal glands in the mid proliferative endometrium and fell to very low levels throughout the tissue in the mid secretory phase. In all cases, HMFG-1 reactivity could be restored by predigestion with keratanase or neuraminidase which removes keratan sulphates and sialic acids, respectively. These observations suggest that regionally restricted glycosylation generates an altered external structure of MUC1. These alterations appear to decrease accessibility to the MUC1 protein core region and are maximal in luminal epithelium at the receptive phase. Due to their large highly extended structures, MUC1 ectodomains are very likely to be among the first cell surface components encountered during human blastocyst attachment to the luminal epithelium. Thus, MUC1 either must be locally removed during the attachment process or functions actually to promote the initial steps in embryo adhesion to the apical surface of the human uterine epithelium.

Adolescent↗

PubMed1998

Complementary expression of HIP, a cell-surface heparan sulfate binding protein, and perlecan at the human fetal-maternal interface.

The human hemochorial placenta is a structure formed by the invasion of cytotrophoblasts into the uterus. Previous studies from our laboratory have demonstrated a role for heparan sulfate proteoglycans (HSPGs) and their binding proteins in interactions between human trophoblastic and uterine cell lines in vitro. In this study, expression of both mRNA and protein of a novel, cell surface, heparin/heparan sulfate interacting protein (HIP), by human trophoblastic cell lines-i.e., JAR, JEG, and BeWo-and by human cytotrophoblast was examined throughout gestation. Immunohistochemistry of the human fetal-maternal interface demonstrated abundant HIP expression in cytotrophoblast cells, with lesser staining in syncytiotrophoblast and little or no staining in surrounding stromal or decidual cells. Staining with antibodies to the basement membrane HSPG, perlecan, demonstrated a pattern of staining complementary to that of HIP. Cytotrophoblasts in the uterine stroma, not affiliated with attached villi, displayed a less intense deposition of perlecan. In vitro binding studies of 125I-perlecan to a 17-amino acid synthetic peptide sequence of HIP, which has a high affinity and specificity for heparin/heparan sulfate, indicates that perlecan binds to the HIP peptide with high affinity (KDapp = 0.6 nM) and in a heparin-inhibitable manner. Furthermore, HIP antibodies inhibited by 61-88% in vitro invasion by trophoblasts in assays using primary cultures of normal human cytotrophoblasts. Consistent with this was the observation that immunohistochemically detectable HIP expression was greatly reduced in pre-eclamptic cytotrophoblasts, a condition in which trophoblast invasion is abnormally shallow. It is suggested that HIP potentiates human cytotrophoblast interactions with HSPGs, in vivo, and facilitates trophoblast invasion processes.

Antibodies↗

PubMed1998

Reduction of mucin-1 expression during the receptive phase in the rat uterus.

Mucin-1 (Muc1), an integral membrane mucin, is expressed on the apical surface of uterine epithelial cells (UE) of various species. Loss of Muc1 is believed to be necessary for embryo attachment. Muc1 expression is markedly reduced in luminal epithelia during the receptive phase in mice, baboons, and pigs. In the present study, we examined Muc1 expression during the rat estrous cycle and at Day 5 of pregnancy, the time of embryo attachment. In contrast to findings in the mouse, indirect immunofluorescence revealed that uterine Muc1 protein expression was unaltered during the estrous cycle. However, similar to what is observed in the mouse and other species, Muc1 protein decreased at Day 5 of pregnancy in luminal UE. The decrease in Muc1 expression was specific to luminal UE and did not occur in glandular UE. A partial cDNA corresponding to the cytoplasmic tail region of rat Muc1 was generated by a reverse transcription-polymerase chain reaction (RT-PCR) strategy. This cDNA sequence is 89% and 91% identical to the corresponding region of mouse Muc1 at the nucleotide and amino acid levels, respectively. The predicted sequence of rat Muc1 protein has 70-90% identity to the Muc1 protein sequence obtained in other species. Semiquantitative RT-PCR experiments indicated that the mRNA encoding rat Muc1 decreased 57% at Day 5 as compared with the levels found at estrus. This value included mRNA from both luminal and glandular UE and so may underestimate the relative decrease in mRNA in the luminal compartment. In conclusion, we have determined that the levels of rat Muc1 protein and mRNA decrease in the luminal UE at the time of implantation, a pattern similar to that seen in the mouse, baboon, and pig. This supports the general theory that reduction of Muc1 expression is necessary for embryo implantation.

Amino Acid Sequence↗

PubMed1998

The occurrence of subsequent malignancy in patients presenting with deep vein thrombosis: results from a historical cohort study.

BACKGROUND: Several studies have reported that patients who present with idiopathic deep vein thrombosis (DVT) have an increased risk of subsequently developing cancer. A clinical trial had previously been conducted examining the optimal duration of oral anticoagulant therapy following initial heparin treatment in patients with proximal DVT. METHODS: A historical cohort study was performed on patients enrolled in the duration of anticoagulant trial. Patients known to have cancer at the time of entry into the trial were excluded. The qualifying DVTs were classified as idiopathic (no known associated risk factors) or secondary without knowledge of subsequent recurrent venous thrombosis or cancer. The patients were then followed for the development of cancer. RESULTS: Thirteen (8.6%) of the 152 patients in the idiopathic cohort subsequently developed cancer compared to eight (7.1%) of 112 patients in the secondary cohort, P = 0.86. Two (5.4%) of 37 patients with recurrent venous thromboembolism and 19 (8.4%) of 227 patients without recurrent thromboembolism developed cancer, P = 0.7. CONCLUSION: Our study did not detect an increased risk of subsequent cancer in patients presenting with idiopathic DVT compared to secondary DVT; nor did we detect an increased incidence of cancer in patients with recurrent venous thromboembolism. Further studies are required prior to pursuing a policy of aggressive screening for cancer in patients with idiopathic venous thromboembolism.

Administration, Oral↗

PubMed1998

A novel antithrombin-heparin covalent complex: antithrombotic and bleeding studies in rabbits.

Heparin has been used extensively for prophylaxis and treatment of deep vein thrombosis. However, heparin has several limitations including a short intravenous half-life, inability to inhibit clot-bound thrombin, and bleeding. We have developed a covalent antithrombin-heparin complex (ATH) that has a longer intravenous half-life and greater anticoagulant activity than heparin. The antithrombotic activity of ATH was tested in a rabbit jugular vein thrombosis treatment model. Administration of ATH caused a 17% reduction in clot weight compared with an increase of 24, 60, 172 and 135% for administration of antithrombin plus heparin, heparin, antithrombin and saline, respectively. Clot weight and fibrin accretion were both significantly lower in the ATH group than in the antithrombin plus heparin group (P < 0.05). The peak anti-factor-Xa activity was fourfold higher in the ATH group than in the antithrombin plus heparin group. Using a rabbit bleeding ear model, there was no significant difference in cumulative blood loss between ATH and antithrombin plus heparin groups, at similar plasma anti-factor-Xa levels. In conclusion, ATH has superior antithrombotic activity and similar bleeding effect compared with heparin on a mass basis. The enhanced antithrombotic activity of ATH may be a result of its increased anticoagulant activity or its longer half life, or both.

Animals↗

PubMed1997

Heparin/heparan sulfate interacting protein expression and functions in human breast cancer cells and normal breast epithelia.

Heparin/heparan sulfate interacting protein (HIP) is a recently identified protein expressed by many normal epithelia and epithelial cell lines. In the present study, we examined expression and potential functions of this protein in a series of human breast cancer cells and in sections of normal and malignant human breast tissue. Four of the five breast cancer cell lines studied (MCF-7, T-47D, MDA-MB468, and BT-549) expressed HIP protein and mRNA at similar levels. In contrast, MDA-MB-231 cells failed to display reactivity with HIP-specific probes in any assay. Cell aggregation assays and cell surface antibody binding studies demonstrated that HIP was expressed on the cell surface. However, HIP expression did not correlate with the number of cell surface [3H]heparin (HP) binding sites. The K(Dapp)s for cell surface HP binding sites were similar in all breast cancer cell lines studied and ranged from 112 to 298 nM. In contrast, cell surface HP binding capacity varied greatly, ranging from 2.3 x 10(5) (MDA-MB-231 and MDA-MB-468) to 99 x 10(5) sites/cell (BT-549). All cell lines tested displayed the ability to bind to a heparan sulfate (HS)-binding synthetic peptide motif of HIP in a HP-inhibitable fashion. Binding to this motif was not inhibited by other glycosaminoglycans including hyaluronic acid, chondroitin sulfates, or keratan sulfate. Furthermore, cell binding to HIP peptide was almost completely lost when intact cells were predigested with heparinases but not chondroitinases. Cell surface HS from breast cancer cells as well as normal human breast epithelia binded to HIP peptide in a HP-inhibitable fashion, demonstrating the ability of these cell surface components to directly interact. HIP was detected in both normal breast epithelia and breast tumors in situ. It is suggested that HIP mediates aspects of HS-dependent interactions of both normal and malignant breast epithelia with other cells and extracellular matrix components.

Animals↗

PubMed1997

Heparin/heparan sulfate (HP/HS) interacting protein (HIP) supports cell attachment and selective, high affinity binding of HP/HS.

Heparin/heparan sulfate (HP/HS), HS proteoglycans, and their binding proteins play important roles in a variety of biological processes. Previously, we identified a novel cell surface HP/HS interacting protein (HIP) from human uterine epithelia and a variety of other human epithelial and endothelial cells and cell lines (Liu, S., Smith, S. E., Julian, J., Rohde, L. H., Karin, N. J., and Carson, D. D. (1996) J. Biol. Chem. 271, 11817-11823; Rohde, L. H., Julian, J., Babaknia, A., and Carson, D. D. (1996) J. Biol. Chem. 271, 11824-11830). In the current studies, we have purified and characterized HIP from HEC cells, a human uterine epithelial cell line, as well as recombinant HIP from a bacterial expression system. HIP supports attachment of the human trophoblastic cell line, JAR, in a HS-dependent fashion. Predigestion of JAR cells with a mixture of heparitinases, but not chondroitinase AC, abolished cell attachment to HIP. In addition, JAR cell attachment to HIP is highly sensitive to HP inhibition and much more selective for HP/HS than other glycosaminoglycans. Dermatan sulfate displays partial inhibitory activity as well, consistent with the observation that chondroitinase ABC digestion partially reduces JAR cell attachment to HIP. Solid-phase binding assays indicate HIP binds [3H]HP with high affinity (apparent KD = 8 nM). Furthermore, HIP bound cell surface/extracellular matrix-associated HS, expressed by RL95 cells, a human uterine epithelial cell line. Anti-HIP antibody generated against a synthetic peptide derived from a putative HP/HS-binding motif resident within HIP inhibited about half of [3H]HP binding to HIP, indicating that this domain is a functional HP-binding domain of HIP. Similarly, this same synthetic peptide motif of HIP could block about 50% of [3H]HP binding to HIP; however, this peptide almost completely inhibited cell attachment to HIP, suggesting a critical role, in this regard. Collectively, these results suggest that HIP can function as a HP/HS-binding cell-cell/cell-matrix adhesion molecule.

Animals↗

PubMed1997

Expression of heparan sulfate proteoglycan (perlecan) in the mouse blastocyst is regulated during normal and delayed implantation.

Previous studies have shown that expression of the heparan sulfate proteoglycan, perlecan, on the external trophectodermal cell surfaces of mouse blastocysts increases during acquisition of attachment competence. However, it is not clear if this change in perlecan protein expression also is reflected at the level of perlecan mRNA expression. In the present investigation, the spatial and temporal patterns of perlecan mRNA expression in the mouse embryo during the periimplantation period were examined by in situ hybridization and reverse transcriptase-polymerase chain reaction. In addition, a delayed implantation model was used to determine the expression of perlecan mRNA and protein in dormant and estrogen-activated hatched blastocysts. The results demonstrate that perlecan mRNA expression is low in morulae, but increases in Day 4 blastocysts, attaining maximal expression in Day 4.5 attachment-competent blastocysts. In contrast, perlecan mRNA is detected in both the dormant and estrogen-activated delayed blastocysts; however, within 12 hr of blastocyst activation by estrogen, both perlecan protein and heparan sulfate chain expression markedly increase. Taken together, these results suggest that during normal development perlecan mRNA expression increases with the acquisition of attachment competence. Moreover, perlecan protein expression also is attenuated during delayed implantation and appears to increase in response to nidatory estrogen, perhaps via the increased translation of preexisting perlecan mRNA.

Animals↗

PubMed1996

Synthesis and intracellular trafficking of Muc-1 and mucins by polarized mouse uterine epithelial cells.

Mucins function as a protective layer rendering the apical surface of epithelial cells nonadhesive to a variety of microorganisms and macromolecules. Muc-1 is a transmembrane mucin expressed at the apical cell surface of mouse uterine epithelial cells (UEC) that disappears as UEC become receptive for embryo implantation (Surveyor, G. A., Gendler, S. J., Pemberton, L., Das, S. K., Chakraborty, I., Julian, J., Pimental, R. A., Wegner, C. W., Dey, S. K., and Carson, D. D. (1995) Endocrinology 136, 3639-3647). In the present study, the kinetics of Muc-1 assembly, cell surface expression, release, and degradation were examined in polarized mouse UEC in vitro. Mucins were identified as the predominant glycoconjugates synthesized, apically expressed, and vectorially released in both wild-type and Muc-1 null mice. When mucins were released, greater than 95% were directed to the apical compartment. Approximately half of the cell-associated mucins lost during a 24-h period were found in the apical compartment. Vectorial biotinylation detected apically disposed, cell-surface mucin and indicated that at least 34% of these mucins are released apically within 24 h. This suggests that release of mucin ectodomains is part of the mechanism of mucin removal from the apical cell surface of UEC. The half-lives of total cell-associated mucins and Muc-1 were 19.5 +/- 1 and 16.5 +/- 0.8 h, respectively. Muc-1 represented approximately 10% of the [3H]glucosamine-labeled, cell-associated mucins. Studies of the kinetics of intracellular transport of Muc-1 indicated transit times of 21 +/- 15 min from the rough endoplasmic reticulum to Golgi apparatus and 111 +/- 28 min from the Golgi apparatus to the cell surface. Collectively, these studies provide the first comprehensive description of Muc-1 and mucin maturation, metabolism, and release by polarized cells, as well as defining a major metabolic fate for mucins expressed by UEC. Normal metabolic processing appears to be sufficient to account for the removal of Muc-1 protein during the transition of UEC to a receptive state.

Animals↗

PubMed1996

A follow-up study of synthetic rubber workers.

Although 1,3-butadiene (BD) has been classified as an animal carcinogen, epidemiologic research has reported inconsistent results on the relationship between BD and lymphopoietic and other cancers in humans. This study evaluated the mortality experience of 15649 men employed for at least 1 year at any of eight North American styrene-butadiene rubber (SBR) plants. About 75% of the subjects were exposed to BD; 83% were exposed to styrene (STY). During 1943-1991, the cohort had a total of 386172 and an average of 25 person-years of follow-up, with 3976 deaths observed compared to 4553 deaths expected based on general population mortality rates (standardized mortality ratio (SMR) = 87, 95% confidence interval (CI) = 85-90). More than expected leukemia deaths occurred in the overall cohort (48 observed/37 expected, SMR = 131, CI = 97-174) and among ever hourly subjects (45/32, SMR = 143, CI = 104-191). The excess was concentrated among ever hourly subjects with 10+ years worked and 20+ years since hire (28/13, SMR = 224, CI = 149-323) and among subjects in polymerization (15/6.0, SMR = 251, CI = 140-414), maintenance labor (13/4.9, SMR = 265, CI = 141-453) and laboratories (10/2.3, SMR = 431, CI = 207-793), three areas with potential for relatively high exposure to BD or STY monomers. Some cohort sub-groups had slight increases in deaths from lymphopoietic cancers other than leukemia, but mortality patterns by race, years worked and process group within the SBR industry did not indicate a causal association with occupational exposures. These results indicate that exposures in the SBR industry cause leukemia.

Butadienes↗

PubMed1996

Leukemia and cumulative exposure to butadiene, styrene and benzene among workers in the synthetic rubber industry.

Retrospective, quantitative estimates of exposure to 1,3-butadiene, styrene and benzene were developed for a follow-up study of leukemia mortality among 16610 subjects employed at six North American styrene-butadiene rubber manufacturing plants (418846 person-years, 58 leukemia deaths). The estimation procedure entailed identifying work areas within each manufacturing process, historical changes in exposure potential and specific tasks involving exposure, and using mathematical models to calculate job- and time-period-specific average exposures. The resulting estimates were linked with the subjects' work histories to obtain cumulative exposure estimates, which were employed in stratified and Poisson regression analyses of mortality rates. Mantel-Haenszel rate ratios adjusted by race, age, and cumulative styrene exposure increase with cumulative butadiene exposure from 1 in the nonexposed category to 4.5 in the category of 80 ppm-years or more (P = 0.01). The risk pattern is less clear and statistically nonsignificant for styrene exposure. A trend of increasing risk with butadiene exposure is still present after exclusion of the nonexposed category (P = 0.03). A parsimonious interpretation of the findings presented here, in light of previous epidemiologic studies, is that exposure to butadiene in the synthetic rubber industry produces a dose-related increase in the occurrence of leukemia.

Benzene↗

PubMed1996

cDNA cloning and expression of HIP, a novel cell surface heparan sulfate/heparin-binding protein of human uterine epithelial cells and cell lines.

Heparan sulfate proteoglycans and their corresponding binding sites have been suggested to play an important role during the initial attachment of murine blastocysts to uterine epithelium and human trophoblastic cell lines to uterine epithelial cell lines. Previous studies on RL95 cells, a human uterine epithelial cell line, had characterized a single class of cell surface heparin/heparan sulfate (HP/HS)-binding sites. Three major HP/HS-binding peptide fragments were isolated from cell surfaces by tryptic digestion, and partial amino-terminal amino acid sequence for each peptide fragment was obtained (Raboudi, N., Julian, J., Rohde, L. H., and Carson, D. D. (1992) J. Biol. Chem. 267, 11930-11939). In the current study, using approaches of reverse transcription-polymerase chain reaction and cDNA library screening, we have cloned and expressed a novel, cell surface HP/HS-binding protein, named HP/HS interacting protein (HIP), from RL95 cells. The full-length cDNA of HIP encodes a protein of 159 amino acids with a calculated molecular mass of 17,754 Da and pI of 11.75. Transfection of HIP full-length cDNA into NIH-3T3 cells demonstrated cell surface expression and a size similar to that of HIP expressed by human cells. Predicted amino acid sequence indicates that HIP lacks a membrane spanning region and has no consensus sites for glycosylation. Northern blot analysis detected a single transcript of 1.3 kilobases in both total RNA and poly(A+) RNA. Examination of human cell lines and normal tissues using both Northern blot and Western blot analyses revealed that HIP is expressed at different levels in a variety of human cell lines and normal tissues but absent in some cell lines and some cell types of normal tissues examined. HIP has relatively high homology (approximately 80% both at the levels of nucleotide and protein sequence) to a rodent ribosomal protein L29. Thus, members of the L29 family may be displayed on cell surfaces where they may participate in HP/HS binding events.

3T3 Cells↗

PubMed1996

Cell surface expression of HIP, a novel heparin/heparan sulfate binding protein, of human uterine epithelial cells and cell lines.

Previous studies established that uterine epithelial cells and cell lines express cell surface heparin/heparan sulfate (HP/HS)-binding proteins (Wilson, O., Jacobs, A. L., Stewart, S., and Carson, D. D. (1990) J. Cell. Physiol. 143, 60-67; Raboudi, N., Julian, J., Rohde, L. H., and Carson, D. D. (1992) J. Biol. Chem. 267, 11930-11939). The accompanying paper (Liu, S., Smith, S. E., Julian, J., Rohde, L. H., Karin, N. J., and Carson, D. D. (1996) J. Biol. Chem. 271, 11817-11823) describes the cloning of a full-length cDNA corresponding to a candidate cell surface HP/HS interacting protein, HIP, expressed by a variety of human epithelia. A synthetic peptide was synthesized corresponding to an amino acid sequence predicted from the cDNA sequence and used to prepare a rabbit polyclonal antibody. This antibody reacted with a protein with an apparent Mr of 24,000 by SDS-polyacrylamide gel electrophoresis that was highly enriched in the 100,000 x g particulate fraction of RL95 cells. This molecular weight is similar to that of the protein expressed by 3T3 cells transfected with HIP cDNA. HIP was solubilized from this particulate fraction with NaCl concentrations > or = 0.8 M demonstrating a peripheral association consistent with the lack of a membrane spanning domain in the predicted cDNA sequence. HIP was not released by heparinase digestion suggesting that the association is not via membrane-bound HS proteoglycans. NaCl-solubilized HIP bound to heparin-agarose in physiological saline and eluted with NaCl concentrations of 0.75 M and above. Furthermore, incubation of 125I-HP with transblots of the NaCl-solubilized HIP preparations separated by two-dimensional gel electrophoresis demonstrated direct binding of HP to HIP. Indirect immunofluorescence studies demonstrated that HIP is expressed on the surfaces of intact RL95 cells. Binding of HIP antibodies to RL95 cell surfaces at 4 degrees C was saturable and blocked by preincubation with the peptide antigen. Single cell suspensions of RL95 cells formed large aggregates when incubated with antibodies directed against HIP but not irrelevant antibodies. Finally, indirect immunofluorescence studies demonstrate that HIP is expressed in both lumenal and glandular epithelium of normal human endometrium throughout the menstrual cycle. In addition, HIP expression increases in the predecidual cells of post-ovulatory day 13-15 stroma. Collectively, these data indicate that HIP is a membrane-associated HP-binding protein expressed on the surface of normal human uterine epithelia and uterine epithelial cell lines.

Amino Acid Sequence↗

PubMed1996

Mucin (Muc-1) expression is differentially regulated in uterine luminal and glandular epithelia of the baboon (Papio anubis).

Changes in the organization and composition of apical cell surface glycoconjugates accompany the transition of luminal epithelial cells from the prereceptive state of the uterus in many species. In spite of the biological and clinical significance of this process, few molecular markers have arisen as useful predictors of uterine receptivity. Recent studies in mice demonstrate that the transmembrane mucin glycoprotein, Muc-1, is abundantly expressed at the apical surface of luminal epithelia under most conditions and is invariably reduced in receptive uteri. These and other observations have led to the suggestion that mucins serve an antiadhesive role and function to maintain a nonreceptive uterine state. A pan-species Muc-1 specific antibody recognizing a peptide motif conserved in the cytoplasmic domain of Muc-1 was used to examine the temporal and spatial expression of cell-associated Muc-1 in baboon uteri under a variety of conditions, including the pre- and perimplantation periods. Muc-1 expression was not driven by estrogen influences alone, but required progesterone action. In animals exposed to both steroids, Muc-1 was expressed at low moderate levels in epithelia of the basalis and functionalis regions. The highest expression of Muc-1 was detected in surface epithelium of the preimplantation phase, i.e., up to Day 8 (Day 0 = day of ovulation), or in ovariectomized animals receiving a steroid hormone regime that mimicked this phase (14 days of estrogen priming followed by 7 days of estrogen plus progesterone). Continued exposure to both hormones, i.e., as seen at Days 10-12 or in ovariectomized baboons given 14 days of estrogen plus progesterone treatment after estrogen priming, resulted in marked reduction of Muc-1 expression in the surface epithelium; however, staining patterns in the glandular epithelium were unchanged by this treatment. The expression of Muc-1 on the surface epithelium during the prereceptive phase was associated with the presence of both estrogen and progestin receptors in these epithelia. Muc-1 expression was reduced by neither antiestrogen treatment during the prereceptive stage nor antiprogestin treatment through to the receptive phase. Furthermore, persistent Muc-1 expression in the functionalis and basalis epithelium correlated with expression of progestin receptors. Thus, Muc-1 expression appeared to be progesterone-dependent rather than estrogen-dependent. It is concluded that Muc-1 expression in surface epithelium serves as a marker of the prereceptive phase in the baboon and that loss of Muc-1 from surface epithelium correlates with generation of a receptive uterine state.

Amino Acid Sequence↗

PubMed1996

Production and characterization of WEG-1, an epidermal growth factor/transforming growth factor-alpha-responsive mouse uterine epithelial cell line.

Uterine epithelial cells (UEC) isolated from 6-week-old CF-1 mice were immortalized using retroviral-mediated transfection of SV40 large T-antigen. One line, WEG-1, retained epithelial morphology and reacted with antibodies to cytokeratins 18, 19, laminin, fibronectin, and beta-catenin. In addition, WEG-1 cells displayed strong nuclear immunoreactivity to SV40 large T-antigen, confirming integration of the retrovirus vector and expression of this gene. WEG-1 cells were negative for nonepithelial markers such as desmin and factor 8. WEG-1 cells did not proliferate in serum-free medium; however, addition of 0.5% FBS supported proliferation to the same extent as 10% FBS. Addition of 50 ng/ml epidermal growth factor to medium containing 0.5% charcoal-stripped FBS restored proliferation comparable with 0.5% whole FBS. Epidermal growth factor or transforming growth factor-alpha (50 ng/ml), but not transforming growth factor-beta, leukemia-inhibiting factor, or fibroblast growth factor, induced the secretion of three proteins (M(r) approximately to 158K, 148K, and 36K). Comparison of protein secretions of WEG-1 cells and UEC showed shared as well as distinct bands. Like UEC, WEG-1 cells secreted PGF2a and PGE2 and expressed PG GH synthase-2. Unlike UEC, WEG-1 cells showed no apical/basal preference for either uptake of methionine or secretion of proteins. The absence of immunoreactive E-cadherin or zona occludens-1 was consistent with the absence of cell polarity in WEG-1 cells. Primary UEC, which polarize in vitro, do not support blastocyst attachment. WEG-1 cells, although not polarized in vitro, also exhibited delayed blastocyst attachment compared with nonuterine cell lines, suggesting that WEG-1 cells partially retained some aspects of UEC function relevant to embryo attachment. WEG-1 cells expressed messenger RNA for Muc-1, an UEC mucin suggested to have antiadhesive properties. Furthermore, WEG-1 cells did not display high affinity heparin binding sites, an activity associated with embryo attachment. WEG-1 cells may provide a model for studying various aspects of UEC function and murine embryo attachment.

Animals↗

PubMed1995

Regulation of multidrug resistance gene mdr1b/mdr1 expression in isolated mouse uterine epithelial cells.

The mammalian uterine epithelium (UE) undergoes drastic physiological and morphological changes during pregnancy. Steady-state levels of murine mdr1b mRNA, transcribed from a multidrug resistance gene encoding a membrane protein which functions as a transporter of lipophilic cytotoxic agents, are low in nonpregnant, cycling UE, but drastically increase (about 1,500- to 2,000-fold) at day 8 of gestation. At day 16 of gestation, levels of mdr1b mRNA are 2,500- to 3,000-fold higher than those in the cycling UE cells. Levels of mdr1b mRNA were elevated to levels comparable to those observed during pregnancy, in the UE of ovariectomized mice following 5-8 days of estrogen and progesterone administration. Withdrawal of these hormones resulted in a drastic reduction of mdr1b mRNA within 36 hr. These results suggested that steroid hormones alone can account for increased mdr1b mRNA expression and do not require the presence of other placenta/embryo-derived factors. Moreover, the hormonal effect on uterine mdr1b mRNA biosynthesis during pregnancy apparently is a delayed phenomenon. Nuclear run-on assays demonstrated that the rate of mdr1b transcription in UE cells prepared from 15-day pregnant mice (d-15 UE cells) was about two- to three-fold higher than that in nonpregnant UE cells. This increased transcription rate alone cannot account for mdr1b mRNA accumulation during pregnancy. mdr1b mRNA expression was investigated in primary cultures of d-15 UE cells. mdr1b mRNA levels decayed by 50% within 3-4 hr of culture and reached a steady-state 0.5-2% of initial levels by 24 hr. The rate of mdr1b mRNA decay in primary d-15 UE cells was decreased by treatment with alpha-amanitin or cycloheximide, suggesting that the decay pathway requires both transcription and de novo protein synthesis. Our results suggest that multiple mechanisms are involved in the maintenance of the high levels of mdr1b mRNA in pregnant UE cells. Furthermore, these data suggest that increased mRNA stability may contribute to the accumulation of mdr1b transcript during pregnancy.

Amanitins↗

PubMed1995

Expression and steroid hormonal control of Muc-1 in the mouse uterus.

Previous studies from our laboratory established that large M(r) mucin glycoproteins are major apically disposed components of mouse uterine epithelial cells in vitro. The present studies demonstrate that Muc-1 represents one of the apically disposed mucin glycoproteins of mouse uterine epithelia, and that Muc-1 protein and messenger RNA (mRNA) expression are regulated in the periimplantation mouse uterus by ovarian steroids. Muc-1 expression is exclusive to the epithelial cells of the uterus under all conditions examined. Muc-1 expression is high in the proestrous and estrous stages and decreases during diestrous. Both Muc-1 protein and mRNA decline to barely detectable levels by day 4 of pregnancy, i.e. before the time of blastocyst attachment. In contrast, Muc-1 expression in the cervix and vagina is maintained during this same period. Delayed implantation was established in pregnant mice by ovariectomy and maintained by the administration of exogenous progesterone (P). Initiation of implantation was triggered by coinjection of P-maintained mice with a nidatory dose of 17 beta-estradiol (E2). Muc-1 levels in the uterine epithelia of P-maintained mice declined to low levels similar to those observed on day 4 of normal pregnancy. Coinjection of E2 did not alter Muc-1 expression, suggesting that down-regulation of Muc-1 is a P-dominated event. This was confirmed in ovariectomized nonpregnant mice, which displayed stimulation of Muc-1 expression after 6 h of E2 injection. E2-Stimulated Muc-1 expression was inhibited by the pure antiestrogen, ICI 164,384. Although P alone had no effect on Muc-1 expression, it antagonized the action of E2. Injection of pregnant mice with the antiprogestin, RU486, a known implantation inhibitor, on day 3 of pregnancy restored high level expression of Muc-1 mRNA on day 4, indicating that down-regulation of Muc-1 is P receptor mediated. Collectively, these data indicate that Muc-1 expression in mouse uterine epithelium is strongly influenced by ovarian steroids. It is suggested that the loss of Muc-1 contributes to generation of a receptive uterine state.

Animals↗

PubMed1994

Regulated expression of prostaglandin endoperoxide synthase-2 by uterine stroma.

Our previous studies demonstrated that interleukin-1 alpha (IL-1 alpha) and soluble factors secreted by polarized uterine luminal epithelial cells (UEC) stimulate prostaglandin (PG) secretion by uterine stromal cells (USC). The present studies were aimed at determining the mechanism by which these agonists stimulate PG secretion by USC. The comoplete inhibition of IL-1 alpha- and UEC-induced PGE2 secretion by cycloheximide and actinomycin-D in the presence of a saturating concentration of arachidonic acid indicated that IL-1 alpha and UEC act to a large extent by inducing de novo expression of PG endoperoxide synthase (PGHS). Western blot analysis of membrane fractions from USC showed a 2- to 4-fold accumulation of the mitogen-inducible isoform of PGHS (PGHS-2), but not the constitutively expressed enzyme (PGHS-1), within 3 h of treatment with IL-1 alpha, UEC-conditioned medium, or serum. Inhibition of UEC-stimulated PGHS-2 expression by anti-IL-1 alpha indicated that IL-1 alpha is one factor secreted by UEC responsible for the synthesis of USC PGHS-2. Expression of PGHS-2, but not PGHS-1, was inhibited by dexamethasone. Dexamethasone also inhibited IL-1 alpha- and UEC-stimulated PGE2 secretion by USC. Immunohistochemical studies demonstrated that PGHS-2 is localized to implantation sites in newly differentiating USC at the time of blastocyst attachment, indicating a potential physiological role for PGHS-2 in early stages of mouse implantation. In contrast, PGHS-1 was localized to UEC during this period. Collectively, these results indicate that enhanced PG secretion by USC in response to IL-1 alpha and soluble factors secreted by UEC is due to selective expression of PGHS-2. In addition, the expression of PGHS-2 by USC in vivo during the periimplantation period may support PG secretion required during early stages of embryo implantation.

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