Expression of the transmembrane mucins, MUC1, MUC4 and MUC16, in normal endometrium and in endometriosis.

STUDY QUESTION: Are the transmembrane mucins, MUC1, MUC4 and MUC16, differentially expressed in endometriosis compared with normal endometrium? SUMMARY ANSWER: This study revealed that transmembrane mucin expression does not vary significantly in normal endometrium during the menstrual cycle and is not altered in endometriosis relative to the epithelial marker, cytokeratin-18 (KRT18). WHAT IS KNOWN ALREADY: Increased serum levels of the transmembrane mucin fragments MUC1, MUC4 and MUC16 that normally dominate the apical surface of simple epithelia are found in several pathological conditions, including endometriosis. Altered mucin expression in gynecologic diseases may promote infertility or endometrial pathologies. STUDY DESIGN, SIZE, DURATION: This was a laboratory-based study of samples from 12 endometriosis patients as well as non-endometriosis control samples obtained from 31 patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Total RNA was isolated from endometrial biopsies of ectopic and eutopic endometrium from women with endometriosis and control patients from different stages of the menstrual cycle. Quantitative (q)-RT-PCR analyses were performed for the mucins, MUC1, MUC4 and MUC16, relative to the epithelial marker, cytokeratin-18 (KRT18), or ß-actin (ACTB). Frozen sections from endometrial biopsies of proliferative and mid-secretory stage women with endometriosis were immunostained for MUC1, MUC4 and MUC16. MAIN RESULTS AND THE ROLE OF CHANCE: qRT-PCR analyses of MUC1 and MUC16 mRNA revealed that these mucins do not vary significantly during the menstrual cycle nor are they altered in women with endometriosis relative to the epithelial marker, KRT18. MUC4 mRNA is expressed at very low levels relative to MUC1 and MUC16 under all conditions. There was little difference in MUC1 and MUC16 expression between eutopic endometrial and ectopic endometriotic tissues. MUC4 expression also was not significantly higher in the ectopic endometriotic tissues. Immunostaining for all three mucins reveals robust expression of MUC1 and MUC16 at the apical surfaces of endometrial epithelia, but little to no staining for MUC4. LIMITATIONS, REASONS FOR CAUTION: qRT-PCR analysis was the main method used for mucin detection. Additional studies with stage III-IV endometriotic tissue would be useful to determine if changes in MUC1 and MUC16 expression occur, or if MUC4 expression increases, at later stages of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: We report a comprehensive comparative profile of the major transmembrane mucins, MUC1, MUC4 and MUC16, relative to the epithelial marker, KRT18, in normal cycling endometrium and in endometriosis, and indicate constitutive expression. Previous studies have profiled the expression of individual mucins relative to ß-actin and indicate accumulation in the luteal phase. Thus, these differences in interpretation appear to reflect the increased epithelial content of endometrium during the luteal phase. STUDY FUNDING: This study was supported by: NIH R01HD29963 to D.D.C.; NIH U54HD007495 to S.M.H.; and NIH R01HD067721 to S.L.Y. and B.A.L. The authors have no competing interests to declare.

MUC1 and Polarity Markers INADL and SCRIB Identify Salivary Ductal Cells.

Current treatments for xerostomia/dry mouth are palliative and largely ineffective. A permanent clinical resolution is being developed to correct hyposalivation using implanted hydrogel-encapsulated salivary human stem/progenitor cells (hS/PCs) to restore functional salivary components and increase salivary flow. Pluripotent epithelial cell populations derived from hS/PCs, representing a basal stem cell population in tissue, can differentiate along either secretory acinar or fluid-transporting ductal lineages. To develop tissue-engineered salivary gland replacement tissues, it is critical to reliably identify cells in tissue and as they enter these alternative lineages. The secreted protein α-amylase, the transcription factor MIST1, and aquaporin-5 are typical markers for acinar cells, and K19 is the classical ductal marker in salivary tissue. We found that early ductal progenitors derived from hS/PCs do not express K19, and thus earlier markers were needed to distinguish these cells from acinar progenitors. Salivary ductal cells express distinct polarity complex proteins that we hypothesized could serve as lineage biomarkers to distinguish ductal cells from acinar cells in differentiating hS/PC populations. Based on our studies of primary salivary tissue, both parotid and submandibular glands, and differentiating hS/PCs, we conclude that the apical marker MUC1 along with the polarity markers INADL/PATJ and SCRIB reliably can identify ductal cells in salivary glands and in ductal progenitor populations of hS/PCs being used for salivary tissue engineering. Other markers of epithelial maturation, including E-cadherin, ZO-1, and partition complex component PAR3, are present in both ductal and acinar cells, where they can serve as general markers of differentiation but not lineage markers.

Vectorial secretion of proteoglycans by polarized rat uterine epithelial cells.

We have studied proteoglycan secretion using a recently developed system for the preparing of polarized primary cultures of rat uterine epithelial cells. To mimic their native environment better and provide a system for discriminating apical from basolateral compartments, we cultured cells on semipermeable supports impregnated with biomatrix. Keratan sulfate proteoglycans (KSPG) as well as heparan sulfate-containing molecules (HS[PG]) were the major sulfated products synthesized and secreted by these cells. The ability of epithelial cells to secrete KSPG greatly increased in parallel with the development of cell polarity. Furthermore, KSPG secretion occurred preferentially to the apical medium in highly polarized cultures. In contrast, HS(PG) secretion did not increase along with development of polarity, although most HS(PG) (85%) were secreted apically as well. Pulse-chase studies indicated that highly polarized cultures secreted 80-90% of the sulfated macromolecules they synthesized, predominantly to the apical secretory compartment. The half-lives for KSPG and HS(PG) secretion were approximately 3 and 4 h, respectively. Parallel studies of cells cultured on tissue culture plastic-coated with biomatrix indicated that neither the state of confluency nor the biomatrix was primarily responsible for inducing the KSPG secretion observed in polarizing cultures. Experiments with uterine strips indicated that the steroid hormone, 17-beta-estradiol, markedly stimulated synthesis and secretion of sulfated macromolecules, but had no preferential effect on KSPG production. The ratio of KSPG to HS(PG) secretion from uterine strips was similar to that found in the apical medium of highly polarized cell cultures. Thus, the pattern of proteoglycan secretion observed in polarized cell cultures mimicked that observed for uterine cells, although the preferential increase in KSPG production by polarized cells could not be attributed to an estrogen response. Collectively, these studies describe the major sulfated molecules secreted by rat uterine epithelial cells under varying conditions and provide evidence for a novel influence of cell polarity on the cell's ability to secrete sulfated glycoconjugates.

Development of morphological and functional polarity in primary cultures of immature rat uterine epithelial cells.

The present study describes a culture environment in which luminal epithelial cells isolated from immature rat uteri and cultured on a matrix-coated permeable surface, with separate apical and basal secretory compartments, proliferate to confluence. Subsequently the cells undergo a process of differentiation accompanied by progressive development of functional polarity. Ultrastructural and immunocytochemical evidence verifies the ability of these primary cultures to regain polar organization, separate membrane domains, and form functional tight junctions as demonstrated by the development of transepithelial resistance. The appearance of uvomorulin is restricted to the lateral cell surface. Coordinated indices of functional polarity that develop progressively in post-confluent cultures include the preferential uptake of [35S]methionine from the basal surface and a rise in uterine epithelial cell secretory activity characterized by a progressive preference for apical secretion. The time dependent development of polarity was characterized by differences in the protein profiles of the apical and basolateral secretory compartments. The maintenance of hormone responsiveness by the cultured cells was validated by the secretion of two proteins identified as secretory markers of estrogen response in the intact uterus. The technique of culturing the cells on a matrix-coated permeable surface with separate secretory compartments produces a uterine epithelial cell that morphologically and functionally resembles its in situ equivalent. The culture method and analytical approach used in this present study may be applied to primary cultures of a variety of natural epithelia, which have hitherto proven resistant to more conventional culture methodologies.

Expression of the heparan sulfate proteoglycan, perlecan, during mouse embryogenesis and perlecan chondrogenic activity in vitro.

Expression of the basement membrane heparan sulfate proteoglycan (HSPG), perlecan (Pln), mRNA, and protein has been examined during murine development. Both Pln mRNA and protein are highly expressed in cartilaginous regions of developing mouse embryos, but not in areas of membranous bone formation. Initially detected at low levels in precartilaginous areas of d 12.5 embryos, Pln protein accumulates in these regions through d 15.5 at which time high levels are detected in the cartilage primordia. Laminin and collagen type IV, other basal lamina proteins commonly found colocalized with Pln, are absent from the cartilage primordia. Accumulation of Pln mRNA, detected by in situ hybridization, was increased in d 14.5 embryos. Cartilage primordia expression decreased to levels similar to that of the surrounding tissue at d 15.5. Pln accumulation in developing cartilage is preceded by that of collagen type II. To gain insight into Pln function in chondrogenesis, an assay was developed to assess the potential inductive activity of Pln using multipotential 10T1/2 murine embryonic fibroblast cells. Culture on Pln, but not on a variety of other matrices, stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stained intensely with Alcian blue and collagen type II antibodies. mRNA encoding chondrocyte markers including collagen type II, aggrecan, and Pln was elevated in 10T1/2 cells cultured on Pln. Human chondrocytes that otherwise rapidly dedifferentiate during in vitro culture also formed nodules and expressed high levels of chondrocytic marker proteins when cultured on Pln. Collectively, these studies demonstrate that Pln is not only a marker of chondrogenesis, but also strongly potentiates chondrogenic differentiation in vitro.

A calcium-binding, asparagine-linked oligosaccharide is involved in skeleton formation in the sea urchin embryo.

We have previously identified a 130-kD cell surface protein that is involved in calcium uptake and skeleton formation by gastrula stage embryos of the sea urchin Strongylocentrotus purpuratus (Carson et al., 1985. Cell. 41:639-648). A monoclonal antibody designated mAb 1223 specifically recognizes the 130-kD protein and inhibits Ca+2 uptake and growth of the CaCO3 spicules produced by embryonic primary mesenchyme cells cultured in vitro. In this report, we demonstrate that the epitope recognized by mAb 1223 is located on an anionic, asparagine-linked oligosaccharide chain on the 130-kD protein. Combined enzymatic and chemical treatments indicate that the 1223 oligosaccharide contains fucose and sialic acid that is likely to be O-acetylated. Moreover, we show that the oligosaccharide chain containing the 1223 epitope specifically binds divalent cations, including Ca+2. We propose that one function of this negatively charged oligosaccharide moiety on the surfaces of primary mesenchyme cells is to facilitate binding and sequestration of Ca+2 ions from the blastocoelic fluid before internalization and subsequent deposition into the growing CaCO3 skeleton.

Heparanase expression and activity influences chondrogenic and osteogenic processes during endochondral bone formation.

Endochondral bone formation is a highly orchestrated process involving coordination among cell-cell, cell-matrix and growth factor signaling that eventually results in the production of mineralized bone from a cartilage template. Chondrogenic and osteogenic differentiation occur in sequence during this process, and the temporospatial patterning clearly requires the activities of heparin binding growth factors and their receptors. Heparanase (HPSE) plays a role in osteogenesis, but the mechanism by which it does so is incompletely understood. We used a combination of ex vivo and in vitro approaches and a well described HPSE inhibitor, PI-88 to study HPSE in endochondral bone formation. In situ hybridization and immunolocalization with HPSE antibodies revealed that HPSE is expressed in the peri-chondrium, peri-osteum, and at the chondro-osseous junction, all sites of key signaling events and tissue morphogenesis. Transcripts encoding Hpse also were observed in the pre-hypertrophic zone. Addition of PI-88 to metatarsals in organ culture reduced growth and suggested that HPSE activity aids the transition from chondrogenic to osteogenic processes in growth of long bones. To study this, we used high density cultures of ATDC5 pre-chondrogenic cells grown under conditions favoring chondrogenesis or osteogenesis. Under chondrogenic conditions, HPSE/Hpse was expressed at high levels during the mid-culture period, at the onset of terminal chondrogenesis. PI-88 addition reduced chondrogenesis and accelerated osteogenesis, including a dramatic up-regulation of osteocalcin levels. In normal growth medium, addition of PI-88 reduced migration of ATDC-5 cells, suggesting that HPSE facilitates cartilage replacement by bone at the chondro-osseous junction by removing the HS component of proteoglycans, such as perlecan/HSPG2, that otherwise prevent osteogenic cells from remodeling hypertrophic cartilage.

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.

Extracellular matrix and androgen receptor expression associated with spontaneous transformation of rat prostate fibroblasts.

Spontaneous transformation in continuous culture of the androgen-sensitive rat prostate fibroblast cell line, NbF-1, resulted in an aggressively tumorigenic nonmetastatic phenotype that coincided with few gross chromosome abnormalities. This study identified transformation-associated alterations in extracellular matrix and androgen receptor expression in the NbF-1 cell line. Substantial levels of procollagens I, III, and IV and fibronectin mRNAs were detected in nontumorigenic NbF-1 cells. Laminin B1 and B2 mRNAs were also detectable, but at lower levels. Expression of all six extracellular matrix mRNAs was nonuniformly lower in tumorigenic NbF-1 cells. This decrease in expression was greatest for alpha 2 procollagen IV mRNA, which was reduced 17-fold. Proteoglycans and glycosaminoglycans synthesized by the NbF-1 cultures were also characterized. The NbF-1 cell line expressed chondroitin sulfate proteoglycans predominantly, and expression was reduced 5- to 10-fold in tumorigenic cultures. In contrast to the extensive alterations in the extracellular matrix, measurement of high-affinity androgen binding and androgen receptor mRNA levels showed substantial expression of androgen receptors in both NbF-1 cultures. Cultures of early and late passage NbF-1 cells demonstrated a mitogenic response to dihydrotestosterone. These data indicate (a) that alterations in expression of extracellular matrix components may represent early markers for tumorigenic transformation in prostatic mesenchymal cells and (b) that these changes can occur without disrupting androgen receptor expression and androgen sensitivity.

Neurotrophin stimulation of human melanoma cell invasion: selected enhancement of heparanase activity and heparanase degradation of specific heparan sulfate subpopulations.

Heparanase is an endo-beta-D-glucuronidase, the enzymatic targets of which are the glycosaminoglycan chains of heparan sulfate proteoglycans. Elevated levels of heparanase are associated with the metastatic potential of melanoma cells. Treatment of murine and human melanoma cells with the prototypic neurotrophin nerve growth factor (NGF) increases the production of heparanase by melanoma cells. We reported previously that physiological concentrations of NGF increased in vitro Matrigel invasion of early-passage human brain-metastatic 70W melanoma cells but not melanoma cells metastatic to other sites or nonmetastatic melanoma cells. Here we found that treatment of 70W melanoma cells with neurotrophin NT-3 increased Matrigel invasion, whereas treatment with neurotrophins other than NGF or NT-3 did not influence invasion. Mutants of NGF that do not bind to the neurotrophin receptor p75NTR or other nonneuronal growth factors were not able to enhance the invasion of 70W melanoma cells. When 70W cells were exposed to antisense oligonucleotides directed against p75NTR mRNA, there was a reduction in NGF and NT-3 binding, and the neurotrophins failed to enhance Matrigel invasion. To study the properties of heparanase in NT-regulated malignant melanoma invasive processes, we developed a sensitive heparanase assay consisting of purified [35S]heparan sulfate subpopulations separated by agarose gel electrophoresis. Incubation of 70W cells with NGF or NT-3, but not brain-derived NT factor, NT-4/5, or mutant NGF, resulted in increased release of heparanase activity that was capable of degrading a subpopulation of heparan sulfate molecules.

Perlecan domain I promotes fibroblast growth factor 2 delivery in collagen I fibril scaffolds.

Perlecan, a heparan sulfate proteoglycan, is widely distributed in developing and adult tissues and plays multiple, important physiological roles. Studies with knockout mouse models indicate that expression of perlecan and heparan sulfate is critical for proper skeletal morphogenesis. Heparan sulfate chains bind and potentiate the activities of various growth factors such as fibroblast growth factor 2 (FGF-2). Previous studies indicate that important biological activities are associated with the heparan sulfate-bearing domain I of perlecan (PlnDI; French et al. J. Bone Miner. Res. 17 , 48, 2002). In the present study, we have used recombinant, glycosaminoglycan-bearing PlnDI to reconstitute three-dimensional scaffolds of collagen I. Collagen I fibrils bound PlnDI much better than native collagen I monomers or heat-denatured collagen I preparations. Heparitinase digestion demonstrated that recombinant PlnDI was substituted with heparan sulfate and that these heparan sulfate chains were critically important not only for efficient integration of PlnDI into scaffolds, but also for FGF-2 binding and retention. PlnDI-containing collagen I scaffolds to which FGF-2 was bound sustained growth of both MG63, an osteoblastic cell line, and human bone marrow stromal cells (hBMSCs) significantly better than scaffolds lacking either PlnDI or FGF-2. Collectively, these studies demonstrate the utility of PlnDI in creating scaffolds that better mimic natural extracellular matrices and better support key biological activities.

MUC1 expression in human prostate cancer cell lines and primary tumors.

MUC1 expression was evaluated in normal prostate epithelial cells (PrEC), and prostate cancer cell lines in response to dihydrotestosterone (DHT), interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) treatment. Expression of MUC1 core protein was stimulated in PrEC and PC-3 cells after cytokine treatment, but was highly and constitutively expressed by DU-145 cells. MUC1 was not expressed by LNCaP, C4-2 or C4-2B cells under any condition. DHT alone or in combination with cytokines had no effect on MUC1 expression in any cell line tested. Using antibodies capable of detecting all isoforms of MUC1 core protein independent of their glycosylation state, immunohistochemical staining of tissue microarrays containing both nontumor and tumor tissue revealed that only 17% of tumor tissues and 41% of nontumor tissues stained positively for MUC1. Staining patterns in tumor tissue varied from focal apical staining to diffuse cytoplasmic staining. Neither the presence of MUC1 core protein nor its subcellular distribution correlated with Gleason grade. These data indicate that MUC1 is a poor marker of prostate cancer progression. Furthermore, IFN-gamma and TNF-alpha strongly induce MUC1 expression in both normal prostate epithelia and certain prostate tumor cell lines and may exacerbate pathologies associated with MUC1-positive prostate cancers.

Cytoskeletal abnormalities in chondrocytes with EXT1 and EXT2 mutations.

The EXT genes are a group of putative tumor suppressor genes that previously have been shown to participate in the development of hereditary multiple exostoses (HME), HME-associated and isolated chondrosarcomas. Two HME disease genes, EXT1 and EXT2, have been identified and are expressed ubiquitously. However, the only known effect of mutations in the EXT genes is on chondrocyte function as evidenced by aberrant proliferation of chondrocytes leading to formation of bony, cartilage-capped projections (exostoses). In this study, we have characterized exostosis chondrocytes from three patients with HME (one with EXT1 and two with EXT2 germline mutations) and from one individual with a non-HME, isolated exostosis. At the light microscopic level, exostosis chondrocytes have a stellate appearance with elongated inclusions in the cytoplasm. Confocal and immunofluorescence of in vitro and in vivo chondrocytes showed that these massive accumulations are composed of actin bundled by 1.5-microm repeat cross-bridges of alpha-actinin. Western blot analysis shows that exostosis chondrocytes from two out of three patients aberrantly produce high levels of muscle-specific alpha-actin, whereas beta-actin levels are similar to normal chondrocytes. These findings suggest that mutations in the EXT genes cause abnormal processing of cytoskeleton proteins in chondrocytes.

Uterine epithelial cell secretion of interleukin-1 alpha induces prostaglandin E2 (PGE2) and PGF2 alpha secretion by uterine stromal cells in vitro.

Uterine epithelial cells (UEC) were isolated from cycling mice and cultured on Matrigel-coated nitrocellulose filters to determine their ability to secrete interleukin-1 alpha (IL-1 alpha) in response to ovarian steroids and induce prostaglandin (PG) secretion by uterine stromal cells (USC). UEC cultured in a polarized manner secreted IL-1 alpha with an 8- to 10-fold apical vs. basal preference, as determined by an enzyme-linked immunosorbent assay. There was no effect of 17 beta-estradiol, progesterone, or 17 beta-estradiol plus progesterone on IL-1 alpha secretion by UEC. The mean total IL-1 alpha secreted to the apical and basal secretory compartments over the 24-h incubation period was 0.8 +/- 0.16 and 0.07 +/- 0.05 ng/2 x 10(5) cells, respectively. Cytokine bioactivity, as determined by [3H]thymidine incorporation into D10 cells in response to UEC-conditioned medium, paralleled the pattern of IL-1 alpha secretion observed using the immunoassay. In addition to the in vitro secretion of IL-1 alpha by polarized UEC, pooled uterine fluid collected from proestrous stage mice contained IL-1 alpha at a concentration of 0.7 ng/ml, indicating that IL-1 alpha is released into the uterine lumen in vivo. Coculture with UEC or treatment with conditioned medium from either the apical or basal UEC secretory compartments induced a several-fold increase in the secretion of PGE2 and PGF2 alpha by USC. Relative to untreated USC, PGE2 was induced to a greater extent than PGF2 alpha. The addition of polyclonal anti-IL-1 alpha significantly inhibited the ability of UEC-conditioned medium and UEC coculture to induce PG secretion by USC. In addition, mouse recombinant IL-1 alpha added at a concentration similar to that secreted by UEC stimulated USC PGE2 and PGF2 alpha secretion in a manner similar to that observed with UEC coculture. Experiments designed to determine the cell type specificity of the induction of PG secretion by USC indicated that conditioned medium from a human UEC line (RL95), a rat prostate epithelial cell line (E4), and a mouse fibroblast cell line (10T1/2) induced PG secretion to an extent that paralleled their ability to induce D10 cell proliferation. The present results demonstrate the ability of UEC to secrete IL-1 alpha in a vectorial manner. Soluble products secreted by UEC are capable of stimulating PGE2 and PGF2 alpha secretion by USC, and IL-1 alpha appears to be a significant factor contributing to this effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Mouse uterine stromal cells secrete a 30-kilodalton protein in response to coculture with uterine epithelial cells.

While uterine stromal cells (USC) appear to modify the function of uterine epithelial cells (UEC) under certain conditions in vivo, relatively little is known about the effect of epithelial cells on stromal cell differentiation and function. To determine if UEC modulate USC function in vitro, highly enriched (> 95%) cultures of polarized UEC were first cultured on Matrigel-coated filters in serum-free medium until confluent, then cocultured with USC for up to 120 h. Subsequently, while maintaining both cell types in physically separate compartments, filters containing UEC were removed, and USC phenotypic markers assayed. Coculture with UEC did not affect the expression of two markers of USC differentiation (desmin and laminin), USC DNA content, [35S]methionine uptake, or total protein synthesis or secretion. However, coculture of USC with UEC or medium conditioned by UEC induced the secretion of a 30-kilodalton protein (p30) from USC as early as 24 h of coculture and through 120 h of coculture. In addition, secretion of a 60-kilodalton protein by USC was frequently observed in response to coculture with UEC. Neither the hormonal stage from which uterine cells were recovered, nor the addition of exogenous progesterone or estradiol modulated UEC-induced p30 secretion. Several purified growth factors (transforming growth factor-beta, epidermal growth factor, interleukin-1 alpha, and fibroblast growth factor) added to the serum-free culture medium failed to induce p30 secretion by USC. The p30-inducing activity in UEC-conditioned medium could not be abolished by either heat or trypsin treatment, suggesting that it is not a protein. Purified prostaglandin E2 or F2 alpha or platelet-activating factor did not induce p30 secretion by isolated USC. Of several epithelial and fibroblastic cell lines tested, UEC and a human uterine adenocarcinoma cell line (RL95-2) were the most effective in inducing p30 secretion by USC. Moreover, UEC also were able to modulate protein secretion by nonuterine murine fibroblast cell lines. Collectively, these data demonstrate that UEC can modulate USC function in vitro via a soluble factor(s).

Polarized rat uterine epithelium in vitro: constitutive expression of estrogen-induced proteins.

The hormonal responsiveness of immature rat primary uterine epithelial (UE) cells, cultured in a serum-free, phenol red-free defined medium, was examined under conditions which allowed the UE cells to reestablish their polarized phenotype. In the absence of estradiol and phenol red UE cells proliferated to confluence, achieving cell densities equal to those reached by UE cells cultured in the presence of estradiol. The expression of marker proteins, characteristic of the in vivo response of the uterus to estrogen, i.e. the adhesion molecule cell CAM 105, complement component C3, the secretory component of the immunoglobulin A receptor, and keratan sulfate proteoglycan, by polarized cultures of UE cells proved to be independent of estrogen in vitro. Polarized UE cells required the presence of estrogen to maintain integrity of their monolayer and did exhibit a dose-dependent response to estradiol in vitro in terms of cell growth (hypertrophy) and the secretion of two proteins not previously described as estrogen response markers. UE cell secretion, in particular apical secretion, was stimulated by estradiol but not by progesterone, dexamethasone, or testosterone. Progesterone failed to down-regulate the polarized UE cell responses to estradiol. Collectively, these observations suggest that many of the responses which nominally characterize the action of estrogen on the UE cell in vivo are likely to be initiated by agents other than estrogen, e.g. growth factors.

Polarized rat uterine epithelium in vitro: responses to estrogen in defined medium.

Previously described procedures for the culture of immature rat uterine epithelium (UE) allowed the cells to proliferate to confluence and develop morphological and functional polarity. The present study describes the transition from culture of UE cells in serum to a serum-free defined medium. This was accomplished with no significant alteration in the ability of UE cells to attain morphological and functional polarity. In defined medium, which contained estrogen (2.5 x 10(-9) M), UE cells proliferated to confluence, demonstrated separation of apical and basal plasma membrane domains, and displayed preferential secretion of proteins and proteoglycans from the apical surface. Apical secretions of polarized cultures contained complement component C3, the secreted portion of the immunoglobulin A receptor and the secretory glycoprotein, USP-1. The cell surface adhesion molecule, CAM 105, could be demonstrated at the apical cell surface. Expression of this profile of secretory and cell surface markers, representative of the in vivo estrogen response of immature rat UE cells, correlated with an in vitro state of non-receptivity of polarized UE cells toward blastocysts which remained viable and competent to attach. We conclude that the polarized UE cell that develops in the described defined medium expresses a phenotype similar to that which characterizes the in vivo uterine estrogen response.

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.

Regulation of N-linked glycoprotein assembly in uteri by steroid hormones.

The effects of the steroid hormones 17 beta-estradiol (E2) and progesterone on N-linked glycoprotein assembly in ovariectomized mice have been examined. Both priming and nidatory E2 markedly stimulate [3H]mannose incorporation (3- to 6-fold) into uterine glycoproteins, whereas uterine bulk protein synthesis is not stimulated under the same conditions. Progesterone alone stimulates glycoprotein synthesis modestly (1.5-fold) over that in oil-injected controls, but antagonizes the action of E2 when coinjected with the estrogen. The E2 effect is not systemic, because livers from these same animals do not display an increase in glycoprotein synthesis. When mice were injected with tamoxifen or clomiphene, two drugs that mimic E2 actions in uteri without inducing the full extent of cell proliferation that normally accompanies E2 treatment, a similar enhancement of uterine glycoprotein synthesis was observed. Although mannosylphosphoryldolichol synthase activity rose in parallel with glycoprotein synthesis during E2 priming, the apparent activities of two other enzymes involved in the assembly of N-linked glycoproteins, namely chitobiosylpyrophosphoryldolichol synthase and oligosaccharyltransferase, remained relatively unchanged. Furthermore, the apparent in vivo rate of dolichol phosphorylation was not altered during E2 priming. Supplementation of uterine tissue slices with dolichylphosphate failed to enhance the rate of protein glycosylation in vivo. In addition, changes in the pool sizes of GDP-mannose did not correlate with changes in the in vivo rate of glycoprotein synthesis. Collectively, these observations indicate that the E2-dependent increase in glycoprotein synthesis is not likely to be due to increased enzyme activities for oligosaccharide assembly or transfer to protein, increased dolichylphosphate availability, or increased sugar nucleotide availability. To study the effects of E2 on the production of specific glycoproteins, the pattern of [3H]mannose-labeled glycoproteins produced as a function of days of E2 priming was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estrogen priming induced the secretion of 9-11 [3H]mannose-labeled glycoproteins by uteri; however, the pattern of tissue-associated glycoproteins remained constant throughout this interval. It appears, therefore, that estrogen priming induces the secretion of a few specific glycoproteins while generally enhancing the production of most tissue-associated glycoproteins. Most (70%) of the [3H]mannose-labeled oligosaccharide chains of these glycoproteins were of the polymannose type.(ABSTRACT TRUNCATED AT 400 WORDS)

Steroid hormones differentially modulate glycoconjugate synthesis and vectorial secretion by polarized uterine epithelial cells in vitro.

Characterization of glycoconjugates synthesized by polarized immature rabbit uterine epithelial (UE) cells in vitro, their vectorial patterns of secretion, and regulation by ovarian steroid hormones are reported. Large (mol wt, greater than 230 kDa) sialomucoglycoproteins and hyaluronate were primarily (86-96%) secreted from the apical cell surface domain, while heparan sulfate proteoglycans were predominantly secreted from the basal cell surface of the polarized UE cells. The polarized UE cells responded to estrogen and progesterone in vitro and exhibited distinct profiles in their synthesis and secretion of different glycoconjugates. Progesterone and/or estrogen reduced the secretion of the mucosialoglycoproteins; however, progesterone caused a 4- to 5-fold accumulation of mucosialoglycoproteins in the cell-associated fraction, suggesting regulation by the hormone at the level of secretion, rather than synthesis. Estrogen and progesterone both stimulated the synthesis and secretion of hyaluronate by the polarized UE cells. Neither hormone substantially altered the synthesis or secretory pattern of heparan sulfate proteoglycans. Collectively, these studies provide the first comprehensive characterization of the major glycoconjugates synthesized and secreted by rabbit UE cells. Furthermore, these observations demonstrate marked differential direct influences of steroid hormones on the production of distinct classes of UE cell glycoconjugates.