Biomedical subjects

D D Carson

Publications and source records attributed to D D Carson.

At least 55 records · Page 3Linked to original sources

PubMed1994

Tenascin is induced at implantation sites in the mouse uterus and interferes with epithelial cell adhesion.

Expression of tenascin, an extracellular matrix protein associated with morphogenetic events and altered states of cellular adhesion, was examined in mouse uterus during the peri-implantation period. A uniform low level expression of tenascin was detected in stromal extracellular matrix during the estrous cycle and days 1 through 4 of early pregnancy. During the period of blastocyst attachment (day 4.5), an intense deposition of tenascin fibrils was located in the extracellular matrix of stroma immediately subjacent to the uterine epithelium surrounding the attaching blastocyst. This localized intensity of tenascin expression was both spatially and temporally restricted. By day 5.5, differentiation of stroma in the immediate area around the embryo to form the primary decidual zone was accompanied by a reduced amount of tenascin expression in the form of fragmented fibrils. Tenascin also could be induced by an artificial stimulus in uterine stroma of mice that had been hormonally prepared for implantation. The ability of artificial stimuli to induce tenascin expression suggested that the tenascin-inducing signals were derived from uterine cells, presumably lumenal epithelium, rather than embryonic cells. Consistent with this, conditioned medium from primary cultures of uterine epithelium was found to induce tenascin expression (2- to 4-fold) in isolated uterine stroma. Artificial stimuli generated a temporal pattern of tenascin expression similar to that observed during early pregnancy; however, in the artificially induced model, tenascin was induced in stroma immediately subjacent to lumenal epithelium along the entire length of the uterus. Purified tenascin and a recombinant tenascin fragment consisting of alternatively spliced fibronectin type III repeats, interfered with maintenance of uterine epithelial cell adhesion to Matrigel. In contrast, other recombinant tenascin fragments or fibronectin had no effect in this regard. Tenascin had no effect on adhesion of uterine stroma. Collectively, these results suggest that stimulation of TN expression in stromal extracellular matrix in vivo occurs via hormonally regulated, epithelial-mesenchymal interactions and serves as an early marker for uterine receptivity and the attachment phase of implantation. Furthermore, tenascin may facilitate embryo penetration by disrupting uterine epithelial cell adhesion to underlying basal lamina.

Animals↗

PubMed1993

Heparin-like glycosaminoglycans participate in binding of a human trophoblastic cell line (JAR) to a human uterine epithelial cell line (RL95).

In vitro studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation. In order to investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were used to model uterine epithelium and embryonal trophectoderm, respectively. A heterologous cell-cell adhesion assay was developed to determine if binding of JAR cells to RL95 cells was heparan sulfate-dependent. Labeled, single cell suspensions of JAR cells attached to confluent monolayers of RL95 cells in a dose- and time-dependent manner. Heparin-like glycosaminoglycans and JAR cell proteoglycans competitively inhibited JAR cell adhesion to RL95 cells by 50% or more. A panel of chemically modified heparins were used to demonstrate that O-sulfation and amino group substitution were critical for inhibition of cell-cell adhesion. Treatment with chlorate, an inhibitor of ATP-sulfurylase, resulted in a 56% reduction in cell-cell binding compared to untreated controls. Heparinase and chondroitinase ABC markedly inhibited JAR-RL95 binding, while chondroitinase AC had no significant effect. These observations indicated that HSPGs as well as dermatan sulfate-containing proteoglycans participated in cell-cell binding. Collectively, these results indicate that initial binding interactions between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAGs) with heparin-like properties (i.e., heparan sulfate and dermatan sulfate). These observations are consistent with an important role for HS and heparin-like GAGs as well as their corresponding binding sites in early stages of human trophoblast-uterine epithelial cell binding.

Cell Adhesion↗

PubMed1993

Heparan sulfate proteoglycan (perlecan) expression by mouse embryos during acquisition of attachment competence.

Cell surface expression of the basement membrane form of heparan sulfate proteoglycan (HSPG), perlecan, basic fibroblast growth factor (bFGF) binding forms of heparan sulfate, and laminin has been examined by immunofluorescence during the attachment and outgrowth phases of mouse embryo development in vitro and in utero. The results indicate that both HSPG and laminin are expressed at the exterior surface of the trophectoderm during the attachment phase of implantation in vivo. HSPG expression correlated with acquisition of attachment competence by embryos in vitro. The intensity of immunofluorescence signal appeared to decrease by the outgrowth stage of development in vitro. It is concluded that expression of HSPG at the trophectodermal surface is coordinated with development of attachment competence by mouse embryos in vitro and in utero.

Animals↗

PubMed1993

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)

Animals↗

PubMed1992

Identification of cell-surface heparin/heparan sulfate-binding proteins of a human uterine epithelial cell line (RL95).

The interaction of heparin (HP) with the cell-surface components of a human uterine epithelial carcinoma cell line (RL95) was studied. Binding of [3H]HP to cell surfaces was saturable in a dose- and time-dependent manner. HP and certain forms of heparan sulfate (HS) efficiently compete for [3H]HP binding. In contrast, other glycosaminoglycans, such as chondroitin sulfate, keratan sulfate, hyaluronic acid, and dermatan sulfate, do not compete for binding to these sites. Scatchard analysis revealed that [3H]HP bound to these sites with an apparent KD of 0.7-0.9 microM and a binding capacity of 9 x 10(6) sites/cell to attached cells. EDTA-detached cells displayed a similar apparent KD, but an approximately 2-fold increase in binding capacity. Protease digestion of cells on ice markedly reduced [3H]HP binding, indicating that these binding sites were associated with proteins. In contrast, heparinase treatment of cells stimulated binding by approximately 2-fold, indicating that a large fraction of these binding sites were occupied with endogenous ligand. We examined the structural features of HP/HS required for HP/HS binding. O-Sulfation, substitution of amino groups, and, to a lesser extent, the presence of carboxyl groups were important recognition features of HP/HS by cell-surface HP/HS-binding sites. N-Sulfation was not required. Photoaffinity labeling with 125I-sulfosuccinimidyl 2-(p-azidosalicylamido)-ethyl-1, 3-dithiopropionate-HP was used to identify HP/HS-binding proteins on RL95 cell surfaces. Proteins with M(r) values of 14,000-18,500 and 31,000 were photolabeled at the surfaces of attached cells. Photolabeling was blocked by the addition of excess HP, but not chondroitin sulfate. Additional proteins with M(r) values greater than 31,000 were photolabeled specifically on EDTA-detached cells. Moreover, the M(r) 14,000-18,500 and 31,000 proteins were retained on the EDTA-detached cells. These observations indicated that certain cell-surface HP/HS-binding proteins were not exposed when cells were attached to substrata. Proteins of similar M(r) values as the photolabeled components as well as many additional proteins were identified by heparin-agarose chromatographic selection of extracts of cells labeled metabolically with [35S]methionine or vectorially with Na125I at the cell surface. Fragments of cell-surface HP/HS-binding proteins were released from intact RL95 and mouse uterine epithelial cells by mild trypsinization and isolated by heparin-agarose affinity chromatography. Three peptides with M(r) values between 6000 and 14,000 required greater than 0.5 M salt for elution from heparin-agarose, retained HP binding activity in a 125I-HP gel overlay assay, and selectively bound [3H]HP in a solid-phase binding assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity Labels↗

PubMed1992

WGA-binding, mucin glycoproteins protect the apical cell surface of mouse uterine epithelial cells.

Expression of apical cell surface proteins and glycoproteins was examined in polarized primary cultures of mouse uterine epithelial cells (UEC). Lectin-gold cytochemistry revealed that wheat germ agglutinin (WGA) bound specifically to the components of the apical glycocalyx as well as intracellular vesicles. Double labeling with the pH sensitive dye 3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine (DAMP) demonstrated the acidic nature of the WGA-staining intracellular vesicles. The enzymatic and chemical sensitivities of the WGA binding sites on the apical cell surface were monitored both by WGA-gold staining as well as by 125I-WGA binding assays. In thin sections, a large fraction of these sites were removed by pronase; however, application of a wide variety of proteases, glycosidases, or chemical treatments to the apical surface of intact UEC failed to reduce WGA binding. In no case did treatments designed to remove sialic acids reduce 125I-WGA binding more than 12%. In contrast, endo-beta-galactosidase as well as a combination of beta-galactosidase with beta-hexosaminidase succeeded in removing 28% and 77% of these sites, respectively. These studies suggested that the majority of the apically disposed WGA binding sites involved N-acetylglucosamine residues rather than sialic acids and included lactosaminoglycans. Many of the proteins detected at the apical cell surface by lactoperoxidase-catalyzed radioiodination were WGA-binding glycoproteins. A major class of these glycoproteins displayed Mr > 200 kDa by SDS-PAGE and was heavily labeled metabolically by 3H-glucosamine or by vectorial labeling at the apical cell surface with galactosyl transferase and UDP-3H-galactose. Analyses of the 3H-labeled oligosaccharides labeled by either procedure indicated that a large fraction of the apically disposed WGA-binding oligosaccharides consisted of neutral, O-linked mucin-type structures with median MW of approximately 1,500. Oligosaccharides in this fraction were partially (15%) sensitive to endo-beta-galactosidase digestion and bound to Datura stramonium agglutinin (68%), demonstrating the presence of lactosaminoglycan sequences. UEC were an extremely effective barrier to attachment or invasion by either a highly invasive melanoma cell line, B16-BL6, or implantation-competent mouse blastocysts. In contrast, neither uterine stromal cells nor a non-polarizing UEC cell line, RL95, prevented B16-BL6 attachment.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Uterine stromal cell chondroitin sulfate proteoglycans bind to collagen type I and inhibit embryo outgrowth in vitro.

Chondroitin sulfate proteoglycans (CSPGs) are the major class of proteoglycans synthesized by mouse uterine stroma in vitro (Jacobs, A. L., and Carson, D. D. (1991). J. Biol. Chem. 266, 15,464-15,473). In the present study, stromal CSPGs were isolated and examined with regard to their ability to bind to specific extracellular matrix (ECM) components. Of a variety of ECM components tested, only collagen type I formed stable complexes with stromal CSPGs in both solid phase and solution binding assays. Proteolytic digestion of the CSPGs did not affect binding and suggested that the protein cores did not participate directly in binding. Furthermore, free chondroitin sulfate polysaccharides do not compete effectively in the binding assays. Therefore, interactions with multiple CS chains and/or the higher charge density afforded by intact CSPGs appear to be required for retention by collagen type I. Intact CSPGs were examined for their ability to modulate embryo attachment and outgrowth in vitro on fibronectin- or collagen type I-coated surfaces. In both cases, intact CSPGs, but not their constituent protein cores or polysaccharides, inhibited both the rate and the extent of outgrowth formation. In addition, embryo outgrowth on stromal ECM was enhanced by predigestion with chondroitinase. Addition of exogenous CSPG markedly retarded embryo outgrowth on stromal matrix. Collectively, these data indicate that stromal cell-derived CSPGs are retained by collagen type I in the stromal interstitial ECM where these molecules may attenuate trophoblast invasive behavior.

Animals↗

PubMed1992

Glycoconjugates as positive and negative modulators of embryo implantation.

A variety of studies indicate that complex glycoproteins participate in or modulate adhesive interactions occurring during embryo implantation. In particular, proteoglycans and proteins that bind proteoglycans are involved at multiple stages of this process. Identification of these binding proteins and the molecular controls over glycoconjugate expression are required to develop a comprehensive understanding of the implantation process.

Animals↗

PubMed1992

Differential temporal and spatial expression of mRNA encoding extracellular matrix components in decidua during the peri-implantation period.

Changes in the temporal and spatial patterns of expression of mRNA encoding uterine extracellular matrix (ECM) proteins were determined during the peri-implantation period. Northern blot hybridization of cDNAs corresponding to laminin (LM) B1, LM B2, entactin, fibronectin, collagen (CL) type IV alpha 1, and CL IV alpha 2 was performed on RNA extracted from either whole mouse uteri or endometrial explants between Day 4, i.e., the day of implantation, and Day 7 of pregnancy, when the decidual response is well established. These analyses revealed a dramatic increase in LM B2, CL IV alpha 1, and CL IV alpha 2 mRNA expression by Day 7 of pregnancy. Relative levels of the mRNA encoding other ECM components, including LM B1, were not altered when compared to changes in the relative level of expression of glyceraldehyde-3-phosphate dehydrogenase mRNA. The differential expression of the B chains of LM appeared to be limited to the stromal cells of the endometrium. In situ hybridization of uterine sections with cRNA probes corresponding to LM B1, LM B2, and CL IV alpha 1 demonstrated that LM B1 was expressed temporally in high amounts in the primary decidual zones (PDZ) and persisted throughout PDZ degeneration. LM B2 mRNA was expressed in both primary and secondary decidual zones and persisted through Day 8 of pregnancy. CL IV alpha 1 mRNA expression mimicked that of LM B2. Oviduct ligation on Day 2 of pregnancy was used to prevent embryo transport to one uterine horn, whereas decidualization and embryo implantation were permitted in the contralateral horn. This experiment demonstrated that the increases in uterine ECM mRNA expression were not due solely to the changing hormonal milieu of the uterus. ECM components, including CL IV, have been shown to bind growth factors such as transforming growth factor-beta (TGF-beta) in an insoluble but biologically active form. The remarkable similarity between the pattern of CL IV and LM B2 expression and previously reported TGF-beta deposition (Tamada et al., Mol Endocrinol 1990; 4:965-972) prompted examination of the effects of this growth factor on blastocyst development in vitro. TGF-beta 1 was tested for its ability to alter embryo outgrowth on LM-coated tissue culture surfaces; however, significant differences in the rate or extent of outgrowth in the presence of TGF-beta were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

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.

Animals↗

PubMed1992

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.

Animals↗

PubMed1992

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.

Animals↗

PubMed1992

Secretion and hormonal regulation of interleukin-6 production by mouse uterine stromal and polarized epithelial cells cultured in vitro.

Uterine stromal (USC) and uterine epithelial (UEC) cells were isolated from immature and mature mice to determine their ability to secrete interleukin-6 (IL-6) in response to ovarian steroids, IL-1 alpha, and soluble products produced by the heterologous cell type. In addition, the effect of IL-6 on embryo attachment and outgrowth in vitro was determined. UEC cultured on nitrocellulose filter inserts in a polarized manner secreted IL-6 with a 2.5- to 5-fold apical vs. basal preference, as determined by a B9 hybridoma cell proliferation assay and enzyme-linked immunosorbent assay. The hormonal status of animals at the time uteri were removed did not influence subsequent secretion of IL-6, as UEC isolated from immature, diestrous, and estrous stage mice exhibited both a similar amount and had a similar apical preference for secretion of IL-6. The addition of 17 beta-estradiol (E) to UEC cultures markedly inhibited total IL-6 secretion, but did not affect vectorial secretion. The inhibitory effect of E on IL-6 secretion by UEC was consistent with an apparent decrease in IL-6 transcript observed by a reverse transcriptase polymerase chain reaction assay. Other transcripts detected by this assay in UEC included IL-1 alpha, but not IL-1 beta or tumor necrosis factor-alpha. Secretion of IL-6 by UEC was not stimulated by IL-1 alpha, conditioned medium from USC, or coculture with USC. USC secreted IL-6, and while this also was inhibited by E, progesterone was more effective in this regard at physiological concentrations. In addition, there was a synergistic effect of E plus progesterone on inhibition of IL-6 secretion by USC. Secretion of IL-6 by USC was stimulated by IL-1 alpha, and coculture studies demonstrated the ability of UEC to stimulate a several-fold increase in IL-6 secretion by USC. The cytokine transcripts detected in USC cultures included IL-6 and IL-1 alpha, but not IL-1 beta. Transcripts for tumor necrosis factor-alpha were present in USC only after culture with IL-1 alpha. IL-6 added to blastocysts on laminin-coated tissue culture wells resulted in a transient inhibition of the rate of blastocyst attachment and, to a greater extent, an inhibition of the rate of embryo outgrowth. In addition, IL-6 inhibited the size of embryo outgrowths at 24 and 48 h of culture.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

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).

Animals↗

PubMed1991

Proteoglycan synthesis and metabolism by mouse uterine stroma cultured in vitro.

Chondroitin sulfate proteoglycans (CSPGs) and hyaluronate have been identified as the predominant glycoconjugates synthesized and secreted by mouse uterine stromal cells (USC) cultured in vitro. CSPGs in both the cell-associated and secreted fractions have identical characteristics with regard to anion exchange chromatographic behavior, sensitivity of the intact molecules and constituent glycosaminoglycans to a variety of chemical and enzymatic digestions, lack of interaction with hydrophobic affinity resins, and density (greater than 1.46 g/ml). Chase labeling studies indicated a metabolic half-life of cell-associated, [35S]sulfate-labeled macromolecules of 5-6 h. Once secreted, CSPGs did not appear to be degraded or endocytosed to a significant extent. In contrast, a large fraction (50%) of the cell-associated CSPGs were degraded to low Mr (less than 3000) products via a chloroquine-sensitive pathway. Studies of the kinetics of intracellular transport indicated that approximately 30 min were required for CSPG core proteins to move from the rough endoplasmic reticulum to the Golgi apparatus and 15-20 min to move from the Golgi to the cell surface, i.e. protease-accessible compartment. There was no significant lag period between the time CSPGs first arrived at the cell surface and the time at which they were first detectable in the medium. Examination of CSPG expression during USC differentiation in utero or in vitro demonstrated that these molecules were produced with similar efficiency by USC under both conditions. Collectively, these studies provide the first comprehensive description of proteoglycan production and metabolism in USC, a uterine cell type intimately involved with embryo implantation processes. Potential functions for CSPGs and hyaluronate as modulators of embryo invasive processes and uterine expansion are proposed.

Animals↗

PubMed1991

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.

Animals↗

PubMed1991

Molecular analysis of a baculovirus regulatory gene.

To better understand the structure and function of a baculovirus regulatory gene, the nucleotide sequence of IE-N expressed by Autographa californica nuclear polyhedrosis virus was determined. The 2.0-kb PstI-EcoRV restriction fragment (97.5 to 98.9 mu) encodes the upstream regulatory sequences, open reading frame, and downstream sequences of the immediate early IE-N gene. Using a convenient restriction site, the 285-bp promoter of IE-N was divided into two functional regions as defined by transient expression assays of mutant sequences. The sequences of IE-N from -1 to -45 nt encoded a minimal promoter capable of directing low levels of transcription. The minimal promoter was fully responsive to positive regulation by IE-N. The upstream region from -46 to -285 nt contains two direct repeats which increased levels of IE-N gene expression. Computer-assisted translation of the IE-N sequence indicates that this fragment of DNA encodes a single long open reading frame with a predicted molecular weight of 47,000. The amino acid sequence of the predicted protein exhibits three motifs common to transcriptional regulators: a serine-threonine rich region, a proline-rich region, and a polyglutamine tract. IE-N autoregulates its own expression and stimulates both IE-1 and IE-0 in transient assays. The stimulation of IE-1 may account for the augmenting activity of IE-N in the IE-1-mediated trans-activation of the 39K promoter.

Amino Acid Sequence↗

PubMed1991

Transient expression of the Autographa californica nuclear polyhedrosis virus immediate-early gene, IE-N, is regulated by three viral elements.

Autographa californica nuclear polyhedrosis virus (AcMNPV) is a double-stranded DNA virus that expresses several immediate-early genes under the control of different promoters. The expression of one of these transcription units, IE-N, is shown here, by a transient expression assay, to be regulated by both cis- and trans-acting viral elements. The steady-state levels of IE-N mRNA were very abundant soon after infection but were nearly undetectable during the late phase of the viral life cycle. Analysis of the transient expression of a reporter construct driven by the IE-N promoter (IE-NCAT) was conducted to define viral elements which regulate IE-N gene expression. Viral enhancer hr1 and two immediate-early genes, IE-1 and IE-N, were shown to affect relative levels of reporter enzyme activity produced by IE-NCAT. The hr1 enhancer stimulated the expression of IE-NCAT, independent of orientation and position relative to the promoter and in the absence of any trans-acting viral factors. Regulation of IE-NCAT expression by the IE-1 and IE-N genes required less than 290 bp of promoter sequences upstream of the site of transcription initiation and was not dependent upon the hr1 enhancer. Coexpression of the IE-N gene had an autostimulatory effect upon IE-NCAT activity, whereas coexpression of the IE-1 gene reduced levels of reporter activity. The levels of reporter activity measured upon coexpression of either immediate-early gene with IE-NCAT linked to the hr1 enhancer appear to be the combined result of both cis- and trans-regulatory elements influencing expression from IE-NCAT. These results suggest that IE-N gene expression in baculovirus infection may be influenced by the concerted activity of three AcMNPV regulatory elements.

Animals↗