Regulation of trophoblast beta1-integrin expression by contact with endothelial cells
© Thirkill et al; licensee BioMed Central Ltd. 2004
Received: 09 March 2004
Accepted: 09 June 2004
Published: 09 June 2004
In human and non-human primates, migratory trophoblasts penetrate the uterine epithelium, invade uterine matrix, and enter the uterine vasculature. Invasive trophoblasts show increased expression of β1 integrin. Since trophoblast migration within the uterine vasculature involves trophoblast attachment to endothelial cells lining the vessel walls, this raises the possibility that cell-cell contact and/or factors released by endothelial cells could regulate trophoblast integrin expression. To test this, we used an in vitro system consisting of early gestation macaque trophoblasts co-cultured on top of uterine microvascular endothelial cells.
When cultured alone, trophoblasts expressed low levels of β1 integrin as determined by quantitative immunofluorescence microscopy. When trophoblasts were cultured on top of endothelial cells for 24 h, the expression of trophoblast β1 integrin was significantly increased as determined by image analysis. β1 Integrin expression was not increased when trophoblasts were cultured with endothelial cell-conditioned medium, suggesting that upregulation requires direct contact between trophoblasts and endothelial cells. To identify endothelial cell surface molecules responsible for induction of trophoblast integrin expression, trophoblasts were cultured in dishes coated with recombinant platelet endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), or αVβ3 integrin. Trophoblast β1 integrin expression (assessed by immunofluorescence microscopy and Western blotting) was increased when PECAM-1 or αVβ3 integrin, but not ICAM-1, was used as substrate.
Direct contact between trophoblasts and endothelial cells increases the expression of trophoblast β1 integrin.
As part of the implantation process and development of the placenta in human and non-human primates, migratory trophoblasts penetrate the uterine epithelium, invade the uterine matrix, and enter the uterine vasculature [1–7]. These invasive trophoblasts show increased expression of β1 and α1 integrins and down-regulation of β4 integrin when compared to non-invasive villous trophoblast cells [8–11]. Integrins are heterodimeric transmembrane proteins that function in cell-matrix and cell-cell adhesion. Integrins also function in cell signaling. Our previous studies suggest a role for trophoblast β1 integrin in trophoblast adhesion to endothelial cells . Beta 1 integrins, and integrins in general, are also known to be involved in cell migratory activity [13–17]. The factors responsible for regulating the acquisition of the migratory trophoblast phenotype, and for controlling integrin expression in these cells, are poorly understood. Trophoblast integrin expression is increased when trophoblast cells are cultured on fibronectin or in the presence of TGF-β [18, 19] and we recently showed that β1 integrin expression by macaque trophoblasts was increased when the cells were exposed to physiological levels of shear stress .
Since trophoblast migration within the uterine vasculature involves trophoblast attachment to endothelial cells lining the vessel walls, this raises the possibility that cell-cell contact and/or factors released by endothelial cells could regulate trophoblast integrin expression. This idea is supported by the analogous upregulation of leukocyte integrins by contact with endothelium [20, 21]. In the present paper we have tested the notion that trophoblast-endothelial cell contact regulates trophoblast integrin expression. The studies use an in vitro system that we have previously described , consisting of macaque trophoblasts co-cultured with human uterine microvascular endothelial cells. The results show that cell-cell contact causes an upregulation of trophoblast β1 integrin. Other data presented here suggest that increased expression of trophoblast β1 integrin is mediated by interaction of trophoblasts with endothelial cell platelet endothelial cell adhesion molecule-1 (PECAM-1) and αVβ3 integrin.
Trophoblast β1 integrin is upregulated by contact with endothelial cells
Trophoblast β1 integrin upregulation is time- and temperature-dependent
Effect of solid-phase recombinant adhesion molecules on trophoblast β1 integrin expression
The results presented here support the idea that the expression of trophoblast β1 integrin is upregulated by direct contact with endothelial cells. We found no evidence that integrin expression was regulated by soluble factor(s) released by endothelial cells or that we were selecting for a population of strongly β1 integrin-expressing trophoblasts. It is well known that the expression of β1 integrin by human and macaque trophoblasts is increased as trophoblasts acquire a migratory phenotype and enter the invasive pathway [8–11]. Invasive trophoblasts migrate within the maternal uterine stroma, although this occurs to a greater extent in the human than in the macaque . In both species, trophoblasts enter superficial venule-like, non-arteriolar vessels [1, 23, 24] and then attach to, and eventually remodel, uterine blood vessel walls. Endovascular trophoblasts express high levels of β1 integrin and α1 integrin compared to villous cytotrophoblasts but show reduced levels of β4 integrin. The in vitro results described here suggest that attachment of trophoblasts to the endothelial surface could contribute to the upregulation of β1 integrin expression seen in vivo. Examination of sections of early gestation human and macaque implantation sites indicates that increased expression of trophoblast β1 integrin begins before the cells enter the vasculature [8–11]. Thus, factors in addition to attachment to endothelial cells are involved in regulating trophoblast integrin expression.
Previous studies have shown that β1 integrin expression by cultured trophoblasts can be increased by extracellular matrix components and by TGF-β . We have also recently shown that trophoblast β1 integrin expression can be increased by fluid flow-derived shear stress . Integrins are involved in cell-cell and cell-extracellular matrix attachment and facilitate cell migration [25–30] and so increased trophoblast β1 integrin expression is likely related to trophoblast adhesion and motility. It is not unreasonable to speculate that the ability of trophoblasts to withstand, and indeed to migrate against, the flow of blood requires a sufficiently high level of integrin expression. Factors that regulate trophoblast integrin expression within the uterine stroma may not be present within the vasculature and so the combined effects of endothelial contact and shear stress would ensure that high levels of integrin expression are maintained in the vascular environment. Since integrins are also involved in signal transduction [31–34], it is possible that increased integrin expression facilitates signaling events that are important for invasive trophoblast survival and function.
The identity of the endothelial cell surface component(s) that are responsible for the induction of trophoblast integrin expression is obviously an important question. While other as yet unidentified molecules could be involved, the studies presented here using recombinant proteins indicate that PECAM-1 and αVβ3 integrin, both of which are major endothelial cell surface molecules, play a role in regulating trophoblast integrin expression. The Western blot data suggest that trophoblast β1 integrin protein amount is increased by contact of trophoblasts with these molecules. Increased protein amount most likely reflects increased protein synthesis but could also reflect decreased degradation. We found no evidence that ICAM-1, another endothelial cell adhesion molecule, is involved in regulating trophoblast integrin expression. PECAM-1 is a member of the immunoglobulin superfamily of adhesion molecules and is expressed by endothelial cells, platelets, and leukocytes. PECAM-1 is believed to play roles in leukocyte extravasation, angiogenesis, cell migration, and cell signaling [35–40]. PECAM is capable of homophilic (PECAM-PECAM) binding as well as heterophilic binding to other molecules such as αVβ3 or CD38. Homophilic interaction between PECAM expressed by neutrophils and endothelial cells is reported to cause upregulation of neutrophil integrin expression . Antibody cross-linking of PECAM also results in activation of several integrins . Homophilic PECAM interactions could also be responsible for the increased expression of β1 integrin seen in the present coculture study since migratory trophoblasts in both the macaque and the human express PECAM-1 [42–44].
αVβ3 integrin is a cell adhesion molecule expressed by several cell types including endothelial cells. αVβ3 integrin binds to vitronectin, fibronectin, osteopontin, and PECAM-1 [45–49]. Upregulation of trophoblast β1 integrin could therefore be the result of interaction between endothelial cell αVβ3 and trophoblast PECAM-1. It could also be the result of endothelial αVβ3 interaction with another as yet unidentified ligand on the trophoblast surface. Clearly, the identity of the signaling pathways responsible for the cell-mediated regulation of integrin expression in trophoblasts warrants further attention.
The Western blot data indicate that trophoblast β1 integrin exists in two forms, distinguishable by slight differences in molecular mass. Studies using various cancer and normal cell lines have demonstrated two different molecular mass forms of β1 integrin [50–54] that appear to be the result of differences in glycosylation. While we have not confirmed that the different β1 integrin forms found in macaque trophoblasts result from differences in glycosylation, the molecular masses correspond to those reported for other cell types. Furthermore, Moss et al  showed that differences in electropheretic mobility of β1 integrin from early and term human cyotrophoblasts resulted from differences in glycosylation. The function of these different β1 integrin isoforms in trophoblasts remains to be elucidated.
The results presented here support the idea that the expression of trophoblast β1 integrin is upregulated by direct contact with endothelial cells. Upregulation was time- and temperature- dependent and no evidence was found to suggest that integrin expression was regulated by soluble factor(s) released by endothelial cells. While additional molecules could be involved, the studies using recombinant proteins indicate that interaction of trophoblast cells with PECAM-1 and/or αVβ3 integrin, both of which are major endothelial cell surface molecules, could play an important role in regulating endovascular trophoblast β1 integrin expression.
It is not unreasonable to speculate that the ability of trophoblasts to withstand, and indeed to migrate against, the flow of blood requires a sufficiently high level of integrin expression. The effects of endothelial contact (as demonstrated here) combined with shear stress would ensure that high levels of trophoblast integrin expression are maintained in the vascular environment. The in vitro data presented here may provide an explanation for the increased expression of β1 integrin that is observed for endovascular trophoblasts in both the human and the macaque. Since integrins are also involved in signal transduction [31–34] it is possible that increased integrin expression facilitates signaling events that are important for invasive trophoblast survival and function.
Trophoblast isolation and culture
We have previously described in detail a procedure used to isolate trophoblast cells from term (165-day) macaque placentas . The same procedure was used in the present case to isolate cells from 40–60 day placental/endometrial tissue. Yields were approximately 3 × 106 cells/g tissue (20–30 × 106 cells per placenta). The cells were subjected to an additional purification step using immunomagnetic microspheres coated with anti-HLA antibodies . This step removes contaminating HLA-positive cells leaving pure (i.e., 100% cytokeratin-positive, HLA-ABC/DR-negative, vimentin-negative) trophoblast cells. FACS analysis of this purified trophoblast population revealed that 75% of the cells were β1 integrin-positive .
Endothelial cells and co-culture conditions
Human uterine myometrial endothelial cells (UtMVEC, passage 4) were purchased from Clonetics Corporation (San Diego, CA) and maintained in endothelial basal medium-2 (Clonetics) supplemented with human recombinant epidermal growth factor, human fibroblast growth factor, vascular endothelial growth factor, ascorbic acid (Vitamin C), hydrocortisone, human recombinant insulin-like growth factor, heparin, gentamicin, amphotercin, and 5% fetal bovine serum. Cells were plated into 8-chamber LabTek slides that had been coated with fibronectin (Becton Dickinson, Bedford, MA). The chambers were incubated at 37°C in humidified 95% air and 5% CO2 for 12–24 hours to allow formation of a near confluent UtMVEC layer. Prior to the addition of trophoblasts, the endothelial cells were incubated for 12 h under serum-free conditions. Trophoblasts were then added and the cocultures were incubated for 24 h. The cocultures were then analyzed by immunocytochemistry as described below. As a control, other trophoblasts were cultured on slides coated with fibronectin and without endothelial cells.
REN mesothelioma cells were provided by S. Albelda (University of Pennsylvania, Philadelphia, PA)
Incubation of trophoblasts with recombinant proteins
A recombinant form of intercellular adhesion molecule-1 (ICAM-1) and a recombinant form of the extracellular domain of human PECAM-1 were obtained from R&D Systems Inc. Minneapolis, MN, and maintained as aqueous stock solutions in PBS. Recombinant αVβ3 integrin was obtained from Chemicon as a stock solution in octylglucoside. The surfaces of LabTek culture chambers were coated with recombinant proteins (10 μg/ml) for 1 h at 37°C. Coating with higher concentrations (up to 40 μg/ml) did not alter the results obtained and so 10 μg/ml was routinely used. The solution was then removed and the chambers were allowed to air dry. The coated chambers were then blocked using bovine serum albumin (BSA; 10 mg/ml) for 1 h at 37°C. Trophoblasts (350,000 cells per cm2) were added to the precoated chambers in Ham's/Waymouth's medium containing BSA (10 mg/ml) and incubated for 24 h. Controls consisted of chambers coated only with BSA. Other control experiments (not shown) confirmed that octylglucoside (carrier for recombinant αVβ3) did not affect trophoblast integrin expression.
Immunocytochemistry and image analysis
Monoclonal antibodies against β1 integrin (clone P4G11) were purchased from Chemicon, Temecula, CA. A polyclonal antibody against cytokeratin (pan) (cat #18-0059) was purchased from Zymed, San Francisco, CA. Oregon Green-labeled goat anti-mouse Ig antibody and TRITC-labeled goat anti-rabbit Ig antibody were purchased from Molecular Probes, Eugene, OR.
Cells in LabTek culture chambers were fixed and permeabilized in ice-cold methanol or fixed in 2% paraformaldehyde (without permeabilization) then stained with primary antibody. Primary antibodies were detected using Oregon Green-labeled or TRITC-labeled goat anti-mouse or goat anti-rabbit Ig. Antibody controls in which cells were incubated with isotype-matched mouse Ig or non-immune rabbit Ig were also included. The stained cells were examined using a Nikon Eclipse E800 epifluorescence microscope. Multiple images from random fields were captured using an Optronics DEI750 CCD camera and Adobe Photoshop software. Identical exposure and brightness level settings were used for test and control samples. Captured digitized images were imported into Image Pro Plus software to determine cellular levels of anti-integrin antibody-associated fluorescence. The software was calibrated using the InSpeck fluorescence Image Intensity Calibration Kit (6 :m beads; Molecular Probes, Eugene OR). Relative cellular fluorescence intensity was determined by reference to a standard curve generated using the calibration beads and is expressed as mean density normalized by area. Background fluorescence (calculated using cells treated with control mouse immunoglobulin instead of the anti-integrin antibody) was subtracted from experimental values. At least 4 random microscope fields were analyzed for each sample well and experiments were repeated at least 3 times.
Cultures were washed with Dulbecco's Modified PBS containing Ca2+ and Mg2+. The cells were then lysed on ice by the addition of M-PER Mammalian Protein Extraction Reagent (Pierce) supplemented with 1% Protease Inhibitor Cocktail (Sigma). The lysate was homogenized by repeated passage through a 27 gauge needle, then mixed with an equal volume of Laemmli sample buffer (BioRad) containing 5% β-mercaptoethanol and heated in a boiling water bath for 5 minutes. The samples were immediately chilled on ice and loaded on to an 8 % SDS-polyacrylamide gel (Gradiopore) at 20 μg per lane. Electrophoresis was performed at 200 V for 45 minutes after which proteins were transferred to PVDF membrane (BioRad) at 100 V on ice for 1 hour. The membrane was blocked for 1 hour in 1% non-fat dried milk (NFDM) followed by overnight incubation with a 1/1000 dilution of mouse anti-β1 integrin antibody (clone JB1A; Chemicon) and 1/2000 dilution of mouse monoclonal antibody cocktail against tubulin (clones DM1A, DMA18, migG1; RDI). Tubulin was used as an internal loading control. The membrane was washed 6X in TBS containing 1%Tween-20 after which it was incubated with goat anti-mouse immunoglobulin conjugated with horseradish peroxidase (BioRad) diluted 1/50,000 in 1% NFDM for 1 h at room temperature. After washing, the membrane was incubated with chemiluminescent substrate (SuperSignal West Dura; Pierce) for 5 min at room temperature. The membrane was then exposed to X-ray film (Pierce). Scanned images of exposed X-ray film were analyzed using Kodak 1D gel analysis software. Band densities were obtained and corrected for background. Densities of bands of interest were expressed relative to the intensity of the loading control (tubulin).
Experiments were repeated at least 3 times using cells from different placentas in each case. Cells from different placentas were not pooled. Statistical analyses were performed by ANOVA followed by Tukey-Kramer multiple comparison post-test using the Prism software program (GraphPad Inc., San Diego, CA). Differences in means were considered significant if p < 0.05.
Early gestation macaque placental tissue was made available to us through the cooperation of the staff at the California Regional Primate Research Center, University of California, Davis. We are particularly indebted to Sara Davis, Dr. Andy Hendryckx, and Katy Lanz. This work was supported by grants from the University of California Davis Health Sciences Research Fund (GCD), Philip Morris USA Inc. (GCD), and NIHRO1HL068035-01A1 (GCD).
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