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Research Article |
University of Alberta Perinatal Research Centre,3 University of Alberta, Edmonton, Alberta, Canada T6G 2E1
Academic Unit of Child Health,4 University of Manchester, Manchester M13 9PT, United Kingdom
Department of Medical Microbiology and Immunology,5 University of Alberta, Edmonton, Alberta, Canada T6G 2H7
ABSTRACT
Cultured human term villous cytotrophoblasts (CT) have been reported to be nonproliferating but differentiate when exposed to epidermal growth factor (EGF). Here we show that CT differentiate into chorionic gonadoptropin (beta-hCG/CGB)-expressing cells when cultured with medium alone. The addition of EGF decreases CGB secretion and prolongs production for up to 13 days. EGF stimulates the phosphorylation (activation) of the signaling intermediate p38 (MAPK11/14), and blocking phosphorylation pharmacologically with either SB203580 or SB202190 strongly inhibited spontaneous and EGF-stimulated secretion of CGB. In addition, EGF-stimulated fusion of cytotrophoblasts into syncytial units was strongly inhibited by SB203580. EGF upregulated trophoblast proliferation (measured by bromodeoxyuridine uptake) and SB203580 increased this proliferation after 5 days. In agreement with these observations, EGF and SB203580 increased expression of the G1-phase-specific gene cyclin-D1 (CCND1) and SB203580 downmodulated its inhibitor p21 (CDKN1A). When added to villous explant cultures, EGF did nothing to the pattern of CGB secretion, but addition of SB203580 prevented the normal surge in secretion during syncytial regeneration over Days 3–7. These data support the hypothesis that EGF-stimulated cytotrophoblast differentiation to syncytium requires MAPK11/14 activation, and that cytotrophoblast proliferation can be stimulated in culture by EGF and enhanced by MAPK11/14 inhibition with a consequent reduction of differentiation.
CGB, culture, EGF, explants, human chorionic gonadotropin, MAPK11, MAPK14, placenta, pregnancy, p38, SB202190, SB203580, syncytialization, syncytiotrophoblast, trophoblast
INTRODUCTION
Culture of isolated term trophoblasts was first reported by Stromberg in 1978 [1]. Modifications have been made [2, 3], culminating in a trophoblast purity in culture of >99.99% [4]. However, primary trophoblasts are reported not to replicate in culture at ambient oxygen levels [5], but they can differentiate into a patchwork of syncytialized cells [6]. Culturing with epidermal growth factor (EGF) or cAMP analogs accelerates the syncytialization process [7, 8].
As its name implies, EGF has both proproliferative and prodifferentiation effects on multiple epithelial cell types [9]. For cultured trophoblasts, EGF increases trophoblast differentiation as marked by cell fusion in culture (see above) and inhibits apoptosis [10, 11]. Expression of human chorionic gonadotropin (ß-hCG, CGB) has also been used as a marker of villous trophoblast differentiation [1]. However, EGF has been shown to both increase [7, 12, 13] and decrease [14] CGB production in cultured trophoblasts.
We have previously shown that EGF promotes resistance to apoptosis by stimulating PI-3 kinase/Akt, ERK kinase, JNK kinase, and sphingosine kinase pathways [15]. Although EGF also stimulates the phosphorylation, and therefore activation, of p38 mitogen-activated protein (MAP) kinase (MAPK11/14), this phosphorylation does not contribute to EGF-stimulated resistance to apoptosis [15]. The MAPK11/14 signaling pathways are involved in a variety of cellular responses, and the outcomes of cellular response are varied and seemingly dependent on cell type. In some tissues, MAPK11/14 signaling promotes cell death [16–19], but it has also been shown that MAPK11/14 cascades enhance survival [20], cell growth [21], and differentiation [22]. As EGF activated MAPK11/14 in trophoblasts, we considered the role of MAPK11/14 in trophoblasts in proliferation and differentiation.
EGF stimulation of trophoblast differentiation is reported to occur through increased CGB production, which acts in an autocrine manner to stimulate differentiation [23]. EGF also stimulates proliferation of very early (4–5 wk) but not later (6–12 wk) placental explants [13]. Further EGF did not greatly prolong the initial proliferation of term villous cytotrophoblasts in culture, while antibody to EGF receptor blocked differentiation as marked by cellular fusion [6]. The role of EGF-stimulated MAPK11/14 activation has not previously been considered in trophoblasts; however, a null mutation in MAPK14 is embryonic lethal due to a deficiency in spongiotrophoblast formation caused by a lack of placental angiogenesis [24, 25]. However, only placental angiogenesis was affected [25], and whether this defect was cause or effect has not been determined. We therefore hypothesized that MAPK11/14 was an intermediate in the chain of events set off by EGF and that its activation culminates in trophoblast differentiation.
The MAPK11 and MAPK14 kinases are inhibited specifically by the pyridinyl imidazole inhibitors SB203580 and SB202190, which bind reversibly to the ATP pocket of the enzymes [26]. We thus tested our hypothesis by culturing purified trophoblasts (>99.99% pure) with and without EGF and with and without the MAPK11/14 inhibitors to determine whether MAPK11/14 inhibition blocked trophoblast differentiation. We monitored differentiation by measuring secretion of CGB and by stimulation of cellular fusion. We also reasoned that blocking differentiation might stimulate proliferation. We found that inhibiting MAPK11/14 blocked EGF-stimulated trophoblast differentiation and prolonged EGF-stimulated proliferation.
MATERIALS AND METHODS
Materials
EGF was obtained from Pepro Tech EC (London, U.K.). The cell-permeable cAMP analog (8-Br-cAMP) was obtained from Sigma (Oakville, ON, Canada). Primary antibodies to cyclin D1/CCND1and p21/CDKN1A (catalog numbers 2926 and 2946; mouse monoclonal) and phosphorylated (p)-MAPK11/14 (catalog number 9211; rabbit polyclonal) were obtained from Cell Signaling Technology (Beverley, MA) and used at a dilution of 1:500. Secondary antibodies for Western blotting were obtained from Bio-Rad Laboratories (Mississauga, ON, Canada) and used at a dilution of 1:2000. SB203580 and SB202190 were obtained from Calbiochem (San Diego, CA) and used at 10 µM. DAPI (4',6-diamidino-2-phenylindole 1.4 µg/ml), Alexa Fluor 488 (1 µg/ml goat anti-mouse Ig conjugate, catalog number A11001), and Alexa Fluor 546 (1 µg/ml anti-mouse Ig conjugate, catalog number A11003) were obtained from Molecular Probes, Invitrogen (Burlington, ON, Canada). Bromodexoxyuridine (BrdU, 10 µg/ml) and antibody to BrdU (1:400 dilution of an ascites fluid, catalog number B2531) were from Sigma.
Isolation and Purification of Trophoblasts
Placentas were obtained with the ethical approval of the Capital Health Authority after elective cesarean or normal term delivery from uncomplicated pregnancies from Royal Alexandra Hospital, Edmonton, AB, Canada. Villous cytotrophoblasts (CT) were isolated by trypsin-DNase digestion of minced chorionic tissue and immunoabsorption on to immunoglobulin (Ig)-coated glass bead columns, as previously described [4, 27], using anti-CD9 (house preparation mAb 50H.19), antimajor histocompatibility complex (MHC) class I (W6/32; Harlan Sera-Lab, Crawley Down, Sussex, U.K., catalog number MAS 1532c), anti-MHC class II (clone 7H3, house preparation) antibodies for immunoelimination. The cells were routinely cryopreserved and, after thawing, were washed twice in Iscove's modified Dulbeco medium (IMDM; Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco) and antibiotics (end concentrations penicillin 100 U/ml, streptomycin 100 µg/ml; Sigma). The cells were seeded at 2.5 x 106 per well per 2 ml of IMDM/FBS in six-well tissue culture dishes (NUNC no. 152795, Gibco) and 1.5 x 106 per well in 96-well dishes (NUNC no.167008, Gibco), and incubated for 4 h at 37°C in a humidified atmosphere of 5% CO2 in air. The nonadherent cells and debris were removed with prewarmed IMDM/FBS, 2 ml IMDM with antibiotics added (the culture was >99.99% cytotrophoblast at this point by absence of vimentin and placental alkaline phosphatase immunohistochemistry [4]), and the incubation continued in humidified 5% CO2 environment with EGF or other drugs added as described.
Isolation and Culture of Placental Explants
Placentas were obtained with local research and ethics committee approval and informed consent at term from the delivery unit of St. Mary's Hospital (Manchester, U.K.) following normal pregnancies, delivered vaginally or by cesarean section. The culture system was developed from procedures of Trowell [28] and Watson et al. [29], as described by Siman et al. [30]. Within 30 min of delivery, chorionic villi were dissected out and carefully rinsed in sterile Dulbecco phosphate-buffered saline with calcium chloride and magnesium chloride (37°C; Sigma) to remove maternal blood. The tissue was cut into pieces weighing ~2 mg. Four such pieces were cultured in individual Costar Netwell (15-mm diameter, 74-µm mesh; Corning, Corning, NY) supports in 1.5 ml of culture medium (CMRL-1066, 5% heat-inactivated fetal bovine serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 1 µg/ml insulin; Sigma). The tissue was supported on the mesh in the liquid-gas interface. Cultures were maintained at 37°C in a humidified gas mixture of 5% CO2 and 95% air, and medium was changed every 24 h. Supernatants were collected and stored at –20°C for CGB analysis. The cultures were maintained for up to 7 days for functional and morphological evaluation. Cultures with apparent bacterial contamination were interrupted and excluded. At the end of the experiment, cultured placental explants were lysed in deionized water and membrane-bound protein content was measured by dissolving the tissue in 4 ml 0.3 M NaOH. Two times 80-µl samples from each dissolved explant were mixed with 320 µl 0.3 M NaOH, 400 µl 0.3 M HCl, and 200 µl Bio-Rad reagent (Bio-Rad Laboratories, Hemel Hempstead, UK) and vigorously mixed. Optical density was measured at 595 nm.
Western Blot Analysis
Sample protein concentrations were determined in duplicate with Micro BCA Reagent (Pierce Chemical Company, Rockford, IL) using a bovine serum albumin stock standard. Sample protein (30 µg/ml) was solubilized in 3x sample buffer (Sigma) by boiling for 5 min and stored until electrophoresis. SDS-PAGE on 10% gels was performed as previously described [15] using a Mini-Protein II gel system (Bio-Rad Laboratories, Inc., Hercules, CA). Following electrophoresis, gels were equilibrated for 15 min in transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol). Proteins were electrophoretically transferred onto nitrocellulose membranes (1.67 h, 60 V), which were then incubated with blocking solution (5% dried skimmed milk in TTBS [100 mM Tris pH 7.5 with 140 mM NaCl and 0.01% Tween 20]) for a minimum of 1 h. The blots were incubated overnight at 4°C with the primary antibodies, washed twice with the blocking and washing solution, incubated with diluted horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, washed extensively in TTBS, and developed using enhanced chemiluminescence (Amersham Pharmacia Biotech, Buckinghamshire, U.K.) on x-ray films. The blots were washed with the blocking solution and reprobed up to three times for proteins of differing molecular weights. The exposed x-ray films were scanned on a GS700 Bio-Rad Densitometer with Molecular Analyst software to quantify band densities.
Apoptosis and Cell-Number Assessment
Treated cells in 96-well plates were gently washed once with PBS, fixed (10 min, ice-cold methanol, –20°C), then washed three times with PBS and stained with DAPI (10 min, at room temperature [RT]), which labels DNA [31]. Apoptosis was assessed by counting brightly labeled cells and dividing by total DAPI-stained nuclei [15].
Cell Proliferation
Cells in 96-well plates were exposed to BrdU on Days 1 and 5 of culture for 12 h. Cells were then washed once with PBS, fixed (10 min, ice-cold methanol, –20°C), washed three times with PBS, and treated with 4 M HCl (30 min at 37°C) to denature (into single strands) DNA. Fixed cells were then incubated with nonimmune goat serum (Zymed Laboratories, Markham, CA) to block nonspecific binding sites for 1 h and exposed to anti-BrdU for 4 h. Following washing with PBS, cells were exposed to goat anti-mouse Alexa Fluor 488 conjugate (Molecular Probes) for 30 min (to both lightly stain all nuclei and to brightly stain BrdU-labeled nuclei), then washed again with PBS.
Digital Photography for Analysis of Apoptosis and BrdU Staining
Fluorescence was visualized with an inverted phase-contrast microscope (model DS-IRB; Leica, Heerbrugg, Switzerland) equipped for epifluorescence with a 100-W high-pressure mercury lamp driven by a Ludl power source (Ludl Electronic Products, Hawthorne, NY). For BrdU analysis, identical digital images of each well were taken with a DAPI filter (blue) and a fluorescein isothiocyanate filter (green) using a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI). Four images, each containing ~400 nuclei, were taken in each of triplicate wells and analyzed using the imaging program Image-Pro Plus (Media Cybernetics, Del Mar, CA).
Assessment of Multinucleation (Fusion)
Treated cells in 96-well plates were washed once with PBS, fixed (10 min, ice-cold methanol, –20°C), and then washed three times with PBS and incubated with nonimmune goat serum (Zymed Laboratories, Markham, CA) to block nonspecific binding sites for 1 h. Cells were then exposed to anti-desmoplakin (IgG1 mAb; ICN ImmunoBiologicals, Costa Mesa, CA, catalog number 69–542, 10 µg/ml) overnight at 4°C. Following washing with PBS, cells were exposed to 1 µg/ml anti-mouse Alexa Fluor 546 (Molecular Probes) for 30 min, then washed again with PBS and counterstained with DAPI (10 min, RT).
Digital Photography and Analysis of Fusion
Images were obtained with an Olympus IX2-UCB microscope with a Roper Scientific camera and a Sutter instruments Lambda DG-4 fluorescent lamp (Olympus, Melville, NY). We used Slidebook 3.0 (Carsen, Markham, ON, Canada) as capture software and Image Pro-Plus (Media Cybernetics, Del Mar, CA) for analysis. Digital images of each well were obtained with a DAPI (blue) and a rhodamine filter (red). Multinucleation was determined by manual counting of nuclei/desmoplakin-stained cells. Mononuclear cells are defined as one nucleus/cell membrane defined by desmoplakin staining, multinuclear as two or more nuclei/cell membrane.
CGB Assays
Trophoblasts in culture. Cell supernatants were collected, microcentrifuged at high speed (10000 x g) for 5 min at 4°C, and frozen at –20°C. A CGB colorometric assay system (DRG diagnostics, Marburg, Germany) was used to determine CGB levels with optical density determined on a 96-well plate reader (Molecular Probes, Eugene, OR).
Placental explants in culture Explant supernatants were collected and frozen at –20°C. CGB was assayed by quantitative immunoradiometric determination using a commercially available kit (hCG solid-phase component system; ICN Pharmaceuticals, Costa Mesa, CA). The CGB assay uses the sandwich technique, where the solid phase binds the alpha subunit of CGB and a radiolabeled antibody in the liquid phase binds to the beta subunit.
Statistical Analysis
Analysis was carried out using GraphPad Prism 4 software (GraphPad Software Inc., San Diego, CA). Data was log transformed to ensure approximate Gaussian distribution and compared using two tailed Student t-tests or ANOVA with Tukey post hoc testing. Significance (P) is indicated where shown, all data represented are means ± SEM.
RESULTS
EGF Decreases CGB Secretion in Cultured Trophoblast Cells
We first resolved the issue of whether EGF up- or downmodulated CGB secretion in culture. Purified trophoblasts were cultured with and without EGF (10 ng/ml with a medium change every 2 days) for 13 days and CGB was measured in the supernatants. We found that, relative to the undifferentiated control cells on Day 1 of culture, EGF-stimulated cells secreted more CGB on all days of culture up to Day 13 (Fig. 1). However, a parallel control culture without EGF showed a peak release of CGB that was over 5-fold greater than the EGF culture. In cultures without EGF, the peak release was rather sharp and rapidly declined so that secretion from EGF-containing cultures was higher after Day 9 of culture. These data indicate that trophoblasts are differentiating into CGB-expressing cells both with and without EGF but that the differentiation with EGF is slower but steadier.
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MAPK11/14 Inhibitors Decrease Differentiation into a CGB-Expressing Cell Type
Using secretion of CGB as a marker, we asked whether the MAPK11/14 inhibitors had an effect on trophoblast differentiation at Day 5 of culture (near the maximum for cultures with and without EGF). We found that both inhibitors almost completely inhibited CGB release from cells cultured with and without EGF (Fig. 2). This indicates that MAPK11/14 activation is required for differentiation to a CGB-expressing state.
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MAPK11/14 Inhibitors Block Fusion of Trophoblasts into Multinucleated Units
Purified term trophoblasts underwent spontaneous membrane fusion when incubated in the presence of serum resulting in 21% ± 5% (n = 8) of cells having two or more nuclei after 5 days (Fig. 3, a and g). Addition of SB203580 and SB202190 alone had no effect on multinucleation (Fig. 3. a, c, d, and g). EGF increased the number of multinucleated cells (n = 8) (Fig. 3, a, b, and g), in agreement with published data [6]. Addition of SB203580 with EGF to trophoblast cultures reversed the increase in multinucleation from 57% ± 5% to 18% ± 2% and SB202190 with EGF reduced it to 16% ± 2% (Fig. 3, b, e–g). Thus, blocking MAPK11/14 kinase activity also inhibited CT differentiation into ST.
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EGF and MAPK11/14 Inhibitors Stimulate Trophoblast Proliferation
Our data shows that EGF is initially inhibiting CGB secretion but also stimulates multinucleation. We next therefore investigated if this was due to EGF stimulating both proliferation and differentiation, as it does in other cell types, and determined the effect of inhibiting MAPK11/14. EGF stimulated trophoblast proliferation both on Day 1 and at Day 5 (Fig. 4). The stimulation was dependent on time in culture (Fig. 4, B and C). Addition of the MAPK11/14 inhibitor enhanced the effect of EGF on proliferation to >60% on Day 1 and >16% on Day 5 and stimulated proliferation alone (although not significantly). These data indicate that EGF stimulates CT proliferation, which was further increased by blocking activation of MAPK11/14.
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Cyclin D1/CCND1 is required for passage of cells from G1 to S phase of the cell cycle [32] and p21/ CDKN1A is a cyclin-dependent kinase inhibitor [33]. Western blot analysis of CCND1 expression showed a stimulation on Day 1 of culture by EGF and SB203580 (Fig. 5, A and B). As expected, CDKN1A was reciprocally expressed (Fig. 5A). Taken together, these data show a stimulation of a proliferative state by EGF and the MAPK11/14 inhibitor, supporting the BrdU incorporation data.
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The Effect of MAPK11/14 Inhibitors on CGB Secretion from Explant Cultures
Explant cultures preserve the in vivo architecture of the placental villous unit with the physiological juxtaposition of ST, CT, and the underlying stromal cells. As described previously [30], we found that CGB secretion from explants initially declined, then increased, and finally started to decline again after 7 days (Fig. 6). This corresponds to degeneration of original syncytium, regeneration of a new syncytium, and then gradual death of the new layer [30]. EGF had no effect on this process, but the MAPK11/14 inhibitor SB203580 completely inhibited CGB secretion during the regeneration phase (Fig. 6) both in the presence and absence of EGF. Thus, inhibiting MAPK11/14 activity appeared to prevent the regeneration of syncytium producing CGB in this model.
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DISCUSSION
We hypothesized that EGF activation of MAPK11/14 (its phosphorylation) mediates EGF-stimulated trophoblast differentiation. By blocking MAPK11/14 kinase activity with two different and specific [34] pyridinyl imidazole compounds (SB203580 and SB202190) [26], we found no significant loss in viability [15] but almost complete (99%) blocking of CT maturation into a CGB-secreting state and 70% inhibition of EGF-stimulated cell fusion. These data support our hypothesis.
Activation of MAPK11/14 is associated with differentiation in other cell types, including chondrocytes [22], adipocytes [35], and osteoblasts [36]. In chondrocytes and adipocytes, downstream action of MAPK11/14 is dependent on activation of CREB1 [35, 37]. The downstream targets of MAPK11/14 in trophoblasts are unknown, but its interaction with CREB1, which upregulates CGB transcription [38], seems likely. How blocking MAPK11/14 inhibits cell fusion is unclear, but activation of MAPK11/14 has been shown to influence the formation of tight junctions [39].
We have found that EGF reduces CGB secretion in cultured trophoblasts but others have published differing results. Huot et al. [14] showed EGF stimulated secretion from the choriocarcinoma cell line JAR but also showed no increase when explants were incubated with EGF (as do we, Fig. 6). Morrish et al. [7] documented increased release from primary human trophoblasts relative to Day 1, which is compatible with our studies. Amemiya et al. [12] cultured primary trophoblasts to show high levels of CGB secreted on Day 1, which decreased and again peaked at Day 4–5, then decreased thereafter. These cultures released CGB over time much as explant cultures do ([30]; this paper, Fig. 6) and the secretion pattern is probably due to heavy contamination with syncytial fragments [40], which our cultures do not have [4]. Our studies with purified term CT in culture show that EGF reduces CGB secretion but prolongs it. Isolated CTs differentiate spontaneously in culture [6], thus start out with very little CGB secretion on Day 1. Relative to Day 1 (when the cells are phenotypically still CT), EGF stimulates secretion. However, relative to Day 5, cells cultured without EGF (which have nonetheless differentiated), they produce less CGB. EGF thus downmodulates a mature cell function, secretion of CGB, but does not eliminate it. However, at the same time, EGF is prolonging CT proliferation. These reciprocal effects of EGF on CT proliferation and ST secretion of CGB have been reported in explant cultures of differing gestational ages [13].
Inhibition of MAPK11/14 does not reverse EGF downmodulation of CGB production and its prolongation. Rather, its inhibition completely blocks EGF-stimulated CGB production, exactly as it does for spontaneous production. Thus, the effects of MAPK11/14 and EGF are separate but overlapping. The effects of EGF are exerted primarily through ErbB1 in trophoblasts and include stimulation of trophoblast survival through four different pathways and stimulation of another (the MAPK11/14 (p38) pathway) [15], which partly regulates trophoblast differentiation (present study). EGF may thus stimulate differentiation triggering CGB expression via the MAPK11/14 pathway but modulate CGB expression by another pathway.
The delayed rise in CGB secretion seen with EGF led us to investigate whether EGF stimulated proliferation as well as differentiation in isolated trophoblast in culture, as it does in epithelial cells of the central nervous system [41]. We examined proliferation by BrdU uptake (a measure of the number of cells in S phase of the cell cycle) and found that EGF stimulates CT proliferation on Day 1 of culture and about 5-fold less on Day 5. We also examined proliferation by expression of cyclin D1/CCND1. EGF upregulates CCND1 at the G1-S boundary of the cell cycle in primary rat hepatocytes [42]. Without EGF, the cells fail to upregulate CCND1 and remain in the G1 phase of the cell cycle. However, transfection with a CCND1 construct allows the hepatocytes to traverse the G1-S boundary in the absence of EGF, indicating the crucial role of CCND1 to EGF's mitogenic action. In agreement with its stimulation of CT proliferation, we show that EGF stimulates CCND1 expression on both Day 1 and 5. We have previously shown that trophoblast proliferation as marked by thymidine incorporation was minimal compared with HELA cells [6], which would be expected to have a much higher rate of proliferation than that of primary cells. In the present study, we also found that proliferation declined, but the decline could be partially reversed by SB203580, further supporting MAPK11/14's role in enhancing trophoblast differentiation. Whether differentiation and proliferation are independently controlled remains to be determined.
Even without EGF stimulation, some cultured trophoblasts undergo proliferation, as demonstrated by untreated CCND1 expression and BrdU incorporation. Spontaneous low levels of proliferation are also a feature of term placental explants [43], which led us to investigate the effects of EGF and MAPK11/14 inhibition on CGB secretion in the explant model. Why EGF does not inhibit explant CGB secretion is unclear because the ST is the exclusive placental producer of CGB [44]. EGF may be being prevented from acting on underlying cytotrophoblasts by the existing syncytiotrophoblast layer, which has been reported to strongly express EGF receptors [45] and may be mopping up EGF at the concentration used. Alternatively, syncytial shielding of cytotrophoblasts may be partially preventing whatever factors are responsible for the activation of prodifferentiation signals in culture conditions, thereby preventing EGF's partial inhibitory effect. This theory fits with observation of syncytial sloughing in the first 2–3 days of explant culture (after which CGB secretion starts to rise) and with the lower levels of secreted CGB secretion of explants as compared with in vitro trophoblasts. Alternatively, it may be that the ST under these physiological conditions have fully occupied EGF receptors, perhaps by TGF
production in CT [46], and that further addition of EGF exerts very little action. We are currently examining varying concentrations of antibodies to EGF, TGF, and EGF receptors to delineate the exact role EGF has on explant function.
FOOTNOTES
1 Correspondence: Larry J. Guilbert, 6–25 HMRC, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2S2. FAX: 780 492 9828; larry.guilbert{at}ualberta.ca 
2 Current address: Academic Unit of Child Health, University of Manchester, Manchester, United Kingdom.
Received: 26 May 2005.
First decision: 25 June 2005.
Accepted: 15 August 2005.
REFERENCES
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