ToxSci Advance Access originally published online on March 2, 2006
Toxicological Sciences 2006 91(2):557-567; doi:10.1093/toxsci/kfj147
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Macaque Trophoblast Migration toward RANTES Is Inhibited by Cigarette Smoke–Conditioned Medium
Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, Davis, California 95616
1 To whom correspondence should be addressed at Department of Cell Biology and Human Anatomy, School of Medicine, Tupper Hall, One Shields Avenue, University of California, Davis, Davis, CA 95616-8643. Fax: (530) 752-8520. E-mail: gcdouglas{at}ucdavis.edu.
Received September 30, 2005; accepted February 22, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Trophoblast migration within the endometrium and uterine vasculature is essential for normal placental and fetal development. We previously demonstrated that macaque trophoblasts express the chemokine receptor CCR5 and that this receptor mediates trophoblast migration toward RANTES (regulated upon activation normal T-cell expressed and secreted). In the present paper we have used primary cultures of early gestation macaque trophoblasts to test the hypothesis that tobacco smoke inhibits trophoblast migration as the result of dysregulation of the RANTES/CCR5 chemotactic axis. Early gestation macaque trophoblasts were incubated in the absence or presence of cigarette smoke–conditioned medium (CSM). Cell migration was quantified using migration chambers. CCR5 and G protein receptor kinase 2 (GRK2) expression was measured by immunofluorescence microscopy and Western blotting. cAMP levels were measured by enzyme-linked immunosorbent assay. Trophoblast migration toward RANTES was reduced when cells were incubated in CSM. Trophoblasts also showed reduced expression of CCR5, increased levels of cAMP, and increased expression of GRK2. Finally, the secretion of RANTES by uterine endothelial cells was reduced by exposing the cells to CSM. These results support the idea that cigarette smoke constituents inhibit directional trophoblast migration by causing increased desensitization of trophoblast CCR5 and inhibiting the secretion of RANTES by endothelial cells.
Key Words: chemokine; CCR5; G protein receptor kinase 2; endothelium; placenta; invasion.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Implantation in human and nonhuman primates is accompanied by invasion of the endometrium by extravillous cytotrophoblast cells (Aplin et al., 1993
; Blankenship et al., 1993a,b; Enders and Blankenship, 1997; Enders and King, 1991; Fisher and Damsky, 1993; Pijnenborg et al., 1981). Migratory cytotrophoblasts also penetrate the uterine vasculature and eventually reach the spiral arteries, which they remodel (Blankenship et al., 1993b). Invasive trophoblasts show increased expression of ß1 and 
1 integrins and downregulation of ß4 integrin compared to villous trophoblasts (Aplin, 1993; Damsky et al., 1994; Soghomonians et al., 2002; Zhou et al., 1997). We have shown that trophoblast attachment to endothelial cells involves Vß3 (online) and ß1 (online) integrins (Douglas et al., 1999; Thirkill and Douglas, 1999). These integrins are also involved in trophoblast migration in vitro (Damsky et al., 1994; Douglas et al., 1999).
While several factors have been shown to be involved in facilitating trophoblast migration, the factors that regulate directional migration are poorly understood. Sato et al. (2003) showed that human first trimester trophoblasts migrated toward the chemokine RANTES (regulated upon activation normal T-cell expressed and secreted; CCL5) and that migration was mediated by the chemokine receptor CCR1. We have demonstrated that macaque trophoblasts express the chemokine receptor CCR5 and that this receptor mediates trophoblast migration toward RANTES (Thirkill et al., 2005). In addition we showed that RANTES induced the expression of trophoblast ß1 (online) integrin and that RANTES was expressed by uterine endothelial cells in the macaque. Chemokines and seven-transmembrane G protein–coupled chemokine receptors are well established as regulators of leukocyte chemotaxis and are also believed to play a role in regulating cell migration during developmental processes (Bonecchi et al., 1999; Fernandis et al., 2004; Firtel and Chung, 2000; Foxman et al., 1997; Rodriguez-Frade et al., 1999; Roth et al., 1995). Therefore, their involvement in the control of directional trophoblast migration is not unreasonable.
Exposure to cigarette smoke during pregnancy is associated with low birth weight, premature delivery, early pregnancy loss, perinatal mortality, and ectopic pregnancy (Shiverick and Salafia, 1999). Toxins present in cigarette smoke affect placental and uterine vascular function and can also alter fetal and placental cell function (Demarini and Preston 2005; Everson et al., 1986; Myers et al., 1996; Sanyal et al., 1993). Smoking subjects the fetus to hypoxia brought about by the transplacental passage of carbon dioxide and nicotine (Soothill et al., 1996). Adverse effects on fetal development and pregnancy outcome may also be related to effects of tobacco smoke components on trophoblast migration. This could be due to direct effects on trophoblast cells and/or to effects on extraneous factors that regulate migration. Villous explants from human chorionic villous samples showed reduced trophoblast cell column formation and reduced trophoblastic outgrowth when exposed to nicotine (Genbacev et al., 1995).
In the present paper we have tested the hypothesis that trophoblast migration is disrupted by the effects of tobacco smoke constituents on the RANTES/CCR5 chemotactic axis. The results show that trophoblast migration toward RANTES is reduced when the cells are incubated in cigarette smoke–conditioned medium (CSM). The cells also showed reduced expression of CCR5, increased levels of cAMP, and increased expression of G protein receptor kinase 2 (GRK2). Finally, the secretion of RANTES by uterine endothelial cells was reduced by exposing the cells to aqueous cigarette smoke extract. These results support the idea that cigarette smoke constituents inhibit trophoblast migration by causing increased desensitization of CCR5 and by inhibiting RANTES secretion by endothelial cells.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Trophoblast isolation and culture.
All procedures involving animals were performed in accordance with the National Institutes of Health Guide for the Care and Use Laboratory Animals and under the approval of the University of California, Davis, Animal Care and Use Committee. We have previously described in detail a procedure used to isolate trophoblast cells from term (165-day) macaque placentas (Douglas and King, 1990
). The same procedure was used in the present case to isolate cells from 40- to 60-day placental/endometrial tissue. Yields were approximately 8 x 106 cells/g tissue. The cells were subjected to an additional purification step using immunomagnetic microspheres coated with anti-HLA antibodies (Douglas and King, 1989). 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 ß1 integrin positive, consistent with migratory trophoblasts (Soghomonians et al., 2002). Trophoblasts were routinely cultured in Ham's/Waymouth's medium (50:50, vol/vol) containing 10% FetalPlex (Gemini Bioproducts, Woodland, CA).
Endothelial cells.
Human uterine microvascular endothelial cells were purchased from Clonetics Corporation (San Diego, CA). Endothelial cells were maintained in Endothelial Basal Medium-2 (EBM, Clonetics Corporation) 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 10% FetalPlex. Cells were plated into eight-chamber LabTek slides that had been coated with type I rat tail collagen (BD Biosciences, Bedford, MA). Cells were not used beyond passage 9.
Cigarette smoke–conditioned medium.
Research cigarettes (1R4F; Tobacco and Health Research Institute, University of Kentucky, Lexington, KY) were smoked in a chamber under conditions to generate sidestream cigarette smoke. The smoking chamber has been described previously (Teague et al., 1994). The concentration of total suspended particulates ranged from 70 to 112 mg/m3. Nicotine levels were 1.25–10 mg/m3. For trophoblast experiments, 200 ml of serum-free culture medium (Ham's/Waymouth's) was placed in the chamber and exposed to smoke for 6 h at room temperature to produce stock CSM. Smoke was not bubbled through the medium. Smoke-conditioned media were stored at 4°C and used within 2 weeks. For experiments, smoke-conditioned media were diluted with unconditioned medium containing FetalPlex (10% final concentration) or bovine serum albumin (BSA; 1% wt/vol, final concentration) as appropriate. For endothelial cell experiments, EBM (Clonetics Corporation) was exposed to cigarette smoke as above. The "stock" medium was then diluted using fresh EBM and supplemented with FetalPlex (10%) or BSA (1%, for serum-free conditions). Preliminary studies established that when diluted to concentrations of 25% or less, CSM was not cytotoxic to cells (based on the release of lactate dehydrogenase (LDH) and by DNA content/cell cycle analysis).
Trophoblast migration assay.
This was performed essentially as described previously (Douglas et al., 1999; Thirkill et al., 2005). The lower surfaces of FluorBlok migration chamber filter inserts (8 µm; BD Biosciences) were coated by placing the inserts on top of 70-µl aliquots of culture medium containing recombinant human RANTES (500nM; R&D Systems Inc., Minneapolis, MN) for 2 h at room temperature. The inserts were removed and allowed to air-dry after which nonspecific sites on both surfaces of the filter were blocked by soaking in 1% heat-denatured BSA. Control inserts received only the BSA coating. The inserts were placed in a cluster dish. The possibility that RANTES bound to filters could subsequently dissociate and generate a soluble gradient was considered. When RANTES-coated inserts were incubated in culture medium, less than 13% of the bound chemokine was detected by enzyme-linked immunosorbent assay (ELISA) in the culture medium after 24 h. Thus, solid-phase RANTES is the major source of RANTES to which the cells are exposed under our culture conditions. Early gestation macaque trophoblasts were labeled with Calcein-AM (Invitrogen, Carlsbad, CA) as we have described previously (Douglas et al., 1999; Thirkill et al., 2005), and 100,000 labeled cells were then added to the upper chambers. The cells were incubated for 24 h at 37°C in the presence or absence of CSM, and then the entire cluster dish containing the inserts was placed in a fluorescence plate reader (Cytofluor 2300, Millipore, Billerica, MA) and the level of fluorescence in the lower chamber was measured. FluorBlok filter inserts do not allow fluorescence in the upper chamber (i.e., nonmigrated cells) to be measured. Fluorescence in the lower chamber therefore provides a direct measure of the extent of cell migration which was expressed as a percentage of the total fluorescence of the input cells. To control for chemokinetic migration, some trophoblasts were incubated in inserts that contained an equal concentration of soluble RANTES (12nM) in both the upper and lower chambers.
Immunocytochemistry and image analysis.
Rabbit polyclonal antibodies against CCR5, CCR1, and CCR3 (sc-13950, sc-7934, and sc-7897) and a polyclonal antibody (sc-562) against human GRK2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A polyclonal antibody against cytokeratin (pan) was purchased from Zymed (San Francisco, CA). Alexa Fluor 488–labeled goat anti-rabbit Ig was purchased from Molecular Probes (Eugene, OR).
Cells in LabTek culture chambers were fixed and permeabilized in ice-cold methanol then stained with primary antibody. Primary antibodies were detected using Alexa Fluor 488–labeled goat anti-rabbit Ig. Antibody controls in which cells were incubated with nonimmune rabbit Ig were always included. 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 Q-Imaging software. Identical exposure and brightness level settings were used for test and control samples.
For image analysis, captured digitized images were imported into Simple PCI software (Compix Inc., Imaging Systems, Cranberry Township, PA) to determine levels of antibody-associated fluorescence intensity as we have described previously (Thirkill et al., 2004). The software was calibrated using the InSpeck fluorescence Image Intensity Calibration Kit (6-µm beads; Molecular Probes). Relative cellular fluorescence intensity in samples was determined by reference to a standard curve generated using the calibration beads. Cells were contoured using the software and the mean pixel density normalized to cell area was calculated. Background fluorescence (determined using cells incubated with control immunoglobulin) was subtracted from sample fluorescence values. The area-normalized fluorescence was taken as an indicator of expression. Four microscope fields (at least 50 objects/field) were analyzed for each sample well, and experiments were repeated three times.
Western blotting.
Cultures were washed with Dulbecco's modified phosphate-buffered saline containing Ca2+ and Mg2+. The cells were then lysed on ice by the addition of Mammalian Protein Extraction Reagent (Pierce Biochemicals, Rockford, IL) supplemented with 1% Protease Inhibitor Cocktail (Sigma-Aldrich Co., St. Louis, MO). The lysate was homogenized by repeated passage through a 27-gauge needle, then mixed with an equal volume of Laemmli sample buffer (BioRad Laboratories, Hercules, CA) containing 5% ß-mercaptoethanol and heated in a boiling water bath for 5 min. The samples were immediately chilled on ice and loaded onto sodium dodecyl sulfate–polyacrylamide gels (Gradiopore; 8%, Promega, Madison, WI) at 20 µg protein/lane. After electrophoresis the proteins were transferred to a nitrocellulose membrane (BioRad Laboratories). The membrane was blocked for 1 h in 1% nonfat dried milk solution. For analysis of CCR5, the blocked membrane was incubated overnight with a goat anti-CCR5 antibody (ab1673; Abcam Inc., Cambridge, MA), then washed and incubated with donkey anti-goat IgG labeled with horseradish peroxidase (HRP) (Santa Cruz Biotechnology). For analysis of GRK2, the membrane was incubated overnight with a rabbit antibody against human GRK2 (Santa Cruz Biotechnology), washed, and then incubated with a goat anti-rabbit IgG labeled with HRP (Pierce Biochemicals). After further washing, the membrane was incubated with chemiluminescent substrate (SuperSignal West Dura; Pierce Biochemicals) for 5 min at room temperature. The membrane was then exposed to an x-ray film (Pierce Biochemicals). Scanned images of exposed x-ray films 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 density of a loading control (either tubulin or GAPDH, detected using monoclonal antibodies from Chemicon (Temecula, CA) and Santa Cruz Biotechnologies, respectively).
RANTES assay.
Endothelial cells were incubated under serum-free (EBM-BSA) conditions for 24 h and preincubated in CSM (also serum free) for 30 min after which the cells were stimulated with TNF- (25 ng/ml) and IFN-
(10 ng/ml) for 24 h. RANTES in cell culture supernatants was then assayed using an ELISA kit (Quantitkine DRN00B, R&D Systems). Results are expressed in picograms of RANTES per microgram of cellular protein. Typical values for RANTES in our endothelial cell culture media before protein normalization ranged from around 80 to 500 pg/ml, depending on treatment.
To ensure that CSM was not directly affecting RANTES stability, a standardized amount of RANTES was incubated in the presence or absence of CSM (25%) (and in the absence of cells) for 24 h at 37°C. Measurement of RANTES levels by ELISA confirmed that RANTES levels did not change with time and were unaffected by incubation in CSM (results not shown).
cAMP assay.
Cells were incubated in the presence or absence of CSM for 24 h. For the last 3 h, serum-free conditions were used. 3-Isobutyl-1-methylxanthine (IBMX; 0.5mM) was added for 30 min followed by forskolin (5µM) for 30 min. RANTES (100nM) was then added and the incubation continued for different times as indicated in the figure legends. The cells were then lysed and cAMP was measured using a chemiluminescence-based ELISA kit purchased from Applied Biosystems (Foster City, CA). cAMP levels were calculated in picomoles of cAMP per microgram of cell protein and then expressed as a percentage of the cAMP level in the presence of forskolin alone.
Cell viability assays.
LDH activity in culture supernatants was determined using the fluorescence-based CytoTox-ONE kit obtained from Promega Corporation(Madison, WI). LDH activity is expressed in arbitrary fluorescence units.
Apoptosis was assessed by DNA content/cell cycle analysis (Hotz et al., 1994; Ormerod, 1992). In this procedure, propidium iodide is used to stain DNA in permeabilized cells. A cell cycle profile is then obtained by flow cytometry. Apoptosis is established by an increase in the number of subdiploid (A0) cells at the expense of the G0/G1 population. Briefly, after experimental treatments, cells were fixed in ice-cold ethanol, washed, and then incubated with DNA extraction buffer (Hotz et al., 1994). The cells were then incubated with propidium iodide (Sigma Chemicals, St. Louis, MO) followed by RNase A (Worthington Biochemicals, Lakewood, NJ). Cell cycle profiles were then obtained by flow cytometry using ModFit software (Verity Software Inc., Topsham, ME) and TreeStar software (Portland, OR). Results are presented as the percentage of cells in the subdiploid (A0) and G0/G1 populations. A positive control consisted of cells in which apoptosis was induced by incubation with hydrogen peroxide (500µM).
Statistical analyses.
Experiments were repeated at least three times. Trophoblast cells were isolated from three different placentas and were not pooled. Data were analyzed by unpaired t-test or two-way ANOVA using the Prism software program (GraphPad Inc., San Diego, CA). Differences were considered significant if p < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
CSM Inhibits Trophoblast Migration
As we reported previously (Thirkill et al., 2005
), trophoblasts show little or no directional migration when soluble RANTES is present at an equal concentration in both the upper and lower compartments of in vitro migration chambers (Fig. 1A, left column) but do show significant migration to the lower chamber when only the underside of the filter is coated with RANTES (Fig. 1A). The results in Figure 1A also show that migration toward RANTES was reduced in a concentration-dependent manner when the cells were incubated with different dilutions of CSM. Using 25% CSM, migration was significantly (p < 0.05) reduced by almost 70% compared to the control. Trophoblast viability, as assessed by LDH release, was not significantly compromised by exposure to CSM (Fig. 1B). Also, incubation in 25% CSM did not induce significant trophoblast apoptosis (Table 1) as measured by DNA content/cell cycle analysis.
|
|
CSM Reduces CCR5 Expression by Trophoblasts
Since macaque trophoblast migration toward RANTES requires expression of the RANTES receptor CCR5 (Thirkill et al., 2005
), we next examined the effect of CSM on the expression of this receptor. Trophoblasts were incubated in CSM for 24 h, and CCR5 expression was assessed by immunofluorescence microscopy. Compared to control cultures (Fig. 2A), cultures exposed to CSM (Fig. 2C) had reduced CCR5 immunofluorescence. Figure 2E shows the immunoglobulin control. Image analysis (Fig. 2G) of micrographs from several experiments confirmed that CCR5 staining was significantly reduced (p = 0.0161) by 23% in cells exposed to CSM. Reduced expression of CCR5 when cells were incubated in CSM was also demonstrated by Western blotting analysis of cell lysates from control (Fig. 2H, lane 1) and CSM-treated (Fig. 2E, lane 2) trophoblast cultures. Densitometric analyses of the bands from several experiments are shown in Figure 2I and confirm that CCR5 protein levels were significantly reduced in cells exposed to CSM compared to controls.
|
Since RANTES can bind to CCR1 and CCR3 in addition to CCR5, we examined CCR1 and CCR3 expression by macaque trophoblasts using immunofluorescence microscopy (Fig. 3). The level of fluorescence in CCR1- and CCR3-stained cultures (Figs. 3A and 3C) was compared to that of CCR5 (Fig. 3E) and an immunoglobulin control (Fig. 3G). CCR1- and CCR3-associated immunofluorescence was low and similar to the immunoglobulin control (Fig. 3G), suggesting little or no expression of these receptors in macaque trophoblasts.
|
We previously showed that RANTES causes an upregulation of trophoblast ß1 integrin expression (Thirkill et al., 2005
) and that this integrin is involved in trophoblast adhesion and migration (Douglas et al., 1999; Thirkill and Douglas, 1999). In order to determine whether the CSM-mediated inhibition of trophoblast migration involved alterations in ß1 integrin expression, cells were preincubated in CSM and then exposed to RANTES for 24 h after which levels of ß1 integrin were quantified by Western blot. No effect of CSM on ß1 integrin levels was found (results not shown).
CSM Abrogates the RANTES-Mediated Reduction in cAMP Levels
While the effect of CSM on trophoblast CCR5 expression was significant, it was modest. We therefore sought to determine whether other aspects of CCR5 function were affected by CSM. Binding of RANTES to CCR5 results in activation of Gi and subsequent inhibition of adenylyl cyclase and reduction in cellular cAMP levels (Ling et al., 1999; Myers et al., 1995). To determine whether the effects of CSM were associated with alterations in RANTES-promoted G protein signaling, trophoblasts were preincubated in the presence or absence of CSM after which RANTES was added and cAMP levels were measured. cAMP levels were increased 17-fold over basal levels by forskolin in control trophoblast cultures (results not shown). As expected, RANTES attenuated the forskolin-induced cAMP accumulation in a time-dependent manner (Fig. 4, lower curve). After 10 min, RANTES had significantly reduced the accumulation of cAMP by almost 50%. However, preincubation of the cells in CSM abrogated the RANTES-mediated decrease in cAMP levels (Fig. 4, upper curve). After 10 min incubation with RANTES, cAMP levels were only reduced by about 10% under these conditions. ANOVA posttesting showed that none of the cAMP values for cultures exposed to CSM were significantly different from the zero time point (forskolin alone), whereas cAMP levels in cultures incubated in the absence of CSM were significantly different (Dunnett's test, p < 0.05) from the zero time point at 10, 15, and 30 min.
|
GRK2 Expression Is Increased after Incubation of Trophoblasts with CSM
As with other G protein–coupled receptors, CCR5 function can be regulated by G protein–coupled receptor kinases (GRKs) and in particular GRK2 (Oppermann, 2004
). We therefore asked whether the expression of GRK2 was altered by incubation of trophoblasts in CSM. The immunofluorescence images in Figure 5 show that trophoblasts incubated for 24 h in CSM (Fig. 5C) had increased GRK2 staining compared to untreated control cells (Fig. 5A). Figure 5E shows cells stained with normal rabbit IgG. Corresponding phase-contrast images (B, D, and F) are also shown beneath each fluorescence image. Quantitation of the fluorescence in multiple images from several experiments by image analysis confirmed the visual impression and suggests that GRK2 expression was significantly increased (p < 0.05) by about 1.6-fold in CSM-treated cells compared to the control cells (Fig. 5G). A significant increase (2.5-fold) in GRK2 expression in CSM-incubated cells compared to control cells was also demonstrated by Western blot analysis of trophoblast cell lysates (Figs. 5H and 5I).
|
Cigarette Smoke Extract Inhibits the Secretion of RANTES by Uterine Endothelial Cells
We have previously shown that uterine endothelial cells express RANTES (Thirkill et al., 2005
) and suggested that this could serve to regulate the directional migration of endovascular trophoblasts. It was therefore of interest to determine the effect of CSM on endothelial RANTES production. When uterine endothelial cells were incubated in the presence of CSM, the secretion of RANTES in response to TNF-/IFN- was significantly reduced compared to untreated control cultures (Fig. 6). Endothlelial cell viability as assessed by DNA content/cell cycle analysis was not affected by incubation in 25% CSM for 24 h (results not shown).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The results presented here show that CSM disrupts the trophoblast chemotactic response to RANTES. The inhibitory effect of CSM correlated with decreased trophoblast CCR5 expression, increased expression of GRK2, and abrogation of the RANTES-mediated decrease in cellular cAMP levels. We previously demonstrated that macaque trophoblasts migrate toward RANTES in vitro and that migration was dependent on the expression of CCR5 and was not due to chemokinesis (Thirkill et al., 2005
). The results presented here confirm the directional, nonchemokinetic, migration of trophoblasts toward RANTES. Because RANTES is predominantly bound to the filter of the migration chamber insert, we conclude that migration is haptotactic under our culture conditions. However, a small chemotactic component cannot be ruled out due to the low amount of RANTES dissociation from the filter. On the basis of these observations and the fact that RANTES is expressed by human and macaque endometrium (Altman et al., 1999; Hornung et al., 1997, 2001; Sato et al., 2003; Thirkill et al., 2005), we suggested that CCR5/RANTES plays a role in regulating trophoblast migration within the endometrium and endometrial vasculature during early gestation. This migratory activity is essential for the establishment of a blood supply to the developing placenta and fetus. The present observations suggest that some of the adverse effects of exposure to cigarette smoke on pregnancy outcome may be related to cigarette smoke–mediated disruption of RANTES-directed trophoblast invasion via haptotactic and/or chemotactic mechanisms. Disruption of invasion would result in placental dysfunction and impairment of fetal development. The soluble components in cigarette smoke responsible for inhibiting trophoblast invasion are not known at this time. While cigarette smoke contains thousands of different chemicals, many of these are not water soluble. An analysis of the water-soluble fraction of cigarette smoke revealed 479 components (Schumacher et al., 1977).
Studies with other cell systems indicate that tobacco smoke or tobacco smoke constituents variously inhibit or stimulate cell migration. For example, nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone stimulated the migration of lung carcinoma cells (Xu and Deng, 2004), whereas sidestream whole tobacco smoke or cigarette smoke extract inhibited the migration of fibroblasts (Nakamura et al., 1995; Wong et al., 2004) and polymorphonuclear leukocytes (Bridges and Hsieh, 1986). The mechanism by which CSM inhibits trophoblast migration is clearly important. The inhibition of trophoblast migration toward RANTES was associated with reduced expression of the RANTES receptor CCR5. Given its well-established role in leukocyte chemotaxis (Fine et al., 2001; Oppermann, 2004) and its involvement in trophoblast migration (Thirkill et al., 2005), reduced expression of CCR5 would be consistent with dysregulation of migration. Thus, reduced CCR5 expression would limit the cell's ability to respond to the chemotactic stimulus presented by RANTES. However, while the reduction in expression under our in vitro conditions was statistically significant, it was modest (about 23% of control values). Nonetheless, it must be remembered that the effects of chronic exposure to cigarette smoke constituents on trophoblast CCR5 expression in vivo may be greater than the effects observed here.
Tobacco smoke has been found to stimulate or inhibit the release of chemotactic factors from different cell types (Masubuchi et al., 1998; Shoji et al., 1995), but we are unaware of any reports of the effects of tobacco smoke on chemokine receptor expression. The mechanism by which CSM reduces CCR5 expression is not known at this time. CCR5 expression can be regulated at several levels. For example, expression of CCR5 at the cell surface can be reduced by increasing the rate of receptor endocytosis (Mueller et al., 2002; Oppermann et al., 1999). CCR5 expression can also be regulated at the transcriptional level (Percherancier et al., 2001; Saccani et al., 2000). While we cannot discount increased endocytosis as a potential mechanism, our Western blot data suggest that total CCR5 protein levels are decreased. Further studies will be required to determine whether this is the result of decreased synthesis or increased degradation. If the results found here for trophoblasts extend to other chemokine receptor–expressing cells, then they might help explain other toxic effects of cigarette smoke both within the pregnant uterus and other organs.
Studies with other cell systems indicate that RANTES activates inhibitory G proteins with resultant inhibition of adenylyl cyclase and stimulation of migration (Aramori et al., 1997; Fine et al., 2001). On the other hand, increased cAMP levels are associated with inhibition of monocyte chemotaxis (Aramori et al., 1997; Fine et al., 2001) and with inhibition of trophoblast migration (McKinnon et al., 2001). The observation reported here that CSM prevents the RANTES-induced reduction in cAMP levels is therefore consistent with the disruption of RANTES/CCR5-coupled G protein signaling and inhibition of migration.
Agonist-dependent activation of CCR5-mediated G protein signaling is followed by agonist-dependent CCR5 desensitization which uncouples G proteins and limits further signaling (Oppermann, 2004). CCR5 desensitization can involve the phosphorylation of CCR5 mediated by GRKs and particularly GRK2 (Aramori et al., 1997; Mueller and Strange, 2004; Oppermann, 2004). Our results show that incubation of trophoblasts in CSM results in increased expression of GRK2. We speculate that this increase in GRK2 expression causes enhanced CCR5 desensitization, loss of G protein activation, and inhibition of migration. The absence of RANTES-induced reduction in cAMP levels in trophoblasts exposed to CSM (as discussed above) is consistent with enhanced CCR5 desensitization. The idea is also supported by the observation that overexpression of GRK2 in smooth muscle cells is associated with increased CCR5 desensitization and inhibition of chemotaxis (Peppel et al., 2002). Reduced expression of GRK2 in T lymphocytes is associated with increased migratory activity (Vroon et al., 2004a). In contrast, deficiency of GRK6 was associated with "increased" neutrophil chemotaxis and "decreased" lymphocyte chemotaxis (Fong et al., 2002; Vroon et al., 2004a,b). These, plus other recent observations, suggest that different GRKs may catalyze phosphorylation of G-protein-coupled receptors at different sites and so elicit different functional responses (Willets et al., 2003). How these different effects are coordinated in cells expressing several GRKs is not known. To our knowledge GRK expression has never been examined in human or macaque trophoblasts. Based on the results presented here, further studies on the regulation of GRK activity in trophoblasts are warranted.
The inhibitory effect of CSM on RANTES secretion by uterine endothelial cells suggests an additional way in which cigarette smoke constituents could dsyregulate trophoblast migration. We have shown that trophoblast migration toward RANTES in vitro is dose dependent (Thirkill et al., 2005) and so diminution in RANTES secretion by endothelial cells (or by stromal cells) would be expected to reduce trophoblast migratory activity. RANTES is known to function as a haptotactic factor by virtue of its ability to bind to cell-surface glycosaminoglycans or to extracellular matrix (Kuschert et al., 1999; Proudfoot et al., 2003). It also functions as a chemotactic factor (Bonecchi et al., 1999; Franitza et al., 1999). Inhibition of RANTES secretion by cigarette smoke constituents could reduce both its haptotactic and chemotactic properties. Previous studies demonstrated that a cigarette smoke condensate inhibits or stimulates the secretion of some cytokines and chemokines by human aortic endothelial cells (Nordskog et al., 2005). While RANTES was not included in this study, the secretion of another CCR5 ligand, MCP-1, was reduced by the cigarette smoke condensate. On the other hand, the secretion of MCP-1 was increased in lung fibroblasts exposed to cigarette smoke condensate (Sato et al., 1999), and lung epithelial cells showed increased secretion of MCP-1 and unidentified chemotactic factors (Masubuchi et al., 1998; Shoji et al., 1995). While we have not tested this, another possibility is that cigarette smoke constituents reduce the production of RANTES by other cells within the uterine stroma. This could reduce interstitial trophoblast invasion.
In summary, the CSM-mediated inhibition of RANTES-directed trophoblast migration and the CSM-mediated inhibition of RANTES secretion by uterine endothelial cells described here suggest two, not necessarily mutually exclusive, means whereby cigarette smoke constituents could adversely affect trophoblast migration within the uterus. These effects could contribute to the negative effects of cigarette smoking on pregnancy outcome.
|
ACKNOWLEDGMENTS |
|---|
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 Philip Morris USA Inc. (G.C.D.) and NIHR01HL068035-01A1 (G.C.D.).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Altman, G. B., Gown, A. M., Luchtel, D. L., and Baker, C. (1999). RANTES production by cultured primate endometrial epithelial cells. Am. J. Reprod. Immunol. 42, 168–174.[Medline]
Aplin, J. D. (1993). Expression of integrin alpha 6 beta 4 in human trophoblast and its loss from extravillous cells. Placenta 14, 203–215.[CrossRef][ISI][Medline]
Aplin, J. D., Vicovac, L., and Sattar, A. (1993). Cell interactions in trophoblast invasion. In Trophoblast Cells. Pathways for Maternal-Embryonic Communication (M. J. Soares, S. Handwerger, and F. Talamantes, Eds.), pp. 92–108. Springer-Verlag, New York.
Aramori, I., Ferguson, S. S., Bieniasz, P. D., Zhang, J., Cullen, B., and Cullen, M. G. (1997). Molecular mechanism of desensitization of the chemokine receptor CCR-5: Receptor signaling and internalization are dissociable from its role as an HIV-1 co-receptor. EMBO J. 16, 4606–4616.[CrossRef][ISI][Medline]
Blankenship, T. N., Enders, A. C., and King, B. F. (1993a). Trophoblastic invasion and modification of uterine veins during placental development in macaques. Cell Tissue Res. 274, 135–144.[CrossRef][ISI][Medline]
Blankenship, T. N., Enders, A. C., and King, B. F. (1993b). Trophoblastic invasion and the development of uteroplacental arteries in the macaque: Immunohistochemical localization of cytokeratins, desmin, type IV collagen, laminin, and fibronectin. Cell Tissue Res. 272, 227–236.[CrossRef][ISI][Medline]
Bonecchi, R., Polentarutti, N., Luini, W., Borsatti, A., Bernasconi, S., Locati, M., Power, C., Proudfoot, A., Wells, T. N., Mackay, C., et al. (1999). Up-regulation of CCR1 and CCR3 and induction of chemotaxis to CC chemokines by IFN-gamma in human neutrophils. J. Immunol. 162, 474–479.
Bridges, R. B., and Hsieh, L. (1986). Effects of cigarette smoke fractions on the chemotaxis of polymorphonuclear leukocytes. J. Leukoc. Biol. 40, 73–85.[Abstract]
Damsky, C. H., Librach, C., Lim, K. H., Fitzgerald, M. L., McMaster, M. T., Janatpour, M., Zhou, Y., Logan, S. K., and Fisher, S. J. (1994). Integrin switching regulates normal trophoblast invasion. Development 120, 3657–3666.[Abstract]
Demarini, D. M., and Preston, R. J. (2005). Smoking while pregnant: Transplacental mutagenesis of the fetus by tobacco smoke. JAMA 293, 1264–1265.
Douglas, G. C., and King, B. F. (1989). Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres. J. Immunol. Methods 119, 259–268.[CrossRef][ISI][Medline]
Douglas, G. C., and King, B. F. (1990). Isolation and morphologic differentiation in vitro of villous cytotrophoblast cells from rhesus monkey placenta. In Vitro Cell. Dev. Biol. 26, 754–758.[ISI][Medline]
Douglas, G. C., Thirkill, T. L., and Blankenship, T. N. (1999). Vitronectin receptors are expressed by macaque trophoblast cells and play a role in migration and adhesion to endothelium. Biochim. Biophys. Acta/General Subjects 1452, 36–45.
Enders, A. C., and Blankenship, T. N. (1997). Modification of endometrial arteries during invasion by cytotrophoblast cells in the pregnant macaque. Acta Anat. (Basel) 159, 169–193.[ISI][Medline]
Enders, A. C., and Blankenship, T. N. (1999). Vascular invasion during implantation and placentation. In Embryo Implantation: Molecular, Cellular, and Clinical Aspects (D. D. Carson, Ed.), pp. 23–38. Springer-Verlag, New York.
Enders, A. C., and King, B. F. (1991). Early stages of trophoblastic invasion of the maternal vascular system during implantation in the macaque and baboon. Am. J. Anat. 192, 329–346.[CrossRef][ISI][Medline]
Everson, R. B., Randerath, E., Santella, R. M., Cefalo, R. C., Avitts, T. A., and Randerath, K. (1986). Detection of smoking-related covalent DNA adducts in human placenta. Science 231, 54–57.
Fernandis, A. Z., Prasad, A., Band, H., Klosel, R., and Ganju, R. K. (2004). Regulation of CXCR4-mediated chemotaxis and chemoinvasion of breast cancer cells. Oncogene 23, 157–167.[CrossRef][ISI][Medline]
Fine, J. S., Byrnes, H. D., Zavodny, P. J., and Hipkin, R. W. (2001). Evaluation of signal transduction pathways in chemoattractant-induced human monocyte chemotaxis. Inflammation 25, 61–67.[CrossRef][ISI][Medline]
Firtel, R. A., and Chung, C. Y. (2000). The molecular genetics of chemotaxis: Sensing and responding to chemoattractant gradients. Bioessays 22, 603–615.[CrossRef][ISI][Medline]
Fisher, S. J., and Damsky, C. H. (1993). Human cytotrophoblast invasion. Semin. Cell Biol. 4, 183–188.[CrossRef][Medline]
Fong, A. M., Premont, R. T., Richardson, R. M., Yu, Y. R., Lefkowitz, R. J., and Patel, D. D. (2002). Defective lymphocyte chemotaxis in beta-arrestin2- and GRK6-deficient mice. Proc. Natl. Acad. Sci. U.S.A. 99, 7478–7483.
Foxman, E. F., Campbell, J. J., and Butcher, E. C. (1997). Multistep navigation and the combinatorial control of leukocyte chemotaxis. J. Cell Biol. 139, 1349–1360.
Franitza, S., Alon, R., and Lider, O. (1999). Real-time analysis of integrin-mediated chemotactic migration of T lymphocytes within 3-D extracellular matrix-like gels. J. Immunol. Methods 225, 9–25.[CrossRef][ISI][Medline]
Genbacev, O., Bass, K. E., Joslin, R. J., and Fisher, S. J. (1995). Maternal smoking inhibits early human cytotrophoblast differentiation. Reprod. Toxicol. 9, 245–255.[CrossRef][ISI][Medline]
Hornung, D., Klingel, K., Dohrn, K., Kandolf, R., Wallwiener, D., and Taylor, R. N. (2001). Regulated on activation, normal T-cell-expressed and -secreted mRNA expression in normal endometrium and endometriotic implants: Assessment of autocrine/paracrine regulation by in situ hybridization. Am. J. Pathol. 158, 1949–1954.
Hornung, D., Ryan, I. P., Chao, V. A., Vigne, J. L., Schriock, E. D., and Taylor, R. N. (1997). Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells. J. Clin. Endocrinol. Metab. 82, 1621–1628.
Hotz, M. A., Gong, J., Traganos, F., and Darzynkiewicz, Z. (1994). Flow cytometric detection of apoptosis: Comparison of the assays of in situ DNA degradation and chromatin changes. Cytometry 15, 237–244.[CrossRef][ISI][Medline]
Kuschert, G. S., Coulin, F., Power, C. A., Proudfoot, A. E., Hubbard, R. E., Hoogewerf, A. J., and Wells, T. N. (1999). Glycosaminoglycans interact selectively with chemokines and modulate receptor binding and cellular responses. Biochemistry (Mosc.) 38, 12959–12968.
Ling, K., Wang, P., Zhao, J., Wu, Y. L., Cheng, Z. J., Wu, G. X., Hu, W., Ma, L., and Pei, G. (1999). Five-transmembrane domains appear sufficient for a G protein-coupled receptor: Functional five-transmembrane domain chemokine receptors. Proc. Natl. Acad. Sci. U.S.A. 96, 7922–7927.
Masubuchi, T., Koyama, S., Sato, E., Takamizawa, A., Kubo, K., Sekiguchi, M., Nagai, S., and Izumi, T. (1998). Smoke extract stimulates lung epithelial cells to release neutrophil and monocyte chemotactic activity. Am. J. Pathol. 153, 1903–1912.
McKinnon, T., Chakraborty, C., Gleeson, L. M., Chidiac, P., and Lala, P. K. (2001). Stimulation of human extravillous trophoblast migration by igf-ii is mediated by igf type 2 receptor involving inhibitory g protein(s) and phosphorylation of mapk. J. Clin. Endocrinol. Metab. 86, 3665–3674.
Mueller, A., Kelly, E., and Strange, P. G. (2002). Pathways for internalization and recycling of the chemokine receptor CCR5. Blood 99, 785–791.
Mueller, A., and Strange, P. G. (2004). Mechanisms of internalization and recycling of the chemokine receptor, CCR5. Eur. J. Biochem. 271, 243–252.[ISI][Medline]
Myers, S. J., Wong, L. M., and Charo, I. F. (1995). Signal transduction and ligand specificity of the human monocyte chemoattractant protein-1 receptor in transfected embryonic kidney cells. J. Biol. Chem. 270, 5786–5792.
Myers, S. R., Spinnato, J. A., Pinorini-Godly, M. T., Cook, C., Boles, B., and Rodgers, G. C. (1996). Characterization of 4-aminobiphenyl-hemoglobin adducts in maternal and fetal blood-samples. J. Toxicol. Environ. Health 47, 553–566.[CrossRef][ISI][Medline]
Nakamura, Y., Romberger, D. J., Tate, L., Ertl, R. F., Kawamoto, M., Adachi, Y., Mio, T., Sisson, J. H., Spurzem, J. R., and Rennard, S. I. (1995). Cigarette smoke inhibits lung fibroblast proliferation and chemotaxis. Am. J. Respir. Crit. Care Med. 151, 1497–1503.[Abstract]
Nordskog, B. K., Fields, W. R., and Hellmann, G. M. (2005). Kinetic analysis of cytokine response to cigarette smoke condensate by human endothelial and monocytic cells. Toxicology 212, 87–97.[CrossRef][ISI][Medline]
Oppermann, M. (2004). Chemokine receptor CCR5: Insights into structure, function, and regulation. Cell. Signalling 16, 1201–1210.[CrossRef][ISI][Medline]
Oppermann, M., Mack, M., Proudfoot, A. E., and Olbrich, H. (1999). Differential effects of CC chemokines on CC chemokine receptor 5 (CCR5) phosphorylation and identification of phosphorylation sites on the CCR5 carboxyl terminus. J. Biol. Chem. 274, 8875–8885.
Ormerod, M. G. (1992). Flow Cytometry: A Practical Approach. Oxford University Press, New York.
Peppel, K., Zhang, L., Huynh, T. T., Huang, X., Jacobson, A., Brian, L., Exum, S. T., Hagen, P. O., and Freedman, N. J. (2002). Overexpression of G protein-coupled receptor kinase-2 in smooth muscle cells reduces neointimal hyperplasia. J. Mol. Cell. Cardiol. 34, 1399–1409.[CrossRef][ISI][Medline]
Percherancier, Y., Planchenault, T., Valenzuela-Fernandez, A., Virelizier, J. L., Arenzana-Seisdedos, F., and Bachelerie, F. (2001). Palmitoylation-dependent control of degradation, life span, and membrane expression of the CCR5 receptor. J. Biol. Chem. 276, 31936–31944.
Pijnenborg, R., Robertson, W. B., Brosens, I., and Dixon, G. (1981). Review article: Trophoblast invasion and the establishment of haemochorial placentation in man and laboratory animals. Placenta 2, 71–92.[ISI][Medline]
Proudfoot, A. E., Handel, T. M., Johnson, Z., Lau, E. K., LiWang, P., Clark-Lewis, I., Borlat, F., Wells, T. N., and Kosco-Vilbois, M. H. (2003). Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proc. Natl. Acad. Sci. U.S.A. 100, 1885–1890.
Rodriguez-Frade, J. M., Vila-Coro, A. J., Martin, A., Nieto, M., Sanchez-Madrid, F., Proudfoot, A. E., Wells, T. N., Martinez, A. C., and Mellado, M. (1999). Similarities and differences in RANTES- and (AOP)-RANTES-triggered signals: Implications for chemotaxis. J. Cell Biol. 144, 755–765.
Roth, S. J., Carr, M. W., and Springer, T. A. (1995). C-C chemokines, but not the C-X-C chemokines interleukin-8 and interferon-gamma inducible protein-10, stimulate transendothelial chemotaxis of T lymphocytes. Eur. J. Immunol. 25, 3482–3488.[ISI][Medline]
Saccani, A., Saccani, S., Orlando, S., Sironi, M., Bernasconi, S., Ghezzi, P., Mantovani, A., and Sica, A. (2000). Redox regulation of chemokine receptor expression. Proc. Natl. Acad. Sci. U.S.A. 97, 2761–2766.
Sanyal, M. K., Li, Y. L., Biggers, W. J., Satish, J., and Barnea, E. R. (1993). Augmentation of polynuclear aromatic hydrocarbon metabolism of human placental tissues of first-trimester pregnancy by cigarette smoke exposure. Am. J. Obstet. Gynecol. 168, 1587–1597.[ISI][Medline]
Sato, E., Koyama, S., Takamizawa, A., Masubuchi, T., Kubo, K., Robbins, R. A., Nagai, S., and Izumi, T. (1999). Smoke extract stimulates lung fibroblasts to release neutrophil and monocyte chemotactic activities. Am. J. Physiol. 277, L1149–L1157.[Medline]
Sato, Y., Higuchi, T., Yoshioka, S., Tatsumi, K., Fujiwara, H., and Fujii, S. (2003). Trophoblasts acquire a chemokine receptor, CCR1, as they differentiate towards invasive phenotype. Development 130, 5519–5532.
Schumacher, J. N., Green, C. R., Best, F. W., and Newell, M. P. (1977). Smoke composition. An extensive investigation of the water-soluble portion of cigarette smoke. J. Agric. Food Chem. 25, 310–320.[CrossRef][ISI][Medline]
Shiverick, K. T., and Salafia, C. (1999). Cigarette smoking and pregnancy I: Ovarian, uterine and placental effects. Placenta 20, 265–272.[CrossRef][ISI][Medline]
Shoji, S., Ertl, R. F., Koyama, S., Robbins, R., Leikauf, G., Von Essen, S., and Rennard, S. I. (1995). Cigarette smoke stimulates release of neutrophil chemotactic activity from cultured bovine bronchial epithelial cells. Clin. Sci. (Lond.) 88, 337–344.[Medline]
Soghomonians, A., Barakat, A. I., Thirkill, T. L., Blankenship, T. N., and Douglas, G. C. (2002). Effect of shear stress on migration and integrin expression in macaque trophoblast cells. Biochim. Biophys. Acta/General Subjects 1589, 233–246.
Soothill, P. W., Morafa, W., Ayida, G. A., and Rodeck, C. H. (1996). Maternal smoking and fetal carboxyhaemoglobin and blood gas levels. Br. J. Obstet. Gynaecol. 103, 78–82.[ISI][Medline]
Teague, S. V., Pinkerton, K. E., Goldsmith, G., Gebremichael, A., Chang, S., Jenkins, R. A., and Moneyhun, J. (1994). A side-stream cigarette smoke generation and exposure system for environmental tobacco smoke studies. Inhal. Toxicol. 6, 79–93.
Thirkill, T. L., and Douglas, G. C. (1999). The vitronectin receptor plays a role in the adhesion of human cytotrophoblast cells to endothelial cells. Endothelium 6, 277–290.[ISI][Medline]
Thirkill, T. L., Hendren, S. R., Soghomonians, A., Mariano, N. F., Barakat, A. I., and Douglas, G. C. (2004). Regulation of trophoblast beta1-integrin expression by contact with endothelial cells. Cell Commun. Signal. 2, 4.[CrossRef][Medline]
Thirkill, T. L., Lowe, K., Vedagiri, H., Blankenship, T. N., Barakat, A. I., and Douglas, G. C. (2005). Macaque trophoblast migration is regulated by RANTES. Exp. Cell Res. 305, 355–364.[CrossRef][ISI][Medline]
Vroon, A., Heijnen, C. J., Lombardi, M. S., Cobelens, P. M., Mayor, F., Jr., Caron, M. G., and Kavelaars, A. (2004a). Reduced GRK2 level in T cells potentiates chemotaxis and signaling in response to CCL4. J. Leukoc. Biol. 75, 901–909.
Vroon, A., Heijnen, C. J., Raatgever, R., Touw, I. P., Ploemacher, R. E., Premont, R. T., and Kavelaars, A. (2004b). GRK6 deficiency is associated with enhanced CXCR4-mediated neutrophil chemotaxis in vitro and impaired responsiveness to G-CSF in vivo. J. Leukoc. Biol. 75, 698–704.
Willets, J. M., Challiss, R. A., and Nahorski, S. R. (2003). Non-visual GRKs: Are we seeing the whole picture? Trends Pharmacol. Sci. 24, 626–633.[CrossRef][Medline]
Wong, L. S., Green, H. M., Feugate, J. E., Yadav, M., Nothnagel, E. A., and Martins-Green, M. (2004). Effects of "second-hand" smoke on structure and function of fibroblasts, cells that are critical for tissue repair and remodeling. BMC Cell Biol. 5, 13.[CrossRef][Medline]
Xu, L., and Deng, X. (2004). Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induces phosphorylation of mu- and m-calpain in association with increased secretion, cell migration, and invasion. J. Biol. Chem. 279, 53683–53690.
Zhou, Y., Fisher, S. J., Genbacev, O., Dejana, E., Wheelock, M., and Damsky, C. H. (1997). Human cytotrophoblasts adopt a vascular phenotype as they differentiate: A strategy for successful endovascular invasion? J. Clin. Investig. 99, 2139–2151.[ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| |||||||