Abstract
Observational studies demonstrate that estradiol and progesterone affect vasoreactivity. In animal studies, progesterone treatment causes immediate relaxation of precontracted arteries with inhibition of calcium influx in vascular endothelial and smooth muscle cells, suggesting a non-genomic mechanism of action. In this study we investigated the presence of novel membrane-bound progesterone receptors in human aortic endothelial cells and correlated the expression with cell-cycle stage. Western blotting analysis with an antibody directed to the hormone-binding domain of the classic progesterone receptors shows predominant bands at 100 and 60 kD, whereas analysis with an antibody to the DNA-binding region shows only the 100-kD band. In contrast, classic nuclear progesterone receptors B and A are identified at 116 and 94 kD in similarly processed T47D cells. Both novel bands localize to the membrane fraction after differential centrifugation. Plasma membrane-bound progesterone receptor was further shown with immunofluorescent antibody and ligand-binding studies in a small percentage of human aortic endothelial cells. Fluorescent activated cell sorting demonstrated that approximately 8% of the human aortic endothelial cells expressed a plasma membrane progesterone receptor and that a greater percentage of the expressing cells were in the G2/M-phase of the cell cycle. Treatment with progesterone conjugated to BSA did not show any significant cell-cycle changes. Plasma membrane-bound progesterone receptor in vascular endothelial cells may regulate the non-genomic actions of progesterone, and expression of the receptor appears to vary with cell cycle stage.
O
The aims of this study were to determine the presence of a PR(s) in the plasma membrane of human aortic endothelial cells (HAECs), to determine if this PR(s) was distinct from the classic nuclear PRs, and to determine if expression of a membrane PR was cell cycle-dependent.
Materials and Methods
Cell Culture
Two cell lines of human aortic endothelial cells (HAECs) obtained from Cascade Biologics (Portland, OR), were used in the study, each derived from a 17-year-old male. Commercial immunohistochemistry (IHC) staining was positive for factor VIII-related antigen and CD31 antigen, and was negative for α-actin.
T47D breast cancer cells were cultured in phenol red-free RPMI 1640 with the addition of 10% FBS (Gibco BRL; Gaithersburg, MD) and HAECs were cultured in phenol red-free Medium 200 with Low Serum Growth Supplement. Both cell lines were grown in a 37C incubator with an atmosphere of 5% CO2. At confluence the flasks were washed with PBS and frozen in liquid nitrogen. HAECs underwent between five and seven passages in culture before experimentation.
Ammonium Sulfate Precipitation and Western Analysis
HAECs or T47D breast cancer cells (positive control) were added to TEMMG buffer (10 mM Tris-HCl, 1.5 mM EDTA, 12 mM monothioglycerol, 10 mM sodium molybdate, 10% glycerol) with protease inhibitors (10 μg/ml leupeptin, 1 mM phenyl-methylsulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml pepstatin). The mixture was homogenized on ice with a Polytron tissue homogenizer. After centrifugation for 20 min at 16,000 × g, ammonium sulfate was added to an aliquot of the supernatant until 30% saturation and rotated overnight at 4C. The mixture was centrifuged at 3000 × g for 30 min. Precipitates were resuspended in TEMMG plus SDS sample buffer and boiled for 2 min before loading.
Samples underwent electrophoresis on a 7.5% polyacrylamide gel and were transferred to a polyvinylidene difluoride (PVDF; Bio-Rad Labs, Hercules, CA) membrane. The membrane was incubated with rabbit polyclonal IgG antihuman progesterone receptor, C19 or C20 (Santa Cruz Biotechnology; Santa Cruz, CA) overnight at 4C. As a control, duplicate lanes were incubated with rabbit serum in place of primary antibody. The membrane was washed and incubated with secondary antibody, peroxidase-conjugated goat anti-rabbit IgG (Cappel; ICN Pharmaceuticals, Costa Mesa, CA). Proteins were detected using an ECL Western blotting detection system (Amersham; Poole, UK) according to manufacturer's directions.
Purification of Cell Membranes
HAECs or T47D cells were suspended in TEMMG buffer with 0.1% Triton X-100 and protease inhibitors. After homogenization on ice, the sample was centrifuged at 12,000 × g for 10 min at 4C and the supernatant removed. The supernatant was then centrifuged at 100,000 × g for 2 hr at 4C. The pellet (membrane fraction) was resuspended in 20 μl TEMMG buffer and SDS sample buffer. The supernatant (cytosolic fraction) was used for ammonium sulfate precipitation. Ammonium sulfate was added until 30% saturated and the mixture rotated overnight at 4C, then centrifuged at 3000 × g for 30 min. Precipitates were resuspended in 20 μl TEMMG plus SDS sample buffer. Western blots were performed as described above.
Ligand Membrane Binding
HAECs were grown until 80% confluence, washed, and incubated for 30 min with buffer A (25 mM HEPES, pH 7.4, 120 mM NaCl, 4.6 mM KCl, 1.25 mM KH2PO4, 25 mM EDTA, 10 mM glucose). Cells were removed from the flask, spun at 1000 × g, and blocked in PBS with 3% bovine serum albumin (PBS/BSA) containing 10% goat serum and 2 μM dexamethasone for 15 min at 4C. All subsequent steps were performed at 4C. Progesterone-BSA-streptavidin-fluorescein isothiocynate (FITC) (Sigma; St Louis, MO) at 20 μg/ml in PBS/BSA was added to part of the cells. For a negative control, BSA-FITC (Sigma) was added at the same concentration to a second portion of the cells. After a 30-min incubation, cells were washed with cold PBS, fixed in 4% formaldehyde, mounted in SlowFade Light (Molecular Probes; Eugene, Oregon), and observed with an Olympus 1×70 microscope using a × 40 lens and a FITC filter of 520 ± 20 nm.
Plasma Membrane Antibody Binding
HAECs were grown until 80% confluence, washed, and incubated for 30 min with buffer A. The cells were removed from the flask, spun, and blocked in PBS/3%BSA containing 10% goat serum for 15 min at 4C. All subsequent steps were performed at 4C. After blocking, the cells were washed in PBS with 1% BSA and the primary antibody, C19, was added for 1 hr. Cells were washed with 1% BSA/PBS and the secondary antibody, a goat anti-rabbit Alexa Fluor 488 antibody (Molecular Probes) was added for 30 min. After three washings, the cells were fixed with 4% formaldehyde for 5 min, mounted in SlowFade Light, and observed as above. Additional cells were visualized with a Zeiss LSM 510 confocal microscope, X40 objective, using the 488-nm spectral line of the He-Ne laser. The specificity of labeling was checked by examination of samples with the primary antibody replaced with the same concentration of normal rabbit IgG.
For permeable reactions, cells were fixed for 25 min in 4% methanol-free formaldehyde in PBS and permeabilized with 0.2% Triton-X 100/PBS for 30 min on ice. The remainder of the steps were as above except performed at room temperature (RT).
Fluorescent Activated Cell Sorting
HAECs were grown to 70–80% confluence in phenol redfree M200 medium supplemented with LSGS, treated for 24 hr, washed and incubated with buffer A. Treatments included progesterone-conjugated BSA (PG-BSA, 10−6 M; Fitzgerald, Concord, MA) or BSA alone for 24 hr. Cells were removed from the flask, spun, washed with PBS, and blocked with PBS/BSA containing 10% goat serum for 15 min at 4C. After washing, the cells were incubated with 10 μg/ml of either C19 antibody or normal rabbit IgG (Santa Cruz Biotechnology) in staining buffer (0.1% BSA + 0.1% sodium azide in PBS). Cells were incubated on ice for 30 min, washed, and incubated with secondary antibody, goat anti-rabbit conjugated with Alexa Fluor 488 (Molecular Probes) for 30 min on ice. After washing, cells were resuspended in 1% paraformaldehyde in PBS for 1 hr on ice, washed again, and resuspended in cold 70% ethanol for 15 min. After spinning, cells were incubated with 0.1 mg/ml RNase A in PBS for 5 min at RT. Propidium iodide (Sigma) was added to the cells at 20 μg/ml for 10 min before FACS analysis. The cells were then analyzed on FACS Calibur (Becton-Dickinson; Richmond, CA). PR-positive cells were gated and the DNA data were collected using CellQuest software. The DNA data and cell cycle stages were then analyzed using ModFit LT 3.0 software.
The percentage of cells in the FACS experiments was reported as mean ± SD. The treatment of PG-BSA was compared to control (BSA) using an independent t-test. Differences in the cell cycle stages between the three groups of total cells, C19-positive cells and C19-negative cells were compared with a one-way analysis of variance. Significant differences were further analyzed with a Bonferroni test. Significance was considered at a level of p≤0.05.
Results
Figure 1 compares the predominant PR proteins in T47D breast cancer cells and HAECs by Western blot analysis after precipitation with 30% ammonium sulfate. The blot was probed with C19, a rabbit polyclonal antibody specific for the C-terminus of the classic PR. Predominant bands are seen in T47D cells at 116 kD and 94 kD, representing classic PR B and A, respectively. In HAECs, predominant bands are present at 100 kD and 60 kD. Western blotting analysis performed with C20, a rabbit polyclonal antibody specific for the DNA-binding region, showed the band at 100 kD with absence of the 60-kD protein. Controls for the Western analyses included replacement of the primary antibody with a nonspecific rabbit IgG, which showed no evidence of nonspecific binding.
Western blotting analysis of HAECs and T47D breast cancer cells. Hybridization with a C19 antibody, directed to the ligandbinding domain of the classic PRs, shows predominant bands at 116 (PR-B), 94 (PR-A) and a faint band of 60 kD in T47D cells. In HAECs, predominant bands are seen at 100 and 60 kD. Hybridization with a C20 antibody, directed to the DNA-binding domain, shows only the 100-kD protein in HAEC cells. To control for nonspecific hybridization, analysis was performed with replacement of the primary antibody with the same concentration of a normal rabbit polyclonal IgG.
In Figure 2, Western analysis was performed on HAECs after differential centrifugation to separate the cell membrane fraction from the cytosol fraction. As seen, the 100- and 60-kD proteins were primarily located in the membrane fraction. Although the majority of the membrane fraction in this technique is composed of plasma membrane, it also contains nuclear membrane, thus not excluding the possibility that the 100- and 60-kD proteins are bound to the nuclear membrane. Similar studies performed with T47D cells show the 116- and 94-kD bands of PR B and A, respectively, to locate to the cytosolic fraction (not shown). To further investigate the presence of a plasma membrane-bound PR in HAECs, we performed fluorescent membrane labeling studies.
Western blotting analysis of HAECs after differential centrifugation to separate the cytosol from the membrane fraction. Proteins of HAECs were separated into a cytosolic and membrane fraction by differential centrifugation. Western blotting analysis with C19 antibody shows the 100- and 60-kD proteins to localize mainly to the membrane fraction. Similar experiments with T47D cells show the classic 116- and 94-kD bands to localize to the cytosolic fraction (not shown). Overdevelopment, compared to Figure 1, shows the less intense 100-kD band present in T47D cells.
Immunofluorescent membrane studies of HAECs with C19 antibody and ligand binding. HAECs were treated at 4C to ensure plasma membrane impermeability, with the primary C19 antibody and a secondary biotinylated goat anti-rabbit. (
The immunofluorescence studies shown in Figure 3 were performed with C19 antibody in the cold at 4C to ensure that the cells remain impermeable to the antibody. Immunofluorescent staining of the plasma membrane is well seen with confocal microscopy. Microscopy shows that not all cells demonstrate positive fluorescence. Less than 10% of the HAECs were estimated to show positive staining with the PR antibody. Controls performed, including use of peptide neutralization of the C19 antibody and replacement of the primary antibody with a normal rabbit IgG, showed the binding to be specific. Staining with the same C19 antibody in permeable cells shows a nuclear pattern consistent with a genomic receptor also present in these cells.
Plasma membrane ligand binding was demonstrated using progesterone conjugated to BSA and FITC. Again, cells were treated at 4C to preclude cell entry. Similar to the antibody studies, less than 10% of HAECs demonstrated fluorescence indicating binding of progesterone to the membrane of the cell. Controls using BSA-conjugated FITC, lacking the progesterone, showed no fluorescence.
FACS was used to determine the percentage of cells staining positive with the C19 antibody and the percentage of cells in the cell stages of G0/G1, G2/M, and S-phase. Treatment groups included BSA alone (control) and progesterone-conjugated BSA (PG-BSA). The percentage of C19-positive cells in the three groups was analyzed in four experiments. There was no significant difference in the percentage of C19-positive cells in the control group (7.9 ± 3.2%) compared to the PG-BSA group (8.5 ± 2.2%). Nonspecific isotype reactions were analyzed for each treatment and ranged from 0.5% to 3%. The percentage of isotypereacted cells was subtracted from the C19-positive cells as a correction.
Figure 4 shows the results of four experiments investigating differences in cell cycle. A greater percentage of C19-positive cells was found in the G2/M stage compared to total or C19-negative cells, and a lesser percentage of C19-positive cells was found in the S-phase compared to total or C19-negative cells. In the population of C19-positive cells, 69.4 ± 8.3% were in the G0/G1 stage, 21.1 ± 9.8% were in the G2/M stage, and 9.4 ± 3.2% were in the S stage. In the population of C19-negative cells, 56 ± 10% were in the G0/G1 stage, 4.7 ± 6.3% were in the G2/M stage, and 39.2 ± 10% were in the S stage. There was no significant effect of PG-BSA treatment in any of the three groups, although total cells tended towards a decrease in the S-phase.
Discussion
Classically, PR-B (116 kD) and PR-A (94 kD) have been considered nuclear receptors controlling gene transcription. PR-A is a truncated version of B, lacking the first 164 amino acids. Binding of progesterone leads to conformational changes with receptor dimerization, autophosphorylation, binding to specific DNA consensus sequences, and gene regulation (Conneely et al. 2000). These nuclear receptors have been identified in human vasculature, including the intima and media of arteries and veins, by IHC (Bergqvist et al. 1993; Mashiah et al. 1999), and transcripts for PR have been demonstrated in human vascular smooth muscle cells (Hodges et al. 1999). Progesterone does appear to regulate transcription within vascular cells. In vascular endothelial cells and vascular smooth muscle cells, progesterone inhibits proliferation by altering the transcription of cyclin E and A, leading to arrest in the G0-G1 stage (Lee et al. 1997; Vazquez et al. 1999). A third PR identified at approximately 60 kD, termed PR-C, has been discussed. This receptor is hypothesized to originate from an in-frame translation start site, and in vitro transcription at this start site with subsequent expression does result in an appropriately sized protein. Yet the naturally occurring transcript for this protein has not been identified, and it is not proven that the 60-kD protein is indeed encoded by this hypothesized start site (Wei and Miner 1994; Wei et al. 1996).
Fluorescent activated cell sorting showed that 7.9 ± 3.2% (mean ± SD) of the cells in the BSA control (▀) and 8.5 ± 2.2% in the PG-BSA treatment (□) reacted with the C19 antibody. The cell cycle stages were compared among the C19-negative, C19-positive and total cells in the control and PG-BSA groups. ANOVA showed significant differences in the G2/M and S stages. In the control group, C19-positive cells had a greater G2/M content compared to C19-negative cells, and this difference approached significance in the PG-BSA group. In the control and PG-BSA groups, the S-phase content was lower in the C19-positive cells compared to total and C19-negative cells. There was no significant difference in the cell stages between the control and PG-BSA treatment groups in C19-positive or C19-negative cells. G2/M, control, C19-positive vs C19-negative, p = 0.04; G2/M, PG-BSA, C19-positive vs C19-negative, p = 0.06; S, control, C19-positive vs C19-negative, p = 0.002; S, control, C19-positive vs total, p = 0.005; S, PG-BSA, C19-positive vs C19-negative, p = 0.014; S, PG-BSA, C19-positive vs total, p = 0.024.
Other actions of progesterone on the vascular system happen too quickly to be due to gene regulation. In animal studies, acute administration of progesterone to anesthetized rats blocks norepinephrineinduced increases in mean arterial pressure (Barbagallo et al. 2001). In vascular tension studies, progesterone causes a dose-dependent relaxation of isolated rat tail artery or aorta precontracted with either norepinephrine, phenylephrine, or KCl (Crews and Khalil 1999a,b; Barbagallo et al. 2001). The regulation of calcium flux in vascular endothelial and smooth muscle cells by progesterone contributes to these observations. The adrenergic agonists norepinephrine and phenylephrine increase vascular smooth muscle calcium by increasing release from intracellular compartments and by increasing calcium influx. KCl treatment primarily increases calcium influx. In contrast, progesterone does not affect arterial contraction induced by caffeine, which causes only the release of calcium from intracellular compartments (Crews and Khalil 1999a,b). These studies suggest a non-genomic inhibition of calcium influx into vascular smooth muscle by progesterone. Fluorescent calcium mobilization studies with in situ vascular endothelial cells yield similar findings. Progesterone treatment decreases the calcium influx induced by the calcium-ATPase inhibitor thapsigargin (Toshima et al. 2000). Other investigations of isolated vascular smooth muscle cells, using a whole-cell pClamp technique to measure inwards currents, show progesterone to inhibit L-type calcium channels (Barbagallo et al. 2001).
Modulation of calcium influx by progesterone has been demonstrated in other tissues and appears to be tissue specific. Progesterone decreases calcium influx in uterine smooth muscle cells (Morishita 1986; Fomin et al. 1999). In contrast, progesterone treatment increases calcium influx into human sperm (Luconi et al. 1998).
Another possible mechanism of non-genomic progesterone action includes activation of cytoplasmic signaling proteins. Boonyaratanakornkit and colleagues (2001) have shown a direct interaction between a polyproline motif on the amino terminal domain of the classic PR and SH3 domains of cytosolic signaling peptides in molecular studies.
These non-genomic actions of progesterone are postulated to occur via membrane-bound PRs that have not previously been identified in vascular endothelial cells. Specific progesterone receptors have been identified in the plasma membrane of human sperm by Western blotting analysis and ligand blot analysis (Sauber et al. 1996; Luconi et al. 1998). Two proteins of 54 kD and 57 kD are recognized after membrane preparation with antibodies directed to the hormonebinding domain (HBD) of the classic PR but are not seen with antibodies to the DNA-binding domain or the amino-terminus domains. Ligand blot analysis demonstrates the hormone-binding capabilities of these two proteins (Luconi et al. 1998). Immunofluorescent binding studies show the presence of membrane-bound PR in approximately 10% of sperm studied (Tesarik et al. 1992).
Our laboratory has recently cloned and expressed a novel 38-kD PR isoform from human aortic and adipose cDNA libraries. This protein appears to be a splice variant of the classic PR containing the hinge and ligand-binding domains but lacking the amino terminal and DNA-binding domains. The localization and function of this isoform continue to be investigated (Saner et al. 2003).
In the present study we have suggested the presence of two novel PRs by Western blotting analysis in HAECs. In comparison to the classic PR-B at 116 kD and PR-A at 94 kD, HAECs contain predominant bands at 100 and 60 kD. The 60-kD protein appears to lack a complete DNA-binding domain because it is not recognized by the C20 antibody directed to this region. In contrast to PR-B and A, these PRs mainly localize to the membrane fraction of HAECs after differential centrifugation. The significant difference in size and the membrane localization suggests that the 100- and 60-kD proteins are distinct from the recognized nuclear PRs. In addition, we have demonstrated the presence of membrane-bound PR(s) by immunofluorescent antibody and ligand binding studies. These experiments are performed at 4C to ensure that the plasma membrane remains impermeable to antibody and ligand. With the above observations we are not able to conclude that the 100- and 60-kD PRs identified with Western blotting are the same proteins identified in the immunofluorescent studies.
Interestingly, we also observed that a minority of HAECs showed immunofluorescence with both antibody and ligand-binding studies. FACS showed approximately 8% of the cell population to be expressing a plasma membrane PR. This population of cells expressing a plasma membrane PR had a significantly different cell cycle-stage distribution compared to the cells not expressing the receptor. A significantly greater percentage of the positive cells are in the G2/M stage, while less are in the S-phase. This suggests that this in vitro cell culture had two different populations of cells. The majority of cells did not express a membrane PR and were characterized by a high S-phase consistent with replicating cell growth. A minority of cells, expressing a membrane PR, were characterized by a low S-phase and high G2/M-phase, consistent with less active replication.
These findings bring into question a possible nongenomic role of progesterone during cell replication. Although a non-genomic role for progesterone in mitosis has not been previously described, there is ample evidence for regulation of meiosis in amphibian oo-cytes by progesterone. In Xenopus laevis, progesterone action via a plasma membrane receptor induces the resumption of meiosis I with germinal vesicle breakdown. Progesterone action is associated with an increase in calcium influx, activation of a phosphatidylinositol diphosphate-specific phospholipase C, and a decrease in cAMP levels (Revelli et al. 1998).
We were not able to demonstrate a significant change in cell-cycle stage with treatment of PG-BSA. PG-BSA treatment tended toward a decrease in S-phase and an increase in G0-G1 stage as has been previously reported with progesterone treatment (Vazquez et al. 1999).
This study yields the first evidence of plasma membrane-bound PR(s) in HAECs. These receptors may be responsible for the non-genomic regulation of calcium flux by progesterone in vascular tissue. In addition, this is the first observation that expression of plasma membrane-bound PR appears to vary during the cell cycle.
Footnotes
Acknowledgements
Supported by grants from the Elsa Pardee Foundation and the MidAtlantic Affiliate of the American Heart Association.
We thank Dr Margaret Jamison for statistical analysis and Ms Lillia Holmes for assistance with FACS.