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Faculty of Agricultural, Food and Environmental Quality Sciences, Institute of Biochemistry, Food Science and Nutrition, The Hebrew University of Jerusalem, PO Box 12, Rehovot 76100, Israel
(Requests for offprints should be addressed to B Schwartz; Email: bschwart{at}agri.huji.ac.il)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The cellular actions of E2 are thought to be mediated through transcriptional regulation of target genes (Halachmi et al. 1994). This process occurs mainly when E2 binds to nuclear estrogen receptor (ER). The resultant complex then binds directly to response elements present on various genes (Nilsson et al. 2001) or modifies transcription through protein–protein interactions prior to DNA binding (Pfahl 1993). When E2 binds to ER, the receptor undergoes a conformational change that results in dimerization, binding to specific DNA elements, and transcriptional regulation of target genes (Nahmias & Strosberg 1995, Csikos et al. 1998). It is becoming increasingly clear, however, that multiple ligands for the steroid receptor superfamily can modulate cell function through nongenomic actions mediated through the plasma-membrane proteins (Blackmore et al. 1991, Nemere et al. 1994, Wehling 1995, Nadal et al. 1998). For example, there is evidence that E2 can trigger a variety of signal-transduction events within seconds to a few minutes. These events include stimulation of adenylate cyclase (Aronica et al. 1994), activation of protein kinase C (Setalo et al. 2005), and triggering of an intracellular calcium spike (Tesarik & Mendoza 1995). Additionally, a putative cell-membrane ER, the existence of which was first reported more than 20 years ago (Pietras & Szego 1977, 1980), appears capable of activating signal-transduction pathways according to more recent investigations (Marin et al. 2003a,b, 2005, Guerra et al. 2004, Gilad et al. 2005, Pietras et al. 2005, Marquez et al. 2006).
The ERK 1/2 cascade has been shown to be involved in cell differentiation, proliferation, and increased cell motility and migration, all responses that can be initiated by estrogens as well. Rapid activation of MAPK by E2 in ROS 17/2.8 cells has provided the first evidence of MAPK activation by E2 through phosphorylation, indicating the involvement of putative plasma-membrane receptors (Castoria et al. 2004). Rapid effects exerted by E2 on growth factor-related signaling pathways have also been demonstrated in neuronal cells and E2 activation of the ERK 1/2 signaling pathways by G protein (Filardo et al. 2002), suggesting a potential mechanism by which E2 might affect the expression of genes with promoters that do not contain strictly estrogen-responsive elements but are responsive to factors acting through other response elements, such as activation protein-1 (AP-1) and serum response elements (Chaban et al. 2004).
Caveolae are flask-shaped structures that serve as platforms for the interaction between a host of signaling proteins in various cell types (Stan 2005). Caveolae actively participate in the regulation of cholesterol trafficking at the plasma membrane, and, in addition, cholesterol has a structural role in the caveolar membrane, contributing to the creation of a specific lipid environment important for protein segregation within rafts (Simons & Ikonen 1997). Caveolin-1, which is a key component of caveola-enriched lipid rafts of the plasma membrane and a structural protein in caveolae, plays a key role in both maintaining the caveolar structure (Rothberg et al. 1992) and binding directly to and interacting with different signaling molecules. A link between cholesterol and caveolae has been well documented (Ikonen & Parton 2000). Caveolin-1 binds Src, Grb7, Raf, Ras, MEK, EGF-R, and ER at the plasma membrane, forming a ‘signalsome’ for rapid activation of intracellular signaling (Couet et al. 1997). Caveolae are thought to be formed by the tissue-specific expression of three caveolin isoforms (caveolin-1, -2, and -3), and they can be induced to form in tissues lacking caveolae following transfection with caveolin-1 (Wharton et al. 2005).
The human embryonic kidney cell line, human embryonic kidney 293 (HEK-293), expresses low to negligible levels of caveolin-1 (Ravid et al. 2005). Following transfection of HEK cell lines with low and high levels of caveolin-1, we obtained cellular models that enabled us to directly dissect the nature of the interactions between E2, caveolar ERß, the MAPK-signaling pathway, and VDR.
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Materials and methods |
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Tissue-culture media and antibiotic antimycotic solution supplements were obtained from Biological Industries Ltd (Beit Haemek, Israel). The PhosphoPlus p42/44 MAPK antibody kit was from New England Biolabs, Inc. (Beverly, MA, USA). Monoclonal human anti-VDR antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Rabbit anti-ERß polyclonal antibody was from Chemicon (Temecula, CA, USA), ER
monoclonal antibody was from Cell Signaling Technology Inc. (Beverly, MA, USA), and monoclonal antibodies to caveolin-1 and caveolin-2 were from BD Transduction Laboratories (Franklin Lakes, NJ, USA). Flourescein isothiocyanate (FITC)-conjugated donkey anti-rabbit immunoglobulin G (IgG) F(ab')2 fragments and Texas Red-conjugated donkey anti-mouse IgG F(ab')2 fragments (Jackson Immunoresearch, West Grove, PA, USA) were used for indirect immunofluorescence staining procedures in confocal microscopy.
Polyclonal antibody to ß-actin was purchased from Sigma Chemical Co. The enhanced chemiluminescence kit was from Amersham Biosciences. The protein determination kit, based on bicinchoninic acid, was from Pierce (Rockford, IL, USA). ICI182 780 was from Tocris (Bristol, UK). All other biochemicals were from Sigma Chemical Co.
Cell lines, culture conditions, and treatments
HEK-293 cells and HT29 colon cancer cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% (v/v) foetal calf serum (FCS), 1% (v/v) L-glutamine, and 0.2% (v/v) antibiotic antimycotic solution 1 and were maintained under a humidified atmosphere and 5% CO2 at 37 °C. MCF-7, a human breast cancer cell line, was cultured similarly, but the medium was also supplemented with 0.2% (w/v) insulin solution. Cells were grown to 80–90% confluence and the medium was replaced every other day.
Stable transfections
To generate stable caveolin-1 HEK-293 cells, we utilized a full-length mouse caveolin-1 cDNA subcloned into a pcDNA3 vector, kindly provided by Dr Mordechai Liscovitch (Weizmann Institute of Science, Rehovot, Israel). DNA (10 µg) was transfected into 6 x 106 cells in a 100 mm dish using lipofectamine-2000 (GIBCO/BRL) according to the manufacturer’s instructions. The selection was carried out in a medium containing 500 µg/ml G418 (Calbiochem, La Jolla, CA, USA) for at least 4 weeks before the experiments. Single colonies were selected usingcloning rings (Falcon, Franklin Lakes, NJ, USA), and each line of HEK-293 cells produced was tested for protein levels of caveolin-1.
Two distinctive clones were developed from this procedure, one with high (clone A) and another with low (clone B) expression levels of caveolin-1. The HEK-293 cell lines expressing different levels of caveolin-1 were grown in DMEM containing 500 µg/ml G418.
A point mutation changing proline 132 to leucine was generated using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s protocol. The P132L caveolin-1 mutant was constructed by PCR using the full-length caveolin-1 cDNA as template and a sense primer containing the desired mutation. The size of the mutated plasmid was verified by restriction enzyme digestion analysis and the correctly sized plasmid was used for further confirmation of the desired mutation by DNA sequencing. The fidelity of the mutation was confirmed by direct sequencing of the plasmid. The mutated vector was stably transfected into HEK-293 cells. The HEK-293 cell lines expressing high levels of caveolin-1-mutated vector (clone C) were selected in DMEM containing 500 µg/ml G418 as above.
Cells were harvested, washed twice by centrifugation in PBS and then cultured in 50 ml flasks or in six-well plates in DMEM with phenol red (PR) or DMEM without PR supplemented with 10% charcoal-stripped FCS, 1% L-glutamine, and 0.2% antibiotic antimycotic solution.
Expression of ERß and of caveolin-1 was determined in the membrane (P) and cytosolic (S) preparations of each of the three clones (A, B, and C) and compared with the wild-type control cells (WT).
Cells from the different clones (A, B, and C) and WT cells were treated with E2 (10–8 M) or respective control cultures which included ethanol at a final concentration of 0.0067% (v/v) in the medium. Cells were exposed to a medium containing the designated treatments for 4 days, the optimal time period as determined in preliminary experiments (data not shown), and VDR protein expression and extent of P-ERK1/ERK2 phosphorylation were assessed.
To study the involvement of the MAPK signal-transduction pathway in clone A, these cells were treated with different concentrations of E2 in the presence or absence of 10 µM UO126 (Calbiochem). To study the role of ER, clone A cells were treated with 1 µM ICI182 780. Drugs were freshly diluted in culture media for each experiment.
To determine whether E2-induced VDR regulation is mediated by the activity of the hormone at the cell membrane, clone A cells were treated with an E2-BSA conjugate that could not traverse the plasma membrane. Before each experiment, stock solutions of BSA conjugate were incubated with charcoal dextran (0.05 mg/ml) and charcoal (50 mg/ml) for 30 min, centrifuged at 3000 g for 10 min and filtered through a 0.22 µm filter to obtain E2-BSA free of unbound E2. E2-BSA was dissolved in phenol-free growth medium at 0.2 mg/ml. The concentration of BSA conjugate was adjusted to the values of the free hormone.
Confocal microscopy: For ERß and caveolin-1 co-staining in the different HEK-293 clones, cells were cultured on chamber slides, fixed with acetone, blocked with 4% fish gelatin, and incubated overnight at 4 °C with rabbit polyclonal anti-ERß antibody and mouse anti-caveolin-1 antibody. After washing, the slides were incubated for 1 h with the appropriate reporter antibodies (goat anti-rabbit-Texas Red (red, caveolin-1) and FITC-conjugated goat anti-mouse anti-mouse IgG (green, ERß)). The slides were viewed on a Zeiss LSM 510 (Carl Zeiss MicroImaging Inc., Thornwood, NY, USA) laser scanning confocal microscope. Images were obtained using LSM510 software.
Isolation of cell membranes
All clones (A, B, and C) and WT HEK-293 cells were grown to confluence, scraped into cold PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4) with 5 mM benzamidine, and pelleted by centrifugation at 1000 g. The cells were resuspended in hypotonic buffer (5 mM Tris (pH 7.5), 1 mM MgCl2, 1 mM EGTA, and 0.1 mM EDTA) supplemented with protease inhibitors, 1 µM aprotinin, 10 µM leupeptin, 1 µM pepstatin, and 5 mM benzamidine. The lysates were incubated on ice for 30 min and passed through a 21 gauge needle ten times. Pellets were collected following low-speed (1000 g) centrifugation to remove nuclear debris. The supernatant lysates were then centrifuged at 133 000 g for 30 min. The membrane pellet was resuspended in PBS containing the protease inhibitors at the concentrations noted earlier.
Cholesterol determinations
Free cholesterol was determined as previously described (Wharton et al. 2005). Briefly, cholesterol was extracted from the different HEK-293 cell clones by the Folch method (Lees et al. 1964). The lipid phase was hydrolyzed with KOH and ethanol, and a colorimetric assay was used for quantitative analyses.
Transient transfections
The 1.5 kb human VDR promoter fragment, inserted into the basic vector pGL2 containing the luciferase reporter gene, was a generous gift from Prof. H F Deluca (Department of Biochemistry, University of Wisconsin, Madison, WI, USA). We also used the AP-1-Luc vector (Clontech), which contains the luciferase gene driven by the TATA box of the thymidine kinase promoter and an AP-1-dependent enhancer element.
In all transient transfections, a vector expressing ß-galactosidase (ß-gal) was always cotransfected in order to standardize the transfection assay. Plasmids were transfected using lipofectamine-2000. A nonmodified pGL2 basic vector with no promoter activity was used as a control. Stimulation of the AP-1-Luc vector and the VDR promoter was induced by treatment of the transfected cells for 48 h with 10–11 M up to 10–7 M E2 in the presence or absence of 10–6 M ICI182 780. Luciferase activity was assessed in each sample and standardized in relation to ß-gal activity. All experiments were performed in triplicate.
For transient transfection of small interfering (si) RNA against ERß, HEK-293 clone A cells were plated in a six-well plates with complete medium. When cells reached 50% confluence, old medium was replaced with fresh medium. To knockdown the expression of ERß, we used commercially available siRNA for ERß from Invitrogen Corporation. Specific siRNA selected and probed for its efficacy at knocking down ERß expression was directed against GCAGACCACAAGCC-CAA (beginning at codon 956). The control scramble sequence containing the same number of nucleotides was GCAACCAACCCGACGAAAT. Mock control cells were HEK-293 clone A cells that underwent the transfection conditions without incubation with any RNA sequence. To knockdown caveolin-1 in HT-29 cells previously demonstrated to express caveolin-1 and ERß (Gilad et al. 2005), we also used commercially available caveolin-1 siRNA from Invitrogen. Specific siRNA selected and probed for its efficacy to knock down caveolin-1 expression was directed against CCGCAT-CAACTTGCAGAAA (beginning at codon 583). The control scramble sequence containing the same number of nucleotides was CCGAACTGTTCGACA-CAAA. Mock control cells were HT-29 cells subjected to the transfection conditions without incubation with any RNA sequence. HT-29 and HEK-293 clone A cells were incubated with lipofectamine-2000 and serum-free medium for 30 min and siRNA was then added and the mixture was incubated for 20 min at room temperature. After 24-h transfection, the expression of ERß and caveolin-1 was detected to measure the effectiveness of the siRNA treatment. The medium of transfected cells was replaced with fresh medium and treated (or not) with E2 for 6 days (HT-29 cells) or 4 days (HEK-293 clone A cells) in order to detect the extent of VDR protein expression.
MEK constructs
The constitutively active MEK construct (EE-MEK) and its catalytically inactive form (KA-MEK) were a gift from Prof. R Seger (Weizmann Institute of Science). HEK-293 cells were transfected with both the 1.5 kb human VDR promoter and the MEK constructs. Luciferase activity was assessed in each sample and standardized relative to ß-gal activity. All experiments were performed in triplicate.
Site-directed mutagenesis of the AP-1-binding site in the VDR promoter
Mutation directed towards the AP-1-binding site of an mVDR-luc plasmid was performed with the Quik-Change site-directed mutagenesis kit from Stratagene as described in the user manual. The sequences of the PCR primer were: forward, 5'-GCTT-TTCTTCTCGAGAGCGTCAGCTTTCCC-3'; reverse, 5'-GGGGAAAGCTGACGCTCTCGAGAAGAAAAG-3'. The size of the mutated plasmid was verified with restriction enzyme digestion analysis and the correctly sized plasmid was used for further confirmation of the desired mutation by DNA sequencing. The mutated vector was transiently transfected into HEK-293 cells, HT29 colon cancer cells and MCF7 cells, and luciferase activity assessed as already described.
Protein determination
Protein concentration in the different cell lysates was determined by a microbicinchoninic acid-based protein assay using BSA as the standard protein.
Western-blot analysis
Cell lysates or cellular subfractions were electrophoresed on a 10% SDS-polyacrylamide gel, transferred to a nylon-transfer membrane (Amersham Biosciences), blocked in 10–3 M Tris-base and 0.1 M sodium chloride, containing 5% (w/v) dry nonfat milk, incubated with monoclonal human anti-VDR antibody, rabbit anti-ERß polyclonal antibody, ER monoclonal antibody or antibodies to caveolin-1 and caveolin-2, and subsequently incubated with a secondary antibody coupled to horseradish peroxidase. Proteins were visualized using an ECL kit (Amersham Biosciences).
To determine ERK 1/2 phosphorylation, cells were plated in six-well plates in DMEM-PR and gradually deprived of FCS as follows: cells were exposed for 2 days to 0.5% charcoal-stripped FCS-DMEM-PR, and then to media devoid of FCS for 24 h including different concentrations of E2. Western blot was performed on cell lysates using a rabbit polyclonal phospho-p42/44 MAPK (Thr202/Tyr204) antibody, or a phospho-Raf antibody, and after stripping the membranes, reactions were performed with their respective antibodies to nonphosphorylated proteins.
RT-PCR analyses
RNA isolation was performed using Tri-Reagent solution (MRC, Cincinnati, OH, USA). RT-PCR assay was performed using the Promega kit assay. The specific selected VDR primers were as follows: 5'-ATGCCATCTG-CATCGTCTC-3' and 5'-GCACCGCACAGGCTGTCCTA-3'. For siRNA, ERß transfectants of HEK-293 clone A cells, the specific ERß primers used were 5'-CAG-CATTCCCAGCAATGTCAC-3' (ERß forward) and 5'-GCAGAAGTCAGCATCCCTCTTTG-3' (ERß reverse) to give a PCR product of 281 bp. To assess the quality and loading of RNA, the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified concomitantly with 5'-GAGCCACATCGCTCAGAC-3' (GAPDH forward) and 5'-AAATCCCATCACCATCTT-3' (GAPDH reverse) to give a PCR product of 250 bp. The PCR protocol for all of these primers was 5 min at 94 °C, then 31 cycles (1 min at 94 °C, 1 min at 54 °C, and 1 min at 72 °C), and finally 10 min at 72 °C.
Statistical analyses
The data presented herein represent means ± standard error (S.E.M). Differences between the control and treatments were evaluated by Student’s t-test.
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Results |
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To demonstrate our cell system’s responsiveness to E2 treatment, we determined whether HEK-293 cells express ERs. Western-blot analyses were performed with specific anti-ER and anti-ERß antibodies. Cells expressed principally ERß and very low levels of ER (Fig. 1A
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shows, by western blotting, the expression levels of the lipid-raft proteins caveolin-1 and caveolin-2 in parental HEK cells and in three cell lines transfected with caveolin-1 cDNA and then selected for high (clone A) and low (clone B) levels of caveolin-1 expression. In clones A and C, caveolin-1 is expressed at significantly higher levels than in clone B or WT HEK-293 cells (P < 0.001). Analysis of the protein lysates revealed that the amount of caveolin-2 in clone A cells expressing high caveolin-1 is also strongly elevated. These data confirm a previous study (Fiucci et al. 2002) in which the overexpression of caveolin-1 induced upregulation of caveolin-2. In contrast, in clone C, expressing genetically disrupted nonfunctional caveolin-1, caveolin-2 was destabilized, resulting in reduced caveolin-2 protein levels, in support of previous findings (Drab et al. 2001, Razani et al. 2001). To determine whether caveolin expression impinges directly upon cholesterol content of transfected HEK-293 cells, we measured free cholesterol in the HEK-293 parental cell line (WT; Table 1) and compared with that in the different HEK-293 clones. High free cholesterol content was measured only in cells from clone A. In contrast to clone A, in cells from clone C expressing the nonfunctional P132L-mutated caveolin-1, the caveolin-1 protein was unable to stabilize caveolin-2 and bind significant amounts of free cholesterol in the cell membrane.
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Subfractionation of cells from clones A, B, and C and the parental WT HEK-293 into pelleted membranous fractions (P) and supernatant cytosolic fractions (S) devoid of nuclei allowed us to detect the expression of the 54 kDa ERß molecule mainly in fraction P of clone A cells. In clone B cells, a different pattern was observed: expression of the ERß molecule was less pronounced and equal in fractions P and S (Fig. 2A). Clone C and WT HEK-293 cells expressed very low levels of ERß in both P and S fractions (Fig. 2A). Caveolin-1 was mainly expressed in the P fractions of clone A and C cells, and to a much lower extent in their S fractions. We concluded that the maximal ERß expression is observed in plasma-membrane fractions expressing the highest functional caveolin-1 levels.
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) and concomitantly enhanced VDR protein expression only in clone A cells, but not in clone B, clone C or WT HEK-293 cells (Fig. 2C).
To determine the ER localization within plasma membranes in each of the clones, we examined cultured cells for possible colocalization of ERß with caveolin-1 by confocal immunofluorescence microscopy (Fig. 3A–I). Images of representative cells from clones A, B, and C were obtained after double immunofluorescent staining for ERß (green) and caveolin-1 (red). Figure 3A, D, and G illustrates ERß staining, Fig. 3B, E, and H illustrates caveolin-1 staining, and Fig. 3C, F, and I show colocalization. As shown in Fig. 3C, there is significant colocalization of ERß (green) with caveolin-1 (red). Yellow labeling indicates an extensive colocalization of the two proteins. This technique allows the detection of the presence of ERß at the cell surface, extensively colocalized with caveolin-1, only in clones expressing caveolin-1. Combined immunostaining (overlapping, indicated in yellow) for ERß and caveolin-1 occurred on the cell membrane. Clone B cells did not exhibit any measurable caveolin-1 expression and clone C cells, expressing high levels of nonfunctional mutated P132L caveolin-1 protein, caveolin-1 is faintly expressed; however, the distribution is completely different from clone A (Fig. 3H). Wild-type HEK-293 cells show identical staining distribution of ERß and caveolin-1 as clone B.
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We further analyzed clone A cells and assessed whether this clone behaves similar to HT-29 colon cancer cells and MCF7 breast cancer cells (Gilad et al. 2005). To assess whether E2 can induce rapid cellular signaling effects in HEK-293 clone A cells, we measured ERK 1/2 phosphorylation. E2 activated ERK 1/2 phosphorylation within 10 min after exposure to a 10–8 M concentration (Fig. 4A), with peak activation at 20 min.
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), indicating that a direct relationship exists between these cellular events. Basal phosphorylation of ERK 1/2 in the control cells (without E2 treatment) was observed in these experiments, most likely because cells were exposed to culture media containing FCS. Effect of E2 on VDR mRNA and protein-expression levels in HEK-293 clone A cells
The effect of different concentrations of E2 on VDR protein expression in HEK-293 clone A cells was assessed by western-blot analysis of whole-cell lysates. The cells were exposed to a medium containing different concentrations of the hormonal treatments for 4 days, which was found to be the optimal time period in preliminary experiments (data not shown). We found that E2 dose-dependently upregulates VDR expression (Fig. 5A). E2 was also effective at upregulating VDR transcription in HEK-293 clone A cells. We evaluated VDR mRNA expression by reverse transcriptase-PCR analyses following the 4-day hormonal treatments. The effect of E2 on VDR mRNA upregulation was found to be similar to its effect on protein expression (Fig. 5B). The specific ER inhibitor ICI182 780 blocked E2-mediated VDR protein upregulation suggesting that E2 mediates VDR expression via a process involving ERs (Fig. 5C).
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To determine whether E2-induced VDR regulation is mediated by the activity of the hormone at the cell membrane where caveolin-1 is located, HEK-293 clone A cells were treated with an E2-BSA conjugate, a molecule unable to traverse the plasma membrane. The effect of E2-BSA mimicked that of E2 on VDR protein expression and ERK 1/2 phosphorylation: following exposure to E2-BSA for 10 min, significant ERK 1/2 phosphorylation was detected, similar in pattern and intensity to that with the nonconjugated hormone (Fig. 6A). E2-BSA at all concentrations tested (10–10 M up to 10–6 M) significantly upregulated VDR expression in HEK-293 cells (Fig. 6B). Clone A cells treated with E2BSA were stained with anti-ERß antibody and visualized with the reporter secondary antibody FITC-conjugated goat anti-mouse IgG (Fig. 6C).
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To demonstrate the role of ERß and caveolin-1 in E2-induced VDR expression, we used RNA interference (RNAi) to block, ERß (siRNA to ERß) in HEK-293 clone A cells or caveolin-1 (siRNA to caveolin-1) in HT29 colon cancer cells, previously shown to express caveolin-1 and be amenable to VDR regulation through ERß (Gilad et al. 2005). The specificity and efficiency of each siRNA were tested in transient transfection experiments, by western-blot or RT-PCR analyses.
Figure 7A demonstrates that the selected siRNA completely blocked caveolin-1 expression and concomitant treatment with E2 did not result in enhanced VDR expression (Fig. 7B), as opposed to scramble- or mock-transfected HT-29 cells. The role of ERß in VDR control by E2 was tested in HEK-293 clone A cells transfected with siRNA ERß. The transfected clones did not express the ERß protein (Fig. 7C) or the RNA transcript (Fig. 7D), demonstrating the efficiency of the siRNA treatment. The mock- and scramble-transfected cells did express the protein and transcript. This treatment demonstrated that VDR is directly dependent on functional ERß expression in HEK-293 clone A cells (Fig. 7E). SiRNA against caveolin-1 and ERß were effective up to 4 days of E2 treatment (not shown).
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We performed transient transfection assays with the luciferase reporter vector pGL2 containing the 1.5 kb region of VDR in HEK-293 clones A, B, and C (bearing the P132L mutation) cells. Transfected cells were treated for 48 h with E2 and luciferase activity was recorded for control and E2-treated cells. The most E2-responsive cells were those from HEK-293 clone A, an effect which was not detectable in cells from clones B and C or from WT HEK-293 cells (Fig. 8A). We again concentrated on clone A cells and treated them with E2 at concentrations of 10–10, 10–8, and 10–6 M. These treatments resulted in upregulation of luciferase activity at all E2 concentrations used (Fig. 8B). Similar to E2-mediated VDR expression activity, the specific ER inhibitor ICI182 780 was able to block E2-mediated VDR promoter upregulation in HEK-293 clone A cells, at all E2 concentrations tested, suggesting that E2-induced VDR promoter activity is mediated by ERs. ICI182 780 therefore significantly inhibited activation of VDR promoter (data not shown).
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HEK-293 clone A cells transiently transfected with the luciferase reporter vector pGL2 containing the 1.5 kb region of VDR were treated for 48 h with E2-BSA at concentrations ranging from 10–10 to 10–7 M. These treatments resulted in upregulation of luciferase activity at all E2-BSA concentrations tested (Fig. 9), suggesting that E2-induced VDR promoter activity is mediated by membrane-bound ERs.
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Using different MEK constructs, we assessed whether MEK can directly activate the VDR promoter in HEK-293 clone A cells. To this end, the constitutively active MEK construct EE-MEK and its catalytically inactive form KA-MEK were transiently transfected into HEK-293 clone A cells concomitantly with the 1.5 kb human VDR promoter luciferase reporter plasmid. A significant fourfold increase in luciferase activity associated with the VDR promoter was detected in the presence of the constitutively active EE-MEK construct, but not in that of the catalytically inactive KA-MEK construct (Fig. 10). This effect was not seen in clone B cells (data not shown).
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The VDR promoter contains three AP-1-binding sites. AP-1 is a transcriptional activator composed of homo-and heterodimers of Jun and Fos proteins. It is involved in the activation of many genes. AP-1 activity is subject to complex regulation both transcriptionally and posttranscriptionally. Transcriptional control of jun and fos gene expression determines the amount and composition of the AP-1 complex. The jun and fos genes are regulated both positively and negatively and are highly inducible in response to extracellular stimuli and to posttranslational control. AP-1 has been shown to play a key role in the nuclear integration of the Ras-ERK phosphorylation pathway. We therefore assessed whether E2 signaling through MAPK pathways integrates at AP-1 sites within the VDR promoter. To this end, we performed transient transfection assays in HEK-293 clone A cells with the AP-1-Luc vector, which contains the luciferase gene driven by the TATA box of the thymidine kinase promoter and an AP-1-dependent enhancer element, and then assessed the effect of E2. Similar to E2-mediated VDR expression activity, E2 activated AP-1-driven promoter activity, while the specific ER inhibitor ICI182 780 was also able to block E2-mediated AP-1 promoter upregulation, suggesting that E2-induced VDR promoter activity is mediated by ER and integrated at AP-1 sites present within the VDR promoter (Fig. 11A).
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). Similar studies in HT-29 and MCF-7 cells demonstrated that mutation of the AP-1-binding site in the VDR-promoter similarly results in downregulation of luciferase activity (Fig. 12A and B). These results provide direct evidence that the AP-1 sequence in the VDR promoter plays a key role in the E2-mediated increase in VDR upregulation.
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Discussion |
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at the plasma membrane. In the present study, we demonstrate a direct functional consequence of caveolin-1 overexpression in HEK-293 cells, i.e., caveolin-1 overexpression in the transfected HEK-293 cells induced caveolin-2 stabilization and expression and coordinately, significantly increased free cholesterol in the cell. Since caveolin-1 is a cholesterol-binding protein and free cholesterol is principally found in the cell membrane, the present findings lent further support to our previous ones in which a direct correlation was obtained between functional caveolin-1 expression and free cholesterol content in the cell.
Cellular fractionation analyses of the different control and E2-treated parental and selected HEK-293 clones demonstrated specific localization of ERß to plasma-membrane domains exclusively in clone A, the clone expressing 18- to 20-fold higher levels of functional caveolin-1 than the parental HEK-293 cells. In HEK-293 clone A cells, we showed that a significant fraction of ERß is expressed in the membrane fraction expressing high caveolin-1 levels. Immunocolocalization shown by confocal microscopy indicated extensive overlap of ERß with caveolin-1 in the whole-cell plasma membrane. This association takes place upon expression of functional caveolin-1 expression. In clone C, expressing the mutated nonfunctional P132L caveolin-1, ERß was not detected in the plasma membrane. In clone B, expressing low caveolin-1 levels similar to those in the parental WT HEK-293 cells, expression of ERß in the plasma-membrane domains was negligible. We assume that caveolin anchors ER proteins to the membrane, similar to that which has been suggested for G-protein -subunit (Couet et al. 1997). Additionally, immunostaining analyses allowed us to demonstrate that in E2-BSA clone A treated cells, ERß is preferentially localized to membrane cellular domains.
The present study directly demonstrates the absolute requirement of functional caveolin-1 expression for E2, following binding to caveolar-membranal ERs, to be able to induce the activation of intracellular signaling that culminates in upregulation of VDR expression.
Several recent studies have indicated that many signaling molecules, such Raf1 and Src family tyrosine kinases, are recruited into caveolae by caveolins, which, through the scaffolding domain, interact with the caveolin-binding motifs in these signal molecules (Kiss et al. 2005). These groups of signal molecules can form ‘preassembled signaling complexes’ on the plasma membrane. Accumulation of receptors together with signal molecules in lipid rafts/caveolae enables them to be in close contact with each other and makes lipid rafts/ caveolae the gateways for signals entering into the cells.
E2 was shown to affect VDR transcription and translation in HEK-293 clone A cells. In addition, ER mediated E2’s effect on VDR, since the ER-specific inhibitor ICI182 780 was extremely effective at abrogating E2-mediated VDR upregulation.
Similar to our previous findings in HT-29 and MCF-7 cells expressing functional caveolae (Gilad et al. 2005), in the present study, E2 induced significant MAPK phosphorylation activities only in clone A, the clone expressing high caveolin-1 levels. These rapid non-genomic effects could take place whether the ER is located within or near the plasma membrane (Watson et al. 1999, Norfleet et al. 2000, Wade et al. 2001, Qi et al. 2002). We demonstrate herein that when HEK-293 clone A cells were treated with an E2-BSA conjugate, a compound unable to traverse the plasma membrane, the conjugate was able to upregulate both VDR expression and ERK phosphorylation, in a fashion that very closely mimicked the effect of the free nonconjugated E2.
The inhibition of VDR protein expression with the specific ERK 1/2 phosphorylation inhibitor UO126 supports the notion that E2 activation through ERK 1/2 modulates VDR expression. These data support the concept that MAPK activation plays a central role in the regulation of VDR expression by E2. To further demonstrate whether a direct relationship exists between ERK 1/2 activation and VDR expression, we used the EE-MEK construct which expresses a constitutively activated MEK and the KA-MEK construct which expresses a catalytically inactive MEK. Cotransfection of the EE-MEK construct with the luciferase reporter VDR promoter resulted in enhanced activation of E2-mediated VDR luciferase activation, in contrast to the catalytically inactive KA-MEK construct which was ineffective in this regard, a finding that further supports the notion that estrogen activation through MEK/ERK 1/2 modulates VDR expression.
The absolute requirement of functional caveola-membrane localization of ERs in HEK-293 cells in the framework of regulation of VDR expression by signaling pathways was further clarified using clone C cells transfected with the mutated nonfunctional P132L caveolin-1 expression vector. These experiments demonstrated that appropriate caveola-membrane organization and detection of ERß in the plasma membrane are directly reflected by MAPK phosphorylation and consequent VDR expression.
Additional convincing results linking caveolin-1 ERß positioning at the plasma membrane and control of VDR expression by E2 were obtained in experiments in which we knocked down ERß with siRNA to ERß in HEK-293 clone A cells and demonstrated that indeed ERß is directly involved in regulating VDR (Fig. 6). In addition, the role of caveolin-1 in positioning ERß to the plasma membrane and thereby allowing signaling to take place along with VDR transcription and translation was demonstrated by knocking down caveolin-1 in the caveolin-1- and ERß-positive colon cancer cell line HT-29.
An additional event downstream of the MAPK phosphorylation reaction is phosphorylation of the nuclear transcription factor c-Jun (Gilad et al. 2005), able to induce transcriptional activation at AP-1 sites. These sites are present within the VDR (Qi et al. 2002) and are involved in regulation of VDR transcription. Luciferase reporter gene assays with the AP-1-Luc vector revealed that AP-1 is dose-dependently activated by E2 and inhibited by the specific ER inhibitor ICI182 780, just like that which occurred with VDR in clone A cells. The human VDR promoter contains AP-1 sites (Qi et al. 2002), and activation of the ERK/MAPK pathway causes induction of fos genes through phosphorylation of ternary complex factors (Kast et al. 2003). Fos heterodimerizes with Jun, or Jun homodimerizes to Jun family members, to form the AP-1 complex, which activates gene transcription by binding to the AP-1 element. Furthermore, Jun/Fos heterodimers can lead to increased c-jun transcription through binding to the AP-1 sites in the c-jun promoter (Shaulian & Karin 2001). Our experimental data suggest that a direct signaling connection exists between E2-ERß, Raf-MAPK pathways, and VDR expression, and that the AP-1 sequence on the VDR promoter plays a key role in mediating this transactivation (Shaulian & Karin 2001), given that point mutations in AP-1 sites resulted in an inactive VDR promoter. These observations were equally demonstrated in HEK-293 clone A cells, HT29, and MCF7 cancer cell lines, indicating that this mechanism is conserved in tumor cells of different origin.
Taken together, our results demonstrate that in VDR regulation by E2, first E2 binds to ERß when this ER is specifically associated with membranal caveolae. Following E2-ERß binding, the Ras-Raf-ERK pathway is triggered. These signaling events activate transcription factors, such as c-Jun or c-Fos, to bind to their specific sequences within the VDR promoter (the AP-1-binding site), activity which culminates in upregulation of VDR gene transcription and expression.
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Received in final form 7 March 2007
Accepted 25 March 2007
Made available online as an Accepted Preprint 29 March 2007
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