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Endocrinology Vol. 145, No. 7 3075-3083
Copyright © 2004 by The Endocrine Society

The Low-Density Lipoprotein Receptor Is Regulated by Estrogen and Forms a Functional Complex with the Estrogen-Regulated Protein Ezrin in Pituitary GH3 Somatolactotropes

Perry M. Smith, Ann Cowan and Bruce A. White

Department of Cell Biology (P.M.S., B.A.W.), Center for Biomedical Imaging Technology (A.C.), University of Connecticut Health Center, Farmington, Connecticut 06030

Address all correspondence and requests for reprints to: Dr. Bruce A. White, Department of Cell Biology, MC 3505, University of Connecticut Health Center, Farmington, Connecticut 06030. E-mail: bwhite{at}nso2.uchc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen regulates the function, growth, and proliferation of lactotropes in the pituitary. We report here that low-density lipoprotein (LDL) receptor (LDLR) gene expression and LDL uptake are strongly up-regulated by estrogen in pituitary somatolactotropic GH3 cells. The uptake of LDL was significantly inhibited by the F-actin-severing drug, swinholide A, indicating that LDL uptake is dependent on the integrity of the cortical actin cytoskeleton in GH3 cells. We examined whether the estrogen-inducible cytoskeletal linker protein, ezrin, interacts with the LDLR. The LDLR coimmunoprecipitated with ezrin, and fluorescently labeled LDL bound to regions of the cell membrane that colocalized with the active, phosphorylated form of ezrin (phosphoezrin). Evidence for a functional interaction between ezrin and the LDLR was obtained by transient transfection experiments using ezrin-green fluorescent protein (GFP) expression constructs. We observed that transient transfection of GH3 cells with an ezrin N terminus-GFP dominant-negative construct prevented the uptake of LDL particles, whereas expression of GFP alone or an ezrin C terminus-GFP construct had no effect on LDL uptake. Transfection with the ezrin N terminus dominant- negative construct had no effect on the endocytosis of transferrin. Thus, estrogen stimulates the expression of ezrin and the LDLR in GH3 cells, which interact physically and functionally to facilitate the endocytosis of LDL. We propose that the up-regulation and interaction of ezrin and the LDLR serves to augment the delivery of cholesterol and other lipids in support of the hypertrophic and proliferative response of cells to estrogen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SPECIFIC FUNCTION of the low-density lipoprotein receptor (LDLR) is to remove cholesterol-rich lipoprotein particles from the circulation (1, 2). In the liver, LDL uptake leads to the excretion of cholesterol. In extrahepatic cells, the LDLR complements the intrinsic cholesterol synthetic pathway, delivering cholesterol-rich LDL to cells that exhibit an elevated demand for cholesterol, such as steroidogenic cells. The LDLR also enhances the uptake LDL particles by extrahepatic cells that are actively undergoing growth and proliferation (3), processes that also increase the demand for cholesterol and other lipids.

LDLR levels are adjusted to meet an increased demand primarily by an intracellular feedback mechanism (4). Hormones and growth factors can superimpose additional regulation on cholesterol synthesis and uptake. The mitogenic steroid, estrogen, increases the ability of cells to synthesize cholesterol in both hepatic and extrahepatic tissues (5, 6), which is consistent with the ability of estrogen to induce cellular hypertrophy and hyperplasia in several reproductive cells and tissues, including the pituitary lactotrope, uterine endometrium, and breast ductal epithelium. A functional estrogen-responsive element has been identified within the promoter of the hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase gene, which encodes the rate-limiting enzyme in cholesterol synthesis. The observation that the rat HMG-CoA reductase promoter was estrogen responsive in peripheral cells, but not in hepatocytes, prompted Beato and colleagues (5) to propose that "in estrogen target cells, such as mammary cells and endometrial cells, the induction of HMG-CoA reductase expression would facilitate cholesterol synthesis that may be required for the proliferative response of these cells to estrogens." In addition to enhancing the ability of cells to make cholesterol, estrogen increases the uptake of cholesterol from the extracellular fluid. There are two primary lipoprotein particles that provide for cholesterol delivery to peripheral cells: 1) high-density lipoproteins (HDLs), via transfer of cholesterol to the cell membrane as mediated by the physiological HDL receptor, the scavenger receptor B-I (SR-BI); and 2) LDL, via LDLR-mediated endocytosis. Estrogen stimulates SR-BI transcription in peripheral, steroidogenic tissue, but represses SR-BI expression in hepatocytes (7). Estradiol (E2) also increases LDLR expression. The stimulation by 17{alpha}-ethinyl E2 of LDLR expression in rat hepatocytes in vivo was reported by Kovanen and colleagues (8) 25 yr ago. Most of the work on estrogen regulation of the LDLR has continued to focus on its actions in hepatocytes, as this is related to how estrogen lowers circulating atherogenic LDL and, consequently, the risk of developing cardiovascular disease. Only a few studies have been published to date on estrogen control of LDLR expression in peripheral tissues (e.g. Refs. 9, 10, 11). These studies have reported that estrogen can increase (in hepatocytes), decrease (in placenta), or have no effect (kidney, intestine) on LDLR expression.

The LDLR is a nutrient cargo that is continually endocytosed and recycled back to the cell membrane. There are several adaptor and accessory proteins that specifically interact with the NPxY (asparagine-proline-any amino acid-tyrosine) internalization motif within the cytoplasmic domain of the LDLR and that increase the efficiency of LDLR clustering into clathrin-coated pits. For example, the autosomal recessive hypercholesterolemia (ARH) protein, which is mutated in ARH (12), is a modular adaptor protein that interacts with several other endocytotic proteins, including the clathrin heavy chain (13, 14). ARH is required for normal LDLR function in hepatocytes and lymphocytes. However, LDLR function appears to be dependent on ß-arrestin2, rather than ARH, in fibroblasts (15). These and other studies (e.g. Ref. 16) have shown that there are multiple, cell-specific proteins that modulate LDLR function, and all are potential targets for hormonal regulation of LDL uptake. However, although progress in the identification of the components of endocytosis and their interactions continues, little is known about the hormonal control of these components.

We have previously reported that estrogen stimulates the expression of the ezrin gene in the rat pituitary tumor GH3 cell line (17) and in the rat pituitary in vivo (18). Ezrin is a scaffolding protein that links membrane proteins to the cortical actin cytoskeleton. Like the LDLR, ezrin is expressed at elevated levels in some highly proliferative cancer cells (19). Although ezrin is involved in numerous cell functions including phagocytosis (20) and regulatory endocytosis of receptors (21), it has not been reported previously to be required for nutritional receptor-mediated endocytosis. In the present study, we show that estrogen strongly up-regulates LDLR expression and LDL uptake in GH3 cells. Furthermore, we provide novel evidence for physical and functional interaction between ezrin and the LDLR. These studies demonstrate that estrogen coordinately regulates the expression of two components that interact to increase LDL delivery to the cell.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
GH3 cells (American Type Culture Collection, Rockville, MD) were maintained in growth medium (GM), composed of DMEM F-12 HAM (Sigma Chemical Co., St. Louis, MO) supplemented with penicillin/streptomycin (2000 U/ml), 5% horse serum, and 1.25% fetal bovine serum (Life Technologies, Inc.-Invitrogen, Grand Island, NY). Cells were grown until a density of more than 50% confluence was reached. At the time of an experiment, cells were washed with estrogen-free, phenol red-free, serum-free medium (SFM) (DMEM:F12) (Sigma Chemical Co.) and treated for 48 h. Water-soluble 17ß E2 was obtained from Sigma Chemical Co., and swinholide A (SWA) was obtained from Calbiochem (La Jolla, CA). The antiestrogen ICI 182780, which is a steroidal antiestrogen known clinically as Faslodex (22), was obtained from Tocris Cookson Inc. (Ballwin, MO).

RNA isolation, cDNA expression array, and Northern blot analysis
The assay of estrogen-responsive genes by cDNA expression array was performed using the rat Atlas array (Clontech, Palo Alto, CA) as described previously (17). For analysis by Northern blot, GH3 cells were cultured in 100-mm dishes for 2 d in SFM only or SFM containing 50 nM ICI 182780, 10 nM E2, or both ICI 182780 and E2. RNA was isolated from each dish using 5 ml Trizol (Invitrogen, Carlsbad, CA) and following Invitrogen’s protocol. RNA (20 µg/sample) was denatured in 50% formamide and 5% formaldehyde, resolved on a 1% agarose-3.7% formaldehyde gel, transferred to Hybond-N+ membrane (Amersham Pharmacia Biotech, Piscataway, NJ), and assayed by Northern blot hybridization using conventional procedures. A rat LDLR cDNA probe was generated from GH3 cell RNA by RT-PCR, using primers to amplify the 3' region encoding the cytoplasmic domain. The sequence of the primers were: sense, 5'-CTGTGGAGGAACTGGCGG-3'; antisense, 5'-CACATCATCCTCCAGGCT-3'. The DNA fragment was gel-purified and 32P-labeled by random priming (Life Technologies, Bethesda, MD). The probe hybridized to a single band of approximately 5.5 kb as expected. The glyceraldehyde-3-phosphate dehydrogenase probe has been described by us previously (23).

Protein analysis
GH3 cells were collected with a cell scraper in 1 ml ice-cold lysis buffer (10 mM Tris-Cl, pH 7.5; 150 mM NaCl; 5 mM EGTA; 1 mM MgCl2; 2.5% Triton X-100; supplemented with 1% each protease inhibitor cocktail and phosphatase inhibitor cocktails I and II) (Sigma Chemical Co.). Lysates were centrifuged for 5 min at 16,000 relative centrifugal force, and protein concentrations determined by bicinchoninic acid assay (Pierce, Rockford, IL). Western blots of cell lysates were performed and nitrocellulose filters were probed with an anti-LDLR antibody (a kind gift of Dr. Joachim Herz, University of Texas Southwestern, Dallas, TX). Molecular weights were determined by comparison with BenchMark prestained protein ladder (Invitrogen).

Immunoprecipitations
Culture dishes of cells were rinsed with PBS and lysed in 500 µl immunoprecipitation buffer (IP buffer: 1% Triton X-100; 150 mM NaCl; 10 mM Tris-Cl, pH 7.4; 1 mM EDTA; 1 mM EGTA, pH 8.0; 0.5% Nonidet P-40) supplemented with 1% protease and phosphatase inhibitor cocktails (Sigma Chemical Co.). Cells were scraped from the dish and passed several times through a 25-gauge syringe to disperse large aggregates. Lysates were clarified by centrifugation of 12,000 x g for 5 min at 4 C. An aliquot of 100 µl lysate was incubated with 3 µg antiezrin antibody (Upstate Biotechnologies Inc., Lake Placid, NY) or normal rabbit IgG (Santa Cruz Biotechnologies, Santa Cruz, CA) under agitation for 1 h at 4 C. After adding 20 µl protein A agarose (Upstate Biotechnologies Inc.), samples were incubated for an additional 30 min. Mixtures were then centrifuged at 12,000 x g at 4 C for 1 min and the supernatant was removed by aspiration. Pellets were washed three times in 1 ml IP buffer, resuspended in 30 µl SDS-PAGE sample buffer, boiled for 5 min, and centrifuged at 12,000 x g for 1 min at 4 C. Supernatant was then analyzed by Western blot.

Immunofluorescence studies
Cells cultured on glass coverslips were fixed in 4% formaldehyde in PBS for 20 min, washed in PBS for 5 min, permeabilized with 0.5% Triton X-100 in PBS for 5 min, blocked with 3% BSA in PBS for 15 min, and washed again in PBS. Fixed cells were incubated with primary antibody (anti-LDLR, described above), anti-phospho-ezrin-radixin-moesin (ERM) (Cell Signaling Technologies, Beverly, MA), or nonimmune control IgG (Santa Cruz Biotechnologies) in 0.1% BSA in PBS for 30 min. Coverslips were washed three times in PBS, incubated in secondary antibody (Alexa 488 chicken antirabbit IgG; Molecular Probes, Eugene, OR) for 30 min, washed three times in PBS, and mounted in Slo-Fade mounting reagent (Molecular Probes). Microscopy was done on a Zeiss (Thornwood, NY) LSM510 confocal laser scanning microscope using a 40 x 1.2 NA (numerical aperture) c-apochromat lens or 100 x 1.4 NA plan-apochromat lens.

LDL and transferrin uptake
Fluorometry.
LDL and transferrin uptake were assayed essentially according to a previously described method (24). Cells were treated with 50 nM ICI 182780 or 10 nM E2 for 2 d in six-well plates, and then incubated with DiI-LDL (10 µg/ml final concentration, Molecular Probes) at 37 C for 5 h. Cells were washed three times in 3% BSA, three times in PBS, lysed in 500 µl of 1% sodium dodecyl sulfate, 1 N NaOH, and quantified by a Cytofluor 4000 fluorometer (excitation {lambda} at 530 nm; emission {lambda} at 580 nm; PerSeptive Biosystems, Foster City, CA). All values were corrected by subtracting fluorescence from a 1% sodium dodecyl sulfate, 1 N NaOH solution.

Images.
Cells were treated for 2 d on coverslips as described. On the second day, cells were washed and incubated in SFM containing DiI-LDL (10 µg/ml) or Alexa Fluor 594-transferrin (50 µg/ml; Molecular Probes). Cells were washed three times in 3% BSA/PBS, three times in PBS, fixed in 10% formalin for 20 min at room temperature, and imaged on a Zeiss 510 confocal microscope as described above.

Transfections
GH3 cells were transfected expression plasmids encoding green fluorescent protein (GFP)-tagged ezrin fragments (a kind gift from Dr. S. Kaul, National Institute of Bioscience and Human Technology, Tsukuba Science City, Japan) by mixing 10 µl lipofectamine (Invitrogen) in 100 µl Opti-MEM SFM (Life Technologies, Inc.-Invitrogen) with 1 µg plasmid DNA in 100 µl Opti-MEM. After 30 min of incubation at room temperature, 800 µl Opti-MEM was added to the DNA-lipofectamine mixture, and then overlaid onto GH3 cells grown on coverslips in six-well plates. After 2 h of incubation at 37 C, the lipofectamine/DNA mixture was removed. Cells were maintained in GM for 48 h before processing for LDL or transferrin uptake by confocal microscopy as described above. DiI-LDL uptake was quantified in transfected cells by selecting a mid-cell section and manually counting red dots, using only transfected cells surrounded by untransfected cells that displayed robust uptake of DiI-LDL.

Statistical analysis
All data are presented as mean ± SEM. Significance was assessed by ANOVA followed by appropriate t test using GraphPad Prism 3.03 software (GraphPad Software, San Diego, CA). P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The LDLR is regulated by E2 in GH3 cells
We initially observed that LDLR expression was stimulated by estrogen in pituitary GH3 cells by comparing gene expression in E2- vs. antiestrogen-treated cells using a cDNA expression array (Fig. 1AGo). This was confirmed by Northern blot hybridization (Fig. 1BGo). GH3 cells that were cultured in an estrogen-free, SFM for 2 d showed low but detectable levels of LDLR mRNA. E2 treatment generated abundant levels of LDLR mRNA, whereas cells treated with the steroidal antiestrogen ICI 182780 had essentially nondetectable levels (Fig. 1BGo). Cotreatment with E2 and ICI 182780 produced intermediate levels of LDLR mRNA.



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  		FIG. 1. Effects of ICI 182780 (ICI) and E2 on LDLR expression. A, Panel D from the Atlas (Clontech) cDNA expression array. GH3 cells were treated with ICI 182780 or E2 (as described in Materials and Methods), and RNA was processed for assay by cDNA expression array according to Clontech protocols. The finger denotes the position of hybridization of 32P-labeled mRNA to duplicate dots of LDLR cDNA. B, Northern blot analysis of LDLR mRNA expression. GH3 cells were treated for 2 d in SFM alone or containing 50 nM ICI 182780, 10 nM E2, or both. Equal amounts of total RNA from duplicate samples for each treatment were resolved by formaldehyde-agarose electrophoresis, transferred to membrane, and hybridized to an LDLR or glyceraldehyde-3-phosphate dehydrogenase cDNA probe as described in Materials and Methods. Results are representative of n = 4. C, Western blot analysis of LDLR protein expression. GH3 cells were treated as in B. Equal amounts of protein from duplicate samples for each treatment were resolved by SDS-PAGE, transferred to nitrocellulose, and stained with an antibody against the rat LDLR. The asterisk indicates a nonspecific, unregulated band that serves as a loading control. Migration of molecular weight markers are indicated on the right of the blot. Results are representative of n = 4.

 
The evidence for pretranslational regulation was paralleled by changes in LDLR protein levels. LDLR protein levels, as assayed by Western blot, showed down-regulation by ICI 182780 and up-regulation by E2 (Fig. 1CGo). Analysis of the LDLR by immunofluorescent confocal microscopy in E2-treated cells revealed abundant LDLR at the cell membrane and in puncta in the cytoplasm, presumably within endocytotic/recycling vesicles (Fig. 2Go). ICI 182780-treated cells showed staining at a level slightly above background. Collectively, these data demonstrate that a high level LDLR expression is promoted by the ER signaling pathway in GH3 cells.



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  		FIG. 2. Distribution of LDLR in GH3 cells. GH3 cells were treated with ICI 182780 (ICI) or E2 as described for Fig. 1Go and processed for confocal microscopy using an antirat LDLR antibody. E2-treated cells (right) show intense staining on the apical edges and numerous bright puncta within the cytoplasm. Bar, 10 µm. Cells stained with secondary antibody alone showed no detectable fluorescence (data not shown). Results are representative of multiple fields observed in duplicate experiments.

 
LDL endocytosis is regulated by E2 in GH3 cells
In patients with mutations in adaptor proteins, certain cell types express the LDLR but do not efficiently take up LDL particles (25, 26). To determine whether GH3 cells are competent to take up cholesterol via receptor-mediated endocytosis, and whether endocytosis of LDL is estrogen sensitive, we assayed the binding and uptake of LDL particles labeled with the fluorophore DiI. GH3 cells were treated with ICI 182780 or E2 as described and incubated in SFM containing 1 µg/ml DiI-LDL for 5 h at 37 C. Lysates were then assayed by fluorometry. As shown in Fig. 3AGo, GH3 cells treated with estrogen displayed about a 3-fold increase in DiI-LDL binding and uptake compared with ICI 182780-treated cells. Binding and uptake of fluorescently labeled transferrin was assayed in a similar manner, and was also found to be up-regulated.



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  		FIG. 3. Estrogen increases LDL and transferrin uptake and binding in GH3 cells. A, GH3 cells in SFM were treated with E2 or ICI 182780 (ICI) for 2 d. Cells were assayed for DiI-LDL or Alexa Fluor 594-transferrin binding and uptake as described in Materials and Methods. Results are expressed as fold change (mean ± SEM) from ICI 182780. The asterisk indicates a significant change (n = 6; P < 0.05). B, Cells were treated as described in A and incubated with DiI-LDL for 10 min at 37 C. Cells were processed for and imaged by confocal microscopy as described in Materials and Methods. Scale bar, 20 µm.

 
LDL endocytosis was also investigated by confocal microscopy. Cells were treated with ICI or E2 in SFM for 2 d, and then were incubated in SFM containing 10 µg/ml DiI-LDL for 10 min at 37 C. Estrogen-stimulated cells showed a robust uptake of cholesterol, as evidenced by the detection of the fluorophore throughout the cytoplasm (Fig. 3BGo). In contrast, cells treated with ICI 182780 showed minimal binding and uptake of cholesterol (Fig. 3BGo).

An intact actin cytoskeleton is necessary for optimal LDL endocytosis in GH3 cells
Although well established in yeast, the role played by the actin cytoskeleton in receptor-mediated endocytosis has yet to be firmly defined in mammals (27). Although some studies have shown receptor-mediated endocytosis to be sensitive to toxins that disrupt the actin cytoskeleton (e.g. Ref. 28), others have reported no effects of F-actin perturbation on receptor-mediated endocytosis (e.g. Ref. 29). To further characterize the nature of LDL uptake in GH3 cells, we tested the effects of the F-actin-severing drug SWA.

GH3 cells were treated for 2 d in SFM containing 10 nM E2. After 24 h, one group was treated with 30 nM SWA. We have previously demonstrated that this concentration of SWA partially disrupts the cytoskeleton, without reducing cell viability (18). SWA reproducibly decreased LDL binding and endocytosis by approximately 50%, indicating some dependency of the endocytotic machinery on F-actin in GH3 cells (Fig. 4Go).



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  		FIG. 4. LDL uptake and binding in GH3 cells is dependent on an intact actin cytoskeleton. GH3 cells incubated for 24 h in SFM containing 10 nM E2 were incubated with or without SWA (30 µM) for an additional 24 h. Cells were then washed and cultured in SFM containing 10 µg/ml DiI-LDL and incubated for 5 h at 37 C. Uptake and binding was analyzed as described in Materials and Methods. The asterisk indicates a significant decrease (n = 6; P < 0.05).

 
Evidence for a physical interaction between ezrin and LDLR
We have previously reported that the expression of the F-actin linker protein ezrin is also stimulated by E2 in GH3 cells and in the pituitary in vivo (18). The possibility that ezrin and the LDLR interact was first examined by colocalization studies, using immunofluorescent confocal microscopy with anti-ezrin antibody and DiI-labeled LDL. The anti-ezrin antibody used here is specific for the phosphorylated, active form of ERM proteins [referred to herein as phosphoezrin, because ezrin represents the major ERM protein expressed in GH3 cells (17)]. As described for other cell types (30), phosphoezrin displayed a pattern of punctate staining on the cell surface of GH3 cells (Fig. 5Go). Diffuse staining of the nucleus with the phosphoezrin antibody was sometimes observed, but the physiological significance of this staining, if any, has yet to be determined. DiI-LDL particles were observed in the cytoplasm and decorating the cell membrane. As shown in Fig. 5Go, DiI-LDL particles at the cell membrane showed a striking colocalization with phosphoezrin. However, LDL particles that had been endocytosed, as evidenced by their location within the cytoplasm, no longer colocalized with ezrin. This observation is consistent with the hypothesis the ezrin interacts with the LDLR at an early stage of clustering and endocytosis.



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  		FIG. 5. Phosphoezrin localizes to areas of LDL uptake. GH3 cells maintained in GM were washed in SFM containing 10 µg/ml DiI-LDL and incubated for 30 m at 37 C. Cells were washed, fixed, and stained with an antibody against phosphoezrin. Left panel, Numerous vesicles containing DiI-LDL are seen within the cytoplasm and along the free cell membrane. Center panel, Phosphoezrin staining is seen on the free surface of the cells. Nonspecific green staining appeared within the nucleus as well (asterisk). Right panel, DiI-LDL (red) and phosphoezrin (green) colocalize extensively along the free cell membrane (yellow arrows), but not within the cytoplasm or nucleus. Scale bar, 5 µm. Results are representative of multiple fields from three experiments.

 
Evidence for physical interaction between the LDLR and adapter molecules (e.g. ß-arrestin) has been obtained by coimmunoprecipitation (15). Immunoprecipitation of ezrin coprecipitated the LDLR (Fig. 6Go). The observations shown in Figs. 5Go and 6Go indicate that ezrin physically interacts, directly or indirectly, with the LDLR.



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  		FIG. 6. Ezrin and the LDLR mutually coimmunoprecipitate. Antibodies against ezrin coimmunoprecipitate the LDLR from GH3 cell lysate. Cells were lysed and processed for immunoprecipitation as described in Materials and Methods, followed by Western blotting against the LDLR. LYSATE, LDLR in whole-cell lysate. (–), LDLR coimmunoprecipitated by normal rabbit IgG. EZRIN, LDLR coimmunoprecipitated by rabbit anti-ezrin antibody (arrow). Results are representative of duplicate experiments.

 
Evidence for a functional interaction between ezrin and LDLR
The ability of ezrin to functionally interact with the LDLR was examined by transient transfection experiments, using plasmids containing GFP alone, GFP linked to the C terminus of ezrin, or GFP linked to the N terminus of ezrin. This last construct is generally considered to act as a dominant- negative and inhibit the function of endogenous ezrin (31). As expected (32), expression of both the C and the N termini-generated membrane ruffles and cell extensions (Fig. 7AGo). Cell prolactin content, as determined by immunofluorescent confocal microscopy, was essentially unchanged by transfection (data not shown).



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  		FIG. 7. A, Dominant-negative ezrin decreases LDL uptake. Cells transfected with GFP alone, GFP-ezrin C terminus (C term-GFP), and GFP-ezrin N terminus (N term-GFP) were maintained in GM for 2 d and incubated with SFM containing DiI-LDL for 30 min at 37 C. Representative fields from three experiments are shown. Cells transfected with GFP alone or C term-GFP internalized LDL particles to the same extent. In contrast, cells expressing the N term-GFP were largely devoid of LDL particles. Scale bar, 10 µm. The bar graph represents tallies of internalized LDL particles from 30 (GFP), 92 (N-Term-GFP), and 33 (C-Term-GFP) cells from three experiments. The asterisk indicates a significant change (P < 0.01). B, Dominant-negative ezrin has no effect on transferrin uptake. GH3 cells transfected with the GFP-ezrin N terminus were maintained for 2 d in GM and incubated for 30 min with Alexa-Fluor 594-conjugated transferrin. The left panel shows two transfected cells (green) adjacent to two nontransfected cells. The right panel shows the same field, in which transfected and nontransfected cells have taken up equivalent amounts of labeled transferrin. Results are representative of three experiments. Scale bar, 20 µm.

 
At 2 d after transfection, GH3 cells were incubated with DiI-labeled LDL particles for 30 min at 37 C, and then analyzed by confocal microscopy. As shown in Fig. 7AGo, neither GFP alone nor ezrin-C terminus-GFP had an effect on LDL uptake. In contrast, LDL uptake in cells transfected with the ezrin N terminus-GFP construct was severely impaired.

The specificity of the effects of estrogen on LDLR-mediated endocytosis, and the role of ezrin in LDLR-mediated endocytosis, was examined by assaying fluorescently labeled transferrin binding and uptake. As previously shown in Fig. 3AGo, E2 significantly increased transferrin binding and uptake in GH3 cells. However, the dominant-negative ezrin N terminus GFP construct had no effect on fluorescently labeled transferrin uptake (Fig. 7BGo). Thus, E2 also increases transferrin delivery to GH3 cells, but not through an ezrin- dependent mechanism.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Estrogen regulates LDLR expression in GH3 cells and in the rat pituitary gland
The molecular mechanism by which estrogen regulates LDLR gene transcription is complex and is consistent with cell-specific regulation. Although the upstream promoter of the LDLR gene does not contain a classical estrogen response element (33), the estrogen receptor has been shown to promote LDLR transcription in hepatocytes by enhancing the binding of the Sp1 transcription factor to the R3 domain of the LDLR promoter (34). It should also be noted that the degradation of LDLR mRNA is a regulated process (35) and, thus, may be influenced by estrogen. The regulation by estrogen of LDLR expression is best established in hepatocytes, in which fairly robust E2-induced increases in LDLR mRNA, LDLR protein levels, and LDL binding have been reported. In contrast, studies in extrahepatic tissues have demonstrated relatively mild or no regulation of LDLR expression by estrogen. An early study showed that levels of 17ß E2 increased 125I-LDL surface binding and expression of LDLR mRNA in HepG2 hepatocytes by more than 2-fold, but had no effect on these parameters in human primary fibroblast cultures (36). A later study found that treatment of ovariectomized rats with 2 mg ethinyl E2 for 7 d increased LDLR mRNA 7-fold in the liver and 2-fold in the intestine (37). However, a 1995 study reported that adult rats treated with 10 mg/kg ethinyl E2 for 4 d showed dramatic changes in LDLR protein and mRNA in the liver, but no change in LDLR protein or mRNA in isolated intestinal villi (11). This same study also showed that E2 had minimal effects on LDLR mRNA levels in the lung, heart and kidney, and adrenals. A more recent study in which female rats were treated with 5 mg/kg 17{alpha}-ethinyl E2 for 5 d reported that LDLR mRNA expression was dramatically increased by estrogen in liver tissue, slightly increased in renal tissue, and unchanged in the adrenal, spleen, and small bowel (38).

We observed a strong regulation of the LDLR mRNA by estrogen in somatolactotropic GH3 cells. This regulation was first detected by cDNA expression array, was confirmed by Northern blot, and extended to show that E2 regulates LDLR protein levels, and LDL binding and uptake. Thus, these findings represent the first description of robust regulation of LDLR expression by estrogen in a nonhepatic cell. Additional work will be required to identify whether estrogen regulates LDLR expression in the pituitary lactotrope in vivo.

It has been suggested by other investigators (5) that estrogen increases cholesterol uptake and/or synthesis in extrahepatic reproductive tissues to meet an increased demand for cholesterol and other lipids. This demand is created by estrogen-induced cellular hypertrophy and proliferation, responses that are exhibited by pituitary lactotropes (39, 40, 41). However, the overall effects of estrogen on cellular cholesterol homeostasis can be complex, involving the regulation of cholesterol synthetic pathways and cholesterol uptake by the LDLR (see above) and the physiological HDL receptor, the SR-BI (42, 43). Estrogen may also diminish proteins that promote cholesterol efflux (i.e. reverse cholesterol transport), such as caveolin (44) or the ATP-binding cassette A1 transporter. Thus, it is possible that estrogen augments cellular cholesterol in some cell types without significantly altering LDLR expression. We show here, however, that the LDLR is a primary target of estrogen in the pituitary GH3 cell.

Actin-binding proteins and endocytosis
There is convincing evidence for a role of the actin cytoskeleton in endocytosis in yeast (27). However, the importance of the actin cytoskeleton in receptor-mediated endocytosis is less well established in mammals. Nevertheless, several proteins that interact directly with F-actin have recently been shown to be involved in receptor-mediated endocytosis. For example, injection of antibodies against the actin-binding protein cortactin suppress both LDL and transferrin uptake in rat liver cells (45). Knocking down mammalian actin-binding protein 1 prevented the uptake of transferrin in HEK-293 cells (46). Huntingtin-interactive protein 1R has been shown to cross-link clathrin cages to F-actin in vitro (47), and knockdown of this protein with RNAi has been reported to disrupt clathrin-coated pits in HeLa cells (27). In the present study, we observed that treating GH3 cells with the actin-severing drug SWA at a concentration (30 nM) previously shown to noticeably disrupt the actin-cytoskeleton without reducing GH3 cell viability (18) significantly inhibited DiI-LDL uptake. The ability of SWA to inhibit LDL uptake, coupled with the ability of the ezrin N terminus dominant-negative construct to essentially obliterate LDL uptake, argue for a important role of the cortical actin cytoskeleton in receptor-mediated endocytosis in rat pituitary GH3 cells.

Ezrin interacts with the LDLR
Our laboratory previously demonstrated that, along with the LDLR, estrogen dramatically increases ezrin gene expression in GH3 cells (17) and in the rat pituitary in vivo (18). Ezrin belongs to the FERM (for "protein 4.1, ezrin, radixin, moesin") superfamily of proteins (48). The ERM subfamily includes ezrin, radixin, and moesin, as well as merlin (schwannomin), which is the tumor suppressor mutated in neurofibromatosis 2. The N terminus of FERM proteins contain a tripartite, cloverleaf structure referred to as the FERM domain (48), which can bind directly to membrane proteins, PtdIns(4, 5)P2 (phosphatidyl inositol 4,5 biphosphate), or PDZ-domain-containing scaffolding proteins. The C terminus of ERM proteins harbors an F-actin-binding site, which confers on these proteins the ability to bridge membrane proteins to the cortical actin cytoskeleton.

The possibility that the ezrin might physically interact with the LDLR was initially suggested to us by the inhibition of LDL uptake by SWA (i.e. evidence for a role of F-actin and F-actin-binding proteins), and the presence of conserved basic residues in the juxtamembrane region of the LDLR that are characteristic of several other ezrin-binding membrane proteins (49, 50). The two proteins coimmunoprecipitated, indicating ezrin and the LDLR physically interact with each other, either directly or indirectly within a protein complex. Only about 10% of ezrin is in the open, active conformation in GH3 cells, and only a fraction of the recycling LDLR is at the cell membrane. Thus, the fact that an interaction between ezrin and the LDLR was detected by the relatively insensitive method of coimmunoprecipitation argues that this interaction involves high-affinity binding that is more stable than the fleeting "kiss and run" interactions described for other components of endocytosis (51).

The cytoplasmic domain of the LDLR interacts in a cell-specific manner with several proteins, which couple the LDLR to the endocytotic/recycling machinery (12, 15, 16). In this light, it is worth noting that the LDLR contains a binding site for the clathrin-coated pit adaptor protein complex, AP-2 (52), and ezrin has been shown to interact with the AP-2 binding site in the cell-adhesion molecule, L1 (53). Very recently, the sorting nexin, SNX17, was shown to interact with the LDLR via both the proximal internalization motif (NPxY) and the distal sorting motif (GYSY). It is striking that SNx17 interacted with the LDLR through its FERM domain (54), a region of homology to N terminus of ERM proteins. Ezrin could also interact indirectly with the LDLR through a scaffolding protein. This possibility is supported by the fact that the ezrin-interacting PDZ-domain containing linker SAP97 (55) has been shown to interact with LDLR family members (56). Thus, several possible modes of ezrin-LDLR interaction exist, which are not mutually exclusive and could display cell-specific variations. The exact nature of ezrin-LDLR interactions in GH3 cells represents the current focus of our research.

The evidence for ezrin-LDLR interaction that was provided by the coimmunoprecipitation studies was supported by the colocalization of LDL particles with ezrin at the cell membrane of GH3 cells, and the striking inhibition of LDL uptake by expression of the ezrin N terminus dominant-negative construct. In the colocalization experiments, the antibody used to detect ezrin was specific for phosphorylated ezrin (termed phosphoezrin), which represents the open, active form (48). This antibody detected puncta of phosphoezrin at the cortex subjacent to free (i.e. nonadhesive) areas of cell membrane. DiI-labeled LDL particles that were bound to membrane LDLRs colocalized very well with phosphoezrin, supporting the existence of a physical interaction between these two proteins within the living cell. Interestingly, as LDL particles were endocytosed into the cytoplasm, they were no longer observed to be associated with ezrin (Fig. 5Go). This suggests that any role that ezrin plays in LDL uptake is involved with either LDL binding to the LDLR (inside-out signaling), or with the early steps in the clustering of the LDLR into clathrin-coated pits. For example, ezrin itself may be recruited to clathrin-coated pits through its interaction with PtdIns(4, 5)P2 (48) and subsequently recruit the LDLR to these regions of enriched endocytotic machinery. It is also clear that ezrin is unlike adaptor proteins such as stonin 2 (57) in that it does not promote all receptor-mediated endocytosis. The specificity of the effect of ezrin on LDL uptake (as indicated by no effect on transferrin uptake) argues for a direct and specific interaction between ezrin, or an ezrin-scaffolding protein complex, with the cytoplasmic domain of the LDLR. Additional in vitro work will be required to establish the details of the ezrin-LDLR interaction.

In summary, we have observed that ezrin and LDLR expression are coordinately stimulated by estrogen, and that these two proteins functionally interact to promote LDL uptake in GH3 cells. Previous studies have noted in passing that both ezrin and the LDLR are up-regulated in chondrocytes after treatment with fibroblast growth factor (58) and that ezrin and the LDLR appear together in membrane ruffles after stimulation of KB cells with epidermal growth factor (59). It is also worth noting that ezrin is elevated in several types of cancer (60), as is LDLR expression (61), and both proteins display particularly high levels in cancers associated with aggressive invasiveness (10, 62). Thus, it is tempting to speculate that the estrogen-induced increase in ezrin and LDLR represents a coordinated physiological response to a mitogenic hormone, which provides an important nutrient (i.e. cholesterol) to growing and dividing cells. We also propose that the elevation of ezrin and the LDLR in cancer cells represents a pathophysiological imbalance that supports the uncontrolled growth and migration of transformed cells. Recently, the coupling of doxorubicin to LDL particles as a chemotherapeutic intervention for liver cancer has been shown to increase both the magnitude and specificity of the antitumor action of the drug (63). Ezrin and estrogen receptor levels might be important parameters to consider in evaluating the efficacy of such treatments.


   Acknowledgments
 
We thank Dr. John Peluso (Department of Cell Biology, University of Connecticut Health Center, Farmington, CT) for his helpful comments.


   Footnotes
 
This work was supported by a grant from the State of Connecticut Department of Public Health (Breast Cancer Research and Education Income Tax Checkoff Fund). Fluorescence microscopy was performed using the facilities of the Center for Biomedical Imaging Technology at the University of Connecticut Health Center, which is supported in part by Public Health Service Grant RR13186.

Abbreviations: ARH, Autosomal recessive hypercholesterolemia; E2, estradiol; ERM, ezrin-radixin-moesin; GFP, green fluorescent protein; GM, growth medium; HDL, high-density lipoprotein; HMG-CoA, hydroxymethylglutaryl coenzyme A; LDL, low-density lipoprotein; LDLR, LDL receptor; SR-BI, scavenger receptor B-I; SWA, swinholide A.

Received February 23, 2004.

Accepted for publication March 15, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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