Cellular functions of plasma membrane estrogen receptors

Steroids 67 (2002) 471– 475

Cellular functions of plasma membrane estrogen receptors Ellis R. Levina,b,* a

Division of Endocrinology, Veterans Affairs Medical Center, Long Beach, 5901 E. 7th St., Long Beach, CA 90822, USA b Departments of Medicine and Pharmacology, University of California, Irvine, CA 92717, USA

Abstract Strong evidence now exists for the presence and importance of plasma membrane estrogen receptors (ER) in a variety of cells that are targets for steroid action. When estradiol (E2) binds cell surface proteins, the initiation of signal transduction triggers downstream signaling cascades that contribute to important functions. These functions include cell growth and survival, migration, and new blood vessel formation. In some instances these effects result from the initiation of gene transcription, upregulated through signaling from the membrane. The membrane ER probably originates from the same gene and transcript that produces the nuclear receptor. In the membrane, ER appear to localize mainly to discrete domains of the plasma membrane, known as caveolae, but the mechanisms by which this small pool of ER translocates to this site are currently unknown. At the caveolae, a cross talk with signaling molecules facilitates E2/ER cell biologic actions. This both includes direct stimulation of signaling via G protein activation, and a cross-activation of the epidermal growth factor receptor (EGFR). This review article highlights some of the important advances in understanding the cell biology of estrogen action that emanates from the membrane. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Plasma membrane; Estrogen receptor; Signaling; Caveolae

1. Introduction In 1977, Pietras and Szego described the rapid generation of cAMP in response to E2, purportedly resulting from E2 binding a receptor protein in cell membranes [1,2]. In the 1980 and 1990s, work from many investigators demonstrated that E2 rapidly activates calcium flux [3], cAMP generation [4], phospholipase C activation [5] and IP3 generation, leading to PKC and PKA activation (reviewed in [6,7]). E2 binding to ER was required in most of these studies. These signaling events are likely to arise from the activation of G proteins by E2, and the activation of Gs␣ and Gq␣ was directly shown in CHO cells expressing either ER␣ or ER␤ [8]. Thus, ER qualify as members of the large family of G-protein coupled receptors (GPCR). It is yet unclear whether the receptor spans the cell membrane, as would be typical for heptahelical receptors of this classification, and in some other way directly contacts the G proteins, or whether ER transactivates another GPCR in the membrane, thereby effecting G protein activation. Upon activating several G proteins (or through other mecha* Corresponding author. Tel.: ⫹1-562-494-5748; fax: ⫹1-562-4945515. E-mail address: [email protected] (E.R. Levin).

nisms), E2/ER can then trigger signaling cascades resulting in cell biologic functions.

2. Signaling pathways and their consequences One signaling pathway that is stimulated by E2, and that has important cell biologic consequences, is the activation of the proline-directed, serine/threonine kinase, extracellular-regulated kinase (ERK). ERK is a member of the MAP kinase family, and is rapidly (2 min) activated by E2 in MCF-7 breast cancer cells. ERK activation results from more proximal kinase activation, including Src, Ras, raf, and MAP kinase (MEK) stimulation [9]. ERK activation via this cascade contributes to both E2-induced proliferation [10] and to the survival of MCF-7 cells [11]. Importantly, specific ER antagonists, such as ICI 182780, generally inhibit E2-activation of this (and many) signal, indicating that ER are necessary for the actions of E2. Recently, Kousteni et al. showed that E2 signaled through this same pathway, to the survival of osteoblasts [12]. Interestingly, these events could also be shown in HeLa cells, mediated by targeting ER␣ to the cell membrane (but not to the nucleus), therefore indicating the role of the plasma membrane ER in this cell action. It must also be appreciated that rapid signaling from

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the ligation of any nuclear steroid receptor superfamily member has not been demonstrated. Stimulation by E2/ER of the cascade leading to ERK and PI3 kinase activation also underlies the up-regulation of nitric oxide production in endothelial cells (EC). This seems to result from the binding of ER␣ receptors in the membrane [13]. Activation of this MAP kinase also is critical to the prevention of glutaminergic, excitotoxicity-induced neuronal necrosis [14], where neuronal salvage is seen in response to E2 or to EGF. Signal transduction through ERK may indirectly influence cell biologic functions via the transactivation of target genes. This was thought to be the exclusive function of the nuclear ER [15,16], but examples are emerging that signaling from the membrane has a similar role. One established mechanism is that growth factor receptors for EGF or IGF can activate ERK, leading to the phosphorylation of Ser118 of the nuclear ER [17,18]. Growth factors also activate pp90rsk-1 via ERK, resulting in Ser 167 phosphorylation of ER [19]. This provides a means by which growth factor signaling transactivates genes, via ER, in the absence of E2. This also partially defines the important cross talk between EGFR and E2 that plays a role in uterine physiology [20]. A second mechanism is illustrated by the fact that E2-augmentation of ERK induces the activation of immediate early genes such as c-fos [21]. This appears to be mediated through ERK effects at the serum response element complex, involving phosphorylation of Elk-1 [22]. Fos serves mainly as a transcription factor, transactivating other genes that are important for the cell biologic effects of this steroid. Signaling may also more directly activate E2-responsive target genes. For instance, E2-induced prolactin gene transcription requires signaling through ERK [23]. It has also been shown that nuclear receptor co-activator proteins can be phosphorlyated by ERK, and that this enhances steroid receptor transactivation functions [24]. Signaling from the membrane ER can also extend to the repression of genes. E2 suppresses the signaling to activator protein 1 (AP-1)-mediated transactivation of the preproendothelin-1 gene, induced by angiotensin II via ERK activity upregulation in endothelial cells [25]. Additional signal transduction pathways have recently been identified as being rapidly responsive to E2, and as originating from the membrane. In EC, this sex steroid, glucocorticoid and thyroid hormones each stimulate phosphoinositiol-3 hydroxy kinase (PI3K). This leads to the activation of Akt kinase, and the generation of NO, inhibited by ICI 182,780 [26]. This pathway was responsible for the ability of E2 to prevent leucocyte accumulation, in an in vivo model of muscle injury following ischemia–reperfusion [26]. In this same cell, E2 stmulates the activity of the p38␤ isoform of the MAP kinase family, leading to activation of the threonine/serine MAPKAP-2 kinase, and the phosphorylation of heat shock protein 27 [27]. E2 was shown to utilize this pathway to protect EC from metabolic disruption of the actin cytoskeleton, hypoxia-induced cell death, and to stimulate angiogenesis.

Additional signaling pathways can be differentially modulated by E2 acting through the two isoforms of ER. In ER-expressing CHO cells, E2 activates a third MAP kinase, c-Jun N-terminal kinase (JNK) via ER␤, but inhibits this kinase via ER␣ [8]. The physiological importance of this observation was recently shown in breast cancer cells. Radiation or chemotherapy treatment kill these cells in large part through inducing apoptosis, and this is mediated through a JNK-dependent mechanism. Recently, we have shown that E2 rapidly blocks JNK activation in this setting, preventing the JNK-induced, inactivating phosphorylation of Bcl2 and Bcl-xl proteins, the subsequent stimulation of the caspase cascade, and cell death [11]. In this way, E2 can act as a survival factor, initiated through membrane signaling. These results are consistent with ER␣ action, based upon the CHO cell signaling model [8], and by the fact that ER␣ is the predominant receptor in most human breast cancer [28]. Consistent with this mechanism, tamoxifen, an estrogen antagonist that prevents the primary occurrence or recurrence of breast cancer, activates apoptosis of breast cancer cells through a JNK-dependent mechanism [29]. In another model, E2 inhibits receptor activator of NF-␬B ligand (RANKL)-induced JNK activation, thereby decreasing osteoclast formation in bone [30].

3. Localization of membrane ER It is important to know the physical structure of the receptor and where it resides within the lipid bilayer, in order to better understand the function of the membrane ER. The endogenous receptor has not yet been isolated and sequenced. However, a variety of antibodies directed against multiple epitopes of the nuclear ER␣, identify an endogenous membrane protein in several cell types [31]. Additionally, expression in CHO cells of a single cDNA for ER␣ (or ER␤) results in both membrane and nuclear pools of receptor [8]. Therefore, data favors the interpretation that the membrane receptor must be very similar to the nuclear receptor, and it may be the same protein translocated to another location. Recent work has begun to clarify the location of this receptor within the plasma membrane. Signaling by growth factor receptor and non growth factortyrosine kinases as well as G protein receptors occurs at least in part after localization to plasma membrane microstructures, known as caveolae [32]. This organelle facilitates signal transduction through the localization of signaling molecules [33], and this interaction is dependent upon the high cholesterol content, and a structural coat protein family, the caveolins. It is believed that caveolin-1 can serve as a scaffold protein, associating with a variety of signaling molecules to organize their activation within the caveolae domains. Although caveolin indirectly facilitates signaling, it may directly inhibit various signal molecules. It is appreciated that caveolin-1 physically associates with eNOS. Upon calcium activation, calmodulin competitively dis-

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places caveolin-1 from binding to eNOS [34], and caveolin-1 moves out of the membrane [34]. These events are necessary for the activation of eNOS. Recently, ER has been shown to localize mainly to caveolae but also to non-caveolar fractions of the EC plasma membrane [35,36] where E2 activates eNOS through ER binding.

4. Complementation of membrane and nuclear ER functions The cell biologic roles of the two receptor pools may be quite complementary even though ER in the membrane and nuclear compartments appear to act by very different mechanisms (signaling versus transcriptional transactivation). There is precedent for E2 to activate gene transcription from both receptor pools, as cited earlier in this article. It may be that kinase signaling can rapidly activate transcription, which can then be sustained by the nuclear receptor. The latter’s action is probably facilitated by the phosphorylation of co-activator proteins, and this could result from ER signaling from the membrane. Signaling from the membrane may also amplify the actions of the nuclear receptor. Furthermore, signaling appears to play an important role in the post-translational modification of proteins that can be upregulated in their synthesis via the nuclear receptor. The important anti-apoptotic protein, Bcl2, serves as an example to illustrate this concept. It has been established that this gene can be transactivated by E2, in part through an Sp-1 site contained within the Bcl2 promoter [37]. As regards the protein, it has recently been shown that the survival function of Bcl2 can be downregulated by phosphorylation within the ‘loop domain’ [38]. E2 prevents the inactivating phosphorylation of this protein by JNK, and thereby enhances breast cancer cell survival [11]. In this way, the activity and concentrations of this protein are modified by discrete cellular pools of ER; this allows both rapid and prolonged regulation of this important protein. Another example is the heat shock protein 27 (Hsp 27). Along with other chaperone family members, this protein is known to associate with ER, with particular relevance in breast cancer [39]. The Hsp27 gene is a target for nuclear ER transcriptional upregulation [40]. Recently, it has been shown that the modulation of Hsp27 protein phosphorylation occurs in response to E2 acting at membrane ER, and that this is critical to the actions of E2 in EC [27]. Again, the membrane and nuclear pools of ER have different but complementary actions to regulate both the short and longerterm cell biologic consequences of Hsp27 function. It is not apparent from examining the amino acid sequence of ER how the membrane receptor signals. Assuming that the structure of this protein is very similar to the nuclear receptor, there are no candidate catalytic or kinase domains present. However, E2 activates multiple signaling events, many emanating from the activation of G proteinrelated pathways [5,6]. E2 rapidly activates membrane ad-

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enylate cyclase (often a Gs␣ function), while IP3 generation and intracellular calcium increases are noted in a variety of cell types [5] (often a Gq␣ or G␤␥ function). The direct evidence that ER can activate G protein alpha subunits and the resulting signaling, comes from studies of CHO cell membranes expressing either ER␣ or ER␤ [8]. We reported that IP3 and cAMP are rapidly generated in response to E2, signaling which is associated with the activation of Gs and Gq␣. Evidence of direct G protein activation in cells expressing endogenous ER, however, has not yet been shown. Since both G proteins and ER exist in caveolae, it is likely that an interaction may take place within this membrane domain. As a possible alternative mechanism, it has recently been reported that E2 can activate a membrane orphan GPCR, GPR30, at least in MCF-7 breast cancer cells expressing endogenous ER [41]. This was reported to lead to the activation of ERK. Curiously, these events were reported to occur independently of ER, through an undefined mechanism. Other GPCR activate ERK in some cell types through the generation of heparan-binding EGF, leading to the subsequent activation and signaling by the EGF receptor [42]. This may contribute to the ability of E2 to activate ERK, especially in MCF-7 cells [41], and would provide an additional cross talk mechanism for the observed interdependance of ER and EGFR in modulating uterine and breast cancer cell biology [20]. Other growth factor receptors may be involved in E2/ER signal transduction. In cells expressing co-transfected ER and the IGF-1 receptor, E2 causes the phosphorylation of IGF-1R and enhanced activation of ERK. The two receptors physically associate in this model, as well [43]. These interactions may be particularly relevant to breast cancer, since E2 augments the ability of IGF-1 to induce cell proliferation [44], and this may be mediated through insulin receptor substrate-1 upregulation [45]. However, in nude mice, MCF-7 cells proliferate despite IGF-1 receptor blockade [46]. Also, IGF-1 may phosphorylate and thus activate the nuclear ER to induce transcription [44]. Additionally, in autocrine fashion, E2 can upregulate PC-cell-derived growth factor production from MCF-7 cells [47], although this may result from nuclear ER action. This novel growth factor in part mediated the ability of E2 to stimulate DNA synthesis in these cells. Finally, E2 may facilitate sex hormone binding globulin (SHBG) signaling from a putative SHBG membrane receptor in prostate cells, by an undefined interaction [48]. The importance of the membrane ER might be intuited from recent discoveries in plants. Arabidopsis, and other flowering plants produce brassinosteroids, that regulate growth and fertility [49]. Brassinosteroids share the basic ring structure with E2. To date, there are no nuclear receptors that have been discovered for these plant steroids. However, a transmembrane, receptor tyrosine kinase has been found to mediate the binding and signaling of brassinosteroids to cell biology [49]. Thus, steroid action at the

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plasma membrane is an ancient and highly conserved function, suggesting its great importance.

5. Perspective Increasing evidence for the importance of the membrane ER in steroid cell biology accumulates. The protein is possibly the same as the nuclear receptor, translocated to a particular location that allows for unique interactions that trigger signaling cascades. The mechanism of translocation needs to be defined, and may afford opportunities to redirect steroid hormone action in both physiological and pathophysiological states. For instance, if the membrane but not the nuclear receptor is critical for discrete cellular actions in breast, bone, uterus, etc., then facilitating the shifting of the receptor pool to a specific location may have merit. Furthermore, developing reagents that affect only one but not a second receptor pool will facilitate our understanding of estrogen biology and may ultimately prove to be the rational way to accomplish sex steroid replacement after the menopause.

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This work was supported by grants from the Research Service of the Department of Veteran’s Affairs, Avon Products Breast Cancer Research Foundation, Department of Defense Breast Cancer Research Program (Grant # BC990915), and the NIH (HL-59890) (to ERL).

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