Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Cell encapsulation wikipedia , lookup
5-Hydroxyeicosatetraenoic acid wikipedia , lookup
Cellular differentiation wikipedia , lookup
NMDA receptor wikipedia , lookup
Purinergic signalling wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
List of types of proteins wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Signal transduction wikipedia , lookup
VLDL receptor wikipedia , lookup
1943 Human Vascular Smooth Muscle Cells Contain Functional Estrogen Receptor Richard H. Karas, MD, PhD; Bruce L. Patterson, MD; Michael E. Mendelsohn, MD Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 Background The decreased incidence of coronary artery disease observed in postmenopausal women given estrogen (E2) replacement demonstrates an atheroprotective effect of E2 that is generally believed to be mediated by indirect, E2-induced changes in cardiovascular risk factor profiles. We hypothesized that the atheroprotective effect of E2 may be in part mediated by a direct effect of E2 on vascular smooth muscle cells (VSMCs). Therefore, a series of experiments was performed to determine whether human VSMCs contain a competent E2 receptor, a ligand-activated transcription factor known to mediate E2-induced effects in nonvascular cells. Methods and Results Ribonuclease protection assays, with a probe derived from the human E2 receptor, were used to demonstrate E2-receptor mRNA in human saphenous vein VSMCs. To show that VSMCs contain E2-receptor protein as well as message, immunoblotting and immunofluorescence studies with a monoclonal anti-E2-receptor antibody were performed, and E2-receptor protein was detected by both methods. Transient transfection assays using a specific E2responsive reporter system were used next to determine whether the VSMC E2 receptor is capable of E2-induced transcriptional transactivation. Initial studies using mammary artery-derived VSMCs resulted in a 2.4-fold increase in reporter activity in response to 10-7 mol/L E2. Subsequent studies using saphenous vein VSMCs demonstrated increasing levels of reporter activation as the concentration of E2 was increased from 10` mol/L (1.3-fold increase; SEM, 0.07; P=.05, n=3) to 10~7 mol/L (1.6-fold increase; SEM, 0.04; P=.002, n=6). The specificity of the E2-induced transactivation of the reporter gene was shown by dose-dependent inhibition of transactivation by the pure E2 antagonist ICI 164,384 and by enhancement of the transactivation by simultaneous overexpression of the E2 receptor. Conclusions We have demonstrated for the first time that human VSMCs express E2-receptor mRNA and protein and that the E2 receptor in VSMCs is capable of estrogendependent gene activation. These data suggest a mechanism by which estrogen may directly alter VSMC function. (Circulation. 1994;89:1943-1950.) Key Words * hormones * smooth muscle * cells . estrogen * genetics C ardiovascular disease is the leading cause of mortality in US women. However, the biology of atherosclerotic disease and ischemic cardiovascular events in women is poorly understood. The low incidence of vascular events in premenopausal women and the marked increase in myocardial infarction following menopause were first recognized more than 50 years ago' and suggested a role for steroid sex hormones in the development of atherosclerosis. Since then, a number of studies have shown that estrogen (E2) administration inhibits the development of experimentally induced atherosclerosis in animal models.2,3 Recent observational studies have demonstrated that oophorectomized women have an increased mortality from cardiovascular disease if they are not given E2 replacement.4 Furthermore, postmenopausal E2 use is associated with a decreased incidence of angiographically documented coronary artery disease,5 myocardial infarction,6 and death from cardiovascular disease.6-9 A recent meta-analysis of the effects of hormonal replacement therapy on mortality in women concluded that postmenopausal E2 replacement reduces the risk of death from cardiac causes by 35%,10 prompting recom- mendations for the widespread use of E2 replacement Received January 19, 1994; revision accepted February 8, 1994. From the Molecular Cardiology Research Center, New England Medical Center, Tufts University School of Medicine, Boston, Mass. Correspondence to Michael E. Mendelsohn, MD, Molecular Cardiology Research Center, New England Medical Center, 750 Washington St, Box 80, Boston, MA 02111. therapy.1,112 The atheroprotective effects of E2 are generally believed to be due to E2-induced changes in classic cardiovascular risk factors. For example, E2 use has been associated with increased high-density lipoprotein levels,5'13,14 decreased low-density lipoprotein levels,5 13 and improved glucose metabolism with decreased serum insulin levels.13 Acute E2 administration has recently been shown to prevent impairment of endothelium-dependent dilation of atherosclerotic arteries in a primate model, suggesting a direct effect of E2 on vascular reactivity.15 However, the mechanism mediating this effect is not yet clear. A number of animal studies have demonstrated specific binding of E2 to vascular cells,16-20 supporting the hypothesis that vascular tissue is E2 sensitive. These studies have relied primarily on detection of intravenously injected radiolabeled hormone in vascular tissues. However, the nature of the E2-binding proteins present, the cells responsible for hormone binding, and direct biological effects of E2 on specific vascular cells have not yet been characterized. Most of the biological effects of steroid hormones are mediated through intracellular hormone receptors that act as ligand-activated transcription factors (reviewed in References 21 through 23). In the presence of ligand, the estrogen receptor (E2 receptor) binds in a highly specific fashion to DNA containing a cis-acting transcriptional enhancer (the E2-response element [ERE]24), resulting in an altered pattern of gene expression (reviewed in 1944 Circulation Vol 89, No S May 1994 Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 References 25 through 27). The E2-receptor gene has been cloned and sequenced28 and codes for a 66-kd zinc finger protein containing hormone binding, DNA binding, and transcriptional activation domains (reviewed in Reference 22). This receptor has been shown to upregulate or downregulate the expression of several important genes, including c-myc,29 cyclin,30 neis31 progesterone receptor,32 and HSP2733 in an E2-dependent fashion. Vascular smooth muscle cells (VSMCs) are one of several cell types within vascular tissue that play a critical role in the pathogenesis of atherosclerosis. VSMC proliferation, induced and sustained by a complex array of growth factors, is the hallmark of myointimal hyperplasia and lesion progression (reviewed in Reference 34). Because of the importance of VSMC proliferation in the development of atherosclerosis and the known growth-regulatory effects of E2 on several cell types,35-39 we hypothesized that the atheroprotective effect of E2 may be in part mediated by a direct effect of E2 on VSMCs. To begin to explore this hypothesis, we undertook a series of experiments to determine whether human VSMCs contain transcriptionally competent E2 receptor. The results we present establish the presence of both E2-receptor mRNA and protein in human VSMCs and demonstrate further, using transient transfection assays, the functional competence of the E2 receptor in VSMCs. Methods Cell Culture Techniques VSMCs were cultured from surgical specimens of human saphenous vein and mammary artery essentially as described.40 For all experiments, VSMCs were grown in media that did not contain phenol red because of the recognized estrogenic effects of this compound.37 Cells were maintained in DMEM containing 10% fetal bovine serum in which the E2 content was determined to be less than 2.6x10-11 mol/L (Hyclone). VSMCs were allowed to migrate from the primary explants and were subsequently passaged at confluence. Cultured cells were a uniform population of smooth muscle cells identified both by their morphology and by immunostaining for smooth muscle-specific a-actin (Sigma Chemical Co; data not shown). VSMCs were harvested at passage 2 for all experiments described below. MCF-7 human breast tumor cells (kindly provided by M. Brown), used as a positive control in several experiments because they are known to express high levels of E2 receptor,41,42 were maintained in DMEM and 10% fetal bovine serum. RNase Protection Assay Total cellular RNA was harvested by a commercially available guanidinium thiocyanate method according to the manufacturer's instructions (Biotecx Laboratories). A radiolabeled RNA probe, complementary to 123 base pairs (bp) of the E region (hormone-binding domain) of the human E2-receptor sequence, was constructed by in vitro transcription (MaxiScript, Ambion) and gel purified on 5% SDS-polyacrylamide gels. RNase protection assays were performed with a commercially available kit (RPAII, Ambion) according to the manufacturer's instructions. The products of the RNase digestion were resolved on 5% SDS-polyacrylamide gels, and relative intensities of protected E2-receptor probe fragments were quantified using the volume integration mode on a PhosphorImager (Molecular Dynamics). Western Blotting VSMCs were suspended by gentle scraping and lysed in the following buffer: 20 mmol/L Tris (pH 7.4), 50 mmol/L NaCl, 50 mmol/L NaF, 50 mmol/L EDTA, 20 mmol/L Na pyrophosphate, 1 mmol/L Na orthovanadate, 1% Triton X-100, 1 mmol/L PMSF, 0.6 mg/mL leupeptin, and 10 ,g/mL aprotinin. The lysis buffer was supplemented with both EDTA and multiple protease inhibitors to prevent the partial proteolysis of the E2-receptor protein observed in preliminary studies. To fractionate subcellular components, total lysates were centri- fuged at 16 OOOg for 10 minutes, and aliquots of the supernatant and cell pellet were solubilized in a high salt buffer (10% SDS, 500 mmol/L NaCI21) and subjected to SDS-PAGE on 10% gels. Immunoblotting was performed with a well-characterized monoclonal anti-E2-receptor antibody (D547), specific for mammalian E2 receptor43 (kindly provided by G. Greene), that was developed by Enhanced Chemiluminescence (ECL, Amersham). Immunofluorescent Staining Immunofluorescent staining of human saphenous VSMCs (HSVSMCs) grown from explants onto glass coverslips was performed using minor modifications of an established protocol.44 Briefly, cells were fixed in 3.7% formaldehyde, incubated for 1 hour with D547 anti-E2-receptor antibody43 (1: 5 to 1:10 dilutions), washed, exposed to FITC-labeled goat anti-rat IgG (Boehringer-Mannheim) (1:20 to 1:100 dilutions), and examined by fluorescence microscopy. Control studies in which primary antibody was omitted were included in all immunofluorescent studies and were consistently negative (data not shown). Transfection Assays Transfection conditions for VSMCs were optimized during preliminary experiments by counting 3-galactosidase-positive cells after transfection with a constitutively expressed (-galactosidase gene (data not shown). The reporter plasmids ERE-Luc (containing three copies of the Xenopus vitellogenin ERE24 proximal to the thymidine kinase promoter driving expression of firefly luciferase cDNA)45 or TK-Luc (identical to ERE-Luc but lacking the ERE; both plasmids were the kind gift of C. Glass) were electroporated (BioRad, Gene Pulser; 220 V, 960 1,F) into cells obtained from one subconfluent T-150 flask. In a subset of experiments, co-transfection of either the expression plasmid pHEGO,46 which contains the wild-type human E2-receptor cDNA driven by an SV-40 enhancer element (kind gift of M. Brown), or an equal quantity of carrier DNA (salmon sperm DNA), was used to determine the effect of overexpression of E2 receptor on the activity of the reporter plasmids. To maintain identical levels of transfection for each experimental condition within an assay, cells pooled from a single electrophoration were aliquoted evenly into a 12-well plate in the presence of 5 mmol/L butyric acid. Thirty-six hours after transfection, the cells were treated with media in the presence or absence of either 17p-estradiol (Sigma Chemical Co) or the pure E2 antagonist ICI 164,38447 (Zeneca Pharmaceuticals; kind gift of A. Wakeling), each dissolved in ethanol. Control cells were treated with ethanol alone to a concentration identical to that of treated cells (final concentration of ethanol, <0.05%). After 24 hours of hormone exposure, luciferase activity was determined in triplicate on a Monolight 2010 luminometer (Analytic Luminescence Laboratories) as described."8 Statistical Analysis Values are reported as mean+SEM. All comparisons were made with the Student's t test. A value of Ps.05 was considered significant. Karas et al Human Vascular SMC Estrogen Receptor ri IG Af - Iges 00 1 1945 l 11 I ER -A' _&_ _,. <_ :; :. -I A.. il. - 1P.11p _. 4, .- ke, 't . h -09 - 3 4, Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 FIG 1. Human saphenous vein smooth muscle cells (HSVSMCs) contain E2-receptor mRNA. Shown is a Phosphorimage from an RNase protection assay demonstrating the presence of E2-receptor mRNA in total RNA from HSVSMCs and MCF-7 breast cancer cells. In each case, a radiolabeled RNA probe from the E region of the human E2-receptor gene is protected (arrowhead). The source and quantity of RNA loaded in each lane are shown above the lane. One of eight similar RNase protection assays is shown. Results HSVSMCs Express the E2-Receptor Gene To demonstrate the presence of E2-receptor message in VSMCs, an RNase protection assay was performed with total RNA prepared from HSVSMCs and a riboprobe from the hormone-binding domain of the E2 receptor. RNase protection assays were used rather than Northern blots because of the anticipated low abundance of the E2-receptor message in nonreproductive tissue.49 In Fig 1, the appearance of a 123-bp protected fragment (arrowhead) demonstrates the presence of E2-receptor mRNA in HSVSMCs. MCF-7 breast cancer cell RNA was included in the assay as a positive control and also protected the riboprobe (Fig 1). Yeast RNA (free of human E2-receptor mRNA) served as a negative control and did not protect the riboprobe (data not shown). Quantification of the intensity of the signal for E2-receptor mRNA demonstrated 42- to 53-fold less E2-receptor message in HSVSMCs than in MCF-7 cells, consistent with the known supraphysiological expression of E2 receptor in these tumor cells.42 HSVSMCs Contain E2-Receptor Protein To demonstrate that VSMCs contain E2-receptor protein as well as message, we analyzed lysates of HSVSMCs by Western blotting with monoclonal anti-E2-receptor antibody. The presence of E2-receptor protein in HSVSMCs is demonstrated in Fig 2. E2-receptor protein was detected consistently by immunoblotting in HSVSMCs derived from both male and female donors and in one study with lysates from VSMCs derived from mammary artery (data not shown). To determine the subcellular distribution of E2-receptor protein in VSMCs, cell lysates were solubilized with high salt conditions used classically to define nuclear and nonnuclear compartmen- FG 2. Human saphenous vein smooth muscle cells (HSVSMCs) contain E2-receptor protein. Total cellular lysate or cytoplasmic (supematant) and nuclear (pellet) fractions from a high salt lysis21 of cultured HSVSMCs were subjected to SDS-PAGE, immunoblotted with anti-E2-receptor antibody, and developed by chemiluminescence techniques. In all such studies, MCF-7 cell protein lysates were used as a positive control in adjacent lanes and produced a band identical in size to that seen in the VSMCs (not shown). The majority of the E2-receptor protein was recovered consistentiy in the cell pellet. One of four similar studies is shown. talization of steroid hormone receptors.21 The E2-receptor protein was recovered predominantly from the nuclear pellet (Fig 2). In all such studies, MCF-7 cell protein lysates were used as a positive control in adjacent lanes and produced a band identical in size to that seen in the VSMCs (data not shown). A larger, 72-kd band of unknown identity was seen in some experiments (Fig 2, nuclear pellet lane), as reported in other cells.50 E2receptor protein was also found by immunoblotting with a different anti-E2-receptor monoclonal antibody (D7543; kindly provided by G. Greene; data not shown). To confirm the subcellular distribution of the E2 receptor observed by immunoblotting, immunofluorescent staining for E2 receptor was also undertaken. Fig 3 shows both the phase-contrast appearance (panel A) and immunofluorescent staining for E2 receptor (panel B) in freshly cultured HSVSMCs. The predominantly nuclear localization of E2 receptor in HSVSMCs is consistent with the pattern observed in similar immunohistochemical studies of E2 receptor in other nonvascular cells.43.5'-53 Control studies in which nonimmune serum was substituted for primary antibody demonstrated only faint and diffuse background staining (data not shown). E2 Receptor in Human VSMCs Is Transcriptionally Competent The data presented thus far demonstrate that VSMCs contain both E2-receptor mRNA and protein but do not establish the functional integrity of the receptor. To determine whether the human VSMC E2 receptor is capable of specific, E2-induced transcriptional transactivation, the E2 responsive reporter plasmid ERE-Luc or the control plasmid TK-Luc was introduced into human VSMCs. Exposure of VSMCs derived from 1946 Circulation Vol 89, No 5 May 1994 Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 FIG 3. Photomicrograph of immunofluorescent identification of E2 receptor in human saphenous vein smooth muscle cells (HSVSMCs). The intracellular distribution of E2 receptor is demonstrated in freshly cultured HSVSMCs. A, Phase-contrast appearance; B, immunofluorescent staining with anti-E2-receptor antibody. Note the predominantly nuclear distribution of the E2 receptor. (x40; bar=2 gum.) mammary arteries to 10` mol/L E2 had no effect on the activity of the TK-Luc plasmid, which lacks the ERE (Fig 4). However, exposure of VSMCs derived from mammary arteries to 10-7 mol/L E2 resulted in a 2.4-fold increase in the activity of the ERE-Luc plasmid (Fig 4). E2 also induced increases in ERE-driven luciferase activity in experiments using saphenous vein VSMCs derived from several different donors. The induction of reporter activity in HSVSMCs increased in a dose-dependent fashion, from 1.3-fold (SEM, 0.07; P=.05, n=3) at 10` mol/L E2 to 1.6-fold (SEM, 0.04; P=.002, n=6) at 10-7 mol/L E2 (Fig 4). These data provide direct evidence for the functional integrity of endogenous E2 receptor in VSMCs. To define further the specificity of the E2-induced activation of the ERE in VSMCs, a separate series of competition experiments was conducted with E2 and the pure antiestrogen ICI 164,384. In mammary arteryderived VSMCs transfected with the ERE-Luc plasmid, 10-7 mol/L E2 activated the ERE 1.5-fold in the presence of low-dose (10-7 mol/L) ICI 164,384 (Fig 5, P=.01). Treatment with increasing doses of ICI 164,384 led to a dose-dependent inhibition of the E2-induced transactivation, which was complete at 10-5 mol/L ICI 164,384 (Fig 5). These data are consistent with the relative potencies of E2 and ICI 164,384 previously reported54 and directly support the specificity of the E2-mediated transactivation of the ERE reporter (see 'Discussion"). Treatment with ICI 164,384 alone had no effect on the activity of the plasmid TK-Luc, which lacks the ERE (data not shown). Taken together, these data demonstrate that human VSMCs contain E2 receptor with transcriptional activation that is specifically regulated by E2, although the magnitude of the transactivation of the reporter system noted in Figs 4 and 5 is relatively modest. To determine whether this result is due to the low abundance of E2 receptor in VSMCs, we examined the effect of increased expression of the E2 receptor on the magnitude of the E2-induced transactivation of the ERE in HSVSMCs. In these studies, increased expression of the E2 receptor was accomplished by co-transfection of an expression plasmid for human wild-type E2 receptor (pHEGO) with either ERE-Ltuc or TK-Luc. In the Mammary- -M _Y ITKTUC IERE-LucI HSV 1 2.5 42.0 - ERE15UC| - -- * _ > W - 0.50 c E2 10-7M c E2 10-7M c E2 E2 10-9M 10-71 FIG 4. Human vascular smooth muscle cells (VSMCs) contain transcriptionally competent E2 receptor. VSMCs derived from either mammary artery or saphenous vein (HSVSMCs) were transiently transfected with a reporter plasmid containing an E2-response element driving expression of the luciferase gene (ERE-Luc) or a control plasmid that lacks the E2-response element (TK-Luc) and subsequently grown for 24 hours in media alone (control state; C), or with either 10-9 mol/L 17f-estradiol (E2 10-9 mol/L) or 10-7 mol/L 17f-estradiol (E2 10-7 mol/L). Bars represent the mean relative luciferase activity with the SEM where appropriate (n=5 mammary artery/TK-Luc; n=2 mammary artery/ERE-Luc; n=3 to 6 HSVSMCs/ERE-Luc). Luciferase activity is shown relative to control cells that were not exposed to hormones. *P=.05, **P=.002 vs HSVSMCs/ERE-Luc control. Karas et al Human Vascular SMC Estrogen Receptor 10-7M 10-7M 10-7M 10-4M 10-SM 0 E2 C -10-7M 10-7M 9 - - - - B 1.50 § 1.25 * 1.00 9 ~~~~~* - - - - - _ _ _ _ __ - 0 ' Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 FIG 5. Activation of the E2-response element reporter in human vascular smooth muscle cells (VSMCs) is competitively inhibited by the antiestrogen ICI 164,384. Mammary artery VSMCs transfected with the ERE-Luc plasmid were grown in the absence or presence of 1 0- mol/L 17p-estradiol (E2) and exposed to various concentrations of the pure antiestrogen ICI 164,384 (IC1). Bars represent the mean luciferase activity relative to control cells that were not exposed to E2 (+SEM; n=3). #P=.01 vs bar A; *P=.05 vs bar B; **P=.001 vs bar B and P=NS vs bar A. absence of overexpression of the E2 receptor, treatment of HSVSMCs with iO-` mol/L E2 again resulted in a 1.5-fold increase in ERE activity (Fig 6; cf Fig 4). However, in HSVSMCs co-transfected with the E2receptor expression plasmid, E2 exposure resulted in a 7.5-fold increase in ERE activity (Fig 6). Of note, co-transfection of the E2-receptor plasmid had no effect on the activity of the TK-Luc (control) plasmid (Fig 6). 8 - -- - - - - - -__ --- ; _ zzj 7-:zz d -I -_ - ___ - 0 ER Wbnmid Reporter E EREL I nwO E + ERE-Luc TK.Luc n= 5 nz10 FiG 6. Overexpression of the E2 receptor in human saphenous vein smooth muscle cells (HSVSMCs) enhances E2-induced transactivation of the E2-response element. HSVSMCs were treated and transiently transfected with a reporter plasmid containing an E2response element driving the expression of the luciferase gene (ERE-Luc) or the control plasmid that lacks the E2-response element (TK-Luc) as in Fig 5. Cells were co-transfected either with an E2-receptor expression plasmid (ER plasmid) or carrier DNA and grown for 24 hours in the absence (hatched bars) or presence (solid bars) of 10-7 mo/L 173-estradiol and assayed for luciferase activity. Bars represent the mean luciferase activity relative to control cells that were not exposed to E2 (+SEM). Note that overexpression of the E2 receptor enhances E2-induced activation of the E2-response element reporter (ERE-Luc) fivefold but has no effect on the control plasmid (TK-Luc) lacking this element. 1947 Discussion The data presented here demonstrate for the first time that human VSMCs contain the biochemical machinery known to mediate E2 effects on the transcription of E2-responsive genes in E2-responsive tissues.55 The results demonstrate E2-receptor mRNA by RNase protection assay and E2-receptor protein by both Western blotting and immunofluorescent staining in HSVSMCs. Furthermore, the majority of VSMC E2 receptor is shown to be localized to the nucleus by both immunoblotting (Fig 2) and immunofluorescence (Fig 3), although some cytosolic receptor was also detected. These findings are consistent with other immunologically based detection methods demonstrating E2 receptor primarily in the nucleus of nonvascular cells.43,51-54 Although the data presented above demonstrate that human VSMCs contain E2 receptor, they also suggest that the receptor is present at a low level. The RNase protection assay demonstrates that E2-receptor mRNA is approximately 50-fold less abundant in HSVSMCs than in MCF-7 breast cancer cells. In addition, several lines of evidence suggest that E2-receptor protein levels were substantially less in HSVSMCs than in MCF-7 cells; preliminary experiments demonstrated that E2receptor protein was difficult to detect by non-ECLbased immunoblotting techniques, and compared with MCF-7 cells, lysates containing 25- to 50-fold more VSMCs were required for detection of E2 receptor by immunoblotting (R.H. Karas, M.E. Mendelsohn, unpublished observations). Finally, the E2-receptor overexpression studies, demonstrating that co-transfection of an E2-receptor expression plasmid increases the magnitude of E2-induced activation of the ERE in VSMCs (Fig 6), further support the conclusion that VSMCs contain a relatively low abundance of functional E2 receptor. Demonstration of E2-receptor mRNA and protein in a cell is not sufficient to establish the functional integrity of the receptor in that cell because of the complex regulatory system controlling transcriptional activation of steroid hormone receptors. A number of intracellular events are known to modulate steroid hormone receptor function. For example, several proteins have been found to associate with estrogen and other steroid receptors.23-56-61 Some such interactions appear to be critical for efficient transactivation of steroid-responsive genes,62 whereas others may inhibit transcriptional activation.57-59 Thus, cells may contain E2 receptor in a transcriptionally inefficient or inactive state due to interactions with and/or absence of associated proteins. The transient transfection assays presented in Figs 4 through 6 demonstrate conclusively that the E2 receptor in human VSMCs is transcriptionally competent. The specificity of the E2-induced transactivation in VSMCs is demonstrated by (1) control studies with the control plasmid TK-Luc, which establish that the ERE is necessary for transactivation of the reporter; (2) the dose-dependent increase in transactivation of ERE-Luc by E2; and (3) dose-dependent inhibition of the E2-induced increase in ERE-driven reporter activity by the specific E2 antagonist ICI 164,384. VSMC proliferation is a central event in atherosclerosis and may have a different biology in women than in men. Demonstration that VSMCs contain functional E2 receptor raises the possibility that E2 may alter patterns 1948 Circulation Vol 89, No 5 May 1994 Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 of gene expression important to VSMC growth. E2 can promote or inhibit proliferation, depending on the type of cell studied. Both neuroblastoma cells and 3T3 fibroblasts transfected with E2-receptor expression vectors are growth suppressed by exposure to E2,35.63 and E2 has been reported to inhibit the growth of VSMCs derived from rabbit aorta,6" rat pulmonary artery,65 and porcine coronary artery.68 These studies and the known atheroprotective effect of estrogen in postmenopausal women are consistent with the hypothesis that in some settings E2 may directly inhibit the proliferation of VSMCs. Although our data demonstrate E2-induced activation of an exogenous ERE in VSMCs, it will be important next to demonstrate that E2 directly activates the expression of endogenous VSMC genes. In nonvascular cells, only a few E2 receptor-activated genes have thus far been identified, including HSP27, the progesterone receptor, c-fos, and c-myc.23'32'67'68 If E2 directly modulates the growth response of VSMCs, the protooncogenes are candidate genes whose expression may be modulated by estrogen. How might the VSMC E2 receptor mediate a growthinhibitory effect of E2? One logical target for E2mediated growth regulation is proto-oncogene expression. E2-dependent regulation of the expression of proto-oncogenes is well described in breast cancer cells.29'31'69 Some of the proto-oncogenes known to be regulated by E2 in breast cancer cells (eg, c-myc29,67) are also important regulators of VSMC proliferation.70,71 Another mechanism by which steroid receptors might participate in growth control is suggested by recent reports demonstrating cross-talk between steroid hormone receptor and cell surface receptor-mediated signaling pathways.39'59'60'72-75 Steroid and cell surface receptor-mediated signaling may interact in several ways. For example, steroid receptors may be activated directly by mitogens that act through membrane-spanning receptors, as in the recent report of epidermal growth factor-induced transactivation of an ERE-reporter system in endometrial adenocarcinoma cells.76 In addition, steroid receptors may have complex interactions with other transcription factors known to regulate proliferation, such as those between the glucocorticoid receptor and the c-fos protein.58-60'68 The E2 receptor has also been shown to interact specifically with AP-1.77,78 Such studies raise the intriguing possibility that E2 receptormediated effects on gene expression in VSMCs may converge in several ways with growth factor-mediated pathways known to regulate the proliferation of VSMCs, a critical component in the pathogenesis of atherosclerosis.4 In summary, we have demonstrated for the first time that human VSMCs express E2-receptor mRNA and protein and have shown further that E2 receptor in VSMCs is capable of transcriptional transactivation. Human VSMCs therefore possess the requisite biochemical machinery to respond directly to E2 exposure. Our model provides a novel system with which to study the role of estrogen and the estrogen receptor in VSMC biology. Given the expanding investigations into the role of antiestrogens in the treatment of breast cancer, the controversy regarding the cardiovascular effects of oral contraceptives, and the growing recognition of the atheroprotective effects of E2, definition of the biolog- ical effects of E2 on VSMCs clinical implications. may also have important Acknowledgments We thank Myles Brown for helpful discussions and critical review of the manuscript. We also thank R. Sanders Williams and Deeb Salem for their support and guidance. We are grateful to Peter Libby for help in establishing human VSMC cultures in our laboratory. We are also grateful to Sarah O'Neill, Wendy Baur, and Laura Tassi for excellent technical support and to Patricia Nayak for expert preparation of the manuscript. We thank Marshall A. Wolf for generous support provided to Bruce Patterson. Richard Karas is the recipient of a Pfizer Postdoctoral Fellowship in Cardiology. This work is dedicated to the memory of Sheldon M. Wolff. References 1. Glendy RE, Levine SA, White PD. Coronary disease in youth: comparison of 100 patients under 40 with 300 persons past 80. JAMA. 1937;109:1775-1781. 2. Stamler J, Pick R, Katz LN. Prevention of coronary atherosclerosis by estrogen-androgen administration in the cholesterol-fed chick. Circ Res. 1953;1:94-98. 3. Weigensberg BI, Lough J, More RH, Katz E, Pugash E, Peniston C. Effects of estradiol on myointimal thickenings from catheter injury and on organizing white mural non-occlusive thrombi. Atherosclerosis. 1984;52:253-265. 4. Colditz GA, Willett WC, Stampfer MJ, Rosner B, Speizer FE, Hennekens CH. Menopause and the risk of coronary heart disease in women. N EngI J Med. 1987;316:1105-1110. 5. Hong MK, Romm PA, Reagan K, Green CE, Rackley CE. Effects of estrogen replacement therapy on serum lipid values and angiographically defined coronary artery disease in postmenopausal women. Am J Cardiol. 1992;69:176-178. 6. Stampfer MJ, Colditz GA, Willett WC, Manson JE, Rosner B, Speizer FE, Hennekens CH. Postmenopausal estrogen therapy and cardiovascular disease. N EngI J Med. 1991;325:756-762. 7. Bush TL, Barrett-Connor E, Cowan LD, Crigui MH, Wallace RB, Suchindran CM, Tyroler HA, Rifkind BM. Cardiovascular mortality and noncontraceptive use of estrogen in women: results from the Lipid Research Clinics Program Follow-up Study. Circulation. 1987;75:1102-1109. 8. Ross RK, Paganini-Hill A, Mack TM, Arthur M, Henderson BE. Menopausal oestrogen therapy and protection from death from ischaemic heart disease. Lancet. 1981;1:858-860. 9. Henderson BE, Paganini-Hill A, Ross RK. Decreased mortality in users of estrogen replacement therapy. Arch Intern Med. 1991;151: 75-78. 10. Grady D, Rubin SM, Petitti DB, Fox CS, Black D, Ettinger B, Ernster VL, Cummings SR. Hormone therapy to prevent disease and prolong life in postmenopausal women. Ann Intern Med. 1992; 117:1016-1037. 11. American College of Physicians. Guidelines for counseling postmenopausal women about preventive hormone therapy. Am Coll Phys. 1992;117:1038-1041. 12. Martin KA, Freeman MW. Postmenopausal hormone-replacement therapy. NEnglJMed. 1993;328:1115-1117. 13. Nabulsi AA, Folsom AR, White A, Patsch W, Geiss G, Wu KK, Szklo M. Association of hormone-replacement therapy with various cardiovascular risk factors in postmenopausal women. N Engl J Med. 1993;328:1069-1075. 14. Cauley JA, LaPorte RE, Kuller LH, Bates M, Sandler RB. Menopausal estrogen use, high density lipoprotein cholesterol subfractions and liver function. Atherosclerosis. 1983;49:31-40. 15. Williams JK, Adams MR, Herrington DM, Clarkson TB. Short-term administration of estrogen and vascular responses of atherosclerotic coronary arteries. J Am Coil Cardiol. 1992;20: 452-457. 16. Horwitz KB, Horwitz LD. Canine vascular tissues are targets for androgens, estrogens, progestins, and glucocorticoids. J Clin Invest. 1982;69:750-759. 17. Nakao J, Chang WC, Murota SI, Orimo H. Estradiol-binding sites in rat aortic smooth muscle cells in culture. Atherosekrosis. 1981; 38:75-80. 18. McGill HC Jr, Sheridan PJ. Nuclear uptake of sex steroid hormones in the cardiovascular system of the baboon. Circ Res. 1981;48:238-244. Karas et al Human Vascular SMC Estrogen Receptor Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 19. Malinow MR, Moguilevsky JA, Lema B, Bur GE. Vascular and extravascular radioactivity after the injection of estradiol-6,7 H3 in the human being. J Clin Endocrinol Metab. 1963;23:306-310. 20. Stumpf WE. Autoradiographic techniques for the localization of hormones and drugs at the cellular and subcellular level. Acta EndocrinoL 1971;153:205-222. 21. Carson-Jurica MA, Schrader WT, O'Malley BW. Steroid receptor family: structure and functions. Endocrinol Rev. 1990;11:201-220. 22. Evans RM. The steroid and thyroid hormone receptor superfamily. Science. 1988;240:889-895. 23. King RJB. Structure and function of steroid receptors. J Endocrinol. 1987;114:341-349. 24. Klein-Hitpass L, Schorpp M, Wagner W, Ryffel GU. An estrogenresponsive element derived from the 5' flanking region of the Xenopus vitellogenin A12 gene functions in transfected human cells. Cell. 1986;46:1053-1061. 25. Wahli W, Martinuzzo M. Superfamily of steroid nuclear receptors: positive and negative regulators of gene expression. FASEB J. 1991;5:2243-2249. 26. Beato M. Gene regulation by steroid hormones. Cell. 1989;56: 335-344. 27. Gronemeyer H. Control of transcription activation by steroid hormone receptors. FASEB J. 1992;6:2524-2529. 28. Walter P, Green S, Krust A, Bornert JM, Jeltsch JM, Staub A, Jensen E, Scrace G, Waterfield M, Chambon P. Cloning of the human estrogen receptor cDNA. Proc Nad Acad Sci USA. 1985; 82:7889. 29. Shiu RPC, Watson PH, Dubik D. c-myc oncogene expression in estrogen-dependent and -independent breast cancer. Clin Chem. 1993;39:353-355. 30. Musgrove EA, Hamilton JA, Lee CSL, Sweeney KJE, Watts CKW, Sutherland RL. Growth factor, steroid, and steroid antagonist regulation of cyclin gene expression associated with changes in T-47D human breast cancer cell cycle progression. Mol Cell Biol. 1993;13:3577-3587. 31. Read LD, Keith D Jr, Slamon DJ, Katzenellenbogen BS. Hormonal modulation of HER-2/neu protooncogene messenger ribonucleic acid and p185 protein expression in human breast cancer cell lines. Cancer Res. 1990;50:3947-3951. 32. Horwitz KB, McGuire WL. Estrogen control of progesterone receptors in human breast cancer: correlations with nuclear processing of estrogen receptors. J Biol Chem. 1978;253:2223-2228. 33. Fuqua SAW, Blum-Salingaros M, McGuire WL. Induction of the estrogen-regulated '24K' protein by heat shock. Cancer Res. 1984; 49:4126-4129. 34. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809. 35. Gaben A-M, Mester J. BALB/C mouse 3T3 fibroblasts expressing human estrogen receptor: effect of estradiol on cell growth. Biochem Biophys Res Commun. 1991;176:1473-1481. 36. Taggart H, Stout RW. Control of DNA synthesis in cultured vascular endothelial and smooth muscle cells. Atherosclerosis. 1980; 37:549-557. 37. Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS. Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proc Nad Acad Sci USA. 1986;83:2496-2500. 38. Darbre P, Yates J, Curtis S, King RJB. Effect of estradiol on human breast cancer cells in culture. Cancer Res. 1983;43:349-354. 39. Berthois Y, Dong XF, Martin PM. Regulation of epidermal growth factor-receptor by estrogen and antiestrogen in the human breast cancer cell line MCF-7. Biochem Biophys Res Commun. 1989;159: 126-131. 40. Libby P, Warner SK, Friedman GB. Interleukin-1: a mitogen for human vascular smooth muscle cells that induces the release of growth-inhibitory prostanoids. J Clin Invest. 1988;81:487-498. 41. Horwitz KB, Zara DT, Thilagar AK, Jensen EM, McGuire WL. Steroid receptor analysis of nine human breast cancer cell lines. Cancer Res. 1978;38:2434-2437. 42. Olea-Serrano N, Devleeschouwer N, Leclercq G, Heuson J-C. Assay for estrogen and progesterone receptors of breast cancer cell lines in monolayer culture. Eur J Cancer Clin OncoL 1985;21: 965-973. 43. Greene GL, Sobel NB, King WJ, Jensen EV. Immunochemical studies of estrogen receptors. J Steroid Biochem Mol Biol. 1984;20: 51-56. 44. Dadabay CY, Patton E, Cooper JA, Pike LU. Lack of correlation between changes in polyphosphoinositide levels and actin/gelsolin complexes in A431 cells treated with epidermal growth factor. J Cell Biol. 1991;112:1151-1156. 1949 45. Glass CK, Holloway JM, Devary OV, Rosenfeld MG. The thyroid hormone receptor binds with opposite transcriptional effects to a common sequence motif in thyroid hormone and estrogen response elements. Cell. 1988;54:313-323. 46. Tora L, Mullick A, Metzger D, Ponglikitmongkol M, Park I, Chambon P. The cloned human oestrogen receptor contains a mutation which alters its hormone binding properties. EMBO J. 1989;8:1981-1986. 47. Wakeling AE, Bowler J. Novel antioestrogen without partial agonist activity. J Steroid Biochem Mol Biol. 1988;31:645-653. 48. DeWet JR, Wood KV, DeLuca M, Helinski DR, Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987;7:725-737. 49. Thomas ML, Xu X, Norfleet AM, Watson CS. The presence of functional estrogen receptors in intestinal epithelial cells. Endocrinology. 1993;132:426-430. 50. Abbondanza C, De Falco A, Nigro V, Medici N, Armetta I, Molinari AM, Moncharmont B, Puca GA. Characterization and epitope mapping of a new panel of monoclonal antibodies to estradiol receptor. Steroids. 1993;58:4-12. 51. Washburn T, Hocutt A, Brautigan DL, Korach KS. Uterine estrogen receptor in vivo: phosphorylation of nuclear specific forms on serine residues. Mol Endocrinol. 1991;5:235-242. 52. Yamashita S, Korach KS. A modified immunohistochemical procedure for the detection of estrogen receptor in mouse tissues. Histochem. 1989;90:325-330. 53. Leiberman JR, van Vroonhoven CCJ, Beckmann I, van der Kwast TH, Wallenburg KCS. Uterine artery estrogen receptors in the nonpregnant and pregnant guinea pig. Am J Obstet Gynecol. 1990; 163:1685-1688. 54. Ali S, Lutz Y, Bellocq J-P, Chenard-Neu M-P, Rouyer N, Metzger D. Production and characterization of monoclonal antibodies recognising defined regions of the human oestrogen receptor. Hybridoma. 1993;12:391-405. 55. Green S, Walter P, Kumar V, Krust A, Bornert J-M, Argos P, Chambon P. Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature. 1986;320:134-139. 56. Mendelsohn ME, Zhu Y, O'Neill S. The 29-kDa proteins phosphorylated in thrombin-activated human platelets are forms of the estrogen receptor-related 27-kDa heat shock protein. Proc Natl Acad Sci USA. 1991;88:11212-11216. 57. Howard KJ, Distelhorst CW. Evidence for intracellular association of the glucocorticoid receptor with the 90-kDa heat shock protein. J Biol Chem. 1988;263:3474-3481. 58. Kerppola TK, Luk D, Curran T. Fos is a preferential target of glucocorticoid receptor inhibition of AP-1 activity in vitro. Mol Cell Biol. 1993;13:3782-3791. 59. Yang-Yen H-F, Chambard J-C, Sun Y-L, Smeal T, Schmidt TJ, Drouin J, Karin M. Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell. 1990;62:1205-1215. 60. Schule R, Rangarajan P, Kliewer S, Ransone U, Bolado J, Yan N, Verma IM, Evans RM. Functional antagonism between oncoprotein c-Jun and the glucocorticoid receptor. Cell. 1990;62: 1217-1226. 61. Smith DF, Toft DO. Steroid receptors and their associated proteins. Mol Endocrinol. 1993;7:4-11. 62. Picard D, Khursheed B, Garabedian MJ, Fortin MG, Lindquist S, Yamamoto KR. Reduced levels of HSP90 compromise steroid receptor action in vivo. Nature. 1990;348:166-168. 63. Ma ZQ, Spreafico E, Pollio G, Santagati S, Conti E, Cattaneo E, Maggi A. Activated estrogen receptor mediates growth arrest and differentiation of a neuroblastoma cell line. Proc Natl Acad Sci USA. 1993;90:3740-3744. 64. Fischer-Dzoga K, Wissler RW, Vesselinovitch D. The effect of estradiol on the proliferation of rabbit aortic medial tissue culture cells induced by hyperlipemic serum. Exp Mol Pathol. 1983;39: 355-363. 65. Farhat MY, Vargas R, Dingaan B, Ramwell PW. In vitro effect of oestradiol on thymidine uptake in pulmonary vascular smooth muscle cell: role of the endothelium. Br J PharmacoL 1992;107: 679-683. 66. Vargas R, Wroblewska B, Rego A, Hatch J, Ramwell PW. Oestradiol inhibits smooth muscle cell proliferation of pig coronary artery. BrJ Pharmacol. 1993;109:612-617. 67. Miller TL, Huzel NJ, Davie JR, Murphy LC. C-myc gene chromatin of estrogen receptor positive and negative breast cancer cells. Mol Cell Endocrinol 1993;91:83-89. 68. Jonat C, Rahmsdorf HJ, Park K-K, Cato ACB, Gebel S, Ponta H, Herrlich P. Antitumor promotion and antiinflammation: down- 1950 69. 70. 71. 72. 73. Circulation Vol 89, No 5 May 1994 modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. CelL 1990.,63:1189-1204. Dubik D, Dembinski TC, Shiu RPC. Stimulation of c-myc oncogene expression associated with estrogen-induced proliferation of human breast cancer cells. Cancer Res. 1987;47: 6517-6521. Biro S, Fu Y-M, Yu Z-X, Epstein SE. Inhibitory effects of antisense oligodeoxynucleotides targeting c-myc mRNA on smooth muscle cell proliferation and migration. Proc NatlAcad Sci USA. 1993;90:654-658. Shi Y, Hutchinson HG, Hall DJ, Zalewskd A. Downregulation of c-m expression by antisense oligonucleotides inhibits proliferation of human smooth muscle cells. C oruzadL 1993;88:1190-1195. Ignar-Trowbridge DM, Nelson KG, Bidwell MC, Curtis SW, Washburn TF, McLachlan JA, Korach KS. Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor. Proc Nat Acad Sci USA. 1992;89:4658-4662. Ignar-Trowbridge DM, Teng CT, Ross KA, Parker MG, Korach KS, McLachlan JA. Peptide growth factors elicit estrogen receptor-dependent transcriptional activation of an estrogenresponsive element. Mol Endxrinwl. 1993;7:992-998. 74. Stewart AJ, Johnson MD, May FEB, Westley BR. Role of insulin-like growth factors and the type I insulin-like growth factor receptor in the estrogen-stimulated proliferation of human breast cancer cells. J Biol Chem. 1990;265:21172-21178. 75. Smith CL, Conneely OM, O'Malley BW. Modulation of the ligandindependent activation of the human estrogen receptor by hormone and antihormone. Proc Natl Acad Sci USA. 1993;90: 6120-6124. 76. Ingar-Trowbridge DM, Teng CT, Ross KA, Parker MG, Korach KS, McLachlan JA. Peptide growth factors elicit estrogen receptor-dependent transcriptional activation of an estrogenresponsive element. Mol Endocrinol. 1993;7:992-998. 77. Philips A, Chalbos D, Rochefort H. Estradiol increases and antiestrogens antagonize the growth factor-induced activator protein-1 activity in MCF7 breast cancer cells without affecting c-fos and c-jun synthesis. J Biol Chem. 1993;268:14103-14108. 78. van der Burg B, de Groot RP, Isbrucker L, Kruijer W, de Laat SW. Stimulation of TPA-responsive element activity by a cooperative action of insulin and estrogen in human breast cancer cells. Mol Endocrinol. 1990;4:1720-1726. Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 Human vascular smooth muscle cells contain functional estrogen receptor. R H Karas, B L Patterson and M E Mendelsohn Circulation. 1994;89:1943-1950 doi: 10.1161/01.CIR.89.5.1943 Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1994 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circ.ahajournals.org/content/89/5/1943 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation is online at: http://circ.ahajournals.org//subscriptions/