Rapid activation of endothelial NO synthase by estrogen: evidence for a steroid receptor fast-action complex (SRFC) in caveolae

Estrogen has important atheroprotective and vasoactive properties related to its capacity to stimulate nitric oxide (NO) production by endothelial NO synthase. Previous work has shown that these effects are mediated by estrogen receptor (ER) alpha functioning in a nongenomic manner via calcium-dependent, MAP kinase-dependent mechanisms. Recent studies have demonstrated that estradiol (E(2)) activates eNOS in isolated endothelial plasma membranes in the absence of added calcium, calmodulin or eNOS cofactors. Studies of blockade by ICI 182,780 and by ER alpha antibody, and also immunoidentification experiments indicate that the process is mediated by a subpopulation of plasma membrane-associated ER alpha. Fractionation of endothelial cell plasma membranes has further revealed that ER alpha protein is localized to caveolae, and that E(2) causes stimulation of eNOS in isolated caveolae which is ER-dependent and calcium-dependent, whereas noncaveolae membranes are insensitive. Furthermore, in intact endothelial cells the activation of eNOS by E(2) is prevented by pertussis toxin, and exogenous GDP beta S inhibits the response in isolated plasma membranes. Coimmunoprecipitation studies have shown that E(2) exposure causes interaction between ER alpha and G(alpha i) on the plasma membrane, and eNOS activation by E(2) is enhanced by overexpression of G(alpha i) and attenuated by expression of a protein regulator of G protein signaling (RGS), RGS4. Thus, a subpopulation of ER alpha is localized to caveolae in endothelial cells, where they are coupled via G(alpha i) to eNOS in a functional signaling module. Emphasizing the dependence on cell surface-associated receptors, these observations provide evidence for the existence of a steroid receptor fast-action complex, or SRFC, in caveolae.     

Functional roles of plasma membrane localized estrogen receptors

A series of emerging data supports the existence and importance of plasma membrane localized estrogen receptors in a variety of cells that are targets for the steroid hormone action. When estradiol (E2) binds to the cell surface protein, the ensuing signal transduction event triggers downstream signaling cascades that contribute to important biological functions. Aside from the classical signaling through nuclear estrogen receptors, we have provided evidence for the functional roles of an estrogen receptor localized in the plasma membrane. This review highlights some of the recent advances made in the understanding of the genomic/non-genomic actions of plasma membrane localized estrogen receptors.     

Estrogen and growth factor receptor interactions in human breast and non-small cell lung cancer cells

Extranuclear estrogen receptors may mediate rapid effects of estradiol that communicate with nuclear receptors and contribute to proliferation of human cancers bearing these signaling proteins. To assess these growth-promoting pathways, we undertook controlled homogenization and fractionation of NIH-H23 non-small cell lung cancer cells. As many breast tumors, NIH-H23 cells express estrogen receptors (ER), with the bulk of specific estradiol binding in nuclear fractions. However, as in breast cells, a significant portion of specific, high-affinity estradiol-17beta binding-sites are also enriched in plasma membranes of lung tumor cells. These estrogen binding-sites co-purify with plasma membrane-marker enzymes and are not significantly contaminated by cytosol or nuclei. On further purification of membrane caveolae from lung tumor cells, proteins recognized by monoclonal antibodies to nuclear ER-alpha and to ER-beta were identified in close association with EGF receptor in caveolae. In parallel studies, ER-alpha and ER-beta are also detected in nuclear and extranuclear sites in archival human breast and lung tumor samples and are noted to occur in clusters at the cell membrane by using confocal microscopy to visualize fluorescent-labeled monoclonal antibodies to ER-alpha. Data on site-directed mutagenesis of cysteine-447 in ER-alpha suggest that association of ER forms with membrane sites may depend on acylation of cysteine by palmitate. Estrogen-induced growth of MCF-7 breast cancer and NIH-H23 lung cancer cells in vitro correlated closely with acute hormonal activation of mitogen-activated protein kinase signaling and was significantly reduced by treatment with Faslodex, a pure anti-estrogen. Further, combination of Faslodex with selected growth factor receptor inhibitors elicited a more pronounced inhibiton of tumor cell growth. Thus, extranuclear forms of ER play a role in promoting downstream signaling for hormone-mediated proliferation and survival of breast, as well as lung, cancers and offer a new target for anti-tumor therapy.     

Plasma membrane estrogen receptors exist and functions as dimers

A small pool of estrogen receptors (ERalpha and -beta) localize at the plasma membrane and rapidly signal to affect cellular physiology. Although nuclear ERs function mainly as homodimers, it is unknown whether membrane-localized ER exists or functions with similar requirements. We report that the endogenous ER isoforms at the plasma membrane of breast cancer or endothelial cells exist predominantly as homodimers in the presence of 17beta-estradiol (E2). Interestingly, in endothelial cells made from ERalpha /ERbeta homozygous double-knockout mice, membrane ERalpha or ERbeta are absent, indicating that the endogenous membrane receptors derive from the same gene(s) as the nuclear receptors. In ER-negative breast cancer cells or Chinese hamster ovary cells, we expressed and compared wild-type and dimer mutant mouse ERalpha. Only wild-type ERalpha supported the ability of E2 to rapidly activate ERK, cAMP, and phosphatidylinositol 3-kinase signaling. This resulted from E2 activating Gsalpha and Gqalpha at the membrane in cells expressing the wild-type, but not the dimer mutant, ERalpha. Intact, but not dimer mutant, ERalpha also supported E2-induced epidermal growth factor receptor transactivation and cell survival. We also confirmed the requirement of dimerization for membrane ER function using a second, less extensively mutated, human ERalpha. In summary, endogenous membrane ERs exist as dimers, a structural requirement that supports rapid signal transduction and affects cell physiology.     

ERs associate with and regulate the production of caveolin: implications for signaling and cellular actions.

Recent evidence supports the existence of a plasma membrane ER. In many cells, E2 activates signal transduction and cell proliferation, but the steroid inhibits signaling and growth in other cells. These effects may be related to interactions of ER with signal-modulating proteins in the membrane. It is also unclear how ER moves to the membrane. Here, we demonstrate ER in purified vesicles from endothelial cell plasma membranes and colocalization of ERalpha with the caveolae structural coat protein, caveolin-1. In human vascular smooth muscle or MCF-7 (human breast cancer) cell membranes, coimmunoprecipitation shows that ER associates with caveolin-1 and -2. Importantly, E2 rapidly and differentially stimulates ER-caveolin association in vascular smooth muscle cells but inhibits association in MCF-7 cells. E2 also stimulates caveolin-1 and -2 protein synthesis and activates a caveolin-1 promoter/luciferase reporter in smooth muscle cells. However, the steroid inhibits caveolin synthesis in MCF-7 cells. To determine a function for caveolin-ER interaction, we expressed caveolin-1 in MCF-7 cells. This stimulated ER translocation to the plasma membrane and also inhibited E2-induced ERK (MAPK) activation. Both functions required the caveolin-1 scaffolding domain. Depending upon the target cell, membrane ERs differentially associate with caveolin, and E2 differentially modulates the synthesis of this signaling-inhibitory scaffold protein. This may explain the discordant signaling and actions of E2 in various cell types. In addition, caveolin-1 is capable of facilitating ER translocation to the membrane.     

Sex-specific effects of 17β-estradiol and dihydrotestosterone (DHT) on growth plate chondrocytes are dependent on both ERα and ERβ and require palmitoylation to translocate the receptors to the plasma membrane

Rat costochondral cartilage growth plate chondrocytes exhibit cell sex-specific responses to 17β-estradiol (E₂), testosterone, and dihydrotestosterone (DHT). Mechanistically, E₂ and DHT stimulate proliferation and extracellular matrix synthesis in chondrocytes from female and male rats, respectively, by signaling through protein kinase C (PKC) and phospholipase C (PLC). Estrogen receptors (ERα; ERβ) and androgen receptors (ARs) are present in both male and female cells, but it is not known whether they interact to elicit sex-specific signaling. We used specific agonists and antagonists of these receptors to examine the relative contributions of ERs and ARs in membrane-mediated E₂ signaling in female chondrocytes and DHT signaling in male chondrocytes. PKC activity in female chondrocytes was stimulated by agonists of ERα and ERβ and required intact caveolae; PKC activity was inhibited by the E₂ enantiomer and by an inhibitor of ERβ. Western blots of cell lysates co-immunoprecipitated for ERα suggested the formation of a complex containing both ERα and ERß with E₂ treatment. DHT and DHT agonists activated PKC in male cells, while AR inhibition blocked the stimulatory effect of DHT on PKC. Inhibition of ERα and ERβ also blocked PKC activation by DHT. Western blots of whole-cell lysates, plasma membranes, and caveolae indicated the translocation of AR to the plasma membrane and specifically to caveolae with DHT treatment. These results suggest that E₂ and DHT promote chondrocyte differentiation via the ability of ARs and ERs to form a complex. The results also indicate that intact caveolae and palmitoylation of the membrane receptor(s) or membrane receptor complex containing ERα and ERβ is required for E₂ and DHT membrane-associated PKC activity in costochondral cartilage cells.     

Initiation of BMP2 signaling in domains on the plasma membrane

Bone morphogenetic protein 2 (BMP2) is a potent growth factor crucial for cell fate determination. It directs the differentiation of mesenchymal stem cells into osteoblasts, chondrocytes, adipocytes, and myocytes. Initiation of BMP2 signaling pathways occurs at the cell surface through type I and type II serine/threonine kinases housed in specific membrane domains such as caveolae enriched in the caveolin-1 beta isoform (CAV1β, caveolae) and clathrin-coated pits (CCPs). In order for BMP2 to initiate Smad signaling it must bind to its receptors on the plasma membrane resulting in the phosphorylation of the BMP type Ia receptor (BMPRIa) followed by activation of Smad signaling. The current model suggests that the canonical BMP signaling pathway, Smad, occurs in CCPs. However, several recent studies suggested Smad signaling may occur outside of CCPs. Here, we determined; (i) The location of BMP2 binding to receptors localized in caveolae, CCPs, or outside of these domains using AFM and confocal microscopy. (ii) The location of phosphorylation of BMPRIa on the plasma membrane using membrane fractionation, and (iii) the effect of down regulation of caveolae on Smad signaling. Our data indicate that BMP2 binds with highest force to BMP receptors (BMPRs) localized in caveolae. BMPRIa is phosphorylated in caveolae and the disruption of caveolae-inhibited Smad signaling in the presence of BMP2. This suggests caveolae are necessary for the initiation of Smad signaling. We propose an extension of the current model of BMP2 signaling, in which the initiation of Smad signaling is mediated by BMPRs in caveolae.     

小窝蛋白-1在细胞衰老及衰老相关疾病中的作用

胞膜小窝(caveolae)是细胞质膜内陷所形成的囊状结构.小窝蛋白(caveolin)是胞膜小窝区别于其它脂筏结构的特征性蛋白分子,维持胞膜小窝的结构和功能,包括3个家族成员小窝蛋白-1、小窝蛋白-2和小窝蛋白-3.其中,小窝蛋白-1是参与胆固醇平衡、分子运输和跨膜信号发放事件的主要结构成分,从而调节细胞的生长、发育和增殖．小窝蛋白-1在细胞衰老中起着重要调控作用,主要通过p53-p21及p16-Rb信号通路抑制细胞增殖、酪氨酸激酶的级联反应,调控粘连信号级联、胰岛素信号及雌激素信号系统等途径调控衰老进程．衰老过程中不同器官小窝蛋白-1变化趋势不尽一致．近年研究还发现,小窝蛋白-1与神经系统退行性疾病、糖尿病、动脉粥样硬化等衰老相关疾病密切相关,通过调节多条信号通路参与这些疾病的发生发展．本文结合最新研究进展,对小窝蛋白-1在细胞衰老进程的作用及参与衰老相关疾病进行综述．     

Estradiol receptors in breast cancer cells: Associated co-factors as targets for new therapeutic approaches

Estrogen receptors α (ERα) and β (ERβ) are nuclear receptors which transduce estradiol (E2) response in many tissues including the mammary gland and breast cancers (BC). They activate or inhibit specific genes involved in cell cycle progression and cell survival through multiple enzyme activities leading to malignant transformation. Hormone therapy (antiestrogens (AEs) and aromatase inhibitors (AIs) have been widely used to block the mitogenic action of E2 in patients with ER-positive BC. ERs act in concert with numerous other proteins outside and inside the nucleus where co-activators such as histone modifying enzymes help reaching optimum gene activation. Moreover, E2-mediated gene regulation can occur through ERs located at the plasma membrane or G protein-coupled estrogen receptor (GPER), triggering protein kinase signaling cascades. Classical AEs as well as AIs are inefficient to block the cascades of events emanating from the membrane and from E2 binding to GPER, leading patients to escape anti-hormone treatments and hormone therapy resistance. Many pathways are involved in resistance, mostly resulting from over-expression of growth factor membrane receptors, in particular the HER2/ErbB2 which can be inhibited by specific antibodies or tyrosine kinases inhibitors. Together with the Hsp90 molecular chaperone machinery, a complex interplay between ERs, co-activators, co-repressors and growth factor-activated membrane pathways represents potent targets which warrant to be manipulated alone and in combination to designing novel therapies. The discovery of new potential targets arising from micro array studies gives the opportunity to activate or inhibit different new ER-modulating effectors for innovative therapeutic interventions.     

Global uterine genomics in vivo: microarray evaluation of the estrogen receptor alpha-growth factor cross-talk mechanism

Cross-talk between growth factor receptors and the estrogen receptor (ER) has been proposed as a signaling mechanism in estrogen target tissues, with ER(alpha) as a direct target of growth factor receptor-activated signals, leading to regulation of estrogen target genes and estrogen-like biological responses to growth factors. We evaluated whether global genomic changes in the mouse uterus in response to epidermal growth factor or IGF-I mimic those of estradiol (E2), reflecting the cross-talk mechanism. Overlapping responses to growth factors and E2 were expected in the wild type (WT) whereas no response was expected in mice lacking ER(alpha) (ER(alpha) knockout). Surprisingly, although most of the E2 response in the WT also occurred after growth factor treatment, some genes were induced only by E2. Second, although E2 did not induce gene changes in the ER(alpha) knockout, the growth factor response was almost indistinguishable from that of the WT. Differences in response of some genes to IGF-I or epidermal growth factor indicated selective regulation mechanisms, such as phosphatidylinositol 3-kinase or MAPK-dependent responses. The robust ER(alpha)-independent genomic response to growth factor observed here is surprising considering that the biological growth response is ER(alpha) dependent. We propose two mechanisms as alternatives to the cross-talk mechanism for uterine gene regulation. First, E2 increases uterine growth factors, which activate downstream signaling cascades, resulting in gene regulation. Second, growth factors and estrogen regulate similar genes. Our results suggest that the estrogen response in the uterus involves E2-specific ER(alpha)-mediated responses as well as responses resulting from convergence of growth factor and ER-initiated activities.     

Estrogen induces vascular wall dilation: mediation through kinase signaling to nitric oxide and estrogen receptors alpha and beta

Estrogen has been shown to affect vascular cell and arterial function in vitro and in vivo. Here we examined the ability of estradiol (E(2)) to cause rapid arterial dilation of elastic and muscular arteries in vivo and the mechanisms involved. E(2) administration caused a rapid increase in the outer wall diameter of both types of arteries in ovariectomized female mice. This resulted from estrogen receptor (ER)-mediated stimulation of nitric oxide production, demonstrated by preinjecting the mice arteries with a soluble inhibitor of nitric oxide (monomethyl l-arginine) and by showing the absence of E(2) action in eNOS-/- mice. Rapid activation of both ERK/MAP kinase and phosphatidylinositol 3-kinase activity was found in the E(2)-exposed arteries, and inhibiting either kinase prevented the vasodilatory action of E(2). Kinase activation and vasodilator responses to E(2) were absent in either ERalpha or ERbeta knock-out mice, implicating both receptor subtypes as mediating this E(2) action. These results indicate that E(2) modulation of arterial tonus through plasma membrane ER and rapid signaling could underlie many previously observed actions of estrogen reported to occur in women.