Membrane association of estrogen receptor alpha and beta influences 17beta-estradiol-mediated cancer cell proliferation

S-Palmitoylation is a widespread post-translational modification of integral and/or peripheral proteins occurring in all eukaryotic cells. The family of S-palmitoylated proteins is large and diverse and recently, estrogen receptor isoforms (ERalpha and ERbeta) belonging to the nuclear receptor superfamily have been added to the palmitoylproteoma. S-Palmitoylation allows ERalpha and ERbeta localization at the plasma membrane, where they associate with caveolin-1. Upon 17beta-estradiol (E2) stimulation, ERalpha dissociates from caveolin-1 allowing the activation of rapid signals relevant for cell proliferation. In contrast to ERalpha, E2 increases ERbeta association with caveolin-1 and activates p38 kinase and the downstream pro-apoptotic cascade (i.e., caspase-3 activation and PARP cleavage). These data highlight the physiological role of palmitoylation in modulating the ERalpha and ERbeta localization at the plasma membrane and the regulation of different E2-induced non-genomic functions relevant for controlling cell proliferation.     

ERbeta shifts from mitochondria to nucleus during estrogen-induced neoplastic transformation of human breast epithelial cells and is involved in estrogen-induced synthesis of mitochondrial respiratory chain proteins

Both estrogen receptors (ER) alpha (ERalpha) and beta (ERbeta) are localized in the nucleus, plasma membrane, and mitochondria, where they mediate the different physiological effects of estrogens. It has been observed that the relative subcellular localization of ERs is altered in several cancer cells. We have demonstrated that MCF-10F cells, the immortal and non-tumorigenic human breast epithelial cells (HBEC) that are ERalpha-negative and ERbeta-positive, are transformed in vitro by 17beta-estradiol (E(2)), generating highly invasive cells that are tumorigenic in severe combined immunodeficient mice. E(2)-transformed MCF-10F (trMCF) cells exhibit progressive loss of ductulogenesis, invasive (bsMCF) and tumorigenic (caMCF) phenotypes. Immunolocalization of ERbeta by confocal fluorescent microscopy and electron microscopy revealed that ERbeta is predominantly localized in mitochondria of MCF-10F and trMCF cells. Silencing ERbeta expression with ERbeta-specific small interference RNA (siRNA-ERbeta) markedly diminishes both nuclear and mitochondrial ERbeta in MCF-10F cells. The ERbeta shifts from its predominant localization in the mitochondria of MCF-10F and trMCF cells to the nucleus of bsMCF cells, becoming predominantly nuclear in caMCF cells. Furthermore, we demonstrated that the mitochondrial ERbeta in MCF-10F cells is involved in E(2)-induced expression of mitochondrial DNA (mtDNA)-encoded respiratory chain (MRC) proteins. This is the first report of an association of changes in the subcellular localization of ERbeta with various stages of E(2)-induced transformation of HBEC and a functional role of mitochondrial ERbeta in mediating E(2)-induced MRC protein synthesis. Our findings provide a new insight into one of the potential roles of ERbeta in human breast cancer.     

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.     

Characterization of estrogen receptor beta2 and expression of the estrogen receptor subtypes alpha, beta1, and beta2 in the protandrous black porgy (Acanthopagrus schlegeli) during the sex change process

Estrogens play an important role in many physiological processes in both female and male vertebrates, mediated by specific nuclear receptor, estrogen receptors (ERs). We have isolated a third ER (ERbeta2), which was found to contain 2004 nucleotides including an open reading frame that encodes 667 amino acids. We have also cloned ERalpha and ERbeta1 from the published information (GenBank accession nos. AY074780 and AY074779) and investigated the expression pattern of these ER subtypes in the gonads during gonad sex change of black porgy by quantitative polymerase chain reaction. Maturity stages can be divided into five stages during the sex change process from immature male to female (immature male, mature male, male of mostly testis, male of mostly ovary and mature female). The expression of ERalpha mRNA was highest in the ovary of mature female, followed by the testis of mature male and testicular portion of mostly testis. ERbeta1 expression was higher in the mature testis and ovary than in the gonads of other maturity stages. In contrast to that, ERbeta2 was highest in the ovary of mature female, and significantly lower levels of ERbeta2 expression were observed in the gonads of the other maturity stages. The present study describes the molecular characterization of ERbeta2, and documents the expression changes of three ER subtypes during sex change process of the protandrous black porgy.     

Differential expression of estrogen receptor beta (ERbeta) and its C-terminal truncated splice variant ERbetacx as prognostic predictors in human prostatic cancer.

Estrogens have been widely used for the treatment of advanced prostatic adenocarcinoma. However, their direct effect to prostatic cancer cells via estrogen receptors remains unclear. We investigated expression of ERalpha, wild-type ERbeta (wtERbeta), and a C-terminal truncated splice variant of ERbeta (ERbetacx) in 50 benign and 100 malignant human prostatic tissue samples by immunohistochemistry. While strong immunostaining of ERalpha was consistently identified in the stromal compartment, wtERbeta was expressed in epithelial cells in both the benign and malignant foci. However, wtERbeta expression was significantly lower in the cancers than in the benign epithelium and inversely correlated with Gleason tumor grade (P < 0.0001 and P = 0.0099, respectively). In contrast, ERbetacx was significantly more expressed in the high-grade cancers (83%) compared with the low-grade tumors (22%) and the benign sites (11%) (P < 0.0001, both). Cancer-specific survival of patients with lower wtERbeta expression was significantly worse than those with higher expression of wtERbeta (P = 0.0018). Conversely, higher ERbetacx expression significantly correlated with poor cancer-specific survival (P = 0.0058). These results suggest that differential expressions of wtERbeta and ERbetacx may be prognostic predictors for prostatic cancer.     

Difference in skeletal muscle function in males vs. females: role of estrogen receptor-beta

Male skeletal muscles are generally faster and have higher maximum power output than female muscles. Conversely, during repeated contractions, female muscles are generally more fatigue resistant and recover faster. We studied the role of estrogen receptor-beta (ERbeta) in this gender difference by comparing contractile function of soleus (mainly slow-twitch) and extensor digitorum longus (fast-twitch) muscles isolated from ERbeta-deficient (ERbeta(-/-)) and wild-type mice of both sexes. Results showed generally shorter contraction and relaxation times in male compared with female muscles, and ERbeta deficiency had no effect on this. Fatigue (induced by repeated tetanic contractions) and recovery of female muscles were not affected by ERbeta deficiency. However, male ERbeta(-/-) muscles were slightly more fatigue resistant and produced higher forces during the recovery period than wild-type male muscles. In fact, female muscles and male ERbeta(-/-) muscles displayed markedly better recovery than male wild-type muscles. Gene screening of male soleus muscles showed 25 genes that were differently expressed in ERbeta(-/-) and wild-type mice. Five of these genes were selected for further analysis: muscle ankyrin repeat protein-2, muscle LIM protein, calsequestrin, parvalbumin, and aquaporin-1. Expression of these genes showed a similar general pattern: increased expression in male and decreased expression in female ERbeta(-/-) muscles. In conclusion, ERbeta deficiency results in increased performance during fatigue and recovery of male muscles, whereas female muscles are not affected. Improved contractile performance of male ERbeta(-/-) mouse muscles was associated with increased expression of mRNAs encoding important muscle proteins.     

Ciliated epithelial-specific and regional-specific expression and regulation of the estrogen receptor-beta2 in the fallopian tubes of immature rats: a possible mechanism for estrogen-mediated transport process in vivo

Several ERbeta isoforms have been identified in human and rodent tissues, but it is unclear whether each isoform has distinctly different cellular targeting characteristics and physiological functions. We have investigated the intracellular localization and regulatory patterns for ERbeta isoforms in rat fallopian tubes. Western blot analysis reveals that two ERbeta isoforms corresponding to ERbeta1 and ERbeta2 are expressed in rat fallopian tubes. However, ERbeta2 is the predominant form of ERbeta in this tissue. High-resolution confocal imaging and immunohistochemical analysis provide ample evidence that ERbeta expression is limited almost exclusively to the ciliated epithelial cells, in contrast to ERalpha, which is widely distributed. Furthermore, within the ciliated epithelial cells, ERbeta is colocalized with beta-tubulin IV at stem portion of the cilia. We show that ERbeta2 protein expression is tightly regulated by E(2) or DPN in a time-dependent manner without changes in ERbeta1 expression. These estrogenic effects are inhibited by an ER antagonist, ICI 182,780. In addition, significant alteration of ERbeta immunoreactivity is detected only histologically in the ampullary region. Since the cilia are considered an essential determinant of tubal transport, we further demonstrate that E(2)- or DPN-induced ERbeta2 activation is associated with alterations in tubal protein expression crucial for the regulation of calcium-dependent ciliary beating. Given the coordinated regulation and interaction of ER and progesterone receptor in the cilia, we hypothesize that tubal ERbeta2 may facilitate the estrogen-mediated transport process by processing protein-protein interaction under physiological and/or pathological conditions. We show for the first time that a previously unrecognized localization of ERbeta isoform in rat fallopian tubes can combine with estrogen to individually control the expression of ER beta-isoforms in normal target tissues.     

Mitochondrial targeting of human DNA glycosylases for repair of oxidative DNA damage.

Oxidative damage to mitochondrial DNA has been implicated in human degenerative diseases and aging. Although removal of oxidative lesions from mitochondrial DNA occurs, the responsible DNA repair enzymes are poorly understood. By expressing the epitope-tagged proteins in COS-7 cells, we examined subcellular localizations of gene products of human DNA glycosylases: hOGG1, hMYH and hNTH1. A gene encoding for hOGG1 which excises 7,8-dihydro-8-oxoguanine (8-oxoG) from DNA generates four isoforms by alternative splicing (types 1a, 1b, 1c and 2). Three tagged isoforms (types 1b, 1c and 2) were localized in the mitochondria. Type 1a protein, which exclusively contains a putative nuclear localization signal, was sorted to the nucleus and lesser amount to the mitochondria. hMYH, a human homolog gene product of Escherichia coli mutY was mainly transported into the mitochondria. hNTH1 protein excising several pyrimidine lesions was transported into both the nucleus and mitochondria. In contrast to the three DNA glycosylases, translocation of the human major AP endonuclease (hAPE) into the mitochondria was hardly observed in COS-7 cells. These results suggest that the previously observed removal of oxidative base lesions in mitochondrial DNA is initiated by the above DNA glycosylases.     

Correspondence regarding Schwend and Gustafsson, "False positives in MALDI-TOF detection of ERbeta in mitochondria"

Recently, Schwend and Gustafsson tried to use the MALDI-TOF methods to confirm one of the results reported by Yang et al., which provided definitive evidences to demonstrate the localization of estrogen receptor beta (ERbeta) in the mitochondria of multiple cell types, using immunocytochemistry, immunoblot, and proteomic approaches. Analysis of the data with the MASCOT database algorithm provided no evidence for the presence of ERbeta in the mouse live mitochondria, in which very low ERbeta expression has been detected in their own report. On the other hand, our MALDI-TOF analysis using human heart mitochondrial protein has identified 7 and 8 sequences that could be potentially from ERbeta and ERbeta3, respectively, but not from ATP synthases. Further, none of the sequences identified by us as those of ERbeta and ERbeta3 shares m/z targeted by Schwend and Gustafsson in their measurements. Therefore, the claim by Gustafsson's laboratory about false positives in MALDI-TOF detection of ERbeta in mitochondria has no relevance to our report.     

Differential distribution of glucocorticoid and estrogen receptor isoforms: localization of GRbeta and ERalpha in nucleoli and GRalpha and ERbeta in the mitochondria of human osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cell lines

The localization of glucocorticoid and estrogen receptors alpha (GRalpha, ERalpha) and beta (GRbeta, ERbeta) in osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cells was studied by immunofluorescence labelling and confocal laser scanning microscopy, as well as by subcellular fractionation and immunoblotting of the proteins of the fractions with respective antibodies. In HepG2 and SaOS-2 cells GRbeta and ERalpha were localized mainly in the nucleus, particularly concentrated in nuclear structures, which on the basis of their staining with antibody against C23-nucleolin, were characterized as nucleoli. A faint, diffuse GRbeta and ERalpha staining was also observed in the cytoplasm. GRalpha and ERbeta were specifically enriched at the site of cell mitochondria, which were visualized by labelling with the vital dye CMX. Immunoblotting experiments corroborated the immunofluorescence labelling distribution of glucocorticoid and estrogen receptor isoforms in the cell lines studied. These findings support the concept of a direct action of steroid/thyroid hormones on mitochondrial functions by way of their cognate receptors and also suggest a direct involvement of GRbeta and ERalpha in nucleolar-related processes in HepG2 and SaOS-2 cells.     

Pivotal role of Akt activation in mitochondrial protection and cell survival by poly(ADP-ribose)polymerase-1 inhibition in oxidative stress

According to the classical view, the cytoprotective effect of inhibitors of poly(ADP-ribose)polymerase (PARP) in oxidative stress was based on the prevention of NAD+ and ATP depletion, thus the attenuation of necrosis. Our previous data on PARP inhibitors in an inflammatory model suggested that PARP-catalyzed ADP-ribosylations may affect signaling pathways, which can play a significant role in cell survival. To clarify the molecular mechanism of cytoprotection, PARP activity was inhibited pharmacologically by suppressing PARP-1 expression by a small interfering RNA (siRNA) technique or by transdominantly expressing the N-terminal DNA-binding domain of PARP-1 (PARP-DBD) in cultured cells. Cell survival, activation of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt system, and the preservation of mitochondrial membrane potential were studied in hydrogen peroxide-treated WRL-68 cells. Our data showed that suppression of the single-stranded DNA break-induced PARP-1 activation by pharmacological inhibitor, siRNA, or by the transdominant expression of PARP-DBD protected cells from oxidative stress and induced the phosphorylation and activation of Akt. Furthermore, prevention of Akt activation by inhibiting PI3-kinase counteracted the cytoprotective effect of PARP inhibition. Microscopy data showed that PARP inhibition-induced Akt activation was responsible for protection of mitochondria in oxidative stress because PI3-kinase inhibitors diminished the protective effect of PARP inhibition. Similarly, Src kinase inhibitors, which decrease Akt phosphorylation, also counteracted the protection of mitochondrial membrane potential supporting the pivotal role of Akt in cytoprotection. These data together with the finding that PARP inhibition in the absence of oxidative stress induced the phosphorylation and activation of Akt indicate that PARP inhibition-induced Akt activation is dominantly responsible for the cytoprotection in oxidative stress.     

Gpx4 protects mitochondrial ATP generation against oxidative damage

Mitochondrial ATP production can be impaired by oxidative stress. Glutathione peroxidase 4 (Gpx4) is an antioxidant defense enzyme found in mitochondria as well as other subcellular organelles that directly detoxifies membrane lipid hydroperoxides. To determine if Gpx4 protects ATP production in vivo, we compared mitochondrial ATP production between wild-type mice and Gpx4 transgenic mice using a diquat model. Diquat (50 mg/kg) significantly decreased mitochondrial ATP synthesis in livers of wild-type mice; however, no decrease in mitochondrial ATP synthesis was detected in Gpx4 transgenic mice after diquat. We observed no differences in activities of mitochondrial respiratory chain complexes between Gpx4 transgenic mice and wild-type mice. However, compared to wild-type mice, diquat-induced loss of mitochondrial membrane potential was attenuated in Gpx4 transgenic mice. Therefore, our results indicate that decreased ATP production under oxidative stress is primarily due to reduced mitochondrial membrane potential and overexpression of Gpx4 maintains mitochondrial membrane potential under oxidative stress.     

Inactivation of Saccharomyces cerevisiae OGG1 DNA repair gene leads to an increased frequency of mitochondrial mutants

The OGG1 gene encodes a highly conserved DNA glycosylase that repairs oxidized guanines in DNA. We have investigated the in vivo function of the Ogg1 protein in yeast mitochondria. We demonstrate that inactivation of ogg1 leads to at least a 2-fold increase in production of spontaneous mitochondrial mutants compared with wild-type. Using green fluorescent protein (GFP) we show that a GFP–Ogg1 fusion protein is transported to mitochondria. However, deletion of the first 11 amino acids from the N-terminus abolishes the transport of the GFP–Ogg1 fusion protein into the mitochondria. This analysis indicates that the N-terminus of Ogg1 contains the mitochondrial localization signal. We provide evidence that both yeast and human Ogg1 proteins protect the mitochondrial genome from spontaneous, as well as induced, oxidative damage. Genetic analyses revealed that the combined inactivation of OGG1 and OGG2 [encoding an isoform of the Ogg1 protein, also known as endonuclease three-like glycosylase I (Ntg1)] leads to suppression of spontaneously arising mutations in the mitochondrial genome when compared with the ogg1 single mutant or the wild-type. Together, these studies provide in vivo evidence for the repair of oxidative lesions in the mitochondrial genome by human and yeast Ogg1 proteins. Our study also identifies Ogg2 as a suppressor of oxidative mutagenesis in mitochondria.     

Selective localization of Bcl-2 to the inner mitochondrial and smooth endoplasmic reticulum membranes in mammalian cells

Bcl-2, an anti-apoptotic protein, is believed to be localized in the outer mitochondrial membrane, endoplasmic reticulum, and nuclear envelope. However, Bcl-2 has also been suggested as playing a role in the maintenance of mitochondrial membrane potential, indicating its possible association with the inner mitochondrial membrane. We therefore further examined the exact localization of Bcl-2 in mitochondria purified from wild-type and bcl-2-transfected PC12 cells and pre- and postnatal rat brains. Double immunostaining demonstrated that Bcl-2 was co-localized with subunit beta of F1F0ATPase in the inner mitochondrial membrane. Biochemical analysis of isolated mitochondria using digitonin and trypsin suggests an association of Bcl-2 with the inner mitochondrial membrane. More interestingly, the majority of Bcl-2 disappeared from the inner membrane of mitochondria when cultured under serum deprivation. These results suggest that Bcl-2 acts as an anti-apoptotic regulator by localizing mainly to the inner mitochondrial and smooth ER membranes.     

The cytoprotective effect of nitrite is based on the formation of dinitrosyl iron complexes

Nitrite protects various organs from ischemia–reperfusion injury by ameliorating mitochondrial dysfunction. Here we provide evidence that this protection is due to the inhibition of iron-mediated oxidative reactions caused by the release of iron ions upon hypoxia. We show in a model of isolated rat liver mitochondria that upon hypoxia, mitochondria reduce nitrite to nitric oxide (NO) in amounts sufficient to inactivate redox-active iron ions by formation of inactive dinitrosyl iron complexes (DNIC). The scavenging of iron ions in turn prevents the oxidative modification of the outer mitochondrial membrane and the release of cytochrome c during reoxygenation. This action of nitrite protects mitochondrial function. The formation of DNIC with nitrite-derived NO could also be confirmed in an ischemia–reperfusion model in liver tissue. Our data suggest that the formation of DNIC is a key mechanism of nitrite-mediated cytoprotection.