Estrogen receptor beta signals to inhibition of cardiac fibrosis.

Cardiac fibrosis evolves from the cardiac hypertrophic state. In this respect, estrogen and estrogen receptor beta (ERß) inhibit the effects of cardiac hypertrophic peptides that also stimulate fibrosis. Here we determine details of the anti-fibrotic functions of ERß. In acutely isolated rat cardiac fibroblasts. E2 or a specific ERß agonist (ßLGND2) blocked angiotensin II (AngII) signaling to fibrosis. This resulted from ERß activating protein kinase A and AMP kinase, inhibiting both AngII de-phosphorylation of RhoA and the resulting stimulation of Rho kinase. Inhibition of Rho kinase from ERß signaling resulted in marked decrease of TGFß expression, connective tissue growth factor production and function, matrix metalloproteinases 2 and 9 expression and activity, and the conversion of fibroblasts to myofibroblasts. Production of collagens I and III were also significantly decreased. Several important aspects were corroborated in-vivo from ßLGND2-treated mice that underwent AngII-induced cardiac hypertrophy. Thus, ERß in cardiac fibroblasts prevents key aspects of cardiac fibrosis development.

Membrane and nuclear estrogen receptor α collaborate to suppress adipogenesis but not triglyceride content.

Estrogen and estrogen receptor (ER)-α suppress visceral fat development through actions in several organs via unclear mechanisms that we sought to identify. Using mice that express only nuclear ER-α [nuclear-only ER-α (NOER) mice] or plasma membrane ER-α [membrane-only ER-α (MOER) mice], we found that 10-wk-old mice that lacked either receptor pool showed extensive abdominal visceral fat deposition and weight gain compared with wild-type (WT) mice. Differentiation of cultured bone marrow stem cells (BMSCs) into the adipocyte lineage was suppressed by 17-ß-estradiol (E2) in WT female mice but not in NOER or MOER mice. This finding correlated with E2 inhibition of prominent differentiation genes in WT BMSCs. In contrast, triglyceride content in differentiated BMSCs or 3T3-L1 cells was suppressed as a result of membrane ER-α signaling through several kinases to inhibit carbohydrate response element-binding protein-α and -ß. We concluded that extranuclear and nuclear ER-α collaborate to suppress adipocyte development, but inhibition of lipid synthesis in mature cells does not involve nuclear ER-α.

Membrane-localized estrogen receptor α is required for normal organ development and function.

Steroid receptors are found in discrete cellular locations, but it is unknown whether extranuclear pools are necessary for normal organ development. To assess this, we developed a point mutant estrogen receptor α (ERα) knockin mouse (C451A) that precludes palmitoylation and membrane trafficking of the steroid receptor in all organs. Homozygous knockin female mice (nuclear-only ERα [NOER]) show loss of rapid signaling that occurs from membrane ERα in wild-type mice. Multiple developmental abnormalities were found, including infertility, relatively hypoplastic uteri, abnormal ovaries, stunted mammary gland ductal development, and abnormal pituitary hormone regulation in NOER mice. These abnormalities were rescued in heterozygous NOER mice that were comparable to wild-type mice. mRNAs implicated in organ development were often poorly stimulated by estrogen only in homozygous NOER mice. We conclude that many organs require membrane ERα and resulting signal transduction to collaborate with nuclear ERα for normal development and function.

Estrogen regulates histone deacetylases to prevent cardiac hypertrophy.

The development and progression of cardiac hypertrophy often leads to heart failure and death, and important modulators of hypertrophy include the histone deacetylase proteins (HDACs). Estrogen inhibits cardiac hypertrophy and progression in animal models and humans. We therefore investigated the influence of 17-ß-estradiol on the production, localization, and functions of prohypertrophic (class I) and antihypertrophic (class II) HDACs in cultured neonatal rat cardiomyocytes. 17-ß-Estradiol or estrogen receptor ß agonists dipropylnitrile and ß-LGND2 comparably suppressed angiotensin II-induced HDAC2 (class I) production, HDAC-activating phosphorylation, and the resulting prohypertrophic mRNA expression. In contrast, estrogenic compounds derepressed the opposite effects of angiotensin II on the same parameters for HDAC4 and 5 (class II), resulting in retention of these deacetylases in the nucleus to inhibit hypertrophic gene expression. Key aspects were confirmed in vivo from the hearts of wild-type but not estrogen receptor ß (ERß) gene-deleted mice administered angiotensin II and estrogenic compounds. Our results identify a novel dual regulation of cardiomyocyte HDACs, shown here for the antihypertrophic sex steroid acting at ERß. This mechanism potentially supports using ERß agonists as HDAC modulators to treat cardiac disease.

Acid-degradable core-shell nanoparticles for reversed tamoxifen-resistance in breast cancer by silencing manganese superoxide dismutase (MnSOD).

Drug resistance acquired by cancer cells is a significant challenge in the clinic and requires impairing the responsible pathological pathway. Administering chemotherapeutics along with silencing resistance-basis activity using RNA interference (RNAi) is expected to restore the activity of the chemotherapeutic and generate synergistic cancer eradication. This study attempted to reverse tamoxifen (TAM)-resistance in breast cancer by silencing a mitochondrial enzyme, manganese superoxide dismutase (MnSOD), which dismutates TAM-induced reactive oxygen species (ROS) (i.e., superoxide) to less harmful hydrogen peroxide and hampers therapeutic effects. Breast cancer cells were co-treated with TAM and MnSOD siRNA-delivering nanoparticles (NPs) made of a siRNA/poly(amidoamine) (PAMAM) dendriplex core and an acid-degradable polyketal (PK) shell. The (siRNA/PAMAM)-PK NPs were designed for the PK shell to shield siRNA from nucleases, minimize detrimental aggregation in serum, and facilitate cytosolic release of siRNA from endosomal compartments. This method of forming the PK shell around the siRNA/PAMAM core via surface-initiated photo-polymerization enables ease of tuning NPs' size for readily controlled siRNA release kinetics. The resulting NPs were notably homogenous in size, resistant to aggregation in serum, and invulnerable to heparan sulfate-mediated disassembly, compared to siRNA/PAMAM dendriplexes. Gel electrophoresis and confocal microscopy confirmed efficient siRNA release from the (siRNA/PAMAM)-PK NPs upon stimuli-responsive hydrolysis of the PK shell. Sensitization of TAM-resistant MCF7-BK-TR breast cancer cells with (MnSOD siRNA/PAMAM)-PK NPs restored TAM-induced cellular apoptosis in vitro and significantly suppressed tumor growth in vivo, as confirmed by biochemical assays and histological observations. This study implies that combined gene silencing and chemotherapy is a promising strategy to overcoming a significant challenge in cancer therapy.

ERß selective agonist inhibits angiotensin-induced cardiovascular pathology in female mice.

Cardiac hypertrophy in humans can progress to cardiac failure if the underlying impetus is poorly controlled. An important direct stimulator of hypertrophy and its progression is the angiotensin II (AngII) peptide. AngII also causes hypertension that indirectly contributes to cardiac hypertrophy. Others and we have shown that estrogens acting through the estrogen receptor (ER)-ß can inhibit AngII-induced or other forms of cardiac hypertrophy in mice. However, the proliferative effects of estrogen in breast and uterus that promote the development of malignancy preclude using the steroid to prevent cardiac disease progression. We therefore tested whether an ERß selective agonist, ß-LGND2, can prevent hypertension and cardiac pathology in female mice. AngII infusion over 3 weeks significantly stimulated systolic and diastolic hypertension, cardiac hypertrophy, and cardiac fibrosis, all significantly prevented by ß-LGND2 in wild-type but not in ERß genetically deleted mice. AngII stimulated the Akt kinase to phosphorylate and inhibit the glycogen synthase kinase-3ß kinase, leading to GATA4 transcription factor activation and hypertrophic mRNA expression. As a novel mechanism, all these actions were opposed by estradiol and ß-LGND2. Our findings provide additional understanding of the antihypertrophic effects of ERß and serve as an impetus to test specific receptor agonists in humans to prevent the worsening of cardiovascular disease.

Estrogen reduces lipid content in the liver exclusively from membrane receptor signaling.

Estrogen induces signal transduction through estrogen receptor α (ERα), which localizes to both the plasma membrane and nucleus. Using wild-type mice, ERα knockout (ERKO) mice, or transgenic mice expressing only the ligand-binding domain of ERα exclusively at the plasma membrane (MOER), we compared the transcriptional profiles of liver tissue extracts after mice were injected with the ERα agonist propyl-pyrazole-triol (PPT). The expression of many lipid synthesis-related genes was comparably decreased in livers from MOER or wild-type mice but was not suppressed in ERKO mice, indicating that only membrane-localized ERα was necessary for their suppression. Cholesterol, triglyceride, and fatty acid content was decreased only in livers from wild-type and MOER mice exposed to PPT, but not in the livers from the ERKO mice, validating the membrane-driven signaling pathway on a physiological level. PPT-triggered activation of ERα at the membrane induced adenosine monophosphate-activated protein kinase to phosphorylate sterol regulatory element-binding factor 1 (Srebf1), preventing its association with and therefore its proteolytic cleavage by site-1 protease. Consequently, Srebf1 was sequestered in the cytoplasm, preventing the expression of cholesterol synthesis-associated genes. Thus, we showed that inhibition of gene expression mediated by membrane-localized ERα caused a metabolic phenotype that did not require nuclear ERα.

Estrogen modulates metabolic pathway adaptation to available glucose in breast cancer cells.

Most cancers use glucose as substrate for aerobic glycolysis in preference to oxidative phosphorylation. However, variable glucose concentrations within the in-vivo tumor microenvironment may necessitate metabolic plasticity. Furthermore, little information exists on a role for estrogen receptors in modulating possible metabolic adaptations in breast cancer cells. Here we find that MCF-7 cells switch between metabolic pathways depending on glucose availability and 17ß-estradiol (E(2)) potentiates adaptation. In high glucose conditions E(2) up-regulates glycolysis via enhanced AKT kinase activity and suppresses tricarboxylic acid cycle activity. After a decrease in extracellular glucose, mitochondrial pathways are activated in preference to glycolysis. In this setting, E(2) suppresses glycolysis and rescues cell viability by stimulating the tricarboxylic acid cycle via the up-regulation of pyruvate dehydrogenase (PDH) activity. E(2) also increases ATP in low glucose-cultured cells, and the novel phosphorylation of PDH by AMP kinase is required for these metabolic compensations. Capitalizing on metabolic vulnerability, knockdown of PDH in the low-glucose state strongly potentiates ionizing radiation-induced apoptosis and reverses the cell survival effects of E(2). We propose that lowering glucose substrate and inhibiting PDH may augment adjuvant therapies for estrogen receptor-positive breast cancer.

Src Inhibition with saracatinib reverses fulvestrant resistance in ER-positive ovarian cancer models in vitro and in vivo.

PURPOSE: More effective, less toxic treatments for recurrent ovarian cancer are needed. Although more than 60% of ovarian cancers express the estrogen receptor (ER), ER-targeted drugs have been disappointing due to drug resistance. In other estrogen-sensitive cancers, estrogen activates Src to phosphorylate p27 promoting its degradation and increasing cell-cycle progression. Because Src is activated in most ovarian cancers, we investigated whether combined Src and ER blockade by saracatinib and fulvestrant would circumvent antiestrogen resistance. EXPERIMENTAL DESIGN: ER and Src were assayed in 338 primary ovarian cancers. Dual ER and Src blockade effects on cell cycle, ER target gene expression, and survival were assayed in ERα+ ovarian cancer lines, a primary human ovarian cancer culture in vitro, and on xenograft growth. RESULTS: Most primary ovarian cancers express ER. Src activity was greater in ovarian cancer lines than normal epithelial lines. Estrogen activated Src, ER-Src binding, and ER translocation from cytoplasm to nucleus. Estrogen-mediated mitogenesis was via ERα, not ERß. While each alone had little effect, combined saracatinib and fulvestrant increased p27 and inhibited cyclin E-Cdk2 and cell-cycle progression. Saracatinib also impaired induction of ER-target genes c-Myc and FOSL1; this was greatest with dual therapy. Combined therapy induced autophagy and more effectively inhibited ovarian cancer xenograft growth than monotherapy. CONCLUSIONS: Saracatinib augments effects of fulvestrant by opposing estrogen-mediated Src activation and target gene expression, increasing cell-cycle arrest, and impairing survival, all of which would oppose antiestrogen resistance in these ER+ ovarian cancer models. These data support further preclinical and clinical evaluation of combined fulvestrant and saracatinib in ovarian cancer.

DHHC-7 and -21 are palmitoylacyltransferases for sex steroid receptors.

Classical estrogen, progesterone, and androgen receptors (ERs, PRs, and ARs) localize outside the nucleus at the plasma membrane of target cells. From the membrane, the receptors signal to activate kinase cascades that are essential for the modulation of transcription and nongenomic functions in many target cells. ER, PR, and AR trafficking to the membrane requires receptor palmitoylation by palmitoylacyltransferase (PAT) protein(s). However, the identity of the steroid receptor PAT(s) is unknown. We identified the DHHC-7 and -21 proteins as conserved PATs for the sex steroid receptors. From DHHC-7 and -21 knockdown studies, the PATs are required for endogenous ER, PR, and AR palmitoylation, membrane trafficking, and rapid signal transduction in cancer cells. Thus the DHHC-7 and -21 proteins are novel targets to selectively inhibit membrane sex steroid receptor localization and function.

Oestrogen prevents cardiomyocyte apoptosis by suppressing p38α-mediated activation of p53 and by down-regulating p53 inhibition on p38ß.

AIMS: we have previously shown that 17-ß-estradiol (E2) protects cardiomyocytes exposed to simulated ischaemia-reperfusion (I/R) by differentially regulating pro-apoptotic p38α mitogen-activated protein kinase (p38α MAPK) and pro-survival p38ß. However, little is known about how E2 modulation of these kinases alters apoptotic signalling. An attractive downstream target is p53, a well-known mediator of apoptosis and a substrate of p38α MAPK. The aim of this study was to determine whether the cytoprotective actions of oestrogen involve regulation of p53 via cardiac p38 MAPKs. METHODS AND RESULTS: cultured rat cardiomyocytes underwent hypoxia followed by reoxygenation (H/R) to simulate I/R. We found that inhibiting p53 significantly reduced apoptosis. Phosphorylation of p53 at serine 15 [p-p53(S15)] increased after H/R in a p38α MAPK- and reactive oxygen species (ROS)-dependent manner. E2 at 10 nM effectively inhibited p-p53(S15) and mitochondrial translocation of p53. Blocking p53 led to augmented p38ß activity and attenuated ROS, suggesting suppression of this antioxidant kinase by p53. The use of a specific agonist for each oestrogen receptor (ER) isoform, ERα and ERß, demonstrated that both isoforms participate in preventing cell death by inhibiting p53 in the mitochondria-centred apoptotic processes. CONCLUSION: our results demonstrate that during H/R stress, cardiomyocytes undergo p53-dependent apoptosis following phosphorylation of p53 by p38α MAPK, leading to p38ß suppression. E2 protects cardiomyocytes by inhibiting p38α-p53 signalling in apoptosis.

Estrogen receptor-beta prevents cardiac fibrosis.

Development of cardiac fibrosis portends the transition and deterioration from hypertrophy to dilation and heart failure. Here we examined how estrogen blocks this important development. Angiotensin II (AngII) and endothelin-1 induce cardiac hypertrophy and fibrosis in humans. and we find that these agents directly stimulate the transition of the cardiac fibroblast to a myofibroblast. AngII and endothelin-1 stimulated TGFß1 synthesis in the fibroblast, an inducer of fibrosis that signaled via c-jun kinase to Sma- and Mad-related protein 3 phosphorylation and nuclear translocation in myofibroblasts. As a result, mesenchymal proteins fibronectin and vimentin were produced, as were collagens I and III, the major forms found in fibrotic hearts. 17ß-Estradiol (E2) or dipropylnitrile, an estrogen receptor (ER)ß agonist, comparably blocked all these events, reversed by estrogen receptor (ER)ß small interfering RNA. E2 and dipropylnitrile signaling through cAMP and protein kinase A prevented myofibroblast formation and blocked activation of c-jun kinase and important events of fibrosis. In the hearts of ovariectomized female mice, cardiac hypertrophy and fibrosis were induced by AngII infusion and prevented by E2 administration to wild type but not ERß knockout rodents. Our results establish the cardiac fibroblast as an important target for hypertrophic/fibrosis-inducing peptides the actions of which were mitigated by E2/ERß acting in these stromal cells.

Heat shock protein 27 is required for sex steroid receptor trafficking to and functioning at the plasma membrane.

Classical sex steroid receptors (SRs) localize at the plasma membranes (PMs) of cells, initiating signal transduction through kinase cascades that contribute to steroid hormone action. Palmitoylation of the SRs is required for membrane localization and function, but the proteins that facilitate this modification and subsequent receptor trafficking are unknown. Initially using a proteomic approach, we identified that heat shock protein 27 (Hsp27) binds to a motif in estrogen receptor alpha (ERalpha) and promotes palmitoylation of the SR. Hsp27-induced acylation occurred on the ERalpha monomer and augmented caveolin-1 interactions with ERalpha, resulting in membrane localization, kinase activation, and DNA synthesis in breast cancer cells. Oligomerization of Hsp27 was required, and similar results were found for the trafficking of endogenous progesterone and androgen receptors to the PMs of breast and prostate cancer cells, respectively. Small interfering RNA (siRNA) knockdown of Hsp27 prevented sex SR trafficking to and signaling from the membrane. These results identify a conserved and novel function for Hsp27 with potential as a target for interrupting signaling from membrane sex SRs to tumor biology in hormone-responsive cancers.

Estrogen inhibits ATR signaling to cell cycle checkpoints and DNA repair.

DNA damage activates the ataxia telangiectasia-mutated and Rad3-related (ATR) kinase signal cascade. How this system is restrained is not understood. We find that in estrogen receptor (ER)-positive breast cancer cells, UV or ionizing radiation and hydroxyurea rapidly activate ATR-dependent phosphorylation of endogenous p53 and Chk1. 17-beta-estradiol (E(2)) substantially blocks ATR activity via plasma membrane-localized ERalpha. E(2)/ER reduces the enhanced association of ATR andTopBP1 proteins that follows DNA damage and strongly correlates to ATR activity. E(2) inhibits ATR activation through rapid PI3K/AKT signaling: AKT phosphorylates TopBP1 at Serine 1159, thereby preventing the enhanced association of ATR with TopBP1 after DNA damage. E(2) also inhibits Claspin:Chk1 protein association via AKT phosphorylation of Chk1, preventing Chk1 signaling to the G2/M checkpoint. ATR-phosphorylation of p53 induces p21 transcription, prevented by E(2)/ER. E(2) delays the assembly and prolongs the resolution of gammaH2AX and Rad51 nuclear foci and delays DNA repair. E(2)/ER also increases the chromosomal damage seen from cell exposure to IR. Therefore, the restraint of ATR cascade activation may be a novel estrogen action relevant to breast cancer.

Developmental phenotype of a membrane only estrogen receptor alpha (MOER) mouse.

Estrogen receptors (ERs) alpha and beta exist as nuclear, cytoplasmic, and membrane cellular pools in a wide variety of organs. The relative contributions of each ERalpha pool to in vivo phenotypes resulting from estrogen signaling have not been determined. To address this, we generated a transgenic mouse expressing only a functional E domain of ERalpha at the plasma membrane (MOER). Cells isolated from many organs showed membrane only localized E domain of ERalpha and no other receptor pools. Liver cells from MOER and wild type mice responded to 17-beta-estradiol (E2) with comparable activation of ERK and phosphatidylinositol 3-kinase, not seen in cells from ERalphaKO mice. Mating the MOER female mice with proven male wild type breeders produced no pregnancies because the uterus and vagina of the MOER female mice were extremely atrophic. Ovaries of MOER and homozygous Strasbourg ERalphaKO mice showed multiple hemorrhagic cysts and no corpus luteum, and the mammary gland development in both MOER and ERalphaKO mice was rudimentary. Despite elevated serum E2 levels, serum LH was not suppressed, and prolactin levels were low in MOER mice. MOER and Strasbourg female mice showed plentiful abdominal visceral and other depots of fat and increased body weight compared to wild type mice despite comparable food consumption. These results provide strong evidence that the normal development and adult functions of important organs in female mice requires nuclear ERalpha and is not rescued by membrane ERalpha domain expression alone.

Estrogen inhibits cardiac hypertrophy: role of estrogen receptor-beta to inhibit calcineurin.

Estrogen has been reported to prevent development of cardiac hypertrophy in female rodent models and in humans. However, the mechanisms of sex steroid action are incompletely understood. We determined the cellular effects by which 17beta-estradiol (E2) inhibits angiotensin II (AngII)-induced cardiac hypertrophy in vivo. Two weeks of angiotensin infusion in female mice resulted in marked hypertrophy of the left ventricle, exacerbated by the loss of ovarian steroid hormones from oophorectomy. Hypertrophy was 51% reversed by the administration of E2 (insertion of 0.1 mg/21-d-release tablets). The effects of E2 were mainly mediated by the estrogen receptor (ER) beta-isoform, because E2 had little effect in ERbeta-null mice but comparably inhibited AngII-induced hypertrophy in wild-type or ERalpha-null mice. AngII induced a switch of myosin heavy chain production from alpha to beta, but this was inhibited by E2 via ERbeta. AngII-induced ERK activation was also inhibited by E2 through the beta-receptor. E2 stimulated brain natriuretic peptide protein expression and substantially prevented ventricular interstitial cardiac fibrosis (collagen deposition) as induced by AngII. Importantly, E2 inhibited calcineurin activity that was stimulated by AngII, related to E2 stimulating the modulatory calcineurin-interacting protein (MCIP) 1 gene and protein expression. E2 acting mainly through ERbeta mitigates the important signaling by AngII that produces cardiac hypertrophy and fibrosis in female mice.

A conserved mechanism for steroid receptor translocation to the plasma membrane.

Multiple steroid receptors (SR) have been proposed to localize to the plasma membrane. Some structural elements for membrane translocation of the estrogen receptor alpha (ER alpha) have been described, but the mechanisms relevant to other steroid receptors are entirely unknown. Here, we identify a highly conserved 9 amino acid motif in the ligand binding domains (E domains) of human/mouse ER alpha and ER beta, progesterone receptors A and B, and the androgen receptor. Mutation of the phenylalanine or tyrosine at position-2, cysteine at position 0, and hydrophobic isoleucine/leucine or leucine/leucine combinations at positions +5/6, relative to cysteine, significantly reduced membrane localization, MAP and PI 3-kinase activation, thymidine incorporation into DNA, and cell viability, stimulated by specific SR ligands. The localization sequence mediated palmitoylation of each SR, which facilitated caveolin-1 association, subsequent membrane localization, and steroid signaling. Palmitoylation within the E domain is therefore a crucial modification for membrane translocation and function of classical sex steroid receptors.

Nature of functional estrogen receptors at the plasma membrane.

Although rapid signaling by estrogen at the plasma membrane is established, it is controversial as to the nature of the receptor protein. Estrogen may bind membrane proteins comparable to classical nuclear estrogen receptors (ERs), but some studies identify nonclassical receptors, such as G protein-coupled receptor (GPR)30. We took several approaches to define membrane-localized estrogen-binding proteins. In endothelial cells (ECs) from ERalpha/ERbeta combined-deleted mice, estradiol (E2) failed to specifically bind, and did not activate cAMP, ERK, or phosphatidyinositol 3-kinase or stimulate DNA synthesis. This is in contrast to wild-type ECs, indicating the lack of any functional estrogen-binding proteins in ERalpha/ERbeta combined-deleted ECs. To directly determine the identity of membrane and nuclear-localized ER, we isolated subcellular receptor pools from MCF7 cells. Putative ER proteins were trypsin digested and subjected to tandem array mass spectrometry. The output analysis identified membrane and nuclear E2-binding proteins as classical human ERalpha. We also determined whether GPR30 plays any role in E2 rapid actions. MCF7 (ER and GPR30 positive) and SKBR-3 (ER negative, GPR30 positive) cells were incubated with E2. Only MCF7 responded with significantly increased signaling. In MCF7, the response to E2 was not different in cells transfected with small interfering RNA to green fluorescent protein or GPR30. In contrast, interfering RNA to ERalpha or ER inhibition prevented rapid signaling and resulting biology in MCF7. In breast cancer and ECs, nuclear and membrane ERs are the same proteins. Furthermore, classical ERs mediate rapid signals induced by E2 in these cells.

Functional estrogen receptors in the mitochondria of breast cancer cells.

Steroid hormones have been reported to indirectly impact mitochondrial functions, attributed to nuclear receptor-induced production of proteins that localize in this cytoplasmic organelle. Here we show high-affinity estrogen receptors in the mitochondria of MCF-7 breast cancer cells and endothelial cells, compatible with classical estrogen receptors ERalpha and ERbeta. We report that in MCF-7, estrogen inhibits UV radiation-induced cytochrome C release, the decrease of the mitochondrial membrane potential, and apoptotic cell death. UV stimulated the formation of mitochondrial reactive oxygen species (mROS), and mROS were essential to inducing mitochondrial events of cell death. mROS mediated the UV activation of c-jun N-terminal kinase (JNK), and protein kinase C (PKC) delta, underlying the subsequent translocation of Bax to the mitochondria where oligomerization was promoted. E2 (estradiol) inhibited all these events, directly acting in mitochondria to inhibit mROS by rapidly up-regulating manganese superoxide dismutase activity. We implicate novel functions of ER in the mitochondria of breast cancer that lead to the survival of the tumor cells.

Estrogen prevents cardiomyocyte apoptosis through inhibition of reactive oxygen species and differential regulation of p38 kinase isoforms.

From human and animal studies, estrogen is known to protect the myocardium from an ischemic insult. However, there is limited knowledge regarding mechanisms by which estrogen directly protects cardiomyocytes. In this report, we employed an in vitro model, in which cultured rat cardiomyocytes underwent prolonged hypoxia followed by reoxygenation (H/R), to study the cardioprotective mechanism of estrogen. 17-beta-estradiol (E2) acting via estrogen receptors inhibited H/R-induced apoptosis of cardiomyocytes. Mitochondrial reactive oxygen species (ROS) generated from H/R activated p38alpha MAPK, and inhibition of p38alpha with SB203580 significantly prevented H/R-induced cell death. E2 suppressed ROS formation and p38alpha activation by H/R and concomitantly augmented the activity of p38beta. Unlike p38alpha, p38beta was little affected by H/R. Dominant negative p38beta protein expression decreased E2-mediated cardiomyocyte survival and ROS suppression during H/R stress. The prosurvival signaling molecule, phosphoinositol-3 kinase (PI3K), has previously been linked to cell survival following ischemia-reperfusion injury. Here, E2-activated PI3K was found to inhibit ROS generated from H/R injury, leading to inhibition of downstream p38alpha. We further linked these signaling pathways in that p38beta was activated by E2 stimulation of PI3K. Thus, E2 differentially modulated two major isoforms of p38, leading to cardiomyocyte survival. This was achieved by signaling through PI3K, integrating cell survival mediators.