Biphasic estradiol-induced AKT phosphorylation is modulated by PTEN via MAP kinase in HepG2 cells

We reported previously in HepG2 cells that estradiol induces cell cycle progression throughout the G1-S transition by the parallel stimulation of both PKC-alpha and ERK signaling molecules. The analysis of the cyclin D1 gene expression showed that only the MAP kinase pathway was involved. Here, the presence of rapid/nongenomic, estradiol-regulated, PI3K/AKT signal transduction pathway, its modulation by the levels of the tumor suppressor PTEN, its cross-talk with the ERK pathway, and its involvement in DNA synthesis and cyclin D1 gene promoter activity have all been studied in HepG2 cells. 17beta-Estradiol induced the rapid and biphasic phosphorylation of AKT. These phosphorylations were independent of each other, being the first wave of activation independent of the estrogen receptor (ER), whereas the second was dependent on ER. Both activations were dependent on PI3K activity; furthermore, the ERK pathway modulated AKT phosphorylation by acting on the PTEN levels. The results showed that the PI3K pathway, as well as ER, were strongly involved in both G1-S progression and cyclin D1 promoter activity by acting on its proximal region (-254 base pairs). These data indicate that in HepG2 cells, different rapid/nongenomic estradiol-induced signal transduction pathways modulate the multiple steps of G1-S phase transition.     

17 beta-estradiol activates PI3K/Akt signaling pathway by estrogen receptor (ER)-dependent and ER-independent mechanisms in endometrial cancer cells

Cellular response to estrogen is mediated both by estrogen receptor (ER) binding to estrogen response element (ERE) and by non-nuclear actions like activation of signal transducing pathways. The main aims are to study if PI3K/Akt signaling pathway can be activated by 17beta-estradiol (E2) via non-nuclear action and to investigate the relationship of the action of E2 and ER in endometrial cancer cells expressing with different status of ER. The levels of phosphorylated Akt (Ser473) (P-Akt) and total Akt were examined by western blot and Akt kinase activity was measured in cells after stimulation with 1 microM E2 at different time points. Inhibitory role of LY294002 on activation of Akt induced by E2 and its estrogen antagonist, ICI182780 were also tested. P-Akt/Akt was used as a measure of activation of Akt. We found that maximum P-Akt/Akt and Akt kinase activity took place at 30 min in Ishikawa cells and 15 min in HEC-1A cells and the activation persisted for at least 2 h after stimulation with 1 microM E2. The activation of Akt elicited gradually with increasing doses of E2. PI3K inhibitor, LY294002, stopped the activating Akt in a dose-dependent manner and 50 microM LY294002 completely blocked the activation of Akt induced by E2. ICI182780 could block the activation of PI3K/Akt in ER-positive Ishikawa cells but not in HEC-1A cells with poor-expressed ER. This study demonstrated that E2 is able to promptly activate PI3K/Akt signal pathway in Ishikawa cells in an ER-dependent manner and ER-independent in HEC-1A cells. Blockage of PI3K/Akt cascade may become a potential and effective way to control endometrial carcinoma, especially in ER-negative cancers, which show no response to endocrinal therapy.     

Phosphorylation of MNAR promotes estrogen activation of phosphatidylinositol 3-kinase

Estrogen actions are mediated by a complex interface of direct control of gene expression (the so-called "genomic action") and by regulation of cell signaling/phosphorylation cascades, referred to as the "nongenomic," or extranuclear, action. We have previously described the identification of MNAR (modulator of nongenomic action of estrogen receptor) as a novel scaffold protein that regulates estrogen receptor alpha (ERalpha) activation of cSrc. In this study, we have investigated the role of MNAR in 17beta-estradiol (E2)-induced activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Consistent with our previous results, a direct correlation was established between MNAR expression levels and E2-induced activation of PI3 and Akt kinases. Endogenous MNAR, ERalpha, cSrc, and p85, the regulatory subunit of PI3 kinase, interacted in MCF7 cells treated with E2. The interaction between p85 and MNAR required activation of cSrc and MNAR phosphorylation on Tyr 920. Consequently, the mutation of this tyrosine to alanine (Y920A) abrogated the interaction between MNAR and p85 and the E2-induced activation of the PI3K/Akt pathway, which was required for the E2-induced protection of MCF7 cells from apoptosis. Nonetheless, the Y920A mutant potentiated the E2-induced activation of the Src/MAPK pathway and MCF7 cell proliferation, as observed with the wild-type MNAR. These results provide new and important insights into the molecular mechanisms of E2-induced regulation of cell proliferation and apoptosis.     

Estradiol rapidly activates Akt via the ErbB2 signaling pathway

Previously, we have demonstrated that the two mitogenic growth factors epidermal growth factor and IGF-I can activate Akt and estrogen receptor-alpha (ERalpha) in the hormone-dependent breast cancer cell line, MCF-7. In this report we now show that estradiol can also rapidly activate phosphatidylinositol 3-kinase (PI 3-K)/Akt and that this effect is mediated by the ErbB2 signaling pathway. Treatment of cells with estradiol resulted in phosphorylation of Akt and a 9-fold increase in Akt activity in 10 min. Akt activation was blocked by wortmannin and LY 294,002, two inhibitors of PI 3-K; by genistein, a protein tyrosine kinase inhibitor and an ER agonist; by AG825, a selective ErbB2 inhibitor; and by the antiestrogens ICI 182,780 and 4-hydroxy-tamoxifen; but not by rapamycin, an inhibitor of the ribosomal protein kinase p70S6K; nor by AG30, a selective epidermal growth factor receptor inhibitor. Akt activation by estradiol was abrogated by an arginine-to-cysteine mutation in the pleckstrin homology domain of Akt (R25C). Growth factors also activated Akt in the ER-negative variant of MCF-7, MCF-7/ADR, but estradiol did not induce Akt activity in these cells. Transient transfection of ERalpha into these cells restored Akt activation by estradiol, suggesting that estradiol activation of Akt requires the ERalpha. Estradiol did not activate Akt in MCF-7 cells stably transfected with an anti-ErbB2-targeted ribozyme, further confirming a role for ErbB2. In vitro kinase assays using immunoprecipitation and anti-Akt1, -Akt2, and -Akt3-specific antibodies demonstrated that Akt1 is activated by estradiol in MCF-7 cells whereas Akt3 is the activated isoform in ER-negative MDA-MB231 cells, implying that selective activation of Akt subtypes plays a role in the actions of estradiol. Taken together, our data suggest that estradiol, bound to membrane ERalpha, interacts with and activates an ErbB dimer containing ErbB2, inducing activation of PI 3-K/Akt.     

Epigenomic reprogramming of the developing reproductive tract and disease susceptibility in adulthood

During development, epigenetic programs are "installed" on the genome that direct differentiation and normal tissue and organ function in adulthood. Consequently, development is also a period of susceptibility to reprogramming of the epigenome. Developmental reprogramming occurs when an adverse stimulus or insult interrupts the proper "install" of epigenetic programs during development, reprogramming normal physiologic responses in such a way as to promote disease later in life. Some of the best examples of developmental reprogramming involve the reproductive tract, where early life exposures to environmental estrogens can increase susceptibility to benign and malignant tumors in adulthood including leiomyoma (fibroids), endometrial, and prostate cancer. Although specific mechanism(s) by which environmental estrogens reprogram the developing epigenome were unknown, both DNA and histone methylation were considered likely targets for epigenetic reprogramming. We have now identified a mechanism by which developmental exposures to environmental estrogens reprogram the epigenome by inducing inappropriate activation of nongenomic estrogen receptor (ER) signaling. Activation of nongenomic ER signaling via the phosphotidylinositol-3-kinase (PI3K) pathway activates the kinase AKT/PKB in the developing reproductive tract, which phosphorylates the histone lysine methyltransferase (HKMT) EZH2, the key "installer" of epigenetic histone H3 lysine 27 trimethylation (H3K27me3). AKT phosphorylation inactivates EZH2, decreasing levels of H3K27 methylation, a repressive mark that inhibits gene expression, in the developing uterus. As a result of this developmental reprogramming, many estrogen-responsive genes become hypersensitive to estrogen in adulthood, exhibiting elevated expression throughout the estrus cycle, and resulting in a "hyper-estrogenized" phenotype in the adult uterus that promotes development of hormone-dependent tumors.     

Differential regulation of signal transduction pathways in wild type and mutated p53 breast cancer epithelial cells by copper and zinc

Previous studies have suggested that cells may differ in their response to metal stress. This study was undertaken to investigate the role of PI3K/Akt signaling pathway in metal resistance in human breast cancer epithelial cells with different p53 and estrogen receptor status. Exposure to copper and zinc increased Akt phosphorylation with its nuclear localization only in MDA-MB-231 cells with no estrogen receptor and mutated p53. Cyclin D1 expression and cell-cycle progression followed the metal-induced Akt phosphorylation. Treatment with LY294002 abrogated these effects, suggesting the essential role of PI3-kinase. In contrast, in MCF-7 cells with wild type p53 and estrogen receptor, there was no change in Akt activation, while suppression of p53 activity by pifithrin-alpha increased phosphorylation of Akt after the treatment with copper. In MCF-7 cells, the metal treatment increased the phosphorylation of p53 at serine 15, up-regulated p21 expression, and resulted in cell-cycle arrest in G1 phase with apoptosis. These results demonstrate that copper-induced apoptosis in MCF-7 cells is p53 dependent, whereas the metal resistance in MDA-MB-231 cells may be due to activation of Akt in the absence of a functional p53.     

17beta-estradiol signaling in skeletal muscle cells and its relationship to apoptosis

17beta-estradiol exerts an antiapoptotic action in skeletal muscle cells through extranuclear ERalpha and beta. This protective action, mainly involves a non-genomic mechanism of ERK1/2 and PI3K/Akt activation and BAD phosphorylation. ERbeta plays a major role in the inhibition of apoptosis by 17beta-estradiol at the level of mitochondria, whereas ERalpha and ERbeta mediate the activation of Akt to the same extent, suggesting differential involvement of ER isoforms depending on the step of the apoptotic/survival pathway involved. The myopathies associated to estrogen deficit states may be related to the mechanisms by which estrogen regulates apoptosis.     

PI3K/Akt-sensitive MEK-independent compensatory circuit of ERK activation in ER-positive PI3K-mutant T47D breast cancer cells

We explored the crosstalk between cell survival (phosphatidylinositol 3-kinase (PI3K)/Akt) and mitogenic (Ras/Raf/MEK/extracellular signal-regulated kinase (ERK)) signaling pathways activated by an epidermal growth factor (EGF) and analyzed their sensitivity to small molecule inhibitors in the PI3K-mutant estrogen receptor (ER)-positive MCF7 and T47D breast cancer cells. In contrast to MCF7 cells, ERK phosphorylation in T47D cells displayed resistance to MEK inhibition by several structurally different compounds, such as U0126, PD 098059 and PD 198306, MEK suppression by small interfering RNA (siRNA) and was also less sensitive to PI3K inhibition by wortmannin. Similar effect was observed in PI3K-wild type ER-positive BT-474 cells, albeit to a much lesser extent.MEK-independent ERK activation was induced only by ErbB receptor ligands and was resistant to inhibition of several kinases and phosphatases that are known to participate in the regulation of Ras/mitogen-activated protein kinase (MAPK) cascade. Although single agents against PDK1 or Akt did not affect EGF-induced ERK phosphorylation, a combination of PI3K/Akt and MEK inhibitors synergistically suppressed ERK activation and cellular growth. siRNA-mediated silencing of class I PI3K or Akt1/2 genes also significantly decreased U0126-resistant ERK phosphorylation.Our data suggest that in T47D cells ErbB family ligands induce a dynamic, PI3K/Akt-sensitive and MEK-independent compensatory ERK activation circuit that is absent in MCF7 cells. We discuss candidate proteins that can be involved in this activation circuitry and suggest that PDZ-Binding Kinase/T-LAK Cell-Originated Protein Kinase (PBK/TOPK) may play a role in mediating MEK-independent ERK activation.     

PS1 activates PI3K thus inhibiting GSK-3 activity and tau overphosphorylation: effects of FAD mutations

Phosphatidylinositol 3-kinase (PI3K) promotes cell survival and communication by activating its downstream effector Akt kinase. Here we show that PS1, a protein involved in familial Alzheimer's disease (FAD), promotes cell survival by activating the PI3K/Akt cell survival signaling. This function of PS1 is unaffected by gamma-secretase inhibitors. Pharmacological and genetic evidence indicates that PS1 acts upstream of Akt, at or before PI3K kinase. PS1 forms complexes with the p85 subunit of PI3K and promotes cadherin/PI3K association. Furthermore, conditions that inhibit this association prevent the PS1-induced PI3K/Akt activation, indicating that PS1 stimulates PI3K/Akt signaling by promoting cadherin/PI3K association. By activating PI3K/Akt signaling, PS1 promotes phosphorylation/inactivation of glycogen synthase kinase-3 (GSK-3), suppresses GSK-3-dependent phosphorylation of tau at residues overphosphorylated in AD and prevents apoptosis of confluent cells. PS1 FAD mutations inhibit the PS1-dependent PI3K/Akt activation, thus promoting GSK-3 activity and tau overphosphorylation at AD-related residues. Our data raise the possibility that PS1 may prevent development of AD pathology by activating the PI3K/Akt signaling pathway. In contrast, FAD mutations may promote AD pathology by inhibiting this pathway.     

The estrogen receptor β-PI3K/Akt pathway mediates the cytoprotective effects of tocotrienol in a cellular Parkinson's disease model

Tocotrienols (T3s) are members of the vitamin E family, have antioxidant properties, and are promising candidates for neuroprotection in the pathogenesis of neurodegenerative disorders such as Parkinson's disease (PD). However, whether their antioxidant capacities are required for their cytoprotective activity remains unclear. In this regard, the antioxidant-independent cytoprotective activity of T3s has received considerable attention. Here, we investigated the signaling pathways that are induced during T3-dependent cytoprotection of human neuroblastoma SH-SY5Y cells, as these cells are used to model certain elements of PD. T3s were cytoprotective against 1-methyl-4-phenylpyridinium ion (MPP⁺) and other PD-related toxicities. γT3 and δT3 treatments led to marked activation of the PI3K/Akt signaling pathway. Furthermore, we identified estrogen receptor (ER) β as an upstream mediator of PI3K/Akt signaling following γT3/δT3 stimulation. Highly purified γT3/δT3 bound to ERβ directly in vitro, and knockdown of ERβ in SH-SY5Y cells abrogated both γT3/δT3-dependent cytoprotection and Akt phosphorylation. Since membrane-bound ERβ was important for the signal-related cytoprotective effects of γT3/δT3, we investigated receptor-mediated caveola formation as a candidate for the early events of signal transduction. Knockdown of caveolin-1 and/or caveolin-2 prevented the cytoprotective effects of γT3/δT3, but did not affect Akt phosphorylation. This finding suggests that T3s and, in particular, γT3/δT3, exhibit not only antioxidant effects but also a receptor signal-mediated protective action following ERβ/PI3K/Akt signaling. Furthermore, receptor-mediated caveola formation is an important event during the early steps following T3 treatment.     

Adaptive hypersensitivity following long-term estrogen deprivation: involvement of multiple signal pathways

Long-term estrogen deprivation causes hypersensitivity of MCF-7 cells to the mitogenic effect of estradiol (E₂) which is associated with activation of mitogen-activated protein kinase (MAPK). However, several lines of evidence indicate that MAPK activation is not the exclusive mechanism for E₂ hypersensitivity and multiple signal pathways might be involved. The current study explores the possible role of the PI3 kinase (PI3K) pathway in development of E₂ hypersensitivity. Basal PI3K activity in long-term estrogen deprived MCF-7 cells (LTED) was elevated as evidenced by increased phosphorylation of three downstream effectors, Akt, p70 S6 kinase, and eukaryotic initiation factor-4E binding protein (4E-BP1), which was blocked by the specific inhibitor of PI3K, LY294002. Dual blockade of both MAPK and PI3K completely reversed E₂ hypersensitivity of LTED cells. Enhancement in aromatase activity is another phenomenon accompanied with E₂ hypersensitivity. In aromatase over-expressing MCF-7 cells, aromatase activity was reduced by inhibitors of MAPK and PI3K suggesting the involvement of protein phosphorylation in the regulation of aromatase activity. Our data suggest that in addition to the MAP kinase pathway, activation of the PI3 kinase pathway is involved in E₂ hypersensitivity, which develops during adaptation of MCF-7 cells to the low estrogen environment.     

Bidirectional interaction between unfolded-protein-response key protein HSPA5 and estrogen signaling in human endometrium

The human endometrium is a dynamic tissue that undergoes cyclic changes under the influence of steroid hormones as well as numerous local paracrine and autocrine factors. Heat shock 70 kDa protein (HSPA5; also known as GRP78/BiP), a molecular chaperone within the endoplasmic reticulum, plays crucial roles in normal cellular processes as well as in stress conditions, in which it is a central regulator for the unfolded protein response (UPR). We hypothesized that HSPA5 expression level is variable throughout the menstrual cycle in human endometrium and that estrogen signaling cross-talks with UPR signaling by interacting with HSPA5. HSPA5 expression throughout the menstrual cycle was evaluated in vivo in normal human endometrium. Using in vitro techniques, we then assessed the bidirectional regulation of HSPA5 and estrogen signaling in human endometrial glandular (Ishikawa) and stromal cells (ESC). HSPA5 immunoreactivity in endometrial glandular and stromal cells was cycle-dependent, and was significantly higher in phases of the menstrual cycle when estradiol (E(2)) levels are known to be the lowest compared with the rest of the cycle (P < 0.001). E(2) did not affect HSPA5 expression after 8-24 h incubation in Ishikawa cells and ESC in vitro. However, tunicamycin-induced HSPA5 expression was significantly lowered in these cells when pretreated with E(2) (P < 0.01 and P < 0.05, respectively). On the other hand, tunicamycin decreased E(2) up-regulated alkaline phosphatase activity (P < 0.001). In conclusion, there is cycle-dependent HSPA5 expression with a possible inverse correlation between HSPA5 expression and E(2) levels in human endometrium. We suggest that estrogen signaling cross-talks with the UPR cascade by interacting with HSPA5, as supported by our in vitro findings.     

Gastrin-releasing peptide activates Akt through the epidermal growth factor receptor pathway and abrogates the effect of gefitinib

Gastrin-releasing peptide (GRP) is a mitogen for lung epithelial cells and initiates signaling through a G-protein-coupled receptor, gastrin-releasing peptide receptor (GRPR). Because GRPR transactivates the epidermal growth factor receptor (EGFR), we investigated induction by GRP of Akt, an EGFR-activated signaling pathway, and examined effects of GRP on viability of non-small cell lung carcinoma (NSCLC) cells exposed to the EGFR tyrosine kinase inhibitor gefitinib. GRP induced Akt activation primarily through c-Src-mediated transactivation of EGFR. Transfection of dominant-negative c-Src abolished GRP-induced EGFR and Akt activation. GRP induced release of amphiregulin, and pre-incubation with human amphiregulin neutralizing antibody eliminated GRP-induced Akt phosphorylation. Pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 completely blocked GRP-initiated Akt phosphorylation. These results suggest that GRP stimulates Akt activation primarily via c-Src activation, followed by extracellular release of the EGFR ligand amphiregulin, leading to the activation of EGFR and PI3K. Pretreatment of NSCLC cells with GRP resulted in an increase in the IC(50) of gefitinib of up to 9-fold; this protective effect was mimicked by the pretreatment of cells with amphiregulin and reversed by Akt or PI3K inhibition. GRP appears to rescue NSCLC cells exposed to gefitinib through release of amphiregulin and activation of the Akt pathway, suggesting GRPR and/or EGFR autocrine pathways in NSCLC cells may modulate therapeutic response to EGFR inhibitors.