Filippo Acconcia

Survival versus apoptotic 17β‐estradiol effect: Role of ERα and ERβ activated non‐genomic signaling

The capability of 17β‐estradiol (E2) to induce the non‐genomic activities of its receptors (ERα and ERβ) and to evoke different signaling pathways committed to the regulation of cell proliferation has been analyzed in different cell cancer lines containing transfected (HeLa) or endogenous (HepG2, DLD1) ERα or ERβ. In these cell lines, E2 induced different effects on cell growth/apoptosis in dependence of ER isoforms present. The E2–ERα complex rapidly activated multiple signal transduction pathways (i.e., ERK/MAPK, PI3K/AKT) committed to both cell cycle progression and apoptotic cascade prevention. On the other hand, the E2–ERβ complex induced the rapid and persistent phosphorylation of p38/MAPK which, in turn, was involved in caspase‐3 activation and cleavage of poly(ADP‐ribose)polymerase, driving cells into the apoptotic cycle. In addition, the E2–ERβ complex did not activate any of the E2–ERα‐activated signal molecules involved in cell growth. Taken together, these results demonstrate the ability of ERβ isoform to activate specific signal transduction pathways starting from plasma membrane that may justify the effect of E2 in inducing cell proliferation or apoptosis in cancer cells. In particular this hormone promotes cell survival through ERα non‐genomic signaling and cell death through ERβ non‐genomic signaling.

Survival versus apoptotic 17beta-estradiol effect: role of ER alpha and ER beta activated non-genomic signaling.

The capability of 17β‐estradiol (E2) to induce the non‐genomic activities of its receptors (ERα and ERβ) and to evoke different signaling pathways committed to the regulation of cell proliferation has been analyzed in different cell cancer lines containing transfected (HeLa) or endogenous (HepG2, DLD1) ERα or ERβ. In these cell lines, E2 induced different effects on cell growth/apoptosis in dependence of ER isoforms present. The E2–ERα complex rapidly activated multiple signal transduction pathways (i.e., ERK/MAPK, PI3K/AKT) committed to both cell cycle progression and apoptotic cascade prevention. On the other hand, the E2–ERβ complex induced the rapid and persistent phosphorylation of p38/MAPK which, in turn, was involved in caspase‐3 activation and cleavage of poly(ADP‐ribose)polymerase, driving cells into the apoptotic cycle. In addition, the E2–ERβ complex did not activate any of the E2–ERα‐activated signal molecules involved in cell growth. Taken together, these results demonstrate the ability of ERβ isoform to activate specific signal transduction pathways starting from plasma membrane that may justify the effect of E2 in inducing cell proliferation or apoptosis in cancer cells. In particular this hormone promotes cell survival through ERα non‐genomic signaling and cell death through ERβ non‐genomic signaling.

Cortactin Promotes Migration and Platelet-derived Growth Factor-induced Actin Reorganization by Signaling to Rho-GTPases

Dynamic actin rearrangements are initiated and maintained by actin filament nucleators, including the Arp2/3-complex. This protein assembly is activated in vitro by distinct nucleation-promoting factors such as Wiskott-Aldrich syndrome protein/Scar family proteins or cortactin, but the relative in vivo functions of each of them remain controversial. Here, we report the conditional genetic disruption of murine cortactin, implicated previously in dynamic actin reorganizations driving lamellipodium protrusion and endocytosis. Unexpectedly, cortactin-deficient cells showed little changes in overall cell morphology and growth. Ultrastructural analyses and live-cell imaging studies revealed unimpaired lamellipodial architecture, Rac-induced protrusion, and actin network turnover, although actin assembly rates in the lamellipodium were modestly increased. In contrast, platelet-derived growth factor-induced actin reorganization and Rac activation were impaired in cortactin null cells. In addition, cortactin deficiency caused reduction of Cdc42 activity and defects in random and directed cell migration. Reduced migration of cortactin null cells could be restored, at least in part, by active Rac and Cdc42 variants. Finally, cortactin removal did not affect the efficiency of receptor-mediated endocytosis. Together, we conclude that cortactin is fully dispensable for Arp2/3-complex activation during lamellipodia protrusion or clathrin pit endocytosis. Furthermore, we propose that cortactin promotes cell migration indirectly, through contributing to activation of selected Rho-GTPases.

Mechanisms of Naringenin‐induced Apoptotic Cascade in Cancer Cells: Involvement of Estrogen Receptor a and ß Signalling

The flavanone naringenin (Nar), especially abundant in the Mediterranean diet, is reported to have anti‐proliferative effects in many cancer cell lines. Antioxidant activities, kinase and glucose uptake inhibition have been proposed as molecular mechanisms for these effects. In addition, an anti‐estrogenic activity has been observed but, at the present, it is poorly understood whether this latter activity could play a role in the Nar anti‐tumoral effects. Here, we tested the ability of Nar to activate a specific, rapid signal transduction pathway committed to the generation of an apoptotic cascade in the presence of one of the two estrogen receptor (ER) isoforms (i.e., ERα or ERβ). Cancer cells containing transfected (human cervix epitheloid carcinoma HeLa cells) or endogenous ERα (human hepatoma HepG2 cells) or ERβ (human colon adenocarcinoma DLD‐1 cells) were used. Our results show that Nar exerts an anti‐proliferative effect only in the presence of ERα or ERβ. Moreover, Nar stimulation induces the activation of p38/MAPK leading to the pro‐apoptotic caspase‐3 activation and to the poly(ADP‐ribose) polymerase cleavage in all cancer cell lines considered. Notably, Nar shows an anti‐estrogenic effect only in ERα containing cells; whereas in ERβ containing cells, Nar mimics the 17β‐estradiol effects. These findings indicate new steps in the mechanism underlying ER‐dependent anti‐proliferative effects of Nar suggesting new potential chemopreventive actions of flavonoids on cancer growth. IUBMB Life, 56: 491‐499, 2004

An inherent role of microtubule network in the action of nuclear receptor

Estrogen receptor α (ERα) functions as both a transcription factor and a mediator of rapid estrogen signaling. Recent studies have shown a role for ERα-interacting membranous and cytosolic proteins in ERα action, but our understanding of the role of the microtubule network in the modulation of ERα signaling remains unclear. Here we found that endogenous ERα associates with microtubules through the microtubule-binding protein hematopoietic PBX-interaction protein (HPIP). Biochemical and RNA-interference studies demonstrated that HPIP influences ERα-dependent rapid estrogen signaling by acting as a scaffold protein and recruits Src kinase and the p85 subunit of phosphatidylinositol 3-kinase to a complex with ERα, which in turn stimulates AKT and MAPK. We also found that ERα interacts with β-tubulin through HPIP. Destabilization of microtubules activated ERα signaling, whereas stabilization of microtubules repressed ERα transcriptional activity in a HPIP-dependent manner. These findings revealed a role for HPIP–microtubule complex in regulating 17β-estradiol–ERα responses in mammalian cells and discovered an inherent role of microtubules in the action of nuclear receptor.

S-palmitoylation modulates estrogen receptor α localization and functions

Abstract 17β-Estradiol (E2) acts as a chemical messenger in target tissues inducing both slow nuclear and rapid extra-nuclear responses. E2 binds to its cognate nuclear receptors (ER) resulting in the activation of target gene transcription in the nucleus. In addition to these genomic effects, E2 modulates cell functions through rapid non-genomic actions. Stimulation of G-proteins, Ca 2+ influx, inositol phosphate generation as well as phospholipase C, ERK/MAPK, and PI3K/AKT activation all occur within seconds to minutes after E2 binding to a small population of ERα located at the plasma membrane. The great impact of these rapid signals on cell physiology renders central the knowledge of the structural bases and mechanisms that mediate extra-nuclear signaling by E2. Several laboratories, including our own, have recently elucidated the structural requirements for localization and function of plasma membrane ERα. This review summarizes the molecular mechanisms of E2-induced rapid non-genomic actions relevant for cell functions, highlighting the role of lipid modification (i.e., palmitoylation) in the ERα localization to and residence at the plasma membrane.

The Effects of 17β-estradiol in Cancer are Mediated by Estrogen Receptor Signaling at the Plasma Membrane.

Two different isoforms of the estrogen receptors (i.e., ERα and ERβ) mediate pleiotropic 17β-estradiol (E2)-induced cellular effects. The ERs are principally localized in the nucleus where they act by globally modifying the expression of the E2-target genes. The premise that E2 effects are exclusively mediated through the nuclear localized ERs has been rendered obsolete by research over the last 15 years demonstrating that ERα and ERβ proteins are also localized at the plasma membranes and in other extra-nuclear organelles. The E2 modulation of cancer cell proliferation represents a good example of the impact of membrane-initiated signals on E2 effects. In fact, E2 via ERα elicits rapid signals driving cancer cells to proliferation (e.g., in breast cancer cells), while E2-induced ERβ rapid signaling inhibits proliferation (e.g., in colon cancer cells). In this review we provide with an overview of the complex system of E2-induced signal transduction pathways, their impact on E2-induced cancer cell proliferation, and the participation of E2-induced membrane-initiated signals in tumor environment.

Palmitoylation Regulates 17β-Estradiol-Induced Estrogen Receptor-α Degradation and Transcriptional Activity

The estrogen receptor-α (ERα) is a transcription factor that regulates gene expression through the binding to its cognate hormone 17β-estradiol (E2). ERα transcriptional activity is regulated by E2-evoked 26S proteasome-mediated ERα degradation and ERα serine (S) residue 118 phosphorylation. Furthermore, ERα mediates fast cell responses to E2 through the activation of signaling cascades such as the MAPK/ERK and phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog 1 pathways. These E2 rapid effects require a population of the ERα located at the cell plasma membrane through palmitoylation, a dynamic enzymatic modification mediated by palmitoyl-acyl-transferases. However, whether membrane-initiated and transcriptional ERα activities integrate in a unique picture or represent parallel pathways still remains to be firmly clarified. Hence, we evaluated here the impact of ERα palmitoylation on E2-induced ERα degradation and S118 phosphorylation. The lack of palmitoylation renders ERα more susceptible to E2-dependent degradation, blocks ERα S118 phosphorylation and prevents E2-induced ERα estrogen-responsive element-containing promoter occupancy. Consequently, ERα transcriptional activity is prevented and the receptor addressed to the nuclear matrix subnuclear compartment. These data uncover a circuitry in which receptor palmitoylation links E2-dependent ERα degradation, S118 phosphorylation, and transcriptional activity in a unique molecular mechanism. We propose that rapid E2-dependent signaling could be considered as a prerequisite for ERα transcriptional activity and suggest an integrated model of ERα intracellular signaling where E2-dependent early extranuclear effects control late receptor-dependent nuclear actions.

Nutritional Flavonoids Modulate Estrogen Receptor α Signaling

Estrogen receptor α (ERα) mediates 17β‐estradiol (E2) actions through the transcription of E2‐sensitive target genes. In addition, rapid non‐genomic signaling (e.g., MAPK/ERK) occurs. It is now well accepted that these rapid membrane‐initiated responses account for E2‐related cancer. Beside many beneficial effects on human health, nutritional flavonoids exert protective and anticarcinogenic effects on E2‐related cancer. The mechanism underlying these effects seems to be related to flavonoids antioxidant properties and/or to their ability to alter signal transduction protein kinases. In addition, an antiestrogenic activity has been proposed but not yet defined. However, the identification and characterization of the responsible mechanisms for flavonoid antitumoral effects is poorly understood. Here, we investigated the possibility that the antimitogenic effects of flavonoids are transduced by modulating ERα‐mediated rapid signaling. The ability of two flavonoids, the flavanone naringenin and the flavanol quercetin, with respect of E2, to induce ERα activities has been studied in the human cervix epitheloid carcinoma cell line (HeLa) devoid of any estrogen receptors and rendered E2‐sensitive by transient transfection with a human ERα expression vector. Our results indicate that flavonoids act as E2 mimetic on ERα transcriptional activity, whereas they impair the activation of rapid signaling pathways committed to E2‐induced proliferation. The resulting decoupling of ERα signal transduction could be proposed as a new mechanism in the protective effects of flavonoids against E2‐related cancer. IUBMB Life, 56: 145‐151, 2004

Phosphorylation-dependent regulation of nuclear localization and functions of integrin-linked kinase

Integrin-linked kinase (ILK) is a phosphorylated protein that regulates physiological processes that overlap with those regulated by p21-activated kinase 1 (PAK1). Here we report the possible role of ILK phosphorylation by PAK1 in ILK-mediated signaling and intracellular translocation. We found that PAK1 phosphorylates ILK at threonine-173 and serine-246 in vitro and in vivo. Depletion of PAK1 decreased the levels of endogenous ILK phosphorylation in vivo. Mutation of PAK1 phosphorylation sites on ILK to alanine reduced cell motility and cell proliferation. Biochemical fractionation, confocal microscopy, and chromatin-interaction analyses of human cells revealed that ILK localizes predominantly in the cytoplasm but also resides in the nucleus. Transfection of MCF-7 cells with point mutants ILK-T173A, ILK-S246A, or ILK-T173A; S246A (ILK-DM) altered ILK localization. Selective depletion of PAK1 dramatically increased the nuclear and focal point accumulation of ILK, further demonstrating a role for PAK1 in ILK translocation. We also identified functional nuclear localization sequence and nuclear export sequence motifs in ILK, delineated an apparently integral role for ILK in maintaining normal nuclear integrity, and established that ILK interacts with the regulatory region of the CNKSR3 gene chromatin to negatively modulate its expression. Together, these results suggest that ILK is a PAK1 substrate, undergoes phosphorylation-dependent shuttling between the cell nucleus and cytoplasm, and interacts with gene-regulatory chromatin.