Ligand-independent Activator Research Articles

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin.

SummaryEstrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ERα) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosin-dependent and reversible by estrogens. Detailed investigations showed that the ERα target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.

Plakophilin-2 induced EGFR phosphorylation: a focus on the intracellular activators of EGFR.

The oncogenic role of EGFR in many tumors has attracted a great deal of attention in the recent years and initiated the development of several potent EGFR inhibitors, which are used clinically for cancer treatment. However, the current therapeutic inhibition of EGFR signaling is limited to monoclonal antibodies that bind to the EGFR extracellular domain or tyrosine kinase inhibitors that block EGFR kinase activation directly. Despite the great promise of these inhibitors, a certain percentage of patients develop resistance to these therapies, highlighting the necessity for alternative therapeutic strategies based on our most current knowledge of the mechanisms of EGFR signaling. We recently reported that Plakofilin-2 (PKP2) is a novel ligand-independent cytoplasmic activator of EGFR signaling. Here we focus on recent studies demonstrating important roles of intracellular EGFR activators, and propose targeted disruption of these activators as a novel avenue of therapeutic intervention to inhibit EGFR-mediated cancer development.

Ligand-independent activation of the arylhydrocarbon receptor by ETK (Bmx) tyrosine kinase helps MCF10AT1 breast cancer cells to survive in an apoptosis-inducing environment

It has been reported that the arylhydrocarbon receptor (AHR) is overexpressed in certain types of breast tumors. However, so far no concrete evidence has been provided yet as to why and how the overexpressed AHR in those cancer cells is functionally activated without exogenous ligands. Here we show that the AHR was functionally activated when estrogen receptor-negative, AHR overexpressing MCF10AT1 human breast cancer cells (designated P20E) were subjected to serum starvation. Transfection of cells with ETK-KQ, a plasmid for kinase-dead epithelial and endothelial tyrosine kinase (ETK), attenuated this AHR activation. Artificial over-expression of ETK in P20E cells through transfection with wild-type ETK plasmid (ETK-wt) caused up-regulation of cytochrome P4501a1 (CYP1A1; a marker of functional activation of AHR). Furthermore, ablation of ETK expression by a specific antisense oligonucleotide or AG879, a specific inhibitor of ETK kinase suppressed activation of AHR induced by omeprazole, a strong ligand-independent activator of AHR. Activation of ETK in those cells conferred them resistance to UVB- as well as doxorubicin-induced apoptosis, both of which were reversed by ETK-KQ. Together, these findings support our conclusion that ETK is the tyrosine kinase responsible for the functional activation of the AHR in these mammary epithelial cells.

Endocrinological Aspects of Proteinuria and Podocytopathy in Diabetes: Role of the Aldosterone/Mineralocorticoid Receptor System

Aldosterone has emerged as a deleterious hormone in the kidney, for example as a potent inducer of proteinuria. We identified the podocyte, the final filtration barrier in the glomerulus, as a novel target of aldosterone. Activation of the mineralocorticoid receptor (MR) in the podocyte disrupts the filtration barrier and induces proteinuria. Recent clinical and experimental studies have shown the efficacy of MR antagonism in reducing albuminuria in patients or rodent models of type 1 and type 2 diabetes. We assessed the pathogenic role of aldosterone in SHR/NDmcr-cp, a rat model of type 2 diabetes/metabolic syndrome. Podocyte injury and proteinuria were early manifestations of nephropathy in this model, and were exacerbated by high-salt feeding. Inappropriate activation of the aldosterone/MR system, possibly via adipocyte-derived aldosterone releasing factors, underlay the renal damage. Furthermore, we identified Rac1, a Rho family small GTPase, as a novel ligand-independent activator of MR. This alternative pathway of MR activation, indeed, contributed to podocyte injury in proteinuric kidney disease. In conclusion, MR can be activated by several different pathways, both aldosterone-dependently and -independently, leading to podocyte impairment and progression of proteinuric kidney disease. MR antagonists are promising anti-proteinuric drugs in diabetes, although hyperkalemia is a concern.

ERα as ligand-independent activator of CDH-1 regulates determination and maintenance of epithelial morphology in breast cancer cells

Estrogen receptor alpha (ERalpha) and E-cadherin are primary markers of luminal epithelial breast cancer cells with E-cadherin being a main caretaker of the epithelial phenotype. E-cadherin repression is needed for cancer cells to acquire motile and invasive properties, and it is known that in ER-positive breast cancer cells, estrogen down-regulate E-cadherin gene transcription. We report here that ERalpha is bound to the E-cadherin promoter in both the presence and the complete absence of estrogen, suggesting an unexpected role for unliganded ERalpha in E-cadherin transcription. Indeed, our data reveal that activation by unliganded ERalpha and repression by estrogen-activated ERalpha require direct binding to a half-estrogen response element within the E-cadherin promoter and exchange from associated coactivators to corepressors. Therefore, these results suggest a pivotal role for unliganded ERalpha in controlling a fundamental caretaker of the epithelial phenotype in breast cancer cells. Here, we show that ERalpha-positive breast cancer T47D cells transduced with the sfRON kinase undergo a full epithelial-mesenchymal conversion and lose E-cadherin and ERalpha expression. Our data show that, although the E-cadherin gene becomes hypermethylated and heterochromatic, kinase inhibitors can restore E-cadherin expression, together with an epithelial morphology in an ERalpha-dependent fashion. Similarly, transfection of ERalpha, in the absence of ligands, was sufficient to restore E-cadherin transcription in both sfRON-T47D and other ERalpha-, E-cadherin-negative cells. Therefore, our results suggest a novel role for the ERalpha that plays the dual role of ligand-independent activator and ligand-dependent repressor of E-cadherin in breast cancer cells.

Modulation of human estrogen receptor α F promoter by a protein kinase C/c-Src-dependent mechanism in osteoblast-like cells

The human estrogen receptor alpha (ERalpha) gene is driven by multiple promoters, of which the F promoter alone is found to be active in primary osteoblasts. The study was aimed at identifying new regulatory pathways affecting transcription of the receptor in this cell lineage. We generated human osteoblast-like cells, Saos-2, stably transfected with a luciferase-reporter gene downstream of the human ERalpha F promoter (Saos F-Luc), and assayed the reporter response to differentiation-related signals. Over-confluence, shown to stimulate osteoblast differentiation, caused a time-dependent increase of F-promoter activity and correlated with an inactivation of protein kinase C alpha (PKCalpha ). PKC downregulation, obtained by long-term treatment with phorbol 12-myristate 13-acetate (PMA), resulted in promoter stimulation at similar levels in sub-confluent cells. The F promoter contains a putative PMA-responsive AP-1 site, but AP-1 activation was unremarkable in over-confluent cells. Treatment with PP1, a specific inhibitor of the non-receptor tyrosine-kinase c-Src, which is a negative regulator of osteoblast differentiation, showed that the activity of this kinase inhibits the F promoter. In PP1-treated cells, F-promoter activity was not further increased by PMA. Treatment with the generic kinase inhibitor 4-dimethylaminopyridine (DMAP) resulted in a dose-dependent induction of the promoter, which matched a parallel decrease of active c-Src. The effect was c-Src dependent, as DMAP caused no further promoter induction in PP1-treated cells. Overexpression of exogenous human ERalpha resulted in modest promoter stimulation, which required the ligand-independent activator function 1 of the receptor. In murine primary osteoblasts, additional ERalpha signal was observed upon induction of F promoter. In conclusion, we demonstrated a robust PKC/c-Src-dependent and estrogen-independent mechanism modulating transcription of ERalpha in osteoblasts, probably affecting estrogen responsiveness during cell differentiation.

Forced dimerization of gp130 leads to constitutive STAT3 activation, cytokine-independent growth, and blockade of differentiation of embryonic stem cells.

The mode of activation of glycoprotein 130 kDa (gp130) and the transmission of the activation status through the plasma membrane are incompletely understood. In particular, the molecular function of the three juxtamembrane fibronectin III-like domains of gp130 in signal transmission remains unclear. To ask whether forced dimerization of gp130 is sufficient for receptor activation, we replaced the entire extracellular portion of gp130 with the c-jun leucine zipper region in the chimeric receptor protein L-gp130. On expression in cells, L-gp130 stimulates ligand-independent signal transducer and activator of transcription (STAT) 3 and extracellular signal-regulated kinase 1/2 phosphorylation. gp130 activation could be abrogated by the addition of a competing peptide comprising the leucine zipper region of c-fos. When stably expressed in the interleukin-3-dependent Ba/F3 murine pre-B-cells, these cells showed constitutive STAT3 activation and cytokine-independent growth over several months. Because gp130 stimulation completely suppressed differentiation of murine embryonic stem cells in vitro, we also stably expressed L-gp130 in these cells, which completely blocked their differentiation in the absence of cytokine stimulation and was consistent with high constitutive expression levels of the stem cell factor OCT-4. Thus, L-gp130 can be used in vitro and in vivo to mimic constitutive and ligand-independent activation of gp130 and STAT3, the latter of which is frequently observed in neoplastic diseases.

New G Protein Interactions

Two groups have uncovered new wrinkles in reactions involving heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins). Ligand binding to G protein-coupled receptors (GPCRs) activates G proteins by catalyzing guanine nucleotide exchange and dissociation of the GTP-bound α subunit (Gα) from the βγ heterodimer (Gβγ). Both subunits are then free to activate various effectors. Ribas et al. partially purified a ligand-independent G-protein activator, NG108-15 G-protein activator (NG-GPA), from the neuroblastoma-glioma hybrid NG108-15. NG-GPA stimulated guanosine 5'-O-thiotriphosphate (GTPγS) binding to purified brain G protein, as well as to recombinant Gαo and Gαi, and inhibited adenylyl cyclase activity in DDT1-MF-2 cell membranes. NG-GPA's stimulation of GTPγS binding, unlike GPCR-stimulated GTPγS binding, was unaffected by treatment with pertussis toxin, which prevents agonist-induced receptor interactions with the Gi family of G proteins, suggesting that NG-GPA interacts with the G protein through a previously unidentified regulatory domain. Dell et al. used a yeast two-hybrid screen to identify new Gβγ effectors and isolated three proteins containing WD40 motifs (which Gβγ also contains) that bound to Gβγ. Gβγ interaction with two of these proteins, receptor for activated C kinase 1 (RACK1) and dynein intermediate chain (DIC), was confirmed by Western analysis of immunoprecipitates. A RACK1 fusion protein bound to both Gβ1γ1 and the heterotrimeric Gαtβ1γ1 transducin heterotrimer but not to Gαt alone; Gαtβ1γ1 had a higher binding affinity than Gβ1γ1. RACK1 interaction with Gαtβ1γ1 was surprising, because few proteins other than GPCRs interact with the heterotrimer. Indeed, all known Gβγ effectors bind to the Gα-interacting region, suggesting a novel mechanism for RACK1-G protein interaction.C. Ribas, A. Takesono, M. Sato, J. D. Hildebrandt, S. M. Lanier, Pertussis toxin-insensitive activation of the heterotrimeric G-proteins Gi/Go by the NG108-15 G-protein activator. J. Biol. Chem. 277, 50223-50225 (2002). [Abstract] [Full Text]E. J. Dell, J. Connor, S. Chen, E. G. Stebbins, N. P. Skiba, D. Mochly-Rosen, H. E. Hamm, The βγ subunit of heterotrimeric G proteins interacts with RACK1 and two other WD repeat proteins. J. Biol. Chem. 277, 49888-49895 (2002). [Abstract] [Full Text]

New G Protein Interactions

Two groups have uncovered new wrinkles in reactions involving heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins). Ligand binding to G protein-coupled receptors (GPCRs) activates G proteins by catalyzing guanine nucleotide exchange and dissociation of the GTP-bound α subunit (Gα) from the βγ heterodimer (Gβγ). Both subunits are then free to activate various effectors. Ribas et al. partially purified a ligand-independent G-protein activator, NG108-15 G-protein activator (NG-GPA), from the neuroblastoma-glioma hybrid NG108-15. NG-GPA stimulated guanosine 5'- O -thiotriphosphate (GTPγS) binding to purified brain G protein, as well as to recombinant Gα o and Gα i , and inhibited adenylyl cyclase activity in DDT 1 -MF-2 cell membranes. NG-GPA's stimulation of GTPγS binding, unlike GPCR-stimulated GTPγS binding, was unaffected by treatment with pertussis toxin, which prevents agonist-induced receptor interactions with the G i family of G proteins, suggesting that NG-GPA interacts with the G protein through a previously unidentified regulatory domain. Dell et al. used a yeast two-hybrid screen to identify new Gβγ effectors and isolated three proteins containing WD40 motifs (which Gβγ also contains) that bound to Gβγ. Gβγ interaction with two of these proteins, receptor for activated C kinase 1 (RACK1) and dynein intermediate chain (DIC), was confirmed by Western analysis of immunoprecipitates. A RACK1 fusion protein bound to both Gβ 1 γ 1 and the heterotrimeric Gα t β 1 γ 1 transducin heterotrimer but not to Gα t alone; Gα t β 1 γ 1 had a higher binding affinity than Gβ 1 γ 1 . RACK1 interaction with Gα t β 1 γ 1 was surprising, because few proteins other than GPCRs interact with the heterotrimer. Indeed, all known Gβγ effectors bind to the Gα-interacting region, suggesting a novel mechanism for RACK1-G protein interaction. C. Ribas, A. Takesono, M. Sato, J. D. Hildebrandt, S. M. Lanier, Pertussis toxin-insensitive activation of the heterotrimeric G-proteins G i /G o by the NG108-15 G-protein activator. J. Biol. Chem. 277 , 50223-50225 (2002). [Abstract] [Full Text] E. J. Dell, J. Connor, S. Chen, E. G. Stebbins, N. P. Skiba, D. Mochly-Rosen, H. E. Hamm, The βγ subunit of heterotrimeric G proteins interacts with RACK1 and two other WD repeat proteins. J. Biol. Chem. 277 , 49888-49895 (2002). [Abstract] [Full Text]

Treatment of rats with 17beta-estradiol or relaxin rapidly inhibits uterine estrogen receptor beta1 and beta2 messenger ribonucleic acid levels.

Estrogen regulates the growth and differentiation of the uterus via binding to estrogen receptors (ERs), members of the nuclear receptor family of transcription factors. Two forms of ER exist: ERalpha and ERbeta. The former is a well-characterized mediator of estrogen-induced transcription, but the function of the latter is unclear. Recent in vitro studies suggest that both splicing forms of ERbeta expressed in rat tissues, beta1 and beta2, may function as inhibitors of ERalpha transcriptional activity. To gain insight into the role of ERbeta in estrogen action, we examined the effects of estrogen and relaxin, a ligand-independent activator of ERs, on the expression of ERbeta1 and ERbeta2 mRNA in the uterus in vivo. Eighteen-day-old female rats were ovariectomized and, after recovery, treated with 17beta-estradiol, relaxin, or vehicle. Quantitative reverse transcription-polymerase chain reaction analyses of uterine RNA from estrogen-treated animals revealed marked decreases in the steady-state levels of the mRNAs for both ERbeta1 and ERbeta2 at 3, 6, and 24 h after treatment. Relaxin induced a similar effect. Neither hormone had any significant effect on ERalpha mRNA levels. To determine if endogenous estrogen exerts this effect, we examined the expression of ERbetas in the uterus during the estrous cycle. Levels of both isoforms were highest at diestrus (low estrogen), were significantly lower at early proestrus (rising estrogen), reached a nadir during late proestrus (peak estrogen), and rebounded at estrus (declining estrogen). These data suggest that down-regulation of ERbeta expression may be required for estrogen to exert its full trophic effects on the uterus.

Pertussis Toxin-insensitive Activation of the Heterotrimeric G-proteins Gi/Go by the NG108-15 G-protein Activator

A ligand-independent activator of heterotrimeric brain G-protein was partially purified from detergent-solubilized extracts of the neuroblastoma-glioma cell hybrid NG108-15. The G-protein activator (NG108-15 G-protein activator (NG-GPA)) increased [(35)S]guanosine 5'-O-(thiotriphosphate) ([(35)S]GTPgammaS) to purified brain G-protein in a magnesium-dependent manner and promoted GDP dissociation from Galpha(o). The NG-GPA also increased GTPgammaS binding to purified, recombinant Galpha(i2), Galpha(i3), and Galpha(o), but minimally altered nucleotide binding to purified transducin. The NG-GPA increased GTPgammaS binding to membrane-bound G-proteins and inhibited basal, forskolin- and hormone-stimulated adenylyl cyclase activity in DDT(1)-MF-2 cell membranes. In contrast to G-protein coupled receptor-mediated activation of heterotrimeric G-proteins in DDT(1)-MF-2 cell membrane preparations, the action of the NG-GPA was not altered by treatment of the cells with pertussis toxin. ADP-ribosylation of purified brain G-protein also failed to alter the increase in GTPgammaS binding elicited by the NG-GPA. Thus, the NG-GPA acts in a manner distinct from that of a G-protein coupled receptor and other recently described receptor-independent activators of G-protein signaling. These data indicate the presence of unexpected regulatory domains on G(i)/G(o) proteins and suggest the existence of pertussis toxin-insensitive modes of signal input to G(i)/G(o) signaling systems.

Characterization of the retinoid orphan-related receptor-alpha coactivator binding interface: a structural basis for ligand-independent transcription.

The retinoid orphan-related receptor-alpha (RORalpha) is a member of the ROR subfamily of orphan receptors and acts as a constitutive activator of transcription in the absence of exogenous ligands. To understand the basis of this activity, we constructed a homology model of RORalpha using the closely related TRbeta as a template. Molecular modeling suggested that bulky hydrophobic side chains occupy the RORalpha ligand cavity leaving a small but distinct cavity that may be involved in receptor stabilization. This model was subject to docking simulation with a receptor-interacting peptide from the steroid receptor coactivator, GR-interacting protein-1, which delineated a coactivator binding surface consisting of the signature motif spanning helices 3-5 and helix 12 [activation function 2 (AF2)]. Probing this surface with scanning alanine mutagenesis showed structural and functional equivalence between homologous residues of RORalpha and TRbeta. This was surprising (given that RORalpha is a ligand-independent activator, whereas TRbeta has an absolute requirement for ligand) and prompted us to use molecular modeling to identify differences between RORalpha and TRbeta in the way that the AF2 helix interacts with the rest of the receptor. Modeling highlighted a nonconserved amino acid in helix 11 of RORalpha (Phe491) and a short-length of 3.10 helix at the N terminus of AF2 which we suggest 1) ensures that AF2 is locked permanently in the holoconformation described for other liganded receptors and thus 2) enables ligand-independent recruitment of coactivators. Consistent with this, mutation of RORalpha Phe491 to either methionine or alanine (methionine is the homologous residue in TRbeta), reduced and ablated transcriptional activation and recruitment of coactivators, respectively. Furthermore, we were able to reconstitute transcriptional activity for both a deletion mutant of RORalpha lacking AF2, and Phe491Met, by overexpression of a GAL-AF2 fusion protein, demonstrating ligand-independent recruitment of AF2 and a role for Phe491 in recruiting AF2.

Mechanism of YC-1-induced activation of soluble guanylyl cyclase.

The signaling molecule nitric oxide (NO) mediates many of its effects by the stimulation of soluble guanylyl cyclase (sGC). The activation process is initiated by high-affinity binding of NO to the enzyme's prosthetic heme group. Despite its poor sGC-activating properties, carbon monoxide (CO) has also been suggested as a physiological activator of sGC. Recently, we have shown that the substance YC-1, a benzyl indazole derivative, stimulates sGC by 10-fold (independently of NO) potentiates the stimulatory effect of NO, and turns CO into a potent activator of sGC. In the present study, we show that activation of sGC by protoporphyrin IX, a ligand-independent activator, was potentiated by YC-1, yet a shift of the concentration-response curve as seen with NO and CO was not observed. YC-1 slowed down the dissociation rates for NO and CO from the activated enzyme as monitored by cGMP accumulation after addition of the NO and CO scavenger oxyhemoglobin. A direct interaction of YC-1 with the heme group can be ruled out because YC-1 did not change the Soret absorption of basal or stimulated sGC and, in addition, still bound to the heme-depleted enzyme. Together, our results indicate that YC-1 increases the maximal catalytic rate and sensitizes the enzyme toward its gaseous activators by binding to an allosteric site on sGC molecules, thereby reducing the ligand dissociation rate from the heme group.

Mutations in the conserved C-terminal sequence in thyroid hormone receptor dissociate hormone-dependent activation from interference with AP-1 activity.

A short C-terminal sequence that is deleted in the v-ErbA oncoprotein and conserved in members of the nuclear receptor superfamily is required for normal biological function of its normal cellular counterpart, the thyroid hormone receptor alpha (T3R alpha). We carried out an extensive mutational analysis of this region based on the crystal structure of the hormone-bound ligand binding domain of T3R alpha. Mutagenesis of Leu398 or Glu401, which are surface exposed according to the crystal structure, completely blocks or significantly impairs T3-dependent transcriptional activation but does not affect or only partially diminishes interference with AP-1 activity. These are the first mutations that clearly dissociate these activities for T3R alpha. Substitution of Leu400, which is also surface exposed, does not affect interference with AP-1 activity and only partially diminishes T3-dependent transactivation. None of the mutations affect ligand-independent transactivation, consistent with previous findings that this activity is mediated by the N-terminal domain of T3R alpha. The loss of ligand-dependent transactivation for some mutants can largely be reversed in the presence of GRIP1, which acts as a strong ligand-dependent coactivator for wild-type T3R alpha. There is excellent correlation between T3-dependent in vitro association of GRIP1 with T3R alpha mutants and their ability to support T3-dependent transcriptional activation. Therefore, GRIP1, previously found to interact with the glucocorticoid, estrogen, and androgen receptors, may also have a role in T3R alpha-mediated ligand-dependent transcriptional activation. When fused to a heterologous DNA binding domain, that of the yeast transactivator GAL4, the conserved C terminus of T3R alpha functions as a strong ligand-independent activator in both mammalian and yeast cells. However, point mutations within this region have drastically different effects on these activities compared to their effect on the full-length T3R alpha. We conclude that the C-terminal conserved region contains a recognition surface for GRIP1 or a similar coactivator that facilitates its interaction with the basal transcriptional apparatus. While important for ligand-dependent transactivation, this interaction surface is not directly involved in transrepression of AP-1 activity.

Function of Nuclear Co-repressor Protein on Thyroid Hormone Response Elements Is Regulated by the Receptor A/B Domain

Recently, a family of nuclear co-repressor proteins (TRACs) have been identified that interact with thyroid hormone (TR) and retinoic acid receptors to mediate ligand-independent repression of gene transcription. In this report, we have cloned and characterized a human TRAC, which when expressed as a truncated protein lacking its repressing domains, can abolish endogenous cellular TRAC activity. Use of this inhibitor has uncovered a differential function of TRACs on negative versus positive thyroid hormone response elements and has demonstrated the importance of the TR A/B domain in modulating TRAC function. Thus, isoform-specific functions of the TR may be mediated by their functional interaction with co-repressor proteins.