Dominant negative ER induces apoptosis in GH(4) pituitary lactotrope cells and inhibits tumor growth in nude mice.

The ER plays an important role in the proliferation and differentiation of lactotrope tumor cells. GH(4) cells were infected with adenoviral vectors (AdL540Q and Ad1-536) to investigate the ability of dominant negative ER mutants to affect the regulation of gene expression and cell growth by endogenous ER. The dominant negative mutants suppressed estradiol stimulation of an estrogen-responsive reporter gene and the PRL promoter in these cells. AdL540Q or Ad1--536 infection also inhibited GH(4) cell growth and induced apoptosis, increasing the expression of the proapoptotic Bax protein and decreasing the expression of antiapoptotic Bcl-2. AdwtER-infected cells also showed decreased Bcl-2 protein. E2-induced activation of p38 MAPK, an enzyme that may participate in apoptosis, was observed in cells infected with AdwtER, AdL540Q, and Ad1--536. Consistent with the apoptotic effects in vitro, infection of GH(4) cells with AdL540Q or Ad1--536 inhibited the ability of the cells to form tumors in nude mice. These results indicate that dominant negative ER mutants induce apoptosis of GH(4) cells and suppress tumor formation and development. The delivery of dominant negative ERs by adenoviral vectors may provide an alternative modality for the targeted therapy of pituitary lactotrope adenomas and other estrogen-responsive tumors.

Estrogen receptor binding to DNA is not required for its activity through the nonclassical AP1 pathway.

In the classical signaling pathway, the estrogen receptor (ER) binds directly to estrogen response elements (EREs) to regulate gene transcription. To test the hypothesis that the nonclassical pathway involves ER interactions with other proteins rather than direct binding to DNA, mutations were introduced into the DNA binding domain (DBD) of the mouse ERalpha. The effects of these DBD mutations were examined in DNA binding assays using reporter constructs containing either EREs (classical) or AP1 (nonclassical) response elements. Using the AP1 reporter, there was a reversal of ER action relative to that seen with the ERE reporter. Estradiol induced suppression, and the antiestrogen ICI 182,780 stimulated transcription of the AP1 reporter. DBD mutations in the proximal (P-box) of the first zinc finger of the ER (E207A/G208A and E207G/G208S) eliminated ERE binding. These mutants were inactive using the ERE reporter but retained partial or full activity with the AP1 reporter. The DBD mutant ERs interacted with Jun when tested in mammalian cell two-hybrid assays. Two mutations (K366D and I362R) in the ER ligand binding domain known to alter coactivator interactions impaired transcriptional responses using either the ERE or AP1 reporters. We concluded that ER action through the AP1 response element involves interactions with other promoter-bound proteins instead of, or in addition to, direct binding to DNA. Interactions with coactivators were required for both pathways. These data supported a model in which ER-mediated transcriptional activation or repression is dependent on the ligand and the nature of the response element in the target gene.

Adenovirus-directed expression of dominant negative estrogen receptor induces apoptosis in breast cancer cells and regression of tumors in nude mice.

BACKGROUND: Estrogen receptors (ER) are expressed in about two thirds of human breast cancer, and are an important pharmacological target for treatment of these tumors. Dominant negative forms of the ER have been suggested as an alternative method to disrupt ER function. In this study, we examined the effect of dominant negative ER mutants (ER1-536 and L540Q) on ER-positive breast cancer cells in vitro and in vivo. MATERIALS AND METHODS: ER-positive T47D breast cancer cells were infected with adenoviral vectors expressing ER1-536 and L540Q to examine the effects of the mutants on gene expression and cell growth. Adenoviral vectors containing the wild type ER (AdwtER) and beta-galactosidase gene (AdGal) were used as controls. RESULTS: Ad1-536 or AdL540Q infection inhibited T47D cell growth and induced apoptosis, increasing Bax protein and phosphorylation of p38 mitogen-activated-protein kinase (MAPK). Consistent with the apoptotic effects in vitro, pre-infection of T47D cells with Ad1-536 or AdL540Q inhibited tumor formation when these cells were introduced into nude mice. In addition, injection of Ad1-536 and AdL540Q into pre-established T47D tumors induced tumor regression. Apoptosis, in conjunction with the activation of caspase-3 and phosphorylation of p38 MAPK, was detected in the shrinking tumors. Overexpression of wild-type ER by AdwtER infection also produced antiproliferative and apoptotic effects, but to a lesser extent than the ER1-536 and L540Q mutants. CONCLUSIONS: These results indicate that dominant negative ER mutants have the potential to induce apoptosis of T47D cells and regression of tumors. The delivery of dominant negative ERs by adenoviral vectors may provide a useful tool for targeted therapy of ER-positive breast cancer.

EGF activates highly selective estrogen-responsive reporter plasmids by an ER-independent pathway.

Epidermal growth factor (EGF) mimics the effects of estrogen on some cells, suggesting that it may activate the estrogen receptor (ER). We examined the ability of EGF to increase expression of several different estrogen-responsive luciferase reporters in MCF-7 breast cancer cells. Although EGF increased reporter activity, this effect was not inhibited by estrogen antagonists and was not dependent on estrogen response elements in the reporter plasmid. Similar results were obtained in BG-1 (ovarian) and Ishikawa (uterine) cells. In ER-negative JEG-3 cells, EGF, but not estradiol, increased reporter activity in the absence of transfected ER. The estrogen antagonist ICI 182780 blocked the ability of estradiol, but not EGF, to stimulate proliferation of T47D breast cancer cells, suggesting that the mitogenic effects of EGF are not mediated by ER. EGF does not appear to activate ER-mediated transcription in these experimental systems, although crosstalk between the estrogen and EGF signaling pathways may occur by other mechanisms.

A fusion protein of the estrogen receptor (ER) and nuclear receptor corepressor (NCoR) strongly inhibits estrogen-dependent responses in breast cancer cells.

Nuclear receptor corepressor (NCoR) mediates repression (silencing) of basal gene transcription by nuclear receptors for thyroid hormone and retinoic acid. The goal of this study was to create novel estrogen receptor (ER) mutants by fusing transferable repressor domains from the N-terminal region of NCoR to a functional ER fragment. Three chimeric NCoR-ER proteins were created and shown to lack transcriptional activity. These fusion proteins silenced basal transcription of the ERE2-tk-Luc reporter gene and inhibited the activity of co-transfected wild-type ER (wtER), indicating that they possess dominant negative activity. One of the fusion proteins (CDE-RD1), containing the ER DNA-binding and ligand-binding domains linked to the NCoR repressor domain (RD1), was selected for detailed examination. Its hormone affinity, intracellular localization, and level of expression in transfected cells were similar to wtER, and it bound to the estrogen response element (ERE) DNA in gel shift assays. Glutathione-S-transferase pull-down assays showed that CDE-RD1 retains the ability to bind to steroid receptor coactivator-1. Introduction of a DNA-binding domain mutation into the CDE-RD1 fusion protein eliminated silencing and dominant negative activity. Thus, the RD1 repressor domain prevents transcriptional activation despite the apparent ability of CDE-RD1 to bind DNA, ligand, and coactivators. Transcriptional silencing was incompletely reversed by trichostatin A, suggesting a histone deacetylase-independent mechanism for repression. CDE-RD1 inhibited ER-mediated transcription in T47D and MDA-MB-231 breast cancer cells and repressed the growth of T47D cells when delivered to the cells by a retroviral vector. These ER-NCoR fusion proteins provide a novel means for inhibiting ER-mediated cellular responses, and analogous strategies could be used to create dominant negative mutants of other transcription factors.

Identification of the cleavage sites of transforming growth factor alpha by insulin-degrading enzymes.

Insulin-degrading enzyme (IDE) is a sulfhydryl-dependent metalloproteinase with a zinc binding site unique to a new class of proteinases. The enzyme is relatively specific for a number of hormones/growth factors, such as insulin, atrial natriuretic peptide, IGF-II, and proinsulin. In this study we have identified the amino-acid bonds cleaved by IDE in transforming growth factor-alpha. High-performance liquid chromatography was used to separate the peptides generated by the degradation of 125I-TGF-alpha. The peptides were then submitted to sequential Edman degradation to determine the peptide bond broken. Cleavage sites were found at amino acids, 10-11 (Asp-Ser), 25-26 (Val-Gln), 28-29 (Asp-Lys), and 30-31 (Pro-Ala). In agreement with studies of cleavage sites of other hormones by this enzyme, no clear amino-acid specificity was seen. However, examination of the sites on a three-dimensional model of TGF-alpha suggest the primary mechanism used by IDE for determining cleavage sites is the tertiary structure of the substrate.

Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor.

The phytochemical resveratrol, which is found in grapes and wine, has been reported to have a variety of anti-inflammatory, anti-platelet, and anti-carcinogenic effects. Based on its structural similarity to diethylstilbestrol, a synthetic estrogen, we examined whether resveratrol might be a phytoestrogen. At concentrations (approximately 3-10 microM) comparable to those required for its other biological effects, resveratrol inhibited the binding of labeled estradiol to the estrogen receptor and it activated transcription of estrogen-responsive reporter genes transfected into human breast cancer cells. This transcriptional activation was estrogen receptor-dependent, required an estrogen response element in the reporter gene, and was inhibited by specific estrogen antagonists. In some cell types (e.g., MCF-7 cells), resveratrol functioned as a superagonist (i.e., produced a greater maximal transcriptional response than estradiol) whereas in others it produced activation equal to or less than that of estradiol. Resveratrol also increased the expression of native estrogen-regulated genes, and it stimulated the proliferation of estrogen-dependent T47D breast cancer cells. We conclude that resveratrol is a phytoestrogen and that it exhibits variable degrees of estrogen receptor agonism in different test systems. The estrogenic actions of resveratrol broaden the spectrum of its biological actions and may be relevant to the reported cardiovascular benefits of drinking wine.

Inducible expression and cellular localization of insulin-degrading enzyme in a stably transfected cell line.

Insulin degrading enzyme (IDE) is an evolutionarily conserved, nonlysosomal metalloprotease that has been implicated in the cellular degradation and processing of insulin. However, the site and the mode of the action of this enzyme are unclear. We have addressed these questions by establishing several Ltk- cell lines that can overexpress human insulin-degrading enzyme (hIDE) upon glucocorticoid induction. The level of overexpression of hIDE protein and transcripts in these lines correlates well with an increase in insulin degradation in both cell lysates and intact cells. Comparison of the deduced amino acid sequences of mammalian and Drosophila IDEs reveals a conserved carboxyl-terminal peroxisomal targeting sequence (A/S-K-L), suggesting that IDE may be localized in peroxisomes. To test this possibility, we determined the cellular location of the stably transfected hIDE by both immunofluorescence and immunocryoelectron microscopy. The overexpressed hIDE predominantly colocalized with catalase in peroxisomes, although IDE was also found in the cytosol at a much lower concentration. These results demonstrate that stably transfected IDE catalyzes a rate-limiting step in cellular insulin degradation and is localized predominantly in peroxisomes.

Functional analysis of conserved residues in the active site of insulin-degrading enzyme.

Insulin-degrading enzyme (IDE), a nonlysosomal metalloprotease involved in metabolizing internalized insulin, has catalytic properties that have been strongly conserved through evolution. Two major properties distinguish IDE from the prototypic metalloprotease thermolysin. 1) It is inhibited by cysteine protease inhibitors as well as metalloprotease inhibitors; 2) it contains an inversion of the HEXXH active site motif of thermolysin, where the histidines coordinate zinc and the glutamate participates in catalysis. Furthermore, cysteine is adjacent to the glutamate residue (HXCEH) in human, rat, and Drosophila IDE, although it is not conserved in their close homologue, Escherichia coli protease III. This cysteine has been postulated to mediate the differential sensitivity of IDE and protease III to cysteine protease inhibitors and chelators. The role of the cysteine in IDE catalysis and inhibitor sensitivity was examined by mutating Cys110 to glycine or serine. To determine whether glutamate in this unusual motif participates in catalysis, we mutated Glu111 to aspartate, valine, or glutamine. Vectors containing wild type or mutant enzymes were transfected into COS cells, and expression was confirmed by Western blotting. Although the glutamate mutants were devoid of insulin degrading activity, the cysteine mutants were indistinguishable from wild type enzyme in both catalytic activity and sensitivity to inhibitors. The loss of activity in the glutamate mutants was not due to gross alterations in tertiary structure, as shown by retention of the ability to bind substrate and by conservative and nonconservative mutation of a neighboring residue with no apparent effect on catalysis. These results demonstrate that the conserved glutamate in the zinc-binding site of human insulin-degrading enzyme is a major catalytic residue, while a conserved cysteine in this region is not essential for catalysis or inhibitor sensitivity.

Mutations in a zinc-binding domain of human insulin-degrading enzyme eliminate catalytic activity but not insulin binding.

Insulin-degrading enzyme is a nonlysosomal metalloprotease that initiates degradation of internalized insulin in some cells. We previously identified a potential catalytic site containing an inversion of the Zn(2+)-binding domain of the thermolysin family (Kuo, W.-L., Gehm, B. D., and Rosner, M. R. (1991) Mol. Endocrinol. 4, 1580-1591). The role of this site in catalysis was examined by mutating one of the presumptive Zn(2+)-coordinating histidines (His108) in human insulin-degrading enzyme to leucine or glutamine, which were predicted to reduce or eliminate Zn2+ binding without substantially altering secondary structure. cDNAs for the mutant and wild-type enzymes were incorporated into an expression vector and transfected into COS cells. Expression of the transfected genes was confirmed by Northern and Western blots. In contrast to the wild-type gene, which increased insulin degradation by cell extracts and intact cells several-fold, the mutated genes had no effect on insulin degradation, indicating a loss of catalytic activity. However, the mutants' ability to bind substrate was unimpaired, as affinity labeling with 125I-insulin was increased compared to the wild type. These results suggest that an intact Zn(2+)-binding domain in human insulin-degrading enzyme is required for catalytic activity and can affect, but is not required for, substrate binding.

Activation of bovine rod outer segment phosphatidylinositol-4,5-bisphosphate phospholipase C by calmodulin antagonists does not depend on calmodulin.

Calmodulin antagonists stimulated phosphatidylinositol-4,5-bisphosphate phospholipase C in soluble and particulate fractions of bovine rod outer segments. Antagonists tested include trifluoperazine, melittin, calmidazolium, compound 48/80, W-13 [N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide], and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide]. All were effective, but W-7 was chosen for further characterization of the effect, which was most pronounced in the soluble fraction. Phospholipase C activity in the soluble fraction did not increase linearly with the quality of enzyme assayed, suggesting the presence of an endogenous inhibitor or an inhibitory self-association of the enzyme. W-7 appeared to counteract this inhibition, resulting in a linear activity-quantity relationship. Stimulation by W-7 was therefore largest when large amounts of crude enzyme were assayed and small or nil when small amounts were assayed. The effect of W-7 was also dependent on [Ca2+], with half-maximal stimulation occurring between 0.1 and 1 microM. W-7 and W-13 were much more effective than their nonchlorinated analogues W-5 and W-12 at increasing phospholipase C activity. While this pattern of effectiveness is typical of calmodulin-mediated processes, the absence of any effect by added calmodulin and the retention of W-7 sensitivity by purified CaM-free enzyme argue against regulation by CaM. Octyl glucoside, a nonionic detergent, mimicked some of the effects of CaM antagonists, suggesting that the antagonists act by interfering with protein-protein interactions. It appears likely that CaM antagonists prevent an inhibitory multimerization or aggregation of at least one form of ROS phospholipase C.

Regulation of insulin degradation: expression of an evolutionarily conserved insulin-degrading enzyme increases degradation via an intracellular pathway.

The insulin-degrading enzyme (IDE) is an evolutionarily conserved enzyme that has been implicated in cellular insulin degradation, but its site of action and importance in regulating insulin degradation have not been clearly established. We addressed this question by examining the effects of overexpressing IDE on insulin degradation in COS cells, using both human IDE (hIDE) and its Drosophila homolog (dIDE). The dIDE, which was recently cloned in our laboratory, has 46% amino acid identity with hIDE, degrades insulin with comparable efficiency, and is readily expressed in mammalian cells. Transient expression of dIDE or hIDE in COS monkey kidney cells led to a 5- to 7-fold increase in the rate of degradation of extracellular insulin, indicating that IDE can regulate cellular insulin degradation. Insulin-degrading activity in the medium was very low and could not account for the difference between transfected and control cells. To further localize the site of IDE action, the fate of insulin after receptor binding was examined. The dIDE-transfected cells displayed increased degradation of prebound insulin compared to control cells. This increase in degradation was observed even when excess unlabeled insulin was added to block reuptake or extracellular degradation. These results indicate that IDE acts at least in part within the cell. The lysosomotropic agents chloroquine and NH4Cl did not affect the increase in insulin degradation produced by transfection with dIDE, indicating that the lysosomal and IDE-mediated pathways of insulin degradation are independent. The results demonstrate that IDE can regulate the degradation of insulin by intact cells via an intracellular pathway.

Regulation of insulin, epidermal growth factor, and transforming growth factor-alpha levels by growth factor-degrading enzymes.

The mechanisms by which growth factors are degraded and the role this process plays in the regulation of cell growth are not well understood. Insulin degradation is believed to be mediated by a specific metalloprotease, insulin-degrading enzyme (IDE). We have previously shown that IDE can also degrade transforming growth factor-alpha (TGF alpha), but not epidermal growth factor (EGF), in vitro. This selectivity was surprising, since TGF alpha and EGF are structurally similar and bind to the same receptor with comparable affinities. Using a spectrum of protease inhibitors, we have now analyzed the degradation of TGF alpha, EGF, and insulin by human hepatoma HepG2 cells. The results suggest that bacitracin-sensitive metalloproteases are involved in the degradation of TGF alpha and EGF as well as insulin, and that the degradation of TGF alpha, but not EGF, is mediated in part by IDE. Inhibiting the activity of these metalloproteases decreased growth factor depletion, suggesting that these enzymes play an important role in the control of extracellular growth factor levels. The existence of separate degradative pathways for EGF and TGF alpha may explain how the two factors exert differential effects in some systems, and degradation of TGF alpha by IDE would provide a possible mechanism for interaction between the insulin and TGF alpha/EGF signalling systems.

Cloning and expression of the cDNA for a Drosophila insulin-degrading enzyme.

We have previously identified and characterized a metalloproteinase from Drosophila that cleaves insulin and transforming growth factor-alpha, but not epidermal growth factor, at physiological concentrations. On the basis of enzymatic properties and substrate specificity, this enzyme was identified as the Drosophila homolog of the mammalian insulin-degrading enzyme (IDE). We now report the cloning and sequencing of the cDNA coding for the Drosophila IDE (dIDE). Northern blot analysis indicates that the dIDE is translated from a 3.6-kilobase transcript similar in size to one of the two human IDE transcripts. The gene for the dIDE has been mapped to chromosome 3L (77B). The sequence of the dIDE is very similar to that of the human IDE, and both enzymes share limited but significant identity with the bacterial metalloproteinase protease III. Indirect studies based upon inhibitors, degradation products, and microinjected antibodies have suggested that the IDE can initiate cellular insulin degradation in mammalian cells. To determine whether dIDE expressed in mammalian cells can also degrade insulin, we transfected the cDNA into murine NIH3T3 cells. Extracts of the transfected cells showed increased insulin-degrading activity, demonstrating that the dIDE can be functionally expressed in mammalian cells. These results indicate that the properties of the IDE are evolutionarily conserved.

Phosphoinositide synthesis in bovine rod outer segments.

Phosphoinositide turnover has been implicated in signal transduction in a variety of cells, including photoreceptors. We demonstrate here the presence of a complete pathway for rapid synthesis of phosphoinositides in isolated bovine retinal rod outer segments (ROS) free of microsomal contaminants. Synthesis was measured by the incorporation of label from radioactive precursors, [gamma-32P]ATP and [3H]inositol. [gamma-32P]ATP also produced large amounts of labeled phosphatidic acid. Incorporation of [3H]inositol required CTP and Mn2+. Mn2+ increased 32P incorporation into phosphatidylinositol 4-phosphate, while spermine increased phosphoinositide labeling generally. ROS that had been washed to remove soluble and peripheral proteins incorporated less label than unwashed ROS into phosphatidic acid and phosphatidylinositol. No effects of light were detected. Inhibitory effects of high concentrations of nonhydrolyzable GTP analogues were probably due to competition with ATP.

Phosphatidylinositol-4,5-bisphosphate phospholipase C in bovine rod outer segments.

Preparations of rod outer segments from cattle retinas contained soluble and particulate phospholipase C activities which hydrolyzed phosphatidylinositol 4,5-bisphosphate (PIP2) and the other phosphoinositides. Ca2+ was required for PIP2 hydrolysis, but high (greater than 300 microM) concentrations were inhibitory. Mg2+ and spermine at low concentrations stimulated the particulate activity but inhibited the soluble. Mn2+ inhibited both. High (greater than 100 microM) concentrations of the nonhydrolyzable GTP analogue guanylyl beta,gamma-methylenediphosphonate inhibited PIP2 hydrolysis by both the soluble and particulate activities, but guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), fluoride, and cholera and pertussis toxins were without effect. Overall phospholipase C activity in ROS was unaffected by light. Evidence was found for multiple forms of the enzyme, requiring isolation and separate characterization before ruling out regulation by light or G-protein.

An evolutionarily conserved enzyme degrades transforming growth factor-alpha as well as insulin.

A single enzyme found in both Drosophila and mammalian cells is able to selectively bind and degrade transforming growth factor (TGF)-alpha and insulin, but not EGF, at physiological concentrations. These growth factors are also able to inhibit binding and degradation of one another by the enzyme. Although there are significant immunological differences between the mammalian and Drosophila enzymes, the substrate specificity has been highly conserved. These results demonstrate the existence of a selective TGF-alpha-degrading enzyme in both Drosophila and mammalian cells. The evolutionary conservation of the ability to degrade both insulin and TGF-alpha suggests that this property is important for the physiological role of the enzyme and its potential for regulating growth factor levels.