In situ hybridization for creatine kinase-B messenger RNA in rat uterus and brain.

Creatine kinase-B (CKB) is present in both uterus and brain, and in uterus its synthesis (protein and mRNA) is regulated by estrogen. In the present study we have used in situ hybridization to detect CKB mRNA in uterus and brain, and to determine whether there is cell type specific induction of CKB by estrogen in these tissues. Tissue was taken from ovariectomized (ovx) rats that had been injected with either estrogen (17 beta-estradiol-3-benzoate and/or 17 beta-estradiol) or vehicle alone, 2, 8, 24 and 72 h previously. The brains and uteri were removed, frozen, cryostat-sectioned, and processed for in situ hybridization histochemistry. The uterine and brain sections were incubated with a tritiated DNA probe complementary to a 3' fragment of CKB mRNA, or a control sense probe to the same 3' fragment. In uterine smooth muscle cells, a 2.5- and 3.5-fold induction of CKB mRNA was observed 2 and 24 h after estrogen administration, respectively, and levels approached ovx controls at 72 h. A smaller induction (1.9-fold, 2 h) was observed in uterine epithelium, with little induction of CKB mRNA in stroma. In the brain CKB mRNA was detected in neurons, but not in clearly identified glia, and only occasionally in ependymal cells. In brain regions containing estrogen receptors there was no evidence of a significant estrogen effect on CKB mRNA levels. Some brain regions had higher neuronal expression than others (e.g. medial septum vs. preoptic area), but expression was widespread and not limited to neuroendocrine sites.

Estrogen control of uterine tissue factor messenger ribonucleic acid levels.

One of the early responses of the immature rat uterus to stimulation by estradiol (E2) is an increase in the specific activity and synthesis of a discrete set of proteins including tissue factor (TF). Increases in TF are associated with stimulation of cell growth in mouse and human fibroblasts and endothelial cells. The increase in TF activity following E2 stimulation of the uterus is due to an increase in TF messenger RNA (mRNA). A 2- to 4-fold increase in mRNA is observed 1 h after injection, reaches a maximum at 3 h, and is reduced at 6 h. The increase is hormone specific and occurs at low levels of E2 (0.66 micrograms/kg). The E2 effect is abolished by actinomycin but not by cycloheximide. The changes in TF mRNA occur in a similar time scale and at similar E2 doses as the increases in the uterine proto-oncogenes c-jun and c-fos.

Delineation of sites mediating estrogen regulation of the rat creatine kinase B gene.

We have previously confirmed the estrogen-induced protein of rat uterus to be creatine kinase B (CKB), and demonstrated a 1.7-kilobase pair fragment encompassing the promoter and adjoining 5'-flank to be capable of conferring estrogen responsiveness in HeLa cells. In this study we find an element at -550, aGGTCAgaaCACCCt, with limited similarity to the estrogen response element consensus, to be involved in conferring estrogen responsiveness on the CKB promoter. This element can bind estrogen receptor (ER) and is flanked by two GC boxes, which we find capable of binding bacterially expressed Sp1. Additional responsiveness is found closely associated with the CKB promoter at high levels of cotransfected ER construct. No potential response element was identified in this region, but we find the ER DNA-binding domain to be required.

Estrogen-stimulation of postconfluent cell accumulation and foci formation of human MCF-7 breast cancer cells.

Foci, nodules of cellular overgrowth, that appear after confluence are an in vitro characteristic of malignant transformation. A well-studied in vitro model of estrogen-dependent tumors is the MCF-7 cell line, derived from a pleural metastasis of a human breast adenocarcinoma. We report that cultivation of MCF-7 cells, using routine methods, results in extensive estrogen-stimulated postconfluent cell accumulation characterized by discrete three-dimensional arrays. Side view Nomarski optical sections revealed these to be principally multicellular foci with occasional domes and pseudoacinar vacuoles. This effect on MCF-7 cell growth occurs in media containing fetal bovine serum but not with calf serum or charcoal-dextran-treated fetal bovine serum unless supplemented with estrogens. Foci formation starts 5-6 days after confluence, and the number of foci generated is a function of the concentration of added estrogens. Foci formation is suppressed by the antiestrogens Tamoxifen and LY 156758. Addition of progesterone, testosterone, or dexamethasone had little or no effect, while various estrogens (ethinyl estradiol, diethylstilbestrol, and moxestrol) induced foci development. Clones derived from single cells of the initial MCF-7 population revealed a wide variance in estrogen-induced foci formation, demonstrating heterogeneity of this tumor cell line. The postconfluent cell growth of the estrogen receptor-deficient cell line, MDA-MB-231, contrasted with MCF-7 by developing an extensive multilayer morphology devoid of discrete structures. The tumorigenic potential of the MCF-7 cells used in our experiments was confirmed by their estrogen-dependent growth in immunosuppressed male BDF1 mice. These data suggest an estrogen receptor-based mechanism for the development of multicellular foci during postconfluent growth of MCF-7 cells. After confluence, foci, in contrast to the quiescent surrounding monolayer, retain proliferating cells. Focus formation, therefore, reflects the heterogeneous responsiveness of these cells to estrogens and should provide a model permitting in vitro comparisons between the progenitor cells of multicellular foci and the monolayer population.

2,3,7,8-Tetrachlorodibenzo-p-dioxin causes an extensive alteration of 17 beta-estradiol metabolism in MCF-7 breast tumor cells.

MCF-7 breast tumor cells form multicellular foci in vitro when supplemented with 17 beta-estradiol (E2). In the presence of E2 and the aryl hydrocarbon-receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), MCF-7 cells grow to confluence but do not form foci. To investigate the role of E2 metabolism in this antiestrogenic effect of TCDD, analyses were performed by capillary GC/MS. The results revealed that pretreatment of MCF-7 cultures with TCDD (10 nM) rapidly depletes E2. In untreated cultures supplemented with 10 nM E2, the concentration of free E2 decreased to 4 nM in the first 12 hr, followed by a slower rate of decline. After 3 days most E2 in the medium was in conjugated form(s); 1.7 nM was present as free E2, and 2.9 nM was released by treatment with glucuronidase/sulfatase. In TCDD-treated cultures, E2 declined to 290 pM in 12 hr and after 2 days was not detected (less than 100 pM) either as free steroid or after treatment with glucuronidase/sulfatase. Intracellular E2 and estrone were likewise depleted by pretreatment with TCDD. Microsomes from TCDD-treated cells showed highly elevated aryl hydrocarbon-hydroxylase activity and catalyzed hydroxylations of E2 at C-2, C-4, C-15 alpha, and C-6 alpha with a combined rate of 0.85 nmol/min per nmol of cytochrome P-450 at saturating E2. These results suggest that depletion of E2 by enhanced metabolism accounts for the antiestrogenic activity of TCDD in MCF-7 cells.

Estrogen regulation of creatine kinase-B in the rat uterus.

Creatine kinase-B (CKB) synthesis is rapidly and specifically induced by estrogen in the uterus of the immature rat. This study indicates that this elevation is due at least in part to increases in the levels of mRNA for CKB. The stimulation of CKB mRNA levels is rapid (a 7- to 10-fold increase is detected 1-3 h after estrogen administration), but transient, as levels return to near control values by 6 h. Analysis of cDNAs to both uterine and brain CKB mRNA indicate that the same sequence is expressed in both tissues despite earlier observations of heterogeneity of the protein isolated from the two tissues. A 1.7-kilobasepair DNA fragment containing the CKB promoter and 5' flanking sequences confers estrogen sensitivity on expression of the bacterial chloramphenicol acetyl transferase gene in HeLa cells on cotransfection with an estrogen-receptor expression vector. However, the CKB promoter sequences lack any motif with convincing similarity to the currently accepted consensus estrogen response element GGTCAnnnTGACC.

Prothrombin levels are increased in the estrogen-treated immature rat uterus.

An estrogen-responsive uterine proenzyme of a proteinase in the immature rat uterus has been known for some time. Its mol wt is 77,000, its N-terminal amino acid sequence is the same as prothrombin's for 15 residues, it contains gamma-carboxyl glutamate residues, its biosynthesis is prevented by warfarin, it cross-reacts with antibodies to human and rat prothrombin, and it can be activated by human factor Xa or a uterine procoagulant. The products of activation, when separated on sodium dodecyl sulfate-gels, react with antibodies to human or rat prothrombin to give bands that have mol wt corresponding to those of the products of activation of prothrombin. These activation intermediates hydrolyze synthetic substrates specific for thrombin and have the same mol wt as the activation products of prothrombin. The proteinase generated in the activation has the following properties of thrombin: it is inhibited by hirudin and PheProArg-chloromethyl ketone, it has kinetic constants similar to those of thrombin with tripeptide p-nitroanilides as substrates, and it digests actin to give the same peptides as thrombin. We conclude that the uterine proenzyme is prothrombin. The time course of the prothrombin response to estrogen suggests that prothrombin enters the uterus as part of the transudation of plasma proteins that occurs after estrogen stimulation. A membrane-bound uterine procoagulant that activates uterine prothrombin also increases in response to estrogen stimulation. We propose that the simultaneous increase in these two activities results in a localized generation of thrombin, a well characterized mitogen in fibroblasts and epithelial cells. Our results suggest that thrombin may have a vital function as a mitogen in the early steps of the estrogen-stimulated hypertrophy and hyperplasia of the immature uterus.

Estrogen regulation of a tissue factor-like procoagulant in the immature rat uterus.

An estrogen-responsive procoagulant activity is present in the plasma membrane fraction of immature rat uterus. This procoagulant has many of the properties of tissue factor, a widely occurring, integral membrane protein which initiates the intrinsic pathway of coagulation. Procoagulant activity was demonstrated to activate prothrombin in rat uterus, to activate human coagulation factor X, and to cause clot formation by human plasma. Procoagulant activity could be solubilized from the plasma membrane by the detergent octyl glucoside and had an apparent mol wt of 20,000-40,000 by gel filtration. Procoagulant activity was increased 4-fold within 3 h after immature rats were injected with estradiol. The increase was tissue- and hormone specific and was not affected by a warfarin-induced vitamin K deficiency. Coagulation factor VII was required for clot formation by the procoagulant. These properties are consistent with identification of the procoagulant as tissue factor. mRNA for tissue factor was increased in the uterus 3 h after estrogen stimulation. In the preceding paper we showed that prothrombin is increased in the immature uterus within 3 h of estrogen stimulation. The presence of increased amounts of a tissue factor-like procoagulant in the same time period suggests a functional relationship between these two proteins and a possible role for both in uterine development. Thrombin is a growth factor in fibroblasts and endothelial cells. We propose that after estrogen stimulation, prothrombin enters the uterus with the influx of plasma proteins and is activated by the procoagulant to thrombin. We suggest that thrombin might act as a paracrine factor early in the estrogen-stimulated development of the uterus.

Estrogen regulation of human breast cancer cell line MCF-7 tissue plasminogen activator.

The human breast cancer cell line MCF-7 produces a number of estrogen-regulated proteins, among which is tissue plasminogen activator (tPA). Increased medium concentrations of PA activity were observed after the addition of 17 beta-estradiol to cultures of MCF-7 cells. However, in the current study these hormone-regulated increases are limited to cultures near or at confluence, but not in the preconfluent period. MCF-7 cell cultures produce either tPA activity alone or in combination with urokinase activators. At confluence, a single exposure to 17 beta-estradiol stimulates a marked transitory rise in tPA activity in the extracellular and cell-associated compartments; the peak increases were at 48 h for medium activity and 24-48 h for cell-associated activity. Sustained exposure to hormone leads to a persistent increase in activity in both compartments. Examination of the structure-function relationships of estrogen agonists, steroidal and nonsteroidal, as well as nonestrogenic steroids indicated that stimulation of PA activity was restricted to estrogen agonists. The increased activity was reflected in enhancement of tissue PA activity when viewed using sodium dodecyl sulfate-polyacrylamide gel zymography. Those cultures expressing both activators revealed no alteration of urokinase activity due to hormone addition. Antiestrogens added to MCF-7 cells not rigorously limited in exogenous estrogens selectively suppressed tissue PA activity, but not that of urokinase. These data indicate that at the point when MCF-7 cell cultures are no longer growing exponentially, addition of estrogen agonists at physiological concentrations elevates tPA activity while not altering expression of urokinase activity. The discussion suggests a possible role that this regulation may subserve in the function of breast epithelial cells.

Enhancement of 2- and 16 alpha-estradiol hydroxylation in MCF-7 human breast cancer cells by 2,3,7,8-tetrachlorodibenzo-P-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin exhibits antiestrogenic activity and induces cytochromes P-450 in estrogen-dependent MCF-7 human breast-cancer cells. To determine whether induction of 2- or 16 alpha-hydroxylation of 17 beta-estradiol has a role in this antiestrogenic activity, MCF-7 cells which were exposed to this xenobiotic for 72 hrs were incubated with either [2-3H] or [16 alpha-3H] 17 beta-estradiol and the extent of tritiated H2O formation, indicative of site-specific hydroxylation, was determined. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-treated MCF-7 cultures showed an 8-fold increase in 2-hydroxylation and a 2-fold increase in 16 alpha-hydroxylation. These results support the suggestion that increased hydroxylation of 17 beta-estradiol may have a role in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells.

Estradiol stimulates a uterine plasma membrane protease activator.

An estrogen-regulated arginine esteropeptidase is present in the immature rat uterus. The enzymatic complex consists of a membrane-bound activator and a soluble proenzyme. The activator is under strong estrogen control; its activity increases 10-fold 3 h after a single dose of 17 beta-estradiol. The subcellular localization of the activator is determined by a radioactive assay of fractions prepared by sucrose density centrifugation. The distribution of activity parallels the distribution of two plasma membrane markers, Mg2+-ATPase and 5'-nucleotidase. Electron micrographic visualization of the gradient fractions containing the activator reveals a population of vesicles 0.2-0.5 micron in diameter.

Suppression of estrogen-regulated extracellular tissue plasminogen activator activity of MCF-7 cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) suppresses the estrogen enhancement of tissue plasminogen activator (t-PA) by MCF-7 breast cancer cells. 17 beta-estradiol treatment of MCF-7 cells was previously shown to enhance t-PA secretion in a receptor-mediated process dependent on RNA and protein synthesis. The current studies demonstrate that treatment with TCDD, at a concentration as low as 10(-11) M, reduces the 17 beta-estradiol-induced enhancement of t-PA secretion in these cells. Treatment of MCF-7 cells with TCDD alone does not alter t-PA activity nor was inhibition of t-PA activity observed when TCDD was added directly to the enzyme assay. Kinetic studies and the lack of inhibition following in vitro mixing of conditioned media from TCDD-treated and control 17 beta-estradiol stimulated MCF-7 cells argue against TCDD induction of a plasminogen activator inhibitor. The related polychlorinated dibenzofuran, 2,3,7,8,-tetrachlorodibenzofuran, while also active, is less potent that TCDD. Other polychlorinated dibenzodioxins, polychlorinated dibenzofurans, and polychlorinated biphenyls do not suppress 17 beta-estradiol induction of t-PA over the concentrations tested. These results are in agreement with the structure-activity relationships established using these compounds in other assay systems. Treatment with TCDD does not alter the number or affinity of 17 beta-estradiol receptors of MCF-7 cells. TCDD treatment does not suppress constitutive t-PA activity in the estrogen independent breast cancer line MDA-MB-231 nor the t-PA induced by 12-O-tetradecanoylphorbol-13-acetate in HeLa cells. These effects suggest that TCDD is not acting directly on expression of the t-PA genome. Induction of aryl hydrocarbon hydroxylase by TCDD, a cytochrome P-450 regulated metabolic enzyme for which TCDD is the most potent known inducer, was observed in MCF-7 cells but not in MDA-MB-231 or HeLa cells. A plausible mechanism for the antiestrogenic activity of TCDD is based on the metabolic conversion of 17 beta-estradiol to less active derivatives by TCDD induced cytochrome P-450 metabolic enzymes.

Separation and identification of two components of an estrogen-responsive, calcium-dependent arginine esteropeptidase.

Arginine esteropeptidase is an estrogen-responsive, calcium-dependent enzyme in rat uterine cytosol. It appears in increased amounts 3 h after administration of physiologic amounts of 17 beta-estradiol to an immature female rat. Its reaction was resolved into two parts: a calcium-dependent activation of the enzyme and a calcium-independent hydrolysis of the substrate. The esteropeptidase was separated by DEAE cellulose chromatography into two components. The properties of component A, the activator, are distinct from those of component B, the enzyme, which has the same response to inhibitors as serine proteinases. Both components are subject to estrogen control. Component A is present in significant amounts only after estrogen stimulation. Component B is increased 3-fold after estrogen stimulation. The responses of the two components to inhibitors, their different molecular weights and chromatographic behavior and the pH optima of their reactions distinguish them from each other and from other uterine proteinases previously described.

Estradiol preferentially enhances extracellular tissue plasminogen activators of MCF-7 breast cancer cells.

MCF-7 human breast cancer cells secrete two immunologic types of plasminogen activator, one related to urokinase, the other unrelated. We have now examined whether estrogen stimulation of secreted plasminogen activator activity reflects an increase in one or both types. Examined semiquantitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoretic zymography, the conditioned media of control cells were seen to contain a major activator band (Mr approximately 54,000) immunologically related to urokinase and a barely discernible doublet (Mr approximately 64,000 and Mr approximately 68,000). Addition of estradiol or, at much higher concentrations, testosterone led to marked enhancement of doublet activity, while the 54-kDa band was invariant. The 64-68-kDa doublet was immunoreactive with antiserum directed against Bowes melanoma tissue plasminogen activator but not with antiurokinase antibodies. Enhancement of doublet activity was correlated with hormone-induced increases in total secreted plasminogen activator activity. Neither progesterone nor dexamethasone increased total activity or the 64-68-kDa zones of lysis. Estradiol and testosterone alterations were blocked by appropriate concentrations of an estrogen antagonist (LY156758), actinomycin D, or cycloheximide. Regulation of MCF-7 cell-secreted tissue plasminogen activators thus appears to be mediated by an estrogen receptor process and to require sustained RNA and protein synthesis.

Esterase A is a proteinase from rat urine that can activate plasminogen.

A proteinase which can activate human, dog and rat plasminogen to plasmin has been isolated from the urine of female rats, using affinity chromatography on benzamidine-coupled Sepharose. Inhibition by diisopropylfluorophosphate, tosyl-L-lysine chloromethylketone and benzamidine classified the enzyme as trypsin-like. The proteinase has weak activity on alpha-casein and hemoglobin, but will not lyse fibrin clots. It readily cleaves arginyl amides, including synthetic substrates specific for human glandular kallikrein and other serine proteinases. A chromogenic substrate for human urokinase (pyro Glu-Gly-Arg-pNA) is a poor substrate for the rat proteinase. Characteristics of the enzyme, such as its molecular weight (25 900), kinetic parameters and inhibition by aprotinin, indicate that this proteinase is esterase A, described by several investigators. Esterase A is shown not to be a true urinary plasminogen activator but rather is a unique arginine-specific proteinase. Urokinase-like and kallikrein-like activity are part of a broader proteolytic activity displayed by this enzyme.

An estrogen-stimulated, calcium-dependent tosylarginine methyl ester (TAME) hydrolase in immature rat uterus.

An arginyl esterase, identified by hydrolysis of tosyl-L-arginine-O-methyl ester (TAME), is present in estrogen-stimulated, immature rat uterine cytosol. The esterase is Ca2+-dependent and appears 3-8 h after injection of 4.5 micrograms of 17 beta-estradiol (E2). The Ca2+-independent TAME hydrolase activity in stimulated uteri decreases as the Ca2+-dependent hydrolase activity increases. The TAME hydrolase activity is not increased by dexamethasone or testosterone, nor is it elevated in liver cytosol of the same E2-treated rats. Rat plasma contains a potent Ca2+-dependent TAME hydrolase activity distinguished from the uterine enzyme by its response to high Ca2+ concentrations and its interaction with goat anti-rat serum immunoglobulin G. The uterine TAME hydrolase is apparently a trypsin-like serine protease, as defined by its pattern of inhibition, e.g. response to diisopropylfluorophosphate. The Ca2+-dependent hydrolase kinetics are complex, with an initial lag phase followed by a dramatic acceleration in the rate of hydrolysis. Both the length of the lag phase and the extent of acceleration vary with the amount of cytosol. The metal requirement is specific for Ca2+. The endogenous substrate of this newly recognized, estrogen-stimulated TAME hydrolase and its role in uterine development remain to be elucidated.

Purification and characterization of creatine kinase, an estrogen-induced uterine protein (IP) from immature rats.

An estrogen-responsive translational product, the induced protein (IP) first described by Notides and Gorski (8), was obtained solely from the target organ, immature rat uterus, and purified to homogeneity in a procedure using two chromatography steps. The purified IP has a molecular weight of 49,000, and the isoelectric point is 5.2. Creatine kinase activity is associated with the homogeneous IP. There are some differences between the uterine enzyme and the creatine kinase BB isoenzyme, including differences in stability, and sensitivity to mercaptans. Estrogen-induced creatine kinase purified by this simple, reproducible method is a useful antigen for further studies on the translation and transcription processes involved in hormone-modulated synthesis.

Oligodeoxynucleotide base recognition by steroid hormone receptors.

Oligodeoxynucleotides covalently linked to cellulose were used as probes of the DNA-binding domains of mouse steroid holoreceptors. With uterine cytosol estrogen receptor (E2R) the relative binding order, in prior studies, was oligo(dG) greater than oligo(dT) greater than or equal to oligo(dC) much greater than oligo(dA) greater than oligo(dI). The binding reactions were salt-sensitive with an optimal KCl concentration of 0.1-0.2 M. There was no enhancement of binding by activation, either temperature- or salt-induced. In the present study, using the oligomer ligands at a lower concentration, oligo(dT) binding was greater than that to oligo(dC). Quantitative differences in oligodeoxynucleotide binding were elicited by a number of inhibitors. These differences are again seen by exposure of E2R to chaotropic salts such as SCN-, ClO-4 and NO3- as well as to putative modifiers of receptor amino acids, ie, iodoacetamide, 1,2 cyclohexanedione, and Rose Bengal. These results, and the quantitative differences following heat and purification, led to a designation of two types of subsites within the DNA-binding domain of uterine E2R. These are stable G sites, which interact with oligo(dG); and labile N sites, which bind to oligo(dT), oligo(dC) and oligo(dA). Stimulation of binding to N sites and stabilization of the holoreceptor was effected by histones H2A and H2B. However, the differential response to incubation at 37 degrees C was not altered by addition of H2B. Treatment of uterine E2R by limited proteolysis also eliminated the stimulatory response to H2B. The above data, as well as prior studies, indicate that steroid holoreceptors can discriminate between the structural features of deoxynucleotide bases and this recognition process can be modulated by accessory proteins.

Immobilized oligodeoxynucleotides as probes of the DNA-binding sites of mouse steroid holoreceptors.

Steroid hormone receptors are DNA-binding proteins with distinct domains for the steroid ligand and for polydeoxyribonucleotides. The characteristics of the polynucleotide-binding sites of the mouse liver dexamethasone receptor, kidney testosterone and estradiol receptors, and uterine estradiol receptor were examined by using oligodeoxynucleotides covalently linked to cellulose as probes. Only dexamethasone receptor binding to oligodeoxynucleotides was dependent upon activation by increased salt concentrations or temperature. The dexamethasone and estradiol receptors required a distinct concentration of KCl for maximal binding to oligodeoxynucleotide-cellulose. In contrast, binding of mouse kidney testosterone receptor was not stimulated by KCl although it was decreased at 0.2 M KCl. The nucleotide affinities of the mouse receptors generally followed the order of binding to oligo(dG)- greater than oligo(dT)- greater than oligo(dC)- greater than oligo(dA)-cellulose. However, the liver dexamethasone receptor bound to oligo(dT)- more avidly than to oligo(dC)-cellulose, while neither the testosterone nor the estradiol receptors clearly differentiated between the oligodeoxypyrimidine celluloses. Although the binding of the kidney testosterone receptor was poorest of those studied, it bound to oligo(dA)-cellulose relatively more efficiently than did the other steroid receptors. Thus, differences were revealed in the requirements for activation, added salt for binding, and relative binding efficiencies to the ligands among different steroid receptors from the same tissue and among the same steroid receptors from different tissues.