Estrogen regulation of an eosinophil chemotactic factor in the immature rat uterus

Associated with the generalized uterine growth stimulated by estradiol in the rat are specific responses including messenger RNA (mRNA) synthesis, protein synthesis, and peroxidase activity. The increase in peroxidase activity, although sensitive to inhibitors of RNA and protein synthesis, results from an estradiol-stimulated influx of eosinophils into the uterus. We postulated the existence of an estradiol-regulated uterine chemotactic factor, testing this by an in vitro chemotactic assay with eosinophils isolated from mature rats. Treatment of immature rats with 1 microgram estradiol for 24 h resulted in a significant increase in eosinophil chemotaxis compared to uterine extracts of vehicle-treated rats. This increase was seen as early as 3 h after estradiol administration and was maximal at 24 h. The magnitude of the chemotactic response was dependent on the dose of estradiol administered (1-100 micrograms). Estrone or estriol treatment resulted in chemotactic activity greater than control but less than estradiol. Direct addition of estradiol to extracts of control animals did not increase chemotaxis. The estradiol-stimulated chemotaxis was blocked by in vivo treatment with the antiestrogen tamoxifen and by inhibitors of RNA and protein synthesis. Analysis of extracts from estradiol-treated uteri shows that the chemotactic factor is heat labile, pronase sensitive, and has a mass of approximately 20 kilodaltons (kDa). These data suggest that the estradiol-stimulated influx of eosinophils into the rat uterus is mediated by the synthesis, modification, or release of a protein whose synthesis is estradiol receptor mediated.     

Estrogen stimulation of DNA-dependent DNA polymerase activity in immature rat uterus

DNA-dependent DNA polymerase activity in the immature rat uterus has been examined in terms of molecular species present and their response to estrogen treatment. The molecular species of DNA polymerase were characterized by DEAE-cellulose chromatography, sucrose density gradient analysis, sulfhydryl reagent sensitivity and optimal assay conditions. DNA polymerase activity was found predominantly in the high-speed cytosol but was also present in 0.4 M KCl nuclear extract preparation. DNA polymerase β activity was only observed in nuclear extracts. Estradiol treatment resulted in a dose-dependent, estrógen-specific increase in DNA polymerase activity, principally due to increases in cytoplasmic DNA polymerase activity. A 3–4-fold maximal increase of DNA polymerase activity occurred 24–30 h after a single hormone treatment in close correspondence to hormone-induced increased DNA synthesis. When hormone treatment was sustained by daily injections of estradiol, the DNA polymerase response became refractory to continued stimulation after two days.     

Estrogen regulation of protein synthesis in the immature rat uterus: the analysis of proteins released into the medium during in vitro incubation

Immature rats were treated with estradiol (E2) or other steroids before their uteri were removed and incubated under in vitro conditions in the presence of [35S]methionine. The analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the radiolabeled proteins synthesized and released into the incubation medium demonstrate that E2 regulates the appearance of two proteins. These two proteins have mol wt of 115,000 and 65,000. The concentration of proteins in the medium increases linearly with time, suggesting that they may be secreted. These two proteins were not produced by several other tissues in response to E2 and appear to be specific to the uterus. They also appear to be increased only by estrogens (E2 greater than estrone greater than estriol) and not by other steroids tested. They are increased in response to a single injection within 6 h, and the maximal concentration of proteins occurs approximately 24 h after E2 administration. The protein concentrations have essentially returned to control values by 72 h after hormone injection. The kinetics of the induction is the same for both proteins, suggesting that their increase may be coordinated. Based on the tissue and hormone specificity of the increase in the 115,000- and the 65,000-dalton proteins, they may serve as reliable markers for the study of the uterine response to E2.     

Estrogen regulation of protein synthesis in the immature rat uterus: the effects of progesterone on proteins released into the medium during in vitro incubations

We have previously identified two major medium proteins secreted from the rat uterus during in vitro incubations that appear to be estrogen regulated. In this study, immature rats were treated with estradiol (E2) progestins, and actinomycin D. Medium proteins were analyzed after incubation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. E2 (1 microgram) increased the synthesis of proteins with mol wt of 115,000 and 65,000. Progesterone inhibited this increase when given in doses of 500 and 250 micrograms and when given within 8 h of estradiol. Lower doses of progesterone were not completely inhibitory. When actinomycin D was given within 6 h of E2, it also inhibited the E2 stimulated increase. This system may provide a useful marker for monitoring hormonal action in the luminal epithelium and may help in understanding hormonal regulation of gene expression.     

Estrogen regulation of alpha 1(I)-procollagen messenger ribonucleic acid in the rat uterus

A cDNA library, prepared from mRNA isolated from the uteri of 3-day estradiol-stimulated immature rats, was constructed in pBR322. From this library an estrogen-regulated clone, pERU3, was isolated. This clone contained sequences complementary to uterine mRNA that migrated during gel electrophoresis as a double band of about 5.0 and 5.8 kilobases. Little of this mRNA was seen in several other tissues examined. An increase in the amount of this RNA in uterus was seen 2 h after estradiol treatment, with maximum hybridization occurring, in different experiments, between 18 and 36 h, followed by a decline. Hybridization of the cDNA insert of the pERU3 plasmid with known probes indicated that it coded for alpha 1(I)-procollagen. This conclusion was supported by in vitro translation experiments in which the hybrid-selected mRNA complementary to pERU3 DNA was shown to code for a collagenase-sensitive protein with a size corresponding to that of alpha 1(I)-procollagen. This system, therefore, provides an additional tool for the study of the estrogen regulation of gene expression in the uterus.     

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 induces CCN5 expression in the rat uterus in vivo

Estrogen plays an important role in the normal physiology as well as various pathologies of the uterus. Given the nature of uterine remodeling during the reproductive cycle and pregnancy, we sought to determine whether CCN5, a gene that we have shown to be important in smooth muscle cell proliferation and migration, is an estrogen-induced gene in the uterus. In the present study, we demonstrate that levels of CCN5 mRNA and protein expression were 5-fold higher in uteri from proestrous females relative to metestrous females, a finding consistent with estrogen induction of the CCN5 gene. Ovariectomized rats treated with exogenous estrogen or estrogen and progesterone exhibited 4- to 8-fold higher levels of CCN5 mRNA and protein than animals treated with either progesterone or vehicle alone. Analysis of rat uterine sections using immunohistochemistry demonstrates CCN5 localization throughout the uterus, including the endometrium and endometrial glands as well as the myometrium. Thus, our data indicate that CCN5 is positively regulated by estrogen in the rat uterus and suggests that this gene may play an important role in maintaining normal uterine physiology.     

Estrogen and androgen regulation of protein synthesis by the immature rabbit epididymis

To obtain evidence of a physiological role for androgens and estrogens in the regulation of the epididymis of sexually immature rabbits, the effects of these hormones on [35S] methionine incorporation into epididymal proteins in vitro were examined. Two-dimensional polyacrylamide gel electrophoresis revealed that short term incubation with estradiol changed the patterns of radiolabeled proteins detected in tissue homogenates of epididymal segments from castrated rabbits compared to those in segments from castrated rabbits that were not exposed to exogenous estradiol. Most of the changes seen in corpus tissue affected proteins with a wide range of pI values and relatively high mol wt (greater than 40K). The effects on caput and cauda tissue proteins were seen over a wide pH and mol wt range. Castration abolished many of the regional differences in protein synthesis; these were restored by incubation with estradiol. Testosterone had little effect on the synthesis of tissue proteins, except for stimulation of the synthesis of a single protein (17K; pI 5.1) in all three segments and stimulation of a small group of proteins (less than 14K; pI 7.0-7.2) in the corpus. Estradiol had little effect on proteins secreted by epididymal segments. Testosterone, however, stimulated the synthesis of a number of unique proteins secreted by the caput and corpus and resulted in a pattern of radiolabeled proteins similar to that obtained with intact animals. Additional secretory proteins could be stimulated in caput, but not corpus, tissue minces from intact rabbits by exogenous testosterone. No androgen-specific synthesis of secretory proteins was detected in the cauda of either castrated or intact rabbits. Estradiol affected the synthesis of both secreted and tissue proteins in terms of influencing which epididymal segment was most active at incorporating [35S]methionine into radiolabeled proteins and which was least active. Testosterone had a similar influence on secreted proteins, but did not have any analogous effect on tissue proteins. These results indicate that testosterone and estradiol influence the synthesis of proteins by the immature rabbit epididymis and that both may, therefore, be important physiological regulators of epididymal development and/or function.     

Hemostasis and cancer: tumor cells induce the expression of tissue factor-like procoagulant activity on endothelial cells

BACKGROUND AND OBJECTIVES: Clotting activation and thromboembolic manifestations are common features in patients with cancer. The two-way interaction between tumor cells and host cells is of crucial importance in this context. In the present study we investigated the effect of tumor cell-endothelial cell co-culture on the expression of procoagulant activity in the mixed cell populations. DESIGN AND METHODS: Human tumor cell lines (HL60 promyelocytic leukemia and HeLa uterine cervical cancer) and human umbilical vein endothelial cells (HUVEC) were cultured in vitro according to standard procedures. Procoagulant activity was studied in a coagulometer and was found to be tissue factor-like. A calibration curve was obtained with decreasing concentrations of rabbit brain thromboplastin (RBT) and the procoagulant activity of both tumor cells and HUVEC was expressed as RBT U/10(5) cells. RESULTS: Before incubation procoagulant activity (means S.E.) was found to be 0.18 +/- 0.04 U in HUVEC, 9.8 +/- 1.9 U in HL60 cells, 11.9 +/- 2.2 U in HeLa cells, 7.2 +/- 1.4 U in a mixed HL60 cell-HUVEC population (ratio 2:1) and 8.5 +/- 2.0 in a mixed HeLa cell-HUVEC population (ratio 2:1). Incubation at 37 degrees C for up to 4 hours of tumor cells or HUVEC alone did not produce any change in procoagulant activity. In contrast, co-incubation of tumor cells with HUVEC for 4 hours was followed by a significant increase in procoagulant activity of the mixed cell populations. Addition of supernatants from tumor cells, HUVEC or tumor cell-HUVEC co-cultures to HUVEC or tumor cells showed that the tissue factor-like procoagulant activity generated during coincubation was localized on HUVEC. INTERPRETATION AND CONCLUSIONS: Our results show that the close interaction of tumor cells with endothelial cells may induce surface expression of tissue factor in the latter. This effect could represent an additional mechanism of clotting activation in patients with cancer.     

Estrogen target gene regulation and coactivator expression in rat uterus after developmental exposure to the ultraviolet filter 4-methylbenzylidene camphor

Because the estrogen receptor (ER) ligand type influences transactivation, it is important to obtain information on molecular actions of nonclassical ER agonists. UV filters from cosmetics represent new classes of endocrine active chemicals, including the preferential ER beta ligands 4-methylbenzylidene camphor (4-MBC) and 3-benzylidene camphor. We studied estrogen target gene expression in uterus of Long Evans rats after developmental exposure to 4-MBC (0.7, 7, 24, and 47 mg/kg x d) administered in feed to the parent generation before mating, during pregnancy and lactation, and to the offspring until adulthood. 4-MBC altered steady-state levels of mRNAs encoding for ER alpha, ER beta, progesterone receptor (PR), IGF-I, androgen receptor, determined by real-time RT-PCR in uterus of 12-wk-old offspring. Western-blot analyses of the same tissue homogenates indicated changes in ER alpha and PR but not ER beta proteins. To assess sensitivity to estradiol (E2), offspring were ovariectomized on d 70, injected with E2 (10 or 50 microg/kg sc) on d 84, and killed 6 h later. Acute up-regulation of PR and IGF-I and down-regulation of ER alpha and androgen receptor by E2 were dose-dependently reduced in 4-MBC-exposed rats. The reduced response to E2 was accompanied by reduced coactivator SRC-1 mRNA and protein levels. Our data indicate that developmental exposure to 4-MBC affects the regulation of estrogen target genes and the expression of nuclear receptor coregulators in uterus at mRNA and protein levels.     

Epidermal growth factor influenced by opioid peptides in immature rat uterus

The aim of the present experiment was to investigate the effect of [D-Met2,Pro5] enkephalinamide (ENK) implantation on the development of the uterus during 8-33 days of age and the involvement of epidermal growth factor (EGF) in the effect. Administration of ENK was attained by osmotic minipumps (5 microg/h) implanted intraperitoneally. ENK resulted in a decrease in the EGF content of the uterus, which was already significant after 48 h of the implantation. The DNA content 24 and 48 h after the treatment decreased, no change at 72 h was found, however the protein/DNA ratio on the effect of ENK treatment was significantly decreased at this time in all examined age groups. High affinity and lower capacity competitive naloxone binding sites were demonstrated in the membrane fraction of the uteri. Seventy-two h after ENK treatment the binding capacity of these sites significantly dropped. The present results suggest a novel multiple interaction between estrogen and two probably paracrine hormones, EGF and opioid peptide, in the regulation of growth and development of the uterus.     

Estrogen regulation of rat anterior pituitary adenylate cyclase

The effect of chronic estrogen treatment on the stimulation and dopamine inhibition of anterior pituitary (AP) adenylate cyclase (AC) activity was examined. Treatment of ovariectomized female rats with estradiol for 21 days resulted in a 450% increase in AP weight compared to ovariectomized controls. Stimulation of AC by guanine nucleotides (GN) (1 nM-0.1 mM) and vasoactive intestinal peptide (1 microM) was reduced by 50%. Stimulation of AC by fluoride ions was unchanged by estradiol treatment. Stimulation above basal by forskolin was reduced by variable amounts (23-50%), and depended on the concentration of forskolin used. Inhibition of AC mediated by D2-dopamine receptors was decreased by 45%. Estrogen treatment had no effect on the toxin-catalyzed incorporation of [32P]ADP into stimulatory and inhibitory GN regulatory proteins. These results indicate that the effect of estrogen on the anterior pituitary include modulation of stimulated, dopamine-inhibited and basal AC activity.     

Estrogen regulation of kallikrein gene expression in the rat anterior pituitary

Using a rat pancreatic kallikrein cDNA probe (pcXP39), previously shown to hybridize to kallikrein mRNA in a variety of tissues, we have explored the control of kallikrein gene expression in rat anterior pituitary. Intact female rats have substantially higher levels of AP kallikrein mRNA than intact males; male levels are unaffected by castration, whereas female levels fall markedly postovariectomy. Administration of estradiol benzoate to intact male or ovariectomized female rats causes an increase in anterior pituitary levels of kallikrein mRNA. Since the pattern of responsiveness parallels that of PRL, we have studied GH3 cells grown in the presence and absence of estradiol; in neither instance was kallikrein mRNA above detection limits. Parallel changes were seen on Northern blots and by hybridization histochemistry; on emulsion autoradiography of pituitary sections, scattered positive cells were seen, but precise definition was not possible. We conclude that whereas in the submaxillary gland kallikrein gene expression appears androgen dependent and in the kidney is postulated to be mineralocorticoid regulated, in the anterior pituitary expression of the gene is under estrogen control; and that the local role(s) of pituitary kallikrein, whether precursor processing, control of blood flow, or other effects, would, in turn, appear to be modulated by estrogen in vivo.     

Expression of tissue factor procoagulant activity: regulation by cytosolic calcium.

Intact bovine fibroblasts, pericytes, and kidney cells manifested significantly less tissue factor procoagulant activity than their disrupted counterparts. Addition of calcium ionophore A23187 rapidly and reversibly enhanced the cell-surface expression of tissue factor in intact cells up to the level achieved by disruption. Inhibitors of calmodulin blocked the ionophore-dependent enhancement of procoagulant activity. Similar kinetic parameters were obtained for factor X hydrolysis by tissue factor-factor VIIa on unperturbed pericytes and phosphatidylcholine vesicles. Increase in Vmax and decrease in apparent Km for this reaction were seen after either disruption or ionophore stimulation of the pericytes. Addition of phosphatidylserine to the reconstituted phospholipid vesicles also increased the Vmax and decreased the apparent Km for factor X hydrolysis. These data agree with the hypothesis that the expression of tissue factor procoagulant activity on cell surfaces is modulated by calcium-mediated changes in the asymmetric distribution of phosphatidylserine in plasma membrane.