Steroid hormone receptors in target cell membranes.

Numerous reports of rapid steroid hormone effects in diverse cell types cannot be explained by the generally prevailing theory that centers on the activity of hormone receptors located exclusively in the nucleus. Cell membrane forms of steroid hormone receptors coupled to intracellular signaling pathways may also play an important role in hormone action. Membrane-initiated signals appear to be the primary response of the target cell to steroid hormones and may be prerequisite to subsequent genomic activation. Recent dramatic advances in this area have intensified efforts to delineate the nature and biologic roles of all receptor molecules that function in steroid hormone-signaling pathways. This work has profound implications for our understanding of the physiology and pathophysiology of hormone actions in responsive cells and may lead to development of novel approaches for the treatment of many cell proliferative, metabolic, inflammatory, reproductive, cardiovascular, and neurologic defects.

Microtubule and plasmalemmal reorganization: acute response to estrogen.

The acute ultrastructural effects of estrogen in endometrial epithelial cells were investigated by transmission electron microscopy (TEM), with special reference to the microtubule (MT) apparatus and the luminal surface. Ovariectomized rats anesthetized with pentobarbitol sodium were injected intravenously with estradiol-17 beta (E2 beta), 0.5 micrograms/100 g body wt. At intervals from approximately 30 s to 30 min thereafter, 70-80 nm cross sections of a uterine horn were prepared for TEM. In placebo controls, cytoplasmic MT were conspicuous in length and number, whereas only a minimal population of short microvilli (MV) was evident. In contrast, the specimens subjected to E2 beta for only 35 s showed a significant decrease in MT number and length, with virtually complete depletion of these organelles by approximately 80 s. Concomitantly, the luminal MV exhibited striking enhancement in length and density. Thereafter, these rapid and reciprocal alterations of MT and MV underwent inversion. Thus MT structures began to reappear within 2 min, increasing progressively so that by 30 min their numbers were again substantial, although lengths remained diminished. During the same interval, the initial surge of luminal MV gradually subsided, to near-control appearance by 30 min. These coordinate, reciprocal, and biphasic responses are consistent with biochemical evidences of abrupt membrane perturbation associated with interception of estrogen at its cellular targets. The resultant modification of the intracellular environment may contribute to limited reorganization of cellular architecture and propagation of the hormonal signal.

Monoclonal antibody toward lysosomal cathepsin B cross-reacts preferentially with distinct histone classes.

1. A set of monoclonal antibodies (Mab) was prepared against cathepsin B (CB) from rat preputial-gland, an organ characterized by rapidly-renewing cell populations, which is a uniquely enriched source of lysosomal enzymes, including CB. Minute amounts of CB are known to be transferred abruptly to the nuclear compartment in a variety of activated cells. 2. Since, on the basis of its stringent substrate requirements, CB was expected to function at limited protein loci in chromatin, Mab Line II-B4 was used to probe Western blots of chromatin fractions and selected proteins. 3. The Mab, which was not directed against the active site of CB, cross-reacted preferentially with histones 3 and 4 (H3 and H4) in acid-soluble fractions of chromatin from rat preputial-gland. Line II-B4 also recognized H3 and H4 selectively in calf thymus histones and among histones purified from a wide range of sources from yeast to man. HMG 1 was minimally immunoreactive among preputial gland constituents and carbonic anhydrase (CA) was also sensitive to the Mab. 4. The common determinants were not shared by any of the H1 series, nor by H2A, H2B, protein A24 or a wide range of natural and synthetic products. 5. Origin of the antigenicity was traced by chemical modifications of H3, H4 and CA to the critical contribution of arginine and hydrophobic amino acid residues in its immediate environment, indicating that Line II-B4 may be directed against an epitope comprising the specific binding-site of CB and its selective substrate(s). 6. These data suggest that certain highly conserved cellular constituents may be uniquely vulnerable to limited proteolysis in preproliferative cells responding to mitotic signals.

Mechanisms of hormone action: parallels in receptor-mediated signal propagation for steroid and peptide effectors.

The purpose of this contribution is to provide in brief form growing evidence in support of an integrated concept of hormone action that appears to shed fresh light on the information gap between the triggering and the effectuation of outcome of the action of given hormones. In accord with these new concepts there has now arisen a substantial body of data from a wide variety of effectors and target cells that demonstrates an astonishing unity in the actions of hormones of widely dissimilar chemical structure. In a nutshell, it now appears that primary recognition sites for both peptide and steroidal agonists occur at the outer cell surface. For steroid hormones, as exemplified by estradiol-17 beta, these sites possess several of the hallmarks of true receptors. Moreover, capture of this ligand is associated with unmistakable signs of membrane perturbation. And at a still very early stage in the signal propagation sequence, activation of a very limited fraction of the cellular lysosomal population may be identified following the application of steroid, as well as peptide, hormones. In turn, there is mounting evidence for cellular entry and even lysosomal uptake of peptidal effectors, the significance of which is still under debate. Likewise, there occur clear signs of limited reorganization of components of the cellular architecture at the surface, in the cytoplasm, and in the nucleus and its subcompartments, which are consistent with minimal recompartmentation of 'microquanta' of lysosomal constituents. These observations may be made within seconds to minutes following application of tropic hormone of either class to its selective targets, and thus, at times preceding those relatively more distal responses of augmented transcriptional and translational activities.

Specific internalization of estrogen and binding to nuclear matrix in isolated uterine cells.

Fractionation of rat uterine cells incubated at 22 degrees C with 0.2 nM [3H]-estradiol-17 beta (E2 beta) was performed to analyze the subcellular distribution of internalized hormone. The postnuclear supernatant of homogenates was resolved in Percoll density gradients into six major fractions defined by enzyme markers. Within 10 s, E2 beta concentrates at the density of plasma membranes and also at a more buoyant density (p = 1.052 +/- 0.001) with peak accumulation of hormone by 2 min. Thereafter, binding in the latter fraction declines concomitantly with appearance of a portion of hormone at higher densities corresponding to Golgi and lysosomes. E2 beta exhibits preferential accumulation in nuclear matrix from 5 to 60 min. Microfiltration and scanning electron microscopy of the buoyant 2-min peak fractions reveal organelles, 50-200 nm. These may represent endocytotic vesicles that serve as vehicles for nuclear transfer of hormone.

Chromatin proteins of rat preputial-gland: acute changes in response to estrogen.

The effects of estradiol-17 beta (E2 beta) at 2 or 15 min in vivo on chromatin proteins of rat preputial-gland were analyzed by a battery of electrophoretic methods. Among histones, E2 beta/control ratios for major bands of H1 decreased substantially between 2 and 15 min. In contrast, ratios of H4 increased (P less than 0.01), whereas, except for losses by 2 min in a H2B-like component and in H3.1, other core histones were unchanged. Among 0.35 M NaCl-soluble proteins, components at 34K-mol. wt and less than 21K -mol wt were increased after 2 min of E2 beta. The bulk of the hormone-responsive low-molecular weight proteins was basic in charge. Electrophoretic correlates of 6 basic lysosomal proteins corresponded to those of low-molecular weight salt-soluble chromatin proteins. Selective proteolysis initiated in vivo by E2 beta depleted some tightly-bound nonhistone proteins.

Estrogen action at endometrial membranes: alterations in luminal surface detectable within seconds.

The morphological effects of estrogen on the luminal surfaces of rat endometrial cells were investigated by scanning electron microscopy. Ovariectomized rats were injected intravenously with estradiol-17 beta (E2 beta), 0.5 micrograms/0.25 ml per 100 g body wt. At various intervals thereafter, the lumen of a uterine horn was flushed with buffered 2% glutaraldehyde and then prepared for scanning electron microscopy by conventional methods. In control rats that had received an equivalent volume of placebo vehicle, the luminal cell surface was characterized by short, sparse microvilli (MV) and, in most cells, a single, central cilium. At 30 s after E2 beta injection, the number of MV was significantly increased. By 1 min, MV density was further increased and MV were frequently clustered; also, the central cilium of many cells was no longer evident. Similar results were obtained after exposure to diethylstilbestrol for 30 s to 1 min, whereas neither a subthreshold dose of E2 beta nor a dose of the relatively inactive congener E2 alpha equivalent to a saturating concentration of E2 beta gave statistically significant responses in surface changes by the present criteria. After 3-7 min of E2 beta exposure, MV had increased greatly in length and density. These effects underwent dramatic regression by 15-30 min after E2 beta treatment, with distinct diminution of microvillar lengths and numbers, reduction of clustering, and reappearance of the central cilium in many cells. This was succeeded at 1 h by a renewed surge of surface activity. These results are consistent with cumulative evidence for rapid alterations of the surface membrane of estrogen-sensitive cells in response to physiological levels of active hormone. Whether these responses in the luminal surfaces are primary, or are secondary reflections of receptor-mediated membrane alterations at the basolateral blood-front, remains to be determined.

Early actions of parathyroid hormone and 1,25-dihydroxycholecalciferol on isolated epithelial cells from rat intestine: I. Limited lysosomal enzyme release and calcium uptake.

Epithelial cells isolated from rat intestine were analyzed for their responsiveness in vitro to parathyroid hormone (PTH) and to 1,25-dihydroxycholecalciferol [1,25-(OH)2D3]. Criteria included determination of whether the agonists promoted extracellular liberation of lysosomal enzyme activities above control values during incubation in Ringer's solution at 22 C. PTH-augmented release of the representative hydrolase activities, cathepsin B and acid phosphatase, to the particle-free supernatant fraction of the medium was evident within 5 min of hormone treatment and was sustained in statistically significant degree for at least 30 min to greater than 20% above control levels. Basal release rarely exceeded 15% of the total cellular content of these enzymic activities. Lactate and succinate dehydrogenase activities were undetectable in the particle-free supernatant fraction under conditions of maximal hormone effect, indicating integrity of the cells and selectivity of the organellar response. Treatment of corresponding cells with 1,25-(OH)2D3 resulted in similar time of onset, magnitude, and duration of response. The most sensitive indicator of limited lysosomal labilization by either hormone was beta-N-acetyl-D-glucosaminidase activity, which underwent accentuated extracellular liberation in the presence of as little as 10(-16) M PTH or 10(-11) M 1,25-(OH)2D3, the latter eliciting a response of greater than 40% above control levels. Parathyroidectomy diminished basal release of the hydrolase activities and sensitized the intestinal cells to the action of PTH vs. preparations from intact or sham-operated animals, as judged by excess liberation of the glycosidase. Nontarget lung cells failed to respond to supramaximal levels of either hormone by the criterion of reduced latency of lysosomal hydrolases. In additional acute experiments with intestinal cells, both PTH and 1,25-(OH)2D3 promoted enhanced 45Ca2+ accumulation above control values. Collectively, these data indicate that PTH is capable of provoking direct effects on intestinal cells, similar in onset and extent to those elicited by 1,25-(OH)2D3.

Antibodies against estradiol-binding lysosomal lipoproteins gain access to the nuclear compartment of preputial gland cells under estrogen influence.

The influence of estrogen in provoking nuclear recompartmentation of lysosomal components in hormone-sensitive cells was investigated by immunological analyses of isolated nuclei from the preputial glands of ovariectomized rats. Fixed smears were prepared from ultrapurified nuclei freed of outer membrane, 2-30 min after iv injection of placebo-control solution or submicrogram amounts of estradiol-17 beta. Cytoplasmic contamination was negligible in such preparations, as monitored by vital staining with acridine orange. On challenge with immunoglobulin G directed toward a group of lysosomal high density lipoproteins which have been shown to bind estradiol-17 beta specifically, control preparations exhibited minimal indirect immunofluorescence that was essentially confined to the nuclear periphery. In contrast, a high proportion of the nuclei exposed for as little as 2 min to estradiol-17 beta but not to the relatively inert 17 alpha-congener, displayed generally more intense immunofluorescence which was distributed over the entire organellar area. Thus, the immunoglobulin becomes accessible to the nuclear interior in vitro as a function of pretreatment in vivo with active hormone. Nuclei from estrogen-pretreated rats were more structurally labile than corresponding controls, as judged by morphological criteria, even when isolated by gentle teasing rather than subjection to the rigorous ultrapurification process. By either method, integrity of the specimens was enhanced somewhat when they were prepared from rats ovariectomized before experiencing even a single estrous cycle. The observations verify and extend independent biochemical and ultrastructural evidence that structural labilization of cellular organelles and enhanced accessibility of limited amounts of lysosomal constituents to the nuclear compartment of specific target cells are early correlates of estrogen action.

Lysosomal cathepsin B-like activity: mobilization in prereplicative and neoplastic epithelial cells.

Extracellular release of acid thiol proteinase activity by prereplicative and neoplastic epithelial cells was studied in serum-free, chemically defined media (CDM) in vitro. Cells isolated from urinary bladder of male bullfrogs and endometrium of ovariectomized rats each showed preferential secretion of cathepsin B-like (CB) activity within 30 min after exposure to carcinogenic nitrosamines (5 X 10(-4) M) or to mitogenic estrogen 1 X 10(-9) M), respectively. In contrast, release of such proteinase, and stimulation of cell proliferation were far less extensive in rat preputial gland cells treated with estradiol-17 beta. Striking secretion of CB was characteristic of neoplastic, but not noncancerous, epithelial cells from human ectocervix. Neoplastic cells with divergent rates of cell-to-cell aggregation were separated by a filtration method. Those cells with high rates of intercellular aggregation also exhibited higher rates of cell proliferation in CDM, as well as in soft gels, and a greater level of CB release than corresponding cancer cells with a relatively low degree of intercellular adhesion. Brief treatment of neoplastic cervical epithelial cells with liposomes containing entrapped leupeptin, a potent inhibitor of CB activity, elicited a sharp reduction in both cellular thiol proteinase activity and cell growth as compared to appropriate controls. These data indicate that mobilization of lysosomal CB activity in prereplicative and malignant cells may play a significant role in the promotion of cell proliferation.

Partial purification and characterization of oestrogen receptors in subfractions of hepatocyte plasma membranes.

To assess the subcellular distribution of oestrogen-binding components in their native state, plasma membrane and other cell fractions were prepared from hepatocytes in the absence of [(3)H]oestradiol-17beta. Cells from livers of ovariectomized rats were disrupted, with submaximal homogenization in buffered isotonic sucrose with CaCl(2) and proteinase inhibitor, and fractionated by using isotonic media. Fractions were characterized by determinations of enzyme activities, biochemical constituents and ligand binding. Specific binding of 2nm-[(3)H]oestradiol-17beta to intact cells and their fractions was detemined after equilibration for 1.5h at 4 degrees C. More than 92% of the radioactivity from representative preparations was verified as authentic oestradiol by thin-layer chromatography. Activities of plasma-membrane marker enzymes as well as binding sites for oestrogen and for wheat germ agglutinin were present principally in particulate fractions, rather than in 105000g-supernatant fractions. However, by using alternative homogenization procedures (i.e. hypotonic media), known to fragment and strip structural components, oestradiol-binding sites and activities of plasma-membrane marker enzymes were distributed predominantly into cytosol. By using the more conservative procedures, plasma membranes of low (rho=1.13-1.16) and high (rho=1.16-1.18) density were purified from crude nuclear fractions. A second low-density subfraction of plasma membrane was prepared from microsome-rich fractions. Activities of plasma-membrane marker enzymes were enriched to about 28 and four times that of the homogenate in plasma membranes of low and high density respectively. Binding sites for wheat germ agglutinin and oestradiol were concentrated in low-density plasma membranes to 46-63 times that of the homogenate. Specific binding of oestrogen in low-density plasma membranes purified from crude nuclei was saturable, with an apparent association constant of 3.5nm. At saturation, such oestradiol receptors corresponded to 526fmol/mg of membrane protein. A Hill plot showed a moderate degree of positive co-operativity in the interaction of hormone with plasma membranes. Specific binding of [(3)H]oestradiol-17beta was reduced by a 200-fold molar excess of unlabelled oestradiol-17beta, oestriol or diethylstilbestrol, but not by oestradiol-17alpha, cortisol, testosterone or progesterone. Binding was also blocked by prior exposure of membranes to trypsin or to 60 degrees C, but remained essentially undiminished by extraction of membranes with either hypotonic or high-salt buffers. Extraction with 0.1% (v/v) Triton X-100 partially solubilized the oestrogen-binding component(s) of plasma membranes. Particle-free extracts were resolved on 5-20% (w/v) sucrose density gradients with either 0.01m- or 0.4m-KCl, and the fractions were analysed by adsorption to hydroxyapatite. In low-salt gradients macromolecule-bound oestrogen sedimented at predominantly 7.4S and binding was 1560 times that of the homogenate. Under high-salt conditions oestradiol-binding activity occurred at both 3.6S and 4.9S.

Luteinizing hormone-accelerated redistribution of lysosome-like organelles preceding dissolution of the nuclear envelope in rat oocytes maturing in vitro.

Maturation of the mammalian oocyte is characterized in part by dissolution of the nuclear envelope, or germinal vesicle breakdown (GVB). By fluorescence microscopy after vital uptake of acridine orange (AO), redistribution and perinuclear accumulation of organelles corresponding to lysosomes occur before GVB in rat oocytes undergoing meiotic maturation in vitro. In follicle-enclosed oocytes explanted during the preovulatory gonadotropin surge (GS) and individually cultured as such in chemically defined medium at approximately 22 degrees C, lysosomes aggregated into disperse clusters after 30 min; by 60 min, perinuclear concentration of lysosomes and their essential disappearance from the cortical ooplasm were observed. GVB occurred within 120 min. In contrast, follicle-enclosed oocytes explanted before the GS displayed a generally homogeneous distribution of lysosomes and intact GV for up to 5 h in culture. In oocytes aspirated from follicles before the GS, partially denuded of granulosa cells, and cultivated without added hormone, most lysosomes concentrated around the GV within 60 min, with GVB occurring generally by 120 min. Luteinizing hormone (LH) added in vitro to the isolated preparation at 3 or 30 x 10(-8) M sharply accelerated these events. The effects of LH, not seen with 1.5 x 10(-8) M hormone, were blocked by anti-LH IgG. Up to 60 x 10(-8) M follicle-stimulating hormone or 80 x 10(-8) M prolactin were ineffective in accelerating lysosome redistribution or GVB. After GVB, lysosomes became once again uniformly dispersed and unresponsive, even to 60 x 10(-8) M added LH, a finding consistent with tachyphylaxis of target cells by independent criteria. The present data, all statistically significant at P less than 0.05, demonstrate that mobilization of lysosomes before GVB is a specific response to factors that promote resumption of meiotic maturation of rat oocytes.

Estrogen-induced membrane alterations and growth associated with proteinase activity in endometrial cells.

Endometrial cells isolated from uteri of ovariectomized rats were treated in vitro with 1 X 10(-9) M estradiol-17 beta (E2beta) to analyze early changes in membrane properties during hormone-induced growth. After 30-min exposure to E2beta at 22 degrees C, cells exhibited an enhanced capacity to bind erythrocytes (hemadsorption) in the presence of concanavalin A (Con A) to 237% of the level in paired controls. Fluorescence microscopy revealted that approximately 25% of cells exposed to E2beta, but not estradiol-17 alpha (E2alpha), showed a redistribution into polar clusters of Con A-binding sites that were dispersed in random patches at the external surfaces of control cells. These hormore-induced membrane alterations were abolished by prior treatment of cells with inhibitors of thiol proteinase activity of the cathepsin B1 (CB1) type, such as leupeptin and iodoacetate. Leupeptin at 4.5 X 10(-7) M also reduced the affinity of [3H]E2beta binding to intact cells but did not influence specific binding of the hormone to macromolecular components of cytosol. A pronounced increase in the availability of endogenous CB1, But not of alkaline phosphatase, succinate, or lactate dehydrogenase, in the extracellular media was elicited within 30 min after E2beta treatment. In cells cultured in chemically defined medium for up to 48 h, E2beta, but not E2alpha, enhanced cell proliferation and stimulated [3H]thymidine incorporation into macromolecular form. These E2beta-induced effects were abolished by prior treatment of cells with liposome-entrapped leupeptin at a final concentration of 7 X 10(-8) M. The net rate of intercellular adhesion among endometrial cells was also enhanced by E2beta. This hormonal response was diminished by prior exposure to leupeptin. Fractionation of cells by selection for adhesiveness due to E2beta exposure for 30 min yielded a subpopulation of rapidly dividing cells which surpassed their less adhesive counterparts in cathepsin secretion and in Con A-mediated hemadsorption. These results indicate that leupeptin-sensitive proteinase activity may contribute to membrane and growth modifications elicited by E2beta treatment in endometrial cells.