Retraction: Prolactin induces release of a factor from membranes capable of stimulating beta-casein gene transcription in isolated mammary cell nuclei.
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Retraction Notice↗
Biomedical subjects
L M Houdebine
Publications and source records attributed to L M Houdebine.
Maintenance of prolactin (PRL) binding sites in rat liver cells in suspension culture: effect of PRL and of inhibitors of various cellular functions.
An in vitro method to study the regulation of PRL receptors has been established using adult rat liver cells cultured in a continuous suspension in L-15 medium. PRL binding averaged 28.2 +/- 1.8% of the added labeled hormone per 10(6) cells in freshly isolated liver cells prepared from female rats treated with 17 beta-estradiol. When these cells were incubated at 37 C, binding rapidly declined by 50% at 10 h and 90% at 48 h. This rapid decline could be counteracted by the inclusion of ovine PRL (50 nM), which maintained initial PRL receptor levels up to 48 h of culture. Higher concentrations of PRL (2.5 microM) induced a rapid down-regulation, apparent at 2 and 10 h of culture. Cycloheximide (50 micrograms/ml) induced a slight diminution of control PRL receptor levels and partially reversed the effect of 50 nM PRL. Approximately 60% of the PRL receptors were resistant to the effect of cycloheximide. On the other hand, actinomycin D (10 micrograms/ml) had no effect on PRL receptor levels in control and only a very slight effect in PRL-treated cells. Dinitrophenol, which blocks metabolic oxidation, also partially reversed the effect of 50 nM PRL although it was without any significant effect on control levels. Chloroquine (100 microM) and colchicine (1 microM) failed to alter PRL binding either in the absence or presence of 50 nM PRL. Our results suggest that the existence of regulatory factors occurring in vivo, which are absent in the culture medium, could be responsible for the decline in PRL receptor levels in the control hepatocytes. PRL itself could be one of these factors. On the other hand, and in agreement with the putative actions of the drugs utilized, the mechanism of the PRL-induced maintenance of receptor levels appears to lie in part with an effect on receptor synthesis at the translational (ribsomal) level but to be independent of the internalization or of lysosomal degradation.
Animals↗
Hormonal action controlling mammary activity.
Mammary gland differentiation includes multiplication of cells, activation of genes specific to milk synthesis, and activation of "house-keeping" genes. These events are controlled by multiple hormones, the roles of which are not known in detail. Prolactin induction of milk synthesis is accompanied by accumulation of casein messenger ribonucleic acid resulting from acceleration of casein gene transcription as well as stabilization of messenger ribonucleic acid. Prolactin also favors translation of casein messenger ribonucleic acid. Glucocorticoids amplify and progesterone inhibits prolactin action on transcription of casein genes.
Animals↗
[Hormonal regulation of the normal mammary gland].
Development and differentiation of mammary gland are controlled by a large number of hormones. In embryo, foetal androgens induce a partial necrosis of mammary epithelium. This action is mediated by cells of the parenchyma which is the real target of androgens. Estrogens are not true growth factor in normal mammary gland: they deliver a message of unknown chemical nature, which allows growth factors to act. The growth factors of mammary cells are only partially known. Clones of the various mammary cell types have been obtained (fibroblasts, adipocytes, myoepithelial and epithelial cells). These clones are a good tool to determine growth factors specific of each cell type. Under the influence of GH, mammary fibroblasts are transformed into preadipocytes which secrete a growth factor (PGE2) which specifically stimulates multiplication of mammary epithelial cells. Other growth factors induced by oestrogens or prolactin have been identified but their exact role remains unknown. Induction of milk synthesis is under the strict dependency of prolactin. The action of this hormone is strongly stimulated by glucocorticoids and insulin at high concentrations and it is inhibited by progesterone and EGF. Prolactin stimulates transcription of milk protein genes and this stimulation is modulated by glucocorticoids and progesterone. Induction of casein and DNA synthesis by prolactin can be mimicked by polyclonal and monoclonal anti-prolactin receptor antibodies. Collagen is necessary for isolated epithelial mammary cells to respond to prolactin signal. The role of other components of the extracellular matrix (proteoglycans, glycoproteins) is partially known. Specific peripheral markers corresponding to the different cell types and to the various stages of mammary gland development have been identified. All these cellular and molecular parameters are compared in normal and tumor mammary cells to point out possible differences.
Animals↗
Purification and biological characterization of chinook salmon prolactin.
Prolactin from chinook salmon pituitaries was purified by acid acetone extraction, saline precipitation, chromatofocusing, and gel filtration. This procedure allowed us to recover highly purified prolactin as demonstrated by the presence of a single NH2-terminal amino acid and a single band in sodium dodecyl sulfate gel electrophoresis. Chinook salmon prolactin appeared to be a basic protein of 22,500 molecular weight. Throughout the purification, prolactin bioactivity was followed by radioreceptor assay for lactogenic hormones. The prolactin character of the purified protein was established by its lactogenic activity in the rabbit mammary gland in vitro and its sodium-retaining activity in hypophysectomized Fundulus heteroclitus.
Amino Acid Sequence↗
Intracellular transformation of prolactin following internalization into rat liver.
We investigated prolactin (PRL) degradation in rat liver lysosomes both in vivo and in vitro. In previous studies we showed that, in addition to the Golgi apparatus, PRL is internalized towards lysosomes and light, lysosome-like vesicles which we identified as 'prelysosomes'. Injected [125I]oPRL that localized in lysosomes and prelysosomes at times varying from 0 to 45 min showed significant differences from fresh and plasma membrane- (PM) or Golgi-bound hormone. First, it was more easily dissociable by 3 M MgCl2 than Golgi- but less than PM-bound [125I]oPRL. Second, it was only in lysosomal fractions that, as time following injection increased, a significant part of dissociable radioactivity became non-TAC-precipitable. When MgCl2-extracted [125I]oPRL was subjected to gel filtration on a Sephadex G-75 fine column, some of the radioactivity, and especially that extracted from prelysosomal or lysosomal fractions, eluted as a high molecular weight (HMW) entity, most co-migrated with fresh [125I]oPRL, and a little was found in small fragments. Only the central peak had any rebinding activity, which was comparable to that of fresh hormone. In an in vitro study we incubated [125I]hGH with lysosomal fractions for 16 h at 25 degrees C. After centrifugation, an aliquot of supernatant hormone was assayed for its binding capacity to standard receptor preparations and the rest subjected to gel filtration. Peak fractions were also tested in binding assay. [125I]hGH that had been in contact with prelysosomes lost almost all of its ability to bind to standard receptors and totally migrated in the HMW peak, at the void volume of the column.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Studies of beta-casein content of normal and neoplastic rat mammary tissues by a homologous radioimmunoassay.
We have developed a sensitive homologous radioimmunoassay for rat beta-casein (r-beta-casein) capable of measuring concentrations of beta-casein as low as 0.2 ng/mg of tissue and have measured casein content of rats under several physiological conditions as well as in experimentally induced mammary tumor explants. Changes in content of beta-casein were followed during pregnancy and lactation. Virgin females had 10 ng beta-casein per micrograms of DNA, with a continuous increase in casein content observed throughout pregnancy. At the outset of lactation, beta-casein increased markedly and remained relatively constant throughout lactation. These variations in beta-casein levels indicate that in the rat the synthesis of this protein is under hormonal control and related to the state of differentiation of the mammary gland. The concentration of beta-casein was very low in mammary tumors (0.6 ng/microgram of DNA). Addition of prolactin to organ culture of NMU-induced tumors did not induce significant changes in beta-casein content. These results indicate that NMU-induced tumors have lost at least one of their hormonally regulated functions of cell differentiation, although their hormonal dependence for growth is maintained.
Animals↗
The interaction of prolactin with its receptors in target tissues and its mechanism of action.
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Affinity Labels↗
Differential biological activities between mono- and bivalent fragments of anti-prolactin receptor antibodies.
Previous studies have established that antibodies against PRL receptors can mimic PRL effects on casein gene expression and on thymidine incorporation into DNA in the mammary gland. In the present work, bivalent F(ab')2 and monovalent Fab' fragments of the anti-PRL receptor antibodies were prepared. Both inhibited the binding of 125I-labeled PRL to rabbit mammary gland membranes. F(ab')2 as well as the unmodified antibodies were able to enhance casein synthesis and thymidine incorporation into DNA in cultured rabbit mammary gland explants. Moreover, when added to isolated membranes, both were able to induce the generation of the PRL relay which specifically stimulates caseins gene transcription in isolated mammary nuclei. In contrast, monovalent fragments were totally devoid of any of these PRL-like activities. However, bivalent and monovalent antibodies were equipotent in inducing a down-regulation of PRL receptors in mammary explants. These data indicate that the biological PRL-like activity of antibodies against PRL receptors is strictly related to their bivalent structure. This fact indicates a possible crucial role of a microaggregation of PRL receptors in the transmission of the PRL message across the membranes. In addition, these experiments reinforce the idea that internalization and down-regulation are not directly related to PRL action on casein or DNA synthesis in mammary gland.
Animals↗
The effect of chloroquine on lysosomal prolactin receptors in rat liver.
PRL receptors have been previously identified in purified rat liver plasma membrane and Golgi vesicle preparations. In this study, we report on PRL receptors located in highly purified lysosome preparations. These lysosomal PRL receptors were characterized using Scatchard analysis and compared to other intracellular and cell surface receptors. We have identified two classes of lysosomes. Lighter lysosome-like vesicles, which are greatly enriched in acid phosphatase activity (the marker enzyme of lysosomes), contain a great deal of binding activity. This PRL binding was only slightly increased by pretreatment of animals with the lysosomotropic agent chloroquine. In contrast, mature lysosomes showed very little binding activity in control animals, but chloroquine treatment increased binding 7- to 8-fold in these mature lysosomes. We suggest that the lysosome-like structures are immature lysosomes (namely prelysosomes) toward which the hormone-receptor complex is internalized: they appear to bear little proteolytic activity. These structures could play a role in PRL receptor recycling. Lysosomal PRL receptors showed curvilinear Scatchard plots, in contrast to plasma membrane and Golgi counterparts, which were linear over the same range of hormone concentrations. The high affinity site in lysosomes had a Kd comparable to the cell surface and Golgi receptors. The number of binding sites per mg protein in prelysosomes and lysosomes was 3 times greater than that in the homogenate, but Golgi preparations were 3 times as rich as lysosomes. The great number of PRL receptors in prelysosomes could be attributed, in large part, to the low affinity sites. The internalization of PRL into rat liver was examined after in vivo injection of [125I]iodoovine PRL. The labeled hormone was found initially in the plasma membrane fraction, after which it localized preferentially in the Golgi fraction, with maximum incorporation 15 min postinjection. Substantial radioactivity was observed in both classes of lysosomes (L-1 and L-2). In contrast to the Golgi fraction, maximum incorporation of [125I]iodoovine PRL in lysosomes occurred at 30 min. This suggests either that during internalization, PRL first reaches Golgi elements and is then transferred to the lysosomal compartment, or that there are two independent pathways of internalization, one rapid toward the Golgi complex (may be a path of receptor recycling) and the other toward lysosomes (probably leading to receptor degradation).
Acid Phosphatase↗
Effect of sodium butyrate on the stimulation of casein gene expression by prolactin.
Sodium butyrate, but not isobutyrate, inhibits prolactin action on the induction of casein synthesis and casein mRNA accumulation in rabbit mammary explants. Sodium butyrate specifically prevents the generation of the prolactin relay which can be released from isolated membranes incubated with prolactin and which stimulates directly casein gene transcription when added to isolated mammary nuclei. This indicates that sodium butyrate exerts its inhibitory action essentially at the membrane level.
Animals↗
In vivo lactogenic effects of anti prolactin receptor antibodies in pseudopregnant rabbits.
Antibodies generated against partially purified prolactin receptors from rabbit mammary gland membranes were tested for their effects on prolactin binding to receptors and for their in vivo biological potencies. These antibodies are able to inhibit prolactin binding to crude rabbit mammary gland membranes. When administered intravenously or intramuscularly to pseudopregnant rabbits, they induce respectively an accumulation of beta-casein or an enhancement of beta-casein synthesis and mRNA concentration in the mammary gland. Moreover the stimulatory effect of these anti-prolactin receptor antibodies on casein synthesis is totally abolished by a simultaneous treatment with progesterone, which is a potent in vivo inhibitor of prolactin action. These results better establish the prolactin-like activities of these antibodies previously observed in vitro and give strong support to the hypothesis that prolactin molecule is not required beyond the initial binding to its receptor to induce hormonal effects.
Animals↗
Comparative measurement of the lactogenic activity of ovine placental lactogen in rabbit and ewe mammary gland.
Ovine placental lactogen is known to bind to prolactin receptors and to initiate milk synthesis in the rabbit mammary gland. However, this hormone exhibited a very low capacity of competing with 125I-labeled human growth hormone for the binding to membranes extracted from ewe mammary gland. Ovine placental lactogen was very efficient in provoking the accumulation of beta-casein mRNA in rabbit mammary explants but was much less active on ewe mammary explants. These data indicate that the placental hormone is not a potent lactogen in the homologous species and that its role in the control of mammary gland development and activity may have been previously overestimated.
Animals↗
Effects of transglutaminase or phospholipase A2 inhibitors on down-regulation of prolactin receptors and stimulation of casein and DNA synthesis in mammary gland explants.
A number of compounds have been found to inhibit the internalization of alpha 2-macroglobulin and/or epidermal growth factor into fibroblasts. Using the same inhibitor we tried to block the down-regulation of prolactin receptors, supposed to mirror internalization of PRL receptors, in order to investigate the possible role of internalization in the mechanism of prolactin action. In rabbit mammary cells it appeared that bacitracin and ethylamine completely block prolactin receptor down-regulation, but dansylcadaverine, the most potent inhibitor of transglutaminase, was without effect. The blockage of phospholipase A2 by chlorpromazine or bromophenacyl bromide (BPB) was without effect on the down-regulation of prolactin receptor. Subcellular distribution of [125I]oPRL has been studied after incubation with or without these various inhibitors after fractionation of mammary gland homogenate on sucrose gradient. Any of these compounds was able to increase the labelling of prolactin receptors located in plasma membrane fractions, suggesting that the rate of internalization of prolactin was not modified. In addition, none of these compounds inhibited the stimulation of beta-casein and DNA synthesis by prolactin. These results suggest that both transglutaminase and phospholipase A2 are not involved in the mechanism of prolactin-induced down-regulation of prolactin receptors, although bacitracin and ethylamine are able to block this phenomenon probably by different mechanism. In all cases, inductions of mitogenesis and beta-casein synthesis by prolactin in the rabbit mammary cells were not modified by the various compounds utilized. We conclude that neither transglutaminase nor phospholipase A2 are involved in the internalization of prolactin-receptor complexes, although bacitracin, ethylamine and quinacrine are able to block the down regulation of prolactin receptors by other means.
Acyltransferases↗
Effects of phorbol esters on multiplication and differentiation of mammary cells.
Three phorbol esters: phorbol-12,13-dibutyrate (PDB), 12-0-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-diacetate (PDA) were added at various concentrations (10(1)-10(4) ng/ml) to the culture medium of rabbit mammary explants. The three phorbol esters inhibited the mitogenic signals of insulin, EGF and prolactin. They also prevented the induction of casein synthesis and casein mRNA accumulation by prolactin. In all cases, the most potent inhibitor was PDB. PDB did not alter the binding of prolactin to its receptors. The three phorbol esters added to mammary cell membranes with prolactin prevented the generation of the hormonal relay which specifically stimulates casein gene transcription when added to isolated nuclei. These data suggest that phorbol esters act directly at the membrane level where they interrupt the transfer of the hormonal messages, through a mechanism which may not be related to their promoter potency.
Animals↗
[Prolactin internalisation by the epithelial mammary cell: effects of lysosomotropic agents and transglutaminase inhibitors].
Prolactin endocytosis was studied by electron microscopy with 125I-prolactin 125I-hGH (human growth hormone) and prolactin-ferritin. Endocytosis and intracellular transit of the labelled hormone proceeded identically in epithelial cells isolated from the mammary glands of pseudopregnant rabbits and in surviving fragments from mammary glands of lactating rabbits. After binding of the hormone to its receptor, the labelled material was rapidly detectable in vesicles showing an homogeneous aspect; 15 min later part of the labelled material was still localized within the same kind of vesicles, but in addition it appeared to have migrated into microvesicles of the Golgi region and into vesicles of heterogeneous aspect tentatively identified with lysosomes. Endocytosis of bovine serum albumin, labelled with ferritin followed the same intracellular pathway. Native ferritin accumulated in vesicles of various sizes, but seemed excluded from the microvesicles of the Golgi zone. In the presence of lysosomotropic agents labelled prolactin accumulated in cytoplasmic vesicles. In the presence of dansylcadaverine, endocytosis of the labelled material proceeded unimpaired. Conversely, in the presence of bacitracin, the internalisation of labelled prolactin seemed to be reduced. These observations show that the endocytosis of the hormone/receptor complex is linked to membrane movements, which eventually lead to its location within both the Golgi apparatus and the lysosomes.
Animals↗
Prolactin-like activity of antiprolactin receptor antibodies in rat mammary tumor explants.
The effects of prolactin and a serum containing antiprolactin receptor antibodies on some lactogenic and mammogenic responses were investigated in nitrosomethylurea-induced mammary tumors in organ cultures. Prolactin was able to induce an increase in lactose synthetase activity, DNA synthesis, and prolactin-binding sites at moderate (i.e., physiological) concentrations of prolactin; at higher concentrations, desensitization of the tissues was observed for DNA synthesis and lactose synthetase activity, whereas the "down-regulation" of prolactin receptors occurred. Prolactin had no effect on casein synthesis. Antiprolactin receptor serum was capable of inducing an increase in lactose synthetase activity, DNA synthesis, and prolactin-binding sites, however, lower than that observed with prolactin, thus mimicking prolactin action. The antiserum did not induce any change in casein synthesis. These findings suggest that, in rat mammary tumors, as it has been observed in the normal mammary gland, the prolactin molecule is not required beyond the initial binding to its receptor for its action to be attained. It appears also that a differentiated function has lost its hormonal dependence in these tumors, although their hormonal dependence for other functions and growth is maintained.
Animals↗
Induction of beta-casein mRNA accumulation by the putative prolactin second messenger added to the culture medium of cultured mammary epithelial cells.
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Animals↗