Prolactin-induced accumulation of casein mRNA in mouse mammary explants: A selective role of glucocorticoid

The role of glucocorticoid in the prolactin-induced accumulation of casein mRNA in mammary explants from midpregnant mice has been studied after an initial 4-day incubation to allow the level of messenger to decline to undetectable levels. Subsequent culture for 3 days: 1) with insulin and glucocorticoid did not result in detectable accumulation of messenger; 2) with insulin and prolactin resulted in a very small accumulation; 3) with insulin, glucocorticoid and prolactin elicited a 20-fold greater accumulation of casein mRNA than the system with only insulin and prolactin. Therefore, although glucocorticoids are not an absolute requirement for casein gene expression in mouse mammary tissue, they are necessary for massive accumulation of casein mRNA induced by prolactin. It appears that this dependence is not a result of either mRNA stabilization or alteration in prolactin receptors. By contrast, stimulation of total epithelial RNA synthesis by prolactin does not have this glucocorticoid dependency.     

Role of prolactin and glucocorticoids in the expression of casein genes in rabbit mammary gland organ culture. Quantification of casein mRNA

Milk synthesis is initiated solely by prolactin in the pseudopregnant rabbit and glucocorticoids potentiate this action of prolactin. In organ culture, prolactin, in the presence or in the absence of insulin, enhances casein synthesis and cortisol (inactive alone) amplifies this action. Measurements of casein mRNA concentration in total cellular RNA, by hybridization with DNA complementary to casein mRNA, revealed that the stimulation of casein synthesis by the glucocorticoid is accompanied by an increase in the amount of casein mRNA. A systematic comparison of variations of these two parameters indicated that the major effect of glucocorticoids on lactogenesis in the rabbit at this stage of mammary gland development is mediated through an increase in the quantity of casein mRNA available for translation. No simultaneous control of casein mRNA translation by cortisol was observed.     

Cortisol induces rapid accumulation of whey acid protein mRNA but not of asl and b-casein mRNA in rabbit mammary explants

Mammary explants from rabbit were cultured in the presence of various combinations of insulin, cortisol and prolactin. The concentration of whey acidic protein (WAP) asl-casein and b-casein mRNA was measured using specific cDNA probes. Medium alone was added to the explants for one day. Prolactin with and without cortisol was then added to the medium. Prolactin alone induced rapidly asl-and b-casein gene but not WAP gene. When cortisol was added with prolactin, the asl- and b-casein genes were induced at the same rate as in the absence of the steroid. In contrast, the WAP mRNA was then rapidly accumulated. This induction process was not altered by cycloheximide for two hours and it was blocked at a later stage. In a second experiment, insulin and prolactin were first added for 24 hours in the culture medium. Cortisol was then added and the concentration of the three mRNA was measured. Cortisol did not significantly modify the level of asl- and b-casein mRNA. On the contrary, the WAP mRNA was rapidly accumulated. These data indicate that the well-established amplificatory effect of glucocorticoids on casein gene expression is a slow process whereas their effect on the WAP gene is rapid. This suggests that glucocorticoids induce casein gene expression through an indirect cellular mechanism not involving a glucocorticoid receptor element in casein gene promoters and that WAP gene is more classically stimulated through the direct binding of the steroid receptor to a glucocorticoid receptor element located in its promoter.     

Cortisol 21-mesylate exerts glucocorticoid action on the induction of milk protein synthesis in cultured mammary gland

Cortisol 21-mesylate, an alkylating derivatives of cortisol, was previously shown to exert an anti-glucocorticoid action in rat hepatoma cell culture (Simons, Thompson and Johnson 1980). In this study the effect of cortisol 21-mesylate on milk protein synthesis induced in cultured mouse mammary gland by glucocorticoid, insulin, and prolactin was investigated. Addition of cortisol 21-mesylate at concentrations ranging from 10(-8) M to 10(-6) M produced no inhibition of casein synthesis that was induced by glucocorticoid, insulin and prolactin in mammary explants from midpregnant mice. On the other hand, cortisol 21-mesylate in combination with insulin and prolactin stimulated casein synthesis in cultured tissue. The potency of cortisol mesylate was about 1/10 to 1/30th of that of cortisol. Cortisol 21-mesylate, like cortisol, also augmented the accumulation of alpha-lactalbumin in midpregnant rat mammary tissue cultured in the presence of insulin and prolactin. A cell-free competition study of glucocorticoid receptors using cytoplasmic extracts from mouse mammary tissue showed that cortisol 21-mesylate competitively inhibited the binding of dexamethasone on glucocorticoid receptors. The apparent affinity of cortisol 21-mesylate for glucocorticoid receptors is about 1/10th of that of cortisol. These results indicate that cortisol 21-mesylate acts as a glucocorticoid but not as an antiglucocorticoid in the mammary gland.     

Correlation between nuclear glucocorticoid receptor levels and casein gene expression in murine mammary gland in vitro

The relationship between nuclear binding of glucocorticoid-receptor complex and casein gene expression was studied in organ culture of the whole mammary gland of the mouse. Pyridoxal 5'-phosphate was used as a modulatory agent for measuring nuclear binding of the receptor complex. Addition of 2 mM and 5mM pyridoxal-5'-P in the medium (Waymouth's MB752/1) resulted in 4- and 12-fold increase of its concentration in the glands incubated with insulin, prolactin, and hydrocortisone. Pyridoxal-5'-P also caused a 52% and 92% inhibition of nuclear binding of [3H]dexamethasone in the glands at 2 mM and 5 mM concentration in the presence of the same hormones in the medium. Corresponding to the reduced nuclear binding of the receptor complex casein mRNA levels, measured by a specific cDNA probe was reduced 86% and over 90% in the glands exposed to 2 mM and 5 mM pyridoxal-5'-P, respectively, in presence of insulin, prolactin, and hydrocortisone in the medium. Withdrawal of pyridoxal-5'-P from the medium restored nuclear binding of the receptor complex near the level of control glands incubated only with the hormones. mRNA casein levels also increased in the gland in the pyridoxal-5'-P-free medium containing the same hormones. This indicates that pyridoxal-5'-P does not alter the specific hormone responsiveness of the mammary cells and its action mediated at the level of the glucocorticoid receptor can influence hormone-inducible expression of the casein genes. Thus, glucocorticoid plays a major role in the multiple hormone regulation of the milk protein gene(s). The findings also suggest that the breast tissue concentration of the vitamin B6 derivative may influence the physiology of lactation in nursing mothers.     

Prolactin induction of casein mRNA in organ culture. A model system for studying peptide hormone regulation of gene expression

The peptide hormone, prolactin, when added to organ explants of rat mammary gland, rapidly (within 1 h) induced the accumulation of casein mRNA. Casein mRNA sequences, as determined by hybridization with a specific cDNA probe, were shown to increase for up to 48 h after prolactin addition. The magnitude of this response was dependent upon the day of pregnancy at which the tissue was placed in culture. Maximal levels of induction (as great as 45-fold) were obtained using tissue from 15-day pregnant rats. Further data indicate that two steroid hormones, hydrocortisone and progesterone, were able to modulate the prolactin-induced accumulation of casein mRNA. The continuous presence of hydrocortisone was not necessary for prolactin induction of casein mRNA. However, the presence of hydrocortisone was required for maximal accumulation of casein mRNA. The induction of casein mRNA by prolactin was inhibited in a dose-dependent manner by the simultaneous addition of progesterone to the organ culture. Thus, hydrocortisone appears to potentiate the prolactin induction of casein mRNA, whereas progesterone is able to prevent casein mRNA accumulation. Since mammary gland organ culture is performed in a serum-free, chemically defined medium, this system allows a detailed examination of the mechanims by which a peptide hormone regulates the rapid accumulation of a specific mRNA.     

Regulation of milk protein synthesis by progesterone in cultured mouse mammary gland

The effect of progesterone on the synthesis of milk proteins, casein and alpha-lactalbumin was investigated by culturing mammary explants from mid-pregnant mice in serum-free medium. The addition of progesterone at concentrations above 10 ng/ml inhibited both the casein and alpha-lactalbumin accumulation that were induced by the synergistic actions of insulin, prolactin and cortisol. The maximal inhibition was attained at a progesterone concentration of 100 ng/ml. The maximal level of inhibition of the alpha-lactalbumin accumulation was about 90% in the presence of insulin and prolactin or insulin, prolactin and 0.01 microgram/ml of cortisol. The inhibition of the casein accumulation by progesterone was about 80% in the presence of insulin and prolactin, and about 40% in the presence of insulin, prolactin and 1 microgram/ml of cortisol, indicating that cortisol partially antagonized the action of progesterone on the casein synthesis. When the inhibitory effect of progesterone on the accumulation of both alpha-lactalbumin and casein was examined in cultured mammary tissues from virgin, early pregnant, mid-pregnant and late pregnant mice, the degree of inhibition was markedly reduced in tissue from late pregnant mice. This indicates that the susceptibility of mammary gland to the inhibitory action of progesterone varies with the developmental stage of the tissue.     

Hormonal regulation of the synthesis of casein and alpha-lactalbumin in a primary mammary cell culture system

The addition of 5 micrograms/ml of both insulin and prolactin, 3 microM cortisol and 5% fetal bovine serum stimulated casein synthesis during a 5 day culture of mammary epithelium from lactating mice using a floating collagen gel as a culture substratum. Omission of any of the three hormones or serum decreased casein synthesis substantially. The use of 10% serum or the attached gel culture system also decreased casein synthesis. Cells cultured with the combination of the three hormones and 5% serum contained a low level of casein mRNA on day 2, but it increased to much higher levels on day 4 and 5, amounting to over 30% of total mRNA on day 5. In contrast to casein synthesis, the maximal increase in alpha-lactalbumin synthesis required the presence of 0.03 microM cortisol. The combination of insulin, prolactin and 3 microM cortisol or insulin and prolactin elicited smaller increases. The translatable mRNA for alpha-lactalbumin in cells cultured with insulin, cortisol and prolactin for 5 days was detected, but not in cells with insulin and cortisol. Both a high and low concentration of cortisol in combination with insulin increased prolactin binding capacity of cultured cells to the same extent, whereas cells cultured with insulin alone contained much lower levels of prolactin binding. The difference in the capacity of prolactin binding between cells cultured with insulin alone and those cultured with insulin and cortisol correlated well with their ability to synthesize casein in response to prolactin.     

Progesterone and prolactin are both required for suppression of the induction of rat alpha-lactalbumin activity

Progesterone prevents lactation during pregnancy. This anti-lactogenic effect includes suppression of the advent of alpha-lactalbumin activity, an effect which prevents the formation of lactose. Alpha lactalbumin activity can be induced to some extent in pregnant rat mammary explants by insulin and hydrocortisone alone, and to a greater extent with prolactin in addition, or with EGF in addition. Physiological levels of progesterone markedly inhibit the induction in the presence of prolactin plus insulin and hydrocortisone, only weakly inhibit in the presence of insulin and hydrocortisone alone, and have no inhibitory effect in the presence of EGF plus insulin and hydrocortisone. Prolactin permits some inhibition in the presence of EGF. The results suggest that progesterone does not subvert the essential insulin or glucocorticoid signals. It also appears that transduction of the prolactin signal is required in order that progesterone effectively block induction of alpha-lactalbumin activity.     

The effect of sodium butyrate on tyrosine aminotransferase induction in primary cultures of normal adult rat hepatocytes

Treatment of primary cultures of adult rat hepatocytes with 5 mM butyrate inhibited the spontaneous decrease in basal activity and mRNA levels of tyrosine aminotransferase (TAT) that occurred during culture (Staecker et al., submitted). We report here that butyrate treatment of primary cultures of rat hepatocytes initially inhibited the induction of TAT. This inhibition was followed by a period of accelerated TAT induction. TAT induction in butyrate-treated primary cultures of adult rat hepatocytes occurred only after metabolism of butyrate by the cultured hepatocytes. The accelerated induction of TAT in hepatocyte cultures treated with sodium butyrate was reflected by increased TAT activity and mRNA levels. Cultured hepatocytes rapidly metabolized butyrate, but the addition of more butyrate into cultures after its initial metabolism resulted in a rapid reduction in TAT activity. These findings indicate that butyrate treatment can affect the expression of TAT in primary hepatocyte cultures in both a positive (increased basal TAT expression) and a negative (inhibition of the induced expression of TAT) manner.     

Regulation of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) gene in the mouse mammary gland differs from that of casein genes

Mouse glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), also known as mC26 and homologous to bovine PP3, is a milk protein synthesized in the mammary gland. Several studies have investigated the regulation of casein, the major milk protein, gene in the mammary gland, but little is known about GlyCAM-1. Here we examined GlyCAM-1 gene expression in mouse mammary epithelial cells. First, we detected GlyCAM-1 expression in mammary epithelial cells in situ by immunohistochemistry; almost all mammary epithelial cells of the lactating mouse expressed GlyCAM-1. Second, mammary epithelial cells were digested with collagenase and cultured with insulin, prolactin and/or glucocorticoid. alpha-Casein and beta-casein genes were expressed following treatment with insulin, prolactin and glucocorticoid. In contrast, GlyCAM-1 expression could not be detected with any combination of these three hormones. We also analyzed changes in the levels of GlyCAM-1 and caseins mRNAs in cultured cells. The addition of hormones to the culture medium increased casein mRNAs, but surprisingly reduced GlyCAM-1 mRNA. Our results suggest that the mechanisms that regulate GlyCAM-1 gene in mammary cells of lactating mice are different from those involved in the regulation of casein genes.     

The differential actions of cortisol on the synthesis and turnover of alpha-lactalbumin and casein and on accumulation of their mRNA in mouse mammary gland in organ culture.

Cortisol was previously shown to exert different, concentration-dependent, effects on the accumulation of casein and alpha-lactalbumin in mammary glands from mid-pregnant mice cultured in the presence of insulin and prolactin [Ono & Oka (1980) Cell 19, 473-480]. The present study demonstrated that the addition of 30nM-cortisol to the medium containing insulin and prolactin resulted in a marked enhancement of the rate of synthesis of both alpha-lactalbumin and casein in cultured tissue. The addition of 3 microM-cortisol in combination with insulin and prolactin caused a marked decrease in the rate of alpha-lactalbumin synthesis, but increased casein synthesis substantially. Similar changes were also observed in the amount of translatable mRNA for alpha-lactalbumin and casein in mammary explants cultured with insulin, prolactin and the two concentrations of cortisol. The study of the turnover of the milk proteins in cultured explants showed that virtually all of the casein synthesized remained intact in tissue explants cultured with 3 microM cortisol, whereas about 45% of casein disappeared in 40h from explants cultured with 30nM-cortisol. In contrast, the two concentrations of cortisol did not differentially affect the disappearance of alpha-lactalbumin, which was about 55% in 40h. These results indicate that the concentration-dependent differential actions of cortisol on the accumulation of alpha-lactalbumin and casein are exerted through its effects on the rate of synthesis and turnover of the two proteins as well as on the accumulation of their mRNA species.     

Effects of glucocorticoids on casein gene expression in the rabbit.

Milk protein synthesis is initiated by prolactin and a glucocorticoid. In the rabbit, prolactin alone is sufficient. However, glucocorticoids potentiate the action of prolactin. The stimulatory effect of glucocorticoids was evaluated after injections of hydrocortisone acetate alone or associated with prolactin by measurements of (a) the total RNA and DNA content of mammary glands, (b) the lactose synthetase activity, (c) casein synthesis, and (d) the concentration of casein mRNA in total cellular RNA and in polysomal RNA by hybridization with its cDNA. The glucocorticoid, totally inactive alone, proved to have a stimulatory effect proportional to the dose injected when prolactin was present. This effect was more evident with low doses of prolactin. Glucocorticoids proceeded by amplifying the capacity of prolactin to enhance the concentration of casein mRNA available for translation. A parallel effect of glucocorticoids on translation of casein mRNA was suspected. Glucocorticoids injected with low doses of prolactin were unable to mimic all the effects of high doses of prolactin alone.     

Hormonal control of casein synthesis in mammary explants from pregnant goats

The effects of insulin, cortisol, prolactin, 3,3',5-triiodo-L-thyronine (L-T3) and progesterone on the synthesis of total protein and casein in mammary explants from pregnant goats were studied. In the absence of hormones and in the presence of insulin plus cortisol the rate of incorporation of 14C-leucine into proteins that were precipitated with the anti-casein antibody decreased during culture. The addition of prolactin to hormonal combination of insulin and cortisol caused large stimulation of rates of casein synthesis. Maximum incorporation of leucine was attained between 3 and 5 days of culture in the presence of 0.5 microgram ml-1 of prolactin. Prolactin stimulated-casein and total protein synthesis were not consistently affected by the addition of L-T3 or progesterone. The inhibition of DNA synthesis by hydroxyurea or cytosine-arabinofuranoside had no effect on casein synthesis in mammary explants from pregnant goats.     

A test for hormonal responsiveness in a mammary epithelial cell line, NMuMg

The NMuMG cell line derived from normal mouse mammary epithelial cells was tested for responsiveness to hormones. The hormones studied included insulin, glucocorticoids (cortisol and dexamethasone), and prolactin. In addition to membrane bound insulin receptors and prolactin receptors, the cells had 2 X 10(4) cytoplasmic glucocorticoid receptors per cell. Morphological changes were observed in response to hormones. Clusters of cells appeared with greatly increased diameter, and the number of cells per plate was reduced. The rate of DNA synthesis, corrected by cell number, indicates that cell division, and hence cell turnover, was increased by the combination of all three hormones. Insulin greatly enhanced protein synthesis, but glucocorticoid and prolactin did not further increase the rate. The combination of three hormones produced a change in the synthesis of histones, consistent with the increase in cell turnover. There were substantial responses of enzyme activities to hormonal treatment of the cells. Insulin by itself induced a doubling of the activity of glyceraldehyde phosphate dehydrogenase and perhaps a modest increase in NADH-cytochrome c reductase. Lactose synthetase activity showed a three- to fourfold induction of both A and B subunits of the enzyme when the cells were treated with insulin, glucocorticoid, and prolactin, and the effect of the latter two hormones was shown to be additional to that of insulin.     

Effects of amiloride on the induction of DNA synthesis and casein gene expression in rabbit mammary explants

Amiloride, an inhibitor of Na+/H+ exchange, was added at various concentrations to the culture medium of rabbit mammary explants. In the concentration range 100-250 microM, amiloride progessively inhibited 14C-thymidine incorporation induced by insulin, EGF or prolactin. Up to 250 microM, amiloride, which did not inhibit basal protein synthesis, was not cytotoxic, but it reduced basal DNA synthesis at the highest concentration. Addition of amiloride to the culture medium of mammary explants also strongly inhibited the induction of casein synthesis and casein mRNA accumulation by prolactin. The inhibition by amiloride is therefore not specific of the mitogenic action of prolactin since this drug also prevented its lactogenic action. The data reported here describe a new inhibitory action of amiloride on the transmission of the lactogenic signals.