Casein mRNA Research Articles

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.

Involvement of collagen formation in the hormonally induced functional differentiation of mouse mammary gland in organ culture.

In mammary gland organ culture from midpregnant mice, synergistic actions of insulin, cortisol, and prolactin stimulate differentiation of mammary epithelium and induce the synthesis of the milk proteins casein and alpha-lactalbumin. In the present study we examined the production of collagen and its function in the hormone-dependent development of mammary gland in vitro. The measurement of collagen production by the hydroxyproline assay and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the accumulation of collagen type I and type III in cultured mammary explants increased with the addition of insulin, cortisol, and prolactin to a chemically defined medium. When an analog of proline, L-azetidine-2-carboxylic acid (LACA), was added at a concentration of 80 microgram/ml with insulin, cortisol, and prolactin at the beginning of culture, collagen production in cultured tissue was inhibited by 75% during a 3-day incubation period. This agent also inhibited the synthesis of casein and alpha-lactalbumin by about 77 and 70%, respectively. The inhibitory effect of LACA could be prevented by L-proline; concomitant addition of L-proline (80 microgram/ml) with LACA (80 microgram/ml) resulted in complete restoration of milk protein synthesis to normal levels. Measurement of the amount of milk protein mRNAs in mammary explants by a cell-free translation assay demonstrated that LACA reduced the hormone-stimulated accumulation of casein mRNA and alpha-lactalbumin mRNA by 79 and 76%, respectively. LACA, however, produced little inhibition of DNA synthesis in cultured tissue. These results suggest that collagen production may be involved in the phenotypic expression of milk protein genes during hormonal induction of mammary epithelial differentiation in vitro.

The interaction of cortisol and prostaglandins on the phenotypic expression of the α-lactalbumin gene in the mouse mammary gland in culture

Addition of cortisol at concentrations above 300 nM selectively inhibited the synthesis of α-lactalbumin and the accumulation of its mRNA in the mouse mammary gland cultured in the presence of insulin and prolactin, whereas the same treatment augmented casein synthesis and the accumulation of casein mRNA. Prostaglandin E 2 or F 2α reversed the inhibitory effects of cortisol in a dose-dependent manner, without affecting casein production. The levels of prostaglandin E 2 or F 2α in tissue explants cultured with insulin and prolactin increased about 2.6-fold over those in uncultured tissue, and the addition of cortisol decreased these levels approximately 2-fold. These results indicate the ability of prostaglandins to counteract the inhibitory effect of cortisol on the α-lactalbumin gene expression in the mouse mammary gland.

Deoxyribonucleic Acid Synthesis-Dependent Casein Gene Expression: Species Differences

Mammary gland explants from mature virgin mice, rats, and rabbits exhibit an increased rate of both DNA and casein syntheses when cultured in the presence of specific combinations of insulin, hydrocortisone, and PRL. If cytosine arabinoside, a potent inhibitor of DNA synthesis, is added to the culture medium, casein synthesis is inhibited in explants from mice but not in those from rats or rabbits. This inhibition is at the level of accumulation of casein mRNA; an 89% reduction of stimulated levels was observed. The nature of this block was investigated further by examining the general response of mature virgin mouse mammary explants to hydrocortisone and PRL, hormones considered essential for casein gene expression in this species. Cytosine arabinoside did not prevent an increment in either hydrocortisone-induced NADH-cytochrome c-reductase or PRL-induced total RNA synthesis. Previous work has shown that certain insulin-induced responses are also unaffected. Taken collectively, these results suggest that the lesion induced by cytosine arabinoside inhibition of DNA synthesis is distal to the receptor for one or more of these hormones. The necessity for coupling DNA synthesis with overt differentiation in the mature virgin mouse, but not in the rat or rabbit, is one of numerous examples of species variation in regard to the interaction of hormones with the mammary gland.

Characterization and cloning of the mRNAs specific for the lactating mouse mammary gland.

We have characterized and cloned the lactation-specific mRNAs of mouse mammary glands. The group of eight milk-protein-specific mRNAs were identified (a) by size and antigenic properties of their translation products in vitro and (b) by characterization of their respective cDNA clones. Two alpha-caseins (43 kDa and 39 kDa) are encoded by mRNAs of 1600 nucleotides and two beta-caseins (26 kDa) are encoded by mRNAs of 1450 nucleotides in length. Three smaller caseins, gamma-casein (23.7 kDa), delta-casein (21 kDa) and epsilon-casein (14.5 kDa) are synthesized by mRNAs of 880, 1150 and 860 nucleotides. Beside these casein mRNAs a mammary specific 620 nucleotide mRNA codes for a novel acidic whey protein (13.7 kDa). cDNA clones corresponding to the mRNAs for the lactation-specific proteins have been isolated from a mammary-specific cDNA library. Cloned alpha-casein cDNA hybridizes to both alpha-casein specific mRNAs and cloned beta-casein cDNAs hybridize with both beta-casein specific mRNAs. By RNA blot analysis we show that the cloned cDNAs for mouse alpha-casein, beta-casein, gamma-casein and epsilon-casein and the acidic whey protein cross-hybridize with mRNAs of the rat, demonstrating partial sequence homology between the corresponding mRNAs of those species.

Correlation between prolactin-receptor interaction, down-regulation of receptors, and stimulation of casein and deoxyribonucleic acid biosynthesis in rabbit mammary gland explants.

Increasing concentrations of PRL were added to culture media of rabbit mammary gland explants. After 24 h of culture, free and total PRL receptors, casein synthesis, casein messenger (m) RNA concentrations and DNA synthesis were estimated. In the PRL concentration range of 0-100 ng/ml, receptors were progressively down-regulated by the hormone, but no occupied desaturable receptors could be detected. At concentrations between 1,000 and 20,000 ng/ml, the PRL receptors were increasingly occupied without being quantitatively down-regulated. There exists a reciprocal correlation between the rate of casein synthesis, casein mRNA concentration, DNA synthesis and the down-regulation of the receptors as a function of PRL concentration. The maximal responses were reached around a PRL concentration of 100 ng/ml. Interestingly, with a large excess of PRL a desensitization process was observed, the responses to the hormone being attenuated. This desensitization was more pronounced for DNA synthesis than for casein synthesis or for casein mRNA accumulation. The data suggest that down-regulation of PRL receptors occurs even at physiological concentrations of PRL. These observations are compatible with the hypothesis that each receptor can generate a limited number of intracellular relays eliciting hormonal action before being irreversibly inactivated and degraded. In addition, high concentrations of PRL induce a refractory state in the mammary gland that is possibly related to a low level of peripheral PRL receptors.

Sequence of rat alpha- and gamma-casein mRNAs: evolutionary comparison of the calcium-dependent rat casein multigene family.

The complete sequences of rat alpha- and gamma-casein mRNAs have been determined. The 1402-nucleotide alpha- and 864-nucleotide gamma-casein mRNAs both encode 15 amino acid signal peptides and mature proteins of 269 and 164 residues, respectively. Considerable homology between the 5' non-coding regions, and the regions encoding the signal peptides and the phosphorylation sites, in these mRNAs as compared to several other rodent casein mRNAs, was observed. Significant homology was also detected between rat alpha- and bovine alpha s1-casein. Comparison of the rodent and bovine sequences suggests that the caseins evolved at about the time of the appearance of the primitive mammals. This may have occurred by intragenic duplication of a nucleotide sequence encoding a primitive phosphorylation site, -(Ser)n-Glu-Glu-, and intergenic duplication resulting in the small casein multigene family. A unique feature of the rat alpha-casein sequence is an insertion in the coding region containing 10 repeated elements of 18 nucleotides each. This insertion appears to have occurred 7-12 million years ago, just prior to the divergence of rat and mouse.

Prolactin-like activity of anti-prolactin receptor antibodies on casein and DNA synthesis in the mammary gland.

Prolactin receptors were partially purified from rabbit mammary gland membranes by using an affinity chromatography technique. Antibodies against this prolactin receptor preparation were obtained in guinea pig and sheep. Both antisera were able to inhibit the binding of 125I-labeled ovine prolactin to rabbit mammary gland membranes. When added to culture media of rabbit mammary explants, the anti-prolactin receptor antiserum inhibited the capacity of prolactin to initiate casein synthesis and casein mRNA accumulation as a function of the antiserum concentration. However, in the absence of prolactin, both antisera (guinea pig and sheep) at moderate concentrations were capable of mimicking prolactin action on casein gene expression and on DNA synthesis. At higher concentrations, the anti-prolactin receptor antibodies inhibited their own actions. Several characteristics of the prolactin effect were also observed with the anti-prolactin receptor antibody: the stimulatory effect of the antibody was amplified by glucocorticoids; colchicine, which was capable of blocking prolactin action, also prevented the induction by the antibody. Lysosomotropic agents, which do not interfere with prolactin action, did not alter the response observed with the antibody. These results indicate that an anti-prolactin receptor antibody can mimic two major actions of prolactin obtained in mammary explant culture and suggests that the prolactin molecule is not required beyond the initial binding to its receptor.

Cloning of mouse β-casein gene sequences

Casein messenger RNAs (mRNAcsn) were purified from lactating mammary glands of BALB/c mice and used as a starting material for cloning of casein gene sequences. Double-stranded casein cDNA (ds-cDNAscn) was prepared and blunt-end ligated to HindIII-specific DNA linker molecules. After digestion with HindIII, the dsDNAcsn was inserted into the HindIII site of plasmid pBR322, using T4 DNA ligase. Escherchia coli strain RH202 was transformed with the hybrid plasmids, and transformants were selected for resistance to ampicillin. Electrophoresis of HindIII-digested hybrid plasmid DNAs, followed by Souther transfer and hybridization to 9[ 32P]cDNAcsn, revealed that one of the hybrid-plasmid-containing colonies, designated pCas51, contained a 400-bp insert which hybridized to the [ 32P]cDNAcsn. Purification of the individual casein mRNAs (mRNAcsn α, β and γ) and solution hybridization of nick-translated insert DNA to each of these revealed that pCas51 contained sequences complementary primarily to mRNAcsn β.

Insulin is essential for accumulation of casein mRNA in mouse mammary epithelial cells.

In the presence of cortisol and prolactin, insulin at concentrations as low as 1 ng/ml significantly stimulates casein synthesis in mammary explants from midpregnant mice; maximal synthesis occurs at 10 ng/ml. However, in the absence of insulin, no detectable immunoprecipitable casein is produced. Insulin also supports enhanced accumulation of casein mRNA in the presence of cortisol and prolactin; neither epidermal growth factor nor somatomedin C has this effect. These inductive actions of insulin are not secondary to a general maintenance effect on the mammary epithelial cell; insulin, epidermal growth factor, and somatomedin C can support the accumulation of RNA in rough endoplasmic reticulum equally well. In addition, these effects do not reflect a specific insulin requirement for prolactin sensitivity; epidermal growth factor can support prolactin-induced total RNA synthesis as well as insulin can. The results demonstrate that, although insulin, epidermal growth factor, and somatomedin C can all function as cell maintenance agents, only insulin, together with cortisol and prolactin, can induce casein mRNA accumulation.

Induction of casein synthesis by prolactin and inhibition by progesterone in the pseudopregnant rabbit treated by colchicine without any simultaneous variations of casein mRNA concentration.

Prolactin injected into pseudopregnant rabbits induced casein synthesis and it provoked an accumulation of casein mRNA and of ribosomal RNA. Colchicine, which has been shown to block the prolactin signal, totally prevented the accumulation of beta-casein mRNA, when injected with the hormone. However, the drug did not hamper the initiation of casein synthesis and the accumulation of ribosomal RNA. The effect of prolactin injected with colchicine on casein synthesis was totally abrogated by progesterone administered simultaneously and it was essentially unmodified by glucocorticoids. These results suggest that prolactin controls casein gene expression by supporting the accumulation of casein mRNAs and by stimulating the translation of these mRNAs, through independent mechanisms. Progesterone which is known to prevent the accumulation of casein mRNAs is also a potent inhibitor of casein mRNA translation whereas glucocorticoids exert their effect essentially by favouring the accumulation of casein mRNA but not by modifying their translation efficiency.

Simultaneous occurrence of pregnancylike lobuloalveolar morphogenesis and casein-gene expression in a culture of the whole mammary gland.

Entire second thoracic mammary glands of estrogen- and progesterone-treated immature virgin BALB/c mice were stimulated to pregnancylike lobuloalveolar morphogenesis after 6 days of incubation with insulin (5 micrograms/ml), aldosterone (1 micrograms/ml), growth hormone (5 micrograms/ml), cortisol (5 micrograms/ml), and prolactin (80 ng/ml, present as a contaminant in 5 micrograms/ml growth hormone). The alveolar growth in the glands, as judged by morphological studies, was accompanied by an increase in cell number as a function of incubation time in the hormonal medium. Hybridization of the total RNA from these glands to the casein mRNA specific complementary DNA probe (cDNAcsn) revealed that the level of casein mRNA rises from 0.00012 to 0.005% between 1 and 6 days of incubation. Estimates showed that the concentration of casein mRNA per cell rises 17-fold from 70 molecules on Day 1 to 1200 molecules on Day 6, whereas the number of epithelial cells increases only twofold during the same incubation time. When the growth hormone preparation was totally replaced by 80 ng of prolactin during the 6-day incubation, casein-mRNA levels were found to be 0.0083%. These results demonstrate that a pregnancy-like morphogenesis and concurrent expression of the casein gene in vitro can be achieved in a controlled hormone environment containing high cortisol and low prolactin concentrations. This one-step mammogenesis-lactogenesis culture model should be useful for studying the mechanisms of hormonal regulation of casein-gene expression observed in prepartum mammary gland in vivo.

Effects of weaning and resuckling on casein messenger RNA levels in the rat mammary gland.

The level of C2-casein mRNA in the mammary gland was estimated after weaning litters from 7 day-lactating rats. After a steep deline (1/34th) within 2 days of weaning, it gradually declined until 4 day-weaning. After different periods of weaning, it was tested whether the mRNA level is restored by resuckling. The mRNA level was increased greatly in a system in which the rats in 1 or 2 day-weaning litters were resuckled for 2 days, suggesting that this system serves as an useful model for investigating regulation mechanisms of casein mRNA levels.

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.

Absolute requirement of glucocorticoid for expression of the casein gene in the presence of prolactin.

Second thoracic mammary glands of immature BALB/c female mice were stimulated to pregnancy-like lobuloalveolar (LA) development aftr 6 days of incubation in a corticosteroid-free step I culture medium containing insulin, prolactin, estradiol, progesterone, and growth hormone. A low basal level (0.0009%) of casein mRNA (mRNAcsn) sequences was detectable in the LA glands by a specific cDNA probe. Subsequent incubation of the LA glands for 3 days in medium containing insulin and prolactin or insulin and cortisol failed to elicit mRNAcsn above the basal level, indicating that neither prolactin nor cortisol alone can support casein gene expression. However, an increase in mRNAcsn levels was observed when the 3-day incubation with insulin and cortisol or insulin and prolactin was followed by 3 days of culture in presence of insulin, prolactin, and cortisol. When a 3-day incubation with insulin and prolactin was followed by 3 days in insulin and cortisol medium, mRNAcsn levels in the gland remained similar to the basal level. However, a 20-fold increase in the mRNAcsn levels ensued when the LA glands were sequentially incubated for 3 days in insulin and cortisol and then for another 3 days in insulin and prolactin medium. After a preincubation in insulin and cortisol medium, the LA glands retained residual cortisol during subsequent incubation in insulin and prolactin medium, and the mRNAcsn levels in these glands were related to the level of residual cortisol present. When mRNAcsn and the residual cortisol level reached a minimum, addition of fresh cortisol to the medium caused a 20-fold increase in the mRNAcsn levels. This indicates that cortisol is a limiting factor in insulin and prolactin medium and its presence is absolutely required for casein gene expression.

Role of prolactin in the transcription of beta-casein and 28-S ribosomal genes in the rabbit mammary gland.

Isolated mammary nuclei were incubated in the presence of HgCTP and the neosynthesized RNA was isolated with a SH-Sepharose column. The concentration of beta-casein mRNA and 28-S ribosomal RNA in the neosynthesized RNA fractions was evaluated using [3H]DNA probes complementary to beta-casein mRNA and 28-S rRNA respectively. In the unstimulated pseudopregnant rabbit, the transcription of both genes was easily detectable. Injections of prolactin progressively enhanced the transcription rate of both genes and preferentially the beta-casein gene. A comparison between the transcription rates and the accumulation of the corresponding gene products in the cell revealed that there is a good correlation between these two parameters for the 28-S rRNA gene. By contrast, the acceleration of beta-casein gene transcription by prolactin is unable to account for the simultaneous accumulation of beta-casein mRNA, indicating that prolactin is a potent stabilizer of casein mRNA. Injections of CB154 into lactating rabbits (a drug which suppresses the secretion of prolactin by hypophysis), induced a rapid drop of beta-casein mRNA concentration and a slow decline of beta-casein gene transcription. Simultaneously the drug was responsible for a marked and rapid decrease of 28-S rRNA gene transcription, while the concentration of the rRNA remained elevated. During weaning the transcription of the beta-casein gene and, to a lower degree, the transcripton of the 28-S rRNA gene proceeded more slowly and this phenomenon was accompanied by a progressive decline of the RNA concentrations.

Casein and α-lactalbumin messenger RNAs during the development of mouse mammary gland: Isolation, partial purification, and translation in a cell-free system

Messenger RNAs for the milk proteins, casein and α-lactalbumin, were isolated and partially purified from lactating mouse mammary glands by oligo(dT)cellulose chromatography followed by sucrose density gradient centrifugation. The translation of poly(A) + mRNA in a wheat germ cell-free system yielded three casein polypeptides and a putative precursor form of α-lactalbumin which were precipitated by specific antibodies and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The casein polypeptides synthesized in vitro had a molecular weight that was no greater than that of the caseins in mouse milk. The presence of individual casein mRNAs coding for these polypeptides was demonstrated by the translation of various fractions of mRNA obtained by sucrose density gradient centrifugation of poly(A) + mRNA. Casein mRNA activity increased about 250-fold between midpregnancy and the 10th–12th days of lactation, amounting to 50–60% of the total mRNA activity in that tissue. A similar study of α-lactalbumin mRNA showed an increase during lactation amounting to 0.2–0.4% of the total mRNA activity, which corresponds to the percentage of α-lactalbumin in total mouse milk protein.

Effet de l'ouabaïne sur l'action lactogene de la prolactine et sur le niveau des récepteurs prolactiniques mammaires

Ouabain added to the culture medium of rabbit mammary gland inhibits prolactin action on the initiation of lactose and casein synthesis. The degree of inhibition is a function of the ouabain concentration in the medium. Likewise, ouabain blocks the accumulation of casein mRNA supported by prolactin. In addition, ouabain provokes a rapid disappearance of prolactin receptors. Conversely prolactin keeps its capacity to enhance the concentration of casein mRNA and the parallel casein synthesis when K+ ions are totally absent from the culture medium. These results suggest that although prolactin induces a modification of the K+/Na+ ratio in the mammary cell and ouabain prevents this effect of prolactin, the inhibitory action of ouabain on lactogenesis can be explained essentially by its effect on the hormone receptors.

Long term effects of perinatal injection of estrogen and progesterone on the morphological and biochemical development of the mammary gland.

The effects of neonatal injection of estrogen and progesterone on the subsequent in vitro hormone responsiveness of murine mammary glands were determined by a coordinated study of the morphological development and the biochemical response of explants. After daily in vivo pretreatment with estrogen and progesterone, explants were cultured for 5 days in a chemically defined medium containing insulin alone or insulin, cortisol, and PRL. After the culture period, the morphological development of alveoli and lobules was rated. In addition, casein mRNA was quantitated using a specific complementary DNA hybridization probe. Perinatal 17 beta-estradiol exposure was found to increase casein mRNA content as well as lobular development. Conversely, perinatal progesterone treatment inhibited both casein mRNA induction and the formation of lobules. The administration of both estradiol and progesterone to newborns resulted in an antagonistic response between these two steroids, and there was no effect on casein mRNA levels or lobular development in comparison with untreated controls. Mammary tissues of perinatally estrogen-treate mice displayed greater lobular development and contained higher levels of casein mRNA in response to an extension of the duration of in vivo pretreatment from 6 to 9 days. A comparison of the PRL dose response in vitro suggested that exposure to estrogen perinatally sensitized the mammary tissue to the subsequent addition of PRL in culture. These studies indicate that injection of newborns with estrogen may enhance the subsequent hormonally regulated differentiation of the mammary gland. Exposure to progesterone may inhibit later development, and both steroids in combination may exert antagonistic effects. These effects were not mediated solely by alterations in the normal endocrine status of the treated animals but were reflected by the subsequent hormonal response of mammary explants in a defined culture medium. This altered sensitive to hormones may be important in the increased incidence of mammary dysplasias observed in these animals.

Prolactin regulation of casein gene expression: possible mediators.

The role of polyamines and cyclic nucleotides as possible mediators of PRL action on casein gene expression has been studied in mammary gland organ culture. The concentration of casein mRNA in insulin and hydrocortiso,ne-treated cultures was decreased upon the addition of spermidine (0.5 mm), whereas when added with insulin alone this polyamine maintained casein mRNA levels similar to those observed in the insulin and hydrocortisone control. The addition of an inhibitor of spermidine synthesis (methylglyoxal bis guanylhydrazone) with PRL decreased the level of casein mRNA, but this effect could not be reversed by the simultaneous addition of spermidine. cGMP, whether in combination with spermidine or alone, when added with insulin and hydrocortisone resulted in a 1.3- to 2.3-fold elevation of casein mRNA levels. This approximate 2- fold response was specific for cGMP and could not be duplicated by butyric acid, GMP, 8-Br-GMP, cAMP, cCMP, or cUMP. This was in contrast to the 6- to 22-fold induction usually...