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.

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.

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.

[Comparison of the mechanism of action of insulin and prolactin]. / Comparaison du mécanisme d'action de l'insuline et de la prolactine.

One of the major actions of prolactin in the mammary cell is to activate the expression of casein genes by enhancing the transcription rate of the genes. Anti-prolactin receptor antibodies can mimic prolactin action when added to mammary cells, suggesting that a relay is formed at the membrane level and transferred to the target genes. None of the classical hormone intracellular relays can account for the transfer of the prolactin message. The incubation of membranes from various tissues containing prolactin receptor with prolactin provokes the release of a factor which specifically accelerates the transcription of the beta-casein gene in isolated mammary nuclei. The factor is thermostable, inactivated by trypsin and is eluted from G-10 Sephadex as a molecule smaller than 1000 daltons. The action of the factor is prevented by phosphatase inhibitors. These results are strikingly reminiscent of those obtained with insulin which activates enzymes via a factor released from the membranes and which dephosphorylates the enzymes. The analogy between the action mechanism of prolactin and insulin is discussed.

[Control of the expression of milk protein genes by prolactin, glucocorticoids and progesterone]. / Le contrôle de l'expression des gènes des protéines du lait par la prolactine, les glucocorticoïdes et la progestérone.

The expression of casein genes is under the control of several hormones of which the most important are prolactin, glucocorticoids and progesterone. In pseudopregnant or mid-pregnant rabbit having partially developped but inactive mammary gland, prolactin induces casein synthesis. The phenomenon is accompanied by an accumulation of casein mRNAs and by a stimulation of their translation. The accumulation of casein mRNAs results from an acceleration of the transcription of the corresponding genes and from a stabilization of the mRNAs. These prolactin effects are amplified by glucocorticoids which are not per se inducers and they are inhibited by progesterone. The essential action of prolactin and glucocorticoids can be obtained in cultured mammary explants and epithelial cells. This induction is accompanied by a transformation of the mammary cell in which are accumulated ribosomes and membranes involved in milk synthesis and exportation. This transformation is favoured by prolactin and inhibited by progesterone. Hence, the abundant milk secretion is triggered only after parturition when the predominence of progesterone is reversed in favour of prolactin. Prolactin incubated with mammary membranes promotes the formation of a factor capable of accelerating beta-casein gene transcription when added to isolated mammary nuclei. This factor is formed only by lactogen hormones and from prolactin receptor containing membranes. The information contained in the factor seems to be understood only by prolactin target genes. The generation of the factor can be provoked by anti-prolactin receptor antibodies and it is inhibited by tubulin binding drugs such as colchicine. The molecule exhibiting prolactin-like activity has a small molecular weight, it is thermostable and inactivated by trypsin. The stimulation of beta-casein gene transcription is abolished when the factor is incubated with nuclei in the presence of phosphatase inhibitors. These facts suggest that prolactin after its binding to its peripheral receptors triggers the release of a small peptide which migrates to nuclei where it activates the transcription of the prolactin target genes through a dephosphorylation of nuclear proteins. This small peptide is a good candidate to the prolactin intracellular relay.

Prolactin induces release of a factor from membranes capable of stimulating beta-casein gene transcription in isolated mammary cell nuclei.

Crude microsomes from lactating rabbit mammary gland were incubated with prolactin. The incubation mixture was centrifuged and the supernatant was incubated with isolated mammary cell nuclei from lactating rabbits treated for 4 days by bromocryptin to antagonize prolactin and to deinduce casein gene transcription. Nuclei were incubated with HgCTP, and the newly synthesized mercurated RNA was isolated on SH-Sepharose columns. The content of beta-casein mRNA sequences in the fraction eluted with 2-mercaptoethanol was estimated with a [(3)H]cDNA probe obtained from partially purified beta-casein mRNA. The supernatant markedly stimulated beta-casein gene transcription but not 28S rRNA transcription. The same effect was obtained with other lactogenic hormones such as human growth hormone and ovine placental lactogen but was not observed with bovine growth hormone, insulin, parathyroid hormone, luteotropic hormone, or epidermal growth factor. Prolactin and human growth hormone were totally inactive when added directly to nuclei. The factor stimulating beta-casein gene transcription was also generated by membranes containing prolactin receptors such as those from liver, ovary, adrenals, and brain but not by membranes from heart, lung, and muscle, which do not bind prolactin. The factor stimulated beta-casein transcription when added to mammary nuclei from pseudopregnant or bromocryptin-treated lactating rabbits, in which the transcription rate is submaximal, but was ineffective on mammary nuclei prepared from untreated fully lactating rabbits. The factor was unable to induce beta-casein gene transcription in nuclei isolated from rabbit liver and reticulocytes. The factor did not stimulate the transcription of globin genes in nuclei isolated from reticulocytes or the transcription of mammary "housekeeping" genes evaluated by a cDNA probe prepared from total mRNA isolated from an unstimulated mammary gland. The transcription of beta-casein genes was abolished by adding alpha-amanitin to the medium in the presence or in the absence of the factor, indicating that the generation of mercurated beta-casein mRNA sequences depended upon the transcriptional activity of RNA polymerase II. The addition of the factor to the incubation mixture did not enhance total and alpha-amanitin-sensitive RNA synthesis. These data suggest that the binding of prolactin to its receptor in vitro induces the formation of a second messager, which specifically stimulates the transcription of prolactin-sensitive genes.

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.

Role of progesterone and glucocorticoids in the transcription of the 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 measured using [3H]cDNA probes complementary to beta-casein mRNA and 28-S rRNA respectively. Prolactin injected into pseudopregnant animals accelerates the transcription of both genes and increases the stability of the beta-casein mRNA but not of the 28-S rRNA. Progesterone injected simultaneously with prolactin reduced considerably all these effects of prolactin, with a lower efficiency when the highest doses of prolactin were injected. These observations suggest that progesterone attenuates the transfer of prolactin information related to the lactogenesis into the mammary cell. Glucocorticoids injected with prolactin amplify the prolactin action on the expression of the beta-casein gene but not of the 28-S rRNA genes. In the absence of prolactin (a situation obtained by injecting simultaneously CB 154, a drug which inhibits pituitary prolactin secretion) glucocorticoids exhibit no effect. In the lactating rabbit glucocorticoids do not delay significantly the drop of beta-casein gene transcription rate provoked by weaning or by prolactin withdrawal obtained by injecting CB 154. A comparison of the beta-casein mRNA accumulation and the transcription rate of the beta-casein gene indicates that glucocorticoids act essentially by amplifying the activation of beta-casein transcription supported by prolactin, but not by enhancing the stability of the beta-casein mRNA.

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.