Bertrand Teyssot

The interaction of prolactin with its receptors in target tissues and its mechanism of action.

Publisher Summary This chapter describes the interaction of prolactin with its receptors in target tissues and with its mechanism of action. Prolactin is a hormone secreted by the anterior pituitary gland, whose primary action involves the development of the mammary gland and production of milk, which is essential for the survival of newborn mammals. The hormonal regulation of prolactin receptors is complex. Prolactin receptors have been localized in a number of different tissues, although they have been most extensively studied in the mammary gland and liver. Receptor numbers differ dependent upon the physiological state of the animal. A large percentage of prolactin receptors are located within the cell in the Golgi and lysosomal compartments. Prolactin can regulate its receptor number both in a positive and negative fashion. A slow upregulation is observed most generally in vivo following extended periods of injection, corresponding to a large extent, to the accumulation of Golgi membranes that occurs during differentiation of the mammary gland. In rat liver cells in suspension culture, this upregulation represents a reduced level of degradation as well as an increased synthesis of receptors. The downregulation of prolactin receptors is a more rapid and reversible process. It can be observed both in vivo and in vitro in rabbit mammary glands and rat liver.

Induction of β-casein mRNA accumulation by the putative prolactin second messenger added to the culture medium of cultured mammary epithelial cells

Milk protein synthesis is under a hormonal control in which a protein hormone, prolactin, plays an essential role. Experiments in several species have shown that the induction of casein synthesis is coincident with an accumulation of the corresponding mRNAs [l-3]. These phenomena can be reproduced using mammary fragments cultured in synthetic media (4-61. Isolated mammary epithelial cells in primary culture have also been shown to retain their capacity to respond to the prolactin stimulus by the accumulation of casein mRNAs [7,8]. Experiments carried out in the rabbit demonstrated that the accumulation of casein mRNAs results from an acceleration of the transcription rate of the casein genes and from a stabilization of the mRNAs [9]. These effects are amplilied by glucocorticoids which per se are not inducers and they are inhibited by progesterone [lo]. Independently, the translation of casein mRNA is activated by prolactin and inhibited by progesterone [I 11. The evaluation of /?-casein gene transcription also proved possible using isolated nuclei [9,10,12]. This technique has been used for the search of the intracellular relay carrying the hormonal information from the receptors located in the plasma membrane to the target genes. It has been observed that the incubation of mammary membranes with prolactin provokes the release of a factor which is a potent stimulator of /?-casein gene transcription when added to isolated nuclei [ 13151. This factor is specifically generated by lactogenic hormones and it stimulates at least one of the prolactin-sensitive genes. The active fraction liberated from mammary membranes by prolactin has a small M,-value [14]. Here, the supernatant from membranes which contain the putative prolactin second messenger were added to the culture medium of isolated mammary epithelial cells, in an attempt to provoke the accumulation of casein mRNA and mimic prolactin action and in order to overcome possible problems encountered with the use of isolated nuclei.

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.

Control of casein gene expression in isolated cultured rabbit epithelial mammary cells

Abstract Epithelial cells were isolated from mammary glands of pseudo-pregnant rabbits after a digestion of the tissue by collagenase and cell fractionation on a percoll gradient. These cells were cultured for various times in the presence of serum until growth was sufficient. The serum was then withdrawn for two days and hormones were added for one more day in the absence of serum. Cells were bound either directly to the plastic of the flask or to collagen spread in one or two layers. The concentration of β-casein mRNA was evaluated at the end of the culture by using a 3 H-DNA complementary to β-casein mRNA. The concentration of β3-casein mRNA was increased when prolactin was present in the culture medium. Cortisol amplified this effect while being totally inefficient alone. This induction was obtained as well when cells were cultured on plastic or on monolayer, bilayer or floating collagen, and also when they were kept in suspension. However, they apparently responded to prolactin only when fibroblasts were eliminated by adding hydroxy-L-proline (allo) or cholera toxin to the culture medium or by using collagen. The lactose synthetase activity and casein synthesis always remained extremely low regardless of the presence of hormones in the culture medium. Cells cultured on collagen tended to become reorganized in a way somewhat similar to that in mammary tissue, whereas cells spread on plastic remained uniformly distributed. These results suggest that isolated rabbit mammary epithelial cells have kept the essence of their capacity to respond to prolactin regardless of the culture technique. Isolated cultured cells thus appear to be a viable tool for further studies on the mechanism of prolactin action.

Prolactin and Casein Gene Expression in the Mammary Cell

The onset of milk synthesis and secretion is the result of complex and multiple processes which operate during pregnancy and at parturition. Before pregnancy, the mammary gland is restricted to a few duct cells. During pregnancy, under the influence of estrogens, progesterone, and growth factors, the secretory cells progressively appear. They are organized in an epithelium forming a large number of alveoli. At the end of pregnancy, many alveolar cells are present and the development of the mammary gland is more or less complete according to species. After parturition, when milk secretion is triggered, the alveolar cells become polarized and hypertrophic. This transformation corresponds to the activation of the cells which have to elaborate and secrete huge amounts of proteins, lipids, and carbohydrates throughout lactation. Thus before being fully active, the mammary gland has been subjected to at least three types of transformation: (1) a cell multiplication which leads to the formation of alveoli, (2) an activation of specific genes directly involved in the elaboration of milk, and (3) an organization of the alveolar cells which become enriched in cellular organelles involved in the bulky production and secretion of milk. After weaning, the alveolar cells disappear until the next pregnancy.