Cultured Mouse Mammary Tissues Research Articles

Effect of prolactin on phosphate transport and incorporation in mouse mammary gland explants.

Inorganic phosphate is present in milk at a concentration that is severalfold higher than in maternal plasma. In cultured mammary tissues from 12- to 14-day-pregnant mice, the intracellular concentration of (32)PO(4) was six times higher than in the culture medium after a 4-h treatment with (32)PO(4). Of the principal lactogenic hormones [insulin (I), cortisol (H), and prolactin (PRL)], only I and PRL (in the presence of H and I) stimulated (32)PO(4) uptake into cultured mammary tissues; H, by itself or in the presence of I or PRL, inhibited (32)PO(4) uptake. All three lactogenic hormones together effected the greatest stimulation of (32)PO(4) uptake. Similar hormone effects were observed with regard to (32)PO(4) incorporation into lipids and trichloroacetic acid-insoluble molecules. In a time course study, the onset of the PRL stimulation of (32)PO(4) uptake and incorporation occurred 8-12 h after PRL addition; in dose-response studies, the PRL effect was manifested with PRL concentrations of 50 ng/ml and above. From kinetic studies, the apparent maximal velocity of PO(4) uptake was determined to be approximately 7.7 mM x h(-1) x l cell water(-1); the apparent Michaelis-Menten constant was approximately 3-5 mM. The PRL effect on (32)PO(4) uptake was abolished when sodium was absent from the uptake medium. These studies thus demonstrate a complex interaction of three hormones (I, H, and PRL) in the regulation of (32)PO(4) uptake and incorporation into macromolecules in cultured mouse mammary tissues.

Effect of prolactin on sodium iodide symporter expression in mouse mammary gland explants.

Iodide accumulates in milk at a concentration that is more than an order of magnitude higher than the iodide concentration in maternal plasma. In earlier studies from our laboratory, we have shown that prolactin (PRL) enhances iodide accumulation by two- to threefold in cultured mammary tissues taken from pregnant mice. In the present studies, we demonstrate via Western blotting techniques that prolactin elevates the quantity of the sodium iodide symporter (NIS) in cultured mouse mammary tissues. In time-course studies, the onset of the PRL effect of NIS accumulation was found to be between 4 and 16 h after addition of PRL to the explants. The lowest PRL concentration that elicited a significant response was 1 ng/ml, and a maximum effect was elicited with PRL concentrations >100 ng/ml. Actinomycin D, cycloheximide, and thiocyanate abolished the PRL effect on NIS accumulation, whereas perchlorate was without effect. These studies suggest that the PRL stimulation of iodide accumulation in milk is mediated, at least in part, by the PRL stimulation of NIS accumulation in mammary gland tissues. These studies further demonstrate that the PRL effect on NIS accumulation occurs via an RNA protein synthesis-dependent mechanism.

The MEK inhibitor PD 098059 inhibits prolactin-induced Nb2 cell mitogenesis but not milk product synthesis in cultured mouse mammary tissues

The MAP kinase pathway has been shown to be active in many growth factor signaling systems, including that of prolactin (PRL). In our studies, the main objective was to examine the possible involvement of MEK kinases (Map/Erk kinase kinases) in PRL-stimulated mitogenic and lactogenic processes. We used the MEK kinase inhibitor PD 098059 to block MEK kinase activation in the Nb2 cell line and mammary gland explants derived from 12- to 14-day pregnancy mice. PD 098059 attenuated PRL-induced Nb2 cell mitogenesis at 10 μM and a maximum inhibition was observed at 100 μM. In cultured mammary tissues, PD 098059 at 100 μM had no effect on the PRL stimulation of lipid, casein and lactose synthesis and iodide uptake. Further, the growth-inhibitory effect of PD 098059 on Nb2 cells was ameliorated when the drug was removed from the culture medium, indicating that PD 098059 acts in a reversible manner. When MEK1 was immunoprecipitated from PD 098059 and/or PRL treated Nb2 cells, PRL-stimulated MEK1 kinase activity was directly inhibited by PD 098059 at concentrations employed in the culture experiments. PRL has no effect on the tyrosyl phosphorylation of MAP kinases in cultured mammary tissues derived from pregnant mice, whereas earlier we found that PRL stimulates the tyrosyl phosphorylation of all four MAP kinases in Nb2 cells. The results suggest that the MAP kinase pathway plays an important role in the PRL stimulation of Nb2 cell mitogenesis but is not involved in the PRL stimulation of milk product synthesis.

Possible involvement of P13K in prolactin-stimulated milk product formation and iodide transport in mouse mammary explants.

The in vitro effect of wortmannin, an inhibitor of P13 kinase, on prolactin (PRL) stimulated p70S6K, iodide transport, and milk product synthesis were investigated in cultured mouse mammary tissues. Mouse mammary gland explants were initially incubated for 24 hr in media M199 containing 1 microg/ml insulin and 10(-7)M cortisol. A subsequent treatment with wortmannin impeded, in a dose-dependent fashion, the PRL stimulation of casein, lipid, and lactose synthesis as well as the PRL stimulation of iodide transport. Rapamycin (25 ng/ml), an inhibitor of p70S6K, also inhibited the effect of PRL on iodide transport; this drug was earlier shown to inhibit PRL effects on milk product synthesis. These results suggest the possible involvement of p70S6K and P13-kinase in PRL-stimulated milk product formation and iodide transport in mouse mammary explants. Since wortmannin caused a diminished cellular content of p70S6K and a reduced extent of P70S6K migration in polyacrylamide gels (likely due to dephosphorylation), P13-kinase likely lies upstream in the PRL signaling pathway for p70S6K activation.

The sodium iodide symporter: its emerging relevance to clinical thyroidology.

The sodium iodide symporter (NIS), an integral basolateral cell membrane protein of thyroid follicular cells, is responsible for active accumulation of iodide within the thyroid gland, a critical step in the biosynthesis of thyroid hormones. After cloning of rat NIS cDNA and of its human counterpart (hNIS), molecular characterization of the human NIS gene has revealed that the hNIS coding region contains 15 exons interrupted by 14 introns, encoding a protein of 643 amino acids. While not definitively resolved, the predicted structure of NIS comprises 12 putative transmembrane domains (1). Analysis by reverse transcriptase (RT)-PCR of hNIS mRNA in various tissues revealed its expression primarily in thyroid tissue as well as in breast, colon and ovary. Compared with normal thyroid tissue, Northern blot analysis has indicated markedly reduced levels of hNIS expression in thyroid carcinomas, which may account for the reduced radioiodide uptake activity observed in scintigraphically cold thyroid nodules and certain thyroid cancers. Interestingly, in follicular thyroid carcinoma cell lines that have lost the capacity to accumulate iodide, treatment with all-trans retinoic acid has recently been shown to upregulate NIS mRNA levels (2). If this leads to reexpression of functional NIS protein, an extended in vivo evaluation of such effects is desirable since it could have therapeutic consequences. Regulation of NIS gene expression has been examined by several groups of investigators. Thyroid-stimulating hormone (TSH) has been shown to increase NIS gene and protein expression in FRTL-5 cells and human thyrocyte monolayers, and this increase was accompanied by an enhanced iodide transport activity (3). However, the presence of abundant amounts of NIS protein in FRTL-5 cells with very low iodide transport activity (i.e. dissociation between NIS expression and function) also suggested that other TSH-regulated events may participate in activation or repression of NIS function. Furthermore, 3- to 4-fold elevated levels of NIS RNA and protein have been observed in Graves’ thyroid tissue compared with normal thyroid tissue, perhaps as a result of stimulation by TSH receptor antibodies that act to stimulate cAMP accumulation and, consequently, NIS expression (4). Interestingly, NIS RNA levels correlated well with thyroperoxidase (TPO) and thyroglobulin (Tg) (but not TSH receptor (TSHR)) gene expression, suggesting a similar mechanism of gene regulation. Further, prolactin stimulation of iodide uptake by cultured mouse mammary tissues has recently been reported (5). Subsequent studies by the same authors have suggested the existence of a prolactin-regulated sodium iodide symporter in mammary gland that shares characteristic features of NIS in the thyroid gland, such as sodium dependence and inhibition of iodide transport by thiocyanate and perchlorate. From these studies, it is apparent that prolactin stimulation of NIS expression by mammary gland tissue provides an important regulatory mechanism for iodide accumulation in milk during lactation (6). In contrast to agents stimulating NIS expression and function, transforming growth factor-b1, a potent inhibitor of growth and DNA synthesis in thyroid cells, has been found to suppress TSH-induced NIS mRNA and protein levels as well as TSH-stimulated iodide uptake activity in FRTL-5 cells (7). Further, in hyperplastic dog thyroid glands that had been stimulated chronically by iodide depletion and revealed increased levels of NIS, TPO and Tg mRNA expression, low doses of potassium iodide acutely inhibited both NIS and TPO, but not TSHR and Tg gene expression (8).

Characteristics of the prolactin stimulation of iodide uptake into mouse mammary gland explants.

We have recently reported that prolactin (PRL) stimulates iodide uptake into cultured mouse mammary tissues. This effect occurs in both TCA soluble and insoluble tissue fractions. The effect of PRL apparently involves an RNA-DNA-dependent mechanism, since actinomycin D and cyclohexamide abolish the PRL stimulation of iodide uptake and its incorporation into protein. Perchlorate and thiocyanate, inhibitors of the iodide transporter, also abolish the PRL effects on iodide uptake and incorporation. Similarly, propylthiouracil and aminotriazole, inhibitors of peroxidase, abolish both effects of PRL. Finally, the extent of iodide uptake in mammary cells is suppressed by about 50% in sodium-free medium. These studies thus suggest the existence of a sodium-iodide symporter in the mammary gland which has characteristics similar to the iodide transporter in the thyroid gland-that is, it is sodium dependent and is inhibited by perchlorate and thiocyanate. The fact that both iodide transporter inhibitors and peroxidase inhibitors abolish PRL-stimulated iodide uptake and incorporation suggests that there may be a coupled mechanism involving the iodide transporter and the peroxidase enzyme.

Effect of rapamycin on prolactin-stimulated S6 kinase activity and milk product formation in mouse mammary explants

The in vitro effects of rapamycin on prolactin (PRL)-stimulated S6 kinase activity and milk product synthesis were investigated in cultured mouse mammary tissues. Mouse mammary gland explants were initially incubated for 24 h in M199 media containing 1 μg/ml insulin and 10 −7 M cortisol. A subsequent treatment of the tissues with 1 μg/ml PRL for 12 h caused a 98% increase in S6 kinase activity in the cytosolic fraction; effects were not observed at earlier times. PRL at or above 500 ng/ml was needed to elicit a maximum stimulation of S6 kinase activity, and this response was specific for lactogenic hormones (PRL, hPL, hGH). Rapamycin, an inhibitor of 70 K S6 kinase, was employed to assess the possible physiological role of S6 kinase in the PRL stimulation of milk product formation. Rapamycin (25–100 ng/ml) impeded, in a dose-dependent fashion, the PRL stimulation of casein, lipid and lactose synthesis in concert with its inhibition of cytoplasmic S6 kinase activity. These results suggest a possible role for the activation of 70 K kinase in the signalling pathway for the PRL regulation of milk product synthesis in the mammary gland.

Effects of prolactin on galactosyl transferase and alpha-lactalbumin mRNA accumulation in mouse mammary gland explants.

Studies were carried out to determine the possible mechanism(s) by which prolactin (PRL) stimulates alpha-lactalbumin and galactosyl transferase activity in cultured mouse mammary tissues. In earlier studies it was shown that the onset of the PRL stimulation of galactosyl transferase activity occurs between 4 and 8 hr after adding prolactin to cultured mouse mammary gland explants, and a maximum effect is evoked by 24 hr. In contrast, an effect of prolactin on alpha-lactalbumin activity occurred 24 hr after adding PRL to mammary gland cultures, but not at earlier times. In the present studies, it is shown that prolactin effects an increased tissue accumulation of mRNAs for both alpha-lactalbumin and galactosyl transferase after a 4 to 6 hr culture with prolactin. The lowest concentration of prolactin that stimulates the accumulation of both galactosyl transferase and alpha-lactalbumin mRNA is approximately 10 ng/ml, and a maximum response is achieved with 200 ng/ml. Thus, it is likely that the effect of PRL on the activity of galactosyl transferase is causally associated with the effect of prolactin on the accumulation of its mRNAs. However, the PRL stimulation of alpha-lactalbumin activity is not temporally associated with the tissue accumulation of the mRNA for alpha-lactalbumin.

Temporal Effects of Prolactin on Casein Kinase Activity, Casein Synthesis and Casein mRNA Accumulation in Mouse Mammary Gland Explants

Studies were carried out to investigate the mechanism by which prolactin stimulates casein synthesis in cultured mouse mammary tissues. The onset of the prolactin stimulation of casein synthesis occurs 8-10 hours after prolactin addition to cultured tissues; a maximum effect is evoked by 16 hours. A similar time-course of response to prolactin was observed when the tissue accumulation of the mRNA for beta-casein was determined. At 16 hours, however, prolactin had no effect on tissue-specific casein kinase activity which is associated with the Golgi apparatus. These studies suggest that the tissue content of casein mRNA is causally related to the onset of the prolactin stimulation of casein synthesis in cultured mouse tissues. In contrast, the activity of the kinase that catalyzes the phosphorylation of the caseins is not altered by prolactin and thus apparently is not a contributing factor to the early prolactin effect on casein synthesis.

Effect of a tyrosine kinase inhibitor, genistein, on the actions of prolactin in cultured mouse mammary tissues

Genistein, an inhibitor of tyrosine kinase, was used to determine the possible role of tyrosine kinase in the prolactin (PRL) stimulation of milk product formation and ornithine decarboxylase (ODC) activation in cultured mouse mammary gland tissue. Genistein (10–200 μM) inhibited in a dose-response fashion the PRL stimulation of casein, lipid and lactose synthesis as well as ODC activation. Genistein, however, did not inhibit the phospholipase C, phorbol myristate acetate or cAMP effects on ODC activation. These results suggest the possible involvement of tyrosine kinase in the mechanism by which PRL expresses its effects in mammary gland tissues.

Temporal Effect of Prolactin on the Activities of Lactose Synthetase, -Lactalbumin, and Galactosyl Transferase in Mouse Mammary Gland Explants

The onset of the prolactin (PRL) stimulation of lactose synthesis is between 4 and 8 hr after adding PRL to cultured mouse mammary tissues. The synthesis of lactose is catalyzed by the enzyme lactose synthetase, which is composed of two parts, alpha-lactalbumin and galactosyl transferase. In time-sequence studies, it was found that the activity of galactosyl transferase is enhanced by PRL in concert with the onset of the PRL stimulation of lactose synthesis. In contrast, the earliest detectable effect of PRL on alpha-lactalbumin activity occurred 24 hr after adding PRL to the cultures. It is, therefore, apparent that the rate-limiting component for the PRL stimulation of lactose synthesis in cultured mouse mammary tissues is galactosyl transferase activity.

Effect of a kinase C inhibitor, gossypol, on the actions of prolactin in cultured mouse mammary tissues

Gossypol, a drug which has been shown to be an inhibitor of kinase C activity in mouse mammary tissues, is shown to abolish several of the actions of prolactin in cultured mouse mammary gland explants. The prolactin effects that are abolished include its stimulatory effects on (a) ornithine decarboxylase activity, (b) the rate of [ 3H]uridine incorporation into RNA, (c) the rate of [ 3H]leucine incorporation into a casein-rich phosphoprotein fraction, and (d) the rate of [ 14C]acetate incorporation into lipids. Since the inhibitory concentrations of gossypol employed in these studies correspond well with the gossypol concentrations required to inhibit kinase C activity, we conclude that ongoing kinase C activity is essential for prolactin to express its differentiative actions in mammary tissues.

Effect of 3-isobutyl-1-methylxanthine on prolactin actions on RNA synthesis, casein synthesis, lipid synthesis and ornithine decarboxylase activity in mouse mammary gland explants.

The effect of 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, was tested on several actions of prolactin in cultured mouse mammary tissues. At concentrations of 0.5 mM and above, IBMX abolished the actions of prolactin on RNA and casein synthesis. IBMX by itself, stimulated ornithine decarboxylase (ODC) activity in a dose-response fashion; but the IBMX at concentrations up to 1 mM had no effect on the magnitude of the prolactin-stimulated ODC activity. IBMX inhibited in a dose-response fashion the rate of [14C]-acetate incorporation into lipids; however, prolactin stimulated lipid biosynthesis in the presence of IBMX concentrations of up to 1 mM.