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.

Prolactin stimulation of tyrosyl phosphorylation of Shc proteins in Nb(2) lymphoma cells, but not mammary tissues.

Prolactin (PRL) stimulates lactogenesis in mammary cells and mitogenesis in a variety of cell types including Nb(2) cells. Studies indicate that a different composite of signaling pathways is involved in the PRL stimulation of mitogenesis as compared to lactogenesis. In the present studies, PRL is shown to stimulate the tyrosyl phosphorylation of all three isoforms of Shc proteins in Nb(2) cells (mitogenesis), but not in the mammary gland. Maximal phosphorylation of the Shc proteins is expressed between 10 and 15 min after a 50-ng/ml PRL treatment. In addition, there is an increased association between the Grb2 protein and Shc proteins upon PRL stimulation. However, no increased association between JAK2 and Shc proteins was observed in either the Nb(2) cells or mammary tissues.

Prolactin stimulation of iodide uptake and incorporation into protein is polyamine-dependent in mouse mammary gland explants.

Previous studies have demonstrated that the prolactin stimulation of most lactational processes (casein, lactose, and triglyceride synthesis) requires an earlier stimulating effect of prolactin on the synthesis of the polyamines. Spermidine appears to be the specific polyamine required for prolactin to enhance milk product synthesis. Inorganic iodide is present in milk at more than an order of magnitude higher concentration than that of the maternal plasma. Since prolactin stimulates iodide accumulation in milk, the goal of these studies was to determine the role of the polyamines in this hormone response. Two drugs were employed in these studies: DFMO (difluoromethylornithine), which inhibits ornithine decarboxylase, and MGBG [methylglyoxal bis(guanyl-hydrazone)], which inhibits S-adenosyl methionine decarboxylase. In mammary gland explants from midpregnant (10-14 days of pregnancy) mice, MGBG at 100 microM abolished the prolactin stimulation of iodide uptake and incorporation into milk proteins, whereas DFMO caused a concentration-dependent inhibition of the PRL response. Selected sensitivity of the MGBG and DFMO inhibitions was validated by a reversal of the drug inhibitions with the addition of 1 mM spermidine to the culture medium. These data suggest that the polyamine signaling pathway is involved in the prolactin stimulation of iodide uptake into milk.

Changes in numbers of prolactin receptors during the cell cycle of Nb2 cells.

Lactogenic hormones including prolactin (PRL) have mitogenic effects on Nb2 cells, a pre-T lymphoma cell line. Previous studies have characterized the PRL stimulation of cellular processes such as RNA/DNA synthesis, signalling molecule activation, and the expression of specific genes. The data presented here explores the fluctuations in plasma membrane PRL receptor (PRLR) number that occur in the Nb2 cells during the course of a 24 h cell cycle. PRLR abundance was determined by measuring specific binding of [I125] oPRL to G1 arrested-intact Nb2 cells in which the cell cycle was initiated by addition of nonradioactive oPRL. Preliminary studies revealed that 1 ng/ml oPRL was the minimum PRL concentration that causes a maximal stimulation of mitogenesis, without interfering with [I125] oPRL binding measurements. Subsequent experiments revealed that upon cell cycle initiation of G1 arrested Nb2 cells with 1 ng/ml oPRL, PRLR number remained constant for the initial 6 h. After 8 h PRLR numbers decreased and at 12 h, the PRLR number was less than 25% of the initial value. After 12 hr, PRLR numbers increased and reach initial values by 18 hr. These studies show that the expression of cell surface PRL receptors is modulated in a sequential fashion during the cell cycle of Nb2 cells.

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 microM and a maximum inhibition was observed at 100 microM. In cultured mammary tissues, PD 098059 at 100 microM 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.

Cyclic AMP impairs the PRL stimulation of iodide uptake into mouse mammary tissues.

In earlier studies the uptake of iodide into mammary cells was found to occur by a mechanism similar to that in thyroid cells (i.e., the uptake occurs via a sodium-iodide symporter that is inhibited by perchlorate and thiocyanate). Although cyclic AMP stimulates the iodide transport mechanism in thyroid cells, the present studies show that cyclic AMP, as well as pharmacological agents that elevate cyclic AMP, impair iodide uptake into mammary cells. In addition, elevated cyclic AMP levels interfere with the PRL stimulation of iodide uptake as well as iodide incorporation into proteins in mammary tissues. Clearly, cyclic AMP has opposite effects on regulating iodide uptake processes in mammary versus thyroid cells.

Lovastatin inhibits prolactin-induced Nb2 cell mitogenesis and milk product synthesis in mouse mammary gland explants.

The p21ras protein 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 further the involvement of ras in prolactin-stimulated mitogenic and metabolic processes. We used the farnesylation inhibitor lovastatin to block effectively ras-dependent signaling in Nb2 cells, a pre-T lymphoma cell line, and mammary gland explants derived from 12- to 14-day pregnant mice. Lovastatin completely inhibited PRL-induced Nb2 cell mitogenesis at 1 micron. In the mammary gland, lovastatin at 0.1 micron inhibited the PRL stimulation of lipid and lactose synthesis; at 2 microns, lovastatin abolished the PRL stimulation of casein production. When p21ras was immunoprecipitated from lovastatin (25 microns)-treated Nb2 cells and mammary gland explants, the unfarnesylated (inactive) form of ras was shown to be redistributed from the cell membrane to the cytosolic compartment of the cell. This suggests that the anchoring of ras to the cell membrane is essential for its action in prolactin signal transduction. Thus, in addition to the well-known active participation of ras in mitogenic growth factor signaling, the presence of functional ras also appears necessary for the PRL stimulation of specific metabolic processes involved in lactation.

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 microgram/ml insulin and 10(-7) M cortisol. A subsequent treatment of the tissues with 1 microgram/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.

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.

Differential tyrosyl-phosphorylation of multiple mitogen-activated protein kinase isoforms in response to prolactin in Nb2 lymphoma cells.

Prolactin (PRL) stimulates mitogenesis and differentiative processes in a variety of cell types. Not all of the molecules involved in PRL signaling, which follows an initial PRL-receptor interaction, have been identified. In the present studies, PRL is shown to stimulate the differential tyrosyl phosphorylation of three isoforms (ERK-1, 2, and 4) of mitogen-activated protein kinases (MAP kinase) in a rat pre-T lymphoma cell line (Nb2). Evidence also suggests that PRL stimulates the tyrosyl phosphorylation of ERK-3, a MAP kinase isoform recently identified. When G1-arrested Nb2 cells are treated with 50 ng/ml oPRL, ERK-1 through 3 become tyrosyl phosphorylated within minutes (an indication of enzyme activation) and then become dephosphorylated within 30 min. Conversely, ERK-4 is rapidly tyrosyl phosphorylated by 5 min, and remains in this state for at least 1 hr.

Prolactin's effects on lipoprotein lipase (LPL) activity and on LPL mRNA levels in cultured mouse mammary gland explants.

The in vitro effects of prolactin (PRL) on lipoprotein lipase (LPL) activity and on LPL mRNA levels were studied in cultured mammary tissues derived from mid-pregnant mice. Mouse mammary gland tissues were initially incubated for 24 hr in M199 media containing 1 microg/ml insulin and 10(-7) M cortisol. A subsequent treatment of the tissues with 1 microg/ml PRL caused a 76% increase in heparin-releasable LPL (hrLPL) activity after 24 hr. A significant increase in LPL activity was detected 16 hr after PRL addition, but not at earlier times. PRL at 100 ng/ml elicited a maximum stimulation of LPL activity. When Northern hybridization techniques were employed, PRL was also found to increase the tissue content of LPL mRNA; this effect was initially detected after a 6-hr PRL treatment employing PRL concentrations of 50 ng/ml and above. Specificity studies revealed that only lactogenic hormones stimulated LPL activity and LPL mRNA accumulation in cultured mammary tissues. PRL also expressed a small (25% increase), but significant, effect on ATP citrate-lyase activity in mammary tissues cultured for more than 6 hr with the hormone.

Characteristics of the prolactin stimulation of c-fos mRNA levels in mouse mammary gland explants.

The signal transduction pathway(s) for the prolactin (PRL) regulation of lactogenic processes in the mammary are not fully known. Recent studies indicate that the PRL occupancy of its receptor activates a tyrosine kinase (JAK-2), which is constitutively associated with the receptor. In the present studies, we have characterized the PRL stimulation of c-fos mRNA accumulation in mouse mammary gland explants that were precultured with insulin and cortisol for 36 hr. In time course studies, an initial effect (2-fold increase) of PRL was apparent after 15 min, a 4-fold increase occurred after 60 min, and a 2-fold increase was apparent after 2 hr. Dose-response experiments indicated an initial effect with 5 ng/ml PRL and a maximum effect with 500 ng/ml PRL. The accumulation of c-fos; mRNA in the cultured mammary tissues was also increased by human GH, recombinant bovine PRL, cycloheximide, and phorbol 12-myristate 13-acetate (TPA). The cycloheximide and PRL responses were additive, thus indicating that cycloheximide did not "superinduce" the PRL stimulation of c-fos mRNA accumulation. Downregulation of protein kinase C (PKC) by pretreating mammary tissues for 1 day with TPA did not affect the magnitude of the PRL stimulation of c-fos mRNA accumulation. The findings from these studies are consistent with c-fos playing a role in the PRL stimulation of lactogenic processes in the mammary gland. PRL had no effect on c-jun mRNA accumulation under any of the experimental conditions employed in the c-fos studies.

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.

Evaluation of prolactin-like activity produced by concanavalin-A stimulated mouse splenocytes.

The prolactin (PRL)-like activity released into synthetic culture medium from concanavalin-A (Con-A)-stimulated mouse splenocytes was bioassayed using 3H thymidine incorporation into Nb2 lymphoma cells. At low cell density (1.0 x 10(6) cells/ml medium) Con-A-stimulated splenocytes from Balb/c mice released more PRL-like activity than did splenocytes from normal C3H/HeJ mice. This difference was maintained with animals of either sex, and for animals of different ages (1, 3 and 5 months). The strain difference was observed both when splenocytes were co-cultured with Nb2 cells, and when postcultured medium from splenocytes was tested. Con-A-stimulated splenocytes also released a substance into the medium which was inhibitory to PRL-induced Nb2 cell proliferation. The concentration of the inhibitory substance in the medium was related to the number of splenocytes in culture and was similar in splenocyte media preparations from both strains of mice. Polyclonal antisera to bovine PRL, rat PRL, mouse placental lactogen II, and mouse PRL did not neutralize the effect of Con-A-stimulated splenocytes to induce Nb2 cell proliferation. Bioassay of the partially purified PRL-like activity found in postcultured medium indicated that the dose-dependent induction of Nb2 cell proliferation was parallel with that of purified mPRL. Using two mouse PRL antisera (Sinha and Talamantes #118) in Western blot analyses, no antibody binding was observed to the partially-purified splenocyte PRL-like material. The data from the antisera neutralization experiments and the SDS/PAGE Western blot analyses clearly indicated that the PRL-like activity produced by Con-A-stimulated splenocytes has little homology with pituitary PRL. Further studies are required to establish the precise molecular identity of the PRL-like activity from Con-A-stimulated splenocytes.

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.

Activation of JAK2 tyrosine kinase by prolactin receptors in Nb2 cells and mouse mammary gland explants.

One of the earliest cellular responses to prolactin (PRL) binding in Nb2 cells, a rat pre-T lymphoma cell line, is an increase in tyrosine phosphorylation of cellular proteins. In this work, immunologic techniques have been used to demonstrate that in Nb2 cells and in mouse mammary gland explants, JAK2, a non-receptor tyrosine kinase, is activated following stimulation with PRL. PRL stimulated tyrosine phosphorylation of JAK2 at times as early as 30 sec and concentrations of PRL as low as 0.5 ng/ml (2.5 pM) in Nb2 cells and 100 ng/ml (5 nM) in mammary gland explants. When JAK2 was immunoprecipitated from solubilized Nb2 cells or mammary gland explants and incubated with [gamma-32P]ATP, 32P was incorporated into a protein migrating with an apparent molecular weight appropriate for JAK2 only when cells had been incubated with PRL, indicating that JAK2 tyrosine kinase activity is exquisitely sensitive to PRL. In Nb2 cells, JAK2 was found to associate with PRL receptor irrespective of whether or not the cells had been incubated with PRL. These results provide strong evidence that JAK2 is constitutively associated with the PRL receptor and that it is activated and tyrosine phosphorylated upon PRL binding to the PRL receptor. These results are consistent with JAK2 serving as an early, perhaps initial, signaling molecule for PRL.

Development of the mammary gland and lactation.

Many hormones, growth factors, and protooncogene products involved in mammary development and lactation have been identified; however, the mechanism of their concerted action remains to be explained. In addition to these regulatory factors, normal mammary development and lactation require the cell-cell interaction of stromal and parenchymal elements in the mammary gland. Recent studies indicate that PRL effects on target cells are likely transmitted into cells via activation of tyrosine kinase associated with a protein-designated JAK-2, while other studies have identified stimulatory and inhibitory factors that interact with the 5' promotor regions of milk-product genes.

Identification of two specific prolactin binding sites on Nb2 rat lymphoma cells.

Several laboratories have earlier identified and characterized a specific prolactin binding site (receptor) on Nb2 cells which has a Kd of about 2 x 10(-10) M and a binding capacity of about 12,000 receptor sites per cell. Using [I125]-labeled human growth hormone (hGH), we have confirmed the results of these earlier reports. In addition, a second, "high-affinity" receptor site for prolactin has been identified. This site has a Kd of about 2 x 10(-12) M and there are an average of 67 sites per cell. Binding of [125]-hGH to the high-affinity receptor site is saturated by 1 min after incubation at 37 degrees C, whereas saturation of the lower affinity site requires a 1-3 hr incubation at 37 degrees C. Specificity of prolactin binding to both the high- and low-affinity "receptors" was established by the fact that several nonlactogenic hormones, including bovine growth hormone and insulin, do not displace [I125]-hGH from the receptor sites. When Nb2 cells were incubated for 1 hr at 0 degrees C with 100 pg/ml [I125]-hGH, no binding to the high-affinity receptor was detected, thus demonstrating the temperature dependence of binding to this receptor. The kinetics of binding of hGH to the high-affinity binding site correlates well with the dose-response effects of lactogenic hormones on mitogenesis in the Nb2 cells. In addition, the rapid binding of hGH to the high-affinity receptor may be related to specific effects of lactogenic-hormones which occur within seconds or minutes after adding these hormones to Nb2 cells.