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The most common and most deadly form of “ovarian”cancer is High-Grade Serous Carcinoma (HGSC), now appreciated to arise from epithelial cells of the fallopian tube/oviduct. The serum analyte with the greatest specificity and sensitivity that correlates with the presence or absence of HGSC is prolactin (PRL). However, unknown is whether elevated PRL initiates disease or is the result of disease, although once cancer is present,it is clear that PRL promotes disease. In order to examine the potential for disease initiation in normal animals, it was first important to determine which cells in the mouse oviduct respond to PRL. In many cell types, the long form of the Prlr mediates increased survival, proliferation and migration, whereas the short forms have the opposite effect. After establishing equivalent efficiencies of primers for the long form (LF) and 3 short forms (SF1-3) of the murine Prlr and the endogenous control gene, Gapdh, the mRNA expression profiles of these forms of the receptor were followed by RT-qPCR as a function of stage of the estrous cycle (proestrus, estrus, metestrus, diestrus - determined by vaginal cytology) and region of the oviduct (isthmus, ampulla and fimbria). Expression of SF1 was not detected. SF2 was essentially consistently expressed throughout the oviduct and at all stages of the estrous cycle. By contrast, the ratio of LF to SF3 varied by region of the tube, with more SF3 towards the fimbria and more LF towards the isthmus. The epithelium of the oviduct is primarily composed of multi-ciliated cells and secretory cells, with more ciliated cells towards the fimbria and more secretory cells towards the isthmus. The RT-qPCR results therefore suggested the possibility that a greater proportion of LF Prlr was present on secretory cells and a greater proportion of SF3 Prlr on ciliated cells. Using antibodies raised against intracellular peptide regions specificto the LF (aa 309-325) and SF3 (aa 281-296), both receptor isoforms were localized by immunofluorescence to apical regions of both epithelial cell types,but the presence of receptors on cilia (clearly demonstrated by 3Dreconstruction and rotation) complicated analysis of relative fluorescence by microscopy. Only the LF Prlr signals via Stat5 and so it was anticipated thatStat5 activation could serve as a substitute marker of the relative presence ofLF Prlr. Following in vivo intraperitoneal injection of PRL (5μg/g, 30 min),activated Stat5 was localized to epithelial cells at the base of, and in between, mucosal folds, thereby suggesting a further regionality to receptor distribution. For the fimbrial region only, which is where HGSC is thought to arise, expression of both the LF and SF3 Prlr changed as a function of the stage of the estrous cycle, with highest mRNA expression at diestrus/proestrus. Ongoing work includes flow cytometry of epithelial subpopulations and spatialanalysis of gene expression.

Even though mutations in the tumor suppressor, BRCA1, markedly increase the risk of breast and ovarian cancer, most breast and ovarian cancers express wild type BRCA1. An important question is therefore how the tumor-suppressive function of normal BRCA1 is overcome during development of most cancers. Because prolactin promotes these and other cancers, we investigated the hypothesis that prolactin interferes with the ability of BRCA1 to inhibit the cell cycle. Examining six different cancer cell lines with wild type BRCA1, and making use of both prolactin and the growth-inhibiting selective prolactin receptor modulator, S179D PRL, we demonstrate that prolactin activation of Stat5 results in the formation of a complex between phospho-Stat5 and BRCA1. Formation of this complex does not interfere with nuclear translocation or binding of BRCA1 to the p21 promoter, but does interfere with the ability of BRCA1 to transactivate the p21 promoter. Overexpression of a dominant-negative Stat5 in prolactin-stimulated cells resulted in increased p21 expression. We conclude that prolactin inhibits a major tumor-suppressive function of BRCA1 by interfering with BRCA1's upregulation of expression of the cell cycle inhibitor, p21.

Prolactin (PRL) has many functions in the CNS, including neuroprotection. During lactation, the dorsal hippocampus is protected from excitotoxic kainic acid (KA)-induced cellular damage. We have previously reported that systemic pre-treatment with ovine PRL had similar protective effects in female rats. Here, we asked (1) whether intracerebral human PRL (hPRL) would have the same action, (2) because phosphorylated PRL is high in lactation, whether a mimic of phosphorylated hPRL, human prolactin in which the normally phosphorylated serine at position 179 is replaced with an aspartate (S179D-PRL), had similar activity, and (3) what signaling pathways mediated the protective effect. Female ovariectomized (OVX, 1 month) rats were implanted with micro-osmotic pumps connected to unilateral icv cannulae directed at the right lateral ventricle. The pumps delivered 0.10 ng/h of hPRL, S179D-PRL, a combination of hPRL+S179D-PRL, or saline vehicle for 7 days prior to a systemic dose of 7.5mg/kg of KA. Rats were sacrificed 48 h after KA injection. Immunostaining for neuronal nuclei (Neu-N) revealed a significant KA-induced decrease in cell number in the CA1, CA3, and CA4 hippocampal areas of rats (∼55% of control). Treatment with either hPRL or S179D-PRL or the combination prevented the damaging effect of KA in these hippocampal regions (∼95% of corresponding control), but was not completely effective at preventing early seizure-related behaviors such as staring and wet dog shakes. Analysis of signals generated by hPRL and S179D-PRL showed no activation of signal transducer and activation of transcription 5 (Stat5) or other signaling molecules in the hippocampus, but activation of extracellular-regulated kinase (ERK)1/2 in the amygdala. These results support a central protective effect of both PRL forms and suggest that PRL could be exerting its protective action by indirectly modulating input signals to the hippocampus and thus regulating excitability.

S179D prolactin (S179D PRL) is a pseudophosphorylated form of human PRL which has potent antitumor and anti-angiogenic activities in vivo. This molecule binds to the same forms of the PRL receptor (PRLR) as unmodified PRL, yet this binding results in different intracellular signaling and biological end points. Since it is now clear that PRLRs are predimerized and therefore that ligand binding must initiate signaling by inducing a conformational change in the receptor dimer, we hypothesized that S179D PRL had an altered conformation compared to unmodified PRL. The conformation of the ligand-receptor ternary complex would therefore also have an altered conformation, and thus, different signaling molecules would be activated. Here we present evidence in support of this hypothesis by demonstrating, in contrast to unmodified PRL, that S179D PRL has reduced nickel and zinc binding capacity and a higher affinity for heparin and DEAE. Conformational changes have occurred since these features are counterintuitive on the basis of the simple substitution of a serine with a negatively charged aspartate residue. To demonstrate that these particular properties of S179D PRL were not due to misfolding of the molecule during production, S179D PRL was expressed in two different mammalian cell lines. Also investigated was the potential for production of S179D PRL as a soluble cytoplasmic, or secreted periplasmic, protein in Escherichia coli.

The rabbit lacrimal gland undergoes an immunophysiological transformation during pregnancy, reminiscent of that of the mammary gland as it prepares to deliver secretory IgA into the nascent fluid product. The contents of TGF-beta and prolactin (PRL) within ductal epithelial cells increase, and their primary localizations shift from the apical to the basal cytoplasm, suggesting a transformation from exocrine to paracrine secretion. Studies with ex vivo acinar cell models demonstrated that elevated PRL suppresses traffic of secretory proteins into the regulated exocrine apparatus and directs them into a novel, induced, regulated paracrine apparatus [Wang, Y., Chiu, C.T., Nakamura, T., Walker, A.M., Petridou, B., Trousdale M.D., Hamm-Alvarez S.F., Schechter J.E., Mircheff A.K., 2007. Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells. Am. J. Physiol. Endocrinol. Metab. 292, E1122-E1134]. However, it was not clear whether PRL itself entered the induced paracrine apparatus. In the present study, confocal immunofluorescence microscopy revealed that natively expressed PRL and over-expressed PRL co-localized with PRL receptors (PRLR); rab11, a marker for the recycling endosome; gamma-adaptin, a marker for the Golgi complex and trans-Golgi network; and rab7, a marker for the autophagic lysosomal apparatus. Natively expressed, over-expressed, and endocytosed PRL also co-localized with rab4 and rab5A, markers for the early endosome, and with rab3D, a marker for regulated exocrine secretory vesicles. Endocytosed PRL was stored in intact form and released in response to stimulation with carbachol. Subcellular fractionation analysis detected relative excesses of PRL over PRLR in fractions that contained fragments of the recycling endosome and fractions that contained both secretory vesicle fragments and prelysosomal and autolysosomal fragments. EM-gold microscopy demonstrated PRL within small vesicles, consistent with endosomes or secondary lysosomes, and in large vesicles, consistent with regulated secretory vesicles. The secretory vesicles were preponderantly localized in the apical cytoplasm of control cells, and in the basal cytoplasm of PRL over-expressing cells. These results indicate that when lacrimal epithelial cells synthesize PRL, and when they endocytose it from their ambient medium, they traffic it both into the endosomes that constitute the constitutive transcytotic paracrine apparatus and also into regulated secretory vesicles, which are associated with the exocrine apparatus at low PRL levels and with the induced paracrine apparatus at high PRL levels.

Increasing evidence suggests an important role for autocrine/paracrine prolactin in breast and prostate cancers and other disease states. Prolactin production in these extrapituitary sites is not governed by dopamine agonists, a finding that has spurred the production of prolactin receptor antagonists. This review focuses on one such antagonist, S179D prolactin, which was produced by mimicking a natural antagonist, phosphorylated prolactin. S179D prolactin is a very effective growth antagonist, partly because it inhibits signalling from unmodified prolactin and partly because it produces its own intracellular signal. This signal results in cell differentiation, cell-cycle arrest or apoptosis depending on dose, duration of treatment and cellular context. S179D prolactin is also a potent antiangiogenic and initial studies have shown it to be a potent anti-inflammatory agent. In light of these additional modes of action, it is suggested that S179D prolactin should now be more aptly referred to as a selective prolactin receptor modulator.

S179D prolactin (PRL) is an experimentally useful mimic of naturally phosphorylated human prolactin. S179D PRL, but not unmodified PRL, was found to be anti-angiogenic in both the chorioallantoic membrane and corneal assays. Further investigation using human endothelial in vitro models showed reduced cell number, reduced tubule formation in Matrigel, and reduced migration and invasion, as a function of treatment with S179D PRL. Analysis of growth factors in human endothelial cells in response to S179D PRL showed: a decreased expression or release of endogenous PRL, heme-oxygenase-1, basic fibroblast growth factor (bFGF), angiogenin, epidermal growth factor and vascular endothelial growth factor; and an increased expression of inhibitors of matrix metalloproteases. S179D PRL also blocked signaling from bFGF in these cells. We conclude that this molecular mimic of a pituitary hormone is a potent anti-angiogenic protein, partly as a result of its ability to reduce utilization of several well-established endothelial autocrine growth loops, partly by its ability to block signaling from bFGF and partly because of its ability to decrease endothelial migration. These findings suggest that circulating levels of phosphorylated PRL may influence the progression of cancer and, furthermore, that S179D PRL may be a useful anti-angiogenic therapeutic.

Prolactin (PRL) is a hormone that contributes to both the growth and differentiation of mammary epithelial cells, activities likely to impact breast cancer in opposite ways. Whether PRL causes growth or differentiation has been solely attributed to the coexisting steroidal environment, with PRL stimulating mammary gland growth during pregnancy, and then milk production after the postpartum drop in estrogen and progesterone. However, previous work from our laboratory has shown that the form of PRL may also be an important factor. During pregnancy, unmodified PRL (U-PRL) promotes mammary growth, while an increase in phosphorylated PRL, or administration of a molecular mimic of phosphorylated PRL (S179D PRL), inhibits growth. Unknown, however, is whether these forms of PRL have opposite effects on growth in the absence of steroids and whether effects are directly on mammary epithelial cells. To mimic the glandular epithelium in vitro, we used contact-inhibited, differentiated cells and showed that even with these minimally growing cells that treatment with U-PRL caused increased expression of cyclin D1 and cyclin-dependent kinase 4, increased activity of both cdk4 and cdk2, while having no effect on the inhibitory protein, p21. S179D PRL, by contrast, had no effect on cyclin D1 and cdk4 expression, but increased p21 expression and expression of the vitamin D receptor (VDR). We conclude that increased U-PRL or decreased phosphorylated PRL can directly affect cell cycle control proteins in relatively differentiated mammary epithelial cells, thereby implicating the balance between these two forms of PRL in the early promotion of breast cancer.

The effects of two major forms of prolactin (PRL) were examined on delayed type hypersensitivity (DTH) responses to Candida albicans . Unmodified PRL (U-PRL) had no effect on the DTH response, whereas a molecular mimic of phosphorylated PRL (S179D PRL) significantly inhibited immune responses to this robust antigen. This effect of S179D PRL was not accompanied by gross alterations in splenic T cell numbers, CD4 / CD8 ratios, or T and B cell activation markers, but did produce a decrease in splenocyte apoptosis. Using gld animals, Fas ligand (FasL) was implicated in the suppressive effects of S179D PRL. Circulating IgG 1 and IgG 2 antibody levels were increased in response to treatment with both forms of PRL, but the effects of S179D PRL were most pronounced. Cytokine changes in the popliteal lymph nodes specific to S179D PRL treatment showed an inhibition of pro-inflammatory cytokines. In conclusion, mice treated with a molecular mimic of phosphorylated prolactin showed a profound inhibition of DTH responses to Candida correlating with an absence of GM-CSF, IL-4, and IL-13 production and a marked reduction in IL-12p70 synthesis.

In this study, we further investigated the mechanisms by which pseudophosphorylated prolactin (S179D PRL) inhibits the growth of human prostate cancer cells. When treated with S179D PRL for 3 days, LnCAP cells responded by increasing expression of the vitamin D receptor (VDR) and the cell cycle regulatory molecule, p21, whereas PC3 and DU145 cells did not. After 5 days of treatment, both PC3 and DU145 cells responded. Untreated LnCAP cells express the short 1b form (SF1b) of the human prolactin receptor, but DU145 and PC3 cells express only low amounts of this receptor until elevated by treatment with S179D PRL. DU145 and PC3 cells become sensitive to the negative effects of S179D PRL on cell number after induction of the SF1b. Transfection of either SF1b or SF1a into PC3 or DU145 cells made them sensitive to S179D PRL in the 3-day time frame, a finding that was not duplicated by transfection with the long form of the receptor. Treatment of LnCAP cells with S179D PRL increased long-term activation of extracellular signal-regulated kinase 1/2 (ERK1/2). This did not occur in PC3 and DU145 cells until transfection with SF1a/SF1b. Blockade of ERK signaling eliminated S179D PRL-stimulated expression of the VDR and p21 in LnCAP cells and transfected PC3 and DU145 cells. We conclude that initiation of alternative splicing to produce SF1b, and subsequent altered signaling, contribute to the growth inhibitory mechanisms of S179D PRL. This is the first indication of a role for short prolactin receptors in the regulation of cell proliferation.

Posttranslational modifications of prolactin (PRL), including phosphorylation, vary with physiologic state and alter biologic activity. In light of the growing evidence for a role for PRL in proliferation in mammary cancer, we examined the ability of a mimic of phosphorylated human PRL, S179D-PRL, to initiate signals to several pathways in mammary tumor cells alone and in combination with unmodified PRL. Unmodified PRL employed multiple pathways to increase cellular proliferation and cyclin D1 levels in PRL-deficient MCF-7 cells. S179D-PRL was a weak agonist compared with unmodified PRL with regard to cellular proliferation, cyclin D1 levels, and phosphorylation of signal transducer and activator of transcription 5 and ERKs. However, S179D-PRL was a potent antagonist of unmodified PRL to these endpoints. In contrast to the reduced levels of the long isoform of the PRL receptor observed in response to a 3-d incubation with unmodified PRL, S179D-PRL up-regulated expression of this isoform, 4-fold. These studies support the utility of this mutant as a PRL antagonist to proliferative signals in mammary epithelial cells, including a potential role in breast cancer therapeutics.

Previous work has shown that naturally phosphorylated prolactin antagonizes the growth-promoting activities of unmodified prolactin (U-PRL) and that this effect is duplicated by a molecular mimic, S179D PRL. At the same time, the S179D PRL is a superagonist with regard to expression of some PRL-regulated genes. We have asked whether the different activities of U-PRL and S179D PRL are the result of differential signaling. HC11 cells (a normal mouse mammary cell line) were grown to confluence, primed with hydrocortisone, and then exposed to the PRLs. A 15 min incubation of PRL-naive cells led to substantial tyrosine phosphorylation of Jak 2 and Stat 5a by U-PRL and an essentially equivalent Jak 2 activation by S179D PRL. The latter, however, was accompanied by reduced tyrosine phosphorylation of Stat 5a. EMSA analysis using a Stat 5 binding site showed both PRLs to cause equivalent binding of nuclear proteins and that most of what bound was complexed through Stat 5a. Phosphoamino acid analysis of Stat 5 showed S179D PRL to double the amount of serine phosphorylation versus that seen with U-PRL. Analysis of the MAP kinase pathway showed U-PRL capable of activation of ERKs 1 and 2 but that signaling via ERKs 1 and 2 was greater with S179D PRL. A 7-day incubation in either PRL increased beta-casein mRNA levels, but S179D PRL caused a 2-fold increase over that seen with U-PRL. The increase, over that seen with U-PRL, was blocked by the MAP kinase inhibitor, PD98059. After 7 days of treatment with S179D PRL, expression of the short PRL receptor was doubled, and signaling showed a greater dependence on the MAP kinase pathway (2.9-fold increase in ERK 1 and 2 activation). We conclude that although both PRLs use both pathways to some extent, U-PRL signals primarily through Jak 2-Stat 5 whereas S179D PRL signals primarily through the MAP kinase pathway especially after prolonged exposure. This is the first demonstration of differential involvement of signaling pathways by different forms of PRL.

There have been few studies aimed at determining the effects of maternal peptide hormones on the developing fetus and even fewer aimed at determining the long-term consequences of abnormalities in maternal hormone exposure. In this study, we have examined the effect of maternal prolactin (PRL) on the production, seeding and long-term function of a T lymphocyte subset for which the precursors are only present during fetal life. Using this system, we can determine long-term consequences of maternal hormone exposure without concern for the subsequent influence of the offspring's endocrine milieu. Recombinant versions of the two major forms of the pituitary hormone, PRL, were administered to rats throughout pregnancy. Administration of a molecular mimic of phosphorylated PRL (PP-PRL) resulted in a marked increase in the level of apoptosis in the thymus of newborn pups, an effect that was not duplicated by administration of unmodified PRL. The increased thymic apoptosis in the animals exposed to PP-PRL resulted in decreased epidermal seeding of gammadeltaT cells and a markedly decreased gammadeltaT cell-modulated epidermal response in the offspring. This decreased gammadeltaT cell modulated response persisted to adulthood. We conclude that maternal PRL composition during pregnancy can have a permanent effect on at least one component of the developing immune system.

We have investigated the individual roles of unmodified prolactin (U-PRL) and a mimic of phosphorylated PRL (S179D PRL) in mammary development. Recombinant versions of the PRLs were delivered to rats throughout pregnancy at a rate of 6 microg/24 h per rat and to non-pregnant females at a rate of 24 microg/24 h per rat. Measurement of progesterone, corticosterone, and estradiol showed no effect of the administered PRLs on the levels of these other mammotropic hormones. Histological and morphometric analysis showed U-PRL to cause mammary growth, whereas S179D PRL inhibited growth. Molecular analysis demonstrated decreased beta-casein expression in the mammary glands of the U-PRL-treated animals at term and increased beta-casein expression in the mammary glands of the S179D PRL-treated animals. Superior beta-casein gene expression in response to S179D PRL versus U-PRL was confirmed in HC11 cells. We conclude that U-PRL is important for growth, whereas S179D PRL promotes at least one measure of differentiated function in the mammary gland.

During rat pregnancy initial high concentrations of prolactin (PRL) decline by about day 9, concomitant with an increase in the ratio of unmodified to phosphorylated PRL. The physiological significance of both the decline in total PRL and the change in ratio of the two PRLs is unknown. To test the importance of each, either unmodified PRL (U-PRL) or a molecular mimic of phosphorylated PRL (PP-PRL) were continuously administered to rats throughout pregnancy. A dose of 6 microg/24 h resulted in circulating concentrations of 50 ng/ml of each administered PRL and had little effect on the pregnancy itself. After birth, pups were killed and various tissues examined. In the pup lungs, exposure to additional PP-PRL caused a reduction in epithelial integrity and an increase in apoptosis, whereas exposure to additional U-PRL had beneficial, anti-apoptotic effects. In the heart, PP-PRL caused an apparent developmental delay, whereas U-PRL promoted tissue compaction. In the blood, U-PRL increased the number of mature red blood cells at the expense of white blood cell production. Within the white blood cell population, myelopoiesis was favored at the expense of lymphopoiesis. PP-PRL, in contrast, had a less dramatic influence on the hematopoietic compartment by promoting red blood cell maturation and granulocyte production. In the thymus, exposure to PP-PRL caused accumulation of apoptotic thymocytes in enlarged glands, whereas exposure to U-PRL resulted in smaller thymi. In the spleen, exposure to U-PRL increased cellularity, with the majority of cells belonging to the erythroid series - a finding consistent with increased red blood cells in the circulation. Exposure to PP-PRL was without discernible effect. In all of these tissues, the contrasting effects of the two PRLs indicate that the absolute concentration of PRL is not crucial, but that the ratio of U-PRL to PP-PRL has a profound effect on tissue development. In brown fat, both PRL preparations decreased the number of lipid droplets. This result is therefore probably a consequence of the increase in total PRL. The results of this study attest to the importance of the U-PRL:PP-PRL ratio normally present during pregnancy and have provided clues as to the possible pathogenesis of a variety of neonatal problems.

The expression of maternal behavior in the newly parturient rat is under endocrine regulation. Blocking endogenous PRL secretion with bromocriptine delays the normal rapid expression of maternal care shown toward foster young in steroid-primed virgin female rats. The recent development of the PRL receptor antagonist S179D-PRL, a mutant of human PRL in which the serine residue at the 179 position is replaced with aspartate, provides a potentially useful tool to examine the role of PRL in neural processing. In the present report, three experiments were conducted that examined the effects of this PRL antagonist on the induction of maternal behavior. In each experiment, ovariectomized, nulliparous rats were treated sequentially with SILASTIC capsules implanted sc with progesterone (days 1–11) and estradiol (days 11–17), a treatment that stimulates a rapid onset of maternal behavior in virgin rats. On day 11, females were implanted with Alzet miniosmotic pumps connected to cannulae directed unilaterally at the...

The purpose of the current study was to determine whether maternal prolactin (PRL) had any effects on bone formation in the developing rat pup. Because the most prevalent forms of PRL in rats are unmodified and phosphorylated PRL, both recombinant PRL and a molecular mimic of phosphorylated PRL (PP-PRL) were administered to pregnant animals. Blood samples from the dams showed normal estrogen and progesterone and no effect of extra PRL on parathyroid hormone (PTH), calcium, or alkaline phosphatase (AP). In newborn pups, however, there was a 30% decrease in blood AP in both PRL-treated groups, whereas PTH and calcium levels were not different from controls. When primary rat osteoblasts were exposed to both PRLs, AP activity was reduced, with PP-PRL being the more potent form of the hormone. Histological examination of pup bone formation showed reduced calvarial bone and reduced endochondral ossification in pups exposed to PP-PRL. These results are the first to show a direct inhibitory effect of PRL on osteoblast function.

We have previously demonstrated that PRL acts as an autocrine growth factor in the pituitary tumor GH3 cell line. In this study we present evidence that the 235-1 cell line also uses PRL as an autocrine growth factor, and that in this case interaction between PRL and its receptor occurs intracellularly. First, the PRL produced by 235-1 cells was shown to be capable of initiating a proliferative response by placement in the Nb2 bioassay. Second, PRL receptors were demonstrated to be present, but, unusually, primarily in the Golgi complex by light and electron microscope immunocytochemistry. Incubation of the cells in anti-PRL had no effect on 235-1 cell proliferation until interleukin-1 treatment caused the PRL receptors to move to the cell surface, whereupon cell proliferation was inhibited. Specific interference with PRL biosynthesis in noninterleukin-1-treated cells also inhibited cell proliferation. Thus, the essential elements of an autocrine loop are present and can be demonstrated to be functional by antibody inhibition of growth after movement of the receptors to the cell surface and by antisense inhibition of growth with the receptors in their normal intracellular location.

Variably-spliced prolactin receptors (PRLRs) and PRL are expressed by the ovarian cancer cell lines, TOV-112D, OV-90 and TOV-21G. Incubation in the PRLR antagonists, G129R- or S179D-PRL, or anti-PRL reduced cell number, indicating a functional autocrine PRL growth loop. Added PRL promoted, and the antagonists decreased, cell migration. When cells were stressed, added PRL decreased apoptosis and increased survival, and the antagonists had the opposite effect. Cells expressing higher long:short PRLR ratios had increased growth, survival and migration in response to PRL. Results suggest that PRLR antagonists may be therapeutically beneficial in ovarian cancer.

Charge isomers of monomer PRL have been described for all species thus far examined. For the rat, cow, and chicken, at least a major proportion of the more acidic isomers have been shown to be phosphorylated. The physiological relevance of this posttranslational phosphorylation, however, remains unclear. In this study we have compared the growth-promoting activities of dephosphorylated and standard rat PRL (partially phosphorylated) in the widely used Nb2 bioassay. Dephosphorylated PRL was produced by treatment of standard rat PRL with acid phosphatase, resulting in the conversion of a 91.9% nonphosphorylated/8.1% phosphorylated preparation to a 98.6% nonphosphorylated preparation. These two preparations were added separately or in combination to stationary Nb2 cells. Cell number was assessed 3 days later using a colorimetric assay. Dephosphorylated PRL showed significantly higher growth-promoting activity than standard PRL at concentrations up to 10 ng/ml. In the 1-5 ng/ml concentration range, dephosphorylated PRL was twice as active. Given that the conversion of 6.7% phosphorylated PRL to nonphosphorylated PRL resulted in a doubling of activity, one can deduce not only that phospho-PRL acted as an antagonist to nonphosphorylated PRL in this assay, but also that it did not do so on an equimolar basis. Titration of the two PRL preparations produced data confirming this latter deduction. These data are of importance in our understanding of PRL-promoted cell proliferation.

In this study we have examined the direct glucoregulation of prolactin secretion from normal anterior pituitary cells in vitro and have found that changes in medium glucose concentration regulate the amount of prolactin released. Nature and/or degree of this response to glucose was influenced by some effect, long-lived in vitro, which was correlatable to serum insulin levels. When the cells were derived from animals with mean low-normal serum insulin levels, there was a stimulation of prolactin secretion by hypoglycemia, the response was rapid, transient, dose-dependent, and could be duplicated by 2-deoxyglucose. When the cells were derived from animals with a higher mean serum insulin level, the prolactin secretion from the cells was slowly, adversely affected by hypoglycemia. Conversely, elevated glucose caused a depression in prolactin secretion in the first group and a stimulation of prolactin secretion in the second. We conclude (1) that modulation of glucose levels in vitro regulates prolactin release from pituitary mammotrophs and (2) that this glucose regulation of prolactin release is in turn coregulated with or regulated by insulin.

Artificial copulomimetic cervical stimulation (CS) induces an immediate release of oxytocin (OT) and prolactin (PRL) followed by a daily PRL rhythm characterized by nocturnal and diurnal surges. Although we have shown that the initial release of PRL is induced by the immediate release of OT, we tested whether the PRL that is released in response to CS is responsible for the initiation and maintenance of the subsequent PRL surges. Thus, we injected OVX rats centrally or peripherally with ovine PRL (oPRL) at 2200 h. Central oPRL induced PRL surges in OVX rats that were similar in size and timing to those of CS rats, whereas peripheral oPRL induced surges that were of smaller amplitude and delayed. We then infused a PRL antagonist (S179D, 0.1 ng/h) centrally into OVX and OVX-CS rats and measured the release of endogenous PRL and the activity of neuroendocrine dopaminergic neurons. Central infusion of S179D did not influence basal PRL secretion in OVX rats but prevented the expression of the CS-induced PRL surges and the accompanying noontime increase of CS-induced dopaminergic activity when continued for 3 d. However, central infusion of S179D only on the day of CS did not prevent the daily rhythm of PRL surges. These results demonstrate that PRL acts centrally to induce the PRL rhythm and that PRL in the brain is essential for the maintenance but not for the initiation of the CS-induced rhythmic PRL surges.

Based on their content of prolactin receptors, osteosarcoma cells were predicted to be responsive to prolactin (PRL), but whether PRL would be beneficial or contribute to pathogenesis was unclear. 1,25(OH)(2) vitamin D(3) [1alpha,25(OH)(2)D(3)] has antiproliferative effects on osteosarcoma cells, and a complex interregulatory situation exists between PRL and 1alpha,25(OH)(2)D(3). Using osteosarcoma cells, Western blot, real time RT-PCR, and promoter-luciferase assays, we have examined the interaction between PRL and 1alpha,25(OH)(2)D(3) and demonstrated that physiological concentrations of PRL block increased osteocalcin and vitamin D receptor (VDR) expression in response to 1alpha,25(OH)(2)D(3.) This blockade was shown to be the result of lack of nuclear accumulation of the VDR in response to 1alpha,25(OH)(2)D(3). Although inhibition of proteasomic degradation with MG132 had no effect on the VDR itself in a 30-min time frame, it relieved the blockade by PRL. Analysis of ubiquitinated proteins brought down by immunoprecipitation with anti-VDR showed PRL regulation of a 250-kDa protein-VDR complex. P250 was identified as the breast cancer tumor suppressor gene product, BRCA1, by Western blot of the VDR immunoprecipitate and confirmed by immunoprecipitation with anti-BRCA1 and blotting for the VDR in the absence and presence of PRL. Knockdown of BRCA1 inhibited nuclear translocation of the VDR and the ability of 1alpha,25(OH)(2)D(3) to induce the VDR. This, to our knowledge, is the first demonstration of a role for BRCA1 in nuclear accumulation of a steroid hormone and the first demonstration that PRL has the potential to affect the cell cycle through effects on BRCA1.

Since the discovery of physiologically-regulated prolactin (PRL) phosphorylation, one focus of the laboratory has been an examination of the different functions of the unmodified and phosphorylated hormone. In the mammary gland, unmodified PRL promotes growth activities, whereas phosphorylated or pseudophosphorylated PRL antagonizes this while also being a superior agonist for changes that favor differentiation. Phosphorylated PRL also increases expression of the short forms of the PRL receptor. These short forms of the receptor have functions beyond the accepted dominant negative and in mammary epithelial cells are capable of generating an intracellular signal leading to increased tight junction formation and beta-casein expression.

Prolactin (PRL) has both stimulatory and inhibitory effects on testicular function, a finding we hypothesized may be related in some part to the form of the hormone present or administered. In the analysis of the pituitary secretion profiles of early pubescent vs. mature male rats, we found PRL released from early pubescent pituitaries had about twice the degree of phosphorylation. Treatment of mature males with either unmodified PRL (U-PRL) or phosphorylated PRL (via the molecular mimic S179D PRL) for a period of 4 wk (circulating level of approximately 50 ng/ml) showed serum testosterone decreased by approximately 35% only by treatment with the phospho-mimic S179D PRL. Given the specificity of this effect, it was initially surprising that both forms of PRL decreased testicular expression of 3beta-hydroxysteroid dehydrogenase and steroidogenic acute regulatory protein. Both forms also increased expression of the luteinizing hormone receptor, but only S179D PRL increased the ratio of short to long PRL receptors. Endogenous PRL and luteinizing hormone levels were unchanged in all groups in this time frame, suggesting that effects on steroidogenic gene expression were directly on the testis. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling analysis combined with staining for 3beta-hydroxysteroid dehydrogenase and morphometric analysis showed that S179D PRL, but not U-PRL, increased apoptosis of Leydig cells, a finding supported by increased staining for Fas and Fas ligand in the testicular interstitium, providing an explanation for the specific effect on testosterone. S179D PRL, but not U-PRL, also increased apoptosis of primary spermatogonia, and U-PRL, but not S179D PRL, decreased apoptosis of elongating spermatids. Thus, in mature males, hyperprolactinemic levels of both forms of PRL have common effects on steroidogenic proteins, but specific effects on the apoptosis of Leydig and germ cells.

Epidermal gamma delta T cells (gammadeltaT) and Langerhans cells (LC) are immune cells altered by exposure to ultraviolet radiation (UVB), a powerful stressor resulting in immune suppression. Prolactin (PRL) has been characterized as an immunomodulator, particularly during stress. In this study, we have asked whether separate administration of the 2 major forms of prolactin, unmodified and phosphorylated, to groups of 15 mice (3 experiments, each with 5 mice per treatment group) affected the number and morphology of these epidermal immune cells under control conditions, and following UV-irradiation. Under control conditions, both PRLs reduced the number of gammadeltaT, but a molecular mimic of phosphorylated PRL (S179D PRL) was more effective, resulting in a 30% reduction. In the irradiated group, however, S179D PRL was protective against a UV-induced reduction in gammadeltaT number and change in morphology (halved the reduction and normalized the morphology). In addition, S179D PRL, but not unmodified (U-PRL), maintained a normal LC:gammadeltaT ratio and sustained the dendritic morphology of LC after UV exposure. These findings suggest that S179D PRL may have an overall protective effect, countering UV-induced cellular alterations in the epidermis.

The prolactin (PRL) receptor antagonist S179D PRL delays the onset of maternal behavior in steroid-primed nulliparous female rats. The present study investigated the role of the neural PRL system in the process of parturition. A preliminary study indicated that S179D PRL treatments administered by ALZET minipump to the lateral ventricle severely disrupted parturition. To examine the likely causes of this disruption, a group of timed-pregnant catheterized rats was continuously infused with S-179D PRL (0.001 and 0.1 ng/h) or vehicle control to the lateral ventricles for 3 days (gestation days 21–23), and serial blood samples were taken throughout this period. Effects of the treatments on parturition were recorded, and blood samples were assayed for PRL, progesterone, and oxytocin. Significantly fewer S179D PRL-treated rats successfully delivered by 1500 h on day 23 of gestation when compared with controls. The higher dose of S179D PRL also significantly suppressed the prepartum rise in PRL throughout the prepartum period, while the lower dose only affected plasma PRL during the first 24 h of treatment. No significant effects of the antagonist on plasma progesterone or oxytocin were detected. We conclude that disruption of parturition by S179D PRL is not caused by significant alterations in the plasma concentrations of progesterone or oxytocin. S179D PRL may indirectly act on parturition through the modulation of prepartum PRL. These findings suggest a previously unrecognized role for PRL in the regulation of parturition.

The aims of this review are threefold: First, to collate what is known about the production and activities of phosphorylated prolactin (PRL), the latter largely, but not exclusively, as illustrated through the use of the molecular mimic, S179D PRL; second, to apply this and related knowledge to produce an updated model of prolactin-receptor interactions that may apply to other members of this cytokine super-family; and third, to promote a shift in the current paradigm for the development of clinically important growth antagonists. This third aim explains the title since, based on results with S179D PRL, it is proposed that agents which signal to antagonistic ends may be better therapeutics than pure antagonists - hence antagonistic agony. Since S179D PRL is not a pure antagonist, we have proposed the term selective prolactin receptor modulator (SPeRM) for this and like molecules.

During pregnancy, lymphocytes infiltrating the rabbit lacrimal gland disperse to the interacinar space from their normal focal concentrations, basal fluid secretion decreases, pilocarpine-induced fluid secretion increases, and stimulated fluid protein concentration decreases. Ductal epithelial cell prolactin (PRL) content increases and redistributes from the apical to the basal-lateral cytoplasm. A replication-incompetent adenovirus vector for rabbit PRL (AdPRL) was used to test the hypothesis that increased intracrine/autocrine PRL signaling alters secretory protein traffic in an ex vivo lacrimal acinar cell model. AdPRL had no discernable influence on microtubules or actin microfilaments or their responses to carbachol (CCh). Endogenous and transduced PRLs exhibited similar, nonpolarized, punctate distributions. Cells secreted PRL consititutively and at increased rates in response to CCh. In contrast, constitutive secretion of β-hexosaminidase was negligible, suggesting that the constitutive pathway for PRL is relatively inaccessible to typical secretory proteins. AdPRL had no significant effect on total secretion of β-hexosaminidase or syncollin-green fluorescent protein (GFP), a chimeric secretory protein construct. However, it reversed the polarized distributions of vesicles containing rab3D and syncollin-GFP. Live-cell imaging indicated that AdPRL redirected CCh-dependent syncollin-GFP exocytosis from the apical plasma membrane to the basal-lateral membrane. Elevated concentrations of exogenous rabbit PRL in the ambient medium elicited similar changes. These observations suggest that elevated PRL, as occurs in the physiological hyperprolactinemia of pregnancy, induces lacrimal epithelial cells to express a mixed exocrine/endocrine phenotype that secretes fluid to the acinus-duct lumen but secretes proteins to the underlying tissue space. This phenotype may contribute to the pregnancy-associated immunoarchitecture.

The secretory activation stage of mammary gland development occurs after parturition and converts inactive lobuloalveoli to active milk secretion. This process is triggered by progestin withdrawal and depends upon augmented prolactin (Prl) signaling. Little is known about the Prl-induced transcriptional changes that occur in the mammary gland to drive this process. To examine changes in the mammary transcriptome responsible for secretory activation, we have used transcript profiling of three mouse models that exhibit failure of secretory activation: knockout of galanin (a regulator of pituitary Prl production and a mammary cell autonomous modulator of Prl action); treatment with S179D Prl (a phosphoprolactin mimic); and knockout of a single Prl receptor allele. A significant reduction in expression was observed in genes belonging to 46 gene ontologies including those representing milk proteins, metabolism, lipid, cholesterol and fatty acid biosynthetic enzymes, immune response, and key transcription factors. A set of 35 genes, commonly regulated in all three models, was identified and their role in lactogenesis was validated by examining their expression in response to Prl stimulation or signal transducer and activator of transcription 5 knockdown in the HC11 mouse mammary cell culture model. The transcript profiles provided by these experiments identify 35 key genes (many for the first time) involved in the secretory activation phase of mammary gland development, show that S179D acts as an antagonist of Prl action, and provide insight into the partial penetrance of failed lactation in Prl receptor heterozygous females.

We have used bioluminescence resonance energy transfer (BRET) to examine the interaction between human prolactins (PRLs) and the long (LF) and two short isoforms (SF1a and SF1b) of the human PRL receptor in living cells. cDNA sequences encoding the LF, SF1a, and SF1b were subcloned into codon-humanized vectors containing cDNAs for either Renilla reniformis luciferase (Rluc) or a green fluorescent protein (GFP2) with a 12- or 13-amino acid linker connecting the parts of the fusion proteins. Transfection into human embryonic kidney 293 cells demonstrated maintained function of Rluc and GFP2 when linked to the receptors, and confocal microscopy demonstrated the localization of tagged receptors in the plasma membrane by 48 h after transfection. All three tagged receptors transduced a signal, with the LF and SF1a stimulating, and SF1b inhibiting, promoter activity of an approximately 2.4-kb β-casein-luc construct. Both unmodified PRL (U-PRL) and the molecular mimic of phosphorylated PRL, S179D PRL, induced BRET with all combinations of long and short receptor isoforms except SF1a plus SF1b. No BRET was observed with the site two-inactive mutant, G129R PRL. This is the first demonstration, 1) that species homologous PRL promotes both homo- and hetero-interaction of most long and short PRLR pairs in living cells, 2) that both U-PRL and S179D PRL are active in this regard, and 3) that there is some aspect of SF1a-SF1b structure that prevents this particular hetero-receptor pairing. In addition, we conclude that preferential pairing of different receptor isoforms is not the explanation for the different signaling initiated by U-PRL and S179D PRL.

In this study, we have tested the hypothesis that unmodified prolactin (U-PRL) and phosphorylated prolactin (P-PRL) have differential roles in the autoregulation of PRL secretion in vivo. Recombinant human U-PRL and a molecular mimic of P-PRL (S179D PRL) were administered to male rats and to female rats in different physiological states and the effect on rat PRL release was measured. Administration of U-PRL elevated rat PRL in all female animals, but was without effect in males. By contrast, S179D PRL was inactive in females, but inhibited PRL release in males. Morphometric and immunohistochemical analyses demonstrated acidophil hypertrophy and evidence of increased PRL secretion in the pituitaries of U-PRL-treated females. Analysis of the two forms of PRL during prolactinoma induction in two differentially susceptible strains of rats found a strong temporal correlation among increased ratios of U-PRL: P-PRL, increased circulating PRL, and increased cell proliferation. We conclude (1). that the autoregulatory mechanism(s) can distinguish between the two major forms of PRL and that higher proportions of U-PRL not only allow for higher circulating levels of PRL, but are also autostimulatory, (2). that the autoregulatory mechanism( s) are set differently in males and females such that females are more sensitive to autostimulation by U-PRL and less sensitive to inhibition by P-PRL, and (3). that U-PRL and P-PRL may also have differential roles in the regulation of pituitary cell proliferation.

BACKGROUND In the current study, we have investigated the individual roles of unmodified, wild-type prolactin (WT PRL) and a molecular mimic of phosphorylated prolactin (S179D PRL) in the normal rat prostate. METHODS In the first animal experiment, recombinant WT PRL and S179D PRL were delivered to adult male rats at a rate of 14 μg/kg per day for 3 weeks. In the second animal experiment, two subcutaneous (200 μg/kg) injections of long-acting forms of the two PRLs were given to adult male rats on day 1 and day 22 for a total of 5.5 weeks of treatment. RESULTS The different forms of PRL had opposite effects on the normal rat prostate, independently of androgens. WT PRL promoted morphologic changes in prostate epithelium consistent with preparation for cell proliferation, whereas S179D PRL produced morphologic evidence of a more differentiated epithelium. Northern blot analysis of expression of the two major prostate specific proteins, prostatein and probasin, showed that WT PRL decreased, whereas S179D PRL increased, the expression of the mRNAs for these two proteins. At the same time, S179D PRL reduced both testosterone and dihydrotestosterone levels. CONCLUSION We conclude that PRL is an important modulator of normal rat prostate biology and that different forms of PRL have specific functions. The molecular mimic of phosphorylated PRL, S179D PRL, is the most important in terms of epithelial cell differentiation. Prostate 54: 25–33, 2003. © 2002 Wiley-Liss, Inc.

In previous work, prolactin (PRL) abnormalities of a lower bioassay (BA) to radioimmunoassay (RIA) ratio were found in schizophrenic patients. This line of research was extended in seven male patients with schizophrenia who were neuroleptic-free; seven male control subjects were also studied. PRL values were assessed by RIA and Nb(2) BA techniques. The schizophrenic group had a significantly lower PRL BA as compared to normal controls and a lower PRL ratio of BA/RIA. The lower ratio is consistent with an earlier finding and suggests that schizophrenic patients have different molecular forms of PRL than control subjects. This difference could be due to a disordered tuberoinfundibular dopamine system or the long-term effects of neuroleptic medications.

Prolactin (PRL) is a hormone recognized as having both proliferative and differentiative activities in the mammary gland. Current theory proposes that it is the coexisting steroidal environment which dictates whether PRL is proliferative or differentiative. Preliminary data, presented as part of the initial proposal and established during year 1, however, suggested that it was the form of PRL released that dictated whether proliferation or differentiation would occur. By form of PRL we mean whether the PRL is released from the pituitary as an unmodified polypeptide or whether it is phosphorylated. To study this issue, we have produced recombinant, unmodified PRL (U-PRL) and a recombinant, molecular mimic of phosphorylated PRL, S179D PRL In the studies conducted during the first and second year, we have demonstrated that U-PRL promotes mammary growth, while Sl79D PRL inhibits growth and promotes differentiation. Further, we have demonstrated that these effects of U-PRL and S179D PRL are produced directly on the mammary gland. In addition, we have been able to show that U-PRL and S179D PRL exert these very different effects by changing the balance of signaling between the two major pathways in mammary epithelium. Thus U-PRL primarily uses the Jak 2-Stat 5a pathway, while Sl%9D PRL increases use of the MAP kinase pathway.

Most prolactin (PRL) in the circulation is produced by the pituitary. However, a wide variety of traditional target tissues of the hormone have also been shown to produce their own prolactin. The amount produced per cell is low, but may well be sufficient for autocrine/paracrine activity. Although dopamine agonists allow one to study the target tissue effects of pituitary PRL, other agents, such as PRL receptor (PRLR) antagonists, are needed to analyze autocrine/paracrine loops. With PRLR antagonists, it should be possible to dissect out the role of extrapituitary prolactin in both the normal physiology, and the pathophysiology of various tissues. In tissues where the locally produced PRL may promote disease, such antagonists have the potential to be important therapeutics. This article briefly, but critically, reviews current understanding of PRL-receptor interactions and initial signaling, and describes the development of both growth hormone (GH) and PRL antagonists within that context. In the final section, results with a very potent PRL antagonist further one theme of the article, which is whether the simple receptor dimerization model explains all signal transduction following PRLR binding.

Previous studies have demonstrated that naturally phosphorylated PRL antagonizes the growth-promoting effects of unmodified PRL in two different PRL-responsive cell lines. In this study our aim was to produce a molecular mimic of phosphorylated PRL by substituting a fairly bulky, negatively charged amino acid (glutamate or aspartate) for the normally phosphorylated serine [serine 179 in human PRL (hPRL)]. In addition, because of the marked effect of phosphorylation on biological activity, we investigated the importance of the unmodified serine in the growth-promoting activity of PRL. hPRL complementary DNA was obtained from the American Type Culture Collection and subcloned into pT7-SCII after site-directed mutagenesis using the deoxyuridine approach. Proteins were expressed in Escherichia coli BL21 (DE3) and were primarily found in inclusion bodies. Agonist and antagonist activities of each serine 179 mutant were assessed using the Nb2 bioassay. Compared with standard hPRL, the recombinant wild-type was more active in the Nb2 assay, attesting to both the absence, or low level, of endotoxin contamination in preparations from these cells and the appropriate folding of the molecule. The aspartate and glutamate mutants had no intrinsic agonist activity, but both antagonized the growth-promoting activity of wild-type PRL, with the aspartate mutant proving to be a very effective antagonist. Two hundred picograms per ml of the aspartate mutant negated 75% of the growth response to 400 pg/ml wild-type PRL. When serine 179 was mutated to alanine or valine, mutant PRLs with 0% and 14% of the biological activity of wild-type PRL, respectively, were produced. These results demonstrate 1) that molecular mimicry of the phosphorylated hormone does produce a PRL antagonist, and 2) that the serine at position 179 is crucial to the growth-promoting activity of PRL. The aspartate mutant can now be used to study many aspects of the physiology of PRL.

PRL in secretory granules is osmotically inert. Previous studies by us and others have suggested that this is due in part to hormone oligomerization. Data suggest intermolecular disulfide bridges and/or intermolecular ionic interactions, as thiols, urea, and chelators increase monomerization of the majority of granule PRL. Because of the inhibitory effect of zinc on PRL release from isolated granules and the effects of zinc on the specific packing of PRL within granules, we examined the possibility that zinc contributed to the stability and/or oligomerization of intragranular PRL. To do this, we first analyzed zinc binding to purified monomeric rat PRL in solution. Zinc binding was demonstrated using the chromogenic chelator 5,5'-nitrilodibarbituric acid (murexide) and was confirmed by matrix-assisted, time of flight mass analysis. Because these spectrophotometric methods were not applicable for intragranular PRL studies, we tested the influence of zinc on granule PRL indirectly. As hormone free thiols were potentially formed during PRL oligomerization and storage, these were possible sites for hormone-divalent cation interactions. By derivatization of thiols with 4-vinyl pyridine and isolation of the carboxyterminal region of granule PRL, we found that a proportion of the cysteines 189 and 197 occurred as thiols and not disulfides. These thiols were only detectable when EDTA was present in the granule incubations. It is proposed that binding of zinc stabilizes the intermolecularly bonded storage form of PRL, in part by protection of hormone free thiols. Removal of the divalent cation and exposure of free thiols could be what initiates the thiol-disulfide interchange necessary for conversion of intermolecular to intramolecular disulfide bonds before exocytosis. Experimentally, both urea and EDTA could remove zinc, thus initiating monomerization and explaining how these agents as well as thiols achieve monomerization.

Annexin 5 is expressed by rat anterior pituitary cells and a depolarizing stimulus results in increased extracellular display and, depending on local calcium concentrations, potential release into the extracellular environment. In order to further investigate the role of annexin 5 in anterior pituitary function, we have examined the intracellular distribution by immunocytochemistry and the effects of annexin 5 on the release of a major secretory product, prolactin. Prolactin was chosen because we could easily monitor effects on basal release and effects on the immediate and sustained phases of thyroid stimulating hormone releasing hormone (TRH)-stimulated release. Immunocytochemical localization of annexin 5 showed staining of the majority of anterior pituitary cells. Labeling was predominantly on the nuclear envelope and plasma membrane. For the chosen secretory product, prolactin, annexin 5 was found in most, but not all prolactin positive cells. When recombinant annexin 5 (50 ng/mL) was added to a 3 h static culture incubation of rat anterior pituitary cells, prolactin release was inhibited by about 30% (p

Several lines of evidence indicate that immunoglobulin-bound prolactin found in human serum is not a conventional complex between an anti-prolactin antibody and prolactin but a different type of association of prolactin with the Fab portion of IgG heavy chains. The complex of prolactin with IgG was purified from serum by anti-human prolactin affinity chromatography and was shown to contain close to 1 mole of N epsilon-(gamma-glutamyl)lysine crosslinks per mole of complex, a characteristic feature in structures crosslinked by transglutaminase. Interestingly, the complex caused a proliferation of cells from a subset of patients with chronic lymphocytic leukemia, while it was inactive in a cell proliferation prolactin bioassay. By contrast, human prolactin stimulated the proliferation of cells in the bioassay but had no effect on the complex-responsive cells from the patients. Competition studies with prolactin and free Fc fragment of IgG demonstrated a necessity for engaging both the prolactin and the immunoglobulin receptors for proliferation. More importantly, competition for the growth response by free prolactin and IgG suggests both possible reasons for the slow growth of this neoplasm as well as avenues for control of the disease.

The literature supports a role for prolactin (PRL) in the development of some cancers through PRL's promotion of the cell cycle and/or inhibition of apoptosis. The selective PRL receptor modulator, S179D PRL, on the other hand, inhibits cancer cell proliferation and promotes differentiation and/or apoptosis. BRCA1 is an important tumor suppressor, shown to play a role in breast (B), ovarian (O), and prostate (P) cancer. The purpose of the current study was to determine the effect of the functionally distinct ligands for the PRL receptor on aspects of BRCA1 biology. Contrary to expectation, both PRL and S179D PRL (at 500 ng/ml) elevated levels of BRCA1 protein (∼3 fold in 24h and up to 10 fold in 48h) in all cancer types examined (B, O, &P). The promoter for the cell cycle inhibitory molecule, p21, has a BRCA1 binding site, but only S179D PRL increased levels of p21 protein as a result of BRCA1 elevation. Using deletion analysis of a p21 promoter-luciferase construct, we confirmed that the BRCA1 binding site (−84 to −129) was crucial to p21 expression in response to S179D PRL. An important question therefore was how elevated BRCA1 in response to each ligand had a different end result in terms of p21 expression. Examination of nuclear translocation of BRCA1 in response to cell stimulation with each ligand showed that both promoted movement into the nucleus. Moreover, both ligands similarly increased BRCA1 interaction with the important region (−84 to −129) of the endogenous promoter of the p21 gene (ChIP assay). A major difference in the signaling resulting from receptor engagement by PRL versus S179D PRL is that only PRL activates Stat5. With PRL, but not S179D PRL, BRCA1 formed a complex with phospho-Stat5 and this complex was present in the nucleus. Since both ligands increase BRCA1 interaction with the p21 promoter, but only PRL results in a BRCA1-phospho-Stat5 complex, it appears that binding of phospho-Stat5 to BRCA1, while not inhibiting its ability to bind to the promoter, negates the ability of BRCA1 to transactivate the p21 promoter. For already synthesized p21, additional studies demonstrated that stimulation of cells with PRL, but not S179D PRL, resulted in phosphorylation of p21, a posttranslational modification that inhibits nuclear translocation and therefore the ability of p21 to inhibit the cell cycle. This study has uncovered two ways in which p21 function is hampered by PRL and hence additional ways in which PRL promotes cell proliferation. At the same time, since S179D PRL inhibits Stat5 activation in response to PRL in addition to itself signaling to p21 elevation, this work also furthers our understanding of the anti-cancer activities of this selective PRL receptor modulator. Supported by California Breast Cancer Research program # 10PB-0127 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 700.

Because PRL has growth factor activities in several tissues, we have asked whether it also has autocrine growth factor activity in pituitary GH3 cells. GH3 cells were grown at increasing densities in the presence or absence of antirat PRL (polyclonal and monoclonal) or nonspecific antibodies. Cell proliferation increased with increasing cell density, as did the concentration of PRL in the medium. Antirat PRL, but not control antibody, markedly inhibited but did not eliminate cell proliferation, and this effect was diminished with increasing PRL concentration in the medium. PRL receptors were demonstrated on 40-50% of the cells by indirect immunofluorescence using a specific antirat PRL receptor monoclonal antibody. Cell surface PRL was colocalized to the same 40-50% of the cells and copatched or cocapped along with the receptors. Absence or presence of PRL receptors did not correlate with stage of the cell cycle, as judged by ethidium bromide dual labeling. Cell surface PRL was found to be on PRL-containing cells. These data have fulfilled four criteria necessary for establishment of a substance as a secreted autocrine growth factor: 1) the factor must be secreted; 2) in log growth phase, increased cell proliferation should occur at increased cell densities; 3) the cells must display a receptor for the factor; and 4) there must be a growth response to the factor. Thus we have established that PRL is an autocrine growth factor for at least 40-50% of the GH3 cell population. This, to our knowledge, is the first example of autocrine growth factor activity of a major hormone normotopically expressed.

As we have recently shown that prolactin secretion in vitro is regulated by extracellular leucine, we wanted to determine whether leucine had a similar regulatory action on prolactin secretion in the intact animal. Leucine was administered to cycling female rats by way of their drinking water (0.5%) for a period of 24 days. At daily intervals during this time, six control and six leucine-treated rats were killed and their trunk blood subsequently assayed for prolactin content. On days 7, 14, and 21 during this period, pituitaries from the sacrificed rats were fixed and processed for electron microscopy. Leucine treatment resulted in an alteration in the normal four-day cyclicity in serum prolactin levels and a stimulation of prolactin synthesis as evidenced by ultrastructural changes in the pituitary mammotrophs. In a parallel set of experiments, daily vaginal smears were taken from nonsynchronized animals for 24 days before and 24 days during leucine administration. Leucine treatment resulted in variable alterations in the estrus cycle depending upon the stage of the cycle when the leucine was first administered. The changes in serum prolactin and the disturbances of the estrus cycle in the leucine-treated animals persisted for approximately 20 of the 24 days. In a shorter control experiment (seven days), alanine treatment (0.5%) was found to have no effect on prolactin levels. It is concluded that a mild elevation in dietary leucine can affect prolactin synthesis and release and the normal progression of the estrus cycle.

p21-activated protein kinase gamma-PAK phosphorylates prolactin (PRL) in rat pituitary secretory granules on Ser-177 and on the equivalent site, Ser-179, in recombinant human PRL. This is shown by comparison of phosphopeptide maps with the human PRL mutant S179D. gamma-PAK is present in rat and bovine granules as identified by in-gel phosphorylation of histone H4, and by immunoblotting. Thus, phosphorylation of PRL by gamma-PAK in granules produces the PRL molecule that has been shown to antagonize the growth-promoting activity of unmodified PRL, and is consistent with the identified role of gamma-PAK in the induction and maintenance of cytostasis.

Pituitary cells maintained in monolayer culture for 48 h were used for double isotope labeling to study the release of newly synthesized vs. old PRL. The intracellular pathway taken by newly synthesized PRL was studied by autoradiography. For double labeling the cells were first incubated in a 14C-labeled amino acid (4 h) and then in a 3H-labeled amino acid (1 h), each followed by a 1-h chase. Total PRL (RIA) and radiolabeled PRL (immunoprecipitation) were determined in both cells and media. Thre rate of release of PRL (RIA) was stable throughout the experimental period. In unstimulated cells the ratio of 3H- to 14C-labeled PRL in the medium at the end of a 1-h incubation following the second chase was twice that in the cells at the beginning of this incubation, indicating that newly synthesized [3H]PRL was preferentially released. Stimulation with TRH resulted in increased release of older [14C]PRL. The earliest that radiolabeled (14C or 3H) PRL could be detected in the medium was 15--30 min after exposure of the cells to isotope. For autoradiography, cells were given a 5-min pulse of [3H]leucine, followed by chase periods of 30--180 min. Grain counts indicated that mammotrophs maintained in culture for 48 h transported and packaged PRL with the same time course as those preparations studied previously. Analysis of the distribution of total grains per cell in mammotrophs revealed the presence of several functional subpopulations with different numbers of total grains per cell, indicating that some cells were manufacturing and secreting PRL at a very fast rate. It is concluded that 1) preferential release of newly synthesized PRL is due to preferential discharge of newly synthesized granules, since release occurs at a time when labeled hormone is already in the granules; 2) there is no evidence that the established secretory pathway is bypassed, since transport, concentration, and granule formation occur; and 3) preferential release of newly synthesized PRL is the result of functional heterogeneity within the mammotroph population. Functional heterogeneity is illustrated by major differences in both synthetic rates and TRH responsiveness.

Cells dissociated from porcine pituitary glands have been used to examine the relationship between cyclic AMP levels and the stimulation of luteinising hormone (LH) secretion by luteinising hormone-releasing hormone (LHRH). It has been shown that while dibutyryl cyclic AMP and theophylline have a modest and, under some conditions, an inhibitory effect on LH release, the phosphodiesterase inhibitors isobutylmethylxanthine and ICI 63197 both induce LH release and enhance LHRH-induced release. The prostaglandins E 1 and E 2 and cholera toxin, which are shown to raise cyclic AMP levels in the dissociated preparation, also induce LH release and increase LHRH-induced LH release. Finally, LHRH is shown to increase the total content of cyclic AMP in the mixed cell preparation by approximately 30% and it is concluded that, in this system, the nucleotide does play a role in coupling LHRH stimulation to LH release.

PRL has been reported to exist as a number of mol wt and charge variants. We have attempted to determine the relationships among the charge and lower mol wt variants by two-dimensional gel analysis of pituitary and pituitary cell extracts. Silver-stained gels of the extracts showed three major charge isoforms of 24,000 mol wt PRL (PRLs 1, 2, and 3, with PRL 3 being the most anodic) and what appeared to be an arc of products originating in the region of PRL 3. Each spot in the arc was distinct and represented a small decrease in size (two to eight residues), with a corresponding increase in net negative charge. When primary cell cultures were labeled with [35S] methionine, the three PRL isoforms and arc products were detectable by autoradiography. Western blots of the two-dimensional gels showed the arc products to be immunologically related to PRL. Treatment of the cell cultures with hydroxynorvaline (5 mM), which inhibits processing of pre-PRL to PRL, resulted in doublet spots in the arc. Elimination of protease inhibitors or an increase in temperature during protein isolation had no effect on the relative concentrations of PRL and arc products. Inclusion of standard PRL or [125I]iodo-PRL in the extraction solution did not increase the size of the spots in the arc or produce labeled arc products, respectively. Treatment of the cell cultures with chloroquine (10(-5) M) before and during radiolabeling had no effect on the production of radiolabeled arc products. Analysis of cell culture medium showed at least some of the arc products to be secreted. We conclude that PRL or a PRL-like molecule is processed intracellularly into a number of smaller derivatives. As the arc products accumulate within the cell and are secreted, we suggest that they may be biologically important relatives of PRL.

In this study we have attempted to determine which of the secreted 24K isoforms was responsible for autocrine regulation of PRL secretion by comparing the isoforms synthesized and secreted by normal cells, which do autoregulate, with those synthesized and secreted by GH3 cells, which do not normally autoregulate. Comparable numbers of cells were washed free of serum and then extracted into Tris-buffered saline by sonication and detergent treatment. Proteins present in these cell extracts and in samples of culture medium were then precipitated with cold acetone (-20 C; 48 h) and subsequently dissolved in urea-lysis buffer for 2-dimensional (2-D) electrophoresis. The 2-D patterns for normal cells showed four 24K PRL isoforms inside the cells and three 24K PRL isoforms (designated 2, 3, and 3') secreted into the medium. The 2-D patterns for GH3 cells showed very little intracellular storage of PRL, but what was present was identified as 24K PRL isoform 2. The GH3 cells secreted large amounts of only 24K PRL isoform 2. Preparations of PRL containing only isoforms 1,2, and 3 (at a total radioimmunoassayable concentration of 5 micrograms/ml PRL) were capable of inducing autoregulation in GH3 cells, as evidence by decreased secretion of prelabeled intracellular PRL. Initiation of autoregulation in GH3 cells caused granulation and the intracellular production of isoform 3. Since a) a preparation containing isoforms 1, 2, and 3 was found to induce autoregulation in GH3 cells, b) isoform 1 is not a secreted form, and c) isoform 2 does not cause autoregulation (at least in GH3 cells), it is deduced that isoform 3 is an autocrine form of PRL. Since initiation of autoregulation in GH3 cells caused those cells to produce isoform 3, it is further deduced that the autoregulatory defect in GH3 cells lies in the actual lack of production of isoform 3 and not in an inherent inability of these cells to produce isoform 3.

A dissociated preparation of normal adult rat pituitary cells has been used to study PRL autoregulation at the level of the mammotroph . Female rat pituitary cells previously cultured for 48 h on polylysine-coated petri dishes were washed to remove serum and accumulated PRL and then incubated in fresh medium in the absence or presence of increasing concentrations of rat PRL. Accurate balance sheets, allowing for degradation and nonspecific adsorption of PRL, showed exogenous PRL to regulate the amount of PRL released by the cells. That this regulation was partly produced by uptake of secreted PRL from the medium was demonstrated by supplementing the medium with [125I]iodo-rat PRL. Inhibition of secretion also played a role and was implied by experiments showing that ease of reversal of the inhibition was inversely proportional to the density of cell culture, which was itself proportional to the amount of PRL in the medium and the duration of autoregulation. These results indicate that normal adult rat pituitary cells in primary culture are capable of regulating the amount of PRL in their external milieu and that uptake of already secreted PRL is an important component of the regulatory mechanism.