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We have used the four core genotypes (FCG) mouse model, which allows a distinction between effects of gonadal secretions and chromosomal complement, to determine when sex differences in the immune system first appear and what influences their development. Using splenic T cell number as a measure that could be applied to neonates with as yet immature immune responses, we found no differences among the four genotypes at postnatal day 1, but by day 7, clear sex differences were observed. These sex differences were unexpectedly independent of chromosomal complement and similar in degree to gonadectomized FCG adults: both neonatal and gonadectomized adult females (XX and XY) showed 2-fold the number of CD4+ and 7-fold the number of CD8+ T cells versus their male (XX and XY) counterparts. Appearance of this long-lived sex difference between days 1 and 7 suggested a role for the male-specific perinatal surge of testicular testosterone. Interference with the testosterone surge significantly de-masculinized the male CD4+, but not CD8+ splenic profile. Treatment of neonates demonstrated elevated testosterone limited mature cell egress from the thymus, whereas estradiol reduced splenic T cell seeding in females. Neonatal male splenic epithelium/stroma expressed aromatase mRNA, suggesting capacity for splenic conversion of perinatal testosterone into estradiol in males, which, similar to administration of estradiol in females, would result in reduced splenic T cell seeding. These sex steroid effects affected both CD4+ and CD8+ cells and yet interference with the testosterone surge only significantly de-masculinized the splenic content of CD4+ cells. For CD8+ cells, male cells in the thymus were also found to express one third the density of sphingosine-1-phosphate thymic egress receptors per cell compared to female, a male characteristic most likely an indirect result of Sry expression. Interestingly, the data also support a previously unrecognized role for non-gonadal estradiol in the promotion of intra-thymic cell proliferation in neonates of both sexes. Microarray analysis suggested the thymic epithelium/stroma as the source of this hormone. We conclude that some immune sex differences appear long before puberty and more than one mechanism contributes to differential numbers and distribution of T cells.

Prolactin promotes a variety of cancers by an array of different mechanisms. Here, we have investigated prolactin's inhibitory effect on expression of the cell cycle-regulating protein, p21. Using a miRNA array, we identified a number of miRNAs upregulated by prolactin treatment, but one in particular that was strongly induced by prolactin and predicted to bind to the 3′UTR of p21 mRNA, miR-106b. By creating a p21 mRNA 3′UTR-luciferase mRNA construct, we demonstrated degradation of the construct in response to prolactin in human breast, prostate and ovarian cancer cell lines. Increased expression of miR-106b replicated, and anti-miR-106b counteracted, the effects of prolactin on degradation of the 3′UTR construct, p21 mRNA levels, and cell proliferation in breast (T47D) and prostate (PC3) cancer cells. Increased expression of miR-106b also stimulated migration of the very epithelioid T47D cell line. By contrast, anti-miR-106b dramatically decreased expression of the mesenchymal markers, SNAIL-2, TWIST-2, VIMENTIN, and FIBRONECTIN. Using signaling pathway inhibitors and the 3′UTR construct, induction of miR-106b by prolactin was determined to be mediated through the MAPK/ERK and PI3K/Akt pathways and not through Jak2/Stat5 in both T47D and PC3 cells. Prolactin activation of MAPK/ERK and PI3K/Akt also activates ERα in the absence of an ERα ligand. 17β-estradiol promoted degradation of the construct in both cell lines and pre-incubation in the estrogen antagonist, Fulvestrant, blocked the ability of both prolactin and 17β-estradiol to induce the construct-degrading activity. Together, these data support a convergence of the prolactin and 17β-estradiol miR-106b-elevating signaling pathways at ERα.

Females have more robust immune responses than males, well-illustrated by the degree of inflammation elicited during delayed-type hypersensitivity (DTH) responses. Here, we have investigated underlying sex differences that may contribute to differential footpad DTH responses using wildtype and four core genotypes (FCG) mice and popliteal lymphnode cellularity and gene expression. DTH responses in XX and XY FCG females showed no role for almost all genes expressed on sex chromosomes. After then filtering-out genes differentially expressed between XX and XY females, only one gene was sexually differentially expressed in wildtype mice, glycosylation-dependent cell adhesion molecule 1 (Glycam1), expressed 7-fold higher in females. Glycam1 facilitates leukocyte entry through high endothelial venules. Consistent with greater Glycam1 expression, female nodes contained twice as many cells. While females had more memory T cells in their nodes, males had a higher percentage of T regulatory cells. This sexual dimorphism in wildtype animals manifested pre-pubertally, was enhanced post-pubertally, and was eliminated by castration. The formation of male gonads is determined by the expression of Sry. Sry overexpression, which does not affect testosterone levels, produced an exaggerated male phenotype. We conclude that Sry expression through formation of the male gonad indirectly negatively impacts the potential for local inflammation.

Immune cells in the mammary gland play a number of important roles, including protection against infection during lactation and, after passing into milk, modulation of offspring immunity. However, little is known about the mechanism of recruitment of immune cells to the lactating gland in the absence of infection. Given the importance of prolactin to other aspects of lactation, we hypothesized it would also play a role in immune cell recruitment. Prolactin treatment of adult female mice for a period equivalent to pregnancy and the first week of lactation increased immune cell flux through the mammary gland, as reflected in the number of immune cells in mammary gland-draining, but not other lymph nodes. Conditioned medium from luminal mammary epithelial HC11 cell cultures was chemo-attractive to CD4+ and CD8+ T cells, CD4+ and CD8+ memory T cells, B cells, macrophages, monocytes, eosinophils, and neutrophils. Prolactin did not act as a direct chemo-attractant, but through effects on luminal mammary epithelial cells, increased the chemo-attractant properties of conditioned medium. Macrophages and neutrophils constitute the largest proportion of cells in milk from healthy glands. Depletion of CCL2 and CXCL1 from conditioned medium reduced chemo-attraction of monocytes and neutrophils, and prolactin increased expression of these two chemokines in mammary epithelial cells. We conclude that prolactin is an important player in the recruitment of immune cells to the mammary gland both through its activities to increase epithelial cell number as well as production of chemo-attractants on a per cell basis.

A comprehensive understanding of prolactin's (PRL's) role in breast cancer is complicated by disparate roles for alternatively-spliced PRL receptors (PRLR) and crosstalk between PRL and estrogen signaling. Among PRLRs, the short form 1b (SF1b) inhibits PRL-stimulated cell proliferation. In addition to ligand-dependent PRLRs, constitutively-active varieties, missing the S2 region of the extracellular domain (ΔS2), naturally occur. Expression analysis of the ΔS2 version of SF1b (ΔS2SF1b) showed higher expression in histologically-normal contiguous tissue versus invasive ductal carcinoma. To determine the function of ΔS2SF1b, a T47D breast cancer line with inducible expression was produced. Induction of ΔS2SF1b blocked estrogen-stimulated cell proliferation. Unlike intact SF1b, induction of ΔS2SF1b had no effect on PRL-mediated activation of Stat5a. However induction inhibited estrogen's stimulatory effects on serine-118 phosphorylation of estrogen receptor α, serine-473 phosphorylation of Akt, serine-9 phosphorylation of GSK3β, and c-myc expression. In addition, induction of ΔS2SF1b increased expression of the cell cycle-inhibiting protein, p21. Thus, increased expression of ΔS2SF1b, such as we demonstrate occurs with the selective PRLR modulator, S179D PRL, would create a physiological state in which estrogen-stimulated proliferation was inhibited, but differentiative responses to PRL were maintained.

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.

Lymphangioleiomyomatosis, a cystic lung disease of women, is characterized by proliferation of smooth muscle-like lymphangioleiomyomatosis cells, which possess mutations in the tuberous sclerosis complex genes, TSC1/TSC2. Growth factors involved in lymphangioleiomyomatosis cell proliferation are unknown. Prolactin, an important reproductive hormone in women, is known to promote cell proliferation and survival in other tissues.To determine the role of prolactin in signaling and proliferation in lymphangioleiomyomatosis.Prolactin levels in the sera of patients with lymphangioleiomyomatosis were correlated with clinical status. Components of prolactin signal transduction pathways were assessed in lymphangioleiomyomatosis lesions from human lung explants by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Prolactin effects on proliferation and signaling were quantified in tuberin-deficient and tuberin-expressing rat cells in vitro.Higher prolactin levels in the sera of patients with lymphangioleiomyomatosis were associated with a faster rate of decline in FEV(1) and an increased history of pneumothorax (P0.01). Higher levels of prolactin and prolactin receptor mRNA and immunoreactivity were found in lymphangioleiomyomatosis lesions when compared with vascular smooth muscle cells in the same region of tissue. This was accompanied by evidence of activation of signal transducer and activator of transcription-1 (STAT1), STAT3, p44/42, and p38 mitogen-activated protein kinase. Tsc2(-/-) Eker rat embryonic fibroblasts expressed more prolactin receptor than did Tsc2(+/+) cells, and responded to prolactin with increased proliferation and activation of the same signaling pathways seen in vivo.Prolactin may be an important growth factor in the pathogenesis of lymphangioleiomyomatosis.

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.

We have demonstrated that S179D prolactin (PRL) is potently antiangiogenic in vivo. Here, we examined apoptosis in human endothelial cells, using procaspase-8 and cytochrome c release as markers of the extrinsic and intrinsic pathways, respectively. Both pathways converge at caspase-3, which is responsible for cleavage of DNA fragmentation factor (DFF45). A 3-d incubation in 50 ng/ml S179D PRL quadrupled the number of early apoptotic cells; this effect was doubled at 100 ng/ml and became maximal at 500 ng/ml. DFF45 and procaspase 8 cleavage were detectable at 100 ng/ml. Cytochrome c, however, was unaffected until 500 ng/ml. The p21 increased at 24 h, whereas a change in p53 required both triple the time and higher doses. The p21 promoter activity was maximal at 50 ng/ml, whereas 500 ng/ml were required to see a significant change in the Bax promoter (a measure of p53 activity). Because S179D PRL and basic fibroblast growth factor (bFGF) have both been shown to activate ERK, the effect of S179D PRL on bFGF-induced ERK signaling was examined. S179D PRL blocked ERK phosphorylation in response to bFGF, whereas continued coincubation caused a delayed and prolonged activation of ERK. PD98059 inhibited this delayed activation of ERK and effects of S179D PRL on all measures except p53 levels or activity of the Bax promoter. We conclude that S179D PRL blocks bFGF-induced ERK signaling and yet uses ERK in a different time frame to elevate p21 and activate the extrinsic pathway. Prolonged incubations and high concentrations additionally activate the intrinsic pathway using an alternate intracellular signal.

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 phosphorylated PRL acts as an antagonist to the Nb2 proliferative activities of unmodified PRL. A molecular mimic of phosphorylated PRL, which substitutes an aspartate residue for the normally phosphorylated serine (serine 179), has the same properties. Because it takes less than one fourth the amount of phosphorylated hormone, or the aspartate mutant, to block the proliferative activity of unmodified hormone, we have investigated whether the high potency of the aspartate mutant is achieved by the production of an alternate and interfering intracellular signal cascade. Nb2 cells were exposed to 5 or 500 ng/ml human NIDDK PRL, wild-type recombinant PRL (unmodified PRL), or aspartate mutant PRL (pseudophosphorylated PRL) for 1, 5, or 10 min at 37 C. At 5 ng/ml and 10 min, wild-type recombinant PRL showed greater activation of Janus kinase 2 (JAK 2) than the NIDDK preparation. This is consistent with a previous report of higher proliferative activity for the wild-type hormone and is primarily a reflection of the presence of some phosphorylated hormone in the NIDDK preparation. At 500 ng/ml and 10 min, saturation eliminated any differences between responses to the two preparations. JAK 2 activation was not seen in response to the aspartate mutant at either concentration. Signal transducer and activator of transcription 5 (STAT 5) activation was, however, just as robust for the aspartate-treated cells as for the other two groups. Time course experiments eliminated the possibility that STAT 5 phosphorylation in response to the aspartate mutant was the result of JAK 2 activation at earlier time points. Experiments in the present study also interestingly showed preassociation of JAK 2 and STAT 5 in the absence of PRL and the absence of detectable phosphorylation of either JAK 2 or STAT 5. Like JAK 2, receptor phosphorylation was absent with the aspartate mutant. A comparison between STAT 5a and STAT 5b activation showed a marked reduction in STAT 5b phosphorylation in response to the aspartate mutant, with concomitant reduction in STAT 5a-STAT 5b heterodimers. STAT 5a activation, however, was indistinguishable between the wild-type and aspartate mutant. We conclude that the nonproliferative aspartate mutant signals and activates STAT 5 without, or with minimal, use of JAK 2 or receptor phosphorylation. The wild-type proliferative PRL, on the other hand, uses receptor phosphorylation and JAK 2 activation.

The purpose of this study was to determine the effects of estrogen (E2) replacement on thermoregulation in ovariectomized rats exposed to heat. Female Sprague-Dawley rats were ovariectomized and splenectomized and implanted with a temperature-sensitive transmitter. Each rat was studied when E2 treated (after an E2 pellet implant) and untreated. Animals were divided into two groups with opposite order of treatment and were studied over a 9-wk period. Measurements of body core temperature (Tc) and evaporative water loss (EWL) were made on unrestrained animals resting at 38 degrees C air temperature. E2-treated animals increased EWL at all levels of Tc, reduced the threshold Tc for onset of saliva spreading, and regulated Tc at a lower level during heat exposure. E2 treatment elevated plasma E2 and reduced hematocrit but did not affect plasma osmolality. These effects of E2 on evaporative cooling and Tc in heat-stressed rats are similar to those that have been reported in human females. The mechanisms of the thermoregulatory effects of E2 remain to be studied.

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.

Prolactin (PRL) in milk influences maturation of gastrointestinal epithelium and development of both the hypothalamo-pituitary and immune systems of offspring. Here, we demonstrate that most PRL in human milk is part of a novel, high-affinity, multicomponent binding complex found on the milk fat globule membrane and not in whey. To examine properties of the complex, a sensitive ELISA was developed such that human PRL (hPRL) binding to the complex was measured by loss of hPRL detectability; thus, as much as 50 ng of hPRL was undetectable in the presence of 10 μl of human milk. Using the same methodology, no comparable complex formation was observed with human serum or amniotic fluid. hPRL complexation in milk was rapid, time dependent, and cooperative. Antibodies to or competitors of the hPRL receptor (placental lactogen and growth hormone) showed the hPRL receptor was not involved in the complex. However, hPRL complexation was antagonized by cyclosporine A and anti-cyclophilins. The complex was very stable, resisting dissociation in SDS, urea, and dithiothreitol. Western analysis revealed an ∼75-kDa complex that included hPRL, cyclophilins A and B, and a 16-kDa cyclophilin A. Compared with noncomplexed hPRL, complexed hPRL in whole milk showed similar activation of STAT5 but markedly delayed activation of ERK. Alteration of signaling suggests that complex formation may alter hPRL biological activity. This is the first report of a unique, multicomponent, high-capacity milk fat reservoir of hPRL; all other analyses of milk PRL have utilized defatted milk.

The discovery of interfering RNAs uncovered a new level of regulation of gene expression. It is now believed that as much as 92% of gene expression may be regulated by interfering RNAs. Interfering RNAs may be micro RNAs (miRNAs) or small interfering RNAs (siRNAs). Our focus is on miRNAs. These are mostly coded in intronic or intergenic regions of DNA and are grouped into families on the basis that they likely evolved from a common ancestral gene. Among the miRNA families, the miR17-92 family has attracted attention because of its oncogenic activity. miRNAs in this family include the miR17-92 cluster and two paralogs, the miR-106a and miR-106b clusters. Expression of these miRNAs is markedly upregulated in several types of cancer, and they are considered oncomirs. The two paralogs derive from an ancient gene duplication event involving the miR17-92 cluster. They therefore share highly similar sequences with miR17-92 family members and each other. As a result, they also work on very similar targets, primarily inhibiting the translation of target mRNAs by binding to the 3’ untranslated region. The miR-106 paralogs are located on different chromosomes from the miR17-92 cluster: miR-106a is intriguingly located on the X chromosome, miR-106b on chromosome 7, and miR17-92 on chromosome 13. Regulation of expression of any of the paralogs can therefore occur without concomitant regulation of the other two. This review examines the thesis that miR-106a in particular may play an important role in the development and progression of breast cancer. Because relatively little attention has yet to be given to miR-106a, the potential role of miR-106a is often suggested on the basis of a known role of a related family member. Similarly, defined roles of miR106a and family members in other neoplasms are used to suggest a role in breast cancer.

Despite the important role of prolactin (PRL) in mammary gland development and function, little is known about the distribution of the different forms of the prolactin receptor (PRLR) under various physiological circumstances. Here, the distribution of the long (LF) and the short (S3 in mouse) receptor common to both mice and rats was determined by immunofluorescence on frozen sections of virgin, pregnant and lactating mouse mammary gland. Myoepithelial cells were consistently and intensely stained for both receptors. For luminal cells at all stages (ducts and alveoli), a large proportion of PRLR staining was unexpectedly present on the apical face. In the non-lactating state, no basal staining of luminal cells was detectable. During lactation, a proportion of both receptors moved to the basolateral surface. In vitro, HC11 cells showed constitutive expression of LF but expression of S3 only upon the formation of adherent junctions. Tight junction formation was accelerated by incubation in pseudo-phosphorylated PRL, as measured by transepithelial resistance and the expression and placement of the tight junction protein, zonula occludens-1. Once an intact monolayer had formed, all LF and S3 receptors were apical (akin to the non-lactating state) and only apical application of PRL activated the Jak2-STAT5 and ERK pathways. By contrast, basolateral application of PRL resulted in a reduction in basal ERK phosphorylation, suggesting an involvement of a dual specificity protein phosphatase. Normal human breast samples also showed apical PRLRs. These results demonstrate important contextual aspects of PRL-PRLR interactions with implications for the analysis of the role of PRL in breast cancer.

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.

The effects of different concentrations of various K-current blockers on prolactin-induced proliferation and membrane K-currents in malignant lymphocytes (Nb2 cells) were investigated. Membrane currents were measured with the whole cell patch-clamp technique, and lymphocyte density was quantified by both spectrophotometric and conventional methods. K-current blockers tested (quinidine, 4-aminopyridine, barium, and tetraethylammonium) exhibited similar rank order potency for K-current block and inhibition of prolactin-induced proliferation of malignant lymphocytes. Because Nb2 cells proliferate independently of a transmembrane Ca-influx, these results suggest that K-currents per se rather than K-current modulation of Ca-influx is an essential event for lymphocyte 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.

BACKGROUND We have shown that treatment of human prostate cancer cells with the selective prolactin (PRL) receptor modulator, S179D PRL, inhibits growth in vitro, and the initiation and growth of xenografts in vivo. S179D PRL treatment also upregulates expression of the short form 1b (SF1b) PRL receptor, activation of which upregulates expression of the cell cycle-regulating protein, p21. METHODS We examined the consequences of long term increased expression and activation of SF1b, at levels comparable to those resulting from treatment with S179D PRL, by creating PC-3-derived stable cell lines expressing a constitutively active form of SF1b, ΔS2 SF1b. RESULTS Increased expression of ΔS2 SF1b decreased growth and migration of the cells. This was accompanied by an increase in cell-matrix interactions, and cell–cell aggregation when cells were plated on basement membrane components. Real-time PCR evaluated the expression of genes related to invasive capacity. Of particular interest was decreased expression of the protease, urokinase-type plaminogen activator, and its receptor, uPAR, and increased expression of its inhibitors, PAI-1 and 2. Also decreased in cells with increased expression of ΔS2 SF1b was expression of basic fibroblast growth factor and vascular endothelial growth factor. CONCLUSION We conclude that at least part of the beneficial effects of S179D PRL is the result of increased expression of SF1b, and that the effects of increased expression and activation of SF1b continue to be of potential benefit in the long term. Prostate 70: 37–47, 2010. © 2009 Wiley-Liss, Inc.

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.

We have previously described a series of C-terminally-clipped forms of PRL, the 21-23.5K PRL-like molecules (PLMs). Because we noted estrogen (E2) induction of PLMs and E2 also induces a pituitary glandular kallikrein, we have investigated the possibility that processing of PRL by kallikrein is responsible for the production of the PLMs. Subcellular fractionation of pituitaries from control or E2-treated female rats showed total kallikrein to be concentrated 1- to 4-fold and 6- to 20-fold in the granules (vs. original homogenate) from control and E2-treated animals, respectively. Cleavage of purified PRL by kallikrein resulted in the formation of large quantities of a number of the PLMs. Incubation of secretory granules derived from control or E2-treated animals under the same conditions showed no cleavage of PRL in the absence of a limiting granule membrane and a small production of the PLMs in the presence of a granule membrane. Production in the latter instance was slightly greater in granules derived from E2-treated animals. Addition of purified kallikrein to secretory granules from control or E2-treated animals in the presence or absence of granule membranes, resulted in the additional production of large amounts of only the smallest PLM (PLM 9), indicating a lack of cleavage of the two most C-terminal sites for the enzyme. Increasing the concentration of beta-mercaptoethanol to 0.64 M, to ensure monomerization of the granule PRL, had no effect on endogenous kallikrein activity of the number of products resulting from the addition of exogenous kallikrein to granules. In summary: 1) purified kallikrein can cleave purified PRL to form products which run with the same isoelectric point and mol wt values as the PLMs; 2) kallikrein is present in PRL secretory granules; 3) some PLM production occurs in a membrane-bound secretory granule fraction, but none occurs after removal of the membrane; this intragranular production, like cleavage of purified PRL with purified kallikrein, is dependent on the presence of a detergent and a reducing agent; 4) cleavage of granular PRL by exogenous kallikrein is limited to a single site and the more C-terminal sites are protected; and 5) protection of the C-terminus is not removed by intermolecular and intramolecular disulfide bond reduction. We conclude: 1) that pituitary glandular kallikrein is a strong candidate for the enzyme responsible for the production of the PLMs, and 2) that there is some element of PRL storage, other than intermolecular disulfide bonds, which involves the C-terminus of the molecule.

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.

BACKGROUND S179D Prolactin (PRL) is a molecular mimic of naturally phosphorylated human PRL which has been shown to inhibit the growth of human prostate cancer cells both in vitro and when grown as tumors in nude mice. METHODS In the current study, we have investigated the potential interplay between S179D PRL and 1,25 dihydroxy vitamin D3 (1,25D) in the inhibition of prostate cancer cell growth by incubating cells under circumstances where each hormone alone has no effect. RESULTS Incubation of DU145 or PC3 cells in 100 pM 1,25D or 10 nM S179D PRL for 3 days showed no effect of each alone on expression of the vitamin D receptor (VDR), or the cell cycle regulatory protein p21, or on cell number. Incubation in both together increased expression of the VDR and p21 two to threefold. This co-operative effect was reproduced when activation of the p21 promoter was analyzed using a p21-luciferase (p21-luc) construct. Elimination of the VDR response element from p21-luc eliminated response to the hormone combination, showing that the effect on p21 was through the VDR. Most importantly, S179D PRL sensitized the cells to 1,25D such that there was a concentration-related reduction in cell number versus controls between 40 and 160 pM. At least part of this effect was via the induction of cell death. CONCLUSIONS These results suggest that combined anti-tumor therapy may be very efficacious and that the dose of 1,25D required may be below the range that results in hypercalcemia. Prostate 67: 1498–1506, 2007. © 2007 Wiley-Liss, Inc.

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.

It is well established that females mount stronger immune responses than males, but only very little is understood about the underlying mechanisms. We have analyzed local cytokine differences among intact females, those that had been ovariectomized (OVX), those receiving estrogen replacement after OVX, and males, both before and after production of delayed-type hypersensitivity (DTH) responses. We report confirmation of a much larger DTH response in females versus males. However, OVX resulted in an even larger response, while estrogen replacement resulted in a smaller response when compared with intact females. In animals exposed for the first time to an antigen (without a DTH response), OVX increased interleukin-6 (IL-6) and estrogen replacement after OVX suppressed IL-6. Of the cytokines that differed between males and females exposed for the first time to an antigen, only IL-6 was higher in females versus males when exposure to antigen occurred for the second time (when the DTH response occurs). Analysis of cytokines with OVX and estrogen replacement after a second exposure to antigen showed that IL-6 did not significantly change. Levels of IL-4; Regulated upon Activation, Normal T-cell Expressed; and Secreted; and thrombopoietin, however, correlated with the DTH response, suggesting direct or indirect positive regulation by estrogen. These results suggest an important role for both IL-6 and IL-4 in determining the degree of DTH response, with IL-6 (which appears negatively regulated by estrogen) increasing and IL-4 (which appears positively regulated by estrogen) decreasing the response. The results further suggest that IL-6 may play a role in predisposing to a larger DTH response, while IL-4 levels seem more important during an active response.

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.

BACKGROUND Membrane-associated human chorionic gonadotropin β (HCGβ) is correlated with a poor prognosis in localized prostate adenocarcinoma. The relationship between HCGβ and metastasis, however, is unclear. METHODS To shed some light on the issue, two stable prostate carcinoma cell lines overexpressing HCGβ, designated DU145 HCGβ and PC3 HCGβ, were created and compared with empty vector stably transfected DU145 and PC3 cells (control cells). RESULTS HCGβ expression resulted in a change in morphology; the cells were more elongated and had multiple pseudopodia, while the control cells were more rounded. This change in morphology was duplicated by incubating control cells in conditioned medium from the DU145 HCGβ or PC3 HCGβ cells, or by adding purified HCGβ to control medium. The DU145 HCGβ and PC3 HCGβ cells were also less adherent than the controls, as assessed by the ease with which trypsin-EDTA could remove them from culture plates. Reduced adherence could be duplicated by incubation of control cells with either conditioned medium or purified HCGβ. Western blot analysis showed that DU145 HCGβ and PC3 HCGβ cells expressed less E-cadherin than control cells and that a change of medium increased expression of E-cadherin. Addition of conditioned medium, or purified HCGβ, to control cells down-regulated E-cadherin. Cell migration and invasion assays showed that DU145 HCGβ and PC3 HCGβ cells were more migratory and invasive than controls and that treatment of control cells with either conditioned medium or purified HCGβ increased their migratory/invasive capacity. CONCLUSION The data indicate that HCGβ is directly responsible for changes in prostate carcinoma cells associated with an increased metastatic phenotype. Cancer 2006. © 2005 American Cancer Society.

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.

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.

Tumor infiltration by T regulatory cells (Tregs) occurs in a wide variety of cancers. Although there is evidence that tumor Tregs decrease immune responses to tumor cells and increase metastasis, there is much to be learned about the mechanisms involved. Tregs constitutively express the prolactin receptor (PRLR), suggesting that PRL is important to function. Essentially all Treg PRLR are the long isoform. To investigate the role that PRL might play in tumor Treg recruitment and function, we used the syngeneic 4T1 orthotopic breast cancer model. Mice were treated systemically with a splice modulating oligomer (SMO) that specifically knocks down expression of only the long isoform (LF) of the PRLR. Treatment with this SMO resulted in decreased tumor Tregs (0.65% of gated tumor cells in PRLRLF knockdown compared to 3.31% in control SMO-treated mice), demonstrating that prolactin (PRL) signaling through the PRLRLF directly or indirectly recruited Tregs to this kind of tumor. Further investigation in vitro and in vivo revealed two different ways that PRL assists in recruitment: 1) PRL directly stimulated tumor cell production of CCL17, a chemokine that attracts Tregs. This was inhibited when PRLRLF was knocked down. Furthermore, the PRLRLF antagonist, S179DPRL, inhibited basal CCL17, demonstrating that tumor cell autocrine PRL could stimulate CCL17 output. 2) PRL signaling is required for CCR4 expression on tumor Tregs. CCR4 is normally constitutively expressed on Tregs and is the receptor that responds to CCL17. Analysis of tumor cell chemokine production in response to estrogen or progesterone in vitro showed estrogen induced CCL22, another ligand for Treg CCR4, while progesterone induced CCL17. All three hormones therefore could promote Treg recruitment, but the ability of PRLRLF knockdown, which has little effect on levels of estrogen and progesterone, to inhibit recruitment suggests the effects of the other two hormones are mediated through PRL. PRL treatment of tumor cells in vitro showed a small decrease in mesenchymal markers consistent with reports in other breast cancer cells. Importantly however, in the presence of Tregs, PRL markedly increased expression of twist-1, snail-2, vimentin and fibronectin. Furthermore, if PRLRLF was knocked down in Tregs, there was no increase in mesenchymal markers in the tumor cells in response to PRL. Cancer stem cells are considered important players in metastatic spread. Analysis of isolated stem cells showed the same PRL and Treg interactions as for bulk tumor cells in terms of mesenchymal markers. Tregs with PRLRLF knockdown showed no lack of suppressive ability in a splenocyte response to lipopolysaccharide test. Taken together, results show that PRLRLF signaling in Tregs is essential for Treg-mediated EMT, but is dispensable for general immune suppressive ability. These effects contributed to an 80% inhibition of metastatic spread coincident with a marked reduction in inflammatory damage from responses to metastatic cells after 40days of PRLRLF knockdown. Citation Format: KuanHui E. Chen, Mrinal K. Ghosh, Tomohiro Yonezawa, Ameae M. Walker. Prolactin drives the recruitment of tumor Tregs and Treg-mediated metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-255. doi:10.1158/1538-7445.AM2014-LB-255

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.