Hyperthyroidism (HyperT) compromises pregnancy and lactation, hindering suckling-induced PRL release. We studied the effect of HyperT on hypothalamic mRNA (RT-qPCR) and protein (Western blot) expression of tyrosine hydroxylase (TH), PRL receptor (PRLR) and signaling pathway members, estrogen-α (ERα) and progesterone (PR) receptors on late pregnancy (days G19, 20 and 21) and early lactation (L2) in rats. HyperT advanced pre-partum PRL release, reduced circulating PRL on L2 and increased TH mRNA (G21 and L2), p-TH, PRLR mRNA, STAT5 protein (G19 and L2), PRLR protein (G21) and CIS protein (G19). PRs mRNAs and protein decreased on G19 but afterwards PRA mRNA (G20), PRB mRNA (G21) and PRA mRNA and protein (L2) increased. ERα protein increased on G19 and decreased on G20. Thus, the altered hypothalamic PRLR, STAT5, PR and ERα expression in hyperthyroid rats may induce elevated TH expression and activation, that consequently, elevate dopaminergic tone during lactation, blunting suckling-induced PRL release and litter growth.

Much evidence suggests that prolactin has an immunoregulatory function and that its effects on cells of the immune system depend on the level and specific forms of the receptors present on the target cells. The effect of administration of prolactin on polyamine catabolism was investigated in thymus of male intact rats by measuring the activities of spermidine/spermine N(1)-acetyltransferase and polyamine oxidase, because of the relationships between polyamines (especially putrescine) and the immune system. The administration of prolactin to rats resulted in the rapid induction of spermidine/spermine N(1)-acetyltransferase activity in the thymus (1.6-times the level of control rats, within 4 h), and in a marked decrease in polyamine oxidase activity at 24 h. The changes in enzyme activities were accompanied by an increase in putrescine concentration and a decrease in spermidine and spermine concentrations. In the spleen, prolactin increased SAT activity only 24 h after administration and was ineffective on PAO activity.

Natural killer (NK) cells are the predominant lymphocytes present in healthy rodent and human implantation sites. In the rat, the expansion, differentiation and subsequent migration of NK cells away from the developing chorioallantoic placenta coincide with the expression of a novel pregnancy- and trophoblast cell-specific cytokine, prolactin (PRL)-like protein A (PLP-A). PLP-A specifically binds to uterine NK cells but does not appear to utilize receptor systems for PRL. In the present report, we show that PLP-A interactions with NK cells are not mediated by receptors utilized by known modulators of NK cell function, including interleukin-2, interleukin-7, interleukin-12, and interleukin-15 (IL-15). Uterine NK cells respond to PLP-A or IL-15 with an increase in intracellular calcium mobilization. In contrast, PLP-A, unlike IL-15, effectively suppresses the ability of NK cells to produce interferon-gamma (IFNgamma), a key mediator of NK cell function. Placental PLP-A expression is reciprocal to mesometrial decidua expression of IFNgamma. Increased expression of PLP-A by the placenta coincides with the decline of IFNgamma content in the mesometrial decidua adjacent to the placenta. In summary, trophoblast cell-derived PLP-A contributes to the regulation of NK cells at the maternal-fetal interface to ensure appropriate embryonic growth and development.

Human prolactin (hPRL) is a pleiotropic and versatile hormone that exercises more than 300 biological activities through binding to its cognate receptors. Recently, multiple studies have implicated hPRL in the development of human breast cancer. As a target of hPRL, both normal and neoplastic human breast cells also synthesize and secrete hPRL, which therefore establishes an autocrine/paracrine action loop in the mammary gland. In contrast to the extensive studies of regulation of hPRL expression in the pituitary gland, regulation of hPRL in mammary tissue and human breast cancer cells has not been extensively addressed. Extrapituitary PRL expression is primarily regulated by a distal promoter located 5.8 kb upstream to the pituitary promoter. As a result of alternative promoter usage, extrapituitary PRL is regulated by different signalling pathways and different hormones, cytokines or neuropeptides compared to regulation in the pituitary. Here, we present evidence that shows estrogen directly induces hPRL gene expression in T47D human breast cancer cells. We have identified a functional, non-canonical estrogen responsive element (ERE) and an AP1 site located in the hPRL distal promoter. Gel shift and chromatin immunoprecipitation assays demonstrated that both estrogen receptor (ER)alpha and ERbeta directly bind to the ERE. However, only ERalpha interacts with AP1 proteins that bind to the AP1 site in the hPRL distal promoter. Promoter-reporter gene studies demonstrate that both ERE and AP1 sites are required for full induction of the promoter activity by estradiol. Our studies suggest that the interactions between estrogens, ERs, the ERE and AP1 transcription factors in regulation of autocrine/paracrine PRL in the human breast may be critical for oncogenesis and may contribute to progression of breast cancer.

Estrogen and dopamine are major opposing regulators of the endocrine functions of pituitary lactotrophs. Dopamine inhibits estrogen-induced changes in the synthesis and secretion of prolactin, and lactotroph proliferation. We studied the mechanism of the inhibitory effects of dopaminergic stimulation on estrogen-induced functional changes of rat lactotrophs in primary culture. The dopaminergic agonist, bromocriptine (BC), suppressed 17β-estradiol-stimulated lactotroph proliferation, prolactin promoter activity, and mRNA expression of some estrogen-responsive genes. In lactotroph-enriched pituitary cells, BC treatment inhibited the estrogen response element (ERE) DNA sequence-mediated estrogen receptor (ER) transcriptional activity. Using a lactotroph-specific ERE transcriptional assay, we found that BC inhibition of the ERE-mediated ER transcriptional activity partly involved D2 dopamine receptor-mediated, pertussis toxin-sensitive G protein-coupled, cAMP/protein kinase A-dependent signaling. BC treatment had no effect on the cellular concentration of ERα or its phosphorylation status at Ser-118. Similar transcriptional inhibition by BC was also found in GH4ZR7 cells, a D2 dopamine receptor-expressing somatomammotrophic cell line. These results suggest that activation of the D2 dopamine receptors inhibits estrogen-dependent lactotroph functions in part via attenuation of ERE-mediated ER transactivation.

Thyroid diseases have deleterious effects on lactation, litter growth and survival, and hinder the suckling-induced hormone release, leading in the case of hyperthyroidism, to premature mammary involution. To determine the effects of hypothyroidism (HypoT) on late lactation, we analyzed the effect of chronic 6-propyl-2-thiouracil (PTU)-induced HypoT on mammary histology and the expression of members of the JAK/STAT/SOCS signaling pathway, milk proteins, prolactin (PRLR), estrogen (ER), progesterone (PR) and thyroid hormone (TR) receptors, markers of involution (such as stat3, lif, bcl2, BAX and PARP) on lactation (L) day 21. HypoT mothers showed increased histological markers of involution compared with control rats, such as adipose/epithelial ratio, inactive alveoli, picnotic nuclei and numerous detached apoptotic cells within the alveolar lumina. We also found decreased PRLR, β-casein and α-lactoalbumin mRNAs, but increased SOCS1, SOCS3, STAT3 and LIF mRNAs, suggesting a decrease in PRL signaling and induction of involution markers. Furthermore, Caspase-3 and 8 and PARP labeled cells and the expression of structural proteins such as β-Actin, α-Tubulin and Lamin B were increased, indicating the activation of apoptotic pathways and tissue remodelation. HypoT also increased PRA (mRNA and protein) and erβ and decreased erα mRNAs, and increased strongly TRα1, TRβ1, PRA and ERα protein levels. These results show that lactating HypoT rats have premature mammary involution, most probably induced by the inhibition of prolactin signaling along with the activation of the LIF-STAT3 pathway.

Human growth hormone (GH) binds and activates GH receptor (GHR) and prolactin (PRL) receptor (PRLR). LNCaP human prostate cancer cells express only GHR. A soluble fragment of IGF-1 receptor (IGF-1R) extracellular domain (sol IGF-1R) interacts with GHR and blocks GH signaling. We now explore sol IGF-1R's specificity for inhibiting GH signaling via GHR vs. PRLR and test GHR and PRLR extracellular domain inhibition determinants. Although T47D human breast cancer cells express GHR and PRLR, GH signaling is largely PRLR-mediated. In T47D, sol IGF-1R inhibited neither GH- nor PRL-induced STAT5 activation. However, sol IGF-1R inhibited GH-induced STAT5 activation in T47D-shPRLR cells, which harbor reduced PRLR. In MIN6 mouse β-cells, bovine GH (bGH) activates mouse GHR, not PRLR, while human GH activates mouse GHR and PRLR. In MIN6, sol IGF-1R inhibited bGH-induced STAT5 activation, but partially inhibited human GH-induced STAT5 activation. These findings suggest sol IGF-1R's inhibition is GHR-specific. Using a cellular reconstitution system, we compared effects of sol IGF-1R on signaling through GHR, PRLR, or chimeras in which extracellular subdomains 2 (S2) of the receptors were swapped. Sol IGF-1R inhibited GH-induced STAT5 activation in GHR-expressing, not PRLR-expressing cells, consistent with GHR specificity of sol IGF-1R. Interestingly, we found that GHR S2 (which harbors the GHR-GHR dimer interface) was required, but not sufficient for sol IGF-1R inhibition of GHR signaling. These results suggest sol IGF-1R specifically inhibits GH-induced GHR-mediated signaling, possibly through interaction with GHR S1 and S2 domains. Our findings have implications for GH antagonist development.