Estrogen receptor alpha is necessary in thymic development and estradiol-induced thymic alterations.

Estrogens affect the development, maturation, and function of multiple organ systems, including the immune system. One of the main targets of estrogens in the immune system is the thymus, which undergoes atrophy and phenotypic alterations when exposed to elevated levels of estrogen. To determine how estrogens influence the thymus and affect T cell development, estrogen receptor alpha (ERalpha) knockout (ERKO) mice were examined. ERKO mice have significantly smaller thymi than their wild-type (WT) littermates. Construction of ER radiation bone marrow chimeras indicated that the smaller thymi were due to a lack of ERalpha in radiation-resistant tissues rather than hemopoietic elements. ERKO mice were also susceptible to estradiol-induced thymic atrophy, but the extent of their atrophy was less than what was seen in WT mice. The estradiol-treated ERKO mice failed, however, to manifest alterations in their thymic CD4/CD8 phenotypes compared with WT mice. Therefore, ERalpha is essential in nonhemopoietic cells to obtain a full-sized thymus, and ERalpha also mediates some of the response of the thymus to elevated estrogen levels. Finally, these results suggest that in addition to ERalpha, another receptor pathway is involved in estradiol-induced thymic atrophy.     

Ligand-independent dimerization of the human prolactin receptor isoforms: functional implications

Prolactin (PRL) contributes to the growth of normal and malignant breast tissues. PRL initiates signaling by engaging the PRL receptor (PRLr), a transmembrane (TM) receptor belonging to the cytokine receptor family. The accepted view has been that PRL activates the PRLr by inducing dimerization of the receptor, but recent reports show ligand-independent dimerization of other cytokine receptors. Using coimmunoprecipitation assays, we have confirmed ligand-independent dimerization of the PRLr in T47D breast cancer and HepG2 liver carcinoma cells. In addition, mammalian cells transfected with differentially epitope-tagged isoforms of the PRLr indicated that long, intermediate, and DeltaS1 PRLrs dimerized in a ligand-independent manner. To determine the domain(s) involved in PRLr ligand-independent dimerization, we generated PRLr constructs as follows: (1) the TM-ICD, which consisted of the TM domain and the intracellular domain (ICD) but lacked the extracellular domain (ECD), and (2) the ECD-TM, which consisted of the TM domain and the ECD but lacked the ICD. These constructs dimerized in a ligand-independent manner in mammalian cells, implicating a significant role for the TM domain in this process. These truncated PRLrs were functionally inert alone or in combination in cells lacking the PRLr. However, when introduced into cells containing endogenous PRLr, the ECD-TM inhibited human PRLr signaling, whereas the TM-ICD potentiated human PRLr signaling. These studies indicate that the ECD-TM and the TM-ICD are capable of modulating PRLr function. We also demonstrated an endogenous TM-ICD in T47D cells, suggesting that these findings are relevant to PRL-signaling pathways in breast cancer.     

Changes in thymic estrogen receptor expression following orchidectomy.

Estrogen receptor levels were measured in cytosols prepared from thymuses of age-matched normal male and female mice. Thymic estrogen receptor concentrations were significantly higher in females than in males. Castration restored the thymic estrogen receptor expression in males to levels nearly equivalent to that of females. The results suggest that androgenic hormones may suppress estrogen receptor expression in thymus and thus account at least in part for the well documented inferior immune response of males. The data further suggest a role for estrogenic hormones in thymic function and thymocyte maturation.     

Prolactin mediates photoperiodic immune enhancement: effects of administration of exogenous prolactin on circulating concentrations, receptor expression, and immune function in steers

Changes in photoperiod can significantly impact the physiology of many species. For example, we have observed an improvement in cellular immune function in cattle on short-day photoperiod (SDPP) relative to long-day photoperiod (LDPP). In addition, prolactin (PRL) and PRL receptor (PRL-R) are affected by photoperiod management. Our hypothesis is that the inverse relationship observed between PRL and PRL-R mRNA expression during photoperiod treatment alters the sensitivity of the animal to PRL, thereby affecting the changes in their cellular immune function. The objective of this study was to determine the effects of exogenous PRL on photoperiodic-mediated immune responses. Eight Holstein steers received each of four treatments: LDPP (16L:8D), SDPP (8L:D), SDom (SDPP plus PRL via osmotic minipump for 10 days), and SDinj (SDPP plus PRL via 3x daily injections for 10 days). Steers on SDPP had decreased PRL relative to the other treatments. Expression of PRL-R mRNA was increased in SDPP animals relative to LDPP, SDom, and SDinj. Prior to PRL treatment, SDPP animals had greater lymphocyte proliferation and neutrophil chemotaxis relative to LDPP animals. Following PRL treatment, cellular immune function of SDom and SDinj animals was reduced to the level of LDPP animals. Addition of PRL to the in vitro lymphocyte proliferation did not alter response of LDPP animals but increased proliferation of lymphocytes from SDPP animals. The results of these experiments suggest that an animal's responsiveness to PRL correlate to changes in cellular immune function that occur with photoperiod manipulation.     

Differential alterations in muscarinic receptor subtypes in Alzheimer's disease: implications for cholinergic-based therapies

Molecular subtypes of muscarinic receptors (m1-m5) are novel targets for cholinergic replacement therapies in Alzheimer's disease (AD). However, knowledge concerning the relative distribution, abundance and functional status of these receptors in human brain and AD is incomplete. Recent data from our laboratory have demonstrated a defect in the ability of the M1 receptor subtype to form a high affinity agonist-receptor-G protein complex in AD frontal cortex. This defect is manifested by decreased M1 receptor-stimulated GTPgammaS binding and GTPase activity and by a loss in receptor-stimulated phospholipase C activity. Normal levels of G proteins suggest that the aberrant receptor-G protein interaction may result from an altered form of the m1 receptor in AD. The combined use of radioligand binding and receptor-domain specific antibodies has permitted the re-examination of the status of muscarinic receptor subtypes in the human brain. In AD, normal levels of m1 receptor [3H]-pirenzepine binding contrasted with diminished m1 immunoreactivity, further suggesting that there is an altered form of the m1 receptor in the disease. Reduced m2 immunoreactivity was consistent with decreased numbers of m2 binding sites. Increased levels of m4 receptors were observed in both binding and immunoreactivity measurements. These findings suggest one possible explanation for the relative ineffectiveness of cholinergic replacement therapies used to date and suggest potential new directions for development of effective therapeutic strategies for AD.     

Stressor-associated alterations in porcine plasma prolactin

Experiments were conducted to determine effects of restraint and thermal stressors on plasma prolactin (PRL) in castrated male pigs. A single 20-min restraining period in a restraining cage which prevented both movement and injury increased (P less than 0.05) plasma PRL when applied at either 0800 or 1600 hr. Exposure to 32 degrees C at 0800-1000 hr or at 1600-1800 hr produced more moderate increases (P less than 0.05). A combination of 20 min restraint and 2 hr at 32 degrees C produced a response similar to restraint alone. Twenty minutes after stressor application plasma PRL concentrations in pigs exposed to restraint or restraint +32 degrees C at 1600 h were greater (P less than 0.05) than concentrations measured in all other treatment groups at that time interval. However, there were no statistically significant differences in additional quantitative indices of the plasma PRL responses (maximal level, maximal change, or integrated response above basal levels) among restraint, 32 degrees C, or restraint +32 degrees C, nor between morning and afternoon applications of treatment. Such data do not provide, therefore, any strong evidence for stressor-dependent or circadian differences in plasma PRL response. A second study subjected castrated male pigs to 20 degrees C (controls), 20 +/- 12 degrees C (cyclic temperature, sine wave variation), 5 degrees C constant, and 5 +/- 12 degrees C cyclic for 20 days. After 6 days exposure to 5 degrees C constant or 5 +/- 12 degrees C cyclic there were decreases (P less than 0.05) of 59 and 67% respectively in plasma PRL when compared either with pretreatment levels or with levels in pigs at 20 or 20 +/- 12 degrees C. There were no differences in PRL responses between cyclic vs constant temperatures. These results are the first to indicate that plasma PRL in pigs is affected by acute restraint and thermal stressors.     

Prolactin receptor antagonism in mouse anterior pituitary: effects on cell turnover and prolactin receptor expression

Since anterior pituitary expresses prolactin receptors, prolactin secreted by lactotropes could exert autocrine or paracrine actions on anterior pituitary cells. In fact, it has been observed that prolactin inhibits its own expression by lactotropes. Our hypothesis is that prolactin participates in the control of anterior pituitary cell turnover. In the present study, we explored the action of prolactin on proliferation and apoptosis of anterior pituitary cells and its effect on the expression of the prolactin receptor. To determine the activity of endogenous prolactin, we evaluated the effect of the competitive prolactin receptor antagonist Δ1-9-G129R-hPRL in vivo, using transgenic mice that constitutively and systemically express this antagonist. The weight of the pituitary gland and the anterior pituitary proliferation index, determined by BrdU incorporation, were higher in transgenic mice expressing the antagonist than in wild-type littermates. In addition, blockade of prolactin receptor in vitro by Δ1-9-G129R-hPRL increased proliferation and inhibited apoptosis of somatolactotrope GH3 cells and of primary cultures of male rat anterior pituitary cells, including lactotropes. These results suggest that prolactin acts as an autocrine/paracrine antiproliferative and proapoptotic factor in the anterior pituitary gland. In addition, anterior pituitary expression of the long isoform of the prolactin receptor, measured by real-time PCR, increased about 10-fold in transgenic mice expressing the prolactin receptor antagonist, whereas only a modest increase in the S3 short-isoform expression was observed. These results suggest that endogenous prolactin may regulate its own biological actions in the anterior pituitary by inhibiting the expression of the long isoform of the prolactin receptor. In conclusion, our observations suggest that prolactin is involved in the maintenance of physiological cell renewal in the anterior pituitary. Alterations in this physiological role of prolactin could contribute to pituitary tumor development.     

The effect of a periovulatory prolactin imbalance on prolactin receptor expression in rat ovary cell

Using the technique of immunohistochemistry in combination with cytophotometry, we have studied the effect of periovulatory hyper- and hypoprolactinemia on the expression of prolactin receptors in various cell types of rat ovaries during early estrus. It has been shown that intense specific staining of oocytes is positively controlled by prolactin. The maximal intensity of specific staining was found in cells of the cumulus and the inner layer of granulosa cells in mature follicles; staining intensity gradually diminished towards the outer boundary cell layer. Postovulatory follicles are distinct from those mature follicles in which there was no ovulation in their more intense manifestation of prolactin receptors in cells of the inner layer and cumulus, as well as in increased positive staining (after prolactin administration) only in the granulosa layer cells closest to theca. In follicles which did not ovulate by the time of the early estrus, prolactin administration leads to a proportional growth of specific immunoreactivity in all cell layers of the granulosa. The administration of bromocryptin, an inhibitor of prolactin secretion, leading to a 10-fold decrease in prolactin level in blood, results in a twofold decrease in the intensity of specific staining of all cell layers of the granulosa in either type of follicle. Corpora lutea of the previous cycle have irregularly positioned luteocytes with weak and strong specific staining, the intensity of which is not changed in response to prolactin and diminishes slightly after the administration of bromocryptin. We conclude that the most intense changes in the content of prolactin receptors under the conditions of imbalance of this hormone during the periovulatory period are observed in those follicles where the oocyte did not ovulate by the time of early estrus.     

Endogenous human prolactin and not exogenous human prolactin induces estrogen receptor alpha and prolactin receptor expression and increases estrogen responsiveness in breast cancer cells

Prolactin (PRL) and estrogen act synergistically to increase mammary gland growth, development, and differentiation. Based on their roles in the normal gland, these hormones have been studied to determine their interactions in the development and progression of breast cancer. However, most studies have evaluated only endocrine PRL and did not take into account the recent discovery that PRL is synthesized by human mammary cells, permitting autocrine/paracrine activity. To examine the effects of this endogenous PRL, we engineered MCF7 cells to inducibly overexpress human prolactin (hPRL). Using this Tet-On MCF7hPRL cell line, we studied effects on cell growth, PRLR, ER alpha, and PgR levels, and estrogen target genes. Induced endogenous hPRL, but not exogenous hPRL, increased ER alpha levels as well as estrogen responsiveness in these cells, suggesting that effects on breast cancer development and progression by estrogen may be amplified by cross-regulation of ER alpha levels by endogenous hPRL. The long PRLR isoform was also upregulated by endogenous, but not exogenous PRL. This model will allow investigation of endogenous hPRL in mammary epithelial cells and will enable further dissection of PRL effects on other hormone signaling pathways to determine the role of PRL in breast cancer.     

Immunoregulation of autocrine prolactin: Suppressing the expression of costimulatory molecules and cytokines in T lymphocytes by prolactin receptor knockdown

Ample evidence indicates that prolactin (PRL) secreted from the pituitary gland plays an important role in a variety of human immune responses. However, the immunoregulation of autocrine PRL in T lymphocytes is not fully understood. To evaluate the role of autocrine PRL in T lymphocyte activation, PRL receptor (PRLR) in Jurkat cells was silenced by lentivirus-mediated stable expression of PRLR shRNAi. Knockdown of PRLR resulted in a considerable reduction of phytohemagglutinin (PHA)-induced T cell proliferation. Moreover, the synthesis and secretion of CD137, CD154, IL-2 and IL-4 were significantly decreased, while the production of CD28, IFN-γ and IL-10 was not affected in PHA-primed PRLR-deficient cells. These results demonstrate the importance of autocrine regulation of the PRL signaling in T lymphocyte growth and activation, and support a mechanism by which autocrine PRL participates in the immunoregulation through selectively influencing the expression of certain critical costimulatory molecules and cytokines.     

Morphine-induced immune alterations in vivo

The high incidence of human immunodeficiency virus (HIV) seropositivity among drug abusers prompted us to examine in an animal model the effects of morphine on aspects of the immune system that may be specifically related to HIV infection. We now report a robust, sustained elevation in the ratio of CD4+/CD8+ cells in the spleen and thymus of mice chronically treated with morphine. Since CD4+ cells have been reported to be target cells for HIV, these alterations, in concert with a marked cellular atrophy that appears to be restricted to organs of the immune system, suggest that opiates may serve as cofactors in altering the immune status of the host and thus contribute to the increased susceptibility to HIV infection and eventual development of AIDS in opiate abusers.     

Developmental alterations in thymocyte sensitivity are actively regulated by MHC class II expression in the thymic medulla

Developing thymocytes are positively selected if they respond to self-MHC-peptide complexes, yet mature T cells are not activated by those same self-complexes. To avoid autoimmunity, positive selection must be followed by a period of maturation when the cellular response to TCR signals is altered. The mechanisms that mediate this postselection developmental tuning remain largely unknown. Specifically, it is unknown whether developmental tuning is a preprogrammed outcome of positive selection or if it is sensitive to ongoing interactions between the thymocyte and the thymic stroma. We probed the requirement for MHC class II-TCR interactions in postselection maturation by studying single positive (SP) CD4 thymocytes from K14/A(beta)(b) mice, in which CD4 T cells cannot interact with MHC class II in the thymic medulla. We report here that SP CD4 thymocytes must receive MHC class II signals to avoid hyperactive responses to TCR signals. This hyperactivity correlates with decreased expression of CD5; however, developmental tuning can occur independently of CD5, correlating instead with differences in the distribution of Lck. Thus, the maturation of postselection SP CD4 thymocytes is an active process mediated by ongoing interactions between the T cell and MHC class II molecules. This represents a novel mechanism by which the thymic medulla prevents autoreactivity.     

Purification, cloning, and expression of the prolactin receptor

The rat liver prolactin receptor has been purified to homogeneity, and partial amino acid sequences have been obtained. The structure of the receptor has been deduced from a single complementary DNA clone. The mature protein of 291 amino acids has a relatively long extracellular region, a single transmembrane segment, and a short (57 amino acids) cytoplasmic domain. With the rat cDNA used as a probe, the prolactin receptor in rabbit mammary gland and human hepatoma cells has also been isolated. These tissues contain a second, longer form of the receptor (592 and 598 amino acids, respectively). Both the short and long forms of the prolactin receptor show regions of strong sequence identity with the human and rabbit growth hormone receptors, suggesting that the prolactin and growth hormone receptors originate from a common ancestor.     

Cloning and expression of the rat prolactin receptor, a member of the growth hormone/prolactin receptor gene family

The primary structure of the rat liver prolactin receptor has been deduced from a single complementary DNA clone. The sequence begins with a putative 19 amino acid signal peptide followed by the 291 amino acid receptor that includes a single 24 amino acid transmembrane segment. In spite of the fact that the prolactin receptor has a much shorter cytoplasmic region than the growth hormone receptor, there is strong localized sequence identity between these two receptors in both the extracellular and cytoplasmic domains, suggesting that the two receptors originated from a common ancestor.     

CRH receptor type 1 mediates continual hypoxia-induced changes of immunoreactive prolactin and prolactin mRNA expression in rat pituitary

We have reported that, in rats, hypoxia (10.8% O2) stimulates prolactin (PRL) release from the pituitary. This study is designed to compare the response of pituitary PRL to acute hypoxia (AH), continual hypoxia (CH), intermittent hypoxia (IH), cold, and restraint, individually and combined with hypoxia. This study also investigates the involvement of the corticotropin-releasing hormone receptor 1 (CRH R1) in the hypoxia-induced PRL response. Hypoxia was induced by exposing the rats to high altitudes of 2 km (16.0% O2) or 5 km (10.8% O2). The PRL levels in the pituitary (iPRL) and in plasma (pPRL) were measured by immunocytochemistry and RIA assay, respectively. The acute hypoxia of 5 km for 2-24 h caused a biphasic change (early decrease and late increase) of PRL. Both CH and IH at 2 or 5 km for 1-5 days markedly increased pPRL but decreased iPRL. Continual severe hypoxia (10.8% O2) for periods of 10, 15, and 25 days significantly enhanced pPRL but this effect was less marked at the lower altitude (16.0% O2) and did not occur during intermittent hypoxia (at both altitudes). The increased pPRL was significantly enhanced by restraint, restraint + hypoxia, hypoxia, and cold + hypoxia exposure. Treatment with a CRH R1 antagonist (CP-154,526) reversed hypoxia-decreased immunoreactive PRL and upregulated PRLmRNA in the pituitary. The data suggest that both CH and IH can stimulate rat PRL release in a time-course- and intensity-dependent manner. However, compared to the relatively low CH-induced response, restraint induced a more powerful response than either cold or hypoxia alone. CRH R1 mediates PRL secretion and PRL mRNA expression in the pituitary under hypoxic exposure. Hypoxia-enhanced PRL response over the lifespan may play a significant role in adaptation to an extreme environment.     

Cocaine decreases expression of neurogranin via alterations in thyroid receptor/retinoid X receptor signaling

The molecule responsible for the enzyme activity plasma membrane (PM) aminophospholipid translocase (APLT), which catalyzes phosphatidylserine (PS) translocation from the outer to the inner leaflet of the plasma membrane, is unknown in mammals. A Caenorhabditis elegans study has shown that ablation of transbilayer amphipath transporter-1 (TAT-1), which is an ortholog of a mammalian P-type ATPase, Atp8a1, causes PS externalization in the germ cells. We demonstrate here that the hippocampal cells of the dentate gyrus, and Cornu Ammonis (CA1, CA3) in mice lacking Atp8a1 exhibit a dramatic increase in PS externalization. Although their hippocampi showed no abnormal morphology or heightened apoptosis, these mice displayed increased activity and a marked deficiency in hippocampus-dependent learning, but no hyper-anxiety. Such observations indicate that Atp8a1 plays a crucial role in PM-APLT activity in the neuronal cells. In corroboration, ectopic expression of Atp8a1 but not its close homolog, Atp8a2, caused an increase in the population (V(max) ) of PM-APLT without any change in its signature parameter K(m) in the neuronal N18 cells. Conversely, expression of a P-type phosphorylation-site mutant of Atp8a1 (Atp8a1*) caused a decrease in V(max) of PM-APLT without significantly altering its K(m) . The Atp8a1*-expressing N18 cells also exhibited PS externalization without apoptosis. Together, our data strongly indicate that Atp8a1 plays a central role in the PM-APLT activity of some mammalian cells, such as the neuronal N18 and hippocampal cells.