Role of Prolactin Receptors in Lymphangioleiomyomatosis

Pulmonary lymphangioleiomyomatosis (LAM) is a rare lung disease caused by mutations in the tumor suppressor genes encoding Tuberous Sclerosis Complex (TSC) 1 and TSC2. The protein product of the TSC2 gene is a well-known suppressor of the mTOR pathway. Emerging evidence suggests that the pituitary hormone prolactin (Prl) has both endocrine and paracrine modes of action. Here, we have investigated components of the Prl system in models for LAM. In a TSC2 (+/-) mouse sarcoma cell line, down-regulation of TSC2 using siRNA resulted in increased levels of the Prl receptor. In human LAM cells, the Prl receptor is detectable by immunohistochemistry, and the expression of Prl in these cells stimulates STAT3 and Erk phosphorylation, as well as proliferation. A high affinity Prl receptor antagonist consisting of Prl with four amino acid substitutions reduced phosphorylation of STAT3 and Erk. Antagonist treatment further reduced the proliferative and invasive properties of LAM cells. In histological sections from LAM patients, Prl receptor immuno reactivity was observed. We conclude that the Prl receptor is expressed in LAM, and that loss of TSC2 increases Prl receptor levels. It is proposed that Prl exerts growth-stimulatory effects on LAM cells, and that antagonizing the Prl receptor can block such effects.     

Tachykinins Induce Secretion of Prolactin from Perifused Rat Anterior Pituitary Cells by Interactions with Two Different Binding Sites

Abstract

Substance P and the two other mammalian tachykinins, neurokinin A and B, are accepted to have direct regulating effects at the anterior pituitary level. We have examined the effects of substance P (SP) and neurokinin B (NKB), alone and in combination, on prolactin release from cultured anterior pituitary cells grown on collagen-coated micro beads and placed in a perfusion system. Prolactin (Prl) secretion was observed within 25 s after exposure to either secretagogue and reached a maximum within 60-80 s. Furthermore, the prolactin response induced by SP and NKB was dose-dependent. Prl secretion remained constant for up to 4 h when SP or NKB were perifused and then fell gradually towards basal levels. Simultaneous addition of submaximal concentrations of SP and NKB resulted in an additive response compared with the responses of either secretagogue alone. Continuous (8 h) perifusion with SP did not prevent a normal prolactin response by NKB or TRH. These results indicate that the tachykinins, substance P and neurokinin B, release Prl from perifused female rat anterior pituitary cells by interaction with two different receptors, possibly the NK1 and NK3 tachykinin receptor subtypes.     

Uterine Notch2 facilitates pregnancy recognition and corpus luteum maintenance via upregulating decidual Prl8a2

The maternal recognition of pregnancy is a necessary prerequisite for gestation maintenance through prolonging the corpus luteum lifespan and ensuring progesterone production. In addition to pituitary prolactin and placental lactogens, decidual derived prolactin family members have been presumed to possess luteotropic effect. However, there was a lack of convincing evidence to support this hypothesis. Here, we unveiled an essential role of uterine Notch2 in pregnancy recognition and corpus luteum maintenance. Uterine-specific deletion of Notch2 did not affect female fertility. Nevertheless, the expression of decidual Prl8a2, a member of the prolactin family, was downregulated due to Notch2 ablation. Subsequently, we interrupted pituitary prolactin function to determine the luteotropic role of the decidua by employing the lipopolysaccharide-induced prolactin resistance model, or blocking the prolactin signaling by prolactin receptor-Fc fusion protein, or repressing pituitary prolactin release by dopamine receptor agonist bromocriptine, and found that Notch2-deficient females were more sensitive to these stresses and ended up in pregnancy loss resulting from abnormal corpus luteum function and insufficient serum progesterone level. Overexpression of Prl8a2 in Notch2 knockout mice rescued lipopolysaccharide-induced abortion, highlighting its luteotropic function. Further investigation adopting Rbpj knockout and DNMAML overexpression mouse models along with chromatin immunoprecipitation assay and luciferase analysis confirmed that Prl8a2 was regulated by the canonical Notch signaling. Collectively, our findings demonstrated that decidual prolactin members, under the control of uterine Notch signaling, assisted pituitary prolactin to sustain corpus luteum function and serum progesterone level during post-implantation phase, which was conducive to pregnancy recognition and maintenance.     

A transplantable rat Leydig cell tumor--1. LH and prolactin receptors and effects of the endocrine status of the host animal

We have examined the binding capacity and properties (affinity, specificity) of LH and prolactin (Prl) receptors in a transplantable rat Leydig cell tumor (H-540) grown in intact, castrated and hypophysectomized rats. LH receptors in adult rat testis and Prl receptors in the rat ventral prostate were examined simultaneously for comparison. The results can be summarized as follows: The qualitative properties (affinity, specificity) of LH and Prl receptors in tumor Leydig cells appear to be identical to those of corresponding receptors in non-tumor tissues. The levels of LH receptors in tumor Leydig cells are only some 1% of that present in normal Leydig cells from adult rats. Tumor Leydig cells grown in hypophysectomized rats had even lower levels of LH receptors; ca. 1/3 of that found in tumors from intact rats. The levels of Prl receptors in the tumor Leydig cells are almost as high as in normal Leydig cells from adult rats. In tumors grown in hypophysectomized rats, the levels of Prl receptors were much lower (ca. 20%) than in tumors from intact or castrated rats. There were great variations in the number of LH and Prl receptors in individual tumors, and there was a positive correlation (r = 0.88; P less than 0.01) between LH and Prl receptors in individual tumors. No differentiation toward a "LH receptor tumor" or "Prl receptor tumor" was observed. Thus, receptors for LH and Prl in tumor cells are qualitatively normal, but the number is greatly (LH) or moderately (Prl) reduced. These receptors in the tumor Leydig cells are stimulated by pituitary hormones.     

Prolactin in the marsupial Macropus eugenii, during the estrous cycle, pregnancy and lactation

An heterologous double antibody radioimmunoassay (RIA) using a guinea-pig antiserum (33-9) raised against human prolactin and 125I-ovine prolactin has been developed to measure prolactin (Prl) in plasma and pituitary preparations of marsupials. In this system, purified tammar and kangaroo Prl preparations showed parallel dose-response curves as did serial dilutions of crude pituitary homogenates of tammar, possum and eastern grey kangaroo. Serial dilutions of plasma from ovariectomized and lactating female and castrate male tammars showed immunoreactivity, and plasma Prl levels increased after injection of TRH. The assay has been used to monitor changes in plasma Prl in female tammars in various reproductive states. Plasma Prl remained at basal concentrations of 20 to 30 ng/ml throughout the estrous cycle, at estrus and during pregnancy. However, just prior to parturition, there was a 2- to 3-fold increase in Prl concentrations which declined to basal levels after birth. During early lactation, Prl levels were low but increased to maximum concentration in the second half of lactation.     

Effect of lipoproteins and luteotrophins on progesterone accumulation by luteal cells from the pregnant pig

The roles of prolactin (Prl) and LH in the maintenance of luteal function in pregnant pigs were investigated. Luteal cells from pigs between days 70 to 95 of pregnancy were dissociated and incubated for 4 h. In the absence of exogenous cholesterol, LH exhibited a dose-dependent stimulatory effect on progesterone secretion. Prl had a mild stimulatory effect on progesterone accumulation and at lower doses Prl potentiated the response to LH. Low density lipoprotein (LDL) but not high density lipoprotein (HDL) had a mild stimulatory effect on progesterone secretion. When exogenous cholesterol was provided as the substrate in the form of LDL or HDL, Prl had a striking stimulatory effect on progesterone secretion. When 25-hydroxycholesterol which bypasses the lipoprotein receptor was provided as the substrate, Prl failed to stimulate progesterone accumulation. The stimulatory effect of LH was potentiated when LDL, HDL, or 25-hydroxycholesterol were present. The results of this study suggest that LH increases the uptake of exogenous cholesterol in the form of lipoproteins and enhances the utilization of internalized cholesterol for progesterone synthesis. Prl appears to stimulate progesterone synthesis by enhancing the uptake of lipoproteins.     

Antiapoptotic effects of erythropoietin in differentiated neuroblastoma SH-SY5Y cells require activation of both the STAT5 and AKT signaling pathways

The hematopoietic cytokine erythropoietin (Epo) prevents neuronal death during ischemic events in the brain and in neurodegenerative diseases, presumably through its antiapoptotic effects. To explore the role of different signaling pathways in Epo-mediated antiapoptotic effects in differentiated human neuroblastoma SH-SY5Y cells, we employed a prolactin receptor (PrlR)/erythropoietin receptor (EpoR) chimera system, in which binding of prolactin (Prl) to the extracellular domain activates EpoR signaling in the cytosol. On induction of apoptosis by staurosporine, Prl supports survival of the SH-SY5Y cells expressing the wild-type PrlR/EpoR chimera. In these cells Prl treatment strongly activates the STAT5, AKT, and MAPK signaling pathways and induces weak activation of the p65 NF-kappaB factor. Selective mutation of the eight tyrosine residues of the EpoR cytoplasmic domain results in impaired or absent activation of either STAT5 (mutation of Tyr(343)) or AKT (mutation of Tyr(479)) or both (mutation of all eight tyrosine residues). Most interestingly, Prl treatment does not prevent apoptosis in cells expressing mutant PrlR/EpoR chimeras in which either the STAT5 or the AKT signaling pathways are not activated. In contrast, ERK 1/2 is fully activated by all mutant PrlR/EpoR chimeras, comparable with the level seen with the wild-type PrlR/EpoR chimera, implying that activation of the MAPK signaling pathway per se is not sufficient for antiapoptotic activity. Therefore, the antiapoptotic effects of Epo in neuronal cells require the combinatorial activation of multiple signaling pathways, including STAT5, AKT, and potentially MAPK as well, in a manner similar to that observed in hematopoietic cells.     

Prolactin heterogeneity in the serum and milk during lactation

Prolactin heterogeneity in serum and milk were separated using Sephadex G-100. Three components were present in serum from lactating women with the following proportions: “void volume” -13.4%, “big” - 26.4%, and “little” - 60.3%. Milk from the same subjects did not contain “big” prolactin. Over 90% of the prolactin found in milk was “little” prolactin. The “little” prolactin in milk may not be similar to the “little” prolactin in the serum.     

Size heterogeneity of pituitary and plasma prolactin: Effect of chronic estrogen treatment

Pituitary homogenates and plasma from untreated and estrogen treated ovariectomized rats were subjected to gel filtration chromatography and the prolactin in fractions collected between the void and total elution volumes of the columns was determined by radio- immunoassay. Three components of prolactin, identified as “void volume”, “big” and “little” according to increasing elution volumes, were observed in pituitary homogenates of ovariectomized rats. These three components accounted for 4, 11 and 85% of the total prolactin activity respectively. Estrogen treatment of ovariectomized rats increased the total prolactin in the pituitary and also selectively increased the “big” component to 21% of the prolactin activity on the column. A smaller increase was also observed in the “void volume” component. Gel filtration of the plasma obtained from estrogen-treated rats before and during the estrogen-induced afternoon surge of prolactin showed that “little” prolactin was the predominate form being secreted and that the “void volume” and “big” components were also released. The release of the components was not in proportion to that observed in the pituitary and the larger components were released in a nonuniform manner. The “void volume” component appeared in the plasma as the surge began but then disappeared as the “big” component appeared at the peak of the surge. The big component decreased as the surge waned leaving primarily “little” component in plasma. The data indicate (1) that estrogen stimulates the formation of the larger components of prolactin in the pituitary (2) that the types of prolactin released into plasma of estrogen-treated ovariectomized rats is not in proportion to that found in the pituitary and (3) that the heterogeneous forms of prolactin are selectively released into plasma during the prolonged secretory episode of the afternoon surge of prolactin induced by estrogen.     

Testicular prolactin receptors and serum growth hormone in golden hamsters: effects of photoperiod and time of day

Investigations were conducted to determine effects of exposure to short photoperiod--with its accompanying reductions in serum prolactin (Prl) concentrations--for various durations on testicular Prl receptors. An additional study investigated the possibility of nyctohemeral fluctuations in testicular Prl receptors and serum growth hormone (GH) concentrations and their alteration by photoperiod. After 10 and 28 days of exposure to a short photoperiod consisting of 5 h of light and 19 h darkness (5L:19D) (and prior to changes in testicular weight), there were progressive and significant reductions in the concentration of testicular Prl receptors (fmol/mg protein) when compared with long-photoperiod controls (14L:10D). After 12 weeks of 5L:19D, when testicular weights were dramatically decreased, Prl receptor concentration was reduced to 39% of long-photoperiod controls in one study, without alteration of affinity of Prl receptors for their labeled ligand. When measured at 6-h intervals in hamsters on 14L:10D, and on 5L:19D for 12 weeks, there were no significant changes in concentration or total content (fmol/testes) of testicular Prl receptors throughout the day. Although serum GH concentrations fluctuated markedly in hamsters on both photoperiods, no definitive nyctohemeral patterns were detected. These data provide indirect evidence for the ability of Prl to regulate its own testicular receptors, and demonstrate that diurnal fluctuations in testicular sensitivity to injected Prl are not a consequence of changes in Prl receptors. The data also suggest the absence of effects of photoperiod on serum GH concentrations in male golden hamsters.     

Interaction of androgens and prolactin on prostatic enzymes of the pyruvate-malate cycle involved in lipogenesis in castrated mature monkey, Macaca radiata.

Effects of androgens, prolactin (Prl) and bromocriptine (Br) on the specific activities of prostatic (caudal and cranial) enzymes of the pyruvate-malate cycle were studied in castrated mature bonnet monkeys. Castration decreased the activity of NADP+ isocitrate dehydrogenase (ICDH), ATP citrate lyase, malate dehydrogenase (MDH), malic enzyme and fatty acid synthase (FAS). Administration of testosterone propionate (TP)/dihydrotestosterone (DHT) increased the activities of all these enzymes in both lobes. Malate dehydrogenase maintained normal activity. Prl also had a stimulatory effect on the enzymes and was further enhanced when Prl was given in combination with TP/DHT. Unlike Prl, bromocriptine treatment inhibited all the enzymes in both lobes. Thus, prolactin was found to have a direct as well as a synergistic effect with androgens on enzymes of the pyruvate-malate cycle in the prostate of castrated mature monkeys.     

Effect of acute intravenous growth hormone-releasing factor on plasma prolactin in short children and patients with growth hormone deficiency

Four normal subjects and 54 growth hormone (GH)-deficient patients including 43 children with growth failure were given an intravenous bolus of growth hormone-releasing factor (GHRF). Plasma prolactin (Prl) and GH after GHRF were studied. Basal plasma Prl was either normal or elevated and could not predict the GH response to GHRF. A correlation was found, within the group with basal hyperprolactinemia, between basal Prl and the net Prl increase after GHRF. No correlation was found between the net GH and the net Prl increase after GHRF. Plasma Prl was significantly, although weakly, increased after GHRF in the normal subjects.     

Differential effects of naloxone on basal and stress-induced release of ACTH and prolactin in the male rat

The effect of i.v. injection of various doses of naloxone (NAL) on plasma adrenocorticotropin (ACTH) and prolactin (Prl) in conscious animals bearing an indwelling intrajugular catheter was assessed. The effects were evaluated in animals which were left undisturbed and in others subjected to either restraint or ether stress. The results revealed that the dose of 3 mg/kg of NAL significantly reduced basal Prl levels, whereas a dose of 6 mg/kg of NAL was required to block completely either ether or restraint stress-induced release of Prl. The behavior of ACTH contrasted with that of Prl. There was no effect whatsoever of the 3 mg/kg dose of NAL on either resting or stress-induced ACTH levels, whereas a 6 mg/kg or 12 mg/kg dose of NAL elevated resting ACTH levels and only partially attenuated the further elevation induced by stress in these animals. The results clearly indicate a NAL sensitive step in the control of resting and stress-induced Prl release but indicate that the control of resting and stress-induced release of ACTH is different in that the predominantly millimicron receptor blocker, NAL, can elevate ACTH at high doses and can only partially block the response to stress. In contrast to Prl where opioid peptide control is solely stimulatory, this control of ACTH secretion appears to have both stimulatory and inhibitory features.     

Autoradiographic analysis of proestrous changes in the binding of 125I-labeled prolactin to the hamster ovary

Autoradiographic histochemistry was employed to examine changes in the binding of 125I-labeled prolactin (Prl) to ovaries from proestrous hamsters before (at 1200 h), during (at 1600 h), and after (at 2000 h) the preovulatory gonadotropin surge. In untreated control hamsters, there was a marked and progressive loss of Prl binding, first in the interstitial cells and follicular thecae by 1600 h, and then in the granulosa cells of the preovulatory follicles by 2000 h. When proestrous hamsters were treated with ergocryptine to significantly lower serum Prl, or injected with exogenous Prl, Prl binding to their ovaries did not differ from controls, suggesting that decreased Prl binding was due to neither increased occupancy of binding sites by endogenous Prl nor down regulation of Prl receptors by Prl itself. Conversely, when proestrous hamsters were treated with phenobarbital to block the luteinizing hormone (LH)/follicle-stimulating hormone (FSH) surge, the loss of Prl binding sites in the ovaries was prevented, suggesting that the LH/FSH surge might initiate a down regulation of Prl receptors in the ovary. Such a down regulation of Prl receptors may serve as a mechanism by which the ability of Prl to affect periovulatory events in the ovary might be regulated.     

Verification of NB2 lymphoma cell bioassay for the measurement of plasma and pituitary prolactin in the Mongolian gerbil (Meriones unguiculatus)

Serum and pituitary glands were taken from male Mongolian gerbils which had received bromocriptine implants, ether stress or no treatment (controls). Pituitary prolactin (Prl) and growth hormone (GH) mRNA were analyzed by Northern hybridization using rat cDNA probes. Pituitary and plasma Prl content were analyzed with the Nb2 lymphoma cell growth bioassay. These assays were sensitive to the decreases in Prl caused by bromocriptine and the elevation of Prl caused by ether stress. The inhibition of pituitary and plasma Prl levels by bromocriptine correlated with a marked inhibition of pituitary Prl mRNA content. In contrast, levels of GH mRNA did not change with treatment, indicating that gerbil GH does not contribute to the lactogenic activity measured in the Nb2 lymphoma cell bioassay. The results indicate that this bioassay is suitable for the measurement of gerbil pituitary and plasma Prl.     

Effect of extracellular matrix on prolactin secretion and mRNA accumulation in GH3 cells

The matrix upon which cells grow affects their morphology, growth rate, response to external stimuli, and protein synthesis. GH3 cells, a well-characterized rat pituitary tumor cell line, synthesize and secrete growth hormone and prolactin (Prl). These cells are rounded, attach loosely, and form clumps when plated on plastic. GH3 cells plated on an extracellular matrix (ECM) from bovine corneal endothelial cells become flattened and strongly adherent to the culture dish, and have an initial increased rate of proliferation. Cells cultured on plastic have a 48-hr lag period before the start of cell division; this can be shortened by increasing the concentration of serum in the medium. Since GH3 cells store little Prl, hormone release is a good index of Prl synthesis. Prl secretion from cells cultured on extracellular matrix is twice as great as from cells cultured on plastic. The increase in Prl secretion from cells grown on extracellular matrix paralleled by a concomitant increase in the accumulation of prolactin mRNA. Cells cultured on plastic secrete more Prl in response to TRH stimulation than do cells cultured on ECM. Cells grown on either surface were unresponsive to dopamine. Thus, culturing cells on ECM may change their morphology and affect the synthesis and regulation of specific cellular proteins and their mRNAs.     

Identification of the mRNA coding for prolactin in the human decidua

Total RNAs extracted from amnion, chorion and decidua of the human second trimester placentas were translated in the cell-free translation system, followed by immunoprecipitation with antiserum to human prolactin (Prl) and electrophoresis in sodium dodecyl sulfate slab gel. A single immunospecific protein with an apparent molecular weight of approximately 25,500, about 3000 daltons larger than authentic Prl, was formed only with RNA from decidua, and it competed with unlabeled Prl but not with unlabeled human placental lactogen (hPL) for binding to the antibody. The electrophoretic patterns of the fragments formed by partial enzymatic proteolysis of it and authentic Prl were similar.     

Regulatory action of prolactin on the in vitro growth of CD34+ve human hemopoietic progenitor cells

The pituitary hormone prolactin (Prl) is known to act as a local regulator of immune cell function, and Prl-binding receptors (Prl-R) have been described to share distinctive features with the members of the newly described cytokine/hemopoietin receptor superfamily. Here we show that the hormone can functionally interact with lineage-specific hemopoietic factors. When highly purified progenitor cells (CD34+ve) were seeded in semisolid methylcellulose cultures in the presence of interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), and erythropoietin (Epo), a selective enhancing effect of Prl on the formation of colony forming unit-granulocyte (CFU-G) and burst forming unit-erythroid (BFU-E) colonies was observed. The effect of the hormone was plotted as a bell shaped curve, with the optimal response at the supraphysiological concentration of 50 ng/ml. Limiting dilution analysis showed that Prl acted directly on hemopoietic progenitors. This was confirmed by the observation on the CD34+ve cells of Prl-binding sites reacting with the specific monoclonal antibodies (mAbs), U5 and PrR-7A. Immunoprecipitation of the metabolically labeled CD34+ve cells with the PrR-7A mAb revealed a structure of 43 kD under reducing conditions. Analysis of the early events associated with the Prl/Prl-R interaction showed an increased number of cells engaged in DNA and hemoglobin synthesis. Enhanced erythroid differentiation of CD34+ve cells in the presence of Prl was secondary to upmodulation of receptors for the lineage-specific factor Epo. Together these data demonstrate the existence of a functional interplay between Prl. and hemopoietic factors. © 1995 Wiley-Liss, Inc.     

Effects of morphine dependence,withdrawal and tolerance on prolactin and growth hormone secretion in the rat

The effects of morphine dependence and withdrawal on prolactin (Prl) and growth hormone (GH) secretion were examined in the rat. Morphine dependence, induced by morphine pellet implantation, had no effect on nonstress concentrations of plasma Prl or GH, but it potentiated the response of Prl secretion to the stress associated with blood collection + injection of saline. Naloxone-induced withdrawal had no demonstrable effect on the changes in Prl and GH secretion produced by stress. In addition, signs of tolerance to both the Prl- and GH-stimulating effects of morphine injection were observed in morphine-dependent rats.