UCP1 is present in porcine adipose tissue and is responsive to postnatal leptin.

Intrauterine growth restriction (IUGR) may be accompanied by inadequate thermoregulation, especially in piglets that are not considered to possess any brown adipose tissue (BAT) and are thus entirely dependent on shivering thermogenesis in order to maintain body temperature after birth. Leptin can stimulate heat production by promoting non-shivering thermogenesis in BAT, but whether this response occurs in piglets is unknown. Newborn female piglets that were characterised as showing IUGR (mean birth weight of approximately 0.98 kg) were therefore administered injections of either saline or leptin once a day for the first 5 days of neonatal life. The dose of leptin was 0.5 mg/kg, which is sufficient to increase plasma leptin by approximately tenfold and on the day of birth induced a rapid increase in body temperature to values similar to those of normal-sized 'control' piglets (mean birth weight of ∼1.47 kg). Perirenal adipose tissue was then sampled from all offspring at 21 days of age and the presence of the BAT-specific uncoupling protein 1 (UCP1) was determined by immunohistochemistry and immunoblotting. UCP1 was clearly detectable in all samples analysed and its abundance was significantly reduced in the IUGR piglets that had received saline compared with controls, but was raised to the same amount as in controls in those IUGR females given leptin. There were no differences in gene expression between primary markers of brown and white adipose tissues between groups. In conclusion, piglets possess BAT that when stimulated exogenously by leptin can promote increased body temperature.

Postnatal leptin promotes organ maturation and development in IUGR piglets.

Babies with intra-uterine growth restriction (IUGR) are at increased risk for experiencing negative neonatal outcomes due to their general developmental delay. The present study aimed to investigate the effects of a short postnatal leptin supply on the growth, structure, and functionality of several organs at weaning. IUGR piglets were injected from day 0 to day 5 with either 0.5 mg/kg/d leptin (IUGRLep) or saline (IUGRSal) and euthanized at day 21. Their organs were collected, weighed, and sampled for histological, biochemical, and immunohistochemical analyses. Leptin induced an increase in body weight and the relative weights of the liver, spleen, pancreas, kidneys, and small intestine without any changes in triglycerides, glucose and cholesterol levels. Notable structural and functional changes occurred in the ovaries, pancreas, and secondary lymphoid organs. The ovaries of IUGRLep piglets contained less oogonia but more oocytes enclosed in primordial and growing follicles than the ovaries of IUGRSal piglets, and FOXO3A staining grade was higher in the germ cells of IUGRLep piglets. Within the exocrine parenchyma of the pancreas, IUGRLep piglets presented a high rate of apoptotic cells associated with a higher trypsin activity. In the spleen and the Peyer's patches, B lymphocyte follicles were much larger in IUGRLep piglets than in IUGRSal piglets. Moreover, IUGRLep piglets showed numerous CD79(+) cells in well-differentiated follicle structures, suggesting a more mature immune system. This study highlights a new role for leptin in general developmental processes and may provide new insight into IUGR pathology.

Postnatal leptin is necessary for maturation of numerous organs in newborn rats.

The postnatal leptin surge, described particularly in rodents, has been demonstrated to be crucial for hypothalamic maturation and brain development. In the present study, the possible general effects of this hormone on maturation of numerous peripheral organs have been explored. To test this hypothesis, we used a leptin antagonist (L39A/D40A/F41A) to investigate the effects of the blockage of postnatal leptin action on neonatal growth and maturation of organs involved in metabolism regulation, reproduction and immunity. For that purpose, newborn female pups were subcutaneously injected from days 2-13 with either saline or leptin antagonist and sacrificed at weaning. Organs were submitted to histological and immunohistochemical analyses. Leptin antagonist treatment clearly impaired the maturation of pancreas, kidney, thymus and ovary. All these alterations, at the organ level, occurred without changes in the whole-body mass of the animals. Leptin antagonist treatment induced: (1) a reduction in b cell area and a concomitant increase of a cells in Langherans islets in the pancreas, (2) a reduction in the number of glomeruli and a persistence of immature glomeruli in kidney, (3) an increase in the thymic cortical layer thickness, reflecting an unmatured stage, (4) a drastic reduction of the pool of primordial follicles, in ovaries. All these results strongly argue for a crucial role of leptin for the achievement of organ maturation, opening new perspectives in the field of leptin physiology and organ development.

Leptin but not ciliary neurotrophic factor (CNTF) induces phosphotyrosine phosphatase-1B expression in human neuronal cells (SH-SY5Y): putative explanation of CNTF efficacy in leptin-resistant state.

Growing evidences suggest that obesity is associated with hypothalamic leptin resistance, leading to the alteration of food intake control. Alternative treatment using ciliary neurotrophic factor (CNTF) has been suggested because CNTF exerts a leptin-like effect, even in leptin-resistant states, but the mechanisms by which CNTF maintains this effect are not yet understood. Both leptin and CNTF act in the hypothalamus through similar signaling pathways including janus kinase-2/signal transducer and activator of transcription (STAT)-3 pathway. To explore the differences and interactions between leptin and CNTF signaling pathways, differentiated human neuroblastoma cells (SH-SY5Y) were exposed to either leptin or CNTF and then challenged for each cytokine. Leptin pretreatment completely abolished leptin-dependent STAT-3 and ERK 1/2 phosphorylations without affecting CNTF action. The lack of cross-desensitization between leptin and CNTF signaling pathways occurred despite the induction of suppressor of cytokine signaling-3 in response to both cytokines. Interestingly, leptin as well as insulin induced the expression of phosphotyrosine phosphatase (PTP)-1B, whereas CNTF treatment did not affect its expression. In addition, acute leptin treatment but not CNTF induced PTP-1B expression in mouse hypothalamic arcuate nucleus. Furthermore, the overexpression of human PTP-1B in SH-SY5Y cells completely abolished leptin- and insulin-dependent janus kinase-2, STAT-3, and ERK 1/2 phosphorylations, but CNTF action was not altered. Collectively, our results suggest that PTP-1B constitutes a key divergent element between leptin/insulin and CNTF signaling pathways at the neuronal level, which may constitute a possible mechanism that explains the efficacy of CNTF in leptin-resistant states.

Long-term consequences of maternal high-fat feeding on hypothalamic leptin sensitivity and diet-induced obesity in the offspring.

Epidemiological and animal studies suggest that the alteration of hormonal and metabolic environment during fetal and neonatal development can contribute to development of metabolic syndrome in adulthood. In this paper, we investigated the impact of maternal high-fat (HF) diet on hypothalamic leptin sensitivity and body weight gain of offspring. Adult Wistar female rats received a HF or a control normal-fat (C) diet for 6 wk before gestation until the end of the suckling period. After weaning, pups received either C or HF diet during 6 wk. Body weight gain and metabolic and endocrine parameters were measured in the eight groups of rats formed according to a postweaning diet, maternal diet, and gender. To evaluate hypothalamic leptin sensitivity in each group, STAT-3 phosphorylation was measured in response to leptin or saline intraperitoneal bolus. Pups exhibited similar body weights at birth, but at weaning, those born to HF dams weighed significantly less (-12%) than those born to C dams. When given the HF diet, males and females born to HF dams exhibited smaller body weight and feed efficiency than those born to C dams, suggesting increased energy expenditure programmed by the maternal HF diet. Thus, maternal HF feeding could be protective against adverse effects of the HF diet as observed in male offspring of control dams: overweight (+17%) with hyperleptinemia and hyperinsulinemia. Furthermore, offspring of HF dams fed either C or HF diet exhibited an alteration in hypothalamic leptin-dependent STAT-3 phosphorylation. We conclude that maternal high-fat diet programs a hypothalamic leptin resistance in offspring, which, however, fails to increase the body weight gain until adulthood.

Messenger RNA expression of leptin and leptin receptors and their prognostic value in 322 human primary breast cancers.

PURPOSE: Leptin and obesity are clearly related, and obesity is associated with an increased risk of breast cancer. We therefore measured the expression of leptin and its two main receptor isoforms, OBR-L and OBR-S, in 322 breast cancers. We analyzed their relations with the classical prognostic factors and with survival to establish their links with breast cancer. EXPERIMENTAL DESIGN: The expression of leptin and its receptors was quantified by real-time reverse transcription-PCR, using TaqMan fluorogenic probes and an ABI PRISM 7700 sequence detector system (Applied Biosystems, Courtaboeuf, France). TATA box binding protein was used to normalize expression. The human breast cancer cell, SK-BR-3, expressing the three targets, was chosen as the calibrator sample (i.e., target expression = 1). RESULTS: All the tumors expressed both receptors, and 318 of 322 expressed leptin. These three variables correlated positively with each other and with estradiol and progesterone receptors, whereas they correlated negatively with histoprognostic grading and tumor diameter. OBR-L/OBR-S expression was inversely correlated with progesterone receptors. Patients with elevated OBR-S expression had longer relapse-free survival (P = 0.008), whereas high OBR-L/OBR-S was associated with a shorter relapse-free survival (P = 0.05). In Cox multivariate analyses, OBR-S maintained its prognostic value (P = 0.02; relative risk, 0.51). CONCLUSIONS: This study shows that (a) almost all of the breast cancers coexpress leptin and its two main isoforms of receptors, suggesting that the human epithelial breast cancer cells respond to leptin acting via an autocrine pathway; (b) high expression levels of leptin and leptin receptors are biological markers of a more differentiated phenotype; and that (c) OBR-S is an independent prognostic factor.

Insulin and leptin induce Glut4 plasma membrane translocation and glucose uptake in a human neuronal cell line by a phosphatidylinositol 3-kinase- dependent mechanism.

The insulin-sensitive glucose transporter Glut4 is expressed in brain areas that regulate energy homeostasis and body adiposity. In contrast with peripheral tissues, however, the impact of insulin on Glut4 plasma membrane (PM) translocation in neurons is not known. In this study, we examined the role of two anorexic hormones (leptin and insulin) on Glut4 translocation in a human neuronal cell line that express endogenous insulin and leptin receptors. We show that insulin and leptin both induce Glut4 translocation to the PM of neuronal cells and activate glucose uptake. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, totally abolished insulin- and leptin-dependent Glut4 translocation and stimulation of glucose uptake. Thus, Glut4 translocation is a phosphatidylinositol 3-kinase-dependent mechanism in neuronal cells. Next, we investigated the impact of chronic insulin and leptin treatments on Glut4 expression and translocation. Chronic exposure of neuronal cells to insulin or leptin down-regulates Glut4 proteins and mRNA levels and abolishes the acute stimulation of glucose uptake in response to acute insulin or leptin. In addition, chronic treatment with either insulin or leptin impaired Glut4 translocation. A cross-desensitization between insulin and leptin was apparent, where exposure to insulin affects leptin-dependent Glut4 translocation and vice versa. This cross-desensitization could be attributed to the increase in suppressor of cytokine signaling-3 expression, which was demonstrated in response to each hormone. These results provide evidence to suggest that Glut4 translocation to neuronal PM is regulated by both insulin and leptin signaling pathways. These pathways might contribute to an in vivo glucoregulatory reflex involving a neuronal network and to the anorectic effect of insulin and leptin.

Preparation of leptin antagonists by site-directed mutagenesis of human, ovine, rat, and mouse leptin's site III: implications on blocking undesired leptin action in vivo.

Six muteins of human, ovine, rat, and mouse leptins mutated to Ala in amino acids 39-41 or 39-42 were prepared by site-directed mutagenesis of the putative site III, which does not affect binding but is necessary for receptor activation, then expressed, solubilized in 4.5 M urea, at pH 11.3 in presence of cysteine, refolded and purified to homogeneity by anion-exchange chromatography on Q-Sepharose or combination of anion-exchange chromatography followed by gel filtration. The overall yields were 400-800 mg from 5 L of fermentation. All proteins were >98% pure as evidenced by SDS-PAGE and contained at least 95% monomers as documented by gel-filtration chromatography under nondenaturing conditions. Circular dichroism analysis revealed that all six muteins have identical secondary structure characteristic of nonmutated leptins, namely 52-63% of alpha helix content. All muteins formed a 1:1 complex with chicken leptin binding domain, (chLBD) and bound chLBD or membrane-embedded leptin receptor with affinity identical to WT leptins. Muteins were devoid of any biological activity in several bioassays but were potent competitive antagonists. Some muteins were pegylated using 40 kDa PEG. Although pegylation decreased the in vitro activity, increasing circulation half-life can recompensate this deficit, so pegylated antagonists are expected to be more potent in vivo.

Fluorescence resonance energy transfer (FRET) microscopy in living cells as a novel tool for the study of cytokine action.

Fluorescence resonance energy transfer (FRET) microscopy was used to study interactions between proteins in intact cells. We showed that growth hormone (GH) causes transient homodimerization of GH receptors tagged with yellow or cyan fluorescent proteins. The peak of FRET signaling occurred 2 to 4 min after hormonal stimulation and was followed by a decrease in FRET signal. Repeating those experiments in cells pretreated with the inhibitor of internalization methyl-beta-cyclodextrin, or in potassium-depleted cells showed no difference in the kinetics of FRET signaling as compared with the non-treated cells, indicating that the decrease in FRET signal does not result from receptor internalization by the pathways inhibited by methyl-beta-cyclodextrin or potassium depleted but might occur by other pathways of internalization. Using a similar methodology, we also demonstrated that ovine placental lactogen (oPL) causes transient heterodimerization of GH and prolactin (PRL) receptors 2.5 to 3 min after oPL application. On the other hand, oGH or oPRL had no effect at all, further substantiating the finding the oPL, which lacks a specific receptor, acts in homologous systems by heterodimerization of GH and PRL receptors. We also demonstrated that both PRL and leptin (LEP) are capable of transactivation of the oncogenic receptors erbB2 and erbB3. Upon PRL or LEP stimulation of HEK-293T cells transfected with LEP or PRL receptors and erbB2 or erbB3, erbB proteins are first phosphorylated and then activate MAPK (erk1/erk2). However, the FRET experiments failed to document any evidence of a direct interaction between erbB2 and the PRL or LEP receptors, suggesting that erbB activation probably occurs via activated JAK2, translocated from the respective receptors to erbB2.

In vivo leptin infusion impairs insulin and leptin signalling in liver and hypothalamus.

Leptin resistance contributes to the pathogenesis of common obesity related metabolic diseases, including insulin resistance. However, the relationship between leptin and insulin resistance is not clearly established. Here, we show that induced hyperleptinemia by leptin infusion alters insulin signalling in rat liver. Leptin infusion clearly reduced the insulin or leptin dependent IRS-1/IRS-2 association to p85 regulatory subunit of PI 3-kinase. Leptin infusion also abolished STAT-3 phosphorylation in response to insulin or leptin and similar results were obtained for MAP-kinase phosphorylation. Hypothalamic leptin resistance was also induced by leptin infusion since leptin was unable to induce STAT-3 phosphorylation. These results provide evidence that induced hyperleptinemia can contribute to the onset of insulin resistance at least at the hepatic level.

Transduction pathways of GH in ovine mammary acini involving regulated and functional growth hormone receptors.

We have investigated the localization and regulation of growth hormone (GH) receptor-related proteins in the ovine mammary gland. Using a new rabbit polyclonal antibody (7122A) directed against the recombinant extracellular domain of GH receptor (GHR-ECD) for western blot assays, we found two bands with apparent molecular weights of 70,000 and 50-60,000 Da in ovine mammary gland solubilized proteins. The 70,000-protein was consistent with a membrane GH receptor form deprived of post-translational modifications such as phosphorylation, glycosylation or ubiquitin binding. The 50-60,000 Da was consistent with soluble GH binding protein, generated by the cleavage of membrane GH receptor. The intensity of related GHR proteins increased slightly throughout mammary gland development and was correlated with the amount of GHR immunoreactivity observed in the mammary gland sections. Moreover, a temporal and spatial regulation of GHR immunoreactivity was found in alveolar epithelial cells. Clearly, marked GHR immunoreactivity was associated with the apical membranes of alveolar epithelial cells at lactation. The up-regulation of related GHR proteins during the differentiation of mammary tissue supports the hypothesis that GH may act specifically via its own receptors. In ovine mammary cells, GH was able to promote a time-dependent activation of MAP kinases such as prolactin (Prl) and placental lactogen (PL). GH was also able to promote slight and transient Stat5 DNA-binding activity. Differences in the time dependence of Stat5 DNA-binding activation by the three different ligands, GH, Prl and PL, were found. All these results emphasize the direct action of GH on ovine mammary cells and highlight the specificity of action of this ligand.

Growth hormone receptor gene expression in the skeletal muscle of normal and double-muscled bovines during foetal development.

The expression of the growth hormone receptor (GHR) gene was investigated in semitendinosus muscle during bovine foetal development in both normal and double-muscled Charolais foetuses which differ with respect to muscle development. Northern-blot analysis of foetal muscle RNA preparations with a GHR cDNA probe identified the 4.5 kb GHR mRNA as early as 130 days post-conception. In double-muscled animals, the expression of GHR mRNA increased from 130 to 210 days of gestation while it stayed stable in normal ones. It was significantly higher (P < 0.05) in double-muscled foetuses compared to normal ones from the second third of gestation. Northern-blot analysis of foetal muscle RNA preparations from both genotypes with a beta-actin cDNA probe, revealed lower beta-actin gene expression in double-muscled foetuses than in normal ones, suggesting a delay in the differentiation of muscle cells. In situ hybridisation revealed the localisation of specific GHR mRNA in muscle cells at all gestation stages analysed (130, 170, 210 days post-conception) but not in connective tissue surrounding the muscle cells. At the adult stage, the hybridisation signal was also very high and observed in muscle cells only. These results show the ontogeny of GHR mRNA in bovine muscle and demonstrate a difference between normal and double-muscled animals.

Identification of the hydrophobic strand in the A-B loop of leptin as major binding site III: implications for large-scale preparation of potent recombinant human and ovine leptin antagonists.

Interaction of leptin with its receptors resembles that of interleukin-6 and granulocyte colony-stimulating factor, which interact with their receptors through binding sites I-III. Site III plays a pivotal role in receptors' dimerization or tetramerization and subsequent activation. Leptin's site III also mediates the formation of an active multimeric complex through its interaction with the IGD (immunoglobulin-like domain) of LEPRs (leptin receptors). Using a sensitive hydrophobic cluster analysis of leptin's and LEPR's sequences, we identified hydrophobic stretches in leptin's A-B loop (amino acids 39-42) and in the N-terminal end of LEPR's IGD (amino acids 325-328) that are predicted to participate in site III and to interact with each other in a beta-sheet-like configuration. To verify this hypothesis, we prepared and purified to homogeneity (as verified by SDS/PAGE, gel filtration and reverse-phase chromatography) several alanine muteins of amino acids 39-42 in human and ovine leptins. CD analyses revealed that those mutations hardly affect the secondary structure. All muteins acted as true antagonists, i.e. they bound LEPR with an affinity similar to the wild-type hormone, had no agonistic activity and specifically inhibited leptin action in several leptin-responsive in vitro bioassays. Alanine mutagenesis of LEPR's IGD (amino acids 325-328) drastically reduced its biological but not binding activity, indicating the importance of this region for interaction with leptin's site III. FRET (fluorescence resonance energy transfer) microscopy experiments have documented that the transient FRET signalling occurring upon exposure to leptin results not from binding of the ligand, but from ligand-induced oligomerization of LEPRs mediated by leptin's site III.

Quantitative FRET imaging of leptin receptor oligomerization kinetics in single cells.

BACKGROUND INFORMATION: Leptin, an adipocyte-secreted hormone, signals through activation of its membrane-embedded receptor (LEPR). To study the leptin-induced events occurring in short (LEPRa) and long (LEPRb) LEPRs in the cell membrane, by FRET (fluorescence resonance energy transfer) methodology, the respective receptors, tagged at their C-terminal with CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein), were prepared. RESULTS: The constructs encoding mLEPRa (mouse LEPRa)-YFP and mLEPRa-CFP, mLEPRb-YFP and mLEPRb-CFP were tested for biological activity in transiently transfected CHO cells (Chinese-hamster ovary cells) and HEK-293T cells (human embryonic kidney 293 T cells) for activation of STAT3 (signal transduction and activators of transcription 3)-mediated LUC (luciferase) activity and binding of radiolabelled leptin. All four constructs were biologically active and were as potent as their untagged counterparts. The localization pattern of the fused protein appeared to be confined almost entirely to the cell membrane. The leptin-dependent interaction between various types of receptors in fixed cells were studied by measuring FRET, using fluorescence lifetime imaging microscopy and acceptor photobleaching methods. CONCLUSIONS: Both methods yielded similar results, indicating that (1) leptin receptors expressed in the cell membrane exist mostly as preformed LEPRa/LEPRa or LEPRb/LEPRb homo-oligomers but not as LEPRb/LEPRa hetero-oligomers; (2) the appearance of transient leptin-induced FRET in cells transfected with LEPRb/LEPRb reflects both a conformational change that leads to closer interaction in the cytosolic part and a higher FRET signal, as well as de novo homo-oligomerization; (3) in LEPRa/LEPRa, exposure to leptin does not lead to any increase in FRET signalling as the proximity of CFP and YFP fluorophores in space already gives maximal FRET efficiency of the preoligomerized receptors.

Cross down-regulation of leptin and insulin receptor expression and signalling in a human neuronal cell line.

Leptin and insulin are major signals to the hypothalamus to regulate energy homoeostasis and body adiposity. IR (insulin receptors) and leptin receptors (long isoform, ObRb) share a number of signalling cascades, such as JAK2/STAT-3 (Janus kinase 2/signal transduction and activator of transcription 3) and PI3K (phosphoinositide 3-kinase); the cross-talk between IR and ObRb have been described previously in non-neuronal cells. Differentiated human neuroblastoma (SH-SY5Y) cells express endogenous ObR and IR, and respond to leptin and insulin with stimulation of STAT-3 and MAPK (mitogen-activated protein kinase) phosphorylation, and PI3K activity. Insulin or leptin pre-treatment of SH-SY5Y cells increased basal STAT-3 phosphorylation, but abolished the acute effect of these hormones, and, interestingly, leptin pre-treatment abolished insulin effect and vice versa. Similar results were obtained for MAPK phosphorylation, but leptin or insulin pre-treatment did not completely abolish the acute effect of insulin or leptin. We have also showed that insulin and leptin are able to activate PI3K through IRS-1 (insulin receptor substrate 1) and IRS-2 respectively. Furthermore, leptin or insulin pre-treatment increased basal PI3K activity and IRS-1 or IRS-2 association with p85 and abolished acute insulin or leptin effect, in addition to the down-regulation of IRS-1 and IRS-2. Finally, insulin pre-treatment reduced leptin binding by approx. 60%, and leptin pre-treatment reduced the expression of insulin receptor by 40% in SH-SY5Y cells, which most likely accounts for the cross down-regulation of leptin and insulin receptors. These results provide evidence to suggest cross down-regulation of leptin and insulin receptors at both receptor and downstream signalling levels. This finding may contribute to the understanding of the complex relationship between leptin resistance and insulin resistance at the neuronal level.

Transactivation of erbB2 by short and long isoforms of leptin receptors.

We generated kinase-positive and kinase-negative erbB2 tagged with YFP and the long form of leptin receptor (LEPRb) tagged with CFP. Both were as active as their untagged analogs. Both short and long isoforms of leptin receptor phosphorylated and thereby activated erbB2 upon leptin binding and enhanced MAPK activity. Our results unveil a novel route by which leptin may provoke erbB2's phosphorylation and thus enhance its oncogenic potential independently of HER family ligands or its overexpression. Using FRET technology in living cells, we found no evidence of complex formation between erbB2 and prolactin or leptin receptors, indicating that the transactivation occurs through an indirect interaction.

Prolactin stimulates cell proliferation through a long form of prolactin receptor and K+ channel activation.

PRL (prolactin) has been implicated in the proliferation and differentiation of numerous tissues, including the prostate gland. However, the PRL-R (PRL receptor) signal transduction pathway, leading to the stimulation of cell proliferation, remains unclear and has yet to be mapped. The present study was undertaken to develop a clear understanding of the mechanisms involved in this pathway and, in particular, to determine the role of K(+) channels. We used androgen-sensitive prostate cancer (LNCaP) cells whose proliferation is known to be stimulated by PRL. Reverse transcriptase PCR analysis showed that LNCaP cells express a long form of PRL-R, but do not produce its intermediate isoform. Patch-clamp techniques showed that the application of 5 nM PRL increased both the macroscopic K(+) current amplitude and the single K(+)-channel open probability. This single-channel activity increase was reduced by the tyrosine kinase inhibitors genistein, herbimycin A and lavandustine A, thereby indicating that tyrosine kinase phosphorylation is required in PRL-induced K(+) channel stimulation. PRL enhances p59( fyn ) phosphorylation by a factor of 2 after a 10 min application in culture. In addition, where an antip59( fyn ) antibody is present in the patch pipette, PRL no longer increases K(+) current amplitude. Furthermore, the PRL-stimulated proliferation is inhibited by the K(+) channel inhibitors alpha-dendrotoxin and tetraethylammonium. Thus, as K(+) channels are known to be involved in LNCaP cell proliferation, we suggest that K(+) channel modulation by PRL, via p59( fyn ) pathway, is the primary ionic event in PRL signal transduction, triggering cell proliferation.

Expression by transgenesis of a constitutively active mutant form of the prolactin receptor induces premature abnormal development of the mouse mammary gland and lactation failure.

Prolactin (PRL) initiates signal transduction by inducing homodimerization of PRL receptor (PRL-R). We have previously developed a mutant form of the PRL-R in which a part of the extracellular domain is deleted. This receptor constitutively activates protein gene transcription. We examined the oligomerization of the mutant PRL-R using two differently epitope-tagged receptors in a coimmunoprecipitation assay. It was shown that mutant receptor dimers were formed in a ligand-independent manner, which may explain the constitutive activity on milk protein gene expression. To study the biological activity of this mutant PRL-R on mammary gland development, we generated two lines of transgenic mice expressing the corresponding cDNA specifically in the mammary epithelial cells. For both transgenic lines, the mammary gland of 8-wk-old virgin mice was overdeveloped with numerous dilated ductal and alveolar structures, whereas only a limited duct network was present in wild-type animals at the same age. During pregnancy, the ducts and alveoli of transgenic mice were more developed than those of control animals. At parturition, the transgenic animals failed to lactate and nourish their offspring, and the involution of the mammary gland was strongly delayed. In conclusion, the expression of a constitutively active PRL-R by transgenesis induces a premature and abnormal mammary development and impairs terminal differentiation and milk production at the end of pregnancy.

Identification of genes differentially expressed in mouse mammary epithelium transformed by an activated beta-catenin.

Beta-catenin is an executor of Wnt signaling and it can control cell fate and specification. Deletion of exon 3 from the endogenous beta-catenin gene in differentiating mammary alveolar epithelium of the mouse results in the generation of an activated protein that lacks amino acids 5-80. This is accompanied by a loss of mammary epithelial differentiation and a transdifferentiation process to squamous metaplasias. To further understand the molecular process of transdifferentiation, the expression of genes in mammary tissue was profiled in the absence and presence of activated of beta-catenin. Microarrays were generated that carry about 8500 cDNA clones with approximately 6000 obtained from mammary tissue. Mutant tissues, which had undergone either partial (TD1) or complete (TD2) squamous transdifferentiation, were compared with wild-type mammary tissue. Four groups of genes were identified. Group 1 contained genes whose expression was induced in both mutant tissues. Groups 2 and 3 contained genes that were active preferentially in TD2 and TD1, respectively. Group 4 contained genes suppressed in both samples. Using this approach, known and unknown genes activated in the transdifferentiation process were identified. A new 20 kDa protein (PANE1) induced upon transdifferentiation was nuclear in nonconfluent cells and cytoplasmic in confluent or dividing cells. Lastly, stabilization of beta-catenin resulted in the retention of differentiated epithelium upon involution and altered activities of several proteases in transdifferentiated mammary epithelium.

Prostaglandin F(2alpha) (PGF(2alpha)) and prolactin signaling: PGF(2alpha)-mediated inhibition of prolactin receptor expression in the Corpus luteum.

It is well established that prolactin (PRL) sustains, whereas prostaglandin F(2alpha) (PGF(2alpha)) curtails, progesterone production by the rodent corpus luteum (CL). We have previously shown that PGF(2alpha) inhibits the expression of several luteal genes stimulated by PRL, whereas it stimulates other genes inhibited by this hormone. We have also found that PGF(2alpha) stimulation of 20alpha-hydroxysteroid dehydrogenase (20alphaHSD), an enzyme that catabolizes progesterone, at the end of pregnancy is accompanied by a dramatic decrease in PRL receptor (PRL-R) expression. These findings, and the fact that the factors that inhibit PRL-R are not known, led us to examine in vivo whether the decline in PRL-R at the end of pregnancy is due to PGF(2alpha) and to also find out whether PGF(2alpha) opposes PRL action by inhibiting PRL-R expression. Using the PGF(2alpha) receptor (PGF(2alpha)-R) knockout, we examined whether the absence of the PGF(2alpha)-R prevents the decline in the expression of both the short and long forms of the PRL-R in the CL. We found that, in sharp contrast to the wild-type mice, in which both forms of the PRL-R decline to low levels between d 18-20 of pregnancy, expression of these receptors remained elevated in the PGF(2alpha)-R null mice. Furthermore, administration of PGF(2alpha) to pregnant rats inhibited PRL-R expression. Time-course analysis revealed that PGF(2alpha) treatment decreases both isoforms of PRL-R within 1 h of treatment in vivo, whereas its stimulatory effect on 20alphaHSD expression was further delayed. Similar results were obtained with luteinized granulosa cells in culture. To examine whether the decline in PRL-R is involved/necessary for PGF(2alpha) action, cells were transfected with a constitutively active PRL-R. The expression of this receptor did not prevent PGF(2alpha) effect on PRL-R or 20alphaHSD expression. Taken together, these results demonstrate that PGF(2alpha) inhibits the expression of the PRL-R and that the decline in both forms of the PRL-R that occurs at the end of pregnancy in the CL is due to PGF(2alpha). The results further suggest that PGF(2alpha)-mediated stimulation of 20alphaHSD is independent from PGF(2alpha) inhibition of PRL signaling in luteal cell.