No description available.

The cessation of egg-laying during the incubation period of the turkey hen is a source of major economic loss to the turkey industry. In August of 2000 there were approximately 2.7 million turkey breeder hens in the United States. Since the value of one fertile turkey egg is $0.62, the loss of only one egg per hen per year would cost the industry $1.7 million. A number of management procedures have been implemented to control egg production and prevent incubation. However, these methods are labor intensive. The anterior pituitary hormone prolactin (PRL) is involved in the onset of incubation in the turkey hen. Levels of circulating PRL and PRL mRNA are 10X greater in photostimulated hens than in photorefractory hens, 20X greater in laying hens, and 100X greater in incubating hens. It would be useful to determine the molecular mechanisms controlling regulation of the turkey (t) PRL gene. This information could be used to modulate the release of PRL and thereby prevent the induction of the incubation period in turkey hens. Approximately 2 kilobases (kb) of the tPRL 5'-flanking region were examined by the electrophoretic mobility shift assay (EMSA) using nuclear extracts from turkey pituitaries and liver. Within this 2 kb fragment, only three regions of the tPRL gene were identified that participate in tissue- and sequence-specific DNA-protein interactions with nuclear extracts from turkey pituitaries. These are the regions from nucleotides (nt) -41 to -73, -105 to -137, and -175 to -199, named tprl-1, tprl-2 and tprl-3, respectively. Three shifted bands were observed using tprl-1 and tprl-2 while two shifted bands were seen using tprl-3. Competition EMSAs done on these three regions showed that in the presence of unlabeled, excess, specific competitor DNA, the proteins bound to competitor DNA and no shifted bands were observed. If the competitor was a nonspecific DNA sequence, then there was no effect on the shifted bands. When using labeled tprl-2 and unlabeled tprl-1 as competitor DNA, no shifted bands were observed. However, when using labeled tprl-1 and unlabeled tprl-2 as competitor DNA, only one of three shifted bands was eliminated. These data indicate that tprl-1 and tprl-2 bind both common and specific pituitary nuclear proteins and have different affinities for pituitary nuclear proteins. A supershift EMSA involving the addition of rabbit-anti-rat Pit-1 indicated that tPit-1 is a common pituitary nuclear protein that is bound to tprl-1 and tprl-2. However, this interaction may not occur in the turkey in vivo. The mapping of transcription factor binding sites in the tPRL 5'-flanking region is the first step toward the identification and isolation of factors that bind to and regulate transcription of the PRL gene. / Master of Science

No description available.

The role of selected hormones in the control of hair follicle activity, fibre growth and moult in cashmere goats was investigated by manipulation of prolactin (PRL), thyroid hormones, and growth hormone (GH) individually or in combination. In experiment 1, the effect of different doses of the anti-thyroid drug "propylthiouracil" (PTU), on thyroxine (T4) and triiodothyronine (T3) profiles and deiodinase enzyme activities in liver, kidney and skin tissues was determined. Types II and III deiodinase enzymes were found to be present in goat skin but not type I. It was concluded that the supply of T3 within the skin was partly independent of circulating hormone profiles. In experiment 2, goats were treated with PTU, triiodothyronine (T3) and bromocriptine (Br) to decrease T3 availability to tissues and circulating PRL concentrations, respectively. Treatment with Br delayed the spring rise in plasma PRL concentrations (P=0.06) and primary (P<0.05) hair follicle activity, and delayed moult onset (P<0.01). PTU treatment did not significantly affect hair follicle activity but generally delayed the time of moult onset (P<0.05). The effects of the treatments were not additive, indicating that the actions of the two hormones were not independent. The effects of PTU and Br treatments were not exerted through changes in IGF-I binding activity in the skin, but binding was greater (P<0.01) in April than November. In experiment 3, treatment with bovine somatotropin (bST), T4 or metoclopramide to increase circulating concentrations of GH, T4 or PRL, failed to prolong the period of anagen in hair follicles, but bST increased fibre growth rate (P<0.05) and this was associated with higher circulating IGF-I concentrations. It is concluded that manipulation of the cycle of the cashmere-producing hair follicle is unlikely to be achieved through manipulation of circulating hormone concentrations alone and that much regulation of hair follicle activity occurs within the skin itself, possibly through changes in enzymes that control the supply of T3 to the follicles, in hormone receptor activity, and in the rate of synthesis of IGF-I and other growth factors within the skin.

S179D prolactina (hPRL) é uma mímica molecular da prolactina humana fosforilada. Demonstrou-se que a S179D-hPRL era anti angiogênica nos ensaios de angiogênese baseados na membrana corialantóica de galinha e na córnea de camundongos. Investigações posteriores realizadas empregando modelos in vitro demonstraram que o tratamento com S179D-hPRL diminuiu o número de células viáveis, reduziu a formação de túbulos em Matrigel e interferiu com a migração e invasão da matriz extracelular. A análise dos fatores de crescimento de células endoteliais humanas tratadas com S179D-hPRL revelou: uma diminuição na expressão ou liberação da PRL endógena, da heme-oxigenase-1, do fator de crescimento de fibroblasto básico (bFGF) e um aumento na expressão de dois inibidores teciduais de metaloproteases. A S179D-hPRL também bloqueou a sinalização provocada por bFGF nessas células. Nós concluímos que essa mímica molecular do hormônio pituitário fosforilado é uma potente proteína anti-angiogênica, em parte devido á sua habilidade de reduzir o estímulo autócrino de fatores de crescimento de células endoteliais de cordão umbilical humano (HUVEC), por sua capacidade de bloquear a sinalização promovida pelo bFGF e por sua habilidade de interferir na migração endotelial. Também foi estudada a influência da S179D-hPRL na apoptose em células endoteliais humanas, empregando caspase-8 como um marcador da via extrínseca, e a liberação de citocromo C como um marcador da via intrínseca. As duas cascatas convergem na ativação da caspase-3, que cliva a fator de fragmentação de DNA (DFF45). Uma incubação de três dias com 50 ng/mL de S179D-hPRL quadruplicou o número de células apoptóticas; esse efeito duplicou-se com uma concentração de 100 ng/mL e atingiu um ápice com 500 ng/mL. A clivagem de DFF45 e da pro-caspase-8 foi detectado com 100 ng/mL. Citocromo C, porém, só foi observado com concentrações de 500 ng/mL. O regulador de ciclo celular p21 (um marcador pró-apoptótico) elevou-se com 100 ng/mL, enquanto que um incremento do supressor tumoral p53 necessitou três vezes o tempo de incubação e 500 ng/mL. A atividade do promotor de p21 foi máxima com 50 ng/mL do análogo de hPRL, enquanto que 500 ng/mL foram necessários para se visualizar uma alteração significativa na atividade do promotor de Bax (um indicador da atividade de p53). Como previamente demonstrado na literatura, S179D-hPRL bloqueou a fosforilação da quinase regulada extracelularmente (ERK) em resposta ao bFGF, mas também causou uma ativação tardia e prolongada da ERK. PD 98059 [inibidor específico da proteína quinase ativada por mitógeno (MAPkinase)] inibiu essa ativação tardia e sustentada assim como outros efeitos da S179D-hPRL, exceto aquele sobre a indução de p53 e ativação do promotor de Bax. Podemos concluir que baixas doses de S179D-hPRL bloqueiam a sinalização de ERK induzida por bFGF e concomitantemente ativam a ERK em um tempo diferente, resultando na elevação de p21 e ativando a via extrínseca de apoptose. Maiores tempos de incubação e concentração, entretanto, ativam a via intrínseca empregando uma cascata intracelular diferente. Esses achados sugerem que níveis circulantes de PRL fosforilada podem inibir a progressão do câncer e, portanto, S179D-hPRL poderia ser um agente anti-angiogênico útil na terapêutica. / S179D-prolactin (hPRL) is an experimentally useful mimic of naturally phosphorylated human prolactin. S179D-hPRL, but not unmodified PRL, was found to be anti-angiogenic in both the chorioallantoic membrane and corneal assays. Further investigation using human endothelial in vitro models showed reduced cell number, reduced tubule formation in Matrigel, and reduced migration and invasion, as a function of treatment with S179D-hPRL. Analysis of growth factors in human endothelial cells in response to S179D-hPRL showed a decreased expression or release of endogenous PRL, heme-oxygenase-1, basic fibroblast growth factor (bFGF), angiogenin, epidermal growth factor and vascular endothelial growth factor and an increased expression of inhibitors of matrix metalloproteases. S179D-hPRL also blocked signaling from bFGF in these cells. We conclude that this molecular mimic of a pituitary hormone is a potent anti-angiogenic protein, partly as a result of its ability to reduce utilization of several well-established endothelial autocrine growth loops, partly by its ability to block signaling from bFGF and partly because of its ability to decrease endothelial migration. We also examined the influence of S179D-hPRL on apoptosis in human endothelial cells, using procaspase-8 as a marker of the extrinsic pathway, and cytochrome C release as a marker of the intrinsic pathway. Both pathways converge at caspase-3, which cleaves DNA fragmentation factor (DFF45). A 3-day incubation with 50 ng/ml S179D-hPRL quadrupled the early apoptotic cells; this effect was doubled at 100 ng/ml and maximal at 500 ng/ml. DFF45 and pro-caspase 8 cleavage were detectable at 100 ng/ml. Cytochrome C, however, was unaffected until 500 ng/ml. p21 increased at 100 ng/ml, whereas a change in p53 activity required both triple the time and 500 ng/ml. p21 promoter activity was maximal at 50 ng/ml, whereas 500 ng/ml were required to see a significant change in the Bax promoter (a measure of p53 activity). As previously shown, S179D-hPRL blocked extracelular regulated kinase (ERK) phosphorylation in response to bFGF, but, in addition, continued co-incubation showed a delayed and prolonged activation of ERK. PD98059 [a specific mitogen-activated protein kinase (MAPkinase) inhibitor] inhibited this delayed activation of ERK and the effects of S179D-hPRL on all parameters except p53, or activity of the Bax promoter. We conclude that low doses of S179D-hPRL block bFGF-induced ERK signaling and yet activates ERK in a different time frame to elevate p21, and activate the extrinsic pathway. Longer incubations and higher concentrations, however, additionally activate the intrinsic pathway using an alternate intracellular signal. These findings suggest that circulating levels of phosphorylated hPRL may reduce the progression of cancer and, furthermore, that S179D-hPRL may be a useful anti-angiogenic therapeutic.

S179D prolactina (hPRL) é uma mímica molecular da prolactina humana fosforilada. Demonstrou-se que a S179D-hPRL era anti angiogênica nos ensaios de angiogênese baseados na membrana corialantóica de galinha e na córnea de camundongos. Investigações posteriores realizadas empregando modelos in vitro demonstraram que o tratamento com S179D-hPRL diminuiu o número de células viáveis, reduziu a formação de túbulos em Matrigel e interferiu com a migração e invasão da matriz extracelular. A análise dos fatores de crescimento de células endoteliais humanas tratadas com S179D-hPRL revelou: uma diminuição na expressão ou liberação da PRL endógena, da heme-oxigenase-1, do fator de crescimento de fibroblasto básico (bFGF) e um aumento na expressão de dois inibidores teciduais de metaloproteases. A S179D-hPRL também bloqueou a sinalização provocada por bFGF nessas células. Nós concluímos que essa mímica molecular do hormônio pituitário fosforilado é uma potente proteína anti-angiogênica, em parte devido á sua habilidade de reduzir o estímulo autócrino de fatores de crescimento de células endoteliais de cordão umbilical humano (HUVEC), por sua capacidade de bloquear a sinalização promovida pelo bFGF e por sua habilidade de interferir na migração endotelial. Também foi estudada a influência da S179D-hPRL na apoptose em células endoteliais humanas, empregando caspase-8 como um marcador da via extrínseca, e a liberação de citocromo C como um marcador da via intrínseca. As duas cascatas convergem na ativação da caspase-3, que cliva a fator de fragmentação de DNA (DFF45). Uma incubação de três dias com 50 ng/mL de S179D-hPRL quadruplicou o número de células apoptóticas; esse efeito duplicou-se com uma concentração de 100 ng/mL e atingiu um ápice com 500 ng/mL. A clivagem de DFF45 e da pro-caspase-8 foi detectado com 100 ng/mL. Citocromo C, porém, só foi observado com concentrações de 500 ng/mL. O regulador de ciclo celular p21 (um marcador pró-apoptótico) elevou-se com 100 ng/mL, enquanto que um incremento do supressor tumoral p53 necessitou três vezes o tempo de incubação e 500 ng/mL. A atividade do promotor de p21 foi máxima com 50 ng/mL do análogo de hPRL, enquanto que 500 ng/mL foram necessários para se visualizar uma alteração significativa na atividade do promotor de Bax (um indicador da atividade de p53). Como previamente demonstrado na literatura, S179D-hPRL bloqueou a fosforilação da quinase regulada extracelularmente (ERK) em resposta ao bFGF, mas também causou uma ativação tardia e prolongada da ERK. PD 98059 [inibidor específico da proteína quinase ativada por mitógeno (MAPkinase)] inibiu essa ativação tardia e sustentada assim como outros efeitos da S179D-hPRL, exceto aquele sobre a indução de p53 e ativação do promotor de Bax. Podemos concluir que baixas doses de S179D-hPRL bloqueiam a sinalização de ERK induzida por bFGF e concomitantemente ativam a ERK em um tempo diferente, resultando na elevação de p21 e ativando a via extrínseca de apoptose. Maiores tempos de incubação e concentração, entretanto, ativam a via intrínseca empregando uma cascata intracelular diferente. Esses achados sugerem que níveis circulantes de PRL fosforilada podem inibir a progressão do câncer e, portanto, S179D-hPRL poderia ser um agente anti-angiogênico útil na terapêutica. / S179D-prolactin (hPRL) is an experimentally useful mimic of naturally phosphorylated human prolactin. S179D-hPRL, but not unmodified PRL, was found to be anti-angiogenic in both the chorioallantoic membrane and corneal assays. Further investigation using human endothelial in vitro models showed reduced cell number, reduced tubule formation in Matrigel, and reduced migration and invasion, as a function of treatment with S179D-hPRL. Analysis of growth factors in human endothelial cells in response to S179D-hPRL showed a decreased expression or release of endogenous PRL, heme-oxygenase-1, basic fibroblast growth factor (bFGF), angiogenin, epidermal growth factor and vascular endothelial growth factor and an increased expression of inhibitors of matrix metalloproteases. S179D-hPRL also blocked signaling from bFGF in these cells. We conclude that this molecular mimic of a pituitary hormone is a potent anti-angiogenic protein, partly as a result of its ability to reduce utilization of several well-established endothelial autocrine growth loops, partly by its ability to block signaling from bFGF and partly because of its ability to decrease endothelial migration. We also examined the influence of S179D-hPRL on apoptosis in human endothelial cells, using procaspase-8 as a marker of the extrinsic pathway, and cytochrome C release as a marker of the intrinsic pathway. Both pathways converge at caspase-3, which cleaves DNA fragmentation factor (DFF45). A 3-day incubation with 50 ng/ml S179D-hPRL quadrupled the early apoptotic cells; this effect was doubled at 100 ng/ml and maximal at 500 ng/ml. DFF45 and pro-caspase 8 cleavage were detectable at 100 ng/ml. Cytochrome C, however, was unaffected until 500 ng/ml. p21 increased at 100 ng/ml, whereas a change in p53 activity required both triple the time and 500 ng/ml. p21 promoter activity was maximal at 50 ng/ml, whereas 500 ng/ml were required to see a significant change in the Bax promoter (a measure of p53 activity). As previously shown, S179D-hPRL blocked extracelular regulated kinase (ERK) phosphorylation in response to bFGF, but, in addition, continued co-incubation showed a delayed and prolonged activation of ERK. PD98059 [a specific mitogen-activated protein kinase (MAPkinase) inhibitor] inhibited this delayed activation of ERK and the effects of S179D-hPRL on all parameters except p53, or activity of the Bax promoter. We conclude that low doses of S179D-hPRL block bFGF-induced ERK signaling and yet activates ERK in a different time frame to elevate p21, and activate the extrinsic pathway. Longer incubations and higher concentrations, however, additionally activate the intrinsic pathway using an alternate intracellular signal. These findings suggest that circulating levels of phosphorylated hPRL may reduce the progression of cancer and, furthermore, that S179D-hPRL may be a useful anti-angiogenic therapeutic.

by Karl Wah-keung Tsim. / Bibliography: leaves 109-119 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1984

Mammary gland development is controlled by hormones such as progesterone and prolactin, which activate a genomic regulatory network. Identification of the components and regulatory links that comprise this network will provide the basis for defining the network's dynamic response during normal development and its perturbation during breast carcinogenesis. This thesis investigates two molecules in detail, Elf5 and KIBRA, which were identified as potential prolactin targets in a transcript profiling screen for key members in this genetic program of mammary morphogenesis. We examined the effect of expression of Elf5, a transcription factor critical in alveolar differentiation, in a 3D culture model of non-transformed mammary epithelial MCF-10A cells. We discovered that Elf5 expression was selectively repressed over time in these cells when cultured on a basement membrane, and that Elf5 overexpression disrupted the architecture of acini resulting in luminal filling. This occurred due to an increase in the expression of epidermal growth factor receptor (EGFR) with repressed the induction of the pro-apoptotic molecule, Bim. We also observed that Elf5 is up-regulated with progesterone treatment, and that suppression of Elf5 expression in T47D breast cancer cells inhibits proliferation. Data obtained from the suppression of Elf5 expression in the presence of progesterone suggested that the role played by Elf5 in the Pg signalling pathway in T47D cells is relatively minor, and that rather than being a major downstream factor, the induction of Elf5 expression is utilised more to influence and potentiate other signalling pathways, such as the Prl pathway. We characterised expression of KIBRA in the mammary gland and breast cancer cell lines, and observed that KIBRA was also up-regulated with progesterone treatment. Using a bioinformatic approach, we identified the tyrosine kinase receptor DDR1 as a binding partner of KIBRA. We have demonstrated that the WW domains of KIBRA bind to a PPxY motif in DDR1, and that these molecules dissociate upon treatment with the DDR1 ligand, collagen. Finally, overexpression and knockdown studies demonstrate that KIBRA promotes the collagen-stimulated activation of the MAPK cascade. Thus KIBRA may play a role in how the reproductive state influences the mammary epithelial cell to respond to changing cell-context information, such as experienced during the tissue remodelling events of mammary gland development. Overall, the data presented in this thesis contributes to our growing knowledge of the genetic program responsible for mammary development and carcinogenesis.

Under normal circumstances, prolactin regulates its own release via a short-loop negative feedback mechanism in which prolactin, secreted from lactotrophs situated within the anterior pituitary gland, stimulates dopaminergic neurons in the hypothalamus to release dopamine into portal blood circulation. Dopamine, in turn, inhibits lactotroph activity. A change in this regulation of prolactin secretion is seen during late pregnancy where tuberoinfundibular dopaminergic (TIDA) neurons no longer respond to elevated levels of placental lactogen (PL), a lactogen structurally and functionally similar to prolactin, allowing a prolonged elevation of prolactin secretion and the induction of an antepartum prolactin surge (Andrews et al., 2001). The mechanisms behind this loss of responsiveness have not yet been determined. Prolactin acts by binding to its receptor on TIDA neurons and activating the signal transducer and activator of transcription 5b (STAT5b). During lactation, prolactin-induced activation of STAT5b is suppressed. This reduction in STAT5b signalling is consistent with a loss in TIDA responsiveness and is correlated with an increase in suppressor of cytokine signalling (SOCS) messenger ribonucleic acid (mRNA) expression within the arcuate nucleus. As SOCS proteins are known to disrupt prolactin signalling by interfering with STAT signalling in other systems, it is likely that the change in TIDA responsiveness to prolactin or PL during late pregnancy occurs at least partially in response to an increase in SOCS proteins at this time. Although prolactin can induce SOCS mRNA expression within the arcuate nucleus, the level of SOCS mRNA expression observed on day 20 of pregnancy is significantly lower to that observed on day 22. As PL is elevated on day 20 of pregnancy, some other factor or a combination of factors unique to the final 2 days of pregnancy induces the change in prolactin signalling. Late pregnancy is associated with elevated levels of estrogen while progesterone significantly declines. The aim of this study was to test the hypothesis that a fall in progesterone in the presence of elevated levels of estrogen during late pregnancy induces the increase in SOCS levels within TIDA neurons. This then results in a disruption of prolactin signalling, a decline in dopamine production and release, and the induction of the antepartum prolactin surge. To determine if ovarian steroid hormones can act directly on TIDA neurons during late pregnancy, expression of progesterone receptors (PR) and estrogen receptors (ER) within TIDA neurons were examined during pregnancy and lactation. Using double-labelled immunohistochemistry, expression of both steroid receptors within arcuate dopaminergic neurons during pregnancy and lactation was confirmed. This is consistent with the hypothesis that changing levels of steroid hormones might directly regulate TIDA activity. Furthermore, as the level of steroid receptor expression within TIDA neurons did not change significantly during pregnancy and lactation, it is likely that changing levels of serum estrogen and progesterone may affect these neurons at this time. To investigate the potential effects of steroid hormones on prolactin-induced and non prolactin-induced expression of SOCS mRNA, ovariectomised rats were treated with bromocriptine to suppress endogenous prolactin, and were then treated with a regime of chronic progesterone and/or estrogen in the presence and absence of an induced prolactin surge. SOCS mRNA expression within the arcuate nucleus was measured using real time quantitative RT-PCR. It was found that both estrogen and prolactin independently induced SOCS mRNA expression within the arcuate nucleus, but high levels of progesterone inhibited this effect. This supported the hypothesis that a change in SOCS mRNA expression within TIDA neurons might occur following the changes in steroid hormone levels observed during late pregnancy. To specifically investigate the role of estrogen and progesterone in regulating SOCS expression during late pregnancy, an animal model was designed to experimentally alter estrogen and progesterone levels during late pregnancy, and then SOCS mRNA expression was examined. In this model, advancing the late pregnant decline in progesterone resulted in a significant advance in the timing of the antepartum prolactin surge and parturition, while delaying the decline in progesterone abolished the antepartum prolactin surge and delayed parturition. Furthermore, within this model, elevated levels of SOCS mRNA expression were always observed following the withdrawal of progesterone. This suggested that following the decline in progesterone during late pregnancy, elevated levels of estrogen (or PL) are able to induce SOCS mRNA expression within the arcuate nucleus. Given that SOCS proteins disrupt cytokine signalling in other systems, the induction of SOCS proteins during late pregnancy would then presumably mediate the change in TIDA responsiveness to prolactin. To determine whether it was possible to change prolactin responses without affecting parturition, it was hoped to specifically alter progesterone and estrogen signalling in the brain. This was done by centrally administering progesterone to maintain progesterone levels during late pregnancy, and the ER antagonist ICI-182,780 (ICI) to block central estrogen levels. To determine the effectiveness of intracerebroventricular (icv) administration of ICI, two central estrogen mediated endpoints were evaluated: estrogen negative feedback on gonadotrophin releasing hormone (GnRH) pulse frequency (as measured by the frequency luteinizing hormone (LH) pulses) and the induction of PR within hypothalamic nuclei. Also, to confirm that central administration of ICI did not have a peripheral effect, estrogen induced uterine proliferation was measured. Although central ICI administration at the maximum possible dose affected estrogen-induced GnRH pulse frequency and partially reduced estrogen-induced PR expression within arcuate dopaminergic neurons, ICI did not affect the antepartum prolactin surge. Furthermore, cental administration of progesterone did not abolish the antepartum prolactin surge. This suggested that central administration of ICI and progesterone as a tool for researching central actions of ovarian steroids is likely to be limited to certain central endpoints, and was not suitable as a model to study central steroid effects on prolactin regulation. Overall, the progression of the findings in this study led to the formulation of a key hypothesis: that during late pregnancy, elevated levels of estrogen and the withdrawal of progesterone allows for the prolactin-induced increase of SOCS proteins within TIDA neurons. Elevated levels of SOCS proteins may then disrupt normal prolactin signalling, mediated via the JAK/STAT pathway. This results in reduced dopamine synthesis and release and, the subsequent induction of the antepartum prolactin surge.

Not available / text