Inhibition of the phosphoinositide 3-kinase pathway for the treatment of patients with metastatic metaplastic breast cancer.

BACKGROUND: Mesenchymal/metaplastic breast cancers (MpBCs) are often triple-negative (TNBC), and chemo-refractory, and can harbor phosphoinositide 3-kinase (PI3kinase) alterations; thus, therapy with mTor inhibitors may demonstrate activity. PATIENTS AND METHODS: Patients with mesenchymal/MpBC treated with temsirolimus-based regimens were evaluated. Mutational analyses [polymerase chain reaction (PCR)-based DNA sequencing method, mass spectrometric detection (Sequenom MassARRAY), or next-generation sequencing] as well as loss of phosphatase and tensin homolog (PTEN) (immunohistochemistry) were performed (archived tissue when available). RESULTS: Twenty-three patients (one of whom was on two separate trials) were treated using temsirolimus-containing regimens: temsirolimus alone (n = 1 patient) or combined with the following: liposomal doxorubicin and bevacizumab (DAT, n = 18); liposomal doxorubicin (DT, n = 1); paclitaxel and bevacizumab (TAT, n = 2); paclitaxel (TT, n = 1); carboplatin and bevacizumab (CAT, n = 1). Response rate [complete response (CR) + partial response (PR)] was 25% across all regimens; 32% in the anthracycline-based regimens [DAT and DT (CR = 2, PR = 4; N = 19)]. An additional two patients achieved stable disease (SD) ≥6 months [total SD ≥6 months/CR/PR = 8 (33%)]. Molecular aberrations in the PI3K pathway were common: PIK3CA mutation = 6/15 (40%), PTEN mutation = 3/11 (27%), and PTEN loss = 2/11 (18%). A point mutation in the NF2 gene (K159fs*16; NF2 alterations can activate mTor) was found in one patient who attained CR (3+ years). Of the eight patients who achieved SD ≥6 months/CR/PR, all 4 patients with available tissue had a molecular aberration that activate the PIK3CA/Akt/mTOR axis: PIK3CA mutation = 2; PTEN loss = 1; NF2 aberration = 1. CONCLUSIONS: DAT has activity in MpBCs including complete CRs. Molecular aberrations that can activate the PI3 K/Akt/mTOR axis are common in MpBC.

INTERACCIÓN DE LA GLUTAMINA SINTETASA (GS) Y EL PÉPTIDO β -AMILOIDE COMO UNA ESTRATEGIA DE PURIFICACIÓN [title language="en"]INTERACTION OF GLUTAMINE SYNTHETASE (GS) AND AMYLOID β-PEPTIDE AS A PURIFICATION STRATEGY / INTERAÇÃO DA GLUTAMINA SINTETASE (GS) E PÉPTIDO β AMILÓIDE COMO UMA ESTRATÉGIA DE PURIFICAÇÃO

La enfermedad de Alzheimer (EA) es la forma de demencia más común en la edad adulta. Se manifiesta con la pérdida progresiva de la memoria a medida que las neuronas en la corteza cerebral y el hipocampo mueren. En todas las formas de EA se evidencia aumento de la expresión de diferentes proteínas, así como la presencia de agregados insolubles de péptido-β-amiloide (PBA). La glutamina sintetasa (GS) es una enzima clave en el metabolismo del glutamato y en la detoxificación de amonio (NH4+). Previamente se ha reportado una posible interacción GS-PBA que puede estar asociada con EA. En este trabajo se realizó la purificación de la enzima cerebral de rata a partir de un extracto sometido a precipitación fraccionada con (NH4)2SO4 del 20-60 % de saturación y posteriormente a través de cromatografías sucesivas de filtración en gel, intercambio iónico y afinidad. El peso molecular del complejo fue calculado en 137 kDa por el orden de elución en la columna de filtración. Se identificó interacción de la enzima con PBA 1-40, lográndose la purificación de una sola banda de 45 kDa, correspondiente a la forma monomérica de la GS. En este trabajo se presenta un nuevo método de purificación de la enzima y se demuestra la interacción de GS con el PBA. Se propone que esta interacción GS-PBA puede ser uno de los procesos que se presentan en la enfermedad al explicar la reducción de la actividad de la enzima en paciente con EA, ya que podría alterar el ciclo glutamato-glutamina y generar cambios en el entorno celular que favorecen excitotoxicidad por glutamato típica de los procesos de neurodegeneración.

Alzheimer's disease (AD) is the most common form of dementia in adulthood; it is manifested by the progressive loss of memory since neurons in both cerebral cortex and hippocampus die. In all the forms of AD is observed the increased expression of different proteins, as well as the presence of insoluble aggregates of β-amyloid peptide (BAP). Glutamine synthetase (GS) is a key enzyme in the metabolism of glutamate and in the detoxification of ammonium (NH4+). A possible interaction GS-PBA has been previously reported and it can be associated with AD. In this work we performed the purification of the enzyme from rat brain extract subjected to fractional precipitation 20-60 % saturation with (NH4)2SO4, and thereafter through successive chromatographies of gel filtration, ion exchange and affinity. The molecular weight of the complex was calculated at 137 kDa by the order of elution in the column filtration. The interaction of the enzyme with 1-40 PBA was identified, achieving the purification of a single band of 45 kDa corresponding to the monomeric form of the GS. In this paper we present a new method of the enzyme purification and we demonstrated the interaction of GS with the PBA. We propose this interaction GS-PBA can be one of the processes that occur in the disease and it could explain the reduction in enzyme activity in patients with AD, since it might alter the glutamate-glutamine cycle and generate changes in the cellular environment which favor glutamate excitotoxicity typical of neurodegeneration processes.

A doença do Alzhéimer (DA) é a forma mais comum de demência na idade adulta, que se manifesta pela perda progressiva da memória, já que os neurónios em córtex cerebral e hipocampo morrem. Em todas as formas de AD é observado o aumento da expressão de proteínas diferentes, bem como a presença de agregados insolúveis de β-amilóide péptido (BAP). Glutamina sintetase (GS) é uma enzima chave no metabolismo do glutamato e na desintoxicação de amónio (NH4+). Uma possível interacção GS-PBA foi relatada anteriormente e pode ser associada com o AD. Neste trabalho, foi realizada a purificação da enzima a partir do extrato do cérebro de rato e foi submetido a precipitação fraccionada com (NH4)2SO4 de 20- 60 % de saturação e, subsequentemente, através de cromatografias sucessivas de filtração em em gel, permuta iónica e de afinidade. O peso molecular do complexo foi calculado em 137 kDa por ordem de eluição na filtração de coluna. A interacção da enzima com 1-40 PBA foi identificada, alcançando a purificação de uma única banda de 45 kDa correspondente à forma monomérica do GS. Neste artigo, apresentamos um novo método de purificação da enzima e demonstramos a interação da GS com o PBA. Propomos que esta interacção GS-PBA pode ser um dos processos que ocorrem na doença e pode explicar a redução na actividade da enzima nos pacientes com o AD, uma vez que poderia alterar o ciclo de glutamatoglutamina e gerar alterações no ambiente celular que favorecem a excitotoxicidade típica do glutamato nos processos de neurodegeneração.

Brk/PTK6 sustains activated EGFR signaling through inhibiting EGFR degradation and transactivating EGFR.

Epidermal growth factor receptor (EGFR)-mediated cell signaling is critical for mammary epithelial cell growth and survival; however, targeting EGFR has shown no or only minimal therapeutic benefit in patients with breast cancer. Here, we report a novel regulatory mechanism of EGFR signaling that may explain the low response rates. We found that breast tumor kinase (Brk)/protein-tyrosine kinase 6 (PTK6), a nonreceptor protein-tyrosine kinase highly expressed in most human breast tumors, interacted with EGFR and sustained ligand-induced EGFR signaling. We demonstrate that Brk inhibits ligand-induced EGFR degradation through uncoupling activated EGFR from casitas B-lineage lymphoma-mediated EGFR ubiquitination. In addition, upon activation by EGFR, Brk directly phosphorylated Y845 in the EGFR kinase domain, thereby further potentiating EGFR kinase activity. Experimental elevation of Brk conferred resistance of breast cancer cells to cetuximab (an EGFR-blocking antibody)-induced inhibition of cell signaling and proliferation, whereas knockdown of Brk sensitized the cells to cetuximab by inducing apoptosis. Our findings reveal a previously unknown role of Brk in EGFR-targeted therapy.

Prognostic impact of discordance between triple-receptor measurements in primary and recurrent breast cancer.

BACKGROUND: We evaluated discordance in expression measurements for estrogen receptor (ER), progesterone receptor (PR), and HER2 between primary and recurrent tumors in patients with recurrent breast cancer and its effect on prognosis. METHODS: A total of 789 patients with recurrent breast cancer were studied. ER, PR, and HER2 status were determined by immunohistochemistry (IHC) and/or FISH. Repeat markers for ER, PR, and HER2 were available in 28.9%, 27.6%, and 70.0%, respectively. Primary and recurrent tumors were classified as triple receptor-negative breast cancer (TNBC) or receptor-positive breast cancer (RPBC, i.e. expressing at least one receptor). Discordance was correlated with clinical/pathological parameters. RESULTS: Discordance for ER, PR, and HER2 was 18.4%, 40.3%, and 13.6%, respectively. Patients with concordant RPBC had significantly better post-recurrence survival (PRS) than discordant cases; patients with discordant receptor status had similarly unfavorable survival as patients with concordant TNBC. IHC scores for ER and PR showed weak concordance between primary and recurrent tumors. Concordance of HER2-FISH scores was higher. CONCLUSIONS: Concordance of quantitative hormone receptor measurements between primary and recurrent tumors is modest consistent with suboptimal reproducibility of measurement methods, particularly for IHC. Discordant cases have poor survival probably due to inappropriate use of targeted therapies. However, biological change in clinical phenotype cannot be completely excluded.

Survival among women with triple receptor-negative breast cancer and brain metastases.

BACKGROUND: The purpose of this study was to determine the incidence of and survival following brain metastases among women with triple receptor-negative breast cancer. PATIENTS AND METHODS: In all, 679 patients with nonmetastatic triple receptor-negative breast cancer diagnosed from 1980 to 2006 were identified. Cumulative incidence of brain metastases was computed. Cox proportional hazards models were fitted to explore factors that predict for development of brain metastases. Survival was computed using the Kaplan-Meier product limit method. RESULTS: Median follow-up was 26.9 months. In all, 42 (6.2%) patients developed brain metastases with a cumulative incidence at 2 and 5 years of 5.6% [95% confidence interval (CI) 3.8% to 7.9%] and 9.6% (95% CI 6.8% to 13%), respectively. A total of 24 (3.5%) patients developed brain metastases as the first site of recurrence with cumulative incidence at 2 and 5 years of 2.0% (95% CI 2.6% to 6.0%) and 4.9% (95% CI 3.2% to 7.0%), respectively. In the multivariable model, no specific factor was observed to be significantly associated with time to brain metastases. Median survival for all patients who developed brain metastases and those who developed brain metastases as the first site of recurrence was 2.9 months (95% CI 2.0-7.6 months) and 5.8 months (95% CI 1.7-11.0 months), respectively. CONCLUSION: In this single-institutional study, patients with nonmetastatic triple receptor-negative breast tumors have a high early incidence of brain metastases associated with poor survival and maybe an ideal cohort to target brain metastases preventive strategies.

Listeria spp., y L. monocytogenes en leche cruda de cabra / Listeria spp. and L. monocytogenes in raw goat’s milk

Objetivo. Detectar Listeria spp. y Listeria monocytogenes en leches crudas de cabras, provenientes del corregimiento de la Garita, Norte de Santander. Materiales y métodos. Se tomaron 90 muestras de leche cruda de cabra, mediante muestreo estratificado, en un período de 4 meses; durante el muestreo se tomó la temperatura de las leches. Para el aislamiento de L. monocytogenes se utilizó la técnica sugerida por el INVIMA y se confirmó la especie por PCR. Resultados. Se encontraron ocho productores de leches de cabra en esta zona, ninguno refrigera, ni pasteuriza la leche. Se encontró una ocurrencia de 3% de L. monocytogenes y 15% de otras especies. Se demostró que la leches obtenidas en esta zona contienen este patógeno que puede llegar a causar Listeriosis en los grupos de riesgo como niños menores de 5 años, mujeres en etapa de gestación, adultos mayores y pacientes inmunocomprometidos. Conclusiones. Se demostró la circulación de este patógeno en la leche de cabra y al ser un producto que se consume directamente por las personas pone en riesgo su salud.

MicroRNA-196a targets annexin A1: a microRNA-mediated mechanism of annexin A1 downregulation in cancers.

Suppression of annexin A1 (ANXA1), a mediator of apoptosis and inhibitor of cell proliferation, is well documented in various cancers but the underlying mechanism remains unknown. We investigated whether decreased ANXA1 expression was mediated by microRNAs (miRNAs), which are small, non-coding RNAs that negatively regulate gene expression. Using Sanger miRBase, we identified miR-584, miR-196a and miR-196b as potential miRNAs targeting ANXA1. Only miRNA-196a showed significant inverse correlation with ANXA1 mRNA levels in 12 cancer cell lines of esophageal, breast and endometrial origin (Pearson's correlation -0.66, P=0.019), identifying this as the candidate miRNA targeting ANXA1. Inverse correlation was also observed in 10 esophageal adenocarcinomas (Pearson's correlation -0.64, P=0.047). Analysis of paired normal/tumor tissues from additional 10 patients revealed an increase in miR-196a in the cancers (P=0.003), accompanied by a decrease in ANXA1 mRNA (P=0.004). Increasing miR-196a levels in cells by miR-196a mimics resulted in decreased ANXA1 mRNA and protein. In addition, miR-196a mimics inhibited luciferase expression in luciferase plasmid reporter that included predicted miR-196a recognition sequence from ANXA1 3'-untranslated region confirming that miR-196a directly targets ANXA1. miR-196a promoted cell proliferation, anchorage-independent growth and suppressed apoptosis, suggesting its oncogenic potential. This study demonstrated a novel mechanism of post-transcriptional regulation of ANXA1 expression and identified miR-196a as a marker of esophageal cancer.

Discordant genetic changes in ovarian and endometrial endometrioid carcinomas: a potential pitfall in molecular diagnosis.

Endometrioid carcinoma simultaneously involving ovaries as well as the uterine corpus may present a diagnostic dilemma because of the difficulty in determining whether the lesions are separate primary tumors or metastases. It has been reported that the detection of clonality using microsatellite markers may be useful in solving this dilemma. To determine the usefulness of this technique, we compared the genetic alterations in microsatellite markers present in matched pairs of ovarian tumors from 12 patients. The study includes four ovarian cancer FIGO stage I and eight stage III/IV patients, and four patients also with independent endometrial carcinoma of the uterus. DNA from paraffin-embedded tissue was extracted and amplified using a multiplex polymerase chain reaction, after which the status of microsatellite instability and loss of heterozygosity in four microsatellite loci (BAT25, BAT26, D17S250, and D5S346) were determined. In the four patients with stage I ovarian cancer, four microsatellite markers were identical in one patient and three were identical in the remaining three patients. In high-stage patients, three markers were identical in at least 4/8 cases. In three of four patients with uterine involvement, three of the four markers were identical in the uterine tumor and one of the corresponding ovarian tumors. These results suggest that genetic discordance does not indicate independent origin or metastasis of the tumor but instead a progression of genetic changes at separate sites probably due to the marked genetic instability existing in these tumors. Because of these discordant genetic changes, great caution should be taken when distinguishing between primary and metastatic tumors on the basis of microsatellite markers.

A single-gene biomarker identifies breast cancers associated with immature cell type and short duration of prior breastfeeding.

The pathogenesis of breast cancers that do not express estrogen receptors or Her-2/neu receptors (ER-/HER2- phenotype) is incompletely understood. We had observed markedly elevated gene expression of gamma-aminobutyric acid type A (GABA(A)) receptor subunit pi (GABApi, GABRP) in some breast cancers with ER-/HER2- phenotype. In this study, transcriptional profiles (TxPs) were obtained from 82 primary invasive breast cancers by oligonucleotide microarrays. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to measure GABApi gene expression in a separate cohort of 121 invasive breast cancers. GABApi gene expression values from TxP and RT-PCR were standardized and compared with clinicopathologic characteristics in the 203 patients. GABApi gene expression was increased in 16% of breast cancers (13/82 TxP, 20/ 121 RT-PCR), particularly in breast cancers with ER-/HER2- phenotype (60%), and breast cancers with basal-like genomic profile (60%). The profile of genes coexpressed with GABApi in these tumors was consistent with an immature cell type. In multivariate linear regression analysis, the level of GABApi gene expression was associated with ER-/HER2- phenotype (P < 0.0001), younger age at diagnosis (P = 0.0003), and shorter lifetime duration of breastfeeding (< or = 6 months) in all women (P = 0.017) and specifically in parous women (P = 0.013). GABApi gene expression was also associated with combinations of high grade with ER-/HER2- phenotype (P = 0.002), and with Hispanic ethnicity (P = 0.036). GABApi gene expression is increased in breast cancers of immature (undifferentiated) cell type and is significantly associated with shorter lifetime history of breastfeeding and with high-grade breast cancer in Hispanic women.

Expression of sigma 1 receptor in human breast cancer.

The sigma 1 receptor (S1R) represents a unique drug-binding site that is distinct from any other receptors. We examined S1R expression in human breast cancer and assessed the activity of S1R ligands in breast cancer cell lines. One-hundred nine breast specimens from normal breast, benign breast disease and cancer were examined with immunohistochemistry or RT-PCR and six different cell lines were also evaluated. S1R mRNA overexpression was detected in 64% of breast cancers compared to normal breast tissue. Immunohistochemistry showed positive epithelial cell staining in 60% of invasive and 41% of in situ cancers, 75% of ductal hyperplasia and in 33% of normal breast. The pattern of expression was more diffuse in invasive breast carcinoma compared to other conditions (p = 0.02). S1R expression was neither a prognostic nor a predictive factor for efficacy of adjuvant chemotherapy but the study only included 58 cancer patients and therefore the statistical power is limited. MDA-MB-361, MDA-MB-435, BT20 and MCF7 cells all expressed S1R protein by Western blot. The non-specific S1R ligands haloperidol, reduced haloperidol and progesterone produced a dose-dependent inhibition of the growth at high (>10 microM) concentrations. Reduced haloperidol also showed additive cytotoxic effects when combined with doxorubicin, vinorelbine , paclitaxel and docetaxel in vitro. The S1R-specific ligand, SKF 10047 demonstrated the least growth inhibitory activity and showed no interaction with chemotherapy. These results demonstrate that some normal and most neoplastic breast epithelial cells and cell lines commonly express S1R. High concentrations of haloperidol inhibit the growth of these cells and potentiate the effect of chemotherapy in vitro.

Modificaciones en el metabolismo del hierro y requerimientos de eritropoyetina tras el paso de gluconato férrico a hierro sacarosa. ¿Compensa el incremento del gasto? / No disponible

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Differential expression of MUC2 and MUC5AC mucin genes in primary ovarian and metastatic colonic carcinoma.

Colonic adenocarcinoma, the most common tumor metastatic to the ovary, may closely mimic primary ovarian adenocarcinoma, especially that of mucinous or endometrioid histology. The differential diagnosis is important for therapeutic considerations. Mucin gene expression is relatively organ-specific and may therefore have use in distinguishing between colonic carcinomas metastatic to the ovary and primary ovarian tumors. In this study, we compared the expression of MUC2 and MUC5AC apomucins in 10 colonic adenocarcinomas metastatic with the ovary, 10 ovarian endometrioid carcinomas (4 primary, 6 metastatic), and 32 primary mucinous ovarian tumors (12 cystadenomas, 10 borderline tumors, and 10 cystadenocarcinomas). Monoclonal antibodies CCP58 and 45M1 were used for immunostains of MUC2 and MUC5AC apomucin, respectively. All but 1 of the 10 metastatic colon adenocarcinomas expressed MUC2, whereas none expressed MUC5AC. None of the 10 endometrioid carcinomas expressed MUC2, and only 2 showed weak immunoreactivity with MUC5AC. All 32 primary mucinous ovarian tumors expressed MUC5AC. The percentages of MUC2-positive immunostaining for cystadenomas, borderline tumors, and cystadenocarcinomas were 0% (0/12), 50% (5/10), and 70% (7/10) respectively. These studies show that MUC2 and MUC5AC are useful markers in the distinction between colonic carcinoma metastatic to the ovary and primary ovarian carcinoma.

The gonadotropin-releasing hormone receptor gene promoter directs pituitary-specific oncogene expression in transgenic mice.

Our previous work has shown that 1.2 kb of the 5' flanking region of the mouse GnRH receptor (mGnRH-R) gene is sufficient to direct tissue-specific expression in vitro. In this study, we have used the cell-specific regulatory sequences of the mGnRH-R gene promoter to target the expression of the simian virus 40 virus T antigen (TAg) to the pituitary gland of transgenic mice. A hybrid transgene, GnRH-R/TAg, was prepared using the -1164/+52 region of the mGnRH-R gene and +2533/+5234 sequences encoding the large T antigen of the simian virus 40. Two founders developed tumors of apparent pituitary origin at 44 (M28, female) and 50 (M25, male) days of age. M28 and M25 mice were about 50% underweight, and their gonads were grossly underdeveloped compared with wild-type litter mates. A third male founder, M29, developed a tumor at a later time (109 days). M29 was able to breed successfully and stably transmit the GnRH-R/TAg transgene. Mice of the M29 transgene line developed tumors at 4-5 months of age. Gross examination showed that the tumors extend from the sella and infiltrate into the inferior surface of the brain. In small tumors collected from young transgenic animals, normal pituitary cells as well as transition areas of increasing cellular atypia are evident. Frankly malignant cells are seen in all tumors. The pituitary tumors express the alpha-, FSHbeta-, and LHbeta-subunits and the GnRH-R messenger RNA, all markers of a gonadotrope but not of other anterior pituitary cell lineages. In summary, our studies indicate that 1.2 kb of the 5'-flanking region of the mGnRH-R gene can be used to target expression specifically to the gonadotropes of the pituitary gland in transgenic mice. The GnRH-R gene promoter-directed expression appears to be cell-specific and results in the formation of tumors that are primarily of gonadotropic origin.

Bacteremia due to streptococcus zooepidemicus associated with an abdominal aortic aneurysm.

Group C streptococci are a common cause of infection in animals but a rare cause of infection in man. To our knowledge, the English literature contains only three cases of arteriosclerotic aneurysms of the abdominal aorta (AAA) secondarily infected with group C Beta-hemolytic Streptococci. Two cases did not survive in spite of adequate antibiotic therapy. One patient responded to aortic repair with a bifurcated woven dacron graft followed by four weeks of high-dose intravenous penicillin. This article describes our clinical experience with a patient who survived an atherosclerotic aneurysm of the abdominal aorta and S. zooepidemicus infection.

The different forms of the prolactin receptor in the rat corpus luteum: developmental expression and hormonal regulation in pregnancy.

The corpora lutea of pregnancy in the rat are highly dependent on the action of PRL and PRL-like hormones to hypertrophy and to produce progesterone needed for the maintenance of gestation. Two forms of the PRL receptor (PRL-R), designated as long (PRL-RL) and short (PRL-RS), have been described in rat tissues. To determine whether both forms are present in the corpus luteum during pregnancy and to examine the developmental and hormonal regulation of their expression, total RNA isolated from corpora lutea at different stages of pregnancy and from highly luteinized granulosa cells subjected to different hormonal treatments were analyzed by semiquantitative RT-PCR. Immunoblotting of luteal proteins from early and late pregnancy was also performed to determine if the pattern of PRL-R proteins follows that of PRL-R messenger RNA (mRNA) expression. In addition, the correlation between the well characterized PRL-regulated gene, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), and PRL-R gene expression was investigated during the time of luteolysis. Both PRL-RL and PRL-RS mRNA and protein were expressed in corpora lutea of pregnancy, with the long form being the most dominant at all stages. Whereas no changes in mRNA level of either PRL-RL or PRL-RS were found until day 20 of gestation, a profound decline in PRL-R mRNA and protein for both receptor types occurred at the end of pregnancy. This drop in PRL-R expression was accompanied by a sharp and abrupt expression of 20alpha-HSD mRNA. Studies performed in vivo and in luteinized cells in culture indicate that PRL can up-regulate the expression of the PRL-RL mRNA, an effect prevented by the tyrosine kinase inhibitor, genistein. PRL-RL mRNA was also selectively increased by cAMP. In summary, the results of this investigation have established that: 1) the corpus luteum of pregnancy expresses both the short and long forms of the PRL-R with the long form being more abundant; 2) the mRNA for both forms of the PRL-R remains at constant levels throughout pregnancy but drops before parturition; 3) the decline in PRL-R mRNA at the end of pregnancy is accompanied by a dramatic rise in 20alpha-HSD; 4) PRL is able to increase the expression of PRL-R mRNA; and that 5) both A kinase and tyrosine kinase mediated pathways appear to participate in the up-regulatory mechanism involved in PRL-R mRNA expression.

PRAP, a prolactin receptor associated protein: its gene expression and regulation in the corpus luteum.

We have recently identified, characterized, and cloned a luteal microsomal 32-kDa phosphoprotein that we named PRAP (for PRL-receptor associated protein), and we have demonstrated that PRAP binds to the intracellular domain of the short but not the long form of the PRL receptor. In this study, we used PRAP cDNA to examine the tissue specificity, the developmental expression, and the hormonal regulation of PRAP gene expression. Northern blot analysis revealed that in the corpus luteum, PRAP cDNA hybridized to multiple transcripts (5.5 kb, 4.3 kb, and 1.8 kb), with the smallest transcript (1.8 kb) corresponding to the size of the cDNA clone. However, none of these transcripts were detected in any other tissues examined. PRAP appears to be tightly regulated by steroids and PRL. When pregnant rats were treated with aminoglutethimide, a steroid synthesis inhibitor, all three PRAP transcripts became barely detectable. Similar results were obtained when all luteotropic support was removed by hypophysectomy and hysterectomy. Estradiol up-regulated PRAP expression and, more specifically, the two lower transcripts. PRL had no stimulatory effect on PRAP messenger RNA (mRNA) expression but caused a substantial increase in the level of PRAP protein when administered to hypophysectomized pregnant rat, suggesting that PRL may stabilize this protein. Similar dissociation between levels of mRNA and protein were observed during luteal development. Although both PRAP mRNA and protein were barely detectable in early pregnancy, their expression increased abruptly from midpregnancy; however, whereas levels of PRAP mRNA declined from day 18, those of the protein remained elevated until parturition. In summary, results of this study have defined the tissue specificity and developmental expression of PRAP mRNA during pregnancy. The data have also revealed that the gene expression of this protein is up-regulated by estradiol, suggesting a pivotal role for PRAP in the synergistic action of estradiol and PRL on the function of the rat corpus luteum.

Transcriptional activation of the gonadotropin-releasing hormone receptor gene by activin A.

It has been reported that activin A stimulates the synthesis of the GnRH receptors (GnRHR) in rat pituitary cultures. However, the role of activin A in the regulation of the GnRHR gene at the molecular level is not known. In the present work, we have studied the regulation of the GnRHR gene by activin A in the gonadotrope cell line, alpha T3-1, where the GnRHR gene is highly expressed. First, we demonstrate that these cells express the mRNAs of three types of activin receptors: I, II, and IIB. Activin A increases GnRHR mRNA levels in a dose-and time-dependent manner, with maximal stimulation (2.5 +/- 0.5-fold) occurring with a dose of 20 ng/ml after 36 h of incubation. To ascertain whether this effect occurs at the transcriptional level, we performed nuclear run-off experiments in alpha T3-1 cells, which demonstrate a 1.6-fold increase in the levels of newly synthesized GnRHR mRNA in response to activin A. To investigate further the effect of activin A on the transcription of the GnRHR gene, alpha T3-1 cells were transiently transfected with a mouse GnRHR promoter/luciferase reporter gene (GnRHR-Luc) and challenged with activin A. Luciferase activity increases in response to activin A to the same extent (2.4 +/- 0.4-fold) and with similar dose-response and time-course profiles as the mRNA levels. Follistatin (100 ng/ml), a well known activin antagonist, completely abolishes the activin A effect on both mRNA levels and GnRHR-Luc activity. Follistatin also decreases the basal expression of the GnRHR gene by 33% as determined by GnRHR-Luc activity. This, together with our demonstration of the presence of the inhibin beta B-subunit mRNA in alpha T3-1 cells, suggests a potential paracrine/autocrine role of endogenous activin B on the regulation of the GnRHR gene in these cells. To provide evidence for biological significance of activin A stimulation of GnRHR gene expression, the response of a human gonadotropin alpha-subunit promoter/luciferase reporter gene (alpha Gon-Luc) to GnRH was assessed in alpha T3-1 cells pretreated with activin A. Activin enhances the stimulation of alpha Gon-Luc activity by GnRH by 1.6 +/- 0.4-fold. These data demonstrate that activin A can stimulate the expression of the GnRHR gene at the transcriptional level. Furthermore, transfection studies localize the activin responsive element to 1.2 kb of the 5'-flanking region of the GnRHR gene. Transcriptional activation of the GnRHR gene by activin A may serve as a mechanism for the modulation of gonadotrope responsiveness to GnRH.