RESUMEN
Metastatic neuroblastoma is an aggressive childhood cancer of neural crest origin. Stathmin, a microtubule destabilizing protein, is highly expressed in neuroblastoma although its functional role in this malignancy has not been addressed. Herein, we investigate stathmin's contribution to neuroblastoma tumor growth and metastasis. Small interfering RNA (siRNA)-mediated stathmin suppression in two independent neuroblastoma cell lines, BE(2)-C and SH-SY5Y, did not markedly influence cell proliferation, viability or anchorage-independent growth. In contrast, stathmin suppression significantly reduced cell migration and invasion in both the neuroblastoma cell lines. Stathmin suppression altered neuroblastoma cell morphology and this was associated with changes in the cytoskeleton, including increased tubulin polymer levels. Stathmin suppression also modulated phosphorylation of the actin-regulatory proteins, cofilin and myosin light chain (MLC). Treatment of stathmin-suppressed neuroblastoma cells with the ROCKI and ROCKII inhibitor, Y-27632, ablated MLC phosphorylation and returned the level of cofilin phosphorylation and cell invasion back to that of untreated control cells. ROCKII inhibition (H-1152) and siRNA suppression also reduced cofilin phosphorylation in stathmin-suppressed cells, indicating that ROCKII mediates stathmin's regulation of cofilin phosphorylation. This data demonstrates a link between stathmin and the regulation of cofilin and MLC phosphorylation via ROCK. To examine stathmin's role in neuroblastoma metastasis, stathmin short hairpin RNA (shRNA)\luciferase-expressing neuroblastoma cells were injected orthotopically into severe combined immunodeficiency-Beige mice, and tumor growth monitored by bioluminescent imaging. Stathmin suppression did not influence neuroblastoma cell engraftment or tumor growth. In contrast, stathmin suppression significantly reduced neuroblastoma lung metastases by 71% (P<0.008) compared with control. This is the first study to confirm a role for stathmin in hematogenous spread using a clinically relevant orthotopic cancer model, and has identified stathmin as an important contributor of cell invasion and metastasis in neuroblastoma.
Asunto(s)
Neoplasias Pulmonares/metabolismo , Neuroblastoma/metabolismo , Interferencia de ARN , Estatmina/genética , Factores Despolimerizantes de la Actina/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Forma de la Célula , Supervivencia Celular , Citoesqueleto/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Ratones , Invasividad Neoplásica , Trasplante de Neoplasias , Neuroblastoma/genética , Neuroblastoma/secundario , Fosforilación , Procesamiento Proteico-Postraduccional , ARN Interferente Pequeño/genética , Estatmina/metabolismo , Carga Tumoral , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
The HER2 (ERBB2) and MYC genes are commonly amplified in breast cancer, yet little is known about their molecular and clinical interaction. Using a novel chimeric mammary transgenic approach and in vitro models, we demonstrate markedly increased self-renewal and tumour-propagating capability of cells transformed with Her2 and c-Myc. Coexpression of both oncoproteins in cultured cells led to the activation of a c-Myc transcriptional signature and acquisition of a self-renewing phenotype independent of an epithelial-mesenchymal transition programme or regulation of conventional cancer stem cell markers. Instead, Her2 and c-Myc cooperated to induce the expression of lipoprotein lipase, which was required for proliferation and self-renewal in vitro. HER2 and MYC were frequently coamplified in breast cancer, associated with aggressive clinical behaviour and poor outcome. Lastly, we show that in HER2(+) breast cancer patients receiving adjuvant chemotherapy (but not targeted anti-Her2 therapy), MYC amplification is associated with a poor outcome. These findings demonstrate the importance of molecular and cellular context in oncogenic transformation and acquisition of a malignant stem-like phenotype and have diagnostic and therapeutic consequences for the clinical management of HER2(+) breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Proto-Oncogénicas c-myc/fisiología , Receptor ErbB-2/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/mortalidad , Carcinoma Ductal de Mama/patología , Línea Celular Tumoral , Proliferación Celular , Femenino , Expresión Génica , Humanos , Ratones , Persona de Mediana Edad , Análisis Multivariante , Trasplante de Neoplasias , Fenotipo , Pronóstico , Análisis de Supervivencia , Transcriptoma , Adulto JovenRESUMEN
Grb2-associated binder 1 (Gab1) is a docking protein that transduces signals from a variety of tyrosine kinases, including Met and the epidermal growth factor receptor (EGFR). Although the related protein Gab2 is strongly implicated in human cancer, a role for Gab1 has been less clear. However, a screen for gene mutations in breast cancer identified two somatic mutations in Gab1, Y83C and T387N. In this paper we describe the functional characterization of these Gab1 mutants. MCF-10A immortalized mammary epithelial cells overexpressing Gab1 Y83C and T387N exhibited a more elongated, fibroblastic phenotype compared with wild-type Gab1 controls. Expression of Gab1 or the mutants promoted epidermal growth factor (EGF)-independent proliferation in monolayer culture to a similar degree. However, in Matrigel culture, both mutants enhanced the formation of acini exhibiting an aberrant, branched morphology. In addition, expression of the mutants modestly increased Erk activation. The two mutants also enhanced branching morphogenesis in a different mammary epithelial cell line, HC11. To gain further insights into the mechanism of action of these mutations, we mapped Gab1 phosphorylation sites by mass spectrometry. This detected phosphorylation of T387 but ;not Y83. Cellular stimulation with EGF or hepatocyte growth factor (HGF) led to a transient, or sustained, induction of T387 phosphorylation, respectively. As T387 corresponds in position to Gab2 T391, which suppresses Gab2 signaling in a phosphorylation-dependent manner, these data support a model in which the T387N mutation abrogates negative-feedback regulation of Gab1. Interrogation of publically-available databases revealed additional cancer-associated mutations at, or in close proximity to, identified serine/threonine phosphorylation sites in other docking proteins. These data indicate that aberrant Gab1 signaling can directly contribute to breast cancer progression, and that negative feedback sites in docking proteins can be targeted by oncogenic mutations.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/metabolismo , Mutación Missense , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Sustitución de Aminoácidos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Transformada , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/farmacología , Femenino , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Proteínas de Neoplasias/genética , Fosforilación/efectos de los fármacos , Fosforilación/genéticaRESUMEN
Prolactin and progesterone act together to regulate mammary alveolar development, and both hormones have been implicated in breast cancer initiation and progression. Here we show that Elf5, a prolactin-induced ETS transcription factor that specifies the mammary secretory cell lineage, is also induced by progestins in breast cancer cells via a direct mechanism. To define the transcriptional response to progestin elicited via Elf5, we made an inducible Elf5 short hairpin-RNA knock-down model in T47D breast cancer cells and used it to prevent the progestin-induction of Elf5. Functional analysis of Affymetrix gene expression data using Gene Ontologies and Gene Set Enrichment Analysis showed enhancement of the progestin effects on cell cycle gene expression. Cell proliferation assays showed a more efficacious progestin-induced growth arrest when Elf5 was kept at baseline levels. These results showed that progestin induction of Elf5 expression tempered the antiproliferative effects of progestins in T47D cells, providing a further mechanistic link between prolactin and progestin in the regulation of mammary cell phenotype.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Progestinas/farmacología , Progestinas/uso terapéutico , Proteínas Proto-Oncogénicas c-ets/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN , Femenino , Humanos , Mifepristona/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , Factores de TranscripciónRESUMEN
Top quartile serum prolactin levels confer a twofold increase in the relative risk of developing breast cancer. Prolactin exerts this effect at an ill defined point in the carcinogenic process, via mechanisms involving direct action via prolactin receptors within mammary epithelium and/or indirect action through regulation of other hormones such as estrogen and progesterone. We have addressed these questions by examining mammary carcinogenesis in transplants of mouse mammary epithelium expressing the SV40T oncogene, with or without the prolactin receptor, using host animals with a normal endocrine system. In prolactin receptor knockout transplants the area of neoplasia was significantly smaller (7 versus 17%; P < 0.001 at 22 weeks and 7 versus 14%; P = 0.009 at 32 weeks). Low-grade neoplastic lesions displayed reduced BrdU incorporation rate (11.3 versus 17% P = 0.003) but no change in apoptosis rate. Tumor latency increased (289 days versus 236 days, P < 0.001). Tumor frequency, growth rate, morphology, cell proliferation and apoptosis were not altered. Thus, prolactin acts directly on the mammary epithelial cells to increase cell proliferation in preinvasive lesions, resulting in more neoplasia and acceleration of the transition to invasive carcinoma. Targeting of mammary prolactin signaling thus provides a strategy to prevent the early progression of neoplasia to invasive carcinoma.
Asunto(s)
Proliferación Celular , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/genética , Receptores de Prolactina/genética , Animales , Antígenos Transformadores de Poliomavirus/genética , Apoptosis , Peso Corporal , Caspasa 3/fisiología , Progresión de la Enfermedad , Femenino , Masculino , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Invasividad Neoplásica , Trasplante de NeoplasiasRESUMEN
Mammalian hair growth is cyclic, with hair-producing follicles alternating between active (anagen) and quiescent (telogen) phases. The timing of hair cycles is advanced in prolactin receptor (PRLR) knockout mice, suggesting that prolactin has a role in regulating follicle cycling. In this study, the relationship between profiles of circulating prolactin and the first post-natal hair growth cycle was examined in female Balb/c mice. Prolactin was found to increase at 3 weeks of age, prior to the onset of anagen 1 week later. Expression of PRLR mRNA in skin increased fourfold during early anagen. This was followed by upregulation of prolactin mRNA, also expressed in the skin. Pharmacological suppression of pituitary prolactin advanced dorsal hair growth by 3.5 days. Normal hair cycling was restored by replacement with exogenous prolactin for 3 days. Increasing the duration of prolactin treatment further retarded entry into anagen. However, prolactin treatments, which began after follicles had entered anagen at 26 days of age, did not alter the subsequent progression of the hair cycle. Skin from PRLR-deficient mice grafted onto endocrine-normal hosts underwent more rapid hair cycling than comparable wild-type grafts, with reduced duration of the telogen phase. These experiments demonstrate that prolactin regulates the timing of hair growth cycles in mice via a direct effect on the skin, rather than solely via the modulation of other endocrine factors.
Asunto(s)
Cabello/crecimiento & desarrollo , Prolactina/farmacología , Receptores de Prolactina/metabolismo , Animales , Biomarcadores/análisis , Depresión Química , Domperidona/farmacología , Antagonistas de Dopamina/farmacología , Femenino , Expresión Génica , Genotipo , Cabello/efectos de los fármacos , Tinturas para el Cabello , Remoción del Cabello , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones SCID , Prolactina/sangre , Prolactina/genética , Antígeno Nuclear de Célula en Proliferación/análisis , ARN Mensajero/análisis , Radioinmunoensayo/métodos , Receptores de Prolactina/análisis , Receptores de Prolactina/genética , Piel/química , Piel/metabolismo , Trasplante de PielRESUMEN
Prolactin is essential for proliferation and differentiation of the developing mammary gland. We have explored a role for Suppressor of Cytokine Signaling 1 (SOCS1) as a modulator of the prolactin response using mice deficient in SOCS1, which were rescued from neonatal death by deletion of the Interferon gamma (IFN gamma) gene. SOCS1(-/-)/IFN gamma(-/-) mice exhibited accelerated lobuloalveolar development in the mammary gland during late pregnancy and precocious lactation. Significantly, the lactogenic defect in prolactin receptor heterozygous females could be rescued by deletion of a single SOCS1 allele. These findings establish a role for SOCS1 as a negative regulator of prolactin signaling and suggest that SOCS1 is required for the prevention of lactation prior to parturition.
Asunto(s)
Mama/crecimiento & desarrollo , Proteínas Portadoras/fisiología , Lactancia/fisiología , Receptores de Prolactina/fisiología , Proteínas Represoras , Alelos , Animales , Proteínas Portadoras/genética , Caseínas/biosíntesis , Línea Celular , Femenino , Expresión Génica , Masculino , Ratones , Ratones Noqueados , Leche/metabolismo , Embarazo , Receptores de Prolactina/genética , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de CitocinasRESUMEN
Pituitary PRL regulates seasonal hair follicle growth cycles in many mammals. Here we present the first evidence implicating PRL in the nonseasonal, wave-like pelage replacement of laboratory mice. In this study we show that messenger RNA transcripts encoding the one long and two short forms of PRL receptor are present in the skin of adult and neonate mice. The receptor protein was immunolocalized to the hair follicle as well as the epidermis and sebaceous glands. Furthermore, PRL messenger RNA was detected within skin extracts, suggesting a possible autocrine/paracrine role. Analysis of the hair growth phenotype of PRL gene-disrupted mice (PRLR(-/-)) revealed a change in the timing of hair cycling events. Although no hair follicle development differences were noted in PRLR(-/-) neonates, observations of the second generation of hair growth revealed PRLR(-/-) mice molted earlier than wild types (PRLR(+/+)). The advance was greater in females (29 days) than in males (4 days), resulting in the elimination of the sexual dimorphism associated with murine hair replacement. Heterozygotes were intermediate between PRLR(-/-) and PRLR(+/+) mice in molt onset. Once initiated, the pattern and progression of the molt across the body were similar in all genotypes. Although all fiber types were present and appeared structurally normal, PRLR(-/-) mice had slightly longer and coarser hair than wild types. These findings demonstrate that PRL has an inhibitory effect on murine hair cycle events. The pituitary PRL regulation of hair follicle cycles observed in seasonally responsive mammals may be a result of pituitary PRL interacting with a local regulatory mechanism.
Asunto(s)
Folículo Piloso/fisiología , Cabello/crecimiento & desarrollo , Periodicidad , Prolactina/fisiología , Receptores de Prolactina/deficiencia , Transducción de Señal , Animales , Animales Recién Nacidos , Peso Corporal , Epidermis/química , Femenino , Cabello/anatomía & histología , Cabello/química , Folículo Piloso/química , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Prolactina/genética , ARN Mensajero/análisis , Receptores de Prolactina/genética , Receptores de Prolactina/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándulas Sebáceas/química , Caracteres Sexuales , Piel/químicaRESUMEN
PURPOSE: To determine whether prolactin receptor is essential for normal development and function of the lacrimal gland and whether hyperprolactinemia can alter lacrimal development. METHODS: Lacrimal gland morphology and function were examined in two genetic mouse models of prolactin action: a prolactin receptor knockout model that is devoid of prolactin action and a transgenic model of hyperprolactinemia. RESULTS: Image analysis of lacrimal and Harderian gland sections was used to quantify glandular morphology. In females, lacrimal acinar area decreased by 30% and acinar cell density increased by 25% over control subjects in prolactin transgenic animals, but prolactin receptor knockout mice showed no changes. In males, transgenic animals showed no changes, but prolactin receptor knockout mice showed a 5% reduction in acinar area and an 11% increase in acinar cell density, which was lost after castration. The morphology of the Harderian glands underwent parallel changes but to a lesser degree. A complete loss of porphyrin accretions was seen in the Harderian glands of male and female knockout animals. No differences in tear protein levels were seen in knockout animals by two-dimensional gels. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the level of secretory component and IgA in knockout mouse tears remained unchanged. There was no change in the predisposition of the 129 mouse strain to conjunctivitis in the knockout animals. CONCLUSIONS: Prolactin plays a small role in establishing the sexual dimorphism of male lacrimal glands. In females, hyperprolactinemia causes a hyperfemale morphology, suggesting a role in dry eye syndromes. Prolactin is required for porphyrin secretion by the Harderian gland but plays no essential role in the secretory immune function of the lacrimal gland.
Asunto(s)
Glándula de Harder/citología , Glándula de Harder/fisiología , Aparato Lagrimal/citología , Aparato Lagrimal/fisiología , Prolactina/fisiología , Animales , Western Blotting , Recuento de Células , Electroforesis en Gel Bidimensional , Ensayo de Inmunoadsorción Enzimática , Proteínas del Ojo/metabolismo , Femenino , Hiperprolactinemia/genética , Hiperprolactinemia/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Receptores de Prolactina/genética , Receptores de Prolactina/fisiología , Caracteres Sexuales , Lágrimas/metabolismoRESUMEN
Prolactin (PRL), synthesized by the anterior pituitary and to a lesser extent by numerous extrapituitary tissues, affects more physiological processes than all other pituitary hormones combined. This hormone is involved in > 300 separate effects in various vertebrate species where its role has been well documented. The initial step in its action is the binding to a specific membrane receptor which belongs to the superfamily of class 1 cytokine receptors. The function of this receptor is mediated, at least in part, by two families of signaling molecules: Janus kinases and signal transducers and activators of transcription. PRL-binding sites have been identified in a number of cells and tissues of adult animals. Disruption of the gene for the PRL receptor has provided a new animal model with which to better understand the actions of PRL on mammary morphogenesis and mammary gland gene expression. The recent availability of genetic mouse models provides new insights into mammary developmental biology and how the action of a hormone at specific stages of development can have effects later in life on processes such as mammary development and breast cancer initiation and progression.
Asunto(s)
Mama , Glándulas Mamarias Animales , Prolactina/fisiología , Receptores de Prolactina/fisiología , Transducción de Señal/fisiología , Animales , Mama/embriología , Mama/fisiología , Femenino , Humanos , Glándulas Mamarias Animales/embriología , Glándulas Mamarias Animales/fisiología , Ratones , Morfogénesis/fisiologíaRESUMEN
PRL, a hormone secreted essentially by the pituitary and other extrapituitary sources such as decidua, has been attributed regulatory roles in reproduction and cell growth in mammals. These effects are mediated by a membrane PRL receptor belonging to the cytokine receptor superfamily. Null mutation of the PRL receptor gene leads to female sterility due to a severely compromised preimplantation development and a complete failure of the implantation of the few embryos reaching the blastocyst stage, strongly implicating PRL in the maternal control of implantation. We measured the hormonal status of -/- mice, which confirmed that the corpus luteum is unable to produce progesterone. Progesterone administration to -/- mice completely rescued the development of preimplantatory eggs and embryo implantation. Pregnancy could be maintained to 19.5 days postcoitum, with about 22% of resulting embryos reaching adulthood. Although progesterone and perhaps PRL appear to facilitate mouse preembryo development throughout the preimplantation stages, other factors as well as a possible direct effect of PRL on the uterus are probably necessary to fully maintain pregnancy. Finally, reduced ductal side-branching in the mammary gland can be rescued by progesterone treatment, but females exhibit reduced alveolar formation. Our model establishes the PRL receptor as a key regulator of reproduction and provides novel insights into the function of lactogenic hormones and their receptor.
Asunto(s)
Implantación del Embrión , Desarrollo Embrionario , Óvulo/fisiología , Progesterona/farmacología , Receptores de Prolactina/deficiencia , Animales , Femenino , Hormonas/sangre , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Noqueados/genética , Óvulo/efectos de los fármacos , Embarazo , Receptores de Prolactina/genéticaRESUMEN
Prolactin (PRL) is the primary lactogenic pituitary hormone that plays an essential role in many aspects of reproduction, from fertilization to mammary gland development and maternal behavior. PRL has also been reported to play a role in immunoregulation. Because initial observations indicated that hypophysectomized rats present abnormalities of the immune system, including increased thymic atrophy and lymphopenia, a number of studies have focused on the potential immunomodulatory roles of PRL. This hormone exerts its biological activities following binding to specific cell surface PRL receptors (PRLRs). In this report, we have characterized the development and function of the immune system in PRLR-deficient mice. Compared with wild-type control mice, PRLR-/- mice demonstrate no alterations in thymic or splenic cellularity or in the composition of the lymphocyte subsets present in primary (bone marrow and thymus) or secondary (spleen and lymph nodes) lymphoid organs. Lymphocytes from PRLR-/- mice are functional in vitro, as they can proliferate normally to mitogens, cytokines, and allogeneic cells. PRLR-/- splenocytes display normal NK-mediated cytotoxicity to YAC-1 target cells. In vivo studies have revealed that PRLR-/- mice are able to 1) generate normal steady-state Ig levels, 2) mount a normal specific Ig response following immunization with a T-dependent Ag, 3) eliminate injected allogeneic tumor cells, and 4) effectively control Listeria monocytogenes infection. Taken together, these results show that immune system development and function proceed normally in the absence of PRL-mediated signaling and suggest that PRLR pathways are not essential for immunomodulation in vivo.
Asunto(s)
Sistema Inmunológico/crecimiento & desarrollo , Receptores de Prolactina/deficiencia , Receptores de Prolactina/genética , Animales , Especificidad de Anticuerpos/genética , Linfocitos B/citología , Linfocitos B/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Pruebas Inmunológicas de Citotoxicidad , Sistema Inmunológico/citología , Sistema Inmunológico/fisiología , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/sangre , Isoantígenos/farmacología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitógenos/farmacología , Linfocitos T/citología , Linfocitos T/inmunología , Timo/citología , Timo/inmunología , Células Tumorales CultivadasRESUMEN
The inactivation of the prolactin receptor gene by homologous recombination has made it possible to investigate the role of prolactin signaling in mammary gland development without resort to ablative surgery of the endocrine glands. In knockout mice lacking the prolactin receptor, mammary development is normal up to puberty. Subsequently, the ducts branch less frequently than those of wild-type animals. While terminal end buds differentiate to alveolar buds in wild-type females by the end of puberty, in knockout females terminal end bud-like structures persist at the ductal ends. To distinguish between the developmental defects that are intrinsic to the epithelium and those that result from systemic endocrine alterations in prolactin receptor knockout mice, mammary epithelium from prolactin receptor knockouts was transplanted into mammary fat pads of wild-type mice. In virgin mice, the knockout epithelial transplants developed normally at puberty, indicating an indirect effect of prolactin on ductal development. Prolactin receptor knockout females are infertile due to multiple reproductive defects, but epithelial transplants allowed us to assess the extent to which the absence of prolactin receptor is limiting, under systemic conditions that allow full mammary gland development. During pregnancy, the prolactin receptor knockout transplants showed normal side branching and the formation of alveolar buds, but no lobuloalveolar development. Thus, prolactin affects mammary morphogenesis in two different ways: it controls ductal side branching and terminal end bud regression in virgin animals via indirect mechanisms, but acts directly on the mammary epithelium to produce lobuloalveolar development during pregnancy.
Asunto(s)
Glándulas Mamarias Animales/crecimiento & desarrollo , Prolactina/farmacología , Receptores de Prolactina/efectos de los fármacos , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Heterocigoto , Histocitoquímica , Glándulas Mamarias Animales/efectos de los fármacos , Ratones , Ratones Noqueados , Embarazo , Transducción de Señal , Trasplante de TejidosRESUMEN
In rodents, the prolactin receptor is expressed as multiple isoforms with identical extracellular and membrane-proximal region sequences but with different 3' sequences, encoding different cytoplasmic regions, and different 5' untranslated region (UTR) sequences. These divergent sequences could be the result of multiple prolactin receptor genes or of a single gene which displays alternative promoter usage and 3'-exon splicing. To investigate the molecular basis for these observations, we have cloned and determined the organization of the mouse prolactin receptor gene. Genomic DNA cloning allowed the arrangement of promoters 1A, 1B, and 1C to be determined. 5'-RACE-PCR from mouse liver identified two novel 5' prolactin receptor sequences, indicating that the gene has at least five different promoters, four of which are active in liver. The remaining nonvariable 5' UTR is encoded by a separate exon (exon 2), while a further 11 coding exons follow, the last 4 of which are alternatively spliced to produce the four isoforms of the receptor. Functional units were found to be exon specific. Thus, the multiple prolactin receptor isoforms are the product of a single gene of >120 kb which displays multiple promoter usage and 3'-exon splicing.
Asunto(s)
Empalme Alternativo/genética , Regiones Promotoras Genéticas/genética , Receptores de Prolactina/genética , Animales , Secuencia de Bases , Clonación Molecular , Evolución Molecular , Exones/genética , Femenino , Dosificación de Gen , Genes/genética , Hígado , Ratones , Datos de Secuencia Molecular , Mapeo Restrictivo , Análisis de Secuencia de ADNRESUMEN
The neuropeptide galanin is predominantly expressed by the lactotrophs (the prolactin secreting cell type) in the rodent anterior pituitary and in the median eminence and paraventricular nucleus of the hypothalamus. Prolactin and galanin colocalize in the same secretory granule, the expression of both proteins is extremely sensitive to the estrogen status of the animal. The administration of estradiol-17beta induces pituitary hyperplasia followed by adenoma formation and causes a 3,000-fold increase in the galanin mRNA content of the lactotroph. To further study the role of galanin in prolactin release and lactotroph growth we now report the generation of mice carrying a loss-of-function mutation of the endogenous galanin gene. There is no evidence of embryonic lethality and the mutant mice grow normally. The specific endocrine abnormalities identified to date, relate to the expression of prolactin. Pituitary prolactin message levels and protein content of adult female mutant mice are reduced by 30-40% compared with wild-type controls. Mutant females fail to lactate and pups die of starvation/dehydration unless fostered onto wild-type mothers. Prolactin secretion in mutant females is markedly reduced at 7 days postpartum compared with wild-type controls with an associated failure in mammary gland maturation. There is an almost complete abrogation of the proliferative response of the lactotroph to high doses of estrogen, with a failure to up-regulate prolactin release, STAT5 expression or to increase pituitary cell number. These data further support the hypothesis that galanin acts as a paracrine regulator of prolactin expression and as a growth factor to the lactotroph.
Asunto(s)
División Celular/fisiología , Galanina/fisiología , Adenohipófisis/metabolismo , Prolactina/metabolismo , Alelos , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Células Cultivadas , Cartilla de ADN , Estrógenos/fisiología , Femenino , Galanina/genética , Ratones , Ratones Mutantes , Adenohipófisis/citologíaRESUMEN
We have studied pup-directed maternal behavior in mice carrying a germ line null mutation of the PRL receptor (PRLR) gene. Homozygous mutant and heterozygous mutant nulliparous females show a deficiency in pup-induced maternal behavior. Moreover, primiparous heterozygous females exhibit a profound deficit in maternal care when challenged with foster pups. Morris maze studies revealed normal configural learning in the heterozygous and homozygous animals. Eating, locomotor activity, sexual behavior, and exploration (all processes regulated by the hypothalamus) are normal in PRLR mutant mice. Olfactory function was tested in an aversive conditioning paradigm, results indicating that heterozygous and homozygous PRLR mutant mice are not anosmic. These studies clearly establish the PRLR as a regulator of maternal behavior.
Asunto(s)
Conducta Materna , Receptores de Prolactina/fisiología , Animales , Cognición , Femenino , Aprendizaje por Laberinto , Ratones , Mutación , Embarazo , Receptores de Prolactina/genética , Olfato , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Prolactin (PRL), secreted by the pituitary, decidua, and lymphoid cells, has been shown to have a regulatory role in reproduction, immune function, and cell growth in mammals. The effects of PRL are mediated by a membrane-bound receptor that is a member of the superfamily of cytokine receptors. Formation of a trimer, consisting of one molecule of ligand and two molecules of receptor, appears to be a necessary prerequisite for biological activity. The function of these receptors is mediated, at least in part, by two families of signaling molecules: Janus tyrosine kinases (JAKs) and signal transducers and activators of transcription (STATs). To study these receptors, we have used two approaches: mutational analysis of their cytoplasmic domains coupled with functional tests and inactivation (knockout) of the receptor gene by homologous recombination in mice. We have produced mice by gene targeting in embryonic stem cells carrying a germline null mutation of the prolactin receptor gene. Heterozygous (+/-) females show almost complete failure to lactate, following their first, but not subsequent pregnancies. Homozygous (-/-) females are infertile as a result of multiple reproductive abnormalities, including ovulation of premiotic oocytes, reduced fertilization of oocytes, reduced preimplantation oocyte development, lack of embryo implantation, and the absence of pseudopregnancy. Half of the homozygous males are infertile or show reduced fertility. In view of the wide-spread distribution of PRL receptors, other phenotypes including those on the immune system, are currently being evaluated in -/- animals. This study establishes the prolactin receptor as a key regulator of mammalian reproduction and provides the first total ablation model to further study the role of the prolactin receptor and its ligands.
Asunto(s)
Sistema Inmunológico/fisiología , Sistemas Neurosecretores/fisiología , Prolactina/fisiología , Animales , Ratones , Ratones Noqueados/genética , Ratones Noqueados/fisiología , Receptores de Prolactina/genética , Receptores de Prolactina/fisiología , Transducción de Señal/fisiología , Linfocitos T/fisiologíaRESUMEN
The GALN gene encodes the preprogalanin protein that is cleaved to liberate the galanin peptide, a neuropeptide and tumor cell mitogen, and the galanin message-associated peptide, which is of unknown function. GALN is located at chromosome 11q13, a frequently amplified locus in diverse tumor types including breast cancer. To determine whether GALN may contribute to the tumor phenotype resulting from 11q13 amplification, we examined GALN amplification and preprogalanin mRNA levels in breast tumors and cell lines. GALN was amplified in a subset of breast tumors and cell lines that carried 11q13 amplifications. Preprogalanin mRNA was expressed in the majority of breast cancer cell lines, but Northern analysis failed to demonstrate a relationship between GALN amplification and preprogalanin mRNA levels. Eight of eight estrogen receptor-positive cell lines expressed detectable preprogalanin mRNA, and further investigation showed that preprogalanin mRNA was increased by treatment with estradiol and progestin and decreased by the removal of serum or treatment with antiestrogens. Thus, GALN amplification is unlikely to contribute to the phenotype conferred by 11q13 amplification in breast cancer, but preprogalanin mRNA is expressed by breast cancer cells and is under steroid hormone control in estrogen receptor-positive cells, opening the wider question of the role of this steroid-regulated neuropeptide in the normal and cancerous breast.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Galanina/biosíntesis , Galanina/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Precursores de Proteínas/biosíntesis , Precursores de Proteínas/genética , Esteroides/farmacología , Northern Blotting , Southern Blotting , Cromosomas Humanos Par 11 , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Dexametasona/farmacología , Dihidrotestosterona/farmacología , Estrógenos/farmacología , Amplificación de Genes , Expresión Génica , Humanos , Progestinas/farmacología , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
The sex steroid hormones and PRL interact synergistically to control the neoplastic growth of the mammary gland. The basis for this hormonal synergy is unknown, but may involve cellular coexpression of the sex steroid and PRL receptors, coupled with receptor cross-regulation. To examine this hypothesis the expression of the sex steroid and PRL receptors was examined in 20 human breast cancer cell lines and 123 primary breast cancers. Regulation of sex steroid receptors by PRL and of the PRL receptor by sex steroids was examined in T-47D and MCF-7 breast cancer cells. Northern analysis of the breast cancer cell lines and tumors indicated that the PRL receptor and the sex steroid receptors were coexpressed. The level of PRL receptor expression in the breast cancer cell lines was linearly related to that of the estrogen and progesterone receptors, but not to that of the androgen receptor. In MCF-7 and T-47D cells, acute treatment with progestins and androgens and long term treatment with estrogens increased PRL receptor levels. Analysis of sex steroid receptor messenger ribonucleic acid and binding activity showed that acute PRL treatment produced a time- and concentration-dependent increase in progesterone receptor and a decrease in androgen receptor. These results indicate that receptors for sex steroids and PRL are coexpressed and are cross-regulated, providing a potential mechanism for the observed synergy among estrogen, progesterone, and PRL in the control of tumor growth.
Asunto(s)
Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Receptores de Prolactina/genética , Receptores de Esteroides/genética , Northern Blotting , Dihidrotestosterona/farmacología , Estradiol/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Progesterona/farmacología , Prolactina/farmacología , Receptores Androgénicos/genética , Receptores de Estrógenos/genética , Receptores de Progesterona/genética , Células Tumorales CultivadasRESUMEN
Mice carrying a germ-line null mutation of the prolactin receptor gene have been produced by gene targeting in embryonic stem cells. Heterozygous females showed almost complete failure of lactation attributable to greatly reduced mammary gland development after their first, but not subsequent, pregnancies. Homozygous females were sterile owing to a complete failure of embryonic implantation. Moreover, they presented multiple reproductive abnormalities, including irregular cycles, reduced fertilization rates, defective preimplantation embryonic development, and lack of pseudopregnancy. Half of the homozygous males were infertile or showed reduced fertility. This work establishes the prolactin receptor as a key regulator of mammalian reproduction, and provides the first total ablation model to further study the role of the prolactin receptor and its ligands.