RESUMEN
In the lactating mammary gland, the plasma membrane calcium ATPase2 (PMCA2) transports milk calcium. Its expression is activated in breast cancers, where high tumor levels predict increased mortality. We find that PMCA2 expression correlates with HER2 levels in breast cancers and that PMCA2 interacts with HER2 in specific actin-rich membrane domains. Knocking down PMCA2 increases intracellular calcium, disrupts interactions between HER2 and HSP-90, inhibits HER2 signaling, and results in internalization and degradation of HER2. Manipulating PMCA2 levels regulates the growth of breast cancer cells, and knocking out PMCA2 inhibits the formation of tumors in mouse mammary tumor virus (MMTV)-Neu mice. These data reveal previously unappreciated molecular interactions regulating HER2 localization, membrane retention, and signaling, as well as the ability of HER2 to generate breast tumors, suggesting that interactions between PMCA2 and HER2 may represent therapeutic targets for breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Receptor ErbB-2/metabolismo , Transducción de Señal , Animales , Neoplasias de la Mama/patología , Calcio/farmacología , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Membrana Celular/metabolismo , Proliferación Celular , Supervivencia Celular , Endocitosis/efectos de los fármacos , Femenino , Técnica del Anticuerpo Fluorescente , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Immunoblotting , Espacio Intracelular/metabolismo , Neoplasias Mamarias Animales , Ratones , Unión Proteica , Transporte de Proteínas , Análisis de SupervivenciaRESUMEN
We examined whether the scaffolding protein sodium-hydrogen exchanger regulatory factor 1 (NHERF1) interacts with the calcium pump PMCA2 and the tyrosine kinase receptor ErbB2/HER2 in normal mammary epithelial cells and breast cancer cells. NHERF1 interacts with the PDZ-binding motif in PMCA2 in both normal and malignant breast cells. NHERF1 expression is increased in HER2-positive breast cancers and correlates with HER2-positive status in human ductal carcinoma in situ (DCIS) lesions and invasive breast cancers as well as with increased mortality in patients. NHERF1 is part of a multiprotein complex that includes PMCA2, HSP90, and HER2 within specific actin-rich and lipid raft-rich membrane signaling domains. Knocking down NHERF1 reduces PMCA2 and HER2 expression, inhibits HER2 signaling, dissociates HER2 from HSP90, and causes the internalization, ubiquitination, and degradation of HER2. These results demonstrate that NHERF1 acts with PMCA2 to regulate HER2 signaling and membrane retention in breast cancers.
Asunto(s)
Neoplasias de la Mama/metabolismo , Fosfoproteínas/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Receptor ErbB-2/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Secuencias de Aminoácidos , Animales , Apoptosis , Neoplasias de la Mama/genética , Calcio/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Proliferación Celular , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ratones , Microscopía Fluorescente , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Magnetic resonance imaging (MRI) of solids is rarely attempted. One of the main reasons is that the broader MR linewidths, compared to the narrow resonance of the hydrogen ((1)H) in free water, limit both the attainable spatial resolution and the signal-to-noise ratio. Basic physics research, stimulated by the quest to build a quantum computer, gave rise to a unique MR pulse sequence that offers a solution to this long-standing problem. The "quadratic echo" significantly narrows the broad MR spectrum of solids. Applying field gradients in sync with this line-narrowing sequence offers a fresh approach to carry out MRI of hard and soft solids with high spatial resolution and with a wide range of potential uses. Here we demonstrate that this method can be used to carry out three-dimensional MRI of the phosphorus ((31)P) in ex vivo bone and soft tissue samples.
Asunto(s)
Imagen por Resonancia Magnética/métodos , Huesos/metabolismo , Isótopos de Fósforo , ProtonesRESUMEN
The modeling of long bone surfaces during linear growth is a key developmental process, but its regulation is poorly understood. We report here that parathyroid hormone-related peptide (PTHrP) expressed in the fibrous layer of the periosteum (PO) drives the osteoclastic (OC) resorption that models the metaphyseal-diaphyseal junction (MDJ) in the proximal tibia and fibula during linear growth. PTHrP was conditionally deleted (cKO) in the PO via Scleraxis gene targeting (Scx-Cre). In the lateral tibia, cKO of PTHrP led to a failure of modeling, such that the normal concave MDJ was replaced by a mound-like deformity. This was accompanied by a failure to induce receptor activator of NF-kB ligand (RANKL) and a 75% reduction in OC number (Pâ ≤â 0.001) on the cortical surface. The MDJ also displayed a curious threefold increase in endocortical osteoblast mineral apposition rate (Pâ ≤â 0.001) and a thickened cortex, suggesting some form of coupling of endocortical bone formation to events on the PO surface. Because it fuses distally, the fibula is modeled only proximally and does so at an extraordinary rate, with an anteromedial cortex in CD-1 mice that was so moth-eaten that a clear PO surface could not be identified. The cKO fibula displayed a remarkable phenotype, with a misshapen club-like metaphysis and an enlargement in the 3D size of the entire bone, manifest as a 40-45% increase in the PO circumference at the MDJ (Pâ ≤â 0.001) as well as the mid-diaphysis (Pâ ≤â 0.001). These tibial and fibular phenotypes were reproduced in a Scx-Cre-driven RANKL cKO mouse. We conclude that PTHrP in the fibrous PO mediates the modeling of the MDJ of long bones during linear growth, and that in a highly susceptible system such as the fibula this surface modeling defines the size and shape of the entire bone.
Asunto(s)
Desarrollo Óseo/fisiología , Peroné/crecimiento & desarrollo , Proteína Relacionada con la Hormona Paratiroidea/fisiología , Periostio/fisiología , Tibia/crecimiento & desarrollo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Eliminación de Gen , Ratones , Ratones Noqueados , Ligando RANK/genéticaRESUMEN
The developing cortical surfaces of long bones are sculpted and modeled by periosteal osteoclasts and osteoblasts. These surfaces also receive the insertions of tendons and ligaments, and these insertion sites too are modeled to form the root systems that anchor them into the cortical bone. The regulatory molecules that control modeling are poorly understood, but recent evidence suggests that parathyroid hormone-related protein (PTHrP) participates in this process. PTHrP functions principally as a paracrine regulatory molecule, and is known to be induced by mechanical loading in a number of sites. The most curious example of developmental modeling of the cortex is the migration of insertion sites such as that of the medial collateral ligament (MCL) along the bone surface during long-bone growth. We report here the mechanisms that mediate MCL migration using a combination of genetic, imaging and histological techniques. We describe a MCL migratory complex that comprises two components. The first is the MCL insertion site itself, which is a prototypical fibrous insertion site with coupled osteoclast and osteoblast activities, and its key feature is that it is anchored early in development, well before initiation of the long-bone growth spurt. Above the insertion site the periosteum is excavated by osteoclasts to form a migratory tract; this is mediated by wholly uncoupled osteoclastic bone resorption and remains as an unmineralized canal on the cortical surface in the adult. Load-induction of PTHrP appears to regulate the osteoclastic activity in both the insertion site and migratory tract.
Asunto(s)
Ligamento Colateral Medial de la Rodilla/crecimiento & desarrollo , Animales , Condrocitos/citología , Articulación de la Rodilla/citología , Articulación de la Rodilla/crecimiento & desarrollo , Ratones , Osteoclastos/citología , Microtomografía por Rayos XRESUMEN
This study's objective was to investigate obtaining high-resolution micro-computed tomography (CT) imaging of the injected arterial circulation of the brains of the dogfish (Squalus acanthias), American bullfrog (Rana catesbeiana), and green iguana (Iguana iguana). No micro-CT images of the arterial morphology of the brains of these vertebrates were previously published. Micro-CT imaging was performed on brains that had the cerebral arterial and ventricular systems injected with a radiopaque barium-gelatin compound in the early 1970s. These specimens were dissected and placed in a preservative fluid for 35 years, until imaged with micro-CT. The obtained micro-CT images were processed with a software program that provided 3D rotational motion rendering, and sequential display of 2D renderings of the micro-CT data. The anatomic information provided by the high-resolution micro-CT is not reproducible by any other radiopaque contrast currently available, without tissue removal corrosion, and enhanced the dissection information. The digital videos of the micro-CT 3D rotational motion rendering and sequential display of 2D renderings of the dogfish, bullfrog, and green iguana, demonstrate the extent of the arterial network within the brain, the arterial segments obscured by overlying structures such as nerves, and identified in the bullfrog the venous cerebral circulation resulting from the centrifugal leptomeningeal arterial capillaries. The rotational 3D images separated superimposed arterial structures, and the sequential display of the 2D renderings clarifies the relationship of cut or overlapped arterial branches. Comparing the brain and arterial morphology of the dogfish, bullfrog, and green iguana demonstrates some of the evolutionary modifications in these vertebrates.
Asunto(s)
Iguanas , Squalus acanthias , Animales , Rana catesbeiana , Cazón , Microtomografía por Rayos XRESUMEN
The calcium-sensing receptor (CaR) regulates transepithelial calcium transport into milk by mammary epithelial cells. Using a genome-wide screening strategy, we identified the plasma membrane calcium ATPase isoform 2 (PMCA2) as a potential downstream target of the CaR. We show that PMCA2 expression in the mouse mammary gland increases during lactation and that PMCA2 is localized solely to the apical plasma membrane of mammary epithelial cells. In milk from deafwaddler mice, which have mutations in the gene encoding PMCA2, calcium concentrations were reduced, confirming its importance in calcium transport into milk. Furthermore, in cultured primary and EpH4 mouse mammary epithelial cells, CaR stimulation up-regulated calcium-dependent ATPase activity in plasma membrane preparations. By small interfering RNA-mediated gene knockdown of PMCA2, we show that PMCA2 accounts for the preponderance of calcium-ATPase activity. We also show that reduction of CaR expression with small interfering RNA eliminates the ability of extracellular calcium to elicit an increase in calcium-dependent ATPase activity in EpH4 cell membranes. These results demonstrate that activation of the CaR increases PMCA2 activity in mouse mammary epithelial cells, providing a mechanism for the regulation of transepithelial calcium transport by calcium in the lactating mouse mammary gland.
Asunto(s)
Calcio/metabolismo , Células Epiteliales/metabolismo , Leche/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Receptores Sensibles al Calcio/fisiología , Animales , Transporte Biológico/efectos de los fármacos , Western Blotting , Calcio/farmacología , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Femenino , Técnica del Anticuerpo Fluorescente , Gadolinio/farmacología , Isoenzimas/genética , Isoenzimas/metabolismo , Lactancia/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Inmunoelectrónica , Proteínas de la Leche/metabolismo , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , Receptores Sensibles al Calcio/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Large amounts of calcium are transferred to offspring by milk. This demand results in negative calcium balance in lactating mothers and is associated with rapid bone loss. The mechanisms of bone loss during lactation are only partly understood. Several studies have suggested that parathyroid hormone-related protein (PTHrP) might be secreted into the circulation by the lactating mammary gland and regulate bone turnover during lactation. Because mammary development fails in the absence of PTHrP, conventional PTHrP knockout mice cannot be used to address this possibility. To examine this hypothesis, we therefore used mice carrying a beta-lactoglobulin promoter-driven Cre transgene, one null PTHrP allele, and one floxed PTHrP allele. Expression of Cre specifically in mammary epithelial cells during late pregnancy and lactation resulted in efficient deletion of the PTHrP gene; mammary gland PTHrP mRNA and milk PTHrP protein were almost completely absent. Removal of PTHrP from the lactating mammary glands resulted in reductions in levels of circulating PTHrP and 1,25-dihydroxy vitamin D and urinary cAMP. In addition, bone turnover was reduced and bone loss during lactation was attenuated. We conclude that during lactation mammary epithelial cells are a source of circulating PTHrP that promotes bone loss by increasing rates of bone resorption.
Asunto(s)
Densidad Ósea , Lactancia/metabolismo , Glándulas Mamarias Animales/química , Proteína Relacionada con la Hormona Paratiroidea/fisiología , Animales , Resorción Ósea/etiología , Calcitriol/sangre , Calcio/metabolismo , Femenino , Homeostasis , Integrasas/genética , Lactoglobulinas/genética , Ratones , Proteína Relacionada con la Hormona Paratiroidea/sangre , Regiones Promotoras Genéticas , Proteínas Virales/genéticaRESUMEN
CONTEXT: The physiology of PTH is well described, but regulation of PTH gene expression remains enigmatic. This is, at least in part, because of a lack of suitable cell culture systems. OBJECTIVE, DESIGN, SETTING, PATIENTS, INTERVENTIONS, AND MAIN OUTCOME MEASURES: We report a case of severe hyperparathyroidism resulting from the ectopic production of PTH by a pancreatic malignancy. Cells from the primary tumor (PEPP1 cells) were established in culture to examine the etiology of ectopic PTH gene expression in this patient. RESULTS AND CONCLUSIONS: We failed to find amplification or rearrangement of the PTH gene but documented hypomethylation of the PTH promoter in tumor tissue. We found that PEPP1 cells support expression of a reporter gene containing regulatory sequences from the human PTH gene promoter. Therefore, this is the first report documenting ectopic PTH production by a tumor as the result of transactivation of the PTH gene. PEPP1 cells may be useful for future studies aimed at elucidating the details of PTH gene regulation.
Asunto(s)
Hiperparatiroidismo/metabolismo , Tumores Neuroendocrinos/metabolismo , Neoplasias Pancreáticas/metabolismo , Glándulas Paratiroides/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/biosíntesis , Proteína Relacionada con la Hormona Paratiroidea/genética , Anciano , Metilación de ADN , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Resultado Fatal , Femenino , Expresión Génica , Humanos , Hipercalcemia/tratamiento farmacológico , Hipercalcemia/genética , Hipercalcemia/metabolismo , Hiperparatiroidismo/tratamiento farmacológico , Hiperparatiroidismo/genética , Tumores Neuroendocrinos/genética , Neoplasias Pancreáticas/genética , Glándulas Paratiroides/fisiología , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Activación TranscripcionalRESUMEN
Providing enough calcium for milk production stresses calcium homeostasis in lactating mammals. A universal response to these demands for calcium appears to be the mobilization of maternal skeletal reserves, and bone loss during lactation has been well documented. However, the regulation of calcium and skeletal metabolism during lactation remains enigmatic. Our study was designed to examine mineral and bone metabolism in lactating mice. We found that mice lose bone rapidly at all sites during lactation. Bone mineral density as determined by dual-energy x-ray absorptiometry was 20 to 30% lower at the spine, femur, and total body in lactating compared with either age-matched virgin or pregnant mice. The decrease in bone mineral density was accompanied by dramatic reductions in bone volume and changes in trabecular architecture. Bone loss was also accompanied by increases in bone turnover as determined by biochemical markers and histomorphometry. PTHrP levels were elevated during lactation and correlated positively with markers of bone resorption and negatively with bone mass at all sites. Estrogen levels were low during lactation and correlated negatively with bone resorption markers. Finally, estrogen and pamidronate treatment lowered rates of bone resorption to baseline virgin levels and mitigated, but did not prevent, bone loss. These data suggest that the combination of estrogen deficiency and elevations in circulating PTHrP during lactation act to stimulate bone resorption and promote bone loss.
Asunto(s)
Resorción Ósea/fisiopatología , Estrógenos/sangre , Lactancia/fisiología , Hormona Paratiroidea/sangre , Animales , Resorción Ósea/sangre , Resorción Ósea/tratamiento farmacológico , Calcio/metabolismo , Difosfonatos/farmacología , Estrógenos/farmacología , Femenino , Ratones , Ratones EndogámicosRESUMEN
Normal breast epithelial cells and breast cancer cells express the calcium-sensing receptor (CaSR), the master regulator of systemic calcium metabolism. During lactation, activation of the CaSR in mammary epithelial cells downregulates parathyroid hormone-related protein (PTHrP) levels in milk and in the circulation, and increases calcium transport into milk. In contrast, in breast cancer cells the CaSR upregulates PTHrP production. A switch in G-protein usage underlies the opposing effects of the CaSR on PTHrP expression in normal and malignant breast cells. During lactation, the CaSR in normal breast cells coordinates a feedback loop that matches the transport of calcium into milk and maternal calcium metabolism to the supply of calcium. A switch in CaSR G-protein usage during malignant transformation converts this feedback loop into a feed-forward cycle in breast cancer cells that may promote the growth of osteolytic skeletal metastases.
Asunto(s)
Mama/metabolismo , Lactancia/metabolismo , Glándulas Mamarias Humanas/metabolismo , Receptores Sensibles al Calcio/metabolismo , Animales , Células Epiteliales/metabolismo , Femenino , Humanos , Glándulas Mamarias Animales/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Receptores Sensibles al Calcio/genética , Transducción de Señal/fisiologíaRESUMEN
The sites that receive ligament and tendon insertions (entheses) on the cortical surfaces of long bones are poorly understood, particularly regarding modeling and regulation. Entheses are classified as either fibrocartilaginous or fibrous based on their structures. Fibrous entheses typically insert into the metaphysis or diaphysis of a long bone, bear a periosteal component, and are modeled during long-bone growth. This modeling forms a root system by which the insertions attach to the cortical surface. In the case of the medial collateral ligament, modeling drives actual migration of the ligament along the cortical surface in order to accommodate linear growth, whereas in other sites modeling may excavate a deep cortical root system (eg, the teres major insertion) or a shallow root system with a large footprint (eg, the latissimus dorsi insertion). We report here that conditionally deleting parathyroid hormone-related protein (PTHrP) in fibrous entheses via Scleraxis-Cre targeting causes modeling to fail in these three iterations of osteoclast-driven enthesis excavation or migration. These iterations appear to represent formes frustes of a common modeling strategy, presumably differing from each other as a consequence of differences in biomechanical control. In sites in which PTHrP is not induced, either physiologically or because of conditional deletion, modeling does not take place and fibrocartilage is induced. These findings represent the initial genetic evidence that PTHrP regulates periosteal/intramembranous bone cell activity on cortical bone surfaces and indicate that PTHrP serves as a load-induced modeling tool in fibrous insertion sites during linear growth.
Asunto(s)
Desarrollo Óseo/fisiología , Fibrocartílago/crecimiento & desarrollo , Modelos Biológicos , Proteína Relacionada con la Hormona Paratiroidea/fisiología , Animales , RatonesRESUMEN
Despite the dramatic bone loss that occurs during lactation, bone mineral density rapidly recovers after offspring are weaned and milk production stops. The goal of this study is to quantify site-specific changes in bone quantity and quality during and after lactation in a mouse model. We used micro computed tomography (µCT), individual trabecula segmentation (ITS), digital topological analysis (DTA)-based tissue mineral density (TMD) analysis, and micro finite element analysis (µFEA) to quantify the effects of lactation and weaning on bone microarchitecture, mineralization, and stiffness at the spine, tibia, and femur. We found a significant decrease in trabecular plate microarchitecture, tissue mineralization of the trabecular surface, trabecular central skeleton, and intervening envelopes, and whole bone stiffness in lactating versus nulliparous mice at all three sites. In recovered mice, all these different aspects of bone quality were comparable to nulliparous mice at the spine. In contrast, trabecular plate microarchitecture and whole bone stiffness at the tibia and femur in recovered mice were lower than nulliparous mice, as were central trabecular tissue mineralization and cortical structure at the femur. These findings are consistent with clinical observations of partial recovery of femoral bone mineral density BMD after lactation in humans. The observed differences in trabecular surface tissue mineralization in nulliparous, lactating, and recovered mice are consistent with prior observations that maternal bone turnover shifts from resorption to formation at the time of pup weaning. The significant differences in trabecular central tissue mineralization during these three states suggest that osteocytes may contribute to the reversible loss of mineral during and after lactation. Future studies are necessary to determine whether differing functions of various bone cells at individual skeletal sites cause site-specific skeletal changes during and after lactation.
Asunto(s)
Huesos/anatomía & histología , Huesos/fisiología , Calcificación Fisiológica/fisiología , Lactancia/fisiología , Destete , Animales , Fenómenos Biomecánicos/fisiología , Densidad Ósea/fisiología , Huesos/diagnóstico por imagen , Femenino , Análisis de Elementos Finitos , Imagenología Tridimensional , Ratones , Microtomografía por Rayos XRESUMEN
The time-honored paradigm for mammary gland transepithelial calcium transport into milk is centered on the view that most, if not all, calcium enters milk through the secretory pathway, and no ionic calcium directly crosses the apical plasma membrane. Data from several recent studies all strongly suggest that most calcium, in fact, is extruded across the apical plasma membrane directly by the plasma membrane calcium-ATPase isoform 2 (PMCA2). In this review we break down transcellular calcium transport into the tasks of calcium entry, calcium sequestration and compartmentalization, and calcium extrusion. We compare and contrast the steps of calcium transport into milk by mammary epithelial cells, and the specific molecules that might perform these tasks, with well-characterized calcium transport mechanisms in other epithelia, such as the kidney, small intestine, and salivary gland. Finally, we suggest an updated model for calcium transport into milk that incorporates calcium transport across the apical plasma membrane.
Asunto(s)
Calcio/metabolismo , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Humanas/metabolismo , Animales , Transporte Biológico , Humanos , Modelos BiológicosRESUMEN
Calcium is an important nutrient that is secreted into milk in quantities that put a considerable stress upon maternal calcium homeostasis. Here we summarize the evidence that two important entities, the extracellular calcium-sensing receptor (CaR) and parathyroid hormone-related protein (PTHrP) are involved in a feedback loop that regulates calcium fluxes to the mammary gland. The CaR may also play a role in regulating milk secretion, and may regulate the proliferation of normal and neoplastic mammary epithelial cells. Finally, the relationship between the CaR and PTHrP in breast cancer cells may promote the formation of osteolytic bone metastases.