RESUMEN
Parathyroid hormone-related protein (PTHrP) has been implicated in many processes during normal and pathological pregnancies. In the human fetal membranes, PTHrP exhibits cytokine-like actions. We have recently shown that inhibitors of the nuclear factor-kappa B (NF-kappaB) and activators of the peroxisome proliferator-activated receptor (PPAR)-gamma signalling pathways down-regulate cytokine release from human gestational tissues. Therefore, the aim of this study was to determine whether NF-kappaB and PPAR-gamma also regulate PTHrP release from human fetal membranes. Human amnion and choriodecidua explants were incubated in the absence (control) or presence of two known NF-kappaB inhibitors (1, 5 and 10 mM sulphasalazine (SASP) or 5, 10 and 15 mM N-acetyl-cysteine (NAC)), and two PPAR-gamma ligands (15 and 30 microM 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) or 15 and 30 microM troglitazone), under basal conditions. After 18 h incubation, the tissues were collected and NF-kappaB p65 DNA binding activity in nuclear extracts was assessed by ELISA, and the incubation medium was collected and the release of PTHrP was quantified by RIA. Treatment of amnion and choriodecidual tissues with SASP concentrations greater than 5 mM, 15 mM NAC, 30 microM 15d-PGJ(2) and 30 microM troglitazone significantly reduced the release of PTHrP (p < 0.05). This study demonstrates that PTHrP release from human fetal membranes is regulated by inhibitors of NF-kappaB, and ligands of PPAR-gamma.
Asunto(s)
Amnios/efectos de los fármacos , Corion/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , PPAR gamma/farmacología , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Prostaglandina D2/análogos & derivados , Acetilcisteína/farmacología , Adulto , Amnios/metabolismo , Células Cultivadas , Corion/metabolismo , Cromanos/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Factores Inmunológicos/farmacología , Ligandos , Embarazo , Prostaglandina D2/farmacología , Sulfasalazina/farmacología , Tiazolidinedionas/farmacología , TroglitazonaRESUMEN
Parathyroid hormone-related protein (PTHrP) has important roles in fetal growth and development through stimulation of placental calcium transport, vasodilatation of the uteroplacental vasculature and regulation of cellular growth and differentiation. The growth restricted spontaneously hypertensive rat (SHR) has reduced fetal plasma, placental and amniotic fluid PTHrP concentrations compared to its progenitor, the Wistar Kyoto (WKY) rat. The aim of this study was to determine whether intrauterine PTHrP infusions can restore PTHrP levels and promote SHR fetal growth. PTHrP(1-34), midmolecule PTHrP(67-94), the PTH/PTHrP receptor antagonist [Asn(10), Leu(11)]-PTHrP(7-34) or vehicle were infused via a mini-osmotic pump between 10 and 20 days of gestation into the uterine lumen of SHR and WKY rats. Uterine, placental, amniotic fluid and plasma (fetal and maternal) PTHrP were measured via N-terminal radioimmunoassay. PTH/PTHrP receptor antagonism and mid-molecule PTHrP(67-94) induced endogenous intrauterine PTHrP production with receptor antagonism eliciting a greater and more wide spread effect. The PTH/PTHrP receptor antagonist [Asn(10), Leu(11)]-PTHrP(7-34) acting through a receptor other than the PTH/PTHrP receptor increased SHR fetal and placental weights above vehicle (P<0.05) to that of the WKY and restored SHR amniotic fluid volume (P<0.05). This was associated with a highly significant up regulation of placental, uterine and plasma (fetal and maternal) PTHrP (P<0.05). Modest increases in placental and uterine PTHrP (P<0.05) following intrauterine infusions of PTHrP(1-34) and PTHrP(67-94) had no effect on WKY and SHR fetal weight. Effective growth promoting actions of increased endogenous PTHrP were observed following PTH/PTHrP receptor antagonism rather than exogenous PTHrP administration. A novel finding was that mid-molecule PTHrP also up regulates endogenous intrauterine N-terminal PTHrP production supporting the existence of a mid-molecule receptor. This study highlights that an increase in endogenous uterine, placental and fetal plasma PTHrP following PTH/PTHrP receptor antagonism was associated with increased SHR fetal growth presumably by improving placental growth and function.
Asunto(s)
Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Hormona Paratiroidea/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Útero/efectos de los fármacos , Líquido Amniótico , Animales , Desarrollo Embrionario y Fetal/efectos de los fármacos , Desarrollo Embrionario y Fetal/fisiología , Femenino , Peso Fetal , Hormona Paratiroidea/antagonistas & inhibidores , Fragmentos de Péptidos/administración & dosificación , Placenta , Embarazo , Proteínas/administración & dosificación , Radioinmunoensayo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptor de Hormona Paratiroídea Tipo 1/antagonistas & inhibidores , Útero/metabolismoRESUMEN
Evidence implicates pivotal roles for parathyroid hormone-related protein (PTHrP) during lactation, including stimulation of mammary and pup growth. As spontaneously hypertensive rat (SHR) pups are growth restricted compared with the control Wistar Kyoto (WKY), we examined the relative roles of pup suckling and maternal lactational environment on pup growth, mammary PTHrP, and milk PTHrP and calcium concentrations. SHR pups were lighter compared with the control from 6 days. SHR mammary PTHrP content and milk PTHrP were lower but maternal plasma PTHrP was raised compared with WKY. SHR mammary morphological development was also impaired compared with control. Cross fostering growth-restricted pups onto WKY mothers increased pup weight in association with normal mammary function and higher milk PTHrP and calcium. Control pups suckling on an SHR mother had reduced body weight. Both cross fostering groups were associated with increased maternal and milk PTHrP concentrations, indicating the importance of suckling, together with a functional mammary gland. The results suggested that impaired SHR mammary function and milk PTHrP are associated with a reduced SHR postnatal growth. Our data also indicated that milk and mammary PTHrP are regulated by different mechanisms but that they are influenced by the maternal lactational environment and the suckling pup.
Asunto(s)
Trastornos del Crecimiento/metabolismo , Lactancia/fisiología , Glándulas Mamarias Animales/metabolismo , Hormonas Peptídicas/metabolismo , Animales , Animales Lactantes , Calcio/análisis , Calcio/sangre , Femenino , Masculino , Glándulas Mamarias Animales/anatomía & histología , Leche/química , Proteína Relacionada con la Hormona Paratiroidea , Hormonas Peptídicas/sangre , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
IL-18 was identified due to its ability to induce interferon-gamma (IFNgamma) production by T cells. It is a pleiotropic factor that shares structural features with IL-1 and functional activities with IL-12. IL-18 has a role in T cell development, where it has been demonstrated to act cooperatively with IL-12 to regulate IFNgamma. In bone, IL-18 is mainly produced by macrophages, but is also expressed by osteoblasts and inhibits osteoclast formation through granulocyte-macrophage colony-stimulating factor (GM-CSF) and not IFNgamma production by T cells. We have investigated the effects of IL-18 on mature osteoclast activity and for potential actions on osteoblasts or chondrocytes. The effects of IL-18 on mature osteoclast activity were determined using two assays: isolated mature osteoclast cell culture and neonatal murine calvarial organ culture. IL-18 did not affect bone resorption in either assay system. The actions of IL-18 on osteogenic cells (primary cell cultures of fetal rat and neonatal mouse osteoblasts, as well as neonatal mouse calvarial organ culture) and primary chondrocytes (canine) were assessed by proliferation assays (quantification of cell numbers and thymidine incorporation). In each assay system, IL-18 acted as a mitogen to the osteogenic and chondrogenic cells. Since IL-18 signal transduction may involve IFNgamma or GM-CSF, we assessed their involvement in the IL-18 response. IL-18 did not induce IFNgamma production by primary osteoblasts, but, of greater significance, IFNgamma had the opposing action to IL-18 in that it inhibited the primary osteoblast cell proliferation. Although IL-18 rapidly induced GM-CSF production by primary osteoblasts, IL-18 was still mitogenic in osteoblast preparations established from GM-CSF-deficient mice. Combined, these studies indicate that IL-18 may have an autocrine/paracrine mitogen role for both osteogenic and chondrogenic cells, independent of the production of IFNgamma or GM-CSF.
Asunto(s)
Condrocitos/citología , Interleucina-18/farmacología , Mitógenos/farmacología , Osteoclastos/citología , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Interferón gamma/genética , Ratones , Técnicas de Cultivo de Órganos , Osteoclastos/efectos de los fármacos , Osteoclastos/fisiología , Ratas , Ratas WistarRESUMEN
Intrauterine parathyroid hormone-related protein (PTHrP) concentrations are reduced in association with growth restriction in the spontaneously hypertensive rat (SHR) compared to those of its normotensive control, the Wistar Kyoto (WKY) rat, implicating PTHrP as a pivotal fetal growth factor. The aim of this study was to examine, by embryo cross-transplanation between SHR and WKY, whether the mother, fetus, or both, are responsible for the suppressed SHR amniotic fluid PTHrP. One-day-old SHR embryos were gestated in either an SHR (SHR-in-SHR) or WKY (SHR-in-WKY) surrogate, similarly one-day-old WKY embryos were gestated in either an SHR (WKY-in-SHR) or WKY (WKY-in-WKY) mother. At 20 days gestation, maternal plasma and amniotic fluid samples were collected and assayed for PTHrP concentrations. Data were analysed by two-way ANOVA (mean+/-sem, n=5-9 mothers/group). There were no differences in litter number or maternal plasma PTHrP concentrations. Fetal weight (P< 0.009), fetal/placental weight ratio (P< 0.004) and amniotic fluid PTHrP concentrations (P< 0.001) were lower and amniotic fluid volume (P< 0.0001) was higher with an SHR fetus compared to the WKY fetus irrespective of maternal strain. Thus, the SHR fetus is growth restricted and has suppressed amniotic fluid PTHrP, which are largely determined by the fetus or gestational tissues and are independent of maternal hypertension or maternal PTHrP. We suggest that the low SHR amniotic fluid PTHrP may play a role in the development of SHR growth restriction.
Asunto(s)
Líquido Amniótico/química , Enfermedades Fetales/metabolismo , Retardo del Crecimiento Fetal/etiología , Hipertensión/complicaciones , Hipertensión/metabolismo , Proteínas/análisis , Amnios/patología , Líquido Amniótico/fisiología , Animales , Presión Sanguínea , Transferencia de Embrión , Membranas Extraembrionarias/patología , Femenino , Edad Gestacional , Tamaño de los Órganos , Proteína Relacionada con la Hormona Paratiroidea , Placenta/patología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Útero/patologíaRESUMEN
We used Hoxa3 knockout mice and other mouse models to study the role of the fetal parathyroids in fetal calcium homeostasis. Hoxa3-null fetuses lack parathyroid glands, and absence of parathyroid hormone (PTH) was confirmed with a rodent PTH immunoradiometric assay. The ionized calcium level of Hoxa3-null fetuses was significantly lower than that of wild-type or heterozygous littermates or of the mother. Both the rate of placental calcium transfer and the plasma PTHrP level were normal in Hoxa3 mutants and their heterozygous siblings. Because we had previously observed an increase in placental calcium transfer in PTH/PTHrP receptor 1-null (Pthr1-null) fetuses, we assayed plasma PTHrP in those mice. Pthr1-null fetuses had plasma PTHrP levels 11-fold higher than those of their littermates. Northern analysis, immunohistochemical, and in situ hybridization studies of Pthr1-null fetuses indicated that liver and placenta had increased expression of PTHRP: In summary, loss of fetal parathyroids in Hoxa3-null fetuses caused marked hypocalcemia but did not alter placental calcium transfer or the circulating PTHrP level. The findings in the Pthr1-null fetuses indicate that several tissues may contribute to the circulating PTHrP level in fetal mice.
Asunto(s)
Calcio/metabolismo , Glándulas Paratiroides/fisiología , Hormona Paratiroidea/metabolismo , Placenta/metabolismo , Animales , Transporte Biológico , Calcitonina/metabolismo , Proteínas de Homeodominio/genética , Ratones , Ratones Noqueados , Glándulas Paratiroides/metabolismo , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/genética , Proteínas/metabolismo , Receptor de Hormona Paratiroídea Tipo 1 , Receptores Sensibles al Calcio , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Hormona Paratiroidea/genética , Receptores de Hormona Paratiroidea/metabolismo , Distribución TisularRESUMEN
OBJECTIVE: Parathyroid hormone-related protein has roles in normal fetal growth, placental calcium transport, and vascular tone regulation; these factors are compromised in growth-restricted fetuses. Our objective was to determine whether intrauterine parathyroid hormone-related protein expression was increased in association with fetal growth restriction. STUDY DESIGN: The expression of parathyroid hormone-related protein was examined in intrauterine tissues from women with idiopathic fetal growth restriction with preterm (n = 8-10) and term (n = 8-10) gestations and from gestation-matched control subjects. The abundance and immunoreactive content of parathyroid hormone-related protein messenger ribonucleic acid were determined by Northern blot and radioimmunoassay, respectively, in the placenta, amnion, and chorion-decidua. RESULTS: The expression of parathyroid hormone-related protein messenger ribonucleic acid was increased in the amnion (placental and reflected) in association with preterm fetal growth restriction (P <.05). Both parathyroid hormone-related protein messenger ribonucleic acid and protein expression were increased in chorion-decidua in association with preterm fetal growth restriction (P <.05). In term gestations both parathyroid hormone-related protein messenger ribonucleic acid and protein expression were greater in amnion over placenta than in reflected amnion (P <.05); these in turn were greater than those in chorion-decidua (P <.05). No significant changes were detected in parathyroid hormone-related protein messenger ribonucleic acid or in protein expression in association with term fetal growth restriction. CONCLUSION: Either parathyroid hormone-related protein messenger ribonucleic acid or protein expression, or both, was increased in the fetal membranes in association with fetal growth restriction in preterm but not term gestations, suggesting that parathyroid hormone-related protein may be involved in the pathogenesis of preterm fetal growth restriction.
Asunto(s)
Membranas Extraembrionarias/química , Retardo del Crecimiento Fetal/metabolismo , Proteínas/genética , Amnios/química , Peso al Nacer , Northern Blotting , Corion/química , Decidua/química , Femenino , Edad Gestacional , Humanos , Tamaño de los Órganos , Proteína Relacionada con la Hormona Paratiroidea , Placenta/anatomía & histología , Placenta/química , Embarazo , ARN Mensajero/análisis , RadioinmunoensayoRESUMEN
The placental syncytiotrophoblast is the site for mineral and nutrient exchange across the maternal-fetal interface. It has been proposed that parathyroid hormone-related protein (PTHrP) is a key factor in the maintenance of a maternal-fetal calcium gradient. Using simultaneously prepared microvillous (maternal facing) and basal (fetal facing) syncytiotrophoblast membranes from term human placentae (n=8), we determined the relative contribution of PTH(1-34), PTHrP(1-34) and PTHrP(67-94) to the regulation of syncytiotrophoblast calcium efflux. The vesicles had correct right-side-out membrane orientation and specific markers validated the fractionation of microvillous and basal membrane vesicles. Calcium efflux was studied by preloading vesicles with calcium-45 in the presence of calcium and magnesium and then incubating the vesicles at 37 degrees C for 15 min with the peptides. In basal membranes, PTHrP(1-! 34) significantly stimulated calcium efflux at a dose of 12.5 nmol/l, whereas PTH(1-34)-stimulated efflux was significant at 50 nmol/l (P<0.05, ANOVA). This efflux was significantly reduced in the presence of the PTH/PTHrP receptor antagonist (PTHrP(7-34)). Midmolecule PTHrP(67-94) had no significant effect on basal membrane calcium efflux. PTH(1-34), PTHrP(1-34) or PTHrP(67-94) had no significant effects on MVM calcium efflux. This study, using the human syncytiotrophoblast in vitro membrane system, demonstrated that PTHrP(1-34) and PTH(1-34) stimulate calcium transport across the basal, but not microvillous, syncytiotrophoblast membrane vesicles, mediated via the PTH/PTHrP receptor.
Asunto(s)
Calcio/metabolismo , Fragmentos de Péptidos/farmacología , Placenta/metabolismo , Proteínas/farmacología , Receptores de Hormona Paratiroidea/metabolismo , Teriparatido/farmacología , Calcio/farmacología , Técnicas de Cultivo , Humanos , Magnesio/farmacología , Proteína Relacionada con la Hormona Paratiroidea , Receptores de Hormona Paratiroidea/antagonistas & inhibidores , Estimulación QuímicaRESUMEN
The parathyroid glands are the only known source of circulating parathyroid hormone (PTH), which initiates an endocrine cascade that regulates serum calcium concentration. Glial cells missing2 (Gcm2), a mouse homologue of Drosophila Gcm, is the only transcription factor whose expression is restricted to the parathyroid glands. Here we show that Gcm2-deficient mice lack parathyroid glands and exhibit a biological hypoparathyroidism, identifying Gcm2 as a master regulatory gene of parathyroid gland development. Unlike PTH receptor-deficient mice, however, Gcm2-deficient mice are viable and fertile, and have only a mildly abnormal bone phenotype. Despite their lack of parathyroid glands, Gcm2-deficient mice have PTH serum levels identical to those of wild-type mice, as do parathyroidectomized wild-type animals. Expression and ablation studies identified the thymus, where Gcm1, another Gcm homologue, is expressed, as the additional, downregulatable source of PTH. Thus, Gcm2 deletion uncovers an auxiliary mechanism for the regulation of calcium homeostasis in the absence of parathyroid glands. We propose that this backup mechanism may be a general feature of endocrine regulation.
Asunto(s)
Neuropéptidos/fisiología , Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/biosíntesis , Transactivadores/fisiología , Animales , Huesos/metabolismo , Huesos/patología , Calcio/metabolismo , Cruzamientos Genéticos , Proteínas de Unión al ADN , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Ratones , Ratones Endogámicos C57BL , Neuropéptidos/deficiencia , Neuropéptidos/genética , Glándulas Paratiroides/anomalías , Glándulas Paratiroides/embriología , Hormona Paratiroidea/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre , Timo/metabolismo , Transactivadores/deficiencia , Transactivadores/genética , Factores de TranscripciónRESUMEN
Evidence implicates pivotal roles for parathyroid hormone-related protein (PTHrP) in stimulating cell growth and differentiation, placental calcium transport, and placental vasodilatation. As spontaneously hypertensive rat (SHR) fetuses are growth restricted compared with those of its normotensive control, the Wistar Kyoto (WKY) rat, we examined intrauterine PTHrP and total and ionic calcium concentrations in these rats. Fetal plasma PTHrP concentrations, but not total calcium concentrations, were lower in the SHR compared with WKY (P < 0.05). SHR placental concentrations of PTHrP were lower than in WKY (P < 0.03) and failed to show the increase observed in WKY near term (P < 0.05). PTHrP concentrations in amniotic fluid from SHR were not raised near term and were lower compared with WKY (P < 0.0005). The increased ionic calcium concentrations in amniotic fluid in the WKY near term (P < 0.05) were not detected in the SHR. Thus SHR fetal plasma, placental, and amniotic fluid PTHrP concentrations were reduced and associated with fetal growth restriction. We suggest that PTHrP may play a role in the etiology of both growth restriction during pregnancy and hypertension later in life.
Asunto(s)
Membranas Extraembrionarias/metabolismo , Sangre Fetal/metabolismo , Retardo del Crecimiento Fetal/metabolismo , Hipertensión/metabolismo , Placenta/metabolismo , Complicaciones Cardiovasculares del Embarazo/metabolismo , Proteínas/metabolismo , Líquido Amniótico/metabolismo , Animales , Calcio/sangre , Membranas Extraembrionarias/anatomía & histología , Membranas Extraembrionarias/química , Membranas Extraembrionarias/citología , Femenino , Edad Gestacional , Masculino , Tamaño de los Órganos , Proteína Relacionada con la Hormona Paratiroidea , Placenta/anatomía & histología , Placenta/química , Embarazo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Útero/química , Útero/metabolismoRESUMEN
Parathyroid hormone-related protein (PTHrP) is present in fetal and gestational tissues, in which its proposed roles include stimulation of epithelial growth and differentiation, vasodilatation of the uteroplacental vasculature, relaxation of uterine muscle and stimulation of placental calcium transport. The aim of this study was to determine whether the release of PTHrP from gestational tissue explants was tissue specific. In addition, PTHrP concentrations were measured in maternal plasma, umbilical artery and vein plasma, and amniotic fluid from term, uncomplicated pregnancies before the onset of labour. PTHrP was detected in low concentrations in the mother, fetus and placental tissue. Amniotic fluid had ten times the PTHrP concentration compared with that in the maternal or fetal circulations. Using late pregnant human gestational tissues in an in vitro explant system, we found that amnion over placenta, choriodecidua, reflected amnion, and placenta released PTHrP into culture medium in progressively greater amounts over 24 h (P<0.05). This release was not associated with a loss of cell membrane integrity, as indicated by measurement of the intracellular enzyme, lactate dehydrogenase, in the incubation media. After 24 h incubation, the fetal membranes released significantly (P<0.05) greater amounts of PTHrP than did the placenta (placenta 3. 7+/-0.5 pmol PTHrP/g protein). Amnion over placenta released significantly more PTHrP (139.3+/- 43.1 pmol PTHrP/g protein) than did reflected amnion (29.0+/-8.3 pmol PTHrP/g protein) (P<0.05). This study unequivocally demonstrated that human gestational tissues release PTHrP and it was concluded that the main contributors to PTHrP in amniotic fluid were the human fetal membranes, particularly amnion over placenta. Fetal membrane-derived and amniotic fluid PTHrP are proposed to have stimulatory effects on epithelial growth and differentiation in fetal lung, gut, skin and hair follicles and paracrine effects on placental vascular tone and calcium transport.
Asunto(s)
Líquido Amniótico/metabolismo , Feto/metabolismo , Proteína Relacionada con la Hormona Paratiroidea , Fragmentos de Péptidos/metabolismo , Proteínas/metabolismo , Amnios/metabolismo , Corion/metabolismo , Técnicas de Cultivo , Decidua/metabolismo , Femenino , Humanos , Placenta/metabolismo , EmbarazoRESUMEN
Breast cancer is the most common malignancy in women, with a worldwide prevalence of 1.5 million throughout the industrialized countries. Its mortality rate is second only to lung cancer in the USA and Europe. Its high incidence and prevalence makes it a major public health problem. Up to one-third of women with early stage breast cancer will eventually succumb to the disease, and most of these will develop bone metastases during the course of the disease. Approximately 70% of patients with breast cancer have bone metastases, with 27% having lung and liver metastases. Hypercalcemia also is very common in advanced breast cancer, manifesting itself in one-third of patients late in the disease. Breast cancer accounts for approximately 25% of the cases of hypercalcemia in cancer. The major skeletal complications of breast cancer--hypercalcemia and bone metastases--almost certainly share certain mechanisms and these will be discussed.
Asunto(s)
Neoplasias Óseas/etiología , Neoplasias de la Mama/complicaciones , Hipercalcemia/etiología , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Citocinas/metabolismo , Femenino , Humanos , Hipercalcemia/metabolismo , Hipercalcemia/patología , Metaloproteinasas de la Matriz/metabolismo , Osteoclastos/fisiología , Proteína Relacionada con la Hormona Paratiroidea , Prostaglandinas/metabolismo , Proteínas/genética , Proteínas/metabolismoRESUMEN
Breast cancer affects approximately one woman in twelve and kills more women than any other cancer. If detected early, patients have a five year survival rate of 66%, but once metastatic disease has developed, there is no effective treatment. About 70% of patients with metastatic disease have bone involvement, while lungs and liver are the other common targets. Bone metastases cause severe pain, pathological fractures and hypercalcaemia and thus are a significant clinical problem. The development of new therapies for metastatic breast carcinoma depends on a better understanding of the mechanism of homing of the tumour cells to bone, liver and lungs and the factors required for their growth in these organs. Research on mechanisms of breast cancer metastasis, particularly to bone, has relied on in vitro studies or on tumour models in which the inoculation route is designed to promote delivery of tumour cells to a specific organ. Metastases in bone are achieved by inoculation into the right ventricle of the heart. To our knowledge there has been no report of a model of metastatic spread from the mammary gland to distant sites which reliably includes bone. In this paper, we describe our recent development of a novel murine model of metastatic breast carcinoma. The new model is unique in that the pattern of metastatic spread closely resembles that observed in human breast cancer. In particular, these murine breast tumours metastasise to bone from the primary breast site and cause hypercalcaemia, characteristics not normally found in murine tumours, but common in human disease. Furthermore, in a preliminary characterisation of this model, we show that secretion of parathyroid hormone-related protein, a role for which has been implicated in breast cancer spread to bone, correlates with metastasis to bone. This model therefore provides an excellent experimental system in which to investigate the factors that control metastatic spread of breast cancer to specific sites, particularly bone. The special advantage of this system is that it involves the whole metastasis process, beginning from the primary site. Existing models consider mechanisms that pertain to growth of tumour once the site has been reached. An understanding of the regulation of these factors by potential therapeutic agents could lead to improvement in therapies designed to combat metastatic disease. For the first time, this development will allow exploration of the molecular basis of site-specific metastasis of breast cancer to bone in a clinically relevant model.
Asunto(s)
Neoplasias Óseas/secundario , Neoplasias Mamarias Experimentales/patología , Proteínas/genética , Animales , Neoplasias Óseas/patología , Calcio/sangre , Femenino , Inmunohistoquímica , Infusiones Intravenosas , Queratinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/análisis , Radioinmunoensayo , Células Tumorales CultivadasRESUMEN
Parathyroid hormone (PTH)-related protein (107-139) (PTHrP(107-139)) and PTHrP(107-111) have been reported to be potent inhibitors of isolated osteoclast activity, and inhibition of bone resorption by PTHrP(107-139) occurs in vivo. However, the actions of C-terminal PTHrP on osteoblast activity has not been studied much. The present study addresses this issue by examining the effect of PTHrP(107-139), PTHrP(107-119), PTHrP(120-139), and PTHrP(107-111) on the proliferation of fetal rat osteoblasts. Treatment with PTHrP(107-139) for 24 h caused a dose-dependent increase in cell number, [3H]thymidine and [3H]phenylalanine incorporation in cultured osteoblasts. The effect was apparent at concentrations of 10-10 M and greater and was sustained over time. PTHrP(107-119) and PTHrP(107-111) had effects on cell number, DNA, and protein synthesis which were comparable to those of PTHrP(107-139), whereas PTHrP(120-139) was without effect. Retroverted PTHrP(107-111) also stimulated all three activities but was only one tenth as potent as PTHrP(107-139). PTHrP(107-139) had no effect on osteoblast apoptosis. It is concluded that PTHrP(107-139) is not only an inhibitor of osteoclastic bone resorption but that it also stimulates osteoblast growth. This activity resides within the pentapeptide fragment PTHrP(107-111). These findings support a possible role for C-terminal fragments of PTHrP in the normal regulation of bone cell function and, possibly, bone mass.
Asunto(s)
Osteoblastos/efectos de los fármacos , Hormona Paratiroidea , Fragmentos de Péptidos/farmacología , Proteínas/farmacología , Animales , División Celular/efectos de los fármacos , Osteoblastos/citología , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/química , Ratas , Estimulación QuímicaRESUMEN
Maternal hypertension, vasoconstriction and placental insufficiency are features of pre-eclampsia. Alterations in calcium homeostasis and in the production of calciotropic hormones and vasoactive agents have also been described in association with pre-eclampsia. Parathyroid hormone-related protein (PTHrP) is abundantly expressed in intrauterine tissues during normal pregnancy and has roles in fetal growth and calcium homeostasis, placental calcium transport and vascular tone regulation. Intrauterine PTHrP mRNA expression and tissue PTHrP content were determined by Northern blot analysis and radio-immunoassay, respectively, in preterm and term pre-eclamptic women. PTHrP mRNA expression and PTHrP content in placenta, amnion over placenta, reflected amnion and choriodecidua from preterm pre-eclamptic women (n=8-10) were not different from preterm controls (n= 10-12). PTHrP mRNA expression and content in amnion over placenta and reflected amnion were significantly greater in term compared to preterm pre-eclamptics (P<0.05). PTHrP mRNA expression was significantly lower in choriodecidua from term pre-eclamptic women (n=8) compared to term controls (n=28, P<0.05), but was not different in placenta or amnion. PTHrP content was not altered in term pre-eclamptic women (n=8) compared to controls (n=25) for any tissue. In summary, PTHrP expression in placenta and amnion was not increased in pre-eclamptic women in association with maternal hypertension, placental insufficiency and vasoconstriction. PTHrP mRNA expression was decreased in choriodecidua in association with term but not preterm pre-eclampsia, however, levels of the protein were not decreased. The data suggest that PTHrP is not involved in the placental pathophysiology of pre-eclampsia in late gestation.
Asunto(s)
Hormona Paratiroidea/metabolismo , Placenta/metabolismo , Preeclampsia/metabolismo , Embarazo/metabolismo , Proteínas/metabolismo , Adulto , Northern Blotting , Femenino , Edad Gestacional , Humanos , Hormona Paratiroidea/genética , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/genética , ARN Mensajero/biosíntesis , RadioinmunoensayoRESUMEN
Skeletal growth is the net product of coordinated bone formation and resorption. Insulin is known to stimulate bone formation by actions on osteoblasts. It is not known whether insulin receptors are present on osteoclasts, or whether insulin regulates osteoclastic function. We present here immunocytochemical evidence of insulin receptor expression by mature mono- and multinucleated murine osteoclast-like cells generated in vitro, and in primary neonatal rat and mouse osteoclasts. Radiolabeled studies indicated that progressive enrichment of osteoclast-like cells in coculture was associated with increased insulin binding. When osteoclast-like cells generated in vitro were plated onto dentine slices, insulin dose-dependently inhibited pit formation by up to 80%, suggesting a role for insulin in osteoclast function. These data are consistent with an effect of insulin on bone resorption in addition to those previously recognized on bone formation, actions that together result in net bone growth.
Asunto(s)
Insulina/farmacología , Osteoclastos/metabolismo , Receptor de Insulina/biosíntesis , Animales , Desarrollo Óseo/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Células Gigantes/efectos de los fármacos , Células Gigantes/metabolismo , Inmunohistoquímica , Ratones , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Osteoclastos/efectos de los fármacos , RatasRESUMEN
Parathyroid hormone-related protein (PTHrP) was detected at 32.8 +/- 3.9 pmol 1-1 in uterine luminal fluid from immature rats treated with oestradiol. As mRNA encoding PTHrP has previously been localized to implantation sites in pregnant rats, the role of luminal PTHrP during pregnancy was explored. Infusion of a parathyroid hormone (PTH)/PTHrP receptor antagonist, [Asn10,Leu11]PTHrP(7-34) amide, into the uterine lumen during pregnancy in rats resulted in excessive decidualization. This effect was also observed after intrauterine infusion of a monoclonal antibody raised against PTHrP. The effect of infusion of PTH/PTHrP receptor antagonist was dependent upon successful implantation, was dose-dependent and confined to the treated horn. A decrease in the number of apoptotic decidual cells in antagonist-infused uterine horns compared with vehicle or non-infused horns was detected immunohistochemically at day 13 of pregnancy, and this decrease is likely to contribute to the 'over-decidualization' observed. In pseudopregnant rats, infusion of PTH/PTHrP receptor antagonist into the uterine lumen resulted in an increase in uterine wet weight of the infused horn compared with the non-infused horn, indicating a direct effect on deciduoma formation. Thus, activation of the PTH/PTHrP receptor by locally produced PTHrP appears to be crucial for normal decidualization during pregnancy in rats.
Asunto(s)
Decidua/efectos de los fármacos , Antagonistas de Hormonas/farmacología , Proteína Relacionada con la Hormona Paratiroidea , Hormona Paratiroidea/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología , Receptores de Hormona Paratiroidea/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Decidua/crecimiento & desarrollo , Femenino , Inmunohistoquímica , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Útero/anatomía & histologíaRESUMEN
Using immunohistology with two specific antisera raised against N-terminal parathyroid hormone-related protein (PTHrP) and in situ hybridization (riboprobe to common coding exon), evidence is provided for the expression of PTHrP by mouse, rabbit, and human osteoclasts derived from several in vitro and in vivo sources. In cocultures of mouse bone marrow and calvarial cells treated with 1,25-dihydroxyvitamin D3, the generated osteoclasts expressed both PTHrP messenger RNA (mRNA) and protein. In addition, PTHrP was detected in the majority of actively resorbing osteoclasts in sections of newborn and adult mouse long bones. Using an in vivo intramembranous bone formation model in rabbits, expression of PTHrP mRNA and protein was demonstrated in osteoclasts at active bone resorption sites as well as in actively synthesizing osteoblasts and bone lining cells. Localization of PTHrP was also demonstrated in osteoclast-like cells of human giant cell tumors from bone. In some of these tumors, a small proportion of the multinucleated cells expressed tartrate resistant acid phosphatase (TRAP), but not PTHrP mRNA or protein. Finally, both mRNA and protein for PTHrP were expressed in osteoclasts in sections of bone or joints from patients with Paget's disease, rheumatoid arthritis, and osteoarthritis. These observations raise the possibility that PTHrP might participate in osteoclast function.
Asunto(s)
Osteoclastos/metabolismo , Hormona Paratiroidea/metabolismo , Proteínas/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Relacionada con la Hormona Paratiroidea , Conejos , Cráneo/citología , Cráneo/metabolismoRESUMEN
The structure of human parathyroid hormone (PTH) related protein (residues 1-34) containing an Ala substituted for an Ile in position 15 was studied by two-dimensional proton nuclear magnetic resonance spectroscopy. This mutant retains quite high levels of adenylate cyclase activity based on slightly reduced PTH receptor binding capacity. Three segments of helix were revealed extending from His5 to Lys11, Lys13 to Arg19, and from Phe22 to Thr33/Ala34, with a decided kink between the first two helices around Gly12. N- and C-terminal helices were stabilized by charged and hydrophobic side chain interactions between His5 and Glu30, Asp17 and both His9 and His25, and between Leu8 and Ala29, resulting in a globular molecule occupying a single conformation. While the structure of the entire mid-molecule region differed greatly from the structure of the native peptide, the structure of both N- and C-terminal regions remains essentially unaltered. The residues responsible for initiating signal transduction in the mutant are located in the vicinity of the residues responsible for receptor binding. The C-terminal amphipathic helix forming the receptor binding site exhibits reduced binding as a result of the closely applied N-terminal signal transduction-activating region. Although not contributing directly to receptor binding, the N-terminal region can sterically affect hormone binding through modifications to certain N-terminal side chains.
Asunto(s)
Proteínas de Neoplasias/química , Proteína Relacionada con la Hormona Paratiroidea , Fragmentos de Péptidos/química , Proteínas/química , Teriparatido/química , Alanina/química , Alanina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Humanos , Isoleucina/química , Isoleucina/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas/metabolismo , Receptores de Hormona Paratiroidea/metabolismo , Transducción de Señal , Soluciones , Relación Estructura-Actividad , Teriparatido/metabolismoRESUMEN
Parathyroid hormone-related protein (PTHrP) is highly expressed in normal skin keratinocytes, and its involvement in growth and differentiation processes in these cells has been implicated by several lines of evidence which include the use of antisense PTHrP (Kaiser et al., 1994, Mol. Endocrinol., 8:139-147). In this study, we have investigated whether PTHrP expression and its subcellular localization is linked to cell cycle progression in a human keratinocyte cell line (HaCat), which constitutively expresses and secretes PTHrP. PTHrP mRNA and immunoreactive PTHrP were assessed in asynchronous dividing cells and in cells blocked at G1 or G2 + M phases of the cell cycle using several different protocols. The response of PTHrP mRNA expression was examined following readdition of serum in the continued presence of cycle blockers, and after release from cell cycle block, or from cell synchronization by serum deprivation. PTHrP expression was greatest in actively dividing cells when cells were in S and G2 + M phases of the cell cycle and were lowest in quiescent G1 cells. Most notable were the high levels of PTHrP mRNA and protein in cells at G2 + M phase of the cell cycle at division. Furthermore, PTHrP was localized to the nucleolus in quiescent cells, but redistributed to the cytoplasm when cells were actively dividing. Taken together, these results support a role for PTHrP in cell division in keratinocytes. In asynchronously growing cells, PTHrP expression fell as cells became confluent at a time when cell growth is inhibited and cells begin to differentiate. Mitogen stimulation of HaCaT cells resulted in a rapid increase in PTHrP mRNA expression, but was dependent upon cells being in the G1 phase of the cell cycle. Cells blocked in G1 responded to mitogen both in the continued presence of aphidicolin or when released from block. Cells blocked at G2 + M with colcemid expressed high levels of PTHrP mRNA and protein, and PTHrP mRNA did not respond further to mitogen in the continued presence of blocker. However, in cells released from block at G2 + M by addition of serum, an increase in PTHrP expression was seen coincident with the progression of cells into G1. In contrast, in a squamous cancer cell line (COLO16), basal PTHrP expression was high and was not altered during the cell cycle or by cell cycle block, consistent with association of its dysregulated expression in malignant cells. The results of this study suggest that PTHrP may have two roles in the cell cycle; one in G1 in response to mitogen, and a second at cell division when its expression is high and it is relocated from the nucleolus to the cytoplasm.