RESUMEN

Estrogen has been closely associated with the genesis and malignant progression of breast cancer. However, the molecular mechanism underlying the effects of estrogen is far from being completely clarified. We previously developed a custom-made cDNA microarray consisting of approximately 200 estrogen-responsive genes in breast cancer cells. Using this system, we found one estrogen-induced gene in various cancer cell lines, including breast cancer MCF-7 cells, which encode a zinc-finger transcription factor, EGR3 (early growth response 3). Northern blot analysis of estradiol-treated MCF-7 cells showed rapid and robust induction of Egr3, and addition of cycloheximide or ICI 182,780 suggested that Egr3 is the bona fide target for the estrogen receptor alpha (ERalpha). Using stable transformants derived from MCF-7 cells which were transfected with expression-controllable Egr3-expression vector, we demonstrated that Nab2 is one of the target genes for EGR3. Microarray analysis of the transformants revealed other candidate EGR3-induced genes. These strategies could be useful for analyzing downstream genes of ERalpha, and may contribute to elucidating the extensive signaling network of estrogen stimuli. Furthermore, a reporter assay using the upstream region of fasL probably involving escape from the immune system revealed that fasL is another target gene for EGR3. The roles of EGR3 in the physiology of breast cancer are discussed.

Asunto(s)

Neoplasias de la Mama/metabolismo , Estrógenos/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Cicloheximida/metabolismo , Proteína 3 de la Respuesta de Crecimiento Precoz , Antagonistas de Estrógenos/metabolismo , Receptor alfa de Estrógeno/metabolismo , Proteína Ligando Fas , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Inhibidores de la Síntesis de la Proteína/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo

RESUMEN

The overexpression of estrogen receptor alpha (ERalpha) is frequently observed in the early stage of breast cancer. We previously reported that the specific promoter of the ERalpha gene is responsible for this enhanced transcription of the gene, and identified the cis-acting elements which play an important role in its transcription. Furthermore, methylation of the ERalpha gene promoters also contribute to the regulation of gene transcription. Elucidation of these mechanisms of ERalpha gene expression may provide useful information for the early detection and chemoprevention of breast cancer. On the other hand, the expression of ERbeta has been reported in breast cancer. We have also assessed the significance and function of ERbeta and its variant types in breast cancer, and suggest that ERbeta and ERbetacx specifically suppress the function of ERalpha through different mechanisms. ERbeta isoforms may be important functional modulators of the estrogen-signaling pathway in breast cancer cells, and might affect the clinical outcome of patients. Moreover, to address the role of these ERs on the estrogen-dependent growth of breast cancer cells and to develop a diagnostic tool, we have analyzed the gene expression profiles of estrogen-responsive genes using cDNA microarray. Based on these results, the expression of several candidate genes in breast cancer tissues were analyzed by real-time RT-PCR and by immunohistochemical techniques, in order to discover new predictive factors for the endocrine therapy of patients with breast cancer. These studies could provide new clues for the elucidation of the estrogen-dependent mechanisms of cancer and the clinical benefits for patients.

Asunto(s)

Neoplasias de la Mama/metabolismo , Receptores de Estrógenos/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Ciclina D1/biosíntesis , Ciclina D1/genética , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Femenino , Humanos , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo

RESUMEN

The higher frequency of human lung adenocarcinoma in females than in males, strongly suggests the involvement of gender dependent factors in the etiology of this disease. This is the first investigation of estrogen receptor (ER) beta in human lung. Immunohistochemical staining revealed ERbeta expression in normal lung and in atypical adenomatous hyperplasia (AAH), considered as a precancerous lesion for adenocarcinomas. Adenocarcinomas showed significantly higher expression of ERbeta than squamous cell carcinomas. On the contrary, ERalpha expression was not detected in all cases. The functional integrity of ERbeta such as the binding ability to estrogen responsive element (ERE) and transcriptional activity was confirmed using a human lung cancer cell line, RERF-LC-OK. Colony formation of this cell was significantly reduced in the presence of pure antiestrogen. We conclude that ERbeta, but not ERalpha, is present in lung tissues with an important physiological function in normal lung. Furthermore, ERbeta may play a role in growth and development of adenocarcinomas.

Asunto(s)

Neoplasias Pulmonares/patología , Receptores de Estrógenos/fisiología , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma/fisiopatología , Anciano , Bronquios/citología , Bronquios/fisiología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/fisiopatología , Adhesión Celular , Receptor beta de Estrógeno , Femenino , Genes Reporteros , Humanos , Hiperplasia , Pulmón/citología , Pulmón/patología , Pulmón/fisiología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatología , Masculino , Persona de Mediana Edad , Receptores de Estrógenos/análisis , Receptores de Estrógenos/genética , Valores de Referencia , Mucosa Respiratoria/citología , Mucosa Respiratoria/fisiología , Transfección , Células Tumorales Cultivadas

RESUMEN

Hemopoietic cells develop in a complex milieu that is made up of diverse components, including stromal cells. Wnt genes, which are known to regulate the fate of the cells in a variety of tissues, are expressed in hemopoietic organs. However, their roles in hemopoiesis are not well characterized. In this study, we examined the roles of Wnt proteins in hemopoiesis using conditioned medium containing Wnt-3a. This conditioned medium dramatically reduced the production of B lineage cells and myeloid lineage cells, except for macrophages in the long-term bone marrow cultures grown on stromal cells, although the sensitivity to the conditioned medium differed, depending on the hemopoietic lineage. In contrast, the same conditioned medium did not affect the generation of B lineage or myeloid lineage cells in stromal cell-free conditions. These results suggested that Wnt proteins exert their effects through stromal cells. Indeed, these effects were mimicked by the expression of a stabilized form of beta-catenin in stromal cells. In this study, we demonstrated that Wnt signaling regulates hemopoiesis through stromal cells with selectivity and different degrees of the effect, depending on the hemopoietic lineage in the hemopoietic microenvironment.

Asunto(s)

Hematopoyesis/fisiología , Proteínas/fisiología , Transducción de Señal/fisiología , Células del Estroma/fisiología , Animales , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/fisiología , Línea Celular , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Proteínas del Citoesqueleto/biosíntesis , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/fisiología , Femenino , Hematopoyesis/genética , Células L , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Proteínas/genética , Transducción de Señal/genética , Células del Estroma/metabolismo , Resistencia a la Tetraciclina/genética , Transactivadores/genética , Transfección , Proteínas Wnt , Proteína Wnt3 , Proteína Wnt3A , beta Catenina

RESUMEN

In a panel of four human melanoma cell lines, equitoxic doses of cisplatin induced the proapoptotic conformation of the Bcl-2 family protein Bak prior to the execution phase of apoptosis. Because cisplatin-induced modulation of the related Bax protein was seen in only one cell line, a degree of specificity in the signal to Bak is indicated. Little is known about upstream regulation of Bak activity. In this study, we examined whether the apoptosis-specific pathway mediated by a kinase fragment of MEKK1 (DeltaMEKK1) is involved in the observed Bak modulation. We report that expression of a kinase-inactive fragment of MEKK1 (dominant negative MEKK [dnMEKK]) efficiently blocked cisplatin-induced modulation of Bak and cytochrome c release and consequently also reduced DEVDase activation and nuclear fragmentation. Accordingly, expression of a kinase-active MEKK1 fragment (dominant positive MEKK) was sufficient to induce modulation of Bak in three cell lines and to induce apoptosis in two of these. dnMEKK did not block cisplatin-induced c-Jun N-terminal kinase (JNK) activation, in agreement with a specifically proapoptotic role for the DeltaMEKK1 pathway. Finally, we show that reduction of Bak expression by antisense Bak reduced cisplatin-induced loss of mitochondrial integrity and caspase cleavage activity in breast cancer cell lines. In summary, we have identified Bak as a cisplatin-regulated component downstream in a proapoptotic, JNK-independent DeltaMEKK1 pathway.

Asunto(s)

Antineoplásicos/metabolismo , Apoptosis , Cisplatino/metabolismo , Quinasa 1 de Quinasa de Quinasa MAP , Proteínas de la Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Antineoplásicos/farmacología , Cisplatino/farmacología , Grupo Citocromo c/metabolismo , Activación Enzimática , Humanos , Proteína Quinasa 8 Activada por Mitógenos , Proteína Quinasa 9 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Células Tumorales Cultivadas , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína X Asociada a bcl-2

RESUMEN

Osteoclasts are derived from hematopoietic stem cells, but the relationship between osteoclast precursors (OCPs) and hematopoietic colony-forming cells (CFCs) has not yet been clarified. Although osteoclasts share certain cell surface markers and growth factor requirements with their macrophage and monocyte cell lineages, osteoclasts are a different lineage with regard to the requirement for signaling via c-Kit. To investigate whether CFCs are able to differentiate into osteoclasts, we performed in vitro studies of osteoclastogenesis. We performed progenitor assays in the presence of hematopoietic colony-stimulating factors. Primary colonies were plucked and examined for their potential to differentiate into osteoclasts. We found that osteoclasts are present in colonies elicited by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kB ligand (RANKL) in semisolid cultures. Moreover, a part of the cells composing the colonies elicited by granulocyte-macrophage colony-stimulating factor (GM-CSF) or M-CSF alone possessed the potential to differentiate into osteoclasts. These OCPs in the colonies were enriched in the c-Fms+ large-sized cell fraction and had a foamy cell morphology, like mature macrophages. A small number of cells in M-CSF-promoted and GM-CSF-promoted colonies formed secondary colonies in the semisolid medium containing these factors. The frequency of OCPs in these secondary colonies elicited by M-CSF was 10 times higher than that elicited by GM-CSF. Multiple origins of OCPs that differentiate into mature osteoclasts are proposed based on the observation that osteoclasts could be generated from OCPs that emerged from CFCs induced under different conditions or developmental stages.

Asunto(s)

Células de la Médula Ósea/efectos de los fármacos , Proteínas Portadoras/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/farmacología , Glicoproteínas de Membrana/farmacología , Osteoclastos/citología , Animales , Biomarcadores , Células de la Médula Ósea/citología , Resorción Ósea , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Células Cultivadas/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Sinergismo Farmacológico , Femenino , Citometría de Flujo , Células Espumosas/citología , Células Madre Hematopoyéticas/clasificación , Células Madre Hematopoyéticas/citología , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-kit/análisis , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Receptor de Factor Estimulante de Colonias de Macrófagos/análisis

RESUMEN

Skin is the major tissue where melanocytes develop, and skin keratinocytes provide the necessary micro-environment for melanocyte survival, proliferation, differentiation, and migration. In this paper, we will discuss the ligands for receptor tyrosine kinases produced as environmental cues to support melanocyte development in the skin.

Asunto(s)

Melanocitos/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Piel/metabolismo , Animales , Senescencia Celular/fisiología , Endotelina-3/fisiología , Factor de Crecimiento de Hepatocito/fisiología , Humanos , Ligandos , Melanoma/fisiopatología , Transducción de Señal/fisiología , Factor de Células Madre/fisiología

RESUMEN

We examined the effects of tranilast on tumor angiogenesis, tumor growth and metastasis in the mouse Lewis lung carcinoma and C57BL mouse system. Tranilast significantly reduced the dense capillary network induced by Lewis lung cancer cells in a mouse dorsal air sac angiogenesis model. Intraperitoneal administration of tranilast at 200 mg/kg/day reduced the tumor size of mouse Lewis lung carcinoma to about 63% of that of the control and suppressed pulmonary metastasis in a spontaneous system. Immunohistochemistry revealed that tranilast reduced the tumor vascularity and increased apoptosis of the tumor cells in vivo. Tranilast potentiated the inhibition of the tumor growth induced by cyclophosphamide, cis-diamminedichloroplatinum(II), adriamycin and vindesine in vivo. These results suggest that tranilast has antiangiogenic and antitumor effects and might have possible therapeutic applications.

Asunto(s)

Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/uso terapéutico , Carcinoma/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , ortoaminobenzoatos/uso terapéutico , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma/irrigación sanguínea , Carcinoma/patología , Cisplatino/farmacología , Cisplatino/uso terapéutico , Ciclohexanos , Ciclofosfamida/farmacología , Ciclofosfamida/uso terapéutico , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , O-(Cloroacetilcarbamoil) Fumagilol , Sesquiterpenos/farmacología , Sesquiterpenos/uso terapéutico , Células Tumorales Cultivadas/efectos de los fármacos , Vindesina/farmacología , Vindesina/uso terapéutico , ortoaminobenzoatos/farmacología

RESUMEN

The cDNA for a novel member of the FGFR family, named HrFGFR, was isolated from a Halocynthia roretzi cDNA library prepared at the mid-tailbud stage. This cDNA was 3507b long, and the deduced amino acid sequence contained a motif characteristic of the vertebrate FGFRs. The existence of a single copy of the FGFR homologue gene in H. roretzi was suggested by restriction site analysis of multiple clones. HrFGFR mRNA was expressed strongly in the posterior region in the epidermis from the middle neurula stage. By contrast, Xenopus FGFR homologues are expressed in the anterior region and are known to induce anterior neural formation. A transition of the region expressing FGFR might have induced the more complicated brain or head formation characteristic of vertebrates.

Asunto(s)

Receptores de Factores de Crecimiento de Fibroblastos/genética , Urocordados/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Hibridación in Situ , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Xenopus

RESUMEN

OBJECTIVE: Osteoclasts are of hematopoietic origin. The mechanism by which hematopoietic stem cells are specified to the osteoclast lineage is unclear. To understand the process of generation and differentiation of this lineage of cells, we performed in vitro studies on the differentiation of embryonic stem cells. MATERIALS AND METHODS: We examined the potential of mutant embryonic stem cell lines harboring targeted deletions of the GATA-1, FOG, SCL/tal-1, or GATA-2 genes to differentiate into osteoclasts and determined when these molecules function in osteoclast development. RESULTS: The lack of GATA-1 or FOG did not affect osteoclastogenesis. In contrast, SCL/tal-1-null embryonic stem cells generated no osteoclasts. In the case of the loss of GATA-2, a small number of osteoclasts were generated. GATA-2-null osteoclasts were morphologically normal and the terminal maturation was not disturbed, but a defect was observed in the generation of osteoclast progenitors. Experiments using specific inhibitors that block the signaling through macrophage colony-stimulating factor and osteoclast differentiation factor/osteoprotegerin ligand suggested that GATA-2 seems to act earlier in osteoclastogenesis than these cytokines. Interestingly, macrophage colony-forming units were not severely reduced by the loss of GATA-2 compared to osteoclast progenitors. CONCLUSION: These results indicate that osteocalsts need SCL/tal-1 at an early point in development, and that GATA-2 is required for generation of osteoclast progenitors but not for the later stages when macrophage colony-stimulating factor and osteoclast differentiation factor/ osteoprotegerin ligand are needed. We also demonstrated that osteoclast progenitors behave as a different population than macrophage colony-forming units.

Asunto(s)

Proteínas Portadoras/fisiología , Proteínas de Unión al ADN/fisiología , Factor Estimulante de Colonias de Macrófagos/fisiología , Glicoproteínas de Membrana/fisiología , Osteoclastos/fisiología , Proteínas Proto-Oncogénicas , Factores de Transcripción/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular , Línea Celular , Ensayo de Unidades Formadoras de Colonias , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Factor de Transcripción GATA2 , Secuencias Hélice-Asa-Hélice , Humanos , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Proteína 1 de la Leucemia Linfocítica T Aguda , Factores de Tiempo

RESUMEN

Preclinical studies in animal models and early results of clinical trials in patients suggest that intramuscular injection of naked plasmid DNA encoding vascular endothelial growth factor (VEGF) can promote neovascularization of ischemic tissues. Such neovascularization has been attributed exclusively to sprout formation of endothelial cells derived from preexisting vessels. We investigated the hypothesis that VEGF gene transfer may also augment the population of circulating endothelial progenitor cells (EPCs). In patients with critical limb ischemia receiving VEGF gene transfer, gene expression was documented by a transient increase in plasma levels of VEGF. A culture assay documented a significant increase in EPCs (219%, P<0.001), whereas patients who received an empty vector had no change in circulating EPCs, as was the case for volunteers who received saline injections (VEGF versus empty vector, P<0.001; VEGF versus saline, P<0.005). Fluorescence-activated cell sorter analysis disclosed an overall increase of up to 30-fold in endothelial lineage markers KDR (VEGF receptor-2), VE-cadherin, CD34, alpha(v)beta(3), and E-selectin after VEGF gene transfer. Constitutive overexpression of VEGF in patients with limb ischemia augments the population of circulating EPCs. These findings support the notion that neovascularization of human ischemic tissues after angiogenic growth factor therapy is not limited to angiogenesis but involves circulating endothelial precursors that may home to ischemic foci and differentiate in situ through a process of vasculogenesis.

Asunto(s)

Células Sanguíneas/citología , Factores de Crecimiento Endotelial/genética , Endotelio Vascular/citología , Técnicas de Transferencia de Gen , Linfocinas/genética , Células Madre/citología , Adulto , Anciano , Moléculas de Adhesión Celular/metabolismo , División Celular/fisiología , Células Cultivadas , Endotelio Vascular/metabolismo , Femenino , Citometría de Flujo , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Transgenes/fisiología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Using the epidermis-specific cytokeratin 14 promoter to deliver HGF exclusively from epidermal keratinocytes, we have examined the potential of hepatocyte growth factor (HGF) secreted from the normal environment to control morphogenesis. The transgenic mice displayed a significant increase of the number of melanocytes and their precursors in embryos starting not later than 16.5 dpc, and then after birth an explosive increase of dermal melanocytes started within 1 week, and these melanocytes were maintained throughout the entire life of the mice. Thus, HGF acts as a paracrine agent to promote survival, proliferation and differentiation of melanocyte precursors in vivo, and eventually causes melanocytosis. Loss of E-cadherin expression in dermal melanocyte precursors suggests that HGF caused dermal localization of melanocytes and their precursors by down-regulation of E-cadherin molecules.

Asunto(s)

Factor de Crecimiento de Hepatocito/genética , Queratinocitos/fisiología , Melanocitos/fisiología , Enfermedades de la Piel/genética , Animales , Animales Recién Nacidos , Cadherinas/genética , Cadherinas/metabolismo , Oído Externo , Regulación del Desarrollo de la Expresión Génica , Factor de Crecimiento de Hepatocito/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Queratinas/genética , Melanocitos/patología , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-kit/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Piel/embriología , Piel/crecimiento & desarrollo , Piel/patología , Enfermedades de la Piel/patología , Pigmentación de la Piel/genética , Factor de Células Madre/metabolismo

RESUMEN

Oncogenic and anti-apoptotic Bcl-2 is expressed much less in estrogen receptor alpha (ERalpha) negative breast cancers, which show more malignant phenotypes, than ERalpha-positive, indicating that some other Bcl-2 family member(s) are involved in the apoptotic balance of the cancer cells. We first analyzed mRNA expression of pro-apoptotic Bak and Bax along with that of anti-apoptotic Bcl-2 and Bcl-xL, using breast cancer specimens of 27 patients. Bak mRNA was expressed much less in ERalpha negative breast cancers, along with reduced expression of Bcl-2. Immunostaining of sections of 108 patients confirmed the observation. Next, stable transformants of MCF-7 cells with sense Bak expression vector showed fewer colonies in soft agar compared with the parental cells, while stable introduction of antisense Bak vector enhanced colony formation at lower estradiol concentrations. The reduction of Bak may play important roles in malignant development of breast cancer to acquire estrogen independency, counteracting the reduced Bcl-2.

Asunto(s)

Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Genes bcl-2 , Proteínas de la Membrana/genética , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/metabolismo , Receptores de Estrógenos/metabolismo , Secuencia de Bases , Neoplasias de la Mama/patología , Cartilla de ADN/genética , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Proteínas de la Membrana/metabolismo , Familia de Multigenes , Neoplasias Hormono-Dependientes/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Transformación Genética , Células Tumorales Cultivadas , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína X Asociada a bcl-2 , Proteína bcl-X

RESUMEN

Adult bone marrow is a major site for hematopoiesis, and reduction of the bone marrow cavity induces hematopoiesis in extramarrow tissues. To investigate the rudimentary intramarrow and the compensatory extramarrow hematopoiesis, particularly B lymphopoiesis, we used 3 osteopetrotic mouse strains [op/op, mi/mi, and Fos (-/-)], which are severely deficient in functional osteoclasts and therefore form inadequate bone marrow cavities. We found that bone marrow in these osteopetrotic mice supports myelopoiesis but not B lymphopoiesis, although cells that have the potential to differentiate into B lineage cells are present in the bone marrow. Although B lymphopoiesis normally occurs both in the spleen and liver of newborn mice, compensatory B lymphopoiesis in adult op/op and mi/mi mice is observed only in the liver, while myelopoiesis is enhanced in both organs. Interestingly, mice lacking the Fos proto-oncogene exhibit B lymphopoiesis in the spleen as well as liver. The amounts of expression of steel factor, Flt3/Flk-2 ligand, and interleukin-7 in the bone marrow, spleen, or liver were not significantly affected in these osteopetrotic mutants. These findings suggest that the volume of the bone marrow cavity regulates B lymphopoiesis without affecting the production of certain hematopoietic growth factors. The splenic microenvironments that support both myelopoiesis and B lymphopoiesis in the neonatal stage are lost in adults and are not reactivated even in the osteopetrotic adults unless the Fos gene is disrupted.

Asunto(s)

Linfocitos B/fisiología , Células de la Médula Ósea/citología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Osteopetrosis/fisiopatología , Envejecimiento , Animales , Linfocitos B/citología , Linfocitos B/patología , Células de la Médula Ósea/patología , Células de la Médula Ósea/fisiología , Genes fos , Hematopoyesis/genética , Células Madre Hematopoyéticas/patología , Células Madre Hematopoyéticas/fisiología , Heterocigoto , Homocigoto , Interleucina-7/genética , Hígado/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteopetrosis/genética , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-fos/deficiencia , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Bazo/inmunología , Factor de Células Madre/genética , Tirosina Quinasa 3 Similar a fms

RESUMEN

The 26S proteasome is a eukaryotic ATP-dependent protease, but the molecular basis of its energy requirement is largely unknown. Ornithine decarboxylase (ODC) is the only known enzyme to be degraded by the 26S proteasome without ubiquitinylation. We report here that the 26S proteasome is responsible for the irreversible inactivation coupled to sequestration of ODC, a process requiring ATP and antizyme (AZ) but not proteolytic activity. Neither the 20S proteasome (catalytic core) nor PA700 (the regulatory complex) by itself contributed to this ODC inactivation. Analysis with a C-terminal mutant ODC revealed that the 26S proteasome recognizes the C-terminal degradation signal of ODC exposed by attachment of AZ, and subsequent ATP-dependent sequestration of ODC in the 26S proteasome causes irreversible inactivation, possibly unfolding, of ODC and dissociation of AZ. These processes may be linked to the translocation of ODC into the 20S proteasomal inner cavity, centralized within the 26S proteasome, for degradation.

Asunto(s)

Adenosina Trifosfato/metabolismo , Ornitina Descarboxilasa/metabolismo , Péptido Hidrolasas/metabolismo , Cisteína Endopeptidasas , Lactonas/farmacología , Modelos Biológicos , Complejos Multienzimáticos , Inhibidores de la Ornitina Descarboxilasa , Complejo de la Endopetidasa Proteasomal , Unión Proteica , Proteínas/farmacología

RESUMEN

Osteoclasts are derived from hematopoietic stem cells and their development is dependent on the products of stromal cells. CD9, a member of the tetraspan transmembrane-superfamily, is expressed on both hematopoietic cells and stromal cells. Addition of antagonistic rat anti-mouse CD9 antibody (KMC8.8) to cultures inhibited osteoclastogenesis on established stromal cell layers. When rat bone marrow cells depleted of adherent stromal cells were cultured on mouse stromal cells, numerous tartrate-resistant acid phosphatase-positive multinuclear cells were observed, and KMC8.8, which recognizes mouse but not rat CD9, completely prevented the generation of osteoclasts, suggesting that the CD9 expressed on the stromal cell is essential for osteoclastogenesis. Possibly for the same reason, KMC8.8 pretreatment of the mouse macrophage-like cell line C7, which is able to differentiate into mature osteoclasts, did not inhibit subsequent C7 cell differentiation, whereas the addition of KMC8.8 to cocultures of C7 cells with stromal cells inhibited the differentiation of C7 cells into osteoclasts. Moreover, we found that blockage of a signal via CD9 on stromal cells reduced transcription of the osteoclast differentiation factor (Odf) gene, which, together with macrophage colony-stimulating factor, is essential for osteoclastogenesis. These results revealed that CD9 molecules on stromal cells play a critical role in osteoclast development, possibly by modulating the expression of Odf.

Asunto(s)

Antígenos CD4/inmunología , Osteoclastos/citología , Células del Estroma/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Antígenos CD4/análisis , Calcitriol/farmacología , Proteínas Portadoras/genética , División Celular , Técnicas de Cocultivo , Dexametasona/farmacología , Regulación de la Expresión Génica/inmunología , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Osteoclastos/inmunología , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Células del Estroma/efectos de los fármacos

RESUMEN

Mice homozygous for the motheaten (Hcph(me)) mutation lack a functional SHP-1 protein tyrosine phosphatase, show severe immunologic dysregulation and die at an early age. Severe pneumonitis in me/me mice is associated with abnormal proliferation of macrophages and granulocytes. Overgrowth of macrophages in long term cultures of me/me bone marrow has prevented analyses of lymphopoiesis in vitro. To establish hematopoietic cell lines from me/me mice, we cultured me/me bone marrow with the PA6 stromal cell line in the presence of antagonistic antibody against the receptor (c-Fms) for macrophage colony stimulating factor (M-CSF). In these cultures, overgrowth of M-CSF-dependent macrophages was suppressed by the antagonistic antibody and other hemopoietic cell lineages were generated efficiently from me/me bone marrow. By using this culture system, we established me/me pro-B cell clones (MEBs) with rearranged DH-JH but not VH-DJH. The growth of MEB clones required IL-7 and c-Kit ligand, corresponding to normal pro-B cells which express SHP-1. MEB cells were sensitive to starvation by either IL-7 or c-Kit ligand, resulting in apoptotic death. The present culture system, which supports hematopoiesis of me/me bone marrow, provides useful tools for the determination of the role of SHP-1 in signal transduction of B lymphopoiesis.

Asunto(s)

Linfocitos B/citología , Linfocitos B/enzimología , Proteínas Tirosina Fosfatasas/genética , Animales , Apoptosis/fisiología , Linfocitos B/metabolismo , Células de la Médula Ósea/citología , Ciclo Celular/fisiología , Línea Celular , Femenino , Reordenamiento Génico de Cadena Pesada de Linfocito B , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Interleucina-7/farmacología , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Endogámicos C3H , Ratones SCID , Mutación , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Proteínas Proto-Oncogénicas c-kit/farmacología , Transducción de Señal/fisiología

RESUMEN

Because hepatocyte growth factor (HGF) is a member of the endothelium-specific growth factors, we hypothesized that HGF may play a role in cardiovascular disease. Therefore we first examined the role of local HGF production in endothelial cell (EC) growth. Addition of anti-HGF antibody to EC resulted in a significant decrease in EC number. Moreover, coculture of vascular smooth muscle cells (VSMC) with EC resulted in an increase in EC number that was completely inhibited by anti-HGF antibody, suggesting that HGF secreted from EC and VSMC regulates EC growth in an autocrine-paracrine manner. Interestingly, transforming growth factor (TGF)-ss significantly decreased HGF secretion from EC, whereas interleukin 6 stimulated immunoreactive HGF secretion. In human VSMC, TGF-ss and angiotensin II suppressed local HGF production in a dose-dependent manner. Interestingly, anti-TGF-beta antibody resulted in significant but not complete inhibition of the decrease in local HGF production. To further study the regulation of local HGF production, we used a coculture system. Coculture of VSMC with EC resulted in a significant decrease in local HGF secretion. The decrease in local HGF production by coculture was significantly attenuated by anti-TGF-beta antibody, suggesting that inhibition of local HGF production in the coculture system was due to TGF-beta activation. Moreover, a further decrease in local HGF production in the coculture system by angiotensin II was also observed. Finally, we studied the role of angiotensin II in the regulation of the local HGF system in vivo by using a balloon injury rat model. Of importance, local HGF production was significantly decreased in balloon-injured arteries compared with intact vessels, accompanied by a reduction of HGF mRNA. An angiotensin-converting enzyme inhibitor (cilazapril) or an angiotensin II type 1 receptor antagonist (E-4177) significantly stimulated local vascular HGF production associated with the inhibition of neointimal formation after balloon injury compared with vehicle. In contrast, hydralazine did not alter local HGF production or neointimal formation despite decreasing blood pressure to a similar level as that in rats treated with cilazapril or E-4177. Overall, local HGF secretion from vascular cells was negatively regulated by TGF-beta and angiotensin II. The present study also demonstrated that blockade of angiotensin II significantly inhibited neointimal formation, accompanied by a significant increase in local vascular HGF production in vivo in the balloon injury model. Given the strong mitogenic activity of HGF on endothelial cells, increased local HGF production by blockade of angiotensin II may enhance reendothelialization after balloon injury. Downregulation of the local vascular HGF system by TGF-beta and vascular angiotensin may play an important role in the pathogenesis of cardiovascular diseases.

Asunto(s)

Angiotensina II/fisiología , Endotelio Vascular/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Músculo Liso Vascular/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Angiotensina II/farmacología , Animales , Comunicación Autocrina/efectos de los fármacos , Enfermedades Cardiovasculares/etiología , Arterias Carótidas/efectos de los fármacos , Traumatismos de las Arterias Carótidas , Células Cultivadas , Citocinas/farmacología , Endotelio Vascular/efectos de los fármacos , Factor de Crecimiento de Hepatocito/sangre , Factor de Crecimiento de Hepatocito/fisiología , Humanos , Masculino , Músculo Liso Vascular/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Ratas , Ratas Endogámicas WKY , Factor de Crecimiento Transformador beta/farmacología , Vasoconstrictores/farmacología

RESUMEN

Osteoclasts are hematopoietic cells essential for bone remodeling and resorption. To understand the process of osteoclast development, we established a macrophage-like cell line C7 that has the potential to differentiate into functional osteoclasts; multinuclear tartrate-resistant acid phosphatase positive cells capable of forming pits on dentin slices. C7 cells share the characteristics of their cell surface molecules and phagocytic activity with macrophages. Generation of osteoclasts from C7 cells was mostly suppressed by the addition of a function-blocking antibody directed to c-Fms, the receptor for macrophage-colony stimulating factor (M-CSF), or by osteoclastogenesis inhibitory factor (OCIF). These responses correspond well with the osteoclast precursors present in bone marrow and peritoneal cavity. Reagents such as bacterial lipopolysaccharide and granulocyte/macrophage-CSF that are known to act as inducers for other cell lineages rather than osteoclasts abolished the potential of osteoclastogenesis in C7 cells. These phenotypes of C7 cells have been stably maintained for more than 2 years. We believe that the cell line established in this study will provide an important tool for osteoclast biology.

Asunto(s)

Diferenciación Celular/fisiología , Macrófagos/metabolismo , Osteoclastos/metabolismo , Receptores Citoplasmáticos y Nucleares , Anticuerpos/inmunología , Anticuerpos/farmacología , Células de la Médula Ósea/metabolismo , División Celular , Línea Celular , Citometría de Flujo , Glicoproteínas/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Sustancias de Crecimiento/farmacología , Lipopolisacáridos/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Osteoclastos/citología , Osteoprotegerina , Fenotipo , Receptor de Factor Estimulante de Colonias de Macrófagos/inmunología , Receptores del Factor de Necrosis Tumoral , Proteínas Recombinantes

RESUMEN

Human T-cell lymphotropic virus type I (HTLV-I) is associated with various clinical disorders including adult T cell leukemia, myelopathy, arthropathy. Hypercalcemia resulting from osteoclast activation and a variety of hematopoietic abnormalities have been also observed in HTLV-I infected patients, however, precise mechanism about initial trigger(s) prior to presenting symptoms is still unknown. In this study, to assess effects of HTLV-I on hematopoiesis, we analysed characteristics of early hematopoietic precursors in HTLV-I env-pX transgenic rats. Progenitor cells for osteoclasts were significantly increased even in the marrow of asymptomatic env-pX rats. Progenitors for B cells were also highly enriched, while colony forming cells (CFC) elicited by GM-CSF(CFU-GM) and M-CSF(CFU-M) were comparable to normal littermates. Following arthritis in env-pX transgenic rats, osteoclastogenesis was further augmented and the CFCs were increased. Bone marrow cells carrying adjuvant-induced arthritis retained a constant number of progenitors for osteoclast and B lymphocytes, whereas the number of CFU-GM and CFU-M increased. These results indicate that the env-pX transgene affect early stages of osteoclast and B-cell lineages prior to developing diseases, in contrast, an increase of the CFCs was caused indirectly by arthritis. This study provides a novel standpoint for the mechanisms of pathogenesis by HTLV-I.

Asunto(s)

Linfocitos B/citología , Células de la Médula Ósea/citología , Leucopoyesis , Osteoblastos/citología , Proteínas Oncogénicas de Retroviridae/metabolismo , Factores de Transcripción , Proteínas del Envoltorio Viral/metabolismo , Animales , Animales Modificados Genéticamente , Artritis Experimental , Diferenciación Celular , Células Cultivadas , Adyuvante de Freund/farmacología , Células Madre Hematopoyéticas/citología , Humanos , Masculino , Ratas , Ratas Endogámicas Lew , Ratas Wistar , Proteínas Oncogénicas de Retroviridae/genética , Proteínas del Envoltorio Viral/genética , Proteínas Reguladoras y Accesorias Virales