RESUMEN
Proliferation of vascular smooth muscle cells (VSMC) represents a key event for the pathogenesis of postangioplasty restenosis. Minichromosome maintenance proteins (MCM) form essential components of the prereplicative complex at DNA replication origins and are regulated by E2F. The present studies were designed to investigate the signal transduction pathways controlling the expression of MCM6 and MCM7 in VSMC in response to mitogenic stimuli. MCM6 and MCM7 expression was substantially increased after stimulation with platelet-derived growth factor-BB and insulin. Pretreatment with PD98059, a specific inhibitor of the extracellular signal-regulated kinases (ERK)-mitogen-activated protein kinase (MAPK), competely inhibited the mitogen-induced MCM6 and MCM7 mRNA and protein expression, demonstrating a critical role for this pathway in transmitting transmembrane signals required for the initiation of DNA replication. The p38MAPK inhibitor SB203580, the phosphatidylinositol 3 kinase (PI3-kinase) pathway inhibitor wortmannin, and the protein kinase C pathway (PKC) inhibitor Gö 6976 did not significantly affect mitogen-induced MCM6 and MCM7 expression. Transient transfection experiments revealed that PD98059 inhibited mitogen-induced MCM6 and MCM7 transcriptional activation. In addition, blockade of ERK/MAPK signaling with PD98059 strongly inhibited phosphorylation of the retinoblastoma protein (Rb) and activity of a luciferase reporter plasmid driven by multiple E2F elements. Inhibition of mitogen-induced MCM6 and MCM7 expression by PD98059 was reversed by ectopic overexpression of E2F, indicating that ERK/MAPK signaling is required for events that occur upstream of E2F release from phosphorylated Rb. In combination, these data demonstrate that the ERK/MAPK signal transduction pathway plays a central role in regulating E2F-dependent MCM expression and DNA replication in VSMC.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Hiperplasia/enzimología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas Nucleares/metabolismo , Becaplermina , Proteínas de Ciclo Celular/genética , División Celular/fisiología , Células Cultivadas , Reestenosis Coronaria/enzimología , Reestenosis Coronaria/fisiopatología , Proteínas de Unión al ADN/genética , Factores de Transcripción E2F , Inhibidores Enzimáticos/farmacología , Oclusión de Injerto Vascular/enzimología , Oclusión de Injerto Vascular/fisiopatología , Humanos , Hiperplasia/fisiopatología , Insulina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Componente 6 del Complejo de Mantenimiento de Minicromosoma , Componente 7 del Complejo de Mantenimiento de Minicromosoma , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/genética , Mitógenos/farmacología , Músculo Liso Vascular/enzimología , Proteínas Nucleares/genética , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-sis , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiologíaRESUMEN
Peroxisome proliferator-activated receptor (PPAR) gamma is activated by thiazolidinediones (TZDs), widely used as insulin-sensitizing agents for the treatment of type 2 diabetes. TZDs have been shown to induce apoptosis in a variety of mammalian cells. In vascular smooth muscle cells (VSMCs), proliferation and apoptosis may be competing processes during the formation of restenotic and atherosclerotic lesions. The precise molecular mechanisms by which TZDs induce apoptosis in VSMCs, however, remain unclear. In the present study, we demonstrate that the TZDs rosiglitazone (RSG), troglitazone (TRO), and a novel non-TZD partial PPARgamma agonist (nTZDpa) induce caspase-mediated apoptosis of human coronary VSMCs. Induction of VSMC apoptosis correlated closely with an upregulation of growth arrest and DNA damage-inducible gene 45 (GADD45) mRNA expression and transcription, a well-recognized modulator of cell cycle arrest and apoptosis. Using adenoviral-mediated overexpression of a constitutively active PPARgamma mutant and the irreversible PPARgamma antagonist GW9662, we provide evidence that PPARgamma ligands induce caspase-mediated apoptosis and GADD45 expression through a receptor-dependent pathway. Deletion analysis of the GADD45 promoter revealed that a 153-bp region between -234 and -81 bp proximal to the transcription start site, containing an Oct-1 element, was crucial for the PPARgamma ligand-mediated induction of the GADD45 promoter. PPARgamma activation induced Oct-1 protein expression and DNA binding and stimulated activity of a reporter plasmid driven by multiple Oct-1 elements. These findings suggest that activation of PPARgamma can lead to apoptosis and growth arrest in VSMCs, at least in part, by inducing Oct-1-mediated transcription of GADD45. The full text of this article is available online at http://www.circresaha.org.
Asunto(s)
Músculo Liso Vascular/metabolismo , Proteínas/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Tiazolidinedionas , Factores de Transcripción/fisiología , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/efectos de los fármacos , Sitios de Unión/genética , Northern Blotting , Inhibidores de Caspasas , División Celular/efectos de los fármacos , Células Cultivadas , Cromanos/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Daño del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Factor C1 de la Célula Huésped , Humanos , Péptidos y Proteínas de Señalización Intracelular , Luciferasas/genética , Luciferasas/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Mutación , Factor 1 de Transcripción de Unión a Octámeros , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/agonistas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Rosiglitazona , Tiazoles/farmacología , Factores de Transcripción/agonistas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Troglitazona , Proteinas GADD45RESUMEN
Using a cDNA array consisting only of cell cycle genes, we found that a novel nonthiazolidinedione partial peroxisome proliferator-activated receptor gamma (PPARgamma) agonist (nTZDpa) inhibited expression of minichromosome maintenance (MCM) proteins 6 and 7 in vascular smooth muscle cells. MCM proteins are required for the initiation and elongation stages of DNA replication and are regulated by the transcription factor E2F. Mitogen-induced MCM6 and MCM7 mRNA expression was potently inhibited by nTZDpa and to a lesser degree by the full PPARgamma agonist, rosiglitazone. Inhibition of MCM6 and MCM7 expression by nTZDpa and rosiglitazone paralleled their effect to inhibit phosphorylation of the retinoblastoma protein and cell proliferation. Transient transfection experiments revealed that the nTZDpa inhibited mitogen-induced MCM6 and MCM7 promoter activity, implicating a transcriptional mechanism. Adenoviral-mediated E2F overexpression reversed the suppressive effect of nTZDpa on MCM6 and MCM7 expression. Furthermore, activity of a luciferase reporter plasmid driven by multiple E2F elements was inhibited by nTZDpa, indicating that their down-regulation by nTZDpa involves an E2F-dependent mechanism. Overexpression of dominant-negative PPARgamma or addition of a PPARgamma antagonist, GW 9662, blocked nTZDpa inhibition of MCM7 transcription. Adenovirus-mediated overexpression of constitutively active PPARgamma inhibited MCM7 expression in a similar manner as the nTZDpa. These findings provide strong evidence that activation of PPARgamma attenuates MCM7 transcription and support the important role of this nuclear receptor in regulating vascular smooth muscle cell proliferation.
Asunto(s)
Proteínas de Ciclo Celular/genética , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Indoles/farmacología , Músculo Liso Vascular/efectos de los fármacos , Proteínas Nucleares/genética , Receptores Citoplasmáticos y Nucleares/agonistas , Sulfuros/farmacología , Tiazolidinedionas/farmacología , Factores de Transcripción/agonistas , Activación Transcripcional/efectos de los fármacos , Animales , Aorta/citología , Proteínas de Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Técnicas In Vitro , Músculo Liso Vascular/metabolismo , Proteínas Nucleares/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Rosiglitazona , Fase S/efectos de los fármacosRESUMEN
Nearly one-half of all hypercalcemic patients with lymphoma present with inappropriately elevated circulating concentrations of the active vitamin D metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D3). However, the cellular source of the vitamin D hormone in lymphomas remains unclear. To address this, we report the case of a 75-year-old man with hypercalcemia associated with raised circulating concentrations of 1,25(OH)2D3 and suppressed parathyroid hormone (PTH) levels. Positron emission tomographic (PET) and computed tomographic (CT) imaging revealed the presence of a large lymphoma that was confined to the spleen; subsequent pathological analysis showed that this was an intermediate grade B-cell lymphoma. After surgical removal of the spleen, serum calcium and 1,25(OH)2D3 levels became normalized within 24 h. Immunolocalization of the vitamin D-activating enzyme 25-hydroxyvitamin D3-1alpha-hydroxylase (la-hydroxylase) in sections of resected spleen showed that staining was negative in the lymphoma cells but positive in neighboring macrophages. This case study indicates that the hypercalcemia associated with lymphomas may be due, in some instances, to excessive extrarenal production of 1,25(OH)2D3. Furthermore, by using immunohistochemistry to assess the distribution of la-hydroxylase, we have been able to show for the first time that tissue macrophages, rather than actual tumor cells, are the most likely ectopic source of this enzyme. Based on this case study, we propose that the abnormal synthesis of 1,25(OH)2D3 associated with some lymphomas is because of paracrine regulation of tumor-associated macrophages.
Asunto(s)
Hipercalcemia/inducido químicamente , Linfoma de Células B/complicaciones , Linfoma de Células B Grandes Difuso/complicaciones , Vitamina D/efectos adversos , Anciano , Humanos , Hipercalcemia/complicaciones , Linfoma de Células B/diagnóstico , Linfoma de Células B Grandes Difuso/diagnóstico , Masculino , Tomografía Computarizada de EmisiónRESUMEN
Rapamycin inhibits vascular smooth muscle cell (VSMC) proliferation and rapamycin-eluting stents represent a novel therapeutic strategy for preventing postangioplasty restenosis. The precise molecular mechanism, for rapamycin-mediated inhibition of VSMC cell cycle progression and DNA replication remain to be elucidated. Minichromosome maintenance proteins (MCM) are essential regulators of DNA replication and the objective of this study was to examine the effect of rapamycin on their expression in rat aortic VSMC. Rapamycin substantially inhibited mitogen-induced MCM6 and MCM7 mRNA and protein expression in a dose-dependent fashion. Transient transfection experiments revealed that rapamycin inhibited MCM6 and MCM7 promoter activity, implicating a transcriptional mechanism. MCM6 and MCM7 transcriptional activation is regulated by E2F and activity of a luciferase reporter plasmid driven by four E2F elements was also significantly inhibited by rapamycin. The inhibitory effect of rapamycin on MCM6 and MCM7 was reversed by overexpression of E2F, indicating that their downregulation by rapamycin involves an E2F-dependent mechanism. These observations suggest that rapamycin inhibits MCM6 and MCM7 expression by blocking their E2F-dependent transactivation which may contribute importantly to the inhibition of VSMC DNA synthesis by rapamycin.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Endotelio Vascular/citología , Músculo Liso/citología , Proteínas Nucleares/metabolismo , Sirolimus/farmacología , Factores de Transcripción/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Northern Blotting , Western Blotting , División Celular , Células Cultivadas , ADN/metabolismo , Regulación hacia Abajo , Factores de Transcripción E2F , Fosforilación , Regiones Promotoras Genéticas , ARN/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Serina/metabolismo , Activación Transcripcional , TransfecciónRESUMEN
We report a case of a virilized 59-yr-old woman with elevated serum testosterone levels and bilateral macronodular adrenal hyperplasia. The patient underwent laparoscopic right adrenalectomy, after which the elevated testosterone level transiently normalized. The immediate postoperative depression of the testosterone level suggested that the process was driven by gonadotropins that were suppressed by the stress of surgery. The excised right adrenal mass contained testosterone by immunohistochemistry and LH receptor mRNA by in situ hybridization. The recurrence of hyperandrogenemia suggested that the enlarged left adrenal was also secreting testosterone. The serum testosterone level increased in response to im injection of human chorionic gonadotropin, suggesting control by aberrant LH receptors. Injection of leuprolide acetate (7.5 mg im) to suppress LH levels resulted in normalization of the testosterone level 12 d later that persisted for several weeks. Ectopic receptors mediating Cushing's syndrome have been described in several cases of bilateral adrenal hyperplasia and adrenal adenoma. This is the first case to our knowledge in which pure androgen overproduction in adrenal hyperplasia has been shown to be controlled by LH receptors. In our patient, the control of androgen secretion by LH may explain the postmenopausal onset of virilization and the transient postoperative normalization of the serum testosterone level.
Asunto(s)
Enfermedades de las Glándulas Suprarrenales/complicaciones , Enfermedades de las Glándulas Suprarrenales/metabolismo , Hormona Luteinizante/metabolismo , Virilismo/etiología , Enfermedades de las Glándulas Suprarrenales/tratamiento farmacológico , Enfermedades de las Glándulas Suprarrenales/cirugía , Glándulas Suprarrenales/metabolismo , Adrenalectomía , Femenino , Humanos , Hiperandrogenismo/etiología , Inyecciones Intramusculares , Leuprolida/administración & dosificación , Leuprolida/uso terapéutico , Hormona Luteinizante/antagonistas & inhibidores , Persona de Mediana Edad , ARN Mensajero/metabolismo , Receptores de HL/genética , Receptores de HL/metabolismo , Testosterona/sangre , Testosterona/metabolismoRESUMEN
BACKGROUND: Fine needle aspiration (FNA) diagnosis of simultaneous medullary and papillary thyroid carcinoma in independent thyroid lobes is exceedingly rare. CASE: A 36-year-old female presented with a one-month history of dysphagia. Thyroid ultrasound revealed a multinodular goiter. She was clinically and biochemically euthyroid. FNA of the right thyroid nodule was consistent with medullary carcinoma, and FNA of the left thyroid lobe was consistent with papillary carcinoma. Immunohistochemistry revealed strong calcitonin and CEA positivity in the right lobe and lack of staining in the left lobe. Conversely, staining for thyroglobulin was negative on the right lobe and positive on the left lobe. CONCLUSION: The patient developed tumors in separate lobes of the thyroid. Immunoreactivity of calcitonin, CEA and thyroglobulin made a sharp distinction between the two tumors. Therefore, we conclude that these tumors were not linked by either embryology or genetics.