RESUMEN
In this study, we demonstrate that B-type natriuretic peptide (BNP) opposed angiotensin II (Ang II)-stimulated de novo cholesterol biosynthesis, cellular cholesterol uptake, cholesterol transfer to the inner mitochondrial membrane, and steroidogenesis, which are required for biosynthesis of steroid hormones such as aldosterone and cortisol in primary human adrenocortical cells. BNP dose-dependently stimulated intracellular cGMP production with an EC(50) of 11 nm, implying that human adrenocortical cells express the guanylyl cyclase A receptor. cDNA microarray and real-time RT-PCR analyses revealed that BNP inhibited Ang II-stimulated genes related to cholesterol biosynthesis (acetoacetyl coenzyme A thiolase, HMG coenzyme A synthase 1, HMG coenzyme A reductase, isopentenyl-diphosphate Delta-isomerase, lanosterol synthase, sterol-4C-methyl oxidase, and emopamil binding protein/sterol isomerase), cholesterol uptake from circulating lipoproteins (scavenger receptor class B type I and low-density lipoprotein receptor), cholesterol transfer to the inner mitochondrial membrane (steroidogenic acute regulatory protein), and steroidogenesis (ferredoxin 1,3beta-hydroxysteroid dehydrogenase, glutathione transferase A3, CYP19A1, CYP11B1, and CYP11B2). Consistent with the microarray and real-time PCR results, BNP also blocked Ang II-induced binding of (125)I-labeled low-density lipoprotein and (125)I-labeled high-density lipoprotein to human adrenocortical cells. Furthermore, BNP markedly inhibited Ang II-stimulated release of estradiol, aldosterone, and cortisol from cultured primary human adrenocortical cells. These findings demonstrate that BNP opposes Ang II-induced steroidogenesis via multiple steps from cholesterol supply and transfer to the final formation of steroid hormones. This study provides new insights into the cellular mechanisms by which BNP modulates Ang II-induced steroidogenesis in the adrenal gland.
Asunto(s)
Corteza Suprarrenal/metabolismo , Angiotensina II/metabolismo , Colesterol/metabolismo , Péptido Natriurético Encefálico/metabolismo , Vasoconstrictores/metabolismo , Corteza Suprarrenal/citología , Corticoesteroides/biosíntesis , Neoplasias de la Corteza Suprarrenal , Carcinoma Corticosuprarrenal , Adulto , Angiotensina II/farmacología , Línea Celular Tumoral , Células Cultivadas , Colesterol/biosíntesis , GMP Cíclico/metabolismo , Interacciones Farmacológicas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Lipoproteínas/metabolismo , Masculino , Persona de Mediana Edad , Péptido Natriurético Encefálico/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esteroides/biosíntesis , Vasoconstrictores/farmacologíaRESUMEN
Transforming growth factor-beta (TGFbeta) is a major mediator of normal wound healing and of pathological conditions involving fibrosis, such as idiopathic pulmonary fibrosis. TGFbeta also stimulates the differentiation of myofibroblasts, a hallmark of fibrotic diseases. In this study, we examined the underlying processes of TGFbetaRI kinase activity in myofibroblast conversion of human lung fibroblasts using specific inhibitors of TGFbetaRI (SD-208) and p38 mitogen-activated kinase (SD-282). We demonstrated that SD-208, but not SD-282, inhibited TGFbeta-induced SMAD signaling, myofibroblast transformation, and collagen gel contraction. Furthermore, we extended our findings to a rat bleomycin-induced lung fibrosis model, demonstrating a significant decrease in the number of myofibroblasts at fibroblastic foci in animals treated with SD-208 but not those treated with SD-282. SD-208 also reduced collagen deposition in this in vivo model. Microarray analysis of human lung fibroblasts identified molecular fingerprints of these processes and showed that SD-208 had global effects on reversing TGFbeta-induced genes involved in fibrosis, inflammation, cell proliferation, cytoskeletal organization, and apoptosis. These studies also revealed that although the p38 pathway may not be needed for appearance or disappearance of the myofibroblast, it can mediate a subset of inflammatory and fibrogenic events of the myofibroblast during the process of tissue repair and fibrosis. Our findings suggest that inhibitors such as SD-208 may be therapeutically useful in human interstitial lung diseases and pulmonary fibrosis.
Asunto(s)
Receptores de Activinas Tipo I/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Fibrosis Pulmonar/etiología , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Receptores de Activinas Tipo I/antagonistas & inhibidores , Diferenciación Celular , Células Cultivadas , Colágeno/metabolismo , Factor de Crecimiento del Tejido Conjuntivo , Citoesqueleto/metabolismo , Fibroblastos/citología , Regulación de la Expresión Génica , Humanos , Proteínas Inmediatas-Precoces/genética , Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Sistema de Señalización de MAP Quinasas , Análisis de Secuencia por Matrices de Oligonucleótidos , Pteridinas/farmacología , Fibrosis Pulmonar/tratamiento farmacológico , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Proteínas Smad/antagonistas & inhibidores , Proteínas Smad/fisiología , Factor de Crecimiento Transformador beta/farmacología , Cicatrización de HeridasRESUMEN
The multiple myeloma (MM) bone marrow (BM) microenvironment plays a critical role in supporting tumor growth and survival as well as in promoting formation of osteolytic lesions. Recent results suggest that the p38 mitogen-activated protein kinase (MAPK) is an important factor in maintaining this activated environment. In this report, we demonstrate that the p38alpha MAPK inhibitor, SCIO-469, suppresses secretion of the tumor-supportive factors IL-6 and VEGF from BM stromal cells (BMSCs) as well as cocultures of BMSCs with MM cells, resulting in reduction in MM cell proliferation. Additionally, we show that SCIO-469 prevents TNFalpha-induced adhesion of MM cells to BMSCs through an ICAM-1- and VCAM-1-independent mechanism. Microarray analysis revealed a novel set of TNFalpha-induced chemokines in BMSCs that is strongly inhibited by SCIO-469. Furthermore, reintroduction of chemokines CXCL10 and CCL8 to BMSCs overcomes the inhibitory effect of SCIO-469 on TNFalpha-induced MM adhesion. Lastly, we show that SCIO-469 inhibits secretion and expression of the osteoclast-activating factors IL-11, RANKL, and MIP-1alpha as well as prevents human osteoclast formation in vitro. Collectively, these results suggest that SCIO-469 treatment can suppress factors in the bone marrow microenvironment to inhibit MM cell proliferation and adhesion and also to alleviate osteolytic activation in MM.
Asunto(s)
Médula Ósea , Adhesión Celular/fisiología , Proliferación Celular , Indoles/metabolismo , Mieloma Múltiple , Osteoclastos/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Médula Ósea/química , Médula Ósea/metabolismo , Proteínas Portadoras/metabolismo , Quimiocinas/metabolismo , Técnicas de Cocultivo , Medios de Cultivo Condicionados , Humanos , Interleucina-6/metabolismo , Glicoproteínas de Membrana/metabolismo , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoclastos/citología , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Células del Estroma/citología , Células del Estroma/metabolismo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Pulmonary fibrosis is characterized by chronic scar formation and deposition of extracellular matrix, resulting in impaired lung function and respiratory failure. Idiopathic pulmonary fibrosis (IPF) is associated with pronounced morbidity and mortality and responds poorly to known therapeutic interventions; there are no known drugs that effectively block or reverse progressive fibrosis. Transforming growth factor beta (TGF-beta) is known to mediate extracellular matrix gene regulation and appears to be a major player in both the initiation and progression of IPF. TGF-beta mediates its biological effects through members of a family of activin receptor-like kinases (ALK). We have used a gene transfer model of progressive TGF-beta1-induced pulmonary fibrosis in rats to study a newly described orally active small molecular weight drug that is a potent and selective inhibitor of the kinase activity of ALK5, the specific TGF-beta receptor. We show that the drug inhibits the induction of fibrosis when administered at the time of initiation of fibrogenesis and, most important, blocks progressive fibrosis when administered transiently to animals with established fibrosis. These data show promise of the development of an effective therapeutic intervention for IPF and that inhibition of chronic progressive fibrosis may be achieved by blocking TGF-beta receptor activation.