RESUMEN
RATIONALE: Recent studies suggest that microRNAs (miRNAs) play important roles in regulation of pulmonary artery smooth muscle cell (PASMC) phenotype and are implicated in pulmonary arterial hypertension (PAH). However, the underlying molecular mechanisms remain elusive. OBJECTIVES: This study aims to understand the mechanisms regulating PASMC proliferation and differentiation by microRNA-17â¼92 (miR-17â¼92) and to elucidate its implication in PAH. METHODS: We generated smooth muscle cell (SMC)-specific miR-17â¼92 and PDZ and LIM domain 5 (PDLIM5) knockout mice and overexpressed miR-17â¼92 and PDLIM5 by injection of miR-17â¼92 mimics or PDLIM5-V5-His plasmids and measured their responses to hypoxia. We used miR-17â¼92 mimics, inhibitors, overexpression vectors, small interfering RNAs against PDLIM5, Smad, and transforming growth factor (TGF)-ß to determine the role of miR-17â¼92 and its downstream targets in PASMC proliferation and differentiation. MEASUREMENTS AND MAIN RESULTS: We found that human PASMC (HPASMC) from patients with PAH expressed decreased levels of the miR-17â¼92 cluster, TGF-ß, and SMC markers. Overexpression of miR-17â¼92 increased and restored the expression of TGF-ß3, Smad3, and SMC markers in HPASMC of normal subjects and patients with idiopathic PAH, respectively. Knockdown of Smad3 but not Smad2 prevented miR-17â¼92-induced expression of SMC markers. SMC-specific knockout of miR-17â¼92 attenuated hypoxia-induced pulmonary hypertension (PH) in mice, whereas reconstitution of miR-17â¼92 restored hypoxia-induced PH in these mice. We also found that PDLIM5 is a direct target of miR-17/20a, and hypertensive HPASMC and mouse PASMC expressed elevated PDLIM5 levels. Suppression of PDLIM5 increased expression of SMC markers and enhanced TGF-ß/Smad2/3 activity in vitro and enhanced hypoxia-induced PH in vivo, whereas overexpression of PDLIM5 attenuated hypoxia-induced PH. CONCLUSIONS: We provided the first evidence that miR-17â¼92 inhibits PDLIM5 to induce the TGF-ß3/SMAD3 pathway, contributing to the pathogenesis of PAH.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Hipertensión Pulmonar/etiología , MicroARNs/fisiología , Proteínas de Microfilamentos/fisiología , Músculo Liso Vascular/citología , Animales , Espectrometría de Masas , Ratones , Ratones Noqueados , Factores de Crecimiento Transformadores/fisiologíaRESUMEN
RATIONALE: Replication deficient adenoviruses (Ad) vectors are common tools in gene therapy. Since Ad vectors are known to activate innate and adaptive immunity, we investigated whether intratracheal administration of Ad vectors alone is sufficient to induce lung injury and pulmonary fibrosis. METHODS: We instilled Ad viruses ranging from 107 to 1.625×109 ifu/mouse as well as the same volume of PBS and bleomycin. 14 and 21 days after administration, we collected bronchoalveolar lavage fluid (BALF) and mouse lung tissues. We measured the protein concentration, total and differential cell counts, and TGF-ß1 production, performed Trichrome staining and Sircol assay, determined gene and protein levels of profibrotic cytokines, MMPs, and Wnt signaling proteins, and conducted TUNEL staining and co-immunofluorescence for GFP and α-SMA staining. RESULTS: Instillation of high dose Ad vectors (1.625×109 ifu/mouse) into mouse lungs induced high levels of protein content, inflammatory cells, and TGF-ß1 in BALF, comparable to those in bleomycin-instilled lungs. The collagen content and mRNA levels of Col1a1, Col1a2, PCNA, and α-SMA were also increased in the lungs. Instillation of both bleomycin and Ad vectors increased expression levels of TNFα and IL-1ß but not IL-10. Instillation of bleomycin but not Ad increased the expression of IL-1α, IL-13 and IL-16. Treatment with bleomycin or Ad vectors increased expression levels of integrin α1, α5, and αv, MMP9, whereas treatment with bleomycin but not Ad vectors induced MMP2 expression levels. Both bleomycin and Ad vectors induced mRNA levels of Wnt2, 2b, 5b, and Lrp6. Intratracheal instillation of Ad viruses also induced DNA damages and Ad viral infection-mediated fibrosis is not limited to the infection sites. CONCLUSIONS: Our results suggest that administration of Ad vectors induces an inflammatory response, lung injury, and pulmonary fibrosis in a dose dependent manner.
Asunto(s)
Adenoviridae/inmunología , Bleomicina/administración & dosificación , Inflamación/inmunología , Lesión Pulmonar/inmunología , Fibrosis Pulmonar/inmunología , Actinas/biosíntesis , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Colágeno Tipo I/biosíntesis , Cadena alfa 1 del Colágeno Tipo I , Modelos Animales de Enfermedad , Inflamación/virología , Integrinas/biosíntesis , Interleucina-13/biosíntesis , Interleucina-16/biosíntesis , Interleucina-1alfa/biosíntesis , Interleucina-1beta/biosíntesis , Lesión Pulmonar/virología , Masculino , Metaloproteinasa 2 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/biosíntesis , Ratones , Ratones Endogámicos C57BL , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Fibrosis Pulmonar/virología , Factor de Crecimiento Transformador beta1/biosíntesis , Proteínas Wnt/biosíntesisRESUMEN
Human pulmonary artery smooth muscle cells (HPASMCs) express both adenosine monophosphate-activated protein kinase (AMPK) α1 and α2. We investigated the distinct roles of AMPK α1 and α2 in the survival of HPASMCs during hypoxia and hypoxia-induced pulmonary hypertension (PH). The exposure of HPASMCs to hypoxia (3% O2) increased AMPK activation and phosphorylation, and the inhibition of AMPK with Compound C during hypoxia decreased their viability and increased lactate dehydrogenase activity and apoptosis. Although the suppression of either AMPK α1 or α2 expression led to increased cell death, the suppression of AMPK α2 alone increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. It also resulted in the decreased expression of myeloid cell leukemia sequence 1 (MCL-1). The knockdown of MCL-1 or MCL-1 inhibitors increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. On the other hand, the suppression of AMPK α1 expression alone prevented hypoxia-mediated autophagy. The inhibition of autophagy induced cell death in HPASMCs. Our results suggest that AMPK α1 and AMPK α2 play differential roles in the survival of HPASMCs during hypoxia. The activation of AMPK α2 maintains the expression of MCL-1 and prevents apoptosis, whereas the activation of AMPK α1 stimulates autophagy, promoting HPASMC survival. Moreover, treatment with Compound C, which inhibits both isoforms of AMPK, prevented and partly reversed hypoxia-induced PH in mice. Taking these results together, our study suggests that AMPK plays a key role in the pathogenesis of pulmonary arterial hypertension, and AMPK may represent a novel therapeutic target for the treatment of pulmonary arterial hypertension.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Hipertensión Pulmonar/metabolismo , Hipoxia/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/metabolismo , Adenosina Monofosfato/farmacología , Animales , Autofagia/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Hipertensión Pulmonar Primaria Familiar , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/metabolismo , Humanos , Ratones , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/enzimología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/enzimología , Pirazoles/farmacología , Pirimidinas/farmacologíaRESUMEN
Lung cancer is the leading cause of cancer-related death worldwide. Hypoxia is known to increase cancer cell migration and invasion. We have previously reported that hypoxia induces epithelial-mesenchymal transition (EMT) in lung cancer cells. However, it is unknown whether hypoxia promotes lung cancer cell migration and invasion via EMT and whether cyclic AMP (cAMP) dependent protein kinase (PKA) plays a role in this process. We found that hypoxia increased PKA activity and induced mRNA and protein expression of PKA catalytic subunit α (PKACA), and regulatory subunits R1A and R1B. Knockdown of HIF-1/2α prevented hypoxia-mediated induction of PKACA mRNA expression and PKA activity. Inhibition of PKA activity with chemical inhibitors prevented EMT induced by hypoxia and tumor growth factor ß1. However, activation of PKA by forskolin and 8-Br-cAMP did not induce EMT. Furthermore, treatment with H89 and knockdown of PKACA prevented hypoxia-mediated, EMT, cell migration, and invasion, whereas overexpression of mouse PKACA rescued hypoxia-mediated migration and invasion in PKACA deficient cancer cells. Our results suggest that hypoxia enhances PKA activity by upregulating PKA gene expression in a HIF dependent mechanism and that PKA plays a key role in hypoxia-mediated EMT, migration, and invasion in lung cancer cells.
Asunto(s)
Hipoxia de la Célula , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Colforsina/farmacología , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/antagonistas & inhibidores , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Isoquinolinas/farmacología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Sulfonamidas/farmacología , Factor de Crecimiento Transformador beta1/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Although von Hippel-Lindau protein (pVHL) is known as a tumor suppressor in kidney and other organs, it remains unclear whether pVHL plays a role in lung cancer development. We investigated the role of pVHL in lung cancer cell proliferation, migration, and colonization using stable A549 cells with knockdown of pVHL. We found that knockdown of pVHL promotes epithelial-mesenchymal transition (EMT) in lung cancer cells. Knockdown of pVHL decreased tumor colonization in a tail-vein injection model and decreased cell proliferation, whereas overexpression of constitutive active HIF increased tumor colonization, suggesting a HIF-independent function of pVHL in lung. Knockdown of pVHL decreased phosphorylation of FAK and expression of integrin, suggesting that pVHL regulates lung cancer development via integrin/FAK signaling pathway.
Asunto(s)
Proliferación Celular , Técnicas de Silenciamiento del Gen , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Animales , Western Blotting , Línea Celular Tumoral , Humanos , RatonesRESUMEN
Pulmonary arterial hypertension (PAH) is a devastating disease, and no effective treatments are available. Hypoxia-induced pulmonary artery remodeling, including smooth muscle cell proliferation, contributes to PAH, but the exact mechanisms underlying this abnormal process are largely undefined. The forkhead box M1 (FoxM1) transcription factor regulates cancer cell growth by modulating gene expression critical for cell cycle progression. Here, we report for the first time, to the best of our knowledge, a novel function of FoxM1 in the hypoxia-stimulated proliferation of human pulmonary artery smooth muscle cells (HPASMCs). Exposure to hypoxia caused a marked up-regulation of FoxM1 gene expression, mainly at the transcription level, and this induction correlated with HPASMC cell proliferation. The knockdown of FoxM1 inhibited the hypoxia-stimulated proliferation of HPASMCs. We found that the knockdown of HIF-2α, but not HIF-1α, diminished FoxM1 induction in response to hypoxia. However, the knockdown of FoxM1 did not alter expression levels of HIF-2α or HIF-1α, suggesting that HIF-2α is an upstream regulator of FoxM1. Furthermore, the knockdown of FoxM1 prevented the hypoxia-induced expression of aurora A kinase and cyclin D1. Collectively, our results suggest that hypoxia induces FoxM1 gene expression in an HIF-2α-dependent pathway, thereby promoting HPASMC proliferation.