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COVID-19/terapia , Respiración Artificial , Centros Médicos Académicos , Anciano , Anciano de 80 o más Años , COVID-19/fisiopatología , Presión de las Vías Aéreas Positiva Contínua , Enfermedad Crítica , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ciudad de Nueva York , Proyectos Piloto , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Tetraspanins (TSPANs) comprise a large family of 4-transmembrane domain proteins. The importance of TSPANs in vascular smooth muscle cells (VSMCs) is unexplored. Given that TGF-ß1 and myocardin (MYOCD) are potent activators for VSMC differentiation, we screened for TGF-ß1 and MYOCD/serum response factor (SRF)-regulated TSPANs in VSMC by using RNA-seq analyses and RNA-arrays. TSPAN2 was found to be the only TSPAN family gene induced by TGF-ß1 and MYOCD, and reduced by SRF deficiency in VSMCs. We also found that TSPAN2 is highly expressed in smooth muscle-enriched tissues and down-regulated in in vitro models of VSMC phenotypic modulation. TSPAN2 expression is attenuated in mouse carotid arteries after ligation injury and in failed human arteriovenous fistula samples after occlusion by dedifferentiated neointimal VSMC. In vitro functional studies showed that TSPAN2 suppresses VSMC proliferation and migration. Luciferase reporter and chromatin immunoprecipitation assays demonstrated that TSPAN2 is regulated by 2 parallel pathways, MYOCD/SRF and TGF-ß1/SMAD, via distinct binding elements within the proximal promoter. Thus, we identified the first VSMC-enriched and MYOCD/SRF and TGF-ß1/SMAD-dependent TSPAN family member, whose expression is intimately associated with VSMC differentiation and negatively correlated with vascular disease. Our results suggest that TSPAN2 may play important roles in vascular disease.-Zhao, J., Wu, W., Zhang, W., Lu, Y. W., Tou, E., Ye, J., Gao, P., Jourd'heuil, D., Singer, H. A., Wu, M., Long, X. Selective expression of TSPAN2 in vascular smooth muscle is independently regulated by TGF-ß1/SMAD and myocardin/serum response factor.
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Músculo Liso Vascular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Factor de Respuesta Sérica/metabolismo , Proteínas Smad/metabolismo , Tetraspaninas/metabolismo , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Diferenciación Celular , Regulación de la Expresión Génica/fisiología , Humanos , Masculino , Miocitos del Músculo Liso/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Factor de Respuesta Sérica/genética , Proteínas Smad/genética , Tetraspaninas/genética , Transactivadores/genética , Transcriptoma , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
OBJECTIVE: Long noncoding RNAs (lncRNA) represent a growing class of noncoding genes with diverse cellular functions. We previously reported on SENCR, an lncRNA that seems to support the vascular smooth muscle cell (VSMC) contractile phenotype. However, information about the VSMC-specific lncRNAs regulated by myocardin (MYOCD)/serum response factor, the master switch for VSMC differentiation, is unknown. APPROACH AND RESULTS: To define novel lncRNAs with functions related to VSMC differentiation, we performed RNA sequencing in human coronary artery SMCs that overexpress MYOCD. Several novel lncRNAs showed altered expression with MYOCD overexpression and one, named MYOcardin-induced Smooth muscle LncRNA, Inducer of Differentiation (MYOSLID), was activated by MYOCD and selectively expressed in VSMCs. MYOSLID was a direct transcriptional target of both MYOCD/serum response factor and transforming growth factor-ß/SMAD pathways. Functional studies revealed that MYOSLID promotes VSMC differentiation and inhibits VSMC proliferation. MYOSLID showed reduced expression in failed human arteriovenous fistula samples compared with healthy veins. Although MYOSLID did not affect gene expression of transcription factors, such as serum response factor and MYOCD, its depletion in VSMCs disrupted actin stress fiber formation and blocked nuclear translocation of MYOCD-related transcription factor A (MKL1). Finally, loss of MYOSLID abrogated transforming growth factor-ß1-induced SMAD2 phosphorylation. CONCLUSIONS: We have demonstrated that MYOSLID, the first human VSMC-selective and serum response factor/CArG-dependent lncRNA, is a novel modulator in amplifying the VSMC differentiation program, likely through feed-forward actions of both MKL1 and transforming growth factor-ß/SMAD pathways.
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Diferenciación Celular , Desarrollo de Músculos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , ARN Largo no Codificante/metabolismo , Factor de Respuesta Sérica/metabolismo , Transactivadores/metabolismo , Transporte Activo de Núcleo Celular , Derivación Arteriovenosa Quirúrgica , Proliferación Celular , Células Cultivadas , Vasos Coronarios/metabolismo , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteínas Nucleares/genética , Fenotipo , Fosforilación , ARN Largo no Codificante/genética , Factor de Respuesta Sérica/genética , Transducción de Señal , Proteína Smad2/metabolismo , Fibras de Estrés/metabolismo , Factores de Tiempo , Transactivadores/genética , Transcripción Genética , Transfección , Factor de Crecimiento Transformador beta1/metabolismo , VasoconstricciónRESUMEN
Zinc is an essential trace element with wide-ranging biological functions, whereas the Hedgehog (Hh) signaling pathway plays crucial roles in both development and disease. Here we show that there is a mechanistic link between zinc and Hh signaling. The upstream activator of Hh signaling, the Hh ligand, originates from Hh autoprocessing, which converts the Hh precursor protein to the Hh ligand. In an in vitro Hh autoprocessing assay we show that zinc inhibits Hh autoprocessing with a Ki of 2 µm. We then demonstrate that zinc inhibits Hh autoprocessing in a cellular environment with experiments in primary rat astrocyte culture. Solution NMR reveals that zinc binds the active site residues of the Hh autoprocessing domain to inhibit autoprocessing, and isothermal titration calorimetry provided the thermodynamics of the binding. In normal physiology, zinc likely acts as a negative regulator of Hh autoprocessing and inhibits the generation of Hh ligand and Hh signaling. In many diseases, zinc deficiency and elevated level of Hh ligand co-exist, including prostate cancer, lung cancer, ovarian cancer, and autism. Our data suggest a causal relationship between zinc deficiency and the overproduction of Hh ligand.