RESUMO
Epitranscriptomic events such as adenosine-to-inosine (A-to-I) RNA editing by ADAR can recode mRNAs to translate novel proteins. Editing of the mRNA that encodes actin crosslinking protein Filamin A (FLNA) mediates a Q-to-R transition in the interactive C-terminal region. While FLNA editing is conserved among vertebrates, its physiological function remains unclear. Here, we show that cardiovascular tissues in humans and mice show massive editing and that FLNA RNA is the most prominent substrate. Patient-derived RNA-Seq data demonstrate a significant drop in FLNA editing associated with cardiovascular diseases. Using mice with only impaired FLNA editing, we observed increased vascular contraction and diastolic hypertension accompanied by increased myosin light chain phosphorylation, arterial remodeling, and left ventricular wall thickening, which eventually causes cardiac remodeling and reduced systolic output. These results demonstrate a causal relationship between RNA editing and the development of cardiovascular disease indicating that a single epitranscriptomic RNA modification can maintain cardiovascular health.
Assuntos
Pressão Sanguínea , Filaminas/metabolismo , Hipertensão/metabolismo , Contração Muscular , Miocárdio/metabolismo , Edição de RNA , Precursores de RNA/metabolismo , Remodelação Vascular , Animais , Filaminas/genética , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Hipertensão/genética , Hipertensão/patologia , Camundongos , Miocárdio/patologia , Precursores de RNA/genética , Análise de Sequência de RNARESUMO
Specificity protein 1 (Sp1) is an important transcription factor implicated in numerous cellular processes. However, whether Sp1 is involved in the regulation of RNA polymerase III (Pol III)-directed gene transcription in human cells remains unknown. Here, we first show that filamin A (FLNA) represses Sp1 expression as well as expression of TFIIB-related factor 1 (BRF1) and general transcription factor III C subunit 2 (GTF3C2) in HeLa, 293T, and SaOS2 cell lines stably expressing FLNA-silencing shRNAs. Both BRF1 promoter 4 (BRF1P4) and GTF3C2 promoter 2 (GTF3C2P2) contain putative Sp1-binding sites, suggesting that Sp1 affects Pol III gene transcription by regulating BRF1 and GTF3C2 expression. We demonstrate that Sp1 knockdown inhibits Pol III gene transcription, BRF1 and GTF3C2 expression, and the proliferation of 293T and HeLa cells, whereas Sp1 overexpression enhances these activities. We obtained a comparable result in a cell line in which both FLNA and Sp1 were depleted. These results indicate that Sp1 is involved in the regulation of Pol III gene transcription independently of FLNA expression. Reporter gene assays showed that alteration of Sp1 expression affects BRF1P4 and GTF3C2P2 activation, suggesting that Sp1 modulates Pol III-mediated gene transcription by controlling BRF1 and GTF3C2 gene expression. Further analysis revealed that Sp1 interacts with and thereby promotes the occupancies of TATA box-binding protein, TFIIAα, and p300 at both BRF1P4 and GTF3C2P2. These findings indicate that Sp1 controls Pol III-directed transcription and shed light on how Sp1 regulates cancer cell proliferation.
Assuntos
RNA Polimerase III/metabolismo , Fator de Transcrição Sp1/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fatores de Transcrição TFIII/metabolismo , Sítios de Ligação , Linhagem Celular , Proliferação de Células , Proteína p300 Associada a E1A/metabolismo , Filaminas/antagonistas & inibidores , Filaminas/genética , Filaminas/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Interferência de RNA , RNA Polimerase III/genética , RNA Interferente Pequeno/metabolismo , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/genética , Fatores Associados à Proteína de Ligação a TATA/antagonistas & inibidores , Fatores Associados à Proteína de Ligação a TATA/genética , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Fatores de Transcrição TFIII/antagonistas & inibidores , Fatores de Transcrição TFIII/genética , Transcrição Gênica , Regulação para CimaRESUMO
An efficient intracellular response to somatostatin analogs (SSA) in pituitary tumors requires filamin A (FLNA). Since cAMP pathway plays an important role in GH-secreting pituitary tumors pathogenesis and FLNA is phosphorylated by PKA on S2152, aim of this study was to investigate in tumoral somatotrophs the impact of cAMP pathway activation and SSA stimulation on FLNA phosphorylation and the consequences on SST2 function. We found a PKA-mediated increase (2-fold) and SST2 agonist-induced decrease (-50%) of FLNA phosphorylation in GH3, GH4C1 and primary somatotroph tumor cells. This modification regulates FLNA function. Indeed, phosphomimetic S2152D FLNA mutant, but not phosphodeficient S2152A, abolished the known SSA antitumoral effects, namely: 1) inhibition of cell proliferation, reduction of cyclin D3 and increase of p27; 2) increase of cell apoptosis; 3) inhibition of cell migration via RhoA activation and cofilin phosphorylation. Coimmunoprecipitation and immunofluorescence assays showed that S2152A FLNA was recruited to activated SST2, whereas S2152D FLNA constitutively bound SST2 on the plasma membrane, but prevented Gαi proteins recruitment to SST2. In conclusion, we demonstrated that FLNA phosphorylation, promoted by cAMP pathway activation and inhibited by SSA, prevented SST2 signaling in GH-secreting tumoral pituitary cells.
Assuntos
AMP Cíclico/metabolismo , Filaminas/metabolismo , Adenoma Hipofisário Secretor de Hormônio do Crescimento/metabolismo , Neoplasias Hipofisárias/metabolismo , Proteínas Quinases/metabolismo , Receptores de Somatostatina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Adenoma Hipofisário Secretor de Hormônio do Crescimento/genética , Adenoma Hipofisário Secretor de Hormônio do Crescimento/patologia , Hormônios/farmacologia , Humanos , Fosforilação/efeitos dos fármacos , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Somatostatina/farmacologia , Somatotrofos/efeitos dos fármacos , Somatotrofos/metabolismo , Células Tumorais CultivadasRESUMO
Bronchopulmonary dysplasia (BPD) is a common long-term complication in premature newborns requiring ventilatory support and is the most common cause of chronic diffuse lung disease in this population. We present the clinical course of a premature newborn with a complicated neonatal respiratory course that was initially thought to be related to BPD, but it did not respond to the typical therapies for this condition. Due to the findings of periventricular nodular heterotopia, the diagnosis of a filamin A gene mutation was eventually made, which explained the respiratory pathology of this patient. When time of onset and clinical course do not correlate with typical BPD, one should consider alternative diagnoses in premature infants, including neonatal diffuse lung disease.