RESUMO
Helper T effector cytokines implicated in asthma modulate the contractility of human airway smooth muscle (HASM) cells. We have reported recently that a profibrotic cytokine, transforming growth factor (TGF)-ß1, induces HASM cell shortening and airway hyperresponsiveness. Here, we assessed whether TGF-ß1 affects the ability of HASM cells to relax in response to ß2-agonists, a mainstay treatment for airway hyperresponsiveness in asthma. Overnight TGF-ß1 treatment significantly impaired isoproterenol (ISO)-induced relaxation of carbachol-stimulated, isolated HASM cells. This single-cell mechanical hyporesponsiveness to ISO was corroborated by sustained increases in myosin light chain phosphorylation. In TGF-ß1-treated HASM cells, ISO evoked markedly lower levels of intracellular cAMP. These attenuated cAMP levels were, in turn, restored with pharmacological and siRNA inhibition of phosphodiesterase 4 and Smad3, respectively. Most strikingly, TGF-ß1 selectively induced phosphodiesterase 4D gene expression in HASM cells in a Smad2/3-dependent manner. Together, these data suggest that TGF-ß1 decreases HASM cell ß2-agonist relaxation responses by modulating intracellular cAMP levels via a Smad2/3-dependent mechanism. Our findings further define the mechanisms underlying ß2-agonist hyporesponsiveness in asthma, and suggest TGF-ß1 as a potential therapeutic target to decrease asthma exacerbations in severe and treatment-resistant asthma.
Assuntos
Asma/fisiopatologia , Músculo Liso/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta2/agonistas , Asma/tratamento farmacológico , Asma/metabolismo , Broncodilatadores/farmacologia , Carbacol/farmacologia , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Isoproterenol/farmacologia , Pulmão/metabolismo , Músculo Liso/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Fosforilação , RNA Interferente Pequeno/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , Fator de Crescimento Transformador beta2/metabolismoRESUMO
Identification and validation of molecular targets are considered as key elements in new drug discovery and development. We have recently demonstrated that a novel synthetic iminoquinone analog, termed [7-(benzylamino)- 1,3,4,8-tetrahydropyrrolo [4,3, 2-de]quinolin-8(1H)-one] (BA-TPQ), has significant anti-breast cancer activity both in vitro and in vivo, but the underlying molecular mechanisms are not fully understood. Herein, we report the molecular studies for BA-TPQ's effects on JNK and its upstream and downstream signaling pathways. The compound up-regulates the JNK protein levels by increasing its phosphorylation and decreasing its polyubiquitination-mediated degradation. It activates ZAK at the MAPKKK level and MKK4 at the MAPKK level. It also up-regulates the TGFß2 mRNA level, which can be abolished by the JNK-specific inhibitor SP600125, but not TGFß pathway-specific inhibitor SD-208, indicating that both JNK and TGFß signaling pathways are activated by BA-TPQ and that the JNK pathway activation precedes TGFß activation. The pro-apoptotic and anti-growth effects of BA-TPQ are significantly blocked by both the JNK and TGFß pathway inhibitors. In addition, BA-TPQ activates the ZAK-MKK4-JNK pathway in MCF7 cells, but not normal MCF10A cells, demonstrating its cancer-specific activities. In conclusion, our results demonstrate that BA-TPQ activates the ZAK-MKK4-JNK-TGFß signaling cascade as a molecular target for its anticancer activity.