RESUMO
Neovascularization, increased basal membrane thickness and increased airway smooth muscle (ASM) bulk are hallmarks of airway remodelling in asthma. In this study, we examined connective tissue growth factor (CTGF) dysregulation in human lung tissue and animal models of allergic airway disease. Immunohistochemistry revealed that ASM cells from patients with severe asthma (A) exhibited high expression of CTGF, compared to mild and non-asthmatic (NA) tissues. This finding was replicated in a sheep model of allergic airways disease. In vitro, transforming growth factor (TGF)-ß increased CTGF expression both in NA- and A-ASM cells but the expression was higher in A-ASM at both the mRNA and protein level as assessed by PCR and Western blot. Transfection of CTGF promoter-luciferase reporter constructs into NA- and A-ASM cells indicated that no region of the CTGF promoter (-1500 to +200 bp) displayed enhanced activity in the presence of TGF-ß. However, in silico analysis of the CTGF promoter suggested that distant transcription factor binding sites may influence CTGF promoter activation by TGF-ß in ASM cells. The discord between promoter activity and mRNA expression was also explained, in part, by differential post-transcriptional regulation in A-ASM cells due to enhanced mRNA stability for CTGF. In patients, higher CTGF gene expression in bronchial biopsies was correlated with increased basement membrane thickness indicating that the enhanced CTGF expression in A-ASM may contribute to airway remodelling in asthma.
Assuntos
Remodelação das Vias Aéreas , Asma/metabolismo , Asma/fisiopatologia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Pulmão/metabolismo , Pulmão/fisiopatologia , Músculo Liso/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Asma/genética , Asma/patologia , Pareamento de Bases/genética , Membrana Basal/metabolismo , Membrana Basal/patologia , Fator de Crescimento do Tecido Conjuntivo/genética , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Regiões Promotoras Genéticas/genética , Pyroglyphidae , Estabilidade de RNA/genética , Ovinos , Adulto JovemRESUMO
On the basis of our recently resolved first cocrystal structure of Mdm4 in complex with a small molecule inhibitor (PDB ID 3LBJ ), we devised an approach for the generation of potential Mdm4 selective ligands. We performed the Ugi four-component reaction (Ugi-4CR) in 96-well plates with an indole fragment, which is specially designed to mimic Trp23, a key amino acid for the interaction between p53 and Mdm4. Generally the reaction yielded mostly precipitates collected by 96-well filter plates. The best hit compound was found to be active and selective for Mdm4 (Ki=5 µM, 10-fold stronger than Mdm2). This initial hit may serve as the starting point for designing selective p53-Mdm4 inhibitor with higher affinity.
