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Title: Amplified expression of dominant-negative transforming growth factor-beta type II receptor inhibits collagen type I production via reduced Smad-3 activity
Author: Hernandez-Canaveral, I.
Gonzalez, J.
Lopez-Casillas, F.
Armendariz-Borunda, J.
Issue Date: 2004
Abstract: Background and Aim: As a pleiotropic protein, transforming growth factor (TGF)-β induces its effects by binding to its Ser/Thr kinase receptor type II and then recruiting and activating receptor type I, which is phosphorylated and activates Smads that transduce the signal to the nucleus. Methods: In this work, the authors blocked TGF-β1 signal transduction pathway via delivery of a dominant-negative receptor-II (ΔCyTbRII)-cDNA lacking Ser/Thr kinase intracytoplasmic domain activity. Thus, Cos-1 and hepatic stellate cells were cotransfected with pCMV5-ΔCyTbRII and pAdTrack-green fluorescent protein using lipofectamine. Results: Fluorescence microscopy demonstrated an average 10% transfection efficiency. Radiolabeled 125I-TGF-β was bound mostly by cell membrane-expressed truncated receptor-II rather than wild-type receptor type II. Electrophoretic mobility shift assays were performed using consensus Smad-2 and -3 sequences rendering a three-fold decrease in DNA-binding activity, reflecting a down-activation in Smad complexes in pCMV5-ΔCyTbRII-transfected cells, but not in mock-transfected cells. The identity of these transcriptional factors was confirmed using irrelevant double-stranded oligonucleotides and specific antibodies to compete for DNA binding. Also, collagen I mRNA expression showed a five-fold decrease, which was reflected at the protein level as a diminished collagen type I production in pCMV5-ΔCyTbRII-transfected Cos-1 cells as measured by [ 3H]proline incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Conclusion: Thus, this could be a useful strategy to downregulate or prevent exacerbated synthesis and deposition of extracellular matrix in a given fibrotic process. © 2004 Blackwell Publishing Asia Pty Ltd.
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