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- Title
HEPATOLOGY Amplified expression of dominant-negative transforming growth factor-beta type II receptor inhibits collagen type I production via reduced Smad-3 activity.
- Authors
Hernandez-Cañaveral, Ivan; González, Jaime; López-Casillas, Fernando; Armendariz-Borunda, Juan
- Abstract
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. 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. 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. Thus, this could be a useful strategy to downregulate or prevent exacerbated synthesis and deposition of extracellular matrix in a given fibrotic process.
- Subjects
TRANSFORMING growth factors-beta; PROTEIN kinases; CELLULAR signal transduction; GREEN fluorescent protein; EXTRACELLULAR matrix proteins; COLLAGEN
- Publication
Journal of Gastroenterology & Hepatology, 2004, Vol 19, Issue 4, p380
- ISSN
0815-9319
- Publication type
Article
- DOI
10.1111/j.1440-1746.2003.03292.x