BACKGROUND AND AIM: As a pleiotropic protein, transforming growth factor (TGF)-beta 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-beta1 signal transduction pathway via delivery of a dominant-negative receptor-II (DeltaCyTbRII)-cDNA lacking Ser/Thr kinase intracytoplasmic domain activity. Thus, Cos-1 and hepatic stellate cells were cotransfected with pCMV5-DeltaCyTbRII and pAdTrack-green fluorescent protein using lipofectamine. RESULTS: Fluorescence microscopy demonstrated an average 10% transfection efficiency. Radiolabeled 125I-TGF-beta 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-DeltaCyTbRII-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-DeltaCyTbRII-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.
BACKGROUND AND AIM: As a pleiotropic protein, transforming growth factor (TGF)-beta 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-beta1 signal transduction pathway via delivery of a dominant-negative receptor-II (DeltaCyTbRII)-cDNA lacking Ser/Thr kinase intracytoplasmic domain activity. Thus, Cos-1 and hepatic stellate cells were cotransfected with pCMV5-DeltaCyTbRII and pAdTrack-green fluorescent protein using lipofectamine. RESULTS: Fluorescence microscopy demonstrated an average 10% transfection efficiency. Radiolabeled 125I-TGF-beta 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-DeltaCyTbRII-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-DeltaCyTbRII-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.
Authors: Anush Oganesian; Sandra Au; Jeremy A Horst; Lars C Holzhausen; Athena J Macy; James M Pace; Paul Bornstein Journal: J Biol Chem Date: 2006-10-03 Impact factor: 5.157