Blockade of the effects of TGF-beta1 on mesangial cells by overexpression of Smad7.
Smad7, a protein induced by transforming growth factor-beta1 (TGF-beta1) in many target cells, inhibits TGF-beta1 signal transduction and is thought to mediate an intracellular negative feedback response that limits TGF-beta1 effects. It is possible that overexpression of Smad7 could block specified effects of TGF-beta1 on mesangial cells, a TGF-beta target in glomerular disease. Smad7 mRNA was induced by TGF-beta1 within 1 h in a concentration-dependent manner in a transformed mouse mesangial cell (MMC) line. Uptake of (14)C-spermidine from the medium by MMC and the transcriptional activity of a segment of the human collagen pro-alpha2 type 1 chain (COL1A2) promoter fused to a luciferase reporter gene were used as indices of TGF-beta1. Treatment with TGF-beta1 increased (14)C-spermidine uptake rate in a time-, concentration-, and temperature-dependent manner. For example, exposure to 1 ng/ml TGF-beta1 for 15 h increased uptake approximately twofold, a response that was attenuated by cycloheximide. Transfection of Smad7 expression vector into MMC abrogated both TGF-beta1-dependent stimulation of spermidine uptake and COL1A2 promoter activity. It is concluded that: (1) TGF-beta1 induces Smad7 in MMC; (2) (14)C-spermidine uptake is a convenient quantitative index of TGF-beta1 effect in these cells; and (3) overexpression of Smad7 is a highly effective method of blocking at least some mesangial cell effects of TGF-beta1 that may warrant evaluation in vivo in experimental glomerular disease.
Chen
Ruihua
R
*Departments of Medicine and Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina; and Medical Specialty Service, Ralph H. Johnson VA Medical Center, Charleston, South Carolina.
Huang
Cancan
C
*Departments of Medicine and Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina; and Medical Specialty Service, Ralph H. Johnson VA Medical Center, Charleston, South Carolina.
Morinelli
Thomas A
TA
*Departments of Medicine and Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina; and Medical Specialty Service, Ralph H. Johnson VA Medical Center, Charleston, South Carolina.
Trojanowska
Maria
M
*Departments of Medicine and Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina; and Medical Specialty Service, Ralph H. Johnson VA Medical Center, Charleston, South Carolina.
Paul
Richard V
RV
*Departments of Medicine and Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina; and Medical Specialty Service, Ralph H. Johnson VA Medical Center, Charleston, South Carolina.
D000818:Animals; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics; D024042:Collagen Type I; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000494:pharmacology*; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000502:physiology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051906:Smad7 Protein; D013095:Spermidine / Q000037:antagonists & inhibitors / Q000493:pharmacokinetics; D015534:Trans-Activators / Q000235:genetics / Q000494:pharmacology*; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1
D024042:Collagen Type I; D004268:DNA-Binding Proteins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; C497806:SMAD7 protein, human; D051906:Smad7 Protein; C497807:Smad7 protein, mouse; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D003094:Collagen; D003513:Cycloheximide; D013095:Spermidine