Transforming growth factor-beta 1 induces collagen IV gene expression in NIH-3T3 cells.
BACKGROUND
While recent studies have implicated transforming growth factor-beta 1 (TGF-beta 1) in the development of glomerular scarring, extraglomerular matrix production is frequently associated with glomerulonephritis and is an important determinant of disease progression. TGF-beta 1 may be an important mediator of extracellular matrix synthesis, both by glomerular and extraglomerular mesenchymal cells. TGF-beta 1-mediated collagen IV gene expression was studied in two mesenchymal cell lines. Initial studies were performed utilizing NIH-3T3 cells, a fibroblast-like line derived from murine embryo that has been used to study regulation of fibrillar collagen (collagen I and collagen III) synthesis by TGF-beta 1. Additional studies were performed using normal rat kidney cells (NRK-49F).


METHODS
Cells were grown in medium supplemented with 0.5% calf serum for 24 hours before treatment with TGF-beta 1. RNA was isolated after the addition of varying amounts of TGF-beta 1 to the cells in culture for varying periods of time, and collagen alpha 1(IV) RNA was quantitated by filter hybridization. Transcription of the alpha 1(IV) and alpha 2(IV) collagen genes was assessed by an in vitro transcription assay. Deposition of collagen IV was identified by immunoblotting.


RESULTS
Induction of alpha 1(IV) gene expression by NIH-3T3 cells and by NRK-49F cells was first seen 2 to 4 hours after TGF-beta 1 treatment, and was maximal after 12 to 18 hours. Maximal induction was observed following addition of 5 ng/ml TGF-beta 1 to NIH-3T3 cells, and following addition of 10 ng/ml of TGF-beta 1 to NRK-49F cells. In the presence of cycloheximide, TGF-beta 1 induction of alpha 1(IV) mRNA was markedly attenuated in both cell lines, suggesting that this effect of TGF-beta 1 requires protein synthesis. TGF-beta 1 increased transcription of both the alpha 1(IV) and alpha 2(IV) collagen genes by NIH-3T3 cells.


CONCLUSIONS
TGF-beta 1 induces collagen IV gene expression in both NIH-3T3 cells and normal rat kidney fibroblasts (NRK-49F cells). Further studies of cytokine-mediated transcriptional regulation of collagen IV, utilizing these cell lines, may provide important information regarding the role of extraglomerular matrix production in the progression of renal disease.
Grande
J
J
Department of Pathology, Mayo Graduate School of Medicine, Rochester, Minnesota.
Melder
D
D
Zinsmeister
A
A
Killen
P
P
D016475:3T3 Cells; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003513:Cycloheximide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects*; D007700:Kinetics; D051379:Mice; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000302:isolation & purification; D012044:Regression Analysis; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D003094:Collagen; D003513:Cycloheximide