Transforming growth factor-β1 induces collagen IV gene expression in NIH- 3T3 cells

Joseph Peter Grande, D. Melder, A. Zinsmeister, P. Killen

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

BACKGROUND: While recent studies have implicated transforming growth factor-β1 (TGF-β1) in the development of glomerular scarring, extraglomerular matrix production is frequently associated with glomerulonephritis and is an important determinant of disease progression. TGF-β1 may be an important mediator of extracellular matrix synthesis, both by glomerular and extraglomerular mesenchymal cells. TGF-β1-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-β1. Additional studies were performed using normal rat kidney cells (NRK-49F). EXPERIMENTAL DESIGN: Cells were grown in medium supplemented with 0.5% calf serum for 24 hours before treatment with TGF-β1. RNA was isolated after the addition of varying amounts of TGF-β1 to the cells in culture for varying periods of time, and collagen α1(IV) RNA was quantitated by filter hybridization. Transcription of the α1(IV) and α2(IV) collagen genes was assessed by an in vitro transcription assay. Deposition of collagen IV was identified by immunoblotting. RESULTS: Induction of α1(IV) gene expression by NIH-3T3 cells and by NRK-49F cells was first seen 2 to 4 hours after TGF- β1 treatment, and was maximal after 12 to 18 hours. Maximal induction was observed following addition of 5 ng/ml TGF-β1 to NIH-3T3 cells, and following addition of 10 ng/ml of TGF-β1 to NRK-49F cells. In the presence of cycloheximide, TGF-β1 induction of α1(IV) mRNA was markedly attenuated in both cell lines, suggesting that this effect of TGF-β1 requires protein synthesis. TGF-β1 increased transcription of both the α1(IV) and α2(IV) collagen genes by NIH-3T3 cells. CONCLUSIONS: TGF-β1 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.

Original languageEnglish (US)
Pages (from-to)387-395
Number of pages9
JournalLaboratory Investigation
Volume69
Issue number4
StatePublished - 1993

Fingerprint

NIH 3T3 Cells
Transforming Growth Factors
Collagen
Gene Expression
Kidney
Cell Line
Disease Progression
Fibroblasts
RNA
Fibrillar Collagens
Cycloheximide
Glomerulonephritis
Immunoblotting
Genes
Cicatrix
Extracellular Matrix
Research Design
Embryonic Structures
Cell Culture Techniques

Keywords

  • Basement membrane
  • Collagen IV
  • Fibroblast
  • Transcription

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

Transforming growth factor-β1 induces collagen IV gene expression in NIH- 3T3 cells. / Grande, Joseph Peter; Melder, D.; Zinsmeister, A.; Killen, P.

In: Laboratory Investigation, Vol. 69, No. 4, 1993, p. 387-395.

Research output: Contribution to journalArticle

Grande, JP, Melder, D, Zinsmeister, A & Killen, P 1993, 'Transforming growth factor-β1 induces collagen IV gene expression in NIH- 3T3 cells', Laboratory Investigation, vol. 69, no. 4, pp. 387-395.
Grande, Joseph Peter ; Melder, D. ; Zinsmeister, A. ; Killen, P. / Transforming growth factor-β1 induces collagen IV gene expression in NIH- 3T3 cells. In: Laboratory Investigation. 1993 ; Vol. 69, No. 4. pp. 387-395.
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abstract = "BACKGROUND: While recent studies have implicated transforming growth factor-β1 (TGF-β1) in the development of glomerular scarring, extraglomerular matrix production is frequently associated with glomerulonephritis and is an important determinant of disease progression. TGF-β1 may be an important mediator of extracellular matrix synthesis, both by glomerular and extraglomerular mesenchymal cells. TGF-β1-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-β1. Additional studies were performed using normal rat kidney cells (NRK-49F). EXPERIMENTAL DESIGN: Cells were grown in medium supplemented with 0.5{\%} calf serum for 24 hours before treatment with TGF-β1. RNA was isolated after the addition of varying amounts of TGF-β1 to the cells in culture for varying periods of time, and collagen α1(IV) RNA was quantitated by filter hybridization. Transcription of the α1(IV) and α2(IV) collagen genes was assessed by an in vitro transcription assay. Deposition of collagen IV was identified by immunoblotting. RESULTS: Induction of α1(IV) gene expression by NIH-3T3 cells and by NRK-49F cells was first seen 2 to 4 hours after TGF- β1 treatment, and was maximal after 12 to 18 hours. Maximal induction was observed following addition of 5 ng/ml TGF-β1 to NIH-3T3 cells, and following addition of 10 ng/ml of TGF-β1 to NRK-49F cells. In the presence of cycloheximide, TGF-β1 induction of α1(IV) mRNA was markedly attenuated in both cell lines, suggesting that this effect of TGF-β1 requires protein synthesis. TGF-β1 increased transcription of both the α1(IV) and α2(IV) collagen genes by NIH-3T3 cells. CONCLUSIONS: TGF-β1 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.",
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AU - Grande, Joseph Peter

AU - Melder, D.

AU - Zinsmeister, A.

AU - Killen, P.

PY - 1993

Y1 - 1993

N2 - BACKGROUND: While recent studies have implicated transforming growth factor-β1 (TGF-β1) in the development of glomerular scarring, extraglomerular matrix production is frequently associated with glomerulonephritis and is an important determinant of disease progression. TGF-β1 may be an important mediator of extracellular matrix synthesis, both by glomerular and extraglomerular mesenchymal cells. TGF-β1-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-β1. Additional studies were performed using normal rat kidney cells (NRK-49F). EXPERIMENTAL DESIGN: Cells were grown in medium supplemented with 0.5% calf serum for 24 hours before treatment with TGF-β1. RNA was isolated after the addition of varying amounts of TGF-β1 to the cells in culture for varying periods of time, and collagen α1(IV) RNA was quantitated by filter hybridization. Transcription of the α1(IV) and α2(IV) collagen genes was assessed by an in vitro transcription assay. Deposition of collagen IV was identified by immunoblotting. RESULTS: Induction of α1(IV) gene expression by NIH-3T3 cells and by NRK-49F cells was first seen 2 to 4 hours after TGF- β1 treatment, and was maximal after 12 to 18 hours. Maximal induction was observed following addition of 5 ng/ml TGF-β1 to NIH-3T3 cells, and following addition of 10 ng/ml of TGF-β1 to NRK-49F cells. In the presence of cycloheximide, TGF-β1 induction of α1(IV) mRNA was markedly attenuated in both cell lines, suggesting that this effect of TGF-β1 requires protein synthesis. TGF-β1 increased transcription of both the α1(IV) and α2(IV) collagen genes by NIH-3T3 cells. CONCLUSIONS: TGF-β1 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.

AB - BACKGROUND: While recent studies have implicated transforming growth factor-β1 (TGF-β1) in the development of glomerular scarring, extraglomerular matrix production is frequently associated with glomerulonephritis and is an important determinant of disease progression. TGF-β1 may be an important mediator of extracellular matrix synthesis, both by glomerular and extraglomerular mesenchymal cells. TGF-β1-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-β1. Additional studies were performed using normal rat kidney cells (NRK-49F). EXPERIMENTAL DESIGN: Cells were grown in medium supplemented with 0.5% calf serum for 24 hours before treatment with TGF-β1. RNA was isolated after the addition of varying amounts of TGF-β1 to the cells in culture for varying periods of time, and collagen α1(IV) RNA was quantitated by filter hybridization. Transcription of the α1(IV) and α2(IV) collagen genes was assessed by an in vitro transcription assay. Deposition of collagen IV was identified by immunoblotting. RESULTS: Induction of α1(IV) gene expression by NIH-3T3 cells and by NRK-49F cells was first seen 2 to 4 hours after TGF- β1 treatment, and was maximal after 12 to 18 hours. Maximal induction was observed following addition of 5 ng/ml TGF-β1 to NIH-3T3 cells, and following addition of 10 ng/ml of TGF-β1 to NRK-49F cells. In the presence of cycloheximide, TGF-β1 induction of α1(IV) mRNA was markedly attenuated in both cell lines, suggesting that this effect of TGF-β1 requires protein synthesis. TGF-β1 increased transcription of both the α1(IV) and α2(IV) collagen genes by NIH-3T3 cells. CONCLUSIONS: TGF-β1 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.

KW - Basement membrane

KW - Collagen IV

KW - Fibroblast

KW - Transcription

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