Grande J, Melder D, Zinsmeister A, Killen P
Department of Pathology, Mayo Graduate School of Medicine, Rochester, Minnesota.
Lab Invest. 1993 Oct;69(4):387-95.
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).
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.
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.
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.
尽管最近的研究表明转化生长因子-β1（TGF-β1）与肾小球瘢痕形成有关，但肾小球外基质的产生常与肾小球肾炎相关，并且是疾病进展的重要决定因素。TGF-β1可能是肾小球和肾小球外间充质细胞合成细胞外基质的重要介质。在两种间充质细胞系中研究了TGF-β1介导的IV型胶原基因表达。最初的研究使用NIH-3T3细胞，这是一种源自鼠胚胎的成纤维细胞样细胞系，已被用于研究TGF-β1对纤维状胶原（I型胶原和III型胶原）合成的调节。另外的研究使用正常大鼠肾细胞（NRK-49F）进行。
在用TGF-β1处理前，细胞在补充有0.5%小牛血清的培养基中培养24小时。在向培养的细胞中加入不同量的TGF-β1并培养不同时间后分离RNA，通过滤膜杂交对α1(IV)胶原RNA进行定量。通过体外转录试验评估α1(IV)和α2(IV)胶原基因的转录。通过免疫印迹鉴定IV型胶原的沉积。
NIH-3T3细胞和NRK-49F细胞在TGF-β1处理后2至4小时首次出现α1(IV)基因表达的诱导，12至18小时后达到最大值。向NIH-3T3细胞中加入5 ng/ml TGF-β1以及向NRK-49F细胞中加入10 ng/ml TGF-β1后观察到最大诱导。在存在放线菌酮时，两种细胞系中TGF-β1对α1(IV) mRNA的诱导均明显减弱，这表明TGF-β1的这种作用需要蛋白质合成。TGF-β1增加了NIH-3T3细胞中α1(IV)和α2(IV)胶原基因的转录。
TGF-β1在NIH-3T3细胞和正常大鼠肾成纤维细胞（NRK-49F细胞）中均诱导IV型胶原基因表达。利用这些细胞系对细胞因子介导的IV型胶原转录调节进行进一步研究，可能会提供有关肾小球外基质产生在肾脏疾病进展中作用的重要信息。
