Solis-Herruzo J A, Brenner D A, Chojkier M
Department of Medicine, Veterans Administration Medical Center, San Diego, California.
J Biol Chem. 1988 Apr 25;263(12):5841-5.
The effects of recombinant human tumor necrosis factor alpha (TNF alpha) on collagen production and gene expression in cultured fibroblasts were studied. Cells were labeled with [3H]proline, and the radioactivity of collagenase-sensitive and -resistant proteins were used to calculate the rates of protein production. The net production of collagen relative to total proteins was inhibited by TNF alpha (0-1.2 nM) in a dose- and time-related manner. The specific activities of the free [3H]proline pool, which were similar in control and TNF alpha-treated cells, were used to calculate the absolute rates of protein production. The absolute rate of collagen production was decreased by 50% in the presence of 1.2 nM TNF alpha during 24-h incubations (851 +/- 104 versus 426 +/- 39 pmol/micrograms of DNA/h; p less than 0.01), whereas noncollagen protein production and the rate of procollagen secretion were unchanged. We found no evidence of cellular toxicity in cultured cells treated with TNF alpha. In addition, TNF alpha did not affect cell proliferation as determined by [6-3H]thymidine incorporation into DNA. Most of the collagen produced by the cultured fibroblasts was type I. Using hybridization with specific DNA probes there was an approximately 50% decrease in the quantity of procollagen alpha 1(I) mRNA, without changes in the quantity of alpha tubulin mRNA or the size of the transcripts, in cells incubated with TNF alpha. Interleukin-1 (2.5 ng/ml) also decreased the levels of procollagen alpha 1(I) mRNA by approximately 50%. Cycloheximide (0.1 mM), an inhibitor of protein synthesis, blocked the inhibitory effect of both TNF alpha and interleukin-1 on procollagen alpha 1(I) mRNA. Nuclear run-off assays demonstrated that TNF alpha decreased procollagen alpha 1(I) transcriptional activity by 50% and had no effects on alpha tubulin gene transcription. Thus, TNF alpha decreases collagen gene transcription, collagen mRNA levels, and collagen production in cultured fibroblasts.
研究了重组人肿瘤坏死因子α（TNFα）对培养的成纤维细胞中胶原蛋白产生和基因表达的影响。用[3H]脯氨酸标记细胞，利用胶原酶敏感和抗性蛋白的放射性来计算蛋白质产生速率。相对于总蛋白的胶原蛋白净产生量受到TNFα（0 - 1.2 nM）的剂量和时间依赖性抑制。对照细胞和TNFα处理细胞中游离[3H]脯氨酸池的比活性相似，用于计算蛋白质产生的绝对速率。在24小时孵育期间，1.2 nM TNFα存在时胶原蛋白产生的绝对速率降低了50%（851±104对426±39 pmol/μg DNA/h；p＜0.01），而非胶原蛋白产生和前胶原分泌速率未改变。我们未发现用TNFα处理的培养细胞存在细胞毒性的证据。此外，如通过[6 - 3H]胸苷掺入DNA所测定，TNFα不影响细胞增殖。培养的成纤维细胞产生的大部分胶原蛋白为I型。使用与特异性DNA探针杂交，在用TNFα孵育的细胞中，前胶原α1（I）mRNA的量减少了约50%，而α微管蛋白mRNA的量或转录本大小未改变。白细胞介素-1（2.5 ng/ml）也使前胶原α1（I）mRNA水平降低了约50%。蛋白质合成抑制剂环己酰亚胺（0.1 mM）阻断了TNFα和白细胞介素-1对前胶原α1（I）mRNA的抑制作用。细胞核转录分析表明，TNFα使前胶原α1（I）转录活性降低了50%，对α微管蛋白基因转录无影响。因此，TNFα降低培养的成纤维细胞中胶原蛋白基因转录、胶原蛋白mRNA水平和胶原蛋白产生。
