Rishikof D C, Kuang P P, Poliks C, Goldstein R H
Pulmonary Center, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118, USA.
Biochem J. 1998 Apr 15;331 ( Pt 2)(Pt 2):417-22. doi: 10.1042/bj3310417.
The steady-state level of alpha1(I) collagen mRNA is regulated by amino acid availability in human lung fibroblasts. Depletion of amino acids decreases alpha1(I) collagen mRNA levels and repletion of amino acids induces rapid re-expression of alpha1(I) mRNA. In these studies, we examined the requirements for individual amino acids on the regulation of alpha1(I) collagen mRNA. We found that re-expression of alpha1(I) collagen mRNA was critically dependent on cystine but not on other amino acids. However, the addition of cystine alone did not result in re-expression of alpha1(I) collagen mRNA. Following amino acid depletion, the addition of cystine with selective amino acids increased alpha1(I) collagen mRNA levels. The combination of glutamine and cystine increased alpha1(I) collagen mRNA levels 6.3-fold. Methionine or a branch-chain amino acid (leucine, isoleucine or valine) also acted in combination with cystine to increase alpha1(I) collagen mRNA expression, whereas other amino acids were not effective. The prolonged absence of cystine lowered steady-state levels of alpha1(I) collagen mRNA through a mechanism involving decreases in both the rate of gene transcription as assessed by nuclear run-on experiments and mRNA stability as assessed by half-life determination in the presence of actinomycin D. The effect of cystine was not mediated via alterations in the level of glutathione, the major redox buffer in cells, as determined by the addition of buthionine sulphoximine, an inhibitor of gamma-glutamylcysteine synthetase. These data suggest that cystine directly affects the regulation of alpha1(I) collagen mRNA.
α1（I）型胶原蛋白mRNA的稳态水平受人类肺成纤维细胞中氨基酸可用性的调节。氨基酸耗竭会降低α1（I）型胶原蛋白mRNA水平，而氨基酸补充则会诱导α1（I）mRNA快速重新表达。在这些研究中，我们研究了单个氨基酸对α1（I）型胶原蛋白mRNA调节的需求。我们发现α1（I）型胶原蛋白mRNA的重新表达严重依赖于胱氨酸，而不依赖于其他氨基酸。然而，单独添加胱氨酸并不会导致α1（I）型胶原蛋白mRNA的重新表达。在氨基酸耗竭后，将胱氨酸与选择性氨基酸一起添加可提高α1（I）型胶原蛋白mRNA水平。谷氨酰胺和胱氨酸的组合使α1（I）型胶原蛋白mRNA水平提高了6.3倍。甲硫氨酸或支链氨基酸（亮氨酸、异亮氨酸或缬氨酸）也与胱氨酸协同作用以增加α1（I）型胶原蛋白mRNA表达，而其他氨基酸则无效。长期缺乏胱氨酸会通过一种机制降低α1（I）型胶原蛋白mRNA的稳态水平，该机制涉及通过核转录实验评估的基因转录速率降低以及通过在放线菌素D存在下半衰期测定评估的mRNA稳定性降低。如通过添加丁硫氨酸亚砜胺（γ-谷氨酰半胱氨酸合成酶的抑制剂）所确定的，胱氨酸的作用不是通过细胞中主要氧化还原缓冲剂谷胱甘肽水平的改变介导的。这些数据表明胱氨酸直接影响α1（I）型胶原蛋白mRNA的调节。
