Bellon G, Chaqour B, Wegrowski Y, Monboisse J C, Borel J P
Laboratory of Biochemistry, CNRS ER X084, Faculty of Medecine, University of Reims, France.
Biochim Biophys Acta. 1995 Sep 21;1268(3):311-23. doi: 10.1016/0167-4889(95)00093-8.
We have previously shown that glutamine stimulates the synthesis of collagen in human dermal confluent fibroblast cultures (Bellon, G. et al. [1987] Biochim. Biophys. Acta, 930, 39-47). In this paper, we examine the effects of glutamine on collagen gene expression. A dose-dependent effect of glutamine on collagen synthesis was demonstrated from 0 to 0.25 mM followed by a plateau up to 10 mM glutamine. Depending on the cell population, collagen synthesis was increased by 1.3-to 2.3-fold. The mean increase in collagen and non-collagen protein synthesis was 63% and 18% respectively. Steady-state levels of alpha 1(I) and alpha 1(III) mRNAs, were measured by hybridizing total RNA to specific cDNA probes at high stringency. Glutamine increased the steady-state level of collagen alpha 1(I) and alpha 1(III) mRNAs in a dose-dependent manner. At 0.15 mM glutamine, collagen mRNAs were increased by 1.7-and 2.3-fold respectively. Nuclear run-off experiments at this concentration of glutamine indicated that the transcriptional activity was increased by 3.4-fold for the pro alpha 1(I) collagen gene. The effect of glutamine on gene transcription was also supported by the measurement of pro alpha 1(I) collagen mRNA half-life since glutamine did not affect its stability. Protein synthesis seemed to be required for the glutamine-dependent induction of collagen gene expression since cycloheximide suppressed the activation. The effect of glutamine appeared specific because analogues and/or derivatives of glutamine, such as acivicin, 6-diazo-5-oxo-L-norleucine, homoglutamine, ammonium chloride and glutamate did not replace glutamine. The influence of amino acid transport systems through plasma membrane was assessed by the use of 2(methylamino)-isobutyric acid and beta 2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid. The glutamine-dependent induction of collagen gene expression was found to be independent of transport system A but dependent on transport system L whose inhibition induced a decrease in pro alpha 1(I) collagen gene transcription by an unknown mechanism. Thus, glutamine, at physiological concentrations, indirectly regulates collagen gene expression.
我们之前已经表明，谷氨酰胺能刺激人真皮汇合的成纤维细胞培养物中胶原蛋白的合成（贝隆，G.等人[1987年]《生物化学与生物物理学报》，930，39 - 47）。在本文中，我们研究了谷氨酰胺对胶原蛋白基因表达的影响。从0到0.25 mM，谷氨酰胺对胶原蛋白合成呈现剂量依赖性效应，随后在高达10 mM谷氨酰胺时达到平台期。根据细胞群体的不同，胶原蛋白合成增加了1.3至2.3倍。胶原蛋白和非胶原蛋白合成的平均增加分别为63%和18%。通过在高严谨度下将总RNA与特异性cDNA探针杂交来测量α1（I）和α1（III）mRNA的稳态水平。谷氨酰胺以剂量依赖性方式增加胶原蛋白α1（I）和α1（III）mRNA的稳态水平。在0.15 mM谷氨酰胺时，胶原蛋白mRNA分别增加了1.7倍和2.3倍。在此谷氨酰胺浓度下进行的核转录实验表明，对于原α1（I）胶原蛋白基因，转录活性增加了3.4倍。谷氨酰胺对基因转录的影响也得到了原α1（I）胶原蛋白mRNA半衰期测量结果的支持，因为谷氨酰胺不影响其稳定性。由于环己酰亚胺抑制了这种激活作用，所以蛋白质合成似乎是谷氨酰胺依赖性诱导胶原蛋白基因表达所必需的。谷氨酰胺的作用似乎具有特异性，因为谷氨酰胺的类似物和/或衍生物，如阿西维辛、6 - 重氮 - 5 - 氧代 - L - 正亮氨酸、高谷氨酰胺、氯化铵和谷氨酸并不能替代谷氨酰胺。通过使用2 - （甲氨基） - 异丁酸和β2 - 氨基双环 - （2.2.1） - 庚烷 - 2 - 羧酸评估了氨基酸通过质膜的转运系统的影响。发现谷氨酰胺依赖性诱导胶原蛋白基因表达独立于转运系统A，但依赖于转运系统L，其抑制通过未知机制导致原α1（I）胶原蛋白基因转录减少。因此，在生理浓度下，谷氨酰胺间接调节胶原蛋白基因表达。
