Kroepelien C F, Knutsen H K, Haugen T B, Hansson V, Eskild W
Institute of Medical Biochemistry, University of Oslo, Norway.
Endocrinology. 1993 Mar;132(3):968-74. doi: 10.1210/endo.132.3.8440198.
This report shows that serum factors dramatically increase the levels of mRNA for cellular retinol-binding protein (CRBP) in cultured rat Sertoli cells. Incubation of rat Sertoli cells (0-24 h) with 10% fetal calf serum (FCS) was associated with a time-dependent increase in CRBP mRNA levels. A significant increase (6-fold) was observed after 3 h of incubation. Maximal levels (15- to 50-fold) were reached after 9-12 h and were maintained for as long as serum was present. The effect was concentration dependent, with maximal induction at 10% FCS. Removal of FCS resulted in a decline in the CRBP mRNA levels, with a t1/2 of approximately 7 h. The CRBP mRNA stimulating activity (CMSA) was completely removed from FCS by precipitation with 5% trichloroacetic acid, but was only partly (50%) inhibited by heating at 100 C or trypsin treatment. Removal of retinol from FCS by repeated ether extractions did not alter the CMSA of FCS. Both the induction and degradation of CRBP mRNA were inhibited by the protein synthesis inhibitor cycloheximide. A nuclear protein binding to the 5'-flanking region of the CRBP gene was detected in nuclear extracts from untreated Sertoli cells, but not in nuclear extracts from Sertoli cells treated with 10% FCS for 3 h. Thus, serum factors, different from retinoids, dramatically stimulate the levels of CRBP mRNA in rat Sertoli cells. This is associated with the loss of protein binding to the 5'-flanking region of the CRBP gene.
本报告显示，血清因子可显著提高培养的大鼠支持细胞中细胞视黄醇结合蛋白（CRBP）的mRNA水平。用10%胎牛血清（FCS）孵育大鼠支持细胞（0 - 24小时），CRBP mRNA水平随时间呈依赖性增加。孵育3小时后观察到显著增加（6倍）。9 - 12小时后达到最高水平（15 - 50倍），并且只要有血清存在就会维持该水平。该效应具有浓度依赖性，在10% FCS时诱导作用最强。去除FCS导致CRBP mRNA水平下降，半衰期约为7小时。通过用5%三氯乙酸沉淀可将FCS中的CRBP mRNA刺激活性（CMSA）完全去除，但在100℃加热或胰蛋白酶处理仅部分（50%）抑制该活性。通过反复乙醚萃取从FCS中去除视黄醇并不改变FCS的CMSA。蛋白质合成抑制剂环己酰亚胺可抑制CRBP mRNA的诱导和降解。在未处理的支持细胞核提取物中检测到一种与CRBP基因5'侧翼区域结合的核蛋白，但在用10% FCS处理3小时的支持细胞核提取物中未检测到。因此，与类维生素A不同的血清因子可显著刺激大鼠支持细胞中CRBP mRNA的水平。这与CRBP基因5'侧翼区域蛋白质结合的丧失有关。
