Hanson R D, Connolly N L, Burnett D, Campbell E J, Senior R M, Ley T J
Department of Medicine Jewish Hospital, Washington University Medical Center, St. Louis, Missouri 63110.
J Biol Chem. 1990 Jan 25;265(3):1524-30.
Cathepsin G is a neutral serine protease that is found in the azurophil granules of neutrophils and monocytes. Previous experiments had demonstrated that cathepsin G is actively produced by the promonocytic U937 cell line, and that 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of these cells toward macrophages resulted in a reduction of cathepsin G activity. In this study, we have analyzed the mechanism of this TPA-induced down-regulatory event. Using in situ hybridization techniques, we demonstrated that cathepsin G mRNA is detectable only at the promyelocytic stage of myeloid development. Using U937 promonocytic cells as a model, we demonstrated; 1) cathepsin G protein levels decline in TPA-treated cells; 2) this decline was due to a nearly complete loss of cathepsin G mRNA in cells treated with TPA for 24 h; and 3) the rate of cathepsin G mRNA loss with TPA treatment was similar to that with actinomycin D. These results suggested that cathepsin G transcription was down-regulated within several hours of TPA addition. This was directly tested by performing nuclear run-off assays of TPA-treated U937 cells; cathepsin G transcription was shown to be strand-specific, and declined within 4 h of TPA addition. Cathepsin G transcription was essentially undetectable 8 or more hours after TPA treatment, suggesting that down-regulation is predominantly transcriptional. Cycloheximide treatment of U937 cells resulted in a partial block of TPA-mediated cathepsin G down-regulation, indicating that continuous protein synthesis is required for down-regulation to occur. A newly synthesized protein or proteins may therefore be required for the transcriptional down-regulation of cathepsin G during the normal development of promyelocytes or promonocytes.
组织蛋白酶G是一种中性丝氨酸蛋白酶，存在于中性粒细胞和单核细胞的嗜天青颗粒中。先前的实验表明，组织蛋白酶G由单核细胞U937细胞系主动产生，并且12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）诱导这些细胞向巨噬细胞分化会导致组织蛋白酶G活性降低。在本研究中，我们分析了这种TPA诱导的下调事件的机制。使用原位杂交技术，我们证明组织蛋白酶G mRNA仅在髓系发育的早幼粒细胞阶段可检测到。以U937单核细胞为模型，我们证明：1）TPA处理的细胞中组织蛋白酶G蛋白水平下降；2）这种下降是由于TPA处理24小时的细胞中组织蛋白酶G mRNA几乎完全丧失；3）TPA处理导致组织蛋白酶G mRNA丧失的速率与放线菌素D处理相似。这些结果表明，在添加TPA后的数小时内，组织蛋白酶G转录被下调。通过对TPA处理的U937细胞进行核转录延伸分析直接验证了这一点；组织蛋白酶G转录显示为链特异性，并且在添加TPA后4小时内下降。TPA处理8小时或更长时间后，组织蛋白酶G转录基本检测不到，表明下调主要是转录水平的。用环己酰亚胺处理U937细胞导致TPA介导的组织蛋白酶G下调部分受阻，表明下调发生需要持续的蛋白质合成。因此，在早幼粒细胞或单核细胞的正常发育过程中，组织蛋白酶G转录下调可能需要一种新合成的蛋白质。
