Expression of Manduca sexta V-ATPase genes mvB, mvG and mvd is regulated by ecdysteroids.
Expression of Manduca sexta V-ATPase genes mvB, mvG and mvd is regulated by ecdysteroids.
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发表时间：
2002-04
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通讯作者：
S. Reineke;H. Wieczorek;H. Merzendorfer
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作者：
S. Reineke;H. Wieczorek;H. Merzendorfer
V- atp酶是由外周atp水解V(1)复合体和膜结合H(+)-易位V(o)复合体组成的复杂蛋白质。烟草角虫（Manduca sexta）中肠的质膜V- atp酶由8个不同的V(1)和4个不同的V(o)亚基组成。在饥饿和蜕皮过程中，由于V(1)复合物与V(o)复合物的分离，V- atp酶活性降低。为了确定在酶失活期间亚基生物合成是否减少，我们测量了v - atp酶基因的转录水平和转录活性。Northern印迹显示饥饿期间几乎所有V-ATPase转录本下调。在换羽过程中，V-ATPase基因mvB、mvG和mvd的转录水平也有所下降，且与20-羟基蜕皮激素（20-HE）滴度呈负相关，与幼体激素（JH）滴度呈正相关。为了测试这些相关性的生物学意义，我们将这两种激素注射到喂养的幼虫中，并在几小时后测量转录水平。注射20-HE后，mRNA水平短期升高，长期降低，而注射JH无显著影响。对中肠上皮的免疫组织化学研究显示，注射20-HE导致杯状细胞形态和V(1)复合体亚细胞分布的变化，与脱毛和饥饿期间的情况相似。在Sf21细胞中使用mvB、mvG和mvd启动子来启动萤火虫荧光素酶的转录，在20-HE孵育细胞后，结果与注射实验中获得的结果相当。这些发现表明，假定的蜕皮激素反应元件存在于所有三种启动子中。综上所述，我们的研究结果表明，v - atp酶基因的表达是由表皮类固醇协调控制的。
V-ATPases are complex proteins consisting of a peripheral, ATP-hydrolysing V(1) complex and a membrane-bound H(+)-translocating V(o) complex. The plasma membrane V-ATPase from the tobacco hornworm (Manduca sexta) midgut is made up of eight different V(1) and four different V(o) subunits. During starvation and moulting, V-ATPase activity decreases as a result of the dissociation of the V(1) complex from the V(o) complex. To determine whether subunit biosynthesis is reduced during periods of enzyme inactivity, we measured the transcript levels and transcriptional activities of V-ATPase genes. Northern blots revealed the downregulation of almost all V-ATPase transcripts during starvation. During moulting, transcript levels of the three V-ATPase genes examined, mvB, mvG and mvd, also decreased, and this decrease was negatively correlated with the titre of 20-hydroxyecdysone (20-HE) and positively correlated with the titre of juvenile hormone (JH). To test the biological significance of these correlations, we injected both hormones into feeding larvae and measured transcript levels several hours later. A short-term increase and a long-term decrease in levels of mRNA were observed after 20-HE injection, whereas JH injection had no significant effect. Immunohistochemical studies of the midgut epithelium revealed that 20-HE injection led to changes in goblet cell morphology and in the subcellular distribution of the V(1) complex comparable with the situation during the moult and during starvation. Reporter gene assays in Sf21 cells using mvB, mvG and mvd promoters to initiate transcription of firefly luciferase led, after incubation of the cells with 20-HE, to results comparable with those obtained in the injection experiments. These findings suggest that putative ecdysone-responsive elements are present in all three promoters. Taken together, our results suggest that the expression of V-ATPase genes is controlled in a coordinated manner by ecdysteroids.