Ismail Rehana, Ul Hussain Mahboob
Department of Biotechnology, Hazratbal Srinagar, University of Kashmir, Jammu and Kashmir, India.
Department of Biotechnology, Hazratbal Srinagar, University of Kashmir, Jammu and Kashmir, India.
Biochimie. 2017 Aug;139:38-45. doi: 10.1016/j.biochi.2017.05.012. Epub 2017 May 22.
Astrocytes cope-up the hypoxia conditions by up regulating the activity of the enzymes catalyzing the irreversible steps of the glycolytic pathway. The phosphofructokinase1 (PFK1), which converts fructose-6-phosphate to fructose-1, 6-bisphosphate, is the major regulatory enzyme of the glycolytic pathway. For this purpose, we investigated the expression regulation of the PFK1 during chemically induced hypoxia.
After 48 h of the chemically induced hypoxia induction of the C6 glioma cells, the PFK1 protein depicted strong up regulation, with no appreciable change in its mRNA levels. The di-cistronic assay indicated the presence of a weak internal ribosome entry site (IRES) element in the 5'UTR of the PFK1 mRNA. Interestingly, the weak IRES element of the PFK1 was strongly up regulated after 48 h of the chemically induced hypoxia, indicative of a possible mechanism responsible for the induction of the PFK1 protein. The authenticity of the hypoxia-regulated IRES element of the PFK1, relative to the presence of the cryptic promoter element and/or the cryptic splicing was established using promoterless di-cistronic assay and the RT-PCR analysis. Moreover, the ectopic expression of the polypyrimidine tract binding (PTB) protein resulted in the enhanced activity of the IRES element of the PFK1. Additionally, it was established that the chemically induced hypoxia resulted in the increased shuttling of the PTB from the cell nucleus to the cytosol.
The presence of a hypoxia responsive IRES element, in the 5'UTR of the PFK1 was established to be the possible mechanism responsible for the up regulation of the PFK1 protein. Our data provides an interesting mechanism that may explain the increased glycolytic capacity of the astrocytes after brain hypoxia.
星形胶质细胞通过上调催化糖酵解途径不可逆步骤的酶的活性来应对缺氧条件。磷酸果糖激酶1（PFK1）将6-磷酸果糖转化为1,6-二磷酸果糖，是糖酵解途径的主要调节酶。为此，我们研究了化学诱导缺氧过程中PFK1的表达调控。
在对C6胶质瘤细胞进行化学诱导缺氧48小时后，PFK1蛋白呈现强烈上调，其mRNA水平无明显变化。双顺反子检测表明PFK1 mRNA的5'UTR中存在一个弱的内部核糖体进入位点（IRES）元件。有趣的是，化学诱导缺氧48小时后，PFK1的弱IRES元件被强烈上调，这表明可能存在一种导致PFK1蛋白诱导的机制。使用无启动子双顺反子检测和RT-PCR分析确定了PFK1缺氧调节的IRES元件相对于隐蔽启动子元件和/或隐蔽剪接的真实性。此外，多嘧啶序列结合（PTB）蛋白的异位表达导致PFK1的IRES元件活性增强。另外，已确定化学诱导缺氧导致PTB从细胞核向细胞质的穿梭增加。
已确定PFK1的5'UTR中存在缺氧反应性IRES元件是导致PFK1蛋白上调的可能机制。我们的数据提供了一个有趣机制，可能解释脑缺氧后星形胶质细胞糖酵解能力增加的原因。
