Departamento de Bioquímica y Biología Molecular, Instituto de Neurociencias de Castilla y León (INCYL), Universidad de Salamanca, Salamanca, Spain.
PLoS One. 2012;7(2):e32448. doi: 10.1371/journal.pone.0032448. Epub 2012 Feb 23.
In previous work we showed that endothelin-1 (ET-1) increases the rate of glucose uptake in astrocytes, an important aspect of brain function since glucose taken up by astrocytes is used to supply the neurons with metabolic substrates. In the present work we sought to identify the signalling pathway responsible for this process in primary culture of rat astrocytes. Our results show that ET-1 promoted an increase in the transcription factor hypoxia-inducible factor-1α (HIF-1α) in astrocytes, as shown in other cell types. Furthermore, HIF-1α-siRNA experiments revealed that HIF-1α participates in the effects of ET-1 on glucose uptake and on the expression of GLUT-1, GLUT-3, type I and type II hexokinase. We previously reported that these effects of ET-1 are mediated by connexin43 (Cx43), the major gap junction protein in astrocytes. Indeed, our results show that silencing Cx43 increased HIF-1α and reduced the effect of ET-1 on HIF-1α, indicating that the effect of ET-1 on HIF-1α is mediated by Cx43. The activity of oncogenes such as c-Src can up-regulate HIF-1α. Since Cx43 interacts with c-Src, we investigated the participation of c-Src in this pathway. Interestingly, both the treatment with ET-1 and with Cx43-siRNA increased c-Src activity. In addition, when c-Src activity was inhibited neither ET-1 nor silencing Cx43 were able to up-regulate HIF-1α. In conclusion, our results suggest that ET-1 by down-regulating Cx43 activates c-Src, which in turn increases HIF-1α leading to the up-regulation of the machinery required to take up glucose in astrocytes. Cx43 expression can be reduced in response not only to ET-1 but also to various physiological and pathological stimuli. This study contributes to the identification of the signalling pathway evoked after Cx43 down-regulation that results in increased glucose uptake in astrocytes. Interestingly, this is the first evidence linking Cx43 to HIF-1, which is a master regulator of glucose metabolism.
在之前的工作中，我们发现内皮素-1（ET-1）可增加星形胶质细胞的葡萄糖摄取率，这是大脑功能的一个重要方面，因为星形胶质细胞摄取的葡萄糖可用于为神经元提供代谢底物。在本研究中，我们试图确定 ET-1 在原代培养的大鼠星形胶质细胞中发挥作用的信号通路。我们的结果表明，ET-1 可促进星形胶质细胞中转录因子缺氧诱导因子-1α（HIF-1α）的转录，这与其他细胞类型的结果一致。此外，HIF-1α-siRNA 实验表明，HIF-1α 参与了 ET-1 对葡萄糖摄取以及 GLUT-1、GLUT-3、I 型和 II 型己糖激酶表达的影响。我们之前报道过，ET-1 的这些作用是通过缝隙连接蛋白 43（Cx43）介导的，Cx43 是星形胶质细胞中的主要缝隙连接蛋白。事实上，我们的结果表明，沉默 Cx43 可增加 HIF-1α，减少 ET-1 对 HIF-1α 的作用，表明 ET-1 对 HIF-1α 的作用是通过 Cx43 介导的。致癌基因（如 c-Src）的活性可上调 HIF-1α。由于 Cx43 与 c-Src 相互作用，我们研究了 c-Src 在该通路中的参与情况。有趣的是，ET-1 处理和 Cx43-siRNA 处理均增加了 c-Src 的活性。此外，当抑制 c-Src 活性时，ET-1 或沉默 Cx43 均不能上调 HIF-1α。总之，我们的结果表明，ET-1 通过下调 Cx43 激活 c-Src，c-Src 又增加 HIF-1α，从而增加星形胶质细胞摄取葡萄糖所需的机制的表达。Cx43 的表达不仅可响应 ET-1 而且可响应各种生理和病理刺激而减少。本研究有助于确定 Cx43 下调后引发的信号通路，该通路可导致星形胶质细胞中葡萄糖摄取增加。有趣的是，这是首次将 Cx43 与 HIF-1 联系起来的证据，HIF-1 是葡萄糖代谢的主要调节因子。
