Functional analysis and regulation of epididymal OCTN2

描述（由申请方提供）：保护细胞免受渗透压胁迫对其存活至关重要，因为如果不受保护，细胞将发生凋亡。附睾腔液是高渗的，因此附睾细胞需要适应较高的渗透压以限制渗透压应激的影响。与肾脏一样，附睾也积累渗透压调节剂以适应渗透压的变化。渗透压调节剂的移动通过特定转运蛋白实现，例如转运渗透压调节剂肌醇的钠-肌醇共转运蛋白。左旋肉碱是另一种渗透压调节剂，在高渗性适应中起关键作用，在附睾腔液中发现高浓度左旋肉碱。因此，本研究将研究L-肉毒碱的转运蛋白，新型有机阳离子转运蛋白2（OCTN 2），如何在附睾细胞适应高渗环境并最终保护自身免受渗透压应激中发挥关键作用。我们的工作假设是附睾细胞的渗透压导致离子内流，从而增加细胞内离子强度。这种增加激活MAPK途径，其继而诱导高渗性应答性转录因子（例如TonEBP）的转录。然后，这些转录因子结合到OCTN 2启动子上的同源结合位点，并允许OCTN 2的转录进行。然后，转运蛋白被翻译，被运输到基底外侧膜，L-肉毒碱被运输到细胞中。渗透剂L-肉毒碱的积累将通过稳定关键蛋白质和DNA来抵消离子的影响。在此阶段，附睾细胞已经适应了新的渗透环境，并提供了自己的保护，免受渗透胁迫。具体而言，将检验以下假设：（1）OCTN 2负责将L-肉毒碱转运到附睾中;（2）OCTN 2启动子受张力响应性转录因子调节;（3）OCTN 2转录和转运活性通过特定信号转导途径受张力变化调节;（4）OCTN 2的缺失导致附睾上皮细胞适应高渗环境的能力丧失，从而导致对渗透应激的易感性，导致附睾功能障碍和男性不育。这项建议的结果将为治疗某些形式的男性不育症和开发男性避孕药提供基本信息。

DESCRIPTION (provided by applicant): Protection of cells from osmotic stress is critical for their survival as cells will undergo apoptosis if not protected. Epididymal luminal fluid is hypertonic and therefore epididymal cells need to adapt to the higher osmolality to limit the effects of osmotic stress. Like the kidney, the epididymis accumulates osmolytes to adapt to changes in osmolality. Movement of osmolytes is achieved by specific transporters, e.g. sodium-myo inositol cotransporter which transports the osmolyte myo-inositol. L-carnitine is another osmolyte that plays a key role in the adaption of hyperosmolality and is found in high concentrations in epididymal luminal fluid. Therefore, this study will examine how the transporter for L-carnitine, Novel Organic Cation Transporter 2 (OCTN2), plays a critical role in allowing epididymal cells to adapt to a hyperosmotic environment and ultimately protecting themselves from osmotic stress. Our working hypothesis is that osmotic stress of epididymal cells results in an influx of ions which increases the intracellular ionic strength. This increase activates the MAPK pathway, which in turn induces transcription of hypertonicity responsive transcription factors, e.g. TonEBP. These transcription factors then bind to their cognate binding sites on the OCTN2 promoter and allow transcription of OCTN2 to proceed. The transport protein is then translated, trafficked to the basolateral membrane and L-carnitine transported into the cell. Accumulation of the osmolyte, L-carnitine, will counteract the effects of ions by stabilizing key proteins and DNA. At this stage the epididymal cells have adapted to the new osmotic enviroment and afforded themselves protection from osmotic stress. Specifically, the following hypotheses will be tested: (1) OCTN2 is responsible for transport of L-carnitine into the epididymis; (2) The OCTN2 promoter is regulated by tonicity responsive transcription factors; (3) OCTN2 transcription and transporting activity are regulated by changes in tonicity via specific signal transduction pathways; (4) Loss of OCTN2 results in the loss of the ability of the epididymal epithelium to adapt to a hyperosmotic enviroment resulting in susceptibility to osmotic stress leading to epididymal dysfunction and male infertility. The findings from this proposal will provide fundamental information for the treatment of certain forms of male infertility and for the development of a male contraceptive.