Functional analysis and regulation of epididymal OCTN2

描述（由申请人提供）：保护细胞免受渗透胁迫对其生存至关重要，因为如果不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，以限制渗透应激的影响。和肾脏一样，附睾也会积累渗透液以适应渗透压的变化。渗透物的运动是由特定的转运体来实现的，例如钠-肌醇共转运体，它运输渗透物肌醇。左旋肉碱是另一种在高渗透压适应中起关键作用的渗透液，在附睾腔液中发现高浓度。因此，本研究将研究左旋肉碱的转运蛋白，新型有机阳离子转运蛋白2 （OCTN2）如何在允许附睾细胞适应高渗环境并最终保护自己免受渗透应激中发挥关键作用。我们的工作假设是附睾细胞的渗透应力导致离子的流入，从而增加细胞内的离子强度。这种增加激活了MAPK通路，进而诱导高渗性应答转录因子的转录，例如TonEBP。然后，这些转录因子结合到OCTN2启动子上的同源结合位点，并允许OCTN2的转录继续进行。然后转运蛋白被翻译，转运到基底外侧膜上，左旋肉碱被转运到细胞内。渗透物左旋肉碱的积累将通过稳定关键蛋白质和DNA来抵消离子的影响。在这个阶段，附睾细胞已经适应了新的渗透环境，并保护自己免受渗透胁迫。具体来说，将测试以下假设：(1)OCTN2负责将左旋肉碱运输到附睾；(2) OCTN2启动子受强直响应转录因子调控；(3) OCTN2的转录和转运活性通过特定的信号转导途径受张力变化的调控；(4) OCTN2缺失导致附睾上皮丧失适应高渗环境的能力，易受渗透应激影响，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具提供基本资料。

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.