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它们的生存至关重要，因为如果不保护细胞，细胞将经历凋亡。附睾腔积液是高渗性的，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，附睾会积累渗透压以适应渗透压的变化。渗透膜的运动是通过特定的转运体实现的，例如，钠-肌醇共转运体运输渗透膜膜的肌醇。L肉碱是另一种在高渗透压适应中起关键作用的渗透压物质，在附睾腔腔液中浓度较高。因此，本研究将探讨L-肉碱的转运蛋白--新型有机阳离子转运蛋白2(OCTN_2)如何在使附睾细胞适应高渗环境并最终保护自身免受渗透胁迫的过程中发挥关键作用。我们的工作假设是，附睾细胞的渗透应激导致离子内流，从而增加细胞内的离子强度。这种增加激活了MAPK通路，进而诱导高张反应转录因子的转录，例如TONEBP。然后，这些转录因子与OCTN2启动子上的同源结合位点结合，允许OCTN2的转录进行。然后，转运蛋白被翻译，运输到基底膜，L-肉碱运输到细胞内。渗透剂L肉碱的积累将通过稳定关键蛋白质和脱氧核糖核酸来抵消离子的影响。在这个阶段，附睾细胞已经适应了新的渗透环境，并对渗透胁迫提供了保护。具体而言，将检验下列假说：(1)OCTN2负责L肉碱向附睾的转运；(2)OCTN2启动子受紧张性反应转录因子的调控；(3)OCTN2的转录和转运活性受特定信号转导途径的紧张性变化的调控；(4)OCTN2的缺失导致附睾上皮丧失适应高渗环境的能力，从而对渗透胁迫敏感，从而导致附睾功能障碍和男性不育。这项建议的结果将为某些形式的男性不育的治疗和男性避孕药的开发提供基本信息。

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.