Further understanding of vesicular pH regulation and its impact on growth factor signaling

真核细胞内pH的调节对于控制包括细胞生长和分裂在内的生理过程是至关重要的。细胞质的pH通过有效的离子质膜运输机制维持在接近中性的水平。细胞空泡的情况则完全不同，细胞空泡的pH值通常在4.5-6.5之间变化很大。虽然我们对质膜转运蛋白的了解很多，但我们对囊泡内离子转运及其对细胞功能的影响的了解目前还处于初级阶段。最近发现的两个囊泡Na+/H+交换器，SLC9A6/NHE6和SLC9A9/NHE9，特异性地定位于细胞内的小泡，可能有助于建立和/或微调小泡内的pH。尽管很重要，但囊泡性NHEs的贡献以及它们的活动与随后的生理相关效应之间的直接联系仍有待确定。我们发现，成纤维细胞在低氧条件下孵育导致细胞内小泡的NHE依赖酸化。这一事件与转化生长因子β(转化生长因子β)信号转导Smad3激活有关。基于这些观察，我们提出了新的假说：1)缺氧等代谢变化对细胞器交换活性的调节是囊泡内pH调节的关键事件；2)参与转化生长因子β信号转导的pH敏感蛋白的功能增强是缺氧影响细胞功能(包括细胞生长)的机制的一部分。在接下来的5年里，我们建议以通用的转化生长因子β系统为模型，确定一些控制液泡pH调节的成分和规则，以及它们在生长因子信号调节中的作用。为了实现这一目标，我们的具体目标是：1)回顾泵-漏模型中涉及的调节囊泡内pH的细胞成分，重点关注新发现的囊泡型NA+/H+交换器；2)通过鉴定结合伙伴和调节器来确定这些交换器如何适应代谢变化，例如缺氧；3)使用pH敏感和pH不敏感的转化生长因子β信号调节器来确定内体pH调节对生长因子信号转导的影响。这些研究的结果将增加我们对囊泡内pH调节所涉及的成分的性质和调节的理解。他们还将揭示内体pH及其调节如何影响生长因子信号通路。这将是一项关键成就，将首次将内体pH调节和细胞生长联系起来。**

The regulation of intracellular pH in all eukaryotic cells is crucial for the control of physiological processes that include cell growth and division. Cytoplasmic pH is maintained close to neutrality by efficient ion plasma membrane transport mechanisms. The situation is quite different in cellular vacuoles, which generally have a rather variable pH in the range of 4.5-6.5. Although much is known about transporters at the plasma membrane, our understanding of intravesicular ion transport and its consequences on cellular functions is at the present rudimentary. Two recently identified vesicular Na+/H+ exchangers, SLC9A6/NHE6 and SLC9A9/NHE9, are specifically located to intracellular vesicles and may contribute to the establishment and/or fine-tuning of intravesicular pH. Although of obvious importance, the contribution of vesicular NHEs as well as a direct link between their activity and an ensuing physiologically relevant effect remains to be established. We have found that incubation of fibroblastic cells under hypoxic conditions resulted in NHE-dependent acidification of intracellular vesicles. This event was associated with the activation of the transforming growth factor beta (TGFβ) signal transducer, Smad3. Based on these observations, we propose the novel hypothesis that 1) the modulation of organellar exchanger activity by metabolic changes such as hypoxia is a key event in intravesicular pH regulation and, 2) enhanced functions of pH-sensitive proteins involved in TGFβ signaling is part of the mechanism by which hypoxia influences cell functions that include cell growth. Over the next 5 years, we propose to identify some of the components and the rules governing vacuolar pH regulation and their role in the regulation of growth factor signaling using the universal TGFβ system as a model. To achieve this, our specific objectives are: 1) to revisit the cellular components involved in the pump-and-leak model for regulation of intravesicular pH with a focus on the newly identified vesicular-type NA+/H+ exchangers; 2) to define how these exchangers adapt to metabolic changes such as hypoxia through identification of binding partners and regulators; 3) to define the impact of endosomal pH regulation on growth factor signaling using pH-sensitive and pH-insensitive TGFβ signal regulators. Results from these studies will increase our understanding on the nature and the regulation of the components involved in intravesicular pH regulation. They will also reveal how endosomal pH and its regulation affect growth factor signaling pathways. This will be a key achievement that will link for the first time endosomal pH regulation and cell growth. **