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

在所有真核细胞中，细胞内pH值的调节对包括细胞生长和分裂在内的生理过程的控制至关重要。细胞质pH值通过有效的离子质膜运输机制保持接近中性。细胞液泡的情况则大不相同，其pH值通常在4.5-6.5之间变化很大。虽然我们对质膜上的转运蛋白了解很多，但我们对囊泡内离子转运及其对细胞功能的影响的理解目前还处于初级阶段。最近发现的两种囊泡Na+/H+交换物SLC9A6/NHE6和SLC9A9/NHE9特异定位于细胞内囊泡，可能有助于囊泡内ph的建立和/或微调。尽管具有明显的重要性，但囊泡NHEs的作用及其活性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，在缺氧条件下培养成纤维细胞会导致细胞内囊泡的nhe依赖性酸化。这一事件与转化生长因子β (TGFβ)信号传感器Smad3的激活有关。基于这些观察结果，我们提出了新的假设：1)代谢变化（如缺氧）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TGFβ信号传导的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）的部分机制。在接下来的5年里，我们将以通用的TGFβ系统为模型，确定一些控制液泡pH调节的成分和规则，以及它们在调节生长因子信号传导中的作用。为了实现这一目标，我们的具体目标是：1)重新审视参与囊泡内pH调节的泵漏模型的细胞成分，重点关注新发现的囊泡型NA+/H+交换剂；2)通过识别结合伙伴和调节因子来确定这些交换物如何适应代谢变化，如缺氧；3)利用pH敏感和pH不敏感的tgf - β信号调节剂确定内体pH调节对生长因子信号传导的影响。这些研究结果将增加我们对参与囊泡内pH调节的成分的性质和调节的理解。他们还将揭示内体pH值及其调控如何影响生长因子信号通路。这将是一项关键的成就，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.