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

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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.