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Misclocalized scaffolding by the Na-H exchanger NHE1 inhibits fibronectin production and assembly

  • Author(s): Karydis, Anastasios
  • Advisor(s): Sheppard, Dean
  • et al.
Abstract

Secretion and assembly of the extracellular matrix protein fibrobnectin (FN) regulates a plethora of normal cell and tissue functions, including development, cell growth, differentiation and cell migration, and is dysregulated in diseases such as fibrosis, diabetes, and cancer. My thesis work revealed that mislocalized scaffolding by the plasma membrane Na-H exchanger NHE1 dominantly suppresses FN expression, secretion, and assembly, and inhibits cleavage of latent to active TGF-beta;. By catalyzing an electroneutral exchange of extracellular Na+ and intracellular H+, NHE1 has recognized functions in intracellular pH and osmotic homeostasis. Recent evidence indicates NHE1 also functions as a plasma membrane anchor for the actin cytoskeleton by binding directly to ERM (ezrin, moesin, radixin) proteins and a PI(4,5)P2-binding scaffold, which are independent of ion translocation by NHE1. Endogenous NHE1 localizes to the distal margin of membrane protrusions in lamellipodia, however a mutantNHE1-KRA2, lacking binding sites for PI(4,5)P2 and the ERM proteins ezrin, radixin and moesin, is mislocalized and distributed uniformly along the plasma membrane. Fibroblasts expressing NHE1-KRA2 but not wild-type NHE1 have impaired FN expression, secretion, and assembly, and reduced active but not latent TGF-beta;. We found that FN production is not regulated by changes in intracellular pH, nor is it attenuated in NHE1-deficient cells, indicating FN expression and TGF-beta; activation are independent of NHE1 activity. However, treating NHE1-KRA2 fibroblasts with recombinant TGF-beta; restores FN secretion and assembly. These data suggest that scaffolding by NHE1-KRA2 sequesters and mislocalizes signals necessary for FN synthesis and TGF-beta; activation. Although the precise signals sequestered by NHE1-KRA2 remain unknown, these findings suggest that NHE1-KRA2 could be a valuable tool for obtaining a mechanistic understanding of how FN production and TGF-beta; activation are regulated, and more speculatively for therapeutic control of increased FN production in pathological conditions such as fibrosis and inflammation.

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