pH-responsive stabilization of glutamate dehydrogenase mRNA in LLC-PK1-F+cells

Abstract

During chronic metabolic acidosis, the adaptive increase in rat renal ammoniagenesis is sustained, in part, by increased expression of mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH) enzymes. The increase in GA activity results from the pH-responsive stabilization of GA mRNA. The 3′-untranslated region (3′-UTR) of GA mRNA contains a direct repeat of an eight-base AU-rich element (ARE) that binds ζ-crystallin/NADPH:quinone reductase (ζ-crystallin) with high affinity and functions as a pH-response element. RNA EMSAs established that ζ-crystallin also binds to the full-length 3′-UTR of GDH mRNA. This region contains four eight-base sequences that are 88% identical to one of the two GA AREs. Direct binding assays and competition studies indicate that the two individual eight-base AREs from GA mRNA and the four individual GDH sequences bind ζ-crystallin with different affinities. Insertion of the 3′-UTR of GDH cDNA into a β-globin expression vector (pβG) produced a chimeric mRNA that was stabilized when LLC-PK1-F+cells were transferred to acidic medium. A pH-responsive stabilization was also observed using a βG construct that contained only the single GDH4 ARE and a destabilizing element from phospho enolpyruvate carboxykinase mRNA. Therefore, during acidosis, the pH-responsive stabilization of GDH mRNA may be accomplished by the same mechanism that affects an increase in GA mRNA.