title: Role of AUF1 and HuR in the pH-responsive stabilization of phosphoenolpyruvate carboxykinase mRNA in LLC-PK₁-F⁺ cells.
abstract: Onset of metabolic acidosis leads to a rapid and pronounced increase in expression of phosphoenolpyruvate carboxykinase (PEPCK) in rat renal proximal convoluted tubules. This adaptive response is modeled by treating a clonal line of porcine LLC-PK(1)-F(+) cells with an acidic medium (pH 6.9, 9 mM HCO(3)(-)). Measurement of the half-lives of PEPCK mRNA in cells treated with normal (pH 7.4, 26 mM HCO(3)(-)) and acidic medium established that the observed increase is due in part to stabilization of the PEPCK mRNA. The pH-responsive stabilization was reproduced in a Tet-responsive chimeric reporter mRNA containing the 3'-UTR of PEPCK mRNA. This response was lost by mutation of a highly conserved AU sequence that binds AUF1 and is the primary element that mediates the rapid turnover of PEPCK mRNA. However, siRNA knockdown of AUF1 had little effect on the basal levels and the pH-responsive increases in PEPCK mRNA and protein. Electrophoretic mobility shift assays established that purified recombinant HuR, another AU element binding protein, also binds with high affinity and specificity to multiple sites within the final 92-nucleotides of the 3'-UTR of the PEPCK mRNA, including the highly conserved AU-rich element. siRNA knockdown of HuR caused pronounced decreases in basal expression and the pH-responsive increases in PEPCK mRNA and protein. Therefore, basal expression and the pH-responsive stabilization of PEPCK mRNA in LLC-PK(1)-F(+) cells, and possibly in the renal proximal tubule, may require the remodeling of HuR and AUF1 binding to the elements that mediate the rapid turnover of PEPCK mRNA.
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA.
authors.forename: Judy
authors.initials: J
authors.lastname: Mufti
authors.forename: Sachin
authors.initials: S
authors.lastname: Hajarnis
authors.forename: Kelly
authors.initials: K
authors.lastname: Shepardson
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Gummadi
authors.forename: Lynn
authors.initials: L
authors.lastname: Taylor
authors.forename: Norman P
authors.initials: NP
authors.lastname: Curthoys
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D003609:Dactinomycin / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D051959:ELAV Proteins / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D018374:LLC-PK1 Cells; D051379:Mice; D008969:Molecular Sequence Data; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine
chemical_list: D020413:3' Untranslated Regions; D000903:Antibiotics, Antineoplastic; D051959:ELAV Proteins; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; C000630988:Hnrnpd protein, rat; C000630987:Hnrpd protein, mouse; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D003609:Dactinomycin; D004317:Doxorubicin; D010729:Phosphoenolpyruvate Carboxykinase (GTP)