Protein-DNA Interactions at the Opossum Npt2a Promoter are Dependent upon NHERF-1.
OBJECTIVE
Phosphate homeostasis is controlled by the renal reabsorption of Pi by the type IIa sodium phosphate cotransporter, Npt2a, which is localized in the proximal tubule brush border membrane. Regulation of Npt2a expression is a key control point to maintain phosphate homeostasis with most studies focused on regulating protein levels in the brush border membrane. Molecular mechanisms that control Npt2a mRNA, however, remain to be defined. We have reported that Npt2a mRNA and protein levels correlate directly with the expression of the Na+/H+ exchanger regulatory factor 1 (NHERF-1) using opossum kidney (OK) cells and the NHERF-1-deficient OK-H cells. The goal of this study was to determine whether NHERF-1 contributes to transcriptional and/or post-transcriptional mechanisms controlling Npt2a mRNA levels.


METHODS
Npt2a mRNA half-life was compared between OK and NHERF-1 deficient OK-H cell lines. oNpt2a promoter-reporter gene assays and electrophoretic mobility shift assays (EMSA) were used identify a NHERF-1 responsive region within the oNpt2a proximal promoter.


RESULTS
Npt2a mRNA half-life is the same in OK and OK-H cells. The NHERF-1 responsive region lies within the proximal promoter in a region that contains a highly conserved CAATT box and G-rich element. Specific protein-DNA complex formation with the CAATT element is altered by the absence of NHERF-1 (OK v OK-H EMSA) although NHERF-1 does not directly contribute to complex formation.


CONCLUSIONS
NHERF-1 helps maintain steady-state Npt2a mRNA levels in OK cells through indirect mechanisms that help promote protein-DNA interactions at the Npt2a proximal promoter.
Departments of Biochemistry and Molecular Genetics, University of Louisville School of Medicine, Louisville, USA.
Barbara J
BJ
Clark
Rebecca D
RD
Murray
Sarah A
SA
Salyer
Samuel C
SC
Tyagi
Cibi
C
Arumugam
Syed J
SJ
Khundmiri
Eleanor D
ED
Lederer
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009893:Opossums; D010710:Phosphates / Q000378:metabolism / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D020871:RNA Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics* / Q000378:metabolism; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa / Q000235:genetics* / Q000378:metabolism
D010710:Phosphates; D010750:Phosphoproteins; D012333:RNA, Messenger; D017923:Sodium-Hydrogen Exchangers; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; C093376:sodium-hydrogen exchanger regulatory factor; D004247:DNA