Protein-DNA Interactions at the Opossum Npt2a Promoter are Dependent upon NHERF-1

Abstract

Background/Aims: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. Conclusion: 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.

Specificity of NHERF1 regulation of GPCR signaling and function in human airway smooth muscle

“…This is consistent with observations by us and others (52), which indicate that, at least in some cell types, c/EBPb is localized in the nucleus, and changes in DNA binding, rather than nuclear translocation, govern transcriptional activity. cAMP-mediated regulation of c/EBP-mediated transcription has long been established (53,54), but only one previous study linked NHERF1 to transcriptional activation at the CCAAT promoter sequences, showing a requirement for NHERF1 expression for the type IIa sodium phosphate cotransporter gene transcription (55). Though additional regulation of cAMP production at later time points has not been excluded, our current study identifies NHERF1 as a critical adapter in cAMP-driven c/EBPb DNA binding, leading to IL-6 gene expression and cytokine production.…”