Chronic effect of parathyroid hormone on Na+/H+ exchanger isoform 3 gene proximal promoter

Abstract

Previous studies have shown that parathyroid hormone (PTH) chronically inhibits NHE3 in opossum kidney cells (OKP) by reducing total NHE3 protein and mRNA levels by modulation of the transcription. The purpose of this study was to elucidate the inhibitory mechanisms of PTH on the NHE3 gene promoter. Different segments of the proximal promoter (−157/+31; −85/+31; −65/+31 and −44/+31) were inserted in the pGL3‐ basic luciferase reporter vector, and point mutations were introduced in some regulatory elements, which were also evaluated by gel shift assays (GMSA). OKP cells were transiently transfect with the vector constructs and kept in serum‐free media for 24 h, then treated with 10−7M PTH for 24 h. PTH decreased the promoter activity of the fragments −65 and −44 (~21% and 31% respectively). Mutations on Sp1/Egr‐1 and AP2 elements completely abolished the PTH inhibitory effect. Inhibition of JAK/STAT pathway abolished the suppressor effect of PTH on fragment −65. In GMSA, PTH reduced the protein‐DNA affinity of the segment −44/+31.In conclusion, these data suggest that the cis‐element(s) required for PTH responsiveness must be localized in the proximal promoter and the JAK/STAT pathway is involved in this inhibitory response.Financial Support: FAPESP and CAPES