Mechanisms of Na+/H+ Exchanger Isoform 3 Gene Proximal Promoter Modulation by Parathyroid Hormone

Abstract

Previous studies have shown that parathyroid hormone (PTH) chronically inhibits NHE3 in opossum kidney cells (OKP) by reducing both, total NHE3 protein and mRNA levels due to transcription modulation. 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. OKP cells were transiently transfect with the vector constructs and kept in serum-free media for 24 h. Cells were then treated with 10-7M PTH for 24 h and promoter activity was determined. These segments were also evaluated by gel shift assays (GMSA). Nuclear expression of the Sp3/EGR-1 transcription factors was also assayed after treatment with 50 μM 1, 2, 3, 4, 5, 6-hexabromocyclohexane (JAK2 autophosphorylation inhibitor), 10 μM Static (STAT3 inhibitor), 1 μM KT5720 (PKA inhibitor) for 24 h.PTH decreased the promoter activity of the fragments −65 and −44 (∼24% and 29% respectively). Inhibition of JAK/STAT and PKA pathways abolished this suppressor effect of PTH. In GMSA, PTH reduced the protein-DNA affinity of the segment −44/+31. Western blot analysis of the transcription factors (Sp3/EGR-1) after treatment with PKA and JAK/STAT signaling pathways inhibitors showed that alone, these inhibitors did not affect their nuclear expression. In conclusion, these data suggest that the cis-element(s) required for PTH responsiveness must be localized in the proximal promoter; the JAK/STAT and PKA pathways may be involved in this inhibitory response; and this effect of PTH on promoter transcription is probably due to changes in protein-DNA affinity without altering transcription factors nuclear expression.