Transcriptomics

Biography

Biography: Nancy Amaral Reboucas

Abstract

The main Na+ reabsorption mechanism in the renal proximal tubules is the Na+/H+ exchanger 3 (NHE3) which is acutely and chronically down regulated by parathyroid hormone (PTH). In rats, continuous administration of PTH to induce hyperparathyroidism reduces the expression of NHE3 both at RNA and protein level with clear decrease of NHE3 at the apical membrane of proximal tubules. Analysis with reporter gene of the rat Nhe3 gene promoter in Opossum Kidney Proximal Tubule (OKP) cells indicated inhibition of transcription by PTH associated to a decrease in NHE3-mRNA stability. We demonstrated that PTH-induced inhibition of Nhe3 gene promoter occurs even in the core promoter. We found that inhibition of the protein kinase A (PKA) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways changed PTH from an inhibitor into an activator of promoter activity as did point mutations in the EGR1, Sp1 and Sp3 binding consensus elements in the promoter. In nuclear extracts of PTH-treated OKP cells, we also observed increased expression of EGR1-mRNA and of some Sp3-protein isoforms. Electrophoretic mobility shift assay showed a super shift of the -61 to -42bp probe with an anti-EGR1 antibody in PTH-treated cells suggesting the EGR1 binding is relevant for the inhibitory activity of PTH. Our results suggest that PTH-induced inhibition of NHE3 transcription is related to higher EGR1 expression to EGR1 binding to the proximal and core promoters and to PKA and JAK/STAT pathway activation. The higher expression of the long isoform (Sp3-li, 90 kDa) and of the short isoform (Sp3-si3) of Sp3 in addition to higher sumoylation of one of the short isoforms of Sp3 induced by PTH could also be related to repression of Nhe3gene expression. As Sp3 was reported to promote RNA polymerase II pause by recruiting phosphatases to the p21CIP1 gene promoter, we hypothesize the PTH might increase RNA Pol II pause at the Nhe3 gene promoter.