Characterization of cis-elements required for osmotic response of rat Na+/H+ exchanger-2 (NHE-2) gene

Liqun Bai, James F. Collins, Yunhua L. Muller, Hua Xu, Pawel R Kiela, Fayez K Ghishan

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

The Na+/H+ exchanger (NHE-2) has been implicated in osmoregulation in the kidney, because it transports Na+ across the cell membrane and efficiently alters intracellular osmolarity. On hyperosmotic stress, NHE-2 mRNA increases in abundance in mouse inner medullary collecting duct (mIMCD- 3) cells, suggesting possible transcriptional regulation. To investigate the molecular mechanism of potential transcriptional regulation of NHE-2 by hyperosmolarity, we have functionally characterized the 5'-flanking region of the gene in mIMCD-3 cells. Transient transfection of luciferase reporter gene constructs revealed a novel cis-acting element, which we call OsmoE (osmotic- responsive element, bp -808 to -791, GGGCCAGTTGGCGCTGGG), and a TonE-like element (tonicity-responsive element, bp - 1201 to - 1189, GCTGGAAAACCGA), which together are shown to be responsible for hyperosmotic induction of the NHE-2 gene. Electrophoretic mobility shift assays suggest that different DNA- protein interactions occur between these two osmotic response elements. However, both DNA sequences were shown to specifically bind nuclear proteins that dramatically increase in abundance under hyperosmotic conditions. Isolation of transacting factors and characterization of their specific interaction with these osmotic response elements will further elucidate the transcriptional mechanisms controlling NHE-2 gene expression under hyperosmolar conditions.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Regulatory Integrative and Comparative Physiology
Volume277
Issue number4 46-4
StatePublished - Oct 1999

Fingerprint

Sodium-Hydrogen Antiporter
Genes
Response Elements
Osmoregulation
5' Flanking Region
Electrophoretic Mobility Shift Assay
Nuclear Proteins
Luciferases
Reporter Genes
Osmolar Concentration
Transfection
Cell Membrane
Kidney
Gene Expression
Messenger RNA
DNA
Proteins

Keywords

  • Gel mobility shift
  • Kidney
  • Mouse inner medullary collecting duct cells
  • OsmoE
  • Transcriptional regulation

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)

Cite this

@article{2d8d6d8898504863a25c82373c983540,
title = "Characterization of cis-elements required for osmotic response of rat Na+/H+ exchanger-2 (NHE-2) gene",
abstract = "The Na+/H+ exchanger (NHE-2) has been implicated in osmoregulation in the kidney, because it transports Na+ across the cell membrane and efficiently alters intracellular osmolarity. On hyperosmotic stress, NHE-2 mRNA increases in abundance in mouse inner medullary collecting duct (mIMCD- 3) cells, suggesting possible transcriptional regulation. To investigate the molecular mechanism of potential transcriptional regulation of NHE-2 by hyperosmolarity, we have functionally characterized the 5'-flanking region of the gene in mIMCD-3 cells. Transient transfection of luciferase reporter gene constructs revealed a novel cis-acting element, which we call OsmoE (osmotic- responsive element, bp -808 to -791, GGGCCAGTTGGCGCTGGG), and a TonE-like element (tonicity-responsive element, bp - 1201 to - 1189, GCTGGAAAACCGA), which together are shown to be responsible for hyperosmotic induction of the NHE-2 gene. Electrophoretic mobility shift assays suggest that different DNA- protein interactions occur between these two osmotic response elements. However, both DNA sequences were shown to specifically bind nuclear proteins that dramatically increase in abundance under hyperosmotic conditions. Isolation of transacting factors and characterization of their specific interaction with these osmotic response elements will further elucidate the transcriptional mechanisms controlling NHE-2 gene expression under hyperosmolar conditions.",
keywords = "Gel mobility shift, Kidney, Mouse inner medullary collecting duct cells, OsmoE, Transcriptional regulation",
author = "Liqun Bai and Collins, {James F.} and Muller, {Yunhua L.} and Hua Xu and Kiela, {Pawel R} and Ghishan, {Fayez K}",
year = "1999",
month = "10",
language = "English (US)",
volume = "277",
journal = "American Journal of Physiology",
issn = "0363-6143",
publisher = "American Physiological Society",
number = "4 46-4",

}

TY - JOUR

T1 - Characterization of cis-elements required for osmotic response of rat Na+/H+ exchanger-2 (NHE-2) gene

AU - Bai, Liqun

AU - Collins, James F.

AU - Muller, Yunhua L.

AU - Xu, Hua

AU - Kiela, Pawel R

AU - Ghishan, Fayez K

PY - 1999/10

Y1 - 1999/10

N2 - The Na+/H+ exchanger (NHE-2) has been implicated in osmoregulation in the kidney, because it transports Na+ across the cell membrane and efficiently alters intracellular osmolarity. On hyperosmotic stress, NHE-2 mRNA increases in abundance in mouse inner medullary collecting duct (mIMCD- 3) cells, suggesting possible transcriptional regulation. To investigate the molecular mechanism of potential transcriptional regulation of NHE-2 by hyperosmolarity, we have functionally characterized the 5'-flanking region of the gene in mIMCD-3 cells. Transient transfection of luciferase reporter gene constructs revealed a novel cis-acting element, which we call OsmoE (osmotic- responsive element, bp -808 to -791, GGGCCAGTTGGCGCTGGG), and a TonE-like element (tonicity-responsive element, bp - 1201 to - 1189, GCTGGAAAACCGA), which together are shown to be responsible for hyperosmotic induction of the NHE-2 gene. Electrophoretic mobility shift assays suggest that different DNA- protein interactions occur between these two osmotic response elements. However, both DNA sequences were shown to specifically bind nuclear proteins that dramatically increase in abundance under hyperosmotic conditions. Isolation of transacting factors and characterization of their specific interaction with these osmotic response elements will further elucidate the transcriptional mechanisms controlling NHE-2 gene expression under hyperosmolar conditions.

AB - The Na+/H+ exchanger (NHE-2) has been implicated in osmoregulation in the kidney, because it transports Na+ across the cell membrane and efficiently alters intracellular osmolarity. On hyperosmotic stress, NHE-2 mRNA increases in abundance in mouse inner medullary collecting duct (mIMCD- 3) cells, suggesting possible transcriptional regulation. To investigate the molecular mechanism of potential transcriptional regulation of NHE-2 by hyperosmolarity, we have functionally characterized the 5'-flanking region of the gene in mIMCD-3 cells. Transient transfection of luciferase reporter gene constructs revealed a novel cis-acting element, which we call OsmoE (osmotic- responsive element, bp -808 to -791, GGGCCAGTTGGCGCTGGG), and a TonE-like element (tonicity-responsive element, bp - 1201 to - 1189, GCTGGAAAACCGA), which together are shown to be responsible for hyperosmotic induction of the NHE-2 gene. Electrophoretic mobility shift assays suggest that different DNA- protein interactions occur between these two osmotic response elements. However, both DNA sequences were shown to specifically bind nuclear proteins that dramatically increase in abundance under hyperosmotic conditions. Isolation of transacting factors and characterization of their specific interaction with these osmotic response elements will further elucidate the transcriptional mechanisms controlling NHE-2 gene expression under hyperosmolar conditions.

KW - Gel mobility shift

KW - Kidney

KW - Mouse inner medullary collecting duct cells

KW - OsmoE

KW - Transcriptional regulation

UR - http://www.scopus.com/inward/record.url?scp=0032726264&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032726264&partnerID=8YFLogxK

M3 - Article

C2 - 10516252

AN - SCOPUS:0032726264

VL - 277

JO - American Journal of Physiology

JF - American Journal of Physiology

SN - 0363-6143

IS - 4 46-4

ER -