Expression of HSD11K (NAD+ dependent 11β-hydroxysteroid dehydrogenase) promoter constructs in renal cell lines

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Abstract

The kidney (11-HSD2 or 11-HSDK) isozyme of 11β-hydroxysteroid dehydrogenase confers specificity for aldosterone on mineralocorticoid receptors in target tissues. In rodent kidney, this isozyme is expressed mainly in cortical collecting ducts and is undetectable in proximal tubules. Using mouse M-1 and rabbit RCD cortical collecting duct cells, we analyzed the 5'-flanking region of the human HSD11K gene encoding this enzyme in an attempt to identify transcriptional regulatory elements responsible for gene expression in the kidney. M-1 and RCD cells had high levels of NAD+ dependent 11-HSD activity with corticosterone as the substrate. Luciferase reporter constructs containing 1785 or 327 nucleotides (nt) upstream of the initiator ATG codon were expressed at similar levels in each cell line, but deletion to 167 nt almost completely abolished expression in both cell types. This region is GC-rich and contain Sp1 binding sites. Electrophoretic mobility shift assays of the region containing the putative Sp1 sites showed several DNA- protein complexes in both the cell types. Mutations of the Sp1 sites decreased transcriptional activity in M-1 cells; however, these mutations had a marginal effect in the RCD cells. These results suggest that elements controlling renal cell type expression are located in the proximal 327 nucleotides of the 5' flanking region of HSD11K.

Original languageEnglish (US)
Pages (from-to)289-302
Number of pages14
JournalEndocrine Research
Volume26
Issue number2
StatePublished - 2000

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11-beta-Hydroxysteroid Dehydrogenases
NAD
Kidney
Cell Line
Nucleotides
5' Flanking Region
Isoenzymes
Transcriptional Regulatory Elements
GC Rich Sequence
Mineralocorticoid Receptors
Mutation
Initiator Codon
Electrophoretic Mobility Shift Assay
Corticosterone
Aldosterone
Luciferases
Rodentia
Binding Sites
Rabbits
Gene Expression

ASJC Scopus subject areas

  • Endocrinology

Cite this

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title = "Expression of HSD11K (NAD+ dependent 11β-hydroxysteroid dehydrogenase) promoter constructs in renal cell lines",
abstract = "The kidney (11-HSD2 or 11-HSDK) isozyme of 11β-hydroxysteroid dehydrogenase confers specificity for aldosterone on mineralocorticoid receptors in target tissues. In rodent kidney, this isozyme is expressed mainly in cortical collecting ducts and is undetectable in proximal tubules. Using mouse M-1 and rabbit RCD cortical collecting duct cells, we analyzed the 5'-flanking region of the human HSD11K gene encoding this enzyme in an attempt to identify transcriptional regulatory elements responsible for gene expression in the kidney. M-1 and RCD cells had high levels of NAD+ dependent 11-HSD activity with corticosterone as the substrate. Luciferase reporter constructs containing 1785 or 327 nucleotides (nt) upstream of the initiator ATG codon were expressed at similar levels in each cell line, but deletion to 167 nt almost completely abolished expression in both cell types. This region is GC-rich and contain Sp1 binding sites. Electrophoretic mobility shift assays of the region containing the putative Sp1 sites showed several DNA- protein complexes in both the cell types. Mutations of the Sp1 sites decreased transcriptional activity in M-1 cells; however, these mutations had a marginal effect in the RCD cells. These results suggest that elements controlling renal cell type expression are located in the proximal 327 nucleotides of the 5' flanking region of HSD11K.",
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T1 - Expression of HSD11K (NAD+ dependent 11β-hydroxysteroid dehydrogenase) promoter constructs in renal cell lines

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AB - The kidney (11-HSD2 or 11-HSDK) isozyme of 11β-hydroxysteroid dehydrogenase confers specificity for aldosterone on mineralocorticoid receptors in target tissues. In rodent kidney, this isozyme is expressed mainly in cortical collecting ducts and is undetectable in proximal tubules. Using mouse M-1 and rabbit RCD cortical collecting duct cells, we analyzed the 5'-flanking region of the human HSD11K gene encoding this enzyme in an attempt to identify transcriptional regulatory elements responsible for gene expression in the kidney. M-1 and RCD cells had high levels of NAD+ dependent 11-HSD activity with corticosterone as the substrate. Luciferase reporter constructs containing 1785 or 327 nucleotides (nt) upstream of the initiator ATG codon were expressed at similar levels in each cell line, but deletion to 167 nt almost completely abolished expression in both cell types. This region is GC-rich and contain Sp1 binding sites. Electrophoretic mobility shift assays of the region containing the putative Sp1 sites showed several DNA- protein complexes in both the cell types. Mutations of the Sp1 sites decreased transcriptional activity in M-1 cells; however, these mutations had a marginal effect in the RCD cells. These results suggest that elements controlling renal cell type expression are located in the proximal 327 nucleotides of the 5' flanking region of HSD11K.

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