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. 2004 Oct;114(8):1146-57.
doi: 10.1172/JCI21647.

Epigenetic regulation of 11 beta-hydroxysteroid dehydrogenase type 2 expression

Rasoul Alikhani-Koopaei  1 , Fatemeh Fouladkou, Felix J Frey, Brigitte M Frey
Affiliations

Epigenetic regulation of 11 beta-hydroxysteroid dehydrogenase type 2 expression

Rasoul Alikhani-Koopaei et al. J Clin Invest. 2004 Oct.

Abstract

The enzyme 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD2) is selectively expressed in aldosterone _target tissues, where it confers aldosterone selectivity for the mineralocorticoid receptor by inactivating 11 beta-hydroxyglucocorticoids. Variable activity of 11 beta HSD2 is relevant for blood pressure control and hypertension. The present investigation aimed to elucidate whether an epigenetic mechanism, DNA methylation, accounts for the rigorous control of expression of the gene encoding 11 beta HSD2, HSD11B2. CpG islands covering the promoter and exon 1 of HSD11B2 were found to be densely methylated in tissues and cell lines with low expression but not those with high expression of HSD11B2. Demethylation induced by 5-aza-2'-deoxycytidine and procainamide enhanced the transcription and activity of the 11 beta HSD2 enzyme in human cells in vitro and in rats in vivo. Methylation of HSD11B2 promoter-luciferase constructs decreased transcriptional activity. Methylation of recognition sequences of transcription factors, including those for Sp1/Sp3, Arnt, and nuclear factor 1 (NF1) diminished their DNA-binding activity. Herein NF1 was identified as a strong HSD11B2 stimulatory factor. The effect of NF1 was dependent on the position of CpGs and the combination of CpGs methylated. A methylated-CpG-binding protein complex 1 transcriptional repression interacted directly with the methylated HSD11B2 promoter. These results indicate a role for DNA methylation in HSD11B2 gene repression and suggest an epigenetic mechanism affecting this gene causally linked with hypertension.

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Figures

Figure 1
Figure 1
CpG plot of the human HSD11B2 gene. The top represents the putative CpG islands along the gene and the middle graph indicates the relative abundance of CpGs (Percentage) as a function of the base number. CpG islands were defined as a region in which the calculated percentage of CpGs over an average range of ten windows was over 50% and the calculated versus the expected CpG distribution higher than 0.6. White boxes in the bottom graph indicate CpG islands identified and investigated. T.S., transcription start site.
Figure 2
Figure 2
The effect of 5-aza-CdR, TSA, or procainamide on the activity and mRNA of HSD11B2. (A and B) The activity of 11βHSD2 is expressed as pmol/mg/h and the abundance of mRNA is normalized to S18 rRNA. The activity and/or mRNA of HSD11B2 increased after the addition of 5-aza-CdR (A) or procainamide (B) in all cells analyzed. TSA increased the activity and/or mRNA when the MCF-7, SW620, and human primary cells were pretreated with 5-aza-CdR (A). The abundance of mRNA paralleled the changes in activity (mRNA in SW620 cells and activity in human primary cells were not measured). The data are given as the mean ± SD of triplicate samples of a representative experiment repeated at least three times.
Figure 3
Figure 3
The effect of 5-aza-CdR on the DNA methylation pattern of HSD11B2 and glucocorticoid-mediated transactivation of MMTV-Luc. (A) Schematic representation of HSD11B2 gene. P1–P6 indicate the stretches of DNA amplified for methylation analyses and refer to parts 1–6 in C. N1-F, N1-R, N2-F, and N2-R are nested primers. (B) Example of bisulfite sequencing (nucleotides –456 to –391). The sequences before (top) and after bisulfite treatment either without (middle) or with (bottom) 5-aza-CdR treatment are presented. After bisulfite treatment, C-to-T conversion did not occur at several CG sites, indicating methylation of those CpG dinucleotides. Treatment with 5-aza-CdR increased unmethylated forms of alleles, as shown by increased T versus C signals (compare middle and bottom). (C) Methylation pattern of the HSD11B2 promoter and exonic and downstream CpG islands in various cells and tissues. The CpG number indicates the number of the CpG dinucleotides along the promoter and CpG position denotes the position of this CpG in relation to the transcription start site. The color of the circle reflects the degree of methylation (key). (D) Effect of 5-aza-CdR on glucocorticoid-mediated MMTV-Luc transactivation in JEG-3 cells. The dose-response curve of cortisol was blunted by 5-aza-CdR. Transfections were performed in triplicate; all results were confirmed by at least two different independent experiments.
Figure 4
Figure 4
Binding of nuclear proteins on methylated or unmethylated putative Arnt- or Sp1/Sp3-binding sites in the HSD11B2 gene. (A and B) The positions of specific complexes (Specific, Sp1/Sp3, and Sp3) or nonspecific bands (N.S.) are indicated along the left margins. Supershifts (S.S.) were performed with specific antibodies (Ab.Arnt, Ab.Ah-Arnt, Ab.Sp1, or Ab.Sp3), combinations (Ab.Sp1/Sp3), or anti-Ap4 as a nonspecific control (Ab.Ap4). For competition studies, a 100-fold (100×) molar excess of cold methylated or unmethylated probe was used.
Figure 5
Figure 5
EMSA with differentially methylated or unmethylated NF1 probes. (A and B) EMSA with fully methylated (Me-NF1) or unmethylated (U-NF1) NF1 probes of the human HSD11B2 promoter was performed on SW620 cell nuclear protein extracts after overexpression of HA-tagged murine NF1 isoforms NF1-A1.1, NF1-B2, NF1-C2, or NF1-X2 (A) and NF1-C2 or NF1-X2 (B). The position of methylation-specific complexes (Meth. Sp. compl.) and NF1-specific complexes (NF1 compl.) or nonspecific bands (N.S.) are indicated along the left margins. Supershifts were performed with HA-specific antibodies (Ab.HA). For competition studies, a 100-fold (×100) molar excess of a cold methylated (Me-), unmethylated (U-) or a consensus unmethylated (NF1-Cons. 100×) NF1 probe was used. The sequences and methylation sites of the probes are given in Table 1. (C–E) EMSA with site-specific methylated probes. EMSA was performed on SW620 cell nuclear protein extracts after overexpression of the HA-tagged murine NF1-X2 isoform. Different DNA probes [Me-NF1(1CG), Me-NF1(2CG), Me-NF1, and U-NF1], including mutated probes [U-NF1(Tg) or Me-NF1(Tg)] and their corresponding cold competitors (×100), were used (see Table 1). Supershifts were performed with specific (Ab.HA, Ab.MBD1) or unrelated (Ab.MBD2 and Ab.Arnt) antibodies. Whereas C demonstrates that the position of CpG methylation and D, that the composition of core neighboring nucleotides affect the binding of NF1, E suggests that MBD1 interacts with NF1 at the NF1 binding site. Sp. compl.; specific complexes.
Figure 6
Figure 6
Relevance of NF1 and/or DNA methylation on HSD11B2 promoter activity and binding of MeCP1-like complex to methylated HSD11B2 promoter probes. (A and B) Repression of promoter-driven HSD11B2 transcription by CpG methylation with and without NF1 overexpression. Different luciferase (Luc) constructs were methylated (filled circles) or mock-methylated (no circles) in vitro and were transfected into SW620 cells. Results are given as luciferase activity normalized to cotransfected pCMV-LacZ activity. The reporter plasmids were cotransfected with pCHNFI A1.1 for NF1 overexpression. Results are the mean ± SE of at least three experiments. (C–E) Binding of MeCP1-like complex to methylated HSD11B2 promoter probes. 32P-labeled mock-methylated (C, U-CG11; D, U-P1; E, U-P2) or methylated (C, Me-CG11; D, Me-P1; E, Me-P2) probes were incubated with MCF-7 nuclear extracts in absence (–) or presence of a 50-fold molar excess (×50) of the corresponding cold unmethylated (U-) or methylated (Me-) probes. Supershifts were performed with N-terminal anti-MBD2 (Ab.MBD2).
Figure 7
Figure 7
Effect of procainamide and 5-aza-CdR on HSD11B2 in rats. (A and B) After treatment with procainamide (PA), the urinary (THB + 5α-THB) / THA ratios declined in all animals (A), indicating increased activity of 11βHSD2. This observation is in line with the elevated mRNA levels in kidney tissue of rats given procainamide (B). The y axis indicates the difference between the cycle threshold values (dCT) of HSD11B2 mRNA and ribosomal RNA as internal control. A low value indicates high content of HSD11B2 mRNA. (C) Schematic representation of rat HSD11B2 CpG islands spanning from nucleotide –358 to nucleotide +763 with respect to the transcription initiation site (arrow at +1). A fragment spanning nucleotides –1,097 to –292 was used for Southern blot analysis. Horizontal bars with numbers (below diagram) indicate the expected size of the hybridized fragments. (D) Methylation-sensitive restriction enzyme analyses. Genomic DNA from different tissues of control rats (–) and 5-aza-CdR–treated rats (+) was double-digested with EcoRI and either HpaII or MspI and was fractionated by agarose gel electrophoresis followed by Southern blotting. For interpretation, see the Results section. (E) Northern blot analyses without and with 5-aza-CdR treatment. An XhoI fragment of HSD11B2 was used as a probe. Expression of HSD11B2 increased after administration of 5-aza-CdR in the three tissues analyzed.
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