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Tumor necrosis factor alpha and phorbol 12-myristate-13-acetate down-regulate human 11ß-hydroxysteroid dehydrogenase type 2 through p50/p50 NF-B homodimers and Egr-1

Department of Nephrology and Hypertension, Department of Clinical Research, University Hospital, Bern, Switzerland

²Correspondence: E-mail: brigitte.frey{at}dkf.unibe.ch

SPECIFIC AIMS

11ß-Hydroxysteroid dehydrogenase type 2 (11ß-HSD2) regulates access of 11ß-hydroxyglucocorticoids to their mineralocorticoid receptor (MR) by reducing the hydroxyl group at position 11 in mineralocorticoid target tissues such as kidney and colon. Reduced activity of 11ß-HSD2 results in overstimulation of the MR by cortisol, causing salt retention with formation of edema and hypertension. Studies in cell cultures have revealed that tumor necrosis factor (TNF-) or phorbol 12-myristate-13-acetate (PMA) down-regulate 11ß-HSD2 mRNA levels and activity. The aim of the present study was to establish whether this effect is present in vivo in transgenic mice overexpressing TNF- (TNF--Tg) and, if so, to elucidate the molecular mechanisms by which TNF- and PMA down-regulate 11ß-HSD2 expression in human colon SW620 cells. We used in vivo methods to investigate stimulus-dependent protein-DNA interactions in the human HSD11B2 promoter including in vivo genomic footprinting and chromatin immunoprecipitations assays (ChIP).

PRINCIPAL FINDINGS

1. Transgenic mice overexpressing TNF- exhibit a reduced expression and activity of 11ß-HSD2 in kidney tissue and an impaired conversion of corticosterone to dehydrocorticosterone
To examine the effect of TNF- on HSD11B2 expression in vivo, we measured renal HSD11B2 mRNA levels in transgenic TNF- (TNF--Tg) overexpressing and wild-type (WT) mice (Fig. 1 A). TNF--Tg mice exhibited a 70% reduced renal HSD11B2 mRNA abundance compared with WT mice. These low HSD11B2 mRNA levels were associated with a 50% decreased 11ß-HSD2 activity as assessed by conversion of corticosterone to 11-dehydrocorticosterone in renal tissue (Fig. 1B ). A 40% increased urinary ratio of corticosterone (Fig. 1B ) and dehydrocorticosterone (Fig 1A ) tetrahydro metabolites (THB+5-THB)/THA was observed in TNF--Tg mice (Fig. 1C ).

View larger version (13K): [in this window] [in a new window]   Figure 1. Reduced mRNA expression and activity of 11ß-HSD2 in TNF--Tg mice. A) Total RNA was isolated from kidneys of WT and TNF--Tg. HSD11B2 mRNA was expressed relative to the mRNA found in WT mice. Data are means ± SE of triplicate measurements (n=5), *P< 0.0001. B) Oxidative activity of 11ß-HSD2 in protein lysates from WT and TNF--Tg kidneys was assessed. Data are means ± SE performed in triplicate, *P< 0.05. C) 11ß-HSD2 activity in animals was assessed in urine by GC-MS. An increased ratio of the metabolites indicates a diminished activity of 11ß-HSD2. Results are means ± SE (n=10), *P< 0.05.

2. TNF- and PMA treatment down-regulates 11ß-HSD2 in SW620 cells
Exposure to TNF- for 24 h decreased HSD11B2 expression and activity by 50% in SW620 cells. A similar decrease was observed when cells were incubated with PMA. Expression (0.5 h) and activity of 11ß-HSD2 (4–6 h) were enhanced during the early phase of treatment with PMA.

3. In vivo genomic footprinting allows identification of stimulus-dependent protein-DNA interactions in B1 (–87/–102), Sp1/Egr-1I (–69/–91), and Sp1/Egr-1II (–43/–70)
To determine in vivo kinetics of inducible protein-DNA interactions at the HSD11B2 promoter, SW620 cells were treated with TNF- or PMA for 0–24 h and analyzed by dimethyl sulfate ligation-mediated PCR in vivo footprinting. This technique permits a view of the gene regulatory regions during transcription in an intact chromosomal environment. We analyzed the proximal promoter and first exon (–220 to +160) of HSD11B2 in cells with and without TNF- or PMA treatment. The pattern of methylated G residues changed at the overlapping B1 and Sp1/Egr-1I regions when in vivo methylated samples of treated vs. untreated cells were compared. Thus, these two regions might be relevant for HSD11B2 transcription. A novel assembly of footprinted guanines was observed within the Sp1/Egr-1II (–43 to –70) site in the absence of stimulation, suggesting a role for this region in basal transcription. We observed some alterations in the pattern of methylated G residues in response to PMA treatment (4 h). These identified regions (B1, Sp1/Egr-1I, and Sp1/Egr-1II) are adjacent to the transcription start of the HSD11B2 promoter.

4. Temporal switch of binding from active p65/p50 to inactive p50/p50 to the B1 site and binding of Egr-1 to the Sp1/Egr-1I and Sp1/Egr-1II sites are essential for TNF- and PMA action at the HSD11B2 promoter
The relevance of B1, Sp1/Egr-1I, and Sp1/Egr-1II sites observed by in vivo footprinting was confirmed by EMSA analyses. A strong binding of active p65/p50 heterodimers to the B1 site was found after 0.5 h of TNF- or PMA treatment. After 4 h of TNF- or PMA treatment p50/p50 appeared to bind to B1 site, gradually displacing p65/p50. At 24 h of stimulation, p50/p50 homodimers bound strongly to the B1 site, competing for binding with the low amounts of p65/p50, correlating with HSD11B2 transcriptional repression. The steady increase in p50 levels after stimulation with TNF- or PMA was confirmed by Western blot analyses with nuclear extracts.

In the absence of treatment, we observed binding of constitutively expressed Sp1 and Sp3 factors to the Sp1/Egr-1I and newly identified Sp1/Egr-1II sites. These factors were found to play a role for basal transcription of the HSD11B2 gene. After 30 min of TNF- and 4 h of PMA treatment, Egr-1 binds to the Sp1/Egr-1I site. Only PMA (4 h) induced short-lived interaction of Egr-1 with the neighboring Sp1/Egr-1II region. Maximal induction of Egr-1 by TNF- was more rapid than that by PMA. The binding kinetics of Egr-1 to the promoter matched well with its inducible expression in the nuclear extracts of treated cells (Western blot analyses).

We used the chromatin immunoprecipitation (ChIP) assay to evaluate the in vivo recruitment of p65, p50, and Egr-1 to the HSD11B2 promoter. Increased occupancy by p65 on the HSD11B2 promoter was seen after 0.5 h of TNF- and 0.5–4 h of PMA treatment, then returned to basal level (24 h) (Fig. 2 ). In contrast, p50 recruitment to the B1 site occurred after 0.5 h and increased for up to 24 h of TNF- or PMA treatment. Augmented recruitment of Egr-1 to HSD11B2 promoter was observed after 0.5 h of TNF- and 4 h of PMA treatment. Sp1 and Sp3 are associated with the HSD11B2 promoter under basal and stimulated conditions. There was no change in the control amplification of input DNA in treated and untreated samples. ChIP assays confirmed the in vivo relevance of the data obtained by EMSA assays.

View larger version (31K): [in this window] [in a new window]   Figure 2. ChIP analyses of the time course of TNF- or PMA-induced binding of p65, p50, Egr-1 at the HSD11B2 promoter. Cross-linked chromatin samples of untreated or treated SW620 cells were immunoprecipitated with anti-p65, anti-p50, anti-Egr-1, anti-Sp1, or anti-Sp3 antibodies; PCR analysis was performed with primers amplifying the region containing B1, Sp1/Egr-1I, and Sp1/Egr-1II sites. DNA products were of the expected size: 210 bp. Amplifications using unprecipitated DNA (no Ab) or nonspecific primers were negative.

5. Overexpression of Egr-1 represses p65/p50 mediated transcriptional activation and p50 inhibits the HSD11B2 promoter activity
High levels of p50 protein in the nuclear extracts and strong binding of p50/p50 homodimers to the HSD11B2 promoter after 24 h of TNF- or PMA stimulation of SW620 cells suggest that inactive p50/p50 homodimers inhibits expression of the HSD11B2 gene. To substantiate these findings, the effect of p50 overexpression on the human HSD11B2 promoter activity was examined in transfection studies. Overexpression of p50 reduced endogenous activity and mRNA abundance of 11ß-HSD2 in SW620 cells and repressed luciferase gene activity driven by various lengths of the HSD11B2 promoter. Since ChIP and EMSA data pointed to the existence of a combined binding of Egr-1 with p65/p50 heterodimers on the HSD11B2 promoter after treatment with inflammatory stimuli, we explored the impact of expressing recombinant transcription factors on this promoter. Results demonstrated clearly an inhibition of the p65/p50-mediated transactivation of the HSD11B2 promoter by overexpressing Egr-1.

6. Point mutations in B1, Sp1/Egr-1I, and Sp1/Egr-1II sites confirmed an important role of NF-B and Egr-1 in regulating HSD11B2 gene transcription
Specific point mutations in p65/p50 B1 binding sites (pmB1) decreased basal 11ß-HSD2 promoter activity by 50%. This may be explained by the fact that the mutation in p50 binding site partially destroys the overlapping binding sites for the constitutive Sp1/Sp3 factors. Promoter activation by p65/p50 and repression by p50 or TNF- were abrogated in the presence of pmB1 vector. When p-220/+117 vector was site-specifically mutated in the Egr-1/Sp1/Sp3 binding sites of the Sp1/Egr-1I and Sp1/Egr-1II regions, a considerable decrease of basal and Egr-1 mediated promoter activity was observed. These results show that all regulatory regions play an important role for TNF- and PMA mediated HSD11B2 transcription.

CONCLUSIONS AND SIGNIFICANCE

Inflammation represents a life-threatening stress stimuli for mammalians. Adrenocorticocosteroids are part of the stress response system. Intracellular access of the adrenal hormone cortisol to the cytoplasmic MR is regulated by the 11ß-HSD2 enzyme, which converts cortisol in humans and corticosterone in rodents to their receptor inactive ketoforms, cortisone, and dehydrocorticosterone, protecting the nonselective MR from occupation by glucocorticoids. Here, we demonstrate for the first time that the proinflammatory mediator TNF- down-regulates 11ß-HSD2 in vivo, as 11ß-HSD2 mRNA abundance and activity in kidney tissue of TNF--Tg mice were decreased. An increased urinary ratio of (THB+5-THB/THA) confirmed the reduced 11ß-HSD2 activity in intact animals. Exposure to TNF- or PMA for 24 h was sufficient to decrease HSD11B2 expression and activity by nearly 50% in SW620 cells. The percentage of this down-regulation of 11ß-HSD2 by proinflammatory stimuli in mice and cell cultures was of the same magnitude as observed in humans with abnormal renal sodium retention and salt-sensitive hypertension.

This investigation provides evidence for the molecular mechanisms by which TNF- and PMA influence expression and activity of 11ß-HSD2. For basal transcription, GC-rich regions Sp1/Egr-1I, Sp1/Egr-1II, Sp1III, and Sp1IV are occupied by Sp1 and Sp3 factors (Fig. 3 ). For TNF- and PMA-mediated transcription, in vivo footprinting and ChIP analyses demonstrated that three overlapping regions are required: B1, Sp1/Egr-1I, and Sp1/Egr-1II. Early stimulation with TNF- and PMA induced binding of the active p65/p50 heterodimers to the B1 site of the promoter. Treatment with TNF- for 0.5 h induced strong binding of Egr-1, which possibly competed with Sp1/Sp3 for binding to the Sp1/Egr-1I region and, by the repressed transcriptional activity of p65/p50, resulting in reduced HSD11B2 transcription. PMA treatment for 0.5 h did not induce high amounts of nuclear Egr-1, allowing an early transcriptional up-regulation of the HSD11B2 by p65/p50 and Sp1/Sp3. However, 4 h of treatment with PMA induced high levels of Egr-1 binding to the Sp1/Egr-1I and, to a lesser extent, to the adjacent Sp1/Egr-1II site, leading to repression of the p65/p50 as reflected by a decline of HSD11B2 transcription. At 24 h treatment with TNF- and PMA, down-regulation of HSD11B2 transcription was associated with predominant binding of inactive p50/p50 homodimers to B1.

View larger version (22K): [in this window] [in a new window]   Figure 3. Time-dependent protein-DNA interactions on the HSD11B2 promoter. Transcription factors = black, gray, and white circles or squares.

We believe this novel molecular mechanism for down-regulation of 11ß-HSD2 by p50 and Egr-1 during inflammation is important for further elucidation of the role of 11ß-HSD2 in linking inflammatory with cardiovascular diseases.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2820fje;

¹ Current address: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

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