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. 2013 Sep;41(17):8045-60.
doi: 10.1093/nar/gkt581. Epub 2013 Jul 1.

Mineralocorticoid Receptor Interaction With SP1 Generates a New Response Element for Pathophysiologically Relevant Gene Expression

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Mineralocorticoid Receptor Interaction With SP1 Generates a New Response Element for Pathophysiologically Relevant Gene Expression

Sandra Meinel et al. Nucleic Acids Res. .
Free PMC article

Abstract

The mineralocorticoid receptor (MR) is a ligand-induced transcription factor belonging to the steroid receptor family and involved in water-electrolyte homeostasis, blood pressure regulation, inflammation and fibrosis in the renocardiovascular system. The MR shares a common hormone-response-element with the glucocorticoid receptor but nevertheless elicits MR-specific effects including enhanced epidermal growth factor receptor (EGFR) expression via unknown mechanisms. The EGFR is a receptor tyrosine kinase that leads to activation of MAP kinases, but that can also function as a signal transducer for other signaling pathways. In the present study, we mechanistically investigate the interaction between a newly discovered MR- but not glucocorticoid receptor- responsive-element (=MRE1) of the EGFR promoter, specificity protein 1 (SP1) and MR to gain general insights into MR-specificity. Biological relevance of the interaction for EGFR expression and consequently for different signaling pathways in general is demonstrated in human, rat and murine vascular smooth muscle cells and cells of EGFR knockout mice. A genome-wide promoter search for identical binding regions followed by quantitative PCR validation suggests that the identified MR-SP1-MRE1 interaction might be applicable to other genes. Overall, a novel principle of MR-specific gene expression is explored that applies to the pathophysiologically relevant expression of the EGFR and potentially also to other genes.

Figures

Figure 1.
Figure 1.
Importance of EGFR as a mediator for other signaling pathways. VSMC of EGFR knockout or wild-type mice were incubated for 5 min with the EGFR ligand EGF (10 µg/l), the PKC activator phorbol-12-myristate-13-acetate (1 µM) or the MR ligand aldosterone (10 nM aldosterone). ERK phosphorylation was assessed by western blot (n = 4–5, data represented as mean ± SEM).
Figure 2.
Figure 2.
MR interaction site on EGFR promoter (A) HEK cells top: Scheme of the EGFR promoter showing localization of MRE1, bottom: HEK cells transiently transfected with MRE1 reporter plasmid and hMR were incubated with 10 nM aldosterone or vehicle for 48 h, and then reporter gene activities were measured and depicted as percentage of control (MRE1 with vehicle) (N = 3–18, n = 9–45, data represented as mean ± SEM), (B) OK cells transiently transfected with MRE1 reporter plasmid and hMR were incubated with 10 nM aldosterone or vehicle for 48 h, and then reporter gene activities were measured and depicted as percentage of control (MRE1.3 with vehicle) (N = 4–16, n = 15–47, data represented as mean ± SEM), (C) Scheme of the different MRE1 promoter deletion constructs tested in the reporter gene assay.
Figure 3.
Figure 3.
MR specificity and dose dependency of MRE1.3 in HEK and OK cells. (A) MR-specificity of MRE1.3 was tested in a reporter gene assay in HEK cells transfected either with MR or GR plus MRE1.3 reporter plasmid and then stimulated with 10 nM aldosterone or 100 nM dexamethasone accordingly (N = 6–8, n = 14–24, data represented as mean ± SEM, *P ≤ 0.05). (B) A dose-response-curve for rising aldosterone concentrations was constructed for MR transactivation activity at MRE1.3 in hMR-HEK cells and EC50 was calculated (N = 3–8, n = 9–27, data represented as mean ± SEM). (C) Experiment (A) was repeated with OK cells to exclude cell-specific effects (N = 4, n = 8–12, data represented as mean ± SEM, *P ≤ 0.05). (D) Experiment (B) was also repeated with OK cells transfected with MR and MRE1.3 or GRE reporter plasmid (N = 3–6, n = 9–18 data represented as mean ± SEM), (E) Influence of eplerenone (antagonist of aldosterone) on MR/aldosterone induced response of MRE1.3 was tested in a reporter gene assay in transiently transfected hMR OK cells after incubation with either vehicle or 10 nM aldosterone after 48 h and depicted as percentage of control (MRE1.3 with vehicle) (N = 3, n = 9, data represented as mean ± SEM), (F) Activation of MRE1.3 by cortisol and aldosterone was analyzed in a reporter gene assay in OK cells transiently transfected with hMR and MRE1.3 and incubation with vehicle, 10 nM aldosterone or 1 µM cortisol for 48 h (N = 3, n = 9, data represented as mean ± SEM).
Figure 4.
Figure 4.
In vitro binding of MR to MRE1 (A) In a transcription factor-binding ELISA, nuclear extracts of hMR- and hGR-HEK cells that had been previously stimulated with vehicle, 10 nM aldosterone or 1 µM hydrocortisone, respectively, were tested for binding of hMR or hGR to biotinylated MRE1. (N = 1–3, n = 4–11, data represented as mean ± SEM, *P ≤ 0.05). (B) Binding of in vitro synthesized MR to MRE1 was tested and compared with unspecific binding of MR to DNA (unspecific = binding of MR to DNA probes of exon of eNOS) and binding of MR to a probe containing three classical GREs, which was used as positive control for this experiment (N = 2–3, n = 4–6, data represented as mean ± SEM, *P ≤ 0.05).
Figure 5.
Figure 5.
Binding of SP1 to MRE1.3, MRE1.3a and MRE1.3c in EMSAs. (A) Sequences of MRE1.3 with in silico predicted SP1-binding sites, different MRE1.3 deletion constructs and constructs with mutated SP1-binding site are depicted. (B) EMSAs for detection of SP1 binding were performed with biotinylated MRE1.3 (lanes 1–3) or MRE1.3a (lanes 4–6) probe. Probes were either incubated with buffer (probe), 300 ng BSA as control for unspecific protein interactions (BSA) or with 300 ng of rhSP1. (C) To identify the relevant SP1-binding site for the interaction, we compared biotinylated MRE1.3c (lanes 1–3) with biotinylated MRE1.3 (lanes 4–6). Probes were either incubated with only buffer (probe), 300 ng of BSA as control for unspecific protein interactions (BSA) or with 300 ng of rhSP1. (D) To exclude unspecific SP1-DNA interactions, EMSAs with biotinylated MRE1.3 (lanes 1–3) were compared with EMSAs with biotinylated CRE (cAMP response element) (lanes 4–6) probe. Probes were either incubated with only buffer (probe), 300 ng of BSA as control for unspecific protein interactions (BSA) or with 300 ng of rhSP1 (N = 6). (E) To verify in vivo binding of SP1 to MRE1.3, we analyzed binding of protein from nuclear extracts to MRE1.3 and MRE1.3 with mutations in the SP1-binding sequence, MRE1.3mut1 and MRE1.3mut2. Biotinylated SP1 consensus sequence was used as a positive control. Probes were either incubated with only buffer (probe) or nuclear extracts (NE) (N = 4).
Figure 6.
Figure 6.
Importance of full-length MRE1.3 and the SP1-binding site (A) Sequences of MRE1.3, different MRE1.3 deletion constructs and constructs with mutated SP1-binding site are depicted. (B) OK cells transiently transfected with hMR and reporter plasmids were incubated with 10 nM aldosterone or vehicle for 48 h and then reporter gene activities were measured and depicted as percentage of control (MRE1.3 with vehicle). MRE1.3mut1 and MRE1.3mut2, both mutated in the SP1-binding site, were compared with MRE1.3 (N = 3–6, n = 9–18, data represented as mean ± SEM). (C) Transiently hMR- and reporter plasmid transfected OK cells were incubated with 10 nM aldosterone or vehicle for 48 h. Reporter gene activities of four different deletion constructs (MRE1.3b, e and f and MRE1.3mut Δ) were measured and compared with full-length MRE1.3 transactivation (N = 3–6, n = 9–18, data represented as mean ± SEM).
Figure 7.
Figure 7.
Effect of SP1 inhibition by WP631 on MR-induced MRE1.3 reporter gene activity. (A) HEK cells transfected with hMR and MRE1.3 reporter plasmid were incubated for 48 h with vehicle or 10 nM aldosterone in the presence and absence of 5 µM WP631, a potent inhibitor of SP1-dependent transcription (N = 3, n = 6–9, data represented as mean ± SEM, *P ≤ 0.05). (B) To exclude unspecific effects on transcription, reporter gene activity was also measured in HEK cells transfected with hMR and classical GRE reporter plasmid after incubating the cells with vehicle or 10 nM aldosterone in the presence and absence of 5 µM WP631 (N = 3, n = 9, data represented as mean ± SEM, *P ≤ 0.05). (C) The effect of rising concentrations of WP631 on aldosterone-induced MRE1.3 promoter activity was tested in OK cells transfected with hMR (N = 1–11, n = 3–33, data represented as mean ± SEM, *P ≤ 0.05), (D) OK cells transfected with hMR and MRE1.3 reporter plasmid were incubated for 48 h with vehicle or 10 nM aldosterone in the presence and absence of 5 µM WP631 (N = 3–4, n = 9–12, data represented as mean ± SEM, *P ≤ 0.05), (E) To exclude unspecific effects on transcription, reporter gene activity was also measured in OK cells transfected with hMR and classical GRE reporter plasmid after incubating the cells with vehicle or 10 nM aldosterone in the presence and absence of 5 µM WP631 (N = 10, n = 30, data represented as mean ± SEM, *P ≤ 0.05).
Figure 8.
Figure 8.
Importance of SP1 for MR-induced MRE1.3 reporter activity and binding of MR to MRE1: (A–C) Effect of SP1 inhibition by SP1-siRNA on MR-induced MRE1.3 reporter gene activity (A) HEK cells were transiently transfected with hMR and with either scramble-siRNA or SP1-specific siRNA. SP1- mRNA levels were then quantified by qPCR 48, 72, and 96 h after transfection and compared to untransfected cells (N = 3, n = 6, data represented as mean ± SEM, *P ≤ 0.05), (B) A representative western blot of SP1 protein levels 96 and 120 h after transfection of HEK cells is shown (N = 3, n = 6). (C) HEK cells transfected with hMR and reporter plasmid were stimulated with vehicle or 10 nM aldosterone. Aldosterone-induced MRE1.3 reporter activity was additionally assessed in the presence of scramble-siRNA and specific SP1-siRNA (N = 3, n = 9, data represented as mean ± SEM, *P ≤ 0.05). (D) Importance of SP1 for MR binding to MRE1 (D) Binding of in vitro synthesized hMR to MRE1 was quantified in the presence and absence of 100 ng of rhSP1 in a transcription factor-binding assay. Binding of in vitro synthesized hGR to MRE1 was also detected in the presence and absence of rhSP1 (N = 2–5, n = 4–12, data represented as mean ± SEM, *P ≤ 0.05).
Figure 9.
Figure 9.
Relevance of SP3 for MR-induced MRE1.3 reporter activity (A) top: Transiently transfected hMR-HEK cells were incubated with either SP3-specific siRNA or scrambled siRNA for 96 h. SP3 protein levels were quantified by western blot and compared with untransfected cells (N = 3, n = 6, data represented as mean ± SEM, *P ≤ 0.05), bottom: representative western blot. (B) HEK cells transfected with hMR and reporter plasmid were stimulated with vehicle or 10 nM aldosterone. Aldosterone-induced MRE1.3 reporter activity was additionally analyzed in the presence of scrambled-siRNA and specific SP3-siRNA (N = 3, n = 6–48), data represented as mean ± SEM, *P ≤ 0.05). (C) Binding of human recombinant SP3 to MRE1.3 and SP1 consensus sequence was analyzed. Probes were either incubated with only buffer (probe), 100 ng of BSA as control for unspecific protein interactions or 100 ng rhSP3 (N = 5).
Figure 10.
Figure 10.
MR domains involved in the interaction with MRE1.3 (A) Deletion constructs of hMR consisting of the domains A and B (AB) or the domains C, D, E, F (CDEF) were tested for their ability to increase MRE1.3 promoter activity in HEK cells (N = 3, n = 9, data represented as mean ± SEM, *P ≤ 0.05). (B) The MRE1.3 promoter activity of the MR deletion constructs used in (A) was also tested in OK cells to exclude cell-specific effects (N = 4–8, n = 12–24, data represented as mean ± SEM, *P ≤ 0.05).
Figure 11.
Figure 11.
Relevance of SP1 for aldosterone-induced EGFR expression in primary culture. (A) HAoSMC were incubated for 24 h with either vehicle, 10 nM aldosterone, 1 µM WP631 or 10 nM aldosterone and 1 µM WP631 and EGFR protein expression was quantified by western blot and densitometric analysis (n = 5, data represented as mean ± SEM, *P ≤ 0.05). (B) Primary rat aortic smooth muscle cells (A7r5) were incubated for 24 h with either vehicle, 10 nM aldosterone, 1 µM WP631 or 10 nM aldosterone and 1 µM WP631 and EGFR protein expression was quantified by western blot and densitometric analysis (n = 3, data represented as mean ± SEM, *P ≤ 0.05).
Figure 12.
Figure 12.
Influence of MR/aldosterone on regulation of promoter regions similar to MRE1.3. An immortalized proximal tubule epithelial cell line from normal adult human kidney (HK2) was transfected with hMR and incubated either with 10 nM aldosterone or vehicle for 24 h. mRNA expression levels of different genes containing promoter regions similar to MRE1.3 and judged to be of potential pathophysiological relevance by literature were detected and depicted as fold induction and ddcq. (N = 3–5, n = 3–15, data represented as mean ± SEM, *P ≤ 0.05).
Figure 13.
Figure 13.
Model of MR-SP1-MRE1.3 Interaction. (A) MR may bind to the DNA adjacent to SP1. (B) SP1 may modulate DNA structure to enable MR binding. (C) MR could bind to SP1.

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