Limits...
Aldosterone regulates a 5 ʹ variant sgk1 transcript via a shared hormone response element in the sgk1 5 ʹ regulatory region

View Article: PubMed Central - PubMed

ABSTRACT

We previously identified a 5ʹ variant alternate transcript of Sgk1 (Sgk1_v3) encoding an NH2‐terminal variant Sgk1 isoform, Sgk1_i3 that, like Sgk1, is expressed in the distal convoluted tubule, connecting tubule and collecting duct and can stimulate epithelial Na+ transport (Am J Physiol Renal Physiol 303: F1527–F1533, 2012). We now demonstrate that, similar to Sgk1, aldosterone and glucocorticoids stimulate Sgk1_v3 expression in cell lines from the collecting duct and airway epithelia. In mice, short term aldosterone infusion and maneuvers that increase endogenous aldosterone secretion including dietary Na+ deprivation and K+ loading increases distal nephron Sgk1_v3 expression in vivo. Although Sgk1_v3 has a different 5ʹ proximal regulatory region from Sgk1, the transcription start sites are less than 1000 bp apart. We cloned the 5ʹ regulatory region for Sgk1 and Sgk_v3 upstream of a luciferase gene and by deletion and reporter gene analysis we localized the corticosteroid regulatory region for Sgk1_v3 to a glucocorticoid response element (GRE) that had previously been identified for Sgk1 (Am J Physiol Endo Metab 283: E971–E979, 2002). We tested this element with MR in an MR‐ cell line and demonstrate that aldosterone stimulates Sgk1 and Sgk1_v3 via this GRE. We conclude that corticosteroids stimulate Sgk1 and Sgk1_v3 expression in epithelial cells via activation of a common conserved GRE in the 5ʹ flanking region of Sgk1.

No MeSH data available.


Identification of cis‐elements required for corticosteroid‐mediated Sgk1_v3 expression. (A) Schematic showing proximal exons (1e, 1d, 1 and 2) and intervening regulatory regions for human Sgk1 gene as well as the sequence present in each of the Sgk1 luciferase (LUC) constructs. (B), (C) and (D) A549 cells transfected with indicated constructs and treated with 100 nmol/L dexamethasone, or vehicle for 24 h. n = 3, mean ± SE** P < 0.01. Constructs that do not contain exon 1d and sequence 5ʹ are not dexamethasone responsive. Selective mutation of the GRE abolishes dexamethasone responsiveness. (E) HT‐29 cells cotransfected with luciferase constructs and rat MR or empty plasmid and treated with 100 nmol/L aldosterone or vehicle for 24 h. Aldosterone increases luciferase activity via the GRE in the Sgk1_v3 5ʹ regulatory region. n = 3, mean ± SE* P < 0.05. This GRE is sufficient to confer aldosterone responsiveness to a heterologous construct.
© Copyright Policy - creativeCommonsBy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5392512&req=5

phy213221-fig-0003: Identification of cis‐elements required for corticosteroid‐mediated Sgk1_v3 expression. (A) Schematic showing proximal exons (1e, 1d, 1 and 2) and intervening regulatory regions for human Sgk1 gene as well as the sequence present in each of the Sgk1 luciferase (LUC) constructs. (B), (C) and (D) A549 cells transfected with indicated constructs and treated with 100 nmol/L dexamethasone, or vehicle for 24 h. n = 3, mean ± SE** P < 0.01. Constructs that do not contain exon 1d and sequence 5ʹ are not dexamethasone responsive. Selective mutation of the GRE abolishes dexamethasone responsiveness. (E) HT‐29 cells cotransfected with luciferase constructs and rat MR or empty plasmid and treated with 100 nmol/L aldosterone or vehicle for 24 h. Aldosterone increases luciferase activity via the GRE in the Sgk1_v3 5ʹ regulatory region. n = 3, mean ± SE* P < 0.05. This GRE is sufficient to confer aldosterone responsiveness to a heterologous construct.

Mentions: Sgk1_v3 is a 5ʹ variant alternate transcript of the Sgk1 gene that has a different transcription start site and a different 5ʹ proximal regulatory region from Sgk1 (Fig. 3A). Others and we have demonstrated that glucocorticoid regulation of Sgk1 is mediated via a GRE in the 5ʹ flanking region of Sgk1 (Webster et al. 1993; Itani et al. 2002). A careful analysis of the 5ʹ flanking region of Sgk1 demonstrated that the GRE that was reported to be 1152 bp upstream of the Sgk1 transcription start site is also 319 bp upstream of the Sgk1_v3 transcription start site since the first exon for Sgk1_v3 (exon 1d) is 834 bp upstream of the first exon for Sgk1 (exon 1). To determine if this GRE functioned to regulate Sgk1 and Sgk1_v3 we cloned this fragment upstream of a luciferase reporter gene and then isolated the 5ʹ flanking region for both exons. A ~2600 bp construct, Sgk1(1 + 3), that included the GRE, the complete exon 1d and the transcription start site for exon 1 conferred glucocorticoid‐responsiveness to the luciferase reporter in A549 cells (Fig. 3B). A ~1600 bp construct, Sgk1(3), that included the GRE and the transcription start site for exon 1d but not downstream sequence was also glucocorticoid‐responsive indicating that this region was sufficient to stimulate transcription of Sgk1_v3 (Fig. 3B). In comparison, a ~500 bp region upstream of exon 1 that excluded exon 1d and sequences 5ʹ to it was not glucocorticoid responsive (Sgk1) indicating that exon 1d or sequences 5ʹ to it are necessary for the glucocorticoid response of the Sgk1 promoter construct (Fig. 3C). To confirm that the GRE mediated the GC responsiveness for exon 1d, we selectively mutated the element from −1234 to −1249 within the promoter reporter construct (Sgk1(3)mutGRE) and demonstrated loss of GC responsiveness (Fig. 3D). To verify that the GRE was sufficient for the GC response we transferred two copies of this GRE to a promoter reporter construct containing the 5ʹ flanking region of αENaC (aENaC(−141 + 55)Sgk2xGRE). We demonstrate that the αENaC construct containing the Sgk1_v3 GRE becomes GC responsive (Fig. 3D).


Aldosterone regulates a 5 ʹ variant sgk1 transcript via a shared hormone response element in the sgk1 5 ʹ regulatory region
Identification of cis‐elements required for corticosteroid‐mediated Sgk1_v3 expression. (A) Schematic showing proximal exons (1e, 1d, 1 and 2) and intervening regulatory regions for human Sgk1 gene as well as the sequence present in each of the Sgk1 luciferase (LUC) constructs. (B), (C) and (D) A549 cells transfected with indicated constructs and treated with 100 nmol/L dexamethasone, or vehicle for 24 h. n = 3, mean ± SE** P < 0.01. Constructs that do not contain exon 1d and sequence 5ʹ are not dexamethasone responsive. Selective mutation of the GRE abolishes dexamethasone responsiveness. (E) HT‐29 cells cotransfected with luciferase constructs and rat MR or empty plasmid and treated with 100 nmol/L aldosterone or vehicle for 24 h. Aldosterone increases luciferase activity via the GRE in the Sgk1_v3 5ʹ regulatory region. n = 3, mean ± SE* P < 0.05. This GRE is sufficient to confer aldosterone responsiveness to a heterologous construct.
© Copyright Policy - creativeCommonsBy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5392512&req=5

phy213221-fig-0003: Identification of cis‐elements required for corticosteroid‐mediated Sgk1_v3 expression. (A) Schematic showing proximal exons (1e, 1d, 1 and 2) and intervening regulatory regions for human Sgk1 gene as well as the sequence present in each of the Sgk1 luciferase (LUC) constructs. (B), (C) and (D) A549 cells transfected with indicated constructs and treated with 100 nmol/L dexamethasone, or vehicle for 24 h. n = 3, mean ± SE** P < 0.01. Constructs that do not contain exon 1d and sequence 5ʹ are not dexamethasone responsive. Selective mutation of the GRE abolishes dexamethasone responsiveness. (E) HT‐29 cells cotransfected with luciferase constructs and rat MR or empty plasmid and treated with 100 nmol/L aldosterone or vehicle for 24 h. Aldosterone increases luciferase activity via the GRE in the Sgk1_v3 5ʹ regulatory region. n = 3, mean ± SE* P < 0.05. This GRE is sufficient to confer aldosterone responsiveness to a heterologous construct.
Mentions: Sgk1_v3 is a 5ʹ variant alternate transcript of the Sgk1 gene that has a different transcription start site and a different 5ʹ proximal regulatory region from Sgk1 (Fig. 3A). Others and we have demonstrated that glucocorticoid regulation of Sgk1 is mediated via a GRE in the 5ʹ flanking region of Sgk1 (Webster et al. 1993; Itani et al. 2002). A careful analysis of the 5ʹ flanking region of Sgk1 demonstrated that the GRE that was reported to be 1152 bp upstream of the Sgk1 transcription start site is also 319 bp upstream of the Sgk1_v3 transcription start site since the first exon for Sgk1_v3 (exon 1d) is 834 bp upstream of the first exon for Sgk1 (exon 1). To determine if this GRE functioned to regulate Sgk1 and Sgk1_v3 we cloned this fragment upstream of a luciferase reporter gene and then isolated the 5ʹ flanking region for both exons. A ~2600 bp construct, Sgk1(1 + 3), that included the GRE, the complete exon 1d and the transcription start site for exon 1 conferred glucocorticoid‐responsiveness to the luciferase reporter in A549 cells (Fig. 3B). A ~1600 bp construct, Sgk1(3), that included the GRE and the transcription start site for exon 1d but not downstream sequence was also glucocorticoid‐responsive indicating that this region was sufficient to stimulate transcription of Sgk1_v3 (Fig. 3B). In comparison, a ~500 bp region upstream of exon 1 that excluded exon 1d and sequences 5ʹ to it was not glucocorticoid responsive (Sgk1) indicating that exon 1d or sequences 5ʹ to it are necessary for the glucocorticoid response of the Sgk1 promoter construct (Fig. 3C). To confirm that the GRE mediated the GC responsiveness for exon 1d, we selectively mutated the element from −1234 to −1249 within the promoter reporter construct (Sgk1(3)mutGRE) and demonstrated loss of GC responsiveness (Fig. 3D). To verify that the GRE was sufficient for the GC response we transferred two copies of this GRE to a promoter reporter construct containing the 5ʹ flanking region of αENaC (aENaC(−141 + 55)Sgk2xGRE). We demonstrate that the αENaC construct containing the Sgk1_v3 GRE becomes GC responsive (Fig. 3D).

View Article: PubMed Central - PubMed

ABSTRACT

We previously identified a 5&#697; variant alternate transcript of Sgk1 (Sgk1_v3) encoding an NH2&#8208;terminal variant Sgk1 isoform, Sgk1_i3 that, like Sgk1, is expressed in the distal convoluted tubule, connecting tubule and collecting duct and can stimulate epithelial Na+ transport (Am J Physiol Renal Physiol 303: F1527&ndash;F1533, 2012). We now demonstrate that, similar to Sgk1, aldosterone and glucocorticoids stimulate Sgk1_v3 expression in cell lines from the collecting duct and airway epithelia. In mice, short term aldosterone infusion and maneuvers that increase endogenous aldosterone secretion including dietary Na+ deprivation and K+ loading increases distal nephron Sgk1_v3 expression in&nbsp;vivo. Although Sgk1_v3 has a different 5&#697; proximal regulatory region from Sgk1, the transcription start sites are less than 1000&nbsp;bp apart. We cloned the 5&#697; regulatory region for Sgk1 and Sgk_v3 upstream of a luciferase gene and by deletion and reporter gene analysis we localized the corticosteroid regulatory region for Sgk1_v3 to a glucocorticoid response element (GRE) that had previously been identified for Sgk1 (Am J Physiol Endo Metab 283: E971&ndash;E979, 2002). We tested this element with MR in an MR&#8208; cell line and demonstrate that aldosterone stimulates Sgk1 and Sgk1_v3 via this GRE. We conclude that corticosteroids stimulate Sgk1 and Sgk1_v3 expression in epithelial cells via activation of a common conserved GRE in the 5&#697; flanking region of Sgk1.

No MeSH data available.