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

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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.

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Effect of corticosteroids on Na+transport and on sgk1_v3 expression. (A) Effect of aldosterone for 4 h on short circuit current in mpkCCDc14 cells. Similar results were obtained for 24 h exposure from several independent experiments. (B) Effect of aldosterone for 24 h on sgk1 and sgk1_v3 mRNA expression in mpkCCDc14 cells. n = 8, mean ± SE from 2 exps. (C) Effect of 100 nm dexamethasone on Sgk1 and Sgk1_v3 mRNA expression in A549 cells for 4 h. Representative of several independent experiments. (D) Effect of 100 nm dexamethasone on Sgk1 and Sgk1_v3 mRNA expression in H441 cells for 2 h. n = 8, mean ± SE from 3 exps. *P < 0.05, **P < 0.01.
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phy213221-fig-0001: Effect of corticosteroids on Na+transport and on sgk1_v3 expression. (A) Effect of aldosterone for 4 h on short circuit current in mpkCCDc14 cells. Similar results were obtained for 24 h exposure from several independent experiments. (B) Effect of aldosterone for 24 h on sgk1 and sgk1_v3 mRNA expression in mpkCCDc14 cells. n = 8, mean ± SE from 2 exps. (C) Effect of 100 nm dexamethasone on Sgk1 and Sgk1_v3 mRNA expression in A549 cells for 4 h. Representative of several independent experiments. (D) Effect of 100 nm dexamethasone on Sgk1 and Sgk1_v3 mRNA expression in H441 cells for 2 h. n = 8, mean ± SE from 3 exps. *P < 0.05, **P < 0.01.

Mentions: We have previously reported the primary structure and expression profile of the alternate 5ʹ variant sgk1 transcript, sgk1_v3 and showed that aldosterone and insulin increased sgk1_v3 expression in mpkCCDc14 cells. We also reported that its encoded protein sgk1_i3 is more stable compared to sgk1 and that it stimulates apical epithelial Na+ transport when expressed in FRT and in mpkCCDc14 cells (Raikwar et al. 2012). Sgk1 and Sgk1_i3 differ only in the NH2 terminus where 25AA in Sgk1 is replaced by 39 AA in Sgk1_i3. To further explore the basis for the corticosteroid stimulation of sgk1_v3 expression, we cultured mpkCCDc14 cells and demonstrated a dose‐dependent increase in short circuit current with aldosterone that correlated with a dose dependent increase in sgk1_v3 mRNA expression in mpkCCDc14 cells (Fig. 1A and B). We also demonstrate that dexamethasone stimulated an increase in Sgk1_v3 in the lung epithelial cell line, A549 and H441 (Fig. 1C, D). These results suggest that mineralocorticoids and glucocorticoids may increase the abundance of Sgk1_v3, similar to Sgk1, via a transcriptional regulatory element.


Aldosterone regulates a 5 ʹ variant sgk1 transcript via a shared hormone response element in the sgk1 5 ʹ regulatory region
Effect of corticosteroids on Na+transport and on sgk1_v3 expression. (A) Effect of aldosterone for 4 h on short circuit current in mpkCCDc14 cells. Similar results were obtained for 24 h exposure from several independent experiments. (B) Effect of aldosterone for 24 h on sgk1 and sgk1_v3 mRNA expression in mpkCCDc14 cells. n = 8, mean ± SE from 2 exps. (C) Effect of 100 nm dexamethasone on Sgk1 and Sgk1_v3 mRNA expression in A549 cells for 4 h. Representative of several independent experiments. (D) Effect of 100 nm dexamethasone on Sgk1 and Sgk1_v3 mRNA expression in H441 cells for 2 h. n = 8, mean ± SE from 3 exps. *P < 0.05, **P < 0.01.
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phy213221-fig-0001: Effect of corticosteroids on Na+transport and on sgk1_v3 expression. (A) Effect of aldosterone for 4 h on short circuit current in mpkCCDc14 cells. Similar results were obtained for 24 h exposure from several independent experiments. (B) Effect of aldosterone for 24 h on sgk1 and sgk1_v3 mRNA expression in mpkCCDc14 cells. n = 8, mean ± SE from 2 exps. (C) Effect of 100 nm dexamethasone on Sgk1 and Sgk1_v3 mRNA expression in A549 cells for 4 h. Representative of several independent experiments. (D) Effect of 100 nm dexamethasone on Sgk1 and Sgk1_v3 mRNA expression in H441 cells for 2 h. n = 8, mean ± SE from 3 exps. *P < 0.05, **P < 0.01.
Mentions: We have previously reported the primary structure and expression profile of the alternate 5ʹ variant sgk1 transcript, sgk1_v3 and showed that aldosterone and insulin increased sgk1_v3 expression in mpkCCDc14 cells. We also reported that its encoded protein sgk1_i3 is more stable compared to sgk1 and that it stimulates apical epithelial Na+ transport when expressed in FRT and in mpkCCDc14 cells (Raikwar et al. 2012). Sgk1 and Sgk1_i3 differ only in the NH2 terminus where 25AA in Sgk1 is replaced by 39 AA in Sgk1_i3. To further explore the basis for the corticosteroid stimulation of sgk1_v3 expression, we cultured mpkCCDc14 cells and demonstrated a dose‐dependent increase in short circuit current with aldosterone that correlated with a dose dependent increase in sgk1_v3 mRNA expression in mpkCCDc14 cells (Fig. 1A and B). We also demonstrate that dexamethasone stimulated an increase in Sgk1_v3 in the lung epithelial cell line, A549 and H441 (Fig. 1C, D). These results suggest that mineralocorticoids and glucocorticoids may increase the abundance of Sgk1_v3, similar to Sgk1, via a transcriptional regulatory element.

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.


Related in: MedlinePlus