Acute activation of NHE3 by dexamethasone correlates with activation of SGK1 and requires a functional glucocorticoid receptor

Abstract

Glucocorticoids stimulate the intestinal absorption of Na(+) and water partly by regulation of the Na(+)/H(+) exchanger 3 (NHE3). Previous studies have shown both genomic and nongenomic regulation of NHE3 by glucocorticoids. Serum and glucocorticoid-inducible kinase 1 (SGK1) has been shown to be part of this cascade, where phosphorylation of NHE3 by SGK1 initiates the translocation of NHE3 to the cell surface. In the present work, we examined a series of changes in SGK1 and NHE3 induced by glucocorticoids using human colonic Caco-2 and opossum kidney cells. We found that dexamethasone rapidly stimulated SGK1 mRNAs, but a significant change in protein abundance was not detected. Instead, there was an increase in SGK1 kinase activity as early as at 2 h. An increase in NHE3 protein abundance was not detected until 12 h of dexamethasone exposure, although the transport activity was significantly stimulated at 4 h. These data demonstrate that the changes of SGK1 precede those of NHE3. Chronic regulation (24 h) of NHE3 was blocked completely by prevention of protein synthesis with cycloheximide or actinomycin D and by the glucocorticoid receptor blocker RU486. The acute effect of dexamethasone was similarly abrogated by RU486, but was insensitive to cycloheximide and actinomycin D. Similarly, the stimulation of SGK1 activity by dexamethasone was blocked by RU486 but not by actinomycin D. Together, these data show that the acute effect of glucocorticoids on NHE3 is mediated by a glucocorticoid receptor dependent mechanism that activates SGK1 in a nongenomic manner.

Cyclic GMP Kinase II (cGKII) Inhibits NHE3 by Altering Its Trafficking and Phosphorylating NHE3 at Three Required Sites

“…663 has been reported to be a specific phosphorylation site for SGK1 and to be necessary for dexamethasone to stimulate NHE3 over short times, not involving transcriptional regulation (39,40). Because our studies showed that cGMP/cGKII phosphorylation of NHE3 at this site inhibited NHE3 activity, we initially confirmed that dexamethasone stimulates NHE3 activity under basal conditions through phosphorylation of Ser 663 and increases NHE3 surface expression (Fig.…”

Human intestinal epithelial cell line SK-CO15 is a new model system to study Na+/H+exchanger 3

“…6B) and NHE3 activity (Fig. 6C), consistent with previous studies using Caco-2 or rabbit intestinal brush-border membrane (35,42).…”

“…The characterization of NHE3 has utilized several cell lines, including fibroblasts, OK, Caco-2, and, to a lesser extent, IEC-6 cells (16,18,35,40,43). Of these, the OK cell line shows robust expression of NHE3, but its renal proximal tubule origin limits its application in the study of intestinal NHEs.…”

“…A clonal variant of Caco-2 cells, Caco-2bbe, has been used, but as for Caco-2 cells, the endogenous level of NHE3 in Caco-2bbe cells is either low or undetected, and the usefulness of the Caco-2bbe clone is largely limited to the characterization of transfected NHE3 (20,21). Alternatively, rat intestinal epithelial IEC-6 has been used, but these cells primarily express NHE1 and lack the endogenous expression of NHE3 (35). Isolation of alternative intestinal epithelial cells suitable for the characterization of NHE3 has been sluggish due to the difficulties in isolation and long-term propagation of primary intestinal epithelial cells.…”

Hormonal Actions and Interactions in Proximal Tubule Cells Associated with the Development of Chronic Kidney Disease

“…They directly stimulate NHE3 through both acute and chronic mechanisms (Bobulescu and Moe 2009). The acute effect of glucocorticoids on cell surface NHE3 is mediated by the activation of HNE3 exocytosis (Bobulescu et al 2005) and a glucocorticoid receptor-dependent mechanism that activates serum and glucocorticoid-inducible kinase1 (SGK1) in a nongenomic manner (Wang et al 2007). Glucocorticoids have also been found to enhance the function of insulin in stimulating NHE3 activity (Klisic et al 2002).…”