The stimulative effect of glucocorticoids on intestinal salt and water absorption has been known for more than two decades. However, molecular mechanisms underlying this activation remain elusive. Previous studies showed that methylprednisolone specifically increased Na(+)/H(+) exchanger isoform (NHE) 3 mRNA in ileum and kidney without affecting NHE1 mRNA levels. These results suggest that glucocorticoids activate NHE3 activity by induction of NHE3 transcripts. We recently found in PS120 and opossum kidney cells that chronic incubation with dexamethasone activated NHE3 independent of gene induction, indicating that the transcriptional activation may not be the only determining factor in the NHE3 activation. Furthermore, dexamethasone activated NHE3 activity only in the presence of a NHE3 regulatory protein, NHERF2, which was previously shown to confer cAMP-dependent inhibition of NHE3. This activation of NHE3 could not be duplicated by NHERF1. We identified serum- and glucocorticoid-induced protein kinase, SGK1, as the protein interacting with PDZ domains of NHERF2 to regulate NHE3 activity. The expression of SGK1 enhanced NHE3 transport in PS120 fibroblasts. In addition, the "kinase-dead" SGK1 blocked activation of NHE3 by dexamethasone in opossum kidney cells. These data demonstrated that glucocorticoid activation of NHE3 requires the activation of SGK1 and the presence of NHERF2 acting as a scaffold protein.

Lysophosphatidic acid (LPA) is a mediator of multiple cellular responses. LPA mediates its effects predominantly through the G protein-coupled receptors LPA₁, LPA₂, and LPA₃. In the present work, we studied LPA₂-mediated signaling using human colon cancer cell lines, which predominantly express LPA₂. LPA₂ activated Akt and Erk1/2 in response to LPA. LPA mediated Akt activation was inhibited by pertussis toxin (PTX), whereas Erk1/2 activation was completely inhibited by a blocker of phospholipase Cβ, U-73122. LPA also induced interleukin-8 (IL-8) synthesis in the colon cancer cells by primarily activating LPA₂ receptor. We also found that LPA₂ interacts with Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2). Activation of Akt and Erk1/2 was significantly attenuated by silencing of NHERF2 expression by RNA interference, suggesting a pivotal role of NHERF2 in LPA₂-mediated signaling. We found that expression of LPA₂ was elevated, whereas expression of LPA₁ downregulated in several types of cancers, including ovarian and colon cancer. We conclude that LPA₂ is the major LPA receptor in colon cancer cells and cellular signals by LPA₂ are largely mediated through its ability to interact with NHERF2.

Background & Aims Diarrhea results from reduced net fluid and salt absorption caused by an imbalance in intestinal absorption and secretion. The bulk of sodium and water absorption in the intestine is mediated by Na⁺/H⁺ exchanger 3 (NHE3), located in the luminal membrane of enterocytes. We investigated the effect of lysophosphatidic acid (LPA) on Na⁺/H⁺ exchanger activity and Na⁺-dependent fluid absorption in the intestine. Methods We analyzed the effects of LPA on fluid absorption in intestines of wild-type mice and mice deficient in Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2; Nherf2^−/−) or LPA₂ (Lpa₂^−/−). Roles of LPA₅ and NHERF2 were determined by analysis of heterologous expression. Results Under basal conditions, LPA increased fluid absorption in an NHE3-dependent manner and restored the net fluid loss in a mouse model of acute diarrhea. Expression of the LPA receptor LPA₅ was necessary for LPA-induced stimulation of NHE3 activity in colonic epithelial cells. Stimulation of NHE3 by the LPA-LPA₅ signaling required coexpression of NHERF2, which interacted with LPA₅. LPA-mediated intestinal fluid absorption was impaired in Nherf2^−/− mice, demonstrating the requirement for NHERF2 in LPA₅ activity. However, fluid absorption was unaltered in Lpa₂^−/− mice. LPA stimulated NHE3 and fluid absorption in part by increasing NHE3 protein abundance at the brush border membrane of intestinal epithelial cells. Conclusions LPA is a potent stimulant of NHE3 and fluid absorption in the intestine, signaling through LPA₅. Regulation by LPA₅ depends on its interaction with NHERF2. LPA might be useful in the treatment of certain diarrheal diseases.

A major ofNa+absorptive process in the proximal part of intestine and kidney is electroneutral exchange ofNa+andH+byNa+/H+exchanger type 3 (NHE3). During the past decade, significant advance has been achieved in the mechanisms of NHE3 regulation. A bulk of the current knowledge onNa+/H+exchanger regulation is based on heterologous expression of mammalianNa+/H+exchangers inNa+/H+exchanger deficient fibroblasts, renal epithelial, and intestinal epithelial cells. Based on the reductionist's approach, an understanding of NHE3 regulation has been greatly advanced. More recently, confirmations of in vitro studies have been made using animals deficient in one or more proteins but in some cases unexpected findings have emerged. The purpose of this paper is to provide a brief overview of recent progress in the regulation and functions of NHE3 present in the luminal membrane of the intestinal tract.

Calcium (Ca²⁺) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca²⁺ regulates the activity of Na⁺/H⁺ exchanger 3 (NHE3). However, the effect of Ca²⁺-dependent signaling on NHE3 activity varies depending on cell types. In this study, we report the identification of IP₃ receptor-binding protein released with IP₃ (IRBIT) as a NHE3 interacting protein and its role in regulation of NHE3 activity. IRBIT bound to the carboxyl-terminal domain of NHE3, which is necessary for acute regulation of NHE3. Ectopic expression of IRBIT resulted in Ca²⁺-dependent activation of NHE3 activity, whereas silencing of endogenous IRBIT resulted in inhibition of NHE3 activity. Ca²⁺-dependent stimulation of NHE3 activity was dependent on the binding of IRBIT to NHE3. Previously Ca²⁺-dependent inhibition of NHE3 was demonstrated in the presence of NHERF2. Co-expression of IRBIT was able to reverse the NHERF2-dependent inhibition of NHE3. We also showed that IRBIT-dependent activation of NHE3 involves exocytic trafficking of NHE3 to the plasma membrane and this activation was blocked by inhibition of calmodulin (CaM) or CaM-dependent kinase II. These results suggest that the overall effect of Ca²⁺ on NHE3 activity is balanced by IRBIT-dependent activation and NHERF2-dependent inhibition.