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Background & Aims Diarrhea is one of the most common illnesses and is often caused by bacterial infection. Recently, we have shown that human Na+/H+ exchanger NHE3 (hNHE3), but not non-human NHE3s, interacts with the E3 ubiquitin ligase Nedd4-2. We hypothesize that this property of hNHE3 contributes to the increased severity of diarrhea in humans. Methods We used humanized mice expressing hNHE3 in the intestine (hNHE3int) to compare the contribution of hNHE3 and mouse NHE3 to diarrhea induced by cholera toxin (CTX) and enteropathogenic Escherichia coli (EPEC). We measured Na+/H+ exchange activity and fluid absorption. The role of Nedd4-2 on hNHE3 activity and ubiquitination was determined by knockdown in Caco-2bbe cells. The effects of protein kinase A (PKA), the primary mediator of CTX-induced diarrhea, on Nedd4-2 and hNHE3 phosphorylation and their interaction were determined. Results The effects of CTX and EPEC were greater in hNHE3int mice than in control wild-type (WT) mice, resulting in greater inhibition of NHE3 activity and increased fluid accumulation in the intestine, the hallmark of diarrhea. Activation of PKA increased ubiquitination of hNHE3 and enhanced interaction of Nedd4-2 with hNHE3 via phosphorylation of Nedd4-2 at S342. S342A mutation mitigated the Nedd4-2–hNHE3 interaction and blocked PKA-induced inhibition of hNHE3. Unlike non-human NHE3s, inhibition of hNHE3 by PKA is independent of NHE3 phosphorylation, suggesting a distinct mechanism of hNHE3 regulation. Conclusions The effects of CTX and EPEC on hNHE3 are amplified, and the unique properties of hNHE3 may contribute to diarrheal symptoms occurring in humans., Graphical abstract

Renewal of the intestinal epithelium is orchestrated by regenerative epithelial proliferation within crypts. Recent studies have shown that lysophosphatidic acid (LPA) can maintain intestinal epithelial renewal in vitro and conditional deletion of

Na(+)/H(+) exchanger NHE3 of human or primates differs from NHE3 of other animals by having a PY motif, which mediates interaction with the E3 ubiquitin (Ub) ligase Nedd4–2. Ub-conjugation of human NHE3 by Nedd4–2 regulates endocytosis of NHE3 but does not affect its cellular expression. Because Ub-conjugation is a reversible process, the aim of this study is to identify deubiquitinating enzyme (DUB) regulating the post-endosomal fate of human NHE3. Using an activity-based chemical screening, we identified ubiquitin specific protease-7 (USP7) and USP10 that bind NHE3. The roles of DUBs in regulation of NHE3 were studied by determining the effects of silencing of USP7 and USP10. Knockdown of USP7 or USP10 resulted in increased NHE3 ubiquitination and decreased NHE3 expression at the surface membrane and cellular level. The endocytic retrieval of NHE3 was promoted by depletion of USP7 or USP10, with increased association of NHE3 with Rab5a and Rab7. Inhibition of USP7 and USP10 by chemical inhibitors or knockdown had an additive effect on NHE3. In addition, NHE3 half-life was reduced accounting for decreased NHE3 protein abundance. NHE3 is inhibited by protein kinase A. Activation of PKA by forskolin decreased the binding of USP7 and USP10 to NHE3, while increasing ubiquitination of NHE3. Knockdown of USP10 had an additive effect on PKA-dependent inhibition of NHE3. These findings demonstrate that USP7 and USP10 are DUBs that regulate NHE3 ubiquitination and expression, and reveal a new mechanism of NHE3 inhibition involving DUBs.

Glycemic control is the key to the management of type 2 diabetes. Metformin is an effective, widely used drug for controlling plasma glucose levels in diabetes, but it is often the culprit of gastrointestinal adverse effects such as abdominal pain, nausea, indigestion, vomiting, and diarrhea. Diarrhea is a complex disease and altered intestinal transport of electrolytes and fluid is a common cause of diarrhea. Na+/H+ exchanger 3 (NHE3, SLC9A3) is the major Na+ absorptive mechanism in the intestine and our previous study has demonstrated that decreased NHE3 contributes to diarrhea associated with type 1 diabetes. The goal of this study is to investigate whether metformin regulates NHE3 and inhibition of NHE3 contributes to metformin-induced diarrhea. We first determined whether metformin alters intestinal water loss, the hallmark of diarrhea, in type 2 diabetic db/db mice. We found that metformin decreased intestinal water absorption mediated by NHE3. Metformin increased fecal water content although mice did not develop watery diarrhea. To determine the mechanism of metformin-mediated regulation of NHE3, we used intestinal epithelial cells. Metformin inhibited NHE3 activity and the effect of metformin on NHE3 was mimicked by a 5′-AMP-activated protein kinase (AMPK) activator and blocked by pharmacological inhibition of AMPK. Metformin increased phosphorylation and ubiquitination of NHE3, resulting in retrieval of NHE3 from the plasma membrane. Previous studies have demonstrated the role of neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2) in regulation of human NHE3. Silencing of Nedd4-2 mitigated NHE3 inhibition and ubiquitination by metformin. Our findings suggest that metformin-induced diarrhea in type 2 diabetes is in part caused by reduced Na+ and water absorption that is associated with NHE3 inhibition, probably by AMPK.

Pharmaceutical drug development rarely addresses a study participant's control of their genomic data, how to return individual incidental findings, and how to make use of genomic data more efficiently for exploratory research purposes. Mutually beneficial solutions to these issues are needed, as whole genome sequencing (WGS) is increasingly adopted in human research and as access to such information could provide impactful health-related information for a participant.In this paper, we offer a novel framework to align a trial sponsor's need for broad exploratory research of the human genome with the study participant's right to data access and access control. The Exploratory Genetic Research Project (EGRP) aims to gather WGS on all participants of a sponsor's clinical trials. It is set up as a separate umbrella protocol to facilitate the consenting process, as well as the delineation between clinical trial endpoints versus exploratory future research.This concept establishes a participant's autonomy regarding access to genomic data and the disclosure of actionable incidental findings. The feasibility of EGRP will be tested and reassessed as it is deployed over the next few years.

Glycemic control is the key to the management of type 2 diabetes. Metformin is an effective, widely used drug for controlling plasma glucose levels in diabetes, but it is often the culprit of gastrointestinal adverse effects such as abdominal pain, nausea, indigestion, vomiting, and diarrhea. Diarrhea is a complex disease and altered intestinal transport of electrolytes and fluid is a common cause of diarrhea. Na

Regeneration of the epithelium by stem cells in the intestine is supported by intrinsic and extrinsic factors. Lysophosphatidic acid (LPA), a bioactive lipid mediator, regulates many cellular functions, including cell proliferation, survival, and cytokine secretion. Here, we identify LPAWe have used genetic mouse models of conditional deletion of Lpar5, Lpar5Conditional loss of Lpar5 induced ablation of the intestinal mucosa, which increased morbidity of Lpar5LPA

Renewal of the intestinal epithelium is orchestrated by regenerative epithelial proliferation within crypts. Recent studies have shown that lysophosphatidic acid (LPA) can maintain intestinal epithelial renewal in vitro and conditional deletion of Lpar5 (Lpar5iKO) in mice ablates the intestinal epithelium and increases morbidity. In contrast, constitutive Lpar5 deletion (Lpar5cKO) does not cause a defect in intestinal crypt regeneration. In this study, we investigated whether another LPA receptor (LPAR) compensates for constitutive loss of LPA5 function to allow regeneration of intestinal epithelium. In Lpar5cKO intestinal epithelial cells (IECs), Lpar2 was upregulated and blocking LPA2 function reduced proliferation and increased apoptosis of Lpar5cKO IECs. Similar to Lpar5cKO mice, the absence of Lpar2 (Lpar2−/−) resulted in upregulation of Lpar5 in IECs, indicating that LPA2 and LPA5 reciprocally compensate for the loss of each other. Blocking LPA2 in Lpar5cKO enteroids reduced phosphorylation of Akt, indicating that LPA2 maintains the growth of Lpar5cKO enteroids through activation of the PI3K-Akt pathway. The present study provides evidence that loss of an LPAR can be compensated by another LPAR. This ability to compensate needs to be considered in studies aimed to define receptor functions or test the efficacy of a LPAR-targeting drug using genetically engineered animal models.

Autotaxin (ATX or ENPP2) is a secreted lysophospholipase D that produces lysophosphatidic acid (LPA), a pleiotropic lipid mediator acting on specific GPCRs. ATX and LPA have been implicated in key (patho)physiologic processes, including embryonic development, lymphocyte homing, inflammation, and cancer progression. Using LPA receptor knockout mice, we previously uncovered a role for LPA signaling in promoting colitis and colorectal cancer. Here, we examined the role of ATX in experimental colitis through inducible deletion of Enpp2 in adult mice. ATX expression was increased upon induction of colitis, whereas ATX deletion reduced the severity of inflammation in both acute and chronic colitis, accompanied by transient weight loss. ATX expression in lymphocytes was strongly reduced in Rag1(−/−) and μMT mice, suggesting B cells as a major ATX-producing source, which was validated by immunofluorescence and biochemical analyses. ATX secretion by B cells from control, but not Enpp2 knockout, mice led to ERK activation in colorectal cancer cells and promoted T cell migration. We conclude that ATX deletion suppresses experimental colitis and that B cells are a major source of ATX in the colon. Our study suggests that pharmacological inhibition of ATX could be a therapeutic strategy in colitis.—Lin, S., Haque, A., Raeman, R., Guo, L., He, P., Denning, T. L., El-Rayes, B., Moolenaar, W. H., Yun, C. C. Autotaxin determines colitis severity in mice and is secreted by B cells in the colon.

Excessive iron increases the incidence of diabetes and worsens diabetic complications. Reciprocally, diabetes induces iron loading, partially attributable to elevated intestinal iron export according to a recent report. Herein, we show that iron uptake and the mRNA expression of iron importer divalent metal transporter 1 (DMT1) were significantly increased in the duodenum of streptozotocin-induced diabetic mice. Immunofluorescence staining of human intestinal biopsies revealed increased brush border membrane (BBM) and decreased cytoplasmic DMT1 expression in patients with diabetes, suggesting translocation of DMT1. This pattern of DMT1 regulation was corroborated by immunoblotting results in diabetic mice showing that BBM DMT1 expression was increased by 210%, in contrast to a 60% increase in total DMT1. PKC mediates many diabetic complications, and PKCα activity was increased in diabetic mouse intestine. Intriguingly, diabetic mice with PKCα deficiency did not show increases in iron uptake and BBM DMT1 expression. High-glucose treatment increased plasma membrane DMT1 expression via the activation of PKCα in cultured IECs. Inhibition of PKCα potentiated the ubiquitination and degradation of DMT1 protein. We further showed that high glucose suppressed membrane DMT1 internalization. These findings demonstrate that PKCα promotes microvillus membrane DMT1 expression and intestinal iron uptake, contributing to diabetic iron loading.—Zhao, L., Bartnikas, T., Chu, X., Klein, J., Yun, C., Srinivasan, S., He, P. Hyperglycemia promotes microvillus membrane expression of DMT1 in intestinal epithelial cells in a PKCα-dependent manner.

Crohn’s disease (CD) is a chronic, relapsing, inflammatory disease that is often associated with malnutrition because of inflammation in the small intestine. Autotaxin (ATX) is a secreted enzyme that produces extracellular lysophosphatidic acid. Increasing evidence suggests that ATX is upregulated during inflammation, and inhibition of ATX has been effective in attenuating chronic inflammatory conditions, such as arthritis and pulmonary fibrosis. This study aims to determine whether inhibition of ATX alleviates CD-associated inflammation and malnutrition by using SAMP1/Fc mice, a model of CD-like ileitis. SAMP1/Fc mice were treated the ATX inhibitor PF-8380 for 4 wk. Inhibition of ATX led to increased weight gain in SAMP1/Fc mice, decreased T helper 2 cytokine expression, including IL-4, IL-5, and IL-13, and attenuated immune cell migration. SAMP1/Fc mice have low expression of Na+-dependent glucose transporter 1 (SGLT1), suggesting impaired nutrient absorption associated with ileitis. PF-8380 treatment significantly enhanced SGLT1 expression in SAMP1/Fc mice, which could reflect the increased weight changes. However, IL-4 or IL-13 did not alter SGLT1 expression in Caco-2 cells, ruling out their direct effects on SGLT1 expression. Immunofluorescence analysis showed that the expression of sucrase-isomaltase, a marker for intestinal epithelial cell (IEC) differentiation, was decreased in inflamed regions of SAMP1/Fc mice, which was partially restored by PF-8380. Moreover, expression of Na+/H+ exchanger 3 was also improved by PF-8380, suggesting that suppression of inflammation by PF-8380 enhanced IEC differentiation. Our study therefore suggests that ATX is a potential target for treating intestinal inflammation and restoration of the absorptive function of the intestine. NEW & NOTEWORTHY This study is the first, to our knowledge, to determine whether autotoxin (ATX) inhibition improves inflammation and body weights in SAMP1/Fc mice, a mouse model of ileitis. ATX inhibition increased body weights of SAMP1/Fc mice and increased Na+-dependent glucose transporter 1 (SGLT1) expression. Increased SGLT1 expression in the inflamed regions was not a direct effect of cytokines but an indirect effect of increased epithelial cell differentiation upon ATX inhibition.

Lysophosphatidic acid (LPA) is a bioactive lipid molecule, which regulates a broad range of pathophysiological processes. Recent studies have demonstrated that LPA modulates electrolyte flux in the intestine, and its potential as an antidiarrheal agent has been suggested. Of six LPA receptors, LPA5 is highly expressed in the intestine. Recent studies by our group have demonstrated activation of Na+/H+ exchanger 3 (NHE3) by LPA5. However, much of what has been elucidated was achieved using colonic cell lines that were transfected to express LPA5. In the current study, we engineered a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC, and investigated the role of LPA5 in NHE3 regulation and fluid absorption in vivo. The intestine of Lpar5ΔIEC mice appeared morphologically normal, and the stool frequency and fecal water content were unchanged compared with wild-type mice. Basal rates of NHE3 activity and fluid absorption and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5. NHE3 activation involves trafficking of NHE3 from the terminal web to microvilli, and this mobilization of NHE3 by LPA was abolished in Lpar5ΔIEC mice. Dysregulation of NHE3 was specific to LPA, and insulin and cholera toxin were able to stimulate and inhibit NHE3, respectively, in both wild-type and Lpar5ΔIEC mice. The current study for the first time demonstrates the necessity of LPA5 in LPA-mediated stimulation of NHE3 in vivo. NEW & NOTEWORTHY This study is the first to assess the role of LPA5 in NHE3 regulation and fluid absorption in vivo using a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC. Basal rates of NHE3 activity and fluid absorption, and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5.

Intestinal epithelial cells form a barrier that is critical in protecting the host from the hostile luminal environment. Previously, we showed that lysophosphatidic acid (LPA) receptor 1 regulates proliferation of intestinal epithelial cells, such that the absence of LPA1 mitigates the epithelial wound healing process. This study provides evidence that LPA1 is important for the maintenance of epithelial barrier integrity. The epithelial permeability, determined by fluorescently labeled dextran flux and transepithelial resistance, is increased in the intestine of mice with global deletion of Lpar1, Lpar1−/− (Lpa1−/−). Serum liposaccharide level and bacteria loads in the intestinal mucosa and peripheral organs were elevated in Lpa1−/− mice. Decreased claudin-4, caudin-7, and E-cadherin expression in Lpa1−/− mice further suggested defective apical junction integrity in these mice. Regulation of LPA1 expression in Caco-2 cells modulated epithelial permeability and the expression levels of junctional proteins. The increased epithelial permeability in Lpa1−/− mice correlated with increased susceptibility to an experimental model of colitis. This resulted in more severe inflammation and increased mortality compared with control mice. Treatment of Caco-2 cells with tumor necrosis factor-α and interferon-γ significantly increased paracellular permeability, which was blocked by cotreatment with LPA, but not LPA1 knockdown cells. Similarly, orally given LPA blocked tumor necrosis factor–mediated intestinal barrier defect in mice. LPA1 plays a significant role in maintenance of epithelial barrier in the intestine via regulation of apical junction integrity.

Na+/H+ exchanger (NHE)3, a major Na+ transporter in the luminal membrane of the proximal tubule, is subject to ANG II regulation in renal Na+/fluid absorption and blood pressure control. We have previously shown that inositol 1,4,5-trisphosphate receptor-binding protein released with inositol 1,4,5-trisphosphate (IRBIT) mediates ANG II-induced exocytosis of NHE3 in cultured proximal tubule epithelial cells. In searching for scaffold protein(s) that coordinates with IRBIT in NHE3 trafficking, we found that NHE regulatory factor (NHERF)1, NHE3, and IRBIT proteins were coexpressed in the same macrocomplexes and that loss of ANG II type 1 receptors decreased their expression in the renal brush-border membrane. We found that NHERF1 was required for ANG II-mediated forward trafficking and activation of NHE3 in cultured cells. ANG II induced a concomitant increase of NHERF1 interactions with NHE3 and IRBIT, which were abolished when the NHERF1 PDZ1 domain was removed. Overexpression of a yellow fluorescent protein-NHERF1 construct that lacks PDZ1, but not PDZ2, failed to exaggerate the ANG II-dependent increase of NHE3 expression in the apical membrane. Moreover, exogenous expression of PDZ1 exerted a dominant negative effect on NHE3 activation by ANG II. We further demonstrated that IRBIT was indispensable for the ANG II-provoked increase in NHERF1-NHE3 interactions and that phosphorylation of IRBIT at Ser68 was necessary for the assembly of the NHEF1-IRBIT-NHE3 complex. Taken together, our findings suggest that NHERF1 mediates ANG II-induced activation of renal NHE3, which requires coordination between IRBIT and the NHERF1 PDZ1 domain in binding and transporting NHE3.

The Na+/H+ exchanger regulatory factor (NHERF) family of proteins is scaffolds that orchestrate interaction of receptors and cellular proteins. Previous studies have shown that NHERF1 functions as a tumor suppressor. The goal of this study is to determine whether the loss of NHERF2 alters colorectal cancer (CRC) progress. We found that NHERF2 expression is elevated in advanced-stage CRC. Knockdown of NHERF2 decreased cancer cell proliferation in vitro and in a mouse xenograft tumor model. In addition, deletion of NHERF2 in Apc Min/+ mice resulted in decreased tumor growth in Apc Min/+ mice and increased lifespan. Blocking NHERF2 interaction with a small peptide designed to bind the second PDZ domain of NHERF2 attenuated cancer cell proliferation. Although NHERF2 is known to facilitate the effects of lysophosphatidic acid receptor 2 (LPA2), transcriptome analysis of xenograft tumors revealed that NHERF2-dependent genes largely differ from LPA2-regulated genes. Activation of β-catenin and ERK1/2 was mitigated in Apc Min/+; Nherf2− /− adenomas. Moreover, Stat3 phosphorylation and CD24 expression levels were suppressed in Apc Min/+; Nherf2− /− adenomas. Consistently, NHERF2 knockdown attenuated Stat3 activation and CD24 expression in colon cancer cells. Interestingly, CD24 was important in the maintenance of Stat3 phosphorylation, whereas NHERF2-dependent increase in CD24 expression was blocked by inhibition of Stat3, suggesting that NHERF2 regulates Stat3 phosphorylation through a positive feedback mechanism between Stat3 and CD24. In summary, this study identifies NHERF2 as a novel oncogenic protein and a potential target for cancer treatment. NHERF2 potentiates the oncogenic effects in part by regulation of Stat3 and CD24.

Lysophosphatidic acid (LPA) is a bioactive lipid molecule, which regulates a broad range of pathophysiological processes. Recent studies have demonstrated that LPA modulates electrolyte flux in the intestine, and its potential as an antidiarrheal agent has been suggested. Of six LPA receptors, LPA(5) is highly expressed in the intestine. Recent studies by our group have demonstrated activation of Na(+)/H(+) exchanger 3 (NHE3) by LPA(5). However, much of what has been elucidated was achieved using colonic cell lines that were transfected to express LPA(5). In the current study, we engineered a mouse that lacks LPA(5) in intestinal epithelial cells, Lpar5(ΔIEC), and investigated the role of LPA(5) in NHE3 regulation and fluid absorption in vivo. The intestine of Lpar5(ΔIEC) mice appeared morphologically normal, and the stool frequency and fecal water content were unchanged compared with wild-type mice. Basal rates of NHE3 activity and fluid absorption and total NHE3 expression were not changed in Lpar5(ΔIEC) mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5(ΔIEC) mice, providing direct evidence for the regulatory role of LPA(5). NHE3 activation involves trafficking of NHE3 from the terminal web to microvilli, and this mobilization of NHE3 by LPA was abolished in Lpar5(ΔIEC) mice. Dysregulation of NHE3 was specific to LPA, and insulin and cholera toxin were able to stimulate and inhibit NHE3, respectively, in both wild-type and Lpar5(ΔIEC) mice. The current study for the first time demonstrates the necessity of LPA(5) in LPA-mediated stimulation of NHE3 in vivo. NEW & NOTEWORTHY This study is the first to assess the role of LPA(5) in NHE3 regulation and fluid absorption in vivo using a mouse that lacks LPA(5) in intestinal epithelial cells, Lpar5(ΔIEC). Basal rates of NHE3 activity and fluid absorption, and total NHE3 expression were not changed in Lpar5(ΔIEC) mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5(ΔIEC) mice, providing direct evidence for the regulatory role of LPA(5).

Lysophosphatidic acid (LPA) is a simple phospholipid with potent mitogenic effects on various cells including colon cancer cells. LPA stimulates proliferation of colon cancer cells by activation of β-catenin or Kruppel-like factor 5 (KLF5), but the functional relationship between these two transcription factors is not clear. Hence, we sought to investigate the mechanism of β-catenin activation by LPA and the role of KLF5 in the regulation of β-catenin by LPA. We found that LPA and Wnt3 additively activated the β-catenin/TCF (T cell factor) reporter activity in HCT116 cells. In addition to phosphorylating glycogen synthase kinase 3β (GSK-3β) at Ser9, LPA resulted in phosphorylation of β-catenin at Ser552 and Ser675. Mutation of Ser552 and Ser675 ablated LPA-induced β-catenin/TCF transcriptional activity. Knockdown of KLF5 significantly attenuated activation of β-catenin/TCF reporter activity by LPA but not by Wnt3. However, nuclear accumulation of β-catenin by LPA was not altered by knockdown of KLF5. β-catenin, TCF, and KLF5 were present in a 250–300 kDa macro-complex, and their presence was enhanced by LPA. LPA simulated the interaction of β-catenin with TCF4, and depletion of KLF5 decreased β-catenin–TCF4 association and the transcriptional activity. In summary, LPA activates β-catenin by multiple pathways involving phosphorylation of GSK-3 and β-catenin, and enhancing β-catenin interaction with TCF4. KLF5 plays a critical role in β-catenin activation by increasing the β-catenin–TCF4 interaction.

Na(+)/H(+) exchanger NHE3 expressed in the intestine and kidney plays a major role in NaCl and HCO3 (-) absorption that is closely linked to fluid absorption and blood pressure regulation. The Nedd4 family of E3 ubiquitin ligases interacts with a number of transporters and channels via PY motifs. A comparison of NHE3 sequences revealed the presence of PY motifs in NHE3s from human and several non-human primates but not in non-primate NHE3s. In this study we evaluated the differences between human and non-primate NHE3s in ubiquitination and interaction with Nedd4-2. We found that Nedd4-2 ubiquitinated human NHE3 (hNHE3) and altered its expression and activity. Surprisingly, rat NHE3 co-immunoprecipitated Nedd4-2, but its expression and activity were not altered by silencing of Nedd4-2. Ubiquitination by Nedd4-2 rendered hNHE3 to undergo internalization at a significantly greater rate than non-primate NHE3s without altering protein stability. Insertion of a PY motif in rabbit NHE3 recapitulated the interaction with Nedd4-2 and enhanced internalization. Thus, we propose a new model where disruption of Nedd4-2 interaction elevates hNHE3 expression and activity.

CXCR4 plays a crucial role in recruitment of inflammatory cells to inflammation sites at the beginning of the disease process. Modulating CXCR4 functions presents a new avenue for anti-inflammatory strategies. However, using CXCR4 antagonists for a long term usage presents potential serious side effect due to their stem cell mobilizing property. We have been developing partial CXCR4 antagonists without such property. A new computer-aided drug design program, the FRESH workflow, was used for anti-CXCR4 lead compound discovery and optimization, which coupled both compound library building and CXCR4 docking screens in one campaign. Based on the designed parent framework, 30 prioritized amide-sulfamide structures were obtained after systemic filtering and docking screening. Twelve compounds were prepared from the top-30 list. Most synthesized compounds exhibited good to excellent binding affinity to CXCR4. Compounds Ig and Im demonstrated notable in vivo suppressive activity against xylene-induced mouse ear inflammation (with 56% and 54% inhibition). Western blot analyses revealed that Ig significantly blocked CXCR4/CXCL12-mediated phosphorylation of Akt. Moreover, Ig attenuated the amount of TNF-α secreted by pathogenic E. coli-infected macrophages. More importantly, Ig had no observable cytotoxicity. Our results demonstrated that FRESH virtual high throughput screening program of targeted chemical class could successfully find potent lead compounds, and the amide-sulfamide pharmacophore was a novel and effective framework blocking CXCR4 function.

The Caco-2 cell line represents absorptive polarized intestinal epithelial cells that express multiple forms of Na+/H+exchanger (NHE) in their plasma membranes. Caco-2 cells express the major apical NHE isoform NHE3, but low NHE3 expression together with inefficient transfection often hamper intended studies. In this study, we examined whether SK-CO15 cells could be used to study NHE3 regulation. SK-CO15 cells grown on Transwell inserts developed polarized epithelial cells with microvilli. The transfection efficiency of SK-CO15 cells was markedly higher compared with Caco-2 cells, an advantage in gene transfer and knockout. SK-CO15 cells expressed NHE1, NHE2, and NHE3. NHE3 expression was significantly greater in these cells than Caco-2, and NHE3 comprised more than half of total NHE activity. Apical expression of NHE3 in SK-CO15 cells was confirmed by confocal immunofluorescence and surface biotinylation. NHE regulatory factors NHERF1 and NHERF2, which are important for regulation of NHE3 activity, were expressed in these cells. Stimulatory response of NHE3 in SK-CO15 cells was assessed by dexamethasone and lysophosphatidic acid (LPA). Treatment with dexamethasone for 24–48 h increased NHE3 expression and activity. Similarly to Caco-2 cells, SK-CO15 cells lacked the expression of the LPA receptor LPA5,but exogenous expression of LPA5resulted in acute stimulation of NHE3. Forskolin acutely inhibited NHE3 activity in SK-CO15 cells, further attesting the validity of these cells. We conclude that SK-CO15 cells with the amenity for transfection and high endogenous NHE3 expression are a new and better cell model for NHE3 regulatory investigation than widely used Caco-2 cells.

Background/Aims: The sodium/bicarbonate transporter NBCn1 plays an essential role in intracellular pH regulation and transepithelial HCO3– movement in the body. NBCn1 also has sodium channel-like activity uncoupled to Na/HCO3 cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD-95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD-95 interaction. Methods: Results: In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C-terminal 131 amino acids of NBCn1 pulled down PSD-95 from rat brain lysates, whereas GST/NBCn1-ΔETSL (deletion of the last four amino acids) and GST/NBCn2 (NCBE) lacking the same ETSL did not. NBCn1 and PSD-95 were coimmunoprecipitated in HEK 293 cells, and their interaction did not affect the efficacy of PSD-95 to bind to the NMDA receptor NR2A. PSD-95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD-95 increased an ionic conductance produced by NBCn1 channel-like activity. This increase was abolished by NBCn1-ΔETSL or by the peptide containing the last 15 amino acids of NBCn1. Conclusion: Our data suggest that PSD-95 interacts with NBCn1 and increases its channel-like activity while negligibly affecting Na/HCO3 cotransport. The possibility that the channel-like activity occurs via an intermolecular cavity of multimeric NBCn1 proteins is discussed.

SGK1 plays an important role in regulation of Na+/H+ exchanger (NHE) 3 in vivo. We show that SGK3 colocalizes with NHE3 in recycling endosomes. These studies identify SGK3 as the effector of the PI3K pathway that activates NHE3 and show that endosomal localization of SGK3 is essential for acute activation of NHE3., Na+/H+ exchanger 3 (NHE3) is the major Na+ transporter in the intestine. Serum- and glucocorticoid-induced kinase (SGK) 1 interacts with NHE regulatory factor 2 (NHERF2) and mediates activation of NHE3 by dexamethasone (Dex) in cultured epithelial cells. In this study, we compared short-term regulation of NHE3 by Dex in SGK1-null and NHERF2-null mice. In comparison to wild-type mice, loss of SGK1 or NHERF2 significantly attenuated regulation of NHE3 by Dex but did not completely obliterate the effect. We show that transfection of SGK2 or SGK3 in PS120 cells resulted in robust activation of NHE3 by Dex. However, unlike SGK1 or SGK2, SGK3 rapidly activated NHE3 within 15 min of Dex treatment in both PS120 and Caco-2bbe cells. Immunofluorescence analysis showed that SGK3 colocalized with NHE3 in recycling endosomes, whereas SGK1 and SGK2 were diffusely distributed. Mutation of Arg-90 of SGK3 disrupted the endosomal localization of SGK3 and delayed NHE3 activation. Activation of SGK3 and NHE3 by Dex was dependent on phosphoinositide 3-kinase (PI3K) and phosphoinositide-dependent kinase 1 (PDK1), and Dex induced translocation of PDK1 to endosomes. Our study identifies SGK3 as a novel endosomal kinase that acutely regulates NHE3 in a PI3K-dependent mechanism.

Trafficking and regulation of the epithelial brush border membrane (BBM) Na+/H+ exchanger 3 (NHE3) in the intestine involves interaction with four different members of the NHERF family in a signal-dependent and possibly segment-specific fashion. The aim of this research was to study the role of NHERF2 (E3KARP) in intestinal NHE3 BBM localization and second messenger-mediated and receptor-mediated inhibition of NHE3. Immunolocalization of NHE3 in WT mice revealed predominant microvillar localization in jejunum and colon, a mixed distribution in the proximal ileum but localization near the terminal web in the distal ileum. The terminal web localization of NHE3 in the distal ileum correlated with reduced acid-activated NHE3 activity (fluorometrically assessed). NHERF2 ablation resulted in a shift of NHE3 to the microvilli and higher basal fluid absorption rates in the ileum, but no change in overall NHE3 protein or mRNA expression. Forskolin-induced NHE3 inhibition was preserved in the absence of NHERF2, whereas Ca2+ ionophore- or carbachol-mediated inhibition was abolished. Likewise, Escherichia coli heat stable enterotoxin peptide (STp) lost its inhibitory effect on intestinal NHE3. It is concluded that in native murine intestine, the NHE3 adaptor protein NHERF2 plays important roles in tethering NHE3 to a position near the terminal web and in second messenger inhibition of NHE3 in a signal- and segment-specific fashion, and is therefore an important regulator of intestinal fluid transport.

Colorectal cancer (CRC) develops through a series of genetic modifications that transforms normal colonic epithelium to an adenoma and then ultimately adenocarcinoma. A body of evidence suggests that lysophosphatidic acid (LPA) is a potent inducer of cancer progression at multiple levels. However, the pathological significance and mechanisms of LPA-mediated effects in the intestinal tract have only recently drawn interests. CRC cells have aberrant LPA receptor expression and LPA enhances proliferation, survival, and invasion of CRC cells, implying LPA and its signaling pathways as potential targets for anti-cancer therapies. The mechanisms of fostering CRC by LPA include dysregulation of transcription factors, activation of mitogen-activated protein kinases, pro-inflammatory cytokines, and angiogenic factors. This review provides a brief summary of recent advance on the effects of LPA on CRC cells.

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.

Growing evidence suggests that pharmacological inhibition of Na/H exchange and Na/HCO(3) transport provides protection against damage or injury in cardiac ischemia. In this study, we examined the contribution of the sodium/bicarbonate cotransporter NBCn1 (slc4a7) to cytotoxicity in cultured hippocampal neurons of rats. In neurons exposed to extracellular pH (pH(o)) ranging from 6.2 to 8.3, NBCn1 protein expression increased by fivefold at pH < 6.5 compared to the expression at pH(o) 7.4. At pH(o) 6.5, the intracellular pH of neurons was approximately 1 unit lower than that at pH 7.4. Immunochemistry showed a marked increase in NBCn1 immunofluorescence in plasma membranes and cytosol of the soma as well as in dendrites, at pH(o) 6.5. NBCn1 expression also increased by 40% in a prolonged Mg(2+)-free incubation at normal pH(o). Knockdown of NBCn1 in neurons had negligible effect on cell viability. The effect of NBCn1 knockdown on cytotoxicity was then determined by exposing neurons to 0.5 mm glutamate for 10 min and measuring lactate dehydrogenase (LDH) release from neurons. Compared to normal incubation (pH(o) 7.2 for 6 h) after glutamate exposure, acidic incubation (pH(o) 6.3 for 6 h) reduced cytotoxicity by 75% for control neurons and 78% for NBCn1-knockdown neurons. Thus, both controls and knockdown neurons showed acidic protection from cytotoxicity. However, in Mg(2+)-free incubation after glutamate exposure, NBCn1 knockdown progressively attenuated cytotoxicity. This attenuation was unaffected by acidic preincubation before glutamate exposure. We conclude that NBCn1 has a dynamic upregulation in low pH(o) and Mg(2+) depletion. NBCn1 is not required for acidic protection, but increases cytotoxicity in Mg(2+)-free conditions.

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.

Lysophosphatidic acids (LPA) exert growth factor-like effects through specific G protein-coupled receptors. The presence of different LPA receptors often determines the specific signaling mechanisms and the physiological consequences of LPA in different environments. Among the four members of the LPA receptor family, LPA(2) has been shown to be overexpressed in colon cancer suggesting that the signaling by LPA(2) may potentiate growth and survival of tumor cells. In this study, we examined the effect of LPA on survival of colon cancer cells using Caco-2 cells as a cell model system. LPA rescued Caco-2 cells from apoptosis elicited by the chemotherapeutic drug, etoposide. This protection was accompanied by abrogation of etoposide-induced stimulation of caspase activity via a mechanism dependent on Erk and PI3K. In contrast, perturbation of cellular signaling mediated by the LPA(2) receptor by knockdown of a scaffold protein NHERF2 abrogated the protective effect of LPA. Etoposide decreased the expression of Bcl-2, which was reversed by LPA. Etoposide decreased the phosphorylation level of the proapoptotic protein Bad in an Erk-dependent manner, without changing Bad expression. We further show that LPA treatment resulted in delayed activation of Erk. These results indicate that LPA protects Caco-2 cells from apoptotic insult by a mechanism involving Erk, Bad, and Bcl-2.

The PDZ-binding protein PDZK1 (CAP70/PDZ-dc-1/NHERF3) in vitro binds to cystic fibrosis transmembrane conductance regulator (CFTR), the anion exchangers SLC26A3 and SLC26A6 and the Na(+)/H(+) exchanger NHE3, all of which are major transport proteins for intestinal anion secretion and salt absorption. This study was undertaken to search for a role of PDZK1 in regulating electrolyte transport in native murine small intestine. Short circuit current (I (SC)) and HCO-(3) secretory rate (J(HCO-)(3)) were measured to assess electrogenic anion secretion; (22)Na(+) fluxes to assess sodium absorption in isolated small intestine. NHE3, CFTR, as well as NHERF1, NHERF2, and PDZK1 messenger RNA (mRNA) expression levels, and NHE3 total enterocyte and brush border membrane (BBM) protein abundance were determined by quantitative polymerase chain reaction (PCR) and Western analysis. NHE3 localization was performed by immunohistochemistry. In pdzk1 -/- jejunal mucosa, basal net Na(+) absorption as well as the inhibition of Na(+) absorption by forskolin was significantly reduced. In pdzk1 -/- duodenal mucosa, identical basal I (SC) and (J(HCO-)(3)) but a significant, yet mild, reduction of forskolin-stimulated Delta(J(HCO-)(3)) and DeltaI (SC) was observed compared to +/+ tissue. Tissue conductance, morphological features, and the DeltaI (SC) and increase in (22)Na(+) absorption in response to luminal glucose was identical in pdzk1 +/+ and -/- small intestine, ruling out a general absorptive defect. While CFTR mRNA expression levels were unchanged, NHE3 mRNA expression levels were significantly increased in small intestinal mucosa of pdzk1 -/- mice. Total enterocyte and BBM abundance was not significantly different, suggesting an increased NHE3 turnover, possibly due to reduced NHE3 membrane retention time. Lack of the PDZ-adapter protein PDZK1 in murine small intestine causes a mild reduction in maximal CFTR activation, but a severe defect in electroneutral Na(+) absorption.

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.

Na+ reabsorption by the kidney is tightly regulated to maintain Na+, fluid and blood pressure homeostasis. Angiotensin II (Ang II) plays a critical role in Na+ reabsorption in the kidney during health and diseases conditions. Na+,K+-ATPase on the basolateral membrane is one of the important targets subjected to Ang II regulation. However, the mechanism by which Ang II activates Na+,K+-ATPase remains poorly understood. We have previously shown that IRBIT (inositol 1,4,5-triphosphate receptor binding protein released with inositol 1,4,5-triphosphate) binds and activates Na+/H+ exchanger (NHE3) in response to Ang II. By mass spectrometry analysis, we identified IRBIT as a novel binding protein of Na+,K+-ATPase, prompting us hypothesize that IRBIT might coordinate transcellular Na+ absorption by regulating apical NHE3 and basolateral Na+,K+-ATPase. Indeed, overexpression of IRBIT resulted in a greater activation of Na+,K+-ATPase activity by Ang II in OKP cells, whereas lentiviral shRNA-mediated knockdown of I...

Na+/H+ exchange by Na+/H+ exchanger 3 (NHE3) is a major route of sodium absorption in the intestine and kidney. We have shown previously that lysophosphatidic acid (LPA), a small phospholipid produced ubiquitously by all types of cells, stimulates NHE3 via LPA5 receptor. Stimulation of NHE3 activity by LPA involves LPA5 transactivating EGF receptor (EGFR) in the apical membrane. EGFR activates proline-rich tyrosine kinase 2 (Pyk2) and ERK, both of which are necessary for NHE3 regulation. However, Pyk2 and ERK are regulated by EGFR via independent pathways and appear to converge on an unidentified intermediate that ultimately targets NHE3. The p90 ribosomal S6 kinase (RSK) family of Ser/Thr protein kinases is a known effector of EGFR and ERK. Hence, we hypothesized that RSK may be the convergent effector of Pyk2 and ERK although it is not known whether Pyk2 regulates RSK. In this study, we show that Pyk2 is necessary for the maintenance of phosphoinositide-dependent kinase 1 (PDK1) autophosphorylation, and knockdown of Pyk2 or PDK1 mitigated LPA-induced phosphorylation of RSK and stimulation of NHE3 activity. Additionally, we show that RSK2, but not RSK1, is responsible for NHE3 regulation. RSK2 interacts with NHE3 at the apical membrane domain, where it phosphorylates NHE3. Alteration of S663 of NHE3 ablated LPA-induced phosphorylation of NHE3 and stimulation of the transport activity. Our study identifies RSK2 as a new kinase that regulates NHE3 activity by direct phosphorylation.

Based on physiological studies, the epithelial brush-border (BB) Na+/H+antiporter3 (NHE3) seems to associate with the actin cytoskeleton both by binding to and independently of the PDZ domain containing proteins NHERF1 and NHERF2. We now show that NHE3 directly binds ezrin at a site in its C terminus between aa 475-589, which is separate from the PSD95/dlg/zonular occludens-1 (PDZ) interacting domain. This is an area predicted to be α-helical, with a positive aa cluster on one side (K516, R520, and R527). Point mutations of these positively charged aa reduced (NHE3 double mutant [R520F, R527F]) or abolished (NHE3 triple mutant [K516Q, R520F, R 527F]) ezrin binding. Functional consequences of these NHE3 point mutants included the following. 1) A marked decrease in surface amount with a greater decrease in NHE3 activity. 2) Decreased surface expression due to decreased rates of exocytosis and plasma membrane delivery of newly synthesized NHE3, with normal total expression levels and slightly reduced endocytosis rates. 3) A longer plasma membrane half-life of mutant NHE3 with normal total half-life. 4) Decreased BB mobile fraction of NHE3 double mutant. These results show that NHE3 binds ezrin directly as well as indirectly and suggest that the former is related to 1) the exocytic trafficking of and plasma membrane delivery of newly synthesized NHE3, which determines the amount of plasma membrane NHE3 and partially determines NHE3 activity, and 2) BB mobility of NHE3, which may increase its delivery from microvilli to the intervillus clefts, perhaps for NHE3-regulated endocytosis.

The constitutive reuptake of albumin from the glomerular filtrate by receptor-mediated endocytosis is a key function of the renal proximal tubules. Both the Cl– channel ClC-5 and the Na+-H+ exchanger isoform 3 are critical components of the macromolecular endocytic complex that is required for albumin uptake, and therefore the cell-surface levels of these proteins may limit albumin endocytosis. This study was undertaken to investigate the potential roles of the epithelial PDZ scaffolds, Na+-H+ exchange regulatory factors, NHERF1 and NHERF2, in albumin uptake by opossum kidney (OK) cells. We found that ClC-5 co-immunoprecipitates with NHERF2 but not NHERF1 from OK cell lysate. Experiments using fusion proteins demonstrated that this was a direct interaction between an internal binding site in the C terminus of ClC-5 and the PDZ2 module of NHERF2. In OK cells, NHERF2 is restricted to the intravillar region while NHERF1 is located in the microvilli. Silencing NHERF2 reduced both cell-surface levels of ClC-5 and albumin uptake. Conversely, silencing NHERF1 increased cell-surface levels of ClC-5 and albumin uptake, presumably by increasing the mobility of NHE3 in the membrane and its availability to the albumin uptake complex. Surface biotinylation experiments revealed that both NHERF1 and NHERF2 were associated with the plasma membrane and that NHERF2 was recruited to the membrane in the presence of albumin. The importance of the interaction between NHERF2 and the cytoskeleton was demonstrated by a significant reduction in albumin uptake in cells overexpressing an ezrin binding-deficient mutant of NHERF2. Thus NHERF1 and NHERF2 differentially regulate albumin uptake by mechanisms that ultimately alter the cell-surface levels of ClC-5.

NHE3 is an intestinal epithelial isoform Na+/H+ exchanger that is present in the brush border of small intestinal, colonic, and gallbladder Na+-absorbing epithelial cells. NHE3 is acutely up- and downregulated in response to some G protein-linked receptors, tyrosine kinase receptors, and protein kinases when studied in intact ileum, when stably expressed in PS120 fibroblasts, and in the few studies reported in the human colon cancer cell line Caco-2. In most cases this is due to changes in V-max of NHE3, although in response to cAMP and squalamine there are also changes in the K'(H+)(i) of the exchanger. The mechanism of the Vmax regulation as shown by cell surface biotinylation and confocal microscopy in Caco-2 cells and biotinylation in PS120 cells involves changes in the amount of NHE3 on the plasma membrane. In addition, in some cases there are also changes in turnover number of the exchanger. In some cases, the change in amount of NHE3 in the plasma membrane is associated with a change in the amount of plasma membrane. A combination of biochemical studies and transport/inhibitor studies in intact ileum and Caco-2 cells demonstrated that the increase in brush border Na+/H+ exchange caused by acute exposure to EGF was mediated by PI 3-kinase. PI 3-kinase was also involved in FGF stimulation of NHE3 expressed in fibroblasts. Thus, NHE3 is another example of a transport protein that is acutely regulated in part by changing the amount of the transporter on the plasma membrane by a process that appears to involve vesicle trafficking and also to involve changes in turnover number.

Glucocorticoids stimulate Na+absorption by activation of the epithelial Na+/H+exchanger NHE3 in the kidney and intestine. It has been thought that glucocorticoid-induced activation of NHE3 is solely dependent on transcriptional induction of the NHE3 gene. While the transcriptional regulation remains an essential part of the chronic effect of glucocorticoids, a previous study by us identified the serum- and glucocorticoid-inducible kinase 1 (SGK1) as an important component of the activation of NHE3 by glucocorticoids. In this work, we have demonstrated phosphorylation of NHE3 by SGK1 as the key mechanism for the stimulation of the transport activity by glucocorticoids. By using in vitro SGK1 kinase assay and site-directed mutagenesis, we have identified Ser663 of NHE3 to be the major site of phosphorylation by SGK1. Ser663 is invariantly conserved in all NHE3 proteins from several species, and the mutation of Ser663 to Ala blocks the effect of dexamethasone, demonstrating the importance of phosphorylation at Ser663. We also show that phosphorylation of NHE3 precedes the changes in NHE3 activity, and the increased activity is associated with an increased amount of NHE3 proteins in the surface membrane. These data reveal that dexamethasone activates NHE3 activity by phosphorylating the NHE3 protein, which initiates trafficking of the protein into the plasma membrane.

Sensory transduction in the mammalian cochlea requires the maintenance of specialized fluid compartments with distinct ionic compositions. This is achieved by the concerted action of diverse ion channels and transporters, some of which can interact with the PDZ scaffolds, Na(+)-H(+) exchanger regulatory factors 1 and 2 (NHERF-1, NHERF-2). Here, we report that NHERF-1 and NHERF-2 are widely expressed in the rat cochlea, and that their expression is developmentally regulated. Reverse transcription/polymerase chain reaction (RT-PCR) and Western blotting initially confirmed the RNA and protein expression of NHERFs. We then performed immunohistochemistry on cochlea during various stages of postnatal development. Prior to the onset of hearing (P8), NHERF-1 immunolabeling was prominently polarized to the apical membrane of cells lining the endolymphatic compartment, including the stereocilia and cuticular plates of the inner and outer hair cells, marginal cells of the stria vascularis, Reissner's epithelia, and tectorial membrane. With maturation (P21, P70), NHERF-1 immunolabeling was reduced in the above structures, whereas labeling increased in the apical membrane of the interdental cells of the spiral limbus and the inner and outer sulcus cells, Hensen's cells, the inner and outer pillar cells, Deiters cells, the inner border cells, spiral ligament fibrocytes, and spiral ganglion neurons (particularly type II). NHERF-1 expression in strial basal and intermediate cells was persistent. NHERF-2 immunolabeling was similar to that for NHERF-1 during postnatal development, with the exception of expression in the synaptic regions beneath the outer hair cells. NHERF-1 and NHERF-2 co-localized with glial fibrillary acidic protein and vimentin in glia. The cochlear localization of NHERF scaffolds suggests that they play important roles in the developmental regulation of ion transport, homeostasis, and auditory neurotransmission.

The epithelial Ca2+ channel TRPV5 (ECaC1) plays a key role in renal and intestinal Ca2+ (re)absorption and is thus regulated by 1,25(OH) 2D3. The present study aims to explore whether TRPV5 is regulated by the serum and glucocorticoid inducible kinase SGK1, a kinase transcriptionally upregulated by 1,25(OH) 2D3. To this end cRNA encoding TRPV5 has been injected into Xenopus oocytes with or without additional injection of SGK1, its isoforms SGK2 and SGK3, constitutively active (S422D)SGK1, inactive (K127N)SGK1, constitutively active (T308D,S473D)PKB and/or the Na+/H+ exchanger regulating factor NHERF2. In Xenopus laevisoocytes expression of TRPV5 increases uptake of tracer Ca(S422D;) and induces a Ca2+ current (ICa). In the presence of Cl-, TRPV5 mediated Ca2+ entry leads to secondary activation of Ca(2+)-sensitive Cl- channels (ICl(Ca)). Coexpression of TRPV5 with both (S422D)SGK1 and NHERF2 stimulates tracer Ca2+ entry, ICa and ICl(Ca). The effect of (S422D)SGK1 on TRPV5 and NHERF2 expressing oocytes is mimicked by SGK1 and SGK3, but not by SGK2, constitutively active (T308D,S473D)PKB or inactive (K127N)SGK1. The observations suggest that SGK1, SGK3 and NHERF2 regulate TRPV5 and are thus likely to participate in the regulation of calcium homeostasis.

The serum- and glucocorticoid- inducible kinase SGK1 stimulates the renal outer medullary K+ channel ROMK1 in the presence of the Na+/H+ exchanger regulating factor NHERF2. SGK1/NHERF2 are effective through enhancement of ROMK1 abundance within the cell membrane. The present study aims to define the molecular requirements for the interaction of ROMK1 with SGK1/NHERF2. Pull down assays reveal that SGK1 interacts with NHERF2 through the second PDZ domain of NHERF2. According to chemiluminescence and electrophysiology, deletion of the second PDZ domain of NHERF2 or the putative PDZ binding motif on ROMK1 abrogates the stimulating effect of SGK1 on ROMK1 protein abundance in the plasma membrane and K+ current.

Histidine residues in Na+/H+ exchangers are believed to participate in proton binding and influence the Na+/H+ exchanger activity. In the present study, the function of three highly conserved histidines in the juxtamembrane cytoplasmic domain of NHE3 was studied. His-479, His-485, and His-499 were mutated to Leu, Gln or Asp and expressed in an Na+/H+ exchanger null cell line and functional consequences on Na+/H+ exchange kinetics were characterized. None of the histidines were essential for NHE3 activity, with all mutated NHE3 resulting in functional exchangers. However, the mutation in His-475 and His-499 significantly lowered NHE3 transport activity, whereas the mutation in H485 showed no apparent effect. In addition, the pH profiles of the H479 and H499 mutants were shifted to a more acidic region, and lowered its set point, the intracellular pH value above which the Na+/H+ exchanger becomes inactive, by approximately 0.3-0.6 pH units. The changes in set point by the mutations were further shifted to more acidic values by ATP depletion, indicating that the mechanism by which the mutations on the histidine residues altered the NHE3 set point differs from that caused by ATP depletion. We suggest that His-479 and His-499 are part of the H+ sensor, which is involved in determining the sensitivity to the intracellular H+ concentration and Na+/H+ exchange rate.

Na+/H+ exchanger regulatory factors, NHERF1 and NHERF2, are structurally related proteins and highly expressed in epithelial cells. These proteins are initially identified as accessory proteins in the regulation of Na+/H+ exchanger isoform 3, NHE3. In addition to regulation of NHE3, recent studies demonstrate the importance of NHERF1 and NHERF2 in recycling and localization of membrane receptors, ion channels and transporters. Recent studies show that serum- and glucocorticoid-induced kinase 1 (SGK1) specifically interacts with NHERF2 but not with NHERF1, adding to the growing number of differences between the two proteins. The association of SGK1 with NHERF2 is necessary for stimulation of NHE3 activity by glucocorticoids. In addition, SGK1 together with NHERF2 stimulates the K+ channel ROMK1, suggesting a broader role of SGK1 in regulation of ion transport.

Mineralocorticoids stimulate Na(+) reabsorption and K(+) secretion in principal cells of connecting tubule and collecting duct. The involved ion channels are ENaC and ROMK1, respectively. In Xenopus oocytes, the serum and glucocorticoid-sensitive kinase SGK1 has been shown to increase ENaC activity by enhancing its abundance in the plasma membrane. With the same method, ROMK1 appeared to be insensitive to regulation by SGK1. On the other hand, ROMK1 has been shown to colocalize with NHERF2, a protein mediating targeting and trafficking of transport proteins into the cell membrane. The present study has been performed to test whether NHERF2 is required for regulation of ROMK1 by SGK1. Coexpression of neither NHERF2 nor SGK1 with ROMK1 increases ROMK1 activity. However, coexpression of NHERF2 and SGK1 together with ROMK1 markedly increases K(+) channel activity. The combined effect of SGK1 and NHERF2 does not significantly alter the I/V relation of the channel but increases the abundance of the channel in the membrane and decreases the decay of channel activity after inhibition of vesicle insertion with brefeldin. Coexpression of NHERF2 and SGK1 does not modify cytosolic pH but leads to a slight shift of pK(a) of ROMK1 to more acidic values. In conclusion, NHERF2 and SGK1 interact to enhance ROMK1 activity in large part by enhancing the abundance of channel protein within the cell membrane. This interaction allows the integration of genomic regulation and activation of SGK1 and NHERF2 in the control of ROMK1 activity and renal K(+) excretion.

Intestinal electroneutral NaCl absorption is mediated by parallel operation of Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange in the enterocyte apical membrane. The ion transporters involved are Na(+)/H(+) exchanger 3 (NHE3) and the down regulated in adenoma (dra) gene product. cAMP-mediated inhibition of NHE3 requires the transporter to bind to the second PDZ (PSD95, disk large, ZO1) domain of the adapter protein NHE3 kinase A regulatory protein (E3KARP). Because the C-terminal four amino acids of dra are ETKF (glutamate-threonine-lysine-phenylalanine), resembling a PDZ interaction motif, we hypothesized that dra may also bind to one of the PDZ domains of E3KARP. In vitro the ETKF motif of dra binds to the second PDZ domain of E3KARP, the affinity being comparable to that of the known ligand CFTR. The C-terminal phenylalanine, which is an unconventional residue in PDZ interaction motifs, can only be substituted by the classical residue leucine, but not by other hydrophobic residues (valine, isoleucine). Immunofluorescence colocalizes dra, NHE3, and E3KARP in the apical compartment of human proximal colon. We suggest a model in which both NHE3 and dra bind to the second PDZ domain of E3KARP and that linking of the transporters occurs through dimerization of E3KARP. In such a model, the first PDZ domain would remain available for instance for signal transduction proteins.

Two PDZ domain-containing proteins, NHERF and E3KARP are necessary for cAMP-dependent inhibition of Na(+)/H(+) exchanger 3 (NHE3). In this study, we demonstrate a specific role of E3KARP, which is not duplicated by NHERF, in Ca(2+)-dependent inhibition of NHE3 activity. NHE3 activity is inhibited by elevation of intracellular Ca(2+) ([Ca(2+)](i)) in PS120 fibroblasts stably expressing E3KARP but not those expressing NHERF. In addition, this Ca(2+)-dependent inhibition requires Ca(2+)-dependent association between alpha-actinin-4 and E3KARP. NHE3 is indirectly connected to alpha-actinin-4 in a protein complex through Ca(2+)-dependent interaction between alpha-actinin-4 and E3KARP, which occurs through the actin-binding domain plus spectrin repeat domain of alpha-actinin-4. Elevation of [Ca(2+)](i) results in oligomerization and endocytosis of NHE3 as well as in inhibition of NHE3 activity. Overexpression of alpha-actinin-4 potentiates the inhibitory effect of ionomycin on NHE3 activity by accelerating the oligomerization and endocytosis of NHE3. In contrast, overexpression of the actin-binding domain plus spectrin repeat domain acts as a dominant-negative mutant and prevents the inhibitory effect of ionomycin on NHE3 activity as well as the oligomerization and internalization of NHE3. From these results, we propose that elevated Ca(2+) inhibits NHE3 activity through oligomerization and endocytosis of NHE3, which occurs via formation of an NHE3-E3KARP-alpha-actinin-4 complex.

The parathyroid hormone 1 receptor (PTH1R) is a class II G-protein-coupled receptor. PTH1R agonists include both PTH, a hormone that regulates blood calcium and phosphate, and PTH-related protein (PTHrP), a paracrine/autocrine factor that is essential for development, particularly of the skeleton. Adenylyl cyclase activation is thought to be responsible for most cellular responses to PTH and PTHrP, although many actions appear to be independent of adenylyl cyclase. Here we show that the PTH1R binds to Na(+)/H(+) exchanger regulatory factors (NHERF) 1 and 2 through a PDZ-domain interaction in vitro and in PTH target cells. NHERF2 simultaneously binds phospholipase C beta 1 and an atypical, carboxyl-terminal PDZ consensus motif, ETVM, of the PTH1R through PDZ1 and PDZ2, respectively. PTH treatment of cells that express the NHERF2 PTH1R complex markedly activates phospholipase C beta and inhibits adenylyl cyclase through stimulation of inhibitory G proteins (G(i/o) proteins). NHERF-mediated assembly of PTH1R and phospholipase C beta is a unique mechanism to regulate PTH signalling in cells and membranes of polarized cells that express NHERF, which may account for many tissue- and cell-specific actions of PTH/PTHrP and may also be relevant to signalling by many G-protein-coupled receptors.