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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

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.

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.

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.

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 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.

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.

A dysfunction of the Na+/H+ exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine. Electronic supplementary material The online version of this article (doi:10.1007/s00424-014-1608-x) contains supplementary material, which is available to authorized users.

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.

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.

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 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.

Na+/H+ exchanger (NHE3) plays a pivotal role in the reabsorption of Na+ and HCO3- in the kidney. The NHE3 activity is tightly regulated by hormones and growth factors to maintain fluid and blood pressure homeostasis. We have previously shown that NHE3 activity and trafficking is stimulated by low dose angiotensin II (Ang II) dependent on IRBIT and CaMKII. However, it remains unclear how IRBIT mediates the exocytosis of NHE3 protein. In this study, we found that IRBIT binds to NHERF1 in OKP cells, which was enhanced by Ang II. Overexpression of IRBIT or Ang II treatment increased NHERF1 and NHE3 binding, whereas knockdown of IRBIT attenuated the interaction. Knockdown of NHERF1 expression by lentiviral shRNA completely blocked the stimulatory effect of Ang II on NHE3 exocytosis and transport activity. Moreover, inhibition of CaMKII by KN-93 abolished the Ang II-induced NHERF1-NHE3 interaction and membrane expression of NHE3, IRBIT and NHERF1. In conclusion, activation of NHE3 exocytosis by Ang II is depend...

Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is a versatile membrane-cytoskeleton linking protein that binds to the COOH-tail of specific integral membrane proteins through its two PDZ domains. These EBP50 binding interactions have been implicated in sequestering interactive sets of proteins into common microdomains, regulating the activity of interacting proteins, and modulating membrane protein trafficking. With only two PDZ domains, it is unclear how EBP50 forms multiprotein complexes. Other PDZ proteins increase their breadth and diversity of protein interactions through oligomerization. Hypothesizing that EBP50 self-associates to amplify its functional capacity, far-Western blotting of cholangiocyte epithelial cell proteins with EBP50 fusion protein revealed that EBP50 binds to a 50-kDa protein. Far-Western blotting of EBP50 isolated by two-dimensional gel electrophoresis or immunoprecipitation demonstrates that the 50-kDa binding partner is itself EBP50. Further, co-transfection/co-precipitation studies show the self-association can occur in an intracellular environment. In vitro analysis of the EBP50-EBP50 binding interaction indicates it is both saturable and of relatively high affinity. Analysis of truncated EBP50 proteins indicates EBP50 self-association is mediated through its PDZ domains. The ability to self-associate provides a mechanism for EBP50 to expand its capacity to form multiprotein complexes and regulate membrane transport events.

Among the phospholipase C that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate, four mammalian phospholipase C-beta (PLC-beta) isotypes (isotypes 1-4) are activated through G protein-coupled receptors (GPCRs). Although the regulation of the PLC-betas by GPCRs and heterotrimeric G proteins has been extensively studied, little is known about the molecular determinants that regulate their activity. The PLC-beta isozymes carry a putative PSD-95/Dlg/ZO-1 (PDZ) binding motif (X(S/T)X(V/L)COOH) at their carboxyl terminus, which is implicated in specific interactions with anchor proteins. Using the yeast two-hybrid system, we identified Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) as a protein that interacted with a C-terminal heptapeptide of PLC-beta3. Immunoprecipitation studies revealed that NHERF2 interacts specifically with PLC-beta3, but not with other PLC-beta isotypes. Furthermore, PLC-beta3 interacted with NHERF2 rather than with other PDZ-containing proteins. This interaction required the COOH-terminal NTQL sequence of PLC-beta3 and the second PDZ domain of NHERF2. Interestingly, NHERF2 potentiated the PLC-beta activation by carbachol in COS7 and HeLa cells, while mutant NHERF2, lacking the second PDZ domain, had no such effect. Taken together, the data suggest that NHERF2 may act as a modulator underlying the process of PLC-beta3-mediated signaling.

The Na/H exchanger regulatory factor (NHE-RE), a recently cloned renal protein, is a necessary cofactor in protein kinase A-mediated inhibition of the renal brush border membrane Na/H exchanger. No studies to date, however, have examined the regulation of NHE-RF itself. The rabbit NHE-RF cDNA and an antibody to rabbit NHE-RF were used to study the effects of serum and cyclic adenosine monophosphate (cAMP) on the steady-state levels of NHE-RF mRNA and on the abundance and intracellular distribution of the protein in OK cells. Incubation of quiescent cells with serum was associated with a significant decrease in steady-state NHE-RF mRNA and protein abundance in the cytosolic and membrane fractions. Incubation of cells with cAMP for 6 h was associated with no change in NHE-mRNA at 24 h. There was, however, a 46% increase in protein abundance in the cytosolic fraction of the cell and a 43% decrease in the membrane fraction. Despite the decrease in membrane-associated NHE-RF in quiescent cells treated with serum of cAMP, there were no differences in either the basal rate of Na/H exchange transport or the inhibitory effect of the acute addition of cAMP on the transporter between experimental and control cells. These studies provide the first description of the regulation of NHE-RF. The results indicate that serum is associated with a decrease in NHE-RF mRNA and protein, while chronic exposure to cAMP is associated with an altered distribution of NHE-RF between the cytosolic and membrane fractions of OK cells.

In this study, we examined the tissue-specific expression of two electroneutral Na/HCO(3) cotransporter (NBCn1) variants that differ from each other by the presence of the N-terminal 123 amino acids (cassette II). A rat Northern blot with the probe to nucleotides encoding cassette II detected a 9 kb NBCn1 mRNA strongly in the heart and weakly in skeletal muscles, but absent from most of the tissues including kidney, brain, and pancreas. In the rat heart, PCR with primers flanking cassette II preferentially amplified a DNA fragment that lacked cassette II. However, in the human heart, PCR preferentially amplified a fragment that contained cassette II. This larger PCR product was found virtually in all regions of the human cardiovascular system with strong amplification in the apex, atrium, and atrioventricular nodes. These findings indicate that the variant containing cassette II is almost absent in tissues including brain, kidney, and pancreas, where NBCn1 has been extensively examined.

NHE3 is the Na + /H + exchanger located on the intestinal and renal brush border membrane, where it functions in transepithelial Na + absorption. The brush border Na + absorptive process is acutely inhibited by activation of cAMP-dependent protein kinase, but the molecular mechanism of this inhibitory effect is poorly understood. We have identified two regulatory proteins, E3KARP and NHERF, that interact with NHE3 to enable cAMP to inhibit NHE3. The two regulatory proteins are structurally related, sharing ≈50% identity in amino acid sequences. It has been previously shown that when NHE3 is transfected into PS120 fibroblasts or Caco-2 cells, cAMP failed to inhibit NHE3 activity. Northern blot analysis showed that both PS120 and Caco-2 cells lacked the expression of both E3KARP and NHERF. In contrast, other cell lines in which cAMP inhibits NHE3, including OK, CHO, and LLC-PK 1 cells, expressed NHERF-related regulatory proteins. To determine their functions in cAMP-dependent inhibition of NHE3, E3KARP and NHERF were transfected into PS120/NHE3 fibroblasts. Transfection in PS120/NHE3 fibroblasts with either NHERF or E3KARP reconstituted cAMP-induced inhibition of NHE3, resulting in 25–30% inhibition in these cells.

Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of the very homologous NHERFs. We studied the association of NHE3, as well as NHERF1 (EBP50), NHERF2 (E3KARP) and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM and their possible association with signaling molecules. NHE3 was found to partially associate with glycosphingolipid-enriched microdomains in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity as well as on second-messenger-dependent regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. A search for other signalling molecules that are implicated in regulating NHE3 through a NHERF interaction, cGMP-dependent kinase II, which together with NHERF2 is essential for guanylin/heat stable enterotoxin of E.coli (STa)-mediated NHE3 inhibition in the intestine, was found exclusively lipid-raft associated. In conclusion, the differential association of the NHERFs, as well as kinases, with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is likely one component of the differential and signal-specific NHE3 regulation by the different NHERFs.

Maintenance of the epithelial barrier in the intestinal tract is necessary to protect the host from the hostile luminal environment. Phospholipase C-β (PLC-β) has been implicated to control myriad signaling cascades. However, the biological effects of selective PLC-β isozymes are poorly understood. We describe novel findings that lysophosphatidic acid (LPA) regulates PLC-β1 and PLC-β2 via two distinct pathways to enhance intestinal epithelial cell (IEC) proliferation and migration that facilitate wound closure and recovery of the intestinal epithelial barrier. LPA acting on the LPA1 receptor promotes IEC migration by facilitating the interaction of Gαq with PLC-β2. LPA-induced cell proliferation is PLC-β1 dependent and involves translocation of Gαq to the nucleus, where it interacts with PLC-β1 to induce cell cycle progression. An in vivo study using LPA1-deficient mice (Lpar1(-/-)) shows a decreased number of proliferating IECs and migration along the crypt-luminal axis. Additionally, LPA enhances migration and proliferation of IECs in an LPA1-dependent manner, and Lpar1(-/-) mice display defective mucosal wound repair that requires cell proliferation and migration. These findings delineate novel LPA1-dependent lipid signaling that facilitates mucosal wound repair via spatial targeting of distinct PLC-βs within the cell.

Rabbit NHE2 and NHE3 are two epithelial isoform Na+/H+ exchangers (NHE), the messages for which are found predominantly and entirely, respectively, in renal, intestinal, and gastric mucosa. The current studies used Western analysis and immunohistochemistry to identify and characterize the apical vs. basolateral membrane distribution of NHE2 and NHE3 in intestinal epithelial cells. Based on Western analysis, NHE2 and NHE3 both are present in brush-border but not basolateral membranes of small intestine. Both NHE2 and NHE3 are 85-kDa proteins. Consistent with Western analysis, NHE2 and NHE3 are immunolocalired to the brush-border but not basolateral membranes of villus epithelial cells, but not goblet cells, in human jejunum and ileum and in surface epithelial cells in the ascending and descending colon and rectum. In addition, NHE2 and NHE3 are present in small amounts in the crypt cell brush border of human jejunum, ileum, ascending and descending colon, and rectum. In rabbit jejunum, ileum, and ascending colon, NHE2 and NHE3 are present in the brush border of epithelial and not goblet cells, again much more in the villus (small intestine)/ surface cells (colon) than the crypt. NHE2 but not NHE3 is present in the brush border of rabbit descending colon surface cells and in small amounts in crypt cells. NHE2 and NHE3 are both human and rabbit small intestinal and colonic epithelial cell brush-border Na+/H+ exchanger isoforms that colocalize in all intestinal segments except rabbit descending colon, which lacks NHE3.

All cloned members of the mammalian Na+/H+ exchanger gene family encode proteins that consist of two functionally distinct domains: a membrane-bound N terminus and a cytoplasmic C terminus, which are required for ion transport and regulation of transport, respectively. Despite their similarity in structure, three members of this family, designated NHE1, NHE2, and NHE3, exhibit different kinetic mechanisms in response to growth factors and protein kinases. For instance, growth factors stimulate NHE1 by a change in the affinity constant for intracellular H+, K'(Hi+), and regulate NHE2 and NHE3 by a change in Vmax. We have constructed chimeric Na+/H+ exchangers by exchanging the N and C termini among three cloned rabbit Na+/H+ exchangers (NHE1 to NHE3) to determine which domain is responsible for the above Vmax-vs.-K'(H(i)+) effect of the Na+/H+ isoforms. All of the chimeras had functional exchange activity and basal kinetic properties similar to those of wild-type exchangers. Studies with serum showed that the N terminus is responsible for the Vmax-vs.-K'(H(i)+) stimulation of the Na+/H+ exchanger isoforms. Moreover, phorbol 12-myristate 13-acetate and fibroblast growth factor altered Na+/H+ exchange only in chimeras that had an epithelial N-terminal domain matched with an epithelial C-terminal domain. Therefore, the protein kinase-induced regulation of Na+/H+ exchangers is mediated through a specific interaction between the N- and C-termini, whcih is restricted so that epithelial N- and epithelial N-and C-terminal portions of the exchangers are required for regulation.

We previously isolated a 1.4-kb partial cDNA from a human kidney cortex library. Using both library screening and reverse transcription-polymerase chain reaction of human kidney RNA, we obtained the entire coding region of the human NHE3 cDNA. The human NHE3 cDNA encoded a protein of 834 amino acids with a calculated relative molecular weight of 92,906. It exhibited 89 and 88% amino acid identity with rat and rabbit NHE3, respectively. The stable transfection of a composite human NHE3 cDNA into Na+/H+ exchanger-deficient PS120 cells established Na+/H+ exchange. Functionally, human NHE3 was similar to the rabbit and rat NHE3 homologues, being relatively resistant to inhibition by amiloride, half-maximal inhibition (IC50) = 49.0 microM, and ethylisopropylamiloride, IC50 = 6.6 microM, and being stimulated by fibroblast growth factor but inhibited by phorbol 12-myristate 13-acetate. However, unlike the rabbit or rat NHE3, human NHE3 message was not restricted to kidney, intestine, stomach, and brain. Northern analysis of multiple human tissues detected NHE3 message, in descending order, as follows: kidney >> small intestine >> testes > ovary > colon = prostate > thymus > peripheral leukocyte = brain > spleen > placenta. Message in the kidney, small intestine, and colon was primarily of 6.7 kb, whereas both 6.7- and 8.9-kb bands were expressed nearly equivalently in the other tissues. No NHE3 message was detected in the human heart, lung, liver, skeletal muscle, or pancreas.

NHE3, a cloned intestinal and renal brush border Na+/H+ exchanger, has previously been shown to be both stimulated and inhibited by different protein kinases/growth factors. For instance, NHE3 is stimulated by serum and fibroblast growth factor (FGF) and inhibited by protein kinase C. In the present study, we used a series of NHE3 C terminus truncation mutants to identify separate regions of the C-terminal cytoplasmic tail responsible for stimulation and inhibition by protein kinases/growth factors. Five NHE3 C terminus truncation mutant stable cell lines were generated by stably transfecting NHE3 deletion cDNAs into PS120 fibroblasts, which lack any endogenous Na+/H+ exchanger. Using fluorometric techniques, the effects of the calcium/calmodulin (CaM) inhibitor W13, calcium/CaM kinase inhibitor KN-62, phorbol myristate acetate, okadaic acid, FGF, and fetal bovine serum on Na+/H+ exchange were studied in these transfected cells. Inhibition of basal activity of full-length NHE3 is mediated by CaM at a site C-terminal to amino acid 756; this CaM effect occurs through both kinase dependent and independent mechanisms. There is another independent inhibitory domain for protein kinase C between amino acids 585 and 689. In addition, there are at least three stimulatory regions in the C-terminal domain of NHE3, corresponding to amino acids 509-543 for okadaic acid, 475-509 for FGF, and a region N-terminal to amino acid 475 for fetal bovine serum. We conclude that separate regions of the C terminus of NHE3 are involved with stimulation or inhibition of Na+/H+ exchange activity, with both stimulatory and inhibitory domains having several discrete subdomains. A conservative model to explain the way these multiple domains in the C terminus of NHE3 regulate Na+/H+ exchange is via an effect on associated regulatory proteins.

A polyclonal antibody (Ab597) was produced in rabbit against a fusion protein of glutathione-S-transferase and the last 87 amino acids of the Na+/H+ exchanger isoform, NHE2. By Western blotting, Ab597 recognized proteins of 75 and 85 kDa in PS120/NHE2 membranes (PS120 cells stably transfected with NHE2), and this antibody did not cross-react with NHE1 and NHE3. When Ab597 was used to immunocytochemically stain PS120/NHE2 cells, permeabilization of the cells was required for staining, confirming the putative membrane topology of NHE2 that the C-terminus is cytoplasmic. NHE1 is N-glycosylated. NHE2 was predicted to be N-glycosylated as it contains one potential N-linked glycosylation site (N350VS), which is conserved among NHE1, NHE3, and NHE4. However, NHE2 was resistant to peptide:N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) digestion, suggesting that NHE2 is not N-glycosylated. In contrast, neuraminidase shifted the mobility of the 85 kDa NHE2 protein in PS120/NHE2 membranes into an 81 kDa band, and O-glycanase further shifted the mobility of the neuraminidase-treated 81 kDa protein to 75 kDa. Incubation of PS120/NHE2 cells with benzyl N-acetyl-alpha-D-galactosaminide (Bz alpha GalNAc), an O-glycosylation inhibitor, decreased the size of the 85 kDa protein to 81 kDa. This treatment had no effect on the initial rate of Na+/H+ exchange of PS120/NHE2 cells. The 75 kDa protein was not affected by the glycosidase treatment of PS120/NHE2 membranes or the Bz alpha GalNAc treatment of PS120/NHE2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Na+/H+exchange was first described by Murer, Hopfer and Kinne [19] in renal brush border membrane vesicles. This process is mediated by Na+/H+exchangers which catalyze the exchange of extracellular Na+for intracellular H with a stoichiometry of 1:1. Na+/H+exchangers have multiple functions, including pH homeostasis, volume regulation, cell proliferation, and transcellular Na+absorption [reviewed in 12]. In no cell is it the only mechanism for any one of these functions. For instance, multiple mechanisms of pH homeostasis are present in most eukaryotic cells including a c┌/HCO3-exchanger, a NaHCO3co-transporter, a Na+- dependent cr/HCO3-exchanger and multiple mechanisms of hT extrusion [reviewed in 15], including the H-K-ATPase pump. In this review, we will focus on recent advances in identification and understanding of the structure/function relationships and acute protein kinase regulation of members of the mammalian Na+/H+exchanger gene family.

Background Metastasis, the spread and growth of tumor cells to distant organ sites, represents the most devastating attribute and plays a major role in the morbidity and mortality of cancer. Inflammation is crucial for malignant tumor transformation and survival. Thus, blocking inflammation is expected to serve as an effective cancer treatment. Among anti-inflammation therapies, chemokine modulation is now beginning to emerge from the pipeline. CXC chemokine receptor-4 (CXCR4) and its ligand stromal cell-derived factor-1 (CXCL12) interaction and the resulting cell signaling cascade have emerged as highly relevant targets since they play pleiotropic roles in metastatic progression. The unique function of CXCR4 is to promote the homing of tumor cells to their microenvironment at the distant organ sites. Methodology/Principal Findings We describe the actions of N,N′-(1,4-phenylenebis(methylene))dipyrimidin-2-amine (designated MSX-122), a novel small molecule and partial CXCR4 antagonist with properties quite unlike that of any other reported CXCR4 antagonists, which was prepared in a single chemical step using a reductive amination reaction. Its specificity toward CXCR4 was tested in a binding affinity assay and a ligand competition assay using 18F-labeled MSX-122. The potency of the compound was determined in two functional assays, Matrigel invasion assay and cAMP modulation. The therapeutic potential of MSX-122 was evaluated in three different murine models for inflammation including an experimental colitis, carrageenan induced paw edema, and bleomycin induced lung fibrosis and three different animal models for metastasis including breast cancer micrometastasis in lung, head and neck cancer metastasis in lung, and uveal melanoma micrometastasis in liver in which CXCR4 was reported to play crucial roles. Conclusions/Significance We developed a novel small molecule, MSX-122, that is a partial CXCR4 antagonist without mobilizing stem cells, which can be safer for long-term blockade of metastasis than other reported CXCR4 antagonists.