Xuhang Li

Na+‐H+ exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3

1 Rabbit ileal Na+‐absorbing cell Na+‐H+ exchanger 3 (NHE3) was shown to exist in three pools in the brush border (BB), including a population in lipid rafts. Approximately 50 % of BB NHE3 was associated with Triton X‐100‐soluble fractions and the other ∼50 % with Triton X‐100‐insoluble fractions; ∼33 % of the detergent‐insoluble NHE3 was present in cholesterol‐enriched lipid microdomains (rafts). 2 The raft pool of NHE3 was involved in the stimulation of BB NHE3 activity with epidermal growth factor (EGF). Both EGF and clonidine treatments were associated with a rapid increase in the total amount of BB NHE3. This EGF‐ and clonidine‐induced increase of BB NHE3 was associated with an increase in the raft pool of NHE3 and to a smaller extent with an increase in the total detergent‐insoluble fraction, but there was no change in the detergent‐soluble pool. In agreement with the rapid increase in the amount of NHE3 in the BB, EGF also caused a rapid stimulation of BB Na+‐H+ exchange activity. 3 Disrupting rafts by removal of cholesterol with methyl‐β‐cyclodextrin (MβCD) or destabilizing the actin cytoskeleton with cytochalasin D decreased the amount of NHE3 in early endosomes isolated by OptiPrep gradient fractionation. Specifically, NHE3 was shown to associate with endosomal vesicles immunoisolated by anti‐EEA1 (early endosomal autoantigen 1) antibody‐coated magnetic beads and the endosome‐associated NHE3 was decreased by cytochalasin D and MβCD treatment. 4 We conclude that: (i) a pool of ileal BB NHE3 exists in lipid rafts; (ii) EGF and clonidine increase the amount of BB NHE3; (iii) lipid rafts and to a lesser extent, the cytoskeleton, but not the detergent‐soluble NHE3 pool, are involved in the EGF‐ and clonidine‐induced acute increase in amount of BB NHE3; (iv) lipid rafts and the actin cytoskeleton play important roles in the basal endocytosis of BB NHE3.

Downregulation of sodium transporters and NHERF proteins in IBD patients and mouse colitis models: Potential contributors to IBD‐associated diarrhea

Background: One of the most common symptoms among patients with inflammatory bowel disease (IBD) is diarrhea, which is thought to be contributed by changes in electrolyte transport associated with intestinal inflammation. This study was designed to test the hypothesis that intestinal Na+‐related transporters/channels and their regulatory proteins may be downregulated as a potential contributor to IBD‐associated diarrhea. Methods: SDS‐PAGE and Western blotting and/or confocal immunomicroscopy were used to examine the expression of Na+/H+‐exchangers 1–3 (NHE1–3), epithelial Na+ channel (ENaC), Na+/K+‐ATPase, the intracellular Cl− channel 5 (ClC‐5), and NHE3 regulatory factors (NHERF1,2) in ileal and colonic pinch biopsies from IBD patients and noninflammatory controls, as well as from colonic mucosa of dextran sodium sulfate (DSS)‐ and TNBS‐induced acute murine IBD models. Results: NHE1,3 (but not NHE2), &bgr;‐ENaC, Na+/K+‐ATPase‐&agr;, ClC‐5, and NHERF1 were all downregulated in sigmoid mucosal biopsies from most cases of active UC and/or CD compared to controls. NHE3 was also decreased in ileal mucosal biopsies of active CD, as well as in ≈50% of sigmoid biopsies from inactive UC or CD. Importantly, similar downregulation of NHE1,3, &bgr;‐ENaC, and NHERF1,2 was also observed in the mouse colon (but not ileum) of DSS‐ and TNBS‐induced colitis. Conclusions: IBD‐associated diarrhea may be due to a coordinated downregulation of multiple Na+ transporter and related regulatory proteins, including NHE1,3, Na+/K+‐ATPase, and ENaC, as well as NHERF1,2, and ClC‐5, all of which are involved directly or indirectly in intestinal Na+ absorption.

D-Glucose Acts via Sodium/Glucose Cotransporter 1 to Increase NHE3 in Mouse Jejunal Brush Border by a Na+/H+ Exchange Regulatory Factor 2–Dependent Process

BACKGROUND & AIMSnOral rehydration solutions reduce diarrhea-associated mortality. Stimulated sodium absorption by these solutions is mediated by the Na(+)/H(+) hydrogen exchanger NHE3 and is increased by Na(+)-glucose co-transport in vitro, but the mechanisms of this up-regulated process are only partially understood.nnnMETHODSnIntracellular pH was measured in jejunal enterocytes of wild-type mice and mice with disrupted Na+/H+ exchange regulatory co-factor 2 (NHERF2-/- mice) by multiphoton microscopy. Diarrhea was induced by cholera toxin. Caco-2BBe cells that express NHE3 and the sodium/glucose cotransporter 1 (SGLT1) were studied by fluorometry, before and after siRNA-mediated knockdown of NHERF1 or NHERF2. NHE3 distribution was assessed by cell-surface biotinylation and confocal microscopy. Brush-border mobility was determined by fluorescence recovery after photobleaching and confocal microscopy.nnnRESULTSnThe nonmetabolized SGLT1 substrate α-methyl-D-Glu (α-MD-G) activated jejunal NHE3; this process required Akt and NHERF2. α-MD-G normalized NHE3 activity after cholera toxin-induced diarrhea. α-MD-G-stimulated jejunal NHE3 activity was defective in NHERF2-/- mice and cells with NHERF2 knockdown, but occurred normally with NHERF1 knockdown; was associated with increased NHE3 surface expression in Caco-2 cells, which also was NHERF2-dependent; was associated with dissociation of NHE3 from NHERF2 and an increase in the NHE3 mobile fraction from the brush border; and was accompanied by a NHERF2 ezrin-radixin-moesin-binding domain-dependent increase in co-precipitation of ezrin with NHE3.nnnCONCLUSIONSnSGLT1-mediated Na-glucose co-transport stimulates NHE3 activity in vivo by an Akt- and NHERF2-dependent signaling pathway. It is associated with increased brush-border NHE3 and association between ezrin and NHE3. Activation of NHE3 corrects cholera toxin-induced defects in Na absorption and might contribute to the efficacy of oral rehydration solutions.

Serum anti-glycan antibody biomarkers for inflammatory bowel disease diagnosis and progression: a systematic review and meta-analysis.

Background: Anti‐glycan antibody serologic markers may serve as a useful adjunct in the diagnosis/prognosis of inflammatory bowel disease (IBD), including Crohns disease (CD) and ulcerative colitis (UC). This meta‐analysis/systemic review aimed to evaluate the diagnostic value, as well as the association of anti‐glycan biomarkers with IBD susceptible gene variants, disease complications, and the need for surgery in IBD. Methods: The diagnostic odds ratio (DOR), 95% confidence interval (CI), and sensitivity/specificity were used to compare the diagnostic value of individual and combinations of anti‐glycan markers and their association with disease course (complication and/or need for surgery). Results: Fourteen studies were included in the systemic review and nine in the meta‐analysis. Individually, anti‐Saccharomyces cervisiae antibodies (ASCA) had the highest DOR for differentiating IBD from healthy (DOR 21.1; 1.8–247.3; two studies), and CD from UC (DOR 10.2; CI 7.7–13.7; seven studies). For combination of ≥2 markers, the DOR was 2.8 (CI 2.2–3.6; two studies) for CD‐related surgery, higher than any individual marker, while the DOR for differentiating CD from UC was 10.2 (CI 5.6–18.5; three studies) and for complication was 2.8 (CI 2.2–3.7; two studies), similar to individual markers. Conclusions: ASCA had the highest diagnostic value among individual anti‐glycan markers. While anti‐chitobioside carbohydrate antibody (ACCA) had the highest association with complications, ASCA and ACCA associated equally with the need for surgery. Although in most individual studies the combination of ≥2 markers had a better diagnostic value as well as higher association with complications and need for surgery, we found the combination performing slightly better than any individual marker in our meta‐analysis. (Inflamm Bowel Dis 2012)

Fractionation of subcellular membrane vesicles of epithelial and non-epithelial cells by OptiPrep™ density gradient ultracentrifugation.

Density gradient ultracentrifugation (DGUC) is widely used for physical isolation (enrichment rather than purification) of subcellular membrane vesicles. It has been a valuable tool to study specific subcellular localization and dynamic trafficking of proteins. While sucrose has been the main component of density gradients, several years ago, synthetic OptiPrep™ (iodixanol) began being used for separation of organelles due to its iso-osmotic property. Here, we describe a detailed protocol for density gradient fractionation of various mammalian subcellular vesicles, including endoplasmic reticulum (ER), Golgi apparatus, endosomes, and lipid rafts, as well as apical and basolateral membranes of polarized epithelial cells.

Elevated Intracellular Calcium Stimulates NHE3 Activity by an IKEPP (NHERF4) Dependent Mechanism

The ileal brush border (BB) contains four evolutionarily related multi-PDZ domain proteins including NHERF1, NHERF2, PDZK1 (NHERF3) and IKEPP (NHERF4). Why multiple related PDZ proteins are in a similar location in the same cell is unknown. However, some specificity in regulation of NHE3 activity has been identified. For example, elevated intracellular Ca2+ ([Ca2+]i) inhibition of NHE3 is reconstituted by NHERF2 but not NHERF1, and involves the formation of large NHE3 complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether the four PDZ domain containing protein IKEPP reconstitutes elevated [Ca2+]i regulation of NHE3. In vitro, IKEPP bound to the F2 region (aa 590-667) of NHE3 in overlay assays, which is the same region where NHERF1 and NHERF2 bind. PS120 cells lack endogenous NHE3 and IKEPP. Treatment of PS120/NHE3/IKEPP cells (stably transfected with NHE3 and IKEPP) with the Ca2+ ionophore, 4-Br-A23187 (0.5μM), stimulated NHE3 Vmax activity by ∼40%. This was associated with an increase in plasma membrane expression of NHE3 by a similar amount. NHE3 activity and surface expression were unaffected by A23187 in PS120/NHE3 cells lacking IKEPP. Based on sucrose density gradient centrifugation, IKEPP was also shown to exist in large complexes, some of which overlap in size with NHE3, and the size of both NHE3 and IKEPP complexes decreased in parallel after [Ca2+]i elevation. FRET experiments on fixed cells demonstrated that IKEPP and NHE3 directly associated at an intracellular site. Elevating [Ca2+]i decreased this intracellular NHE3 and IKEPP association. In summary: (1) In the presence of IKEPP, elevated [Ca2+]i stimulates NHE3 activity. This was associated with increased expression of NHE3 in the plasma membrane as well as a shift to smaller sizes of NHE3 and IKEPP containing complexes. (2) IKEPP directly binds NHE3 at its F2 C-terminal domain and directly associates with NHE3 in vivo (FRET). (3) Elevated [Ca2+]i decreased the association of IKEPP and NHE3 in an intracellular compartment. Based on which NHERF family member is expressed in PS120 cells, elevated [Ca2+]i stimulates (IKEPP), inhibits (NHERF2) or does not affect (NHERF1) NHE3 activity. This demonstrates that regulation of NHE3 depends on the nature of the NHERF family member associating with NHE3 and the accompanying NHE3 complexes.

Alterations in the proteome of the NHERF1 knockout mouse jejunal brush border membrane vesicles

Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCβ3, E-cadherin, p120, β-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.

PLC-γ directly binds activated c-Src, which is necessary for carbachol-mediated inhibition of NHE3 activity in Caco-2/BBe cells

Elevated levels of intracellular Ca(2+) ([Ca(2+)]i) inhibit Na(+)/H(+) exchanger 3 (NHE3) activity in the intact intestine. We previously demonstrated that PLC-γ directly binds NHE3, an interaction that is necessary for [Ca(2+)]i inhibition of NHE3 activity, and that PLC-γ Src homology 2 (SH2) domains may scaffold Ca(2+) signaling proteins necessary for regulation of NHE3 activity. [Ca(2+)]i regulation of NHE3 activity is also c-Src dependent; however, the mechanism by which c-Src is involved is undetermined. We hypothesized that the SH2 domains of PLC-γ might link c-Src to NHE3-containing complexes to mediate [Ca(2+)]i inhibition of NHE3 activity. In Caco-2/BBe cells, carbachol (CCh) decreased NHE3 activity by ∼40%, an effect abolished with the c-Src inhibitor PP2. CCh treatment increased the amount of active c-Src as early as 1 min through increased Y(416) phosphorylation. Coimmunoprecipitation demonstrated that c-Src associated with PLC-γ, but not NHE3, under basal conditions, an interaction that increased rapidly after CCh treatment and occurred before the dissociation of PLC-γ and NHE3 that occurred 10 min after CCh treatment. Finally, direct binding to c-Src only occurred through the PLC-γ SH2 domains, an interaction that was prevented by blocking the PLC-γ SH2 domain. This study demonstrated that c-Src 1) activity is necessary for [Ca(2+)]i inhibition of NHE3 activity, 2) activation occurs rapidly (∼1 min) after CCh treatment, 3) directly binds PLC-γ SH2 domains and associates dynamically with PLC-γ under elevated [Ca(2+)]i conditions, and 4) does not directly bind NHE3. Under elevated [Ca(2+)]i conditions, PLC-γ scaffolds c-Src into NHE3-containing multiprotein complexes before dissociation of PLC-γ from NHE3 and subsequent endocytosis of NHE3.

The NHERF2 sequence adjacent and upstream of the ERM-binding domain affects NHERF2-ezrin binding and dexamethasone stimulated NHE3 activity.

The microvillar localization of Na+-H+ exchanger regulatory factor (NHERF)1/2 requires not only ezrin, radixin and moesin (ERM)-binding domain (EBD) but also a newly defined ERM-binding regulatory sequence (EBRS) that modulates NHERF1/2–ezrin binding. NHERF2 EBRS is also regulated by phosphorylation, which affects NHE3 (Na+-H+ exchanger 3) stimulation by dexamethasone.