Diarrhea is one of the troublesome complications of diabetes, and the underlying causes of this problem are complex. Here, we investigated whether altered electrolyte transport contributes to diabetic diarrhea. We found that the expression of Na+/H+ exchanger NHE3 and several scaffold proteins, including NHE3 regulatory factors (NHERFs), inositol trisphosphate (IP3) receptor-binding protein released with IP3 (IRBIT), and ezrin, was decreased in the intestinal brush border membrane (BBM) of mice with streptozotocin-induced diabetes. Treatment of diabetic mice with insulin restored intestinal NHE3 activity and fluid absorption. Molecular analysis revealed that NHE3, NHERF1, IRBIT, and ezrin form macrocomplexes, which are perturbed under diabetic conditions, and insulin administration reconstituted these macrocomplexes and restored NHE3 expression in the BBM. Silencing of NHERF1 or IRBIT prevented NHE3 trafficking to the BBM and insulin-dependent NHE3 activation. IRBIT facilitated the interaction of NHE3 with NHERF1 via protein kinase D2-dependent phosphorylation. Insulin stimulated ezrin phosphorylation, which enhanced the interaction of ezrin with NHERF1, IRBIT, and NHE3. Additionally, oral administration of lysophosphatidic acid (LPA) increased NHE3 activity and fluid absorption in diabetic mice via an insulin-independent pathway. Together, these findings indicate the importance of NHE3 in diabetic diarrhea and suggest LPA administration as a potential therapeutic strategy for management of diabetic diarrhea.

The two members of the group I metabotropic glutamate receptor family, mGluR1 and mGluR5, both couple to Gq to mediate rises in intracellular calcium. The alternatively spliced C termini (CT) of mGluRs 1 & 5 are known to be critical for regulating receptor activity and to terminate in motifs suggestive of potential interactions with PDZ domains. We therefore screened the CTs of both mGluR1a and mGluR5 against a PDZ domain proteomic array. Out of 96 PDZ domains examined, the domain that bound most strongly to mGluR5-CT was the second PDZ domain of the Na+/H + exchanger regulatory factor 2 (NHERF-2). This interaction was confirmed by reverse overlay, and a single point mutation to the mGluR5-CT was found to completely disrupt the interaction. Full-length mGluR5 robustly associated with full-length NHERF-2 in cells, as assessed by co-immunoprecipitation and confocal microscopy experiments. In contrast, mGluR1a was found to bind NHERF-2 in vitro with a weaker affinity than mGluR5, and furthermore mGluR1a did not detectably associate with NHERF-2 in a cellular context. Immunohistochemical experiments revealed that NHERF-2 and mGluR5 exhibit overlapping patterns of expression in mouse brain, being found most abundantly in astrocytic processes and postsynaptic neuronal elements. In functional experiments, the interaction of NHERF-2 with mGluR5 in cells was found to prolong mGluR5-mediated calcium mobilization and to also potentiate mGluR5-mediated cell death, whereas co-expression of mGluR1a with NHERF-2 had no evident effects on mGluR1a functional activity. These observations reveal that NHERF-2 can selectively modulate mGluR5 signaling, which may contribute to cell-specific regulation of mGluR5 activity.

The group II metabotropic glutamate receptors mGluR2 and mGluR3 are key modulators of glutamatergic neurotransmission. In order to identify novel Group II metabotropic glutamate receptor (mGluR)-interacting partners, we screened the C-termini of mGluR2 and mGluR3 for interactions with an array of PDZ domains. These screens identified the Na+/H+ exchanger regulatory factors 1 and 2 (NHERF-1 & -2) as candidate interacting partners. Follow-up co-immunoprecipitation studies demonstrated that both mGluR2 and mGluR3 can associate with NHERF-1 and NHERF-2 in a cellular context. Functional studies revealed that disruption of PDZ interactions with mGluR2 enhanced receptor signaling to Akt. However, further studies of mGluR2 and mGluR3 signaling in astrocytes in which NHERF expression was reduced by gene knockout (KO) and/or siRNA knockdown techniques revealed that the observed differences in signaling between WT and mutant mGluR2 were likely not due to disruption of interactions with the NHERF proteins. Electron microscopic analyses revealed that Group II mGluRs were primarily expressed in glia and unmyelinated axons in WT, NHERF-1 and NHERF-2 KO mice, but the relative proportion of labeled axons over glial processes was higher in NHERF-2 KO mice than in controls and NHERF-1 KO mice. Interestingly, our anatomical studies also revealed that loss of either NHERF protein results in ventriculomegaly, which may be related to the high incidence of hydrocephaly that has previously been observed in NHERF-1 KO mice. Together, these studies support a role for NHERF-1 and NHERF-2 in regulating the distribution of Group II mGluRs in the murine brain, while conversely the effects of the mGluR2/3 PDZ-binding motifs on receptor signaling are likely mediated by interactions with other PDZ scaffold proteins beyond the NHERF proteins.

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.

The intestinal epithelium interacts dynamically with the immune system to maintain its barrier function to protect the host, while performing the physiological roles in absorption of nutrients, electrolytes, water and minerals. The importance of lysophosphatidic acid (LPA) and its receptors in the gut has been progressively appreciated. LPA signaling modulates cell proliferation, invasion, adhesion, angiogenesis, and survival that can promote cancer growth and metastasis. These effects are equally important for the maintenance of the epithelial barrier in the gut, which forms the first line of defense against the milieu of potentially pathogenic stimuli. This review focuses on the LPA-mediated signaling that potentially contributes to inflammation and tumor formation in the gastrointestinal tract.

Background: SR-BI mediates selective delivery of lipoprotein-CE to the liver, adrenals, and gonads for product formation. Results: NHERFs interact with and down-regulate SR-BI protein levels to inhibit selective CE uptake and steroidogenesis. Conclusion: Two protein domains (PDZ and MERM) are required for NHERF1/2-mediated inhibition of SR-BI expression and function. Significance: This work reveals a novel mechanism of translational/posttranslational regulation of SR-BI. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Growing evidence suggests that pharmacological inhibition of Na/H exchange and Na/HCO3 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 (pHo) ranging from 6.2 to 8.3, NBCn1 protein expression increased by fivefold at pH < 6.5 compared to the expression at pHo 7.4. At pHo 6.5, the intracellular pH of neurons was ~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 pHo 6.5. NBCn1 expression also increased by 40% in a prolonged Mg2+-free incubation at normal pHo. 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 (pHo 7.2 for 6 h) after glutamate exposure, acidic incubation (pHo 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 Mg2+-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 pHo and Mg2+ depletion. NBCn1 is not required for acidic protection, but increases cytotoxicity in Mg2+-free conditions.

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

Background/Aims: Enterocytes express a number of NHE isoforms with presumed localization in the apical (NHE2, 3 and 8) or basolateral (NHE1) membrane. Functional activity and localization of enterocyte NHE isoforms were assessed using fully differentiated Caco-2BBe cells, whose genetic expression profile closely resembles mature enterocytes. Methods: The activity of the different NHEs was analyzed by fluorometric pHi-metry in a perfusion chamber with separate apical and basolateral perfusion, using specific inhibitors and shRNA knockdown of NHE2. The expression of the NHEs and of other relevant acid extrusion transporters was quantified by qPCR. Results: Quantitative comparison of the mRNA expression levels of the different NHE isoforms in 14 day-differentiated Caco-2BBe cells showed the following order: NHE2>NHE8>NHE3>NHE1. Acid-activated NHE exchange rates in the basolateral membrane were >6-fold higher than in the apical membrane. 79 ± 3 % of the acid-activated basolateral Na+/H+ exchange rate displayed a NHE1-typical inhibitor profile, and no NHE2/3/8 typical activity could be observed. Analysis of the apical Na+/H+ exchange rates revealed that approximately 51 ± 3 % of the total apical activity displayed a NHE2/8-typical inhibitor profile and 31 ± 6 % a NHE3-typical inhibitor profile. Because no selective NHE2 inhibitor is available, a stable NHE2 knockdown cell line (C2NHE2KD) was generated. C2NHE2KD displayed a reduced NHE2-typical apical Na+/H+ exchange rate and maintained a lower steady-state pHi, despite high expression levels of other acid extruders, in particular NBCn1 (Slc4a7). Conclusion: Differentiated Caco-2BBe cells display particularly high mRNA expression levels of NHE2, which can be functionally identified in the apical membrane. Although at low intracellular pH, NHE2 transport rate was far lower than that of NHE1. NHE2 activity was nevertheless essential for the maintenance of the steady-state pHi of these cells.

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.