NHERF1 Expression Research Articles

NHERF-1: Modulator of Glioblastoma Cell Migration and Invasion

The invasive nature of malignant gliomas is a clinical problem rendering tumors incurable by conventional treatment modalities such as surgery, ionizing radiation, and temozolomide. Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1) is a multifunctional adaptor protein, recruiting cytoplasmic signaling proteins and membrane receptors/transporters into functional complexes. This study revealed that NHERF-1 expression is increased in highly invasive cells that reside in the rim of glioblastoma multiforme (GBM) tumors and that NHERF-1 sustains glioma migration and invasion. Gene expression profiles were evaluated from laser capture-microdissected human GBM cells isolated from patient tumor cores and corresponding invaded white matter regions. The role of NHERF-1 in the migration and dispersion of GBM cell lines was examined by reducing its expression with small-interfering RNA followed by radial migration, three-dimensional collagen dispersion, immunofluorescence, and survival assays. The in situ expression of NHERF-1 protein was restricted to glioma cells and the vascular endothelium, with minimal to no detection in adjacent normal brain tissue. Depletion of NHERF-1 arrested migration and dispersion of glioma cell lines and caused an increase in cell-cell cohesiveness. Glioblastoma multiforme cells with depleted NHERF-1 evidenced a marked decrease in stress fibers, a larger cell size, and a more rounded shape with fewer cellular processes. When NHERF-1 expression was reduced, glioma cells became sensitized to temozolomide treatment resulting in increased apoptosis. Taken together, these results provide the first evidence for NHERF-1 as a participant in the highly invasive phenotype of malignant gliomas and implicate NHERF-1 as a possible therapeutic target for treatment of GBM.

Differential desensitization of Na coupled transporters with chronic parathyroid hormone (PTH) stimulation

PTH regulates renal proximal tubule apical (BBM) and basolateral membrane (BLM) transporters including Npt2a, Na+, K+ ATPase (Na‐K), sodium hydrogen exchangers 1 and 3 (NHE1, NHE3). We have shown that the acute inhibitory effect of PTH on proximal tubule Na‐K activity disappears with chronic PTH treatment, but the inhibitory effect on Npt2a activity persists. We hypothesize that chronic PTH results in desensitization of PTH actions on Na but not phosphate transport. To address this hypothesis, we compared the effect of acute and chronic PTH stimulation on expression and function of transport proteins and signaling pathways in Sprague Dawley rat kidney and in OK cells. Treatment with PTH (5 µg/kg) for 6 h decreased Na‐K activity and Na‐K, NHE1, and NHE3 expression in rat BLM, while chronic treatment with PTH (4 days) increased Na‐K‐activity and expression above control levels. BBM Npt2a expression significantly decreased with acute and chronic PTH treatment. PTH did not affect BLM or BBM PTH receptor expression, while BLM expression of NHERF1 significantly increased. Similar findings were seen in OK cells. After chronic PTH, selective stimulation of OK cell BBM with PTH did not stimulate additional PKA and PKC activity, while stimulation of BLM significantly increased PKC activity. BBM or BLM stimulation with 8‐bromo‐cAMP resulted in further PKA activation; stimulation with phorbol myristate acetate resulted in further PKC activation. We conclude that the effects of PTH on Na transport undergo desensitization at a post‐receptor level while the effects of PTH on phosphate transport do not desensitize. The role of differential NHERF1 regulation in modulating PTH receptor‐transporter signaling is unknown. Support from VAMR.

MiR-8 microRNAs regulate the response to osmotic stress in zebrafish embryos

MicroRNAs (miRNAs) are highly conserved small RNAs that act as translational regulators of gene expression, exerting their influence by selectively targeting mRNAs bearing complementary sequence elements. These RNAs function in diverse aspects of animal development and physiology. Because of an ability to act as rapid responders at the level of translation, miRNAs may also influence stress response. In this study, we show that the miR-8 family of miRNAs regulates osmoregulation in zebrafish embryos. Ionocytes, which are a specialized cell type scattered throughout the epidermis, are responsible for pH and ion homeostasis during early development before gill formation. The highly conserved miR-8 family is expressed in ionocytes and enables precise control of ion transport by modulating the expression of Nherf1, which is a regulator of apical trafficking of transmembrane ion transporters. Ultimately, disruption of miR-8 family member function leads to an inability to respond to osmotic stress and blocks the ability to properly traffic and/or cluster transmembrane glycoproteins at the apical surface of ionocytes.

NHERF1 Regulates Parathyroid Hormone Receptor Desensitization: Interference with β-Arrestin Binding

Type 1 parathyroid hormone receptor (PTH1R) activation, desensitization, internalization, and recycling proceed in a cyclical manner. The Na(+)/H(+) exchange regulatory factor 1 (NHERF1) is a cytoplasmic adapter protein that regulates trafficking and signaling of several G protein-coupled receptors (GPCRs) including the PTH1R. The mineral ion wasting and bone phenotype of NHERF1-null mice suggests that PTH1R may interact with NHERF1. The objective of this study was to examine the effect of NHERF1 on PTH1R desensitization. Using rat osteosarcoma T6-N4 cells expressing the endogenous PTH1R, in which NHERF1 expression could be induced by tetracycline, PTH1R desensitization was assessed by measuring adenylyl cyclase activity after successive PTH challenges. PTH1R-mediated adenylyl cyclase responses were desensitized by repetitive PTH challenges in a concentration-dependent manner, and desensitization was inhibited by NHERF1. NHERF1 blocked PTH-induced dissociation of the PTH1R from Galpha(s). Blocking PTH1R endocytosis did not mitigate PTH1R desensitization. Reducing constitutive NHERF1 levels in human osteosarcoma SAOS2 cells, which express both endogenous PTH1R and NHERF1, with short hairpin RNA directed against NHERF1 restored PTH1R desensitization. Mutagenesis of the PDZ-binding domains or deletion of the NHERF1 MERM domain demonstrated that both are required for inhibition of receptor desensitization. A phosphorylation-deficient PTH1R exhibited reduced desensitization and interaction with beta-arrestin2 compared with wild-type PTH1R. NHERF1 inhibited beta-arrestin2 binding to wtPTH1R but had no effect on beta-arrestin2 association with pdPTH1R. Such an effect may protect against PTH resistance or PTH1R down-regulation in cells harboring NHERF1.

Regulation of Protein Expression and Function of OCTN2 in Forskolin-Induced Syncytialization in BeWo Cells

Placental OCTN2 is a high-affinity carnitine transporter that can interact with a number of therapeutic agents. The process of syncytialization is associated with the expression of a variety of genes. However, the association between syncytialization and OCTN2 expression is not yet clear. Given that forskolin induces BeWo cells to undergo biochemical and morphological differentiation, the purpose of the present study was to investigate whether the function and expression of OCTN2 are influenced by forskolin treatment during syncytialization.The forskolin-induced differentiation of BeWo cells was validated by secretion of β-human chorionic gonadotropin (β-hCG) and syncytin expression. Cellular localization of OCTN2 was analyzed by confocal microscopy. Expression of OCTN2 and the modular proteins PDZK1, PDZK2, NHERF1 and NHERF2 was analyzed by Western blotting and carnitine uptake by BeWo cells was estimated and the kinetic properties of uptake measured. The results showed that forskolin treatment increased β-hCG secretion and syncytin expression, suggesting induction of syncytialization. Confocal images of BeWo cells showed the localization of OCTN2 in the brush-border membrane. OCTN2 protein expression was upregulated in isolated brush-border membranes by long-term forskolin treatment, but the Vm for carnitine uptake was unchanged, although the Km increased. PDZK1, NHERF1 and NHERF2 protein expression in the brush-border membrane was downregulated by forskolin treatment, whereas PDZK2 levels remained unchanged.In conclusion, protein expression and function of OCTN2 in BeWo cells can be regulated by forskolin treatment. While the presence of forskolin results in an increase in OCTN2 protein expression, the increase in uptake capacity may be compensated by the decreased expression of PDZK1, NHERF1 or NHERF2.

Involvement of NHERF1 in apical membrane localization of MRP4 in polarized kidney cells

Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette protein superfamily, confers resistance to nucleoside and nucleotide analogs as well as camptothecin derivatives. MRP4 also mediates the efflux of certain cyclic nucleotides, eicosanoids, conjugated steroids, and uric acid. Depending on the cell type, MRP4 may localize to either apical or basolateral membranes in polarized cells. The adaptor protein NHERF1 has previously been implicated in MRP4 internalization in non-polarized cells. We have now found that NHERF1 levels are very low in polarized MDCKI cells which express MRP4 on basolateral membranes relative to polarized LLC-PK1 cells which express MRP4 on apical membranes. Furthermore, ectopic expression of FLAG-tagged NHERF1 in MDCKI cells and in MDCKI cells stably expressing eGFP-tagged MRP4 causes endogenous MRP4 and eGFP-MRP4, respectively, to traffic to the apical membranes. These data establish NHERF1 as a major determinant of MRP4 trafficking to apical membranes of mammalian kidney cells.

β‐Oestradiol rescues ΔF508CFTR functional expression in human cystic fibrosis airway CFBE41o− cells through the up‐regulation of NHERF1

CF (cystic fibrosis) is a disease caused by mutations within the CFTR (CF transmembrane conductance regulator) gene. The most common mutation, DeltaF508 (deletion of Phe-508), results in a protein that is defective in folding and trafficking to the cell surface but is functional if properly localized in the plasma membrane. We have recently demonstrated that overexpression of the PDZ protein NHERF1 (Na(+)/H(+)-exchanger regulatory factor 1) in CF airway cells induced both a redistribution of DeltaF508CFTR from the cytoplasm to the apical membrane and the PKA (protein kinase A)-dependent activation of DeltaF508CFTR-dependent chloride secretion. In view of the potential importance of the targeted up-regulation of NHERF1 in a therapeutic context, and since it has been demonstrated that oestrogen treatment increases endogenous NHERF1 expression, we tested the hypothesis that oestrogen treatment can increase NHERF1 expression in a human bronchiolar epithelial CF cell line, CFBE41o(-), with subsequent rescue of apical DeltaF508CFTR chloride transport activity. We found that CFBE41o(-) cells do express ERs (oestrogen receptors) in the nuclear fraction and that beta-oestradiol treatment was able to significantly rescue DeltaF508CFTR-dependent chloride secretion in CFBE41o(-) cell monolayers with a peak between 6 and 12 h of treatment, demonstrating that the DeltaF508CFTR translocated to the apical membrane can function as a cAMP-responsive channel, with a significant increase in chloride secretion noted at 1 nM beta-oestradiol and a maximal effect observed at 10 nM. Importantly, knock-down of NHERF1 expression by transfection with siRNA (small interfering RNA) for NHERF1 inhibited the beta-oestradiol-dependent increase in DeltaF508CFTR protein expression levels and completely prevented the beta-oestradiol-dependent rescue of DeltaF508CFTR transport activity. These results demonstrate that beta-oestradiol-dependent up-regulation of NHERF1 significantly increases DeltaF508CFTR functional expression in CFBE41o(-) cells.

718 Enhanced Cytoskeletal Association of Ezrin By ESPF Is Involved in EPEC-Mediated Suppression of NHE3 Activity

Sodiumhydrogen exchanger 3 (NHE3) plays an important role in sodium and fluid absorption throughout the intestine. Our previous work showed that enteropathogenic Escherichia coli (EPEC) significantly decreases NHE3 activity in human intestinal epithelial Caco-2 cells and PS120 fibroblasts transfected with NHE3. Mutation of the translocated effector protein EspF (∆espF) prevented the change in NHE3 activity. The aim of this study was to determine the mechanism by which EspF reduces NHE3 activity. NHE3 activity was assayed as 22Na+ uptake utilizing NHE inhibitors EIPA and HOE-694. EspF has several effects on intestinal epithelial cells including induction of cell death, alteration of vesicle trafficking via interactions with sorting nexin 9 (SNX9) and enhancing the cytoskeletal association of ezrin. An EspF point mutant that prevents cell death, L16E was used to determine if this phenotype was related to the decrease in NHE3. Complementation of ∆espF with the L16E mutation restored the decrease in NHE3 activity suggesting that cell death is not the cause of reduced NHE3 activity (WT EPEC -35%, ∆espF +3%, L16E -29%, p<0.05, n=15, NHE3 activity as percent change from uninfected). EspF interacts with SNX9 via proline rich repeats (PRR) and alters vesicle trafficking. As NHE3 is regulated by apical recycling, we tested the effect of EspFD3, a strain harboring point mutations in each PRR that is impaired for SNX9 binding. EspF-D3 restored the decrease in NHE3 activity in a ∆espF mutant (WT EPEC -52%, ∆espF 0%, +espF -38%, +espF-D3 -45%, n=15). Thus, interference with vesicle trafficking is not involved in EspF-mediated modulation of NHE3 activity. We published previously that EspF enhances the cytoskeletal association of ezrin. Ezrin regulates NHE3 activity by binding directly to NHE3 or to NHERF1/2. Expression of the scaffolding proteins NHERF1 and NHERF2 in PS120 fibroblasts enhances the NHE3 response to EPEC (NHE3 only -36%, +NHERF1 -56%, +NHERF2 -65%, n=9 p<0.05). To determine if ezrin was involved in EPEC-induced decrease in NHE3 activity, the effect of expressing full-length versus dominant negative ezrin was studied. While Caco-2 cells expressing full-length ezrin exhibited the expected 2-fold decrease in NHE3 activity in response to EPEC infection, expression of dominant negative ezrin prevented this response causing a minor but not significant, increase in NHE3 activity (p<0.01, n=9). Our data suggest that EspF reduces NHE3 activity, at least in part, via its effects on ezrin. These events likely contribute to the diarrhea associated with EPEC infection.

NHERF1: molecular brake on the PI3K pathway in breast cancer

The adaptor protein NHERF1/EBP50 (Na/H exchanger regulatory factor 1/ezrin-radixin-moesin-binding phosphoprotein 50) emerged recently as an important player in breast cancer progression. Consisting of two tandem PDZ domains linked to a carboxyl-terminal ezrin-binding region, NHERF1 assembles macromolecular complexes at the apical membrane of epithelial cells in many epithelial tissues, including the mammary gland. Involved initially in trafficking and regulation of transmembrane ion transporters and G protein-coupled receptors, NHERF1 also couples molecules involved in cell growth, such as the platelet-derived growth factor receptor (PDGFR) and PTEN (phosphatase and tensin homolog deleted on chromosome 10). In the previous issue of Breast Cancer Research, Pan and colleagues show an inhibitory action of NHERF1 on the phosphoinositide-3 kinase (PI3K)/Akt pathway in breast cancer cells via interaction of NHERF1 with PTEN, the physiological antagonist of the PI3K. Additionally, they show that NHERF1 expression confers susceptibility to PDGFR pharmacological inhibition depending on the presence of PTEN tumor suppressor.

Physiological role of NHERF family proteins in the regulation of CFTR

In epithelial cells, salt‐water balance is maintained by the expression and function of CFTR. The NHERF family PDZ proteins have been shown to interact with the c‐terminus of CFTR. To elucidate the functional role in human lung epithelia, we have used Calu‐3 cells derived from the serous cells of submucosal glands, as a model system. We have used RNA interference techniques to generate stable NHERF1 and NHERF2 knockdown (KD) cell lines. In these cells, the expression of NHERF1 and NHERF2 ranged from 10‐40% of Calu‐3NT cells (control cells). In these KD cells, steady‐state CFTR protein level was greatly reduced, whereas its apical localization was unaffected. We also found structural defects in the apical microvilli. In polarized NHERF1‐KD cells, membrane‐permeable cAMP analogs were unable to stimulate CFTR mediated short‐circuit currents at lower concentration. However, at higher concentration of analogs, their response was nearly normal despite reduced CFTR expression. Examinations of β‐adrenergic‐and adenosine‐receptor signaling pathways suggest that NHERF1, but not NHERF2 plays a unique role in organizing the signaling pathway.

Novel regulatory function for NHERF-1 in Npt2a transcription

Several lines of evidence show that sodium/hydrogen exchanger regulatory factor 1 (NHERF-1) regulates the expression and activity of the type IIa sodium-dependent phosphate transporter (Npt2a) in renal proximal tubules. We have previously demonstrated that expression of a COOH-terminal ezrin binding domain-deficient NHERF-1 in opossum kidney (OK) cells decreased expression of Npt2a in apical membranes but did not affect responses to parathyroid hormone. We hypothesized that NHERF-1 regulates apical membrane expression of Npt2a in renal proximal tubule cells. To address this hypothesis, we compared regulation of Npt2a expression and function in NHERF-deficient OK cells (OK-H) and wild-type cells (OK-WT). In OK-H cells, phosphate uptake and expression of Npt2a protein in apical membranes were significantly lower than in OK-WT cells. Transient transfection of green fluorescent protein-tagged Npt2a cDNA into OK-H cells resulted in aberrant localization of an Npt2a fragment to the cytosol but not to the apical membrane. OK-H cells also exhibited a marked decrease in Npt2a mRNA expression. As demonstrated by luciferase assay, Npt2a promoter activity was significantly decreased in OK-H cells compared with that shown in OK-WT cells. Transfection of OK-H cells with human NHERF-1 restored Npt2a expression at both the protein and mRNA levels and regulation by parathyroid hormone. Expression of NHERF-1 constructs with mutations in the PDZ domains or the ezrin binding domain in OK-H cells suggested that the PDZ2 domain is critical for apical translocation of Npt2a and for expression at the mRNA level. Our data demonstrate for the first time that NHERF-1 regulates Npt2a transcription and membrane insertion.

Role of the scaffold protein RACK1 in apical expression of CFTR

Previous studies from this laboratory demonstrated a role for protein kinase C (PKC)epsilon in the regulation of cAMP-dependent cystic fibrosis transmembrane regulator (CFTR) Cl channel function via binding of PKCepsilon to RACK1, a receptor for activated C kinase, and of RACK1 to human Na(+)/H(+) exchanger regulatory factor (NHERF1). In the present study, we investigated the role of RACK1 in regulating CFTR function in a Calu-3 airway epithelial cell line. Confocal microscopy and biotinylation of apical surface proteins demonstrate apical localization of RACK1 independent of actin. Mass spectrometric analysis of NHERF1 revealed copurification of tubulin, which, in in vitro binding assays, selectively binds to NHERF1, but not RACK1, via a PDZ1 domain. In binding and pulldown assays, we show direct binding of a PDZ2 domain to NHERF1, pulldown of endogenous NHERF1 by a PDZ2 domain, and inhibition of NHERF1-tubulin binding by a PDZ1 domain. Downregulation of RACK1 using double-stranded silencing RNA reduced the amount of RACK1 by 77.5% and apical expression of biotinylated CFTR by 87.4%. Expression of CFTR, NHERF1, and actin were not altered by treatment with siRACK1 or by nontargeting control silencing RNA, which, in addition, did not affect RACK1 expression. On the basis of these results, we model a RACK1 proteome consisting of PKCepsilon-RACK1-NHERF1-NHERF1-tubulin with a role in stable expression of CFTR in the apical plasma membrane of epithelial cells.

Down regulation of small intestinal ion transport in PDZK1- (CAP70/NHERF3) deficient mice

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.

Regulation of Expression and Localisation of the Na +/H + Exchanger (NHE) 3 and the NHE Regulatory Factor 2 in Baboon Placental Syncytiotrophoblast by Oestrogen

Our understanding of the regulation of the expression of the sodium hydrogen exchangers (NHE) and their regulatory factors (NHERF), which play important roles in fetal–placental homeostasis, is incomplete. We previously showed that the expression and localisation of NHE3 and NHERF2 in the juxtanuclear compartment of the placental syncytiotrophoblast were markedly decreased between mid and late baboon pregnancy. In the current study, immunocytochemical fluorescence localisation and level of NHE3/NHE1 and NHERF1/NHERF2 proteins were determined in late gestation in baboons untreated or treated throughout the second half of gestation with an aromatase inhibitor CGS 20267 alone (reduced oestrogen levels by >95%) or with oestradiol to determine whether oestrogen regulated antiporter developmental expression. The immunocytochemical expression of NHE3 and NHERF2 in the juxtanuclear compartment was minimal in baboons untreated or treated with CGS 20267 plus oestradiol (i.e. oestrogen-replete) but extensive in oestrogen-suppressed animals. Moreover, the abundant expression of NHERF2 in fetal vascular endothelium of oestrogen-replete baboons was decreased in oestrogen-suppressed animals. In contrast, expression and localisation of NHE1 and NHERF1 in the placental syncytiotrophoblast were not altered by oestrogen deprivation in baboons. Based on our current and previous findings, we propose that oestrogen plays an important role in regulating localisation and expression of components of the NHE system within and consequently development and function of the primate placental syncytiotrophoblast.

The NHERF1 PDZ2 Domain Regulates PKA–RhoA–p38-mediated NHE1 Activation and Invasion in Breast Tumor Cells

Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na(+)/H(+) exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1alpha expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na(+)/H(+) exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling.

Knockdown of NHERF1 Enhances Degradation of Temperature Rescued ΔF508 CFTR from the Cell Surface of Human Airway Cells

ΔF508 CFTR can be functionally restored in the plasma membrane by exposure of the cell to lower temperature. However, restored ΔF508 CFTR has a much shorter half-life than normal. We studied whether NHERF1, which binds to the PDZ motif of CFTR, might be a critical mediator in the turnover of ΔF508 CFTR from the cell surface. We used RNAi to reduce the expression of NHERF1 in human airway epithelial cells. Knockdown of NHERF1 reversibly reduces surface expression of WT-CFTR without altering its total expression. As expected, temperature correction increased mature C band ΔF508 CFTR (rΔF508) but unexpectedly allowed immature B band of rΔF508 to traffic to the cell surface. Both surface and total expression of rΔF508 in NHERF1 knockdown cells were reduced and degradation of surface localized rΔF508 was even faster in NHERF1 knockdown cells. Proteasomal and lysosomal inhibitor treatments led to a significant decrease in the accelerated degradation of surface rΔF508 in NHERF1 knockdown cells. These results indicate that NHERF1 plays a role in the turnover of CFTR at the cell surface, and that rΔF508 CFTR at the cell surface remains highly susceptible to degradation.

Na+–H+ exchanger regulatory factor 1 is a PDZ scaffold for the astroglial glutamate transporter GLAST

Glutamate is a key neurotransmitter and its levels in the synaptic cleft are tightly regulated by reuptake mechanisms that primarily involve transporters in astrocytes. This requires that the glutamate transporters be spatially constrained to effect maximum glutamate transport. GLAST (EAAT1) is the predominant astroglial transporter and contains a class I PDZ-binding consensus (ETKM) in its C-terminus. The epithelial Na(+)/H(+) exchanger regulatory factors NHERF1 and NHERF2 are PDZ proteins that contain two tandem PDZ domains and a C-terminal domain that binds members of the ERM (ezrin-radixin-moesin) family of membrane-cytoskeletal adaptors. NHERF proteins have been extensively characterized in renal epithelia and their expression in brain has recently been reported; however, their function in the brain remains unknown. The aims of the current study were to (1) determine the distribution of NHERF1/2 in the rodent brain and (2) investigate whether GLAST was a physiological ligand for NHERF1/2. Immunohistochemistry revealed that NHERF1 expression was widespread in rat brain (abundant in cerebellum, cerebral cortex, hippocampus, and thalamus) and primarily restricted to astrocytes whereas NHERF2 expression was primarily restricted to endothelial cells of blood vessels and capillaries. Importantly, NHERF1 distribution closely matched that of GLAST and confocal imaging demonstrated co-localization of the two proteins. Co-immunoprecipitation demonstrated that GLAST, NHERF1, and ezrin associate in vivo. In vitro binding assays showed that GLAST bound directly to the PDZ1 domain of NHERF1 via the C-terminal ETKM motif of GLAST. These findings implicate the GLAST-NHERF1 complex in the regulation of glutamate homeostasis in astrocytes.

Adenoviral expression of NHERF-1 in NHERF-1 null mouse renal proximal tubule cells restores Npt2a regulation by low phosphate media and parathyroid hormone

Sodium-dependent phosphate transport in NHERF-1(-/-) proximal tubule cells does not increase when grown in a low phosphate media and is resistant to the normal inhibitory effects of parathyroid hormone (PTH). The current experiments employ adenovirus-mediated gene transfer in primary cultures of mouse proximal tubule cells from NHERF-1 null mice to explore the specific role of NHERF-1 on regulated Npt2a trafficking and sodium-dependent phosphate transport. NHERF-1 null cells have decreased sodium-dependent phosphate transport compared with wild-type cells. Infection of NHERF-1 null cells with adenovirus-GFP-NHERF-1 increased phosphate transport and plasma membrane abundance of Npt2a. Adenovirus-GFP-NHERF-1 infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP demonstrated increased phosphate transport and Npt2a abundance in the plasma membrane when grown in low phosphate (0.1 mM) compared with high phosphate media (1.9 mM). PTH inhibited phosphate transport and decreased Npt2a abundance in the plasma membrane of adenovirus-GFP-NHERF-1-infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP. Interestingly, phosphate transport is inhibited by activation of protein kinase A and protein kinase C in wild-type proximal tubule cells but not in NHERF-1(-/-) cells. Together, these results highlight the requirement for NHERF-1 for physiological control of Npt2a trafficking and suggest that the Npt2a/NHERF-1 complex represents a unique PTH-responsive pool of Npt2a in renal microvilli.

Postnatal developmental expression of the PDZ scaffolds Na+-H+ exchanger regulatory factors 1 and 2 in the rat cochlea

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.

Parathyroid Hormone Regulation of Na+,K+-ATPase Requires the PDZ 1 Domain of Sodium Hydrogen Exchanger Regulatory Factor-1 in Opossum Kidney Cells

It was demonstrated that expression of murine sodium hydrogen exchanger regulatory factor (NHERF-1) lacking the ezrin-binding domain blocks parathyroid hormone (PTH) regulation of Na+,K+-ATPase in opossum kidney (OK) cells. The hypothesis that the NHERF-1 PDZ domains contribute to PTH regulation of Na+,K+-ATPase was tested by comparison of PTH regulation of Na+,K+-ATPase in wild-type OK (OK-WT) cells, NHERF-deficient OKH cells, OK-WT transfected with siRNA for NHERF (NHERF siRNA OK-WT), and OKH cells that were stably transfected with full-length NHERF-1 or constructs with mutated PDZ domains. OKH cells and NHERF siRNA OK-WT showed decreased expression of NHERF-1 but equivalent expression of ezrin and Na+,K+-ATPase alpha1 subunit when compared with OK-WT cells. PTH decreased Na+,K+-ATPase activity and stimulated phosphorylation of the Na+,K+-ATPase alpha1 in OK-WT cells but not in NHERF-deficient cells. Rubidium (86Rb) uptake was equivalent in OK-WT, OKH, and OKH cells that were transfected with all but the double PDZ domain mutants. PTH decreased 86Rb uptake significantly in OK-WT but not in OKH cells. PTH also significantly inhibited 86Rb uptake in OKH cells that were transfected with full-length NHERF-1 or NHERF-1 with mutated PDZ 2 but not in OKH cells that were transfected with mutated PDZ 1. Transfection with NHERF expressing both mutated PDZ domains resulted in diminished basal 86Rb uptake that was not inhibited further by PTH. PTH stimulated protein kinase Calpha activity and alpha1 subunit phosphorylation in OK-WT but not in NHERF-deficient cells. Transfection of OKH cells with NHERF constructs that contained an intact PDZ1 domain restored PTH-stimulated protein kinase Calpha activity and alpha1 subunit phosphorylation. These results demonstrate that NHERF-1 is necessary for PTH-mediated inhibition of Na+,K+-ATPase activity and that the inhibition is mediated through the PDZ1, not PDZ2, domain.