Ayhan Cinar

The Role of the NHERF Family of PDZ Scaffolding Proteins in the Regulation of Salt and Water Transport

The four members of the NHERF (Na+/H+ exchanger regulatory factor) family of PDZ adapter proteins bind to a variety of membrane transporters and receptors and modulate membrane expression, mobility, interaction with other proteins, and the formation of signaling complexes. All four family members are expressed in the intestine. The CFTR (cystic fibrosis transmembrane regulator) anion channel and the Na+/H+ exchanger NHE3 (Na/H exchanger‐ isoform 3) are two prominent binding partners to this PDZ‐adapter family, which are also known key players in the regulation of intestinal electrolyte and fluid transport. Experiments in heterologous expression systems have provided a number of mechanistic models how NHERF protein interactions can affect the function of their targets at the molecular level. Recently, NHERF1, 2, and 3 knockout mice have become available, and this review summarizes the reports on electrolyte and fluid transport regulation in the native intestine of these mice.

NHE3 inhibition by cAMP and Ca2+ is abolished in PDZ-domain protein PDZK1-deficient murine enterocytes.

The PDZ‐binding protein PDZK1 (NHERF3/CAP70/PDZ‐dc‐1) in vitro binds to NHE3, but its role in the regulation of NHE3 activity in native enterocytes is unknown. This study was undertaken to understand the physiological role of PDZK1 in regulating NHE3 activity in native murine colonic enterocytes. NHE3 transport rates were assessed fluorometrically in BCECF‐loaded colonic crypts in the NHE3‐expressing cryptal openings by measuring acid‐activated, Na+‐dependent, Hoe 642‐insensitive proton efflux rates. NHE3 mRNA expression levels and NHE3 total enterocyte and brush border membrane (BBM) protein abundance were determined by quantitative PCR and Western analysis and immunohistochemistry. In pdzk1−/− colonic surface cells, acid‐activated NHE3 transport rates were strongly reduced, and the inhibitory effect of forskolin and ionomcyin was virtually abolished. Hyperosmolarity, on the other hand, still had an inhibitory effect. In addition, the NHE3‐selective inhibitor S1611 inhibited acid‐activated NHE3 activity in pdzk1−/− and +/+ mice, suggesting that functional NHE3 is present in pdzk1‐deficient colonocytes. NHE1 and NHE2 activity was not altered in pdzk1−/− colonic crypts. Immunohistochemistry revealed apical NHE3 staining in pdzk1−/− and +/+ proximal colon, and Western blot analysis revealed no difference in NHE3 abundance in colonic enterocyte homogenate as well as brush border membrane. Lack of the PDZ‐adaptor protein PDZK1 in murine proximal colonic enterocytes does not influence NHE3 abundance or targeting to the apical membrane, but abolishes NHE3 regulation by cAMPergic and Ca2+ ‐dependent pathways. It leaves NHE3 inhibition by hyperosmolarity intact, suggesting an important and selective role for PDZK1 in the agonist‐mediated regulation of intestinal NHE3 activity.

Defective jejunal and colonic salt absorption and alteredNa +/H+ exchanger 3 (NHE3) activity in NHE regulatory factor 1 (NHERF1) adaptor protein-deficient mice

We investigated the role of the Na+/H+ exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na+ absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.

Preserved Na(+)/H(+) exchanger isoform 3 expression and localization, but decreased NHE3 function indicate regulatory sodium transport defect in ulcerative colitis.

Background: A major causative factor of diarrhea in ulcerative colitis (UC) patients is the loss of Na+ absorptive capacity of the inflamed colonic mucosa. Potential contributing mechanisms include reduced driving force for active transport, and impaired expression, mislocalization, or defective transport function of Na+ absorptive proteins. We therefore studied the expression, brush border membrane (BBM) localization, and transport capacity of the major intestinal Na+ absorptive protein, the Na+/H+ exchanger isoform 3 (NHE3) in biopsies from UC patients. Methods: In UC and control biopsies, inflammation was graded histologically, NHE3, tumor necrosis factor alpha (TNF‐&agr;), villin, as well as other housekeeping genes were analyzed by quantitative real‐time polymerase chain reaction (PCR), BBM localization of NHE3 determined by immunohistochemistry, and confocal microscopy. Na+ absorptive capacity was assessed by 22Na+ isotope fluxes and NHE3 transport activity measured microfluorometrically in BCECF‐loaded surface colonocytes within isolated crypts. Results: In mildly, moderately, and severely inflamed sigmoid colon of UC patients, neither NHE3 mRNA expression nor the abundance of NHE3 in the BBM was significantly altered compared to other structural components of the BBM. However, Na+ absorption was strongly reduced by ≈80% and acid‐activated NHE3 transport activity was significantly decreased in the surface cells of sigmoid colonic crypts even in moderately inflamed mucosa. Conclusions: In the colonic mucosa of patients with active UC, NHE3 transport capacity was found significantly decreased despite correct NHE3 location and abundance in the brush border, independent of current treatment. These findings suggest functional NHE3 transport as a novel factor for inflammatory diarrhea in UC patients. (Inflamm Bowel Dis 2010)

Molecular Mechanisms of Disturbed Electrolyte Transport in Intestinal Inflammation

Abstract:  Diarrhea is the hallmark of both ulcerative colitis (UC) and Crohns disease. Loss of resorptive area, destruction of epithelial cells, leaky tight junctions, and release of inflammatory mediators and products from immune cells that stimulate fluid secretion all have been implicated in the pathogenesis of inflammatory diarrhea. Very early studies in patients, however, have pinpointed the overwhelming transport abnormality in inflamed intestinal mucosa: a virtually complete loss of sodium resorptive capacity. Recently, tools have become available to study the molecular basis of disturbances in the major electrolyte transport systems during intestinal inflammation. This review gives a brief overview of the historical development of research related to electrolyte transport in inflammatory bowel disorders, focusing on the studies performed in humans, and highlights recent understanding of the molecular mechanisms that may help explain the origin of diarrhea in intestinal inflammation.

Loss of PDZ‐adaptor protein NHERF2 affects membrane localization and cGMP‐ and [Ca2+]‐ but not cAMP‐dependent regulation of Na+/H+ exchanger 3 in murine intestine

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.

Knockout mouse models for intestinal electrolyte transporters and regulatory PDZ adaptors: new insights into cystic fibrosis, secretory diarrhoea and fructose-induced hypertension.

Knockout mouse models have provided key insights into the physiological significance of many intestinal electrolyte transporters. This review has selected three examples to highlight the importance of knockout mouse technology in unravelling complex regulatory relationships important for the understanding of human diseases. Genetic ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) has created one of the most useful mouse models for understanding intestinal transport. Recent work has provided an understanding of the key role of the CFTR anion channel in the regulation of HCO3− secretion, and the important consequences that a defect in HCO3− output may have on the viscoelastic properties of mucus, on lipid absorption and on male and female reproductive function. The regulation of CFTR activity, and also that of the intestinal salt absorptive transporter NHE3, occurs via the formation of PSD95‐Drosophila homologue Discs‐large‐tight junction protein ZO‐1 (PDZ) adaptor protein‐mediated multiprotein complexes. The recent generation of knockout mice for three members of the sodium‐hydrogen regulatory factor (NHERF) family of PDZ adaptor proteins, namely NHERF1 (EBP50), NHERF2 (E3KARP) and NHERF3 (PDZK1), has helped to explain why NHERF1 is essential for both normal and mutant CFTR function. In addition, they have provided new insight into the molecular mechanisms of secretory diarrhoeas. Genetic ablation of members of the recently discovered Slc26 anion transporter gene family not only reproduced the phenotype of the genetic diseases that led to the discovery of the gene family, but also resulted in new insights into complex human diseases such as secretory diarrhoea, fructose‐induced hypertension and urolithiasis.

Evidence for a causal link between adaptor protein PDZK1 downregulation and Na + /H + exchanger NHE3 dysfunction in human and murine colitis

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.

Essential role of the electroneutral Na+–HCO3− cotransporter NBCn1 in murine duodenal acid–base balance and colonic mucus layer build-up in vivo

•  In the upper intestinal tract, luminal acidity due to intermittent release of gastric juice needs to be counteracted by basolateral HCO3− import. In the lower gastrointestinal tract, the build‐up of a thick mucus layer is a major defence mechanism against pathogens, and HCO3− is of the utmost importance for this process. The pathways for HCO3− transport that play a role in these mucosal defence strategies are, however, unknown. •  We recently identified the electroneutral Na+–HCO3− cotransporter NBCn1 as a major regulator of intracellular pH in duodenal villous enterocytes. The present study shows that the murine duodenocytes, whose intracellular pH was monitored by in vivo two‐photon confocal microscopy in anaesthetized NBCn1 knock‐out mice, are unable to recover rapidly from intracellular acidification imposed by a short pulse of low‐pH solution in the duodenal lumen. Likewise, they are not able to respond to contact of the surface with low pH by a protective HCO3− secretory response. •  The cotransporter NBCn1 is also expressed in the basolateral membrane of colonic crypt cells, many of which stain positive for mucin granules. We found only a minor role for NBCn1 in colonic epithelial HCO3− secretion, but the build‐up of a mucus layer, measured in the exteriorized colon of anaesthetized mice by in vivo microscopy, was significantly delayed in the absence of NBCn1 expression. •  Therefore, NBCn1 plays major but different roles in mucosal protective functions in the upper and lower intestine.

T1841 Differential Effect of NHE3 Kinase a Regulatory Protein (E3karp/NHERF2) Knockout on cAMP-, cGMP-, or [Ca2+]-Induced Inhibition of Na+/H+ Exchanger 3 (NHE3) Activity Along the Murine Intestinal Tract

G A A b st ra ct s of calcitonin on ion transport in IECs. Human colonic T84 cells grown on Transwell inserts were utilized as an in-vitro model of IECs. Chloride secretion was assessed by the measurement of short circuit current (Isc) across T84 monolayers mounted in Ussing chambers. We first examined the expression of CTR in IECs. Real time QRT-PCR and western blot analysis demonstrated the expression of CTR in T84 cells. Exposure of T84 cells to calcitonin from the basolateral but not apical side significantly increased Isc (a change of 287 ± 48 μA/cm2 from the baseline). Stimulation of Isc by calcitonin was dose-dependent (1-100 nM) and was completely blocked by the CTR antagonist, CT8-32. In addition, the increase in Isc was blocked by replacing chloride in the bath solutions with equal amount of gluconate (95 ± 6% reduction, P < 0.01) and was significantly inhibited by 10 μM of the specific CFTR inhibitor, CFTR127inh, (88 ± 9% reduction, P < 0.01) indicating the involvement of CFTR chloride channel in CT-induced effects. To further investigate the signaling pathways involved, we examined calcitonin-induced Isc in the presence of the chelator of [Cai], BAPTA-AM and PKA inhibitor, H89. Preincubation with 20 μMof BAPTAAM or 10μM H89 for 45 minutes significantly reduced calcitonin-induced Isc indicating the involvement of both Ca++and PKA-dependent pathways (58±13% reduction, P < 0.01 and 55±7%, P < 0.01, respectively). Simultaneous inhibition of both Ca++ and PKA pathways had an additive effect on the CT-induced stimulation of Isc (78 ± 8% reduction, P < 0.01). In conclusion, calcitonin-induced short circuit current appears to be mediated by CTR via direct activation of the CFTR channel and is Ca++ and PKA-dependent. This novel study showing the expression of calcitonin receptor in intestinal epithelial cells provides further insights into the molecular mechanisms underlying calcitonin-induced diarrhea. (Supported by NIDDK and Dept of Veteran Affairs).