Epithelial Inducible Nitric-oxide Synthase Is an Apical EBP50-binding Protein That Directs Vectorial Nitric Oxide Output

Abstract

Nitric oxide (NO), produced via inducible NO synthase (iNOS), can modulate polarized epithelial processes such as solute transport. Given the high reactivity of NO, we hypothesized that optimal NO regulation of polarized epithelial functions is achieved through compartmentalization of iNOS, allowing local NO delivery to its molecular targets. Here, we show that iNOS localizes to the apical domain of epithelial cells within a submembranous protein complex tightly bound to cortical actin. We further show that iNOS can bind to the apical PDZ protein, EBP50 (ezrin-radixin-moesin-binding phosphoprotein 50), an interaction that is dependent on the last three COOH-terminal amino acids of iNOS, SAL, but requires the presence of additional unknown cellular proteins. Mutation of these three COOH-terminal residues abolishes the iNOS-EBP50 interaction and disrupts the apical association of iNOS in transfected cells, showing that this COOH-terminal motif is essential for the correct localization of iNOS in epithelial cells. Apically localized iNOS directs vectorial NO production at the apical proximal tubule epithelial cell surface. These studies define human epithelial iNOS as an apical EBP50-binding protein and suggest that the physical association of iNOS with EBP50 might allow precise NO modulation of EBP50-associated protein functions. Nitric oxide (NO), produced via inducible NO synthase (iNOS), can modulate polarized epithelial processes such as solute transport. Given the high reactivity of NO, we hypothesized that optimal NO regulation of polarized epithelial functions is achieved through compartmentalization of iNOS, allowing local NO delivery to its molecular targets. Here, we show that iNOS localizes to the apical domain of epithelial cells within a submembranous protein complex tightly bound to cortical actin. We further show that iNOS can bind to the apical PDZ protein, EBP50 (ezrin-radixin-moesin-binding phosphoprotein 50), an interaction that is dependent on the last three COOH-terminal amino acids of iNOS, SAL, but requires the presence of additional unknown cellular proteins. Mutation of these three COOH-terminal residues abolishes the iNOS-EBP50 interaction and disrupts the apical association of iNOS in transfected cells, showing that this COOH-terminal motif is essential for the correct localization of iNOS in epithelial cells. Apically localized iNOS directs vectorial NO production at the apical proximal tubule epithelial cell surface. These studies define human epithelial iNOS as an apical EBP50-binding protein and suggest that the physical association of iNOS with EBP50 might allow precise NO modulation of EBP50-associated protein functions. inducible nitric-oxide synthase cystic fibrosis transmembrane regulator ezrin-radixin-moesin-binding phosphoprotein 50 glutathioneS-transferase high speed pellet high speed supernatant low speed supernatant monoclonal antibody(s) Madin-Darby canine kidney cells Na+/H+ exchanger l-N6-(1-iminoethyl)lysine proximal tubule epithelial cell tetramethylrhodamine isothiocyanate zona occludens-1 Inducible nitric-oxide synthase (iNOS)1 produces high concentrations of the ubiquitous signaling molecule, NO (1Nathan C. FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4158) Google Scholar). iNOS induction has been demonstrated in several epithelial cell types, including those of renal and bronchial origin (2Markewitz B.A. Michael J.R. Kohan D.E. J. Clin. Invest. 1993; 91: 2138-2143Crossref PubMed Scopus (188) Google Scholar, 3Meng Q.H. Polak J.M. Edgar A.J. Chacon M.R. Evans T.J. Gruenert D.C. Bishop A.E. J. Pathol. 2000; 190: 126-132Crossref PubMed Scopus (20) Google Scholar). The role of high NO output from iNOS in epithelial cells is unclear, although there is increasing evidence that epithelial NO exerts its effects on polarized cell functions. For example, NO modulates the activity of the cystic fibrosis transmembrane regulator (CFTR) (4Dong Y.J. Chao A.C. Kouyama K. Hsu Y.P. Bocian R.C. Moss R.B. Gardner P. EMBO J. 1995; 14: 2700-2707Crossref PubMed Scopus (54) Google Scholar) and the apical renal proximal tubule epithelial cell (PTEC) Na+/H+exchanger (NHE3) (5Liang M. Knox F.G. Am. J. Physiol. 2000; 278: R1117-R1124Crossref PubMed Google Scholar). NO is highly diffusible and reactive within cells and with neighboring cells and molecules (6Lancaster J.R.J. Nitric Oxide. 1997; 1: 18-30Crossref PubMed Scopus (400) Google Scholar). We therefore hypothesized that endogenous NO-dependent regulation of apical epithelial functions, such as solute transport, might optimally be achieved through localization of iNOS to the apical cell compartment, allowing local NO delivery to its molecular targets. How might iNOS be targeted to the apical cell compartment? Recent studies have shown that PDZ domain-containing proteins play an important role in trafficking, sorting, and retention of several apical proteins (7Fanning A.S. Anderson J.M. J. Clin. Invest. 1999; 103: 767-772Crossref PubMed Scopus (401) Google Scholar). PDZ domains mediate protein-protein interactions via binding to specific amino acid sequences at the COOH terminus of target proteins (8Songyang Z. Fanning A.S., Fu, C., Xu, J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1222) Google Scholar). One such protein, ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50), contains two PDZ domains that recognize the COOH-terminal sequence (S/T)XL (9Hall R.A. Ostedgaard L.S. Premont R.T. Blitzer J.T. Rahman N. Welsh M.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8496-8501Crossref PubMed Scopus (375) Google Scholar). EBP50 is highly abundant within apical epithelial microvilli (10Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (518) Google Scholar) and retains several apical proteins via PDZ domain-mediated interactions, including NHE3 and CFTR (11Weinman E.J. Minkoff C. Shenolikar S. Am. J. Physiol. 2000; 279: F393-F399Crossref PubMed Google Scholar, 12Short D.B. Trotter K.W. Reczek D. Kreda S.M. Bretscher A. Boucher R.C. Stutts M.J. Milgram S.L. J. Biol. Chem. 1998; 273: 19797-19801Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). EBP50 also exerts regulatory effects on its binding partners; for example, EBP50 confers cyclic AMP-mediated regulation of NHE3 (11Weinman E.J. Minkoff C. Shenolikar S. Am. J. Physiol. 2000; 279: F393-F399Crossref PubMed Google Scholar). Inspection of the last three COOH-terminal residues of human iNOS shows that they contain a potential PDZ domain-binding motif, SAL (13Charles I.G. Palmer R.M. Hickery M.S. Bayliss M.T. Chubb A.P. Hall V.S. Moss D.W. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11419-11423Crossref PubMed Scopus (283) Google Scholar). One possibility may be, therefore, that iNOS could interact with a PDZ domain via its three COOH-terminal amino acids. In this study, we show for the first time that iNOS localizes to the apical domain of polarized epithelial cells in association with the cortical actin cytoskeleton. Further, we show that iNOS can bind to the PDZ protein EBP50 via its three COOH-terminal amino acids. These residues are also found to be essential for the correct apical localization of iNOS within epithelial cells. We demonstrate that iNOS produces NO in a vectorial manner predominantly at the apical cell surface. This work defines epithelial iNOS as a polarized apical protein and provides a mechanism whereby locally delivered NO may influence polarized epithelial cell functions. iNOS, EBP50, E-cadherin, and caveolin-1 monoclonal antibodies were from Transduction Laboratories. The mAb to β1-integrin was from Chemicon International. Zona occludens-1 (ZO-1) polyclonal antibody was from Zymed Laboratories Inc.. EBP50 anti-sera was a gift from Prof. A. Bretscher (Cornell University, Ithaca, NY; described in Ref. 10Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (518) Google Scholar). iNOS polyclonal Ig was made as described (14Evans T.J. Buttery L.D. Carpenter A. Springall D.R. Polak J.M. Cohen J. Proc. Natl. Acad. Sci. 1996; 93: 9553-9558Crossref PubMed Scopus (271) Google Scholar) and also was obtained from Transduction Laboratories. Biotinylated secondary antibodies, fluorescein avidin, and Vectashield® were from Vector Laboratories. Cy3-conjugated anti-rabbit Ig was from Jackson ImmunoResearch Laboratories. Interleukin 1α, tumor necrosis factor-α, and interferon-γ were from Peprotech. pGEX-6P-1 was fromAmersham Biosciences. Unless stated otherwise, all other chemicals were from Sigma-Aldrich. Primary human PTEC cultures were established in serum-free medium as described (15Glynne P.A. Evans T.J. Septic Shock: Methods and Protocols. Humana Press, Totowa, NJ2000: 197-205Google Scholar). Human bronchial epithelial 16HBE14o− cells (a gift from Dr D. Gruenert, University of California, San Francisco), grown in RPMI 1640/10% FCS, were stably transfected with human iNOS cDNA (a gift from Dr. I. Charles, Wellcome Laboratories) cloned into theXbaI sites of the expression vector pCI-neo (Promega) using Perfectlipid 6 (Invitrogen). iNOS-expressing and control cells were cloned by limiting dilution in the presence of G418. iNOS expression in 50% of the cells was achieved after 48 h of incubation with 5 mmn-butyric acid. A similar approach was used to derive stable iNOS transfectants in MDCK II cells (a kind gift from K. Mostov, University of California). For each construct ≥2 independent clones were analyzed with identical results. Cells were polarized by growth on a semipermeable support with separate apical and basolateral chambers (Anapore filters, Nunc, Abingdon, UK) NO was measured as nitrate + nitrite (NOx) using the Griess assay following reduction of nitrate to nitrite (16Archer S. FASEB J. 1993; 7: 349-360Crossref PubMed Scopus (854) Google Scholar). Vectorial NO output was measured by the hemoglobin method (17Hevel J.M. Marletta M.A. Methods Enzymol. 1994; 233: 250-258Crossref PubMed Scopus (424) Google Scholar). iNOS activity was assayed by arginine to citrulline conversion (NOSdetect™, Stratagene). NO production was inhibited by the iNOS inhibitorl-N6-(1-iminoethyl)lysine (l-NIL; Calbiochem). Cells grown on Anopore filters (Nalgene Nunc) were immunostained as described (14Evans T.J. Buttery L.D. Carpenter A. Springall D.R. Polak J.M. Cohen J. Proc. Natl. Acad. Sci. 1996; 93: 9553-9558Crossref PubMed Scopus (271) Google Scholar). iNOS was visualized using the appropriate biotinylated secondary antibody (1 h) and fluorescein avidin (1 h) (both diluted 1:200). ZO-1 and EBP50 were visualized using Cy3-conjugated secondary antibody (1:500). Cells were mounted in Vectashield® and analyzed by confocal microscopy (Bio-Rad MRC 1024 running LaserSharp software). iNOS-expressing cells were quantified as the percentage of the area of the epithelial sheet in a given micrograph using NIH Image software, version 1.62, to measure the area of stained cells with pixel intensity above the threshold level. Apical iNOS staining was quantified by determining the mean fluorescence intensities of apical and cytoplasmic areas of the cell and then subtracting the background value measured over the nucleus. The product of the mean fluorescent intensity and the measured area was used to determine the relative amounts of iNOS in each compartment. All protocols were performed at 4 °C. PTEC were washed three times in PBS, scraped into lysis buffer (50 mm Tris, pH 7.5, 0.3 μm aprotinin, 2 μm leupeptin, 3 μm pepstatin, 1 mm phenylmethylsulfonyl fluoride), and lysed by Dounce homogenization and sonication. After the were at for the low speed supernatant was at for 1 h to a high speed supernatant and pellet analyzed for iNOS by were using and quantified by The was in 1 of lysis buffer and a over a at for h at 4 were from the of the and analyzed by The was in mm in lysis were at for and and at for 1 and and were analyzed by The COOH-terminal residues of human iNOS from were cloned in pGEX-6P-1 after and at for The construct was made by the three COOH-terminal SAL, with were by proteins were produced in and on The COOH terminus of iNOS was to using with the and using iNOS cDNA as The product was and and this was into iNOS in with the The was then cloned into pCI-neo and by sequence The from cells lysed in lysis buffer + was with of protein for 2 h at 4 with proteins were washed times with lysis in and analyzed by A similar approach was used to the binding of EBP50 cDNA (a kind gift of Dr. A. was cloned into the expression vector This construct was transfected into cells using and the protein was from cell h using The protein was as by staining of protein on an are as the The of of from was using We first established optimal for iNOS induction in human of PTEC with 1α, and tumor necrosis for h produced with cells cells this was used in all PTEC NO synthase activity was in cell and after with cells iNOS induction was by of a protein in PTEC by These that NO production from PTEC was In there is iNOS activity in cells. The of iNOS was by using two iNOS antibodies, and identical were obtained with each iNOS staining was in iNOS was in a of PTEC value of iNOS in separate microscopy that iNOS localized to the apical domain and above the ZO-1 the the apical and basolateral domains of a polarized epithelial in these cells that ZO-1 staining is to a area at the cells that to the of the cells by the in In iNOS was found to with the apical EBP50 This of apical staining was in all iNOS cells analyzed in polarized PTEC kidney The of iNOS within quantified by that of iNOS was in the apical compartment. The apical localization of iNOS is also in epithelial cells stably transfected with iNOS expression These were for MDCK cells and the epithelial 16HBE14o− In all iNOS localized to the apical Cells were stained for of epithelial were and 2 and and β1-integrin and of polarized 16HBE14o− cells, areas of cells to two cells In these iNOS was also localized to the apical of the cells Given the of of confocal microscopy we were to iNOS was with the in the apical was localized to an submembranous We therefore a approach to further the association of iNOS with the of from PTEC demonstrated that iNOS is in and cell that of cellular iNOS was from the a similar to that found for the protein EBP50 iNOS activity was in and with the of cellular iNOS The percentage of iNOS in the assayed by is that by confocal this may of iNOS from this on cell lysis and The iNOS activity in the may these that a of cell iNOS is and within a We further analyzed the of iNOS within the by the in lysis buffer and then to by iNOS in similar to the of actin and the of the protein iNOS was from a protein and of were also found in 3 and but were from This of the within the shows that these a into these that iNOS within the is within a but may be in a submembranous protein complex with the cortical cytoskeleton. further the of this we the with a of and We found that high concentrations to were in iNOS 2 a that bound proteins E.J. J. Cell Biol. 1997; 139: PubMed Scopus Google iNOS iNOS was by that is an The protein in the presence of and 6 and but was by + mm and These a interaction with the although as as for proteins such as E.J. J. Cell Biol. 1997; 139: PubMed Scopus Google Scholar). Given the apical localization of iNOS and the that iNOS is with the we the iNOS and cortical actin. microscopy demonstrated that iNOS and with cortical actin and in polarized PTEC in iNOS was into the following actin by that iNOS is with cortical actin via a Given its potential COOH-terminal PDZ binding motif we iNOS might to the apical domain via an interaction with We this using with to the COOH-terminal residues of iNOS a COOH-terminal sequence incubation of with 16HBE14o− cell a and two were in the as shown on the in These were 16HBE14o− were with of the that the to human EBP50 The two molecular were also by the that they might EBP50 In EBP50 was in the of 16HBE14o− cell with using PTEC an identical specific interaction EBP50 and but binding of EBP50 to the COOH-terminal of human iNOS to EBP50, an interaction that is dependent on the three COOH-terminal amino is also that iNOS may interact with other of the EBP50 such as with a similar on has a EBP50 and is found within the proximal tubule cells used in the above J. Reczek D. Bretscher A. J. Cell Biol. PubMed Scopus Google Scholar). shown in iNOS and EBP50 within epithelial cells at the apical of the We to demonstrate an interaction within cells by iNOS and EBP50 from epithelial cells. we were to this from cells. of iNOS in stably transfected cells was found in of the cell and following the for a of iNOS to molecular of these may the to interactions in a cellular the were performed in they a interaction of iNOS with this we EBP50 in human cells. this we were to demonstrate a interaction EBP50 and iNOS a assay that could amounts of This that interaction of iNOS and EBP50 the presence of additional proteins found in cell as used for the the potential interaction of the COOH terminus of iNOS with the PDZ domain-containing protein EBP50 play a role in iNOS to the apical for this possibility we an expression vector for iNOS the COOH-terminal from to that the interaction with EBP50 as shown in the We stable transfectants of MDCK cells with and COOH-terminal and the of iNOS in each cell shown in iNOS localized to the apical of MDCK cells. The COOH-terminal a with of its association with the apical This was from that with iNOS the three COOH-terminal amino acids are essential for correct localization of iNOS in the apical of epithelial cells. with the other a for this be an interaction of iNOS with EBP50 to its apical and association with the cortical actin cytoskeleton. determine iNOS NO locally at the apical we measured apical and NO output from PTEC of NO in these two NO in the apical with output Apical NO μm in the h after with the NO output of μm These show that apical compartmentalization of iNOS in vectorial NO at the apical PTEC surface. We the that iNOS in is localized to the apical and directs of NO in a vectorial at the apical epithelial surface. We demonstrate that iNOS is tightly with cortical actin and has a COOH-terminal domain that can interact with the apical submembranous protein, This domain is essential for the correct localization of iNOS to the apical compartment. These show that iNOS, in with the other J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar) and Chao K. 1995; Full Text PDF PubMed Scopus Google is targeted to a specific cellular that compartmentalization of NO is important in limiting NO production to the of the cell Given the high reactivity of NO, be to the cell to the of high NO in to its of limiting to other cell This the of NO delivery to its such as apical and potential in the One possibility is that NO may modulate the of other proteins with EBP50, such as the apical NHE3 the protein, The show that iNOS is localized in to the apical of and epithelial cells iNOS be produced in the human kidney following with we have been to the of iNOS in as renal is from for example, in iNOS might be to be given the of epithelial cell in we this we suggest that this is a of iNOS within the localization of iNOS to within the to of the of iNOS within epithelial cells shows that is tightly bound to a cell with that suggest association with the actin cytoskeleton. This is by the of iNOS and cortical actin that is by the actin We found a similar association of iNOS and cortical actin within D.W. Evans T.J. PubMed Scopus Google that a similar association in these cells. We show that the COOH terminus of iNOS can mediate an interaction with the PDZ protein EBP50 This is an important that has a of binding its interaction with is to the actin (10Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (518) Google Scholar). In to the apical NHE3 S. M. D. S. E.J. M. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google the CFTR D.B. Trotter K.W. Reczek D. Kreda S.M. Bretscher A. Boucher R.C. Stutts M.J. Milgram S.L. J. Biol. Chem. 1998; 273: 19797-19801Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, S. M. 1998; PubMed Scopus Google the protein Kreda S.M. Boucher R.C. M. Stutts M.J. Milgram S.L. J. Cell Biol. 1999; PubMed Scopus Google the R.A. Premont R.T. Blitzer J.T. A. L.S. Shenolikar S. 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Full Text Full Text PDF PubMed Scopus Google Scholar). In the possibility for interactions by EBP50 is and the relative of these interactions may on the presence of other proteins bound within the The for the three COOH-terminal amino acids of iNOS for apical localization to the apical domain that the interaction of iNOS with EBP50 is important in A of might for this polarized on the iNOS iNOS may be to apical and basolateral but is in the apical by binding to the COOH-terminal domain may interact with a PDZ protein within the to to the apical compartment. This might be EBP50, PDZ domain-containing protein such as is localized within the and has been shown to bind to CFTR and modulate its the COOH-terminal domain may be a to iNOS to the apical compartment. is to these We have that in cells, iNOS is localized within an within the In cells this is into the A. and J. that through the could determine localization of of the three amino acids of iNOS to the localization of iNOS within this although the precise effects on localization have been iNOS is also localized to the of in human The molecular mechanism in the apical of iNOS within epithelial cells other the that is dependent on the COOH-terminal three amino acids of In we have shown that human epithelial iNOS localizes to the apical domain of polarized cells, tightly bound to cortical actin. Further, we show that iNOS to the apical PDZ protein EBP50 via its three COOH-terminal amino Apical localization of iNOS within cells requires the three COOH-terminal amino showing the of this interaction in iNOS directs vectorial NO production at the apical cell surface. This work has important for the of for NO in the regulation of apical epithelial cell functions. The precise of the iNOS-EBP50 interaction and its for correct iNOS localization and within the apical of polarized epithelial cells are to be and be the of