Role of Na-K-ATPase in the assembly of tight junctions - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:45 |只看该作者 |倒序浏览 |打印

作者：Ayyappan K. Rajasekaran and Sigrid A. Rajasekaran作者单位：Department of Pathology and Laboratory Medicine, David Geffen School ofMedicine at UCLA, University of California, Los Angeles, California90095




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      【摘要】
   Na-K-ATPase, also known as the sodium pump, is a crucial enzyme thatregulates intracellular sodium homeostasis in mammalian cells. In epithelialcells Na-K-ATPase function is also involved in the formation of tightjunctions through RhoA GTPase and stress fibers. In this review, a newtwo-step model for the assembly of tight junctions is proposed: step1, an E-cadherin-dependent formation of partial tight junction strandsand of the circumferential actin ring; and step 2, active actinpolymerization-dependent tethering of tight junction strands to formfunctional tight junctions, an event requiring normal function of Na-K-ATPase in epithelial cells. A new role for stress fibers in the assembly of tightjunctions is proposed. Also, implications of Na-K-ATPase function on tightjunction assembly in diseases such as cancer, ischemia, hypomagnesemia, andpolycystic kidney disease are discussed.   f4 L' y% o* `4 J
      【关键词】 Ecadherin stress fibers ischemia( W2 w" v- i4 I+ F4 d9 O4 r
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THE VECTORIAL TRANSPORT FUNCTION (directional transport of molecules across an epithelial cell layer) of epithelial cells largely dependson the transepithelial flow of sodium ions (Na ). Na enters the cell down its electrochemical gradient through channels,exchangers, and cotransporters localized to the apical plasma membrane and ispumped out of the cell by Na-K-ATPase localized to the basolateral plasmamembrane. Na-K-ATPase catalyzes an ATP-dependent transport of threeNa ions out and two K ions into the cell per pumpcycle, thereby generating the transmembrane Na gradient across theplasma membrane that is crucial to regulate the vectorial transport functionof epithelial cells.0 p( I# d4 k9 w) |! n, X( i+ c
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Na-K-ATPase is a heterodimer, consisting of an -and -subunit (reviewed in Ref. 56 ), butrecent studies have demonstrated the presence of an additional -subunit( 25, 67 ). The -subunit ( 112 kDa) is the catalytic subunit whereas both the -subunit (50-60kDa) and the -subunit ( 7 kDa) have a modulatory role inNa-K-ATPase activity ( 4, 31, 64, 96, 97 ). Of the four -subunit and the three -subunit isoforms known, 1 and 1 are expressed in most epithelialtissues (reviewed in Refs. 56, 66, 69, and 94 ). In addition to the roleof Na-K-ATPase in transepithelial transport, recent studies have demonstrated a role for Na-K-ATPase function in the assembly of junctional complexes inepithelial cells ( 83 ). Thisshort review will focus on insights into how Na-K-ATPase, an ion pump, isinvolved in the assembly of tight junctions.

STRUCTURE AND MOLECULAR COMPOSITION OF TIGHT JUNCTIONS

Tight junctions (zonula occludens) form a continuous belt at the boundarybetween the apical and lateral plasma membrane domains of neighboringepithelial cells ( Fig.1 A ) and are structurally characterized by the closeapposition of contiguous plasma membranes( 23 ). They selectivelyregulate the passage of molecules across the paracellular space( Fig. 1 B ) (gate function) ( 21 ) and passivelyseparate molecules into the apical and basolateral plasma membrane domains(fence function) ( 59 ). A tightjunction is crucial in maintaining the polarized phenotype and the vectorialtransport functions of epithelial cells. The current knowledge of tightjunctions is consistent with a view that tight junctions are specializedmembrane microdomains that might function as a molecular platform involved incell signaling, vesicle protein docking, actin organization, and cell polarityin epithelial cells (reviewed in Refs. 68, 98, 99, and 109 ).

Fig. 1. Schematic diagram of tight junctions in epithelial cells. A :localization of tight junction at the apicolateral side of polarizedepithelial cells. B : organization of tight junction strands, eachstrand consisting of tight junction particles (beads). C : enlargedview of a tight junction particle showing membrane proteins connected to thecytoplasmic plaque proteins and actin cytoskeleton. For simplicity, onlyzonula occludens (ZO)-1 and ZO-2 are shown. -Ct, -catenin.7 ~4 h! u8 W, a

In freeze-fracture electron micrographs, tight junctions appear asanastomosing intramembranous particle strands localized at the apicolateralside of polarized epithelial cells. The contacts between the tight junctionstrands of adjacent cells are in such close apposition as to effectivelyeliminate any intercellular space. These areas of intimate contact can be visualized as membrane contact points (kisses) in transmission electronmicrographs ( Fig. 1 B ).Each particle of the tight junction strand is composed of transmembraneproteins and cytoplasmic plaque proteins connected to the actin cytoskeleton( Fig. 1 C ). Occludins,claudins, and the junctional adhesion molecule (JAM) are the threetransmembrane proteins localized to the tight junctions. Occludin was thefirst tight junction transmembrane protein identified( 28 ) but appears to beunnecessary for the formation of tight junction strands( 86 ). The JAM, a member of theimmunoglobulin superfamily, appears to have roles in cell adhesion andextravasation of immune cells across tight junctions( 7 ). Recently, claudins havebeen identified as membrane proteins localized to tight junctions and havebeen shown to be involved in the formation of tight junction strands ( 26, 30 ). Until now, 24 members ofthe claudin family have been described in various epithelial cells( 98 ). Identification ofoccludin and claudins has tremendously increased our understanding of thestructure and barrier function of tight junctions. Both occludin and claudinhave four transmembrane domains and are involved in creating the paracellularbarrier ( 2, 6, 18, 27, 63, 101, 105 ). The intracellular COOHterminus of both occludin ( 22, 29 ) and claudin( 43 ) associates withcytoplasmic plaque proteins such as zonula occludens-1 (ZO-1)( 92 ), ZO-2 ( 46 ), and ZO-3( 40 ). ZO-1 and ZO-2 associatewith the actin binding proteins -catenin( 44, 45, 80 ) and spectrin( 61 ) ( Fig. 1 C ) and link thetight junction plaques to the actin cytoskeleton. Additional plaque proteinsidentified include cingulin( 16 ), symplekin( 49 ), AF-6( 106 ), and 7H6( 110 ). Proteins involved insignal transduction (c-src and c-yes), proteins involved in membrane traffic(VAP-33, Rab3b, Rab13, Rab 8, Sec6, and Sec8), and cell polarity-relatedproteins (Par3 and Par6) are present in the vicinity of tight junctions (reviewed in Refs. 68, 98, and 109 ). The structure, protein composition, and regulation of paracellular permeability of tight junctionshave been recently described in further detail in excellent reviews( 12, 58, 68, 75, 91, 98, 99 ).
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N A -K-ATP ASE ENZYME ACTIVITY IS REQUIRED FORTIGHT JUNCTION FORMATION
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The formation of tight junctions is a critical event in the biogenesis ofpolarized epithelial cells during early vertebrate development (reviewed inRef. 24 ), during tubular andductal development in epithelial tissues, and during recovery from tissuedamage after ischemic or toxic injury. To understand the molecular events thattake place during the formation of junction complexes in epithelial cells,Madin-Darby canine kidney (MDCK) cells have been extensively used (reviewed in Refs. 12 and 20 ). ACa 2 -switch assay( 32 ) is a widely utilized method for investigating the mechanisms involved in the formation of tightjunctions. In this approach, single-cell suspensions are allowed to attach tothe substratum in a normal-Ca 2 -containing medium for 30 min. During this time, the cells are still round and do not establishpolarity ( Fig. 2 ). The attachedcells are then transferred to a low-Ca 2 medium (& H9 d/ b8 R6 e
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Fig. 2. Illustration of the calcium-switch assay used to study the role ofNa-K-ATPase in the assembly of tight junctions.7 k2 M& F* p5 A1 {8 |( s2 ]' L
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With the use of these approaches, a recent study demonstrated thatNa-K-ATPase activity is necessary for the formation of tight junctions anddevelopment of polarity in MDCK cells ( 83 ). Inhibition of Na-KATPaseactivity by ouabain (a specific inhibitor) or by K depletion[inhibition due to the lack of the steep K gradient necessary forpump function ( 10, 77, 78 )] in MDCK cells subjectedto a Ca 2 switch prevented the formation of tightjunctions and the development of epithelial polarity. Tight junctions formedrapidly, and polarity was established after reactivation of Na-KATPase pumpfunction by restoring the initial K concentration (K repletion), indicating that the effects of Na-K-ATPase inhibition werereversible. Inhibition of tight junction formation correlated with increased intracellular Na concentration levels([Na ] i ) generated by the inhibition of Na-K-ATPase, andthe Na ionophore gramicidin, which increases[Na ] i, mimicked the effects of Na-K-ATPase inhibitionon tight junction formation and epithelial polarity. Moreover, treatment ofcells with ouabain in Na -free medium did not affect tight junctionformation. Because Na-KATPase function controls a variety of ions andmetabolite transport systems, inhibition of this enzyme might induce multiplebiochemical changes in cells, including altered Ca 2 signaling, cell volume, cell pH, and membrane potential. However, this studydemonstrated that Na-K-ATPase enzyme activity is involved in the formation oftight junctions in epithelial cells. It is well established that cell-cell andcell-substratum contacts are involved in the establishment of tight junctionsand polarity in epithelial cells (reviewed in Ref. 108 ). Lack of tight junctionassembly and polarity in Na-K-ATPase-inhibited cells suggests that the intracellular ionic gradient maintained by Na-K-ATPase is also involved in theassembly of tight junctions and generation of polarity in epithelialcells.7 `$ j! m+ [: W& g. u

SYNERGISM BETWEEN N A -K-ATP ASE AND E-CADHERIN INTHE ASSEMBLY OF TIGHT JUNCTIONS
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The cell-cell contact mediated by the cell adhesion molecule E-cadherin hasbeen implicated in the formation of tight junctions in MDCK cells. E-cadherinis a single transmembrane protein localized to the basolateral plasma membranein polarized epithelial cells. The extracellular domain of E-cadherin containsfive IgG-like repeats that mediate cell-cell contact between epithelial cellsby homophilic interaction in a Ca 2 -dependent manner (reviewed in Ref. 95 ). Thecytoplasmic tail of E-cadherin associates with -, -, and -catenins ( 76 ) andp120 ctn ( 89 ). -Catenin either directly or through -actinin links theE-cadherin complex to the actin cytoskeleton, which is crucial for the celladhesion function of E-cadherin( 51, 85 ).

The function of E-cadherin seems to be necessary for the translocation oftight junction proteins to the plasma membrane and for the formation of tightjunction complexes. Inhibition of E-cadherin's cell-cell adhesion function byextracellular domain-specific antibodies prevented the assembly of tightjunctions ( 37 ). In cellsmaintained in a low-Ca 2 medium, ZO-1 is localized inthe cytoplasm. On transfer of the cells to a normal-Ca 2 medium, ZO-1 rapidly translocated to the plasma membrane in control cells,whereas in the presence of anti-E-cadherin extracellular domain antibody thistranslocation was drastically reduced ( 37 ).8 O; N+ _; }8 [! b0 J
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In contrast, in Na-K-ATPase-inhibited cells post-Ca 2 switch, ZO-1 was localized to the plasma membrane but failed to show acontinuous staining pattern normally seen in MDCK cells ( 83 ). This suggests thatNa-K-ATPase inhibition does not affect E-cadherin function. Moreover, adherensjunctions, the formation of which requires functional E-cadherin( 107 ), were present inouabain-treated and K -depleted cells( 83 ). Therefore, plasmamembrane localization of tight junction proteins such as ZO-1 and occludinseems to be due to the presence of functional E-cadherin present inNa-K-ATPase-inhibited cells, but the discontinuous ZO-1 staining patternsuggested that Na-K-ATPase function is necessary for formation of thecontinuous ZO-1 staining pattern in cells with established tight junctions. Consistent with this view, another study showed that in Moloney sarcomavirus-transformed MDCK cells (MSV-MDCK), a cell line with highly reducedE-cadherin and Na-K-ATPase -subunit levels, ectopic expression ofE-cadherin resulted in the localization of ZO-1 to the plasma membrane yetwith a discontinuous staining pattern( 80, 84 ). Only ectopic expressionof E-cadherin combined with restored Na-K-ATPase function (through ectopicexpression of Na-K-ATPase -subunit) resulted in a continuous ZO-1staining pattern and the formation of functional tight junctions,substantiating a hypothesis for a synergistic function of E-cadherin andNa-K-ATPase in the formation of tight junctions in epithelial cells. At thispoint, however, we cannot exclude the possibility that the -subunit ofNa-K-ATPase itself might play a role in the synergistic function ofNa-K-ATPase and E-cadherin in establishing tight junctions and polarity inepithelial cells.( ?: `  P1 g8 G# E! c9 z0 U3 b
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REGULATION OF TIGHT JUNCTION FORMATION BYN A -K-ATP ASE THROUGH MAPK

The MAPK pathway has been implicated in tight junction assembly in variouscell lines. Chen et al. ( 15 )have shown that downregulation of MAPK signaling in ras-transformed MDCK cellsrestored epithelial morphology and tight junctions. In pig thyrocytes,activation of MAPK by EGF and transforming growth factor (TGF)- 1resulted in the loss of tight junctions, and the inhibition of MAPK activation by MEK inhibitor prevented this tight junction loss( 35 ). In salivary glandepithelial cells, activation of MAPK by constitutive expression of Rafresulted in the reduction of occludin levels and disruption of tight junctions( 55 ). In endothelial cells, H 2 O 2 -mediated activation of ERK1/ERK2 leads to thedisruption of endothelial tight junctions( 50 ). In these cell lines, low MAPK activity seems to be associated with formation of tight junctions andepithelial polarization, whereas increased MAPK activity leads to thedisruption of tight junctions. Interestingly, inhibition of Na-K-ATPaseactivity by ouabain activated MAPK in cardiac myocytes and other cell types( 38 ) as well as in polarizedepithelial cells (Rajasekaran SA, Espineda C, and Rajasekaran AK and Landon Iand Rajasekaran AK, unpublished observations). It is tempting to speculatethat increased MAPK activity induced by Na-K-ATPase inhibition has aninhibitory role on the assembly of functional tight junctions. Signaling pathways induced by the inhibition of Na-KATPase function and their impact ontight junction assembly will be an important area to pursue in the future.

ROLE OF STRESS FIBERS IN THE ASSEMBLY OF TIGHT JUNCTIONS

Several earlier studies showed that tight junction structure andpermeability are regulated by the perijunctional actomyosin ring (reviewed inRefs. 3, 58, and 99 ). This ring is located atthe apical pole of polarized epithelial cells and can be visualized by lightmicroscopy as a circumferential actin ring that colocalizes with tightjunction and adherens junction proteins. Earlier studies showed thatdisruption of tight junction structure and permeability correlated withdisruption of the circumferential actin ring( 3, 58, 99 ). However, inNa-K-ATPase-inhibited MDCK cells that did not form tight junctions, noapparent change in the circumferential actin ring was detected at the light microscopic level ( 83 ).Although less prominent, it has also been shown that stress fibers projectingfrom the perijunctional actin ring interface at the cytoplasmic surface oftight junction membrane contact points( 41, 57 ).
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A role for RhoA GTPase (a small GTP-binding protein) involved in theformation of stress fibers( 39, 47, 100 ) has been demonstrated tobe involved in the regulation of tight junction structure function( 42, 48, 74 ). Interestingly, theimpediment of tight junction assembly in Na-K-ATPase-inhibited MDCK cells wasaccompanied by a drastic reduction in stress fibers and correlated withdiminished RhoA activity ( 83 ).Exogenous overexpression of wild-type RhoA GTPase bypassed the inhibitoryeffect of Na-K-ATPase on tight junction formation, indicating that RhoA GTPaseis an essential component downstream of Na-K-ATPase function linkingNa-K-ATPase to the formation of functional tight junctions( 83 ). A similar effect onstress fibers, RhoA GTPase, and tight junction assembly was found in cellstreated with gramicidin, a Na ionophore that, like inhibition ofNa-K-ATPase, increases  i. This indicated that intracellularNa homeostasis regulated by Na-K-ATPase function is involved inthe formation of stress fibers and regulation of RhoA GTPase in MDCK cells. Inpolarized monolayers of retinal pigment epithelial cells, inhibition ofNa-KATPase resulted in an increase in tight junction permeability. Thisincreased permeability correlated with decreased tight junction membranecontact points and reduced actin stress fibers without an apparent change inthe circumferential actin ring( 82 ). Therefore, it seemslikely that both the perijunctional actin ring and stress fibers are involvedin the assembly and function of tight junctions. The perijunctional actin ring might regulate paracellular permeability and provide stability to tightjunctions, whereas stress fibers might be involved in more dynamic functionsrelated to the assembly and function of tight junctions. These dynamicfunctions during tight junction assembly may include proper molecularalignment of tight junction proteins at the tight junction region andtethering of tight junction strands to establish functional tight junctions(see model below). In cells with established tight junctions, the stressfibers might have a role in maintaining the tight junction membrane contactpoints through their association with tight junction membrane and cytoplasmicproteins. Reduced stress fibers associated with minimal change in thecircumferential actin ring in Na-K-ATPase-inhibited cells highlighted for the first time that stress fibers are also involved in the regulation of tightjunction assembly and function.

How Na-K-ATPase function is involved in the regulation of tight junctionassembly through RhoA GTPase activity and stress fiber formation in epithelialcells is not currently known. Rho function is modulated by a set of regulatoryproteins. Rho is activated through GDP-GTP exchange, which is promoted byguanine nucleotide exchange factors and is inactivated throughGTPase-activating proteins (reviewed in Refs. 39 and 100 ). Stabilization of theinactive GDP-bound form of Rho is mediated by Rho guanine nucleotidedissociation inhibitors ( 39, 100 ). It is possible thatinhibition of Na-K-ATPase function may either inhibit the function of guaninenucleotide exchange factors, resulting in the accumulation of the inactiveform of Rho (Rho-GDP), or promote the functions of GTPase-activating proteinsor Rho guanine nucleotide dissociation inhibitors, affecting the activation ofRho. Na-K-ATPase-mediated signaling mechanisms involved in the regulation ofRhoA GTPase and actin assembly will be an important area of future researchthat will provide more insights into how ion homeostasis might regulate therole of stress fibers in the assembly and function of tight junctions.

TWO-STEP MODEL FOR THE ASSEMBLY OF TIGHT JUNCTIONS
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From these recent observations on the Na-K-ATPase function in tightjunction assembly, we propose a two-step model for the formation of tightjunctions in epithelial cells ( Fig.3 ). Step 1 is dependent on the cell-cell adhesionfunction of E-cadherin. On E-cadherin-mediated cell-cell interactions, tightjunction proteins are translocated from the cytoplasm to the plasma membrane and circumferential actin ring and the adherens junctions are established( Fig. 3, A and C ). The translocation of ZO-1 might be due to itsassociation with catenins that, in turn, are associated withE-cadherin-containing transport vesicles( 80 ). Inhibition ofE-cadherin-mediated cell-cell adhesion function by extracellulardomain-specific anti-E-cadherin antibodies inhibited the translocation of ZO-1to the plasma membrane as well as prevented the formation of theperijunctional actin ring( 37 ). Also, during thisinitial step of tight junction assembly, ZO-1 association with the actincytoskeleton as well as with occludin and claudin might take place to form thediscontinuous tight junction strands, as seen in Na-K-ATPase-inhibited cells. Formation of discontinuous tight junction strands after the Ca 2 switch has been described earlier( 32 ). Step 2 of tight junction assembly is dependent on active actin polymerization probablyregulated by RhoA GTPase. Active polymerization of actin filaments associatedwith ZO-1, ZO-2, ZO-3, or occludin ( 104 ) might provide thepropulsive force to mobilize discontinuous tight junction strands in the planeof the membrane and facilitate tethering of tight junction strands at theapicolateral region to establish functional tight junctions( Fig. 3, B and D ). Thus this model predicts that both stress fibers andcircumferential actin are involved in the assembly and function of tightjunctions. Because inhibition of Na-K-ATPase reduced RhoA GTPase activity andprevented tight junction assembly, we suggest that normal intracellular ionichomeostasis maintained by NA-K-ATPase is necessary for step 2 of thetight junction assembly. Future experiments are necessary to identifymolecular components and signaling pathways involved in these two steps oftight junction assembly described in this model.# K- V2 C7 F9 |9 v4 s5 y( V) o

Fig. 3. Two-step model for the assembly of tight junctions in epithelial cells. A : diagrammatic representation of tight junction assembly mediated byE-cadherin function. Note the partial tight junction strands (red), thecircumferential actin ring (grey), and adherens junction (blue) B :fully formed tight junction strands at the apicolateral side of epithelialcells (red). Note stress fibers (black) localized at tight junction membranecontact points. C : immunofluorescence of ZO-1 after 3 h ofCa 2 switch in ouabain-treated cells. Note the gaps inthe ZO-1 staining pattern (arrows). D : immunofluorescence of ZO-1after 3 h of calcium switch in control cells. Bar, 10 µm.
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N A -K-ATP ASE, TIGHT JUNCTIONS, AND DISEASE
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Cancer

In carcinoma (cancer derived from epithelial cells), the polarized epithelial phenotype is lost( 9 ). Events that lead to the loss of tight junctions and epithelial polarity might eventually lead toproliferation and metastasis of carcinoma cells. Interestingly, in patienttumor samples of renal clear cell carcinoma, an invasive and aggressive formof renal cancer, expression of Na-K-ATPase -subunit and Na-K-ATPaseactivity are highly reduced ( 81 ). A morphometric analysisof tight junctions in these tumor tissues revealed the loss of tight junctionsearly during the development of cancer (Kim G, Thomas G, Rajasekaran SA, Rosen E, Shintaku P, Lassman C, Said J, and Rajasekaran AK, unpublished observations). It is tempting to speculate that reduced Na-K-ATPase activityor its subunit expression might play a role in the loss of tight junctions andpossibly in the transition from the polarized epithelial phenotype to a moremesenchymal phenotype (epithelial-mesenchymal transition) often found incancer ( 79 ). In addition,reduced Na-K-ATPase activity might activate MAPK signaling, which is known toinduce cell proliferation in a wide variety of cells( 13 ). In fact, partialinhibition of Na-K-ATPase by ouabain in endothelial cells( 5 ) and epithelial cells(Rajasekaran SA, Espineda C, and Rajasekaran AK, unpublished observations)activated MAPK and increased cell proliferation. It is tempting to speculatethat functional tight junctions once formed might transmit cell proliferationinhibitory signals in normal epithelial cells. Diminished Na-K-ATPase function might lead to loss of tight junctions and increased cell proliferation inaffected cells.- {3 K% t& u& n7 u! R8 u1 E6 w
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Ischemia# h  U: x. v# b/ E5 c
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Ischemia is a condition caused by the deficiency of oxygenation of a bodypart caused by an obstruction in or the constriction of a blood vessel.Ischemic events in the kidney in vivo( 53, 70, 71 ) and in vitro( 11, 59 ) are associated with theloss of tight junction integrity. A role of Na-K-ATPase in re-establishing tight junctions and epithelial polarity might have important clinicalimplications during recovery from ischemic injury. During renal ischemia, theATP content of the affected epithelial cells is rapidly depleted, resulting inthe inhibition of Na-K-ATPase function( 17, 60, 87 ). The consequences of renalischemia in vivo can be mimicked in cultured epithelial cells by depleting ATPby glycolytic (2-deoxy- D -glucose) or oxidative phosphorylation (antimycin A) inhibitors ( 20, 33, 59 ). Recent studies revealed striking similarities between inhibition of tight junction formation afterNa-K-ATPase inhibition and after ATP depletion. First, expression ofconstitutively active RhoA GTPase in ATP-depleted MDCK cells prevented tightjunction disassembly induced by ATP depletion( 34 ). Second, the levels ofstress fibers and of active RhoA GTPase were drastically reduced inATP-depleted cells, followed by disruption of tight junctions and cellpolarity. Third, fewer changes were observed at the light microscopic level inthe organization of the circumferential actin ring (Dr. S. J. Atkinson,Indiana School of Medicine, Indianapolis, IN, personal communication). It ispossible that these effects on tight junction integrity observed on ATPdepletion are due, at least in part, to the inhibition of Na-KATPase. In fact, decreased surface levels of Na-KATPase have been reported in ischemia-inducedacute renal failure ( 54 ) andduring postischemic renal injury( 52 ). In addition, duringischemia Na-K-ATPase relocates to the apical plasma membrane due to itsdetachment from the cytoskeleton( 72 ), and heat shockprotein-70 is involved in the restoration of the cytoskeletal linkage of theNa-K-ATPase ( 8 ). BecauseNa-K-ATPase is known to associate with the actin cytoskeleton( 73 ), it is possible thatalterations in the actin cytoskeleton might affect the localization of thisprotein. We recently observed a change in the polarity of Na-KATPase inNa-K-ATPase-inhibited cells( 82 ). In retinal pigmentepithelial cells, Na-K-ATPase is predominantly localized to the apical plasmamembrane. Inhibition of Na-K-ATPase resulted in an increased basolaterallocalization of Na-K-ATPase. A change in polarity of the Na-K-ATPasecorrelated with dramatic changes in the amount of actin stress fibers,suggesting that Na-K-ATPase function somehow regulates its localizationthrough its association with the actin cytoskeleton. The inhibition ofNa-K-ATPase during ischemia might be one of the mechanisms involved in the loss of tight junction integrity and polarity in ischemic cells. An impedimentin the reestablishment of Na-KATPase function during recovery from ischemic ortoxic injury may inhibit the process of reestablishing tight junctions andthus may delay the healing process or may even lead to hyperplasia of the affected tissue. Defining the role of Na-K-ATPase on tight junction disassembly during ischemia and tight junction reassembly during ischemicrecovery should provide insights into therapeutic modalities for the treatmentof ischemia.

Hypomagnesemia

In dominant renal hypomagnesemia, a dominant negative mutation in theNa-K-ATPase -subunit (FXYD2) has been linked to the loss ofMg 2 associated with this disease( 65 ). A conserved glycine-41within the putative transmembrane domain is mutated to arginine in thisdisease. The -subunit mutant protein localized intracellularly and didnot codistribute with - and -subunits at the plasma membrane.Whether the mutant-expressing cells have reduced Na-K-ATPase activity thatcontributes to increased tight junction permeability toMg 2 remains to be tested. Interestingly, claudin-16mutations have been demonstrated as the basis of recessive hypomagnesemia inhumans ( 90 ). Whether afunctional link exists between claudin-16 and the Na-K-ATPase -subunit remains to be seen.$ L/ a8 x& }. i; v' d1 D
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Polycystic Kidney Disease
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Polycystic kidney disease is characterized by the appearance offluid-filled cysts within the parenchyma of the kidney, eventually leading tokidney failure. Autosomal dominant polycystic kidney disease (ADPKD) is themajor hereditary type and is characterized by the loss of renal function inthe fifth or sixth decade of life. Mutations in PKD1 and PKD2 genes encoding polycystin-1 and polycystin-2 have been linked to the onset of this disease(reviewed in Ref. 93 ). One ofthe characteristic features of this disease is the loss of polarity of several proteins (reviewed in Ref. 102 ). For example,Na-K-ATPase is mislocalized to the apical plasma membrane yet is fully functional ( 102, 103 ). A recent studydemonstrated that the integrity of tight junctions is not altered in ADPKD( 14 ). Whether the integrity ofthe tight junctions is due to the presence of functional Na-K-ATPase remainsto be seen.# }5 n  {0 M2 R" q/ r, w( n
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MDCK cells grown on collagen gels form microcysts and have been utilized asa model for studying mechanisms involved in fluid secretion in ADPKD( 62 ). MDCK microcysts arefilled with fluid, and the cells in the monolayer lining are polarized, withthe apical surfaces facing the lumen. Inhibition of Na-K-ATPase by ouabaindecreased fluid secretion and caused stratification of cells within the cysts( 36 ). Electron micrographs ofthe ouabain-treated cysts showed increased intercellular spaces between thecells. It is not clear whether these cells have tight junctions or whether thetight junction permeability is altered in these cells. Future research usingthis model should provide more insights into the role of Na-K-ATPase on tightjunction structure and function in polycystic kidney disease.

CONCLUSIONS+ w+ g" v2 v5 H6 V. o) u3 o; U2 ^

Recent studies on the implications of Na-K-ATPase in tight junction formation strongly suggest that Na-K-ATPase may not only regulate vectorialtransport function but also, directly or indirectly, the cell structure ofepithelial cells. In addition to the new role in epithelial cell structure,several new functions of Na-K-ATPase have been reported in the last threeyears (reviewed in Ref. 88 ),including a signaling role in NF-kB activation ( 1 ) and MAPK activation( 38 ), a role in the inductionof cell proliferation through EGF receptor transactivation( 5, 38 ), and a role in cellsubstratum attachment ( 19 ).These recent findings indicate that in addition to its ionic pump function, Na-KATPase might have multiple functions in epithelial cells. Themultifunctional nature of Na-K-ATPase might be due to its enzymatic activityin controlling intracellular Na homeostasis. In addition, the -, -, and -subunits themselves may have individual rolesin cell regulation independent of their role in pump function. Understandingthe multiple functions of Na-K-ATPase is crucial to obtaining more insightinto the role of Na-K-ATPase in normal and disease states.. B' C1 _+ l6 c, s& A# }. [1 M

DISCLOSURES
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This work was supported by National Institute of Diabetes and Digestive andKidney Diseases Grant DK-56216. S. A. Rajasekaran is supported by U.S.Department of Health and Human Services Institutional National ResearchService Award T32CA09056.
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ACKNOWLEDGMENTS# y2 w+ v) A% x8 H3 P+ U
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We thank our collaborators, Drs. Lawrence Palmer, Alejandro Peralta-Soler,Jeffrey Harper, Gerard Apodaca, Yi Zheng, William James Ball, Jr., and NeilBander, for help in carrying out these studies. We thank Drs. Ernie Wright,Alexander Van der Bliek, and Michael Meranze for critical reading of themanuscript.

Address for reprint requests and other correspondence: A. K. Rajasekaran,Dept. of Pathology and Laboratory Medicine, Rm. 13-344 CHS, Univ. ofCalifornia, Los Angeles, CA 90095 (E-mail: arajasekaran{at}mednet.ucla.edu 4 [# G1 k+ H# L: K( t+ z# {  I3 T
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