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Multiple Functions of Na,K-Atpase in Epithelial Cells Sigrid A

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Multiple Functions of Na,K-Atpase in Epithelial Cells Sigrid A Multiple Functions of Na,K-ATPase in Epithelial Cells Sigrid A. Rajasekaran, Sonali P. Barwe, and Ayyappan K. Rajasekaran The Na,K-adenosine triphosphatase (ATPase), or sodium pump, has been well studied for its role in the regulation of ion homeostasis in mammalian cells. Recent studies suggest that Na,K-ATPase might have multiple functions such as a role in the regulation of tight junction structure and function, induction of polarity, regulation of actin dynamics, control of cell movement, and cell signaling. These functions appear to be modulated by Na,K- ATPase enzyme activity as well as protein–protein interactions of the ␣ and ␤ subunits. In this review we attempt to differentiate functions associated with enzyme activity and subunit interactions. In addition, the consequence of impaired Na,K-ATPase function or reduced subunit expression levels in kidney diseases such as cancer, tubulointerstitial fibrosis, and ischemic nephropathy are discussed. Semin Nephrol 25:328-334 © 2005 Elsevier Inc. All rights reserved. KEYWORDS Na,K-ATPase, tight junction, cell motility, actin, E-cadherin a,K-adenosine triphosphatase (ATPase), also known as erties differ. The isoforms predominantly expressed in kid- ␣ ␤ 15,16 ␣ ␤ the sodium pump, is an ubiquitous plasma membrane ney are 1 and 1. The and subunits referred to in this N ␣ ␤ protein complex extensively studied for its function in ion review are the 1 and 1 isoforms of Na,K-ATPase. homeostasis. It couples the hydrolysis of 1 molecule of ATP The low intracellular Naϩ concentration maintained by the to the translocation of 2 Kϩ into and 3 Naϩ out of the cell pump is crucial for the efficient functioning of various Naϩ- against their electrochemical gradient. Thus, the Na,K- coupled transport systems and the sodium gradient provides ATPase is crucial for maintaining the normally low Naϩ and the primary energy for uptake and extrusion of a wide variety high Kϩ concentrations of eukaryotic cells (for comprehen- of solutes. Furthermore, in epithelia the transepithelial Naϩ sive reviews see Kaplan1 and Lingrel and Kuntzweiler2). The gradient generated by the basolaterally localized Na,K-ATPase is enzyme complex consists of ubiquitous and essential ␣ and ␤ vital for the directional transport of solutes across the epithe- subunits3,4 and an optional ␥ subunit whose expression is lial cell layer (vectorial transport). Although the transport restricted to certain tissues.5 The ␣ subunit is a polytopic function of the Na,K-ATPase has been investigated exten- membrane protein with the catalytic activity of the enzyme sively, recent evidence suggests that the sodium pump might and the binding sites for Naϩ,Kϩ, and ATP.4 The ␤ subunit be a multifunctional protein involved in the induction of has been shown to be important for the efficient translation of epithelial cell tight junctions and polarity,17-20 cell motility,17 the ␣ subunit on the endoplasmic reticulum,6 the correct cell signaling,21 actin dynamics,17,18,20 and cancer.22-25 This folding and membrane insertion of the ␣ subunit, and the review summarizes some of the recent work on these novel expression of the enzyme on the plasma membrane.7-9 The ␤ functions associated with Na,K-ATPase in kidney epithelial subunit, as well as the ␥ subunit, also has been suggested to cells. be a modulator of the pump function.8,10,11 In mammals, at least 4 isoforms of the ␣ subunit12-14 and 3 isoforms of the ␤ subunit2,15 have been described. These isoforms are ex- Na,K-ATPase and the pressed in a tissue-specific manner and their functional prop- Establishment of Tight Junctions and Epithelial Polarity From the Department of Pathology and Laboratory Medicine, UCLA David Epithelial cells line the inner and outer surfaces of the body. Geffen School of Medicine, Los Angeles, CA. Their functions include the formation of a barrier between 2 Supported by DK56216. biological compartments and the control of the exchange of Address reprint requests to Ayyappan K. Rajasekaran, PhD, Department of Pathology and Laboratory Medicine, Room 13-344 CHS, University of molecules between these compartments via regulated secre- California, Los Angeles, CA 90095. E-mail: arajasekaran@mednet. tion and absorption. To serve these functions, epithelial cells ucla.edu have their plasma membrane divided into functionally and 328 0270-9295/05/$-see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.semnephrol.2005.03.008 Multi-functionality of Na,K-ATPase 329 function appears dispensable once the cells have established tight junctions.33 However, Na,K-ATPase function appears to be involved in both the formation and maintenance of tight junctions. Inhibition of Na,K-ATPase function in polarized monolayers of human retinal pigment epithelial cells resulted in increased tight junction permeability to both ions and nonionic molecules. These permeability changes were ac- companied by a reduction in tight junction membrane con- tact points (kissing points) at the apicolateral plasma mem- Figure 1 Schematic presentation of the multiple functions of Na,K- brane border without complete destruction of the tight ATPase in epithelial cells. Functions that are mediated by Na,K- junctions.20 Similar results were obtained in HPAF cells, a ATPase activity are indicated by solid arrows. Dashed arrows indi- well-differentiated pancreatic tumor cell line that forms tight cate functions of Na,K-ATPase that possibly involve protein–protein junctions in culture. Interestingly, inhibition of Na,K-ATPase interactions of Na,K-ATPase ␣ or ␤ subunit with other cellular resulted in an increase in the permeability of ionic and non- proteins. ionic molecules without altering the localization of tight junction proteins (our unpublished data). It is possible that in epithelial cells with established tight junctions, Na,K- biochemically distinct apical and basolateral domains sepa- ATPase function is necessary to maintain tight junction mem- rated by tight junctions. This unique structural phenotype brane contact points and thus the permeability of tight junc- also is referred to as a polarized phenotype or well-differentiated tions. 26-28 phenotype. Given the ubiquitous presence of Na,K-ATPase in mam- Tight junctions are multiprotein complexes located at the malian cells and its fundamental role in cell functions, it is boundary of apical and basolateral plasma membrane do- tempting to speculate that the sodium pump might have a mains. Characterized by the close apposition of contiguous conserved role in the regulation of tight junction structure plasma membranes, they prevent the mixing of molecules and functions in epithelial cells. Indeed, Genova and Fehon34 between the apical and basolateral domains and regulate the reported an essential role of Na,K-ATPase in the barrier func- passage of molecules across the paracellular space. Despite tion of septate junctions in Drosophila. Septate junctions in tight junctions being crucial for the epithelial phenotype and Drosophila generally are considered analogous to tight junc- functioning of epithelia, the molecular mechanisms that reg- tions in mammalian cells.35 In Drosophila, both Na,K-ATPase ulate the formation of these specialized membrane microdo- ␣ subunit (Atp␣) and Nervana 2 (Nrv2) (which encodes the ␤ mains are understood poorly (for comprehensive reviews see subunit of Na,K-ATPase) were identified as essential for the Anderson and Van Itallie,29 Tsukita et al,30 and Schneeberger barrier function of the septate junction. Similarly, Paul et al36 and Lynch31). Although previous studies implicated the reported that mutations in ATP␣ and Nrv2 disrupt the stable function of E-cadherin, a cell-adhesion molecule, in the for- formation of septate junctions in Drosophila. These recent mation and maintenance of junctional complexes and the studies identified an essential function of Na,K-ATPase in epithelial phenotype,32 studies from our laboratory revealed establishing and maintaining the primary paracellular barrier a crucial role for Na,K-ATPase in maintaining the integrity in invertebrate as well as in mammalian epithelial cells. and functions of tight junctions.17,18,23 Inhibition of Na,K- ATPase activity by ouabain (a specific inhibitor of Na,K- ϩ ATPase ␣ subunit) or by K depletion in Madin-Darby canine Na,K-ATPase and the kidney (MDCK) cells subjected to a Ca2ϩ-switch experiment (an assay to study the formation of junctional complexes), Cortical Actin Cytoskeleton prevented the formation of tight junctions. Consequently, The actin cytoskeleton plays an important role in the struc- the lack of functional tight junctions in these cells also pre- ture and functions of epithelial cells. The interaction of the vented the establishment of polarity. Because the Naϩ iono- ϩ actin cytoskeleton with cell adhesion molecules is thought to phore gramicidin that increases intracellular Na concentra- strengthen cell–cell contacts, providing a scaffolding plat- tions mimicked the effect of Na,K-ATPase inhibition, it is form for signaling molecules involved in epithelial polariza- likely that the intracellular ionic gradient maintained by the tion and to promote further the assembly of junctional com- sodium pump is involved in the assembly of tight junctions plexes in epithelial cells including adherens junctions and 18 and generation of polarity in epithelial cells (Fig. 1). tight junctions.37,38 These junctions are associated with the cortical actin cytoskeleton also referred to as the perijunctional Na,K-ATPase as a Regulator actomyosin ring. Earlier studies showed that tight junction of Tight Junction Permeability structure and permeability are regulated by the perijunc- tional actomyosin ring,29,39,40 and it has been proposed that Molecular mechanisms involved in the formation of tight contraction of perijunctional actin filaments and the resulting junctions might be distinct from the mechanisms required to centrifugal traction on tight junction membrane regulates maintain tight junctions. For example, although E-cadherin tight junction permeability.39 function is critical for the formation of tight junctions,32 its Inhibition of Na,K-ATPase pump function did not alter 330 S.A. Rajasekaran, S.P.
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