Role of Na-K-ATPase in the assembly of tight junctions
Ayyappan K. Rajasekaran and
Sigrid A. Rajasekaran
Department of Pathology and Laboratory Medicine, David Geffen School of
Medicine at UCLA, University of California, Los Angeles, California
90095
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ABSTRACT
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Na-K-ATPase, also known as the sodium pump, is a crucial enzyme that
regulates intracellular sodium homeostasis in mammalian cells. In epithelial
cells Na-K-ATPase function is also involved in the formation of tight
junctions through RhoA GTPase and stress fibers. In this review, a new
two-step model for the assembly of tight junctions is proposed: step
1, an E-cadherin-dependent formation of partial tight junction strands
and of the circumferential actin ring; and step 2, active actin
polymerization-dependent tethering of tight junction strands to form
functional tight junctions, an event requiring normal function of Na-K-ATPase
in epithelial cells. A new role for stress fibers in the assembly of tight
junctions is proposed. Also, implications of Na-K-ATPase function on tight
junction assembly in diseases such as cancer, ischemia, hypomagnesemia, and
polycystic kidney disease are discussed.
E-cadherin; stress fibers; ischemia
THE VECTORIAL TRANSPORT FUNCTION (directional transport of
molecules across an epithelial cell layer) of epithelial cells largely depends
on 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 is
pumped out of the cell by Na-K-ATPase localized to the basolateral plasma
membrane. Na-K-ATPase catalyzes an ATP-dependent transport of three
Na+ ions out and two K+ ions into the cell per pump
cycle, thereby generating the transmembrane Na+ gradient across the
plasma membrane that is crucial to regulate the vectorial transport function
of epithelial cells.
Na-K-ATPase is a heterodimer, consisting of an
-and
-subunit
(reviewed in Ref. 56), but
recent studies have demonstrated the presence of an additional
-subunit
(25,
67). The
-subunit
(
112 kDa) is the catalytic subunit whereas both the
-subunit (50-60
kDa) and the
-subunit (
7 kDa) have a modulatory role in
Na-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 epithelial
tissues (reviewed in Refs. 56,
66,
69, and
94). In addition to the role
of Na-K-ATPase in transepithelial transport, recent studies have demonstrated
a role for Na-K-ATPase function in the assembly of junctional complexes in
epithelial cells (83). This
short review will focus on insights into how Na-K-ATPase, an ion pump, is
involved in the assembly of tight junctions.
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STRUCTURE AND MOLECULAR COMPOSITION OF TIGHT JUNCTIONS
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Tight junctions (zonula occludens) form a continuous belt at the boundary
between the apical and lateral plasma membrane domains of neighboring
epithelial cells (Fig.
1A) and are structurally characterized by the close
apposition of contiguous plasma membranes
(23). They selectively
regulate the passage of molecules across the paracellular space
(Fig. 1B) (gate
function) (21) and passively
separate molecules into the apical and basolateral plasma membrane domains
(fence function) (59). A tight
junction is crucial in maintaining the polarized phenotype and the vectorial
transport functions of epithelial cells. The current knowledge of tight
junctions is consistent with a view that tight junctions are specialized
membrane microdomains that might function as a molecular platform involved in
cell signaling, vesicle protein docking, actin organization, and cell polarity
in epithelial cells (reviewed in Refs.
68,
98,
99, and
109).
In freeze-fracture electron micrographs, tight junctions appear as
anastomosing intramembranous particle strands localized at the apicolateral
side of polarized epithelial cells. The contacts between the tight junction
strands of adjacent cells are in such close apposition as to effectively
eliminate any intercellular space. These areas of intimate contact can be
visualized as membrane contact points (kisses) in transmission electron
micrographs (Fig. 1B).
Each particle of the tight junction strand is composed of transmembrane
proteins and cytoplasmic plaque proteins connected to the actin cytoskeleton
(Fig. 1C). Occludins,
claudins, and the junctional adhesion molecule (JAM) are the three
transmembrane proteins localized to the tight junctions. Occludin was the
first tight junction transmembrane protein identified
(28) but appears to be
unnecessary for the formation of tight junction strands
(86). The JAM, a member of the
immunoglobulin superfamily, appears to have roles in cell adhesion and
extravasation of immune cells across tight junctions
(7). Recently, claudins have
been identified as membrane proteins localized to tight junctions and have
been shown to be involved in the formation of tight junction strands
(26,
30). Until now, 24 members of
the claudin family have been described in various epithelial cells
(98). Identification of
occludin and claudins has tremendously increased our understanding of the
structure and barrier function of tight junctions. Both occludin and claudin
have four transmembrane domains and are involved in creating the paracellular
barrier (2,
6,
18,
27,
63,
101,
105). The intracellular COOH
terminus of both occludin (22,
29) and claudin
(43) associates with
cytoplasmic plaque proteins such as zonula occludens-1 (ZO-1)
(92), ZO-2
(46), and ZO-3
(40). ZO-1 and ZO-2 associate
with the actin binding proteins
-catenin
(44,
45,
80) and spectrin
(61)
(Fig. 1C) and link the
tight junction plaques to the actin cytoskeleton. Additional plaque proteins
identified include cingulin
(16), symplekin
(49), AF-6
(106), and 7H6
(110). Proteins involved in
signal transduction (c-src and c-yes), proteins involved in membrane traffic
(VAP-33, Rab3b, Rab13, Rab 8, Sec6, and Sec8), and cell polarity-related
proteins (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 junctions
have been recently described in further detail in excellent reviews
(12,
58,
68,
75,
91,
98,
99).
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NA-K-ATPASE ENZYME ACTIVITY IS REQUIRED FOR
TIGHT JUNCTION FORMATION
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The formation of tight junctions is a critical event in the biogenesis of
polarized epithelial cells during early vertebrate development (reviewed in
Ref. 24), during tubular and
ductal development in epithelial tissues, and during recovery from tissue
damage after ischemic or toxic injury. To understand the molecular events that
take 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). A
Ca2+-switch assay
(32) is a widely utilized
method for investigating the mechanisms involved in the formation of tight
junctions. In this approach, single-cell suspensions are allowed to attach to
the substratum in a normal-Ca2+-containing medium for
30 min. During this time, the cells are still round and do not establish
polarity (Fig. 2). The attached
cells are then transferred to a low-Ca2+ medium (<5
µM Ca2+) for
12 h to allow for the formation of
dense monolayers. The cells are then switched to a
normal-Ca2+ medium, in which tight junctions and cell
polarity will be established within 3 h. Thus the
Ca2+-switch assay allows for the rapid monitoring of
molecular events that occur during tight junction development
(Fig. 2). Techniques including
immunofluorescence localization of tight junction proteins, electron
microscopy, and transepithelial electrical resistence (TER), an indicator of
the paracellular permeability function of tight junctions, are used to monitor
tight junction development.

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Fig. 2. Illustration of the calcium-switch assay used to study the role of
Na-K-ATPase in the assembly of tight junctions.
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With the use of these approaches, a recent study demonstrated that
Na-K-ATPase activity is necessary for the formation of tight junctions and
development of polarity in MDCK cells
(83). Inhibition of Na-KATPase
activity by ouabain (a specific inhibitor) or by K+ depletion
[inhibition due to the lack of the steep K+ gradient necessary for
pump function (10,
77,
78)] in MDCK cells subjected
to a Ca2+ switch prevented the formation of tight
junctions and the development of epithelial polarity. Tight junctions formed
rapidly, and polarity was established after reactivation of Na-KATPase pump
function by restoring the initial K+ concentration (K+
repletion), indicating that the effects of Na-K-ATPase inhibition were
reversible. Inhibition of tight junction formation correlated with increased
intracellular Na+ concentration levels
([Na+]i) generated by the inhibition of Na-K-ATPase, and
the Na+ ionophore gramicidin, which increases
[Na+]i, mimicked the effects of Na-K-ATPase inhibition
on tight junction formation and epithelial polarity. Moreover, treatment of
cells with ouabain in Na+-free medium did not affect tight junction
formation. Because Na-KATPase function controls a variety of ions and
metabolite transport systems, inhibition of this enzyme might induce multiple
biochemical changes in cells, including altered Ca2+
signaling, cell volume, cell pH, and membrane potential. However, this study
demonstrated that Na-K-ATPase enzyme activity is involved in the formation of
tight junctions in epithelial cells. It is well established that cell-cell and
cell-substratum contacts are involved in the establishment of tight junctions
and polarity in epithelial cells (reviewed in Ref.
108). Lack of tight junction
assembly and polarity in Na-K-ATPase-inhibited cells suggests that the
intracellular ionic gradient maintained by Na-K-ATPase is also involved in the
assembly of tight junctions and generation of polarity in epithelial
cells.
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SYNERGISM BETWEEN NA-K-ATPASE AND E-CADHERIN IN
THE ASSEMBLY OF TIGHT JUNCTIONS
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The cell-cell contact mediated by the cell adhesion molecule E-cadherin has
been implicated in the formation of tight junctions in MDCK cells. E-cadherin
is a single transmembrane protein localized to the basolateral plasma membrane
in polarized epithelial cells. The extracellular domain of E-cadherin contains
five IgG-like repeats that mediate cell-cell contact between epithelial cells
by homophilic interaction in a Ca2+-dependent manner
(reviewed in Ref. 95). The
cytoplasmic tail of E-cadherin associates with
-,
-, and
-catenins (76) and
p120ctn (89).
-Catenin either directly or through
-actinin links the
E-cadherin complex to the actin cytoskeleton, which is crucial for the cell
adhesion function of E-cadherin
(51,
85).
The function of E-cadherin seems to be necessary for the translocation of
tight junction proteins to the plasma membrane and for the formation of tight
junction complexes. Inhibition of E-cadherin's cell-cell adhesion function by
extracellular domain-specific antibodies prevented the assembly of tight
junctions (37). In cells
maintained in a low-Ca2+ medium, ZO-1 is localized in
the cytoplasm. On transfer of the cells to a normal-Ca2+
medium, ZO-1 rapidly translocated to the plasma membrane in control cells,
whereas in the presence of anti-E-cadherin extracellular domain antibody this
translocation was drastically reduced
(37).
In contrast, in Na-K-ATPase-inhibited cells post-Ca2+
switch, ZO-1 was localized to the plasma membrane but failed to show a
continuous staining pattern normally seen in MDCK cells
(83). This suggests that
Na-K-ATPase inhibition does not affect E-cadherin function. Moreover, adherens
junctions, the formation of which requires functional E-cadherin
(107), were present in
ouabain-treated and K+-depleted cells
(83). Therefore, plasma
membrane localization of tight junction proteins such as ZO-1 and occludin
seems to be due to the presence of functional E-cadherin present in
Na-K-ATPase-inhibited cells, but the discontinuous ZO-1 staining pattern
suggested that Na-K-ATPase function is necessary for formation of the
continuous ZO-1 staining pattern in cells with established tight junctions.
Consistent with this view, another study showed that in Moloney sarcoma
virus-transformed MDCK cells (MSV-MDCK), a cell line with highly reduced
E-cadherin and Na-K-ATPase
-subunit levels, ectopic expression of
E-cadherin resulted in the localization of ZO-1 to the plasma membrane yet
with a discontinuous staining pattern
(80,
84). Only ectopic expression
of E-cadherin combined with restored Na-K-ATPase function (through ectopic
expression of Na-K-ATPase
-subunit) resulted in a continuous ZO-1
staining pattern and the formation of functional tight junctions,
substantiating a hypothesis for a synergistic function of E-cadherin and
Na-K-ATPase in the formation of tight junctions in epithelial cells. At this
point, however, we cannot exclude the possibility that the
-subunit of
Na-K-ATPase itself might play a role in the synergistic function of
Na-K-ATPase and E-cadherin in establishing tight junctions and polarity in
epithelial cells.
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REGULATION OF TIGHT JUNCTION FORMATION BY
NA-K-ATPASE THROUGH MAPK
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The MAPK pathway has been implicated in tight junction assembly in various
cell lines. Chen et al. (15)
have shown that downregulation of MAPK signaling in ras-transformed MDCK cells
restored epithelial morphology and tight junctions. In pig thyrocytes,
activation of MAPK by EGF and transforming growth factor (TGF)-
1
resulted in the loss of tight junctions, and the inhibition of MAPK activation
by MEK inhibitor prevented this tight junction loss
(35). In salivary gland
epithelial cells, activation of MAPK by constitutive expression of Raf
resulted in the reduction of occludin levels and disruption of tight junctions
(55). In endothelial cells,
H2O2-mediated activation of ERK1/ERK2 leads to the
disruption of endothelial tight junctions
(50). In these cell lines, low
MAPK activity seems to be associated with formation of tight junctions and
epithelial polarization, whereas increased MAPK activity leads to the
disruption of tight junctions. Interestingly, inhibition of Na-K-ATPase
activity by ouabain activated MAPK in cardiac myocytes and other cell types
(38) as well as in polarized
epithelial cells (Rajasekaran SA, Espineda C, and Rajasekaran AK and Landon I
and Rajasekaran AK, unpublished observations). It is tempting to speculate
that increased MAPK activity induced by Na-K-ATPase inhibition has an
inhibitory role on the assembly of functional tight junctions. Signaling
pathways induced by the inhibition of Na-KATPase function and their impact on
tight junction assembly will be an important area to pursue in the future.
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ROLE OF STRESS FIBERS IN THE ASSEMBLY OF TIGHT JUNCTIONS
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Several earlier studies showed that tight junction structure and
permeability are regulated by the perijunctional actomyosin ring (reviewed in
Refs. 3,
58, and
99). This ring is located at
the apical pole of polarized epithelial cells and can be visualized by light
microscopy as a circumferential actin ring that colocalizes with tight
junction and adherens junction proteins. Earlier studies showed that
disruption of tight junction structure and permeability correlated with
disruption of the circumferential actin ring
(3,
58,
99). However, in
Na-K-ATPase-inhibited MDCK cells that did not form tight junctions, no
apparent 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 projecting
from the perijunctional actin ring interface at the cytoplasmic surface of
tight junction membrane contact points
(41,
57).
A role for RhoA GTPase (a small GTP-binding protein) involved in the
formation of stress fibers
(39,
47,
100) has been demonstrated to
be involved in the regulation of tight junction structure function
(42,
48,
74). Interestingly, the
impediment of tight junction assembly in Na-K-ATPase-inhibited MDCK cells was
accompanied by a drastic reduction in stress fibers and correlated with
diminished RhoA activity (83).
Exogenous overexpression of wild-type RhoA GTPase bypassed the inhibitory
effect of Na-K-ATPase on tight junction formation, indicating that RhoA GTPase
is an essential component downstream of Na-K-ATPase function linking
Na-K-ATPase to the formation of functional tight junctions
(83). A similar effect on
stress fibers, RhoA GTPase, and tight junction assembly was found in cells
treated with gramicidin, a Na+ ionophore that, like inhibition of
Na-K-ATPase, increases [Na]i. This indicated that intracellular
Na+ homeostasis regulated by Na-K-ATPase function is involved in
the formation of stress fibers and regulation of RhoA GTPase in MDCK cells. In
polarized monolayers of retinal pigment epithelial cells, inhibition of
Na-KATPase resulted in an increase in tight junction permeability. This
increased permeability correlated with decreased tight junction membrane
contact points and reduced actin stress fibers without an apparent change in
the circumferential actin ring
(82). Therefore, it seems
likely that both the perijunctional actin ring and stress fibers are involved
in the assembly and function of tight junctions. The perijunctional actin ring
might regulate paracellular permeability and provide stability to tight
junctions, whereas stress fibers might be involved in more dynamic functions
related to the assembly and function of tight junctions. These dynamic
functions during tight junction assembly may include proper molecular
alignment of tight junction proteins at the tight junction region and
tethering of tight junction strands to establish functional tight junctions
(see model below). In cells with established tight junctions, the stress
fibers might have a role in maintaining the tight junction membrane contact
points through their association with tight junction membrane and cytoplasmic
proteins. Reduced stress fibers associated with minimal change in the
circumferential actin ring in Na-K-ATPase-inhibited cells highlighted for the
first time that stress fibers are also involved in the regulation of tight
junction assembly and function.
How Na-K-ATPase function is involved in the regulation of tight junction
assembly through RhoA GTPase activity and stress fiber formation in epithelial
cells is not currently known. Rho function is modulated by a set of regulatory
proteins. Rho is activated through GDP-GTP exchange, which is promoted by
guanine nucleotide exchange factors and is inactivated through
GTPase-activating proteins (reviewed in Refs.
39 and
100). Stabilization of the
inactive GDP-bound form of Rho is mediated by Rho guanine nucleotide
dissociation inhibitors (39,
100). It is possible that
inhibition of Na-K-ATPase function may either inhibit the function of guanine
nucleotide exchange factors, resulting in the accumulation of the inactive
form of Rho (Rho-GDP), or promote the functions of GTPase-activating proteins
or Rho guanine nucleotide dissociation inhibitors, affecting the activation of
Rho. Na-K-ATPase-mediated signaling mechanisms involved in the regulation of
RhoA GTPase and actin assembly will be an important area of future research
that will provide more insights into how ion homeostasis might regulate the
role of stress fibers in the assembly and function of tight junctions.
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TWO-STEP MODEL FOR THE ASSEMBLY OF TIGHT JUNCTIONS
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From these recent observations on the Na-K-ATPase function in tight
junction assembly, we propose a two-step model for the formation of tight
junctions in epithelial cells (Fig.
3). Step 1 is dependent on the cell-cell adhesion
function of E-cadherin. On E-cadherin-mediated cell-cell interactions, tight
junction 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 its
association with catenins that, in turn, are associated with
E-cadherin-containing transport vesicles
(80). Inhibition of
E-cadherin-mediated cell-cell adhesion function by extracellular
domain-specific anti-E-cadherin antibodies inhibited the translocation of ZO-1
to the plasma membrane as well as prevented the formation of the
perijunctional actin ring
(37). Also, during this
initial step of tight junction assembly, ZO-1 association with the actin
cytoskeleton as well as with occludin and claudin might take place to form the
discontinuous tight junction strands, as seen in Na-K-ATPase-inhibited cells.
Formation of discontinuous tight junction strands after the
Ca2+ switch has been described earlier
(32). Step 2 of tight
junction assembly is dependent on active actin polymerization probably
regulated by RhoA GTPase. Active polymerization of actin filaments associated
with ZO-1, ZO-2, ZO-3, or occludin
(104) might provide the
propulsive force to mobilize discontinuous tight junction strands in the plane
of the membrane and facilitate tethering of tight junction strands at the
apicolateral region to establish functional tight junctions
(Fig. 3, B and
D). Thus this model predicts that both stress fibers and
circumferential actin are involved in the assembly and function of tight
junctions. Because inhibition of Na-K-ATPase reduced RhoA GTPase activity and
prevented tight junction assembly, we suggest that normal intracellular ionic
homeostasis maintained by NA-K-ATPase is necessary for step 2 of the
tight junction assembly. Future experiments are necessary to identify
molecular components and signaling pathways involved in these two steps of
tight junction assembly described in this model.

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Fig. 3. Two-step model for the assembly of tight junctions in epithelial cells.
A: diagrammatic representation of tight junction assembly mediated by
E-cadherin function. Note the partial tight junction strands (red), the
circumferential actin ring (grey), and adherens junction (blue) B:
fully formed tight junction strands at the apicolateral side of epithelial
cells (red). Note stress fibers (black) localized at tight junction membrane
contact points. C: immunofluorescence of ZO-1 after 3 h of
Ca2+ switch in ouabain-treated cells. Note the gaps in
the ZO-1 staining pattern (arrows). D: immunofluorescence of ZO-1
after 3 h of calcium switch in control cells. Bar, 10 µm.
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NA-K-ATPASE, 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 to
proliferation and metastasis of carcinoma cells. Interestingly, in patient
tumor samples of renal clear cell carcinoma, an invasive and aggressive form
of renal cancer, expression of Na-K-ATPase
-subunit and Na-K-ATPase
activity are highly reduced
(81). A morphometric analysis
of tight junctions in these tumor tissues revealed the loss of tight junctions
early 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 activity
or its subunit expression might play a role in the loss of tight junctions and
possibly in the transition from the polarized epithelial phenotype to a more
mesenchymal phenotype (epithelial-mesenchymal transition) often found in
cancer (79). In addition,
reduced Na-K-ATPase activity might activate MAPK signaling, which is known to
induce cell proliferation in a wide variety of cells
(13). In fact, partial
inhibition 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 speculate
that functional tight junctions once formed might transmit cell proliferation
inhibitory signals in normal epithelial cells. Diminished Na-K-ATPase function
might lead to loss of tight junctions and increased cell proliferation in
affected cells.
Ischemia
Ischemia is a condition caused by the deficiency of oxygenation of a body
part 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 the
loss of tight junction integrity. A role of Na-K-ATPase in re-establishing
tight junctions and epithelial polarity might have important clinical
implications during recovery from ischemic injury. During renal ischemia, the
ATP content of the affected epithelial cells is rapidly depleted, resulting in
the inhibition of Na-K-ATPase function
(17,
60,
87). The consequences of renal
ischemia in vivo can be mimicked in cultured epithelial cells by depleting ATP
by 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 after
Na-K-ATPase inhibition and after ATP depletion. First, expression of
constitutively active RhoA GTPase in ATP-depleted MDCK cells prevented tight
junction disassembly induced by ATP depletion
(34). Second, the levels of
stress fibers and of active RhoA GTPase were drastically reduced in
ATP-depleted cells, followed by disruption of tight junctions and cell
polarity. Third, fewer changes were observed at the light microscopic level in
the organization of the circumferential actin ring (Dr. S. J. Atkinson,
Indiana School of Medicine, Indianapolis, IN, personal communication). It is
possible that these effects on tight junction integrity observed on ATP
depletion 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-induced
acute renal failure (54) and
during postischemic renal injury
(52). In addition, during
ischemia Na-K-ATPase relocates to the apical plasma membrane due to its
detachment from the cytoskeleton
(72), and heat shock
protein-70 is involved in the restoration of the cytoskeletal linkage of the
Na-K-ATPase (8). Because
Na-K-ATPase is known to associate with the actin cytoskeleton
(73), it is possible that
alterations in the actin cytoskeleton might affect the localization of this
protein. We recently observed a change in the polarity of Na-KATPase in
Na-K-ATPase-inhibited cells
(82). In retinal pigment
epithelial cells, Na-K-ATPase is predominantly localized to the apical plasma
membrane. Inhibition of Na-K-ATPase resulted in an increased basolateral
localization of Na-K-ATPase. A change in polarity of the Na-K-ATPase
correlated with dramatic changes in the amount of actin stress fibers,
suggesting that Na-K-ATPase function somehow regulates its localization
through its association with the actin cytoskeleton. The inhibition of
Na-K-ATPase during ischemia might be one of the mechanisms involved in the
loss of tight junction integrity and polarity in ischemic cells. An impediment
in the reestablishment of Na-KATPase function during recovery from ischemic or
toxic injury may inhibit the process of reestablishing tight junctions and
thus 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 ischemic
recovery should provide insights into therapeutic modalities for the treatment
of ischemia.
Hypomagnesemia
In dominant renal hypomagnesemia, a dominant negative mutation in the
Na-K-ATPase
-subunit (FXYD2) has been linked to the loss of
Mg2+ associated with this disease
(65). A conserved glycine-41
within the putative transmembrane domain is mutated to arginine in this
disease. The
-subunit mutant protein localized intracellularly and did
not codistribute with
- and
-subunits at the plasma membrane.
Whether the mutant-expressing cells have reduced Na-K-ATPase activity that
contributes to increased tight junction permeability to
Mg2+ remains to be tested. Interestingly, claudin-16
mutations have been demonstrated as the basis of recessive hypomagnesemia in
humans (90). Whether a
functional link exists between claudin-16 and the Na-K-ATPase
-subunit
remains to be seen.
Polycystic Kidney Disease
Polycystic kidney disease is characterized by the appearance of
fluid-filled cysts within the parenchyma of the kidney, eventually leading to
kidney failure. Autosomal dominant polycystic kidney disease (ADPKD) is the
major hereditary type and is characterized by the loss of renal function in
the 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 of
the 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 study
demonstrated that the integrity of tight junctions is not altered in ADPKD
(14). Whether the integrity of
the tight junctions is due to the presence of functional Na-K-ATPase remains
to be seen.
MDCK cells grown on collagen gels form microcysts and have been utilized as
a model for studying mechanisms involved in fluid secretion in ADPKD
(62). MDCK microcysts are
filled with fluid, and the cells in the monolayer lining are polarized, with
the apical surfaces facing the lumen. Inhibition of Na-K-ATPase by ouabain
decreased fluid secretion and caused stratification of cells within the cysts
(36). Electron micrographs of
the ouabain-treated cysts showed increased intercellular spaces between the
cells. It is not clear whether these cells have tight junctions or whether the
tight junction permeability is altered in these cells. Future research using
this model should provide more insights into the role of Na-K-ATPase on tight
junction structure and function in polycystic kidney disease.
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CONCLUSIONS
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Recent studies on the implications of Na-K-ATPase in tight junction
formation strongly suggest that Na-K-ATPase may not only regulate vectorial
transport function but also, directly or indirectly, the cell structure of
epithelial cells. In addition to the new role in epithelial cell structure,
several new functions of Na-K-ATPase have been reported in the last three
years (reviewed in Ref. 88),
including a signaling role in NF-kB activation
(1) and MAPK activation
(38), a role in the induction
of cell proliferation through EGF receptor transactivation
(5,
38), and a role in cell
substratum attachment (19).
These recent findings indicate that in addition to its ionic pump function,
Na-KATPase might have multiple functions in epithelial cells. The
multifunctional nature of Na-K-ATPase might be due to its enzymatic activity
in controlling intracellular Na+ homeostasis. In addition, the
-,
-, and
-subunits themselves may have individual roles
in cell regulation independent of their role in pump function. Understanding
the multiple functions of Na-K-ATPase is crucial to obtaining more insight
into the role of Na-K-ATPase in normal and disease states.
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DISCLOSURES
|
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This work was supported by National Institute of Diabetes and Digestive and
Kidney Diseases Grant DK-56216. S. A. Rajasekaran is supported by U.S.
Department of Health and Human Services Institutional National Research
Service Award T32CA09056.
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ACKNOWLEDGMENTS
|
---|
We thank our collaborators, Drs. Lawrence Palmer, Alejandro Peralta-Soler,
Jeffrey Harper, Gerard Apodaca, Yi Zheng, William James Ball, Jr., and Neil
Bander, for help in carrying out these studies. We thank Drs. Ernie Wright,
Alexander Van der Bliek, and Michael Meranze for critical reading of the
manuscript.
Address for reprint requests and other correspondence: A. K. Rajasekaran,
Dept. of Pathology and Laboratory Medicine, Rm. 13-344 CHS, Univ. of
California, Los Angeles, CA 90095 (E-mail:
arajasekaran{at}mednet.ucla.edu).
 |
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