From the Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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Polarized epithelial cells have highly developed
tight junctions (TJ) to maintain an impermeant barrier and segregate
plasma membrane functions, but the mechanisms that promote TJ formation and maintain its integrity are only partially defined. Treatment of
confluent monolayers of Madin-Darby canine kidney (MDCK) cells with
AlF4 (activator of
heterotrimeric G protein
subunits) results in a 3-4-fold increase
in transepithelial resistances (TER), a reliable indicator of TJ
integrity. MOCK cells transfected with activated G
0 (Q205L) have acclerated TJ formation (Denker, B. M.,
Saha, C., Khawaja, S., and Nigam, S. J. (1996) J. Biol.
Chem. 271, 25750-25753). G
i2 has been
localized within the tight junction, and a role for G
i2
in the formation and/or maintenance of the tight junction was studied
by transfection of MDCK cells with vector without insert (PC), wild
type G
i2, or a GTPase-deficient mutant (constitutively activated), Q205L
i2. Tryptic conformational analysis
confirmed expression of a constitutively active G
i2 in
Q205L
i2-MDCK cells, and confocal microscopy showed a
similar pattern of G
i2 localization in the three cell
lines. Q205L
i2-MDCK cells had significantly higher
base-line TER values than wild type G
i2- or PC-MDCK
cells (1187 ± 150 versus 576 ± 89 (G
i2);
377 ± 52
·cm2 (PC)), and both
G
i2- and Q205L
i2-transfected cell lines
more rapidly develop TER in the Ca2+ switch, a model widely
used to study the mechanisms of junctional assembly. Treatment of cells
with AlF4
during the
Ca2+ switch had little effect on the kinetics of TER
development in G
i2- or Q205L
i2-MDCK
cells, but PC cells reached half-maximal TER significantly sooner in
the presence of AlF4
(similar times
to G
i2-transfected cells). Base-line TER values obtained
after the switch were significantly higher for all three cell lines in
the presence of AlF4
. These findings
indicate that G
i2 is important for both the maintenance
and development of the TJ, although additional G
subunits are likely
to play a role.
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INTRODUCTION |
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Polarized epithelia have developed highly specialized membrane functions enabling vectorial transport across the cellular layer. The junctional complex of epithelial cells includes gap junctions, adherens junctions, and tight junctions. The tight junction (TJ)1 is the most apical component of the junctional complex and provides two essential functions: (i) the permeability barrier to paracellular fluxes and (ii) the "fence" separating the apical and basolateral membrane domains. In developing tissues as well as cell culture models, the critical signaling events important to junction formation appear to be quite different from mechanisms that maintain junctional integrity. The TJ is composed of a complex of proteins that includes occludin, the only transmembrane protein identified so far (1). There are several peripherally attached membrane proteins found in the TJ including the zona occludens family (ZO-1, -2, and -3) (2-4). ZO proteins are members of the MAGUK (membrane associated guanylate kinase) superfamily that are often found at sites of cell-cell contact and may function to couple extracellular signaling pathways with the cytoskeleton. Other proteins found in or near the TJ include cingulin, 7H6, symplekin, unidentified phosphoproteins, and a series of signal transduction molecules (reviewed in Ref. 5).
MDCK cells are a cultured epithelial cell line that has been extensively utilized for studies of epithelial polarity, targeting of proteins, and the study of intercellular junctions (6). The Ca2+ switch model of TJ formation in MDCK cells has been widely utilized to gain insights into the function of polarized epithelial cells (7-11) and recapitulates many of the critical molecular events of epithelial morphogenesis. MDCK cells cultured in low calcium (µM) lack cell-cell contact, polarity, and junctions. "Switching" to normal calcium medium (NC) triggers a series of molecular events that leads to establishment of the polarized phenotype with characteristics of a tight transporting epithelium. Tight junction development can be followed by measuring the transepithelial resistance (TER), a rapid and reproducible assessment of tight junction integrity. Because MDCK cells are clonal and TJ development can be synchronized in the Ca2+ switch, the role of specific proteins on TJ biogenesis can be studied in this system by cDNA transfections.
The critical role of calcium in the formation of intercellular
junctions is well established. Extracellular calcium is required for
homotypic interactions of E-cadherin and is likely to be the initial
event of junctional complex formation (12). Regulated intracellular
calcium stores are also important for tight junction biogenesis. There
are local increases in intracellular calcium concentration at the
points of cell-cell contact (9), and chelation of intracellular calcium
perturbs TER development (13). Thapsigargin depletes intracellular
endoplasmic reticulum stores of calcium, and thapsigargin treatment of
MDCK cells prior to initiation of cell-cell contact prevents TER
development and the sorting of ZO-1 to the TJ (7). The signaling events
important for TJ biogenesis are complex and utilize a variety of
pathways. Phosphorylation events are important as several proteins
become phosphorylated in the TJ, and protein kinase C (PKC) isoforms
translocate to the TJ during biogenesis. PKC inhibitors markedly
inhibit the development of TER in the calcium switch, and PKC agonists
stimulate ZO-1 translocation to the membrane. The importance of PKC in
tight junction biogenesis, as well as regulated calcium stores,
suggests important roles for heterotrimeric G proteins. The proximity
of several G proteins to the TJ also suggests they may have potential roles in regulating the development and/or maintenance of the TJ.
PKC and PKC
, have also been localized in the vicinity of the TJ
(14-17). We previously demonstrated that expressing a constitutively activated G
o (Q205L) in MDCK cells significantly
accelerated TJ biogenesis without affecting base-line resistances.
Although G
o is a member of the G protein family
inhibited by pertussis toxin (~80% similar to
G
i1-3), its receptors and effectors are distinct, and
furthermore, G
o is not detected in renal epithelia or
MDCK cells (16, 18, 19). Several G
i family members are expressed in epithelial cells, and G
i2 has been shown to
overlap with the tight junction in epithelial cell lines (16, 17). Taken together, these observations raise the possibility that G
i2 may be an important regulator of tight junctions. To
test this hypothesis, we initially looked for effects of
AlF4
(activator of G
subunits) on
tight junctions in control cell lines and then established MDCK cell
lines overexpressing wild type G
i2 and a constitutively
activated G
i2 (GTPase-deficient, Q205L
i2). We find that
AlF4
significantly increases TER in
control cells and accelerates TER development during the
Ca2+ switch. The effects of
AlF4
can be reproduced in MDCK cells
expressing activated G
i2, indicating that this G
subunit is critical to the development and maintenance of tight
junctions.
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EXPERIMENTAL PROCEDURES |
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Plasmid Construction and Cell Culture--
Rat
Gi2 cDNA was cloned into Bluescript (Stratagene) as
described previously (20) and recloned into the EcoRI and
ApaI sites of pcDNA3 (Invitrogen).
Q205L
i2 was provided by Dr. Gary Johnson and cloned into
Bluescript using HindIII sites and then into pcDNA3
using XhoI and XbaI sites. MDCK cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
antibiotics plus 5% fetal calf serum. Transfected cell lines were
maintained in G418 (500 µg/ml; Life Technologies, Inc.)
Transfection--
Subconfluent MDCK cells (ATCC, Manassas, VA)
were transfected with 10 µg of linearized plasmid by calcium
phosphate precipitation method as described previously (16).
G418-resistant colonies were analyzed for increased Gi2
expression by Western blot using a rabbit polyclonal antibody directed
toward the C terminus of G
i2 (AS7; NEN Life Science
Products). Control cells were obtained by transfecting pcDNA3
without insert, and all cell lines were established in parallel.
Tryptic Analysis of Transfected Clones--
Confluent PC-,
Gi2-, or Q205L
i2 -MDCK cells were washed
twice with PBS and then scraped into buffer A (50 mM
Tris-HCl, pH 7.5, 6 mM MgCl2, 75 mM
sucrose, 1 mM dithiothreitol, 1 mM EDTA). Cells
were frozen and thawed three times and triturated ten times through a
27 gauge needle. All samples were incubated at 30 °C with no added
nucleotide or 100 µM GTP
S. Samples were immediately placed on ice, and trypsin was added (20 pmol of
L-1-(tosylamido)-2-phenylethyl chloromethyl ketone-treated
trypsin (Sigma). All samples were incubated at 30 °C for 20 min, and
digestion was terminated by the addition of SDS-polyacrylamide gel
electrophoresis sample buffer followed by boiling for 5 min. Samples
were then analyzed by SDS-polyacrylamide gel electrophoresis and
Western blot using AS7 anti-G
i2 rabbit polyclonal
antibody (1:1,000) and ECL (Pierce) with goat anti-rabbit horseradish
peroxidase (1:10,000).
Immunohistochemistry--
PC-, Gi2-, or
Q205L
i2-MDCK cells were grown on coverslips or Transwell
filters (12 mm) (Costar), rinsed with PBS, and fixed with methanol
(100%,
70 °C) for 10 min. Cells were then washed with PBS and
blocked as described previously (16). Samples were incubated with
rabbit polyclonal G
i2 (AS7, from NEN Life Science Products) at several dilutions and rat monoclonal to ZO-1 (undiluted supernatant; courtesy of D. Goodenough) for 1 h. Cells were washed with PBS three times at 5-min intervals and incubated with secondary antibodies (fluorescein- or Texas Red-conjugated goat anti-rabbit or
anti-rat IgG; Jackson Immuno Research, West Grove, PA) at 1:100 with
for 1 h. Coverslips were visualized on a Nikon Labphot-2 immunofluorescence microscope or a Bio-Rad 1024 confocal microscope using the 63× oil immersion objective. Images were processed in Adobe
Photoshop (Adobe, CA) and figure compiled in Adobe Illustrator (Adobe,
CA).
Ca2+ Switch and Measurement of TER--
MDCK cells
were plated on 12-mm transwell filter (Costar) at confluence (~3 × 105 cells) and allowed to attach for 24-36 h to form a
tight monolayer in normal Ca2+ containing medium (NC).
Cells were placed in low Ca2+ (1-4 µM)
medium (low calcium) for 1 h followed by switch to NC medium. TER
was measured using a Millipore (Bedford, MA) electrical resistance
system, and the results are expressed in ·cm2. TER was
measured in stable monolayers and during Ca2+ switch in the
presence and absence of aluminum fluoride
(AlF4
; 3 mm NaF + 50 µM
AlCl3, Sigma).
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RESULTS AND DISCUSSION |
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Several lines of evidence have suggested the involvement of
heterotrimeric G proteins in tight junction formation. Early studies with G protein modulators such as pertussis toxin, cholera toxin, AlF4, and a variety of other agents
showed variable effects on TJ biogenesis (10). Several confocal studies
have localized G
i2, G
i3, and
G
12 in the vicinity of the tight junction (16, 17, 19,
21). Recently, we demonstrated that G
o (a member of the G
family inhibited by pertussis toxin) expressed in MDCK cells localizes to the subapical lateral membrane overlapping with ZO-1 in
the tight junction (16), and this was subsequently confirmed in another
study (19). A constitutively active mutant of G
o (Q205L)
also localizes in this region, and cells expressing G
o showed no differences in base-line junctional properties as determined by transepithelial resistance. However, in the Ca2+ switch,
MDCK cells expressing activated G
o
(Q205L
o-MDCK) developed tight junctions at twice the
rate and reached significantly higher peak TER values than either
G
o-MDCK or PC-MDCK cells. Although G
o is
not normally expressed in epithelia, this observation raises the
possibility that one of the G
subunits normally found in this
location could have a fundamental role in regulating the development
and/or maintenance of the TJ.
To further examine the role of G protein subunits affecting the TJ,
we studied the effects of the G protein activator
AlF4
on TJ formation in wild type (not
transfected; WT-MDCK) and vector (pcDNA3) transfected MDCK cells
(PC-MDCK). AlF4
has no known effects
on small GTP binding proteins but activates heterotrimeric G
subunits. Crystal structures of G
i1 obtained with GDP
complexed with AlF4
reveal that the
position of the
-phosphate is occupied by
AlF4
. AlF4
in this
position prevents catalysis by immobilizing Gln204 and
Arg178 (22). Fig. 1 shows
that wild type MDCK cells and PC transfected cells cultured on filters
develop significantly higher TER in the presence of
AlF4
. Untreated steady state TER
values were similar between the cell lines, and
AlF4
reproducibly increased TER values
3-4-fold. This finding is consistent with activation of one or more
endogenous G
subunits that results in enhanced steady state
resistances.
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Because AlF4 activates all G
subunits in MDCK cells and several studies have placed
G
i2 in close proximity to the TJ, we tested the
hypothesis that G
i2 was important to this process by
stably expressing G
i2 and a constitutively activated
G
i2 (Q205L
i2) in MDCK cells. The amount
of transfected G
i2 was determined relative to the levels
of endogenous G
i2 in PC-MDCK cells. Western blots of the
three cell lines using identical amounts of total protein were analyzed
(not shown) using NIH image (Wayne Rasband, NIH). Relative to PC-MDCK
cells, the level of G
i2 in G
i2-MDCK cells
was 3.9 ± 0.4-fold (n = 7) increased, and for
Q205L
i2-MDCK the level was of G
i2 was
1.8 ± 0.2-fold (n = 7) above PC-MDCK cells. To
confirm that constitutively activated G
i2 was expressed in these transfected MDCK cells, we utilized a tryptic cleavage analysis of G
i2 (Fig. 2).
This technique has been widely utilized as an indicator of G
subunit
conformation (23, 24) and is based on the observation that G
subunits have a different cleavage pattern depending on whether they
are folded into an active or inactive conformation. In the active
conformation (GTP-liganded), there is only a single tryptic site
accessible near the N terminus (approximately Arg21)
resulting in a slightly truncated protein (39 kDa instead of 41 kDa for
G
i2). In the inactive (GDP-liganded) conformation, an
additional site becomes accessible in the
2 helix or switch region
(near Arg209) resulting in peptides of approximately 25 and
17 kDa. Fig. 2 demonstrates the tryptic cleavage patterns of cell
homogenates from each of the transfected cell lines (PC-,
G
i2-, and Q205L
i2-MDCK cells). In PC- and
G
i2-transfected cells, untreated G
i2
migrates at 41 kDa (first lane of each set) and is
stabilized in the active conformation (39 kDa) if preincubated with the
nonhydrolyzable GTP analogue, GTP
S (last lane of each
set). However, in the absence of added nucleotide (middle
lane of each set) the G
subunits should be GDP bound from
endogenous GTP/GDP in the cell, and G
i2 is cleaved into
25- and 17-kDa fragments with no detectable 39-kDa peptide. The 25-kDa
fragment (derived from the N terminus) is not detectable with the AS7
antibody, and the 17-kDa fragment is more labile (25) and consequently
is not well visualized on these blots (not shown). Tryptic digestion of
Q205L
i2-transfected cells shows that in the absence of
added nucleotide (middle lane), there is a fraction of
G
i2 that is tryptic-resistant, indicating persistence of
G
i2 in an "active" conformation. The amount of G
i2 in the active conformation of
Q205L
i2-MDCK cells is a small fraction of the starting
material. This is due, in part, to the observation that G
subunits
with mutations that result in an active conformation are more sensitive
to proteolysis than wild type G
subunits activated with GTP
S
(20). In addition, it is necessary to do these studies on whole cell
lysates in the absence of protease inhibitors. The Western blots were
deliberately overexposed to look for bands migrating at 39 kDa. This
analysis confirms expression of constitutively activated
G
i2 in Q205L
i2-MDCK cells.
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We have previously demonstrated that WT-MDCK cells express some
Gi2 in the subapical lateral membrane overlapping with
ZO-1 (16). To eliminate the possibility that the transfection process affects G
i2 localization, PC-MDCK cells were
characterized by confocal microscopy. Fig.
3A shows a confocal image of
PC-MDCK cells costained with antibodies to ZO-1 and G
i2
(used at 1:25 dilution). The confocal images reveal that
G
i2 partially colocalizes with ZO-1 at the level of the
tight junction. There is significant intracellular staining that was
also seen in WT-MDCK cells (16). To determine whether transfected
G
i2 subunits were localized in a similar manner to the
endogenous G
i2, the G
i2 antibody (AS7)
was diluted to a point where the endogenous G
i2 was
barely detectable. Fig. 3B shows a confocal analysis of PC-,
G
i2-, and Q205L
i2-MDCK stained and
analyzed under identical conditions using a 1:100 dilution of the
G
i2 antibody. In panel a, PC-MDCK cells only
demonstrate faint intracellular staining, but in panels b
and c, transfected G
i2 and
Q205L
i2 can be visualized in the subapical lateral
membrane overlapping with the TJ marker, ZO-1. Again, there is
intracellular staining that is similar to the endogenous
G
i2 (Fig. 3A). Overall the pattern of
transfected G
i2 and Q205L
i2 is very
similar to that seen with the endogenous G
i2 subunits.
These results confirm that transfected G
i2 and Q205L
i2 partition between the lateral membrane
overlapping with the TJ and intracellular compartments. This finding is
similar to our prior findings with G
o-transfected MDCK
cells (16).
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Because transfected Gi2 and Q205L
i2 were
localized in a manner similar to that of the endogenous
G
i2, we next determined whether G
i2
localization in the TJ had any functional consequences for the tight
junction. PC-, G
i2-, and Q205L
i2-MDCK
cells were simultaneously analyzed under steady state conditions and
also by using the Ca2+ switch. TJ integrity was followed by
measurement of transepithelial resistance. Fig.
4A demonstrates the base-line
resistances in these cells and the pattern of TER development after the
Ca2+ switch. Overexpressing G
i2 had a small
but insignificant (p = 0.07) effect on base-line
resistances in comparison with PC-MDCK cells (576 ± 89 versus 377 ± 52; n = 12), but
Q205L
i2-MDCK cells had significantly higher base-line
TER values (1187 ± 150
·cm2; p < 0.001). The base-line TER values for G
i2- and PC-MDCK
cells were similar to reported values of G
o (16), and
several clones were analyzed with no significant differences seen among
the clones. To gain insight into the mechanism of higher TER values
observed in Q205L
i2-MDCK cells, all three cell lines
were simultaneously analyzed in the Ca2+ switch. The
elevated TER in Q205L
i2-MDCK cells could be achieved by
differences in the kinetics of TER development. Nonlinear regression analysis of the TER data between 0-12 h for all of the cell lines (Fig. 4A) indicates an asymptotic approach to peak TER.
Although the data do not precisely fit standard kinetic models, the
kinetics of TER development in these cells is similar to what has been reported in other studies (15, 26). The time to half-maximal TER is a
useful value for discussing the effects of G
i2
expression on TER biogenesis, and these values were calculated for each
cell line in the presence and absence of
AlF4
(Table
I). Table I shows that the time to
half-maximal TER (T50) was significantly more
rapid in G
i2- and Q205L
i2-MDCK cells
(0.8 ± 0.3 and 1.2 ± 0.3 h, respectively) in
comparison with PC cells (3.0 ± 0.5 h).
AlF4
had no significant on
G
i2-transfected cells but significantly shortened
T50 for PC cells (1.1 ± 0.7 h, a
value similar to that of G
i2 cells). The observation
that PC-MDCK cells treated with AlF4
develop TER nearly as rapidly as Q205L
i2-MDCK cells
suggests that G
i2 may be the predominant G
subunit
critical in TER development. Fig. 4B shows that the
base-line TER values for the three cell lines on the day following the
Ca2+ switch (26 h) are significantly higher in the
presence of ALF4
. In contrast to
AlF4
effects on the rate of TER
development, all three cell lines had significantly increased base-line
TER at 26 h in the presence of
AlF4
. Similar effects of
AlF4
were seen with the three cell
lines cultured in the steady state (not shown). This raises the
possibility that AlF4
activates
additional G
subunits in the steady state that enhances transepithelial resistance.
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Taken together, these studies offer direct evidence that
Gi2 is a critical regulator of tight junction biogenesis
and affects base-line characteristics of the tight junction. The
protein composition of the TJ is complex with one integral membrane
protein identified so far (occludin), several peripherally attached
proteins with partially defined functions (including ZO-1, -2, and -3)
and a variety of signal transduction molecules including PKC isoforms, G
subunits, and tyrosine kinases (see Ref. 5 for review). How these
diverse proteins function to maintain and regulate the development of
tight junctions is not well understood. G proteins could be activated
within the TJ through a classical seven-transmembrane receptor
(although none yet identified in the TJ) or alternatively through a
modulatory protein that promotes GDP release or slows GTP hydrolysis.
Additional transmembrane proteins must exist within the TJ (27), and
there are multiple examples of modulatory proteins that affect G
protein function. GTPase activating proteins (RGS proteins;
regulators of G protein signaling;
reviewed in Ref. 28) interact with G
subunits, and nucleotide
exchange factors that promote GDP release have been described for many
small G proteins such as Ras (29). Although analogous proteins for G
subunits have not yet been identified, such proteins may exist and
could provide mechanisms for activation of G
subunits in the TJ or
within intracellular compartments (30). Our findings that
G
i2 is important for both the maintenance and
development of the TJ does not exclude roles for other G
subunits,
and in fact the effects of AlF4
on the
steady state TER suggests that other G
subunits are likely to
enhance this barrier.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM55223 (to B. M. D.) and DK53507 (to S. K. N.) and a March of Dimes Basil O'Connor Award (to B. M. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a National Research Service Award.
§ Established Investigator of the American Heart Association.
¶ To whom correspondence should be addressed: Harvard Inst. of Medicine, 77 Ave. Louis Pasteur, Boston, MA 02115. Tel.: 617-525-5809; Fax: 617-525-5830; E-mail: bdenker{at}rics.bwh.harvard.edu.
The abbreviations used are:
TJ, tight junction; MDCK, Madin-Darby canine kidney; G protein, guanine nucleotide-binding
protein; ZO, zona occludens; TER, transepithelial resistance; PKC, protein kinase C; NC, normal calcium; PBS, phosphate-buffered saline; GTPS, guanosine 5'-3-O-(thio)triphosphate.
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REFERENCES |
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