Departments of 1 Pharmaceutical Sciences, 2 Molecular Pharmacology and Toxicology, 3 Physiology and Biophysics, 4 Biomedical Engineering, and 5 Medicine, 6 Will Rogers Institute Pulmonary Research Center, and 7 Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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Etk/Bmx is a member of the Tec family of cytoplasmic
non-receptor tyrosine kinases known to express in epithelial cells. We demonstrate herein that Etk activation in stably Etk-transfected epithelial Pa-4 cells resulted in a consistently increased
transepithelial resistance (TER). After 24 h of hypoxic (1%
O2) exposure, the TER and equivalent active ion transport
rate (Ieq) were reduced to <5% of the normoxia
control in Pa-4 cells, whereas both TER and Ieq
were maintained at comparable and 60% levels, respectively, relative
to their normoxic controls in cells with Etk activation. Moreover, Pa-4
cells exhibited an abundant actin stress fiber network with a diffuse
distribution of -catenin at the cell periphery. By contrast,
Etk-activated cells displayed a redistribution of actin to an
exclusively peripheral network, with a discrete band of
-catenin
also concentrated at the cell periphery, and an altered occludin
distribution profile. On the basis of these findings, we propose that
Etk may be a novel regulator of epithelial junctions during
physiological and pathophysiological conditions.
signal transduction; adaptive response
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INTRODUCTION |
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AN IMPORTANT CHARACTERISTIC of the epithelium is that it forms a functional barrier to prevent the permeation of noxious agents to the internal milieu and, at the same time, to allow passive vectorial transepithelial transport of nutrients, electrolytes, and small solutes. Several disease-associated states, such as ischemic and hypoxic injuries, effectively disrupt the permeability barrier as well as enhance the movement of ions, large solutes, and inflammatory cells across tight epithelial structures (7, 13, 17, 24, 48). Hence, a better understanding of the mechanism underlying the regulation of epithelial barrier function is of potential physiological and pharmacological significance. Although the structure and function of junctional barriers have been extensively studied, the molecular signaling pathways that modulate assembly, disassembly, and maintenance of junctional integrity under various health and disease states appear to be multifactorial and rather complex.
The epithelium contains at least four classes of intercellular
junctions: tight junctions (TJs), adherens junctions (AJs), desmosomes,
and gap junctions. Among these classes, TJs are the most critical in
forming barriers to the diffusion of solute through the paracellular
pathway, whereas AJs play a key role in the formation of tight
junctions (10). TJs are also essential for the
polarization of epithelial cells, since they form a boundary between
apical and basolateral plasma membrane domains. Structurally, TJs
comprise several transmembrane proteins, such as occludin and members
of the claudin family, and peripheral membrane proteins, such as zonula
occludens (ZO)-1, ZO-2, ZO-3, cingulin, 7H6, and symplekin (15). The peripheral membrane protein ZO-1 binds to actin
filaments, directly or through a linking protein, serving thus as a
candidate for coupling perijunctional actin to the paracellular barrier (15). The basic components of AJs in epithelial cells
include transmembrane protein E-cadherin and the cytoplasmic proteins -,
-, and
-catenins, which link E-cadherin to the actin
cytoskeleton (9, 21). Among them, E-cadherin is
responsible for the correct establishment and maintenance of AJs
through a Ca2+-dependent homophilic interaction with
adjacent cells. Blocking the function of E-cadherin results in a
destruction of AJs and subsequent disassembly of TJs (31).
On the other hand, catenins tether the cadherin complexes to actin
cytoskeleton and have often been investigated as potential cytoplasmic
targets for regulation of AJs (19). Together, the
expression and modification of these TJ and AJ molecules determine the
permeability properties of epithelial barriers toward hydrophilic
solutes and plasticity of intercellular junctions.
In addition to the structural interdependence between TJs and AJs, agents that disrupt the actin cytoskeleton can also lead to the disassembly of TJs (2, 22). This observation is consistent with a model in which the establishment of appropriate actin cytoarchitecture is a key factor in the formation of TJs (15). This notion is further supported by the observation that TJs appear to be tethered to the actin filaments (12). Hence, both actin filaments and AJs appear to participate directly or indirectly in the formation and/or maintenance of TJs. While AJ complexes are primarily involved in maintaining cell-cell adhesions between adjacent epithelial cells, TJ structures modulate epithelial barrier function and paracellular permeability.
The results from many laboratories have suggested that a complex set of
signal transduction pathways is likely to target and control the
junctional properties of epithelial cells. The barrier function of TJs
is reportedly influenced by growth factors, extra- and intra-cellular
Ca2+ levels, protein kinase C, receptor and non-receptor
tyrosine kinases, and phospholipase C in different types of epithelial cells (3, 4, 6, 16, 19, 27, 28, 42, 54, 55). In terms of
AJs, significant tyrosine phosphorylation of -catenin,
-catenin,
and p120-catenin is detected in proliferating epithelial cells
(35). As epithelial cells reach confluence and undergo the
process of contact inhibition, tyrosine phosphorylation of catenins
decreases. This observed decrease in tyrosine phosphorylation is
correlated with an increased tyrosine phosphatase activity (5,
11). Many receptor and non-receptor tyrosine phosphatases have
been coimmunoprecipitated with cadherin-catenin complexes. Thus
components of TJs, AJs, plasma membranes, and cytoskeleton are all
potential targets for these already-identified or to-be-identified kinases and phosphatases. The biochemical basis of these modulations by
signaling molecules is only beginning to be unraveled. The focus of
this study is the characterization of the effects of a novel epithelial
tyrosine kinase, Etk, on paracellular permeability and junctional
protein complexes of a model epithelial barrier, Pa-4.
Etk, also named Bmx, belongs to a new class of cytoplasmic non-receptor tyrosine kinases, Btk/Tec, members of which are expressed in both hematopoietic and nonhematopoietic cells. This family consists of Btk (46, 50), Itk (18, 39), Tec (30), and Etk/Bmx (34, 43) tyrosine kinases, which share homologous structures, including the NH2-terminal pleckstrin homology (PH) domain, followed by Tec homology (TH), Src homology (SH) 3, SH2, and tyrosine kinase domains. These Tec kinases have been demonstrated to participate in signaling pathways involving a variety of cytokine receptors and antigen receptors. Etk, unlike other members of the Tec family kinases that are mostly hematopoietic cell specific, is preferentially expressed in epithelial cells (34). We have previously demonstrated that Etk directly activates signal transducer and activator of transcription (STAT) 1, STAT3, and STAT5 in salivary epithelial cells by using an estrogen (E2)-inducible Etk construct (52). To date, the precise biological function of Etk in epithelial cells has not been defined.
To better understand the molecular nature of salivary epithelial
cellular responses to the activation of Etk, we have established a
model system by stably transfecting rat salivary epithelial Pa-4 cells
with an inducible Etk-estrogen receptor (ER) chimeric construct
(Etk:ER), which is activated in cells by the ER ligand,
-estradiol. Through a combined approach in search of Etk-mediated biological events, we identify here a series of effects of Etk on
transepithelial resistance (TER) in parallel with components of AJs and
TJs. We have demonstrated that Etk activation is sufficient to elicit
an increase in TER, a common gauge of junctional tightness between
epithelial cells, and have further shown that this increase was
sustained in response to hypoxic challenge. The increased TER elicited
by Etk activation was accompanied by changes in actin filaments
organization and in the recruitment and/or changes in protein
properties of several peripheral and integral membrane proteins
involved in TJ and AJ regulation. Together, we postulate that the
functional and biochemical modulation of TJ and AJ during normoxia and
hypoxia appears to be dependent on an Etk-mediated signaling cascade.
Moreover, our results shown herein suggest that the cells with stable
expression of
Etk:ER represent a unique and useful tool to elucidate
the role of Etk in modulating TJ/AJ assembly/disassembly and subsequent
responses to hypoxic challenge in salivary epithelial cells as well as
in other cell types. These observations are significant in
understanding the physiological regulation of epithelial permeability
and discerning the mechanism leading to epithelial permeability
dysfunction(s) associated with many disease states.
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MATERIALS AND METHODS |
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Reagents.
Rhodamine-phalloidin, phenylmethylsulfonyl fluoride (PMSF), aprotinin,
pepstatin A, N-tosyl-L-phenylalanine
chloromethyl ketone, leupeptin,
N--p-tosyl-L-lysine chloromethyl
ketone, and N-
-p-tosyl-L-arginine methyl ester were all obtained from Sigma (St. Louis, MO). Goat anti-mouse horseradish peroxidase-conjugated secondary antibody and
enhanced chemiluminescence (ECL) reagents were from Amersham (Arlington
Heights, IL). The cell culture media, sera, and antibiotics were from
Life Technologies (Rockville, MD).
Cell culture.
The rat parotid epithelial cell line Pa-4, also known as parotid C5
cells (26), was plated on Primaria culture dishes (Falcon) in Dulbecco's modified Eagle's/Ham's F-12 (1:1) medium supplemented with 2.5% fetal calf serum, insulin (5 µg/ml), transferrin (5 µg/ml), epidermal growth factor (25 ng/ml), hydrocortisone (1.1 µM), glutamate (5 mM), and kanamycin monosulfate (60 µg/ml) and was
maintained in a humidified atmosphere of 5% CO2-95% air
at 35°C. The Pa-4Etk:ER cells were established by stably
transfecting Pa-4 cells with
Etk:ER. The tyrosine kinase activity of
Etk:ER in Pa-4
Etk:ER cells can be further induced by the addition
of 1 µM estrogen receptor agonist,
-estradiol, to the culture
medium, as demonstrated by the autophosphorylation of Tyr-566 of Etk
(52). The Pa-4
Etk:ER cells were maintained with
geneticin (G418; 600 µg/ml) and Dulbecco's modified Eagle's/Ham's
F-12 (1:1, phenol red free) medium supplemented with 2.5%
charcoal-stripped fetal calf serum plus the aforementioned ingredients.
Madin-Darby canine kidney (MDCK)
Etk:ER cell clones were established
and screened as described previously (52).
Measurements of TER.
Epithelial cells were grown on permeable membranes (Clearwell;
Costar-Corning, San Francisco, CA) that allow visual monitoring growth
of polarized epithelial cells to confluence. Bioelectric parameters of
cell monolayers were monitored at predesignated time intervals with a
MilliCell ERS screening device (Millipore, Bedford, MA) that can
measure spontaneous potential difference (SPD; expressed in mV, taking
the apical aspect as reference) and TER (expressed in
k · cm2) with chopstick-style electrodes.
Background potential difference (PD) arising from the asymmetry of
voltage-sensing electrodes and electrical resistance contributed by
both the bathing fluids and the filter membrane were measured and
averaged by using the values observed at the beginning and end of each
set of SPD and TER measurements from two blank filters bathed with the
same medium utilized in cultivation of epithelial cells. These
background PD and electrical resistance values were subtracted from the
raw data of SPD and TER, respectively. With the use of the corrected SPD and TER, equivalent active ion transport rate
(Ieq; equivalent short-circuit current) is
estimated as SPD/TER, assuming the prevalence of Ohm's law for a given
epithelial cell monolayer system.
Confocal fluorescence microscopy.
Pa-4 and Pa-4Etk:ER cells were cultured and exposed to 1 µM
E2 for 4 h before they were rinsed with Dulbecco's
PBS (DPBS). For
-catenin detection, cells were processed by
following the procedures reported by Woo et al. (55).
Briefly, cells were fixed with 2% paraformaldehyde in PBS, followed by
exposure to PBS supplemented with 50 mM NH4Cl for 5 min,
and then permeabilized for 10 min in PBS supplemented with 0.5% Triton
X-100 (Tx-100) before being blocked and exposed to anti-
-catenin
antibody and an appropriate secondary antibody. Rhodamine-phalloidin
was used to detect actin filaments. For analysis of the effects of
hypoxia on F-actin and
-catenin, confluent Pa-4 and Pa-4
Etk:ER
cells were exposed to 1 µM estradiol for 4 h before being
exposed to hypoxia for 16 h. Cells were then fixed and processed
as described above for detection of F-actin and
-catenin.
Isolation and Western blot analysis of soluble and insoluble
protein fractions.
Cells were cultured on petri dishes, exposed to 1 µM E2
for 4 h, and then washed twice with warm DPBS. These washed cells were incubated in a cell lysis buffer solution (pH 6.75) containing 0.1 M PIPES, 1 mM EGTA, 1 mM MgSO4, 2 M glycerol, 1% Tx-100, 1 mM PMSF, 1 µg/ml pepstatin A, 10 µg/ml
N-tosyl-L-phenylalanine chloromethyl ketone, 1 µg/ml leupeptin, 1 mM sodium orthovanadate, 10 µg/ml
N--p-tosyl-L-lysine chloromethyl
ketone, and 10 µg/ml N-
-p-tosyl-L-arginine methyl ester
for 10-15 min. Some experiments utilized the buffer above
containing 0.1% Nonidet P-40 (NP-40) in place of Tx-100 to isolate
detergent-soluble fractions (51). However, results were
comparable when either detergent was used to isolate cytosol and
detergent-soluble membranes. After the soluble (cytosolic and
detergent-soluble membrane proteins) fraction of cell lysates was
harvested by pipetting, the remaining cellular materials on the dish
representing the insoluble fraction were scraped into RIPA composed of
1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate
(SDS), 1 mM PMSF, 20 µg/ml aprotinin, and 1 mM sodium orthovanadate
in PBS. Unless indicated, equal amounts (between 10 and 30 µg) of soluble and insoluble fractions of cell lysates were diluted
with 2× SDS sample buffer, resolved on 7.5% polyacrylamide gels, and
electroblotted onto Immobilon-P (Millipore) or nitrocellulose
membranes. Immunoprecipitation of
Etk:ER with an anti-ER antibody
(HC-20; Santa Cruz Biotechnology, Santa Cruz, CA) was carried out as
described previously (25), followed by immunoblot
analyses using anti-phosphotyrosine antibody (4G10; Upstate
Biotechnology). Blots were probed with appropriate primary antibodies
(occludin, phosphotyrosine, or estrogen receptor) and goat anti-rabbit
or anti-mouse secondary antibody, where appropriate, conjugated to
horseradish peroxidase before visualization with an ECL detection system.
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RESULTS |
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Activated Etk:ER is translocated to a detergent insoluble pool.
The Btk/Tec family of kinases is so far the only known tyrosine kinase
family to carry an NH2-terminal PH domain. The versatile roles of Btk/Tec kinases are reflected by their protein-protein and
protein-lipid interaction through the PH domain (for reviews, see Refs.
29 and 58). Accumulating evidence has suggested that PH
domains of Btk/Tec family members are able to interact with F-actin
(59). The actin cytoskeleton plays an essential role in a
variety of cellular processes including cell division, shape, and
motility, to name a few. Hence, we explored the possibility of
translocation of
Etk:ER from the cytoplasm to the membrane upon its
activation to further study the role of Etk activation in epithelial
cell signaling.
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Phenotypic manifestation of epithelial cells that express Etk.
To investigate the role of Etk in the regulation of epithelial cell
physiology, both Pa-4Etk:ER and parental Pa-4 cells were grown to
form confluent monolayers on polyester Clearwell, and their TER values
were measured. As shown in Fig. 2, TER in
stably transfected and E2-stimulated Pa-4
Etk:ER as
well as MDCK
Etk:ER epithelial cells reached a higher
level than that in corresponding parental cells. The
corresponding Ieq for
E2-stimulated parental and Pa-4
Etk:ER cell monolayers
were 1.85 ± 0.08 and 1.08 ± 0.09 µA/cm2, respectively. Because the measurements of both
potential difference (SPD) and TER in MDCK cells were too low to give
reproducible calculations of Ieq, the calculated
Ieq values from MDCK and MDCK
Etk:ER cells are not shown. The observed difference in TER between parental and Etk-activated cells was persistent throughout an 8-day period of
measurement (data not shown). This suggests tightening of the paracellular seals upon Etk activation. The TER of parental Pa-4 and
MDCK cells are quite different in that Pa-4 cells exhibit intrinsically
higher TER than do the MDCK cells (Fig. 2). Thus Etk activation
enhances TER in epithelial barrier of either leaky or tight nature. The
epithelial barrier to the diffusion of hydrophilic solutes through the
paracellular pathway is afforded by TJ. Permeation across TJ is not
static but is dynamically regulated under physiological environment and
under pathophysiological conditions, such as hypoxia. In particular,
epithelial cells have been reported to respond to hypoxic stress,
rendering the depletion of ATP and causing the loss of TER
(12).
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Etk-induced enhancement of TER in response to hypoxia involves regulation of the actin cytoskeleton. One of the injurious effects of hypoxia on cells is to induce actin depolymerization (23, 37). Because the ability of the TJ to form a seal is dependent on the actin filaments organization (for a review, see Ref. 15), we investigated whether the hypoxia-induced reduction in TER might be mimicked by disassembly of actin and, further, whether Etk activation could preserve TER under conditions of actin filaments loss. Latrunculin B, an actin-depolymerizing agent (20), was utilized for this purpose.
As shown in Fig. 4, during 24-h treatment of latrunculin B, the TER values of E2-treated Pa-4 and Pa-4
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Etk activation reduces the mobility of occludin on SDS-PAGE.
To investigate whether comparable changes in elements of TJs were
also elicited by Etk activation, we next examined the cellular distribution of tight junctional proteins, occludin, claudin, and ZO-1
in Etk-activated Pa-4Etk:ER and parental Pa-4 cells. Immunofluorescence studies of these cell monolayers demonstrated that
occludin was localized at the cell membrane in a comparable manner in both monolayers (Fig. 7). The
tight junctional peripheral protein ZO-1 and the transmembrane protein
claudin also exhibited a similar distribution in both Pa-4 and
Pa-4
Etk:ER cells, where no marked changes in ZO-1 or claudin
localization were detected as a result of Etk activation (data not
shown). Hence, the localization of occludin, ZO-1, and claudin did not
appear to be significantly modulated by Etk activation.
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Increased TER after Etk activation is dependent on tyrosine kinase
activity.
Etk is a non-receptor tyrosine kinase. To establish the mechanistic
role of Etk in the demonstrated enhancement of TER in Etk:ER-transfected cells, as shown in Fig. 2, we utilized a widely used tyrosine kinase inhibitor, genistein (Fig.
9). Treatment of genistein caused
decreases of TER in Pa-4, Pa-4
Etk:ER, and E2-stimulated
Pa-4
Etk:ER cell monolayers over the 24-h measurements. The rate and
extent of genistein-induced TER decreases were barely distinguishable
between Pa-4 and Pa-4
Etk:ER monolayers in the absence of
E2-treatment. However, decrease of TER in
E2-activated Pa-4
Etk:ER cell monolayers in response to
genistein at both 1 µM and 50 µM was more pronounced than in those
cell monolayers without E2-activation (Fig. 9). Moreover,
genistein-elicited TER decreases in both Pa-4 and Pa-4
Etk:ER cell
monolayers, except for exposure to extremely high concentrations (e.g.,
200 µM) of genistein, were reversible after 16 h of treatment
(data not shown). Although genistein is not a specific Etk inhibitor , our results demonstrate that the catalytic activity of tyrosine
kinase(s) is necessary to maintain TER in both Pa-4 and Pa-4
Etk:ER
cell monolayers. The observation that Pa-4
Etk:ER cells (with
E2) are more sensitive to genistein than the parental Pa-4
and Pa-4
Etk:ER cells (without E2) is also consistent
with our hypothesis that Etk activation enhances epithelial barrier
function and that Etk may be the critical tyrosine kinase involved in
this signaling pathway in epithelial cells.
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DISCUSSION |
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We sought to develop a cell system that could serve as a useful
model to provide insights into the function of Etk and to determine
cellular events following Etk activation in both physiological and
pathophysiological conditions. To this end, we demonstrated conclusively that Etk activation increases TER in both Pa-4 and MDCK
epithelial cells under normoxia (Fig. 2). We also showed that prolonged
hypoxia without reoxygenation results in a loss of TER and
Ieq in the parental Pa-4 epithelial cells (Fig.
3). By contrast, Etk activation resulted in an enhanced TER and, thus, cell protection against injury from prolonged hypoxia, allowing the
Pa-4Etk:ER cells to maintain their TER and
Ieq under hypoxic stress (Fig. 3).
The increased TER shown in Pa-4Etk:ER cells was associated with
changes in the organization and localization of components of AJs,
including actin filaments and
-catenin (Fig. 5). Moreover, the
hypoxia-induced changes in F-actin and
-catenin were prevented by
prior activation of Etk. Although no prominent changes in cellular localization of constituents of TJs, such as occludin and ZO-1, were
detected in Pa-4
Etk:ER (Fig. 7 ), the mobility of occludin prepared
from Pa-4
Etk:ER cells was markedly reduced on SDS-PAGE (Fig. 8),
suggesting that Etk activation renders increased occludin phosphorylation.
We also demonstrated that elements involved in the maintenance of TER
in both Pa-4 and Pa-4Etk:ER cells are sensitive to the treatment of
a known tyrosine kinase inhibitor, genistein (Fig. 9). The literature
on the role of tyrosine phosphorylation in TJ and AJ
assembly/disassembly is somewhat controversial and inconclusive. For
example, as epithelial cells reach confluence and undergo the process
of contact inhibition, tyrosine phosphorylation of catenins decreases.
This observed decrease in tyrosine phosphorylation in catenins is
correlated with an increased association of tyrosine phosphatase
activity (5, 11). Considerable circumstantial evidence
also implicates tyrosine phosphorylation in the disassembly of
cadherin-mediated cell-cell adhesion. Specifically, expression of
constitutively activated v-Src oncoproteins, which induce tyrosine phosphorylation of
- and p120-catenins and E-cadherin, leads to the
loss or weakening of AJs (32, 41). However, very little information is available on the regulation of AJs by other tyrosine kinases such as Etk that may be more involved in the modulation of cell
function, rather than proliferation and differentiation afforded by Src
tyrosine kinase activation.
Occludin has been a prime target for a number of signaling pathways
involved in the regulation of TJs, and the level of occludin tyrosine
phosphorylation has been reported to be correlated with the TER level
(8, 47). In these reports, increased occludin tyrosine
phosphorylation is shown to be associated with the reassembly of TJs
after ATP depletion and TJ restoration following "rescue" from
Ras-mediated transformation in MDCK cells. We have now demonstrated by
functional, immunocytochemical, and biochemical analyses that Etk-activated cascades enhance TJ/AJ assembly under normoxia and hypoxia. However, it is plausible that other phosphorylation targets in
addition to occludin exist in the AJ and TJ complexes that may also be
putative. The positioning of these junctions is coordinated and
stabilized through an association with a continuous band of bundled
actin filaments, known as an adhesion belt (27). However, it is unclear how the expression and function of each TJ and/or AJ
molecule(s) are regulated to confer the overall epithelial barrier
function. A profound Etk-induced reorganization of actin cytoskeleton
into bundles of peripherally localized filaments may influence TER, as
has been previously suggested (12, 22). In fact, we
propose that reorganization and stabilization of actin filaments may be
one of the principal functions of Etk, whether or not additional direct
regulation of AJ or TJ components occurs. The proposal that Etk serves
as a major regulator of actin cytoskeleton is derived from the
observations of 1) the dramatically improved barrier
function against hypoxic stress (Fig. 3), 2) the observed reorganization of F-actin and -catenin by Etk (Fig. 5),
3) the blockage of effects on F-actin and
-catenin in
cells exposed to hypoxia (Fig. 6), and 4) the preservation
of TER from latrunculin B-treated Pa-4
Etk:ER cells (Fig. 4).
Moreover, Btk, closely related to Etk, has been shown to colocalize
with actin filaments upon stimulation (49, 59). In a
separate study, the activated Bmx-green fluorescent protein (GFP) has
been demonstrated to be localized in the membrane, while the resting
Bmx-GFP is restricted to the cytoplasm (14), further
supporting our notion.
Several studies have explored the relationship between hypoxia and disassembly of actin filaments. For instance, hypoxia induces dephosphorylation and activation of actin depolymerization factor (ADF)/cofilin, an actin regulatory protein that mediates cellular actin dynamics (37). Cofilin dephosphorylation is associated with acceleration of actin exchange on filament polymerization as well as loss of F-actin. Enhancing or preserving ADF/cofilin phosphorylation is, therefore, one way of preserving cellular F-actin. Recent work has implicated two LIM kinases in regulation of ADF/cofilin phosphorylation and actin dynamics: LIM- kinase 1 via a Rac-mediated pathway (1, 57) and LIM-kinase 2 via a Rho and/or Cdc42-mediated pathway (40). If Etk-induced protection from hypoxic injury involves prevention of ADF/cofilin-induced actin disassembly, this could occur either by direct activation of LIM-kinases or indirectly through actions on Rho-, Rac- or Cdc42-based signaling pathways. Etk may also act to alter effectors of actin filaments assembly other than ADF/cofilin. The membrane association of activated Etk also implicates the possibility that Etk is involved in Rho/Rac/Cdc42-mediated signaling pathways.
Hypoxic injury results in a disturbance in intra- and intercellular homeostasis ranging from the depletion of intracellular ATP to development of intracellular acidosis, decreased redox buffer capacity, and rearrangement of actin-based cytoskeleton (37). Theoretically, such processes could compromise the integrity of many epithelia, rendering an inability to function as an effective barrier to prevent leakages of small solutes to HMW proteins and to modulate the passage of inflammatory cells. For example, ATP depletion resulting from hypoxia or through usage of metabolic inhibitors in polarized epithelial cells has been demonstrated to cause perturbations in the actin cytoskeleton as well as disruption of TJ structures (38). Although several candidate genes and their regulatory mechanisms identified in response to oxidative stresses induced by intracellular oxygen radical production or cell penetration by oxidants are known, eukaryotic signal transduction and gene regulatory pathways that respond to hypoxia have only begun to unfold. In addition to altering epithelial cell behavior and function, hypoxia initiates a novel gene expression program (38) that culminates in a variety of changes in downstream events. Presumably, the balance between phosphorylation and dephosphorylation status is one of the means to render the ultimate biological manifestation as a result of hypoxia. However, little information on the kinase(s) or phosphatase(s) involved in hypoxia-related responses is available to date.
On the basis of our results, we hypothesize that the Etk activation in
Pa-4 and also possibly in MDCK cells may "prime" these cells
against hypoxic injury. In particular, Etk activation may upregulate
the ATP-producing pathway(s) by improving their stoichiometric efficiency via phosphorylation/activation modalities or by inducing the
expression of genes for ATP-supplying glycolytic enzymes. Alternatively, Etk activation may repress the activity or expression of
the less required enzymes or pathways that consume ATP. These working
hypotheses are consistent with our observations that 1) Ieq was sustained under prolonged hypoxic
conditions in cells with activated Etk (Fig. 3B),
2) the injurious effect on TER elicited by latrunculin B was
rapidly ameliorated by Etk activation (Fig. 4), and 3) the
organization of actin-based cytoskeleton and the assembly of TJ were
altered concomitantly with the augmentation of sealing function of TJs
in the stimulated Pa-4Etk:ER cells (Figs. 2, 3, and 8). Moreover,
our preliminary results demonstrated that Etk activation enhances
hypoxia-response element-dependent gene activation (C. Chau and D. K. Ann, unpublished observation), further supporting our notion. The
reported role of Etk/Bmx protein in cell survival and apoptosis
suggests rather complex functions for this protein. For example, Etk
was shown to be essential for interleukin-6-induced neuroendocrine
differentiation (34) and for protection against
therapy-induced apoptosis (56) in prostate cancer
cells. On the other hand, overexpression of Bmx in 32D myeloid
progenitor cells resulted in apoptosis in the presence of granulocyte colony-stimulating factor, while cells expressing a
kinase dead mutant of Bmx differentiated into mature granulocytes (14). Moreover, Bmx was shown to reconstitute
apoptosis in Btk-deficient chicken B cells (44)
and to link Src to STAT3 activation in Src-mediated cell transformation
(45). Together, these findings imply that Etk/Bmx is more
than likely to have distinct functions in different cell types, even in
the same cell lineage during stages of development and differentiation
or exposure to external environmental stimuli. In accordance with our
present results, Etk/Bmx activation may be involved in modulating cell
response to extreme environmental conditions, such as maintaining TER
during prolonged hypoxic condition, in addition to regulating cell
proliferation and differentiation.
In summary, this study provides the first direct evidence demonstrating
that activated Etk enhances TER under resting conditions and that it
sustains TER as well as maintains barrier function under prolonged
hypoxia. On the basis of the data presented herein, we envision two
potential signaling pathways by which Etk activation and its downstream
events enhance and stabilize epithelial barrier function. As depicted
in Fig. 10, we postulate that Etk
restores epithelial TER properties by preventing the disassembly of
intercellular TJ through a novel signaling pathway, even in the face of
hypoxic insult. In the first pathway, tight junctional seal and, thus, TER are enhanced and/or maintained by Etk activation via stabilization of the actin cytoskeleton, as noted above. In the second pathway, in
response to hypoxia, Etk acts directly on elements of TJs such as
occludin to enhance TJ integrity. To our knowledge, this is the first
report on a non-receptor tyrosine kinase, Etk, that enhances epithelial
barrier function by biochemical modulation of TJ/AJ components in
epithelial cells. Specific mechanisms and target proteins involved in
the possible regulation of actin filaments and in the regulation of TJ
by Etk activation remain future challenges.
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ACKNOWLEDGEMENTS |
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This work is supported in part by National Institutes of Health Grants DE-10742, EY-11386, HL-38658, HL-64365, and DK-48522 and by American Heart Association Grant-in-Aid 9950442N.
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FOOTNOTES |
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* A. Chang and Y. Wang contributed equally to this work.
Address for reprint requests and other correspondence: D. K. Ann, Univ. of Southern California, PSC-210B, Health Sciences Campus, 1985 Zonal Ave., Los Angeles, CA 90033 (E-mail: ann{at}hsc.usc.edu).
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.
Received 16 November 2000; accepted in final form 12 February 2001.
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