1 Intestinal Disease Research Programme and 2 Infection and Immunity Programme, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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Signal transducers and
activators of transcription (STATs) are critical intracellular
signaling molecules for many cytokines. We compared the ability of T84
epithelial cells to activate STATs in response to cytokines
[interferon- (IFN-
), interleukin (IL)-4, IL-10, and tumor
necrosis factor-
(10 ng/ml)] and conditioned medium from
superantigen [Staphylococcus aureus enterotoxin B (SEB)]-activated peripheral blood mononuclear cells (PBMC) using electrophoretic mobility shift assays (EMSA). Of the cytokines tested,
only IFN-
caused a STAT-1 response. Exposure to SEB-PBMC-conditioned medium resulted in STAT-1 or STAT-1/3 activation, and inclusion of
anti-IFN-
antibodies in the conditioned medium abolished the STAT-1
signal. Cells treated with transcription factor decoys, DNA
oligonucleotides bearing the STAT-1 recognition motif, and then
SEB-PBMC-conditioned medium displayed a reduced STAT-1 signal on EMSA,
yet this treatment did not prevent the drop in transepithelial resistance (measured in Ussing chambers) caused by SEB-PBMC-conditioned medium. In contrast, the phosphatidylinositol 3'-kinase (PI 3-K) inhibitor LY-294002 significantly reduced the drop in transepithelial resistance caused by SEB-PBMC-conditioned medium. Thus data are presented showing STAT-1 (±STAT-3) and PI 3-K activation in epithelial cells in response to immune mediators released by superantigen immune
activation. Although the involvement of STAT-1/-3 in the control of
barrier function remains a possibility, PI-3K has been identified as a
regulator of T84 paracellular permeability.
intestine; Staphylococcus aureus enterotoxin B; phosphatidylinositol 3'-kinase
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INTRODUCTION |
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CYTOKINE LIGATION OF
the appropriate receptor can mobilize one, or more, intracellular
signaling pathways, including activation of Janus kinase (JAK)-signal
transducers and activators of transcription (STAT), nuclear factor-
(NF-
) mitogen-activated protein kinases (MAPK),
sphingomyelinase-ceramide, and phosphatidylinositol 3'-kinase (PI 3-K), all of which can eventually result in regulation of
gene transcription. Thus, although cytokine effects on epithelia (cell
lines in vitro and to a lesser extent studies with tissue segments ex
vivo) are well documented (26), the intracellular signaling cascades that mediate the responses are less well defined, particularly in terms of the integration of responses to multiple messengers (3).
Recently, the JAK-STAT pathway has been highlighted as an important
membrane-to-nucleus signaling pathway for many cytokines (15). Briefly, binding of specific cytokines to their
receptors results in JAK phosphorylation followed by the recruitment,
tyrosine phosphorylation, and dimerization of cytosolic STAT monomers. The STAT dimers rapidly translocate to the nucleus, where they bind to
promoter regions of the DNA and regulate gene transcription (15). Although it is intuitive to accept that exposure of
epithelia to cytokines will generate stereotypical JAK-STAT responses,
this must nevertheless be tested. Indeed, precise definition of the kinetics of the induction of STAT signaling is crucial if modulation of
their function is to be a rational therapy for enteric inflammatory or
secretory disease (14). Descriptive data are beginning to emerge on STAT protein activation in a variety of epithelia. For instance, the levels of STAT-1 and STAT-6 in nuclear extracts are
increased in human airway epithelium in response to interferon (IFN)- and interleukin (IL)-4, respectively (13).
Similarly, IFN-
treatment evoked a JAK-2/STAT-1 response in human
salivary epithelial cells, and IL-4 stimulation of the human colonic
HT-29 epithelial cell line resulted in phosphorylation of JAK-2
(29, 46). A recent report suggests, somewhat unexpectedly,
that epidermal growth factor resulted in STAT-2 translocation to the
nucleus in the absence of tyrosine phosphorylation in rat IEC-6 cells (17); this does not fit with current dogma
(15) and raises the possibility of unique STAT regulatory
mechanisms in gut epithelium. The JAK-STAT pathway is central to the
mediation of many cytokine and growth factor responses; however, as
noted above, alternative signaling pathways are also available.
Additionally, a recent study with macrophages presents a model in which
the STAT and PI 3-K pathways may be linked (37). PI 3-K
activity is involved in vesicle trafficking and cytoskeletal regulation
(10) and as such presents itself as a possible candidate
molecule involved in the regulation of epithelial tight junction
activity (i.e., rate-limiting step in controlling paracellular
permeability). Indeed, we have presented data indicating that
IL-4-induced decreases in T84 monolayer transepithelial resistance can
be elevated significantly by cotreatment with inhibitors of PI 3-K
activity (8).
We have shown that the mixed mediator milieu produced by immune cells
activated by bacterial superantigens can lead to increased epithelial
permeability across monolayers of the human colonic T84 epithelial cell
line (27). The aim of this study was twofold: 1) to assess STAT protein activation in gut epithelia in
response to single recombinant cytokines (principally IFN-) and the
conditioned medium (CM) from immune cells activated by the
superantigen, Staphylococcus aureus enterotoxin B
(SEB); and 2) given the observation that CM from
superantigen-activated immune cells increases T84 monolayer permeability, we wanted to assess the putative involvement of STAT
proteins and PI 3-K in the regulation of epithelial paracellular permeability. The findings indicate that immune mediators released from
superantigen-activated immune cells elicit a STAT-1 or a STAT-1/3
response and activate PI 3-K in gut epithelium. Although the role of
the STAT proteins in the control of epithelial barrier function remains
a possibility, our pharmacological studies provide evidence for PI 3-K
involvement in the control of epithelial paracellular permeability.
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MATERIALS AND METHODS |
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Human Epithelial Cell Culture and Stimulation
Human colonic T84 cells were used primarily throughout this study (some experiments used human Caco-2 or HT-29 epithelial cells). Epithelial cells (3 × 106) were seeded on 6.0-cm-diameter petri dishes and were cultured at 37°C for 7 days as previously described (27). Cells were treated with human recombinant IFN-Lamina propria lymphocytes (LPL) were isolated from sections of resected human gut using standard protocols (9) and were exposed to SEB (1 µg/106 LPLs; 24 h). The ability of the SEB-LPL CM to evoke a STAT response in T84 cells was then examined. (Surgical specimens were supplied and used according to the guidelines of the Departments of Surgery and Pathology at McMaster University/Hamilton Health Sciences Cooperation.)
Pharmacological Inhibitors
The following four additional experiments were performed: 1) T84 cells were pretreated for 1 h with the tyrosine phosphorylation inhibitor genistein (1-10 µM; Sigma Chemical) and then were treated with SEB-PBMC CM for 12 h plus genistein [the inactive analog of genistein, diazein (Sigma Chemical), was included as a control]; 2) T84 cells were exposed to transcription factor decoys (TFDs; 10 µM) for 20 h in serum-free medium. [TFDs are double-stranded DNA oligonucleotides that contain the STAT binding site (see below) and have been phosphorothioated to enhance entry to the cells and render them less susceptible to degradation. In theory, TFDs will capture the STAT dimers and prevent gene regulation (6, 8, 44).] Cells were rinsed (3 times), cultured in serum containing 10% FCS for 1 h, and then exposed to SEB-PBMC CM for 30 min or 24 h; 3) T84 cells were pretreated with LY-294002 [20 µM; Sigma Chemical (10)], a specific inhibitor of PI 3-K activity, for 10 min and then were exposed to SEB-PBMC CM + LY-294002 for 30 min or 24 h; and 4) T84 cells were pretreated with SB-203580 [1 or 10 µM; Sigma Chemical (19)], a specific inhibitor of p38 MAPK activity, for 1 h and then were exposed to SEB-PBMC CM + SB-203580 for 24 h.Electrophoretic Mobility Shift Assay
After treatment (15, 30, and 60 min and 4, 12, 24, and 48 h), epithelial nuclear extracts were obtained according to Andrews and Faller (2) with the addition of the enzyme inhibitors aprotinin (10 µg/ml), pepstatin A, leupeptin (both at 2 µg/ml), and phenylmethysulfonyl fluoride (20 µg/ml; all from Sigma Chemical). Electrophoretic mobility shift assays (EMSAs) were conducted using a published protocol (35). Briefly, 5-15 µg of nuclear protein extract were reacted in binding buffer with [Samples of nuclear extracts were reacted with nonlabeled hSIE (100 ng double-stranded oligonucleotide; 15-20 min on ice) as a cold competitor before exposure to the radiolabeled probe or with a mutant variant of the hSIE sequence that does not bind STAT-1 (42). Supershift EMSAs were performed in which samples were preincubated with antibodies against STAT-1 (2 different polyclonal antibodies were used during this study), tyrosine 701-phosphorylated STAT-1 (monoclonal antibody), or STAT-3 (polyclonal antibody; 2 µg for 20 min on ice; Santa Cruz Biotechnology, Santa Cruz, CA) before reaction with the hSIE probe. An irrelevant IgG-matched antibody (i.e., anti-STAT-6) was used as a specificity control. Finally, EMSAs were also conducted under identical experimental conditions, except that the nuclear extracts were exposed to double-stranded DNA oligonucleotide probes known to bind either STAT-5 or STAT-6 (for sequences see Refs. 20 and 32).
Epithelial Paracellular Permeability
T84 monolayers grown on filter supports (27) were cultured with 25 or 50% SEB-PBMC CM (placed in the basal compartment of the culture well) for 24 h with or without pretreatment with TFDs (10 µM), genistein (5 µM), LY-294002 (20 µM), or SB-203580 (1 or 10 µM). T84 monolayers were then mounted in Ussing chambers and clamped at 0 V, and permeability (transepithelial ion resistance measured by the differential pulse method) was assessed [IFN-Data Presentation and Analysis
EMSA analyses were conducted on at least three separate epithelial preparations for each experimental condition. Data from the epithelial physiology studies are presented as means ± SE, where n is the number of experiments (with 2 monolayers per condition/experiment). Data were normalized based on control responses within each experiment and were analyzed by one-way ANOVA followed by post hoc Newman-Keuls comparisons (27). P < 0.05 was accepted as a level of statistically significant difference. ![]() |
RESULTS |
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IFN- Evokes Epithelial STAT-1 Activation
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SEB-PBMC CM Elicits Epithelial STAT-1 and STAT-3 Signals
Exposure of T84 cells to SEB only (or the related superantigen, Staphylococcus aureus enterotoxin A) did not elicit a detectable STAT response. This would be expected since T84 cells have very low constitutive expression of major histocompatibility class (MHC) II, the ligand for superantigens. Treatment with SEB-PBMC CM resulted in a time- and dose-dependent induction of STAT activation. STAT-1 activation occurred within 5 min of treatment (data not shown); like IFN-The level of IFN- in the 50% SEB-PBMC CM was approximately fourfold
less than that used in the recombinant cytokine studies (2.78 ± 0.53 ng/ml; n = 4). Neutralization of IFN-
via
anti-IFN-
antibodies significantly reduced the intensity of the
STAT-1 signal observed after 30 min of exposure to 50% and abolished
the signal elicited in response to 25% SEB-PBMC CM (Fig.
3). Inclusion of the control
isotype-matched anti-TNF-
antibody did not interfere with the T84
STAT response to SEB-PBMC CM (data not shown).
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When examining enteric epithelial responses to immune mediators, it is
appropriate to conduct studies with gut-derived lymphocytes whenever
they are available. Thus, as shown in Fig.
4, exposure to CM from SEB-activated LPL
obtained from resected ileal or colonic tissue from patients with
active Crohn's disease (n = 3) or colonic cancer
(n = 1) resulted in STAT-1 activation in T84 cells.
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Pharmacological Assessment of SEB-PBMC CM Evoked Drop in Transepithelial Resistance
Having shown a clear activation of STAT-1 (±STAT-3), we proceeded to examine the putative involvement of STATs in the SEB-PBMC CM-evoked drop in transepithelial resistance. Also, since an interaction of STAT proteins with PI 3-K has been suggested (37), we assessed the effect of PI 3-K inhibition on SEB-PBMC CM effects on T84 resistance.Genistein.
STAT activation is dependent on tyrosine phosphorylation; thus, we
first employed a general inhibitor of tyrosine phosphorylation. Addition of genistein to T84 cells did not evoke a STAT response (Fig.
5A). In contrast, genistein
treatment of T84 cells concomitantly exposed to SEB-PBMC CM led to
reduced STAT-1- and STAT-3 DNA-binding activity on EMSA (Fig.
5A; n = 2). Figure 5B shows that
genistein (5 µM), but not the inactive isomer daidzein, partially
prevented the SEB-PBMC CM-induced drop in epithelial monolayer
resistance.
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TFDs.
EMSAs conducted with the TFDs as a cold competitor (i.e., added to the
reaction mixture and not treatment of the epithelial cells directly)
confirmed that the phosphorothioation process did not significantly
impair the ability of the TFD to bind the STAT proteins (see Fig.
6A, lane 3). Subsequently, T84 cells were exposed
to TFDs for 20 h and then were treated with 25% SEB-PBMC CM for
30 min. The T84 cells transfected with the TFDs showed a significant
reduction in, but not a complete block of, the STAT-1 activation
elicited by a subsequent exposure to SEB-PBMC CM (n = 3; STAT identification on EMSA blocked by ~40-70%; Fig.
6B). However, with the use of
the same treatment regime and SEB-PBMC CM from the same blood cell
donors (+3 additional experiments), the TFDs had no effect on the drop
in transepithelial resistance evoked by a 24-h exposure to SEB-PBMC CM
(Fig. 6C). Use of a mutated sequence of the hSIE probe
revealed that the mutant TFD did not function as a cold competitor on
EMSA (data not shown) and did not affect transepithelial resistance
(Fig. 6C). These data neither support nor conclusively
refute STAT-1/-3 modulation of epithelial paracellular permeability, so
we utilized a pharmacological approach to assess the possible role of
PI 3-K and p38 MAPK in the SEB-PBMC CM-induced drop in transepithelial
resistance.
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LY-294002.
LY-294002 is recognized as a specific inhibitor of PI 3-K activity, a
molecule that has been implicated in cytokine signaling and in
regulation of the cytoskeleton (10, 41). Pretreatment with
this agent significantly prevented the increase in epithelial permeability induced by 24 h of exposure to 25% SEB-PBMC CM (Fig. 7; n = 6). Also, the
STAT-1 signal elicited by 1 h of exposure to IFN- (10 ng/ml)
was not affected by cotreatment with LY-294002 (personal observation).
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SB-203580.
Finally, the specific p38 MAPK inhibitor SB-203580 was employed. These
experiments served the following two roles: 1) to examine the role of p38 MAPK in the control of paracellular permeability and
2) to serve as a pharmacological control for comparison with PI 3-K inhibition. With the use of doses and treatment times defined in
the literature (19), SB-203580 had no significant effect on the reduction in transepithelial resistance caused by exposure to
25% SEB-PBMC CM for 24 h [control = 1,339 ± 194;
SB-20358 (10 µM) only = 1,274 ± 172; SEB-PBMC CM = 777 ± 135 (P < 0.05 compared with control);
SEB-PBMC CM + SB-20358 = 599 ± 123 (P < 0.05 compared with control) /cm2 (n = 4 monolayers from 2 experiments)].
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DISCUSSION |
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Interference with cytokine intercellular signaling cascades allows
the modulation of physiological or pathophysiological processes by
administration, or inhibition of, the appropriate cytokine. An
alternative strategy is the extrinsic regulation of the cytokine's target cell. For example, adenoviral gene transfer of a superrepressor inhibitor protein- B has been used to inhibit NF-
B activity and
consequently the production of proinflammatory cytokines by epithelial
cells (16). In the present study, we show that IFN-
, the prototypic activator of STAT-1, and SEB-PBMC CM activate STAT proteins in model gut epithelia. Pharmacological interventions suggest
that the disruption of epithelial barrier function caused by exposure
to SEB-PBMC CM occurs, at least in part, via a PI 3-K pathway, and we
are unable to provide conclusive evidence in support of or refuting
STAT-1 (or STAT-3) involvement in the regulation of epithelial
paracellular permeability.
Activation of the JAK-STAT signaling pathway has piqued the interest of
the epithelial biologist (13, 17, 21, 29, 35). IFN-
mobilizes STAT-1 only (15), and, since IFN-
regulates many epithelial functions including permeability and expression of MHC
II (26, 39), we first examined STAT-1 induction by IFN-
in gut-derived epithelia. Exposure to IFN-
resulted in detectable
STAT-1 proteins in nuclear extracts 15 min posttreatment, and, although
the intensity of the signal was considerably diminished, nuclear STAT-1
proteins were still apparent after 24 h. The significance of the
longevity of this response is unclear but could be linked to the 24- to
72-h period required to observe the IFN-
(>10 ng/ml)-induced increase in T84 epithelial permeability (24). The
specificity of the IFN-
response was shown by inhibition with a cold
competitor, and inclusion of STAT-1-specific antibodies in the EMSAs
confirmed the identity of the detected band as authentic STAT-1. IL-4
and TNF-
do not mobilize STAT-1, although both can affect epithelial permeability, and T84 cells treated with either cytokine showed no
STAT-1 signal when nuclear extracts were reacted with the hSIE probe
(indicates specificity of the probe and the IFN-
effect). IL-10 can
activate STAT-1 and STAT-3 (37) but did not elicit a
signal in T84 cells. Direct effects of IL-10 on T84 cells have been
reported, albeit at a high dose (100 ng/ml) administered daily for 7 days (25). The discrepancy between these data and our
findings suggests that either >10 ng/ml of IL-10 are required to
elicit detectable STAT-1 or STAT-3 signals in T84 cells or that the
IL-10 effect in the former study was via a STAT-independent mechanism,
perhaps analogous to that proposed for IL-10 inhibition of
lipopolysaccharide activation of macrophages (31).
These studies underscore the need to precisely define STAT signaling in
multiple cell types (cell lines and primary isolates) if
pharmacological STAT regulation is to be a therapeutic goal
(14).
Analysis of the effects of recombinant cytokines on epithelia has been
complemented by coculture studies comprising immune cells (±specific
stimuli) or CM (18, 27, 40). We showed that superantigen
activation of T cells caused increased T84 permeability that was
apparent 12 h after the initiation of the coculture and was
maximal by 24 h (11, 27). In the present study, we
extended these observations by assessing STAT protein activation in
response to SEB-PBMC CM. EMSA revealed that SEB-PBMC CM treatment of
T84 cells resulted in STAT-1 activation, but not STAT-5 or STAT-6, and
this signal was still evident 24 h posttreatment. The kinetics (i.e., intensity of STAT bands, induction and longevity of the response) of this response were similar to those elicited by
recombinant IFN-, and neutralization of IFN-
completely abolished
the epithelial STAT-1 response to SEB-PBMC CM. These data are
consistent with in vivo and in vitro data showing that superantigen
immune activation is initially dominated by IFN-
-driven events
(5, 12). In addition, we found that exposure of T84 cells
to SEB-LPL CM also resulted in an increase in STAT-1 DNA binding, and
this may have disease ramifications (e.g., active participation in
modulation of mucosal immunity) should superantigens gain access to the
gut mucosa.
Exposure of T84 cells to some, but not all, SEB-PBMC CM resulted in
STAT-1 and STAT-3 activation; STAT-3 mediates some of the effects of
IL-6-type cytokines, IL-2, and IL-10 (15). IL-6 levels are
increased in SEB-PBMC CM; however, neutralizing IL-6 antibodies failed
to abrogate the loss of epithelial barrier function evoked by SEB-PBMC
CM (27). The functional significance of induction of a
STAT-1 vs. a STAT-1/-3 signal in response to SEB-PBMC CM is unclear.
However, it is intriguing to speculate that the pattern of STAT protein
responses elicited in an epithelium may be a determinant in the host's
overall response to immune activation, such as the susceptibility to
infection, time to recovery, etc. Increased activation of STAT-1 has
been shown in bronchial epithelium from patients with asthma compared
with controls, and this occurred in the absence of any significant
increase in tissue levels of IFN- or IFN-
-producing cells
(38). This finding is complemented by the present
examination of enteric epithelium showing that exposure to IFN-
or
SEB-PBMC CM resulted in a nuclear STAT-1 signal that was still
detectable 24 h posttreatment.
Analysis of STAT responses must be coupled to functional studies. With the exception of MHC II and intercellular adhesion molecule-1 expression (28, 43), there is little firm evidence relating to epithelial functions that are directly regulated by STAT proteins. Here, T84 cells treated with the tyrosine phosphorylation inhibitor genistein showed reduced STAT-1/-3 activation and a partial inhibition of the drop in transepithelial resistance caused by SEB-PBMC CM. Similarly, recent studies found that genistein or the unrelated tyrosine kinase inhibitor herbimycin can reduce immune-mediated disruption of epithelial barrier function (4, 7, 40); STAT proteins were not examined in these studies.
The correlation of genistein's ability to reduce the disruption in
epithelial barrier function and STAT signaling elicited by SEB-PBMC CM
suggests but does not prove that the two events are causally linked.
Indeed, it is feasible that inhibition of tyrosine phosphorylation of
non-STAT proteins contributes to the maintenance of epithelial barrier
integrity. Endogenous off-signals for STAT proteins are being
identified (30); however, specific pharmacological
inhibitors of STAT proteins are not available. Therefore, we attempted
to block STAT signaling using TFDs following methods that have been
shown to block, or reduce, the effects of STAT protein activation or
NF-B signaling (6, 8, 44). T84 epithelia pretreated
with TFDs showed reduced STAT-1 activation when subsequently challenged
with SEB-PBMC CM, but the disruption in barrier function remained
unaffected. At least three possibilities explain these findings.
1) The amount of STAT-1 that escaped the TFDs was enough to
affect gene transcription, leading to opening of the epithelial tight
junctions; 2) transepithelial resistance is a sensitive
assay of permeability, and since TFD transfection will have been
random, responses from cells that did not incorporate a significant
amount of TFD could account for the ~50% drop in resistance; and
3) STAT-1 is not involved in the modulation of paracellular
permeability. The investigative technique used here provides no
conclusive evidence in support of, or refuting, STAT-1 involvement in
the regulation of epithelial permeability. To unequivocally address
this issue requires the development of an epithelium containing a
stable (and preferably inducible) dominant-negative STAT-1 gene (46) that is also suitable for Ussing chamber/flux studies.
IFN- does directly (24), or in the context of CM
(11), affect epithelial permeability, and, although
research has focused on STAT-1 as the major molecule that transduces
IFN-
effects, data are emerging showing IFN-
- and
STAT-1-independent signaling mechanisms (34). A common
pathway in intracellular cytokine signaling involves the activation of
PI 3-K (41). This molecule has been implicated in
vesicular trafficking, modulation of the cytoskeleton (a determinant of
tight junction activity; see Refs. 10 and
23), and in IL-13 regulation of HT-29 epithelial cell survival (45). Moreover, recent data from our laboratory
(8) indicate that IL-4 disruption of T84 permeability is
significantly ablated when the cells are cotreated with inhibitors of
PI 3-K activity. Thus we assessed the ability of the specific PI 3-K inhibitor LY-294002 to affect SEB-PBMC CM-induced increases in epithelial permeability; LY-294002 consistently and significantly reduced the increased permeability caused by exposure to SEB-PBMC CM.
These findings fit with the effects of genistein in the physiological experiments because PI 3-K activity is dependent on tyrosine
phosphorylation. Moreover, we (personal observation and Ref.
8) and others (1) have shown that PI 3-K
inhibitors do not prevent STAT activation as monitored by EMSA and
Western blotting. Also, inhibition of p38 MAPK activity did not alter
T84 responses to SEB-PBMC CM. This observation is noteworthy in the
context of a recent report showing stimulus-dependent cross-talk
between the STAT-1 and MAPK pathways in macrophages (19).
Collectively, our pharmacological studies provide data in support of
SEB-PBMC CM-induced activation of PI 3-K and indicate that a PI
3-K-dependent mechanism is at least partially responsible for the
regulation of epithelial permeability in this model system. Additional
studies are required to test this hypothesis and provide a fuller
understanding of the kinetics of PI 3-K activity in enteric epithelia
and downstream events after PI 3-K activation.
In summary: 1) IFN- caused a rapid and specific induction
of STAT-1 in three human colonic epithelial cell lines; 2)
exposure to CM from SEB-activated PBMC or LPL evoked a STAT-1 or a
STAT-1/-3 (but not STAT-5 or STAT-6) response in T84 cells, and we
suggest that epithelial signal transduction is important in the
homeostatic balance between physiological and pathophysiological
events; and 3) regulation of epithelial paracellular
permeability is at least partially dependent on a PI 3-K pathway, and
the putative involvement of STAT-1 in the modulation of epithelial
barrier function requires additional experimentation. The latter two
observations raise important issues for future studies aimed at
understanding the kinetics of epithelial STAT protein activation and
the physiological events that they control and for elucidation of the
full role of PI 3-K in the modulation of epithelial tight junction activity.
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ACKNOWLEDGEMENTS |
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The technical assistance of J. Lu and J. Brokenshire is gratefully acknowledged.
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FOOTNOTES |
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This work was funded by Medical Research of Canada grants to D. M. McKay and C. D. Richards and in part by a Crohn's and Colitis Foundation of Canada grant to D. M. McKay.
Portions of this work were presented at the American Association of Gastroenterology Meeting in New Orleans (May 1998).
Address for reprint requests and other correspondence: D. M. McKay, Intestinal Disease Research Programme, HSC-3N5, McMaster Univ., 1200 Main St. W., Hamilton, Ontario, Canada L8N 3Z5 (E-mail:mckayd{at}fhs.mcmaster.ca).
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 22 November 1999; accepted in final form 8 June 2000.
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