(Received for publication, September 9, 1996, and in revised form, January 30, 1997)
From the Department of Pharmacology, Bath University, Claverton Down, Bath, Avon BA2 7AY, United Kingdom
The human colonic epithelial cell line HT-29 can
be induced by a combination of the cytokines interleukin (IL)-1,
tumor necrosis factor
, and interferon-
to express the inducible
form of nitric-oxide synthase (iNOS; Kolios, G., Brown, Z., Robson, R.,
Robertson, D. A. F., & Westwick, J. (1995) Br. J. Pharmacol.
116, 2866-2872). IL-13 is a potent inhibitor of cytokine-induced
iNOS mRNA expression and nitric oxide generation in HT-29 cells via
an unknown mechanism. We report here that in HT-29 cells, IL-13 induces
a concentration and time-dependent increase in the
formation of the lipid products of phosphatidylinositol (PtdIns)
3-kinase, namely phosphatidylinositol (3,4)-bisphosphate and
phosphatidylinositol (3,4,5)-trisphosphate. IL-13 also induces a
parallel concentration and time-dependent increase in the
in vitro lipid kinase activity present in
immunoprecipitates of the p85 regulatory subunit of PtdIns 3-kinase. In
addition, we also demonstrate that IL-13 stimulates the tyrosine
phosphorylation of the adaptor molecule insulin receptor substrate 1, which may facilitate receptor coupling to PtdIns 3-kinase. Both the
increases in D-3 phosphatidylinositol lipids and the increased in
vitro lipid kinase activity of p85 immunoprecipitates were
inhibited by wortmannin and LY294002. Inhibition of the PtdIns 3-kinase activity was paralleled by a reversal of the ability of IL-13 to
inhibit iNOS mRNA expression and nitrite generation in HT-29 cells.
These data demonstrate that the activation of PtdIns 3-kinase by IL-13
is a key signal that is responsible for the inhibition of iNOS
transcription in activated epithelial cells.
Interleukin-13 (IL-13)1 is a
pleiotropic cytokine secreted by activated Th-2 T lymphocytes that
regulates a variety of immune target cells (1, 2). In B lymphocytes,
IL-13 induces proliferation and differentiation and promotes CD23
expression and production of certain immunoglobulins such as IgG4 and
IgE (3-5). In monocytes, IL-13 induces morphological changes (2),
up-regulates expression of members of the integrin superfamily and
major histocompatibility complex class II antigen expression, and
down-regulates expression of CD14 and FcR receptors (6). In
lipopolysaccharide-stimulated monocytes, IL-13 also acts as a
suppressor of proinflammatory cytokines (e.g. tumor necrosis
factor (TNF) type
, IL-1, and IL-6), chemokines (e.g.
IL-8, macrophage inflammatory protein 1
), and hematopoietic growth
factors (e.g. granulocyte/macrophage-colony stimulating
factor, granulocyte-colony stimulating factor) expression by activated
monocytes/macrophages or endothelial cells (1, 6). Another target of
IL-13 is epithelial cells, and we have recently demonstrated that IL-13
can modulate IL-8 generation from the human colonic epithelial cell
line HT-29 (7).
Little is currently known about IL-13 signal transduction, although
evidence points to similarities with IL-4 signal transduction. For
instance, IL-13 can cross-compete with IL-4 for binding, leading to the
suggestion that their receptors (IL-4R and IL-13R) share a common
component (8). Both the IL-4R chain and the common
chain subunit
have been proposed as likely shared components (9-11). More recently,
it has been proposed that the IL-13R may comprise the recently cloned
IL-13R
chain (12), an IL-13-binding protein related to the IL-5
receptor
chain (13) and the IL-4R
chain (12). A common receptor
may explain the observations that IL-4 can mimic every cellular
response mediated by IL-13. In contrast, human T cells and mouse T and
B cells respond to IL-4 but not to IL-13 (14). Unlike other cytokines,
IL-4 and IL-13 do not induce tyrosine phosphorylation of the adaptor
protein Shc or its association with Grb-2, and they do not activate the
mitogen-activated family of protein kinases such as extracellular
signal regulated kinases 1 and 2 (11). However, Janus family kinases
and STAT proteins have been shown to be significant signal transduction components of cytokine receptors (15). Indeed, the IL-4R
chain associates with JAK1, whereas the common
chain associates with JAK3, and both are required for IL-4 activation of STAT-6 (15-17). JAK1, TYK2, and STAT-6 are also activated by IL-13, but JAK3 is not
(11).
IL-4 and IL-13 share the ability to induce phosphorylation of several
cellular proteins including the IL-4R chain itself (11, 18) and in
particular, a p170 protein (11, 19) recently identified as IRS-2 (20).
IRS-2, like its homolog, IRS-1, contains multiple specific
YXXM motifs, which after tyrosine phosphorylation may bind
the SH2 domains of the p85 regulatory subunit of phosphatidylinositol (PtdIns) 3-kinase, which associates through SH2 domains in the p85
subunit with proteins that are tyrosine-phosphorylated by protein-tyrosine kinases (20, 21). Indeed, several groups have shown
that phosphorylation of the 170-kDa protein results in its tight
association with PtdIns 3-kinase after IL-4 and IL-13 treatment (11,
19, 22). PtdIns 3-kinase belongs to a growing family of lipid kinases
(23) and can potentially generate three products:
phosphatidylinositol-(3)-monophosphate (PtdIns(3)P), phosphatidylinositol-(3,4)-bisphosphate (PtdIns(3,4)P2),
and phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns(3,4,5)P3), which are referred to collectively as
D-3 phosphatidylinositol lipids (reviewed in Ref. 24). PtdIns 3-kinase
may in fact be a multifunctional molecule because the different lipid
products formed by the p110 catalytic subunit (24) may mediate
different functions such as trafficking of cell-surface receptors
(e.g. PtdIns(3)P) and cell signaling (e.g.
PtdIns(3,4)P2 and PtdIns(3,4,5)P3) (24).
Moreover, the p85 subunit may act as an adaptor molecule that
facilitates protein-protein interactions via its SH2 and SH3 domains
and/or its proline-rich regions (24, 25). The significance of the
association of PtdIns 3-kinase with IRS-2 after IL-4 and IL-13
treatment has yet to be established, but IL-4 treatment of mast cells,
myeloid cells, and T cells results in the accumulation of D-3
phosphatidylinoistol lipids (26). This implies that the function of
PtdIns 3-kinase is probably not restricted to that of a p85
subunit-mediated adaptor function and that the putative signaling
cascade regulated by PtdIns(3,4,5)P3 contributes to
IL-4 signaling. Recent advances in understanding the role of PtdIns
3-kinase as an upstream element in an important signaling pathway have
largely been made possible by the availability of inhibitors of
the enzyme, in particular the fungal metabolite wortmannin (27) and
the structurally unrelated synthetic quercetin derivative
LY294002 (28). Wortmannin irreversibly inhibits, at
concentrations up to 100 nM (Ki 1-10
nM), both the lipid kinase and serine kinase activity of
PtdIns 3-kinase through covalent interaction with the p110 catalytic
subunit (29). With the possible exception of a soluble PtdIns 4-kinase
(30) and phospholipase A2 (31), PtdIns 3-kinase is the only
high-affinity target for wortmannin in mammalian cells, but higher
concentrations (>1 µM) have been shown to inhibit other
enzymes and signaling pathways (25, 27). Because wortmannin inhibits
several cellular responses and biochemical events initiated by a
variety of receptors in numerous cell systems (reviewed in Ref. 25),
PtdIns 3-kinase has been implicated in the regulation of a growing
number of diverse physiological events, in particular cell growth and
proliferation (25).
We have recently demonstrated that epithelial cells of the colonic
mucosa derived from patients with ulcerative colitis are a rich source
of the nitric oxide-generating enzyme
iNOS,2 which belongs to a family of
nitric-oxide synthases (32) responsible for the production of nitric
oxide, an important molecular messenger that has been implicated in
sepsis, ischemia, and inflammation (33). Interestingly, the human
colonic epithelial cell line HT-29 can be used to express iNOS
mRNA, protein, and nitrite in response to a mixture of the
proinflammatory cytokines IL-1, TNF-
, and interferon-
(IFN-
) (34). Furthermore, iNOS expression can be inhibited by
pretreatment of the HT-29 cells with IL-13 or IL-4, but not by
IL-10.2 Thus, we have used the colonic epithelial cell line
HT-29 to explore the signaling mechanism of IL-13-induced suppression
of iNOS induction. In this report, we describe the activation of the
PtdIns 3-kinase signaling pathway after IL-13 treatment in HT-29 cells
and that inhibition of this signaling pathway reverses the suppressive
effect of IL-13 on iNOS expression and activity that is induced in
response to a combination of IL-1
, TNF-
, and IFN-
. Thus,
PtdIns 3-kinase may be a pivotal pathway in determining the
anti-inflammatory actions of IL-13.
Human recombinant IL-13 was kindly provided by Dr
A. Minty (Sanofi Elf Bio Recherches, Laberges, France). Human
recombinant IL-1 (specific activity, 5 × 107
units/mg) and TNF-
(specific activity, 6 × 107
units/mg) were generous gifts from Glaxo (Greenford, United Kingdom) and Bayer (Slough, United Kingdom), respectively. Human recombinant IFN-
(specificity, >2.0 × 107 units/mg) was
purchased from Boehringer Mannheim. Mouse p85
monoclonal antibody
(mAb) was a gift from Doreen Cantrell (Imperial Cancer Research Fund,
London, United Kingdom). 4G10 phosphotyrosine mAb (
PY) as well as
the rabbit IRS-1 and IRS-2 polyclonal antibodies were purchased from
Upstate Biotechnology. All cell culture reagents were from Life
Technologies, Inc. 2,3-Diaminonapthalene was purchased from Lancaster
Synthesis Ltd. 5
-Digoxigenin-labeled probe for iNOS was a mixture
containing 3 antisense 30-mer oligonucleotides purchased from R & D
Systems (Abingdon, United Kingdom). Wortmannin and standard
phosphatidylinositol lipids were purchased from Sigma. [
-32P]ATP (3000 Ci/mmol) and
[32P]orthophosphate (8500-9120 Ci/mmol) were from DuPont
NEN. LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) was
purchased from Affiniti (Exeter, United Kingdom).
The human colon epithelial carcinoma cell line HT-29 was obtained from the European Collection of Animal Cell Cultures. Cells were cultured in humidified incubators at 37 °C, 5% CO2 in McCoy's 5A medium supplemented with 10% (v/v) fetal bovine serum, 10 units/ml penicillin, 10 µg/ml streptomycin, and 0.5 µg/ml fungizone. For experiments, HT-29 cells were seeded at 2-3 × 104 cells/ml until confluent. Confluent cells were washed and cultured in fresh medium without fetal bovine serum 24 h before stimulation. Growth-arrested cells were treated with the appropriate concentrations of stimuli in medium without serum and incubated as described above. Cell counting and viability were checked by trypan blue exclusion at the beginning and the end of each experiment using representative wells. Cell viability was always greater than 95%.
Fluorometric Assay for Nitric OxideNitric oxide production by HT-29 cells was determined by measuring the stable end-product nitrite in the cell culture supernatants by flourometric assay that is based upon the reaction of nitrite with 2,3-diaminonapthalene to form the fluorescent product 1-(H)-naphthotriazole, as described previously (35). 200 µl of freshly prepared 2,3-diaminonapthalene (0.05 mg/ml in 0.62 M HCl) were added in 2 ml of sample and mixed immediately. After 10 min of incubation at room temperature in the dark, the reaction was terminated with 100 µl of 2.8 N NaOH. The samples were measured using a Photon Technology International Inc. dual-wavelength spectrofluorometer (excitation at 365 nm and emission at 405 nm) and compared with known concentrations of sodium nitrite. The sensitivity of the assay is 10 nM.
Northern Blot AnalysisTotal cellular RNA from HT-29 cells was isolated as described previously (34, 36). Briefly, HT-29 monolayers were solubilized in a solution containing 25 mM Tris (pH 8.0), 4 M guanidine isothiocyanate, 0.5% (v/v) Sarcosyl, and 0.1 M 2-mercaptoethanol. After homogenization, the above-described suspension was added to an equal volume of 100 mM Tris (pH 8.0), 10 mM EDTA, and 1% (w/v) SDS. The RNA was then extracted with chloroform-phenol (1:1, v:v) and chloroform-isoamyl (24:1, v:v). The total RNA was alcohol-precipitated, and the pellet was dissolved in diethyl pyrocarbonate-treated water. The concentration of RNA was measured by obtaining the absorbance at A260 and A280 nm, and 10 µg of RNA was loaded into each well of the agarose gel. Total RNA was separated by electrophoresis using formaldehyde and 1% (w/v) agarose gels and transferred overnight to nylon membrane (Boehringer Mannheim) by capillary blotting, and iNOS mRNA was detected using a digoxigenin luminescent detection kit (Boehringer Mannheim). The blots were baked at 120 °C for 20 min and then hybridized with digoxigenin-labeled oligonucleotide probes (10 ng/ml) for iNOS according to the manufacturer's instructions. Bound probes were detected using anti-digoxigenin Fab fragments conjugated to alkaline phosphatase with lumigen PPD (Boehringer Mannheim) as the chemiluminescent substrate. Blots were exposed after hybridization to x-ray film. Equivalent amounts of total RNA loaded/lane were assessed by monitoring 18 and 28 S RNA (34).
Cell Lysis and In Vitro Lipid Kinase Assays3.5 × 106 cells/ml were stimulated and incubated at 37 °C in
RPMI 1640 medium as indicated. Reactions were terminated by the addition of 1 ml of ice-cold lysis buffer (1% (v/v) Nonidet P-40, 100 mM NaCl, 20 mM Tris (pH 7.4), 10 mM
iodoacetamide, 10 mM NaF, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml antipain,
1 µg/ml chymostatin, 1 µg/ml pepstatin A, and 1 mM
sodium orthovanadate). Lysates were incubated at 4 °C for 15 min,
followed by centrifugation at 14,000 rpm. The supernatant was
precleared with protein G-Sepharose beads for 1 h at 4 °C.
Immunoprecipitates were performed for 2 h at 4 °C as described
(37) using p85 mAb (1 µg/ml) precoupled to protein G-Sepharose
beads (Pharmacia Biotech Inc.). Immunoprecipitates were washed and
subjected to in vitro lipid kinase assays as described (37)
using a lipid mixture of 100 µl of 0.1 mg/ml PtdIns and 0.1 mg/ml
phosphatidylserine dispersed by sonication in 20 mM HEPES,
pH 7.0, and 1 mM EDTA. The reaction was initiated by the
addition of 20 µCi of [
-32P]ATP (3000 Ci/mmol;
DuPont NEN) and 100 µM ATP to the immunoprecipitates suspended in 80 µl of kinase buffer. The reaction was terminated after 15 min, and phospholipids were then separated by TLC (37).
When appropriate, cells were lysed after stimulation by IL-13 as already described. Immunoprecipitates were generated using either anti-p85 (1 µg/ml), anti-IRS-1 (1 µl/ml), or anti-IRS-2 (1 µl/ml) antibodies, and the immunoprecipitated samples for immunoblotting were electrophoresed through 10% acrylamide gels by SDS-polyacrylamide gel electrophoresis and transferred by electroblotting onto nitrocellulose (Schleicher & Schull) as described previously (11, 38). Blots were probed with the anti-phosphotyrosine mAb 4G10 (0.5 µg/ml), and proteins were visualized by a chemiluminescence detection system (ECL; Amersham) with a goat anti-mouse Ig (0.1 µg/ml) conjugated with horseradish peroxidase as a secondary antibody (Dako). When appropriate, blots were completely stripped of antibodies by incubation at 55 °C for 60 min with stripping solution (62.5 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, and 100 mM 2-mercaptoethanol). After extensive washing, blots were reblocked before reprobing.
D-3 Phosphatidylinositol Lipid Labeling, Extraction, and HPLC Separation4 × 108 HT-29 cells were labeled with 1 mCi of [32P]orthophosphate (8500-9120 Ci/mmol; DuPont NEN) as described (39). 32P-labeled HT-29 cells were aliquoted at 107 cells/120 µl and stimulated as described in the figure legends, and phospholipids were extracted with 750 µl of chloroform:methanol:H2O (32.6:65.3:2.1%, v/v/v, respectively) as described (39). The samples were deacylated and analyzed by anion exchange HPLC analysis using a Partisphere SAX column (Whatman) (39). The eluate was fed into a Canberra Packard A-500 Flo-One on-line radiodetector, and the results were analyzed by the Flo-One data program (Radiomatic). Eluted peaks were compared with retention times for standards prepared from 3H-labeled phosphatidylinositol lipids (Amersham) and 32P-labeled D-3 phosphatidylinositols as described elsewhere (39).
To investigate the signaling mechanisms activated by IL-13
treatment, 32P-labeled HT-29 cells were stimulated with
IL-13, and the outcome on PtdIns 3-kinase activation was determined by
measuring the accumulation of D-3 phosphatidylinositol lipids.
Treatment of HT-29 cells with IL-13 (0.3-30 ng/ml) resulted in the
concentration-dependent accumulation of
PtdIns(3,4,5)P3 (Fig. 1A).
Furthermore, the accumulation of PtdIns(3,4,5)P3 was
transient, with maximum accumulation occurring at 30 s post-IL-13
treatment (Fig. 1B). The levels of
PtdIns(3,4,5)P3 had declined back to basal levels within 10 min of IL-13 treatment. In addition, IL-13 also induced the
accumulation of another D-3 phosphatidylinositol, namely
PtdIns(3,4)P2. However, the accumulation of
PtdIns(3,4)P2 occurred with slower kinetics, such that
maximum accumulation of PtdIns(3,4)P2 occurred at 1 min
post-IL-13 treatment (Fig. 1B), although the levels of
PtdIns(3,4)P2 also declined back to basal levels within 10 min. The apparent lag time for the accumulation of
PtdIns(3,4)P2 compared with the accumulation of
PtdIns(3,4,5)P3 is consistent with the proposal that
PtdIns(3,4)P2 is the metabolic breakdown product of
PtdIns(3,4,5)P3 (24).
IL-13 Enhances the in Vitro Lipid Kinase Activity of p85 Immunoprecipitates
PtdIns 3-kinase activation in HT-29 cells was
also determined by assaying the in vitro lipid kinase
activity present in immunoprecipitates of the p85 regulatory subunit.
Treatment of HT-29 cells with IL-13 (0.3-30 ng/ml) resulted in a
concentration-dependent increase in the in vitro
lipid kinase activity present in p85 immunoprecipitates (Fig.
2A). As was observed for the IL-13-induced
accumulation of PtdIns(3,4,5)P3 and
PtdIns(3,4)P2, the IL-13-induced increase in in
vitro lipid kinase activity was transient, with maximum lipid
kinase activity present in p85 immunoprecipitates derived from HT-29
cells stimulated for 30 s with IL-13 (Fig. 2B). The p85-associated lipid kinase activity declined back to basal levels in
p85 immunoprecipitates derived from cells treated for 10 min with IL-13
(Fig. 2B).
IL-13 Induces Tyrosine Phosphorylation of IRS-1 and Association with p85
To define more clearly the mechanism of interaction of
the IL-13R with PtdIns 3-kinase, we performed experiments to
investigate whether or not the coupling of the IL-13R to PtdIns
3-kinase involved the adaptor molecules IRS-1 and/or IRS-2. Both IRS-1
and IRS-2 have previously been implicated in IL-4R- and IL-13R-mediated signal transduction events (11, 19, 40, 41), and both can associate
with PtdIns 3-kinase via specific phosphotyrosine-containing sequences
that bind the p85 subunit (20, 42). Accordingly, immunoprecipitations
were performed on cell lysates derived from IL-13-stimulated HT-29
cells using antibodies directed against either the p85 subunit of
PtdIns 3-kinase, IRS-1, or IRS-2 (Fig. 3). The resulting
precipitates were immunoblotted with the 4G10 anti-phosphotyrosine mAb.
Two co-immunoprecipitating tyrosine-phosphorylated proteins migrating
at Mr 165,000-170,000, consistent with the molecular weights of IRS-1 and IRS-2, respectively, were precipitated by the p85 mAb (Fig. 3A). Immunoblotting of the p85
immunoprecipitates with the polyclonal anti-IRS-1 at 1:2000 revealed a
time-dependent increase in the association of IRS-1 with
p85 that correlated with the kinetics of tyrosine phosphorylation of
the proteins migrating at 165-170 kDa (data not shown). Furthermore,
immunoblotting of the IRS-1 immunoprecipitates from IL-13-stimulated
HT-29 cells with the 4G10 anti-phosphotyrosine mAb confirmed that IL-13
stimulation induced the strong tyrosine phosphorylation of IRS-1 (Fig.
3B). However, similar experiments using IRS-2
immunoprecipitates (Fig. 3C) revealed no detectable increase
in IRS-2 tyrosine phosphorylation after IL-13 stimulation. Each blot
was stripped and reprobed with 0.5 µg/ml anti-p85 (Fig.
3A) polyclonal anti-IRS-1 at 1:2000 (Fig. 3B) and
polyclonal anti-IRS-2 at 1:4000 (Fig. 3C) to confirm
efficiency of immunoprecipitation in each case (data not
shown).
Inhibition of IL-13-induced Activation of PtdIns 3-Kinase by Wortmannin and LY294002
The accumulation of
PtdIns(3,4,5)P3 (Fig. 4, A
and B) and PtdIns(3,4)P2 (data not shown) after
IL-13 treatment was inhibited by pretreatment with the PtdIns 3-kinase
inhibitors wortmannin (100 nM) (27) and the structurally
unrelated LY294002 (1-30 µM) (28) 10 min before the
addition of IL-13. Moreover, the IL-13-induced increase in lipid kinase
activity present in p85 immunoprecipitates was also inhibited by 10 min
pretreatment with wortmannin (Fig. 4C) and LY294002 (data
not shown).
Wortmannin and LY294002 inhibit IL-13-induced
activation of PtdIns 3-kinase.
[32P]orthophosphate-labeled HT-29 cells (2 × 107 cells/120 µl) were incubated with (A) 100 nM wortmannin and (B) 1-30 µM
LY294002 for 10 min at 37 °C. Cells were stimulated with 30 ng/ml
IL-13 for 30 s. PtdIns(3,4,5)P3 was extracted and
deacylated, and the glycerophosphorylinositol derivative of
PtdIns(3,4,5)P3 (GroPIns(3,4,5)P3)
was analyzed by HPLC separation as described under "Experimental
Procedures." C, HT-29 cells (3.5 × 106
cells/120 µl) were incubated for 10 min with wortmannin at the concentrations indicated, followed by treatment with IL-13 (30 ng/ml)
for 30 s. The HT-29 cells were lysed, and the lysates were subjected to immunoprecipitation with anti-p85 mAb. The
immunoprecipitates were washed and analyzed for PtdIns kinase activity
using PtdIns as a substrate. Extraction and TLC separation of the lipid
products were performed as described under "Experimental
Procedures." Lipids were detected by exposure to film at 70 °C
(upper panel) and quantitated by densitometry (lower
panel). Data are from a single experiment representative of at
least three others.
IL-13 Suppression of Proinflammatory Cytokine-induced iNOS Expression and Nitrite Production Is Prevented by PtdIns 3-Kinase Inhibitors
As shown previously (34), growth-arrested HT-29
monolayers stimulated with the proinflammatory cytokines IL-1 (10 ng/ml), TNF-
(100 ng/ml), and IFN-
(300 units/ml) result in the
optimal generation of nitrite and expression of iNOS mRNA (Fig.
5). Both the expression of iNOS mRNA (Fig.
5A) and the generation of nitrite (Fig. 5B)
induced by the proinflammatory cytokine mixture can be prevented by
pretreatment of HT-29 cells with IL-13 (0.3-30 ng/ml)2 for
1 h before the addition of the cytokine mixture. However, addition
of wortmannin (Fig. 5) or LY294002 (data not shown) to HT-29 cells 10 min before IL-13 treatment prevents the inhibitory effects of IL-13 on
iNOS mRNA expression (Fig. 5A) and nitrite production
(Fig. 5B).
In this report we demonstrate that IL-13 activates PtdIns 3-kinase in a human colonic epithelial cell line as determined by the accumulation of D-3 phosphatidylinositol lipids in vivo and increased lipid kinase activity present in immunoprecipitates of the p85 regulatory subunit of PtdIns 3-kinase. In addition, we also demonstrate that IL-13 stimulates the tyrosine phosphorylation of the adaptor molecule IRS-1, which has previously been reported to facilitate receptor coupling to PtdIns 3-kinase (42). This is the first evidence that IL-13 can stimulate the increased lipid kinase activity of PtdIns 3-kinase and is an important observation given that D-3 phosphatidylinositol lipids are increasingly thought to act as important regulatory molecules utilized by a plethora of receptors involved in diverse outcomes (reviewed in Ref. 24).
Accordingly, we have used the two structurally unrelated PtdIns
3-kinase inhibitors wortmannin and LY294002 to demonstrate that the
IL-13-mediated activation of PtdIns 3-kinase and the resulting
accumulation of D-3 phosphatidylinositol lipids is necessary for the
IL-13-induced suppression of iNOS mRNA expression and nitrite
production induced by the mixture of proinflammatory cytokines IL-1,
TNF-
, and IFN-
. The ability of IL-13 to inhibit nitric oxide
production and iNOS activity in macrophages (43, 44), mesangial cells
(45), and HT-29 carcinoma cells2 induced by proinflammatory
cytokines seems to be an important function of IL-13. Until now,
however, the signaling cascades that mediate the effects of IL-13 on
iNOS have not previously been characterized. Our demonstration that
PtdIns 3-kinase inhibitors prevent the effects of IL-13 on iNOS is
similar to a previous report that demonstrated that insulin-induced
inhibition of phosphoenolpyruvate carboxykinase is dependent on PtdIns
3-kinase activation (46) and further demonstrates an important role for
PtdIns 3-kinase in the negative regulation of the induction of specific
mRNA.
To date, at least three forms of mammalian PtdIns 3-kinase have been
identified, and these include various isoforms of the p85/p110
heterodimer (47), the G protein-coupled PtdIns 3-kinase (48), and
the PtdIns-specific 3-kinase (49). The p85/p110 heterodimer is the best
studied of these lipid kinases, and reagents to either p85 or p110 are
readily available and reliable. In contrast, studies relating to PtdIns
3-kinase
and PtdIns 3-kinase are hampered by a lack of reliable and
commercially available antibodies. Immunoblotting experiments revealed
that HT-29 cells predominantly expressed the p85
subunit of PtdIns
3-kinase, and we were unable to detect p85
in these
cells.3 Furthermore, in vitro
assays for associated lipid kinase activity revealed that IL-13
treatment increased the amount of lipid kinase activity present in
p85
immunoprecipitates. These data strongly indicate that the
p85/p110 heterodimeric PtdIns 3-kinase is coupled to and activated by
the IL-13R. However, coupling of the IL-13R to other PtdIns 3-kinase
family members cannot be entirely discounted, and these lipid kinases
may potentially contribute to the production of total
PtdIns(3,4,5)P3 extracted from IL-13-stimulated
32P-labeled HT-29 cells. Although the precise nature of the
IL-13R is unclear, the known p85 binding motifs have not so far been identified in the intracellular domain of the related IL-4R.
Nevertheless, the IL-4R has been demonstrated to associate with and/or
activate PtdIns 3-kinase (22, 26, 50), and this coupling is thought to
to occur via an intermediate adaptor such as IRS-2, which does contain
several YXXM motifs (20). IRS-2 is tyrosine-phosphorylated after IL-4 treatment, and this phosphorylation may be required for
association of IRS-2 with PtdIns 3-kinase (20). IRS-2 has also been
demonstrated to be tyrosine-phosphorylated and to associate with PtdIns
3-kinase after IL-13 treatment (11, 19), suggesting that IRS-2 may also
facilitate the coupling of the IL-13R to the putative signaling
cascades regulated by PtdIns 3-kinase. Interestingly, this study
revealed that IL-13 was not able to induce detectable tyrosine
phosphorylation of IRS-2 in HT-29 cells, although it did stimulate a
rapid and strong tyrosine phoshorylation of IRS-1 within 1 min of
poststimulation. Generally, the kinetics of IL-13-stimulated IRS-1
tyrosine phosphorylation correlate with the kinetics of IL-13-stimulated PtdIns 3-kinase activation. Data indicate that IRS-1
may potentially have an important role in coupling the IL-13R to
PI3-kinase in HT-29 cells. However, a role for IRS-2 in the coupling of
IL-13R to PtdIns 3-kinase in HT-29 cells cannot be entirely ruled out
because low stoichiometry tyrosine phosphorylation of IRS-2 after IL-13
stimulation may be sufficient to allow IRS-2 to recruit PtdIns 3-kinase
to the IL-13R.
Our demonstration that IL-13 differentially stimulates the tyrosine
phosphorylation of IRS-1 but not IRS-2 correlates with previous
observations that IRS-1 is a major phosphoprotein of nonhematopoietic
cells, whereas IRS-2 phosphorylation is induced principally in murine
hematopoietic cell types by various growth factors and cytokines (40,
51-55). Other evidence exists to indicate considerable heterogeneity
in both receptor structure and signal transduction in different cell
types for both IL-4 and IL-13. For instance, in immune cells IL-4 is a
growth and differentiation factor, and the common chain is
associated with the IL-4R (14). However, the common
chain is not
expressed in colon carcinoma cells in which IL-4 mediates a growth
inhibitory effect (56). Moreover, both IL-4 and IL-13 induce the
tyrosine phosphorylation of JAK2 tyrosine kinase in colon carcinoma
cells (56, 57) but are unable to tyrosine-phosphorylate JAK2 in immune
cells (11, 56, 58-60). Similarly, IL-4 is unable to phosphorylate JAK3
in colon carcinoma cells (56) but is able to phosphorylate JAK3 in
immune cells (11, 58, 60). In addition to phosphorylation of cellular
proteins, IL-4 has also been shown to trigger a unique second messenger
pathway in human but not mouse B cells. This is characterized by a
rapid, transient production of inositol (1,4,5)-trisphosphate and
mobilization of Ca2+, followed after a brief lag period by
an increase in intracellular cAMP (61). This pathway is also activated
by IL-13 in human monocytes and is required for the IL-13-mediated
inhibition of protein kinase C-triggered respiratory burst (62). It is
interesting to note, however, that we were unable to detect any changes
in intracellular Ca2+ concentration in the HT-29 cells
after IL-13 stimulation.3 The diversity and heterogeneity
of both receptors and signaling pathways activated by IL-4 and IL-13
may therefore facilitate the different functional effects of these
related cytokines. Given the possible heterogeneity of signal
transduction pathways coupled to the IL-13R, it will be necessary to
determine whether IL-13 is able to activate PtdIns 3-kinase in other
cell types such as immune cells and to investigate the relevance of
PtdIns 3-kinase to other IL-13 functional events. This will undoubtedly
help in evaluating the potential of the PtdIns 3-kinase pathway as a
new and specific therapeutic target for intestinal inflammation.
We have therefore provided the first evidence to demonstrate that PtdIns 3-kinase activation is an important signal in determining the IL-13R-mediated inhibition of iNOS mRNA expression in response to inflammatory cytokines in the epithelial cell line HT-29. Additional experiments are necessary to determine the precise downstream events that occur after PtdIns 3-kinase activation in this system. For instance, it will be necessary to determine the effect of IL-13 on the activity of known downstream effectors of PtdIns 3-kinase such as protein kinase B and p70 S6 kinase (25) and to determine whether constitutively active mutants of PtdIns 3-kinase and known downstream effectors can mimic the effects of IL-13 on iNOS expression.
We are grateful to Richard Parry for assistance with HPLC separations and to Melanie Welham for helpful comments and discussions.