(Received for publication, December 19, 1996, and in revised form, April 23, 1997)
From the Laboratory of Molecular Tumor Biology,
Division of Cellular and Gene Therapies, Center for Biologics
Evaluation and Research, Food and Drug Administration, Bethesda,
Maryland 20892 and the ¶ Division of Neurosurgery, Department of
Surgery, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033
Interleukin (IL)-13 and IL-4 are cytokine
products of TH2 cells which exert similar effects in
a variety of cell types. We recently described IL-13R expression on
human renal cell and colon carcinoma cells and demonstrated that
c is not a component of IL-13R or IL-4R systems in these
cells. In lymphoid cells such as B cells and monocytes, which respond
to IL-13,
c is a component of IL-4R but does not appear
to be a component of IL-13R. Furthermore, while significant IL-13
binding is observed on carcinoma cells, IL-13 barely binds these
lymphoid cells and the binding characteristics are different. To better
understand the role of
c in IL-13 binding and signaling,
we have transfected a renal cell carcinoma cell line with
c and examined IL-13 and IL-4 binding and signaling. IL-13 binding as well as IL-13 and IL-4 signaling through the JAK-STAT
signaling pathway were severely inhibited. This inhibition was
paralleled by a loss of expression of one of the IL-13R chains and
intercellular cell adhesion molecule-1. Thus, although
c has been shown to enhance IL-4 binding and function in some cell types,
its influence on IL-13R function in tumor cells appear to be largely
negative.
Interleukin-13 (IL-13)1 and IL-4 are pleiotropic immune regulatory cytokines, which are predominantly produced by activated lymphocytes. Both cytokines mediate similar effects on B cells and monocytes; however, IL-13 has not been shown to have direct effects on T cells (1-3). The effects of both cytokines are mediated by cell surface receptors (R) that are specific for each of these ligands (4, 5). The receptor for IL-4 has been extensively investigated and its structure appears to vary with cell types. We have proposed three models for the structure of the IL-4R complex (6). However, despite their structural differences, all of the identified forms of the IL-4R complex are quite effective in transducing signals in response to IL-4 binding.
Unlike the IL-4 receptor system, the receptor for IL-13 has not been
well characterized. We have demonstrated that RCC cells express a large
number of IL-13 binding sites and that these cells respond to IL-13 (4,
7). Cross-linking studies indicate that the IL-13R is predominantly
composed of a ~65-70-kDa protein (4). RCC cells also express IL-4R,
and 125I-IL-4 cross-links to IL-4R (the p140 chain of
IL-4R) and a 65-70-kDa IL-4-binding protein. The inhibition of IL-4
binding to both proteins by IL-13 supports the notion that IL-4 and
IL-13 receptors are structurally and functionally interrelated (4,
7-10). We recently proposed several models to account for the complex
interactions among IL-13, IL-4, and the receptors for these ligands as
they are expressed in different cell types. We proposed that IL-13R shares IL-4R
(p140) and IL-4R
(p70) with the IL-13R system in certain cell types (11). We had also predicted that IL-13 binds to a
heterodimer consisting of two ~70-kDa proteins, one of which binds
IL-13 alone (we termed this IL-13R
) while the other binds IL-13 as
well as IL-4 (we termed this IL-13R
) (7, 11). The genes for a
murine IL-13-binding protein, its human homologue, and a second human
IL-13-binding protein were subsequently identified and characterized
(12-14). The two human IL-13R genes encode 65-70-kDa proteins with
different IL-13 binding characteristics. One of these (corresponding to
our IL-13R
) requires IL-4R
to bind and transduce IL-13 signal
(14), while the other (corresponding to our IL-13R
) can bind IL-13
in the absence of IL-4R
, but its role in IL-13 signaling remains
unclear (13). Whether other as yet unidentified proteins are required
to constitute a functional high affinity IL-13R remains to be
resolved.
The IL-2R chain termed
c is shared by receptors for
IL-4, IL-7, IL-9, and IL-15 on immune cells (15-18). It was
hypothesized that IL-2R
c is also a component of the
IL-13R system (15). However, we have reported that an
anti-
c antibody did not immunoprecipitate any
125I-IL-13-bound protein from RCC (4) or colon carcinoma
(6) cell lysates, indicating that the
c protein may not
be directly involved in IL-13 binding. The
c protein was
not expressed in these cells. However, whether
c
affects IL-13 binding in cells that normally express it is not known.
It is also not known whether
c affects IL-13R structure
and signal transduction.
In this study, we have examined the effect of the c
chain on IL-13 and IL-4 binding and signaling. We have transfected
ML-RCC cells with the
c cDNA and examined its
influence on certain biological responses of these cells to IL-13 and
IL-4. We present evidence that
c severely decreased the
IL-13 binding capacity of these cells and prevented the expression of
the
chain of the IL-13 receptor as well as ICAM-1. Furthermore,
although
c had no significant effect on the binding of
IL-4 to its receptors, its presence altered IL-13 and IL-4 signaling
pathways in these cells in several ways, including the inhibition of
IL-13- and IL-4-induced STAT-6 activation.
Recombinant human IL-13 (rhIL-13)
was expressed in Escherichia coli and purified as described
previously (4). Recombinant human IL-4 was kindly provided by Dr.
Michael Widmer of Immunex Corp., Seattle, WA. Antibodies to JAKs 1 and
2 and biotin-anti-phosphotyrosine (4G10) were purchased from Upstate
Biotechnology, Inc. (Lake Placid, NY). Antibodies to c,
JAK3, Tyk2, and STATs 3, 4, and 6 were purchased from Santa Cruz (Santa
Cruz, CA). Antibodies to STAT1 and STAT5 were provided by Dr. David
Finbloom (Center for Biologics Evaluation and Research, Food and Drug
Administration (CBER, FDA), Bethesda, MD).
The ML-RCC (synonym: MA-RCC) renal cell carcinoma cell line was established in our laboratory as described previously (19) from primary surgical tissues and was maintained in HEPES-buffered Dulbecco's modified Eagle's medium with high glucose supplemented with glutamine plus 10% fetal calf serum and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin).
Transfection of ML-RCC CellsML-RCC cells (3 × 105) were plated in 12-well tissue culture plates and
cultured overnight. They were then transfected with a mixture of
cDNAs encoding the genes for the c subunit of the IL-2 receptor or neomycin transferase or both. Transfection was effected by the calcium phosphate method using the PROFECTANT kit
(Promega) according to the manufacturer's instruction.
Neomycin-resistant transfectants were selected by incubating the
transfected cells in 0.8 mg/ml active G418 for 10 days. Selected cells
were examined by Northern blot analysis to identify clones containing
the
c gene in addition to the neomycin resistance gene.
Two such clones isolated from the ML-RCC cell line were identified as
ML
c. Clones transfected with the neomycin resistance but not the
c gene (MLneo) were used as negative
controls for the double transfectants. Additional negative control
transfectants (i.e. neomycin-resistant,
c
(
)) were generated by using a recombination deficient retroviral
vector to introduce the neomycin resistance gene into the target ML-RCC cells. This was achieved by infecting target cells with supernatant generated by the amphotropic retroviral vector-producing PA317 cell
line (20), kindly provided by Dr. Carolyn Wilson (CBER, FDA). Identical
results were observed when all negative control and parental cell lines
were used in all tests. Similarly the two
c-positive
clones (ML
c) were identical in their responses to all
tests performed.
Cells were harvested from culture
with Versene (Bio-Whittaker, Walkersville, MD), washed, and total RNA
isolated in a one-step procedure using Trizol (Life Technologies, Inc.)
according to manufacturer's instructions. Aliquots of total RNA were
electrophoresed through agarose (0.8%)/formaldehyde denaturing gel,
transferred to a nylon membrane (S & S Nytran; Schleicher & Schuell) by
capillary action and immobilized by ultraviolet cross-linking
(Stratagene, Inc., La Jolla, CA). The membrane was then prehybridized
overnight at 42 °C and hybridized with a 32P-labeled
cDNA probe for c at 42 °C for 18 h. The
membrane was subsequently exposed to an X-AR film (Eastman Kodak Co.)
to obtain an autoradiograph. To evaluate the amount of RNA loaded per
lane, the blots were stripped and reprobed with 32P-labeled
GAPDH cDNA.
rhIL-13 and rhIL-4 were labeled with 125I (Amersham Research Products) by using IODO-GEN reagent (Pierce) according to the manufacturer's instructions. The specific activity of the radiolabeled cytokines was estimated to range from 20 to 80 µCi/µg of protein for 125I-IL-4 and 40-120 µCi/µg for 125I-IL-13. Binding experiments were performed as described previously (4). Typically, 1 × 106 cells were incubated at 4 °C for 3-5 h with 125I-IL-13 (100-500 pM) or 125I-IL-4 (100-500 pM) in the presence or absence of increasing concentrations (up to 1000 nM) of unlabeled IL-13 or IL-4. The data were analyzed with the LIGAND (21) program to determine the number of binding sites/cell as well as binding affinity.
Flow CytometryMLc RCC cells as well as
MLneo control cells were cultured at 1 × 105 cells/ml in medium containing IL-4 or IL-13 (0-10
ng/ml) over a 48-h period. Cells were washed and incubated with a
fluorescein isothiocyanate (FITC)-conjugated anti-human ICAM-1 antibody
(Immunotech/Coulter) at a concentration of 40 µg/ml in FACS staining
buffer (phosphate-buffered saline containing 0.1% fetal bovine serum,
2.5 mM EDTA, and 0.1% sodium azide) at 4 °C for 60 min.
Control cells were incubated in FACS staining buffer alone or
FITC-conjugated IgG1 (isotype control antibody). The cells
were subsequently washed and fluorescence data collected on a
FACScan/C32 equipment (Becton Dickinson, San Jose, CA). The data were
analyzed with a LYSIS II software program, and fluorescence intensity
was expressed as mean channel number on a 256-channel/104
log scale.
Cells (5-10 × 106) were incubated with 125I-IL-13 in the presence or absence of excess IL-13 or IL-4 for 2 h at 4 °C. The bound ligand was cross-linked to its receptor with disuccinimidyl suberate (Pierce) at a final concentration of 2 mM for 30 min. Cells were lysed in lysis buffer consisting of 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 0.02 mM leupeptin, 5.0 µM trypsin inhibitor, 10 mM benzamidine HCl, 1 mM phenanthroline, iodoacetamide, 50 mM aminocaproic acid, 10 µg/ml pepstatin, and 10 µg/ml aprotinin. The cell lysates were precleared and cleared by boiling in sample buffer containing 2-mercaptoethanol and analyzed by electrophoresis through an 8% SDS-polyacrylamide gel. The gel was subsequently dried and autoradiographed. In some instances, the receptor-ligand complex was captured from the precleared total cell lysate on protein A-Sepharose beads previously incubated with specific antibodies. The complex was washed in lysis buffer, boiled in sample buffer, and analyzed by SDS-PAGE.
Immunoprecipitation and ImmunoblottingML-RCC cells (2 × 107) were incubated with 250 ng/ml IL-13 or 50 ng/ml IL-4 or left untreated as control for 10 min at 37 °C. The reactions were quenched by addition of 10 ml of PBS containing 1 mM sodium vanadate, 25 mM NaF, 10 mM sodium pyrophosphate, and 1 mM EDTA. The cells were lysed at 4 °C with 1% Nonidet P-40, 300 mM NaCl, 50 mM Tris (pH 7.4), leupeptin (10 mg/ml), aprotinin (10 mg/ml), phenylmethylsulfonyl fluoride (1 mM), 1 mM sodium vanadate, 25 mM NaF, 10 mM sodium pyrophosphate, and 1 mM EDTA. The cell lysates were precleared by incubation with protein A-Sepharose beads (Sigma). They were then incubated with protein A-Sepharose beads that had been preincubated with primary antibody for 2 h at 4 °C. The resulting complex was washed five times in lysis buffer, resuspended in loading buffer (NOVEX), and separated through 8% SDS-polyacrylamide gel. Separated proteins were transferred to PVDF membrane (NOVEX) for 1 h. The blots were washed three times in TBS-T buffer (20 mM Tris-HCl, 137 mM NaCl, 0.1% Tween 20), followed by 1 h of incubation in horseradish peroxidase-conjugated goat anti-rabbit or strepto-avidin (Amersham), depending on the nature of the primary antibody, followed by three washes. The blots were incubated with RENAISSANCETM chemiluminescence substrate mixture according to the manufacturer's instructions (NEN Life Science Products) for 1 min and exposed to x-ray film for 1-30 min.
Electrophoretic Mobility Shift Assay (EMSA)After treatment
with/without IL-13 (250 ng/ml) or IL-4 (50 ng/ml), cells were washed in
cold PBS and solubilized with cold whole cell extraction buffer (1 mM MgCl2, 20 mM HEPES, pH 7.0, 10 mM KCl, 300 mM NaCl, 0.5 mM
dithiothreitol, 0.1% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, and 20% glycerol). DNA-binding
proteins were identified by EMSA by using bandshift kit from Pharmacia
Biotech Inc. Briefly, 30 µg of protein samples were incubated for 20 min at room temperature with 1 ng of 32P-labeled
oligonucleotide probe consisting of double-stranded GRR
(5-AGCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAG-3
) of the promoter of the
FcR
1 gene in 20 µl of binding buffer (10 mM Tris-HCl
(pH 7.5), 50 mM NaCl, 0.5 mM dithiothreitol,
10% glycerol, 0.05% Nonidet P-40, 0.05 mg/ml poly(dI-dC)·(dI-dC)).
A 200-fold excess of cold GRR probe was added as a competitor. Before
loading, 10 × loading dye was added to each sample and separated
in a 5% non-reducing polyacrylamide gel at 150 V for 2.5 h. Gels
were dried for 2 h and autoradiographed overnight at room
temperature. For the supershift assay, cell lysates were preincubated
with preimmune rabbit serum as a control or STAT1, 3, 4, 5, and 6 antibodies on ice for 30 min, standard EMSA was then performed as
described above.
Two
c mRNA-positive (+) clones of ML-RCC
(ML
c) were selected from five neomycin-resistant
(MLneo) clones. As shown in Fig. 1A, alternatively spliced
c mRNA (16) was detected in ML
c but
not in MLneo cells. Both of the ML
c clones
were tested in all experiments shown, and both yielded identical
results. Similarly, all the negative control clones (MLneo)
yielded identical results in tests performed. To confirm that the
c mRNA being transcribed was also being properly
translated into its protein in ML
c cell lines, we
examined total cell lysates from these
c
mRNA+ cell lines for the presence of
c
protein by immunoprecipitation and Western blot analysis. The results
indicate that ML
c cells expressed the
c
protein as detected by anti-
c antibody, whereas the
control and untransfected parental cell lines were negative (Fig.
1B). We have also demonstrated
c protein
expression on the surface of these
c
mRNA+ cells by flow cytometry (22). Following
c transfection, the ML
c clones took on a
different morphology, appearing smaller and more elongated than the
neomycin-resistant control cell lines (data not shown).
A and B, expression of
c mRNA and protein in ML
c cells. Ten
micrograms of total RNA from ML-RCC cells transfected with
c and/or neomycin phosphotransferase cDNA as
described under "Experimental Procedures" was fractionated over
formaldehyde denaturing agarose gel, transferred to a nylon membrane support, and hybridized with a 32P-labeled
c cDNA probe as described
previously (4). Blots were stripped and rehybridized with
32P-labeled cDNA probe for GAPDH (lower
panel, A). ML
c and MLneo cells (10 × 106) were lysed at 4 °C with 1%
Nonidet P-40 containing protease inhibitors as described under
"Experimental Procedures." Proteins in the cell lysate were
separated by SDS-PAGE and transferred to a PVDF membrane (NOVEX) for
Western blotting using a
c specific antibody. Detection
was achieved by chemiluminescence using RENAISSANCE reagents (NEN Life
Science Products). Molecular size standards are shown on the
left of the gel (B). C, affinity
cross-linking and immunoprecipitation. ML
c and
MLneo cells (7 × 107cells/ml) were
incubated with 5 nM 125I-IL-13 for 2 h at
4 °C. 125I-IL-13 was cross-linked to its receptor with
disuccinimidyl suberate (2 mM), and total cell lysate was
prepared from the cells, precleared in protein A-Sepharose beads. Some
lysates were incubated with protein A-Sepharose beads previously
incubated with
c-specific antibody. The
immunoprecipitated complex (IP) as well as the
125I-IL-13·IL-13R complex were analyzed by SDS-PAGE as
described (4).
Association of Transfected
Since c modulated the binding of
IL-13 to its plasma membrane receptors, it was important to determine
whether the
c protein expressed in ML-RCC cells
transfected with the
c cDNA is associated with IL-13
receptor proteins. We cross-linked 125I-IL-13 to its
receptors on
c-transfected and control cells. The cell
lysate from each group was subsequently incubated with anti-
c antibody, and the resulting antigen-antibody
complex was analyzed by SDS-PAGE. The gel was overexposed (5 days) to
ensure visualization of the IL-13/
c band. The results
(Fig. 1C) indicate the formation of an
125I-IL-13·IL-13R complex of 58-65 kDa in
ML
c cells. Furthermore, the
c protein
could be immunoprecipitated from this band in ML
c as a
faint ~60-kDa band (Fig. 1C) but not from the control
MLneo cell line (data not shown).
Since c has previously been shown to
increase the binding affinity of certain cytokine ligands to their
receptor (15-18), we examined its effect on IL-13 binding to
ML
c and MLneo cells by performing
125I-IL-13 equilibrium binding assays. The results (Table
I, Fig. 2,
A and B) show that the
neoR+ control RCC cells (MLneo)
significantly bound 125I-IL-13, and this binding was
specific as it was displaced by increasing concentrations of the
unlabeled ligand (Fig. 2). However, introduction of the
c chain into these cells resulted in a drastic reduction
of 125I-IL-13 binding. For example, assuming a 1:1 ratio of
radiolabeled ligand to cell surface receptor, an analysis of the
125I-IL-13 binding data with the LIGAND program (21)
indicates that neoR+ control MLneo
cell lines express 480 ± 86 × 103 (mean ± S.D.) IL-13 binding sites/cell, and the number of binding sites/cell on
the parental cell line, ML-RCC was determined to be 351 ± 90 × 103. However, in the
c-transfected
ML
c, 125I-IL-13 binding was dramatically
reduced (by up to 2 log orders) to 4080 ± 1890 binding
sites/cell. It is interesting that, in addition to reducing the level
of ligand binding,
c induced high affinity
125I-IL-13 binding to RCC cells (Fig. 2B). For
example, ML and MLneo cells bound 125I-IL-13
with a dissociation constant (Kd) ranging from 7.5 to 20.5 nM (mean ± S.D. = 13.4 ± 4.6 nM), but in ML
c, the Kd
value (mean ± S.D.) was 0.23 nM ± 0.14 (n = 4) (Table I). In some experiments, analysis of the
binding data with the LIGAND program (21) indicated a concurrent
expression of a lower affinity IL-13R (Kd = 7.5 nM) (Fig. 2B).
|
125I-IL-13 binding in
MLc and MLneo cells. Equilibrium
binding studies were performed on
c-transfected
(ML
c) and control (MLneo) ML-RCC cells by
incubating 1 × 106 cells with 100-500 pM
125I-IL-13 for 2-5 h. Increasing concentrations (0-1000
nM) of unlabeled IL-13 were added to compete the binding of
the radiolabeled ligand. Cell-bound radioactivity was determined with a
counter and the data analyzed with the LIGAND program (21). Binding
data are shown in A, while a Scatchard plot of the data is
shown in B. IL-4R and IL-13R expression in control
(MLneo) and
c-transfected (ML
c) cells was also analyzed by direct binding to 2 nM
125I-IL-4 or 125I-IL-13. Nonspecific binding was
determined by including excess unlabeled IL-4 or IL-13 in the binding
tube and was subtracted from total binding values to obtain specific
binding data (C).
Since IL-4R expression is much more limited in ML-RCC cells, we
compared the effect of c on IL-4 binding in
MLneo and ML
c cells by a direct binding
assay using a saturating concentration of 125I-IL-4 and
125I-IL-13. Our results (Fig. 2C) show that in
comparison to the profound decrease in IL-13R expression induced by
c, IL-4R expression is not significantly affected.
Since c inhibited IL-13 binding
and IL-13R
and IL-13R
chains were recently cloned, we were
interested to examine whether one or both chains were modulated by the
presence of
c in ML-RCC. By Northern analysis, we found
that IL-13R
mRNA is expressed in MLneo but not in
ML
c cells (Fig. 3,
upper panel). The expression of IL-13R
mRNA was
reduced in ML
c in comparison to MLneo (Fig. 3, middle panel). When we normalized IL-13R
mRNA
band to the GAPDH mRNA band in each cell type for comparison, we
observed that IL-13R
expression is about 3 times higher in
MLneo than in ML
c.
Modulation of ICAM-1 Expression by
Since
c modulated IL-13 binding and appeared to alter the
morphology of
c-transfected cells, we further
investigated its role in ML-RCC cells by examining the effect of IL-13
and IL-4 on the surface expression of ICAM-1 molecules. Consistent with our previous findings (4, 23), the control MLneo cell line had considerable baseline expression of ICAM-1 (Fig.
4A, left panel),
which was significantly enhanced by IL-13 or IL-4 treatment. However,
transfection of
c inhibited the basal level of ICAM-1 expression in ML
c and neither IL-13 nor IL-4 could
reverse this inhibition (Fig. 4A, right panel).
Additionally, we examined the effect of
c on ICAM-1
mRNA expression by Northern analysis. Our results show that in the
c-positive ML
c cells (Fig. 4B,
right lane) ICAM-1 mRNA is not expressed, but it is
expressed in the
c-negative MLneo and the
parental ML-RCC cells (two left lanes).
Effect of
It has been shown that a number of cytokines
including IL-13 and IL-4 can induce phosphorylation and activation of
the Janus family of kinase (24-27). We recently demonstrated that
colon carcinoma cells express JAK1, JAK2, and Tyk2 (6, 24) and that
these kinases are phosphorylated by IL-4 and IL-13. JAK3 was not
expressed and was not phosphorylated in these cells (24). In the
present study, we examined the impact of the presence of
c chain on the IL-13- and IL-4-induced phosphorylation
of JAK tyrosine kinase in ML-RCC cell lines. As shown in Fig.
5A, IL-4 induced the
phosphorylation of JAK1 and JAK2 tyrosine kinase in ML cells. JAK1
kinase was weakly phosphorylated in response to IL-13, but in contrast
to our previous observation in colon carcinoma cells (24), IL-13 did
not induce phosphorylation of JAK2 in ML-RCC cells. Tyk2 kinase was
constitutively phosphorylated, and this level of phosphorylation was
not enhanced by either interleukin. In the
c(+) ML
c cells, JAK1 was
constitutively phosphorylated and the level of phosphorylation was not
affected by IL-4 or IL-13 treatment. JAK2 was weakly phosphorylated in
unstimulated as well as in IL-13- or IL-4-treated groups and Tyk2
kinase was not detected. As previously observed in colon carcinoma
cells (24), JAK3 kinase was neither expressed nor phosphorylated in
MLneo and ML
c cells, although it was
expressed and phosphorylated in the EBV-B cell line used as a positive
control (Fig. 5B and Ref. 24). Thus, in
c-transfected ML
c cells, Tyk2 kinase was not
expressed and IL-4 and IL-13 could not enhance or induce the
phosphorylation of JAK1 and JAK2 otherwise seen in control ML
cells.
Effect of
We next investigated the effect of
c expression on STAT activation by performing EMSA on
lysates from these cells. As shown in Fig.
6A, in
c-negative cells (ML and MLneo), NF (nuclear
factor)-IL-4 was activated and bound to the labeled GRR probe in
response to IL-13 or IL-4. However, in the
c-positive
ML
c cells, NF-IL-4 was not activated in response to
IL-13 or IL-4. We ascertained the specificity of the assay by
incubating the labeled probe with a 200-fold excess unlabeled probe
before running it on the gel. No NF-IL-4/GRR bands were observed in
lanes containing these samples.
Effect of c on IL-13- and
IL-4-induced STAT phosphorylation and activation in ML-RCC cells.
A, after treatment with medium, IL-13 (250 ng/ml), or IL-4
(50 ng/ml), cells (ML, ML
c, MLneo) were
washed in cold PBS and solubilized with cold whole cell extraction
buffer. DNA binding activity was determined by EMSA. Briefly, 30 µg
of protein samples were incubated for 20 min at room temperature with
1 ng of 32P-labeled GRR probe in 20 µl of binding
buffer. A 200-fold excess of cold GRR probe was added as a competitor.
For the supershift assay (B), cell lysates were preincubated
with preimmune rabbit serum as a control or STAT1, 3, 4, 5, and 6 antibodies on ice for 30 min; standard EMSA was then performed as
described above. C, following IL-13 and IL-4 treatment,
cells were lysed in lysing buffer and STAT6 protein was
immunoprecipitated (IP) with specific antibody. The complex
was separated by SDS-PAGE and transferred onto PVDF membrane. STAT6
phosphorylation was detected by anti-phosphotyrosine antibody (4G10)
(upper panel). STAT6 protein expression was confirmed by
reprobing the stripped blot with STAT6 antibody (lower
panel).
To confirm the identity of the STAT protein which was activated in
response to IL-4 and IL-13 in ML and MLneo RCC cells, we next performed the supershift assay. The results (Fig. 6B)
show that the binding of NF-IL-4 in the IL-4-treated ML group is not affected by preincubation in normal serum or in antibodies specific for
STATs 1, 3, 4, or 5, whereas the band representing the bound NF-IL-4
was not visible when the lysate was preincubated in anti-STAT6 antibody. These results indicate that NF-IL-4 corresponds to STAT6 and
the other STATs were not activated in response to IL-4 in ML cells. In
the c-positive cells (ML
c), no STAT
protein was activated in response to IL-4.
To further investigate the mechanism underlying the c
inhibition of STAT6 activation, we examined the phosphorylation of STAT6 protein from
c(+) and
c(
) ML-RCC cells in response to IL-13 and
IL-4 by immunoprecipitation and immunoblotting. As shown in Fig.
6C, while IL-13 and IL-4 treatment induced STAT6
phosphorylation in the parental ML and the control
neoR+ transfectant (MLneo) cell
lines, STAT6 phosphorylation was not induced in the
c(+) ML
c cell line
(left panel). These results indicate that
c
inhibited the IL-4- and IL-13-mediated activation of STAT6 by
inhibiting its phosphorylation.
In our initial characterization of IL-13 binding, we noted that
while IL-13 was able to displace or inhibit IL-4 binding to RCC cells,
which lack c expression, it did not inhibit IL-4 binding in Raji B and MLA144 T lymphoid cells in which
c is
highly expressed. Furthermore, we noted that B cells and monocytes
which are highly responsive to IL-13 express very few IL-13R (4). This
led us to further explore the role of
c in IL-13
binding. In this report, we have demonstrated that the
c
protein can exert a profound influence on the binding of IL-13 to its
receptors and on the signal transduction pathways of IL-13 and IL-4
following ligand binding. Expression of
c in ML-RCC
profoundly inhibited IL-13 binding, IL-13-induced signaling, and ICAM-1
expression. Although IL-4 binding was not affected by
c
expression, IL-4-induced signaling was also impaired in the
c-transfected cell line and IL-4 failed to induce ICAM-1
expression in these cells.
Our results regarding the expression of c in cells that
co-express IL-4R and IL-13R demonstrate that
c
expression is disruptive to the expression of the
chain of the
IL-13R while diminishing IL-13R
expression, and may be responsible
for the reduction in IL-13 binding. On a broader scale, this may
explain why
c-positive cells such as B cells, monocytes,
and TF-1 cell lines, which are very responsive to IL-13, have been
shown to express low levels of IL-13R. Similarly, the failure of IL-13
to compete for the binding of IL-4 on MLA 144 and Raji B lymphoblastoid
cell line (4) otherwise seen in other cell types (4, 8, 10, 25) may be
due to the high
c content of these cells and the
consequent deficiency in IL-13R
expression.
It is of interest that the c protein affected IL-13R
rather than IL-4R in this manner because, although previous studies have shown that
c is a natural component of IL-4R in a
number of cell types, it has not been identified as a natural component of IL-13R in any cell type examined thus far (11). In a study carried
out with COS-7 cells, the inhibition of IL-13 but not IL-4 binding by
c was demonstrated (13). However, the biologic consequences of the observation were not addressed. Our results demonstrate that
c has the potential to physically
interact with components of native IL-13 signaling pathway and exert a
downstream effect. Consistent with this concept,
c
expression altered the pattern of intracellular signaling in response
to IL-13. For example, IL-13 has been shown to cause the
phosphorylation and activation of JAK1 and Tyk2 tyrosine kinase in
hematopoietic cells and JAK1, JAK2, and Tyk2 in non-hematopoietic cells
(6, 24-31). However, in the current study, phosphorylation of JAK2 and
Tyk2 was not seen in
c-transfected ML
c
cells. Similarly, IL-13 failed to induce the phosphorylation of JAK-1
kinase in
c-transfected cells, although IL-13 induced
the phosphorylation of JAK1 in control cells. Tyk2 was constitutively
phosphorylated in control cells, and IL-13 did not modulate its
phosphorylation. In
c-transfected cells, neither
constitutive nor IL-13-induced phosphorylation of Tyk2 was observed.
These results suggest that the expression of
c leads to
an inhibition of IL-13-mediated activation of JAK kinase in ML-RCC cell
line and that IL-13R
may play a pivotal role in these signaling
events.
The activation of JAK kinase has been shown to cause the
phosphorylation and activation of STAT proteins. We therefore evaluated phosphorylation and activation of STAT proteins in control and c-transfected ML
c cells as well as the
effects of IL-13 on these events. It is of interest to note that IL-13
phosphorylated and activated STAT6 protein in control cells but not in
c-transfected ML
c cells. These data
suggest that
c expression and the accompanying disruption of IL-13R
and/or diminution of IL-13R
expression inhibited not only the proximal events that result from IL-13 activation but the distal/terminal IL-13 and IL-4 signaling events as
well. The results also suggest that IL-13R
and IL-13R
may be
essential for the transduction of certain IL-4 and IL-13 signals supporting previous suggestions that the signal pathways for both cytokines converge at one or more points.
The reason for the profound decrease in IL-13 binding to
c-transfected ML cells is not completely known. However,
our results suggest that IL13R
is predominantly essential for IL-13
binding and its apparent displacement by
c in the IL-13R
complex may be related to the drastic reduction in IL-13 binding. This
structural modification of the IL-13R complex by
c may
also have affected the affinity of its binding to IL-13. In this study,
we evaluated IL-13 binding by 125I-IL-13 displacement
assays and our results suggest that the affinity of IL-13 binding is
lower in MLneo than in ML
c. However, in
other studies,2 we have
proposed a different model for the analysis of IL-13 binding to these
cells, which may be more appropriate for addressing the apparent
cluster distribution of IL-13R on these cells. While
c
may have favored high affinity IL-13 binding, it may also have disrupted necessary interactions between the different chains of IL-4R
and IL-13R through the sequestration of IL-4R
by
c.
The loss of IL-4 and IL-13 signaling in MLc may also
have resulted from a disruption of necessary interactions between the intracellular domains of the receptor for IL-4 and IL-13. It was recently shown that the IL-4R
protein undergoes homodimerization for
IL-4 signaling and that
c is not required for the
phosphorylation and activation of STAT6 protein (32, 33). Although
there is no direct evidence that the different intracellular domains of IL-4R and IL-13R interact, IL-13 has been shown to phosphorylate IL-4R
(24). Thus it is quite likely that the introduction of
c interferes with the assembly of a functional IL-4R
complex, resulting in an impaired intracellular signaling by IL-4 and
IL-13. It is also possible that the introduction of
c
may have activated phosphatases whose activities could have resulted in
the disruption of IL-13 and IL-4 signaling. Additional studies are
necessary to address these possibilities.
We next examined the effect of c on the functional
response of ML-RCC cells to IL-4 and IL-13 stimulation. Surprisingly, in
c-transfected ML
c cells, there was a
distinct change in morphology accompanied by a drastic diminution of
ICAM-1 expression, and neither interleukin reversed these changes. The
mechanism(s) underlying these changes is not clear but our results
suggest that the disruption of IL-13R
is directly or indirectly
involved in a structural modification of the cytoskeletal framework of
the cell to alter its shape and size. Whether this morphological effect
is related to the loss of ICAM-1 expression is not clear.
Alternatively, the introduction of
c into the cells may
have turned off ICAM-1 gene expression and cellular responsiveness to
IL-13 and IL-4. Additional studies are needed to improve our
understanding of these events.
In summary, we have provided evidence for a novel role for
c protein in ML-RCC cells. Not only did this protein
inhibit the binding of IL-13 to its receptor but, more importantly, it
also inhibited intracellular signaling induced by IL-13 and IL-4. In addition,
c modified cellular function in these cells by
inhibiting constitutive IL-13R
and ICAM-1 expression, and this
inhibition was not restored by IL-4 or IL-13. Our results suggest that
an abnormal expression of
c may have significant disease
implications with or without IL-13 and IL-4 involvement and that
additional studies are needed to better understand the role of
c in tumor cell function.
We acknowledge the expert assistance provided
by Howard Mostowski (flow cytometry) and invaluable technical support
provided by Pam Leland, both of CBER, FDA. We are grateful for useful
discussions of the manuscript with Drs. Priti Mehrotra and Angus Grant
(CBER, FDA). We also thank Dr. Warren Leonard (NHLBI, NIH) for kindly providing cDNAs for c and IL-13R
chain.