(Received for publication, September 21, 1995; and in revised form, October 20, 1995)
From the
We have previously reported on the expression of interleukin-4
receptors (IL-4R) on many human epithelial cancer cells; however, the
binding characteristics, structure, function, and signal transduction
through the IL-4R in cancer cells is not known. IL-4 binding
characteristics were determined in human colon carcinoma cell lines by
a I-IL-4 binding assay, which demonstrated that the HT-29
and WiDr colon cancer cell lines expressed high affinity IL-4R (K
= 200 pM).
Cross-linking experiments revealed a major band of 140 kDa and a broad
band at 70 kDa. While the common
chain of IL-2R is associated
with IL-4R in immune cells and is similar in size to the 70-kDa
protein, this chain was not expressed in these colon cancer cells.
Interestingly, IL-13, which has many functions similar to IL-4,
inhibited
I-IL-4 binding to both the 140- and 70-kDa
molecules. Next, we investigated the mechanism of IL-4-induced signal
transduction in colon cancer cells. After stimulation with IL-4, a
170-kDa band was primarily phosphorylated within 1 min of exposure and
was identified as insulin receptor substrate-1. In addition, by
immunoprecipitation assay, three other phosphorylated bands were
identified as JAK1, JAK2, and Tyk2 tyrosine kinases. The
phosphorylation of JAK1 and JAK2 was induced by IL-4 stimulation;
however, Tyk2 was constitutively phosphorylated, and IL-4 treatment
further augmented this phosphorylation. The kinetics and in vitro kinase assays demonstrated that JAK1, JAK2, and Tyk2 were
phosphorylated within minutes and that JAK1 and JAK2 were activated
after IL-4 exposure. Contrary to observations in immune cells, JAK3
mRNA was neither detected in colon cancer cells nor did IL-4 treatment
cause phosphorylation of JAK3. These data indicate that in colon
carcinoma cells JAK1, JAK2, Tyk2, and insulin receptor substrate-1 are
phosphorylated after IL-4 stimulation. In addition, as is the case in
lymphoid cells, IL-4 activated and phosphorylated signal transducers
and activators of transcription (IL-4-STAT or STAT-6) protein in both
colon cancer cell lines. These results indicate that the IL-4R complex
is composed of different subunits in different tissues and shares a
component with the IL-13R complex. In addition, we demonstrate for the
first time that like its family members (e.g. IL-3 and
GM-CSF), IL-4 can phosphorylate and activate JAK-2 kinase.
IL-4 ()is a growth and differentiation factor of
human B- and T-lymphocytes(1, 2, 3) . In
contrast to its growth stimulatory effects on lymphocytes, IL-4 has a
growth inhibitory effect on many human carcinoma cells. We (4, 5) and others (6, 7, 8) have reported that IL-4 can inhibit
the growth of human melanoma, colon, breast, and renal cell carcinomas
in addition to cells of hematologic
malignancies(9, 10, 11) . It has been shown
that IL-4 receptors are expressed on a variety of cell types (12, 13, 14) and that IL-4 functions by
signaling through its receptors(15) . However, the mechanism
for the opposing biological activities elicited by IL-4 is not clear.
While the structure of the IL-4R has been studied extensively, it
has not been fully characterized. Cross-linking studies using I-IL-4 have revealed that, on human cells, radiolabeled
IL-4 cross-linked to one major protein of 140 kDa, and in some cases,
one or two additional bands were cross-linked (70-80 and
65-70 kDa)(16, 17, 18, 19) .
In COS-7 cells transfected with the human IL-4R cDNA, radiolabeled IL-4
bound to a 140-kDa protein(15, 20) ; however, this
binding was not sufficient to cause IL-4 signaling(20) .
Cotransfection of the IL-2R
chain (a 64-kDa protein, termed
) into these COS-7 cells (20) caused
IL-4-induced phosphorylation of insulin receptor substrate-1 (IRS-1).
These data suggest that
is associated with the
140-kDa protein of IL-4R, and this association is necessary for
signaling in these cells. Subsequently, the
chain has
been shown to be utilized in other receptor systems, such as those for
IL-7, IL-9, and
IL-15(20, 21, 22, 23, 24) .
The identity of the 70-80-kDa IL-4 cross-linked species is still
not clear. Previously, it was thought to be a breakdown product of the
140-kDa protein(25) , although recent studies have identified a
low affinity 70-kDa IL-4R protein(26) .
More recent studies
have examined the mechanism of IL-4 signaling in different cell types.
As a member of the hematopoietin family and cytokine receptor
family(27) , IL-4R has no consensus sequence motifs for
tyrosine and/or serine/threonine kinases in its intracellular
domain(15, 28) . However, IL-4R has been reported to
associate with tyrosine kinases and induce tyrosine phosphorylation of
110-, 140-, and 170-kDa proteins in murine cell
lines(29, 30) . The 140-kDa protein has been shown to
be an IL-4 binding receptor protein(31, 32) , and the
170-kDa protein was designated 4PS, which is similar to
IRS-1(32, 33) . It was demonstrated that IRS-1 or 4PS
expression is necessary for efficient IL-4 and insulin-mediated
mitogenic signaling in 32D cells (34, 51) . In
addition, it has been shown that the 140-kDa IL-4R protein associates
with and activates, by phosphorylation, members of the Janus kinase
family (JAK)(35) . The JAK family consists of four members,
JAK1, JAK2, JAK3, and Tyk2. Recently, it has been shown that IL-4
stimulated the proliferation of D10 T cells and that this proliferation
was correlated with the phosphorylation of the JAK1 and IRS-1 (36) proteins upon ligand-receptor interaction. Association of
the JAK3 kinase with IL-2R and IL-4R complexes has also been
demonstrated (37, 38, 39, 40) .
Furthermore, it has been suggested that as association between
and JAK3 is essential for signaling through IL-2R
system, the malfunction of
-JAK3 pathway is believed
to be tied to X-linked severe combined immune deficiency syndrome or
XCID(23) . However, the steps involved in the signaling
pathways leading to growth inhibition of tumor cells triggered by IL-4
are not known.
In the current study, we examined the binding
characteristics, structure, function, and signaling through the IL-4R
complex on human colon carcinoma cells. Our data indicate that the
IL-4R complex on colon carcinoma cells is composed of a predominant
140-kDa protein and a diffuse band suggesting a 70-kDa protein. By
Northern analysis, cross-linking, and immunoprecipitation, it was
demonstrated that while the common is not associated
with the IL-4R system on colon cancer cells, these IL-4 receptors were
functional because IL-4 caused phosphorylation of signaling proteins
and inhibited the growth of these cells in tissue culture. To
characterize the IL-4 signaling pathways in colon carcinoma cells, we
examined the patterns of protein phosphorylation upon stimulation with
IL-4. We report here that IL-4 induced tyrosine phosphorylation of
JAK1, JAK2, Tyk2, and IRS-1 but not JAK3 proteins in colon cancer
cells. We further demonstrate that the STAT-6 protein was
phosphorylated and activated by IL-4 treatment.
For immunoprecipitation, I-IL-4
IL-4R cross-linked complex was
immunoprecipitated from the lysate overnight at 4 °C by incubating
with protein A-Sepharose beads, which had been preincubated with
anti-
antibody. The resulting conjugate was washed
twice with solubilizing buffer, diluted with reducing buffer, boiled
for 5 min, and analyzed by SDS-PAGE as described above. The gel was
dried and autoradiographed.
Figure 1: Expression of high affinity IL-4R on HT-29 and WiDr cells. Displacement curve (A) and Scatchard analyses (B) were generated from the binding data using the LIGAND program.
To examine whether the IL-4 receptors expressed on colon cancer
cells were functional, we investigated the effect of IL-4 on the
proliferation of these cells by [H]thymidine
incorporation assay. IL-4 inhibited tumor cell growth in both cell
lines in a dose-dependent manner. Maximal growth inhibition (
50%)
occurred at
10 ng/ml (data not shown); however, the IL-4-induced
growth inhibitory effects were less pronounced in the WiDr colon cancer
cell line. It is possible that the lower number of IL-4 binding sites
on WiDr cells compared to HT-29 cells explain the lower responsiveness
to IL-4.
Figure 2:
IL-4 receptor structure analysis on colon
cancer cell lines. A, HT-29 and WiDr cells (5
10
) were labeled with
I-IL-4 in the absence (lanes 1 and 4) or presence of excess unlabeled IL-4 (lanes 2 and 5) or IL-13 (lanes 3 and 6). Molecular weight markers are shown on the left. Upper arrow on the left of A corresponds to
140 kDa, and the lower arrow corresponds to 70-kDa proteins. B, immunoprecipitation of
I-IL-4R complex with
anti-
antibody. Note the lower arrow in this
figure corresponds to the 63-kDa protein. C, Northern blot
analysis for
. Total RNA (20 µg) from cell lines
was electrophoresed in formaldehyde/agarose gels, transferred to
membranes, and probed with cDNA of
. Equivalent RNA
loading was ascertained when this blot was rehybridized with
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. The
positions of 28 and 18 S RNAs are shown on left.
When I-IL-4
IL-4R cross-linked complexes were
immunoprecipitated with anti-
antibody, no bands were
detected on SDS-PAGE and autoradiography in both cell lines (Fig. 2B, lanes 2 and 3). However,
anti-
antibody immunoprecipitated 140- and 65-kDa
(
) bands in phytohemagglutinin-activated peripheral
blood lymphocytes (Fig. 2B, lane 1). These
data indicated that
is not associated with IL-4R on
both colon carcinoma cell lines. The absence of
was
further confirmed by Northern analysis, which did not detect common
mRNA in both HT-29 and WiDr cell lines. The human T
cell line (H9) used as positive control expressed significant level of
mRNA (Fig. 2C).
Figure 3:
Protein tyrosine phosphorylation in HT-29
and WiDr colon carcinoma cells induced by IL-4. A, HT-29 and
WiDr cells were serum starved and stimulated with IL-4. The total cell
lysates of 1 10
cells/sample were separated by 8%
SDS-PAGE, transferred to PVDF membranes, and immunoblotted with
antiphosphotyrosine antibody (4G10). B, same membrane was
stripped and reimmunoblotted with IRS-1 antibody and visualized by ECL. C, HT-29 cell lysates were immunoprecipitated with anti-IL-4R
(p7) (lane 1, IL-4 treated; lane 2, control) and
IRS-1 (lane 3, IL-4 treated; lane 4, control)
antibodies and immunoblotted with antiphosphotyrosine antibody. The
same blots were stripped and reprobed with anti-IL-4R and IRS-1
antibodies. D, WiDr cells were immunoprecipitated with
anti-IRS-1, immunoblotted with antiphosphotyrosine, stripped, and
reblotted with anti-IRS-1 antibody. The positions of the molecular
weight markers are shown on left, and proteins of interest are
shown on right.
To determine the identity of the 170-kDa band, we reimmunoblotted the membrane with IRS-1 antibody. Our results indicate that the phosphorylated 170-kDa protein in both colon cancer cell lines is the IRS-1 protein (Fig. 3B). Immunoprecipitation with anti-IRS-1 antibody before immunoblotting with 4G10 revealed that IRS-1 was constitutively phosphorylated in the cultured cells (RPMI plus 10% fetal calf serum) (Fig. 3C, lane 4), and IL-4 treatment of HT-29 cells further increased this phosphorylation (Fig. 3C, lane 3). Similarly, IRS-1 was constitutively phosphorylated in WiDr cells, and IL-4 stimulation further increased this phosphorylation (Fig. 3D). These data further confirmed that IRS-1 is phosphorylated in response to IL-4.
We next investigated whether IL-4 induced phosphorylation of IL-4R p140 protein. HT-29 cells were either left untreated (Fig. 3C, lane 2) or treated with IL-4 for 5 min (Fig. 3C, lane 1), cell lysate was immunoprecipitated with anti-IL4R p140 specific antibody, and immunoblotting was performed with 4G10. As shown, IL-4 induced phosphorylation of the IL-4R p140 protein.
The identity of the kinase involved in the phosphorylation of both proteins, IRS-1 and IL-4R 140 kDa, is not known. Yin et al.(36) reported that in T cells JAK1 forms complexes with IL-4R p140 and IRS-1/4PS proteins, indicating that JAK1 may phosphorylate both IRS-1/4PS and IL-4R p140 proteins.
Figure 4:
Tyrosine phosphorylation of JAK1, JAK2,
and Tyk2 kinases and time course. HT-29 and WiDr cells were stimulated
with IL-4 for 5 min or not stimulated (A) or HT-29 cells were
stimulated for various periods of time (B-D). Cell
lysates (2 10
cells/sample) were immunoprecipitated
with indicated antibodies as described in Fig. 3.
Immunoprecipitates were separated by 8% SDS-PAGE, transferred to PVDF
membranes, and immunoblotted with 4G10 antibody. Then, membranes were
stripped and reimmunoblotted with anti-JAK1, JAK2, and Tyk2 and
visualized by ECL. Control and IL-4-pretreated HT-29 cells were
subjected to anti-JAK1, Tyk2, and JAK2 immunoprecipitation for in
vitro kinase assay (E). Cell lysates from 20
10
cells/lane were utilized. Blots were stripped and
reblotted with anti-JAK1, Tyk2, and JAK2 antibodies. As seen, band
intensities were darker in IL-4+ATP-treated lanes compared to IL-4
and no ATP lanes for JAK1 and JAK2 tyrosine kinases; however, no change
in band intensity was observed with Tyk2
kinase.
The kinetic studies were next undertaken, which indicated that the tyrosine phosphorylation of JAK1 and JAK2 occurred within 1 min following incubation with IL-4. This IL-4-induced phosphorylation reached a maximum at 5 min for JAK1 (Fig. 4B) and 5-10 min for JAK2 (Fig. 4C). The constitutive phosphorylation of Tyk2 was also increased within 1 min of IL-4 treatment, and this increase reached a maximum at 5 min (Fig. 4D).
Figure 5: IL-4 does not phosphorylate JAK2 in T cells. H9 cells were stimulated with IL-4 for 5 min, and cell lysates were immunoprecipitated with anti-JAK2, electrophoresed on 8% SDS-PAGE, and immunoblotted with antiphosphotyrosine antibody. The blot was stripped and reblotted with anti-JAK2 and JAK3 antibodies. The positions of the molecular weight markers are shown on the left, and proteins of interest are shown on right.
Figure 6: Phosphorylation of IL-4 STAT. HT-29 and WiDr cells were treated with IL-4 for 5 min, and then cell lysates from an equal number of control and IL-4 stimulated cells were immunoprecipitated with anti-STAT-6 antibody. Samples were electrophoresed, and blot was hybridized with anti-phosphotyrosine antibody. Molecular weight markers are shown on the left, and proteins of interest are on the right. In many experiments, even though we used equal number of cells in control and IL-4 treated cells, still unequal amounts of STAT-6 were detected by blotting with anti-STAT-6 antibody. However, in all experiments, no phosphorylation of STAT-6 protein was observed in control cells, but IL-4 treated cells showed significant phosphorylation.
In this study, we demonstrate that human colon carcinoma cell
lines express high affinity IL-4R and that these receptors are
functional since IL-4 inhibited their growth in tissue culture. The
IL-4R on these colon cancer cells seemed to be composed of two major
proteins with a molecular mass of 140 and 70 kDa. The 140-kDa IL-4R
protein has been well characterized(28) ; however, the exact
identity of the-70 kDa protein is not clear. It was previously thought
that the 70-kDa protein was a proteolytically degraded product of the
larger 140-kDa protein(19) . However, other studies (20, 21) have demonstrated that the IL-2R common
chain (64 kDa), which is similar in size to the 70-kDa protein, is a
component of the IL-4R complex in immune cells. Thus, it is possible
that the common
chain is also a component of the IL-4R complex in
colon cancer cells. To determine the identity of the 70-kDa IL-4R
subunit, two types of experiments were performed. First, cell lysates
were immunoprecipitated with antibody to
(23) and analyzed on SDS-PAGE. These data demonstrated
that although monoclonal antibody to
immunoprecipitated a 64- and 140-kDa protein in
phytohemagglutinin-activated T cells, no bands were immunoprecipitated
in the colon cancer cell lines examined (Fig. 2, A and B). Second, by Northern analysis, mRNA for
was not detected in either colon cancer cell line. Thus, unlike
human B and T cells, the IL-4R complex on colon cancer cells does not
utilize the common
chain for the function of IL-4.
The identity of the 70-kDa IL-4R protein is still unknown. It is of
interest to note that IL-13, a recently discovered cytokine that has
many activities similar to IL-4(46, 47) , inhibited
the appearance of both I-IL-4 cross-linked p70 and p140
bands on SDS-PAGE as did unlabeled IL-4 (Fig. 2). These data
agree with our recent report, which indicated that IL-13 competes for
the binding of IL-4 to its receptors on human renal cell carcinoma
(RCC) cell lines (41) . In addition, our data agree with
another previous report, which demonstrated that IL-13 competitively
inhibited the binding of human IL-4 to cells that respond to both IL-4
and IL-13(48) . Taken together, these results suggest that the
IL-4R complex shares a component with the IL-13R complex. Since, as
shown previously,
I-IL-13 cross-linked to a
58-69-kDa protein in RCC cell lines(41) , and this size
is similar to the 70-kDa protein of the IL-4R complex, it is
hypothesized that this 70-kDa protein is also a component of the IL-13R
complex.
It is also of interest to note that the structure of the IL-4R complex on colon cancer cell lines appears to be similar to that of RCC cells (18, 41) . Both types of human cancers originate from different precursor cell types, yet they seem to express two similar 140- and 70-kDa IL-4 binding proteins.
To understand the mechanism of IL-4-induced inhibition of tumor cell growth in vitro, we investigated the mechanism of signal transduction by IL-4 in colon cancer cells. We have demonstrated that IL-4-induced signaling events in colon carcinoma cell lines are different from those reported on immune cells(36, 39, 40, 50) . In both colon carcinoma cell lines, JAK1, JAK2, Tyk2, and 4PS/IRS-1 proteins were tyrosine phosphorylated in response to IL-4. These data partially agree with recent reports in which IL-4 was found to phosphorylate JAK1 and 4PS/IRS-1 in T cells, NK cells, and myeloid cells(36, 37, 39, 40) . Similarly, in a recent study Tyk2 was also shown to be phosphorylated after IL-4 stimulation of human erythroleukemia (TF-1) and murine plasmacytoma cell lines (B9)(40) . However, in contrast to previous reports utilizing immune cells(39, 40, 50) , JAK3 kinase was not phosphorylated in response to IL-4 in colon cancer cells. These data corroborated with Northern analysis and immunoprecipitation data and showed that both colon cancer cell lines studied did not express JAK3 mRNA (data not shown). In addition, the phosphorylation of JAK2 protein by IL-4 was not seen in the reports using immune cells, e.g. CTLL T lymphocytes, D10 T lymphocytes, TF-1, and FD-5 cell lines(36, 39, 40, 50) , whereas we demonstrate JAK2 utilization by IL-4 in colon cancer lines. Our data provide the first report that shows that, like the other members of the IL-4 family of lymphokines (e.g. GM-CSF and IL-3)(56, 57) , IL-4 can induce phosphorylation and activation of JAK2 tyrosine kinase.
It has been previously reported
that the common chain is required for tyrosine
phosphorylation of IRS-1 in response to IL-4 in immune
cells(20) . It has also been reported that expression of the
common
chain, in addition to IL-4R p140, is required
for the proliferation of mouse F7 cells in response to IL-4 (60) . However, colon carcinoma cells did not express
chain, yet the phosphorylation of IRS-1/4PS was
observed. Our data suggest that another protein, the 70-kDa protein
identified here, may function instead of
in HT-29
cells to help phosphorylate IRS-1 in response to IL-4.
In previous
studies, IRS-1 was shown to be phosphorylated in response to IL-4 only
in murine cells(32, 33, 36) , and no
phosphorylation was observed in human and murine cells transfected only
with human IL-4R 140-kDa protein(20, 52) . However, a
recent study reported that IL-4 caused phosphorylation of IRS-1 on the
human TF-1 cell line, which expressed (53) .
Our results agree with this report and provide direct evidence that
IL-4 can also cause phosphorylation of IRS-1/4PS in human colon cancer
cells without the presence of
. The type of kinase
that utilizes IRS-1/4PS as a substrate is still unclear. Since JAK1 and
IRS-1/4PS have been shown to associate with the IL-4R p140 protein in
murine T cells(36) , it is possible that JAK1 phosphorylates
IRS-1/4PS. Furthermore, since Tyk2 and JAK2 were also phosphorylated by
IL-4 in colon cancer cells, it is possible that these kinases also
utilize IRS-1/4PS as a substrate.
Following the Janus kinase family activation, tyrosine phosphorylation and activation of the STAT family of transcription factors may follow (54) . Recently, growth factors and cytokines including IL-4 have been shown to activate specific STAT proteins(43, 44, 45) . For example, IL-4 has been reported to activate IL-4 STAT/STAT-6 in immune cells(44, 45) . Similarly, in the present study, we found that IL-4 was able to activate STAT-6 in both colon carcinoma cell lines.
It is clear that IL-4 has contrasting effects on the
growth of different cell types. In antigen-specific T helper
lymphocytes (D10) and other human immune
cells(36, 55) , IL-4 has potent growth stimulatory
effects; however, in colon carcinoma cells and other solid human
carcinoma
cells(4, 5, 6, 7, 8, 55) ,
it has growth inhibitory effect. The reason for the contrasting effects
of IL-4 on cell growth is not clear. Some of the differential effects
may be attributable to the differential receptor structure on different
cell types (Fig. 7). One of these differences identified in this
and other studies (41) is the lack of in
cancer cells. It is possible that the absence of
may
change the way cell responds to IL-4. However, we have recently
observed that RCC cell lines stably transfected with the
chain
still showed IL-4 growth inhibitory effects as did untransfected
control cells. (
)Thus,
chain does not seem
to contribute directly in the growth inhibitory effects of IL-4. We
therefore hypothesized that the differential signal transduction
pathway may be responsible for the contrasting effects of IL-4. In
colon cancer cells, IL-4 caused the growth inhibition and
phosphorylation of JAK2; however, JAK3 was not phosphorylated. IL-4
induced the proliferation of lymphohemopoietic cells including, T cells
and TF-1 and B9 cells, and induced the phosphorylation of JAK3 but not
JAK2 kinase in these
cells(36, 39, 40, 50) . Based on
these observations, it is tempting to speculate that the absence of the
JAK3 complex or the presence of the 70-kDa/JAK2
activation may determine the type of response that cells generate.
However, further evidence is needed to support this speculation.
Experiments are ongoing to address these possibilities.
Figure 7:
Schematic model showing that the IL-4R
structure is different in various cell types. This model summarizes
data from this and previous studies. No correlation is made between the
structure and function of IL-4R. Some examples of cell types, which
express different chains of IL-4R, are shown below the figure.
Tumor cells (18, 41) and some monocytic cell lines (58, 59) do not express , however,
tumor cells express 140-kDa (termed IL-4R
) and 70-kDa (termed
IL-4R
) proteins. It has been suggested that the IL-4R
protein
may be a component of the IL-13R(41) . T cells and NK cells do
not express IL-13R, but they do express
. On the other
hand, B cells and monocytes respond to IL-13 and express
IL-13R(41) , and thus IL-4R in these cells may be composed of
three components (IL-4R
, IL-4R
, and
).
Our data demonstrate that the IL-4R complex on human colon
carcinoma cells is different from that expressed on immune cells (see schematic model in Fig. 7). Unlike immune cells, IL-4R
on colon cancer cells do not utilize the common otherwise shared between the receptors for IL-2, IL-4, IL-7,
IL-9, and IL-15. Based on our data and published information, we
propose that the IL-4R system may be composed of two to three subunits.
In some cells, IL-4R may share
, while in other cells
it may share a chain with the IL-13R complex; yet still in other cell
types all three chains may be present. The latter model of IL-4R may
resemble that of the IL-2R system, which is also composed of a
trimolecular complex.
Finally, even with the contrasting effects on immune and tumor cells, IL-4 induced rapid phosphorylation of JAK1, Tyk2, 4PS/IRS-1, and STAT-6 proteins in both types of cells. However, JAK3 was neither present nor phosphorylated in these tumor cells. Furthermore, we report for the first time that JAK2 is phosphorylated and activated in response to IL-4. Thus, differences in the subunit structure of the IL-4 receptor and IL-4 signaling pathways exist between tumor cells and immune cells. Additional studies are necessary to determine whether one or both of these differences may be responsible for contrasting functional effects of IL-4.