From the Department of Molecular Genetics and
Microbiology, Robert Wood Johnson Medical School, Piscataway, New
Jersey 08854-5635 and the ¶ Division of Therapeutic Proteins,
Center for Biologics Evaluation and Research, Food and Drug
Administration, Bethesda, Maryland 20892
Received for publication, August 28, 2000, and in revised form, October 10, 2000
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ABSTRACT |
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Interleukin-10 (IL-10)-related T cell-derived
inducible factor (IL-TIF; provisionally designated IL-22) is a cytokine
with limited homology to IL-10. We report here the identification of a
functional IL-TIF receptor complex that consists of two receptor chains, the orphan CRF2-9 and IL-10R2, the second chain of the IL-10
receptor complex. Expression of the CRF2-9 chain in monkey COS cells
renders them sensitive to IL-TIF. However, in hamster cells both
chains, CRF2-9 and IL-10R2, must be expressed to assemble the
functional IL-TIF receptor complex. The CRF2-9 chain (or the IL-TIF-R1
chain) is responsible for Stat recruitment. Substitution of the CRF2-9
intracellular domain with the IFN- Six new ligands with limited sequence homology (19-27% identity)
to IL-101 have been recently
identified (1-5). One of these IL-10 homologs is a viral protein,
whereas others are encoded in the genome. Cytomegalovirus-encoded
IL-10, designated cmvIL-10 (5), demonstrates only 27% identity to
human IL-10. Despite this limited homology, cmvIL-10 binds to and
signals through the canonical IL-10 receptor complex (5, 6). cmvIL-10
is produced by cytomegalovirus-infected cells and is likely to play a
role in immune evasion helping virus to avoid clearance by the host
immune system (5, 7). Another IL-10 homolog was cloned as a protein
whose expression is elevated in terminally differentiated human
melanoma cells and was designated mda-7, for melanoma
differentiation-associated gene 7 (3). The expression of the rat mda-7
analog was linked to wound healing (8) (the protein was designated
c49a) and to ras transformation (9) (the protein was
designated mob-5). The expression of rat mda-7 (c49a) was localized
primarily to fibroblast-like cells at the wound edge and base. During
wound healing the level of c49a mRNA was transiently elevated 9- to
12-fold above unwounded controls (8). In addition, expression of rat
mda-7 (mob-5) was demonstrated to be induced by expression of oncogenic
ras. Moreover, mob-5 and its putative receptor are oncogenic
ras-specific targets; mob-5 binds to the cell surface of
ras-transformed cells but not of parental untransformed
cells (9).
Another IL-10 homolog, designated ak155, was cloned as a protein
expressed by Herpesvirus saimiri-transformed T
lymphocytes (4). Transcription of the gene of a fourth IL-10 homolog,
designated IL-19, was demonstrated to be induced in monocytes by LPS
treatment. The appearance of IL-19 mRNA in LPS-stimulated monocytes
coincided with the expression of IL-10 mRNA (2). An additional
protein with homology to IL-10 was designated Zcyto10 (GenBankTM
accession number AF224266), but there is no published information
available about its activities or expression.
Finally, an IL-10 homolog, designated IL-TIF (IL-10-related T
cell-derived inducible factor), is expressed by IL-9-treated murine T
cells (1). Its human analog (human IL-TIF or, provisionally, IL-22) was
recently reported (10, 11). Murine IL-TIF expression can be induced by
IL-9 in thymic lymphomas, T cells, and mast cells in vitro
and by LPS in various organs in vivo. It was also demonstrated that IL-TIF injection induced production of acute-phase reactants in mouse liver, suggesting involvement of IL-TIF in the
inflammatory response (10). IL-TIF can induce activation of Stat
proteins (Stat1, Stat3, and Stat5) in several cell lines, including
mesangial MES13, neuronal PC12, and hepatoma HepG2 cell lines (1, 10).
In addition, there are data linking IL-TIF to allergy and asthma.
IL-TIF is induced by IL-9, a Th2 cytokine active on T and B
lymphocytes, mast cells, and eosinophils, and potentially involved in
allergy and asthma (12-14). The IL-TIF gene (and also the ak155 gene)
is located on human chromosome 12q, where several loci potentially
linked to asthma and atopy have been identified by genetic studies,
particularly in the 12q13.12-q23.3 region (for review see Ref. 15). The
strongest evidence for linkage is in a region near the gene encoding
IFN- Cytokines exert their actions by binding to specific cell
surface receptors that leads to the activation of cytokine-specific signal transduction pathways. The functional IL-10 receptor complex consists of two chains (6), the ligand binding IL-10R1 subunit (21) and
the second IL-10R2 subunit that supports signaling through the IL-10R1
chain (6). Both chains belong to the class II cytokine receptor family
(22, 23), which also includes two receptor chains for type I
interferons (IFNs), two receptor chains for type II IFN, and the tissue
factor that binds coagulation factor VIIa (for review see Ref. 24). In
addition, there are currently at least five orphan receptors CRF2-8,
CRF2-9, CRF2-10, CRF2-11, and CRF2-12 (cytokine
receptor family class II members) and the
extracellular domains of CRF2-8, CRF2-9, and CRF2-10 are mostly
homologous to the IL-10R1 extracellular domain
(24).2
In this study we demonstrate that the functional IL-TIF receptor
complex consists of two receptor chains, the orphan CRF2-9 chain and
the IL-10R2 chain, which we demonstrate to be a common shared chain
between the IL-TIF and the IL-10 receptor complexes.
Plasmid Construction--
Primers
5'-CCGGTACCAATGGCCGCCCTGCAGAAATCTG-3' and
5'-GCGAATTCAAATGCAGGCATTTCTCAG-3' (tif1) and total RNA isolated from
PBMCs obtained from a healthy donor were used for reverse
transcription-PCR to clone the human IL-TIF cDNA into
plasmid pcDEF3 (25) with the use of KpnI and
EcoRI restriction endonucleases, resulting in plasmid
pEF-IL-TIF. The PCR product obtained with primers
5'-CCGGATCCACAGGGAGGAGCAGCTGCGCCC-3' (tif2) and tif1 and plasmid
pEF-IL-TIF as a template was digested with BamHI and
EcoRI restriction endonucleases and cloned into corresponding sites of the pEF-SPFL vector (5), resulting in plasmid
pEF-SPFL-IL-TIF. This plasmid encodes IL-TIF tagged at its N terminus
with the FLAG epitope (FL-IL-TIF). The PCR product obtained with
primers tif2 and
5'-CCGAATTCATGCGACTGACGCTCGTCGAATGCAGGCATTTCTCAGAGAC-3' and plasmid
pEF-IL-TIF as a template was digested with BamHI and EcoRI restriction endonucleases and cloned into
corresponding sites of the pEF-SPFL vector, resulting in plasmid
pEF-SPFL-IL-TIF-P. This plasmid encodes FL-IL-TIF tagged at its C
terminus with the Arg-Arg-Ala-Ser-Val-Ala sequence
(FL-IL-TIF-P), which contains the consensus amino acid sequence
recognizable by the catalytic subunit of the cAMP-dependent
protein kinase (26-29).
Primers 5'-CCGGTACCGATGAGGACGCTGCTGACCATC-3' and
5'-GGCGCTAGCAAGGTCCATGTCCGGTCTGGCAGTG-3' and a library containing
cDNA isolated from human fetal liver (CLONTECH,
catalog no. HL4029AH) were used for PCR to clone the extracellular
domain of the CRF2-9 protein (24) into plasmid pEF3-IL-10R1/
A "tandem vector" encoding two receptors, the CRF2-9/
The nucleotide sequences of the modified regions of all constructs were
verified in their entirety by DNA sequencing.
Cells, Transfection, and Cytofluorographic Analysis--
The
16-9 hamster x human somatic cell hybrid line is the Chinese hamster
ovary cell (CHO-K1) hybrid containing a translocation of the long arm
of human chromosome 6 encoding the human IFNGR1 (Hu-IFN-
Leukocytes were obtained from a normal donor by leukapheresis.
Peripheral blood mononuclear cells (PBMCs) were then isolated by
density centrifugation with polysucrose and sodium diatrizoate according to the manufacturer's suggested protocol (Sigma,
HISTOPAQUE-1077).
To detect cytokine-induced MHC class I antigen (HLA-B7) expression,
cells were treated with COS cell supernatants or purified recombinant
proteins as indicated in the text for 72 h and analyzed by flow
cytometry. Cell surface expression of the HLA-B7 antigen was detected
by treatment with mouse anti-HLA (W6/32) (31) monoclonal antibody
followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG
(Santa Cruz Biotechnology Inc., catalog no. SC-2010). The cells then
were analyzed by cytofluorography as described previously (32).
Electrophoretic Mobility Shift Assays and Western and Nothern
Blotting--
Cells were starved overnight in serum-free media and
then treated with IL-10 or IL-TIF as indicated in the text for 15 min at 37 °C and used for EMSA experiments to detect activation of Stat1, Stat3, and Stat5 as described (6). EMSAs were performed with a
22-base pair sequence containing a Stat1
Three days after transfection, conditioned media from COS-1 cells
transiently transfected with expression plasmids was collected and
subjected to Western blotting with anti-FLAG epitope-specific M2
monoclonal antibody (Sigma) as described (5).
Northern blotting was performed with two blots
(CLONTECH, catalog nos. 7757-1 and 7780-1) and a
CRF2-9 probe corresponding to the coding region of the CRF2-9 cDNA
as described (2). The RNA loading was adjusted by the manufacturer with
a Cross-linking--
The FL-IL-TIF-P protein was transiently
expressed in COS cells and purified from conditioned media by
immunoaffinity chromatography with the anti-FLAG M1 gel (Sigma)
according to the manufacturer's suggested protocols. FL-IL-TIF-P was
labeled with [32P]ATP and used for cross-linking as
described (28, 29, 33).
Ligands, Receptors, and Their Derivatives--
The following
ligands and receptors and their derivatives were created and used in
this study. Human IL-TIF (IL-21) (GenBankTM accession no.
AJ277247) is a cytokine with limited homology to IL-10 (10, 11).
Three expression vectors were created (Fig. 1A) encoding intact human
IL-TIF, N-terminal FLAG-tagged IL-TIF (FL-IL-TIF), or FL-IL-TIF with
the consensus amino acid sequence Arg-Arg-Ala-Ser-Val-Ala
(phosphorylatable site, P), recognizable by the catalytic subunit of
the cAMP-dependent protein kinase (26-29) fused to its C
terminus (FL-IL-TIF-P). COS cells were transiently transfected with
the expression vectors, and 3 days later conditioned media containing
FL-IL-TIF or FL-IL-TIF-P were tested by Western blotting with anti-FLAG
antibody for protein expression (Fig. 1B, lanes 3 and 4). Western blotting revealed that FL-IL-TIF was secreted from COS cells and migrated on the SDS-PAGE gel as several bands in the region of about 25-40 kDa, suggesting possible
glycosylation of the protein. Indeed, there are three potential sites
for N-linked glycosylation (Asn-Xaa-Thr/Ser) in human
IL-TIF. Treatment of the conditioned media with
peptide:N-glycosidase F (PNGase F) resulted in the
disappearance of the higher bands and enhancement of a band in the
region of 21 kDa (Fig. 1B, lane 4), consistent with glycosylation of the 25-40 kDa proteins. FL-IL-TIF-P, purified by
affinity column chromatography, was also analyzed by Western blotting
with anti-FLAG antibody (Fig. 1B, lane 5). The
32P-labeled FL-IL-TIF-P ([32P]FL-IL-TIF-P)
was also resolved on the gel and autoradiographed (Fig. 1B,
lane 6). Human IL-10 tagged with the FLAG epitope at the N
terminus and with the phosphorylation site at the C terminus was used
as a control (Fig. 1B, lane 2).
CRF2-9 is an orphan human receptor from the class II cytokine receptor
family as shown in Fig. 1D (24). We constructed expression vectors encoding intact CRF2-9 and a chimeric CRF2-9/ Experiments in COS Cells--
COS cells were transfected with the
expression plasmid encoding CRF2-9, and transfectants were selected by
growth in 350 µg/ml G418 for 3 weeks and pooled. To test for
responsiveness to IL-TIF, pooled cells were treated with conditioned
media from COS cells expressing FL-IL-TIF or left untreated as control,
and the detergent-free total cellular lysates were prepared for
electrophoretic mobility shift assays (EMSAs). The formation of Stat
DNA-binding complexes was detected in FL-IL-TIF-treated COS cells
transfected with the plasmid expressing the CRF2-9 chain and not in
untreated cells or in FL-IL-TIF-treated control COS cells transfected
with the blank expression vector (Fig.
2). The DNA-binding complexes (Fig. 2)
were shown to consist mainly of two Stats with anti-Stat1 and anti-Stat3 antibodies: Stat1 Experiments in Hamster Cells--
We hypothesized that the IL-TIF
receptor complex might be structurally homologous to the IL-10 receptor
complex, and to consist of two receptor chains with one common chain
shared between these two receptor complexes. It has been demonstrated
that, in hamster cells, unlike COS cells, hamster IL-10R2 failed to
support signaling by the human IL-10R1 chain (6). To determine whether
a similar situation holds for the human IL-TIF receptor complex
expressed in hamster cells, hamster cells were transfected with the
chimeric CRF2-9/ Cross-linking of IL-TIF with Membrane-bound Receptors--
To
further characterize the interaction between IL-TIF and its receptors,
cross-linking experiments with radiolabeled IL-TIF and the IL-TIF
receptor chains expressed in hamster cells were performed (Fig.
4). COS cell-expressed FL-IL-TIF-P was
purified to homogeneity and radiolabeled (Fig. 1). Parental hamster
cells, cells expressing either the chimeric human CRF2-9/ Expression of the CRF2-9 mRNA--
Nothern blots containing
RNA from multiple human tissues and human cancer cell lines were used
to assess expression of the CRF2-9 mRNA (Fig.
5). In normal tissues a transcript in the
region of 3.0 kb was detected in kidney and liver (Fig. 5B).
The size of the transcript is comparable with the size of the CRF2-9
cDNA 2.8 kb. Among tested cancer cell lines, expression of the
CRF2-9 mRNA was detected in three solid tumor cell lines,
colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma
G361, and not in promyelocytic leukemia HL-60, epitheloid carcinoma HeLa S3, lymphoblastic leukemia MOLT-4, and Burkitt's lymphoma Raji
(Fig. 5A). The CRF2-9 mRNA was also expressed by the
hepatoma cell line HepG2 and the renal carcinoma cell line Caki-1 (data not shown). A549 cells were responsive to IL-TIF as demonstrated by
IL-TIF-induced Stat activation determined by EMSA (data not shown). It
is noteworthy that the five cell lines expressing the CRF2-9 mRNA
(SW480, A549, G-361, HepG2, and Caki) are nonhematopoietic tumor cell
lines. In addition, the fact that the CRF2-9 gene is expressed in
normal liver and kidney tissue correlates with a recent report
demonstrating that IL-TIF functions as an hepatocyte-stimulating factor
(10).
The IL-10R2 chain is ubiquitously expressed, whereas the IL-10
activity is restricted mainly to cells of hematopoietic origin (35, 36). This raised the question of why the second chain of the IL-10
receptor complex is widely expressed when its function was required
only in limited cellular subsets. One hypothesis is that the IL-10R2
chain is shared by receptors for ligands other than IL-10. If such
ligands existed, they should demonstrate homology to IL-10. Thus, we
investigated whether such ligands existed. Our initial screening of the
GeneBankTM EST and genomic data bases resulted in identification of
the cytomegalovirus-encoded IL-10 homolog (cmvIL-10) (5), which can
bind and signal through the canonical IL-10 receptor complex (6)
despite the low homology between cmvIL-10 and IL-10 (27% identity).
The discovery of cmvIL-10 demonstrated that only 27% identity is
sufficient to allow ligands to share both chains of the IL-10 receptor
complex and, thus, suggested that ligands of lower homology might share
only one receptor chain, particularly the IL-10R2 chain.
Human IL-TIF was cloned and expressed with a FLAG tag at its N terminus
(FL-IL-TIF) in COS cells to enable detection of the protein in COS
cell-conditioned media. The anti-FLAG antibody recognized several
proteins of about 25-40 kDa, suggesting possible glycosylation of
FL-IL-TIF (Fig. 1). Treatment of conditioned media with PNGase F
resulted in the disappearance of the higher molecular mass bands and
appearance of a 21-kDa band comparable in size to FL-IL-10 (Fig. 1)
demonstrating glycosylation of the 25-40 kDa protein forms. FL-IL-TIF
does not utilize the canonical IL-10 receptor complex for signaling as
demonstrated by the inability of human IL-TIF to induce MHC class I
antigen expression and Stat1 activation in hamster cells expressing the
chimeric IL-10R1/ Because IL-10 receptor chains belong to the class II cytokine receptor
family, new ligands that have homology to IL-10 might signal through
receptors from the same family. Several orphan receptors from the class
II cytokine receptor family were identified by searching available
public EST and genomic data bases with the sequence of the IL-10R1
extracellular domain as the query sequence (24).2 We
examined whether one of them was a subunit of the IL-TIF receptor complex.
Our initial experiments demonstrated that, in COS cells expressing the
CRF2-9 chain, IL-TIF induced formation of Stat DNA-binding complexes
characteristic of IL-TIF signaling in MES-13 and PC-12 cells (1). Thus,
the expression of the CRF2-9 chain alone in COS cells is sufficient to
render the cells responsive to IL-TIF. We hypothesized that the second
IL-TIF receptor chain, likely the IL-10R2 chain, is expressed in COS
cells analogous to the results with IL-10 (6, 21). It was previously
demonstrated that the IL-10R2 chain has limited species specificity:
The IL-10R2 chain of mouse, human, or monkey origin can support
signaling through the IL-10R1 chain of these species (6, 21). However, the hamster IL-10R2 chain can support signaling only through mouse IL-10R14 but not through
human IL-10R1 (6). In addition, to enable us to detect IL-TIF-induced
biological activities, we utilized the chimeric receptor approach that
we used to characterize other receptor complexes (5, 6, 32). The human
CRF2-9 extracellular domain was fused to the transmembrane and
intracellular domains of the human IFN- We then repeated a similar series of experiments in hamster cells. We
did not observe the IL-TIF-induced activation of Stats in hamster cells
expressing the chimeric CRF2-9/ These experiments demonstrate that IL-TIF specifically binds to and
signals through the CRF2-9 chain and that the second chain of the IL-10
receptor complex, the IL-10R2 chain, also functions as the IL-TIFR2
chain. It is possible that the IL-10R2 chain may be shared by receptors
for the other IL-10 homologs: ak155, mda-7, IL-19, and Zcyto10. The
fact that all IL-10 homologs have most of their identical residues
located in the C-terminal half of the protein, with the highest
homology in the region of the helix F (37), suggests that this region
is involved in the interaction with the same receptor component. New
ligands are likely to possess their own unique ligand binding chains
(like CRF2-9 for IL-TIF), sharing the second IL-10R2 for signaling.
Because experiments with the orphan receptors CRF2-8 and CRF2-11 failed
to demonstrate their interaction with IL-TIF (data not shown), they are
receptor candidates for other IL-10 homologs. The conserved residues in helix F of IL-10 homologs may be involved in the interaction with the
common second chain of their receptor complexes. Thus, it is tempting
to propose that the IL-10R2 chain serves also as the second chain for
the other IL-10 homologs and can be designated R2c for
common receptor two chain.
The IL-TIF receptor (Fig. 6) is likely to
be structurally homologous to the IL-10 and IFN-R1 intracellular domain changes
the pattern of IL-TIF-induced Stat activation. The CRF2-9 gene
is expressed in normal liver and kidney, suggesting a possible role for
IL-TIF in regulating gene expression in these tissues. Each chain,
CRF2-9 and IL-10R2, is capable of binding IL-TIF independently and can
be cross-linked to the radiolabeled IL-TIF. However, binding of IL-TIF
to the receptor complex is greater than binding to either receptor
chain alone. Sharing of the common IL-10R2 chain between the IL-10 and
IL-TIF receptor complexes is the first such case for receptor complexes
with chains belonging to the class II cytokine receptor family,
establishing a novel paradigm for IL-10-related ligands similar to the
shared use of the gamma common chain (
c) by
several cytokines, including IL-2, IL-4, IL-7, IL-9, and
IL-15.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(16-19). However, the gene for IFN-
appears to be highly
conserved (no sequence variations were detected in 265 individuals),
suggesting that mutations of the IFN-
gene are unlikely to be a
significant cause of inherited asthma (20). The IL-TIF and ak155 genes
are positioned next to the IFN-
gene on the bacterial artificial
chromosome BAC RPCI11-444B24 (GenBankTM accession number
AC007458) and, thus, are possible candidates for linkage to asthma.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
R1 with
the use of KpnI and NheI restriction
endonucleases, resulting in plasmid pEF-CRF2-9/
R1. Primers
5'-GGCGCTAGCCTCCGGAGCCTTCCTGTTCTCCATG-3' and
5'-CCGAATTCAGGACTCCCACTGCCAGTCAG-3' and the same library were used for
PCR to clone the CRF2-9 intracellular domain into plasmid
pEF-CRF2-9/
R1 with the use of NheI and EcoRI restriction endonucleases, resulting in plasmid pEF-CRF2-9.
R1 and the
IL-10R2 chains, in which the expression of each receptor is controlled
by separate promoters and polyadenylation signals was created as
follows. The fragment containing the EF-1
promoter, the IL-10R2
coding sequence, and the bovine growth hormone polyadenylation signal
was released from the pEF-CRF (or pEF-IL-10R2) vector (6) by digestion
with BsaI and BssHII restriction endonucleases
and ligated into the BsaI and MluI sites of the
pEF-CRF2-9/
R1 plasmid. The resulting plasmid was designated
pEF-CRF2-9/
R1+IL-10R2.
R1) gene and
a transfected human HLA-B7 gene (30). The cells were maintained in F-12
(Ham) medium (Sigma) containing 5% heat-inactivated fetal bovine serum
(Sigma). COS-1 cells, an SV40-transformed fibroblast-like simian CV-1
cell line, were maintained in Dulbecco's modified Eagle's medium
(Life Technologies, Inc.) with 10% heat-inactivated fetal bovine
serum. Cells were transfected as described previously (5, 6) except
that stable COS cell transfectants were selected with 350 µg/ml G418.
COS cell supernatants were collected at 72 h as a source of the
expressed proteins.
-binding site corresponding
to the GAS (IFN-
activation sequence) element in the promoter
region of the human IRF-1 gene (5'-GATCGATTTCCCCGAAATCATG-3') as
described (33, 34).
-actin signal.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Ligands and receptors. A,
intact human IL-TIF and its two derivatives. IL-TIF was tagged at the N
terminus with the FLAG epitope (FL-IL-TIF) and was tagged at
the C terminus with the Arg-Arg-Ala-Ser-Val-Ala sequence that
contains the consensus amino acid motif recognizable by the catalytic
subunit of the cAMP-dependent protein kinase
(FL-IL-TIF-P). B, expression of FL-IL-TIF and
FL-IL-TIF-P in COS cells. COS cells were transiently transfected with
the pEF-SPFL (lane 1, mock), the pEF-SPFL-IL-TIF
(lane 3, FL-IL-TIF), and the pEF-SPFL-IL-TIF-P
(lane 5, FL-IL-TIF-P) expression vectors. Three
days later, 20 µl of the conditioned media was subjected to Western
blotting experiments with anti-FLAG antibody. FL-IL-10-P was used as a
control (lane 2). Conditioned medium containing FL-IL-TIF
was treated with peptide:N-glycosidase F (PNGase F) to
demonstrate that the protein is glycosylated (lane 4).
FL-IL-TIF-P was purified from conditioned media by affinity
chromatography and evaluated by Western blotting with anti-FLAG
antibody (lane 5). Lane 6 represents an
autoradiograph of the SDS-PAGE gel containing radiolabeled FL-IL-TIF-P.
The molecular weight markers are shown on the left.
C, expression vectors encoding the intact CRF2-9 chain, an
orphan receptor from the class II cytokine receptor family, and the
chimeric CRF2-9/ R1 receptor that has the CRF2-9 extracellular domain
fused to the transmembrane and intracellular domains of the IFN-
R1
chain were constructed. IL-10R2 is the intact second chain of the human
IL-10 receptor complex (6). D, predicted amino acid sequence
of CRF2-9. The comparison of sequences of the extracellular domains of
CRF2-9 and other receptors from the class II cytokine receptor family
demonstrates that the CRF2-9 chain belongs to this receptor family and
is most similar to the IL-10R1 chain and the orphan receptor CRF2-8
(24). Amino acid residues of the putative signal peptide and of the
putative transmembrane domain of the CRF2-9 are boxed.
Tyrosine residues are underlined (Y). Stat3
recruitment or docking sites are also underlined
(YXXQ motif). Potential glycosylation sites are noted by
lines over these sequences.
R1 receptor that has the CRF2-9 extracellular domain fused to the transmembrane and
intracellular domains of the human IFN-
R1 chain (Fig.
1C). The previously constructed pEF-CRF (or pEF-IL-10R2)
vector (6) was also utilized in this study. In addition, to express
both receptor chains in a single transfected cell the tandem vector encoding two receptors, the CRF2-9/
R1 and the IL-10R2 chains, in
which expression of each receptor is controlled by separate set of
promoter and polyadenylation signal was constructed.
and Stat3. Thus, the pattern of IL-TIF-induced Stat DNA-binding complexes observed in COS cells expressing CRF2-9 correlates with the pattern of Stat activation demonstrated for IL-TIF signaling in PC-12 or MES-13 cells (1). COS
cells were also stably transfected with an expression vector encoding
the chimeric CRF2-9/
R1 receptor with the CRF2-9 extracellular domain
fused to the transmembrane and the intracellular domains of the
IFN-
R1 chain (Fig. 1C). This chimeric receptor was made to enable us to detect IFN-
-like biological activities induced by
IL-TIF. Because IL-TIF-specific biological activities are not well
characterized and may be restricted to specific cell types, and because
we expect that the CRF2-9 receptor complex structurally mimics the
IL-10 receptor complex, we followed the same approach that was used to
create the chimeric IL-10-IFN-
receptor complex (6). We predicted
that, in cells expressing the chimeric CRF2-9/
R1 receptor, IL-TIF
would induce IFN-
-specific biological activities. As expected, in
COS cells expressing the chimeric CRF2-9/
R1 chain, IL-TIF treatment
induced activation of Stat1 DNA-binding complexes as demonstrated by
EMSA with anti-Stat1 antibody (Fig. 2).
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Fig. 2.
IL-TIF-induced Stat activation in monkey COS
cells and hamster 16-9 cells. Electrophoretic mobility-shift assay
(EMSA) was used to determine Stat DNA-binding complexes activated by
IL-TIF treatment in COS or hamster cells transfected with different
receptors as indicated on the figure. COS or hamster cells were
transfected with blank vector or with vectors encoding receptors as
indicated on the figure, grown in the presence of G418 for 3-4 weeks,
and G418-resistant clones were pooled and used for EMSA. Cells were
incubated with conditioned media from COS cells expressing either
FL-IL-TIF or FL-IL-10 (100 µl per 106 cells in 1 ml).
Cellular lysates were prepared and assayed for Stat activation in the
EMSA as described previously (6). Positions of Stat DNA-binding
complexes are indicated by arrows. Antibodies against Stat1
and Stat3 were added as indicated to reduce the mobility of complexes
containing these proteins. To detect a comparable amount of Stat
DNA-binding complexes in the EMSA the portion of the gel with samples
obtained from COS cells was exposed 2 days, whereas the portion of the
gel with samples from hamster cells was exposed for 8 h.
R1 receptor. In these cells IL-TIF treatment failed
to induce Stat activation (Fig. 2) or MHC class I antigen expression (Fig. 3B). In contrast,
hamster cells transfected with the tandem vector encoding both the
CRF2-9/
R1 and the IL-10R2 chains responded to IL-TIF treatment by
activation of Stat1 DNA-binding complexes (Fig. 2) and up-regulation of
MHC class I antigen expression (Fig. 3D). Parental hamster
cells and cells expressing the human IL-10R2 chain (6) did not respond
to the IL-TIF treatment (Figs. 2, 3A, and 3C).
FL-IL-TIF also failed to induce activation of Stat1 DNA-binding
complexes (Fig. 2) and up-regulation of MHC class I antigen expression
(Fig. 3E) in hamster cells expressing both chains of the
human IL-10 receptor. By comparison, IL-10 was capable of inducing
Stat1 activation and MHC class I antigen expression in these cells as
previously demonstrated (5, 6) but not in parental hamster cells or
cells expressing IL-10R2 alone, CRF2-9, or both receptors together
(Fig. 3). By flow cytometry with anti-FLAG antibody, low levels of
binding of FL-IL-TIF were observed with cells expressing either CRF2-9
or IL-10R2 alone or both receptors together, but not with parental
cells (data not shown). In all these experiments clonal populations
were used that were selected based on their ability to bind FL-IL-TIF
or to induce MHC class I antigen expression in response to IL-TIF
treatment (Fig. 3).
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Fig. 3.
Ligand binding and MHC class I antigen
induction. Row I, Schematic of five cell lines (clonal
populations) used in these experiments: the parental Chinese hamster
16-9 cells, cells expressing the intact IL-10R2 chain (6), the chimeric
CRF2-9/ R1 chain, or both receptors together, and cells expressing
the modified IL-10 receptor complex containing the intact IL-10R2 chain
and the chimeric IL-10R1/
R1 chain (5, 6). Row II,
A-E, the ability of IL-TIF or IL-10 to induce MHC class I
antigen expression was demonstrated by flow cytometry as described
previously (6). The cells described in row I were left
untreated (open areas, thick lines) or treated
with conditioned media (100 µl) from COS cells transfected with the
pEF-SPFL-IL-TIF plasmid (shaded areas, thin
lines) or with Hu-IL-10 (100 units/ml; open areas,
thin lines). The ordinate represents relative
cell number, and the abscissa represents relative
fluorescence.
R1 chain,
the intact human IL-10R2 chain, or both, and cells expressing the human
IL-10R1/
R1 and the IL-10R2 chains together were incubated with
radiolabeled FL-IL-TIF-P. The cells were washed to remove unbound
ligand, then bound ligand was cross-linked to the cells. After
cross-linking, cells were lysed and cross-linked complexes were
resolved on 7.5% SDS-PAGE (Fig. 4). The appearance of several labeled
cross-linked complexes was observed in all cell lines except parental
hamster cells, and the specificity of binding was shown by competition
with an excess of unlabeled IL-TIF (Fig. 4). In cells expressing the
CRF2-9/
R1 chain, major cross-linked complexes migrated in the region
of 115 kDa, with less intense bands in the region of 60-85 kDa. In
cells expressing the IL-10R2 chain, major cross-linked complexes
migrated in the region of 105 kDa, with possibly three additional lower
molecular mass bands. These additional bands may represent ligand
oligomers not cross-linked to receptors. The cross-linking pattern in
cells expressing both the IL-10R1/
R1 and the IL-10R2 chains was
identical to the pattern obtained with cells expressing the IL-10R2
chain alone. We did not observe any cross-linked complexes in parental
hamster cells. Major cross-linked complexes from cells expressing both
the CRF2-9/
R1 chain and the IL-10R2 chain migrated on SDS-PAGE in
the region of 105 and 115 kDa, corresponding in size to the complexes
obtained with cells expressing either chain alone. The amount of each
sample loaded on the gel was normalized to a constant number of cells used for cross-linking experiments. Thus, it appears that there is more
IL-TIF binding to cells expressing both CRF2-9 and IL-10R2 chains than
to cells expressing each chain alone. This is consistent with our
preliminary direct binding data that indicate the increased binding of
IL-TIF to cells with both chains is due to increased affinity rather
than to an increase in the number of binding
sites.3 The faster migrating
species were also seen with cells expressing both chains of the IL-TIF
receptor complex. Moreover, cross-linked complexes migrating in the
region of 200 kDa appeared only in cells expressing both chains of the
IL-TIF receptor complex and not in cells expressing either chain alone.
These complexes are likely to contain oligomers of IL-TIF and both
receptor chains, formed as a result of the association of the IL-TIF
receptor chains induced by ligand binding. The cross-linking
experiments provide direct strong evidence that IL-TIF can bind to each
chain of the IL-TIF receptor complex independently.
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Fig. 4.
Cross-linking. The hamster cells
described in Fig. 3, row I, were incubated with
32P-labeled FL-IL-TIF-P with or without addition of a
100-fold excess of unlabeled IL-TIF (competitor), washed, harvested,
and cross-linked. The extracted cross-linked complexes were analyzed on
7.5% SDS-PAGE. The molecular weight markers are shown on the
right.
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Fig. 5.
The expression of the CRF2-9 mRNA.
Northern blotting was performed on two blots containing mRNA
isolated from: A, human cancer cell lines (promyelocytic
leukemia HL-60, epitheloid carcinoma HeLa S3, lymphoblastic leukemia
MOLT-4, Burkitt's lymphoma Raji, colorectal adenocarcinoma SW480, lung
carcinoma A549, and melanoma G361); and B, normal human
tissues (brain, heart, skeletal muscle, colon, thymus, spleen, kidney,
liver, small intestine, placenta, lung, peripheral blood leukocytes
(PBL)). The arrow points to the CRF2-9
transcript. Equal RNA loading was assessed by evaluating the expression
of the -acting gene.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
R1 chain and the intact IL-10R2 chain, whereas
human IL-10 did (Figs. 2 and 3) (5, 6). IL-TIF also failed to induce
Stat DNA-binding complexes in PBMCs (data not shown), whereas IL-10 did
(6). Thus, we hypothesized that IL-TIF requires its own specific
receptor complex for signaling, but might share the IL-10R2 chain with the IL-10 receptor complex.
R1 chain resulting in the
chimeric CRF2-9/
R1 chain. We hypothesized that, in cells expressing
the modified IL-TIF receptor complex, in which the intact CRF2-9 is
replaced with the chimeric CRF2-9/
R1 chain, IL-TIF would induce
IFN-
-like biological activities such as MHC class I antigen
expression and Stat1 activation, which can be readily measured. To test
this hypothesis we expressed the chimeric CRF2-9/
R1 chain in COS
cells. Indeed, the pattern of IL-TIF-induced Stat activation changed to
that characteristic of IFN-
signaling (Fig. 2); only Stat1 DNA-binding complexes were observed in IL-TIF-treated COS cells expressing the chimeric CRF2-9/
R1 chain. These experiments also demonstrate that the activation of Stats is mediated by the CRF2-9 intracellular domain, because the substitution of the CRF2-9
intracellular domain by the IFN-
R1 intracellular domain caused a
change in the pattern of Stat activation (Fig. 2).
R1 chain (Fig. 2). Because we
hypothesized that the functional IL-TIF receptor complex might contain
both CRF2-9 and the IL-10R2 chain, we expressed both the chimeric
CRF2-9/
R1 chain and the intact IL-10R2 chain in hamster cells. In
these cells IL-TIF was able to induce Stat1 activation as measured by
EMSA (Fig. 2). Moreover, IL-TIF treatment causes up-regulation of class
I MHC antigen expression only in cells expressing both receptors (Fig.
3D) and failed to do so in cells expressing each of the
receptors alone or in parental cells (Fig. 3, A-C). We also
used hamster cells expressing various receptor combinations to
demonstrate ligand binding. By cross-linking we demonstrated that
IL-TIF did not bind to parental cells but was bound to hamster cells
expressing either the CRF2-9/
R1 or the IL-10R2 chain, and to cells
expressing both chains (Fig. 4). Low levels of binding of FL-IL-TIF to
cells expressing either receptor alone or both together, but not to
parental hamster cells, were also detected by flow cytometry with
anti-FLAG antibody (data not shown). The ability of the IL-10R2 alone
to bind IL-TIF was a surprising result, because, when expressed alone,
this chain is unable to bind IL-10 (5, 6). Furthermore, other ligands (IFN-
and IFN-
) signaling through receptor complexes whose chains belong to the class II cytokine receptor family do not bind to their
"second" chains with high affinity (24). Whether this unusual
binding is of functional significance remains to be determined. Because
IL-10R2 is ubiquitously expressed but unable to transduce a signal
without an additional chain (IL-10R1 or CRF2-9), it is possible that
secreted IL-TIF will be retained at the site of secretion by being
bound to the IL-10R2 chain, providing local action but preventing its
action at remote sites.
receptor complexes
(24). IL-TIF binding is likely to induce oligomerization of two CRF2-9
(or IL-TIF-R1) chains and two IL-10R2 (or IL-10hR2c)
chains. A distinct feature of the IL-TIF receptor complex is that both
chains can independently bind ligand, whereas in the IL-10 and IFN-
receptor complexes, only one chain (the R1 chain) can bind ligand in
the absence of the other. In all of these receptors, the second (R2) chains are necessary for signaling.
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Fig. 6.
Model of the IL-22 (IL-TIF) receptor complex
and signal transduction. The functional IL-22 (IL-TIF)
receptor complex consists of two receptor chains, the IL-22R1
(IL-TIF-R1 or CRF2-9) chain (24) and the IL-10R2 chain (6) and is
likely to be structurally homologous to the functional IL-10 receptor
complex (C). The IL-10R2 chain is a shared common chain for
at least two receptor complexes, the IL-10 receptor complex, and the
IL-22 receptor complex and is likely to be a shared receptor chain with
receptor complexes for other IL-10 homologs. Thus, this chain can be
designated receptor two (2) common chain (R2c). Both
chains of the IL-22 receptor complex are ligand binding chains,
however, none of them are capable of transducing IL-22-signaling alone
(A). Both chains are necessary to assemble the functional
receptor complex able to induce signaling after binding IL-22
(B). The IL-10R2 chain is associated with Tyk2 (6, 34). Both
cytokines, IL-10 and IL-22, activate a similar combination of Stat
proteins, Stat1, Stat3, and Stat5 (1, 40, 41).
Identification of the receptor for a particular cytokine provides information about possible signal transduction cascades. It has been demonstrated that IL-TIF induces Stat1, Stat3, and Stat5 activation (1). There are several Tyr residues in the CRF2-9 intracellular domain that are potential sites for phosphorylation (Fig. 1D). Analysis of amino acids surrounding Tyr residues within the CRF2-9 intracellular domain reveals the presence of four potential Stat3 recruitment sites, phospho-Tyr-Xaa-Xaa-Gln sequence (Fig. 1, YXXQ motif). It remains to be determined how Stat1 and Stat5 are recruited to the IL-TIF receptor complex and also whether all four Stat3 docking sites are active or only a subset of them.
A recent report (11) is in agreement with our results, although
the authors did not demonstrate that the expression of the CRF2-9
receptor (they named IL-22R) in COS cells rendered them sensitive to
IL-TIF. In our experiments, COS cells expressing intact or modified
CRF2-9 are responsive to IL-TIF treatment; thus, the conclusion about
the necessity of the R2c chain to assemble the functional
IL-TIF receptor complex required experiments in hamster cells. Our data
demonstrate that the endogenous IL-10R2 in COS cells can support
signaling through the human IL-TIF receptor complex as we previously
demonstrated for the IL-10 receptor complex (6, 24). The discrepancy
can be explained by the fact that we used stable transfectants of COS
cells, whereas Xie et al. (11) used transiently transfected
cells. Transient expression results in overexpression of the CRF2-9
chain so that most of the CRF2-9 chains do not interact with the
limited level of the endogenous IL-10R2 chain resulting in
nonfunctional CRF2-9-IL-TIF complexes (38, 39). Our experiments in
hamster cells, in which the hamster IL-10R2 chain cannot support
signaling through the human IL-10R1 chain (6, 24), demonstrate the
requirement for both CRF2-9 (IL-TIF-R1) and IL-10R2 chains for
reconstitution of a functional IL-TIF receptor complex and that IL-10R2
(R2c) serves as a common receptor chain for both IL-10 and
IL-TIF.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Jean-Christophe Renauld for recombinant IL-TIF and the sequence of the human IL-TIF cDNA, Kevin Moore and Satwant Narula for IL-10, Jerome Langer and Michael Newlon for the critical review of the text, and Eleanor Kells for assistance in the preparation of this manuscript.
![]() |
FOOTNOTES |
---|
* This study was supported by American Heart Association Grant AHA-9730247N and by State of New Jersey Commission on Cancer Research Grant 799-021 (to S. V. K.), by United States Public Health Services Grants RO1-CA46465 and 1P30-CA72720 from the National Cancer Institute, by Grants RO1-AI36450 and RO1-AI43369 from NIAID, National Institutes of Health, and by an award from the Milstein Family Foundation (to S. P.).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.
§ To whom correspondence may be addressed: Tel.: 732-235-4830; Fax: 732-235-5223; E-mail: kotenkse@umdnj.edu.
To whom correspondence may be addressed: Tel.: 732-235-4567;
Fax: 732-235-5223; E-mail: pestka@mbcl.rutgers.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M007837200
2 S. V. Kotenko and S. Pestka, unpublished data.
3 L. S. Izotova, S. Kotenko, and S. Pestka, unpublished data.
4 S. V. Kotenko, Wen He, and S. Pestka, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: IL-10, interleukin-10; Stat, signal transducers and activators of transcription; PBMC, peripheral blood mononuclear cells; LPS, lipopolysaccharide; IL-TIF, IL-10-related T cell-derived inducible factor; IFN, interferon; PCR, polymerase chain reaction; MHC, major histocompatibility complex; EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; PNGase F, peptide:N-glycosidase F; cmvIL-10, cytomegalovirus-encoded IL-10.
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