Molecular and Cellular Biology, October 2001, p. 6369-6386, Vol. 21, No. 19
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.19.6369-6386.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
ková,
í
Nehyba, andSection of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712-1095
Received 8 March 2001/Returned for modification 22 April 2001/Accepted 27 June 2001
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ABSTRACT |
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The avian homologue of the interferon regulatory factor 4 (IRF-4)
and a novel splice variant lacking exon 6, IRF-4
E6, were isolated
and characterized. Chicken IRF-4 is expressed in lymphoid organs, less
in small intestine, and lungs. IRF-4
E6 mRNA, though less abundant
than full-length IRF-4, was detected in lymphoid tissues, with the
highest levels observed in thymic cells. IRF-4 is highly expressed in
v-Rel-transformed lymphocytes, and the expression of IRF-4 is increased
in v-Rel- and c-Rel-transformed fibroblasts relative to control cells.
The expression of IRF-4 from retrovirus vectors morphologically
transformed primary fibroblasts, increased their saturation density,
proliferation, and life span, and promoted their growth in soft agar.
IRF-4 and v-Rel cooperated synergistically to transform fibroblasts.
The expression of IRF-4 antisense RNA eliminated formation of soft agar
colonies by v-Rel and reduced the proliferation of v-Rel-transformed
cells. v-Rel-transformed fibroblasts produced interferon 1 (IFN1),
which inhibits fibroblast proliferation. Infection of fibroblasts with
retroviruses expressing v-Rel resulted in an increase in the mRNA
levels of IFN1, the IFN receptor, STAT1, JAK1, and 2',5'-oligo(A)
synthetase. The exogenous expression of IRF-4 in v-Rel-transformed
fibroblasts decreased the production of IFN1 and suppressed the
expression of several genes in the IFN transduction pathway. These
results suggest that induction of IRF-4 expression by v-Rel likely
facilitates transformation of fibroblasts by decreasing the induction
of this antiproliferative pathway.
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INTRODUCTION |
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v-rel, which is
derived from the c-rel proto-oncogene, is the acutely
transforming oncogenic member of the Rel/NF-
B family (32,
42). The Rel/NF-
B family of transcription factors regulate gene expression from promoters or enhancers containing
B
binding sites (GGGRNNYYCC, where R is a purine, Y is a pyrimidine, and N is any nucleotide) (7, 85). Rel/NF-
B
proteins coordinate the expression of genes involved in natural and
acquired immunity (56). In addition, the Rel/NF-
B
proteins participate in the regulation of other processes such as
apoptosis and embryonic development (8, 15, 29, 34, 36,
51). The function of Rel/NF-
B family members has been highly
conserved during vertebrate evolution, and essentially the same five
transcription factors
RelA, RelB, c-Rel, NF-
B1 (p50), and NF-
B2
(p52)
are present in birds and mammals (30).
Altered regulation of Rel/NF-
B activity has been linked to several
pathological processes, including oncogenesis (25, 90). While rearrangements and amplifications of Rel/NF-
B genes have been
found in human tumors, c-Rel is the only family member for which acute
oncogenesis has been demonstrated (43, 59, 78). Mutations
are necessary to activate c-Rel's oncogenic potential. The most highly
oncogenic c-Rel mutant, v-Rel, transforms two different cell types,
fibroblasts and cells of hematopoietic origin (26, 62).
v-Rel-transformed fibroblasts acquire a distinct morphology, are
capable of limited growth in soft agar, have a prolonged life span, and
induce solid tumors (26, 59, 72, 74). Transformed
hematopoietic cells become immortalized, and most of them form
aggressive lymphomas in vivo. The ability of v-Rel to transform a
particular cell type depends on its ability to induce or repress a
specific set of genes. Approximately two dozen genes which exhibit
elevated expression in v-Rel-transformed cells have been identified
(32, 42). These genes encode cytokines, cell surface
receptors, chaperones, genes involved in oxidative metabolism,
apoptosis, and various transcription factors.
Interferon regulatory factors (IRFs) are transcription factors which
bind to promoter elements that contain an AANNGAAA consensus sequence and either stimulate or repress transcription of these target
genes (27). IRFs, originally identified as regulators of
interferons (IFNs) and IFN-stimulated genes, are involved in the
regulation of natural immunity against viruses, acquired
immunity, apoptosis, and embryogenesis, functions also
attributed to the Rel/NF-
B family (40, 66, 83, 88).
Nine IRF family members have been described in mammals: IRF-1 through
7, IRF-8, also called IFN consensus sequence-binding protein (ICSBP),
and IRF-9, originally described as IFN-stimulated gene factor 3-
(ISGF3-
). All IRF proteins possess a highly conserved DNA-binding
domain with five regularly spaced tryptophans at their N termini. The C
termini contain sequences involved in transcriptional activation and
repression (66, 83). The IRF members except IRF-1 and
IRF-2 have a conserved IRF association domain, which mediates
interactions with both other IRF proteins and transcription factors of
other families (83).
IRFs transactivate multiple genes that encode proteins with diverse
effects on cell proliferation and differentiation, including genes of
the IFN family. IFNs are multifunctional cytokines that play an
important role in the induction of antiviral responses, cell growth,
differentiation, and immunomodulation (40, 103). Type I
IFNs (mammalian IFN-
, -
, -
, and -
as well as avian IFN1 and
IFN2) are produced by a variety of cell types, while type II IFNs
(IFN-
) are secreted primarily by T cells and natural killer cells
(101, 102). IFN type I cytokines are the major IFNs that
respond to virus infection. Viral infection activates IRF, Rel/NF-
B,
and bZIP transcription factors, leading to IFN synthesis
(66). Secreted IFNs exert their effect by binding to cell
surface receptors and activating the JAK/STAT signal transduction pathway, leading to DNA binding by a complex composed of STAT1, STAT2,
and IRF-9 (103). This results in activation of the
IFN-stimulated genes, which exert an antiviral and, in many cell types,
antiproliferative effect (49).
Several IRF proteins have been associated with the development of cancer, and viral IRF homologues are present in oncogenic herpesviruses (28). IRF-1 and IRF-8 may function as antioncogenes, while IRF-2 and IRF-4 have some characteristics of oncogenes (39, 41, 47, 97). IRF-4 is activated by Tax in adult T-cell leukemia and is overexpressed in multiple myelomas as a result of a translocation of the IRF-4 gene into the immunoglobulin M (IgM) heavy-chain locus (47, 108, 110). In contrast to other IRF family members, IRF-4 is not induced by viral infection or IFNs but by proliferative stimuli (13, 68, 108). Furthermore, while most IRFs are expressed in a variety of cell types, IRF-4 is expressed predominantly in B cells and in activated T cells (66). Mice deficient in IRF-4 have defects principally in the development and function of B and T lymphocytes (71). However, IRF-4 is also detected in macrophages, lens cells, and melanocytes, suggesting that it may also be involved in the regulation of cells other than lymphocytes (35, 63, 64, 67, 94). IRF-4 inhibits the expression of some IFN-induced genes, presumably by interfering with the action of other IRFs (12, 68, 108).
In this work, we demonstrate that IRF-4 overexpression transforms primary chicken embryonic fibroblasts (CEFs). IRF-4 also synergistically cooperates with v-Rel in transformation of CEFs, while antisense IRF-4 expression inhibits v-Rel-mediated transformation. v-Rel transformation of CEFs is associated with the induction of expression of IFN1, the IFN1 receptor, STAT1, JAK1, and the IFN target gene 2',5'-oligo(A) synthetase (OAS). IRF-4 expression, which is induced in CEFs following infection by v-Rel- and c-Rel-expressing viruses, alters the antiviral gene expression program and likely contributes to transformation of fibroblasts by decreasing the negative antiproliferative effect of the v-Rel-induced antiviral program.
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MATERIALS AND METHODS |
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Cloning of chicken IRF-4 cDNA. An 858-bp fragment of chicken IRF-4 was cloned by reverse transcription (RT)-PCR from chicken splenic mRNA. First-strand cDNA synthesis was carried out with avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, Wis.) and a locking-docking primer (LDP) specific for poly(A) RNA (5'-AATCTAGAATGTCGACATGCGCGCTTTTTTTTTTTTTTTTVN-3', where V is an A, C, or G) at 42°C. Degenerate PCR primers I4-1 (5'-CACCTCGAGAAYGAYTTYGARGARYTNGT-3') and I4-2 (5'-GCAGCGCGCGGRAAYTCYTCNCCRAARCA-3') were designed based on the similarity between the amino acid sequences of human and mouse IRF-4 as well as differences in the sequence of the closest IRF family member, chicken IRF-8 (50). Two rounds of PCR were carried out with these primers using Taq DNA polymerase (Gibco-BRL Life Technologies, Grand Island, N.Y.). The second round used a 1-µl aliquot of the first-round PCR synthesis as a template. Each round consisted of 35 cycles of 1 min of denaturation at 94°C, 1 min of annealing, and 2 min of extension at 72°C. The annealing temperature was 55°C in the first round and 45°C in the second. The resulting 858-bp PCR fragment was cloned into the pGEM-T vector (Promega), creating pIRF858, and sequenced. The sequence of the 858-bp fragment, which corresponded to amino acids 105 to 392 of human IRF-4, was used to design an additional primer (5'-GGTACCAGGTTGCTCTGTGCTTCGGAGAG-3') to amplify the 3' region (1,920 bp) of the IRF-4 cDNA from chicken bursal mRNA by 3'SMART RACE PCR technology (Clontech Laboratories, Palo Alto, Calif.). The PCR products were cloned into the pGEM-T Easy vector (Promega), creating pIRF4CT, and sequenced.
In order to obtain the 5' end of IRF-4 cDNA, the combined sequences of both PCR fragments and the N-terminal protein sequence of human IRF-4 were used to screen chicken expressed sequence tag (EST) databases (2, 106). Among the several positive ESTs, we identified one clone, pat.pk0027.c10.f (accession number AI980577), isolated from a concanavalin A (ConA)-activated chicken splenic T-cell library, which encoded the N terminus of chicken IRF-4. The complete sequencing of this cDNA clone determined that its 3' region is 99.7% identical to the sequences of our PCR clones, with one notable exception. The pat.pk0027.c10.f clone was missing a region encoding 36 amino acids in the middle of the open reading frame (ORF) of IRF-4, suggesting that it may represent an alternatively spliced IRF-4 variant. To obtain the full ORF of IRF-4 including these 36 amino acids, primers which flanked the ORF on both sides (5'-GAGCTGGTCACAGAGGTGGTGCTCAG-3' and 5'-TGCTATCCAGATCAGCTCCTCCACTC-3') were used to PCR-amplify the cDNA from bursa as well as from ConA-activated splenic cells. Plasmids containing the cloned PCR fragments were designated pGE.IRF4-1 and pGE.IRF4-2 and sequenced.Plasmids. The pATH-I4#6 bacterial expression plasmid encodes a TrpE/IRF-4 fusion protein. This plasmid was created by cloning the PvuII-BssHII (828 nucleotides [nt]) fragment of pIRF858 between the SmaI and HindIII sites of pATH1 using a double-stranded adapter (5'-CGCGCGCTGAGTGACTGAGCTCA-3' and 5'-AGCTTGAGCTCAGTCACTCAGCG-3') containing a BssHII site, three stop codons in all reading frames, and a HindIII site (54).
pTZ-IRF-4
E6 was created by cloning the
Eco72I-BglII fragment of pat.pk0027.c10
into pTZ-18R which was modified by the introduction of a
double-stranded oligonucleotide adapter
(5'-AATTGCTCGAGCACGTGGAATTCAGATCTATTCGCCATTCCTCTATTCAAGATAAGCGCGC-3' and
5'-TCGAGCGCGCTTATTCTTGAATAGAGGAATGGCGAATAGATCTGAATT CCAGTGCTCGAGC-3') between the EcoRI and HindIII sites. The
adapter contained an XhoI site, an Eco72I site,
the sequence encoding amino acids 436 to 445 of IRF-4 (containing a
BglII site) followed by a stop codon, and a
BssHII site. pTZ-IRF-4, which contains the full-length
variant of IRF-4, was obtained by replacing the
ApaI-PstI region of pTZ-IRF-4
E6 with an
ApaI-PstI fragment from the pIRF858 clone.
The REV-T-based pREV-0 and pREV-TW (containing v-rel)
reticuloendotheliosis virus (REV)-based retroviral vectors have been described previously (44, 81). The retroviral vector pRSN was created by cloning an adapter containing NotI,
XhoI, MfeI, and SpeI sites
(5'-TCGACGCGGCCGCCTCGAGCAATTGACTAGTG-3' and
5'-CGCGCACTAGTCAATTGCTCGAGGCGGCCGCG-3') between the
XhoI and BssHII sites in pREV-0. The pCSV11S3
plasmid encodes an infectious genome of chicken syncytial virus (CSV) (23). The pREV-IRF-4 and pREV-IRF-4
E6 retroviral
vectors were obtained by cloning the XhoI-BssHII
fragments of pTZ-IRF-4 and pTZ-IRF-4
E6 into pREV-0. The retroviral
vector pREV-antiIRF-4, which expresses IRF-4 in the antisense
orientation, was obtained by cloning an XhoI-SpeI
fragment containing nucleotides 73 to 1429 of the IRF-4 cDNA from
pTZ-IRF-4 into the retroviral vector pRSN (Fig.
1A). The pRCAS plasmid encodes a
replication-competent avian sarcoma-leukemia virus (46).
pDS3 and pREP-A plasmids were used to prepare DS3 virus
(84). Plasmids were cut with SalI and ligated
to form concatemers before transfection of CEFs. pRCAS, pDS3, and
pREP-A plasmids are derived from the same genomic clone of
Schmidt-Ruppin Rous sarcoma virus (20). pRCASc-Rel was
obtained by cloning of an HpaII-HpaII
c-rel fragment from pBSc-rel#29 into a ClaI site
in the RCAS vector (16). pDSv-Rel was constructed by
insertion of v-rel into pDS3 as described previously (58). To prepare DSv-Rel virus, pDSv-Rel and pREP-A
plasmids were cut with SalI and ligated to form concatemers
before transfection of CEFs. Plasmid pcDNAchIFN1 was used for the
production of IFN1 in COS-1 cells as described (99, 102).
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Chickens, cell lines, and tissue culture. Embryonated eggs of specific-pathogen-free White Leghorn chickens were obtained from Hy-Vac, Adel, Iowa (SPF-SC strain), and Charles River SPAFAS, North Franklin, Conn. (SPAFAS strain). Most of the work presented in this article was done with fibroblasts and animals of SPF-SC strain. Cells from SPAFAS birds were used only for the cloning of pGE.IRF4-2 cDNA clone, the detection of IRF-4 protein in DSv-Rel-transformed cells, and the experiment shown in Fig. 9B. CEFs were prepared from 10- or 11-day-old embryos. Cells were cultured with Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% newborn calf serum (Atlanta Biologicals, Norcross, Ga.), 5% chicken serum (Gibco-BRL Life Technologies, Grand Island, N.Y.), 100 U of penicillin, and 50 µg of streptomycin per ml. Secondary cultures of CEFs were used for transfection of plasmid DNA by a modified calcium phosphate method as described previously (44). pREV-0-based plasmids (30 µg) together with pCSV11S3 (1 µg), pRCAS-based plasmids (30 µg), pDS3- or pDSv-rel-pREP-A concatemers (4 µg), or pCSV11S3 (1 µg) alone were used for transfection per 100-mm-diameter tissue culture dish of CEFs. Viruses were harvested between 5 and 7 days after transfection, and the infectious titers of Rel-expressing viruses and CSV were determined by an immunochemical titration assay (44, 104).
The simian COS-1 cell line was used for production of recombinant IFN1 (69). QT6 is a quail fibroblast cell line obtained by chemical mutagenesis (76). The 1757 duck cell line was derived from a duck sarcoma induced by avian leukosis virus (ALV) (82). All other cell lines used are of chicken origin. DT40 is a B-cell line established from an ALV-infected chicken bearing a bursa-derived lymphoid tumor (6). DT40 cells are transformed bursal stem cells which are continuously undergoing immunoglobulin gene diversification (53). The DT95 cell line is also derived from a chicken with an ALV-induced lymphoid leukosis and exhibits a more mature phenotype, including secretion of IgM (6). MSB-1 and RP-1 are T-cell lines established from a Marek's disease virus-induced lymphoma, 123/12 is a v-rel-transformed B-cell line, 160/2 is a v-rel-transformed T-cell line, and 123/6T is a macrophage-like v-rel-transformed cell line (3, 44, 80). AEV-1 is an avian erythroblastosis virus-transformed erythroid cell line (89). BM-2 is a macrophage-like cell line derived by transformation of yolk sac cells by AMV (77). C4-1 cells are a lymphoid non-virus-producing S2A3v-rel-transformed cell line (93, 111). The fibroblastoid cell line 26T6 was derived from a sarcoma induced by REV-Cgi, which expresses a c-rel mutant (43).Transformation assay. Transformed and control fibroblasts (105) were plated in 5 ml of DMEM containing 15% chicken serum and 0.35% Noble agar per 60-mm-diameter dish containing a bottom layer of 0.75% agar. Cells were refed with additional soft agar medium every 10 days. Plates were scored for the development of colonies 3 weeks after plating.
Preparation and treatment of white blood cells, lymphocytes, and bone marrow cells. Peripheral white blood cells and lymphocyte-enriched fractions from bursa, thymus, and spleen used to prepare RNA for Northern and RT-PCR analyses were isolated using Histopaque (Sigma Chemical Co., St. Louis, Mo.). A single-cell suspension of bursal lymphocytes for the experiment shown in Fig. 3 was obtained by passing minced tissue through a nylon mesh followed by a nylon cell strainer (100 µm; Becton Dickinson Labware, Franklin Lakes, N.J.). Cells were stimulated with phorbol-12-myristate-13-acetate (PMA) (1 µg/µl) (Sigma). Bone marrow was isolated from femur and tibiotarsus bones. The bones were cut longitudinally with a razor blade, and the entire contents of the bone cavity were scraped out and used for RNA preparation.
Antisera. The AI4-6 polyclonal antiserum was raised in a rabbit immunized with a TrpE/IRF-4 fusion protein purified from C600 bacteria transformed by the pATH-I4#6 expression plasmid. The TrpE/chIRF-4 fusion protein contains amino acids 112 to 386 of chicken IRF-4. Monoclonal antibody HY87 is specific for avian c-Rel and v-Rel (44). The goat anti-mouse IgG1 and goat anti-rabbit IgG biotinylated antibodies were purchased from Southern Biotechnology Associates, Inc., Birmingham, Ala. The monoclonal antibody 8A9, which recognizes chIFN1 but not chIFN2, was a generous gift from P. Staeheli (88a).
Western analysis. Western analysis was performed as described previously (44). Briefly, harvested cells were washed, resuspended, and boiled in sodium dodecyl sulfate (SDS) sample buffer, and proteins were separated on an SDS-polyacrylamide gel using a Mini-Protean II apparatus (Bio-Rad Laboratories, Hercules, Calif.). Proteins were transferred to a PolyScreen polyvinylidene difluoride membrane (NEN Life Science Products, Inc., Boston, Mass.) and sequentially reacted with rabbit polyclonal antiserum AI4-6 or monoclonal antibody HY87, goat anti-mouse IgG1 or goat anti-rabbit IgG biotinylated antibodies, and streptavidin-linked alkaline phosphatase (Roche Molecular Biochemicals, Indianapolis, Ind.). Proteins were visualized by an enzymatic reaction using 5-bromo-4-chloro-3-indolylphosphate and 4-nitro blue tetrazolium chloride as substrates (Roche Molecular Biochemicals). Calibrated prestained molecular size markers from Bio-Rad Laboratories were used. Nuclear and cytoplasmic fractions of CEFs were prepared in hypotonic buffer (50 mM Tris-HCl [pH 8], 1.1 mM MgCl2, 0.5% Triton X-100) as previously described (75).
Northern analysis and probes.
Total RNA was isolated by
RNAwiz (Ambion, Austin, Tex.). RNA was separated by electrophoresis in
a 1% agarose gel in 20 mM MOPS
(3-[N-morpholino]propanesulfonic acid) buffer and
transferred to a HybondN+ membrane (Amersham Pharmacia Biotechnology,
Piscataway, N.J.). Parallel samples with ethidium bromide were analyzed
under identical conditions, and the gel was photographed. Filters were hybridized with [
-32P]dCTP-labeled DNA
fragments using UltraHyb solution (Ambion) at 55°C. The probes used
for hybridization were labeled by nick translation and are listed in
Table 1.
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Semiquantitative RT-PCR.
Total RNA (5 µg) together with 1 µl of 10 µM LDP
(5'-AATCTAGAATGTCGACATGCGCGCTTTTTTTTTTTTTTTTVN-3') and 1 µl of 10 µM Smart II primer (Clontech Laboratories, Inc.) was
denatured for 10 min at 70°C in 11 µl of water. First-strand cDNA
synthesis was carried out with 15 U of ThermoScript RT (Gibco-BRL Life
Technologies), 2 µl of 10 µM deoxynucleoside triphosphate (dNTP)
mix, and 2 µl of 100 µM dithiothreitol at 55°C for 1 h. For
detection of IRF-4 and IRF-4
E6 by RT-PCR, 2.5 µl of the
first-strand synthesis reaction was used together with 1 µl of
Advantage cDNA polymerase mix (Clontech Laboratories, Inc.) and primers
FP572 (5'-AGGAGCAGCCATTGATGAACC-3') and BP878
(5'-CCTCTGGATAAGGGAAGATGACTTG-3'). PCRs were performed in a
DNA thermal cycler 480 (Perkin-Elmer, Inc., Boston, Mass.) as follows:
five cycles of 30 s at 94°C and 3 min at 72°C; five cycles of 30 s at 94°C, 30 s at 70°C, and 3 min at
72°C; and 25 to 30 cycles of 30 s at 94°C, 30 s at
65°C, and 3 min at 72°C.
Nucleotide and protein sequence analysis. The nucleotide sequence of the chicken IRF-4 clones was compared with the databases of GenBank, EMBL, DDBJ, and PDB utilizing a Blast 2.0 search engine (http://www.ncbi.nlm.nih.gov/BLAST) (4, 5). The same search engine was used to compare the protein encoded by the ORF of chicken IRF-4 with the protein sequence databases. The sequences of all IRFs similar to chicken IRF-4 were retrieved, and their amino acid homology was more precisely evaluated using the ClustalX program coupled with the MacBoxshade 2.15 analysis tool (48). The following protein sequence files from Swiss-Prot, translated GenBank, and DDJB databases were compared to the chicken IRF-4 sequence: sp Q90876, sp P10914, sp P15314, sp P23570, sp Q98925, sp P14316, sp P23906, sp Q14653, sp P70671, sp Q15306, sp Q64287, sp Q13568, sp P56477, sp O14896, gb AAB36714.1, dbj BAA24349.1, sp Q92985, sp P70434, sp Q90871, sp Q02556, sp P23611, sp Q00978, sp Q61179, and sp Q90643. Nucleotide sequences in the 5' and 3' untranslated regions (UTRs) of chicken IRF-4 cDNA were compared to sequences of mammalian IRF-4 (GenBank U52682 and U11692) using FASTA and Macaw 2.0.5 alignment tools to determine homology (61, 86, 98). Possible PEST regions were evaluated by the PEST-FIND program (available at http://www.at.embnet.org/embnet/tools/bio/PESTfind/) (92).
Nucleotide sequence accession numbers.
The sequence of
chicken IRF-4 submitted to GenBank under accession number AF320331 is a
composite of two cDNA clones, pat.pk0027.c10 and pGE.IRF4-2. The
sequence of the IRF-4
E6 splice variant is based on the
pat.pk0027.c10 clone and was submitted in a separate file with
accession number AF320332. The four cDNA clones (pIRF858, pIRF4CT, and
pGE.IRF4-1 and -2) that were obtained by RT-PCR differed in overlapping
regions at a few nucleotide positions from pat.pk0027.c10 and from each
other. Because at least some of these differences are probably caused
by the low fidelity inherent to PCR, the sequence of pat.pk0027.c10 was
taken to be the standard, as this plasmid was isolated directly from a
plasmid-based cDNA library without a PCR amplification step. The
fidelity of the sequence in the exon 6 region, which is not present in
pat.pk0027.c10, is supported by the identity of this sequence in
two independently isolated PCR clones, pGE.IRF4-2 and pIRF858.
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RESULTS |
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Cloning and sequence analysis of chicken IRF-4.
Mammalian
IRF-4 has been previously cloned and characterized (22, 35, 68,
108). Several of its properties suggest that IRF-4
expression may be important in v-Rel-mediated transformation. IRF-4
is expressed in lymphocytes, the hematopoietic target cells of v-Rel,
and is induced by proliferative stimuli (62, 68). Furthermore, mice deficient in IRF-4, like those deficient in c-Rel, have defects in B-cell proliferation (55, 71).
These similarities, together with the presence of
B sites in the
IRF-4 promoter, suggest that IRF-4 expression may potentially
be regulated by c-Rel and its oncogenic form, v-Rel (68).
E6, has not been described for mammalian IRF-4;
however, a different type of alternative splicing has been detected in mouse and human IRF-4. About 50% of the mammalian IRF-4 clones contain an additional glutamine residue at the boundary between the
fourth and fifth exons (35, 68). Four independently
isolated clones of chicken IRF-4 did not contain this glutamine
residue, suggesting that this form may not exist in the chicken.
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Characterization of IRF-4 protein.
The protein product of
the cloned IRF-4 cDNA was detected by Western analysis, confirming
that the cDNA encodes a protein corresponding in size to the protein
produced by the endogenous IRF-4 gene (Fig.
3). The coding region of IRF-4 was
inserted into a retroviral vector and expressed in CEF cultures, in
which no detectable IRF-4 protein is normally present. This protein
was then compared with endogenous IRF-4 from the chicken C4-1
B-cell line transformed by the S2A3 v-rel oncogene (Fig.
3A). IRF-4 was expressed at comparable levels in both cell types
and was detected as two closely migrating species with molecular
weights corresponding to the theoretical molecular weight of IRF-4.
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E6 protein variant had a similar subcellular localization as full-length IRF-4 (Fig. 3B, lanes 5 and
6). In bursal lymphocytes, very small amounts of the two IRF-4
isoforms were found, and most of the IRF-4 protein was located in
the nucleus as the faster-migrating form. Both IRF-4 cytoplasmic isoforms dramatically increased in abundance, and the level of nuclear
IRF-4 was elevated after stimulation with PMA (Fig. 3C, lanes 3 and
4). The presence of two IRF-4 species in fibroblasts and lymphoid
cells is most likely the result of posttranslational modification,
which may function to regulate nucleocytoplasmic transport. Regulation
of subcellular localization by phosphorylation has been described for
several other IRF family members (60, 66). Collectively,
these experiments showed that exogenously expressed IRF-4 in CEF
cultures has a similar migration pattern and subcellular distribution
as the endogenous IRF-4 found in v-Rel-transformed or
PMA-stimulated B cells.
Expression of IRF-4 in normal and transformed cells.
To
determine whether chicken IRF-4 is expressed principally in
lymphoid cells, as described for its mammalian counterpart, Northern
analysis was performed using total RNA from a variety of organs of
adult chickens, including spleen, bursa, thymus, bone marrow,
peripheral blood, lungs, liver, intestine, gizzard, kidney, gonads,
brain, muscles, heart, and skin (Fig. 4
and data not shown). High expression of chicken IRF-4 mRNA was
detected exclusively in tissues of hematopoietic origin, with the
highest level found in cells of the avian B-cell-specific organ, the
bursa of Fabricius, followed by spleen, peripheral white blood cells, thymus, and bone marrow (Fig. 4A, upper panel). Higher expression was
detected in purified lymphocyte populations than in the intact organ.
Low levels of IRF-4 were also detected in the small intestine and
lungs. Two mRNAs of different size were detected in the small intestine. In addition, Northern blot analysis revealed that the tissue-specific expression of c-rel correlates with that of
IRF-4 (Fig. 4A, lower panel).
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E6, RNA isolated
from bursal, thymic, splenic, and peripheral blood lymphocytes and from
bone marrow was analyzed by RT-PCR (Fig. 4B). IRF-4
E6 mRNA
was detected at various levels in all of these tissues, with the
highest expression detected in thymocytes, followed by lymphocytes from
spleen and peripheral blood. In all tissues, the spliced form
represented the minor fraction of the total IRF-4 mRNA.
Northern blot analysis of IRF-4 expression in immortalized avian
cell lines demonstrated that its expression is highest in v-Rel-transformed cell lines of B, T, and non-T, non-B phenotypes (Fig.
4C, lanes 7 to 9). High expression was also detected in the Marek's
disease virus-derived T-cell lines MSB-1 and RP-1 and in B-cell lines
DT40 and DT95, derived from ALV-induced B-cell lymphomas. Low levels of
IRF-4 expression were detected in the macrophage cell line BM-2
after longer exposure (data not shown). IRF-4 expression was not
detected by Northern blot analysis in the AEV-1 cell line. However,
IRF-4 was detected in all cell lines, including the one transformed
by AEV-1, by RT-PCR (Fig. 4D). IRF-4
E6 was also detected as a
minor component of total IRF-4 mRNA in all the cell lines. The
highest expression of the spliced form was found in v-rel-
and Marek's disease virus-transformed cell lines.
v-Rel and c-Rel induce expression of IRF-4 in transformed
fibroblasts.
Both v-Rel and c-Rel transform CEFs (1, 26, 45,
59, 72, 74). To define whether IRF-4 may contribute to
v-Rel- and c-Rel-mediated transformation, the steady-state level of
IRF-4 mRNA in normal fibroblasts and transformed fibroblasts
was determined by Northern blot analysis (Fig.
5). Though IRF-4
mRNA was not detected in normal CEFs, IRF-4 was expressed in
both v-Rel- and c-Rel-transformed CEF cultures (Fig. 5A). RT-PCR
revealed that expression of both the full-length and spliced IRF-4
variants was induced in transformed cells. As observed in the lymphoid cell lines, full-length IRF-4 was the predominant form (Fig. 5B, lanes 1 to 3). Although not detected by Northern analysis, a
significant amount of IRF-4 mRNA was detected by RT-PCR in
normal chicken fibroblasts (Fig. 5B, lane 4).
|
IRF-4 transforms primary chicken fibroblasts and participates
in transformation with v-Rel.
Preliminary experiments showed that
fibroblasts which express IRF-4 or IRF-4
E6 from retroviral
vectors become morphologically transformed. In order to characterize
these transformed cells and analyze the contribution of IRF-4 and
IRF-4
E6 to v-Rel-mediated transformation, CEF cultures were
infected with retroviruses expressing v-Rel, IRF-4, or
IRF-4
E6 or coinfected by viruses expressing v-Rel and IRF-4
or v-Rel and IRF-4
E6. The infected cells expressed high levels
of the exogenously expressed proteins (Fig.
6A). Both v-Rel- and
IRF-4-overexpressing cells became morphologically transformed 1 week after viral infection. The shape and orientation of cells expressing v-Rel were, however, distinct from those of cells expressing IRF-4 (Fig. 6B). v-Rel-transformed fibroblasts were rounded, with irregular edges, and disorganized. The size of these cells varied greatly, and the presence of giant cells was observed.
IRF-4-transformed cells retained a more regular growth pattern and
did not vary in size. At low density, they assumed a short cylindrical
shape which at confluence changed to small, rounded, very densely
packed cells. Cells overexpressing both genes had a morphology which was intermediate between those of v-Rel- and IRF-4-transformed cells: the cells had the disorganized growth pattern of
v-Rel-transformed cells and, like IRF-4-transformed cells, grew to
high density. Control cultures expressing the helper virus CSV or empty
vector were morphologically indistinguishable from uninfected cells
(data not shown). Cells expressing IRF-4
E6 or coexpressing v-Rel
and IRF-4
E6 were morphologically similar to cells expressing
IRF-4 or v-Rel and IRF-4, respectively. The morphological
changes in these cells were, however, less pronounced than in those
expressing full-length IRF-4 (data not shown).
|
E6 or coinfected by these viruses was
analyzed 30 to 40 days after infection (Table
2). These transformation characteristics
were compared with parameters determined for uninfected and DS3- and
CSV-infected control cells. v-Rel-transformed cells did not proliferate
more rapidly than control cells and grew to the same saturation density
as control cells. However, in contrast to control cells,
v-Rel-transformed cells formed colonies in soft agar. Cell cultures
expressing IRF-4 or IRF-4
E6 proliferated approximately twice
as rapidly as control cells. These cultures also reached a saturation
density approximately two to three times higher than that of
v-Rel-expressing and control cells. IRF-4 and IRF-4
E6, like
v-Rel, induced the limited growth of colonies in soft agar. v-Rel and
IRF-4 functioned synergistically to increase the proliferation
rate, saturation density, and colony formation ability of the
fibroblasts. Cells coexpressing v-Rel and either IRF-4 or
IRF-4
E6 proliferated about four times more rapidly than v-Rel-infected cells and two times more rapidly than IRF-4- or IRF-4
E6-infected cells. Fibroblasts coexpressing v-Rel and
IRF-4 (but not IRF-4
E6) grew to a saturation density four
times higher than that of v-Rel-expressing cells and stopped growing
when they reached 30 × 106 cells on
60-mm-diameter dishes (1.4 × 106/cm2). The coexpression
of IRF-4 and v-Rel also significantly enhanced soft agar colony
formation relative to fibroblast cells expressing either v-Rel or
IRF-4. These cells produced about 12 times more colonies, which in
turn grew to a diameter about three times greater than that of
colonies formed by cells expressing v-Rel or IRF-4 alone (Table 2,
Fig. 6B).
|
E6 began to proliferate more rapidly
than uninfected cultures after 1 week and continued to do so until 60 days after infection. Two weeks after infection, cells expressing
IRF-4 or IRF-4
E6 also began to proliferate more rapidly than
control cultures, while v-Rel-expressing cultures entered crisis at
approximately 10 days, during which time they grew more slowly than
control cells. When v-Rel-expressing cultures overcame this crisis,
they began to proliferate at the same rate as uninfected cells or
control cells infected with the empty vector. Finally, cells
overexpressing both v-Rel and IRF-4 or IRF-4
E6 reached two
times the number of generations of control cells or v-Rel-transformed
cells (Table 2). Cells overexpressing IRF-4 or IRF-4
E6 alone
doubled less frequently than these cells but still doubled
approximately 30 more times than control or v-Rel-expressing cells.
IRF-4 therefore cooperates synergistically with v-Rel in the
transformation of CEFs.
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Antisense IRF-4 RNA decreases soft agar colony formation and
proliferation of v-Rel-transformed fibroblasts.
To determine if
the induction of endogenous levels of IRF-4 in v-Rel-expressing
fibroblasts contributes to their transformation, we used antisense RNA
technology as a complementary approach to the coexpression studies
described above. Western blot analysis of C4-1 cells demonstrated that
the expression of IRF-4 in the antisense orientation reduced the
levels of IRF-4 protein in v-Rel-transformed cells to less than
50% of wild-type levels (Fig. 8).
v-Rel-transformed fibroblasts and lymphoid cells were infected with
retroviruses expressing the IRF-4 gene in the antisense
orientation. Both types of cells began to grow significantly more
slowly (three to six times) than control cells infected with the CSV
helper virus (Table 3). Interestingly,
the growth of normal fibroblasts was also inhibited by the expression
of the antisense IRF-4 construct. The ability of v-Rel-transformed
fibroblasts expressing antisense IRF-4 to proliferate and to form
colonies in soft agar was then determined (Table 3). In contrast to
v-Rel-transformed fibroblasts, cells coexpressing v-Rel and antisense
IRF-4 did not form colonies in soft agar. Collectively, the
inability of v-Rel-transformed cells expressing antisense IRF-4 to
form colonies in soft agar, the efficient transformation of fibroblasts
by IRF-4 alone, and the cooperation of v-Rel and IRF-4 in
fibroblast transformation suggest that increased levels of IRF-4 in
v-Rel-transformed fibroblasts contribute to their transformation.
|
|
IRF-4 represses v-Rel-induced expression of the negative cell
proliferation regulator IFN1.
v-Rel-transformed fibroblasts do not
proliferate more rapidly than control cells and enter crisis at 2 weeks
after infection, at which time their proliferation rate decreases
relative to control cells (Table 2). This is perplexing because
v-Rel-transformed fibroblasts not only have increased levels of
IRF-4 but also constitutively express elevated levels of c-Jun and
c-Fos, which are known to induce cell proliferation (58).
The expression of IRF-4 in v-Rel-transformed cells rescues
v-Rel-transformed cells from their proliferation defect (Table 2).
Since Rel/NF-
B, Jun/ATF-2, and IRF family members are known to
cooperatively regulate the expression of type I IFNs, we determined
whether v-Rel induces IFN, which in turn suppresses the proliferation
of v-Rel-transformed cells (107). The expression of avian
IFN1 mRNA in v-Rel-transformed cells was strongly increased
relative to that in control cells (Fig.
9A, lanes 1 and 4).
|
E6). Therefore, the increased levels of IRF-4
in v-Rel-transformed cells may partially eliminate the negative effects
of IFN1 on cell proliferation and permit v-Rel-transformed cells to
proliferate at rates equivalent to control cells. To test this
hypothesis directly, culture fluids from v-Rel-transformed cells, cells
transformed by v-Rel/IRF-4, and control cells were applied to CEF
cultures, and their proliferation rate was evaluated (Fig. 9B).
Supernatant fluids from v-Rel-transformed cells contained an
inhibitor which strongly interfered with the proliferation of
normal fibroblasts. This antiproliferative activity was partially
reduced by preincubation with anti-IFN1 antibody. Cells exposed to
supernatant fluids obtained from v-Rel/IRF-4-transformed cells
proliferated significantly better than those exposed to culture fluids
from v-Rel-transformed cells. These results indicate that
v-Rel-transformed cells produce IFN1, which strongly inhibits cell
proliferation, and that IRF-4 expression decreases the production
of this inhibitor.
IRF-4 modulates expression of genes involved in IFN
transduction pathway in v-Rel-transformed fibroblasts.
To
determine whether the induction of IFN1 expression in v-Rel-transformed
cells is coordinated with the expression of other components of the IFN
pathway, the levels of mRNA encoding the IFN receptor (IFNaR1 and
IFNaR2), the STAT1 transcription factor and its regulatory kinase,
JAK1, and the IFN target gene OAS were analyzed (Fig. 9C, lanes 1, 2, and 5). The expression of all of these genes was elevated, indicating
that the type I IFN signal transduction pathway is activated in
v-Rel-transformed fibroblasts. In contrast, the expression of IFNaR1
and JAK1 was decreased in cells expressing IRF-4 alone, while the
expression of STAT1 and OAS was increased in these cells (Fig. 9C, lane
3). The possibility that IRF-4 expression may alter the expression
of these genes in v-Rel-transformed cells was explored (Fig. 9C, lanes
1 to 4, 6, and 7). As with IFN1, the expression of IFNaR1 and -2 and
JAK1 was downregulated in cells coexpressing v-Rel and IRF-4s
relative to cells expressing v-Rel alone. While the expression of STAT1 and OAS was also decreased, significant levels of OAS mRNA were still present in cells expressing both v-Rel and IRF-4.
Collectively, these results suggest that the expression of IRF-4,
and to a lesser extent IRF-4
E6, not only influences the
expression of IFN1 but also modulates the expression of other genes in
the IFN transduction pathway, likely decreasing the sensitivity of
cells to the antiproliferative effects of IFN1. IRF-4, however,
does not appear to be a general repressor of genes of the IFN
transduction pathway, but is rather a modulator of their relative
expression levels.
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DISCUSSION |
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In this report we described the molecular cloning of chicken IRF-4 and the distribution of IRF-4 expression in tissues and cell lines and identified a novel splice variant of this gene. We further demonstrated that IRF-4 transforms primary fibroblast cells and that its expression is induced in fibroblasts by v-Rel and c-Rel and established a role for IRF-4 in the transformation of fibroblasts by v-Rel.
Alternative splice variant of IRF-4.
A differentially
spliced variant of IRF-4 mRNA (IRF-4
E6) was isolated
that does not contain exon 6 and consequently has a deletion of 36 amino acids immediately preceding the IAD. The amino acid sequence
encoded by exon 6 is highly conserved between mammalian and avian
IRF-4 family members, suggesting that this region may be
functionally important. This region was described earlier as a
potential PEST sequence (22). PEST sequences, which are
rich in proline, glutamic acid, serine, and threonine amino acids, have
been identified as destabilizing motifs that target proteins for rapid
degradation (92). However, analysis of the IRF-4
sequence using the PEST motif prediction algorithm suggests that it is
unlikely that this sequence serves as a PEST sequence (see legend to
Fig. 2B). While the IRF-4
E6 protein could be overexpressed to
slightly higher steady-state levels than IRF-4 in fibroblasts, this
difference does not suggest a dramatically different half-life. Previously, a 96-amino-acid-long domain in mouse IRF-4 was
identified that appears to mask IRF-4 transactivation activity
(79). This domain includes all 36 residues encoded by exon
6, suggesting that the exon 6-encoded region may serve as a regulator
of transactivation function. Various levels of IRF-4
E6 mRNA
were detected by RT-PCR analysis in several lymphoid and hematopoietic
tissues, albeit at lower abundance than full-length IRF-4. The
levels of IRF-4
E6 varied in different tissues, with the highest
levels detected in thymus cells. Collectively, these results suggest
that IRF-4
E6 is differentially regulated and has overlapping but
distinct functions compared with full-length IRF-4. Interestingly,
a splice variant of human IRF-7, IRF-7B, was described that is lacking
29 amino acids encoding a region corresponding to exon 6 of IRF-4
(112). Since IRF-7 is not closely related to IRF-4, it
is likely that alternative splicing of the region between the
transactivation domain and IAD is used by additional members
of the IRF family.
IRF-4 transforms primary fibroblast cells. Several reports suggested that IRF-4 may function as an oncogene. IRF-4 was found to be translocated next to the immunoglobulin heavy-chain locus in 20% of multiple myeloma cell lines, and IRF-4 overexpression induced the formation of soft agar colonies in the permanent fibroblastoid cell line Rat-1 (47, 110). Furthermore, it was suggested that IRF-4 plays an important role in adult T-cell leukemia (108). Other reports, however, failed to show the ability of IRF-4 to induce leukemogenesis (95). The results presented in this article show that IRF-4 functions as a very potent oncogene in fibroblasts. The expression of IRF-4 in primary fibroblasts increases their saturation density and permits them to form colonies in soft agar. Moreover, our results demonstrated new characteristics associated with IRF-4 transformation: dramatic alteration of cell morphology, decrease of the doubling time to half that of normal fibroblasts, and significant increases in life span.
The fact that IRF-4 is involved in human oncogenesis is largely accepted, but how IRF-4 acts as an oncogene has not been explored. Low but significant levels of IFN1 mRNA, as well as mRNA for several components of the IFN signaling pathway, have been detected in our experiments in normal fibroblasts. It is possible that IRF-4 may, at least partly, regulate fibroblast proliferation by modulating expression of these genes. Our hypothesis is also consistent with the observation that overexpression of antisense IRF-4 reduces the proliferation rate of normal fibroblasts. Interestingly, the recent report of the isolation of a third viral IRF, vIRF-3, from human herpesvirus 8 (HHV-8) suggested a similar mechanism (65). HHV-8 is the etiological agent of Kaposi's sarcoma and also plays an important role in the pathogenesis of AIDS-associated body cavity-based lymphoma. vIRF-3, which is most homologous to IRF-4, has the ability to repress transactivation of the IFNA (IFN-
) gene normally
caused by virus-mediated activation of IRF-3- and IRF-7.
v-Rel- and c-Rel-induced expression of IRF-4 in fibroblasts
contributes to cell transformation.
The increased expression of
IRF-4 found in v-Rel- and c-Rel-transformed fibroblasts suggests
that IRF-4 is under the control of Rel transcription factors in
CEFs. This is supported by a report by Grumont and Gerondakis,
published while this study was in progress, which demonstrated that
IRF-4 is transcriptionally regulated by c-Rel in murine B
lymphocytes via
B sites present in the IRF-4 promoter
(37). Therefore, it seems likely that IRF-4 is also a
direct transcriptional target of v-Rel and c-Rel in chicken fibroblasts. The expression of about a dozen genes has been found to be
changed in v-Rel-transformed cells (32, 42). However, only
a few of these genes were connected with the transformation phenotype
of cells. The AP-1 family of transcription factors and the chemokine
mip-1
have been shown to play a functional role in
v-Rel-mediated transformation of fibroblasts (58, 87). The
ability of IRF-4 to transform fibroblasts suggested that an increased level of IRF-4 may also contribute to the transformed phenotype of these cells. To test this hypothesis, we performed experiments in which the level of IRF-4 in v-Rel-expressing
fibroblasts was increased or decreased. Coexpression of v-Rel and
IRF-4 synergistically increased transformation of fibroblasts, and
expression of antisense IRF-4 RNA in v-Rel-transformed fibroblasts
prevented the formation of soft agar colonies and reduced the
proliferation of these cells. These results establish that IRF-4
contributes to transformation of fibroblasts by v-Rel.
v-Rel induces expression of genes with antiviral and antiproliferative functions in fibroblasts. The expression of v-Rel results in the development of a cell type-specific cytopathic effect (81, 100). In most instances, v-Rel-expressing embryonic fibroblasts, despite their transformed morphology and ability to grow in soft agar, do not proliferate more rapidly than control cells (26, 74). REV-T-transformed fibroblast cultures produce less virus over time, suggesting that there may be a selective pressure against fibroblasts expressing high levels of v-Rel (12). Our studies provide a possible explanation for these observations. The expression of IFN1, which has an antiproliferative effect, is induced in v-Rel-transformed fibroblasts. Simultaneously with the expression of IFN1, the expression of IFNaR1, IFNaR2, STAT1, and JAK1 is elevated in v-Rel-transformed cells. The induction of expression of these genes of the IFN transduction pathway suggests that not only is the level of IFN1 increased, but the sensitivity of CEF cultures to its action is also increased. As a result, the increased expression of the IFN target gene OAS was also detected in v-Rel-transformed cells. The expression of IFN-induced genes has been shown in many instances to correlate with decreased cell proliferation (9).
v-Rel likely interacts directly with the promoters of at least some genes of the IFN/IRF signal transduction cascade. IFN1 itself may be a v-Rel target gene. It has been shown that the Rel/NF-
B family
members cooperate with IRF-3, IRF-