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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1668-1676
A Janus Kinase Inhibitor, JAB, Is an
Interferon- -Inducible Gene and Confers Resistance to
Interferons
By
Hiroshi Sakamoto,
Hideo Yasukawa,
Masaaki Masuhara,
Shyu Tanimura,
Atsuo Sasaki,
Kentaro Yuge,
Motoaki Ohtsubo,
Akira Ohtsuka,
Takasi Fujita,
Tsunetaka Ohta,
Yusuke Furukawa,
Satsuki Iwase,
Hisashi Yamada, and
Akihiko Yoshimura
From the Institute of Life Science, Kurume University, Aikawamachi,
Kurume; the Department of Tumor Cell Biology, Tokyo Metropolitan
Institute of Medical Science, Bunkyoku, Tokyo; the Fujisaki Institute,
Hayashibara Biochemical Laboratories, Inc, Okayama; the Division of
Hemopoiesis, Institute of Hematology, Jichi Medical School, Tochigi;
and the Departments of Internal Medicine (IV) and Molecular Genetics,
Institute of DNA Medicine, Jikei University School of Medicine, Tokyo,
Japan.
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ABSTRACT |
It has been shown that interferons (IFNs) exert their signals
through receptor-associated Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). However, molecular
mechanism of regulation of IFN signaling has not been fully understood.
We have reported novel cytokine-inducible SH2 protein (CIS) and JAK
binding protein (JAB) family genes that can potentially modulate
cytokine signaling. Here we report that JAB is strongly induced
by IFN- but not by IFN- in mouse myeloid leukemia M1
cells and NIH-3T3 fibroblasts. NIH-3T3 cells ectopically expressing JAB
but not CIS3 lost responsiveness to the antiviral effect of IFN- and
IFN-. M1 leukemic cells stably expressing JAB were also resistant to
IFN- and IFN--induced growth arrest. In both NIH-3T3 and M1
transformants expressing JAB, IFN- did not induce tyrosine
phosphorylation and DNA binding activity of STAT1. Moreover,
IFN--induced activation of JAK1 and JAK2 and IFN--induced JAK1
and Tyk2 activation were inhibited in NIH-3T3 JAB transformants. These
results suggest that JAB inhibits IFN signaling by blocking JAK
activity. We also found that IFN-resistant clones derived from LoVo
cells and Daudi cells expressed high levels of JAB without stimulation.
In IFN-resistant Daudi cells, IFN-induced STAT1 and JAK phosphorylation
was partially reduced. Therefore, overexpression of JAB could be, at
least in part, a mechanism of IFN resistance.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
A DIRECT SIGNAL transduction pathway from
the interferon (IFN) receptor to the nucleus has been
discovered.1,2 A novel subfamily of protein tyrosine
kinases, known as Janus kinases (JAKs), was found to play an
important role in IFN receptor signaling. JAKs associate with IFN
receptors and are stimulated when IFNs bind to their cognate receptors.
In particular, IFN- / activates JAK1 and Tyk2, whereas IFN-
activates JAK1 and JAK2.1,2 The activated JAKs in turn
convert latent cytoplasmic transcription factors, known as signal
transducer and activator of transcription (STATs), into active forms by
tyrosine phosphorylation. The tyrosine-phosphorylated STATs form
homodimers or heterodimers and translocate into the nucleus, where they
bind to their specific target sequences. STAT1 and STAT2 are
phosphorylated in response to IFN-/ and heterotrimerize together
with a 48-kD protein, then bind to the IFN--specific DNA element, IFN-stimulated response element (ISRE). STAT1 is activated
by IFN-, homodimerized, and then binds to a different DNA element,
IFN--activated site (GAS). Many cytokines including interleukins
and colony-stimulating factors use similar JAK-STAT pathways.3
IFNs, especially IFN-, have been used for treatment of hairy cell
leukemia, melanoma, and chronic myelogenous leukemia (CML). However,
patients in the late chronic phase, accelerated phase, or blastic phase
of CML are usually resistant to the antiproliferative effects of
IFNs.4 Efforts to understand the molecular basis of the
cellular resistance to IFN have been made, using somatic cell mutants
resistant to IFNs. Stark et al identified genes necessary for IFN signaling and have shown that defects of any signaling molecule, including receptors, JAKs, and STATs, could be mechanisms of
IFN unresponsiveness and resistance.1,2 In fact, marked reduction in the level of STAT1 was found in IFN-resistant melanoma patients.5 Such IFN resistance should be a recessive
phenotype. However, the molecular basis of the dominant phenotype of
IFN resistance is not clear.6
Negative regulation of JAK-STAT signaling has only just begun to be
studied. Selective degradation or dephosphorylation of receptors and
STATs has been reported.7-10 Naturally occurring dominant
negative variants of STAT5 and a specific STAT3 inhibitor, PIAS3, have
also been found.11-13 Deregulation of the negative feedback
of the JAK-STAT pathway could be involved in IFN resistance. We cloned
a cytokine-inducible SH2 protein, CIS.14 CIS gene is a
direct target of STAT5, and its product tightly binds to the
tyrosine-phosphorylated interleukin-3 (IL-3) receptor and erythropoietin (EPO) receptor. It partially suppresses STAT5
activation, probably through masking of STAT5 docking sites on the
receptor.15 We recently cloned another CIS family member,
JAB, which directly binds to the JAK2 tyrosine kinase domain and
inhibits tyrosine kinase activity.16 Overexpression of JAB
resulted in the suppression of all cytokine signaling using JAKs. We
also found five additional CIS family members (CIS2-CIS6), and the
original CIS has been referred to as CIS1.17 CIS3 binds to
JAK2-JH1 tyrosine kinase domain like JAB; however, the interaction
between JH1 and CIS3 seems to be weaker than that between JH1 and
JAB.17 Forced expression of CIS3 and JAB at physiological
levels inhibited IL-6 or leukemia inhibitory factor
(LIF)-mediated growth arrest of M1 leukemia cells. Two other groups
have reported on related genes. One referred to JAB, CIS2, and CIS3 as
SOCS-1, SOCS-2, and SOCS-3,18 respectively, and the other
as SSI-1, SSI-2, and SSI-3, respectively.19,20 Because the
CIS family genes (CISs) seem to be involved in regulation of cytokine
signaling, we examined their role in IFN signaling. We found that
forced expression of JAB but not CIS1, CIS2, and CIS3 in M1 leukemia
cells or NIH-3T3 cells conferred resistance to the action of IFNs.
Because elevated expression of JAB was found in two independent
IFN-resistant cell lines, JAB could potentially be involved in IFN
resistance or reduced response to IFNs.
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MATERIALS AND METHODS |
Cells and transformants.
M1 myelogenous leukemia cells were cultured in Dulbecco's
modified Eagle's medium (DMEM) containing 10% horse serum. M1
transformants were obtained by electroporation with pcDNA3 carrying
Myc-tagged full-length CISs and JAB and maintained in the presence of
0.5 mg/mL G418 as described previously.17 NIH-3T3 cells
were cultured in DMEM containing 10% calf serum (CS). NIH-3T3
transformants expressing CIS1 were obtained by electroporation with
pEFneo-CIS1 (without epitope tag). Other transformants were obtained by
infection of retrovirus vector pLXSN carrying Myc-tagged CIS2, CIS3, or JAB.16 NIH-3T3 transformants were selected with 0.7 mg/mL
G418, and two to three positive clones for each transfection were
maintained in DMEM containing 0.5 mg/mL G418. Isolation and
characterization of IFN--resistant Daudi clone (DaudiR)
will be described elsewhere. Briefly, it was obtained by long-term exposure of parental cells to IFN- (Sumitomo Pharmaceutical
Co, Osaka, Japan) and cloning with colony formation. The
resistant clone was 10,000 times more resistant to IFN- than
parental cells. Daudi and its IFN--resistant clone were cultured in
RPMI containing 10% fetal calf serum (FCS). LoVo and its IFN-resistant
clones were maintained in Ham's F12 containing 10%
FCS.6 IFN-resistant phenotype of Daudi-and LoVo-derived
clones remained in the absence of IFNs for more than 1 year.
IFN-induced growth arrest of M1 cells.
Two to four independent clones were tested for IFN-- (a gift from
TORAY Research Lab, Kamakura, Japan) or IFN--
(Hayashibara Biochemical Lab, Okayama, Japan) induced growth arrest.
Briefly, 0.5 × 104 parental M1 cells and
transformants expressing Myc-tagged CISs or JAB were cultured in medium
containing 10% horse serum supplemented with or without the indicated
amount of IFNs for 6 days, then the viable cells were determined by
Trypan blue exclusion and counted in a hemocytometer.
IFN-induced activation of JAKs and STAT1.
Immunoblotting with antiphosphotyrosine, anti-STAT1 (C20;
Santa Cruz, Santa Cruz, CA), or
anti-tyrosine-phosphorylated STAT1 (New England BioLabs, Beverly, MA)
was performed as described15-17 after cells were stimulated
with either saline or 1,000 IU/mL IFNs for 15 minutes to 18 hours at
37°C. Electrophoretic mobility shift assay (EMSA) was performed as
described.21 Immunoprecipitation with anti-JAK1 and JAK2
(UBI) from 2 × 106 cells and immunoblotting with
antiphosphotyrosine were performed as described.15
Northern hybridization.
Cells were stimulated with 1,000 IU/mL IFN- or IFN- for indicated
periods. For Northern blotting, total RNA (5 µg) was separated on
1.0% agarose gels containing 2.4% formaldehyde, then transferred to
positively charged nylon membranes. After fixation under calibrated ultraviolet irradiation, the membranes were hybridized with DIG-labeled riboprobes and visualized using alkaline-phosphatase-labeled anti-DIG antibody according to the manufacturer's instructions (Boehringer, Mannheim, Germany). Probe cDNAs for CIS3, JAB, IRF-1,
FcRI, and G3PDH have been described previously.17,22
Antiviral assay.
NIH3T3 cells (5,000 cells/well) were plated in 96-well microplates.
After IFN- and IFN- treatments for 24 hours, the medium was
changed to that containing vesicular stomatitis virus (VSV) at 8,000 plaque-forming units (PFU) per well. At 72 hours
postinfection, the cytopathic effects were estimated by measuring the
number of living cells using the methylthiotetrazole
(MTT) assay as described.23 The cytopathic effects were
expressed as the percentage of MTT reduction activity of IFN-treated
cells to that of untreated, uninfected cells.
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RESULTS |
Induction of JAB expression by IFN-.
Because CIS family members have shown to be a cytokine-inducible gene,
we examined induction of CIS1, 2, 3, and JAB by IFNs using Northern
hybridization. CIS1 and CIS2 were not induced in M1 leukemic cells and
NIH-3T3 fibroblast cells by either IFN- or IFN- (data not shown).
IFN- did not induce CIS3 and JAB in either cell line
(Fig 1A and B). IFN- induced JAB but not
CIS3 in M1 cells, whereas CIS3 and JAB were induced by IFN- in
NIH-3T3 cells. Expression of JAB was detected as long as IFN- was
present for at least 24 hours after stimulation in both M1 and NIH-3T3 cells, whereas induction of CIS3 in NIH-3T3 cells was rapid and transient. We examined several cell lines and cytokines including IL-6,
LIF, and granulocyte-macrophage colony-stimulating factor (GM-CSF) and
found that JAB was most frequently and strongly induced by IFN-
(data not shown). Although LIF and IL-6 have shown to induce JAB
expression in M1 cells,18,19 JAB induction by IFN- was
much higher (Fig 2). Thus, JAB seemed
to be an IFN--inducible gene.
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| Fig 1.
Expression of endogenous JAB or CIS3 with IFNs. M1 (A)
and NIH3T3 (B) cells were stimulated with IFNs (1,000 IU/mL) for the
indicated time, and the total RNA was subjected to Northern blotting
with probe for JAB, CIS3, or control G3PDH.
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| Fig 2.
Expression of JAB in M1 and 3T3 transformants. Parental
M1 or NIH-3T3 cells were stimulated without (N.S.) or with 1,000 IU/mL
IFN-, 1,000 IU/mL IFN-, or 10 ng/mL LIF for 12 hours, and then
the total RNA was isolated. Total RNA was also isolated from untreated
M1 transformant expressing JAB (M1-JAB), NIH-3T3 transformant
(NIH3T3-JAB), and CTLL2 cells. The RNAs were subjected to Northern
blotting with probe for JAB and control G3PDH. The arrowheads indicate
JAB message derived from transfected cDNA in pLXSN (LXSN-JAB) in
NIH3T3-JAB transformant, that in pcDNA3 (pcDNA3-JAB) in M1-JAB
transformant, and endogenous JAB (end-JAB).
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Forced expression of JAB conferred resistance to IFNs on M1 and
NIH-3T3 cells.
We examined the effect of forced expression of JAB and CISs on the
biological action of IFNs in M1 leukemia cells and NIH-3T3 fibroblastic
cells. In M1 transformants, the expression level of transfected JAB was
comparable to or less than that of endogenous JAB in IFN--treated
parental M1 or NIH-3T3 cells (Fig 2). The expression level of JAB in
NIH-3T3 transformants was as high as that in IFN--treated parental
cells or in CTLL2 cells (Fig 2). Immunoblotting with anti-Myc antibody
revealed that the expression levels of Myc-CIS1, CIS2, CIS3, and JAB
were similar as described.17 In NIH-3T3 transformants, the
expression level of each CIS was about fivefold higher than that of M1
cells as judged by immunoblotting (data not shown).
First, we examined the antiproliferative effect of IFNs in M1 cells,
because our NIH-3T3 exhibited only weak growth arrest in response to
IFNs. As shown in Fig 3B, IFN- induced
growth arrest of parental M1 cells and CIS3 transformants, whereas JAB transformants were highly resistant to the antiproliferative effect of
IFN-. Although IFN- required higher concentrations for growth inhibition than IFN-, the growth of parental M1 cells and CIS3 transformants was also suppressed by IFN- (Fig 3A). JAB
transformants were 10 to 30 times more resistant to IFN- than
parental M1 cells and CIS3 transformants.
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| Fig 3.
Effect of forced expression of JAB and CIS3 on
IFN-induced growth inhibition of M1 cells. Parental M1 cells and CIS3
or JAB transformants (5,000 cells/well) were plated in 24-well plates
and cultured for 6 days in the presence of the indicated amount of
IFN- (A) or IFN- (B). Then the viable cells were counted by
Trypan blue exclusion. The viability of the cells is expressed as the
percentage of the number of IFN-treated cells to that of untreated
cells. The numbers of untreated cells (100%) are 2.4 × 105 (parental M1 cells), 2.4 × 105 (JAB
transformant), and 2.1 × 105 (CIS3 transformant).
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Next, we examined the IFN-induced antiviral effect using NIH-3T3
transformants. Transformants expressing CIS1, CIS2, and CIS3 were as
sensitive as parental NIH-3T3 cells to both IFN- and IFN-,
whereas JAB transformants were almost completely insensitive to the
IFN-induced antiviral effect (Fig 4). Thus,
forced overexpression of JAB conferred IFN resistance on both
hematopoietic and fibroblastic cells.
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| Fig 4.
Effect of CIS family members and JAB on the antiviral
effect of IFNs. Parental NIH3T3 cells and transformants were infected
with VSV for 3 days after incubation with the indicated amount of
IFN- (A) or IFN- (B) for 24 hours. The viable cells were measured
by MTT assay. The optical density (OD) value at 420 nm
that represents viable cell number is shown.
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Inhibition of STAT1 activation by forced expression of JAB.
To elucidate the mechanism of inhibition of IFN--mediated growth
arrest and the antiviral effect by JAB, we first examined the
IFN--dependent gene expression (Fig 5A
and B). IFN-induced growth arrest of M1 cells has been shown to be
accompanied by induction of interferon regulatory factor (IRF)-1.
FcRI has been used as a marker of IFN- action. Promoter regions
of IRF-1 and FcRI contain GAS sequences and could be primary targets
of STAT1 and STAT3. IFN- induced the expression of IRF-1 in both
parental M1 and NIH-3T3 cells within 1 hour, and a high level of
expression was maintained thereafter. Similar induction of IRF-1 was
observed in CIS3 transformants. In contrast, IRF-1 expression was only marginally elevated in M1 and NIH-3T3 transformants expressing JAB.
FcRI expression was gradually induced from 1 hour after stimulation
in parental M1 cells and CIS3 transformants, whereas it was not induced
within 6 hours in JAB transformants. We could not detect FcRI
expression in NIH-3T3 cells. These data suggest that JAB but not CIS3
can efficiently inhibit IFN--induced STAT1 activation.
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| Fig 5.
Effect of JAB and CIS3 on IFN- inducible genes. The
cells (A; M1-derived cells, B; NIH3T3-derived cells) were
stimulated with 1,000 IU/mL IFN- for the indicated periods (h) and
analyzed with Northern hybridization using IRF1, FcRI, and control
G3PDH probes. FcRI was not detected in parental NIH3T3 cells (not
shown).
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Next, we examined IFN--induced STAT1 activation directly. Parental
M1 and NIH-3T3 cells or their transformants expressing CIS3 or JAB were
stimulated with or without IFN-, then cell extracts were blotted
with antibody specific to tyrosine-phosphorylated STAT1
(Fig 6; PY-STAT1) or to STAT1 (Fig 6;
STAT1). STAT1 was tyrosine phosphorylated in response to IFN- in
parental M1 cells and transformants expressing CIS3 for over 24 hours.
On the other hand, the tyrosine phosphorylation of STAT1 in NIH-3T3
cells was transient. In both M1 and NIH-3T3 JAB transformants,
IFN--induced tyrosine phosphorylation of STAT1 was largely reduced.
Interestingly, STAT1 content was markedly increased after stimulation
with IFN- in parental M1 cells, NIH-3T3 cells, and their CIS3
transformants, suggesting a positive feedback regulation of STAT1
synthesis. No such increase in STAT1 content in response to IFN- was
evident in JAB transformants. The DNA binding activity of STATs was
assessed by EMSA using the IRF-1 GAS probe21 (Fig 6; EMSA).
STAT1/DNA binding complexes appeared after stimulation with IFN- for
60 minutes in parental cells and transformants expressing CIS3. DNA binding activity of STAT1 in M1 cells lasted over 24 hours, whereas that in NIH-3T3 cells was transient, which is consistent with the time
course of STAT1 phosphorylation. IFN--induced DNA binding activity
of STAT1 was not detected in either M1 or NIH-3T3 transformants expressing JAB (Fig 6; EMSA).
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| Fig 6.
Inhibition of IFN--induced STAT1 activation by JAB.
Cells (A; M1-derived cells, B; NIH3T3-derived cells) were stimulated
with or without IFN- (1,000 IU/mL) for the indicated periods and
lysed. The postnuclear supernatant was subjected to immunoblotting with
anti-tyrosine-phosphorylated STAT1 (PY-STAT1) or anti-STAT1
(STAT1) and to EMSA with IRF-1 probe (EMSA).
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Impaired JAK activation in JAB transformants in response to IFN-
and IFN-.
To elucidate further molecular basis of the inhibition of STAT1
activation by JAB, we examined IFN--induced activation of JAK1 and
JAK2 and IFN--induced activation of JAK1 and Tyk2 in NIH-3T3 cells.
Tyrosine phosphorylation of JAKs in response to IFNs in M1 cells was
not very evident, probably because of very low content of JAKs or
receptors. After stimulation of parental NIH-3T3 cells or JAB
transformants with IFN- for 15 minutes, cells were lysed and then
immunoprecipitated with anti-JAK1 or JAK2 antibody and probed with
antiphosphotyrosine antibody (Fig 7A and
B). Tyrosine phosphorylation of JAK1 and JAK2 was largely impaired in
JAB transformants. Similarly, tyrosine phosphorylation of JAK1 and Tyk2
in response to IFN- was largely reduced in JAB transformants (Fig 7C
and D). As shown previously,16 fibroblast growth factor (FGF) induced tyrosine phosphorylation and activation of
the FGF receptor normally, suggesting that the inhibition of tyrosine
kinase activity in JAB transformants was specific to JAKs. Thus, the
IFN unresponsiveness of JAB transformants can be explained by
inhibition of activation of JAKs by JAB.
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| Fig 7.
Inhibition of IFN-- and IFN--induced JAK
activation by JAB. NIH3T3 parental cells (parent) and JAB transformants
(JAB) were stimulated with 1,000 IU/mL IFN- (A and B) or IFN- (C
and D) for 15 minutes and lysed. The postnuclear supernatant was
immunoprecipitated with anti-JAK1 (A and C) anti-JAK2 (B), or anti-Tyk2
(D). The immunoprecipitates were blotted with antiphosphotyrosine
antibody (upper panels), then the membrane was stripped and reprobed
with anti-JAK1 (A and C), anti-JAK2 (B), or anti-Tyk2 (C) antibodies
(lower panels).
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Elevated expression of JAB in IFN-resistant cell lines.
To extend the potential involvement of JAB expression in the
IFN-resistant phenotype, we examined JAB expression in IFN-resistant somatic cell mutants derived from a human Burkitt's lymphoma cell line, Daudi, and a colon adenocarcinoma cell line, LoVo. Daudi cells
are highly sensitive to IFN--induced growth arrest, although IFN- exhibits no effect. An IFN--resistant clone was obtained by
colony formation in the presence of IFN-. Two IFN--resistant mutants, IGR-5 and IGR-53, with dominant phenotypes were obtained from
LoVo cells previously.6 IGR-5 is highly resistant to
IFN- and weakly to IFN-, and IGR-53 is highly resistant to both.
IGR-53 cells lack functional IFN- receptors, whereas IGR-5 possesses IFN- receptors. The molecular mechanism of IFN resistance of IGR-5
has not been clarified. We examined JAB and CIS3 expression in these
mutants. As shown in Fig 8,
IFN--resistant Daudi cells and IGR-5, but not IGR-53, exhibited
elevated expression of JAB. The level of JAB in DaudiR
cells was comparable to that in CTLL2 cells (data not shown). IFN--induced tyrosine phosphorylation of JAK1 and Tyk2 as well as
that of STAT1 were reduced to about 50% in IFN--resistant Daudi
cells compared with parental Daudi cells
(Fig 9). Remarkable reduction in tyrosine
phosphorylation of JAK1, JAK2, and STAT1 was observed in response to
IFN- in IGR-5 cells (Fig 10).
Therefore, overexpression of JAB could be, at least in part, a
mechanism of IFN resistance in vitro.
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| Fig 8.
Expression of JAB and CIS3 mRNA in IFN-resistant clones
from Daudi and LoVo cells. Total RNAs from exponentially growing
parental LoVo cells (LoVo), IFN--resistant IGR-5 cells
[IGR-5()], IFN-- and IFN--resistant IGR-53 cells
[IGR-53(,)], parental Daudi cells (Daudi), and
IFN--resistant Daudi cells [DaudiR()] were
extracted and analyzed with Northern hybridization using JAB, CIS3, and
G3PDH probes.
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| Fig 9.
IFN--induced tyrosine phosphorylation of JAK1, Tyk2,
and STAT1 in Daudi and its IFN-resistant clone. Parental Daudi cells
(Daudi) or IFN-resistant cells (DaudiR) were stimulated
with IFN- (1,000 IU/mL) for 15 minutes and lysed. The postnuclear
supernatants were immunoprecipitated with anti-JAK1 (A) or anti-Tyk2
(B). The immunoprecipitates were blotted with antiphosphotyrosine
antibody (upper panels), then the membrane was stripped and reprobed
with anti-JAK1 (A) or anti-Tyk2 (B) antibodies (lower panels). (C)
Total cell extracts were also blotted with anti-phospho STAT1
(PY-STAT1) and anti-STAT1 (STAT1).
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| Fig 10.
IFN--induced tyrosine phosphorylation of JAK1, JAK2,
and STAT1 in LoVo and IGR-5. Parental LoVo cells (LoVo) or
IFN-resistant cells (IGR-5) were stimulated with IFN- (1,000 IU/mL)
for indicated periods (h) and lysed. The postnuclear supernatants were
immunoprecipitated with anti-JAK1 (A) or anti-JAK2 (B). The
immunoprecipitates were blotted with antiphosphotyrosine antibody
(upper panels, PY), then the membrane was stripped and reprobed with
anti-JAK1 (A) or anti-JAK2 (B) antibodies (lower panels). (C) Total
cell extracts were also blotted with anti-phospho STAT1 (PY-STAT1)
and anti-STAT1 (STAT1).
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DISCUSSION |
In this study, we found that JAB is an IFN--inducible gene and that
forced expression of JAB conferred resistance not only to IFN- but
also to IFN- in murine cell lines. The molecular mechanism of the
inhibition of IFN signaling by JAB is most likely to be the inhibition
of activation of JAKs, thereby blocking STATs activation and target
gene expression. Several lines of evidence suggest a direct inhibition
of tyrosine kinase activity by binding of JAB to JAKs. As shown
previously, coexpression of JAB efficiently inhibits tyrosine
phosphorylation of JAK as well as cellular proteins in 293 cells.16 LIF- or IL-6-induced tyrosine phosphorylation of
STAT3 and gp130 was largely impaired in M1 cells expressing JAB.18-20 However, in M1 cells it has been very difficult
to show tyrosine phosphorylation of JAKs in response to LIF or IFNs. In the present study, we showed that in NIH-3T3 cells expressing JAB that
do not respond to IFNs, IFN-induced tyrosine phosphorylation of JAK1,
JAK2, and Tyk2 was strongly inhibited. These inhibitions probably lead
further inhibition of STAT1 activation, IRF-1 expression, and induction
of an antiviral state. Recently, we showed inhibition of JAK2 tyrosine
kinase activity in vitro using purified recombinant JAK2 and JAB
(Yasukawa et al, unpublished data, February 1998). Thus,
JAB is probably an intrinsic inhibitor of JAKs.
Starr et al reported that in mouse bone marrow cells, SOCS-1/JAB is
highly induced by GM-CSF, IL-13, and IFN-.18
We also observed that JAB was induced by GM-CSF in UT7 myeloid leukemia cells and by EPO in F36E erythroleukemia cells.17 However,
as far as we ascertained, IFN- is the most potent inducer of JAB in
a wide variety of cell lines. The promoter region of JAB contains GAS
motif,24 suggesting the involvement of STAT1 in the
induction of JAB by IFN-. Previously, JAB/SOCS1 was reported to be
induced by IL-6 in mouse liver and M1 cells and to inhibit IL-6
signaling in M1 cells, suggesting that JAB is a negative feedback
regulator of IL6/STAT3. However, our experiments suggest that IL-6 or
LIF induces expression of JAB in M1 cells much less efficiently than IFN- (Fig 2 and data not shown). We found that IFN- induces JAB
expression in M1 cells at a level probably high enough to inhibit not
only IL-6 but also IFN- signaling (see Fig 2). Thus, JAB could be a
negative feedback regulator of IFN-/STAT1 and also could be involved
in IFN--induced downregulation of other cytokine responsiveness. We
also found that JAB expression is very high in CTLL2 cells without
IFN- (see Fig 2). As expected, CTLL2 did not respond to IFN- and
IFN- for growth inhibition (Sakamoto et al,
unpublished data, March 1998). It also has been shown
that exogenously expressed EPO receptor and granulocyte colony-stimulating factor (G-CSF) receptor are not functional in this
cell line.25,26 Because the molecular basis of such a
selective inhibition of particular cytokine receptor signaling has not
been elucidated, JAB could be a good candidate for the factor that is
involved in this cytokine unresponsiveness in CTLL2. Although EPO does
not induce tyrosine phosphorylation of JAK2 in CTLL2 cells expressing
exogenous EPO receptor,27 it is not clear why CTLL2 still
can respond to IL-2. JAB may have a lower affinity to JAK3 than to JAK1
and JAK2, thereby allowing activation of IL-2 signaling but not EPO or
G-CSF signaling.
If JAB is a negative feedback regulator, why cannot JAB induced by
IFN- inhibit further IFN- signaling to produce an antiviral effect? The signaling events induced by early JAK activation until the
appearance of JAB (usually these inductions take a few hours) may be
essential and sufficient for further irreversible changes inside cells.
Indeed, tyrosine phosphorylation of STAT1 in response to IFN- is
usually transient even in the continuous presence of IFN- (see Fig
6B). JAK activity is also transient in most cases. JAB mRNA is detected
at 1 to 3 hours and remains after 24 hours of stimulation (Fig 1).
Thus, JAB expression could be a mechanism of downmodulation of
IFN--induced JAK/STAT1 activity. However, in M1 cells, activation
of STAT1 induced by IFN- lasted for more than 24 hours. Similarly,
prolonged activation and increased levels of STAT3 were observed in M1
cells after stimulation with IL-6.28 The molecular
mechanism of such prolonged activation of STATs in M1 cells has not
been elucidated but may be partly due to the increase of the levels of
STAT1 (Fig 6) and STAT3.28 Similar IFN- "priming"
effect for IFN--induced STAT1 and STAT2 tyrosine phosphorylation
has been reported.29 However, the efficiency of STAT1
phosphorylation compared with the level of STAT1 content apparently
decreased in M1 cells after continuous IFN- exposure, suggesting a
downregulation of JAK activation even in M1 cells (Fig 6).
Our present study suggests disregulated overexpression of JAB can
confer IFN resistance on cells. JAB could be a mechanism of IFN
resistance with dominant phenotype because resistance appeared on
constitutive expression. Indeed, two independently isolated IFN-resistant clones expressed elevated levels of JAB. IFN resistance with recessive phenotype has been well described. Mutations in the
receptors, JAKs, and STATs have been found in IFN-resistant mutants.1,2 Recently, marked reduction of STAT1 was
consistently observed in IFN-resistant melanoma cells from
patients.5 Exogenous expression of STAT1 improved
responsiveness to IFN- in an IFN-resistant cell line.5
These are examples of IFN resistance with recessive phenotype. On the
other hand, IFN resistance with dominant phenotype has not been well
documented. As far as we know, IGR-5 and IGR-53 are the only examples
of dominant resistant phenotypes.6 However, Raiph et al
reported that resistance of melanoma cell lines to IFNs correlates with
reduction of IFN-induced tyrosine phosphorylation of cellular
proteins.30 Thus, as shown here, reduced
activation of JAKs by JAB overexpression could be, at least in part, a
mechanism of IFN resistance with dominant phenotype. At present, we do
not know what kind of mutations can elevate JAB expression. However, constitutive activation of STATs by oncogenic tyrosine kinases such as
Bcr-Abl can potentially be involved in high JAB expression. Although
partial inhibition of JAK/STAT pathway by JAB may not be able to
explain 10,000 times resistance to IFN- of DaudiR cells,
overexpression of JAB could contribute to some feature of IFN
resistance obtained from sensitive cell lines by in vitro selection.
Probably, DaudiR cells have multiple mutations that confer
strong resistance to IFN--induced apoptosis and growth arrest. Our
study strongly encourages the retrospective search of JAB expression in
cells from patients, which may elucidate the involvement of JAB in
actual IFN resistance in cancer or viral diseases.
IFN- is known as an inhibitory cytokine and is known to suppress
many cytokine actions or cellular responses. For example, IL-4-induced
IgE synthesis and germline  transcription was suppressed in the
presence of IFN- in B cells.31 JAB could be a part of the mechanism of the antagonistic effect of IFN- on other cytokines. JAB is also most abundantly expressed in thymus,18 which
suggests a regulatory role for JAB in cytokine actions in T cells.
IFN- has been shown to play an important role in immune responses, including the development of Th1 cells.32 IFN--treated
Th1 cells become resistant to growth inhibition by IFNs, which has been
explained by loss of the IFN- receptor chain.33 However, such loss of the receptor
may need long-term incubation of cells with IFN-. Induction of JAB
may partly explain early downregulation of IFN- responsiveness in
Th1 cells. The physiological role of JAB on IFN- activity may be
defined by the creation of mice lacking the JAB gene.
| |
FOOTNOTES |
Submitted February 3, 1998;
accepted April 20, 1998.
Supported in part by grants from the Ministry of Science, Education and
Culture of Japan, Ryoichi Naito Medical Foundation, Suzuken Memorial
Foundation, TORAY Research Foundation, and Uehara Memorial Foundation.
Address reprint requests to Akihiko Yoshimura, Institute of Life
Science, Kurume University, Aikawamachi 2432-3, Kurume 839-0861, Japan;
e-mail: yosimura{at}lsi.kurume-u.ac.jp.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Ms H. Ohgusu for excellent technical assistance.
| |
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Abstract
Introduction
Abstract