(Received for publication, August 26, 1996, and in revised form, January 17, 1997)
From the Department of Biochemistry, Kyoritsu College
of Pharmacy, Shibakoen, Minato-ku, Tokyo 105, Japan, the
¶ Division of Cell Differentiation, Kumamoto University School of
Medicine, Kumamoto-shi 860, Japan, and the
Department of
Pharmacology, Institute of Cancer Research, Kanazawa University,
Ishikawa-ken 960, Japan
Most types of cells can produce interleukin
(IL)-8 in response to various inflammatory stimuli. To study the role
of protein phosphatases in the signal transduction leading to IL-8
production, a subline of HL-60 (C-15) was treated with okadaic acid
(OA) and sodium orthovanadate (VA), inhibitors of
phosphoserine/phosphothreonine phosphatase and phosphotyrosine
phosphatase, respectively. Both OA and VA dramatically increased IL-8
secretion up to 200-fold in the HL-60 cells. OA and VA stimulation was
accompanied by a marked increase in IL-8 mRNA expression and also
by activation of a transcription factor, NF-B. In addition, an
essential role of the NF-
B site in the IL-8 gene activation was
confirmed by the chloramphenicol acetyltransferase assay. IL-8
production by OA or VA was inhibited by protein kinase inhibitors,
including staurosporine, H-7, K252a, herbimycin A, and genistein.
Both OA and VA induced significant tyrosine phosphorylation of p44,
which was presumed to be Erk1, a member of the mitogen-activated protein kinase family, with concomitant activation of the
mitogen-activated protein kinase activity. In parallel, rapid
degradation of IB-
, an inhibitory component of NF-
B, was
observed. Since OA-activated Erk1 phosphorylated recombinant I
B-
in vitro, we assumed that Erk1 is involved in the
phosphorylation and subsequent degradation of I
B-
, thus leading
to the activation of IL-8 gene transcription.
Interleukin (IL)1-8 is a cytokine that
has chemotactic activity on neutrophils as well as T cells and
basophils (1-3). IL-8 belongs to a chemokine family and is produced by
a variety of cells stimulated with lipopolysaccharide (LPS) or
proinflammatory cytokines, such as IL-1 or tumor necrosis factor-
(TNF-
) (4-8). Production of IL-8 is regulated at the
transcriptional level through the activation of NF-
B complexes as
well as C/EBP/NF-IL-6 or AP-1 complexes (9-11). NF-
B, originally
identified as a transcription factor necessary for Ig
gene
expression (12), is a pleiotropic transcription factor that regulates
the activation of various inflammatory genes (13-15). Recently,
Ishikawa et al. (16, 17) reported the activation of a
NF-
B complex in a cell-free system using the NF-
B binding site in
the IL-8 gene and claimed that a staurosporine-sensitive kinase as well
as tyrosine kinase is involved in the LPS-mediated NF-
B
activation.
The phosphorylation-dephosphorylation of cellular proteins appears to
be a major regulatory mechanism for cytokine production. In fact,
inhibitors of protein phosphatases including okadaic acid (OA) or
calyculin A (CA) are known to be good stimulators for IL-1 or TNF-
(18, 19). We therefore attempted to investigate the role of protein
phosphorylation and/or dephosphorylation in IL-8 induction in a human
promyelocytic cell line HL-60 subline, using OA and sodium
orthovanadate (VA), specific inhibitors of phosphoserine/phosphothreonine phosphatase and tyrosine phosphatase, respectively. Our finding demonstrated that both OA and VA induced high
levels of IL-8 production and its mRNA expression. Further, we
analyzed the molecular mechanism of IL-8 gene activation by OA or VA in
the HL-60 (C-15) cell line. We observed that mutation of the NF-
B
binding site abolished the induction of CAT activity upon stimulation
with OA or VA, indicating the essential role of NF-
B site also for
the phosphatase inhibitor-induced IL-8 gene activation. In addition, an
electrophoretic mobility shift assay (EMSA) revealed that OA or VA
induced the formation of the NF-
B complexes. We also investigated
the relationship of mitogen-activated protein kinase (MAPK) and
I
B-
degradation during the IL-8 gene activation.
OA, staurosporine, and genistein were obtained
from Research Biochemicals International. VA was obtained from Sigma.
Herbimycin A, H-7, H-8, KN62, and KN252a were obtained from Wako Pure
Chemical Inc. (Osaka, Japan). [-32P]ATP, enhanced
chemiluminescence reagents, and a p42/p44 MAPK enzyme assay system were
obtained from Amersham (Tokyo, Japan). Monoclonal anti-phosphotyrosine
antibody (mAb 4G10) was purchased from Upstate Biotechnology, Inc.
(Lake Placid, NY). Rabbit polyclonal anti-MAPK (Erk-1/2) and
anti-phospho-MAPK Abs were purchased from Biolabs, Inc. Anti-Erk1 mAb
was obtained from Transduction Laboratories (Lexington, KY).
Horseradish peroxidase-conjugated secondary Ab was purchased from DAKO
(Denmark).
An eosinophil-committed subline of the human promyelocytic leukemia cell line, HL-60 (C-15) (American Type Culture Collection (ATCC) CRL 1964, a gift of Dr. Steven Fischkoff) (20, 21) was used, since this subline produced higher levels of IL-8 than several other HL-60 cell lines. The HL-60 cell line was maintained in RPMI 1640 supplemented with 5% fetal calf serum (Hyclone, Logan, UT), 2 mM L-glutamine.
Measurement of Cytokine Production and Northern Blot AnalysisImmunoreactive IL-8 was quantitated using an ELISA method as described elsewhere (22). This ELISA method detected at least 30 pg/ml of IL-8 and did not cross-react with other known members of the CXC and CC chemokine families. Total RNA was extracted from the cultured cells by 4 M guanidine thiocyanate. Northern blot analysis was performed as described previously (23).
Preparation of the Cell Lysates and ImmunoprecipitationCells (106) were lysed with lysis
buffer (50 mM HEPES (pH 7.5), 0.5% Triton X-100, 100 mM NaF, 10 mM sodium phosphate, 4 mM EDTA, 2 mM sodium vanadate, 2 mM
sodium molybdate, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml
phenylmethylsulfonyl fluoride) by stirring for 1 h at 4 °C.
After centrifugation for 20 min at 15,000 rpm at 4 °C, protein
concentration was determined, and the samples were stored at
80 °C. For the immunoprecipitation, 5 × 106
cells were suspended in 1 ml of lysis buffer, and the lysate was
centrifuged for 20 min at 15,000 rpm. Twenty µl of a slurry of
pansorbin (Calbiochem) and 3 µg of anti-I
B-
Ab (kindly provided by Dr. Nancy Rice, NCI-Frederick Research and Development Center, Frederick, MD) were added to the supernatant and incubated overnight at
4 °C. The precipitates spun down were washed three times with lysis
buffer and were then boiled for 5 min in Laemmli sample buffer. The
sample was electrophoresed in 10% polyacrylamide gel and transferred
onto a membrane filter followed by the Western blot using
anti-I
B-
Ab and peroxidase-labeled anti-rabbit IgG, as described
elsewhere (16, 17).
An equal volume of 2 × Laemmli sample buffer was added to the cell lysate prepared as described above. Samples were boiled for 10 min, and equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked in 3% bovine serum albumin in phosphate-buffered saline for 1 h and then incubated with anti-phosphotyrosine or anti-MAPK or anti-phospho-MAPK Abs for 1 h at room temperature. After incubation with secondary Ab coupled to horseradish peroxidase, detection was made using the enhanced chemiluminescence system. Molecular sizes were determined by the relative mobilities of prestained molecular weight markers.
MAPK AssayMAPK activity was measured using the p42/44 MAPK
enzyme assay system according to the manufacturer's instruction
(Amersham). Cell lysates (15 µl) prepared as described above were
incubated with a substrate peptide (human EGF receptor) in 10 µl of
kinase buffer. The reaction was initiated of 5 µl of
[-32P]ATP (6 nmol, 37 kBq). The reaction mixture was
incubated at 30 °C for 20 min. The reaction was stopped by
transferring the reaction mixture onto p81 filter paper. The filters
were washed three times in 75 mM phosphoric acid to remove
nonreacted [
-32P] ATP, and air-dried. Radioactivity
was determined by liquid scintillation counting.
CAT expression vectors harboring
the 5-flanking region of the IL-8 gene spanning the
133 region and
C/EBP, AP-1, and NF-
B mutants were constructed as described
previously (9-11). For transient expression studies, 2 × 106 HL-60 (C-15) cells were transfected with 10 µg of
plasmid DNA by the DEAE-dextran (200 µg/ml) method. After 5 h,
cells were incubated with or without stimulants for 2 days. The CAT
activity in cell extract was determined as described elsewhere (9-11). Transfection and CAT assay were performed at least three times with
each CAT expression vector, and representative results are shown here.
We performed CAT assays using several different concentrations of
proteins so that the assay is within linear range.
108
cells were stimulated with OA or VA for 30 min, and nuclear
extracts were prepared. Protein concentration of nuclear extracts was
determined and assayed for NF-B activity or stored at
80 °C until further use. EMSA was performed essentially as described elsewhere (9-11, 16). The sequences of NF-
B oligonucleotide and
mutated NF-
B used in this EMSA were 5
-CGTGGAATTTCCTCTG-3
and
5
-CGTTAACTTTCCTCTG-3
, respectively.
A kinase assay was performed
using immunoprecipitates of anti-Erk1 mAb. The immune complex was
washed twice with kinase buffer (25 mM HEPES, pH 7.4, 25 mM MgCl2, 2 mM dithiothreitol). The
assays were initiated by the addition of 1 µg of substrate protein
and 50 µM [-32P]ATP (220TBq/mmol) in a
final volume of 25 µl. Reactions were terminated after 30 min at
30 °C by the addition of Laemmli sample buffer. The phosphorylation
of the substrate protein was examined after SDS-polyacrylamide gel
electrophoresis, followed by autoradiography. GST-I
B-
fusion
protein was prepared as described elsewhere (16, 17).
When HL-60 was
stimulated with OA or VA, a significant amount of IL-8 was produced in
the culture supernatants. HL-60 cultured without any exogenous stimuli
released a marginal level of IL-8 (<0.5 ng/ml). It should be noted
that both OA and VA were effective at a narrow range of concentration
with maximal activation at 100 nM and 80 µM,
respectively (Fig. 1). The effect of OA and VA was
limited by their toxicity, which became apparent at concentrations greater than 100 nM (OA) and 200 µM (VA).
Since induction of IL-8 was greatest at 100 nM (OA) or 80 µM (VA), these concentrations were used in further
experiments to study the effect of these stimulants. As determined by
kinetic analysis, IL-8 production induced by OA or VA was detectable at
3-6 h with maximal levels at 12-24 h (data not shown). OA increased
IL-8 both in the supernatants and in cell lysate, with each 200- and
100-fold increase above unstimulated control. The effect of VA on IL-8
production was similar to that of OA, although the magnitude of IL-8
production was lower than that of OA. IL-8 production induced by OA or
VA was still much higher than the levels induced by TNF- (Fig.
2). These results indicated that both OA and VA by
themselves are effective stimulants for IL-8 production. When the
simultaneous addition of OA and VA was tested, no significant
synergism, only an additive effect, was observed (data not shown).
Although we studied the direct effect of phosphatase inhibitors on the
undifferentiated HL-60 cells, TNF- (but not IL-1
/
) induced
only a moderate level of IL-8 by this cell line. Therefore, we examined
whether phosphatase inhibitors modulate TNF-
-induced IL-8
production. As shown in Fig. 2, additive or weak synergistic effects
were observed between TNF-
(20 ng/ml) and lower doses of OA (20 nM) and VA (10 µM), suggesting that
phosphatase inhibitor did not interfere with TNF-
- induced IL-8
production, but rather acted cooperatively.
We also examined the effect of OA and VA on the normal human blood monocytes. As shown in Table I, both OA and VA increased IL-8 production only marginally, with a 1.3-1.7-fold increase above control, in which unstimulated monocytes spontaneously produced high levels of IL-8. Rather, higher doses of OA (50-100 nM), which were optimal for IL-8 production by HL-60 cells, inhibited IL-8 production, suggesting the differential effects of phosphatase inhibitors on the primary monocytes and leukemic cell line.
|
To confirm
whether the increase of IL-8 was accompanied by the transcription of
the IL-8 gene, we examined the expression of IL-8 mRNA. Northern
blot analysis showed that IL-8 mRNA was detected as early as 1 h, and OA elicited a dramatic induction of IL-8 mRNA at 6-12 h
(Fig. 3). The increase of IL-8 mRNA peaked at
12 h of OA treatment and declined at 24 h. Similarly, VA
elicited a moderate but significant induction of IL-8 mRNA without
a significant decline of IL-8 mRNA at 24 h.
To determine if OA and VA contributes to IL-8 mRNA stabilization, we measured the half-life of IL-8 mRNA, by incubating the cells with OA or VA for 3 h, followed by the treatment with actinomycin D. The half-life (t1/2) of IL-8 mRNA from unstimulated cells was approximately 5 h, while those of OA- or VA-treated cells were prolonged more than 10 h (data not shown). These results unambiguously indicated that both OA and VA increased stability of IL-8 mRNA markedly, thus contributing to the sustained expression of IL-8 mRNA.
Effects of Protein Kinase Inhibitors on IL-8 Induction by OA or VATo investigate which type of protein kinase(s) was involved in
the stimulation with OA or VA, HL-60 cells were treated with staurosporine, a nonspecific protein kinase inhibitor. Staurosporine at
100 nM completely blocked the increase of IL-8 by OA or VA (Fig. 4), suggesting that a basal level of protein
phosphorylation is necessary for the action of OA or VA. Protein kinase
C inhibitors, H-7 and K252a, markedly blocked IL-8 production induced
by OA or VA with an IC50 of H-7 and K252a at 10 and 20 µM, respectively (Fig. 4). HA1004, an analog of H-7, a
weak protein kinase C inhibitor, was inactive even at 100 µM (data not shown). In addition, the tyrosine kinase
inhibitors, genistein and herbimycin A, significantly inhibited the
increase of IL-8 production by OA or VA, with the IC50 of
genistein at 6-10 µM and that of herbimycin A at 1 µM, indicating that tyrosine phosphorylation is essential
for the IL-8 induction by OA or VA. On the other hand, treatment with KN-62, a calmodulin-dependent kinase inhibitor, had no
effect. Similarly, H-8, PKA, and cGMP-dependent protein
kinase inhibitors showed only marginal inhibitory effects on OA and VA
stimulation.
These results suggested that protein kinase C as well as tyrosine kinase are involved in the stimulation of IL-8 production by OA or VA. However, no direct evidence was obtained on the involvement of the cAMP- and cGMP-dependent protein kinases and calmodulin-dependent kinases.
Effect of OA and VA on Tyrosine PhosphorylationTo delineate
the target proteins involved in protein phosphorylation, lysates of OA-
or VA-stimulated cells were prepared, electrophoresed, and
immunoblotted with an anti-phosphotyrosine Ab (clone 4G10). Stimulation
with OA or VA for 1 h induced increased tyrosine phosphorylation
of several proteins, particularly a protein with an apparent molecular
mass of 44 kDa (p44) (Fig. 5). It should be of note that
no significant tyrosine phosphorylation of p44 was detected in lysates
of unstimulated cells. Assuming that p44 might be a member of the MAPK
family, which plays a crucial role in signal transduction from receptor
tyrosine kinases (24), cell lysates were subjected to immunoblotting
with anti-MAPK or phospho-MAPK Abs. When lysates of OA or VA-stimulated
cells were electrophoresed and immunoblotted with rabbit anti-MAPK (rat
Erk1/2) Ab, 44-kDa (Erk1) and 42-kDa (Erk2) bands were consistently
detected 0.5, 1, and 3 h after the stimulation with OA or VA (Fig.
6A). Immunoblotting using anti-phospho-MAPK
Ab, which recognizes phosphorylated MAPK (phosphorylated Erk1 and -2),
revealed that a phosphorylated 44-kDa protein was detected 30 min after
stimulation with OA or VA and increased gradually until 3 h (Fig.
6B), indicating that tyrosine-phosphorylated p44 is likely
to be an Erk1 of the MAPK family.
OA or VA Stimulation Induces MAPK Activity
In accordance with
the above finding, we investigated whether MAPK activity was induced by
OA and VA treatment, i.e. lysates from OA- or VA-stimulated
cells were prepared and assayed for MAPK activity. Time kinetics of the
MAPK activity by 100 nM OA or 80 µM VA
stimulation were shown in Fig. 7. After a 30-min
stimulation with OA or VA, MAPK activity rapidly increased, reaching a
maximal level at 1 h, which was maintained thereafter. Thus, a
good correlation was observed between OA- or VA-induced tyrosine
phosphorylation and MAPK activity. It should be noted that herbimycin A
(5 µg/ml), an inhibitor of tyrosine kinases, inhibited MAPK activity
almost completely (data not shown).
Requirement of NF-
Since it has been demonstrated that both TNF-- and
IL-1-induced IL-8 gene activation requires the NF-
B site in the
5
-flanking region of the IL-8 gene (9-11), we determined whether the
NF-
B site as well as AP-1 and C/EBP sites are prerequisite as well for the OA- or VA-induced IL-8 gene activation. Since CAT activity transfected with
133-CAT expression vectors was normally induced by
OA or VA (Fig. 8B), the mutants of the AP-1,
C/EBP, or NF-
B sites were constructed as shown in Fig.
8A. Mutation of AP-1 or C/EBP/NF-IL-6 had little effect on
the induction of CAT activity upon stimulation with OA or VA,
indicating that these sites were not essential for gene regulation by
these agents. On the other hand, mutation of the NF-
B site
completely abolished the induction of CAT activity upon stimulation
with OA or VA (Fig. 8B), indicating the essential role of
the NF-
B site for IL-8 gene activation by these phosphatase
inhibitors, as was the case in IL-1-induced IL-8 production in the
glioblastoma cell line T98G (11).
Induction of NF-
To
delineate the role of a nuclear factor, NF-B, in the OA- and
VA-induced IL-8 mRNA transcription, we performed an EMSA using
nuclear extracts from the OA- or VA-stimulated HL-60 cells. Nuclear
extracts from OA-treated HL-60 cells contained NF-
B complex as
indicated, utilizing an authentic NF-
B oligo probe (Fig.
9, lane 2), whereas no specific NF-
B·DNA
complex formation was observed in extracts from unstimulated cells
(lane 1). The specificity of the complex was confirmed by
competition experiments using an unlabeled NF-
B probe or an
unlabeled mutated NF-
B probe. The band disappeared in the presence
of an excess amount of an unlabeled NF-
B probe (lane 4),
whereas the band did not disappear in the presence of an excess amount
of unlabeled mutated NF-
B probe (lane 3). Moreover, a
labeled mutated NF-
B probe failed to form a NF-
B complex
(lane 5). VA-stimulated nuclear extracts gave results
similar to those of OA. These results indicated that a specific NF-
B
complex occurred following OA or VA stimulation.
Effect of OA and VA on the Phosphorylation and Degradation of I
Finally, we investigated the fate of IB-
after
the treatment of cells with OA or VA. Anti-I
B-
Ab detected a
38-kDa band (I
B-
) on Western blots in extracts from unstimulated
HL-60 cells (Fig. 10), while no corresponding band was
seen with the control antibody (data not shown). Within 10-20 min
after OA stimulation, the I
B-
band almost disappeared, indicating
degradation of this component. In contrast, the decrease of I
B-
following treatment of VA was slower and not complete as compared with
OA-treatment.
In addition, we examined whether IB-
could serve as a substrate
of Erk1. Erk1 was prepared by immunoprecipitating OA-stimulated HL-60
cells and was used to phosphorylate recombinant GST-I
B-
protein.
In an immune complex kinase assay, Erk1 effectively phosphorylated GST-I
B-
(Fig. 11A, lane 2)
but not control GST (Fig. 10A, lane 1). A time
course study showed phosphorylation peaking at 20 min after OA
stimulation (Fig. 11B). These results indicated that
GST-I
B-
could be a substrate of Erk1. Since Erk1 prepared from
OA-stimulated, but not from unstimulated, HL-60 cells markedly enhanced
phosphorylation of GST-I
B-
, it is quite possible that OA induces
activation of Erk1 to phosphorylate I
B-
, followed by the
degradation of I
B-
.
We have demonstrated in this paper that both OA and VA, two potent protein phosphatase inhibitors, were effective stimulants for IL-8 production in HL-60 cells both at transcriptional and post-transcriptional levels. In addition, a nonspecific protein kinase inhibitor, staurosporine, blocked OA- or VA-induced IL-8 production, indicating the requirement of a basal level of protein phosphorylation for this activity. Because there were no essential differences between VA and OA stimulation and no synergistic actions were observed between these stimuli, both stimuli appear to activate the same or a very similar pathway resulting in the induction of IL-8 production.
Numerous exogenous and endogenous stimuli such as endotoxin, stresses,
viruses, and bacteria as well as IL-1 and TNF- stimulate IL-8 gene
activation in a variety of cells (1, 5, 7-10) in which transcriptional
activation of the IL-8 gene is fairly well understood (9-11). The CAT
assay experiments revealed that, in the HL-60 cell line as well, the
NF-
B site was prerequisite for the appropriate activation of the
IL-8 gene by OA or VA stimulation, similar to TNF-
and IL-1
stimulation, as has been described elsewhere (9-11). In addition, EMSA
revealed that OA or VA induced the formation of the NF-
B complexes.
However, the role of protein phosphorylation in IL-8 gene activation
has not yet been clearly defined. Of interest is the finding that
inhibitors of protein phosphatases including OA or calyculin A induce
IL-1 or TNF-
in various cell systems (18, 19). Furthermore, it has
been shown that OA treatment results in immediate phosphorylation of a
variety of proteins in many cell types including fibroblasts and that
OA mimics gene expression induced by IL-1 and TNF-
(25). In this
study, we demonstrated that OA or VA caused tyrosine phosphorylation of several proteins in HL-60 cells. We have assumed that tyrosine phosphorylation of MAPK is one of the key signal transduction pathways
leading to IL-8 gene activation in HL-60 cells. To prove the
assumption, we tried to identify a tyrosine-phosphorylated 44-kDa
protein in OA- or VA-stimulated HL-60 cells. Immunoblots using
anti-phosphotyrosine, MAPK, and phospho-MAPK Abs indicated that the
tyrosine-phosphorylated 44-kDa protein was Erk1, a member of the MAPK
family (24). The involvement of c-Jun N-terminal kinase/stress-activated protein kinase or p38 MAPK homologues, which
also are members of MAPK family (26-28), cannot be completely ruled
out in this study, although the anti-phospho-MAPK Ab used detects
tyrosine-phosphorylated MAPK and does not cross-react with the
corresponding phosphorylated tyrosine of either c-Jun N-terminal
kinase/stress-activated protein kinase or p38 MAPK. More detailed study
is required to elucidate the involvement of the c-Jun N-terminal
kinase/stress-activated protein kinase or p38 MAPK family.
Members of the MAPK family are active only when they are concomitantly phosphorylated on both tyrosine and threonine residues (29). While only tyrosine phosphorylation was measured in this report, the finding that MAPK activity was increased by OA or VA strongly suggests that the threonine residue was also phosphorylated. Nishida et al. (30) have demonstrated that MAPK activation from the ligand-receptor interaction involves Ras, Raf, and MAPK kinase with subsequent activation of MAPK. We have found that OA- or VA-induced tyrosine phosphorylation of p44 correlates with increased MAPK activity. Moreover, herbimycin A, a tyrosine kinase inhibitor, caused a decrease in MAPK activity. Thus, MAPK activity is thought to be down-regulated by phosphotyrosine dephosphorylation. It is known that both the Thr-183 and Tyr-185 residues in Erk1/Erk2 are phosphorylated and activated by various stimulations (31-32). Phospho-Thr-183 of Erk1/Erk2 may be dephosphorylated by phosphatase 2A, leaving phospho-Tyr-185, which is dephosphorylated by an unknown phosphotyrosine phosphatase (33). One of the targets of OA and VA action might be the inhibition of the dephosphorylation of this Erk1/2. Alternatively, OA- or VA-induced alteration in IL-8 gene expression may be due to the phosphorylation of transcription factors and the subsequent alteration in their DNA binding properties. Some transcription factors (Elk-1, Sap-1) are good substrates for Erk1/Erk2 in vitro (34, 35). Identification of the substrates of MAPK in this cell system will provide important information concerning the mechanism of IL-8 production.
In this study, a close association of MAPK activity with IB-
degradation was observed, leading to NF-
B activation. NF-
B is
retained in an inactive form by being associated with its inhibitor, I
B, in the cytoplasm in most types of cells. Stimulation of cells with LPS or with proinflammatory cytokines such as IL-1
/
and TNF-
causes phosphorylation of I
B, and the subsequent release of
NF-
B from I
B permits translocation of NF-
B to the nucleus and
binds to the NF-
B binding site (36-38). However, protein kinases and proteases involved in this I
B degradation remain to be
investigated. Chen et al. (39) recently found that I
B-
was phosphorylated by a I
B kinase complex containing ubiquitin. On
the other hand, several groups reported that protein kinase C (40) and
double-stranded RNA-dependent protein kinase phosphorylate
I
B-
in vitro (41). Furthermore, Kuno et al.
(17) identified a novel 42-kDa I
B-
-kinase, which is distinct from
MAPK. We found that Erk1 phosphorylated a GST-I
B-
protein
in vitro. From these studies, it is probable that there
exist several pathways following different stimuli leading to the
phosphorylation of I
B-
.
Induction of IL-8 mRNA expression by OA or VA and the complete
inhibition of mRNA synthesis by actinomycin D show that the regulation of IL-8 by OA or VA in HL-60 is primarily transcriptional. Furthermore, OA and VA increased the half-life of IL-8 mRNA. It has
been reported that a 32-kDa protein has been identified that binds to
the AU-rich 3-untranslated region of short-lived cytokine genes such
as TNF-
(42). Whether this protein is activated or induced by OA or
VA and thereby selectively modulates mRNA stability remains to be
determined. Inasmuch as OA and VA inhibit phosphoserine/phoshothreonine
phosphatase and phosphotyrosine phosphatase, it is conceivable that OA
and VA thus may prolong the half-life of phosphorylated proteins (MAPK
and others) with regard to their signal transduction, leading to
sustained IL-8 gene activation. Further experiments will be directed
toward identifying the kinases that are involved in the induction of
IL-8 gene activation.
We are grateful to Chieko Inaba, Chie
Satokawa, Yuko Haga, and Yuki Tanabe for technical assistance. We
express great thanks to Dr. Nancy Rice, for providing anti-IB
Ab,
and to Dr. Howard A. Young (NCI-Frederick Research and Development
Center) for critical reading of the manuscript.