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*
Division of Allergy and Clinical Immunology, and
Division of Nephrology, Departments of Medicine and Immunology, University of Colorado Health Sciences Center, Denver, CO 80262
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1, JNK2
1, and JNK22. The p54 JNK that is
phosphorylated in LPS-stimulated, PMA-differentiated THP-1 cells is
most likely JNK22 because 97% of the p54 JNK-encoding messages code
for JNK22. By analogous reasoning, the p46 JNKs that are not heavily
phosphorylated, but account for approximately half of the N-terminal
c-jun kinase enzymatic activity, are most likely either JNK11 or
JNK21 because they account for 98% of the messages that can code
for 46kDa JNKs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is greatly enhanced (1). Thus,
differentiated THP-1 cells serve as an excellent model system for
identification of pathways that are important in LPS-mediated gene
activation.
LPS, a major outer membrane component of Gram-negative bacteria, causes
tissue injury and shock by activation of monocytes and macrophages and
subsequent release of multiple proinflammatory cytokines such as IL-1,
IL-6, IL-8, and TNF-, arachidonic acid products such as
PGE2, PGF2, and
leukotriene C4, and increased expression of
adhesion molecules (2, 3, 4, 5). LPS bound to a plasma protein,
the LPS-binding protein, interacts with CD14, a GPI-linked surface
protein that transduces its signal by association with Toll-like
receptors (e.g., TLR2). Responses to LPS are characterized by
activation of multiple intracellular pathways, including
mitogen-activated protein kinases
(MAPKs)3 and NF-
B
(5, 6, 7).
Members of the MAPK family of protein kinases include the c-Jun
N-terminal kinases (JNKs), the p42 and p44 extracellular signal
receptor kinases (ERKs), and the p38 kinases (8, 9, 10). All
MAPKs are activated by phosphorylation of conserved threonine and
tyrosine residues that are present in distinct triads in kinase domain
VII: TPY for JNKs, TEY for ERKs, and TGY for p38 kinases (9, 11). The MAPKs are likely associated with their upstream
activators and their downstream targets via molecular scaffolds that
enhance transduction of signals from the plasma membrane to the nucleus
and may be further regulated by association with phosphatases
(12). The JNKs, ERKs, and p38 kinases influence gene
transcription based on their ability to phosphorylate and activate
multiple transcription factors including c-Jun, JunD, ATF-2,
Sap-1a, and Elk-1 (8, 10, 12, 13, 14, 15, 16, 17, 18, 19). In combination with
each other and with other transcription factors (e.g., NF-B), these
proteins bind to critical regions in the promoters of genes that are
important for cell growth and for inflammatory responses (11, 14).
Ten separate JNK isoforms have been identified as the products of three
genes with two alternative splice sites in jnk1 and jnk2 and one splice
site in jnk3 (see Ref. 13). The first splice site, which
is present in jnk1 and jnk2, determines whether the resulting protein
will be the or the isoform. The second splice site, which is
present in all three jnk genes, dictates whether the p46 or p54
isoforms are encoded. The smaller p46 JNKs are referred to as JNK11,
JNK11, JNK21, JNK21, and JNK31. The p54 JNK proteins are
referred to as JNK12, JNK12, JNK22, JNK22, and JNK32.
Messenger RNA for JNK1 and 2 is expressed in all tissues, whereas JNK3
is reported to be predominately found in the brain and testis
(20). Although JNK1 and JNK2 are ubiquitously expressed,
the relative expression of the different splice variants has not been
reported. Although the JNK isoforms may share functions in some
settings, there is experimental evidence that the JNK isoforms are not
redundant. For example, JNK21 and JNK22 bind c-jun 5–10 times
more strongly than do JNK11 and JNK12, suggesting that these
isoforms may target different groups of substrates in vivo
(10). Furthermore, JNK1 but not JNK2 rescues the HOG1
osmotic stress pathways in yeast (21), an inhibitory
mutant of JNK1 but not of JNK2 reduces UV radiation-induced apoptosis
in small cell lung cancer cells (22), and expression of an
inhibitory mutant of JNK2 but not JNK1 sensitizes renal epithelial
cells to hypertonic stress (23). Antisense JNK2 inhibits
epidermal growth factor-induced growth of A549 lung carcinoma cells
(24). There is preferential activation of JNK22 and
JNK32 in PC-12 cells by UV radiation (25), and there is
preferential activation of a p46 isoform of JNK in murine macrophages
activated by TNF- (26, 27). The p38 inhibitor SB203580
has differential effects on JNK isoforms, inhibiting the but not
the splice variants of JNK2 at 2–10 µM (28, 29).
Finally, jnk1 and jnk2 knockout mice have distinct defects in T cell
function (30, 31, 32). CD4 T cells from jnk1 knockout mice
have markedly reduced JNK activity and preferentially differentiate
into Th2 cells with increased accumulation of Th2 cytokines
(30). Jnk2 knockout mice have reduced JNK activity (after
anti-CD3) in CD4+ Th1 cells and normal JNK
activity in Th2 cells (31).
Given the possibility that JNK isoforms have specific roles, we have examined the isoforms of jun kinases in THP-1 cells and find evidence of unexpected restriction in both their expression and activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Soluble and agarose-linked rabbit polyclonal Abs to p46 JNK1 and JNK3 (JNK1/3; C-17) and to p54 JNK2 (N-18 and D-2) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-JNK was from New England Biolabs (Beverly, MA). HRP-labeled goat anti-rabbit IgG was purchased from Bio-Rad (Hercules, CA). A bacterially expression construct encoding the fusion protein GST c-jun1–79 was a gift from Gary L. Johnson (University of Colorado Health Sciences Center, Denver, CO) and was used to produce GST-c-jun1–79 immobilized on reduced glutathione-agarose beads (22). LPS derived from Eschericia coli 055:B5 (Difco, Detroit, MI) was stored at -20°C at 1 mg/ml and was vortexed vigorously before use. Nonidet P-40 was obtained as a 10% solution from Pierce (Rockford, IL). PMA and most other reagents were purchased from Sigma (St. Louis, MO).
Cells
THP-1 cells were obtained from the American Type Culture Collection (Manassas, VA) and grown according to their instructions. The cells were plated at 1–2 x 105/ml in LPS-free media and allowed to grow for 2–3 days in tissue culture dishes before differentiation with 10-8 M PMA for 42–48 h when >99% of the cells were adherent. Cells treated with DMSO alone did not differentiate. After exposure to PMA, cells were treated with LPS at 37°C at doses and times noted. All further steps were performed on ice or in the cold room unless otherwise noted.
Lysis of cells
Adherent cells (
3 x 106 in a 10
cm2 dish) were washed twice with ice-cold PBS and
lysed. All steps were performed at 4°C. Cells were lysed in either
JNK lysis buffer (25 mM HEPES, 20 mM -glycerophosphate, 300 mM NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.1%
Triton X-100, 1 mM PMSF, 4 µg/ml aprotinin, 2 µg/ml leupeptin, and
0.1 mM Na3VO4; Ref.
22) or a modified RIPA buffer (1x PBS, 1% Nonidet P-40,
0.1% SDS, 0.25% sodium deoxycholate, 1 mM PMSF, 6 µg/ml aprotinin,
2 mM Na3VO4). The modified
lysis buffer was necessary to obtain effective immunoprecipitation with
preservation of kinase activity. Lysates were passed through a 22-gauge
needle three times, clarified by centrifugation, and assayed for
protein content (Pierce).
Clearing by immunoprecipitation
Each lysate was exposed to specific Abs linked to agarose beads in the modified RIPA buffer for 2 h at 4°C with rocking. Immunoprecipitates were removed by brief centrifugation and the resulting "cleared" supernatants analyzed as described in the text.
In vitro kinase assays
Protein (50 µg) in 500 µl of JNK lysis buffer was incubated
with 20 µl of a 1:1 slurry of GST-c-jun1–79
agarose beads for 2 h. The beads were washed and transferred to
JNK assay buffer containing [
-32P]ATP, and
JNK activity was determined (33). For the experiments
shown in Fig. 3
, A and B,
32P incorporated into
GST-c-jun1–79 was quantitated with a
phosphoimager and ImageQuant software (Molecular Dynamics,
Sunnyvale, CA).
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Lysates containing 50–100 µg of protein (as noted) were boiled in sample buffer for 5 min, run on 12% SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were blocked with 5% blotto in TBS as recommended by NEB, probed with Abs as noted, and developed with ECL (Santa Cruz Biotechnology). Quantitation of exposed photographic film was performed with Nucleotech GelExpert software (Nucleotech, San Mateo, CA). Films chosen for quantitation were relatively underexposed to keep the bands within the relatively narrow dynamic range of the ECL method.
Generation of cDNA standards for the JNK isoforms
The cDNAs for human JNK11, JNK12, JNK11, JNK12,
JNK21, JNK22, JNK21, and JNK22 isoforms were cloned using
primers based on published sequences or constructed in the expression
vector, LNCX, using procedures described previously (25).
cDNAs for rat JNK31 and JNK32 were as described previously
(25). Compared with human JNK3s, rat JNK3s are 89%
homologous in the splice regions chosen for the primer pairs and 100%
homologous in the region chosen for the TaqMan probe. The cloned cDNAs
were checked by sequencing.
RT-PCR
Quantitative, fluorescent PCR was performed using the TaqMan
system (ABI 7700; PE Applied Biosystems, Foster City, CA). Sequences
for human JNKs were obtained from GenBank and aligned with MacDNAISIS
Pro version 3.5 (Hitachi Software Engineering, South San Francisco,
CA). We chose forward primers and reverse primers to overlay the
and splice sites for jnk1 and jnk2 and to overlay the splice
site and a conserved 3' untranslated region for jnk3. TaqMan probes
were chosen to be used with these primers with Primer Express version.
1.0 (PE Applied Biosystems). Forward and reverse primers were made by
Operon (Alameda, CA), whereas TaqMan probes were made by PE Applied
Biosystems. The primers and probes used in these studies are shown
(Table I).
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PCRs for the individual JNKs and for ribosomal RNA were performed in
duplicate in 25 µl of total reaction volumes with 175 nM TaqMan
probe, 1.25 U AmpliTaq gold (PE Applied Biosystems), 1x TaqMan PCR
buffer (PE Applied Biosystems), 200 µM dNTP, plus forward primers,
reverse primers, and variable amounts of magnesium optimized for each
primer set (Table II). Primers and probes
for ribosomal RNA, purchased from PE Applied Biosystems, served as an
internal control. Thermal cycling was performed with 10-min
denaturation at 95°C, followed by 50 cycles of 95°C for 30 s,
60°C for 20 s, then 72°C for 1 min in the ABI Prism 7700
detection system (PE Applied Biosystems).
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1 and JNK32.
Qualitative PCR was performed by using the same conditions but without
the TaqMan probe. Statistics
Data were analyzed with unpaired, two-tailed t tests using the statistical software supplied with GraphPad Prism, version 2.0b (GraphPad, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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THP-1 cells, after differentiation with PMA, produce large amounts
of TNF- after treatment with LPS (1). We were
interested in discovering if these cells were a useful system in which
to study activation of the JNK pathway and found that the level of the
total JNK activity in response to LPS is dramatically enhanced after
differentiation with PMA (Fig. 1).
Furthermore, there is a shift in the dose response toward lower doses
of LPS for maximal activation and in the time course toward earlier
times. These observations demonstrate that differentiated THP-1 cells
are a useful model to explore the expression and activation of JNK
proteins in human monocyte/macrophages.
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We next asked whether there is increased ability of LPS to cause
phosphorylation of JNKs in the differentiated vs the undifferentiated
cells. Although it is known that PMA up-regulates CD14 expression in
these cells, we also wanted to know if there are changes in the
expression of p46 and/or p54 JNKs after differentiation.
Undifferentiated and differentiated THP-1 cells were triggered with LPS
and lysates and were analyzed on Western blots with Abs to JNK1/3,
JNK2, and phosphorylated JNK (Fig. 2).
Treatment of differentiated THP-1 cells with LPS leads to
phosphorylation of a dominant JNK polypeptide at 54 kDa (pp54) and
several more faintly phosphorylated polypeptides in the 46-kDa (pp46)
range (Fig. 2, upper panel). This phosphorylation is
dramatically more than that seen in undifferentiated cells and is
compatible with the increase in JNK activity (see Fig. 1
). A similar
preponderance of pp54 over pp46 was seen with the murine monoclonal
anti-phospho-JNK Ab from Santa Cruz Biotechnology (data not shown).
These blots were stripped and reprobed with Abs to JNK1/3 (C-17) and to
JNK2 (N-18; Fig. 2, lower panel) revealing the
presence of p46 JNK1/3 and p54JNK2. With longer exposure, a p54JNK1/3
band and a p46 JNK2 band can be seen (not shown in this figure). The
p54 phospho-JNK band aligned with the p54 JNK2 band and not with the
p54JNK1/3 band (data not shown). Of note, with differentiation, there
is no increase in the mass of either p46 JNK1/3, p54 JNK1/3, p46 JNK2,
or p54 JNK2. However, there is loss of a p46 JNK1/3 band (Fig. 2, lower panel, lanes 1 and 2).
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The anti-JNK1/3 (C-17) used in this analysis is reported to
predominantly recognize p46 isoforms of JNK1 and JNK3, whereas the
anti-JNK2 Abs (N-18 and D-2) predominantly recognizes p54 JNK2
(35). To demonstrate that these Abs recognize the
requisite family of JNK proteins, lysates from differentiated THP-1
cells stimulated with LPS were subjected to clearing by
immunoprecipitation with either anti-JNK1/3 (C-17)-agarose or with
anti-JNK2 (D-2)-agarose, and the resultant supernatants were probed
for JNK1/3 and JNK2 (Fig. 3, A
and B, top two panels). To analyze the results of
these experiments, the density of bands on lightly exposed ECL-detected
blots were measured by semiquantitative scanning. As can be seen, in
the top two panels of Fig. 3B, the C-17 anti-JNK1/3 Ab
removed 99% of the predominant p46 band and a less prominent p54 band
that are recognized by the C-17 Ab (p < 0.001)
and 20% the p54 band that is recognized by the N-18 anti-JNK2 Ab
(not statistically significant). The D-2 anti-JNK2 Ab also was
effective, removing 70% of the predominant p54 band that is recognized
by the N-18 Ab (p < 0.002) but was less
specific, removing 50% the p46 and p54 bands recognized by the
anti-JNK1/3 Ab (p = 0.02). Of note, the
anti-JNK2 (D-2) Ab was more efficient in clearing the JNK2 proteins
from lysates of cells that had not been activated with LPS (data not
shown).
Anti-phospho-JNK Western blots are not significantly changed after clearing of JNK isoforms by immunoprecipitation.
The same cleared lysates used in the Western blots in Fig. 3A were probed in parallel for reactivity with
anti-phospho JNK. As can be seen in Fig. 3A and in the
summary data from three to four separate experiments in Fig. 3B, there is little diminution in the pJNK signal after
clearing of JNK isoforms by immunoprecipitation, even with both Abs.
None of these changes were statistically significant even though
significant amounts of JNK1/3 and JNK2 were consistently removed.
JNK1 proteins account for approximately half of the JNK activity
The increased phosphorylation of the p54 JNK proteins compared
with the p46 proteins and the observation that the phospho-JNK band
aligned best with the p54 JNK2 band suggested that there may be a
disproportionate contribution of JNK2 to the JNK enzymatic activity. To
address this question, lysates from the LPS-treated cells described
above were assayed for c-jun N-terminal kinase activity. Compared with
the uncleared lysates, the JNK enzymatic activity was reduced by 46%
(p < 0.01) by removing the 99% of the JNK1/3
isoforms (p < 0.001) and further depleted
after clearing by immunoprecipitation with both Abs (72%,
p < 0.01; Fig. 3, A and B,
lower panels). The ability of the D-2
Ab alone to clear N-terminal c-jun kinase activity was variable in our
four experiments and did not achieve statistical significance.
RT-PCR of JNK isoforms
The precise nature of the proteins detected as p46 and p54 JNKs is
unknown, as both the p46 and p54 bands, based on predicted protein size
derived from the primary amino acid sequences, could each contain
proteins of up to five different JNK isoforms. With the limitation that
expression of mRNA does not always correlate with expression of
protein, we felt that analysis of mRNA for each of the JNKs may shed
some light on the nature of the JNK isoforms expressed in
differentiated THP-1 cells. To do this, we elected to examine, first
qualitatively and then quantitatively, the levels of mRNA for each of
the JNK isoforms in differentiated THP-1 cells. Control data that
demonstrate our ability under the conditions used for quantitative
(TaqMan) PCR to amplify cDNA for each JNK isoform are shown (Fig. 4). This figure shows each primer/probe
combination as listed in Table I under conditions listed in Table II to
amplify bona fide message and to not amplify cDNA for the two other JNK
isoforms most likely to show cross-reactivity. Additional experiments
demonstrated that there was no cross-reactivity with the other JNK
isoforms with less similar nucleotide sequences (data not shown). The
only significant cross-reactivity is the amplification of JNK21 by
the JNK22 probes. Although this is a concern in qualitative RT-PCR,
it is easily handled in the quantitative approach (see below).
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1, JNK11, JNK21, and JNK22 (Fig. 5). Notably, only one of the five
potential p54 JNKs, JNK22, is detected. There is little or no mRNA
for JNK12, JNK12, JNK21, JNK22, JNK32, or JNK32,
although these species are readily amplified from bona fide cDNAs.
Therefore, it is likely that the predominant phosphorylated p54 JNK
(Fig. 2) is JNK22. Furthermore, amplified DNA is easily detected for
three of the five potential p46 JNKs: JNK11, JNK11, and
JNK21.
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1 and JNK22,
the two isoforms that were the most difficult to resolve are shown
(Fig. 6). Copy number for JNK 21 can
be measured without interference from JNK22, whereas measurement of
JNK22 requires a correction for JNK21 that is incorrectly
detected by the JNK22 primer/probe combination (print over of
40%). This was determined from data such as that shown in Fig. 6, where the primers and probe for JNK22 are able to detect JNK 21
sequences (Fig. 6B). Therefore, the number of copies of
JNK22 needs to be reduced by 37% of the number of copies of
JNK21, determined independently with the JNK21 primers and probe
that do not detect JNK22 (Fig. 6A).
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). These values are proportionate and
not absolute because reverse transcription is not 100% efficient.
Surprisingly, only 3 (JNK11, JNK21, and JNK22) of 10 possible
isoforms account for 98% of the JNK message in differentiated THP-1
cells and only one isoform, JNK22, accounts for 97% of the message
for the p54 JNKs. Although JNK11 is seen on qualitative RT-PCR (Fig. 5), it accounts for only 0.6% of the total JNK mRNA (Table III). An
analysis of JNK expression in undifferentiated cells reveals similar
data except that there is more expression of JNK11 (3.6% of the p46
JNKs and 6.3% of the JNK1 p46 message, data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). We next proceeded to examine the
nature of the JNK polypeptides at protein, phosphoprotein, and activity
levels and then attempted to further understand the contributions of
various isoforms by analyzing the expression of these isoforms at the
mRNA level.
The anti-phospho-JNK Abs used in these studies are predicted to
recognize equally the phosphorylated versions of p46 and p54 JNK in
that there is 100% homology among the JNKs in the region of the
critical phosphorylated threonine and tyrosine (13, 27).
Therefore, these Abs are able to identify phosphorylated p46 and p54
JNKs but cannot distinguish among the JNK1, JNK2, and JNK3 proteins. We
found that after activation of either the undifferentiated or the
differentiated cells with LPS, there is predominant phosphorylation of
a p54 JNK band and that this is dramatically increased in the
differentiated cells (Fig. 2). This mirrors the changes in
LPS-activated total JNK activity (Fig. 1). This increased
phophorylation of JNKs and increased JNK activity is likely
attributable, at least in part, to up-regulation of CD14 (Ref.
1) and our data, not shown) and is not attributable to
increased expression of JNK proteins (Fig. 2). Other possibilities that
have not been addressed include increased expression of MAPK kinase
(MKK) 4 and/or MKK7 and increased expression of MKK kinases or
scaffolding proteins that could integrate signaling by LPS.
Distinction among the jun kinase isoforms within cells and in cellular
extracts is both confusing and inexact at the present time. Part of the
confusion arises from the initial descriptions of JNK1 as a 46-kDa
protein and JNK2 as a 54-kDa protein. The appreciation that there are
p54 JNK1s as well as p46 JNK2s arose from molecular cloning that
occurred after the commercially available Abs used in these studies
were described. Thus, the anti-JNK1/3 (C-17) Ab from Santa Cruz
Biotechnology is said to identify predominantly p46 JNK1 and p46 JNK3
and the anti-JNK2 Abs (D-2 and N-18) are said to identify
predominantly p54 JNK2. In our hands, there is some cross-recognition
in immunoprecipitation experiments of JNK1/3 by the anti-JNK2 Abs
(D-2) and of JNK2 by the anti-JNK1/3 Ab (C-17; Fig. 3). On Western
blots, the anti-JNK1/3 Ab (C-17) recognizes a p54 kDa band that
migrates slightly slower than the JNK2 p54 band recognized by the N-18
anti-JNK2 Ab. This could be JNK12, JNK12, or JNK32.
Because mRNA for JNK32 is detected but not for JNK12 or JNK12,
we conclude that this is most likely JNK32. In a similar fashion,
the anti-JNK2 Ab (N-18) recognizes a faint p46 band that migrates
slightly faster than the p46 JNK1 band recognized by the C-17
anti-JNK1/3 Ab. This could be JNK21 or JNK21. Because there
are many more copies of JNk21 than for JNK21, we conclude this is
most likely JNK21.
We have attempted to identify the isoform(s) responsible for the JNK
activity in LPS-stimulated, PMA-differentiated THP-1 cells. We first
examined the phosphorylated JNK bands by stripping a large-format,
phospho-JNK Western blot (Fig. 2) and reprobing it with anti-JNK1
or anti-JNK2. This reveals that the phosphorylated p54 band
comigrates with the p54JNK2 band and not the p54 band seen with the
anti-JNK1/3 Ab (data not shown). Unexpectedly, after clearing of
lysates with immunoprecipitation of JNK1/3 and/or JNK2, there is a poor
relationship between the level of phosphorylated p54 JNK seen on
anti-phospho-JNK Western blot and the JNK activity (Fig. 3).
Possible explanations for this observation include lack of linearity of
the anti-phospho-JNK immunoblot, lack of equivalent binding of the
Ab with different phospho-JNKs, and existence of a JNK isoform that is
not reactive with either the anti-JNK1/3 or anti-JNK2 used in
this study.
However, although it is generally accepted that the level of
phosphorylation of JNKs correlates with their activity, a lack of
concordance has been reported previously in murine macrophages
activated with TNF-. In these cells, there was near equal
phosphorylation of p46 and p54 JNKs after treatment with TNF-, but
the kinase activity was predominantly associated with the p46 JNKs
(27). This is compatible with our findings that the p46
JNKs are less strongly phosphorylated than are the p54 JNKs, but the
JNK1 species, which are predominantly p46, account for 50% of the
JNK enzymatic activity (Fig. 3).
Delineation of the individual JNK isoforms at the level of the first
splice site ( vs ) is inexact because there currently are no
reagents available to distinguish the from the isoforms at the
protein level. To approach this problem, we used RT-PCR in an attempt
to make some reasonable statements regarding the possible components of
the p46 and p54 JNK species. The data in Fig. 4 demonstrate that the
primer pairs and probes we chose, with the exception of the JNK 22
reagents, had the needed specificity. We first performed qualitative
RT-PCR and found that only 4 of the possible 10 isoforms are expressed
(Fig. 5). Then we performed real-time, quantitative PCR with the TaqMan
technique. Although our analysis includes numbers of copies for each
isoform, there was significant variability among our five experiments,
so we chose to focus on the relative amounts of each isoform. Analysis
of mRNA by real-time, RT-PCR reveals that JNK22 mRNA is the
predominant (97%) of the p54 isoform, with JNK22 the next
most-prevalent p54 isoform, representing 1.5% (Table III). The only
p54 mRNA for that encodes for a protein that could be recognized by the
C-17 anti-JNK1/3 Ab is JNK32 that represents 1% of the total
p54 message. These data are compatible with the Western blot data (Fig. 2 and Fig. 3A) where the JNK1/3 p54 band is much more faint
than the JNK1/3 p46 band.
Regarding the p46 species, the data from real-time RT-PCR are
compatible with the presence of two predominant species, JNK11 and
JNK21. It is unknown whether the faintness of the p46 JNK2 band seen
on the anti-JNK2 Western (Fig. 3) is attributable to lack of
JNK21 protein (translational regulation) or to lower affinity of the
Ab for p46 JNK2 compared with p54 JNK2. Finally, the p46 species that
is seen in the undifferentiated cells but not after differentiation
(Fig. 2, lower panel), is likely JNK11 because its
expression decreases with differentiation (TaqMan RT PCR, data not
shown).
In summary, we have demonstrated that the THP-1 cell line, after
differentiation with PMA, is an excellent model system for studying the
LPS-mediated activation of the JNK pathway in human
monocyte/macrophages. We have determined that whereas both p46 and p54
polypeptides are phosphorylated after treatment of these cells with
LPS, only selected isoforms are present and activated. Based on the
RT-PCR data, it is likely that the p54 JNK molecules are predominantly
JNK22 and the p46 molecules are predominantly JNK11 and JNK21.
This is particularly interesting because these isoforms are the ones
reported to bind c-jun most strongly (13). Finally, we
have shown that p46 JNK1, although not strongly phosphorylated as
defined with the anti-phospho-JNK Ab, accounts for 50% of the
LPS-stimulated c-jun N-terminal kinase activity in these cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephen C. Dreskin, University of Colorado Health Sciences Center, Denver, CO 80262.
3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; JNK, c-jun N-terminal kinase; ERK, extracellular receptor kinase; MKK, MAPK kinase.
Received for publication February 15, 2000. Accepted for publication February 16, 2001.
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(TNF-) translation: glucocorticoids inhibit TNF- translation by blocking JNK/SAPK. Mol. Cell Biol. 17:6274.[Abstract]
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. Proc. Natl. Acad. Sci. USA 94:13169.This article has been cited by other articles:
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C. Lecaroz, M. J. Blanco-Prieto, M. A. Burrell, and C. Gamazo Intracellular killing of Brucella melitensis in human macrophages with microsphere-encapsulated gentamicin J. Antimicrob. Chemother., September 1, 2006; 58(3): 549 - 556. [Abstract] [Full Text] [PDF]
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S. Tsuboi A Complex of Wiskott-Aldrich Syndrome Protein with Mammalian Verprolins Plays an Important Role in Monocyte Chemotaxis. J. Immunol., June 1, 2006; 176(11): 6576 - 6585. [Abstract] [Full Text] [PDF]
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S. R. Himes, D. P. Sester, T. Ravasi, S. L. Cronau, T. Sasmono, and D. A. Hume The JNK Are Important for Development and Survival of Macrophages J. Immunol., February 15, 2006; 176(4): 2219 - 2228. [Abstract] [Full Text] [PDF]
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 H. Shiratsuchi and M. D. Basson Activation of p38 MAPK{alpha} by extracellular pressure mediates the stimulation of macrophage phagocytosis by pressure Am J Physiol Cell Physiol, May 1, 2005; 288(5): C1083 - C1093. [Abstract] [Full Text] [PDF]
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H. Shiratsuchi and M. D. Basson Extracellular pressure stimulates macrophage phagocytosis by inhibiting a pathway involving FAK and ERK Am J Physiol Cell Physiol, June 1, 2004; 286(6): C1358 - C1366. [Abstract] [Full Text] [PDF]
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M. Utsugi, K. Dobashi, T. Ishizuka, K. Endou, J. Hamuro, Y. Murata, T. Nakazawa, and M. Mori c-Jun N-Terminal Kinase Negatively Regulates Lipopolysaccharide-Induced IL-12 Production in Human Macrophages: Role of Mitogen-Activated Protein Kinase in Glutathione Redox Regulation of IL-12 Production J. Immunol., July 15, 2003; 171(2): 628 - 635. [Abstract] [Full Text] [PDF]
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 B. Su, P. J. M. Ceponis, S. Lebel, H. Huynh, and P. M. Sherman Helicobacter pylori Activates Toll-Like Receptor 4 Expression in Gastrointestinal Epithelial Cells Infect. Immun., June 1, 2003; 71(6): 3496 - 3502. [Abstract] [Full Text] [PDF]
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 V. Levresse, L. Marek, D. Blumberg, and L. E. Heasley Regulation of Platinum-Compound Cytotoxicity by the c-Jun N-Terminal Kinase and c-Jun Signaling Pathway in Small-Cell Lung Cancer Cells Mol. Pharmacol., September 1, 2002; 62(3): 689 - 697. [Abstract] [Full Text] [PDF]
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Y. C. Xu, R. F. Wu, Y. Gu, Y.-S. Yang, M.-C. Yang, F. E. Nwariaku, and L. S. Terada Involvement of TRAF4 in Oxidative Activation of c-Jun N-terminal Kinase J. Biol. Chem., July 26, 2002; 277(31): 28051 - 28057. [Abstract] [Full Text] [PDF]
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, JNK2
, JNK2