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, ß, 
, and 
in Inflammatory Cell Lineages
Department of Biology, Amgen, Boulder, CO 80301
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß, 
, 
)
have been described. To understand the role of p38 family members in
inflammation, we determined their relative expression in cells that
participate in the inflammatory process. Expression was measured at the
level of mRNA by reverse-transcriptase PCR and protein by Western blot
analysis. p38 was the dominant form of p38 in monocytes; expression
of p38 was low and p38ß was undetected. In macrophages, p38 and
p38 were abundant, but p38ß was undetected. p38 and p38 were
also expressed by neutrophils, CD4+ T cells, and
endothelial cells. Again, p38ß was not detected in neutrophils,
although low amounts were present in CD4+ T cells. In
contrast, p38ß was abundant in endothelial cells. p38 protein was
not detected in any cell type, although p38 mRNA was present in
endothelial cells. Immunokinase assays showed a strong activation of
p38 and a lesser activation of p38 in LPS-stimulated macrophages.
Abs specific for mono- and dual-phophorylated forms of p38 suggested
that LPS induces dual phosphorylation of p38, but primarily
mono-phosphorylation of p38. IL-1ß activated p38 and p38ß in
endothelial cells. However, p38 was the more activated form based on
kinase assays and phosphorylation analysis. Expression and activation
patterns of p38 in macrophages and endothelial cells suggest that
p38 plays a major role in the inflammatory response. Additional
studies will be needed to define the contribution of p38 to
macrophage, neutrophil, and T cell functions, and of p38ß to
signaling in endothelial cells and T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four genes encode the known members of the p38 family, p38 (1),
p38ß (4, 5), p38 (6, 7), and p38 (8, 9). In addition to high
homology, the family is defined by a TGY motif within kinase subdomain
seven. Dual phosphorylation of this domain in p38, ß, , and
is catalyzed by the kinase, MKK6, and is required for p38 activation
(10, 11, 12). With the exception of p38ß, each p38 may also be activated
by MKK3 (10, 11, 12). p38, ß, , and each phosphorylate
activating transcription factor 2 (ATF2) (4, 5, 6, 7, 8, 9, 10, 11, 12). Even so, distinct
functions for the kinases are suggested by the ability of p38 and
p38ß, but not p38 and p38, to phosphorylate MAPK-activated
protein kinase-2 (MAPKAPK2) (8, 10, 12). Conversely, p38 and p38,
but not p38 and p38ß, favor stathmin as a substrate (13), and an
unidentified 12-kDa protein in muscle is a selective substrate for
p38 (7).
The widely used pyridinyl imidazole inhibitors of p38, SB203580 and
SB202190, are nearly equipotent against p38 and p38ß, but do not
inhibit p38 or p38 (4, 5, 8, 9, 12). These and similar compounds
reduce the severity of murine collagen-induced arthritis and rat
adjuvant arthritis, as well as hyperangiogenesis associated with murine
air pouch granuloma (2, 3). The antiinflammatory effects can be
attributed, in part, to the ability of the inhibitors to suppress
monocyte/macrophage production of TNF-, IL-1ß, and other cytokines
(14, 15, 17). Numerous other antiinflammatory activities have been
elucidated, including suppression of IL-1-induced prostaglandin H
synthase-2 expression by endothelial cells (18), FMLP-induced
neutrophil chemotaxis (19), and IL-2- and IL-7-induced lymphocyte
proliferation (20).
Because the available compounds inhibit both p38 and p38ß, it has
remained unclear which antiinflammatory activities can be attributed to
p38 and which can be attributed to p38ß. It is also possible that
the function of the two kinases may be redundant in some cells.
Furthermore, although the compounds do not block p38 or p38,
similarities in the activation of p38 family members suggest roles for
p38 and p38 in inflammation. Specifically, MKK6 activates each
p38, and when overexpressed by cell transfection, each p38 can be
activated in cells treated with IL-1ß and TNF- (10, 11, 12). However,
the contributions of p38 and p38 to inflammatory processes are
largely unexplored.
The tissue distribution of mRNAs encoding each p38 family member has
been evaluated by Northern and dot blot analysis (4, 5, 6, 7, 8, 9). Only p38
exhibited a limited tissue distribution, with high expression of mRNA
in skeletal muscle. p38, p38ß, and p38 mRNAs are more widely
distributed. However, analysis of tissue mRNA does not yield
information on expression within specific cell lineages. Knowledge of
kinase expression within a cell type is essential for understanding the
function of a kinase, and the expression of the p38 family members
within cell lineages has been largely unexplored. Furthermore, the
activation of endogenous p38 family members by relevant environmental
stimuli has not yet received much attention.
To investigate the role(s) of p38 family members in inflammation, we
determined the expression profile of p38, ß, , and in
several cell lineages that participate in the inflammatory response.
Lineage- and differentiation-specific expression was observed.
Furthermore, differential activation of p38 members was observed in
endothelial cells and macrophages responding to inflammatory stimuli.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Escherichia coli K235 LPS was purchased from Sigma
(St. Louis, MO). Calyculin A and calcitriol were from Biomol (Plymouth
Meeting, PA). Cytokines were purchased from R&D Systems (Minneapolis,
MN). p38, ß, , and plasmids and recombinant protein
were prepared as described (10).
Isolation and culture of human cells
Neutrophils were isolated from blood of normal volunteers by
density centrifugation using 1-Step Polymorphs gradient (Accurate
Chemical, Westbury, NY). Monocytes were isolated by centrifugal
elutriation, as described (17), and were 
95% CD14 positive. To
prepare CD4+ T cells, blood mononuclear cells were isolated
by density-gradient centrifugation using Ficoll-Paque Plus (Pharmacia,
Uppsala, Sweden). Mononuclear cells were resuspended to 5 x
107/ml in ice-cold PBS containing 0.5% BSA, 5 mM EDTA, and
50 µl/ml anti-CD4 Dynabeads (Miltenyi Biotec, Auburn, CA). After
15 min, the cells were washed and resuspended to 1 ml, and
CD4+ T cells (>95% CD4+) were isolated using
a Miltenyi VS+ Positive Selection Column (Miltenyi Biotec).
Macrophages were derived by culture of adherent monocytes. For this, blood mononuclear cells were resuspended to 5 x 106/ml in serum-free RPMI 1640 medium, and 17 ml was dispensed into TC75 Falcon flasks (Becton Dickinson, Franklin Lakes, NJ). Following 2 h of culture (6% CO2, 37°C) to allow the monocytes to adhere, nonadherent cells were removed by washing, and the media were replaced with RPMI 1640 containing 5% FCS, 5% human AB serum (Gemini BioProducts, Calabasas, CA), 50 nM calcitriol, 2 mM glutamine, 100 U/ml penicillin-G, 100 µg/ml streptomycin, and 0.6 µg/ml fungizone. Cells were fed with fresh media every 2 days, and in vitro differentiated macrophages were used for experimentation on day 7.
Primary HUVEC and human coronary artery endothelial cells purchased from Clonetics (San Diego, CA) were cultured in TC75 Falcon flasks in endothelial cell growth medium (Clonetics). Cells were used at passage four or five.
Analysis of p38, ß, , and mRNA levels by reverse-
transcriptase PCR (RT-PCR)
Cells were lysed in RNAzol (Teltext, Friendswood, TX), and total
RNA was isolated according to the manufacturer’s instructions. RNA
integrity was verified by agarose gel electrophoresis and ethidium
bromide staining. Relative levels of p38, ß, , and mRNA
were determined using an RT-PCR approach based on the work of Holland
et al. (21) and described in detail in Protocol Part Number 402823 (PE
Applied Biosystems, Foster City, CA). Sample RNA (20 ng) was added to a
50 µl RT-PCR reaction mix (TaqMan PCR Core Reagent Kit; PE Applied
Biosystems) that included specific forward and reversed primers and a
specific probe sequence. The gene-specific primers provided templates
for the RT reaction (48° x 30 min). Next, AmpliTaq Gold polymerase
was heat activated (10 min x 95°C), and PCR was performed in
the same reaction mix. Probes were complementary to a region amplified
by the primer set and contained a 5'-reporter dye, 6-carboxyfluorescein
(FAM), which was quenched by the presence of a 3'-quencher dye,
6-carboxytetramethylrhodamine (TAMRA). Taq polymerase has a
5'-nucleolytic activity and digests probe that binds cDNA during
amplification. The released reporter dye fluoresces, and the
accumulation of dye was measured in real time using a ABI Prism 7700
Sequence Detection System. The data were analyzed using Sequence
Detector v1.6 software (PE Applied Biosystems).
Relative mRNA levels were calculated using a variant of the Comparative
CT Method (22). Using this technology, several PCR cycles
are required before the specific fluorescent signal emerges above
background fluorescence (see Fig. 2
for examples). The number of PCR
cycles (CT) required to generate a threshold of 0.03
fluorescence units was determined in triplicate for each test RNA
sample. During the exponential phase of PCR, cDNA is amplified by 1 +
AE per cycle, in which AE is the amplification efficiency. AE values
were calculated as described elsewhere (23), and ranged from 0.96 to 1
for p38, ß, , , and 18S rRNA. Thus, each cycle results in an
approximate doubling of cDNA, and so it can be estimated that a sample
yielding a CT of 10 has about 8 times the mRNA of a sample
with a CT of 13. More precisely, the amount of a mRNA
transcript in a sample is proportional to 1/(1 + AE)CT.
Relative 18S rRNA values were determined and used to normalize samples
for RNA content. 18S rRNA was selected because the proportion of total
RNA that is ribosomal RNA does not vary substantially between cell
types (24). Sample calculations are provided in Table I.
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, (5') AAC CTG TCT CCA GTG GGC TCT, (3') CGT AAC
CCC GTT TTT GTG TCA, (probe) 6-FAM-CGC CTA TGG CTC TGT GTG TGC TGC
TT-TAMRA; p38ß, (5') CAC CCA GCC CTG AGG TTC T, (3') AGA TGC TGC TCA
GGT CCT TCT, (probe) 6-FAM-ACA CGC CCG GAC ATA TAT CCA GTC CC-TAMRA;
p38, (5') CGC CTC CGG CTG AGT TT, (3') GCT TGC ATT GGT CAG GAT AGA,
(probe) 6-FAM-CAG AGC GAT GAG GCC AAG AAC TAC ATG AA-TAMRA; p38,
(5') TGC TCG GCC ATC GAC AA, (3') TGG CGA AGA TCT CGG ACT GA, (probe)
6-FAM-AAG GTG GCC ATC AAG AAG CCG AGC-TAMRA. Generation and purification of polyclonal Abs specific to p38 family members
Polyclonal antisera were prepared in rabbits to the following
peptides: p38 (amino acids (aa) 341–360), p38ß (aa 346–364),
p38 (aa 321–339), and p38 (aa 262–280). Peptides were
conjugated to keyhole limpet hemacyanin and injected with CFA. Rabbit
polyclonal antisera were also generated to full-length recombinant
p38 and to GST-flag-p38. For immunoprecipitation studies, IgG was
purified from antiserum using the ImmunoPure IgG (protein A)
Purification Kit (Pierce, Rockford, IL).
Western blot analysis
Freshly isolated monocytes, CD4+ T cells, and
neutrophils were resuspended at 5 x 107 cells/ml of
lysis buffer (50 mM Tris, pH 7.5, 50 mM KCl, 1 mM EDTA, 0.5% Nonidet
P-40, 1 mM Na3VO4, 50 nM calyculin A, 100 µM
TPCK (L-1-tosylamido-2-phenylethyl chloromethyl
ketone), 1 µg/ml pepstatin, and 1 mM PMSF). Confluent cultures
of macrophages, HUVEC, and human coronary artery endothelial cells in
TC75 flasks were washed twice with ice-cold PBS and resuspended with
scraping into 2 ml of lysis buffer. All cell types were allowed to lyse
5 min on ice, and the soluble portion of the lysates was obtained by
centrifugation 2 min x 14,000 g. Three volumes of
lysate were transferred to fresh tubes containing 1 vol of 4x Laemmli
buffer and heated to 95°C for 5 min. Portions of lysate were also
used to determine protein content using the Pierce BCA assay. Samples
(25 µg protein) were resolved using 10% SDS-PAGE (Novex, San Diego,
CA), and transferred to Immobilon-P membranes (Millipore,
Bedford, MA). Blots were blocked 1 h in wash buffer (20 mM
Tris-HCl, pH 7.5, 137 mM NaCl, and 0.1% Tween-20) containing 2.5%
gelatin and 5% milk protein. Blots were next incubated with primary
Ab/antiserum diluted into wash buffer containing 5% milk protein. Abs
included 0.5 µg/ml anti-p38 (C20) (Santa Cruz Biotechnology,
Santa Cruz, CA), 1/1000 dilutions of rabbit anti-dual-phospho-p38
(lot 5) or anti-mono-phospho-p38 (lot 2) (New England Biolabs,
Beverly, MA), or 1/2700 dilutions of antiserum raised against
p38ß, , or peptides. Blotting with anti-p38 (C20) or
anti-phospho-p38 (dual or mono) was performed for 2 h at room
temperature. Other primary Abs were blotted at 4°C overnight. Blots
were then washed and incubated with a 1/3000 dilution of goat
anti-rabbit horseradish peroxidase (Dako, Carpenteria, CA). Binding
of secondary Ab was detected using enhanced chemoluminescence (Pierce).
Positive controls for Western blot detection of p38 family members
included recombinant p38, flag-p38ß, p38, and GST-flag-p38
prepared in E. coli, as described previously (10). Molecular
weights of proteins were estimated based on log-linear plots using
SeaBlue protein standards (Novex).
Immune depletion assays
Macrophages and HUVEC lysates were prepared as described above
for Western blotting. A total of 100 µg lysate protein adjusted to
250 µl with lysis buffer was supplemented with 5 µg of purified
IgG. IgG included anti-full-length p38, anti-p38ß
(peptide), anti-full-length p38, and preimmune IgG. Lysates were
then rocked 6 h at 4°C. Immune complexes were bound by addition
of 30 µl of protein A-agarose (Calbiochem-Novabiochem, La Jolla, CA).
Lysates were rocked 2 h, and bound agarose was removed by
centrifugation (15 s x 14,000 x g). The cleared
supernatants were subjected to Western blot analysis, as described
above.
Immunokinase assays
p38 family members were immunodepleted from cell lysates, as
described above. The bound agarose was washed twice in lysis buffer and
twice in PBS and then resuspended to 50 µl in PBS adjusted to contain
10 mM MgCl2, 0.5 mM EGTA, 4 mM DTT, 2.5 µM cold ATP, 10
µCi [-32P]ATP, 6000 Ci/mmol (Amersham), and 2 µg
of ATF2 (11-109). Reactions (28°C x 30 min) were terminated by
addition of 4x Laemmli load buffer and boiled 5 min. Samples were
resolved by 10% SDS-PAGE. The gels were dried, and phosphorylated ATF2
(11-109) was visualized by autoradiography and quantified using a
PhosphorImager.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our objective was to measure the expression of p38, ß, ,
and in cell types important to inflammation. For this purpose,
primer pairs were designed to measure the expression of each form of
p38 mRNA by RT-PCR. The primer pairs were first tested in PCR reactions
containing 1 ng of purified plasmids encoding p38, ß, , or
(Fig. 1). Each primer pair was shown to
be absolutely specific for its targeted p38 family member.
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, and peptides. An excellent
IgG against p38 was already available (Santa Cruz Biotechnology).
The anti-p38 IgG and p38ß, , and antisera were first
tested on Western blots containing 4 ng of recombinant p38, ß,
, and (Fig. 1
). Each antiserum/IgG was absolutely specific for
its target p38.
p38, ß, , and mRNA levels in inflammatory cell lineages
To measure relative p38, ß, , and mRNA expression, we
used a recently developed RT-PCR approach that utilizes the
5'-exonuclease activity of Taq polymerase (21). The
exonuclease activity cleaves a reporter dye from an oligonucleotide
probe that hybridizes to the amplified cDNA sequence (23). The release
of the dye is catalyzed coincident with cDNA amplification and can be
followed in real time by fluorometric measurement. Primers and PCR
conditions are optimized to yield amplification efficiencies
approaching 100%. For this reason, it is possible to determine the
relative expression of different mRNAs within the same sample. Fig. 2 illustrates the fluorescence generated
versus cycle number for RT-PCR reactions with p38, ß, , and
primers and probes and macrophage RNA. Expression of 18S rRNA was also
measured to normalize for RNA input. Using this technology, several PCR
cycles are required before the specific fluorescent signal emerges
above background fluorescence. The p38 amplification plot was the
first p38 plot to emerge above background and was closely followed by
p38. The p38ß and p38 amplification plots lagged behind the
p38 plot by >9 and >11 cycles, respectively. The data indicated
that p38 mRNA was the most abundant p38 mRNA in this macrophage
sample. p38 mRNA was about 30% as abundant as p38 mRNA, and
p38ß and p38 mRNAs were expressed at very low levels. Table I
shows the calculations used to convert amplification plots to relative
expression values.
Expression of p38, ß, , and mRNAs was next evaluated in RNA
from three to four independent isolates/cultures of monocytes,
macrophages, endothelial cells, CD4+ T cells, and
neutrophils (Table II). p38 mRNA was
by far the dominant form of p38 mRNA expressed by monocytes. p38
mRNA was undetectable, p38ß mRNA was negligible, and p38 mRNA was
expressed at 10% the level of p38 mRNA. In striking contrast to
monocytes, p38 as well as p38 mRNA was abundant in macrophages,
neutrophils, and CD4+ T cells. Neutrophils resembled
monocytes and macrophages in very low or absent expression of p38ß
and p38 mRNA. CD4+ T cells expressed somewhat higher
amounts of p38ß mRNA. p38 expression in endothelial cells was
markedly different from that found in leukocytes. Distinguishing
endothelial cells was the robust expression of p38ß mRNA and the
readily detectable expression of p38 mRNA. p38 and p38 mRNAs
were also present in endothelial cells. Expression of p38 family mRNAs
was similar in umbilical vein and coronary artery endothelial cells
(data not shown).
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Western blot analysis was used to investigate the expression of
p38 protein in the five cell types (Fig. 3). p38 protein levels (ng p38/µg cell
protein) were estimated by comparing lysates against dilutions of
recombinant p38 proteins (dilution series not shown). The correlation
between protein and mRNA expression was good. For example, p38 was
the dominant form of p38 protein in monocytes and was estimated at 0.5
ng/µg protein. p38 expression in monocytes was estimated at 0.05
ng/µg, and p38ß was undetectable (<0.015 ng/µg). The faint, low
m.w. bands observed when monocyte lysates were probed with p38ß
antiserum were nonspecific, i.e., these bands were still detected when
blots were probed with p38ß antiserum in the presence of excess
ß-peptide (not shown). In macrophages, p38 and p38 were both
expressed strongly, but, again, p38ß was undetectable. p38 was
abundant in neutrophils and CD4+ T cells with somewhat less
expression in endothelial cells. CD4+ T cells and
neutrophils also expressed p38. In neutrophils, p38 antiserum
detected a doublet. The minor (lower) band, but not the major (upper)
band, proved to be p38 based on specific competition with
peptide (not shown). p38ß expression was low in CD4+ T
cells and undetectable in neutrophils. In contrast to the leukocyte
lineages, p38ß protein was abundant (
0.3 ng/µg) in both
umbilical vein and coronary artery endothelial cells. p38 was also
detectable upon prolonged exposures in both types of endothelial cells.
Although p38 expressed in lysates of murine muscle was readily
detected using the p38 antiserum, p38 was undetectable (<0.005
ng/µg) in the five cell types (data not shown). These data contrasted
with the expression of p38 mRNA in endothelial cells.
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When overexpressed in transfected cells, each p38 family member
can be activated by TNF- or IL-1ß (12). Therefore, we investigated
the activation of endogenously expressed p38 family members in response
to inflammatory stimuli. For this, macrophages were stimulated with
LPS, TNF-, or GM-CSF for timed intervals, lysed, and subjected to
Western blot analysis using an Ab raised against p38 residues
171–186 in which Y182 was phosphorylated (Fig. 4). A 41- and a 43.5-kDa form of p38 was
phosphorylated following LPS and TNF- stimulation, but not following
GM-CSF stimulation. Similar blots were stripped and reprobed with an Ab
raised against p38 residues 171–186 in which both T180 and Y182 were
phosphorylated (Fig. 5). Remarkably, this
dual-phospho-p38 Ab recognized the 41-kDa and a very faint 44-kDa band,
but it did not detect the 43.5-kDa band that was detected using the
mono-phospho-p38 Ab. When the phosphatase inhibitor calyculin A was
included as stimuli, the 44-kDa phospho-p38 was enhanced markedly. The
reduced mobility exhibited by the 44-kDa band and the ability of
phosphatase inhibitor to increase its intensity were consistent with
the 43.5- and 44-kDa proteins representing mono- and
dual-phosphorylated proteins, respectively.
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and
p38, respectively (data not shown). The identity of the 41- and
43.5/44-kDa phosphoproteins was confirmed by selective immunodepletion
analysis. Ab raised against full-length p38 efficiently
immunodepleted all p38 and all 41-kDa phospho-p38 from macrophage
lysates (Fig. 6). Ab raised against
full-length p38 depleted most of the p38 present in the
macrophage lysates along with most of the 43.5-kDa phosphoprotein.
Similar experiments confirmed that the 44-kDa phosphoprotein induced in
the presence of calyculin A was p38 (data not shown).
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and p38 correlated with their activation. Macrophages were
treated with or without LPS for 10 min and lysed, and p38 and p38
were selectively immunoprecipitated from the lysates. The p38 bound in
the immunoprecipitates was used to phosphorylate ATF2 (Fig. 7). LPS induced p38 activity >12-fold
and p38 activity >8-fold above background. Induced p38 activity
was 3.5-fold higher than induced p38 activity.
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Activation of p38 family members in endothelial cells was of
particular interest due to the relative abundance of p38ß in this
cell lineage. HUVECs were treated with IL-1ß for timed intervals
and lysates were evaluated for phosphorylation of p38 members (Fig. 8A). A 41-kDa form of p38 was
phosphorylated in response to IL-1ß. Immunodepletion experiments
identified the phosphoprotein as p38 (Fig. 8B).
Anti-full-length p38 selectively eliminated p38 from the lysates
and removed all of the 41-kDa phosphoprotein. The p38ß peptide Ab and
the anti-full-length p38 Ab selectively eliminated p38ß and
p38, respectively, but did not reduce the major 41-kDa
phosphoprotein.
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, ß, and were next immunoprecipitated from lysates of
IL-1ß-stimulated HUVEC and analyzed by immunokinase assay using ATF2
as substrate. Fig. 9A confirms
a strong activation (>10-fold) of p38 by IL-1ß. However, p38ß
was also activated, although to a lesser extent than p38. When ATF2
was used as substrate, p38 was >6-fold more active than p38ß in
IL-1ß-stimulated HUVEC. The authenticity of p38ß activity was
confirmed. Neutralization of p38ß antiserum with excess ß peptide
reduced the kinase activity measured in anti-p38ß
immunoprecipitates to prestimulation levels (Fig. 9B).
Immunokinase assays did not detect activation of p38 following
IL-1ß treatment of HUVEC (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, but did not express p38ß or p38. Expression of p38 was
low, but detectable. However, the low expression of p38 was probably
overestimated since monocytes purified by elutriation contain 5%
with lymphocytes, and lymphocytes express high levels of p38. We
found that p38ß expression was under strong lineage-specific control.
With the exception of low expression of p38ß in CD4+ T
cells, the p38ß gene was essentially silent in leukocytes. In
contrast, p38ß was expressed strongly in endothelial cells. p38
was also silent in leukocytes, and although p38 mRNA was expressed
by endothelial cells, p38 protein was undetectable, consistent with
previous reports that p38 is a muscle-specific protein (6, 7).
Interestingly, p38 expression was regulated during cellular
differentiation within the myeloid lineage. As blood monocytes
differentiated into macrophages, a striking induction of p38 mRNA
and protein occurred so that p38 protein became at least as abundant
as p38.
When p38, ß, , and are overexpressed in transfected cells,
each p38 family member can be activated by cotransfecting cells with
MKK6 or by stimulating the cells with TNF- or IL-1ß (7, 8, 9, 10, 11, 12). These
data suggest that the activation of p38 family members may be regulated
coordinately. However, to date, only a few papers have dealt with the
activation of endogenous p38ß, , or (9, 25), and one report
showed the activation of p38ß to be independent of p38 activation
in a neuronal cell line (25).
Herein, immunokinase assays were used to demonstrate that both p38
and p38 are activated in macrophages stimulated with LPS. However,
p38 was activated to a greater extent, and phosphorylation analysis
suggested that p38 and p38 were regulated distinctly. Following
LPS activation of macrophages, p38 and p38 were both recognized
by Ab against p38 (171–186), in which only Y182 was phosphorylated.
Following LPS activation, p38 was also recognized by Ab against p38
(171–186), in which both T180 and Y182 were phosphorylated, whereas
p38 was poorly recognized by this Ab. Thus, p38 appeared to be
preferentially phosphorylated on tyrosine versus threonine in
LPS-activated macrophages. Differential phosphorylation of tyrosine may
reflect the bimolecular interactions known to occur between MKK and
MAPK level kinases. Phosphorylation occurs by an ordered addition of
phosphate first to tyrosine and then to threonine with MKK dissociation
from MAPK between each step (26). Our data suggest phosphorylation of
p38 T180 may be relatively inefficient in LPS-activated macrophages,
or, conversely, dephosphorylation of p38 T180 may be relatively
efficient. We speculate that p38 T180 represents a site of
regulation by stimuli that may prime macrophages for full activation of
p38. p38 activity was increased 25-fold in macrophages treated
with LPS and calyculin A versus LPS alone (C. Manthey, unpublished
results), underscoring the potential for signals that down-regulate
phosphatase activity to synergize with LPS for p38 activation.
We report herein that endothelial cells express comparable levels of
p38 and p38ß. Immunokinase assays were used to demonstrate that
both forms are activated in endothelial cells stimulated with IL-1ß.
However, p38 activity was >6-fold more than p38ß activity. When
lysates of IL-1ß-treated cells were evaluated by Western blot, the
major phospho-p38 was 41 kDa and was identified as p38 by
immunodepletion analysis. Upon prolonged exposures, a 42.5-kDa
phospho-p38 was also observed that comigrated with p38ß (C. Manthey,
unpublished). Attempts to identify this faint band by immunodepletion
were unsuccessful because it was no longer detectable in lysates after
immunodepletion even when Ab was omitted (presumably due to
dephosphorylation during the extended procedure).
By understanding the expression and activation patterns of p38 members,
insights into their functions are obtained. For example, the p38
inhibitors, SB202190 and SB203580, can block TNF- production by
monocytes by >95% (14, 15, and C. Manthey, unpublished data). These
compounds block p38 and p38ß with nearly equal potency, but do not
inhibit p38 or p38 (8, 9, 12). Thus, either p38 or p38ß
might mediate TNF- production. However, we show herein that p38
protein was expressed abundantly in leukocytes. In contrast, p38ß
protein expression was undetectable in monocytes, macrophages, and
neutrophils, and a highly sensitive RT-PCR approach confirmed that
p38ß mRNA was absent or present in extremely low amounts in these
cells. Thus, p38, but not p38ß, appears to contribute to TNF-
production by these lineages. Similarly, SB203580 was shown recently to
block FMLP-induced neutrophil chemotaxis and superoxide production
(19). Because expression of p38 is abundant in neutrophils and
p38ß was undetectable, it is likely that p38 also mediates these
functions.
Inhibitors of p38 block IL-1ß-induced endothelial cell expression of
IL-6, and prostaglandin H synthase-2 (18). Although IL-1ß activated
p38 to a greater extent than p38ß, our data do not exclude the
possibility that p38ß yet plays a role in these responses. Recently,
p38 inhibitors were shown to block vascular endothelial cell growth
factor-induced actin filament formation and migration (27), and more
work is needed to investigate the role of p38ß in responses to this
and other stimuli. CD4+ T cells also expressed p38ß.
Although we did not investigate activation of p38ß in this lineage, a
contribution of p38ß should be considered in lymphocyte responses
suppressed by SB203580, e.g., IL-2- and IL-7-induced lymphocyte
proliferation.
The role for p38 in macrophage function is not known. The increase
in p38 upon macrophage differentiation suggests a role in functions
gained as monocytes mature into macrophages, such as increased
phagocytic activity. In this regard, it is notable that p38, but not
p38, phosphorylates stathmin (13), a ubiquitous 18-kDa cytosolic
protein involved in microtubule dynamics (28). Also, we have found that
SB202190 inhibits maximally 65% of LPS-induced TNF- production
by macrophages (C. Manthey, unpublished data). Since SB202190 does not
inhibit p38 (8, 9), we speculate that p38 may mediate the 35%
of TNF- production that is insensitive to SB202190. A role for
p38 in inflammation was suggested recently by Jiang et al. (9), who
reported the activation of renal p38 during glomerulonephritis in
rats.
In the brief time since the discovery of p38, a considerable effort has
been mounted to develop p38 inhibitors for inflammatory diseases.
Compounds described in the literature inhibit both p38 and p38ß.
As reported herein, p38 is the major p38 expressed by monocytes, and
p38 is the most active p38 in LPS-treated macrophages and
IL-1ß-treated endothelial cells. The data suggest that p38 may be
more important relative to p38ß in the inflammatory response.
Development of inhibitors with greater selectivity for p38 will
permit the testing of this hypothesis and may result in compounds with
more precise and focused in vivo pharmacology.
|
Acknowledgments |
|---|
, ß, , and
protein and ATF2 (11-109); Tom Zamborelli for p38, ß, , and
peptides; David Andrew for purified CD4+ T cells; and
Stephen Kinney for purified neutrophils. |
Footnotes |
|---|
2 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; AE, amplification efficiency; ATF2, activating transcription factor-2; CT, polymerase chain reaction cycle to threshold fluorescence; FAM, 6-carboxyfluorescein; GM-CSF, granulocyte-macrophage CSF; MKK, MAPK kinase; RT, reverse transcriptase; TAMRA, 6-carboxytetramethylrhodamine.
Received for publication October 23, 1998. Accepted for publication January 6, 1999.
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S. Zhang and M. H. Kaplan The p38 Mitogen-Activated Protein Kinase Is Required for IL-12-Induced IFN-{gamma} Expression J. Immunol., August 1, 2000; 165(3): 1374 - 1380. [Abstract] [Full Text] [PDF]
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 Q. Jing, S.-M. Xin, W.-B. Zhang, P. Wang, Y.-W. Qin, and G. Pei Lysophosphatidylcholine Activates p38 and p42/44 Mitogen-Activated Protein Kinases in Monocytic THP-1 Cells, but Only p38 Activation Is Involved in Its Stimulated Chemotaxis Circ. Res., July 7, 2000; 87(1): 52 - 59. [Abstract] [Full Text] [PDF]
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A. Mocsai, Z. Jakus, T. Vantus, G. Berton, C. A. Lowell, and E. Ligeti Kinase Pathways in Chemoattractant-Induced Degranulation of Neutrophils: The Role of p38 Mitogen-Activated Protein Kinase Activated by Src Family Kinases J. Immunol., April 15, 2000; 164(8): 4321 - 4331. [Abstract] [Full Text] [PDF]
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M. Allen, L. Svensson, M. Roach, J. Hambor, J. McNeish, and C. A. Gabel Deficiency of the Stress Kinase P38{alpha} Results in Embryonic Lethality: Characterization of the Kinase Dependence of Stress Responses of Enzyme-Deficient Embryonic Stem Cells J. Exp. Med., March 6, 2000; 191(5): 859 - 870. [Abstract] [Full Text] [PDF]
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 D. A. Partrick, E. E. Moore, P. J. Offner, D. R. Meldrum, D. Y. Tamura, J. L. Johnson, and C. C. Silliman Maximal Human Neutrophil Priming for Superoxide Production and Elastase Release Requires p38 Mitogen-Activated Protein Kinase Activation Arch Surg, February 1, 2000; 135(2): 219 - 225. [Abstract] [Full Text] [PDF]
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P. Kovarik, D. Stoiber, P. A. Eyers, R. Menghini, A. Neininger, M. Gaestel, P. Cohen, and T. Decker Stress-induced phosphorylation of STAT1 at Ser727 requires p38 mitogen-activated protein kinase whereas IFN-gamma uses a different signaling pathway PNAS, November 23, 1999; 96(24): 13956 - 13961. [Abstract] [Full Text] [PDF]
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J. P. Lian, R. Huang, D. Robinson, and J. A. Badwey Activation of p90RSK and cAMP Response Element Binding Protein in Stimulated Neutrophils: Novel Effects of the Pyridinyl Imidazole SB 203580 on Activation of the Extracellular Signal-Regulated Kinase Cascade J. Immunol., October 15, 1999; 163(8): 4527 - 4536. [Abstract] [Full Text] [PDF]
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S.-C. Hsu, M. A. Gavrilin, M.-H. Tsai, J. Han, and M.-Z. Lai p38 Mitogen-activated Protein Kinase Is Involved in Fas Ligand Expression J. Biol. Chem., September 3, 1999; 274(36): 25769 - 25776. [Abstract] [Full Text] [PDF]
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S. R. Dashti, T. Efimova, and R. L. Eckert MEK7-dependent Activation of p38 MAP Kinase in Keratinocytes J. Biol. Chem., March 9, 2001; 276(11): 8059 - 8063. [Abstract] [Full Text] [PDF]
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S. Zhuang, J. T. Demirs, and I. E. Kochevar p38 Mitogen-activated Protein Kinase Mediates Bid Cleavage, Mitochondrial Dysfunction, and Caspase-3 Activation during Apoptosis Induced by Singlet Oxygen but Not by Hydrogen Peroxide J. Biol. Chem., August 18, 2000; 275(34): 25939 - 25948. [Abstract] [Full Text] [PDF]
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R. A. Ward, M. Nakamura, and K. R. McLeish Priming of the Neutrophil Respiratory Burst Involves p38 Mitogen-activated Protein Kinase-dependent Exocytosis of Flavocytochrome b558-containing Granules J. Biol. Chem., November 17, 2000; 275(47): 36713 - 36719. [Abstract] [Full Text] [PDF]
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G. Alonso, C. Ambrosino, M. Jones, and A. R. Nebreda Differential Activation of p38 Mitogen-activated Protein Kinase Isoforms Depending on Signal Strength J. Biol. Chem., December 15, 2000; 275(51): 40641 - 40648. [Abstract] [Full Text] [PDF]
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 T. Matsumoto, I. Turesson, M. Book, P. Gerwins, and L. Claesson-Welsh p38 MAP kinase negatively regulates endothelial cell survival, proliferation, and differentiation in FGF-2-stimulated angiogenesis J. Cell Biol., January 7, 2002; 156(1): 149 - 160. [Abstract] [Full Text] [PDF]
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