National Creative Research Initiatives Center for ARS Network, College of Pharmacy, Seoul National University, Korea
Correspondence: Sunghoon Kim, National Creative Research Initiatives Center for ARS Network, College of Pharmacy, Seoul National University, San 56-1, Shillim-dong, Kwanak-ku, Seoul, 151-742, Korea. E-mail: sungkim{at}snu.ac.kr
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Key Words: intercellular adhesion molecule-1 • mitogen-activated protein kinase • cell-cell adhesion • integrin ß2
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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EMAP II is equivalent to the C-terminal domain of p43, which is a noncatalytic protein associated with macromolecular aminoacyl-tRNA synthetase complex [7 ]. For this reason, EMAP II was thought to be a cleaved product of p43. However, we recently found that p43, but not EMAP II, is secreted from the intact cells to exert cytokine activities and induces various proinflammatory signaling molecules in the monocytic THP-1 cell [8 ]. Among p43-induced genes, expression of intercellular adhesion molecule-1 (ICAM-1) was more than tenfold increased by the treatment of p43.
ICAM-1 is a surface-adhesion molecule playing a role in lymphocyte extravasation by the mechanisms of cell-cell adhesion [9 ]. The homo- and heterotypic cell adhesions are involved in a variety of physiological and pathophysiological processes including inflammation and atherosclerosis [10 ]. ICAM-1 mediates cell-cell adhesion via its interaction with the activated lymphocyte function-associated antigen-1 (LFA-1) and/or Mac-1, called the integrin ß2 family members [11 , 12 ]. The cell-cell adhesion is also induced by the increased level of ICAM-1 expression [13 14 15 16 ].
Mitogen-activated protein kinases (MAPKs) and other signaling molecules
including phosphatidylinositol 3-kinase (PI3-K) are considered to play
important roles in cell-cell adhesions [11
,
17
]. Because p43 stimulates the activity of nuclear
factor-
B (NF-B) and MAPK family members, extracellular
signal-regulating kinase (ERK), c-Jun N-terminal kinase (JNK), and p38
MAPK [8
], we thought that these molecules might be
involved in p43-induced expression of intercellular adhesion molecule-1
(ICAM-1) and cell adhesion. Here, we investigated whether p43-induced
expression of ICAM-1 is actually responsible for homo- and heterotypic
adhesion of monocytes and determined the related signal pathway.
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Construction and purification of p43 deletions
The plasmids expressing p43-(1-312), p43-(1-147), and
p43-(148-312) were described previously [18
]. To
construct p43-(1-108), pET28a (Novagen, Madison, WI) containing the
full-length p43 was digested with Asp718 and
SalI. The large fragment was treated with the Klenow
fragment to fill up the DNA ends, and the resulting DNAs were
religated. The DNA fragments coding for p43-(1-90), p43-(91-170),
p43-(91-256), p43-(91-312), p43-(218-312), and p43-(257-312) regions
were synthesized by polymerase chain reaction (PCR) with specific
primer sets. (The primer sequences will be available upon request.) The
specific PCR products were digested with EcoRI and
XhoI and ligated into pET28a cut with the same enzymes.
Each of the full-length p43 and p43-deleted constructs was expressed as His-tag fusion protein in Escherichia coli BL21 (DE3) and purified by nickel affinity chromatography and Mono Q or S ion-exchange chromatography as described previously [18 ]. To remove lipopolysaccharide (LPS), the protein solution was dialyzed in pyrogen-free buffer (10 mM potassium phosphate buffer, pH 6.0, 100 mM NaCl). After dialysis, the protein was loaded to polymyxin resin (Bio-Rad, Hercules, CA), pre-equilibrated with the same buffer, incubated for 20 min, and eluted. The concentration of the residual LPS was below 20 pg/ml when determined using the Limulus Amebocyte Lysate QCL-1000 kit (Bio-Whittaker, Walkersville, MD).
Western blots
The cells treated with p43 were harvested by
centrifugation at 600 g for 5 min and lysed with 0.25 ml lysis
buffer [50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM vanadate, 1 mM
dithiothreitol, 1.0% Triton X-100, and 0.1 mM phenylmethylsulfonyl
fluoride (PMSF)]. For p38 MAPK assay, cells were lysed by
sonication in radioimmune precipitation buffer (RIPA). The proteins
in the lysates were resolved by 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto
a polyvinylidene difluoride (PVDF) membrane (Millipore, Milford, MA),
and probed with specific antibodies. The antibodies bound to
specific proteins were reacted with the secondary antibody conjugated
with horseradish peroxidase and then detected with an enhanced
chemiluminescence substrate kit (Amersham, Arlington Heights, IL).
PI3-K assay
After THP-1 cells were treated with p43 (100 nM), the cells were
harvested at various time points. Then the cells were lysed with PI3-K
lysis buffer [15 mM Tris, pH 7.50, 130 mM NaCl, 1 mM
MgCl2, 1 mM CaCl2, 10% glycerol, 1% Nonidet
P-40 (NP-40), 5 mM sodium orthovanadate, and 0.1 mM PMSF]. The soluble
lysates (500 µg) were then incubated with antibodies specific for the
PI3-K subunit p85
for 3 h at 4°C, followed by additional
incubation for 1 h with protein G-agarose beads (Gibco BRL-Life
Technologies, Grand Island, NY). Then, the immune complex was washed
four times with the PI3-K lysis buffer and resolved by 10% SDS-PAGE.
Proteins on the gel were electrotransferred to a PVDF membrane
(Millipore). The membranes were immunoblotted with antiphosphotyrosine
antibody.
Homotypic cell-cell adhesion
THP-1 cells were inoculated at 2 x 106
cells/ml in 24-well plates. The cells were treated with each inhibitor
at the indicated concentrations for 1 h. Then, each of p43 or its
deletion mutants was added at 100 nM to the cells and incubated for
20 h. The homotypic adhesion of THP-1 cells was observed by
microscope at 100x magnification or quantified by counting the
adhesive cells in the representative aliquots from each culture on a
hematocytometer as a modified method of Takeda et al.
[19
]. Briefly, after the THP-1 cells were treated with
none or p43, the THP-1 cells in aggregates coagulated with more than
three cells were counted on the hematocytometer as the adhesive cells.
We counted the adhesive cells in at least 600 cells for each execution.
Degree of aggregation was calculated by the ratio of the number of the
adhesive cells to the number of total cells.
For neutralization, the cells were pretreated with mouse anti-CD11a (MAB1386; Chemicon, El Segundo, CA), anti-CD11b (MAB1380; Chemicon), and anti-ICAM-1 (Chemicon) antibodies 1 h before p43 treatment. Quantification was performed as described above.
Assay of gene expression by cDNA array analysis
The Atlas Human cDNA Expression Array 1.2 (Clontech, Palo
Alto, CA) was used for cDNA array analysis. Total and polyadenylated
RNAs were prepared from the control or p43-treated THP-1 cells by the
Atlas Pure Total RNA labeling system (Clontech) as recommended by the
manufacturer. Polyadenylated RNA (1 µg) isolated from the control or
p43-treated cells was converted to radioactive cDNA by reverse
transcription (RT) in the presence of [-32P]-dATP. The
radioactively labeled cDNA was then denatured and hybridized to the
cDNA expression arrays as recommended by the manufacturer. The
radioactivity on the membranes was quantified by a phosphoimager. We
calculated the change in gene expression after p43 treatment as the
percentage of the untreated cells, using three of the internal controls
recommended by the manufacturer for normalization to ensure the
comparability of the control and p43-treated samples.
Quantitative RT-PCR analysis
RNA (2 µg) was converted to cDNA by RT using
Moloney murine leukemia virus (M-MLV) RT (Gibco BRL) and the
anchored oligo-dT primer set. The cDNA was then amplified by PCR using
the ICAM-1 primers: sense, 5'-TCACATGACTAAGCCAAGAG-3'; antisense,
5'-CTGAGTGTCATTGTGAACAC-3'.
The reaction was run at 94°C (1 min), 58°C (40 s), and 72°C (50 s) for 35 cycles, which was within the linear reaction window. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the same cDNA was also measured as control by RT-PCR using the GAPDH primers: sense, 5'-TCATTGACCTCAACTACATG-3'; antisense, 5'-CCAAAGTTGTCATGGATGAC-3'. After amplification, the PCR products were resolved by agarose-gel electrophoresis.
Fluorescein-activated cell sorter (FACS) analysis of ICAM-1 surface
expression on THP-1 cells
THP-1 cells (2x105/ml) grown in six-well plates
were treated with none or 100 nM p43 for 18 h. Cells were then
harvested, washed, and stained with 10 µg fluorescein isothiocyanate
(FITC)-conjugated anti-CD54 antibody (Chemicon) for 3 h at 4°C.
After washing, stained cells were analyzed by flow cytometry using
Becton-Dickinson (San Jose, CA) FACS Caliber.
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Figure 1. Induction of homotypic adhesion of monocytic THP-1 cells by p43. THP-1
cells were untreated (Control) or treated with 100 nM p43. The cell
adhesion was monitored microscopically and shown on the top panel.
After quantification, the dose curve and the time course of p43-induced
cell adhesion were plotted as a bar and line graph, respectively. For
the dose curve, the cells were treated with p43 for 20 h. The bar
and line graphs show the means ± SE obtained from
four independent experiments.
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Figure 2. ICAM-1 expression was up-regulated by the treatment of THP-1 with p43.
(A) THP-1 cells were treated with 100 nM p43 for the indicated time
points, and the expression of the eight different cell adhesion
molecules was determined by the hybridization analysis of cDNA array.
Expression of each gene was quantified by a phosphoimager and
normalized based on three housekeeping genes (cytoplasmic ß-actin,
60S ribosomal protein L13A, and 40S ribosomal protein S9). The bar
graph is representative of four independent experiments. (B) The
expression of ICAM-1 mRNA was monitored by RT-PCR, after the THP-1
cells were treated with 100 nM p43 for indicated time points. GAPDH
mRNA was also monitored as a control. ICAM-1 and GAPDH mRNAs were
resolved by agarose gel electrophoresis. The gel picture is
representative of three independent experiments. (C) The protein
amounts of ICAM-1 were detected by Western blot analysis after THP-1
cells were treated with 100 nM p43 for indicated time points. Tubulin
was also blotted for a control. The gel picture shown here was
representative of three independent experiments. (D) ICAM-1 surface
expression on THP-1 cells was measured by FACS analysis. After
treatment with none or p43, cells were harvested, washed, and stained
with 10 µg FITC-conjugated anti-ICAM-1 antibody for 3 h at
4°C. After washing, stained cells were analyzed by flow cytometry
(Becton-Dickinson FACS Caliber). The mean fluorescence intensity (MFI)
of p43-untreated cells with anti-p43 antibody to MFI of p43-treated
cells with anti-p43 antibody is 1.5. The number of experiments with
similar results = 3.
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Figure 3. ICAM-1, LFA-1, and Mac-1 play a critical role in p43-induced cell
adhesion. (A) Cells were preincubated with each of the indicated
antibodies and then treated with 100 nM p43. The homotypic adhesion was
detected under the microscope. (B) The homotypic adhesion was
quantified as described in Materials and Methods. The bar graphs
represent the means ± SE obtained in four independent
experiments.
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Figure 4. THP-1 cells were pretreated with none or 5 µg/ml cycloheximide 30 min
before incubation with 100 nM p43. The cells were aggregated by
treatment with p43 alone (•), but the cell adhesion was not seen in
cells treated with cycloheximide alone ( ) or p43 + pretreatment
with cycloheximide ( ).
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B
[8
]. We examined which signaling molecules among them
are actually involved in the up-regulation of ICAM-1 and p43-induced
cell-cell adhesion. In Figure 5 A
, p43-induced cell adhesion was inhibited by pretreatment of
PD98059 (PD), which specifically blocks the activation of ERK
[27
], or the p38 MAPK inhibitor, SB202190 (SB)
[28
]. To see a more specific effect of ERK, the cells
were treated with U0126, a more specific inhibitor for MEK-1, and
p43-induced cell-cell adhesion was inhibited by the treatment
(unpublished results). Because PI3-K has been shown to mediate the
adhesion of human neutrophils [17
], we have also tested
whether it is involved in p43-induced adhesion of THP-1 cells. The
cells were pretreated with PI3-K inhibitors, wortmannin (WT) or
LY294002 (LY) [29
], and subsequently with p43.
p43-Induced cell adhesion was blocked by the presence of these
inhibitors, indicating that PI3-K is also involved in this process
(Fig. 5)
. We then tested whether the inhibitors block the up-regulation
of ICAM-1 in response to p43. THP-1 cells were pretreated with the ERK
or p38 MAPK inhibitor, and their effect on p43-induced ICAM-1
expression was monitored by the cDNA array and a Western blot (Fig. 5B
and 5C) . p43-Induced up-regulation of ICAM-1 expression was blocked by
PD98059, SB202190 [28
, 30
] (Fig. 5C)
, or
U0126 (unpublished results). These results suggest that ERK and p38
MAPK are involved in ICAM-1 expression induced by p43. ICAM-1
expression in THP-1 cells was also blocked by the presence of the PI3-K
inhibitor (Fig. 5B
and 5C)
, consistent with the effect of PI3-K
inhibitors on THP-1 cell adhesion.
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Figure 5. PI3-K, ERK, and p38 MAPK inhibitors blocked p43-induced THP-1 cell
adhesion and expression of ICAM-1. (A) THP-1 cells were preincubated
with none, WT (100 nM), LY (10 µM), PD (10 µM), or SB (10 µM) and
then treated with 100 nM p43 for 20 h. Homotypic cell adhesion was
monitored as in Figure 1
. (B) THP-1 cells were pretreated LY (10 µM),
PD (10 µM), or SB (10 µM) and incubated for 2 h in the absence
or presence of p43. ICAM-1 gene expression was quantified as described
in Figure 2A
. CD11a gene expression was seen as a control. (C) ICAM-1
expression in the THP-1 cells treated consecutively with none or each
inhibitor (2 h), and p43 (20 h) was detected by Western blot analysis
as in Figure 2C
.
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, which is the regulatory subunit of PI3-K
[31
]. p85 was precipitated with its specific
antibody, and its phosphorylation was detected by Western blots using
phosphotyrosine antiboby. Tyrosine phosphorylation of p85 was
increased by the treatment of p43 in a time-dependent manner
(Fig. 6 A
). We also observed the time course of PI3-K activation induced by
p43, corresponding to the time course of p85 phosphorylation
(unpublished results).
 View larger version (42K): [in a new window] |
Figure 6. ERK activation is mediated by p43-induced PI3-K. (A) The THP-1 cells
treated with 100 nM p43 were harvested, and the activity of PI3-K was
determined as described in Experimental Procedures. PI3-K was
precipitated with the antibody specific to PI3-K subunit, p85, and
its phosphorylation was determined with antiphosphotyrosine antibody.
(B) The THP-1 cells were preincubated with WT (100 nM) or LY (10 µM)
for 1 h and incubated for 1 h in the absence or presence of
p43. The proteins extracted from the treated cells were resolved by
SDS-PAGE, and the activities of ERK and p38 MAPK were determined by
phosphorylation of ERK and p38 MAPK, respectively. (C) Quantification
of Western blot for phosphorylation of ERK has been performed by
densitometry. The bar graph shows the mean ± SE
(n=3). The solid and open bars stand for the activities of ERK in the
presence and absence of p43, respectively.
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Deletion mapping of p43
Because the full length of p43 had a stimulatory effect on cell
adhesion, we investigated which region of p43 is responsible for cell
adhesion. The full-length p43 and its deletion mutants were expressed
as recombinant proteins and purified to homogeneity. After removing
trace amount of the contaminating LPS, each of the purified proteins
was used for the experiments. THP-1 cells were treated with each of the
purified proteins, and cell aggregates were observed 20 h after
the treatment. The active domain was determined by monitoring the
degree of the induced homotypic cell aggregation. Many purified p43
fragments were able to induce the homotypic cell adhesion, and the
C-terminal fragments of p43 composed of residues 218–312 and 257–312
were inactive (Fig. 7
). It is interesting that the full length or the peptides spanning
outside of the EMAP II domain (residues 147–312) showed higher cell
adhesion activity.
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Figure 7. Schematic diagram of p43 and its deletion mutants and their activities
to induce the homotypic cell-cell adhesion. Each peptide was added at
100 nM. The bar graphs show the mean ± SE
(n=3).
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Figure 8. A proposed signaling pathways leading to p43-induced cell adhesion. ERK
and p38 MAPK are activated by a PI3-K-dependent and -independent
manner, respectively. The activation of these kinases up-regulates the
expression of ICAM-1, which is directly involved in cell-cell
adhesion.
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L/integrin ß2) and CD11b/CD18 (Mac-1;
integrin M/integrin ß2) for cell adhesion.
p43-Induced cell adhesion appears to result from up-regulation of
ICAM-1 surface expression, making a firm association with LFA-1 and
Mac-1, integrin ß2 family members (Fig. 3)
. The activity
of p43 in integrin ß2-mediated homotypic cell adhesion
indicates its contribution to inflammatory responses
[25
]. The function of p43 is not clear as to whether p43 activates integrin ß2 family members. Because p43 activates upstream signaling molecules, PI3-K and ERK, for integrin ß2 family members [17 , 39 ], p43 is possible to enhance the avidity and/or binding activity of integrin ß2 family members. However, up-regulation of ICAM-1 induced by p43 is critical in THP-1 homotypic cell adhesion, because kinetics for cell adhesion is corresponding to the time course of ICAM-1 expression (Figs. 2 and 3) , and more importantly, p43-induced cell adhesion was completely prevented by a protein synthesis inhibitor (Fig. 4) . The results indicate that p43-induced cell adhesion requires up-regulation of ICAM-1 expression, but this work does not rule out a possible activity of p43 to alter the activity and/or the avidity of adhesion molecules.
We also investigated how p43 induces the expression of ICAM-1. Three
MAPK family members and NF-B were shown previously to be activated
by p43 [8
]. These signaling molecules are involved in
intercellular adhesion of various cell types. NF-B mediates cellular
adhesion by the regulation of ICAM-1 expression in human fibroblasts
and pancreatic acinar cells [13
, 14
]. ERK
and PI3-K have been shown to be upstream regulators for integrin
ß2-mediated cellular adhesion [17
,
39
]. Here, we dissected the p43-induced signaling pathway
in a more systematic and detailed manner. PI3-K, ERK, and p38 MAPK have
been found to regulate the homotypic THP-1 cell adhesion. These kinases
are organized into PI3-K/ERK-dependent and p38 MAPK-dependent pathways
(Fig. 8)
. A bifurcating point of the two parallel signaling pathways
lies upstream of PI3-K. NF-B remains to be studied as to whether it
is involved in p43-dependent cell adhesion. p43-Dependent multiple
signaling pathways determined here would give an important insight to
understand the working mechanism of this novel cytokine. In conclusion,
the results of this work identified that p43 stimulates different MAPKs
via PI3-K-dependent and -independent ways, leading to cell-cell
adhesion of monocytes (Fig. 8)
, and implicates p43 for the potent role
in inflammatory and atherosclerotic processes.
Received July 7, 2001; revised September 11, 2001; accepted October 5, 2001.
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