© 1999 American Heart Association, Inc.
Vascular Biology |
Stat6 Activation Is Essential for Interleukin-4 Induction of P-Selectin Transcription in Human Umbilical Vein Endothelial Cells
From the Hanson Centre for Cancer Research, Division of Human Immunology, Institute of Medical and Veterinary Science, Adelaide, Australia.
Correspondence to Yeesim Khew-Goodall, PhD, Hanson Centre for Cancer Research, Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia. E-mail yeesim.khew-goodall{at}imvs.sa.gov.au
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Chronic upregulation of P-selectin expression on the surface of the endothelium has been observed in and likely contributes to a number of chronic inflammatory diseases, including atherosclerosis. Agonists of P-selectin expression fall into 2 categories: those that induce a very rapid, transient increase, lasting only hours, and those that induce prolonged upregulation lasting days. It is the latter group, which includes interleukin-4 (IL-4), that is likely to be a mediator of chronic P-selectin upregulation. The increase in P-selectin expression induced by IL-4 results from increased transcriptional activation of the P-selectin gene. The aim of this study was to deduce the postreceptor signaling pathway(s) giving rise to the prolonged increase in P-selectin expression induced by IL-4. We demonstrate the existence of 2 functional signal transducer and activator of transcription 6 (Stat6) binding sites on the P-selectin promoter and further demonstrate, by functional analysis of the P-selectin promoter, that binding of activated Stat6 to at least 1 site is essential for IL-4-induction of P-selectin transcription. Site 1 (nucleotide[nt] -142) bound Stat6 with a higher affinity than did site 2 (nt -229), and this difference was reflected functionally as constructs in which only site 1 was functional showed full IL-4 inducibility, whereas constructs in which only site 2 was functional showed only 40% of maximal IL-4 inducibility. IL-4 also induced prolonged activation of Stat6, which was contingent on the continuous presence of IL-4. The sustained activation of Stat6 induced by IL-4 is likely to be a key factor leading to the prolonged activation of the P-selectin promoter, thereby resulting in prolonged P-selectin upregulation.
Key Words: P-selectin • interleukin-4 • Stat6 • inflammation • chronic effects
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The role of inflammation in the development of atherosclerosis is clearly established. The adhesion of circulating leukocytes, in particular, monocytes, to the endothelium and their subsequent migration through the endothelial barrier is a requisite step in the formation of fatty streaks, the precursors to atherosclerotic plaques. Members of the selectin family of adhesion receptors, when expressed on the surface of activated endothelial cells (ECs) during inflammation, mediate the capture of fast- flowing leukocytes, leading to leukocyte rolling on the vessel wall, a step necessary for the subsequent firm adhesion and extravasation of leukocytes (reviewed in References 1 and 21 2 ).
P-selectin, a member of the selectin family of adhesion receptors, is a
140-kDa glycoprotein stored in the Weibel-Palade bodies of
ECs3 4 and the 
-granules of platelets and
megakaryocytes.5 The importance of P-selectin in
inflammation is aptly demonstrated by the decrease in numbers of
rolling leukocytes in P-selectin–deficient mice, resulting in delayed
and decreased leukocyte extravasation during acute
inflammation.6 Increased expression of P-selectin in the
blood vessels at sites of inflammation has been observed in a number of
chronic inflammatory diseases such as rheumatoid
arthritis,7 Graves disease,8 nasal
polyps,9 and atherogenic plaques.10 In a
recent study of P-selectin–null mice crossed with low density
lipoprotein receptor–null mice (an established model of familial
hypercholesterolemia11 ), the
resulting double-knockout mice showed significantly smaller and fewer
fatty streaks with delayed onset compared with their low density
lipoprotein receptor–null parents. These results suggest that
P-selectin plays an essential role in the initiation and perhaps also
the progression of atherogenic plaques. Collectively, these
observations suggest that a sustained increase in
endothelial P-selectin expression may play a
significant role in the maintenance of a chronic inflammatory
state that could underpin the development of some chronic inflammatory
diseases such as atherosclerosis. Little, however, is
known about the mechanism(s) that leads to the prolonged upregulation
of P-selectin expression at sites of chronic inflammation.
Two categories of agonist that bring about cell-surface elaboration of
P-selectin have been described to date. Most known
activators result in peak cell-surface expression in 10
minutes (eg, thrombin, histamine, and phorbol 12-myristate
13-acetate)12 13 or 2 to 4 hours (oxygen
radicals14 and tumor necrosis factor- [TNF-] in
the mouse15 ). The half-life of the protein on the cell
surface induced by agonists such as thrombin or phorbol
12-myristate 13-acetate is short, with a return to basal
expression levels within several hours, even in the sustained presence
of the agonist. This observation implies that there is a refractory
period during which the cells are not responsive to further stimulation
by the same agonist and also suggests that this class of agonist is
more likely to be a mediator of acute inflammation.
Recently, another group of agonists were discovered that induced
cell-surface expression with kinetics very different from that induced
by the agonists described earlier. These are the cytokines
interleukin-3 (IL-3), IL-4, and oncostatin-M (OsM), products of
activated T cells and monocytes usually present at chronic
inflammatory sites. We showed that IL-3 can upregulate cell-surface
P-selectin expression on cultured human umbilical vein ECs (HUVECs)
for a prolonged period, beginning 
16 hours after stimulation and
lasting at least 4 days.16 The increase in cell-surface
P-selectin expression was accompanied by an equivalent persistent
increase in its mRNA level. IL-4 and OsM also caused prolonged
expression of both P-selectin mRNA and cell-surface P-selectin on ECs
with similar kinetics to IL-3 (Reference 1717 and this study). Although
Yao et al17 could not confirm the induction of P-selectin
mRNA expression by IL-3, we have found it to be a consistent
observation. Although the increase in mRNA level induced by IL-3 was
less than that induced by IL-4, it was similar to that induced by OsM
(2-fold; Y.K-G. et al, unpublished observations, 1998).
The unusual kinetics of induction of P-selectin expression by IL-3, OsM, and IL-4 suggests that they are likely to be mediators of increased P-selectin expression seen in chronic inflammatory diseases and raise intriguing questions regarding the nature of the intracellular signaling pathway(s) utilized. Binding of IL-4 to its receptor results in activation of several intracellular signaling pathways, including the phosphatidylinositol-3-kinase18 and the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) pathways. Signaling through the Jak/STAT pathway occurs by a tyrosine phosphorylation cascade19 whereby Jak tyrosine kinases activated by tyrosine phosphorylation in turn phosphorylate the STAT family of transcription factors, leading to STAT dimerization and ultimately, translocation into the nucleus.
IL-4 stimulation results in activation of Stat620 in most
cell types, including HUVECs.21 22 IL-4 suppression of
TNF-–induced E-selectin expression on HUVECs also occurs through
Stat6 activation.23 We therefore hypothesized that
P-selectin gene transcription in response to IL-4 stimulation would
probably occur through activation of Stat6. The proximal P-selectin
promoter contains 2 putative Stat6- binding sites, and in this study,
we demonstrate that they are both functional and that the binding of
Stat6 to at least 1 Stat6 site is essential for IL-4 induction of
P-selectin gene transcription. We also found that IL-4 not only induced
the prolonged expression of P-selectin but, surprisingly, also induced
a similarly prolonged activation of the transcription factor Stat6.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Cell Culture, Reagents, and Antibodies (Abs)
HUVECs were extracted by collagenase treatment according to a modified version of the procedure of Wall et al24 as previously described.16 Cells were either used 2 to 5 days after establishment of culture by harvesting with trypsin (0.05%)-EDTA (0.02%) (Gibco-BRL, Life Technologies) for replating or passaged further. After replating, EC growth supplement (Collaborative Research) at 25 mg · mL-1 and heparin were included in the culture medium. All reagents used in the growth, passaging, and stimulation of HUVECs were made up under endotoxin-free conditions and contained between 10 and 100 pg · mL-1 endotoxin as ascertained by the Limulus amoebocyte assay. Typically cells between passages 1 and 4 were used.
Stat6 antiserum was a gift from Dr James Ihle, St Jude Children's Hospital, Memphis Tenn; recombinant human (rh) IL-4 was a gift of Immunex Corp, Seattle Wash; rhIL-3 was a gift from Dr Christopher Bagley; rhOsM was a gift from Drs Richard Simpson and Robert Moritz of the Ludwig Institute, Melbourne, Australia; and affinity- purified polyclonal P-selectin antibody (Ab) was a gift from Dr Michael Berndt, Baker Medical Research Institute, Prahran, Australia. The anti–active mitogen-activated protein kinase (MAPK) polyclonal Ab was obtained from Promega, and the MAPK polyclonal Ab (anti-ERK1 and 2) was from Zymed.
Synthetic Oligonucleotides and Plasmid
Constructs
The following oligonucleotides were used as
probes or competitors in electrophoretic mobility shift assays (EMSAs):
(1) site 1 probe, 5'-agcttTAACTTCATGGGAAGGGCg-3'; (2) site 2 probe,
5'-agcttCATCTTCCATGGAAGGGGg-3'; (3) I
,
5'-agcttGCAGTTCCCAAGAACATGg-3'; (4) mut,
5'-agcttGCAGTTCATGGGCTCATCg-3'; and (5) ß-cas,
5'-agcttAGATTTCTAGGAATTCAAATCCg-3'. (The lower- case letters
represent the sequences added to generate an Hind
III restriction site at the 5' end and an Sal I site at the
3' end.) Site 1 and 2 sequences were from the P-selectin promoter, and
the I core Stat6 sequence (italicized) was from the immunoglobulin
(I) promoter that was previously shown to bind
Stat6.25 The mut sequence contained mutations in the
site 1 core sequence that rendered it unable to bind Stat6 (data not
shown). The ß-cas sequence is the interferon-
activation
site–like sequence (GAS) element from the ß- casein promoter, which
binds Stat526 and acts as a general STAT- binding site
(Y.K-G. et al, unpublished observations, 1998, and Reference 2727 ).
The P-selectin promoter fragment from nucleotides (nt's) -270 to -13 was generated by polymerase chain reaction (PCR) amplification of genomic DNA and specific 5' and 3' primers based on a previously published sequence28 and subcloned into the chloramphenicol acetyltransferase (CAT) reporter vector, pBLCAT3, for use in transient transfection assays. Mutations introduced into the Stat6 sites were also generated by PCR with oligonucleotides containing the mutated sequence as primers. All constructs were sequenced to ensure that no unintended mutations had been introduced.
Nuclear Extract Preparation and EMSA
Nuclear extracts were prepared from
2x106 cells at 4°C according to the protocol
of Mui et al.29 EMSAs were performed in a total volume of
15 µL containing 10 mmol/L Tris-HCl (pH 8.0), 100 mmol/L
KCl, 5 mmol/L MgCl2, 1 mmol/L DTT, 0.5
mg/mL BSA, 10% glycerol, 1 mmol/L PMSF, 2 µg poly(dI-dC), and
2x105 counts per minute (0.8 ng) of
32P-labeled probe. The reactions were initiated
by the addition of 9 µg of nuclear extract (in a volume of 2 to 5
µL) and allowed to incubate at room temperature for 20 minutes before
electrophoretic separation on a 5% polyacrylamide gel in
0.25x Tris-borate-EDTA buffer. In competition assays, unlabeled
oligonucleotides were preincubated with nuclear extract
in the reaction mix 10 minutes before the addition of labeled probe.
Similarly, in Ab supershift assays, anti-Stat6 or preimmune serum was
preincubated with nuclear extract in the reaction mix for 1 hour at
4°C before the addition of labeled probe.
Transient Transfections and CAT Assays
HUVECs were transiently transfected using lipofectin (BRL-Gibco,
Life Technologies), according to the manufacturer's recommendations,
with 6 µL lipofectin reagent and 1 µg plasmid in 1 mL Opti-MEM
medium for 105 cells per well in 6-well
tissue-culture dishes. Cells were harvested 48 hours after
transfection, and the CAT assays were then performed. CAT assays were
carried out as previously described.30 The various
acetylated forms of
[14C]chloramphenicol were detected by using a
PhosphorImager (Molecular Dynamics) and quantitated by using ImageQuant
software (Molecular Dynamics).
Flow Cytometry to Detect Cell-Surface P-Selectin
Expression
To determine cell-surface expression of P-selectin, IL-4
stimulation was carried out in a defined medium (Opti-MEM) containing
2% FCS instead of HUVEC medium containing 20% FCS, which has been
used in all other experiments described. Stimulating HUVECs in the
presence of a high FCS concentration resulted in a 3- to 4-fold higher
basal P-selectin expression and hence, apparently less IL-4
stimulation.
Flow cytometry was used to detect cell-surface P-selectin expression as previously described16 using a rabbit polyclonal Ab against human platelet P-selectin at 10 mg mL-1. Before cell fixation, all solutions used with HUVECs contained 0.2% (wt/vol) NaN3 to prevent reinternalization of P-selectin from the cell surface. The polyclonal P-selectin Ab had previously been shown to bind specifically to P-selectin and did not cross-react with E-selectin expressed on the surface of Chinese hamster ovary cells transfected with the E-selectin cDNA (data not shown). Cells were washed twice at 4°C before the addition of FITC-conjugated sheep anti-rabbit IgG Fab2 secondary Ab (Silenus, Victoria, Australia) at a 1:80 dilution. The cells were incubated on ice for 30 minutes; washed once; and then fixed in 1% formaldehyde, 2% (wt/vol) glucose, and 0.02% (wt/vol) NaN3 in PBS. Flow cytometry was carried out using an EPICS Profile II (Coulter); 10 000 cells per group were analyzed. The background mean fluorescence intensity was determined by using rabbit nonimmune serum.
Western Blotting to Detect MAPK Activation
Untreated or cytokine- treated HUVECs
(2x105 cells) were washed in PBS containing
1 mmol/L sodium vanadate and then lysed in lysis buffer (20
mmol/L Tris-Cl, pH 8.0; 160 mmol/L NaCl; 1 mmol/L
CaCl2 ; 1% Triton X-100; 1 µmol/L
leupeptin; 200 µmol/L sodium vanadate; 1 mmol/L Na F;
2 mmol/L PMSF; 25 mmol/L benzamide; and 5 mmol/L
ß-glycerophosphate). The cell lysate was collected and
centrifuged. The supernatant was added to a 1/3 volume
of 20 mmol/L Tris-Cl, pH 8.0, containing 20% SDS, 6% sucrose,
and 10 mmol/L ß-mercaptoethanol. The proteins were separated by
electrophoresis on a 7.5% polyacrylamide gel and transferred
to a polyvinylidene difluoride membrane. Active MAPK was
detected with a 1/20 000 dilution of anti–active MAPK Ab. The filters
were later stripped and reprobed with an anti-ERK Ab to detect total
MAPK.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
P-Selectin Promoter Binds Stat6 Induced by IL-4
Two putative Stat6- binding sites (Figure 1
), fitting the consensus sequence
TTCNNNNGAA,27 were identified in the proximal promoter of
the P-selectin gene28 at nt -142 (denoted as site 1) and
-229 (site 2). Scanning 5 kb of the upstream flanking sequence failed
to reveal additional Stat6 sites. The binding characteristics of these
putative Stat6 sites were investigated by EMSA using either site 1 or
site 2 oligonucleotide probes with either unstimulated
or IL-4–treated HUVEC nuclear extracts (Figure 2). Both putative Stat6 sites bound a
factor that was induced by treatment with IL-4 for 15 minutes. Two
lines of evidence suggest that this factor is Stat6. First, the binding
was completely abolished by the addition of a cold competitor
oligonucleotide (denoted I) containing the
Stat6-binding site of the I promoter, which had previously been
demonstrated to efficiently bind Stat6.25 In contrast, an
oligonucleotide probe with a mutation in the Stat6
consensus sequence (denoted mut) was unable to compete for binding to
either site 1 or site 2 probes even when added at 100-fold molar
excess. This finding suggests that the IL-4–inducible factor was
specifically binding to both of the canonical Stat6 sequences of the
P-selectin promoter. Second, incubation in the presence of an
anti-Stat6–specific Ab resulted in a supershift that was not observed
when nonimmune serum was used (Figure 2). This result was again
observed with both site 1 and site 2 probes, confirming that the factor
binding to both sites was indeed Stat6.
|
|
During EMSA with the site 1 and 2 probes, it was noted that the site 1
probe gave a more intense band than did the site 2 probe (see Figures 2
and 3), suggesting that site 1
might bind Stat6 with a higher affinity than site 2. This was
investigated further by using either site 1 or site 2 as a probe and
unlabeled site 1 or site 2 oligonucleotide as a
competitor. The site 2 oligonucleotide was a very poor
competitor of the site 1 probe, reducing Stat6 binding by only 27% at
a 50-fold molar excess (Figure 3a). In contrast, a 5-fold molar
excess of site 1 oligonucleotide reduced Stat6 binding
to the site 1 probe by 72%, and a 25-fold molar excess completely
abolished binding. By comparison, a 5-fold molar excess of either site
1 or site 2 oligonucleotide abolished binding to the
site 2 probe (Figure 3b). Similarly, site 1 was a more efficient
competitor than site 2 when the I Stat6 sequence was used as a probe
(data not shown). These observations are consistent with site
1's having a greater affinity for activated Stat6 than has
site 2.
|
At Least 1 Stat6 Site on the P-Selectin Promoter Is Required for
IL-4 Induction
The induction of cell-surface P-selectin by IL-4 has previously
been shown to be accompanied by an increase in transcriptional activity
of the P-selectin gene.17 Having established that the
P-selectin promoter has 2 Stat6- binding sites, their role in mediating
IL-4–induced transcription of the P-selectin gene was investigated. A
set of promoter-CAT reporter constructs (Figure 4a) was made and transiently transfected
into HUVECs, and the CAT activity in transfectants that were either
untreated or treated with IL-4 was determined. Transfectants carrying
the parent construct p-270-CAT, which contains the proximal P-selectin
promoter from -270 to -13 and includes both Stat6- binding sites,
showed a 2.5±0.4-fold (mean±SEM) increase in CAT activity when
treated with IL-4 compared with untreated transfectants (Figure 4b). The increase in CAT activity observed in transfectants
stimulated with IL-4 suggests that the P-selectin promoter from -270
to -13 contains the elements that confer IL-4 responsiveness. In
contrast, transfectants with reporter-CAT constructs driven by an
unrelated promoter, such as the thymidine kinase promoter, did not
exhibit any increase in CAT activity when stimulated with IL-4 (Figure 4b).
|
To establish that the Stat6-binding sites are critical for mediating
IL-4 induction of P-selectin promoter activity, mutations that rendered
them unable to bind activated Stat6 were introduced into either
1 or both Stat6 sites. The mutations introduced were at the 3' end of
the consensus sequence, altering it from TTCNNNNGAA to TTCNNNNCTT.
Owing to 2 different problems that arose, 2 sets of constructs
containing wild- type Stat6 sites and 2 sets containing site 1
mutations were made. First, the cloning strategy used to obtain site 1
and the double mutants resulted in a 2-bp (CG) insertion at nt -120 of
the P-selectin promoter. A control construct (p-270CG-CAT) was
therefore generated that contained the 2-bp insertion and wild- type
Stat6 sites to ensure that the 2-bp insertion itself did not impair
IL-4 responsiveness. Table 1 shows that
the 2-bp insertion had no significant effect on IL-4 induction of
P-selectin promoter activity. Because introduction of the site 1
mutation [in pS6(1)3'-CAT] also resulted in mutation of an
overlapping putative ets transcription factor–binding site (see Figure 1), a second site 1 mutant construct [pS6(1)5'-CAT] with the
mutations introduced at the 5' end of the Stat6- binding site
(AAGNNNNGAA) was made. A comparison of the IL-4 inducibility showed no
significant difference between the 2 site 1 mutants (Table 2).
|
|
In the overall analysis (Figure 4), transfection data
obtained from the 2 constructs with wild- type Stat6 sites and the 2
site 1 mutants were pooled and collectively referred to as p-270-CAT
and pS6(1)-CAT, respectively. Simultaneously mutating both
Stat6 sites [pS6(1/2)-CAT] resulted in complete loss of IL-4
induction of P-selectin promoter activity. Mutation of site 1 alone
[pS6(1)-CAT] led to a significant but partial loss of IL-4
stimulation (1.6±0.1-fold, P<0.05). This value
represents a 60% decrease in IL-4 inducibility compared with
p-270-CAT, which contained both authentic Stat6 sites. However,
mutation to site 2 alone had no significant effect on IL-4 stimulation
of P-selectin promoter activity (2.7±0.4-fold, P=0.7).
These data suggest that IL-4 stimulation of P-selectin promoter
activity was dependent on having at least 1 active Stat6 binding site
on the P-selectin promoter. They also suggest that site 1 alone is
sufficient for full stimulation by IL-4, whereas site 2 alone results
in only partial stimulation.
IL-4 Induces Both Prolonged Cell-Surface P-Selectin Expression and
Stat6 Activation
The time course of IL-4 induction of cell-surface P-selectin
expression was examined by flow cytometry using a polyclonal Ab raised
against P-selectin. IL-4 induced a 3-fold increase in cell-surface
P-selectin expression that peaked 24 hours after IL-4 stimulation
and was sustained for at least 72 hours after stimulation (see Figure 6b). Therefore, the kinetics of IL-4–induced Stat6 activation
was examined by EMSA using the site 1 oligonucleotide
probe from the P-selectin promoter. Figure 5 shows that activation of Stat6 by IL-4
was very rapid, occurring within 15 minutes of IL-4 addition.
Surprisingly, activation of Stat6, like that of cell-surface P-selectin
expression, was also prolonged, lasting at least 48 hours (Figure 5).
|
|
Prolonged Activation of Stat6 and Prolonged P-Selectin Expression
Both Require the Continuous Presence of IL-4
The prolonged activation of Stat6 after IL-4 stimulation suggested
incomplete receptor downregulation after ligand binding or a long-lived
signal generated after IL-4 binding to its receptor. To differentiate
between these 2 possibilities, we treated HUVECs with IL-4 for 23
hours, after which IL-4 was thoroughly removed by 3 changes of medium.
The cells were then further incubated in the absence of IL-4 for 1 or 3
hours before nuclear extracts were made. The presence of
activated Stat6 was detected with an
oligonucleotide probe to site 1. After 23 hours of IL-4
stimulation, there was a significant level of activated Stat6
present (Figure 6a). One hour after
removal of IL-4, there was little change in the level of
activated Stat6 remaining, but 3 hours after removal of IL-4,
no detectable levels of activated Stat6 were observed. In the
controls to which IL-4 was added again for a further 3 hours after the
wash, there was no detectable decrease in activated Stat6
(Figure 6a). These results suggest that the continuous presence
of IL-4 is necessary for prolonged activation of Stat6.
A similar approach was followed to assess whether removal of IL-4 also
results in a shortened duration of P-selectin expression on the cell
surface. Removal of IL-4 after 24 hours of stimulation resulted in a
75% loss of cell-surface P-selectin and a complete return to basal
expression 48 hours afterward (Figure 6b). Thus, like Stat6
activation, continuous stimulation by IL-4 for 72 hours or replacement
of IL-4 into the medium for a further 48 hours after washing out the
initial stimulant is necessary for maintenance of prolonged
cell-surface expression. However, unlike downregulation of
activated Stat6, a complete return to basal levels was not
observed until at least 24 hours after removal of IL-4. This delay in
the kinetics of P-selectin downregulation compared with the
downregulation of activated Stat6 is most likely due to the
long half-life of P-selectin mRNA.17
OsM and IL-3 Do Not Activate Stat6
Because OsM and IL-3 also induce expression of P-selectin on
HUVECs,16 17 we investigated whether these
cytokines utilize the same signaling pathway as IL-4. HUVECs
were treated for 30 minutes with OsM or IL-3, and the activation of
Stat transcription factors was analyzed by EMSA with the GAS
element of the ß- casein promoter. The ß- casein GAS element
contains the consensus sequence TTCNNNGAA, which is a general Stat-
binding sequence.27 OsM was found to induce 1 prominent
complex (Figure 7a) that was identified
to be Stat1 by a Stat1 Ab supershift (Figure 7b) and by its
relative mobility on gels (data not shown). Occasionally, a weaker
Stat3 band was observed after OsM stimulation (Figure 7b), but
Stat1-Stat3 heterodimers have not been observed. Stat1 and Stat5 both
bind to a GAS element with the canonical sequence TTCNNNGAA, of which
the Stat6- binding site is a variant. Because there were no sequence
motifs in the P-selectin promoter fitting the canonical sequence of the
Stat1-Stat5 GAS element, we investigated whether the 2 Stat6 sites
present could bind Stat1 or Stat5. Figure 7a shows that
although OsM induced a large activation of Stat1, none of it was able
to bind to either of the Stat6 sites. IL-3 was found to
activate Stat5, which also did not to bind to either Stat6 site
(data not shown). Thus, OsM and IL-3 induce P-selectin expression by a
pathway different from that used by IL-4 and that does not involve
Stat6 activation.
|
In addition to the Jak/STAT pathway, both OsM and IL-3 also
activate the ras-MAPK pathway in several different cell
types.31 32 To investigate whether OsM or IL-3 also
activated MAPK in HUVECs, an Ab specific for active MAPK
(phosphorylated p44Erk and
p42Erk) was used in Western blot
analysis. Figure 7c shows that both OsM and IL-3 induced
MAPK activation 15 minutes after their addition, with residual active
MAPK still detectable 24 hours afterward, although the levels
were markedly reduced. In contrast, this was not observed when cells
were stimulated with IL-4.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
P-selectin on ECs has been shown to be a major determinant of inflammatory responses. Two mechanisms of control of cell-surface P-selectin expression have been delineated that appear to function at 2 extremes of the time course: 1 that results in rapid upregulation within minutes of stimulation, utilizing only preformed protein stored in Weibel-Palade bodies and lasting only several hours, and the other, taking several hours to increase P-selectin expression, through a sustained increase in gene transcription lasting several days. Recently, we and others have identified a number of cytokines (References 16, 17, and 3316 17 33 and this study) as possible mediators of the latter mechanism of chronic P-selectin expression on endothelium by virtue of the following: (1) they are products of activated T cells and monocytes, cells characteristic of chronic inflammatory sites and (2) they induce prolonged expression of P-selectin on ECs in vitro by stimulating P-selectin gene transcription. One such cytokine is IL-4, a product of activated T cells, mast cells, and eosinophils that is present at sites of chronic and allergic inflammation.
The signal transduction pathway(s) leading to prolonged P-selectin expression is largely unknown. One pathway common to all 3 cytokines that induce prolonged P-selectin expression is the Jak/STAT pathway. The identification of 2 canonical Stat6-binding sequences on the proximal P-selectin promoter at positions -229 and -142 led us to postulate that IL-4 stimulation of P-selectin transcription might occur through activation of Stat6. We established, by EMSA and with the use of unlabeled oligonucleotide competitors and an anti-Stat6–specific Ab, that both of these putative Stat6 sites could indeed specifically bind Stat6 activated by IL-4. Our data also demonstrated that Stat6 binding to at least 1 Stat6- binding site was obligatory for inducing transcription of the P-selectin gene after IL-4 stimulation, since simultaneous inactivation of both Stat6 sites led to complete loss of IL-4 inducibility.
Northern blot analysis (data not shown) showed a 2- to 3-fold
increase in P-selectin mRNA as early as 3 hours after IL-4 addition.
This is consistent with the increase in P-selectin promoter
activity (Figure 4b) induced by IL-4 and also with the kinetics
of Stat6 activation. The steady-state level of P-selectin mRNA
continued to increase linearly over time up to between 16 and 24 hours,
when a plateau was reached. The amount of mRNA at 24 hours varied
between 8- and 10-fold above basal levels. Yao et al17 had
previously investigated the kinetics of induction of P-selectin mRNA
after IL-4 stimulation and found a similar response, except that no
increase was observed between the 3- to 7-hour time point. This result,
together with the requirement for protein synthesis to upregulate
P-selectin mRNA, could be indicative of an indirect effect of IL-4.
However, the long, slow, linear increase in steady-state mRNA to the
plateau level, as we have observed, is consistent with a small
increase in transcriptional rate induced by IL-4 coupled with a slow
turnover rate of mRNA. This, together with the data showing that Stat6
binding to the P-selectin promoter is essential for IL-4 induction of
P-selectin transcription, suggests that IL-4–activated Stat6
is acting directly on the P-selectin promoter to increase its
transcription rate.
Activation of Stat6 occurs through tyrosyl phosphorylation and is thus independent of de novo protein synthesis. Indeed, the presence of cycloheximide for 24 hours did not significantly alter the amount of Stat6 binding to the site 1 probe (data not shown). Yao et al17 had previously reported and our data (not shown) confirm that the increase in mRNA induced by IL-4 was cycloheximide-sensitive, suggesting that de novo protein synthesis was also essential for induction of mRNA synthesis. We therefore postulate that in addition to Stat6 activation, de novo synthesis of a second transcription factor is also required for IL-4 induction of P-selectin transcription.
The 2 Stat6-binding sites on the P-selectin promoter have different properties. They differ by 3 nt's in the variable region of the canonical sequence, which results in site 1's having a much greater affinity for Stat6 than does site 2. This suggests that Stat6 would preferentially occupy site 1 in this promoter. The difference in Stat6 binding affinity is also reflected in the function of the 2 sites: mutations that left only site 1 functional showed full IL-4 inducibility compared with the native promoter, whereas when site 2 only was functional, IL-4 inducibility was reduced to <50%. It is unclear what the role of site 2 is. One possibility is that Stat6 bound to site 2 could interact with other transcription factors to affect their activity. Alternatively, we hypothesize that it could be a binding site for other Stat proteins and thereby participate in transducing signals from other cytokine receptors to the P-selectin promoter.
IL-316 and OsM17 are 2 other cytokines that induce P-selectin expression and also activate the Jak/STAT pathway. IL-3 activates Stat5 in HUVECs (E.I. Korpelainen, et al, unpublished observations, 1995), whereas OsM predominantly activated Stat1, with lower levels of Stat3 observed occasionally. Stats 1, 3, and 5 bind a TTCNNNGAA consensus sequence that is not present in the P-selectin promoter. We therefore investigated whether they could bind to site 1 and 2 sequences and found no complexes binding to either Stat6 site when HUVECs were stimulated with IL-3 or OsM. The lack of binding sites for Stats1 and 5 on the P-selectin promoter and the lack of activation of Stat3 by OsM suggests that upregulation of P-selectin expression by IL-3 and OsM is unlikely to occur through Stat activation. In addition to activation of the Jak/STAT pathway, both IL-3 and OsM also activated MAPK in HUVECs. One transcription factor that is a potential downstream target after gp130–ras-MAPK activation is nuclear factor (NF)–IL-6.34 The canonical NF—IL-6 binding sequence is TT/GNNGNAAT/G,35 and a sequence fitting this consensus is present at -118 on the P-selectin promoter. It remains to be seen whether this is the mechanism by which IL-3 and OsM upregulate P-selectin expression.
Three different observations are particularly relevant in the sustained increase in P-selectin expression induced by IL-4. First, we show here that IL-4 induced prolonged activation of Stat6 itself, with elevated levels of Stat6 detectable by EMSA as long as 48 hours after IL-4 stimulation. This was somewhat surprising because the activation of STATs has often been observed to be rapid and transient. The consequence of sustained activation of Stat6 after IL-4 stimulation would be a prolonged duration of increased synthesis of P-selectin mRNA. Second, P-selectin mRNA itself has an intrinsically long half-life.17 Third, our studies also showed that both prolonged Stat6 activation and prolonged P-selectin expression are contingent on the continuous presence of IL-4. Removal of IL-4 after stimulation led to a rapid loss of activated Stat6 followed by a slower loss of cell-surface P-selectin expression. The requirement for a continuous presence of IL-4 to sustain Stat6 activation suggests that continuous receptor-mediated events were necessary. It also suggests that removal of IL-4 or its source may be an effective way of downmodulating P-selectin expression at sites of chronic inflammation.
Finally, IL-4 appears to have different immunomodulatory effects on the
endothelium that are mediated through different
signaling pathways and mechanisms. IL-4 upregulates the expression of
P-selectin and augments TNF-–induced vascular cell adhesion
molecule-1 (VCAM-1) expression36 but inhibits TNF-
induction of E-selectin expression.37 IL-4 upregulates
P-selectin expression by increasing transcription through the Jak/Stat6
pathway (this study), whereas the VCAM-1 promoter does not contain any
Stat6-binding sites. VCAM-1 upregulation occurs by stabilization of its
mRNA36 through an as-yet-unidentified signaling pathway.
On the other hand, IL-4 also inhibits TNF- induction of E-selectin
expression through a Jak/Stat6 pathway,23 but this is
effected through competition with NF-
B binding. The Stat6-binding
site on the E-selectin promoter overlaps the NF-B site, which
mediates TNF- induction, such that Stat6 binding results in
competition with NF-B binding. In the E-selectin promoter, Stat6
binding after IL-4 stimulation of HUVECs does not result in
transactivation of promoter activity. The reason for this is unknown,
but 1 suggestion is that the necessary component for Stat6 to interact
with the basal transcriptional machinery is not present. It is
presently unclear whether transactivation is a prerequisite for
IL-4 induction of P-selectin gene transcription or whether the increase
in transcription occurs because Stat6 binding is competing away a
repressor.
The reasons for the different immunomodulatory effects of IL-4 are not
entirely understood, but they may serve to determine the leukocyte
subtypes that extravasate at different stages of the inflammatory
response. In the acute stages of inflammation, when TNF- is
present, upregulation of E-selectin expression and IL-8
production may facilitate neutrophil extravasation. As
inflammation progresses to the transition stages when T-cell
infiltration occurs, IL-4 is produced, switches the selectin
elaboration on the endothelium from E- to P-selectin,
and also enhances and prolongs VCAM-1 expression. The combination of
P-selectin and VCAM-1 would facilitate monocytic infiltration over a
more prolonged period. At later stages, if the source of IL-4 persists,
then there exists the possibility for chronic inflammation to develop.
However, sites of inflammation are often complex, with a plethora of
cytokines and immunomodulatory molecules being expressed, which
then establishes a complex signaling network. Unraveling the signaling
networks and how cross-talk between different pathways can influence
the final outcome will be an important step toward the next generation
of therapeutics.
|
Acknowledgments |
|---|
This work was supported by a program grant (No. 973204) from the National Health and Medical Research Council of Australia. We thank the staff of the delivery wards of the Women's and Children's Hospital and Burnside Memorial Hospital, Adelaide, for the collection of umbilical cords. We also thank Immunex Corp, Seattle, Wash, for the generous gift of rhIL-4, Drs Richard Simpson and Robert Moritz for rhOsM, Dr Christopher Bagley for rhIL-3, Dr James Ihle for the antiserum to Stat6, Dr Michael Berndt for the polyclonal Ab to P-selectin, and Jenny Drew for preparation of HUVECs.
Received September 29, 1998; accepted November 25, 1998.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Butcher EC. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell. 1991;67:1033–1036.[Medline] [Order article via Infotrieve]
2. Lawrence MB, Springer TA. Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell. 1991;65:859–873.[Medline] [Order article via Infotrieve]
3.
McEver RP, Beckstead JH, Moore KL, Marshall-Carlson L,
Bainton DF. GMP-140, a platelet -granule membrane protein,
is also synthesized by vascular endothelial cells and
is localized in Weibel-Palade bodies. J Clin Invest. 1989;84:92–99.
4.
Bonfanti R, Furie BC, Furie B, Wagner DD. PADGEM
(GMP140) is a component of Weibel-Palade bodies of human
endothelial cells. Blood. 1989;73:1109–1112.
5.
Stenberg PE, McEver RP, Shuman MA, Jacques YV, Bainton
DF. A platelet -granule membrane protein (GMP-140) is
expressed on the plasma membrane after activation. J Cell
Biol. 1985;101:880–886.
6. Mayadas TN, Johnson RC, Rayburn H, Hynes RO, Wagner DD. Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice. Cell. 1993;74:541–554.[Medline] [Order article via Infotrieve]
7. Grober JS, Bowen BL, Ebling H, Athey B, Thompson CB, Fox DA, Stoolman LM. Monocyte-endothelial adhesion in chronic rheumatoid arthritis: in situ detection of selectin and integrin-dependent interactions [see comments]. J Clin Invest.. 1993;91:2609–2619.
8. Miyazaki A, Mirakian R, Bottazzo GF. Adhesion molecule expression in Graves' thyroid glands; potential relevance of granule membrane protein (GMP-140) and intercellular adhesion molecule-1 (ICAM-1) in the homing and antigen presentation processes. Clin Exp Immunol. 1992;89:52–57.[Medline] [Order article via Infotrieve]
9.
Symon FA, Walsh GM, Watson SR, Wardlaw AJ. Eosinophil
adhesion to nasal polyp endothelium is
P-selectin-dependent. J Exp Med. 1994;180:371–376.
10. Johnson-Tidey RR, McGregor JL, Taylor PR, Poston RN. Increase in the adhesion molecule P-selectin in endothelium overlying atherosclerotic plaques: coexpression with intercellular adhesion molecule-1. Am J Pathol. 1994;144:952–961.[Abstract]
11. Johnson RC, Chapman SM, Dong ZM, Ordovas JM, Mayadas TN, Herz J, Hynes RO, Schaefer EJ, Wagner DD. Absence of P-selectin delays fatty streak formation in mice. J Clin Invest. 1997;99:1037–1043.[Medline] [Order article via Infotrieve]
12.
Hattori R, Hamilton KK, Fugate RD, McEver RP, Sims PJ.
Stimulated secretion of endothelial von
Willebrand factor is accompanied by rapid redistribution to the
cell surface of the intracellular granule membrane protein GMP-140.
J Biol Chem. 1989;264:7768–7771.
13. Geng JG, Bevilacqua MP, Moore KL, McIntyre TM, Prescott SM, Kim JM, Bliss GA, Zimmerman GA, McEver RP. Rapid neutrophil adhesion to activated endothelium mediated by GMP-140. Nature. 1990;343:757–760.[Medline] [Order article via Infotrieve]
14.
Patel KD, Zimmerman GA, Prescott SM, McEver RP,
McIntyre TM. Oxygen radicals induce human endothelial
cells to express GMP-140 and bind neutrophils. J Cell
Biol. 1991;112:749–759.
15.
Weller A, Isenmann S, Vestweber D. Cloning of the mouse
endothelial selectins: expression of both E- and
P-selectin is inducible by tumor necrosis factor-. J Biol
Chem. 1992;267:15176–15183.
16.
Khew-Goodall Y, Butcher CM, Litwin MS, Newlands S,
Korpelainen EI, Noack LM, Berndt MC, Lopez AF, Gamble JR, Vadas MA.
Chronic expression of P-selectin on endothelial cells
stimulated by the T-cell cytokine, interleukin-3.
Blood. 1996;87:1432–1438.
17.
Yao L, Pan J, Setiadi H, Patel KD, McEver RP.
Interleukin 4 or oncostatin M induces a prolonged increase in
P-selectin mRNA and protein in human endothelial cells.
J Exp Med. 1996;184:81–92.
18.
Imani F, Rager KJ, Catipovic B, Marsh DG. Interleukin-4
(IL-4) induces phosphatidylinositol 3-kinase (p85)
dephosphorylation: implications for the role of
shp-1 in the il-4-induced signals in human b cells. J Biol
Chem. 1997;272:7927–7931.
19. Schindler C, Darnell JE Jr. Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu Rev Biochem. 1995;64:621–651.[Medline] [Order article via Infotrieve]
20. Mikita T, Campbell D, Wu P, Williamson K, Schindler U. Requirements for interleukin-4-induced gene expression and functional characterization of Stat6. Mol Cell Biol. 1996;16:5811–5820.[Abstract]
21.
Palmer-Crocker RL, Hughes CC, Pober JS. IL-4 and IL-13
activate the JAK2 tyrosine kinase and Stat6 in cultured human
vascular endothelial cells through a common pathway
that does not involve the c chain. J Clin
Invest. 1996;98:604–609.[Medline]
[Order article via Infotrieve]
22.
Lugli SM, Feng N, Heim MH, Adam M, Schnyder B, Etter H,
Yamage M, Eugster HP, Lutz RA, Zurawski G, Moser R. Tumor necrosis
factor- enhances the expression of the interleukin (IL)-4
receptor -chain on endothelial cells increasing IL-4
or IL-13-induced Stat6 activation. J Biol Chem. 1997;272:5487–5494.
23.
Bennett BL, Cruz R, Lacson RG, Manning AM.
Interleukin-4 suppression of tumor necrosis factor -stimulated
E-selectin gene transcription is mediated by STAT6 antagonism of
NF-B. J Biol Chem. 1997;272:10212–10219.
24. Wall RT, Harker LA, Quadracci LJ, Striker GE. Factors influencing endothelial cell proliferation in vitro. J Cell Physiol. 1978;96:203–213.[Medline] [Order article via Infotrieve]
25.
Fenghao X, Saxon A, Nguyen A, Ke Z, Diaz-Sanchez D, Nel
A. Interleukin 4 activates a signal transducer and
activator of transcription (Stat) protein which interacts
with an interferon- activation site-like sequence upstream of
the I epsilon exon in a human B cell line: evidence for the involvement
of Janus kinase 3 and interleukin-4 Stat. J Clin
Invest. 1995;96:907–914.
26. Gouilleux F, Pallard C, Dusanter-Fourt I, Wakao H, Haldosen LA, Norstedt G, Levy D, Groner B. Prolactin, growth hormone, erythropoietin and granulocyte-macrophage colony stimulating factor induce MGF-Stat5 DNA binding activity. EMBO J. 1995;14:2005–2013.[Medline] [Order article via Infotrieve]
27.
Seidel HM, Milocco LH, Lamb P, Darnell JE Jr,
Stein RB, Rosen J. Spacing of palindromic half sites as a determinant
of selective STAT (signal transducers and activators of
transcription) DNA binding and transcriptional activity. Proc
Natl Acad Sci U S A. 1995;92:3041–3045.
28.
Pan J, McEver RP. Characterization of the promoter for
the human P-selectin gene. J Biol Chem. 1993;268:22600–22608.
29. Mui AL, Wakao H, O'Farrell AM, Harada N, Miyajima A. Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs. EMBO J. 1995;14:1166–1175.[Medline] [Order article via Infotrieve]
30. Khew-Goodall Y, Mayer RE, Maurer F, Stone SR, Hemmings BA. Structure and transcriptional regulation of protein phosphatase 2A catalytic subunit genes [published erratum appears in Biochemistry.. 1991;30:5328]. Biochemistry. 1991;30:89–97.
31. Faris M, Ensoli B, Stahl N, Yancopoulos G, Nguyen A, Wang S, Nel AE. Differential activation of the extracellular signal-regulated kinase, Jun kinase and Janus kinase-Stat pathways by oncostatin M and basic fibroblast growth factor in AIDS-derived Kaposi's sarcoma cells. AIDS. 1996;10:369–378.[Medline] [Order article via Infotrieve]
32. Stancato LF, Sakatsume M, David M, Dent P, Dong F, Petricoin EF, Krolewski JJ, Silvennoinen O, Saharinen P, Pierce J, Marshall CJ, Sturgill T, Finbloom DS, Larner AC. ß-Interferon and oncostatin M activate Raf-1 and mitogen-activated protein kinase through a JAK1-dependent pathway. Mol Cell Biol. 1997;17:3833–3840.[Abstract]
33. Modur V, Feldhaus MJ, Weyrich AS, Jicha DL, Prescott SM, Zimmerman GA, McIntyre TM. Oncostatin M is a proinflammatory mediator: in vivo effects correlate with endothelial cell expression of inflammatory cytokines and adhesion molecules. J Clin Invest. 1997;100:158–168.[Medline] [Order article via Infotrieve]
34. Akira S. IL-6-regulated transcription factors. Int J Biochem Cell Biol. 1997;29:1401–1418.[Medline] [Order article via Infotrieve]
35.
Faisst S, Meyer S. Compilation of vertebrate-encoded
transcription factors. Nucleic Acids Res. 1992;20:3–26.
36.
Iademarco MF, Barks JL, Dean DC. Regulation of vascular
cell adhesion molecule-1 expression by IL-4 and TNF- in
cultured endothelial cells. J Clin
Invest. 1995;95:264–271.
37.
Thornhill MH, Haskard DO. IL-4 regulates
endothelial cell activation by IL-1, tumor necrosis
factor, or IFN-. J Immunol. 1990;145:865–872[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  K. Yamaji-Kegan, Q. Su, D. J. Angelini, and R. A. Johns IL-4 Is Proangiogenic in the Lung under Hypoxic Conditions J. Immunol., May 1, 2009; 182(9): 5469 - 5476. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.R.S. Girn, N.M. Orsi, and S. Homer-Vanniasinkam An overview of cytokine interactions in atherosclerosis and implications for peripheral arterial disease Vascular Medicine, November 1, 2007; 12(4): 299 - 309. [Abstract] [PDF]
|
|
|
|
 |
|
 Y. Miyazaki, T. Satoh, K. Nishioka, and H. Yokozeki STAT-6-Mediated Control of P-Selectin by Substance P and Interleukin-4 in Human Dermal Endothelial Cells Am. J. Pathol., August 1, 2006; 169(2): 697 - 707. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. K. Fritz, C. Kerr, L. Tong, D. Smyth, and C. D. Richards Oncostatin-M Up-Regulates VCAM-1 and Synergizes with IL-4 in Eotaxin Expression: Involvement of STAT6 J. Immunol., April 1, 2006; 176(7): 4352 - 4360. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S G Baidya and Q-T Zeng Helper T cells and atherosclerosis: the cytokine web Postgrad. Med. J., December 1, 2005; 81(962): 746 - 752. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Kizer, D. A. Zisman, N. P. Blumenthal, R. M. Kotloff, S. E. Kimmel, R. M. Strieter, S. M. Arcasoy, V. A. Ferrari, and J. Hansen-Flaschen Association Between Pulmonary Fibrosis and Coronary Artery Disease Arch Intern Med, March 8, 2004; 164(5): 551 - 556. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Chen, R. Lund, T. Aittokallio, M. Kosonen, O. Nevalainen, and R. Lahesmaa Identification of Novel IL-4/Stat6-Regulated Genes in T Lymphocytes J. Immunol., October 1, 2003; 171(7): 3627 - 3635. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Von der Thusen, J. Kuiper, T. J. C. Van Berkel, and E. A. L. Biessen Interleukins in Atherosclerosis: Molecular Pathways and Therapeutic Potential Pharmacol. Rev., March 1, 2003; 55(1): 133 - 166. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Woltmann, C. A. McNulty, G. Dewson, F. A. Symon, and A. J. Wardlaw Interleukin-13 induces PSGL-1/P-selectin-dependent adhesion of eosinophils, but not neutrophils, to human umbilical vein endothelial cells under flow Blood, May 15, 2000; 95(10): 3146 - 3152. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Yao, H. Setiadi, L. Xia, Z. Laszik, F. B. Taylor, and R. P. McEver Divergent Inducible Expression of P-Selectin and E-Selectin in Mice and Primates Blood, December 1, 1999; 94(11): 3820 - 3828. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. B. Ehret, P. Reichenbach, U. Schindler, C. M. Horvath, S. Fritz, M. Nabholz, and P. Bucher DNA Binding Specificity of Different STAT Proteins. COMPARISON OF IN VITRO SPECIFICITY WITH NATURAL TARGET SITES J. Biol. Chem., February 23, 2001; 276(9): 6675 - 6688. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||