CHAPTER 1: INTRODUCTION
1.2 TNF-D signaling in regulating endothelial inflammation
1.2.1 TNF-D induces cell adhesion molecules expression on endothelium
TNF-D is a pleiotropic cytokine which initiates a wide range of diverse cellular
responses including cell survival, activation, differentiation and proliferation, and cell rcecececececececececececcee cccccccccccccccaaaanananaanaanaaaaaaa leleleleleadadad ttttto o oo o ththththththththththhhhhhhhhhhee e e eee e e ee e
4
death.8 Upon interaction with receptors on the endothelium, TNF-D induced signal
transduction initiates pro-inflammatory changes, including expression of adhesion
molecules and increase of leukocyte adhesion for transendothelial migration.8, 10
Endothelial cells respond to TNF by releasing chemokines and displaying in a distinct
temporal, spatial and anatomical pattern adhesion molecules, including E-selectin,
intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1
(VCAM-1). It has been well-characterized that endothelial ICAM-1 plays an essential
role in neutrophil recruitment at the site of acute inflammation.11-14 ICAM-1 is a cell
surface glycoprotein of 505 amino acids with a molecular weight ranging from 76 to
114 kDa, depending upon extent of tissue-specific glycosylation.15, 16 It belongs to
immunoglobulin superfamily and is characterized by the presence of five extracellular
Ig-like domains, a hydrophobic transmembrane domain and a short cytoplasmic domain
of 28 amino acids.17 ICAM-1 functions as a ligand for E2 (CD11/CD18)-integrin and
associates with lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18) and
macrophage-1 antigen (Mac-1, CD11b/CD18) on neutrophils through Ig-like domain1
and 3 respectively.18, 19 Due to a strong bond between ICAM-1 and E2-integrin, TNF-D-induced ICAM-1 facilitates neutrophil forming firm adhesion to the
endothelium and further migrate across the endothelial barrier.20
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5
1.2.2 TNF-DD activates canonical NF-NB pathway to regulate ICAM-1 expression
TNF-D regulates ICAM-1 expression mainly through activating canonical nuclear
factor (NF)-NB-dependent transcriptional pathway.21 The NF-NB family of transcription
factors consists of RelA (p65), c-Rel, RelB, NF-NB1, and NF-NB2. Activation of the
canonical NF-NB pathway results in the degradation of bound inhibitor of NF-NB
(INB)-D, INB-E, or INB-H in the cytoplasm, which leads to the translocation of NF-NB to
the nucleus to mediate transcriptional events.22 Analysis of the 5’ flanking region of ICAM-1 gene revealed two NF-NB binding sites (upstream, -533 bp and downstream,
-223 bp).23 Site-directed mutagenesis and gel supershift assays demonstrated that ICAM-1 expression requires NF-NB p65 (RelA) binding to the downstream NF-NB site
of the ICAM-1 promoter.24, 25These findings were further supported by the identification
of consensus motifs on the promoter regions of ICAM-1 gene that is specifically targeted by the NF-NB dimers.26
1.2.3 TNF-D-induced MAPKs activation in endothelial inflammation
Except activating of NF-NB pathway, TNF-D stimulation also activates
mitogen-activated protein kinases (MAPKs) pathway in vascular endothelium.27 The
MAPKs family includes the p38 MAPK, c-Jun N-terminal kinase (JNK) and the
extracellular signaling-regulated kinase (ERK). It has been proposed that crosstalk M
6
between individual MAPK and NF-NB pathways may play a key role in
TNF-D-dependent pro-inflammatory responses.27, 28Some studies have implicated a role
of p38 MAPK and JNK in regulating TNF-D-induced ICAM-1 expression.29-31 However,
ERK seems to function as negative regulator in NF-NB mediated ICAM-1 expression.
One report showed that constitutively active ERK pathway inhibited NF-NB-driven
transcription, suggesting a negative role of ERK in regulating NF-NB activity.32 A
subsequent study demonstrated that suppression of ERK signaling enhanced NF-NB-dependent transcription,33 further suggesting that ERK inactivates NF-NB
pathway. Importantly, experiments using human endothelium have identified an
anti-inflammatory function of ERK, one in which it suppresses the expression of ICAM-1 by inhibiting NF-NB activity in TNF-D signaling.34 Collectively, these findings
indicate that inhibitory effect of ERK on NF-NB-directed transcriptional activation
would be essential in the context of TNF-D-mediated endothelial inflammation, as
evidenced by inducible ICAM-1 expression and neutrophil recruitment. Therefore, we assume that under TNF-D signaling there should have some phosphatases activate to
inhibit ERK activity, thus promoting TNF-D-induced endothelial inflammation.
a
7
1.3 Role of DUSPs in regulating MAP kinase and cell inflammation
MAP kinases activation requires phosphorylation on a threonine and tyrosine
residue at TXY motif located on the activation loop of kinase domain. Dual specificity
phosphatases (DUSPs) is a subclass of the protein tyrosine phosphatase (PTP)
superfamily,35 which dephosphorylate the critical phosphotyrosine and
phosphothreonine residues within MAP kinase.36 The expression of DUSPs is induced
by growth factors and cellular stress, and is restricted to a subset of tissue types and
localized to different subcellular compartments.37 Due to the catalytic activation of
DUSPs after tight binding of its amino-terminal to the target MAP kinase, some DUSPs
have high selective for inactivating distinct MAP kinase isoforms and hence are also
referred to as MAP kinase phosphatase (MKPs).38 DUSPs regulate activity of MAPK
through TXY motif dephosphorylation as well as represent particularly important
negative regulators.39 In addition to their dephosphorylating capacity, DUSPs serve to
anchor or shuttle MAP Kinases and control their subcellular localization.40, 41
Some members of DUSPs have been reported to regulate MAP kinase as well as
cell inflammatory response. In study of macrophages, DUSP1/MKP1 serves to limit the
inflammatory reaction by inactivating JNK and p38, thus preventing multiorgan failure
caused by exaggerated inflammatory responses.42, 43 DUSP2 is a positive regulator of
inflammatory cell signaling and cytokines functions. DUSP2 deficiency in macrophages
l
8
leads to increased JNK activity but impairment of ERK and p38 activity.44 Overexpression of DUSP4/MKP2 enhances TNF-D-induced adhesion molecules
expression (ICAM-1 and VCAM-1) and protects against apoptosis in HUVECs.45Mice
lacking the DUSP4/MKP2 gene had a survival advantage over wild-type mice when
challenged with intraperitoneal lipopolysaccharide (LPS) or a polymicrobial infection
via cecal ligation and puncture.46 DUSP10/MKP5 protects mice from sepsis-induced
acute lung injury.47 Mice lacking DUSP10/MKP5 displayed severe lung tissue damage
following LPS challenge, characterized with increased neutrophil infiltration and edema
compared with wild-type (WT) controls. Phosphorylation of p38 MAPK, JNK, and
ERK were enhanced in DUSP10/MKP5-deficient macrophages upon LPS stimulation.
Collectively, above findings suggest that DUSPs may participate in regulating cell
inflammation and immune response by controlling MAP kinase intensity and duration.
Therefore, DUSPs are promising drug targets for manipulating MAPK-dependent
immune response, to suppress infectious diseases or inflammatory disorders.48However,
except DUSP4 (MKP2) which was performed in HUVECs, most of functional
characterizations were performed in macrophages not in endothelium. We need further
studies to know the function of DUSPs in regulating endothelial inflammation.
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9
1.4 Study the role of DUSPs targeting on ERK to regulate endothelial inflammation
It was found that transiently activated ERK is down-regulated before the start of a relatively slow process of NF-NB-dependent ICAM-1 expression in endothelium
stimulated with TNF-D.49 These results suggest that the immediate response of ERK
signaling must be switched off in order to promote vascular inflammation through the canonical NF-NB pathway. We hypothesize that DUSPs, in particular ERK-specific
cytoplasmic phosphatase DUSP6/MKP-3,38might target the TEY motif in the activation
loop of ERK and dephosphorylate both Thr and Tyr residues, hence down-regulating
ERK activity in endothelium undergoing pro-inflammatory reaction. In fact, DUSP6 has
been identified as an early response gene whose expression is rapidly induced by
various extracellular stimuli or stresses.50, 51 Therefore, it is likely that DUSP6 is transiently expressed in endothelium exposed to TNF-D. If the initial ERK activity
could be terminated by endothelial DUSP6, NF-NB-dependent transcription for ICAM-1
expression could be switched on, allowing TNF-D-induced neutrophil adhesion to
commence.
This study investigated whether and how DUSP6 might promote expression of
ICAM-1 on the surface of endothelium under pro-inflammatory stimulation. We
examined the mechanism through which DUSP6 controls the crosstalk between ERK
a
10
and NF-NB signaling pathways in primary human endothelial cells treated with TNF-D.
Using knockout mice, we inspected further the in vivo function of DUSP6 in vascular
inflammation, and explored the regulatory role that endothelial DUSP6 plays in
pulmonary neutrophil recruitment during experimental sepsis induced by LPS, a process depending on the interaction between ICAM-1 and E2 (CD11/CD18)-integrin.52, 53
ea
11
CHAPTER 2: MATERIALS AND METHODS
12
2.1 Reagents
Collagenase, Low glucose Dulbecco’s modified Eagle’s medium (DMEM), M199
medium, RPMI-1640 medium, fetal bovine serum (FBS), glutamine, penicillin and
streptomycin were purchased from Gibco. Endothelial cell growth supplements (ECGS)
and Neon Transfection System was purchased from Invitrogen. Heparin, gelatin,
actinomycin D, cycloheximide and LPS (from E. coli serotype O55:B5) were purchased from Sigma. TNF-D was purchased from R&D system. PD184352 was purchased from
Biovision. PD98059 and U0126 were purchased from Cell signaling. BAY-117082 was
purchased from Calbiochem. Small interfering RNA oligonucleotides (siRNA) were
purchased from Dharmacon Thermo Scientific.
For stable isotope labeling by amino acids in cell culture (SILAC): SILAC DMEM
medium was from Gibico. Sequence grade trypsin and Lys-C protease were from
Promega. TiO2 bead was from GL Sciences, Japen. L-13C615N4-arginine (Arg10),
L-13C6-lysine (Lys6), iodoacetamide (IAM) and PT66 antibody were from sigma. 4G10
agarose bead was from Millipore. C18StageTip was from PROXEON.
2.2 Cell culture and transient transfection
2.2.1 Culture conditions for each cell line
The EAhy926 endothelial cells (ATCC) were maintained in low glucose DMEM, m (D(D(D(D(D(D(D(D(D((D(D((DDDDMMMEMEMEMEMEMEMMMMMMMMMMMMMM M)M)M)M)M)M), , M1M1M1M1M1M 99999999999999999999999999999999999
ne peniiiiiiiiiiciciciciccccciccillilllllllliiininininininininininn and
13
supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin and 100 Pg/ml
streptomycin. Human umbilical vein endothelial cells (HUVECs) were obtained from
collagenase-digested umbilical veins as described previously2 and subsequently
maintained in M199 medium, supplemented with 20% FBS, 25 U/ml heparin and 30 Pg/ml ECGS, 2 mM glutamine, 100 U/ml penicillin and 100 Pg/ml streptomycin in
gelatin-coated plates. HUVECs between the third and the fifth passage were used for
experiments. U937 cells (ATCC) were cultured in RPMI-1640 medium, supplemented
with 10% FBS.
2.2.2 Transient cell transfection
For direct exposure to TNF-Dor co-treatment with chemical inhibitors (actinomycin
D, cycloheximide, PD184352, PD98059, U0126, BAY-117082), ECs were plated in
medium containing FBS for 16 hours and then serum-starved for 6 hours before
treatment. Small interfering RNA oligonucleotides (siRNA) or expression plasmids
were delivered to ECs by electroporation using Neon Transfection System (Invitrogen)
according to manufacturer’s instructions. Briefly, ECs (2x105 cells per reaction for
siRNA transfection or 3.5x105cells per reaction for expression vector transfection) were
suspended in the Resuspension Buffer R (included with Neon Kits) together with the
siRNA duplexes targeting DUSPs (siRAN oligonucleotides obtained from Dharmacon, linininininininininininnn aaaaaaaaaaaaaaaanndndndndnndndnnnnnnnn 1111100000000 PPPPPg/g/g/g/g/mmlmlmmmmlmlmmlmlmlmmlmlmlmlmlmmm
weeeeeeeeeeeeeeeeerererererrerererererreeeeee oooooooooooooooooooooobtbtbtbtbttaiaiaaaianeneneneneneddd dd frfrfrfrfromomomomommmmmmmmmmmmmmmmm
and subsbsbbbsbsbsbsbsbbbsbsbsseqeqeqeqqqqqqquuueuuuuu ntly
14
and their sequences are shown in Table 2) or DUSP6 expression vectors. After
electroporation, cells were seeded in a single well of 12-well culture plate and then
incubated in normal culture condition without antibiotics for 16 hours, followed by
serum deprivation for an additional 6 hours prior to stimulation with inhibitor and/or TNF-D. For re-expression of the wild type form, C/S mutant form or KIM mutant form
of DUSP6 in HUVECs in which endogenous DUSP6 was ablated, shRNA constructs
bearing 3’-UTR sequence of DUSP6 (from the National RNAi Core Facility, Academia
Sinica, Taiwan) were tested initially by lentivirus-mediated infection. According to the
knockdown efficiency of DUSP6 by shRNA constructs, a specific clone
(TRCN0000355536) was chosen. Due to the poor viability of virus-infected HUVECs,
an alternative approach of transfection was established. The sequence of selected
shRNA clone targeting the DUSP6 3’-UTR was used as a template for synthesis of
siRNA duplexes (Dharmacon), which were ultimately applied to
electroporation-mediated transient transfection for knocking down only endogenous
DUSP6 but not re-expressed DUSP6.
2.3 Immunoblotting and antibodies
Aliquots of total lysates (15-20 Pg) were subjected to SDS-PAGE and transferred to
nitrocellulose membranes, and then incubated with antibodies recognizing caspase8 ioooooooooooooooonnn nn n nn nnn nnnnnnn vvvvevevevevvvvevvvvvvv ctctctctctorororoo s.s.s. AAAAAftftftfftftftftttttttererererererererereeeeeeeeeerrr
15
(9746), caspase3 (9662), phospho-p38 MAPK (9211), p38 MAPK (9212),
phospho-JNK (9251), JNK (9525), phospho-ERK (9101), ERK (9102), human ICAM-1 (4915), phospho-NF-NB (3033), NF-NB (3034), INB-D (9242) above all from Cell
Signaling; VCAM-1 (sc-13160) and ERK5 (sc-1284) from Santa Cruz; Tubulin (T5168),
Flag (F3165) both from Sigma and DUSP6 (a gift from Stephen Keyse and described
previously54). The specific signals were visualized by ECL Reagents (GE Healthcare).
2.4 RNA extraction and quantitative real-time PCR
Total RNA was isolated from EAhy926 cells or HUVECs using High Pure RNA
Isolation Kit (Roche) according to manufacturer’s instructions. The cDNA was
synthesized from total RNA with Transcriptor reverse transcriptase (Roche) using
oligo(dT)15 primer (Promega) according to manufacturer’s instructions. The mRNA
expression levels were quantified by real-time PCR using a LightCycler instrument
(Roche) with the SYBR Green PCR Master Mix (Qiagen) in a one-step reaction
according to manufacturer’s instructions. Primers (sequences are shown in Table 1)
were designed as described previously.55 The sequences for the house keeping gene,
hydroxymethylbilane synthase (HMBS), were 5’-AGTATTCGGGGAAACCT-3’
(forward) and, 5’-AAGCAGAGTCTCGGGA-3’ (reverse). The mRNA levels of target
genes were normalized to the relative amounts of HMBS.
M
16
2.5 Monocyte adhesion assay
Endothelial-monocyte adhesion assay was performed following the protocol
described previously.56 HUVECs (2x105 cells per reaction for siRNA transfection or
3.5x105 cells per reaction for expression vector transfection) were transiently
transfected with siRNA or expression vectors by electroporation, and then subsequently
seeded on a 24-well plate for overnight. Once reaching to confluence, cells were treated with TNF-D 10 ng/ml) for 4 hours. At the meantime, monocytic U937 (4.5x105) were
labeled with 10 Pg/ml of BCECF-AM (Invitrogen) at 37 ɗ for 30 minutes in dark,
subsequently washed twice with PBS to remove free dye, and then suspended in
HUVEC culture medium ready for use. Fluorescence dye-labeled U937 cells were added onto a monolayer of TNF-D-treated HUVECs and then incubated for 1 hour.
Non-adherent U937 cells were removed by two gentle washes with penol-red free M199
medium (Gibco). The fraction of HUVEC-associated U937 cells was quantified by a
fluorescence analyzer (Infinite F200, Tecan) using excitation and emission wavelength
at 485 and 535 nm, respectively. The images of adherent U937 cells on HUVEC
monolayer were captured using a fluorescence microscope (BX50, Olympus).
2.6 DUSP6 expression plasmids and luciferase reporter constructs
The full-length DUSP6 cDNA was obtained by reverse transcription of total mRNA in
17
isolated from HUVECs and subcloned into an N-terminal pFlag-CMV2 vector (Sigma).
The phosphatase dead C293S mutant of DUSP6 was generated by site-directed
mutagenesis according to the standard procedure. The DUSP6 KIM mutant construct (a
gift from Stephen Keyse) was generated as described previously41 and then subcloned
into an N-terminal pFlag-CMV2 vector. The ICAM-1 and VCAM-1-luciferase reporter construct were generated by insertion of NF-NB binding element to a pGL4.27
[luc2P/minP/Hygro] firefly luciferase vector (Promega), which contains a multiple
cloning region for insertion of a response element of interest upstream of a minimal
promoter and the luciferase reporter gene luc2P. The DNA duplex sequences
5’-TGGAAATTCC-3’ located at -187 bp of the ICAM-1 promoter, and
5’-GGGTTTCCCCTTGAAGGGATTTCCC-3’ located at -72 bp of the VCAM-1
promoter, were synthesized with a flanking restriction enzyme site KpnI/BglII. The
KpnI/BglII digested-DNA duplex was then inserted into KpnI/BglII digested-pGL4.27
vector. All expression clones were verified by sequencing.
2.7 NF-NNB reporter assay
HUVECs (3.5x105 cells per reaction in a single well of 12-well culture plate) were transiently transfected with 0.5 Pg of the reporter plasmid and 0.025 Pg of the pRL-null
vector (Renilla internal control reporter vector, Promega) by electroporation using the V2
18
Neon Transfection System (Invitrogen) according to manufacturer’s instructions. Cells were seeded on a 12-well plate for overnight and then treated with TNF-D 10 ng/ml)
for 4 hours. An aliquot of total lysates was subjected to specific luciferase activity and
was analyzed using the Dual-Luciferase Reporter Assay System (Promega) with a
luminometer (Luminoskan Ascent, Thermo Scientific).
2.8 RNA extraction and Gene expression profiling
Total RNA was isolated from HUVECs using High Pure RNA Isolation Kit (Roche)
according to manufacturer’s instructions. 300 ng total RNA were used for cDNA
synthesis, labeled by in vitro transcription followed by fragmentation according to the
manufacturer’s suggestion (GeneChip Expression Analysis Technical Manual rev5, Affymetrix). 11 Pg labeled samples were hybridized to Human Genome U133 Plus 2.0
Array (Affymetrix) at 45ɗ for 16.5 hours. The wash and staining were performed by
Fluidic Station-450 and the array were scanned with Affymetrix GeneChip Scanner 7G.
2.9 Animal studies
2.9.1 Mice housing
DUSP6-null mice (B6;129X1-Dusp6tm1Jmol/J,57 stock number 009069, backcrosses
number=1) and their appropriate control mice (B6129SF2/J, stock number 101045, in
19
recommended by the manufacture http://jaxmice.jax.org/strain/009069.html) were
purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were bred and
maintained in a specific pathogen-free (SPF) animal facility in a room subjected to a
12-hours light/dark cycle and maintained at constant temperature (22ɗ) and humidity
(55%). Mice received normal rodent chow and water ad libitum. All experimental
procedures were performed in accordance with the guidelines of the Institutional
Animal Care and Utilization Committee (IACUC) of Academia Sinica.
2.9.2 Genotyping
The genomic DNA was extracted from the tail tissue of mouse by the KAPA Express
Extract kit (KAPK Biosystem) according to the manufacturer’s instructions. A common
forward primer A (5’-CCT TCT CCT GCA GCT CGA C-3’, #12227), the wild type
mouse reverse primer B (5’-ATG GCA GAT TCG ATG TGT GA-3’, #12226) and
Dusp6-/- mouse reverse primer C (5’-CCG CTT CAG TGA CAA CGT C-3’, #12228,
catalog numbers provided by The Jackson Laboratory) were used for standard PCR in a
mixture of the KAPA2G Robust HotStart reagent (KAPK Biosystem) according to
manufacturer’s instruction.
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20
2.9.3 Tail vein injection with TNF-DD
In order to induce endothelium inflammatory response, male mice (10-12 weeks old) were injected with 5 Pg/kg of TNF-D diluted in PBS (Sigma) to a total volume of 100
Pl) into the lateral tail vein. Control mice were injected with an equal volume of PBS.
After 16 hours, mice were sacrificed. Vessels (containing aorta and inferior vena cava
(IVC)) were removed and processed for immunohistochemical staining.
2.9.4 Immunohistochemstry staining and image quantification
Organs from TNF-D-, LPS- or PBS-treated mice were harvested, rinsed in ice-cold
PBS, fixed in 4% paraformaldehyde and then embedded in paraffin. For
immnunohistochemistry staining, tissue sections were blocked with 10% goat serum
(005-000-001, Jackson Immunoresearch) for 2 hours and then incubated for overnight
with anti-mouse ICAM-1 antibodies (14-0542) or isotype control (14-4321, both from
eBioscience) at a dilution of 1:50. After three washes in PBS, the samples were treated
with goat anti-rat IgG secondary antibody (A9037, Sigma) at a dilution of 1:200 for 1.5
hours at room temperature. Bound antibody was detected using a DAB kit (Vector
Laboratories). Sections were counterstained with hematoxylin and eosin (H & E, both
from Sigma-Aldrich), dehydrated, treated with xylene substitute (Fluka) and
subsequently mounted with entellan (Merck). Images of the whole aorta and IVC were (1
21
captured using a microscope (BX50, Olympus) with 60x magnification, and images of
lung were captured with 40x magnification. For quantification, images were processed
and analyzed using software Image-Pro Plus 6.0 (Media Cybernetics).
2.9.5 LPS-induced experimental sepsis and neutrophil adoptive transfer
2.9.5 LPS-induced experimental sepsis and neutrophil adoptive transfer