୯ҥᆵεᏢғڮࣽᏢଣғϯࣽᏢࣴز܌
റγፕЎ
Graduate Institute of Biochemical Sciences College of Life Science
National Taiwan University Doctoral Dissertation
ᚈ܄ᕗለНှ䁙ჹܭՈᆅϣҜಒझวݹϸᔈޑፓ
The Role of Inducible Dual-Specificity Phosphatases in Vascular Endothelial Inflammation
లޱ Shu-Fang Hsu
ࡰᏤ௲Ǻۏηߙ റγ Advisor: Tzu-Ching Meng, Ph.D.
ύ҇୯104ԃ7Д July, 2015
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ࣴز ز ز ز
زز ز ز ز ز ز ز ز ز ز ز ز ز ز ز ز ز ز ز܌ ܌ ܌ ܌ ܌ ܌
i
ᇞ ᇞᖴ
!
!!!!ಖܭ܌ԖޑոΚӧ೭څϯࣁჴǴҁፕЎளаֹԋाགᖴӭΓޑбрǶ २ӃགᖴࡰᏤ௲ۏηߙԴৣӭԃٰޑႴᓰᆶЈ௲ᏤǴᡣךԾࣴزշޑғࢲ ύགڙډՉࣴزޑ፪ǴԶԖ߿ࡷᏯόёૈޑҺ୍.ֹԋറγᏢՏ૽ግǶ೭
ၡوٰǴଯեଆҷǴགᖴۏԴৣޑவόܫకǴ૽ግךᡄᒠࡘԵᆶՉࣽᏢࣴز ޑૈΚǶ܌Ԗޑ௲ᇧךᙣܭЈǴ٠ոΚჴ፬ǴӧԜठനుϪޑགᖴǶӕਔག ᖴЦჱԴৣǵ့଼҅ԴৣǵᙼܿԴৣаϷߋᝩ፵Դৣӧα၂ය໔ჹҁፕЎගٮ ᝊޑཀـᆶࡰᏤǴᡣךᕇؼӭǴΨᡣҁፕЎ׳ᖿֹ๓Ƕ!
!!!!ќѦǴགᖴէᄪᐥԴৣӧךைޑਔংǴऐЈޑ᠋ᆶਔޑࡰЇǴᡣךό ठъԶቲǹགᖴᎄལོᙴৣаᖏفࡋ܌ගٮޑፏӭᝊཀـǹགᖴऍ࣓ᙴ
ৣคدӦނჴᡍаϷಔᙃϪТࢉՅޑᅿᅿޕǶᗋाགᖴՏමӅ٣ ޑӕՔॺǴᏃᆅӧךޑࣴزғఱύ੮ΠుభόޑىၞǴՠ೭ഉՔΑך ޑԋߏǶགᖴӕࣁ໒୯ϡԴޑሎՕǵℱǵमדǴࡐ໒ЈεৎԖӚԾޑТϺ<
གᖴᒃஏᏯ϶Նզǵ✎⊭ǵߞണᆶាઔǴؒԖգॺҁፕЎคݤֹԋ<གᖴൣ॥ଌཪ ޑྷᗪǵڄǵદჱǵࡏⷺᆶ BcjsbnjǹགᖴගٮӚԄፕޑഋറ.܃ܵ<གᖴࡏᆺ ޑຬសዕԋ܌ගٮޑЈᡫҬࢬ<གᖴਜࢋ๏ϒޑѕᓲᆶᜢᚶǶ׳ाགᖴ Fmgz ޑऍ კᆶ Effqb ЈঅुךޑमЎቪբǶाགᖴޑΓϼӭǴคݤಒኧǴӧԜᗫ ଇགᖴ܌ԖΓჹܭךޑႴᓰᆶྣ៝Ƕ!
!!!!നࡕǴགᖴךᒃངৎΓޑЍǴคፕࢂচғৎ܈ࢂӧޑৎΓǶЀځགᖴ ךޑќъ KbdlǴӧךᏢޑ೭ࢤය໔คค৷ޑбрᆶхǴيঋኧᙍޑᔅך
ֹԋ܌Ԗεελλޑ٣ǴΨࢂךനख़ाޑЍࢊǶགᖴჹёངٽζ.Bmmfo!'!BooǴ ԖΑգॺᡣך׳ԖΚࣁΑܴϺԶոΚǶ!
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!!!!ᙣаԜፕЎ๏ךനལངޑРᒃ௵ϻӃғǶ!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!లޱ!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!215 ԃ 8 Дܭύࣴଣғϯ܌!
!
ᖴӭӭӭӭӭӭӭӭӭӭӭӭӭӭӭӭӭӭӭӭΓޑΓޑΓޑΓޑΓޑΓޑΓޑΓޑΓޑΓΓޑΓޑΓޑΓޑΓޑΓޑΓޑΓޑΓޑΓޑΓΓޑΓޑΓޑޑбрбрбрбрбрбрбрбрбрбрбрбрбрбрбрбрбрбрбрбрбрбрбрррǶǶ Ծࣴزշޑޑޑޑޑޑޑޑޑғࢲ
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ᄔ ᄔा
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!!ဍዦᚯԝӢηD(Tumor necrosis factor-D, TNF-D)ࢂᅿӭфૈ܄ޑߦݹੱ
ಒझᐟનǴӧགࢉ܌ԋޑಒझཞύჹܭӃϺխࣝس(innate immune system) Ԗख़ाޑፓբҔǶӕਔ TNF-DڈᐟՈᆅϣҜಒझ(vascular endothelial cell)ᙖ җፄᚇޑಒझϣߞ৲ፓԋಒझวݹϸᔈࣗԿࢂેๆރฯϯ(atherothrombosis) аϷวݹ܄੯ੰ(inflammatory disease)ޑวғǶӧวݹϸᔈၸำύǴTNF-D௴
ೱՍಒझᐟ䁙(kinase)ޑૻ৲ሀǴࢲϯਡᙯᒵӢη(nuclear factor NB, NF-NB)Ǵߦ
ಒझ߄य़ᗹϩη(adhesion molecule)ޑ߄аϷࡕុқՈౚಒझ(leukocyte)ޑ ߕǶӧԜၸำύǴΓॺჹܭಒझᐟ䁙ޑфૈԖ࣬ჹޑΑှǴҞךॺ٠όమཱ
ೈқ፦ᕗለНှ䁙(protein phosphatase)ࢂցӕኬୖᆶፓ TNF-Dԋޑૻ৲ሀǶ ӧҁፕЎύǴךॺᚈ܄ᕗለНှ䁙(dual specificity phosphatases, DUSPs) ӧ TNF-DፓϣҜಒझวݹϸᔈύޑفՅתᄽǶᙖҗୀෳ୷Ӣ߄ޑmRNA֖ໆǴ ӧΓᜪϣҜಒझਲ਼ EAhy926 ύךॺפډဂ TNF-DᇨᏤ߄ޑ DUSPsǶךॺΨ ว TNF-DᇨᏤ߄ޑಒझᗹϩη(intercellular adhesion molecule-1, ICAM-1)ӧ
༾λ RNA υᘋ(RNAi)ԋޑ DUSP6 ୷ӢকନჴᡍύǴ߄ໆܴᡉΠफ़ǹࡕុ
ൂਡౚ(monocyte)ӧϣҜಒझ߄य़ߕޑኧໆΨᒿϐΠफ़ǴᡉҢ DUSP6 ӧፓว ݹϸᔈԖ҅ӛޑբҔǶךॺௗճҔΓᜪ߃жᙏᓉેϣҜಒझ(human umbilical vein endothelial cells, HUVECs)ٰࣴزፓᐒڋǶ่݀ᡉҢǴӧ TNF-Dڈᐟޑ HUVEC ಒ झ ύ Ǵ DUSP6 ᙖ җ ڋ ಒ झ Ѧ ૻ ဦ ፓ ᐟ 䁙 (extracellular signaling-regulated kinase, ERK)ޑࢲ ܄Զߦ NF-NB ޑᙯᒵࢲ܄аϷځΠෞ
ICAM-1 ޑ߄ǶӧλႵޑՈᆅಔᙃϪТࢉՅ(immunohistochemistry, IHC)ύǴךॺ
Ψᢀჸډ ICAM-1 ޑ߄ໆӧ DUSP6 ୷Ӣকନ(Dusp6-/-)λႵեܭഁғࠠλႵǴ
ჴ DUSP6 ዴჴתᄽߦՈᆅϣҜวݹޑفՅǶԜѦǴ࣬ၨܭഁғࠠλႵޑ௵གǴ DUSP6 ୷ӢকନλႵჹܭિӭᗐϣࢥન(lipopolysaccharide, LPS)܌ԋޑ௳Ո܄
ޤཞԖၨ٫ޑܢᑇૈΚǶ೭٤่݀ჴΑ DUSQ7 ԖߦϣҜಒझวݹϸᔈϷ วݹ࣬ᜢੰၸำޑཥᑉفՅǴᡉҢځբࣁݯᕍวݹ܄੯ੰᛰނ໒วޑཥࠨᐒǶ!
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ᜢᗖຒ:!ဍዦᚯԝӢηDǵϣҜಒझวݹϸᔈǵᚈ܄ᕗለНှ䁙ǵಒझ߄य़ᗹ
ϩηǵқՈౚǵ௳Ոੱǵޤཞ
фૈૈૈૈૈૈૈૈૈૈૈૈૈૈૈૈૈૈૈૈૈૈ܄ޑ܄ޑ܄ޑ܄ޑ܄ޑ܄ޑ܄ޑ܄ޑ܄ޑ܄܄ޑ܄ޑ܄ޑ܄ޑ܄ޑ܄ޑ܄ޑ܄ޑ܄ޑ܄ޑ܄ޑ܄ޑ܄ޑ܄ޑߦߦߦߦߦݹੱݹੱݹੱݹੱݹੱݹੱݹੱݹੱݹੱݹੱݹੱݹੱݹੱݹੱݹੱݹੱݹݹੱݹੱݹੱݹݹੱݹੱݹੱੱ e immununununununununununununnneeeeeeeeeee sysysysysysyyyyyyyyystststststststststsststeemeeee )
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ABSTRACT
Tumor necrosis factor alpha (TNF-D) is a proinflammatory cytokine that directs multiple events of the innate immune system during infection of cell injury. Meanwhile, TNF-D activates a diverse array of signaling pathways in vascular endothelial cells (ECs), leading to the inflammatory phenotype that contributes to the pathogenesis of atherothrombosis and inflammatory diseases. In a typical inflammatory response, TNF-D initiates a kinase-dependent signaling cascade, which activates nuclear factor (NF)-NB, leading to inducible expression of adhesion molecules and recruitment of leukocytes. In contrast to the known function of kinases in this context, it is not clear whether protein phosphatases participate in the regulation of TNF-D signaling. In the present study, we have investigated the role of dual specificity phosphatases (DUSPs) in TNF-D-induced inflammatory response. Using human endothelia, EAhy926, for screening of mRNA levels, we identified a group of DUSPs to be inducibly expressed under the TNF-D stimulation. Among them, DUSP6 functioned as a prominent positive regulator of the inflammatory response, evidenced by a clear decrease of TNF-D-induced expression of intercellular adhesion molecule-1 (ICAM-1) and a drastic reduction of monocyte adhesion on the surface of endothelia when DUSP6 was ablated via RNAi. We further examined the underlying mechanism controlled by DUSP6 using primary human umbilical vein endothelial cells (HUVECs). Our data showed that inducible DUSP6 promoted canonical NF-NB-dependent increase of adhesion molecules exclusively through inhibition of extracellular signaling-regulated kinase (ERK) in TNF-D-stimulated human ECs. The role that DUSP6 plays in facilitating endothelial inflammation in aorta and vein was confirmed by in vivo experiments using Dusp6-/- mice. Furthermore, genetic deletion of Dusp6 significantly reduced the susceptibility to inflammatory responses in a mouse model of lung sepsis. These results are the first to demonstrate a novel function of DUSP6 in the regulation of vascular inflammatory response and the underlying mechanism through which DUSP6 promotes endothelial inflammation-mediated pathological process. Our findings suggest that inhibition of DUSP6 holds great potential for the treatment of inflammatory diseases.
Keywords: tumor necrosis factor-D (TNF-D), endothelial inflammation, dual specificity phosphatases 6 (DUSP6), intercellular adhesion molecule-1 (ICAM-1), neutrophil, sepsis, lung injury
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ABBREVIATIONS
CD cluster of differentiation DUSPs dual specificity phosphatases ECs endothelial cells
ERK extracellular signaling-regulated kinase HUVECs human umbilical vein endothelial cells ICAM-1 intercellular adhesion molecules 1 IHC immunohistochemistry
INNB-D inhibitor of NB D IKK INB kinase IVC inferior vena cava JNK c-jun N-terminal kinase KIM kinase interacting motif LPS lipopolysaccharide
MAPKs mitogen-activated protein kinases MEK MAP kinase/ERK kinase
MKP MAP kinase phosphatase MPO myeloperoxidase NF-NB nuclear factor NB p38 MAPK p38 MAP kinase
PP4 protein phosphatase 4 TBP TATA-binding protein TNF-D tumor necrosis factor D
VCAM-1 vascular cell adhesion molecule 1
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TABLE OF CONTENTS
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ᇞᖴ ... i!
ᄔा ... ii!
ABSTRACT ... iii
ABBREVIATION ... iv
TABLE OF CONTENTS ... v
LIST OF FIGURES ... ix
LIST OF SCHEMES ... xii
LIST OF TABLES ... xii
CHAPTER 1: INTRODUCTION
... 11.1 The endothelium function and endothelial inflammation ... 2
1.2 TNF-D signaling in regulating endothelial inflammation ... 3
1.2.1 TNF-D induces cell adhesion molecules expression on endothelium ... 3
1.2.2 TNF-D activates canonical NF-NB pathway to regulate ICAM-1 expression .. 5
1.2.3 TNF-D-induced MAPKs activation in endothelial inflammation ... 5
1.3 Role of DUSPs in regulating MAP kinase and cell inflammation ... 7
1.4 Study the role of DUSPs targeting on ERK to regulate endothelial inflammation ... 9
CHAPTER 2: MATERIALS AND METHODS
...112.1 Reagents ... 12
2.2 Cell culture and transient transfection ... 12
2.2.1 Culture conditions for each cell line ... 12 .................................................................................................................................................................iiiiiiiiii
vi
2.2.2 Transient cell transfection ... 13
2.3 Immunoblotting and antibodies ... 14
2.4 RNA extraction and quantitative real-time PCR... 15
2.5 Monocyte adhesion assay ... 16
2.6 DUSP6 expression plasmids and luciferase reporter constructs ... 16
2.7 NF-NNB reporter assay ... 17
2.8 RNA extraction and Gene expression profiling ... 18
2.9 Animal studies ... 18
2.9.1 Mice housing ... 18
2.9.2 Genotyping ... 19
2.9.3 Tail vein injection with TNF-D ... 20
2.9.4 Immunohistochemstry staining and image quantification ... 20
2.9.5 LPS-induced experimental sepsis and neutronphil adoptive transfer ... 21
2.9.6 Neutrophil isolation from mouse blood ... 22
2.9.7 Flow cytometry analysis ... 22
2.10 Exploring DUSP6-mediated phosphorylation network in TNF-D-activated HUVECs by MS analyss ... 23
2.10.1 Sample preparation for MS/MS analysis ... 23
2.10.2 In-solution protein digestion ... 23
2.10.3 TiO2 beads enrichment ... 24
2.10.4 Immunoprecipitation for phosphotyrsine peptide enrichment ... 24
2.10.5 Shotgun proteomic identifications ... 25
2.10.6 Data analysis ... 26
2.11 Statistical analysis ... 26 ...............................................................................................1111111111111111113333 3333333333333333 .
. . . . . . . . . . .
..................................................................................1111144444 ...............................................................................................................................................................................1111111111111111111111115 55555555555555
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CHAPTER 3: RESULTS
... 27 3.1 TNF-DDtreatment triggers MAPKs transient activation rather than cellapoptosis in endothelial EAhy926 cells ... 28 3.2 DUSPs are inducibly expressed in endothelial cell exposed to TNF-D and
function as MKPs ... 29 3.3 DUSP6 involves in TNF-D-induced endothelial inflammation by regulating
intercellular adhesion molecules 1 (ICAM-1) expression ... 31 3.4 Inducible DUSP6 regulates TNF-D-directed inflammatory responses in
primary endothelial HUVECs ... 32 3.5 DUSP6-mediated termination of ERK activity is essential for TNF-D-induced
inflammatory response in endothelium ... 34 3.6 Inhibition of ERK by DUSP6 promotes NF-NB transcriptional activation
in endothelium exposed to TNF-D ... 36 3.7 TNF-D-induced ICAM-1expression on the endothelial layer of aorta and
vein is attenuated in Dusp6-/-mice ... 39 3.8 Deficiency of DUSP6 protects mice from acute lung injuries during
experimental sepsis ... 41 3.9 Pulmonary endothelial DUSP6 is essential for LPS-induced neutrophil
recruitment in mice ... 42 3.10 Exploring DUSP6-mediated phosphorylation network in TNF-D-activated
HUVECs by MS analysis ... 44
CHAPTER 4: DISCUSSION
... 48CHAPTER 5: FUTURE PERPECTIVES
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viii
CHAPTER 6: FIGURES
... 59CHAPTER 7: REFERENCES
... 96APPENDIX
... 104 List of identified phosphoproteins altered in DUSP6-ablated HUVECs............................................................................................................. . 595959595595959595959595959999999
.......................................................................................................................................................... ......9696969696969699969696999699699696969969996
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LIST OF FIGURES
Figure 1. TNF-DD treatment in EAhy926 does not trigger caspases activation
and cell apoptosis ... 60 Figure 2. Mitogen-activated kinases (MAPKs) were transiently activated in
endothelial EAhy926 cells stimulated with TNF-D ... 61 Figure 3. In EAhy926 cells, TNF-D regulates the activity of MAPKs through
transcriptional and translational regulation mechanism ... 62 Figure 4. Based on quantitative real-time PCR analysis, 12 DUSPs, typical
MKP, were classified to three groups by gene expression pattern upon
TNF-D stimulation ... 63
Figure 5. Based on RNA interference knockdown technique, DUSP6, 8,
and 16 were identified as both ERK and JNK phosphatases... 64 Figure 6. Inducible DUSP6 promotes expression of ICAM-1 in endothelial
EAhy926 cells stimulated with TNF-D ... 66 Figure 7. Transient expression of DUSP6 in HUVECs stimulated with TNF-D ... 67 Figure 8. Inducible DUSP6 is essential for expression of ICAM-1 in HUVECs
stimulated with TNF-D ... 68 Figure 9. The catalytic activity of DUSP6 is required for inducible ICAM-1
expression in HUVECs stimulated with TNF-D ... 69 Figure 10. Inducible DUSP6 is essential for endothelial leukocyte interaction
in HUVECs stimulated with TNF-D ... 70 Figure 11. DUSP6 functions as ERK phosphatase in HUVECs stimulated
with TNF-D ... 71 Figure 12. Inactivation of ERK by chemical inhibitors promoted ICAM-1
expression in HUVECs stimulated with TNF-D ... 72 ac
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Figure 13. Ablation of ERK by RNA interference promoted ICAM-1 expression in HUVECs stimulated with TNF-DD ... 73 Figure 14. Inactivation of ERK restored ICAM-1 expression in DUSP6
RNAi-ablated HUVECs stimulated with TNF-D ... 74 Figure 15. Inactivation of ERK by DUSP6 is required for inducible expression
of ICAM-1 in HUVECs stimulated with TNF-D ... 75 Figure 16. DUSP6 regulates ICAM-1 expression in a transcriptional-dependent
manner in HUVECs stimulated with TNF-D ... 76 Figure 17. NF-NB is major regulator of ICAM-1 expression and DUSP6 ablation
does not affect NF-NB activation in HUVECs stimulated with TNF-D ... 77 Figure 18. NF-NB-directed transcriptional activation of ICAM-1 gene depends
on termination of ERK signaling by inducible DUSP6 in HUVECs stimulated with TNF-D ... 78 Figure 19. Inactivation of ERK by DUSP6 is required for inducible expression
of ICAM-1 in HUVECs stimulated with TNF-D ... 79 Figure 20. Ablation of DUSP6 reduced endothelial ICAM-1 expression in vitro
after prolonged TNF-D treatment ... 80
Figure 21. A loss of DUSP6 expression and an increased phosphorylation of ERK in liver and lung isolated from Dusp6-/-mice ... 81 Figure 22. Specific ICAM-1 staining was observed on aorta and inferior vena
cava (IVC) in the wild type (WT) mice treated with TNF-D ... 82 Figure 23. Ablation of DUSP6 reduced endothelial ICAM-1 expression in vivo
after prolonged TNF-D treatment ... 83
Figure 24. Deficiency of DUSP6 reduced lung injury and pulmonary neutrophil infiltration during experimental sepsis ... 84
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. . . . . . . . . . . .
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xi
Figure 25. The effect of irradiation on leukocyte removal in mice ... 85 Figure 26. Purification of polymorphonuclear leukocytes (PMNs) for the
adoptive transfer of neutrophils in lung during experimental sepsis ... 86 Figure 27. DUSP6 deficiency-reduced neutrophil infiltration in lung is
pulmonary endothelium intrinsic ... 87 Figure 28. TNF-DD-induced mRNA profile of DUSPs in HUVECs by microarray
analysis ... 88 Figure 29. NF-NB-directed transcriptional activation of VCAM-1 gene depends
on termination of ERK signaling by inducible DUSP6 in HUVECs stimulated with TNF-D ... 89
Figure 30. Proposed model for the functional role of endothelial DUSP6 in
regulating vascular inflammation ... 90 Figure 31. Sub-network indicates proteins involved in cell junction and focal
adhesion derived from IPA analysis ... 92 ............................................................................................. .. 858585858585858585858858585858585555 fo
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xii
LIST OF SCHEME
Scheme 1. Quantitative phosphoproteomic workflow ... 91
LIST OF TABLES
Table 1. Primers used for quantitative real-time PCR analysis ... 93 Table 2. Oligonucleotides of siRNA used for dusp6 knockdown ... 94 Table 3. List of the idenfified up-regulated phosphoproteind in
DUSP6-ablated HUVECs ... 95 ................................................................................................999991 1 1 1 1
1
CHAPTER 1: INTRODUCTION
2
1.1 The endothelium function and endothelial inflammation
As a semipermeable barrier lining on the internal surface of blood vessels, the
endothelium regulates vascular tone as well as the exchange of fluids and solutes
between the blood and interstitial space, thus maintaining physiological homeostasis.1
Vascular endothelium also exerts anticoagulant, antiplatelet, antiproliferation of smooth
muscle cells and fibrinolytic properties. Therefore, a healthy endothelium not only
controls vasodilation, but also suppresses vascular inflammation, thrombosis, and
hypertrophy.2
In addition, the endothelium is an integral component of host innate immune
response. Vascular endothelia are uniquely situated to detect the presence of pathogens
within the vasculature as they are in direct and constant contact with the circulating
blood.3, 4 When microbial infections or tissue injury occurs, a large amount of
damage-associated molecular patterns (DAMPs) are released and they stimulate the
pattern-recognition receptors (PRRs) on immune cells. The activated immune cells
release excessive amount of pro-inflammatory cytokines to induce nearby endothelial
cells inflammation, causing the up regulation of cell adhesion molecules on endothelial
cell surface to recruit and activate leukocytes at sites of inflammation.5 Although the
leukocyte adhesion cascade ultimately helps to clear the infectious agents and to repair
damaged tissues, during disseminated infections or inflammatory disorders the
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activation of the endothelium at sites remote from the inciting source can lead to the
dysregulation of a variety of microvascular functions, causing organ failure and
subsequent death.6, 7
Tumor necrosis factor (TNF)-D is a pro-inflammatory cytokine, which is
synthesized primarily by immune cells such as macrophages, dendritic cells, monocytes
and T lymphocytes, to induce endothelial inflammation.8 Accumulating evidence suggests that TNF-D plays a pivotal role in disrupting macrovascular and microvascular
circulation both in vivo and in vitro, which causes endothelial inflammation and
vascular dysfunction and eventually contributes to pathogenesis of many chronic
inflammatory disease.9 Anti-TNF-D treatment has been applied to a range of
inflammatory conditions, including rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease and psoriasis, highlighted the role of TNF-D in infectious
diseases.10 Understanding the molecular machine in how TNF-D regulates endothelial
inflammatory response may provide further opportunity to treat inflammatory diseases.
1.2 TNF-D 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
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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
M M M M M M M M M M M M M M
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ily of trannnnnnnscscsccriririririiiiiiiiption
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 a a a a a a a a a a a a a a a a a
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ssion2929999999999999-3131313131331313313131313313111HHHHHHHHHHoooooooowever
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 l l l l l l l l
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ni ni ni ni ni ni ni ni ni ni ni nii ni ni ni n
nnnennenennnennennnenenenn aaaaandndndndndnd ttttttyyyryry ososoososo inininininnnnnnnnnnnnnnne eee eeeee e e
n Duallllllll ssssssspepeppepeppppppecicicicicicciciciciciiiififififififififfiffffcity
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.
ndddddddddddddddd ppppppppppppppppppp333838383338383333333 aaaaactctctivivivivivititititityyyyyyyyyy...44444444444
he he he he he he he he he he he hee he he he h
hesisisissisisssssioooonononononoooooooooooooo mmmmmolololololececcccculululululeseeeeseseseeseeeeseeseesees ss
n HUVECECEECECECECECECECCCCCCssssssss44545454545454555MMMMMMMMMiMMMMMce
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 a a a a a a a a a a a a a
at t t t t t t t t t t t te e e e e e e e e e e e e e e e e e ee e e e e e e e e e e e e e e e e e e e e e e e e en n n n n n nd d d d do o o o ot t th h h he e e e el l l l li i i i ia a a a a a a a a al l l l l l l l l l l l l l l l l
efore thehehhhhhhehh sssssstatatataaaaaaaartrtrtrtrrrrttttttt of 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
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DU DU DU DU DU DUU DU DU DU DUUUU DU DU DUU
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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
u u u u u u u u u u u u u
urerererererererererereeeeeee pppppppppppppppppppppplalalalalallll tetetetetete aaaaaandnndnnn tttttheheheheheeeeeeeeeeeeeennnnnnnnnnnn
hours foffffff lllllllllllllllllllllowowowowowowowwwwwwwededededededddddd by
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.
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2))))))))))))))))),,,huhuhuhuhuhuhuhuhuhuhuhuuuuuuuuuuuuumamamamamammmmann n n ICICICICICICAMAMAMAMAMA ---1111111111111
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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
in in in in inn in in in innnn in in in
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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
V2 V2 V2 V2 V2 V2 V2 V2 V2 V2 V2 V2 V2 V2
V22 vvvvvvvvvvvvvvvvvveeeecececeeceeceeeeeee tototototor r rr r (S(S(Sigigigigigmamamamamaaaaaaaaaaa).).).).).).).).).).).))))))).
d d d d d d d d d d d d d
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mutant cononoonononononnnnnstststttttrrururururrrrrur ct (a
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
in in in in in in in in in in inn
instststststststststststststssstsstrurururururururrurrrururrurrr ctctctctctioioioioionsnsns.... CeCeCeCeCelllllllllllllllllllllllllllllss s s sss s s ss s
T T T T T T T T T T T T T T T T
TNFNFNFNFNFNFNFNFNFNFNFNNFNNFNFNNFFFFF---DDDDD 101010000 nng/g/g/g/g/g/mlmlmlmlmlmlmlmlmlmlmlmlmlllllllll) ) )) ) ) ))) ) ))) ))
ferase acccctitititttititittttiivivivivviiiiiiitytttytytytytytytytt and
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.
0 0 0 0 0 0 0 0 0 0 0 0 0
09090909090909090909090909069000000006969696969696969696696966666969.h99.h.h.h.htmtmtmtmtmlll) ) ) wewewewewereeeeeererererrererrererrrrrrrreeee
ceeeeeeeeeeeeeeeee wwwwwwwwwwwwwwwwwererererereeeeee e e e eee brbrbrbrbrbrededededed aaaaandndndndndndndndndndndndndndndnnndddd
om subjbjbjbjbjbjbjbbjbjjeececececeeecececcteteteteeeeeeeddddddddddddddd to a
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
(1 (1 (1 (1 (1 (11 (11 (1 ( ( (11 ( (1
( 0-0-0-0-0-0-0-000-0-121212121211111111 weweweweweweekekekekeke ssssssololololdldddddddddddddddddddd) ) )) ) ) ))) ) ))) ))
otal vollummmmmmmmeofofofofoffffffffff 100
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
For induction of the experimental sepsis, male mice (5-8 weeks old) were injected intraperitoneally with 0.1 mg/kg of TNF-D or 10 mg/kg of LPS in a total volume of 200
Pl. After 24 hours, mice were sacrificed. Lung were isolated and processed for
myeloperoxidase (MPO) determination by MPO-specific enzyme-linked
immunosorbent assay (ELISA; HyCult Biotechnology) according to manufacturer’s
instruction. For neutrophil adoptive transfer, male mice (10-12 weeks old) were utilized.
The endogenous polymorphonuclear leukocytes (PMNs, mainly neutrophils) of
recipient mice were removed by irradiation (9 Gy) exposure. After recovery for 24
hours, 1x107 purified neutrophils in PBS (total volume 200 Pl) were adoptively
transferred to recipient mice by intravenous injection, followed by intraperitoneal injection with 10 mg/kg of LPS in a total volume of 200 Pl. After 4 hours, mice were
sacrificed. Lung were isolated and processed for H&E staining, immunohistochemistry
staining with anti-ICAM-1 antibody and MPO assay.
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22
2.9.6 Neutrophil isolation from mouse blood
The procedure of neutrophil isolation was performed according to the protocol
described previously.58 In brief, whole blood from adult donor mouse was collected in
tubes containing EDTA and then mixed with an equal volume of PBS. The cells were
separated onto a three-layer Percoll gradient of 78, 69, and 52% Percoll diluted in PBS
through centrifugation at 1500x g for 35 min at room temperature. The fraction of
neutrophils at the 69/78% interface were harvested and washed with PBS containing 1%
BSA once. The residual red blood cells were then eliminated by RBC Lysis Buffer
(Becton Dickinson) at 37ɗ for 3 min. After two times of wash with PBS containing
1% BSA, the purified neutrophils were suspended in PBS and used immediately. The
purity and viability of purified neutrophils was confirmed by Ly6G/CD11b double
staining and trypan blue (Sigma) exclusion, respectively.
2.9.7 Flow cytometry analysis
Cells were incubated with Ly6G-FITC (11-5931), CD11b-PerCP-Cyanine5.5
(45-0112) or isotype control antibodies (11-4031 and 45-4031, all from eBioscience)
against cell surface antigens in the dark for one hour on ice. Cytofluorimetry was
performed with a BD Calibur cytometer (Becton Dickinson) equipped with FL1
(533/30), FL3 (650LP) filters. Neutrophils were identified by characteristic forward/side g
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23
scatter and Ly6G/CD11b positivity. Data were analyzed and presented using the BD
CellQuest Pro software (Becton Dickinson).
2.10 Exploring DUSP6-mediated phosphorylation network in TNF-D D-activated HUVECs by MS analysis
2.10.1 Sample preparation for MS/MS analysis
HUVECs were cultured in ready-to-use SILAC DMEM medium containing 13C
labeled arginine (L-13C615
N4-arginine, Arg10) and lysine (L-13C6-lysine, Lys6) amino
acids for five cell division cycles before performing DUSP6 knockdown. 24 hours after knocking down, cells were treated with TNF-D for 1.5 hours then harvested and lysed in
1% NP40 buffer.
2.10.2 In-solution protein digestion
Equal amount (3 mg) of total cell lysates from normal (light) and DUSP6-KD (heavy)
HUVECs were combined into one pool. Lysate mixture was reduced with 1 mM
dithiothreitol (DTT) for 1 hour at room temperature (RT) and alkylated with 5.5 mM
iodoacetamide (IAM) for 1 hour at RT in the dark. Excess detergent, DTT, and IAM
were removed by Amicon Ultra-4 10K centrifugal filter unit, and buffer was exchanged
to 8 M urea. Proteins were digested for 3 hours with the protease Lys-C (1:100 nt
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24
enzyme/substrate) at 37°C. Sample was diluted with 50 mM ammonium bicarbonate to
reduce urea concentration less than 2 M, and trypsin (1:100) was added for further
digestion at 37°C overnight. The peptide mixture was acidified by adding trifluoroacetic
acid (TFA) to a final concentration of 2.5%.
2.10.3 TiO2beads enrichment
Twenty percent of digested peptide pool was mixed with loading buffer (1:6 v/v, 30
mg/ml 2,5 dihydrobenzoic acid and 80% acetonitrile in water) and incubated with 5 mg
TiO2 beads for 30 minutes at RT for twice. TiO2 beads were washed with washing
solution I (30% acetonitrile/3% TFA) and II (80% acetonitrile/0.1% TFA).
Phosphopeptides were eluted 2 times with 100 Pl elution solution I (1% of NH4OH in
20% acetonitrile) and 1 time with 100 Pl elution solution II (1% of NH4OH in 40%
acetonitrile). Eluates were dried and then resuspended in 1% acetonitrile/0.5% TFA.
2.10.4 Immunoprecipitation for phosphotyrsine peptide enrichment
Eighty percent of digested peptide pool was dried and resuspended in IP buffer (50
mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCl). Peptide mixture was
incubated with PT66 and 4G10 agarose beads at 4°C overnight. Beads were washed 3
times with IP buffer, followed by 2 washes with water. Phosphotyrsine peptides were iumumumumumumumumumumumumummmmmmmmm bbbbbbbbbbbbbbbbbbicicicicicarararararbobobobb nananananatetetetetetttttttttttttttttoo o o ooo o o oo o
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25
eluted by adding two times 50 Pl of 0.15% TFA for 10 min at room temperature. Eluted
peptides were then desalted and concentrated on C18StageTip and resuspended in 1%
acetonitrile/0.5% TFA.
2.10.5 Shotgun proteomic identifications
1DQR/&íQDQR(6L-MS/MS analysis was performed on a nanoAcquity system (Waters,
Milford, MA) connected to an LTQ-Orbitrap XL hybrid mass spectrometer (Thermo
Fisher Scientific, Bremen, Germany) equipped with a nanospray interface (Proxeon, Odense, Denmark). Peptide mixtures were loaded onto a 75 Pm ID, 25 cm length C18
BEH column (Waters, Milford, MA) packed with 1.7 Pm particles with a pore width of
130 Å and were separated using a segmented gradient in 120 min from 5% to 40%
solvent B (acetonitrile with 0.1% formic acid) at a flow rate of 300 nl/min and a column
temperature of 35°C. Solvent A was 0.1% formic acid in water. The mass spectrometer
was operated in the data-dependent mode. Briefly, survey full scan MS spectra were
acquired in the orbitrap (m/z 350–1600) with the resolution set to 60,000 at m/z 400 and
automatic gain control (AGC) target at 106. The 10 most intense ions were sequentially
isolated for CID MS/MS fragmentation and detection in the linear ion trap (AGC target
at 7000) with previously selected ions dynamically excluded for 90 s. Ions with singly
and unrecognized charge state were also excluded. For TiO2 enriched samples, e
e e e e
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26
“multistage activation” at 97.97, 48.99, and 32.66 Thomson (Th) relative to the
precursor ion was enabled in all MS/MS events to improve the fragmentation spectra of
the phosphopeptides. All the measurements in the orbitrap were performed with the lock
mass option for internal calibration.
2.10.6 Data analysis
Phosphopeptides with false discovery rate under 1% were identified and quantified
by MaxQuant (version 1.2.2.5). Only high confident phosphopeptides with the
localization probability of phosphorylation (pSTY) greater than 0.75 from the two
enrichment methods were retained and a list of phosphoproteins from the
phosphopeptide results was generated for functional annotation. The proteomics data
analyzed by LTQ-Orbitrap XL hybrid mass spectrometer were performed by the
Academia Sinica Common Mass Spectrometry Facilities located at the Institute of
Biological Chemistry.
2.11 Statistical analysis
Values were expressed as means ± SD. Statistical significance was determined using
a Student’s t-test. A P-value <0.05 was considered significant.
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