comparison of il262induced stat1 activation in different by luckboy

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									106

ISSN1007 - 8738 ˇ‚ߺ•ˆ§

( Chin J Cell Mol Immunol) 2003 , 19 ( 2)

: 1007 - 8738 ( 2003) 02 - 106 - 03

Department of Molecular Immunology , Institute of Basic Medical Sciences , Academy of Military Medical Sciences , Beijing 100850

ferent target cells.

fect on the tyro sine2pho sphorylation of STAT1. On the other †»˝‹•·ƒ—ˇ‚ß—

ner in M1 , R2 and U937 cells which underwent growth arre st and The se re sults sugge st that IL262induced STAT1 activation might Keywords : IL26 ; STAT1 ; tyro sine2pho sphorylation

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”˝¿„–¸Æ`¸Æ»fl „ß : IL26 ¿·‰ł U937 ˇ‚ß º˚–…— ¡£ ¶ł IL26 „…·˚ ˝‹˜œ˛§—§ƒ ¡£ —˝…•”¯

¶ STA T1 ˜ –¸Æ`¸Æ»fl¸fi˘‰ˆ»—ˆ ˇ ˇ ¡£ 3 ˇ‚ß—ø¿…⁄»

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Aut hor : ZHAN G Ji2yan ( 19722) , female , born in Heilongjiang Province signaling mechanism. Tel. (010) 66931325 , Email :shenbf @mx. cei. gov. cn Corresponding aut hor : SHEN Bei2fen.

help to explain why IL26 exerts distinct biological effects in dif2

macrophage differentiation upon IL26 induction. CONCL USION :
Received date : 2002 - 11 - 18 ; ¡¡ revised date : 2002 - 12 - 03

hand , IL26 activated STAT1 in a do se2 and time2dep endent man2 be a ssociated with growth2inhibitory effects , and our data might

and activator of transcription 1 ( STAT1) induced by interlerkin26

analysis wa s p erformed with antibodie s against STAT1 and tyro2 growth2promoting effects on 7TD1 and TF1 cells but had little ef2 sine2pho sphorylated STAT1. RESUL TS : It¡fl found that IL26 had s

Comparison of IL262induced STAT1 activation in different responsive cells
Abstract
AIM : To inve stigate the activation status of signal transducer ( IL26 ) in different re sponsive cells. METHODS : Immuno blot
Fund : This project is supported by national fund for distinguished young scholars(No . 39925019)

Z HA N G Ji2yan , L I Y an , R EN Y u n2f ang , S H EN Bei2f en

in a wide variety of tissues and organs , including t he immune subunit shared by t he receptors for t he IL26 family of cytokines. nucleus , and regulate a set of genes[ 1 ] . maintains pluripotent proliferation of embryonic stem cells[ 4 ] .

hematopoietic and nervous systems. gp130 is a signal transducing The binding of IL26 to its receptor induces dimerization of gp130

, Ł ¡¡

, ¨˛•…

: ‰†»˝‹•·ƒ—ˇ‚ß—ˇ‚߉ظ

1 ( STA T1) ˜…⁄» ¡£ STA T1 ˜¿„

7 TD1 ”˝ TF1 ˇ‚ßœ‡⁄

: IL26 ; STA T1 ; –¸Æ`¸Æ»fl

: R392. 1 ¡¡¡¡¡¡¡¡˛˜ˇ–Œ˚¶´º

nehe county , associate research , Ph. D ; majoring in cytokine

IL26 …ˇ´œ‡⁄˝£„†¢ˇ ˚ˇ‚ß•‰ˇ•»fl

‰Æ´: STA T1 ˜»»fl¿˜º ˘—§ƒ—„ ¡£

IL26 ¶ ST AT1 …⁄»˜–¨‰ˇ
, –¶•
, ––' ( ˚´‰§¿˘§”»ø 100850) •‰•¤: ߈¿„ , ‰ł——ˆ ¡…£•˛ ¡£ , « `‰ˇ‚ß— IL26 †»˝‹—§ƒ—ˇ‚ß——†» :A

STA T1 , ˙…⁄»—§ƒ—…``¿

6 ( IL26 ) ¶—¯”¯“

STA T1

‰Æ

observed in M1 cells[ 2 ] .

M1 ¡¢ ”˝ R2

,

of IL26. Tyrosine2p hosp horylated STA Ts form dimers , enter t he the signaling mechanism through gp130. It¡fl found that activation of s of STA T3 activation varied between t hem[ 527 ] . These data sug2 gest t hat t he heterogeneity of STA T activation might contribute ated wit h growt h2inhibitory effects. to t he multifuctionality of IL26. In t his work , we furt her investi2 data suggest t hat IL262induced STA T1 activation might be associ2 BAF2B03 [ 3 ] and for t he L IF2mediated signaling pat hway t hat inhibitory factor ( L IF ) 2induced growt h arrest and macrop hage alt hough t he cells respond to IL26 quite heterogeneously. In our subline L T12) , 7 TD1 ( mouse2mouse B cell hybridoma) and TF1 monoblast leukemia cell line) , R2 ( human IL26 receptor ƒ` gene duces t he same activation status of STA Ts in different target cells in M1 ( murine myeloblast leukemia cell line) , U937 ( human transformants of t he Brown Norway acute myelocytic leukemia nisms and biological functions , it is not clear whet her IL26 in2 ( human eryt hroleukemia cell line) cells. IL262induced activation required for gp1302mediated survival of t he mouse pro2B cell line STA T3 and STA T5a is absolutely necessary for IL26/ leukemia The biological roles of STA T1 in t he signaling of IL26 are not as In spite of t he extensive studies of STA T activation mecha2

clear as STA T3 , and no evident roles of activated STA T1 were

previous studies , we determined t he activation status of STA T3

of STA T3 was observed in all of t he five cell lines but t he duration

way and Ras/ MAP K ( mitogen activated protein kinases) pat h2 way , which are controlled by distinct regions of gp130. It is well known t hat STA Ts play essential roles in t he signal transduction

and activation of gp1302associated JA Ks (Janus tyrosine kinases) activation of multiple signal transduction pat hways such as JA K/

t hrough transp horylation , which results in t he p hosp horylation of

tyrosine residues on gp130 and JA Ks. These events lead to t he STA Ts ( signal transducers and activators of transcription) pat h2

differentiation of M1 leukemia cells[ 2 ] . Furt hermore , STA T3 is

gated t he activation status of STA T1 in t he five cell lines. Our

¡¡¡¡ Interleukin26 ( IL26) is a pleiotropic cytokine. It plays pivotal roles

Much has been done in studying the biological roles of ST ATs in

ISSN1007 - 8738 ˇ‚ߺ•ˆ§

( Chin J Cell Mol Immunol) 2003 , 19 ( 2)

107

MATERIALS AND METHODS
Cell culture ¡¡ M1 ¡¢ 937 ¡¢ U TF1 and 7 TD1 cells were obtained from lished by Ren YF et al . t hrough transferring human IL26 recep2 torƒ` chain cDNA into rat leukemia cells L T12
[8] ¡¡ .

STA T1 in t he five cell lines , immunoblot analysis was performed p horylation of STA T1 was determined. It¡fl found t hat IL262in2 s duction had little effect on t he protein amount of STA T1 in 7 TD1 and TF1 cells. There was a basal level of tyrosine2p hosp horylation ( Fig 2) . t he cells were stimulated wit h IL26 for 5 minutes , a significant Fig 1 ¡¡ Effects of IL26 on growt h of 7 TD1 , TF1 ( A ) , M1 , U937 and R2 (B) cells in 7 TD1 and TF1 cells IL262induced activation of STAT1 in M1 , R2 and U937 cells ¡¡ ¡¡ ¡¡ƒ (rh IL26) / mg¡⁄L - 1 ¡¡¡¡¡¡ ¡¡¡¡¡¡
ƒ

cultured in RPM I1640 medium supplemented wit h 100 ml/ L IL26 ( 10
10

centration of 5 ¡` 10

ort hovanadate , 0. 5 mmol/ L PMSF , 10 mmol/ L NaF , and 3 mg/ L each of pepstatin , leupeptin , chymostatin and aprotinin : Sigma) . The lysates were centrifuged at 12 000 r/ min for 10

lysis buffer ( 5 ml/ L N P2 40 , 10 mmol/ L Tris2HCl , p H 7. 4 , membranes were blocked wit h 50 g/ L skimmed milk for 1 hour at STA T1 or tyrosine2p hosp horated STA T1 ( Santa Cruz ) . After 37 ¡ , and t hen were incubated wit h polyclonal antibodies against veloped using t he ECL chemiluminescene reagents.

in 100 ml/ L FCS2RPM I1640 and seeded into 962well plate and (48 hours for TF1) . Afterwards , 10 ƒ L M T T ( Sigma , 5 g/ L ) mazan. The samples were measured at A 570 nm value by an EL ISA reader ( Dynatech Laboratories , Inc. USA) . Immunoblot analysis ¡¡ After IL26 induction , t he cells ( 5 ¡` 105 ) cells underwent growt h arrest upon IL26 induction ( Fig 1B) . Different effects of IL26 on growth of different target cells ¡¡ 7 TD1 and TF1 cells ¡¡ To investigate t he activation status of were collected , washed and lysed for 20 min in 20 ƒ L ice2cold was added to each well. 4 hours later , 100 g/ L SDS210 mmol/ L t hey could be divided into two groups. IL26 promoted growt h of subjected to 100 g/ L SDS2PA GE. Proteins on gel were t hen

cultured in t he presence of varying doses of rh IL26 for 72 hours min. After protein determination , t he supernatants were boiled in gated goat2anti2rabbit antibody and washed again. Blots were de2 7 TD1 and TF1 cells ( Fig 1A ) . However , M1 , U937 and R2 Effects of IL26 on the tyrosine2phosphorylation of STAT1 in HCl of equal volume was added to dissolve t he blue crystals of for2

cells , ƒ´2mercaptoet hanol and recombinant human granulocyte2 added into t he medium to an eventual concentration of 5 ¡` 10 mol/ L and 5 ƒ g/ L , respectively. 2 ¡` reducing SDS loading buffer of equal volume for 10 min , and washing , t he membranes were incubated wit h peroxidase2conju2 macrop hage colony stimulating factor ( GM2CSF , 1010 U/ g) were
5

150 mmol/ L NaCl , 0. 4 mmol/ L ED TA , 1 mmol/ L sodium

transferred to nitrocellulose membranes at 40 V for 3 hours. The

t he American Type Culture Collection. R2 cell line was estab2 The cells were MTT incorporation ¡¡ The cells were starved in serum2free RPM I1640 for 5 hours. Then t he cells were washed , resuspended

FCS. For 7 TD1 cells ,ƒ´2mercaptoet hanol and recombinant human

RESUL TS

These five cell lines responded to IL26 quite heterogeneously , but

U/ g) were added into t he medium to an eventual con2
- 5

mol/ L and 5 ƒ g/ L , respectively. For TF1

ƒ g/ L of IL26 , and t he level of tyrosine2phosphorylation was aug2 thermore , the duration of ST 1 activation were also investigated. AT duced activation of ST 1 lasted over 60 minutes too ( Fig 3) . AT mented when the dose of IL26 increased. However , only high dose of

of STA T1 in 7 TD1 cells , but not in TF1 cells. And IL26 had lit2
p2STA T1 : Tyrosine2phosphorylated STA T1.

protein amount of STA T1 in M1 , R2 and U937 cells. There was a basal level of tyrosine2p hosp horylation of STA T1 in R2 cells , but not in M1 and U937 cells. In M1 and R2 cells , it¡fl found s t hat IL26 activated STA T1 in a dose2dependent manner. When

increase of tyrosine2p hosp horylation of STA T1 was detected at 1

tion with 100 ƒ g/ L IL26. The maximal level maintained until 60 tectable at 5 minutes after stimulation with 100ƒ g/ L IL26 , IL262in2 duced ST 1 activation lasted over 60 minutes. In R2 cells , IL 62in2 AT 2 minutes. In U937 cells , ST 1 activation was rapid and already de2 AT

tle effect on t he tyrosine2p hosp horylation of STA T1 in t he two Fig 2 ¡¡ Effects of IL26 on t he tyrosine2p hosp horylation of STA T1
(rh IL26) / mg¡⁄L 1

cell lines , even if t he incubation time was prolonged up to 6 hours

and time/ dose course of changes in IL262induced tyrosine2p hos2

As in 7 TD1 and TF1 cells , IL262induction had little effect on t he rapid and the maximal level was observed at 5 minutes after stimula2 IL26 (100 ƒ g/ L ) could barely activate ST 1 in U937 cells. Fur2 AT

In M1 cells , IL262induced tyrosine2phosphorylation of ST 1 was AT

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ISSN1007 - 8738 ˇ‚ߺ•ˆ§

( Chin J Cell Mol Immunol) 2003 , 19 ( 2)

ferent from anot her.

fect on t he tyrosine2p hosp horylation of STA T1. On t he ot her ‰˝˜‚†»˜†œœØ–¸Æ tion might be associated with growth2inhibitory effects. The biological however , its roles in the signaling mechanism through gp130 are not Why IL26 exerts distinct biological effects in different target , †˛º†¡¶¿¯`£˜—˛‡ ¡£ CT ˇ , »„˚¿„˜˚ N1N2 „ƒ˜˙ł , †»˜‚—¨ˇ‚œ ¡£¨»”‰«¿„›»ø , —‚—¨˜`ƒ ¡£·¸˝¤„ N1N2 »ø¨”ˇ„„‰¤¿„›¿ ¡£»—–¿„›ºˇƒ¿„‰Æ N1N2 „ƒ˜ , ˚œ†¯»‚· ( mDHFR) »¥†„˘‹¶˛ , •–º¿„› , »—–¿„ mDHFR »ø•‡¶‚˘‹¶˛ , ¶‚ mDHFR »ø˘‹¶˛»¥†„—˛‡—» DHFR , ˇ‚߆¯˜·» ¡£ ¢ ¿„› LexA ˜ Vp16 †¡¶“´……⁄»˙ł , »•·fi ¡£—' „†“¨¨–ˇ—˝‰˝˜‚ˇ‚ß ,« LexA DNA ‰Æ”ˇ˛»ªˇ´˛”‹ , ˜˘¶fl clear , our work has provided helpful information. vide some helpful information. IL26 induction results in different another. Eventually different cell lines exhibit different reactivities activation status of STA T3 and STA T1 in different res ponsive cells[ 527 ] . Thus , t he equilibrium relationship between promoting

p2STA T1 : Tyrosine2phosphorylated STA T1. ns : Nonspecific ladder.

D ISCUSSION

duction[ 527 ,9 ] . These data suggest that IL262induced ST 1 activa2 AT

cells is an important question yet to be resolved. Our results pro2

accordingly , t he activation status of STA T1 in one cell line is dif2 However , similarities exist .

( ˇ‰ 105 ‡ ) ¨»ø¶¸˜„ƒ˜˙ł CT ”‹—·'˜⁄ˆ“¶¤˙ł
œˆ»— º ”ˇ”

‚ø»ªŁ…˘‰«¿„¿„˚

¡æ—‚—¨¿ˆ‰ —¿„› ¡¢„ ¡£ ¢˜¿¶¿‚¡ ¿ ……˚ı ‚ˆ•‰•¤˚˙»ø ˚¶˙¶¸Æ»„›ˆ‚ , ˜»‚· ¡£Ł…˘‰«˚ ›ºˇƒ¿„‰Æ”ˇ˚– —˜

»ø”˝¿„»ø¨”ˇ„„‰¤‡¶‚¿ ¡£„†“¨ˇ‚ß

‰«—¿„›¿¸´¡·‡ƒ‚¸œ ‰¤¿„›¿ ¡£¿„»ø¿¸´¡ „„‰¤¿„¿

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growt h2promoting effects on 7 TD1 and TF1 cells and has little ef2

undergo growth arrest and macrophage differentiation upon IL26 in2

roles of ST 1 in interferon signaling have been well established , AT

signals and inhibitory signals in a certain cell line differs from one

hand , ST 1 is activated by IL26 in M1 , R2 and U937 cells which AT

Fig 3 ¡¡ Effects of IL26 on t he tyrosine2p hosp horylation of STA T1 in M1 , R2 and U937 cells These five cell lines respond to IL26 quite heterogeneously ,

2 ≠ ∑

DNA ‰Æ”ˇ 3¡ ¶¸

his »ø˜

IL26 has

to IL26. However , furt her investigations are needed to make clear target cells.

why IL26 induces different activation status of STA Ts in different

REFERENCES :

[1 ] Hirano T , Nakajima K , Hibi M. Signaling mechanism t hrough gp130 : a model of t he cytokine system [ J ] . Cytoki ne & Grow t h
Factor Rev , 1997 , 8 (4) : 2412252.

[ 2 ] Nakajima K , Yamanaka Y , Nakee K , et al . A central role for Stat3 in IL262induced regulation of growt h and differentiation in M1 leukemia cells[J ] . EMB O J , 1996 , 15 (14) : 365123658. 17 (8) : 4492 452. 14 (4) : 2622264. 1994 , 2 (1) : 352 41.
m unol) , 2001 , 17 (1) :16219. ( Chi n J I m m unol) , 2000 , 16 (4) : 1732177.

[ 3 ] Shirogane T , Fukada T , Muller J M M , et al . Synergistic roles for apoptosis[J ] . I m m unity , 1999 , 11 (6) : 7092719. (6) : 120721217.

≈ , ˚ˇ¨ ≥
, „„ 5¡ ¶¸ , ,

his3

[ 7 ] Zhang J Y , Li Y , Shen BF. IL26 selectively activates STA T3 in U937 [ 8 ] Ren YF , Guo N , Zhang HQ , et al . Introduction and expression of duces differentiation of R2 cells of rat acute myeloid leukemia[J ] . Xi

[ 6 ] Zhang J Y , Shen B F , Li Y , et al . Activation status of IL262related nuclear factors in acute myeloid leukemia cells and t heir biological roles[J ] . Zhongguo Mianyi x ue Zaz hi ( Chi n J I m m unol ) , 2001 , cell[J ] . Zhongguo S hiyan X ueyex ue Zaz hi ( Chi n J Ex p Hem atol) ,

[ 9 ] Zhang J Y , Ren YF , Shen BF. Interleukin 6 inhibits growt h and in2
B ao Y u Fen Zi Mian Yi X ue Za Zhi ( J Cell Mol I m m unol) , 1998 ,

[ 5 ] Zhang J Y , Shen B F , Li Y , et al . Comparison of IL262activated nu2 clear factors in M1 and 7 TD1 cells[J ] . Zhongguo Mianyi x ue Zaz hi

[ 4 ] Boeuf H , Hauss C , De Graeve F , et al . L IF2dependent transcription activation in embryonic stem cells [ J ] . J Cell Biology , 1997 , 138

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