Effects of PD on IL -6- induced Growth Arrest and Term ina l D ifferen tia tion of M 1 Cells
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1 ISSN CN gQ Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy 17 (1): PD IL -6 M 1, 3,,, (, ) IL 26 M 1 R asgm A PK, M EK PD R asgm A PK, PD IL 26 M 1 PD M 1, IL 26 M 1 PD IL 26 M 1 CD 11b, IL 2 6 M 1 R asgm A PK, JA K2STA T s, IL 26 R asgm A PK, PD ,, R 392 Effects of PD on IL -6- induced Growth Arrest and Term ina l D ifferen tia tion of M 1 Cells ZHAN G J i2yan, SH EN Bei2fen 3,L I Yan, SUN Y ing2xun, YU M ing (Institu te of B asic M ed ical S ciences, A cad emy of M ilitary M ed ical S ciences, B eij ing , Ch ina) Abstract To investigate the b io logical ro les of R asgm A PK pathw ay in IL 262induced M 1 leukem ia cells, the con stitu tive and inducib le activation of R asgm A PK pathw ay w as b locked w ith M EK specific inh ib ito r PD and the effects of PD on IL 262induced grow th arrest and term inal differen tiation ofm 1 cells w ere determ ined. PD w as found to inh ib it the grow th of M 1 cells by itself and enhance IL 262induced grow th arrest of M 1 cells. A lthough PD had little effects on the m o rpho logical changes and CD 11b exp ression of M 1 cells induced by IL 26, it reduced IL 262induced p hagocy t ic act ivity of M 1 cells sign ifican t ly. T hu s the con st itu t ive and inducib le activation of R asgm A PK pathw ay w as im po rtan t in IL 262induced m acrophage differen tiation of M 1 cellṡ Key words R asgm A PK pathw ay, PD , grow th, differen tiation [ 4 R asgm A PK ] STA T 3 RNA, STA T 3 STA T 3, IL 26 M 1 [ 1 ] IL 26 R asgm A PK (ER K1 ER K2) gn F2IL 6 [ 2 ], R asgm A PK IL 26 : , : (N o ) 3 T el: (010) , Fax: (010) IL 26 M 1 R asg M A PK,M 1 IL 26,, ,, Received:M arch 24, 2000 A ccep ted: June 19, 2000 [ 3 ], Suppo rted by N ational Science Foundation fo r distinguished young IL 26 Scho lars, N o Co rresponding autho r T el: (010) N akajim a STA T 3 IL 26 M 1 Fax: (010) ,
2 116 17, IL 26 M 1 R asg m in M A PK IL 26? 116, R asgm A PK, PD [ 5 ] R asgm A PK (2 lgm l, 0160 m ) 37, 5% CO M 1 A TCC, 10% FCS PD B io lab s, DM SO, 20 mm o lgl ER K2 San ta C ruz 118 W r ight s F ITC CD 11b [4 ] Imm uno tech rh IL 26 ( 011% BSA PBS ) 10 7 U gm g, rh IL m in, PBS 1 RN ase A PM SF g, (20 mm o lgl T ris2c l ph 810, Sigm a, dn T P K lenow P rom ega, [Α2 32 P ]da T P 1% N P240, 1 mm o lgl PM SF, 10 m ggl pep statin, PA GE A, 10 m ggl leupep tin), 4 30 m in g 113 (EM SA) rh IL m in, 4 2 h, P ro tein A 2agaro se 4 K lenow 5 (20 mm o lgl T ris2c l ph 8. 0, [ Α2 32 P ]da T P (3 000 C igmm o l) 137 mm o lgl N ac l, 10% ) 4 6, (10 20 g ) 10 fm o l (cpm 5 000) 10% SD S2PA GE. 20 m in 4 ER K2, 15%, 0125 TA E (1 TA E 40 mm o lgl T ris2c l ph 7. 8, 1. 1 mm o lgl ED TA, 37 mm o lgl ), : A PR E ( STA T 3) = 5 2 cgat CT TCT GGGAA T TCCTA 23 [ 6 ] ; A T FgN F2IL 6 ( N F2IL 6 ) = 5 2ctgaGA T GA CGTA GT T T T GGCGCT 23 [ 7 ] ;, M A PK (ER K1 ER K2) gn F2IL PD , IL 26 STA T 3; R asgm A PK M 1 96, g PD h rh IL 26; M 1 R asgm A PK 72 h, M T T, 4 h, 10% SD S20101 m o lgl HC l, A CD 11b 4 h, Ca 2+ M g 2+ PBS 3, %, 10 6 gm l, 4 18 h, PBS P I P I 50 m ggl, 01002% T riton X U gm l RN ase A PBS, 30 m in mm o lgl N ac l, 10%, 2 mm o lgl N a3vo 4, 15 m in, ER K2 SD S 10 m in, PD Ra sgm APK IL 26 JA K2STA T s R asg, JA K2STA T s [ 2, IL 26 N F2IL 6 ], M A PK N F2 IL 6 DNA, R asgm A PK, STA T 3 IL 26, 1 g F ITC, STA T 3, M A PK CD 11b 30 N F2IL 6 DNA
3 1 : PD IL 26 M (F ig. 1, 2), IL 26 JA K2STA T s R asg M A PK, IL 26 STA T 3, N F2IL 6 IL 26 (F ig. 1) F ig. 1 Do se course of nuclear facto rs activated by incubation w ith rh IL 26 fo r 15 m in F ig. 2 Effects of PD on the tyro sine pho spho rylation of M A PK (A ) and on the DNA binding ability of STA T 3 and N F2IL 6 (B) IP: imm unop recip itation; IB: imm unoblo tting; pt yr: pho spho tyro sine PD R asgm A PK R asgm A PKgN F2IL 6 M EK, PD IL 26 M 1 [ 5 M EK M EK ],, IL 26 IL 2 R asgm A PK 6, PD IL 26, 100 m o lgl PD ER K2 M A PK (F ig. 2A ) ; EM SA, A % ) ; IL 26 PD N F2IL 6 DNA, 100 ggl, PD IL 26 M 1 STA T 3 DNA (F ig. 2B ),, PD , PD R asg IL 26 N F2IL 6 M A PKgN F2IL 6, (F ig. 3B ) JA K2STA T s PD M PD M 1 ( PD ggl IL 26 M 1 M 1 PD M 1,, 50% S,, (F ig. 3A ), M 1 (< 1% ) 100 m o lgl PD
4 F ig. 3 Effects of PD on the grow th (A ) and IL 262induced grow th arrest (B)of M 1 cells PD 0 mo lgl ; PD 50 mo lgl ; PD 100 mo lgl ; PD 200 mo lgl F ig. 4 Effect of PD on the cell cycle redistribution of M 1 cells induced by 100 ggl rh IL 26 S, M A PK (F ig. 4, 3 ), M 1 PD m o lgl, R asgm A PK, R asg ( ) F ig. 3 M A PK IL 26 PD , PD IL PD IL -6 M 1 IL 26, PD PD M ggl IL d m o lgl, PD IL 26 PD IL ggl, IL 2 M 1, ; 6 ( ) IL 26 IL 26,, 100 ggl, 100 m o lgl PD , 100 m o lgl PD IL 26 IL 26 S, (F ig. 5A ), (F ig. 4, ), R asg
5 1 : PD IL 26 M F ig. 5 Effects of PD on the mo rpho logical changes (A ) and CD 11b exp ression (B) of M 1 cells induced by 100 ggl rh IL 26 M ggl IL d CD 11b 100 m o lgl PD M 1 CD 11b, ; 100 ggl IL 26, CD 11b, (F ig. 5B ) 3, 3 t, (t= 1, P > 0105) M 1, IL 26 M 1, 100 ggl IL 26 4 d, 80% M 1 PD IL 26 M 1,,, R asgm A PK 100 m o lgl PD M 1 (F ig. 6), F ig. 6 Effect of PD on the phagocytic activity of M 1 cells
6 IL 26, IL 26 PD ? JA K2STA T s R asgm A PK JA K2STA T s, IL 26 STA T 3, JA K1 STA T 3 N F2IL 6 ino S, IL 26 [ 2 ] PD M EK STA T 1 STA T 5 STA T N F2IL 6,,, R asgm A PK, IL 26 R asgm A PK (Grb2gSO S R as R af21 M EK M A PK) N F2IL 6 STA T IL 26 R asgm A PK,,, [ 2, 8 R asgm A PK ], STA T 3 c2m yc c2m yb JunB IR F1 P 19 IN K4D P 21WA F1gC IP1, [ 8 ] STA T 3 RNA N akajim a STA T 3 IL 2 [ 4 6 M 1 ] IL 26 M 1 R asg M A PK, IL 26 M 1 R asgm A PK?, R asgm A PK PD M 1, IL 26 M 1 (F ig. 3, 4), R asgm A PK, STA T 3 ;, R asgm A PK,M 1 IL 26, CD 11b (F ig. 5), (F ig. 6), R asgm A PK, R asgm A PK STA T 3 PD STA T 3 DNA (F ig. 2), R asgm A PK STA T 3, STA T 3 IL 26 M 1, STA T 3, STA T 3 R asgm A PK R asgm A PK JA K2STA T s, IL 26, N F2IL 6 knock ou t,, ino S,, (References) 1 Sugium R, Toda T, D hut S, Shuntoh H, Kuto T. T he M A PK kinase Pek I acts as a pho spho rylation2dependent mo lecular sw itch. N atu re, 1999, 399 (6735) : A k ira S. IL 262regulated transcrip tion facto rs. Int J B iochem Cell B iol, 1997, 12: ,,,. IL 26 M 1. (Zhang J i2yan, Shen Bei2fen, W ang J ian2an, Zhang Xue2m in. A pop to sis of M 1 cells is induced by IL 26 after the cells undergo differentiation and its mo lecular m echanism. Imm unol J ), 2000, 16 (1) : N akajim a K, Yam anaka Y, N akee K, Ko jim a H, Ich ibam, Kiuch i N, Kitaoka T, Fukada T, H ibi M, H irano T. A central ro le fo r STA T 3 in IL 262induced regulation of grow th and differentiation in M 1 leukem ia cellṡ EM B O J, 1996, 15: Sengup ta T K, T albo t E S, Scherle P A, Ivashk iv L B. Rap id inh ibition of interleuk in26 signaling and Stat3 activation m ediated by m itogen2activated p ro tein k inases. P roc N atl. A cad S ci U SA, 1998, 95: W egenka U M, Buschm ann J, L utticken C, L tticken C, H einri2 ch PC, Ho rn F. A cute2phase responsive facto r, a nuclear facto r binding to acute2phase responsive elem ents, is rap idly activated by interleukin26 at the po sttranslational level. M ol Cell B iol, 1993, 13: H su W, Kerppo la T K, Chen P L, Curran T, Chen2Kiang S. Fo s and Jun rep ress transcrip tion activation by N F2IL 6 through association at the basic zipper region. M ol Cell B iol, 1994, 14: H irano T, N akajim a K, H ibi M. Signaling m echanism th rough gp130: a model of the cytokine system. Cy tok ine & G row th f actor R ev, 1997, 8 (4) : ,
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