IRUCAA@TDC : 骨芽細胞におけるテロメア結合タンパクおよびタンキラーゼの局在性

全文

(1)Title. 骨芽細胞におけるテロメア結合タンパクおよびタンキラーゼの局在性. Author(s). 遠藤, 隆行; 佐藤, 大輔; 澁川, 義幸; 石原, 和幸; 橋本, 貞充; 吉成, 正雄; 松坂, 賢一; 田﨑, 雅和; 井上, 孝. Journal URL. 歯科学報, 108(2): 117-121 http://hdl.handle.net/10130/475. Right. Posted at the Institutional Resources for Unique Collection and Academic Archives at Tokyo Dental College, Available from http://ir.tdc.ac.jp/.

(2) 77=. . ¢. |d~¤@/2HT]YKo{SÛO /ECS^NZ$RAl a!y'&(& }¡'&)& °gw'&(& r®w'&*& k¥ '&+& h¨'&,& q`'&)& "e®'&(& _. v'&)&. #¯«A¦x=-HT]YKB. VU@gF"l{omusµ©Z¦["³À. TTAGGG AmG 5¬0F?G"T]YKo{. YLA"àÛLPéíkõRT$ßkòBMg"A. SÛO%TRF1"TRF2& >o{5<.H!S^N. jcg QOL Z¾äk>ÕLT Î#Yµ©MgJ. Z$RB3A TRF JW[ ADP [VQ\cJ6H. VVb¹CdB!JZPaY["ÆIÝZ`kûã. z=-H!¥pi=B"nuSÛOJª.<. VMgZU[XH"íúY¥Jg"í6 #X¸ã. |d~¤©~¤ MC3T3-E1 @/2H TRF2 >S^. kïLdB"VAB-ób1CU@iBVÂCT. NZ$RAlJD:!PLK^nuSÛO. Ag!. %CFP& >bnu S ^ U O %YFP& J TRF2 >. OJU:unr}ZSVLT"_N[L. S^NZ$RAX$M$>5<"8I9Iª.:!. ZÍ9)tsYSAT!^T`gJVVLP!. TRF2-CFP >S^NZ$R-YFP Jjs47:|. Í97$öæ"l}'tvXW["Í9)YD. d~¤©~¤ MC3T3-E1 @/.<"TRF2 Bf. AT@&XzrÚFô»ÏAkDAMgJVY. @l5"`£S^NZ$RB~¤@. dRT"Í9¸ãk-¸Mg!LELXFe"L. 5<.H3>1§F0@?;:!. ZÍ9)ts[=eEYXRTAXA!. . California ü Z McCullough V Kelly [ L k. t. JB"¦LTAg!. FÈZ<;ÌäZÆIé½[866<+Y864Yý. +Aging is a complex biological process that is a. LP!8686+Y[8;4"MXjQÈ;Z:éYé. consequence of both intrinsic or genetically pro-. [ÆIÝVAB"ESTWZêZWZÈb®¶L. grammed aging that occurs with time"and ex-. XERPÆIÜk°CgJVF?øKhTAg!. trinsic aging caused by environmental factors# ,. JZdBX§ùXÆIZè%Ydf"FÈYD. MXjQ"/LV[1:Ú#Dd]&#Y. IgL+Ö XWZ£KGZáCëF_M_MÆ. sKhP"×¢®V«Y¥JgL2Dd. _RTGTAg!IØú[þLb.IehXAJ. ]"1:Ú#YU[XH"¡¬ÓYdf¥JgL 2 "OZFÝZ±¥Jg"5ÒXí6 #. q''~%|l±Çzr"zq'y"¯¼ zr"I 'ªÖü ½Ã µ©xz' HRC77."íE Å Ë8."H â ´ É µ © Ù9."/ í 6 Å Ë:."2 E Å Ë;."ÖE¿ ÅË<. -866=+78²7?*Þ4. -866>+7²7;*ÞE. 8Ñî¨ð%58<7)>;68 ñBÔ03çÊ73838 'ªÖü íE ÅË (!Ä. xvU@g! 0VABbZU@g7.! JZdBX5ÒX¸ãU@gLVAB¸ãk7 Ó ZpwkÿÐMgPaY"Fs '[|lVABÁ÷6"Dd]OZ¤,k _N=MgJVVLP! }ZìåúÍ9[<ºY[7JUGN";6*=6 7JMh\7JUGXHXg!JZ·[|. ( 89 (.

(3) 118. ¨V" @$TRF Tr&~XÊí-. jXFÿW<e! jY5(BXxGW<ep. iÈGK PARP TJRìuZW?íKeGT. Þ á < S < e!DNA B k 0 I f e T C") /. B{cAWVPO21(25*!r&~Y:xG¤. DNA êX5xGYKÎWe\RFD"KÎê5x. W PARP kiKe21"26*!. GYk0ÝWlÍf"ÓÊ")/ê#KÎêT`. ò¯#W@=R"ë`¥dRV×%Y¼è. k0HTW5xGB50(150©ÅLQFÿKeXS. XS<g>!ò²iÛQJR=eò9èBNX. <e!IcW" jY"¹wWjq&u. Ç_iP¬IM"òýfVUX¦ðT^æPR". IfR=eGTBIcfR@d"ìulÆW@E. ò²DiÈGK]SW1úKeGTBADXå. e5(BlWþ VÇ_i°OJR=e ![. ùW@=RÂïIfe!NGS"éµ±%Y". X jY"5-TTAGGG-3XÌdoJÀ. yuX´{X]L¥qTJR"èÏìuW@. cVe DNA Wbdá.If"DNA &z"5. Ee TRF"r&~XÊí-iM\O!. 2*. (Bæ"5(BXgQiÈGKb>V5(B. "!#$. xGX!iv=S=e3(5*! [SY"ìul"Z=RYWQfR jXFÿaÇ_&·Bb×Ke. éµ±%Yè³ìuW@Eeyui®ñ. GTAc". Ke}RS"TRF2 £Tð@b[r&~£. ìuXuW jBÃJR=eT. TðX:xGWÐàt÷£TðiQVFO. â?cfR=e. t&iî0JR"aè³ìu¾ MC3T3-E1 ì. 6(11*. !GfcXGTAc"+ . 12" 13*. jxk|Ylµ,iÑ=KelôÑS<e, ". uWY\JO!. + jFÿBlüzXÙ¶iÒ_e,TØh. Ë^"tvAcÐàt÷BbÖIf"N. feGT`<e!]O"lm&$ßÍ)Werner. X£TðBEIfOGTWbd"GXt. syndrome* "#so&8$. ÷" GFP)Green fluorescent protein* Bkpk&. )Progeria* "&$ßÍ)Bloom syndrome* ". zutX¥stTJRADIfeb>WVP. | ì Ô Á ¸ L - § W õ M $)Ataxia telangecta-. O!IcWm ¡ Y \ W b d BFP)Blue fluorescent. sia* "4 S - » ¡ ' $)Dyskeratosis congenita*. protein *" CFP) Cyan fluorescent protein *" YFP. VUXö¿SY"Õ'+We\RNX j. )Yellow fluorescent protein* VUXq&n&. XFÿ>UB>D"8ÆWBYIfe!Gf. y`îcfO27*!NGS";xG.TRF2.x. cX8Æ$ßÍSYÉOJR5(BXgQ. kjÐàt.:xGXt&iáJJ. " jNXFÿ"ÄWCKeb$Àû-*. O!TRF2 Open Biosystems cDNA t & "ID. BrçIfR=eGTAc" jB5(BX¢. 4346819"Accesion No. BC024890i K Î T J"5. óaìuXÄSþ V~½i°OJR=eGTB. Hind""3 BamH!xkiQE"{w. ÚBS`º]IfR=e !. i"D CDS )753bp* i PCR ;jJO!;jHni. 14*. jhX DNA YFêci`Sá.IfR =R"GXh T:Üîiã> j DNA Ó. pPS-CFP2-N t&)evrogen ø* XXxkW in frame S7\JO!. æt ÷ B telomere-repeat-binding factor )T-. ]O";xG.r&~.«(Ðàt. RF* TzyIfR=e!TRF Y jNXM3. .:x G X t & ` á J J O! r & ~. WÃJR=e15(17*!. Open Biosystems cDNA t&"ID4374347"A-. ADP yY" ADP &{æ. ccesion No. BF979695i K Î T J"5 Sac!"3. .ä6)PARP* Bwjjzt. Age!xkiQE"{wi"D CDS. p)NAD * iÅ÷TJR"ADP &{ij. )3981bp* i PCR ;jJO!;jHni pPS-YFP-N. t}&t÷WÓæIM"IcW2d!I. t&)evrogen ø* XXxkW in frame S7. fO ADP &{i α1/2uwyÓæWb. \JO!. -. dþæIMeä6cªS<e. !r&~T. GfcX{inti=. =>t÷B TRF WÓæJ ADP y. Raè³ìu¾ MC3T3-E1 ìuW{m. 18(20*. ' 24 '.

(4) ºú. 図１. Vol#+*2"No#,(,**2). 骨芽細胞 MC3T3-E1 に TRF2-CFP とタンキラーゼ-YFP を共発現させ，サイアン蛍光タンパク質を励起，観察したもの。バー：４０μm. 図２. ++3. 骨芽細胞 MC3T3-E1 に TRF2-CFP とタンキラーゼ-YFP を共発現させ，黄色蛍光タンパク質を励起，観察したもの。バー：４０μm. ? I 5 :"± û ½ H ë ! J ¾ ÷ J R ² A. `cuzZ«ED!. W-."-/)!gz_v%fKNK²L TRF HKÔ. %$!#"&'. ¦³"Z#@"CXJL TRF K NLS ls^z:. ©¢òJTW´JTV"ï$¯±û. À#IþZD@F5W,2)!É~èJ NLS ls. MC3T3-E1 ±ûJ8=W TRF2 8TMgz_v%. ^zZ@Dgz_v%fZð£@D±ûJ85. fK²Ì:ýU9JIED!TRF2 L±ûJ. FLiys\KÈã:QUXD-0)!. ²@(Ê+) "gz_v%fL±ûÒÛJõô@F5. °§>KtjxZ%"@"!$IÆ G'gz. D(Ê,) !Ñ Â K ä V"TRF2 L/TTAGGG3. o`½Kh^mrci]ZâOF5;D5Hª7F. KVø@î)HAW>HR">KZ¸». 5W!. AWRKH·YXW!. PD"íÃZð£@Diys\L\qk%c. gz_v%fL±û¾J}Ù@F""ÏÇ. eJTW±û¹Z>A-1'-3)!¿EF"°§L\q. Û"bxdÕßHCK|ßK²:ö@F5<>. k%ceKcamxgzo`½HKÔRâO. H:"HeLa ±û8TMÐ±ûJ85Fú®?. F5;D5Hª7F5W!. XF5W. !>XUK>H9UR"gz_v%. (HIU[G"üÌ "å*ÌÜá "üÌ. fKê;Liys\K»J&PUI5>H:ËØ. GL"±û"ÜáÄñ±ûSÄñ±ûKi. ?XW!CX¥"gz_v%fKíLiys\ã. ys\ÝÁ:ú®?X"üÌ¼JTWiys\K. KâÎJ¤UXF5I5RKHª7UXF5W!. ÝÁ:ú®?XD.*)!ï$ÍÅ±ûS±ûGLi. ,0" ,2'-*). iys\Liysv%fH56¬ÓJTEFù¨. ys\ÝÁ:ÏÇÛìKé¶Kó{çÌZ×?. ?XW>HRÞUXF5W !gz_v%fJTW. BW>H:ÞUXF5W:"üÌ¼GRë!G4. TRF Kqw ADP wpcxL"iysv%fK. W>H:ª7UXW!°§L?UIW¡H@F". DNA íZæ?BW. TRF Sgz_v%fKSüÌ¼HKYVR. -+). !. ,+'-,). Ú<K"¾÷J²PDLNK±ûìÖZ. ¡@F5;D5!. ð£AWgzo`½L"CK\rnµî)àJ ²camx(NLS) ls^zZÿ@F5W--)!gz_ v%fLCK\rnµî)àJ NLS ls^zZÿ & ,/ &. % ( ï$¯±û MC3T3-E1 ±ûJ85F"TRF2.

(5) -.,. zÑ" Ä$TRF Jivbr'hN ». OàM D"ivbr'hOàÀÅMÝÚ DH7YAJ<ãW;JLGF!Oå&Lì| £"é9P¢|fmtfU¨¾"_vi'u_ bvLKMVGH"·N.¯Nivpc¬<ì| NàÒafd'nJLY;K8;\ÊQH7>! M"® Ã I O 6 Y <".,,1Ô N Lancet © M"ÖâUyLKN¼³<kuq]Ë\È> EYJ78<ÕÙCZ"Éä\´SH7 Y0-+!ë|\¼=YFSMT"å&LÝ¤¼ Ü ÌL`w^lvfT±´D"QOL N·MÏ SH7>Q=I6YJ¥[ZY! ANOÐª~Æ ~ gvi' HRC7 NÞ Û~ ¶o_kcse'jgvi'«MVY×Nßµ \°?F! áíÞNç¦O"Ç.40Ðª~Æ Â*.,,3Ô-, .,Ó"½æ+ M:7HÕÙD"Ëº¿\°?FTNI6 Y!. " ! \²9YM6FX"áM:?YxÍ¤Á¡MB¹è {C7RDFÐª~Æ Ø¼Ü îÎ}§M> ê¸D·@RE!. ,. +. -+McCullough, JL, Kelly, KM : Prevention and treatment of skin aging ; Ann NY Acad Sci "!'($/./)//-".,,2# .+de Lange, T : Human telomeres are attached to the nuclear matrix ; EMBO J ""$3-3)3.0"-55.# /+Counter, CM, Avilion, AA, LeFeuvre, CE, Stewart, NG, Greider, CW, Harley, CB, Bacchetti, S : Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity ; EMBO J ""$-5.-)-5.5"-55.# 0+Blasco, MA, Lee, HW, Hande, MP, Samper, E, Lansdorp, PM, De Pinho, RA, Greider, CW : Telomere shortening and tumor formation by mouse cells lacking telomerase RNA ; Cell *"$.1)/0"-553# 1+Hande, MP, Samper, E, Lansdorp, P, Blasco, MA : Telomere length dynamics and chromosomal instability in cells derived from telomerase null mice; J Cell Biol "%%$ 145)2,-"-555# 2+Blackburn, EH : Structure and function of telomeres ; Nature $&!$125)13/"-55-# 3+de Lange, T : Telomeres and senescence : ending the debate ; Science #(*$//0)//1"-554# 4+Dandjinou, AT, Dionne, I, Gravel, S, LeBel, C, Parenteau, J, Wellinger, RJ : Cytological and functional aspects of telomere maintenance ; Histol Histopat Hol "%$1-3) 1.0"-555# 5+ Campisi, J : The biology of replicative senescence ; Eur J Cancer $$$3,/)3,5"553# -,+Sedivy, JM : Can ends justify the means% : telomeres. and the mechanisms of replicative senescence and immortalization in mammalian cells ; Proc Natl Acad Sci USA *&$5,34)5,4-"-554# --+Sherr, CJ, DePinho, RA : Cellular senescence : mitotic clock or culture shock% ; Cell "!#$0,3)0-,".,,,# -.+Harley, CB, Futcher, AB, Greider, CW : Telomeres shorten during ageing of human fibroblasts ; Nature $%&$014)02,"-55,# -/+Harley, CB : Telomere loss : mitotic clock or genetic time bomb% ; Mutat Res #&'$.3-).4."-55-# -0+Blasco, MA : Telomeres and human disease : ageing, cancer and beyond ; Nature Reviews Genetics '$2--) 2..".,,1# -1+Bilaud, T, Brun, C, Ancelin, K, Koering, CE, Laroche, T, Gilson, E : Telomeric localization of TRF2, a novel human telobox protein ; Nat Genet "($./2)./5"-553# -2+Broccoli, D, Smogorzewska, A, Chong, L, de Lange, T : Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2 ; Nat Genet "($./-)./1"-553# -3+Smogorzewska, A, de Lange, T : Regulation of telomerase by telomeric proteins ; Annu Rev Biochem 3/$ -33).,4".,,0# -4+Jeggo, PA : DNA repair, PARP-another guardian angel% ; Curr Biol )$R05)R1-"-554# -5+Lindahl, T, Satoh, MS, Poirier, GG, Kiungland, A : Posttranslational modification of poly *ADP-ribose+ polymerase induced by DNA strand breaks ; Trends Biochem Sci #!$0,1)0--"-551# .,+Bouchard, VJ, Rouleau, M, Poirier, GG : PARP-1, a determinant of cell survival in response to DNA damage ; Exp Hematol $"$002)010".,,/# .-+Smith, S, Giriat, I, Schmitt, A, de Lange, T : Tankyrase, a poly *ADP-ribose+ polymerase at human telomeres ; Science #)#$-040)-043"-554# ..+Kaminker, PG, Kim, SH, Taylor, RD, Zebarjadian, Y, Funk, WD, Morin, GB, Yaswen, P, Campisi, J : TANK2, a new TRF1-associated poly *ADP-ribose+ polymerase, causes rapid induction of cell death upon overexpression ; J Biol Chem #('$/145-)/1455".,,-# ./+Cook, BD, Dynek, JN, Chang, W, Shostak, G, Smith, S : Role for the related poly *ADP-Ribose+ polymerase tankyrase 1 and 2 at human telomeres ; Mol Cell Biol ##$ //.)/0.".,,.# .0+Rippmann, JF, Damm, K, Schnapp, A : Functional characterization of the poly *ADP-ribose+ polymerase activity of tankyrase 1, a potential regulator of telomere length ; J Mol Biol $#$$.-3)..0".,,.# .1+Sbodio, JI, Lodish, HF, Chi, NW : Tankyrase-2 oligomerizes with tankyrase-1 and binds to both TRF1*telomererepeat-binding factor 1+and IRAP *insulin-responsive aminopeptidase+; Biochem J $'"$01-)015".,,.# .2+Chi, NW, Lodish, HF : Tankyrase is a Golgi-associated mitogen-activated protein kinase substrate that interacts with IRAP in GLUT4 vesicles ; J Biol Chem #(&$/40/3 )/4000".,,,# .3+Tsien, RY : The green fluorescent protein ; Ann Rev Biochem '($1,5)100"-554# .4+Smith, S, de Lange, T : Cell cycle dependent localization of the telomeric PARP, tankyrase, to nuclear pore complexes and centrosomes ; J Cell Sci ""#$/205) /212"-555# .5+van Steensel, B, Smogorzewska, A, de Lange, T : TRF2. ( .2 (.

(6) 4235. Vol")(0!No"*&*((0'. protects human telomeres from end-to-end fusions ; Cell *##,()%,)+!)110" +('Karlseder, J, Broccoli, D, Dai, Y, Hardy, S, de Lange, T : p53 and ATM-dependent apoptosis induced by telomeres lacking TRF2 ; Science #)$#)+*)%)+*-!)111" +)'Blackburn, EH : Telomeres ; Trends Biochem Sci "'# +/0%+0)!)11)" +*'Loayza, D, de Lange, T : POT1 as a terminal transducer of TRF1 telomere length control ; Nature %#$# )()+%)()0!*((+" ++'Hodel, MR, Corbett, AH, Hodel, AE : Dissection of a nuclear localization signal ; J Biol Chem #('#)+)/% )+*-!*(()" +,'DAmours, D, Desnoyers, S, DSilva, I, Poirier, GG : Poly &ADP-ribosyl' ation reactions in the regulation of nuclear functions ; Biochem J $%##*,1%*.0!)111" +-'Seimiya, H, Smith, S : The telomeric poly &ADP-ribose' polymerase, tankyrase 1, contains multiple binding sites for telomeric repeat binding factor & 1 TRF1' and a novel acceptor, 182-kDa tankyrase-binding protein &TAB182'; J Biol Chem #((#),)).%),)*.!*((*" +.'Smith, S, de Lange, T : Tankyrase promotes telomere elongation in human cells ; Curr Biol "!#)*11%)+(*!. )*). *(((" +/'Hahn, WC, Stewart, SA, Brooks, MW, York, SG, Eaton, E, Kurachi, A, Beijersbergen, RL, Knoll, JH, Meyerson, M, Weinberg, RA : Inhibition of telomerase limits the growth of human cancer cells; Nat Med &#)).,%))/(!)111" +0'Herbert, B, Pitts, AE, Baker, SI, Hamilton, SE, Wright, WE, Shay, JW, Corey, DR : Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death ; Proc Natl Acad Sci USA *'#),*/.%),*0)!)111" +1'Karlseder, J, Broccoli, D, Dai, Y, Hardy, S, de Lange, T : p53 and ATM-dependent apoptosis induced by telomeres lacking TRF2 ; Science #)$#)+*)%)+*-!)111" ,('OSullivan, IJN, Bronner, MP, Brentnall, TA, Finley, JC, Shen, WT, Emerson, S, Emond, MJ, Gollahon, KA, Moskovitz, AH, Crispin, DA, Potter, JD, Rabinovitch, PS : Chromosomal instability in ulcerative colitis is related to telomere shortning ; Nat Genet $##*0(%*0,!*((*" ,)'Valdes, AM, Andrew, T, Gardner, JP, Kimura, M, Oelsner, E, Cherkas, LF, Aviv, A, Spector, TD : Obesity, cigarette smoking, and telomere length in women ; Lancet +..#..*%..,!*((-". Localization of TRF and tankyrase in osteoblast-like MC3T3-E1 cells Takayuki ENDOH, Daisuke SATO, Yoshiyuki SHIBUKAWA Kazuyuki ISHIHARA, Sadamitsu HASHIMOTO, Masao YOSHINARI Kenichi MATSUZAKA, Masakazu TAZAKI, Takashi INOUE Oral Health Science Center HRC7, Department of Physiology, Tokyo Dental College Key words : Telomere-repeat-binding factor, Tankyrase, Green fluorescent protein, Aging Short running head : TRF and tankyrase in bone cells. Telomeres of mammalian chromosomes are composed of long tandem repeats TTAGGG which bind in a sequence-specific manner two proteins-telomere-repeat-binding factor!TRF1 and TRF2" Tankyrase was a poly &ADP-ribose' -polymerase that is initially identified through its interaction with TRF" In this study!we analyzed the subcellular localization of TRF2 and tankyrase in living osteoblast-like MC3T3-E1 cells by using fusion proteins with different spectral variants of green fluorescent protein &GFP' " Two fluorescent proteins!cyan fluorescent protein &CFP' and yellow fluorescent protein &YFP' were used as markers to visualize TRF2 and tankyrase!respectively" Osteoblast-like MC3T3-E1 cells co-transfected with TRF2-CFP and tankyrase-YFP showed that TRF2CFP appeared to be concentrated at distinct subcellular structures &presumably the telomeres' !whereas tankyrase-YFP was uniformly distributed throughout the cytoplasm" &The Shikwa Gakuho!"!)#))/%)*)!2008'. $ */ $.

(7)