N: nucleus
FFFiiiggg...222...IIInnncccrrreeeaaassseee ooofffttthhheee ppp---SSSTTTAAATTT333 ppprrrooottteeeiiinnn wwwaaasss aaaccccccooommmpppaaannniiieeeddd wwwiiittthhh ttthhheee iiinnncccrrreeeaaassseee ooofff ppp---SSSTTTAAATTT333 ppprrrooottteeeiiinnn iiinnn ttthhheee nnnuuucccllleeeuuusss... NIH3T3 cells were transfected with Trk-Metsm and harvested following treatment with NGF(100ng/ml).Thecellswerefractionated intothecytoplasmicand nuclear portion,andtheSTAT3proteinwasmeasuredbywesternblot analysiswith anti-phosphoSTAT3antibody(Ser727).Theblotswerestrippedandre-probed withanti-tubulin(cytoplasmicmarker).
FFFiiiggg... 333... TTThhheee dddeeelllaaayyyeeeddd ppphhhooosssppphhhooorrryyylllaaatttiiiooonnn ooofff SSSTTTAAATTT333 wwwaaasss bbbllloooccckkkeeeddd bbbyyy ppprrreeetttrrreeeaaatttmmmeeennnttt wwwiiittthhh cccyyyccclllooohhheeexxxiiimmmiiidddeee iiinnn TTTrrrkkk---MMMeeetttsssmmm tttrrraaannnsssfffeeecccttteeeddd NNNIIIHHH333TTT333 ccceeellllllsss...CulturedNIH3T3cellswereeitherpretreatedwith 10ug/mlor15ug/ml cycloheximide for 1hour and then incubationed NGF(100ng/ml) for 2hour.
Normalized wholecellextractswereused forWestern blotanalysiswith an anti-phosphoSTAT3antibodyrecognizingTyr705oranti-STAT3antibody.
FFFiiiggg...444...TTTrrreeeaaatttmmmeeennntttooofffaaaccctttiiinnnooommmyyyccciiinnnDDD aaabbbrrrooogggaaattteeesssSSSTTTAAATTT333ppphhhooosssppphhhooorrryyylllaaatttiiiooonnn... Cultured NIH3T3cellswereeitherpretreated with 5ug/mlactinomycin D for 30 min and then incubationed NGF(100ng/ml)for 2hour.Normalized whole cellextracts were used for western blot analysis with an anti-phospho STAT3antibody recognizing Tyr705andanti-STAT3antibody orp-Erkand Erkantibody.
FFFiiiggg 555... TTThhheee cccooonnndddiiitttiiiooonnneeeddd mmmeeedddiiiaaa tttrrreeeaaattteeeddd wwwiiittthhh llliiigggaaannnddd iiinnnddduuuccceeeddd ppphhhooosssppphhhooorrryyylllaaatttiiiooonnn ooofffSSSTTTAAATTT333 jjjuuussstttwwwiiittthhhiiinnn 111555 mmmiiinnnuuuttteeesss wwwhhheeennn aaappppppllliiieeeddd tttooo aaa nnneeewww ccceeellllll...(A)NIH3T3CellstransfectedTrk-Metsm weretreatedwith NGF (100ng/ml)for2hours.Andthen theconditionedmediaweretreated newly prepared NIH3T3 cells for 15 minute.(B)Chang Cells were treated with HGF(100unit/ml)for6 hour.And then the conditioned media were treated newly prepared Chang cells for 15 minute.Normalized whole cellextracts wereusedfor Western blotanalysis with an anti-phosphoSTAT3antibody recognizingTyr705andanti-STAT3antibody.
FFFiiiggg...666...NNNGGGFFF iiisss nnnoootttnnneeeccceeessssssaaarrryyy fffooorrrppphhhooosssppphhhooorrryyylllaaatttiiiooonnn ooofffSSSTTTAAATTT333...NIH3T3 CellstransfectedTrk-Metsm weretreatedwithNGF (100ng/ml)for2hours.
And then the conditioned media were collected and neutralized with NGF antibody (1:500,1:5000)for4 hourin CO2incubator.Normalized whole cell extractswereusedfor Westernblotanalysis withananti-phosphoSTAT3 antibodyrecognizingTyr705,anti-STAT3antibody,p-ErkandErk.
FFFiiiggg...777...IIInnnddduuuccctttiiiooonnn ooofffIIILLL---666mmmRRRNNNAAA wwwaaasssppprrrooommmiiinnneeennntttaaafffttteeerrrttthhheeetttrrreeeaaatttmmmeeennntttooofff cccooorrrrrreeessspppooonnndddiiinnnggg llliiigggaaannndddsss...Cells were treated with (A)NGF(100ng/ml)or(B) HGF(100unit/ml).RT-PCR was performed with molecules known to induce activationofSTATs.TotalRNA wasisolatedfrom respectiveconfluentcells andanalyzedbygelrunningonan1.5% agarosegels.
FFFiiiggg...888...RRReeecccooommmbbbiiinnnaaannnttthhhuuummmaaannn IIILLL---666 iiinnnddduuuccceeeddd ppphhhooosssppphhhooorrryyylllaaatttiiiooonnn ooofffSSSTTTAAATTT333 jjjuuussstttwwwiiittthhhiiinnn111555mmmiiinnnuuuttteeesssiiinnnCCChhhaaannnggg ccceeellllll...Normalizedwholecellextractswere used for Western blot analysis with an anti-phosphoSTAT3 antibody recognizingTyr705 andanti-STAT3antibody.
FFFiiiggg... 999... IIILLL---666 nnneeeuuutttrrraaallliiizzziiinnnggg aaannntttiiibbbooodddyyy iiinnnhhhiiibbbiiittteeeddd dddeeelllaaayyyeeeddd SSSTTTAAATTT333 ppphhhooosss-- -ppphhhooorrryyylllaaatttiiiooonnn...Chang cellsweretreatedwithHGF(100unit/ml)for8handthen the conditioned media was incubated with controlIgG orIL-6 neutralizing antibody for 2h in the 37℃ incubator.Newly prepared Chang cells were treated with the conditioned media for 15min. The lysates were immunoblottedwithp-STAT3andSTAT3antibody.
Ⅳ Ⅳ Ⅳ. . .고 고 고 찰 찰 찰
conditioned media로 2시간동안 배양시킨 다음 새로이 준비된 Chang 세포에 15 분 처리 했을때,STAT3의 인산화가 현저히 저해된 것을 볼수 있었다.이를 정 리해 보면 STAT3의 지연형 인산화는 HGF/Metsignaling에 의해 IL-6가 발현 되고 이후에 나타나는 이차적인 신호전달임을 관찰하였다.사실,IL-6가 유도되 어 autocrine하게 작용하는 것은 최근에 나온 보고에서도 볼 수 있다.cAMP로 C6Glioma세포를 astrocyte로 분화 시켰을때,IL-6가 autocrine하게 작용한다 는 보고가 있다.이때도 STAT3가 24시간이 되어 인산화가 이루어지고 cAMP에 의해 IL-6가 발현되고, 이것을 항 IL-6 중화항체를 가지고 저해 시켰더니 STAT3의 인산화가 저해된 것을 관찰하였다.(Takanaga등,2004)이것으로 본 실험에서는 HGF/Met신호전달에 의해 STAT3의 지연형 인산화를 관찰하였고, 중간 매개물질이 IL-6라는 것을 밝혀 내었다.이러한 신호전달이 생리적으로 어 떠한 의미를 갖을 지는 더 연구를 해 봐야 겠다.
Ⅴ Ⅴ Ⅴ. . .결 결 결 론 론 론
본 연구를 통해 HGF/Met활성화에 의해 생성된 STAT3의 지연형 인산화 현상을 확인하였고,이를 유발하는 세포의 분비 물질이 IL-6라는 것을 알게 되었 다.STAT3의 지연형 인산화가 일어나는데,HGF/Met세포 신호전달에 의해 세 포 분비 물질이 발현되고,IL-6에 의해 이차적인 STAT3세포 신호전달을 이룬 다는 것이 어떠한 의미를 갖을 지 더 연구를 해 봐야 겠다.