劉友平， 嚴(yán)冬梅， 陳川寧， 段春燕， 陳紹坤， 李 洪， 代榮陽(yáng)△

(瀘州醫(yī)學(xué)院1生物化學(xué)教研室，2醫(yī)學(xué)生物學(xué)與遺傳學(xué)教研室, 四川 瀘州 646000)

PI3K/Akt調(diào)控內(nèi)質(zhì)網(wǎng)應(yīng)激對(duì)GRP78的誘導(dǎo)*

劉友平1， 嚴(yán)冬梅1， 陳川寧1， 段春燕1， 陳紹坤2， 李 洪1， 代榮陽(yáng)1△

(瀘州醫(yī)學(xué)院1生物化學(xué)教研室，2醫(yī)學(xué)生物學(xué)與遺傳學(xué)教研室, 四川 瀘州 646000)

目的研究?jī)?nèi)質(zhì)網(wǎng)應(yīng)激條件下PI3K/Akt信號(hào)通路對(duì)HEK293細(xì)胞中葡萄糖調(diào)節(jié)蛋白78(GRP78)表達(dá)水平的調(diào)控作用。方法采用PI3K抑制劑LY294002、Akt1失活型突變載體Akt1(K179M)及Akt siRNAs阻斷內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的Akt活化，采用Akt激活型突變載體Myr-Akt過度激活內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的Akt活化，并利用RT-PCR和Western blotting技術(shù)分析內(nèi)質(zhì)網(wǎng)應(yīng)激條件下PI3K/Akt信號(hào)途徑對(duì)HEK293細(xì)胞中GRP78表達(dá)水平的調(diào)控作用。結(jié)果LY294002、Akt1(K179M)及Akt1 siRNA均明顯抑制了內(nèi)質(zhì)網(wǎng)應(yīng)激對(duì)GRP78的誘導(dǎo)。Myr-Akt1明顯促進(jìn)內(nèi)質(zhì)網(wǎng)應(yīng)激對(duì)GRP78的誘導(dǎo)。Myr-Akt2/3及Akt2/3 siRNA對(duì)GRP78的誘導(dǎo)均無影響。PI3K/Akt信號(hào)通路阻斷或過度激活對(duì)GRP78 mRNA水平的誘導(dǎo)無影響，但是對(duì)GRP78的降解有顯著影響。結(jié)論HEK293細(xì)胞中，PI3K/Akt通過蛋白穩(wěn)定性調(diào)節(jié)促進(jìn)內(nèi)質(zhì)網(wǎng)應(yīng)激對(duì)GRP78的誘導(dǎo)。

PI3K/Akt； 葡萄糖調(diào)節(jié)蛋白質(zhì)78； 內(nèi)質(zhì)網(wǎng)應(yīng)激； HEK293細(xì)胞

內(nèi)質(zhì)網(wǎng)(endoplasmic reticulum，ER)是細(xì)胞膜蛋白和分泌蛋白加工成熟的主要場(chǎng)所，內(nèi)質(zhì)網(wǎng)功能紊亂將導(dǎo)致錯(cuò)誤折疊和未折疊蛋白在內(nèi)質(zhì)網(wǎng)腔內(nèi)聚集，進(jìn)而引起內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress，ER stress)。內(nèi)質(zhì)網(wǎng)應(yīng)激時(shí)，細(xì)胞通過啟動(dòng)未折疊蛋白反應(yīng)(unfolded protein response，UPR)特異信號(hào)系統(tǒng)抑制蛋白翻譯、促進(jìn)未折疊蛋白加工成熟和促進(jìn)錯(cuò)誤折疊蛋白降解，減輕內(nèi)質(zhì)網(wǎng)蛋白負(fù)荷并促進(jìn)細(xì)胞重建內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)[1-3]。未折疊蛋白信號(hào)系統(tǒng)包括RNA激活蛋白激酶的內(nèi)質(zhì)網(wǎng)類似激酶/真核細(xì)胞翻譯起始因子2α (RNA-activated protein kinase-like endoplasmic neticulum kinase/eukaryotic translation initiation factor 2 alpha,PERK/eIF2α)、激活轉(zhuǎn)錄因子6(activating transcription factor 6，ATF6)和需肌醇酶1/X盒結(jié)合蛋白1 (inositol-requiring enzyme 1/ X-box-binding protein 1,IRE1/XBP1)3條信號(hào)途徑[4，5]。未折疊蛋白反應(yīng)對(duì)應(yīng)激細(xì)胞恢復(fù)正常功能起重要作用，但是當(dāng)細(xì)胞面臨持續(xù)性內(nèi)質(zhì)網(wǎng)應(yīng)激刺激時(shí)，未折疊蛋白反應(yīng)則會(huì)啟動(dòng)凋亡信號(hào)清除不能恢復(fù)功能的細(xì)胞。

PI3K/Akt是調(diào)節(jié)細(xì)胞增殖、分化、凋亡和衰老的關(guān)鍵信號(hào)途徑[6]。已有研究表明PI3K/Akt途徑對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激細(xì)胞起重要保護(hù)作用[7]，但其具體機(jī)制還很不清楚。葡萄糖調(diào)節(jié)蛋白78(glucose-regulated protein 78，GRP78)屬于熱休克蛋白70家族成員，其在促進(jìn)蛋白加工、成熟以及維持內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)中發(fā)揮重要作用[8,9]。多種擾亂內(nèi)質(zhì)網(wǎng)功能的刺激均能夠誘導(dǎo)GRP78表達(dá)。在未折疊蛋白反應(yīng)過程中，GRP78的誘導(dǎo)表達(dá)對(duì)應(yīng)激細(xì)胞抵抗凋亡和恢復(fù)正常功能具有重要意義[8,10]。本研究旨在探討PI3K/Akt通路在內(nèi)質(zhì)網(wǎng)應(yīng)激對(duì)GRP78的誘導(dǎo)中的調(diào)控作用。

材 料 和 方 法

1材料

1.1細(xì)胞系 人胚腎293細(xì)胞株(human embryonic kidney 293 cell line, HEK293 celltine)購(gòu)于中國(guó)科學(xué)院上海生命科學(xué)研究院。

1.2試劑 毒胡蘿卜素(thapsigargin, TG)和二硫蘇糖醇(dithiothreitol, DTT)購(gòu)自Sigma；PI3K抑制劑LY294002購(gòu)自Merck；抗GRP78Ⅰ抗購(gòu)自Santa Cruz；抗β-actin、HA-tag、p-Akt(Ser473) 和AktⅠ抗購(gòu)自Cell Signaling Technology；Ⅱ抗購(gòu)自Santa Cruz。Akt突變激活載體HA-myr-Akt、突變失活載體HA-Akt1(K179M)和空載體pCMV(作對(duì)照)由Jin Q. Cheng教授惠贈(zèng)。

2方法

2.1細(xì)胞培養(yǎng)及處理 HEK293細(xì)胞株用DMEM 完全培養(yǎng)基(含10 % FBS、20 mmol/L NaHCO3、20 mmol/L HEPES、1×105U/L 青霉素和100 μg/L 鏈霉素)在5 % CO2、37 ℃ 孵箱內(nèi)培養(yǎng)，根據(jù)具體情況更換培養(yǎng)基。用TG(1 μmol/L)和DTT(2.5 mmol/L)誘導(dǎo)細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激，按對(duì)應(yīng)時(shí)點(diǎn)收集細(xì)胞并作相應(yīng)檢測(cè)。

2.2Western blotting分析 SDS-PAGE電泳分離蛋白質(zhì)后，采用半干式電轉(zhuǎn)移將蛋白分子轉(zhuǎn)移到PVDF膜，電轉(zhuǎn)移結(jié)束后PVDF膜用洗膜緩沖液TBST洗滌3次(5 min/次)，封阻緩沖液(5%BSA)在室溫下密閉輕搖動(dòng)封阻1 h。洗膜緩沖液洗滌3次(5 min/次)后，PVDF膜與Ⅰ抗稀釋液室溫下輕搖動(dòng)孵育2 h，洗膜緩沖液洗滌3次(5 min/次)。PVDF膜與II抗稀釋液于室溫下輕搖動(dòng)孵育1 h，洗膜緩沖液洗滌3次(5 min/次)。加入ECL 化學(xué)發(fā)光試劑A、B液各0.5 mL，混勻，潤(rùn)透PVDF膜后室溫作用1 min。暗室中將PVDF膜迅速封入保鮮膜中，Kodak X-ray film 壓片，放射自顯影5 min。X-ray film 置于顯影液中15-30 s，定影液中1.5 min，清水沖洗晾干。

2.3RT-PCR 參照我們的前述方法利用Trizol法提取細(xì)胞總RNA，根據(jù)操作流程用M-MLV逆轉(zhuǎn)錄試劑盒進(jìn)行逆轉(zhuǎn)錄反應(yīng)得到cDNA[11]。用2% 或4%的瓊脂糖凝膠分離PCR產(chǎn)物。RT-PCR引物序列如下：GRP78上游引物為5’-ATC ACG CCG TCC TAT GTC GC-3’，下游引物為5’-TCT CCC CCT CCC TCT TAT CC-3’；XBP1上游引物為5’-CCT TGT AGT TGA GAA CCA GG-3’，下游引物為5’-GGG GCT TGG TAT ATA TGT GG-3’；18S上游引物為5’-GGG AGG TAG TGA CGA AAA AT-3’，下游引物為5’-ACC AAC AAA ATA GAA CCG CG-3’。

3統(tǒng)計(jì)學(xué)處理

結(jié) 果

1HEK293細(xì)胞中內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)Akt持續(xù)活化

DTT(2.5 mmol/L)和TG(1 μmol/L)刺激顯著誘導(dǎo)HEK293細(xì)胞中葡萄糖調(diào)節(jié)蛋白78的表達(dá)(圖1A)和XBP1 mRNA的活化剪切(圖1B)，表明DTT和TG能有效誘導(dǎo)HEK293細(xì)胞發(fā)生未折疊蛋白反應(yīng)。圖1C表明DTT和TG刺激均能使Akt在HEK293細(xì)胞中保持較長(zhǎng)時(shí)間的高水平磷酸化，而我們前期的結(jié)果已經(jīng)表明內(nèi)質(zhì)網(wǎng)應(yīng)激能夠較快引起肝癌細(xì)胞中Akt磷酸化水平下降[11]，提示在HEK293細(xì)胞中，PI3K/Akt通路對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激可能有特殊調(diào)控作用。

2阻斷PI3K/Akt通路抑制內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的GRP78誘導(dǎo)

已經(jīng)有研究指出PI3K/Akt能夠通過抑制生長(zhǎng)阻滯和DNA損傷誘導(dǎo)蛋白153 (growth arrest and DNA damage-inducible protein 153, GADD153)的誘導(dǎo)對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激細(xì)胞起保護(hù)作用[12,13]，而我們的結(jié)果提示在HEK293細(xì)胞中，PI3K/Akt對(duì)GADD153的誘導(dǎo)無明顯調(diào)控作用(未發(fā)表資料)。值得注意的是，在HEK293細(xì)胞中，PI3K抑制劑LY294002預(yù)處理明顯抑制了DTT和TG刺激介導(dǎo)的GRP78誘導(dǎo)表達(dá),見圖2,提示在HEK293細(xì)胞中，PI3K/Akt通路對(duì)GRP78的誘導(dǎo)表達(dá)具有重要調(diào)控作用。

Figure 1. Effect of ER stress on the phosphorylation of Akt. A: effect of DTT and TG on GRP78 induction in HEK 293 cells;B: effect of DTT and TG on XBP1 mRNA splicing in HEK 293 cell; C: effect of DTT and TG on the phosphorylation of Akt in HEK 293 cells.

3Akt1調(diào)控內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的GRP78誘導(dǎo)

鑒于Akt包括Akt1、Akt2和Akt3三種高度保守的同工酶，我們分析了Akt1、Akt2和Akt3在GRP78誘導(dǎo)調(diào)控中的作用。圖3A表明，HA-myr-Akt1瞬時(shí)轉(zhuǎn)染明顯促進(jìn)DTT和TG對(duì)GRP78的誘導(dǎo)，而HA-myr-Akt2和HA-myr-Akt3瞬時(shí)轉(zhuǎn)染則對(duì)GRP78的誘導(dǎo)表達(dá)無影響。為了進(jìn)一步證實(shí)Akt1在GRP78表達(dá)調(diào)控中的作用，我們分析了突變失活載體Akt1 (K179M)和Akt1 siRNA對(duì)GRP78誘導(dǎo)表達(dá)的影響。結(jié)果表明Akt1(K179M)和Akt1 siRNA均能顯著抑制DTT和TG對(duì)GRP78誘導(dǎo)，見圖3B、C，而Akt2 siRNA和Akt3 siRNA對(duì)GRP78的表達(dá)均無影響(未發(fā)表資料)。上述結(jié)果表明在HEK293細(xì)胞中，PI3K/Akt通路中的Akt1在內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的GRP78誘導(dǎo)中起重要作用。

Figure 2. LY294002 inhibits ER stress-induced GRP78 accumulation. ±s.n=6. *Plt;0.05 vs control group (0 h) ; #Plt;0.05 vs DTT group or TG group.

Figure 3. Effect of Akt1 on ER stress-induced GRP78 accumulation. A: effect of myr-Akt1/2/3 on GRP78 induction in DTT- and TG-treated HEK293 cells;B: effect of Akt 1(K179M) on GRP78 induction in DTT- and TG-treated HEK293 cells;C: effect of Akt1 siRNA on GRP78 induction in DTT- and TG-treated HEK293 cells±s.n=6.*Plt;0.05 vs control group (0 h) ; #Plt;0.05 vs DTT group or TG group .

4PI3K/Akt調(diào)控GRP78蛋白的穩(wěn)定性

由于內(nèi)質(zhì)網(wǎng)應(yīng)激對(duì)GRP78的誘導(dǎo)表達(dá)主要體現(xiàn)在轉(zhuǎn)錄水平，我們進(jìn)一步檢測(cè)了PI3K/Akt對(duì)GRP78轉(zhuǎn)錄水平的影響。結(jié)果表明,LY294002預(yù)處理和myr-Akt1瞬時(shí)轉(zhuǎn)染均對(duì)GRP78 mRNA水平無明顯影響(圖4A、B)，這提示Akt對(duì)GRP的調(diào)控發(fā)生在蛋白水平。圖4C表明蛋白酶體抑制劑MG132有效阻斷了LY294002對(duì)GRP78誘導(dǎo)的抑制作用。上述結(jié)果表明PI3K/Akt通過促進(jìn)蛋白穩(wěn)定性發(fā)揮對(duì)GRP78誘導(dǎo)表達(dá)的調(diào)控作用。

討 論

內(nèi)質(zhì)網(wǎng)應(yīng)激過程中細(xì)胞形成了高度保守的自我保護(hù)信號(hào)轉(zhuǎn)導(dǎo)通路，該信號(hào)通路系統(tǒng)稱為未折疊蛋白反應(yīng)。未折疊蛋白反應(yīng)通過抑制蛋白合成、促進(jìn)錯(cuò)誤折疊蛋白降解等機(jī)制保護(hù)發(fā)生內(nèi)質(zhì)網(wǎng)應(yīng)激的細(xì)胞[1-3]。GRP78的誘導(dǎo)表達(dá)對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激細(xì)胞起關(guān)鍵保護(hù)作用[8,10]。作為未折疊蛋白反應(yīng)活化的經(jīng)典標(biāo)志分子，GRP78不僅是未折疊蛋白反應(yīng)啟動(dòng)與否的分子開關(guān)，還是促進(jìn)未折疊蛋白加工成熟和維持內(nèi)質(zhì)網(wǎng)內(nèi)穩(wěn)態(tài)的核心分子[8,14,15]。內(nèi)質(zhì)網(wǎng)應(yīng)激對(duì)GRP78的誘導(dǎo)主要是在轉(zhuǎn)錄水平，ATF6、XBP1和活化轉(zhuǎn)錄因子4(activating transcription factor 4, ATF4)均能誘導(dǎo)GRP78 mRNA表達(dá)， 其中ATF6是目前所知最關(guān)鍵的調(diào)控因子[16-18]。

Figure 4. Effect of PI3K/Akt on the stability of GRP78 protein. A: effect of LY294002 on GRP78 mRNA level in DTT- and TG-treated HEK293 cells;B: effect of myr-Akt1 on GRP78 mRNA level in DTT- and TG-treated HEK293 cells;C: effect of MG132 on LY294002-mediated GRP78 induction inhibition in DTT- and TG-treated HEK293 cells. ±s.n=6. *Plt;0.05 vs control group (0 h).LY:LY294002.

PI3K/Akt信號(hào)通路不僅在細(xì)胞代謝、增殖和存活等多種生物學(xué)過程中起關(guān)鍵調(diào)控作用，也對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激細(xì)胞起重要保護(hù)作用[9]。雖然已經(jīng)明確PI3K/Akt信號(hào)通路在多種細(xì)胞抵抗內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)的凋亡中起重要作用，但是其分子機(jī)制還不太清楚。在已經(jīng)報(bào)道的多種細(xì)胞中，內(nèi)質(zhì)網(wǎng)應(yīng)激能夠引起Akt磷酸化水平迅速下降，而在內(nèi)質(zhì)網(wǎng)應(yīng)激性HEK293細(xì)胞中，Akt的活化持續(xù)時(shí)間較長(zhǎng)，這提示Akt對(duì)HEK293細(xì)胞的內(nèi)質(zhì)網(wǎng)應(yīng)激可能有特殊調(diào)控作用。本實(shí)驗(yàn)采用PI3K/Akt信號(hào)通路的小分子抑制劑、小RNA干擾和質(zhì)粒轉(zhuǎn)染技術(shù)發(fā)現(xiàn)PI3K/Akt信號(hào)途徑對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)的GRP78有重要調(diào)控作用。阻斷PI3K/Akt信號(hào)通路顯著抑制內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)的GRP78表達(dá)，而上調(diào)PI3K/Akt信號(hào)通路活性則促進(jìn)了內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)的GRP78表達(dá)。而且，進(jìn)一步的實(shí)驗(yàn)結(jié)果證明，發(fā)揮GRP78誘導(dǎo)調(diào)控作用的是Akt1，而Akt2和Akt3則無調(diào)控作用。雖然現(xiàn)有報(bào)道表明內(nèi)質(zhì)網(wǎng)應(yīng)激對(duì)GRP78的誘導(dǎo)主要體現(xiàn)在轉(zhuǎn)錄水平，但我們的結(jié)果卻發(fā)現(xiàn)PI3K/Akt通路對(duì)GRP78誘導(dǎo)的調(diào)控發(fā)生在蛋白水平，并且是通過抑制蛋白酶體對(duì)GRP78蛋白的降解，促進(jìn)其穩(wěn)定性增加以發(fā)揮其調(diào)控作用。

總之，本實(shí)驗(yàn)發(fā)現(xiàn)在HEK293細(xì)胞中，PI3K/Akt信號(hào)通路通過調(diào)控蛋白穩(wěn)定性，在內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)GRP78表達(dá)增加中發(fā)揮重要作用，其具體分子機(jī)制有待進(jìn)一步研究。

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PI3K/Aktregulatesendoplasmicreticulumstress-mediatedGRP78induction

LIU You-ping1, YAN Dong-mei1, CHEN Chuan-ning1, DUAN Chun-yan1, CHEN Shao-kun2, LI Hong1, DAI Rong-yang1

(1DepartmentofBiochemistry,2DepartmentofBiologyandGenetics,LuzhouMedicalCollege,Luzhou646000,China.E-mail:dryrun2502@163.com)

AIM: To investigate the effect of PI3K/Akt pathway on endoplasmic reticulum (ER) stress-mediated glucose-regulated protein 78 (GRP78) induction in human embryonic kidney 293 cells (HEK293) cells.METHODSPI3K inhibitor LY294002, dominant negative kinase-dead mutant vector for HA-Akt (K179M) and Akt1 siRNAs were used to block the PI3K/Akt pathway under ER stress. Constitutively active expression vectors for Akt (myr-HA-Akt) were used to up-regulate Akt activity under ER stress. The effects of PI3K/Akt on ER stress-mediated GRP78 induction in HEK293 cells were determined by Western blotting and RT-PCR.RESULTSGRP78 induction was inhibited by LY294002, Akt1 (K179M) and Akt1 siRNA, but was increased by myr-Akt1 in dithiothreitol-and thapsigargin-treated HEK293 cells. However, both myr-Akt2/3 and Akt2/3 siRNA had no effect on GRP78 induction in HEK293 cells under ER stress. Furthermore, the PI3K/Akt pathway didn’t regulated GRP78mRNA induction but increased GRP78 protein stability.CONCLUSIONPI3K/Akt promotes GRP78 accumulation through increasing the stability of GRP78 protein in HEK293 cells under ER stress.

PI3K/Akt; Glucose-regulated protein 78; Endoplasmic reticulum stress; HEK293 cells

1000-4718(2011)04-0749-06

R735.7; Q255

A

10.3969/j.issn.1000-4718.2011.04.024

2010-10-18

2011-01-19

國(guó)家自然科學(xué)基金資助項(xiàng)目(No.81000886)；四川省教育廳科技基金資助項(xiàng)目(No.09ZA050)；四川省衛(wèi)生廳科技基金資助項(xiàng)目(No.2007-431)

△通訊作者 Tel:0830-3160283；E-mail: dryrun2502@163.com