DNA損傷反應(yīng)誘導(dǎo)炎癥和衰老通過抑制GATA4自噬

1、The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4DNA損傷反應(yīng)通過抑制GATA4自噬誘導(dǎo)炎癥和老化introduction：細(xì)胞衰老是一個由多重應(yīng)激導(dǎo)致基因表達(dá)變異和擴(kuò)增的過程。雖然這也是一種潛在的腫瘤抑制機(jī)制，但衰老機(jī)制也參與了一些病理過程，包括老化，年齡相關(guān)的疾病，甚至與致瘤機(jī)制有關(guān)。衰老細(xì)胞能分泌一些衰老相關(guān)分泌表型SASP影響自身的微環(huán)境，這些SASP包括前炎性細(xì)胞因子，趨化因子，生長因子和蛋白酶。SASP啟動和維持的機(jī)制以轉(zhuǎn)錄因子NF-B和C / E

2、BPb為特征的經(jīng)典炎癥調(diào)節(jié)機(jī)制。rational：P53和p16INK4a/ RB是衰老過程的兩個重要核心調(diào)控通道。 不依賴于p53或p16INK4a的另一種獨(dú)立衰老調(diào)控通路能調(diào)節(jié)SASP。根據(jù)miR-146a的啟動子片段開發(fā)了一種綠色熒光蛋白標(biāo)記的衰老信號，可以檢測到人類成纖維細(xì)胞衰老過程中的miR-146a。 衰老誘導(dǎo)刺激物能激活SASP，包括復(fù)制疲憊，DNA損傷，致癌RAS激活。RESULTS:通過對miR-146a啟動子的分析，我們定位衰老誘導(dǎo)活化的關(guān)鍵區(qū)域并確定了轉(zhuǎn)錄調(diào)控因子GATA4在衰老調(diào)控中的作用。正常情況下， GATA4 與P62自噬體結(jié)合發(fā)生選擇性自噬而被降解。在衰老反應(yīng)中

3、，GATA4和P62反應(yīng)減弱可以抑制這種自噬反應(yīng)。GATA4通過誘導(dǎo)能激活NF-kB通路的因子啟動和維持SASP，從而可以促進(jìn)衰老過程，這些因子包括腫瘤壞死因子受體相關(guān)蛋白2和IL-1A。GATA4通路的活化同p53 和p16INK4a通路活化機(jī)制相似，需要DDR激酶ATM和ATR的活化。不同的是，GATA4通路是不同于p53和p16INK4a的另外的獨(dú)立通路。GATA4蛋白大量存在與衰老刺激物誘導(dǎo)的老鼠、正常老化的老鼠及人的多個組織中，包括腦細(xì)胞，這些發(fā)現(xiàn)均提示GATA4通路參與年齡相關(guān)性炎癥反應(yīng)的過程。CONCLUSION: 我們的結(jié)果說明GATA4通過TRAF3IP2和IL-1A激活的N

4、F-B通路參與自噬反應(yīng)、DDR所致的衰老及炎癥過程。GATA4通過DDR調(diào)控衰老是不同于P53和p16INK4a通路的獨(dú)立通路的關(guān)鍵。我們認(rèn)為衰老細(xì)胞中大量積聚的GATA4能通過炎癥反應(yīng)促進(jìn)老化和疾病，而抑制GATA4通路可能為疾病的治療提供了一個新方向。細(xì)胞老化是應(yīng)激反應(yīng)的最終階段，由P53和p16INK4a腫瘤抑制蛋白調(diào)控。老化的顯著特征是一種與腫瘤生長和老化相關(guān)的前炎性反應(yīng)，SASP。已經(jīng)證實(shí)轉(zhuǎn)錄因子GATA4可以調(diào)控老化和SASP。正常情況下，GATA4能通過P62介導(dǎo)的選擇性自噬作用降解，而衰老反應(yīng)則可使GATA4保持恒定。Text：在應(yīng)激狀態(tài)下，細(xì)胞老化是一種防止異常細(xì)胞進(jìn)一步擴(kuò)增

5、的機(jī)制。細(xì)胞老化抑制細(xì)胞和組織的再生能力，衰老細(xì)胞的消除有助于誘導(dǎo)老化模型鼠的老化相關(guān)表型的表達(dá)。而如何接收衰老信號啟動老化反應(yīng)的具體調(diào)節(jié)機(jī)制并不清楚。多種衰老刺激信號能引起哺乳動物細(xì)胞進(jìn)入不可逆的生長阻滯期，這些信號包括復(fù)發(fā)擴(kuò)散導(dǎo)致的端粒的縮短、DNA損傷以及活化癌基因的表達(dá)。衰老細(xì)胞除了能引起細(xì)胞生長阻滯外，在基因表達(dá)中也有很大的作用，包括SASP的表達(dá)。在生理狀態(tài)下，SASP因子可以通過自分泌和旁分泌的方式加強(qiáng)衰老細(xì)胞的生長阻滯，老化細(xì)胞能刺激癌前細(xì)胞或癌變細(xì)胞擴(kuò)增形成腫瘤。另一方面，SASP激活免疫系統(tǒng)可以抑制腫瘤，而當(dāng)衰老細(xì)胞在發(fā)揮作用被移除后可以促進(jìn)受損組織的修復(fù)。SASP可以直接

6、或間接地促進(jìn)與一些年齡相關(guān)性疾病的慢性炎性因子的分泌。盡管SASP由很廣泛的生物學(xué)活性，但是對于SASP產(chǎn)生的NF-kB經(jīng)典途徑以外的作用卻是有限的，比如CCAAT/ 增強(qiáng)子結(jié)合蛋白 b (C/EBPb)，IL-1a和P38MAPK。在老化刺激下NF-kB炎性反應(yīng)被激活的機(jī)制亦是未知的。與老化生長停滯相比，P53和p16INK4a/Rb腫瘤抑制途徑有重要的作用，SASP并不依賴于P53和p16INK4a/Rb腫瘤抑制途起作用，而是有自己獨(dú)立的衰老調(diào)控通路。與急劇的正常炎性反應(yīng)相比，SASP反應(yīng)很緩慢，到老化細(xì)胞生長阻滯通常需要幾天的時間，這將提供一個衰老特異性的激活機(jī)制。除了轉(zhuǎn)錄調(diào)控外，自噬通

7、過target of rapamycin(TOR) autophagy spatial coupling compartment(TASCC)影響SASP的形成。TASCC通過mTOR與自噬溶酶體氨基酸殘基結(jié)合而起到促進(jìn)分泌蛋白質(zhì)合成的作用。也有研究表明抑制自噬在某些條件下能促進(jìn)老化。因此，自噬和老化之間的關(guān)系暫時還不清楚。GATA4, a novel senescence regulator我們分析了microRNA在非老化細(xì)胞和衰老人成纖維細(xì)胞的表達(dá)，通過復(fù)制疲憊誘導(dǎo)老化并發(fā)現(xiàn)miR-146a在老化細(xì)胞中高度表達(dá)。我們猜測miR-146a調(diào)控主要發(fā)生在轉(zhuǎn)錄水平的初始階段。我們將1.5-kb

8、 miR-146a啟動子片段融合如綠色熒光蛋白中PmiR-146a-GFP。miR-146a在一些老化誘導(dǎo)因子作用下表達(dá)增加，包括復(fù)制疲憊、電離輻射及致癌基因RAS 的表達(dá)。通過ECB瀏覽器，我們發(fā)現(xiàn)miR-146a有兩個高度進(jìn)化的保守域，ECR1和ECRU2。ECR2對基因活性有重要的作用，因為ECR2有NF-B結(jié)合部位，而NF-B能調(diào)控miR-146a，我們從藥理學(xué)和遺傳學(xué)角度抑制了完全表達(dá)SASP的衰老細(xì)胞的NF-B通路。而衰老細(xì)胞中只有部分NF-B被抑制，這個結(jié)果表明其它的轉(zhuǎn)錄因子也作用于激活miR-146a關(guān)聯(lián)的細(xì)胞老化。為了確定其它轉(zhuǎn)錄因子的活性，我們搜查了那些被預(yù)測能結(jié)合ECR2

9、的轉(zhuǎn)錄因子。我們逐一將搜查13個轉(zhuǎn)錄因子過度表達(dá)并檢測miR-146a的活性，只有GATA4符合條件。在衰老細(xì)胞中，siRNA GATA4被耗盡。ChIP-qPCR顯示GATA4表達(dá)與miR-146a ECR2直接連接。GATA4是一種鋅指轉(zhuǎn)錄因子，在各種器官的發(fā)育中是必不可少的，包括心臟，睪丸，前腸，肝，和腹側(cè)胰腺。為了檢測GATA4的功能，我們研究了異位表達(dá)或缺乏在人二倍體成纖維細(xì)胞老化反應(yīng)中的影響。GATA4異位表達(dá)誘導(dǎo)人包皮成纖維細(xì)胞和IMR-90成纖維細(xì)胞的老化，通過增加SA-b-Gal的活性及減少BrdU的合成。更重要的是，通過穩(wěn)定表達(dá)shRNA導(dǎo)致GATA4耗盡能部分下調(diào)IR誘導(dǎo)

10、的SA-B-Gal的活性和延遲復(fù)制性衰老。這是由于 GATA4發(fā)生CRISPR突變。這些數(shù)據(jù)表明GATA4對老化有正性調(diào)節(jié)作用。在衰老中GATA4有調(diào)控作用，而在肺，結(jié)腸癌，前列腺癌，卵巢癌和乳腺癌中，則保持沉默。Selective autophagy suppresses GATA4 and senescence在檢測GATA4 siRNA的效率時，我們發(fā)現(xiàn)GATA4的量在衰老細(xì)胞中增加。事實(shí)上，豐富的GATA4蛋白，而不是mRNA，能增加IR-和癌基因誘導(dǎo)的衰老和復(fù)制老化。這是由于蛋白質(zhì)穩(wěn)定性提高，通過放線菌酮和全球蛋白質(zhì)穩(wěn)定性分析檢測蛋白質(zhì)的穩(wěn)定性。在真核細(xì)胞中有兩個主要蛋白質(zhì)降解途徑：

11、泛素蛋白酶體和自噬溶酶體途徑。用MG-132抑制蛋白酶體，MG-132是一種蛋白酶體抑制劑，對GATA4沒有影響，而GATA4蛋白在溶酶體抑制劑（溶酶素A、E64d、胃蛋白酶抑制劑）存在的細(xì)胞中穩(wěn)定表達(dá)。這些發(fā)現(xiàn)提示自噬溶酶體途徑能調(diào)控GATA4。自噬部件ATG5或ATG7同樣能上調(diào)GATA4蛋白。自噬可以通過特異性自噬受體介導(dǎo)途徑選擇性地降低某些基物。事實(shí)上，自噬受體P62的耗盡能增加GATA4蛋白。衰老過程中外源性表達(dá)和內(nèi)源性GATA4與p62相互作用減弱。因此，在正常條件下，GATA4與P62發(fā)生特異性作用發(fā)生自噬，而在老化中，可能與P62的相互作用減弱，GATA4變得穩(wěn)定了。衰老與自噬

12、之間的關(guān)系目前還不清楚。自噬在老化或抑制老化過程中是必不可少的。我們結(jié)果可以解釋這些矛盾。選擇性自噬可能會通過抑制衰老調(diào)控因子如GATA4來防止老化。然而，衰老誘導(dǎo)刺激物可以引起GATA4逃脫選擇性自噬。隨后，非選擇性自噬可能激活有助于老化。因此，GATA4的選擇性自噬可能對老化的起到負(fù)性調(diào)節(jié)作用。如果是這樣，瞬時抑制自噬可引起衰老。為了驗證這一點(diǎn)，我們利用多西環(huán)素（阿霉素）誘導(dǎo)的shRNA耗盡ATG5或ATG7和瞬時抑制自噬，使GATA4增加，然后通過去除多西環(huán)素恢復(fù)自噬，使細(xì)胞達(dá)到一個可老化的狀態(tài)。短暫抑制自噬（高濃度GATA4，自噬on）誘導(dǎo)的衰老比持續(xù)性抑制自噬（高濃度GATA4，自噬

13、off）更有效，這種效果，至少在一部分取決于GATA4。連續(xù)和短暫抑制自噬使獲得同樣的GATA4濃度，但連續(xù)抑制未能誘導(dǎo)衰老。在正常狀態(tài)下，P62缺乏比自噬調(diào)節(jié)受體ATG7或ATG5的缺乏更有效率的誘導(dǎo)老化。因此，選擇性自噬可以成為GATA4的抗衰老機(jī)制，而非選擇性自噬則是促衰老機(jī)制。GATA4 regulates the SASP為了確定GATA4調(diào)節(jié)老化的機(jī)制，我們探究了GATA4是如何影響人成纖維細(xì)胞基因表達(dá)的。GATA4異位表達(dá)時可以誘導(dǎo)老化，我們利用阿霉素誘導(dǎo)GATA4載體在GATA4表達(dá)之前和之后對RNA轉(zhuǎn)錄普進(jìn)行測序。我們根據(jù)基因本體論系統(tǒng)描述了GATA4是如何影響細(xì)胞進(jìn)程的。基

14、因表達(dá)GATA4的增加對一些應(yīng)答是有意義的，如免疫應(yīng)答、炎癥反應(yīng)、創(chuàng)傷反應(yīng)，而GATA4的表達(dá)減少則與細(xì)胞周期的生理過程有關(guān)。我們比較了GATA4基因集和與增殖老化基因集的區(qū)別，無論基因是上調(diào)還是下調(diào)均有意義，而基因的上調(diào)更具統(tǒng)計學(xué)意義，說明GATA4有轉(zhuǎn)錄激活因子的作用。結(jié)果說明GATA4可能激活一部分老化相關(guān)基因。在GATA4調(diào)節(jié)的老化相關(guān)基因中我們發(fā)現(xiàn)了很多SAPA基因，這些基因能編碼IL6, IL8, CXCL1, 粒-巨細(xì)胞集落刺激因子 (GM-CSF), 細(xì)胞外基質(zhì)蛋白激酶和抑制劑。炎癥應(yīng)答和免疫應(yīng)答中的細(xì)胞因子和趨化因子由老化細(xì)胞分泌，可以改變細(xì)胞內(nèi)微環(huán)境，增強(qiáng)老化阻滯，GATA

15、4通過SASP可能直接調(diào)節(jié)其他的老化表型，尤其是生長阻滯。GATA4的異位表達(dá)通過Rt-qPCR可以誘導(dǎo)SASP相關(guān)的基因表達(dá)。更為重要的是，在建立老化過程中，GATA4的過度消耗可以抑制很多SASP基因的表達(dá)，說明GATA4控制了很多SASP基因而GATA家族的另一成員GATA3并不能增加SASP相關(guān)基因的表達(dá)，即使預(yù)測說它是一種很強(qiáng)的腫瘤抑制因子。同樣的，GATA3的表達(dá)不能增加腫瘤壞死因子相關(guān)受體蛋白2 TRAF3IP2的表達(dá)。GATA4 regulates NF-kBNF-kB在調(diào)控SASP上有至關(guān)重要的作用，但對于NF-kB是如何激活衰老過程的人們知道得很少。 為了檢測GATA4與N

16、F-B在調(diào)節(jié)SASP的關(guān)系，我們測試了當(dāng)NF-kB的重要成分RELA受到抑制時，是如何影響GATA4誘導(dǎo)SASP。 RELA耗盡能夠抑制GATA4調(diào)節(jié)的SASP相關(guān)基因表達(dá)。GATA4的表達(dá)觸發(fā)NF-kB的激活，而GATA4缺乏在老化過程中抑制NF-KB的活化; 這些研究結(jié)果表明，GATA4在調(diào)節(jié)SASP過程中作用于NF-B的上游。為了了解GATA4是如何激活NF-B的，我們收索了在基因組芯片實(shí)驗中發(fā)現(xiàn)的約束GATA4的啟動子，據(jù)此發(fā)現(xiàn)并檢驗與激活NF-kB具有同樣功能的基因是如何被GATA4調(diào)控的。GATA4減少TRAF3IP2的表達(dá)，一種TRAF6的泛素連接酶和TRAF3IP2的缺乏能部分

17、阻滯GATA4激活NF-kB，可以通過SASP基因的表達(dá)進(jìn)行評估。TRAF3IP2異位表達(dá)能部分緩解在IR誘導(dǎo)老化細(xì)胞中由于GATA4缺乏導(dǎo)致的SASP減少。SASP因子能抵抗TRAF3IP2消耗，如IL6 和CXCL3，結(jié)果表明GATA4的下游基因通過TRAF3IP2通路對SASP起到調(diào)控作用。RELA消耗能完全阻滯GATA4調(diào)控的SASP的活化，除了IL1A外。IL1A在SASP調(diào)控中，對NF-KB有正反饋調(diào)控作用。因此，SASP在GATA4依賴的途徑中，IL1A可能與TRAF3IP2有協(xié)同作用。通過評估SASP基因的表達(dá)，IL1A缺乏能減少GATA活化的NF-KB途徑。此外，將SASP相

18、關(guān)的活化基因轉(zhuǎn)移到缺乏GATA4反應(yīng)的細(xì)胞中，由GATA4誘導(dǎo)的細(xì)胞通過條件培養(yǎng)液獲得。因此，GATA4通過TRAF3IP2 and IL1A激活NF-KB途徑作用于SASP。TRAF3IP2缺失也部分阻滯GATA4誘導(dǎo)的衰老，SA-B-Gal的活性。不同于GATA4的表達(dá)，TRAF3IP2表達(dá)不足以誘導(dǎo)的SA-B-Gal的活化，卻能激活SASP相關(guān)的基因表達(dá)。此外，TRAF3IP2異位表達(dá)與IL1A一起仍然不足以誘導(dǎo)SA-B-Gal活化，卻能激活GATA4。因此，該SASP可能不是GATA4調(diào)控衰老的唯一機(jī)制。事實(shí)上，此前確定的老化調(diào)控基因，如早幼粒白血病蛋白（PML）和YPEL3都屬于GA

19、TA4誘導(dǎo)的基因。GATA4, a new branch of the senescence regulatory pathway為了更全面地了解GATA4調(diào)控衰老的機(jī)制，我們研究了兩個核心衰老的調(diào)節(jié)通路中，p53和p16INK4a通路。GATA4對老化的影響是獨(dú)立于 p53和p16INK4a/ Rb途徑的。GATA4激活產(chǎn)生能誘導(dǎo)老化的IR劑量，能保持完整的細(xì)胞中缺乏的p53或p53基因和Rb。此外，沒有活化的P53和p16INK4a中，GATA4同樣被激活。因此，GATA4是獨(dú)立于 p53-和p16INK4a的途徑。DNA損傷應(yīng)答（DDR）作用于該途徑的上游。事實(shí)上，抑制DDR調(diào)控因子AT

20、M和ATR能抑制老化的GATA4途徑。因此，GATA4途徑誘導(dǎo)的SASP似乎是在DDR的一個獨(dú)立分支。DDR是如何抑制自噬基礎(chǔ)上GATA4降解仍然是今后研究的核心問題。GATA4s role in senescence in vivo為了測試GATA4活性和調(diào)控作用在體內(nèi)同樣發(fā)生，我們檢測了存在氧生理條件下小鼠胚胎成纖維細(xì)胞通過IR誘導(dǎo)的老化的GATA4表達(dá)。Gata4增加能引起衰老誘導(dǎo)的IR增加，需要幾個GATA4靶基因的作用，包括那些編碼Traf3ip2和SASP因子的基因，如IL6，IL1A，和CXCL1因子。因此，GATA4調(diào)節(jié)老化表型的途徑也能存在與小鼠中。為了確定GATA4在小鼠體

21、內(nèi)對老化應(yīng)答是否有作用，我們檢測了多種組織均老化的輻射鼠的GATA4水平。在皮膚可肝臟中，GATA4增加引起老化誘導(dǎo)的IR增加，因此，GATA4在體內(nèi)積聚能通過DNA損傷誘導(dǎo)老化。衰老細(xì)胞隨著小鼠和人年齡的增長而堆積。我們研究了年輕和老的小鼠（分別是6月齡和22月）并發(fā)現(xiàn)年齡大的小鼠的腎臟和肝臟中GATA4積聚增加：這種積累與p16INK4a基因相關(guān)。而且，老化小鼠肝臟表明NF-KB通過RELAX磷酸化被活化，這個結(jié)果與我們培養(yǎng)的細(xì)胞一致。我們也研究了人腦中的GATA4，老年人的額葉皮質(zhì)中，GATA4和p16INK4a均增加。為了證實(shí)這些結(jié)果，我們通過免疫熒光微鏡研究了年輕人和老年人額葉皮質(zhì)G

22、ATA4和p16INK4a基因之間的空間關(guān)系。GATA4和p16INK4a在老年人大腦中的表達(dá)更多。此外，我們在少突膠質(zhì)細(xì)胞、椎體神經(jīng)元、星形細(xì)胞中發(fā)現(xiàn)了GATA4和p16INK4a的一個顯著空間相關(guān)性，進(jìn)一步支持GATA4在人衰老過程中的老化作用。 因此，在鼠和人衰老過程中，GATA4可能促進(jìn)細(xì)胞衰老和炎癥反應(yīng)。我們的研究結(jié)果表明，自噬對衰老有正性和負(fù)性調(diào)節(jié)作用。In response to IR，這些衰老表型依賴于DDR信號激酶ATM和ATR，和衰老相關(guān)基因p53和p16INK4a的表達(dá)類似。然而，GATA4途徑是獨(dú)立于p53和p16INK4a通路的，從而建立了一個新的DDR途徑。 GAT

23、A4調(diào)節(jié)細(xì)胞衰老一部分是通過影響SASP起作用的。同樣，GATA4可以作為TRAF3IP2 and IL1A 介導(dǎo)的NF-B的上游調(diào)節(jié)因子啟動和維持NF-B活性的調(diào)節(jié)因子。GATA4也激活的miR-146a的表達(dá)，從而降低NF-KB的活性。 GATA4-MIR-146A-NF-kB通路形成了一個無規(guī)律的正反饋途徑，在SASP基因表達(dá)風(fēng)暴形成之后，能抑制炎癥反應(yīng)。 然而，這并不終止炎癥反應(yīng)，盡管在高水平的miR-146A存在下，衰老細(xì)胞也能維持SASP的水平。miR-146a可能防止意外激活該途徑以至于GATA4或其它因子在正常狀態(tài)下隨機(jī)波動。 總之，我們的結(jié)果表明，GATA4是一衰老表型的關(guān)鍵

24、條件因子，并通過IL1A和TRAF3IP2激活NF-B途徑參與自噬和DDR所致的老化過程。細(xì)胞老化被證明是把雙刃劍。這個途徑可以促進(jìn)正常的發(fā)育和傷口的愈合。然而，衰老細(xì)胞的積累可作為識別生物體年齡，能促進(jìn)老化相關(guān)的表型與衰老相關(guān)的疾病，包括癌癥和神經(jīng)退行性變。因此，了解潛在的對老化的控制途徑對人體健康有重要意義。調(diào)節(jié)GATA4途徑可能為治療年齡相關(guān)性疾病提供了一個新思路。GATA4, a novel senescence regulatorIn an effort to develop new markers for senescence, we analyzed microRNA expre

25、ssion in non senescent and senescent human fibroblasts (strain IMR-90 from fetal lung). We induced senescence by replicative exhaustion and found miR-146a to be highly expressed by senescent but not non senescent cells (fig.S1A),a result also reported for human foreskin fibroblasts (HCA2)(34). Given

26、 its abundant expression, we suspected that miR-146a regulation occurs primarily at the level of transcription. We therefore generated a 1.5-kb miR-146a promoter fragment fused to green fluorescent protein (PmiR-146a-GFP). Expression of this reporter construct was significantly increased in response

27、 to several senescence-inducing stimuli, including replicative exhaustion, ionizing radiation (IR; 12 Gy), and expression of oncogenic RAS (fig. S1B). Using the Evolutionarily Conserved Regions browser (35), we foundthat the miR-146a promoter contains two evolutionarily conserved regions, ECR1 and E

28、CR2. ECR2 was critical for reporter activity (fig. S1C). Because ECR2 has a putative NF-kB binding site and NF-kB is known to regulate miR-146a (36), we inhibited NF-kB either pharmacologically (with an inhibitor of IkB kinase, Bay11-7082) or genetically with short interfering RNAs (siRNAs) targetin

29、g the NF-kB family member RELA(p65) in fully senescent SASP-expressing cells. NF-kB inhibition only partially decreased reporter activity in senescent cells (fig. S1D). These results indicate that other transcription factors also contribute to senescence dependent activation of the miR-146a reporter

30、. To identify additional transcription factors responsible for the reporter activity, we searched for ones that were predicted to bind to ECR2(see Materials and Methods). We individually overexpressed each candidate and examined reporter activity. Of 13 transcription factors selected, only GATA4, bu

31、t not other GATA family members, activated the reporter (Fig. 1A and fig. S2, A and B). Depletion of GATA4 with siRNAs showed that it was required for reporter activation during senescence (Fig. 1B). Chromatin immunoprecipitation combined with quantitative polymerase chainreaction(ChIP-qPCR) reveale

32、d that exogenously expressed GATA4 bound directly the miR-146a ECR2 region(fig.S2C). These results indicate that GATA4 contributes to activation of the miR-146a promoter in senescent cells. GATA4 is a zinc finger transcription factor essential for the development of various organs, including heart,

33、testis, foregut, liver, and ventral pancreas (37). To examine whether GATA4 functions in senescence, we examined the effects of ectopic expression or depletion during the senescence response of human diploid fibroblasts. Ectopic expression of GATA4 induced senescence in human foreskin fibroblasts (s

34、train BJ;Fig.1C)and IMR-90 fibroblasts(fig.S3,Aand B), as shown by increased senescence-associated b-galactosidase (SA-b-Gal) activity and decreased 5-bromo-2-deoxyuridine (BrdU) in corporation. More important, depletion of GATA4 with stably expressed short hair pinRNAs (shRNAs) partially decreased

35、IR-induced SA-b-Gal activity (Fig. 1D and fig. S3C) and delayed replicative senescence (Fig. 1E). We confirmed these results by CRISPR mutation of GATA4 (fig. S3D). These data indicate that GATA4 is a positive regulator of senescence. Consistent with a regulatory role in senescence, GATA4 is frequen

36、tly silenced in lung, colon,prostate,ovarian,and breast cancer (38,39). Selective autophagy suppresses GATA4 and senescence While examining the efficiency of GATA4 siRNAs (Fig. 1B), we noticed that the amount of GATA4 increased in senescent cells. Indeed, abundance of the GATA4 protein, but not mRNA

37、, increased during IR- and oncogene-induced senescence and replicative senescence (Fig. 2A). This increase was primarily due to increased protein stability, as indicated by the measurement of protein stability in the presence of cycloheximide (Fig. 2B) and by global protein stability profiling (40)

38、(fig. S4A). Two major pathways in eukaryotic cells mediate protein degradation: the ubiquitin-proteasome and autophagy-lysosome pathways.Inhibition of the proteasome by MG-132, a proteasome inhibitor,had no effect on GATA4 abundance, whereas GATA4 protein was stabilized in cells treated with distinc

39、t lysosomal inhibitors known to block autophagy: bafilomycin A1 (a selective inhibitor of the vacuolar-type H+adenosine triphosphatase) or E64dandpepstatin(lysosomalproteaseinhib-itors)(Fig.2,CandD).Thesefindings suggest the autophagy-lysosome pathway, not the ubiquitin proteasome pathway,regulatesG

40、ATA4.Consistent with this interpretation, depletion of the autophagy components ATG5 or ATG7 also increased the abundance of GATA4 protein (Fig. 2E). Autophagy can selectively degrade certain substrates, mediated by specific autophagic adaptors (4143). Indeed, depletion of the autophagic adaptor p62

41、 increased the abundance of GATA4 protein(Fig.2F). Exogenously expressed and endogenous GATA4 physically interacted with p62, and the interaction was reduced during senescence (Fig. 2G and fig. S4B). Thus, GATA4 appears to be targeted for autophagic degradation through its association with p62 under

42、 normal conditions but becomes stabilized during senescence, possibly through reduced association with p62. The relationship between senescence and autophagy is unclear. Autophagy is reported to be required for the establishment of senescence (31, 32, 44) or to inhibit senescence (33). Our results c

43、an reconcile these conflicting reports. Selective autophagy might prevent senescence by suppressing positive senescence regulators such as GATA4. However, senescence-inducing stimuli cause GATA4 to escape selective autophagy. Subsequently, general autophagy may be activated, which then contributes t

44、o establishment of senescence. Hence, selective autophagy for GATA4 may be a negative senescence regulator, whereas general autophagy is a positive regulator of senescence. If so, transiently inhibiting autophagy should induce senescence. To test this, we used doxycycline(Dox)inducible shRNAs to dep

45、lete ATG5 or ATG7 and transiently inhibit autophagy (45), allowing GATA4 to accumulate, and then restored autophagy by removing Dox, returning the cell to a senescence permissive state. Transient inhibition of autophagy (GATA4 abundance high, autophagy on) induced senescence more effectively than di

46、d continuous inhibition (GATA4 abundance high, autophagy off); this effect, at least in part, depended on GATA4 (Fig. 2, H and I, and fig.S4C). Continuous and transient inhibition of autophagy increased GATA4 abundance to similar extents, but continuous inhibition failed to induce senescence (Fig. 2

47、I). Consistent with this finding, p62 depletion induced senescence even more effectively than did depletion of the core autophagy regulators ATG7 or ATG5 under normal conditions(fig.S4D). Thus,selective autophagy for GATA4 appears to function as an antisenescence mechanism, whereas general autophagy

48、 functions as a pro-senescence mechanism.GATA4 regulates the SASPTo investigate the mechanisms through which GATA4 regulates senescence, we explored how GATA4 influences gene expression in human fibroblasts. Because GATA4 induces senescence when ectopically expressed, we performed transcriptional pr

49、ofiling with RNA sequencing(RNA-seq) before and after GATA4 expression using a Dox inducible GATA4 vector. We used Gene Ontology(GO) analysis to systematically characterize the cellular processes affected by GATA4 (46). Genes showing increased expression in response to GATA4 showed significant enric

50、hment for theterms “immune response,” “inflammatory response,” and “response to wounding,” whereas genes with decreased expression were mostly enriched for biological processes related to the cell cycle, which correlated well with terms previously linked to senescence(Fig.3AandtableS1). We compared

51、the GATA4-regulated gene set (GATA4-regulated set) with a gene set differentially regulated during replicative senescence(Senescent set). Both up-regulated and down-regulated genes overlapped significantly, with greater statistical significance for the up-regulated genes (P =2.46 × 1040), consi

52、stent with the fact that GATA4 acts mostly as a transcriptional activator (Fig. 3B). These results suggest that GATA4 might activate a major portion of senescence-associated genes. Among the GATA4-regulated, senescence-associated genes, we found several SASP genes, including those encoding IL6, IL8,

53、 C-X-C motif ligand 1 (CXCL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and extracellular matrix(ECM) proteases and inhibitors(7).Because inflammatory and immune-modulatory cytokines and chemokines secreted by senescent cells can reinforce senescence arrest and alter the microenvir

54、onment (1, 2, 10), GATA4 might indirectly regulate other senescent phenotypes, notably growth arrest, through the SASP. We confirmed that ectopic expression of GATA4 induces the expression of genes associated with the SASP by reverse transcription qPCR (RT-qPCR) (Fig. 3C). More important, depletion

55、of GATA4 suppressed the expression of several SASP genes during the establishment of senescence (Fig.3D), indicating that GATA4 indeed controls many SASP genes. Ectopic expression of GATA3another GATAfamily member predicted to be a strong tumor suppressor(47,48) did not increase expression of genes

56、associated with the SASP. Likewise, ectopic expression of GATA3 did not increase expression of TRAF3IP2 tumor necrosis factor receptor associated factor(TRAF)interactingprotein2,a key GATA4 downstream target (see below), although hit is functionally active, as shown by its ability to activate its we

57、ll-known target IL13 (fig. S5A). These results support a specific role for GATA4 in SASP regulation. However, we cannot rule out the possibility that other GATA factors including GATA3 may have a similar role in other cell types.GATA4 regulates NF-kBNF-kB has a crucial role in controlling the SASP (

58、18, 19, 49) (Fig. 3D), yet little is known about how NF-kB is activated during senescence. To examine the relationship between GATA4 and NF-kB in regulating the SASP, we tested how suppression of the essential NF-kB component RELA affected the GATA4-induced SASP. RELA depletion inhibited the express

59、ion of genes associated with the SASP in response to GATA4(Fig. 4A). GATA4 expression triggered NF-kB activation, and GATA4 depletion inhibited NF-kB activation during senescence (Fig. 4B); these findings suggest that GATA4 acts upstream of NF-kB in regulating the SASP. To understand how GATA4 activates NF-kB, we searched promoters bound by GATA4 in genome wide ChIP experiments (50) to find associated genes that function as NF-kB activators, andexamined their regulation by GATA4. GATA4 induced the