Smurf1对增生性瘢痕纤维化形成的影响及分子机制

摘要 目的：探究Smurf1对增生性瘢痕形成中纤维化进程的影响及分子机制。方法：收集2021年6月至2022年6月空军军医大学第二附属医院烧伤整形科行增生性瘢痕切除手术患者的瘢痕组织及正常皮肤标本各12例,采用HE和Masson染色进行病理学检查。取增生性瘢痕组织无菌处理后,采取组织块法分离培养获取人增生性瘢痕成纤维细胞（HSF）。将HSF细胞按照实验方案分组,（1）Smurf1过表达分组：对照1组（Con-1组）,空载体组（Vector组）和Smurf1过表达组（OE-Smurf1组）;（2）Smurf1干扰表达分组：对照2组（Con-2组）,阴性组（si-NC组）,Smurf1干扰表达组（si-Smurf1组）。再分别将pcDNA3.1空质粒、pcDNA3.1+Smurf1质粒、si-NC和si-Smurf1转染至Vector组、OE-Smurf1组、si-NC组和si-Smurf1组HSF细胞。通过qRT-PCR检测Smurf1表达水平,Western blot检测蛋白表达水平,CCK-8检测细胞增殖水平,流式细胞术检测细胞凋亡水平,Transwell实验检测细胞侵袭水平,细胞划痕实验检测细胞迁移水平。结果：HE染色和Masson染色结果显示,与正常皮肤组织相比,增生性瘢痕组织的真皮层厚度显著增加,真皮层中存在大量被染成蓝色的胶原纤维,排列紊乱且致密。与正常皮肤组织相比,增生性瘢痕组织中TGF-β1、α-SMA、COL1、COL3、TβR-I、p-Smad3和Smad7的蛋白表达水平均升高（P<0.05）,Smurf1表达水平降低（P<0.05）。与Con-1或Vector组比较,OE-Smurf1组HSF细胞中TGF-β1、α-SMA、COL1、COL3、TβR-I、p-Smad3和Smad7蛋白的表达水平均降低（P<0.05）,HSF细胞增殖、侵袭和迁移水平降低（P<0.05）,凋亡水平升高（P<0.05）。与Con-2组或si-NC组比较,si-Smurf1组HSF细胞中TGF-β1、α-SMA、COL1、COL3、TβR-I、p-Smad3和Smad7蛋白的表达水平均升高（P<0.05）,HSF细胞增殖、侵袭和迁移水平升高（P<0.05）,凋亡水平降低（P<0.05）。结论：Smurf1可能通过抑制TGF-β1/Smad通路,进而抑制增生性瘢痕的纤维化进程。     

黄芩苷对慢性萎缩性胃炎模型鼠OPG/RANKL轴的影响

摘要 目的：探讨黄芩苷对慢性萎缩性胃炎模型鼠OPG/RANKL轴的影响。方法：将建模成功的大鼠(n=42)平分为三组-模型组、雷尼替丁组与黄芩苷组,黄芩苷组灌胃6.3 g/kg体重的黄芩苷溶液(5 mg?kg^-1),雷尼替丁组灌胃6.3 g/kg体重的雷尼替丁生理盐水溶液(150 mg?kg^-1),模型组灌胃与同容积的生理盐水,记录不同时间点OPG/RANKL轴表达变化情况。结果：雷尼替丁组与黄芩苷组治疗后2 w与4 w的体重高于模型组(P<0.05),黄芩苷组高于雷尼替丁组(P<0.05)。雷尼替丁组与黄芩苷组治疗后2 w与4 w的胃黏膜组织评分低于模型组(P<0.05),黄芩苷组低于雷尼替丁组(P<0.05)。雷尼替丁组与黄芩苷组治疗后2 w与4 w的血清NO与SOD含量高于模型组(P<0.05),黄芩苷组高于雷尼替丁组(P<0.05)。雷尼替丁组与黄芩苷组治疗后2 w与4 w的胃窦组织OPG、RANKL蛋白相对表达水平高于对照组,黄芩苷组高于雷尼替丁组(P<0.05)。结论：黄芩苷治疗慢性萎缩性胃炎模型鼠能激活OPG/RANKL轴,提高血清NO与SOD含量,能减少胃黏膜组织损伤,提高大鼠体重。     

HSP70、eIF4E、DNMT1在宫颈癌中的表达及意义

摘要 目的：探讨热休克蛋白70（HSP70）、真核细胞翻译起始因子4E（eIF4E）、DNA甲基转移酶1（DNMT1）在宫颈癌中的表达及意义。方法：选择2015年2月至2017年2月我院接诊的40例女性宫颈癌患者为本研究对象,收集所有患者手术切除病理组织制作石蜡切片,并选择我院同期接受其他手术的35例标本作为对照组,使用SP免疫组织化学法观察乳腺癌组织中HSP70、eIF4E、DNMT1染色结果,并分析其和临床病理因素之间的关系。结果：在免疫组化学法结果中显示,40例宫颈癌组织中,HSP70阳性表达率为65.00%（26/40）,eIF4E阳性表达率为67.50%（27/40）,DNMT1阳性表达率为72.50%（29/40）,均显著高于对照组（P＜0.05）;在宫颈癌组织中,HSP70、eIF4E、DNMT1和分化程度、临床分期及淋巴转移均有密切关系,（P＜0.05）;将分化程度、临床分期、淋巴转移、HSP70、eIF4E、DNMT1进行相关分析,结果显示,HSP70、eIF4E、DNMT1和分化程度、临床分期及淋巴转移之间均呈正相关（P＜0.05）,且HSP70和eIF4E、DNMT1均呈正相关（P＜0.05）,eIF4E和DNMT1呈正相关（P＜0.05）。结论：在宫颈癌组织中HSP70、eIF4E、DNMT1的高表达和临床病理之间存在着密切关系。     

奥曲肽联合黄芩素抑制胰腺神经内分泌肿瘤细胞增殖和迁移

摘要 目的：探讨奥曲肽（Octreotide，Oct）联合黄芩素（Baicalein，BE）对胰腺神经内分泌肿瘤（Pancreatic neuroendocrine neoplasms, pNENs）的影响。方法：用不同浓度的奥曲肽和黄芩素处理pNENs来源的QGP-1细胞，CCK8法检测细胞的存活率，以其半抑制浓度（IC₅₀）的比值作为浓度梯度进行联合用药，并用Compusyn软件进行联合指数（CI）分析，取细胞毒性效应适中且CI值较低的浓度（奥曲肽150 μmol/L，黄芩素20 μmol/L）进行表型实验。将QGP-1细胞分为四组：对照组、奥曲肽组、黄芩素组、奥曲肽和黄芩素联用组。通过克隆形成实验、EdU增殖检测、划痕实验、细胞凋亡和周期检测评价四组细胞的增殖、迁移能力以及凋亡和细胞周期的情况。结果：药物处理24 h后，奥曲肽和黄芩素均能够呈浓度依赖性抑制QGP-1细胞增殖（P<0.05），奥曲肽的IC₅₀为329.90 μmol/L，黄芩素的IC₅₀为42.86 μmol/L，两者IC₅₀的比值约为7.5:1，联用组细胞活性明显低于单药组（P<0.05），且CI值随着药物浓度的增加而降低。克隆形成实验和EdU增殖检测结果表明，奥曲肽和黄芩素均可抑制QGP-1细胞增殖（P<0.05），且联用组的克隆形成率和EdU阳性细胞百分比均低于单药组（P<0.05）。划痕实验结果表明，奥曲肽和黄芩素均可降低QGP-1细胞的迁移率（P<0.05），且联用组的迁移率低于单药组（P<0.05）。细胞凋亡和周期检测结果表明，黄芩素可促进QGP-1细胞的凋亡（P<0.05），并使QGP-1细胞阻滞于G0/G1期（P<0.05），而奥曲肽对QGP-1细胞的凋亡和细胞周期无明显影响（P>0.05），两药联用对其凋亡和周期无协同作用。结论：奥曲肽和黄芩素联合用药可协同抑制QGP-1细胞增殖和迁移，对细胞凋亡和周期无明显协同作用。     

黄芩素对人口腔鳞癌细胞增殖和侵袭的作用及其机制

目的：研究黄芩素（BAI）抑制口腔鳞癌细胞（TCA8113）增殖与侵袭及与ERK-FAK的可能关系。方法：本研究分为两次实验,每次实验有4个组：control,20 μmol/L BAI,40 μmol/L BAI,80 μmol/L BAI;control,40 μmol/L BAI,MEK抑制剂（0.33 nmol/L）,MEK抑制剂（0.33 nmol/L）+40 μmol/L BAI。每组3个复孔,各组分别处理24 h和48 h。利用CCK8实验检测黄芩素对口腔鳞癌细胞的增殖抑制作用;半定量PCR及Western blot分析黄芩素对E-cadherin和Vimentin的影响;利用Western blot分析黄芩素对ERK,p-ERK,FAK以及p-FAK的调节作用;采用MEK抑制剂（U0126）,利用Western blot分析其对黄芩素的调控作用。结果：黄芩苷处理组的细胞增殖率明显低于对照组（P<0.01）;黄芩素处理组对E-cadherin的mRNA和蛋白水平的上调作用明显高于对照组,对Vimentin的mRNA和蛋白水平的下调作用也明显低于对照组（P<0.01）;黄芩素处理组的p-ERK和p-FAK的蛋白水平明显低于对照组（P<0.01）,但总的ERK与FAK的蛋白水平与对照组相比没有明显的差别（P<0.05）;MEK抑制剂处理组的E-cadherin的蛋白水平明显高于对照组（P<0.01）,Vimentin,p-ERK和p-FAK的蛋白水平明显低于对照组（P<0.01）,但总的ERK与FAK的蛋白水平与对照组相比没有明显的差别（P<0.05）。结论：黄芩素能够抑制口腔鳞癌细胞的增殖以及侵袭,其机制可能与黄芩素抑制ERK-FAK信号通路有关。     

碱性成纤维细胞生长因子对激光烧伤大兔皮肤愈合的影响分析

摘要 目的：探讨碱性成纤维细胞生长因子对激光烧伤大兔皮肤愈合的影响分析。方法：通过热辐射仪激光灼烧对大兔耳朵进行烧伤处理,根据实验需求,将其随机分为三组：对照组,bFGF组,bFGF + DAPT组。通过ImageJ软件测量伤口面积和疤痕组织的厚度,并定期计算残余伤口面积率和疤痕指数。通过组织学分析大兔伤口愈合的新血管生成量。通过蛋白印迹分析Notch1、Jagged1和Hes1的蛋白表达。通过免疫荧光分析愈合后的皮肤中α-SMA,Col I和Col III的相对蛋白水平。结果：bFGF组较对照组的疤痕指数降低（P<0.05）,bFGF+DAPT组较bFGF组疤痕指数升高（P<0.05）。bFGF组较对照组的愈合面积增加（P<0.05）,bFGF + DAPT组较bFGF组愈合面积降低（P<0.05）。与对照组相比,bFGF组的愈合时间明显缩短,较低的残余伤口面积和较低的疤痕指数（P<0.05）,而bFGF + DAPT组表现出明显的愈合延迟和较高的疤痕指数（P<0.05）。bFGF组较对照组的新血管生成量增加（P<0.05）,bFGF + DAPT组较bFGF组心血管生成量减少（P<0.05）。bFGF组较对照组的肉芽组织平均厚度增加,表皮间隙的闭合百分比升高（P<0.05）,bFGF + DAPT组较bFGF组肉芽组织平均厚度增加、表皮间隙的闭合百分比减少（P<0.05）。H＆E染色进行组织学分析发现,与对照组和bFGF + DAPT组相比,bFGF组出现明显的再上皮化和新血管形成,愈合效率较高（P<0.05）。bFGF组较对照组Notch1、Jagged1和Hes1的蛋白表达升高（P<0.05）,bFGF + DAPT组较bFGF组Notch1、Jagged1和Hes1的蛋白表达降低（P<0.05）。bFGF组较对照组?琢-SMA,Col I和Col III的蛋白表达降低（P<0.05）,bFGF + DAPT组较bFGF组表达升高（P<0.05）。结论：bFGF可以通过促进ESC的增殖并通过激活Notch1 / Jagged1途径抑制其向肌成纤维细胞（Myofibroblasts,MFB）的分化加快伤口愈合,减少疤痕形成。     

Rho激酶抑制剂Y-27632对C3H10T1/2细胞增殖与成脂分化的作用

探究Rho激酶抑制剂Y-27632对间充质干细胞（mesenchymal stem cells,MSCs）C3H10T1/2增殖和成脂分化的影响.实验分为对照组、成脂诱导组和Y-27632处理组（Y-27632+成脂诱导）. 利用MTT检测细胞增殖情况,油红O染色,异丙醇萃取法检测细胞成脂分化情况,半定量RT-PCR检测过氧化物酶体增殖物激活受体γ(peroxisome proliferator activiated receptor γ, PPARγ)和CCAAT增强子结合蛋白α (CCAAT enhancer binding protein α, C/EBPα)基因表达. 结果表明,Y-27632能够显著抑制C3H10T1/2细胞的增殖(P<0.05),并呈一定的浓度依赖性;高浓度Y-27632对C3H10T1/2细胞成脂分化具有显著抑制作用（P<0.05）;半定量RT-PCR结果显示,成脂诱导处理组PPARγ和C/EBPα表达量在第3 d、5 d和7 d显著低于成脂诱导组（P<0.05）. 综上所述,Y-27632能够抑制C3H10T1/2细胞增殖与成脂分化.     

降钙素基因相关肽对肺纤维化大鼠肺组织eIF3a、p27表达的影响

目的：观察降钙素基因相关肽（CGRP）对肺纤维化大鼠肺组织真核翻译起始因子3a （eIF3a）、p27表达的影响,探讨CGRP在肺纤维化中的作用及机制。方法：雄性SD大鼠,体重180~220 g,随机分为3组（n=8）：对照组、博莱霉素组、博莱霉素+辣椒素组。采用气管内注射博莱霉素（5 mg/kg）诱导肺纤维化大鼠模型。造模前4 d大鼠皮下注射辣椒素（Capsaicin）（50 mg/kg·d）,造模后第28天处死动物,颈动脉采血ELISA法测定血浆CGRP含量。细胞实验分6组（n=9）：Control组,转化生长因子-β1（TGF-β1）组,CGRP （1、10、100 nmol/L）组,CGRP8-37 1 μmol/L和CGRP 100 nmol/L组。细胞用CGRP和（或） CGRP8-37预处理1 h,再用TGF-β1（5 ng/ml）处理48 h。5-溴脱氧尿嘧啶核苷（BrdU）法检测细胞增殖。免疫组化、real-time PCR和（或） Western blot检测eIF3a、p27、α-平滑肌肌动蛋白（α-SMA）、collagen Ⅰ mRNA及蛋白表达。结果：博莱霉素诱发肺纤维化动物肺组织eIF3a、α-SMA及Ⅰ胶原表达增高,CGRP及p27的表达明显降低。外源性CGRP可剂量依赖性的抑制TGF-β1诱导的肺成纤维细胞增殖,明显抑制eIF3a、α-SMA、Ⅰ胶原的表达,上调p27的表达,这些作用可以被CGRP阻断剂CGRP8-37所取消。结论：CGRP在博莱霉素诱导的肺纤维化中起着重要作用,可能通过抑制eIF3a、上调p27的表达而抑制肺成纤维细胞的增殖,进而抑制肺纤维化的形成与发展。     

IL-33/HMGB-1与胎鼠皮肤伤口的瘢痕形成的相关性研究

摘要 目的：探究IL-33/HMGB-1在胎鼠伤口愈合中的作用。方法：构建小鼠伤口愈合模型,并随机分组为注射PBS组、注射重组蛋白IL-33组和重组蛋白HMGB-1组。通过免疫组织化学、DAB和苏木精复染方法检测IL-33/HMGB-1的表达及定位;结合Axiovision软件计算MOMA-2阳性巨噬细胞、波形蛋白阳性成纤维细胞和血管密度;通过Masson''s三色染色评估伤口胶原蛋白的沉积情况和愈合情况。结果：E15和E18胎鼠未损伤皮肤的基底角质形成细胞核均呈阳性染色;与E15胎鼠相比,E18胎鼠皮肤中HMGB-1和IL-33的表达水平升高（P<0.05）。处理0 h-48 h,E15和E18胎鼠伤口边缘附近角质形成细胞的核染色呈降低,IL-33和HMGB-1表达水平均降低（P<0.05）。Masson三色染色结果显示,与PBS组相比,当采取200 ng或400 ng HMGB-1或IL-33处理,E15胎鼠伤口愈合形成疤痕的数量均显著增加（P<0.05）,且疤痕大小呈剂量依赖性增加（P<0.05）。创伤后7 d,与PBS组相比,HMGB-1和IL-33处理的E15胎鼠伤口和瘢痕中波形蛋白阳性成纤维细胞、MOMA-2阳性巨噬细胞的数量和PECAM阳性血管密度均显著升高（P<0.01）。结论：IL-33/HMGB-1可以促进胎鼠伤口瘢痕的形成,其可能机制包括对成纤维细胞的直接刺激,以及与伤口中血管生成和巨噬细胞募集增加有关。     

4-1BBL通过激活Wnt/β-catenin信号通路促进人胃癌细胞的增殖和迁移

4-1BB和4-1BB配体(4-1BBL),又被称为CD137和CD137配体,分别属于肿瘤坏死因子(TNF)受体和配体家族的成员。4-1BBL 与4-1BB相互作用可以激活T细胞免疫应答。因此,4-1BBL一直在抗肿瘤免疫应答中发挥经典的免疫共刺激分子作用。近期研究发现,4-1BBL在肿瘤细胞中另有其他的生物学功能,但4-1BBL在胃癌进展过程中的功能尚不明确。本文探讨了4-1BBL在人胃癌细胞中的生物学功能和分子作用机制。首先,通过检索TCGA和Kaplan Meier plotter数据库发现,4-1BBL在胃癌组织中的表达显著高于癌旁组织（P<0.001）,且4-1BBL的高表达与胃癌的不良预后正相关（P<0.05）。细胞生物学的结果显示,敲除4-1BBL明显抑制胃癌细胞的增殖（P<0.05）、侵袭和迁移（P<0.05）,促进胃癌细胞的凋亡（P<0.05）;另外,蛋白质免疫印迹结果表明,敲除4-1BBL可使β-联蛋白、c-Myc和细胞周期蛋白D1(cyclin D1)的蛋白质表达水平下降,抑制Wnt/β-catenin信号通路。相反,过表达4-1BBL则显著促进胃癌细胞增殖（P<0.05）、侵袭和迁移（P<0.05）,减少胃癌细胞的凋亡（P<0.05）;且过表达4-1BBL促进β-联蛋白(β-catenin)、c-Myc和细胞周期蛋白D1的蛋白质表达,激活Wnt/β-catenin信号通路。综上所述,4-1BBL可通过激活Wnt/β-catenin信号通路促进人胃癌细胞的增殖和迁移。     

Human cytomegalovirus infection induces rapamycin-insensitive phosphorylation of downstream effectors of mTOR kinase

Signaling mediated by the cellular kinase mammalian target of rapamycin (mTOR) activates cap-dependent translation under normal (nonstressed) conditions. However, translation is inhibited by cellular stress responses or rapamycin treatment, which inhibit mTOR kinase activity. We show that during human cytomegalovirus (HCMV) infection, viral protein synthesis and virus production proceed relatively normally when mTOR kinase activity is inhibited due to hypoxic stress or rapamycin treatment. Using rapamycin inhibition of mTOR, we show that HCMV infection induces phosphorylation of two mTOR effectors, eucaryotic initiation factor 4E (eIF4E) binding protein (4E-BP) and eIF4G. The virally induced phosphorylation of eIF4G is both mTOR and phosphatidylinositol 3-kinase (PI3K) independent, whereas the phosphorylation of 4E-BP is mTOR independent, but PI3K dependent. HCMV infection does not induce mTOR-independent phosphorylation of a third mTOR effector, p70S6 kinase (p70S6K). We show that the HCMV-induced phosphorylation of eIF4G and 4E-BP correlates with the association of eIF4E, the cap binding protein, with eIF4G in the eIF4F translation initiation complex. Thus, HCMV induces mechanisms to maintain the integrity of the eIF4F complex even when mTOR signaling is inhibited.     

The splicing factor SF2/ASF regulates translation initiation by enhancing phosphorylation of 4E-BP1

The SR protein SF2/ASF has been initially characterized as a splicing factor but has also been shown to mediate postsplicing activities such as mRNA export and translation. Here we demonstrate that SF2/ASF promotes translation initiation of bound mRNAs and that this activity requires the presence of the cytoplasmic cap-binding protein eIF4E. SF2/ASF promotes translation initiation by suppressing the activity of 4E-BP, a competitive inhibitor of cap-dependent translation. This activity is mediated by interactions of SF2/ASF with both mTOR and the phosphatase PP2A, two key regulators of 4E-BP phosphorylation. These findings suggest the model whereby SF2/ASF functions as an adaptor protein to recruit the signaling molecules responsible for regulation of cap-dependent translation of specific mRNAs. Taken together, these data suggest a novel mechanism for the activation of translation initiation of a subset of mRNAs bound by the shuttling protein SF2/ASF.     

Perillyl alcohol and genistein differentially regulate PKB/Akt and 4E-BP1 phosphorylation as well as eIF4E/eIF4G interactions in human tumor cells

Previously we demonstrated that secondary products of plant mevalonate metabolism called isoprenoids attenuate 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA translational efficiency and cause tumor cell death. Here we compared effects of "pure" isoprenoids (perillyl alcohol and gamma-tocotrienol) and a "mixed" isoprenoid-genistein-on the PKB/Akt/mTOR pathway that controls mRNA translation and m(7)GpppX eIF4F cap binding complex formation. Effects were cell- and isoprenoid-specific. Perillyl alcohol and genistein suppressed 4E-BP1(Ser65) phosphorylation in prostate tumor cell lines, DU145 and PC-3, and in Caco2 adenocarcinoma cells. Suppressive effects were similar to or greater than that observed with a PI3 kinase inhibitor or rapamycin, an mTOR inhibitor. 4E-BP1(Thr37) phosphorylation was reduced by perillyl alcohol and genistein in DU145, but not in PC-3. Conversely, perillyl alcohol but not genistein decreased 4E-BP1(Thr37) phosphorylation in Caco2. PKB/Akt activation via Ser473 phosphorylation was enhanced in DU145 by perillyl alcohol and in PC-3 by gamma-tocotrienol, but was suppressed by genistein. Importantly, perillyl alcohol disrupted interactions between eIF4E and eIF4G, key components of eIF4F (m(7)GpppX) cap binding complex. These results demonstrate that "pure" isoprenoids and genistein differentially impact cap-dependent translation in tumor cell lines.     

4E-BP1, a multifactor regulated multifunctional protein

Eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) is a member of a family of translation repressor proteins, and a well-known substrate of mechanistic target of rapamycin (mTOR) signaling pathway. Phosphorylation of 4E-BP1 causes its release from eIF4E to allow cap-dependent translation to proceed. Recently, 4E-BP1 was shown to be phosphorylated by other kinases besides mTOR, and overexpression of 4E-BP1 was found in different human carcinomas. In this review, we summarize the novel findings on mTOR independent 4E-BP1 phosphorylation in carcinomas. The implications of overexpression and possible multi-function of 4E-BP1 are also discussed.     

Functional interaction between RAFT1/FRAP/mTOR and protein kinase cdelta in the regulation of cap-dependent initiation of translation

Hormones and growth factors induce protein translation in part by phosphorylation of the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). The rapamycin and FK506-binding protein (FKBP)-target 1 (RAFT1, also known as FRAP) is a mammalian homolog of the Saccharomyces cerevisiae target of rapamycin proteins (mTOR) that regulates 4E-BP1. However, the molecular mechanisms involved in growth factor-initiated phosphorylation of 4E-BP1 are not well understood. Here we demonstrate that protein kinase Cdelta (PKCdelta) associates with RAFT1 and that PKCdelta is required for the phosphorylation and inactivation of 4E-BP1. PKCdelta-mediated phosphorylation of 4E-BP1 is wortmannin resistant but rapamycin sensitive. As shown for serum, phosphorylation of 4E-BP1 by PKCdelta inhibits the interaction between 4E-BP1 and eIF4E and stimulates cap-dependent translation. Moreover, a dominant-negative mutant of PKCdelta inhibits serum-induced phosphorylation of 4E-BP1. These findings demonstrate that PKCdelta associates with RAFT1 and thereby regulates phosphorylation of 4E-BP1 and cap-dependent initiation of protein translation.     

Seasonal and state-dependent changes of eIF4E and 4E-BP1 during mammalian hibernation: implications for the control of translation during torpor

Mammalian hibernation involves cessation of energetically costly processes typical of homeostatic regulation including protein synthesis. To further elucidate the mechanisms employed in depressing translation, we surveyed key eukaryotic initiation factors [eIF2, eIF4B, eIF4E, eIF4GI and -II, and 4E-binding protein-1 (4E-BP1), -2, and -3] for their availability and phosphorylation status in the livers of golden-mantled ground squirrels (Spermophilus lateralis) across the hibernation cycle. Western blot analyses indicated only one significant locus for regulation of translational initiation in ground squirrel liver: control of eIF4E. We found seasonal variation in a potent regulator of eIF4E activity, 4E-BP1. Summer squirrels lack 4E-BP1 and apparently control eIF4E activity through direct phosphorylation. In winter, eIF4E is regulated through binding with 4E-BP1. During the euthermic periods that separate bouts of torpor (interbout arousal), 4E-BP1 is hyperphosphorylated to promote initiation. However, during torpor, 4E-BP1 is hypophosphorylated and cap-dependent initiation of translation is restricted. The regulation of cap-dependent initiation of translation may allow for the differential expression of proteins directed toward enhancing survivorship.     

A hypoxia-controlled cap-dependent to cap-independent translation switch in breast cancer

Translational regulation is critical in cancer development and progression. Translation sustains tumor growth and development of a tumor vasculature, a process known as angiogenesis, which is activated by hypoxia. Here we first demonstrate that a majority of large advanced breast cancers overexpress translation regulatory protein 4E-BP1 and initiation factor eIF4G. Using model animal and cell studies, we then show that overexpressed 4E-BP1 and eIF4G orchestrate a hypoxia-activated switch from cap-dependent to cap-independent mRNA translation that promotes increased tumor angiogenesis and growth at the level of selective mRNA translation. Elevated levels of 4E-BP1 trigger hypoxia inhibition of cap-dependent mRNA translation at high-oxygen levels and, with eIF4G, increase selective translation of mRNAs containing internal ribosome entry sites (IRESs) that include key proangiogenic, hypoxia, and survival mRNAs. The switch from cap-dependent to cap-independent mRNA translation facilitates tumor angiogenesis and hypoxia responses in animal models.     

Eukaryotic translation initiation is controlled by cooperativity effects within ternary complexes of 4E-BP1, eIF4E,and the mRNA 5′ cap

Initiation is the rate-limiting step during mRNA 5′ cap-dependent translation, and thus a target of a strict control in the eukaryotic cell. It is shown here by analytical ultracentrifugation and fluorescence spectroscopy that the affinity of the human translation inhibitor, eIF4E-binding protein (4E-BP1), to the translation initiation factor 4E is significantly higher when eIF4E is bound to the cap. The 4E-BP1 binding stabilizes the active eIF4E conformation and, on the other hand, can facilitate dissociation of eIF4E from the cap. These findings reveal the particular allosteric effects forming a thermodynamic cycle for the cooperative regulation of the translation initiation inhibition.     

Effects of simian virus 40 large and small tumor antigens on mammalian target of rapamycin signaling: small tumor antigen mediates hypophosphorylation of eIF4E-binding protein 1 late in infection

We report that late in a simian virus 40 (SV40) infection in CV-1 cells, there are significant decreases in phosphorylations of two mammalian target of rapamycin (mTOR) signaling effectors, the eIF4E-binding protein (4E-BP1) and p70 S6 kinase (p70S6K). The hypophosphorylation of 4E-BP1 results in 4E-BP1 binding to eIF4E, leading to the inhibition of cap-dependent translation. The dephosphorylation of 4E-BP1 is specifically mediated by SV40 small t antigen and requires the protein phosphatase 2A binding domain but not an active DnaJ domain. Serum-starved primary African green monkey kidney (AGMK) cells also showed decreased phosphorylations of mTOR, 4E-BP1, and p70S6K at late times in infection (48 h postinfection [hpi]). However, at earlier times (12 and 24 hpi), in AGMK cells, phosphorylated p70S6K was moderately increased, correlating with a significant increase in phosphorylation of the p70S6K substrate, ribosomal protein S6. Hyperphosphorylation of 4E-BP1 at early times could not be determined, since hyperphosphorylated 4E-BP1 was present in mock-infected AGMK cells. Elevated levels of phosphorylated eIF4G, a third mTOR effector, were detected in both CV-1 and AGMK cells at all times after infection, indicating that eIF4G phosphorylation was induced throughout the infection and unaffected by small t antigen. The data suggest that during SV40 lytic infection in monkey cells, the phosphorylations of p70S6K, S6, and eIF4G are increased early in the infection (12 and 24 hpi), but late in the infection (48 hpi), the phosphorylations of mTOR, p70S6K, and 4E-BP1 are dramatically decreased by a mechanism mediated, at least in part, by small t antigen.