Functional characterization of growth arrest-specific genes in primary fibroblasts

**0*0*1*439*2507*麦克马斯特大学*20*5*2941*14.0***正常*0***FALSE**EN-US*JA*X-NONE******/**mso-tstyle-rowband-size：0；*mso-tstyle-colband-size：0；*mso-style-noshow：是；*mso-style-first：99；*mso-style-parly：“”；*mso-pending-alt：0 cm 5.4pt 0 cm 5.4pt；*mso-para-mark-top：0 cm；*mso-para-edge-right：0 cm；*mso-para-mark-Bottom：10.0pt；*mso-para-边距-Left：0 cm；*mso-Page：寡妇-孤儿；*字体大小：12.0pt；*FONT-Family：Cambria；*mso-ascii-FONT-Family：Cambria；*mso-ascii-主题-FONT：Minor-拉丁语；*mso-hansi-FONT-Family：Cambria；*mso-hansi-heme-FONT：Minor-拉丁语；*mso-fareast-Language：JA；}*面临营养耗竭(饥饿)的细胞退出细胞周期，进入可逆的生长停滞状态，称为G0或静止。正常的贴壁细胞，如成纤维细胞，也会由于接触抑制而在汇合处退出细胞周期。接触抑制的信号和机制仍然不清楚。以鸡胚成纤维细胞(CEF)为模型，我们最近发现低氧(氧气限制)是密度滞留细胞的一个特征，足以在亚融合的CEF中诱导G0。与这一发现一致的是，接触抑制诱导了几个基因对低氧的反应。其中一个基因p20K Lipocalin基因的特征表明，p20K在接触抑制细胞的生存中发挥了作用。事实上，shRNA介导的p20K下调导致了脂质过氧化和细胞凋亡的发生。初步的脂质体分析表明，在低氧和/或接触抑制期间，几种游离脂肪酸(FFA)和游离胆固醇积累。P20K的下调进一步显著地促进了FFA的积累，而不是胆固醇的积累，这与这种Lipocalin在脂质动态平衡中的作用一致。基因图谱显示，几种脂结合蛋白或脂代谢调节因子(包括磺基转移酶1E1、蛋白激酶Cη和ABCA1转运蛋白)的基因在接触抑制下被激活，而不是饥饿，这表明在融合性CEF中，脂质和膜的动态平衡是GAS基因的关键靶点。蛋白激酶Cη的初步鉴定表明，它与内质网高尔基体有关，存在于低氧或接触抑制的CEF中的胞外囊泡中。*基于这些观察，我们提出了以下假设：*“接触抑制调节GAS基因表达的程序是对极限氧浓度的适应性反应的一部分。这种反应促进了生长停滞、脂质/膜稳态和细胞存活。*为了检验这一假说，我们提出了以下目标：*1-表征p20K在脂质稳态中的作用。*2-通过研究基因图谱分析中确定的脂结合因子，在生长受阻的CEF中建立“脂质过氧化/膜应激反应”的概念。*3-确定新的信号因子和生物过程的作用，包括外切体的释放，在对接触抑制和氧气限制的反应中。*通过类比饥饿和内质网应激引起的“未折叠蛋白反应”(UPR)，接触抑制了细胞Fa。

* * 0* 0* 1* 439* 2507* McMaster University* 20* 5* 2941* 14.0* * * * *** * Normal* 0* * * * * false* false* false* * EN-US* JA* X-NONE* * * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * **** /* Style Definitions */*table.MsoNormalTable* {mso-style-name:"Table Normal";* mso-tstyle-rowband-size:0;* mso-tstyle-colband-size:0;* mso-style-noshow:yes;* mso-style-priority:99;* mso-style-parent:"";* mso-padding-alt:0cm 5.4pt 0cm 5.4pt;* mso-para-margin-top:0cm;* mso-para-margin-right:0cm;* mso-para-margin-bottom:10.0pt;* mso-para-margin-left:0cm;* mso-pagination:widow-orphan;* font-size:12.0pt;* font-family:Cambria;* mso-ascii-font-family:Cambria;* mso-ascii-theme-font:minor-latin;* mso-hansi-font-family:Cambria;* mso-hansi-theme-font:minor-latin;* mso-fareast-language:JA;}****Cells facing nutrient depletion (starvation) exit the cell cycle and enter a state of reversible growth arrest known as G0 or quiescence. Normal adherent cells such as fibroblasts also exit the cell cycle at confluence as a result of contact inhibition. The signals and mechanisms of contact inhibition remain poorly defined. Using chicken embryo fibroblasts (CEF) as a model, we showed recently that hypoxia (oxygen limitation) is a feature of density-arrested cells and is sufficient to induce G0 in sub-confluent CEF. In agreement with this finding, several genes of the response to hypoxia were induced by contact inhibition. The characterization of one of these genes, the p20K lipocalin gene, pointed to a role for p20K in the survival of contact inhibited cells. Indeed, the down-regulation of p20K mediated by a shRNA led to increased lipid peroxidation and the onset of apoptosis. Preliminary lipidome analyses, indicated that several free fatty acid (FFA) species and free cholesterol accumulate during hypoxia and/or contact inhibition. The accumulation of FFA, but not cholesterol, was further and drastically enhanced by the down-regulation of p20K consistent with a role for this lipocalin in lipid homeostasis. Gene profiling revealed that genes for several lipid-binding proteins or regulators of lipid metabolism (including sulfo-transferase 1E1, Protein Kinase C η and ABCA1 transporter) are activated by contact inhibition but not starvation suggesting that lipid and membrane homeostasis is a key target of gas genes in confluent CEF. Preliminary characterization of PKCη revealed that it is associated with the ER-Golgi network and is present in extra-cellular vesicles known as exosomes in hypoxic or contact inhibited CEF. *** ***Based on these observations, we propose the following HYPOTHESIS:*** ***“The program of gas gene expression regulated by contact inhibition is part of an adaptive response to limiting oxygen concentrations. This response promotes growth arrest, lipid/membrane homeostasis and cell survival.”***** ***To test this hypothesis, we propose the following OBJECTIVES:*** ***1- To characterize the role of p20K in lipid homeostasis.*** ***2- To establish the concept of “Lipid Peroxidation/Membrane Stress Response” in growth-arrested CEF through the study of lipid binding factors identified in gene profiling analyses.*** ***3- To determine the role of novel signaling factors and biological processes, including the release of exosomes, in the response to contact inhibition and oxygen limitation.*** ***By analogy to the “Unfolded Protein Response” (UPR) induced in response to starvation and ER stress, contact inhibited cells fa