Functional characterization of growth arrest-specific genes in primary fibroblasts

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* * 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