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M. Mulhern, K. Ikesugi, R. Yamamoto, N. Fatma, D.P. Singh, T. Shinohara; ATF4 Binds to and Regulates the Promoter of the LEDGF Gene During the Unfolded Protein Response . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3516.
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Cataracts can be induced by the unfolded protein response (UPR), which initiates diversified pathways including the production of reactive oxygen species (ROS) and apoptosis. In the UPR pathway, cell death is induced by the activation of CHOP, a member of the C/EBP family of transcriptional factors, which suppresses the expression of Bcl2 and subsequently activates caspases. CHOP is activated by activating transcriptional factor 4 (ATF4) in the UPR. The UPR also activates the expression of the ledgf gene. Lens epithelium derived growth factor (LEDGF) previously has been defined as a survival factor and activates several antiapoptotic enzymes. The purpose of this study is to determine whether LEDGF is under the control of ATF4 and CHOP in the UPR pathway.
Human lens epithelial cells (LECs) were cultured with ER stressors such as 5mM homocysteine, 1uM calcium ionophore (A23187), or 5uM tunicamycin for 24 hrs. Total protein and RNA were extracted from the resultant cells. Protein blot analysis with antibody to UPR enzymes was conducted. In vitro transcriptional assays with the LEDGF promoter and CAT constructs as well as electrophoretic mobility shift assays (EMSA) were conducted.
Two highly conserved regulatory elements for ATF4 binding and C/EBP binding were found in the center of the LEDGF promoter. ATF4 bound to one of the elements, and we observed an up–regulation of the LEDGF promoter constructs. We also detected increased levels of CHOP in the early phase of the UPR (4–8 hrs) and of LEDGF in the late phase of the UPR (24–48 hrs). These results suggest that ATF4 and CHOP regulate the transcription of the ledgf gene. Resultant higher levels of LEDGF activate the transcription of catalase in addition to other antiapoptotic enzymes to protect cells from the UPR dependent apoptosis.
These results suggest that the following timeline of sequential gene activations can be induced in the UPR: the UPR activates ATF4 and subsequently CHOP, and the resultant CHOP and ATF4 together activate the transcription of the ledgf gene.
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