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T. Shinohara, K. Ikesugi, R. Yamamoto, M.L. Mulhern; The Unfolded Protein Response (UPR); ATF4 Directly Controls the Expression of the LEDGF Gene in LECs . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3606.
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Purpose: Misfolded protein aggregates recognized in the endoplasmic reticulum (ER) induce ER stress and activate the unfolded protein response (UPR). The UPR initiates diversified pathways and an ER resident protein kinase (PERK) is activated. Activated PERK phosphorylates the translational initiation factor (elF2α) and subsequent transcriptional activation of transcriptional factor 4 (ATF4) occurs. LEDGF, a member of the HDGF family of proteins, is a survival factor that rescues many cell types under stress. Although live cells have LEDGF, apoptotic cells lose LEDGF from the nucleus. We hypothesize that ATF4 regulates the expression of the ledgf gene. Methods: Human LECs were cultured with 5mM homocysteine, 1µM Ca++ Ionophore (A23187), or 5µM tunicamycin for 24 hrs. Total protein and RNA were extracted from the resultant cells. Protein blot analysis with Ab to the UPR enzymes was conducted. Levels of mRNA were evaluated by RT–PCR. An in vitro transcriptional assay with the LEDGF promoter and CAT constructs was conducted. Results: We found a duplicated ATF4 binding element in the center of the LEDGF promoter. ATF4 bound to the elements and regulated the expression of LEDGF. Levels of eIF2α and ATF4 were activated during the early phase of the UPR induced by homocysteine, tunicamycin, and A23187. In the apoptotic phase of the UPR, ATF4 and LEDGF were suppressed. Thus, levels of ATF4 and not phosphorylation control the levels of LEDGF transcript. These results suggest that a timeline of events occurs in the UPR; PERK phosphorylates elF2α, which activates ATF4. ATF4 regulates the expression of CHOP and LEDGF. The cell fate might be determined by delicate balance of these factors in the UPR cascades. Conclusions: We found that ATF4 directly regulates the expression of a survival factor, LEDGF in the UPR pathway.
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