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K. Zaniolo, S. Leclerc, S.L. Guérin; Expression of the Poly(ADP-Ribose) Polymerase (PARP) Gene in Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):858.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Poly (ADP-ribose) polymerase (PARP) is a nuclear enzyme that, by post-translationally modifying various proteins, is involved in several important cellular functions, including DNA repair, DNA transcription, carcinogenesis and apoptosis. The transcriptional activity directed by the PARP gene promoter is mainly dictated through its recognition by the transcription factors Sp1 and Sp3. Considering that the level of expression of both Sp1 and Sp3 is deeply altered by the state of cell density in primary cultures of rabbit corneal epithelial cells (RCECs), and that PARP may play a protective function during wound healing, we investigated whether: i) PARP was expressed in RCECs, and ii) whether its pattern of expression followed that of Sp1/Sp3 at varying cell densities and upon cell passages. Methods: Western blot analyses were conducted to follow the expression of both PARP and Sp1/Sp3 in subconfluent and postconfluent RCECs. Similarly, recombinant constructs bearing various lengths from the PARP promoter fused to the CAT reporter gene were transiently transfected in RCECs under similar culture conditions. Electrophoretic mobility shift assays (EMSAs) were conducted to assess the expression of both Sp1 and Sp3 in nuclear extracts from both confluent and subconfluent cells, as well as from RCECs that have been subjected to serial passages in culture. Results: Subconfluent RCECs were shown to express high levels or PARP whereas no PARP expression was observed in two or five-days postconfluent cells. This pattern of PARP expression was identical to that of Sp1/Sp3: high levels of these transcription factors were observed in subconfluent but not confluent RCECs. Consistent with these results, the activity directed by the PARP promoter was found to be elevated in subconfluent but not confluent RCECs. In addition, expression of both Sp1 and Sp3, as well as that of PARP, was shown to be dramatically reduced as cells are passaged in culture and progress toward a more terminally differentiated phenotype. Conclusion: Expression of the PARP gene appears to be coordinated with that of the transcription factors Sp1 and Sp3 in primary cultured RCECs. PARP may therefore play a protective function during the proliferative phase that characterize wound healing of corneal epithelial cells. CR: None Supported by a grant from the Canadian Institutes of Health Research (CIHR)
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