Expression of the gene encoding poly(ADP-ribose) polymerase-1 is modulated by fibronectin during corneal wound healing.
OBJECTIVE
Poly(ADP-ribose) polymerase (PARP)-1 is a nuclear enzyme essential in several cellular functions such as DNA repair, DNA transcription, carcinogenesis, and apoptosis. Expression of the PARP-1 gene is mainly dictated by the transcription factor Sp1. Fibronectin (FN), a component from the extracellular matrix transiently expressed at high levels during wound healing of the corneal epithelium, was reported to exert a positive influence on expression of the alpha5 integrin subunit gene promoter by altering the state of Sp1 phosphorylation, a process that depended on the activation of the ERK signaling pathway. The present study was undertaken to investigate whether PARP-1 gene expression might be similarly regulated by FN through the same signaling pathways and attempted to link expression of this gene to corneal wound healing in vitro.


METHODS
Expression of PARP-1, Sp1/Sp3, ERK1/2, phospho-ERK1/2, P38 and phospho-P38 was monitored by Western blot in cultures of rabbit corneal epithelial cells (RCECs) grown on FN in the presence of inhibitors of the MAPK, PI3K, and P38 signaling pathways. Electrophoretic mobility shift assays (EMSAs) were conducted to assess the binding of Sp1 and Sp3 in nuclear extracts from RCECs grown on FN in the presence of inhibitors. Plasmids bearing the PARP-1 promoter fused to the CAT reporter gene were also transfected into RCECs grown under similar culture conditions to assess the influence of these inhibitors on PARP-1 promoter activity.


RESULTS
Expression of PARP-1, Sp1, and Sp3 increased considerably in RCECs grown on FN and translated into increased binding of Sp1 and Sp3 to their DNA target sites. In addition, FN increased PARP-1 promoter activity in a cell-density-dependent manner. Inhibition of both the MAPK and the PI3K pathways entirely abolished these properties.


CONCLUSIONS
PARP-1 gene expression was strongly activated by FN through alterations in the phosphorylation state of Sp1 and Sp3 that resulted from the activation of the MAPK and PI3K signaling pathways, thereby suggesting that PARP-1 may play a critical function during the highly proliferative phase that characterizes wound healing of the corneal epithelium.
Zaniolo
Karine
K
Oncology and Molecular Endocrinology Research Center, Universitaire de Québec and Laval University, Québec, Canada.
Gingras
Marie-Eve
ME
Audette
Marie
M
Guérin
Sylvain L
SL
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D019573:Epithelium, Corneal / Q000166:cytology / Q000293:injuries / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D039081:Integrin alpha5beta1 / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics; D015398:Signal Transduction; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor / Q000378:metabolism; D014162:Transfection; D014945:Wound Healing / Q000502:physiology*
D005353:Fibronectins; D039081:Integrin alpha5beta1; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; C499322:Parp1 protein, rat; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D019869:Phosphatidylinositol 3-Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases