p27

purposep27

methodsPhotoreceptor damage was induced in wild-type (WT) and p27 knockout (KO) mice with N-methyl-N-nitrosourea (MNU) treatment. Damage-induced responses of the RPE were investigated with bromodeoxyuridine (BrdU) incorporation assays, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays at different stages after MNU treatment. Subcellular localization of p27 in the WT RPE was also analyzed in vivo and in vitro.

resultsMNU treatment induced photoreceptor-specific degeneration in the WT and KO retinas. BrdU incorporation assays revealed virtually no proliferation of RPE cells in the WT retinas while, in the KO retinas, approximately 16% of the RPE cells incorporated BrdU at day 2 after MNU treatment. The RPE in the KO retinas developed aberrant protrusions into the outer nuclear layer in response to photoreceptor damage and engulfed outer segment debris, as well as TUNEL-positive photoreceptor cells. Increased phosphorylation of myosin light chains and their association with rhodopsin-positive phagosomes were observed in the mutant RPE, suggesting possible deregulation of cytoskeletal dynamics. In addition, WT RPE cells exhibited evidence of the epithelial-mesenchymal transition (EMT), including morphological changes, induction of α-smooth muscle actin expression, and attenuated expression of tight junction protein ZO-1 while these changes were absent in the KO retinas. In the normal WT retinas, p27 was localized to the nuclei of RPE cells while nuclear and cytoplasmic p27 was detected in RPE cells undergoing EMT, suggesting a role for cytoplasmic p27 in the phenotype changes of RPE cells.

conclusionsp27 loss promoted proliferation and phagocytic activity of RPE cells while preventing EMT after photoreceptor damage. These findings provide evidence for the role of p27 in the control of RPE responses to retinal damage.