Caspase-Activated DNase (CAD), p27 and Prox1 are Key Downstream Signaling Targets of Caspase-3 in Lens Differentiation Initiation

Abstract

Purpose : Mitochondrial depolarization in the lens equatorial zone induces a low-level activation of caspase-3 required for initiation of lens differentiation. This pathway is shared by many cell types, is shown to involve activation of CAD, and leads to genetic reprogramming events associated with cell-cycle withdrawal and changes in a cell’s differentiation state. We are now investigating the functional link between the caspase-3/CAD pathway in the developing lens and the expression of genes required for lens differentiation, focusing on the cell cycle inhibitor p27^Kip1, and Prox1, a transcription factor important to early stages of lens cell differentiation.

Methods : The correlation between lens epithelial cell cycle withdrawal and import of p27 and Prox1 to the nucleus was examined in E8 chick embryo lenses in lens organ culture using the EDU click-it assay to identify proliferating lens cells and immunolabeling for p27 and Prox1. To determine whether the activation of caspase-3 is required for the expression and nuclear localization of CAD, p27, and Prox1, caspase-3 activity was blocked in E8 lenses in organ cultures with the caspase-3-specific inhibitor DEVD. Results were analyzed by immunoblot after microdissection into lens epithelial and fiber cell fractions and by immunolocalization analysis.

Results : Nuclear localization of p27 and Prox1 were closely linked to the transition of proliferating lens cells to a post-mitotic state, marking one of the earliest stages of lens differentiation. Expression of p27 and Prox1 is suppressed when caspase-3 activity is inhibited, and confocal image analysis of lens sections immunolabeled for CAD, p27 or Prox1 and co-labeled for nuclei showed their nuclear import is impaired when caspase-3 activation is blocked. Both p27 and Prox1 are missing from the anterior-most regions of the equatorial zone where the lens epithelial cells typically withdraw from the cell cycle. In addition, these studies revealed that blocking caspase-3 activity negatively impacts the nuclear localization of p27 and Prox1 in nascent fiber cells.

Conclusions : Consistent with our discovery that low-levels of caspase-3 activity signal lens fiber cell differentiation initiation, we find that the mechanism involves nuclear localization of CAD, and induction of the transit of the lens differentiation determinants p27 and Prox1 to the nucleus.

This is a 2020 ARVO Annual Meeting abstract.