Abstract
Purpose :
FOXOs, a family of transcription factors sharing a conserved forkhead DNA-binding domain, are known to be regulated by PI3K/Akt signaling. Akt phosphorylation of FOXOs at three conserved sites is associated with their cytoplasmic localization and their dephosphorylation with retention in the nucleus where they bind to chromatin to regulate transcription. We examined how Akt inhibition impacts FOXO localization in different regions of the lens and differentiation initiation, and the potential function of FOXO1 in formation of the lens Organelle Free Zone.
Methods :
At E12, prior to formation of the OFZ, embryonic chick lenses were exposed in organ culture to the PI3K/Akt-specific inhibitor MK-2206 for 24 hrs with vehicle DMSO as control. Lenses were microdissected into differentiation-state specific regions and cytoplasmic, nucleoplasmic, and chromatin fractions isolated for immunoblot analysis or fixed, cryoprotected and 25mm cryosections prepared for immunolocalization studies using antibodies to FOXO1, FOXO4, and their transcriptional target the cell cycle inhibitor p27. For inhibitor studies E12 chick lenses were exposed in organ culture to the inhibitor of FOXO1 transcriptional activity AS1842856 or its vehicle DMSO for 24 hrs. Lenses were either microdissected for immunoblot analysis or cryosectioned for immunolocalization analysis using antibodies to molecular intermediates in autophagy like Beclin, and molecular indicators associated with chromatin cleavage that could be regulated by the FOXOs.
Results :
FOXO1 and FOXO4 association with chromatin increased in response to inhibition of the PI3K/Akt signaling axis in lens epithelial, cortical fiber and nuclear fiber cells. This outcome was directly correlated with increased expression of the FOXO1/4 transcriptional target p27, its premature expression in lens epithelial cells and more widespread expression across the lens fiber cell region. Inhibition of FOXO1 transcriptional activity suppresses expression of beclin1, an initiator of autophagy, in cortical fiber cells. Inhibiting FOXO1 transcriptional activity also suppressed cleavage of the caspase-3 target PARP1 and blocked DNA cleavage in central fiber cell nuclei, shown by immunolabeling for pH2AX.
Conclusions :
The inhibition of PI3K/Akt induced FOXO1/4 chromatin association and the early induction of withdrawal of lens cells from the cell cycle. FOXO1 plays a role in autophagy induction in lens fiber cells and chromatin cleavage.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.