Abstract
Purpose :
Selective autophagy, a highly regulated pathway specifically degrades certain cytosolic components that are recognized by selective adaptors. For example, selective autophagy degrades Notch1 protein via specific adaptors p62, which leads to inhibition of stem/progenitor cell differentiation in various tissues including neural cells. However, the role of selective autophagy in corneal/limbal epithelia remains unclear.
Methods :
To determine the differentiation status of limbal epithelium, expression of differentiation markers (e.g., mucin1, PAI-2) as well as putative limbal epithelial stem cell markers (e.g., N-cad, K15) were determined by immunoblotting and immunostaining. 3D organotypic raft cultures of HLECs, a stratified limbal epithelial equivalent, were used to investigate the expression of differentiation markers (e.g., IVL, mucin1).
Results :
Stem/TA cell-enriched HLECs were induced to differentiate by increasing the calcium concentration from 0.07mM to 1.6mM. Such calcium modulation reduced the expression of LC3II, as well as increased p62, indicating an inhibition of autophagy flux. Knockdown of autophagy-related genes (e.g., Beclin1, Atg5) enhanced HLEC differentiation as evidenced by increased expression of mucin1 and PAI-2 and reduced N-cad. This observation was confirmed using 3D organotypic raft cultures, which demonstrated that activation of autophagy by rapamycin inhibited the expression of IVL and mucin 1. To explore how autophagy regulates stem/TA cell-enriched HLEC differentiation, our single cell RNA seq investigation identified a group of novel stem/TA cell regulators including Bmal1 and Wdfy1. Inhibition of autophagy decreased stem cell regulators, Bmal1 and Wdfy1 in vitro and in vivo. Furthermore, knockdown of Bmal1 or Wdfy1 enhanced HLEC differentiation. Significantly, inhibition of Notch signaling, which is specifically and negatively regulated by selective autophagy, increased Wdfy1 expression. This suggests that attenuated autophagy induces differentiation of stem/TA cell-enriched limbal epithelial basal cells via controlling novel stem/TA cell regulators including Bmal1, Notch/Wdfy1.
Conclusions :
Our observations suggest that a decrease in selective autophagy triggers the differentiation of stem/TA cell-enriched limbal epithelium into more differentiated limbal epithelial superficial cells.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.