Abstract
Purpose :
Autophagy plays critical roles in maintenance of tissue homeostasis and is an important protective factor in tissues governed by stem cells. The autophagic process in corneal epithelial stem cells is under studied
Methods :
We used antagomirs (antagos) to knock-down miR-103, -107 or -124 (irrelevant-antago) in cultured human limbal epithelial keratinocytes (HLEKs) and a LC3-GFP mouse. To characterize the vacuoles, we used contrast light, immunofluorescence, and transmission electron microscopy. To determine the origin of the vacuoles, pharmacological and genetic inhibition at different stages of autophagy was performed. Bioinformatic analysis combined with luciferase assays and biochemistry identified autophagy-related miRNA targets
Results :
In HLEKs, antagos-103/107 cause the formation of large hybrid vacuoles, which are positive for lysosomal (LAMP1 and lysotrackor), autophagosomal (LC3), and macropinosomal (Rabankyrin-5) markers. Such vacuoles were rescued by inhibiting autophagosomal-lysosomal fusion, indicative that vacuole accumulation occurred after autophagolysosome formation and is due to a failure of end-stage autophagy. An increase in LC3II and p62 was noted indicating an attenuation of autophagy flux by antago treatment. More LC3-GFP puncta (a measure of autophagy) were observed in limbal (miRs-103/107-enriched) versus corneal (miRs-103/107-low) basal epithelia, indicative of a high degree of autophagy in the stem cell-enriched limbal epithelium. Active autophagy in this compartment is consistent with autophagy being a stem cell protective factor. In support of this idea, a decrease of holoclone (stem cell-derived) colonies, was detected in HLEKs with compromised autophagic capability. Dynamin, an essential protein for hybrid vacuoles recycling in end stage autophagy, is phosphorylated (inactive) by PKC and p35. Phosphatidylcholine-specific phospholipase D 1/2 and sphingomyelin synthase 2, upstream activators of PKC signaling, were shown to be direct targets of miRs-103/107, as was p35. Inhibition of PKC and p35 pathways decreased p-dynamin and rescued the accumulation of hybrid vacuoles
Conclusions :
miRs-103/107 are critical for regulating proliferation, proliferative capacity and cell-cell communication in the limbal epithelium. The addition of autophagy to the regulatory portfolio of miRs-103/107 highlights the importance of this family in stem cell biology
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.