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Jong Kook Park, Han Peng, Wending Yang, Congcong He, Robert M Lavker; NUS1 positively regulates autophagic activity in the stem cell-enriched limbal epithelium. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4238.
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© ARVO (1962-2015); The Authors (2016-present)
Autophagy is a highly evolutionary conserved cellular process by which cytoplasmic material is segregated into autophagosomes that fuse with lysosomes for degradation. Blocking autophagy diminishes keratinocyte proliferative capacity. High proliferative capacity is a feature of epithelial stem cells. Despite its importance, how autophagic activity is regulated in the limbal and corneal epithelia is largely understudied. We propose that nuclear undecaprenyl pyrophosphate synthase 1 (NUS1), which is regulated by microRNA-184 (miR-184), preserves autophagic activity.
Eyes of GFP-LC3 transgenic mice were dissected and the GFP punctas in the corneal/limbal epithelia were counted. NUS1 expression was analyzed by immunofluorescence staining of sections of human limbal/corneal epithelium. Loss-of-function experiments in primary human cultured limbal epithelial keratinocytes (HLEKs) were conducted using siRNA against the NUS1. WST-1 cell proliferation assay was used to analyze HLEK proliferation. To validate NUS1 as a target of miR-184, we used luciferase reporter assays and immunoblotting.
LC3 is an accepted marker of autophagy. Monitoring autophagic activity using GFP-LC3 trangenic mice demonstrated that limbal epithelial basal cells had significantly greater amounts of LC3-positive puncta than corneal epithelial basal cells. NUS1 was highly expressed in limbal epithelium and minimally detected in corneal epithelium. Knockdown of NUS1 in HLEKs resulted in a downregulation of p-Erk1/2 and LC3II protein levels concomitant with a decrease in proliferation. Reporter assays and immunoblotting indicated that NUS1 is a direct target of miR-184. A marked increase in LC3-positive puncta was detected in mouse eyes treated with antago-184 in ex vivo organ culture. There was a reciprocal association of miR-184 and LC3 expression in the corneal epithelium. miR-184 is primarily detected in corneal epithelial basal cells, with little expression in wing and superficial cells. Conversely, LC3 pucta are low to absent in basal cells and high in wing and superficial cells.
Our findings provide strong evidence that NUS1 functions to maintain autophagic activity in the limbal keratinocytes and that miR-184 may suppress this activity via targeting NUS1. Our data suggest that a NUS1/miR-184 axis can regulate keratinocyte proliferative capacity via modulation of autophagy.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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