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Robert M Lavker, Jong Kook Park, Julia Katsnelson, Wending Yang, CongCong He, Han Peng; miRs-103/107 regulate macropinocytosis in limbal/corneal epithelia. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Macropinocytosis, by which cells ingest large amounts of fluid, can induce non-apoptotic cell death. Alternatively, macropinocytosis can function metabolically as a supply route for amino acids. The role(s) of macropinocytosis in limbal/corneal epithelia has not been investigated.
We used antagomirs (antagos) to knock-down miR-103, -107 or -124 (irrelevant-antago) in primary human limbal epithelial keratinocytes (HLEKs). To characterize vacuoles, we used light, immunofluorescence (IF), and transmission electron microscopy (TEM). To determine the vacuole origin, macropinocytosis was pharmacologically and genetically inhibited. Bioinformatic analysis combined with luciferase assays and biochemistry identified miRNA targets related to macropinocytosis.
In HLEKs, which are enriched in miRs-103/107, large hybrid vacuoles (HVs) were formed after removal of this miRNA family. TEM revealed that the HVs were electron lucent, devoid of cellular material and enveloped by a single membrane. On the internal surface of the vacuole membrane, material was organized into small vesicles or myelin-like figures. IF microscopy showed that HVs were positive for macropinosomal markers Rabankyrin-5 and lucifer yellow, indicative that these vacuoles originated via a macropinocytotic process. Loss of miRs-103/107 activate Src and Ras, two critical genes that induce macropinocytosis. Interestingly, NEDD9, and Src homology 2 domain-containing transforming protein C3 (SHC3), which are negative regulators of Src and Ras activation, were shown to be direct targets of miRs-103/107. HVs induced by antagos-103/107 were rescued by pharmacologically and genetically inhibiting: (i) Src and Ras; or (ii) NEDD9 and SHC3. Thus loss of miRs-103/107 induces macropinocytosis via upregulating NEDD9/SHC3. Antagos-103/107-induced macropinocytosis did not cause cell death in HLEKs. mRNA profiling combined with bioinformatic analysis suggested that miRs-103/107 play a role in regulating metabolic processes, such as macropinocytosis.
miRs-103/107 play a negative role in macropinocytosis, which is critical for providing nutrients in high volume. We postulate that due to the highly vascularized stroma beneath the limbal epithelium, there is less need for active macropinocytosis, Conversely in the avascular cornea, macropinocytosis may be a mechanism by which corneal epithelium enhances its metabolic needs.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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