With respect to digestion, while we are beginning to understand how this process is regulated in the limbal epithelium, there still are several areas that need attention. For example, knowledge of the early stages of autophagy has not been defined from either a regulatory or biochemical perspective. Does the degradation of intracellular and extracellular material follow the canonical autophagy pathway or the phagocytic pathway? There is emerging evidence for an autophagosome-independent role for autophagy proteins in lysosome fusion and turnover of extracellular substrates (see the report of Florey et al.
54 and references therein). Since corneal and limbal epithelia have distinct physiologies, are the degradation processes similar for both epithelia? As mentioned previously, based on LC3 expression, corneal epithelial basal cells appear to be less active than their limbal counterparts.
44 In contrast, high expression of LC3 was noted in the corneal wing and superficial cell layers. These regions are in relatively close approximation to the surface and are the first areas to experience environmental stresses. Thus, it is not surprising that autophagy would be active in wing and superficial cells. Another consideration is the idea that autophagy might have a role as a mediator of early differentiation. In many tissues, autophagy is highly active during differentiation (see the study of Mizushima and Levine,
81 and references therein). Thus, we reason that the marked LC3 expression in superficial cells may, in part, reflect a role for autophagy-related proteins as initiators of early differentiation in these cells. When HaCaT cells (a keratinocyte cell line) were induced to differentiate, release of Beclin 1 and enhancement of ATG12 and LC3II were noted, suggestive that autophagy might have a role in the early stages of differentiation.
82 Recently, the molecular machinery involved in the removal of nuclei (nucleophagy) during the end-stages of keratinization have been detailed and this form of autophagy was demonstrated to be induced when keratinocytes differentiate.
83,84 Taken together these observations raise questions of how autophagy is regulated in the corneal epithelium and the role of autophagy and nucleophagy in corneal epithelial differentiation. We have preliminary evidence implicating a regulatory role for miR-184, which is the most highly expressed corneal epithelial miRNA.
38 miR-184 was initially shown to function in the corneal epithelium by inhibiting miR-205, which targeted the tumor suppresser
SHIP2.
40 By preserving SHIP2 levels in the corneal epithelium, proper Akt signaling was assured, which maintained corneal epithelial survival.
40 More recently, miR-184 was shown to have angiostatic properties and, thus, functioned in maintaining corneal avascularity.
85,86 Other functions for miR-184 in the corneal epithelium have been lineage specification,
39 controlling familial severe keratoconus as well as cataract formation.
87,88 We now have evidence (unpublished observations) that miR-184 targets the Nogo-B receptor (NUS1). The Nobo-B receptor stabilizes Neimann-Pick Type C2 protein (NPC2),
89 which is required for the proper clearance of autophagosomes and, thus, has a role in regulation of autophagy-lysosomal activity.
90,91 We posit that miR-184 targeting of
NUS1 leads to a failure of NPC2 to maintain proper autophagic flux, which is detected by a decrease in LC3 activity in corneal epithelial basal cells.
44 In support of this idea is the 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.
38 Conversely, LC3 pucta are low to absent in basal cells and high in wing and superficial cells. It is important to remember that all cells have autophagic capability, including corneal epithelial basal cells. A major gap in our knowledge is the mechanism(s) that corneal epithelial basal cells use to invoke autophagy during normal and pathologic conditions.