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James V Jester, Yilu Xie, Geraint J Parfitt, Donald J Brown; Localization and Characterization of Meibocyte Differentiation Specific Proteins.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5661.
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© ARVO (1962-2015); The Authors (2016-present)
While the cellular and molecular mechanisms underlying meibocyte differentiation and holocrine secretion are poorly understood, recent RNA Seq analysis of young and old mouse meibomian glands point to several genes that may play important roles in this process. The purpose of this study was to validate the protein expression of these identified genes in human and mouse meibomian glands and begin to characterize their potential role in meibocyte differentiation in culture.
Mouse and post-surgical human eyelid tissue were obtained with approval from the UCI IACUC and IRB. Tissue was fresh frozen, sectioned and immunostained for proteins specific for 1) age (DKKL1 and Caspase 14), 2) apoptosis (Caspase 3a, 9) and 3) autophagy (BECN1 and ULK1). In addition, lysates from rosiglitazone stimulated meibocyte cultures (human and mouse) were evaluated for protein localization associated with lipogenesis.
Antibodies to age-related proteins, DKKL1 and Caspase 14 both showed strong meibocyte immunostaining, with anti-DKKL1 showing staining of the ductal epithelium while anti-Caspase 14 was specific for the suprabasal acinar meibocytes, very superficial ductal epithelium and nuclei of keratinized epidermis. Antibodies to apoptosis related proteins, Caspase 3a and 9, also showed strong staining specific for suprabasal meibocytes. However, TUNEL labeling of the meibomian gland was limited to the ductal epithelium and possibly the very distal, degenerated meibocytes. Antibodies to autophagy related proteins showed strong acinar staining with anti-BECN1 localized to both the cytoplasm and suprabasal nuclei, while anti-ULK1 showed only suprabasal meibocyte staining. Meibocyte cells in culture treated with 50 μM rosiglitazone to induce lipid synthesis showed increased anti-DKKl1 staining with nuclear localization in non-proliferating cells containing lipid vacuoles.
These results indicate proteins associated with age, apoptosis and autophagy genes identified by RNA seq analysis are expressed in human and mouse meibomian glands. Both age-related proteins are known to be involved in epithelial differentiation suggesting that atrophy of meibomian glands may involve loss of meibocyte differentiation potential. The finding that both apoptosis and autophagy related proteins appear highly expressed in meibocytes also suggests that holocrine secretion of lipid may involve both pathways.
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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