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James Jester, Yilu Xie, Jonathan Sanghoon Kang, Paul Quoc Hung Nguyen, Vickie Thi Bui, Kelly Huynh, Donald J. Brown; PPARγ Regulates Meibocyte Differentiation and Lipid Synthesis of Cultured Human Meibomian Gland Epithelial (hMGE) Cells.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4393.
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© ARVO (1962-2015); The Authors (2016-present)
PPARγ is a lipid sensitive nuclear receptor that regulates expression of genes involved in lipogenesis. In recent studies we have shown that PPARγ signaling is down regulated in aging human and mouse meibomian glands and up-regulates lipogenesis of mouse meibocytes in culture. The purpose of this study was to evaluate the role of PPARγ in regulating meibocyte differentiation and lipid synthesis in cultured hMGE cells.
Telomerized hMGE cells were cultured in DMEM/F12 supplemented with 10ng/ml EGF and exposed to the PPARγ agonist, Rosiglitazone (Rosi), at 10-50 μM. Cultures were also exposed to PPARγ antagonist, GW9662, to block Rosi effects. Cells were then fixed for staining with LipidTox to measure lipid synthesis, Ki67 to measure cell cycling, and antibodies to meibocyte differentiation proteins, PPARγ, ADFP, caspase 9, caspase 14 and ULK1 s556. Proteins were also collected for western blotting. All experiments were run in triplicate.
Rosi induced a significant (P<.001) dose and time dependent increase in neutral lipid synthesis from 20-50 μM that peaked with 30 μM at day 6 after treatment (P<.001), increasing over 80 fold. Treating cells with the inhibitor, GW9662, at 20 μM and 50μM significantly blocked lipid synthesis induced by the concomitant treatment of 20 μM Rosi for 5 days by 64% (P<.001) and 89% (P<.001) respectively. Treatment with Rosi also induced significant (P<.001) loss of Ki67 labeling, which was reduced by 77%, 86% and 96% following 2 days of treatment with 10 μM, 30 μM and 50 μM respectively. Expression of PPARγ was also significantly increased ~4 fold following treatment with 30 μM and 50 μM Rosi (P<.025) as well as other differentiation markers including caspase 9, caspase 14, ADFP and ULK1 s556 identified by western blotting.
These results indicate that PPARγ receptor signaling specifically induces cell cycle exit of hMGE cells and meibocyte differentiation involving expression of meibocyte specific proteins and lipid synthesis. Overall, these findings support the hypothesis that PPARγ is a master regulator of meibocyte differentiation.
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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