Sun Woong Kim1, Chang Rae Rho2, Jinseor Kim3, Yilu Xie3, Richard C Prince4, Khawla Mustafa5, Eric O Potma6, Donald J Brown3, James V Jester7. 1. Department of Ophthalmology, Yonsei University Wonju College of Medicine, Wonju, South Korea. 2. Department of Ophthalmology, Daejeon St Mary Hospital, Daejeon, South Korea; Gavin Herbert Eye Institute, University of California, Irvine, Irvine, CA, USA. 3. Gavin Herbert Eye Institute, University of California, Irvine, Irvine, CA, USA. 4. Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA. 5. Department of Chemistry, University of California, Irvine, Irvine, CA, USA. 6. Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA; Department of Chemistry, University of California, Irvine, Irvine, CA, USA. 7. Gavin Herbert Eye Institute, University of California, Irvine, Irvine, CA, USA. Electronic address: JJester@uci.edu.
Abstract
PURPOSE: The purpose of this study was to access the ability of the natural PPAR agonist, eicosapentaenoic acid (EPA), to activate PPAR gamma (γ) signaling leading to meibocyte differentiation in human meibomian gland epithelial cell (hMGEC). METHODS: HMGEC were exposed to EPA, alone and in combination with the specific PPARγ antagonist, T0070907, to selectively block PPARγ signaling. Expression of PPARγ response genes were evaluated by qPCR. Effect on cell cycle was evaluated using Ki-67 labelling and western blots. During differentiation, autophagy was monitored using the Autophagy Tandem Sensor (ATS) and LysoTracker. Lipid accumulation was characterized by Stimulated Raman Scattering microscopy (SRS) and neutral lipid staining in combination with ER-Tracker, LysoTracker, and ATS. Autophagy was also investigated using western blotting. Seahorse XF analysis was performed to monitor mitochondrial function. RESULTS: EPA specifically upregulated expression of genes related to lipid synthesis and induced cell cycle exit through reduced cyclin D1 expression and increased p21 and p27 expression. EPA also induced accumulation of lipid droplets in a time and dose dependent manner (P < 0.05) by specific PPARγ signaling. Lipid analysis identified both de novo synthesis and extracellular transport of lipid to form lipid droplets that were localized to the ER. PPARγ signaling also induced activation of AMPK-ULK1 signaling and autophagy, while inhibition of autophagy induced mitochondrial crisis with no effect on lipid accumulation. CONCLUSIONS: EPA induces meibocyte differentiation through PPARγ activation that is characterized by cell cycle exit, de novo and transported lipid accumulation in the ER, and autophagy.
PURPOSE: The purpose of this study was to access the ability of the natural PPAR agonist, eicosapentaenoic acid (EPA), to activate PPAR gamma (γ) signaling leading to meibocyte differentiation in human meibomian gland epithelial cell (hMGEC). METHODS: HMGEC were exposed to EPA, alone and in combination with the specific PPARγ antagonist, T0070907, to selectively block PPARγ signaling. Expression of PPARγ response genes were evaluated by qPCR. Effect on cell cycle was evaluated using Ki-67 labelling and western blots. During differentiation, autophagy was monitored using the Autophagy Tandem Sensor (ATS) and LysoTracker. Lipid accumulation was characterized by Stimulated Raman Scattering microscopy (SRS) and neutral lipid staining in combination with ER-Tracker, LysoTracker, and ATS. Autophagy was also investigated using western blotting. Seahorse XF analysis was performed to monitor mitochondrial function. RESULTS: EPA specifically upregulated expression of genes related to lipid synthesis and induced cell cycle exit through reduced cyclin D1 expression and increased p21 and p27 expression. EPA also induced accumulation of lipid droplets in a time and dose dependent manner (P < 0.05) by specific PPARγ signaling. Lipid analysis identified both de novo synthesis and extracellular transport of lipid to form lipid droplets that were localized to the ER. PPARγ signaling also induced activation of AMPK-ULK1 signaling and autophagy, while inhibition of autophagy induced mitochondrial crisis with no effect on lipid accumulation. CONCLUSIONS: EPA induces meibocyte differentiation through PPARγ activation that is characterized by cell cycle exit, de novo and transported lipid accumulation in the ER, and autophagy.
Authors: Jerry R Paugh; Alba Alfonso-Garcia; Andrew Loc Nguyen; Jeffrey L Suhalim; Marjan Farid; Sumit Garg; Jeremiah Tao; Donald J Brown; Eric O Potma; James V Jester Journal: Ocul Surf Date: 2018-10-11 Impact factor: 5.033
Authors: William P Esler; Gregory J Tesz; Marc K Hellerstein; Carine Beysen; Raja Sivamani; Scott M Turner; Steven M Watkins; Paul A Amor; Santos Carvajal-Gonzalez; Frank J Geoly; Kathleen E Biddle; Julie J Purkal; Mark Fitch; Clare Buckeridge; Annette M Silvia; David A Griffith; Matthew Gorgoglione; Lauren Hassoun; Suzana S Bosanac; Nicholas B Vera; Timothy P Rolph; Jeffrey A Pfefferkorn; Gabriele E Sonnenberg Journal: Sci Transl Med Date: 2019-05-15 Impact factor: 17.956
Authors: R Verheijen; H J Kuijpers; R O Schlingemann; A L Boehmer; R van Driel; G J Brakenhoff; F C Ramaekers Journal: J Cell Sci Date: 1989-01 Impact factor: 5.285