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James V. Jester, Chyong Nien, Ginna Kim, Donald J. Brown; Cytoplasmic Localization of PPAR in Mouse Meibomian Glands and Cultured Meibocytes Associated with Enhanced Lipid Synthesis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6702.
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Previous reports suggest that age-related meibomian gland atrophy is associated with loss of cytoplasmic/vesicular localization of the lipid sensitive nuclear receptor PPARγ suggesting altered PPARγ signaling in older meibomian glands. The purpose of this study was to confirm the loss of cytoplasmic PPARγ in older mouse meibomian glands and identify the role of PPARγ in modulating meibocyte lipid synthesis.
Cytoplasmic and nuclear fractions from meibomian glands of young (2M) and old (2Y) C57Bl6 mice were probed using antibodies specific for PPARγ. Mouse meibomian glands were also cultured, immortalized using a SV40 lentiviral vector and cloned. Cloned cells were evaluated for lipid synthesis using LipidTox staining for neutral lipids and CARS/Raman spectroscopy. Lipid synthesizing clones were then tested for the effects of PPARγ agonist, rosiglitazone, on lipid synthesis, PPARγ localization and induction of PPARγ response genes, adiponectin (ADP) and adipocytes differentiation related protein (ADFP) by real-time PCR.
Nuclear fractions from 2M old mice showed a single 50 kDa PPARγ band that was reduced by 40% in older glands. The cytoplasmic fraction in young mice contained both a 72 kDa PPARγ band in addition to the 50 kDa band that was reduce by 89% and 100%, respectively, in older mice. One immortalized meibocyte clone, CN-G2, produced neutral lipid that contained equal amounts of wax and sterol esters similar to mouse meibum as identified by Raman. Rosiglitazone (10-50 µM) treatment of CN-G2 cells increased lipid synthesis compared to serum starved cells (2% fbs) by over 700%. Rosiglitazone also dose dependently increased cytoplasmic localization of PPARγ and induced expression of PPARγ, ADP and ADFP by 1.4, 3.0 and 4.5 fold respectively.
This study confirms earlier findings that there is a loss of cytoplasmic PPARγ localization in older, atrophic mouse meibomian glands. More importantly, our finding show that increased cytoplasmic PPARγ is associated with increased lipid synthesis induced by PPARγ agonists and suggest that lipid synthesis in older mice is down regulated.
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