Abstract

Volume.124 Number.1

Original article : Basic science

Differentiation of Cultured Meibocytes through the Activation of Peroxisome Proliferator-activated Receptor γ
Yoko Karasawa1, Masataka Ito2, Yoshiaki Nishio1, Masaru Takeuchi1
1 Department of Ophthalmology, National Defense Medical College
2 Developmental Anatomy and Regenerative Biology, National Defense Medical College

Purpose: Acinar cells of the meibomian gland stop proliferating during differentiation, and lipid droplets accumulate in the cells. Thereafter, the cells collapse and secrete accumulated fat as meibum along with the cytoplasm. Activation of peroxisome proliferator-activated receptor γ (PPARγ) has been reported to induce cell differentiation and accumulation of lipid droplets in adipocytes and sebocytes. The effect of rosiglitazone (RSG), a PPARγ agonist, on the differentiation of immortalized cultured human meibocytes was investigated.
Methods: Cell growth, cell death, and lipid droplet formation were evaluated using subconfluent cultured cells induced to differentiate in a differentiation medium supplemented with RSG. Cells synthesizing DNA were counted by immunocytochemical staining following BrdU labeling. Cell death was assessed by measuring the activity of lactate dehydrogenase released into the medium from dead cells. The lipid droplets in cells were observed by trigly ceride staining with fluorescent dye. Among PPARγ target genes, the gene expression levels of CCAAT enhancer-binding protein α (CEBPA), angiopoietin like 4 (ANGPTL4), and perilipin2 (PLIN2), which are known to increase during the differentiation of adipocytes and sebocytes, were measured by reverse-transcription quantitative polymerase chain reaction.
Results: The addition of 50 μM RSG resulted in a significant decrease in the number of proliferating cells and an increase in the number of dead cells; however, no significant differences were observed at concentrations of 20 μM or less. Fine lipid droplets accumulated in cells depending on RSG concentration. Lipid droplets were also present in cells that were killed and floating in a 50 μM RSG-supplemented medium. The expression levels of CEBPA, ANGPTL4, and PLIN2 increased proportionally with RSG concentration.
Conclusion: These results suggest that signal transduction as a result of the activation of PPARγ is involved in the differentiation of meibocytes.Nippon Ganka Gakkai Zasshi (J Jpn Ophthalmol Soc) 124: 7-14, 2020.

Key words
Meibocytes, Cell differentiation, Peroxisome proliferator-activated receptor γ (PPARγ), Cell death, Gene expression
Reprint requests to
Yoko Karasawa, M. D. Department of Ophthalmology, National Defense Medical College. 3-2 Namiki, Tokorozawa-shi 359-8513, Japan