Eicosapentaenoic acid (EPA) activates PPARγ signaling leading to cell cycle exit, lipid accumulation, and autophagy in human meibomian gland epithelial cells (hMGEC)
Introduction
Over the past several decades, increased interest has focused on meibomian gland dysfunction (MGD) as being the cause of an evaporative dry eye disease (EDED) that commonly afflicts older individuals [1] and patients on Accutane therapy [2]. EDED patients exhibit shortened tear film break-up time, increased tear osmolarity, and corneal epithelial damage and inflammation leading to symptoms of chronic pain and discomfort that is thought to be due to the loss or atrophy of the meibomian glands with altered quantity or quality of the lipid (meibum) excretions in the presence of a normal functioning lacrimal gland [2]. Despite heightened interest in EDED and MGD, our knowledge regarding the molecular and cellular mechanisms controlling meibocyte differentiation and holocrine secretion are limited.
Past studies have shown that during aging in the human and mouse meibomian gland there is a significant decrease in the expression and post-translational modification of the lipid sensitive nuclear receptor, peroxisome proliferator activated receptor gamma (PPARγ) [3,4]. While PPARγ is ubiquitously expressed in many cell types, it is known to play a critical role in lipid synthesis and storage in adipocytes and sebocytes [[5], [6], [7]]. During meibomian gland morphogenesis, expression of PPARγ coincides with the initial synthesis of lipid around post-natal day 3 [8]. Furthermore, PPARγ activation by the synthetic agonist, rosiglitazone, is associated with up-regulation of PPARγ response genes, PPARγ sumoylation, and cytoplasmic export leading to lipid accumulation in both cultured human and mouse acinar cells or meibocytes [[9], [10], [11]]. These observations suggest that PPARγ plays an important role in the differentiation and holocrine secretion of the meibomian gland.
Although a synthetic agonist was useful to investigate the role of PPARγ signaling, endogenous ligands may have broader specificity and activity. Unfortunately, endogenous ligands for PPARγ signaling of the meibomian gland have not been identified, although various fatty acid derivatives including eicosanoids have shown to have the ability to enhance lipid production by meibocytes in culture [12]. In this study, we hypothesized that Eicosapentaenoic acid (EPA), which belongs to omega-3 fatty acids, could serve as a PPARγ ligand in immortalized human meibomian gland epithelial cells (hMGEC). Since previous studies have shown that dietary supplementation of omega-3 fatty acids improved signs and symptoms of dry eye disease including changes in meibum quality [13,14], investigating the effect of EPA on PPARγ signaling and lipid synthesis in meibocytes would be valuable.
In this report, we establish that the natural PPARγ ligand, EPA, similar to the synthetic agonist, rosiglitazone, induces cell cycle exit mediated by decreased expression of cyclin D1 and increased expression of p21 and p27. Furthermore, both agonists lead to the accumulation of lipid that is synthesized de novo and/or transported to lipid droplets within the smooth endoplasmic reticulum (ER). PPARγ agonist also enhance autophagolysosome formation that appears critical for the maintenance of mitochondrial function and potentially meibocyte disintegration. Together these findings support the hypothesis that PPARγ signaling controls meibocyte differentiation and lipid secretion in the meibomian gland.
Section snippets
Cultivation and differentiation of meibocytes
Immortalized human meibomian gland epithelial cells (hMGEC), a generous gift from Dr. Sullivan (Schepens Eye Research Institute), were grown in Keratinocyte Serum Free Media (KSFM, Invitrogen-Gibco, Grand Island, NY) as previously described [10]. At 80% confluence, differentiation was induced by culturing cells in DMEM-F12 (Gibco, Grand Island, NY) supplemented with Epidermal Growth Factor (EGF, 10 ng/ml, Sigma) and Bovine serum albumin (fatty acid free-BSA, 3 mg/ml, Sigma) with or without
EPA induced upregulation of lipogenic genes by activating PPARγ signaling
Previously we have shown that the specific, synthetic PPARγ agonist, rosiglitazone (Rosi), upregulates the expression of PPARγ response genes related to lipid synthesis at the transcriptional level [10,11]. In this experiment, we evaluated the effects of EPA, a potential PPARα, β/δ, γ agonist, on expression of PPARγ response genes. As shown in Fig. 1, EPA significantly upregulated mRNAs in hMGEC encoding PLIN2 (Fig. 1A), ELOVL4 (Fig. 1B), and SOAT1 (Fig. 1D) by 2.7, 3.0, and 2.1 folds on
Discussion
This paper expands upon our previous studies of the role of PPARγ in regulating meibocyte differentiation [10], and evaluates the effect of the natural PPAR ligand, EPA, that broadly activates PPAR signaling through α, β/δ and γ isoforms [12,39]. In this study we confirm that EPA, through specific PPARγ receptor signaling induces cell cycle exit, lipogenic gene expression and the accumulation of neutral lipids, similar to that of the specific, synthetic PPARγ agonist, rosiglitazone [10]. While
Conclusion
This study shows that PPARγ activation, induced by both rosiglitazone and EPA, leads to cell cycle exit, lipid production, and autophagy during differentiation. Autophagy seems to play a role for maintaining mitochondrial homeostasis and underlying mechanism how PPARγ signaling and autophagy lead to cellular disintegration need to be further studied.
Disclosure
The authors have no commercial interest in any concept or product discussed in this article.
Funding
This work was supported in part by NIH/NEI EY021510, NIH/NGMS GM132506, an Unrestricted Grant from Research to Prevent Blindness, Inc. RPB-203478, and the Skirball program in Molecular Ophthalmology and basic science research program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2017R1D1A3B03036549).
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2022, Pharmacology and TherapeuticsCitation Excerpt :Accordingly, senescence marker protein 30 (SMP30) deficiency significantly inhibited CCl4-induced liver fibrosis through downstream signaling of p-smad2/3 and TGFβ.Whereas, induced expression of PPARγ was pivotal factor in attenuation of liver fibrosis on SMP30 knockout mice (Park et al., 2010). Recently, a study established a link among upregulation of p21, p27, downregulation of cyclin D1 and AMPK/ Unc-51 Like Autophagy Activating Kinase 1 (ULK1) to cell cycle abrogation and autophagy, followed by induced PPARγ expression (Kim et al., 2020). Likewise, PPARγ-regulated expression of Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN), NFκB, p65 and JAK/STATs further modulate inflammatory factors and autophagy, and exert hepatoprotective effects (Chen, Chen, He, & Stiles, 2018; Gump & Thorburn, 2011; Lee et al., 2005; Weber et al., 2010; Xiang, Chen, Xu, Wang, & Guo, 2020).