Elsevier

The Ocular Surface

Volume 18, Issue 3, July 2020, Pages 427-437
The Ocular Surface

Eicosapentaenoic acid (EPA) activates PPARγ signaling leading to cell cycle exit, lipid accumulation, and autophagy in human meibomian gland epithelial cells (hMGEC)

https://doi.org/10.1016/j.jtos.2020.04.012Get rights and content

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.

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).

References (65)

  • B.P. Gaber et al.

    On the quantitative interpretation of biomembrane structure by Raman spectroscopy

    Biochim Biophys Acta

    (1977)
  • H.A. Rinia et al.

    Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy

    Biophys J

    (2008)
  • Y. Liu et al.

    One man's poison is another man's meat: using azithromycin-induced phospholipidosis to promote ocular surface health

    Toxicology

    (2014)
  • H.S. Hwang et al.

    Meibocyte differentiation and renewal: insights into novel mechanisms of meibomian gland dysfunction (MGD)

    Exp Eye Res

    (2017)
  • F. Echeverria et al.

    Long-chain polyunsaturated fatty acids regulation of PPARs, signaling: relationship to tissue development and aging

    Prostaglandins Leukot Essent Fatty Acids

    (2016)
  • R.F. Morrison et al.

    Role of PPARgamma in regulating a cascade expression of cyclin-dependent kinase inhibitors, p18(INK4c) and p21(Waf1/Cip1), during adipogenesis

    J Biol Chem

    (1999)
  • M. Dahlhoff et al.

    PLIN2, the major perilipin regulated during sebocyte differentiation, controls sebaceous lipid accumulation in vitro and sebaceous gland size in vivo

    Biochim Biophys Acta

    (2013)
  • M. Kutsuna et al.

    Presence of adipose differentiation-related protein in rat meibomian gland cells

    Exp Eye Res

    (2007)
  • I.A. Butovich

    Meibomian glands, meibum, and meibogenesis

    Exp Eye Res

    (2017)
  • U. Hampel et al.

    In vitro effects of docosahexaenoic and eicosapentaenoic acid on human meibomian gland epithelial cells

    Exp Eye Res

    (2015)
  • J.M. Duerden et al.

    Lipid synthesis in vitro by rabbit Meibomian gland tissue and its inhibition by tetracycline

    Biochim Biophys Acta

    (1990)
  • H. Wang et al.

    How lipid droplets "TAG" along: glycerolipid synthetic enzymes and lipid storage

    Biochim Biophys Acta Mol Cell Biol Lipids

    (2017)
  • C.L. Monteleon et al.

    Lysosomes support the degradation, signaling, and mitochondrial metabolism necessary for human epidermal differentiation

    J Invest Dermatol

    (2018)
  • J. Caddy et al.

    Rosiglitazone induces the unfolded protein response, but has no significant effect on cell viability, in monocytic and vascular smooth muscle cells

    Biochem Biophys Res Commun

    (2010)
  • A.J. Bron et al.

    The contribution of meibomian disease to dry eye

    Ocul Surf

    (2004)
  • W.D. Mathers et al.

    Meibomian gland morphology and tear osmolarity: changes with Accutane therapy

    Cornea

    (1991)
  • C.J. Nien et al.

    Effects of age and dysfunction on human meibomian glands

    Arch Ophthalmol

    (2011)
  • R.L. Rosenfield et al.

    Peroxisome proliferator-activated receptors and skin development

    Horm Res

    (2000)
  • C.J. Nien et al.

    The development of meibomian glands in mice

    Mol Vis

    (2010)
  • M.S. Macsai

    The role of omega-3 dietary supplementation in blepharitis and meibomian gland dysfunction (an AOS thesis)

    Trans Am Ophthalmol Soc

    (2008)
  • G. Giannaccare et al.

    Efficacy of omega-3 fatty acid supplementation for treatment of dry eye disease: a meta-analysis of randomized clinical trials

    Cornea

    (2019)
  • R.C. Prince et al.

    Stimulated Raman scattering: from bulk to nano

    Chem Rev

    (2017)
  • Cited by (27)

    • PPARγ signaling in hepatocarcinogenesis: Mechanistic insights for cellular reprogramming and therapeutic implications

      2022, Pharmacology and Therapeutics
      Citation 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).

    View all citing articles on Scopus
    View full text