Elsevier

Experimental Eye Research

Volume 181, April 2019, Pages 223-231
Experimental Eye Research

Sirt3 regulates mitophagy level to promote diabetic corneal epithelial wound healing

https://doi.org/10.1016/j.exer.2019.02.011Get rights and content

Highlights

  • High glucose conditions inhibate the expression of Sirt3 in the TKE2 cells and corneal epithelia of type 1 diabetic mice.

  • The activation of mitophagy is downregulated by Sirt3 under HG conditions.

  • Sirt3 regulates mitophagy to promote diabetic corneal epithelial wound healing via the Foxo3a/PINK1-Parkin Pathway.

Abstract

We aim to investigate how Sirt3 (silent mating type information regulation 2 homolog 3) promoting diabetic corneal epithelial wound healing by regulating mitophagy.

The effect of HG(High Glucose, 25 mM D-glucose) on Sirt3 and LC3B(light chain 3 beta)which representing of mitophagy were investigated in TKE2 cells (a murine limbal/corneal epithelium-derived progenitor cell line) and corneal epithelium from C57BL/6J-Ins2Akita (Ins2Akita/+) mice using RT-PCR and Western blotting. How overexpression of Sirt3 promoting diabetic corneal epithelial wound healing was investigated with cell migration assay、immunofluorescence、 immunofluorescence colocalization and corneal injury model. We found that HG reduced the expression of Sirt3 as well the mitophagy both in TKE2 cells and corneas from Ins2Akita/+ mice. And overexpression of Sirt3 prominently promoted wound healing speed under HG condition via upregulating the level of mitophagy. Mitophagy level was increased dramatically when the Foxo3a (Forkhead box O3)/PINK1(PTEN Induced putative kinase protein 1)-Parkin pathway was activated by Sirt3 overexpression which suggested that the mitophagy was involved in cell injury under HG condition. This study demonstrated the mechanism of Sirt3 regulating mitophagy to promote diabetic corneal epithelial wound healing in vivo and in vitro, which suggested that Sirt3 may positively impact diabetic keratopathy(DK).

Introduction

Diabetes mellitus (DM) is a widespread chronic diseases in the world. Previous research indicated that the prevalence of diabetes was estimated to be 2.8% in 2000 and will be 4.4% in 2030 for all age groups (Wild et al., 2004). DK was first reported in 1981 by Schultz RO et al. (Schultz et al., 1981) Recent research has demonstrated that corneal problems are frequent, affecting up to 70% of examined diabetic patients (Abdelkader et al., 2011; Vieira-Potter et al., 2016)and it is a sign of peripheral neuropathy (Bikbova et al., 2016). Manifestations of it include delayed wound healing, compromised barrier function, persistent epithelial defects and ulcerations, recurrent erosions, epithelial edema, decreasing corneal sensitivity, neurotrophic corneal ulcers, and stem cell dysfunction (Chen et al., 2009; Herse, 1988; Wang et al., 2014). Potential mechanisms including: abnormal adhesion between epithelium cells and basement membrane(BM) (Gul et al., 2008), enhanced activation of proteolytic enzymes(MMPs),for example MMP-10、MMP-3 (Saghizadeh et al., 2001; Takahashi et al., 2000), diminished signaling axis of epidermal growth factor (EGF) -phosphatidylinositol-3-kinase (PI3K) – Akt kinase (Saghizadeh et al., 2005; Zieske et al., 2000), accumulaition of advanced glycation end products (AGEs) and ROS (Kim et al., 2011). Lateraly researches further revealed that miRNAs(h-miR-146a、 h-miR-424) were upregulated in diabetic corneas and leading to slower wound healing rate (Funari et al., 2013; Winkler et al., 2014). In addition, it was uncovered that miR-204–5p combined with Sirt1 and cyclin D1 affecting the wound healing in diabetic mice (Gao et al., 2015a). Recently, a creasing more attention is on the nonnegligible corneal damages in DM, but the precise mechanism is unclear.

Mitophagy is a highly selective autophagy process that removes dysfunctional or excess mitochondria during hypoxia or other stimulating conditions (Lemasters, 2005a).Recent studies have suggested a potential functional role for mitophagy in the development of diabetes and diabetic complications (Kobayashi and Liang, 2015; Czajka et al., 2015; Xu et al., 2013). It has been reported that the P53 and Clec16a genes are involved in the pathogenesis of DM by regulating the mitophagy process (Meissner et al., 2011a; Narendra et al., 2010). In addition, evidence shows that a reduction in PINK1 and Parkin proteins plays a potential role in the development of diabetic cardiomyopathy(DC) and diabetic nephropathy(DN) (Chan et al., 2011; Michiorri et al., 2010). Accroding to the classic regulation pathway of mitophagy,the accumulation of PINK1 on damaged mitochondria is necessary for Parkin recruitment to damaged mitochondria. The accumulate of Parkin on dysfunctional mitochondria trigging the mitophagy (Meissner et al., 2011b; Springer and Kahle, 2011). Based on these findings, it is worthwhile to investigate the effects of mitophagy on DK. A recent manuscript demonstrated that mitophagy regulates the DC process via the PINK1-Parkin pathway. The same report suggests that Sirt3 appears to be the upstream regulator (Yu et al., 2017).

Sirt3 mainly locates at the mitochondria and is important for acetylating mitochondrial proteins (Lombard et al., 2007a). It was reported that Sirt3 maintains mitochondrial membrane potential and intracellular pH to prevent cell death in response to hypoxic stress. And inhibition of Sirt3 increases hypoxia and staurosporine-induced cell death by stimulating mitophagy (Pellegrini et al., 2012). Moreover, under hypoxic conditions, the forkhead box class transcription factor Foxo3a is deacetylated by Sirt3. Sirt3-mediated deacetylation of Foxo3a results in reduced Foxo3a phosphorylation, ubiquitination and degradation (Tseng et al., 2014). Furthermore, in mammalian cells, Foxo3a can be deacetylated by Sirt1, which improves resistance against oxidative stress and inhibits Foxo3a-mediated cell death (Brunet et al., 2004). Recent study on osteoarthritis revealed that inhibition of Sirt3 by the 3-TYP decreasing the expression of PINK1 and Parkin (Wang et al., 2018). Laterly,an important research revealed that the Sirt3 interaction with Sirt1 modulates mitophagy through the Foxo3a/PINK1-Parkin signaling network and enhances antiaging properties (Das et al., 2014). Additionaly, knocking out Sirt3 aggravates the suppression of mitophagy and cardiac dysfunction in STZ-induced DM rodent models (Yu et al., 2017). The expression levels of Foxo3a and Parkin, however, were decreased by Sirt3 KO. In contrast, these effects were facilitated by Sirt3 OE under HG settings. 28We hypothesized that the overexpression of Sirt3 increases deacetylated Foxo3a and enhances mitophagy by activating the PINK1-Parkin pathway, thereby contributing to corneal epithelial wound healing. Our results suggest that Sirt3 and mitophagy might play a potential role not only in the mechanism but also as a target for attenuating epithelial wound healing in DK.

Section snippets

Cell culture and treatment

The TKE2 cells were maintained in keratinocyte serum-free medium (KSFM; Life Technologies, Shanghai, China) supplemented with 2.5 ng/mL human recombinant epidermal growth factor (EGF), 25 μg/mL bovine pituitary extract (BPE), and 1% penicillin in a humidified 5% CO2 incubator at 37 °C (Wang et al., 2013). The TKE2 cells were maintained in media containing 5 mM D-glucose (normal glucose NG), 25 mM D-glucose (high glucose HG). The osmotic pressure of the NG medium was adjusted to the HG medium by

HG envioronment suppress the expression of Sirt3,LC3B as well as the activation of Foxo3a/PINK1-Parkin Pathway in vivo and in vitro

Firtstly,by treating TKE2 cells separately with NG and HG treament,we found that contrast to NG treatment, Sirt3 was reduced in HG conditions. (Fig. 1 A). Then, we investigated the expression of Sirt3 in diabetic animal models. Contrast to control Ins2+/+ mice, Sirt3 was dramatically declined in the corneal epithelia from Ins2Akita/+ mice (Fig. 1 B). As expected,the Sirt3 was exactly located at the corneal epithelium (Fig. 1 C). Then we dicovered that LC3B protein was also decreseaed in the HG

Discussion

Our study investigates the effect of Sirt3 on regulating mitophagy in diabetic corneal epithelial wound healing. We use TKE2 cells as the in vitro model, and the Ins2Akita/+ mice as the in vivo model. The findings demonstrated that hyperglycemia reducing Sirt3 expression and downregulating both mitophagy and the Foxo3a/PINK1-Parkin signaling activiation, likely accounting for impaired diabetic corneal epithelial wound healing. Using the adenoviral system to overexpress Sirt3,we revealed that

Disclosure

J. Hu, None; T. Kan, None; X. Hu, None.

Acknowledgments

The authors thank Qinjun Zhou and Ye Wang (Shandong Eye Institute,Qingdao, Shandong,China) for lab technical support. Supported in part by the National Natural Science Foundation of China,Beijing,China (grant nos.: 81870636), the Science and Technology Innovation Joint Fund Project of Fujian Province, Fuzhou, China (grant nos.: 2016Y9013), and Natural Science Foundation of Fujian Province, Fuzhou, China (grant nos.: 2017J01280).

References (59)

  • R. Tao et al.

    Sirt3-mediated deacetylation of evolutionarily conserved lysine 122 regulates MnSOD activity in response to stress

    Mol. Cell

    (2010 Dec 22)
  • X. Xu et al.

    Diminished autophagy limits cardiac injury in mouse models of type 1 diabetes

    J. Biol. Chem.

    (2013 Jun 21)
  • W. Yu et al.

    SIRT3 protein deacetylates isocitrate dehydrogenase 2 (IDH2) and regulates mitochondrial redox status

    J. Biol. Chem.

    (2012 Apr 20)
  • W. Yu et al.

    Sirt3 deficiency exacerbates diabetic cardiac dysfunction: role of Foxo3a-Parkin-mediated mitophagy

    Biochim. Biophys. Acta

    (2017 Aug)
  • H. Abdelkader et al.

    New therapeutic approaches in the treatment of diabetic keratopathy: a review

    Clin. Exp. Ophthalmol.

    (2011 Apr)
  • G. Bikbova et al.

    Neuronal changes in the diabetic cornea: perspectives for neuroprotection

    BioMed Res. Int.

    (2016)
  • A. Brunet et al.

    Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase

    Science (New York, N.Y.)

    (2004 Mar 26)
  • N.C. Chan et al.

    Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy

    Hum. Mol. Genet.

    (2011 May 1)
  • Y. Cheng et al.

    Therapeutic targeting of autophagy in disease: biology and pharmacology

    Pharmacol. Rev.

    (2013 Aug 13)
  • S. Das et al.

    Antiaging properties of a grape-derived antioxidant are regulated by mitochondrial balance of fusion and fission leading to mitophagy triggered by a signaling network of Sirt1-Sirt3-Foxo3-PINK1-PARKIN

    Oxid. Med. Cell Longev.

    (2014)
  • V.A. Funari et al.

    Differentially expressed wound healing-related microRNAs in the human diabetic cornea

    PLoS One

    (2013)
  • J. Gao et al.

    MicroRNA-204-5p-Mediated regulation of SIRT1 contributes to the delay of epithelial cell cycle traversal in diabetic corneas

    Invest. Ophthalmol. Vis. Sci.

    (2015)
  • J. Gao et al.

    MicroRNA-204-5p-Mediated regulation of SIRT1 contributes to the delay of epithelial cell cycle traversal in diabetic corneas

    Invest. Ophthalmol. Vis. Sci.

    (2015 Jan 22)
  • C.A. Gautier et al.

    Loss of PINK1 causes mitochondrial functional defects and increased sensitivity to oxidative stress

    P Natl Acad Sci USA

    (2008 Aug 12)
  • M. Gul et al.

    Protective effects of melatonin and aminoguanidine on the cornea in streptozotocin-induced diabetic rats

    Cornea

    (2008)
  • P.R. Herse

    A review of manifestations of diabetes mellitus in the anterior eye and cornea

    Am. J. Optom. Physiol. Opt.

    (1988 Mar)
  • K.M. Jacobs et al.

    SIRT3 interacts with the daf-16 homolog FOXO3a in the mitochondria, as well as increases FOXO3a dependent gene expression

    Int. J. Biol. Sci.

    (2008 Sep 5)
  • J. Kim et al.

    Involvement of advanced glycation end products, oxidative stress and nuclear factor-kappaB in the development of diabetic keratopathy

    Graefe's archive for clinical and experimental ophthalmology Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie

    (2011)
  • I.K. Law et al.

    Identification and characterization of proteins interacting with SIRT1 and SIRT3: implications in the anti-aging and metabolic effects of sirtuins

    Proteomics

    (2009 May)
  • Cited by (0)

    1

    First co-authors: these authors have contributed equally to the project.

    View full text