Sirt3 regulates mitophagy level to promote diabetic corneal epithelial wound healing
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).
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First co-authors: these authors have contributed equally to the project.