Casein Kinase 2-Interacting Protein-1 Alleviates High Glucose-Reduced Autophagy, Oxidative Stress, and Apoptosis in Retinal Pigment Epithelial Cells via Activating the p62/KEAP1/NRF2 Signaling Pathway

Background Casein kinase 2-interacting protein-1 (CKIP-1) has been proved to be associated with complications of diabetes. Diabetic retinopathy is a main diabetic complication which usually leads to blindness. The current study aims to investigate the role of CKIP-1 in high glucose-treated retinal pigment epithelial (RPE) cells which is a component of blood-retinal barriers. Methods The RPE cells, ARPE-19, are treated with high glucose to mimic the diabetic stimulation. CKIP-1 was overexpressed in ARPE-19 cells to evaluate its effects on autophagy, oxidative stress, and apoptosis induced by high glucose treatment, using Western blot, immunofluorescence, and flow cytometry assays, respectively. Results CKIP-1 was expressed at a lower level in high glucose-treated cells than in normal glucose cells. Overexpression of CKIP-1 enhanced the Nrf2 translocation to the nucleus. Furthermore, high glucose-induced autophagy, oxidative stress, and apoptosis were inhibited after overexpression of CKIP-1. Also, CKIP-1 regulates the p62/Keap1/Nrf2 signaling, which might be the potential mechanism in this model. Conclusion In conclusion, CKIP-1 may be a potential therapeutic target that protects RPE cells from injury and subsequent diabetic retinopathy induced by high glucose.


Introduction
Diabetic retinopathy (DR) is one of the common microvascular complications of diabetes leading to vision impairment and even blindness [1]. At the early stages of DR, ischemia, vascular leakage, and diabetic macular edemainduced central vision loss occur, accompanied by secondary angiogenesis and hemorrhage in the retina [2]. e bloodretinal barriers (BRB) mainly consisted of retinal endothelial cells and retinal pigment epithelial (RPE) cells, which can retain the completeness of retinal tissues. Alteration of BRB is essential for the development of retinal diseases [3]. Inflammation and oxidative stress are two risk factors for DR induction and progression [4,5]. e RPE is a restrictive layer to prevent some molecules or charge transport and even maintain the balance of permeability of BRB [6]. Research has reported that continuing stimulation of hyperglycemia on RPE cells induces oxidative stress and cell apoptosis [7][8][9], which promotes BRB injury and subsequent DR progression.
Casein kinase 2-interacting protein-1 (CKIP-1), initially found as an interacting protein of casein kinase 2 (CK2) α subunit, is vital for apoptosis and oxidative stress [10]. A lot of diseases have been reported to be modulated by CKIP-1 expressions such as atherosclerosis, osteoporosis, and cardiac remodeling [11][12][13]. Only one study showed that CKIP-1 was associated with DR [14]. CKIP-1 upregulated Nrf2 by inhibiting Keap1 in hypoxia-induced cardiomyocyte injury [15]. Activating CKIP-1 promoted the Nrf2-ARE antioxidative pathway in the kidneys of high glucose-induced diabetic mice [16]. Upregulation of CKIP-1 also suppressed apoptosis and oxidative stress by inhibiting Keap1 and activating Nrf2/ARE signaling in hippocampal neurons [10]. e autophagic adaptor p62 was reported to physically interact with Keap1, an Nrf2 inhibitor, resulting in increased activation of Nrf2 and oxidative stress in hepatocellular carcinoma [17]. e p62-Keap1-Nrf2-ARE pathway plays a critical role in prion disease via regulating autophagy, oxidative stress, and mitochondrial dysfunction [18]. We supposed that CKIP-1 may also regulate oxidative stress, apoptosis, and autophagy by regulating p62/KEAP1/NRF2 signaling in high glucose-induced retinal pigment epithelial cells. is study identified a potential biomarker of BRB for the understanding of the correlation between epithelial activation and retinal diseases. To study the role of CKIP-1 in HG-induced ARPE-19 cells, CKIP-1 was overexpressed in ARPE-19 cells by transfection with pcDNA3.1-CKIP-1 using Lipofectamine 3000 (Invitrogen) for 48 h. e ORF sequence of human CKIP-1 (GenBank No. NM_016274) cDNA was amplified by RT-PCR and subsequently cloned into vector pcDNA3.1 (Invitrogen, USA). e pcDNA3.1-empty vector plasmids were transfected as negative control.

Cell Viability
Analysis. ARPE-19 cells were seeded in 96well plates with 2000 cells/well in 100 μl DF-12 medium with 10% FBS. After cells were adherent to the bottom of plates, ARPE-19 cells were starved with serum-free low glucose DMEM culture medium or high glucose DMEM medium or overexpressed by CKIP-1 and high glucose for 48 h. e cell viability was then evaluated by cell counting kit-8 assay (CCK-8; Beyotime, China). e optical density value was measured at 450 nm by a microplate reader (SpectraMax Gemini UVmax; Molecular Devices, USA).

Analysis of Oxidative
Stress. 1 × 10 6 ARPE-19 cells per well in 6-well plates were cultured with 2 ml DF-12 medium with 10% FBS. Cells were treated as previously indicated. For ROS assay (Nanjing Jiancheng Bioengineering Institute, E004-1), cells after digestion were prepared into single-cell suspension and divided into negative, positive, and tested groups. e negative group was suspended with 0.01 mol/L PBS, and the positive group was suspended with DCFH-DA together with hydrogen peroxide to induce production of ROS. e tested groups were suspended with DCFH-DA alone. Cells in all groups were suspended into 1 × 10 6 ∼2 × 10 7 cells/ml. After suspension, cells were incubated in 37°C for 30 min. After that, cells were centrifuged under 1000 g for 5 min and washed with PBS once. After removal of the supernatant, cells were resuspended with PBS, and the fluorescence intensity was tested under the fluorescence spectrophotometer.
For SOD assay (Nanjing Jiancheng Bioengineering Institute, A001-3), cells after digestion were prepared into single-cell suspension and washed by PBS once before being smashed in the ultrasonic cell breaker. e mixture was centrifuged at 1000 g at 4°C for 5 min, and supernatant was used for the assay. Reagents and samples were mixed in a 96-well plate as described in the manual. e activity of SOD (U/ml) � 100% × ((OD (control) − OD (blank of control) ) − (OD (test) − OD (blank of test) ))/(OD (control) − OD (blank of control) ).
For MDA assay (Nanjing Jiancheng Bioengineering Institute, A003-1), cells after digestion were prepared into single-cell suspension and washed by PBS once before being smashed in the ultrasonic cell breaker. e mixture was centrifuged at 1000 g at 4°C for 5 min, and the supernatant was used for the assay. Reagents and samples were mixed in a 1.5 ml tube as described in the manual. After mixing, the mixture was heated at 95°C for 40 min and cooled down to room temperature. After centrifugation at 4000 g for 10 min, the supernatant was read at 532 nm. e OD values of the standard and test were corrected by OD of blank. Solutions 1, 2, and 3 were provided by the kit.
2.6. Cell Apoptosis. Cell apoptosis was determined by an Annexin V-FITC-PI apoptosis detection kit (Vazyme, China). Cells were harvested after treatment with indicated time at a concentration of 1-5 × 10 5 cells. 100 μl 1 × binding buffer was added to resuspend the cells. 5 μl of V-FITC and 5 μl of PI staining were gently added into the cells and incubated in the dark. After 15 min of incubation, 400 μl 1 × binding buffer was added and mixed, and cells were then detected in 1 h.
e Annexin V-FITC-positive and PInegative cells were recorded as early apoptotic cells; the Annexin V-FITC-positive and PI-positive cells were recorded as later apoptotic cells. e apoptotic cells included the early apoptotic cells and later apoptotic cells.

2.7.
Immunofluorescence. 1 × 10 4 ARPE-19 cells were plated on a coverslip and treated as previously indicated. 4% paraformaldehyde was used to fix the cells for 10 min, and 0.1% Triton X-100 was used to permeabilize the cells for 5 min. e goat serum was used to block for 1 h, and rabbit anti-LC3-II mAb (Alexa Fluor 488 conjugate; Cell signaling, #3868) was incubated with cells overnight at 4°C. 4′, 6′diamidino-2-phenylindole (DAPI) was used to stain the nuclei for 3 min.

Statistical Analysis.
e data are presented as mean ± SD of the number of determinations. Statistical difference analysis was calculated using GraphPad Prism 6.0 software. e differences in multiple groups were detected using oneway ANOVA with Tukey's test. P value less than 0.05 was treated as a significant difference.

Results
High glucose treatment triggered the reduction of CKIP-1 levels and the activation of oxidative stress and autophagy in retinal pigment epithelial cells.
Nrf2 translocates from the cytosol to the nucleus to respond to the oxidative stress, and p62-Keap1 interaction promotes the translocation of Nrf2 to the nucleus [19]. e results manifested that CKIP-1 overexpression enhanced the p62 accumulation in high glucose-treated ARPE-19 cells (Figure 3(a)). High glucose treatment did not affect the KEAP1 expression, but overexpression of CKIP-1 dramatically reduced the KEAP1 expression in ARPE-19 cells treated with high glucose (Figure 3(b)). In addition, the total Nrf2 levels were not changed in the cells both treated with high glucose or high glucose plus CKIP-1 overexpression. However, the level of nuclear Nrf2 was decreased in high glucose-treated cells, and CKIP-1 overexpression reenhanced the Nrf2 expression in the nucleus of ARPE-19 cells, but the cytoplasmic Nrf2 expression presented the opposite trend (Figure 3(c)), indicating that CKIP-1 might promote the translocation of Nrf2 to the nucleus.
Due to high glucose induced autophagy, we next studied the effects of CKIP-1 on the autophagic pathway by Western blot. Overexpression of CKIP-1 prominently increased the p62 accumulation and inhibited the ratio of LC3-II/I (Figures 4(a) and 4(b)). In addition, the Beclin-1 expression was also decreased by CKIP-1 (Figure 4(a)). e results from immunofluorescence for the measurement of LC3-II were in accordance with the protein changes shown by Western blot (Figure 4(c)). Besides, the Nrf2 inhibitor, ML385, reversed the inhibitory effect of CKIP-1 on autophagy (Figure 4).

CKIP-1 Overexpression Suppresses High Glucose-Induced
Oxidative Stress. Nrf2 cascade signaling can serve as an antioxidant response to regulate oxidative stress. To investigate the protective effects of CKIP-1, ROS was first accumulated by high glucose treatment. en, the downregulated expression of ROS induced by CKIP-1 overexpression was observed in high glucose-induced ARPE-19 cells (Figure 5(a)). Increased CKIP-1 expression exerted a similar protective effect by dramatically decreasing the lipid peroxidation product, MDA levels, and recovered the antioxidant enzyme SOD level, revealing the role of CKIP-1 in alleviating tissue damage mediated by oxidative stress (Figures 5(b) and 5(c)). Furthermore, the Nrf2 inhibitor reactivated the oxidative stress in high glucosetreated cells transfected with pcDNA3.1-CKIP-1 (Figures 5(a) and 5(c)).
High glucose-triggered apoptosis is blocked in ARPE-19 cells with overexpression of CKIP.
To further set forth the protective mechanism of CKIP-1 on RPE cells, we evaluated the relationship between cell death and CKIP-1. High glucose treatment for 48 h significantly induced cytotoxicity of ARPE-19 cells, which was reduced by CKIP-1 overexpression (Figure 5(d)). Furthermore, we found that CKIP-1 mainly reduced high glucose-induced apoptosis in ARPE-19 cells (Figures 6(a) and 6(b)). Concomitant with the apoptotic assay, Western blot showed similar results that high glucose enhanced the levels of proapoptotic proteins including cleaved-caspase3, cleaved-caspase7, Bax, and Bad inhibited by CKIP-1 (Figures 6(c) and 6(d)). Meanwhile, the level of antiapoptotic protein Bcl-2 was enhanced by CKIP-1 overexpression (Figure 6(d)). erefore, the apoptosis induced by high glucose is blocked in ARPE-19 cells with overexpression of CKIP.

Discussion
Researchers have found that activation of CKIP-1 protects against diabetic renal fibrosis [20,21]. Besides, evidence supported that CKIP-1 was dramatically downregulated in DR tissues and high glucose-treated human retinal endothelial cells [14]. e blood-retinal barrier (BRB) is composed of the tight junctions of the inner retinal endothelial cells and the outer retinal pigment epithelial cells, which regulate ion, protein, and water flux in and out of the retina [3]. BRB breakdown is thought to be one of the characteristics of DR, and the pathology is mainly associated with oxidative stress and inflammation [22]. In the current study, CKIP-1 was also downregulated in retinal pigment epithelial (RPE) cells under high glucose condition. Furthermore, overexpression of CKIP-1 in high glucose-treated RPE cells enhanced the ROS and MDA generation, as well as reduced SOD activity. Hence, we found that overexpression of CKIP-1 suppressed apoptosis. In DR progression, oxidative stress, endoplasmic reticulum stress-induced expression of death receptors, and mitochondrial damage are the major reasons that initiate apoptosis-related cell death [23]. e hyperglycemia promotes overproduction of ROS which induces mitochondrial dysfunction and apoptosis [24]. e results revealed the role of CKIP-1 in oxidative stress and apoptosis in DR progression, providing evidence that CKIP-1 can be a promising therapeutic target for DR therapy.
Autophagy can be a two-edged sword during DR progression. Activated autophagy can protect the cells from the occurrence of diabetes, endless autophagy; however, it will lead to autophagy dysfunction that elicits cell death [25,26]. In the present study, high glucose induced the upregulation of ratio of LC3-II/I and the expression of Beclin-1 and downregulation of p62, indicating the activation of autophagy upon HG stimulation in RPE cells. However, CKIP-1 overexpression significantly decreased the expression of LC3-II/I and Beclin-1, but enhanced p62 expression. Emerging evidence has confirmed that autophagy is involved significantly in the progression of DR [27]. In the course of autophagy, LC3-I is transformed by the addition of a group of LC3-II which permits the combination of the protein to autophagosome membranes, and Beclin-1 plays a part to the initial autophagic vesicles development [27]. CKIP-1 has been reported to augment autophagy in steatotic hepatocytes [28]. Our research was the first to show that overexpression of CKIP-1 can inhibit the autophagy activation in high glucose-treated RPE cells. Excessive ROS production is found in mammalian cells to promote autophagy [29]. Besides, the antiapoptotic protein Bcl-2 inhibits the Beclin-1-dependent autophagy [30]. Overexpression of CKIP-1 suppressed not only the ROS level but also Bcl-2 expression, indicating that autophagy may mediate the role   of CKIP-1 in high glucose-induced oxidative stress and apoptosis. e p62 deficiency or overexpression in the autophagy process through transcriptional (in the nucleus) and posttranscriptional (in the cytoplasm) regulation has been investigated. Previous investigation has indicated that p62 assembly led to activation of Nrf2 through suppression of Keap1 [31,32]. Our data showed that CKIP-1 overexpression enhanced the expression of p62 and nuclear Nrf2, but suppressed Keap1. In addition, the protective effects of CKIP-1 in high glucose-induced autophagy, oxidative stress, and apoptosis were reversed by the Nrf2 inhibitor, ML385, demonstrating that CKIP-1 shows that protective effects on high glucose-induced injury might be mediated by the p62/ Keap1/Nrf2 signaling.

Conclusion
In conclusion, the current investigation proved that CKIP-1 overexpression could suppress high glucose-induced RPE cell autophagy, oxidative stress, and apoptosis by regulating the p62/Keap1/Nrf2 signaling pathway, which helps the exploration of new therapeutic strategy for DR.

Data Availability
All data generated or analyzed during this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
XZ and YB contributed to conception and design; XZ, JW, and PL contributed to acquisition of data; XZ and LT contributed to analysis and interpretation of data; YB involved in drafting the manuscript or revising it critically for important intellectual content; all authors have given final approval of the version to be published.