MIND4-17 protects retinal pigment epithelium cells and retinal ganglion cells from UV

Nrf2 activation would efficiently protect retinal cells from UV radiation (UVR). Recent studies have developed a Nrf2-targeting thiazole-containing compound MIND4-17, which activates Nrf2 through blocking its association with Keap1. In the current study, we demonstrated that pretreatment with MIND4-17 efficiently protected retinal pigment epithelium (RPE) cells (RPEs) and retinal ganglion cells (RGCs) from UVR. UVR-induced apoptosis in the retinal cells was also largely attenuated by MIND4-17 pretreatment. MIND4-17 presumably separated Nrf2 from Keap1, allowing its stabilization and accumulation in retinal cells, which then translocated to cell nuclei and promoted transcription of ARE-dependent anti-oxidant genes, including HO1, NQO1 and GCLM. Significantly, shRNA-mediated knockdown of Nrf2 almost completely abolished MIND4-17-induced cytoprotection against UVR. Further studies showed that MIND4-17 largely ameliorated UVR-induced ROS production, lipid peroxidation and DNA damages in RPEs and RGCs. Together, MIND4-17 protects retinal cells from UVR by activating Nrf2 signaling.

Recent studies have developed a Nrf2-targeting thiazole-containing compound, namely MIND4-17 [19,20]. This small molecule compound covalently modified a critical stress-sensor cysteine (C151) of Keap1, leading to Nrf2's departure from the ubiquitin E3 ligase complex. This would lead to Nrf2 Research Paper stabilization, accumulation and nuclear translocation [19,20]. Thus, treatment of this compound would induce profound Nrf2 activation. In the current study, we demonstrate that MIND4-17 efficiently protects RPEs and RGCs from UVR.
Lactate dehydrogenase (LDH) release is often tested as a marker of cell death. Following UVR, the LDH level in the conditional medium of ARPE-19 cells was significantly increased ( Figure 1C), indicating cell death. Pretreatment with MIND4-17 at 1-10 μM largely attenuated UVR-induced ARPE-19 cell death (LDH release, Figure 1C). Retinal ganglion cells (RGCs) are also main UVR-targeting cells in the retina [21,22]. Here, we demonstrated that UVR similarly induced viability loss (CCK-8 OD reduction, Figure 1D) and cell death (LDH release, Figure 1E) in primary human RGCs [21,22]. Importantly, such effects by UVR were largely attenuated with pretreatment of MIND4-17 (5 μM) ( Figure 1D and 1E). It should be noted that treatment with MIND4-17 alone at tested concentration failed to change viability and death of the retinal cells ( Figure 1B-1E). Together, these results and were further cultured for 48 hours; Cell viability was tested by the CCK-8 assay (B and D); Cell death was examined by LDH release in the conditional medium (C and E). For each assay, n = 5. "C" stands for untreated control cells. *p < 0.05 vs. "C" cells. # p < 0.05 vs. "UVR" only cells. Experiments in this figure were repeated four times to insure consistency of results. demonstrate that MIND4-17 protects human RPEs and RGCs from UVR.
On the other hand, Nrf2 mRNA level was unchanged before and after t MIND4-17 treatment ( Figure 3D). Nrf2 protein level was yet significantly increased in MIND4-17-treated RPE cells, suggesting Nrf2 stabilization ( Figure 3E). Protein expressions of HO1, NQO1 and GCLM were also boosted

Nrf2 is required for MIND4-17-mediated retinal cytoprotection against UVR
In order to test that Nrf2 signaling activation is required for MIND4-17-mediated cytoprotection, short hairpin RNA (shRNA) method was employed to knockdown Nrf2 in retinal cells. As described [7,24], the Nrf2 shRNA-containing lentiviral particles (purchased from Santa Cruz Biotech) were added to ARPE-19 cells. Stable cells were then selected by puromycin. The applied Nrf2 shRNA dramatically downregulated Nrf2 mRNA expression in the stable cells ( Figure 4A). Since basal Nrf2 protein level was low in the ARPE-19 cells (see Figure 3), we stimulated cells with MIND4-17 (5 μM, 3 hours). Western blotting assay results confirmed that the applied Nrf2 shRNA also dramatically downregulated Nrf2 protein in MIND4-17-treated ARPE-19 cells ( Figure 4B). Downstream HO1 expression was also largely inhibited ( Figure 4B). Remarkably, UVR-induced cell death (CCK-8 OD reduction, Figure 4C) and apoptosis (ssDNA ELISA OD increase, Figure 4D) were intensified in Nrf2-shRNAexpressing ARPE-19 cells. These results suggest that Nrf2 activation might also be important for the protection against UVR, which is consistent with previously findings [6,7]. Significantly, MIND4-17-mediated cytoprotection against UVR was almost completely abolished in Nrf2silenced cells (Figure 4C and 4D). MIND4-17 was also most ineffective against UVR in Nrf2-shRNA expressing cells ( Figure 4C and 4D). The similar results were also obtained in the primary human RGCs (Data not shown). These results suggest that Nrf2 is required for MIND4-17mediated retinal cytoprotection against UVR.

MIND4-17 efficiently attenuates UVR-induced oxidative stress in retinal cells
Reactive oxygen species (ROS) production and following oxidative stress are major causes of cell death , GAPDH mRNA was tested as the internal control); Listed proteins in total cell lysates (E) and nuclear fraction lysates (F) were also tested by Western blotting assay (GAPDH was tested as the loading control, which was absent in the nuclear fractions). "C" stands for untreated control cells. *p < 0.05 vs. "C" cells. Experiments in this figure were repeated three times to insure consistency of results. by UVR [26,27]. Nrf2 signaling is a well-established antioxidant signaling [10,12,17,28]. In line with previous findings [6,7,29,30], UVR radiation to ARPE-19 cells induced significant ROS production, which was tested by increase of DCFH-DA intensity ( Figure 5A). UVRinduced ROS production was followed by increase of DNA damages (γ-H2AX intensity increase, Figure  5B) and lipid peroxidation (TBAR intensity increase, Figure 5C). Remarkably, such effects by UVR were largely attenuated by pretreatment of MIND4-17 (5 μM) ( Figure 5A-5C). In the primary human RGCs, MIND4-17 (5 μM) pretreatment also inhibited ROS production by UVR ( Figure 5D). These results demonstrate that MIND4-17 efficiently attenuates UVR-induced oxidative stress in retinal cells.
Recent studies have characterized MIND4-17 as a unique and highly selective small molecule activator of Nrf2 [19,20]. MIND4-17-induced Nrf2 activation is initially through covalent modification of the Keap1 sensor-cysteine C151 [19,20]. This modification mimics the effects of oxidative stress, which causes Keap1 conformational change and arrests of the Keap1-Nrf2 complex [19,20]. This would release Nrf2 from the from the ubiquitin E3 ligase complex, causing its stabilization and accumulation in the cytoplasm. It is followed by nuclear translocation of Nrf2 and de novo synthesis of ARE-response genes [19,20]. Unlike other known Nrf2 activators, this small molecule compound directly and uniquely disassociates the Nrf2-Keap1 complex. It therefore activates Nrf2 signaling at an extremely high efficiency [19,20].
In the present study, we show that MIND4-17 treatment presumably separated Nrf2 from Keap1, thus allowing its stabilization and accumulation in retinal cells. Stabilized Nrf2 by MIND4-17 translocated to cell nuclei, causing transcription and expression of multiple AREdependent anti-oxidant genes, including HO1, NQO1 and GCLM. Subsequently, activation of Nrf2 by MIND4-17 largely ameliorated UVR-induced ROS production, lipid peroxidation and DNA damages in RPEs and RGCs.
Retinal cell death and apoptosis by UVR were also dramatically alleviated by MIND4-17. Importantly, we demonstrate that Nrf2 is required for MIND4-17's actions in retinal cells. shRNA-mediated knockdown of Nrf2 almost completely reversed MIND4-17-induced retinal cytoprotection against UVR.

Reagents, chemicals and antibodies
Based on the structure in previous studies [19,20] (also see Figure 1A), MIND4-17 was synthesized and verified by Minde Biotech (Suzhou, China). Puromycin scramble control shRNA ("sh-sc") or Nrf2 shRNA ("sh-Nrf2"), were treated with MIND4-17 (5 μM) for 3 hours, Nrf2 mRNA (A) and listed proteins (B) were tested by RT-qPCR assay (GAPDH mRNA was tested as the internal control) and Western blotting assay (GAPDH was tested as the loading control), respectively; Cells were exposed UV radiation (UVR, UVA2 + B, 30 mJ/cm 2 ) and cultured for applied time; Cell survival and apoptosis were tested by CCK-8 assay (C) and ssDNA ELISA assay (D), respectively. "Ctrl" stands for parental ARPE-19 cells (A and B). For each assay, n = 5. *p < 0.05 vs. "sh-sc" cells. Experiments in this figure were repeated three times to insure consistency of results. www.impactjournals.com/oncotarget was obtained from Sigma-Aldrich (Nanjing, China). The reagents for cell culture were provided by Gibco (Guangzhou, China). Antibodies against Nrf2, HO1, NQO1, GCLM and GAPDH were purchased from Santa Cruz Biotech (Beijing, China). Antibodies for cleavedcaspase-3 and cleaved-PARP were provided by Cell Signaling Tech (Nanjing, China).

Culture of RPEs
The established ARPE-19 cells were provided as a gift from Dr. Jiang [6,35,36]. The DMEM plus fetal bovine serum (FBS) medium was employed for ARPE-19 cell culture.

Culture of primary human RGCs
The primary-cultured human RGCs were also provided by Dr. Jiang [21]. The primary human cells were maintained as described previously [21]. Primary human RGCs at passage 3-8 were utilized for further experiments.

Cell viability and cell death assays
Testing of cell viability by the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) [37] as well as cell death assay by lactate dehydrogenase (LDH) release assay (Takara, Tokyo, Japan) were extensively described in early studies [38]. CCK-8 optic density at 450 nm was recorded. LDH content in the conditional medium was normalized to total LDH [38,39].
ssDNA ELISA assay of cell apoptosis DNA formamide denaturation and subsequent the production of single strand DNA (ssDNA) is characteristic marker of cell apoptosis, which was tested by the ApoStrandTM ELISA apoptosis detection kit (BIOMOL International, Plymouth Meeting, PA). ELISA OD at 450 nm was recorded.

Assessment of apoptosis by Annexin V FACS assay
Following the applied treatment, retinal cells were harvested, washed and resuspended in binding buffer with 1 μL of Annexin V-FITC and 1 μL of propidium iodide (PI) (Biyuntian, Wuxi, China). After incubation for 20 min at room temperature, cells were then tested by flow cytometry via CellQuest software (BD Biosciences, Shanghai, China). Annexin V ratio was recorded [8,39]. , cells were then subjected to UV radiation (UVR, UVA2 + B, 30 mJ/cm 2 ) and were further cultured for applied time; ROS production was tested by DCFH-DA intensity assay (A and D); DNA damages and lipid peroxidation were tested by γ-H2AX assay (B) and TBAR activity assay (C), respectively. "C" stands for untreated control cells. *p < 0.05 vs. "C" cells. #p < 0.05 vs. "UVR" only cells. Experiments in this figure were repeated three times to insure consistency of results. www.impactjournals.com/oncotarget

Western blotting assay
Cells were lysed via the described lysis buffer [41]. Determination of the protein concentration was though the Bio-Rad Protein Assay (Bio-Rad, Shanghai, China). Protein concentration (μg/mL) was measured at 690 nm. The detailed protocol for Western blotting assay and data quantification were described previously [42,43]. Cell nuclei were isolated by the "cell nuclei isolation kit" (Sigma) [8].

Real-time quantitative PCR
Total RNA was extracted by TRIzol reagents (Invitrogen, Shanghai, China) and then reversely transcribed using a PrimeScript RTreagent kit (Takara Biotechnology, Japan). Real-time quantitative PCR ("RT-qPCR") assay was performed by a SYBR Premix Ex TaqTM kit on the ABI-7700 fast PCR system (Takara Biotechnology, Japan). mRNA primers for human HO-1, NQO1 and GLCM and GAPDH were described previously [44]. GAPDH mRNA was tested as the reference gene. The 2 -ΔΔCt method was employed to calculate relative expression of targeted mRNAs.

Nrf2 shRNA
The Nrf2 shRNA lentiviral particles (sc-37030-V, Santa Cruz Biotech, Shanghai, China) and the scramble control shRNA lentiviral particles (sc-108080, Santa Cruz Biotech) were both commercial available. The shRNA lentivirus was added to ARPE-19 cells for 12 hours. Afterwards, puromycin (2.5 μg/mL) was added to select stable cells for 5-6 passages. Nrf2 knockdown was verified by both Western blotting assay and RT-qPCR assay.

Reactive oxygen species (ROS) assay
ROS intensity in the control and UVR-treated retinal cells was measured by the carboxy-H2DCF-DA dye (Invitrogen, Shanghai, China) assay [45]. Following the treatment, cells were incubated with 1 μM of carboxy-H2-DCFDA for 120 min under the dark, which were then washed with warm PBS and immediately tested by a spectrofluorometer at excitation and emission wavelengths of 485 and 535 nm, respectively. The results were expressed as increase in fluorescence with respect to control cells.

Lipid peroxidation assay
Thiobarbituric acid reactive substances (TBAR) level was examined to reflect the production of toxic aldehyde resulting from oxidative fatty acyl degradation, the malondialdehyde (MDA). The detailed protocol was described previously [45,46].
γ-H2AX assay of DNA damages. ARPE-19 cells were fixed in ice-cold ethanol, which were then incubated with a mouse monoclonal anti-γ-H2AX antibody (Santa Cruz Biotech). Afterwards, the FITC-conjugated antimouse secondary antibody (Santa Cruz) was then added. Cells were then subjected to FACS assay to determine the γ-H2AX percentage, reflecting DNA damage intensity [47].

Statistics
The results were presented as the mean ± standard deviation (SD). One-way ANOVA was employed to examine the significant differences between groups using SPSS 17.0 (SPSS, Chicago, CA). Values of p < 0.05 were considered statistically significant.

CONCLUSIONS
Together, we conclude that targeting Nrf2 by MIND4-17 efficiently protects RPEs and RGCs from UVR.

Author contributions
All the listed authors carried out the experiments, participated in the design of the study and performed the statistical analysis, participated in its design and coordination and helped to write the manuscript and proof readings.