Activation of Nrf2 by MIND4-17 protects osteoblasts from hydrogen peroxide-induced oxidative stress

MIND4-17 is a recently developed NF-E2-related factor 2 (Nrf2) activator, which uniquely causes Nrf2 disassociation from Keap1. Here, we showed that pretreatment with MIND4-17 significantly inhibited hydrogen peroxide (H2O2)-induced viability reduction of primary osteoblasts and OB-6 osteoblastic cells. Meanwhile, MIND4-17 inhibited both apoptotic and non-apoptotic osteoblast cell death by H2O2. MIND4-17 treatment induced Keap1-Nrf2 disassociation, causing Nrf2 stabilization, accumulation and nuclear translocation in osteoblasts, leading to transcription of several Nrf2-dependent genes, including heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), γ-glutamylcysteine synthetase modifier subunit (GCLM) and catalytic subunit (GCLC). Additionally, MIND4-17 largely attenuated H2O2-reactive oxygen species (ROS) production, lipid peroxidation and DNA damages. Nrf2 knockdown by targeted short hairpin RNA (shRNA) exacerbated H2O2-induced cytotoxicity in OB-6 osteoblastic cells, and nullified MIND4-17-mediated cytoprotection against H2O2. Meanwhile, Keap1 shRNA took over MIND4-17′s actions and protected OB-6 cells from H2O2. Together, MIND4-17 activates Nrf2 signaling and protects osteoblasts from H2O2.

OB-6 cells were also treated with H 2 O 2 at other concentrations, from 50-400 μM. With the increase of H 2 O 2 's concentration, more cell death was noticed ( Figure  1C and 1D). Pretreatment with 3 μM of MIND4-17 was again cytoprotective, and inhibited cytotoxicity by H 2 O 2 at all tested concentrations ( Figure 1C and 1D). We also tested the effect of this novel Nrf2 inducer in other osteoblasts. As displayed, in both primary human osteoblasts ( Figure 1E) and primary murine osteoblasts ( Figure 1F)

Nrf2 knockdown abolishes MIND4-17-mediated cytoprotection against H 2 O 2
If Nrf2 activation is required for MIND4-17-mediated cytoprotection against H 2 O 2 , Nrf2 depletion should then abolish MIND4-17's actions in osteoblasts. To test this hypothesis, we utilized short hairpin RNA (shRNA) method to inhibit Nrf2 expression in OB-6 cells. A set of two lentiviral shRNAs, against independent and non-overlapping   for applied time; The association between Nrf2 and Keap1 was examined by the co-immunoprecipitation ("Co-IP") assay (A, left panel); Expressions of the indicated proteins in total cell lysates (A, right panel, and D) as well as in the nuclear lysates (B, Lamin-B was a marker protein) were presented; mRNA expressions of listed genes were tested by quantitative real-time PCR ("qRT-PCR") assay (C). Data were presented as mean (n=5) ± standard deviation (SD). * p<0.05 vs. "C" cells. Experiments in this figure were repeated for three times, and similar results were obtained. human Nrf2 sequence (from Dr. Jiang [31]), were utilized. Puromycin was added to select the stable cells. Western blotting assay confirmed that the two applied Nrf2 shRNA ("-1/-2" [31]) each potently downregulated Nrf2 protein in MIND4-17 (3 μM, 3 hour)-treated OB-6 cells ( Figure 5A), resulting in over 90% Nrf2 protein depletion ( Figure 5A). Consequently, MIND4-17-induced protein ( Figure 5A) and mRNA ( Figure 5B and 5C) expressions of HO1 and NQO1 was also largely inhibited by Nrf2 shRNAs. CCK-8 assay results in Figure 5D and Histone DNA apoptosis ELISA assay results in Figure 5E displayed that H 2 O 2 -induced viability reduction and apoptosis were significantly boosted in Nrf2-silenced cells. More importantly, MIND4-17-induced anti-H 2 O 2 activity was almost completely nullified in Nrf2 shRNA-expressing OB-6 cells ( Figure 5D and 5E). These results indicate that Nrf2 activation is required for MIND4-17-mediated cytoprotection in OB-6 cells.

DISCUSSION
Increased ROS production and subsequent oxidative stress is major cause of osteoblast cell injuries. Activation of Nrf2 signaling could efficiently protect osteoblasts [17,33,34] and other cells from oxidative stress. Keap1, a BTB-Kelch protein, is the upstream suppressor protein of Nrf2. Keap1 maintains Nrf2's subcellular localization  and steady-state level. It works as an E3 ubiquitin ligase complex with Nrf2, causing Nrf2 ubiquitin conjugation and proteasomal degradation [28,35,36].
In this study, we propose that activation of Nrf2 is required for MIND4-17-mediated cytoprotection against

Culture of osteoblasts and osteoblastic cells
The OB-6 human osteoblastic cells were cultured and differentiated as described in our previous studies [37,38]. The isolation and primary culture of murine osteoblasts derived from the trabecular bone of C57/B6 mice were described previously [39][40][41][42], with the animal protocol approved by Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University. C57/B6 mice, 5-6 week old, all female, were obtained from the Experimental Animal Center of Nanjing Medical University (Nanjing, China). The detailed protocol for the culture of primary human osteoblasts was also described previously. Briefly, the trabecular bone fragments were minced and were then digested (2 mg/mL collagenase type II, (300 U/mg; Sigma). The primary cells were thereafter placed in culture flasks with the described medium [43]. Medium was changed two-three times a week until cells reached confluence. The protocols using human tissues/cells were conducted according to the principles of Declaration of Helsinki, and were approved by the Ethics Review Board (ERB) of Nanjing Medical University.

Cell apoptosis assay
In line with our previous studies [39,40], the histone-DNA ELISA plus kit (Roche, Palo Alto, CA) was utilized, and ELISA OD at 450 nm was recorded to quantitatively reflect cell apoptosis intensity [39,40].

Fluorescence activated cell sorting (FACS) assay
Osteoblasts/osteoblastic cells with applied treatment were washed and resuspended in binding buffer together with 2.5 μL of Annexin V-FITC (Invitrogen, Shanghai, China) and 2.5 μL of propidium iodide (PI) (Invitrogen). Osteoblasts/osteoblastic cells were then examined by flow cytometry using the CellQuest software (BD Biosciences, Shanghai, China). Annexin V positive cells were labeled as apoptotic cells [44,45]. Annexin V negative and PI positive cells were labeled as non-apoptotic dead cells [44,45].

Western blotting assay
The detailed protocol of Western blotting assay has been extensively discussed in our previous studies [37,[39][40][41][42]. The protocol of isolation of nuclear proteins was described in detail in the previous studies [44,46].

Co-immunoprecipitation (Co-IP) assay
As described [44,46], the Co-IP assay was performed to test the association between Keap1-Nrf2. In brief, OB-6 www.impactjournals.com/oncotarget osteoblastic cells with applied treatment were lysed [44]. To the cleared lysates, 0.25 μg of Nrf2 antibody (Santa Cruz Biotech) was added per 0.8 mg of total cellular lysate proteins, and the immune complex formed by rotation for 24 hours at 4 °C. The protein A/G-Sepharose (25 μL, Sigma) was then added and the incubation continued for additional 12 hours. The resulting immuno-precipitates captured with protein A/G-Sepharose were washed four times with CHAPS-containing buffer and analyzed by Western blotting assay.

RNA isolation and qRT-PCR
Total RNA in osteoblasts/osteoblastic cells was extracted by the RNeasy Midi Kit (Qiagen, Wuxi, China). Five-hundred ng of total RNA per treatment was reversetranscribed through the RT-PCR kit (TOYOBO, Japan). Quantitative real-time PCR ("qRT-PCR") was performed through employing the SYBR green kit [47,48], under the ABI-7600 Fast-PCR system (Applied Biosystems, Shanghai, China). The mRNA primers of Nrf2 pathways genes, including Nrf2, HO-1, GCLC, GCLM and NQO1, and GAPDH were previously described [31,44,46,49]. GAPDH was always tested as the internal control gene. The 2 -ΔΔCt method was utilized to calculate relative expression of indicated mRNA. Its value was normalized to that of control cells.

shRNA and stable cell selection
The two lentiviral Nrf2 shRNAs ("Seq-1/2", with unique and non-overlapping sequences against human Nrf2) were provided by Dr. Jiang [31]. The shRNAcontaining lentivirus was added to cultured osteoblasts/ osteoblastic cells at 10 μL virus/1 mL medium. After 12 hours, puromycin (0.5 μg/mL) was added to select stable cells for additional 96 hours. Nrf2 knockdown in the resulting stable cells was verified by the qRT-PCR assay and Western blotting assay. Control cells were infected with lentiviral scramble control shRNA [37]. Keap1 shRNA and stable cell selection was described in the previous study [50].

Lipid peroxidation assay
Cellular TBAR (Thiobarbituric acid reactive substances) content was tested to examine the production of toxic aldehyde resulting from oxidative fatty acyl degradation, the malondialdehyde (MDA). TBAR intensity was then an indicator of lipid peroxidation. The detailed protocol for testing TBAR was described previously [51,52]. The TBAR absorbance was measured at 532 nm. The lipid peroxide level was expressed as MDA/mg of protein. The value of treatment cells was normalized to that of control cells.

Statistical analysis
Comparisons between groups were performed via one-way ANOVA and the Newman-Keuls test (SPSS 18.0

CONCLUSION
We conclude that MIND4-17 activates Nrf2 signaling and protects osteoblasts from H 2 O 2. Dex and other glucocorticoids shall exert direct injuries to human osteoblasts, which is an important contributor of osteoporosis and osteonecrosis. The results of our study indicate that MIND4-17 and possible other Nrf2 signaling activators could possibly be further tested to treat Dex-associated bone damages.

Author contributions
All 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 draft the manuscript.

CONFLICTS OF INTEREST
The listed authors have no conflicts of interest.

FUNDING
This work is supported by the National Natural Science Foundation (81672170).