PGK1 depletion activates Nrf2 signaling to protect human osteoblasts from dexamethasone

Abstract

Activation of nuclear-factor-E2-related factor 2 (Nrf2) cascade can alleviate dexamethasone (DEX)-induced oxidative injury and death of human osteoblasts. A recent study has shown that phosphoglycerate kinase 1 (PGK1) inhibition/depletion will lead to Kelch-like ECH-associated protein 1 (Keap1) methylglyoxal modification, thereby activating Nrf2 signaling cascade. Here, in OB-6 osteoblastic cells and primary human osteoblasts, PGK1 silencing, by targeted shRNA, induced Nrf2 signaling cascade activation, causing Nrf2 protein stabilization and nuclear translocation, as well as increased expression of ARE-dependent genes (HO1, NQO1, and GCLC). Functional studies demonstrated that PGK1 shRNA largely attenuated DEX-induced oxidative injury and following death of OB-6 cells and primary osteoblasts. Furthermore, PGK1 knockout, by the CRISPR/Cas9 method, similarly induced Nrf2 signaling activation and protected osteoblasts from DEX. Importantly, PGK1 depletion-induced osteoblast cytoprotection against DEX was almost abolished by Nrf2 shRNA. In addition, Keap1 shRNA mimicked and nullified PGK1 shRNA-induced anti-DEX osteoblast cytoprotection. At last we show that PGK1 expression is downregulated in human necrotic femoral head tissues of DEX-taking patients, correlating with HO1 depletion. Collectively, these results show that PGK1 depletion protects human osteoblasts from DEX via activation of Keap1-Nrf2 signaling cascade.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1. PGK1 silencing activates Nrf2 signaling in human osteoblasts.

Expression of listed genes (mRNAs and proteins) in stable OB-6 osteoblastic cells, with applied PGK1 shRNA (“sh-PGK1-S1/S2”) or the non-sense control shRNA (“sh-C”), were tested by qPCR and western blotting assays (a–c, f). The relative NQO1 activity was tested as well (g). OB-6 cells were treated with MG-132 (10 μM) or plus sh-PGK1-S1 infection (“+sh-PGK1-S1”), after 24 h Nrf2 and Erk1/2 (the loading control) protein expression in total cell lysates was shown (d). Stable OB-6 cells with sh-PGK1-S1 were treated with cycloheximide (CHX, 100 μg/mL) for 12 and 24 h, Nrf2 and Erk1/2 protein expression in total cell lysates was shown (e). The primary human osteoblasts were transduced with lentiviral PGK1 shRNA (“sh-PGK1-S1”) or the lentiviral non-sense control shRNA (“sh-C”), expression of listed genes (h–j) and the relative NQO1 activity (k) were tested. Expression of listed proteins was quantified, normalized to the loading control (a, c–e, h, i). Data were expressed as mean ± standard deviation (SD, n = 5). *p < 0.05 vs. “sh-C” cells. Experiments were repeated four times, with similar results obtained.

Fig. 2. PGK1 silencing protects human osteoblasts from DEX-induced death and apoptosis.

Stable OB-6 osteoblastic cells (a–f) or primary human osteoblasts (g–i) with applied PGK1 shRNA (“sh-PGK1-S1/S2”) or the non-sense control shRNA (“sh-C”) were treated with or without DEX (1 μM) for applied time periods, cell viability (MTT assay; a and g) and cell death (LDH medium release; b and h) were tested; Cell apoptosis activation was tested by the listed assays (c–f, i). Expression of listed proteins was quantified, normalized to the loading control (d). Data were expressed as mean ± standard deviation (SD, n = 5). “Ctrl” represents untreated control group (Same for all Figures). *p < 0.05 vs. “sh-C” cells with “Ctrl” treatment. #p < 0.05 vs. “sh-C” cells with DEX treatment. Experiments in this figure were repeated four times, and similar results were obtained.

Fig. 3. PGK1 silencing attenuates DEX-induced oxidative stress and programmed necrosis in human osteoblasts.

Stable OB-6 human osteoblastic cells (a–f) or primary human osteoblasts (g, h) with applied PGK1 shRNA (“sh-PGK1-S1/S2”) or the non-sense control shRNA (“sh-C”), were treated with or without DEX (1 μM) for applied time periods, oxidative stress (a, b, g) and programmed necrosis (c–f, h) were tested by the listed assays mentioned in the text. For mitochondrial depolarization assay, JC-1 green intensity was examined at 550 nm via a fluorescence spectrofluorometer (results normalized to the control level), and the representative JC-1 fluorescence images, merging both green and red fluorescence pictures, were presented (c, d). Expression of listed proteins was quantified, normalized to the loading control (e, f). Data were expressed as mean ± standard deviation (SD, n = 5). *p < 0.05 vs. “sh-C” cells with “Ctrl” treatment. #p < 0.05 vs. “sh-C” cells with DEX treatment. Experiments in this figure were repeated four times, and similar results were obtained. Bar = 100 μm (c).

Fig. 4. PGK1 knockout activates Nrf2 signaling and protects osteoblasts from DEX.

Stable OB-6 osteoblastic cells (a–g) or primary human osteoblasts (h–k) with the CRISPR/Cas9-PGK1 knockout construct (“ko-PGK1”) or the CRISPR/Cas9 sgRNA control construct (“sg-C”) were established, expression of listed genes (a, b, h) was shown. The relative NQO1 activity was also tested (c). Cells were further treated with or without DEX (1 μM) for applied time periods, cellular superoxide levels were shown (d); cell viability (e, i), death (f, j), and apoptosis (g, k) were examined by MTT, LDH release, and Histone-DNA ELISA assays, respectively. Expression of listed proteins was quantified, normalized to the loading control (a, h). Data were expressed as mean ± standard deviation (SD, n = 5). *p < 0.05 vs. “sg-C” cells with “Ctrl” treatment. #p < 0.05 vs. “sg-C” cells with DEX treatment. Experiments in this figure were repeated three times, and similar results were obtained.

Fig. 5. PGK1 depletion-induced osteoblast cytoprotection against DEX is through activation of Keap1-Nrf2 cascade.

OB-6 cells with the CRISPR/Cas9-PGK1-knockout construct (“ko-PGK1”) were further transduced with Nrf2 shRNA (“+sh-Nrf2”) or the non-sense control shRNA (“+sh-C”), stable cells were established via selection by puromycin, relative expression of Nrf2 pathway genes was shown (a, b). The NQO1 activity was also tested (c). Above cells and the control cells (with CRISPR/Cas9 sgRNA control construct/sg-C) were further treated with or without DEX (1 μM) for applied time periods, cell viability (d), death (e) and apoptosis (f) were examined by MTT, LDH release, and Histone-DNA ELISA assays, respectively. Expression of the listed genes in the stable OB-6 cells with the non-sense control shRNA (“sh-C”), the lentiviral Keap1 shRNA (“sh-Keap1”), or together with the PGK1 shRNA (“sh-PGK1-S1”, sh-Keap1 + sh-PGK1) was shown (g, h), the relative NQO1 activity was tested as well (i). The above cells were treated with or without DEX (1 μM) for applied time periods, cell viability (j), death (k), and apoptosis (l) were tested similarly. Expression of listed proteins was quantified, normalized to the loading control (b, g). Data were expressed as mean ± standard deviation (SD, n = 5). *p < 0.05 vs. “ko-PGK1” cells (a, c); *p < 0.05 (d–f); #p < 0.05 vs. “sh-C” cells (h–l). *p < 0.05 vs. “Ctrl” treatment (j–l). p > 0.05 stands for no statistical difference. Experiments in this figure were repeated three times, and similar results were obtained.

Fig. 6. PGK1 downregulation in human necrotic femoral head tissues correlates with HO1 depletion.

mRNA (a, b) and protein (c–e) expression of PGK1 and HO1 in necrotic femoral head tissues (“N”) and surrounding normal femoral head tissues (“S”) of 12 different DEX-taking patients was tested by qPCR and western blotting assays. f The proposed signaling carton of this study. Data were expressed as mean ± standard deviation (SD, n = 12). *p < 0.05 vs. “S” tissues. Experiments in this figure were repeated three times, and similar results obtained.