Nrf2 epigenetic derepression induced by running exercise protects against osteoporosis

Abstract

Osteoporosis (OP) is a common skeletal disease involving low bone mineral density (BMD) that often leads to fragility fracture, and its development is affected by multiple cellular pathologies and associated with marked epigenetic alterations of osteogenic genes. Proper physical exercise is beneficial for bone health and OP and reportedly possesses epigenetic modulating capacities; however, whether the protective effects of exercise on OP involve epigenetic mechanisms is unclear. Here, we report that epigenetic derepression of nuclear factor erythroid derived 2-related factor-2 (Nrf2), a master regulator of oxidative stress critically involved in the pathogenesis of OP, mediates the significant osteoprotective effects of running exercise (RE) in a mouse model of OP induced by ovariectomy. We showed that Nrf2 gene knockout (Nfe2l2^-/-) ovariectomized mice displayed a worse BMD reduction than the controls, identifying Nrf2 as a critical antiosteoporotic factor. Further, femoral Nrf2 was markedly repressed with concomitant DNA methyltransferase (Dnmt) 1/Dnmt3a/Dnmt3b elevations and Nrf2 promoter hypermethylation in both patients with OP and ovariectomized mice. However, daily 1-h treadmill RE significantly corrected epigenetic alterations, recovered Nrf2 loss and improved the femur bone mass and trabecular microstructure. Consistently, RE also normalized the adverse expression of major osteogenic factors, including osteoblast/osteoclast markers, Nrf2 downstream antioxidant enzymes and proinflammatory cytokines. More importantly, the RE-conferred osteoprotective effects observed in the wild-type control mice were largely abolished in the Nfe2l2^-/- mice. Thus, Nrf2 repression due to aberrant Dnmt elevation and subsequent Nrf2 promoter hypermethylation is likely an important epigenetic feature of the pathogenesis of OP, and Nrf2 derepression is essential for the antiosteoporotic effects of RE.

Fig. 1

Nrf2 is a critical antiosteoporotic factor suppressed in osteoporotic femurs. Representative photomicrographs of (a) H&E-stained (the upper panel, the arrows indicate trabeculae) and micro-CT-scanned (μ-CT, the lower panel) distal femur sections and (b) micro-CT 3D trabecular architecture of distal femurs from the Nrf2 knockout mice (Nfe2l2^−/−) and wild-type littermates (WT) that underwent sham treatment or ovariectomy (Ovx) for 5 weeks (6 mice in each group). c Quantitative analysis of the ratio of sectional trabeculae over total area (Trabe/Total), microstructural bone mineral density (BMD, mg·ccm⁻¹), the ratio of bone volume to tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) of the μ-CT-scanned distal femurs in Fig. 1b. Data are presented as the mean ± SEM. *P < 0.05, two-way ANOVA. d Western blot analysis of Nrf2 and collagen 1 (Col 1) from the femoral homogenates of 3 healthy controls/4 patients with OP, sham/ovariectomized female mice, and young (3 months)/old (25 months) male mice (6 mice in each group, three or two randomly selected samples are shown). β-actin served as the internal control. WB quantifications are shown underneath the blots. Relative values are presented as the mean ± SEM of all human and mouse samples. *P < 0.05, one-way ANOVA. e Representative femur sections of the sham and ovariectomized mice stained for Nrf2 by immunohistochemistry (IHC). The lower panel images are enlarged frames in the upper panel. The positively stained cells in brown are indicated by arrows

Fig. 2

The Nrf2 promoter is hypermethylated in the femurs of osteoporotic patients and ovariectomized mice. a Schematic diagrams of human and murine Nrf2 promoters. The positions of CpG islands (gray area) and MSP/BSP primers are depicted relative to the transcription start site. b Representative agarose gel analyses of MSP products (methylated, unmethylated and input PCR) from the femurs of 4 control individuals and 3 patients with OP (the left panel) and the sham or ovariectomized mice (the right panel, 5 weeks after Ovx, 6 mice in each group, two representative samples are shown). c Quantifications of MSPs of Fig. 2b. Values are presented as percentage ± SEM of methylated/unmethylated PCR over total PCR products after adjustment with input controls from all human samples and experimental mice. *P < 0.05, one-way ANOVA. d Western blots of femoral Dnmt1, Dnmt3a, and Dnmt3b from human and mouse femurs (three randomly selected mouse samples are shown). e Quantifications of Fig. 2d. *P < 0.05, one-way ANOVA. f Representative mouse femur sections stained for Dnmt1, Dnmt3a, and Dnmt3b by immunohistochemistry (IHC). The lower panel images are the enlarged frames of the upper panel. The positively stained cells in brown are indicated by arrows

Fig. 3

Running exercise demethylates the Nrf2 promoter and inhibits aberrant femoral Dnmt expression. Female ICR mice were treated and divided into the sham, Ovx, RE, and RE/Ovx groups (6 mice in each group) as described in the methods. a MSP assay. Representative agarose gel analysis of MSP products. Two samples from each group are shown. b Quantifications of MSP products in Fig. 3a. Values are presented as percentage changes of methylated/unmethylated PCR over total PCR products after adjustment with input controls, *P < 0.05, two-way ANOVA. c BSP assay. Three randomly selected mice (M1, M2, and M3) from each group were analyzed by BSP (the primer positions on the Nrf2 promoter are shown in Fig. 2a, right panel). The PCR products were cloned, and five clones from each PCR were sequenced. One box represents one mouse. Each row of dots in the box represents one single sequenced clone, and each dot represents one CpG site. Empty or dark dots indicate unmethylated or methylated CpGs, respectively. d Quantification of Fig. 3c. Data are presented as the mean percentage ± SEM of methylated CpGs over total CpGs in each group. *P < 0.05, two-way ANOVA. e Western blotting of mouse femoral Dnmt1, Dnmt3a and Dnmt3b. Three randomly selected samples from each group are shown. f Quantifications of Fig. 3e. ^*P < 0.05, one-way ANOVA

Fig. 4

Running exercise derepresses Nrf2 and protects mice from osteoporosis. Female ICR mice were divided into the sham, RE, Ovx and RE/Ovx groups as described in the methods (6 mice in each group). a Representative photomicrographs of H&E-stained (the upper panel), micro-CT-scanned distal femoral trabecula (the middle panel) and cortex (the lower panel). b Three-dimensional microstructural analyses of distal femurs in Fig. 4a. Bone mineral density (BMD, mg·ccm⁻¹), the ratio of bone volume to tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and cortical thickness (Ct.Th) were assessed. Data are presented as the mean ± SEM, *P < 0.05, two-way ANOVA. c Western blotting of Nrf2, collagen 1 (Col 1) and Runx2 from femur homogenates of the experimental mice. Two or three representative samples from each group are shown. d Quantification of Fig. 4c. Data are presented as the mean ± SEM of all experimental mice. *P < 0.05, one-way ANOVA. e Representative microphotographs of immunohistochemistry staining for TRAP and osteocalcin from mouse femur sections. The positively stained cells are indicated by arrows

Fig. 5

Running exercise mitigates the suppression of antioxidant enzymes. a Western blotting of femoral catalase and SOD2 from 3 healthy controls and 4 patients with OP, as well as the sham, ovariectomized and RE/ovariectomized mice (6 mice in each group, two randomly selected samples from each group are shown). b Quantifications of samples in Fig. 5a. *P < 0.05, one-way ANOVA. c The enzyme activities of GPX and SOD were examined from mouse femoral homogenates of the sham, RE, Ovx and RE/Ovx groups. Data are presented as the mean ± SEM of all experimental mice. *P < 0.05, two-way ANOVA

Fig. 6

Nrf2 is essential for the antiosteoporotic effects of running exercise. Nfe2l2^−/− mice and the control littermates (WT) were subjected to the sham, RE, Ovx and RE/Ovx treatments as described in the methods (6 mice in each group). a Representative photomicrographs of micro-CT-scanned femoral trabeculae and cortex. b Quantitation of the samples in Fig. 6a. BMD (mg·ccm⁻¹), the ratio of bone volume to tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and cortical thickness (Ct.Th) were assessed. *P < 0.05, three-way ANOVA with Tukey’s post hoc test. P values for the effect of genotype (P1), running intervention (P2), and the interaction between genotype and running intervention (P3) are indicated. c Western blotting of femoral Nrf2, osteocalcin (OCN), Runx2, TRAP, catalase and SOD2. Two representative samples from each group are shown. d Quantification of the samples in Fig. 6c. e The enzyme activities of GPX and SOD were examined in mouse femoral homogenates. d and e Data are presented as the mean ± SEM of all experimental mice. *P < 0.05, two-way ANOVA with Tukey’s post hoc test

Fig. 7

A schematic diagram of mouse running exercise, Nrf2 promoter demethylation and protection against osteoporosis (OP). Ovariectomized mice display increased femoral Dnmt1/3a/3b (Dnmts), Nrf2 promoter hypermethylation and Nrf2 suppression, which promote oxidative stress (OS), osteoclastogenesis (OCG) and OP (dashed line). Running exercise (RE, solid line) normalizes Dnmt aberrations, resulting in Nrf2 promoter demethylation, Nrf2 recovery and reduced femoral osteoporotic pathologies