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. 2020 Apr;53(4):e12789.
doi: 10.1111/cpr.12789. Epub 2020 Mar 11.

Oestrogen-activated autophagy has a negative effect on the anti-osteoclastogenic function of oestrogen

Liang Cheng  1 , Yunrong Zhu  2 , Dianshan Ke  1 , Denghui Xie  1
Affiliations

Oestrogen-activated autophagy has a negative effect on the anti-osteoclastogenic function of oestrogen

Liang Cheng et al. Cell Prolif. 2020 Apr.

Erratum in

Abstract

Objectives: Oestrogen is known to inhibit osteoclastogenesis, and numerous studies have identified it as an autophagic activator. To date, the role of oestrogen in the autophagy of osteoclast precursors (OCPs) during osteoclastogenesis remains unclear. This study aimed to determine the effect of autophagy regulated by the biologically active form of oestrogen (17β-estradiol) on osteoclastogenesis.

Materials and methods: After treatment with 17β-estradiol in OCPs (from bone marrow-derived macrophages, BMMs) and ovariectomy (OVX) mice, we measured the effect of 17β-estradiol on the autophagy of OCPs in vitro and in vivo. In addition, we studied the role of autophagy in the OCP proliferation, osteoclast differentiation and bone loss regulated by 17β-estradiol using autophagic inhibitor or knock-down of autophagic genes.

Results: The results showed that direct administration of 17β-estradiol enhanced the autophagic response of OCPs. Interestingly, 17β-estradiol inhibited the stimulatory effect of receptor activator of nuclear factor-κB ligand (RANKL) on the autophagy and osteoclastogenesis of OCPs. Moreover, 17β-estradiol inhibited the downstream signalling of RANKL. Autophagic suppression by pharmacological inhibitors or gene silencing enhanced the inhibitory effect of 17β-estradiol on osteoclastogenesis. In vivo assays showed that the autophagic inhibitor 3-MA not only inhibited the autophagic activity of the OCPs in the trabecular bone of OVX mice but also enhanced the ability of 17β-estradiol to ameliorate bone loss.

Conclusions: In conclusion, our study showed that oestrogen directly enhanced the autophagy of OCPs, which inhibited its anti-osteoclastogenic effect. Drugs based on autophagic inhibition may enhance the efficacy of oestrogen on osteoporosis.

Keywords: LC3; RANKL; autophagy; oestrogen; osteoclastogenesis.

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Conflict of interest statement

The authors have no competing interests to disclose.

Figures

FIGURE 1
FIGURE 1
Treatment with 17β‐estradiol enhances the autophagic activity of OCPs. A, Following administration of different concentrations of 17β‐estradiol (0, 1, 5 or 10 nmol/L) for 8 h in the absence of RANKL, the Atg5, Atg7 and Beclin1 proteins in BMM‐derived OCPs were detected using Western blot analyses. B, The ratio of LC3II/I in the OCPs treated with 17β‐estradiol (5 nmol/L) for 8 h in the presence or absence of E64d plus PEPS A. C, After the OCPs were treated with the reagents as described for (B) for 12 h, LC3 puncta (red arrows) were imaged using immunofluorescence staining and then observed under fluorescence microscopy. Scale bar, 20 μm. D, Statistical diagram showing the percentages of cells with LC3 puncta in C (≥5 dots, 50 cells per field, n = 5). Data are expressed as the mean ± SEM from three independent experiments. *P < .05. Cont, control group; E, E64D; E2, 17β‐estradiol; ns, no significance; P, PEPS A
FIGURE 2
FIGURE 2
Treatment with 17β‐estradiol inhibits the RANKL‐induced autophagy of OCPs. A, Following administration of different concentrations of 17β‐estradiol (0, 1, 5 or 10 nmol/L) for 8 h in the presence of RANKL, the Atg5, Atg7 and Beclin1 proteins in OCPs were detected using Western blot analyses. B, The efficiency of lentivirus (LV‐BECN1) transduction was verified by detecting BECN1 mRNA level. C, The ratio of LC3II/I and the soluble p62, insoluble p62 proteins in the OCPs treated with 17β‐estradiol (5 nmol/L) and/or RANKL for 8 h after lentiviral transduction. D, Images of LC3 puncta (red arrows) in the OCPs treated with the above reagents for 12 h after lentiviral transduction. Scale bar, 20 μm. E, The percentages of cells with LC3 puncta in D. Data are expressed as the mean ± SEM from three independent experiments. *P < .05. BE, lentivirus encoding BECN1; Cont, control group; E2, 17β‐estradiol; ns, no significance; R, RANKL
FIGURE 3
FIGURE 3
Treatment with 17β‐estradiol suppresses the TRAF6‐MAPK signalling pathway downstream of RANKL. A and B, OCPs were treated with or without RANKL for the indicated times. TRAF6, p‐ERK, p‐JNK and p‐P38 were detected using Western blot analyses. The histograms represent the comparisons of the expression levels of each protein between the RANKL‐lacking group and the RANKL group at the indicated time points. The expression of phosphorylated proteins is represented by the ratio of phosphorylated protein to total protein. C, OCPs were treated with or without RANKL for 2 h. Cell lysates were extracted for Co‐IP with anti‐RANK or anti‐TRAF6 antibody, and subsequently, precipitates were detected using Western blots with anti‐TRAF6 or anti‐RANK antibody, respectively. Data are expressed as the mean ± SEM from three independent experiments. *P < .05. E2, 17β‐estradiol; IB, the antibody for immunoblot; IP, the antibody for immunoprecipitation; ns, no significance; R, RANKL
FIGURE 4
FIGURE 4
Suppression of autophagic activity promotes the inhibitory effect of 17β‐estradiol on osteoclastogenesis. A, The proliferation of the OCPs treated with 17β‐estradiol (0, 1, 5 or 10 nmol/L) for the indicated times was detected by CCK‐8 assays. B, The proliferation of the OCPs treated with 17β‐estradiol (0, 1, 5 or 10 nmol/L) along with PBS or 3‐MA (0.5 μmol/L) for 24 h was detected by CCK‐8 assays. C, The OCPs were treated with M‐CSF plus RANKL along with PBS, 3‐MA (0.5 μmol/L) and/or 17β‐estradiol (5 nmol/L) for 5 d. Representative images of TRAP‐positive multinucleated cells in each group. Scale bar, 200 μm. D, The quantitative results showed the number of TRAP‐positive multinucleated cells in C. E, The Atg5, Atg7 and BECN1 mRNA levels in the OCPs infected with lentiviruses encoding Atg5‐shRNA, Atg7‐shRNA, or BECN1‐shRNA (LV‐sh‐Atg5, LV‐sh‐Atg7, LV‐sh‐BECN1) or control viruses (LV‐sh‐cont). F, Following lentiviral transduction, the ratio of LC3II/I in the OCPs treated with 17β‐estradiol (5 nmol/L) in the presence of RANKL for 8 h was detected using Western blot analyses. G, Typical images of TRAP staining of the mature osteoclasts derived from the OCPs transduced with lentiviruses followed by M‐CSF plus RANKL along with PBS or 17β‐estradiol (5 nmol/L) treatment for 5 days. Scale bar, 200 μm. H and I, The quantitative results regarding mature osteoclasts (more than 3 nuclei) or large osteoclasts (more than 5 nuclei) in G. Data are expressed as the mean ± SEM from three independent experiments. *P < .05. E2, 17β‐estradiol; M, M‐CSF; ns, no significance; R, RANKL
FIGURE 5
FIGURE 5
Treatment with 3‐MA increases the inhibitory effect of 17β‐estradiol on LC3 fluorescence intensity in trabecular OCPs in OVX mice. The OVX‐operated 10‐wk‐old female mice were treated with PBS or 3‐MA (30 mg/kg, i.p. for 60 d) and/or 17β‐estradiol (20 μg/kg, s.c. for 60 d). A, Tibial sections were stained with green and red fluorochromes for LC3B and RANK, respectively, and observed using fluorescence microscopy. The overlapping staining of LC3B and RANK are indicated with red arrows (yellow fluorescence). Scale bar, 2.5 μm. B, Statistical diagram indicates the quantitative results of IF assays (the ratio of RANK+ LC3+ cells/RANK+ cells; 100 cells per mouse were counted). C, Images of LC3 puncta (red arrows) in bone marrow RANK+ CD11b+ cells sorted by flow cytometry. D, The percentages of cells with LC3 puncta in C (N = 3). E2, 17β‐estradiol; OVX, ovariectomized mice
FIGURE 6
FIGURE 6
Treatment with 3‐MA enhances the ability of 17β‐estradiol to ameliorate bone loss and decrease osteoclastogenesis in OVX mice. The OVX‐operated 10‐wk‐old female mice were treated with PBS or 3‐MA (30 mg/kg, i.p. for 60 d) and/or 17β‐estradiol (20 μg/kg, s.c. for 60 d). A, Representative 3D micro‐CT reconstructed images of the tibiae from each group. Scale bar, 100 μm. B, Representative H&E‐stained tibial sections from each group. Scale bar, 20 μm. C, Representative TRAP‐stained tibial sections from each group (red arrows indicate TRAP+ cells). Scale bar, 5 μm. D, Representative IHC‐stained tibial sections of OCN from each group. E‐H, The trabecular bone parameters, including BMD, BV/TV, Tb.N and Tb.Sp, were analysed using micro‐CT (N = 8). I, The trabecular bone parameter, Tb.Ar, was analysed by H&E staining and using the IPP system (N = 8). J, The number of osteoclasts per millimetre of trabecular bone surface was counted (N = 8). K, Statistical diagram indicates the quantitative results of IHC assays (N = 6). Data are expressed as the mean ± SEM. *P < .05. E2, 17β‐estradiol; ns, no significance; OVX, ovariectomized mice
FIGURE 7
FIGURE 7
The working model regarding oestrogen‐regulated autophagy during osteoclastogenesis. In brief, autophagy plays a significant role in the formation of mature osteoclasts derived from OCPs. Oestrogen directly activates autophagy in OCPs, which contributes to OCP proliferation. However, oestrogen inhibits osteoclast differentiation by inhibiting RANKL downstream signalling, in which the inhibition of autophagy activated by RANKL signalling is also an important pathway. Accordingly, autophagy inhibition enhances the inhibitory effect of oestrogen on osteoclastogenesis. OC, osteoclast; OCP, osteoclast precursor; formula image promote; formula image inhibit; formula image synergy
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