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. 2020 Mar 27:11:360.
doi: 10.3389/fphar.2020.00360. eCollection 2020.

Pregnenolone Inhibits Osteoclast Differentiation and Protects Against Lipopolysaccharide-Induced Inflammatory Bone Destruction and Ovariectomy-Induced Bone Loss

Xiaochen Sun  1 , Chenxi Zhang  2 , Huan Guo  2 , Jiao Chen  2 , Yali Tao  2 , Fuxiao Wang  2 , Xixi Lin  1 , Qian Liu  1 , Li Su  2 , An Qin  1   3
Affiliations

Pregnenolone Inhibits Osteoclast Differentiation and Protects Against Lipopolysaccharide-Induced Inflammatory Bone Destruction and Ovariectomy-Induced Bone Loss

Xiaochen Sun et al. Front Pharmacol. .

Abstract

Osteolytic bone disease is characterized by excessive osteoclast bone resorption leading to increased skeletal fragility and fracture risk. Multinucleated osteoclasts formed through the fusion of mononuclear precursors are the principle cell capable of bone resorption. Pregnenolone (Preg) is the grand precursor of most if not all steroid hormones and have been suggested to be a novel anti-osteoporotic agent. However, the effects of Preg on osteoclast biology and function has yet to be shown. Here we examined the effect of Preg on receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast formation and bone resorption in vitro, and potential therapeutic application in inflammatory bone destruction and bone loss in vivo. Our in vitro cellular assays demonstrated that Preg can inhibit the formation of TRAP+ve osteoclast formation as well as mature osteoclast bone resorption in a dose-dependent manner. The expression of osteoclast marker genes CTSK, TRAP, DC-STAMP, ATP6V0d2, and NFATc1 were markedly attenuated. Biochemical analyses of RANKL-induced signaling pathways showed that Preg inhibited the early activation of extracellular regulated protein kinases (ERK) mitogen-activated protein kinase (MAPK) and nuclear factor-κB, which consequently impaired the downstream induction of c-Fos and NFATc1. Using reactive oxygen species (ROS) detection assays, we found that Preg exhibits anti-oxidant properties inhibiting the generation of intracellular ROS following RANKL stimulation. Consistent with these in vitro results, we confirmed that Preg protected mice against local Lipopolysaccharide (LPS)-induced inflammatory bone destruction in vivo by suppressing osteoclast formation. Furthermore, we did not find any observable effect of Preg on osteoblastogenesis and mineralization in vitro. Finally Preg was administered to ovariectomy (OVX)-induced bone loss and demonstrated that Preg prevented systemic OVX-induced osteoporosis. Collectively, our observations provide strong evidence for the use of Preg as anti-osteoclastogenic and anti-resorptive agent for the potential treatment of osteolytic bone conditions.

Keywords: ERK; LPS (lipopolysaccharide); OVX model; RANKL (receptor activator of nuclear factor kappa-B ligand); osteoclast (OCs).

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Figures

Figure 1
Figure 1
Cytotoxicity of pregnenolone (Preg) on osteoclast precursor cells (A) Chemical structure of Preg. (B, C) BMM cell viability as assessed by CCK-8 assay following treatment without or with indicated concentrations of Preg for (B) 48 and (C) 96 h. Data presented as mean ± standard deviation (n = 3).
Figure 2
Figure 2
Pregnenolone (Preg) dose-dependently inhibits RANKL-induced osteoclast differentiation and gene expression in vitro. (A) Representative TRAP stained images of BMM-derived osteoclasts stimulated with RANKL for without or with indicated concentrations of Preg. The (B) number and (C) size (cell spread area) of TRAP+ve multinucleated osteoclasts with three or more nuclei were quantified. (D) The relative expression of osteoclast marker genes (TRAP, CTSK, DC-STAMP, NFATc1, ATP6V0d2, and c-Fos) following Preg treatment were quantified by real time PCR. Values presented as the mean ± standard deviation (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3
Figure 3
Pregnenolone (Preg) attenuated RANKL-induced osteoclast fusion and bone resorption. (A) Representative fluorescence images of actin stained BMM-derived osteoclasts stimulated with RANKL for 7 d with RANKL without or with 10 and 20 μM Preg. Actin cytoskeleton were stained Phalloidin-iFluor 488 (green) and nuclei with DAPI (blue). The (B) average cell size (cell spread area based on actin podosomal belt) and (C) average number of nuclei per osteoclasts were quantified. (D) Representative images of bone resorption by mature osteoclasts in the absence or presence of Preg. Pre-osteoclasts stimulated with RANKL for 3 d were reseeded onto hydroxyapatite-coated OsteoAssay plates and then treated without or with indicated concentrations of Preg for further 3 d. Cells were removed with sodium hypochlorite, and resorption pits were imaged under light microscopy. (E) The resorption area expressed as a percentage of total well area for each condition were quantified. Values presented as the mean ± standard deviation (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4
Figure 4
Pregnenolone (Preg) suppressed RANKL-induced intracellular ROS production. (A, B) BMMs were stimulated with RANKL in the absence or presence of indicated concentrations of Preg for 3 d and intracellular ROS was detected by the intracellular conversion of non-fluorescent DCFH-DA to highly fluorescent DCF. BMMs stimulated with M-CSF only was used as negative control. (C) The number of ROS positive cells were quantified. (D) The mean value of DCFH-DA were calculated. Values presented as the mean ± standard deviation (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5
Figure 5
Pregnenolone (Preg) inhibited the RANKL-induced activation of ERK and NF-κB signaling cascades, and the downstream induction of c-Fos and NFATc1. (A) To examine early RANKL signaling events, BMMs were serum-starved for 1 h, pre-treated with 20 μM Preg for 3 h, and then stimulated with RANKL for the indicated time. Total cellular protein were extracted and subjected to western blot analyses using specific antibodies against ERK and p-ERK, p38 and p-p38, JNK and p-JNK, IκBα, NF-κB p65 and p-p65, and β-actin. (B–F) Quantitative densitometric analysis of phosphorylated protein to total protein counterpart, or normalized to β-actin were conducted. (G) To examine downstream (late stage) RANLK signaling events, BMMs were treated with RANKL for 0, 1, 3, and 5 d. M-CSF-dependent BMMs were stimulated with RANKL without or with 10 and 20 μM Preg for 72 h. Total cellular proteins were extracted and subjected to western blot analyses using specific antibodies against NFATc1, c-Fos, and GAPDH. (H, I) Quantitative densitometric analysis of (h) NFATc1 and (i) c-Fos normalized to GAPDH were conducted. Values presented as the mean ± standard deviation (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6
Figure 6
Pregnenolone (Preg) protects against LPS-induced inflammatory osteolysis of mouse calvarium in vivo. (A) Representative 3D μCT reconstructions of mouse calvarium from each treatment group. All subcutaneous injections were conducted over the sagittal midline suture of the calvarium. sham and LPS (5 mg/kg body weight) received injections of PBS and LPS respectively. Preg treatment groups received injections of LPS and Preg (low—1 mg/kg or high—10 mg/kg) together. Injection were carried out every day over a 7 d period after which all mice were sacrificed. (B) Representative hematoxylin-eosin staining (H&E) (100X magnification) and TRAP (100X magnification) stained sections from each treatment group. Calvarial bones were decalcified in 10% EDTA for 2 weeks, embedded in paraffin for histological sectioning, and then stained with H&E and TRAP. (C–E) Quantitative analysis of (C) bone volume to total tissue volume (BV/TV), (D) percentage of porosity, and (E) the number of TRAP+ve cells were quantified. Values presented as the mean ± standard deviation (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 7
Figure 7
Pregnenolone (Preg) does not no affect the osteogenic differentiation and mineralization of BMSC-derived osteoblasts. (A) BMSC cell viability as assessed by CCK-8 assay following treatment without or with indicated concentrations of Preg for 48 and 96 h. (B) ALP and alizarin red S staining of BMSC-derived osteoblasts following osteogenic differentiation without or with 10 and 20 μM Preg for 7 and 21 d respectively. (C, D) ALP and mineralization activity relative to untreated controls were quantified. (E) The relative expression of OPG and RANKL in bone samples were quantified by real time PCR. Values presented as the mean ± standard deviation (n = 3); *p < 0.05.
Figure 8
Figure 8
Pregnenolone (Preg) prevents bone loss in ovariectomized (OVX) mice in vivo. (A) Representative 3D μCT reconstructions of mouse tibial bone from sham (PBS injection), OVX (PBS injection), OVX with 1 mg/kg Preg (low dose), and OVX with 10 mg/kg Preg (high dose). (B) Quantitative bone morphometric parameters of bone volume to total tissue volume (BV/TV), bone surface to tissue volume (BS/TV, mm−1), trabecular spacing (Tb.Sp., mm), trabecular number (Tb.N., mm−1), cortical thickness (Ct.Th), and cortical bone mineral content (Ct.BMC) were measured. (C) Representative histological assessment of tibial bone sections stained for H&E and TRAP activity (40X and 100X magnification) to assess osteoclast activity. Tibial bone samples were decalcified in 10% EDTA for 2 weeks, embedded in paraffin blocks, sectioned and then stained with H&E and TRAP. (D, E) Quantitative assessment of (D) the total number of TRAP+ve cells and (E) the number of osteoclast per bone surface were conducted. Values presented as the mean ± standard deviation (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.
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