Andrographolide prevents bone loss via targeting estrogen-related receptor-alpha-regulated metabolic adaption of osteoclastogenesis

Background and Purpose: Metabolic adaptation driven by oestrogen-related receptor-alpha (ERR alpha/NR3B1) is required to meet the increased energy demand during osteoclast differentiation. Here, we hypothesize that natural product, andrographolide, acts as an ERR alpha inverse agonist to inhibit osteoclastogenesis. Experimental Approach: Virtual docking and site-directed mutagenesis analysis were employed to study the binding mode of andrographolide to ERR alpha. Co-immunoprecipitation, luciferase reporter assay, real-time polymerase chain reaction (PCR) and immunoblot analyses were performed to identify andrographolide as an ERR alpha inverse agonist. The pharmacological effects of andrographolide in vivo were assessed in mice models of osteopenia induced by either a high-fat diet in male or ovariectomy in female mice. Key Results: ERR alpha-dependent expression of glutaminase, a rate-limiting enzyme of mitochondrial glutamine anaplerosis, is required for ex vivo bone marrow osteoclast differentiation. Andrographolide inhibited glutaminase expression induced by ERR alpha and co-activator peroxisome proliferator-activated receptor gamma co-activator-1 beta (PGC-1 beta), leading to reduction in osteoclastogenesis. Andrographolide acted as an inverse agonist of ERR alpha by disrupting its interaction with co-activator PGC-1 beta. Phenylalanine 232, valine 395 and phenylalanine 399 of ERR alpha ligand-binding domain were confirmed to be essential for this effect. In contrast, glutaminase overexpression restored the impairment triggered by andrographolide. Accordingly, andrographolide suppressed osteoclastic bone resorption and attenuated bone loss in vivo. Conclusions and Implications: These findings demonstrate that andrographolide acts as an ERR alpha inverse agonist for perturbation of ERR alpha/PGC-1 beta/glutaminase axis-driven metabolic adaption during osteoclast differentiation, implying that andrographolide may be a promising natural compound for preventing physiological and pathological bone loss.