Inhibition of Atg13-mediated autophagy enhances the anti-osteoclastogenic effect of sirolimus by counteracting its pro-autophagic activity

Abstract

Sirolimus can inhibit osteoclastogenesis. But sirolimus-activated autophagy is a favorable factor for osteoclastogenesis. This study aimed to explore the significance of autophagy in sirolimus-regulated osteoclastogenesis. Our results confirmed that sirolimus inhibited osteoclastic differentiation (including the number and size of osteoclasts as well as the expression of osteoclastic genes) and promotes osteoclast precursor (OCP) autophagy (including LC3 conversion and autophagosome/autolysosome formation). As expected, OCP autophagy (including LC3 conversion and LC3-puncta formation) promoted by sirolimus was reversed by autophagy inactivation with 3-MA or Atg13 silencing. Importantly, compared with single intervention of sirolimus, the combination of sirolimus and 3-MA or Atg13 silencing more effectively inhibited osteoclastic differentiation and OCP proliferation. In vivo experiments also demonstrated that the combination of sirolimus and Atg13-silencing adeno-associated viruses (AAVs) was more effective than sirolimus alone in improving decreased bone density and damaged bone microstructure (including Micro-CT imaging results, bone tissue parameters and trabecular area), and attenuating osteoclastic activity (including the abundance of osteoclasts in trabecular bones and the production of osteoclastic markers in serum) in ovariectomized (OVX) mice. In conclusion, repressing Atg13-related autophagy can effectively enhance the function of sirolimus in inhibiting osteoclastogenesis by counteracting its pro-autophagic activity. Therefore, the combination of sirolimus and Atg13-targeting therapy is expected to enhance the efficacy of sirolimus in ameliorating osteoclastic osteoporosis.