SIS3 suppresses osteoclastogenesis and ameliorates bone loss in ovariectomized mice by modulating Nox4-dependent reactive oxygen species

Osteoporosis is a metabolic disorder of reduced bone mass, accompanied by the deterioration of the bone microstructure, resulting in increased brittleness and easy fracture. Its pathogenesis can be explained by mainly excessive osteoclast formation or bone resorption hyperfunction. Oxidative stress is intricately linked with bone metabolism, and the maturation and bone resorption of osteoclasts respond to intracellular ROS levels. SIS3 is a small-molecule compound that selectively suppresses SMAD3 phosphorylation in the TGF-β/Smad signaling pathway and attenuates the ability to bind to target DNA. Several studies have reported that SMAD3 plays a significant role in bone metabolism. However, whether SIS3 can modulate bone metabolism by affecting osteoclastogenesis and the specific molecular mechanisms involved remain unknown. Here, we demonstrated that SIS3 could suppress osteoclastogenesis and ameliorate bone loss in ovariectomized mice. Mechanistically, SIS3 inhibited SMAD3 phosphorylation in BMMs, and the deficiency of phosphorylated SMAD3 downregulated ROS production and Nox4-dependent expression during osteoclast formation, thereby blocking MAPK phosphorylation and the synthesis of downstream osteoclast marker proteins. Similarly, NOX4 plasmid transfection significantly alleviated osteoclast formation inhibited by SIS3. In addition, we identified the interaction region between SMAD3 and NOX4 by ChIP and dual luciferase reporter assays. Collectively, we found that SIS3 could inhibit SMAD3 phosphorylation, reduce Nox4-dependent ROS generation induced by RANKL, and prevent osteoclast differentiation and maturation, making it a promising alternative therapy for osteoporosis.