ERK inhibits osteoclast differentiation in RAW 264.7 cells through the osteoprotegerin-mediated autophagy

Abstract

Osteoclasts (OCs) are bone-resorbing cells derived from the monocyte/macrophage lineage. The extracellular signal-regulated kinase (ERK) pathway controls cellular responses such as proliferation, differentiation, and survival, including those of OCs. In the present study, ERK inhibitors reduced the proliferation of bone marrow-derived macrophages (BMMs) and RAW 264.7 cells. However, ERK inhibitors decreased OC differentiation in BMMs but increased it in RAW 264.7 cells. ERK downregulation using small interfering RNA transfection also increased the OC differentiation and the expression of receptor activator of nuclear factor-κB, OC-specific markers, and OC-associated transcription factors in RAW 264.7 cells. These findings suggest ERK regulates OC differentiation in RAW 264.7 cells differently than in BMMs. Thus, we further investigated the mechanism by which ERK negatively regulates OC differentiation in RAW 264.7 cells. ERK inhibition decreased the expression of osteoprotegerin (OPG), a negative regulator of OC differentiation. OPG knockdown increased OC formation. ERK inhibitors activated the Akt/mammalian target of the rapamycin (mTOR) signaling pathway while inhibiting unc-51-like autophagy activating kinase 1 (ULK1). This resulted in decreased levels of microtubule-associated protein 1A/1B-light chain 3-II (LC3-II) and increased levels of p62, thereby reducing autophagy. In addition, OPG knockdown reduced autophagy by activating Akt/mTOR and inhibiting ULK1, resulting in decreased LC3-II and accumulated p62. Therefore, ERK inhibition promoted OC differentiation by downregulating OPG-mediated inhibition of osteoclastogenesis and autophagy in RAW 264.7 cells. These findings highlight ERK's complex role in OC differentiation and suggest that understanding ERK's dual impact on OC differentiation can provide insights into novel treatment strategies for bone-related disorders.