Inhibition of SLC40A1 represses osteoblast formation via inducing iron accumulation and activating the PERK/ATF4/CHOP pathway mediated oxidative stress

Objective: This study aimed to investigate the effects of solute carrier family 40 member 1 (SLC40A1) on iron accumulation, oxidative stress and differentiation in osteoblasts and potential mechanisms.

Methods: Mouse preosteoblastic MC3T3-E1 cells were transfected with the SLC40A1 overexpression vector (oeSLC40A1) and siRNA (siSLC40A1), then cell differentiation was induced via ascorbic acid and β-glycerophosphate. Besides, Ferrostatin-1 (Ferroptosis inhibitor) and GSK2606414 (PERK Inhibitor) were added with siSLC40A1.

Results: Fe²⁺, malondialdehyde (MDA), and Reactive Oxygen Species (ROS) were higher but reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio was lower after siSLC40A1 transfection, while reduced Fe²⁺ and ROS but elevated GSH/GSSG ratio was observed after oeSLC40A1 transfection. Alkaline Phosphatase (ALP) staining, Alizarin Red S (ARS) staining, Osteopontin (OPN) and Bone Morphogenetic Protein 2 (BMP2) were lower after siSLC40A1 transfection but were greater after oeSLC40A1 transfection. Furthermore, SLC40A1 negatively regulated the PERK/ATF4/CHOP pathway. Further exploration revealed that Fe²⁺, MDA, ROS, and the PERK/ATF4/CHOP pathway were attenuated, while GSH/GSSG ratio, ALP staining, ARS staining, and OPN expression were increased after ferrostatin-1 treatment in the siSLC40A1-transfected cells. Similar trends were observed with respect to GSK2606414 treatment with siSLC40A1.

Conclusion: SLC40A1 inhibition suppresses osteoblast formation by facilitating iron accumulation and activating the PERK/ATF4/CHOP pathway-mediated oxidative stress.