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

Redox Rep. 2024 Dec;29(1):2428147. doi: 10.1080/13510002.2024.2428147. Epub 2024 Nov 28.

Abstract

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.

Keywords: GSK2606414; PERK/ATF4/CHOP pathway; SLC40A1; ferrostatin-1; iron accumulation; osteoblast differentiation; osteoblast formation; oxidative stress.

MeSH terms

  • Activating Transcription Factor 4* / genetics
  • Activating Transcription Factor 4* / metabolism
  • Animals
  • Cation Transport Proteins* / genetics
  • Cation Transport Proteins* / metabolism
  • Cell Differentiation / drug effects
  • Ferroportin
  • Iron* / metabolism
  • Mice
  • Osteoblasts* / drug effects
  • Osteoblasts* / metabolism
  • Oxidative Stress* / drug effects
  • Reactive Oxygen Species / metabolism
  • Signal Transduction / drug effects
  • Transcription Factor CHOP* / genetics
  • Transcription Factor CHOP* / metabolism
  • eIF-2 Kinase* / genetics
  • eIF-2 Kinase* / metabolism

Substances

  • Activating Transcription Factor 4
  • Iron
  • Ferroportin
  • eIF-2 Kinase
  • Transcription Factor CHOP
  • Cation Transport Proteins
  • Reactive Oxygen Species
  • PERK kinase
  • Atf4 protein, mouse
  • Ddit3 protein, mouse

Grants and funding

This study was supported by Inner Mongolia Autonomous Region Natural Science Foundation (2022MS08058), Inner Mongolia Autonomous Region Higher Education Scientific Research Project (NJZZ22614), Inner Mongolia Medical University Joint Project (YKD2021LH005), The Affiliated Hospital of Inner Mongolia Medical University Doctoral Scientific Research Startup Fund (2022NYFYBS003), Inner Mongolia Autonomous Region Health Science and Technology Plan Project (202202160), Inner Mongolia Autonomous Region Natural Science Foundation (2022MS08012), Inner Mongolia Medical University Joint Project (YKD2021LH026), Inner Mongolia Medical University Science and Technology Innovation Team (YKD2022TD034), Inner Mongolia Medical University Scholarly Talent Support Program (ZY20243117).