Volume 51, March 2025, Pages 312-328Author links open overlay panel, , , , , , , , , , , , , Highlights

•Through human proteomics, COPB1 has been identified as a potential key target for osteoporosis caused by iron accumulation.

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COPB1 in osteoblasts has a fundamental role in bone homeostasis and its deletion induces osteopenia.

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COPB1-ATF6-SLC7A11 is a key pathway linking endoplasmic reticulum stress and ferroptosis.

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Overexpression of COPB1 can prevent osteopenia caused by iron accumulation or ovariectomy.

AbstractBackground

Osteoporosis (OP) is a systemic bone metabolic disease that results from an imbalance between bone formation and bone resorption. The accumulation of iron has been identified as an independent risk factor for osteoporosis. Ferroptosis, a novel form of programmed cell death, is driven by iron-dependent lipid peroxidation. Nevertheless, the precise role of ferroptosis in iron accumulation-induced osteoporosis remains uncertain.

Methods

We utilized proteomics and ELISA to screen key regulatory molecules related to iron accumulation in osteoporosis populations. HE staining was used to assess osteocyte changes in Hamp knockout (KO) iron accumulation mouse models. Western Blot, qPCR, ALP staining, and Alizarin Red staining were employed to explore the effects of siRNA-mediated gene knockdown on osteogenic differentiation in the MC3T3 cell line. ELISA, micro-CT, von Kossa staining, toluidine blue staining, TRAP staining, and calcein analysis were used to study the bone phenotype of conditional gene knockout mice. RNA-seq, endoplasmic reticulum activity probes, transmission electron microscopy (TEM), Western Blot, co-immunoprecipitation (Co-IP), flow cytometry, and ChIP-seq were employed to investigate the regulatory mechanisms of the target gene in osteogenic differentiation. OVX and Hamp KO mice were used to establish osteoporosis models, and AAV-mediated overexpression was employed to explore the intervention effects of the target gene on osteoporosis.

Results

The experiments demonstrate that iron accumulation can lead to changes in COPB1 expression levels in bone tissue. Cellular and animal experiments revealed that COPB1 deficiency reduces the osteogenic ability of osteoblasts. Transcriptome analysis and phenotypic experiments revealed that COPB1 deficiency induces ferroptosis and endoplasmic reticulum stress in cells. Further investigation confirmed that COPB1 plays a key role in endoplasmic reticulum stress by inhibits SLC7A11 transcription via ATF6. This reduces cystine uptake, ultimately inducing ferroptosis. Overexpression of COPB1 can restore osteogenic function in both cells and mice.

Conclusion

This study elucidated the essential role of COPB1 in maintaining bone homeostasis and highlights it as a potential therapeutic target for treating iron accumulation-related osteoporosis.

The translational potential of this article

Our data elucidate the critical role of COPB1 in maintaining bone homeostasis and demonstrate that COPB1 can directly promote bone formation, making it a potential therapeutic target for the future treatment of osteoporosis.

Graphical abstractDownload: Download high-res image (214KB)Download: Download full-size imageKeywords

Osteoblast

Ferroptosis

Er stress

Proteomics

Targeted therapy

Abbreviations(ALP)

Alkaline Phosphatase

(ATF6)

Activating Transcription Factor 6

(α-MEM)

Alpha Minimum Essential Medium

(BMP2)

Bone Morphogenetic Protein 2

(Cas8)

Cysteine-aspartic Acid Protease 8

(CCK-8)

Cell Counting Kit-8

(COPB1)

Coatomer Protein Complex Subunit Beta 1

(COPI)

Coat Protein Complex I

(Cl-Cas3)

Cleaved Cysteine-aspartic Acid Protease 3

(ELISA)

Enzyme-Linked Immunosorbent Assay

(ER)

Endoplasmic Reticulum

(FAC)

Ferri Ammonii Citras

(GPX4)

Glutathione Peroxidase 4

(HBSS)

Hank's Balanced Salt Solution

(H&E)

Hematoxylin and eosin staining

(KEGG)

Kyoto Encyclopedia of Genes and Genomes

(LC3A/B)

Microtubule-Associated Protein 1 Light Chain 3A/B

(PBS)

Phosphate-Buffered Saline

(Ptgs2)

Prostaglandin-Endoperoxide Synthase 2

(P-IRE1)

Phosphorylated Inositol-Requiring Enzyme 1

(P-PERK)

Phosphorylated Protein Kinase RNA-like Endoplasmic Reticulum Kinase

(PS)

Penicillin-Streptomycin

(P-SMAD1/5)

Phosphorylated SMAD Family Member 1/5

(qPCR)

quantitative Polymerase Chain Reaction

(RIPA)

Radio-Immunoprecipitation Assay

(ROS)

Reactive Oxygen Species

(RUNX2)

Runt-related Transcription Factor 2

(SLC7a11)

Solute Carrier Family 7 Member 11

(SMAD5)

SMAD Family Member 5

(SP7/OSX/OSTERIX)

Sp7 Transcription Factor

(TRIzol)

Total RNA Isolation Reagent

© 2025 The Authors. Published by Elsevier B.V. on behalf of Chinese Speaking Orthopaedic Society.