COPB1 deficiency triggers osteoporosis with elevated iron stores by inducing osteoblast ferroptosis

Abstract

Background: 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.

Keywords: Er stress; Ferroptosis; Osteoblast; Proteomics; Targeted therapy.

Graphical abstract

Fig. 1

Correlation between Copb1 and iron accumulation in osteoporosis. a. Schematic diagram representing bone tissue and serum in the osteoporosis with elevated iron stores group and the normal iron and bone mass group. b. GO pathway enrichment analysis of differentially expressed proteins between the IOP and P groups (n = 5). c. Volcano plot showing differentially expressed proteins between the IOP group and the N group (n = 5). d. Correlation analysis of hip joint T-scores, serum ferritin, and COPB1 protein levels (Pearson correlation analysis) (n = 10). e. Elisa analysis of serum COPB1 levels between the IOP group (n = 10) and the N group (n = 15). f. qPCR analysis of COPB1 compared to the control group (n = 3). g. Representative images and quantification of femoral COPB1 IHC staining in Hamp-ko mice and WT mice (n = 3) (scale bar, bottom right).ns = not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 2

Copb1 deficiency inhibits in vitro osteogenesis. a-d. qPCR analysis of osteogenesis-related genes (Alpl, Runx2, Osterix, Ocn) in MC3T3-E1 cells in the control group and knockdown group after 7 days of osteogenic differentiation induction. e-f. Western Blot analysis and quantitative statistical analysis of osteogenesis-related proteins (ALPL, RUNX2, OSTERIX, OCN) in MC3T3-E1 cells in the control group and knockdown group after 7 days of osteogenic differentiation induction. g-h. Western Blot analysis and quantitative statistical analysis of osteogenesis-related pathway proteins (BMP2, SMAD5, P-SMAD1/5) in MC3T3-E1 cells in the control group and knockdown group after 7 days of osteogenic differentiation induction. i. Representative images of alkaline phosphatase staining of MC3T3-E1 cells in the control group and knockdown group after 7 days of osteogenic differentiation induction (scale bar 200 μm). j. Representative images of alizarin red staining of MC3T3-E1 cells in the control group and knockdown group after 14 days of osteogenic differentiation induction, with quantitative analysis performed using a microplate reader after dissolving the stain (scale bar 200 μm). n = 3. Compared to the control group, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 3

COPB1 deletion in osteoblasts leads to bone loss. a. Schematic diagram of COPB1 CKO mouse breeding. b. Serum levels of type I collagen amino-terminal propeptide (P1NP) in 2-month-old and 6-month-old COPB1 CKO and WT male mice (n = 3). c-d. Quantitative analysis of maximal load, fracture energy, and stiffness in the three-point bending test of tibia bone midshafts from 2-month-old and 6-month-old male mice (n = 3). e, g. Histomorphometric analysis of trabecular bone in 2-month-old and 6-month-old COPB1 CKO and WT male mice, including bone mineral density (BMD), bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) (n = 6). f. Representative micro-CT images of whole femurs (bottom) and trabecular bone (bottom) from 6-month-old COPB1 CKO and WT male mice. h. Representative images and quantitative analysis of Von Kossa staining in 6-month-old COPB1 CKO and WT male mice (n = 3) (scale bar in the bottom left). i. Representative images of toluidine blue staining in 6-month-old COPB1 CKO and WT male mice (n = 3) (scale bar in the bottom left). j. Representative images and quantitative analysis of calcein double labeling in 6-month-old COPB1 CKO and WT male mice (n = 3) (scale bar 100 μm).Compared to the control group, ns = not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 4

COPB1 deficiency induces endoplasmic reticulum stress and ferroptosis. a. Volcano plot showing differentially expressed genes in MC3T3-E1 cells between the control group and the COPB1 knockdown group(n = 3). b. KEGG pathway enrichment analysis of differentially expressed proteins between the NC and si-COPB1 groups (n = 3). c. qPCR analysis of the ferroptosis-related gene (Ptgs2) and CCK-8 assay for live cell count after 3 days of Copb1 knockdown in MC3T3-E1 cells (n = 3). d, f. Endoplasmic reticulum fluorescence intensity detected and quantitatively analyzed using an endoplasmic reticulum fluorescent probe in MC3T3-E1 cells after 3 days of Copb1 knockdown (n = 3, blue indicates nucleus, red indicates endoplasmic reticulum). e-f. Western Blot analysis and quantitative statistical analysis of GPX4, ATF6, P-IRE1, and P-PERK expression levels in MC3T3-E1 cells in the control and knockdown groups (n = 3). g. Microplate reader detection of MDA content in MC3T3-E1 cells in the control and knockdown groups (n = 3). h. Transmission electron microscopy observation of endoplasmic reticulum and mitochondrial morphology in MC3T3-E1 cells after 3 days of Copb1 knockdown (n = 3) (scale bar 200 nm, 500 nm). i. Flow cytometry detection and quantitative analysis of LPO content in MC3T3-E1 cells in the control and knockdown groups (n = 3). j. Flow cytometry detection and quantitative analysis of ROS content in MC3T3-E1 cells in the control and knockdown groups (n = 3). Compared to the control group, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001.

Fig. 5

COPB1 induces ferroptosis through endoplasmic reticulum stress. a. Western Blot analysis and quantitative statistical analysis of SLC7A11 expression levels in MC3T3-E1 cells in the control and knockdown groups (n = 3). b. Microplate reader detection of cystine transport efficiency in MC3T3-E1 cells in the control and knockdown groups (n = 3). c. CO-IP results of COPB1 using ATF6 antibody. d. Western Blot analysis and quantitative statistical analysis of SLC7A11 expression levels in MC3T3-E1 cells after ATF6 knockdown (n = 3). e. CHIP-seq peaks of ATF6 enrichment in the DNA region of SLC7A11. f. Western Blot analysis and quantitative statistical analysis of SLC7A11 and GPX4 expression levels in MC3T3-E1 cells in the control, knockdown, and rescue groups (n = 3). g, i. Flow cytometry detection and quantitative analysis of live and dead cell numbers in MC3T3-E1 cells after overexpression of COPB1 and addition of Erastin (n = 3). (NC represents the control group, OE represents the COPB1 overexpression group, Era represents the addition of Erastin, and Era + OE represents the group with both addition of Erastin and COPB1 overexpression.)h, i. Flow cytometry detection and quantitative analysis of live and dead cell numbers in MC3T3-E1 cells after overexpression of COPB1 and addition of RSL3 (n = 3). (NC represents the control group, OE represents the COPB1 overexpression group, RSL3 represents the addition of RSL3, RSL3+OE represents the group with both addition of RSL3 and COPB1 overexpression.).

Fig. 6

Overexpression of COPB1 restores bone loss induced by iron accumulation. a, c. Western Blot analysis and quantitative statistical analysis of ALPL, RUNX2, and OSX expression levels in MC3T3-E1 cells in the control, COPB1 overexpression, iron accumulation, and groups (n = 3). (NC represents the control group, OE represents the COPB1 overexpression group, FAC represents the intracellular iron accumulation group, and FAC + OE represents the group with both intracellular iron accumulation and COPB1 overexpression.)b, c. Western Blot analysis and quantitative statistical analysis of BMP2, P-SMAD1/5, and SMAD5 expression levels in MC3T3-E1 cells in the control, overexpression, iron accumulation, and rescue groups (n = 3). (NC represents the control group, OE represents the COPB1 overexpression group, FAC represents the intracellular iron accumulation group, and FAC + OE represents the group with both intracellular iron accumulation and COPB1 overexpression.)d. Schematic diagram of the rescue experiment in Hamp−/− mice. e. Representative micro-CT images of whole femurs (bottom) and trabecular bone (bottom) from 4-month-old Ctrl, Hamp−/−, and Rescue male mice(Rescue represents COPB1 overexpression.). f. Histomorphometric analysis of trabecular bone in 4-month-old Ctrl, Hamp−/−, and Rescue male mice, including bone mineral density (BMD), bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) (n = 6) (Rescue represents COPB1 overexpression.).