Journal: Blood

Article Title: Ferric carboxymaltose increases fracture risk in patients and reduces bone formation in mice with iron deficiency anemia

doi: 10.1182/blood.2025031806

Figure Lengend Snippet: FCM inhibits DMP1 binding to its potential cell surface receptor αVβ3-integrin and signaling. (A) Effect of 200 μg/mL of each IV iron formulation (FCM, FDI, or LMWID) on dose-dependent DMP1 binding to αVβ3-integrin. DMP1 bound to immobilized αVβ3-integrin was quantified immunometrically. Observed concentrations (y-axis) were plotted against increasing DMP1 concentrations (x-axis, ng/mL). (B) Binding of a constant DMP1 concentration (200 ng/mL) to immobilized αVβ3-integrin in the presence of increasing concentrations of FCM, FDI, or LMWID (10-200 μg/mL). (C) DMP1 binding to αVβ3 at pH 6.9, 7.4, and 7.9 in the additional presence of 200 μg/mL FCM or FDI. Binding is shown as a percentage of maximal αVβ3-DMP1 binding obtained at the highest DMP1 concentration in the absence of IV iron for each pH concentration. All data are shown as mean, n = 2. In panels A-C, a linear regression was applied, whereas in panel B, a nonlinear regression model was used. All data are shown as mean ± SD (n = 3). (D) Schematic illustration of the experimental approach used to assess recombinant murine DMP1-mediated signaling in MC3T3-E1 murine osteoblastic precursor cells by phosphoproteomics analysis. D created with BioRender.com . Wagner SA. (2026) https://BioRender.com/enm3jta . Cells were stimulated with 500 ng/mL DMP1 for 5 minutes, harvested in 100mM ammonium bicarbonate buffer, and processed for phosphoproteomics. (E) Phosphoproteomics data were analyzed with PANTHER Pathways. Enriched proteins were compared with the Mus musculus reference list, and the most significantly activated pathways are listed. Pathways further illustrated in panel F are marked in green (integrin signaling pathway) and blue (cytoskeletal regulation by Rho GTPase). (F) Illustration of the MAPK pathway and cytoskeletal regulation by Rho GTPase activated by DMP1 binding to αVβ3-integrin based on phosphoproteomics results. The MAPK signaling pathway and actin polymerization are initiated by the phosphorylation of focal adhesion kinase (FAK). F created with BioRender.com . Wagner SA. (2026) https://BioRender.com/enm3jta . (G) Activation of the MAPK pathway in MC3T3-E1 cells by DMP1 stimulation. Cells were incubated for 5 minutes with 500, 300, and 100 ng/mL of DMP1. FAK inhibitor (FAKi, 1 and 10μM) was used to block the phosphorylation cascade. Cell lysates were analyzed for phosphor-ERK1/2 (P-ERK) and total ERK 1/2 by western blot. α-Tubulin served as a loading control. (H) Representative western blot analysis of P-ERK, total ERK, and α-tubulin in MC3T3-E1 cell lysates after stimulation with 350 ng/mL DMP1 and 200 μg/mL IV iron formulations (FDI, FCM, LMWID, and IS) for 15 minutes. (I) Quantification of the western blot in panel H. (J) Western blot analysis of P-ERK, ERK, and α-tubulin in cell lysates after stimulation of MC3T3-E1 cells with 50 and 100 ng/mL FGF2 with or without 200 μg/mL IV iron formulations (FDI, FCM, and LMWID) for 15 minutes. (K) Representative western blot analysis of P-ERK, total ERK, and α-tubulin in U2OS cell lysates after stimulation with 500 ng/mL recombinantly expressed human C-terminal DMP1 and 200 μg/mL IV iron formulations (FDI, FCM, and LMWID) for 15 minutes. (L) RNA sequencing analysis of femora (bone marrow free) from mice aged 13 weeks, 1 week after 0.5 mg IV iron treatment, comparing Cntrl, IDA + NaCl, IDA + FCM, and IDA + FDI. Gene ontology analysis identified the integrin pathway as differentially regulated between groups. The heat map represents detected genes within this pathway, shown as z-scores and compared across all groups (mean of n = 3). Gene expression per mouse is shown in H. Arp2/3, actin related proteins 2 and 3; Cntrl, control; ERK1/2, extracellular signal-regulated kinase 1 and 2; Fe-NTA, iron-nitriloacetic acid; GRB2, growth factor receptor-bound protein 2; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MEK, MAPK/ERK kinase; P-ERK, phosphorylated ERK; PI3K, phosphoinositide 3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; Raf, rapidly accelerated fibrosarcoma–kinase; Ras-GTP, rat sarcoma bound to guanosine triphosphate; SD, standard deviation; SOS, son of sevenless homolog; TCA, tricarboxylic acid.

Article Snippet: Cells were stimulated for 15 minutes with 350 ng/mL murine DMP1 (R&D Systems, 4386-DM) or 50 ng/mL and 100 ng/mL FGF2 (Miltenyi Biotec; 130-105-787) with or without 200 μg/mL of FCM, FDI, or LMWID.

Techniques: Binding Assay, Cell Surface Receptor Assay, Formulation, Concentration Assay, Recombinant, Phospho-proteomics, Activation Assay, Incubation, Blocking Assay, Western Blot, Control, RNA Sequencing, Gene Expression, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Standard Deviation