Objective
Iron plays a significant role in multiple biological processes. The purpose of this study was to measure whether iron mediated osteoclast differentiation through regulation of triggering receptor expressed in myeloid cells‐2 (Trem‐2) expression and the PI3K/Akt signaling pathway.
Methods
The effects of six different concentrations of ferric ammonium citrate (FAC) (100, 80, 40, 20, 10 and 0 μmol/L) on RAW 264.7 cells proliferation were assessed by Cell Counting Kit‐8 (CCK‐8) gassay. Tartrate resistant acid phosphatase (TRAP) assay was performed to detect the effects of FAC on osteoclast formation. The expression of osteoclast differentiation‐related (TRAP, NFATc‐1, and c‐Fos) and Trem‐2 mRNA and proteins was analyzed by reverse transcription‐polymerase chain reaction and western blot, respectively. Si‐Trem‐2 was constructed and transfected to RAW264.7 to measure the effects of Trem‐2 on FAC‐mediated osteoclast formation. TRAP assay and osteoclast differentiation‐related gene analyses were further performed to identify the role of Trem‐2 in osteoclastogenesis. The Search Tool for the Retrieval of Interacting Genes (STRING) was used to explore the target genes of Trem‐2. Trem‐2‐related gene ontology and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were used for further in‐depth analysis. PI3K/Akt pathway‐related proteins were detected by immunofluorescence and western blot.
Results
In groups with FAC concentration of 10 (102.5 ± 3.1), 20 (100.5 ± 1.5), and 40 μmol/L (98.7 ± 3.1), compared with the control group (100.1 ± 2.2), cell viability was not significantly different from the control (P > 0.05). When the concentration of FAC exceeded 80 μmol/L, cell viability was significantly decreased (87.5 ± 2.8 vs 100.1 ± 2.2, P < 0.05). FAC promotes Trem‐2 expression and osteoclast differentiation in a dose‐response manner (P < 0.05). The number of osteoclast‐like cells was found to be reduced following transfection with the siRNA of Trem‐2 (42 ± 3 vs 30 ± 5, P < 0.05). We observed that most of Trem‐2 target genes are primarily involved in response to organic substance, regulation of reactive oxygen species metabolic process, and regulation of protein phosphorylation. The STRING database revealed that Trem‐2 directly target two gene nodes (Pik3ca and Pik3r1), which are key transcriptional cofactors of the PI3K/Akt signaling pathway. KEGG pathways include the “PI3K‐Akt signaling pathway,” the “thyroid hormone signaling pathway”, “prostate cancer,” the “longevity regulating pathway,” and “insulin resistance.” Expression of p‐PI3K and p‐Akt protein, measured by immunofluorescence and western blotting, was markedly increased in the FAC groups. Trem‐2 siRNA caused partial reduction of these two proteins (p‐PI3K and p‐Akt) compared to the FAC alone group.
Conclusion
The FAC promoted osteoclast differentiation through the Trem‐2‐mediated PI3K/Akt signaling pathway. However, its regulation osteoclastogenesis should be verified through further in vivo studies.