Ferric Ion Induction of Triggering Receptor Expressed in Myeloid Cells-2 Expression and PI3K/Akt Signaling Pathway in Preosteoclast Cells to Promote Osteoclast Differentiation

Abstract

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.

Keywords: Ferric ammonium citrate; Osteoclast differentiation; PI3K/Akt; Trem-2.