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.

sTREM-1 promotes the phagocytic function of microglia to induce hippocampus damage via the PI3K–AKT signaling pathway

“…TREM-1 induces inflammation and lipid accumulation in human hepatoma cell line HepG2 and primary mouse hepatocytes by activating the PI3K-AKT pathway 74 . TREM-2 promotes the differentiation of osteoclasts, increases the production of inflammatory factors, and enhances the killing effect of bacteria in the murine macrophage cell line RAW264.7 through the PI3K–AKT pathway 75 , 110 , 111 . sTREM-2 is dependent on the PI3K–AKT pathway to induce inflammation of microglia, promoting their survival and enhancing its phagocytic function in vivo and in vitro 73 , 105 .…”

Traditional Chinese decoction Si Zhi Wan attenuates ovariectomy (OVX)-induced bone loss by inhibiting osteoclastogenesis and promoting apoptosis of mature osteoclasts

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miR-4739/ITGA10/PI3K signaling regulates differentiation and apoptosis of osteoblast

“…Liu et al. also found that the ferric ammonium citrate could promote osteoclast differentiation through the Trem-2-mediated PI3K/AKT signaling pathway [ 39 ]. Based on the important role of this pathway in bone-related diseases, we further studied the effect of miR-4739 on the PI3K/AKT pathway in osteoblasts.…”