Background/Purpose: Osteoclasts (OC) are bone-resorbing, multinuclear cells that originate from myeloid progenitor cells through repetitive cycles of cell-cell fusion. Dendritic cell-specific transmembrane protein (DC-STAMP) is essential for cell-cell fusion and formation of fully functional OC, resulting in mild-moderate osteopetrosis in DC-STAMP^-/- mice; however the molecular mechanisms of its action is not well understood. We examined how the complete absence of DC-STAMP in the osteogenic progenitor cells (OCPs) affects their ability to participate in cell-cell fusion, alters calcium (Ca²⁺) signaling, and gene and protein expression in response to osteoclastogenic stimuli: macrophage colony-stimulating factor (MCSF) and  receptor activator of nuclear factor kappa-B ligand (RANKL).

Methods: We isolated bone marrow macrophages (BMMs) from wild type (WT) and DC-STAMP knockout (KO) mice. To analyze cell-cell fusion, we labeled DCSTAMP^+/+ and DCSTAMP^-/- BMMs with red (CellVue® Red) and green (CellVue® Jade) membrane dyes respectively, cultured them with MCSF (30 – 50 ng/ml) and RANKL (30 – 50 ng/ml) and monitored cell-cell fusion with live cell imaging. Calcium signaling was examined using microspectrofluorimetry of Fura-2 loaded OC precursors. Activation of osteoclastogenic transcription factor NFATc1 was assessed using immunoblotting and immunofluorescence. The effect of DC-STAMP knockout on gene and protein expression was examined using RNAseq, quantitative PCR (qPCR), and immunoblotting.

Results: DCSTAMP^+/+ cells were essential to initiate cell-cell fusion events; however DC-STAMP^-/- BMMs were successfully incorporated into forming OC. RANKL-induced Ca²⁺ oscillations were still present in DC-STAMP^-/- precursors, and increased in intensity compared to DC-STAMP^+/+ OCPs during the later stages of differentiation. NFATc1 protein levels in DCSTAMP^-/- OCPs increased with time following RANKL exposure and were comparable to WT. However, nuclear translocation of NFATc1 was significantly decreased in DCSTAMP^-/- osteoclast precursors at day 3 of differentiation. We further observed decreased expression of key osteoclastogenic genes (CTSK, ATP6V0D2, ACP5) in DCSTAMP^-/- cells. RNAseq analysis of DCSTAMP^-/- and WT OCPs identified differentially expressed genes indicating alterations in key molecular pathways, including RELA, NFκB, FOS, PKC, and TREM1 signaling.

Conclusion: Our findings indicate that while DC-STAMP^-/- OCPs cannot form multinuclear OCs, they do fuse with DCSTAMP^+/+ OCPs and are incorporated into maturing OCs. We demonstrate that even though calcium oscillations are present in DC-STAMP^-/- OCPs, NFATc1 nuclear translocation is deficient, suggesting that DC-STAMP acts in the  NFATc1 pathway, but downstream of calcium signaling. Our RNAseq analysis identified multiple differentially expressed genes that are potentially involved in cell-cell fusion and maturation of OCs.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-mechanism-of-dc-stamp-mediated-signaling-in-cell-cell-fusion-and-osteoclast-maturation/