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 (DCSTAMP) is a transmembrane protein essential for cell-cell fusion and formation of fully functional OC although the molecular mechanisms are not well understood. Utilizing RAW cell lines we previously demonstrated that during OC differentiation, heterogeneity in membrane expression levels of DCSTAMP correlated with efficient cell-cell fusion during multinuclear OC formation. Optimal fusion was observed when DCSTAMP^low and DCSTAMP^high cells interact. Herein, we examined how complete absence of DC-STAMP in the osteogenic progenitor cells (OCPs) controls their ability to participate in cell-cell fusion events required for efficient multinuclear OC formation.

Methods:

We isolated bone marrow macrophages (BMMs) from wild type (WT) and DCSTAMP knockout (KO) mice. DCSTAMP^-/- mice show mild osteopetrosis. To analyze cell-cell fusion, we labeled DCSTAMP^+/+ and DCSTAMP^-/- BMMs obtained from WT and KO mice, respectively, with red (CellVue® Red) and green (CellVue® Jade) membrane dyes, cultured them with MCSF (30 ng/ml) and RANKL (30 ng/ml), and monitored cell-cell fusion with live cell imaging. Moreover, we examined the expression dynamics and fate of DCSTAMP protein in forming OC employing retroviral-mediated expression of GFP-tagged DCSTAMP protein in DCSTAMP^-/- cells. In addition, we investigated how DC-STAMP alters expression levels of key osteoclast-related genes in WT and KO cells.

Results:

We find that DCSTAMP^-/- BMMs are incorporated into forming OC, however, DCSTAMP^+/+ cells are essential to initiate cell-cell fusion events. Following retroviral vector-mediated complementation of GFP-tagged DCSTAMP protein expression in DCSTAMP^-/- BMMs, we noted that during OC formation, DC-STAMP expression level remained high but progressively declined during several rounds of cell-cell fusion and levels were low or absent in mature OCs. Interestingly, we noted that the absence of DC-STAMP did not alter mRNA expression levels of NFATc1 and other key genes involved in cell-cell fusion and other OC differentiation pathways such as ACP5, CTSK and ATP6V0D2. Unexpectedly we further noted that mRNA expression levels of ACP5, and CTSK genes were more than 2 fold higher in RANKL activated DC-STAMP^-/-cells compared to WT cells after 120 hours in culture.

Conclusion:

Our findings indicate that DCSTAMP expression levels in OCP are high during the early cell-cell fusion events but progressively decline and are absent or low in mature OCs. While DCSTAMP^-/- OCPs cannot form multinuclear OCs, they do fuse with DCSTAMP^+/+ OCPs and are incorporated into maturing OCs. These data indicate that DCSTAMP membrane expression is not required for cell-cell fusion in the presence of other cells expressing this molecule. The absence of DCSTAMP in OCPs does not alter CTSK, ACP5, ATP6V0D2 mRNA expression levels during early fusion events but these genes were elevated in later stages in KO cells. DC-STAMP may regulate expression of key OC-specific genes in the late stages of OC differentiation.

« Back to 2018 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/elucidation-of-the-function-of-dendritic-cell-specific-transmembrane-protein-dcstamp-in-osteoclast-differentiation/