Background/Purpose:

Osteoclasts (OC) are the only cell type known to erode bone. Many bone diseases including osteoporosis and arthritis are caused by excessive OC activity. The molecular mechanisms underlying cell-cell fusion, a key step in osteoclastogenesis (OCgenesis), are not well understood. Our studies focused on DC-STAMP (Dendritic Cell-Specific Transmembrane Protein), a 7-pass transmembrane protein which is expressed on the cell surface of OC precursors (OCPs) and is essential for OCgenesis. DC-STAMP knock-out (KO) mice form only mono-nucleated OC, and manifest mild osteopetrosis due to the absence of functional polykaryon OC. Previously, we identified an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) on the cytoplasmic tail of DC-STAMP. By engineering a DC-STAMP:rhodopsin photactivatable chimeric molecule that can be activated by light, we demonstrated that the ITIM is a functional motif that alters intracellular Ca⁺²flux, suggesting the potential role of DC-STAMP in signaling. Herein, we further examined the effect of ITIM deletion on bone erosion, cell mobility, and NFATc1 nuclear translocation.

Methods:

The wild-type (WT) and ITIM-deleted (TD) versions of DC-STAMP were tagged with GFP, and introduced into DC-STAMP-/- cells isolated from the DC-STAMP KO mice. DC-STAMP-/- cells which overexpress WT- or TD- DC-STAMP were tested for complementation to OC-forming deficiency by in vitro culture, bone erosion activity by bone wafer assay, cell surface distribution by confocal microscopy, and cell mobility by the scanning electron microscopy (SEM). The dynamic changes of NFATc1 protein after introduction of the WT DC-STAMP into DC-STAMP-/- cells were examined by Western blotting.

Results:

Deletion of the DC-STAMP ITIM was associated with decreased OCgenesis and altered downstream signaling as summarized by the following key observations. (1) Complementation: The OC-forming deficiency of DC-STAMP-/- cells was complemented when cells were infected with WT- but not TD- DC-STAMP; (2) Cell-cell fusion: Most of the TD-DC-STAMP-expressing cells were mononuclear, whereas a minority fused twice with 3 maximal nuclei per cell. As expected, TD cells showed smaller cell volumes than WT-DC-STAMP expressing cells due to the limited frequency of cell-cell fusion; (3) Bone erosion: TD-DC-STAMP-expressing cells showed decreased bone erosion activity as evidenced by smaller and shallower erosion pits (7.2 & 1.5 mm²/pit area for WT & TD, respectively), which is associated with a reduced cell mobility revealed by SEM; (4) NFATc1 nuclear translocation: in contrast to WT-DC-STAMP, the NFATc1 proteins in TD-DC-STAMP-expressing cells failed to translocate to the nuclei.

Conclusion:

The ITIM on DC-STAMP is a functional motif that regulates OCgenesis through the Ca²⁺/NFATc1 axis. Deletion of ITIM on DC-STAMP resulted in decreased bone erosion activity, impaired osteoclast mobility, and diminished nuclear translocation of NFATc1. This is the first demonstration that DC-STAMP regulates the translocation of NFATc1, a master transcription factor of OCgenesis. Targeted inhibition of the ITIM on DC-STAMP may prove an effective strategy to prevent pathologic bone in inflammatory and metabolic bone disorders.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dc-stamp-regulates-osteoclastogenesis-through-the-ca2-nfatc1-axis/