Background/Purpose

DC-STAMP (Dendritic Cell-Specific Transmembrane Protein), a 7-pass transmembrane protein essential for cell-to-cell fusion during osteoclast (OC) differentiation, is expressed on the cell surface of OC precursors (OCP). DC-STAMP knock-out (KO) mice form only mononuclear OC and have mild osteopetrosis due to the absence of functional multi-nucleated OC with bone erosion activity. Previously, we identified an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) on the cytoplasmic tail of DC-STAMP, which suggested the potential role of DC-STAMP in signaling during osteoclastogenesis. A major hurdle in DC-STAMP research, however, is the absence of a known ligand. To overcome this barrier, we engineered DC-STAMP:Rhodopisn chimeric molecules that can be activated by light (photo-activatable). This approach enabled us to investigate the downstream events of DC-STAMP activation, and examine the importance and function of the ITIM in osteoclastogenesis.

Methods

DNA constructs were generated in which the four extracellular domains of DC-STAMP were replaced with the photo-activatable extracellular domains of rhodopsin. The functionality of two DC-STAMP chimeras (WT: original ITIM; TD: ITIM-deleted) was confirmed by: (1) activation of a calcium signal by 505-nm light, a wavelength that specifically excites rhodopsin; and (2) expression of mCherry protein (red), which was fused to the C’-terminus of DC-STAMP during engineering. These 2 chimeras were transfected into either murine 293T cells, or mouse bone marrow-derived macrophages (BMM) isolated from the DC-STAMP KO mice. To assess the role of the ITIM in signaling, we assessed the dynamic intracellular Ca²⁺flux known to occur during normal osteoclastogenesis between the WT and TD transfected cells. Bone erosion was assessed by the bone wafer assay.

Results

The studies with the transfected chimeric molecules revealed the following. First, deletion of ITIM on DC-STAMP resulted in a significant and continuous (non-pulsatile) elevation (3-fold >WT DC-STAMP) in the intracellular Ca²⁺ level after light activation. Second, ITIM-deleted DC-STAMP did not complement the deficiency of cell-cell fusion in DC-STAMP KO cells, which was fully corrected and restored by the WT DC-STAMP. Most cells expressing ITIM-deleted DC-STAMP were unable to fuse, although rare cells with 3 nuclei were observed. Third, In contrast to an even distribution of wild-type DC-STAMP on the cell surface, ITIM-deleted DC-STAMP was expressed on the cell surface in a clustered and punctate distribution fashion. Fourth, bone resorption was decreased in cells expressing TD compared to WT DC-STAMP.

Conclusion

Our results suggest that the ITIM on the cytoplasmic tail of DC-STAMP functions (1) to induce pulsatile intracellular Ca²⁺ flux required for osteoclastogenesis after DC-STAMP activation; (2) to trigger cell-to-cell fusion between OCPs and form multinucleated mature OC; (3) to maintain an even distribution of DC-STAMP on the cell surface; (4) to support bone erosion activity of OC. Blocking the ITIM on DC-STAMP by targeted inhibition might serve as a novel strategy to inhibit pathologic bone resorption by OC in inflammatory and metabolic bone disorders.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/deletion-of-the-inhibitory-receptor-motif-itim-on-dc-stamp-alters-osteoclast-differentiation-and-function/