Regulation of cytoskeleton and adhesion signaling in osteoclasts by tetraspanin CD82

Abstract

We used a myeloid-specific Cre to conditionally delete CD82 in mouse osteoclasts and their precursors. In contrast to global loss of CD82 (gKO), conditional loss of CD82 (cKO) in osteoclasts does not affect cortical bone, osteoblasts, or adipocytes. CD82 loss results in greater trabecular volume and trabecular number but reduced trabecular space in 6-month old male mice. Though this trend is present in females it did not reach significance; whereas there was an increase in osteoclast numbers and eroded surface area only in female cKO mice. In vitro, there is an increase in osteoclast fusion and defects in actin assembly in both gKO and cKO mice, irrespective of sex. This is accompanied by altered osteoclast morphology and decreased release of CTX in vitro. Integrin αvβ3 expression is reduced, while integrin β1 is increased. Signaling to Src, Syk, and Vav are also compromised. We further discovered that expression of Clec2 and its ligand, Podoplanin, molecules that also signal to Syk and Vav, are increased in differentiated osteoclasts. Loss of CD82 reduces their expression. Thus, CD82 is required for correct assembly of the cytoskeleton and to limit osteoclast fusion, both needed for normal osteoclast function.

Fibroblastic Reticular Cell Response to Dendritic Cells Requires Coordinated Activity of Podoplanin, CD44 and CD9

“…CLEC-2 hi platelets may potentially bind tumour cells and CAFs to upregulate podoplanin expression, influencing tumour progression. Furthermore, we find that CD82 is required for podoplanin expression in FRCs; a more dramatic phenotype than reported in CD82 KO osteoclasts which showed reduced podoplanin expression (45). Our data suggests that CD82 controls podoplanin trafficking to the plasma membrane, which is a well-known function of tetraspanin family members (44).…”

“…Tetraspanins control a variety of cellular processes, including cell-cell interactions, cell migration, and signalling events (38,39). We find that tetraspanin CD82 is essential for podoplanin membrane localisation in FRCs; a more dramatic phenotype than reported in CD82 KO osteoclasts which also showed reduced podoplanin expression (42). Our data suggests that CD82 controls podoplanin trafficking to the plasma membrane.…”

“…Tetraspanins are master-organisers of the plasma membrane, binding to and controlling expression of membrane proteins (38), but can also regulate protein trafficking (44). One candidate for controlling podoplanin expression is tetraspanin CD82 as it was recently reported that loss of CD82 in osteoclasts decreases podoplanin protein expression (45). To investigate the function of CD82 in podoplanin expression in FRCs, we generated CD82 knock-out (KO) FRCs using CRISPR/Cas9 editing and selected single-cell clones by quantitative RT-PCR ( Supplementary Fig.…”

“…Podoplanin drives actomyosin contrac-tility in FRCs (24,25). Tetraspanin CD82 also controls cytoskeletal structures via RhoGTPase signalling (42)(43)(44)(45), and loss of CD82 in osteoclasts decreases the level of podoplanin expression (42). We hypothesized that tetraspanin CD82 may regulate podoplanin-driven actomyosin contractility in FRCs.…”

“…CLEC-2 binding to FRCs drives podoplanin clustering into cholesterol-rich domains (24), but the function of these podoplanin clusters is unknown. Another protein potentially involved in podoplanindriven actomyosin contractility is tetraspanin CD82, since osteoclasts lacking CD82 expression show disrupted actin structures due to defects in RhoGTPase signalling, and a dramatic decrease in podoplanin expression (42). In prostate cancer cells, CD82 expression controls RhoGTPase activity and lamellipodia protrusions (43).…”