Phosphoglycerol dihydroceramide, a distinctive ceramide produced by Porphyromonas gingivalis, promotes RANKL-induced osteoclastogenesis by acting on non-muscle myosin II-A (Myh9), an osteoclast cell fusion regulatory factor

Abstract

Among several virulence factors produced by the periodontal pathogen Porphyromonas gingivalis (Pg), a recently identified novel class of dihydroceramide lipids that contains a long acyl-chain has the potential to play a pathogenic role in periodontitis because of its higher level of tissue penetration compared to other lipid classes produced by Pg. However, the possible impact of Pg ceramides on osteoclastogenesis is largely unknown. In the present study, we report that the phosphoglycerol dihydroceramide (PGDHC) isolated from Pg enhanced osteoclastogenesis in vitro and in vivo. Using RAW264.7 cells, in vitro assays indicated that PGDHC can promote RANKL-induced osteoclastogenesis by generating remarkably larger TRAP+ multinuclear osteoclasts compared to Pg LPS in a TLR2/4-independent manner. According to fluorescent confocal microscopy, co-localization of non-muscle myosin II-A (Myh9) and PGDHC was observed in the cytoplasm of osteoclasts, indicating the membrane-permeability of PGDHC. Loss- and gain-of-function assays using RNAi-based Myh9 gene silencing, as well as overexpression of the Myh9 gene, in RAW264.7 cells showed that interaction of PGDHC with Myh9 enhances RANKL-induced osteoclastogenesis. It was also demonstrated that PGDHC can upregulate the expression of dendritic cell-specific transmembrane protein (DC-STAMP), an important osteoclast fusogen, through signaling that involves Rac1, suggesting that interaction of PGDHC with Myh9 can elicit the cell signal that promotes osteoclast cell fusion. Taken together, our data indicated that PGDHC is a Pg-derived, cell-permeable ceramide that possesses a unique property of promoting osteoclastogenesis via interaction with Myh9 which, in turn, activates a Rac1/DC-STAMP pathway for upregulation of osteoclast cell fusion.

Myosins in Osteoclast Formation and Function

“…These results suggest that high levels of Myo2a inhibit osteoclast fusion, and that its loss triggers the fusion process. Other studies have confirmed these findings [51,52] and have likewise shown that reducing myosin II activity by inhibition of myosin light chain kinase similarly triggers osteoclast fusion [53]. Precisely how Myo2a achieves this inhibition is unclear, but it may be due to production of intracellular tension at defined locations within the cells.…”

Periodontal reconstruction by heparan sulfate mimetic-based matrix therapy in Porphyromonas gingivalis-infected mice

“…P. gingivalis factors promote bone resorption [ 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. In this study, bone resorption was evaluated along the socket of the third molar; the resorption provoked by P. gingivalis infection added to the physiological osteoclastic resorption related to the physiological tooth drift [37] .…”

Osteoclast Multinucleation: Review of Current Literature

“…Kanzaki et al have reported that phosphoglycerol dihydroceramide (PGDHC), a ceramide produced by the peridontal pathogen Porphyromonas gingivalis , is the virulence factor promoting the formation of larger osteoclasts. PGDHC directly binds to non-muscle myosin IIA (Myh9), elevating the expression of DC-STAMP by activating Rac1 GTPase [ 195 ].…”