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.
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.
The Forsyth Institute, Department of Immunology and Infectious Diseases, Cambridge, MA, USA; Tsurumi University, School of Dental Medicine, Department of Orthodontics, Kanagawa, Japan. Electronic address: kanzaki-h@tsurumi-u.ac.jp.
Hiroyuki
H
Kanzaki
The Forsyth Institute, Department of Immunology and Infectious Diseases, Cambridge, MA, USA. Electronic address: amovila@forsyth.org.
Alexandru
A
Movila
Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia. Electronic address: rkayal@kau.edu.sa.
Rayyan
R
Kayal
Laboratory of Immunology and Molecular Biology, São Leopoldo Mandic Institute and Research Center, São Paulo, Brazil. Electronic address: marcelo.napimoga@gmail.com.
Marcelo H
MH
Napimoga
Department of Immunology and Infectious Diseases, The Forsyth Institute, Cambridge, MA, USA; LION Corporation, Research and Development Headquarters, Odawara, Kanagawa, Japan.
Kenji
K
Egashira
The Forsyth Institute, Department of Microbiology, Cambridge, MA, USA; Harvard School of Dental Medicine, Department of Oral Medicine, Infection and Immunity, Boston, MA, USA. Electronic address: FDewhirst@forsyth.org.
Floyd
F
Dewhirst
The Forsyth Institute, Department of Immunology and Infectious Diseases, Cambridge, MA, USA; Harvard School of Dental Medicine, Department of Oral Medicine, Infection and Immunity, Boston, MA, USA. Electronic address: HSasaki@forsyth.org.
Hajime
H
Sasaki
Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia. Electronic address: mhowait@icloud.com.
Mohammed
M
Howait
Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia. Electronic address: aaldharab@kau.edu.sa.
Ayman
A
Al-Dharrab
Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia. Electronic address: amira@kau.edu.sa.
Abdulghani
A
Mira
The Forsyth Institute, Department of Immunology and Infectious Diseases, Cambridge, MA, USA; Harvard School of Dental Medicine, Department of Oral Medicine, Infection and Immunity, Boston, MA, USA. Electronic address: XHan@forsyth.org.
Xiaozhe
X
Han
The Forsyth Institute, Department of Immunology and Infectious Diseases, Cambridge, MA, USA; Harvard School of Dental Medicine, Department of Oral Medicine, Infection and Immunity, Boston, MA, USA. Electronic address: MTaubman@forsyth.org.
Martin A
MA
Taubman
Department of Oral Health and Diagnostic Sciences, University of Connecticut School of Dental Medicine, Farmington, CT, USA. Electronic address: Nichols@nso.uchc.edu.
Frank C
FC
Nichols
The Forsyth Institute, Department of Immunology and Infectious Diseases, Cambridge, MA, USA; Department of Immunology and Infectious Diseases, The Forsyth Institute, Cambridge, MA, USA; NOVA Southeastern University College of Dental Medicine, Department of Periodontology, Fort Lauderdale, FL, USA. Electronic address: tkawai@forsyth.org.
Toshihisa
T
Kawai
D000818:Animals; D002450:Cell Communication / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics; D002518:Ceramides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing; D020404:Glycerophospholipids / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D018995:Myosin Heavy Chains; D009419:Nerve Tissue Proteins / Q000235:genetics; D024507:Nonmuscle Myosin Type IIA / Q000235:genetics* / Q000378:metabolism; D010010:Osteoclasts / Q000378:metabolism / Q000473:pathology; D010012:Osteogenesis / Q000235:genetics; D010518:Periodontitis / Q000235:genetics* / Q000382:microbiology / Q000473:pathology; D016966:Porphyromonas gingivalis / Q000378:metabolism* / Q000472:pathogenicity; D053245:RANK Ligand / Q000378:metabolism; D000067996:RAW 264.7 Cells; D015398:Signal Transduction / Q000235:genetics; D020830:rac1 GTP-Binding Protein / Q000235:genetics
D002518:Ceramides; C499636:DC-STAMP protein, mouse; D020404:Glycerophospholipids; D008565:Membrane Proteins; C525088:Myh9 protein, mouse; D009419:Nerve Tissue Proteins; D053245:RANK Ligand; C505689:Tnfsf11 protein, mouse; C109343:dihydroceramide; D024507:Nonmuscle Myosin Type IIA; D018995:Myosin Heavy Chains; D020830:rac1 GTP-Binding Protein
DC-STAMP; Non-muscle myosin II-A; Phosphoglycerol dihydroceramide; Porphyromonas gingivalis; RANKL-induced osteoclastogenesis; Rac1 small GTPase