RANKL-induced expression of tetraspanin CD9 in lipid raft membrane microdomain is essential for cell fusion during osteoclastogenesis.
We showed that CD9, a member of tetraspanin superfamily proteins, is expressed in a specific membrane microdomain, called "lipid raft," and is crucial for cell fusion during osteoclastogenesis after activation of the RANK/RANKL system.


BACKGROUND
Osteoclasts are bone-resorbing multinuclear polykaryons that are essential for bone remodeling and are formed through cell fusion of mononuclear macrophage/monocyte lineage precursors. Although osteoclastogenesis has been shown to be critically regulated by the RANK/RANKL system, the mechanism how precursor cells fuse with each other remains unclear. We examined the function of CD9, a member of tetraspanin superfamily, which has previously been shown to form macromolecular membrane microdomains and to regulate cell-cell fusion in various cell types.


METHODS
We used RAW264.7, a macrophage/monocyte lineage cell line, which can differentiate into osteoclast-like polykaryons on the application of RANKL. Expression and distribution of CD9 was assessed by Western blotting, fluorescence-assorted cell sorting (FACS) and immunohistochemistry with light and electron microscopy. A specific neutralizing antibody and RNA interference were used to inhibit the function of CD9, and green fluorescent protein (GFP)-CD9 was exogenously expressed to enhance the effect of CD9. The distribution of CD9 in lipid microdomain was examined by biochemical (sucrose density gradient) isolation and imaging technique.


RESULTS
CD9 is expressed on cell surfaces of RAW264.7, which is enhanced by RANKL. Targeted inhibition of CD9 decreases the number of osteoclast-like cells. On the other hand, overexpression of CD9 promotes spontaneous cell fusion even in the absence of RANKL. CD9 is localized in detergent-insoluble "lipid raft" microdomain in RANKL stimulation, and disruption of lipid rafts markedly reduces the formation of osteoclast-like polykaryons. Immunohistochemical studies of bone tissues revealed the expression of CD9 in osteoclasts in vivo.


CONCLUSIONS
These data suggest that function of tetraspanin CD9 and its expression in lipid rafts are crucial for cell fusion during osteoclastogenesis.
Ishii
Masaru
M
Department of Clinical Research, Osaka Minami Medical Center, Osaka, Japan. mishii@ommc-hp.jp
Iwai
Kaori
K
Koike
Masato
M
Ohshima
Shiro
S
Kudo-Tanaka
Eriko
E
Ishii
Taeko
T
Mima
Toru
T
Katada
Yoshinori
Y
Miyatake
Kunio
K
Uchiyama
Yasuo
Y
Saeki
Yukihiko
Y
D000818:Animals; D015703:Antigens, CD / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D001842:Bone and Bones / Q000166:cytology / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002459:Cell Fusion; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology / Q000648:ultrastructure; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D060245:Tetraspanin 29; D015854:Up-Regulation
D015703:Antigens, CD; D002352:Carrier Proteins; C554870:Cd9 protein, mouse; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D060245:Tetraspanin 29; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse