Expression and function of transmembrane-4 superfamily (tetraspanin) proteins in osteoclasts: reciprocal roles of Tspan-5 and NET-6 during osteoclastogenesis.
BACKGROUND
Osteoclasts are bone-resorbing multinuclear polykaryons essential for bone remodeling, formed through cell fusion of mononuclear macrophage/monocyte lineage precursor cells upon stimulation by the RANK/RANKL system. Recent studies have revealed that a family of tetraspanin proteins, such as CD9, is critically involved in the cell fusion/polykaryon formation of these cell types. Until now, however, there is limited knowledge about the types of tetraspanins expressed in osteoclasts and their precursors.


METHODS
The expression of different tetraspanin proteins in a monocyte/macrophage-lineage osteoclast precursor cell line, RAW264.7, was cyclopedically investigated using RT-PCR with specific primers and quantitative real-time RT-PCR. The function of two kinds of tetraspanins, Tspan-5 and NET-6, whose expression pattern was altered by RANKL stimulation, was examined by transfecting gene-specific short-interfering RNAs into these cell types.


RESULTS
Of the 17 tetraspanins in mammalian hematopoietic cells, RAW264.7 cells express mRNA for 12 different kinds of tetraspanins, namely, CD9, CD37, CD53, CD63, CD81, CD82, CD151, NAG-2, NET-6, SAS, Tspan-3, and Tspan-5. Interestingly, during their maturation into osteoclasts upon RANKL stimulation, the transcript for Tspan-5 is up-regulated, whereas that for NET-6 is down-regulated. Targeted inhibition of Tspan-5 by using gene-specific RNA interference suppressed RANKL-induced cell fusion during osteoclastogenesis, whereas inhibition of NET-6 augmented the osteoclastogenesis itself. These results suggest that Tspan-5 and NET-6 have a reciprocal function during osteoclastogenesis, i.e., positive and negative regulation by Tspan-5 and NET-6, respectively. RANKL regulates osteoclastogenesis by altering the balances of these tetraspanin proteins.


CONCLUSIONS
These data indicate that a diversity of tetraspanins is expressed in osteoclast precursors, and that cell fusion during osteoclastogenesis is regulated by cooperation of distinct tetraspanin family proteins such as Tspan-5 and NET-6. This study indicates that functional alterations of tetraspanin family proteins may have therapeutic potential in diseases where osteoclasts play a major role, such as rheumatoid arthritis and osteoporosis.
Iwai
Kaori
K
Department of Clinical Research, National Hospital Organization Osaka Minami Medical Center, Osaka, Japan.
Ishii
Masaru
M
Ohshima
Shiro
S
Miyatake
Kunio
K
Saeki
Yukihiko
Y
D000818:Animals; D002450:Cell Communication / Q000235:genetics; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D015536:Down-Regulation / Q000235:genetics; D020869:Gene Expression Profiling; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology; D051379:Mice; D005810:Multigene Family; D010010:Osteoclasts / Q000166:cytology* / Q000378:metabolism*; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D059470:Tetraspanins; D015854:Up-Regulation / Q000235:genetics
D008565:Membrane Proteins; D034741:RNA, Small Interfering; D059470:Tetraspanins; C528335:Tspan13 protein, mouse; C435756:Tspan5 protein, mouse