Proteomic profile of osteoclast membrane proteins: identification of Na+/H+ exchanger domain containing 2 and its role in osteoclast fusion.
Osteoclast formation and bone resorption are multiple processes that involve the participation of specialized membrane structures and their associated proteins. In this study, we used an MS to analyze the profile of proteins associated with osteoclast membranes and focused on the function of channel proteins in osteoclast differentiation and function. We filtered out with a SEQUEST score greater than 10 and a peptide hit number of more than 2, resulting in the identification of 499 proteins that were commonly found in both macrophages and osteoclasts, 96 proteins selectively found in osteoclasts, and 179 proteins selectively found in macrophages. The proteins that were selectively found in osteoclasts were classified based on their localizations: plasma membrane (17%), ER/Golgi and lysosome/endosome (15%), mitochondrion (18%), nucleus (13%), cytosol (19%), and unknown (18%). Proteins associated with osteoclast function such as v-ATPase, IGF2R, TRAP, and cathepsin K were found in osteoclasts as previously shown. We found several ion channel proteins such as Ank and Nhedc2 and signaling molecules such as Dock5 and RAB-10 in osteoclasts. Inhibition of the Na(+)/H(+) exchanger family by amiloride suppressed RANKL-induced osteoclast fusion and bone resorption. In addition, shRNA for Nhedc2 inhibited osteoclast differentiation. Our results provide a proteomic profile of osteoclast membrane proteins and identify Nhedc2, which is probably associated with proton transport in osteoclasts, as a regulator of osteoclast function.
Ha
Byung Geun
BG
Skeletal Diseases Genomic Research Center, Kyungpook National University Hospital, Jung-gu, Daegu, Republic of Korea.
Hong
Jung Min
JM
Park
Ju-Yong
JY
Ha
Mi-Hyun
MH
Kim
Tae-Ho
TH
Cho
Je-Yeol
JY
Ryoo
Hyun-Mo
HM
Choi
Je-Yong
JY
Shin
Hong-In
HI
Chun
So Young
SY
Kim
Shin-Yoon
SY
Park
Eui Kyun
EK
D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000502:physiology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002458:Cell Fractionation; D002459:Cell Fusion; D002460:Cell Line; D002853:Chromatography, Liquid; D003470:Culture Media / Q000494:pharmacology; D007202:Indicators and Reagents / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology; D008565:Membrane Proteins / Q000032:analysis* / Q000145:classification / Q000502:physiology; D051379:Mice; D008813:Mice, Inbred ICR; D010010:Osteoclasts / Q000166:cytology* / Q000502:physiology*; D017434:Protein Structure, Tertiary; D053245:RANK Ligand / Q000494:pharmacology; D012394:Rosaniline Dyes / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D000071681:Tartrate-Resistant Acid Phosphatase; D013997:Time Factors
D003470:Culture Media; D007202:Indicators and Reagents; D007527:Isoenzymes; D008565:Membrane Proteins; D053245:RANK Ligand; D012394:Rosaniline Dyes; D017923:Sodium-Hydrogen Exchangers; D016173:Macrophage Colony-Stimulating Factor; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase; C004692:coomassie Brilliant Blue