Elucidation of CPX-1 involvement in RANKL-induced osteoclastogenesis by a proteomics approach.
To identify proteins potentially involved in osteoclast differentiation, we conducted a proteomics-based analysis using the osteoclastogenesis model cell line RAW264.7. Total proteins from undifferentiated cells, committed pre-osteoclasts, and differentiated osteoclasts were resolved by two-dimensional gel electrophoresis. Protein spots showing differential expression levels were processed for peptide mass fingerprinting. Among them, we identified the metallocarboxypeptidase CPX-1, which was prominently increased in pre-osteoclasts and then decreased in mature osteoclasts. Results of reverse transcription polymerase chain reaction, Western blot, and confocal microscopy were in agreement with the proteomics data. Notably, the forced overexpression of CPX-1 led to the inhibition of osteoclast formation, but not pre-osteoclast generation. Therefore, the transient up-regulation pattern of CPX-1 expression may be important for the successful progression from pre-osteoclasts to mature osteoclasts.
Chang
Eun-Ju
EJ
Department of Cell and Developmental Biology, Dental Research Institute, and Brain Korea 21 Program, College of Dentistry, Seoul National University, Seoul 110-749, South Korea.
Kwak
Han Bok
HB
Kim
Hyunsoo
H
Park
Joo-Cheol
JC
Lee
Zang Hee
ZH
Kim
Hong-Hee
HH
D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000494:pharmacology* / Q000502:physiology*; D002454:Cell Differentiation; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000502:physiology*; D045727:Metalloexopeptidases; D051379:Mice; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects; D010449:Peptide Mapping; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015854:Up-Regulation
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D011506:Proteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D045727:Metalloexopeptidases; C117650:Cpxm1 protein, mouse; D008666:Metalloendopeptidases