Anti-osteoactivin antibody inhibits osteoblast differentiation and function in vitro.
Osteoactivin (OA) is a novel protein identified by mRNA differential display using bone from osteopetrotic versus normal rats. Bioinformatic analysis showed that OA cDNA has an open reading frame of 1716 bp encoding a protein of 572 aa, the first 21 aa constitute a signal peptide. OA sequence analysis also demonstrated 13 putative N-glycosylation sites suggestive of a heavily glycosylated protein. In this study, we localized OA protein in primary osteoblast culture by immunofluorescent staining and Western blot analysis. Primary osteoblast cultures pass through three stages: proliferation from day 1 to 7, matrix formation from day 7 to 14, and matrix mineralization from day 14 to 21. OA protein was detected at all stages examined, with maximal expression at 3 weeks when osteoblasts are terminally differentiated. Using the Chariot transfection reagent as a vehicle to deliver anti-OA antibody into the cells, we demonstrated that anti-OA antibody significantly inhibited osteoblast differentiation markers, including alkaline phosphatase activity, nodule formation, osteocalcin production, and calcium deposition, without affecting cell proliferation or viability. These data suggest that OA is an osteoblast-related protein that plays an important role in the regulation of osteoblast differentiation and function.
Selim
Abdulhafez A
AA
Department of Anatomy and Cell Biology, Temple University, School of Medicine, Philadelphia, PA 19140, USA.
Abdelmagid
Samir M
SM
Kanaan
Reem A
RA
Smock
Steven L
SL
Owen
Thomas A
TA
Popoff
Steven N
SN
Safadi
Fayez F
FF
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D000906:Antibodies / Q000737:chemistry*; D015153:Blotting, Western; D001842:Bone and Bones / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D019295:Computational Biology; D018076:DNA, Complementary / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D020869:Gene Expression Profiling; D006031:Glycosylation; D008562:Membrane Glycoproteins; D008856:Microscopy, Fluorescence; D016366:Open Reading Frames; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D015675:Osteocalcin / Q000378:metabolism; D021382:Protein Sorting Signals; D011506:Proteins / Q000737:chemistry* / Q000276:immunology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D000906:Antibodies; D018076:DNA, Complementary; C503043:Gpnmb protein, rat; D008562:Membrane Glycoproteins; D021382:Protein Sorting Signals; D011506:Proteins; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013844:Thiazoles; D015675:Osteocalcin; D000469:Alkaline Phosphatase; C022616:thiazolyl blue; D002118:Calcium