Osteoclast stimulatory transmembrane protein (OC-STAMP), a novel protein induced by RANKL that promotes osteoclast differentiation
Yang, Meilheng ; Birnbaum, Mark J. ; MacKay, Carole A. ; Mason-Savas, April ; Thompson, Benjamin ; Odgren, Paul R.
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Keywords
Amino Acid Sequence
Animals
Antibodies
Bone Marrow Cells
Bone and Bones
Cell Differentiation
Cell Nucleus
Cells, Cultured
Immunohistochemistry
Isoenzymes
Membrane Proteins
Mice
Mice, Knockout
Microarray Analysis
Molecular Sequence Data
Osteoclasts
RANK Ligand
RNA, Messenger
RNA, Small Interfering
Rats
Reverse Transcriptase Polymerase Chain Reaction
Sequence Analysis
Transfection
Up-Regulation
Cell Biology
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Abstract
Microarray and real-time RT-PCR were used to examine expression changes in primary bone marrow cells and RAW 264.7 cells in response to RANKL. In silico sequence analysis was performed on a novel gene which we designate OC-STAMP. Specific siRNA and antibodies were used to inhibit OC-STAMP RNA and protein, respectively, and tartrate-resistant acid phosphatase (TRAP)+ multinucleated osteoclasts were counted. Antibodies were used to probe bone tissues and western blots of RAW cell extracts +/- RANKL. cDNA overexpression constructs were transfected into RAW cells and the effect on RANKL-induced differentiation was studied. OC-STAMP was very strongly up-regulated during osteoclast differentiation. Northern blots and sequence analysis revealed two transcripts of 2 and 3.7 kb differing only in 3'UTR length, consistent with predictions from genome sequence. The mRNA encodes a 498 amino acid, multipass transmembrane protein that is highly conserved in mammals. It has little overall homology to other proteins. The carboxy-terminal 193 amino acids, however, are significantly similar to the DC-STAMP family consensus sequence. DC-STAMP is a transmembrane protein required for osteoclast precursor fusion. Knockdown of OC-STAMP mRNA by siRNA and protein inhibition by antibodies significantly suppressed the formation of TRAP+, multinucleated cells in differentiating osteoclast cultures, with many TRAP+ mononuclear cells present. Conversely, overexpression of OC-STAMP increased osteoclastic differentiation of RAW 264.7 cells. We conclude that OC-STAMP is a previously unknown, RANKL-induced, multipass transmembrane protein that promotes the formation of multinucleated osteoclasts.
Source
J Cell Physiol. 2008 May;215(2):497-505. Link to article on publisher's site