Osteoclast stimulatory transmembrane protein (OC-STAMP), a novel protein induced by RANKL that promotes osteoclast differentiation.
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.
Yang
Meiheng
M
Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Birnbaum
Mark J
MJ
MacKay
Carole A
CA
Mason-Savas
April
A
Thompson
Benjamin
B
Odgren
Paul R
PR
D000135:Acid Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D001842:Bone and Bones / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002467:Cell Nucleus / Q000648:ultrastructure; D002478:Cells, Cultured; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D046228:Microarray Analysis; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D053245:RANK Ligand / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017421:Sequence Analysis; D000071681:Tartrate-Resistant Acid Phosphatase; D014162:Transfection; D015854:Up-Regulation
D000906:Antibodies; D007527:Isoenzymes; D008565:Membrane Proteins; C527519:OC-STAMP protein, mouse; D053245:RANK Ligand; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase