Alpha9beta1: a novel osteoclast integrin that regulates osteoclast formation and function.
We identified a previously unknown integrin, alpha(9)beta(1), on OCLs and their precursors. Antibody to alpha(9) inhibited OCL formation in human marrow cultures, and OCLs from alpha(9) knockout mice had a defect in actin ring reorganization and an impaired bone resorption capacity.


BACKGROUND
Integrins play important roles in osteoclast (OCL) formation and function. Mature OCLs mainly express alpha(v)beta(3) integrin, a heterodimer adhesion receptor that has been implicated in osteoclastic bone resorption. We identified ADAM8, a disintegrin and metalloproteinase, as a novel stimulator of OCL differentiation and showed that the disintegrin domain of ADAM8 mediated its effects on OCL formation. Because the disintegrin domain of ADAM8 does not bind Arg-Gly-Asp (RGD) sequences, we determined which integrin bound ADAM8 and characterized its role in OCL formation and activity.


METHODS
Chinese hamster ovary cells (CHO) expressing different integrin subunits were tested for their capacity to bind the disintegrin domain of ADAM8. Mouse or human bone marrow cells and purified OCL precursors were tested for alpha(9)beta(1) integrin expression by Western blot, immunocytochemistry, and real-time RT-PCR. A monoclonal antibody to human alpha(9) was used to block alpha(9)beta(1) on OCL precursors stimulated by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] or RANKL. Vertebrae of 7-day-old alpha(9)(-/-) mice and wildtype (WT) littermates were compared using bone histomorphometry and 3D microCT analysis.


RESULTS
Alpha(9) integrin was expressed by mouse and human bone marrow-derived OCLs and their precursors. Importantly, the anti-alpha(9) antibody inhibited human OCL formation stimulated by 1alpha,25(OH)(2)D(3) or RANKL dose-dependently. Furthermore, analysis of OCLs formed in marrow cultures from alpha(9)(-/-) mice showed that the OCLs formed were more contracted and formed significantly less bone resorption pits on dentin slices. Histologic analysis of alpha(9)(-/-) vertebrae showed thickened trabecular regions and retained cartilage within vertebral bodies of alpha(9)(-/-) mice. 3D microCT analysis of alpha(9)(-/-) vertebrae also showed a significant increase in trabecular bone volume/total tissue volume and a tendency for decreased trabecular separation compared with WT mice.


CONCLUSIONS
These results support a previously unknown role for alpha(9)beta(1) integrin in OCL formation and function.
Rao
Hongwei
H
Medicine-Hematology/Oncology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Lu
Ganwei
G
Kajiya
Hiroshi
H
Garcia-Palacios
Veronica
V
Kurihara
Noriyoshi
N
Anderson
Judy
J
Patrene
Ken
K
Sheppard
Dean
D
Blair
Harry C
HC
Windle
Jolene J
JJ
Choi
Sun Jin
SJ
Roodman
G David
GD
D051722:ADAM Proteins / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001862:Bone Resorption; D016466:CHO Cells; D002454:Cell Differentiation; D006224:Cricetinae; D003599:Cytoskeleton / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D016023:Integrins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010010:Osteoclasts / Q000166:cytology / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D015703:Antigens, CD; D039302:Integrin alphaVbeta3; D016023:Integrins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C113256:integrin alpha 9 beta 1; D051722:ADAM Proteins; C489402:Adam8 protein, mouse