Critical role of beta3 integrin in experimental postmenopausal osteoporosis.
We show that mice lacking beta3 integrin are protected from OVX-induced bone loss. Using a lentiviral-based strategy to express beta3 mutants in beta3(-/-) mice, we also show that beta3(S752), but not beta3(Y747/Y759), is important for osteoclastic bone resorption in vivo.


BACKGROUND
Mice lacking the beta3 integrin have dysfunctional osteoclasts and therefore accumulate bone mass with age. Thus, the alphavbeta3 integrin is a potential anti-osteoporosis target. Identifying components of the beta3 integrin that determine its function in vivo is essential for therapeutically exploiting the antiresorptive properties of alphavbeta3.


METHODS
We used DXA and histomorphometry to assess bone loss after ovariectomy in wildtype and beta3 integrin null mice. We used lentiviral vectors carrying various human beta3 (hbeta3) integrin constructs to transduce beta3(-/-) bone marrow and reconstituted lethally irradiated beta3(-/-) mice with the transduced marrow. The expressed constructs include the intact integrin and two mutants, namely hbeta3(Y747F/Y759F) and hbeta3(S752P), each of which induces the bleeding dyscrasia, Glanzmann's thrombasthenia, in humans. Two months after transplantation, the expression of hbeta3 was measured by flow cytometry of marrow-derived macrophages. Osteoclast differentiation and function were assessed ex vivo by TRACP and actin-ring staining, respectively. Reconstituted mice were ovariectomized, and bone loss was assessed by DXA, histomorphometry, and serum TRACP5b assay.


RESULTS
beta3(-/-) mice are protected from ovariectomy-induced bone loss, showing no difference in BMD compared with sham-operated controls. We successfully expressed hbeta3 integrins in beta3(-/-) hosts using lentiviral transduction of bone marrow. Two months after transplantation, 25-35% of marrow-derived macrophages expressed the hbeta3 constructs. Similar to its effect in vitro, hbeta3(WT) completely rescued the osteoclast and platelet phenotype of beta3(-/-) mice. Whereas platelet function remained deranged in beta3(-/-) mice overexpressing hbeta3(Y747F/Y759F), osteoclast function was fully restored. In contrast, beta3(-/-) mice expressing hbeta3(S752P) continued to exhibit prolonged bleeding times and dysfunctional osteoclasts in vitro and ex vivo. Most importantly, hbeta3(WT) and hbeta3(Y747F/Y759F) transplanted mice underwent equivalent ovariectomy-induced bone loss, whereas, like those bearing the control vector, hbeta3(S752P) transplanted mice were protected.


CONCLUSIONS
Functional beta3 integrin is required for ovariectomy-induced bone loss. beta3(S752), but not beta3(Y747/Y759), is critical for osteoclast function in vivo.
Zhao
Haibo
H
Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri, USA.
Kitaura
Hideki
H
Sands
Mark S
MS
Ross
F Patrick
FP
Teitelbaum
Steven L
SL
Novack
Deborah Veis
DV
D000135:Acid Phosphatase / Q000097:blood / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D001760:Bleeding Time; D001792:Blood Platelets / Q000502:physiology; D015519:Bone Density / Q000235:genetics; D001854:Bone Marrow Cells / Q000378:metabolism; D016026:Bone Marrow Transplantation; D001862:Bone Resorption / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002352:Carrier Proteins / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D004195:Disease Models, Animal; D005260:Female; D005270:Femur Head / Q000737:chemistry; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D039661:Integrin beta3 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D007527:Isoenzymes / Q000097:blood / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D008159:Lumbar Vertebrae / Q000737:chemistry; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation / Q000235:genetics; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015663:Osteoporosis, Postmenopausal / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D010052:Ovariectomy; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D000071681:Tartrate-Resistant Acid Phosphatase; D013977:Tibia / Q000737:chemistry; D014162:Transfection
D000199:Actins; D002352:Carrier Proteins; D039661:Integrin beta3; D007527:Isoenzymes; D008562:Membrane Glycoproteins; 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; D016173:Macrophage Colony-Stimulating Factor; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase