Critical Role of β3 Integrin in Experimental Postmenopausal Osteoporosis

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JOURNAL OF BONE AND MINERAL RESEARCH Volume 20, Number 12, 2005 Published online on July 25, 2005; doi: 10.1359/JBMR.050724 © 2005 American Society for Bone and Mineral Research

Critical Role of ␤3 Integrin in Experimental Postmenopausal Osteoporosis Haibo Zhao,1 Hideki Kitaura,1 Mark S Sands,2 F Patrick Ross,1 Steven L Teitelbaum,1 and Deborah Veis Novack1,2

ABSTRACT: We show that mice lacking ␤3 integrin are protected from OVX-induced bone loss. Using a lentiviral-based strategy to express ␤3 mutants in ␤3−/− mice, we also show that ␤3S752, but not ␤3Y747/Y759, is important for osteoclastic bone resorption in vivo. Introduction: Mice lacking the ␤3 integrin have dysfunctional osteoclasts and therefore accumulate bone mass with age. Thus, the ␣v␤3 integrin is a potential anti-osteoporosis target. Identifying components of the ␤3 integrin that determine its function in vivo is essential for therapeutically exploiting the antiresorptive properties of ␣v␤3. Materials and Methods: We used DXA and histomorphometry to assess bone loss after ovariectomy in wildtype and ␤3 integrin null mice. We used lentiviral vectors carrying various human ␤3 (h␤3) integrin constructs to transduce ␤3−/− bone marrow and reconstituted lethally irradiated ␤3−/− mice with the transduced marrow. The expressed constructs include the intact integrin and two mutants, namely h␤3Y747F/Y759F and h␤3S752P, each of which induces the bleeding dyscrasia, Glanzmann’s thrombasthenia, in humans. Two months after transplantation, the expression of h␤3 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: ␤3−/− mice are protected from ovariectomy-induced bone loss, showing no difference in BMD compared with sham-operated controls. We successfully expressed h␤3 integrins in ␤3−/− hosts using lentiviral transduction of bone marrow. Two months after transplantation, 25–35% of marrow-derived macrophages expressed the h␤3 constructs. Similar to its effect in vitro, h␤3WT completely rescued the osteoclast and platelet phenotype of ␤3−/− mice. Whereas platelet function remained deranged in ␤3−/− mice overexpressing h␤3Y747F/Y759F, osteoclast function was fully restored. In contrast, ␤3−/− mice expressing h␤3S752P continued to exhibit prolonged bleeding times and dysfunctional osteoclasts in vitro and ex vivo. Most importantly, h␤3WT and h␤3Y747F/Y759F transplanted mice underwent equivalent ovariectomy-induced bone loss, whereas, like those bearing the control vector, h␤3S752P transplanted mice were protected. Conclusions: Functional ␤3 integrin is required for ovariectomy-induced bone loss. ␤3S752, but not ␤3Y747/Y759, is critical for osteoclast function in vivo. J Bone Miner Res 2005;20:2116–2123. Published online on July 25, 2005; doi: 10.1359/JBMR.050724 Key words: osteoporosis, osteoclasts, bone resorption, ␣v␤3 integrin, ovariectomy

INTRODUCTION

T

HE ADULT SKELETON is a dynamic organ that undergoes continuous renewal involving two specialized cellular activities: bone resorption, mediated by osteoclasts, and bone formation, carried out by osteoblasts. Progressive loss of bone mass, as seen in disorders such as postmenopausal osteoporosis, periodontal disease, and rheumatoid arthritis, reflects an imbalance in skeletal remodeling in which bone resorption exceeds bone formation because of an increase in osteoclast differentiation and/or function.(1)

The authors have no conflict of interest.

The osteoclast is a specialized polykaryon derived from the monocyte/macrophage lineage under the control of two essential cytokines: macrophage-colony stimulating factor (M-CSF) and RANKL.(2,3) Bone degradation by mature osteoclasts is a multistep process initiated by the attachment of osteoclasts to mineralized bone matrix. Hence, the direct interaction of osteoclast surface molecules with bone matrix is a rate-limiting step in the cell’s function. Several lines of evidence posit the ␣v␤3 integrin as a key mediator of this interaction.(4,5) ␣v␤3 integrin is highly expressed in osteoclasts of all species examined.(6) Experiments using function blocking antibodies, ligand-containing disintegrins, and peptidomi-

1 Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri, USA; 2Department of Medicine, Washington University School of Medicine, St Louis, Missouri, USA.

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␤3 INTEGRIN IN POSTMENOPAUSAL OSTEOPOROSIS metic antagonists suggest ␣v␤3 is essential to the resorptive process both in vitro and in vivo.(7–10) Most importantly, ␤3 integrin knockout mice have dysfunctional osteoclasts and become progressively osteosclerotic as they age.(11) Although these findings indicate a fundamental role of ␣v␤3 integrin in osteoclast function, the underlying mechanism of action at the molecular level is still not fully understood. With this in mind, we previously performed in vitro structure–function analysis of the ␤3 integrin cytoplasmic domain mediating osteoclast function, using retroviral transduction of ␤3−/− osteoclast precursors.(12) Of a series of human ␤3 integrin constructs bearing point mutations known to affect ␤ integrin function, only one, h␤3S752P, representing a mutation characterizing the human bleeding disorder, Glanzmann’s thrombasthenia, failed to rescue ␤3−/− osteoclast function in vitro. Although Y747 and Y759, in combination, are also essential for platelet function,(13) the ␤3Y747F/Y759F mutant behaves like intact ␤3 in regard to the osteoclasts in vitro. These in vitro experiments suggest that ␣v␤3 is a promising antiresorptive therapeutic target, but optimizing this strategy will require an understanding of the role of this integrin in the context of pathophysiology in vivo. To this end, we have used the ovariectomy (OVX) model in ␤3−/− mice to confirm the critical role of ␤3 in pathological bone loss. Additionally, we have tested the function of the ␤3 mutants, in vivo, by reconstituting lethally irradiated ␤3−/− mice with bone marrow cells expressing various human ␤3 constructs and assessing their response to OVX.

MATERIALS AND METHODS Animal protocol All animal procedures were approved by the Animal Studies Committee of Washington University. After body weight measurements, ␤3−/− female mice in C57BL/6 × 129 background were anesthetized and underwent bilateral OVX using the dorsal approach. Sham-operated mice had the ovaries exteriorized but not removed. Uterine weight was determined at death to verify the successful removal of the ovaries.

Lentiviral vector production The third generation of self-inactivating (SIN) lentivirus vector and all packaging plasmids were kindly provided by Cell Genesys (San Francisco, CA, USA). Wildtype or point mutated forms of full-length of human ␤ 3 integrin cDNA(14) were released from ⌬U3 retroviral vectors by digestion with XbaI and subcloned into the shuttle vector, SP73 (Promega, Madison, WI, USA). The SP73-h␤3 vectors were digested with BglII/SalI, and the inserts were cloned into the BamHI/SalI sites of the pptpgkGFP transfer vector, replacing the GFP gene with h␤3. Vesicular stomatitis virus G protein–pseudotyped viruses were produced by calcium phosphate–mediated transient transfection of 293T cells in high-glucose DMEM (Hyclone, Logan, UT, USA) with the transfer construct and the three packing plasmids pMDLg/pRRE, pMD.G, and pRSV-REV as previously described.(15,16) The medium was refreshed after 16 h, and

2117 viral supernatant was collected after another 24 h and passed through 0.45-␮m cellulose acetate syringe (Corning) filters. Titers of all vector preparations were determined by transducing NIH-3T3 cells with serial dilutions of vector supernatants, followed by the measurement of the expression of GFP and ␤3 integrin (by fluorescence activated cell sorting [FACS] analysis) 2 days later.

Transduction and bone marrow transplantation Whole bone marrow cells were harvested from 2- to 3-month-old female ␤3 integrin knockout mice pretreated with 150 mg/kg of 5-fluorouracil administered by intraperitoneal injection. Red blood cells were lysed by incubation for 5 minutes with an ammonium chloride lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4). The remaining mononuclear marrow cells were resuspended in RPMI medium (Hyclone) containing 20% FBS. Cells were transduced by two rounds of centrifugation with lentiviral stock at a multiplicity of infection (MOI) of 30 (i.e., 30 3T3 transducing units per target cell).(17) The transduced marrow cells were resuspended in Hank’s buffered saline (Invitrogen, Carlsbad, CA, USA). A total of 2 × 106 transduced cells were injected by tail vein into 8-week-old female ␤3−/− mice that had been lethally irradiated with 1000 cGy of ␥ radiation from a 137Cs source 16 h previously.

Flow cytometric analysis Two months after transplantation, bone marrow cells were harvested from the femurs and tibias. Red blood cells were lysed, and the remaining cells were cultured with 1/10 vol of CMG 14–12 culture supernatant, equivalent to 130 ng/ml of recombinant M-CSF(18) for 3–4 days. Bone marrow macrophages (BMMs) were suspended in blocking buffer (0.5% BSA/2 mM EDTA/PBS) and incubated on ice for 20 minutes. Surface human ␤3 was labeled with 1 ␮g/ml monoclonal antibody 1A2 for 30 minutes on ice. Cells were washed with blocking buffer twice and were incubated with FITC-labeled goat anti-mouse antibody (Jackson Laboratories) for 20 minutes on ice. Cells were washed twice and resuspended in 500 ␮l blocking buffer. Flow cytometry was conducted on BD FACS Calibur system (BD Biosciences, San Jose, CA, USA) with Cell Quest software.

BMM isolation and osteoclast culture BMMs were prepared as described previously with slight modification.(16,18) Whole bone marrow was extracted from femora of mice with ␣-MEM and cultured overnight in ␣-MEM (Sigma, St Louis, MO, USA) containing 10% heatinactivated FBS, 100 IU/ml penicillin, and 100 ␮g/ml streptomycin (␣10 medium). The nonadherent cells were collected by centrifugation and replated in a new 10-cm petri dish in ␣10 medium with 1/10 vol of CMG 14–12 culture supernatant. Cells were incubated at 37°C in 6% CO2-94% air for 4 days. Fresh media and M-CSF were supplemented every other day. Cells were washed with PBS, lifted with 1× Trypsin/EDTA (Invitrogen) in PBS, and seeded at 1.5 × 104 cells per well of 48-well tissue culture dish. Mature osteoclasts were generated after a 5-day culture of BMMs with 1/100 vol of CMG 14–12 culture supernatant and 100 ng/ml

2118 recombinant RANKL. The cells cultured on plastic dishes were fixed with 3% paraformaldehyde/PBS for 10 minutes at room temperature followed by washing with PBS twice for 5 minutes. TRACP was stained with a commercial kit (387-A; Sigma). For cells cultured on whale dentine slices, filamental actin was labeled with Alexa-488 conjugated phalloidin (Molecular Probes, Eugene, OR, USA), as previously described.(16,19) Photographs were taken under a conventional microscope equipped with a CCD camera (NikonUSA, Melville, NY, USA) or with a laser confocal scanning microscope (Biorad 2000; Biorad, Hercules, CA, USA).

BMD measurement BMD of whole body, lumbar vertebrae, and proximal femora was measured in vivo in anesthetized mice at baseline and 4 weeks after surgery by DXA using a PIXI2 bone densitometer (GE Medical System; Lunar, Madison, WI, USA). Data were presented as percent change from baseline. BMD of isolated tibia was also determined in vitro with the same machine.

Histology and histomorphometry Both tibias of mice were fixed with 10% neutral buffered formalin overnight, followed by decalcification in 14% EDTA for 4–5 days. The samples were dehydrated through increasing concentrations of ethanol. Paraffin-embedded sections were prepared and stained with TRACP to quantitate osteoclasts. Cancellous bone volume (BV/TV) and osteoclast number per millimeter of trabecular surface (OCs/BS) at the proximal metaphysic of the tibia were measured and analyzed using Osteomeasure (OsteoMetrics, Atlanta, GA, USA) in a blinded fashion.

Serum TRACP 5b assay Blood was collected retro-orbitally under anesthesia immediately before death. Serum TRACP 5b, a specific marker of osteoclastic bone resorption, was measured using a mouse specific ELISA assay (SB-TR103; Immuno Diagnostic Systems, Fountain Hills, AZ, USA) according to the protocols from manufacturer.

Bleeding time assays The bleeding time measurement was performed as described(20) by immersing the severed tip of the tail in 0.9% isotonic saline at 37°C. The bleeding time was defined as the time required for the stream of bleeding to cease in the absence of spontaneous clotting. All experiments were stopped at 10 minutes by cauterizing the tail.

Statistical analysis All data are presented as mean ± SD. Significance of differences was calculated by unpaired Student’s t-test. p values
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