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. 2001 May;107(9):1137-44.
doi: 10.1172/JCI12040.

A Glanzmann's mutation in beta 3 integrin specifically impairs osteoclast function

X Feng  1 , D V Novack, R Faccio, D S Ory, K Aya, M I Boyer, K P McHugh, F P Ross, S L Teitelbaum
Affiliations

A Glanzmann's mutation in beta 3 integrin specifically impairs osteoclast function

X Feng et al. J Clin Invest. 2001 May.

Abstract

Osteoclastic bone resorption requires cell-matrix contact, an event mediated by the alpha v beta 3 integrin. The structural components of the integrin that mediate osteoclast function are, however, not in hand. To address this issue, we generated mice lacking the beta 3 integrin gene, which have dysfunctional osteoclasts. Here, we show the full rescue of beta 3(-/-) osteoclast function following expression of a full-length beta 3 integrin. In contrast, truncated beta 3, lacking a cytoplasmic domain (h beta 3c), is completely ineffective in restoring function to beta 3(-/-) osteoclasts. To identify the components of the beta 3 cytoplasmic domain regulating osteoclast function, we generated six point mutants known, in other circumstances, to mediate beta integrin signaling. Of the six, only the S(752)P substitution, which also characterizes a form of the human bleeding disorder Glanzmann's thrombasthenia, fails to rescue beta 3(-/-) osteoclasts or restore ligand-activated signaling in the form of c-src activation. Interestingly, the double mutation Y(747)F/Y(759)F, which disrupts platelet function, does not affect the osteoclast. Thus similarities and distinctions exist in the mechanisms by which the beta 3 integrin regulates platelets and osteoclasts.

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Figures

Figure 1
Figure 1
Efficient hβ3 integrin surface expression is obtained after retroviral transduction of primary osteoclast precursors. (a) Schematic of full-length and truncated hβ3 proteins produced by retroviral transduction of macrophages with ΔU3-hβ3 and ΔU3-hβ3Δc constructs. Black boxes, transmembrane domain; hatched box, cytoplasmic domain. (b) Flow cytometric analysis of β3–/– murine marrow macrophages transduced with virus bearing β-gal, hβ3, or hβ3Δc. The cells were subjected to FACS analysis using a mAb (1A2) recognizing human but not murine β3, and a FITC-conjugated secondary Ab (1A2 + 2oAb). An internal negative control using secondary antibody alone (2oAb) is shown in each panel. Cultures transduced with β-gal virus show no staining above background, while those transduced with either hβ3 or hβ3Δc show 80–90% of cells with significant integrin expression.
Figure 2
Figure 2
The β3 integrin cytoplasmic domain is essential for osteoclast spreading and resorptive activity. (a) TRAP-stained osteoclast cultures derived from WT and β3–/– marrow macrophages, and from β3–/– macrophages transduced with virus encoding hβ3 or hβ3Δc, in coculture with ST2 stromal cells (arrows, osteoclasts; arrowheads, ST2 stromal cells; scale bar = 100 μm). (b) β3–/– BMMs, either virgin or hβ3- or hβ3Δc-transduced, were cultured in osteoclastogenic conditions for 9 days with M-CSF and RANKL. Cultures were then immunostained with anti-hβ3 external domain mAb and incubated with Alexa488-phalloidin to visualize fibrillar actin. (c) Osteoclasts were generated as in a, on whale dentin. Eight days later, resorption lacunae were examined by scanning electron microscopy.
Figure 3
Figure 3
β3 integrin S752 uniquely regulates osteoclast spreading. (a) Sequences of six point mutants of β3 integrin cytoplasmic domain. (b) Osteoclasts derived from β3–/– macrophages infected with virus encoding WT hβ3 or mutations (detailed in a) were stained for TRAP activity after 8 days of ST2 coculture. Scale bar = 100 μm. (c) Percent surface area of culture covered by spread osteoclasts for the experiment shown in b. Results are typical of those seen in four separate experiments. AP < 0.001 compared with hβ3 (without mutation); error bars represent SEM.
Figure 4
Figure 4
hβ3(S752P) is effectively expressed by β3–/– osteoclasts and their precursors. (a) Flow cytometric analysis of β3–/– BMMs nontransduced or transduced with virus encoding hβ3(D723A), hβ3(S752P), or hβ3(Y747F/Y759F), for hβ3 expression using 1A2 (1A2 + 2oAb). All mutants are expressed at approximately equivalent levels. An internal negative control using secondary antibody alone (2oAb) is shown in each panel. (b) Mature osteoclasts, generated from the same transduced β3–/– precursors shown in a, were analyzed for expression of the various hβ3 mutants by immunofluorescence using anti-hβ3 external domain mAb.
Figure 5
Figure 5
β3 integrin S752P uniquely regulates osteoclast resorptive activity. (a) β3–/– marrow macrophages, either nontransduced (β3–/–) or transduced with retrovirus encoding hβ3 or its D723A, S752P, or Y747F/Y759F mutants were cultured in osteoclastogenic conditions with ST2 cells on slices of whale dentin for 8 days. Resorption lacunae were examined by scanning electron microscopy. (b) Pit density and resorbed area were determined by examination of Coomassie brilliant blue–stained dentin slices using light microscopy. Pit depth was determined by confocal microscopy. AP < 0.001 compared with WT in all panels; BP < 0.01 compared with both WT and β3–/–); error bars represent SEM.
Figure 6
Figure 6
Activation of c-src requires the cytoplasmic tail of β3, and is abrogated by the S752P mutation but not the Y747F/Y759F mutation. β3–/– BMMs transduced with hβ3, hβ3Δc, or the S752P and Y747F/Y759F mutants were grown in M-CSF and RANKL for 3 days to generate early (not fully spread) osteoclasts. Following overnight starvation, cells were lifted with EDTA, and either kept in suspension (S) or adhered to vitronectin-coated plates (A) for 1 hour. As a control, cleared lysates were analyzed by immunoblot for total c-src. Activation of c-src was determined by immunoprecipitation of equal amounts of lysates with the anti-phosphotyrosine Ab PY99, followed by immunoblot with anti-pY416src. The ratio of pY416src band intensity between adherent and suspension cells (A/S) is indicated, normalized to total c-src levels.
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