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Osteoclast fusion is based on heterogeneity between fusion partners.

Hobolt-Pedersen AS, Delaissé JM, Søe K - Calcif. Tissue Int. (2014)

Bottom Line: These stainings revealed heterogeneous localization patterns of all three factors within a given culture of osteoclasts.CD47 was found to be localized primarily in small osteoclasts and preosteoclasts, which were also positive for DC-STAMP but negative for cathepsin K expression.Considering the in vivo environment in which osteoclasts develop and fuse, our findings seem very applicable and provide novel, important insight into key issues in bone and fusion research.

View Article: PubMed Central - PubMed

Affiliation: Clinical Cell Biology, Vejle Hospital/Lillebaelt Hospital, Institute of Regional Health Research, University of Southern Denmark, Kabbeltoft 25, 7100, Vejle, Denmark.

ABSTRACT
Bone-resorbing osteoclasts are formed through fusion of mononucleated precursors. Their choice of partners during the fusion process remains unclear. We hypothesized that osteoclasts are selective in their choice of fusion partner and that this selectivity is based on heterogeneity among the cells with respect to their maturation stage and their expression and cellular organization of fusion factors. Support for this hypothesis was found from immunofluorescence staining of the osteoclast fusion factors CD47, dendritic cell-specific transmembrane protein (DC-STAMP), and syncytin-1. These stainings revealed heterogeneous localization patterns of all three factors within a given culture of osteoclasts. CD47 was found to be localized primarily in small osteoclasts and preosteoclasts, which were also positive for DC-STAMP but negative for cathepsin K expression. A role of CD47 in the early osteoclast fusion steps was also suggested from experiments with a CD47 blocking antibody, which resulted in an inhibition of the fusion of small osteoclasts. Conversely, blocking of connexin 43 affected the fusion of larger osteoclasts with four or more nuclei. The suggestion that different fusion factors function at different stages of osteoclast fusion supports the idea of heterogeneity in the osteoclast population; our results suggest that osteoclast fusion is indeed based on heterogeneity. Considering the in vivo environment in which osteoclasts develop and fuse, our findings seem very applicable and provide novel, important insight into key issues in bone and fusion research.

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DC-STAMP and syncytin-1 are heterogeneously localized in (pre)OCs. Visualization of DC-STAMP and syncytin-1 in (pre)OCs (1 × 105 per well) differentiated for 5 days with RANKL. Nuclei were visualized with DAPI (blue). a Staining of DC-STAMP (red) and F-actin (green). b Staining of syncytin-1 (red) and F-actin (green). Note pattern of heterogeneous localization of both these proteins in cells in close proximity and opposite polarization of syncytin-1 (arrows)
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Fig5: DC-STAMP and syncytin-1 are heterogeneously localized in (pre)OCs. Visualization of DC-STAMP and syncytin-1 in (pre)OCs (1 × 105 per well) differentiated for 5 days with RANKL. Nuclei were visualized with DAPI (blue). a Staining of DC-STAMP (red) and F-actin (green). b Staining of syncytin-1 (red) and F-actin (green). Note pattern of heterogeneous localization of both these proteins in cells in close proximity and opposite polarization of syncytin-1 (arrows)

Mentions: The heterogeneity in the presence of CD47 among cells at different differentiation stages in (pre)OC culture and the clear presentation of this protein at the interface of fusing preOCs support the idea that CD47 is an important factor in OC formation (Figs. 2, 3, 4). These findings led to related studies of the localization of other known OC fusion factors. For this purpose, the factors DC-STAMP and syncytin-1 were chosen because of their documented involvement in OC fusion [5, 15–17]. Interestingly, heterogeneity was also found in the expression and localization of these factors. IF staining of F-actin, together with either DC-STAMP or syncytin-1, revealed complementary protein localization patterns in our (pre)OC cultures in both cases. Several of the DC-STAMP-stained cells exerted a pattern of either absence or of clear presence of this protein in the cellular membrane. The results in Fig. 5a show examples of such DC-STAMP-positive and -negative cells. The top pictures illustrate two cells in close proximity. The DC-STAMP-positive partner has a conformation resembling a phagocytic cup indicative of fusion [5]. Likewise, staining of syncytin-1 showed heterogeneity in the localization of this protein. In this case, opposite polarization of syncytin-1 in two fusion partners was observed in several cells (Fig. 5b), which supports our previously published findings [5]. Collectively, these results indicate a pattern of diversity in the localization of several known fusion factors between individual cells in a (pre)OC culture.Fig. 5


Osteoclast fusion is based on heterogeneity between fusion partners.

Hobolt-Pedersen AS, Delaissé JM, Søe K - Calcif. Tissue Int. (2014)

DC-STAMP and syncytin-1 are heterogeneously localized in (pre)OCs. Visualization of DC-STAMP and syncytin-1 in (pre)OCs (1 × 105 per well) differentiated for 5 days with RANKL. Nuclei were visualized with DAPI (blue). a Staining of DC-STAMP (red) and F-actin (green). b Staining of syncytin-1 (red) and F-actin (green). Note pattern of heterogeneous localization of both these proteins in cells in close proximity and opposite polarization of syncytin-1 (arrows)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4048669&req=5

Fig5: DC-STAMP and syncytin-1 are heterogeneously localized in (pre)OCs. Visualization of DC-STAMP and syncytin-1 in (pre)OCs (1 × 105 per well) differentiated for 5 days with RANKL. Nuclei were visualized with DAPI (blue). a Staining of DC-STAMP (red) and F-actin (green). b Staining of syncytin-1 (red) and F-actin (green). Note pattern of heterogeneous localization of both these proteins in cells in close proximity and opposite polarization of syncytin-1 (arrows)
Mentions: The heterogeneity in the presence of CD47 among cells at different differentiation stages in (pre)OC culture and the clear presentation of this protein at the interface of fusing preOCs support the idea that CD47 is an important factor in OC formation (Figs. 2, 3, 4). These findings led to related studies of the localization of other known OC fusion factors. For this purpose, the factors DC-STAMP and syncytin-1 were chosen because of their documented involvement in OC fusion [5, 15–17]. Interestingly, heterogeneity was also found in the expression and localization of these factors. IF staining of F-actin, together with either DC-STAMP or syncytin-1, revealed complementary protein localization patterns in our (pre)OC cultures in both cases. Several of the DC-STAMP-stained cells exerted a pattern of either absence or of clear presence of this protein in the cellular membrane. The results in Fig. 5a show examples of such DC-STAMP-positive and -negative cells. The top pictures illustrate two cells in close proximity. The DC-STAMP-positive partner has a conformation resembling a phagocytic cup indicative of fusion [5]. Likewise, staining of syncytin-1 showed heterogeneity in the localization of this protein. In this case, opposite polarization of syncytin-1 in two fusion partners was observed in several cells (Fig. 5b), which supports our previously published findings [5]. Collectively, these results indicate a pattern of diversity in the localization of several known fusion factors between individual cells in a (pre)OC culture.Fig. 5

Bottom Line: These stainings revealed heterogeneous localization patterns of all three factors within a given culture of osteoclasts.CD47 was found to be localized primarily in small osteoclasts and preosteoclasts, which were also positive for DC-STAMP but negative for cathepsin K expression.Considering the in vivo environment in which osteoclasts develop and fuse, our findings seem very applicable and provide novel, important insight into key issues in bone and fusion research.

View Article: PubMed Central - PubMed

Affiliation: Clinical Cell Biology, Vejle Hospital/Lillebaelt Hospital, Institute of Regional Health Research, University of Southern Denmark, Kabbeltoft 25, 7100, Vejle, Denmark.

ABSTRACT
Bone-resorbing osteoclasts are formed through fusion of mononucleated precursors. Their choice of partners during the fusion process remains unclear. We hypothesized that osteoclasts are selective in their choice of fusion partner and that this selectivity is based on heterogeneity among the cells with respect to their maturation stage and their expression and cellular organization of fusion factors. Support for this hypothesis was found from immunofluorescence staining of the osteoclast fusion factors CD47, dendritic cell-specific transmembrane protein (DC-STAMP), and syncytin-1. These stainings revealed heterogeneous localization patterns of all three factors within a given culture of osteoclasts. CD47 was found to be localized primarily in small osteoclasts and preosteoclasts, which were also positive for DC-STAMP but negative for cathepsin K expression. A role of CD47 in the early osteoclast fusion steps was also suggested from experiments with a CD47 blocking antibody, which resulted in an inhibition of the fusion of small osteoclasts. Conversely, blocking of connexin 43 affected the fusion of larger osteoclasts with four or more nuclei. The suggestion that different fusion factors function at different stages of osteoclast fusion supports the idea of heterogeneity in the osteoclast population; our results suggest that osteoclast fusion is indeed based on heterogeneity. Considering the in vivo environment in which osteoclasts develop and fuse, our findings seem very applicable and provide novel, important insight into key issues in bone and fusion research.

Show MeSH
Related in: MedlinePlus