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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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CD47 is preferentially found in (pre)OCs, which are small both with respect to size and number of nuclei. a Staining of CD47 (red) and F-actin (green) in (pre)OCs differentiated for 5 days with RANKL. Nuclei were visualized with DAPI (blue). b The cells (seeding density: 5 × 104 per well) were assed for CD47 status. Then the area of each cell in 8 culture wells was measured in square pixels, and the sizes of the CD47-positive and CD47-negative cells were compared. Paired t test, P = 0.0004. c Relative distribution of CD47-positive and CD47-negative OCs according to their number of nuclei was calculated as described under Fig. 1b. Fisher’s exact test of the relation between CD47 status and proportion of OCs with 2 nuclei compared to those with 3 or more, P = 0.0209
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Fig2: CD47 is preferentially found in (pre)OCs, which are small both with respect to size and number of nuclei. a Staining of CD47 (red) and F-actin (green) in (pre)OCs differentiated for 5 days with RANKL. Nuclei were visualized with DAPI (blue). b The cells (seeding density: 5 × 104 per well) were assed for CD47 status. Then the area of each cell in 8 culture wells was measured in square pixels, and the sizes of the CD47-positive and CD47-negative cells were compared. Paired t test, P = 0.0004. c Relative distribution of CD47-positive and CD47-negative OCs according to their number of nuclei was calculated as described under Fig. 1b. Fisher’s exact test of the relation between CD47 status and proportion of OCs with 2 nuclei compared to those with 3 or more, P = 0.0209

Mentions: To visualize the presence of CD47 in fusing (pre)OCs in culture, and to supplement the results obtained by the blocking of CD47, IF staining of CD47 and F-actin was performed. Cells were fixed and stained at a point in time at which fusion was ongoing and where both preOCs and OCs at various fusion stages were observed. The staining showed the presence of CD47 both in preOCs and OCs; however, the most striking finding was that the cells presenting CD47, either in the whole cell or at the cell membrane, appeared to be relatively small (Fig. 2a). Many of these CD47-positive cells were rather compact, whereas the large OCs with a widespread flat appearance were often CD47 negative. In order to quantify the relevance of this indicative finding, (pre)OCs were categorized according to their CD47 status. A measurement of the area of (pre)OCs positive for CD47 revealed that they had a significantly smaller area than the CD47-negative cells (Fig. 2b). In support of this finding, we also discovered that CD47-positive OCs had significantly fewer nuclei than the CD47-negative OCs (Fig. 2c). Together, these results indicate that the CD47-presenting (pre)OCs are smaller than the CD47-negative cells in terms of both their size and their number of nuclei.Fig. 2


Osteoclast fusion is based on heterogeneity between fusion partners.

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

CD47 is preferentially found in (pre)OCs, which are small both with respect to size and number of nuclei. a Staining of CD47 (red) and F-actin (green) in (pre)OCs differentiated for 5 days with RANKL. Nuclei were visualized with DAPI (blue). b The cells (seeding density: 5 × 104 per well) were assed for CD47 status. Then the area of each cell in 8 culture wells was measured in square pixels, and the sizes of the CD47-positive and CD47-negative cells were compared. Paired t test, P = 0.0004. c Relative distribution of CD47-positive and CD47-negative OCs according to their number of nuclei was calculated as described under Fig. 1b. Fisher’s exact test of the relation between CD47 status and proportion of OCs with 2 nuclei compared to those with 3 or more, P = 0.0209
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048669&req=5

Fig2: CD47 is preferentially found in (pre)OCs, which are small both with respect to size and number of nuclei. a Staining of CD47 (red) and F-actin (green) in (pre)OCs differentiated for 5 days with RANKL. Nuclei were visualized with DAPI (blue). b The cells (seeding density: 5 × 104 per well) were assed for CD47 status. Then the area of each cell in 8 culture wells was measured in square pixels, and the sizes of the CD47-positive and CD47-negative cells were compared. Paired t test, P = 0.0004. c Relative distribution of CD47-positive and CD47-negative OCs according to their number of nuclei was calculated as described under Fig. 1b. Fisher’s exact test of the relation between CD47 status and proportion of OCs with 2 nuclei compared to those with 3 or more, P = 0.0209
Mentions: To visualize the presence of CD47 in fusing (pre)OCs in culture, and to supplement the results obtained by the blocking of CD47, IF staining of CD47 and F-actin was performed. Cells were fixed and stained at a point in time at which fusion was ongoing and where both preOCs and OCs at various fusion stages were observed. The staining showed the presence of CD47 both in preOCs and OCs; however, the most striking finding was that the cells presenting CD47, either in the whole cell or at the cell membrane, appeared to be relatively small (Fig. 2a). Many of these CD47-positive cells were rather compact, whereas the large OCs with a widespread flat appearance were often CD47 negative. In order to quantify the relevance of this indicative finding, (pre)OCs were categorized according to their CD47 status. A measurement of the area of (pre)OCs positive for CD47 revealed that they had a significantly smaller area than the CD47-negative cells (Fig. 2b). In support of this finding, we also discovered that CD47-positive OCs had significantly fewer nuclei than the CD47-negative OCs (Fig. 2c). Together, these results indicate that the CD47-presenting (pre)OCs are smaller than the CD47-negative cells in terms of both their size and their number of nuclei.Fig. 2

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