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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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Blocking of fusion factors CD47 and Cx43 affects fusion of OCs at different fusion stages. a Light microscopy images, at equal magnification, of Giemsa-stained (pre)OC cultures incubated without additives or with CD47-blocking antibody. b Relative distribution of OCs according to treatment and their number of nuclei. For each culture condition, counts from 3 different cultures were pooled, ranked according to the number of nuclei per OC, and the percentages of OCs with a given number of nuclei calculated. Fisher’s exact test of the relation between culture condition and proportion of OCs with 6 or more nuclei compared to those with fewer nuclei: CD47 antibody–treated OCs versus both of the controls, P < 0.0001 in both tests; isotype control vs. control without additives, P = not significant. c Relative distribution of OCs in an 18α-GA-treated (pre)OC culture and control according to their number of nuclei. Quantifications were done as in b. Fisher’s exact test of the relation between culture condition and proportion of OCs with 2 nuclei compared to those with 3 or more nuclei: 18α-GA-treated OCs vs. control, P = 0.0007
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Fig1: Blocking of fusion factors CD47 and Cx43 affects fusion of OCs at different fusion stages. a Light microscopy images, at equal magnification, of Giemsa-stained (pre)OC cultures incubated without additives or with CD47-blocking antibody. b Relative distribution of OCs according to treatment and their number of nuclei. For each culture condition, counts from 3 different cultures were pooled, ranked according to the number of nuclei per OC, and the percentages of OCs with a given number of nuclei calculated. Fisher’s exact test of the relation between culture condition and proportion of OCs with 6 or more nuclei compared to those with fewer nuclei: CD47 antibody–treated OCs versus both of the controls, P < 0.0001 in both tests; isotype control vs. control without additives, P = not significant. c Relative distribution of OCs in an 18α-GA-treated (pre)OC culture and control according to their number of nuclei. Quantifications were done as in b. Fisher’s exact test of the relation between culture condition and proportion of OCs with 2 nuclei compared to those with 3 or more nuclei: 18α-GA-treated OCs vs. control, P = 0.0007

Mentions: To evaluate the function of CD47 and Cx43 in human OC fusion, CD47 and Cx43 inhibitors were added to cultures of preOC at an early differentiation stage, at which time fusion had just started. Incubation of the preOCs with CD47-blocking antibody resulted in an increased formation of large nuclei-rich OCs, which were easily identified by light microscopy (Fig. 1a). This finding was consistent in four experiments. A comparison of the OCs treated with CD47 antibody with those of control cultures revealed an interesting distribution of the number of nuclei per OC: the proportion of OCs with six or more nuclei was significantly different from the proportion of OCs with fewer nuclei. While 26 and 29 % of the OCs in the control cultures had six or more nuclei, this proportion reached 52 % of the OCs treated with CD47 antibody (Fig. 1b).Fig. 1


Osteoclast fusion is based on heterogeneity between fusion partners.

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

Blocking of fusion factors CD47 and Cx43 affects fusion of OCs at different fusion stages. a Light microscopy images, at equal magnification, of Giemsa-stained (pre)OC cultures incubated without additives or with CD47-blocking antibody. b Relative distribution of OCs according to treatment and their number of nuclei. For each culture condition, counts from 3 different cultures were pooled, ranked according to the number of nuclei per OC, and the percentages of OCs with a given number of nuclei calculated. Fisher’s exact test of the relation between culture condition and proportion of OCs with 6 or more nuclei compared to those with fewer nuclei: CD47 antibody–treated OCs versus both of the controls, P < 0.0001 in both tests; isotype control vs. control without additives, P = not significant. c Relative distribution of OCs in an 18α-GA-treated (pre)OC culture and control according to their number of nuclei. Quantifications were done as in b. Fisher’s exact test of the relation between culture condition and proportion of OCs with 2 nuclei compared to those with 3 or more nuclei: 18α-GA-treated OCs vs. control, P = 0.0007
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048669&req=5

Fig1: Blocking of fusion factors CD47 and Cx43 affects fusion of OCs at different fusion stages. a Light microscopy images, at equal magnification, of Giemsa-stained (pre)OC cultures incubated without additives or with CD47-blocking antibody. b Relative distribution of OCs according to treatment and their number of nuclei. For each culture condition, counts from 3 different cultures were pooled, ranked according to the number of nuclei per OC, and the percentages of OCs with a given number of nuclei calculated. Fisher’s exact test of the relation between culture condition and proportion of OCs with 6 or more nuclei compared to those with fewer nuclei: CD47 antibody–treated OCs versus both of the controls, P < 0.0001 in both tests; isotype control vs. control without additives, P = not significant. c Relative distribution of OCs in an 18α-GA-treated (pre)OC culture and control according to their number of nuclei. Quantifications were done as in b. Fisher’s exact test of the relation between culture condition and proportion of OCs with 2 nuclei compared to those with 3 or more nuclei: 18α-GA-treated OCs vs. control, P = 0.0007
Mentions: To evaluate the function of CD47 and Cx43 in human OC fusion, CD47 and Cx43 inhibitors were added to cultures of preOC at an early differentiation stage, at which time fusion had just started. Incubation of the preOCs with CD47-blocking antibody resulted in an increased formation of large nuclei-rich OCs, which were easily identified by light microscopy (Fig. 1a). This finding was consistent in four experiments. A comparison of the OCs treated with CD47 antibody with those of control cultures revealed an interesting distribution of the number of nuclei per OC: the proportion of OCs with six or more nuclei was significantly different from the proportion of OCs with fewer nuclei. While 26 and 29 % of the OCs in the control cultures had six or more nuclei, this proportion reached 52 % of the OCs treated with CD47 antibody (Fig. 1b).Fig. 1

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