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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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Related in: MedlinePlus

Similar to the OC marker DC-STAMP, surface localization of CD47 is primarily found among preOCs and OCs at early differentiation stages (seeding density: 1 × 105 per well). a Distribution of CD47- or DC-STAMP-positive (pre)OCs according to their number of nuclei. Counts of (pre)OCs from 4 different CD47 and DC-STAMP double-stained cultures were pooled, ranked according to the number of nuclei per cell, and percentages calculated of (pre)OCs, with a given number of nuclei, presenting CD47 or DC-STAMP in the cellular membrane. Statistics: 2-way ANOVA, P = not significant. b Distribution of all (pre)OCs analyzed according to their CD47 and DC-STAMP status and their number of nuclei. Statistics: 2-way ANOVA test of correlation between nuclei distribution and cell status among 4 variable CD47/DC-STAMP cell phenotypes (±), (+/+), (∓), and (−/−), P < 0.0001. c Distribution of CD47- or CatK-positive (pre)OCs according to their number of nuclei. Data were pooled from countings of (pre)OCs in 4 different CD47 and CatK double-stained cultures. Cells were ranked according to their number of nuclei and percentages calculated of CD47- or CatK-positive cells. Statistics: 2-way ANOVA, P = 0.0005. d Distribution of all (pre)OCs analyzed according to their CD47 and CatK status and their number of nuclei. Statistics: 2-way ANOVA test of the correlation between nuclei distribution and cell status among the 4 variable CD47/CatK cell phenotypes (±), (+/+), (∓), and (−/−), P < 0.0001. Bonferroni posttests: statistical significance was defined as *P < 0.05, and ***P < 0.001
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Fig3: Similar to the OC marker DC-STAMP, surface localization of CD47 is primarily found among preOCs and OCs at early differentiation stages (seeding density: 1 × 105 per well). a Distribution of CD47- or DC-STAMP-positive (pre)OCs according to their number of nuclei. Counts of (pre)OCs from 4 different CD47 and DC-STAMP double-stained cultures were pooled, ranked according to the number of nuclei per cell, and percentages calculated of (pre)OCs, with a given number of nuclei, presenting CD47 or DC-STAMP in the cellular membrane. Statistics: 2-way ANOVA, P = not significant. b Distribution of all (pre)OCs analyzed according to their CD47 and DC-STAMP status and their number of nuclei. Statistics: 2-way ANOVA test of correlation between nuclei distribution and cell status among 4 variable CD47/DC-STAMP cell phenotypes (±), (+/+), (∓), and (−/−), P < 0.0001. c Distribution of CD47- or CatK-positive (pre)OCs according to their number of nuclei. Data were pooled from countings of (pre)OCs in 4 different CD47 and CatK double-stained cultures. Cells were ranked according to their number of nuclei and percentages calculated of CD47- or CatK-positive cells. Statistics: 2-way ANOVA, P = 0.0005. d Distribution of all (pre)OCs analyzed according to their CD47 and CatK status and their number of nuclei. Statistics: 2-way ANOVA test of the correlation between nuclei distribution and cell status among the 4 variable CD47/CatK cell phenotypes (±), (+/+), (∓), and (−/−), P < 0.0001. Bonferroni posttests: statistical significance was defined as *P < 0.05, and ***P < 0.001

Mentions: To relate these findings to OC maturity in terms of differentiation stage, we correlated the presence of CD47 to that of OC differentiation markers DC-STAMP and CatK in the individual (pre)OCs. Membrane-associated DC-STAMP is well documented to be a marker of early OC differentiation stages [26, 29], while CatK is a marker for rather late stages [30]. We consider multinucleation of the OCs to be an indicator of progressed differentiation, and thereby we regard cells with several nuclei as more mature than those with few. The (pre)OCs were analyzed for their membrane localization of CD47 and DC-STAMP (Fig. 3a, b), or their CD47 status was compared to CatK expression (Fig. 3c, d). Analyses were done for each individual cell using the described categorization. The results revealed that the cells show comparable patterns of CD47 and DC-STAMP presentation, with the highest prevalence of both proteins in preOCs. The proportion of cells presenting CD47 or DC-STAMP at their membrane declined among the binucleated OCs and further among those with three or more nuclei (Fig. 3a). Consistently, double staining of CD47 and DC-STAMP showed that these factors are primarily presented by the same (pre)OCs (Fig. 3b). Actually, 77 % of the CD47-positive (pre)OCs were also positive for surface DC-STAMP (data not shown). On the contrary, the corresponding comparison between CD47 and CatK in the cells showed significantly different patterns of (pre)OCs distribution according to number of nuclei. Because the presence of CatK in the cells became more frequent with their differentiation, the proportions of CatK presenting (pre)OCs reached the highest level among those cells with three or more nuclei (Fig. 3c). Consistently, only a little overlap between CD47 and CatK presentation in the individual (pre)OCs was found (Fig. 3d). Less than 28 % of all CD47-positive cells in the analysis showed concurrent expression of CatK (data not shown). Thus, the pattern of CD47 presence among the (pre)OCs closely resembled that described for their surface expression of DC-STAMP, and thereby CD47 is characteristically related to (pre)OCs early in their differentiation.Fig. 3


Osteoclast fusion is based on heterogeneity between fusion partners.

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

Similar to the OC marker DC-STAMP, surface localization of CD47 is primarily found among preOCs and OCs at early differentiation stages (seeding density: 1 × 105 per well). a Distribution of CD47- or DC-STAMP-positive (pre)OCs according to their number of nuclei. Counts of (pre)OCs from 4 different CD47 and DC-STAMP double-stained cultures were pooled, ranked according to the number of nuclei per cell, and percentages calculated of (pre)OCs, with a given number of nuclei, presenting CD47 or DC-STAMP in the cellular membrane. Statistics: 2-way ANOVA, P = not significant. b Distribution of all (pre)OCs analyzed according to their CD47 and DC-STAMP status and their number of nuclei. Statistics: 2-way ANOVA test of correlation between nuclei distribution and cell status among 4 variable CD47/DC-STAMP cell phenotypes (±), (+/+), (∓), and (−/−), P < 0.0001. c Distribution of CD47- or CatK-positive (pre)OCs according to their number of nuclei. Data were pooled from countings of (pre)OCs in 4 different CD47 and CatK double-stained cultures. Cells were ranked according to their number of nuclei and percentages calculated of CD47- or CatK-positive cells. Statistics: 2-way ANOVA, P = 0.0005. d Distribution of all (pre)OCs analyzed according to their CD47 and CatK status and their number of nuclei. Statistics: 2-way ANOVA test of the correlation between nuclei distribution and cell status among the 4 variable CD47/CatK cell phenotypes (±), (+/+), (∓), and (−/−), P < 0.0001. Bonferroni posttests: statistical significance was defined as *P < 0.05, and ***P < 0.001
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Fig3: Similar to the OC marker DC-STAMP, surface localization of CD47 is primarily found among preOCs and OCs at early differentiation stages (seeding density: 1 × 105 per well). a Distribution of CD47- or DC-STAMP-positive (pre)OCs according to their number of nuclei. Counts of (pre)OCs from 4 different CD47 and DC-STAMP double-stained cultures were pooled, ranked according to the number of nuclei per cell, and percentages calculated of (pre)OCs, with a given number of nuclei, presenting CD47 or DC-STAMP in the cellular membrane. Statistics: 2-way ANOVA, P = not significant. b Distribution of all (pre)OCs analyzed according to their CD47 and DC-STAMP status and their number of nuclei. Statistics: 2-way ANOVA test of correlation between nuclei distribution and cell status among 4 variable CD47/DC-STAMP cell phenotypes (±), (+/+), (∓), and (−/−), P < 0.0001. c Distribution of CD47- or CatK-positive (pre)OCs according to their number of nuclei. Data were pooled from countings of (pre)OCs in 4 different CD47 and CatK double-stained cultures. Cells were ranked according to their number of nuclei and percentages calculated of CD47- or CatK-positive cells. Statistics: 2-way ANOVA, P = 0.0005. d Distribution of all (pre)OCs analyzed according to their CD47 and CatK status and their number of nuclei. Statistics: 2-way ANOVA test of the correlation between nuclei distribution and cell status among the 4 variable CD47/CatK cell phenotypes (±), (+/+), (∓), and (−/−), P < 0.0001. Bonferroni posttests: statistical significance was defined as *P < 0.05, and ***P < 0.001
Mentions: To relate these findings to OC maturity in terms of differentiation stage, we correlated the presence of CD47 to that of OC differentiation markers DC-STAMP and CatK in the individual (pre)OCs. Membrane-associated DC-STAMP is well documented to be a marker of early OC differentiation stages [26, 29], while CatK is a marker for rather late stages [30]. We consider multinucleation of the OCs to be an indicator of progressed differentiation, and thereby we regard cells with several nuclei as more mature than those with few. The (pre)OCs were analyzed for their membrane localization of CD47 and DC-STAMP (Fig. 3a, b), or their CD47 status was compared to CatK expression (Fig. 3c, d). Analyses were done for each individual cell using the described categorization. The results revealed that the cells show comparable patterns of CD47 and DC-STAMP presentation, with the highest prevalence of both proteins in preOCs. The proportion of cells presenting CD47 or DC-STAMP at their membrane declined among the binucleated OCs and further among those with three or more nuclei (Fig. 3a). Consistently, double staining of CD47 and DC-STAMP showed that these factors are primarily presented by the same (pre)OCs (Fig. 3b). Actually, 77 % of the CD47-positive (pre)OCs were also positive for surface DC-STAMP (data not shown). On the contrary, the corresponding comparison between CD47 and CatK in the cells showed significantly different patterns of (pre)OCs distribution according to number of nuclei. Because the presence of CatK in the cells became more frequent with their differentiation, the proportions of CatK presenting (pre)OCs reached the highest level among those cells with three or more nuclei (Fig. 3c). Consistently, only a little overlap between CD47 and CatK presentation in the individual (pre)OCs was found (Fig. 3d). Less than 28 % of all CD47-positive cells in the analysis showed concurrent expression of CatK (data not shown). Thus, the pattern of CD47 presence among the (pre)OCs closely resembled that described for their surface expression of DC-STAMP, and thereby CD47 is characteristically related to (pre)OCs early in their differentiation.Fig. 3

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