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CD45 regulates retention, motility, and numbers of hematopoietic progenitors, and affects osteoclast remodeling of metaphyseal trabecules.

Shivtiel S, Kollet O, Lapid K, Schajnovitz A, Goichberg P, Kalinkovich A, Shezen E, Tesio M, Netzer N, Petit I, Sharir A, Lapidot T - J. Exp. Med. (2008)

Bottom Line: Moreover, immature CD45 knockout (KO) cells showed defective motility, including reduced homing (both steady state and in response to stromal-derived factor 1) and reduced granulocyte colony-stimulating factor mobilization.Consequently, CD45KO mice had less primitive cells in the BM and increased numbers of these cells in the spleen, yet with reduced homing and repopulation potential.Uncoupling environmental and intrinsic defects in chimeric mice, we demonstrated that CD45 regulates progenitor movement and retention by influencing both the hematopoietic and nonhematopoietic compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
The CD45 phosphatase is uniquely expressed by all leukocytes, but its role in regulating hematopoietic progenitors is poorly understood. We show that enhanced CD45 expression on bone marrow (BM) leukocytes correlates with increased cell motility in response to stress signals. Moreover, immature CD45 knockout (KO) cells showed defective motility, including reduced homing (both steady state and in response to stromal-derived factor 1) and reduced granulocyte colony-stimulating factor mobilization. These defects were associated with increased cell adhesion mediated by reduced matrix metalloproteinase 9 secretion and imbalanced Src kinase activity. Poor mobilization of CD45KO progenitors by the receptor activator of nuclear factor kappaB ligand, and impaired modulation of the endosteal components osteopontin and stem cell factor, suggested defective osteoclast function. Indeed, CD45KO osteoclasts exhibited impaired bone remodeling and abnormal morphology, which we attributed to defective cell fusion and Src function. This led to irregular distribution of metaphyseal bone trabecules, a region enriched with stem cell niches. Consequently, CD45KO mice had less primitive cells in the BM and increased numbers of these cells in the spleen, yet with reduced homing and repopulation potential. Uncoupling environmental and intrinsic defects in chimeric mice, we demonstrated that CD45 regulates progenitor movement and retention by influencing both the hematopoietic and nonhematopoietic compartments.

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Defective maturation of CD45KO osteoclasts in vitro involving impaired expression of MMPs, DC-STAMP, and Src kinase. (A) BM-derived osteoclasts from WT (top) and CD45KO (bottom) mice immunolabeled for CD45 expression (green), and stained for polymerized actin (red) and nuclear DNA (blue). Bars, 20 μm. (B) TRAP staining (purple) of BM-derived WT (top) and CD45KO (bottom) osteoclasts. Bars, 200 μm. (C and D) Semiquantitative PCR analysis for DC-STAMP mRNA levels in WT and CD45KO BM-derived cells. (C) Representative PCR image. (D) Summary of four independent experiments showing the ratio between DC-STAMP and GAPDH mRNA expression (±SE; *, P < 0.01). (E) Conditioned medium of WT versus CD45KO BM-derived osteoclasts was tested for the activity of secreted MMP-9 in a gelatin zymography assay. (F) WT and CD45KO BM-derived osteoclasts cultured with or without G-CSF were immunolabeled for expression of MT1-MMP (green), and stained for polymerized actin (red) and nuclear DNA (blue). Bar, 10 μm. (G) TRAP staining (purple) of BM-derived WT (top) and CD45KO (bottom) osteoclasts cultured in vitro in the presence of DMSO vehicle (left) or the Src inhibitor PP2 (right). Bars, 200 μm. (H) Src activity assay for osteoclast precursors incubated with 1 μM PP2 or DMSO (ctrl) for 5 d. Values indicate the fold changes of WT control mice ± SE (indicated by a horizontal line), showing a representative experiment.
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fig5: Defective maturation of CD45KO osteoclasts in vitro involving impaired expression of MMPs, DC-STAMP, and Src kinase. (A) BM-derived osteoclasts from WT (top) and CD45KO (bottom) mice immunolabeled for CD45 expression (green), and stained for polymerized actin (red) and nuclear DNA (blue). Bars, 20 μm. (B) TRAP staining (purple) of BM-derived WT (top) and CD45KO (bottom) osteoclasts. Bars, 200 μm. (C and D) Semiquantitative PCR analysis for DC-STAMP mRNA levels in WT and CD45KO BM-derived cells. (C) Representative PCR image. (D) Summary of four independent experiments showing the ratio between DC-STAMP and GAPDH mRNA expression (±SE; *, P < 0.01). (E) Conditioned medium of WT versus CD45KO BM-derived osteoclasts was tested for the activity of secreted MMP-9 in a gelatin zymography assay. (F) WT and CD45KO BM-derived osteoclasts cultured with or without G-CSF were immunolabeled for expression of MT1-MMP (green), and stained for polymerized actin (red) and nuclear DNA (blue). Bar, 10 μm. (G) TRAP staining (purple) of BM-derived WT (top) and CD45KO (bottom) osteoclasts cultured in vitro in the presence of DMSO vehicle (left) or the Src inhibitor PP2 (right). Bars, 200 μm. (H) Src activity assay for osteoclast precursors incubated with 1 μM PP2 or DMSO (ctrl) for 5 d. Values indicate the fold changes of WT control mice ± SE (indicated by a horizontal line), showing a representative experiment.

Mentions: We continued by investigating the involvement of CD45 in osteoclast development and function. First, we confirmed that mature multinucleated osteoclasts derived from the BM of WT mice express CD45 (Fig. 5 A, top left), whereas, as expected, CD45KO osteoclasts did not express this molecule (Fig. 5 A, bottom left). Next, we observed that CD45KO osteoclasts cultured and developed in vitro demonstrated abnormal morphology (Fig. 5 B). Appearance of the phosphatase TRAP indicates the maturation status and functional stage of bone-resorbing osteoclasts. TRAP staining of CD45KO osteoclasts grown in vitro showed their inability to acquire the flattened, spread morphology and assemble the typical sealing zone that can be seen as a purple ring in the perimeter of WT osteoclasts. In addition, these CD45KO osteoclasts appeared smaller, with a reduced ability to form multinucleated cells. Transcriptional assessment of DC-STAMP mRNA expression revealed that CD45KO BM cells exhibited reduced expression of this receptor (Fig. 5, C and D), which may lead to defects in cell fusion and maturation. We tested additional factors involved in osteoclast development and function, and examined expression of MMP-9 and MT1-MMP expressed by CD45KO osteoclasts. Notably, these cells secreted lower amounts of MMP-9 compared with their WT counterparts (Fig. 5 E), implying reduced osteoclast motility and activity. Moreover, G-CSF, which was shown to activate osteoclast development (30), induced up-regulation of MT1-MMP in WT osteoclasts, whereas expression of this enzyme in CD45KO osteoclasts remained low, as in the steady state (Fig. 5 F). CD45 deficiency is associated with hyperphosphorylation and activation of its substrate, Src, leading to impaired cell movement (Fig. 3). We therefore assessed osteoclast development in the presence of the Src inhibitor PP2. Src inhibition impaired the development and organization of WT osteoclasts (Fig. 5 G, top), resembling the Src−/− phenotype (31). Conversely, PP2 treatment restored the normal morphology and sealing zone assembly of CD45KO osteoclasts (Fig. 5 G, bottom). Src activity assay confirmed that treating CD45KO osteoclast precursors with PP2 reduced their hyperactive Src to levels equivalent to WT cells (Fig. 5 H), enabling the formation of CD45KO osteoclasts exhibiting a normal phenotype. These results suggest that the expression and function of CD45 and its downstream target Src in monocyte-derived osteoclasts are required for the regulation of normal osteoclast development.


CD45 regulates retention, motility, and numbers of hematopoietic progenitors, and affects osteoclast remodeling of metaphyseal trabecules.

Shivtiel S, Kollet O, Lapid K, Schajnovitz A, Goichberg P, Kalinkovich A, Shezen E, Tesio M, Netzer N, Petit I, Sharir A, Lapidot T - J. Exp. Med. (2008)

Defective maturation of CD45KO osteoclasts in vitro involving impaired expression of MMPs, DC-STAMP, and Src kinase. (A) BM-derived osteoclasts from WT (top) and CD45KO (bottom) mice immunolabeled for CD45 expression (green), and stained for polymerized actin (red) and nuclear DNA (blue). Bars, 20 μm. (B) TRAP staining (purple) of BM-derived WT (top) and CD45KO (bottom) osteoclasts. Bars, 200 μm. (C and D) Semiquantitative PCR analysis for DC-STAMP mRNA levels in WT and CD45KO BM-derived cells. (C) Representative PCR image. (D) Summary of four independent experiments showing the ratio between DC-STAMP and GAPDH mRNA expression (±SE; *, P < 0.01). (E) Conditioned medium of WT versus CD45KO BM-derived osteoclasts was tested for the activity of secreted MMP-9 in a gelatin zymography assay. (F) WT and CD45KO BM-derived osteoclasts cultured with or without G-CSF were immunolabeled for expression of MT1-MMP (green), and stained for polymerized actin (red) and nuclear DNA (blue). Bar, 10 μm. (G) TRAP staining (purple) of BM-derived WT (top) and CD45KO (bottom) osteoclasts cultured in vitro in the presence of DMSO vehicle (left) or the Src inhibitor PP2 (right). Bars, 200 μm. (H) Src activity assay for osteoclast precursors incubated with 1 μM PP2 or DMSO (ctrl) for 5 d. Values indicate the fold changes of WT control mice ± SE (indicated by a horizontal line), showing a representative experiment.
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fig5: Defective maturation of CD45KO osteoclasts in vitro involving impaired expression of MMPs, DC-STAMP, and Src kinase. (A) BM-derived osteoclasts from WT (top) and CD45KO (bottom) mice immunolabeled for CD45 expression (green), and stained for polymerized actin (red) and nuclear DNA (blue). Bars, 20 μm. (B) TRAP staining (purple) of BM-derived WT (top) and CD45KO (bottom) osteoclasts. Bars, 200 μm. (C and D) Semiquantitative PCR analysis for DC-STAMP mRNA levels in WT and CD45KO BM-derived cells. (C) Representative PCR image. (D) Summary of four independent experiments showing the ratio between DC-STAMP and GAPDH mRNA expression (±SE; *, P < 0.01). (E) Conditioned medium of WT versus CD45KO BM-derived osteoclasts was tested for the activity of secreted MMP-9 in a gelatin zymography assay. (F) WT and CD45KO BM-derived osteoclasts cultured with or without G-CSF were immunolabeled for expression of MT1-MMP (green), and stained for polymerized actin (red) and nuclear DNA (blue). Bar, 10 μm. (G) TRAP staining (purple) of BM-derived WT (top) and CD45KO (bottom) osteoclasts cultured in vitro in the presence of DMSO vehicle (left) or the Src inhibitor PP2 (right). Bars, 200 μm. (H) Src activity assay for osteoclast precursors incubated with 1 μM PP2 or DMSO (ctrl) for 5 d. Values indicate the fold changes of WT control mice ± SE (indicated by a horizontal line), showing a representative experiment.
Mentions: We continued by investigating the involvement of CD45 in osteoclast development and function. First, we confirmed that mature multinucleated osteoclasts derived from the BM of WT mice express CD45 (Fig. 5 A, top left), whereas, as expected, CD45KO osteoclasts did not express this molecule (Fig. 5 A, bottom left). Next, we observed that CD45KO osteoclasts cultured and developed in vitro demonstrated abnormal morphology (Fig. 5 B). Appearance of the phosphatase TRAP indicates the maturation status and functional stage of bone-resorbing osteoclasts. TRAP staining of CD45KO osteoclasts grown in vitro showed their inability to acquire the flattened, spread morphology and assemble the typical sealing zone that can be seen as a purple ring in the perimeter of WT osteoclasts. In addition, these CD45KO osteoclasts appeared smaller, with a reduced ability to form multinucleated cells. Transcriptional assessment of DC-STAMP mRNA expression revealed that CD45KO BM cells exhibited reduced expression of this receptor (Fig. 5, C and D), which may lead to defects in cell fusion and maturation. We tested additional factors involved in osteoclast development and function, and examined expression of MMP-9 and MT1-MMP expressed by CD45KO osteoclasts. Notably, these cells secreted lower amounts of MMP-9 compared with their WT counterparts (Fig. 5 E), implying reduced osteoclast motility and activity. Moreover, G-CSF, which was shown to activate osteoclast development (30), induced up-regulation of MT1-MMP in WT osteoclasts, whereas expression of this enzyme in CD45KO osteoclasts remained low, as in the steady state (Fig. 5 F). CD45 deficiency is associated with hyperphosphorylation and activation of its substrate, Src, leading to impaired cell movement (Fig. 3). We therefore assessed osteoclast development in the presence of the Src inhibitor PP2. Src inhibition impaired the development and organization of WT osteoclasts (Fig. 5 G, top), resembling the Src−/− phenotype (31). Conversely, PP2 treatment restored the normal morphology and sealing zone assembly of CD45KO osteoclasts (Fig. 5 G, bottom). Src activity assay confirmed that treating CD45KO osteoclast precursors with PP2 reduced their hyperactive Src to levels equivalent to WT cells (Fig. 5 H), enabling the formation of CD45KO osteoclasts exhibiting a normal phenotype. These results suggest that the expression and function of CD45 and its downstream target Src in monocyte-derived osteoclasts are required for the regulation of normal osteoclast development.

Bottom Line: Moreover, immature CD45 knockout (KO) cells showed defective motility, including reduced homing (both steady state and in response to stromal-derived factor 1) and reduced granulocyte colony-stimulating factor mobilization.Consequently, CD45KO mice had less primitive cells in the BM and increased numbers of these cells in the spleen, yet with reduced homing and repopulation potential.Uncoupling environmental and intrinsic defects in chimeric mice, we demonstrated that CD45 regulates progenitor movement and retention by influencing both the hematopoietic and nonhematopoietic compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
The CD45 phosphatase is uniquely expressed by all leukocytes, but its role in regulating hematopoietic progenitors is poorly understood. We show that enhanced CD45 expression on bone marrow (BM) leukocytes correlates with increased cell motility in response to stress signals. Moreover, immature CD45 knockout (KO) cells showed defective motility, including reduced homing (both steady state and in response to stromal-derived factor 1) and reduced granulocyte colony-stimulating factor mobilization. These defects were associated with increased cell adhesion mediated by reduced matrix metalloproteinase 9 secretion and imbalanced Src kinase activity. Poor mobilization of CD45KO progenitors by the receptor activator of nuclear factor kappaB ligand, and impaired modulation of the endosteal components osteopontin and stem cell factor, suggested defective osteoclast function. Indeed, CD45KO osteoclasts exhibited impaired bone remodeling and abnormal morphology, which we attributed to defective cell fusion and Src function. This led to irregular distribution of metaphyseal bone trabecules, a region enriched with stem cell niches. Consequently, CD45KO mice had less primitive cells in the BM and increased numbers of these cells in the spleen, yet with reduced homing and repopulation potential. Uncoupling environmental and intrinsic defects in chimeric mice, we demonstrated that CD45 regulates progenitor movement and retention by influencing both the hematopoietic and nonhematopoietic compartments.

Show MeSH
Related in: MedlinePlus