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Development of severe skeletal defects in induced SHP-2-deficient adult mice: a model of skeletal malformation in humans with SHP-2 mutations.

Bauler TJ, Kamiya N, Lapinski PE, Langewisch E, Mishina Y, Wilkinson JE, Feng GS, King PD - Dis Model Mech (2010)

Bottom Line: Induced deletion of SHP-2 resulted in impaired hematopoiesis, weight loss and lethality.Skeletal malformations were associated with alterations in cartilage and a marked increase in trabecular bone mass.The model is predicted to be of further use in understanding how SHP-2 regulates skeletal morphogenesis, which could lead to the development of novel therapies for the treatment of skeletal malformations in human patients with SHP-2 mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-5620, USA.

ABSTRACT
SHP-2 (encoded by PTPN11) is a ubiquitously expressed protein tyrosine phosphatase required for signal transduction by multiple different cell surface receptors. Humans with germline SHP-2 mutations develop Noonan syndrome or LEOPARD syndrome, which are characterized by cardiovascular, neurological and skeletal abnormalities. To study how SHP-2 regulates tissue homeostasis in normal adults, we used a conditional SHP-2 mouse mutant in which loss of expression of SHP-2 was induced in multiple tissues in response to drug administration. Induced deletion of SHP-2 resulted in impaired hematopoiesis, weight loss and lethality. Most strikingly, induced SHP-2-deficient mice developed severe skeletal abnormalities, including kyphoses and scolioses of the spine. Skeletal malformations were associated with alterations in cartilage and a marked increase in trabecular bone mass. Osteoclasts were essentially absent from the bones of SHP-2-deficient mice, thus accounting for the osteopetrotic phenotype. Studies in vitro revealed that osteoclastogenesis that was stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) was defective in SHP-2-deficient mice. At least in part, this was explained by a requirement for SHP-2 in M-CSF-induced activation of the pro-survival protein kinase AKT in hematopoietic precursor cells. These findings illustrate an essential role for SHP-2 in skeletal growth and remodeling in adults, and reveal some of the cellular and molecular mechanisms involved. The model is predicted to be of further use in understanding how SHP-2 regulates skeletal morphogenesis, which could lead to the development of novel therapies for the treatment of skeletal malformations in human patients with SHP-2 mutations.

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Blocked M-CSF signal transduction in induced SHP-2-deficient mice. (A) Bone marrow cells from a ptpn11fl/fl ert2-cre mouse and a littermate ptpn11fl/fl mouse (both treated with tamoxifen 10 days beforehand at 7 weeks of age) were cultured in wells of a 24-well plate (1×106 cells/well) in the presence of M-CSF. After 5 days, macrophages in wells were harvested and counted. Depicted is the mean number of macrophages ± 1 s.e.m. (n=4). Results are representative of four repeat experiments. Statistical significance was determined by two sample Student’s t-test. **P<0.005. (B) Western blots showing expression of SHP-2 in bone marrow cells (freshly isolated or after culture in M-CSF for 5 days) from a ptpn11fl/fl ert2-cre mouse and a littermate ptpn11fl/fl mouse (both treated with tamoxifen 3 weeks beforehand at 6 weeks of age). NS, non-specific band. Blots were stripped and reprobed with an anti-GAPDH antibody to verify equivalent protein loading. Similar results were obtained in five independent experiments. (C) Lineage-negative bone marrow cells from a ptpn11fl/fl ert2-cre mouse and a littermate ptpn11fl/fl mouse (both treated with tamoxifen 10 days beforehand at 6 weeks of age) were stimulated with M-CSF for the indicated times (in minutes). Activation of AKT was determined by western blotting of whole-cell lysates using a phospho-specific anti-AKT antibody. Blots were reprobed with an anti-AKT antibody to verify equal loading. Similar results were obtained in three repeat experiments.
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f7-0040228: Blocked M-CSF signal transduction in induced SHP-2-deficient mice. (A) Bone marrow cells from a ptpn11fl/fl ert2-cre mouse and a littermate ptpn11fl/fl mouse (both treated with tamoxifen 10 days beforehand at 7 weeks of age) were cultured in wells of a 24-well plate (1×106 cells/well) in the presence of M-CSF. After 5 days, macrophages in wells were harvested and counted. Depicted is the mean number of macrophages ± 1 s.e.m. (n=4). Results are representative of four repeat experiments. Statistical significance was determined by two sample Student’s t-test. **P<0.005. (B) Western blots showing expression of SHP-2 in bone marrow cells (freshly isolated or after culture in M-CSF for 5 days) from a ptpn11fl/fl ert2-cre mouse and a littermate ptpn11fl/fl mouse (both treated with tamoxifen 3 weeks beforehand at 6 weeks of age). NS, non-specific band. Blots were stripped and reprobed with an anti-GAPDH antibody to verify equivalent protein loading. Similar results were obtained in five independent experiments. (C) Lineage-negative bone marrow cells from a ptpn11fl/fl ert2-cre mouse and a littermate ptpn11fl/fl mouse (both treated with tamoxifen 10 days beforehand at 6 weeks of age) were stimulated with M-CSF for the indicated times (in minutes). Activation of AKT was determined by western blotting of whole-cell lysates using a phospho-specific anti-AKT antibody. Blots were reprobed with an anti-AKT antibody to verify equal loading. Similar results were obtained in three repeat experiments.

Mentions: Defective osteoclastogenesis in tamoxifen-treated ptpn11fl/fl ert2-cre mice could be explained by impaired M-CSF or RANKL signaling, or both. Evidence that at least M-CSF signaling is blocked in the absence of SHP-2 was provided by experiments in which bone marrow cells were treated with this cytokine alone (Fig. 7). Treatment of control bone marrow cells with M-CSF for 5 days promoted the development of abundant numbers of macrophages, as expected. By contrast, considerably fewer macrophages grew out from cultures of ptpn11fl/fl ert2-cre bone marrow treated with M-CSF for the same time (Fig. 7A). Furthermore, of those macrophages that did develop in ptpn11fl/fl ert2-cre bone marrow cultures, these were found to express the same levels of SHP-2 as macrophages that had grown out from control cultures (Fig. 7B). These findings suggest that SHP-2 is essential for M-CSF signal transduction in macrophage-osteoclast precursor cells, i.e. only those small number of precursors that retain expression of SHP-2 in tamoxifen-treated ptpn11fl/fl ert2-cre mice are able to respond to M-CSF. In itself, this impaired response to M-CSF would account for the defective osteoclastogenesis in induced SHP-2-deficient mice.


Development of severe skeletal defects in induced SHP-2-deficient adult mice: a model of skeletal malformation in humans with SHP-2 mutations.

Bauler TJ, Kamiya N, Lapinski PE, Langewisch E, Mishina Y, Wilkinson JE, Feng GS, King PD - Dis Model Mech (2010)

Blocked M-CSF signal transduction in induced SHP-2-deficient mice. (A) Bone marrow cells from a ptpn11fl/fl ert2-cre mouse and a littermate ptpn11fl/fl mouse (both treated with tamoxifen 10 days beforehand at 7 weeks of age) were cultured in wells of a 24-well plate (1×106 cells/well) in the presence of M-CSF. After 5 days, macrophages in wells were harvested and counted. Depicted is the mean number of macrophages ± 1 s.e.m. (n=4). Results are representative of four repeat experiments. Statistical significance was determined by two sample Student’s t-test. **P<0.005. (B) Western blots showing expression of SHP-2 in bone marrow cells (freshly isolated or after culture in M-CSF for 5 days) from a ptpn11fl/fl ert2-cre mouse and a littermate ptpn11fl/fl mouse (both treated with tamoxifen 3 weeks beforehand at 6 weeks of age). NS, non-specific band. Blots were stripped and reprobed with an anti-GAPDH antibody to verify equivalent protein loading. Similar results were obtained in five independent experiments. (C) Lineage-negative bone marrow cells from a ptpn11fl/fl ert2-cre mouse and a littermate ptpn11fl/fl mouse (both treated with tamoxifen 10 days beforehand at 6 weeks of age) were stimulated with M-CSF for the indicated times (in minutes). Activation of AKT was determined by western blotting of whole-cell lysates using a phospho-specific anti-AKT antibody. Blots were reprobed with an anti-AKT antibody to verify equal loading. Similar results were obtained in three repeat experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3046097&req=5

f7-0040228: Blocked M-CSF signal transduction in induced SHP-2-deficient mice. (A) Bone marrow cells from a ptpn11fl/fl ert2-cre mouse and a littermate ptpn11fl/fl mouse (both treated with tamoxifen 10 days beforehand at 7 weeks of age) were cultured in wells of a 24-well plate (1×106 cells/well) in the presence of M-CSF. After 5 days, macrophages in wells were harvested and counted. Depicted is the mean number of macrophages ± 1 s.e.m. (n=4). Results are representative of four repeat experiments. Statistical significance was determined by two sample Student’s t-test. **P<0.005. (B) Western blots showing expression of SHP-2 in bone marrow cells (freshly isolated or after culture in M-CSF for 5 days) from a ptpn11fl/fl ert2-cre mouse and a littermate ptpn11fl/fl mouse (both treated with tamoxifen 3 weeks beforehand at 6 weeks of age). NS, non-specific band. Blots were stripped and reprobed with an anti-GAPDH antibody to verify equivalent protein loading. Similar results were obtained in five independent experiments. (C) Lineage-negative bone marrow cells from a ptpn11fl/fl ert2-cre mouse and a littermate ptpn11fl/fl mouse (both treated with tamoxifen 10 days beforehand at 6 weeks of age) were stimulated with M-CSF for the indicated times (in minutes). Activation of AKT was determined by western blotting of whole-cell lysates using a phospho-specific anti-AKT antibody. Blots were reprobed with an anti-AKT antibody to verify equal loading. Similar results were obtained in three repeat experiments.
Mentions: Defective osteoclastogenesis in tamoxifen-treated ptpn11fl/fl ert2-cre mice could be explained by impaired M-CSF or RANKL signaling, or both. Evidence that at least M-CSF signaling is blocked in the absence of SHP-2 was provided by experiments in which bone marrow cells were treated with this cytokine alone (Fig. 7). Treatment of control bone marrow cells with M-CSF for 5 days promoted the development of abundant numbers of macrophages, as expected. By contrast, considerably fewer macrophages grew out from cultures of ptpn11fl/fl ert2-cre bone marrow treated with M-CSF for the same time (Fig. 7A). Furthermore, of those macrophages that did develop in ptpn11fl/fl ert2-cre bone marrow cultures, these were found to express the same levels of SHP-2 as macrophages that had grown out from control cultures (Fig. 7B). These findings suggest that SHP-2 is essential for M-CSF signal transduction in macrophage-osteoclast precursor cells, i.e. only those small number of precursors that retain expression of SHP-2 in tamoxifen-treated ptpn11fl/fl ert2-cre mice are able to respond to M-CSF. In itself, this impaired response to M-CSF would account for the defective osteoclastogenesis in induced SHP-2-deficient mice.

Bottom Line: Induced deletion of SHP-2 resulted in impaired hematopoiesis, weight loss and lethality.Skeletal malformations were associated with alterations in cartilage and a marked increase in trabecular bone mass.The model is predicted to be of further use in understanding how SHP-2 regulates skeletal morphogenesis, which could lead to the development of novel therapies for the treatment of skeletal malformations in human patients with SHP-2 mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-5620, USA.

ABSTRACT
SHP-2 (encoded by PTPN11) is a ubiquitously expressed protein tyrosine phosphatase required for signal transduction by multiple different cell surface receptors. Humans with germline SHP-2 mutations develop Noonan syndrome or LEOPARD syndrome, which are characterized by cardiovascular, neurological and skeletal abnormalities. To study how SHP-2 regulates tissue homeostasis in normal adults, we used a conditional SHP-2 mouse mutant in which loss of expression of SHP-2 was induced in multiple tissues in response to drug administration. Induced deletion of SHP-2 resulted in impaired hematopoiesis, weight loss and lethality. Most strikingly, induced SHP-2-deficient mice developed severe skeletal abnormalities, including kyphoses and scolioses of the spine. Skeletal malformations were associated with alterations in cartilage and a marked increase in trabecular bone mass. Osteoclasts were essentially absent from the bones of SHP-2-deficient mice, thus accounting for the osteopetrotic phenotype. Studies in vitro revealed that osteoclastogenesis that was stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) was defective in SHP-2-deficient mice. At least in part, this was explained by a requirement for SHP-2 in M-CSF-induced activation of the pro-survival protein kinase AKT in hematopoietic precursor cells. These findings illustrate an essential role for SHP-2 in skeletal growth and remodeling in adults, and reveal some of the cellular and molecular mechanisms involved. The model is predicted to be of further use in understanding how SHP-2 regulates skeletal morphogenesis, which could lead to the development of novel therapies for the treatment of skeletal malformations in human patients with SHP-2 mutations.

Show MeSH
Related in: MedlinePlus