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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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Impaired osteoclastogenesis in induced SHP-2-deficient mice. (A) Femur sections of tamoxifen-injected moribund ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate controls were stained for TRAP to visualize osteoclasts (red color). Images are from 11-week-old mice injected with tamoxifen 5 weeks previously. The location of osteoclasts beneath growth plates (g) and within the secondary spongiosa are indicated with blue arrowheads. Note the paucity of osteoclasts in ptpn11fl/fl ert2-cre mice. Scale bars: top panels, 500 μm; bottom panels, 200 μm. (B) Bone marrow cells from ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate control mice, both treated with tamoxifen 3 weeks previously (at 7 weeks of age), were cultured with M-CSF and RANKL for 7 days on glass coverslips. Osteoclasts were identified by TRAP staining. Note the abundance of multinucleated osteoclasts in control cultures and absence from ptpn11fl/fl ert2-cre cultures. Scale bars: 100 μm. (C) Shown are the mean numbers of osteoclasts + 1 s.e.m. per field identified in bone sections (in situ) and in vitro osteoclast differentiation experiments described in A and B. For in situ analysis, the field size was as shown in the top panels of A and encompassed the growth plate and secondary spongiosa regions. Data are derived from randomly selected fields of femur heads from moribund ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate controls injected with tamoxifen at 6 weeks of age (n=3 in each genotype). Mice were 10–11 weeks of age at the time of analysis. Size of fields in in vitro experiments are as indicated in B and were selected randomly on coverslips. Bone marrow was derived from ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate mice that were injected with tamoxifen at 6–8 weeks of age (n=6 in each genotype). Osteoclast differentiation experiments were initiated 1–3 weeks thereafter. Statistical significance was determined by paired Student’s t-test. **P<0.005.
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f6-0040228: Impaired osteoclastogenesis in induced SHP-2-deficient mice. (A) Femur sections of tamoxifen-injected moribund ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate controls were stained for TRAP to visualize osteoclasts (red color). Images are from 11-week-old mice injected with tamoxifen 5 weeks previously. The location of osteoclasts beneath growth plates (g) and within the secondary spongiosa are indicated with blue arrowheads. Note the paucity of osteoclasts in ptpn11fl/fl ert2-cre mice. Scale bars: top panels, 500 μm; bottom panels, 200 μm. (B) Bone marrow cells from ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate control mice, both treated with tamoxifen 3 weeks previously (at 7 weeks of age), were cultured with M-CSF and RANKL for 7 days on glass coverslips. Osteoclasts were identified by TRAP staining. Note the abundance of multinucleated osteoclasts in control cultures and absence from ptpn11fl/fl ert2-cre cultures. Scale bars: 100 μm. (C) Shown are the mean numbers of osteoclasts + 1 s.e.m. per field identified in bone sections (in situ) and in vitro osteoclast differentiation experiments described in A and B. For in situ analysis, the field size was as shown in the top panels of A and encompassed the growth plate and secondary spongiosa regions. Data are derived from randomly selected fields of femur heads from moribund ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate controls injected with tamoxifen at 6 weeks of age (n=3 in each genotype). Mice were 10–11 weeks of age at the time of analysis. Size of fields in in vitro experiments are as indicated in B and were selected randomly on coverslips. Bone marrow was derived from ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate mice that were injected with tamoxifen at 6–8 weeks of age (n=6 in each genotype). Osteoclast differentiation experiments were initiated 1–3 weeks thereafter. Statistical significance was determined by paired Student’s t-test. **P<0.005.

Mentions: To further investigate the basis for the skeletal phenotype observed in tamoxifen-injected ptpn11fl/fl ert2-cre mice, we enumerated bone osteoclasts, the cell type that is responsible for resorption of bone and for maintenance of bone homeostasis. To identify osteoclasts, bone sections were stained for tartrate-resistant acid phosphatase (TRAP), an osteoclast-specific marker. Osteoclasts were readily identified in the metaphyses of ptpn11fl/fl control bone as well as in the secondary spongiosa (Fig. 6A,C). By contrast, much fewer osteoclasts were found in the same regions of ptpn11fl/fl ert2-cre bones.


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)

Impaired osteoclastogenesis in induced SHP-2-deficient mice. (A) Femur sections of tamoxifen-injected moribund ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate controls were stained for TRAP to visualize osteoclasts (red color). Images are from 11-week-old mice injected with tamoxifen 5 weeks previously. The location of osteoclasts beneath growth plates (g) and within the secondary spongiosa are indicated with blue arrowheads. Note the paucity of osteoclasts in ptpn11fl/fl ert2-cre mice. Scale bars: top panels, 500 μm; bottom panels, 200 μm. (B) Bone marrow cells from ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate control mice, both treated with tamoxifen 3 weeks previously (at 7 weeks of age), were cultured with M-CSF and RANKL for 7 days on glass coverslips. Osteoclasts were identified by TRAP staining. Note the abundance of multinucleated osteoclasts in control cultures and absence from ptpn11fl/fl ert2-cre cultures. Scale bars: 100 μm. (C) Shown are the mean numbers of osteoclasts + 1 s.e.m. per field identified in bone sections (in situ) and in vitro osteoclast differentiation experiments described in A and B. For in situ analysis, the field size was as shown in the top panels of A and encompassed the growth plate and secondary spongiosa regions. Data are derived from randomly selected fields of femur heads from moribund ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate controls injected with tamoxifen at 6 weeks of age (n=3 in each genotype). Mice were 10–11 weeks of age at the time of analysis. Size of fields in in vitro experiments are as indicated in B and were selected randomly on coverslips. Bone marrow was derived from ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate mice that were injected with tamoxifen at 6–8 weeks of age (n=6 in each genotype). Osteoclast differentiation experiments were initiated 1–3 weeks thereafter. Statistical significance was determined by paired Student’s t-test. **P<0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6-0040228: Impaired osteoclastogenesis in induced SHP-2-deficient mice. (A) Femur sections of tamoxifen-injected moribund ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate controls were stained for TRAP to visualize osteoclasts (red color). Images are from 11-week-old mice injected with tamoxifen 5 weeks previously. The location of osteoclasts beneath growth plates (g) and within the secondary spongiosa are indicated with blue arrowheads. Note the paucity of osteoclasts in ptpn11fl/fl ert2-cre mice. Scale bars: top panels, 500 μm; bottom panels, 200 μm. (B) Bone marrow cells from ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate control mice, both treated with tamoxifen 3 weeks previously (at 7 weeks of age), were cultured with M-CSF and RANKL for 7 days on glass coverslips. Osteoclasts were identified by TRAP staining. Note the abundance of multinucleated osteoclasts in control cultures and absence from ptpn11fl/fl ert2-cre cultures. Scale bars: 100 μm. (C) Shown are the mean numbers of osteoclasts + 1 s.e.m. per field identified in bone sections (in situ) and in vitro osteoclast differentiation experiments described in A and B. For in situ analysis, the field size was as shown in the top panels of A and encompassed the growth plate and secondary spongiosa regions. Data are derived from randomly selected fields of femur heads from moribund ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate controls injected with tamoxifen at 6 weeks of age (n=3 in each genotype). Mice were 10–11 weeks of age at the time of analysis. Size of fields in in vitro experiments are as indicated in B and were selected randomly on coverslips. Bone marrow was derived from ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate mice that were injected with tamoxifen at 6–8 weeks of age (n=6 in each genotype). Osteoclast differentiation experiments were initiated 1–3 weeks thereafter. Statistical significance was determined by paired Student’s t-test. **P<0.005.
Mentions: To further investigate the basis for the skeletal phenotype observed in tamoxifen-injected ptpn11fl/fl ert2-cre mice, we enumerated bone osteoclasts, the cell type that is responsible for resorption of bone and for maintenance of bone homeostasis. To identify osteoclasts, bone sections were stained for tartrate-resistant acid phosphatase (TRAP), an osteoclast-specific marker. Osteoclasts were readily identified in the metaphyses of ptpn11fl/fl control bone as well as in the secondary spongiosa (Fig. 6A,C). By contrast, much fewer osteoclasts were found in the same regions of ptpn11fl/fl ert2-cre bones.

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