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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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Induced SHP-2 deficiency in adult mice results in weight loss and rapid mortality. (A) Kaplan-Meier plot depicting the survival of ptpn11fl/fl ert2-cre mice (n=38) and pooled ptpn11+/+, ptpn11fl/+, ptpn11fl/+ ert2-cre and ptpn11fl/fl littermate control mice (n=75) following tamoxifen injection at 6–8 weeks of age. (B) Weight of tamoxifen-injected ptpn11fl/fl ert2-cre mice (n=19) expressed as a mean percentage ± 1 s.e.m. of the weight of tamoxifen-injected littermate controls (n=33) at time points at and prior to the time that ptpn11fl/fl ert2-cre mice appear moribund. Mice were injected with tamoxifen at 6–8 weeks of age. Statistical significance was determined using a one sample Student’s t-test. In A and B, all control mice were similar with regards to survival and weight following tamoxifen injection and no effect of haploinsufficiency of SHP-2 in ptpn11fl/+ ert2-cre mice was observed. For ptpn11fl/fl ert2-cre mice, no influence of mouse gender upon survival or weight loss was apparent. *P<0.05; **P<0.005. (C) Western blots showing expression of SHP-2 in the indicated organs from ptpn11fl/fl ert2-cre (+) and littermate control ptpn11fl/fl mice, both injected with tamoxifen 15 days previously (at 6–8 weeks of age). SM, skeletal muscle; He, heart; Liv, liver; Kid, kidney; Lu, lung; Sp, spleen; BM, bone marrow. Blots were stripped and reprobed with an anti-GAPDH antibody to demonstrate equivalent protein loading. Shown are representative experiments of three repeats.
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f1-0040228: Induced SHP-2 deficiency in adult mice results in weight loss and rapid mortality. (A) Kaplan-Meier plot depicting the survival of ptpn11fl/fl ert2-cre mice (n=38) and pooled ptpn11+/+, ptpn11fl/+, ptpn11fl/+ ert2-cre and ptpn11fl/fl littermate control mice (n=75) following tamoxifen injection at 6–8 weeks of age. (B) Weight of tamoxifen-injected ptpn11fl/fl ert2-cre mice (n=19) expressed as a mean percentage ± 1 s.e.m. of the weight of tamoxifen-injected littermate controls (n=33) at time points at and prior to the time that ptpn11fl/fl ert2-cre mice appear moribund. Mice were injected with tamoxifen at 6–8 weeks of age. Statistical significance was determined using a one sample Student’s t-test. In A and B, all control mice were similar with regards to survival and weight following tamoxifen injection and no effect of haploinsufficiency of SHP-2 in ptpn11fl/+ ert2-cre mice was observed. For ptpn11fl/fl ert2-cre mice, no influence of mouse gender upon survival or weight loss was apparent. *P<0.05; **P<0.005. (C) Western blots showing expression of SHP-2 in the indicated organs from ptpn11fl/fl ert2-cre (+) and littermate control ptpn11fl/fl mice, both injected with tamoxifen 15 days previously (at 6–8 weeks of age). SM, skeletal muscle; He, heart; Liv, liver; Kid, kidney; Lu, lung; Sp, spleen; BM, bone marrow. Blots were stripped and reprobed with an anti-GAPDH antibody to demonstrate equivalent protein loading. Shown are representative experiments of three repeats.

Mentions: To examine the effect of global deletion of SHP-2 in adults, we generated ptpn11 exon 4 floxed (ptpn11fl/fl) mice that carry an ubiquitin-promoter-driven ert2-cre transgene. Administration of the estrogen antagonist tamoxifen to these mice was predicted to result in nuclear translocation of the ERT2-Cre fusion protein and recombination at the floxed ptpn11 locus to yield a ptpn11 allele in all tissues. 6- to 8-week-old ptpn11fl/fl ert2-cre mice were administered tamoxifen via intraperitoneal injection on two consecutive days. By 4 weeks post-injection, we observed 50% mortality of ptpn11fl/fl ert2-cre mice, with 100% mortality by 20 weeks post-injection (Fig. 1A). By contrast, essentially no death was observed in any of the tamoxifen-injected ptpn11+/+, ptpn11fl/+, ptpn11fl/+ ert2-cre and ptpn11fl/fl littermate controls over the course of several months.


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)

Induced SHP-2 deficiency in adult mice results in weight loss and rapid mortality. (A) Kaplan-Meier plot depicting the survival of ptpn11fl/fl ert2-cre mice (n=38) and pooled ptpn11+/+, ptpn11fl/+, ptpn11fl/+ ert2-cre and ptpn11fl/fl littermate control mice (n=75) following tamoxifen injection at 6–8 weeks of age. (B) Weight of tamoxifen-injected ptpn11fl/fl ert2-cre mice (n=19) expressed as a mean percentage ± 1 s.e.m. of the weight of tamoxifen-injected littermate controls (n=33) at time points at and prior to the time that ptpn11fl/fl ert2-cre mice appear moribund. Mice were injected with tamoxifen at 6–8 weeks of age. Statistical significance was determined using a one sample Student’s t-test. In A and B, all control mice were similar with regards to survival and weight following tamoxifen injection and no effect of haploinsufficiency of SHP-2 in ptpn11fl/+ ert2-cre mice was observed. For ptpn11fl/fl ert2-cre mice, no influence of mouse gender upon survival or weight loss was apparent. *P<0.05; **P<0.005. (C) Western blots showing expression of SHP-2 in the indicated organs from ptpn11fl/fl ert2-cre (+) and littermate control ptpn11fl/fl mice, both injected with tamoxifen 15 days previously (at 6–8 weeks of age). SM, skeletal muscle; He, heart; Liv, liver; Kid, kidney; Lu, lung; Sp, spleen; BM, bone marrow. Blots were stripped and reprobed with an anti-GAPDH antibody to demonstrate equivalent protein loading. Shown are representative experiments of three repeats.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-0040228: Induced SHP-2 deficiency in adult mice results in weight loss and rapid mortality. (A) Kaplan-Meier plot depicting the survival of ptpn11fl/fl ert2-cre mice (n=38) and pooled ptpn11+/+, ptpn11fl/+, ptpn11fl/+ ert2-cre and ptpn11fl/fl littermate control mice (n=75) following tamoxifen injection at 6–8 weeks of age. (B) Weight of tamoxifen-injected ptpn11fl/fl ert2-cre mice (n=19) expressed as a mean percentage ± 1 s.e.m. of the weight of tamoxifen-injected littermate controls (n=33) at time points at and prior to the time that ptpn11fl/fl ert2-cre mice appear moribund. Mice were injected with tamoxifen at 6–8 weeks of age. Statistical significance was determined using a one sample Student’s t-test. In A and B, all control mice were similar with regards to survival and weight following tamoxifen injection and no effect of haploinsufficiency of SHP-2 in ptpn11fl/+ ert2-cre mice was observed. For ptpn11fl/fl ert2-cre mice, no influence of mouse gender upon survival or weight loss was apparent. *P<0.05; **P<0.005. (C) Western blots showing expression of SHP-2 in the indicated organs from ptpn11fl/fl ert2-cre (+) and littermate control ptpn11fl/fl mice, both injected with tamoxifen 15 days previously (at 6–8 weeks of age). SM, skeletal muscle; He, heart; Liv, liver; Kid, kidney; Lu, lung; Sp, spleen; BM, bone marrow. Blots were stripped and reprobed with an anti-GAPDH antibody to demonstrate equivalent protein loading. Shown are representative experiments of three repeats.
Mentions: To examine the effect of global deletion of SHP-2 in adults, we generated ptpn11 exon 4 floxed (ptpn11fl/fl) mice that carry an ubiquitin-promoter-driven ert2-cre transgene. Administration of the estrogen antagonist tamoxifen to these mice was predicted to result in nuclear translocation of the ERT2-Cre fusion protein and recombination at the floxed ptpn11 locus to yield a ptpn11 allele in all tissues. 6- to 8-week-old ptpn11fl/fl ert2-cre mice were administered tamoxifen via intraperitoneal injection on two consecutive days. By 4 weeks post-injection, we observed 50% mortality of ptpn11fl/fl ert2-cre mice, with 100% mortality by 20 weeks post-injection (Fig. 1A). By contrast, essentially no death was observed in any of the tamoxifen-injected ptpn11+/+, ptpn11fl/+, ptpn11fl/+ ert2-cre and ptpn11fl/fl littermate controls over the course of several months.

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