Development of severe skeletal defects in induced SHP-2 ... · SHP-2 (encoded by PTPN11) is a broadly expressed Src homology-2 domain (SH2)-containing protein tyrosine phosphatase

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INTRODUCTIONSHP-2 (encoded by PTPN11) is a broadly expressed Src homology-2 domain (SH2)-containing protein tyrosine phosphatase (PTP) thathas been implicated in signal transduction initiated by multiplegrowth factor and cytokine receptors (Chong and Maiese, 2007;Matozaki et al., 2009). The exact mechanism by which SHP-2participates in receptor signal transduction is uncertain and is likelyto vary from receptor to receptor. However, commonly, SHP-2seems to be required for activation of the Ras signaling pathway,which drives several different cell responses, including growth,survival, proliferation and differentiation (Chong and Maiese, 2007;Dance et al., 2008; Matozaki et al., 2009). In addition, SHP-2 might

also function as a negative regulator of receptor-mediated signaltransduction, e.g. during cytokine receptor signaling, acting todephosphorylate STAT transcription factors (Chong and Maiese,2007).

In humans, autosomal dominant germline mutations of thePTPN11 gene result in different clinical syndromes with manyoverlapping features. Noonan syndrome type I (NS) is caused bygain-of-function PTPN11 mutations that disrupt intramolecularfold-mediated inhibition of the PTP domain of SHP-2, resulting inincreased PTP activity (Fragale et al., 2004; Keilhack et al., 2005;Noonan, 1968; Tartaglia et al., 2001). LEOPARD syndrome (LS),by contrast, is caused by PTPN11 mutations that result in reducedPTP activity. The mutant PTP is then thought to act in a dominant-negative fashion to inhibit SHP-2 expressed from the normalPTPN11 allele (Digilio et al., 2002; Gorlin et al., 1969; Hanna et al.,2006; Kontaridis et al., 2006; Tartaglia et al., 2006). Clinical featuresof these syndromes that have been recognized include facialdysmorphia, short stature, cardiovascular defects, pulmonarystenosis, lentigines and skeletal abnormalities including lateralcurvature (scoliosis) and dorsal curvature (kyphosis) of the spine.How mutations that have opposing effects upon SHP-2 activityresult in essentially identical diseases is currently unknown,although it has been proposed that dysregulated activation of theRas-mitogen-activated protein kinase (MAPK) signaling pathwayis the underlying cause (Edouard et al., 2007; Gelb and Tartaglia,2006). In support of this, it has recently been demonstrated thatCostello syndrome and cardio-facio-cutaneous syndrome, both ofwhich share clinical features with NS and LS, are caused bygermline mutations in genes that encode components of this

Disease Models & Mechanisms 4, 228-239 (2011) doi:10.1242/dmm.006130

1Department of Microbiology and Immunology, University of Michigan MedicalSchool, Ann Arbor, MI 48109-5620, USA2Department of Biologic and Materials Sciences, University of Michigan School ofDentistry, Ann Arbor, MI 48109-1078, USA3Center for Excellence in Hip Disorders, Texas Scottish Rite Hospital for Children,Dallas, TX 75219, USA4Unit for Laboratory Animal Medicine, University of Michigan Medical School, AnnArbor, MI 48109-5620, USA5Department of Pathology, University of California San Diego, San Diego,CA 92093, USA*Author for correspondence ([email protected])

Received 26 May 2010; Accepted 22 September 2010

© 2011. Published by The Company of Biologists LtdThis is an Open Access article distributed under the terms of the Creative Commons AttributionNon-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), whichpermits unrestricted non-commercial use, distribution and reproduction in any medium providedthat the original work is properly cited and all further distributions of the work or adaptation aresubject to the same Creative Commons License terms

SUMMARY

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

Development of severe skeletal defects in inducedSHP-2-deficient adult mice: a model of skeletalmalformation in humans with SHP-2 mutationsTimothy J. Bauler1, Nobuhiro Kamiya2,3, Philip E. Lapinski1, Eric Langewisch1, Yuji Mishina2, John E. Wilkinson4, Gen-Sheng Feng5

and Philip D. King1,*

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SHP-2 in skeletal remodeling RESEARCH ARTICLE

pathway (Estep et al., 2006; Gripp et al., 2006; Niihori et al., 2006;Rodriguez-Viciana et al., 2006).

Studies of genetically engineered SHP-2-deficient mice have thepotential to shed light upon the molecular and cellular basis forthe clinical abnormalities observed in humans with PTPN11mutations. Mice that are homozygous for a ptpn11-null allele dieat day 10 of embryonic development as a result of defectivegastrulation, which, at least in part, can be accounted for bysuboptimal fibroblast growth factor (FGF) receptor signaling(Saxton et al., 1997). To circumvent this early lethality, differentgroups generated mice that harbor conditional loxP-flanked (floxed)ptpn11 alleles that were then crossed with tissue-specific Crerecombinase transgenic mice (Fornaro et al., 2006; Zhang et al.,2004). Accordingly, mice that lack SHP-2 specifically in neuronalcells, neuronal progenitors, liver, pancreas, striated and cardiacmuscle, mammary gland and T cells have now been generated(Bard-Chapeau et al., 2006; Fornaro et al., 2006; Hagihara et al.,2009; Ke et al., 2006; Ke et al., 2007; Kontaridis et al., 2008;Krajewska et al., 2008; Nguyen et al., 2006; Princen et al., 2009;Zhang et al., 2004; Zhang et al., 2009). Studies of these mice haveconfirmed an essential role for SHP-2 in signal transductionthrough a number of cell surface receptors in these tissues that isnecessary for normal tissue functioning.

To further understand the role of SHP-2 in tissue homeostasis,we generated homozygote ptpn11 floxed mice that carry aubiquitously expressed tamoxifen-inducible estrogen receptor-2(ert2)-cre transgene (Ruzankina et al., 2007; Zhang et al., 2004).Induced loss of SHP-2 expression in this model resulted in multipleabnormalities, foremost amongst which was the development ofsevere skeletal malformations. Therefore, the model is expected tobe useful in understanding the cellular basis and molecular basisfor the role of SHP-2 in skeletal growth and remodeling, and theetiology of skeletal abnormalities in humans with PTPN11mutations.

RESULTSInduced deletion of SHP-2 from adult mice results in early lethalityTo examine the effect of global deletion of SHP-2 in adults, wegenerated ptpn11 exon 4 floxed (ptpn11fl/fl) mice that carry anubiquitin-promoter-driven ert2-cre transgene. Administration ofthe estrogen antagonist tamoxifen to these mice was predicted toresult in nuclear translocation of the ERT2-Cre fusion protein andrecombination at the floxed ptpn11 locus to yield a null ptpn11allele in all tissues. 6- to 8-week-old ptpn11fl/fl ert2-cre mice wereadministered tamoxifen via intraperitoneal injection on twoconsecutive days. By 4 weeks post-injection, we observed 50%mortality of ptpn11fl/fl ert2-cre mice, with 100% mortality by 20weeks post-injection (Fig. 1A). By contrast, essentially no death wasobserved in any of the tamoxifen-injected ptpn11+/+, ptpn11fl/+,ptpn11fl/+ ert2-cre and ptpn11fl/fl littermate controls over thecourse of several months.

ptpn11fl/fl ert2-cre mice exhibited marked weight loss prior todeath (Fig. 1B). Weight loss was precipitous and was evident 1 weekbefore death. To determine the efficiency of SHP-2 deletion, organlysates from tamoxifen-injected ptpn11fl/fl ert2-cre mice andcontrols were prepared and examined for expression of SHP-2 bywestern blotting. In the majority of tissues examined a substantialreduction in SHP-2 protein was observed (Fig. 1C).

Pathology of induced SHP-2-deficient micePostmortem analysis did not identify an obvious cause of deathof tamoxifen-injected ptpn11fl/fl ert2-cre mice. Elevated serumlevels of blood urea nitrogen (BUN), creatinine and phosphorous(Fig. 2A) suggested kidney dysfunction, although uponhistological analysis no obvious lesions in the kidneys were noted(not shown). Furthermore, although mice showed reducedalkaline phosphatase (ALKP) and a trend towards increasedaspartate transaminase (AST) in serum (Fig. 2A), no liver

Fig. 1. Induced SHP-2 deficiency in adult mice results in weight loss andrapid mortality. (A)Kaplan-Meier plot depicting the survival of ptpn11fl/fl ert2-cre mice (n38) and pooled ptpn11+/+, ptpn11fl/+, ptpn11fl/+ ert2-cre andptpn11fl/fl littermate control mice (n75) following tamoxifen injection at 6-8weeks of age. (B)Weight of tamoxifen-injected ptpn11fl/fl ert2-cre mice (n19)expressed as a mean percentage ± 1 s.e.m. of the weight of tamoxifen-injectedlittermate controls (n33) at time points at and prior to the time that ptpn11fl/fl

ert2-cre mice appear moribund. Mice were injected with tamoxifen at 6-8weeks of age. Statistical significance was determined using a one sampleStudent’s t-test. In A and B, all control mice were similar with regards tosurvival and weight following tamoxifen injection and no effect ofhaploinsufficiency of SHP-2 in ptpn11fl/+ ert2-cre mice was observed. Forptpn11fl/fl ert2-cre mice, no influence of mouse gender upon survival or weightloss was apparent. *P<0.05; **P<0.005. (C)Western blots showing expressionof SHP-2 in the indicated organs from ptpn11fl/fl ert2-cre (+) and littermatecontrol ptpn11fl/fl mice, both injected with tamoxifen 15 days previously (at 6-8weeks 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 arerepresentative experiments of three repeats.

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abnormalities were detected upon histological analysis ofptpn11fl/fl ert2-cre mice. Considering the high frequency ofcardiac defects in NS and LS patients as well as in mice withcardiac- or muscle-specific deletion of SHP-2, hearts fromptpn11fl/fl ert2-cre mice were also examined for lesions. Nolesions in cardiac valves or walls were detected. This might beexplained by a requirement for SHP-2 in cardiac developmentbut not in continued cardiac function in adults. Alternatively,the absence of heart abnormalities in this model might beexplained by the relatively poor deletion of SHP-2 in this tissue(Fig. 1C).

Further postmortem analysis of ptpn11fl/fl ert2-cre mice did notreveal lesions in any tissues that might account for death. However,in skin, a marked epidermal hyperplasia (acanthosis) and thickeningof the outermost layer of dead keratinocytes (hyperkeratosis) wasobserved (Fig. 2C). Furthermore, moribund mice exhibited anemiaas evidenced by a reduced peripheral blood hematocrit (Fig. 2B).However, reticulocytogenesis is apparently intact in these mice. Thepercentage representation of reticulocytes in peripheral blood waselevated, as was the reticulocyte index (Fig. 2C and data not shown).Other abnormalities in ptpn11fl/fl ert2-cre mice included impairedhematopoiesis and skeletal malformations. Both of these aredescribed in detail below.

Impaired hematopoiesis in induced SHP-2-deficient miceThymi isolated from tamoxifen-injected moribund ptpn11fl/fl ert2-cre mice were exceptionally small and showed much-reducedcellularity (Fig. 3A). Numbers of each of CD4–CD8– double-negative (DN), CD4+CD8+ double-positive (DP) and CD4+CD8–and CD4–CD8+ single-positive (SP) T cells were dramaticallyreduced in the thymi of ptpn11fl/fl ert2-cre mice (Fig. 3A). Spleensshowed considerable variability in size and cellularity (Fig. 3B). In

Fig. 2. Altered levels of serum biomarkers, anemia and skin abnormalitiesin induced SHP-2-deficient mice. (A)Shown are the mean levels of theindicated biomarkers in serum ± 1 s.e.m. of tamoxifen-injected (at 6-8 weeks)moribund ptpn11fl/fl ert2-cre mice and littermate control mice (n6 eachgenotype). Age range of mice at the time of analysis was 9-12 weeks. ALKPand AST concentrations are shown in U/l; BUN, mg/dl; phosphorous,g/ml;creatinine,g/cl. Statistical significance was determined using a two sampleStudent’s t-test. *P<0.05; **P<0.005. (B)Depicted are mean hematocrit(percent blood volume) and reticulocyte counts (percent blood cells) ± 1 s.e.m.performed on heparinized whole blood from mice in A. Statistical significancewas determined using a two sample Student’s t-test. (C)Representative H&E-stained skin section (n6) of a tamoxifen-injected (at 6 weeks of age)moribund ptpn11fl/fl ert2-cre mouse and a littermate ptpn11+/+ control showinghyperkeratosis (k) and acanthosis (a) in the former. Analysis was performed 4weeks after tamoxifen injection. Scale bars: 200m.

Fig. 3. Altered hematopoiesis in induced SHP-2-deficient mice.(A)Numbers of total thymocytes (left) and indicated thymocytesubpopulations (right) in thymi isolated from tamoxifen-injected moribundptpn11fl/fl ert2-cre mice (n8) and littermate controls (n8). DN, CD4–CD8–double negative; DP, CD4+CD8+ double positive; SP, CD4+CD8– andCD4–CD8+ single positive T cells. (B)Numbers of total splenocytes (left) andindicated splenocyte subpopulations (right) in spleens isolated fromtamoxifen-injected moribund ptpn11fl/fl ert2-cre mice (n12) and littermatecontrols (n14). T cells (T), B cells (B), macrophages (Mac) and neutrophils(Neut) were identified as TCR+, B220+, CD11bint/GR1int and CD11bhi/GR1hi,respectively. Ter119+ cells represent erythrocyte precursors. (C)Total numbersand numbers of indicated lineage-positive cells (left) and numbers ofindicated lineage-negative progenitor cells (right) in bone marrow oftamoxifen-injected moribund ptpn11fl/fl ert2-cre mice and littermate controls(for each genotype, n11 for lineage-positive and n5 for lineage-negative).LSK, Lin–Sca-1+c-Kit+ cells, which are enriched for HSCs; CLP, Lin–Sca-1loc-KitloCD127+; CMP, Lin–Sca-1–c-Kit+CD34+CD16/32lo; GMP, Lin–Sca-1–c-Kit+CD34+CD16/32hi; MEP, Lin–Sca-1–c-Kit+CD34–CD16/32–/lo. In scatter plots,each symbol represents an individual mouse. In bar graphs, the mean ± 1s.e.m. is depicted. All mice were injected with tamoxifen at 6-8 weeks of age.Age range of mice at time of analysis was 10-16 weeks. Statistical significancewas determined by two sample Student’s t-test. *P<0.05; **P<0.005.

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mice in which splenomegaly was apparent, this could be accountedfor by a large increase in the number of cells that express Ter119,which is a marker of late-stage erythroid precursors. This increaseis indicative of extra-medullary hematopoiesis. Consistent withreduced thymic cellularity, the total number of T cells in spleenswas reduced. Likewise, numbers of B220+ B cells in spleen weresubstantially reduced. By contrast, normal numbers of macrophagesand neutrophils were found in spleens of ptpn11fl/fl ert2-cre mice(Fig. 3B).

We also examined the number of hematopoietic cells in bonemarrow of ptpn11fl/fl ert2-cre mice (Fig. 3C). A significant decreasein the number of B220+ B cells and neutrophils, and a significantincrease in the number of macrophages, was apparent. By contrast,there were normal numbers of T cells and Ter119+ cells. Regardinghematopoietic precursor cells, total numbers of Lin–Sca-1+c-kit+

(LSK) cells, a population enriched in hematopoietic stem cells(HSCs), remained unchanged (Fig. 3C). However, numbers ofcommon lymphoid progenitor cells (CLPs) were significantlyreduced. Therefore, this reduced number of CLP might, at least inpart, account for the reduced number of T cells in thymus andspleen, and also the reduced number of B cells in bone marrowand spleen. Notably, numbers of common myeloid progenitors

(CMPs), granulocyte-macrophage progenitors (GMPs) andmegakaryocyte-erythroid progenitors (MEPs) were found to benormal in ptpn11fl/fl ert2-cre mice (Fig. 3C).

Skeletal abnormalities in induced SHP-2-deficient miceOne of the most striking features of tamoxifen-injected ptpn11fl/fl

ert2-cre mice is the development of skeletal abnormalities. Visualinspection of live and deceased mice revealed pronounced kyphosisand scoliosis (Fig. 4A). These skeletal abnormalities were apparentas soon as 2 weeks after tamoxifen injection and were observed inall mice to greater or lesser degrees prior to euthanasia or naturaldeath. Kyphosis and scoliosis were confirmed by X-ray analysis ofspines (Fig. 4B). Furthermore, curvature of humeri was detected(Fig. 4B). X-ray analysis also revealed an increased radiodensity ofall vertebral bodies in the spine of ptpn11fl/fl ert2-cre mice. Inaddition, increased radiodensity was apparent in metaphyses ofhumeri and femora and in all rib bones of ptpn11fl/fl ert2-cre mice(Fig. 4B). These findings suggest that bone malformations andosteopetrosis affect the entire skeleton of ptpn11fl/fl ert2-cre mice.

To understand spinal curvature in three dimensions and toquantitate the increased bone radiodensity in ptpn11fl/fl ert2-crespines, we scanned thoracic and lumbar vertebral bones using

Fig. 4. Spinal curvature and increased bone mineral content in induced SHP-2-deficient mice. (A)Gross morphology of moribund ptpn11fl/fl ert2-cre miceand ptpn11fl/fl littermate control mice. Note lateral spinal curvature (dotted line) and hump (arrowhead) in the ptpn11fl/fl ert2-cre mouse. (B)X-rays of tamoxifen-injected moribund ptpn11fl/fl ert2-cre mice showing kyphosis (top left) and scoliosis (top right), compared with the indicated littermate controls. Note alsoincreased radiodensity of vertebral bodies of spines, metaphyses of femur (bottom left) and humerus (middle right), and entire rib bones (bottom right) of theptpn11fl/fl ert2-cre mice (all indicated with arrowheads). In A and B, mice were treated with tamoxifen 5 weeks previously, at 6-8 weeks of age. (C)CT images ofthe isosurfaces of spines of ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate control mice injected with tamoxifen 5 weeks previously, at 7 weeks of age. Imagesshow scoliosis with rotated vertebral bodies. (D)Mean bone mineral content ± 1 s.e.m. of individual thoracic (T) and lumbar (L) vertebrae from ptpn11fl/fl ert2-cremice and littermate controls determined byCT scanning (n4 for each mouse strain). All mice were treated with tamoxifen 5 weeks previously, at 7 weeks ofage. Statistical significance was determined by paired Student’s t-test. *P<0.05.

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micro-computed tomography (CT). In these experiments, wecompared spines from a cohort of ptpn11fl/fl ert2-cre and littermatecontrol mice that had been injected with tamoxifen 5 weekspreviously (Fig. 4C,D). Results of CT analyses demonstrated severescoliosis with a rotation of vertebral bodies and osteophyte-likeectopic calcifications on the spinal column (Fig. 4C). In addition,increased bone mineral content in vertebrae of induced SHP-2-deficient mice was confirmed (Fig. 4D). Increased bone mineralcontent was especially apparent in lower thoracic and upper lumbarvertebrae, i.e. in the region where kyphosis and scoliosis is mostfrequently observed. CT scanning also revealed several otherskeletal abnormalities in ptpn11fl/fl ert2-cre mice, including increasedrib thickness, evidence of previous rib fractures and the presenceof ectopic calcified growths on ribs (Fig. 4C and data not shown).

Bone histologyHistological analysis confirmed that bones from tamoxifen-injectedptpn11fl/fl ert2-cre mice were osteopetrotic compared withlittermate control mice (Fig. 5). Vertebrae and metaphyses of femoraand humeri showed marked increases in the amount of trabecularbone (Fig. 5, top panels, and data not shown). Changes in theamount of cortical bone were less apparent. However, the uniform

organization of remodeled cortical bone layers was clearly disruptedin SHP-2-deficient long bones, with regions of remodeled corticalbone showing ultrastructural features of trabecular bone (Fig. 5,right middle panels). Presumably, this trabecular-like cortical boneis formed after the time of tamoxifen administration.

In addition to abnormalities in bone, cartilage abnormalitieswere noted in ptpn11fl/fl ert2-cre mice. Cartilaginous growth platesof vertebrae, femora and humeri were disorganized (Fig. 5, topand left middle panels, and data not shown). Alcian blue stainingconfirmed the disorganization of the columnar growth plate andthe absence of a columnar morphology of differentiatingchondrocytes in ptpn11fl/fl ert2-cre mice (Fig. 5, bottom panels).In addition, the remains of ectopic cartilaginous elements intrabecular bone were identified in ptpn11fl/fl ert2-cre mice (Fig.5, top left, middle left and bottom left panels). Interestingly,ectopic cartilage was also identified next to growth plates invertebrae and long bones (Fig. 5, top right panels, and data notshown). Lastly, the area of hypertrophic chondrocytes was muchexpanded in ptpn11fl/fl ert2-cre mice (Fig. 5, bottom right panels).These findings suggest that the process of bone remodelingthrough endochondral bone formation was largely disrupted byloss of SHP-2.

Fig. 5. Increased and disorganized bone and cartilage in induced SHP-2-deficient mice. Shown are representative images of L4 vertebrae and femora ofmoribund tamoxifen-injected ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate controls. Mice were injected with tamoxifen at 7 weeks of age and analysis wasperformed at 12 weeks of age. Top and middle panels are stained with H&E, and bottom panels are stained with Alcian blue to highlight cartilage. Scale bars:1 mm in top panels, 500m in L4 vertebrae middle panels, and 200m in femora middle panels and all bottom panels. The amount of trabecular bone (t) isdramatically increased in ptpn11fl/fl ert2-cre vertebrae and femora. A region of remodeled (r) cortical bone (c) in femora of ptpn11fl/fl ert2-cre mice showsdisorganized bone in this region compared with the same region in the control. Remains of ectopic cartilaginous elements (e) were identified in the trabecularbone region and ectopic cartilage formation (cf ) was identified next to growth plates (g) in ptpn11fl/fl ert2-cre mice. The columnar formation of growth plates wasdisorganized in ptpn11fl/fl ert2-cre mice and the area of hypertrophic chondrocytes (hc) was elongated.

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Induced loss of SHP-2 results in impaired osteoclastogenesisTo further investigate the basis for the skeletal phenotype observedin tamoxifen-injected ptpn11fl/fl ert2-cre mice, we enumerated boneosteoclasts, the cell type that is responsible for resorption of boneand for maintenance of bone homeostasis. To identify osteoclasts,bone sections were stained for tartrate-resistant acid phosphatase(TRAP), an osteoclast-specific marker. Osteoclasts were readilyidentified in the metaphyses of ptpn11fl/fl control bone as well asin the secondary spongiosa (Fig. 6A,C). By contrast, much fewerosteoclasts were found in the same regions of ptpn11fl/fl ert2-crebones.

Osteoclasts are of hematopoietic origin and are derivedprincipally from GMPs (Asagiri and Takayanagi, 2007; Menaa etal., 2000; Takayanagi, 2007; Yavropoulou and Yovos, 2008). Cultureof whole bone marrow cells in vitro with the cytokines macrophagecolony stimulating factor (M-CSF) and receptor activator of nuclearfactor kappa B ligand (RANKL) leads to selective outgrowth of thiscell type (Asagiri and Takayanagi, 2007). Therefore, to determinewhether the paucity of osteoclasts in bones of ptpn11fl/fl ert2-cremice could be explained by defective osteoclastogenesis, and at thesame time understand whether any defective osteoclastogenesiswere intrinsic to the hematopoietic compartment, we examinedosteoclastogenesis in vitro. Whole bone marrow cells fromptpn11fl/fl ert2-cre and control mice were cultured with M-CSF andRANKL for 7 days, after which time osteoclasts were identified byTRAP staining (Fig. 6B,C). Under these conditions, multinucleated

TRAP+ osteoclasts were readily identified in control cultures. Bycontrast, much fewer osteoclasts were identified in cultures fromptpn11fl/fl ert2-cre mice. Therefore, osteoclastogenesis is impairedin ptpn11fl/fl ert2-cre mice. Furthermore, this seems to be anintrinsic property of hematopoietic cells and not secondary to otherfactors such as reduced bone marrow volume.

Impaired M-CSF signal transduction in the absence of SHP-2Defective osteoclastogenesis in tamoxifen-treated ptpn11fl/fl ert2-cre mice could be explained by impaired M-CSF or RANKLsignaling, or both. Evidence that at least M-CSF signaling isblocked in the absence of SHP-2 was provided by experiments inwhich bone marrow cells were treated with this cytokine alone(Fig. 7). Treatment of control bone marrow cells with M-CSF for5 days promoted the development of abundant numbers ofmacrophages, as expected. By contrast, considerably fewermacrophages grew out from cultures of ptpn11fl/fl ert2-cre bonemarrow 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 thesame levels of SHP-2 as macrophages that had grown out fromcontrol cultures (Fig. 7B). These findings suggest that SHP-2 isessential for M-CSF signal transduction in macrophage-osteoclastprecursor cells, i.e. only those small number of precursors thatretain expression of SHP-2 in tamoxifen-treated ptpn11fl/fl ert2-cre mice are able to respond to M-CSF. In itself, this impaired

Fig. 6. Impaired osteoclastogenesis in induced SHP-2-deficient mice. (A)Femur sections of tamoxifen-injected moribund ptpn11fl/fl ert2-cre mice andptpn11fl/fl littermate controls were stained for TRAP to visualize osteoclasts (red color). Images are from 11-week-old mice injected with tamoxifen 5 weekspreviously. The location of osteoclasts beneath growth plates (g) and within the secondary spongiosa are indicated with blue arrowheads. Note the paucity ofosteoclasts in ptpn11fl/fl ert2-cre mice. Scale bars: top panels, 500m; bottom panels, 200m. (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: 100m. (C)Shown are the mean numbers of osteoclasts + 1 s.e.m. per field identified in bone sections (in situ) and in vitro osteoclast differentiationexperiments 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 secondaryspongiosa regions. Data are derived from randomly selected fields of femur heads from moribund ptpn11fl/fl ert2-cre mice and ptpn11fl/fl littermate controlsinjected with tamoxifen at 6 weeks of age (n3 in each genotype). Mice were 10-11 weeks of age at the time of analysis. Size of fields in in vitro experiments areas 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 wereinjected with tamoxifen at 6-8 weeks of age (n6 in each genotype). Osteoclast differentiation experiments were initiated 1-3 weeks thereafter. Statisticalsignificance was determined by paired Student’s t-test. **P<0.005.

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response to M-CSF would account for the defectiveosteoclastogenesis in induced SHP-2-deficient mice.

The two principal ways in which M-CSF is thought to promotethe proliferation and survival of macrophage-osteoclast precursorsis through activation of MAPK and the antiapoptotic protein kinaseAKT (Ross and Teitelbaum, 2005; Takayanagi, 2007). SHP-2 hasbeen previously implicated in receptor-mediated activation ofboth these protein kinases (Dance et al., 2008; Hakak et al., 2000;Ivins Zito et al., 2004). Therefore, we investigated whether M-CSF-induced activation of MAPK or AKT was impaired in macrophage-osteoclast precursors in the absence of SHP-2. For this purpose,we examined M-CSF-induced responses in lineage-negative bonemarrow cells, a relatively large percentage of which are GMP (Fig.3C). As determined in western blot experiments, M-CSF inducedonly weak and inconsistent activation of MAPK in lineage-negativecells, even in control mice (not shown). Therefore, it was notpossible to reliably determine whether activation of MAPK wasimpaired in lineage-negative cells in the absence of SHP-2. Bycontrast, activation of AKT in lineage-negative cells was readilydetected in control mice after stimulation with M-CSF (Fig. 7C).However, in tamoxifen-treated ptpn11fl/fl ert2-cre mice, M-CSFessentially failed to induce activation of AKT in lineage-negativecells (Fig. 7C). Thus, these findings provide a molecular basis forblocked osteoclastogenesis in ptpn11fl/fl ert2-cre mice.

DISCUSSIONPrevious studies of conditional SHP-2-deficient mice have revealedan important physiological role for this PTP in multiple differenttissues, including the central nervous system, cardiac and striatedmuscle, liver, pancreas, mammary gland, and thymus (Bard-Chapeau et al., 2006; Fornaro et al., 2006; Hagihara et al., 2009; Keet al., 2006; Ke et al., 2007; Kontaridis et al., 2008; Krajewska et al.,2008; Nguyen et al., 2006; Princen et al., 2009; Zhang et al., 2004;Zhang et al., 2009). These mice serve as models with which tounderstand the role of SHP-2 in tissue homeostasis that would berelevant to an understanding of the etiology of certain clinicalmanifestations in humans with SHP-2 mutations. However, not allfeatures of NS and LS are recapitulated in these mouse models, in

part owing to the tissue-restricted nature of SHP-2 deletion. In thecurrent studies, therefore, we have attempted to address this issueby examining the effect of global deletion of SHP-2 de novo in adultmice.

Systemic loss of SHP-2 resulted in the early demise of mice thatwas associated with abnormalities of liver and kidney function, andmetabolic abnormalities suggested by precipitous weight loss.However, a definitive cause of death was not identified. Onepossibility is that death results from lung or cardiac compressionsecondary to skeletal alterations in this model (see below). Onenovel finding associated with induced SHP-2 loss in this model wasskin epidermal acanthosis and hyperkeratosis. This finding pointsto a role for SHP-2 as a regulator of keratinocyte proliferation anddifferentiation. It is tempting to speculate that SHP-2 regulateskeratinocyte proliferation and differentiation through its ability topromote activation of the Ras-MAPK signaling pathway. In thisregard, augmented Ras-MAPK signaling has been shown previouslyto result in terminal differentiation and inhibition of proliferationof keratinocytes (Lin and Lowe, 2001; Roper et al., 2001). However,published literature on the role of the Ras-MAPK signaling pathwayin the regulation of keratinocyte proliferation is conflicting (Cai etal., 2002). Nonetheless, the finding here of skin abnormalities ininduced SHP-2-deficient mice is consistent with the occurrence ofcutaneous lentigines in LS patients (Digilio et al., 2002; Gorlin etal., 1969).

Induced systemic deletion of SHP-2 was also found to result indisorders of hematopoiesis. The role of SHP-2 in hematopoiesis invivo in mice has not been properly examined before. Most studiesthat have addressed this issue have used SHP-2-deficient embryonicstem (ES) cells (Chan et al., 2003; Feng, 2007; Qu et al., 2001; Zouet al., 2006). SHP-2 is required for ES cell differentiation tomesoderm and mesoderm differentiation to hemangioblasts (Quet al., 2001; Chan et al., 2003). Therefore, it has not been possibleto examine the effect of loss of SHP-2 upon HSC renewal ordifferentiation of HSCs into hematopoietic lineages by using SHP-2-deficient ES cells. As an alternative approach, Chan et al.examined the influence of SHP-2 haploinsufficiency upon HSCfunction in competitive repopulation studies wherein heterozygote

Fig. 7. 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. (n4). Results are representative offour repeat experiments. Statistical significance was determined by two sample Student’s t-test. **P<0.005. (B)Western blots showing expression of SHP-2 inbone 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 withtamoxifen 3 weeks beforehand at 6 weeks of age). NS, non-specific band. Blots were stripped and reprobed with an anti-GAPDH antibody to verify equivalentprotein loading. Similar results were obtained in five independent experiments. (C)Lineage-negative bone marrow cells from a ptpn11fl/fl ert2-cre mouse and alittermate 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-AKTantibody to verify equal loading. Similar results were obtained in three repeat experiments.

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ptpn11+/– and wild-type HSCs were co-transferred into irradiatedwild-type recipients (Chan et al., 2006). Despite the finding thatthe number of HSCs was not different in the bone marrow ofptpn11+/– and wild-type mice, these studies showed a clear reducedrepopulating activity of the ptpn11+/– HSCs that was associatedwith diminished HSC self renewal. In the current studies, in whichdeletion of SHP-2 was induced de novo in adult mice, we also didnot observe a reduction in HSC number. However, this does notnecessarily challenge the view that SHP-2 is required for HSC self-renewal, and this might be revealed in transplantation experiments.In addition to normal HSC numbers being detected, the numberof CMPs, MEPs and GMPs were found to be normal in bonemarrow following induced loss of SHP-2. This suggests that SHP-2 is not essential for differentiation of HSCs into these committedprecursors. In contrast to this, the number of CLPs was substantiallyreduced in induced SHP-2-deficient mice, thus demonstrating arole for SHP-2 in the differentiation of HSCs into CLPs and/or CLPsurvival.

A reduced number of CLPs is consistent with the finding ofreduced numbers of thymocytes and of T and B cells in peripherallymphoid organs. However, the severe thymic atrophy that isobserved upon loss of SHP-2 points to additional roles for SHP-2in the generation of T cells. As revealed with the use of a (LCK)-promoter-driven Cre transgene that is active at the late DN stageof T cell development, SHP-2 is required for normal pre-T-cell-receptor (pre-TCR) signaling in developing thymocytes (Nguyenet al., 2006). Signal transduction through the pre-TCR triggers aproliferative burst in DN cells, culminating in the development ofDP cells. Therefore, a reduced number of CLPs together with factorssuch as impaired pre-TCR signal transduction probably accountfor the observed near complete block in T cell development.Presumably, T cells that exist in peripheral lymphoid organs ininduced SHP-2-deficient mice exited the thymus prior to the timeof SHP-2 loss. In contrast to bone marrow, loss of SHP-2 proteinexpression in whole spleen was only modest in induced SHP-2-deficient mice (Fig. 1C). Therefore, changes in the cellularcomposition of spleen in these animals can most likely be explainedsolely by altered hematopoiesis. Alternatively, SHP-2 might initiallybe deleted in a more substantial fraction of mature splenocytes butconfer a survival disadvantage to deleted cells that results in theirrapid clearance.

Aside from lymphopenia, induced SHP-2-deficient mice alsodeveloped anemia. This finding is consistent with previous reportsthat physical association of SHP-2 with the erythropoietin receptoris required for full proliferative activity of erythrocyte precursorsin vitro (Wojchowski et al., 1999). However, the number of Ter119+erythrocyte precursors were found to be normal in bone marrowof induced SHP-2-deficient mice and were elevated in spleen ofthese animals, which is indicative of extramedullary hematopoiesis.Furthermore, numbers of reticulocytes were elevated in theperipheral blood. Therefore, only the terminal stages of erythrocytedevelopment may be affected by SHP-2 loss in vivo. Alternatively,the anemia that is observed might not be intrinsic to thehematopoietic compartment but instead be caused by extrinsic, yet-to-be-determined, factors.

Despite the hematological and immunological abnormalitiesfound in this model, similar disorders have not been reported inNS and LS patients as far as we are aware. In NS, there is increased

susceptibility to juvenile myelomonocytic leukemia (JMML) and,in non-NS patients with JMML, PTPN11 somatic missensemutations are frequent (Tartaglia et al., 2003). Furthermore,genome-wide association studies have revealed strong linkage ofPTPN11 polymorphisms to autoimmune and inflammatorydisorders in humans, such as type I diabetes and Crohn’s disease(Todd et al., 2007). The fact that there is discordance with regardsthese actual or implied phenotypes in humans and the mousephenotype described here most probably reflects differences in thenature of the mutations involved.

Skeletal malformation is the most striking phenotype resultingfrom induced loss of SHP-2 in adult animals. The majority of micedeveloped kyphosis and/or scoliosis within weeks of SHP-2 loss.This is clinically significant because similar skeletal malformationshave been documented in NS patients (Noonan, 2006). In one studyof Korean NS patients, as many as 30% of sufferers were found todevelop kyphoses or scoliosis (Lee et al., 2001). Skeletal defects inhumans with SHP-2 mutations, however, are not limited to thespine: chest deformity in the form of pectus carinatum and pectusexcavatum are observed in both NS and LS patients (Sarkozy etal., 2008; Sharland et al., 1992). Thus, a normal range of Rassignaling seems to be required for normal skeletal structure inhumans because excessive Ras signaling, as in NS, and reduced Rassignaling, as in LS, both cause skeletal abnormalities. Consistentwith this, patients with neurofibromatosis, which is caused bygermline mutations in the neurofibromin 1 (NF1) gene, whichencodes a negative regulator of Ras, also develop scoliosis(Schindeler and Little, 2008).

In induced SHP-2-deficient mice, skeletal abnormalities areassociated with osteopetrosis and cartilage alterations includingaccumulation of collagenous matrix (osteoid) in bone. Asdocumented by X-ray analysis, CT scanning and histology,osteopetrosis affects the entire skeleton, including vertebrae, longbones and ribs. Also, ectopic calcified growths were frequentlyobserved on the spine and ribs. Histological analysis revealed apaucity of osteoclasts in vertebrae and long bones of induced SHP-2-deficient mice and in vitro M-CSF- and RANKL-induceddifferentiation of osteoclasts from bone marrow precursor cellswas shown to be severely impaired. Therefore, a defect inosteoclastogenesis that is intrinsic to osteoclast precursor cellsseems to be a major cause of the osteopetrosis in induced SHP-2-deficient mice. In addition, osteoid formation in induced SHP-2-deficient mice could contribute to low osteoclast number andinterruption of the normal process of bone resorption in vivo.

Another possible cause of osteopetrosis in induced SHP-2-deficient mice is increased osteoblast activity. The function of theRas-MAPK pathway in osteogenesis is controversial, with somestudies showing a role for this pathway in the promotion ofosteoblast development and others showing an inhibitory role(reviewed by Schindeler and Little, 2006). Nonetheless, increasedosteoblast activity in induced SHP-2-deficient mice would beconsistent with the finding that NF1 is required for the properdifferentiation and function of osteoblasts (Kolanczyk et al., 2007;Yu et al., 2005). Indeed, we determined that expression of the boneformation markers alkaline phosphatase, integrin-bindingsialoprotein and Runx2 was increased in induced SHP-2-deficientmice as soon as 1 week after tamoxifen administration(supplementary material Fig. S1). Increased activity of osteoblasts,

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therefore, together with defective osteoclastogenesis, wouldaccelerate the increase in bone mass in this model.

The ability of M-CSF to drive differentiation of macrophage-osteoclast precursor cells is blocked following induced loss ofSHP-2. This finding, therefore, accounts for the defectiveosteoclastogenesis, although we cannot rule out the possibility thatRANKL signaling is also impaired in these precursors.Mechanistically, we determined that AKT is not activated properlyin response to M-CSF stimulation. This is significant because M-CSF-induced activation of AKT provides essential survival signalsto macrophage-osteoclast precursors necessary for their expansionand differentiation (Ross and Teitelbaum, 2005; Takayanagi, 2007).During the course of M-CSF signal transduction, SHP-2 has thepotential to promote activation of AKT directly or indirectlythrough the regulation of Ras. Regardless, the notion that impairedactivation of AKT in osteoclast precursors contributes to theosteopetrotic phenotype of induced SHP-2-deficient mice issupported by findings in ovariectomized female NF1+/– mice.Parallel to the phenotype described here, ovariectomized NF1+/–

mice develop osteopenia that is at least in part a consequence ofincreased generation of osteoclasts. This increased osteoclastgeneration represents an intrinsic osteoclast defect that iscoincident with augmented activation of AKT (Yan et al., 2008;Yang et al., 2006).

Despite the finding that M-CSF signaling is blocked in theabsence of SHP-2, the number of macrophages in bone marrowand peripheral lymphoid organs of induced SHP-2-deficient micewere not reduced. There are two potential explanations for thisfinding. First, macrophage differentiation might proceedindependently of M-CSF in whole animals. For instance, M-CSF-deficient and M-CSFR-deficient mice, although both severelyosteopetrotic, show varying degrees of macrophage depletion,depending upon the tissue examined and age (Dai et al., 2002;Wiktor-Jedrzejczak et al., 1982; Yoshida et al., 1990). In some tissues,macrophage numbers might be reduced by less than 50% of thosefound in corresponding tissues of littermate wild-type mice (Daiet al., 2002). Presumably, this reflects the action of othermacrophage-differentiation-promoting cytokines such as IL-3 andgranulocyte-macrophage colony-stimulating factor (GM-CSF).Second, the longevity of tissue macrophages is considerably greaterthan that of osteoclasts. The estimated half-life of murine tissuemacrophages ranges from weeks to months, again depending upontissue (Murphy et al., 2008; Papadimitriou and Ashman, 1989). Bycontrast, the estimated half-life of murine osteoclasts is around 1.3days (Marshall and Davie, 1991). In the model described herein,the majority of induced SHP-2-deficient mice succumbed withinweeks of gene deletion, at which point macrophage numbers weredetermined. Within this time frame, substantial numbers ofmacrophages that had developed prior to the time of SHP-2deletion would remain in tissues. Thus, the relatively long half-life oftissue macrophages and M-CSF-independent-driven macrophagedifferentiation are probably significant contributing factors thatexplain the lack of diminution of macrophage numbers followinginduced loss of SHP-2.

In summary, we report here a model in which induced systemicdeletion of SHP-2 in adult mice results in early lethality, metabolic,skin and hematologic disorders, and, most strikingly, thedevelopment of severe skeletal abnormalities. We anticipate that

the model will be of further use in dissecting the role of SHP-2 asa regulator of skeletal morphogenesis. Knowledge gained is likelyto yield insight into the etiology of skeletal malformations inhumans with SHP-2 mutations and provide a rational basis for thedevelopment of therapies for the prevention and treatment of thiscondition.

METHODSMiceptpn11fl/fl mice and ubiquitin-promoter-driven ert2-cre transgenicmice have been described (Ruzankina et al., 2007; Zhang et al.,2004). Mice were intercrossed to generate ptpn11fl/fl ert2-cre miceand littermate ptpn11+/+, ptpn11fl/+, ptpn11fl/+ ert2-cre andptpn11fl/fl control mice. Mice were injected twice with tamoxifen(Sigma; 200 g/g body weight in corn oil on 2 consecutive days)at 6-8 weeks of age. Animal health was monitored daily. Initialobservations established that, once ptpn11fl/fl ert2-cre mice becamemoribund, they died within 24 hours. Thereafter, moribund micewere euthanized immediately upon identification. Moribundeuthanized ptpn11fl/fl ert2-cre mice are included in survival andweight loss analyses. Unless otherwise stated all other analyses wereperformed upon ptpn11fl/fl ert2-cre mice and control mice at thetime that the former became moribund. All research was performedin compliance with University of Michigan guidelines and wasapproved by the University Committee on the Use and Care ofAnimals.

Clinical chemistrySpin hematocrits and methylene blue reticulocyte counts wereperformed on heparinized whole blood. Levels of biomarkers inserum were analyzed on a Vettest Chemistry Analyzer Model 8008(Idexx).

HistologyTissues were fixed in 10% buffered formalin, transferred to 70%ethanol and embedded in paraffin. Bone samples were decalcifiedover a period of 2 weeks in 10% EDTA-ammonium hydroxide, pH7.2, prior to embedding. Sections (8 m for bone, 5 m for all othertissues) were stained with H&E, Alcian blue with an eosin-orangeG counterstain, or for TRAP using a TRAP staining kit (Sigma).

Flow cytometrySingle cell suspensions of thymocytes, splenocytes and hind-limb-derived bone marrow cells were stained with fluorochrome-labeledmonoclonal antibodies to TCR, CD3, CD19, CD4, CD8, CD11c,NK1.1, B220, CD11b, GR-1 and Ter119 (all BD Biosciences) andanalyzed by flow cytometry on a FacsCanto (BD Biosciences) toenumerate different leukocyte subpopulations. In addition, bonemarrow cells were additionally stained with labeled antibodies toSca-1, c-Kit, CD127, CD34 and CD16/32 (BD Biosciences) toenumerate lineage-negative precursor populations.

X-ray and CTX-ray images were acquired using a microradiography machine(Faxitron Corporation). CT scanning was performed using a conebeam CT system (GE Healthcare Biosciences). Imagereconstruction was performed on 25-m voxels, a threshold wasgenerated to define mineralized tissue and regions of interest were

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analyzed using the MicroView Analysis program (GE HealthcareBiosciences).

OsteoclastogenesisBone marrow cells were cultured overnight on plastic Petri dishesin minimal essential medium containing 10% fetal calf serum,antibiotics, 25 ng/ml recombinant murine M-CSF and 100 ng/mlrecombinant murine RANKL (R&D Systems). Nonadherent cellswere then replated into wells of 24-well plastic plates containingglass coverslips. After 7 days, cells on coverslips were fixed andstained for TRAP.

SHP-2 expressionTo prepare tissue lysates, organs were crushed and lysed in 1% NP-40 lysis buffer. Lysates were run on 10% SDS-PAGE gels andanalyzed by western blotting using an anti-SHP-2 rabbit polyclonalantibody (Cell Signaling) before stripping and reprobing with aGAPDH antibody (Santa Cruz) as a loading control.

In other experiments, bone marrow cells were cultured in wellsof 24-well plastic plates in the same medium used for in vitroosteoclastogenesis minus RANKL. After 5 days, macrophageswere lysed in wells and analyzed for expression of SHP-2 andGAPDH as above. Comparisons were made with freshly isolatedbone marrow cells.

AKT activationBone marrow cells were depleted of TCR-, CD3-, CD19-, CD4-,CD8-, CD11c-, NK1.1-, B220-, GR-1- and Ter119-expressinglineage-positive cells by negative selection using MACS columns(Miltenyi). Cells were then stimulated with M-CSF (100 ng/ml) forthe indicated times before lysis. Activation of AKT was determinedby western blotting using a phospho-specific (T308) anti-AKTantibody (Cell Signaling). Blots were stripped and reprobed withan anti-AKT antibody to ascertain equal loading (Cell Signaling).

Statistical analysisStatistical significance was determined using Student’s one sample,two sample or paired sample t-tests as indicated. In one sample t-tests, in the case that more than one control mouse was availablefor each test mouse, a mean control value was calculated for usein statistical calculations. *P<0.05; **P<0.005.

ACKNOWLEDGEMENTSWe thank Eric Brown (University of Pennsylvania) for providing ubiquitin ert2-cremice. This work was supported by National Institutes of Health grant AI050699 toP.D.K.

COMPETING INTERESTSThe authors declare no competing financial interests.

AUTHOR CONTRIBUTIONST.J.B., N.K., Y.M. and P.D.K. designed experiments, T.J.B., N.K., P.E.L., E.L., J.E.W. andP.D.K. performed experiments, G.-S.F. contributed SHP-2 floxed mice, and T.J.B., N.K.and P.D.K. analyzed data and wrote the paper.

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.006130/-/DC1

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TRANSLATIONAL IMPACT

Clinical issueIn humans, gain-of-function and loss-of-function mutations of the SHP-2 proteintyrosine phosphatase (PTP) cause Noonan syndrome (NS) and LEOPARDsyndrome (LS), respectively. Noonan syndrome is relatively common, affectingbetween 1 in 1000 and 1 in 5000 live births, whereas LEOPARD syndrome ismuch rarer. Both are autosomal dominant diseases with variable penetrance. Thesyndromes have several overlapping features, including skeletal abnormalitiessuch as growth retardation and chest malformations; in NS, sufferers often haveadditional elbow and hand malformations and spinal curvature. How mutationsin SHP-2 lead to skeletal malformation is unknown.

ResultsThe development of an inducible systemic SHP-2-deficient mouse modelwould enable greater understanding of the role of SHP-2 in skeletal growthand remodeling. Here, the authors report that induced broad deletion of theptpn11 gene (which encodes SHP-2) in adult mice results in multiple tissueabnormalities. The most prominent of these is severe curvature of the spine,causing both scoliosis (side-to-side curvature) and kyphosis (hunched back).These skeletal malformations are associated with osteopetrosis and cartilagealterations. A principal cause of the osteopetrosis in this model is defectiveosteoclastogenesis (differentiation of the cells that resorb bone). In SHP-2-deficient osteoclast precursor cells, the growth factor macrophage colony-stimulating factor (M-CSF) cannot induce activation of AKT protein kinase,which is essential for osteoclast precursor survival and differentiation.

Implications and future directionsLoss of function of SHP-2 in mice causes skeletal abnormalities in part byinhibiting bone remodeling, and it is likely that the same process is disruptedin humans with LS. The model described here will be useful for identifying anddissecting the signaling pathways that are controlled by SHP-2 during skeletalmorphogenesis, and might also yield new information about mechanisms ofbone regulation in general. Such studies will aid the rational design oftherapeutics for treatment of skeletal disorders in humans with inheritedSHP-2 mutations.

doi:10.1242/dmm.007013

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Development of severe skeletal defects in induced SHP-2 ... · SHP-2 (encoded by PTPN11) is a broadly expressed Src homology-2 domain (SH2)-containing protein tyrosine phosphatase