SHP2 ablation mitigates osteoarthritic cartilage degeneration by promoting chondrocyte anabolism through SOX9.

Articular cartilage phenotypic homeostasis is crucial for life-long joint function, but the underlying cellular and molecular mechanisms governing chondrocyte stability remain poorly understood. Here, we show that the protein tyrosine phosphatase SHP2 is differentially expressed in articular cartilage (AC) and growth plate cartilage (GPC) and that it negatively regulates cell proliferation and cartilage phenotypic program. Postnatal SHP2 deletion in Prg4+ AC chondrocytes increased articular cellularity and thickness, whereas SHP2 deletion in Acan+ pan-chondrocytes caused excessive GPC chondrocyte proliferation and led to joint malformation post-puberty. These observations were verified in mice and in cultured chondrocytes following treatment with the SHP2 PROTAC inhibitor SHP2D26. Further mechanistic studies indicated that SHP2 negatively regulates SOX9 stability and transcriptional activity by influencing SOX9 phosphorylation and promoting its proteasome degradation. In contrast to published work, SHP2 ablation in chondrocytes did not impact IL-1-evoked inflammation responses, and SHP2's negative regulation of SOX9 could be curtailed by genetic or chemical SHP2 inhibition, suggesting that manipulating SHP2 signaling has translational potential for diseases of cartilage dyshomeostasis.

A method for colocalizing lineage tracing reporter and RNAscope signals on skeletal tissue section.

Fluorescent reporters have been widely used in modern biology as a powerful tool in cell lineage tracing during development and in studying the pathogenesis of diseases. RNAscope is a recently developed RNA in situ hybridization method with high specificity and sensitivity. Combined application of these two techniques on skeletal tissue is difficult and has not been done before; the reporter fluorophores in the tissue specimen bleach quickly and mRNAs degrade rapidly due to the decalcification process typically used in processing skeletal samples. Therefore, we developed a method that can simultaneously detect and colocalize both the fluorescent lineage tracing reporter signal and the RNAscope signal in the same skeletal section without compromising the fidelity, sensitivity, and specificity of lineage tracing and RNAscope. This was achieved by cryosectioning bone and cartilage tissue without decalcification, thus allowing the fluorescent reporter signal and RNA in the sections to be well-preserved so that RNAscope can be carried out in situ, and these two signals can be colocalized. Our method of colocalization has versatile applications, e.g., determination of gene knockout efficacy at the mRNA level in a specific cell lineage in situ, detection of alterations in target gene transcripts in reporter-positive cells caused by a specific gene mutation, studies of the disease pathology by examining the transcript-level expression of genes of interest in the cell lineage in vivo.

SHP2 regulates the development of intestinal epithelium by modifying OSTERIX+ crypt stem cell self-renewal and proliferation.

The protein tyrosine phosphatase SHP2, encoded by PTPN11, is ubiquitously expressed and essential for the development and/or maintenance of multiple tissues and organs. SHP2 is involved in gastrointestinal (GI) epithelium development and homeostasis, but the underlying mechanisms remain elusive. While studying SHP2's role in skeletal development, we made osteoblast-specific SHP2 deficient mice using Osterix (Osx)-Cre as a driver to excise Ptpn11 floxed alleles. Phenotypic characterization of these SHP2 mutants unexpectedly revealed a critical role of SHP2 in GI biology. Mice lacking SHP2 in Osx+ cells developed a fatal GI pathology with dramatic villus hypoplasia. OSTERIX, an OB-specific zinc finger-containing transcription factor is for the first time found to be expressed in GI crypt cells, and SHP2 expression in the crypt Osx+ cells is critical for self-renewal and proliferation. Further, immunostaining revealed the colocalization of OSTERIX with OLFM4 and LGR5, two bona fide GI stem cell markers, at the crypt cells. Furthermore, OSTERIX expression is found to be associated with GI malignancies. Knockdown of SHP2 expression had no apparent influence on the relative numbers of enterocytes, goblet cells or Paneth cells. Given SHP2's key regulatory role in OB differentiation, our studies suggest that OSTERIX and SHP2 are indispensable for gut homeostasis, analogous to SOX9's dual role as a master regulator of cartilage and an important regulator of crypt stem cell biology. Our findings also provide a foundation for new avenues of inquiry into GI stem cell biology and of OSTERIX's therapeutic and diagnostic potential.

GNAI1 and GNAI3 Reduce Colitis-Associated Tumorigenesis in Mice by Blocking IL6 Signaling and Down-regulating Expression of GNAI2.

BACKGROUND & AIMS: Interleukin 6 (IL6) and tumor necrosis factor contribute to the development of colitis-associated cancer (CAC). We investigated these signaling pathways and the involvement of G protein subunit alpha i1 (GNAI1), GNAI2, and GNAI3 in the development of CAC in mice and humans. METHODS: B6;129 wild-type (control) or mice with disruption of Gnai1, Gnai2, and/or Gnai3 or conditional disruption of Gnai2 in CD11c+ or epithelial cells were given dextran sulfate sodium (DSS) to induce colitis followed by azoxymethane (AOM) to induce carcinogenesis; some mice were given an antibody against IL6. Feces were collected from mice, and the compositions of microbiomes were analyzed by polymerase chain reactions. Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) isolated from spleen and colon tissues were analyzed by flow cytometry. We performed immunoprecipitation and immunoblot analyses of colon tumor tissues, MDSCs, and mouse embryonic fibroblasts to study the expression levels of GNAI1, GNAI2, and GNAI3 and the interactions of GNAI1 and GNAI3 with proteins in the IL6 signaling pathway. We analyzed the expression of Gnai2 messenger RNA by CD11c+ cells in the colonic lamina propria by PrimeFlow, expression of IL6 in DCs by flow cytometry, and secretion of cytokines in sera and colon tissues by enzyme-linked immunosorbent assay. We obtained colon tumor and matched nontumor tissues from 83 patients with colorectal cancer having surgery in China and 35 patients with CAC in the United States. Mouse and human colon tissues were analyzed by histology, immunoblot, immunohistochemistry, and/or RNA-sequencing analyses. RESULTS: GNAI1 and GNAI3 (GNAI1;3) double-knockout (DKO) mice developed more severe colitis after administration of DSS and significantly more colonic tumors than control mice after administration of AOM plus DSS. Development of increased tumors in DKO mice was not associated with changes in fecal microbiomes but was associated with activation of nuclear factor (NF) κB and signal transducer and activator of transcription (STAT) 3; increased levels of GNAI2, nitric oxide synthase 2, and IL6; increased numbers of CD4+ DCs and MDSCs; and decreased numbers of CD8+ DCs. IL6 was mainly produced by CD4+/CD11b+, but not CD8+, DCs in DKO mice. Injection of DKO mice with a blocking antibody against IL6 reduced the expansion of MDSCs and the number of tumors that developed after CAC induction. Incubation of MDSCs or mouse embryonic fibroblasts with IL6 induced activation of either NF-κB by a JAK2-TRAF6-TAK1-CHUK/IKKB signaling pathway or STAT3 by JAK2. This activation resulted in expression of GNAI2, IL6 signal transducer (IL6ST, also called GP130) and nitric oxide synthase 2, and expansion of MDSCs; the expression levels of these proteins and expansion of MDSCs were further increased by the absence of GNAI1;3 in cells and mice. Conditional disruption of Gnai2 in CD11c+ cells of DKO mice prevented activation of NF-κB and STAT3 and changes in numbers of DCs and MDSCs. Colon tumor tissues from patients with CAC had reduced levels of GNAI1 and GNAI3 and increased levels of GNAI2 compared with normal tissues. Further analysis of a public human colorectal tumor DNA microarray database (GSE39582) showed that low Gani1 and Gnai3 messenger RNA expression and high Gnai2 messenger RNA expression were significantly associated with decreased relapse-free survival. CONCLUSIONS: GNAI1;3 suppresses DSS-plus-AOM-induced colon tumor development in mice, whereas expression of GNAI2 in CD11c+ cells and IL6 in CD4+/CD11b+ DCs appears to promote these effects. Strategies to induce GNAI1;3, or block GNAI2 and IL6, might be developed for the prevention or therapy of CAC in patients.

A ERK/RSK-mediated negative feedback loop regulates M-CSF-evoked PI3K/AKT activation in macrophages.

Activation of the RAS/ERK and its downstream signaling components is essential for growth factor-induced cell survival, proliferation, and differentiation. The Src homology-2 domain containing protein tyrosine phosphatase 2 (SHP2), encoded by protein tyrosine phosphatase, non-receptor type 11 ( Ptpn11), is a positive mediator required for most, if not all, receptor tyrosine kinase-evoked RAS/ERK activation, but differentially regulates the PI3K/AKT signaling cascade in various cellular contexts. The precise mechanisms underlying the differential effects of SHP2 deficiency on the PI3K pathway remain unclear. We found that mice with myelomonocytic cell-specific [ Tg(LysM-Cre); Ptpn11fl/fl mice] Ptpn11 deficiency exhibit mild osteopetrosis. SHP2-deficient bone marrow macrophages (BMMs) showed decreased proliferation in response to M-CSF and decreased osteoclast generation. M-CSF-evoked ERK1/2 activation was decreased, whereas AKT activation was enhanced in SHP2-deficient BMMs. ERK1/2, via its downstream target RSK2, mediates this negative feedback by negatively regulating phosphorylation of M-CSF receptor at Tyr721 and, consequently, its binding to p85 subunit of PI3K and PI3K activation. Pharmacologic inhibition of RSK or ERK phenotypically mimics the signaling defects observed in SHP2-deficient BMMs. Furthermore, this increase in PI3K/AKT activation enables BMM survival in the setting of SHP2 deficiency.-Wang, L., Iorio, C., Yan, K., Yang, H., Takeshita, S., Kang, S., Neel, B.G., Yang, W. An ERK/RSK-mediated negative feedback loop regulates M-CSF-evoked PI3K/AKT activation in macrophages.

Ptpn11 deletion in a novel progenitor causes metachondromatosis by inducing hedgehog signalling.

The tyrosine phosphatase SHP2, encoded by PTPN11, is required for the survival, proliferation and differentiation of various cell types. Germline activating mutations in PTPN11 cause Noonan syndrome, whereas somatic PTPN11 mutations cause childhood myeloproliferative disease and contribute to some solid tumours. Recently, heterozygous inactivating mutations in PTPN11 were found in metachondromatosis, a rare inherited disorder featuring multiple exostoses, enchondromas, joint destruction and bony deformities. The detailed pathogenesis of this disorder has remained unclear. Here we use a conditional knockout (floxed) Ptpn11 allele (Ptpn11(fl)) and Cre recombinase transgenic mice to delete Ptpn11 specifically in monocytes, macrophages and osteoclasts (lysozyme M-Cre; LysMCre) or in cathepsin K (Ctsk)-expressing cells, previously thought to be osteoclasts. LysMCre;Ptpn11(fl/fl) mice had mild osteopetrosis. Notably, however, CtskCre;Ptpn11(fl/fl) mice developed features very similar to metachondromatosis. Lineage tracing revealed a novel population of CtskCre-expressing cells in the perichondrial groove of Ranvier that display markers and functional properties consistent with mesenchymal progenitors. Chondroid neoplasms arise from these cells and show decreased extracellular signal-regulated kinase (ERK) pathway activation, increased Indian hedgehog (Ihh) and parathyroid hormone-related protein (Pthrp, also known as Pthlh) expression and excessive proliferation. Shp2-deficient chondroprogenitors had decreased fibroblast growth factor-evoked ERK activation and enhanced Ihh and Pthrp expression, whereas fibroblast growth factor receptor (FGFR) or mitogen-activated protein kinase kinase (MEK) inhibitor treatment of chondroid cells increased Ihh and Pthrp expression. Importantly, smoothened inhibitor treatment ameliorated metachondromatosis features in CtskCre;Ptpn11(fl/fl) mice. Thus, in contrast to its pro-oncogenic role in haematopoietic and epithelial cells, Ptpn11 is a tumour suppressor in cartilage, acting through a FGFR/MEK/ERK-dependent pathway in a novel progenitor cell population to prevent excessive Ihh production.

Gain-of-function mutations of Ptpn11 (Shp2) cause aberrant mitosis and increase susceptibility to DNA damage-induced malignancies.

Gain-of-function (GOF) mutations of protein tyrosine phosphatase nonreceptor type 11 Ptpn11 (Shp2), a protein tyrosine phosphatase implicated in multiple cell signaling pathways, are associated with childhood leukemias and solid tumors. The underlying mechanisms are not fully understood. Here, we report that Ptpn11 GOF mutations disturb mitosis and cytokinesis, causing chromosomal instability and greatly increased susceptibility to DNA damage-induced malignancies. We find that Shp2 is distributed to the kinetochore, centrosome, spindle midzone, and midbody, all of which are known to play critical roles in chromosome segregation and cytokinesis. Mouse embryonic fibroblasts with Ptpn11 GOF mutations show a compromised mitotic checkpoint. Centrosome amplification and aberrant mitosis with misaligned or lagging chromosomes are significantly increased in Ptpn11-mutated mouse and patient cells. Abnormal cytokinesis is also markedly increased in these cells. Further mechanistic analyses reveal that GOF mutant Shp2 hyperactivates the Polo-like kinase 1 (Plk1) kinase by enhancing c-Src kinase-mediated tyrosine phosphorylation of Plk1. This study provides novel insights into the tumorigenesis associated with Ptpn11 GOF mutations and cautions that DNA-damaging treatments in Noonan syndrome patients with germ-line Ptpn11 GOF mutations could increase the risk of therapy-induced malignancies.

SHP2 regulates chondrocyte terminal differentiation, growth plate architecture and skeletal cell fates.

Loss of PTPN11/SHP2 in mice or in human metachondromatosis (MC) patients causes benign cartilage tumors on the bone surface (exostoses) and within bones (enchondromas). To elucidate the mechanisms underlying cartilage tumor formation, we investigated the role of SHP2 in the specification, maturation and organization of chondrocytes. Firstly, we studied chondrocyte maturation by performing RNA-seq on primary chondrocyte pellet cultures. We found that SHP2 depletion, or inhibition of the ERK1/2 pathway, delays the terminal differentiation of chondrocytes from the early-hypertrophic to the late-hypertrophic stage. Secondly, we studied chondrocyte maturation and organization in mice with a mosaic postnatal inactivation of Ptpn11 in chondrocytes. We found that the vertebral growth plates of these mice have expanded domains of early-hypertrophic chondrocytes that have not yet terminally differentiated, and their enchondroma-like lesions arise from chondrocytes displaced from the growth plate due to a disruption in the organization of maturation and ossification zones. Furthermore, we observed that lesions from human MC patients also display disorganized chondrocyte maturation zones. Next, we found that inactivation of Ptpn11 in Fsp1-Cre-expressing fibroblasts induces exostosis-like outgrowths, suggesting that loss of SHP2 in cells on the bone surface and at bone-ligament attachment sites induces ectopic chondrogenesis. Finally, we performed lineage tracing to show that exostoses and enchondromas in mice likely contain mixtures of wild-type and SHP2-deficient chondrocytes. Together, these data indicate that in patients with MC, who are heterozygous for inherited PTPN11 loss-of-function mutations, second-hit mutations in PTPN11 can induce enchondromas by disrupting the organization and delaying the terminal differentiation of growth plate chondrocytes, and can induce exostoses by causing ectopic chondrogenesis of cells on the bone surface. Furthermore, the data are consistent with paracrine signaling from SHP2-deficient cells causing SHP2-sufficient cells to be incorporated into the lesions.

SHP2 regulates osteoclastogenesis by promoting preosteoclast fusion.

Genes that regulate osteoclast (OC) development and function in both physiologic and disease conditions remain incompletely understood. Shp2 (the Src homology-2 domain containing protein tyrosine phosphatase 2), a ubiquitously expressed cytoplasmic protein tyrosine phosphatase, is implicated in regulating M-CSF and receptor activator of nuclear factor-κB ligand (RANKL)-evoked signaling; its role in osteoclastogenesis and bone homeostasis, however, remains unknown. Using a tissue-specific gene knockout approach, we inactivated Shp2 expression in murine OCs. Shp2 mutant mice are phenotypically osteopetrotic, featuring a marked increase of bone volume (BV)/total volume (TV) (+42.8%), trabeculae number (Tb.N) (+84.1%), structure model index (+119%), and a decrease of trabecular thickness (Tb.Th) (-34.1%) and trabecular spacing (Tb.Sp) (-41.0%). Biochemical analyses demonstrate that Shp2 is required for RANKL-induced formation of giant multinucleated OCs by up-regulating the expression of nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1), a master transcription factor that is indispensable for terminal OC differentiation. Shp2 deletion, however, has minimal effect on M-CSF-dependent survival and proliferation of OC precursors. Instead, its deficiency aborts the fusion of OC precursors and formation of multinucleated OCs and decreases bone matrix resorption. Moreover, pharmacological intervention of Shp2 is sufficient to prevent preosteoclast fusion in vitro. These findings uncover a novel mechanism through which Shp2 regulates osteoclastogenesis by promoting preosteoclast fusion. Shp2 or its signaling partners could potentially serve as pharmacological targets to regulate the population of OCs locally and/or systematically, and thus treat OC-related diseases, such as periprosthetic osteolysis and osteoporosis.

Shp2-dependent ERK signaling is essential for induction of Bergmann glia and foliation of the cerebellum.

Folding of the cortex and the persistence of radial glia (RG)-like cells called Bergmann glia (BG) are hallmarks of the mammalian cerebellum. Similar to basal RG in the embryonic neocortex, BG maintain only basal processes and continuously express neural stem cell markers. Past studies had focused on the function of BG in granule cell migration and how granule cell progenitors (GCP) regulate cerebellar foliation. The molecular control of BG generation and its role in cerebellar foliation are less understood. Here, we have analyzed the function of the protein tyrosine phosphatase Shp2 in mice by deleting its gene Ptpn11 in the entire cerebellum or selectively in the GCP lineage. Deleting Ptpn11 in the entire cerebellum by En1-cre blocks transformation of RG into BG but preserves other major cerebellar cell types. In the absence of BG, inward invagination of GCP persists but is uncoupled from the folding of the Purkinje cell layer and the basement membrane, leading to disorganized lamination and an absence of cerebellar folia. In contrast, removing Ptpn11 in the GCP lineage by Atoh1-cre has no effect on cerebellar development, indicating that Shp2 is not cell autonomously required in GCP. Furthermore, we demonstrate that Ptpn11 interacts with Fgf8 and is essential for ERK activation in RG and nascent BG. Finally, expressing constitutively active MEK1 rescues BG formation and cerebellar foliation in Shp2-deficient cerebella. Our results demonstrate an essential role of Shp2 in BG specification via fibroblast growth factor/extracellular signal-regulated protein kinase signaling, and reveal a crucial function of BG in organizing cerebellar foliation.

Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development.

Mechanical stress regulates development by modulating cell signaling and gene expression. However, the cytoplasmic components mediating mechanotransduction remain unclear. In this study, elimination of muscle contraction during chicken embryonic development resulted in a reduction in the activity of mammalian target of rapamycin (mTOR) in the cartilaginous growth plate. Inhibition of mTOR activity led to significant inhibition of chondrocyte proliferation, cartilage tissue growth, and expression of chondrogenic genes, including Indian hedgehog (Ihh), a critical mediator of mechanotransduction. Conversely, cyclic loading (1 Hz, 5% matrix deformation) of embryonic chicken growth plate chondrocytes in 3-dimensional (3D) collagen scaffolding induced sustained activation of mTOR. Mechanical activation of mTOR occurred in serum-free medium, indicating that it is independent of growth factor or nutrients. Treatment of chondrocytes with Rapa abolished mechanical activation of cell proliferation and Ihh gene expression. Cyclic loading of chondroprogenitor cells deficient in SH2-containing protein tyrosine phosphatase 2 (Shp2) further enhanced mechanical activation of mTOR, cell proliferation, and chondrogenic gene expression. This result suggests that Shp2 is an antagonist of mechanotransduction through inhibition of mTOR activity. Our data demonstrate that mechanical activation of mTOR is necessary for cell proliferation, chondrogenesis, and cartilage growth during bone development, and that mTOR is an essential mechanotransduction component modulated by Shp2 in the cytoplasm.

Essential role for Ptpn11 in survival of hematopoietic stem and progenitor cells.

Src homology 2 domain-containing phosphatase 2 (Shp2), encoded by Ptpn11, is a member of the nonreceptor protein-tyrosine phosphatase family, and functions in cell survival, proliferation, migration, and differentiation in many tissues. Here we report that loss of Ptpn11 in murine hematopoietic cells leads to bone marrow aplasia and lethality. Mutant mice show rapid loss of hematopoietic stem cells (HSCs) and immature progenitors of all hematopoietic lineages in a gene dosage-dependent and cell-autonomous manner. Ptpn11-deficient HSCs and progenitors undergo apoptosis concomitant with increased Noxa expression. Mutant HSCs/progenitors also show defective Erk and Akt activation in response to stem cell factor and diminished thrombopoietin-evoked Erk activation. Activated Kras alleviates the Ptpn11 requirement for colony formation by progenitors and cytokine/growth factor responsiveness of HSCs, indicating that Ras is functionally downstream of Shp2 in these cells. Thus, Shp2 plays a critical role in controlling the survival and maintenance of HSCs and immature progenitors in vivo.

LRP5, Bone Mass Polymorphisms and Skeletal Disorders.

The formation and maintenance of the gross structure and microarchitecture of the human skeleton require the concerted functioning of a plethora of morphogenic signaling processes. Through recent discoveries in the field of genetics, numerous genotypic variants have been implicated in pathologic skeletal phenotypes and disorders arising from the disturbance of one or more of these processes. For example, total loss-of-function variants of LRP5 were found to be the cause of osteoporosis-pseudoglioma syndrome (OPPG). LRP5 encodes for the low-density lipoprotein receptor-related protein 5, a co-receptor in the canonical WNT-ß-catenin signaling pathway and a crucial protein involved in the formation and maintenance of homeostasis of the human skeleton. Beyond OPPG, other partial loss-of-function variants of LRP5 have been found to be associated with other low bone mass phenotypes and disorders, while LRP5 gain-of-function variants have been implicated in high bone mass phenotypes. This review introduces the roles that LRP5 plays in skeletal morphogenesis and discusses some of the structural consequences that result from abnormalities in LRP5. A greater understanding of how the LRP5 receptor functions in bone and other body tissues could provide insights into a variety of pathologies and their potential treatments, from osteoporosis and a variety of skeletal abnormalities to congenital disorders that can lead to lifelong disabilities.

Protein tyrosine phosphatases in skeletal development and diseases.

Skeletal development and homeostasis in mammals are modulated by finely coordinated processes of migration, proliferation, differentiation, and death of skeletogenic cells originating from the mesoderm and neural crest. Numerous molecular mechanisms are involved in these regulatory processes, one of which is protein posttranslational modifications, particularly protein tyrosine phosphorylation (PYP). PYP occurs mainly through the action of protein tyrosine kinases (PTKs), modifying protein enzymatic activity, changing its cellular localization, and aiding in the assembly or disassembly of protein signaling complexes. Under physiological conditions, PYP is balanced by the coordinated action of PTKs and protein tyrosine phosphatases (PTPs). Dysregulation of PYP can cause genetic, metabolic, developmental, and oncogenic skeletal diseases. Although PYP is a reversible biochemical process, in contrast to PTKs, little is known about how this equilibrium is modulated by PTPs in the skeletal system. Whole-genome sequencing has revealed a large and diverse superfamily of PTP genes (over 100 members) in humans, which can be further divided into cysteine (Cys)-, aspartic acid (Asp)-, and histidine (His)-based PTPs. Here, we review current knowledge about the functions and regulatory mechanisms of 28 PTPs involved in skeletal development and diseases; 27 of them belong to class I and II Cys-based PTPs, and the other is an Asp-based PTP. Recent progress in analyzing animal models that harbor various mutations in these PTPs and future research directions are also discussed. Our literature review indicates that PTPs are as crucial as PTKs in supporting skeletal development and homeostasis.

Altered glucose homeostasis in mice with liver-specific deletion of Src homology phosphatase 2.

The Src homology 2 domain-containing protein-tyrosine phosphatase Shp2 has been implicated in a variety of growth factor signaling pathways, but its role in insulin signaling has remained unresolved. In vitro studies suggest that Shp2 is both a negative and positive regulator of insulin signaling, although its physiological function in a number of peripheral insulin-responsive tissues remains unknown. To address the metabolic role of Shp2 in the liver, we generated mice with either chronic or acute hepatic Shp2 deletion using tissue-specific Cre-LoxP and adenoviral Cre approaches, respectively. We then analyzed insulin sensitivity, glucose tolerance, and insulin signaling in liver-specific Shp2-deficient and control mice. Mice with chronic Shp2 deletion exhibited improved insulin sensitivity and increased glucose tolerance compared with controls. Acute Shp2 deletion yielded comparable results, indicating that the observed metabolic effects are directly caused by the lack of Shp2 in the liver. These findings correlated with, and were most likely caused by, direct dephosphorylation of insulin receptor substrate (IRS)1/2 in the liver, accompanied by increased PI3K/Akt signaling. In contrast, insulin-induced ERK activation was dramatically attenuated, yet there was no effect on the putative ERK site on IRS1 (Ser(612)) or on S6 kinase 1 activity. These studies show that Shp2 is a negative regulator of hepatic insulin action, and its deletion enhances the activation of PI3K/Akt pathway downstream of the insulin receptor.

An Shp2/SFK/Ras/Erk signaling pathway controls trophoblast stem cell survival.

Little is known about how growth factors control tissue stem cell survival and proliferation. We analyzed mice with a null mutation of Shp2 (Ptpn11), a key component of receptor tyrosine kinase signaling. Null embryos die peri-implantation, much earlier than mice that express an Shp2 truncation. Shp2 null blastocysts initially develop normally, but they subsequently exhibit inner cell mass death, diminished numbers of trophoblast giant cells, and failure to yield trophoblast stem (TS) cell lines. Molecular markers reveal that the trophoblast lineage, which requires fibroblast growth factor-4 (FGF4), is specified but fails to expand normally. Moreover, deletion of Shp2 in TS cells causes rapid apoptosis. We show that Shp2 is required for FGF4-evoked activation of the Src/Ras/Erk pathway that culminates in phosphorylation and destabilization of the proapoptotic protein Bim. Bim depletion substantially blocks apoptosis and significantly restores Shp2 null TS cell proliferation, thereby establishing a key mechanism by which FGF4 controls stem cell survival.

Leukemogenic Ptpn11 causes fatal myeloproliferative disorder via cell-autonomous effects on multiple stages of hematopoiesis.

PTPN11, which encodes the tyrosine phosphatase SHP2, is mutated in approximately 35% of patients with juvenile myelomonocytic leukemia (JMML) and at a lower incidence in other neoplasms. To model JMML pathogenesis, we generated knockin mice that conditionally express the leukemia-associated mutant Ptpn11(D61Y). Expression of Ptpn11(D61Y) in all hematopoietic cells evokes a fatal myeloproliferative disorder (MPD), featuring leukocytosis, anemia, hepatosplenomegaly, and factor-independent colony formation by bone marrow (BM) and spleen cells. The Lin(-)Sca1(+)cKit(+) (LSK) compartment is expanded and "right-shifted," accompanied by increased stem cell factor (SCF)-evoked colony formation and Erk and Akt activation. However, repopulating activity is decreased in diseased mice, and mice that do engraft with Ptpn11(D61Y) stem cells fail to develop MPD. Ptpn11(D61Y) common myeloid progenitors (CMPs) and granulocyte-monocyte progenitors (GMPs) produce cytokine-independent colonies in a cell-autonomous manner and demonstrate elevated Erk and Stat5 activation in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation. Ptpn11(D61Y) megakaryocyte-erythrocyte progenitors (MEPs) yield increased numbers of erythrocyte burst-forming units (BFU-Es), but MEPs and erythrocyte-committed progenitors (EPs) produce fewer erythrocyte colony-forming units (CFU-Es), indicating defective erythroid differentiation. Our studies provide a mouse model for Ptpn11-evoked MPD and show that this disease results from cell-autonomous and distinct lineage-specific effects of mutant Ptpn11 on multiple stages of hematopoiesis.

SHP-2 activates signaling of the nuclear factor of activated T cells to promote skeletal muscle growth.

The formation of multinucleated myofibers is essential for the growth of skeletal muscle. The nuclear factor of activated T cells (NFAT) promotes skeletal muscle growth. How NFAT responds to changes in extracellular cues to regulate skeletal muscle growth remains to be fully defined. In this study, we demonstrate that mice containing a skeletal muscle-specific deletion of the tyrosine phosphatase SHP-2 (muscle creatine kinase [MCK]-SHP-2 null) exhibited a reduction in both myofiber size and type I slow myofiber number. We found that interleukin-4, an NFAT-regulated cytokine known to stimulate myofiber growth, was reduced in its expression in skeletal muscles of MCK-SHP-2-null mice. When SHP-2 was deleted during the differentiation of primary myoblasts, NFAT transcriptional activity and myotube multinucleation were impaired. Finally, SHP-2 coupled myotube multinucleation to an integrin-dependent pathway and activated NFAT by stimulating c-Src. Thus, SHP-2 transduces extracellular matrix stimuli to intracellular signaling pathways to promote skeletal muscle growth.

Mouse model of Noonan syndrome reveals cell type- and gene dosage-dependent effects of Ptpn11 mutation.

Noonan syndrome is a common human autosomal dominant birth defect, characterized by short stature, facial abnormalities, heart defects and possibly increased risk of leukemia. Mutations of Ptpn11 (also known as Shp2), which encodes the protein-tyrosine phosphatase Shp2, occur in approximately 50% of individuals with Noonan syndrome, but their molecular, cellular and developmental effects, and the relationship between Noonan syndrome and leukemia, are unclear. We generated mice expressing the Noonan syndrome-associated mutant D61G. When homozygous, the D61G mutant is embryonic lethal, whereas heterozygotes have decreased viability. Surviving Ptpn11(D61G/+) embryos ( approximately 50%) have short stature, craniofacial abnormalities similar to those in Noonan syndrome, and myeloproliferative disease. Severely affected Ptpn11(D61G/+) embryos ( approximately 50%) have multiple cardiac defects similar to those in mice lacking the Ras-GAP protein neurofibromin. Their endocardial cushions have increased Erk activation, but Erk hyperactivation is cell and pathway specific. Our results clarify the relationship between Noonan syndrome and leukemia and show that a single Ptpn11 gain-of-function mutation evokes all major features of Noonan syndrome by acting on multiple developmental lineages in a gene dosage-dependent and pathway-selective manner.