Endothelial deletion of SHP2 suppresses tumor angiogenesis and promotes vascular normalization.

SHP2 mediates the activities of multiple receptor tyrosine kinase signaling and its function in endothelial processes has been explored extensively. However, genetic studies on the role of SHP2 in tumor angiogenesis have not been conducted. Here, we show that SHP2 is activated in tumor endothelia. Shp2 deletion and pharmacological inhibition reduce tumor growth and microvascular density in multiple mouse tumor models. Shp2 deletion also leads to tumor vascular normalization, indicated by increased pericyte coverage and vessel perfusion. SHP2 inefficiency impairs endothelial cell proliferation, migration, and tubulogenesis through downregulating the expression of proangiogenic SRY-Box transcription factor 7 (SOX7), whose re-expression restores endothelial function in SHP2-knockdown cells and tumor growth, angiogenesis, and vascular abnormalization in Shp2-deleted mice. SHP2 stabilizes apoptosis signal-regulating kinase 1 (ASK1), which regulates SOX7 expression mediated by c-Jun. Our studies suggest SHP2 in tumor associated endothelial cells is a promising anti-angiogenic target for cancer therapy.

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.

SHP2 knockdown ameliorates liver insulin resistance by activating IRS-2 phosphorylation through the AKT and ERK1/2 signaling pathways.

Diabetes is a chronic metabolic disease characterized by insulin resistance (IR). SHP2 has previously been identified as a potential target to reduce IR in diabetes. Here, we examined the effects of SHP2 on glucose consumption (GC), IR level and the expression of insulin receptor substrate (IRS), AKT and extracellular signal-regulated kinase (ERK)1/2 proteins in a cellular and animal model of diabetes. IR was induced in hepatocellular carcinoma (HCC) cells, and SHP2 was up-regulated or down-regulated in cells. Diabetic rats were treated with SHP2 inhibitor. GC of cells, and the weight, total cholesterol, triglycerides, fasting blood glucose, fasting insulin, homeostasis model assessment-IR index and insulin sensitivity (ISI) of the rats were analyzed. The levels of SHP2 and the activation of IRS-2, AKT and ERK1/2 in cells and rats were measured by quantitative real-time PCR (qRT-PCR) or western blot. GC was reduced, but expression of SHP2 was enhanced in IR HCC cells. Phosphorylation of IRS-2 and AKT in IR HCC cells and diabetic rats was decreased, whereas phosphorylation of ERK1/2 was enhanced. In both the cell and animal models, SHP2 knockdown enhanced GC, ameliorated IR, activated IRS-2 and AKT, and inhibited ERK1/2 phosphorylation, in contrast with the effects of SHP2 overexpression. SHP2 knockdown may enhance GC and ameliorate IR through phosphorylation of IRS-2 via regulating AKT and ERK1/2 in liver.

SHP2 deficiency promotes Staphylococcus aureus pneumonia following influenza infection.

OBJECTIVES: Secondary bacterial pneumonia is common following influenza infection. However, it remains unclear about the underlying molecular mechanisms. MATERIALS AND METHODS: We established a mouse model of post-influenza S aureus pneumonia using conditional Shp2 knockout mice (LysMCre/+ :Shp2flox/flox ). The survival, bacterial clearance, pulmonary histology, phenotype of macrophages, and expression of type I interferons and chemokines were assessed between SHP2 deletion and control mice (Shp2flox/flox ). We infused additional KC and MIP-2 to examine the reconstitution of antibacterial immune response in LysMCre/+ :Shp2flox/flox mice. The effect of SHP2 on signal molecules including MAPKs (JNK, p38 and Erk1/2), NF-κB p65 and IRF3 was further detected. RESULTS: LysMCre/+ :Shp2flox/flox mice displayed impaired antibacterial immunity and high mortality compared with control mice in post-influenza S aureus pneumonia. The attenuated antibacterial ability was associated with the induction of type I interferon and suppression of chemo-attractants KC and MIP-2, which reduced the infiltration of neutrophils into the lung upon secondary bacterial invasion. In additional, Shp2 knockout mice displayed enhanced polarization to alternatively activated macrophages (M2 phenotype). Further in vitro analyses consistently demonstrated that SHP2-deficient macrophages were skewed towards an M2 phenotype and had a decreased antibacterial capacity. Moreover, SHP2 modulated the inflammatory response to secondary bacterial infection via interfering with NF-κB and IRF3 signalling in macrophages. CONCLUSIONS: Our findings reveal that the SHP2 expression enhances the host immune response and prompts bacterial clearance in post-influenza S aureus pneumonia.

Shp2 in myocytes is essential for cardiovascular and neointima development.

Mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase Shp2, cause Noonan syndrome and LEOPARD syndrome, inherited multifaceted diseases including cardiac and vascular defects. However, the function of Shp2 in blood vessels, especially in vascular smooth muscle cells (VSMCs), remains largely unknown. We generated mice in which Shp2 was specifically deleted in VSMCs and embryonic cardiomyocytes using the SM22α-Cre transgenic mouse line. Conditional Shp2 knockout resulted in massive hemorrhage, cardiovascular defects and embryonic lethality at the late embryonic developmental stage (embryonic date 16.5). The thinning of artery walls in Shp2-knockout embryos was due to decreased VSMC number and reduced extracellular matrix deposition. Myocyte proliferation was decreased in Shp2-knockout arteries and hearts. Importantly, cardiomyocyte-specific Shp2-knockout did not cause similar vascular defects. Shp2 was required for TGFß1-induced expression of ECM components, including collagens in VSMCs. In addition, collagens were sufficient to promote Shp2-inefficient VSMC proliferation. Finally, Shp2 was deleted in adult mouse VSMCs by using SMMHC-CreERT2 and tamoxifen induction. Shp2 deletion dramatically inhibited the expression of ECM components, proliferation of VSMCs and neointima formation in a carotid artery ligation model. Therefore, Shp2 is required for myocyte proliferation in cardiovascular development and vascular remodeling through TGFß1-regulated collagen synthesis.

Platelet Shp2 negatively regulates thrombus stability under high shear stress.

Essentials Shp2 negatively regulates thrombus stability under pathological shear rate. Shp2 suppresses TXA2 receptor-mediated platelet dense granule secretion. Through αIIbß3 outside-in signaling, Shp2 targets calmodulin-dependent activation of Akt. Shp2 may serve to prevent the formation of unwanted occlusive thrombi. SUMMARY: Background Perpetuation is the final phase of thrombus formation; however, its mechanisms and regulation are poorly understood. Objective To investigate the mechanism of Shp2 in platelet function and thrombosis. Methods and results We demonstrate that the platelet-expressed Src homology region 2 domain-containing protein tyrosine phosphatase Shp2 is a negative regulator of thrombus stability under high shear stress. In a ferric chloride-induced mesenteric arteriole thrombosis model, megakaryocyte/platelet-specific Shp2-deficient mice showed less thrombi shedding than wild-type mice, although their occlusion times were comparable. In accordance with this in vivo phenotype, a microfluidic whole-blood perfusion assay revealed that the thrombi formed on collagen surfaces by Shp2-deficient platelets were more stable under high shear rates than those produced by wild-type platelets. Whereas Shp2 deficiency did not alter platelet responsiveness towards thrombin, ADP and collagen stimulation, Shp2-deficient platelets showed increased dense granule secretion when stimulated by the thromboxane A2 analog U46619. Shp2 appears to act downstream of integrin αIIb ß3 outside-in signaling, inhibiting the phosphorylation of Akt (Ser473 and Thr308) and dense granule secretion. Calmodulin was also shown to bind both Shp2 and Akt, linking Shp2 to Akt activation. Conclusions Platelet Shp2 negatively regulates thrombus perpetuation under high shear stress. This signaling pathway may constitute an important mechanism for the prevention of unwanted occlusive thrombus formation, without dramatically interfering with hemostasis.

Mutant KRAS-driven cancers depend on PTPN11/SHP2 phosphatase.

The ubiquitously expressed non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, is involved in signal transduction downstream of multiple growth factor, cytokine and integrin receptors1. Its requirement for complete RAS-MAPK activation and its role as a negative regulator of JAK-STAT signaling have established SHP2 as an essential player in oncogenic signaling pathways1-7. Recently, a novel potent allosteric SHP2 inhibitor was presented as a viable therapeutic option for receptor tyrosine kinase-driven cancers, but was shown to be ineffective in KRAS-mutant tumor cell lines in vitro8. Here, we report a central and indispensable role for SHP2 in oncogenic KRAS-driven tumors. Genetic deletion of Ptpn11 profoundly inhibited tumor development in mutant KRAS-driven murine models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. We provide evidence for a critical dependence of mutant KRAS on SHP2 during carcinogenesis. Deletion or inhibition of SHP2 in established tumors delayed tumor progression but was not sufficient to achieve tumor regression. However, SHP2 was necessary for resistance mechanisms upon blockade of MEK. Synergy was observed when both SHP2 and MEK were targeted, resulting in sustained tumor growth control in murine and human patient-derived organoids and xenograft models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. Our data indicate the clinical utility of dual SHP2/MEK inhibition as a targeted therapy approach for KRAS-mutant cancers.

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.

Loss of Shp2 within radial glia is associated with cerebral cortical dysplasia, glial defects of cerebellum and impaired sensory­motor development in newborn mice.

Radial glia are key neural progenitors involved in the development of the central nervous system. Tyrosine-protein phosphatase non­receptor type 11 (Shp2) is a widely expressed intracellular enzyme with multiple cellular functions. Previous studies have revealed the critical role of Shp2 in a variety of neural cell types; however, further investigation into the function of Shp2 within radial glia is required. In the present study, a conditional knockout mouse was generated using a human glial fibrillary acidic protein (hGFAP)­Cre driver, in which the Shp2 genes were deleted within radial glia. Loss of Shp2 within radial glia was associated with developmental retardation, postnatal lethality, reduced brain size and thinner cerebral cortices in newborn mice. Deletion of Shp2 also led to an increase in gliogenesis, a reduction in neural genesis and extracellular signal­regulated kinase signaling within the cerebral cortex. Furthermore, glial cell defects within the cerebellum of Shp2 mutants were observed, with abnormal granular cell retention and glial cell alignment in the external granular layer. In addition, Shp2 mutants exhibited impaired sensory­motor development. The results of the present study suggested that Shp2 may have an important role within radial glia, and regulate cerebral cortical and cerebellar development in newborn mice.

T lymphocyte SHP2-deficiency triggers anti-tumor immunity to inhibit colitis-associated cancer in mice.

Nonresolving inflammation is involved in the initiation and progression process of tumorigenesis. Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) is known to inhibit acute inflammation but its role in chronic inflammation-associated cancer remains unclear. The role of SHP2 in T cells in dextran sulfate sodium (DSS)-induced colitis and azoxymethane-DSS-induced colitis-associated carcinogenesis was examined using SHP2CD4-/- conditional knockout mice. SHP2 deficiency in T cells aggravated colitis with increased level of pro-inflammatory cytokines including IFN-γ and IL-17A. In contrast, the SHP2CD4-/- mice developed much fewer and smaller tumors than wild type mice with higher level of IFN-γ and enhanced cytotoxicity of CD8+ T cells in the tumor and peritumoral areas. At the molecular level, STAT1 was hyper-phosphorylated in T cells lacking SHP2, which may account for the increased Th1 differentiation and IFN-γ secretion. IFN-γ neutralization or IFN-γ receptor knockout but not IL-17A neutralization, abrogated the anti-tumor effect of SHP2 knockout with lowered levels of perforin 1, FasL and granzyme B. Finally, the expression of granzyme B was negatively correlated with the malignancy of colon cancer in human patients. In conclusion, these findings suggest a new strategy to treat colitis-associated cancer via targeting SHP2.

SHP2-Deficiency in Chondrocytes Deforms Orofacial Cartilage and Ciliogenesis in Mice.

Congenital orofacial abnormalities are clinically seen in human syndromes with SHP2 germline mutations such as LEOPARD and Noonan syndrome. Recent studies demonstrate that SHP2-deficiency leads to skeletal abnormalities including scoliosis and cartilaginous benign tumor metachondromatosis, suggesting that growth plate cartilage is a key tissue regulated by SHP2. The role and cellular mechanism of SHP2 in the orofacial cartilage, however, remains unknown. Here, we investigated the postnatal craniofacial development by inducible disruption of Shp2 in chondrocytes. Shp2 conditional knockout (cKO) mice displayed severe deformity of the mandibular condyle accompanied by disorganized, expanded cartilage in the trabecular bone region, enhanced type X collagen, and reduced Erk production. Interestingly, the length of primary cilia, an antenna like organelle sensing environmental signaling, was significantly shortened, and the number of primary cilia was reduced in the cKO mice. The expression levels of intraflagellar transports (IFTs), essential molecules in the assembly and function of primary cilia, were significantly decreased. Taken together, lack of Shp2 in orofacial cartilage led to severe defects of ciliogenesis through IFT reduction, resulting in mandibular condyle malformation and cartilaginous expansion. Our study provides new insights into the molecular pathogenesis of SHP2-deficiency in cartilage and helps to understand orofacial and skeletal manifestations seen in patients with SHP2 mutations.

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.

Shp2 plays an important role in acute cigarette smoke-mediated lung inflammation.

Cigarette smoke (CS), the major cause of chronic obstructive pulmonary disease, contains a variety of oxidative components that were implicated in the regulation of Src homology domain 2-containing protein tyrosine phosphatase 2 (Shp2) activity. However, the contribution of Shp2 enzyme to chronic obstructive pulmonary disease pathogenesis remains unclear. We investigated the role of Shp2 enzyme in blockading CS-induced pulmonary inflammation. Shp2 levels were assessed in vivo and in vitro. Mice (C57BL/6) or pulmonary epithelial cells (NCI-H292) were exposed to CS or cigarette smoke extract (CSE) to induce acute injury and inflammation. Lungs of smoking mice showed increased levels of Shp2, compared with those of controls. Treatment of lung epithelial cells with CSE showed elevated levels of Shp2 associated with the increased release of IL-8. Selective inhibition or knockdown of Shp2 resulted in decreased IL-8 release in response to CSE treatment in pulmonary epithelial cells. In comparison with CS-exposed wild-type mice, selective inhibition or conditional knockout of Shp2 in lung epithelia reduced IL-8 release and pulmonary inflammation in CS-exposed mice. In vitro biochemical data correlate CSE-mediated IL-8 release with Shp2-regulated epidermal growth factor receptor/Grb-2-associated binders/MAPK signaling. Our data suggest an important role for Shp2 in the pathological alteration associated with CS-mediated inflammation. Shp2 may be a potential target for therapeutic intervention for inflammation in CS-induced pulmonary diseases.

SH2 domain-containing phosphatase 2 is a critical regulator of connective tissue mast cell survival and homeostasis in mice.

Mast cells require KIT receptor tyrosine kinase signaling for development and survival. Here, we report that SH2 domain-containing phosphatase 2 (SHP2) signaling downstream of KIT is essential for mast cell survival and homeostasis in mice. Using a novel mouse model with shp2 deletion within mature mast cells (MC-shp2 knockout [KO]), we find that SHP2 is required for the homeostasis of connective tissue mast cells. Consistently with the loss of skin mast cells, MC-shp2 KO mice fail to mount a passive late-phase cutaneous anaphylaxis response. To better define the phenotype of shp2-deficient mast cells, we used an inducible shp2 knockout approach in bone marrow-derived mast cells (BMMCs) or cultured peritoneal mast cells and found that SHP2 promotes mast cell survival. We show that SHP2 promotes KIT signaling to extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase and downregulation of the proapoptotic protein Bim in BMMCs. Also, SHP2-deficient BMMCs failed to repopulate mast cells in mast cell-deficient mice. Silencing of Bim partially rescued survival defects in shp2-deficient BMMCs, consistent with the importance of a KIT → SHP2 → Ras/ERK pathway in suppressing Bim and promoting mast cell survival. Thus, SHP2 is a key node in a mast cell survival pathway and a new potential therapeutic target in diseases involving mast cells.

Tyrosine phosphatase SHP2 regulates the expression of acyl-CoA synthetase ACSL4.

Acyl-CoA synthetase 4 (ACSL4) is implicated in fatty acid metabolism with marked preference for arachidonic acid (AA). ACSL4 plays crucial roles in physiological functions such as steroid synthesis and in pathological processes such as tumorigenesis. However, factors regulating ACSL4 mRNA and/or protein levels are not fully described. Because ACSL4 protein expression requires tyrosine phosphatase activity, in this study we aimed to identify the tyrosine phosphatase involved in ACSL4 expression. NSC87877, a specific inhibitor of the tyrosine phosphatase SHP2, reduced ACSL4 protein levels in ACSL4-rich breast cancer cells and steroidogenic cells. Indeed, overexpression of an active form of SHP2 increased ACSL4 protein levels in MA-10 Leydig steroidogenic cells. SHP2 has to be activated through a cAMP-dependent pathway to exert its effect on ACSL4. The effects could be specifically attributed to SHP2 because knockdown of the phosphatase reduced ACSL4 mRNA and protein levels. Through the action on ACSL4 protein levels, SHP2 affected AA-CoA production and metabolism and, finally, the steroidogenic capacity of MA-10 cells: overexpression (or knockdown) of SHP2 led to increased (or decreased) steroid production. We describe for the first time the involvement of SHP2 activity in the regulation of the expression of the fatty acid-metabolizing enzyme ACSL4.

Ptpn11/Shp2 acts as a tumor suppressor in hepatocellular carcinogenesis.

The human gene Ptpn11, which encodes the tyrosine phosphatase Shp2, may act as a proto-oncogene because dominantly activating mutations have been detected in several types of leukemia. Herein we report a tumor-suppressor function of Shp2. Hepatocyte-specific deletion of Shp2 promotes inflammatory signaling through the Stat3 pathway and hepatic inflammation/necrosis, resulting in regenerative hyperplasia and development of tumors in aged mice. Furthermore, Shp2 ablation dramatically enhanced diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) development, which was abolished by concurrent deletion of Shp2 and Stat3 in hepatocytes. Decreased Shp2 expression was detected in a subfraction of human HCC specimens. Thus, in contrast to the leukemogenic effect of dominant-active mutants, Ptpn11/Shp2 has a tumor-suppressor function in liver.

Kit-Shp2-Kit signaling acts to maintain a functional hematopoietic stem and progenitor cell pool.

The stem cell factor (SCF)/Kit system has served as a classic model in deciphering molecular signaling events in the hematopoietic compartment, and Kit expression is a most critical marker for hematopoietic stem cells (HSCs) and progenitors. However, it remains to be elucidated how Kit expression is regulated in HSCs. Herein we report that a cytoplasmic tyrosine phosphatase Shp2, acting downstream of Kit and other RTKs, promotes Kit gene expression, constituting a Kit-Shp2-Kit signaling axis. Inducible ablation of PTPN11/Shp2 resulted in severe cytopenia in BM, spleen, and peripheral blood in mice. Shp2 removal suppressed the functional pool of HSCs/progenitors, and Shp2-deficient HSCs failed to reconstitute lethally irradiated recipients because of defects in homing, self-renewal, and survival. We show that Shp2 regulates coordinately multiple signals involving up-regulation of Kit expression via Gata2. Therefore, this study reveals a critical role of Shp2 in maintenance of a functional HSC/progenitor pool in adult mammals, at least in part through a kinase-phosphatase-kinase cascade.

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

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.

Shp2 suppresses PyMT-induced transformation in mouse fibroblasts by inhibiting Stat3 activity.

We have examined the effect of expression of the protein tyrosine phosphatase Shp2 on transformation by the mouse polyoma virus middle T antigen (PyMT). Gain-of-function mutations in Shp2 indicate that it may serve as an oncogene in several types of human leukemia. Paradoxically, however, some catalytically dominant-negative mutations of Shp2 have also been identified in leukemia and neuroblastomas. In this study, we show that Shp2 suppresses transformation induced by PyMT, the major polyoma viral oncoprotein known to act through binding and activation of pp60(c-src). Over-expression of a catalytically inactive Shp2 mutant in NIH3T3 cells significantly enhanced PyMT-induced transformation. Conversely, re-introduction of Shp2 into Shp2-deficient cells strongly inhibited PyMT-induced transformation and tumorigenesis. Short hairpin RNA (shRNA)-mediated Shp2 knockdown potentiated PyMT-induced transformation. Finally, we present evidence that the transformation-suppressive effects of Shp2 are mediated at least partially through the inhibition of signal transducers and activators of transcription 3.