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.

Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells.

The phosphatase Shp-2 was implicated in NK cell development and functions due to its interaction with NK inhibitory receptors, but its exact role in NK cells is still unclear. Here we show, using mice conditionally deficient for Shp-2 in the NK lineage, that NK cell development and responsiveness are largely unaffected. Instead, we find that Shp-2 serves mainly to enforce NK cell responses to activation by IL-15 and IL-2. Shp-2-deficient NK cells have reduced proliferation and survival when treated with high dose IL-15 or IL-2. Mechanistically, Shp-2 deficiency hampers acute IL-15 stimulation-induced raise in glycolytic and respiration rates, and causes a dramatic defect in ERK activation. Moreover, inhibition of the ERK and mTOR cascades largely phenocopies the defect observed in the absence of Shp-2. Together, our data reveal a critical function of Shp-2 as a molecular nexus bridging acute IL-15 signaling with downstream metabolic burst and NK cell expansion.

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.

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.

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.

Macrophage-Restricted Shp2 Tyrosine Phosphatase Acts as a Rheostat for MMP12 through TGF-ß Activation in the Prevention of Age-Related Emphysema in Mice.

Persistent activation of macrophages in lungs plays a critical role in the production of matrix metalloproteinases (MMPs) that contributes to the destruction of alveolar walls, a hallmark for pulmonary emphysema. Dysregulated TGF-ß1 signaling has been an essential determinant in the elevation of MMPs during the development of emphysema. Nevertheless, the mechanism for this MMP-dependent pathogenesis has yet to be clearly investigated. Recently, we identified an important role for tyrosine phosphatase Src homology domain-containing protein tyrosine phosphatase 2 (Shp2) in regulating the activation of alveolar macrophages. Over a long-term observation period, mice with Shp2 deletion in macrophages (LysMCre:Shp2fl/fl ) develop spontaneous, progressive emphysema-like injury in the lungs, characterized by massive destruction of alveolar morphology, interstitial extracellular matrix degradation, and elevated levels of MMPs, particularly, significant increases of macrophage elastase (MMP12) in aged mice. Further analysis demonstrated that MMP12 suppression by TGF-ß1 activation was apparently abrogated in LysMCre:Shp2fl/fl mice, whereas the TGF-ß1 concentration in the lungs was relatively the same. Mechanistically, we found that loss of Shp2 resulted in attenuated SMAD2/3 phosphorylation and nuclear translocation in response to TGF-ß activation, thereby upregulating MMP12 expression in macrophages. Together, our findings define a novel physiological function of Shp2 in TGF-ß1/MMP12-dependent emphysema, adding insights into potential etiologies for this chronic lung disorder.

Protein tyrosine phosphatase Shp2 deficiency in podocytes attenuates lipopolysaccharide-induced proteinuria.

Podocytes are specialized epithelial cells that play a significant role in maintaining the integrity of the glomerular filtration barrier and preventing urinary protein leakage. We investigated the contribution of protein tyrosine phosphatase Shp2 to lipopolysaccharide (LPS)-induced renal injury. We report increased Shp2 expression in murine kidneys and cultured podocytes following an LPS challenge. To determine the role of podocyte Shp2 in vivo, we generated podocyte-specific Shp2 knockout (pod-Shp2 KO) mice. Following administration of LPS, pod-Shp2 KO mice exhibited lower proteinuria and blood urea nitrogen concentrations than controls indicative of preserved filter integrity. In addition, renal mRNA and serum concentrations of inflammatory cytokines IL-1ß, TNFα, INFγ and IL-12 p70 were significantly decreased in LPS-treated knockout mice compared with controls. Moreover, the protective effects of podocyte Shp2 deficiency were associated with decreased LPS-induced NF-κB and MAPK activation, nephrin phosphorylation and attenuated endoplasmic reticulum stress. These effects were recapitulated in differentiated E11 murine podocytes with lentiviral-mediated Shp2 knockdown. Furthermore, Shp2 deficient podocytes displayed reduced LPS-induced migration in a wound healing assay. These findings identify Shp2 in podocytes as a significant contributor to the signaling events following LPS challenge and suggest that inhibition of Shp2 in podocytes may present a potential therapeutic target for podocytopathies.

Increased Src Family Kinase Activity Disrupts Excitatory Synaptic Transmission and Impairs Remote Fear Memory in Forebrain Shp2-Deficient Mice.

Src homolog domain-containing phosphatase 2 (Shp2) signals a variety of cellular and physiological functions including learning and memory. Dysregulation of ERK signaling is known to be responsible for the cognitive deficits associated with gain-of-function mutated Shp2 mimicking Noonan syndrome. However, here, we report that CaMKIIα-cre induced knockout (CaSKO) of Shp2 in hippocampal pyramidal neurons resulted in increased Src activity, upregulated phosphorylation of N-methyl-D-aspartate receptors (NMDARs) at Y1325 of GluN2A and at Y1472 of GluN2B, disrupted the balance of synaptic transmission, and impaired long-term potentiation and remote contextual fear memory. Administration of PP2, a specific Src family kinase inhibitor, reversed the tyrosine phosphorylation of NMDARs, restored basal synaptic transmission, and rescued the contextual fear memory deficit in CaSKO mice without altering the phospho-ERK level. Taken together, our results reveal a novel role of Shp2 in NMDAR-dependent synaptic function and fear memory via the Src signaling pathway rather than the ERK pathway, and suggest a complicated mechanism for Shp2-associated cognitive deficits.

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.

Manipulating the air-filled zebrafish swim bladder as a neutrophilic inflammation model for acute lung injury.

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are life-threatening diseases that are associated with high mortality rates due to treatment limitations. Neutrophils play key roles in the pathogenesis of ALI/ARDS by promoting the inflammation and injury of the alveolar microenvironment. To date, in vivo functional approaches have been limited by the inaccessibility to the alveolar sacs, which are located at the anatomical terminal of the respiratory duct in mammals. We are the first to characterize the swim bladder of the zebrafish larva, which is similar to the mammalian lung, as a real-time in vivo model for examining pulmonary neutrophil infiltration during ALI. We observed that the delivery of exogenous materials, including lipopolysaccharide (LPS), Poly IC and silica nanoparticles, by microinjection triggered significant time- and dose-dependent neutrophil recruitment into the swim bladder. Neutrophils infiltrated the LPS-injected swim bladder through the blood capillaries around the pneumatic duct or a site near the pronephric duct. An increase in the post-LPS inflammatory cytokine mRNA levels coincided with the in vivo neutrophil aggregation in the swim bladder. Microscopic examinations of the LPS-injected swim bladders further revealed in situ injuries, including epithelial distortion, endoplasmic reticulum swelling and mitochondrial injuries. Inhibitor screening assays with this model showed a reduction in neutrophil migration into the LPS-injected swim bladder in response to Shp2 inhibition. Moreover, the pharmacological suppression and targeted disruption of Shp2 in myeloid cells alleviated pulmonary inflammation in the LPS-induced ALI mouse model. Additionally, we used this model to assess pneumonia-induced neutrophil recruitment by microinjecting bronchoalveolar lavage fluid from patients into swim bladders; this injection enhanced neutrophil aggregation relative to the control. In conclusion, our findings highlight the swim bladder as a promising and powerful model for mechanistic and drug screening studies of alveolar injuries.

SHP-2 Phosphatase Prevents Colonic Inflammation by Controlling Secretory Cell Differentiation and Maintaining Host-Microbiota Homeostasis.

Polymorphisms in the PTPN11 gene encoding for the tyrosine phosphatase SHP-2 were described in patients with ulcerative colitis. We have recently demonstrated that mice with an intestinal epithelial cell-specific deletion of SHP-2 (SHP-2(IEC-KO) ) develop severe colitis 1 month after birth. However, the mechanisms by which SHP-2 deletion induces colonic inflammation remain to be elucidated. We generated SHP-2(IEC-KO) mice lacking Myd88 exclusively in the intestinal epithelium. The colonic phenotype was histologically analyzed and cell differentiation was determined by electron microscopy and lysozyme or Alcian blue staining. Microbiota composition was analyzed by 16S sequencing. Results show that innate defense genes including those specific to Paneth cells were strongly up-regulated in SHP-2-deficient colons. Expansion of intermediate cells (common progenitors of the Goblet and Paneth cell lineages) was found in the colon of SHP-2(IEC-KO) mice whereas Goblet cell number was clearly diminished. These alterations in Goblet/intermediate cell ratio were noticed 2 weeks after birth, before the onset of inflammation and were associated with significant alterations in microbiota composition. Indeed, an increase in Enterobacteriaceae and a decrease in Firmicutes were observed in the colon of these mice, indicating that dysbiosis also occurred prior to inflammation. Importantly, loss of epithelial Myd88 expression inhibited colitis development in SHP-2(IEC-KO) mice, rescued Goblet/intermediate cell ratio, and prevented NFκB hyperactivation and inflammation. These data indicate that SHP-2 is functionally important for the maintenance of appropriate barrier function and host-microbiota homeostasis in the large intestine. J. Cell. Physiol. 231: 2529-2540, 2016. © 2016 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.

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.

LEOPARD syndrome without hearing loss or pulmonary stenosis: a report of 2 cases.

LEOPARD syndrome is an autosomal dominant disease caused by germline mutations in the RAS-MAPK (mitogen-activated protein kinase) pathway. LEOPARD is an acronym for the main manifestations of the syndrome, namely, multiple Lentigines, Electrocardiographic conduction abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormalities of genitalia, Retardation of growth, and sensorineural Deafness. None of these characteristic features, however, are pathognomonic of LEOPARD syndrome, and since they are highly variable, they are often not present at the time of diagnosis. We describe 2 cases of LEOPARD syndrome without hearing loss or pulmonary stenosis in which diagnosis was confirmed by identification of a mutation in the PTPN11 gene. Regular monitoring is important for the early detection of complications, as these can occur at any time during the course of disease.

Shp2 signaling in POMC neurons is important for leptin's actions on blood pressure, energy balance, and glucose regulation.

Previous studies showed that Src homology-2 tyrosine phosphatase (Shp2) is an important regulator of body weight. In this study, we examined the impact of Shp2 deficiency specifically in proopiomelanocortin (POMC) neurons on metabolic and cardiovascular function and on chronic blood pressure (BP) and metabolic responses to leptin. Mice with Shp2 deleted in POMC neurons (Shp2/Pomc-cre) and control mice (Shp2(flox/flox)) were implanted with telemetry probes and venous catheters for measurement of mean arterial pressure (MAP) and leptin infusion. After at least 5 days of stable control measurements, mice received leptin infusion (2 µg·kg(-1)·day(-1) iv) for 7 days. Compared with Shp2(flox/flox) controls, Shp2/Pomc-cre mice at 22 wk of age were slightly heavier (34 ± 1 vs. 31 ± 1 g) but consumed a similar amount of food (3.9 ± 0.3 vs. 3.8 ± 0.2 g/day). Leptin infusion reduced food intake in Shp2(flox/flox) mice (2.6 ± 0.5 g) and Shp2/Pomc-cre mice (3.2 ± 0.3 g). Despite decreasing food intake, leptin infusion increased MAP in control mice, whereas no significant change in MAP was observed in Shp2/Pomc-cre mice. Leptin infusion also decreased plasma glucose and insulin levels in controls (12 ± 1 to 6 ± 1 µU/ml and 142 ± 12 to 81 ± 8 mg/100 ml) but not in Shp2/Pomc-cre mice. Leptin increased V̇o2 by 16 ± 2% in controls and 7 ± 1% in Shp2/Pomc-cre mice. These results indicate that Shp2 signaling in POMC neurons contributes to the long-term BP and antidiabetic actions of leptin and may play a modest role in normal regulation of body weight.

Cytoplasmic tyrosine phosphatase Shp2 coordinates hepatic regulation of bile acid and FGF15/19 signaling to repress bile acid synthesis.

Bile acid (BA) biosynthesis is tightly controlled by intrahepatic negative feedback signaling elicited by BA binding to farnesoid X receptor (FXR) and also by enterohepatic communication involving ileal BA reabsorption and FGF15/19 secretion. However, how these pathways are coordinated is poorly understood. We show here that nonreceptor tyrosine phosphatase Shp2 is a critical player that couples and regulates the intrahepatic and enterohepatic signals for repression of BA synthesis. Ablating Shp2 in hepatocytes suppressed signal relay from FGFR4, receptor for FGF15/19, and attenuated BA activation of FXR signaling, resulting in elevation of systemic BA levels and chronic hepatobiliary disorders in mice. Acting immediately downstream of FGFR4, Shp2 associates with FRS2α and promotes the receptor activation and signal relay to several pathways. These results elucidate a molecular mechanism for the control of BA homeostasis by Shp2 through the orchestration of multiple signals in hepatocytes.