Shp2 gain-of-function mutants negatively regulate Stat3 activation in murine bone marrow cells and peripheral blood nucleated cells from individuals with NS. A, panel a, Western blot analyses examining GM-CSF-stimulated (20 ng/ml) Stat3 activation in the mouse bone marrow-derived macrophage progenitors transduced with MIEG3 (empty vector), WT Shp2, or Shp2E76K. Panel b, density quantification of protein band in A, panel a; B, panel a, representative flow cytometric analysis showing reduced Stat3 tyrosine phosphorylation in peripheral blood cells from individuals with NS in both the quiescent state and after IL-6 stimulation. Panel b, statistical analysis of six NS patients with confirmed PTPN11 mutations and six sex- and age-matched normal controls (p < 0.04, statistics performed using unpaired Student's t test). Panel c, Western blot analyses examining basal and IL-6- (50 ng/ml) stimulated STAT3 phosphorylation and total STAT3 protein level in nucleated peripheral blood cells from control and individuals with NS. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Error bars represent S.D. Asterisks indicate the difference is statistically significant.

Conditional deletion of Stat3 with nestin-cre induces pulmonary stenosis due to the thickening of leaflets. A, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside(X-gal) staining of nestin-cre/Rosa26R mouse embryos and newborns indicates the cre activity in the developing mouse embryos. A, panels a and b, central nervous system is positive for cre activity in the mouse embryos at E10.5. Panels c and d, cre activity was found in the pulmonary valves (PV) and the aortic valves (AV) at E14.5 (A, panel c) and P0 (B, panel d). B, ablation of Stat3 with nestin-cre leads to pulmonary stenosis caused by the thickening and enlargement of leaflets. B, panel a, newborn of Stat3^f/f (f/f) and Stat3^f/f/Nestin-cre (c;f/f) mice. B, panel b, hematoxylin and eosin staining of the transverse section of heart from control (f/f) and STAT3^f/f/Nestin-cre (c;f/f) newborn mice (P0). Note that Stat3 ablation leads to right ventricle dilation in Stat3^f/f/Nestin-cre mice. B, panels d–g, hematoxylin and eosin staining of sections of the pulmonary valve and aorta valve regions in control (B, panels d and f) and Stat3^f/f/Nestin-cre mutant (B, panels e and g) newborn mice. LV, left ventricle; RV, right ventricle. B, panel h, quantification of pulmonary valve thickness in Stat3^f/f/Nestin-cre and control hearts (P0) (n = 6, p < 0.03, Student's paired t test; 2 tails).

Stat3 overexpression reduces the sensitivity to GM-CSF stimulation in Shp2E76K-transduced cells. A, bone marrow low density mononuclear cells were isolated and co-transduced with WT Shp2 or Shp2E76K pMIEG3-EGFP in combination with pMIEG3-hCD4 or pMIEG3-Stat3CA-hCD4. Following transduction, cells were sorted for EGFP or human CD4 antigen expression using FACS to enrich for transduced double positive cells. B, Western blotting showing Shp2 and Stat3 overexpression in virus-transduced and FACS-enriched EGFP and CD4 expressing cells. C, methylcellulose-based colony forming assay demonstrating that Stat3CA expression corrects the gain-of-function Shp2-induced hematopoietic progenitor hypersensitivity to GM-CSF. Shown are representative data from two independent experiments that demonstrate similar results. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Error bars represent S.D.

Ablation of Stat3 in bone marrow cells gives rise to skewed myeloid cell differentiation and hypersensitivity to GM-CSF. A, morphological, histological, and flow cytometric analyses of spleens from Stat3^f/f/btie2-cre and Stat3^f/f littermate control mice. Panel a, splenomagaly was found in Stat3^f/f/btie2-cre mice; panel b, comparison of spleen weight to body weight ratio of Stat3^f/f and Stat3^f/f/btie2-cre mice, p < 0.01 (Student's t test, 2 tails); panels c and d, disrupted spleen architecture in Stat3^f/f/btie2-cre mice (d) when compared with Stat3^f/f controls (c); panel e, both F480⁺Mac1⁺ and GR-1⁺Mac1⁺ populations were significantly increased in Stat3^f/f/btie2-cre spleens compared with the Stat3^f/f control spleens. B, bone marrow F480⁺Mac1⁺ and GR-1⁺Mac1⁺ populations were increased in the Stat3^f/f/btie2-cre mice compared with Stat3^f/f control mice. C, methylcellulose-based colony forming assays demonstrated increased bone marrow progenitor-derived colonies in response to increasing doses of GM-CSF from the Stat3^f/f/btie2-cre mice compared with the Stat3^f/f mice. D, increased proliferative activity of Stat3^f/f/btie2-cre bone marrow cells in response to various cytokines. The final concentration of all cytokines, except IL-3 (100u/ml), was 30 ng/ml. E, decreased apoptosis in Stat3^f/f/btie2-cre bone marrow cells when cultured in a low serum in the presence of 0.1 or 1 ng/ml of GM-CSF. Error bars represent S.D.

Stat3 activation in valvulogenesis, and Shp2 gain-of-function mutants inhibit the activation of Stat3 in response to EGF. A, immunohistochemistry staining of activated Stat3 (pY Stat3) in developing and mature PV of wild type mice. Nuclear brown signals are positive staining. Activated Stat3 is apparent in E14.5 embryonic heart and is restricted to the developing leaflets (A, panels a to c), and remains into adult stage (panel d). Activated Stat3 is present in the PV of control embryo hearts at E17.5 (panel e), absent in PV from Stat3^f/f/nestin-cre embryo heart at the same stage (panel f). B, panel a, after serum deprivation overnight, Western blot analysis was used to examine basal and EGF-stimulated (50 ng/ml) Stat3 tyrosine phosphorylation in NIH3T3 cells expressing Shp2 mutants (N308D and E76K) or controls (MIEG3 or WT Shp2). Panel b, density quantification of the protein band in B, panel a. C, EMSA to examine Stat3 DNA binding activity using the m67-SIE probe and nuclear extracts from NIH3T3 cells transduced with MIEG3 (M), WT Shp2 (WT), Shp2N308D (N308D), or Shp2E76K (E76K). Error bars represent S.D.

Shp2 dephosphorylates Tyr(P)-Stat3 and Shp2-Stat3-mediated signaling critically contributes to the pathogenesis of NS. A, Western blot analysis of Stat3-deficient bone marrow-derived macrophage progenitors treated with GM-CSF to assess Stat3 and ERK activation. B, panel a, purified constitutively active Shp2 protein (catalytic domain) was capable of dephosphorylating Tyr(P)-Stat3. Stat3 protein was immunoprecipitated from IL-6-treated Raw 264.7 cell extract, and then incubated with purified recombinant Shp2 protein (active form, 2 μg) for the indicated time with or without preincubation of the small molecule Shp2 phosphatase inhibitor IIB-08 (100 μm). Panel b, Shp2 inhibitor IIB-08 inhibits ERK activation, whereas enhancing the Tyr(P)-Stat3 level, in IL-6 induced Raw 264.7 cells. Raw 264.7 cells were pretreated with the Shp2 inhibitor IIB-08 (10 μm), or dimethyl sulfoxide (DMSO) for 60 min, and then incubated with IL-6 (20 ng/ml) for 60 min before being subjected to lysis and Western blot analysis.

Schematic diagram of dual-signaling pathways regulated by Shp2. In normal physiological conditions, Stat3 activity is under the positive and negative regulation of Janus kinases and Shp2, respectively (A). Shp2 gain-of-function mutations lead to the hyperactivation of the RAS/ERK signaling pathway and the excessive inactivation of Stat3, which synergistically promotes the pathogenesis of Noonan syndrome and/or JMML (B).