Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia
2009; Elsevier BV; Volume: 284; Issue: 33 Linguagem: Inglês
10.1074/jbc.m109.020495
ISSN1083-351X
AutoresWenjun Zhang, Rebecca J. Chan, Hanying Chen, Zhenyun Yang, Yantao He, Xian Zhang, Yong Luo, Fuqing Yin, Akira Moh, Lucy Miller, R. Mark Payne, Zhong-Yin Zhang, Xin-Yuan Fu, Weinian Shou,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoNoonan syndrome (NS) is an autosomal dominant congenital disorder characterized by multiple birth defects including heart defects and myeloproliferative disease (MPD). Approximately 50% of NS patients have germline gain-of-function mutations in PTPN11, which encodes the protein-tyrosine phosphatase, Shp2. We provide evidence that conditional ablation of Stat3 in hematopoietic cells and cardiac valvular tissues leads to myeloid progenitor hyperplasia and pulmonary stenosis due to the leaflet thickening, respectively. Consistently, STAT3 activation is significantly compromised in peripheral blood cells from NS patients bearing Shp2-activating mutations. Biochemical and functional analyses demonstrate that activated Shp2 is able to down-regulate Tyr(P)-Stat3 and that constitutively active Stat3 rescues activating mutant Shp2-induced granulocyte-macrophage colony-stimulating factor hypersensitivity in bone marrow cells. Collectively, our work demonstrates that Stat3 is an essential signaling component potentially contributing to the pathogenesis of NS and juvenile myelomonocytic leukemia caused by PTPN11 gain-of-function mutations. Noonan syndrome (NS) is an autosomal dominant congenital disorder characterized by multiple birth defects including heart defects and myeloproliferative disease (MPD). Approximately 50% of NS patients have germline gain-of-function mutations in PTPN11, which encodes the protein-tyrosine phosphatase, Shp2. We provide evidence that conditional ablation of Stat3 in hematopoietic cells and cardiac valvular tissues leads to myeloid progenitor hyperplasia and pulmonary stenosis due to the leaflet thickening, respectively. Consistently, STAT3 activation is significantly compromised in peripheral blood cells from NS patients bearing Shp2-activating mutations. Biochemical and functional analyses demonstrate that activated Shp2 is able to down-regulate Tyr(P)-Stat3 and that constitutively active Stat3 rescues activating mutant Shp2-induced granulocyte-macrophage colony-stimulating factor hypersensitivity in bone marrow cells. Collectively, our work demonstrates that Stat3 is an essential signaling component potentially contributing to the pathogenesis of NS and juvenile myelomonocytic leukemia caused by PTPN11 gain-of-function mutations. Noonan syndrome (NS) 4The abbreviations used are: NSNoonan syndromeJMMLjuvenile myelomonocytic leukemiaMAPKmitogen-activated protein kinaseILinterleukinFACSfluorescence-activated cell sortingGM-CSFgranulocyte-macrophage colony stimulating factorEMSAelectrophoretic mobility shift assayEGFPenhanced green fluorescent proteinPVpulmonary valveStat3CAconstitutively active Stat3ERKextracellular signal-regulated kinaseSTATsignal transducers and activators of transcriptionPTPprotein-tyrosine phosphataseWTwild type. 4The abbreviations used are: NSNoonan syndromeJMMLjuvenile myelomonocytic leukemiaMAPKmitogen-activated protein kinaseILinterleukinFACSfluorescence-activated cell sortingGM-CSFgranulocyte-macrophage colony stimulating factorEMSAelectrophoretic mobility shift assayEGFPenhanced green fluorescent proteinPVpulmonary valveStat3CAconstitutively active Stat3ERKextracellular signal-regulated kinaseSTATsignal transducers and activators of transcriptionPTPprotein-tyrosine phosphataseWTwild type. is an autosomal dominant disorder characterized by unusual facial characteristics, short stature, heart defects, skeletal malformations, bleeding problems (1Tartaglia M. 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Signal transducer and activator of transcription 3 (Stat3) belongs to a family of transcription factors that regulates a broad range of biological processes. In general, STAT activation is through the phosphorylation of a single tyrosine residue (Tyr705) that results in Src homology 2 domain-mediated dimerization, nucleus localization, DNA binding, and ultimately transcription activation or repression (20Bromberg J. Darnell Jr., J.E. Oncogene. 2000; 19: 2468-2473Crossref PubMed Scopus (1039) Google Scholar). Stat3 can be activated by receptor-tyrosine kinases, such as epidermal growth factor receptor (EGFR), cytokine receptor-associated Janus kinases or Src kinases. It plays a convergent role in integrating multiple signaling pathways. Ablation of Stat3 in mice gave rise to defects in embryonic pattern formation and early lethality (21Levy D.E. Lee C.K. J. Clin. Invest. 2002; 109: 1143-1148Crossref PubMed Scopus (745) Google Scholar). 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Previously, we generated mutant mice in which Stat3 was ablated specifically in the hematopoietic cell lineage using btie2-cre mice (Stat3f/f/btie2-cre) (24Welte T. Zhang S.S. Wang T. Zhang Z. Hesslein D.G. Yin Z. Kano A. Iwamoto Y. Li E. Craft J.E. Bothwell A.L. Fikrig E. Koni P.A. Flavell R.A. Fu X.Y. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 1879-1884Crossref PubMed Scopus (336) Google Scholar, 25Laouar Y. Welte T. Fu X.Y. Flavell R.A. Immunity. 2003; 19: 903-912Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). Interestingly, our initial characterization revealed that the Stat3f/f/btie2-cre mice demonstrated an increase in autonomous myeloid cell proliferation (24Welte T. Zhang S.S. Wang T. Zhang Z. Hesslein D.G. Yin Z. Kano A. Iwamoto Y. Li E. Craft J.E. Bothwell A.L. Fikrig E. Koni P.A. Flavell R.A. Fu X.Y. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 1879-1884Crossref PubMed Scopus (336) Google Scholar), similar to that observed in the Shp2D61G mutant mice (8Araki T. Mohi M.G. Ismat F.A. Bronson R.T. Williams I.R. Kutok J.L. Yang W. Pao L.I. Gilliland D.G. Epstein J.A. Neel B.G. Nat. Med. 2004; 10: 849-857Crossref PubMed Scopus (341) Google Scholar), indicating that loss of Stat3 function contributes to phenotypic anomalies observed in NS. To follow up this initial observation and to further delineate the potential function of Stat3 in the pathogenesis of NS, we conducted a series of analyses on conditional Stat3 mutant mice, as well as examined the biochemical regulation of Stat3 by Shp2 phosphatase. Collectively, our data demonstrate that Stat3 is a direct target for Shp2 and is potentially an essential signaling component in PTPN11 mutation-associated NS and JMML. Stat3f/f/btie2-cre and Stat33f/f/nestin-cre mice were generated as previously described (24Welte T. Zhang S.S. Wang T. Zhang Z. Hesslein D.G. Yin Z. Kano A. Iwamoto Y. Li E. Craft J.E. Bothwell A.L. Fikrig E. Koni P.A. Flavell R.A. Fu X.Y. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 1879-1884Crossref PubMed Scopus (336) Google Scholar, 26Gao Q. Wolfgang M.J. Neschen S. Morino K. Horvath T.L. Shulman G.I. Fu X.Y. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 4661-4666Crossref PubMed Scopus (314) Google Scholar). Embryos were harvested by cesarean section. Embryos and isolated hearts were fixed in 10% neutral buffered formalin, paraffin embedded and sectioned (6 μm), and stained with hematoxylin and eosin. To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer舗s instructions. Peripheral blood samples were collected in sodium heparin tubes. The Institutional Review Board at the Indiana University School of Medicine approved all protocols. Informed consent was obtained from donors or from the guardian of children less than 18 years of age in accordance with the Declaration of Helsinki. Single cell suspensions from BM or spleen were prepared as previously described (24Welte T. Zhang S.S. Wang T. Zhang Z. Hesslein D.G. Yin Z. Kano A. Iwamoto Y. Li E. Craft J.E. Bothwell A.L. Fikrig E. Koni P.A. Flavell R.A. Fu X.Y. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 1879-1884Crossref PubMed Scopus (336) Google Scholar). Aliquots of cells (1–2 × 106) were stained with fluorescence-conjugated antibodies specifically against Mac-1 (CD11b), Gr-1, or F480 (Pharmingen). For human peripheral blood tyrosine-phosphorylated STAT3 staining, 300 μl of human whole blood were incubated with IL-6 for 30 min, followed by treatment with fix/lysis and permeabilization buffer III (Pharmingen) according to manufacturer舗s instructions. Cells were then stained with phycoerythrin-conjugated antibody against Tyr(P)705-STAT3 (Pharmingen). Flow cytometry was performed using a FACScalibur (BD Biosciences), and data were analyzed using CELLQUEST software. The retroviral vectors pMIEG3, pMIEG3-WT Shp2, pMIEG3-N308D, pMIEG3-E76K, and pMIEG3-hCD4 have been reported previously (27Chan R.J. Leedy M.B. Munugalavadla V. Voorhorst C.S. Li Y. Yu M. Kapur R. Blood. 2005; 105: 3737-3742Crossref PubMed Scopus (131) Google Scholar, 28Chang H.C. Zhang S. Thieu V.T. Slee R.B. Bruns H.A. Laribee R.N. Klemsz M.J. Kaplan M.H. Immunity. 2005; 22: 693-703Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). pMIEG3- Stat3CA-hCD4 was constructed by insertion of Stat3CA into the EcoRI site of pMIEG3-hCD4. Ecotropic retroviral supernatants were prepared using Eco-Phoenix packaging cells. Bone marrow low density mononuclear cells were isolated and transduced with pMIEG3, pMIEG3-WT Shp2, pMIEG3-N308D, pMIEG3-E76K, pMIEG3-hCD4, and pMIEG3-Stat3CA-hCD4, as previously described (27Chan R.J. Leedy M.B. Munugalavadla V. Voorhorst C.S. Li Y. Yu M. Kapur R. Blood. 2005; 105: 3737-3742Crossref PubMed Scopus (131) Google Scholar). Following transduction, cells were sorted for EGFP alone or EGFP and human CD4 antigen co-expression using fluorescence-activated cell sorting (FACS) to enrich for transduced cells (supplemental Fig. 6). The expression of Stat3 and Shp2 were further confirmed by Western blot analysis (supplemental Fig. 6). BM single cell suspensions were prepared from Stat3f/f and Stat3f/f/ btie2-cre as previously described (24Welte T. Zhang S.S. Wang T. Zhang Z. Hesslein D.G. Yin Z. Kano A. Iwamoto Y. Li E. Craft J.E. Bothwell A.L. Fikrig E. Koni P.A. Flavell R.A. Fu X.Y. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 1879-1884Crossref PubMed Scopus (336) Google Scholar). For the BM cell proliferation assay, 7.5 × 104 cells of each genotype were plated per well in 96-well plates, subjected to growth factor and serum deprivation for 5 h, cultured with various cytokines for 16 h, pulsed with 1.0 μCi (0.037 MBq) of [3H]thymidine for 24 h, and harvested using an automated 96-well cell harvester (Brandel, Gaithersburg, MD) for counting thymidine incorporation (counts/min). To assay cell survival, cells were deprived of serum and growth factor for 5 h followed by treatment with granulocyte-macrophage colony stimulating factor (GM-CSF) at 0.1 and 1 ng/ml for 24 h and analyzed for apoptosis staining with Annexin-V (BD Biosciences). Colony assays were performed using MethoCult M3434 (Stem Cell Technology, Vancouver) in the absence or presence of increasing concentrations of GM-CSF (Peprotech, Rocky Hill, NJ), as previously described (27Chan R.J. Leedy M.B. Munugalavadla V. Voorhorst C.S. Li Y. Yu M. Kapur R. Blood. 2005; 105: 3737-3742Crossref PubMed Scopus (131) Google Scholar). Western blots were performed using standard protocols. Phospho-Stat3 (Tyr(P)705) and phospho-p44/42 (Thr202/Tyr404) antibodies were from Cell Signaling Technologies (Beverly, MA). Anti-Stat3 (C-20) was from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-glyceraldehyde-3-phosphate dehydrogenase was from Biodesign International (Saco, ME). Stat3 EMSA was performed according the protocol previously described (29Asao H. Fu X.Y. J. Biol. Chem. 2000; 275: 867-874Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The small molecule Shp2 inhibitor IIB-08 was identified from a small focused library of benzofuran salicylic acid derivatives. The design and synthesis of the library, and the identification and characterization of IIB-08 as a Shp2 inhibitor will be described elsewhere. IIB-08 exhibits an IC50 of 5.5 μm for Shp2 with little activity against a panel of mammalian PTPs, including Lyp, CD45, Cdc14, MKP3, and VHR. Raw 264.7 (mouse monocyte macrophage cell line) cells were treated with IL-6 (20 ng/ml) for 30 min before being subjected to lysis in a solution containing 10 mm Hepes, pH 7.4, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 10 mm sodium fluoride, 1 mm Na3VO4. After centrifugation, cell extract was incubated with anti-Stat3 C-20 antibody (Santa Cruz Biotechnology) for Stat3 protein isolation. Stat3 immunoprecipitates were washed three times with 50 mm 3,3-dimethylglutarate buffer, pH 7.0. Two μg of recombinant constitutively active Shp2 protein (catalytic domain) was added to each sample and the reactions were incubated at 37 °C for the indicated time in 50 mm 3,3-dimethylglutarate buffer, pH 7.0. Reactions were quenched by adding SDS-loading buffer and boiling at 95 °C for 10 min. Samples were separated by SDS-PAGE and Stat3 dephosphorylation was determined by Western blotting analysis using anti-Tyr(P)705-Stat3 antibodies. To further confirm and evaluate the role of Stat3 in myeloid cell function, we re-examined the hematopoietic compartment of Stat3f/f/btie2-cre mice. Stat3f/f/btie2-cre newborns were normal in size at birth, but became visibly smaller compared with littermates by 2 weeks of age, rapidly became sick, and died between 4 and 6 weeks of age. By gross anatomical analysis, we found that Stat3f/f/btie2-cre mice had dramatically enlarged spleens (Fig. 1A, a and b). Histology of these enlarged spleens revealed disruption of the white and red pulp architecture (Fig. 1A, c and d). Flow cytometric analysis of 3-week-old spleens demonstrated a significant expansion of both neutrophil (Mac-1+Gr-1+ cells, a 5.59-fold increase) and macrophage (Mac-1+F480+ cells, a 13.95-fold increase) in the Stat3f/f/btie2-cre spleens when compared with controls (Fig. 1A, e). Similarly, the frequency of Mac-1+F480+ and Mac-1+Gr-1+ cells in Stat3f/f/btie2-cre bone marrow was also significantly increased (Fig. 1B). Peripheral blood progenitor cells from JMML patients and murine hematopoietic progenitors ectopically expressing gain-of-function Shp2 mutants demonstrate increased sensitivity to GM-CSF (8Araki T. Mohi M.G. Ismat F.A. Bronson R.T. Williams I.R. Kutok J.L. Yang W. Pao L.I. Gilliland D.G. Epstein J.A. Neel B.G. Nat. Med. 2004; 10: 849-857Crossref PubMed Scopus (341) Google Scholar, 27Chan R.J. Leedy M.B. Munugalavadla V. Voorhorst C.S. Li Y. Yu M. Kapur R. Blood. 2005; 105: 3737-3742Crossref PubMed Scopus (131) Google Scholar, 30Emanuel P.D. Bates L.J. Castleberry R.P. 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Akashi K. Gilliland D.G. Neel B.G. Cancer Cell. 2005; 7: 179-191Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar), Stat3f/f/btie2-cre bone marrow low density mononuclear cells had significantly increased sensitivity to GM-CSF at concentrations as low as 0.1 ng/ml for colony-forming units granulocyte-macrophage formation (Fig. 1C). Because Stat3f/f/btie2-cre mutants showed a dramatic increase in myeloid progenitor cell population, we further tested the responses of Stat3f/f/btie2-cre and control bone marrow cells to a panel of cytokines (SCF, IL-3, G-CSF, and GM-CSF) in vitro. [3H]Thymidine incorporation assays demonstrated that Stat3f/f/btie2-cre mutant cells had A significantly higher labeling index in response to growth factor stimulation, especially to GM-CSF and IL-3 (Fig. 1D). In the presence of low concentrations of GM-CSF (0.1 and 1 ng/ml), Stat3f/f/btie2-cre mutant cells also demonstrated a reduction in apoptosis when compared with control cells (Fig. 1E). These data strongly suggest that the hematopoietic phenotypic is similar between NS and Stat3f/f/btie2-cre mutants. Previous studies have shown that ablation of Shp2 enhances Stat3 activation (33Ohtani T. Ishihara K. Atsumi T. Nishida K. Kaneko Y. Miyata T. Itoh S. Narimatsu M. Maeda H. Fukada T. Itoh M. Okano H. Hibi M. Hirano T. Immunity. 2000; 12: 95-105Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 34Zhang E.E. Chapeau E. Hagihara K. Feng G.S. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 16064-16069Crossref PubMed Scopus (205) Google Scholar, 35Chan R.J. Johnson S.A. Li Y. Yoder M.C. Feng G.S. Blood. 2003; 102: 2074-2080Crossref PubMed Scopus (81) Google Scholar). To test whether Shp2 negatively regulates Stat3 activation in hematopoietic progenitors, we retrovirally transduced low density mononuclear cells with empty vector (MIEG3), WT Shp2, or Shp2E76K, one of the most common activating PTPN11 mutations observed in JMML (9Tartaglia M. Niemeyer C.M. Fragale A. 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Genet. 2006; 78: 279-290Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar), sorted for transduced enhanced green fluorescent protein (EGFP)-expressing cells, and cultured into macrophage progenitors as previously described (27Chan R.J. Leedy M.B. Munugalavadla V. Voorhorst C.S. Li Y. Yu M. Kapur R. Blood. 2005; 105: 3737-3742Crossref PubMed Scopus (131) Google Scholar). Cells were serum-deprived, stimulated with GM-CSF, and analyzed for activation of Stat3 (Tyr(P)705) and expression of total Stat3. At baseline and upon GM-CSF stimulation, Shp2E76K-expressing macrophage progenitors expressed significantly lower total Stat3 as well as activated Stat3 when compared with MIEG3- or WT Shp2-transduced cells (Fig. 2A). This inhibition of Stat3 expression and function in Shp2E76K-transduced cells suggests that Shp2 plays a critical negative regulatory role in Stat3-mediated signaling in hematopoietic cells. To further investigate whether reduced STAT3 function is associated with the pathogenesis of NS in humans, we collected peripheral blood samples from five NS patients with sequence confirmed PTPN11 gain-of-function mutations and six age- and sex-matched individuals lacking any congenital disease diagnosis and assayed baseline and IL-6-stimulated Tyr(P)-STAT3 (Tyr705) levels using flow cytometric and Western blot analysis. In the quiescent state, Tyr(P)-STAT3 is significantly higher in cells obtained from control individuals compared with that from NS patients, which persisted after 30 min of IL-6 treatment (Fig. 2B). These findings demonstrate a tight association between significantly lower activated STAT3 levels and the NS phenotype in patients bearing PTPN11 mutations. Taken together with previous work showing Stat3 activation is enhanced in Shp2-deficient cells (33Ohtani T. Ishihara K. Atsumi T. Nishida K. Kaneko Y. Miyata T. Itoh S. Narimatsu M. Maeda H. Fukada T. Itoh M. Okano H. Hibi M. Hirano T. Immunity. 2000; 12: 95-105Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 34Zhang E.E. Chapeau E. Hagihara K. Feng G.S. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 16064-16069Crossref PubMed Scopus (205) Google Scholar, 35Chan R.J. Johnson S.A. Li Y. Yoder M.C. Feng G.S. Blood. 2003; 102: 2074-2080Crossref PubMed Scopus (81) Google Scholar), these findings strongly suggest that Shp2 serves as an upstream phosphatase to negatively regulate Stat3 activation. Given the apparent role of Stat3 in the hematopoietic phenotype of NS patients, we next investigated the potential role of Stat3 in cardiac valve development because NS patients commonly manifest congenital heart defects such as pulmonic valve stenosis. Using immunohistological staining to examine the cellular distribution of activated Stat3 in valvular tissues during normal development, we found that Tyr(P)-Stat3 expression is highly restricted to the developing valvular tissues in normal mouse embryos (Fig. 3A), suggesting that dysregulated Stat3 expression or function may contribute to the abnormal valve development. Signaling from the EGFR has been shown to play a critical role in valvulogenesis and Shp2 has been shown to play a positive role in EGFR-stimulated regulation of cellular proliferation and apoptosis during valvular remodeling (37Iwamoto R. Mekada E. Cell Struct. Funct. 2006; 31: 1-14Crossref PubMed Scopus (101) Google Scholar, 38Chen B. Bronson R.T. Klaman L.D. Hampton T.G. Wang J.F. Green P.J. Magnuson T. Douglas P.S. Morgan J.P. Neel B
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