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1.
Cardiovasc Drugs Ther ; 36(4): 589-604, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-33689087

RESUMO

PURPOSE: Noonan syndrome with multiple lentigines (NSML) is an autosomal dominant disorder presenting with hypertrophic cardiomyopathy (HCM). Up to 85% of NSML cases are caused by mutations in the PTPN11 gene that encodes for the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP2). We previously showed that low-dose dasatinib protects from the development of cardiac fibrosis in a mouse model of NSML harboring a Ptpn11Y279C mutation. This study is performed to determine the pharmacokinetic (PK) and pharmacodynamic (PD) properties of a low-dose of dasatinib in NSML mice and to determine its effectiveness in ameliorating the development of HCM. METHODS: Dasatinib was administered intraperitoneally into NSML mice with doses ranging from 0.05 to 0.5 mg/kg. PK parameters of dasatinib in NSML mice were determined. PD parameters were obtained for biochemical analyses from heart tissue. Dasatinib-treated NSML mice (0.1 mg/kg) were subjected to echocardiography and assessment of markers of HCM by qRT-PCR. Transcriptome analysis was performed from the heart tissue of low-dose dasatinib-treated mice. RESULTS: Low-dose dasatinib exhibited PK properties that were linear across doses in NSML mice. Dasatinib treatment of between 0.05 and 0.5 mg/kg in NSML mice yielded an exposure-dependent inhibition of c-Src and PZR tyrosyl phosphorylation and inhibited AKT phosphorylation. We found that doses as low as 0.1 mg/kg of dasatinib prevented HCM in NSML mice. Transcriptome analysis identified differentially expressed HCM-associated genes in the heart of NSML mice that were reverted to wild type levels by low-dose dasatinib administration. CONCLUSION: These data demonstrate that low-dose dasatinib exhibits desirable therapeutic PK properties that is sufficient for effective target engagement to ameliorate HCM progression in NSML mice. These data demonstrate that low-dose dasatinib treatment may be an effective therapy against HCM in NSML patients.


Assuntos
Cardiomiopatia Hipertrófica , Síndrome LEOPARD , Animais , Cardiomiopatia Hipertrófica/tratamento farmacológico , Cardiomiopatia Hipertrófica/genética , Dasatinibe/farmacologia , Dasatinibe/uso terapêutico , Modelos Animais de Doenças , Síndrome LEOPARD/tratamento farmacológico , Síndrome LEOPARD/genética , Síndrome LEOPARD/metabolismo , Camundongos , Mutação
2.
Orphanet J Rare Dis ; 14(1): 252, 2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31722741

RESUMO

BACKGROUND: Animal studies suggested that blocking the activation of the mammalian target of rapamycin (mTOR) pathway might be effective to treat cardiac hypertrophy in LEOPARD syndrome (LS) caused by PTPN11 mutations. RESULTS: In the present study, mTOR pathway activity was examined in human myocardial samples from two patients with LS, four patients with hypertrophic cardiomyopathy (HCM), and four normal controls. The two patients with LS had p.Y279C and p.T468 M mutations of the PTPN11 gene, respectively. Although PTPN11 mutation showed initially positive regulation on phosphoinositide 3-kinase, overall the mTOR complex 1 pathway showed widely attenuated activity in LS. This included mildly hypophosphorylated mTOR and ribosomal protein S6 kinase and significantly hypophosphorylated Akt308 and ribosomal protein S6, which is similar to HCM. Akt473 is a basal molecule of the mTOR complex 2 pathway. Akt473 was less affected and showed hyperactivity in LS compared with HCM and normal controls. Additionally, MAPK/ERK kinase and ERK1/2 were significantly more phosphorylated in both HCM and LS than normal controls. CONCLUSIONS: In LS, the mTOR signaling pathway shows similar activity to HCM and is attenuated compared with normal controls. Thus, caution should be applied when using rapamycin to treat heart hypertrophy in LS.


Assuntos
Cardiomegalia/etiologia , Cardiomegalia/metabolismo , Síndrome LEOPARD/complicações , Síndrome LEOPARD/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Adolescente , Adulto , Animais , Cardiomegalia/tratamento farmacológico , Humanos , Síndrome LEOPARD/tratamento farmacológico , Masculino , Modelos Animais , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirolimo/uso terapêutico , Adulto Jovem
3.
Sci Signal ; 11(522)2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29559584

RESUMO

Catalytically activating mutations in Ptpn11, which encodes the protein tyrosine phosphatase SHP2, cause 50% of Noonan syndrome (NS) cases, whereas inactivating mutations in Ptpn11 are responsible for nearly all cases of the similar, but distinct, developmental disorder Noonan syndrome with multiple lentigines (NSML; formerly called LEOPARD syndrome). However, both types of disease mutations are gain-of-function mutations because they cause SHP2 to constitutively adopt an open conformation. We found that the catalytic activity of SHP2 was required for the pathogenic effects of gain-of-function, disease-associated mutations on the development of hydrocephalus in the mouse. Targeted pan-neuronal knockin of a Ptpn11 allele encoding the active SHP2 E76K mutant resulted in hydrocephalus due to aberrant development of ependymal cells and their cilia. These pathogenic effects of the E76K mutation were suppressed by the additional mutation C459S, which abolished the catalytic activity of SHP2. Moreover, ependymal cells in NSML mice bearing the inactive SHP2 mutant Y279C were also unaffected. Mechanistically, the SHP2 E76K mutant induced developmental defects in ependymal cells by enhancing dephosphorylation and inhibition of the transcription activator STAT3. Whereas STAT3 activity was reduced in Ptpn11E76K/+ cells, the activities of the kinases ERK and AKT were enhanced, and neural cell-specific Stat3 knockout mice also manifested developmental defects in ependymal cells and cilia. These genetic and biochemical data demonstrate a catalytic-dependent role of SHP2 gain-of-function disease mutants in the pathogenesis of hydrocephalus.


Assuntos
Mutação com Ganho de Função , Hidrocefalia/genética , Síndrome LEOPARD/genética , Síndrome de Noonan/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Animais , Biocatálise , Epêndima/citologia , Epêndima/metabolismo , Predisposição Genética para Doença/genética , Humanos , Hidrocefalia/metabolismo , Síndrome LEOPARD/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Neurais/metabolismo , Síndrome de Noonan/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo
4.
PLoS One ; 12(6): e0178905, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28582432

RESUMO

Noonan Syndrome with Multiple Lentigines (NSML, formerly LEOPARD syndrome) is an autosomal dominant "RASopathy" disorder manifesting in congenital heart disease. Most cases of NSML are caused by catalytically inactivating mutations in the protein tyrosine phosphatase (PTP), non-receptor type 11 (PTPN11), encoding the SH2 domain-containing PTP-2 (SHP2) protein. We previously generated knock-in mice harboring the PTPN11 mutation Y279C, one of the most common NSML alleles; these now-termed SHP2Y279C/+ mice recapitulate the human disorder and develop hypertrophic cardiomyopathy (HCM) by 12 weeks of age. Functionally, heart and/or cardiomyocyte lysates from SHP2Y279C/+ mice exhibit increased basal and agonist-induced AKT and mTOR activities. Here, we sought to determine whether we could reverse the hypertrophy in SHP2Y279C/+ mice using ARQ 092, an oral and selective allosteric AKT inhibitor currently in clinical trials for patients with PI3K/AKT-driven tumors or Proteus syndrome. We obtained echocardiographs of SHP2Y279C/+ and wildtype (SHP2+/+) littermates, either in the presence or absence of ARQ 092 at 12, 14, and 16 weeks of age. While SHP2Y279C/+ mice developed significant left ventricular hypertrophy by 12 weeks, as indicated by decreased chamber dimension and increased posterior wall thickness, treatment of SHP2Y279C/+ mice with ARQ 092 normalized the hypertrophy in as early as 2 weeks following treatment, with hearts comparable in size to those in wildtype (SHP2+/+) mice. In addition, we observed an increase in fractional shortening (FS%) in SHP2Y279C/+ mice, an effect of increased compensatory hypertrophy, which was not apparent in SHP2Y279C/+ mice treated with ARQ 092, suggesting functional improvement of HCM upon treatment with the AKT inhibitor. Finally, we found that ARQ 092 specifically inhibited AKT activity, as well as its downstream effectors, PRAS and S6RP in NSML mice. Taken together, these data suggest ARQ 092 may be a promising novel therapy for treatment of hypertrophy in NSML patients.


Assuntos
Aminopiridinas/farmacologia , Cardiomiopatia Hipertrófica/tratamento farmacológico , Cardiotônicos/farmacologia , Imidazóis/farmacologia , Síndrome LEOPARD/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Alelos , Animais , Cardiomiopatia Hipertrófica/diagnóstico por imagem , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Modelos Animais de Doenças , Ecocardiografia , Regulação da Expressão Gênica , Humanos , Síndrome LEOPARD/diagnóstico por imagem , Síndrome LEOPARD/genética , Síndrome LEOPARD/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Domínios Proteicos , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
5.
Biochem Biophys Res Commun ; 469(4): 1133-9, 2016 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-26742426

RESUMO

SHP2, encoded by the PTPN11 gene, is a protein tyrosine phosphatase that plays a key role in the proliferation of cells via RAS-ERK activation. SHP2 also promotes Wnt signaling by dephosphorylating parafibromin. Germline missense mutations of PTPN11 are found in more than half of patients with Noonan syndrome (NS) and LEOPARD syndrome (LS), both of which are congenital developmental disorders with multiple common symptoms. However, whereas NS-associated PTPN11 mutations give rise to gain-of-function SHP2 mutants, LS-associated SHP2 mutants are reportedly loss-of-function mutants. To determine the phosphatase activity of LS-associated SHP2 more appropriately, we performed an in vitro phosphatase assay using tyrosine-phosphorylated parafibromin, a biologically relevant substrate of SHP2 and the positive regulator of Wnt signaling that is activated through SHP2-mediated dephosphorylation. We found that LS-associated SHP2 mutants (Y279C, T468M, Q506P, and Q510E) exhibited a substantially reduced phosphatase activity toward parafibromin when compared with wild-type SHP2. Furthermore, each of the LS-associated mutants displayed a differential degree of decrease in phosphatase activity. Deviation of the SHP2 catalytic activity from a certain range, either too strong or too weak, may therefore lead to similar clinical outcomes in NS and LS, possibly through an imbalanced Wnt signal caused by inadequate dephosphorylation of parafibromin.


Assuntos
Síndrome LEOPARD/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Via de Sinalização Wnt , Animais , Células COS , Catálise , Chlorocebus aethiops , Ativação Enzimática , Humanos , Síndrome LEOPARD/genética , Mutação/genética , Ligação Proteica , Especificidade por Substrato
6.
Acta Derm Venereol ; 95(8): 978-84, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25917897

RESUMO

LEOPARD syndrome (LS) is an autosomal dominant condition with multiple anomalies, including multiple lentigines. LS is caused by mutations in PTPN11, encoding the protein tyrosine phosphatase, SHP-2. We report here 2 unrelated Japanese cases of LS with different PTPN11 mutations (p.Y279C and p.T468P). To elucidate the pathogenesis of multiple lentigines in LS, ultrastructural and immunohistochemical analyses of lentigines and non-lesional skin were performed. Numerous mature giant melanosomes in melanocytes and keratinocytes were observed in lentigines. In addition, the levels of expression of endothelin-1 (ET-1), phosphorylated Akt, mTOR and STAT3 in the epidermis in lentigines were significantly elevated compared with non-lesional skin. In in vitro assays, melanin synthesis in human melanoma cells expressing SHP-2 with LS-associated mutations was higher than in cells expressing normal SHP-2, suggesting that LS-associated SHP-2 mutations might enhance melanin synthesis in melanocytes, and that the activation of Akt/mTOR signalling may contribute to this process.


Assuntos
Queratinócitos/ultraestrutura , Síndrome LEOPARD/genética , Síndrome LEOPARD/patologia , Melanócitos/ultraestrutura , Melanoma/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Adolescente , Endotelina-1/análise , Feminino , Humanos , Síndrome LEOPARD/metabolismo , Melaninas/biossíntese , Melanócitos/metabolismo , Melanoma/genética , Melanossomas/ultraestrutura , Mutação , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogênicas c-akt/análise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/análise , Transdução de Sinais , Pele/química , Pele/ultraestrutura , Serina-Treonina Quinases TOR/análise , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais Cultivadas , Adulto Jovem
7.
Am J Physiol Heart Circ Physiol ; 308(9): H1086-95, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25724491

RESUMO

Noonan syndrome with multiple lentigines (NSML) is primarily caused by mutations in the nonreceptor protein tyrosine phosphatase SHP2 and associated with congenital heart disease in the form of pulmonary valve stenosis and hypertrophic cardiomyopathy (HCM). Our goal was to elucidate the cellular mechanisms underlying the development of HCM caused by the Q510E mutation in SHP2. NSML patients carrying this mutation suffer from a particularly severe form of HCM. Drawing parallels to other, more common forms of HCM, we hypothesized that altered Ca(2+) homeostasis and/or sarcomeric mechanical properties play key roles in the pathomechanism. We used transgenic mice with cardiomyocyte-specific expression of Q510E-SHP2 starting before birth. Mice develop neonatal onset HCM with increased ejection fraction and fractional shortening at 4-6 wk of age. To assess Ca(2+) handling, isolated cardiomyocytes were loaded with fluo-4. Q510E-SHP2 expression increased Ca(2+) transient amplitudes during excitation-contraction coupling and increased sarcoplasmic reticulum Ca(2+) content concurrent with increased expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase. In skinned cardiomyocyte preparations from Q510E-SHP2 mice, force-velocity relationships and power-load curves were shifted upward. The peak power-generating capacity was increased approximately twofold. Transmission electron microscopy revealed that the relative intracellular area occupied by sarcomeres was increased in Q510E-SHP2 cardiomyocytes. Triton X-100-based myofiber purification showed that Q510E-SHP2 increased the amount of sarcomeric proteins assembled into myofibers. In summary, Q510E-SHP2 expression leads to enhanced contractile performance early in disease progression by augmenting intracellular Ca(2+) cycling and increasing the number of power-generating sarcomeres. This gives important new insights into the cellular pathomechanisms of Q510E-SHP2-associated HCM.


Assuntos
Sinalização do Cálcio , Síndrome LEOPARD/metabolismo , Força Muscular , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Sarcômeros/metabolismo , Animais , Modelos Animais de Doenças , Acoplamento Excitação-Contração , Feminino , Genótipo , Síndrome LEOPARD/genética , Síndrome LEOPARD/patologia , Síndrome LEOPARD/fisiopatologia , Masculino , Camundongos Transgênicos , Mutação , Miócitos Cardíacos/ultraestrutura , Fenótipo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Sarcômeros/ultraestrutura , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Volume Sistólico
8.
Mol Cell Biol ; 34(15): 2874-89, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24865967

RESUMO

Noonan syndrome (NS) is an autosomal dominant disorder caused by activating mutations in the PTPN11 gene encoding Shp2, which manifests in congenital heart disease, short stature, and facial dysmorphia. The complexity of Shp2 signaling is exemplified by the observation that LEOPARD syndrome (LS) patients possess inactivating PTPN11 mutations yet exhibit similar symptoms to NS. Here, we identify "protein zero-related" (PZR), a transmembrane glycoprotein that interfaces with the extracellular matrix to promote cell migration, as a major hyper-tyrosyl-phosphorylated protein in mouse and zebrafish models of NS and LS. PZR hyper-tyrosyl phosphorylation is facilitated in a phosphatase-independent manner by enhanced Src recruitment to NS and LS Shp2. In zebrafish, PZR overexpression recapitulated NS and LS phenotypes. PZR was required for zebrafish gastrulation in a manner dependent upon PZR tyrosyl phosphorylation. Hence, we identify PZR as an NS and LS target. Enhanced PZR-mediated membrane recruitment of Shp2 serves as a common mechanism to direct overlapping pathophysiological characteristics of these PTPN11 mutations.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Síndrome LEOPARD/genética , Síndrome de Noonan/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Transdução de Sinais/genética , Peixe-Zebra/genética , Animais , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Síndrome LEOPARD/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Células NIH 3T3 , Síndrome de Noonan/metabolismo , Fosforilação/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Peixe-Zebra/metabolismo
9.
Cell Commun Adhes ; 21(3): 129-40, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24738885

RESUMO

The importance of desmosomes in tissue homeostasis is highlighted by natural and engineered mutations in desmosomal genes, which compromise the skin or heart and in some instances both. Desmosomal gene mutations account for 45-50% of cases of arrhythmogenic right ventricular cardiomyopathy, and are mutated in an array of other disorders such as striate palmoplantar keratoderma, hypotrichosis with or without skin vesicles and lethal acantholytic epidermolysis bullosa. Recently, we reported loss-of-function mutations in the human ADAM17 gene, encoding for the 'sheddase' ADAM17, a transmembrane protein which cleaves extracellular domains of substrate proteins including TNF-α, growth factors and desmoglein (DSG) 2. Patients present with cardiomyopathy and an inflammatory skin and bowel syndrome with defective DSG processing. In contrast, the dominantly inherited tylosis with oesophageal cancer appears to result from gain-of-function in ADAM17 due to increased processing via iRHOM2. This review discusses the heterogeneity of mutations in desmosomes and their regulatory proteins.


Assuntos
Desmossomos/genética , Desmossomos/metabolismo , Síndrome LEOPARD/genética , Síndrome LEOPARD/metabolismo , Dermatopatias/genética , Dermatopatias/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM17 , Desmoplaquinas/genética , Desmoplaquinas/metabolismo , Desmossomos/química , Humanos , Síndrome LEOPARD/patologia , Mutação , Dermatopatias/patologia
10.
Cell Commun Adhes ; 21(1): 3-11, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24460197

RESUMO

The classic cardiocutaneous syndromes of Naxos and Carvajal are rare. The myocardial disorder integral to their pathology - arrhythmogenic cardiomyopathy - is arguably not uncommon, with a prevalence of up to 1 in 1,000 despite almost certain under-recognition. Yet the study of cardiocutaneous syndromes has been integral to evolution of the contemporary perspective of arrhythmogenic cardiomyopathy - its clinical course, disease spectrum, genetics, and cellular and molecular mechanisms. Here we discuss how recognition of the association of hair and skin abnormalities with underlying heart disease transformed our conception of a little-understood but important cause of sudden cardiac death.


Assuntos
Displasia Arritmogênica Ventricular Direita/genética , Síndrome LEOPARD/genética , Animais , Displasia Arritmogênica Ventricular Direita/metabolismo , Displasia Arritmogênica Ventricular Direita/patologia , Desmocolinas/genética , Desmocolinas/metabolismo , Desmoplaquinas/genética , Desmoplaquinas/metabolismo , Desmossomos/metabolismo , Estudos de Associação Genética , Humanos , Síndrome LEOPARD/metabolismo , Síndrome LEOPARD/patologia , Mutação , gama Catenina/genética , gama Catenina/metabolismo
11.
Oncogene ; 32(45): 5292-301, 2013 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-23318428

RESUMO

Shp2 is a positive regulator for Erk activation downstream of receptor tyrosine kinases for growth factors. It has been controversial how Shp2 induces Erk activation. We here demonstrate that EphA2 is responsible for Shp2-mediated Erk activation by phosphorylating Tyr542 and Tyr580 of Shp2 in the cells stimulated with growth factors. In NMuMG mammary epithelial cells stimulated with hepatocyte growth factor (HGF), HGF-dependent Erk phosphorylation was prolonged only in the presence of EphA2. This Erk activation paralleled the phosphorylation of Tyr542/580 of Shp2 and the association of Grb2 with Shp2, suggesting the positive signal involving Grb2 signal to activate Ras-Erk pathway. Immunohistochemical studies of mammary cancer specimens revealed that the cancer progression was associated with both Tyr580 phosphorylation of Shp2 and increased expression of EphA2, which were also correlated with increased Erk phosphorylation. Overexpression of either Shp2Thr468Met (a phosphatase-defective mutant found in Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Retardation of growth and sensorineural Deafness (LEOPARD) syndrome) or Shp2Asn308Asp (a phosphatase-active mutant found in Noonan syndrome) with EphA2 exhibited comparable activation of Erk and stronger activation than wild-type Shp2, suggesting the phosphatase-independent Erk activation. Expression of Shp2Thr468Met with Tyr542/580Phe mutations resulted in the suppression of Erk activation. Phosphatase-active and -inactive, and wild-type Shp2s bound equally to Grb2, suggesting that phosphorylation of Tyr542/580 of Shp2 was essential but not sufficient for Shp2-mediated Erk activation. We found that Gab1 (Grb2-associated binder 1) was involved in the mutant Shp2-mediated Erk activation. Zebrafish injected with Shp2Thr468Met mRNA showed cardiac edema, whereas those depleted of EphA2b showed less phenotype, suggesting that EphA2 might partly account for the phenotype of LEOPARD syndrome. Collectively, tyrosine phosphorylation of Shp2 by EphA2 contributes to the phosphatase-independent Shp2-mediated activation of Erk and might be involved in Shp2-associated diseases.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína Adaptadora GRB2/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Receptor EphA2/metabolismo , Animais , Edema Cardíaco , Ativação Enzimática , Fator de Crescimento de Hepatócito , Humanos , Síndrome LEOPARD/genética , Síndrome LEOPARD/metabolismo , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Fosforilação , Transdução de Sinais/genética , Peixe-Zebra
13.
J Mol Cell Cardiol ; 51(1): 4-15, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21440552

RESUMO

RAS activation is implicated in physiologic and pathologic cardiac hypertrophy. Cross-talk between the Ras and calcineurin pathways, the latter also having been implicated in cardiac hypertrophy, has been suspected for pathologic hypertrophy. Our recent discovery that germ-line mutations in RAF1, which encodes a downstream RAS effector, cause Noonan and LEOPARD syndromes with a high prevalence of hypertrophic cardiomyopathy provided an opportunity to elaborate the role of RAF1 in cardiomyocyte biology. Here, we characterize the role of RAF1 signaling in cardiomyocyte hypertrophy with an aim of identifying potential therapeutic targets. We modeled hypertrophic cardiomyopathy by infecting neonatal and adult rat cardiomyocytes (NRCMs and ARCMs, respectively) with adenoviruses encoding wild-type RAF1 and three Noonan/LEOPARD syndrome-associated RAF1 mutants (S257L, D486N or L613V). These RAF1 proteins, except D486N, engendered cardiomyocyte hypertrophy. Surprisingly, these effects were independent and dependent of mitogen activated protein kinases in NRCMs and ARCMs, respectively. Inhibiting Mek1/2 in RAF1 overexpressing cells blocked hypertrophy in ARCMs but not in NRCMs. Further, we found that endogenous and heterologously expressed RAF1 complexed with calcineurin, and RAF1 mutants causing hypertrophy signaled via nuclear factor of activated T cells (Nfat) in both cell types. The involvement of calcineurin was also reflected by down regulation of Serca2a and dysregulation of calcium signaling in NRCMs. Furthermore, treatment with the calcineurin inhibitor cyclosporine blocked hypertrophy in NRCMs and ARCMs overexpressing RAF1. Thus, we have identified calcineurin as a novel interaction partner for RAF1 and established a mechanistic link and possible therapeutic target for pathological cardiomyocyte hypertrophy induced by mutant RAF1. This article is part of a Special Issue entitled 'Possible Editorial'.


Assuntos
Cardiomegalia/prevenção & controle , Cardiomiopatia Hipertrófica/prevenção & controle , Ciclosporina/farmacologia , Síndrome LEOPARD , Síndrome de Noonan , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Adenoviridae/genética , Animais , Animais Recém-Nascidos , Calcineurina/metabolismo , Sinalização do Cálcio , Cardiomiopatia Hipertrófica/genética , Modelos Animais de Doenças , Síndrome LEOPARD/genética , Síndrome LEOPARD/metabolismo , Síndrome LEOPARD/patologia , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Síndrome de Noonan/patologia , Ratos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/biossíntese , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/metabolismo , Proteínas ras/metabolismo
14.
Nature ; 465(7299): 808-12, 2010 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-20535210

RESUMO

The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth and deafness), an autosomal-dominant developmental disorder belonging to a relatively prevalent class of inherited RAS-mitogen-activated protein kinase signalling diseases, which also includes Noonan syndrome, with pleomorphic effects on several tissues and organ systems. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSCs have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LEOPARD syndrome iPSCs are larger, have a higher degree of sarcomeric organization and preferential localization of NFATC4 in the nucleus when compared with cardiomyocytes derived from human embryonic stem cells or wild-type iPSCs derived from a healthy brother of one of the LEOPARD syndrome patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signalling pathways that may promote the disease phenotype.


Assuntos
Células-Tronco Pluripotentes Induzidas/patologia , Síndrome LEOPARD/patologia , Modelos Biológicos , Medicina de Precisão , Adulto , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Ativação Enzimática , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Células-Tronco Pluripotentes Induzidas/enzimologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome LEOPARD/tratamento farmacológico , Síndrome LEOPARD/metabolismo , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fosfoproteínas/análise , Reação em Cadeia da Polimerase , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Fatores de Transcrição SOXB1/genética
15.
Mol Cell Biol ; 30(10): 2498-507, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20308328

RESUMO

LEOPARD syndrome (LS), a disorder with multiple developmental abnormalities, is mainly due to mutations that impair the activity of the tyrosine phosphatase SHP2 (PTPN11). How these alterations cause the disease remains unknown. We report here that fibroblasts isolated from LS patients displayed stronger epidermal growth factor (EGF)-induced phosphorylation of both AKT and glycogen synthase kinase 3beta (GSK-3beta) than fibroblasts from control patients. Similar results were obtained in HEK293 cells expressing LS mutants of SHP2. We found that the GAB1/phosphoinositide 3-kinase (PI3K) complex was more abundant in fibroblasts from LS than control subjects and that both AKT and GSK-3beta hyperphosphorylation were prevented by reducing GAB1 expression or by overexpressing a GAB1 mutant unable to bind to PI3K. Consistently, purified recombinant LS mutants failed to dephosphorylate GAB1 PI3K-binding sites. These mutants induced PI3K-dependent increase in cell size in a model of chicken embryo cardiac explants and in transcriptional activity of the atrial natriuretic factor (ANF) gene in neonate rat cardiomyocytes. In conclusion, SHP2 mutations causing LS facilitate EGF-induced PI3K/AKT/GSK-3beta stimulation through impaired GAB1 dephosphorylation, resulting in deregulation of a novel signaling pathway that could be involved in LS pathology.


Assuntos
Fator de Crescimento Epidérmico/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Síndrome LEOPARD , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Células Cultivadas , Embrião de Galinha , Ativação Enzimática , Fibroblastos/citologia , Fibroblastos/fisiologia , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Síndrome LEOPARD/genética , Síndrome LEOPARD/metabolismo , Síndrome LEOPARD/patologia , Mutação , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
17.
Hum Mol Genet ; 17(13): 2018-29, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18372317

RESUMO

Missense PTPN11 mutations cause Noonan and LEOPARD syndromes (NS and LS), two developmental disorders with pleiomorphic phenotypes. PTPN11 encodes SHP2, an SH2 domain-containing protein tyrosine phosphatase functioning as a signal transducer. Generally, different substitutions of a particular amino acid residue are observed in these diseases, indicating that the crucial factor is the residue being replaced. For a few codons, only one substitution is observed, suggesting the possibility of specific roles for the residue introduced. We analyzed the biochemical behavior and ligand-binding properties of all possible substitutions arising from single-base changes affecting codons 42, 139, 279, 282 and 468 to investigate the mechanisms underlying the invariant occurrence of the T42A, E139D and I282V substitutions in NS and the Y279C and T468M changes in LS. Our data demonstrate that the isoleucine-to-valine change at codon 282 is the only substitution at that position perturbing the stability of SHP2's closed conformation without impairing catalysis, while the threonine-to-alanine change at codon 42, but not other substitutions of that residue, promotes increased phosphopeptide-binding affinity. The recognition specificity of the C-SH2 domain bearing the E139D substitution differed substantially from its wild-type counterpart acquiring binding properties similar to those observed for the N-SH2 domain, revealing a novel mechanism of SHP2's functional dysregulation. Finally, while functional selection does not seem to occur for the substitutions at codons 279 and 468, we point to deamination of the methylated cytosine at nucleotide 1403 as the driving factor leading to the high prevalence of the T468M change in LS.


Assuntos
Substituição de Aminoácidos , Síndrome LEOPARD/genética , Síndrome de Noonan/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Simulação por Computador , Análise Mutacional de DNA , Células HeLa , Humanos , Síndrome LEOPARD/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Síndrome de Noonan/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo
18.
Nat Genet ; 39(8): 1007-12, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17603483

RESUMO

Noonan and LEOPARD syndromes are developmental disorders with overlapping features, including cardiac abnormalities, short stature and facial dysmorphia. Increased RAS signaling owing to PTPN11, SOS1 and KRAS mutations causes approximately 60% of Noonan syndrome cases, and PTPN11 mutations cause 90% of LEOPARD syndrome cases. Here, we report that 18 of 231 individuals with Noonan syndrome without known mutations (corresponding to 3% of all affected individuals) and two of six individuals with LEOPARD syndrome without PTPN11 mutations have missense mutations in RAF1, which encodes a serine-threonine kinase that activates MEK1 and MEK2. Most mutations altered a motif flanking Ser259, a residue critical for autoinhibition of RAF1 through 14-3-3 binding. Of 19 subjects with a RAF1 mutation in two hotspots, 18 (or 95%) showed hypertrophic cardiomyopathy (HCM), compared with the 18% prevalence of HCM among individuals with Noonan syndrome in general. Ectopically expressed RAF1 mutants from the two HCM hotspots had increased kinase activity and enhanced ERK activation, whereas non-HCM-associated mutants were kinase impaired. Our findings further implicate increased RAS signaling in pathological cardiomyocyte hypertrophy.


Assuntos
Cardiomiopatia Hipertrófica/genética , Síndrome LEOPARD/genética , Mutação de Sentido Incorreto , Síndrome de Noonan/genética , Proteínas Proto-Oncogênicas c-raf/genética , Animais , Células COS , Cardiomiopatia Hipertrófica/metabolismo , Chlorocebus aethiops , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Síndrome LEOPARD/metabolismo , Síndrome de Noonan/metabolismo , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais , Transfecção , Proteínas ras/metabolismo
19.
Hum Mol Genet ; 15 Spec No 2: R220-6, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16987887

RESUMO

Noonan syndrome is a relatively common, genetically heterogeneous Mendelian trait with a pleiomorphic phenotype. Prior to the period covered in this review, missense mutations in PTPN11 had been found to account for nearly 50% of Noonan syndrome cases. That gene encodes SHP-2, a protein tyrosine kinase that plays diverse roles in signal transduction including signaling via the RAS-mitogen activated protein kinase (MAPK) pathway. Noonan syndrome-associated PTPN11 mutations are gain-of-function, with most disrupting SHP-2's activation-inactivation mechanism. Here, we review recent information that has elucidated further the types and effects of PTPN11 defects in Noonan syndrome and compare them to the related, but specific, missense PTPN11 mutations causing other diseases including LEOPARD syndrome and leukemias. These new data derive from biochemical and cell biological studies as well as animal modeling with fruit flies and chick embryos. The discovery of KRAS missense mutation as a minor cause of Noonan syndrome and the pathogenetic mechanisms of those mutants is discussed. Finally, the elucidation of gene defects underlying two phenotypically related disorders, Costello and cardio-facio-cutaneous syndromes is also reviewed. As these genes also encode proteins relevant for RAS-MAPK signal transduction, all of the syndromes discussed in this article now can be understood to constitute a class of disorders caused by dysregulated RAS-MAPK signaling.


Assuntos
Sistema de Sinalização das MAP Quinases/genética , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Animais , Animais Geneticamente Modificados , Embrião de Galinha , Drosophila/genética , Genes ras , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Síndrome LEOPARD/genética , Síndrome LEOPARD/metabolismo , Camundongos , Modelos Biológicos , Modelos Moleculares , Mutação , Conformação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética
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