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Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) constitute members of the dual-specificity family of protein phosphatases that dephosphorylate the MAPKs. MKP-5 dephosphorylates the stress-responsive MAPKs, p38 MAPK and JNK, and has been shown to promote tissue fibrosis. Here, we provide insight into how MKP-5 regulates the transforming growth factor-ß (TGF-ß) pathway, a well-established driver of fibrosis. We show that MKP-5-deficient fibroblasts in response to TGF-ß are impaired in SMAD2 phosphorylation at canonical and non-canonical sites, nuclear translocation, and transcriptional activation of fibrogenic genes. Consistent with this, pharmacological inhibition of MKP-5 is sufficient to block TGF-ß signaling, and that this regulation occurs through a JNK-dependent pathway. By utilizing RNA sequencing and transcriptomic analysis, we identify TGF-ß signaling activators regulated by MKP-5 in a JNK-dependent manner, providing mechanistic insight into how MKP-5 promotes TGF-ß signaling. This study elucidates a novel mechanism whereby MKP-5-mediated JNK inactivation is required for TGF-ß signaling and provides insight into the role of MKP-5 in fibrosis.
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Protein tyrosine phosphorylation and dephosphorylation are key regulatory mechanisms in eukaryotes. Protein tyrosine phosphorylation and dephosphorylation are catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), respectively. The combinatorial action of both PTKs and PTPs is essential for properly maintaining cellular functions. In this unit, we discuss different novel methods to identify PTP substrates. PTPs depend on specific invariant residues that enable binding to tyrosine-phosphorylated substrates and aid catalytic activity. Identifying PTP substrates has paved the way to understanding their role in distinct intracellular signaling pathways. Due to their high specific activity, the interaction between PTPs and their substrates is transient; therefore, identifying the physiological substrates of PTPs has been challenging. To identify the physiological substrates of PTPs, various PTP mutants have been generated. These PTP mutants, named "substrate-trapping mutants," lack catalytic activity but bind tightly to their tyrosine-phosphorylated substrates. Identifying the substrates for the PTPs will provide critical insight into the function of physiological and pathophysiological signal transduction. In this chapter, we describe interaction assays used to identify the PTP substrates.
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Proteínas Tirosina Fosfatasas , Transducción de Señal , Proteínas Tirosina Fosfatasas/genética , Fosforilación , Proteínas Tirosina Quinasas , TirosinaRESUMEN
Hypertension is caused by a combination of genetic and environmental factors. Angiotensinogen (AGT) is a component of RAAS, that regulates blood pressure. The human angiotensinogen (hAGT) gene has -6A/-6G polymorphism and -6A variant is associated with human hypertension. In this study, we have investigated the epigenetic regulation of the hAGT. To understand transcriptional regulation of the hAGT, we have made transgenic animals containing -6A. We show that HS affects DNA methylation and modulates transcriptional regulation of this gene in liver and kidney. High salt (HS) increases hAGT gene expression in -6A TG mice. We have observed that the number of CpG sites in the hAGT promoter is decreased after HS treatment. In the liver, seven CpG sites are methylated whereas after HS treatment, only three CpG sites remain methylated. In the kidney, five CpG sites are methylated, whereas after HS treatment, only three CpG sites remain methylated. These results suggest that HS promotes DNA demethylation and increasing AGT gene expression. RT-PCR and immunoblot analysis show that hAGT gene expression is increased by HS. Chip assay has shown that transcription factors bind strongly after HS treatment. RNA-Seq identified differentially expressed genes, novel target genes associated with hypertension, top canonical pathways, upstream regulators. One of the plausible mechanisms for HS induced up-regulation of the hAGT gene is through IL-6/JAK/STAT3/AGT axis.
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The RAS/mitogen-activated protein kinase (MAPK) pathway controls a plethora of developmental and post-developmental processes. It is now clear that mutations in the RAS-MAPK pathway cause developmental diseases collectively referred to as the RASopathies. The RASopathies include Noonan syndrome, Noonan syndrome with multiple lentigines, cardiofaciocutaneous syndrome, neurofibromatosis type 1, and Costello syndrome. RASopathy patients exhibit a wide spectrum of congenital heart defects (CHD), such as valvular abnormalities and hypertrophic cardiomyopathy (HCM). Since the cardiovascular defects are the most serious and recurrent cause of mortality in RASopathy patients, it is critical to understand the pathological signaling mechanisms that drive the disease. Therapies for the treatment of HCM and other RASopathy-associated comorbidities have yet to be fully realized. Recent developments have shown promise for the use of repurposed antineoplastic drugs that target the RAS-MAPK pathway for the treatment of RASopathy-associated HCM. However, given the impact of the RAS-MAPK pathway in post-developmental physiology, establishing safety and evaluating risk when treating children will be paramount. As such insight provided by preclinical and clinical information will be critical. This review will highlight the cardiovascular manifestations caused by the RASopathies and will discuss the emerging therapies for treatment.
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Síndrome de Costello , Displasia Ectodérmica , Cardiopatías Congénitas , Síndrome de Noonan , Niño , Humanos , Cardiopatías Congénitas/tratamiento farmacológico , Cardiopatías Congénitas/genética , Síndrome de Noonan/tratamiento farmacológico , Síndrome de Noonan/genética , Síndrome de Costello/genética , Insuficiencia de Crecimiento/tratamiento farmacológico , Insuficiencia de Crecimiento/genética , Displasia Ectodérmica/genéticaRESUMEN
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.
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Cardiomiopatía Hipertrófica , Síndrome LEOPARD , Animales , Cardiomiopatía Hipertrófica/tratamiento farmacológico , Cardiomiopatía Hipertrófica/genética , Dasatinib/farmacología , Dasatinib/uso terapéutico , Modelos Animales de Enfermedad , Síndrome LEOPARD/tratamiento farmacológico , Síndrome LEOPARD/genética , Síndrome LEOPARD/metabolismo , Ratones , MutaciónRESUMEN
Noonan syndrome with multiple lentigines (NSML) is a rare autosomal dominant disorder that presents with cardio-cutaneous-craniofacial defects. Hypertrophic cardiomyopathy (HCM) represents the major life-threatening presentation in NSML. Mutations in the PTPN11 gene that encodes for the protein tyrosine phosphatase (PTP), SHP2, represents the predominant cause of HCM in NSML. NSML-associated PTPN11 mutations render SHP2 catalytically inactive with an "open" conformation. NSML-associated PTPN11 mutations cause hypertyrosyl phosphorylation of the transmembrane glycoprotein, protein zero-related (PZR), resulting in increased SHP2 binding. Here we show that NSML mice harboring a tyrosyl phosphorylation-defective mutant of PZR (NSML/PZRY242F) that is defective for SHP2 binding fail to develop HCM. Enhanced AKT/S6 kinase signaling in heart lysates of NSML mice was reversed in NSML/PZRY242F mice, demonstrating that PZR/SHP2 interactions promote aberrant AKT/S6 kinase activity in NSML. Enhanced PZR tyrosyl phosphorylation in the hearts of NSML mice was found to drive myocardial fibrosis by engaging an Src/NF-κB pathway, resulting in increased activation of IL-6. Increased expression of IL-6 in the hearts of NSML mice was reversed in NSML/PZRY242F mice, and PZRY242F mutant fibroblasts were defective for IL-6 secretion and STAT3-mediated fibrogenesis. These results demonstrate that NSML-associated PTPN11 mutations that induce PZR hypertyrosyl phosphorylation trigger pathophysiological signaling that promotes HCM and cardiac fibrosis.
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Cardiomiopatía Hipertrófica/patología , Modelos Animales de Enfermedad , Péptidos y Proteínas de Señalización Intracelular/fisiología , Síndrome LEOPARD/complicaciones , Mutación , Fosfoproteínas/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Tirosina/metabolismo , Animales , Cardiomiopatía Hipertrófica/etiología , Cardiomiopatía Hipertrófica/metabolismo , Femenino , Síndrome LEOPARD/genética , Síndrome LEOPARD/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , FosforilaciónRESUMEN
Angiotensinogen (AGT) is the precursor of one of the most potent vasoconstrictors, peptide angiotensin II. Genome-wide association studies have shown that two A/G polymorphisms (rs2493134 and rs2004776), located at +507 and +1164 in intron I of the human AGT (hAGT) gene, are associated with hypertension. Polymorphisms of the AGT gene result in two main haplotypes. Hap-I contains the variants -217A, -6A, +507G, and +1164A and is pro-hypertensive, whereas Hap-II contains the variants -217G, -6G, +507A, and +1164G and does not affect blood pressure. The nucleotide sequence of intron I of the hAGT gene containing the +1164A variant has a stronger homology with the hepatocyte nuclear factor 3 (HNF3)-binding site than +1164G. Here we found that an oligonucleotide containing +1164A binds HNF3ß more strongly than +1164G and that Hap-I-containing reporter gene constructs have increased basal and HNF3- and glucocorticoid-induced promoter activity in transiently transfected liver and kidney cells. Using a knock-in approach at the hypoxanthine-guanine phosphoribosyltransferase locus, we generated a transgenic mouse model containing the human renin (hREN) gene and either Hap-I or Hap-II. We show that transgenic animals containing Hap-I have increased blood pressure compared with those containing Hap-II. Moreover, the transcription factors glucocorticoid receptor, CCAAT enhancer-binding protein ß, and HNF3ß bound more strongly to chromatin obtained from the liver of transgenic animals containing Hap-I than to liver chromatin from Hap-II-containing animals. These findings suggest that, unlike Hap-II variants, Hap-I variants of the hAGT gene have increased transcription rates, resulting in elevated blood pressure.
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Angiotensinógeno/metabolismo , Presión Sanguínea , Factor Nuclear 3-beta del Hepatocito/metabolismo , Angiotensinógeno/sangre , Angiotensinógeno/genética , Animales , Sitios de Unión , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Cromatina/metabolismo , Células Hep G2 , Humanos , Intrones , Desequilibrio de Ligamiento , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Polimorfismo de Nucleótido Simple , Unión Proteica , ARN Mensajero/metabolismo , Receptores de Glucocorticoides/metabolismo , Renina/genéticaRESUMEN
BACKGROUND: The contribution of single nucleotide polymorphisms in transcriptional regulation of the human angiotensin receptor type I (hAT1R) gene in age-related chronic pathologies such as hypertension and associated renal disorders is not well known. The hAT1R gene has single nucleotide polymorphisms in its promoter that forms 2 haplotypes (Hap), Hap-I and Hap-II. Hap-I of AT1R gene is associated with hypertension in Caucasians. We have hypothesized here that age will alter the transcriptional environment of the cell and will regulate the expression of hAT1R gene in a haplotype-dependent manner. This could likely make subjects with Hap-I increasingly susceptible to age-associated, AT1R-mediated complications. METHOD: We generated transgenic (TG) mice with Hap-I and Hap-II. Adults (10-12 weeks) and aged (20-24 months) TG male mice containing either Hap-I or Hap-II were divided into 4 groups to study (i) the age-associated and haplotype-specific transcriptional regulation of hAT1R gene and (ii) their physiological relevance. RESULTS: In aged animals, TG mice with Hap-I show increased expression of hAT1R and higher blood pressure (BP); suppression of antioxidant defenses (hemoxygenase, superoxide dismutase) and antiaging molecules (ATRAP, Klotho, Sirt3); increased expression of pro-inflammatory markers (IL-6, TNFα, CRP, NOX1); and increased insulin resistance. In vivo ChIP assay shows stronger binding of transcription factor USF2 to the chromatin of Hap-I mice. CONCLUSION: Our results suggest that in aged animals, as compared with Hap-II, the TG mice with Hap-I overexpress hAT1R gene due to the stronger transcriptional activity, thus resulting in an increase in their BP and associated renal disorders.