RESUMO
The inhibitory-kappaB kinases (IKKs) IKKα and IKKß play central roles in regulating the non-canonical and canonical NF-κB signalling pathways. Whilst the proteins that transduce the signals of each pathway have been extensively characterised, the clear dissection of the functional roles of IKKα-mediated non-canonical NF-κB signalling versus IKKß-driven canonical signalling remains to be fully elucidated. Progress has relied upon complementary molecular and pharmacological tools; however, the lack of highly potent and selective IKKα inhibitors has limited advances. Herein, we report the development of an aminoindazole-pyrrolo[2,3-b]pyridine scaffold into a novel series of IKKα inhibitors. We demonstrate high potency and selectivity against IKKα over IKKß in vitro and explain the structure-activity relationships using structure-based molecular modelling. We show selective target engagement with IKKα in the non-canonical NF-κB pathway for both U2OS osteosarcoma and PC-3M prostate cancer cells by employing isoform-related pharmacodynamic markers from both pathways. Two compounds (SU1261 [IKKα Ki = 10 nM; IKKß Ki = 680 nM] and SU1349 [IKKα Ki = 16 nM; IKKß Ki = 3352 nM]) represent the first selective and potent pharmacological tools that can be used to interrogate the different signalling functions of IKKα and IKKß in cells. Our understanding of the regulatory role of IKKα in various inflammatory-based conditions will be advanced using these pharmacological agents.
Assuntos
Desenho de Fármacos , Quinase I-kappa B , NF-kappa B , Inibidores de Proteínas Quinases , Transdução de Sinais , Quinase I-kappa B/metabolismo , Quinase I-kappa B/antagonistas & inibidores , Humanos , NF-kappa B/metabolismo , NF-kappa B/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Linhagem Celular Tumoral , Piridinas/farmacologia , Piridinas/química , Piridinas/síntese química , Indazóis/farmacologia , Indazóis/química , Indazóis/síntese química , Modelos MolecularesRESUMO
Mutations in the lamin A/C gene (LMNA) encoding the nuclear intermediate filament proteins lamins A and C cause a group of tissue-selective diseases, the most common of which is dilated cardiomyopathy (herein referred to as LMNA cardiomyopathy) with variable skeletal muscle involvement. We previously showed that cardiomyocyte-specific overexpression of dual specificity protein phosphatase 4 (DUSP4) is involved in the pathogenesis of LMNA cardiomyopathy. However, how mutations in LMNA activate Dusp4 expression and whether it is necessary for the development of LMNA cardiomyopathy are currently unknown. We now show that female LmnaH222P/H222P mice, a model for LMNA cardiomyopathy, have increased Dusp4 expression and hyperactivation of extracellular signal-regulated kinase (ERK) 1/2 with delayed kinetics relative to male mice, consistent with the sex-dependent delay in the onset and progression of disease. Mechanistically, we show that the H222P amino acid substitution in lamin A enhances its binding to ERK1/2 and increases sequestration at the nuclear envelope. Finally, we show that genetic deletion of Dusp4 has beneficial effects on heart function and prolongs survival in LmnaH222P/H222P mice. These results further establish Dusp4 as a key contributor to the pathogenesis of LMNA cardiomyopathy and a potential target for drug therapy.
Assuntos
Cardiomiopatias/genética , Lamina Tipo A/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteínas Tirosina Fosfatases/genética , Substituição de Aminoácidos/genética , Animais , Cardiomiopatias/fisiopatologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Regulação da Expressão Gênica , Humanos , Lamina Tipo A/economia , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , MutaçãoRESUMO
BACKGROUND: As the survival of castration-resistant prostate cancer (CRPC) remains poor, and the nuclear factor-κB (NF-κB) pathways play key roles in prostate cancer (PC) progression, several studies have focused on inhibiting the NF-κB pathway through generating inhibitory κB kinase subunit α (IKKα) small molecule inhibitors. However, the identification of prognostic markers able to discriminate which patients could benefit from IKKα inhibitors is urgently required. The present study investigated the prognostic value of IKKα, IKKα phosphorylated at serine 180 (p-IKKα S180) and threonine 23 (p-IKKα T23), and their relationship with the androgen receptor (AR) and Ki67 proliferation index to predict patient outcome. METHODS: A cohort of 115 patients with hormone-naïve PC (HNPC) and CRPC specimens available were used to assess tumor cell expression of proteins within both the cytoplasm and the nucleus by immunohistochemistry. The expression levels were dichotomized (low vs high) to determine the associations between IKKα, AR, Ki67, and patients'Isurvival. In addition, an analysis was performed to assess potential IKKα associations with clinicopathological and inflammatory features, and potential IKKα correlations with other cancer pathways essential for CRPC growth. RESULTS: High levels of cytoplasmic IKKα were associated with a higher cancer-specific survival in HNPC patients with low AR expression (hazards ratio [HR], 0.33; 95% confidence interval [CI] log-rank, 0.11-0.98; P = .04). Furthermore, nuclear IKKα (HR, 2.60; 95% CI, 1.27-5.33; P = .01) and cytoplasmic p-IKKα S180 (HR, 2.10; 95% CI, 1.17-3.76; P = .01) were associated with a lower time to death from recurrence in patients with CRPC. In addition, high IKKα expression was associated with high levels of T-cells (CD3+ P = .01 and CD8+ P = .03) in HNPC; however, under castration conditions, high IKKα expression was associated with high levels of CD68+ macrophages (P = .04), higher Gleason score (P = .01) and more prostate-specific antigen concentration (P = .03). Finally, we identified crosstalk between IKKα and members of the canonical NF-κB pathway in the nucleus of HNPC. Otherwise, IKKα phosphorylated by noncanonical NF-κB and Akt pathways correlated with members of the canonical NF-κB pathway in CRPC. CONCLUSION: The present study reports that patients with CRPC expressing high levels of nuclear IKKα or cytoplasmic p-IKKα S180, which associated with a lower time to death from recurrence, may benefit from IKKα inhibitors.
Assuntos
Quinase I-kappa B/metabolismo , Neoplasias de Próstata Resistentes à Castração/enzimologia , Neoplasias da Próstata/enzimologia , Idoso , Biomarcadores Tumorais/metabolismo , Núcleo Celular/enzimologia , Estudos de Coortes , Citoplasma/enzimologia , Humanos , Quinase I-kappa B/imunologia , Imunidade Inata , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Masculino , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Prognóstico , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/imunologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais , Taxa de SobrevidaRESUMO
Mitogen-activated protein kinases (MAPKs) regulate normal brain functioning, and their dysfunction is implicated in a number of brain disorders. Thus, there is great interest in understanding the signalling systems that control MAPK functioning. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in foetal development, the immune system, cancer and synaptic plasticity and memory. In the present study, we performed an unbiased investigation using MKP-2-/- mice to assess whether MKP-2 plays a global role in modulating brain function. Local cerebral glucose utilization is significantly increased in the ventral tegmental area (VTA) of MKP-2-/- mice, with connectivity analysis revealing alterations in VTA functional connectivity, including a significant reduction in connectivity to the nucleus accumbens and hippocampus. In addition, spontaneous excitatory postsynaptic current frequency, but not amplitude, onto putative dopamine neurons in the VTA is increased in MKP-2-/- mice, which indicates that increased excitatory drive may account for the increased VTA glucose utilization. Consistent with modified VTA function and connectivity, in behavioural tests MKP-2-/- mice exhibited increased sucrose preference and impaired amphetamine-induced hyperlocomotion. Overall, these data reveal that MKP-2 plays a role in modulating VTA function and that its dysfunction may contribute to brain disorders in which altered reward processing is present.
Assuntos
Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Proteínas Tirosina Fosfatases/genética , Área Tegmentar Ventral , Anfetamina , Animais , Deleção de Genes , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Fosfatase 1 , Recompensa , Área Tegmentar Ventral/metabolismoRESUMO
Protease-activated receptor-2 (PAR2) has been extensively studied since its discovery in the mid-1990. Despite the advances in understanding PAR2 pharmacology, it has taken almost 25 years for the first inhibitor to reach clinical trials, and so far, no PAR2 antagonist has been approved for human use. Research has employed classical approaches to develop a wide array of PAR2 agonists and antagonists, consisting of peptides, peptoids and antibodies to name a few, with a surge in patent applications over this period. Recent breakthroughs in PAR2 structure determination has provided a unique insight into proposed PAR2 ligand binding sites. Publication of the first crystal structures of PAR2 resolved in complex with two novel non-peptide small molecule antagonists (AZ8838 and AZ3451) revealed two distinct binding pockets, originally presumed to be allosteric sites, with a PAR2 antibody (Fab3949) used to block tethered ligand engagement with the peptide-binding domain of the receptor. Further studies have proposed orthosteric site occupancy for AZ8838 as a competitive antagonist. One company has taken the first PAR2 antibody (MEDI0618) into phase I clinical trial (NCT04198558). While this first-in-human trial is at the early stages of the assessment of safety, other research into the structural characterisation of PAR2 is still ongoing in an attempt to identify new ways to target receptor activity. This review will focus on the development of novel PAR2 modulators developed to date, with an emphasis placed upon the advances made in the pharmacological targeting of PAR2 activity as a strategy to limit chronic inflammatory disease.
Assuntos
Desenho de Fármacos , Receptor PAR-2/metabolismo , Sítio Alostérico , Animais , Anticorpos/química , Química Farmacêutica/métodos , Ensaios Clínicos como Assunto , Humanos , Inflamação , Concentração Inibidora 50 , Ligantes , Segurança do Paciente , Peptídeos/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Receptor PAR-2/antagonistas & inibidoresRESUMO
BACKGROUND: Mitogen-activated protein kinase phosphatase-2 (MKP-2) is a type 1 nuclear dual specific phosphatase (DUSP-4). It plays an important role in macrophage inflammatory responses through the negative regulation of Mitogen activated protein kinase (MAPK) signalling. However, information on the effect of MKP-2 on other aspect of macrophage function is limited. METHODS: We investigated the impact of MKP-2 in the regulation of several genes that are involved in function while using comparative whole genome microarray analysis in macrophages from MKP-2 wild type (wt) and knock out (ko) mice. RESULTS: Our data showed that the lack of MKP-2 caused a significant down-regulation of colony-stimulating factor-2 (Csf2) and monocyte to macrophage-associated differentiation (Mmd) genes, suggesting a role of MKP-2 in macrophage development. When treated with macrophage colony stimulating factor (M-CSF), Mmd and Csf2 mRNA levels increased but significantly reduced in ko cells in comparison to wt counterparts. This effect of MKP-2 deletion on macrophage function was also observed by cell counting and DNA measurements. On the signalling level, M-CSF stimulation induced extracellular signal-regulated kinases (ERK) phosphorylation, which was significantly enhanced in the absence of MKP-2. Pharmacological inhibition of ERK reduced both Csf2 and Mmd genes in both wild type and ko cultures, which suggested that enhanced ERK activation in ko cultures may not explain effects on gene expression. Interestingly other functional markers were also shown to be reduced in ko macrophages in comparison to wt mice; the expression of CD115, which is a receptor for M-CSF, and CD34, a stem/progenitor cell marker, suggesting global regulation of gene expression by MKP-2. CONCLUSIONS: Transcriptome profiling reveals that MKP-2 regulates macrophage development showing candidate targets from monocyte-to-macrophage differentiation and macrophage proliferation. However, it is unclear whether effects upon ERK signalling are able to explain the effects of DUSP-4 deletion on macrophage function.
Assuntos
Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Macrófagos/metabolismo , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Deleção de Sequência , Transdução de Sinais , Animais , Biomarcadores , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Imunofenotipagem , Lipopolissacarídeos/imunologia , Sistema de Sinalização das MAP Quinases , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Análise em MicrossériesRESUMO
Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2(-/-) mice), we show that long-term potentiation is impaired in MKP-2(-/-) mice compared with MKP-2(+/+) controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2(-/-) mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2(-/-) mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling.
Assuntos
Deleção de Genes , Hipocampo/metabolismo , Memória/fisiologia , Plasticidade Neuronal/fisiologia , Proteínas Tirosina Fosfatases/deficiência , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Proteínas Tirosina Fosfatases/genéticaRESUMO
OBJECTIVE: Proteinase-activated receptor 2 (PAR2) deficiency protects against cartilage degradation in experimental osteoarthritis (OA). The wider impact of this pathway upon OA-associated pathologies such as osteophyte formation and pain is unknown. Herein, we investigated early temporal bone and cartilage changes in experimental OA in order to further elucidate the role of PAR2 in OA pathogenesis. METHODS: OA was induced in wild-type (WT) and PAR2-deficient (PAR2-/-) mice by destabilisation of the medial meniscus (DMM). Inflammation, cartilage degradation and bone changes were monitored using histology and microCT. In gene rescue experiments, PAR2-/- mice were intra-articularly injected with human PAR2 (hPAR2)-expressing adenovirus. Dynamic weight bearing was used as a surrogate of OA-related pain. RESULTS: Osteophytes formed within 7â days post-DMM in WT mice but osteosclerosis was only evident from 14â days post induction. Importantly, PAR2 was expressed in the proliferative/hypertrophic chondrocytes present within osteophytes. In PAR2-/- mice, osteophytes developed significantly less frequently but, when present, were smaller and of greater density; no osteosclerosis was observed in these mice up to day 28. The pattern of weight bearing was altered in PAR2-/- mice, suggesting reduced pain perception. The expression of hPAR2 in PAR2-/- mice recapitulated osteophyte formation and cartilage damage similar to that observed in WT mice. However, osteosclerosis was absent, consistent with lack of hPAR2 expression in subchondral bone. CONCLUSIONS: This study clearly demonstrates PAR2 plays a critical role, via chondrocytes, in osteophyte development and subchondral bone changes, which occur prior to PAR2-mediated cartilage damage. The latter likely occurs independently of OA-related bone changes.
Assuntos
Artrite Experimental/patologia , Osso e Ossos/patologia , Cartilagem Articular/patologia , Osteoartrite/patologia , Receptor PAR-2/metabolismo , Animais , Artralgia/etiologia , Artralgia/patologia , Artrite Experimental/etiologia , Condrócitos/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Osteoartrite/etiologia , Osteócitos/metabolismoRESUMO
Since the identification of the proteinase-activated receptor (PAR) family as mediators of serine protease activity in the 1990s, there has been tremendous progress in the elucidation of their pathophysiological roles. The development of drugs that target PARs has been the focus of many laboratories for the potential treatment of thrombosis, cancer and other inflammatory diseases. Understanding the mechanisms of PAR activation and G protein signalling pathways evoked in response to the growing list of endogenous proteases has yielded great insight into receptor regulation at the molecular level. This has led to the development of new selective modulators of PAR activity, particularly PAR1. The mixed success of targeting PARs has been best exemplified in the context of inhibiting PAR1 as a new antiplatelet therapy. The development of the competitive PAR1 antagonist, vorapaxar (Zontivity), has clearly shown the value in targeting PAR1 in acute coronary syndrome (ACS); however the severity of associated bleeding with this drug has limited its use in the clinic. Due to the efficacy of thrombin acting via PAR1, strategies to selectively inhibit specific PAR1-mediated G protein signalling pathways or to target the second thrombin platelet receptor, PAR4, are being devised. The rationale behind these alternative approaches is to bias downstream thrombin activity via PARs to allow for inhibition of pro-thrombotic pathways but maintain other pathways that may preserve haemostatic balance and improve bleeding profiles for widespread clinical use. This review summarizes the structural determinants that regulate PARs and the modulators of PAR activity developed to date.
Assuntos
Inibidores da Agregação Plaquetária/farmacologia , Receptores Ativados por Proteinase/efeitos dos fármacos , Humanos , Hidrólise , Lactonas/farmacologia , Lactonas/uso terapêutico , Ligantes , Inibidores da Agregação Plaquetária/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Receptores Ativados por Proteinase/metabolismo , Transdução de Sinais , Trombose/tratamento farmacológicoRESUMO
The dual specific phosphatase, MAP kinase phosphatase-2 (MKP-2) has recently been demonstrated to negatively regulate macrophage arginase-1 expression, while at the same time to positively regulate iNOS expression. Consequently, MKP-2 is likely to play a significant role in the host interplay with intracellular pathogens. Here we demonstrate that MKP-2(-/-) mice on the C57BL/6 background have enhanced susceptibility compared with wild-type counterparts following infection with type-2 strains of Toxoplasma gondii as measured by increased parasite multiplication during acute infection, increased mortality from day 12 post-infection onwards and increased parasite burdens in the brain, day 30 post-infection. MKP-2(-/-) mice did not, however, demonstrate defective type-1 responses compared with MKP-2(+/+) mice following infection although they did display significantly reduced serum nitrite levels and enhanced tissue arginase-1 expression. Early resistance to T. gondii in MKP-2(+/+), but not MKP-2(-/-), mice was nitric oxide (NO) dependent as infected MKP-2(+/+), but not MKP-2(-/-) mice succumbed within 10 days post-infection with increased parasite burdens following treatment with the iNOS inhibitor L-NAME. Conversely, treatment of infected MKP-2(-/-) but not MKP-2(+/+) mice with nor-NOHA increased parasite burdens indicating a protective role for arginase-1 in MKP-2(-/-) mice. In vitro studies using tachyzoite-infected bone marrow derived macrophages and selective inhibition of arginase-1 and iNOS activities confirmed that both iNOS and arginase-1 contributed to inhibiting parasite replication. However, the effects of arginase-1 were transient and ultimately the role of iNOS was paramount in facilitating long-term inhibition of parasite multiplication within macrophages.
Assuntos
Arginase/metabolismo , Macrófagos/parasitologia , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Toxoplasma/fisiologia , Toxoplasmose/parasitologia , Animais , Arginase/antagonistas & inibidores , Arginase/genética , Western Blotting , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Toxoplasmose/metabolismoRESUMO
Increased adult cardiac fibroblast proliferation results in an increased collagen deposition responsible for the fibrosis accompanying pathological remodelling of the heart. The mechanisms regulating cardiac fibroblast proliferation remain poorly understood. Using a minimally invasive transverse aortic banding (MTAB) mouse model of cardiac hypertrophy, we have assessed fibrosis and cardiac fibroblast proliferation. We have investigated whether calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) regulates proliferation in fibroblasts isolated from normal and hypertrophied hearts. It is known that CaMKIIδ plays a central role in cardiac myocyte contractility, but nothing is known of its role in adult cardiac fibroblast function. The MTAB model used here produces extensive hypertrophy and fibrosis. CaMKIIδ protein expression and activity is upregulated in MTAB hearts and, specifically, in cardiac fibroblasts isolated from hypertrophied hearts. In response to angiotensin II, cardiac fibroblasts isolated from MTAB hearts show increased proliferation rates. Inhibition of CaMKII with autocamtide inhibitory peptide inhibits proliferation in cells isolated from both sham and MTAB hearts, with a significantly greater effect evident in MTAB cells. These results are the first to show selective upregulation of CaMKIIδ in adult cardiac fibroblasts following cardiac hypertrophy and to assign a previously unrecognised role to CaMKII in regulating adult cardiac fibroblast function in normal and diseased hearts.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomegalia/fisiopatologia , Fibroblastos/fisiologia , Angiotensina II/farmacologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/biossíntese , Proliferação de Células/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismoRESUMO
Neovascular age-related macular degeneration (nAMD) remains a major cause of visual impairment and puts considerable burden on patients and health care systems. L-DOPA-treated Parkinson Disease (PD) patients have been shown to be partially protected from nAMD, but the mechanism remains unknown. Using murine models, combining 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD and laser-induced nAMD, standard PD treatment of L-DOPA/DOPA-decarboxylase inhibitor, or specific dopamine receptor inhibitors, we here demonstrate that L-DOPA treatment-induced increase of dopamine mediated dopamine receptor D2 (DRD2) signaling inhibits choroidal neovascularization independently of MPTP-associated nigrostriatal pathway lesion. Analyzing a retrospective cohort of more than two hundred thousand nAMD patients receiving anti-VEGF treatment from the French nationwide insurance database, we show that DRD2-agonist treated (PD) patients have a significantly delayed age of onset for nAMD (81.4 (±7.0) vs 79.4 (±8.1) years old, respectively, p<0.0001) and reduced need for anti-VEGF therapies (-0.6 injections per 100 mg/day daily dose of DRD2 agonists the second year of treatment), similar to the L-DOPA treatment. While providing a mechanistic explanation for an intriguing epidemiological observation, our findings suggest that systemic DRD2 agonists might constitute an adjuvant therapy to delay and reduce the need for anti-VEGF therapy in nAMD patients.
RESUMO
Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit ß-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR â A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of ß-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.
Assuntos
Membrana Celular/metabolismo , Multimerização Proteica/fisiologia , Receptor PAR-2/metabolismo , Receptores de Trombina/metabolismo , Transdução de Sinais/fisiologia , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Membrana Celular/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Sinais Direcionadores de Proteínas/fisiologia , Transporte Proteico/fisiologia , Receptor PAR-2/genética , Receptores de Trombina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
In this study, we examined the activation of non-canonical nuclear factor Kappa B (NFκB) signalling in U2OS cells, a cellular metastatic bone cancer model. Whilst Lymphotoxin α1ß2 (LTα1ß2) stimulated the expected slow, delayed, sustained activation of serine 866/870 p100 phosphorylation and increased cellular expression of p52 NFκB, we found that canonical agonists, Interleukin-1ß (IL-1ß) and also Tumour necrosis factor-α (TNFα) generated a rapid transient increase in pp100, which was maximal by 15-30 min. This rapid phosphorylation was also observed in other cells types, such as DU145 and HCAECs suggesting the phenomenon is universal. IKKα deletion using CRISPR/Cas9 revealed an IKKα-dependent mechanism for serine 866/870 and additionally serine 872 p100 phosphorylation for both IL-1ß and LTα1ß2. In contrast, knockdown of IKKß using siRNA or pharmacological inhibition of IKKß activity was without effect on p100 phosphorylation. Pre-incubation of cells with the NFκB inducing-kinase (NIK) inhibitor, CW15337, had no effect on IL-1ß induced phosphorylation of p100 however, the response to LTα1ß2 was virtually abolished. Surprisingly IL-1ß also stimulated p52 nuclear translocation as early as 60 min, this response and the concomitant p65 translocation was partially reduced by IKKα deletion. Furthermore, p52 nuclear translocation was unaffected by CW15337. In contrast, the response to LTα1ß2 was essentially abolished by both IKKα deletion and CW15337. Taken together, these finding reveal novel forms of NFκB non-canonical signalling stimulated by ligands that activate the canonical NFκB pathway strongly such as IL-1ß.
Assuntos
Quinase I-kappa B , Interleucina-1beta , NF-kappa B , Transdução de Sinais , Humanos , Linhagem Celular Tumoral , Quinase I-kappa B/metabolismo , Interleucina-1beta/metabolismo , NF-kappa B/metabolismoRESUMO
Mitogen-activated protein kinase phosphatase-2 (MKP-2) is a type 1 nuclear dual specific phosphatase (DUSP) implicated in a number of cancers. We examined the role of MKP-2 in the regulation of MAP kinase phosphorylation, cell proliferation, and survival responses in mouse embryonic fibroblasts (MEFs) derived from a novel MKP-2 (DUSP-4) deletion mouse. We show that serum and PDGF induced ERK-dependent MKP-2 expression in wild type MEFs but not in MKP-2(-/-) MEFs. PDGF stimulation of sustained ERK phosphorylation was enhanced in MKP-2(-/-) MEFs, whereas anisomycin-induced JNK was only marginally increased. However, marked effects upon cell growth parameters were observed. Cellular proliferation rates were significantly reduced in MKP-2(-/-) MEFs and associated with a significant increase in cell doubling time. Infection with adenoviral MKP-2 reversed the decrease in proliferation. Cell cycle analysis revealed a block in G(2)/M phase transition associated with cyclin B accumulation and enhanced cdc2 phosphorylation. MEFs from MKP-2(-/-) mice also showed enhanced apoptosis when stimulated with anisomycin correlated with increased caspase-3 cleavage and γH2AX phosphorylation. Increased apoptosis was reversed by adenoviral MKP-2 infection and correlated with selective inhibition of JNK signaling. Collectively, these data demonstrate for the first time a critical non-redundant role for MKP-2 in regulating cell cycle progression and apoptosis.
Assuntos
Divisão Celular/fisiologia , Embrião de Mamíferos/enzimologia , Fibroblastos/enzimologia , Fase G2/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Caspase 3/genética , Caspase 3/metabolismo , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Ciclina B/genética , Ciclina B/metabolismo , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fase G2/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Histonas/genética , Histonas/metabolismo , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Tirosina Fosfatases/genéticaRESUMO
OBJECTIVE: Proteinase-activated receptor-2 (PAR(2)) has been implicated in inflammatory articular pathology. Using the collagen-induced arthritis model (CIA) the authors have explored the capacity of PAR(2) to regulate adaptive immune pathways that could promote autoimmune mediated articular damage. METHODS: Using PAR(2) gene deletion and other approaches to inhibit or prevent PAR(2) activation, the development and progression of CIA were assessed via clinical and histological scores together with ex vivo immune analyses. RESULTS: The progression of CIA, assessed by arthritic score and histological assessment of joint damage, was significantly (p<0.0001) abrogated in PAR(2) deficient mice or in wild-type mice administered either a PAR(2) antagonist (ENMD-1068) or a PAR(2) neutralising antibody (SAM11). Lymph node derived cell suspensions from PAR(2) deficient mice were found to produce significantly less interleukin (IL)-17 and IFNγ in ex vivo recall collagen stimulation assays compared with wild-type littermates. In addition, substantial inhibition of TNFα, IL-6, IL-1ß and IL-12 along with GM-CSF and MIP-1α was observed. However, spleen and lymph node histology did not differ between groups nor was any difference detected in draining lymph node cell subsets. Anticollagen antibody titres were significantly lower in PAR(2) deficient mice. CONCLUSION: These data support an important role for PAR(2) in the pathogenesis of CIA and suggest an immunomodulatory role for this receptor in an adaptive model of inflammatory arthritis. PAR(2) antagonism may offer future potential for the management of inflammatory arthritides in which a proteinase rich environment prevails.
Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Fatores Imunológicos/metabolismo , Receptor PAR-2/metabolismo , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Citometria de Fluxo , Fatores Imunológicos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Receptor PAR-2/imunologiaRESUMO
In this study we generated a novel dual specific phosphatase 4 (DUSP4) deletion mouse using a targeted deletion strategy in order to examine the role of MAP kinase phosphatase-2 (MKP-2) in immune responses. Lipopolysaccharide (LPS) induced a rapid, time and concentration-dependent increase in MKP-2 protein expression in bone marrow-derived macrophages from MKP-2(+/+) but not from MKP-2(-/-) mice. LPS-induced JNK and p38 MAP kinase phosphorylation was significantly increased and prolonged in MKP-2(-/-) macrophages whilst ERK phosphorylation was unaffected. MKP-2 deletion also potentiated LPS-stimulated induction of the inflammatory cytokines, IL-6, IL-12p40, TNF-α, and also COX-2 derived PGE(2) production. However surprisingly, in MKP-2(-/-) macrophages, there was a marked reduction in LPS or IFNγ-induced iNOS and nitric oxide release and enhanced basal expression of arginase-1, suggesting that MKP-2 may have an additional regulatory function significant in pathogen-mediated immunity. Indeed, following infection with the intracellular parasite Leishmania mexicana, MKP-2(-/-) mice displayed increased lesion size and parasite burden, and a significantly modified Th1/Th2 bias compared with wild-type counterparts. However, there was no intrinsic defect in MKP-2(-/-) T cell function as measured by anti-CD3 induced IFN-γ production. Rather, MKP-2(-/-) bone marrow-derived macrophages were found to be inherently more susceptible to infection with Leishmania mexicana, an effect reversed following treatment with the arginase inhibitor nor-NOHA. These findings show for the first time a role for MKP-2 in vivo and demonstrate that MKP-2 may be essential in orchestrating protection against intracellular infection at the level of the macrophage.
Assuntos
Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Leishmania mexicana/patogenicidade , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/prevenção & controle , Macrófagos/imunologia , Proteínas Tirosina Fosfatases/fisiologia , Animais , Arginase/metabolismo , Western Blotting , Medula Óssea/imunologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Células Cultivadas , Feminino , Leishmaniose Cutânea/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Óxido Nítrico/metabolismo , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The MKPs (mitogen-activated protein kinase phosphatases) are a family of at least ten DUSPs (dual-specificity phosphatases) which function to terminate the activity of the MAPKs (mitogen-activated protein kinases). Several members have already been demonstrated to have distinct roles in immune function, cancer, fetal development and metabolic disorders. One DUSP of renewed interest is the inducible nuclear phosphatase MKP-2, which dephosphorylates both ERK (extracellular-signal-regulated kinase) and JNK (c-Jun N-terminal kinase) in vitro. Recently, the understanding of MKP-2 function has been advanced due to the development of mouse knockout models, which has resulted in the discovery of novel roles for MKP-2 in the regulation of sepsis, infection and cell-cycle progression that are distinct from those of other DUSPs. However, many functions for MKP-2 still await to be characterized.
Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Animais , Núcleo Celular/enzimologia , Citoplasma/enzimologia , Humanos , Isoenzimas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Dax-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital region on X-chromosome gene 1) blocks 17ß-estradiol biosynthesis and its knockdown would be expected to increase 17ß-estradiol production. We hypothesized that knockdown of Dax-1 in a conditionally immortalized neural stem cell (NSC) line, MHP36, is a useful approach to increase 17ß-estradiol production. Short hairpin (sh) RNA targeted to Dax-1 in NSCs, namely MHP36-Dax1KD cells, resulted in the degradation of Dax-1 RNA and attenuation of Dax-1 protein expression. In vitro, MHP36-Dax1KD cells exhibited overexpression of aromatase and increased 17ß-estradiol secretion compared to MHP36 cells. As 17ß-estradiol has been shown to promote the efficacy of cell therapy, we interrogated the application of 17ß-estradiol-enriched NSCs in a relevant in vivo disease model. We hypothesized that MHP36-Dax1KD cells will enhance functional recovery after transplantation in a stroke model. C57BL/6 male adult mice underwent ischemia/reperfusion by left middle cerebral artery occlusion for 45 min using an intraluminal thread. Two days later male mice randomly received vehicle, MHP36 cells, MHP36-Dax1KD cells, and MHP36 cells suspended in 17ß-estradiol (100 nm) or 17ß-estradiol alone (100 nm) with serial behavioral testing over 28 days followed by post-mortem histology and blinded analysis. Recovery of sensorimotor function was accelerated and enhanced, and lesion volume was reduced by MHP36-Dax1KD transplants. Regarding mechanisms, immunofluorescence indicated increased synaptic plasticity and neuronal differentiation after MHP36-Dax1KD transplants. In conclusion, knockdown of Dax-1 is a useful target to increase 17ß-estradiol biosynthesis in NSCs and improves functional recovery after stroke in vivo, possibly mediated through neuroprotection and improved synaptic plasticity. Therefore, targeting 17ß-estradiol biosynthesis in stem cells may be a promising therapeutic strategy for enhancing the efficacy of stem cell-based therapies for stroke.
RESUMO
OBJECTIVE: Increasing evidence implicates serine proteinases in pathologic tissue turnover. The aim of this study was to assess the role of the transmembrane serine proteinase matriptase in cartilage destruction in osteoarthritis (OA). METHODS: Serine proteinase gene expression in femoral head cartilage obtained from either patients with hip OA or patients with fracture to the neck of the femur (NOF) was assessed using a low-density array. The effect of matriptase on collagen breakdown was determined in cartilage degradation models, while the effect on matrix metalloproteinase (MMP) expression was analyzed by real-time polymerase chain reaction. ProMMP processing was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis/N-terminal sequencing, while its ability to activate proteinase-activated receptor 2 (PAR-2) was determined using a synovial perfusion assay in mice. RESULTS: Matriptase gene expression was significantly elevated in OA cartilage compared with NOF cartilage, and matriptase was immunolocalized to OA chondrocytes. We showed that matriptase activated proMMP-1 and processed proMMP-3 to its fully active form. Exogenous matriptase significantly enhanced cytokine-stimulated cartilage collagenolysis, while matriptase alone caused significant collagenolysis from OA cartilage, which was metalloproteinase-dependent. Matriptase also induced MMP-1, MMP-3, and MMP-13 gene expression. Synovial perfusion data confirmed that matriptase activates PAR-2, and we demonstrated that matriptase-dependent enhancement of collagenolysis from OA cartilage is blocked by PAR-2 inhibition. CONCLUSION: Elevated matriptase expression in OA and the ability of matriptase to activate selective proMMPs as well as induce collagenase expression make this serine proteinase a key initiator and inducer of cartilage destruction in OA. We propose that the indirect effects of matriptase are mediated by PAR-2, and a more detailed understanding of these mechanisms may highlight important new therapeutic targets for OA treatment.