ABSTRACT
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.
Subject(s)
Drug Design , I-kappa B Kinase , NF-kappa B , Protein Kinase Inhibitors , Signal Transduction , I-kappa B Kinase/metabolism , I-kappa B Kinase/antagonists & inhibitors , Humans , NF-kappa B/metabolism , NF-kappa B/antagonists & inhibitors , Signal Transduction/drug effects , Structure-Activity Relationship , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Cell Line, Tumor , Pyridines/pharmacology , Pyridines/chemistry , Pyridines/chemical synthesis , Indazoles/pharmacology , Indazoles/chemistry , Indazoles/chemical synthesis , Models, MolecularABSTRACT
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.
ABSTRACT
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.
Subject(s)
I-kappa B Kinase , Interleukin-1beta , NF-kappa B , Signal Transduction , Humans , Cell Line, Tumor , I-kappa B Kinase/metabolism , Interleukin-1beta/metabolism , NF-kappa B/metabolismABSTRACT
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.
ABSTRACT
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.
Subject(s)
I-kappa B Kinase/metabolism , Prostatic Neoplasms, Castration-Resistant/enzymology , Prostatic Neoplasms/enzymology , Aged , Biomarkers, Tumor/metabolism , Cell Nucleus/enzymology , Cohort Studies , Cytoplasm/enzymology , Humans , I-kappa B Kinase/immunology , Immunity, Innate , Immunohistochemistry , Ki-67 Antigen/metabolism , Male , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Prognosis , Prostatic Neoplasms/immunology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Prostatic Neoplasms, Castration-Resistant/immunology , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Androgen/metabolism , Signal Transduction , Survival RateABSTRACT
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.
Subject(s)
Drug Design , Receptor, PAR-2/metabolism , Allosteric Site , Animals , Antibodies/chemistry , Chemistry, Pharmaceutical/methods , Clinical Trials as Topic , Humans , Inflammation , Inhibitory Concentration 50 , Ligands , Patient Safety , Peptides/chemistry , Protein Binding , Protein Conformation , Protein Domains , Receptor, PAR-2/antagonists & inhibitorsABSTRACT
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.
Subject(s)
Mitogen-Activated Protein Kinase Phosphatases/genetics , Protein Tyrosine Phosphatases/genetics , Ventral Tegmental Area , Amphetamine , Animals , Gene Deletion , Mice , Mice, Knockout , Mitogen-Activated Protein Kinases/metabolism , Protein Phosphatase 1 , Reward , Ventral Tegmental Area/metabolismABSTRACT
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.
Subject(s)
Gene Expression Regulation , Genome-Wide Association Study , Macrophages/metabolism , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolism , Sequence Deletion , Signal Transduction , Animals , Biomarkers , Computational Biology/methods , Gene Expression Profiling , Immunophenotyping , Lipopolysaccharides/immunology , MAP Kinase Signaling System , Macrophage Activation/genetics , Macrophage Activation/immunology , Macrophages/immunology , Mice , Mice, Knockout , Microarray AnalysisABSTRACT
This study was conducted to examine the cytotoxic effects of Nubein6.8 isolated from the venom of the Egyptian Spitting Cobra Naja nubiae on melanoma (A375) and ovarian carcinoma cell lines and to reveal its mode of action. The size of Nubein6.8 (6801.8â¯Da) and its N-terminal sequence are similar to cytotoxins purified from the venom of other spitting cobras. Nubein6.8 showed a high significant cytotoxic effect on A375â¯cell line and moderate effect on A2780. A clonogenic assay showed that Nubein6.8 has a significant long-term potency on A375â¯cell survival when compared to A2780. The molecular intracellular signaling pathways of Nubein6.8 have been investigated using Western blotting analysis, flow cytometry, and microscale protein labeling. This data revealed that Nubein6.8 has DNA damaging effects and the ability to activate apoptosis in both tumor cell lines. Cellular uptake recordings revealed that the labeled-Nubein6.8 was intracellularly present in A375â¯cells while A2780 displayed resistance against it. SEM examination showed that Nubein6.8 was found to have high accessibility to malignant melanoma cells. The apoptotic effect of Nubein6.8 was confirmed by TEM examination that revealed many evident characteristics for Nubein6.8 apoptotic efficacy on A375â¯cell sections. Also, TEM reflected many resistant characteristics that faced Nubein6.8 acquisition through ovarian carcinoma cell sections. Accordingly, the snake venom peptide of Nubein6.8 is a promising template for developing potential cytotoxic agents targeting human melanoma and ovarian carcinoma.
Subject(s)
Antineoplastic Agents/chemistry , Elapid Venoms/chemistry , Elapid Venoms/toxicity , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line , Cell Line, Tumor , Cell Survival/drug effects , Colony-Forming Units Assay , DNA Damage/drug effects , Elapid Venoms/metabolism , Humans , Naja , Neoplasms/drug therapy , Peptides/chemistry , Peptides/metabolism , Peptides/toxicity , Signal TransductionABSTRACT
Objectives: We have previously shown mitogen-activated protein kinase phosphatase 2 (MKP-2) to be a key regulator of proinflammatory cytokines in macrophages. In the study presented here, we investigated the role of MKP-2 in inflammatory arthritis with a particular focus on neutrophils. Methods: To achieve this, we subjected MKP-2 deficient and wild type mice to collagen antibody induced arthritis, an innate model of arthritis, and determined disease pathology. To further our investigation, we depleted neutrophils in a prophylactic and therapeutic fashion. Last, we used chemotaxis assays to analyse the impact of MKP-2 deletion on neutrophil migration. Results: MKP-2-/- mice showed a significant increase in disease pathology linked to elevated levels of proarthritic cytokines and chemokines TNF-α, IL-6 and MCP-1 in comparison to wild type controls. This phenotype is prevented or abolished after administration of neutrophil depleting antibody prior or after onset of disease, respectively. While MCP-1 levels were not affected, neutrophil depletion diminished TNF-α and reduced IL-6, thus linking these cytokines to neutrophils. In vivo imaging showed that MKP-2-/- mice had an increased influx of neutrophils into affected joints, which was higher and potentially prolonged than in wild type animals. Furthermore, using chemotaxis assays we revealed that MKP-2 deficient neutrophils migrate faster towards a Leukotriene B4 gradient. This process correlated with a reduced phosphorylation of ERK in MKP-2-/- neutrophils. Conclusions: This is the first study to show a protective role for MKP-2 in inflammatory arthritis.
Subject(s)
Arthritis/etiology , Protein Tyrosine Phosphatases/genetics , Animals , Arthritis/metabolism , Arthritis/pathology , Arthritis, Experimental , Cytokines/metabolism , Disease Models, Animal , Disease Susceptibility , Genetic Association Studies , Inflammation Mediators/metabolism , Male , Mice , Mice, Knockout , Optical Imaging/methods , Protein Tyrosine Phosphatases/metabolismABSTRACT
Recurrent broad-scale heterozygous deletions are frequently observed in human cancer. Here we tested the hypothesis that compound haploinsufficiency of neighboring genes at chromosome 8p promotes tumorigenesis. By targeting the mouse orthologs of human DOK2 and DUSP4 genes, which were co-deleted in approximately half of human lung adenocarcinomas, we found that compound-heterozygous deletion of Dok2 and Dusp4 in mice resulted in lung tumorigenesis with short latency and high incidence, and that their co-deletion synergistically activated MAPK signaling and promoted cell proliferation. Conversely, restoration of DOK2 and DUSP4 in lung cancer cells suppressed MAPK activation and cell proliferation. Importantly, in contrast to downregulation of DOK2 or DUSP4 alone, concomitant downregulation of DOK2 and DUSP4 was associated with poor survival in human lung adenocarcinoma. Therefore, our findings lend in vivo experimental support to the notion that compound haploinsufficiency, due to broad-scale chromosome deletions, constitutes a driving force in tumorigenesis.
Subject(s)
Adaptor Proteins, Signal Transducing , Cell Transformation, Neoplastic , Haploinsufficiency , Lung Neoplasms , Neoplasm Proteins , Phosphoproteins , Protein Tyrosine Phosphatases , Adaptor Proteins, Signal Transducing/biosynthesis , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Line, Tumor , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Down-Regulation , Female , Gene Expression Regulation, Neoplastic , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , MAP Kinase Signaling System/genetics , Male , Mice , Mice, Knockout , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Phosphoproteins/biosynthesis , Phosphoproteins/genetics , Protein Tyrosine Phosphatases/biosynthesis , Protein Tyrosine Phosphatases/geneticsABSTRACT
Previous research from our laboratory has demonstrated a novel phenomenon whereby GPCRs play a role in inhibiting cytokine-mediated c-Jun N-terminal kinase (JNK) signalling. So far this novel phenomenon seems to have been vastly overlooked, with little research in the area. Therefore, in this study we explored this further; by assessing the potential of P2YRs to mediate inhibition of cytokine-mediated JNK signalling and related functional outcomes in human endothelial cells. We utilised primary endothelial cells, and employed the use of endogenous activators of P2YRs and well characterised pharmacological inhibitors, to assess signalling parameters mediated by P2YRs, Interleukin-1ß (IL-1ß), TNFα and JNK. Activation of P2YRs with adenosine tri-phosphate (ATP) resulted in a time- and concentration-dependent inhibition of IL-1ß-mediated phosphorylation of JNK and associated kinase activity. The effect was specific for cytokine-mediated JNK signalling, as ATP was without effect on JNK induced by other non-specific activators (e.g. sorbitol, anisomycin), nor effective against other MAPK pathways such as p38 and the canonical NFκB cascade. Pharmacological studies demonstrated a role for the P2Y11 receptor in mediating this effect, but not the P2Y1 nor the adenosine receptors (A1, A2A, A2B & A3). The novel Gαq/11 inhibitor YM254890 and a protein kinase A (PKA) inhibitor H89 both partially reversed ATP-mediated inhibition of IL-1ß-stimulated JNK indicating involvement of both Gαq/11 and Gαs mediated pathways. ATP also partially reversed IL-1ß-mediated induction of cyclooxygenase-2 (COX-2) and E-selectin. Collectively, these studies indicate the potential for activation of purinergic receptors to protect the endothelium from inflammatory driven JNK activation and may be a new target for inflammatory disease therapy.
Subject(s)
Coronary Vessels , Endothelial Cells/metabolism , MAP Kinase Signaling System/drug effects , Purinergic P2Y Receptor Antagonists/pharmacology , Receptors, Purinergic P2/metabolism , Adenosine Triphosphate/metabolism , Cells, Cultured , Coronary Vessels/cytology , Coronary Vessels/metabolism , Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells , Humans , Interleukin-1beta/metabolism , Receptors, Purinergic P1/metabolism , Receptors, Purinergic P2Y1/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
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.
Subject(s)
Cardiomyopathies/genetics , Lamin Type A/genetics , Mitogen-Activated Protein Kinase 3/genetics , Protein Tyrosine Phosphatases/genetics , Amino Acid Substitution/genetics , Animals , Cardiomyopathies/physiopathology , Disease Models, Animal , Disease Progression , Female , Gene Expression Regulation , Humans , Lamin Type A/economics , MAP Kinase Signaling System/genetics , Male , Mice , MutationABSTRACT
As a target, the JNK pathway has been implicated in roles including cell death, proliferation, and inflammation in variety of contexts which span cardiovascular disease, neurodegenerative pathologies, and cancer. JNK1 and JNK2 have recently been demonstrated to function independently, highlighting a new parameter in the study of the JNK pathway. In order for JNK1 and JNK2-specific roles to be defined, better tools need to be employed. Previous studies have relied upon the broad spectrum JNK inhibitor, SP600125, to characterize the role of JNK signaling in a number of cell lines, including the breast cancer cell line MCF-7. In line with previous literature, our study has demonstrated that SP600125 treatment inhibited c-Jun and JNK phosphorylation and MCF-7 proliferation. However, in addition to targeting JNK1, JNK2, and JNK3, SP600125 has been previously demonstrated to suppress the activity of a number of other serine/threonine kinases, making SP600125 an inadequate tool for JNK isoform-specific roles to be determined. In this study, lentiviral shRNA was employed to selectively knockdown JNK1, JNK2, and JNK1/2 in MCF-7 cells. Using this approach, JNK phosphorylation was fully inhibited following stable knockdown of respective JNK isoforms. Interestingly, despite suppression of JNK phosphorylation, MCF-7 cell proliferation, cell cycle progression, or cell death remained unaffected. These findings raise the question of whether JNK phosphorylation really is pivotal in MCF-7 cell growth and death or if suppression of these events is a result of one of the many off-targets cited for SP600125.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Janus Kinase Inhibitors/pharmacology , MAP Kinase Signaling System/drug effects , Anthracenes/pharmacology , Anthracenes/therapeutic use , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Apoptosis/genetics , Breast Neoplasms/pathology , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Survival/drug effects , Cell Survival/genetics , Female , Gene Knockdown Techniques , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/metabolism , JNK Mitogen-Activated Protein Kinases/genetics , JNK Mitogen-Activated Protein Kinases/metabolism , Janus Kinase Inhibitors/therapeutic use , MAP Kinase Signaling System/genetics , MCF-7 Cells , Molecular Targeted Therapy/methods , Phosphorylation/genetics , RNA, Small Interfering/metabolismABSTRACT
Increasing evidence implicates serine proteinases in the proteolytic cascades leading to the pathological destruction of extracellular matrices such as cartilage in osteoarthritis (OA). We have previously demonstrated that the type II transmembrane serine proteinase (TTSP) matriptase acts as a novel initiator of cartilage destruction via the induction and activation of matrix metalloproteinases (MMPs). Hepsin is another TTSP expressed in OA cartilage such that we hypothesized this proteinase may also contribute to matrix turnover. Herein, we demonstrate that addition of hepsin to OA cartilage in explant culture induced significant collagen and aggrecan release and activated proMMP-1 and proMMP-3. Furthermore, hepsin directly cleaved the aggrecan core protein at a novel cleavage site within the interglobular domain. Hepsin expression correlated with synovitis as well as tumour necrosis factor α expression, and was induced in cartilage by a pro-inflammatory stimulus. However, a major difference compared to matriptase was that hepsin demonstrated markedly reduced capacity to activate proteinase-activated receptor-2. Overall, our data suggest that hepsin, like matriptase, induces potent destruction of the extracellular matrix whilst displaying distinct efficiencies for the cleavage of specific substrates.
Subject(s)
Extracellular Matrix/metabolism , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 3/metabolism , Serine Endopeptidases/metabolism , Aggrecans/metabolism , Animals , Cartilage, Articular/cytology , Cartilage, Articular/metabolism , Cattle , Cells, Cultured , Collagen/metabolism , Humans , Matrix Metalloproteinase 1/chemistry , Matrix Metalloproteinase 3/chemistry , Mice , Mice, Inbred C57BL , Molecular Dynamics Simulation , Osteoarthritis/metabolism , Osteoarthritis/pathology , Protein Structure, Tertiary , Receptor, PAR-2/metabolism , Serine Endopeptidases/chemistry , Synovitis/pathology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
IKKß plays a central role in the canonical NF-kB pathway, which has been extensively characterized. The role of IKKα in the noncanonical NF-kB pathway, and indeed in the canonical pathway as a complex with IKKß, is less well understood. One major reason for this is the absence of chemical tools designed as selective inhibitors for IKKα over IKKß. Herein, we report for the first time a series of novel, potent, and selective inhibitors of IKKα. We demonstrate effective target engagement and selectivity with IKKα in U2OS cells through inhibition of IKKα-driven p100 phosphorylation in the noncanonical NF-kB pathway without affecting IKKß-dependent IKappa-Bα loss in the canonical pathway. These compounds represent the first chemical tools that can be used to further characterize the role of IKKα in cellular signaling, to dissect this from IKKß and to validate it in its own right as a target in inflammatory diseases.
Subject(s)
I-kappa B Kinase/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Animals , Biomarkers, Pharmacological/metabolism , Cell Line, Tumor , Drug Design , Humans , I-kappa B Kinase/chemistry , Mice , Molecular Docking Simulation , Molecular Dynamics Simulation , NF-kappa B p52 Subunit/metabolism , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/chemistry , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Pyrroles/chemical synthesis , Pyrroles/chemistry , Signal Transduction/drug effects , Structure-Activity RelationshipABSTRACT
Mitogen-activated protein kinase phosphatases (MKPs) play key roles in inflammation and immune mediated diseases. Here we investigated the mechanisms by which MKP-2 modulates central nervous system (CNS) inflammation in experimental autoimmune encephalomyelitis (EAE). Our results show that MKP-2 mRNA levels in the spinal cord and lymphoid organs of EAE mice were increased compared with naive controls, indicating an important role for MKP-2 in EAE development. Indeed, MKP-2-/- mice developed reduced EAE severity, associated with diminished CNS immune cell infiltration, decreased proinflammatory cytokine production and reduced frequency of CD4+ and CD8+ T cells in spleens and lymph nodes. In addition, MKP-2-/- CD11c+ dendritic cells (DCs) had reduced expression of MHC-II and CD40 compared with MKP-2+/+ mice. Subsequent experiments revealed that CD4+ T cells from naïve MKP-2-/- mice had decreased cell proliferation and IL-2 and IL-17 production relative to wild type controls. Furthermore, co-culture experiments showed that bone marrow derived DCs of MKP-2-/- mice had impaired capability in antigen presentation and T cell activation. While MKP-2 also modulates macrophage activation, our study suggests that MKP-2 is essential to the pathogenic response of EAE, and it acts mainly via regulating the important antigen presenting DC function and T cell activation.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Lymphocyte Activation , Protein Tyrosine Phosphatases/deficiency , Animals , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/pathology , Dendritic Cells/pathology , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/pathology , Interleukin-17/genetics , Interleukin-17/immunology , Interleukin-2/genetics , Interleukin-2/immunology , Mice , Mice, Knockout , Protein Tyrosine Phosphatases/immunologyABSTRACT
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.
Subject(s)
Platelet Aggregation Inhibitors/pharmacology , Receptors, Proteinase-Activated/drug effects , Humans , Hydrolysis , Lactones/pharmacology , Lactones/therapeutic use , Ligands , Platelet Aggregation Inhibitors/therapeutic use , Pyridines/pharmacology , Pyridines/therapeutic use , Receptors, Proteinase-Activated/metabolism , Signal Transduction , Thrombosis/drug therapyABSTRACT
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.
Subject(s)
Gene Deletion , Hippocampus/metabolism , Memory/physiology , Neuronal Plasticity/physiology , Protein Tyrosine Phosphatases/deficiency , Animals , Excitatory Postsynaptic Potentials/physiology , Male , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Organ Culture Techniques , Protein Tyrosine Phosphatases/geneticsABSTRACT
Protease-activated receptors (PARs) are a novel family of G-protein coupled receptors (GPCRs) whose activation requires the cleavage of the N-terminus by a serine protease. However, recent evidence reveals that alternative routes of activation also occur, that PARs signal via multiple pathways and that pathway activation is activator- dependent. Given our increased understanding of PAR function both under physiological and pathophysiological conditions, one aspect that has remained constant is the link between PAR2 and inflammation. PAR2 is expressed in immune cells of both the innate and adaptive immune system and has been shown to play a role in several peripheral inflammatory conditions. PAR2 is similarly expressed on astrocytes and microglia within the CNS and its activation is either protective or detrimental to CNS function depending on the conditions or disease state investigated. With a clear similarity between the function of PAR2 on both immune cells and CNS glial cells, here we have reviewed their roles in both these systems. We suggest that the recent development of novel PAR2 modulators, including those that show biased signalling, will further increase our understanding of PAR2 function and the development of potential therapeutics for CNS disorders in which inflammation is proposed to play a role.