RESUMEN
In this issue honoring the contributions of Greg Lemke, the Earp and Graham lab teams discuss several threads in the discovery, action, signaling, and translational/clinical potential of MERTK, originally called c-mer, a member of the TYRO3, AXL, and MERTK (TAM) family of receptor tyrosine kinases. The 30-year history of the TAM RTK family began slowly as all three members were orphan RTKs without known ligands and/or functions when discovered by three distinct alternate molecular cloning strategies in the pre-genome sequencing era. The pace of understanding their physiologic and pathophysiologic roles has accelerated over the last decade. The activation of ligands bridging externalized phosphatidylserine (PtdSer) has placed these RTKs in a myriad of processes including neurodevelopment, cancer, and autoimmunity. The field is ripe for further advancement and this article hopefully sets the stage for further understanding and therapeutic intervention. Our review will focus on progress made through the collaborations of the Earp and Graham labs over the past 30 years.
Asunto(s)
Neoplasias , Tirosina Quinasa c-Mer , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/antagonistas & inhibidores , Tirosina Quinasa c-Mer/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Animales , Terapia Molecular Dirigida , Transducción de Señal/efectos de los fármacos , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidoresRESUMEN
BACKGROUND: Atherosclerosis is a chronic inflammatory disease causing a fatal plaque rupture, and its key aspect is a failure to resolve inflammation. We hypothesize that macrophage-targeted near-infrared fluorescence emitting photoactivation could simultaneously assess macrophage/lipid-rich plaques in vivo and facilitate inflammation resolution. METHODS: We fabricated a Dectin-1-targeted photoactivatable theranostic agent through the chemical conjugation of the near-infrared fluorescence-emitting photosensitizer chlorin e6 and the Dectin-1 ligand laminarin (laminarin-chlorin e6 [LAM-Ce6]). Intravascular photoactivation by a customized fiber-based diffuser after administration of LAM-Ce6 effectively reduced inflammation in the targeted plaques of atherosclerotic rabbits in vivo as serially assessed by dual-modal optical coherence tomography-near-infrared fluorescence structural-molecular catheter imaging after 4 weeks. RESULTS: The number of apoptotic macrophages peaked at 1 day after laser irradiation and then resolved until 4 weeks. Autophagy was strongly augmented 1 hour after the light therapy, with the formation of autophagolysosomes. LAM-Ce6 photoactivation increased the terminal deoxynucleotidyl transferase dUTP (deoxyuridine triphosphate) nick end labeling/RAM11 (rabbit monocyte/macrophage antibody)- and MerTK (c-Mer tyrosine kinase)-positive cells in the plaques, suggesting enhanced efferocytosis. In line with inflammation resolution, photoactivation reduced the plaque burden through fibrotic replacement via the TGF (transforming growth factor)-ß/CTGF (connective tissue growth factor) pathway. CONCLUSIONS: Optical coherence tomography-near-infrared fluorescence imaging-guided macrophage Dectin-1-targetable photoactivation could induce the transition of macrophage/lipid-rich plaques into collagen-rich lesions through autophagy-mediated inflammation resolution and TGF-ß-dependent fibrotic replacement. This novel strategy offers a new opportunity for the catheter-based theranostic strategy.
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Clorofilidas , Imagen Multimodal , Fármacos Fotosensibilizantes , Placa Aterosclerótica , Porfirinas , Tomografía de Coherencia Óptica , Animales , Placa Aterosclerótica/diagnóstico por imagen , Conejos , Imagen Multimodal/métodos , Tomografía de Coherencia Óptica/métodos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Macrófagos/metabolismo , Nanomedicina Teranóstica/métodos , Ratones , Masculino , Autofagia , Tirosina Quinasa c-Mer/metabolismo , ApoptosisRESUMEN
Ewing sarcoma is a pediatric bone and soft tissue tumor treated with chemotherapy, radiation, and surgery. Despite intensive multimodality therapy, ~50% patients eventually relapse and die of the disease due to chemoresistance. Here, using phospho-profiling, we find Ewing sarcoma cells treated with chemotherapeutic agents activate TAM (TYRO3, AXL, MERTK) kinases to augment Akt and ERK signaling facilitating chemoresistance. Mechanistically, chemotherapy-induced JAK1-SQ phosphorylation releases JAK1 pseudokinase domain inhibition allowing for JAK1 activation. This alternative JAK1 activation mechanism leads to STAT6 nuclear translocation triggering transcription and secretion of the TAM kinase ligand GAS6 with autocrine/paracrine consequences. Importantly, pharmacological inhibition of either JAK1 by filgotinib or TAM kinases by UNC2025 sensitizes Ewing sarcoma to chemotherapy in vitro and in vivo. Excitingly, the TAM kinase inhibitor MRX-2843 currently in human clinical trials to treat AML and advanced solid tumors, enhances chemotherapy efficacy to further suppress Ewing sarcoma tumor growth in vivo. Our findings reveal an Ewing sarcoma chemoresistance mechanism with an immediate translational value.
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Péptidos y Proteínas de Señalización Intercelular , Janus Quinasa 1 , Proteínas Tirosina Quinasas Receptoras , Sarcoma de Ewing , Transducción de Señal , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Sarcoma de Ewing/genética , Humanos , Janus Quinasa 1/metabolismo , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/genética , Línea Celular Tumoral , Animales , Transducción de Señal/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Ratones , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Tirosina Quinasa del Receptor Axl , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Neoplasias Óseas/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Fosforilación/efectos de los fármacos , Femenino , Factor de Transcripción STAT6RESUMEN
A series of bifunctional compounds have been discovered for their dual functionality as MER/AXL inhibitors and immune modulators. The furanopyrimidine scaffold, renowned for its suitability in kinase inhibitor discovery, offers at least three distinct pharmacophore access points. Insights from molecular modeling studies guided hit-to-lead optimization, which revealed that the 1,3-diketone side chain hybridized with furanopyrimidine scaffold that respectively combined amino-type substituent and 1H-pyrazol-4-yl substituent on the top and bottom of the aryl regions to produce 22 and 33, exhibiting potent antitumor activities in various syngeneic and xenograft models. More importantly, 33 demonstrated remarkable immune-modulating activity by upregulating the expression of total T-cells, cytotoxic CD8+ T-cells, and helper CD4+ T-cells in the spleen. These findings underscored the bifunctional capabilities of 33 (BPR5K230) with excellent oral bioavailability (F = 54.6%), inhibiting both MER and AXL while modulating the tumor microenvironment and highlighting its diverse applicability for further studies to advance its therapeutic potential.
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Antineoplásicos , Tirosina Quinasa del Receptor Axl , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas , Proteínas Tirosina Quinasas Receptoras , Microambiente Tumoral , Tirosina Quinasa c-Mer , Animales , Microambiente Tumoral/efectos de los fármacos , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Antineoplásicos/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Tirosina Quinasa c-Mer/antagonistas & inhibidores , Tirosina Quinasa c-Mer/metabolismo , Ratones , Línea Celular Tumoral , Relación Estructura-Actividad , Descubrimiento de Drogas , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Femenino , Ensayos Antitumor por Modelo de Xenoinjerto , Ratones Endogámicos BALB C , Proliferación Celular/efectos de los fármacosRESUMEN
Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an outstanding contribution from the team of Prof. Greg Lemke. MerTK activity is implicated in a wide variety of functions involving the elimination of apoptotic cells and has recently been linked to cancers, auto-immune diseases, and atherosclerosis/stroke. In the retina, MerTK is required for the circadian phagocytosis of oxidized photoreceptor outer segments by the retinal-pigment epithelial cells, a function crucial for the long-term maintenance of vision. We previously showed that MerTK ligands carry the opposite role in vitro, with Gas6 inhibiting the internalization of photoreceptor outer segments while Protein S acts conversely. Using site-directed mutagenesis and ligand-stimulated phagocytosis assays on transfected cells, we presently demonstrate, for the first time, that Gas6 and Protein S recognize different amino acids on MerTK Ig-like domains. In addition, MerTK's function in retinal-pigment epithelial cells is rhythmic and might thus rely on the respective stoichiometry of both ligands at different times of the day. Accordingly, we show that ligand bioavailability varies during the circadian cycle using RT-qPCR and immunoblots on retinal and retinal-pigment epithelial samples from control and beta5 integrin knockout mice where retinal phagocytosis is arrhythmic. Taken together, our results suggest that Gas6 and Protein S might both contribute to refine the acute regulation of MerTK in time for the daily phagocytic peak.
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Péptidos y Proteínas de Señalización Intercelular , Fagocitosis , Proteína S , Tirosina Quinasa c-Mer , Animales , Ratones , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/genética , Ritmo Circadiano/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Ligandos , Proteína S/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citologíaRESUMEN
Oligodendrocyte death is common in aging and neurodegenerative disease. In these conditions, dying oligodendrocytes must be efficiently removed to allow remyelination and to prevent a feedforward degenerative cascade. Removal of this cellular debris is thought to primarily be carried out by resident microglia. To investigate the cellular dynamics underlying how microglia do this, we use a single-cell cortical demyelination model combined with longitudinal intravital imaging of dual-labeled transgenic mice. Following phagocytosis, single microglia clear the targeted oligodendrocyte and its myelin sheaths in one day via a precise, rapid, and stereotyped sequence. Deletion of the fractalkine receptor, CX3CR1, delays the microglial phagocytosis of the cell soma but has no effect on clearance of myelin sheaths. Unexpectedly, deletion of the phosphatidylserine receptor, MERTK, has no effect on oligodendrocyte or myelin sheath clearance. Thus, separate molecular signals are used to detect, engage, and clear distinct sub-compartments of dying oligodendrocytes to maintain tissue homeostasis.
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Receptor 1 de Quimiocinas CX3C , Microglía , Oligodendroglía , Fagocitosis , Tirosina Quinasa c-Mer , Animales , Oligodendroglía/metabolismo , Microglía/metabolismo , Receptor 1 de Quimiocinas CX3C/metabolismo , Receptor 1 de Quimiocinas CX3C/genética , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/genética , Ratones , Vaina de Mielina/metabolismo , Ratones Transgénicos , Ratones Endogámicos C57BL , Muerte CelularRESUMEN
Liebold et al. recently revealed how the identity of dying cells drives distinct changes to the macrophages which engulf and clear them, a process known as efferocytosis. During infection with the helminth Schistosoma mansoni, liver macrophages recapitulate these phenotypes, mediated by Axl/MerTK receptors and regulating egg burdens.
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Macrófagos , Fagocitosis , Schistosoma mansoni , Animales , Macrófagos/inmunología , Macrófagos/parasitología , Schistosoma mansoni/fisiología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/parasitología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/inmunología , Humanos , Hígado/parasitología , Hígado/inmunología , Tirosina Quinasa del Receptor Axl , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/fisiología , EferocitosisRESUMEN
Introduction: TAM receptor-mediated efferocytosis plays an important function in immune regulation and may contribute to antigen tolerance in the lungs, a site with continuous cellular turnover and generation of apoptotic cells. Some studies have identified failures in efferocytosis as a common driver of inflammation and tissue destruction in lung diseases. Our study is the first to characterize the in vivo function of the TAM receptors, Axl and MerTk, in the innate immune cell compartment, cytokine and chemokine production, as well as the alveolar macrophage (AM) phenotype in different settings in the airways and lung parenchyma. Methods: We employed MerTk and Axl defective mice to induce acute silicosis by a single exposure to crystalline silica particles (20 mg/50 µL). Although both mRNA levels of Axl and MerTk receptors were constitutively expressed by lung cells and isolated AMs, we found that MerTk was critical for maintaining lung homeostasis, whereas Axl played a role in the regulation of silica-induced inflammation. Our findings imply that MerTk and Axl differently modulated inflammatory tone via AM and neutrophil recruitment, phenotype and function by flow cytometry, and TGF-ß and CXCL1 protein levels, respectively. Finally, Axl expression was upregulated in both MerTk-/- and WT AMs, confirming its importance during inflammation. Conclusion: This study provides strong evidence that MerTk and Axl are specialized to orchestrate apoptotic cell clearance across different circumstances and may have important implications for the understanding of pulmonary inflammatory disorders as well as for the development of new approaches to therapy.
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Tirosina Quinasa del Receptor Axl , Homeostasis , Pulmón , Macrófagos Alveolares , Ratones Noqueados , Proteínas Proto-Oncogénicas , Proteínas Tirosina Quinasas Receptoras , Silicosis , Tirosina Quinasa c-Mer , Animales , Ratones , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Silicosis/metabolismo , Silicosis/inmunología , Silicosis/patología , MasculinoRESUMEN
Introduction: The protein growth arrest-specific 6 (Gas6) and its tyrosine kinase receptors Tyro-3, Axl, and Mer (TAM) are ubiquitous proteins involved in regulating inflammation and apoptotic body clearance. Multiple sclerosis (MS) is the most common inflammatory demyelinating disease of the central nervous system leading to progressive and irreversible disability if not diagnosed and treated promptly. Gas6 and TAM receptors have been associated with neuronal remyelination and stimulation of oligodendrocyte survival. However, few data are available regarding clinical correlation in MS patients. We aimed to evaluate soluble levels of these molecules in the cerebrospinal fluid (CSF) and serum at MS diagnosis and correlate them with short-term disease severity. Methods: In a prospective cohort study, we enrolled 64 patients with a diagnosis of clinical isolated syndrome (CIS), radiological isolated syndrome (RIS) and relapsing-remitting (RR) MS according to the McDonald 2017 Criteria. Before any treatment initiation, we sampled the serum and CSF, and collected clinical data: disease course, presence of gadolinium-enhancing lesions, and expanded disability status score (EDSS). At the last clinical follow-up, we assessed EDSS and calculated MS severity score (MSSS) and age-related MS severity (ARMSS). Gas6 and TAM receptors were determined using an ELISA kit (R&D Systems) and compared to neurofilament (NFLs) levels evaluated with SimplePlex™ fluorescence-based immunoassay. Results: At diagnosis, serum sAxl was higher in patients receiving none or low-efficacy disease-modifying treatments (DMTs) versus patients with high-efficacy DMTs (p = 0.04). Higher CSF Gas6 and serum sAXL were associated with an EDSS <3 at diagnosis (p = 0.04; p = 0.037). Serum Gas6 correlates to a lower MSSS (r2 = -0.32, p = 0.01). Serum and CSF NFLs were confirmed as disability biomarkers in our cohort according to EDSS (p = 0.005; p = 0.002) and MSSS (r2 = 0.27, p = 0.03; r2 = 0.39, p = 0.001). Results were corroborated using multivariate analysis. Conclusions: Our data suggest a protective role of Gas6 and its receptors in patients with MS and suitable severity disease biomarkers.
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Tirosina Quinasa del Receptor Axl , Biomarcadores , Péptidos y Proteínas de Señalización Intercelular , Esclerosis Múltiple , Proteínas Tirosina Quinasas Receptoras , Tirosina Quinasa c-Mer , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Péptidos y Proteínas de Señalización Intercelular/sangre , Péptidos y Proteínas de Señalización Intercelular/líquido cefalorraquídeo , Esclerosis Múltiple/diagnóstico , Esclerosis Múltiple/líquido cefalorraquídeo , Esclerosis Múltiple/sangre , Pronóstico , Estudios Prospectivos , Proteínas Proto-Oncogénicas/sangre , Proteínas Proto-Oncogénicas/líquido cefalorraquídeo , Proteínas Tirosina Quinasas Receptoras/sangre , Proteínas Tirosina Quinasas Receptoras/líquido cefalorraquídeo , Índice de Severidad de la EnfermedadRESUMEN
Triple-negative breast cancer (TNBC) is characterized by the absence of the estrogen receptor, progesterone receptor, and receptor tyrosine kinase HER2 expression. Due to the limited number of FDA-approved targeted therapies for TNBC, there is an ongoing need to understand the molecular underpinnings of TNBC for the development of novel combinatorial treatment strategies. This study evaluated the role of the MerTK receptor tyrosine kinase on proliferation and invasion/metastatic potential in TNBC. Immunohistochemical analysis demonstrated MerTK expression in 58% of patient-derived TNBC xenografts. The stable overexpression of MerTK in human TNBC cell lines induced an increase in proliferation rates, robust in vivo tumor growth, heightened migration/invasion potential, and enhanced lung metastases. NanoString nCounter analysis of MerTK-overexpressing SUM102 cells (SUM102-MerTK) revealed upregulation of several signaling pathways, which ultimately drive cell cycle progression, reduce apoptosis, and enhance cell survival. Proteomic profiling indicated increased endoglin (ENG) production in SUM102-MerTK clones, suggesting that MerTK creates a conducive environment for increased proliferative and metastatic activity via elevated ENG expression. To determine ENG's role in increasing proliferation and/or metastatic potential, we knocked out ENG in a SUM102-MerTK clone with CRISPR technology. Although this ENG knockout clone exhibited similar in vivo growth to the parental SUM102-MerTK clone, lung metastasis numbers were significantly decreased ~4-fold, indicating that MerTK enhances invasion and metastasis through ENG. Our data suggest that MerTK regulates a unique proliferative signature in TNBC, promoting robust tumor growth and increased metastatic potential through ENG upregulation. Targeting MerTK and ENG simultaneously may provide a novel therapeutic approach for TNBC patients.
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Proliferación Celular , Neoplasias de la Mama Triple Negativas , Tirosina Quinasa c-Mer , Humanos , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/genética , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Animales , Femenino , Ratones , Línea Celular Tumoral , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Endoglina/metabolismo , Endoglina/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Metástasis de la Neoplasia , Transducción de Señal , Apoptosis/genéticaRESUMEN
Organ fibrosis is a devastating medical challenge that is collectively responsible for an estimated 45% of all deaths in developed countries and poses a substantial health and economic burden. The process of fibrosis has common characteristics that can occur in various organs, such as the liver, kidney, lung, and skin. Currently, there is a paucity of effective treatments available for fibrosis. Therefore, it is crucial to identify new approaches to find potential therapeutic targets. Genetic studies have shown great promise in advancing the drug development process. Mer tyrosine kinase (MERTK) was recently identified as a crucial regulator of fibrosis that specifically controls the activity of transforming growth factor beta (TGFß). In this brief review, we provide an overview of the potential role of MERTK as a targeted and valuable approach for treating organ fibrosis.
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Fibrosis , Tirosina Quinasa c-Mer , Humanos , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/genética , Animales , Factor de Crecimiento Transformador beta/metabolismo , Terapia Molecular DirigidaRESUMEN
Greg Lemke's laboratory was one of the pioneers of research into the TAM family of receptor tyrosine kinases (RTKs). Not only was Tyro3 cloned in his laboratory, but his group also extensively studied mice knocked out for individual or various combinations of the TAM RTKs Tyro3, Axl, and Mertk. Here we primarily focus on one of the paralogs-MERTK. We provide a historical perspective on rodent models of loss of Mertk function and their association with retinal degeneration and blindness. We describe later studies employing mouse genetics and the generation of newer knockout models that point out incongruencies with the inference that loss of MERTK-dependent phagocytosis is sufficient for severe, early-onset photoreceptor degeneration in mice. This discussion is meant to raise awareness with regards to the limitations of the original Mertk knockout mouse model generated using 129 derived embryonic stem cells and carrying 129 derived alleles and the role of these alleles in modifying Mertk knockout phenotypes or even displaying Mertk-independent phenotypes. We also suggest molecular approaches that can further Greg Lemke's scintillating legacy of dissecting the molecular functions of MERTK-a protein that has been described to function in phagocytosis as well as in the negative regulation of inflammation.
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Ratones Noqueados , Fagocitosis , Tirosina Quinasa c-Mer , Animales , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/genética , Ratones , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Modelos Animales de Enfermedad , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Humanos , Inflamación/genética , Inflamación/metabolismoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: HuoXueTongFu Formula (HXTF) is a traditional Chinese herbal formula that has been used as a supplement and alternative therapy for intraperitoneal adhesion (IA). However, its specific mechanism of action has not been fully understood. AIM OF THE STUDY: In surgery, IA presents an inevitable challenge, significantly impacting patients' physical and mental well-being and increasing the financial burden. Our previous research has confirmed the preventive effects of HXTF on IA formation. However, the precise mechanism of its action still needs to be understood. METHODS: In this study, the IA model was successfully established by using the Ischemic buttons and treated with HXTF for one week with or without Mer Tyrosine Kinase (MerTK) inhibitor. We evaluated the pharmacodynamic effect of HXTF on IA mice. The MerTK/phosphoinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway-associated proteins were detected by Western blotting. Neutrophil extracellular traps (NETs) were detected by immunofluorescence. Macrophage phenotype was assessed by immunohistochemistry and flow cytometry. Inflammatory cytokines were detected by Real Time Quantitative PCR and Western blotting. RESULTS: HXTF reduced inflammatory response and alleviated IA. HXTF significantly enhanced MerTK expression, increased the number of M2c macrophages, and decreased the formation of NETs. In addition, the MerTK/PI3K/AKT pathway was significantly activated by HXTF. However, after using MerTK inhibitors, the role of HXTF in inducing M2c macrophage through activation of the PI3K/AKT pathway was suppressed and there was no inhibitory effect on NETs formation and inflammatory responses, resulting in diminished inhibition of adhesion. CONCLUSION: HXTF may improve IA by activating the MerTK/PI3K/AKT pathway to induce M2c polarization, which removes excess NETs and attenuates the inflammatory response.
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Medicamentos Herbarios Chinos , Macrófagos , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Tirosina Quinasa c-Mer , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Medicamentos Herbarios Chinos/farmacología , Ratones , Tirosina Quinasa c-Mer/metabolismo , Transducción de Señal/efectos de los fármacos , Masculino , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Adherencias Tisulares/prevención & control , Adherencias Tisulares/metabolismo , Trampas Extracelulares/efectos de los fármacos , Trampas Extracelulares/metabolismo , Modelos Animales de EnfermedadRESUMEN
The trigeminal system is key to the pathophysiology of migraine and cluster headache, two primary headache disorders that share many features. Recently, MER proto-oncogene tyrosine kinase (MERTK), a cell surface receptor, was strongly associated with cluster headache through genetic studies. Further, the MERTK ligand galectin-3 has been found to be elevated in serum of migraine patients. In this study, MERTK and MERTK ligands were investigated in key tissue to better understand their potential implication in the pathophysiology of primary headache disorders. Immunohistochemistry was used to map MERTK and galectin-3 expression in rat trigeminal ganglia. RT-qPCR was used to assess MERTK gene expression in blood, and ELISA immunoassays were used for MERTK ligand quantification in serum from study participants with and without cluster headache. MERTK gene expression was elevated in blood samples from study participants with cluster headache compared to controls. In addition, MERTK ligand galectin-3 was found at increased concentration in the serum of study participants with cluster headache, whereas the levels of MERTK ligands growth arrest specific 6 and protein S unaffected. MERTK and galectin-3 were both expressed in rat trigeminal ganglia. Galectin-3 was primarily localized in smaller neurons and to a lesser extent in C-fibres, while MERTK was found in satellite glia cells and in the outer membrane of Schwann cells. Interestingly, a strong MERTK signal was found specifically in the region proximal to the nodes of Ranvier. The overexpression of MERTK and galectin-3 in tissue from study participants with cluster headache, as well as the presence of MERTK in rat peripheral satellite glia cells and Schwann cells in the trigeminal ganglia, further highlights MERTK signalling as an interesting potential future therapeutic target in primary headache.
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Cefalalgia Histamínica , Ganglio del Trigémino , Tirosina Quinasa c-Mer , Animales , Cefalalgia Histamínica/metabolismo , Cefalalgia Histamínica/sangre , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/genética , Ganglio del Trigémino/metabolismo , Humanos , Masculino , Ratas , Femenino , Proto-Oncogenes Mas , Adulto , Persona de Mediana Edad , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Sanguíneas , GalectinasRESUMEN
Chronic lymphocytic leukemia (CLL) is a chronic lymphoproliferative disorder characterized by monoclonal B cell proliferation. Studies carried out in recent years suggest that extracellular vesicles (EVs) may be a potential biomarker in cancer. Tyro3-Axl-Mertk (TAM) Receptor Tyrosine Kinases (RTKs) and Phosphatidylserine (PS) have crucial roles in macrophage-mediated immune response under normal conditions. In the tumor microenvironment, these molecules contribute to immunosuppressive signals and prevent the formation of local and systemic antitumor immune responses. Based on this, we aimed to evaluate the amount of PS and TAM RTK in plasma and on the surface of EVs in CLL patients and healthy volunteers in this study. In this study, 25 CLL (11 F/14 M) patients in the Rai (O-I) stage, newly diagnosed or followed up without treatment, and 15 healthy volunteers (11 F/4 M) as a control group were included. For all samples, PS and TAM RTK levels were examined first in the plasma and then in the EVs obtained from the plasma. We detected a significant decrease in plasma PS, and TAM RTK levels in CLL patients compared to the control. Besides, we determined a significant increase in TAM RTK levels on the EV surface in CLL, except for PS. In conclusion, these receptor levels measured by ELISA in plasma may not be effective for the preliminary detection of CLL. However, especially TAM RTKs on the surface of EVs may be good biomarkers and potential targets for CLL therapies.
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Vesículas Extracelulares , Leucemia Linfocítica Crónica de Células B , Fosfatidilserinas , Proteínas Tirosina Quinasas Receptoras , Humanos , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Femenino , Fosfatidilserinas/metabolismo , Fosfatidilserinas/sangre , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/sangre , Masculino , Persona de Mediana Edad , Anciano , Tirosina Quinasa del Receptor Axl , Proteínas Proto-Oncogénicas/sangre , Proteínas Proto-Oncogénicas/metabolismo , Adulto , Tirosina Quinasa c-Mer/metabolismo , Anciano de 80 o más AñosRESUMEN
Exposure to fine particulate matter (PM2.5) and polycyclic aromatic hydrocarbons (PAHs) is known to be associated with the polarization of pro-inflammatory macrophages and the development of various cardiovascular diseases. The pro-inflammatory polarization of resident cardiac macrophages (cMacs) enhances the cleavage of membrane-bound myeloid-epithelial-reproductive receptor tyrosine kinase (MerTK) and promotes the formation of soluble MerTK (solMER). This process influences the involvement of cMacs in cardiac repair, thus leading to an imbalance in cardiac homeostasis, myocardial injury, and reduced cardiac function. However, the relative impacts of PM2.5 and PAHs on human cMacs have yet to be elucidated. In this study, we aimed to investigate the effects of PM2.5 and PAH exposure on solMER in terms of myocardial injury and left ventricular (LV) systolic function in healthy children. A total of 258 children (aged three to six years) were recruited from Guiyu (an area exposed to e-waste) and Haojiang (a reference area). Mean daily PM2.5 concentration data were collected to calculate the individual chronic daily intake (CDI) of PM2.5. We determined concentrations of solMER and creatine kinase MB (CKMB) in plasma, and hydroxylated PAHs (OH-PAHs) in urine. LV systolic function was evaluated by stroke volume (SV). Higher CDI values and OH-PAH concentrations were detected in the exposed group. Plasma solMER and CKMB were higher in the exposed group and were associated with a reduced SV. Elevated CDI and 1-hydroxynaphthalene (1-OHNa) were associated with a higher solMER. Furthermore, increased solMER concentrations were associated with a lower SV and higher CKMB. CDI and 1-OHNa were positively associated with CKMB and mediated by solMER. In conclusion, exposure to PM2.5 and PAHs may lead to the pro-inflammatory polarization of cMacs and increase the risk of myocardial injury and systolic function impairment in children. Furthermore, the pro-inflammatory polarization of cMacs may mediate cardiotoxicity caused by PM2.5 and PAHs.
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Contaminantes Atmosféricos , Material Particulado , Hidrocarburos Policíclicos Aromáticos , Humanos , Hidrocarburos Policíclicos Aromáticos/toxicidad , Material Particulado/toxicidad , Niño , Masculino , Femenino , Preescolar , Contaminantes Atmosféricos/toxicidad , Tirosina Quinasa c-Mer , Función Ventricular Izquierda/efectos de los fármacos , Exposición a Riesgos Ambientales/estadística & datos numéricos , Macrófagos/efectos de los fármacosRESUMEN
Background: MER proto-oncogene tyrosine kinase (MerTK) is a key receptor for efferocytosis, a process for the clearance of apoptotic cells. MerTK is mainly expressed in macrophages and immature dendritic cells. There are very limited reports focused on MerTK biology in aortic endothelial cells (ECs). It remains unclear for the role of blood flow patterns in regulating MerTK-mediated efferocytosis in aortic ECs. This study was designed to investigate whether endothelial MerTK and EC efferocytosis respond to blood flow patterns during atherosclerosis. Methods: Big data analytics, RNA-seq and proteomics combined with our in vitro and in vivo studies were applied to reveal the potential molecular mechanisms. Partial carotid artery ligation combined with AAV-PCSK9 and high fat diet were used to set up acute atherosclerosis in 4 weeks. Results: Our data showed that MerTK is sensitive to blood flow patterns and is inhibited by disturbed flow and oscillatory shear stress in primary human aortic ECs (HAECs). The RNA-seq data in HAECs incubated with apoptotic cells showed that d-flow promotes pro-inflammatory pathway and senescence pathway. Our in vivo data of proteomics and immunostaining showed that, compared with WT group, MerTK-/- aggravates atherosclerosis in d-flow areas through upregulation of endothelial dysfunction markers (e.g. IL-1ß, NF-κB, TLR4, MAPK signaling, vWF, VCAM-1 and p22phox) and mitochondrial dysfunction. Interestingly, MerTK-/-induces obvious abnormal endothelial thickening accompanied with decreased endothelial efferocytosis, promoting the development of atherosclerosis. Conclusions: Our data suggests that blood flow patterns play an important role in regulating MerTK-mediated efferocytosis in aortic ECs, revealing a new promising therapeutic strategy with EC efferocytosis restoration to against atherosclerosis.
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Aorta , Aterosclerosis , Células Endoteliales , Fagocitosis , Tirosina Quinasa c-Mer , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Humanos , Células Endoteliales/metabolismo , Animales , Aorta/metabolismo , Aorta/patología , Ratones , Apoptosis , Proto-Oncogenes Mas , Masculino , Ratones Endogámicos C57BL , Dieta Alta en Grasa , Células Cultivadas , EferocitosisRESUMEN
Transforming growth factor-ß (TGFß) drives fibrosis and disease progression in a number of chronic disorders, but targeting this ubiquitously expressed cytokine may not yield a viable and safe antifibrotic therapy. Here, we sought to identify alternative ways to inhibit TGFß signaling using human hepatic stellate cells and macrophages from humans and mice in vitro, as well as mouse models of liver, kidney, and lung fibrosis. We identified Mer tyrosine kinase (MERTK) as a TGFß-inducible effector of fibrosis that was up-regulated during fibrosis in multiple organs in three mouse models. We confirmed these findings in liver biopsy samples from patients with metabolic dysfunction-associated fatty liver disease (MAFLD). MERTK also induced TGFß expression and drove TGFß signaling resulting in a positive feedback loop that promoted fibrosis in cultured cells. MERTK regulated both canonical and noncanonical TGFß signaling in both mouse and human cells in vitro. MERTK increased transcription of genes regulating fibrosis by modulating chromatin accessibility and RNA polymerase II activity. In each of the three mouse models, disrupting the fibrosis-promoting signaling loop by reducing MERTK expression reduced organ fibrosis. Pharmacological inhibition of MERTK reduced fibrosis in these mouse models either when initiated immediately after injury or when initiated after fibrosis was established. Together, these data suggest that MERTK plays a role in modulating organ fibrosis and may be a potential target for treating fibrotic diseases.
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Hígado , Proteínas Tirosina Quinasas , Animales , Humanos , Ratones , Tirosina Quinasa c-Mer/metabolismo , Modelos Animales de Enfermedad , Fibrosis , Hígado/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
MERTK and AXL are members of the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases that are aberrantly expressed and have been implicated as therapeutic targets in a wide variety of human tumors. Dual MERTK and AXL inhibition could provide antitumor action mediated by both direct tumor cell killing and modulation of the innate immune response in some tumors such as nonsmall cell lung cancer. We utilized our knowledge of MERTK inhibitors and a structure-based drug design approach to discover a novel class of macrocyclic dual MERTK/AXL inhibitors. The lead compound 43 had low-nanomolar activity against both MERTK and AXL and good selectivity over TYRO3 and FLT3. Its target engagement and selectivity were also confirmed by NanoBRET and cell-based MERTK and AXL phosphorylation assays. Compound 43 had excellent pharmacokinetic properties (large AUC and long half-life) and mediated antitumor activity against lung cancer cell lines, indicating its potential as a therapeutic agent.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa del Receptor Axl , Proteínas Proto-Oncogénicas/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Línea Celular TumoralRESUMEN
Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under physiological conditions. In recent years, Mertk has also been linked to pathophysiological roles in cancer, whereby, in several cancer types, including solid cancers and leukemia/lymphomas. Mertk contributes to oncogenic features of proliferation and cell survival as an oncogenic tyrosine kinase. In addition, Mertk expressed on macrophages, including tumor-associated macrophages, promotes immune evasion in cancer and is suggested to act akin to a myeloid checkpoint inhibitor that skews macrophages towards inhibitory phenotypes that suppress host T-cell anti-tumor immunity. In the present study, to better understand the post-translational regulation mechanisms controlling Mertk expression in monocytes/macrophages, we used a PMA-differentiated THP-1 cell model to interrogate the regulation of Mertk expression and developed a novel Mertk reporter cell line to study the intracellular trafficking of Mertk. We show that PMA treatment potently up-regulates Mertk as well as components of the ectodomain proteolytic processing platform ADAM17, whereas PMA differentially regulates the canonical Mertk ligands Gas6 and Pros1 (Gas6 is down-regulated and Pros1 is up-regulated). Under non-stimulated homeostatic conditions, Mertk in PMA-differentiated THP1 cells shows active constitutive proteolytic cleavage by the sequential activities of ADAM17 and the Presenilin/γ-secretase complex, indicating that Mertk is cleaved homeostatically by the combined sequential action of ADAM17 and γ-secretase, after which the cleaved intracellular fragment of Mertk is degraded in a proteasome-dependent mechanism. Using chimeric Flag-Mertk-EGFP-Myc reporter receptors, we confirm that inhibitors of γ-secretase and MG132, which inhibits the 26S proteasome, stabilize the intracellular fragment of Mertk without evidence of nuclear translocation. Finally, the treatment of cells with active γ-carboxylated Gas6, but not inactive Warfarin-treated non-γ-carboxylated Gas6, regulates a distinct proteolytic itinerary-involved receptor clearance and lysosomal proteolysis. Together, these results indicate that pleotropic and complex proteolytic activities regulate Mertk ectodomain cleavage as a homeostatic negative regulatory event to safeguard against the overactivation of Mertk.