RESUMO
The establishment of axon-dendrite polarity is fundamental for radial migration of neurons, cortical patterning, and formation of neuronal circuits. Here, we show that the receptor tyrosine kinases, Ltk and Alk, are required for proper neuronal polarization. In isolated primary mouse embryonic neurons, the loss of Ltk and/or Alk causes a multiple axon phenotype. In mouse embryos and newborn pups, the absence of Ltk and Alk delays neuronal migration and subsequent cortical patterning. In adult cortices, neurons with aberrant neuronal projections are evident and axon tracts in the corpus callosum are disrupted. Mechanistically, we show that the loss of Alk and Ltk increases the cell-surface expression and activity of the insulin-like growth factor 1 receptor (Igf-1r), which activates downstream PI3 kinase signaling to drive the excess axon phenotype. Our data reveal Ltk and Alk as new regulators of neuronal polarity and migration whose disruption results in behavioral abnormalities.
Assuntos
Neurônios , Receptores Proteína Tirosina Quinases , Animais , Camundongos , Axônios/metabolismo , Polaridade Celular , Neurogênese/genética , Neurônios/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de SinaisRESUMO
AIMS: Kinase fusion-positive soft tissue tumors represent an emerging, molecularly defined group of mesenchymal tumors with a wide morphologic spectrum and diverse activating kinases. Here, we present two cases of soft tissue tumors with novel LTK fusions. METHODS AND RESULTS: Both cases presented as acral skin nodules (big toe and middle finger) in pediatric patients (17-year-old girl and 2-year-old boy). The tumors measured 2 and 3 cm in greatest dimension. Histologically, both cases exhibited bland-looking spindle cells infiltrating adipose tissue and accompanied by collagenous stroma. One case additionally displayed perivascular hyalinization and band-like stromal collagen. Both cases exhibited focal S100 staining, and one case had patchy coexpression of CD34. Targeted RNA-seq revealed the presence of novel in-frame MYH9::LTK and MYH10::LTK fusions, resulting in upregulation of LTK expression. Of interest, DNA methylation-based unsupervised clustering analysis in one case showed that the tumor clustered with dermatofibrosarcoma protuberans (DFSP). One tumor was excised with amputation with no local recurrence or distant metastasis at 18-month follow-up. The other case was initially marginally excised with local recurrence after one year, followed by wide local excision, with no evidence of disease at 10 years of follow-up. CONCLUSIONS: This is the first reported case series of soft tissue tumors harboring LTK fusion, expanding the molecular landscape of soft tissue tumors driven by activating kinase fusions. Furthermore, studies involving a larger number of cases and integrated genomic analyses will be warranted to fully elucidate the pathogenesis and classification of these tumors.
Assuntos
Neoplasias de Tecido Conjuntivo e de Tecidos Moles , Proteínas de Fusão Oncogênica , Neoplasias Cutâneas , Neoplasias de Tecidos Moles , Adolescente , Criança , Feminino , Humanos , Masculino , Antígenos CD34/metabolismo , Biomarcadores Tumorais/genética , Neoplasias de Tecido Conjuntivo e de Tecidos Moles/genética , Neoplasias de Tecido Conjuntivo e de Tecidos Moles/patologia , Receptores Proteína Tirosina Quinases , Neoplasias Cutâneas/patologia , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/patologia , Proteínas de Fusão Oncogênica/genética , Cadeias Pesadas de Miosina/genética , Miosina não Muscular Tipo IIB/genéticaRESUMO
Neural crest cells migrate long distances throughout the embryo and rely on extracellular signals that attract, repel and/or stimulate survival to ensure proper contribution to target derivatives. Here, we show that leukocyte receptor tyrosine kinase (LTK), an ALK-type receptor tyrosine kinase, is expressed by neural crest cells during early migratory stages in chicken embryos. Loss of LTK in the cranial neural crest impairs migration and results in increased levels of apoptosis. Conversely, midkine, previously proposed as a ligand for ALK, is secreted by the non-neural ectoderm during early neural crest migratory stages and internalized by neural crest cells in vivo Similar to loss of LTK, loss of midkine reduces survival of the migratory neural crest. Moreover, we show by proximity ligation and co-immunoprecipitation assays that midkine binds to LTK. Taken together, these results suggest that LTK in neural crest cells interacts with midkine emanating from the non-neural ectoderm to promote cell survival, revealing a new signaling pathway that is essential for neural crest development.
Assuntos
Movimento Celular , Midkina/metabolismo , Crista Neural/citologia , Crista Neural/enzimologia , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Padronização Corporal , Sobrevivência Celular , Embrião de Galinha , Ectoderma/metabolismo , Ligação ProteicaRESUMO
Mutations in anaplastic lymphoma kinase (ALK) are implicated in somatic and familial neuroblastoma, a pediatric tumor of neural crest-derived tissues. Recently, biochemical analyses have identified secreted small ALKAL proteins (FAM150, AUG) as potential ligands for human ALK and the related leukocyte tyrosine kinase (LTK). In the zebrafish Danio rerio, DrLtk, which is similar to human ALK in sequence and domain structure, controls the development of iridophores, neural crest-derived pigment cells. Hence, the zebrafish system allows studying Alk/Ltk and Alkals involvement in neural crest regulation in vivo. Using zebrafish pigment pattern formation, Drosophila eye patterning, and cell culture-based assays, we show that zebrafish Alkals potently activate zebrafish Ltk and human ALK driving downstream signaling events. Overexpression of the three DrAlkals cause ectopic iridophore development, whereas loss-of-function alleles lead to spatially distinct patterns of iridophore loss in zebrafish larvae and adults. alkal loss-of-function triple mutants completely lack iridophores and are larval lethal as is the case for ltk null mutants. Our results provide in vivo evidence of (i) activation of ALK/LTK family receptors by ALKALs and (ii) an involvement of these ligand-receptor complexes in neural crest development.
Assuntos
Citocinas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Quinase do Linfoma Anaplásico , Animais , Linhagem Celular Tumoral , Drosophila , Olho/metabolismo , Humanos , Linfoma/enzimologia , Crista Neural/enzimologia , Células PC12 , Pigmentação , Ratos , Peixe-ZebraRESUMO
Tyrosine kinases are signaling molecules that are common to all metazoans and are involved in the regulation of many cellular processes such as proliferation and survival. While most attention has been devoted to tyrosine kinases signaling at the plasma membrane and the cytosol, very little attention has been dedicated to signaling at endomembranes. In this review, I will discuss recent evidence that we obtained on signaling of tyrosine kinases at the surface of the endoplasmic reticulum (ER), as well as in the lumen of this organelle. I will discuss how tyrosine kinase signaling might regulate ER proteostasis and the implication thereof to general cell physiology.
Assuntos
Retículo Endoplasmático/enzimologia , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Citosol/enzimologia , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Camundongos , Fosforilação/fisiologia , Proteostase/fisiologia , Resposta a Proteínas não DobradasRESUMO
The outcomes of refractive surgical procedures to improve uncorrected vision in patients-including photorefractive keratectomy (PRK), laser in-situ keratomileusis (LASIK), Small Incision Lenticule Extraction (SMILE) and corneal inlay procedures-is in large part determined by the corneal wound healing response after surgery. The wound healing response varies depending on the type of surgery, the level of intended correction of refractive error, the post-operative inflammatory response, generation of opacity producing myofibroblasts and likely poorly understood genetic factors. This article details what is known about these specific wound healing responses that include apoptosis of keratocytes and myofibroblasts, mitosis of corneal fibroblasts and myofibroblast precursors, the development of myofibroblasts from keratocyte-derived corneal fibroblasts and bone marrow-derived fibrocytes, deposition of disordered extracellular matrix by corneal fibroblasts and myofibroblasts, healing of the epithelial injury, and regeneration of the epithelial basement membrane. Problems with epithelial and stromal cellular viability and function that are altered by corneal inlays are also discussed. A better understanding of the wound healing response in refractive surgical procedures is likely to lead to better treatments to improve outcomes, limit complications of keratorefractive surgical procedures, and improve the safety and efficiency of refractive surgical procedures.
Assuntos
Cirurgia da Córnea a Laser/métodos , Lasers de Excimer/uso terapêutico , Miopia/cirurgia , Refração Ocular/fisiologia , Humanos , Ceratomileuse Assistida por Excimer Laser In Situ/métodos , Ceratoplastia Penetrante/métodos , Miopia/fisiopatologia , Ceratectomia Fotorrefrativa/métodosAssuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/cirurgia , Neoplasias Pulmonares/tratamento farmacológico , Antineoplásicos/uso terapêutico , Quinase do Linfoma Anaplásico , Inibidores de Proteínas Quinases/uso terapêutico , Aminopiridinas/uso terapêutico , Lactamas Macrocíclicas/uso terapêutico , Receptores Proteína Tirosina QuinasesRESUMO
BACKGROUND: Most preclinical studies assess vaccine effectiveness in single-pathogen infection models. This is unrealistic given that humans are continuously exposed to different commensals and pathogens in sequential and mixed infections. Accordingly, complications from secondary bacterial infection are a leading cause of influenza-associated morbidity and mortality. New vaccination strategies are needed to control infections on simultaneous fronts. METHODS: We compared different anti-influenza vaccines for their protective potential in a model of viral infection with bacterial superinfection. Mice were immunized with H1N1/A/California/7/2009 subunit vaccines, formulated with different adjuvants inducing either T-helper type 1 (Th1) (MF59 plus CpG)-, Th1/2 (MF59)-, or Th17 (LTK63)-prone immune responses and were sequentially challenged with mouse-adapted influenza virus H1N1/A/Puerto Rico/8/1934 and Staphylococcus aureus USA300, a clonotype emerging as a leading contributor in postinfluenza pneumonia in humans. RESULTS: Unadjuvanted vaccine controlled single viral infection, yet mice had considerable morbidity from viral disease and bacterial superinfection. In contrast, all adjuvanted vaccines efficiently protected mice in both conditions. Interestingly, the Th1-inducing formulation was superior to Th1/2 or Th17 inducers. CONCLUSIONS: Our studies should help us better understand how differential immunity to influenza skews immune responses toward coinfecting bacteria and discover novel modes to prevent bacterial superinfections in the lungs of persons with influenza.
Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/imunologia , Superinfecção/prevenção & controle , Adjuvantes Imunológicos/administração & dosagem , Animais , Toxinas Bacterianas/administração & dosagem , Enterotoxinas/administração & dosagem , Proteínas de Escherichia coli/administração & dosagem , Feminino , Humanos , Imunização , Vacinas contra Influenza/administração & dosagem , Influenza Humana/complicações , Influenza Humana/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/administração & dosagem , Polissorbatos/administração & dosagem , Organismos Livres de Patógenos Específicos , Esqualeno/administração & dosagem , Infecções Estafilocócicas/complicações , Infecções Estafilocócicas/microbiologia , Superinfecção/microbiologiaRESUMO
Accurate thermal models for the cornea of the eye support the development of thermal techniques for reshaping the cornea and other scientific purposes. Heat transfer in the cornea must be quantified accurately so that a thermal treatment does not destroy the endothelial layer, which cannot regenerate, and yet is responsible for maintaining corneal transparency. We developed a custom apparatus to measure the thermal conductivity of ex vivo porcine corneas perpendicular to the surface and applied a commercial apparatus to measure thermal conductivity parallel to the surface. We found that corneal thermal conductivity is 14% anisotropic at the normal state of corneal hydration. Small numbers of ex vivo feline and human corneas had a thermal conductivity perpendicular to the surface that was indistinguishable from the porcine corneas. Aqueous humor from ex vivo porcine, feline, and human eyes had a thermal conductivity nearly equal to that of water. Including the anisotropy of corneal thermal conductivity will improve the predictive power of thermal models of the eye.
Assuntos
Córnea/fisiologia , Condutividade Térmica , Animais , Anisotropia , Humor Aquoso/fisiologia , Gatos , Humanos , Modelos Teóricos , SuínosRESUMO
Autoimmune disorders and some types of blood cancer originate when B lymphocytes malfunction. In particular, when B cells produce antibodies recognizing the body's proteins, it leads to various autoimmune disorders. Additionally, when B cells of various developmental stages transform into cancer cells, it results in blood cancers, including multiple myeloma, lymphoma, and leukemia. Thus, new methods of targeting B cells are required for various patient groups. Here, we used protein kinase inhibitors alectinib, brigatinib, ceritinib, crizotinib, entrectinib, and lorlatinib previously approved as drugs treating anaplastic lymphoma kinase (ALK)-positive lung cancer cells. We hypothesized that the same inhibitors will efficiently target leukocyte tyrosine kinase (LTK)-positive, actively protein-secreting mature B lymphocytes, including plasma cells. We isolated CD19-positive human B cells from the blood of healthy donors and used two alternative methods to stimulate cell maturation toward plasma cells. Using cell proliferation and flow cytometry assays, we found that ceritinib and entrectinib eliminate plasma cells from B cell populations. Alectinib, brigatinib, and crizotinib also inhibited B cell proliferation, while lorlatinib had no or limited effect on B cells. More generally, we concluded that several drugs previously developed to treat ALK-positive malignant cells can be also used to treat LTK-positive B cells.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Crizotinibe/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Antineoplásicos/farmacologia , Quinase do Linfoma Anaplásico/uso terapêutico , Neoplasias Pulmonares/patologia , Receptores Proteína Tirosina Quinases , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Lactamas Macrocíclicas , Linfócitos BRESUMO
The high morbidity and mortality of non-small cell lung cancer (NSCLC) have always been major threats to people's health. With the identification of carcinogenic drivers in non-small cell lung cancer and the clinical application of targeted drugs, the prognosis of non-small cell lung cancer patients has greatly improved. However, in a large number of non-small cell lung cancer cases, the carcinogenic driver is unknown. Identifying genetic alterations is critical for effective individualized therapy in NSCLC. Moreover, targeted drugs are difficult to apply in the clinic. Cancer drug resistance is an unavoidable obstacle limiting the efficacy and application of targeted drugs. This review describes the mechanisms of targeted-drug resistance and newly identified non-small cell lung cancer targets (e.g., KRAS G12C, NGRs, DDRs, CLIP1-LTK, PELP1, STK11/LKB1, NFE2L2/KEAP1, RICTOR, PTEN, RASGRF1, LINE-1, and SphK1). Research into these mechanisms and targets will drive individualized treatment of non-small cell lung cancer to generate better outcomes.
RESUMO
This review commemorates the 10-year anniversary of the discovery of physiological ligands Augα (Augmentor α; ALKAL2; Fam150b) and Augß (Augmentor ß; ALKAL1; Fam150a) for anaplastic lymphoma kinase (ALK) and leukocyte tyrosine kinase (LTK), previously considered orphan receptors. This manuscript provides an in-depth review of the biophysical and cellular properties of ALK family receptors and their roles in cancer, metabolism, pain, ophthalmology, pigmentation, central nervous system (CNS) function, and reproduction. ALK and LTK receptors are implicated in the development of numerous cancers, and targeted inhibition of their signaling pathways can offer therapeutic benefits. Additionally, ALK family receptors are involved in regulating body weight and metabolism, modulating pain signaling, and contributing to eye development and pigmentation. In the CNS, these receptors play a role in synapse modulation, neurogenesis, and various psychiatric pathologies. Lastly, ALK expression is linked to reproductive functions, with potential implications for patients undergoing ALK inhibitor therapy. Further research is needed to better understand the complex interactions of ALK family receptors and Aug ligands and to repurpose targeted therapy for a wide range of human diseases.
Assuntos
Neoplasias , Receptores Proteína Tirosina Quinases , Humanos , Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Neoplasias/tratamento farmacológico , Neoplasias/genética , Dor , LigantesRESUMO
BACKGROUND: In recent years, several studies have demonstrated that bacterial ABC transporters present relevant antigen targets for the development of vaccines against bacteria such as Streptococcus pneumoniae and Enterococcus faecalis. In Streptococcus mutans, the glutamate transporter operon (glnH), encoding an ABC transporter, is associated with acid tolerance and represents an important virulence-associated factor for the development of dental caries. RESULTS: In this study, we generated a recombinant form of the S. mutans GlnH protein (rGlnH) in Bacillus subtilis. Mice immunized with this protein antigen elicited strong antigen-specific antibody responses after sublingual administration of a vaccine formulation containing a mucosal adjuvant, a non-toxic derivative of the heat-labile toxin (LTK63) originally produced by enterotoxigenic Escherichia coli (ETEC) strains. Serum anti-rGlnH antibodies reduced adhesion of S. mutans to the oral cavity of naïve mice. Moreover, mice actively immunized with rGlnH were partially protected from oral colonization after exposure to the S. mutans NG8 strain. CONCLUSIONS: Our results indicate that S. mutans rGlnH is a potential target antigen capable of inducing specific and protective antibody responses after immunization. Overall, these observations raise the prospect of the development of mucosal anti-caries vaccines.
Assuntos
Cárie Dentária , Streptococcus mutans , Camundongos , Animais , Streptococcus mutans/genética , Cariostáticos/metabolismo , Anticorpos Antibacterianos , Proteínas de Transporte/metabolismo , Ácido Glutâmico/metabolismo , Cárie Dentária/prevenção & controle , Cárie Dentária/metabolismo , Saliva/metabolismo , Proteínas/metabolismoRESUMO
Neuroblastoma is a neural crest-derived paediatric cancer that is the most common and deadly solid extracranial tumour of childhood. It arises when neural crest cells fail to follow their differentiation program to give rise to cells of the sympathoadrenal lineage. These undifferentiated cells can proliferate and migrate, forming tumours mostly found associated with the adrenal glands. Activating mutations in the kinase domain of anaplastic lymphoma kinase (ALK) are linked to high-risk cases, where extensive therapy is ineffective. However, the role of ALK in embryonic development, downstream signal transduction and in metastatic transformation of the neural crest is poorly understood. Here, we demonstrate high conservation of the ALK protein sequences among vertebrates. We then examine alk mRNA expression in the frog models Xenopus laevis and Xenopus tropicalis. Using in situ hybridisation of Xenopus embryos, we show that alk is expressed in neural crest domains throughout development, suggesting a possible role in neuroblastoma initiation. Lastly, RT-qPCR analyses show high levels of alk expression at tadpole stages. Collectively, these data may begin to elucidate how alk functions in neural crest cells and how its deregulation can result in tumorigenesis.
Assuntos
Quinase do Linfoma Anaplásico/genética , Crista Neural/metabolismo , Proteínas de Xenopus/genética , Quinase do Linfoma Anaplásico/metabolismo , Animais , Crista Neural/embriologia , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Aberrant activation of anaplastic lymphoma kinase (ALK) has been described in a range of human cancers, including non-small cell lung cancer and neuroblastoma (Hallberg and Palmer, 2013). Vertebrate ALK has been considered to be an orphan receptor and the identity of the ALK ligand(s) is a critical issue. Here we show that FAM150A and FAM150B are potent ligands for human ALK that bind to the extracellular domain of ALK and in addition to activation of wild-type ALK are able to drive 'superactivation' of activated ALK mutants from neuroblastoma. In conclusion, our data show that ALK is robustly activated by the FAM150A/B ligands and provide an opportunity to develop ALK-targeted therapies in situations where ALK is overexpressed/activated or mutated in the context of the full length receptor.
Assuntos
Citocinas/metabolismo , Ativação Enzimática , Receptores Proteína Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico , Linhagem Celular , Humanos , Dados de Sequência Molecular , Ligação Proteica , Análise de Sequência de DNARESUMO
Dorso-ventral pigment pattern differences are the most widespread pigmentary adaptations in vertebrates. In mammals, this pattern is controlled by regulating melanin chemistry in melanocytes using a protein, agouti-signalling peptide (ASIP). In fish, studies of pigment patterning have focused on stripe formation, identifying a core striping mechanism dependent upon interactions between different pigment cell types. In contrast, mechanisms driving the dorso-ventral countershading pattern have been overlooked. Here, we demonstrate that, in fact, zebrafish utilize two distinct adult pigment patterning mechanisms - an ancient dorso-ventral patterning mechanism, and a more recent striping mechanism based on cell-cell interactions; remarkably, the dorso-ventral patterning mechanism also utilizes ASIP. These two mechanisms function largely independently, with resultant patterns superimposed to give the full pattern.
Assuntos
Pigmentação , Peixe-Zebra/fisiologia , Animais , Contagem de Células , Regulação da Expressão Gênica no Desenvolvimento , Larva/genética , Melanóforos/metabolismo , Pigmentação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND: A lot of pathogens enter the body via the nasal route. The construction of non-toxic mutants of heat labile Escherichia coli enterotoxin (LT), which is a potent mucosal adjuvant, represents a major breakthrough for the development of mucosal vaccines. OBJECTIVE: This study was undertaken to critically evaluate the adjuvanticity of the mutant of LT (LTK63) on the cellular immune responses to intranasally co-administered recombinant measles virus nucleoprotein (rMVNP). METHODS: Groups of CBA mice were immunized intranasally with rMVNP with or without LT or LTK63 as adjuvants. Another group was immunized subcutaneously with rMVNP in Freund's adjuvant. rMVNP and measles virus (MV) were used in a proliferation assay to test the LTK63 potentiating ability to induce T cell responses. Subsequently MVNP synthetic peptides spanning the length of the N protein were used with a proliferation assay to identify the T cell epitopes. RESULTS: Splenocytes from mice immunized intranasally with rMVNP plus LT or LTK63, showed strong dose dependent proliferative responses to both the MVNP and MV. However, proliferative responses from the latter group were significantly lower than the former group (P < 0.05). Splenocytes tested recognized peptides 20, 21, 28, 31, 39, 40 and 50, suggesting these to be among important epitopes. Subcutaneous route was not effective in priming for T cell responses to rMVNP. CONCLUSION: These data further demonstrate the great potential of LTK63 as a safe mucosal vaccine adjuvant.