RESUMO
TRiC/CCT is a chaperonin complex required for the folding of cytoplasmic proteins. Although mutations in each subunit of TRiC/CCT are associated with various human neurodegenerative diseases, their impact in mammalian models has not yet been examined. A compound heterozygous mutation in CCT2 (p.[Thr400Pro]; p.[Arg516His]) is causal for Leber congenital amaurosis. Here, we generate mice carrying each mutation and show that Arg516His (R516H) homozygosity causes photoreceptor degeneration accompanied by a significant depletion of TRiC/CCT substrate proteins in the retina. In contrast, Thr400Pro (T400P) homozygosity results in embryonic lethality, and the compound heterozygous mutant (T400P/R516H) mouse showed aberrant cone cell lamination and died 2 weeks after birth. Finally, CCDC181 is identified as a interacting protein for CCTß protein, and its localization to photoreceptor connecting cilia is compromised in the mutant mouse. Our results demonstrate the distinct impact of each mutation in vivo and suggest a requirement for CCTß in ciliary maintenance.
Assuntos
Chaperonina com TCP-1 , Modelos Animais de Doenças , Amaurose Congênita de Leber , Animais , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/metabolismo , Amaurose Congênita de Leber/patologia , Camundongos , Chaperonina com TCP-1/genética , Chaperonina com TCP-1/metabolismo , Mutação , Heterozigoto , Camundongos Endogâmicos C57BLRESUMO
With significant advances in analytical technologies, research in the field of cancer immunotherapy, such as adoptive T cell therapy, cancer vaccine, and immune checkpoint blockade (ICB), is currently gaining tremendous momentum. Since the efficacy of cancer immunotherapy is recognized only by a minority of patients, more potent tumor-specific antigens (TSAs, also known as neoantigens) and predictive markers for treatment response are of great interest. In cancer immunity, immunopeptides, presented by human leukocyte antigen (HLA) class I, play a role as initiating mediators of immunogenicity. The latest advancement in the interdisciplinary multiomics approach has rapidly enlightened us about the identity of the "dark matter" of cancer and the associated immunopeptides. In this field, mass spectrometry (MS) is a viable option to select because of the naturally processed and actually presented TSA candidates in order to grasp the whole picture of the immunopeptidome. In the past few years the search space has been enlarged by the multiomics approach, the sensitivity of mass spectrometers has been improved, and deep/machine-learning-supported peptide search algorithms have taken immunopeptidomics to the next level. In this review, along with the introduction of key technical advancements in immunopeptidomics, the potential and further directions of immunopeptidomics will be reviewed from the perspective of cancer immunotherapy.
Assuntos
Neoplasias , Humanos , Neoplasias/terapia , Antígenos de Histocompatibilidade Classe I , Antígenos de Neoplasias , Espectrometria de Massas/métodos , ImunoterapiaRESUMO
Numerous omics studies, primarily genomics analyses, have been conducted to fully understand the molecular biological characteristics of cancer. In recent years, the depth of proteomic analysis, which comprehensively analyzes proteins and molecules that function directly in vivo, has increased dramatically. Proteomics using mass spectrometry (MS) is a promising technology to directly examine proteoforms, including post-translational modifications and variants originating from genomic aberrations. Recent advances in MS-based proteomics have enabled direct, in depth, and quantitative analysis of the expression levels of various cancer-related proteins, as well as their cancer-specific proteoforms, and proteins that fluctuate with cancer initiation and progression in cell lines and tissue samples. Additionally, the integration of proteomic data with genomic, epigenomic, and transcriptomic data has formed the growing field of proteogenomics, which is already yielding new biological and diagnostic knowledge. Deep proteomic profiling provides clinically useful information in various aspects, including understanding the mechanisms of cancer development and progression and discovering targets for diagnosis and drug development. Furthermore, it is expected to make a significant contribution to the promotion of personalized medicine. In this review, recent advances and impacts in MS-based clinical proteomics are highlighted with a focus on oncology.
Assuntos
Neoplasias , Proteogenômica , Humanos , Proteômica/métodos , Genômica/métodos , Proteogenômica/métodos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/terapia , Espectrometria de Massas/métodosRESUMO
Normal-tension glaucoma (NTG) is a heterogeneous disease characterized by retinal ganglion cell (RGC) death leading to cupping of the optic nerve head and visual field loss at normal intraocular pressure (IOP). The pathogenesis of NTG remains unclear. Here, we describe a single nucleotide mutation in exon 2 of the methyltransferase-like 23 (METTL23) gene identified in 3 generations of a Japanese family with NTG. This mutation caused METTL23 mRNA aberrant splicing, which abolished normal protein production and altered subcellular localization. Mettl23-knock-in (Mettl23+/G and Mettl23G/G) and -knockout (Mettl23+/- and Mettl23-/-) mice developed a glaucoma phenotype without elevated IOP. METTL23 is a histone arginine methyltransferase expressed in murine and macaque RGCs. However, the novel mutation reduced METTL23 expression in RGCs of Mettl23G/G mice, which recapitulated both clinical and biological phenotypes. Moreover, our findings demonstrated that METTL23 catalyzed the dimethylation of H3R17 in the retina and was required for the transcription of pS2, an estrogen receptor α target gene that was critical for RGC homeostasis through the negative regulation of NF-κB-mediated TNF-α and IL-1ß feedback. These findings suggest an etiologic role of METTL23 in NTG with tissue-specific pathology.
Assuntos
Glaucoma , Histonas , Animais , Camundongos , Modelos Animais de Doenças , Glaucoma/metabolismo , Histonas/genética , Histonas/metabolismo , Pressão Intraocular/genética , Metilação , Mutação , Células Ganglionares da Retina/metabolismoRESUMO
Understanding the properties of human leukocyte antigen (HLA) peptides (immunopeptides) is essential for precision cancer medicine, while the direct identification of immunopeptides from small biopsies of clinical tissues by mass spectrometry (MS) is still confronted with technical challenges. Here, to overcome these hindrances, high-field asymmetric waveform ion mobility spectrometry (FAIMS) is introduced to conduct differential ion mobility (DIM)-MS by seamless gas-phase fractionation optimal for scarce samples. By established DIM-MS for immunopeptidomics analysis, on average, 42.9 mg of normal and tumor colorectal tissues from identical patients (n = 17) were analyzed, and on average 4921 immunopeptides were identified. Among these 44,815 unique immunopeptides, two neoantigens, KRAS-G12V and CPPED1-R228Q, were identified. These neoantigens were confirmed by synthetic peptides through targeted MS in parallel reaction monitoring (PRM) mode. Comparison of the tissue-based personal immunopeptidome revealed tumor-specific processing of immunopeptides. Since the direct identification of neoantigens from tumor tissues suggested that more potential neoantigens have yet to be identified, we screened cell lines with known oncogenic KRAS mutations and identified 2 more neoantigens that carry KRAS-G12V. These results indicated that the established FAIMS-assisted DIM-MS is effective in the identification of immunopeptides and potential recurrent neoantigens directly from scarce samples such as clinical tissues.
Assuntos
Neoplasias Colorretais , Espectrometria de Mobilidade Iônica , Neoplasias Colorretais/genética , Antígenos de Histocompatibilidade Classe I , Humanos , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Mutação , Peptídeos/química , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Inherited optic neuropathies are rare eye diseases of optic nerve dysfunction that present in various genetic forms. Previously, mutation in three genes encoding mitochondrial proteins has been implicated in autosomal recessive forms of optic atrophy that involve progressive degeneration of optic nerve and retinal ganglion cells (RGC). Using whole exome analysis, a novel double homozygous mutation p.L81R and pR212W in malonyl CoA-acyl carrier protein transacylase (MCAT), a mitochondrial protein involved in fatty acid biosynthesis, has now been identified as responsible for an autosomal recessive optic neuropathy from a Chinese consanguineous family. MCAT is expressed in RGC that are rich in mitochondria. The disease variants lead to structurally unstable MCAT protein with significantly reduced intracellular expression. RGC-specific knockdown of Mcat in mice, lead to an attenuated retinal neurofiber layer, that resembles the phenotype of optic neuropathy. These results indicated that MCAT plays an essential role in mitochondrial function and maintenance of RGC axons, while novel MCAT p.L81R and p.R212W mutations can lead to optic neuropathy.
Assuntos
Proteína de Transporte de Acila S-Maloniltransferase/genética , Genes Recessivos , Mitocôndrias/patologia , Doenças do Nervo Óptico/patologia , Nervo Óptico/patologia , Células Ganglionares da Retina/patologia , Proteína de Transporte de Acila S-Maloniltransferase/química , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Sequência de Aminoácidos , Animais , Criança , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Mutação , Nervo Óptico/metabolismo , Doenças do Nervo Óptico/etiologia , Doenças do Nervo Óptico/metabolismo , Linhagem , Conformação Proteica , Células Ganglionares da Retina/metabolismo , Homologia de Sequência , Sequenciamento do ExomaRESUMO
Purpose: The compound heterozygous mutations in the ß subunit of chaperonin containing TCP-1 (CCT), encoded by CCT2, lead to the Leber congenital amaurosis (LCA). In this study, a cct2 mutant line of zebrafish was established to investigate the role of CCT2 mutations in LCA in vertebrates. Methods: A cct2 mutant zebrafish line was produced using the CRISPR-Cas9 system. Changes in the eyes of developing wild-type and mutant larvae were monitored using microscopy, immunostaining, TUNEL, and EdU assays. Phenotypic rescue of mutant phenotype was investigated by injection of CCT2 RNA into zebrafish embryos. Results: The cct2 mutation (L394H-7del) led to the synthesis of a mutated cctß protein with the L394H replacement and deletion of 7 amino acid residues (positions 395-401). The homozygous cct2-L394H-7del mutant exhibited a small eye phenotype at 2 days post fertilization (dpf) and was embryonically lethal after 5 dpf. In homozygous cct2-L394H-7del mutants, the retinal ganglion cell differentiation was attenuated, retinal cell cycle was affected, and the neural retinal cell death was significantly increased at 2 dpf compared with wild-type. Injection of RNA encoding wild-type human CCTß rescued the small eye phenotype, reduced retinal cell death, and restored the levels of CCTß protein and the major client protein Gß1 that were significantly reduced in the homozygous cct2-L394H-7del mutant compared with wild-type. These results indicate that cct2 plays an essential role in retinal development by regulating the cell cycle. Conclusions: The retinal pathology observed in the homozygous cct2-L394H-7del mutants resembles the retinal pathology of human LCA patients.
Assuntos
Apoptose/genética , Ciclo Celular/genética , Chaperonina com TCP-1/genética , Amaurose Congênita de Leber/genética , Retina/patologia , Deleção de Sequência , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Western Blotting , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Microscopia , Dados de Sequência Molecular , Fenótipo , RNA/genética , Peixe-ZebraRESUMO
Glaucoma is one of the leading causes of bilateral blindness, affecting nearly 57 million people worldwide. Glaucoma is characterized by a progressive loss of retinal ganglion cells and is often associated with intraocular pressure (IOP). Normal tension glaucoma (NTG), marked by normal IOP but progressive glaucoma, is incompletely understood. In 2002, Sarfarazi et al. identified FIP-2 gene mutations responsible for hereditary NTG, renaming this gene "optineurin" (OPTN). Further investigations by multiple groups worldwide showed that OPTN is involved in several critical cellular functions, such as NF-κB regulation, autophagy, and vesicle transport. Recently, OPTN mutations were found to cause amyotrophic lateral sclerosis (ALS). Surprisingly, a mutation in the OPTN interacting protein, i.e., the duplication of TANK binding protein 1 (TBK1) gene, also can cause both NTG and ALS. These phenotypically distinct neuronal diseases are now merging into one common pathological mechanism by these two genes. TBK1 inhibition has emerged as a potential therapy for NTG. In this manuscript, we focus on the OPTN E50K mutation, the most common mutation for NTG, to describe the molecular mechanism of NTG by expressing a mutant Optn gene in cells and genetically modified mice. Patient iPS cells were developed and differentiated into neural cells to observe abnormal behavior and the impact of the E50K mutation. These in vitro studies were further extended to identify the inhibitors BX795 and amlexanox, which have the potential to reverse the disease-causing phenomenon in patient's neural cells. Here we show for the first time that amlexanox protects RGCs in Optn E50K knock-in mice.
Assuntos
DNA/genética , Glaucoma/genética , Mutação , Fator de Transcrição TFIIIA/genética , Animais , Proteínas de Ciclo Celular , Análise Mutacional de DNA , Glaucoma/metabolismo , Humanos , Proteínas de Membrana Transportadoras , Fator de Transcrição TFIIIA/metabolismoRESUMO
Leber congenital amaurosis (LCA) is a hereditary early-onset retinal dystrophy that is accompanied by severe macular degeneration. In this study, novel compound heterozygous mutations were identified as LCA-causative in chaperonin-containing TCP-1, subunit 2 (CCT2), a gene that encodes the molecular chaperone protein, CCTß. The zebrafish mutants of CCTß are known to exhibit the eye phenotype while its mutation and association with human disease have been unknown. The CCT proteins (CCT α-θ) forms ring complex for its chaperon function. The LCA mutants of CCTß, T400P and R516H, are biochemically instable and the affinity for the adjacent subunit, CCTγ, was affected distinctly in both mutants. The patient-derived induced pluripotent stem cells (iPSCs), carrying these CCTß mutants, were less proliferative than the control iPSCs. Decreased proliferation under Cct2 knockdown in 661W cells was significantly rescued by wild-type CCTß expression. However, the expression of T400P and R516H didn't exhibit the significant effect. In mouse retina, both CCTß and CCTγ are expressed in the retinal ganglion cells and connecting cilium of photoreceptor cells. The Cct2 knockdown decreased its major client protein, transducing ß1 (Gß1). Here we report the novel LCA mutations in CCTß and the impact of chaperon disability by these mutations in cellular biology.
Assuntos
Proliferação de Células/genética , Chaperonina com TCP-1 , Células-Tronco Pluripotentes Induzidas , Amaurose Congênita de Leber , Mutação , Animais , Chaperonina com TCP-1/genética , Chaperonina com TCP-1/metabolismo , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/metabolismo , Amaurose Congênita de Leber/patologia , Estabilidade Proteica , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Glaucoma is the leading cause for blindness affecting 60 million people worldwide. The optineurin (OPTN) E50K mutation was first identified in familial primary open-angle glaucoma (POAG), the onset of which is not associated with intraocular pressure (IOP) elevation, and is classified as normal-tension glaucoma (NTG). Optineurin (OPTN) is a multifunctional protein and its mutations are associated with neurodegenerative diseases such as POAG and amyotrophic lateral sclerosis (ALS). We have previously described an E50K mutation-carrying transgenic (E50K-tg) mouse that exhibited glaucomatous phenotypes of decreased retinal ganglion cells (RGCs) and surrounding cell death at normal IOP. Further phenotypic analysis of these mice revealed persistent reactive gliosis and E50K mutant protein deposits in the outer plexiform layer (OPL). Over-expression of E50K in HEK293 cells indicated accumulation of insoluble OPTN in the endoplasmic reticulum (ER). This phenomenon was consistent with the results seen in neurons derived from induced pluripotent stem cells (iPSCs) from E50K mutation-carrying NTG patients. The E50K mutant strongly interacted with TANK-binding kinase 1 (TBK1), which prohibited the proper oligomerization and solubility of OPTN, both of which are important for OPTN intracellular transition. Treatment with a TBK1 inhibitor, BX795, abrogated the aberrant insolubility of the E50K mutant. Here, we delineated the intracellular dynamics of the endogenous E50K mutant protein for the first time and demonstrated how this mutation causes OPTN insolubility, in association with TBK1, to evoke POAG.
Assuntos
Glaucoma de Ângulo Aberto/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição TFIIIA/genética , Animais , Proteínas de Ciclo Celular , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Glaucoma de Ângulo Aberto/metabolismo , Gliose , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Transgênicos , Pirimidinas/efeitos adversos , Retina , Tiofenos/efeitos adversos , Fator de Transcrição TFIIIA/química , Fator de Transcrição TFIIIA/metabolismoRESUMO
Overexpression of the ErbB2/HER2 receptor tyrosine kinase contributes to tumorigenesis. However, mechanisms regulating ErbB2 protein levels remain largely unclear. Here, we identified novel mechanisms of ErbB2 downregulation. ErbB2 constitutively binds to an adaptor protein FRS2ß. We found that FRS2ß bound to CD2AP and CIN85, which induces endosomal trafficking that targets lysosomes. FRS2ß colocalized with CIN85 in the cytoplasm. Expression of wild type FRS2ß but not its CIN85 non-binding mutant, downregulated the ErbB2 protein and inhibited anchorage-independent cell growth. Moreover, the E3 ubiquitin-protein ligase Cbl was contained within a complex of FRS2ß and CIN85. Knockdown of both CIN85 and CD2AP or of Cbl, or treatment with lysosomal degradation inhibitors diminished FRS2ß downregulation of ErbB2. In addition, knockdown of endogenous FRS2ß caused upregulation of ErbB2 in primary neural cells. Finally, immunohistochemical analysis showed that human breast cancer tissues that overexpress ErbB2 expressed low levels of FRS2ß. Thus, an FRS2ß-CIN85/CD2AP-Cbl axis for downregulation of ErbB2 may regulate ErbB2 protein levels in physiological and pathological settings. Molecular targeting drugs that can increase or stabilize the ErbB2-FRS2ß-CIN85/CD2AP-Cbl axis may have promise for the control of ErbB2-overexpressing tumors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Neoplasias da Mama/metabolismo , Células Cultivadas , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Neurônios/metabolismo , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: The widespread use of proton pump inhibitors (PPIs) is known to cause sporadic gastric fundic gland polyps (FGPs). Altered expression and localization of the water or ion transport proteins might contribute to the excess fluid secretion into the cystic lumen for the development of FGPs. AIMS: We investigated the alteration of the murine gastric fundic mucosa after PPI treatment, and examined the expression of water channel aquaporin-4 (AQP4) and potassium channel KCNQ1, which are expressed only in the parietal cells in the gastric mucosa. METHODS: Male 5-week-old C57BL/6J mice were administered lansoprazole (LPZ) by subcutaneous injection for 8 weeks. The expression of AQP4 and KCNQ1 were investigated by Western blotting, quantitative RT-PCR, and immunohistochemistry. The expression of mucin-6 (Muc6), pepsinogen, and sonic hedgehog (Shh) were also investigated as mucosal cell lineage markers. RESULTS: Gastric mucosal hyperplasia with multiple cystic dilatations, exhibiting similar histological findings to the FGPs, was observed in the LPZ-treated mice. An increase in the number of AQP4-positive parietal cells and KCNQ1-positive parietal cells was observed. The extension of the distribution of AQP4-positive cells toward the surface of the fundic glands was also observed. The expression levels of AQP4 mRNA and protein were significantly enhanced. The expression of KCNQ1 mRNA was correlated with that of AQP4 mRNA in the LPZ-treated mice. Mucous neck-to-zymogenic cell lineage differentiation was delayed in association with decreased expression of Shh in the LPZ-treated mice. CONCLUSIONS: PPI administration increased the number of parietal cells with enhanced expression of AQP4 and KCNQ1.
Assuntos
2-Piridinilmetilsulfinilbenzimidazóis/farmacologia , Aquaporina 4/metabolismo , Ácido Gástrico/metabolismo , Canal de Potássio KCNQ1/metabolismo , Células Parietais Gástricas/metabolismo , Inibidores da Bomba de Prótons/farmacologia , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Celulas Principais Gástricas/fisiologia , Hiperplasia , Imuno-Histoquímica , Lansoprazol , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Parietais Gástricas/patologiaRESUMO
Helicobacter pylori (HP)-eradication therapy increases Runt domain transcription factor 3 (RUNX3) expression in the glandular epithelial cells in enlarged-fold gastritis. The aim of this study is to evaluate expression of the RUNX3 protein, the product of a gastric tumor suppression gene, and mutagenic oxidative stress in human gastric mucosal specimens obtained from patients with HP-induced enlarged-fold gastritis. Methods. RUNX3 expression was immunohistochemically scored and the degree of the mucosal oxidative stress was directly measured by the chemiluminescense (ChL) assay in the biopsy specimens. Results. RUNX3 expression was detected in the gastric epithelial cells. HP-eradication significantly increased RUNX3 expression in the glandular epithelium of the corpus, however, no change was observed in those of the antrum. A fourfold higher mucosal ChL value was observed in the corpus as compared with that in the antrum. HP-eradication significantly decreased the mucosal ChL values in both portions of the stomach to nearly undetectable levels. Conclusion. The glandular epithelium is exposed to a high level of carcinogenic oxidative stress and shows low levels of expression of the tumor suppressive molecule, RUNX3; however, this expression was restored after HP-eradication, suggesting the high risk of carcinogenesis associated with HP-induced enlarged-fold gastritis of the corpus.
Assuntos
Alanina/análogos & derivados , Antiulcerosos/farmacologia , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Proteínas Hedgehog/biossíntese , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori , Quinolonas/farmacologia , Alanina/farmacologia , Animais , Gerbillinae , HumanosRESUMO
BACKGROUND AND AIM: Sonic hedgehog (Shh) is a morphogen involved in the homeostasis of the gastric fundic glands. Alterations of gastric mucosal Shh expression after eradication of Helicobacter pylori were examined. METHOD: Mongolian gerbils were inoculated with H. pylori at the age of 5 weeks. H. pylori eradication was then carried out at 12, 24 or 48 weeks after the inoculation, and the gerbils were examined at 10 weeks after the eradication. Gastric inflammation was evaluated by the tissue myeloperoxidase activity and the histological scoring. Immunohistochemistry and in situ hybridization were performed for determining the Shh expression. RESULTS: Significant decrease of the myeloperoxidase activity and scores for acute and chronic inflammation as well as atrophy were observed in the H. pylori-eradicated gerbils as compared with the findings in the non-H. pylori-eradicated gerbils. Significant increase of the horizontal length of the area positive for Shh expression was noted in the H. pylori-eradicated gerbils as compared with that in the non-H. pylori-eradicated gerbils. Earlier eradication promoted better restoration of Shh expression. 50% of the animals of the 24-week eradication group and all animals in the 48-week eradication group exhibited heterotopic proliferative glands. In the animals showing heterotopic proliferative glands, the front line of Shh regeneration was cut off at the point of development of heterotopic proliferative glands. CONCLUSION: H. pylori-associated deregulation of Shh expression that could be linked to gastric atrophy and the associated preneoplastic transformation appears to be reversible with early H. pylori eradication.
Assuntos
Anti-Infecciosos/uso terapêutico , Mucosa Gástrica/metabolismo , Proteínas Hedgehog/metabolismo , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/fisiologia , Animais , Mucosa Gástrica/microbiologia , Expressão Gênica , Gerbillinae , Interações Hospedeiro-Patógeno , Masculino , RNA Mensageiro/metabolismoRESUMO
The fibroblast growth factor receptor substrate (FRS)-2 protein family comprises FRS2alpha, a well-known central mediator for fibroblast growth factor signaling, and FRS2beta, whose endogenous expression pattern and function are not yet defined. Immunohistochemical analysis revealed that expression of FRS2beta was restricted to neural tissues and it colocalized with Tuj1, a neuronal marker. There are two distinct patterns of FRS2beta expression in neural cells: punctate and cup/ring-shaped; moreover, some particles colocalized with lysosomes. Stimulation with brain-derived neurotrophic factor (BDNF) enhanced FRS2beta phosphorylation and the cup/ring-shaped pattern. These results suggest a probable role of FRS2beta in the intracellular degradation systems of neural cells, which involves lysosomes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Vesículas Citoplasmáticas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Anticorpos Monoclonais/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Mamíferos , Técnica Direta de Fluorescência para Anticorpo , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos ICRRESUMO
BACKGROUND: Ghrelin, an appetite-promoting peptide secreted from the stomach, is reported to enhance preprandial acid output, possibly through stimulation of the cephalic phase. The present study was designed to clarify the dynamics of ghrelin in H(2) receptor (H(2)R)-null mice by genetic H(2)R knockout. METHODS: Fifteen-week- and 54-week-old H(2)R-null mice and their littermates were used. After evaluating the levels of food intake and body-weight increments, mice were killed, and the plasma and gastric active and total ghrelin levels were examined by radioimmunoassay, and gastric preproghrelin mRNA expression was examined by quantitative reverse transcriptase-polymerase chain reaction. Furthermore, each stomach specimen was evaluated by immunohistochemistry and transmission electron microscopy for ghrelin. RESULTS: The levels of food intake and body-weight gain of the H(2)R-null mice were higher than those of wild-type mice. The gastric pH of the 54-week-old H(2)R-null mice was lower than that of the 15-week-old mice. Gastric preproghrelin mRNA expression, plasma ghrelin level, and density of ghrelin-immunoreactive cells in the gastric mucosa of the H(2)R-null mice were significantly increased compared with those of the wild-type mice. Ghrelin-positive immunogold density seen in the electron micrograph was significantly reduced in A-like cells of the H(2)R-null mouse stomach. CONCLUSIONS: Ghrelin production and secretion from A-like cells in the gastric fundus are upregulated in H(2)R-null mice, a genetic H(2)R knockout model.
Assuntos
Células Enterocromafins/metabolismo , Ácido Gástrico/metabolismo , Expressão Gênica , Grelina/genética , RNA Mensageiro/genética , Animais , Peso Corporal/fisiologia , Células Enterocromafins/ultraestrutura , Determinação da Acidez Gástrica , Grelina/biossíntese , Antagonistas dos Receptores H2 da Histamina/farmacologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , RNA Mensageiro/biossíntese , Radioimunoensaio , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND/AIMS: Sonic hedgehog (Shh) is a morphogen involved in many aspects of patterning of the gut during embryogenesis and in gastric fundic gland homeostasis in the adult. Shh expression is reportedly to be reduced in Helicobacterpylori-associated gastritis. The aim of this study was to assess the restoration of Shh expression after H. pylori eradication. METHODOLOGY: Twenty H. pylori-positive patients who underwent upper gastrointestinal endoscopy before and after the eradication were studied. Biopsy specimens were taken from the greater curvature of the middle third of the stomach body. The specimens were evaluated for the severity of acute and chronic inflammation and for that of mucosal atrophy, based on the updated Sydney system. Immunohistochemistry for Shh and H(+)-, K(+)-ATPase was also performed; the percentages of positively stained epithelial cells for the two molecules were expressed as the Shh index and H(+)-, K(+)-ATPase index, respectively. RESULTS: There was a significant decrease in the acute and chronic inflammation scores and also in the mucosal atrophy score following the eradication. The Shh and H(+)-, K(+)-ATPase index were significantly increased following the eradication. CONCLUSIONS: Suppressed Shh expression in the gastric mucosa by H. pylori infection was significantly restored following eradication of the infection.
Assuntos
Mucosa Gástrica/metabolismo , Proteínas Hedgehog/metabolismo , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori , Gastropatias/tratamento farmacológico , Adulto , Idoso , Quimioterapia Combinada , Feminino , ATPase Trocadora de Hidrogênio-Potássio/análise , Proteínas Hedgehog/análise , Proteínas Hedgehog/antagonistas & inibidores , Infecções por Helicobacter/metabolismo , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/isolamento & purificação , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Estômago/microbiologia , Estômago/patologia , Gastropatias/microbiologia , Gastropatias/patologiaRESUMO
Sonic hedgehog (Shh) is a morphogen involved in many aspects of patterning of the gut during embryogenesis and in gastric fundic gland homeostasis in the adult. Intestinal metaplastic change of the gastric epithelium is associated with the loss of Shh expression, and mice that lack Shh expression show intestinal transformation of the gastric mucosa. The present study was designed to investigate the alteration of Shh expression in the stomach of an experimental model of Helicobacter pylori (H. pylori) colonization. Male Mongolian gerbils were inoculated with H. pylori and examined 4 and 51 weeks later. The level of Shh mRNA expression was determined by quantitative RT-PCR and in situ hybridization. Shh protein expression was determined by immunoblotting and immunohistochemistry. Shh was expressed in the parietal cells, zymogenic cells, and mucous neck cells of the gastric fundic glands of gerbils. Prolonged colonization by H. pylori led to extension of the inflammation from the antrum to the corpus of the stomach, with loss of Shh expression. Loss of Shh expression correlated with loss of parietal cells, disturbed maturation of the mucous neck cell-zymogenic cell lineage, and increased cellular proliferation. Shh expression is significantly reduced in H. pylori-associated gastritis. These data show for the first time that H. pylori infection leads to down-regulation of the expression of a morphogen with an established role (Shh) in gastric epithelial differentiation.
Assuntos
Mucosa Gástrica/metabolismo , Gastrite Atrófica/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori , Transativadores/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Modelos Animais de Doenças , Regulação para Baixo , Mucosa Gástrica/microbiologia , Gastrite Atrófica/genética , Gastrite Atrófica/microbiologia , Gerbillinae , Proteínas Hedgehog , Infecções por Helicobacter/genética , Técnicas Imunoenzimáticas , Hibridização In Situ , Masculino , Dados de Sequência Molecular , RNA Mensageiro/genética , Transativadores/genéticaRESUMO
Ghrelin, produced and secreted by the A-like cells of the stomach, stimulates growth hormone secretion, gastric motility, and food intake. Cysteamine inhibits the release of somatostatin and induces the formation of duodenal ulcers in rats. The present study was conducted to investigate the dynamics of ghrelin secretion in rats treated with cysteamine. Male Wistar rats (7 wk old) were administered three doses of cysteamine (400 mg/kg) orally; at 50 h after the first dose, duodenal ulcers were induced, and the plasma level of somatostatin and gastric density of somatostatin-immunoreactive cells were significantly reduced. The plasma total and active ghrelin levels were significantly higher in the cysteamine-treated rats than in the control rats, whereas the gastric ghrelin levels, number of gastric ghrelin-immunoreactive cells, and preproghrelin mRNA expression levels were significantly lower. Even at the time points of 2 and 10 h after the first dose of cysteamine, at which time no significant ulcer formation or antral neutrophil accumulation was yet noted, a significant increase in the plasma ghrelin level and decrease in the gastric ghrelin level were observed. Furthermore, although lansoprazole treatment attenuated the duodenal ulceration induced by cysteamine, the increase in the plasma level of ghrelin could still be demonstrated. Because an inverse correlation was found between the plasma ghrelin and somatostatin levels, the inhibition of somatostatin secretion may be associated with the increased ghrelin secretion. In conclusion, an increase in the plasma ghrelin level precedes the formation of duodenal ulcers in rats treated with cysteamine.