RESUMO
The emergence of drug-resistant Mycobacterium tuberculosis (M.tb) has led to the development of novel anti-tuberculosis (anti-TB) drugs. Common methods for testing the efficacy of new drugs, including two-dimensional cell culture models or animal models, have several limitations. Therefore, an appropriate model representative of the human organism is required. Here, we developed an M.tb infection model using human lung organoids (hLOs) and demonstrated that M.tb H37Rv can infect lung epithelial cells and human macrophages (hMφs) in hLOs. This novel M.tb infection model can be cultured long-term and split several times while maintaining a similar number of M.tb H37Rv inside the hLOs. Anti-TB drugs reduced the intracellular survival of M.tb in hLOs. Notably, M.tb growth in hLOs was effectively suppressed at each passage by rifampicin and bedaquiline. Furthermore, a reduction in inflammatory cytokine production and intracellular survival of M.tb were observed upon knockdown of MFN2 and HERPUD1 (host-directed therapeutic targets for TB) in our M.tb H37Rv-infected hLO model. Thus, the incorporation of hMφs and M.tb into hLOs provides a powerful strategy for generating an M.tb infection model. This model can effectively reflect host-pathogen interactions and be utilized to test the efficacy of anti-TB drugs and host-directed therapies.
Assuntos
Antituberculosos , Pulmão , Mycobacterium tuberculosis , Organoides , Humanos , Organoides/microbiologia , Mycobacterium tuberculosis/efeitos dos fármacos , Pulmão/microbiologia , Pulmão/patologia , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia , Macrófagos/microbiologia , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Células Epiteliais/microbiologiaRESUMO
Alternative pre-mRNA splicing is key to proteome diversity; however, the biological roles of alternative splicing (AS) in signaling pathways remain elusive. Here, we focus on TEA domain transcription factor 1 (TEAD1), a YAP binding factor in the Hippo signaling pathway. Public database analyses showed that expression of YAP-TEAD target genes negatively correlated with the expression of a TEAD1 isoform lacking exon 6 (TEAD1ΔE6) but did not correlate with overall TEAD1 expression. We confirmed that the transcriptional activity and oncogenic properties of the full-length TEAD1 isoform were greater than those of TEAD1ΔE6, with the difference in transcription related to YAP interaction. Furthermore, we showed that RNA-binding Fox-1 homolog 2 (RBFOX2) promoted the inclusion of TEAD1 exon 6 via binding to the conserved GCAUG element in the downstream intron. These results suggest a regulatory mechanism of RBFOX2-mediated TEAD1 AS and provide insight into AS-specific modulation of signaling pathways.
Assuntos
Proteínas de Ligação a DNA , Fatores de Transcrição , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Processamento Alternativo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/metabolismoRESUMO
Under various cellular stress conditions, including exposure to toxic chemicals, RNA-binding proteins (RBPs), including Ras GTPase-activating protein-binding protein 1 (G3BP1), aggregate and form stress granule complexes, which serve as hallmarks of cellular stress. The existing methods for analyzing stress granule assembly have limitations in the rapid detection of dynamic cellular stress and ignore the effects of constitutively overexpressed RBP on cellular stress and stress-related processes. Therefore, to overcome these limitations, we established a G3BP1-GFP reporter in a human lung epithelial cell line using CRISPR/Cas9-based knock-in as an alternative system for stress granule analysis. We showed that the G3BP1-GFP reporter system responds to stress conditions and forms a stress granule complex similar to that of native G3BP1. Furthermore, we validated the stress granule response of an established cell line under exposure to various household chemicals. Overall, this novel G3BP1-GFP reporter human lung cell system is capable of monitoring stress granule dynamics in real time and can be used for assessing the lung toxicity of various substances in vitro.
Assuntos
DNA Helicases , Pulmão , RNA Helicases , Grânulos de Estresse , Humanos , DNA Helicases/metabolismo , Pulmão/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Grânulos de Estresse/metabolismo , Proteínas de Fluorescência Verde , Genes ReporterRESUMO
Inhibition of lipid synthesis in sebocytes is essential for acne treatments. The effects of natural product-derived substances on lipid synthesis are unknown. This study investigated the effects of water extract of Mangifera indica leaves (WEML) on lipid synthesis in human sebocytes. Sebocyte differentiation in low serum conditions increased lipid accumulation and proliferator-activated receptor γ expression. WEML treatment significantly inhibited lipid accumulation and adipogenic mRNA expression in sebocytes. Mangiferin, a bioactive compound in WEML, also reduced lipid accumulation and adipogenic mRNA expression via the AKT pathway. Thus, WEML and mangiferin effectively inhibit lipid synthesis in sebocytes, showing promise for acne treatment.
RESUMO
Alternative pre-mRNA splicing is a critical mechanism that generates multiple mRNA from a single gene, thereby increasing the diversity of the proteome. Recent research has highlighted the significance of specific splicing isoforms in cellular processes, particularly in regulating cell numbers. In this review, we examine the current understanding of the role of alternative splicing in controlling cancer cell growth and discuss specific splicing factors and isoforms and their molecular mechanisms in cancer progression. These isoforms have been found to intricately control signaling pathways crucial for cell cycle progression, proliferation, and apoptosis. Furthermore, studies have elucidated the characteristics and functional importance of splicing factors that influence cell numbers. Abnormal expression of oncogenic splicing isoforms and splicing factors, as well as disruptions in splicing caused by genetic mutations, have been implicated in the development and progression of tumors. Collectively, these findings provide valuable insights into the complex interplay between alternative splicing and cell proliferation, thereby suggesting the potential of alternative splicing as a therapeutic target for cancer.
RESUMO
The autophagy-mediated lysosomal pathway plays an important role in conferring stress tolerance to tumor cells during cellular stress such as increased metabolic demands. Thus, targeted disruption of this function and inducing lysosomal cell death have been proved to be a useful cancer therapeutic approach. In this study, we reported that octyl syringate (OS), a novel phenolic derivate, was preferentially cytotoxic to various cancer cells but was significantly less cytotoxic to non-transformed cells. Treatment with OS resulted in non-apoptotic cell death in a caspase-independent manner. Notably, OS not only enhanced accumulation of autophagic substrates, including lapidated LC3 and sequestosome-1, but also inhibited their degradation via an autophagic flux. In addition, OS destabilized the lysosomal function, followed by the intracellular accumulation of the non-digestive autophagic substrates such as bovine serum albumin and stress granules. Furthermore, OS triggered the release of lysosomal enzymes into the cytoplasm that contributed to OS-induced non-apoptotic cell death. Finally, we demonstrated that OS was well tolerated and reduced tumor growth in mouse xenograft models. Taken together, our study identifies OS as a novel anticancer agent that induces lysosomal destabilization and subsequently inhibits autophagic flux and further supports development of OS as a lysosome-targeting compound in cancer therapy. ⢠Octyl syringate, a phenolic derivate, is preferentially cytotoxic to various cancer cells. ⢠Octyl syringate destabilizes the lysosomal function. ⢠Octyl syringate blocks the autophagic flux. ⢠Octyl syringate is a potential candidate compound for cancer therapy.
Assuntos
Antineoplásicos , Neoplasias , Camundongos , Animais , Humanos , Apoptose , Antineoplásicos/farmacologia , Morte Celular , Autofagia , Lisossomos/metabolismo , Neoplasias/metabolismoRESUMO
Human lung organoids (hLOs) are useful for disease modelling and drug screening. However, a lack of immune cells in hLOs limits the recapitulation of in vivo cellular physiology. Here, we generated hLOs containing alveolar macrophage (AMφ)-like cells derived from pluripotent stem cells (PSC). To bridge hLOs with advanced human lung high-resolution X-ray computed tomography (CT), we acquired quantitative micro-CT images. Three hLO types were observed during differentiation. Among them, alveolar hLOs highly expressed not only lung epithelial cell markers but also AMφ-specific markers. Furthermore, CD68+ AMφ-like cells were spatially organized on the luminal epithelial surface of alveolar hLOs. Bleomycin-treated alveolar hLOs showed upregulated expression of fibrosis-related markers and extracellular matrix deposits in the alveolar sacs. Alveolar hLOs also showed structural alterations such as excessive tissue fraction under bleomycin treatment. Therefore, we suggest that micro-CT analyzable PSC-derived alveolar hLOs are a promising in vitro model to predict lung toxicity manifestations, including fibrosis.
Assuntos
Células-Tronco Pluripotentes , Fibrose Pulmonar , Células Epiteliais Alveolares , Bleomicina/metabolismo , Humanos , Pulmão , Macrófagos Alveolares , Organoides , Células-Tronco Pluripotentes/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Microtomografia por Raio-XRESUMO
Early-life exposure to bisphenol A (BPA), synthetic compound used in polycarbonate plastic, is associated with altered cognitive and emotional behavior later in life. However, the brain mechanism underlying the behavioral deficits is unknown. Here, we show that maternal BPA exposure disrupted self-renewal and differentiation of neural progenitors during cortical development. The BPA exposure reduced the neuron number, whereas it increased glial cells in the cerebral cortex. Also, synaptic formation and transmission in the cerebral cortex were suppressed after maternal BPA exposure. These changes appeared to be associated with autophagy as a gene ontology analysis of RNA-seq identified an autophagy domain in the BPA condition. Mouse behavioral tests revealed that maternal BPA caused hyperactivity and social deficits in adult offspring. Together, these results suggest that maternal BPA exposure leads to abnormal cortical architecture and function likely by activating autophagy.
Assuntos
Compostos Benzidrílicos , Efeitos Tardios da Exposição Pré-Natal , Animais , Compostos Benzidrílicos/toxicidade , Córtex Cerebral , Camundongos , Neurogênese , Fenóis/toxicidade , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamenteRESUMO
Polyhexamethylene guanidine phosphate (PHMG-p), a humidifier disinfectant, is known to cause lung toxicity, including inflammation and pulmonary fibrosis. In this study, we aimed to investigate the effect of PHMG-p on human lung tissue models (2D epithelial cells and 3D organoids) under conditions of oxidative stress and viral infection. The effect of PHMG-p was studied by evaluating the formation of stress granules (SGs), which play a pivotal role in cellular adaptation to various stress conditions. Under oxidative stress and respiratory syncytial virus (RSV) infection, exposure to PHMG-p remarkably increased eIF2α phosphorylation, which is essential for SG-related signalling, and significantly increased SG formation. Furthermore, PHMG-p induced fibrotic gene expression and caused cell death due to severe DNA damage, which was further increased under oxidative stress and RSV infection, indicating that PHMG-p induces severe lung toxicity under stress conditions. Taken together, toxicity evaluation under various stressful conditions is necessary to accurately predict potential lung toxicity of chemicals affecting the respiratory tract.
Assuntos
Infecções por Vírus Respiratório Sincicial , Grânulos de Estresse , Guanidinas/toxicidade , Humanos , Pulmão , OrganoidesRESUMO
Bisphenol A (BPA) is a well-known endocrine-disrupting chemical that is widely used in a variety of products, including plastics, medical equipment and receipts. Hence, most people are exposed to BPA through the skin, via inhalation and via the digestive system, and such exposure has been linked to cardiovascular diseases including coronary artery disease, hypertension, atherosclerosis, and myocardial infarction. However, the underlying mechanisms of cardiac dysfunction caused by BPA remain poorly understood. In this study, we found that BPA exposure altered cardiac function in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Acute BPA exposure in hiPSC-CMs resulted in reduced field potential, as measured by multielectrode array (MEA). Furthermore, we observed that BPA dose-dependently inhibited ICa, INa or IKr channels. In addition, BPA exposure dose-dependently inhibited calcium transients and contraction in hiPSC-CMs. Our findings suggest that BPA exposure leads to cardiac dysfunction and cardiac risk factors such as arrhythmia.
Assuntos
Poluentes Ocupacionais do Ar/toxicidade , Compostos Benzidrílicos/toxicidade , Cardiotoxinas/toxicidade , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Fenóis/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologiaRESUMO
Magnesium is well known as a biodegradable biomaterial that has been reported to promote bone remodeling in several studies; however, the underlying biological mechanism remains unclear. In the present study, the role of magnesium ions in the migration of U-2 OS cells, which are osteoblast-like cell lines, was investigated. Magnesium treatment did not significantly alter the global transcriptome of U-2 OS cells, but increased the protein expression level of SNAI2, an epithelial-mesenchymal transition (EMT) marker. In addition, it was confirmed that the junctional site localization of Zona-occludens 1 (ZO-1), a representative tight junction protein, was destroyed by magnesium treatment; furthermore, it was determined that cytoplasmic localization increased, and alkaline phosphatase (ALP) activity increased. The obtained results on the mechanism by which magnesium is involved in osteoblast migration, which is important for fracture healing, will contribute to the understanding of the bone-formation process in patients with osteoporosis and musculoskeletal injury.
Assuntos
Cloreto de Magnésio/farmacologia , Osteoblastos/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Transição Epitelial-Mesenquimal/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Microscopia de Fluorescência , Osteoblastos/citologia , Osteoblastos/metabolismo , Análise de Sequência de RNA , Fatores de Transcrição da Família Snail/metabolismo , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Calcific tendinopathy (CT), developed due to calcium hydroxyapatite deposition in the rotator cuff tendon, mostly affects women in their 40 s and 50 s and causes severe shoulder pain. However, the molecular basis of its pathogenesis and appropriate treatment methods are largely unknown. In this study, we identified 202 differentially expressed genes (DEGs) between calcific and adjacent normal tendon tissues of rotator cuff using RNA sequencing-based transcriptome analysis. The DEGs were highly enriched in extracellular matrix (ECM) degradation and inflammation-related processes. Further, matrix metalloproteinase 9 (MMP9) and matrix metalloproteinase 13 (MMP13), two of the enzymes associated with ECM degradation, were found to be highly upregulated 25.85- and 19.40-fold, respectively, in the calcific tendon tissues compared to the adjacent normal tendon tissues. Histopathological analyses indicated collagen degradation and macrophage infiltration at the sites of calcific deposit in the rotator cuff tendon. Our study acts as a foundation that may help in better understanding of the pathogenesis associated with CT, and thus in better management of the disease.
Assuntos
Calcinose/genética , Matriz Extracelular/patologia , Manguito Rotador/patologia , Análise de Sequência de RNA , Tendinopatia/genética , Feminino , HumanosRESUMO
BACKGROUND: The selection of reference genes is essential for quantifying gene expression. Theoretically they should be expressed stably and not regulated by experimental or pathological conditions. However, identification and validation of reference genes for human cancer research are still being regarded as a critical point, because cancerous tissues often represent genetic instability and heterogeneity. Recent pan-cancer studies have demonstrated the importance of the appropriate selection of reference genes for use as internal controls for the normalization of gene expression; however, no stably expressed, consensus reference genes valid for a range of different human cancers have yet been identified. RESULTS: In the present study, we used large-scale cancer gene expression datasets from The Cancer Genome Atlas (TCGA) database, which contains 10,028 (9,364 cancerous and 664 normal) samples from 32 different cancer types, to confirm that the expression of the most commonly used reference genes is not consistent across a range of cancer types. Furthermore, we identified 38 novel candidate reference genes for the normalization of gene expression, independent of cancer type. These genes were found to be highly expressed and highly connected to relevant gene networks, and to be enriched in transcription-translation regulation processes. The expression stability of the newly identified reference genes across 29 cancerous and matched normal tissues were validated via quantitative reverse transcription PCR (RT-qPCR). CONCLUSIONS: We reveal that most commonly used reference genes in current cancer studies cannot be appropriate to serve as representative control genes for quantifying cancer-related gene expression levels, and propose in this study three potential reference genes (HNRNPL, PCBP1, and RER1) to be the most stably expressed across various cancerous and normal human tissues.
Assuntos
Pesquisa Biomédica , Regulação Neoplásica da Expressão Gênica , Genes , Neoplasias/genética , Proteínas Adaptadoras de Transporte Vesicular , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Humanos , Glicoproteínas de Membrana , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Calcific tendinopathy (CT) is characterized by deposits of calcium, most commonly found in the shoulder tendons. The exact cause and pathogenesis of CT are not fully understood. This study analyzed the expression pattern of RNA-binding protein fox-1 homolog 2 (RBFOX2), a crucial splicing regulator in tissue differentiation. METHODS: Normal and calcific tendons were compared for RBFOX2 mRNA level using quantitative reverse-transcription polymerase chain reaction. Intracellular localization of RBFOX2 protein was investigated using immunofluorescence microscopy. Normal and calcific tendon cDNAs were used to clone RBFOX2. Sequencing analysis identified coding sequences of the RBFOX2 isoform. RESULTS: The intracellular localization of RBFOX2 protein differed with disease status, with RBFOX2 localized in the cytoplasm in calcific tendons and the nucleus in normal tendons. Analysis of the RBFOX2 protein-coding sequence showed that exon 10, responsible for nuclear localization, was absent in calcific tendons. Splicing of RBFOX2 target genes CHD2 and MBNL1 was significantly affected by cytoplasmic localization of RBFOX2 in calcific tendons. DISCUSSION: Given the function of RBFOX2 as a splicing regulator in the nucleus, cytoplasmic localization of RBFOX2 protein in calcific tendons may have affected overall splicing events and altered gene expression. These results provide insights for comprehension of CT pathogenesis.
Assuntos
Processamento Alternativo , Citoplasma/genética , Fatores de Processamento de RNA/genética , Proteínas Repressoras/genética , Tendinopatia/genética , Idoso , Sequência de Aminoácidos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Éxons/genética , Feminino , Células HeLa , Humanos , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteínas Repressoras/metabolismo , Homologia de Sequência de Aminoácidos , Tendinopatia/diagnóstico , Tendinopatia/metabolismo , Tendões/metabolismo , Tendões/patologiaRESUMO
Claudin 1, a major tight junction protein, is highly expressed in various types of tumors such as thyroid, breast, and colorectal cancers. Moreover, claudin 1 is frequently found in the cytoplasm in various types of tumor cells. However, the cytoplasmic function of claudin 1 in tumors still remains largely unknown. Here, we investigated the novel function of cytoplasmic claudin 1 in autophagy. The mRNA expression level of claudin 1 was higher in several types of tumors than in normal tissues. Furthermore, colon tumor tissues showed increased autophagy compared with the adjacent normal tissues. Both endogenous and exogenous claudin 1 showed a cytoplasmic punctate staining pattern and were co-stained with the lysosome-associated membrane protein 1 (LAMP1). Importantly, autophagy-induced conditions, including starvation, increased the protein stability of claudin 1. Moreover, the increased level of claudin 1 stimulated autophagy by decreasing the level of the autophagy substrate, sequestosome1/p62 (SQSTM1), under autophagy-inducing conditions; activation of AMP-activated protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR). Taken together, we demonstrate that the novel function of cytoplasmic claudin 1 is related to autophagy. This study is the first to show a cytoplasmic function of claudin 1 as an autophagy regulator and provides the evidence that claudin 1-mediated autophagy regulation is an integral part of the mechanism by which claudin 1 regulates cancer progression.
Assuntos
Autofagia/fisiologia , Hipóxia Celular , Claudina-1/metabolismo , Glucose/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína Sequestossoma-1/metabolismo , Citoplasma/metabolismo , Células HeLa , Humanos , Células MCF-7 , Regulação para CimaRESUMO
Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by in vivo crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing.
Assuntos
Processamento Alternativo , Antígenos Nucleares/genética , Epigênese Genética , Histonas/genética , Proteínas do Tecido Nervoso/genética , Fator de Processamento Associado a PTB/genética , Fatores de Processamento de RNA/genética , Proteínas Repressoras/genética , Antígenos Nucleares/metabolismo , Cromatina/química , Cromatina/metabolismo , Reagentes de Ligações Cruzadas/química , Células HeLa , Histonas/metabolismo , Humanos , Imunoprecipitação , Metilação , Proteínas do Tecido Nervoso/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Spliceossomos/genética , Spliceossomos/metabolismo , Succinimidas/químicaRESUMO
Rbfox family of proteins that consists of Rbfox1, Rbfox2, and Rbfox3 in mammals regulates alternative pre-mRNA splicing in various tissues via direct binding to their RNA binding element. Although many studies have indicated the splicing activity of each member of the Rbfox family, the interactions of Rbfox family proteins are largely unknown. Here, we have investigated interactions among Rbfox family proteins. Co-immunoprecipitation (Co-IP) and GST-pull down assays confirmed that Rbfox proteins form homo and hetero complexes. Moreover, in vivo crosslinking using disuccinimidyl suberate treatment indicated that the Rbfox proteins form a dimer which then assembles with other proteins to form a large multiprotein complex. Duolink in situ proximity ligation (PLA) assay revealed that neuron specific Rbfox3 protein interacts with other Rbfox family proteins. This study is the first to provide an evidence that Rbfox family proteins form homo- and hetero-oligomeric complexes in vivo.
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Neurônios/química , Neurônios/metabolismo , Multimerização Proteica/fisiologia , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Animais , Encéfalo/metabolismo , Química Encefálica , Células Cultivadas , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Atopic dermatitis (AD) is a chronic inflammatory skin disease. Many studies investigating AD pathogenesis and its therapy have been conducted but none have been successful. One of the causes of AD is dysfunction of tight junctions through reduction of claudin 1 expression in the epidermal barrier of the skin. In the present study, we investigated the role of bortezomib (BTZ) in the restoration of the reduced expression of claudin 1. Immunoblot and immunofluorescence analyses revealed that BTZ increased the protein expression level of claudin 1 in the human keratinocyte cell line HaCaT, thereby forming paracellular barriers. Furthermore, repeated application of BTZ alleviated atopic symptoms on the backs and ears of 2, 4-dinitrochlorobenzene (DNCB)-induced AD mice, and led to the formation of normal tight junctions in the epidermal barrier of DNCB-induced mice skin. Taken together, these results demonstrate that BTZ-induced claudin 1 expression may be a valuable therapeutic approach for AD.
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Bortezomib/administração & dosagem , Claudina-1/metabolismo , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/metabolismo , Inibidores de Proteassoma/administração & dosagem , Junções Íntimas/efeitos dos fármacos , Animais , Linhagem Celular , Dermatite Atópica/patologia , Relação Dose-Resposta a Droga , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Junções Íntimas/patologia , Resultado do TratamentoRESUMO
Magnesium (Mg) ions are the most abundant intracellular divalent cations and play a pivotal role in numerous cellular processes. Biodegradable Mg-containing materials, including scaffolds, are promising candidates for orthopedic applications. Here, we investigated the effect of Mg ions on the cellular properties of osteoblasts. Cytotoxicity tests on osteoblasts confirmed that no cytotoxic effects were found up to a supplementing Mg ion concentration of 10 mM. Mg ions at a concentration of 5 mM increased the migration and invasiveness of osteoblasts. To investigate the stimulatory effect of Mg ions on cell motility in scaffolds, we fabricated 10 wt% Mg ion-containing polycaprolactone (PCL) scaffolds, using the wire-network molding (WNM) technique. Mg ion-containing scaffolds persistently released Mg ions at a concentration of 5 mM in the media after pre-incubation. Furthermore, increased cell motility was confirmed in Mg ion-containing scaffolds by quantification of genomic DNA and protein content. Our results provide an important basis for the function of Mg ions and their effect on cell motility, and propose a novel role for Mg ions in scaffold applications.
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Movimento Celular/efeitos dos fármacos , Magnésio/química , Osteoblastos/metabolismo , Alicerces Teciduais/química , Materiais Biocompatíveis/farmacologia , Substitutos Ósseos/farmacologia , Cátions , DNA/química , Humanos , Teste de Materiais , Microscopia de Fluorescência , Osteogênese/efeitos dos fármacos , Poliésteres/química , Porosidade , Engenharia Tecidual/métodosRESUMO
Western blotting (WB) analysis is the most popular and widely used methodology for protein detection and characterization over recent decades. In accordance with the advancement of the technologies for the acquisition of WB signals, a quantitative value is used to present the abundance of target proteins in a complex sample, thereby requiring the use of specific proteins as internal references that represent total proteins. Heretofore, proteins encoded by housekeeping genes such as GAPDH, ß-tubulin and ß-actin have been commonly used as loading controls without any hesitation because their mRNA expression levels tend to be high and constant in many different cells and tissues. Experimentally, however, some of the housekeeping reference proteins are often displayed with inconsistent expression levels in both homogeneous and heterogeneous tissues, and, in terms of mRNA levels, they have a weak correlation to the abundance of proteins. To estimate accurate, reliable, and reproducible protein quantifications, it is crucial to define appropriate reference controls. For this paper, we explored the recently released large-scale, human proteomic database ProteomicsDB including 16 857 liquid chromatography tandem-mass-spectrometry data from 27 human tissues, and suggest 20 ubiquitously- and constitutively-expressed, putative internal-reference controls for the quantification of differential protein expressions. Intriguingly, the most commonly used, known housekeeping genes were entirely excluded in our newly defined candidates. Although the applications of the candidates under many different biological conditions and in other organisms are yet to be empirically verified, we propose reliable, potential loading controls for a WB analysis in this paper.