RESUMO
Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) has been frequently overexpressed in many types of malignancy, suggesting its oncogenic function. It recognizes phosphorylated serine or threonine (pSer/Thr) of a target protein and isomerizes the adjacent proline (Pro) residue, thereby altering folding, subcellular localization, stability, and function of target proteins. The oncogenic transcription factor, Nrf2 harbors the pSer/Thr-Pro motif. This prompted us to investigate whether Pin1 could bind to Nrf2 and influence its stability and function in the context of implications for breast cancer development and progression. The correlation between Pin1 and Nrf2 in the triple-negative breast cancer cells was validated by RNASeq analysis as well as immunofluorescence staining. Interaction between Pin1 and Nrf2 was assessed by co-immunoprecipitation and an in situ proximity ligation assay. We found that mRNA and protein levels of Pin1 were highly increased in the tumor tissues of triple-negative breast cancer patients and the human breast cancer cell line. Genetic or pharmacologic inhibition of Pin1 enhanced the ubiquitination and degradation of Nrf2. In contrast, the overexpression of Pin1 resulted in the accumulation of Nrf2 in the nucleus, without affecting its transcription. Notably, the phosphorylation of Nrf2 at serine 215, 408, and 577 is essential for its interaction with Pin1. We also identified phosphorylated Ser104 and Thr277 residues in Keap1, a negative regulator of Nrf2, for Pin1 binding. Pin1 plays a role in breast cancer progression through stabilization and constitutive activation of Nrf2 by competing with Keap1 for Nrf2 binding.
Assuntos
Neoplasias da Mama/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Neoplasias da Mama/genética , Feminino , Células HEK293 , Humanos , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Peptidilprolil Isomerase de Interação com NIMA/genética , Proteínas de Neoplasias/genética , Ligação Proteica , Estabilidade Proteica , Proteólise , UbiquitinaçãoRESUMO
Alveolar organoids (AOs), derived from human pluripotent stem cells (hPSCs) exhibit lung-specific functions. Therefore, the application of AOs in pulmonary disease modeling is a promising tool for understanding disease pathogenesis. However, the lack of immune cells in organoids limits the use of human AOs as models of inflammatory diseases. In this study, we generated AOs containing a functional macrophage derived from hPSCs based on human fetal lung development using biomimetic strategies. We optimized culture conditions to maintain the iMACs (induced hPSC-derived macrophages) AOs for up to 14 days. In lipopolysaccharide (LPS)-induced inflammatory conditions, IL-1ß, MCP-1 and TNF-α levels were significantly increased in iMAC-AOs, which were not detected in AOs. In addition, chemotactic factor IL-8, which is produced by mononuclear phagocytic cells, was induced by LPS treatment in iMACs-AOs. iMACs-AOs can be used to understand pulmonary infectious diseases and is a useful tool in identifying the mechanism of action of therapeutic drugs in humans. Our study highlights the importance of immune cell presentation in AOs for modeling inflammatory pulmonary diseases.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Diferenciação Celular , Humanos , Lipopolissacarídeos/farmacologia , Pulmão , Macrófagos , OrganoidesRESUMO
The hypoxia-inducible factor (HIF-1α) functions as a master regulator of oxygen homeostasis. Oxygen-dependent hydroxylation of HIF-1α is tightly regulated by prolyl hydroxylase domain containing proteins (PHD1, PHD2, and PHD3). The prolyl hydroxylation facilitates the recruitment of the von Hippel-Lindau (VHL) protein, leading to ubiquitination and degradation of HIF-1α by the proteasomes. Besides prolyl hydroxylation, phosphorylation of HIF-1α is another central post-translational modification, which regulates its stability under hypoxic conditions as well as normoxic conditions. By use of LC/MS/MS-based analysis, we were able to identify a specific serine residue (Ser451) of HIF-1α phosphorylated under hypoxic conditions. Using plasmids expressing wild type (WT), non-phosphorylatable mutant HIF-1α (S451A), and phosphomimetic mutant HIF-1α (S451E), we demonstrated that the phosphorylation at Ser451 is important in maintaining the HIF-1α protein stability. Notably, phosphorylation at S451 interrupts the interaction of HIF-1α with PHD and pVHL. A phosphomimetic construct of HIF-1α at Ser451 (S451E) is significantly more stable than WT HIF-1α under normoxic conditions. Cells transfected with unphosphorylatable HIF-1α exhibited significantly lower HIF-1 transcriptional activity than WT cells and markedly reduced tumor cell migration. Further, tumors derived from the phosphomimetic mutant cells grew faster, whereas the tumors derived from non-phosphorylatable mutant cells grew slower than the control tumors, suggesting that the phosphorylation of HIF-1α at the Ser451 site is critical to promote tumor growth in vivo. Taken together, our data suggest an alternative mechanism responsible for the regulation of HIF-1α stability.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Substituição de Aminoácidos , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Hipóxia Celular , Células HCT116 , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Modelos Biológicos , Mutagênese Sítio-Dirigida , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fosforilação , Prolil Hidroxilases/química , Prolil Hidroxilases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Serina/química , Proteína Supressora de Tumor Von Hippel-Lindau/química , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
PIN1, the peptidyl-prolyl isomerase (PPIase), is an enzyme that changes the conformation of phosphoproteins. The conformational change induced by PIN1 alters the function and stability of the target proteins. PIN1 is overexpressed in many different types of malignancies, including breast, lung, cervical, brain and colorectal tumors. PIN1 overexpression has been associated with activation of multiple oncogenic signaling pathways during tumor development. Hypoxia-inducible factor 2α (HIF-2α), a transcription factor activated in hypoxia, plays a role in erythropoiesis, glycolysis, tissue invasion, metastasis and angiogenesis. In this study, we found the direct interaction between HIF-2α and PIN1 in colorectal cancer HCT116 cells. Notably, serine 16 and lysine 63 residues of PIN1 were critical for its interaction with HIF-2α. When PIN1 protein was silenced by transient transfection of PIN1 short interfering RNA, the expression of HIF-2α was attenuated under a hypoxic condition. Moreover, genetic and pharmacologic inhibition of PIN1 abrogated the expression of vascular endothelial growth factor and angiogenesis. The cycloheximide chase experiment revealed the stabilization of HIF-2α by PIN1. Both WW and PPIase domains of PIN1 appear to be critical for its interaction with HIF-2α.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Neovascularização Patológica/etiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Embrião de Galinha , Feminino , Células HCT116 , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Células NIH 3T3 , Peptidilprolil Isomerase de Interação com NIMA/química , Peptidilprolil Isomerase de Interação com NIMA/genética , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Interferência de RNA , RNA Interferente Pequeno/genética , Hipóxia Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Aberrant activation of H-Ras is often associated with tumor aggressiveness in breast cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) is a unique enzyme that interacts with phosphorylated serine or threonine of a target protein and isomerizes the adjacent proline residue. Pin1 is prevalently overexpressed in human cancers, and its overexpression correlates with poor prognosis. Nuclear factor E2-related factor 2 (Nrf2) is a master regulator of cellular redox homeostasis. The sustained activation/accumulation of Nrf2 has been observed in many different types of human malignancies, conferring an advantage for growth and survival of cancer cells. The activated form of H-Ras (GTP-H-Ras) is highly overexpressed in human breast cancer tissues. In our present study, silencing of H-Ras decreased the invasiveness of MDA-MB-231 human breast cancer cells and abrogated the interaction between Pin1 and Nrf2 in these cells. Pin1 knockdown blocked the accumulation of Nrf2, thereby suppressing proliferation and clonogenicity of MCF10A-Ras human mammary epithelial cells. We found that Pin1 binds to Nrf2 which stabilizes this transcription factor by hampering proteasomal degradation. In conclusion, H-Ras activation in cooperation with the Pin1-Nrf2 complex represents a novel mechanism underlying breast cancer progression and constitutive activation of Nrf2 and can be exploited as a therapeutic target.
Assuntos
Neoplasias da Mama/metabolismo , Genes ras/fisiologia , Fator 2 Relacionado a NF-E2/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Genes ras/genética , Células HEK293 , Humanos , Fator 2 Relacionado a NF-E2/genética , Peptidilprolil Isomerase de Interação com NIMA/genéticaRESUMO
Helicobacter pylori (H. pylori) infection has been known to be implicated in human gastric carcinogenesis. Snail, the zinc-finger transcription factor known as a key inducer of changes in the cell shape and morphogenetic movement, is aberrantly overexpressed and correlates with lymph node metastasis in gastric cancer. In the present study, we investigated whether H. pylori could induce Snail activation to provoke these changes. Using a cell scatter assay, we noticed that human gastric cancer AGS cells infected with H. pylori underwent morphological changes as well as disruption of cell-cell interaction, which was then reversed by silencing of Snail by use of small interfering RNA (siRNA). In addition, infection with H. pylori resulted in an increased intracellular level of Snail in gastric cancer cells, which was abrogated in the presence of U0126 and LY294002, inhibitors of MEK/Erk and PI3K/Akt pathways, respectively. Cycloheximide pulse-chase experiments coupled with immunocytochemical analysis revealed that the induction of Snail by H. pylori was regulated at multiple levels, including increased transcription of Snail mRNA, inhibition of protein degradation, and enhancement of nuclear translocation of Snail. Pre-treatment of AGS cells with N-acetylcysteine, a well-known reactive oxygen species (ROS) scavenger, attenuated the H. pylori-induced activation of Erk, its binding to Snail promoter, inactivation of GSK-3ß, and accumulation of Snail. Collectively, these findings suggest that the upregulation of Snail expression induced by H. pylori and transformation to a spindle-like shape as a consequence in gastric cancer cells are attributable to ROS-mediated activation of Erk and the inhibition of GSK-3ß signaling. © 2016 Wiley Periodicals, Inc.
Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Infecções por Helicobacter/complicações , Helicobacter pylori/fisiologia , Sistema de Sinalização das MAP Quinases , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição da Família Snail/genética , Neoplasias Gástricas/virologia , Regulação para Cima , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/virologia , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismoRESUMO
BACKGROUND: Recent studies have shown that Helicobacter pylori (H. pylori) activates signal transducer and activator of transcription 3 (STAT3) that plays an important role in gastric carcinogenesis. However, the molecular mechanism underlying H. pylori-mediated STAT3 activation is still not fully understood. In this study, we investigated H. pylori-induced activation of STAT3 signaling in AGS human gastric cancer cells and the underlying mechanism. MATERIALS AND METHODS: AGS cells were cocultured with H. pylori, and STAT3 activation was assessed by Western blot analysis, electrophoretic mobility shift assay and immunocytochemistry. To demonstrate the involvement of reactive oxygen species (ROS) in H. pylori-activated STAT3 signaling, the antioxidant N-acetylcysteine was utilized. The expression and production of interleukin-6 (IL-6) were measured by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. The interaction between IL-6 and IL-6 receptor (IL-6R) was determined by the immunoprecipitation assay. RESULTS: H. pylori activates STAT3 as evidenced by increases in phosphorylation on Tyr(705) , nuclear localization, DNA binding and transcriptional activity of this transcription factor. The nuclear translocation of STAT3 was also observed in H. pylori-inoculated mouse stomach. In the subsequent study, we found that H. pylori-induced STAT3 phosphorylation was dependent on IL-6. Notably, the increased IL-6 expression and the IL-6 and IL-6R binding were mediated by ROS produced as a consequence of H. pylori infection. CONCLUSIONS: H. pylori-induced STAT3 activation is mediated, at least in part, through ROS-induced upregulation of IL-6 expression. These findings provide a novel molecular mechanism responsible for H. pylori-induced gastritis and gastric carcinogenesis.
Assuntos
Helicobacter pylori/imunologia , Interleucina-6/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Western Blotting , Linhagem Celular Tumoral , Técnicas de Cocultura , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Imuno-Histoquímica , Imunoprecipitação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação TranscricionalRESUMO
Under hypoxia, hypoxia-inducible factor (HIF)-1 regulates hypoxia-inducible genes, such as vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2. It is an oxygen-dependent transcriptional activator that plays a crucial role in tumor angiogenesis and mammalian embryo development. It is a heterodimeric protein comprising a constitutively expressed HIF-1ß subunit and the highly regulated HIF-1α subunits. Using CRISPR-Cas9 genome editing, we generated biallelic HIF-1α mutants in human induced pluripotent stem cells (hiPSCs). The HIF-1α homozygous-knockout hiPSCs retained their normal morphology, gene expression, and in vivo differentiation potential.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Diferenciação Celular , Técnicas de Inativação de Genes , Hipóxia Celular , Sistemas CRISPR-Cas , Linhagem Celular , Edição de Genes , AnimaisRESUMO
Programmed Cell Death Ligand 1 (PD-L1) is a trans-membrane protein that attenuates the host immune response to tumor cells. PD-L1 ligand on the surface of the cancer binds to PD-1 transmembrane receptors on T cells and exhausts T cells function. Using CRISPR-Cas9 genome editing, we generated biallelic PD-L1 mutants in human induced pluripotent stem cells (hiPSCs). The PD-L1 homozygous-knockout hiPSCs retained their normal morphology, gene expression, and in vivo differentiation potential.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Ligantes , Sistemas CRISPR-Cas/genética , Linhagem Celular TumoralRESUMO
Patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (FLUAV) coinfections were associated with severe respiratory failure and more deaths. Here, we developed a model for studying SARS-CoV-2 and FLUAV coinfection using human pluripotent stem cell-induced alveolar type II organoids (hiAT2). hiAT2 organoids were susceptible to infection by both viruses and had features of severe lung damage. A single virus markedly enhanced the susceptibility to other virus infections. SARS-CoV-2 delta variants upregulated α-2-3-linked sialic acid, while FLUAV upregulated angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). Moreover, coinfection by SARS-CoV-2 and FLUAV caused hyperactivation of proinflammatory and immune-related signaling pathways and cellular damage compared to a respective single virus in hiAT2 organoids. This study provides insight into molecular mechanisms underlying enhanced infectivity and severity in patients with co-infection of SARS-CoV-2 and FLUAV, which may aid in the development of therapeutics for such co-infection cases.
Assuntos
COVID-19 , Coinfecção , Influenza Humana , Células-Tronco Pluripotentes , Humanos , SARS-CoV-2 , Influenza Humana/metabolismo , Pulmão , Replicação Viral , OrganoidesRESUMO
The TLR family plays a fundamental function in the recognition of pathogens and activation of innate immunity. TLR2 is a membrane protein that is expressed on the surface of certain cells and recognizes foreign substances; it initiates a cascade of innate immune responses. TLR2 recognizes many bacterial, fungal, and viral components, as well as certain endogenous substances. We generated biallelic mutants of the TLR2 gene in human induced pluripotent stem cells (hiPSCs) using a CRISPR-Cas9 genome editing method. The TLR2 heterozygous-knockout hiPSCs retained normal morphology, gene expression, and in vivo differentiation potential.
RESUMO
The Toll-like receptor (TLR) family plays an important role in the recognition of pathogens, including bacteria, viruses, fungi, and parasites, followed by the activation of innate immunity. TLR3 recognizes double-stranded RNA, a form of genetic material produced by positive-strand RNA viruses and DNA viruses, and is activated by viral infection. Upon recognition, TLR3 promotes the activation of interferon regulatory factor 3 to enhance the expression and secretion of type I interferons that signal other cells to enhance their antiviral defenses. We generated biallelic mutants of the TLR3 gene using a CRISPR-Cas9 genome editing method in human induced pluripotent stem cells (hiPSCs). TLR3 homozygous-knockout hiPSCs retained normal morphology, gene expression, and in vivo differentiation potential.
Assuntos
Células-Tronco Pluripotentes Induzidas , Receptor 3 Toll-Like , Sistemas CRISPR-Cas/genética , Edição de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , RNA de Cadeia Dupla , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismoRESUMO
Macrophages exhibit high plasticity to achieve their roles in maintaining tissue homeostasis, innate immunity, tissue repair and regeneration. Therefore, macrophages are being evaluated for cell-based therapeutics against inflammatory disorders and cancer. To overcome the limitation related to expansion of primary macrophages and cell numbers, human pluripotent stem cell (hPSC)-derived macrophages are considered as an alternative source of primary macrophages for clinical application. However, the quality of hPSC-derived macrophages with respect to the biological homogeneity remains still unclear. We previously reported a technique to produce hPSC-derived macrophages referred to as iMACs, which is amenable for scale-up. In this study, we have evaluated the biological homogeneity of the iMACs using a transcriptome dataset of 6,230 iMACs obtained by single-cell RNA sequencing. The dataset provides a valuable genomic profile for understanding the molecular characteristics of hPSC-derived macrophage cells and provide a measurement of transcriptomic homogeneity. Our study highlights the usefulness of single cell RNA-seq data in quality control of the cell-based therapy products.
RESUMO
The loss of cell-matrix interactions induces apoptosis, known as anoikis. For successful distant metastasis, circulating tumor cells (CTCs) that have lost matrix attachment need to acquire anoikis resistance in order to survive. Cell aggregate formation confers anoikis resistance, and CTC clusters are more highly metastatic compared to single cells; however, the molecular mechanisms underlying this aggregation are not well understood. In this study, we demonstrated that cell detachment increased cell aggregation and upregulated fibronectin (FN) levels in lung and breast cancer cells, but not in their normal counterparts. FN knockdown decreased cell aggregation and increased anoikis. In addition, cell detachment induced cell-cell adhesion proteins, including E-cadherin, desmoglein-2, desmocollin-2/3, and plakoglobin. Interestingly, FN knockdown decreased the levels of desmoglein-2, desmocollin-2/3, and plakoglobin, but not E-cadherin, suggesting the involvement of desmosomal junction in cell aggregation. Accordingly, knockdown of desmoglein-2, desmocollin-2, or plakoglobin reduced cell aggregation and increased cell sensitivity to anoikis. Previously, we reported that NADPH oxidase 4 (Nox4) upregulation is important for anoikis resistance. Nox4 inhibition by siRNA or apocynin decreased cell aggregation and increased anoikis with the downregulation of FN, and, consequently, decreased desmoglein-2, desmocollin-2/3, or plakoglobin. The coexpression of Nox4 and FN was found to be significant in lung and breast cancer patients, based on cBioPortal data. In vivo mouse lung metastasis model showed that FN knockdown suppressed lung metastasis and thus enhanced survival. FN staining of micro tissue array revealed that FN expression was positive for human lung cancer (61%) and breast cancer (58%) patients. Furthermore, the expression levels of FN, desmoglein-2, desmocollin-2, and plakoglobin were significantly correlated with the poor survival of lung and breast cancer patients, as per the Kaplan-Meier plotter analysis. Altogether, our data suggest that FN upregulation and enhanced desmosomal interactions are critical for cell aggregation and anoikis resistance upon cell detachment.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fibronectinas/biossíntese , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células A549 , Animais , Anoikis/fisiologia , Neoplasias da Mama/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Agregação Celular/fisiologia , Linhagem Celular Tumoral , Fibronectinas/genética , Fibronectinas/metabolismo , Xenoenxertos , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , NADPH Oxidase 4/biossíntese , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Metástase Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise Serial de Tecidos , Regulação para CimaRESUMO
Benzylideneacetophenone analogues are known to have several significant biological activities, including antiinflammatory, antitumor, antibacterial, antiviral, and gastric-protective activities. However, the antiproliferative effects of benzylideneacetophenone analogues on vascular smooth muscle cells (VSMCs) are unknown. The aim of this study was to elucidate the antiproliferative effects and molecular mechanism of BST406, a newly synthesized benzylideneacetophenone derivative, on platelet-derived growth factor (PDGF)-BB-stimulated rat aortic VSMCs. BST406 inhibited [(3)H]-thymidine incorporation into DNA in VSMCs following treatment with PDGFBB 25 ng/ml. PDGF-BB-stimulated DNA synthesis was significantly reduced. Moreover, pretreatment with BST406 (0-10microM) suppressed the proliferation of PDGF-BB-stimulated cells in a concentration-dependent manner. We also investigated the mechanism of the antiproliferative effects of BST406 in PDGF-BB-stimulated VSMCs. In Western blot analysis, PDGF-BB-stimulated (25 ng/ml) phospholipase-C (PLC)gamma1 and Akt phosphorylation was inhibited by BST406 (0-10microM). However, BST406 did not inhibit the PDGF-receptor beta-chain (PDGF-Rbeta) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation induced by PDGF-BB. To confirm that the inhibitory effects of BST406 are mediated through the inhibition of PLCgamma1 or Akt, the effects of inhibitors on cell viability were examined. U73122 completely inhibited PDGF-BB-induced proliferation of VSMCs. However, LY294002 10microM had no significant effects on PDGF-BB-induced proliferation. These findings suggest that the inhibitory effects of BST406 on the proliferation of PDGF-BB-stimulated VSMCs are mediated by suppression of the PLCgamma1 signaling pathways. Our observations may explain, in part, the mechanistic basis for the prevention of cardiovascular disease (such as atherosclerosis and restenosis after coronary angioplasty) by BST406.
Assuntos
Anisóis/farmacologia , Fármacos Cardiovasculares/farmacologia , Doenças Cardiovasculares/prevenção & controle , Proliferação de Células/efeitos dos fármacos , Cetonas/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Anisóis/síntese química , Aorta/citologia , Western Blotting , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Isótopos , Cetonas/síntese química , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Fosfolipase C gama/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Coloração e Rotulagem , Timidina/metabolismoRESUMO
Human pluripotent stem cells (hPSCs) have promising therapeutic applications due to their infinite capacity for self-renewal and pluripotency. Genomic stability is imperative for the clinical use of hPSCs; however, copy number variation (CNV), especially recurrent CNV at 20q11.21, may contribute genomic instability of hPSCs. Furthermore, the effects of CNVs in hPSCs at the whole-transcriptome scale are poorly understood. This study aimed to examine the functional in vivo and in vitro effects of frequently detected CNVs at 20q11.21 during early-stage differentiation of hPSCs. Comprehensive transcriptome profiling of abnormal hPSCs revealed that the differential gene expression patterns had a negative effect on differentiation potential. Transcriptional heterogeneity identified by single-cell RNA sequencing (scRNA-seq) of embryoid bodies from two different isogenic lines of hPSCs revealed alterations in differentiated cell distributions compared with that of normal cells. RNA-seq analysis of 22 teratomas identified several differentially expressed lineage-specific markers in hPSCs with CNVs, consistent with the histological results of the altered ecto/meso/endodermal ratio due to CNVs. Our results suggest that CNV amplification contributes to cell proliferation, apoptosis, and cell fate specification. This work shows the functional consequences of recurrent genetic abnormalities and thereby provides evidence to support the development of cell-based applications.
Assuntos
Biomarcadores Tumorais/genética , Diferenciação Celular , Aberrações Cromossômicas , Cromossomos Humanos Par 20/genética , Variações do Número de Cópias de DNA , Células-Tronco Pluripotentes/patologia , Teratoma/patologia , Animais , Biomarcadores Tumorais/metabolismo , Células Cultivadas , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Pluripotentes/metabolismo , Análise de Sequência de RNA , Teratoma/genética , Teratoma/metabolismo , TranscriptomaRESUMO
Helicobacter pylori (H. pylori) infection is considered as one of the principal risk factors of gastric cancer. Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) plays an important role in inflammation-associated gastric carcinogenesis. In the canonical STAT3 pathway, phosphorylation of STAT3 on Tyr705 is a major event of STAT3 activation. However, recent studies have demonstrated that STAT3 phosphorylated on Ser727 has an independent function in mitochondria. In the present study, we found that human gastric epithelial AGS cells infected with H. pylori resulted in localization of STAT3 phosphorylated on Ser727 (P-STAT3Ser727), predominantly in the mitochondria. Notably, H. pylori-infected AGS cells exhibited the loss of mitochondrial integrity and increased expression of the microtubule-associated protein light chain 3 (LC3), the autophagosomal membrane-associated protein. Treatment of AGS cells with a mitophagy inducer, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), resulted in accumulation of P-STAT3Ser727 in mitochondria. In addition, the elevated expression and mitochondrial localization of LC3 induced by H. pylori infection were attenuated in AGS cells harboring STAT3 mutation defective in Ser727 phosphorylation (S727A). We also observed that both P-STAT3Ser727 expression and LC3 accumulation were increased in the mitochondria of H. pylori-inoculated mouse stomach.
Assuntos
Autofagia/fisiologia , Células Epiteliais/microbiologia , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/metabolismo , Fator de Transcrição STAT3/metabolismo , Estômago/microbiologia , Animais , Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/microbiologia , FosforilaçãoRESUMO
Cudrania tricuspidata has been proposed to possess anti-inflammatory, antioxidant, hepatoprotective, and antitumor activities. Although cudraflavone B, isolated from the root bark of C. tricuspidata, has a variety of pharmacological effects, its effects on rat aortic smooth muscle cells (RASMCs) are unclear. In the present study, cudraflavone B was found to inhibit cell proliferation and DNA synthesis in cultured RASMCs. Pretreatment with cudraflavone B (0.1-4 microM) suppressed platelet-derived growth factor-BB (PDGF-BB)-stimulated cell number in a concentration-dependent manner. The inhibition percentages were 19.7%, 36.4%, 52.3%, and 99.1% at concentrations of 0.1, 1, 2, and 4 microM, respectively. Moreover, cudraflavone B inhibited [H]-thymidine incorporation into DNA in RASMCs in response to 25 ng/mL PDGF-BB. PDGF-BB-stimulated DNA synthesis was significantly reduced by 15.9%, 31.7%, 43.1%, and 78.2% at concentrations of 0.1, 1, 2, and 4 muM, respectively. Thus, cudraflavone B blocked the PDGF-BB-inducible progression through G0/G1 to S phase of the cell cycle in synchronized cells. Furthermore, PDGF-BB-induced phosphorylation of retinoblastoma protein (pRb), the hyperphosphorylation of which is a hallmark of the G1-S transition in the cell cycle, was significantly inhibited by cudraflavone B. Because pRb phosphorylation is regulated by cyclin-dependent kinases (CDKs), we investigated the expression of CDK2, CDK4, cyclin E, and cyclin D1 and the CDK inhibitors p21 and p27. Treatment with cudraflavone B downregulated the cyclins and CDKs and upregulated the expression of p21 and p27, a CDK inhibitor. These findings suggest that cudraflavone B inhibits RASMC proliferation via the induction of p21 and p27 expression and subsequent cell cycle arrest with reduction of pRb phosphorylation at the G1-S phase.
Assuntos
Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Flavonoides/farmacologia , Moraceae/química , Miócitos de Músculo Liso/efeitos dos fármacos , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Aorta/citologia , Células Cultivadas , Meios de Cultura Livres de Soro , Relação Dose-Resposta a Droga , Flavonoides/química , Flavonoides/isolamento & purificação , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Casca de Planta/química , Raízes de Plantas/química , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ratos , Fatores de TempoRESUMO
Toll Like Receptor (TLR) family plays an important role in the activation of innate immunity against pathogens. TLR7 mediates the recognition of single-stranded RNA viruses, such as human immunodeficiency virus, hepatitis C virus, and influenza virus in endosomes. Here, we generated a TLR7 homozygous knockout human induced pluripotent cell (hiPSC) line, hiPSC-TLR7KO-A59, using the CRISPR/Cas9 genome editing method. The hiPSC-TLR7KO-A59 line maintains normal morphology, pluripotency, and differentiation capacity into three germ layers.
Assuntos
Sistemas CRISPR-Cas/genética , Células-Tronco Pluripotentes Induzidas/citologia , Receptor 7 Toll-Like/genética , Animais , Linhagem Celular , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Edição de Genes , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Cariótipo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Teratoma/patologiaRESUMO
The Toll like Receptor (TLR) family plays an essential role in pathogen recognition and innate immunity activation. TLR8, an endosomal receptor, can recognize single-stranded RNA viruses, such as influenza virus, Sendai virus, Coxsackie B virus, HIV, and HCV. TLR8 binding to the viral RNA recruits MyD88 and leads to activation of the transcription factor NF-kB and antiviral response. We generated biallelic mutants of the TLR8 gene using a CRISPR-Cas9 genome editing method in human induced pluripotent stem cells (hiPSCs). The TLR8 homozygous-knockout hiPSCs retained normal morphology, gene expression, and in vivo differentiation potential.