RESUMEN
The Pcdhg gene cluster encodes 22 γ-Protocadherin (γ-Pcdh) cell adhesion molecules that critically regulate multiple aspects of neural development, including neuronal survival, dendritic and axonal arborization, and synapse formation and maturation. Each γ-Pcdh isoform has unique protein domains-a homophilically interacting extracellular domain and a juxtamembrane cytoplasmic domain-as well as a C-terminal cytoplasmic domain shared by all isoforms. The extent to which isoform-specific versus shared domains regulate distinct γ-Pcdh functions remains incompletely understood. Our previous in vitro studies identified protein kinase C (PKC) phosphorylation of a serine residue within a shared C-terminal motif as a mechanism through which γ-Pcdh promotion of dendrite arborization via myristoylated alanine-rich C-kinase substrate (MARCKS) is abrogated. Here, we used CRISPR/Cas9 genome editing to generate two new mouse lines expressing only non-phosphorylatable γ-Pcdhs, due either to a serine-to-alanine mutation (PcdhgS/A) or to a 15-amino acid C-terminal deletion resulting from insertion of an early stop codon (PcdhgCTD). Both lines are viable and fertile, and the density and maturation of dendritic spines remain unchanged in both PcdhgS/A and PcdhgCTD cortex. Dendrite arborization of cortical pyramidal neurons, however, is significantly increased in both lines, as are levels of active MARCKS. Intriguingly, despite having significantly reduced levels of γ-Pcdh proteins, the PcdhgCTD mutation yields the strongest phenotype, with even heterozygous mutants exhibiting increased arborization. The present study confirms that phosphorylation of a shared C-terminal motif is a key γ-Pcdh negative regulation point and contributes to a converging understanding of γ-Pcdh family function in which distinct roles are played by both individual isoforms and discrete protein domains.
Asunto(s)
Proteínas Relacionadas con las Cadherinas , Cadherinas , Corteza Cerebral , Dendritas , Proteína Quinasa C , Animales , Corteza Cerebral/metabolismo , Corteza Cerebral/citología , Cadherinas/metabolismo , Cadherinas/genética , Fosforilación/fisiología , Dendritas/metabolismo , Ratones , Proteína Quinasa C/metabolismo , Proteína Quinasa C/genética , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Secuencias de Aminoácidos/fisiología , Ratones TransgénicosRESUMEN
BACKGROUND: Hepatocellular carcinoma is the fourth most common cause of cancer-related death. However, the cross-talk between tumor immune microenvironment and hepatocellular carcinoma (HCC) remains unclear. MATERIAL AND METHODS: We analyzed the expression of miR-143-3p in exosomes from different HCC cell lines. Differentially expressed genes (DEGs) in Tumor-associated macrophages (TAMs) co-cultured with HCC cell lines were overlapped with miR-143-3p target genes. We used the Oncomine, Kaplan-Meier plotter, and The Cancer Genome Atlas (TCGA) databases to assess Myristoylated alanine-rich C-kinase substrate (MARCKS) expression in various types of cancers. The relationship between patient clinicopathological characteristics and MARCKS expression level was identified using the Kaplan-Meier plotter database. Last, we analyzed how MARCKS expression correlated with immune infiltration makers using the TCGA database, Tumor IMmune Estimation Resource (TIMER), and Gene Expression Profiling Interactive Analysis (GEPIA). RESULTS: Exosomal miR-143-3p was elevated after IL-6 treatment in the HCC cell line. MARCKS, a target gene of miR-143-3p, was up-regulated in Tumor-associated macrophages co-cultured with high-metastatic-potential HCC cell line. MARCKS expression was identified as significantly correlated with outcome in multiple types of cancer, especially in HCC. High MARCKS expression level was associated with poorer overall survival (OS), Progress-free survival (PFS), and also with patient gender, race, hepatitis virus background, stage, grade, AJCC_T, and vascular invasion. MARCKS was positively associated with levels of T follicular helper cells (TFH) (R = .48, p < .001), T helper type 2 (Th2) cells (R = .47, p < .001), macrophages (R = .41, p ≤ .001), T helper cells (R = .40, p < .001), T helper type 1 (Th1) cells (R = .38, p < .001), T cells (R = .34, p < .001), NK CD56bright cells (R = .34, p < .001) and immature DC (iDC) (R = .33, p < .001), and negatively associated with levels of T helper 17 (Th17) cells. Also, MARCKS may influence the M2 polarization and immune escape. CONCLUSION: The present study suggests that MARCKS on TAMs is associated with poor prognosis and immune cell infiltration in HCC.
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Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Macrófagos Asociados a Tumores/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Exosomas/genética , Células Hep G2 , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , MicroARNs/genética , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células THP-1RESUMEN
Cells can switch between Rac1 (lamellipodia-based) and RhoA (blebbing-based) migration modes, but the molecular mechanisms regulating this shift are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from their common inhibitor RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation; instead, DGKζ stimulates RhoA release via a kinase-independent scaffolding mechanism. The molecular determinants that mediate the selective targeting of DGKζ to Rac1 or RhoA signaling complexes are unknown. Here, we show that protein kinase Cα (PKCα)-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification also enhanced DGKζ interaction with the scaffold protein syntrophin. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the RhoA-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells also required the PDZ-binding motif, suggesting that syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism whereby DGKζ phosphorylation by PKCα plays a role in the interconversion between Rac1 and RhoA signaling pathways that underlie different cellular migration modes.
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Movimiento Celular , Diacilglicerol Quinasa/fisiología , Proteínas Asociadas a la Distrofina/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Neuropéptidos/metabolismo , Proteína Quinasa C-alfa/farmacología , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Diglicéridos/metabolismo , Proteínas Asociadas a la Distrofina/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Ratones , Ratones Noqueados , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Neuropéptidos/genética , Dominios Proteicos , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Glioblastoma (GBM) is an aggressive malignancy with limited effectiveness of standard of care therapies including surgery, radiation, and temozolomide chemotherapy necessitating novel therapeutics. Unfortunately, GBMs also harbor several signaling alterations that protect them from traditional therapies that rely on apoptotic programmed cell death. Because almost all GBM tumors have dysregulated phosphoinositide signaling as part of that process, we hypothesized that peptide mimetics derived from the phospholipid binding domain of Myristoylated alanine-rich C-kinase substrate (MARCKS) could serve as a novel GBM therapeutic. Using molecularly classified patient-derived xenograft (PDX) lines, cultured in stem-cell conditions, we demonstrate that cell permeable MARCKS effector domain (ED) peptides potently target all GBM molecular classes while sparing normal human astrocytes. Cell death mechanistic testing revealed that these peptides produce rapid cytotoxicity in GBM that overcomes caspase inhibition. Moreover, we identify a GBM-selective cytolytic death mechanism involving plasma membrane targeting and intracellular calcium accumulation. Despite limited relative partitioning to the brain, tail-vein peptide injection revealed tumor targeting in intracranially implanted GBM PDX. These results indicate that MARCKS ED peptide therapeutics may overcome traditional GBM resistance mechanisms, supporting further development of similar agents.
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Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Fragmentos de Péptidos/farmacología , Animales , Astrocitos , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/patología , Caspasas/metabolismo , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Resistencia a Antineoplásicos/efectos de los fármacos , Glioblastoma/patología , Humanos , Ratones , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/uso terapéutico , Dominios Proteicos/genética , Transducción de Señal/efectos de los fármacos , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Phosphatidylinositol 4,5-bisphosphate (PIP2) acts as substrate and unmodified ligand for Gq-protein-coupled receptor signalling in vascular smooth muscle cells (VSMCs) that is central for initiating contractility. The present work investigated how PIP2 might perform these two potentially conflicting roles by studying the effect of myristoylated alanine-rich C kinase substrate (MARCKS), a PIP2-binding protein, on vascular contractility in rat and mouse mesenteric arteries. Using wire myography, MANS peptide (MANS), a MARCKS inhibitor, produced robust contractions with a pharmacological profile suggesting a predominantly role for L-type (CaV1.2) voltage-gated Ca2+ channels (VGCC). Knockdown of MARCKS using morpholino oligonucleotides reduced contractions induced by MANS and stimulation of α1-adrenoceptors and thromboxane receptors with methoxamine (MO) and U46619 respectively. Immunocytochemistry and proximity ligation assays demonstrated that MARCKS and CaV1.2 proteins co-localise at the plasma membrane in unstimulated tissue, and that MANS and MO reduced these interactions and induced translocation of MARCKS from the plasma membrane to the cytosol. Dot-blots revealed greater PIP2 binding to MARCKS than CaV1.2 in unstimulated tissue, with this binding profile reversed following stimulation by MANS and MO. MANS evoked an increase in peak amplitude and shifted the activation curve to more negative membrane potentials of whole-cell voltage-gated Ca2+ currents, which were prevented by depleting PIP2 levels with wortmannin. This present study indicates for the first time that MARCKS is important regulating vascular contractility and suggests that disinhibition of MARCKS by MANS or vasoconstrictors may induce contraction through releasing PIP2 into the local environment where it increases voltage-gated Ca2+ channel activity.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Músculo Liso Vascular/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Vasoconstricción , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Arteria Mesentérica Superior/metabolismo , Ratones de la Cepa 129 , Músculo Liso Vascular/efectos de los fármacos , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/antagonistas & inhibidores , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Fragmentos de Péptidos/farmacología , Ratas Wistar , Transducción de Señal , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacologíaRESUMEN
MiR-142-3p as one key molecule in oncogenesis and inflammation plays crucial roles in hepatic fibrosis, hepatocellular carcinoma and other liver disease. However, there have no literatures to report its effects on hepatic ischemia-reperfusion (HI/R) injury. In the present work, hypoxia reoxygenation (H/R) models on AML12 and HepG2 cells, and ischemia/reperfusion model in mice were established. The methods of real-time PCR, dual luciferase reporter, mimic, inhibitor, agomir, antagomir and siRNA transfection assays were used. The expression levels of miR-142-3p were decreased in model groups in vitro and in vivo compared with control group or Sham group, which directly targeted MARCKS to regulate its expression. Then, MARCKS activated p38/JNK signal, up-regulated NF-κB expression to accelerate inflammation, and inhibited PI3K/AKT signal to promote apoptosis. Moreover, miR-142-3p mimic in vitro and agomir in vivo lowered the expression levels of MARCKS, thereby alleviating apoptosis and inflammation to relieve HI/R injury. Furthermore, miR-142- 3p inhibitor in vitro and antagomir in vivo up-regulated the expression levels of MARCKS to aggravate HI/R damage via promoting inflammation and apoptosis. Consistently, MARCKS siRNA markedly inhibited HI/R injury by restraining apoptosis and inflamm- ation in mice. MiR-142-3p played a considerable part in adjusting HI/R injury by targeting MARCKS, and miR-142-3p/MARCKS should be a new therapeutic target for HI/R injury.
Asunto(s)
Apoptosis , Hepatopatías/metabolismo , Hígado/metabolismo , MicroARNs/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Daño por Reperfusión/metabolismo , Animales , Hipoxia de la Célula , Modelos Animales de Enfermedad , Células Hep G2 , Humanos , Mediadores de Inflamación/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Hígado/patología , Hepatopatías/genética , Hepatopatías/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , MicroARNs/genética , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Daño por Reperfusión/genética , Daño por Reperfusión/patología , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Alzheimer's disease (AD) is the most common neurodegenerative disorder caused by neuronal loss that results in cognitive and functional impairment. Formation of neurofibrillary tangles composed of abnormal hyperphosphorylation of tau protein is one of the major pathological hallmarks of AD. Importantly, several neurodegenerative disorders, including AD, are associated with abnormal protein phosphorylation events. However, little is known thus far on global protein phosphorylation changes in AD. We report a phosphoproteomics study examining the frontal gyrus of people with AD and age-matched cognitively normal subjects, using tandem mass tag (TMT) multiplexing technology along with immobilized metal affinity chromatography to enrich phosphopeptides. We identified 4631 phosphopeptides corresponding to 1821 proteins with liquid chromatography-mass spectrometry (MS)/MS analysis on an Orbitrap Fusion Lumos Tribrid mass spectrometer. Of these, 504 phosphopeptides corresponding to 350 proteins were significantly altered in the AD brain: 389 phosphopeptides increased whereas 115 phosphopeptides decreased phosphorylation. We observed significant changes in phosphorylation of known as well as novel molecules. Using targeted parallel reaction monitoring experiments, we validated the phosphorylation of microtubule-associated protein tau and myristoylated alanine-rich protein kinase C substrate (MARCKS) in control and AD (Control = 6, AD = 11) brain samples. In conclusion, our study provides new evidence on alteration of RNA processing and splicing, neurogenesis and neuronal development, and metabotropic glutamate receptor 5 (GRM5) calcium signaling pathways in the AD brain, and it thus offers new insights to accelerate diagnostics and therapeutics innovation in AD.
Asunto(s)
Enfermedad de Alzheimer/genética , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Fosfoproteínas/genética , Proteoma/genética , Receptor del Glutamato Metabotropico 5/genética , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Autopsia , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Masculino , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Fosfoproteínas/metabolismo , Fosforilación , Corteza Prefrontal/metabolismo , Corteza Prefrontal/patología , Proteoma/metabolismo , Proteómica/métodos , Receptor del Glutamato Metabotropico 5/metabolismoRESUMEN
Overexpression of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) is implicated in drug resistance and progression of multiple myeloma (MM). The basis for MARCKS induction and impact on MM are not known. Here we show that microRNA-34a (miR-34a), regulates MARCKS translation and is under-expressed in drug-resistant MM cells, leading to increased MARCKS protein level. Over-expression of miR-34a reduces MARCKS expression and sensitizes resistant cells to anti-myeloma drugs. A MARCKS peptide inhibitor (MPS) exerts a dose dependent cytotoxic effect on drug-resistant MM cells with minimal cytotoxicity to normal hematopoietic cells. MPS synergizes with the proteasomal-inhibitor bortezomib to effectively kill drug-resistant MM cells both in vitro and in a xenograft model of MM. While MARCKS inhibition killed MM cells, it also enhanced a pro-survival autophagic pathway that sustained growth following MARCKS inhibition. In accordance, combined treatment with MARCKS antagonists, bortezomib and the autophagy inhibitor, chloroquine, significantly diminished tumor growth in drug-resistant MM cell lines as well as primary MM cells. This study uncovers a mechanism of drug resistance involving miR-34a-MARCKS autoregulatory loop and provides a framework for a potentially new therapeutic strategy to overcome drug resistance in multiple myeloma.
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Autofagia/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Bortezomib/administración & dosificación , Bortezomib/farmacología , Línea Celular Tumoral , Cloroquina/administración & dosificación , Cloroquina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones SCID , MicroARNs/genética , Mieloma Múltiple/patología , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Male breast cancer (MBC) is a rare and aggressive disease. Thus, identification of new therapeutic targets is crucial. OBJECTIVE: Our objective was to evaluate the protein expression of MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate) in MBC and to investigate its prognostic value. MATERIALS AND METHODS: MARCKS protein expression in tumor and stromal cells was analyzed by immunohistochemistry (IHC) in a retrospective series of 96 pre-chemotherapy MBC samples and 80 normal breast samples, from Tunisian patients treated at Salah Azaiez Institute. Correlations were searched between MARCKS expression and clinicopathological features including overall survival (OS). RESULTS: MARCKS was overexpressed in epithelial tumor cells in 66% of the MBC samples versus 26% of normal samples (p= 1.40 × 10-7). Such positive MARCKS expression in epithelial tumor cells was associated with positive HER2 status (p= 4.0 × 10-3). It was associated with shorter OS in uni-and multivariate analysis. By contrast, stromal IHC MARCKS expression was correlated only with tumor grade. CONCLUSION: MARCKS tumor cell overexpression might in part explain the aggressiveness and the poor prognosis of MBC. MARCKS can represent a potential therapeutic target for MBC.
Asunto(s)
Neoplasias de la Mama Masculina/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama Masculina/genética , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Pronóstico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
Targeting activated fibroblasts, including myofibroblast differentiation, has emerged as a key therapeutic strategy in patients with idiopathic pulmonary fibrosis (IPF). However, there is no available therapy capable of selectively eradicating myofibroblasts or limiting their genesis. Through an integrative analysis of the regulator genes that are responsible for the activation of IPF fibroblasts, we noticed the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding protein, myristoylated alanine-rich C-kinase substrate (MARCKS), as a potential target molecule for IPF. Herein, we have employed a 25-mer novel peptide, MARCKS phosphorylation site domain sequence (MPS), to determine if MARCKS inhibition reduces pulmonary fibrosis through the inactivation of PI3K/protein kinase B (AKT) signaling in fibroblast cells. We first observed that higher levels of MARCKS phosphorylation and the myofibroblast marker α-smooth muscle actin (α-SMA) were notably overexpressed in all tested IPF lung tissues and fibroblast cells. Treatment with the MPS peptide suppressed levels of MARCKS phosphorylation in primary IPF fibroblasts. A kinetic assay confirmed that this peptide binds to phospholipids, particularly PIP2, with a dissociation constant of 17.64 nM. As expected, a decrease of phosphatidylinositol (3,4,5)-trisphosphate pools and AKT activity occurred in MPS-treated IPF fibroblast cells. MPS peptide was demonstrated to impair cell proliferation, invasion, and migration in multiple IPF fibroblast cells in vitro as well as to reduce pulmonary fibrosis in bleomycin-treated mice in vivo. Surprisingly, we found that MPS peptide decreases α-SMA expression and synergistically interacts with nintedanib treatment in IPF fibroblasts. Our data suggest MARCKS as a druggable target in pulmonary fibrosis and also provide a promising antifibrotic agent that may lead to effective IPF treatments.-Yang, D. C., Li, J.-M., Xu, J., Oldham, J., Phan, S. H., Last, J. A., Wu, R., Chen, C.-H. Tackling MARCKS-PIP3 circuit attenuates fibroblast activation and fibrosis progression.
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Fibroblastos/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Fosfatidilinositoles/metabolismo , Fibrosis Pulmonar/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Antibióticos Antineoplásicos/toxicidad , Bleomicina/toxicidad , Proliferación Celular , Células Cultivadas/efectos de los fármacos , Células Cultivadas/fisiología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Fosfatidilinositoles/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fibrosis Pulmonar/inducido químicamenteRESUMEN
OBJECTIVES: It has been confirmed that morphine was detrimental to patients with cancers. Hence, we aimed to reveal a certain mechanism of morphine in cancer development. METHODS: Microarray and GSEA analysis were utilized to seek for differently expressed genes and pathway. KEY FINDINGS: Bioinformatics analysis identified that downregulation of MARCKS and upregulation of miR-543 in samples treated with morphine. FcγR-mediated phagocytosis pathway was illustrated to be upregulated in the control. PANC-1 and DU145 cell viability was increased but apoptosis was declined as morphine concentration went up from 10-8 to 10-6 mol/l. On the other curve, the viability was reduced and apoptosis was elevated from 10-6 to 10-5 mol/l. The expression of miR-543 ran the same trend as cell viability. Assays in vivo and in vitro validated that miR-543 facilitated cell viability, tumour growth, levels of CA199 and PSA, whereas inhibited apoptosis. MARCKS could target and inhibit miR-543 expression, which exhibited an opposite effect on cancer progression. MiR-543 blocked but MARCKS activated FcγR-mediated phagocytosis pathway. CONCLUSIONS: Morphine at 10-6 mol/l could benefit miR-543 expression to inhibit MARCKS expression, consequently, blocking FcγR-mediated phagocytosis pathway, which contributed to the cancer progression in vitro and in vivo.
Asunto(s)
MicroARNs/genética , Morfina/farmacología , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Fagocitosis/efectos de los fármacos , Receptores de IgG/genética , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Biología Computacional/métodos , Regulación hacia Abajo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Long noncoding RNAs (lncRNAs) can regulate target gene expression by acting in cis (locally) or in trans (non-locally). Here, we performed genome-wide expression analysis of Toll-like receptor (TLR)-stimulated human macrophages to identify pairs of cis-acting lncRNAs and protein-coding genes involved in innate immunity. A total of 229 gene pairs were identified, many of which were commonly regulated by signaling through multiple TLRs and were involved in the cytokine responses to infection by group B Streptococcus We focused on elucidating the function of one lncRNA, named lnc-MARCKS or ROCKI (Regulator of Cytokines and Inflammation), which was induced by multiple TLR stimuli and acted as a master regulator of inflammatory responses. ROCKI interacted with APEX1 (apurinic/apyrimidinic endodeoxyribonuclease 1) to form a ribonucleoprotein complex at the MARCKS promoter. In turn, ROCKI-APEX1 recruited the histone deacetylase HDAC1, which removed the H3K27ac modification from the promoter, thus reducing MARCKS transcription and subsequent Ca2+ signaling and inflammatory gene expression. Finally, genetic variants affecting ROCKI expression were linked to a reduced risk of certain inflammatory and infectious disease in humans, including inflammatory bowel disease and tuberculosis. Collectively, these data highlight the importance of cis-acting lncRNAs in TLR signaling, innate immunity, and pathophysiological inflammation.
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Regulación de la Expresión Génica , Inmunidad Innata/inmunología , Inflamación/inmunología , Macrófagos/inmunología , ARN Largo no Codificante/metabolismo , Infecciones Estreptocócicas/microbiología , Receptores Toll-Like/metabolismo , Células Cultivadas , Citocinas/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Genoma Humano , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Humanos , Inflamación/genética , Inflamación/microbiología , Macrófagos/metabolismo , Macrófagos/microbiología , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Regiones Promotoras Genéticas , ARN Largo no Codificante/genética , Infecciones Estreptocócicas/inmunología , Streptococcus agalactiae/aislamiento & purificación , Receptores Toll-Like/genéticaRESUMEN
Myristoylated alaninerich Ckinase substrate (MARCKS) serves an important role in various pathological processes in several malignancies. However, little is known about the specific role and molecular mechanism of MARCKS in glioblastoma (GBM). In the present study, it was found that the expression of MARCKS was significantly upregulated in GBM, and was associated with a poor clinical outcome in patients with GBM. Knockdown of MARCKS suppressed the migration and invasion of GBM cells in vitro. Western blotting showed that the knockdown of MARCKS reduced the expression of phosphorylated phosphoinositide 3kinase and protein kinase B, as well as zinc finger protein SNAI1 expression, thereby modulating the expression of its downstream epithelialmesenchymal transition (EMT)associated factors, including Ecadherin, vimentin, Ncadherin and ßcatenin in GBM cells. These results indicate that MARCKS functioned in the migration and invasion of GBM, and therefore may provide a potential therapeutic target in GBM therapy.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Glioblastoma/patología , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Adulto , Anciano , Encéfalo/patología , Encéfalo/cirugía , Línea Celular Tumoral , Movimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , Femenino , Estudios de Seguimiento , Técnicas de Silenciamiento del Gen , Glioblastoma/genética , Glioblastoma/mortalidad , Glioblastoma/cirugía , Humanos , Masculino , Persona de Mediana Edad , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Invasividad Neoplásica/patología , Pronóstico , ARN Interferente Pequeño/metabolismo , Transducción de Señal/genética , Análisis de Supervivencia , Regulación hacia Arriba , Adulto JovenRESUMEN
Over eighty million people in the United States have cardiovascular disease that can affect the heart causing myocardial infarction; the carotid arteries causing stroke; and the lower extremities leading to amputation. The treatment for end-stage cardiovascular disease is surgical-either endovascular therapy with balloons and stents-or open reconstruction to reestablish blood flow. All interventions damage or destroy the protective inner lining of the blood vessel-the endothelium. An intact endothelium is essential to provide a protective; antithrombotic lining of a blood vessel. Currently; there are no agents used in the clinical setting that promote reendothelialization. This process requires migration of endothelial cells to the denuded vessel; proliferation of endothelial cells on the denuded vessel surface; and the reconstitution of the tight adherence junctions responsible for the formation of an impermeable surface. These processes are all regulated in part and are dependent on small GTPases. As important as the small GTPases are for reendothelialization, dysregulation of these molecules can result in various vascular pathologies including aneurysm formation, atherosclerosis, diabetes, angiogenesis, and hypertension. A better understanding of the role of small GTPases in endothelial cell migration is essential to the development for novel agents to treat vascular disease.
Asunto(s)
Proteínas de Unión al GTP Monoméricas/metabolismo , Enfermedades Vasculares/etiología , Enfermedades Vasculares/metabolismo , Animales , Biomarcadores , Movimiento Celular , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Humanos , Proteínas de Unión al GTP Monoméricas/genética , Familia de Multigenes , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Túnica Íntima/metabolismo , Túnica Íntima/patología , Enfermedades Vasculares/patologíaRESUMEN
Failure of neural tube closure results in severe birth defects and can be induced by high glucose levels resulting from maternal diabetes. MARCKS is required for neural tube closure, but the regulation and of its biological activity and function have remained elusive. Here, we show that high maternal glucose induced MARCKS acetylation at lysine 165 by the acetyltransferase Tip60, which is a prerequisite for its phosphorylation, whereas Sirtuin 2 (SIRT2) deacetylated MARCKS. Phosphorylated MARCKS dissociates from organelles, leading to mitochondrial abnormalities and endoplasmic reticulum stress. Phosphorylation dead MARCKS (PD-MARCKS) reversed maternal diabetes-induced cellular organelle stress, apoptosis and delayed neurogenesis in the neuroepithelium and ameliorated neural tube defects. Restoring SIRT2 expression in the developing neuroepithelium exerted identical effects as those of PD-MARCKS. Our studies reveal a new regulatory mechanism for MARCKS acetylation and phosphorylation that disrupts neurulation under diabetic conditions by diminishing the cellular organelle protective effect of MARCKS.
Asunto(s)
Diabetes Mellitus Experimental/complicaciones , Enfermedades Fetales/patología , Lisina Acetiltransferasa 5/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Defectos del Tubo Neural/patología , Sirtuina 2/metabolismo , Transactivadores/metabolismo , Acetilación , Animales , Glucemia/metabolismo , Línea Celular , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/inducido químicamente , Embrión de Mamíferos , Estrés del Retículo Endoplásmico , Femenino , Enfermedades Fetales/sangre , Enfermedades Fetales/etiología , Humanos , Lisina Acetiltransferasa 5/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/patología , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Defectos del Tubo Neural/etiología , Neurulación , Fosforilación , Embarazo , Embarazo en Diabéticas/sangre , Sirtuina 2/genética , Estreptozocina/toxicidad , Transactivadores/genéticaRESUMEN
BACKGROUND Accumulating evidence suggests a connection of Myristoylated alanine-rich C-kinase substrate (MARCKS) with several physiological and pathological processes. However, the relevance of MARCKS in gastric cancer (GC) needs to be elucidated. MATERIAL AND METHODS The abundance of MARCKS in GC tissues was assessed using techniques of immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR). Moreover, the MARCKS expression profile in the TCGA database was analyzed through an online website analysis. We also investigated MARCKS function using cell wounding and Matrigel invasion assays. RESULTS TCGA analysis and our data suggest that transcript abundance and protein level of MARCKS was higher in GC tumor samples compared with peri-tumor tissues. There was a remarkable association of upregulated MARCKS with the cell differentiation (P<0.001), T stage (P=0.034), and N stage (P=0.002) followed by advanced TNM phase (P=0.008). Furthermore, it was predicted that higher expression of MARCKS is linked to poor overall survival (P=0.015) and disease-free survival (P=0.020), and that high levels of MARCKS function as an independent prognostic marker, as shown by multivariate Cox regression analysis in prediction of poor overall (HR=0.408; 95% confidence interval=0.247-0.674; P<0.001) and disease-free survival rates (HR=0.525; 95% confidence interval=0.216-0.584; P<0.001). GC cells showed significant reduction in cell migration and invasion upon depletion of MARCKS as noted through Matrigel invasion and cell wounding assays. Further analyses showed that silencing MARCKS impeded the epithelial-mesenchymal transition (EMT). CONCLUSIONS Our study indicates that elevated expression of MARCKS is significantly associated with metastatic capability of GC cells, and MARCKS overexpression can serve as a biomarker of GC poor prognosis.
Asunto(s)
Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/fisiología , Neoplasias Gástricas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , China , Supervivencia sin Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Humanos , Inmunohistoquímica/métodos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Estimación de Kaplan-Meier , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Desnudos , Persona de Mediana Edad , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Fosforilación , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Neoplasias Gástricas/genética , Neoplasias Gástricas/fisiopatología , Activación Transcripcional , Transcriptoma/genética , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The prognosis of patients with advanced-stage lung squamous cell carcinoma (LUSQ) is poor, and effective treatment protocols are limited. Our continuous analyses of antitumor microRNAs (miRNAs) and their oncogenic targets have revealed novel oncogenic pathways in LUSQ. Analyses of our original miRNA expression signatures indicated that both strands of miR-144 (miR-144-5p, the passenger strand; miR-144-3p, the guide strand) showed decreased expression in cancer tissues. Additionally, low expression of miR-144-5p significantly predicted a poor prognosis in patients with LUSQ by The Cancer Genome Atlas database analyses (overall survival, P = 0.026; disease-free survival, P = 0.023). Functional assays revealed that ectopic expression of miR-144-5p and miR-144-3p significantly blocked the malignant abilities of LUSQ cells, eg, cancer cell proliferation, migration, and invasion. In LUSQ cells, 13 and 15 genes were identified as possible oncogenic targets that might be regulated by miR-144-5p and miR-144-3p, respectively. Among these targets, we identified 3 genes (SLC44A5, MARCKS, and NCS1) that might be regulated by both strands of miR-144. Interestingly, high expression of NCS1 predicted a significantly poorer prognosis in patients with LUSQ (overall survival, P = 0.013; disease-free survival, P = 0.048). By multivariate analysis, NCS1 expression was found to be an independent prognostic factor for patients with LUSQ patients. Overexpression of NCS1 was detected in LUSQ clinical specimens, and its aberrant expression enhanced malignant transformation of LUSQ cells. Our approach, involving identification of antitumor miRNAs and their targets, will contribute to improving our understanding of the molecular pathogenesis of LUSQ.
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Carcinoma de Células Escamosas/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Femenino , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , MicroARNs , Persona de Mediana Edad , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Proteínas Sensoras del Calcio Neuronal/genética , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , PronósticoRESUMEN
BACKGROUND: The Myristoylated Alanine-Rich C-kinase Substrate (MARCKS) and MARCKS-like protein 1 (MARCKSL1) have a wide range of functions, ranging from roles in embryonic development to adult brain plasticity and the inflammatory response. Recently, both proteins have also been identified as important players in regeneration. Upon phosphorylation by protein kinase C (PKC) or calcium-dependent calmodulin-binding, MARCKS and MARCKSL1 translocate from the membrane into the cytosol, modulating cytoskeletal actin dynamics and vesicular trafficking and activating various signal transduction pathways. As a consequence, the two proteins are involved in the regulation of cell migration, secretion, proliferation and differentiation in many different tissues. MAIN BODY: Throughout vertebrate development, MARCKS and MARCKSL1 are widely expressed in tissues derived from all germ layers, with particularly strong expression in the nervous system. They have been implicated in the regulation of gastrulation, myogenesis, brain development, and other developmental processes. Mice carrying loss of function mutations in either Marcks or Marcksl1 genes die shortly after birth due to multiple deficiencies including detrimental neural tube closure defects. In adult vertebrates, MARCKS and MARCKL1 continue to be important for multiple regenerative processes including peripheral nerve, appendage, and tail regeneration, making them promising targets for regenerative medicine. CONCLUSION: This review briefly summarizes the molecular interactions and cellular functions of MARCKS and MARCKSL1 proteins and outlines their vital roles in development and regeneration.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/genética , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Vertebrados/fisiología , Animales , Movimiento Celular , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Regeneración , Vertebrados/genética , Vertebrados/crecimiento & desarrolloRESUMEN
Upregulation of B-cell CLL/lymphoma (BCL)2 expression following lithium treatment is seemingly well established and has been related to the neuroprotective property of the drug. However, while demonstrated by some (but not all) studies based on low-throughput techniques (e.g. qPCR) this effect is not reflected in high-throughput studies, such as microarrays and RNAseq. This manuscript presents a systematic review of currently available reports of lithium's effect on BCL2 expression. To our surprise, we found that the majority of the literature does not support the effect of lithium on BCL2 transcript or protein levels. Moreover, among the positive reports, several used therapeutically irrelevant lithium doses while others lack statistical power. We also noticed that numerous low-throughput studies normalized the signal using genes/proteins affected by lithium, imposing possible bias. Using wet bench experiments and reanalysis of publicly available microarray data, here we show that the reference gene chosen for normalization critically impacts the outcome of qPCR analyses of lithium's effect on BCL2 expression. Our findings suggest that experimental results might be severely affected by the choice of normalizing genes, and emphasize the need to re-evaluate stability of these genes in the context of the specific experimental conditions.