RESUMEN
Prostasin and matriptase are extracellular membrane serine proteases with opposing effects in solid epithelial tumors. Matriptase is an oncoprotein that promotes tumor initiation and progression, and prostasin is a tumor suppressor that reduces tumor invasion and metastasis. Previous studies have shown that a subgroup of Burkitt lymphoma have high levels of ectopic matriptase expression but no prostasin. Reducing the matriptase level via small interfering RNAs in B lymphoma cells impeded tumor xenograft growth in mice. Here, we report a novel approach to matriptase regulation in B cancer cells by prostasin via exosomes to initiate a prostasin-matriptase protease activation cascade. The activation and shedding of matriptase were monitored by measuring its quantity and trypsin-like serine protease activity in conditioned media. Sustained activation of the protease cascade in the cells was achieved by the stable expression of prostasin. The B cancer cells with prostasin expression presented phenotypes consistent with its tumor suppressor role, such as reduced growth and increased apoptosis. Prostasin exosomes could be developed as an agent to initiate the prostasin-matriptase cascade for treating B lymphoma with further studies in animal models.
RESUMEN
Dopamine (DA) is an important neurotransmitter, which is essential for transmitting signals in neuronal communications. The deficiency of DA release from neurons is implicated in neurological disorders. There has been great interest in developing new optical probes for monitoring the release behavior of DA from neurons. H-aggregates of organic dyes represent an ordered supramolecular structure with delocalized excitons. In this paper, we use the self-assembly of 3,3'-diethylthiadicarbocyanine iodide (DiSC2(5)) in ammonia solution to develop crystalline H-aggregate nanoparticles, in which DiSC2(5) molecules show long-range π-π stacking. The crystalline H-aggregate nanoparticles are stable in cell culture medium and can serve as an efficient photo-induced electron transfer (PET) probe for the detection of DA with the concentration as low as 0.1 nM in cell culture medium. Furthermore, the crystalline H-aggregate nanoparticle-based PET probe is used to detect the release behavior of DA from the M17 human neuroblastoma cells. We find that the DA release from the cells is enhanced by nicotine stimulations. Our results highlight the potential of crystalline H-aggregate nanoparticle-based PET probes for diagnosing nervous system diseases and verifying therapies.
Asunto(s)
Ditiazanina , Nanopartículas , Neuroblastoma , Amoníaco , Colorantes , Dopamina/farmacología , Humanos , Yoduros , Neuroblastoma/diagnóstico por imagen , Neurotransmisores , NicotinaRESUMEN
The serine protease prostasin is a negative regulator of lipopolysaccharide-induced inflammation and has a role in the regulation of cellular immunity. Prostasin expression in cancer cells inhibits migration and metastasis, and reduces epithelial-mesenchymal transition. Programmed death-ligand 1 (PD-L1) is a negative regulator of the immune response and its expression in cancer cells interferes with immune surveillance. The aim of this study was to investigate if prostasin regulates PD-L1 expression. We established sublines over-expressing various forms of prostasin as well as a subline deficient for the prostasin gene from the Calu-3 human lung cancer cells. We report here that PD-L1 expression induced by interferon-gamma (IFNg) is further enhanced in cells over-expressing the wild-type membrane-anchored prostasin. The PD-L1 protein was localized on the cell surface and released into the culture medium in extracellular vesicles (EVs) with the protease-active prostasin. The epidermal growth factor-epidermal growth factor receptor (EGF-EGFR), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) participated in the prostasin-mediated up-regulation of PD-L1 expression. A Gene Set Enrichment Analysis (GSEA) of patient lung tumors in The Cancer Genome Atlas (TCGA) database revealed that prostasin and PD-L1 regulate common signaling pathways during tumorigenesis and tumor progression.
RESUMEN
The serine protease prostasin is a negative regulator of lipopolysaccharide-induced inflammation and has a role in the regulation of cellular immunity. Prostasin expression in cancer cells inhibits migration and metastasis, and reduces epithelial-mesenchymal transition. Programmed death-ligand 1 (PD-L1) is a negative regulator of the immune response and its expression in cancer cells interferes with immune surveillance. The aim of the present study was to investigate if prostasin regulates PD-L1 expression. We established sublines overexpressing various forms of prostasin as well as a subline deficient for the prostasin gene from the Calu-3 human lung cancer cells. We report here that PD-L1 expression induced by interferon-γ (IFNγ) is further enhanced in cells overexpressing the wildtype membrane-anchored prostasin. The PD-L1 protein was localized on the cell surface and released into the culture medium in extracellular vesicles (EVs) with the protease-active prostasin. The epidermal growth factor-epidermal growth factor receptor (EGF-EGFR), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) participated in the prostasin-mediated up-regulation of PD-L1 expression. A Gene Set Enrichment Analysis (GSEA) of patient lung tumors in The Cancer Genome Atlas (TCGA) database revealed that prostasin and PD-L1 regulate common signaling pathways during tumorigenesis and tumor progression.
Asunto(s)
Adenocarcinoma del Pulmón/enzimología , Antígeno B7-H1/metabolismo , Vesículas Extracelulares/enzimología , Neoplasias Pulmonares/enzimología , Serina Endopeptidasas/metabolismo , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/inmunología , Antígeno B7-H1/genética , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/genética , Vesículas Extracelulares/inmunología , Regulación Neoplásica de la Expresión Génica , Humanos , Interferón gamma/farmacología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Quinasa C/metabolismo , Serina Endopeptidasas/genética , Transducción de Señal , Regulación hacia ArribaRESUMEN
OBJECTIVE: The type-II transmembrane extracellular serine protease matriptase was shown to cleave at Arg-102 in the amino-terminal region of the amyloid precursor protein (APP). In this study we determined matriptase cleavage sites in the amyloid-beta (Aß) peptide region of APP (Asp-597 to Ala-638 in the APP695 isoform). A recombinant human matriptase protease domain was used to cleave a synthetic human Aß1-42 peptide. The human APP695 or mutants at the candidate matriptase cleavage sites was co-expressed with the human matriptase or its protease-dead mutant in HEK293 cells to evaluate matriptase cleavage of APP. Overexpression of matriptase in the M17 human neuroblastoma cells was performed to determine the effect of matriptase expression on endogenous APP. RESULTS: The human Aß1-42 peptide can be cleaved by the matriptase serine protease domain, at Arg-5, Lys-16, and Lys-28, as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Co-expression of matriptase but not its protease-dead mutant with APP695 resulted in site-specific cleavages of the latter. Replacement of Arg-601 (Arg-5 in Aß1-42) by Ala in APP695 prevented matriptase cleavage at this site. Overexpression of matriptase but not its protease-dead mutant in the M17 cells resulted in a significant reduction of the endogenous APP quantity.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Fragmentos de Péptidos/metabolismo , Serina Endopeptidasas/metabolismo , Línea Celular Tumoral , Células HEK293 , HumanosRESUMEN
Although cigarette smoke is known to alter immune responses, whether and how CD4 T cells are affected is not well-described. We aimed to characterize how exposure to cigarette smoke extract impacts CD4 T cell effector generation in vitro under Th1-polarizing conditions. Our results demonstrate that cigarette smoke directly acts on CD4 T cells to impair effector expansion by decreasing division and increasing apoptosis. Furthermore, cigarette smoke enhances Th1-associated cytokine production and increases expression of the transcription factor T-bet, the master regulator of Th1 differentiation. Finally, we show that exposure to cigarette smoke extract during priming impairs the ability of effectors to form memory cells. Our findings thus demonstrate that cigarette smoke simultaneously enhances effector functions but promotes terminal differentiation of CD4 T cell effectors. This study may be relevant to understanding how smoking can both aggravate autoimmune symptoms and reduce vaccine efficacy.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Memoria Inmunológica/inmunología , Activación de Linfocitos/inmunología , Nicotiana/química , Humo , Células TH1/inmunología , Animales , Apoptosis/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/inmunología , División Celular/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Humanos , Ratones Endogámicos BALB C , Ratones Transgénicos , Células TH1/metabolismoRESUMEN
The epithelial extracellular membrane-associated serine proteases matriptase, hepsin, and prostasin are proteolytic modifying enzymes of the extracellular domain (ECD) of the epidermal growth factor receptor (EGFR). Matriptase also cleaves the ECD of the vascular endothelial growth factor receptor 2 (VEGFR2) and the angiopoietin receptor Tie2. In this study we tested the hypothesis that these serine proteases may cleave the ECD of additional receptor tyrosine kinases (RTKs). We co-expressed the proteases in an epithelial cell line with Her2, Her3, Her4, insulin receptor (INSR), insulin-like growth factor I receptor (IGF-1R), the platelet-derived growth factor receptors (PDGFRs) α and ß, or nerve growth factor receptor A (TrkA). Western blot analysis was performed to detect the carboxyl-terminal fragments (CTFs) of the RTKs. Matriptase and hepsin were found to cleave the ECD of all RTKs tested, while TMPRSS6/matriptase-2 cleaves the ECD of Her4, INSR, and PDGFR α and ß. Prostasin was able to cleave the ECD of Her3 and PDGFRα. Matriptase cleaves phosphorylated Her2 at Arg558 and Arg599 and the Arg599 cleavage produces a CTF not recognized by the monoclonal antibody trastuzumab/Herceptin. Her2 cleavages by matriptase can be inhibited by the hepatocyte growth factor activator inhibitor 1 (HAI-1) in the MDA-MB-231 human breast cancer cells. Matriptase silencing in the Her2, matriptase, and HAI-1 triple-positive SKBR3 human breast cancer cells enhanced Her2 protein down-regulation induced by a sustained exposure to phorbol 12-myristate 13-acetate (PMA), which down-regulated matriptase protein. The novel Her2 cleavage and expression regulation mechanisms mediated by matriptase may have potential impacts in Her2-targeting therapies.
RESUMEN
BACKGROUND: The glycosylphosphatidylinositol-anchored extracellular membrane serine protease prostasin is expressed in normal bladder urothelial cells. Bladder inflammation reduces prostasin expression and a loss of prostasin expression is associated with epithelial-mesenchymal transition (EMT) in human bladder transitional cell carcinomas. Non-steroidal anti-inflammatory drugs (NSAIDs) decrease the incidence of various cancers including bladder cancer, but the molecular mechanisms underlying the anticancer effect of NSAIDs are not fully understood. METHODS: The normal human bladder urothelial cell line UROtsa, the normal human trophoblast cell line B6Tert-1, human bladder transitional cell carcinoma cell lines UM-UC-5 and UM-UC-9, and the human breast cancer cell line JIMT-1 were used for the study. Expression changes of the serine proteases prostasin and matriptase, and cyclooxygenases (COX-1 and COX-2) in these cells following ibuprofen treatments were analyzed by means of reverse-transcription/quantitative polymerase chain reaction (RT-qPCR) and immunoblotting. The functional role of the ibuprofen-regulated prostasin in epithelial tight junction formation and maintenance was assessed by measuring the transepithelial electrical resistance (TEER) and epithelial permeability in the B6Tert-1 cells. Prostasin's effects on tight junctions were also evaluated in B6Tert-1 cells over-expressing a recombinant human prostasin, silenced for prostasin expression, or treated with a functionally-blocking prostasin antibody. Matriptase zymogen activation was examined in cells over-expressing prostasin. RESULTS: Ibuprofen increased prostasin expression in the UROtsa and the B6Tert-1 cells. Cyclooxygenase-2 (COX-2) expression was up-regulated at both the mRNA and the protein levels in the UROtsa cells by ibuprofen in a dose-dependent manner, but was not a requisite for up-regulating prostasin expression. The ibuprofen-induced prostasin contributed to the formation and maintenance of the epithelial tight junctions in the B6Tert-1 cells. The matriptase zymogen was down-regulated in the UROtsa cells by ibuprofen possibly as a result of the increased prostasin expression because over-expressing prostasin leads to matriptase activation and zymogen down-regulation in the UROtsa, JIMT-1, and B6Tert-1 cells. The expression of prostasin and matriptase was differentially regulated by ibuprofen in the bladder cancer cells. CONCLUSIONS: Ibuprofen has been suggested for use in treating bladder cancer. Our results bring the epithelial extracellular membrane serine proteases prostasin and matriptase into the potential molecular mechanisms of the anticancer effect of NSAIDs.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Ibuprofeno/farmacología , Serina Endopeptidasas/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Células CACO-2 , Línea Celular , Ciclooxigenasa 2/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Serina Endopeptidasas/genética , Uniones Estrechas/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/genéticaRESUMEN
BACKGROUND: Because of the faltering sensitivity and/or specificity, urine-based assays currently have a limited role in the management of patients with bladder cancer. The aim of this study was to externally validate our previously reported protein biomarker panel from multiple sites in the United States and Europe. METHODS: This multicenter external validation study included a total of 320 subjects (bladder cancer = 183). The 10 biomarkers (IL8, MMP9, MMP10, SERPINA1, VEGFA, ANG, CA9, APOE, SDC1, and SERPINE1) were measured using commercial ELISA assays in an external laboratory. The diagnostic performance of the biomarker panel was assessed using receiver operator curves (ROC) and descriptive statistical values. RESULTS: Utilizing the combination of all 10 biomarkers, the area under the ROC for the diagnostic panel was noted to be 0.847 (95% confidence interval, 0.796-0.899), outperforming any single biomarker. The multiplex assay at optimal cutoff value achieved an overall sensitivity of 0.79, specificity of 0.79, positive prediction value of 0.73, and negative prediction value of 0.84 for bladder cancer classification. Sensitivity values of the diagnostic panel for high-grade bladder cancer, low-grade bladder cancer, muscle invasive bladder cancer, and non-muscle invasive bladder cancer were 0.81, 0.90, 0.95, and 0.77, respectively. CONCLUSIONS: Urinary levels of the biomarker panel enabled discrimination of patients with bladder cancer and controls, and the levels of biomarker subsets were associated with advancing tumor grade and stage. IMPACT: If proven to be reliable, urinary diagnostic biomarker assays can detect bladder cancer in a timely manner such that the patient can expect improvements in overall survival and quality of life.
Asunto(s)
Biomarcadores de Tumor/orina , Proteinuria/orina , Neoplasias de la Vejiga Urinaria/orina , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Europa (Continente)/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Proteinuria/epidemiología , Proteinuria/patología , Reproducibilidad de los Resultados , Estados Unidos/epidemiología , Neoplasias de la Vejiga Urinaria/epidemiología , Neoplasias de la Vejiga Urinaria/patología , Adulto JovenRESUMEN
Intravesical Bacillus Calmette-Guérin (BCG) has been shown to induce a specific immunologic response (i.e., activation of IL-2 and effector T-cells), while preclinical studies using ALT-803 (mutated IL-15 analogue combined with IL-15Rα-Fc fusion) have shown promising results by prolonging the agent's half-life and stimulating CD8+ T-cells. Based on these results, we hypothesized that the intravesical administration of ALT-803 along with BCG will generate an immunologic response leading to significant bladder tumor burden reduction. Using a well-established carcinogen induced rat non-muscle invasive bladder cancer (NMIBC) model, we studied the effects of intravesical ALT-803 with and without BCG. Rat tissues were evaluated to document treatment response. Intravesical ALT-803 was safe and well tolerated alone and in combination with BCG. As a single treatment agent, ALT-803 reduced tumor burden by 35% compared to control whereas BCG alone only reduced tumor burden by 15%. However, the combination of ALT-803 plus BCG reduced tumor burden by 46% compared to control. Immune monitoring suggested that the antitumor response was linked to the production and secretion of IL-1α, IL-1ß and RANTES, which in turn, induced the proliferation and activation of NK cells. Lastly, tumoral responses of the combinational treatment were associated with 76% reduction in angiogenesis, which is significantly higher than when assessed with either agent alone. The enhanced therapeutic index seen with this duplet provides justification for the development of this regimen for future clinical trials.
Asunto(s)
Antineoplásicos/uso terapéutico , Citocinas/biosíntesis , Células Asesinas Naturales/inmunología , Mycobacterium bovis/inmunología , Proteínas/uso terapéutico , Carga Tumoral/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Administración Intravesical , Animales , Antineoplásicos/farmacología , Modelos Animales de Enfermedad , Femenino , Proteínas/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/inmunología , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/inducido químicamente , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
UNLABELLED: Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates intracellular signaling networks for inhibition of apoptosis. Tetraspanin (CD63), a cell surface binding partner for TIMP-1, was previously shown to regulate integrin-mediated survival pathways in the human breast epithelial cell line MCF10A. In the current study, we show that TIMP-1 expression induces phenotypic changes in cell morphology, cell adhesion, cytoskeletal remodeling, and motility, indicative of an epithelial-mesenchymal transition (EMT). This is evidenced by loss of the epithelial cell adhesion molecule E-cadherin with an increase in the mesenchymal markers vimentin, N-cadherin, and fibronectin. Signaling through TIMP-1, but not TIMP-2, induces the expression of TWIST1, an important EMT transcription factor known to suppress E-cadherin transcription, in a CD63-dependent manner. RNAi-mediated knockdown of TWIST1 rescued E-cadherin expression in TIMP-1-overexpressing cells, demonstrating a functional significance of TWIST1 in TIMP-1-mediated EMT. Furthermore, analysis of TIMP-1 structural mutants reveals that TIMP-1 interactions with CD63 that activate cell survival signaling and EMT do not require the matrix metalloproteinase (MMP)-inhibitory domain of TIMP-1. Taken together, these data demonstrate that TIMP-1 binding to CD63 activates intracellular signal transduction pathways, resulting in EMT-like changes in breast epithelial cells, independent of its MMP-inhibitory function. IMPLICATIONS: TIMP-1's function as an endogenous inhibitor of MMP or as a "cytokine-like" signaling molecule may be a critical determinant for tumor cell behavior.
Asunto(s)
Neoplasias de la Mama/genética , Transición Epitelial-Mesenquimal/genética , Proteínas Nucleares/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/genética , Proteína 1 Relacionada con Twist/metabolismo , Apoptosis/genética , Neoplasias de la Mama/patología , Cadherinas/genética , Cadherinas/metabolismo , Proliferación Celular/genética , Supervivencia Celular/genética , Células Epiteliales , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Nucleares/genética , Transducción de Señal , Tetraspanina 30 , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Proteína 1 Relacionada con Twist/genéticaRESUMEN
Several aspects of HIV-1 virulence and pathogenesis are mediated by the envelope protein gp41. Additionally, peptides derived from the gp41 ectodomain have been shown to induce chemotaxis in monocytes and neutrophils. Whereas this chemotactic activity has been reported, it is not known how these peptides could be produced under biological conditions. The heptad repeat 1 (HR1) region of gp41 is exposed to the extracellular environment and could therefore be susceptible to proteolytic processing into smaller peptides. Matriptase is a serine protease expressed at the surface of most epithelia, including the prostate and mucosal surfaces. Here, we present evidence that matriptase efficiently cleaves the HR1 portion of gp41 into a 22-residue chemotactic peptide MAT-1, the sequence of which is highly conserved across HIV-1 clades. We found that MAT-1 induced migration of primary neutrophils and monocytes, the latter of which act as a cellular reservoir of HIV during early stage infection. We then used formyl peptide receptor 1 (FPR1) and FPR2 inhibitors, along with HEK 293 cells, to demonstrate that MAT-1 can induce chemotaxis specifically using FPR2, a receptor found on the surface of monocytes, macrophages and neutrophils. These findings are the first to identify a proteolytic cleavage product of gp41 with chemotactic activity and highlight a potential role for matriptase in HIV-1 transmission and infection at epithelial surfaces and within tissue reservoirs of HIV-1.
Asunto(s)
Quimiotaxis de Leucocito , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/metabolismo , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismo , Serina Endopeptidasas/metabolismo , Células HEK293 , Proteína gp41 de Envoltorio del VIH/inmunología , Células HL-60 , Humanos , Fragmentos de Péptidos/biosíntesisRESUMEN
Cigarette smoking is the single most important epidemiological risk factor for bladder cancer but it is not known whether exposure of urothelial cells to the systemic soluble contents of cigarette smoke is directly causative to bladder cancer and the associated epigenetic changes such as tumor suppressor gene hypermethylation. We undertook this study to investigate if long-term treatment of human urothelial cells with cigarette smoke extract (CSE) results in tumor suppressor gene hypermethylation, a phenotype that was previously associated with long-term constant CSE treatment of airway epithelial cells. We chronically treated an immortalized human urothelial cell line UROtsa with CSE using a cyclic daily regimen but the cells were cultured in CSE-free medium between daily treatments. Bisulfite sequencing and real-time PCR array-based methylation profiling were employed to evaluate methylation changes at tumor suppressor gene loci in the chronically CSE-treated cells versus the passage-matched untreated control cells. The RUNX3 tumor suppressor gene promoter was hypomethylated with a significant increase in proportion of the completely unmethylated haplotype after the long-term CSE treatment; whereas RUNX3 promoter hypermethylation was previously reported for bladder cancers of smokers. Hypomethylation induced by the long-term CSE treatment was also observed for the IGF2-H19 locus. The methylation status at the PRSS8/prostasin and 16 additional loci however, was unaffected by the chronic CSE treatment. Transient CSE treatment over 1 daily regimen resulted in transcriptional down-regulation of RUNX3 and H19, but only the H19 transcription was down-regulated in the chronically CSE-treated urothelial cells. Transcription of a key enzyme in one-carbon metabolism, dihydrofolate reductase (DHFR) was greatly reduced by the long-term CSE treatment, potentially serving as a mechanism for the hypomethylation phenotype via a reduced supply of methyl donor. In conclusion, chronic cyclic CSE treatment of urothelial cells induced hypomethylation rather than hypermethylation at specific loci.
Asunto(s)
Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Metilación de ADN/genética , Exposición a Riesgos Ambientales , Sitios Genéticos/genética , Factor II del Crecimiento Similar a la Insulina/genética , Fumar/efectos adversos , Urotelio/citología , Línea Celular Transformada , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Epigénesis Genética , Humanos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Factores de Tiempo , Urotelio/metabolismoRESUMEN
Maternal cigarette smoking has adverse effects on pregnancy outcomes. The granulocyte-macrophage colony-stimulating factor (GM-CSF) is an essential cytokine for a normal pregnancy. We investigated the impact of cigarette smoke extract (CSE) on GM-CSF expression in human cytotrophoblast cells and suggested a cellular mechanism underlying the CSE-induced GM-CSF expression. An immortalized normal human trophoblast cell line (B6Tert-1) was treated with CSE. The viability and proliferation of the CSE-treated B6Tert-1 cells were evaluated, and the expression of GM-CSF in these cells was quantified at the mRNA and the protein levels by means of reverse-transcription and quantitative polymerase chain reaction (RT-qPCR); and enzyme-linked immunosorbent assay (ELISA), respectively. Human trophoblast cells treated with CSE had an increased expression of GM-CSF at both the mRNA and the protein levels. The CSE-induced GM-CSF expression was synergistically enhanced by the addition of the proteasome inhibitor MG-132, but inhibited by AG-1478, an inhibitor of the epidermal growth factor receptor (EGFR) kinase. Furthermore, CSE treatment increased the phosphorylation of the extracellular-signal regulated kinases (ERK1/2) in the trophoblast cells. The expression of other growth factors such as heparin-binding epidermal growth factor-like growth factor (HB-EGF) and vascular endothelial growth factor (VEGF) was also evaluated. Our data suggested that cigarette smoking and proteasome inhibition synergistically up-regulate GM-CSF cytokine expression by activating the EGFR signaling pathway.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Nicotiana/química , Inhibidores de Proteasoma/farmacología , ARN Mensajero/metabolismo , Humo/efectos adversos , Trofoblastos/metabolismo , Western Blotting , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Receptores ErbB/antagonistas & inhibidores , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Leupeptinas/farmacología , Microscopía Fluorescente , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Embarazo , Quinazolinas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tirfostinos/farmacologíaRESUMEN
Membrane-associated serine protease matriptase has been implicated in human diseases and might be a drug target. In the present study, a novel class of matriptase inhibitors targeting zymogen activation is developed by a combination of the screening of compound library using a cell-based matriptase activation assay and a computer-aided search of commercially available analogues of a selected compound. Four structurally related compounds are identified that can inhibit matriptase activation with IC(50) at low micromolar concentration in both intact-cell and cell-free systems, suggesting that these inhibitors target the matriptase autoactivation machinery rather than the intracellular signaling pathways. These activation inhibitors can also inhibit prostasin activation, a downstream event that occurs in lockstep with matriptase activation. In contrast, the matriptase catalytic inhibitor CVS-3983 at a concentration 300-fold higher than its K(i) fails to inhibit activation of either protease. Our results suggest that inhibiting matriptase activation is an efficient way to control matriptase function.
Asunto(s)
Precursores Enzimáticos/metabolismo , Pirroles/química , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/síntesis química , Línea Celular , Sistema Libre de Células , Activación Enzimática/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Ensayos Analíticos de Alto Rendimiento , Humanos , Oligopéptidos/farmacología , Proteolisis , Pirroles/farmacología , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Relación Estructura-ActividadRESUMEN
We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called "SEVI" and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity.
Asunto(s)
VIH-1/fisiología , Fragmentos de Péptidos/farmacología , Proteínas Tirosina Fosfatasas/farmacología , Semen/virología , Fosfatasa Ácida , Amiloide , Relación Dosis-Respuesta a Droga , Realizador del VIH , VIH-1/efectos de los fármacos , Humanos , Antígeno Prostático Específico/metabolismo , Serina Endopeptidasas/metabolismoRESUMEN
Matriptase, a membrane-tethered serine protease, plays essential roles in epidermal differentiation and barrier function, largely mediated via its activation of prostasin, a glycosylphosphatidylinositol-anchored serine protease. Matriptase activity is tightly regulated by its inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) such that free active matriptase is only briefly available to act on its substrates. In the current study we provide evidence for how matriptase activates prostasin under this tight control by HAI-1. When primary human keratinocytes are induced to differentiate in a skin organotypic culture model, both matriptase and prostasin are constitutively activated and then inhibited by HAI-1. These processes also occur in HaCaT human keratinocytes when matriptase activation is induced by exposure of the cells to a pH 6.0 buffer. Using this acid-inducible activation system we demonstrate that prostatin activation is suppressed by matriptase knockdown and by blocking matriptase activation with sodium chloride, suggesting that prostatin activation is dependent on matriptase in this system. Kinetics studies further reveal that the timing of autoactivation of matriptase, prostasin activation, and inhibition of both enzymes by HAI-1 binding are closely correlated. These data suggest that, during epidermal differentiation, the matriptase-prostasin proteolytic cascade is tightly regulated by two mechanisms: 1) prostasin activation temporally coupled to matriptase autoactivation and 2) HAI-1 rapidly inhibiting not only active matriptase but also active prostasin, resulting in an extremely brief window of opportunity for both active matriptase and active prostasin to act on their substrates.
Asunto(s)
Diferenciación Celular/fisiología , Epidermis/enzimología , Queratinocitos/enzimología , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Serina Endopeptidasas/metabolismo , Células 3T3 , Animales , Diferenciación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Ratones , Proteínas Inhibidoras de Proteinasas Secretoras/farmacologíaRESUMEN
We recently demonstrated that tissue kallikrein (TK) promotes keratinocyte migration through activation of protease-activated receptor-1 (PAR(1)) and transactivation of the epi-dermal growth factor receptor (EGFR). In this study, we investigated the potential role of PAR(1) in mediating the effect of TK on cancer cell migration, invasion and proliferation. Our results show that TK promotes DU145 prostate cancer cell migration in a concentration-dependent manner, but has no effect on A549 lung cancer cells. Active TK markedly increases DU145 cell migration and invasion, which are blocked by aprotinin but minimally affected by icatibant; kinin treatment has little effect. TK-induced cell migration and invasion are abolished by inhibition of PAR(1) using a pharmacological inhibitor or RNA interference. The effect of TK on cell migration and invasion are also blocked by inhibitors of protein kinase C, c-Src, matrix metalloproteinase, EGFR and extracellular signal-regulated kinase (ERK). Moreover, TK stimulates ERK phosphorylation, which is inhibited by an EGFR antagonist. Additionally, TK but not kinin stimulates DU145 cell proliferation through activation of the kinin B2 receptor, but not PAR(1) and EGFR. These results indicate differential signaling pathways mediated by TK in promoting prostate cancer cell migration and invasion via PAR(1) activation, and proliferation via kinin B2 receptor stimulation.
Asunto(s)
Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptor PAR-1/metabolismo , Calicreínas de Tejido/metabolismo , Aprotinina/farmacología , Bradiquinina/análogos & derivados , Bradiquinina/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Cininas/farmacología , Masculino , Invasividad Neoplásica , Receptor PAR-1/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Calicreínas de Tejido/antagonistas & inhibidores , Calicreínas de Tejido/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
Most of the existing techniques cannot be used to detect molecular biomarkers contained in complex fluids due to issues such as enzyme inhibition or signal interference. We have developed a nanoparticle-based scanning calorimetric method for the highly sensitive detections of multiple DNA biomarkers contained in cell lysate and milk by using solid-liquid phase change nanoparticles as thermal barcodes. The detection is based on the principle that the temperature of solid will not rise above the melting temperature unless all solid is molten, thus nanoparticles have sharp melting peaks during the thermal scan process. A one-to-one correspondence can thus be created between one type of nanoparticles and one type of biomarker, i.e., multiple biomarkers can be detected at the same time using a combination of nanoparticles. The melting temperature and the heat flow reflect the type and the concentration of the biomarker, respectively. The target oligonucleotides at low concentration in cell lysate (80 pM) have been detected through thermal signal transduction. The melting temperature of nanoparticles can be designed to avoid interference from coexisting species contained in the fluids, bringing simultaneously high sensitivity and multiplicity, as well as sample preparation benefits to biomarker detections.
Asunto(s)
Biomarcadores/análisis , Calorimetría/métodos , ADN/análisis , Secuencia de Bases , Microscopía Electrónica de Transmisión , Nanopartículas , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Prostasin is a glycosylphosphatidylinositol-anchored extracellular serine protease with a role in epidermal growth factor receptor (EGFR) signal modulation. EGFR signaling has been shown to be important for regulating cytotrophoblast (CT) cell proliferation in human placenta. We investigated the impact of prostasin expression regulation on this cellular function as well as the molecular mechanisms involved in human cytotrophoblastic cells. METHODS: An immortalized normal human CT cell line (B6Tert-1) was used as an in vitro cell model. Prostasin expression in B6Tert-1 cells was knocked down by transfection of a short interfering RNA. Lentivirus-mediated expression of recombinant human prostasin under tetracycline regulation was performed to obtain stable B6Tert-1 cell sublines that over-expressed prostasin. Changes in cell proliferation and EGFR signaling were evaluated by immunocytochemistry for Ki67 and western blot analysis, respectively, in B6Tert-1 cells with knocked-down or increased prostasin expression. RESULTS: Prostasin knock-down in B6Tert-1 cells resulted in inhibition of cell proliferation, in association with down-regulated EGFR protein expression (both P < 0.05 versus control) as well as reduced phosphorylation of c-raf, mitogen-activated protein kinase (MAPK) kinases (MEK1/2) and extracellular signal-regulated kinases (Erk1/2) (all P < 0.05 versus control). Over-expression of prostasin led to up-regulation of the EGFR protein, but had no effect on cell proliferation or phosphorylation of MAPK signaling molecules in the B6Tert-1 cells. CONCLUSIONS: Prostasin may regulate trophoblast cell proliferation via modulating the EGFR-MAPK signaling pathway.