RESUMEN
Programmed death-1 homolog (PD-1H) is a coinhibitory molecule that negatively regulates T cell-mediated immune responses. In this study, we determined whether ablation of T cell-associated PD-1H could enhance adoptive T cell therapy in experimental tumor models. The expression of PD-1H is upregulated in activated and tumor-infiltrating CD8+ T cells. Activated CD8+ T cells from PD-1H-deficient (PD-1H-KO) mice exhibited increased cell proliferation, cytokine production, and antitumor activity in vitro. Adoptive transfer of PD-1H-KO CD8+ T cells resulted in the regression of established syngeneic mouse tumors. Similar results were obtained when PD-1H was ablated in T cells by CRISPR/Cas9-mediated gene silencing. Furthermore, ablation of PD-1H in CAR-T cells significantly improved their antitumor activity against human xenografts in vivo. Our results indicate that T cell-associated PD-1H could suppress immunity in the tumor microenvironment and that targeting PD-1H may improve T cell adoptive immunotherapy.
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Traslado Adoptivo/métodos , Linfocitos T CD8-positivos/inmunología , Inmunoterapia Adoptiva/métodos , Proteínas de la Membrana , Microambiente Tumoral/inmunología , Animales , Línea Celular Tumoral , Proliferación Celular , Citocinas/biosíntesis , Silenciador del Gen , Marcación de Gen/métodos , Humanos , Inmunidad Celular/genética , Inmunidad Celular/inmunología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/inmunología , Ratones , Neoplasias Experimentales/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal carcinoma (EC) in China. Although the PD-1 inhibitor pembrolizumab has been approved to treat patients with EC, its therapeutic efficacy is limited. Thus, additional immunotherapeutic targets for EC treatment are needed. Programmed Death-1 Homolog (PD-1H) is a negative checkpoint regulator that inhibits antitumor immune responses. Here, PD-1H expression in 114 patients with ESCC was evaluated by immunohistochemistry. Next, 12 randomly selected tumor tissue sections were used to assess the colocalization of PD-1H protein and multiple immune markers by multiplex immunohistochemistry. Our results demonstrated that PD-1H was expressed at high frequency in ESCC tumor tissues (85.1%). PD-1H protein was predominantly expressed in CD68+ tumor-associated macrophages and expressed at low levels in CD4+ T cells and CD8+ T cells in ESCC tumor tissues. Furthermore, based on ESCC data in The Cancer Genome Atlas (TCGA), the gene expression levels of PD-1H were positively associated with the infiltration levels of immune-activated cells especially CD8+ cytotoxic T cells. In contrast, the gene expression levels of PD-1H were negatively correlated with myeloid-derived suppressor cells (MDSCs). Importantly, PD-1H expression in tumor sites was significantly correlated with favorable overall survival in patients with ESCC. Collectively, our findings first provided direct information on the PD-1H expression pattern and distribution in ESCC, and positive correlation of PD-1H expression with overall survival suggested PD-1H expression levels could be a significant prognostic indicator for patients with ESCC. Future studies need to explore the immunoregulatory of PD-1H in the tumor microenvironment of ESCC.
RESUMEN
The application of chimeric antigen receptor (CAR)-T cell therapy in patients with advanced solid tumors remains a significant challenge. Simultaneously targeting antigen and the solid tumor microenvironment are two major factors that greatly impact CAR-T cell therapy outcomes. In this study, we engineered CAR-T cells to specifically target B7-H3, a protein commonly found in solid human tumors, using a single-chain variable fragment (scFv) derived from an anti-B7-H3 monoclonal antibody. We tested the antitumor activity of B7-H3 CAR-T cells in mouse models with solid human tumors and determined that B7-H3 CAR-T cells exhibited potent antitumor activity against B7-H3+ tumor cells in vitro and in vivo. In addition, PD-1 decoy receptors were engineered to include extracellular PD-1 fused to the intracellular stimulatory domain of either CD28 or IL-7 receptor, respectively, which were then introduced into B7-H3 CAR-T cells. As a result, these newly modified, superior CAR-T cells exhibited more persistent antitumor activity in B7-H3+/B7-H1+ tumors in vivo. Our findings indicate that B7-H3 specific CAR-T cells have the potential to treat multiple types of advanced solid tumors.
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Inmunoterapia Adoptiva , Neoplasias Experimentales/terapia , Receptores Quiméricos de Antígenos , Animales , Antígeno B7-H1 , Línea Celular Tumoral , Humanos , Ratones , Receptor de Muerte Celular Programada 1 , Receptores Quiméricos de Antígenos/genética , Linfocitos T , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The oncogenic epidermal growth factor receptor (EGFR) is commonly overexpressed in solid cancers. The tyrosine kinase activity of EGFR has been a major therapeutic target for cancer; however, the efficacy of EGFR tyrosine kinase inhibitors to treat cancers has been challenged by innate and acquired resistance at the clinic. Accumulating evidence suggests that EGFR possesses kinase-independent pro-survival functions, and that cancer cells are more vulnerable to reduction of EGFR protein than to inhibition of its kinase activity. The molecular mechanism underlying loss-of-EGFR-induced cell death remains largely unknown. In this study, we show that, unlike inhibiting EGFR kinase activity that is known to induce pro-survival non-selective autophagy, downregulating EGFR protein, either by siRNA, or by a synthetic EGFR-downregulating peptide (Herdegradin), kills prostate and ovarian cancer cells via selective mitophagy by activating the mTORC2/Akt axis. Furthermore, Herdegradin induced mitophagy and inhibited the growth of orthotopic ovarian cancers in mice. This study identifies anti-mitophagy as a kinase-independent function of EGFR, reveals a novel function of mTORC2/Akt axis in promoting mitophagy in cancer cells, and offers a novel approach for pharmacological downregulation of EGFR protein as a potential treatment for EGFR-positive cancers.
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The metastatic potential of osteosarcoma cells is inversely correlated to cell surface FAS expression. Downregulation of FAS allows osteosarcoma cells to escape FAS ligand-mediated apoptosis when they enter a FAS ligand-positive microenvironment such as the lung. We have previously demonstrated that miR-20a, encoded by the miR-17-92 cluster, downregulates FAS expression in osteosarcoma. We further demonstrated an inverse correlation between FAS expression and miR-20a expression. However, the mechanism of FAS regulation by miR-20a was still unclear. The purpose of the current study was to evaluate the mechanism of FAS regulation by miR-20a in vitro and test the effect of targeting miR-20a in vivo We investigated whether miR-20a's downregulation of FAS was mediated by binding to the 3'-untranslated region (3'-UTR) of FAS mRNA with the consequent induction of mRNA degradation or translational suppression. We identified and mutated two miR-20a binding sites on the FAS mRNA 3'-UTR. Using luciferase reporter assays, we demonstrated that miR-20a did not bind to either the wild-type or mutated FAS 3'-UTR. In contrast, overexpression of miR-20a resulted in downregulation of FAS promoter activity. Similarly, the inhibition of miR-20a increased FAS promoter activity. The critical region identified on the FAS promoter was between -240 bp and -150 bp. Delivery of anti-miR-20a in vivo using nanoparticles in mice with established osteosarcoma lung metastases resulted in upregulation of FAS and tumor growth inhibition. Taken together, our data suggest that miR-20a regulates FAS expression through the modulation of the FAS promoter and that targeting miR-20a using anti-miR-20a has therapeutic potential. Mol Cancer Ther; 17(1); 130-9. ©2017 AACR.
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Neoplasias Pulmonares/secundario , MicroARNs/genética , MicroARNs/metabolismo , Osteosarcoma/genética , Receptor fas/biosíntesis , Animales , Línea Celular Tumoral , Células HEK293 , Células HeLa , Xenoinjertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Osteosarcoma/metabolismo , Osteosarcoma/patología , Regiones Promotoras Genéticas , Transfección , Receptor fas/genéticaRESUMEN
There is a crucial need for a new therapeutic approach for osteosarcoma (OS) lung metastasis since this disease remains the main cause of mortality in OS. We previously demonstrated that natural killer (NK) cell therapy has minimal efficacy against OS metastasis. This study determined whether the histone deacetylase inhibitor entinostat could immunosensitize OS cells to NK cell lysis and increases the efficacy of NK cell therapy for OS lung metastasis. Entinostat upregulated ligands for NK cell-activating receptors (major histocompatibility complex [MHC] class I polypeptide-related chain A [MICA] and B [MICB]; UL16 binding proteins 1, 2, 5, and 6; and CD155) on OS cells both in vitro and in vivo and led to more susceptibility to NK cell-mediated cytotoxicity in vitro. Importantly, entinostat did not change NK cell viability, receptor expression, or function within the 24-h treatment. We also demonstrated two potential mechanisms by which entinostat enhanced expression of MICA and MICB on OS cells. Although entinostat upregulated ligands for the NK cell activating receptor on OS lung metastasis, it failed to augment the efficacy of NK cell therapy in our nude mouse human OS lung metastasis model. This can be partly explained by our finding that although the infused NK cells were active and functional and could penetrate into the lungs, they failed to infiltrate into the lung nodules. These challenges regarding cellular immunotherapy against solid tumors may be overcome by combination therapy, such as adding a NK cell-activating cytokine (IL-2 or IL-21).
RESUMEN
The lungs are the most common site for the metastatic spread of osteosarcoma. Success in using chemotherapy to improve overall survival has reached a plateau. Understanding the biologic properties that permit osteosarcoma cells to grow in the lungs may allow the identification of novel therapeutic approaches-the goal being to alter the tumor cells' expression of cell surface proteins so that there is no longer compatibility with the metastatic niche. We have demonstrated that the Fas Ligand positive (FasL(+)) lung microenvironment eliminates Fas(+) osteosarcoma cells that metastasize to the lungs. Indeed, osteosarcoma lung metastases from patients are Fas(-), similar to what we found in several different mouse models. The Fas(+) cells are cleared from the lungs through apoptosis induced by the Fas signaling pathway following interaction of Fas on the tumor cell surface with the lung FasL. Blocking the Fas signaling pathway interferes with this process, allowing the Fas(+) cells to grow in the lungs. Our investigations show that Fas expression in osteosarcoma cells is regulated epigenetically by the micro-RNA miR-20a, encoded by the miR-17-92 cluster. Our studies support the feasibility of finding agents that can re-induce Fas expression as a novel therapeutic approach to treat osteosarcoma patients with lung metastases. We have identified two such agents, the histone deacetylase inhibitor entinostat and the chemotherapeutic agent gemcitabine (GCB). Aerosol GCB and oral entinostat induce the upregulation of Fas and the regression of established osteosarcoma lung metastases. Aerosol GCB was not effective in the FasL-deficient gld mouse confirming that the lung microenvironment was central to the success of this therapy. Our studies establish the critical role of the lung microenvironment in the metastatic process of osteosarcoma to the lungs and suggest an alternative focus for therapy, that is, incorporating the lung microenvironment as part of the treatment strategy against established osteosarcoma disease in the lungs.
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Neoplasias Óseas/genética , Proteína Ligando Fas/genética , Neoplasias Pulmonares/genética , Osteosarcoma/genética , Microambiente Tumoral/efectos de los fármacos , Receptor fas/genética , Animales , Antineoplásicos/farmacología , Benzamidas/farmacología , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Epigénesis Genética , Proteína Ligando Fas/agonistas , Proteína Ligando Fas/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/metabolismo , Osteosarcoma/secundario , Piridinas/farmacología , ARN Largo no Codificante , Transducción de Señal , Receptor fas/agonistas , Receptor fas/metabolismo , GemcitabinaRESUMEN
BACKGROUND: TC-71 Ewing sarcoma cells overexpress vascular endothelial growth factor (VEGF) with a shift from the 189 to the 165 isoform. METHODS: The effect of CAPER-α on the expression of the VEGF isoforms, tumor growth, and vessel density was analyzed after transfection of TC-71 cells with CAPER-α cDNA or siRNA. RESULTS: CAPER-α correlated inversely with the VEGF(165) /VEGF(189) mRNA ratio. Up-regulation of CAPER-α resulted in decreased tumor growth, tumor vessel density, and chemotactic activity of the cell's supernatant. CAPER-α expression was regulated by EWS/FLI-1 through a protein-protein interaction. CONCLUSIONS: Increased VEGF(165) expression is secondary to the down-regulation of CAPER-α by EWS/FLI-1. CAPER-α mediates alternative splicing and controls the shift from VEGF(189) to VEGF(165) .
Asunto(s)
Empalme Alternativo , Proteínas Nucleares/genética , Proteínas de Unión al ARN/genética , Sarcoma de Ewing/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
The ability of osteosarcoma cells to form lung metastases has been inversely correlated to cell surface Fas expression. Downregulation of Fas allows osteosarcoma cells to circumvent FasL-mediated apoptosis upon entrance into the FasL(+) lung microenvironment. However, the mechanism of Fas regulation remains unclear. Here, we show that miRNA plays a role in the downregulation of Fas expression in osteosarcoma. Expression levels of several members of the miR-17-92 cluster including miR-20a and miR-19a were found to be higher in metastatic low-Fas-expressing LM7 cells than in the parental nonmetastatic high-Fas-expressing SAOS-2 cells. We also found an inverse correlation between Fas and miR-20a expression in all 8 cell lines derived from patient samples. Overexpression of miR-20a consistently resulted in the downregulation of Fas expression in SAOS-2 cells and thus in decreased sensitivity to FasL. Conversely, inhibiting miR-20a in LM7 cells increased Fas expression and their sensitivity to FasL. Mice injected with LM7 stably transfected with anti-miR-20a had fewer metastases than those with control plasmids. Taken together, our findings suggest that miR-20a, encoded by miR-17-92, downregulates Fas expression in osteosarcoma, thus contributing to the metastatic potential of osteosarcoma cells by altering the phenotype and allowing survival in the FasL(+) lung microenvironment.
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Neoplasias Óseas/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/secundario , MicroARNs/fisiología , Osteosarcoma/genética , Osteosarcoma/secundario , Receptor fas/metabolismo , Animales , Neoplasias Óseas/genética , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Pulmonares/genética , Ratones , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Osteosarcoma/patología , ARN Largo no Codificante , TransfecciónRESUMEN
The treatment of osteosarcoma pulmonary metastases remains a challenge. T cells genetically modified to express a chimeric antigen receptor (CAR), which recognizes a tumor-associated antigen, have shown activity against hematopoietic malignancies in clinical trials, but this requires the identification of a specific receptor on the tumor cell. In the current study, we found that interleukin (IL)-11Rα was selectively expressed on 14 of 16 osteosarcoma patients' lung metastases and four different human osteosarcoma cell lines, indicating that IL-11Rα may be a novel target for CAR-specific T-cell therapy. IL-11Rα expression was absent or low in normal organ tissues, with the exception of the gastrointestinal tract. IL-11Rα-CAR-specific T cells were obtained by non-viral gene transfer of Sleeping Beauty DNA plasmids and selectively expanded ex vivo using artificial antigen-presenting cells derived from IL-11Rα + K562 cells genetically modified to coexpress T-cell costimulatory molecules. IL-11Rα-CAR(+) T cells killed all four osteosarcoma cell lines in vitro; cytotoxicity correlated with the level of IL-11Rα expression on the tumor cells. Intravenous injection of IL-11Rα-CAR(+) T cells into mice resulted in the regression of osteosarcoma pulmonary metastases with no organ toxicity. Together, the data suggest that IL-11Rα-CAR T cells may represent a new therapy for patients with osteosarcoma pulmonary metastases.
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Neoplasias Óseas/patología , Subunidad alfa del Receptor de Interleucina-11/metabolismo , Neoplasias Pulmonares/secundario , Osteosarcoma/patología , Linfocitos T/metabolismo , Animales , Línea Celular Tumoral , Electroporación , Citometría de Flujo , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , PlásmidosRESUMEN
Fas expression in osteosarcoma (OS) cells is inversely correlated with the metastatic potential of OS to the lung. The purpose of this study was to determine whether loss of Fas expression in metastatic OS cells is secondary to DNA methylation of CpG islands in the Fas gene. SAOS-2 cells have high levels of Fas expression and do not form lung metastases when injected intravenously, whereas LM7 cells have low levels of Fas expression and do produce lung metastases. Using the endonucleases HpaII and MspI and a polymerase chain reaction-based methylation assay, we found that all four CpG sites in the CCGG sequence in the Fas promoter region were unmethylated in both SAOS-2 and LM7 cells. We performed detailed analysis of the 28 and 46 CpG sites in the Fas promoter and first intron region, respectively, using bisulfite-modified genomic DNA sequencing. More than 99.8% of the examined CpG sites were unmethylated and there was no difference of CpG methylation in SAOS-2 and LM7 cells as well as LM7 metastatic lung tumor tissue samples. Treatment of LM7 cells and another OS cell line, DLM8 with low levels of Fas expression, with demethylation agent, 5-azadeoxycitidine (AzadC), did not change the Fas expression and did not increase sensitivity of AzadC-treated cells to Fas ligand (FasL) treatment. In conclusion, our data indicate that decreased Fas expression in OS cells is not secondary to DNA methylation of CpG islands in the Fas gene and that Fas expression cannot be increased by using demethylation agents.
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Islas de CpG/genética , Metilación de ADN/fisiología , Regulación Neoplásica de la Expresión Génica , Osteosarcoma/metabolismo , Receptor fas/genética , Azacitidina/análogos & derivados , Azacitidina/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Metilasas de Modificación del ADN/antagonistas & inhibidores , Decitabina , Inhibidores Enzimáticos/farmacología , Proteína Ligando Fas/farmacología , Humanos , Intrones/genética , Metástasis de la Neoplasia , Osteosarcoma/genética , Regiones Promotoras Genéticas/genética , Receptor fas/metabolismoRESUMEN
Chemoresistance is a major reason that patients with osteosarcoma fail to achieve a lasting chemotherapy response, and it contributes to disease relapse, progression, and death. Human glutathione S-transferase P1 (GSTP1), a phase II detoxification enzyme, contributes to chemoresistance in many cancers. However, the role of GSTP1 in osteosarcoma chemoresistance is ill defined. We hypothesized that GSTP1 has cytoprotective effects in human osteosarcoma. To assess this possibility, we used GSTP1 cDNA transfection or RNA interference to overexpress or suppress GSTP1 in osteosarcoma cells, and assessed the cytotoxic effect of chemotherapeutic agents on these cells. Our results showed that GSTP1 expression was up-regulated in osteosarcoma cells when they were treated with doxorubicin or cisplatin. GSTP1 overexpression in SAOS-2 osteosarcoma cells caused the cells to be more resistant to doxorubicin and cisplatin. In contrast, GSTP1 suppression in HOS cells caused more apoptosis and extensive DNA damage in response to doxorubicin and cisplatin. The cytotoxicity assay also showed that GSTP1 suppression caused a 2.5-fold increase in cell growth inhibition resulting from doxorubicin and cisplatin treatments [the IC(50)s are approximately 0.16 micromol/L (doxorubicin) and 1.8 micromol/L (cisplatin) for parental HOS versus 0.06 micromol/L (doxorubicin) and 0.75 micromol/L (cisplatin) for GSTP1-silenced HOS]. Moreover, GSTP1 suppression decreased the activation of extracellular signal-regulated kinase 1/2, which is induced by cisplatin and doxorubicin. Taken together, these findings show that GSTP1 contributes to doxorubicin and cisplatin resistance in osteosarcoma, which may be mediated in part by the activation of extracellular signal-regulated kinase 1/2. Targeting of GSTP1 combined with chemotherapy may have synergistic therapeutic effects on osteosarcoma.
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Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica , Gutatión-S-Transferasa pi/biosíntesis , Gutatión-S-Transferasa pi/genética , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/enzimología , Apoptosis , Línea Celular Tumoral , Ensayo Cometa , Activación Enzimática , Humanos , Concentración 50 Inhibidora , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
The first intracellular loop (iLP1, residues 39-51) of human prostacyclin receptor (IP) was proposed to be involved in signaling via its interaction with the Galphas protein. First, evidence of the IP iLP1 interaction with the C-terminus of the Galphas protein was observed by the fluorescence and NMR spectroscopy using the synthetic peptide (Galphas-Ct) mimicking the C-terminal 11 residues of the Galphas protein in the presence of a constrained synthetic peptide mimicking the IP iLP1. Then, the residues (Arg42, Ala44, and Arg45) in the IP iLP1 peptide possibly involved in contacting the Galphas-Ct peptide were initially assigned by observation of the significant proton resonance shifts of the side chains of the constrained IP iLP1 peptide using 2D (1)H NMR spectroscopy. The results of the NMR studies were used as a guide for further identification of the residues in the IP important to the receptor signaling using a recombinant protein approach. A profile of the residues in the IP iLP1, including the residues observed from the NMR studies involved in the Galphas mediated signaling, was mapped out by mutagenesis. According to our results, it can be predicted that the seven residues (Arg42-Ala48) with the conserved Arg45 at the center will form an epitope with a specific conformation involved in the Galphas mediated signaling. The conservation of the basic residues (Arg45 in the IP) in all of the prostanoid receptors suggests that the iLP1 regions of the other prostanoid receptors may also contain the epitopes important to their signaling.
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Subunidades alfa de la Proteína de Unión al GTP Gs/fisiología , Receptores de Epoprostenol/química , Secuencia de Aminoácidos , Secuencia Conservada , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Estructura Secundaria de Proteína , Receptores de Epoprostenol/genética , Receptores de Epoprostenol/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
The conformation of a constrained peptide mimicking the putative first intracellular domain (iLP1) of thromboxane A(2) receptor (TP) was determined by (1)H 2D NMR spectroscopy. Through completed assignments of TOCSY, DQF-COSY, and NOESY spectra, a NMR structure of the peptide showed a beta-turn in residues 56-59 and a short helical structure in the residues 63-66. It suggests that residues 63-66 may be part of the second transmembrane domain (TM), and that Arg60, in an exposed position on the outer surface of the loop, may be involved in signaling through charge contact with Gq protein. The sequence alignment of Lys residue in the same position of other prostanoid receptors mediates different G protein couplings, suggesting that the chemical properties of Arg and Lys may also affect the receptor signaling activity. These hypotheses were supported by mutagenesis studies, in which the mutant of Arg60Leu completely lost activity in increasing intracellular calcium level through Gq coupling, and the mutant of Arg60Lys retained only about 35% signaling activity. The difference between the side chain functions of Lys and Arg in effecting the signaling was discussed.
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Receptores de Tromboxano A2 y Prostaglandina H2/química , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Arginina/genética , Arginina/metabolismo , Células COS , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Dicroismo Circular , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Tromboxano A2 y Prostaglandina H2/antagonistas & inhibidores , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transfección , Vasoconstrictores/farmacologíaRESUMEN
The second extracellular loop (eLP2) of the thromboxane A(2) receptor (TP) had been proposed to be involved in ligand binding. Through two-dimensional (1)H NMR experiments, the overall three-dimensional structure of a constrained synthetic peptide mimicking the eLP2 had been determined by our group (Ruan, K.-H., So, S.-P., Wu, J., Li, D., Huang, A., and Kung, J. (2001) Biochemistry 40, 275-280). To further identify the residues involved in ligand binding, a TP receptor antagonist, SQ29,548 was used to interact with the synthetic peptide. High resolution two-dimensional (1)H NMR experiments, NOESY, and TOCSY were performed for the peptide, SQ29,548, and peptide with SQ29,548, respectively. Through completed (1)H NMR assignment and by comparing the different spectra, extra peaks were observed on the NOESY spectrum of the peptide with SQ29,548, which implied the contacts between residues of eLP2 at Val(176), Leu(185), Thr(186), and Leu(187) with SQ29,548 at position H2, H7, and H8. Site-directed mutagenesis was used to confirm the possible ligand-binding sites on native human TP receptor. Each of the four residues was mutated to the residues either in the same group, with different structure or different charged. The mutated receptors were then tested for their ligand binding activity. The receptor with V176L mutant retained binding activity to SQ29,548. All other mutations resulted in decreased or lost binding activity to SQ29,548. These mutagenesis results supported the prediction from NMR experiments in which Val(176), Leu(185), Thr(186), and Leu(187) are the possible residues involved in ligand binding. This information facilitates the understanding of the molecular mechanism of thromboxane A(2) binding to the important receptor and its signal transduction.
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Receptores de Tromboxanos/metabolismo , Secuencia de Aminoácidos , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes , Células COS , Ácidos Grasos Insaturados , Hidrazinas/metabolismo , Ligandos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Receptores de Tromboxanos/química , Receptores de Tromboxanos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
BACKGROUND: It was recently discovered that prostacyclin constituted 40-50% of prostaglandins (PG) produced by minced human oviduct. It is well established that prostacyclin relaxes vascular smooth muscle, but whether oviductal smooth muscle synthesizes prostacyclin and whether its contraction is affected by prostacyclin remain unclear. METHODS: Smooth muscle microdissected from human oviducts was used for the study. The expression of prostacyclin synthase (PGIS) and prostacyclin receptor (IP) was confirmed by Western blot analysis. Metabolites of [(3)H]PGH(2) were analysed for prostacyclin. Functional coupling of IP to adenyl cyclase was assessed by the accumulation of intracellular cAMP upon prostacyclin challenge. The presence of saturable, specific binding sites for prostacyclin was confirmed by binding assay. The identity of IP was further confirmed by RT-PCR and nucleotide sequence analysis. Finally, the effects of prostacyclin on muscle contraction were studied. RESULTS: Human oviductal smooth muscle expresses functionally active PGIS and IP. The IP expressed is the same as that cloned from human lung tissue. The ED(50) of prostacyclin to increase intracellular cAMP was 16 nmol/l. Prostacyclin dose-dependently decreased the amplitude of muscle contraction. CONCLUSIONS: Human oviductal smooth muscle produces prostacyclin, which, in turn, decreases its contractility. Prostacyclin may regulate embryo transport.