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Alain Mauviel - One of the best experts on this subject based on the ideXlab platform.

  • smad3 4 dependent transcriptional activation of the human type vii collagen gene col7a1 promoter by transforming growth factor β
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: Laurence Vindevoghel, Robert J Lechleider, Atsushi Kon, Mark P De Caestecker, Jouni Uitto, Anita B Roberts, Alain Mauviel
    Abstract:

    The human type VII collagen gene (COL7A1) recently has been identified as an immediate-early response gene for transforming growth factor β (TGF-β)/SMAD signaling pathway. In this study, by using MDA-MB-468 SMAD4−/− breast carcinoma cells, we demonstrate that expression of SMAD4 is an absolute requirement for SMAD-mediated promoter activity. We also demonstrate that the SMAD binding sequence (SBS) representing the TGF-β response element in the region −496/−444 of the COL7A1 promoter functions as an enhancer in the context of a heterologous promoter. Electrophoretic mobility-shift assays with nuclear extracts from COS-1 cells transfected with expression vectors for SMADs 1–5 indicate that SMAD3 forms a Complex with a migration similar to that of the endogenous TGF-β-specific Complex observed in fibroblast extracts. Electrophoretic mobility-shift assays using recombinant glutathione S-transferase-SMAD fusion Proteins indicate that both SMAD4 and C-terminally truncated SMAD3, but not SMAD2, can bind the COL7A1 SBS. Coexpression of SMAD3 and SMAD4 in COS-1 cells leads to the formation of two Complexes: a DNA/Protein Complex containing SMAD3 alone and another slower-migrating Complex containing both SMAD3 and SMAD4, the latter Complex not being detected in fibroblasts. Maximal transactivation of COL7A1 SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant overexpression of SMAD3 and SMAD4. These data may represent the first identification of a functional homomeric SMAD3 Complex regulating a human gene.

  • smad3 4 dependent transcriptional activation of the human type vii collagen gene col7a1 promoter by transforming growth factor beta
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: Laurence Vindevoghel, Robert J Lechleider, Atsushi Kon, Mark P De Caestecker, Jouni Uitto, Anita B Roberts, Alain Mauviel
    Abstract:

    The human type VII collagen gene (COL7A1) recently has been identified as an immediate-early response gene for transforming growth factor beta (TGF-beta)/SMAD signaling pathway. In this study, by using MDA-MB-468 SMAD4-/- breast carcinoma cells, we demonstrate that expression of SMAD4 is an absolute requirement for SMAD-mediated promoter activity. We also demonstrate that the SMAD binding sequence (SBS) representing the TGF-beta response element in the region -496/-444 of the COL7A1 promoter functions as an enhancer in the context of a heterologous promoter. Electrophoretic mobility-shift assays with nuclear extracts from COS-1 cells transfected with expression vectors for SMADs 1-5 indicate that SMAD3 forms a Complex with a migration similar to that of the endogenous TGF-beta-specific Complex observed in fibroblast extracts. Electrophoretic mobility-shift assays using recombinant glutathione S-transferase-SMAD fusion Proteins indicate that both SMAD4 and C-terminally truncated SMAD3, but not SMAD2, can bind the COL7A1 SBS. Coexpression of SMAD3 and SMAD4 in COS-1 cells leads to the formation of two Complexes: a DNA/Protein Complex containing SMAD3 alone and another slower-migrating Complex containing both SMAD3 and SMAD4, the latter Complex not being detected in fibroblasts. Maximal transactivation of COL7A1 SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant overexpression of SMAD3 and SMAD4. These data may represent the first identification of a functional homomeric SMAD3 Complex regulating a human gene.

Hiroyasu Inoue - One of the best experts on this subject based on the ideXlab platform.

  • bradykinin b2 receptor mediates nf κb activation and cyclooxygenase 2 expression via the ras raf 1 erk pathway in human airway epithelial cells
    Journal of Immunology, 2004
    Co-Authors: Bing Chang Chen, Hui Chieh Lei, Ming Shyan Chang, Ming J Hsu, Chuen L Huang, Mei Chieh Chen, Joen Rong Sheu, Tseng F Chen, Ta Liang Chen, Hiroyasu Inoue
    Abstract:

    In this study, we investigated the signaling pathways involved in bradykinin (BK)-induced NF-κB activation and cyclooxygenase-2 (COX-2) expression in human airway epithelial cells (A549). BK caused concentration- and time-dependent increase in COX-2 expression, which was attenuated by a selective B2 BK receptor antagonist (HOE140), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), an NF-κB inhibitor (pyrrolidine dithiocarbate), and an IκB protease inhibitor (l-1-tosylamido-2-phenylethyl chloromethyl ketone). The B1 BK receptor antagonist (Lys-(Leu8)des-Arg9-BK) had no effect on COX-2 induction by BK. BK-induced increase in COX-2-luciferase activity was inhibited by cells transfected with the κB site deletion of COX-2 construct. BK-induced Ras activation was inhibited by manumycin A. Raf-1 phosphorylation at Ser338 by BK was inhibited by manumycin A and GW 5074. BK-induced ERK activation was inhibited by HOE140, manumycin A, GW 5074, and PD 098059. Stimulation of cells with BK activated IκB kinase αβ (IKKαβ), IκBα phosphorylation, IκBα degradation, p65 and p50 translocation from the cytosol to the nucleus, the formation of an NF-κB-specific DNA-Protein Complex, and κB-luciferase activity. BK-mediated increase in IKKαβ activity and formation of the NF-κB-specific DNA-Protein Complex were inhibited by HOE140, a Ras dominant-negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Our results demonstrated for the first time that BK, acting through B2 BK receptor, induces activation of the Ras/Raf-1/ERK pathway, which in turn initiates IKKαβ and NF-κB activation, and ultimately induces COX-2 expression in human airway epithelial cell line (A549).

  • bradykinin b2 receptor mediates nf κb activation and cyclooxygenase 2 expression via the ras raf 1 erk pathway in human airway epithelial cells
    Journal of Immunology, 2004
    Co-Authors: Bing Chang Chen, Hui Chieh Lei, Ming Shyan Chang, Ming J Hsu, Chuen L Huang, Mei Chieh Chen, Joen Rong Sheu, Tseng F Chen, Ta Liang Chen, Hiroyasu Inoue
    Abstract:

    In this study, we investigated the signaling pathways involved in bradykinin (BK)-induced NF-kappaB activation and cyclooxygenase-2 (COX-2) expression in human airway epithelial cells (A549). BK caused concentration- and time-dependent increase in COX-2 expression, which was attenuated by a selective B2 BK receptor antagonist (HOE140), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), an NF-kappaB inhibitor (pyrrolidine dithiocarbate), and an IkappaB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone). The B1 BK receptor antagonist (Lys-(Leu8)des-Arg9-BK) had no effect on COX-2 induction by BK. BK-induced increase in COX-2-luciferase activity was inhibited by cells transfected with the kappaB site deletion of COX-2 construct. BK-induced Ras activation was inhibited by manumycin A. Raf-1 phosphorylation at Ser338 by BK was inhibited by manumycin A and GW 5074. BK-induced ERK activation was inhibited by HOE140, manumycin A, GW 5074, and PD 098059. Stimulation of cells with BK activated IkappaB kinase alphabeta (IKKalphabeta), IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 and p50 translocation from the cytosol to the nucleus, the formation of an NF-kappaB-specific DNA-Protein Complex, and kappaB-luciferase activity. BK-mediated increase in IKKalphabeta activity and formation of the NF-kappaB-specific DNA-Protein Complex were inhibited by HOE140, a Ras dominant-negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Our results demonstrated for the first time that BK, acting through B2 BK receptor, induces activation of the Ras/Raf-1/ERK pathway, which in turn initiates IKKalphabeta and NF-kappaB activation, and ultimately induces COX-2 expression in human airway epithelial cell line (A549).

Bing Chang Chen - One of the best experts on this subject based on the ideXlab platform.

  • phorbol 12 myristate 13 acetate upregulates cyclooxygenase 2 expression in human pulmonary epithelial cells via ras raf 1 erk and nf κb but not p38 mapk pathways
    Cellular Signalling, 2005
    Co-Authors: Ming Shyan Chang, Bing Chang Chen, Joen Rong Sheu, Tseng F Chen, Chienhuang Lin
    Abstract:

    Abstract In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release by phorbol 12-myristate 13-acetate (PMA), a Protein kinase C (PKC) activator, in human pulmonary epithelial cells (A549). PMA-induced COX-2 expression was attenuated by PKC inhibitors (Go 6976 and Ro 31-8220), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), and an NF-κB inhibitor (PDTC), but not by a tyrosine kinase inhibitor (genistein) or a p38 MAPK inhibitor (SB 203580). PMA also caused the activation of Ras, Raf-1, and ERK1/2. PMA-induced activation of Ras and Raf-1 was inhibited by Ro 31-8220 and manumycin A. PMA-mediated activation of ERK1/2 was inhibited by Ro 31-8220, manumycin A, GW 5074, and PD 098059. Stimulation of cells with PMA caused IκBα phosphorylation, IκBα degradation, and the formation of a NF-κB-specific DNA–Protein Complex. The PMA-mediated increase in κB-luciferase activity was inhibited by Ro 31-8220, manumycin A, GW5074, PD 098059, and PDTC. Taken together, these results indicate that PMA might activate PKC to elicit activation of the Ras/Raf-1/ERK1/2 pathway, which in turn initiates NF-κB activation, and finally induces COX-2 expression and PGE2 release in A549 cells.

  • bradykinin b2 receptor mediates nf κb activation and cyclooxygenase 2 expression via the ras raf 1 erk pathway in human airway epithelial cells
    Journal of Immunology, 2004
    Co-Authors: Bing Chang Chen, Hui Chieh Lei, Ming Shyan Chang, Ming J Hsu, Chuen L Huang, Mei Chieh Chen, Joen Rong Sheu, Tseng F Chen, Ta Liang Chen, Hiroyasu Inoue
    Abstract:

    In this study, we investigated the signaling pathways involved in bradykinin (BK)-induced NF-κB activation and cyclooxygenase-2 (COX-2) expression in human airway epithelial cells (A549). BK caused concentration- and time-dependent increase in COX-2 expression, which was attenuated by a selective B2 BK receptor antagonist (HOE140), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), an NF-κB inhibitor (pyrrolidine dithiocarbate), and an IκB protease inhibitor (l-1-tosylamido-2-phenylethyl chloromethyl ketone). The B1 BK receptor antagonist (Lys-(Leu8)des-Arg9-BK) had no effect on COX-2 induction by BK. BK-induced increase in COX-2-luciferase activity was inhibited by cells transfected with the κB site deletion of COX-2 construct. BK-induced Ras activation was inhibited by manumycin A. Raf-1 phosphorylation at Ser338 by BK was inhibited by manumycin A and GW 5074. BK-induced ERK activation was inhibited by HOE140, manumycin A, GW 5074, and PD 098059. Stimulation of cells with BK activated IκB kinase αβ (IKKαβ), IκBα phosphorylation, IκBα degradation, p65 and p50 translocation from the cytosol to the nucleus, the formation of an NF-κB-specific DNA-Protein Complex, and κB-luciferase activity. BK-mediated increase in IKKαβ activity and formation of the NF-κB-specific DNA-Protein Complex were inhibited by HOE140, a Ras dominant-negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Our results demonstrated for the first time that BK, acting through B2 BK receptor, induces activation of the Ras/Raf-1/ERK pathway, which in turn initiates IKKαβ and NF-κB activation, and ultimately induces COX-2 expression in human airway epithelial cell line (A549).

  • bradykinin b2 receptor mediates nf κb activation and cyclooxygenase 2 expression via the ras raf 1 erk pathway in human airway epithelial cells
    Journal of Immunology, 2004
    Co-Authors: Bing Chang Chen, Hui Chieh Lei, Ming Shyan Chang, Ming J Hsu, Chuen L Huang, Mei Chieh Chen, Joen Rong Sheu, Tseng F Chen, Ta Liang Chen, Hiroyasu Inoue
    Abstract:

    In this study, we investigated the signaling pathways involved in bradykinin (BK)-induced NF-kappaB activation and cyclooxygenase-2 (COX-2) expression in human airway epithelial cells (A549). BK caused concentration- and time-dependent increase in COX-2 expression, which was attenuated by a selective B2 BK receptor antagonist (HOE140), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), an NF-kappaB inhibitor (pyrrolidine dithiocarbate), and an IkappaB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone). The B1 BK receptor antagonist (Lys-(Leu8)des-Arg9-BK) had no effect on COX-2 induction by BK. BK-induced increase in COX-2-luciferase activity was inhibited by cells transfected with the kappaB site deletion of COX-2 construct. BK-induced Ras activation was inhibited by manumycin A. Raf-1 phosphorylation at Ser338 by BK was inhibited by manumycin A and GW 5074. BK-induced ERK activation was inhibited by HOE140, manumycin A, GW 5074, and PD 098059. Stimulation of cells with BK activated IkappaB kinase alphabeta (IKKalphabeta), IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 and p50 translocation from the cytosol to the nucleus, the formation of an NF-kappaB-specific DNA-Protein Complex, and kappaB-luciferase activity. BK-mediated increase in IKKalphabeta activity and formation of the NF-kappaB-specific DNA-Protein Complex were inhibited by HOE140, a Ras dominant-negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Our results demonstrated for the first time that BK, acting through B2 BK receptor, induces activation of the Ras/Raf-1/ERK pathway, which in turn initiates IKKalphabeta and NF-kappaB activation, and ultimately induces COX-2 expression in human airway epithelial cell line (A549).

Ming Shyan Chang - One of the best experts on this subject based on the ideXlab platform.

  • phorbol 12 myristate 13 acetate upregulates cyclooxygenase 2 expression in human pulmonary epithelial cells via ras raf 1 erk and nf κb but not p38 mapk pathways
    Cellular Signalling, 2005
    Co-Authors: Ming Shyan Chang, Bing Chang Chen, Joen Rong Sheu, Tseng F Chen, Chienhuang Lin
    Abstract:

    Abstract In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release by phorbol 12-myristate 13-acetate (PMA), a Protein kinase C (PKC) activator, in human pulmonary epithelial cells (A549). PMA-induced COX-2 expression was attenuated by PKC inhibitors (Go 6976 and Ro 31-8220), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), and an NF-κB inhibitor (PDTC), but not by a tyrosine kinase inhibitor (genistein) or a p38 MAPK inhibitor (SB 203580). PMA also caused the activation of Ras, Raf-1, and ERK1/2. PMA-induced activation of Ras and Raf-1 was inhibited by Ro 31-8220 and manumycin A. PMA-mediated activation of ERK1/2 was inhibited by Ro 31-8220, manumycin A, GW 5074, and PD 098059. Stimulation of cells with PMA caused IκBα phosphorylation, IκBα degradation, and the formation of a NF-κB-specific DNA–Protein Complex. The PMA-mediated increase in κB-luciferase activity was inhibited by Ro 31-8220, manumycin A, GW5074, PD 098059, and PDTC. Taken together, these results indicate that PMA might activate PKC to elicit activation of the Ras/Raf-1/ERK1/2 pathway, which in turn initiates NF-κB activation, and finally induces COX-2 expression and PGE2 release in A549 cells.

  • bradykinin b2 receptor mediates nf κb activation and cyclooxygenase 2 expression via the ras raf 1 erk pathway in human airway epithelial cells
    Journal of Immunology, 2004
    Co-Authors: Bing Chang Chen, Hui Chieh Lei, Ming Shyan Chang, Ming J Hsu, Chuen L Huang, Mei Chieh Chen, Joen Rong Sheu, Tseng F Chen, Ta Liang Chen, Hiroyasu Inoue
    Abstract:

    In this study, we investigated the signaling pathways involved in bradykinin (BK)-induced NF-κB activation and cyclooxygenase-2 (COX-2) expression in human airway epithelial cells (A549). BK caused concentration- and time-dependent increase in COX-2 expression, which was attenuated by a selective B2 BK receptor antagonist (HOE140), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), an NF-κB inhibitor (pyrrolidine dithiocarbate), and an IκB protease inhibitor (l-1-tosylamido-2-phenylethyl chloromethyl ketone). The B1 BK receptor antagonist (Lys-(Leu8)des-Arg9-BK) had no effect on COX-2 induction by BK. BK-induced increase in COX-2-luciferase activity was inhibited by cells transfected with the κB site deletion of COX-2 construct. BK-induced Ras activation was inhibited by manumycin A. Raf-1 phosphorylation at Ser338 by BK was inhibited by manumycin A and GW 5074. BK-induced ERK activation was inhibited by HOE140, manumycin A, GW 5074, and PD 098059. Stimulation of cells with BK activated IκB kinase αβ (IKKαβ), IκBα phosphorylation, IκBα degradation, p65 and p50 translocation from the cytosol to the nucleus, the formation of an NF-κB-specific DNA-Protein Complex, and κB-luciferase activity. BK-mediated increase in IKKαβ activity and formation of the NF-κB-specific DNA-Protein Complex were inhibited by HOE140, a Ras dominant-negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Our results demonstrated for the first time that BK, acting through B2 BK receptor, induces activation of the Ras/Raf-1/ERK pathway, which in turn initiates IKKαβ and NF-κB activation, and ultimately induces COX-2 expression in human airway epithelial cell line (A549).

  • bradykinin b2 receptor mediates nf κb activation and cyclooxygenase 2 expression via the ras raf 1 erk pathway in human airway epithelial cells
    Journal of Immunology, 2004
    Co-Authors: Bing Chang Chen, Hui Chieh Lei, Ming Shyan Chang, Ming J Hsu, Chuen L Huang, Mei Chieh Chen, Joen Rong Sheu, Tseng F Chen, Ta Liang Chen, Hiroyasu Inoue
    Abstract:

    In this study, we investigated the signaling pathways involved in bradykinin (BK)-induced NF-kappaB activation and cyclooxygenase-2 (COX-2) expression in human airway epithelial cells (A549). BK caused concentration- and time-dependent increase in COX-2 expression, which was attenuated by a selective B2 BK receptor antagonist (HOE140), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), an NF-kappaB inhibitor (pyrrolidine dithiocarbate), and an IkappaB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone). The B1 BK receptor antagonist (Lys-(Leu8)des-Arg9-BK) had no effect on COX-2 induction by BK. BK-induced increase in COX-2-luciferase activity was inhibited by cells transfected with the kappaB site deletion of COX-2 construct. BK-induced Ras activation was inhibited by manumycin A. Raf-1 phosphorylation at Ser338 by BK was inhibited by manumycin A and GW 5074. BK-induced ERK activation was inhibited by HOE140, manumycin A, GW 5074, and PD 098059. Stimulation of cells with BK activated IkappaB kinase alphabeta (IKKalphabeta), IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 and p50 translocation from the cytosol to the nucleus, the formation of an NF-kappaB-specific DNA-Protein Complex, and kappaB-luciferase activity. BK-mediated increase in IKKalphabeta activity and formation of the NF-kappaB-specific DNA-Protein Complex were inhibited by HOE140, a Ras dominant-negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Our results demonstrated for the first time that BK, acting through B2 BK receptor, induces activation of the Ras/Raf-1/ERK pathway, which in turn initiates IKKalphabeta and NF-kappaB activation, and ultimately induces COX-2 expression in human airway epithelial cell line (A549).

Aziz Sancar - One of the best experts on this subject based on the ideXlab platform.

  • assembly subunit composition and footprint of human DNA repair excision nuclease
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: Mitsuo Wakasugi, Aziz Sancar
    Abstract:

    The assembly and composition of human excision nuclease were investigated by electrophoretic mobility shift assay and DNAse I footprinting. Individual repair factors or any combination of up to four repair factors failed to form DNA–Protein Complexes of high specificity and stability. A stable Complex of high specificity can be detected only when XPA/RPA, transcription factor IIH, XPC⋅HHR23B, and XPG and ATP are present in the reaction mixture. The XPF⋅ERCC1 heterodimer changes the electrophoretic mobility of the DNA–Protein Complex formed with the other five repair factors, but it does not confer additional specificity. By using Proteins with peptide tags or antibodies to the repair factors in electrophoretic mobility shift assays, it was found that XPA, replication Protein A, transcription factor IIH, XPG, and XPF⋅excision repair cross-complementing 1 but not XPC⋅HHR23B were present in the penultimate and ultimate dual incision Complexes. Thus, it appears that XPC⋅HHR23B is a molecular matchmaker that participates in the assembly of the excision nuclease but is not present in the ultimate dual incision Complex. The excision nuclease makes an assymmetric DNAse I footprint of ≈30 bp around the damage and increases the DNAse I sensitivity of the DNA on both sides of the footprint.

  • reconstitution of human DNA repair excision nuclease in a highly defined system
    Journal of Biological Chemistry, 1995
    Co-Authors: David Mu, Chi Hyun Park, Tsukasa Matsunaga, Joyce T Reardon, Aziz Sancar
    Abstract:

    Abstract Xeroderma pigmentosum is a hereditary disease caused by defective DNA repair. Somatic cell genetics and biochemical studies with cell-free extracts indicate that at least 16 polypeptides are required to carry out the repair reaction proper, i.e. the removal of the lesion from the DNA by the dual incisions of the damaged strand. To find out if these Proteins are necessary and sufficient for excision repair, they were obtained at a high level of purity in five fractions. The mixture of these five fractions reconstituted the excision nuclease (excinuclease) activity. Using the reconstituted excinuclease, we found that the excised fragment remains associated with the post-incision DNA-Protein Complex, suggesting that accessory Proteins are needed to release the excised oligomer.