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First published online 27 June 2006
doi: 10.1242/jcs.03014

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NFAT3 is specifically required for TNF--induced cyclooxygenase-2 (COX-2) expression and transformation of Cl41 cells

Yan Yan^1,*, Jingxia Li^1,*, Weiming Ouyang^1,*, Qian Ma¹, Yu Hu¹, Dongyun Zhang¹, Jin Ding¹, Qingshan Qu¹, Kotha Subbaramaiah² and Chuanshu Huang^1,

¹ Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987, USA
² Department of Medicine F-203A, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA

View larger version (27K): [in a new window]   Fig. 1. Induction of COX-2 and iNOS by TNF- in Cl41 cells. (A,B) 8x103 Cl41-COX-2 mass1 (A), and Cl41-iNOS mass1 (B) cells were seeded into each well of a 96-well plate. After being cultured at 37°C overnight, the cells were treated with 20 U/ml TNF- for various time periods as indicated. The cells were then extracted with lysis buffer, and the luciferase activity was measured as described in the Materials and Methods. The results were expressed as COX-2 or iNOS relative luciferase activity (RLU). Each bar indicates the mean and standard deviation of triplicate wells. (C) Cl41 cells (2x105) were seeded into each well of a 6-well plate, and cultured in 5% FBS MEM at 37°C. When the cell density reached 70-80%, the culture medium was replaced with 0.1% FBS MEM. After being cultured for 24 hours, the cells were exposed to various concentrations of TNF- as indicated. The cells were then washed once with ice-cold PBS and extracted with SDS-sample buffer. The western blot was carried out with specific antibodies against COX-2 and GAPDH, respectively, as described in Materials and Methods. GAPDH was used as the protein loading control. (D,E) After being exposed to various concentrations of TNF- for 24 h, Cl41 cells were extracted with Trizol reagent for the total RNA isolation. COX-2 (D) and iNOS (E) were amplified with their specific primers by RT-PCR for 30 cycles. ß-actin was used as an internal control. (F) 8x103 of Cl41 cells were seeded into each well of a 96-well plate. After being cultured at 37°C overnight, the cells were treated with 10 and 20 U/ml of TNF- for 48 hours. The PGE2 levels in the 48 hour culture medium were determined by ELISA according to the manufacturer's instructions. The results were expressed as the mean ± s.d. of triplicate samples.

View larger version (20K): [in a new window]   Fig. 2. Activation of NFAT3 by TNF- in Cl41 cells. (A) 8x103 Cl 41-NFAT Luc cells were seeded into each well of a 96-well plate. After being cultured at 37°C overnight, cells were treated with 5 or 20 U/ml of TNF- in combination with or without 10 µM CsA for 24 hours. The luciferase activity was measured as indicated in Fig. 1A. The results were expressed as the relative NFAT activity. Each bar indicates the mean and standard deviation of triplicate wells. (B) Cl41 cells (2x105) were seeded into each well of a 6-well plate, and cultured in 5% FBS MEM at 37°C. When the cell density reached 70-80%, the cells were exposed to various concentrations of TNF- for 24 hours, and then washed once with ice-cold PBS and extracted with SDS-sample buffer. The NFAT3 protein levels in the extracts were detected by western blot. (C) Cl41 cells were treated with 20 U/ml TNF- for 12 hours, and then extracted, as described in Materials and Methods. The NFAT3 protein levels in cytoplasm (C) and nuclear extracts (N) were detected by western blot.

View larger version (25K): [in a new window]   Fig. 3. Requirement of NFAT3 for COX-2 but not iNOS induction, by TNF- in Cl41 cells. (A,B) 2x105 cells of Cl41 transfectants were seeded into each well of a 6-well plate, and the cells were extracted with Tris-Glycine SDS sample buffer after the cell density reached 90-100%. Western blotting was carried out with specific antibodies as indicated. NF-B and c-Jun were used as specific controls for NFAT3 siRNA, and GAPDH was used as the protein loading control. (C-F) Cl41-COX-2 mass1, Cl41-siNFAT3/COX-2 mass1, Cl41-iNOS mass1 cells and Cl41-siNFAT3/iNOS mass2 (8x103), were seeded into each well of a 96-well plate. The cells were treated with TNF- for 72 hours and the luciferase activities were determined. The results were expressed as COX-2 (C,E) and iNOS (D,F) transcriptional induction relative to medium control (relative COX-2 induction or relative iNOS induction). Each bar indicates the mean and standard deviation of triplicate wells. (G) Cl41 COX-2 mass1 and Cl41 siNFAT3/COX-2 mass1 (2x105) were seeded into each well of a 6-well plate, and cultured in 5% FBS MEM at 37°C. When the cell density reached 70-80%, the cells were exposed to various concentrations of TNF- as indicated for 24 hours and 48 hours. The induction of COX-2 at protein level was detected by western blot.

View larger version (41K): [in a new window]   Fig. 4. Essential role of NFAT3 in TNF--induced anchorage-independent growth of Cl41 cells. (A,C) Cl41-COX-2 mass1, Cl41-siNFAT3/COX-2 mass2 (1x104) were exposed to indicated concentrations of TNF- in 1 ml of 0.33% MEM agar containing 5% FBS over 2.5 ml of 0.5% MEM agar containing 5% FBS in each well of a 6-well plate. After being cultured in a 37°C, 5% CO2 incubator for 3 weeks, the cell colonies were counted under microscopy, and colonies with more than 16 cells were scored. Each bar indicates the mean and s.d. from triplicate assays. (B,D) Schematic diagrams outlining the anchorage-independent cell growth in Fig. 4A,C. (E) The soft agar assay was performed as indicated in Fig. 3A, 2x104 cells were exposed to 20 U/ml of TNF- in combination with various concentrations of CsA as indicated for 3 weeks. (F) Schematic diagram outlining the anchorage-independent cell growth in Fig. 4E.

View larger version (41K): [in a new window]   Fig. 5. Involvement of COX-2 in NFAT3-mediated anchorage-independent growth of Cl41 cells induced by TNF-. (A) Cl41-mock vector and Cl41-siCOX-2 cells (2x105) were seeded into each well of a 6-well plate, and cultured in 5% FBS MEM at 37°C. When the cell density reached 70-80%, the cells were exposed to 20 U/ml of TNF- for 24 hours. The induction of COX-2 at the protein level was detected by western blot. (B) The soft agar assay was performed as indicated in Fig. 4A; 2x104 of Cl41-mock vector and Cl41-siCOX-2 cells were exposed to 20 U/ml of TNF- for 3 weeks. (C) Schematic diagram outlining the anchorage-independent cell growth shown in panel B. (D) The soft agar assay was performed as indicated in Fig. 4A, 2x104 of Cl41 cells were exposed to 20 U/ml of TNF- with various concentrations of NS398 for 3 weeks. (E) Schematic diagram outlining the anchorage-independent cell growth shown in panel D. (F) The proposed NFAT pathway involved in TNF--induced anchorage-independent cell growth.