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First published online 14 April 2008
doi: 10.1242/jcs.019018

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Downregulation by lipopolysaccharide of Notch signaling, via nitric oxide

Mi-Yeon Kim^1,*, Ji-Hye Park^1,*, Jung-Soon Mo¹, Eun-Jung Ann¹, Seung-Ok Han¹, Sang-Hyun Baek¹, Kyoung-Jin Kim², Suhn-Young Im², Jeen-Woo Park³, Eui-Ju Choi⁴ and Hee-Sae Park^1,

¹ Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Yongbong-dong, Buk-ku, Gwangju, 500-757, Republic of Korea
² Department of Biological Sciences, College of Natural Sciences, Chonnam National University, Yongbong-dong, Buk-ku, Gwangju, 500-757, Republic of Korea
³ Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu, Republic of Korea
⁴ School of Life Science and Biotechnology, Korea University, Seoul, Republic of Korea

View larger version (28K): [in this window] [in a new window]   Fig. 1. LPS inhibits Notch1 transcriptional activity. (A) RAW264.7 cells were transfected for 24 hours with the expression vector for 4xCSL-Luc and Notch1-IC, along with lacZ, and then exposed to the indicated amount of LPS for 24 hours. (B-D) RAW264.7 cells transfected for 24 hours with the indicated combinations of expression vector for 4xCSL-Luc (B), Hes1-Luc (C) or Hes5-Luc (D) and Notch1-IC along with lacZ, and then exposed to 5 µg/ml LPS for 24 hours. The cells were then lysed and assayed for luciferase activity. The activity of the luciferase reporter in each of the samples was then normalized according to the β-galactosidase activity measured in the same sample. (E) The cell lysates were also subjected to immunoblotting analysis with the anti-Hes1 or anti-Hes5 antibody (top panels). THP-1 cells were treated with IL-1 (5 ng/ml) for 16 hours (middle panel). Jurkat T cells were exposed to UV light (60 J/m2) (bottom panel). The cells were then treated with 200 µM SNAP for 8 hours. The cell lysates were subjected to immunoblotting analysis with the anti-Mn-SOD or anti-CD95L antibody. These results represent the means ± average deviation of triplicates from one of three independent experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 2. LPS suppresses Notch1-IC transcriptional activity through NO in macrophage cells. (A-H) RAW264.7 (A,C,E,G) or alveolar macrophage (B,D,F,H) cells were transfected for 24 hours with the indicated combinations of expression vector for 4xCSL-Luc with Notch1-IC along with lacZ. (A-J) The cells were pre-treated with 100 µM L-NIL (A,B,E,F) or 2 mM L-NNA (C,D,G,H,I,J) for 30 minutes and then exposed to 5 µg/ml LPS for 24 hours. (A-D) NO released into the culture medium was then determined by the Griess method and represents nitrate+nitrite formation per 1x106 cells. (E-H) Cells were lysed and assayed for luciferase activity. The activity of the luciferase reporter in each of the samples was then normalized according to the β-galactosidase activity measured in the same sample. These results represent the means ± average deviation of triplicates from one of three independent experiments. (I,J) The cell lysates were also subjected to immunoblotting analysis with anti-Hes1 or anti-Hes5 antibody. (K,L) RAW264.7 or alveolar macrophage cells were pre-treated with 2 mM L-NNA for 30 minutes and then exposed to 5 µg/ml LPS for 24 hours. (M) HEK293 cells were transfected with expression vector for Notch1-IC and RBP-Jk. The cells were then treated with 200 µM SNAP for 8 hours. (N) HEK293-neo or HEK293-bNOS cells were transfected with expression vector for Notch1-IC and RBP-Jk. The cells were pre-treated with 2 mM L-NNA for 30 minutes and then exposed to 20 mM L-Arg for 16 hours. The cells were crosslinked with formaldehyde and DNA was immunoprecipitated with the indicated antibodies. The immunoprecipitated DNA was analyzed by PCR using primers recognizing the Hes1 or Hes5 promoters. As a negative control, we also tested a sample with vehicle only and pre-immune IgG, and included an input sample.

View larger version (31K): [in this window] [in a new window]   Fig. 3. NO modulates Notch transcriptional activity. (A,B) NIH3T3 cells were transfected for 40 hours with the indicated combinations of expression vector for 4xCSL-Luc, Hes1-Luc or Hes5-Luc and Notch1-IC along with lacZ. The cells were then treated with 200 µM SNAP (A) or 200 µM NAP (B) for 8 hours. The cell lysates were subjected to immunoblotting analysis with the anti-Hes1 or anti-Hes5 antibody. (C,D) HEK293-neo, -eNOS or -bNOS cells were transfected for 32 hours with 4xCSL-Luc and Notch1-IC along with lacZ. The cells were pre-treated with 2 mM L-NNA for 1 hour and then exposed to 20 mM L-Arg for 16 hours. (C) NO released into the culture medium was determined by the Griess method and represents nitrate+nitrite formation per 1x106 cells. (D) Cells were lysed and assayed for luciferase activity. The activity of the luciferase reporter in each of the samples was then normalized according to the β-galactosidase activity measured in the same sample. These results represent the means ± average deviation of triplicates from one of three independent experiments. (E) HEK293-neo, HEK293-eNOS and HEK293-bNOS cells were transfected with expression vector for EN1 and then exposed to 20 mM L-Arg for 16 hours. The cell lysates were subjected to immunoblotting analysis with the anti-V1744 antibody.

View larger version (11K): [in this window] [in a new window]   Fig. 4. NO-mediated cyclic GMP signaling is not involved in the suppression of Notch signaling. (A) NIH3T3 cells were transfected with the 4xCSL-Luc promoter, plus/minus Notch1-IC, along with lacZ. The cells were pre-treated with 100 µM ODQ for 1 hour prior to treatment with 200 µM SNAP, or were exposed to 100 µM 8-Bromo-cGMP for 8 hours. (B) Rat primary alveolar macrophage cells were transfected for 24 hours with the indicated combinations of expression vector for 4xCSL-Luc and Notch1-IC along with lacZ. The cells were pre-treated with 100 µM ODQ for 1 hour prior to treatment with exposure to 5 µg/ml LPS for 24 hours, or were exposed to 100 µM 8-Bromo-cGMP for 24 hours. The cells were lysed and assayed for luciferase activity. The activity of the luciferase reporter in each of the samples was then normalized according to the β-galactosidase activity measured in the same sample. These results represent the means ± average deviation of triplicates from one of three independent experiments.

View larger version (41K): [in this window] [in a new window]   Fig. 5. NO prevents the physical interaction between Notch1-IC and RBP-Jk both in vitro and in vivo. (A,B) HEK293 cells were transfected with Myc–Notch1-IC and Flag–RBP-Jk, and then the cells were exposed to 200 µM SNAP for 8 hours. After 48 hours, the cells were lysed and immunoprecipitated against anti-Myc (A) or anti-Flag (B) monoclonal antibody. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-Flag monoclonal antibody. (C,D) HEK293 cells were transfected with Myc–Notch1-IC and Flag-RBP-Jk. The cells were lysed and immunoprecipitated against anti-Flag (C) or anti-Myc (D) monoclonal antibody, and then exposed to 200 µM SNAP for 1 hour on ice. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-Myc monoclonal antibody. (A-D) The expression of Notch1-IC or RBP-Jk was analyzed via immunoblotting using anti-Myc or anti-Flag monoclonal antibody, respectively. (E) Recombinant GST and GST–Notch1-IC proteins, immobilized on glutathione-agarose beads, were exposed to 200 µM SNAP for 1 hour on ice, and extensively washed to remove remnant SNAP. HEK293 cells were transfected with RBP-Jk, and then the cells were lysed and added to the immobilized proteins. The beads were extensively washed, eluted and analyzed using SDS-PAGE immunoblotting against anti-Flag monoclonal antibody. Coomassie blue staining represents immobilized proteins. (F) RAW264.7 cells were pre-treated with 2 mM L-NNA for 30 minutes and then exposed to 5 µg/ml LPS for 24 hours. The cells were lysed and immunoprecipitated against anti-Notch1-IC antibody. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-RBP-Jk antibody. (G) Primary alveolar macrophage cells from 5 µg/ml LPS-injected rats were lysed and immunoprecipitated against anti-Notch1-IC antibody. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-RBP-Jk antibody. (F,G) The expression of Notch1-IC or RBP-Jk was analyzed via immunoblotting using anti-Notch1-IC or anti-RBP-Jk antibody, respectively. Probing with an antibody to β-actin was used as a loading control. (H) HEK293 cells were transfected for 40 hours with the indicated combinations of expression vector for Myc–Notch1-IC, Flag–RBP-Jk and HA-Mastermind, and then the cells were exposed to 200 µM SNAP for 8 hours. After 48 hours, the cells were lysed and immunoprecipitated against anti-Myc monoclonal antibody. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-Flag or anti-HA monoclonal antibody. The expression of Notch1-IC, RBP-Jk or Mastermind (Mam) was analyzed via immunoblotting using anti-Myc, anti-Flag or anti-HA monoclonal antibody, respectively.

View larger version (30K): [in this window] [in a new window]   Fig. 6. Nitration of the Notch tyrosine residue 1905 mediated a conformational change. (A) HEK293 cells were transfected with Myc–Notch1-IC, after which the cells were exposed to 200 µM SNAP for 8 hours. The cells were lysed and immunoprecipitated against anti-Myc monoclonal antibody. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-nitrocysteine or anti-nitrotyrosine antibodies. (B) Various concentrations of purified Notch1-IC (0, 50, 75, 100, 125, 150 or 200 µM) were pre-treated with 200 µM SNAP for 30 minutes on ice and then incubated with 0.20 mM DTNB. Maximum absorbance (Abs) was obtained at 412 nm. (C) The purified Notch1-IC proteins were exposed to 200 µM SNAP and dissolved in 50 mM Tris-HCl at a pH of 7.4. The intrinsic fluorescence spectra were acquired at an excitation wavelength of 278 nm, and excitation and emission slits of 5 nm. We conducted an emission wavelength scan from 300 nm to 400 nm. (D) RAW264.7 cells were pre-treated with 1 mM L-NNA for 30 minutes and then exposed to 5 µg/ml LPS for 24 hours. The cells were lysed and immunoprecipitated against anti-Notch1-IC antibody. (E) Primary alveolar macrophage cells from 1 mM L-NNA- and 5 µg/ml LPS-injected rats were lysed and immunoprecipitated against anti-Notch1-IC antibody. (D,E) The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-nitrotyrosine antibody. The expression of Notch1-IC or RBP-Jk was analyzed via immunoblotting using anti-Notch1-IC or anti-RBP-Jk antibody, respectively. Probing with an antibody to β-actin was used as a loading control.

View larger version (29K): [in this window] [in a new window]   Fig. 7. Mapping of nitrated tyrosine residues in Notch1-IC. (A) HEK293 cells were transfected with Flag–Notch1-IC, Flag–Notch1-IC-N, Flag–Notch-IC-NC or Flag–Notch1-IC-C, after which the cells were exposed to 200 µM SNAP for 8 hours. The cells were lysed and immunoprecipitated against anti-Flag antibody and immunoblotted against anti-nitrotyrosine antibodies. (B) RAW264.7 cells were transfected for 24 hours with the indicated expression vectors for Flag–Notch1-IC (Y1905/1928F), Flag–Notch1-IC (Y1928/2064F) or Flag–Notch1-IC (Y1905/2064F) and then exposed to 5 µg/ml LPS for 24 hours. The cells were lysed and immunoprecipitated against anti-Flag antibody. The immunocomplexes were analyzed via SDS-PAGE and immunoblotting against anti-nitrotyrosine or anti-Flag monoclonal antibody. (C) NIH3T3 and RAW264.7 cells were transfected with the indicated expression vector for 4xCSL-Luc, β-galactosidase and Notch1-IC (Y1905F), and then exposed to 200 µM SNAP for 8 hours or 5 µg/ml LPS for 24 hours, respectively. The cells were then lysed and assayed for luciferase activity. The activity of the luciferase reporter in each of the samples was normalized according to the β-galactosidase activity measured in the same sample. These results represent the means ± average deviation of triplicates from one of three independent experiments. (D) HEK293 cells were transfected with Flag–Notch1-IC (Y1905F) and Myc–RBP-Jk, after which the cells were exposed to 200 µM SNAP for 8 hours. The cells were lysed and immunoprecipitated against anti-Flag or anti-Myc antibody and immunoblotting was conducted against anti-Myc or anti-Flag antibody.

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