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First published online September 19, 2007
doi: 10.1242/10.1242/jcs.007872

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Notch3 and IL-1 exert opposing effects on a vascular smooth muscle cell inflammatory pathway in which NF-B drives crosstalk

Nathalie Clément^1,*, Marie Gueguen^1,*, Martine Glorian¹, Régis Blaise^1,2, Marise Andréani¹, Christel Brou², Pedro Bausero¹ and Isabelle Limon^1,

¹ UMR 7079 de Physiologie et Physiopathologie, Université Pierre et Marie Curie, CNRS, 7 quai Saint-Bernard, 75252 Paris, France
² Unité de Signalisation Moléculaire et Activation Cellulaire, URA 2582, CNRS, Institut Pasteur, 75724 Paris Cedex 15, France

View larger version (23K): [in this window] [in a new window]   Fig. 1. Inhibition of -secretase increases the positive effect of IL-1 on the biosynthesis pathway of lipid mediators. Serum-starved cells were treated daily for 6, 24 or 48 hours with IL-1 (10 ng/ml) or vehicle (control) and/or with DAPT (0.5 µM). (A) PLA2 secretion was expressed as a percentage of that of control cells (in pmol/min/µg RNA: 6.7±0.1 at 24 hours; 4.7±0.1 at 48 hours). (B) 15 µg of total proteins were separated by electrophoresis (6.5% SDS PAGE). COX-2 and -actin were immunodetected with appropriate antibodies. The autoradiogram is representative of three independent experiments. The histogram represents values obtained from scanning COX-2 bands normalized to that of -actin and results are expressed as a percentage of those from IL-1-treated cells. (C,D) PLA2-IIA (C) and COX-2 (D) transcripts were assayed by RT-PCR. Results are expressed as a percentage of the PLA2-IIA or COX-2 mRNA level of control cells and represent the mean ± s.e. of three independent experiments. (E) PGE2 secretion was expressed as a percentage of the basal PGE2 secretion of control cells (in pg/ml: 16.7±2.1 at 24 hours; 19.1±0.5 at 48 hours). For PLA2 and PGE2 assays, the data represent the mean ± s.e.m. of five or six independent experiments. C, control; IL, IL-1; D, DAPT; D+IL, DAPT plus IL-1 versus IL-1; ns, non-significant; *, P<0.05; **, P<0.01; ***, P<0.001.

View larger version (66K): [in this window] [in a new window]   Fig. 2. Inhibition of -secretase enhances the effect of IL-1 on the contractile apparatus. Serum-starved cells were treated daily for 1 day (A) or 3 days (B,C) with IL-1 (10 ng/ml) or vehicle (control) and/or with DAPT (0.5 µM). (A) -Actin, SM-22, myocardin and calponin transcripts were assayed by RT-PCR. Results were expressed as a percentage of the mRNA level of control cells and represent the mean ± s.e.m. of three independent experiments. D+IL, DAPT plus IL-1 versus IL-1. *, P<0.05; **, P<0.01. (B) Immunostaining of -actin stress fibers was performed using a monoclonal antibody against -actin and a secondary antibody coupled to FITC (green-stain, 20x or 63x). Cell nuclei were stained with Hoechst (blue). Images are representative of four independent experiments. (C) 15 µg of total proteins were separated by electrophoresis (10% SDS PAGE). -Actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were immunodetected with appropriate antibodies. The autoradiogram is representative of three independent experiments. The histogram represents values obtained from scanning -actin bands normalized to that of GAPDH and are expressed as a percentage of control cells. C, control; IL, IL-1; D, DAPT; D+IL, DAPT plus IL-1 versus IL-1; **, P<0.01; ***, P<0.001; , P<0.05.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Effect of activated Notch on the prostanoid biosynthesis pathway and expression of -actin. (A) Cells were treated for 48 hours with IL-1 (10 ng/ml) or vehicle (control) and conditioned media collected from wild-type (WT CM) or Delta1-Fc-transfected (1-Fc CM) confluent HEK 293 cells. The medium was assayed for secreted PLA2 and PGE2. Both parameters were expressed as a percentage of that of control cells (3.5±0.5 pmol/min/µg RNA and 27.9±8.7 pg/ml, respectively). The data represent the mean ± s.e.m. of three independent experiments. IL + 1-Fc CM, IL-1 plus 1-Fc CM versus IL-1 plus WT CM; *, P<0.05; **, P<0.01. (B) Cells were transfected with Notch3 intracellular domain (N3-ICD), Notch1 intracellular domain (N1-ICD) or empty (mock) plasmids and treated for 6, 24 or 48 hours with IL-1 (10 ng/ml) and/or vehicle (control). The levels of PLA2 and PGE2 secretion were expressed as a percentage of those of control cells. The basal PLA2 secretion from mock-transfected cells was 7.3±0.3 and 4±1 pmol/min/µg RNA 24 and 48 hours after transfection, respectively. For N3-ICD-transfected cells, this parameter was 5.7±1.7 and 5.2±1.1 pmol/min/µg RNA and for N1-ICD-transfected cells, 4.8±0.8 and 5.1±1 pmol/min/µg RNA. The basal PGE2 secretion from mock-transfected cells was 18.2±0.6 pg/ml 48 hours after transfection. For N3-ICD- and N1-ICD-transfected cells, this parameter was 17.8±0.5 pg/ml. The data represent the mean ± s.e. of three to six independent experiments. PLA2-IIA transcripts were assayed by RT-PCR. The results are expressed as a percentage of the PLA2-IIA mRNA level of control cells and represent the mean ± s.e. of three independent experiments. *, P<0.05; **, P<0.01; ns, non-significant. (C) 15 µg of total proteins from mock, N3-ICD- or N1-ICD-transfected cells treated for 48 hours with IL-1 (10 ng/ml) and/or vehicle (control) were separated by electrophoresis (10% SDS PAGE). -Actin and GAPDH were immunodetected with appropriate antibodies. The autoradiogram is representative of three independent experiments. The histogram represents values obtained from scanning -actin bands normalized to that of GAPDH and are expressed as a percentage of each control cell. C, control; IL, IL-1; *, P<0.05; ns, non-significant.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Effect of the overexpression of the RBP-J mutant on the prostanoid biosynthesis pathway. Cells were transfected with RBP-J DN (a mutated form of RBP-J) or empty (mock) plasmids and treated for 6, 24 or 48 hours with IL-1 (10 ng/ml) or vehicle (control). (A) PLA2-IIA transcripts were assayed by RT-PCR. Results are expressed as a percentage of the PLA2-IIA mRNA level of control cells and represent the mean ± s.e.m. of three independent experiments. (B) Secretion of PLA2 and PGE2 was expressed as a percentage of that of control cells. The basal PLA2 secretion from mock-transfected cells was 3.6±0.9 and 4.1±0.6 pmol/min/µg RNA, 24 and 48 hours after transfection, respectively. For RBP-J DN-transfected cells, this parameter was 4.9±0.7 and 4.5±0.8 pmol/min/µg RNA. The basal PGE2 secretion levels from mock and RBP-J-DN-transfected cells were 16.9±1.5 and 18.3±0.9 pg/ml, respectively. The data represent the mean ± s.e.m. of four to five independent experiments. RBP-J DN versus mock: *, P<0.05; **, P<0.01.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Regulation of Notch-family gene expression in response to IL-1. (A) Serum-starved cells were treated for 6 hours with IL-1 (10 ng/ml) or vehicle (control). Transcripts encoding Notch1, Notch3, Jagged1, Hes1, HRT1 and HRT2 were assayed by RT-PCR. Results are expressed as the mRNA level in IL-1-treated cells normalized to that of control cells. Values represent the mean ± s.e.m. of three independent experiments and were compared with the baseline. (B) Cells were transfected with IB32-36 (a mutated form of IB) or empty (mock) plasmids and treated for 6 hours with IL-1 (10 ng/ml) or vehicle (control). Results are expressed as a percentage of the mRNA level in control cells and represent the mean ± s.e.m. of three independent experiments. Values of the mRNA expression from IL-1-treated IB32-36-transfected cells were compared with those of IL-1-treated mock-transfected cells. C, control; IL, IL-1; *, P<0.05; **, P<0.01; ***, P<0.001; ns, non-significant. Results are expressed as the mean ± s.e. of at least four independent experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Effect of the overexpression of Notch1 or Notch3 intracellular domains on IB expression. Cells were transfected with N3-ICD, N1-ICD or empty (mock) plasmids and treated for 6 hours with IL-1 (10 ng/ml) or vehicle (control). IB transcripts were assayed by RT-PCR. Results are expressed as a percentage of the IB mRNA level from control cells and represent the mean ± s.e.m. of three independent experiments. C, control; IL, IL-1; *, P<0.05; ** P<0.01.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Putative model for Notch and IL-1 crosstalk to regulate vascular smooth muscle inflammation and differentiation. See Discussion for details. HDAC, histone deacetylase complex; N-CoR, nuclear co-repressor receptor.

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