Effect of RCAN1 overexpression on the NGF-induced activation of NFAT and the upregulation of PAI-1.

<p>PC12 cells that conditionally overexpress RCAN1 (A,B and C) were treated with NGF (50 ng/ml) for the times indicated. Doxycycline was added overnight before NGF treatment in the cells indicated as NGF/RCAN1. A, Western blot analysis showing RCAN1 overexpression. B, Quantification of NFAT promoter activity by luciferase assays. C, Quantification of the cellular PAI-1 protein levels by immunoblotting. D, PC12 cells were treated with NGF (50 ng/ml) for 2.5 h and the RCAN1.4 transcript levels were quantified by qRT-PCR. *, p<0.05; C, control. Means ± SEM from 3 independent experiments are presented. The treatment effects in panels B and C were statistically significant as judged by comparison of the area under the curve (p<0.05; Student’s t test).</p

DYRK1A overexpression reduces the NGF effect on PAI-1 expression.

<p>PC12 cells that allow conditional overexpression of DYRK1A under the control of doxycyclin were generated. DYRK1A overexpression was induced by treating the cells with doxycyclin overnight. Cells were treated with NGF (50 ng/ml) for 2.5 h (C) or for 3 h (B) or for the indicated times (D). FK506 (10 nM) was added 30 min before NGF treatment. A, Western blot analysis showing DYRK1A overexpression. B, Effect of DYRK1A overexpression on NFAT-driven promoter activity. C, Effect of DYRK1A overexpression on PAI-1 transcripts levels. D, DYRK1A kinase activity was determined by immunocomplex kinase assays in untreated PC12 cells (C) or after cells were treated with NGF for 15, 60 or 150 min. Doxycyclin-induced PC12 cells overexpressing DYRK1A were used as a positive control (D1A). Column diagrams present the means ± SEM from 3 independent experiments. C, control; D1A, DYRK1A overexpression was induced by overnight treatment with doxycyclin; FK, FK506; NS, not significant; *, p<0.05; ***, p<0.001.</p

Scheme illustrating the proposed pathway by which NGF induces PAI-1 expression and its negative regulation by DYRK1A and RCAN1.

<p>NGF activates the calcineurin/NFAT pathway via the TrkA receptor and subsequently the activated NFAT induces the transcription of PAI-1. RCAN1 inhibits the dephosphorylation/activation of NFAT by the protein phosphatase calcineurin. DYRK1A reduces the NGF effect by phosphorylation/deactivation of NFAT. In parallel, NGF induces PAI-1 transcription by activating the PKC pathway. The dashed arrows suggest the possible ways of PKC implication in the induction of PAI-1 transcription.</p

Activation of NFAT is not sufficient for maximal upregulation of PAI-1.

<p>PC12 cells were treated with NGF (50 ng/ml), ATP (300 µM) or calcimycin (10 µM) for 3 h before cell lysis. Cell lysates were assayed for NFAT dependent reporter gene (A), PAI-1 mRNA levels (B) or cellular PAI-1 protein levels (C). The diagrams show the means ± SEM from 3 independent experiments. C, Control; NS, not significant; **, p<0.01; ***, p<0.001.</p

Implication of PKC in the regulation of PAI-1 by NGF.

<p>PC12 cells were treated with NGF (50 ng/ml) for 2.5 h (B) or 3 h (A, C). Gö6976 (7.5 µM) was added 30 min before NGF treatment. A, Cellular PAI-1 levels (upper panel) and autophosphorylation of PKC isoforms (lower panel). B, PAI-1 mRNA levels. C, NFAT dependent reporter gene activity. Diagrams present means ± SEM from 3 independent experiments. C, Control; NS, not significant; *, p<0.05.</p

NGF upregulates PAI-1 <i>via</i> the calcineurin/NFAT pathway.

<p>PC12 cells were maintained in the presence or absence of NGF (50 ng/ml) for 3 h. When indicated, FK506 (10 nM) or VIVIT (concentrations are indicated) were added 30 min before NGF. Cell lysates were assayed for NFAT-driven luciferase activity (A,E) or cellular PAI-1 levels (B,C). PAI-1 mRNA was determined by qRT-PCR (D). The diagrams in panels A, B, C and E present means ± SEM from 3 independent experiments and the graph in panel D shows one representative experiment of 2 replicates. C, Control; *, p<0.05; **, p<0.01.</p