Notch2 signaling sensitizes endothelial cells to apoptosis by negatively regulating the key protective molecule survivin - PubMed (original) (raw)

Notch2 signaling sensitizes endothelial cells to apoptosis by negatively regulating the key protective molecule survivin

Thibaut Quillard et al. PLoS One. 2009.

Abstract

Background: Notch signaling pathway controls key functions in vascular and endothelial cells (ECs) where Notch4 plays a major role. However, little is known about the contribution of other Notch receptors. This study investigated regulation of Notch2 and further examined its implication in EC dysfunction.

Methodology/principal findings: Here, we provide evidence for a novel link between Notch and TNF signaling, where Notch2 is upregulated and activated in response to TNF. Forced expression of Notch2 intracellular domain in cultured ECs promotes apoptosis and allows the significant downregulation of several cell-death-related transcripts in a dose-dependent manner. In particular, activation of Notch2 led to a rapid decrease in survivin mRNA and protein expression, while survivin upregulation was obtained by the selective knockdown of Notch2 in ECs, indicating that survivin expression is controlled at the Notch level. Moreover, Notch2 silencing and ectopic expression of survivin, but not XIAP or Bcl2, rescued ECs from TNF and Notch2-mediated apoptosis, respectively.

Conclusions/significance: In conclusion, TNF signaling activates Notch2 that sensitizes ECs to apoptosis via modulation of the key apoptosis regulator survivin. Overall, our findings also indicate that specific Notch receptors control distinct functions in vascular cells and inflammatory cytokines contribute to this specificity.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Regulation of Notch2 signaling in response to TNF.

(A) RT-PCR for Notch1-4 in ECs treated with TNF. A western blotting analysis (B) and its quantification showing the expression of the native Notch2 (C) and the activated cleaved form (cNotch2) in response to TNF (D). (E) RT-PCR for Notch2 and hey1 in ECs treated with TNF. (F) qRT-PCR for hey1 in EC stimulated with TNF and DAPT. *p<0.05 versus controls.

Figure 2

Figure 2. Notch2 activation elicits EC apoptosis.

(A) Quantification of EC viability 0–72 h post-infection with AdGFP, AdNull or AdN2ICD using a DNA content assay. (B) Apoptosis detection 72 h post-transduction after AnnexinV and Propidium Iodide (PI) staining. (C) PhiPhiLux™ system to detect real-time activation of caspase3/7 in live ECs after 48 h post-infection for 18 hours, A representative image after imaging is shown in panel C. Caspase3/7 positive cells/field (in %) were counted every 2 hours during the experiment (D). A western blot analysis for cleaved-forms of caspase-3, -7 and PARP in response to Notch2 NICD (E). (F) A colorimetric assay for caspase 3 and 7 activation 48 h after transduction.

Figure 3

Figure 3. TNF and PDTC treatment induces EC mortality.

EC death induction by DNA content assay (A) and activated cleaved-Notch2 expression by Western Blot (B) after TNF and PDTC treatment. Results are representative of 3 independent experiments. *p<0.05 versus controls.

Figure 4

Figure 4. Notch2 signaling downregulates apoptosis mediators.

(A) A schematic representation of apoptosis-related transcripts repressed (red) or induced (green) by N2ICD or TNF determined by a dedicated PCR array. Means are shown as fold changes compared to AdGFP or untreated ECs, respectively. (B) Validation of transcripts regulation by qRT-PCR in ECs transduced with AdN2NICD (moi 20 and 40) in comparison to controls (non infected (NI) and AdGFP) (B–C). (D) Dose-response effect of AdN2ICD on Survivin protein expression by western blot. Results are representative of 5 independent experiments. *p<0.05 versus controls.

Figure 5

Figure 5. Notch2 knockdown increases survivin and rescues ECs from apoptosis.

(A) Validation of Notch2 silencing in ECs by qRT-PCR. (B) qRT-PCR for survivin mRNA in ECs transfected with a scramble siRNA or siRNAs targeting Notch2. (C) Survivin and Notch2 expression by western blot. (D) DNA content analysis of siRNA transfected ECs after induction of apoptosis by TNF and PDTC (NT: non transfected cells). Results are means±SEM of 3 independent experiments.*p<0.05 versus scramble.

Figure 6

Figure 6. Survivin prevents Notch2-mediated apoptosis.

ECs were transduced or not (NT) with AdGFP (5 moi) or AdN2ICD (30 moi) after transfection with a plasmid encoding survivin, XIAP or Bcl2 full cDNAs or the empty vector (mock) as a control. Cell death was measured by DNA content (A). Corresponding means±SEM of 7 independent experiments (B). *p<0.05 versus mock. A representative western blotting analysis for Notch2, survivin and cleaved-caspase 7 expression in ECs non infected (NI) or transduced with AdN2ICD (30 moi) 24 h prior to transfection with mock or survivin plasmids. Cell lysates were collected 48 h after infection (representative of 3 independent experiments).

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