Activation of an endothelial Notch1-Jagged1 circuit induces VCAM1 expression, an effect amplified by interleukin-1β - PubMed (original) (raw)

. 2015 Dec 22;6(41):43216-29.

doi: 10.18632/oncotarget.6456.

Laura Adesso 1, Isabelle Limon 2, Anna Alisi 3, Marie Gueguen 2, Nadia Panera 3, Ezio Giorda 4, Lavinia Raimondi 1, Roberta Ciarapica 1, Antonio F Campese 5, Isabella Screpanti 5, Stefano Stifani 6, Jan Kitajewski 7, Lucio Miele 8, Rossella Rota 1, Franco Locatelli 1 9

Affiliations

Activation of an endothelial Notch1-Jagged1 circuit induces VCAM1 expression, an effect amplified by interleukin-1β

Federica Verginelli et al. Oncotarget. 2015.

Abstract

The Notch1 and Notch4 signaling pathways regulate endothelial cell homeostasis. Inflammatory cytokines induce the expression of endothelial adhesion molecules, including VCAM1, partly by downregulating Notch4 signaling. We investigated the role of endothelial Notch1 in this IL-1β-mediated process. Brief treatment with IL-1β upregulated endothelial VCAM1 and Notch ligand Jagged1. IL-1β decreased Notch1 mRNA levels, but levels of the active Notch1ICD protein remained constant. IL-1β-mediated VCAM1 induction was downregulated in endothelial cells subjected to pretreatment with a pharmacological inhibitor of the γ-secretase, which activates Notch receptors, producing NotchICD. It was also downregulated in cells in which Notch1 and/or Jagged1 were silenced.Conversely, the forced expression of Notch1ICD in naïve endothelial cells upregulated VCAM1 per se and amplified IL-1β-mediated VCAM1 induction. Jagged1 levels increased and Notch4 signaling was downregulated in parallel. Finally, Notch1ICD and Jagged1 expression was upregulated in the endothelium of the liver in a model of chronic liver inflammation.In conclusion, we describe here a cell-autonomous, pro-inflammatory endothelial Notch1-Jagged1 circuit (i) triggering the expression of VCAM1 even in the absence of inflammatory cytokines and (ii) enhancing the effects of IL-1β. Thus, IL-1β regulates Notch1 and Notch4 activity in opposite directions, consistent with a selective targeting of Notch1 in inflamed endothelium.

Keywords: IL-1β; Notch1; Notch4; Pathology Section; VCAM1; endothelial cells.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no conflict of interest to declare.

Figures

Figure 1

Figure 1. IL-1β modulates the expression of components of the Notch pathway in human aortic endothelial cells (HAECs)

Confluent primary HAECs were treated with IL-1β (10 ng/ml) for 6 h or left untreated. A. mRNA levels (Sybr Green method, as reported in the Methods section) for Notch ligands were normalized with respect to β-actin and are expressed as the cDNA copy number (x103) (see Methods section). B. mRNA levels (Sybr Green method) for the Notch receptors Notch1, Notch2, Notch4 and for the Notch target genes Hes1 and Hey1 were normalized with respect to β-actin and are expressed as the cDNA copy number (x103) (see Methods section). A. and B.: Mean±SD. C. Representative western blot showing levels of Jagged1, full-length Notch1 (FL) and the Notch1 intracellular domain (Notch1ICD) (left), Notch2ICD and Notch4ICD (right) in HAECs treated for 6 h with IL-1β or left untreated. β-Actin is the loading control. D. mRNA levels for Jagged1 in HAECs treated with IL-1β or left untreated for the reported times, quantified by the 2(−ΔΔCt) method (see Methods section) after normalization with respect to β-actin, expressed as a fold-change with respect to untreated sample at 0.5 h (100 arbitrary units). Mean±SEM. E. Representative western blot showing levels of Jagged1 in HAECs treated with IL-1β for the reported times. β-actin is the loading control. All experiments were performed in duplicate and repeated independently at least 3 times. *P < 0.05, **P < 0.01, ***P < 0.001, _t_-test.

Figure 2

Figure 2. Pharmacological inhibition of Notch signaling impairs IL-1β-induced VCAM1 upregulation in human aortic endothelial cells (HAECs)

Confluent HAECs were subjected to pretreatment for 16 h with either the γ-secretase inhibitor DAPT (5 μM) or vehicle (DMSO), and were then treated for six hours with DAPT and IL-1β (10 ng/ml) or with DAPT alone and subjected to flow cytometry analysis. (A., left) A representative histogram shows overlays of VCAM1 expression analyzed by flow cytometry: Vehicle (gray solid curve), DAPT (dotted line), IL-1 β + Vehicle (gray line) and IL-1β + DAPT (black line). (A., right) The histogram depicts the quantification of VCAM1 expression in IL-1β-treated cells, expressed as a mean fluorescence intensity (MFI), in arbitrary values. Mean±SD. B. mRNA levels of Hey1 (left) and Hes1 (right) in cells 16 h after treatment with either DAPT (5 μM) or vehicle (DMSO) were quantified by the 2(−ΔΔCt) method (see Methods section) after normalization with respect to β-actin, and are expressed as a fold-change relative to vehicle-treated cells (100 arbitrary unit). Mean±SEM. All the experiments were performed in duplicate and repeated independently at least 3 times. *P < 0.05, **P < 0.01, ***P < 0.001, _t_-test.

Figure 3

Figure 3. The silencing of Notch1 and Jagged1 impairs IL-1β-induced VCAM1 upregulation in human aortic endothelial cells (HAECs)

(A., left) HAECs were transiently transfected with either small interfering oligo RNAs (siRNAs) targeting Jagged1 (375 nM) or non-targeting control siRNAs (Scramble siRNA) and, 48 h later, they were treated with IL-1β (10 ng/ml) for 6 h or left untreated. The cells were then harvested and analyzed by flow cytometry. Representative histogram showing overlays of VCAM1 expression analyzed by flow cytometry: Scramble siRNA (gray solid curve), Jagged1 siRNA (dotted line), IL-1β + Scramble siRNA (gray line) and IL-1β + Jagged1 siRNA (black line). (A., right) The histogram depicts the quantification of VCAM1 expression in IL-1β-treated cells analyzed by flow cytometry and expressed as a mean fluorescence intensity (MFI) in arbitrary values. Mean±SD. B. Representative western blot showing levels of Jagged1 and Notch1 intracellular domain (Notch1ICD) in HAECs treated as in A. β-actin is the loading control. (C., left) HAECs were transiently transfected with either a siRNA targeting Notch1 (375 nM) or non-targeting control siRNAs (Scramble siRNA) and, 48 h later, they were treated with IL-1β (10 ng/ml) or left untreated for 6 h, harvested and analyzed by flow cytometry. A representative histogram showing overlays of VCAM1 expression analyzed by flow cytometry: Scramble siRNA (gray solid curve), Notch1 siRNA (dotted line), IL-1β + Scramble siRNA (gray line) and IL-1β + Notch1 siRNA (black line). (C., right) The histogram depicts the quantification of VCAM1 expression in IL-1β-treated cells analyzed by flow cytometry and expressed as a mean fluorescence intensity (MFI) in arbitrary values. Mean±SD. D. Representative western blot showing levels of Jagged1 and Notch1 intracellular domain (Notch1ICD) in HAECs treated as in C. β-actin is the loading control. (E., left) HAECs were transiently cotransfected with siRNAs targeting either Jagged1 (125 nM) or Notch1 (250 nM), or with non-targeting control siRNAs (Scramble siRNA) and, 48 h later, they were treated with IL-1β (10 ng/ml) for 6 h or left untreated. They were then harvested and analyzed by flow cytometry. A representative histogram showing overlays of VCAM1 expression analyzed by flow cytometry: Scramble siRNA (gray solid curve), Jagged1 siRNA + Notch1 siRNA (dotted line), IL-1β + Scramble siRNA (gray line) and IL-1β + Jagged1 siRNA + Notch1 siRNA (black line). (E., right) The histogram depicts the quantification of VCAM1 expression, analyzed by flow cytometry, in IL-1β-treated cells and expressed as a mean fluorescence intensity (MFI), in arbitrary values. Mean±SD. F. Representative western blot showing levels of Jagged1 and Notch1 intracellular domain (Notch1ICD) in HAECs treated as in E. β-actin expression was used as the loading control. All experiments were performed in triplicate and repeated independently at least 3 times. *P < 0.05, **P < 0.01, ***P < 0.001, _t_-test

Figure 4

Figure 4. Forced expression of Notch1ICD increases VCAM1 expression in human aortic endothelial cells (HAECs)

HAECs were co-nucleofected with a plasmid encoding the Notch1 intracellular domain (Notch1ICD) and a plasmid encoding the green fluorescent protein (GFP) (3:1 molar proportion) and, 48 h later, they were treated with IL-1β (10 ng/ml) for 6 h or left untreated. They were then harvested and analyzed by flow cytometry. An empty vector (EV) was used as a control. (A., left) Representative histogram showing overlays of VCAM1 expression analyzed by flow cytometry: Empty Vector (EV) (gray solid curve), Notch1ICD (dotted line), Empty Vector + IL-1β (gray line) and Notch1ICD + IL-1β (black line). (A., right) The histogram depicts the quantification of VCAM1 expression, analyzed by flow cytometry and expressed as a mean fluorescence intensity (MFI) in arbitrary values. Mean±SD. B. Representative western blot showing levels of Jagged1, Notch1 intracellular domain (Notch1ICD) and VCAM1 in HAECs treated as in A. β-actin is the loading control. **P < 0.01, ***P < 0.001, ANOVA (Bonferroni correction)

Figure 5

Figure 5. Notch1ICD-mediated VCAM1 induction is partly counteracted by NF-kB inhibition in human umbilical vein endothelial cells (HUVECs)

HUVECs were transduced with either a retroviral vector co-expressing a flag-tagged murine Notch1ICD (Notch1ICD*) and the enhanced green fluorescent protein (eGFP) or an eGFP vector (EV*) as a control. After 48 h from infection, cells were subjected to pretreatment with the NF-kB inhibitor BAY 11-7082 (20 μM) for 1 h or with vehicle (DMSO) and they were then treated with IL-1β for 1 h or left untreated. A. Levels of VCAM1 mRNA were quantified by the 2(−ΔΔCt) method (see Materials and Methods section) after normalization with respect to β-actin, and expressed as a fold-change relative to EV*-vehicle-treated cells (1 arbitrary unit). Mean±SD. B. Representative western blot showing levels of intracellular domain (Notch1ICD), Jagged1 and VCAM1 in HUVECs treated as in A. α-tubulin is the loading control. The experiments were performed independently and repeated at least twice. **P < 0.01, ***P < 0.001, ANOVA (Bonferroni correction). C. Proposed model of Notch1-dependent induction of endothelial VCAM1. In endothelial cells, IL-1β reduces the transcription of the Notch4 gene in an NF-kB-dependent manner, leading to downregulation of both the active Notch4ICD form and expression of the Notch4 target gene Hes1. Both these both phenomena are responsible, at least in part, for the upregulation of VCAM1 in the endothelium. Concomitantly, IL-1β induces VCAM1 expression in an NF-kB-dependent manner. In parallel, IL-1β decreases Notch1 transcript levels and upregulates expression of the Notch ligand Jagged1 via a mechanism involving NF-kB. Jagged1 binds to Notch1, leading to the cleavage of the receptor and the sustained formation of the activated form Notch1ICD, in turn favoring Jagged1 expression. At the same time, Notch1ICD amplifies the NF-kB-dependent VCAM1 expression induced by IL-1β. Possibly due to Notch1-inducing VCAM1 upregulation in the absence of IL-1β, the activation of Notch1 also decreases Notch4 and Hes1 expression.

Figure 6

Figure 6. Jagged1 and Notch1 are upregulated in the liver vessels of rats with low-grade chronic inflammation

Rats were fed ad libitum with either a high-fat diet (HFD) or standard chow (SD) (control rats) and were analyzed after 3 months. A. Representative semi-quantitative RT-PCR for IL-1β mRNA, on total RNA from the livers of SD- or HFD-fed rats. Following the preliminary setting up of PCR conditions, each sample was analyzed during the linear phase of amplification and PCR products were visualized under UV illumination with gel red (Biotium Inc., Hayward, CA), after electrophoresis in a 1.5% agarose gel. Primer sequences are listed in Table 1. HPRT was used as the housekeeping gene. B. Quantification by an enzyme-linked immunosorbent assay (ELISA) for TNFα in the serum of SD- and HFD-fed rats. C. Immunohistochemical staining showing CD31 (top) and Jagged1 (bottom) expression on endothelial cells from serial sections of liver samples from SD- and HFD-fed rats. Positive cells are indicated by brown staining. The arrows indicate the endothelial cells lining the blood vessels. 600x magnification. D. Double-labeling immunofluorescence analysis of Notch1ICD (green), with an anti-Val1744 antibody that detects only the cleaved form of the receptor, and CD31 (red) on serial sections of liver from SD- and HFD-fed rats. Nuclei are counterstained with DRAQ5. Blue arrows indicate Notch1ICD-negative nuclei (SD rats), yellow arrows indicate Notch1ICD-positive nuclei and pink arrows indicate Notch1ICD-positive cytoplasmic staining (HFD rats). A white arrow indicates a Notch1ICD-positive liver parenchymal nucleus. Top panels, 600 x magnification; lower panels are a higher magnification (2400 x magnification) of the squared area in the top panels. A representative set of images is shown. **P < 0.01 _t_-test.

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