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

Journal of Cell Science 120, 3352-3361 (2007)
Published by The Company of Biologists 2007
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Research Article

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

Nathalie Clément1,*, Marie Gueguen1,*, Martine Glorian1, Régis Blaise1,2, Marise Andréani1, Christel Brou2, Pedro Bausero1 and Isabelle Limon1,{ddagger}

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

{ddagger} Author for correspondence (e-mail: Isabelle.Limon-Boulez{at}snv.jussieu.fr)

Accepted 20 July 2007


   Summary
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 Summary
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Atherogenesis begins with the transfer of monocytes from the lumen to the intimal layer of arteries. The paracrine activity acquired by these monocytes shifts vascular smooth muscle cells from a contractile-quiescent to a secretory-proliferative phenotype, allowing them to survive and migrate in the intima. Transformed and relocated, they also start to produce and/or secrete inflammatory enzymes, converting them into inflammatory cells. Activation of the Notch pathway, a crucial determinant of cell fate, regulates some of the new features acquired by these cells as it triggers vascular smooth muscle cells to grow and inhibits their death and migration. Here, we evaluate whether and how the Notch pathway regulates the cell transition towards an inflammatory or de-differentiated state. Activation of the Notch pathway by the notch ligand Delta1, as well as overexpression of the active form of Notch3, prevents this phenomenon [initiated by interleukin 1beta (IL-1beta)], whereas inhibiting the Notch pathway enhances the transition. IL-1beta decreases the expression of Notch3 and Notch target genes. As shown by using an I{kappa}B{alpha}-mutated form, the decrease of Notch3 signaling elements occurs subsequent to dissociation of the NF-{kappa}B complex. These results demonstrate that the Notch3 pathway is attenuated through NF-{kappa}B activation, allowing vascular smooth muscle cells to switch into an inflammatory state.

Key words: Vascular smooth muscle cells, Notch, Inflammation


   Introduction
Top
 Summary
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
In atherosclerosis, pathological changes in vessel structure arise, at least in part, from changes in signaling pathways that govern the transition of contractile smooth muscle cells towards a phenotype that allows them to migrate from the medial to the intimal layer and survive (Lusis, 2000Go; Ross, 1999Go). Once transformed and relocated, they are able to synthesize and/or secrete several extracellular matrix proteins (collagen, elastin, proteoglycans) that are required to generate the fibrous cap that ultimately surrounds foam cells, as well as cell-adhesion molecules (e.g. VCAM) and mitosis factors such as cyclin D, Cdk2, Cdk4 and Cdk6 (for a review, see Lusis, 2000Go). They also start to produce and/or secrete inflammatory enzymes such as phospholipase A2 (PLA2) and cyclooxygenase-2 (COX-2), which results in a massive increase in intracellular levels of arachidonic acid and prostanoids (Nakano et al., 1990Go; Rimarachin et al., 1994Go). This transition towards an inflammatory state is mainly instigated by pro-inflammatory cytokines released by infiltrating macrophages. The released cytokines include tumor necrosis factor {alpha} (TNF-{alpha}) and IL-1beta, which act through the NF-{kappa}B pathway.

Recent ex vivo data, mostly obtained from the rat, demonstrate that the Notch signaling pathway regulates some of these new features acquired by vascular smooth muscle cells during atherogenesis. Indeed, Notch1 and Notch3 regulate cell fate in vascular smooth muscle cells and control their growth and migration in response to growth factor stimulation following vascular injury (Sweeney et al., 2004Go); Notch3 also promotes resistance to apoptosis by intimal smooth muscle cells, which is a prominent feature of the response to injury, and regulates the consequent formation of the neointima (Campos et al., 2002Go; Sweeney et al., 2004Go; Wang et al., 2002bGo); Notch signaling represses myocardin-dependent smooth muscle cell differentiation and maintenance of the contractile phenotype of smooth muscle cells (Proweller et al., 2005Go). Although it has been shown that pro-inflammatory molecules, acting through NF-{kappa}B, can repress Notch target genes (Espinosa et al., 2003Go), the possibility that the Notch pathway modulates the cytokine-mediated transition towards an inflammatory state has not been explored until now.

Genes of the Notch family encode a series of type I transmembrane receptors used by metazoan cells to control cell fate through local cell interactions. Mammals have four Notch genes, homologous to the single notch gene of invertebrates, each encoding a Notch receptor subtype (1 to 4). Notch activation starts with the binding of Notch to the extracellular domains of transmembrane ligands (named Delta and Jagged) of emitting cells. This interaction allows Notch to undergo a first cleavage in its extracellular domain, catalyzed by the metalloprotease TNF-{alpha}-converting enzyme (TACE, ortholog of invertebrate ADAM17). The membrane-tethered Notch product of the first cleavage is itself a substrate for a proteolytic activity of a {gamma}-secretase-like complex. This complex cleaves Notch and releases the intracellular domain, which translocates into the nucleus, where it converts the DNA-binding RBP-J{kappa} protein (also known as CSL, for CBF1/Su(H)/Lag1) from a transcription repressor to a transcription activator (Kopan, 2002Go). The target genes upregulated by Notch activation encode transcription factors of the Enhancer of Split group, which in mammals are Hairy and enhancer of split (Hes) and Hairy-related transcription factor (HRT) proteins. Using pharmacological and molecular biology tools, the present study establishes that the Notch3 and IL-1beta pathways exert opposite effects on the organisation of the contractile apparatus of vascular smooth muscle cells and their transition towards an inflammatory state. As an initial approach to study the mechanism of crosstalk between both pathways, we investigated the role of the NF-{kappa}B complex.


   Results
Top
 Summary
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Inhibition of the Notch signaling pathway enhances the effect of IL-1beta on the biosynthesis pathway of lipid mediators and on the contractile apparatus
To determine whether Notch activation could regulate the transition of vascular smooth muscle cells towards an inflammatory state, we first treated control and IL-1beta-induced cells with the {gamma}-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and assayed the secretion of PLA2 and the prostaglandin PGE2 as well as expression of COX-2. DAPT (0.5 µM) treatment greatly enhances the secretion of PLA2 (Fig. 1A) only when cells are induced by IL-1beta (10 ng/ml). This concentration of IL-1beta is commonly used to allow smooth muscle cells to fully de-differentiate (Clément et al., 2006Go). This DAPT effect becomes significant when compared with that induced by IL-1beta alone at 48 hours of treatment (5383±696% versus 2364±677%, P<0.05) and persists or increases during the following 24 hours (5999±495% versus 2271±567%, P<0.01). As illustrated by western blot (Fig. 1B), the inhibition of the {gamma}-secretase-like activity also triggers a potentiation of the COX-2 expression induced by IL-1beta. DAPT similarly affects COX-2 expression at both time periods studied. Although the Notch pathway is activated in control or untreated cells as shown by DAPT-induced downregulation of Notch target gene expression in these cells (data not shown), DAPT alone is unable to trigger the expression of the inflammatory markers PLA2 and COX-2. Of note, when measuring transcript levels, similar results were obtained after only 6 hours of treatment (Fig. 1C,D). DAPT also enhances the secretion of PGE2, the principal prostanoid released during inflammation (Wen et al., 1998Go; Wohlfeil and Campbell, 1999Go; Yamamoto et al., 1999Go), after 24 and 48 hours of IL-1beta treatment (Fig. 1E).


Figure 1
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  		Fig. 1. Inhibition of {gamma}-secretase increases the positive effect of IL-1beta on the biosynthesis pathway of lipid mediators. Serum-starved cells were treated daily for 6, 24 or 48 hours with IL-1beta (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 beta-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 beta-actin and results are expressed as a percentage of those from IL-1beta-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-1beta; D, DAPT; D+IL, DAPT plus IL-1beta versus IL-1beta; ns, non-significant; *, P<0.05; **, P<0.01; ***, P<0.001.

 
The transition to the secretory-inflammatory state initiated by IL-1beta is accompanied by a loss of organized stress fibers (Clément et al., 2006Go) resulting, in particular, in a downregulation of expression of {alpha}-actin. Therefore, we also investigated whether a similar potentiation effect of DAPT is observed on the decrease of levels of several contractile proteins, including {alpha}-actin. When 10 ng/ml of IL-1beta is combined with DAPT and applied for 24 hours, the expression of the genes studied (encoding {alpha}-actin, SM-22, myocardin and calponin) is significantly downregulated in comparison with that measured in the presence of IL-1beta alone (Fig. 2A). Immunofluorescence experiments using an antibody against {alpha}-actin revealed that a 72-hour treatment with both compounds (IL-1beta and DAPT) prevents immunolabeling of cells (only the nuclei are visible with the Hœchst coloration; Fig. 2B, 20x magnification). When the same concentration of IL-1beta is used alone, only half the cells are unlabeled. DAPT–IL-1beta-treated cells that do stain positively for {alpha}-actin present a diffused staining pattern (clearly visualized at 63x magnification), instead of staining being restricted to the short filaments, as observed in IL-1beta-treated cells. As a consequence of this filament disorganization, most of the DAPT–IL-1beta-treated cells display a `wrinkly' organization (particularly evident at 20x magnification), whereas, when treated with IL-1beta alone, a significant fraction of cells remain spindle shaped. Control or untreated cells, and those treated with DAPT alone, were all positively immuno-stained by the antibody against {alpha}-actin and always display a well-organized network of actin stress fibers as well as a spindle shape. As shown by western blot, the DAPT-enhanced disorganization of {alpha}-actin filaments reflects a pronounced decrease in {alpha}-actin expression in comparison with that displayed by cells treated with IL-1beta alone (Fig. 2C). Here again, DAPT alone has no effect on {alpha}-actin expression, which remains similar to that of untreated or control cells (Fig. 2C). Taken together, the DAPT potentiation effects depicted in Figs 1 and 2 demonstrate that the proteolytic product(s) generated by the {gamma}-secretase complex reduces the IL-1beta-mediated transitions towards an inflammatory or de-differentiated state.


Figure 2
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  		Fig. 2. Inhibition of {gamma}-secretase enhances the effect of IL-1beta on the contractile apparatus. Serum-starved cells were treated daily for 1 day (A) or 3 days (B,C) with IL-1beta (10 ng/ml) or vehicle (control) and/or with DAPT (0.5 µM). (A) {alpha}-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-1beta versus IL-1beta. *, P<0.05; **, P<0.01. (B) Immunostaining of {alpha}-actin stress fibers was performed using a monoclonal antibody against {alpha}-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). {alpha}-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 {alpha}-actin bands normalized to that of GAPDH and are expressed as a percentage of control cells. C, control; IL, IL-1beta; D, DAPT; D+IL, DAPT plus IL-1beta versus IL-1beta; **, P<0.01; ***, P<0.001; {dagger}, P<0.05.

 
The Notch3-activated pathway decreases PLA2 and PGE2 secretion and enhances the expression of contractile markers in IL-1beta-treated cells
Published evidence suggests that the {gamma}-secretase products most likely to fulfill such an inhibitory role are the Notch intracellular domains. Indeed, Notch1 or Notch3 activation regulates some of the other characteristics acquired by vascular smooth muscle cells during the pathogenesis of vascular diseases, such as migration, proliferation and apoptosis (Sweeney et al., 2004Go). Nevertheless, the {gamma}-secretase complex also cleaves some other transmembrane proteins (distinct from Notch), leading to additional proteolytic products (Parks and Curtis, 2007Go). To confirm such a role for Notch receptor(s), we stimulated this pathway. To do so, we used conditioned medium (CM) obtained from confluent HEK 293T cells transfected with a construct encoding a chimeric protein including the extracellular domain of rat Delta1, one of the endogenous ligands of Notch. The secretion of the fusion protein [referred to as Delta1-Fc (Hicks et al., 2002Go)] was revealed by immunoblotting with an antibody against Fc, as the chimeric protein also contains the human IgG Fc sequence lacking the hinge region (supplementary material Fig. S1A). The addition of conditioned medium obtained from transfected HEK 293T cells (Delta1-Fc CM) significantly reduces secretion of PLA2 and PGE2 in IL-1beta-treated vascular smooth muscle cells (Fig. 3A) while enhancing the expression of Notch target genes (supplementary material Fig. S1B). No effect was observed with conditioned medium collected from wild-type HEK 293T cells (referred to as WT CM). We also transfected our cell model with an expression plasmid encoding Notch3 and Notch1 constitutively active intracellular domains. Transfection efficiency was verified by real-time PCR (RT-PCR) and western blot in control cells (supplementary material Fig. S2A). The functionality of both Notch intracellular domains is attested by the fact that Notch downstream effector transcripts (encoding Hes1 and HRT1) are upregulated by ~2-5 fold in comparison with cells transfected with control plasmids (referred to as `mock', supplementary material Fig. S2B). Of note, Hes1 appears to be specifically synthesized in response to Notch3 activation, whereas HRT1 is more likely a Notch1 target. Overexpression of the Notch3 intracellular domain reduces the influence of IL-1beta on the mRNA level, and secretion of, PLA2, whereas the Notch1 intracellular domain produced no or little effect (Fig. 3B). When PGE2 was assayed, we observed a similar pattern of results (Fig. 3B). The somewhat lower mean value of IL-1beta-induced PGE2 secretion obtained in the presence of the Notch1 intracellular domain was not significantly different from that measured with the control plasmid (1414±191% versus 1955±142%, P>0.05). Of note, the extent of the inhibitory influence of the Notch3 intracellular domain on mRNA levels and PLA2 secretion is dependent on its level of expression; conversely, increasing the level of the Notch1 intracellular domain has no effect on these parameters (data not shown). When measuring expression of {alpha}-actin, the IL-1beta-induced downregulation, visualized in cells transfected with control plasmid (mock), was significantly reversed by the introduction of the intracellular domain of Notch3 (Fig. 3C). Although partial, this reversion reaches 60% of the {alpha}-actin expression of cells not treated with IL-1beta but overexpressing Notch3. By contrast, the Notch1 intracellular domain behaves inefficiently (Fig. 3C). No significant effect was observed on {alpha}-actin expression after overexpressing Notch3 or Notch1 in control cells. This set of experiments demonstrates that activated Notch3 prevents, at least partially, some of the IL-1beta effects that lead cells to adopt an inflammatory state and rescues the deleterious effect of IL-1beta on the expression of contractile proteins. This phenomenon occurs through Notch target genes. Indeed, the forced expression of a dominant-negative form of RBP-J{kappa} (shown by RT-PCR and western blot, supplementary material Fig. S3), which prevents transcription of Notch target genes in response to Notch activation (Iso et al., 2002Go; Waltzer et al., 1995Go), significantly increases the levels of PLA2-IIA mRNA induced by treatments for 6 or 24 hours with 10 ng/ml IL-1beta (Fig. 4A) and increases the subsequent secretion of PLA2 and PGE2 (Fig. 4B). Of note, these experiments provide results similar to those obtained with DAPT in IL-1beta-treated cells.


Figure 3
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  		Fig. 3. Effect of activated Notch on the prostanoid biosynthesis pathway and expression of {alpha}-actin. (A) Cells were treated for 48 hours with IL-1beta (10 ng/ml) or vehicle (control) and conditioned media collected from wild-type (WT CM) or Delta1-Fc-transfected ({Delta}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 + {Delta}1-Fc CM, IL-1beta plus {Delta}1-Fc CM versus IL-1beta 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-1beta (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-1beta (10 ng/ml) and/or vehicle (control) were separated by electrophoresis (10% SDS PAGE). {alpha}-Actin and GAPDH were immunodetected with appropriate antibodies. The autoradiogram is representative of three independent experiments. The histogram represents values obtained from scanning {alpha}-actin bands normalized to that of GAPDH and are expressed as a percentage of each control cell. C, control; IL, IL-1beta; *, P<0.05; ns, non-significant.

 

Figure 4
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  		Fig. 4. Effect of the overexpression of the RBP-J{kappa} mutant on the prostanoid biosynthesis pathway. Cells were transfected with RBP-J{kappa} DN (a mutated form of RBP-J{kappa}) or empty (mock) plasmids and treated for 6, 24 or 48 hours with IL-1beta (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{kappa} 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{kappa}-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{kappa} DN versus mock: *, P<0.05; **, P<0.01.

 

Crosstalk between the IL-1beta and Notch pathways
If Notch3 activation partially inhibits IL-1beta effects, then a question arising is: does IL-1beta attenuate Notch signaling to switch vascular smooth muscle cells into an inflammatory, or de-differentiated, state? To address this question, we first investigated whether IL-1beta regulates the expression of Notch subtypes 1 and 3. We approached this issue by using RT-PCR. As shown in Fig. 5A, a concentration of 10 ng/ml of IL-1beta, applied for 6 hours, reduces Notch3 mRNA expression (by a factor of 2-3 in comparison with untreated or control cells, P<0.001). IL-1beta also decreases the mRNA level of at least one of the ligands (Jagged1) of the Notch receptor family and reduces expression of three of the Notch targets [HRT1 and HRT2, also known as cardiovascular helix-loop-helix factors 1 and 2, CHF1 and CHF2 (Chin et al., 2000Go), and Hes1]. Conversely, it increases the level of transcripts encoding Notch1 (threefold, P<0.01). Recent data established that the NF-{kappa}B complex, which is activated by several factors, including IL-1beta, can also repress gene expression of central elements of the Notch signaling system (Espinosa et al., 2003Go). As an initial approach to elucidate the mechanism by which the IL-1beta pathway could downregulate expression of Notch3 and its targets (Hes1, HRT1 and HRT2) in vascular smooth muscle cells, we prevented NF-{kappa}B activation by blocking its release from I{kappa}B{alpha} by overexpressing a mutated, non-phosphorylatable, form of this protein (I{kappa}B{alpha}32-36) and measuring the impact of this on mRNA levels. Overexpression of this mutant reverses the downregulation mediated by IL-1beta on Notch3 and Hes1 expression (visualized in cells transfected with control plasmid, Fig. 5B). Conversely, the IL-1beta-induced effect on Notch1, HRT1 and HRT2 expression persists. Of note, a control for transfection efficiency was the demonstration that the overexpression of I{kappa}B{alpha}32-36 significantly decreases the expression of the NF-{kappa}B-regulated gene encoding secreted PLA2 (supplementary material Fig. S4). These results demonstrate that IL-1beta downregulates the expression of the Notch3 signaling pathway through NF-{kappa}B activation in vascular smooth muscle cells.


Figure 5
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  		Fig. 5. Regulation of Notch-family gene expression in response to IL-1beta. (A) Serum-starved cells were treated for 6 hours with IL-1beta (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-1beta-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 I{kappa}B{alpha}32-36 (a mutated form of I{kappa}B{alpha}) or empty (mock) plasmids and treated for 6 hours with IL-1beta (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-1beta-treated I{kappa}B{alpha}32-36-transfected cells were compared with those of IL-1beta-treated mock-transfected cells. C, control; IL, IL-1beta; *, 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.

 

The phenotypic change of vascular smooth muscle cells is associated with a decrease of cytoplasmic I{kappa}B{alpha} expression (Ganguli et al., 2005Go). Activation of the NF-{kappa}B complex is a likely Notch regulatory mechanism as it has been shown recently that: (1) basal expression of I{kappa}B{alpha} is controlled by RBP-J{kappa} and its activator Notch1 (Oakley et al., 2003Go); (2) Notch1 augments NF-{kappa}B activity by facilitating its nuclear retention (Shin et al., 2006Go); (3) Notch3 regulates multiple NF-{kappa}B activation pathways (Bellavia et al., 2003Go; Vacca et al., 2006Go). Therefore, it was crucial to evaluate the role of activated Notch on the expression of smooth muscle I{kappa}B{alpha}. In control and IL-1beta-treated cells (Fig. 6), overexpression of the Notch1 intracellular domain decreases both the basal and stimulated expression of I{kappa}B{alpha} transcripts (100±1% versus 44±9%, P<0.05; 327±30% versus 182±54%, P<0.05). No significant effect of the Notch3 intracellular domain was observed regardless of whether cells were treated. Similar results were obtained at the protein level (data not shown).


Figure 6
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  		Fig. 6. Effect of the overexpression of Notch1 or Notch3 intracellular domains on I{kappa}B{alpha} expression. Cells were transfected with N3-ICD, N1-ICD or empty (mock) plasmids and treated for 6 hours with IL-1beta (10 ng/ml) or vehicle (control). I{kappa}B{alpha} transcripts were assayed by RT-PCR. Results are expressed as a percentage of the I{kappa}B{alpha} mRNA level from control cells and represent the mean ± s.e.m. of three independent experiments. C, control; IL, IL-1beta; *, P<0.05; ** P<0.01.

 

   Discussion
Top
 Summary
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Several reports have established that the Notch pathway tightly regulates the vascular smooth muscle cell response to several stress factors through growth, migration, apoptosis and differentiation (Campos et al., 2002Go; Noseda et al., 2006Go; Sweeney et al., 2004Go; Wang et al., 2002aGo). Surprisingly, none of these studies investigated whether and how Notch could regulate one of the major events occurring during atherogenesis: the transition of vascular smooth muscle cells towards an inflammatory state. As an initial approach to this issue, we first inhibited Notch signaling by using two strategies, one blocking the cleavage of the Notch intracellular domain and the other preventing the transcriptional activity of this domain. As shown by these two sets of experiments, inhibition of Notch signaling significantly enhances the inflammation state of vascular smooth muscle cells. Conversely, activating this pathway with a secreted form of the Notch ligand Delta1 decreases inflammation. This is the first evidence linking the alteration of the Notch pathway and the acquisition of the inflammatory state by vascular smooth muscle cells. Results obtained from IL-1beta-treated vascular smooth muscle cells overexpressing Notch1 or Notch3 intracellular domains are consistent with an anti-inflammatory role for Notch3.

The transfection of Notch1 or Notch3 intracellular domains prevents the effect of IL-1beta on the expression of contractile markers [{alpha}-actin (Fig. 3) and SM-22 (data not shown)], whereas the inhibition of the Notch pathway by DAPT enhances the effect of IL-1beta. Our results are in line with those of Domenga and colleagues (Domenga et al., 2004Go) demonstrating that, in Notch3-knockout mice, the vascular smooth muscle cells do not complete their differentiation, and with those of Doi and colleagues (Doi et al., 2006Go) showing that Notch3 transforms fibroblasts into contractile cells and enhances human aortic smooth muscle cell differentiation. Nevertheless, our results contradict those of Morrow and colleagues (Morrow et al., 2005Go) that suggest that Notch1 and Notch3 decrease the expression of several contractile markers ({alpha}-actin, myosin, calponin, smoothelin) in human vascular smooth muscle cells. Interestingly, in the latter study, the de-differentiating effect of Notch, although significant, is moderate and would more likely result from the Notch subtype 1. Indeed, HRT gene expression is preferentially induced by Notch1, whereas Hes1 is mainly controlled by Notch3 (supplementary material Fig. S2B) and is likely able to induce the decrease of myosin and smoothelin expression (Doi et al., 2005Go; Proweller et al., 2005Go). Taken together, these studies and the present study suggest that the Notch subtypes 1 and 3 could have different, or even opposite, regulatory roles on transdifferentiation of vascular smooth muscle cells (i.e. their conversion into noncontractile/inflammatory cells), acting perhaps on specific target genes. This assumption is in line with the findings of Ong and colleagues (Ong et al., 2006Go) showing that the four Notch receptors do not equally activate Notch target genes. Nevertheless, this needs clarification as several different studies establish that both Notch1 and Notch3 can have similar regulatory effects on vascular smooth muscle cell differentiation (Doi et al., 2006Go; Morrow et al., 2005Go; Noseda et al., 2006Go; Sweeney et al., 2004Go).

The observation that IL-1beta induces an inhibitory effect on Notch3 and Jagged1 similar to the effect of platelet-derived growth factor (PDGF) and angiotensin II suggested that such downregulation might be a common feature of all the factors activated in vessel injury and repair (Campos et al., 2002Go). If this is the case, then the coordinate downregulation of the major components of the Notch pathway can be regarded as one of the mechanisms facilitating the inflammatory response. To establish a clear cause-and-effect relationship between these two molecular events, it would be of interest to compare the levels of inflammatory markers produced by smooth muscle cells from Notch3-knockout mice (Krebs et al., 2003Go) with those of wild-type mice after a balloon injury or after induction of atherosclerosis. Taking advantage of the capacity of IL-1beta to knock-down Notch3 receptor expression, we re-evaluated Notch regulation of inflammatory markers by using DAPT on vascular smooth muscle cells pre-treated with IL-1beta. Under these conditions, the inhibition of the Notch pathway by DAPT significantly decreased PLA2 secretion and did not affect PGE2 secretion induced by IL-1beta (supplementary material Fig. S5), rather than enhancing these secretions when both compounds are introduced simultaneously into the cell medium (as shown in Fig. 1A,E). This is consistent with other pro-inflammatory Notch pathway component(s) continuing to be expressed. Also, it supports the idea that Notch3, the major Notch receptor in differentiated vascular smooth muscle cells (Villa et al., 2001Go), plays an anti-inflammatory role.

The regulation of Notch signaling components is likely to be a direct consequence of IL-1beta-dependent signaling events. Indeed, this cytokine modulates Notch signaling components and expression of inflammatory enzymes simultaneously (Couturier et al., 1999Go; Jiang et al., 2004Go), and a mutated form of I{kappa}B{alpha} blocking dissociation of the NF-{kappa}B complex prevents the IL-1beta-induced inhibition of Notch3 and Hes1 gene expression (Fig. 5B). A putative molecular mechanism for the regulation induced by IL-1beta on Notch signaling components is given in Fig. 7. The nuclear co-repressor receptor from the NF-{kappa}B complex, once free in the cytosol, migrates to the nucleus and interacts with promoters of various genes including some Notch target genes such as Hes1 by associating with RBP-J{kappa} [as described previously by Espinosa and colleagues (Espinosa et al., 2002Go)]. The IL-1beta-induced inhibition of the expression of the gene encoding Notch3 is likely to reinforce nuclear co-repressor receptor association to RBP-J{kappa} in preventing the conversion of RBP-J{kappa} into an activator of transcription.


Figure 7
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  		Fig. 7. Putative model for Notch and IL-1beta crosstalk to regulate vascular smooth muscle inflammation and differentiation. See Discussion for details. HDAC, histone deacetylase complex; N-CoR, nuclear co-repressor receptor.

 

As IL-1beta-induced regulation of the genes encoding HRT1, HRT2 and Notch1 is not affected by overexpression of the mutated form of I{kappa}B{alpha}, we conclude that they are regulated by a mechanism different from that described above for Hes1 and Notch3. Interestingly, it has been shown that HRT expression can be downregulated by the ERK pathway in cultured vascular smooth muscle cells (Wang et al., 2002aGo). As IL-1beta also signals through ERK, it is possible that the NF-{kappa}B-independent downregulation of HRT2 induced by IL-1beta occurs by means of ERK.

Our work also showed that preventing the transcription of Notch target genes (by the use of a mutated form of the nuclear protein RBP-J{kappa}; data not shown) upregulates the expression of PLA2 and COX-2 that is initially induced by IL-1beta. Therefore, it is possible that at least some of the Notch target gene products are able to repress the genes encoding these two enzymes. Consistent with this idea, the Notch-Hes1 signal transcriptionally regulates the gene encoding lipocalin-type prostaglandin D synthase (involved in prostanoid biosynthesis) by interacting with an E-box motif (Fujimori et al., 2003Go); the E-box is present in the promoter of the genes encoding secreted PLA2 and COX-2 (Antonio et al., 2002Go; Thomas et al., 2000Go). Regarding these data, it is likely that Notch3-specific target gene products bind to the E-box within the promoter of the genes encoding PLA2 and COX-2. Therefore, to complete the scheme presented in Fig. 7, we suggest that the decreased expression of Hes1 repressors could liberate the E-box of PLA2 and COX-2 promoters, thus allowing the NF-{kappa}B-dependent transcription of the genes encoding these two inflammatory enzymes. Additionally, the Notch1 upregulation mediated by IL-1beta could facilitate NF-{kappa}B activation, thus downregulating I{kappa}B{alpha} expression. These data agree with the notion that Notch1 and Notch3 could have opposite effects on the inflammatory phenotype. Outside of any inflammatory environment, the Notch3 intracellular domain translocates to the nucleus, binds to the DNA-binding protein RBP-J{kappa} and enhances the expression of Hes1 (Fig. 7). The increased expression of Notch target genes could, together with the absence of NF-{kappa}B translocation, further block PLA2 and COX-2 expression through their interaction with the E-box, prohibiting any biosynthesis of these pro-inflammatory lipid mediators.

In summary, we demonstrate for the first time that Notch3 and IL-1beta signals exert opposite effects on the transition of mature vascular smooth muscle cells towards an inflammatory or de-differentiated state and suggest a mechanism of crosstalk between these two pathways. Future investigations on Notch-knockout mice and animals that develop arteriopathies, such as ApoE–/– mice and/or mice receiving a high-fat diet, could provide new insights into the molecular mechanisms of vascular complications.


   Materials and Methods
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 Results
 Discussion
 Materials and Methods
References
 
Reagents
Dulbecco's Modified Eagle's Medium (DMEM), type I collagen from calf skin, glutamine, penicillin, streptomycin, fatty-acid-free bovine serum albumin and type II secreted PLA2 from bee venom were purchased from Sigma Aldrich (Saint Quentin Fallavier, France). Foetal calf serum and collagenase were from Gibco BRL (Cergy Pontoise, France). Elastase, protease inhibitors and LightCycler-DNA Master Plus SYBR Green were obtained from Roche Diagnostics (Meylan, France). Coverslips used for immunocytochemistry experiments were from Fisher Bioblock Scientific (Illkirch, France). Oligonucleotides were sourced from MWG Biotech AG (Courtaboeuf, France). Kits for RNA extraction (RNeasy Mini kit) were obtained from QiaGen (Courtaboeuf, France). RT-MMLV and RNAsin were obtained from Invitrogen (Cergy Pontoise, France). Protran nitrocellulose membranes were from Schleicher and Schuell (Dassel, Germany). ECL reagent kit was from Amersham Pharmacia Biotech (les Ulis, France). The fluorescent substrate 1-hexadecanoyl-2-(1-pyrenldecanoyl)-sn-glycero-3-phosphoglycerol was from Interchim (Montluçon, France). Interleukin-1beta (IL-1beta) was from Santa Cruz Biotechnology. Basic nucleofector kit for primary smooth muscle cells was purchased from Amaxa (Gaithersburg, MD). PGE2 enzyme-linked immunosorbent assay (EIA) kit was from Cayman Chemical (Montigny le Bretonneux, France). The {gamma}-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was from Calbiochem (La Jolla, CA). The mouse Notch3 intracellular domain expression vector was a gift from U. Lendahl (Medical Nobel Institute, Stockholm, Sweden). The mouse Notch1 intracellular domain expression vector was obtained as described previously (Gupta-Rossi et al., 2001Go). The expression vectors encoding the mutated form of human I{kappa}B{alpha} (I{kappa}B{alpha}32-36) and human RBP-J{kappa} [RBP-J{kappa} dominant-negative (DN)] were gifts from A. Israel (Institut Pasteur, Paris, France) and A. Sergeant (Ecole Normale Supérieure, Lyon, France), respectively. Delta1-Fc conditioned medium was prepared as previously described (Hicks et al., 2002Go).

Isolation and culture of rat aortic smooth muscle cells
Rat vascular smooth muscle cells were isolated by enzymatic digestion of thoracic aortic media from male Wistar rats and cultured as described previously (Clément et al., 2006Go). All experiments were performed on cells at passages ranging from two to eight. 24 hours before any treatment, confluent cells were made quiescent by culturing them in a serum-free medium. All experiments were performed in serum-free medium. Conditions of cell treatment are as indicated in the figure legends. All incubations were performed at 37°C, 5% CO2. During treatments, medium was renewed every 24 hours. When drugs (DAPT) were solubilized in DMSO, control cells received the same final dilution of vehicle (1:100,000).

Transfection by electroporation
Smooth muscle cells were transfected by electroporation in the Amaxa electroporation device using the D-33 program. Briefly, 1 million cells were resuspended in 100 µl of Amaxa electroporation transfection solution and 2 µg of Notch intracellular domain 1 or 3 expression vectors or 4 µg of other vectors (I{kappa}B{alpha}32-36, RBP-J{kappa} DN) were added. After electroporation, transfected cells were plated in two wells of a six-well plate, each containing 1 ml of cell culture medium. 24 hours after plasmid transfection, the cells were starved in serum-free medium. The following day, the cells were treated as indicated in the figure legends. Medium, RNA or proteins were collected as described below.

Measurement of PLA2 activity and PGE2 concentration
Secreted PLA2 activity was assayed on the cell culture medium using a fluorimetric assay as described previously (Couturier et al., 1999Go). Arbitrary fluorescence units were converted to picomoles of substrate by total hydrolysis of 1 nanomole of substrate, 1-hexadecanoyl-2-(1-pyrenldecanoyl)-sn-glycero-3-phosphoglycerol, with an excess of bee venom PLA2. PLA2 activity was normalized to the amount of total RNA extracted using Chomczynski and Sacchi's modified method (Chomczynski and Sacchi, 1987Go) and expressed as pmol/min/µg RNA. The secreted PGE2 concentration was assayed on the same samples of cell culture medium using an EIA system. Results are expressed as pg/ml of medium.

Immunocytochemistry experiments and immunoblots
Immunocytochemistry was carried out as described previously (Clément et al., 2006Go). Briefly, cells were incubated for 1 hour with primary antibody (mouse monoclonal anti-{alpha}-actin) (DakoCytomation, diluted 1:100 in PBS plus 3% BSA) followed by 20 minutes with the secondary antibody fluorescein isothiocyanate (FITC)-goat anti-mouse, diluted 1:300 in PBS. Cell nuclei were stained with Hoechst. Slides were then mounted with mowiol and analyzed by fluorescent microscopy. Images were obtained from an Axioskop Zeiss microscope (20x and 63x oil immersion Zeiss plan-neofluar objectives). Pictures were captured with a Canon powershot G5.

For immunoblots, total proteins were extracted in 50 mM Tris pH 7.9, 2 mM NaVO3, 10 mM Na pyrophosphate, 400 mM NaCl, 1% NP40 and a cocktail of proteases inhibitors. The protein concentration of each sample was determined using DC Protein Assay (Bio-Rad). Proteins were loaded on 6.5 or 10% SDS-PAGE and transferred to nitrocellulose membranes (Hybond, Amersham). After blocking with 5% nonfat dried milk for 1 hour, membranes were incubated overnight at 4°C with primary antibodies: mouse monoclonal anti-{alpha}-actin: 1:5000 (DakoCytomation), goat polyclonal anti-COX-2: 1:250 (Santa Cruz Biotechnology), rabbit polyclonal anti-I{kappa}B{alpha}: 1:1000 (Santa Cruz Biotechnology), rabbit polyclonal anti-cleaved Notch1 (Val1744): 1:1000 (Cell Signaling), mouse monoclonal anti-Flag: 1:1000 (Sigma Aldrich), goat polyclonal anti-GAPDH: 1:400 (Santa Cruz Biotechnology) and mouse monoclonal anti-beta-actin: 1:5000 (Sigma Aldrich). Appropriate secondary antibodies conjugated to horseradish peroxidase were used: 1:1000.

Reverse transcription and RT-PCR
Total RNA was isolated from confluent vascular smooth muscle cells using a RNeasy Mini kit according to the manufacturer's instructions. After annealing oligodT (1 µM) to template RNAs (0.5 µg) at 70°C for 5 minutes, primer extension was initiated by adding the 1 unit (U) RT-MMLV enzyme plus 0.5 mM dNTP, 1 U RNAsin and 10 mM dithiothreitol, and carried out for 45 minutes at 37°C. PCR amplification was performed using a LightCycler instrument (Roche Diagnostics) as previously described (Clément et al., 2006Go). Results are expressed as relative induction (versus control) after normalization with the level of mRNA encoding hypoxanthine phosphoribosyltransferase (HPRT). The single-stranded oligo-deoxynucleotide primers used to selectively amplify the genes encoding Hes1, HRT1, HRT2, Jagged1, Notch1, Notch2, Notch3, {alpha}-actin, SM-22, myocardin, calponin, PLA2-IIA, COX-2, I{kappa}B{alpha} and HPRT are presented in Table 1. When RT-PCR was used to screen cDNA expression in transfected cells, the resulting PCR products obtained after 30 cycles were resolved on a 2% agarose gel containing 0.5 µg/ml ethidium bromide and DNA bands were visualized by UV fluorescence. Gels are shown as negative images of agarose gels stained with ethidium bromide. The forward and reverse primers used to selectively amplify the cDNAs encoding mouse Notch1 intracellular domain, mouse Notch3 intracellular domain and human RBP-J{kappa} DN are presented in Table 1.


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  		Table 1. Summary of PCR primers

 

Statistical analysis
Data are reported as means ± s.e.m. Unpaired Students' t-test was used to compare the mean values between groups with the GraphPad InStat version (GraphPad Software, San Diego, CA).


   Acknowledgments
 
We thank Anne-Sophie Le Port for her precious technical contribution. We are grateful to P. Lazarow and M. Raymondjean for helpful discussions. We thank G. Weinmaster for authorizing us to use HEK 293 T cells transfected with the construct encoding Delta1-Fc.


   Footnotes
 
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/120/19/3352/DC1

* These authors contributed equally to this work Back


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 Introduction
 Results
 Discussion
 Materials and Methods
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