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Notch 1 and 3 receptor signaling modulates vascular smooth muscle cell growth, apoptosis, and migration via a CBF-1/RBP-Jk dependent pathway

CATHERINE SWEENEY^*, DAVID MORROW^*, YVONNE A. BIRNEY^*, SEAMUS COYLE^*, COLM HENNESSY^*, AGNIESZKA SCHELLER^*, PHILIP M. CUMMINS^*, DERMOT WALLS, EILEEN M. REDMOND and PAUL A. CAHILL^*,1

^* Vascular Health Research Centre, Faculty of Science and Health,
School of Biotechnology and the National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland; and
Department of Surgery, University of Rochester Medical Center, Rochester, New York, USA

¹Correspondence: Vascular Health Research Centre, Faculty of Science and Health, Dublin City University, Dublin 9, Ireland. E-mail: paul.cahill{at}dcu.ie

SPECIFIC AIMS

Vascular smooth muscle cell (SMC) fate decisions (cell growth, migration, and apoptosis) are fundamental features in the pathogenesis of vascular disease. The aim of the study was to examine the role of activated Notch 1 and Notch 3 receptors (Notch IC, the cleaved intracellular domains) in controlling SMC fate in vitro. Using adult rat SMC in culture, we examined the role of CBF-1/RBP-Jk-dependent gene expression in mediating the effects of Notch 1 and 3 IC signaling on SMC growth, apoptosis, and migration.

PRINCIPAL FINDINGS

1. Notch 1 and 3 IC receptors activate notch target genes in a CBF-1-dependent manner in SMC
Assessment of Notch signaling in rat SMC in vitro using Western blot analysis, luciferase reporter assays, and real-time PCR revealed that Hairy Enhancer of Split (hes-1 and -5) and related hrt’s (hrt-1, -2, and -3) are direct downstream target genes of Notch 1 and 3 IC in SMC and identified an essential role for the nuclear protein CBF-1/RBP-Jk in their regulation. Overexpression of constitutively active Notch 1 and 3 IC resulted in a significant up-regulation of CBF-1/RBP-Jk-dependent promoter activity and Notch target gene expression, an effect that was blocked after coexpression with delta RAM deleted Notch IC (mNotch 1 IC), the Epstein Barr virus encoded gene product, RPMS-1, which inhibits CBF-1/RBP-Jk transcriptional activity, and after treatment with pharmacological inhibitors of Notch IC receptor trafficking from Golgi apparatus brefeldin A and monensin. Inhibition of endogenous Notch IC-dependent, CBF-1/RBP-Jk-regulated gene expression using pharmacological and molecular interventions resulted in a significant inhibition of Notch 1 and 3 IC-dependent Notch target gene promoter activity and mRNA levels.

2. Notch 1 and 3 receptor activation promotes SMC proliferation in vitro in a CBF-1/RBP-Jk-dependent manner
Serum stimulation of quiesced SMC caused a temporal increase in Notch target gene expression and CBF-1/RBP-Jk-dependent promoter activity, an effect that was also inhibited with RPMS-1, mNotch 1 IC, brefeldin A, and monensin. The functional importance of Notch 1 and 3 IC expression on SMC fate was confirmed by determining the effects on SMC growth. In quiesced cells, there was a significant temporal decrease in serum-stimulated SMC proliferation after inhibition of CBF-1/RBP-Jk-dependent gene expression with RPMS-1 (28±2%, P<0.05 n=3). Constitutive expression of Notch 1 and 3 IC significantly increased RVSMC proliferation (47.6±5% and 49±5%, P<0.05 n=3, respectively) and the expression of proliferating cell nuclear antigen expression (pCNA), an effect also significantly inhibited by coexpression with RPMS-1. This increase in serum-stimulated RVSMC proliferation was mirrored in clonal proliferation assays where Notch 1 and 3 IC promoted clonal proliferation, an effect also inhibited by coexpression of RPMS-1.

3. Notch 1 and 3 receptor activation attenuates RVSMC apoptosis in vitro in a CBF-1/RBP-Jk-dependent manner
As SMC growth reflects the balance between cell proliferation and death, the functional importance of Notch 1 and 3 IC expression on SMC fate was further confirmed by determining their effects on SMC apoptosis (Fig. 1 a). There was a significant increase in annexin V binding in the subpopulation of propidium iodide-negative cells after inhibition of CBF-1/RPB-Jk-dependent Notch signaling with RPMS-1 (4.3±2.7 to 16.2±1.4% apoptotic nuclei, P<0.05 n=3, P<0.05). In a similar manner, constitutive expression of mNotch 1 IC or treatment with the pharmacological inhibitor brefeldin A resulted in a significant increase in annexin V binding (Fig. 1a ). When SMC apoptosis was induced after serum deprivation for 72 h, there was a significant increase in annexin V binding (2.5±0.6% vs. 67.8±4.5% apoptotic nuclei, P<0.05 n=3, P<0.05). Constitutive expression of Notch 1 and 3 IC resulted in a significant decrease in annexin V binding (38.5±2% and 39.2±3.8% apoptotic nuclei, P<0.05 n=3), a reduction that was significantly reversed after inhibition of CBF-1/RBP-Jk-dependent gene expression with RPMS-1 and mNotch 1 IC, respectively (Fig. 1b ). The inhibition of apoptosis by Notch IC was further confirmed using acridine orange (AO)/ethidium bromide staining and caspase-3 activity. Serum deprivation for 72 h resulted in a significant increase in the number of cells staining with AO (4±1% to 71±7% apoptotic nuclei, P<0.05 n=3); this effect was significantly reversed after constitutive expression of Notch 1 and 3 IC. Serum depletion was further associated with a significant increase in caspase 3 activity. Inhibition of CBF-1/RBP-Jk gene expression with RPMS-1 resulted in a significant increase in caspase 3 activity whereas constitutive expression of Notch 3 IC resulted in a significant decrease in caspase-3 activity in these cells.

View larger version (28K): [in this window] [in a new window]   Figure 1. Notch signaling inhibits SMC apoptosis through CBF-1/RBP-Jk signaling. a) Representative histochemical analysis and cumulative data on the % annexin V/propidium iodide-positive cells (apoptotic cells) in serum-stimulated SMC after inhibition of CBF-1/RBP-Jk activity with RPMS-1, delta RAM deleted Notch 1 IC (mNotch 1 IC), and brefeldin A (0.1 mg/mL, 24 h). b) Representative histochemical analysis and cumulative data on the % annexin V/propidium iodide-positive (apoptotic cells) in serum-deprived SMC after constitutive expression of Notch 3 IC (CMX-Notch 3 IC) with or without coexpression of RPMS-1 and the delta RAM deleted Notch 1 IC (mNotch 1 IC).

4. Notch 1 and 3 receptor activation attenuates RVSMC migration in vitro in a CBF-1/RBP-Jk-dependent manner
A functional role for Notch IC in controlling SMC migration was examined. At lower cell densities, constitutive expression of Notch 1 and 3 IC failed to significantly alter the low level of baseline migration. However, inhibition of CBF-1/RBP-Jk-dependent signaling with RPMS-1 significantly enhanced SMC migration, which was reversed after coexpression with Notch IC (Fig. 2 a). At higher densities, where significant baseline migration was observed, constitutive expression of Notch 1 and 3 IC resulted in a significant inhibition of baseline migration. Moreover, inhibition of CBF-1/RBP-Jk signaling with RPMS-1 significantly enhanced baseline SMC migration, an effect that was reversed after constitutive expression of Notch 1 and 3 IC (Fig. 2b ).

View larger version (68K): [in this window] [in a new window]   Figure 2. Notch signaling inhibits SMC migration through CBF-1/RBP-Jk signaling. a) Stimulation of SMC transmigration after constitutive expression of Notch 1 IC or inhibition of CBF-1/RBP-Jk gene expression with RPMS-1. Representative transwell migration assay on the number of cells seeded at 1 x 104 cells/50 mm2 that migrated through the filter per 4 hpf after constitutive expression of Notch 1 IC with or without coexpression of RPMS-1. Cumulative data on the number of cells seeded at 1 x 104 cells/50 mm2 migrated through the filter/4 hpf after constitutive expression of Notch 1 and 3 IC with or without coexpression of RPMS-1 and the delta RAM deleted Notch 1 IC (mNotch 1 IC). b) Representative transwell migration assay and cumulative data on number of cells seeded at 2 x 104 cells/50 mm2 that have migrated through the filter per 4 hpf after constitutive expression of Notch 3 IC with or without coexpression of RPMS-1. Data represent mean values from 3 independent experiments ±SE, *P < 0.05 compared with mock controls, ¶P < 0.05 vs. RPMS-1 alone.

CONCLUSIONS AND SIGNIFICANCE

In the present study, substantial evidence is provided that Notch 1 and 3 IC receptors control SMC fate in vitro through CBF-1/RBP-Jk pathways. We established the role of Notch 1 and 3 IC signaling in controlling adult SMC fate by demonstrating that Hairy Enhancer of Split (hes-1 and -5) and related hrt’s (hrt-1, -2, and -3) are direct downstream target genes of Notch in SMC and identified an essential role for nuclear protein CBF-1/RBP-Jk in their regulation. Constitutive expression of Notch 1 and 3 IC resulted in a significant up-regulation of CBF-1/RBP-Jk-dependent promoter activity and Notch target gene expression concomitant with significant increases in SMC growth while concurrently inhibiting SMC apoptosis and migration. Moreover, inhibition of CBF-1/RBP-Jk-regulated gene expression resulted in a significant decrease in cell growth while concomitantly increasing SMC apoptosis and migration.

Recent evidence has demonstrated that the Notch pathway is involved in multiple aspects of vascular development including proliferation, endothelial migration, smooth muscle differentiation, angiogenic processes, and arterial-venous differentiation. Whereas previous studies have demonstrated that Notch 3 IC receptors and hrt-1 control SMC growth in stably transfected embryonic A7r5 cells and that Notch 3 IC is associated with enhanced CBF-1/RBP-Jk-dependent promoter activity in these cells, the current study establishes that inhibition of Notch activation of CBF-1/RBP-Jk-dependent signaling results in significant changes in SMC fate in adult SMC in vitro. In particular, our study describes for the first time the regulation of SMC growth, apoptosis, and migration by Notch 1 IC through CBF-1/RBP-Jk-dependent pathways. This work adds considerably to the work published on Notch 3 IC in SMC by defining a novel role for Notch 1 IC and 3 IC receptors in regulating SMC migration in vitro and extends our knowledge of the role of both receptors in controlling SMC growth and apoptosis in vitro (Fig. 3 ).

View larger version (23K): [in this window] [in a new window]   Figure 3. Schematic. Stimulation of Notch 1 and 3 IC receptors leads to activation of Notch target genes. Under resting conditions, Notch signaling is repressed with the repressor CBF-1/RBP-Jk complex inhibiting Notch target gene transcription. Upon activation, the Notch receptor translocates to the nucleus to de-repress target gene expression and drive changes in SMC fate.

These findings suggest that Notch receptors and downstream target genes control vascular cell fate in vitro. In light of our data on Notch 1 IC, Notch signaling therefore represents a novel therapeutic target for disease states in which changes in vascular cell fate occur in vivo.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1700fje; doi: 10.1096/fj.04-1700fje

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