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. 2015 Jun 26;290(26):16226-37.
doi: 10.1074/jbc.M115.655548. Epub 2015 May 8.

Differential Regulation of NOTCH2 and NOTCH3 Contribute to Their Unique Functions in Vascular Smooth Muscle Cells

Jeremy T Baeten  1 , Brenda Lilly  2
Affiliations

Differential Regulation of NOTCH2 and NOTCH3 Contribute to Their Unique Functions in Vascular Smooth Muscle Cells

Jeremy T Baeten et al. J Biol Chem. .

Abstract

Notch signaling is a key regulator of vascular smooth muscle cell (VSMC) phenotypes, including differentiation, proliferation, and cell survival. However, the exact contribution of the individual Notch receptors has not been thoroughly delineated. In this study, we identify unique roles for NOTCH2 and NOTCH3 in regulating proliferation and cell survival in cultured VSMCs. Our results indicate that NOTCH2 inhibits PDGF-B-dependent proliferation and its expression is decreased by PDGF-B. In contrast, NOTCH3 promotes proliferation and receptor expression is increased by PDGF-B. Additionally, data show that NOTCH3, but not NOTCH2 protects VSMCs from apoptosis and apoptosis mediators degrade NOTCH3 protein. We identified three pro-survival genes specifically regulated by NOTCH3 in cultured VSMCs and in mouse aortas. This regulation is mediated through MAP kinase signaling, which we demonstrate can be activated by NOTCH3, but not NOTCH2. Overall, this study highlights discrete roles for NOTCH2 and NOTCH3 in VSMCs and connects these roles to specific upstream regulators that control their expression.

Keywords: Notch receptor; apoptosis; differentiation; mitogen-activated protein kinase (MAPK); proliferation; vascular smooth muscle cells.

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Figures

FIGURE 1.
FIGURE 1.
NOTCH2, but not NOTCH3 receptor expression is decreased by PDGF-B. A and B, HAoSMCs were serum-starved for 48 h, then treated with 20 ng/ml PDGF-B and collected to isolate mRNA for qPCR (A), or protein for Western blot analysis (B), at designated timepoints. qPCR shown as expression relative to GAPDH, n = 5. NOTCH2 and NOTCH3 band intensity quantified relative to tubulin, n = 6. C, serum-starved HAoSMCs were treated with/without 20 ng/ml PDGF-B, and vehicle control, 5 μm PDGF-receptor inhibitor (PDGFRI), 10 μm MEK inhibitor (U0126), or 10 μm JNK inhibitor (JNKII) for 48 h. qPCR shown as expression relative to GAPDH, n = 4. Significance determined by one-way ANOVA. *, p < 0.05. n.s., not significant.
FIGURE 2.
FIGURE 2.
NOTCH2 and NOTCH3 have opposing effects on PDGF-B-dependent smooth muscle cell proliferation. HAoSMCs with siRNA knockdown of (si)NOTCH2 and (si)NOTCH3, or lentiviral overexpression of NOTCH2 (NICD2) and NOTCH3 (NICD3) intracellular domains. A–D, cells were serum-starved for 48 h and treated with 20 ng/ml PDGF-B for proliferation assays, n = 3. “*” indicates significant difference compared with GFP or siRNA (siControl) controls. Significance determined by two-way ANOVA, p < 0.05, ± S.E. E and F, protein extracts collected for Western blots after 24 h to measure proliferation marker MKI67 expression. Significance determined by one-way ANOVA, p < 0.05.
FIGURE 3.
FIGURE 3.
Genetic deletion of Notch2 or Notch3 has unique effects on proliferation of mouse aortic smooth muscle cells. SMCs were isolated from aortas of wild-type (WT), Notch2 (Notch2fl/fl; MCC+/−), and Notch3 (Notch3−/−)-deficient mice and used in methylene blue proliferation assays. A, comparison of WT and Notch2-null cells. B, comparison of WT and Notch3-null cells, n = 4. “*” indicates significant difference compared with WT in the same PDGF-B treatment group determined by two-way ANOVA, p < 0.05, ± S.E.
FIGURE 4.
FIGURE 4.
NOTCH3 is uniquely regulated by inducers of apoptosis. HAoSMCs were treated with increasing concentrations of H2O2 for 24 h or exposed to indicated levels of UV radiation and collected 12 h later for RNA and protein expression analysis. A and B, qPCR expression analysis of NOTCH2 and NOTCH3, shown relative to GAPDH, n = 3. C and D, Western blots of NOTCH2 and NOTCH3 expression with tubulin as a loading control. E, HAoSMCs were exposed to UV radiation and cultured for 12 h with 10 μm MG-132 proteasome inhibitor or with vehicle control. Western blots of NOTCH3 expression with tubulin as a loading control, n = 3. F, HAoSMCs with lentivirally overexpressed FLAG-tagged NICD2 or NICD3 were UV-irradiated and cultured for 9 h with 10 μm MG-132. Western blots to detect FLAG-tagged proteins, relative to tubulin, n = 3. Significance determined by one-way ANOVA, *, p < 0.05, n.s., not significant.
FIGURE 5.
FIGURE 5.
NOTCH3 protects smooth muscle cells from cell death. HAoSMCs with lentivirally overexpressed NICD2, NICD3, and GFP, or siRNA knockdown of NOTCH2 (siN2), NOTCH3 (siN3), and control (siCTL) were exposed to 50 μJ/cm2 UV radiation and collected for protein 9 h later. A and B, Western blots to detect NOTCH2 and NOTCH3 expression, and level of total and cleaved caspase3 with tubulin used as loading control, n = 3. C and D, caspase3 enzymatic activity assay using fluorescent caspase substrate AFC-DEVD, n = 4. Significance determined by one-way ANOVA; *, p < 0.05.
FIGURE 6.
FIGURE 6.
NOTCH3 promotes transcription of cell survival genes via activation of MAPK. HAoSMCs with lentiviral overexpression of GFP (control), NICD2 and NICD3, or siRNA knockdown of (si)NOTCH2, (si)NOTCH3 and control (siCTL). A, RNA expression of putative pro-survival genes were measured by qPCR with relative expression compared with GAPDH, n = 3. B, HAoSMCs with lentiviral-overexpressed NICDs were treated with 10 μm U0126 or vehicle control for 24 h, and RNA was isolated to measure pro-survival gene expression, n = 3. C and D, Western blots to measure total and phosphorylated (phospho)-ERK, with tubulin as loading control, n = 3. Significance determined by one-way ANOVA; *, p < 0.05.
FIGURE 7.
FIGURE 7.
Genetic deletion of Notch3 has unique effects on the survival of mouse aortic smooth muscle cells. A, mRNA expression of pro-survival genes in aortas of wild-type (WT), Notch2-deficient (Notch2fl/fl; MCC+/−), and Notch3-mutant (Notch3−/−) adult mice. qPCR with relative expression compared with GAPDH, n = 4. B, SMCs were isolated and cultured from aortas of wild-type, Notch2, and Notch3-deficient mice and used in enzymatic caspase3 activity assays, n = 4. C, total and phosphorylated (phospho)-ERK was measured in cultured smooth muscle cells following serum challenge, with tubulin used as loading control, n = 4. Significance determined by one-way ANOVA, *, p < 0.05.
FIGURE 8.
FIGURE 8.
NOTCH2 and NOTCH3 have unique roles in proliferation and cell survival related to their expression.
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