Cellular Biology

A Receptor-Specific Function for Notch2 in Mediating Vascular Smooth Muscle Cell Growth Arrest Through Cyclin-dependent Kinase Inhibitor 1BNovelty and Significance

Joshua M. Boucher, Anne Harrington, Bahman Rostama, Volkhard Lindner, Lucy Liaw
https://doi.org/10.1161/CIRCRESAHA.113.301272
Circulation Research. 2013;113:975-985
Originally published September 26, 2013

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Abstract

Rationale: Deregulated vascular smooth muscle cell (VSMC) proliferation contributes to multiple vascular pathologies, and Notch signaling regulates VSMC phenotype.

Objective: Previous work focused on Notch1 and Notch3 in VSMC during vascular disease; however, the role of Notch2 is unknown. Because injured murine carotid arteries display increased Notch2 in VSMC as compared with uninjured arteries, we sought to understand the impact of Notch2 signaling in VSMCs.

Methods and Results: In human primary VSMCs, Jagged-1 (Jag-1) significantly reduced proliferation through specific activation of Notch2. Increased levels of p27kip1 were observed downstream of Jag-1/Notch2 signaling and were required for cell cycle exit. Jag-1 activation of Notch resulted in increased phosphorylation on serine 10, decreased ubiquitination, and prolonged half-life of p27kip1. Jag-1/Notch2 signaling robustly decreased S-phase kinase–associated protein, an F-box protein that degrades p27kip1 during G1. Overexpression of S-phase kinase–associated protein before Notch activation by Jag-1 suppressed the induction of p27kip1. Additionally, increased Notch2 and p27kip1 expression was colocalized to the nonproliferative zone of injured arteries as indicated by co-staining with proliferating cell nuclear antigen, whereas Notch3 was expressed throughout normal and injured arteries, suggesting Notch2 may negatively regulate lesion formation.

Conclusions: We propose a receptor-specific function for Notch2 in regulating Jag-1–induced p27kip1 expression and growth arrest in VSMCs. During vascular remodeling, colocalization of Notch2 and p27kip1 to the nonproliferating region supports a model where Notch2 activation may negatively regulate VSMC proliferation to lessen the severity of the lesion. Thus, Notch2 is a potential target for control of VSMC hyperplasia.

Introduction

Vascular smooth muscle cells (VSMCs) exhibit tremendous phenotype plasticity in response to injury, remodeling, sheer stress, and other environmental cues.1 VSMC express contractile proteins necessary for maintaining vessel tone and function, including α-smooth muscle actin (SM actin), calponin1, SM22α, smooth muscle myosin heavy chain, and smoothelin B. In response to vascular injury, VSMCs downregulate contractile proteins and begin proliferating and migrating in response to secreted cytokines, potentially leading to the development and progression of neointimal hyperplasia, pulmonary hypertension, and other vascular diseases.2

An intact Notch pathway is critical for normal vascular development and remodeling in response to vascular pathology.3 Notch3 was characterized as a major regulator of VSMC development4 and is mutated in human cerebral autosomal dominant arteriopathy with sub-cortical infarcts and leukoencephalopathy.5 We now understand that signaling through Notch2 and Notch1 also activate pathways required during embryonic vascular development and vascular repair. For example, mice with a homozygous hypomorphic mutation in Notch2 undergo abnormal development of the heart and eye vasculature,6 and Notch2 has been implicated in the development of aortic VSMC.7 Both Notch2 and Notch3 are required for normal VSMC development during embryogenesis where they play compensatory, yet nonoverlapping, roles.8 Several reports have investigated potential functions of Notch or Jagged-1 (Jag-1) in neointimal lesion formation during pathological remodeling in response to injury9 and demonstrated their regulation of VSMC phenotype. Interestingly, however, there have not been unique signaling functions identified downstream of Notch1, Notch2, or Notch3 in VSMCs.

Our work using human VSMCs identifies a novel, Notch2-specific signaling role in the regulation of VSMC proliferation. Although human VSMCs additionally express Notch1 and Notch3, and all can interact with Jag-1, only Notch2 signals to mediate cell cycle exit. We propose that this specific cell cycle regulatory pathway mediated by Jag-1 interaction with Notch2 is an important negative regulatory mechanism to prevent excessive VSMC proliferation in injured arteries.

Methods

An expanded Methods section is available in the Online Data Supplement.

Surgical Procedures

All mouse studies were performed with strict adherence to protocols approved by the Institutional Animal Care and Use Committee at Maine Medical Center. For the arterial ligation model, 8-week-old FVB male mice were subjected to common carotid artery ligation.10 Control mice received a sham operation. Carotid arteries were collected after 14 days.

Immunohistochemistry

Paraffin-embedded mouse carotid arteries were cut into 5 μm sections immediately adjacent to the site of injury. Sections were rehydrated, and antigen retrieval was performed using citric acid buffer and heat, permeabilized with 1% Triton X-100 for 30 minutes and blocked for 2 hours in a solution of 2% bovine serum albumin and 2% goat serum. Antibodies against Notch1, Notch2, Notch3, p27kip1 (Cell Signaling), PCNA (Santa Cruz), SM-actin (Sigma), or platelet-endothelial cell adhesion molecule 1 (PECAM1, CD31, from Abcam) were diluted 1:200–1:500 and incubated with sections overnight at 4°C. After washing in Tris-buffered saline containing 0.01% tween-20 (TBS-T, pH=7.4), sections were either incubated with HRP-conjugated goat antirabbit IgG for 2 hours, reacted with diaminobenzidine and counterstained with hematoxylin, or incubated with Alexa-fluor conjugated secondary antibodies (Invitrogen) for 2 hours, washed with TBS-T, counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and coverslipped.

Cell Culture

Human primary aortic VSMCs (Lonza) were used between passages 5 and 7. Human pulmonary arterial VSMCs and coronary artery VSMCs (Lonza) were used at passage 5. To activate Notch, VSMCs were plated on dishes precoated with 3 μg recombinant rat Jag-1 fused to human Fc (R&D Systems) or with a human Fc control protein (Millipore) as described.11,12 Small-interfering RNAs or scrambled control (Qiagen) were transfected into VSMCs using the Amaxa nucleofector.12

Cell Cycle Analysis

Human aortic VSMCs were harvested by trypsinization, spun down, and washed in PBS before resuspension in ice-cold 70% ethanol and incubation at −20°C overnight. The next day, the cells were centrifuged, washed in ice-cold PBS, and resuspended in MUSE cell cycle reagent (Millipore), a propidium iodide-based staining kit compatible with the MUSE cell analyzer. DNA content was analyzed using the MUSE cell analyzer.

Statistical Analysis

F-scores were generated for experiments containing multiple comparisons using ANOVA. Student’s 2-tailed t test was used for pairwise analysis. Statistical significance was considered at P<0.05.

Results

Notch2 Expression Is Increased in VSMC of Remodeling Arteries

To determine the levels of Notch receptors in VSMCs of normal and injured vessels, we used the carotid artery ligation model as a reproducible means to generate neointimal lesion formation.10 Carotid arteries from 8-week-old FVB male mice were studied 14 days after left carotid artery ligation or sham surgery. Expression of Notch3 was localized to the media of sham arteries, whereas Notch1 and Notch2 were undetectable (Figure 1A, left columns). Consistent with previous studies,13 vascular injury resulted in robust upregulation of Notch2, predominantly localized to the medial VSMCs (arrowheads). Notch3 expression was high in both the medial and neointimal VSMCs, whereas Notch1 was marginally elevated 14 days after vascular injury (Figure 1A, right columns). Cells with increased Notch2 protein in the ligated artery were also positive for smooth muscle actin and SM22α, markers of VSMCs (data not shown). This expression pattern in injured arteries suggests an enhanced function for Notch2 in response to vascular remodeling.

Figure 1.
Figure 1.

Notch2 is produced in remodeling vascular smooth muscle cells (VSMCs) and responsive to Jag-1 ligand. A, Immunohistochemical analysis of Notch1, Notch2, or Notch3 receptors in sham control and injured murine carotid arteries 14 days after ligation. Slides were counterstained with hematoxylin. NC indicates, nonimmune control; IEL, internal elastic lamina; and EEL, external elastic lamina. Scale bar=100 μm for columns 1 and 3, and 50 μm for columns 2 and 4, n=5 per group. B, Immunoblot of whole-cell lysates for the intracellular domain (ICD) of Notch1 (N1ICD), Notch2 (N2ICD), or Notch3 (N3ICD) in human aortic VSMCs plated on Fc or Jag-1 Fc for 6 hours. C, VSMCs were plated on Fc or Jag-1 Fc for 15 hours, and Notch2 was analyzed using immunofluorescence (green). DAPI and phalloidin were used to visualize cell nuclei and actin filaments, respectively. Scale bar=50 μm.

Previous studies found that Jag-1 activation of Notch3 in VSMCs leads to maturation and quiescence.14 To determine whether Jag-1 also signals through other Notch receptors, we activated VSMCs with recombinant Jag-1 fused to a human Fc domain12 and analyzed whole-cell lysates by immunoblot for Notch. Notch1, Notch2, and Notch3 were detected in cultured human aortic VSMCs; however, only Notch2 and Notch3 intracellular domains (ICD) were increased by stimulation with Jag-1 as compared with Fc (Figure 1B). Notch2 activation after Jag-1 stimulation was further verified by immunostaining (Figure 1C). Before ligand treatment, Notch2 was localized to the cell membrane (arrowheads) but was predominantly nuclear after Jag-1 stimulation. These experiments confirm accumulation of Notch2 in VSMCs after vascular injury and its expression and activation in cultured human aortic VSMCs.

Jag-1-Selective Activation of Notch2 Is Required to Inhibit VSMC Proliferation

Proliferation of VSMCs contributes to neointimal hyperplasia during pathological remodeling, and antiproliferative agents have proven efficacious in reducing restenosis.15 We previously reported that Jag-1 activation of Notch receptors in VSMCs significantly reduced cell proliferation in addition to inducing differentiation.12 To identify the receptor mediating the cell proliferation effect, we silenced Notch1, Notch2, or Notch3 in VSMCs using small-interfering (si) RNA. Confirmation of Notch1, Notch2, or Notch3 knockdown was performed by immunoblot analysis for ICD compared with nontargeting RNA (ntRNA) control (Figure 2A). We found each siRNA to reduce its Notch target specifically and effectively. We then analyzed Notch target gene Hes1 by quantitative reverse transcription polymerase chain reaction after Jag-1 stimulation to validate suppression of Notch signaling (Online Figure IA–C). Knockdown of each Notch receptor significantly reduced the level of Hes1 transcript induced by Jag-1 stimulation. Finally, we assessed the effect of Notch knockdown on the VSMC differentiated phenotype. Jag-1–induced SM-actin transcripts were significantly reduced with knockdown of Notch1, Notch2, or Notch3 (Online Figure ID). Additionally, reduction in the basal levels of Notch2 and Notch3 was sufficient to reduce the ability of cells to contract a collagen gel (Online Figure IE).

Figure 2.
Figure 2.

Jag-1-selective activation of Notch2 is required to inhibit VSMC proliferation. A, Human aortic vascular smooth muscle cells (VSMCs) were transfected with 200 pmol of nontargeting RNA (ntRNA), or siNotch, siNotch2, or siNotch3, and whole-cell lysates were collected 48 hours later for immunoblot analysis of each Notch intracellular domain (ICD) to validate specificity and efficiency of knockdown. VSMCs with selective Notch receptor knockdown were plated on Fc or Jag-1 Fc for 42 hours, then pulsed with 10 μM 5-bromo-2′-deoxyuridine (BrdU) for 6 hours. Cells were processed for immunostaining to detect and quantify the percentage of BrdU-positive nuclei in Notch1 (B), Notch2 (C), or Notch3 (D) knockdown cells. Graphed are means±SD. NS indicates not significant. (E and F) VSMCs transfected with ntRNA, siNotch1, siNotch2, or siNotch3 probes were plated on Fc or Jag-1 Fc, and whole-cell lysates were collected after 48 hours for immunoblot to detect phospho-histone H3 (p-H3). Quantification of p-H3 protein relative to β-tubulin is shown (F). G, Representative phase micrographs of these VSMCs are shown after stimulation with either Fc or Jag-1 Fc for 48 hours. Scale bar=200 μm.

VSMCs transfected with ntRNA or siRNA probes against Notch1, Notch2, or Notch3 were activated with Jag-1 Fc for 48 hours and analyzed for cell proliferation. Cells were pulsed in the last 6 hours on ligand with 5-bromo-2′-deoxyuridine (BrdU) to label cells undergoing DNA synthesis. As previously reported, Jag-1 Fc decreased cell proliferation (Figure 2B–D). Although inhibition of Notch1 (Figure 2B) and Notch3 (Figure 2D) did not change this, knockdown of Notch2 inhibited this suppression as compared with Fc control (Figure 2C). We also assessed levels of phosphorylated histone H3 on serine 10 (p-H3), a marker of mitotic cells,16 in Notch knockdown VSMCs activated with Jag-1 Fc. Consistent with BrdU experiments, p-H3 levels were reduced by Jag-1 in control, Notch1 and Notch3 knockdown cells as compared with Fc; however, no change was observed in Notch2 knockdown cells (Figure 2E and 2F). The suppression in cell proliferation correlated with cell number. Cells transfected with ntRNA, siNotch1, or siNotch3 had significantly reduced cell number, whereas transfected siNotch2 populations had high cell density (Figure 2G). These data show that Jag-1 signals exclusively through Notch2 to inhibit VSMC proliferation in vitro.

Nuclear Notch2 ICD Is Downregulated During Entry Into S-Phase

Because Jag-1–specific activation of Notch2 is required to inhibit VSMC proliferation, we analyzed whether endogenous Notch2ICD expression varies during cell cycle progression. We used propidium iodide (PI) staining of total DNA content and analyzed the cells to quantify proportions in different phases of the cell cycle. To validate our system, VSMCs were plated on Fc or Jag-1 Fc for 48 hours, and the cell cycle was analyzed using PI staining (Online Figure IIA). Quantification of cells activated by Jag-1 Fc as compared with Fc revealed ≈14% increase in the G0/G1 population, whereas the S-phase and G2/M populations were reduced by ≈5% and ≈7%, respectively (Online Figure IIB). To study Notch2ICD expression throughout cell cycle progression, VSMCs were serum starved for 30 hours to synchronize the cells in G017 and then released using freshly prepared SmGM medium. Cells were harvested at 0 (30 hours starvation time point), 12, 15, 18, 24, and 30 hours after releasing and were simultaneously processed for cell cycle analysis (Figure 3A–G) or nuclear and cytoplasmic fractionation for Notch2ICD levels (Figure 3H and 3I). Nuclear and cytoplasmic levels of Notch2ICD were at their lowest from 12 to 15 hours after release, concomitant with entry of the G0/G1 population into S-phase (Figure 3G). At 18 hours after release, the S-phase population began moving into G2/M, and simultaneous upregulation of nuclear Notch2ICD was observed (Figure 3I, blue line). After increased nuclear Notch2ICD expression at 18 hours, the population of cells in S-phase rapidly and steadily declined until 24 hours. Nuclear Notch2 steadily decreased through 30 hours as the cells normalized their proliferation rates. Steadily decreasing Notch2ICD coincided with a steady increase in Notch2ICD in the cytoplasm, suggesting nuclear export of the protein after transition of the population from S-phase to G2/M at 18 hours. Thus, nuclear Notch2ICD in VSMC changes during progression through the cell cycle, is lowest during entry into S-phase, and peaks during exit from S-phase.

Figure 3.
Figure 3.

Nuclear Notch2 intracellular domain (ICD) is downregulated during entry into S-phase. Vascular smooth muscle cells (VSMCs) at passage 5 were synchronized in serum-free smooth muscle basal medium (SmBM) for 30 hours to induce G0. Freshly prepared smooth muscle growth media were added, and the cells were harvested for cell cycle analysis at 0 (A), 12 (B), 15 (C), 18 (D), 24 h (E), and 30 hours (F). G, Graphical representation of the percentage of cells in G0/G1, S, and G2/M at each time point was analyzed. Graphed are means±SD. H, VSMCs were harvested at the same time points after release from starvation and fractionated into their nuclear (left) and cytoplasmic (right) constituents for immunoblot analysis of Notch2ICD. Total histone H3 and pyruvate kinase PKM 1/2 were used as loading controls for nuclear and cytoplasmic fractions, respectively. Notch2ICD was normalized to loading control and quantified for each time point (I). Graphed are means±SD.

Selective Regulation of p27kip1 by Jag-1/Notch2 Signaling Inhibits VSMC Proliferation

To identify cell cycle regulatory proteins targeted by Jag-1 via Notch2, we analyzed p27kip1, p21cip1/waf1, cyclin E1, and its associated cyclin-dependent kinase 2 (CDK2), all important regulators of VSMC cell cycle.18,19 Although p21cip1/waf1 was slightly downregulated by activation with Jag-1 Fc for 48 hours, p27kip1 levels doubled (Figure 4A). Additionally, Jag-1 Fc activation inhibited expression of cyclin-dependent kinase 2 and cyclin E1. One function of p27kip1 is to bind cyclin E1/cyclin-dependent kinase 2 complexes and prevent cell cycle progression.20 To determine whether Jag-1 Fc promotes increased nuclear levels of p27kip1, we stimulated VSMC with Jag-1 Fc or Fc for 48 hours before fractionating the cells into nuclear and cytoplasmic components. Immunoblot analysis to detect p27kip1 protein showed increases in both nuclear and cytoplasmic levels in response to Jag-1 Fc (Figure 4B and 4C), suggesting that enhanced nuclear p27kip1 expression may mediate the cell cycle inhibitory effects.

Figure 4.
Figure 4.

Selective regulation of p27kip1 by Jag-1/Notch2 signaling inhibits vascular smooth muscle cell (VSMC) proliferation. A, VSMCs were plated on Fc or Jag-1 Fc for 48 hours, and cell lysates were collected for immunoblot to detect total p27kip1, p21cip1/waf1, cyclin E1, and cyclin-dependent kinase 2 (CDK2). B, Cell lysates were also fractionated into a nuclear and cytoplasmic pool, and immunoblotted to detect total p27kip1. Pyruvate kinase (PKM1/2) and TATA box binding protein (TBP) were used as cytoplasmic and nuclear markers, respectively. C, Quantification of nuclear and cytoplasmic expression of p27kip1 in response to activation by Fc or Jag-1 Fc for 48 hours. D, VSMCs were transfected with 125 pmol of small-interfering targeting p27kip1 (si-p27kip1), and efficiency was analyzed by immunoblot (D and E). F, VSMCs transfected with nontargeting RNA or si-p27kip1 were plated on Fc or Jag-1 Fc for 42 hours before pulsing with 10 μM 5-bromo-2′-deoxyuridine (BrdU) for 6 hours and quantifying the percentage of BrdU-positive nuclei. G–J, Immunoblot analysis and quantification of p27kip1 levels after 48 hours activation by Fc or Jag-1 Fc in control, Notch1, Notch2, or Notch3 knockdown cells, respectively. Graphed are means±SD.

To determine whether p27kip1 is required for Jag-1 to suppress VSMC proliferation, we used an siRNA targeting p27kip1 (si-p27kip1) to suppress the induction by Jag-1 signaling. Quantification of knockdown efficiency showed that 125 pmol of si-p27kip1 reduced levels of total p27kip1 and p-p27kip1 S10 by ≈38% and 45%, respectively (Figure 4D and 4E). Phosphorylation of p27kip1 on S10 is known to promote its stability and considerably increase its half-life.21 Using this system, we seeded ntRNA and si-p27kip1–transfected VSMCs on Fc or Jag-1 Fc for 42 hours before pulsing with BrdU for 6 hours. Quantification of BrdU–positive nuclei showed a significant reduction in proliferation in ntRNA receiving cells plated on Jag-1 Fc at 48 hours as compared with Fc (Figure 4F), whereas even a moderate reduction in p27kip1 protein rescued the Jag-1–induced suppression of proliferation. These results were confirmed using propidium iodide staining in conjunction with cell cycle analysis (data not shown). These data show that the increase in p27kip1 is required for Jag-1 to suppress VSMC proliferation.

Because Notch2 selectively mediates Jag-1 signaling to reduce cell proliferation, we tested whether regulation of p27kip1 is also specific to Notch2. VSMCs were transfected with ntRNA, siNotch1, siNotch2, or siNotch3, and plated on Jag-1 Fc for 48 hours before harvesting to analyze p27kip1 protein. We found that Jag-1 Fc upregulated expression of p27kip1 in control, Notch1 and Notch3 knockdown cells but not in cells lacking Notch2 (Figure 4G–J). These data indicate that Jag-1 induces p27kip1 expression exclusively through Notch2 to promote cell cycle arrest.

Jag-1 Signaling via Notch2 Stabilizes p27kip1 Protein in VSMCs

Canonical Notch signaling involves cleavage and translocation of NotchICD to the nucleus where it alleviates repression of the transcriptional regulator C promoter binding factor-1 (CBF-1) to promote transcriptional activation. We evaluated the levels of p27kip1 transcript after 24- and 48-hour activation by Jag-1 in VSMCs. Using quantitative reverse transcription polymerase chain reaction, we did not observe any significant changes in p27kip1 mRNA levels after Jag-1 stimulation (Figure 5A). Another common level of p27kip1regulation is post-translational, including phosphorylation events at specific residues that affect protein stability. S10 phosphorylated p27kip1 is the primary form in G0/G1 cells, representing ≈70% of the phosphorylated protein.21 Importantly, S10 phosphorylation increases the stability of p27kip1 in vivo.22 In addition, p27kip1 can be phosphorylated on threonine (T)187, which promotes its turnover.23 To determine whether Jag-1 signaling affects levels of these phosphorylated forms, we plated VSMCs on Fc or Jag-1 Fc for 48 hours and analyzed whole-cell lysates for total and phosphorylated forms of p27kip1. Jag-1 activation resulted in increased p-p27kip1 S10, a decrease in p-p27kip1 T187 and, as anticipated, increased total p27kip1 at 48 hours (Figure 5B). A profile of decreased phosphorylation on T187 and increased phosphorylation on S10 is expected to increase the stability of the protein. To determine whether the overall increase in p27kip1 was indeed a result of protein stabilization, we measured its half-life. VSMCs were plated on Fc or Jag-1 Fc for 24 hours before adding 200 μM of final concentration cycloheximide (CHX) to inhibit de novo protein synthesis or vehicle (DMSO). Lysates were collected at 0, 8, or 15 hours to quantify p27kip1. Representative immunoblots (Figure 5C) indicated that the normal half-life of p27kip1 was within 8 hours. However, after Jag-1 Fc stimulation, the protein level was unchanged at 8 hours, and had an extended half-life, decreasing only by 15 hours. These data demonstrate that Jag-1 activation of Notch2 likely results in stabilization of the existing pool of p27kip1.

Figure 5.
Figure 5.

Jag-1 signaling via Notch2 stabilizes p27kip1 in vascular smooth muscle cell (VSMC). A, VSMCs were stimulated with Jag-1 Fc or Fc for 24 or 48 hours, and RNA were collected for quantitative reverse transcription polymerase chain reaction analysis of p27kip1 transcript. Graphed are the fold changes relative to Fc. B, Lysates were collected from VSMCs stimulated with Fc or Jag-1 Fc for 48 hours and analyzed by immunoblot for phosphorylated and total p27kip1. C, VSMCs were plated on Fc or Jag-1 Fc for 24 hours before addition of dimethyl sulfoxide (DMSO) or cycloheximide (CHX). Lysates were collected at indicated times. D–G, Control, Notch1, Notch2, or Notch3 knockdown cells were plated on Fc or Jag-1 Fc for 48 hours, and lysates were collected for immunoblot analysis of p-p27kip1 S10 (top) and respective quantification relative to β-tubulin (bottom). H, VSMCs were stimulated with Fc or Jag-1 Fc for 48 hours, and cell lysates were immunoprecipitated (IP) to pull down total p27kip1. Analysis of whole-cell lysates and remaining supernatant after p27kip1 IP confirms robust upregulation of p27kip1 by Jag-1 Fc at 48 hours and subsequent depletion of p27kip1 after IP (H, input and supernatant, respectively). I, Immunoprecipitated p27kip1 was analyzed by immunoblot for ubiquitin, and levels were normalized to immunoprecipitated p27kip1 for quantification (J). Graphed are means±SD.

We then tested whether increased levels of p-p27kip1 S10 stimulated by Jag-1 Fc was mediated via Notch2 signaling. Similar to total p27kip1 expression (Figure 4G–J) Jag-1 requires Notch2 to increase p-p27kip1 S10 protein. Suppression of Notch1 or Notch3 did not affect the ability of Jag-1 Fc to increase p-p27kip1 S10 (Figure 5D–G). Ubiquitination of p27kip1 marks the protein for turnover.24 Because of enhanced phosphorylation on S10 and prolonged half-life in response to Jag-1 Fc, we analyzed ubiquitination of p27kip1. VSMCs were activated with Jag-1 Fc or Fc for 48 hours before immunoprecipitation (IP) of p27kip1. Before and after IP, a small amount of whole-cell lysate from Fc and Jag-1 Fc conditions was analyzed by immunoblot for p27kip1 (Figure 5H, input and supernatant, respectively). Although significantly more p27kip1 was immunoprecipitated from Jag-1 activated cells as compared with Fc, relatively equal levels of ubiquitin were detected (Figure 5I). Normalization of ubiquitin to immunoprecipitated p27kip1 suggested an ≈70% reduction in ubiquitinated p27kip1 in response to activation by Jag-1 Fc (Figure 5J), further explaining the increased half-life of p27kip1 observed in Figure 5C. These experiments suggest that Jag-1/Notch2 signaling does not regulate p27kip1 by inducing de novo transcription, but instead, stabilizes the existing species by promoting extensive post-transcriptional modifications. Increased S10 phosphorylation and decreased ubiquitination likely account for enhanced p27kip1 stability and VSMC cell cycle arrest.

Jag-1/Notch2 Regulation of p27kip1 Is via Downregulation of Skp2

Skp2 is a potent regulator of p27kip1 levels via ubiquitination and proteosomal degradation.23 Notch signaling regulates Skp2 expression in T-cell acute lymphoblastic leukemia cell lines25 and cell cycle progression via Skp2-dependent regulation of p27kip1 in adult stem cells.26 Additionally, Skp2-mediated ubiquitination of p27kip1 regulates VSMC proliferation in culture and in response to vascular injury.27,28 In light of reduced p27kip1 ubiquitination (Figure 5I–J), and the regulation of p27kip1 by Skp2 in VSMC, we investigated whether Jag-1/Notch2 signaling regulates Skp2. VSMCs were stimulated with Jag-1 Fc for 24 and 48 hours, and Skp2 mRNA and protein levels were analyzed. Although no change in Skp2 transcript was apparent at either time (Figure 6A), Skp2 protein was robustly suppressed (Figure 6B). In Fc-stimulated cells, Skp2 expression was mainly nuclear, and although Jag-1 did not affect the localization of Skp2, it significantly reduced its levels after 24 and 48 hours (Figure 6C, arrowheads). Reduced Skp2 expression in the nucleus is consistent with increased nuclear p27kip1 (Figure 4B and 4C).

Figure 6.
Figure 6.

Jag-1/Notch2 regulation of p27kip1 is via downregulation of Skp2. Vascular smooth muscle cells (VSMCs) were plated on Fc or Jag-1 Fc for 24 and 48 hours and harvested for quantitative reverse transcription polymerase chain reaction (A) and immunoblot analysis for Skp2 (B). Graphed are the fold changes relative to Fc. C, To study localization of Skp2, VSMCs were treated as in (B) and processed for immunofluorescence analysis of Skp2 (green). F-actin and nuclei are stained in red (phalloidin) and blue (DAPI), respectively. Scale bar=25 μm. D, VSMCs were transfected with ntRNA, or small-interfering RNA (siRNA) targeting Notch1, Notch2, or Notch3, and plated on Fc or Jag-1 Fc for 48 hours. Cell lysates were collected for immunoblot of p27kip1 and Skp2. E, Protein levels from (D) were quantified relative to Fc. F–H, VSMCs were transduced with ≈2000vp/cell human adenovirus encoding Skp2. After 48 hours, whole-cell lysates were harvested for analysis of Skp2 and p27kip1 (F). Skp2 antibody was used to confirm overexpression. G, VSMCs were transduced as in (F), stimulated for 48 hours with Fc or Jag-1 Fc and analyzed by immunoblot for p27kip1. H, Quantification of p27kip1 relative to Fc from immunoblot in (G). Graphed are means±SD.

To determine whether Jag-1 regulates Skp2 expression via Notch2 exclusively, we plated control, Notch1, Notch2, or Notch3 knockdown cells on Fc or Jag-1 Fc for 48 hours and analyzed expression of Skp2 and p27kip1 by immunoblot (Figure 6D and 6E). Knockdown of Notch2 rescued suppression of Skp2 by Jag-1 observed in control, Notch1, and Notch3 knockdown cells. In addition, decreased Skp2 by Jag-1 was associated with increased p27kip1 under all conditions except when Notch2 receptors were silenced.

VSMC response to stimuli varies depending on the source from which they are derived and can even vary within the same artery because of differential origins during development.29 To determine whether Jag-1 regulation of Skp2 and p27kip1 is a common pathway in VSMC derived from other vascular beds, primary human pulmonary artery or coronary artery VSMC was plated on Fc or Jag-1 Fc for 48 hours and assessed for levels of p27kip1, p-p27kip1 S10, and Skp2 (Online Figure III). Consistent with human aorta-derived VSMCs, VSMCs from these sources responded to Jag-1 with increased total p27kip1, p-p27kip1 S10, and decreased Skp2 protein compared with Fc. Thus, Jag-1 regulation of Skp2 and p27kip1 may be a common pathway in human VSMCs from multiple origins. We also tested the effect of overexpression of a constitutively active Notch1ICD, Notch2ICD, or Notch3ICD on Skp2, p27kip1, and proliferation. Unlike the receptor-specific functions observed by endogenous activation of Notch by Jag-1, overexpression of V5-tagged constitutively active forms of Notch1, Notch2, or Notch3 caused increased p27kip1, decreased Skp2, and decreased proliferation as indicated by Ki67 staining (Online Figure IVA and IVB). These findings show that expression of high levels of constitutively active Notch can mask receptor-specific functions compared with ligand stimulation at a more physiologically relevant level. Notch signaling is also activated by the Dll ligands, and we tested the activity of Dll-1 in p27kip1 regulation. VSMCs were plated on Dll-1 Fc for 48 hours and analyzed by immunoblot. No discernible changes were observed in p27kip1 in response to Dll-1 (Online Figure IVC), suggesting that regulation is specific to Jag-1.

As a final evaluation, we asked whether Skp2 overexpression could hinder the induction of p27kip1 in Jag-1-activated VSMCs. A Skp2 adenoviral (Ad) expression construct30 was titrated to maintain expression while minimizing potential off-target effects (data not shown). A titer of ≈2000vp/cell resulted in robust expression of Skp2 as compared with green fluorescent protein (GFP) control but had minimal effect on endogenous p27kip1 levels (Figure 6F). The absence of endogenous Skp2 in the Ad-GFP samples is attributable to the short exposure time required to resolve a band in the Ad-Skp2 expressing cells (<5 s). Endogenous Skp2 was detected in Ad-GFP transduced cells with longer exposure times (≈30 s), however this resulted in overexposure of the Ad-Skp2 lane (data not shown). Using this approach, VSMCs were transduced with Ad-GFP or Ad-Skp2 (≈2000vp/cell), and given 24 hour recovery before stimulation with Fc or Jag-1 Fc for 48 hours. Immunoblot analysis of p27kip1 showed ≈4 fold induction by Jag-1 Fc in the Ad-GFP transduced cells that could be suppressed by using the expression construct to maintain Skp2 levels in the presence of Jag-1 stimulation (Figure 6G and 6H). This effect was also observed using 4000vp/cell (data not shown). Interestingly, Jag-1–stimulated Ad-Skp2 transduced cells still showed downregulation of Skp2 as compared with Fc, exemplifying the potent inhibitory action of Jag-1 on Skp2 levels. Thus, Jag-1 inhibits Skp2 levels in VSMC via activation of Notch2 exclusively, and suppression of Skp2 is required for Notch2-specific regulation of p27kip1.

Notch2 and p27kip1 Co-localize to the Non-proliferative Zone of Injured Carotid Arteries

Notch signaling is involved in regulating neointimal lesion formation in response to vascular injury.13,31 Although Notch2 is required for VSMC differentiation and maturation during development,7,8 its function in vascular injury is poorly understood. Because of our observations that Notch2 increases in response to vascular injury, and inhibits VSMC proliferation via regulation of p27kip1, we studied the expression and colocalization of Notch receptors and p27kip1 in injured and normal arteries. Wild-type, 8-week-old male FVB mice were subjected to complete ligation of the left common carotid artery directly adjacent to the bifurcation or a sham surgery. After 14 days, carotid arteries were harvested for immunohistochemistry analysis. Histological staining revealed extensive vascular remodeling characterized by neointima formation and reduced lumen size in the ligated artery compared with sham surgery (Figure 7A). At higher magnification, the subendothelial neointima appears ≈8 to 12 layers thick, and the medial layer is hypertrophic (Figure 7B).

Figure 7.
Figure 7.

Notch2 and p27kip1 co-localize to the non-proliferative zone of injured carotid arteries. Eight-week-old male FVB mice underwent complete ligation of the left common carotid artery (injury) or sham control surgery. Carotid arteries were harvested after 14 days, embedded in paraffin, and sectioned for H&E analysis (A). B, Higher magnification of control and injured arteries to visualize internal elastic lamina (IEL), intima (IN), neointima (NI), media (M), and adventitia (AD). Injured artery sections were processed immunofluorescence analysis of proliferating cell nuclear antigen (PCNA)/Notch1 (C), PCNA/Notch2 (D), PCNA/Notch3 (E), PCNA/p27kip1 (F), SMA/PECAM1 (G) or antibody control (H) and counterstained with DAPI to visualize nuclei. Green and red channels indicated to visualize individual protein stains, and were overlaid with DAPI to show colocalization in the artery. Dotted line separates regions of high and low PCNA staining, white arrows indicate high Notch2 and p27kip1 staining in the medial layer (n=3).

To study Notch and p27kip1 expression in the proliferative and nonproliferative regions of the remodeling artery, we used costaining with proliferating cell nuclear antigen (PCNA) to label proliferating cells.32 In injured arteries after 14 days, PCNA staining was predominantly localized to the neointimal VSMCs (Figure 7C–F, green channel, dotted white line marks the internal elastic lamina), whereas no PCNA positive cells were present in the sham arteries (Online Figure VA–D, green channel). Additionally, Notch1, Notch2, and p27kip1 expression was undetectable in sham arteries; however, prominent Notch3 levels were observed in the medial VSMCs (Online Figure VA–D). Staining for smooth muscle marker SM-actin and endothelial marker CD31 was performed to identify vessel structure and composition, and a negative control for antibody specificity was used (Online Figure VE and VF). In injured arteries, Notch1 was detectable in the endothelium and trace amounts in neointimal VSMCs (Figure 7C). In stark contrast to uninjured arteries, Notch2 levels were high in the medial VSMCs (Figure 7D, white arrows). Interestingly, Notch2 expression was high in the nonproliferating VSMC as indicated by staining in regions that were negative for PCNA staining (Figure 7D, overlay). Only trace amounts of Notch2 were detectable in the endothelium and neointimal VSMCs, whereas Notch3 was expressed throughout the injured vascular wall (Figure 7E). Similar to Notch2 protein, high levels of p27kip1 were localized to the medial VSMCs (Figure 7F white arrows) and outside of the proliferative zone. SM-actin and CD31 staining are shown to indicate cell type(s) and vessel structure (Figure 7G). This localization of Notch receptors is consistent with our model that Notch2 and p27kip1 are upregulated and colocalized to the nonproliferative VSMCs of the vascular wall after injury. Notch2 may be one regulator of p27kip1 expression in the injured vasculature that leads to re-establishment of vascular quiescence during remodeling.

Discussion

Proper Notch signaling is required for the maturation of the cardiovascular system during development, and in humans, mutations of components of the Notch pathway lead to vascular disease (reviewed in Boucher et al3). Quiescent VSMCs in vivo express high levels of Notch3 and Jag-1, whereas injury or pathology promotes expression of Notch1 and Notch2 within the VSMCs13 (Figure 1). The specific roles and signaling functions of each of the 4 Notch receptors is not well understood. Our study is the first to identify a Notch2-specific signaling function in human vascular cells, which when activated, is predicted to suppress smooth muscle hyperproliferation. Because of the association of impaired Notch signaling and vascular disorders, there is an appreciation for targeting the Notch pathway in the treatment of cardiovascular diseases.33 The most widely used Notch antagonist is γ-secretase inhibitor, which is being tested in clinical trials of patients with cancer. However, the lack of specificity of this enzyme for the Notch pathway34 presents a complex challenge when targeting diseases where multiple Notch receptors are active. Previous studies suggest that inhibition of some Notch pathways, including Notch1, may be effective in decreasing neointimal lesion formation.13,31 However, our findings suggest that selectively enhancing Notch2 function under conditions of VSMC hyperproliferation may promote cell cycle exit. Current attempts are indeed geared toward more selective targeting of individual Notch receptors.35 We suggest that it is critical to understand the roles of each Notch receptor in specific disease processes to successfully apply targeted therapeutic interventions.

We identified a specific requirement for Notch2 in negatively regulating VSMC proliferation downstream of Jag-1. Although cooperative roles can be shared between receptors, our data suggest Notch2-specific signaling roles that are unique (Figure 8). To our knowledge, this is the first study to identify a receptor-specific function for Notch2 in VSMCs. Notch2 is required for Jag-1–induced VSMC differentiation via targeting of Skp2 and p27kip1 to decrease cell proliferation (Figure 8). This cell cycle regulation is not mediated via either Notch1 or Notch3 receptors, although both of those receptors can respond to a Jag-1 signal. Thus, we hypothesize that one function of Notch2 is to provide crucial negative feedback on VSMC proliferation in response to vascular injury. Loss of differentiation of medial VSMCs and subsequent migration and proliferation to the subendothelial compartment in response to injury have been reported.36 Based on the in vitro mechanisms presented in this report, and the enhanced expression and colocalization of Notch2 and p27kip1 to medial VSMCs after injury, one could speculate that Notch2 activation antagonizes excessive proliferation of medial VSMCs to the neointimal layer, thereby acting to regulate lesion formation and vascular occlusion negatively.

Figure 8.
Figure 8.

Proposed model for Notch2-specific regulation of vascular smooth muscle cell (VSMC) proliferation during vascular remodeling. During vascular homeostasis, Notch2 is not present in arterial VSMCs, whereas Notch3 is primarily expressed. After vascular injury or the progression of disease, cytokine release and changes in the microenvironment lead to the induction of Skp2, which suppresses p27kip1. These factors contribute to loss of the contractile phenotype and VSMC proliferation. Notch2 is induced during this remodeling process in the VSMCs. Unlike Notch1 and Notch3, Notch2 expression colocalized with p27kip1 to the nonproliferative medial VSMCs. Our in vitro data support a mechanism whereby Jag-1 activation of Notch2 inhibits Skp2, resulting in reduced ubiquitination and decreased turnover of p27kip1 in VSMCs. As a result of increased stability, total p27kip1 and p-p27 S10 accumulate in the cell, inhibiting cyclinE/cyclin-dependent kinase 2 (CDK2) functions. We hypothesize that reduced expression of cyclinE/CDK2 is a result of increased p27kip1 levels and accounts for dramatically decreased levels of p-p27kip1 T187. This pathway is required for Jag-1 inhibition of VSMC proliferation in vitro. Notch2 function is required for the full VSMC maturation because suppression of either Notch2 or Notch3 suppresses their differentiated, contractile phenotype.

Little is known about the contributions of Notch2 signaling during VSMC development and in response to vascular injury. Proliferation of VSMCs derived from cardiac neural crest cells requires Notch2 signaling.7 This result is in contrast to our model that Notch2 suppresses proliferation in VSMCs from the adult injured vessel. It is possible that Notch2 proliferative signals are sensed differently in an embryonic vascular progenitor cell versus an adult differentiated VSMCs. Also embryonically, a delay in VSMC differentiation is observed in developing blood vessels of Notch2-deficient mice, and these effects are severely exacerbated by dual knockout of Notch2 and Notch38. Constitutive expression of Notch2 and Notch3 are in the neointimal and medial VSMCs after injury, and Notch1 expression is the highest in neointimal VSMCs.13 Although associated with many pathologies, pulmonary stenosis is often observed in patients with Allagile syndrome, caused by mutations in Jag-1 or Notch2 in humans,37 and is consistent with Jag-1/Notch2 negatively regulating VSMC proliferation. Although there are many overlapping functions for Notch receptors, their differences in expression in time and space in response to vascular injury suggest the possibility of distinct receptor-specific functions. The diverse origin of VSMC progenitors during development may also strongly influence the nonoverlapping functions of Notch receptors in VSMCs, and sensitivity to Notch2 signaling could vary during homeostasis or remodeling, and at different anatomic sites. Further identification of receptor-specific roles for Notch in large elastic arteries and smaller arterioles will be required to gain a more comprehensive picture of vascular function. Our findings are important in advancing our understanding of the molecular events contributing to arterial stenosis and other proliferative vascular disorders.

Acknowledgments

We are thankful for the donation of the Skp2 adenoviral expression construct generously provided by Dr. Dennis Bruemmer and colleagues at the University of Kentucky School of Medicine. We are also grateful for the resources provided by the core facilities at Maine Medical Center Research Institute in support of this project, including the Histopathology Core for tissue processing and sectioning, the Mouse Transgenic Facility for mouse surgical support, the Genomics and Bioinformatics Core for equipment for quantitative gene expression, and the Viral Vector Core for large-scale purification of our adenoviral expression constructs.

Sources of Funding

This work was supported by the National Institutes of Health1 (NIH) grants R01HL070865 and R01HL109652 (Dr Liaw). J. Boucher was partially supported by a Predoctoral Fellowship from the American Heart Association (10PRE3680003). The Mouse Transgenic Facility and the Viral Vector Core are supported by 5P30GM103392 (Principal Investigator: R. Friesel), and the Genomics and Bioinformatics Core is supported by 8P20GM103465 (Principal Investigator: D. Wojchowski). Both of these NIH Center of Biomedical Research Excellence awards jointly support the Histopathology Core.

Disclosures

None.

Footnotes

  • In July 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.24 days.

  • Nonstandard Abbreviations and Acronyms
    BrdU
    5-bromo-2′-deoxyuridine
    CBF-1
    C promoter binding factor-1
    DAPI
    4′,6-diamidino-2-phenylindole
    Jag-1
    Jagged-1
    NotchICD
    Notch intracellular domain
    Skp2
    S-phase kinase–associated protein
    VSMC
    vascular smooth muscle cell
    PCNA
    proliferating cell nuclear antigen

  • Received February 27, 2013.
  • Revision received August 20, 2013.
  • Accepted August 21, 2013.

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Novelty and Significance

What Is Known?

  • Jagged-1 activation of Notch signaling in vascular smooth muscle cells (VSMCs) promotes their differentiation and maturation.

  • The Notch pathway is reactivated in the vessel wall in response to injury and during vascular disease.

What New Information Does This Article Contribute?

  • Jagged-1 activates Notch1, Notch2, and Notch3 in human VSMCs; however, only Notch2 induces stabilization and nuclear accumulation of p27kip1, causing cell cycle exit.

  • S-phase associated kinase (Skp2) is suppressed by Jagged-1/Notch2 signaling in VSMCs and is required for the induction of p27kip1.

  • Notch2 is induced in VSMCs after vascular injury, and colocalizes with p27kip1 in nonproliferative regions of the remodeling artery.

Arterial vascular smooth muscle cells (VSMCs) contribute to vascular tone and homeostasis. During vascular obstructive diseases, VSMC hyperproliferation leads to lumen occlusion. Notch signaling regulates lesion formation after injury; however, specific signaling mechanisms for each of the 4 Notch proteins are not clear. We identified a unique function of Notch2 in VSMCs, downstream of Jagged-1 stimulation. We found that Notch2 specifically suppresses proliferation via induction of the cell cycle inhibitor p27kip1, a function that cannot be mediated via Notch1 or Notch3. The mechanism includes the suppression of Skp2, a negative regulator of p27kip1. This corresponds to the stabilization and nuclear accumulation of p27kip1, leading to reduced proliferation in VSMCs. During homeostasis, Notch2 is not expressed in the vessel wall but is induced after vascular injury. Unlike widespread expression of Notch1 and Notch3 in injured VSMCs, Notch2 is expressed in a subpopulation of VSMCs coincident with p27kip1in areas of nonproliferating VSMCs. These findings support the notion that selective activation of Notch2 in VSMCs suppresses proliferation and provide a potential means to reduce VSMCs hyperproliferation during vascular disease.

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  • A Receptor-Specific Function for Notch2 in Mediating Vascular Smooth Muscle Cell Growth Arrest Through Cyclin-dependent Kinase Inhibitor 1BNovelty and Significance
    Joshua M. Boucher, Anne Harrington, Bahman Rostama, Volkhard Lindner and Lucy Liaw
    Circulation Research. 2013;113:975-985, originally published September 26, 2013
    https://doi.org/10.1161/CIRCRESAHA.113.301272
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