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J. Biol. Chem., Vol. 277, Issue 24, 21723-21729, June 14, 2002

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Notch3 Signaling in Vascular Smooth Muscle Cells Induces c-FLIP Expression via ERK/MAPK Activation

RESISTANCE TO Fas LIGAND-INDUCED APOPTOSIS^*

Wenli Wang, Chengyu Z. Prince, Yongshan Mou, and Matthew J. Pollman^§

From the Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, Georgia 30310

Received for publication, March 6, 2002, and in revised form, March 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mutations in the Notch3 receptor result in the cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephelopathy (CADASIL) syndrome, a heritable arteriopathy predisposing to early onset stroke. Based upon clinical evidence that CADASIL arteriopathy results in degeneration and loss of vascular smooth muscle cells (VSMC) from the arterial wall, we postulated that Notch3 signaling is a critical determinant of VSMC survival. We initially established that both transient and constitutive Notch3 signaling promoted VSMC survival in response to the proapoptotic Fas ligand (FasL). Resistance to FasL-induced apoptosis was associated with the induction of c-FLIP, a primary inhibitor of the FasL signaling pathway. We determined that Notch3's regulation of c-FLIP was independent of the activity of the classical DNA-binding protein, RBP-Jk, but dependent upon cross-talk activation of the ERK/MAPK pathway. We extended our observations to the in vivo context by determining a coordinate regulation of Notch3 and c-FLIP within the arterial wall in response to injury. Furthermore, we defined that expression levels of Notch3 and c-FLIP are coordinately up-regulated within the neointima of remodeled arteries. Taken together, these findings provide initial evidence that Notch3 signaling may be a critical determinant of VSMC survival and vascular structure by modulating the expression of downstream mediators of apoptosis via signaling cross-talk with the ERK/MAPK pathway.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

It is postulated that pathological changes in vessel structure seen in conditions such as hypertensive arteriopathy, atherosclerosis, and restenosis are induced in part by signaling pathways that govern cell growth, death, differentiation, and matrix production (1, 2). However, the factors that regulate these programs within the vasculature remain poorly defined.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephelopathy (CADASIL)¹ is a heritable syndrome characterized by a predisposition to stroke due to an underlying arteriopathy. This diffuse arteriopathy is characterized by prominent degeneration and eventual loss of VSMC from the vessel wall (3, 4). Genetic linkage analyses have documented mutations in Notch3 as the etiologic basis of the CADASIL syndrome (3-7). Furthermore, Notch3 expression is largely confined to VSMC in adulthood (7). These findings suggest that the Notch pathway may be an important determinant of VSMC fate and vascular structure in human health and disease.

The Notch family of receptors has been characterized as critical determinants of cell fate in a variety of organisms. In mice, Notch1 and Notch2 gene deletions are characterized by perturbations in organogenesis that result in embryonic lethality (8, 9). Mechanistic studies performed in cell culture models in other cell types indicate that the Notch pathway influences cell fate by regulating programs governing growth, apoptosis, and differentiation (10-14). However, the functional role of the Notch signaling pathway in VSMC in vitro and in vivo remains to be defined.

The Notch receptor family is activated via a proteolytic cleavage of the intracellular domain (IC) of Notch. In certain contexts, the Notch IC portion translocates to the nucleus together with Suppressor of Hairless (Su(H)) (mammalian orthologue, RBP-Jk/CBF-1). RBP-Jk provides DNA binding specificity through recognition of the consensus sequence, whereas Notch IC functions as an activation domain. In support of this notion, several studies have demonstrated the utility of overexpressing a dominant negative RBP-Jk (associates with Notch IC but lacks DNA binding) in the context of Notch IC expression to determine whether Notch-induced cellular events occur via an RBP-Jk-dependent or independent transcriptional pathway (15-17).

In the classic model, in response to Notch signaling, RBP-Jk activates transcription of basic helix-loop-helix transcription factors such as hairy-and-enhancer of split 1 (18) and the Hairy-related transcription factor (HRT) genes (19). Zebrafish embryos harboring a mutation in Gridlock, an orthologue of HRT2, show dramatic impairment of vascular formation (20). Furthermore, recent evidence has established that Gridlock expression is a critical determinant of arterial versus venous cell fate within the developing vasculature (21, 22).

In addition to the classical model of Notch signaling via RBP-Jk-dependent transcriptional events, Notch may engage other signaling cascades in a cross-talk fashion. Recent studies indicate that in certain contexts, the Notch signaling pathway may modulate Src and Ras signal transduction (23-25). However, the elucidation of Notch3 signaling via RBP-Jk-dependent versus signal transduction cross-talk and the activation of downstream target genes in adult VSMC remains to be defined.

Studies from our laboratory and several other laboratories have suggested that apoptosis may play an essential role in atherogenesis and vascular remodeling (26-28). VSMC apoptosis is a prominent feature of the response to injury and the consequent formation of the neointima. A growing body of evidence indicates that the selection and accumulation of intimal VSMC in a context involving a coordinate up-regulation of antiapoptotic genes and a down-regulation of proapoptotic mediators might be an essential survival mechanism for maintaining intimal lesion stability and progression over the long term course of vascular disease (29). Fas is ubiquitously expressed in various tissues including the vessel wall (30). Activation of Fas by its ligand (FasL) rapidly induces cell death through recruitment and activation of caspase-8 via the adapter protein Fas-associated death domain protein (31). c-FLIP competitively inhibits binding of caspase-8 to the Fas receptor complex, thus shuffling off the downstream Fas-signaling pathway. It is reported that c-FLIP is widely expressed in the normal vessel wall and may contribute to an apoptosis-resistant state of VSMC and that down-regulation of c-FLIP may render VSMC susceptible to apoptosis (32). c-FLIP is up-regulated in the intima and media after arterial injury and remodeling (33). Furthermore, there are additional reports suggesting that the extracellular signal-regulated kinase (ERK) cascade functions as a survival pathway by regulating c-FLIP expression (34-36).

The present study tested the hypothesis that Notch3 signaling is a critical determinant of VSMC survival. We employed both in vitro and in vivo model systems to investigate the relationship between the Notch3 signaling pathway and the inhibition of FasL-induced apoptosis in VSMC and define downstream mediators responsible for the survival-promoting function. In accord with this hypothesis, our findings indicate that Notch3 signaling inhibits FasL-induced cell death through the up-regulation of c-FLIP via an ERK/MAPK-dependent pathway, suggesting a mechanism through which Notch3 in a cross-talk fashion governs VSMC fate and ultimately vascular structure.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture-- Rat embryonic aorta A7r5 cells (ATCC, passages 3-20) and primary rat aortic smooth cells (passages 4-15) were used in our study. Stable cell lines overexpressing the Notch3 intracytoplasmic domain (N3IC; pCMX-PL2-N3IC; a kind gift from Dr. U. Lendahl, Karolinska Institute, Stockholm, Sweden) and HRT1 (PIRES2-EGFP-HRT1; a kind gift from Dr. C. C. W. Hughes, University of California, Irvine, CA) were generated from A7r5 cells by transduction with a retroviral vector (pLNCX2; CLONTECH) and selection in the presence of geneticin (500 µg/ml; GLT).

Rat Carotid Artery Balloon Injury-- Male Sprague-Dawley rats (350-400 g) were balloon-injured using previously described methods (44) in accordance with a protocol approved by the Standing Committee on Animals, Morehouse School of Medicine. Rats were anesthetized with an intraperitoneal injection of xylazine (5 mg/kg of body weight) and katamine hydrochloride (90 mg/kg of body weight). The left common carotid artery was injured with a 2-French Fogarty embolectomy balloon catheter, and vessels were harvested 14 (n = 5) and 28 days later for mRNA and/or protein analysis. Injured vessels were compared with their contralateral controls.

Quantitative Real Time Reverse Transcription-PCR (QRTPCR)-- Total RNA from cell pellets or pulverized arteries was extracted (Rneasy kit; Qiagen Inc., Valencia, CA), and a reverse transcriptase reaction (Advantage RT for PCR kit; CLONTECH) was performed with 0.5-1 µg of DNase I (Ambion, Austin, TX)-treated RNA. QRTPCR was carried out using the LightCycler thermocycler and the SYBR green I kit (Roche Diagnostics Corp.), according to the manufacturer's recommendations. Cycle numbers obtained at the log-linear phase of the reaction were plotted against a standard curve prepared with serially diluted control samples. Expression levels of target genes were normalized by GAPDH mRNA levels measured concurrently.

Immunoblots-- Ten to 60 µg of protein/sample from cell cultures or rat carotid arteries were analyzed by SDS-PAGE. Goat anti-rat Notch3 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-FLIP (F9800; Sigma), and phospho-p44/42 MAPK (Thr²⁰²/Tyr²⁰⁴) antibody (9101; Cell Signaling Technology, Beverly, MA) as well as anti-pan-ERK (610123; BD Transduction Laboratories, San Diego, CA) were employed for the immunoblots. Membranes were developed through the enhanced chemiluminescence method (Ecl-Luminol kit; Santa Cruz Biotechnology). Protein loading was systematically verified by Ponceau S staining and/or staining rat actin immunoblotting.

Determination of Apoptosis-- Quantitative nuclear chromatin morphology was employed for the apoptosis counting. Cells to be analyzed for apoptosis by nuclear chromatin morphology were stained with Hoechst 33342 and assessed for the characteristic condensed, coalesced chromatin pattern of apoptotic cells as previously described by our laboratory (27, 28, 37). For Fas ligand-induced apoptosis, 70% confluent cells in 2% FBS were treated with 25 ng/ml Fas ligand (Upstate Biotechnology, Inc., Lake Placid, NY) versus vehicle control for 24 h prior to harvest and determination of percentage of apoptotic nuclei as described above.

Plasmid Preparation and Cloning-- N3IC and HRT1 were released from their expression vectors (pCMX-PL2-N3IC and PIRES2-EGFP-HRT1 were kind gifts from Dr. U. Lendahl and Dr. C. C. W. Hughes, respectively.) and subcloned to a retroviral vector, pLNCX2 (CLONTECH), and a mammalian expression vector, pcDNA 3.1 (Invitrogen). A dominant negative construct (R218H) for RBP-Jk was also employed in this study. It carries an arginine-to-histidine substitution at position 218, which is critical for the DNA binding activity of RBP-Jk. R218H in pCMX was a kind gift from Dr. Tasuku Honjo (Department of Medical Chemistry, Kyoto University).

Transfection of Cultured Cells-- Native A7r5 cells were transfected with N3IC versus empty vector control (Effectene transfection reagent; Qiagen) according to the manufacturer's instructions for 24 h prior to the apoptosis experiments. In a similar manner, A7r5 cells overexpressing N3IC versus empty cassette control were transfected with the dominant negative construct for RBP-Jk, R218H, or empty vector control 24 h prior to the determination of HRT1 and c-FLIP expression levels by QRTPCR.

Protocols-- The mRNA expression levels of Notch3 as well as c-FLIP in rat carotid arteries were studied by QRTPCR at 5 and 28 days post-balloon injury. For determining the mediator role of the ERK/MAPK pathway, cells in serum-free medium for 6 h were exposed to U0126 (10 µmol/liter; Biomol Research Laboratories, Inc., Plymouth Meeting, PA) versus vehicle control for an additional 1 h prior to harvest for c-FLIP mRNA and protein expression analysis.

Statistical Analysis-- All experiments, including the immunoblots, were independently repeated at least three times. Comparisons between two groups were analyzed via a Student's t test, and values of p < 0.05 were considered to be significant. Results were presented as means ± S.E. At least three different samples were analyzed in each experimental group.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Notch3 Signaling Promotes VSMC Survival-- To define the functional role of Notch3 signaling in VSMC, we initially established an in vitro model system. We generated a VSMC stable cell line expressing the constitutively active intracellular portion of Notch3 (N3IC) by retroviral transfection and marker selection. The selected cell line, N3ICSMC, exhibited a 35-fold up-regulation of Notch3 mRNA compared with its control cell line (Fig 1, left) and a correspondingly dramatic increase in Notch3 IC protein (Fig. 1, right). In addition, we confirmed that N3ICSMC exhibited significant tonic up-regulation of the HRT1 and -2 genes, previously described as downstream target genes of Notch signaling in other cell types (data not shown). To determine the functional role of Notch3 signaling on VSMC fate, we investigated the effect of Notch3 on promoting VSMC survival. We defined the antiapoptotic effect of Notch3 signaling in N3ICSMC by quantitative nuclear chromatin morphology analysis as previously described by our laboratory (27, 28, 37). Initially, we determined that constitutive Notch3 signaling in the N3ICSMC line promoted VSMC survival in response to serum deprivation (data not shown). To better define distinct apoptotic pathways that Notch3 signaling may modulate, we examined the effect of Notch3 signaling on mediating FasL-induced apoptosis. We demonstrated that constitutive Notch3 signaling in the N3ICSMC promoted a 2-fold increase in VSMC survival in response to FasL-induced apoptosis versus its control cell line (Fig. 2A). We confirmed these findings in native A7r5 by transient transfection of Notch3 IC. In accord with the data obtained in the stable N3ICSMC cell line, an approximate 2-fold survival increase in response to FasL was observed in VSMC transiently transfected with N3IC versus empty vector control (Fig. 2B). Fig. 2C depicts representative ultraviolet fluorescent photomicrographs of VSMC transfected with control (panels I and II) versus N3IC (panels III and IV) plasmid in 2% FBS in the absence (panels I and III) or presence of FasL (panels II and IV). Taken together, these data suggest that Notch3 signaling modulates VSMC response to the well defined Fas signaling pathway.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   N3IC is constitutively up-regulated in a retroviral stably transfected VSMC line (N3ICSMC). Increased expression of N3IC mRNA (left) and protein (right) in N3ICSMC compared with the empty vector control cell line (pLNCXSMC) by QRTPCR (n = 3; **, p < 0.01; normalized to GAPDH) and Western blotting, respectively.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Notch3 signaling promotes VSMC survival in response to FasL. A, constitutive Notch3 intracellular domain (N3IC) signaling in a retroviral stably transfected VSMC line (N3ICSMC) promotes survival in response to FasL-induced apoptosis compared with the empty vector control cell line, pLNCXSMC. Nearly confluent cells in 2% FBS were exposed to FasL (25 ng/ml) versus vehicle for 24 h prior to determination of percent apoptotic nuclei by Hoechst 33342 staining and UV microscopy. B, transient expression of Notch3 intracellular domain (N3IC) expression inhibits FasL-induced cell death in A7r5 VSMC compared with cells transfected with empty vector control. Experimental conditions were as outlined above. **, p < 0.01 compared with 2% FBS control, n = 12. C, representative ultraviolet fluorescent photomicrographs of VSMC transfected with control (panels I and II) versus N3IC (panels III and IV) plasmid in 2% FBS in the absence (panels I and III) or presence of FasL (25 ng/ml) (panels II and IV). After treatment, cells were stained with the DNA chromatin binding dye, Hoechst 33342, harvested, and viewed under UV light at ×200. *, cells exhibiting the brightly fluorescent, condensed, and coalesced chromatin staining pattern characteristic of apoptosis.

Notch3 Signaling Induces c-FLIP Expression in VSMC-- To define the potential mechanism by which Notch3 signaling confers resistance to FasL-induced apoptosis, we investigated the expression levels of several putative mediators of apoptosis and FasL signaling in VSMC. In the N3ICSMC cells, we examined the mRNA and protein expression levels of c-FLIP, IAP, and Bcl-2 compared with the control cell line. We established that resistance to FasL-induced apoptosis was associated with the induction of c-FLIP, a primary inhibitor of the Fas signaling pathway, as quantitated by real time RT-PCR and immunoblotting (Fig. 3). As depicted in Fig. 3, steady state mRNA levels of c-FLIP were up-regulated about 2-fold in N3ICSMC versus the control cell line. Accordingly, the Notch3-induced up-regulation of c-FLIP was defined by a corresponding increase in protein expression confirmed by immunoblotting (Fig. 3, right panel).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Notch3 signaling induces c-FLIP expression in VSMC. Left, histogram of c-FLIP mRNA expression levels in retroviral stably transfected VSMC line (N3ICSMC) versus empty vector control cell line (pLNCXSMC) by QRTPCR. Results were normalized to GAPDH (n = 4; *, p < 0.05). Right, immunoblot detection of elevated c-FLIP (55 kDa) protein expression levels in N3ICSMC versus pLNCXSMC. Nearly confluent cells in 10% FBS were harvested prior to mRNA and protein analysis. Results are representative of three repetitions.

Notch3 Induces c-FLIP Expression through an RBP-Jk-independent Mechanism-- After establishing that Notch3 signaling induced c-FLIP expression in association with promoting VSMC survival, it remained to be determined whether c-FLIP was a direct downstream target gene of the classic Notch-RBP-Jk signaling in VSMC. To address this question, we performed transient overexpression experiments with a well described dominant negative inhibitor of RBP-Jk, R218H, and determined the effect of inhibiting the tonic Notch3RBP-Jk signaling in the N3ICSMC stable cell line. The mRNA expression levels of c-FLIP and a previously established downstream target gene, HRT1, were determined by QRTPCR. As depicted in Fig. 4, blockade of RBP-Jk activity with the dominant negative inhibitor R218H markedly attenuated both the basal mRNA expression levels of HRT1 in the control cell line and the induced HRT1 mRNA expression levels in the N3ICSMC cell line. In contrast, inhibition of Notch3RBP-Jk activity had no effect on either the basal or the induced expression levels of c-FLIP. Therefore, these results suggest that Notch3 modulated c-FLIP expression through an RBP-Jk-independent pathway.

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Notch3 signaling induces c-FLIP expression in VSMC through an RBP-Jk-independent pathway. Shown is a histogram of the HRT1 and c-FLIP expression levels determined by QRTPCR in retroviral stably transfected VSMC line (N3ICSMC) versus empty vector control cell line (pLNCXSMC) after transient transfection with dominant negative RBP-Jk (R218H) versus empty vector control (pcDNA). Results indicate that blockade of RBP-Jk activity with the dominant-negative inhibitor R218H markedly attenuated both the basal mRNA expression levels of HRT1 in the control cell line (pLNCXSMC) and the induced HRT1 mRNA expression levels in the N3ICSMC cell line. In contrast, inhibition of Notch3-RBP-Jk activity had no effect on either the basal or the induced expression levels of c-FLIP. *, p < 0.01 compared with the base-line expression control levels (pLNCXSMC, pcDNA); n = 6; representative results of three repetitive experiments.

Notch3 Signaling Induces c-FLIP Expression through Cross-talk Activation of the ERK/MAPK Pathway-- After determining that Notch3 modulated c-FLIP through an apparent RBP-Jk-independent pathway, we investigated the potential role of other putative mediator pathways. Based upon previous reports in other cell types suggesting that the ERK/MAPK cascade may modulate c-FLIP expression (34, 35), we postulated that Notch3 signaling activates the ERK/MAPK pathway in a cross-talk fashion. To test this postulate, we initially determined the steady state protein expression levels of both the total and active (phosphorylated) forms of p42/p44 ERK/MAPK in the N3ICSMC stable cell line via immunoblotting. As depicted in Fig. 5, without affecting the levels of total ERK/MAPK, constitutive Notch3 signaling promotes a marked increase in ERK/MAPK pathway activation in VSMC via an undefined cross-talk mechanism.

View larger version (52K): [in this window] [in a new window]   Fig. 5.   Notch3 signaling in VSMC induces ERK/MAPK activation. Immunoblot analysis of p44/p42 versus pan-ERK/MAPK levels in retroviral stably transfected VSMC line (N3ICSMC) compared with empty vector control cell line (pLNCXSMC). Nearly confluent cells were placed in reduced serum conditions for 6 h prior to analysis. The experiment was independently repeated three times.

To establish the mediator role of the Notch3-induced ERK/MAPK activation in regulating c-FLIP expression in VSMC, we examined the effect of inhibiting ERK/MAPK pathway activation on the Notch3-induced c-FLIP mRNA expression by QRTPCR analysis. As demonstrated in the histogram in Fig. 6, steady state mRNA expression levels of c-FLIP in the control cell line are reduced in the absence of serum stimulation but are not further reduced after blockade of ERK/MAPK pathway activation via administration of U0126, a specific inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. In contrast, while c-FLIP expression levels were moderately reduced in the absence of serum stimulation, tonic Notch3 signaling in the N3ICSMC cells promoted the preservation of elevated c-FLIP expression levels compared with the control cells. However, the elevated c-FLIP expression levels in the N3ICSMC cells were reduced to base-line control levels after blockade of the previously defined tonic ERK/MAPK pathway activation. Similar results were obtained in the setting of transient expression of Notch3 IC in VSMC (data not shown). Furthermore, we determined that inhibition of the ERK/MAPK pathway specifically attenuated c-FLIP expression, while levels of other antiapoptotic factors such as IAP, Bcl-2, and Bcl-xL were unaffected (data not shown). Taken together, these results suggest that Notch3 mediates c-FLIP expression via cross-talk activation of the ERK/MAPK pathway in VSMC.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Notch3 signaling modulates c-FLIP expression in VSMC through an ERK/MAPK-dependent mechanism. Histogram of mRNA expression levels of c-FLIP in retroviral stably transfected VSMC line (N3ICSMC) compared with empty vector control cell line (pLNCXSMC) in 10% FBS and after 6-h reduced serum conditions in the presence of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor U0126 (10 µM) versus vehicle control (1 h). mRNA expression levels were monitored by QRTPCR. Results were normalized by GAPDH (n = 6; *, p < 0.05 compared with its 10% FBS control).

Notch3 and c-FLIP Are Coordinately Regulated in Response to Arterial Injury-- After defining that Notch3 signaling promotes VSMC survival by inducing c-FLIP expression in vitro, we further postulated that the well described modulation of VSMC fate in vascular lesion formation involves a coordinate regulation of Notch3 and c-FLIP. To test this postulate, we examined the coordinate expression levels of Notch3 and c-FLIP in rat carotid arteries following standard balloon injury. As depicted in Fig. 7A, as we have previously reported (38), Notch3 is acutely down-regulated within 1 week postinjury, exhibiting approximately a 6-fold change in mRNA expression levels at day 5. This down-regulation is seen at both the mRNA and protein level by QRTPCR and immunoblotting, respectively. When we examined the expression levels of c-FLIP at the same time point, we observed a similar, coordinate down-regulation of mRNA and protein by QRTPCR and immunoblotting, respectively (Fig. 7B). Similar findings with Notch3 and c-FLIP were observed at day 3 postinjury. By day 7, the expression levels of both Notch3 and c-FLIP genes returned to preinjury levels (data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Coordinate down-regulation of Notch3 and c-FLIP expression in rat carotid arteries 5 days post-balloon injury. A, Notch3 mRNA and protein (90-kDa active intracellular domain) expression levels in rat carotid arteries 5 days post-balloon injury versus contralateral uninjured control arteries. B, c-FLIP mRNA and protein (55 kDa) expression levels in rat carotid arteries 5 days post-balloon injury versus contralateral uninjured control arteries. mRNA levels were determined by QRTPCR and normalized to GAPDH. n = 5; *, p < 0.01. Immunoblot analysis is representative of four matched vessel pairs.

We further postulated that an up-regulation of Notch3 expression is a characteristic of the altered growth and apoptosis phenotype associated with neointimal cells. Previous reports indicate that c-FLIP expression is elevated within intimal cells of remodeled arteries (26). Accordingly, as indicated in Fig. 8A, c-FLIP mRNA and protein levels were increased within tissue selectively isolated from the intima of carotid arteries 4 weeks after carotid balloon injury compared with the paired contralateral uninjured vessel. When we examined Notch3 mRNA and protein levels within the same neointimal tissue, we observed significantly elevated expression levels coordinate with increased c-FLIP expression (Fig. 8B). Taken together, these findings establish a coordinate pattern of Notch3 and c-FLIP expression in response to arterial injury and within intimal cells of remodeled arteries.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Coordinate up-regulation of Notch3 and c-FLIP expression in neointimal tissue at day 28 post-balloon injury. A, Notch3 mRNA and protein (90-kDa active intracellular domain) expression levels from selectively isolated neointima in rat carotid arteries 28 days post-balloon injury versus contralateral uninjured control arteries. B, c-FLIP mRNA and protein (55 kDa) expression levels from selectively isolated neointima in rat carotid arteries 28 days post-balloon injury versus contralateral uninjured control arteries. mRNA levels were determined by QRTPCR and normalized to GAPDH. n = 9; *, p < 0.05. Immunoblot analysis is representative of four matched vessel pairs.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Although it is well documented that the Notch pathway is a potent modulator of cell function and fate during organogenesis and vascular ontogeny, the role of this signaling pathway within the adult vasculature remains to be elucidated. Intriguingly, the human CADASIL syndrome, caused by mutations in the Notch3 receptor, is characterized by a systemic arteriopathy featuring degeneration and eventual loss of VSMC. Based upon these distinguishing clinicopathological features, we postulated that Notch3 signaling may be a critical determinant of VSMC survival. This present study provides initial insight into Notch3 signaling and function as a determinant of VSMC fate through the modulation of critical molecular mediators of VSMC apoptosis. In addition, the study provides an initial description of elevated Notch3 expression as a characteristic feature of neointimal cells. We have previously shown that neointimal cells are relatively resistant to the induction of apoptosis in association with the up-regulation of antiapoptotic mediators (27-29).

Utilizing models of transient and stable transgene expression, we demonstrated that constitutive Notch3 receptor activation induced an antiapoptotic phenotype in VSMC. It is recognized that the regulation of apoptosis is a complex process potentially involving a number of different cellular mediator pathways. It is conceivable that Notch3 may inhibit VSMC apoptosis by pathways independent of c-FLIP. In support of this notion, our study suggests that Notch3 signaling promotes survival in response to serum deprivation in addition to the administration of FasL. However, the mechanisms of serum deprivation-induced apoptosis are complex and poorly defined. Numerous, plausible antiapoptotic mechanisms can be hypothesized: interaction with nuclear receptors of the steroid/thyroid/retinoid/orphan superfamily; regulation of the expression of antiapoptotic members of the Bcl family; regulation of NF-B family members' expression or function; and regulation of c-Jun N-terminal kinase activity. It is entirely possible that several or all of these mechanisms may be used by a Notch receptor, simultaneously or alternatively, depending on the cellular context (39).

Intriguingly, our studies establish that Notch3 signaling promotes resistance to FasL-induced apoptosis. Increasing evidence suggests that Fas-mediated death plays a critical role in VSMC biology and pathobiology in vitro and in vivo (40-42). Based upon this finding, we postulated that Notch3 signaling in VSMC regulates the expression of antiapoptotic genes known to modulate the Fas signaling pathway. The phenotype of Fas resistance in VSMC may result from reduced expression of proapoptotic proteins involved in Fas signaling, including FasL, Fas-associated death domain protein, and caspase-3, -7, and -8, and increased expression of antiapoptotic proteins such as c-FLIP, Bcl-2, and c-IAP-1 (28, 29, 40, 43). We investigated the expression levels of the above antiapoptotic proteins as well as the Fas receptor.

Our findings further establish that Notch3 signaling promotes the expression of the antiapoptotic mediator, c-FLIP, a direct inhibitor of the Fas signal transduction pathway. Intriguingly, it is reported that c-FLIP is expressed in the neointima in animal models and in human atherosclerosis (32, 33). In accord with these previous descriptions, the present study demonstrates that c-FLIP is coordinately up-regulated with Notch3 within the neointima. Whereas this observation suggests a possible mechanistic link between Notch3 signaling and c-FLIP expression in vivo, further in vivo studies are necessary to define this relationship. Taken together, these studies are the first to establish the essential survival-promoting role of Notch3 and its modulation of c-FLIP as a putative downstream effector of this function in VSMC.

The present study investigated potential mechanisms by which Notch3 signaling modulates c-FLIP expression in VSMC. In addition to the classical model of Notch signaling via RBP-Jk-dependent transcriptional events, Notch may engage other signaling cascades in a cross-talk fashion (23-25). However, the elucidation of Notch3 signaling via RBP-Jk-dependent versus signal transduction cross-talk and the activation of downstream target genes in adult VSMC is poorly defined. Our findings suggest that Notch3 modulates c-FLIP expression in a manner largely independent of RBP-Jk activity. The well described dominant negative mutant of RBP-Jk, R218H, inhibited VSMC expression of an established downstream Notch-RBP-Jk target gene, HRT1, but failed to inhibit the expression of c-FLIP. Furthermore, preliminary studies in our laboratory indicate that VSMC expression of HRT1 does not promote the induction of c-FLIP. Taken together, these studies provide supportive evidence that suggests Notch3 modulates c-FLIP through a mechanism largely independent of the RBP-JkHRT pathway.

Our findings indicate that Notch3 signaling modulates c-FLIP expression through activation of the ERK/MAPK pathway in a yet undefined cross-talk fashion. In support of this notion, recent studies in other cell types indicate that in certain contexts, the Notch signaling pathway may modulate Src and Ras signal transduction by as yet undefined mechanisms (23-25). Future studies are needed to define the means by which Notch3 signaling cross-talks with the ERK/MAPK signal transduction cascade. Possible mechanisms include a direct interaction of the Notch3 receptor with upstream elements of the ERK cascade or an indirect autocrine/paracrine induction of ERK-activating growth factors. We speculate that cross-talk activation of the ERK/MAPK pathway may represent an additional novel mechanism through which Notch3 signaling determines VSMC fate.

In summary, these studies are the first to establish the essential survival-promoting role of Notch3 signaling in VSMC. The working model defined by our data suggests that as part of a survival-promoting phenotype in response to Fas activation, Notch3 induces c-FLIP expression through an ERK/MAPK-dependent mechanism. It is intriguing that mutations of the Notch3 receptor are postulated to result in a loss of Notch3 function primarily affecting the fate of VSMC in the CADASIL syndrome (3, 4). We speculate that a loss of the survival-promoting influence of Notch3 may render VSMC more susceptible to signals from Fas and other related proapoptotic signaling pathways. Conversely, increased Notch3 signaling may induce resistance to proapoptotic signals and contribute to the abnormal accumulation of VSMC seen within the intima. It is anticipated that further elucidation of Notch3 signaling, target gene induction, and function in VSMC will provide new insights into the molecular mechanisms of vascular disease and complications.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by the National Institutes of Health (NIH)-Minority Biomedical Research Support program.

^§ Supported by grants from the NIH-MBRS program, the Research Centers in Minority Institutions program, and an NIH-K08 award. To whom correspondence should be addressed. Tel.: 404-752-1545; Fax: 404-752-1042; E-mail: address: mpollman{at}msm.edu.

Published, JBC Papers in Press, March 29, 2002, DOI 10.1074/jbc.M202224200

	ABBREVIATIONS

The abbreviations used are: CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephelopathy; IC, intracellular domain; VSMC, vascular smooth muscle cell(s); ERK, extracellular signal regulated kinase; MAPK, mitogen-activated protein kinase; FBS, fetal bovine serum; QRTPCR, quantitative real time reverse transcription-PCR; HRT, Hairy-related transcription factor; FasL, Fas ligand; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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