Coordinate Notch3-Hairy-related Transcription Factor Pathway Regulation in Response to Arterial Injury

The Notch family of receptors and downstream effectors plays a critical role in cell fate determination during vascular ontogeny. Moreover, the human cerebralautosomal dominant artriopathy withsubcortical infarcts andleukoencephalopathy (CADASIL) syndrome of premature stroke and dementia is a heritable arteriopathy with alterations in vascular smooth muscle cells (VSMCs) resulting from mutations within Notch3. However, the expression and regulation of the Notch and hairy-related transcription factor (HRT) pathway in adult VSMCsin vitro and in vivo remain poorly characterized. The present study documents that the well-described modulation of VSMC fate in response to vascular injury and growth factor activation involves a coordinate regulation of the Notch and HRT pathways. Furthermore, platelet-derived growth factor promotes a similar coordinate down-regulation of the Notch receptors andHRT genes in cultured VSMCs via an ERK-dependent signaling pathway. Moreover, we established that HRT1 and HRT2 are direct downstream target genes of Notch3 signaling in VSMCs and determined that the activity of the nuclear protein RBP-Jk is essential for their regulation. These findings provide initial insight into the context- and cell type-dependent coordinate regulation ofNotch3 and downstream HRT transcriptional pathway effector genes in VSMCs in vitro and in vivo that may have important implications for understanding the role of Notch signaling in human health and vascular disease.

It is postulated that pathological changes in vessel structure seen in conditions such as hypertensive arteriopathy 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 artriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a heritable syndrome characterized by a predisposition to stroke due to an underlying arteriopathy. Genetic linkage analyses have documented mutations in Notch3 as the etiologic basis of the CADASIL syndrome (3)(4)(5)(6). Furthermore, Notch3 expression is largely confined to VSMCs 1 in adulthood (3). These findings suggest that the Notch pathway may be an important determinant of vascular structure in human health and disease. The Notch family receptors have been characterized as critical determinants of cell fate in a variety of organisms. In mice, Notch-1 and Notch-2 gene deletions are characterized by perturbations in organogenesis that result in embryonic lethality (7,8). 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 (9 -13). However, the role of the Notch signaling pathway in VSMCs 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/C promoter binding factor (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 (14 -16).
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 (HES-1) (17) and HES-related protein-1 (HESR-1 (HRT-1)) (18), which may mediate the effects on cell fate. Recent studies have established that the Hairy-related transcription factor (HRT) genes, HRT1, HRT2, and HRT3, are downstream targets for transcriptional activation by Notch signaling. The HRT genes share expression periodicity within the vasculature with components of the Notch signaling pathways. HRT genes are highly expressed in the embryonic vasculature, including the outflow tract of the heart and the aortic sac (14,19). Studies performed by Chin and co-workers (20,21) investigating the potential role of CHF2 (HRT1) and CHF1 (HRT2) in muscle cells suggest that they may function as molecular regulators of terminal differentiation. Moreover, zebrafish embryos harboring a mutation in Gridlock, an orthologue of HRT2, show dramatic impairment of vascular formation (22). Furthermore, recent evidence has established that Gridlock expression is a critical determinant of arterial versus venous cell fate within the developing vasculature (23,24).
In most cell types examined, expression of Notch1IC leads to increased CBF-1/RBP-Jk-dependent activity. Conversely, evidence suggests that the role of Notch3 is context-and cell type-specific (15). Studies performed in other cell types have indicated that Notch3IC expression inhibits Notch1IC-RBP-Jkmediated HES activation (15). These results suggest that Notch3 may act as an antagonist of the Notch1-RBP-Jk signaling pathway. Therefore, the expression and regulation of the Notch3 signaling pathway in VSMCs in vivo and in vitro and the elucidation of the Notch3-RBP-Jk signaling pathway and downstream target genes in adult VSMCs remain to be defined.
The present study tested the hypothesis that the well-described modulation of VSMC fate in response to vascular injury and growth factor activation involves a coordinate regulation of the Notch pathway. In accord with this hypothesis, our findings indicate that the Notch family of receptors and the downstream HRT effector genes are coordinately down-regulated in response to vascular injury. It is well established that growth factors such as PDGF are important mediators of vascular remodeling and lesion formation (25)(26)(27)(28). We postulated that pathological modifications of vessel structure induced by PDGF may be mediated by the coordinate modulation of the Notch-HRT pathway. Consistent with this hypothesis, our findings indicate that PDGF promotes a similar coordinate down-regulation of the Notch receptors and HRT genes in cultured VSMCs via an ERK-dependent signaling pathway.
We further established that Notch3 signaling in VSMCs does not inhibit but indeed promotes RBP-Jk-mediated signaling and induces the expression of the downstream target genes, HRT1 and HRT2. Taken together, this study documents the coordinate regulation of the Notch and HRT genes in VSMCs in vitro and in vivo in a context-and cell type-dependent manner. In addition, it has defined that HRT1 and HRT2 are direct downstream target genes of Notch3-RBP-Jk signaling in VSMCs. These findings provide initial insight into the context-and cell type-dependent coordinate regulation of Notch3 and downstream HRT target genes in VSMCs and define a potential mediator signaling pathway, ERK/MAPK, in vitro and in vivo.

MATERIALS AND METHODS
Cell Culture-Primary isolated rat aortic smooth cells (RASMCs; passages 4 -15) and the rat embryonic aortic A7r5 cell line (ATCC) were used in our study. Unless otherwise specified, cells were maintained in Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal bovine serum (Invitrogen).
Rat Carotid Artery Balloon Injury-Male Sprague-Dawley rats (350 -400 g) were balloon-injured using previously described methods (29) 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 body weight) and katamine hydrochloride (90 mg/kg body weight). The left common carotid artery was injured with a 2-French Fogarty embolectomy balloon catheter, and vessels were harvested at 1 (n ϭ 5), 3 (n ϭ 6), 5 (n ϭ 3), and 7 (n ϭ 5) days after injury for mRNA analysis, and the right carotid artery was used as control. Both uninjured and injured vessels were washed in ice-cold phosphate-buffered saline, and the adventitial layer was removed. In addition, the endothelium of the control vessels was denuded by cotton swab sweeping twice. The samples for protein analysis were harvested in an identical manner.
Quantitative Real-time Reverse Transcription-PCR (QRTPCR)-Total RNA from cell pellets or pulverized arteries was extracted (Rneasy kit; Qiagen Inc., Valencia, CA), and reverse transcription reaction (Advantage RT for PCR kit; CLONTECH) was performed with 0.5-1 g of DNase I (Ambion, Austin, TX)-treated RNA. QRTPCR was carried out using the LightCycler thermocycler and the SYBR green I kit (Roche Diagnostics) according to the manufacturer's recommendations. Primer sequences and reaction conditions can be found in Table I. 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 concurrently measured GAPDH mRNA levels.
Western Blots-10 -60 g of protein per sample from cell cultures or rat carotid arteries were analyzed by SDS-PAGE. Goat anti-rat Notch3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was employed for the immunoblots. Membranes were developed using an enhanced chemiluminescence method (Ecl-Luminol kit; Santa Cruz Biotechnology  RBP-Jk-responsive elements (3 ϫ 5Ј-CTGGTGTAAACA CGCCGTGG-GAAAAAATTT-3Ј) were inserted into the PGL3-promoter luciferase reporter plasmid (Promega) upstream of the thymidine kinase promoter.
Transfection of Cultured Cells-Experiments were performed using Nucleofector technology. The electroporations were performed using the human aortic smooth muscle cell Nucleofector kit (Amaxa Biosystems, Koeln, Germany) according to the manufacturer's instructions. Simply, for each reaction, 1 ϫ 10 6 cells were harvested by trypsin-EDTA and mixed with a total of 7 g of plasmid DNA in 100 l of human aortic smooth muscle cell Nucleofector solution. After electroporation, the samples were transferred into 6-well plates and incubated at 37°C in 5% CO 2 . Gene expression analysis and luciferase assays were performed 24 h after electroporation.
Luciferase Assays-A7r5 VSMCs were transiently co-transfected with the genes of interest and a reporter plasmid using the Nucleofector electroporation system as described above. Luciferase activity was measured 24 h after transfection using a commercially available kit (Promega) and normalized by green fluorescent protein fluorescence levels.
Experimental Procedures-The expression levels of Notch signaling pathway elements in rat carotid arteries were investigated by QRTPCR at different time points within 1 week after balloon injury. Expression levels of Notch-1, Notch-3, HRT1, and HRT2 were analyzed in RASMCs treated with PDGF (20 ng/ml; Sigma) by QRTPCR. Postconfluent RASMCs were kept quiescent in OPTI-MEM I (Invitrogen) for 48 h before PDGF administration for the time points indicated. Protein level changes were confirmed by immunoblot with available reagents. The mediator role of the ERK, p38, and phosphatidylinositol 3-kinase pathways was evaluated in PDGF-treated RASMCs after 12 h of treatment with the following inhibitors, respectively: U0126 (10 mol/liter; Biomol Research Laboratories, Inc., Plymouth Meeting, PA), SB202190 (25 mol/liter; Biomol Research Laboratories, Inc.), and LY294002 (50 mol/liter; Sigma). The efficacy of pathway blockade at these doses has been confirmed in our laboratory as described previously by others (30 -32).
Statistical Analysis-All experiments, including the immunoblots, were independently repeated a minimum of three times. Comparisons between two groups were analyzed via Student's t test, and values of p Ͻ 0.05 were considered to be significant. Results were presented as means Ϯ S.E. At least three different samples were analyzed in each experimental group.

Notch Receptors and HRT Transcription Factors Are Coordinately Regulated in Response to
Arterial Injury in Vivo-We initiated our study by defining the basal in vitro and in vivo mRNA expression levels of the Notch receptors, Notch1-4, and the HRT gene family, HRT1-3, by QRTPCR. As indicated in Table II, each of the four Notch receptors and the three HRT genes was detected in adult rat carotid arteries. With the exception of Notch4, whose expression levels were undetectable in vitro, similar relative expression of the Notch receptors and HRT genes was detected in cultured RASMCs under confluent, quiescent culture conditions. After defining the basal expression of the Notch-HRT pathway in VSMCs in vivo and in vitro, we hypothesized that the well-described modulation of VSMC fate in response to vascular injury and growth factor activation involves a coordinate regulation of the Notch-HRT pathway. To test this hypothesis, we examined the relative mRNA expression levels of Notch1-3 and HRT1-3 by QRTPCR in rat carotid arteries after standard balloon injury. As seen in Fig. 1A, each of the Notch receptors was markedly down-regulated between 2-and 3-fold within the first 24 h after injury. The initial down-regulation was maintained at days 3 and 5, with a trend toward recovery of relative preinjury levels by day 7. The most dramatic down-regulation was observed with the Notch3 receptor. We further examined the protein expression levels of the Notch3 receptor at day 3 by immunoblotting. As noted in Fig. 1B, the Notch3 receptor protein levels were dramatically down-regulated in a fashion similar to the mRNA expression levels.
We next examined the expression levels of the HRT gene family at the identical time points after arterial injury. QRT-PCR analysis of the mRNA expression levels of HRT1-3 (Fig.  1C), revealed a similar pattern of acute down-regulation, suggesting a possible coordinate regulation of the Notch receptors and HRT genes. The most dramatic down-regulation was observed with the HRT1 gene, reaching ϳ13-fold at day 3. By day 7, the expression levels of the HRT genes had returned to preinjury levels.

PDGF Down-regulates the Notch-HRT Pathway in
VSMCs-It is well established that growth factors such as PDGF are important mediators of vascular remodeling and lesion formation (25)(26)(27)(28). We postulated that pathological modifications of vessel structure induced by PDGF may be mediated by the coordinate modulation of the Notch-HRT pathway.
We observed that PDGF induced a time-dependent downregulation of mRNA levels of the Notch receptors Notch1 and Notch3 in RASMCs within 6 -12 h of administration ( Fig. 2A). The most dramatic regulation was again observed with Notch3. A similar trend was observed for Notch2 (data not shown). We further defined the PDGF-induced down-regulation of the Notch3 receptor by establishing a dose-dependent relationship. As seen in Fig. 2B, PDGF induced a dose-dependent downregulation of the Notch3 receptor at the level of steady-state protein. This down-regulation was observed for both the fulllength form of the receptor (300 kDa) as well as the intracellular domain portion of the receptor (90 kDa).
We further hypothesized that the coordinate regulation of the Notch receptors and HRT transcription factors observed in vivo may be simulated by PDGF administration in cultured VSMCs. To address this question, we performed QRTPCR analysis of the mRNA expression levels of HRT1 and HRT2 after PDGF administration. We observed a similar pattern of downregulation within the 6 -12-h time period, again suggesting a coordinate regulation of Notch receptors and HRT transcrip-tion factors (Fig. 2C). Similar to the results obtained after arterial injury, the most dramatic down-regulation was seen with the gene HRT1.
ERK Signaling Pathway Mediates Notch Receptor and HRT Gene Expression in Vitro-To examine the cellular mechanisms that link PDGF to the regulation of the Notch-HRT pathway, we assessed the mediator role of ERK/MAPK. QRTPCR analysis demonstrated that the PDGF-induced down-regulation of Notch3 could be abolished by ERK blockade with the pharmacologic MEK inhibitor U0126 (Fig. 3A). We further demonstrated the specificity of the ERK pathway as a mediator of Notch3 inhibition by establishing that blockade of neither the p38 nor phosphatidylinositol 3-kinase pathways reversed the PDGF-induced downregulation (Fig. 3A). To further define the role of the ERK pathway, we examined its overall effect on Notch3 protein expression at 12 h. As shown in Fig. 3B, the MEK inhibitor (U0126) prevented the PDGF-induced down-regulation of Notch3 protein levels in RASMCs.
We further hypothesized that the PDGF-induced coordinate down-regulation of HRT1 and HRT2 was mediated by the ERK signaling pathway. QRTPCR analysis demonstrated that the PDGF-induced down-regulation of HRT1 and HRT2 could similarly be abolished specifically by blockade of the ERK pathway, and not the p38 or phosphatidylinositol 3-kinase pathways (Fig. 3, C and D). As additional control experiments, we established that only blockade with the MEK inhibitor (U0126) and not the p38 (SB203580) or the phosphatidylinositol 3-kinase (LY294002) inhibitors affected the levels of ERK activation in our in vitro model system (Fig. 3E).
HRT1 and HRT2 Are Direct Downstream Target Genes of Notch3 Signaling in VSMCs-Whereas the studies performed above established a coordinate regulation of the Notch receptors and HRT transcription factors in vitro and in vivo, it remained to be determined whether the HRT genes were directly downstream of Notch signaling in VSMCs.
In most cell types examined, expression of Notch1IC leads to increased CBF-1/RBP-Jk-dependent activity. Conversely, evidence suggests that the role of Notch3 is context-and cell type-specific (15). Studies performed in other cell types have indicated that Notch3IC expression inhibits Notch1IC-RBP-Jkmediated HES activation (15). These results suggest that Notch3 may act as antagonist of the Notch1-RBP-Jk signaling pathway. The effects of Notch3 signaling on RBP-Jk activity and downstream target genes within VSMCs remained to be determined.
To begin to examine Notch3 signal transduction in VSMCs, we used promoter-reporter assays and observed that transient co-transfections of Notch3IC with the 3ϫ wild-type RBP-Jkreporter construct markedly induced luciferase activity (Fig.  4A). To further define the critical role of RBP-Jk in this process, we established that co-transfection of either the wild-type or R218H RBP-Jk blocked the Notch3IC-induced luciferase activity (Fig. 4A). These data are in accord with observations in other cell types that define RBP-Jk as a suppressor of transcriptional activity in the absence of an association with the intracytoplasmic domain of a Notch receptor. As an additional control, we established by QRTPCR analysis that co-expression of either RBP-Jk or dominant negative mutant R218H RBP-Jk had no effect on the expression levels of Notch3IC (Notch3IC ϩ control plasmid ϭ 1 Ϯ 0.011, Notch3IC ϩ RBP-Jk ϭ 0.94 Ϯ 0.15, Notch3IC ϩ R218H ϭ 0.97 Ϯ 0.18, normalized values relative to vimentin; n ϭ 3; p Ͼ 0.05). We further determined that co-expression of Notch3IC and Notch1IC promotes RBP-Jk-dependent transcriptional activation to a greater degree than expression of Notch1IC alone (data not shown). Taken together, these studies suggest that in contrast to Notch3 sig- nal transduction in other cell types, Notch3 signaling in VSMCs is not an antagonist of Notch1 but a potent inducer of RBP-Jk-dependent transcriptional activity.
After establishing that Notch3 signaling induced RBP-Jk-dependent transcriptional activation, we examined whether HRT1 and HRT2 were direct downstream target genes of the Notch3-RBP-Jk transcriptional pathway in VSMCs. We initially established the transfection efficiency within our in vitro model system using the Amaxa Biosystems Nucleofector electroporation technique. Utilizing green fluorescent protein-reporter plasmid constructs and UV microscopy, we defined an approximate 70% transfection efficiency within our VSMC in vitro model system within 24 -48 h of transfection. After defin-  HRT1 and HRT2, in VSMCs. A, histogram of luciferase activity after co-transfection of 3ϫ RBP-Jk-reporter constructs with Notch3IC and increasing levels of wild-type or dominant negative RBP-Jk (R218H) expression plasmids versus empty cassette control plasmid. Wild-type RBP-Jk and R218H attenuated the Notch3-induced luciferase activity in a dose-dependent manner. A7r5 VSMCs were co-transfected with green fluorescent protein expression vectors for normalization (mean Ϯ S.E.; n ϭ 6; p Ͻ 0.01). B and C, histograms of HRT1 and HRT2 mRNA induction by transient co-transfection of Notch3IC expression construct with increasing levels of wild-type or dominant negative mutant RBP-Jk (R218H) versus empty cassette control plasmids. Wild-type RBP-Jk and R218H attenuated Notch3-induced up-regulation of HRT1 and HRT2 in a dose-dependent manner. The expression levels of HRT1 and HRT2 were detected by QRTPCR after electroporation, and the results were normalized by GAPDH (mean Ϯ S.E.; n ϭ 6; p Ͻ 0.01).
ing the transfection efficiency, we established that transient transfection of VSMCs with Notch3IC markedly induced the expression of both HRT1 and HRT2 mRNA expression levels as determined by QRTPCR (Fig. 4, B and C). Furthermore, in accord with our data using the RBP-Jk reporter construct above, the Notch3-induced expression of these genes could be inhibited by co-expression of either the wild-type or dominant negative RBP-Jk. Taken together, these studies establish that HRT1 and HRT2 are direct downstream target genes of Notch3-RBP-Jk activity in VSMCs. DISCUSSION It is well established that the pathogenesis of vascular disease is characterized by the activation of signaling pathways and transcriptional programs that modulate cell proliferation, differentiation, apoptosis, migration, and extracellular matrix modification. These transcriptional programs are reminiscent of the genetic programs determining organogenesis during ontogeny. Given that the Notch family of receptors and downstream transcriptional effectors are highly conserved cell fate determinants involved in organogenesis, we were intrigued by the finding that a heritable arteriopathy involves mutations within the Notch3 receptor. This study tested the hypothesis that cell fate determinants such as Notch3 are modulated by growth factors expressed in the context of vascular injury and repair in adult VSMCs.
Although the role of Notch receptors and downstream effectors in vasculogenesis is being well characterized, the potential influence on vascular function and structure in the adult animal remained to be defined. The present study documents that major components of the Notch signaling pathway, including the Notch receptors (Notch1-3) and the downstream effector genes (HRT1-3), are constitutively expressed in adult VSMCs both in vitro and in vivo. Furthermore, our findings establish a coordinate regulation of the Notch receptors and downstream HRT effectors in the context of vascular injury. To our knowledge, these findings are the first description of HRT gene regulation within the vasculature of the adult animal. These findings complement previous studies that have documented the expression of Jagged-1 (33), Notch-4 (34), and HESR-1 (35) in endothelial cells. In addition, Lindner et al. (36), using in situ hybridization of en face tissue preparations, have recently reported that all four Notch receptors are expressed in the adult vasculature and regulated in the endothelium and adjacent intima in the context of vascular injury. However, the expression levels of the Notch family of receptors and the downstream HRT effector genes within VSMCs of the vessel wall remained to be determined.
It is postulated that although the HRT genes may be downstream and regulated in large part by the activity of the Notch receptors, there may be other regulatory mechanisms involved. Support for this notion is seen in our data depicting the time course of recovery of the Notch receptor and HRT gene expression levels. Our data suggest that recovery of HRT gene expression is observed before the recovery of Notch receptor expression. These data are in accord with observations and discussions in other in vitro and in vivo model systems that lead to the postulate that other mechanisms such as positive and negative feedback loops, as well as other yet to be determined mediators, modulate HRT expression levels in addition to Notch-RBP-Jk activity (14,24). Taken together, our data indicate that many of the critical elements of the Notch receptor signal transduction pathway are present within the adult vasculature, and the expression levels of these mediators are modulated in response to vascular injury.
The observation that expression of Notch signaling pathway elements is modulated by vascular injury led us to postulate an interplay between growth factor stimulation and the activity of this cell fate program. It is well established by multiple investigators that vascular injury promotes PDGF and PDGF receptor expression as well as ERK activation (37)(38)(39)(40). Our findings indicate that PDGF acutely induces a coordinate down-regulation of Notch1, Notch3, HRT1, and HRT2 in cultured VSMCs. Furthermore, we showed that an ERK-dependent pathway mediates the down-regulation of Notch and HRT expression by PDGF. To our knowledge, this is the first demonstration that the expression of these Notch receptors and the downstream HRT effectors is modulated by the ERK signaling pathway in mammalian cells. This finding suggests a possible nexus by which the activation of a growth-regulatory signaling pathway is coupled to the Notch cell fate program. Additional studies are needed to better define the mechanism by which ERK signal transduction regulates the expression of the Notch pathway elements.
We were intrigued by the apparent coordinate down-regulation of both Notch1 and Notch3 and the downstream HRT genes by growth factor stimulation. These data suggested to us that the HRT genes may be directly downstream of both Notch1 and Notch3 signaling in VSMCs. These results were intriguing because the signal transduction of Notch3 within VSMCs remained to be determined and indeed could exhibit cell type specificity. In most cell types examined, expression of Notch1IC leads to increased CBF-1/RBP-Jk-dependent activity. Conversely, evidence suggests that the role of Notch3 is context-and cell type-specific (15). Studies performed in other cell types have indicated that Notch3IC expression inhibits Notch1IC-RBP-Jk-mediated HES activation (15). These results suggest that Notch3 may act as an antagonist of the Notch1-RBP-Jk signaling pathway. Therefore, the effects of Notch3 signaling on RBP-Jk activity and downstream target genes within VSMCs remained to be determined.
Using the RBP-Jk promoter-reporter assay system, we initially defined that Notch3 signal transduction in VSMCs induced RBP-Jk activity that could be inhibited by either wildtype or dominant negative RBP-Jk. The finding that expression of either the wild-type or dominant negative RBP-Jk inhibited Notch3-induced luciferase activity is in accord with previous observations in other cell types with Notch1-mediated activity. RBP-Jk alone acts as a transcriptional repressor in the absence of interactions with the intracytoplasmic domain of Notch1. Furthermore, wild-type RBP-Jk acts to inhibit both endogenous NotchIC-RBP-Jk and overexpressed NotchIC-RBP-Jkmediated activity by complexing with NotchIC but not binding to the DNA recognition sequence (14 -16).
The findings of this study suggest that in contrast to Notch3 signal transduction in other cell types, Notch3 signaling in VSMCs is a potent inducer of RBP-Jk-dependent transcriptional activity in a cell type-specific manner. Furthermore, we established that HRT1 and HRT2 were direct downstream target genes of the Notch3-induced RBP-Jk activity in VSMCs. To our knowledge, this is the first description establishing Notch3-RBP-Jk activity inducing the direct downstream HRT target effector genes in VSMCs.
Although it is well documented that the Notch pathway is a potent modulator of cell function and fate in various model systems, the role of this pathway in VSMCs remains to be fully elucidated. Our findings that this pathway is regulated in the context of vascular injury and growth factor stimulation lead us to postulate that this pathway may be coupled to VSMC growth and/or differentiation regulation. Consistent with this notion are studies performed by Chin and co-workers (20,21) suggesting that CHF2 (HRT1) and CHF1 (HRT2) may function as molecular regulators of muscle cell terminal differentiation.
In preliminary studies, we have demonstrated an intriguing bifunctional effect of constitutive Notch3 receptor activation on the regulation of VSMC growth (41). Additional studies are needed to better define the functional role of Notch receptor signaling and downstream HRT effector gene activity in VSMCs.
Studies of Notch-1 knockout mice have documented striking abnormalities in vasculogenesis in addition to other developmental defects (42). Similarly, a striking vasculopathy in zebrafish reminiscent of aortic coarctation is due to a mutation of the downstream Notch effector gene HRT2 (23). Furthermore, it is established that HRT2 activity is critical for arterial versus venous fate specification during the fashioning of the first zebrafish artery. In addition, it is noteworthy that mutations in the Notch3 pathway are associated with an arteriopathy in humans that predisposes them to early-onset stroke (3,6,43). We speculate that changes in the expression and activity of the Notch signaling pathway may also contribute to the pathogenesis of acquired arteriopathies that predispose individuals to stroke. It is anticipated that future investigation of the regulation of the Notch signaling pathway in VSMCs will provide new insights into the molecular mechanisms of vascular complications.