1Vascular Health Research Centre, Faculty of Science and Health, and 2School of Biotechnology and the National Centre for Sensor Research, Dublin City University, Dublin, Ireland; and 3Department of Surgery, University of Rochester Medical Center, Rochester, New York
Submitted 26 April 2005 ; accepted in final form 25 June 2005
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ABSTRACT |
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basic helix-loop-helix; cyclic strain; myosin; smoothelin
Studies in cultured VSMCs have implicated a large number of factors in regulating VSMC differentiation, including mechanical forces (5, 17), contractile agonists, extracellular matrix components, neuronal factors, reactive oxygen species, and endothelial cell-VSMC interactions, all of which promote expression of VSMC marker genes in vitro (18). Surprisingly, despite the facts that cultured VSMC lines are highly modulated and that phenotypic modulation is a critical process in atherogenesis (29) and vascular injury repair (3), few factors and/or pathways have been identified that selectively and directly promote phenotypic modulation of the VSMC, with the exception of platelet-derived growth factor (PDGF)-BB. Researchers in a recent study (19) suggested that activation of Notch signaling mediated through members of the hairy enhancer of split (HES) gene and Hes-related family of transcription (Hrt) factors of basic helix-loop-helix (bHLH) represses smooth muscle cell (SMC) differentiation and the expression of genes encoding SMC contractile markers in a reporter cell line and in embryonic SMCs.
Notch receptor-ligand interactions are a highly conserved mechanism that were originally described in developmental studies using Drosophila. These interactions regulate intercellular communication and direct individual vascular cell fate decisions (10, 28). The intracellular (IC) Notch receptor (Notch IC) is translocated to the nucleus, where it interacts with the CSL {Cp-binding factor 1 (CBF-1)/recombination signal sequence-binding protein-J (RBP-J
), Suppressor of hairless [Su(h)], and Lag-1} family of transcription factors to become a transcriptional activator that can then modulate the expression of Notch target genes that regulate cell fate decisions (10, 28). These include the HES gene and Hrt factors that are critically involved in mammalian cell differentiation (28). Notch receptor-ligand interactions are known to regulate intercellular communication and direct individual cell fate decisions during the development of the embryonic vasculature (10, 28). We and others have recently shown that Notch receptors and downstream target genes (HES and HRT) are crucial in controlling the modulation of SMC growth, migration, and apoptosis in vitro and in vivo (23, 26, 27). Moreover, we have established that equibiaxial cyclic strain inhibits SMC proliferation while enhancing SMC apoptosis through inhibition of Notch receptor and downstream target gene expression (16). In the present study, we have addressed the role of Notch receptors in controlling human VSMC (hVSMC) phenotype and describe for the first time that Notch 1 and Notch 3 receptors repress SMC differentiation in a CBF-1/RBP-J
-dependent manner in vitro. In addition, equibiaxial cyclic strain downregulates SMC Notch receptor expression while promoting SMC differentiation.
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MATERIALS AND METHODS |
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Antibodies.
Polyclonal rabbit anti-Notch 1 IC and Notch 3 IC antibodies were obtained from Upstate Cell Signaling Solutions (Milton Keynes, UK). Polyclonal anti-smoothelin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-myosin (smooth) clone hSM-V, anti--smooth muscle actin clone 1A4, and anti-calponin clone hCP antibodies were obtained from Sigma-Aldrich. Alexa Fluor 488-conjugated antibodies of rabbit anti-
-actin and goat anti-mouse IgG were purchased from Molecular Probes (Leiden, The Netherlands). Peroxidase-conjugated anti-rabbit, anti-mouse, and anti-goat IgG, as well as anti-mouse and anti-goat fluorescein isothiocyanate (FITC) conjugates, were obtained from Amersham Biosciences (Little Chalfont, UK) and Sigma-Aldrich, respectively.
Expression vectors.
Vectors expressing hemagglutinin (HA)-tagged Notch 1 IC (pCMV-ED1-HA) were generously donated by B. Kempkes (8). The Notch 3 IC expression construct (pCMX-Notch 3 IC-HA) was a kind gift from U. Lendahl (12). RPMS-1 (pcDNA3-RPMS-1) was a gift from P. Farrell (22). RPMS-1 is an Epstein-Barr virus (EBV)-encoded viral gene product that specifically inhibits CBF-1/RBP-J signaling in VSMCs. We previously demonstrated the potency of expression of RPMS-1 (23). The product of the EBV RPMS-1 open-reading frame has been shown to regulate the activity of Notch IC negatively by specifically binding to CBF-1/RBP-J
and the CBF-1/RBP-J
-associated corepressors Sin3A and conserved inverted repeat (CIR) and to partially reverse Notch IC-mediated inhibition of differentiation in muscle cells by blocking relief of CBF-1-mediated repression and interfering with Ski-interacting protein (SKIP)-CIR interactions (17). RPMS-1 is therefore a specific negative regulator of Notch IC trans-activation of Notch target genes through interactions with proteins in the Notch corepressor complex. The plasmid pGK3-puro encoding puromycin resistance (25) was a kind gift from P. Ling.
Cell culture and cyclic strain.
Human aortic SMCs were purchased from Cell Applications (San Diego, CA). Cells were maintained in a 37°C humidified atmosphere of 5% CO2-95% air in RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg of streptomycin. Cells were routinely subcultured (passages 318) after treatment for 5 min with 0.125% trypsin-EDTA at 37°C. For cyclic strain studies, hVSMCs were seeded into six-well Bioflex plates (Dunn Labortechnik, Asbach, Germany) at a density of 5 x 105 cells/well. Bioflex plates contain a pronectin-coated silicon membrane bottom that enables the precise deformation of cultured cells with the use of a microprocessor-controlled vacuum (2). Once 90% confluence was reached, cells were exposed to a physiological level of equibiaxial cyclic strain (10% strain, 60 cycles/min, 24 h, cardiac simulation waveform) using a Flexercell Tension Plus FX-4000T unit (Flexcell International, Hillsborough, NC).
Plasmid preparation and transient transfection.
Plasmids were prepared for transfection according to the manufacturer's instructions using a Qiagen plasmid midi kit (Qiagen, Crawley, UK). Cells were plated onto six-well plates 2 days before transfection at a density of 1 x 105 cells/well and transfected at 70% confluence. Plasmid transfection was performed using Lipofectamine reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. The cells were transfected with various expression constructs, with the addition of a total of 2.0 µg of DNA/well. Cells were harvested 1624 h posttransfection using 1x reporter lysis buffer (Promega, Madison, WI). Transfection efficiency was verified by performing -galactosidase assays, and Western blot analysis was used to confirm overexpression of effector proteins. For constitutively active Notch IC transient overexpression studies, cells transfected with vectors expressing Notch 1 IC, Notch 3 IC, and RPMS-1 were cotransfected with pGK3-puro and selected after treatment of cells with 0.8 mg/ml puromycin for 48 h (16, 23). At this concentration of puromycin, >80% of cells that survived after 48 h were transfected with the plasmid of interest.
Preparation of cell lysates.
Harvested cells were pelleted by performing low-speed centrifugation. The cell pellet was placed in ice-cold lysis buffer [20 mM Tris, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100 (vol/vol), 2.5 mM sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM sodium orthovanadate, and 1 µg/ml leupeptin, pH 7.5] and subjected to ultrasonication with a sonic dismembrator (Vibracell; Sonics and Materials, Gland, Switzerland). Samples were divided into aliquots and stored at 80°C before use. Protein concentration was determined using the method of Bradford and BSA as a standard.
Western blot analysis.
Proteins from cell lysates (1020 µg) were analyzed by SDS-PAGE with the use of 7% or 12% resolving gels, followed by transfer to nitrocellulose membranes. Equal protein loading and transfer efficiency were determined by staining the nitrocellulose membranes with Ponceau S (Amersham Biosciences). Membranes were rinsed in wash buffer (PBS containing 0.05% Tween 20) and blocked for 60 min in wash buffer containing 5% nonfat dried milk at room temperature and gently agitated. After three 15-min washes in wash buffer, membranes were incubated overnight at 4°C with primary antibody (-actin, 1:2,000 dilution; calponin or myosin, 1:500 dilution) in PBS/Tween 20/milk. The dilution factor for each antibody was determined empirically. After three 10-min washes in wash buffer, membranes were incubated with the appropriate secondary antibody (1:1,000 dilution for calponin and myosin; 1:2,000 dilution for
-actin) in PBS/Tween 20/milk for 34 h at room temperature on an orbital shaker. After three final 15-min washes, the ECL detection reagent (Amersham Biosciences) was placed on the membranes for 5 min before they were exposed to ECL HyperFilm. The signal intensity of the appropriate bands on the autoradiogram was calculated using the EDAS 120 system (Kodak, Rochester, NY).
Immunocytochemistry.
hVSMCs were seeded onto six-well plates 2 days before staining at 2 x 105 cells/well. Cells were stained for differentiation markers or Notch signaling pathway component protein expression at 8090% confluence. In brief, confluent hVSMCs were permeabilized, fixed with ice-cold methanol for 10 min, and subsequently rehydrated with PBS containing 3% BSA for 10 min. Cells were then incubated in the appropriate primary antibody (1:200 dilution for -actin; 1:50 dilution for calponin, myosin, and smoothelin in PBS/3% BSA) at 4°C overnight with gentle agitation. After five 10-min washes in PBS, cells were incubated in the appropriate secondary antibody (1:200 dilution in PBS/3% BSA using FITC or anti-mouse Alexa Fluor) for 2 h at 37°C in a humidified chamber. Cells were then washed three times in PBS before visualization using an Olympus DP-50 fluorescent microscope (with appropriate excitation and emission spectra at x200 and x400 magnification) coupled with Studio Life software.
Quantitative real-time RT-PCR.
For quantitative measurement of mRNA, we used quantitative real-time RT-PCR (QRT-PCR) with GAPDH mRNA levels as an internal control. Total RNA from cell pellets was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's specifications before 1 µg of total RNA was reverse transcribed in a reaction containing 1x Moloney murine leukemia virus (MMLV) reverse transcriptase buffer (Promega, Madison, WI), 5 µM oligo(dT) 12-18 (Invitrogen), 1 mM 2-deoxynucleotide 5'-triphosphate, 2 µg of acetylated BSA, and 200 U of MMLV reverse transcriptase (Promega) at 37°C for 1 h before 2 U/µl RNase H (Promega) was added for 20 min at 37°C. QRT-PCR was performed using the Rotor Gene (RG-3000) and the SYBR Green PCR kit (Qiagen) as described by the manufacturer. PCR was performed using the following specific primers (MWG Biotech, Ebersberg, Germany): -actin reverse primer, 5'-GTA CGT CCA GAG GCA TAG AG-3', forward primer, 5'-ATC TGG CAC CAC TCT TTC TA-3'; myosin forward primer, 5'- GGAGGATGAGATCCTGGTCA-3', reverse, 5'-TTAGCCGCACTTCCAGTTCT-3'; and smoothelin forward primer, 5' GGCAGTGTCACTCACGTCAC-3', reverse, 5' CTGATCCAGCATCTTGTCCA-3'. The conditions for amplification were 5 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 58°C, and 2 min at 72°C. The specificity of PCR products was also validated by performing electrophoresis on 2% agarose gels and visualized using ethidium bromide staining.
siRNA transfection. For gene silencing studies, Lipofectamine 2000 reagent (Invitrogen, The Netherlands) was used for transient transfection of SMCs with gene-specific small interfering RNA (siRNA) duplexes as previously described (16). The siRNA duplexes for Hrt-1, Hrt-2, and Hrt-3 were as follows: scrambled, aa-auucua ucacuagcgugac; Hrt-1, aa-gacggagaggcaucaucga; Hrt-2, aa-ccaccucucaga uuauggc; and Hrt-3 aa-gcgcagagggaucauagag. All duplexes were acquired from MWG Biotech (Milton Keynes, UK).
Data analysis. Results are expressed as means ± SE. Experimental points were performed in triplicate, with a minimum of three independent experiments. An unpaired Student's t-test and a Wilcoxon signed-rank test were used for comparison of two groups. P < 0.05 was considered statistically significant.
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RESULTS |
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DISCUSSION |
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Recent studies have significantly advanced the understanding of the effect of Notch signaling on vascular development (1, 9, 7, 13). During the early stages of vasculogenesis, SMCs are highly migratory, undergo rapid cell proliferation, and exhibit high rates of synthesis of ECM components (17, 18). It is known that the loss of Notch signaling in zebra fish embryos leads to molecular defects in arteriovenous differentiation, including loss of artery-specific markers within the dorsal aorta (14). Moreover, Notch mutations are linked to some late-onset hereditary vascular pathological conditions such as CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), which further suggests the important contribution of this signaling pathway to vascular homeostasis (11). In contrast, in adult blood vessels, SMCs show a low rate of proliferation and/or turnover, are largely nonmigratory, show a low rate of synthesis of ECM components, and are focused primarily on contractile function (21). The model that has emerged for SMCs within adult mammals is a highly plastic cell population that is capable of rather profound alterations in phenotype in response to changes in local environmental cues that are important for differentiation (17, 18).
Our study is the first to demonstrate in adult human arterial SMCs that Notch decreases SMC differentiation marker expression in a CBF-1/RBP-J-dependent manner. Our findings in hVSMCs are that inhibition of endogenous Notch IC with RPMS-1 or Hrt knockdown enhances SMC differentiation marker expression, while Notch activation directly decreases SMC contractile phenotype and concomitantly increases cell proliferation (23, 26, 27). This notion supports a potentially critical role for Notch signaling in mediating the response of VSMCs to arterial injury by regulating the expression of genes encoding contractile proteins and promoting cell proliferation. Indeed, investigators at our laboratory (16, 23) and others (26, 27) have previously reported that Notch regulates the growth, migration, and death of SMCs in vitro through a CBF-1/RBP-J
-dependent pathway, a response that is mirrored in animal models of vascular injury in which Notch receptors are differentially regulated postinjury as changes in vascular cell fate occur unabated (15, 26). These findings strongly suggest a possible nexus in which the activation of Notch signaling is coupled to the control of SMC differentiation marker expression and phenotype in vitro and is mirrored in vivo.
The mechanisms of how Notch controls SMC phenotype are currently unknown, but recent studies have highlighted the role of Notch signaling in myocardin-dependent transcription of SMC-restricted genes in a reporter cell line (C3H10T1/2 fibroblasts) and in embryonic rat SMCs (19). Consistent with our findings in hVSMCs, forced expression of Hrt-2 in embryonic A10 cells inhibited myocardin-induced expression of SMC-restricted genes and the activity of SMC-restricted transcriptional regulatory elements (19). In addition, the repressive function of Hrt-2 was not mediated via the capacity of the bHLH to bind SMC CArG [CC(AT) 6 GG] elements or by disruption of myocardin-serum response factor protein complexes. Using selective knockdown of HRT, we have demonstrated, in agreement with these findings, that Hrt-2 is an important bHLH factor in mediating Notch IC inhibition of SMC myosin expression. We have further demonstrated that Hrt-1 and Hrt-2 regulated the expression of myosin and smoothelin to varying degrees, suggesting a further complex interaction between individual bHLH factors and their respective downstream targets. Furthermore, a preliminary report by Berrou et al. (4) suggested that Notch 3 modulation of the -actin cytoskeleton in SMCs, despite RhoA involvement in SMC phenotypic modulation, was RhoA independent. Collectively, these data are all consistent with a model whereby Notch receptor activation through CBF-1/RBP-J
-dependent mechanisms represses SMC differentiation and maintenance of the contractile SMC phenotype.
In the present study, equibiaxial cyclic strain reduced both Notch 1 and Notch 3 activation concomitant with a significant increase in SMC -actin, calponin, myosin, and smoothelin expression. The strain-induced decrease in Notch activation in human cells mirrors that observed in animal cells (16) and in vivo (15). Moreover, the cyclic strain-induced increase in SMC differentiation marker expression was further enhanced after inhibition of CBF-1/RBP-J
-dependent signaling using RPMS-1. These data are in accord with those from previous studies that documented a dual effect of strain on SMC phenotype characteristics by potentiating SMC proliferation, an attribute of a dedifferentiated phenotype, while concomitantly increasing the expression of the high molecular weight form of caldesmon, considered a marker of a differentiated SMC state (5). Consistent with the current findings, cyclic strain increased
-actin protein expression and promoter activity through a mechanism involving MAPK family (Erk, JNK, and p38) (24). Because the strain-induced decrease in Notch IC expression and activity in SMC is Erk dependent (16), it is clear that cyclic strain may modulate SMC phenotype through similar mechanisms that promote changes in SMC growth (i.e., proliferation and apoptosis). Collectively, these studies indicate that in adult SMCs, mechanical strain leads to increased expression of smooth muscle differentiation markers, resulting in a more contractile phenotype. Moreover, biomechanical activation of SMCs enhances SMC differentiation in part through inhibition of Notch activation in these cells. Moreover, these data suggest that Notch, in addition to its role in vascular development, may also play a significant role in determining adult cell fate in vascular cells subjected to various environmental cues.
In conclusion, we have shown for the first time that Notch promotes changes in hVSMC phenotype via activation of CBF-1/RBP-J-dependent pathways in vitro and contributes to the phenotypic response of SMCs to cyclic strain. Future studies will address the regulation of SMC differentiation downstream of activation of Notch target genes in an effort to provide new insights into the molecular mechanisms underlying changes in vascular cell fate that underlie vascular proliferative disease.
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GRANTS |
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FOOTNOTES |
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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.
* D. Morrow and A. Scheller contributed equally to this work.
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