Smooth Muscle α-Actin Is a Direct Target of Notch/CSL
Intercellular signaling mediated by Notch receptors is essential for proper cardiovascular development and homeostasis. Notch regulates cell fate decisions that affect proliferation, survival, and differentiation of endothelial and smooth muscle cells. It has been reported that Jagged1–Notch interactions may participate in endocardial cushion formation by inducing endothelial-to-mesenchymal transformation. Here, we show that Notch directly regulates expression of the mesenchymal and smooth muscle cell marker smooth muscle α-actin (SMA) in endothelial and vascular smooth muscle cells via activation of its major effector, CSL. Notch/CSL activation induces SMA expression during endothelial-to-mesenchymal transformation, and Notch activation is required for expression of SMA in vascular smooth muscle cells. CSL directly binds a conserved cis element in the SMA promoter, and this consensus sequence is required for Notch-mediated SMA induction. This is the first evidence of the requirement for Notch activation in the regulation of SMA expression.
- endothelial cells
- smooth muscle cells
- smooth muscle actin
- endothelial-to-mesenchymal transformation
Either loss or gain of function of the Notch pathway causes defects in cardiovascular development in human, mouse, and zebrafish.1,2 Notch mediates intercellular signals that affect proliferation, survival, and differentiation of endothelial and vascular smooth muscle cells (SMC).3–7 We and others have recently shown that Jagged1–Notch interactions may participate in endocardial cushion formation by inducing endothelial-to-mesenchymal transformation (EMT).3,8
Engagement of Notch receptors by their ligands results in a 2-step cleavage that releases the intracellular domain (NotchIC), permitting translocation to the nucleus. Presenilin-dependent γ-secretase activity is essential for the ultimate intramembrane clip that releases NotchIC.9 Following nuclear localization, NotchIC interacts with the DNA-binding factor CSL (also known as RBP-Jκ and CBF1), resulting in transactivation of various promoters, such as those of the HES and HEY families.10,11
Notch-mediated mesenchymal transformation results in loss of endothelial markers and induction of mesenchymal proteins such as smooth muscle α-actin (SMA).3 However, the mechanism of Notch-induced SMA expression has not been studied. SMA is the most abundant protein in SMC and appears to play an important role in mechanotransduction and generation of traction forces in SMC as well as myofibroblasts.12 Here we demonstrate that Notch-mediated upregulation of SMA is directly dependent on the activation and binding of CSL to the SMA promoter. Importantly, not only is Notch/CSL-dependent induction of SMA involved in EMT, but it is also required for SMA expression in SMC.
Materials and Methods
The human microvascular endothelial cell line HMEC-1 (HMEC), human aortic endothelial cells (HAEC), and human umbilical vein endothelial cells (HUVEC) were obtained as previously described.3,5 Primary human foreskin fibroblasts (HFF) were provided by Dr C. Sherlock (St. Paul’s Hospital, Vancouver, BC). Human aortic SMC (HASMC) were purchased from Cascade Biologics. Culture conditions are described in the expanded Materials and Methods section available in the online data supplement at http://circres.ahajournals.org.
Plasmids, Gene Transfer, and RNA Interference
Cells were transduced as previously described.3,5 For a description of plasmids and details on small interfering RNA (siRNA), see the online data supplement.
Immunoblotting and Immunofluorescence Staining
Immunoblotting, immunostaining, and image acquisition were performed as described previously.5 Antibodies are listed in the online data supplement.
The SMA-promoter luciferase construct (gift of F. Dandre and G. K. Owens, University of Virginia Health Sciences Center, Charlottesville) has been previously described.3,13 See the online data supplement for more details.
Chromatin Immunoprecipitation Assay
HMEC transduced with LNCX or vector expressing Flag-tagged CSL were fixed in 1% formaldehyde, lysed, and sonicated. One percent of total chromatin was used as positive control for PCR (online data supplement).
Results and Discussion
We first confirmed that Notch1IC induces SMA expression in endothelial cells by immunoblotting (Figure 1A). Activated Notch1 also induced expression of SMA in primary human fibroblasts (Figure 1B). Given that SMA is a marker of differentiation for SMC and that these cells express endogenous Notch1, Notch2, and Notch3, as well as the ligand Jagged1, we tested whether activated Notch and Jagged1-induced Notch activation would induce upregulation of SMA in HASMC.5,14 Immunoblotting demonstrated that both Notch1IC and Jagged1-mediated Notch activation induced expression of SMA in SMC (Figure 1C and 1D). To determine whether Notch regulates SMA expression in SMC through endogenous Notch ligand–receptor interactions, HASMC transduced with vector alone (HASMC-vector) or Jagged1 (HASMC-Jagged1) were treated with vehicle or a γ-secretase inhibitor (GSI).5,8 Inhibition of Notch processing blocked expression of SMA in control cells as well as in Jagged1 cocultures, indicating that in SMC the endogenous Notch pathway participates in the maintenance of SMA expression and that Jagged1-mediated induction of SMA is dependent on Notch activation (Figure 1E). These results indicate that the Notch pathway is a major regulator of the expression of SMA not only during EMT but also in fibroblasts and importantly in SMC.
Notch activation also induced expression of SMA mRNA and activated the SMA promoter as seen in a promoter-luciferase assay in HMEC and 293T cells (supplemental Figure I and data not shown), suggesting that Notch induces SMA through transcriptional activation.3,13 The human SMA promoter contains a CSL consensus binding site (TGGGAA) beginning at −64 from the cap site that is conserved in apes and rodents (supplemental Figure II).3,13 We thus tested whether CSL activation was sufficient to induce SMA expression. Transduction of constitutively active CSL (engineered by fusing CSL with the transcriptional activation domain of the herpes viral protein 16 [CSL-VP16]) was sufficient to induce expression of SMA in endothelial cells and fibroblasts (Figure 2A).15 In addition, transfection of CSL-VP16 was sufficient to activate the SMA promoter in endothelial cells, as demonstrated by promoter-luciferase assay (Figure 2B).
To determine whether CSL is necessary for Notch-mediated SMA induction, HFF were double-transduced with Notch1IC and CSL-DN (dominant-negative CSL) or the empty vectors. Notch-induced morphological changes were lost in cells coexpressing Notch1IC and CSL-DN (data not shown). Immunoblotting showed decreased expression of SMA in cells transduced with CSL-DN and Notch1IC compared with cells transduced with Notch1IC and the empty vector (Figure 2C). Secondly, we used lentiviral transduction of short-hairpin RNAs to generate siRNAs targeting CSL (siCSL). Cells were infected with empty vector or Notch1IC and 1 of 2 lentiviral constructs targeting 2 different sequences of CSL (siCSLa or siCSLb) or a nonsilencing control (siRandom). Knock-down of CSL was confirmed by RT-PCR (Figure 2D). Cells infected with siCSLa or siCSLb and Notch1IC showed reduced expression of SMA compared with control cells (Figure 2D). Thus, Notch-mediated induction of SMA is mediated through activation of CSL.
To test whether the putative CSL-binding site in the SMA promoter is required for its activation, the consensus sequence was disrupted by site-directed mutagenesis. Endothelial cells were cotransfected with NotchIC or control vector and either the wild-type (SMA-WT) or CSL-binding site–mutated (SMA-mut) SMA-promoter luciferase constructs. Results show complete inhibition of Notch-dependent luciferase activation when the CSL-binding site is mutated (Figure 3A). To confirm a role for endogenous Notch/CSL signaling in regulation of the SMA promoter in SMC, we transfected HASMC with SMA-WT or SMA-mut (Figure 3B). Luciferase assays demonstrate that mutation of the CSL-binding site significantly reduces activation of the SMA promoter in SMC, suggesting a critical role for endogenous CSL activity in inducing and maintaining expression of SMA. To confirm that CSL directly binds the SMA promoter, chromatin immunoprecipitation assay (ChIP) assays were performed in HMEC transduced with vector encoding Flag-tagged CSL or the empty vector. PCR of Flag immunoprecipitated DNA using primers flanking the CSL consensus site confirmed that CSL directly binds the SMA promoter (Figure 3C).
In summary, this study shows that Notch/CSL signaling directly regulates expression of SMA by a transcriptional mechanism that requires binding of CSL to the SMA promoter. Of note, Notch/CSL-mediated induction of SMA is involved in EMT and fibroblast acquisition of SMA, as well as in the maintenance of SMA expression in SMC. These data also trigger more questions regarding the role of Notch in the vasculature. For instance, what are the factors that determine cell-type and context-specific effects of Notch in the endothelial and mural compartments? Notch1 and Jagged1 have been detected in both endothelial cells and SMC, and whether specific Notch or ligand expression (eg, Notch3 in SMC and Notch4 and Dll4 in endothelial cells) contributes to these decisions remains to be investigated. On a broader perspective, given that Notch appears to play a role in tissue regeneration and that myofibroblasts and SMC appear to use SMA to transmit mechanical forces through the cell, our data provide a potential explanation of the role of Notch in wound healing and vascular remodeling.12,16,17
We thank Denise McDougal for assistance with cell sorting and Dana Wong for technical help.
Sources of Funding
This research was supported by grants to A.K. from the Heart and Stroke Foundation of British Columbia and the Yukon and the Canadian Institutes of Health Research. M.N. was supported by a fellowship from the Canadian Institutes of Health Research and a Research Trainee Award from the Michael Smith Foundation for Health Research. Y.F. and K.N. are supported by Research Trainee Awards from the Michael Smith Foundation for Health Research. A.K. is a scholar of the Michael Smith Foundation for Health Research.
Original received March 23, 2006; revision received April 27, 2006; accepted May 18, 2006.
Noseda M, McLean G, Niessen K, Chang L, Pollet I, Montpetit R, Shahidi R, Dorovini-Zis K, Li L, Beckstead B, Durand RE, Hoodless PA, Karsan A. Notch activation results in phenotypic and functional changes consistent with endothelial-to-mesenchymal transformation. Circ Res. 2004; 94: 910–917.
MacKenzie F, Duriez P, Wong F, Noseda M, Karsan A. Notch4 inhibits endothelial apoptosis via RBP-Jkappa-dependent and -independent pathways. J Biol Chem. 2004; 279: 11657–11663.
Noseda M, Chang L, McLean G, Grim JE, Clurman BE, Smith LL, Karsan A. Notch activation induces endothelial cell cycle arrest and participates in contact inhibition: role of p21Cip1 repression. Mol Cell Biol. 2004; 24: 8813–8822.
Domenga V, Fardoux P, Lacombe P, Monet M, Maciazek J, Krebs LT, Klonjkowski B, Berrou E, Mericskay M, Li Z, Tournier-Lasserve E, Gridley T, Joutel A. Notch3 is required for arterial identity and maturation of vascular smooth muscle cells. Genes Dev. 2004; 18: 2730–2735.
Campos AH, Wang W, Pollman MJ, Gibbons GH. Determinants of Notch-3 receptor expression and signaling in vascular smooth muscle cells: implications in cell-cycle regulation. Circ Res. 2002; 91: 999–1006.
Timmerman LA, Grego-Bessa J, Raya A, Bertran E, Perez-Pomares JM, Diez J, Aranda S, Palomo S, McCormick F, Izpisua-Belmonte JC, de la Pompa JL. Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation. Genes Dev. 2004; 18: 99–115.
Shimizu RT, Blank RS, Jervis R, Lawrenz-Smith SC, Owens GK. The smooth muscle alpha-actin gene promoter is differentially regulated in smooth muscle versus nonsmooth muscle cells. J Biol Chem. 1995; 270: 7631–7643.
Noseda M, Niessen K, McLean G, Chang L, Karsan A. Notch-dependent cell cycle arrest is associated with downregulation of minichromosome maintenance proteins. Circ Res. 2005; 97: 102–104.
Raya A, Koth CM, Buscher D, Kawakami Y, Itoh T, Raya RM, Sternik G, Tsai HJ, Rodriguez-Esteban C, Izpisua-Belmonte JC. Activation of Notch signaling pathway precedes heart regeneration in zebrafish. Proc Natl Acad Sci U S A. 2003; 100 (suppl 1): 11889–11895.