Initial studies have established expression of low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) in vascular smooth muscle cells (VSMCs). We hypothesized that LRP6 is a critical mediator governing the regulation of the canonical Wnt/β-catenin/T cell factor 4 (Tcf-4) cascade in the vasculature. This hypothesis was based on our previous work demonstrating a role for the β-catenin/Tcf-4 pathway in vascular remodeling as well as work in other cell systems establishing a role for LRP family members in the Wnt cascade. In line with our hypothesis, LRP6 upregulation significantly increased Wnt-1-induced Tcf activation. Moreover, a dominant interfering LRP6 mutant lacking the carboxyl intracellular domain (LRP6ΔC) abolished Tcf activity. LRP6-induced stimulation of Tcf was blocked in VSMCs harboring constitutive expression of a dominant negative Tcf-4 transgene lacking the β-catenin binding domain, suggesting that LRP6-induced activation of Tcf was mediated through a β-catenin-dependent signal. Expression of the dominant interfering LRP6ΔC transgene was sufficient to abolish the Wnt-induced survival as well as cyclin D1 activity and cell cycle progression. In conclusion, these findings provide the first evidence of a role for an LDL receptor-related protein in the regulation of VSMC proliferation and survival through the evolutionary conserved Wnt signaling cascade.
- cyclin D1
- T cell factor-4
regulation of vascular smooth muscle cell (VSMC) proliferation and apoptosis plays a critical role in the pathogenesis of vascular disease and remodeling (4, 11, 12, 45). Recent work from our lab has identified a role for the evolutionary conserved Wnt signaling pathway in governing VSMC proliferation and survival in the context of vascular injury (54).
Wnts are a family of secreted glycoproteins that bind to a class of Frizzled receptors (3, 9, 21, 40). The conserved Wnt cascade is known to govern cell fate, proliferation, differentiation, and polarity (3, 9, 21, 40). Wnt binding to the Frizzled family of receptors stabilizes and translocates β-catenin to the nucleus and activates a family of T cell factor/lymphoid enhancing factors (Tcf/Lef) (3, 40). We demonstrated a robust temporal upregulation of β-catenin in intimal VSMCs that led to activation of the transcription factor Tcf-4 (54). We further demonstrated that activation of the Wnt cascade led to transactivation of the Tcf-responsive gene cyclin D1, an increase in VSMC proliferation, and inhibition of apoptosis (54). Loss of Tcf-4 function abolished both proliferative and survival promoting effects (54). The objective of this study was to extend these findings and assess the previously undefined role of low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) in the regulation of Wnt signaling in the vasculature.
LRP6 and LRP5 are members of the LRP superfamily that have recently been found to be required for activation of the canonical Wnt signaling pathway (35, 37, 42, 50, 56). LRP5 and LRP6 are single transmembrane proteins that contain four epidermal growth factor-like repeats and three LDL receptor type A repeats (7, 17). LRP6 knockout mice develop multiple developmental defects that resemble a combination of several Wnt mutant phenotypes, namely, Wnt-1, Wnt-3a, and Wnt-7a (42). Several groups have identified LRP1 expression in VSMCs (18, 28–32, 55); however, LRP6 expression has not been previously characterized in VSMCs.
Our objective was to test the hypothesis that LRP6 plays an integral role in Wnt signaling in VSMCs. Our findings support this hypothesis and provide the first evidence of a well-defined pathway linking an LDL receptor-related protein to VSMC proliferation and survival.
MATERIALS AND METHODS
The following plasmids were utilized in this study: mouse Wnt-1, human LRP6 and LRP5, well-characterized dominant interfering LRP6 (LRP6ΔC; which lacks the carboxyl intracellular domain necessary to stimulate Wnt signaling) (all gifts from X. He) (50), dominant negative Tcf-4 construct, Tcf-4ΔN31 [Tcf-4(N31); which lacks the amino terminal 31 amino acids] (E. Fearon) (23), β-catenin mutant construct (in which the conserved serine/threonine residues in the amino terminus were mutated to alanines) (D. Kimelman), the reporter constructs Topflash and Fopflash (which contained either three copies of the optimal Tcf motif CCTTTGATC or the mutant motif CCTTTGGCC upstream of a minimal c-Fos promoter driving luciferase expression) (B.Vogelstein) (24), cyclin D1 luciferase reporter construct (R. Pestell) (8), enhanced green fluorescent protein (EGFP) expression vector (pEGFP-C1; Clontech; Palo Alto, CA), mito tracker red expression vector (pDsRed1-Mito) and pDsRed1-Mito (all from Clontech), and pcDNA3.1 expression vector (Invitrogen).
C57BL6 mice were euthanized in accordance with an Institutional Animal Care and Use Committee-approved protocol at the University of Minnesota. Tissues were harvested for RNA isolation and quantitation of LRP6 expression.
The clonal A7r5 rat aortic VSMCs were purchased from American Type Culture Collection. Human aortic VSMCs were purchased from Clonetics and used between passages 2 and 4.
A7r5 VSMCs were transiently transfected with Effectene (Qiagen) as previously described with a total of 0.3 μg DNA/well according to the manufacturer's directions. Transfection efficiency is ∼20%. Rat A7r5 VSMCs were cultured in 12-well plates for transient transfection. The following day, the cells had achieved ∼60% confluency and were transiently transfected according to the manufacturer's directions. The ratio of DNA utilized was as follows: 3 μg of total DNA were transfected per well (0.125 μg for Wnt or LRP6, 0.025 μg Tcf reporter, 0.025 μg EGFP, and pCDNA3.1 as a control). Cells were harvested 48 h posttransfection.
Real-time quantitative PCR, RT-PCR, and product sequencing.
Cells were exposed to trypsin-EDTA and harvested. Snap-frozen rat vessels and murine tissues were pulverized in liquid nitrogen. Total RNA was isolated with RNeasy columns with RNase-free DNase treatment. One microgram of total RNA was used for the RT reactions using oligo(dT)18 according to the manufacturer's directions (Clontech) as previously described (54). Real-time quantitative PCR (RTQPCR) analysis was performed with a Light Cycler (Roche Diagnostics). Reactions were prepared in the presence of the fluorescent dye SYBR green I for specific detection of double-stranded DNA as previously described (54). Cumulative fluorescence was measured at the end of the extension phase of each cycle. Primer sequences and reaction parameters are depicted in Table 1. Primers for LRP6 and LRP5 were designed to a conserved region in the mouse, rat, and human. Product-specific amplification was confirmed by a melting curve analysis and agarose gel electrophoresis analysis. Quantification was performed at the log-linear phase of the reaction, and cycle numbers obtained at this point were plotted against a standard curve prepared with serially diluted samples. Results were normalized to GAPDH or, in the case of LRP6 expression, in mouse tissue normalized to identical amounts of RNA given the difficulty of finding a housekeeper gene that is expressed at similar levels across all tissues. RT-PCR was carried out using a MJ thermocycler.
For sequence analysis, 2 μl of cDNA were used for PCR with primers. Primer sequences and reaction parameters are depicted in Table 2. PCR products were run on a 2% DNA agarose gel, visualize with ethidium bromide, and then photographed under ultraviolet light illumination. For product sequencing of LRP6 and LRP5 from both human VSMCs and the murine aorta, the amplified products were purified using the PCR purification kit (catalog no. 28104, Qiagen) and then sequenced. Sequence alignment was obtained by BLAST searching against the GenBank database [human LRP6 (AF074264), human LRP5 (AF064548), mouse LRP6 (BC060704.1), and mouse LRP5 (NM_008513)].
LRP6ΔC was subcloned to the shuttle vector pacAd5CMVK-Np. Viruses were prepared and titrated by the Roy J. and Lucille A. Carver College of Medicine Gene Vector Core Lab at the University of Iowa (http://www.uiowa.edu/∼gene/index.htm) as previously described (1). The control adenovirus used was pacAd5CMV-GFP. Virus stocks were aliquoted and stored at −80°C. VSMCs were incubated for 6 h at 37°C with 0.5 ml of serum-free culture medium containing 4–100 plaque-forming units (pfu)/cell and then placed in fresh DMEM + 10% FBS. Preliminary studies confirmed that this dose range of control adenovirus had no significant effect on VSMC proliferation, apoptosis, or morphology.
Quantitation of apoptosis.
Apoptosis was assessed by staining with Hoechst 33342 (H33342) and quantitating the percentage of apoptotic nuclei (300 cells counted/sample) in the transfected subset by identifying cells cotransfected with pDsRed1-Mito as previously described (13–15, 54). We have extensively cross-validated the use of H33342 staining with other apoptotic indexes (15, 45).
Cell cycle analysis.
Cell cycle analysis was assessed by FACS in VSMCs infected with an EGFP adenovirus or the dominant interfering LRP6 mutant (4 pfu/cell) as previously described (54). Twenty-four hours postadenoviral infection, cells were exposed to 1% FBS for 8 h to induce a quiescent state, followed by stimulation with 1% or 10% FBS for 18 h.
Comparisons between two groups were analyzed via a Student's t-test (P < 0.05), whereas comparisons between three groups were analyzed by ANOVA with a Student-Newman-Keuls post hoc test (P < 0.05). Data are presented as means ± SE.
We utilized RTQPCR to define relative mRNA expression of LRP6 in the aorta compared with other tissues including the brain, heart, liver, and skeletal muscle in the mouse (Fig. 1A). LRP6 expression in the aorta was comparable to that in the brain, less than LRP6 expression in the heart and liver, and greater than expression in skeletal muscle. LRP6 expression levels were also compared in rat and human VSMCs as well as the rat carotid artery (Fig. 1B). The expression level of LRP6 was similar between the rat and human. Specificity of the product was determined by a stepwise melting curve analysis and confirmed by assessing the size of the PCR product (466 bp) on a gel. Sequence verification of LRP6 PCR products from human primary VSMCs and the mouse aorta revealed 99% homology with GenBank sequences for human LRP6 (AF074264) and mouse LRP6 (BC060704.1) (Fig. 1C).
Given the close homology between LRP5 and LRP6, we also determined the expression levels of LRP5 in both rat and human VSMCs using RTQPCR. LRP6 expression in rat VSMCs normalized to GAPDH was 0.90 ± 0.05, whereas LRP5 expression was 0.02 ± 0.01 (n = 4). LRP6 expression in human VSMCs normalized to GAPDH was 1.05 ± 0.01, whereas LRP5 was 0.98 ± 0.02 (n = 4). The potential differences in efficiencies of RTQPCR preclude any definitive conclusions regarding the absolute abundances of LRP6 and LRP5. However, a rough estimate of the ratio of LRP6 to LRP5 was ∼45:1 in rat aortic clonal VSMCs and ∼1:1 in primary human aortic VSMCs. Sequence verification of LRP5 PCR products from human primary VSMCs and the mouse aorta revealed 99% and 96% homology with human LRP5 (AF064548) and mouse LRP5 (NM_008513) (Fig. 1D).
In line with our previous findings, upregulation of Wnt-1 or a degradation-resistant β-catenin transgene (DR β-cat) resulted in significant activation of the well-characterized Tcf/Lef reporter gene Topflash (Fig. 2A). Specificity of the response was demonstrated by the lack of a significant response with the mutated reporter Fopflash. Cotransfection of LRP6 with Wnt-1 resulted in a potent 15-fold increase in Tcf/Lef activity. Interestingly, LRP6 alone had no significant effect on Tcf 4 activation. RT-PCR was used to verify enhanced expression of the transgenes for these experiments (Fig. 2B). LRP5 had similar effects on Tcf/Lef activation (Fig. 2C).
To test our hypothesis that LRP6 is necessary for Wnt-1-stimulated activation of Tcf/Lef, we utilized an adenoviral strategy to dose dependently upregulate a well-defined dominant interfering LRP6 transgene lacking the carboxyl intracellular domain (LRP6ΔC) (50). In line with our hypothesis, increasing amounts of LRP6ΔC competitively inhibited the potentiation of Tcf/Lef activity induced by wild-type LRP6 (Fig. 2D). In addition, LRP6ΔC also dose dependently inhibited Wnt-induced Tcf/Lef activation in the absence of exogenously added LRP6, suggesting a role for endogenous LRP6 in regulating Tcf activity (Fig. 2E). Similar inhibition of Topflash was seen in transient transfection experiments (data not shown).
Previous work from our laboratory has identified that upregulation of β-catenin in VSMCs led to specific activation of Tcf-4 (54). To test the hypothesis that LRP6 potentiation of Wnt-induced Tcf/Lef activation was mediated through β-catenin binding to Tcf-4, we utilized previously characterized VSMCs with constitutive expression of a mutant Tcf-4 gene lacking a β-catenin binding domain [Tcf-4(N31)] (23, 54). In line with our hypothesis, loss of this β-catenin binding domain in Tcf-4 significantly inhibited both Wnt-1- and Wnt-1/LRP6-mediated Tcf transactivation (Fig. 3).
Earlier work from our laboratory demonstrated that stimulation of the Wnt/β-catenin cascade promoted VSMC survival (54). We hypothesized that LRP6 was necessary in promoting this prosurvival pathway. To test this, we utilized LRP6ΔC. Indeed, upregulation of the dominant negative LRP6 transgene significantly inhibited Wnt-1-mediated survival (Fig. 4). To define the role of LRP6 on VSMC survival in the absence of transient upregulation of Wnt1, we infected VSMCs with LRP6ΔC adenovirus and exposed the cells to serum withdrawal. VSMCs harboring LRP6ΔC were significantly more susceptible to serum withdrawal-induced apoptosis (control transfected, 21 ± 2% apoptotic nuclei; LRP6ΔC, 31 ± 2%, n = 5, P < 0.001). Taken together, these findings suggest that LRP6 plays a significant role in VSMC survival.
The cell cycle regulatory protein cyclin D1 contains a Tcf-responsive element in its promoter region (52). We previously demonstrated that activation of the Wnt cascade in VSMCs significantly enhanced cyclin D1 transactivation (54). We hypothesized that LRP6 would synergistically stimulate Wnt-induced cyclin D1 activity. Indeed, cotransfection of LRP6 with Wnt-1 synergistically increased cyclin D1 transcriptional activity, whereas blockade of LRP6 with LRP6ΔC abolished Wnt-1- and Wnt-1/LRP6-induced cyclin D1 promoter activity (Fig. 5).
To further define the role of LRP6 in cell cycle progression, we infected VSMCs with the dominant negative LRP6 adenovirus and asked whether this was sufficient to inhibit serum-induced cell cycle stimulation. VSMCs were infected with control or LRP6ΔC adenovirus and stimulated with 1% or 10% serum. Loss of LRP6 function led to a significant increase in the percentage of VSMCs in G0/G1 concomitant with a significant decrease in the percentage of cells in the S phase in VSMCs exposed to both 1% and 10% serum, with more robust inhibition seen in 1% serum (Fig. 6). Thus, in summary, LRP6 appears to play a role in cyclin D1 transcriptional activation and cell cycle progression.
The major findings of this study are the identification of LRP6 in VSMCs and its integral role in the Wnt signaling pathway. Our previous work demonstrated that the highly conserved Wnt cascade plays a critical regulatory role in remodeling of the vessel wall in response to balloon injury by promoting VSMC growth and survival through activation of the transcription factor Tcf-4 (54). In this study, we have extended these findings and demonstrated a role for LRP6 in Wnt-induced VSMC proliferation and survival.
The LRP family is well known for its ability to bind multiple ligands including apolipoprotein E (ApoE), very LDL remnants, lipoprotein lipase, tissue-type and urokinase-type plasminogen activators, and thrombospondin I (25, 26, 39). Work to date has identified only LRP5 and LRP6 as coreceptors for the Wnt signaling cascade (42, 50, 56). Although LRP6 shares a small degree of similarity to other LRP family members, it is clear that LRP5 and LRP6 are distinct from other family members (7). LRP6 is a single-pass transmembrane protein that contains four extracellular epidermal growth factor type repeats and three LDLR repeats (7). The cytoplasmic tail contains the axin binding region and has been shown to be critical in activating the Wnt signaling pathway (37, 50). Our studies further confirm the important role of the cytoplasmic region of LRP6 in promoting Wnt activation.
We utilized both gain and loss of function approaches to demonstrate a role for LRP6 as a critical factor regulating Wnt signaling in VSMCs. Upregulation of LRP6 synergistically activated Wnt-induced activation of Tcf/Lef. This is in agreement with several recent publications (19, 37). Interestingly, our work and that of others has consistently found that LRP5/6 synergistically activates the canonical Wnt pathway but has little effect on the pathway by itself (37). Published ligands for LRP6 include Wnt family members, Dickkhopf, and recently connective tissue growth factor (CTGF) (6, 16, 35, 38, 42, 47, 48). We did not detect any changes in Dickkhopf expression in the conditions utilized in this experiment. Recent work from LRP5 and LRP6 double-knockout mice suggests that the pattern of defective gastrulation in these mice closely resembles those of FGF8 and FGF1 mutants (20). Recent published work also suggests that CTGF is also a ligand for LRP6 (38). The recent demonstration that CTGF binds to LRP6 is intriguing and is likely to be important in the context of vascular remodeling (38). Recent work by Liu et al. (27) demonstrates that LRP6 forms an inactive dimmer through interactions in its extracellular domain and that Wnts induce an intracellular conformational switch that relieves allosteric inhibition. Our findings are consistent with these. We further demonstrated that LRP6 potentiation of Tcf/Lef activity was significantly inhibited in VSMCs harboring a dominant negative Tcf-4 transgene lacking the β-catenin binding domain. These experiments suggested that the ability of LRP6 to stimulate Wnt-mediated activation of Tcf-4 was mediated mainly through β-catenin. It is noteworthy that Wnt-1/LRP6 activation of Tcf/Lef was not completely inhibited in VSMCs harboring the dominant negative Tcf-4 transgene lacking a β-catenin binding domain. Because the establishment of this dominant negative Tcf-4 construct, several spliced isoforms of the Tcf family have been identified that harbor β-catenin binding domains (2), which may explain the reason we saw only partial inhibition. It is also possible that LRP6 may potentiate Tcf-4 through a β-catenin-independent mechanism and/or that LRP6 may stimulate other Tcf isoforms and/or Lef. To test whether LRP6 was necessary for Wnt-1-induced Tcf/Lef activity in VSMCs, we utilized a well-characterized dominant interfering LRP6 mutant to perform loss of function experiments. In accord with our hypothesis, loss of LRP6 abolished Wnt-1-induced Tcf/Lef activity. Furthermore, loss of LRP6 function inhibited cyclin D1 transactivation and significantly inhibited cell cycle progression in VSMCs. The effects of loss of LRP6 were more pronounced under conditions of low serum. The reason for this is unclear but may reflect the effects of serum on other ligands for LRP6, alterations in LRP5 expression, cobinding partners for Tcf-4, or potentially that LRP6 is working through multiple transcription factors governing cell cycle proteins. It is also possible that the more pronounced effect in 1% serum reflects cell loss due to apoptosis. However, the significant difference under conditions of 10% serum, along with significant LRP6-mediated activation of cyclin D1, still suggest a role for LRP6 on cell cycle progression. These findings are in agreement with our previous work demonstrating that loss of Tcf-4 activity significantly inhibited [3H]thymidine uptake in VSMCs in response to serum. To our knowledge, these studies are the first to demonstrate a role for LRP6 and the Wnt cascade in regulating cell cycle.
Prior work from our laboratory and others has demonstrated that mitogens, nutrient signals, or receptor ligand systems that induce a proliferative response also have the capability of promoting VSMC survival (11–13, 15, 44, 45). In line with these findings, we have demonstrated that activation of the Wnt cascade promotes VSMC proliferation as well as survival. Furthermore, we demonstrated that LRP6 was a critical mediator in Wnt-induced cyclin D1 activation and cell cycle progression. Recent work in cancer literature also suggests that cyclin D1 acts as a prosurvival factor (51). Thus we hypothesized that LRP6 plays an important role in inhibiting apoptosis. In line with our hypothesis, LRP6 significantly potentiated Wnt-1-mediated survival, whereas loss of LRP6 function resulted in attenuation of the survival promoting effects.
LRP6 function is regulated by several extracellular antagonists, including Frizzled-related proteins (46), Cerberus (5), WNT inhibitory factor-1 (25), Kremen proteins, and the Dickkopf family (34, 35). Recent work suggests that Dickkopfs interact with LRP6 and inhibit Wnt signaling by disrupting the binding of LRP6 to the Wnt/Frizzled ligand-receptor complex (Fig. 7) (34, 35, 48, 57). Recently, Mao et al. (34, 35) identified two transmembrane proteins, Kremen1 and Kremen2. The Kremen proteins are high-affinity receptors that functionally cooperate with Dickkopf to block Wnt/β-catenin signaling. Evidence currently suggests that Dickkopf interacts with its receptor Kremen to form a ternary complex with LRP6, which promotes endocytosis and prevents the interaction of LRP6 with Wnt-1 (34). In addition to identified antagonists of LRP6, Groucho and CREB have been identified as corepressor proteins that are recruited to the enhancer region of Tcf target genes to inhibit transcription in the absence of sufficient amounts of β-catenin in the nucleus [as reviewed by Barker et al. (3)]. The role and regulation of Groucho and CREB to inhibit Tcf-4 also remain to be defined in VSMCs.
Kim et al. (22) demonstrated that LRP5 is able to recognize the ligand ApoE. Interestingly, LRP5 has been shown to increase in cholesterol-laden foam cells within an atherosclerotic lesion (22), whereas LRP5 expression in VSMCs within the media was nearly undetectable (22). Recent work also suggests a potentiation of atherosclerotic lesions in LRP5 null mice backcrossed to an ApoE null background (33). The ability of LRP6 to recognize ApoE has not yet been defined. Interestingly, evidence suggests that both LRP5 and LRP6 are candidate genes for conferring susceptibility to diabetes (7, 10, 17, 33, 41, 53). Given that the risk of developing vascular disease among the diabetic population is significantly elevated, characterization of the role of polymorphisms in the regulation of LRP5 and LRP6, and the elucidation of how these polymorphisms may translate into altered regulation of the Wnt cascade is an area of research we are currently pursuing.
In conclusion, we identified a role of LRP6 in the regulation of VSMC proliferation and survival. This is the first evidence demonstrating a direct role for a LRP in governing VSMC growth and fate. Furthermore, we demonstrated that LRP6 exerts its effects through the evolutionary conserved Wnt cascade.
This study was supported by American Heart Association Postdoctoral Fellowship Award 025503Z (to X. Wang), by American Heart Association Scientific Development Grant 0030136N (to J. L. Hall), and by the Lillehei Heart Institute at the University of Minnesota.
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.
- Copyright © 2004 by the American Physiological Society