The Role of ?-Transducin Repeat-Containing Protein (?-TrCP) in the Regulation of NF-B in Vascular Smooth Muscle Cells
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《动脉硬化血栓血管生物学》
From the Lillehei Heart Institute, Division of Cardiology, Department of Medicine, University of Minnesota, Minneapolis.
Correspondence to Jennifer L. Hall, PhD, Assistant Professor of Medicine Director, Cardiovascular Genomics Cardiovascular Division Lillehei Heart Institute, University of Minnesota, 420 Delaware St, Minneapolis, MN 55455. E-mail Hallx068@umn.edu
Abstract
Objective— Degradation of IB is an essential step in nuclear factor (NF)-B activation. However, the determinants regulating this process have not been defined in vascular smooth muscle cells (VSMCs). We hypothesized that the E3-ligase, ?-transducin repeat-containing protein 1 (?-TrCP1), was a rate-determining mediator that regulates the ubiquitin-mediated degradation of IB (in VSMC).
Methods and Results— Upregulation of ?-TrCP1 accelerated the rate of IB degradation, leading to increased NF-B activity. In contrast, VSMCs harboring a dominant-negative ?-TrCP1 transgene lacking the F-box domain exhibited a reduction in serum-stimulated NF-kB activity but no alteration in response to tumor necrosis factor (TNF). These findings suggest that ?-TrCP1 increases the rate of NF-B activation but is not rate-limiting in response to TNF in VSMCs. Endogenous ?-TrCP1 expression was regulated through the conserved Wnt cascade. Upregulation of Wnt1 resulted in ?-catenin–mediated activation of Tcf-4, leading to increased ?-TrCP1 expression and NF-B activity. Furthermore, VSMCs harboring a Tcf-4 mutant lacking a ?-catenin binding domain exhibited a significant reduction in ?-TrCP1 expression along with abolishment of NF-B activity.
Conclusions— We provide the first evidence of crosstalk between the Wnt cascade and NF-B signaling in VSMCs. This crosstalk is mediated through the E3-ligase, ?-TrCP1.
Key Words: nuclear factor-B ? ?-TrCP1 ? muscle, vascular, smooth ? Wnt ? ?-catenin
Introduction
Nuclear factor-B (NF-B) acts as a central intersecting node regulating proliferation, survival, and inflammation in vascular smooth muscle cells (VSMCs) in response to cytokines, infectious agents, dyslipidemia, hypertension, angina, vascular injury, and atherosclerosis.1,2 The degradation of IB as an essential step in NF-B activation has been well described.3–6 However, the molecular determinants regulating this process have not been well defined in VSMCs.
NF-B is maintained in an inactive form by sequestration in the cytoplasm through interaction with the IB family of proteins.5,6 This IB family of proteins binds to the Rel homology domain of NF-B members and masks the nuclear localization sequence of NF-B. Stimuli, including tumor necrosis factor- (TNF-) and lipopolysaccharide (LPS), stimulate the phosphorylation of IB on serine residues 32 and 36.5–7 Phosphorylation of these residues is thought to target IB for ubiquitination by the stem cell factor (SCF) ubiquitin/ligase complex and to induce rapid transient degradation.3,5,6,8–10 Although the majority of studies suggest that phosphorylation of IB is a necessary step in the activation of NF-B, it is clear that phosphorylation alone is not sufficient and that the ubiquitin-dependent degradation of IB is a necessary step in NF-B activation.6,11
Work in other cell systems has identified a SCF ubiquitin/ligase complex, which contains the proteins Skp1, Cdc53/Cul1, and an F-box protein, that is thought to recognize phosphorylated IB and regulate its ubiquitination.7 The ubiquitination process requires 3 proteins, E1, E2, and E3. Briefly, ubiquitin is activated as a thiol-ester on E1 in an ATP-dependent reaction and transferred to an E2 as a thiol ester.8,9,12,13 The F-box/WD proteins, designated E3s, can be roughly categorized as HECT, RING finger, and U-box–containing proteins or multiprotein complexes. The F-box is the substrate-recognition component of multiprotein E3s and normally requires a RING finger protein for catalytic activity. The E3 protein responsible for recognizing the phosphorylated motif on IB (DS[PO3]GXS[PO3]), where represents a hydrophobic residue) has recently been identified as ?-TrCP/E3RS/Fwd1.14–16 ?-TrCP binds to lysine resides on phosphorylated IB, leading to recognition by the 26S proteasome and degradation.8,9,12,17,18
?-TrCP also recognizes an identical phosphorylated motif on the proto-oncogene ?-catenin.5,19,20 ?-Catenin is a key mediator in the Wnt pathway. Activation of the Wnt cascade results in the stabilization and translocation of ?-catenin to the nucleus, where it activates a family of transcription factors referred to as T-cell factors/lymphoid-enhancing factors that collectively include Lef-1, Tcf-1, Tcf-3, and Tcf-4.21,22 Work from our laboratory has recently demonstrated that ?-catenin plays a critical role in vascular remodeling by stimulating VSMC proliferation and inhibiting apoptosis through the activation of the downstream transcription factor Tcf-4.23 Recent work highlighting the dual role of ?-TrCP in both Wnt and NF-B signaling pathways prompted us to extend our findings and test the hypothesis that the evolutionary conserved Wnt cascade was instrumental in the control of NF-B signaling in VSMCs through the regulation of ?-TrCP.
Methods
Plasmids
The plasmids were constitutively active IKK?S177 E plasmid (S. Fuchs),19,20 ?-TrCP wild type, and ?-TrCP (F-box deletion mutant) expression vectors (R. Benarous);14 a degradation-resistant ?-catenin construct in which the conserved serine/threonine residues in the N-terminus of ?-catenin were mutated to alanines (D. Kimelman);24 a dominant-negative form of Tcf-4, known as Tcf-4N31 (Tcf4 [N31]) lacking the N-terminal 31 aa (E. Fearon);25 IB(S32A/S36A) expression construct (Upstate Biotechnology); pNFB-Luc vector containing 4 tandem copies of the NF-B consensus sequence fused to a TATA-like promoter (PTAL) region from the herpes simplex virus thymidine kinase (HSV-TK) promoter; and the enhanced green fluorescent protein expression vector (pEGFP-C1, Clontech), Topflash and Fopflash, containing either 3 copies of the optimal Tcf motif CCTTTGATC or 3 copies of the mutant motif CCTTTGGCC upstream of a minimal c-Fos promoter driving luciferase expression (B.Vogelstein).26
Cell Culture
The clonal A7r5 rat aortic VSMCs were purchased from ATCC. Experiments were conducted with cells between passages 5 to 20. Human aortic VSMCs were purchased from Clonetics and used between passages 2 and 5.
Transfection and Reporter Assays
Transient transfection was performed with Effectene according to the manufacturer’s directions. Luciferase activity and EGFP fluorescence were determined according to the manufacturer’s directions, and the data were expressed as fold activation over control.
Retrovirus-Mediated Transfection
Wild-type ?-TrCP and the dominant-negative ?-TrCP mutant lacking the F-box domain were subcloned into the pLNCX2 retroviral expression vector (Clontech) as previously described.23 Characterization of the VSMCs harboring the Tcf-4N31 transgene has been previously described.23 The degradation-resistant ?-catenin transgene was subcloned into the pTRE expression vector (Clontech), and VSMCs harboring the inducible degradation-resistant ?-catenin transgene or empty vector were established. Characterization of these cells by polymerase chain reaction (PCR) confirmed that ?-catenin was constitutively upregulated after treatment with 1 μg/mL doxycycline for 48 hours.
Quantitative Real-Time Reverse Transcription PCR
RNA isolation and reverse transcription (RT) were performed as we have previously described.23 Changes in mRNA levels were compared by quantitative real-time RT-PCR analysis, using the Light Cycler thermocycler (Roche Diagnostics) as we have described.23 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 control samples.
Electrophoretic Mobility Shift Assay
TNF- was infused into the mouse vasculature via cardiac puncture. Forty minutes after TNF- infusion, mouse aortas were harvested and immediately frozen in liquid nitrogen. Nuclear extracts were isolated for comparison. Nuclear proteins were isolated by a modified cell lysis and centrifugation method by using CelLyte NuClear Extraction kit (Sigma). An electrophoretic mobility shift assay (EMSA) was performed by using the manufacturer’s instructions (Gel Shift Assay Kit, Promega) except where noted. Nuclear extracts (10 μg) were incubated with32P-labeled consensus oligonucleotides (1x107 cpm) specific for NF-B binding (5'-AGTTGAGGGGACTTTCCCAGG-3') and binding buffer.6 Supershift was completed with anti p65 (Santa Cruz).
Western Blot Analysis
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were performed as described23,27 by using the IB (C-21) antibody (sc-371) and goat anti-rabbit-HRP (sc-2030, Santa Cruz Biotechnology), ?-catenin, (?-catenin E-5, Santa Cruz Biotechnology), ?-TrCP (?-TrCP H-300, Santa Cruz Biotechnology), and ?-actin and vinculin (Sigma). ?-TrCP expression experiments were performed by immunoprecipitating 1 mg protein from cell lysates with 20 μg ?-TrCP antibody overnight at 4°C, followed by incubation with protein G-agarose beads (Santa Cruz Biotech) for 2 hours, centrifugation, washing twice with cold phosphate-buffer solution, and Western analysis. The membrane was probed for ?-TrCP with the aforementioned antibody.
Immunofluorescence
VSMCs were stained with the fluorescein isothiocyanate-labeled active p65 antibody, recognizing the epitope overlapping the nuclear localization signal (MAB3026, Chemicon International, 1:50 dilution) according to the manufacturer’s directions, and the secondary antibody was Alexa Fluor 488 rabbit anti-mouse (Molecular Probes, 1:500 dilution). ?-Catenin staining was performed with an anti-mouse ?-catenin antibody (Sigma, 1:50 dilution) and Alexa Fluor555 goat anti-mouse secondary (Molecular Probes, 1:1000 dilution). For experiments with lithium chloride (LiCl), human VSMCs were treated with 50 mmol/L LiCl in serum-free conditions for 6 hours.
Statistical Analysis
Comparisons between 2 groups were analyzed via a Student’s t test (P<0.05), and comparisons between 3 groups were analyzed by ANOVA with a Student-Newman-Keuls post-hoc test (P<0.05). Data are presented as mean±SE.
Results
TNF induced a significant degradation of IB in rat VSMCs at 20 minutes (Figure 1A). In parallel with degradation of IB, TNF- induced a significant increase in NF-B transcriptional activity as measured with an NF-B reporter construct to levels reported in earlier publications (baseline, 1.00±0.04 normalized luciferase units/EGFP; TNF-, 1.79±0.07; n=6, P<0.01).28,29 To establish the relevance of this finding in vivo, we performed EMSA on aortas after delivery of TNF- (please see online Figure I, available at http://atvb.ahajournals.org). These findings reconfirmed the ability of TNF- to stimulate NF-B in the vessel wall.
Figure 1. Representative Western blot demonstrating that TNF (40 ng/mL) degrades IB in rat VSMCs (20 minutes after TNF exposure). The proteasome inhibitor MG132 (30 μmol/L) blocked TNF-induced IB degradation. The immunoblot was reprobed with ?-actin.
MG132, a proteosomal inhibitor, efficiently blocked TNF-–induced degradation of IB (Figure 1A) and abolished NF-B activity, as measured with the NF-B reporter construct (vehicle treated, 1.44±0.07 normalized luciferase units/EGFP; MG132, 0.65±0.04; n=4, P<0.001). To demonstrate the necessary role of IB phosphorylation as a prerequisite for NF-B activation, we used a well-characterized IB mutant transgene in which the serine phosphorylation sites were changed to alanines (S32A/S36A). Mutation of serine residues 32 and 36 abolished TNF-–induced NF-B activity (control, 1.79±0.07 normalized luciferase units/EGFP; IBS32A/S36A, 1.06±0.04; n=6, P<0.01). Taken together, these studies systematically verified the ability of TNF- to stimulate NF-kB activity in cultured VSMCs and the intact aorta and demonstrated that serine phosphorylation and ubiquitin-dependent degradation of IB are necessary steps in the activation of NF-B transcriptional activity in VSMCs.
Expression of the E3 ligase ?-TrCP was confirmed in VSMCs and the aorta (Figure 2A). To test our hypothesis that ?-TrCP1 is a critical mediator governing NF-B activation in VSMCs, we established rat VSMCs with constitutive upregulation of ?-TrCP1. The increase in ?-TrCP protein expression in these cells was confirmed by Western blotting in Figure 2A. In line with our hypothesis, ?-TrCP1 upregulation resulted in a significant increase in both baseline and induced NF-B activity (Figure 2B). Baseline experiments were conducted in serum-containing media. NF-B was induced by transiently expressing the IKKS177E/S181E (IKKSE) active mutant that constitutively phosphorylates IB, or by treating cells with TNF-. The ability of ?-TrCP1 to significantly increase NF-B transactivation in the presence of serum alone suggests that there may be a constitutively low level of IB phosphorylated in VSMCs in culture. This has been suggested previously by Bourcier et al30 and recently supported by work demonstrating a role for growth factors in NF-B activity.31 In parallel with these findings, upregulation of ?-TrCP1 resulted in a decrease in IB at baseline (no TNF-) compared with control transfected cells (please see online Figure II, available at http://atvb.ahajournals.org). Furthermore, the expression levels of IB were significantly less at each time point after TNF- exposure in VSMCs harboring increased expression of ?-TrCP1 (please see online Figure II).
Figure 2. A, Western blot demonstrating ?-TrCP expression in the aorta and VSMCs. Lane 1 indicates aorta; lane 2, rat VSMC; and lane 3, rat VSMC with constitutive upregulation of wild-type ?-TrCP. B, Constitutive upregulation of ?-TrCP1 significantly increases NF-B activity. Rat VSMCs harboring constitutive expression of ?-TrCP1 or a control transgene were transiently transfected with the NF-B reporter gene and EGFP under baseline conditions, in the presence of an IKKS177E/S181E (IKKSE) active mutant or TNF-; (n=6, P<0.001), *Significantly different from baseline; #significantly different from control transfected VSMCs.
Next, rat VSMCs harboring constitutive expression of a well-characterized dominant-negative ?-TrCP1 construct lacking the F-box domain were established. Serum-stimulated NF-B activity was decreased in these cells, as measured by the absence of nuclear localization of p65 (please see online Figure III, available at http://atvb.ahajournals.org). NF-B activity as measured with the NF-B reporter was also significantly downregulated (please see online Figure III). However, TNF-induced NF-B activity was not affected (please see online Figure III).
Given that ?-TrCP recognizes a similar phosphorylated motif in ?-catenin, a proto-oncogene in the canonical Wnt signaling cascade, we hypothesized that ?-TrCP may serve as a bridge allowing cross-talk between the Wnt and NF-B signaling pathways in the vasculature, similar to previous work in 293 cells. Indeed, stimulation of the Wnt cascade through the upregulation of Wnt1 or a degradation-resistant ?-catenin gene significantly activated NF-B at baseline and in response to TNF- (Figure 3). Further studies demonstrated a dose-dependent relationship between the upregulation of Wnt1 and NF-B activation (control transfected, 1.00±0.03; Wnt1 [0.125 μg], 1.16±0.13; Wnt1 [0.225 μg], 1.67±0.17; n=6, P<0.001). The activation of NF-B correlated with a significant increase in ?-TrCP1 expression in VSMCs harboring expression of a degradation-resistant ?-catenin gene (please see online Figure IV, available at http://atvb.ahajournals.org). GAPDH values were not significantly different between groups.
Figure 3. The Wnt pathway significantly increases NF-B transactivation at baseline and in response to TNF-. VSMCs were transiently transfected with Wnt1, the degradation-resistant ?-catenin transgene, or a control vector, the NF-B reporter gene, and EGFP. NF-B activity was determined at baseline and in response to TNF- (20 ng/mL). Data are expressed as fold activation of luciferase/EGFP over control cells (n=6, P<0.001). *Significantly different from control transfected cohort; #significantly different from baseline condition.
We next assessed the level of endogenous ?-catenin protein in VSMCs harboring the ?-TrCP F-box deletion mutant. If this mutant was competing with and blocking the ability of endogenous ?-TrCP to degrade ?-catenin, endogenous ?-catenin protein levels would be expected to increase in these cells. Indeed, ?-catenin expression was elevated in the ?-TrCP F-box deletion mutant VSMCs compared with untransfected control VSMCs (please see online Figure V, available at http://atvb.ahajournals.org). In contrast, VSMCs expressing wild-type ?-TrCP1 exhibited a loss of ?-catenin (please see online Figure V).
Wnt-induced activation of NF-B was associated with a significant elevation in transactivation of Tcf/Lef, a well-characterized transcription factor in the Wnt cascade (please see online Figure VI, available at http://atvb.ahajournals.org). Tcf/Lef activity was measured with the well-characterized Tcf/Lef reporter gene Topflash. Lack of a significant luciferase induction in VSMCs transfected with the mutated reporter Fopflash demonstrated the specificity of the response (please see online Figure VI). To address the role of Tcf-4 in the regulation of NF-B, we established rat VSMCs with constitutive upregulation of a Tcf-4 dominant-negative mutant transgene lacking the ?-catenin binding domain (Tcf4 [N31]).23,25 In line with our hypothesis, blockade of ?-catenin binding to Tcf-4 resulted in a decrease in expression of ?-TrCP1 transcript (please see online Figure VI). Values were normalized to GAPDH expression values that were not significantly different between the groups. Finally, blockade of ?-catenin binding to Tcf-4 resulted in a near complete loss of NF-B activity induced by Wnt1 activation or TNF- (Figure 4).
Figure 4. Loss of ?-catenin binding to Tcf-4 abolished NF-B activity. VSMCs with constitutive upregulation of the Tcf-4 dominant-negative mutant or a control gene were transiently transfected with Wnt1 or a control vector along with the NF-B reporter gene and were treated with TNF- (n=7, P<0.001). *Significantly different from baseline conditions; #significantly different from control transfected VSMCs; and *significantly different from control transfected VSMCs.
To demonstrate the relevance of ?-TrCP1 regulation of NF-B to the human vasculature, ?-catenin translocation to the nucleus was induced in human aortic VSMCs with the well-characterized pharmacological agent LiCl. This prompted a parallel induction of nuclear p65 staining (please see online Figure VII, available at http://atvb.ahajournals.org). In line with the working hypothesis, the Wnt stimulated increase in NF-B activity was accompanied by a significant decrease in IB protein expression (please see online Figure VII). The membrane was reprobed with vinculin to assure equal protein loading.
Taken together, these studies provide the first evidence that the E3 ligase, ?-TrCP1, plays a critical role in NF-B activation in VSMCs. Furthermore, we have demonstrated that ?-TrCP1 expression is mediated in part through the evolutionary conserved Wnt pathway, thus providing a cross-talk mechanism whereby the Wnt cascade can stimulate NF-B signaling in the vasculature (Figure 5).
Figure 5. Schematic summarizing the coupling of ?-catenin and NF-B pathways in VSMCs.
Discussion
The purposes of the present study were (1) to investigate the role of ?-TrCP in the regulation of IB degradation and NF-B activation in the vasculature and (2) to define the key regulatory steps governing ?-TrCP expression in VSMCs. In line with our hypothesis, ?-TrCP1 significantly enhanced the rate of IB degradation and potentiated NF-B activity. However, expression of a dominant-negative ?-TrCP1 transgene lacking the F-box domain had no significant effect on TNF-–induced NF-B activity. This suggests that other ?-TrCP isoforms, including ?-TrCP2, may be playing a role in the degradation of IB.
We further demonstrated that ?-TrCP1 is regulated through the transcription factor Tcf-4 and the evolutionary conserved Wnt signaling pathway, thus providing a mechanistic link between the Wnt cascade and NF-B activation in the vasculature. A schematic in Figure 5 illustrates our current understanding of the coupling between the Wnt and NF-kB signaling pathways. Activation of the canonical Wnt cascade leads to ?-catenin binding to Tcf/Lef. Transactivation of Tcf/Lef results in the upregulation of the E3 ligase, ?-TrCP1. ?-TrCP recognizes identical phosphorylated motifs on ?-catenin and IB, binds to these phosphorylated proteins, and results in the tethering of the ubiquitin ligase machinery and subsequent degradation of IB by the 26S proteosome. Binding of ?-TrCP to ?-catenin provides a negative feedback loop switching off the ?-catenin/Tcf pathway, whereas ?-TrCP binding to IB results in NF-B activation.
?-TrCP was first identified in Xenopus as an orphan WD protein 32 and has since been described in Drosophila (Slimb), human, and mouse.13,31 Human and mouse ?-TrCP share 98% sequence identity.13 Our data reaffirm work in Hela and 293 cells, suggesting that ?-TrCP is the E3 ligase linking the ubiquitin complex to IB.12,19,20,33 Furthermore, our findings support previous work demonstrating a role for Tcf-4 in the regulation of ?-TrCP1 and NF-B activation.5,9,19,20,34 Recent work from the laboratory of Dr. Ben-Neriah suggests that endogenous ?-TrCP1 is bound to a pseudosubstrate, hnRNP-U, an abundant nuclear phosphoprotein, and is predominantly expressed in the nucleus under resting conditions.35 Work from other groups further supports the nuclear localization of ?-TrCP1.36 The amino acid sequence of ?-TrCP1 does not contain an apparent nuclear localization motif, suggesting that hnRNP-U may serve this function. Certainly, localization of ?-TrCP1 may serve as a critical control point governing its regulation of NF-B activity.
Furthermore, the role of ?-TrCP1 appears to be tissue specific, given that activation of the ?-catenin/Tcf pathway had no effect on ?-TrCP1 expression in fibroblasts,19 in contrast to our work in VSMCs and work in 293 cells.19 Furthermore, data in cancer cell lines suggest that ?-catenin is able to bind to NF-B and reduce NF-B DNA binding.37 Finally, recent work suggests that IKK and IKK? phosphorylate ?-catenin,38 and that RelA may suppress ?-catenin/Tcf transcription.39 Collectively, these findings highlight the divergent interactions of the ?-catenin and NF-B pathways and further emphasize the importance of characterizing their interactive role in the vasculature. The tissue-specific role of ?-TrCP may be related to recent work identifying a second isoform of ?-TrCP termed HOS/?TrCP2, which shares 85% similarity to ?-TrCP.5,19,33 Why we do not see a significant decrease in TNF-stimulated NF-B activity in VSMCs harboring the F-box ?-TrCP mutant is likely to be explained by an overlapping role for ?-TrCP2. The ?-TrCP1 F-box mutant we used significantly decreased serum-stimulated NF-B activity, as well as led to a significant increase in endogenous ?-catenin protein expression as expected in VSMCs, thus ruling out the mutant itself is not functionally active. Our data that demonstrated that upregulation of ?-TrCP1 is sufficient to increase both uninduced and induced NF-B activity whereas the dominant-negative ?-TrCP1 transgene only blocks uninduced NF-B activity but does not affect induced NF-B activity provide evidence that ?-TrCP2 is playing a role in the regulation of IB degradation. Current data are mixed on the binding affinity of ?-TrCP2 to pIB5,40 and have not yet been resolved. The publication of two recent articles that demonstrate that deletion of the ?-TrCP1 allele in mice does not fully block IB degradation and NF-B activation support a role for ?-TrCP2 and/or other yet-identified factors.41,42
Previous work in our laboratory identified that significant activation of the Wnt pathway in the process of vascular remodeling directly governed both VSMC proliferation and survival.23 Our findings demonstrating near complete loss of TNF-induced NF-B activity in VSMCs harboring the dominant-negative Tcf-4 transgene lacking a ?-catenin binding domain is a novel finding and suggests that ?-catenin binding to Tcf-4 is a critical regulatory point in NF-B activity.
Taken together, we have identified a role for the E3 ligase, ?-TrCP, in the regulation of IB degradation and NF-B activation in VSMCs. Furthermore, we have provided evidence demonstrating that the evolutionary conserved Wnt pathway plays a critical role in NF-B signaling through the E3 ligase ?-TrCP.
Acknowledgments
This work was supported by a Scientific Development Grant from the American Heart Association (J.H., 0030136N), a postdoctoral fellowship award from the American Heart Association (X.H.W., 025503Z) and the Lillehei Heart Institute at the University of Minnesota (J.H.).
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Deng J, Miller SA, Wang HY, Xia W, Wen Y, Zhou BP, Li Y, Lin SY, Hung MC. ?-Catenin interacts with and inhibits NF-B in human colon and breast cancer. Cancer Cell. 2002; 2: 323–334.
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Masui O, Ueda Y, Tsumura A, Koyanagi M, Hijikata M, Shimotohno K. RelA suppresses the Wnt/?-catenin pathway without exerting trans-acting transcriptional ability. Int J Mol Med. 2002; 9: 489–493.
Suzuki H, Chiba T, Suzuki T, Fujita T, Ikenoue T, Omata M, Furuichi K, Shikama H, Tanaka K. Homodimer of two F-box proteins ?TrCP1 or ?TrCP2 binds to IB for signal-dependent ubiquitination. J Biol Chem. 2000; 275: 2877–2884.
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Nakayama K, Hatakeyama S, Maruyama S, Kikuchi A, Onoe K, Good RA, Nakayama KI. Impaired degradation of inhibitory subunit of NF-B (IB) and ?-catenin as a result of targeted disruption of the ?-TrCP1 gene. Proc Natl Acad Sci U S A. 2003; 100: 8752–8757.(Xiaohong Wang; Neeta Adhi)
Correspondence to Jennifer L. Hall, PhD, Assistant Professor of Medicine Director, Cardiovascular Genomics Cardiovascular Division Lillehei Heart Institute, University of Minnesota, 420 Delaware St, Minneapolis, MN 55455. E-mail Hallx068@umn.edu
Abstract
Objective— Degradation of IB is an essential step in nuclear factor (NF)-B activation. However, the determinants regulating this process have not been defined in vascular smooth muscle cells (VSMCs). We hypothesized that the E3-ligase, ?-transducin repeat-containing protein 1 (?-TrCP1), was a rate-determining mediator that regulates the ubiquitin-mediated degradation of IB (in VSMC).
Methods and Results— Upregulation of ?-TrCP1 accelerated the rate of IB degradation, leading to increased NF-B activity. In contrast, VSMCs harboring a dominant-negative ?-TrCP1 transgene lacking the F-box domain exhibited a reduction in serum-stimulated NF-kB activity but no alteration in response to tumor necrosis factor (TNF). These findings suggest that ?-TrCP1 increases the rate of NF-B activation but is not rate-limiting in response to TNF in VSMCs. Endogenous ?-TrCP1 expression was regulated through the conserved Wnt cascade. Upregulation of Wnt1 resulted in ?-catenin–mediated activation of Tcf-4, leading to increased ?-TrCP1 expression and NF-B activity. Furthermore, VSMCs harboring a Tcf-4 mutant lacking a ?-catenin binding domain exhibited a significant reduction in ?-TrCP1 expression along with abolishment of NF-B activity.
Conclusions— We provide the first evidence of crosstalk between the Wnt cascade and NF-B signaling in VSMCs. This crosstalk is mediated through the E3-ligase, ?-TrCP1.
Key Words: nuclear factor-B ? ?-TrCP1 ? muscle, vascular, smooth ? Wnt ? ?-catenin
Introduction
Nuclear factor-B (NF-B) acts as a central intersecting node regulating proliferation, survival, and inflammation in vascular smooth muscle cells (VSMCs) in response to cytokines, infectious agents, dyslipidemia, hypertension, angina, vascular injury, and atherosclerosis.1,2 The degradation of IB as an essential step in NF-B activation has been well described.3–6 However, the molecular determinants regulating this process have not been well defined in VSMCs.
NF-B is maintained in an inactive form by sequestration in the cytoplasm through interaction with the IB family of proteins.5,6 This IB family of proteins binds to the Rel homology domain of NF-B members and masks the nuclear localization sequence of NF-B. Stimuli, including tumor necrosis factor- (TNF-) and lipopolysaccharide (LPS), stimulate the phosphorylation of IB on serine residues 32 and 36.5–7 Phosphorylation of these residues is thought to target IB for ubiquitination by the stem cell factor (SCF) ubiquitin/ligase complex and to induce rapid transient degradation.3,5,6,8–10 Although the majority of studies suggest that phosphorylation of IB is a necessary step in the activation of NF-B, it is clear that phosphorylation alone is not sufficient and that the ubiquitin-dependent degradation of IB is a necessary step in NF-B activation.6,11
Work in other cell systems has identified a SCF ubiquitin/ligase complex, which contains the proteins Skp1, Cdc53/Cul1, and an F-box protein, that is thought to recognize phosphorylated IB and regulate its ubiquitination.7 The ubiquitination process requires 3 proteins, E1, E2, and E3. Briefly, ubiquitin is activated as a thiol-ester on E1 in an ATP-dependent reaction and transferred to an E2 as a thiol ester.8,9,12,13 The F-box/WD proteins, designated E3s, can be roughly categorized as HECT, RING finger, and U-box–containing proteins or multiprotein complexes. The F-box is the substrate-recognition component of multiprotein E3s and normally requires a RING finger protein for catalytic activity. The E3 protein responsible for recognizing the phosphorylated motif on IB (DS[PO3]GXS[PO3]), where represents a hydrophobic residue) has recently been identified as ?-TrCP/E3RS/Fwd1.14–16 ?-TrCP binds to lysine resides on phosphorylated IB, leading to recognition by the 26S proteasome and degradation.8,9,12,17,18
?-TrCP also recognizes an identical phosphorylated motif on the proto-oncogene ?-catenin.5,19,20 ?-Catenin is a key mediator in the Wnt pathway. Activation of the Wnt cascade results in the stabilization and translocation of ?-catenin to the nucleus, where it activates a family of transcription factors referred to as T-cell factors/lymphoid-enhancing factors that collectively include Lef-1, Tcf-1, Tcf-3, and Tcf-4.21,22 Work from our laboratory has recently demonstrated that ?-catenin plays a critical role in vascular remodeling by stimulating VSMC proliferation and inhibiting apoptosis through the activation of the downstream transcription factor Tcf-4.23 Recent work highlighting the dual role of ?-TrCP in both Wnt and NF-B signaling pathways prompted us to extend our findings and test the hypothesis that the evolutionary conserved Wnt cascade was instrumental in the control of NF-B signaling in VSMCs through the regulation of ?-TrCP.
Methods
Plasmids
The plasmids were constitutively active IKK?S177 E plasmid (S. Fuchs),19,20 ?-TrCP wild type, and ?-TrCP (F-box deletion mutant) expression vectors (R. Benarous);14 a degradation-resistant ?-catenin construct in which the conserved serine/threonine residues in the N-terminus of ?-catenin were mutated to alanines (D. Kimelman);24 a dominant-negative form of Tcf-4, known as Tcf-4N31 (Tcf4 [N31]) lacking the N-terminal 31 aa (E. Fearon);25 IB(S32A/S36A) expression construct (Upstate Biotechnology); pNFB-Luc vector containing 4 tandem copies of the NF-B consensus sequence fused to a TATA-like promoter (PTAL) region from the herpes simplex virus thymidine kinase (HSV-TK) promoter; and the enhanced green fluorescent protein expression vector (pEGFP-C1, Clontech), Topflash and Fopflash, containing either 3 copies of the optimal Tcf motif CCTTTGATC or 3 copies of the mutant motif CCTTTGGCC upstream of a minimal c-Fos promoter driving luciferase expression (B.Vogelstein).26
Cell Culture
The clonal A7r5 rat aortic VSMCs were purchased from ATCC. Experiments were conducted with cells between passages 5 to 20. Human aortic VSMCs were purchased from Clonetics and used between passages 2 and 5.
Transfection and Reporter Assays
Transient transfection was performed with Effectene according to the manufacturer’s directions. Luciferase activity and EGFP fluorescence were determined according to the manufacturer’s directions, and the data were expressed as fold activation over control.
Retrovirus-Mediated Transfection
Wild-type ?-TrCP and the dominant-negative ?-TrCP mutant lacking the F-box domain were subcloned into the pLNCX2 retroviral expression vector (Clontech) as previously described.23 Characterization of the VSMCs harboring the Tcf-4N31 transgene has been previously described.23 The degradation-resistant ?-catenin transgene was subcloned into the pTRE expression vector (Clontech), and VSMCs harboring the inducible degradation-resistant ?-catenin transgene or empty vector were established. Characterization of these cells by polymerase chain reaction (PCR) confirmed that ?-catenin was constitutively upregulated after treatment with 1 μg/mL doxycycline for 48 hours.
Quantitative Real-Time Reverse Transcription PCR
RNA isolation and reverse transcription (RT) were performed as we have previously described.23 Changes in mRNA levels were compared by quantitative real-time RT-PCR analysis, using the Light Cycler thermocycler (Roche Diagnostics) as we have described.23 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 control samples.
Electrophoretic Mobility Shift Assay
TNF- was infused into the mouse vasculature via cardiac puncture. Forty minutes after TNF- infusion, mouse aortas were harvested and immediately frozen in liquid nitrogen. Nuclear extracts were isolated for comparison. Nuclear proteins were isolated by a modified cell lysis and centrifugation method by using CelLyte NuClear Extraction kit (Sigma). An electrophoretic mobility shift assay (EMSA) was performed by using the manufacturer’s instructions (Gel Shift Assay Kit, Promega) except where noted. Nuclear extracts (10 μg) were incubated with32P-labeled consensus oligonucleotides (1x107 cpm) specific for NF-B binding (5'-AGTTGAGGGGACTTTCCCAGG-3') and binding buffer.6 Supershift was completed with anti p65 (Santa Cruz).
Western Blot Analysis
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were performed as described23,27 by using the IB (C-21) antibody (sc-371) and goat anti-rabbit-HRP (sc-2030, Santa Cruz Biotechnology), ?-catenin, (?-catenin E-5, Santa Cruz Biotechnology), ?-TrCP (?-TrCP H-300, Santa Cruz Biotechnology), and ?-actin and vinculin (Sigma). ?-TrCP expression experiments were performed by immunoprecipitating 1 mg protein from cell lysates with 20 μg ?-TrCP antibody overnight at 4°C, followed by incubation with protein G-agarose beads (Santa Cruz Biotech) for 2 hours, centrifugation, washing twice with cold phosphate-buffer solution, and Western analysis. The membrane was probed for ?-TrCP with the aforementioned antibody.
Immunofluorescence
VSMCs were stained with the fluorescein isothiocyanate-labeled active p65 antibody, recognizing the epitope overlapping the nuclear localization signal (MAB3026, Chemicon International, 1:50 dilution) according to the manufacturer’s directions, and the secondary antibody was Alexa Fluor 488 rabbit anti-mouse (Molecular Probes, 1:500 dilution). ?-Catenin staining was performed with an anti-mouse ?-catenin antibody (Sigma, 1:50 dilution) and Alexa Fluor555 goat anti-mouse secondary (Molecular Probes, 1:1000 dilution). For experiments with lithium chloride (LiCl), human VSMCs were treated with 50 mmol/L LiCl in serum-free conditions for 6 hours.
Statistical Analysis
Comparisons between 2 groups were analyzed via a Student’s t test (P<0.05), and comparisons between 3 groups were analyzed by ANOVA with a Student-Newman-Keuls post-hoc test (P<0.05). Data are presented as mean±SE.
Results
TNF induced a significant degradation of IB in rat VSMCs at 20 minutes (Figure 1A). In parallel with degradation of IB, TNF- induced a significant increase in NF-B transcriptional activity as measured with an NF-B reporter construct to levels reported in earlier publications (baseline, 1.00±0.04 normalized luciferase units/EGFP; TNF-, 1.79±0.07; n=6, P<0.01).28,29 To establish the relevance of this finding in vivo, we performed EMSA on aortas after delivery of TNF- (please see online Figure I, available at http://atvb.ahajournals.org). These findings reconfirmed the ability of TNF- to stimulate NF-B in the vessel wall.
Figure 1. Representative Western blot demonstrating that TNF (40 ng/mL) degrades IB in rat VSMCs (20 minutes after TNF exposure). The proteasome inhibitor MG132 (30 μmol/L) blocked TNF-induced IB degradation. The immunoblot was reprobed with ?-actin.
MG132, a proteosomal inhibitor, efficiently blocked TNF-–induced degradation of IB (Figure 1A) and abolished NF-B activity, as measured with the NF-B reporter construct (vehicle treated, 1.44±0.07 normalized luciferase units/EGFP; MG132, 0.65±0.04; n=4, P<0.001). To demonstrate the necessary role of IB phosphorylation as a prerequisite for NF-B activation, we used a well-characterized IB mutant transgene in which the serine phosphorylation sites were changed to alanines (S32A/S36A). Mutation of serine residues 32 and 36 abolished TNF-–induced NF-B activity (control, 1.79±0.07 normalized luciferase units/EGFP; IBS32A/S36A, 1.06±0.04; n=6, P<0.01). Taken together, these studies systematically verified the ability of TNF- to stimulate NF-kB activity in cultured VSMCs and the intact aorta and demonstrated that serine phosphorylation and ubiquitin-dependent degradation of IB are necessary steps in the activation of NF-B transcriptional activity in VSMCs.
Expression of the E3 ligase ?-TrCP was confirmed in VSMCs and the aorta (Figure 2A). To test our hypothesis that ?-TrCP1 is a critical mediator governing NF-B activation in VSMCs, we established rat VSMCs with constitutive upregulation of ?-TrCP1. The increase in ?-TrCP protein expression in these cells was confirmed by Western blotting in Figure 2A. In line with our hypothesis, ?-TrCP1 upregulation resulted in a significant increase in both baseline and induced NF-B activity (Figure 2B). Baseline experiments were conducted in serum-containing media. NF-B was induced by transiently expressing the IKKS177E/S181E (IKKSE) active mutant that constitutively phosphorylates IB, or by treating cells with TNF-. The ability of ?-TrCP1 to significantly increase NF-B transactivation in the presence of serum alone suggests that there may be a constitutively low level of IB phosphorylated in VSMCs in culture. This has been suggested previously by Bourcier et al30 and recently supported by work demonstrating a role for growth factors in NF-B activity.31 In parallel with these findings, upregulation of ?-TrCP1 resulted in a decrease in IB at baseline (no TNF-) compared with control transfected cells (please see online Figure II, available at http://atvb.ahajournals.org). Furthermore, the expression levels of IB were significantly less at each time point after TNF- exposure in VSMCs harboring increased expression of ?-TrCP1 (please see online Figure II).
Figure 2. A, Western blot demonstrating ?-TrCP expression in the aorta and VSMCs. Lane 1 indicates aorta; lane 2, rat VSMC; and lane 3, rat VSMC with constitutive upregulation of wild-type ?-TrCP. B, Constitutive upregulation of ?-TrCP1 significantly increases NF-B activity. Rat VSMCs harboring constitutive expression of ?-TrCP1 or a control transgene were transiently transfected with the NF-B reporter gene and EGFP under baseline conditions, in the presence of an IKKS177E/S181E (IKKSE) active mutant or TNF-; (n=6, P<0.001), *Significantly different from baseline; #significantly different from control transfected VSMCs.
Next, rat VSMCs harboring constitutive expression of a well-characterized dominant-negative ?-TrCP1 construct lacking the F-box domain were established. Serum-stimulated NF-B activity was decreased in these cells, as measured by the absence of nuclear localization of p65 (please see online Figure III, available at http://atvb.ahajournals.org). NF-B activity as measured with the NF-B reporter was also significantly downregulated (please see online Figure III). However, TNF-induced NF-B activity was not affected (please see online Figure III).
Given that ?-TrCP recognizes a similar phosphorylated motif in ?-catenin, a proto-oncogene in the canonical Wnt signaling cascade, we hypothesized that ?-TrCP may serve as a bridge allowing cross-talk between the Wnt and NF-B signaling pathways in the vasculature, similar to previous work in 293 cells. Indeed, stimulation of the Wnt cascade through the upregulation of Wnt1 or a degradation-resistant ?-catenin gene significantly activated NF-B at baseline and in response to TNF- (Figure 3). Further studies demonstrated a dose-dependent relationship between the upregulation of Wnt1 and NF-B activation (control transfected, 1.00±0.03; Wnt1 [0.125 μg], 1.16±0.13; Wnt1 [0.225 μg], 1.67±0.17; n=6, P<0.001). The activation of NF-B correlated with a significant increase in ?-TrCP1 expression in VSMCs harboring expression of a degradation-resistant ?-catenin gene (please see online Figure IV, available at http://atvb.ahajournals.org). GAPDH values were not significantly different between groups.
Figure 3. The Wnt pathway significantly increases NF-B transactivation at baseline and in response to TNF-. VSMCs were transiently transfected with Wnt1, the degradation-resistant ?-catenin transgene, or a control vector, the NF-B reporter gene, and EGFP. NF-B activity was determined at baseline and in response to TNF- (20 ng/mL). Data are expressed as fold activation of luciferase/EGFP over control cells (n=6, P<0.001). *Significantly different from control transfected cohort; #significantly different from baseline condition.
We next assessed the level of endogenous ?-catenin protein in VSMCs harboring the ?-TrCP F-box deletion mutant. If this mutant was competing with and blocking the ability of endogenous ?-TrCP to degrade ?-catenin, endogenous ?-catenin protein levels would be expected to increase in these cells. Indeed, ?-catenin expression was elevated in the ?-TrCP F-box deletion mutant VSMCs compared with untransfected control VSMCs (please see online Figure V, available at http://atvb.ahajournals.org). In contrast, VSMCs expressing wild-type ?-TrCP1 exhibited a loss of ?-catenin (please see online Figure V).
Wnt-induced activation of NF-B was associated with a significant elevation in transactivation of Tcf/Lef, a well-characterized transcription factor in the Wnt cascade (please see online Figure VI, available at http://atvb.ahajournals.org). Tcf/Lef activity was measured with the well-characterized Tcf/Lef reporter gene Topflash. Lack of a significant luciferase induction in VSMCs transfected with the mutated reporter Fopflash demonstrated the specificity of the response (please see online Figure VI). To address the role of Tcf-4 in the regulation of NF-B, we established rat VSMCs with constitutive upregulation of a Tcf-4 dominant-negative mutant transgene lacking the ?-catenin binding domain (Tcf4 [N31]).23,25 In line with our hypothesis, blockade of ?-catenin binding to Tcf-4 resulted in a decrease in expression of ?-TrCP1 transcript (please see online Figure VI). Values were normalized to GAPDH expression values that were not significantly different between the groups. Finally, blockade of ?-catenin binding to Tcf-4 resulted in a near complete loss of NF-B activity induced by Wnt1 activation or TNF- (Figure 4).
Figure 4. Loss of ?-catenin binding to Tcf-4 abolished NF-B activity. VSMCs with constitutive upregulation of the Tcf-4 dominant-negative mutant or a control gene were transiently transfected with Wnt1 or a control vector along with the NF-B reporter gene and were treated with TNF- (n=7, P<0.001). *Significantly different from baseline conditions; #significantly different from control transfected VSMCs; and *significantly different from control transfected VSMCs.
To demonstrate the relevance of ?-TrCP1 regulation of NF-B to the human vasculature, ?-catenin translocation to the nucleus was induced in human aortic VSMCs with the well-characterized pharmacological agent LiCl. This prompted a parallel induction of nuclear p65 staining (please see online Figure VII, available at http://atvb.ahajournals.org). In line with the working hypothesis, the Wnt stimulated increase in NF-B activity was accompanied by a significant decrease in IB protein expression (please see online Figure VII). The membrane was reprobed with vinculin to assure equal protein loading.
Taken together, these studies provide the first evidence that the E3 ligase, ?-TrCP1, plays a critical role in NF-B activation in VSMCs. Furthermore, we have demonstrated that ?-TrCP1 expression is mediated in part through the evolutionary conserved Wnt pathway, thus providing a cross-talk mechanism whereby the Wnt cascade can stimulate NF-B signaling in the vasculature (Figure 5).
Figure 5. Schematic summarizing the coupling of ?-catenin and NF-B pathways in VSMCs.
Discussion
The purposes of the present study were (1) to investigate the role of ?-TrCP in the regulation of IB degradation and NF-B activation in the vasculature and (2) to define the key regulatory steps governing ?-TrCP expression in VSMCs. In line with our hypothesis, ?-TrCP1 significantly enhanced the rate of IB degradation and potentiated NF-B activity. However, expression of a dominant-negative ?-TrCP1 transgene lacking the F-box domain had no significant effect on TNF-–induced NF-B activity. This suggests that other ?-TrCP isoforms, including ?-TrCP2, may be playing a role in the degradation of IB.
We further demonstrated that ?-TrCP1 is regulated through the transcription factor Tcf-4 and the evolutionary conserved Wnt signaling pathway, thus providing a mechanistic link between the Wnt cascade and NF-B activation in the vasculature. A schematic in Figure 5 illustrates our current understanding of the coupling between the Wnt and NF-kB signaling pathways. Activation of the canonical Wnt cascade leads to ?-catenin binding to Tcf/Lef. Transactivation of Tcf/Lef results in the upregulation of the E3 ligase, ?-TrCP1. ?-TrCP recognizes identical phosphorylated motifs on ?-catenin and IB, binds to these phosphorylated proteins, and results in the tethering of the ubiquitin ligase machinery and subsequent degradation of IB by the 26S proteosome. Binding of ?-TrCP to ?-catenin provides a negative feedback loop switching off the ?-catenin/Tcf pathway, whereas ?-TrCP binding to IB results in NF-B activation.
?-TrCP was first identified in Xenopus as an orphan WD protein 32 and has since been described in Drosophila (Slimb), human, and mouse.13,31 Human and mouse ?-TrCP share 98% sequence identity.13 Our data reaffirm work in Hela and 293 cells, suggesting that ?-TrCP is the E3 ligase linking the ubiquitin complex to IB.12,19,20,33 Furthermore, our findings support previous work demonstrating a role for Tcf-4 in the regulation of ?-TrCP1 and NF-B activation.5,9,19,20,34 Recent work from the laboratory of Dr. Ben-Neriah suggests that endogenous ?-TrCP1 is bound to a pseudosubstrate, hnRNP-U, an abundant nuclear phosphoprotein, and is predominantly expressed in the nucleus under resting conditions.35 Work from other groups further supports the nuclear localization of ?-TrCP1.36 The amino acid sequence of ?-TrCP1 does not contain an apparent nuclear localization motif, suggesting that hnRNP-U may serve this function. Certainly, localization of ?-TrCP1 may serve as a critical control point governing its regulation of NF-B activity.
Furthermore, the role of ?-TrCP1 appears to be tissue specific, given that activation of the ?-catenin/Tcf pathway had no effect on ?-TrCP1 expression in fibroblasts,19 in contrast to our work in VSMCs and work in 293 cells.19 Furthermore, data in cancer cell lines suggest that ?-catenin is able to bind to NF-B and reduce NF-B DNA binding.37 Finally, recent work suggests that IKK and IKK? phosphorylate ?-catenin,38 and that RelA may suppress ?-catenin/Tcf transcription.39 Collectively, these findings highlight the divergent interactions of the ?-catenin and NF-B pathways and further emphasize the importance of characterizing their interactive role in the vasculature. The tissue-specific role of ?-TrCP may be related to recent work identifying a second isoform of ?-TrCP termed HOS/?TrCP2, which shares 85% similarity to ?-TrCP.5,19,33 Why we do not see a significant decrease in TNF-stimulated NF-B activity in VSMCs harboring the F-box ?-TrCP mutant is likely to be explained by an overlapping role for ?-TrCP2. The ?-TrCP1 F-box mutant we used significantly decreased serum-stimulated NF-B activity, as well as led to a significant increase in endogenous ?-catenin protein expression as expected in VSMCs, thus ruling out the mutant itself is not functionally active. Our data that demonstrated that upregulation of ?-TrCP1 is sufficient to increase both uninduced and induced NF-B activity whereas the dominant-negative ?-TrCP1 transgene only blocks uninduced NF-B activity but does not affect induced NF-B activity provide evidence that ?-TrCP2 is playing a role in the regulation of IB degradation. Current data are mixed on the binding affinity of ?-TrCP2 to pIB5,40 and have not yet been resolved. The publication of two recent articles that demonstrate that deletion of the ?-TrCP1 allele in mice does not fully block IB degradation and NF-B activation support a role for ?-TrCP2 and/or other yet-identified factors.41,42
Previous work in our laboratory identified that significant activation of the Wnt pathway in the process of vascular remodeling directly governed both VSMC proliferation and survival.23 Our findings demonstrating near complete loss of TNF-induced NF-B activity in VSMCs harboring the dominant-negative Tcf-4 transgene lacking a ?-catenin binding domain is a novel finding and suggests that ?-catenin binding to Tcf-4 is a critical regulatory point in NF-B activity.
Taken together, we have identified a role for the E3 ligase, ?-TrCP, in the regulation of IB degradation and NF-B activation in VSMCs. Furthermore, we have provided evidence demonstrating that the evolutionary conserved Wnt pathway plays a critical role in NF-B signaling through the E3 ligase ?-TrCP.
Acknowledgments
This work was supported by a Scientific Development Grant from the American Heart Association (J.H., 0030136N), a postdoctoral fellowship award from the American Heart Association (X.H.W., 025503Z) and the Lillehei Heart Institute at the University of Minnesota (J.H.).
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