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Cardiovascular Research Advance Access first published online on October 13, 2008
This version [Corrected Proof] published online on October 30, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn278
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org.

MMP-9 and -12 cause N-cadherin shedding and thereby β-catenin signalling and vascular smooth muscle cell proliferation

Amrita Dwivedi, Sadie C. Slater{dagger} and Sarah J. George*

Bristol Heart Institute, University of Bristol, Level 7, Bristol Royal Infirmary, Bristol BS2 8HW, UK

* Corresponding author. Tel: +44 117 9283154; fax: +44 117 9283581.E-mail address: s.j.george{at}bris.ac.uk

Received 2 May 2008; revised 10 September 2008; accepted 9 October 2008

Time for primary review: 22 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: Vascular smooth muscle cell (VSMC) proliferation contributes to intimal thickening in restenosis and atherosclerosis. Previously, we demonstrated that matrix-degrading metalloproteinase (MMP)-dependent shedding of the extracellular portion of N-cadherin increased VSMC proliferation via elevation of β-catenin signalling and cyclin D1 expression. In this study, we aimed to determine whether MMP-2, -9, -12, or -14 regulates VSMC proliferation via N-cadherin shedding.

Methods and results: N-cadherin shedding was significantly impaired in proliferating mouse aortic VSMCs deficient in MMP-9 (MMP-9–/–) and MMP-12 (MMP-12–/–) compared with wild-type controls (1.1 ± 0.7- and 1.0 ± 0.1- vs. 2.0 ± 0.2-fold). Furthermore, proliferating VSMCs subjected to MMP-9 or -12 siRNA knockdown or deficient in MMP-9 or -12 showed significantly increased cellular levels of N-cadherin compared with controls (1.7 ± 0.2-, 2.7 ± 0.6-, and 3.5 ± 1.6-, 1.7 ± 0.2-fold, respectively). Incubation of VSMCs with active MMP-9 or -12 independently increased N-cadherin cleavage. Additionally, β-catenin signalling was significantly reduced by 52 ± 17 and 81 ± 12% in MMP-9–/– and -12–/– proliferating VSMCs, respectively, and this was corroborated by siRNA knockdown of MMP-9 and -12. Decreased β-catenin signalling coincided with significantly reduced proliferation and cyclin D1 protein levels in MMP-9–/– and -12–/– cells. Little or no additive effect was observed with combined modulation of MMP-9 and -12 in all experiments. In contrast, N-cadherin shedding and VSMC proliferation were not modulated by MMP-2 and -14.

Conclusion: In conclusion, we propose that MMP-9 and -12 promote intimal thickening by independent cleavage of N-cadherin, which elevates VSMC proliferation via β-catenin signalling.

KEYWORDS MMP; N-cadherin; Intimal thickening; Smooth muscle cell; Proliferation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Homophilic cell–cell adhesions are mediated by cadherins that interact with β-catenin to form a link with the actin cytoskeleton. These cell–cell contacts are essential for normal tissue architecture, embryogenesis, and morphogenesis. β-Catenin also participates in the Wnt signalling pathway, which plays an important role in many biological processes such as tumorigenesis and metastasis.1 β-Catenin binds to the DNA-binding factor and T-cell factor; together this complex activates gene targets including cell cycle activators such as cyclin D12 and c-myc,3 and other genes including matrix-degrading metalloproteinases (MMPs) such as MMP-7,4 -14,5 and fibronectin.6 Consequently, the cadherin:catenin complex regulates adhesion and cell signalling and thereby modulates cell behaviour, including proliferation.7,8

Vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of atherosclerosis and restenosis after injury.9 We previously demonstrated that VSMC proliferation is regulated by N-cadherin via modulation of β-catenin signalling.10,11 Loss of N-cadherin and translocation of β-catenin to the nucleus increased β-catenin signalling, which resulted in elevated expression of cyclin D1 and thereby VSMC proliferation.10,11

Additionally, we demonstrated that the loss of N-cadherin from the cell surface was mediated by MMPs.10 In this study, we have examined whether MMP-2, -9, -12, and -14 is involved in regulation of VSMC proliferation via cleavage of N-cadherin and β-catenin signalling. Together, the MMP family can degrade all of the components of the extracellular matrix as well as non-matrix proteins including cadherins.12 MMPs have been implicated in the regulation of VSMC proliferation and intimal thickening.13 However, non-selective synthetic MMP inhibitors have been ineffective in the reduction of intimal thickening.14–17 Consequently, a greater understanding of the properties of individual MMPs may be helpful in the design of MMP inhibitors to reduce intimal thickening. We focussed on MMP-2, -9, -12, and -14, as these MMPs have been implicated in the regulation of VSMC proliferation and intimal thickening.18–20


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
2.1 Vascular smooth muscle cell culture
Aortas were obtained from MMP-9 (MMP-9–/–) and MMP-12 (MMP-12–/–) deficient mice (a kind gift from Professor Steve Shapiro, Harvard Medical School, Boston, MA, USA) and strain matched wild-type controls and TOPGAL transgenic mice containing the TOPFLASH reporter (a kind gift of Dr Yingzi Yang, National Human Genome Research Institute, Bethesda, MD, USA). The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). VSMCs were grown from aortic explants as described previously11 and used at passage 3–9. VSMCs were quiesced in DMEM supplemented with antibiotics and L-glutamine for 24 h. Proliferation was stimulated with 10% (v/v) FCS or 20 ng/mL PDGF-BB (R&D Systems) in DMEM supplemented with antibiotics and L-glutamine. In some cases, VSMCs were cultured with 2 µM MMP-2 inhibitor (444244, Calbiochem), or 2.12 µg/mL MMP-14 neutralizing antibody (AB8102, Chemicon) as described previously.21

2.2 siRNA knockdown of MMP-2, -9, -12, and -14
Silencing RNA oligonucleotides (siRNA) for MMP-2 (SI01314019, SI01314033, Qiagen), MMP-9 (SI02715328, SI00206920, Qiagen), MMP-12 (SI00177737, SI02733808, Qiagen), MMP-14 (sc-41566, Santa Cruz), and GFP (VSC-1001, Amaxa, Inc., Cologne, Germany) were purchased. Transfection was performed with a Nucleofector device and VSMC kit (Amaxa, Inc.) following the manufacturer's instructions. Briefly 8–10 x 105 cells were subjected to nucleofection with 250 pmol of MMP or GFP siRNAs using A-33 program and analysed 24 h after nucleofection.

2.3 Cleavage of N-cadherin by active MMPs
Pro-MMP-12 (R&D Systems) was activated by incubation for 30 h at 37°C in TCNB buffer [50 mM Tris, 10 mM CaCl2, 0.15 M NaCl2, and 0.05% (w/v) Brij], resulting in 100% activation of MMP-12 (data not shown). To determine whether MMP-9 and -12 cleave cellular and purified N-cadherin, active MMP-9 (R&D systems), or active MMP-12 was incubated with VSMCs and 2 nM of the extracellular portion of N-cadherin bound to Fc (SNC-Fc), respectively. SNC-Fc was generated by amplifying the Fc domain, containing mutations in the IgG-R and complement binding domains, by PCR from an IL-10-Fc fusion plasmid (generously provided by Dr Terry Storm, Harvard University, MA, USA). The extracellular domain of mouse N-cadherin was cloned with the Fc domain into pDC515 (Microbix) and recombined with the adenovirus genomic plasmid by co-transfection into 293 cells. CHO cells were infected with 50 pfu/cell of adenovirus and conditioned media collected at 3 and 6 days. SNC-Fc was purified with protein A columns (Amersham Biosciences) and protein concentration determined using the Bradford Protein assay (Sigma).

2.4 RNA extraction and quantitative polymerase chain reaction
Total RNA was extracted from VSMCs using RNAeasy kit (Qiagen). After reverse transcription, cDNA was subjected to quantitative PCR for N-cadherin using Quantitect primers (QT00148106, Qiagen) as described in the manufacturer's instructions.

2.5 Western blotting
VSMC proteins were extracted by SDS lysis and the protein concentration determined using a bicinchoninic acid protein assay kit (Perbio Science, Tattenhall, UK) as described previously.22 Equal protein concentrations were subjected to western blotting. Samples were loaded on 3–8% Novex Tris-acetate gels (Invitrogen, Paisley, UK) and transferred to nitrocellulose. Blots were blocked in 5% (w/v) skimmed milk powder and incubated overnight at 4°C with 0.1 µg/mL mouse anti-N-cadherin antibody (610920, clone 32, BD Biosciences, which recognizes the intracellular domain of N-cadherin, was used for cellular N-cadherin), 1 µg/mL rabbit anti-N-cadherin (sc-7939, H-63, Santa Cruz, which recognizes the extracellular domain, was used for soluble N-cadherin), 0.2 µg/mL mouse anti-MMP-12 (AB19051, Chemicon), 10 µg/mL rabbit anti-MMP-14 (AB8221, Chemicon), and 1 µg/mL mouse anti-glyceraldehyde-3-phosphate dehydrogenase (368662, Calbiochem) in Starting block (Pierce). Bound antibodies (and SNC-Fc) were detected by rabbit anti-mouse horseradish peroxidise-conjugated antibodies (Dako) and enhanced chemiluminescence (Amersham International, Chalfont St Giles, UK). Detected bands were quantified using a Bio-Rad GS-690 scanning densitometer (Bio-Rad, Hemel Hempstead, UK) by measuring the optical density of each band (OD x mm2) and normalized by GAPDH values.

2.6 Zymography
MMP-2 and -9 were detected by gelatin zymography as described previously.23

2.7 β-Catenin localization
VSMCs grown on glass coverslips were quiesced for 24 h prior to the addition of FCS-containing media for 4 h. VSMCs were fixed with ice-cold 4% (w/v) paraformaldehyde for 20 min. After incubation in 1% (v/v) Triton X-100 at 4°C for 5 min, VSMCs were incubated with 2.5 µg/mL β-catenin antibodies (610153, clone 14, BD Biosciences) or non-immune mouse IgG (Sigma) in 1% BSA (w/v) in PBS for 2 h at room temperature. VSMCs were incubated for 45 min with biotinylated goat anti-mouse IgG (Dako) diluted 1:200 in 1% BSA (w/v) in PBS and then for 30 min with ExtravidinTM-FITC diluted 1:200 in 1% (w/v) BSA in PBS. Coverslips were mounted on glass slides with Vectashield® mounting medium (Vector Laboratories, Peterborough, UK).

2.8 β-Catenin reporter assay
β-Catenin activity was quantified in MMP-9–/– and -12–/– VSMCs transfected with TOPFLASH plasmid and normalized by Renilla luciferase as described previously,11 after 24 h with FCS. β-Catenin activity was quantified in TOPGAL VSMCs subjected to siRNA treatment as described previously,11 24 h after the treatment with siRNA.

2.9 Cell proliferation
Cell proliferation was assessed by Trypan Blue exclusion cell counts and bromodeoxyuridine (BrdU) incorporation as described previously.24

2.10 Immunohistochemsitry for MMP-12
Immunohistochemistry was performed on balloon-injured rat carotid paraffin sections at 0, 2, and 10 days after injury and human saphenous vein 0 and 14 days after culture using 1 µg/mL rabbit anti-human MMP-12 antibodies (AB19051, Chemicon) as described previously.22

2.11 Statistics
Experiments were carried out at least three times with VSMCs from different aortas. Data were analysed by one sample t-test or ANOVA and Student–Newman–Keuls post-test and a significant difference accepted when P < 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 N-cadherin cleavage by MMP-9 and -12
Stimulation of proliferation with 20 ng/mL PDGF-BB significantly reduced cellular N-cadherin and significantly increased cleaved (soluble) N-cadherin in the conditioned media in wild-type VSMCs (Figure 1A), as previously observed in human VSMCs.10 However, in MMP-9–/– and -12–/– VSMCs, the addition of 20 ng/mL PDGF-BB did not significantly affect the amount of cellular N-cadherin or the amount of soluble N-cadherin in the conditioned media (Figure 1A). In fact, the amount of cellular N-cadherin protein was significantly higher in proliferating in MMP-9–/– and -12–/– VSMCs compared with wild-type VSMCs (1.18 ± 0.16 and 1.84 ± 0.42 vs. 0.74 ± 0.11 OD x mm2). Furthermore, addition of active MMP-9 or -12 to MMP-9–/– and -12–/– VSMCs for 2 h resulted in significantly lower levels of cellular N-cadherin (Figure 1B). Quantitative PCR demonstrated that the expression of N-cadherin mRNA was not significantly different in MMP-9–/–, -12–/–, and wild-type VSMCs (data not shown). This indicates that regulation is via protein stability rather than modulation of mRNA expression, corroborating our previous findings in human VSMCs.10


Figure 1
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  		Figure 1 Effect of MMP-9 and -12 on N-cadherin cleavage. (A) Representative western blots of quiescent (Q) and proliferating (P: PDGF) mouse wild-type (control), MMP-9–/– and MMP-12–/– VSMCs for cellular N-cadherin, cleaved (soluble) N-cadherin, and GAPDH (loading control). Densitometric analysis of N-cadherin protein expressed as fold change from quiescent cells. (B) Representative western blots of quiescent MMP-9–/– and -12–/–VSMCs treated with active MMP-9 and -12, respectively, for 2 h to detect cellular N-cadherin and GAPDH. Densitometric analysis of N-cadherin protein normalized by GAPDH levels. *Significant difference from quiescent VSMCs (n = 4).

 
Efficient siRNA knockdown of MMP-9 (74 ± 6%) and MMP-12 (70 ± 11%) was demonstrated by zymography and western blotting, respectively (see Supplementary material online, Figure S1). siRNA for MMP-9 did not affect MMP-12 protein and vice versa (see Supplementary material online, Figure S1). Combined siRNA knockdown of MMP-9 and -12 did not affect the efficiency of MMP-9 and -12 knockdown, 69 ± 7 and 74 ± 8%, respectively (see Supplementary material online, Figure S1). siRNA knockdown of MMP-9 and -12 expression significantly increased the amount of cellular N-cadherin protein compared with control VSMCs treated with control (GFP) siRNA (Figure 2), confirming the findings from the MMP-deficient VSMCs. Simultaneous knockdown of MMP-9 and -12 had a significantly greater effect on N-cadherin protein than knockdown of an MMP-9 alone but not MMP-12 alone (Figure 2).


Figure 2
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  		Figure 2 Effect of MMP-9 and -12 siRNA knockdown on N-cadherin protein expression. Representative western blots for N-cadherin and GAPDH of proliferating (FCS) VSMCs treated with siRNA for GFP, MMP-9, -12, and MMP-9 and -12. Densitometric analysis of N-cadherin protein normalized by GAPDH and expressed as a percentage of GFP control, *Significant difference from GFP control and $significant difference from MMP-9 alone (P < 0.05, n = 5).

 
Efficient siRNA knockdown of MMP-2 and -14 (>80%) was demonstrated by gelatin zymography and western blotting, respectively (see Supplementary material online, Figure S2A). However, knockdown of MMP-2 and -14 did not affect cellular N-cadherin protein or the presence of soluble N-cadherin in the conditioned media (see Supplementary material online, Figure S2B). Additionally, culture with a synthetic MMP-2 inhibitor or neutralizing MMP-14 antibody did not affect cellular N-cadherin levels (see Supplementary material online, Figure S2C).

Addition of active MMP-9 and -12 to purified soluble N-cadherin (SNC-Fc) for 30 min caused a dose-dependent and time-dependent cleavage (Figure 3A and B). Significant cleavage was observed with 20–2000 pM and at 30 min and 2 h. Combined addition of active MMP-9 and -12 caused a significantly greater cleavage than MMP-9 alone (Figure 3B). Addition of active MMP-9 or -12 to quiescent VSMCs also caused a significant reduction in the amount of N-cadherin present in cell lysates at 6 and 24 h after treatment (Figure 3C and D). However, despite an apparent trend, the combined addition of these MMPs did not significantly reduce the amount of N-cadherin (Figure 3C). To determine whether activation of MMP-9 by MMP-12 or vice versa was required for cleavage, we incubated MMP-9–/– and -12–/– VSMCs with active MMP-12 and -9, respectively. In both cases, the cleavage of N-cadherin was similar to that seen in the wild-type control VSMCs (Figure 3E), indicating that activation of the other MMP was not required for cleavage and therefore cleavage by both of these MMPs is direct.


Figure 3
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  		Figure 3 Cleavage of cellular and purified N-cadherin by MMP-9 and -12. (A) Representative western blots of SNC-Fc after incubation with active MMPs for 30 min. Densitometric analysis of SNC-Fc expressed as a percentage of the untreated control. *Significant difference from control (n = 3). (B) Representative western blots for SNC-Fc 30 min after the addition of 2 nM active MMP-9, -12, or MMP-9 and -12. Densitometric analysis of SNC-Fc at 30 min and 2 h expressed as a percentage of the untreated control. *Significant difference from control and $significant difference from MMP-9 treated (n = 4). (C) Representative western blots for cellular N-cadherin in quiescent wild-type control VSMC lysates 6 h after the addition of 2 nM active MMP-9, -12, or MMP-9 and -12. Densitometric analysis of N-cadherin expressed as a percentage of the untreated control. *Significant difference from 0 h control (n = 3). (D) Representative western blots for cellular N-cadherin in quiescent wild-type control VSMC lysates at various time-points after the addition of 2 nM active MMP-9 or -12. Densitometric analysis of N-cadherin expressed as a percentage of the untreated control. *Significant difference from control (n = 5). (E) Representative western blots for cellular N-cadherin in quiescent MMP-9 and -12 deficient VSMC lysates 6 h after the addition of 2 nM active MMP-9, -12, or MMP-9 and -12, n = 3.

 
3.2 β-Catenin localization and signalling
Localization of β-catenin performed by immunocytochemistry 4 h after stimulation with FCS revealed less nuclear β-catenin in MMP-9–/– and -12–/– VSMCs compared with wild-type VSMCs (Figure 4A), suggestive of less β-catenin signalling in the MMP-deficient VSMCs. Indeed, TOPFLASH reporter assays revealed that MMP-9 and -12 deficiency significantly reduced β-catenin signalling (Figure 4B). Similarly, MMP-9 and -12 knockdown with siRNA significantly reduced β-catenin signalling in proliferating TOPGAL VSMCs (Figure 4C). We previously demonstrated that cyclin D1 expression is up-regulated by β-catenin signalling in proliferating VSMCs,11 and therefore we examined cyclin D1 expression in MMP-9–/– and MMP-12–/– VSMCs and compared with wild-type VSMCs. A significant induction (~3.5-fold) in cyclin D1 expression was observed in wild-type VSMCs after stimulation with FCS (Figure 5A). However, in MMP-9–/– and -12–/– VSMCs, this increase in cyclin D1 was significantly attenuated (Figure 5A). Furthermore, addition of active MMP-9 and -12 significantly increased cyclin D1 levels in MMP-9–/– and -12–/– VSMCs, respectively.


Figure 4
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  		Figure 4 Effect of MMP-9 and -12 on β-catenin localization and signalling. (A) Fluorescent immunocytochemistry (green) for β-catenin in wild-type, MMP-9–/– and -12–/– VSMCs 4 h after stimulation with FCS. Scale bar representing 20 µm applies to all panels. Arrows indicate some VSMCs with nuclear β-catenin. (B) β-Catenin signalling quantified by TOPFLASH activity 24 h after the addition of FCS and expressed as a percentage of the wild-type control VSMCs. *Significant difference from wild-type control, n = 4. (C) β-Catenin signalling quantified in quiescent and PDGF-stimulated TOPGAL VSMCs 24 h after treatment with siRNA for GFP, MMP-9, -12, and MMP-9 and -12 and expressed as a percentage of the GFP control VSMCs. *Significant difference from GFP PDGF-stimulated control (n = 3).

 


Figure 5
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  		Figure 5 Effect of MMP-9 and -12 on cyclin D1 protein. (A) Representative western blots for cyclin D1 of quiescent and proliferating wild-type control, MMP-9–/– and -12–/– VSMCs 24 h after the addition of FCS. Densitometric analysis of results is expressed as a percentage of quiescent (Q) control (n = 7; *significant difference from quiescent control). (B) Representative western blots for cyclin D1 of quiescent MMP-9–/– and -12–/– VSMCs treated with 2 nM active MMP-9 and -12 for 6 h. Densitometric analysis shows cyclin D1 normalized by GAPDH (n = 3; *significant difference from quiescent control).

 
3.3 Vascular smooth muscle cell proliferation
Proliferation of VSMCs deficient in MMP-9 and -12 determined by BrdU incorporation was significantly less than that observed in wild-type control VSMCs (Figure 6A). Similar findings were observed by cell counting (data not shown). Knockdown of MMP-9 and -12 significantly reduced VSMC proliferation assessed by BrdU incorporation (Figure 6B). Furthermore, addition of active MMP-9 and -12 to MMP-9–/– and -12–/– VSMCs, respectively, significantly increased proliferation (Figure 6C). In contrast, siRNA knockdown of MMP-2 and -14 did not affect VSMC proliferation (see Supplementary material online, Figure S2D).


Figure 6
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  		Figure 6 Effect of MMP-9 and -12 on VSMC proliferation. Proliferation was assessed by BrdU incorporation in: (A) wild-type, MMP-9–/–, and -12–/– VSMCs 24 h after treatment with serum free media (quiescent) or FCS (proliferating). *Significant difference from quiescent control and $significant difference from wild-type control (n = 3). (B) Wild-type VSMCs 24 h after treatment with siRNA for GFP, MMP-9, -12, and MMP-9 and -12. *Significant difference from GFP control (n = 4). (C) MMP-9–/– and -12–/– VSMCs 24 h after addition of 2 nM active MMP-9 or -12. *Significant difference from GFP control (n = 4).

 
3.4 MMP-12 expression during intimal thickening
Immunohistochemistry revealed balloon injury of the rat carotid caused increased MMP-12 expression in the media at 2 days and in the intima at 10 days (see Supplementary material online, Figure S3). Similarly, MMP-12 protein was increased during intimal thickening in human saphenous vein organ cultures (see Supplementary material online, Figure S3).

3.5 Effect of plasmin activators on N-cadherin cleavage
Deficient in urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) or adenoviral over-expression of plasminogen activator inhibitor-1 (PAI-1) did not affect cellular N-cadherin protein in VSMCs (see Supplementary material online, Figure S4).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
We have previously demonstrated that dismantling of N-cadherin:β-catenin complexes modulates VSMC proliferation.10 Furthermore, we observed that this is mediated at least in part by MMP-dependent proteolysis. However, the identification of the MMP responsible for this proteolysis was unknown. In this study, we have demonstrated that MMP-9 and -12 are involved in the regulation of N-cadherin protein. In addition, this provides a novel mechanism for regulation of VSMC proliferation by these MMPs.

As reported in several cell types including VSMCs,19,25,26 we observed a link between MMP-9 and proliferation in this study. However, the mechanism by which MMP-9 regulates entry into the cell cycle has not been fully elucidated. In this study, therefore, we examined the possibility that MMP-9 regulates VSMC proliferation by disrupting cell–cell adhesion. We examined the effect of MMP-9 using two approaches: deficiency in MMP-9 using VSMCs from MMP-9 knockout mice and siRNA-mediated knockdown of MMP-9. We observed that lack of MMP-9 led to elevated levels of cellular N-cadherin protein and decreased nuclear β-catenin, β-catenin signalling, cyclin D1 levels, and proliferation. This suggests that MMP-9 regulates VSMC proliferation by disrupting cell–cell adhesion and increasing β-catenin signalling.

The findings of this study have implications for several cardiovascular pathologies including restenosis and atherosclerosis. In animal models of restenosis including the mouse carotid ligation27 and electric injury28 models and after balloon injury of rat and porcine carotids,29–31 elevated expression of MMP-9 has been observed. In addition, MMP-9 expression is elevated in models of vein graft restenosis.23,32 We suggest that these elevated levels of MMP-9 may lead to loss of N-cadherin from the cell surface and increased β-catenin signalling, which participates in the stimulation of VSMC proliferation that occurs during restenosis. This may also regulate proliferation of VSMCs during atherosclerotic plaque formation. Choi et al.33 demonstrated that reduced migration and proliferation may be responsible for the reduced accumulation of VSMCs occurs in atherosclerotic plaque in apolipoprotein E/MMP-9 deficient mice. It is possible therefore that increased N-cadherin levels as a result of MMP-9 deficiency may therefore retard VSMC proliferation in these plaques.

A role for MMP-12 in VSMC proliferation is less well established. However, Wu et al.18 previously demonstrated that stimulation of proliferation of a VSMC cell line with PDGF-BB caused a concurrent significant increase in MMP-12 expression. Additionally, increased expression of MMP-12 has been detected in intimal VSMCs in human34 and rabbit35 atherosclerotic plaques. We have confirmed that MMP-12 is expressed in mouse VSMCs and is elevated in intima lesions in the rat balloon injury model and human saphenous veins. In this study, we observed an association of MMP-12 with VSMC proliferation. In a similar manner to MMP-9, deficiency or knockdown of MMP-12 led to elevated levels of N-cadherin protein, nuclear β-catenin, β-catenin signalling, cyclin D1 levels, and proliferation. This suggests that MMP-12 also can regulate VSMC proliferation by disrupting cell–cell adhesion and increasing β-catenin signalling.

To determine whether MMP-9 was responsible for activation of MMP-12 or vice versa, we incubated MMP-deficient VSMCs with the opposite active MMP. In both cases, cleavage of N-cadherin was similar to that observed in the wild-type VSMCs, indicating that activation of one MMP by the other is not involved. To determine whether these MMPs are capable of direct cleavage of N-cadherin, we incubated purified N-cadherin with active MMPs. We observed that both MMPs cleaved the purified protein in 30 min, indicating that cleavage of N-cadherin is direct and does not require the presence of other proteins. In all experiments, it appeared that MMP-12 was more effective at cleaving N-cadherin and this may result in the greater observed effect on proliferation. In particular, MMP-12 was more effective at cleavage of SNC-Fc than MMP-9. It is therefore possible that MMP-9 requires a cellular component to maximize proteolytic activity, such as CD44 or {alpha}4β1 integrin, which facilitates binding of MMP-9 to the cell surface.36,37 In general, very little additional effect was observed when both MMPs were combined. This may indicate that there is a common site of cleavage by these two MMPs or that a sustainable threshold of cleavage exists.

It is possible that increased β-catenin signalling leads to elevated transcription of other β-catenin target genes in addition to cyclin D1. To date, at least 54 mammalian β-catenin target genes have been identified (see http://www.stanford.edu/~rnusse/wntwindow.html for a current list). Although it was beyond the scope of this current study to determine which of the other β-catenin target genes are regulated as a result of MMP-9 and -12 activity, we are currently undertaking additional studies in our laboratory to determine whether other β-catenin target genes are involved in the stimulation of VSMC proliferation.

Although this study substantiates existing evidence that MMPs are involved in proteolytic cleavage of cadherins,10,38–43 this is the first observation to our knowledge that MMP-9 and -12 can cleave N-cadherin. Our study does not exclude the involvement of other MMPs in addition to MMP-9 and -12 and further studies are required to examine the involvement of other MMPs. However, from this study, we can eliminate the involvement of MMP-2 and -14, two other MMPs associated with activated VSMCs.44,45 Our results with MMP-2 support previous findings with NRK cells, but the lack of effect with MMP-14 is in contrast to observations in ischaemic NRK cells,21 which may indicate cell type or stimulus specific effects. Furthermore, it should be noted that other proteases are capable of cleavage of cadherins. It is reported that plasmin,46 calpain,47 presenilin/{gamma}-secretase complex,40 ADAMs,48,49 and caspases50 are all capable of cleavage of cadherins. We observed no effect of deficiency in uPA and tPA or over-expression of PAI-1 on N-cadherin cleavage in VSMCs, suggesting a lack of involvement of these proteases and plasmin. It is possible that in different cell types other proteases may modulate proliferation via shedding of cadherins and during various cellular processes cleavage of cadherins may be mediated by different proteases. It is also possible that more than one class of protease is required for complete cleavage of the cadherin protein from the cell surface and release of β-catenin into the cytoplasm. For example, the extracellular domain of the cadherin may be released through MMP-dependent proteolysis mediated while the remainder of the cadherin protein is further processed by different protease, as seen in neurons with ADAM10 and {gamma}-secretase, respectively.48 Our observation that deficiency of MMP-9 and -12 is sufficient to significantly reduce the loss of N-cadherin suggests that proteolysis by MMP-9 and -12 may be involved in the initiating proteolytic steps.

In conclusion, our findings demonstrate that MMP-9 and -12 regulate β-catenin signalling via modulation of the N-cadherin:β-catenin complex and result in increased cyclin D1 protein and VSMC proliferation. The cleavage of N-cadherin by these MMPs may be significant for the regulation of cell–cell adhesion and VSMC proliferation during intimal thickening in vascular pathologies.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
British Heart Foundation (RG/04/009).


   Acknowledgements
 
We are very grateful to Mrs Jill Tarlton for her excellent technical assistance. We thank Jason Johnson for assistance with genotyping of the MMP-9 and MMP-12 deficient mice and Cressida Beeching for generating SNC-Fc.

Conflict of interest: none declared.


   Notes
 
{dagger} Present address. Paul O'Gorman Lifeline Centre, Southmead Hospital, Bristol BS10 5NB, UK. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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