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


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, beta-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 beta-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.


CONCLUSIONS
In conclusion, we propose that MMP-9 and -12 promote intimal thickening by independent cleavage of N-cadherin, which elevates VSMC proliferation via beta-catenin signalling.
Bristol Heart Institute, University of Bristol, Level 7, Bristol Royal Infirmary, Bristol BS2 8HW, UK.
Amrita
A
Dwivedi
Sadie C
SC
Slater
Sarah J
SJ
George
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism*; D002448:Cell Adhesion; D049109:Cell Proliferation; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism; D053508:Matrix Metalloproteinase 12 / Q000378:metabolism*; D053511:Matrix Metalloproteinase 14 / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology*; D051176:beta Catenin / Q000378:metabolism*
D015820:Cadherins; C489083:Cdh2 protein, mouse; D034741:RNA, Small Interfering; D051176:beta Catenin; D019938:Cyclin D1; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D053508:Matrix Metalloproteinase 12; D053511:Matrix Metalloproteinase 14