MMP-7 mediates cleavage of N-cadherin and promotes smooth muscle cell apoptosis - PubMed (original) (raw)

MMP-7 mediates cleavage of N-cadherin and promotes smooth muscle cell apoptosis

Helen Williams et al. Cardiovasc Res. 2010.

Abstract

Aims: Vascular smooth muscle cell (VSMC) apoptosis can lead to thinning of the fibrous cap and plaque instability. We previously showed that cell-cell contacts mediated by N-cadherin reduce VSMC apoptosis. This study aimed to determine whether matrix-degrading metalloproteinase (MMP)-dependent N-cadherin cleavage causes VSMC apoptosis.

Methods and results: Induction of human VSMC apoptosis using different approaches, including 200 ng/mL Fas ligand (Fas-L) and culture in suspension, caused N-cadherin cleavage and resulted in the appearance of a C-terminal fragment of N-cadherin (approximately 35 kDa). Appearance of this fragment during apoptosis was inhibited by 47% with the broad-spectrum MMP inhibitor BB-94. We observed retarded cleavage of N-cadherin after treatment with Fas-L in aortic mouse VSMCs lacking MMP-7. Furthermore, VSMC apoptosis, measured by quantification of cleaved caspase-3, was 43% lower in MMP-7 knockout mouse VSMCs compared with wild-type VSMCs following treatment with Fas-L. Addition of recombinant active MMP-7 increased the amount of N-cadherin fragment by 82% and augmented apoptosis by 53%. The involvement of MMP-7 was corroborated using human cells, where a MMP-7 selective inhibitor reduced the amount of fragment formed by 51%. Importantly, we observed that treatment with Fas-L increased levels of active MMP-7 by 80%. Finally, we observed significantly increased cleavage of N-cadherin, MMP-7 activity, and apoptosis in human atherosclerotic plaques compared with control arteries, and a significant reduction in apoptosis in atherosclerotic plaques from MMP-7 knockout mice.

Conclusion: This study demonstrates that MMP-7 is involved in the cleavage of N-cadherin and modulates VSMC apoptosis, and may therefore contribute to plaque development and rupture.

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Figures

Figure 1

Figure 1

N-cadherin cleavage occurs during VSMC apoptosis. Representative western blot for full-length (FL, 130 kDa) and ∼35 kDa C-terminal fragment of N-cadherin protein in human VSMCs after induction of apoptosis with Fas-L at various time-points (A), full-length N-cadherin at 4 h (B), and ∼35 kDa C-terminal fragment of N-cadherin after 4 h (C). Bar charts show densitometric analysis of western blots (B and C) and * indicates a significant difference (P < 0.05) from control, n = 5. (D) Representative western blot for full-length (130 kDa) and ∼35 kDa C-terminal fragment of N-cadherin protein in human VSMCs after induction of apoptosis with 50 µM Bcl2 antagonist (Bcl2 antag) for 18 h, 100 µM H2O2, or 50 ng/mL TNFα for 22 h, and suspension culture for 24 h. β-Tubulin is shown as loading control.

Figure 2

Figure 2

N-cadherin cleavage during apoptosis is mediated by MMPs. (A) Representative western blots (n = 6) for N-cadherin protein in human VSMCs 4 h after the addition of Fas-L and 1 µM BB-94 or DMSO (vehicle control). (B) Representative western blots (n = 3) N-cadherin protein in mouse wild-type VSMCs 4 h after the addition of Fas-L with or without 1 µM BB-94 or DMSO vehicle control. (C) Representative western blots (n = 3) for N-cadherin protein in mouse wild-type or MMP-3, -7, -9, or -12 knockout VSMCs 4 h after the addition of Fas-L. (D) Representative western blots (n = 3) for N-cadherin protein in mouse MMP-7 knockout VSMCs 4 h after the addition of Fas-L with or without 1 µM BB-94 or DMSO vehicle control. Bar charts (A, B, and C) show densitometric analysis of western blots for ∼35 kDa C-terminal fragment of N-cadherin, data are shown as percent of control, and * and $ indicate a significant difference (P < 0.05) from control and Fas-L + DMSO, respectively. β-Tubulin is shown as loading control. (E) Cleaved caspase-3 activity expressed as LAU in mouse wild-type or MMP-3, -7, -9, or -12 knockout (KO) VSMCs 8 h after the addition of Fas-L. * indicates a significant difference (P < 0.05) from wild-type control, n = 6.

Figure 3

Figure 3

MMP-7 promotes N-cadherin cleavage and apoptosis. Representative Western blots for full-length N-cadherin (A), and ∼35 kDa C-terminal fragment of N-cadherin (B) at 4 h after addition of 60 nM recombinant MMP-7. β-Tubulin is shown as loading control. Bar charts show densitometric analysis of western blots (A and B), data are shown as % of control and * indicates a significant difference (P < 0.05) from control, n = 6. Cleaved caspase-3 activity in human VSMCs treated with MMP-7 for 24 h (C), * indicates a significant difference (P < 0.05) from control, n = 3. MMP-7 activity in human VSMC conditioned media with or without Fas-L for 4 h (D), * indicates a significant difference (P < 0.05) from control, n = 4. (E) Representative western blot for Fc domain of mouse IgG after 30 min incubation of 2 nM MMP-7 with 2 nM recombinant N-cadherin (SNC-Fc).

Figure 4

Figure 4

MMP-7 inhibitor (MMP-7i) retards N-cadherin cleavage and VSMC apoptosis. (A) Representative western blot (n = 3) for full length and ∼35 kDa C-terminal fragment of N-cadherin protein in human VSMCs 4 h after the addition of Fas-L with or without 10 nM MMP-7 inhibitor (MMP-7i). (B) Representative western blot (n = 3) for full length and ∼35 kDa C-terminal fragment of N-cadherin protein in mouse wild-type VSMCs grown in suspension (Susp) to induce apoptosis and treated with 10 nM MMP-7 inhibitor. Bar charts (A and B) show densitometric analysis of western blots, data are shown as percent of control and * indicates a significant difference (P < 0.05) from Fas-L or suspension culture alone. (C) Cleaved caspase-3 activity in human VSMCs treated with Fas-L with or without 10 nM MMP-7 inhibitor for 8 h, * indicates a significant difference (P < 0.05) from Fas-L alone, n = 4. (D) Representative western blots (n = 3) for pAkt, and total Akt. Bar chart shows densitometric analysis of western blots, data are shown as percent of control and * and $ indicate a significant difference (P < 0.05) from control and Fas-L, respectively. β-Tubulin is shown as loading control. All control samples were supplemented with DMSO to act as a vehicle control.

Figure 5

Figure 5

N-cadherin, MMP-7, and apoptosis in human IMA and atherosclerotic plaques. Representative western blot for N-cadherin (A) and cleaved caspase-3 (B) in IMA and coronary and carotid atherosclerotic plaques, n = 3 per group. β-Tubulin is shown as loading control. Bar chart in (A) shows densitometric analysis of N-cadherin western blots. (C) MMP activity in IMA and atherosclerotic plaques, n = 15 and 20. * indicates a significant difference from IMA.

Figure 6

Figure 6

MMP-7 and apoptosis in atherosclerotic plaques. In situ zymography of coronary atherosclerotic plaque in the absence (A) and presence (B) of 10 nM MMP-7 inhibitor. Green colour indicates MMP activity and nuclei are stained blue with DAPI. Dual detection of cleaved caspase-3 (red) and MMP activity (green) in coronary plaque (C) and IMA (D). (E) High power image of marked area in (C), arrows indicate co-location of apoptosis and MMP activity. (F) Quantification of the percentage of apoptotic (ISEL positive) cells in atherosclerotic plaques from ApoE−/−/MMP-7+/+ and ApoE−/−/MMP-7−/−. * indicates a significant difference from ApoE−/−/MMP-7+/+. ISEL staining of atherosclerotic plaques from ApoE−/−/MMP-7+/+ (G) and ApoE−/−/MMP-7−/− (H) mice. ISEL positive apoptotic cells are green and nuclei are stained blue (DAPI).

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