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First published online 9 December 2008
doi: 10.1242/jcs.035279

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MT1-MMP promotes vascular smooth muscle dedifferentiation through LRP1 processing

Kaisa Lehti^1,*, Nina F. Rose^1,, Sara Valavaara¹, Stephen J. Weiss² and Jorma Keski-Oja¹

¹ Departments of Pathology and Virology, Haartman Institute, University of Helsinki and Helsinki University Hospital, Biomedicum Helsinki, FIN-00014 Helsinki, Finland
² Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA

View larger version (54K): [in this window] [in a new window]   Fig. 1. Correlation between MT1-MMP expression and VSMC contractile-protein expression. (A) MT1-MMP levels were assessed by immunoblotting protein extracts from the aortas of 4-day-old wild-type (+/+) and MT1-MMP–/– (–/–) mice, and from the corresponding isolated VSMCs at the indicated passages using antibodies against the catalytic domain of MT1-MMP. Extracts from human HT-1080 fibrosarcoma cells that expressed constitutively activated MT1-MMP were analyzed as controls side-by-side with the tenth-passage VSMCs. β-tubulin served as a loading control. Mean values of relative MT1-MMP levels are expressed below each lane (n=2). Relative mobilities of the molecular-mass markers are indicated in kDa. (B) Aortic frozen sections of 4-day-old wild-type (MT1-MMP+/+) and MT1-MMP–/– mice, and the corresponding isolated passage-1 and -10 VSMCs from the aortas of wild-type and MT1-MMP–/– mice, were analyzed for SMA by immunofluorescence. Nuclei were visualized by DAPI. Note the high SMA expression in the tenth-passage MT1-MMP–/– VSMCs. Scale bars: 20 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 2. MT1-MMP promotes PDGFRβ- and PDGF-BB-dependent suppression of VSMC contractile proteins. (A) Quantitative assessment of SMA and calponin mRNA expression by real-time PCR. mRNA was isolated from subconfluent tenth-passage VSMCs cultured with the wide-spectrum MMP inhibitor BB-94 (10 µM), PDGFRβ kinase inhibitor AG1296 (10 µM) and EGFR tyrosine-kinase inhibitor AG1478 (10 µM) as indicated. The expression data are normalized against mouse TBP and presented relative to the mRNA expression in mock (0.1% DMSO)-treated wild-type cells (mean ±1 s.d., n=3). (B) Early-passage wild-type (MT1+/+) and MT1-MMP–/– VSMCs were cultured on polymerized collagen I in the presence of TIMP2 (4 µg/ml) for 48 hours as indicated, and exposed to PDGF-BB (25 ng/ml) under serum-free conditions. After 48 hours, the cells were lysed and the lysates analyzed by immunoblotting for the relative levels of calponin, SMA and vimentin. β-tubulin served as a loading control. Mean values of the ratios between normalized SMA and vimentin protein levels are presented below each lane (n=3). Relative mobilities of the molecular-mass markers are indicated in kDa. (C) Calponin expression was assessed by immunofluorescence staining of cells exposed to PDGF-BB as above. Filamentous actin was visualized with phalloidin (F-actin) and nuclei with DAPI. (D) MT1-MMP–/– VSMCs were transfected with EGFP expression-vector alone (Mock), or with either expression construct for wild-type MT1-MMP (MT1-MMP) or the catalytically inactive mutant (MT1-E240A) as indicated. Calponin expression (red) was assessed by immunofluorescence after 48 hours of treatment with PDGF-BB.

View larger version (49K): [in this window] [in a new window]   Fig. 3. MT1-MMP induces LRP1 processing. (A) Wild-type (MT1+/+) and MT1-MMP–/– (MT1–/–) VSMCs were cultured on polymerized collagen I for 48 hours in the presence of MMP inhibitor (GM6001, 10 µM) as indicated and treated with PDGF-BB (25 ng/ml) under serum-free conditions. Total cell lysates were analyzed by immunoblotting for both the - and β-chains of LRP1. (B) The cells were treated with PDGF-BB for 48 hours. For the last 24 hours of PDGF-BB treatment, the cells were labeled with [35S]-methionine (50 µCi/ml) as indicated. Soluble LRP1 fragments were detected from the conditioned medium by immunoprecipitation with polyclonal rabbit anti-LRP antibodies followed by immunoblotting with mouse monoclonal anti-LRP antibodies (LRP) or by autoradiography ([S35]-meth). Relative mobilities of the molecular-mass markers are indicated in kDa.

View larger version (50K): [in this window] [in a new window]   Fig. 4. MT1-MMP promotes interactions between PDGFRβ, β3 integrin and LRP1. (A) VSMCs were treated with PDGF-BB for 30 minutes and lysed. The lysates were subjected to immunoprecipitation with polyclonal rabbit anti-LRP1 antibodies. Tyrosine-phosphorylated proteins in the immunoprecipitates were detected by anti-phosphotyrosine antibodies (pY) and LRP1 by mouse monoclonal anti-LRPβ and anti-LRP antibodies. Arrow indicates the phosphorylated protein that co-precipitates with LRP1 and most probably represents PDGFRβ. (B) LRPβ protein levels, as detected by immunoblotting, in the lysates of VSMCs that were treated with PDGF-BB for 3 hours. β-tubulin served as a loading control. Arrow indicates the cleaved fragment of LRP1 β-chain. (C) Wild-type (MT1+/+) and MT1-MMP-null (MT1–/–) VSMCs were cultured on polymerized collagen I and were serum starved for 24 hours. The cells were then treated with PDGF-BB (10 ng/ml) for 30 minutes and lysed. The levels of β1- and β3-integrins in the cell lysates were detected by immunoblotting. β-tubulin served as a loading control. (D) The cell lysates were prepared as above and subjected to immunoprecipitation with polyclonal rabbit anti-β1- and β3-integrin antibodies. PDGFRβ, LRPβ, MT1-MMP, and β1- and β3-integrin in the immunoprecipitates were detected by immunoblotting.

View larger version (64K): [in this window] [in a new window]   Fig. 5. MT1-MMP is required for efficient ligand-induced internalization of PDGFRβ in VSMCs. (A) VSMCs on polymerized collagen I were fixed and permeabilized for immunofluorescence staining using antibodies against PDGFRβ and caveolin 1. PDGFRβ colocalized with caveolin 1 (caveolae) in both wild-type (+/+) and MT1-MMP-null (–/–) cells, but specific intracellular caveolin-1 structures did not contain PDGFRβ in MT1-MMP–/– VSMCs. (B) To detect internalization, quiescent VSMCs were labeled with anti-PDGFRβ antibodies for 1 hour at 4°C. Unbound antibodies were removed and the cells shifted to 37°C for 30 minutes in the presence of PDGF-BB (10 ng/ml). After blocking the anti-PDGFRβ antibodies that remained on the cell surface with unlabelled secondary antibodies, the cells were fixed, permeabilized and stained with anti-caveolin-1 antibodies followed by secondary Alexa-Fluor-488- and Alexa-Fluor-594-conjugated antibodies. The merged high-magnification image depicts colocalization between internalized PDGFRβ and caveolin 1 in yellow. Note the efficient PDGFRβ internalization in caveolin-positive structures of wild type (+/+) VSMCs in contrast to minor PDGFRβ internalization in MT1-MMP-null (–/–) cells.

View larger version (71K): [in this window] [in a new window]   Fig. 6. PDGFRβ remains stable in PDGF-BB-induced and -internalized LRP1 complexes in MT1-MMP-expressing cells. (A) Control (+/+) and MT1-MMP-null (–/–) VSMCs on collagen were treated with PDGF-BB for 30 minutes and the subcellular localization of LRP1 and PDGFRβ analyzed by immunofluorescence. Note the intracellular colocalization of these receptors in PDGF-BB-treated wild-type VSMCs. (B) VSMCs were surface biotinylated and shifted to 37°C for 30 minutes in the presence of PDGF-BB to allow PDGFRβ internalization. The biotinylated PDGFRβ remaining on the cell surface was left intact (biotinylated) or removed by reduction (internalized). Total biotinylated and internalized receptors were then detected in PDGFRβ immunoprecipitates by streptavidin conjugate. Total and ubiquitylated PDGFRβ in precipitates was detected by immunoblotting. (C) VSMCs were stimulated with PDGF-BB for the indicated periods of time (minutes) and lysed for analysis. Total PDGFRβ protein levels were assessed by immunoblotting. Relative mobilities of the molecular-mass markers are indicated in kDa.

View larger version (47K): [in this window] [in a new window]   Fig. 7. LRP1 knockdown by siRNA rescues PDGF-BB-mediated dedifferentiation of MT1-MMP–/– VSMCs. (A) Wild-type (MT1+/+) and MT1-MMP–/– (MT1–/–) VSMCs were plated on type I collagen and transfected with control, β3-integrin (right panels) or LRP1 (left panels) siRNAs as indicated. The cells were then exposed to PDGF-BB (25 ng/ml) under serum-free conditions for 48 hours and lysed. The relative levels of LRP1, β3 integrin and PDGFRβ in the lysates were assessed by immunoblotting. β-tubulin served as a loading control. (B) The relative levels of calponin and SMA were assessed by immunoblotting. Similar results were obtained with two different siRNA oligomers targeting LRP1 [LRP(1) and LRP(2)] and β3 integrin. (C) Quantitative assessment of SMA and calponin protein levels normalized against β-tubulin. The data are expressed as relative values (mean ±1 s.d., n=3).

View larger version (46K): [in this window] [in a new window]   Fig. 8. Schematic representation of the MT1-MMP-dependent regulation of PDGFRβ-membrane interactions and VSMC gene expression. Our results support the mechanism of VSMC-phenotype regulation in which MT1-MMP induction after vascular injury or exposure to growth stimuli results in LRP processing and the dynamic association of complexes of PDGFRβ, β3 integrin and LRP1. These complexes can be actively mobilized through endocytosis coincidently with efficient signal transduction through downstream pathways to the nuclei (Tallquist and Kazlauskas, 2004). There, the altered interactions of transcription factors, including serum response factor and myocardin, lead to the suppression of contractile genes, and to the induction of pro-migratory and pro-mitogenic genes (Kawai-Kowase and Owens, 2007).

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