Regulation of membrane type-1 matrix metalloproteinase activity and intracellular localization in clinical thoracic aortic aneurysms

Abstract

Objective: Membrane type-1 matrix metalloproteinase (MT1-MMP) is elevated during thoracic aortic aneurysm (TAA) development in mouse models, and plays an important role in the activation of matrix metalloproteinase (MMP)-2 and the release of matrix- bound transforming growth factor-β. In this study, we tested the hypothesis that MT1-MMP is subject to protein kinase C (PKC)-mediated regulation, which alters intracellular trafficking and activity with TAAs.

Methods: Levels of MMP-2, native and phosphorylated MT1-MMP, and PKC-δ were measured in aortic tissue from patients with small TAAs (<5 cm; n = 8) and large TAAs (>6.5 cm; n = 8), and compared with values measured in normal controls (n = 8). Cellular localization of green fluorescent protein (GFP)-tagged MT1-MMP was assessed in aortic fibroblasts isolated from control and 4-week TAA mice. The effects of PKC-mediated phosphorylation on MT1-MMP cellular localization and function (active MMP-2 vs phospo-Smad2 abundance) were assessed after treatment with a PKC activator (phorbol-12-myristate-13-acetate [PMA], 100 nM) with and without a PKC-δ-specific inhibitor (röttlerin, 3 μM).

Results: Compared with controls, MT1-MMP abundance was increased in aortas from both TAA groups. Active MMP-2 was increased only in the large TAA group. The abundances of phosphorylated MT1-MMP and activated PKC-δ were enhanced in the small TAA group compared with the large TAA group. MT1-MMP was localized on the plasma membrane in aortic fibroblasts from control mice and in endosomes from TAA mice. Treatment with PMA induced MT1-MMP-GFP internalization, enhanced phospho-Smad2, and reduced MMP-2 activation, whereas röttlerin pretreatment inhibited these effects.

Conclusions: Phosphorylation of MT1-MMP mediates its activity through directing cellular localization, shifting its role from MMP-2 activation to intracellular signaling. Thus, targeted inhibition of MT1-MMP may have therapeutic relevance as an approach to attenuating TAA development.

Keywords: MT1-MMP; aneurysm; protein kinase C; remodeling; thoracic aorta.

Figure 1. MT1-MMP and MMP-2 abundance in clinical TAA specimens

A. Top. Representative immunoblot showing MT1-MMP protein abundance in normal aorta (n=8), small TAA (4.0 – 4.9 cm; n=8), and large TAA (6.5 – 8.0 cm; n=8). Bottom. Densitometric quantitation of MT1-MMP protein abundance determined by immunoblotting. Results are expressed as a mean percent change of normal aorta. Individual data points in each group overlay box plots. The box defines the 25^th–75^th interquartile range, and both the median (red line) and mean (green line) values are shown within the box. * p<0.05 versus Normal (ANOVA with Tukey’s wsd). B. Top. Representative zymogram showing the protein abundance of the latent form (72kD band) and active form (64kD band) of MMP-2 in normal aorta (n=7), small TAA (4.0 – 4.9 cm; n=8), and large TAA (6.5 – 8.0 cm; n=8). Bottom. Densitometric quantitation of active (64kD band) MMP-2 protein abundance determined by gelatin zymography. Results are expressed as a mean percent change of normal aorta. Individual data points in each group overlay box plots. The box defines the 25^th–75^th interquartile range, and both the median (red line) and mean (blue line) values are shown within the box. * p<0.05 versus Normal, # p<0.05 versus Small TAA (ANOVA with Tukey’s wsd).

Figure 2. Phosphorylation of MT1-MMP in clinical TAA specimens

A. MT1-MMP was immunoprecipitated from normal aorta, small TAA (4.0 – 4.9 cm), and large TAA (6.5 – 8.0 cm). The immunoprecipitated protein was fractionated by electrophoresis and immunoblotted for phospho-Threonine and for MT1-MMP to confirm immunoprecipitation. Tissue homogenates from the same sample set were also immunoblotted for total PKC-δ, and phosphor-PKC-δ. Representative images are shown. B. Densitometric quantitation of MT1-MMP-specific phospho-Threonine residues in normal aorta (n=6), small TAA (4.0 – 4.9 cm; n=5), and large TAA (6.5 – 8.0 cm; n=5). Results are expressed as a mean percent change of normal aorta. Individual data points in each group overlay box plots. The box defines the 25^th–75^th interquartile range, and both the median (red line) and mean (green line) values are shown within the box. * p<0.05 versus Normal (ANOVA with Tukey’s wsd). C. Densitometric quantitation of the ratio of phospho-PKC-δ (activated):total PKC-δ from normal aorta (n=6), small TAA (4.0 – 4.9 cm; n=5), and large TAA (6.5 – 8.0 cm; n=4). Results are expressed as a mean percent change of normal aorta. Individual data points in each group overlay box plots. The box defines the 25^th–75^th interquartile range, and both the median (red line) and mean (purple line) values are shown within the box. * p<0.05 versus Normal, # p<0.05 versus Small TAA (ANOVA with Tukey’s wsd).

Figure 3. MT1-MMP abundance and localization in primary aortic fibroblasts

A. Protein abundance of MT1-MMP in normal and 4-week TAA aortic fibroblasts (n=4 each group). Results are expressed as a mean percent change of Control aorta. Individual data points in each group overlay box plots. The box defines the 25^th–75^th interquartile range, and both the median (red line) and mean (green line) values are shown within the box. * p<0.05 versus Control (unpaired Student’s t-test). B. Representative images from normal and 4-week TAA aortic fibroblasts transfected with MT1-MMP-GFP. At steady-state MT1-MMP-GFP was localized predominantly at the plasma membrane in the normal cells, but was almost exclusively localized in endosomal vesicles in the 4-week TAA cells.

Figure 4. PKC-δ-mediated MT1-MMP translocation in normal aortic fibroblasts

Representative images of primary aortic fibroblasts transfected with MT1-MMP-GFP and treated with or without PMA, in the presence or absence of Röttlerin. Images show MT1-MMP-GFP predominantly at the plasma membrane in controls. Upon activation of PKC with PMA, MT1-MMP translocated to intracellular endosomal vesicles. When the cells were pretreated with Röttlerin, a PKC-δ-specific inhibitor, PMA-induced translocation was inhibited. Röttlerin alone was sufficient to lock MT1-MMP at the plasma membrane.

Figure 5. PMA induced changes in MT1-MMP function

A. Primary aortic fibroblasts from normal mice (n=4) were treated with or without PMA, in the presence or absence of Röttlerin for 18 h (n=4 each treatment group). Cell homogenates were fractionated by gel electrophoresis and immunoblotted for latent and active MMP2. Results are expressed as a mean percent change in each cell line from its respective steady-state control (vehicle treated; dotted line). Individual data points in each group overlay box plots. The box defines the 25^th–75^th interquartile range, and both the median (red line) and mean (blue line) values are shown within the box. * p<0.05 versus control (ANOVA with Tukey’s wsd). B. Primary aortic fibroblasts from normal mice (n=4) were treated with or without PMA (30 min), in the presence or absence of Röttlerin (3 h pre-incubation, plus 30 min concurrent; n=4 each treatment group). Cell homogenates were fractionated by gel electrophoresis and immunoblotted for phosphor-Smad-2. Results are expressed as a mean percent change in each cell line from its respective steady-state control (vehicle treated; dotted line). Individual data points in each group overlay box plots. The box defines the 25^th–75^th interquartile range, and both the median (red line) and mean (orange line) values are shown within the box. * p<0.05 versus control, # p<0.05 versus PMA (ANOVA with Tukey’s wsd).