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TIMP-2 Interaction with MT1-MMP Activates the AKT Pathway and Protects Tumor Cells from Apoptosis.

Valacca C, Tassone E, Mignatti P - PLoS ONE (2015)

Bottom Line: In addition, MT1-MMP activates intracellular signaling through proteolysis-dependent and independent mechanisms.Here we show that in MT1-MMP expressing cells TIMP-2 also induces rapid and sustained activation of AKT in a dose- and time-dependent manner and by a mechanism independent of the proteolytic activity of MT1-MMP.Conversely, TIMP-2 upregulates apoptosis induced by three-dimensional type I collagen in epithelial cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiothoracic Surgery, New York University School of Medicine, New York, New York, United States of America.

ABSTRACT
Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades a variety of extracellular matrix (ECM) components. In addition, MT1-MMP activates intracellular signaling through proteolysis-dependent and independent mechanisms. We have previously shown that binding of tissue inhibitor of metalloproteinases-2 (TIMP-2) to MT1-MMP controls cell proliferation and migration, as well as tumor growth in vivo by activating the Ras-extracellular signal regulated kinase-1 and -2 (ERK1/2) pathway through a mechanism that requires the cytoplasmic but not the proteolytic domain of MT1-MMP. Here we show that in MT1-MMP expressing cells TIMP-2 also induces rapid and sustained activation of AKT in a dose- and time-dependent manner and by a mechanism independent of the proteolytic activity of MT1-MMP. Fibroblast growth factor receptor-1 mediates TIMP-2 induction of ERK1/2 but not of AKT activation; however, Ras activation is necessary to transduce the TIMP-2-activated signal to both the ERK1/2 and AKT pathways. ERK1/2 and AKT activation by TIMP-2 binding to MT1-MMP protects tumor cells from apoptosis induced by serum starvation. Conversely, TIMP-2 upregulates apoptosis induced by three-dimensional type I collagen in epithelial cancer cells. Thus, TIMP-2 interaction with MT1-MMP provides tumor cells with either pro- or anti-apoptotic signaling depending on the extracellular environment and apoptotic stimulus.

No MeSH data available.


Related in: MedlinePlus

Downregulation of MT1-MMP blocks TIMP-2 activation of AKT in MT1-MMP expressing MDA-MB-435 cells.Western blotting analysis of AKT activation (p-AKT) and MT1-MMP expression in MDA-MB-435 cells transiently transfected with MT1-MMP siRNA (siMT1-MMP) or control, scrambled siRNA (siControl), and incubated with TIMP-2 (100 ng/ml) for 15 min. Total AKT and β-tubulin (TUB) are shown as loading controls. The lower panel shows the densitometric analysis of the MT1-MMP and p-AKT bands normalized to the corresponding TUB and AKT controls, respectively. *, p ≤ 0.05; + TIMP-2 vs. the corresponding – TIMP-2 sample. This experiment was repeated twice with comparable results.
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pone.0136797.g002: Downregulation of MT1-MMP blocks TIMP-2 activation of AKT in MT1-MMP expressing MDA-MB-435 cells.Western blotting analysis of AKT activation (p-AKT) and MT1-MMP expression in MDA-MB-435 cells transiently transfected with MT1-MMP siRNA (siMT1-MMP) or control, scrambled siRNA (siControl), and incubated with TIMP-2 (100 ng/ml) for 15 min. Total AKT and β-tubulin (TUB) are shown as loading controls. The lower panel shows the densitometric analysis of the MT1-MMP and p-AKT bands normalized to the corresponding TUB and AKT controls, respectively. *, p ≤ 0.05; + TIMP-2 vs. the corresponding – TIMP-2 sample. This experiment was repeated twice with comparable results.

Mentions: To investigate whether TIMP-2 activation of AKT is a unique feature of MCF-7 cells or results from the high levels of MT1-MMP expressed by the transfected cells, we characterized the effect of TIMP-2 on AKT activation in human MDA-MB-435 melanoma cells, which constitutively express MT1-MMP. To analyze the requirement for MT1-MMP we transfected these cells with MT1-MMP siRNA or control, scrambled siRNA. Forty-eight hours later the cells were treated with TIMP-2, and analyzed for MT1-MMP expression and AKT activation. MT1-MMP expression was reduced by approximately 80% in the cells transfected with MT1-MMP siRNA relative to the control siRNA transfectants (Fig 2). TIMP-2 addition to the culture medium of control siRNA transfectants resulted in increased level of MT1-MMP, an effect that reflects the stabilization and accumulation of the relatively low amount of active MT1-MMP on the cell surface [39]. Consistent with our previous results, addition of TIMP-2 to the culture medium of control siRNA-transfected cells induced rapid (15 min) activation of AKT. Conversely, TIMP-2 had no such effect in MT1-MMP siRNA transfectants, which expressed almost undetectable levels of MT1-MMP (Fig 2).


TIMP-2 Interaction with MT1-MMP Activates the AKT Pathway and Protects Tumor Cells from Apoptosis.

Valacca C, Tassone E, Mignatti P - PLoS ONE (2015)

Downregulation of MT1-MMP blocks TIMP-2 activation of AKT in MT1-MMP expressing MDA-MB-435 cells.Western blotting analysis of AKT activation (p-AKT) and MT1-MMP expression in MDA-MB-435 cells transiently transfected with MT1-MMP siRNA (siMT1-MMP) or control, scrambled siRNA (siControl), and incubated with TIMP-2 (100 ng/ml) for 15 min. Total AKT and β-tubulin (TUB) are shown as loading controls. The lower panel shows the densitometric analysis of the MT1-MMP and p-AKT bands normalized to the corresponding TUB and AKT controls, respectively. *, p ≤ 0.05; + TIMP-2 vs. the corresponding – TIMP-2 sample. This experiment was repeated twice with comparable results.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4558019&req=5

pone.0136797.g002: Downregulation of MT1-MMP blocks TIMP-2 activation of AKT in MT1-MMP expressing MDA-MB-435 cells.Western blotting analysis of AKT activation (p-AKT) and MT1-MMP expression in MDA-MB-435 cells transiently transfected with MT1-MMP siRNA (siMT1-MMP) or control, scrambled siRNA (siControl), and incubated with TIMP-2 (100 ng/ml) for 15 min. Total AKT and β-tubulin (TUB) are shown as loading controls. The lower panel shows the densitometric analysis of the MT1-MMP and p-AKT bands normalized to the corresponding TUB and AKT controls, respectively. *, p ≤ 0.05; + TIMP-2 vs. the corresponding – TIMP-2 sample. This experiment was repeated twice with comparable results.
Mentions: To investigate whether TIMP-2 activation of AKT is a unique feature of MCF-7 cells or results from the high levels of MT1-MMP expressed by the transfected cells, we characterized the effect of TIMP-2 on AKT activation in human MDA-MB-435 melanoma cells, which constitutively express MT1-MMP. To analyze the requirement for MT1-MMP we transfected these cells with MT1-MMP siRNA or control, scrambled siRNA. Forty-eight hours later the cells were treated with TIMP-2, and analyzed for MT1-MMP expression and AKT activation. MT1-MMP expression was reduced by approximately 80% in the cells transfected with MT1-MMP siRNA relative to the control siRNA transfectants (Fig 2). TIMP-2 addition to the culture medium of control siRNA transfectants resulted in increased level of MT1-MMP, an effect that reflects the stabilization and accumulation of the relatively low amount of active MT1-MMP on the cell surface [39]. Consistent with our previous results, addition of TIMP-2 to the culture medium of control siRNA-transfected cells induced rapid (15 min) activation of AKT. Conversely, TIMP-2 had no such effect in MT1-MMP siRNA transfectants, which expressed almost undetectable levels of MT1-MMP (Fig 2).

Bottom Line: In addition, MT1-MMP activates intracellular signaling through proteolysis-dependent and independent mechanisms.Here we show that in MT1-MMP expressing cells TIMP-2 also induces rapid and sustained activation of AKT in a dose- and time-dependent manner and by a mechanism independent of the proteolytic activity of MT1-MMP.Conversely, TIMP-2 upregulates apoptosis induced by three-dimensional type I collagen in epithelial cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiothoracic Surgery, New York University School of Medicine, New York, New York, United States of America.

ABSTRACT
Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades a variety of extracellular matrix (ECM) components. In addition, MT1-MMP activates intracellular signaling through proteolysis-dependent and independent mechanisms. We have previously shown that binding of tissue inhibitor of metalloproteinases-2 (TIMP-2) to MT1-MMP controls cell proliferation and migration, as well as tumor growth in vivo by activating the Ras-extracellular signal regulated kinase-1 and -2 (ERK1/2) pathway through a mechanism that requires the cytoplasmic but not the proteolytic domain of MT1-MMP. Here we show that in MT1-MMP expressing cells TIMP-2 also induces rapid and sustained activation of AKT in a dose- and time-dependent manner and by a mechanism independent of the proteolytic activity of MT1-MMP. Fibroblast growth factor receptor-1 mediates TIMP-2 induction of ERK1/2 but not of AKT activation; however, Ras activation is necessary to transduce the TIMP-2-activated signal to both the ERK1/2 and AKT pathways. ERK1/2 and AKT activation by TIMP-2 binding to MT1-MMP protects tumor cells from apoptosis induced by serum starvation. Conversely, TIMP-2 upregulates apoptosis induced by three-dimensional type I collagen in epithelial cancer cells. Thus, TIMP-2 interaction with MT1-MMP provides tumor cells with either pro- or anti-apoptotic signaling depending on the extracellular environment and apoptotic stimulus.

No MeSH data available.


Related in: MedlinePlus