title: Tissue inhibitor of metalloproteinase 2 inhibits activation of the β-catenin signaling in melanoma cells.
abstract: The tissue inhibitor of metalloproteinase (TIMP) family, including TIMP-2, regulates the activity of multifunctional metalloproteinases in pathogenesis of melanoma. The Wnt/β-catenin pathway is constitutively activated and plays a critical role in melanoma progression. However, the relationship between TIMP-2 expression and β-catenin activity is still unclear. We hypothesize that TIMP-2 over expression inhibits the activation of the Wnt/β-catenin pathway in melanoma cells. Protein expression, distribution, and transcriptional activity of β-catenin were assayed in established stable melanoma cell lines: parental A2058 expressing, A2058 T2-1 over-expressing (T2-1), and A2058 T2R-7 under-expressing (T2R-7) TIMP-2. Compared to T2-1 cells at the basal level, T2R-7 showed significantly lower amount protein and weaker immunofluorescence staining of β-catenin. This regulation is through posttranslational level via ubiquitination. Functionally, proliferation and cell growth were lower in T2R-7 compared to A2058 and T2-1. Lithium treatment was used to mimics activation of the Wnt/β-catenin pathway. In T2R-7 cells under-expressing TIMP2, lithium significantly increased total β-catenin, nuclear β-catenin, and its downstream protein phosphor-c-Myc (S62). Nuclear β-catenin staining was enhanced in T2R-7. Beta-catenin transcriptional activity and cell proliferation were also increased significantly. Axins inhibit β-catenin pathway via GSK-3 β. We further found the ratio of p-GSK-3 β (S9) to β-catenin and protein levels of Axins were significantly lower, whereas downstream Wnt 11 was high in T2R-7 treated with lithium. Collectively, the high level of TIMP-2 protein inhibits the activation of the Wnt/β-catenin pathway, thus suppressing proliferation. Insights in the molecular mechanisms of TIMP-2 may provide promising opportunities for anti-proliferative therapeutic intervention.
authors.affiliation: a Department of Biochemistry; Rush University ; Chicago , IL , USA.
authors.forename: Yuxuan
authors.initials: Y
authors.lastname: Xia
authors.forename: Shaoping
authors.initials: S
authors.lastname: Wu
mesh_terms: D060466:Axin Protein / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D008094:Lithium / Q000494:pharmacology; D008545:Melanoma / Q000378:metabolism / Q000503:physiopathology*; D008954:Models, Biological; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism*; D054875:Ubiquitination; D051176:beta Catenin / Q000378:metabolism*
chemical_list: D060466:Axin Protein; C560823:TIMP2 protein, human; D051176:beta Catenin; D019716:Tissue Inhibitor of Metalloproteinase-2; D008094:Lithium
keywords: APC, Adenomatous polyposis coli; GSK-3b, Glycogen synthase kinase 3b; MMPs, Metalloproteinases; TCF/LEF, T-cell factor/L-lymphoid enhancer factor; TIMP, Tissue inhibitor of metalloproteinases; TIMP-2; Wnt; axin1; axin2; lithium; melanoma; β-catenin
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