Maspin is physically associated with 1 integrin regulating cell adhesion in mammary epithelial cells

Baylor College of Medicine, Department of Molecular and Cellular Biology, ALKEK Bldg., Rm. N630, One Baylor Plaza, Houston, Texas 77030, USA.
The FASEB Journal (Impact Factor: 5.04). 08/2006; 20(9):1510-2. DOI: 10.1096/fj.05-5500fje
Source: PubMed


Maspin is a tumor-suppressor serpin (serine protease inhibitor), which inhibits cell invasion and migration. Here, we analyzed maspin function in cell adhesion in nontransformed mammary epithelial cells and investigated the underlying mechanism involved in this process. We report that maspin acts in the early steps in the cell adhesion process. Addition of recombinant maspin rapidly increased MCF-10A cell adhesion to the endogenously deposited matrix, and conversely both an antimaspin antibody (Ab) and maspin knockdown by RNA interference resulted in decreased cell adhesion. Mutation analyses revealed that a region of 86 amino acids located between aa 139 and aa 225 was responsible for maspin effect on adhesion. In addition, we show that maspin is associated with detergent-insoluble cortical cytoskeleton elements. Collectively, these results suggest that maspin is part of the supramolecular structure of the adhesion plaque and it modulates cell adhesion via a beta1 integrin-dependent mechanism.

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Available from: Khatri Latha, Mar 17, 2014
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    • "Parasite strain and cell culture T. cruzi Y strain was used throughout and was grown and maintained as described by Andrews and Colli (1982). MCF- 10A human mammary cells (purchased from BCRJ/UFRJ) were cultivated as monolayers in Dulbecco's modified Eagle's medium (DMEM)/F12 (Life Technologies) containing 20 % donor horse serum, 20 ng/ml epidermal growth factor (EGF), 100 ng/ml cholera toxin, 10 ng/ml insulin, 500 ng/ml hydrocortisone , 50 U/ml penicillin, and 50 μg/ml streptomycin at 37 °C and 5 % CO 2 in a humidified air atmosphere (Cella et al. 2006). The membrane-enriched fraction was obtained by treatment of 5 × 10 7 MCF-10A cells with lysis buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 10 mM EGTA, 0.25 mM sucrose, protease inhibitor cocktail (Sigma-Aldrich), and centrifugation at 100,000g for 40 min. "
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    • "Therefore, we suspect maspin tyrosine phosphoforms are not efficiently immunoprecipitated by the commercially available maspin antibodies. Since maspin is present in multiple cellular compartments and it is also detected in the Triton X-100 insoluble cytoskeleton fraction [6], cells lysates were prepared in a strongly denaturing and chaotropic solution containing 8 M urea (see Section 2), to reassure that maspin associated with the different cellular components would be extracted . However, this extraction solution precludes the phosphatase treatment of the extract. "
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