Identification of CD63 as a tissue inhibitor of metalloproteinase-1 interacting cell surface protein

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Abstract
This study identified CD63, a member of the tetraspanin family, as a TIMP‐1 interacting protein by yeast two‐hybrid screening. Immunoprecipitation and confocal microscopic analysis confirmed CD63 interactions with TIMP‐1, integrin β1, and their co‐localizations on the cell surface of human breast epithelial MCF10A cells. TIMP‐1 expression correlated with the level of active integrin β1 on the cell surface independent of cell adhesion. While MCF10A cells within a three‐dimensional (3D) matrigel matrix form polarized acinar‐like structures, TIMP‐1 overexpression disrupted breast epithelial cell polarization and inhibited caspase‐mediated apoptosis in centrally located cells, necessary for the formation and maintenance of the hollow acinar‐like structures. Small hairpin RNA (shRNA)‐mediated CD63 downregulation effectively reduced TIMP‐1 binding to the cell surface, TIMP‐1 co‐localization with integrin β1, and consequently reversed TIMP‐1‐mediated integrin β1 activation, cell survival signaling and apoptosis inhibition. CD63 downregulation also restored polarization and apoptosis of TIMP‐1 overexpressing MCF10A cells within a 3D‐matrigel matrix. Taken together, the present study identified CD63 as a cell surface binding partner for TIMP‐1, regulating cell survival and polarization via TIMP‐1 modulation of tetraspanin/integrin signaling complex.

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