Effects of Clusterin Overexpression on TNFα- and TGFβ-Mediated Death of L929 Cells

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Abstract
Clusterin is a widely distributed and highly conserved protein for which many functions have been proposed. We used transfected L929 cells to study the effect of clusterin expression on the regulation of cell death signals. We showed that high levels of clusterin expression, about 0.2 pg clusterin secreted per cell per 48 h period, specifically protected L929 cells from TNFα-mediated cytotoxicity, while low expression (about 4 fg/cell/48 h) had no effect. However, clusterin expression did not provide transfected L929 cells with protection against death mediated by colchicine, staurosporine or azide. High level expression of clusterin in transfected L929 cells also potentiated the cytotoxicity of TGFβ. It had previously been shown that exposure of L929 cells to TGFβ provides protection against TNFα. We showed that this protective effect is not additive to that of clusterin expression. One interpretation of this data is that it suggests that clusterin and TGFβ may act via a common mechanism to provide protection against the cytotoxicity of TNFα. Our results indicate that an intracellular action of clusterin protein is responsible for protection against TNFα cytotoxicity. Exposure to TNFα induces an increase in the level of cell-associated clusterin and specifically in the level of a novel clusterin molecule, which when analyzed under reducing conditions by SDS/PAGE and immunoblotting appears as two closely spaced bands at about 36 and 38.5 kDa. When analyzed under the same conditions, the normal form of intracellular clusterin, which is present with or without exposure to TNFα, appears as two poorly resolved bands at about 43−45 kDa. Since the novel form of clusterin is also expressed in cells exposed to TGFβ, colchicine, staurosporine, and azide, it may result from toxin-induced disruption of processes of normal cellular protein production.