EFFECTS OF CLUSTERIN OVEREXPRESSION ON TNF-ALPHA-MEDIATED AND TGF-BETA-MEDIATED DEATH OF L929 CELLS

Clusterin is a widely distributed and highly conserved protein for whi ch many functions have been proposed. We used transfected L929 cells t o study the effect of clusterin expression on the regulation of cell d eath signals. We showed that high levels of clusterin expression, abou t 0.2 pg clusterin secreted per cell per 48 h period, specifically pro tected L929 cells from TNF alpha-mediated cytotoxicity, while low expr ession (about 4 fg/cell/48 h) had no effect. However, clusterin expres sion did not provide transfected L929 cells with protection against de ath mediated by colchicine, staurosporine or azide. High level express ion of clusterin in transfected L929 cells also potentiated the cytoto xicity of TGF beta. It had previously been shown that exposure of L929 cells to TGF beta provides protection against TNF alpha. We showed th at this protective effect is not additive to that of clusterin express ion. One interpretation of this data is that it suggests that clusteri n and TGF beta may act via a common mechanism to provide protection ag ainst the cytotoxicity of TNF alpha. Our results indicate that an intr acellular action of clusterin protein is responsible for protection ag ainst TNF alpha cytotoxicity. Exposure to TNF alpha induces an increas e in the level of cell-associated clusterin and specifically in the le vel of a novel clusterin molecule, which when analyzed under reducing conditions by SDS/PAGE and immunoblotting appears as two closely space d bands at about 36 and 38.5 kDa. When analyzed under the same conditi ons, the normal form of intracellular clusterin, which is present with or without exposure to TNF alpha, appears as two poorly resolved band s at about 43-45 kDa. Since the novel form of clusterin is also expres sed in cells exposed to TGF beta, colchicine, staurosporine, and azide , it may result from toxin-induced disruption of processes of normal c ellular protein production.