A reexamination of the role of clusterin as a complement regulator.

Clusterin is a highly conserved glycoprotein which has been proposed to protect host cells against complement-mediated cytolysis. We tested the hypothesis that clusterin is a complement regulator using erythrocytes and cells which had been stably transfected with a membrane-anchored form of clusterin as targets for complement-mediated cytolysis. Clusterin gave dose-dependent protection of antibody-coated sheep erythrocytes against complement-mediated lysis by diluted normal human serum. There was a linear relationship between the concentration of clusterin giving 50% protection and the concentration of serum; extrapolation of this to the case of undiluted human serum showed that a clusterin concentration at least two orders of magnitude greater than its physiological plasma concentration would be needed to confer protection against complement-mediated cytolysis under physiological conditions. Physiological concentrations of clusterin did not protect rabbit erythrocytes against alternative complement pathway-mediated lysis using dilute human serum. Exogenous clusterin had no effect on lysis of human erythrocytes triggered by the addition of inulin to autologous human serum. Induction of cell-surface clusterin expression by L929 (murine fibroblast) cells which had been stably transfected with cDNA for human clusterin linked to DNA coding for the 44 C-terminal amino acid residues of CD55 did not protect the cells against complement-mediated lysis by either normal or clusterin-depleted human serum. These data suggest that clusterin may not be a physiologically relevant regulator of complement activation.

Effects of clusterin overexpression on TNFalpha- and TGFbeta-mediated death of L929 cells.

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 TNFalpha-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 TGFbeta. It had previously been shown that exposure of L929 cells to TGFbeta provides protection against TNFalpha. 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 TGFbeta may act via a common mechanism to provide protection against the cytotoxicity of TNFalpha. Our results indicate that an intracellular action of clusterin protein is responsible for protection against TNFalpha cytotoxicity. Exposure to TNFalpha 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 TNFalpha, appears as two poorly resolved bands at about 43-45 kDa. Since the novel form of clusterin is also expressed in cells exposed to TGFbeta, colchicine, staurosporine, and azide, it may result from toxin-induced disruption of processes of normal cellular protein production.