Effects of fibrillar C-terminal fragment of cleaved α1-antitrypsin on cholesterol homeostasis in HepG2 cells

Amyloid fibrils of diverse origin are known to disturb vital cellular functions and induce cell death. In this study, the effects of amyloid fibrils from the C-terminal fragment (C-36) of cleaved alpha(1)-antitrypsin (AAT) on low-density lipoprotein (LDL) metabolism were investigated in HepG2 cells. Treatment of the cells with C-36 fibrils (10 mu mol/L) enhanced I-125-LDL binding and uptake 10 to 15 times, and highly up-regulated levels of LDL receptor mRNA, as compared with control cells. Competition experiments using excess of unlabeled LDL and blockage experiments with a monoclonal LDL receptor antibody diminished or completely abolished the stimulatory effects of fibrils on LDL binding and LDL receptor mRNA levels, suggesting that fibrils act via the LDL receptor pathway. However, C-36 fibrils had no significant effect on [2-C-14]acetate incorporation into cholesterol biosynthesis and cholesterol ester formation, but inhibited I-125-LDL degradation by 20% and reduced bile acid biosynthesis up to 48% in a dose-dependent manner. Preincubation of the cells with fibrils before the addition of LDL totally abolished the LDL inhibitory effect on unesterified cholesterol synthesis, fur ther confirming the LDL receptors to be the target for C-36 fibrils. Moreover, the expression of sterol regulatory element binding protein-1 (SREBP-1) was found to increase twofold and more after 24 hours of incubation of the cells with several concentrations of C-36 fibrils. Our study suggests that the cytotoxicity of C-36 fibrils on HepG2 cells is associated with perturbed intracellular cholesterol homeostasis, induced through fibril-stimulated expression of the LDL receptors via the sterol-responsive element.