Abstract
Leflunomide, used for the treatment of rheumatoid arthritis, has been reported to cause severe liver problems and liver failure; however, the underlying mechanisms are not clear. In this study, we used multiple approaches including genomic analysis to investigate and characterize the possible molecular mechanisms of the cytotoxicity of leflunomide in hepatic cells. We found that leflunomide caused endoplasmic reticulum (ER) stress and activated an unfolded protein response, as evidenced by increased expression of related genes including CHOP and GADD34; and elevated protein levels of typical ER stress markers including CHOP, ATF-4, p-eIF2α, and spliced XBP1. The secretion of Gaussia luciferase was suppressed in cells treated with leflunomide in an ER stress reporter assay. Inhibition of ER stress with an ER stress inhibitor 4-phenylbutyrate, and knockdown of ATF-4 and CHOP genes partially protected cells upon leflunomide exposure. In addition, both genomic and biochemical analyses revealed that JNK and ERK1/2 of MAPK signaling pathways were activated, and both contributed to the leflunomide-induced cytotoxicity. Inhibiting JNK activation using a JNK inhibitor attenuated the ER stress and cytotoxicity of leflunomide, whereas inhibiting ERK1/2 using an ERK1/2 inhibitor or ERK1/2 siRNA increased the adverse effect caused by leflunomide, suggesting opposite roles for the two pathways. In summary, our data indicate that both ER stress and the activation of JNK and ERK1/2 contribute to leflunomide-induced cytotoxicity.