CYPZE1-mediated oxidative stress induces collagen type I expression in rathepatic stellate cells

Hepatic stellate cells (HSCs) are a major source of extracellular matrix, w hich, during fibrogenesis, undergo a process of "activation" characterized by increased proliferation and collagen synthesis. Oxidative stress can sti mulate HSC proliferation and collagen synthesis in vitro. Cytochrome P4502E 1 (CYP2E1) is an effective producer of reactive oxygen species. To study ho w intracellular oxidative stress modulates alpha 2 collagen type I (COL1A2) gene induction, a rat HSC line (HSC-T6) was transfected with human CYP2E1 complementary DNA in the sense and antisense orientation and with empty vec tor, and stable cell lines were generated. The cells expressing CYP2E1 disp layed elevated production of reactive oxygen species and showed a 4-fold in crease in COL1A2 messenger RNA (mRNA) levels; expression of this mRNA among different clones appeared to correlate with the level of CYP2E1. COL1A2 ex pression was decreased by vitamin E treatment or transfection with manganes e superoxide dismutase, and was further increased after treatment with L-bu thionine sulfoximine (BSO) to lower GSH levels, Thus, CYP2E1-dependent oxid ative stress plays a major role in the elevation of COL1A2 mRNA levels in t his system. Nuclear run-on assay showed a 3-and-a-half-fold increase in COL 1A2 transcription in the cells expressing CYP2E1; stabilization of COL1A2 m RNA was also observed. These results indicate that under oxidative stress c onditions, COL1A2 mRNA expression is regulated both transcriptionally and t hrough mRNA stabilization. The CYP2E1-expressing HSC appear to be a valuabl e model for the sustained generation of reactive oxygen species and may all ow the elucidation of signaling pathways responsible for oxidant stress-med iated collagen gene induction.