Abstract
Cadmium (Cd) is an environmental contaminant that has been classified as a human carcinogen. Exposure of Cd leads to cell death or malignant transformation. Metallothioneins (MTs) and Glutathione (GSH) are two major branches involved in protecting cells from Cd toxicity. Previous studies showed that c-Myc transcriptionally regulates γ-glutamyl-cysteine synthetase (γ-GCS), the rate-limiting enzyme catalyzing GSH biosynthesis. In addition, c-Myc was reported to associate with MT gene expression. Cd is known to activate c-Myc proto-oncogene. However, the mechanism has not been explored. We investigated here the mechanism of c-Myc expression under Cd treatment in HepG2 cells. The c-Myc protein and mRNA levels increased with dose- and time-dependent manners after Cd treatment. This increase was not associated with promoter activity and protein stability of c-Myc but was due to an increase in c-Myc mRNA stability. To explore the mechanism involved in enhancing the mRNA stability, several cellular signaling factors that evoked by Cd treatment were analyzed. PI3K, p38, ERK and JNK were activated after Cd treatment. However, ERK did not participate in the Cd-induced c-Myc expression. Further analysis revealed that mTORC2 was a downstream factor of p38. PI3K, JNK and p38/mTORC2 coordinately activated Akt. Akt was phosphorylated at Thr450 in the untreated cells. Cd treatment led to additional phosphorylation at Thr308 and Ser473. Blocking any of the three signaling factors resulted in the reduction of phosphorylation level at all three Akt sites. The activated Akt phosphorylated Foxo1 and allowed the modified protein to translocate into the cytoplasm. Overexpression of Foxo1 reduced the Cd-induced c-Myc mRNA and protein levels, and knockdown of Foxo1 increased c-Myc mRNA level. These results suggest that Cd-induced accumulation of c-Myc requires the activation of PI3K、JNK and p38/mTORC2 signaling pathways. The signals act coordinately for Akt activation and drive the Foxo1 from the nucleus to the cytoplasm. Reduction of Foxo1 in the nucleus reduces the transcription of its target genes that may affect c-Myc mRNA stability, resulting in a higher accumulation of the c-Myc proteins. PI3K、JNK、p38 inhibitors and c-Myc knockdown enhanced Cd-induced cell death. These inhibitors and c-Myc knockdown also reduced Cd-induced γ-GCS mRNA level and GSH content, but did not affect MT2A mRNA level. These results suggest that Cd-induced c-Myc level may regulate GSH synthesis and provide protection from Cd-induced toxicity.