metallothionein and phorbol-12-myristate

Previously we have identified a distal region of the rainbow trout (Oncorhynchus mykiss) metallothionein-A (rtMT-A) enhancer region, being essential for free radical activation of the rtMT-A gene. The distal promoter region included four activator protein 1 (AP1) cis-acting elements and a single nuclear factor interleukin-6 (NF-IL6) element. In the present study we used the rainbow trout hepatoma (RTH-149) cell line to further examine the involvement of NF-IL6 and AP1 in rtMT-A gene expression following exposure to oxidative stress and tumour promotion.. Using enhancer deletion studies we observed strong paraquat (PQ)-induced rtMT-A activation via NF-IL6 while the AP1 cis-elements showed a weak but significant activation. In contrast to mammals the metal responsive elements were not activated by oxidative stress. Electrophoretic mobility shift assay (EMSA) mutation analysis revealed that the two most proximal AP1 elements, AP11,2, exhibited strong binding to the AP1 consensus sequence, while the more distal AP1 elements, AP13,4 were ineffective. Phorbol-12-myristate-13-acetate (PMA), a known tumor promoter, resulted in a robust induction of rtMT-A via the AP1 elements alone. To determine the conservation of regulatory functions we transfected human Hep G2 cells with the rtMT-A enhancer constructs and were able to demonstrate that the cis-elements were functionally conserved. The importance of NF-IL6 in regulation of teleost MT is supported by the conservation of these elements in MT genes from different teleosts. In addition, PMA and PQ injection of rainbow trout resulted in increased hepatic rtMT-A mRNA levels.. These studies suggest that AP1 primarily is involved in PMA regulation of the rtMT-A gene while NF-IL6 is involved in free radical regulation. Taken together this study demonstrates the functionality of the NF-IL6 and AP-1 elements and suggests an involvement of MT in protection during pathological processes such as inflammation and cancer.

Topics: Animals; Binding Sites; Carcinoma, Hepatocellular; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Enhancer Elements, Genetic; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Metallothionein; Mutation; Oncorhynchus mykiss; Oxidative Stress; Paraquat; Phorbol Esters; Promoter Regions, Genetic; Transcription Factor AP-1; Transfection

Metallothioneins (MTs), a group of small, cystein-rich proteins, possess various functions, including metal detoxification and homeostasis. We here report new findings on the participation of MT in cellular differentiation processes. MT isogene transcription was significantly increased in phorbol-12-myristate-13-acetate (PMA) incubated leukemic DAMI cells, which supports its role in cellular differentiation. To further address this possibility, we constructed one stable MT-2A overexpressing DAMI cell line. Increase of cell size, intracellular granulation and megakaryocytic specific antigen expression such as CD41 and CD42, and arresting cell proliferation have validated the role of MT in differentiation in this cell line.

Topics: Animals; Biomarkers; Cell Differentiation; Humans; Leukemia; Megakaryocytes; Metallothionein; Phorbol Esters; Tumor Cells, Cultured