Epidermal growth factor induces tumour marker AKR1B10 expression through activator protein-1 signalling in hepatocellular carcinoma cells

AKR1B10 (aldo-keto reductase 1B10) is overexpressed in liver and lung cancer, and plays a critical role in tumour development and progression through promoting lipogenesis and eliminating cytotoxic carbonyls. AKR1B10 is a secretory protein and potential tumour marker; however, little is known about the regulatory mechanism of AKR1B10 expression. The present study showed that AKR1B10 is induced by mitogen EGF (epidermal growth factor) and insulin through the AP-1 (activator protein-1) signalling pathway. In human HCC (hepatocellular carcinoma) cells (HepG2 and Hep3B), EGF (50?ng/ml) and insulin (10?nM) stimulated endogenous AKR1B10 expression and promoter activity. In the AKR1B10 promoter, a putative AP-1 element was found at bp -222 to -212. Deletion or mutation of this AP-1 element abrogated the basal promoter activity and response to EGF and AP-1 proteins. This AP-1 element bound to nuclear proteins extracted from HepG2 cells, and this binding was stimulated by EGF and insulin in a dose-dependent manner. Chromatin immunoprecipitation showed that the AP-1 proteins c-Fos and c-Jun were the predominant factors bound to the AP-1 consensus sequence, followed by JunD and then JunB. The same order was followed in the stimulation of endogenous AKR1B10 expression by AP-1 proteins. Furthermore, c-Fos shRNA (short hairpin RNA) and AP-1 inhibitors/antagonists (U0126 and Tanshinone IIA) inhibited endogenous AKR1B10 expression and promoter activity in HepG2 cells cultured in vitro or inoculated subcutaneously in nude mice. U0126 also inhibited AKR1B10 expression induced by EGF. Taken together, these results suggest that AKR1B10 is up-regulated by EGF and insulin through AP-1 mitogenic signalling and may be implicated in hepatocarcinogenesis.     

Activation of c-Jun N-terminal kinase 1 by UV irradiation is inhibited by wortmannin without affecting c-iun expression

Activation of c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases is an early response of cells upon exposure to DNA-damaging agents. JNK-mediated phosphorylation of c-Jun is currently understood to stimulate the transactivating potency of AP-1 (e.g., c-Jun/c-Fos; c-Jun/ATF-2), thereby increasing the expression of AP-1 target genes. Here we show that stimulation of JNK1 activity is not a general early response of cells exposed to genotoxic agents. Treatment of NIH 3T3 cells with UV light (UV-C) as well as with methyl methanesulfonate (MMS) caused activation of JNK1 and an increase in c-Jun protein and AP-1 binding activity, whereas antineoplastic drugs such as mafosfamide, mitomycin C, N-hydroxyethyl-N-chloroethylnitrosourea, and treosulfan did not elicit this response. The phosphatidylinositol 3-kinase inhibitor wortmannin specifically blocked the UV-stimulated activation of JNK1 but did not affect UV-driven activation of extracellular regulated kinase 2 (ERK2). To investigate the significance of JNK1 for transactivation of c-jun, we analyzed the effect of UV irradiation on c-jun expression under conditions of wortmannin-mediated inhibition of UV-induced stimulation of JNK1. Neither the UV-induced increase in c-jun mRNA, c-Jun protein, and AP-1 binding nor the activation of the collagenase and c-jun promoters was affected by wortmannin. In contrast, the mitogen-activated protein kinase/ERK kinase inhibitor PD98056, which blocked ERK2 but not JNK1 activation by UV irradiation, impaired UV-driven c-Jun protein induction and AP-1 binding. Based on the data, we suggest that JNK1 stimulation is not essential for transactivation of c-jun after UV exposure, whereas activation of ERK2 is required for UV-induced signaling leading to elevated c-jun expression.     

Tissue-specific regulation of the ecto-5'-nucleotidase promoter. Role of the camp response element site in mediating repression by the upstream regulatory region.

We have isolated the 5' region of the ecto-5'-nucleotidase (low K(m) 5'-NT) gene and established that a 969-base pair (bp) fragment confers cell-specific expression of a CAT reporter gene that correlates with the expression of endogenous ecto-5'-NT mRNA and enzymatic activity. A 768-bp upstream negative regulatory region has been identified that conferred lymphocyte-specific negative regulation in a heterologous system with a 244-bp deoxycytidine kinase core promoter. DNase I footprinting identified several protected areas including Sp1, Sp1/AP-2, and cAMP response element (CRE) binding sites within the 201-bp core promoter region and Sp1, NRE-2a, TCF-1/LEF-1, and Sp1/NF-AT binding sites in the upstream regulatory region. Whereas the CRE site was essential in mediating the negative activity of the upstream regulatory region in Jurkat but not in HeLa cells, mutation of the Sp1/AP-2 site decreased promoter activity in both cell lines. Electrophoretic mobility shift assay analysis of proteins binding to the CRE site identified both ATF-1 and ATF-2 in Jurkat cells. Finally, phorbol 12-myristate 13-acetate increased the activity of both the core and the 969-bp promoter fragments, and this increase was abrogated by mutations at the CRE site. In summary, we have identified a tissue-specific regulatory region 5' of the ecto-5'-NT core promoter that requires the presence of a functional CRE site within the basal promoter for its suppressive activity.