SB203580, a pharmacological inhibitor of p38 MAP kinase transduction pathway activates ERK and JNK MAP kinases in primary cultures of human hepatocytes.

Mitogen-activated protein kinases (MAPKs) were extensively studied in cancer-derived cell lines; however, studies in non-transformed human cells are scarce. In the current paper, we studied the effect of SB203580, a pharmacological inhibitor of p38 MAPK, on activation and inhibition of p38 MAPK transduction partway in primary human hepatocytes (in vitro model of differentiated cells) in comparison with several tumor cell lines (proliferating non-differentiated in vitro model). In addition, we analyzed the effect of SB203580 on extracellular-regulated protein kinase (ERK) and c-jun-N-terminal kinase (JNK) pathways both in primary human hepatocytes and tumor cell lines employing primary antibodies detecting phosphorylated kinases. We show that SB203580 activates ERK and JNK in primary cultures of human hepatocytes. The levels of ERK-P(Thr202/Tyr204), JNK-P(Thr183/Tyr185) and c-Jun-P(Ser63/73), a target down-stream protein of JNK, were increased by SB203580. In contrast, SB203580 activated ERK but not JNK in HepG2, HL-60, Saos-2 and HaCaT human cancer cell lines. We tested, whether the effects of SB203580 are due to metabolism. Using liquid chromatography/mass spectrometry, we found one minor metabolite in human liver microsomes but not in HepG2 cells. These data imply that biotransformation could be responsible for the effects of SB203580 in human hepatocytes. This study is the first report on the effects of MAPK activators (sorbitol, anisomycin, EGF) and MAPK inhibitors in primary human hepatocytes. We observed differential effects of these compounds in primary human hepatocytes and in cancer cells, implying the cell-type specificity and the essential differences between the role and function of MAPKs in normal and cancer cells.

Role of mitogen-activated protein kinases in aryl hydrocarbon receptor signaling.

Human populations are increasingly exposed to a number of environmental pollutants such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls and dioxins. These compounds are activators of the aryl hydrocarbon receptor (AhR) that controls the expression of many genes including those for detoxification enzymes. The regulatory mechanisms of AhR are multi-factorial and include phosphorylation by various protein kinases. Significant progress in the research of mitogen-activated protein kinases (MAPKs) has been achieved in the last decade. Isolated reports have been published on the role of MAPKs in AhR functions and vice versa, with activation of MAPKs by AhR ligands. This mini-review summarizes current knowledge on the mutual interactions between MAPKs and AhR. The majority of studies has been done on cancer-derived cell lines that have impaired cell cycle regulation and lacks the complete detoxification apparatus. We emphasize the importance of the future studies that should be done on non-transformed cells to distinguish the role of MAPKs in cancer and normal cells. Primary cultures of human or rodent hepatocytes that are equipped with a fully functional biotransformation battery or xenobiotics-metabolizing extra-hepatic tissues should be the models of choice, as the results in our experiments confirm.

JNK inhibitor SP600125 is a partial agonist of human aryl hydrocarbon receptor and induces CYP1A1 and CYP1A2 genes in primary human hepatocytes.

SP600125, a specific inhibitor of c-Jun-N-Terminal kinase (JNK), was reported as a ligand and antagonist of aryl hydrocarbon receptor (AhR) [Joiakim A, Mathieu PA, Palermo C, Gasiewicz TA, Reiners Jr JJ. The Jun N terminal kinase inhibitor SP600125 is a ligand and antagonist of the aryl hydrocarbon receptor. Drug Metab Dispos 2003;31(11):1279-82]. Here we show that SP600125 is not an antagonist but a partial agonist of human AhR. SP600125 significantly induced CYP1A1 and CYP1A2 mRNAs in primary human hepatocytes and CYP1A1 mRNA in human hepatoma cells HepG2. This effect was abolished by resveratrol, an antagonist of AhR. Consistent with the recent report, SP600125 dose-dependently inhibited CYP1A1 and CYP1A2 genes induction by a prototype AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in human hepatocytes. Moreover, SP600125 displayed typical behavior of a partial agonist in HepG2 cells transiently transfected with a reporter plasmid containing two inverted repeats of the dioxin responsive element or with a plasmid containing 5'-flanking region of human CYP1A1 gene. SP600125 transactivated the reporter plasmids with EC(50) of 0.005 and 1.89 microM, respectively. On the other hand, TCDD-dependent transactivation of the reporter plasmids was inhibited by SP600125 with IC(50) values of 1.54 and 2.63 microM, respectively. We also tested, whether the effects of SP600125 are due to metabolism. Using liquid chromatography/mass spectrometry approach, we observed formation of two minor monohydroxylated metabolites of SP600125 in human hepatocytes, human liver microsomes but not in HepG2 cells. These data imply that biotransformation is not responsible for the effects of SP600125 on AhR signaling. In conclusion, we demonstrate that SP600125 is a partial agonist of human AhR, which induces CYP1A genes.