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Abstract: In this work, we investigated a potential mechanism behind the observation of increased aminotransferase levels in a phase I clinical trial using a lipid-lowering drug, the peroxisome proliferator-activated receptor (PPAR) α agonist, AZD4619. In healthy volunteers treated with AZD4619, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were elevated without an increase in other markers for liver injury. These increases in serum aminotransferases have previously been reported in some patients receiving another PPARα agonist, fenofibrate. In subsequent in vitro studies, we observed increased expression of ALT1 protein and mRNA in human hepatocytes after treatment with fenofibric acid. The PPAR effect on ALT1 expression was shown to act through a direct transcriptional mechanism involving at least one PPAR response element (PPRE) in the proximal ALT1 promoter, while no effect of fenofibrate and AZD4619 was observed on the ALT2 promoter. Binding of PPARs to the PPRE located at −574 bp from the transcriptional start site was confirmed on both synthetic oligonucleotides and DNA in hepatocytes. These data show that intracellular ALT expression is regulated by PPAR agonists and that this mechanism might contribute to increased ALT activity in serum. [Copyright &y& Elsevier]

Abstract: Serum alanine aminotransferase (ALT) is used as a clinical marker of hepatotoxicity. Two forms of ALT have been identified, ALT1 and ALT2, encoded by separate genes. The cellular and tissue distribution of the different ALT proteins has not been characterized in humans, and their relative contribution to serum is unknown. Here, we describe the development of novel isoenzyme specific ALT1 and ALT2 antibodies and the expression of the enzymes in human cells and organs. In normal human tissue, high expression of ALT1 was found in liver, skeletal muscle and kidney and low levels in heart muscle and not detectable in pancreas. High ALT2 reactivity was detected in heart and skeletal muscle, while no ALT2 expression was found in liver or kidney. Using immunohistochemistry, strong ALT1 reactivity was found in hepatocytes, renaltubular epithelial cells and in salivary gland epithelial cells, while ALT2 was expressed in adrenal gland cortex, neuronal cell bodies, cardiac myocytes, skeletal muscle fibers and endocrine pancreas. Immunoprecipitation using ALT antibodies on normal human serums showed ALT1 to be mainly responsible for basal ALT activity. Together, the results points to a differential expression of ALT1 and ALT2 in human organs and substantiate a need for investigations regarding the possible impacts on ALT measurements. [Copyright &y& Elsevier]

Abstract: Glucocorticoid (GC) effects are mediated via the GC-receptor (GR), which either stimulates or represses gene expression. Repression of target genes often involves negative cross-talk between the GR and other transcription factors e.g. NF-κB, important for gene activation. Using HEK293 cells we here describe that repression of NF-κB requires functions of the GR that are dependent on the signaling pathways employed to activate NF-κB. While a GR mutant was able to repress NF-κB activity following activation by TNFα, it did not so following activation by the phorbol ester TPA. In these cells, TPA stimulation but not TNFα, activated extracellular signal-regulated kinase (ERK). We demonstrated that the ability of the dexamethasone activated GR mutant to repress TPA-induced NF-κB activity was restored in conjunction with ERK1/2 inhibition. Previous reports have shown GC-mediated inhibition of ERK1/2 phosphorylation to involve GC induction of MAPK phosphatase-1 (MKP-1). Here, we demonstrated that the GRR488Q mutant was incapable of inducing gene expression of endogenous MKP-1 following dexamethasone treatment, in contrast to the GRwt. However, TPA treatment alone resulted in much stronger MKP-1 expression in both GRwt and GRR488Q containing cells than that of dexamethasone suggesting that the inability of GRR488Q to inhibit TPA-induced NF-κB activity did not involve a lack of MKP-1 expression. In line with this, RNAi targeted towards MKP-1 did not abolish or inhibit the ability of the GRwt to repress NF-κB activity. Importantly, we observed no difference in activated ERK1/2 (phospho-ERK1/2) expression over time between GRwt and GRR488Q containing cells following co-treatment with TPA and dexamethasone. Based on these results we suggest that GRwt does not directly regulate ERK1/2 but rather alters ERK1/2-mediated effects allowing it to repress NF-κB activity, a capacity lacked by the GRR488Q mutant. [Copyright &y& Elsevier]