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Serum alanine aminotransferase (ALT) is used as a clinical marker of hepatotoxicity. Three forms of human ALT have been identified, ALT1 and 2 and an alternative splice variant of ALT2 (herein called ALT2_2). The standard ALT activity assay does not discriminate between ALT from different organs, or the isoforms measured in the plasma. Here, we show that ALT1 and 2 possess similar enzymatic activity for alanine and pyruvate but with different Km and kcat values, while recombinant ALT2_2 protein does not possess any enzymatic activity. Isolation of organelles from cultured human skeletal muscle cells, showed localisation of ALT2 to the mitochondrial fraction and endoplasmatic reticulum (ER), but not to the cytosol. In human hepatocytes, on the other hand, ALT1 was only localised to the cytosol and ER, with no detection in mitochondria. ALT2 was not detected in cultured human hepatocytes, liver extract or tissue using Western blotting or immunohistochemistry. The islet of Langerhans and cardiomyocytes were other examples of cells with high expression of catalytic ALT2. A clinical method for selective measurement of ALT1 and 2 in human plasma is described, and both ALT1 and 2 were immunoprecipitated from human plasma and structurally detected using Western blotting techniques.

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-kappaB, important for gene activation. Using HEK293 cells we here describe that repression of NF-kappaB requires functions of the GR that are dependent on the signaling pathways employed to activate NF-kappaB. While a GR mutant was able to repress NF-kappaB activity following activation by TNFalpha, it did not so following activation by the phorbol ester TPA. In these cells, TPA stimulation but not TNFalpha, activated extracellular signal-regulated kinase (ERK). We demonstrated that the ability of the dexamethasone activated GR mutant to repress TPA-induced NF-kappaB 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-kappaB 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-kappaB 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-kappaB activity, a capacity lacked by the GRR488Q mutant.

Glucocorticoid hormones (GCs) exert an antiproliferative effect on most cells. However, the molecular mechanism is still largely unclear. We investigated the antiproliferative mechanism by GCs in human embryonic kidney 293 cells with stably introduced glucocorticoid receptor (GR) mutants that discriminate between cross-talk with nuclear factor-(kappa)B (NF-(kappa)B) and activator protein-1 signaling, transactivation and transrepression, and antiproliferative vs. non-antiproliferative responses. Using the GR mutants, we here demonstrate a correlation between repression of NF-(kappa)B signaling and antiproliferative response. Gene expression profiling of endogenous genes in cells containing mutant GRs identified a limited number of genes that correlated with the antiproliferative response. This included a GC-mediated up-regulation of the NF-(kappa)B-inhibitory protein I(kappa)B(alpha), in line with repression of NF-(kappa)B signaling being important in the GC-mediated antiproliferative response. Interestingly, the GC-stimulated expression of I(kappa)B(alpha) was a direct effect despite the inability of the GR mutant to transactivate through a GC-responsive element. Selective expression of I(kappa)B(alpha) in human embryonic kidney 293 cells resulted in a decreased percentage of cells in the S/G2/M phase and impaired cell proliferation. These results demonstrate that GC-mediated inhibition of NF-(kappa)B is an important mechanism in the antiproliferative response to GCs.