CONICET | Buscador de Institutos y Recursos Humanos

Starvation or reduction of fetal bovine serum (FBS) in culture medium arrest cells in a quiescent state or in G0 phase. In these cells, addition of serum induced numerous genes and proteins phosphorylation. Hepatic delta aminolevulinic synthase (ALAS1), the firstand regulatory enzyme of heme pathway, is inducible at transcriptional and/or post-transcriptional levels through processes involving participation of tyrosines phosphorylation. FBS in chicken embryo hepatocytes culture medium affected ALAS1 expression forcing medium modifications to obtain a reproducible model. The aim of this study was to investigate the effect of serum on ALAS1 expression. In the in vitro assays C3A cells were incubated in DMEM medium with different concentrations of FBS. mRNA and protein levels were determined at different time points by qPCR and western blot respectively. FBS fasting (18h) provoked a significant drop in ALAS1 protein levels that increased when FBS was restored. Different concentrations of FBS (1, 2, 5 and 10%; 2h) increased ALAS1 protein levels progressively reaching an 800% increment at FBS 10% compared to control (C) group, nevertheless, mRNA levels were significantly lower than those observed in group C. Time course of FBS effect (FBS 2%; 5, 15, 30, 60 and 120min) showed increased ALAS1 protein levels up to 300% at 2h, whereas treatment with FBS+cycloheximide (0.1mM) didn´t modify ALAS1 levels respect to time 0. Pretreatment with a tyrosine phosphatases inhibitor, phenylarsine oxide (PAO) (5 and 40 uM; 30 min), and subsequent addition of FBS 2%, 2 h resulted in a decrease of ALAS1 35% and 80% at both PAO concentrations respectively. These results suggest that FBS affected ALAS1 content due to an increase in protein translation and tyrosine phosphatases are involvedin this induction