This observation strengthens the idea that Flvcr1a deletion/down-regulation leads to the coordinated induction of heme degradation and down-regulation of the heme biosynthetic pathway. To evaluate this point, we analyzed HO-1 as well as ALAS1 protein and activity in the liver of Flvcr1afl/fl;alb-cre and Flvcr1afl/fl mice, treated with
dexamethasone or Be(a)P. After the stimulation of CYP synthesis, HO-1 and ALAS1 expression were induced in the liver of both Flvcr1afl/fl;alb-cre and Flvcr1afl/fl mice ( Figure 6A and B). HO-1 induction was significantly higher and ALAS1 expression was markedly reduced in the liver of Flvcr1afl/fl;alb-cre mice compared with Flvcr1afl/fl counterparts. This correlated with the enzymatic activities of HO-1 and ALAS1, which were respectively GSK-3 inhibitor higher and lower in the liver of Flvcr1afl/fl;alb-cre mice than in that of Flvcr1afl/fl animals ( Figure 6A and B). HO-1 induction as well as ALAS1 inhibition were likely mediated by heme overload occurring in Flvcr1afl/fl;alb-cre mice. Consistently, after the stimulation of CYP synthesis, heme accumulated to a higher extent in the cytosolic fraction of Flvcr1afl/fl;alb-cre
mice compared with Flvcr1afl/fl controls. On the Selleckchem Volasertib other hand, heme content was significantly lower in the microsomal fraction of Flvcr1afl/fl;alb-cre mice than in that of Sclareol Flvcr1afl/fl
animals ( Figure 6C). As microsomal heme reflects the heme fraction contained in CYPs, we measured mRNA and protein expression and enzymatic activity of CYP3A and CYP1A1 in the livers of our mice. In agreement with heme levels, CYP3A and CYP1A1 mRNA, protein levels and activities were significantly lower in the livers of dexamethasone- and Be(a)P-treated Flvcr1afl/fl;alb-cre mice than in those of treated-Flvcr1afl/fl animals ( Figure 6D and E). Similar results were obtained when mice were treated with imidazole ( Supplementary Results; Supplementary Figure 10). On the enhancement of heme demand, Flvcr1a deletion resulted in an expansion of the cytosolic heme pool that stimulates heme degradation and inhibits heme and CYP synthesis. To test whether the main determinant for CYP expression/function was the size of heme pool or the rate of heme synthesis, both impaired in Flvcr1a-deleted liver, we treated wild-type mice with dexamethasone or Be(a)P alone or together with hemin, to mimic heme overload occurring in Flvcr1afl/fl;alb-cre mice, or with succinylacetone or DL-penicillamine, 2 inhibitors of heme biosynthesis. As expected, dexamethasone and Be(a)P treatment caused a marked increase in ALAS1 activity as well as in CYP expression/activity, and HO-1 expression/activity was only slightly induced ( Figure 7A and B).