Drugs Mediate the Transcriptional Activation of the 5-Aminolevulinic Acid Synthase (ALAS1) Gene via the Chicken Xenobiotic-sensing Nuclear Receptor (CXR)

Abstract

Heme is an essential component in oxygen transport and metabolism in living systems. In non-erythropoietic cells, 5-aminolevulinate synthase (ALAS1) is the first and rate-limiting enzyme in the heme biosynthesis pathway. ALAS1 expression and heme levels are increased in vivo by drugs and other chemical inducers of cytochrome P450 hemoproteins through mechanisms that are poorly understood. In the present studies, a chicken genomic cosmid library was employed to isolate a major portion of the ALAS1 gene. Two drug-responsive enhancer sequences, 176 and 167 base pairs in length, were identified in the 5-flanking region of the gene in reporter gene assays in the hepatoma cell line LMH. The relative potency of inducers to activate these enhancers corresponds to induction of ALAS1 mRNA levels in LMH cells. Analysis of putative transcription factor binding sites within the enhancers revealed DR5 and DR4 type recognition sequences for nuclear receptors. Drug activation of the enhancer elements was reduced at least 60% after mutagenesis of individual nuclear receptor binding sites and was virtually eliminated following alteration of both recognition sites within the respective elements. Electrophoretic mobility shift assays and transactivation studies demonstrate direct interactions between the nuclear receptor binding sites and the recently described chicken xenobiotic-sensing receptor, (CXR) implicating drug activation mechanisms for ALAS1 similar to those found in inducible cytochrome(s) P450. This is the first report describing direct transcriptional activation of ALAS1 by drugs via drug-responsive enhancer sequences.

Aquisition, Mobilization and Utilization of Cellular Iron and Heme: Endless Findings and Growing Evidence of Tight Regulation

“…The level of intracellular uncommitted heme is extremely low since increased levels of free heme are toxic, so heme biosynthesis is tightly regulated at the step of ALAS1 by multiple mechanisms such as (1) preventing the transfer of ALAS1 precursor to the mitochondria, (2) decreasing the stability of ALAS1 mRNA, and (3) repressing the transcription of ALAS1 mRNA in mammals (Munakata et al 2004). On the other hand, the drug-responsive elements that mediate the direct activation of the transcription were identified in the promoter region of the murine, chicken and human ALAS1 genes (Fraser et al 2002;Podvinec et al 2004). The binding of nuclear receptors such as constitutive androstane receptor and pregnane X receptor to the drug-responsive elements led to activation of the transcription, the mechanisms being similar to those for the transcriptional activation of druginducible cytochrome P-450s (Podvinec et al 2004).…”

Identification of the xenosensors regulating human 5-aminolevulinate synthase

“…Our studies of human, chicken (13), and mouse (14) ALAS1 regulation, combined with recent studies in transgenic mice and human hepatocytes (34)(35)(36), reveal a complex picture. Loss-of-function experiments in mice lacking either PXR or CAR clearly showed involvement of PXR in hepatic ALAS1 induction, which was decreased in PXR-deficient mice.…”

“…Accordingly, we found drug-responsive sequences within the chicken and murine ALAS1 genes, and we tested them in regard to their interaction with nuclear receptors (NRs) (13,14). The central role of orphan NRs in drug-induced expression of CYPs is well established (see refs.…”

Polycyclic Aromatic Hydrocarbons (PAHs) Mediate Transcriptional Activation of the ATP Binding Cassette Transporter ABCB6 Gene via the Aryl Hydrocarbon Receptor (AhR)

“…Hence, heme demand is higher in animals exposed to PAH (16,46,47). When heme demand is high, the preferred response of cells to increased heme demand is to increase heme synthesis.…”