XENOBIOTIC REGULATION OF THE ATP BINDING CASSETTE TRANSPORTER ABCB6 AND ITS SIGNIFICANCE TO HEPATIC HEME HOMEOSTASIS

Abstract

Heme is indispensable for mammalian life. It is an essential component of numerous heme proteins, with functions including oxygen transport and storage, energy metabolism, drug and steroid metabolism and signal transduction. Under normal physiological conditions intracellular free heme levels are extremely low because increased levels of free heme are cytotoxic and accordingly, heme biosynthesis is tightly regulated. Although, 5-aminolevulinic acid synthase (ALAS) mediated regulation of heme synthesis is considered the key step in heme biosynthesis, recent reports have identified a second regulatory step in heme biosynthesis mediated by the mitochondrial ATP binding cassette transporter b6 (Abcb6). Abcb6 expression is directly related to enhanced de novo porphyrin biosynthesis, and Abcb6 overexpression activates the expression of genes important for heme biosynthesis. Thus, Abcb6 represents a previously unrecognized rate-limiting step in heme biosynthesis. The dissertation outlines the progress made since its initiation in understanding the mechanism(s) that regulate Abcb6 expression and the significance of Abcb6 expression to cellular heme homeostasis. Exposure to therapeutic drugs and environmental contaminants leads to an increase in heme demand to compensate for the increased expression of the heme-dependent cytochrome P450s (P450s) detoxifying enzymes. Cells respond to this increasing heme demand by increasing heme synthesis. Thus, exposure to environmental contaminants serves as an optimal in vivo and in vitro model system to study mechanisms that regulate heme synthesis. In this model, Abcb6 expression was induced in response to exposure to xenobiotics [polyaromatic hydrocarbons (PAHs), 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and pregnenolone 16alpha -carbonitrile (PCN)] suggesting a co-ordinate induction of Abcb6 to support the increased heme synthesis. Increased Abcb6 expression in response to cellular heme demands was mediated by the xenobiotic sensing nuclear receptors aryl-hydrocarbon receptor (AhR), the constitutive androstane receptor (CAR), and the pregnane-X receptor (PXR). Exposure to environmental contaminants also leads to the generation of oxidative stress, a primary mechanism by which these compounds cause cellular damage. Cells respond to this increased oxidative stress by activating anti-oxidant defense mechanisms, whose principal components include hemo-proteins (such as catalase, superoxide dismutase, etc). Arsenic, an environmental contaminant and a major hazard following occupational exposure exerts its chronic toxicity through the generation of reactive oxygen species. Of importance, exposure to arsenic also activates the antioxidant defense mechanism. Thus, exposure to arsenic serves as a good model system to evaluate in vivo and in vitro oxidative stress response. In this model system, sodium arsenite induced Abcb6 expression in a dose-dependent manner both in mice fed sodium arsenite in drinking water and in cells exposed to sodium arsenite in vitro. Arsenite-induced Abcb6 expression was transcriptionally regulated but was not mediated by the redox sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2). The significance of Abcb6 expression to cellular heme homeostasis under conditions of heme demand was evaluated in vitro by both gain of function (cells engineered to overexpress Abcb6) and loss of function (cells where endogenous Abcb6 expression was knocked down using Abcb6 specific ShRNA) analysis. Loss of Abcb6 expression in these in vitro model systems significantly compromises the ability of cells to respond to increased heme demand and the ability to protect against oxidative stress following exposure to environmental contaminants. To understand the significance of Abcb6 function to heme homeostasis in vivo, we generated mice carrying homozygous deletion of the Abcb6 allele (Abcb6 null mice). Abcb6 null animals appear phenotypically normal with a trend towards decreased hepatic heme levels, although, the decreased heme levels did not appear to be statistically significant. Interestingly however, Abcb6 null mice demonstrate genotypic changes that suggest a role for Abcb6 in lipid and cholesterol homeostasis. Abcb6 null mice have increased fasting serum cholesterol and increased accumulation of androstone metabolites. Abcb6 null mice also show decreased expression and activity of a specific set of P450s suggesting a role for Abcb6 in drug metabolism and disposition. Mitochondrial ABC transporters are difficult to study because of the two-membrane architecture of mitochondria, problems associated with analyzing transport process, and the high abundance of other ATPases and carriers/transporters. Thus, the development of an in vitro system with pure and active protein is a prerequisite toward understanding the mechanistic relationships between ATP binding and hydrolysis and coupling of these events to translocation of substrates across the lipid membranes. Towards this end, we developed an in vitro liposomal transport system with pure and active Abcb6 protein. Reconstitution of Abcb6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate stimulated ATPase activity (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III and (iii) transport kinetics of substrates identified using a HTS assay. In summary, this dissertation provides insight into the mechanisms that regulate Abcb6 expression in response to increasing heme demand and the in vitro significance of Abcb6 to cellular heme homeostasis. Development of the Abcb6-null mice suggests that loss of Abcb6 does not severely affect heme-dependent functions in the liver probably because of the activation of compensatory mechanisms that balance the loss of Abcb6. More interestingly, Abcb6-null mice show a phenotype that is characteristic of the deficiency of a protein that is involved in cholesterol and lipid homeostasis. Development of the Abcb6-null mice and the development of an in vitro system with purified Abcb6 should serve as useful tools to understand the transport function of Abcb6 and its role in normal and patho-physiology.