Laboratory research is directed to improving our understanding of several aspects of hepatic biochemistry and pathophysiology, including studies on the regulation of metabolism of porphyrins, heme, bile pigments, cholesterol, and bile acids, and the roles of cytokines and T lymphocytes in chronic viral hepatitis.
We use several systems as experimental models, including primary cultures of liver cells, cell lines derived from hepatomas, and intact rodents (guinea pigs, mice, rats, rabbits).
Regulation of Hepatic Porphyrin, Heme, and Bile Pigment Metabolism
The rate-controlling enzymes for heme synthesis and breakdown, respectively, are 5-aminolevulinate (ALA) synthase and heme oxygenase (HO). They are both regulated by heme, but in opposite ways: increased heme in liver cells down-regulates ALA synthase, chiefly by decreasing the stability of its mRNA, whereas it up-regulates HO, chiefly by increasing gene transcription. Several other chemical and physical agents induce or repress activities of these two key enzymes. An understanding of the factors that regulate expression of these enzymes is of fundamental importance for understanding disorders of porphyrin, heme, and bile pigment metabolism (porphyrias, hyperbilirubinemias) and the possible roles of carbon monoxide and bile pigments (products of heme breakdown) in health and disease. The laboratory of Dr. Bonkovsky was among the first to clone and sequence the cDNA and gene of HO, and we are now studying the 5'-regulatory region of the gene to identify the major regulatory elements and the mechanisms whereby several inducers of HO function at a molecular level.
To aid in this work, we have prepared several constructs of selected portions of the 5'-regulatory region of HO linked to reporter genes (CAT, luciferase) and have transfected primary cultures and hepatocyte cell lines with these constructs. We have shown that heme and other inducers of HO require different parts of the 5-regulatory region. In collaboration with Roger J. Davis, a Howard Hughes Investigator and Professor of Biochemistry and Molecular Biology at UMASS, we have found that several inducers (cobalt, arsenite, heat shock), but not heme, induce kinase pathways that mediate the "stress response" of cells. The key questions we plan to answer are: "What is the molecular mechanism for induction of HO by heme or by other types of inducers?" and "How is induction of HO related to repression of ALA synthase?"
To advance our understanding of the pathogenesis of porphyria cutanea tarda, the most common form of human porphyria, we are preparing a mouse model in which the gene for uroporphyrinogen decarboxylase (Uro-D) has been "knocked out." In order to develop the model, we first had to clone, sequence, and characterize the murine Uro-D gene. Then we inserted a small piece of foreign DNA (a neomycin resistant element) into the middle of the gene and attached a second piece containing the thymidine kinase gene of the herpes simplex virus (HSV-tk) to one end (Figure 1). The insertion disrupts the normal expression of the gene and also confers neomycin or G418 resistance to cells into which the altered gene has been introduced. The addition of HSV-tk renders transfected cells sensitive to gancyclovir, an anti-viral compound that requires tk to be phosphorylated and activated. These additions allow us to select embryonic stem cells from mice in which, by homologous recombination, the altered Uro-D gene has been integrated into the genome in place of the normal gene (Figure 1). Such stem cells will be used to generate mice in which one or both Uro-D alleles have been "knocked out." These mice will be bred and characterized carefully. They will provide a unique model of the inherited forms of PCT and will be of great use for elucidating pathogenesis and treatment of the disease. For example, we will use this model to ask such questions as: "What are the crucial effects of iron and other metals on hepatic heme metabolism? How does iron exacerbate the human disease porphyria cutanea tarda (PCT)? What is the role of forms of cytochrome P-450 in producing disease?"
FIGURE 1. Development of a "knockout" Mouse for UROD. The General Structure of an Altered Gene for Uroporphyrinogen Decarboxylase (UROD) and its Use for Selection of Cells into Which it has been Integrated
Bonkovsky HL, et al. Purification and characterization of heme oxygenase from chick liver. Comparison of the avian and mammalian enzymes. Eur J Biochem 189: 155-166, 1990.
Evans C-O, et al. Cloning, sequencing and expression of cDNA for chick liver haem oxygenase. Biochem J 273: 659-666, 1991.
Bonkovsky HL. Key relationships of hepatic heme and iron metabolism. New insights into functions of two old molecules. Viewpoints Dig Dis 23: 19-25, 1991.
Bonkovsky HL, Cable EE, Cable JW, Donohue SE, White EC, Greene YJ, Lambrecht RW, et al. Porphyrogenic properties of the terpenes camphor, pinene, and thujone (with a note on historic implications for absinthe and the illness of Vincent van Gogh). Biochem Pharmacol 43: 2359-2368, 1992.
Cable EE, Cable JW, Bonkovsky HL. Repression of hepatic ¶-aminolevulinate synthase by heme and metalloporphyrins: relationship to inhibition of heme oxygenase. Hepatology 18: 119-127, 1993.
Srivastava KK, Cable EE, Donohue SE, Bonkovsky HL. Molecular basis for heme-dependent induction of heme oxygenase in primary cultures of chick hepatocytes: demonstration of acquired refractoriness to heme. Eur J Biochem 213: 909-917, 1993.
Bonkovsky HL. Advances in understanding and treating 'the little imitator,' acute porphyria. Gastroenterology 105: 590-594, 1993.
Russo S, Pepe JA , Cable EE , Lambrecht RW, Bonkovsky HL. Synergistic repression of ALA synthase by heme and zinc-mesoporphyrin in a chick embryo liver cell culturemodel of acute porphyria. Eur J Clin Invest 24: 406-415, 1994.
Cable EE, Pepe JA, Karamitsios NC, Lambrecht RW, Bonkovsky HL. Differential effects of metalloporphyrins on mRNA levels of -aminolevulinate synthase and heme oxygenase: studies in cultured chick embryo liver cells. J Clin Invest 94: 649-654, 1994.
Cable EE, Pepe JA, Donohue SE, Lambrecht RW, Bonkovsky HL. Effects of RU-486 on heme metabolism and cytochromes P-450 in cultured chick embryo liver cells: Possible Implications for acute porphyria. Eur J Biochem 225: 651-657, 1994.
Gabis KK, Gildemeister OS, Pepe JA, Lambrecht RW, Bonkovsky HL. Induction of heme oxygenase-1 in LMH cells. Comparison of LMH cells to primary cultures of chick embryo liver cells. Biochim Biophys Acta, in press, 1995.