The overall objective of this study is to elucidate the controls regulating expression of rat liver cytochromes P450b and P450e (collectively referred to as P450b/e), a gene family of microsomal hemoproteins that catalyze the oxidative metabolism of many lipophilic chemicals. Despite the determined efforts of many laboratories no one has been able to demonstrate the mechanism by which P450b/e gene transcription is increased by a heterogeneous collection of drugs, e.g., phenobarbital (PB), pesticides and lipophilic chemicals. Studies have been hampered by the lack of a cell culture system that retains P450b/e gene expression. My studies are based on the fact that I have discovered a way to preserve cytochrome P450b/e gene expression in rat hepatocytes by culturing them on a basement membrane extract """"""""matrigel"""""""". Using specific antibodies and cDNAs as molecular probes, I will first define whether transcriptional or post-transcriptional mechanism are involved in increasing the accumulation of P450b/e mRNA and proteins in response to PB in rat and mouse hepatocytes cultured on matrigel. Second, I will define the extracellular regulatory factors that allow expression of P450b/e in hepatocytes cultured on matrigel by determining whether the effect of matrigel is a function of its composition, the physical state of the matrix or a consequence of its effect on hepatocyte cell shape. The third approach is to identify DNA regulatory sequences within the 5' flanking region of the P450e gene. Established techniques will be used to identify DNAse hypersensitive sites, protein-DNA binding domains and regulatory elements of the P450b/e gene. Finally, I will determine whether these putative DNA regulatory sequences contribute to P450e expression in vivo by transfecting recombinant P450e genes (already formed by cloning progressive deletion fragments of the putative regulatory region of the P450e gene upstream of the reporter gene chloramphenicol acetyltransferase) by electroporation into cultured hepatocytes, treating with various inducers and examining CAT activity. This study will provide a better understanding of liver cell physiology and how the extracellular matrix signals intracellular gene expression. Moreover, this study will provide new and important information on regulation of P450b/e by bringing together for the first time the recombinant P450e plasmids, a P450b/e responsive cell culture system, and a suitable method for introducting biologically active DNA into hepatocytes (electroporation).
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