Structural and functional analysis of human lung-specific cytochrome P450 4B1 5'flanking regulatory region

Abstract

The homeostatic mechanism for the respiratory system involves the tight control of expression of several gene products. One such gene is a member of the multi-substrate oxidase system called cytochrome P450 (P450). Pulmonary P450s may be involved in the tissue-specific detoxification, and/or bio-activation of chemical agents. Additionally, pulmonary cytochrome P450s, particularly the human iso-form 4B1, could play an important role in the development and homeostasis of the pulmonary tissue by removing androgens that cause deleterious effects on lung maturation. Human cytochrome P450 4B1 (CYP 4B1) is a predominant cytochrome P450 activity in the lung. CYP 4B1 protein is reported to bio-activate some pulmonary toxicants. Therefore it may mediate chemical-induced damage to that organ. However, the high expression levels of CYP 4B1 gene products in the lung (approximately 80% of the total P450 activity) may suggest the involvement of the iso-form in normal physiological functions for pulmonary tissue. On the other hand, the results of this and other laboratories have shown an apparent species-specific difference in the catalytic activity of 4B1 protein in man versus rodents. For instance, the rodent iso-form is potent at activating pro-carcinogens and devoid of androgen hydrolyzing activities towards androgen metabolism. The human 4B1 protein has higher 6-beta-testosterone hydroxylase activity while the rodent iso-form is devoid of this action. Furthermore, the manner in which the 4B1 gene is regulated appears to be species-specific. Therefore, my studies have been directed toward further defining the genetic mechanism of action that regulates the human 4B1 gene expression levels. My research objectives have been divided into three specific aims: to determine the presence or absence of cis-acting elements located in the 3.0 Kbp upstream fragment of the human 4B1 gene; to determine the DNA structure of the 4.5 Kbp fragment that overlaps and lies further upstream to the 3.0 Kbp regulatory region of the human 4B1 gene; and to determine the actions of a contiguous 6.5 Kbp fragment containing the entire putative upstream region of 4B1 gene on transcription of the luciferase reporter gene in human lung cells. The results of my studies show the presence of multiple promoter elements in addition to the TATA-box that are contained in the CYP 4B 13.0 Kbp upstream fragment. Nucleotide sequence analysis of the 4.5 Kbp upstream fragment that contains 1.0 Kbp overlapping fragment with the 3.0 Kbp fragment based on restriction mapping confirms that it is indeed part of the contiguous region of the CYP 4B1 gene. A number of consensus DNA regulatory motifs have been identified. (Abstract shortened by UMI.)