| Description |
The cytochrome P450 enzyme system in the human lung is an essential component in lung-directed damage caused by several inhaled xenobiotics. However, the biochemical mechanisms responsible for selective expression of these P450s, especially in lung, have not been adequately characterized. Members of the P4503A subfamily are major contributors to the metabolism of a variety of compounds. The human CYP3A subfamily has four members: CYP3A4, CYP3A5, CYP3A7 and CYP3A43. In contrast to the other CYP3A enzymes, CYP3A5 is widely expressed in various tissues, including the human adult liver, kidney, lung, and blood. Surprisingly, the CYP3A4 and CYP3A5 promoters share over 90% homology within the first 500 base pairs, and yet show remarkable differences in expression within the human lung; CYP3A4 is not expressed in liver. Therefore, the hypothesis of this dissertation was that the selective expression of the P450 gene CYP3A5 in human lung epithelial cells is driven by factors that are distinct from those responsible for the expression of CYP3A4 within the human liver. The goal of the proposed research was to precisely determine the mechanisms that regulate the differential expression of CYP3A4 and CYP3A5 in human lung tissues. The following results were obtained: (1) cloned the promoters of CYP3A4 and CYP3A5, characterized their 5'-regulatory regions, and identified known cis-regulatory elements.; (2) compared the functionality of the 5'-flanking promoter regions of CYP3A4 and CYP3A5 using luciferase reporter constructs transfected into human lung A549 cells and human liver hepatocarcinoma HepG2 cells; and (3) detected and characterized transcription factor proteins that regulate the expression of CYP3A4 and CYP3A5 using electrophoretic mobility shift assays (EMSA). The major difference between the 3A4 and 3A5 promoters is a 57 base pair insertion in the 3A4 promoter (-71 to -127). Deletion constructs that included this insert significantly repressed luciferase activity. EMSA analyses of this region suggested that two E-box motifs were the cis-acting elements. These results demonstrate that a regulatory motif in the 5'-upstream region of 3A4 represses expression in liver. However, the wild-type 3A5 gene is expressed in liver because this insert is not present in the 5'-upstream region of the 3A5 gene. |
