Received for publication March 8, 2005.
Revised May 10, 2005.
Accepted for publication May 13, 2005.

Molecular Mechanisms Regulating Human CYP4B1 Lung-Selective Expression

Abstract

Lung-selective cytochrome P450 expression is well recognized, however, little is known regarding regulatory mechanisms. To address this knowledge gap, transient expression of CYP4B1/luciferase constructs was used to identify: a proximal, positively acting regulatory element, position -139 to -45 that functioned in all cells examined, a negatively acting element, position -457 to -216 that only functioned in HepG2 hepatoblastoma cells, and a distal, positively acting element, position -1087 to -1008 that functioned in A549 or BEAS-2B lung-derived cells, but not HepG2 cells or 293 kidney-derived cells. Competitive electrophoretic mobility shift assays further localized specific A549, but not HepG2, nuclear protein binding to two sites within the distal element, CYP4B1 position -1052 to -1042 and -1026 to -1008. Several potential lung-selective transcription factor recognition sequences were identified within these elements. However, attempts to identify specific factor(s) were unsuccessful. In contrast, in vitro DNA/protein binding assays combined with transient expression and mutagenesis studies identified two functional Sp/XKLF sites within the proximal element (position -118 to -114 and position -77 to -73) that bound both Sp1 and Sp3 in vitro. Further, Sp1-dependent synergistic regulation was observed in A549 cells involving the proximal and distal regulatory elements. Chromatin immunoprecipitation assays demonstrated binding of neither Sp1 nor Sp3 to the CYP4B1 proximal element in human liver tissue, while selective Sp1 binding was observed in human lung tissue. Thus, the composite findings are consistent with both the proximal Sp1 elements and the distal regulatory element acting to synergistically control CYP4B1 lung-selective expression.

Key words: CYP4, cytochrome P450, cytochrome P450 regulation, extrahepatic cytochrome P450, gene regulation, human CYP enzymes, lung cytochrome P450, regulation of gene expression, transcriptional regulation