Myeloid Expression of Cytochrome P450 4F3 Is Determined by a Lineage-specific Alternative Promoter

Abstract

The cytochrome P450 4F3 (CYP4F3) gene encodes two functionally distinct enzymes that differ only by the selection of exon 4 (CYP4F3A) or exon 3 (CYP4F3B). CYP4F3A inactivates leukotriene B 4 , a reaction that has significance for controlling inflammation. CYP4F3B converts arachidonic acid to 20-hydroxyeicosatetraenoic acid, a potent activator of protein kinase C. We have previously shown that mRNAs coding for CYP4F3A and CYP4F3B are generated from distinct transcription start sites in neutrophils and liver. We therefore investigated mechanisms that regulate the cell-specific expression of these two isoforms. Initially, we analyzed the distribution of CYP4F3 in human leukocytes and determined a lineage-specific pattern of isoform expression. CYP4F3A is expressed in myeloid cells and is coordinate with myeloid differentiation markers such as CD11b and myeloperoxidase during development in the bone marrow. In contrast, CYP4F3B expression is restricted to a small population of CD3؉ T lymphocytes. We identified distinct transcriptional features in myeloid, lymphoid, and hepatic cells that indicate the presence of multiple promoters in the CYP4F3 gene. The hepatic promoter depends on a cluster of hepatocyte nuclear factor sites 123-155 bp upstream of the initiator ATG codon. The myeloid promoter spans 400 bp in a region 468 -872 bp upstream of the ATG codon; it is associated with clusters of CACCT sites and can be activated by ZEB-2, a factor primarily characterized as a transcriptional repressor in cells that include lymphocytes. ZEB-2 interacts with C-terminal binding protein and Smads, and this would provide opportunities for integrating environmental signals in myelopoiesis and inflammation.The ability of cells to attain differentiated stages in development and to alter their functional phenotype in inflammatory settings depends on lineage-specific and cytokine-dependent transcription factors that either activate or repress target genes. Distinct patterns of transcription factors are associated with the myeloid and lymphoid lineages in hematopoiesis (1) and with the induction of pro-inflammatory genes in host defense (2). Transcription factor activity is context-dependent; a repressor of one gene can activate a different gene associated with an opposing function or alternative cell lineage (3). This has implications for the coordinated induction and repression of sets of genes that are functionally related. ZEB-1 (␦EF1, Zfhx1a) and ZEB-2 (SIP1, Zfhx1b) are recently characterized two-handed zinc finger transcription factors (4, 5) that may function as both gene silencers and activators. ZEBs repress transcription of genes that include IL-2, CD4, and GATA-3 in lymphocytes (6 -8), whereas their role in myeloid cells has not been determined. The C-terminal binding protein (CtBP) 1 is a corepressor for ZEBs and other factors but does not bind DNA directly (9). Recent evidence suggests that ZEBs can activate the vitamin D 3 receptor gene in colon carcinoma cells (10).Cytochrome P450 4F3 (CYP4F3) functions to control i...

CYP4F3B Expression Is Associated with Differentiation of HepaRG Human Hepatocytes and Unaffected by Fatty Acid Overload

“…HNF4␣ modulates the expression of a large number of genes involved in the uptake of lipoproteins, synthesis of cholesterol and triglycerides, and very low-density lipoprotein processing (Hwang-Verslues and Sladek, 2010). A potential binding site for HNF4␣ was identified in the CYP4F3B isoform promoter in HepG2 cells (Christmas et al, 2003). Although CYP4F3B and CYP3A4 were expressed once HepaRG cells expressed HNF4␣, we did not observe a correlation between HNF4␣ activation and the regulation of CYP4F3B expression.…”

Role of Cytochrome P450s in Inflammation

“…A different pattern of expression is observed for human CYP4F3A in neutrophils, a short-lived cell associated with the early stages of inflammation. CYP4F3A is expressed at a high constitutive level in neutrophils (Christmas et al, 2003), both before and after stimulating the cells with LTB 4 . Pro-and anti-inflammatory controls probably operate simultaneously during the activation stage of inflammation to control the magnitude of the response (Nathan, 2002).…”

Cytochrome P-450 4F18 Is the Leukotriene B4 ω-1/ω-2 Hydroxylase in Mouse Polymorphonuclear Leukocytes

“…The cells were then blocked in 10% goat serum for 30 min and labeled with affinity-purified rabbit anti-CYP4F3 (10 g/ml) for 1 h at room temperature. The affinity purified antibody to CYP4F3 has been described previously (32,34), and it reacts with both human (CYP4F3) and mouse (CYP4F18) proteins. The secondary antibody was fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Jackson Laboratories), used at a 1:250 dilution in PBS for 1 h at room temperature.…”