Recently we cloned and characterized the gene for cytochrome P450 4F3 (CYP4F3). The gene contains 14 exons and 13 introns, and undergoes alternative splicing to generate two splice forms containing either exon 3 or exon 4. These isoforms catalyze omega-hydroxylation of different substrates. The isoform expressing exon 4 (CYP4F3A) utilizes the inflammatory mediator leukotriene B4 (LTB4) as its substrate and renders it inactive for pro-inflammatory functions such as neutrophil chemoattraction. LTB4 has been implicated as a pathological mediator in inflammatory disorders such as inflammatory bowel disease, glomerulonephritis, and asthma. The uniquely low Km of CYP4F3A for LTB4, and its unique localization among CYP4 enzymes to myeloid cells, suggest that its expression plays a central role in the control of LTB4-mediated inflammation. The isoform of CYP4F3 expressing exon 3 (CYP4F3B) utilizes arachidonic acid as a substrate and generates 20-HETE, an intracellular activator of protein kinase C and Ca2+/calmodulin-dependent kinase II. 20-HETE is a preeminent eicosanoid in the kidney where it performs a vasoactive and natriuretic function, but it is also active in many other tissues. Preliminary data suggests that the range of CYP4F3 expression is extended beyond myeloid cells by alternative promoter usage. The hypothesis of this proposal is that coordinated tissue-specific expression and splicing determine whether the CYP4F3 gene functions to inactivate a bioactive eicosanoid (LTB4) in inflammation, or generate a bioactive eicosanoid (20-HETE). The goals of the proposal are to determine how the expression of the different functional forms of CYP4F3 are regulated.
In Specific Aim 1, the tissue-specific regulation of CYP4F3 gene expression will be investigated. Luciferase reporter constructs, DNase I footprinting, EMSA, and site-directed mutagenesis will be employed to identify promoter elements that regulate transcription in myeloid cells, and non-hematopoietic tissues such as liver. An initial characterization of DNA elements that regulate splicing will be performed using minigene constructs in splicing assays.
In Specific Aim 2, the functional consequences of CYP4F3 expression will be studied. The possibility of additional roles for CYP4F3A in inflammatory signal transduction pathways will be investigated. We will determine if reactive oxygen species generated during CYP4F3A-dependent LTB4 metabolism can activate stress-activated protein kinases (SAPKs) via the dissociation of ASK1 and thioredoxin. The tissue localization of CYP4F3 gene products in the gastrointestinal tract and kidney will be analyzed by immunohistochemistry, in situ hybridization, and isoform-specific PCR.
