Massachusetts General Hospital

Peter Christmas, PhD 
Instructor of Medicine 
149 13th Street
Charlestown, MA 02129
Phone: (617) 726-4336
Fax: (617) 726-5669
Email: pchristmas@partners.org

 The overall objective of our research is to investigate the role of cytochrome P450s (CYPs) in the regulation of bioactive lipids called eicosanoids. Some CYP enzymes participate in arachidonic acid metabolism and eicosanoid biosynthesis. These enzymes are analogous in function to the cyclooxygenases which generate prostaglandins, and lipoxygenases which generate leukotrienes. Other CYPs have the capacity to inactivate eicosanoids, and therefore down-regulate activities dependent on these lipid mediators. We cloned the gene for human CYP4F3 and identified two distinct enzymes that are generated by alternative splicing; one splice form (CYP4F3A) inactivates leukotriene B₄ (LTB₄), whereas the alternative splice form (CYP4F3B) converts arachidonic acid to 20-HETE, a potent eicosanoid in tissues such as kidney. The opposing functions of CYP4F3 isoforms to generate an active mediator (20-HETE) or inactivate one (LTB₄) provides an unusual system which has interesting implications for dysregulated expression in disease.

We currently focus on the role of CYP4F3 in the regulation of LTB₄-dependent activities. LTB₄ is an inflammatory mediator which functions as a chemoattractant and activator of myeloid cells such as neutrophils and monocytes; it has been implicated as a pathological mediator in diseases which include glomerulonephritis, inflammatory bowel disease, and asthma. CYP4F3A inactivates LTB₄, and is expected to play a role in limiting the magnitude and duration of LTB₄-dependent inflammation. To investigate this hypothesis, we identified the mouse homologue of CYP4F3A as CYP4F18, and we are breeding mice with targeted deletions in the CYP4F18 gene. The inflammatory response in wild type and CYP4F18 knockout (or conditional knockout) mice will be compared using established models of inflammation. We are currently using a mouse model of renal ischemia-reperfusion injury, and are taking both in vitro and in vivo approaches to define the mechanism of action of CYP4F18 and identify the cell types affected.

We are also investigating the mechanisms that regulate human CYP4F3 gene expression and substrate specificity. We have determined that alternative promoters are employed in myeloid cells and liver/kidney, and we are identifying the response elements and transcription factors required for tissue-specific expression of CYP4F3 under basal conditions or in response to inflammatory signals. Alternative splicing involves selection between two exons of identical size, and CYP4F3 constructs have been created to identify cis- and trans-elements that regulate selection of one exon in myeloid cells (to generate CYP4F3A), or the second mutually exclusive exon in liver and kidney (to generate CYP4F3B). The unusual combination of alternative tissue-specific promoters and mutually exclusive exon splicing provides a novel model for studying regulation of gene expression. We will explore the consequences of alternative splicing in humans, and its implications for inflammatory disease.

References:

1. Christmas P, Ursino SR, Fox JW, and Soberman RJ.  Expression of the CYP4F3 gene. tissue-specific splicing and alternative promoters generate high and low K_m forms of leukotriene B₄ w-hydroxylase.   J. Biol. Chem. 1999; 274: 21191-21199.
2. Christmas P, Fox JW, Ursino SR, and Soberman RJ.  Differential localization of 5- and 15-lipoxygenases to the nuclear envelope in RAW macrophages.  J. Biol. Chem. 1999; 274:25594-25598.
3. Christmas P, Jones JP, Patten CJ, Rock DA, Zheng Y, Cheng SM, Weber BM, Carlesso N, Scadden DT, Rettie AE, and Soberman RJ. Alternative splicing determines the function of CYP4F3 by switching substrate specificity.  J. Biol. Chem. 2001; 276: 38166-38172.
4. Christmas P, Weber BM, McKee M, Brown D, and Soberman RJ. Membrane localization and topology of leukotriene C₄ synthase. J. Biol. Chem. 2002; 277: 28902-28908.
5. Christmas P, Carlesso N, Shang H, Cheng SM, Weber BM, Preffer FI, Scadden DT, and Soberman RJ. Myeloid expression of cytochrome P450 4F3 is determined by a lineage-specific alternative promoter.  J. Biol. Chem. 2003; 278: 25133-25142.
6. Soberman RJ and Christmas P.  The organization and consequences of eicosanoid signaling.  J. Clin. Invest. 2003; 111: 1107-1113.
7. Mandal AK, Skoch J, Bacskai BJ, Hyman BT, Christmas P, Miller D, Yamin TD, Xu S, Wisniewski D, Evans JF, and Soberman RJ. The membrane organization of leukotriene synthesis. Proc. Natl. Acad. Sci. 2004; 101: 6587-6592.
8. Soberman RJ and Christmas P. Revisiting prostacyclin: new directions in pulmonary fibrosis and inflammation. Am. J. Physiol. 2006; 291: L142-L143.
9. Christmas P, Tolentino K, Primo V, Zemski Berry K, Murphy RC, Chen M, Lee DM, and Soberman RJ. Cytochrome P-450 4F18 is the leukotriene B₄ w-1/w-2 hydroxylase in mouse polymorphonuclear leukocytes. Identification as the functional orthologue of human polymorphonuclear leukocyte CYP4F3A in the down-regulation of responses to LTB₄. J. Biol. Chem. 2006; 281: 7189-7196.
10. Mandal AK, Jones PB, Bair AM, Christmas P, Miller D, Yamin TT, Wisniewski D, Menke J, Evans JF, Hyman BT, Backsai B, Chen M, Lee DM, Nikolic B, and Soberman RJ. The nuclear membrane organization of leukotriene synthesis. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 20434-20439.