Biochemical and molecular studies on COX, PG-synthases and NOS
We will investigate the path of oxygen movement during COX2 reaction. Previous modeling of the molecular dynamics of oxygen movement in COX2 suggested a targeted transport of oxygen from the reaction buffer into the active site. Random oxygen diffusion into the enzyme appears rather unlikely. In this project we attempt to gradually block oxygen penetration by site directed mutagenesis of the enzyme. These studies are expected to lead to mutant enzyme species exhibiting a reduced oxygen affinity (higher KM for oxygen). These studies will be carried out in collaboration (molecular dynamics) with Dr. J. Saam and Holzhütter from our institution.

Biochemical and molecular studies on LOX-isoforms, LTA-hydrolase and LTC-synthase
In this work package we will similar experiments on the rabbit 15-LOX1. Here again, previous modeling of the molecular dynamics of oxygen movement in this enzyme suggested a targeted transport of oxygen from the reaction buffer into the active site. In fact, detailed simulation of oxygen movement in 15-LOX1 suggested a separate oxygen access channel, which will be targeted by site directed mutagenesis. These experiments are also expected to lead to mutant enzyme species exhibiting a reduced oxygen affinity (higher KM for oxygen). Molecular dynamics modeling will be carried out in collaboration (molecular dynamics) with Dr. J. Saam and Holzhütter from our institution.

Studies on single nucleotide polymorphism in human 15-LOX1
15-LOX1 has been implicated in the pathogenesis of several diseases via the formation of 15-HETE. However, the positional specificity of the enzyme can strongly be altered just by the substitution of a single amino acid and these “hotspots” in the primary structure (positional determinants) have been identified in the past. In this sub-project we will investigate whether there is a single nucleotide polymorphism in the human gene that impacts the positional specificity of the enzyme. If so, we will attempt to correlate this SNP to the pathogenesis of different diseases.

LOX products as mediators in cardiovascular disease
LOX isoforms have been implicated in the pathogenesis of atherosclerosis but the precise role of these enzymes still remains elusive. The formation of lipid laden foam cells is an early event in atherogenesis. Foam cells develop from macrophages by taking up modified lipoproteins and depositing cholesterol esters as lipid droplet in the cytosol. We have developed an in vitro foam cells assay and intent to investigate the impact of 12/15-LOX expression on intracellular lipid deposition. For this purpose human and murine macrophages (J774, U937) will stably be transfected with 12/15-LOX and intracellular lipid deposition will be assayed by HPLC after the cells have been stimulated with modified LDL.

Eicosanoids and NO in the CNS
Little is known about expression of eicosanoid synthesizing enzymes, in particular of the various LOX-isoforms, in the brain. It has, however, been reported that lipid peroxidation is minimal in normal brain but is strongly after cerebral ischemia and after entorhinal cortex lesion. During the first project period we will profile the expression pattern of eicosanoid synthesizing enzymes (COX-, LOX- and Gpx-isoforms) in various parts of normal and lesional brain by immunohistochemistry and RT-PCR. In this context cellular and subcellular distribution of these enzymes will be studied (immunocytochemistry, confocal and electron microscopic) and we would also like to explore the expression profile of the anti-oxidative phGPx. Similar experiments will be carried out in cell cultures using neuronal and non-neuronal cells cultured under normoxic and hyperoxic conditions
During later stages of the project we would like to address the question of whether or not regulatory events, which have been implicated in expression regulation of LOX and phGPx in somatic cells may also be important for cells of the CNS. Interleukins-4 and –13 are potent upregulators of 12/15-LOX expression in peripheral monocytes but nothing is known whether these cytokines may also impact LOX expression in the brain. To address this question we will take advantage of IL4 overexpressing transgenic mice and will compare the expression of eicosanoid synthesizing enzymes and of phGPx in the brain of these animals with that of the corresponding control animals.

COX, PG-synthase and NO in oncogenesis
Transgenic mice that overexpress COX-2 under the control of the keratin 5 promoter have been created. These animals strongly overexpress COX-2 in the epidermis and develop hyperplastic and dysplastic lesions in the skin and bladder. The major aim of the first project period is to study in a combined genomic/proteomic approach expression of tumor related gene products on the mRNA and protein level. Using the methodological approach it is intended identify early biomarkers for carcinogenesis in these particular tissues. If such tumor markers are discovered we will apply the above described analytical scheme to laser-microdissected human bladder tissues.

LOX-isoenzymes and tumor development
Different LOX-isoforms have been implicated in carcinogenesis but their precise role is rather elusive. In order to shed light on this topic we will employ transgenic mouse models that overexpress various LOX isoforms (endothelial specific expression of 15-LOX1, epidermis-specific expression of 12S-LOX). After induction of carcinogenesis tumor growth will be monitored in these animal models and compared to normal mice. To analyze the cross-talk between LOX isozymes an inducible keratinocyte expression system (Tet on/off) has been established and during the first project period the impact of the forced expression of a selected LOX-isoform (platelet-type 12-LOX or 12/15-LOX) on the expression level of other LOXs will be determined. Basic cell physiological parameters with potential importance for carcinogenesis (proliferation rate, degree of differentiation, sensitivity for apoptosis) will be determined.
In additional experiments expression of other LOX-isoforms (15-LOX2, 12R-LOX) in different stages of tumor development will be investigated.

Project related recent publications
Anning, P.B., Coles, B., Bermudez-Fajardo, A., Martin, P.E., Levison, B.S., Hazen, S.L., Funk, C.D., Kuhn, H., O'donnell,´V.B. Elevated endothelial nitric oxide bioactivity and resistance to Angiotensin-dependent hypertension in 12/15-lipoxygenase knockout mice. Am. J. Pathol. 166, 653-662 (2005).

Kühn, H. Lipoxygenases in the cardiovascular system. Circ. Res. 94, 1527-1529 (2004)

Walther, M., Wiesner, R. and Kühn, H. Investigations into calcium-dependent membrane association of 15-lipoxygenase-1. Mechanistic role of surface exposed hydrophobic amino and calcium. J. Biol. Chem. 279, 3717-3725 (2004)

Bräuer, A.U., Savaskan, N.E., Kühn, H., Prehn, S.,Ninnemann, O., Nitsch, R. Identification of a novel lipid phosphate phosphatase PRG-1 (plasticity related gene-1) involved in axonal outgrowth and regenerative sprouting. Nat. Neuroscience 6, 572-578 (2003).

Kuhn, H., Anton, M., Gerth, C., Habenicht, A. Amino acid differences in the deduced 5-lipoxygenase sequence of CAST atherosclerosis- resistant mice confer impaired activity when introduced into the human ortholog. Arterioskl. Thromb. Vasc. Biol. 23, 1072-1076 (2003).

Walther, M., Anton, M., Wiedmann, M., Fletterick, R. and Kühn, H. The N-terminal domain of the reticulocyte-type 15-lipoxygenase is not essential for enzymatic activity but contains determinants for membrane binding.
J. Biol. Chem. 277, 27360-27366 (2002).

Kühn, H. and Borchert, A. Regulation of enzymatic lipid peroxidation: the interplay of peroxidizing and peroxide reducing enzymes. Free Rad. Biol. Med. 33, 154-172 (2002).

Spanbroek, R., M. Hildner, A. Kohler, A. Muller, F. Zintl, H. Kuhn, O. Radmark,B. Samuelsson, A. J.Habenicht. IL-4 determines eicosanoid formation in dendritic cells by down-regulation of 5-lipoxygenase and up-regulation of 15-lipoxygenase 1 expression. Proc. Natl. Acad. Sci USA. 98, 5152-5157 (2001).

M. Walther, I. Ivanov, G. Myagkova, H. Kuhn. Alterations of lipoxygenase specificity by targeted substrate modification and site-directed mutagenesis. Chem. Biol. 115, 1-13 (2001).