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Arachidonate 8(S)-lipoxygenase
Prostaglandins & other Lipid Mediators 68–69 (2002) 235–243
Arachidonate 8(S)-lipoxygenase Gerhard Fürstenberger∗ , Friedrich Marks, Peter Krieg Research Program Tumor Cell Regulation, Deutsches Krebsforschungszentrum, Heidelberg, Germany
Abstract The recently identified mouse 8(S)-lipoxygenase almost exclusively directs oxygen insertion into the 8(S) position of arachidonic acid and, with lower efficiency, into the 9(S) position of linoleic acid. The protein of 677 amino acids displays 78% sequence identity to human 15(S)-lipoxygenase-2 which is considered to be its human orthologue. The 8(S)-lipoxygenase gene, Alox15b, consisting of 14 exons and spanning 14.5 kb is located within a gene cluster of related epidermis-type lipoxygenases at the central region of mouse chromosome 11. 8(S)-Lipoxygenase is predominantly expressed in stratifying epithelia of mice, constitutively in the hair follicle, forestomach, and footsole and inducible in the back skin with strain-dependent variations. The expression is restricted to terminally differentiating keratinocytes, in particular the stratum granulosum and 8(S)-lipoxygenase activity seems to be involved in terminal differentiation of mouse epidermis. Tumor-specific upregulation of 8(S)-lipoxygenase expression and activity indicate a critical role of this enzyme in malignant progression during tumor development in mouse skin. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Epidermal lipoxygenase; Arachidonic acid; Carcinogenesis; Keratinocyte differentiation
1. Introduction Mammalian lipoxygenases have been identified inserting oxygen into the 5-, 8-, 12- and 15-positions of arachidonic acid with varying stereoconfiguration (S or R) [1]. Among them, the 8(S)-lipoxygenase has been described only recently [2,3]. 8-Hydroxy-5,9,11, 14-eicosatetraenoic acid (8-HETE) was first observed to be formed as minor product in human neutrophils [4], in psoriatic skin [5], in human tracheal epithelial cells [6], and squamous head and neck carcinomas [7]. 8-HETE from these sources, however, is most probably not the product of an 8-lipoxygenase because this enzyme has not yet been found in human tissues (see below). Instead, 8-HETE may be either generated by non-enzymatic lipid peroxidation [8] or by P450 monooxygenase-catalyzed arachidonic acid metabolism [9]. ∗
Corresponding author. Tel.: +49-6221-42-4504; fax: +49-6221-42-4406. E-mail address: [email protected] (G. Fürstenberger).
0090-6980/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 0 - 6 9 8 0 ( 0 2 ) 0 0 0 3 3 - 3
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8-HETE as a major arachidonic acid metabolite was first detected in phorbol ester-treated mouse skin [10] and characterized as an 8(S)-lipoxygenase-derived metabolite by identifying 8-hydroperoxy-5,9,11,14-eicosatetraenoic acid [8-H(P)ETE] as precursor product [11] and 8-HETE as a pure S-enantiomer [12].
2. Molecular biology of 8(S)-lipoxygenase 2.1. cDNA cloning and protein structure The 8(S)-lipoxygenase cDNA was cloned from phorbol ester-treated mouse skin by RT-PCR strategies using degenerate primers based on highly conserved sequences in mammalian lipoxygenases and, subsequently, by screening skin-derived cDNA libraries. The open reading frame of the 3.2 kb transcript encodes a protein of 677 amino acids with a calculated molecular weight of 76.23 kDa and a pI of 6.72 [2,3]. 8(S)-Lipoxygenase is most closely related to human and bovine 15(S)-lipoxygenase-2 exhibiting 78 and 75% amino acid identity, respectively. Based on this high sequence identity and on the structure and chromosomal localization of the corresponding gene (see below) 15(S)-lipoxygenase-2 is regarded to represent the human orthologue of mouse 8(S)-lipoxygenase [13]. Sequence comparison with other mammalian lipoxygenases shows about 54% amino acid identity with 12(R)-lipoxygenases, 50% with epidermis-type lipoxygenases-3, 43% with 5(S)-lipoxygenases, but only 38% identity with the 12(S)- and 12/15(S)-lipoxygenases. The relatively large molecular weight of 8(S)-lipoxygenase is mainly due to an unique eight amino acid insertion within the -barrel domain (positions 74–81 of 8(S)-lipoxygenase). In a phylogenetic tree which displays subfamilies and relative distances between the individual members of the mammalian lipoxygenases 8(S)-lipoxygenase and its human and bovine orthologue 15(S)-lipoxygenase-2 are placed together with other skin-derived isoenzymes including 12(R)-lipoxygenases and epidermis-type lipoxygenases-3 in a separate subfamily of epidermal lipoxygenases (Fig. 1). 8(S)-Lipoxygenase contains the characteristic well-conserved amino acids found in all lipoxygenases including the histidines of the so-called lipoxygenase motif and the putative iron-binding histidine residues at positions 374, 379, and 554 as well as the C-terminal isoleucine. The putative fifth iron-ligand at amino acid position 558 which is either a histidine or an asparagine in all other lipoxygenases has been thought to be a serine in mouse 8(S)-lipoxygenase and human 15(S)-lipoxygenase-2. Upon exchange of this serine residue by asparagine or histidine the mutant 8(S)-lipoxygenases exhibited equivalent or slightly increased catalytic activities as compared with the wild-type enzyme, and in addition, some 15(S)-lipoxygenase activity. Significant 8(S)-lipoxygenase activity was still retained when serine 558 was exchanged by alanine, however, questioning a critical function of this amino acid in the active site of this enzyme [14]. The fact that the human orthologue of the mouse 8(S)-lipoxygenase encodes a lipoxygenase with different positional specificity is unique among lipoxygenases. In this context it is intriguing that the exchange of only two amino acids, tyrosine 603 and histidine 604, of 8(S)-lipoxygenase by the corresponding amino acids of 15(S)-lipoxygenase-2, i.e. asparagine and valine, converts the positional specificity from 8(S) to 15(S) [15]. The
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Fig. 1. Phylogenetic tree of mammalian lipoxygenases. Multiple sequence alignments were performed using PileUp programs and a phylogenetic tree was created from a distance matrix using the GrowTree program of the Heidelberg Unix Sequence Analysis Resources (HUSAR) software programs. Abbreviations and sources are as follows: p12(S)-LOX, platelet-type 12(S)-lipoxygenase (accession numbers: mouse: S80446, human: M62982, bovine: Y08829); l12(S)-LOX, leukocyte-type 12(S)-lipoxygenase (rat: L06040, mouse: U04331, rabbit: Z97654, bovine: M81320, porcine: A35087); 15(S)-LOX, 15(S)-lipoxygenase (rabbit: M27214, human: M23892); e12(S)-LOX, epidermis-type 12(S)-lipoxygenase (X99252); 5(S)-LOX, 5(S)-lipoxygenase (mouse: L42198, rat: J03960, hamster: U43333, human: J03600); 8(S)-LOX, 8(S)-lipoxygenase (Y14696); 15(S)-LOX-2, 15(S)-lipoxygenase-2 (human: U78294, bovine: AF107263); 12(R)-LOX, 12(R)-lipoxygenase (mouse: Y14334, human: AF038461); e-LOX-3, epidermis-type lipoxygenase-3 (mouse: Y14695, human: AJ269499).
observation that both products exhibit S chirality indicates that this shift can not only be due to a differential penetration of arachidonic acid into the substrate binding pocket of the enzyme. According to recently developed models predicting the positional and enantiomeric specificity of lipoxygenases [16–20] the formation of either 8(S)- or 15(S)-H(P)ETE by the
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wild-type and mutant 8(S)-lipoxygenase, respectively, is only conceivable with a switch in the head to tail binding of the substrate accepting arachidonic acid “head-first” to generate 8(S)-H(P)ETE by the wild-type enzyme and “tail-first” for the formation of 15(S)-H(P)ETE by the mutant enzyme [15]. 2.2. Gene structure and chromosomal localization The gene encoding mouse 8(S)-lipoxygenase is designated Alox15b referring to the previously annotated human orthologous gene ALOX15B. Like the genes of most other
Fig. 2. Comparison of the genomic organization of the mouse 8S-lipoxygenase and the human 15-lipoxygenase-2 genes. The linear arrangement of a mouse gene cluster encoding 8(S)-lipoxygenase (Alox15b), 12(R)-lipoxygenase (Alox12b), and epidermis-type lipoxygenase3 (Aloxe3) and their orientation towards one another (indicated by arrows) are shown in the upper panel. The syntetic human gene cluster containing in addition the 15-lipoxygenase pseudogene (ALOX15P) is depicted in the lower panel. The exon/intron organization of the mouse 8(S)-lipoxygenase gene and the human 15(S)-lipoxygenase-2 gene is drawn to scale. Exons are numbered and depicted by boxes. 5 - and 3 -non-coding sequences are indicated by open boxes.
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mammalian lipoxygenases Alox15b is split into 14 exons with exon/intron boundaries at highly conserved positions. It is approximately 14.5 kb in length exhibiting a very similar exon clustering as the human ALOX15B gene (unpublished observations; Fig. 2). The gene is located at the central region of mouse chromosome 11 adjacent to the genes encoding 12(R)-lipoxygenase (Alox12b) and epidermis-type lipoxygenase3 (Aloxe3), which are arranged in a head-to-tail fashion approximately 13 kb downstream to Alox15b on the opposite DNA strand (Fig. 2). The genomic organization of this lipoxygenase gene cluster has been found highly conserved as a syntenic group at the human chromosome 17p13.1 [21].
3. Species and tissue distribution of 8(S)-lipoxygenase To our knowledge 8(S)-lipoxygenase expression or activity has been detected in tissues of mice and rats only [2,3,22] whereas 15(S)-lipoxygenase-2 was found to be expressed in bovine and human tissues [13,23]. In rats, an 8(S)-HETE generating activity has been localized to corneal epithelium [22]. In NMRI mice, Northern blot analyses showed a low constitutive expression of (8S)-lipoxygenase in lung, colon, brain, but not in back or ear skin [24]. However, a body-site specific expression could be observed at epithelial sites exposed to mechanical stress such as pressure (footsole and tail) or stretch (forestomach) [2,11,24]. In black Swiss mice constitutive 8(S)-lipoxygenase expression was detected in hair follicles [2]. In mouse skin 8(S)-lipoxygenase mRNA and protein as well as activity can be induced by phorbol ester application. The strongest induction has been found inducible in the epidermis of 6–10-day-old NMRI mice [3,10,11]. Induced 8(S)-lipoxygenase protein and activity were restricted to the post-mitotic compartment of epidermis, i.e. in particular to the stratum granulosum, showing a correlation of enzyme expression and terminal differentiation of keratinocytes [2,11,24]. However, major differences of both the constitutive and inducible expression of 8(S)-lipoxygenase are known to occur between different mouse strains [25]. Similar as NMRI mice the skin of black Swiss mice showed a low constitutive but strongly inducible 8(S)-lipoxygenase expression upon phorbol ester treatment whereas 6–10-day-old Sencar mice exhibited a high level of constitutive expression but low inducibility by phorbol esters [15]. No measurable induction of 8(S)-lipoxygenase activity by phorbol esters was seen in skin of C57BL/6J mice [26]. Interestingly, the orthologous 15(S)-lipoxygenase-2 was also found to be expressed in skin and cornea of man [13,27]. In human skin, however, the expression of the protein was restricted to basal keratinocytes in contrast to 8(S)-lipoxygenase [2,11,24] indicating different functions of the two enzymes in mouse and human skin.
4. Enzymatic activity of 8(S)-lipoxygenase The mouse 8(S)-lipoxygenase was found to metabolize arachidonic acid and linoleic acid exclusively to 8(S)-H(P)ETE and 9(S)-hydroperoxy-octadeca-10,12-dienoic acid [9(S)-H(P) ODE], respectively, both as native enzyme and recombinant protein expressed in HEK293,
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Table 1 Substrate specificity of recombinant 8(S)-lipoxygenase Fatty acid substrate
Enzyme activity (ectopic expression) (pmol/mg protein/min)
Reference
Arachidonic acid
127 (COS-7) 896 (HEK293 cells)
[29] [28]
Linoleic acid
22 (COS-7) 71 (HEK293 cells) 136 (COS-7) 111 (COS-7) 56 (COS-7) 76 (COS-7)
[29] [28] [29] [29] [29] [29]
␣-Linolenic acid Docosahexaenoic acid ␥-Linolenic acid Eicosapentaenoic acid
10, 000 × g supernatants of HEK293 cells [28] or crude lysates of COS7 cells [29] transfected with 8(S)-lipoxygenase expression plasmids were incubated with various substrates at 37 ◦ C for 12 or 45 min. Products were separated by reverse or straight phase HPLC. The products were identified and quantified by comparing retention times and peak areas with those of authentic external standards.
COS-7, vaccinia-infected HeLa cells, and as His-tagged fusion protein in bacteria (see Table 1) [2,11,28,29]. Arachidonic acid was consistently more efficient as a substrate than linoleic acid. With arachidonic acid the enzyme activity was found to be linear for up to 45 min, exhibited a pH optimum of 8 and did not depend on the presence of Ca2+ ions [11,28,29]. 8(S)H(P)ETE formation was shown to be associated with the stereo-selective removal of the proR hydrogen from the 10-carbon and the exclusive oxygen insertion into the 8(S)position [12]. (-6) fatty acids such as ␣-linolenic acid and docosahexaenoic acid were oxygenated with a similar rate as arachidonic acid whereas the (-3) fatty acids including ␥-linolenic acid and eicosapentaenoic acid only with about half the efficiency of arachidonic acid (Table 1) [28,29]. Methyl and cholesteryl esters of linoleic acid or arachidonic acid were not accepted as substrates (unpublished observations). The purified enzyme was shown to convert 5-H(P)ETE into leukotriene A4 as indicated by the formation of 6-trans-leukotriene B4 epimers being non-enzymatic degradation products of the former. With 5-H(P)ETE as a substrate 8(S)-lipoxygenase activity decreased within 10 min pointing to a suicide inactivation of the enzyme [29].
5. Biologic function of 8(S)-lipoxygenase 5.1. Terminal differentiation of keratinocytes 8(S)-Lipoxygenase expression and activity do not only correlate with terminal differentiation of keratinocytes but they are causally related. Transgenic mice with 8(S)-lipoxygenase targeted to keratinocytes by using a loricrin promoter showed highly differentiated skin epidermis, tongue, and forestomach phenotypes correlating with aberrant expression of the differentiation-specific keratin-1. The strong hyperkeratosis of the epidermis of transgenic mice was found to be matched by a compensatory epidermal hyperproliferation in
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transgenics [30]. 8(S)-HETE is known to specifically bind to and activate the transcription factor peroxisome proliferator activator receptor-␣ (PPAR-␣) and has indeed been shown to stimulate keratinocyte differentiation by inducing keratin 1 via a PPAR-␣-dependent pathway [30,31]. The human 15(S)-lipoxygenase-2, on the other hand, was found to be selectively expressed in differentiating sebocytes generating 15(S)-HETE which is known to be an activating PPAR-␥ ligand [27,32,33]. Taken into account that synthetic PPAR-␥ activators have been shown to induce sebocyte differentiation it may be concluded that 15(S)-HETE is an endogenous mediator of this process [33]. Thus, depending on the species, differentially expressed 8(S)-lipoxygenase and 15(S)-lipoxygenase-2 appear to induce cell type-specific differentiation processes through activation of individual PPAR isoforms. This indicates a functional equivalence of the two lipoxygenases encoded by orthologous genes. 5.2. Chemically induced tumor development in mouse skin Constitutive 8(S)-lipoxygenase expression and activity leading to a massive accumulation of 8(S)-HETE has been detected in papillomas, i.e. benign epidermal tumors, generated according to the initiation-promotion-protocol of mouse skin carcinogenesis. In addition, large amounts of 12(S)-HETE have been found in the tumors being due to a constitutive overexpression of platelet-type 12(S)-lipoxygenase [25,26]. Upon malignant progression of papillomas to carcinomas 8(S)-lipoxygenase expression and activity becomes downregulated [28,35]. Similarly, 15(S)-lipoxygenase-2 expression was found to be reduced in human basal cell carcinoma, prostate adenocarcinoma, and sebaceous carcinomas as compared to premalignant states such as sebaceous adenomas or the parent normal tissues [27,34]. A hint as to the pathological role of 8(S)-HETE in mouse skin tumors is given by the observation that their amount correlates with etheno-adduct formation in DNA isolated from epidermal tumors. These DNA adducts are known to be pre-mutagenic DNA lesions giving rise to chromosomal alterations [36]. Moreover, both 8-H(P)ETE and 8-HETE as well as the corresponding 12-regioisomers have been shown to induce clastogenic effects and chromosomal aberrations in mouse keratinocytes in cultures, an effect that was suppressed by lipoxygenase inhibitors [37,38]. In this context it is intriguing that malignant progression of papillomas occurs spontaneously, i.e. in the absence of any further treatment, and is indeed accompanied by an accumulation of chromosomal alterations [39,40] indicating that endogenous factors may be responsible for the genotoxicity. Thus, 8- and 12-lipoxygenase-derived products may contribute to the endogenous genotoxic potential of the premalignant lesions and, therefore, are thought to be responsible for genetic instability. Tumor induction in mice constitutively overexpressing 8(S)-lipoxygenase under the control of a loricrin promoter lent support to this hypothesis. Using the initiation-promotion-protocol the incidence and multiplicity of papillomas was admittedly similar in both transgenic and wild-type mice. Development of carcinomas, however, was strongly accelerated and increased in transgenic mice finally leading to spindle cell carcinomas, i.e. a highly malignant poorly differentiated tumor type rarely observed in wild-type animals. These results point to a critical role of constitutively over-expressed 8(S)-lipoxygenase in malignant progression [41].
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Acknowledgements This work was supported by the Deutsche Krebshilfe, Bonn, Germany and by a grant from the Deutsche Forschungsgemeinschaft, Bonn, Germany. The excellent technical assistance by Dagmar Kucher, Ina Kutchera and Brigitte Steinbauer is gratefully acknowledged. References [1] Brash AR. Lipoxygenases: occurrence functions, catalysis, and acquisition of substrate. J Biol Chem 1999;274:23679. [2] Jisaka M, Kim RB, Boeglin WE, Nanney LB, Brash AR. Molecular cloning and functional expression of a phorbol ester-inducible 8S-lipoxygenase from mouse skin. J Biol Chem 1997;272:24410. [3] Krieg P, Kinzig A, Heidt M, Marks F, Fürstenberger G. cDNA cloning of an 8-lipoxygenase and a novel epidermis-type lipoxygenase from phorbol ester-treated mouse skin. Biochim Biophys Acta 1998;1391:7. [4] Goetzl EJ, Sun FF. Generation of unique mono-hydroxy-eicosatetraenoic acids from arachidonic acid by human neutrophils. J Exp Med 1979;150:406. [5] Camp RD, Mallet AI, Woollard PM, Brain SD, Black AK, Greaves MW. The identification of hydroxy fatty acids in psoriatic skin. Prostaglandins 1983;26:431. [6] Hunter JA, Finkbeiner WE, Nadel JA, Goetzl EJ, Holtzman MJ. Predominant generation of 15-lipoxygenase metabolites of arachidonic acid by epithelial cells from human trachea. Proc Natl Acad Sci USA 1985;82:4633. [7] El-Attar TM, Lin HS, Vanderhoek JY. Biosynthesis of prostaglandins and hydroxy fatty acids in primary squamous carcinomas of head and neck in humans. Cancer Lett 1985;27:255. [8] Guido DM, McKenna R, Mathews WR. Quantitation of hydroperoxy-eicosatetraenoic acids and hydroxy-eicosatetraenoic acids as indicators of lipid peroxidation using gas chromatography-mass spectrometry. Anal Biochem 1993;209:123. [9] Capdevila J, Yadagiri P, Manna S, Falck JR. Absolute configuration of hydroxyeicosatetraenoic acids (HETEs) formed during catalytic oxygenation of arachidonic acid by cytochrome P-450. Biochem Biophys Res Commun 1986;141:1007. [10] Gschwendt M, Fürstenberger G, Kittstein W, Besemfelder E, Hull WE, Hagedorn H, et al. Generation of the arachidonic acid metabolite 8-HETE by extracts of mouse skin treated with phorbol ester in vivo; identification by 1H-NMR and GC–MS spectroscopy. Carcinogenesis 1986;7:449. [11] Fürstenberger G, Hagedorn H, Jacobi T, Besemfelder E, Stephan M, Lehmann WD, et al. Characterization of an 8-lipoxygenase activity induced by the phorbol ester tumor promoter 12-Otetradecanoylphorbol-13-acetate in mouse skin in vivo. J Biol Chem 1991;266:15738. [12] Hughes MA, Brash AR. Investigation of the mechanism of biosynthesis of 8-hydroxeicosatetraenoic acid in mouse skin. Biochim Biophys Acta 1991;1081:347. [13] Brash AR, Boeglin WE, Chang MS. Discovery of a second 15S-lipoxygenase in humans. Proc Natl Acad Sci USA 1997;95:6148. [14] Jisaka M, Boeglin WE, Kim RB, Brash AR. Site-directed mutagenesis studies on a putative fith iron ligand of mouse 8S-lipoxygenase: retention of catalytic activity on mutation of Serine-558 to asparagine, histidine, or alanine. Arch Biochem Biophys 2001;386:136. [15] Jisaka M, Kim RB, Boeglin WE, Brash AR. Identification of amino acid determinants of the positional specificity of mouse 8S-lipoxygenase and human 15 S-lipoxygenase-2. J Biol Chem 2000;275:1287. [16] Hamberg M, Hamberg G. On the mechanism of the oxygenation of arachidonic acid by human platelet lipoxygenase. Biochem Biophys Res Commun 1980;95:1090. [17] Kühn H, Sprecher H, Brash AR. On singular or dual positional specificity of lipoxygenases .The number of chiral products varies with alignment of methylene groups at the active site of the enzyme. J Biol Chem 1990;265:16300. [18] Boyington JC, Gaffney BJ, Amzel LM. The three-dimensional structure of an arachidonic acid 15-lipoxygenase. Science 1993;260:1482.
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[19] Gillmor SA, Villasenor A, Fletterick A, Sigal E, Browner MF. The structure of mammalian 15-lipoxygenase reveals similarity to the lipases and the determinants of substrate specificity. Nat Struct Biol 1997;2:1003. [20] Solomon EI, Zhou J, Neese F, Pavel EG. New insights from spectroscopy into structure/function relationships of lipoxygenases. Chem Biol 1997;4:795. [21] Krieg P, Marks F, Fürstenberger G. A gene cluster encoding human epidermis-type lipoxygenases at chromosome 17p13.1: cloning, physical mapping, and expression. Genomics 2001;73:323. [22] Yamada M, Proia AD. 8(S)-Hydroxyeiocsatetaenoic acid is the lipoxygenase metabolite of arachidonic acid that regulates epithelial cell migration in the rat cornea. Cornea 2000;19(3 Suppl):S13. [23] Liminga M, Oliw E. cDNA cloning of 15-lipoxygenase type 2 and 12-lipoxygenases of bovine corneal epithelium. Biochim Biophys Acta 1999;1437:124. [24] Heidt M, Fürstenberger G, Vogel S, Marks F, Krieg P. Diversity of mouse lipoxygenases: identification of a subfamily of epidermal isozymes exhibiting a differentiation-dependent mRNA expression pattern. Lipids 2000;35:701. [25] Fischer SM, Fürstenberger G, Marks F, Slaga TJ. Events associated with mouse skin tumor promotion with respect to arachidonic acid metabolism: a comparison between SENCAR and NMRI mice. Cancer Res 1987;47:3174. [26] Fischer SM, Baldwin JK, Jasheway DW, Patrick KE, Cameron GS. Phorbol ester induction of 8-lipoxygenase in inbred SENCAR (SSIN) but not C57BL/6J mice correlated with hyperplasia, edema, and oxidant generation but not ornithine decarboxylase induction. Cancer Res 1988;48:658. [27] Shappel SB, Keeney DS, Zhang J, Page R, Olson SJ, Brash AR. 15-Lipoxygenase-2 expression in benign and neoplastic sebaceous glands and other cutaneous adnexa. J Invest Dermatol 2001;117:36. [28] Bürger F, Krieg P, Kinzig A, Schurich B, Marks F, Fürstenberger G. Constitutive expression of 8-lipoxygenase in papillomas and clastogenic effectes of lipoxygenase-derived arachidonic acid metabolites in keratinocytes. Mol Carcinogenesis 1999;24:108. [29] Qiao N, Takahashi Y, Takamatsu H, Yoshimoto T. Leukotriene A synthase activity of purified mouse skin arachidonate 8-lipoxygenase expressed in Escherichia coli. Biochim Biophys Acta 1999;1438:131. [30] Muga SJ, Thuillier P, Pavone A, Rundhaug JE, Boeglin WE, Jisaka M, et al. 8(S)-Lipoxygenase products activate peroxisome proliferator-activated receptor alpha and induce differentiation in murine keratinocytes. Cell Growth & Differentiation 2000;11:447. [31] Yu K, Bayona W, Kallen CB, Harding HP, Ravera CP, McMahon G. Differential activation of peroxisome proliferator-activated receptors by eicosanoids. J Biol Chem 1995;270:23975. [32] Huang JT, Welch JS, Ricote M, Binder CJ, Wilson TM, Kelly C, et al. Interleukin-4-dependent production of PPAR-gamma ligands in macrophages by 12/15-lipoxygenase. Nature 1999;400:378. [33] Rosenfield RL, Deplewski D, Kentsis A, Cilett N. Mechanism of androgen induction of sebocyte differentiation. Dermatology 1998;196:43. [34] Shappell SB, Boeglin WE, Olson SJ, Kasper S, Brash AR. 15-Lipoxygenase-2 (15-LOX-2) is expressed in benign prostatic epithelium and reduced in prostate adenocarcinoma. Am J Pathol 1999;155:235. [35] Lehmann WD, Stephan M, Fürstenberger G. Profiling assay for lipoxygenase products of linoleic and arachidonic acid by gas chromatography-mass spectrometry. Anal Biochem 1992;204:158. [36] Nair J, Furstenberger G, Burger F, Marks F, Bartsch H. Promutagenic etheno-DNA adducts in multistage mouse skin carcinogenesis: correlation with lipoxygenase-catalyzed arachidonic acid metabolism. Chem Res Toxicol 2000;13:703. [37] Furstenberger G, Schurich B, Kaina B, Petrusevska RT, Fusenig NE, Marks F. Tumor induction in initiated mouse skin by phorbol esters and methyl methanesulfonate: correlation between chromosomal damage and conversion (stage I of tumor promotion)in vivo. Carcinogenesis 1989;10:749. [38] Petrusevska RT, Fürstenberger G, Marks F, Fusenig NE. Cytogenetic effects caused by the phorbol ester tumor promoters in primary mouse keratinocyte cultures:correlation with the convertogenic activity of TPA in multistage carcinogenesis. Carcinogenesis 1988;9:1207. [39] Aldaz CM, Conti CJ. The premalignant nature of mouse skin papillomas: histopathologic, cytogenetic, and biochemical evidence. In: Conti CJ, Slaga TJ, Klein-Szanto, AJP, editors. Skin tumors, experimental and clinical aspects. New York: Raven Press, 1989. p. 227. [40] Brown K, Burns PA, Balmain A. Transgenic approaches to understanding the mechanisms of chemical carcinogenesis in mouse skin. Toxicol Lett 1995;82/83:123. [41] Muga SJ, Kim EJ, Fischer SM. Regulation of 8S-lipoxygenase in mouse skin. Proc Am Assoc Cancer Res 2000;41:84.
Arachidonate 8(S)-lipoxygenase Gerhard Fürstenberger∗ , Friedrich Marks, Peter Krieg Research Program Tumor Cell Regulation, Deutsches Krebsforschungszentrum, Heidelberg, Germany
Abstract The recently identified mouse 8(S)-lipoxygenase almost exclusively directs oxygen insertion into the 8(S) position of arachidonic acid and, with lower efficiency, into the 9(S) position of linoleic acid. The protein of 677 amino acids displays 78% sequence identity to human 15(S)-lipoxygenase-2 which is considered to be its human orthologue. The 8(S)-lipoxygenase gene, Alox15b, consisting of 14 exons and spanning 14.5 kb is located within a gene cluster of related epidermis-type lipoxygenases at the central region of mouse chromosome 11. 8(S)-Lipoxygenase is predominantly expressed in stratifying epithelia of mice, constitutively in the hair follicle, forestomach, and footsole and inducible in the back skin with strain-dependent variations. The expression is restricted to terminally differentiating keratinocytes, in particular the stratum granulosum and 8(S)-lipoxygenase activity seems to be involved in terminal differentiation of mouse epidermis. Tumor-specific upregulation of 8(S)-lipoxygenase expression and activity indicate a critical role of this enzyme in malignant progression during tumor development in mouse skin. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Epidermal lipoxygenase; Arachidonic acid; Carcinogenesis; Keratinocyte differentiation
1. Introduction Mammalian lipoxygenases have been identified inserting oxygen into the 5-, 8-, 12- and 15-positions of arachidonic acid with varying stereoconfiguration (S or R) [1]. Among them, the 8(S)-lipoxygenase has been described only recently [2,3]. 8-Hydroxy-5,9,11, 14-eicosatetraenoic acid (8-HETE) was first observed to be formed as minor product in human neutrophils [4], in psoriatic skin [5], in human tracheal epithelial cells [6], and squamous head and neck carcinomas [7]. 8-HETE from these sources, however, is most probably not the product of an 8-lipoxygenase because this enzyme has not yet been found in human tissues (see below). Instead, 8-HETE may be either generated by non-enzymatic lipid peroxidation [8] or by P450 monooxygenase-catalyzed arachidonic acid metabolism [9]. ∗
Corresponding author. Tel.: +49-6221-42-4504; fax: +49-6221-42-4406. E-mail address: [email protected] (G. Fürstenberger).
0090-6980/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 0 - 6 9 8 0 ( 0 2 ) 0 0 0 3 3 - 3
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8-HETE as a major arachidonic acid metabolite was first detected in phorbol ester-treated mouse skin [10] and characterized as an 8(S)-lipoxygenase-derived metabolite by identifying 8-hydroperoxy-5,9,11,14-eicosatetraenoic acid [8-H(P)ETE] as precursor product [11] and 8-HETE as a pure S-enantiomer [12].
2. Molecular biology of 8(S)-lipoxygenase 2.1. cDNA cloning and protein structure The 8(S)-lipoxygenase cDNA was cloned from phorbol ester-treated mouse skin by RT-PCR strategies using degenerate primers based on highly conserved sequences in mammalian lipoxygenases and, subsequently, by screening skin-derived cDNA libraries. The open reading frame of the 3.2 kb transcript encodes a protein of 677 amino acids with a calculated molecular weight of 76.23 kDa and a pI of 6.72 [2,3]. 8(S)-Lipoxygenase is most closely related to human and bovine 15(S)-lipoxygenase-2 exhibiting 78 and 75% amino acid identity, respectively. Based on this high sequence identity and on the structure and chromosomal localization of the corresponding gene (see below) 15(S)-lipoxygenase-2 is regarded to represent the human orthologue of mouse 8(S)-lipoxygenase [13]. Sequence comparison with other mammalian lipoxygenases shows about 54% amino acid identity with 12(R)-lipoxygenases, 50% with epidermis-type lipoxygenases-3, 43% with 5(S)-lipoxygenases, but only 38% identity with the 12(S)- and 12/15(S)-lipoxygenases. The relatively large molecular weight of 8(S)-lipoxygenase is mainly due to an unique eight amino acid insertion within the -barrel domain (positions 74–81 of 8(S)-lipoxygenase). In a phylogenetic tree which displays subfamilies and relative distances between the individual members of the mammalian lipoxygenases 8(S)-lipoxygenase and its human and bovine orthologue 15(S)-lipoxygenase-2 are placed together with other skin-derived isoenzymes including 12(R)-lipoxygenases and epidermis-type lipoxygenases-3 in a separate subfamily of epidermal lipoxygenases (Fig. 1). 8(S)-Lipoxygenase contains the characteristic well-conserved amino acids found in all lipoxygenases including the histidines of the so-called lipoxygenase motif and the putative iron-binding histidine residues at positions 374, 379, and 554 as well as the C-terminal isoleucine. The putative fifth iron-ligand at amino acid position 558 which is either a histidine or an asparagine in all other lipoxygenases has been thought to be a serine in mouse 8(S)-lipoxygenase and human 15(S)-lipoxygenase-2. Upon exchange of this serine residue by asparagine or histidine the mutant 8(S)-lipoxygenases exhibited equivalent or slightly increased catalytic activities as compared with the wild-type enzyme, and in addition, some 15(S)-lipoxygenase activity. Significant 8(S)-lipoxygenase activity was still retained when serine 558 was exchanged by alanine, however, questioning a critical function of this amino acid in the active site of this enzyme [14]. The fact that the human orthologue of the mouse 8(S)-lipoxygenase encodes a lipoxygenase with different positional specificity is unique among lipoxygenases. In this context it is intriguing that the exchange of only two amino acids, tyrosine 603 and histidine 604, of 8(S)-lipoxygenase by the corresponding amino acids of 15(S)-lipoxygenase-2, i.e. asparagine and valine, converts the positional specificity from 8(S) to 15(S) [15]. The
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Fig. 1. Phylogenetic tree of mammalian lipoxygenases. Multiple sequence alignments were performed using PileUp programs and a phylogenetic tree was created from a distance matrix using the GrowTree program of the Heidelberg Unix Sequence Analysis Resources (HUSAR) software programs. Abbreviations and sources are as follows: p12(S)-LOX, platelet-type 12(S)-lipoxygenase (accession numbers: mouse: S80446, human: M62982, bovine: Y08829); l12(S)-LOX, leukocyte-type 12(S)-lipoxygenase (rat: L06040, mouse: U04331, rabbit: Z97654, bovine: M81320, porcine: A35087); 15(S)-LOX, 15(S)-lipoxygenase (rabbit: M27214, human: M23892); e12(S)-LOX, epidermis-type 12(S)-lipoxygenase (X99252); 5(S)-LOX, 5(S)-lipoxygenase (mouse: L42198, rat: J03960, hamster: U43333, human: J03600); 8(S)-LOX, 8(S)-lipoxygenase (Y14696); 15(S)-LOX-2, 15(S)-lipoxygenase-2 (human: U78294, bovine: AF107263); 12(R)-LOX, 12(R)-lipoxygenase (mouse: Y14334, human: AF038461); e-LOX-3, epidermis-type lipoxygenase-3 (mouse: Y14695, human: AJ269499).
observation that both products exhibit S chirality indicates that this shift can not only be due to a differential penetration of arachidonic acid into the substrate binding pocket of the enzyme. According to recently developed models predicting the positional and enantiomeric specificity of lipoxygenases [16–20] the formation of either 8(S)- or 15(S)-H(P)ETE by the
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wild-type and mutant 8(S)-lipoxygenase, respectively, is only conceivable with a switch in the head to tail binding of the substrate accepting arachidonic acid “head-first” to generate 8(S)-H(P)ETE by the wild-type enzyme and “tail-first” for the formation of 15(S)-H(P)ETE by the mutant enzyme [15]. 2.2. Gene structure and chromosomal localization The gene encoding mouse 8(S)-lipoxygenase is designated Alox15b referring to the previously annotated human orthologous gene ALOX15B. Like the genes of most other
Fig. 2. Comparison of the genomic organization of the mouse 8S-lipoxygenase and the human 15-lipoxygenase-2 genes. The linear arrangement of a mouse gene cluster encoding 8(S)-lipoxygenase (Alox15b), 12(R)-lipoxygenase (Alox12b), and epidermis-type lipoxygenase3 (Aloxe3) and their orientation towards one another (indicated by arrows) are shown in the upper panel. The syntetic human gene cluster containing in addition the 15-lipoxygenase pseudogene (ALOX15P) is depicted in the lower panel. The exon/intron organization of the mouse 8(S)-lipoxygenase gene and the human 15(S)-lipoxygenase-2 gene is drawn to scale. Exons are numbered and depicted by boxes. 5 - and 3 -non-coding sequences are indicated by open boxes.
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mammalian lipoxygenases Alox15b is split into 14 exons with exon/intron boundaries at highly conserved positions. It is approximately 14.5 kb in length exhibiting a very similar exon clustering as the human ALOX15B gene (unpublished observations; Fig. 2). The gene is located at the central region of mouse chromosome 11 adjacent to the genes encoding 12(R)-lipoxygenase (Alox12b) and epidermis-type lipoxygenase3 (Aloxe3), which are arranged in a head-to-tail fashion approximately 13 kb downstream to Alox15b on the opposite DNA strand (Fig. 2). The genomic organization of this lipoxygenase gene cluster has been found highly conserved as a syntenic group at the human chromosome 17p13.1 [21].
3. Species and tissue distribution of 8(S)-lipoxygenase To our knowledge 8(S)-lipoxygenase expression or activity has been detected in tissues of mice and rats only [2,3,22] whereas 15(S)-lipoxygenase-2 was found to be expressed in bovine and human tissues [13,23]. In rats, an 8(S)-HETE generating activity has been localized to corneal epithelium [22]. In NMRI mice, Northern blot analyses showed a low constitutive expression of (8S)-lipoxygenase in lung, colon, brain, but not in back or ear skin [24]. However, a body-site specific expression could be observed at epithelial sites exposed to mechanical stress such as pressure (footsole and tail) or stretch (forestomach) [2,11,24]. In black Swiss mice constitutive 8(S)-lipoxygenase expression was detected in hair follicles [2]. In mouse skin 8(S)-lipoxygenase mRNA and protein as well as activity can be induced by phorbol ester application. The strongest induction has been found inducible in the epidermis of 6–10-day-old NMRI mice [3,10,11]. Induced 8(S)-lipoxygenase protein and activity were restricted to the post-mitotic compartment of epidermis, i.e. in particular to the stratum granulosum, showing a correlation of enzyme expression and terminal differentiation of keratinocytes [2,11,24]. However, major differences of both the constitutive and inducible expression of 8(S)-lipoxygenase are known to occur between different mouse strains [25]. Similar as NMRI mice the skin of black Swiss mice showed a low constitutive but strongly inducible 8(S)-lipoxygenase expression upon phorbol ester treatment whereas 6–10-day-old Sencar mice exhibited a high level of constitutive expression but low inducibility by phorbol esters [15]. No measurable induction of 8(S)-lipoxygenase activity by phorbol esters was seen in skin of C57BL/6J mice [26]. Interestingly, the orthologous 15(S)-lipoxygenase-2 was also found to be expressed in skin and cornea of man [13,27]. In human skin, however, the expression of the protein was restricted to basal keratinocytes in contrast to 8(S)-lipoxygenase [2,11,24] indicating different functions of the two enzymes in mouse and human skin.
4. Enzymatic activity of 8(S)-lipoxygenase The mouse 8(S)-lipoxygenase was found to metabolize arachidonic acid and linoleic acid exclusively to 8(S)-H(P)ETE and 9(S)-hydroperoxy-octadeca-10,12-dienoic acid [9(S)-H(P) ODE], respectively, both as native enzyme and recombinant protein expressed in HEK293,
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Table 1 Substrate specificity of recombinant 8(S)-lipoxygenase Fatty acid substrate
Enzyme activity (ectopic expression) (pmol/mg protein/min)
Reference
Arachidonic acid
127 (COS-7) 896 (HEK293 cells)
[29] [28]
Linoleic acid
22 (COS-7) 71 (HEK293 cells) 136 (COS-7) 111 (COS-7) 56 (COS-7) 76 (COS-7)
[29] [28] [29] [29] [29] [29]
␣-Linolenic acid Docosahexaenoic acid ␥-Linolenic acid Eicosapentaenoic acid
10, 000 × g supernatants of HEK293 cells [28] or crude lysates of COS7 cells [29] transfected with 8(S)-lipoxygenase expression plasmids were incubated with various substrates at 37 ◦ C for 12 or 45 min. Products were separated by reverse or straight phase HPLC. The products were identified and quantified by comparing retention times and peak areas with those of authentic external standards.
COS-7, vaccinia-infected HeLa cells, and as His-tagged fusion protein in bacteria (see Table 1) [2,11,28,29]. Arachidonic acid was consistently more efficient as a substrate than linoleic acid. With arachidonic acid the enzyme activity was found to be linear for up to 45 min, exhibited a pH optimum of 8 and did not depend on the presence of Ca2+ ions [11,28,29]. 8(S)H(P)ETE formation was shown to be associated with the stereo-selective removal of the proR hydrogen from the 10-carbon and the exclusive oxygen insertion into the 8(S)position [12]. (-6) fatty acids such as ␣-linolenic acid and docosahexaenoic acid were oxygenated with a similar rate as arachidonic acid whereas the (-3) fatty acids including ␥-linolenic acid and eicosapentaenoic acid only with about half the efficiency of arachidonic acid (Table 1) [28,29]. Methyl and cholesteryl esters of linoleic acid or arachidonic acid were not accepted as substrates (unpublished observations). The purified enzyme was shown to convert 5-H(P)ETE into leukotriene A4 as indicated by the formation of 6-trans-leukotriene B4 epimers being non-enzymatic degradation products of the former. With 5-H(P)ETE as a substrate 8(S)-lipoxygenase activity decreased within 10 min pointing to a suicide inactivation of the enzyme [29].
5. Biologic function of 8(S)-lipoxygenase 5.1. Terminal differentiation of keratinocytes 8(S)-Lipoxygenase expression and activity do not only correlate with terminal differentiation of keratinocytes but they are causally related. Transgenic mice with 8(S)-lipoxygenase targeted to keratinocytes by using a loricrin promoter showed highly differentiated skin epidermis, tongue, and forestomach phenotypes correlating with aberrant expression of the differentiation-specific keratin-1. The strong hyperkeratosis of the epidermis of transgenic mice was found to be matched by a compensatory epidermal hyperproliferation in
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transgenics [30]. 8(S)-HETE is known to specifically bind to and activate the transcription factor peroxisome proliferator activator receptor-␣ (PPAR-␣) and has indeed been shown to stimulate keratinocyte differentiation by inducing keratin 1 via a PPAR-␣-dependent pathway [30,31]. The human 15(S)-lipoxygenase-2, on the other hand, was found to be selectively expressed in differentiating sebocytes generating 15(S)-HETE which is known to be an activating PPAR-␥ ligand [27,32,33]. Taken into account that synthetic PPAR-␥ activators have been shown to induce sebocyte differentiation it may be concluded that 15(S)-HETE is an endogenous mediator of this process [33]. Thus, depending on the species, differentially expressed 8(S)-lipoxygenase and 15(S)-lipoxygenase-2 appear to induce cell type-specific differentiation processes through activation of individual PPAR isoforms. This indicates a functional equivalence of the two lipoxygenases encoded by orthologous genes. 5.2. Chemically induced tumor development in mouse skin Constitutive 8(S)-lipoxygenase expression and activity leading to a massive accumulation of 8(S)-HETE has been detected in papillomas, i.e. benign epidermal tumors, generated according to the initiation-promotion-protocol of mouse skin carcinogenesis. In addition, large amounts of 12(S)-HETE have been found in the tumors being due to a constitutive overexpression of platelet-type 12(S)-lipoxygenase [25,26]. Upon malignant progression of papillomas to carcinomas 8(S)-lipoxygenase expression and activity becomes downregulated [28,35]. Similarly, 15(S)-lipoxygenase-2 expression was found to be reduced in human basal cell carcinoma, prostate adenocarcinoma, and sebaceous carcinomas as compared to premalignant states such as sebaceous adenomas or the parent normal tissues [27,34]. A hint as to the pathological role of 8(S)-HETE in mouse skin tumors is given by the observation that their amount correlates with etheno-adduct formation in DNA isolated from epidermal tumors. These DNA adducts are known to be pre-mutagenic DNA lesions giving rise to chromosomal alterations [36]. Moreover, both 8-H(P)ETE and 8-HETE as well as the corresponding 12-regioisomers have been shown to induce clastogenic effects and chromosomal aberrations in mouse keratinocytes in cultures, an effect that was suppressed by lipoxygenase inhibitors [37,38]. In this context it is intriguing that malignant progression of papillomas occurs spontaneously, i.e. in the absence of any further treatment, and is indeed accompanied by an accumulation of chromosomal alterations [39,40] indicating that endogenous factors may be responsible for the genotoxicity. Thus, 8- and 12-lipoxygenase-derived products may contribute to the endogenous genotoxic potential of the premalignant lesions and, therefore, are thought to be responsible for genetic instability. Tumor induction in mice constitutively overexpressing 8(S)-lipoxygenase under the control of a loricrin promoter lent support to this hypothesis. Using the initiation-promotion-protocol the incidence and multiplicity of papillomas was admittedly similar in both transgenic and wild-type mice. Development of carcinomas, however, was strongly accelerated and increased in transgenic mice finally leading to spindle cell carcinomas, i.e. a highly malignant poorly differentiated tumor type rarely observed in wild-type animals. These results point to a critical role of constitutively over-expressed 8(S)-lipoxygenase in malignant progression [41].
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