Functional expression and cellular localization of a mouse epidermal lipoxygenase.
Three distinct murine lipoxygenase genes have been functionally characterized: 5-lipoxygenase (Chen, X.-S., Naumann, T. A., Kurre, U. , Jenkins, N. A., Copeland, N. G., and Funk, C. D. (1995) J. Biol. Chem. 270, 17993-17999), platelet-type 12-lipoxygenase and leukocyte-type 12-lipoxygenase (Chen, X.-S., Kurre, U., Jenkins, N. A., Copeland, N. G., and Funk, C. D. (1994) J. Biol. Chem. 269, 13979-13987). Here, we describe the cloning and functional characterization of a fourth lipoxygenase gene in mice. Using a polymerase chain reaction-based approach together with partial sequence information from a genomic clone, we isolated a novel lipoxygenase cDNA from the RNA of 3-6-day-old mouse epidermis. The open reading frame predicts a 662-amino acid lipoxygenase that displays 60% identity with both murine 12-lipoxygenase isozymes and 40% identity to 5-lipoxygenase; the sequence is identical to a genomic sequence reported recently (van Dijk, K. W., Steketee, K., Havekes, L., Frants, R., and Hofker, M. (1995) Biochim. Biophys. Acta 1259, 4-8). A full-length clone was expressed in human embryonic kidney 293 cells and homogenates from disrupted cells produced 12-hydroxyeicosatetraenoic acid (12-HETE) and minor amounts of 15-HETE from arachidonic acid. Chiral phase analysis indicated that the 12-HETE is exclusively the 12S enantiomer. In situ hybridization revealed highly specific expression of epidermal lipoxygenase in differentiated keratinocytes of the epidermis and in restricted regions of the root sheath and bulb of hair follicles. High expression was also detected in conjunctiva of the eyelid and in cells of Meibomian and preputial (sebaceous) glands. A 2. 4-kilobase mRNA was detected in mouse epidermis by Northern blot analysis and its abundance was not affected by phorbol ester treatment. The epidermal lipoxygenase gene (Aloxe) resides on mouse chromosome 11 closely linked with the two 12-lipoxygenase genes (Alox12p and Alox12l).
Funk
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CD
Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, USA.
Keeney
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Oliw
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Boeglin
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Brash
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AR
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004817:Epidermis / Q000201:enzymology*; D017403:In Situ Hybridization; D015603:Keratinocytes / Q000201:enzymology*; D008084:Lipoxygenase / Q000145:classification / Q000302:isolation & purification / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D008084:Lipoxygenase