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Functional expression and cellular localization of a mouse epidermal lipoxygenase
C D Funk1, D S Keeney, E H Oliw
1Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, USA.
The Journal of Biological Chemistry
|September 20, 1996
Summary
Researchers identified a fourth lipoxygenase gene in mice, termed epidermal lipoxygenase (Aloxe). This gene is specifically expressed in skin cells and produces 12S-HETE, a key eicosanoid in epidermal function.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Three murine lipoxygenase genes (5-LOX, platelet-type 12-LOX, leukocyte-type 12-LOX) were previously characterized.
- Lipoxygenases are key enzymes in the metabolism of polyunsaturated fatty acids, producing signaling molecules.
- Understanding the diversity of lipoxygenase genes is crucial for elucidating their roles in biological processes.
Purpose of the Study:
- To clone and functionally characterize a novel, fourth lipoxygenase gene in mice.
- To determine the enzymatic activity and product specificity of the newly identified lipoxygenase.
- To investigate the tissue-specific expression pattern of this epidermal lipoxygenase.
Main Methods:
- Polymerase chain reaction (PCR) and genomic sequence information were used to isolate a novel lipoxygenase cDNA from mouse epidermis.
- The full-length cDNA was expressed in human embryonic kidney 293 cells for functional analysis.
- Enzymatic activity was assessed by measuring the production of hydroxyeicosatetraenoic acids (HETEs) from arachidonic acid, with chiral phase analysis for enantiomer determination.
- In situ hybridization and Northern blot analysis were employed to determine gene expression patterns and mRNA levels.
Main Results:
- A novel lipoxygenase cDNA, termed epidermal lipoxygenase (Aloxe), was cloned and characterized.
- Expressed Aloxe produced 12-hydroxyeicosatetraenoic acid (12S-HETE) as the primary product from arachidonic acid, with minor 15-HETE.
- Aloxe expression was highly specific to differentiated keratinocytes in the epidermis, hair follicles, conjunctiva, and sebaceous glands.
- A 2.4-kilobase mRNA transcript was detected in mouse epidermis, unaffected by phorbol ester treatment.
- The Aloxe gene is located on mouse chromosome 11, closely linked to the two known 12-lipoxygenase genes.
Conclusions:
- A fourth lipoxygenase gene, epidermal lipoxygenase (Aloxe), has been identified and functionally characterized in mice.
- This enzyme specifically produces 12S-HETE and is predominantly expressed in epidermal and related skin structures.
- Aloxe represents a significant addition to the known lipoxygenase family, suggesting specialized roles in skin biology and potentially other tissues.