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Basal Transepidermal Water Loss is Increased in Platelet-Type 12-Lipoxygenase Deficient Mice

1999; Elsevier BV; Volume: 112; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.1999.00595.x

1523-1747

Eric N. Johnson, Lillian B. Nanney, Jyoti Virmani, John A. Lawson, Colin Funk,

Contact Dermatitis and Allergies

The roles of fatty acids in the skin have been under investigation since early reports of the phenotypic abnormalities of mice fed a diet deficient in essential fatty acids. Little is known about the functional significance of fatty acid metabolism by lipoxygenases in epidermis. Here, we have examined the role of platelet-type 12-lipoxygenase which converts arachidonic acid to the oxygenated metabolite 12-hydroperoxyeicosatetraenoic acid, in the skin using platelet-type 12-lipoxygenase-deficient mice generated by gene targeting. Platelet-type 12-lipoxygenase in wild-type mice was localized to the stratum granulosum by immunohistochemical analysis. Platelet-type 12-lipoxygenase-deficient mice lacked immunodetectable platelet-type 12-lipoxygenase in platelets and epidermis, appeared grossly normal, and exhibited an increase in basal transepidermal water loss without alteration in basal mitotic activity. Water loss and mitotic activity in mice with an acetone-disrupted membrane barrier were normal. No defect in ultrastructural properties or content of major fatty acids in dorsal skin or ear inflammation response was apparent in platelet-type 12-lipoxygenase-deficient mice. These results indicate that the platelet-type 12-lipoxygenase pathway in mice is partly responsible for normal permeability barrier function but the mechanism awaits further elucidation. The roles of fatty acids in the skin have been under investigation since early reports of the phenotypic abnormalities of mice fed a diet deficient in essential fatty acids. Little is known about the functional significance of fatty acid metabolism by lipoxygenases in epidermis. Here, we have examined the role of platelet-type 12-lipoxygenase which converts arachidonic acid to the oxygenated metabolite 12-hydroperoxyeicosatetraenoic acid, in the skin using platelet-type 12-lipoxygenase-deficient mice generated by gene targeting. Platelet-type 12-lipoxygenase in wild-type mice was localized to the stratum granulosum by immunohistochemical analysis. Platelet-type 12-lipoxygenase-deficient mice lacked immunodetectable platelet-type 12-lipoxygenase in platelets and epidermis, appeared grossly normal, and exhibited an increase in basal transepidermal water loss without alteration in basal mitotic activity. Water loss and mitotic activity in mice with an acetone-disrupted membrane barrier were normal. No defect in ultrastructural properties or content of major fatty acids in dorsal skin or ear inflammation response was apparent in platelet-type 12-lipoxygenase-deficient mice. These results indicate that the platelet-type 12-lipoxygenase pathway in mice is partly responsible for normal permeability barrier function but the mechanism awaits further elucidation. 5-bromo-2′-deoxyuridine essential fatty acid hydroxyeicosatetraenoic acid hydroperoxyeicosatetraenoic acid platelet-type 12-lipoxygenase homozygous P-12LO deficient wild-type P-12LO transepidermal water loss One primary function of the epidermis is to form a barrier between an organism and the outside hostile environment designed to avert the invasion of bacteria and other foreign entities while simultaneously preventing the escape of water required for terrestrial life. Immediately exterior to the stratum granulosum is the hydrophobic region of epidermis where the lipids reside in an intercellular milieu. These lipids originate from lamellar bodies, organelles synthesized in the spinous and granular cells. The lamellar contents undergo a reorganization into a system of broad sheets which fill the intercellular space of the stratum corneum (Schurer and Elias, 1991Schurer N.Y. Elias P.M. The biochemistry and function of stratum corneum lipids.Adv Lipid Res. 1991; 24: 27-56Crossref PubMed Google Scholar). The function of these lipids was first examined many years ago when essential fatty acids (EFA) were shown to be critical in the maintenance of the water permeability barrier as EFA-deficient mice develop scaly skin, loss of hair, and necrosis of the tail (Burr and Burr, 1929Burr G.O. Burr M.M. A new deficiency disease produced by the rigid exclusion of fat from the diet.J Biol Chem. 1929; 82: 345-367Abstract Full Text PDF Google Scholar;Menton, 1970Menton D.N. The effects of essential fatty acid deficiency on the fine structure of mouse skin.J Morphol. 1970; 132: 181-206Crossref PubMed Scopus (16) Google Scholar). The addition of certain fatty acids, such as linoleic acid, to the diet could reverse some of the cutaneous symptoms of EFA deficiency and those acids were termed "essential" (for review seeZiboh, 1996Ziboh V.A. The significance of polyunsaturated fatty acids in cutaneous biology.Lipids. 1996; 31: S249-S253Crossref PubMed Scopus (49) Google Scholar). Further studies revealed this phenotype to be the result of decreased internal lamella; EFA-deficient lamellar body contents failed to coalesce into the broad neutral lipid-rich sheets that fill the stratum corneum interstices of control animals (Elias and Brown, 1978Elias P.M. Brown B.E. The mammalian cutaneous permeability barrier. Defective barrier function in essential fatty acid deficiency correlated with abnormal intercellular lipid deposition.Lab Invest. 1978; 39: 574-583PubMed Google Scholar). Disruption of the barrier by either acetone treatment or tape stripping causes a wide variety of responses including the secretion of lamellar body contents (Menon et al., 1992Menon G.K. Feingold K.R. Elias P.M. Lamellar body secretory response to barrier disruption.J Invest Dermatol. 1992; 98: 279-289Crossref PubMed Scopus (232) Google Scholar) and epidermal synthesis of lipid and DNA (Proksch et al., 1993Proksch E. Holleran W.M. Menon G.K. Elias P.M. Feingold K.R. Barrier function regulates epidermal lipid and DNA synthesis.Br J Dermatol. 1993; 128: 473-482Crossref PubMed Scopus (193) Google Scholar), which may be signaled by water loss itself (Grubauer et al., 1989Grubauer G. Elias P.M. Feingold K.R. Transepidermal water loss: the signal for recovery of barrier structure and function.J Lipid Res. 1989; 30: 323-333Abstract Full Text PDF PubMed Google Scholar). Normal barrier recovery requires fatty acid biosynthesis as indicated by diminished recovery in animals treated with 5-(tetradecyloxy)-2-furancarboxylic acid, an inhibitor of acetyl coenzyme A carboxylase required for fatty acid synthesis (Mao-Qiang et al., 1993Mao-Qiang M. Elias P.M. Feingold K.R. Fatty acids are required for epidermal permeability barrier function.J Clin Invest. 1993; 92: 791-798Crossref PubMed Scopus (139) Google Scholar). Restoration of various abnormal cutaneous parameters has been demonstrated upon oral, parenteral, or topical administration of linoleic acid (for review seeProttey, 1976Prottey C. Essential fatty acids and the skin.Br J Dermatol. 1976; 94: 579-585Crossref PubMed Scopus (83) Google Scholar andZiboh, 1996Ziboh V.A. The significance of polyunsaturated fatty acids in cutaneous biology.Lipids. 1996; 31: S249-S253Crossref PubMed Scopus (49) Google Scholar). In liver and brain, linoleic acid can be converted to arachidonic acid, the substrate for several enzyme systems including the cyclooxygenases and lipoxygenases via desaturase and elongase activities; however, the lack of relevant desaturase activity in the epidermis prevents this conversion and arachidonic acid must be synthesized elsewhere and delivered to the skin (for review seeZiboh, 1994Ziboh V.A. Essential fatty acids/eicosanoid biosynthesis in the skin: biological significance.Proc Soc Exp Biol. 1994; 205: 1-11Crossref PubMed Scopus (14) Google Scholar). Despite this, arachidonic acid metabolites have been implicated in many physiologic and pathologic skin functions including ultraviolet light-induced abnormalities, cell proliferation, and tumorigenesis (for review seeZiboh, 1994Ziboh V.A. Essential fatty acids/eicosanoid biosynthesis in the skin: biological significance.Proc Soc Exp Biol. 1994; 205: 1-11Crossref PubMed Scopus (14) Google Scholar). Topical application of arachidonic acid has been shown to induce ear inflammation as a result of metabolism by both the 5-lipoxygenase (Chen et al., 1994aChen X.-S. Sheller J.B. Johnson E.N. Funk C.D. Role of leukotrienes revealed by targeted disruption of the 5-lipoxygenase gene.Nature. 1994; 372: 179-182Crossref PubMed Scopus (340) Google Scholar) and cyclooxygenase-1 (Langenbach et al., 1995Langenbach R. Morham S. Tiano H. et al.Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and and indomethacin-induced gastic ulceration.Cell. 1995; 83: 483-492Abstract Full Text PDF PubMed Scopus (1002) Google Scholar) pathways. The metabolism of EFA by lipoxygenases in normal and diseased skin has been the subject of many experiments (Nugteren et al., 1985Nugteren D.H. Christ-Hazelhof E. van der Beek A. Houtsmuller U.M. Metabolism of linoleic acid and other essential fatty acids in the epidermis of the rat.Biochim Biophys Acta. 1985; 834: 429-436Crossref PubMed Scopus (113) Google Scholar;Nugteren and Kivits, 1987Nugteren D.H. Kivits G.A.A. Conversion of linoleic and arachidonic acid by skin epidermal lipoxygenases.Biochim Biophys Acta. 1987; 921: 135-141Crossref PubMed Scopus (55) Google Scholar). There are several lipoxygenases known to exist in mammalian skin and hair follicles including two 15-LO, four 12-lipoxygenase, an 8-lipoxygenase, and a 5-lipoxygenase (Sun et al., 1998Sun D. McDonnell M. Chen X.-S. et al.Human 12 (R) -lipoxygenase and the mouse ortholog. Molecular cloning, expression, and gene chromosomal assignment.J Biol Chem. 1998; 273: 33540-33547Crossref PubMed Scopus (63) Google Scholar;Funk et al., 1996Funk C.D. Keeney D.S. Oliw E.H. Boeglin W.E. Brash A.R. Functional expression and cellular localization of a mouse epidermal lipoxygenase.J Biol Chem. 1996; 271: 23338-23344Crossref PubMed Scopus (81) Google Scholar;Jisaka et al., 1997Jisaka M. Kim R.B. Boeglin W.E. Nanney L.B. Brash A.R. Molecular cloning and functional expression of a phorbol ester-inducible 8S-lipoxygenase from mouse skin.J Biol Chem. 1997; 272: 24410-24416Crossref PubMed Scopus (91) Google Scholar). Evidence suggests distinct cell-specific expression patterns for the different lipoxygenases within the epidermis. Whereas the well-delineated chemical reaction for each of these enzymes is the oxygenation of a specific carbon of arachidonic acid, the biologic implications of this function are not well understood (Funk, 1996Funk C.D. The molecular biology of lipoxygenases and the quest for eicosanoid functions using lipoxygenase-deficient mice.Biochim Biophys Acta. 1996; 1304: 65-84Crossref PubMed Scopus (230) Google Scholar). Functional assays, immunoprecipitation, and reverse transcriptase–polymerase chain reaction have localized the platelet-type 12-lipoxygenase (P-12LO) to human epidermis (Takahashi et al., 1993Takahashi Y. Reddy G.R. Ueda N. Yamamoto S. Arase S. Arachidonate 12-lipoxygenase of platelet-type in human epidermal cells.J Biol Chem. 1993; 268: 16443-16448Abstract Full Text PDF PubMed Google Scholar) whereas immunohistochemistry has indicated expression specifically in keratinocytes of normal skin, with an increased expression in keratinocytes of psoriatic skin (Hussain et al., 1994Hussain H. Shornick L.P. Shannon V.R. Wilson J.D. Funk C.D. Pentland A.P. Holtzman M.J. Epidermis contains platelet-type 12-lipoxygenase that is overexpressed in germinal layer keratinocytes in psoriasis.Am J Physiol. 1994; 266: C243-C253PubMed Google Scholar). Many investigators have speculated on the enzyme's role in normal and diseased skin, but the lack of specific inhibitors has hampered progress in this area. In order to circumvent this limitation, we have studied mice with a disruption in the P-12LO gene. The resultant mice completely lack P-12LO in skin as demonstrated by western blot analysis. Comparisons between these mice and wild-type controls have demonstrated that a lack of P-12LO produces a phenotype that results in increased basal transepidermal water loss. Epidermis from 3 to 6 d old mice was removed from the skin by incubation in a 50°C water bath for 30 s followed by an ice bath for 30 s (Murray et al., 1980Murray A.W. Solaki V. Froscio M. Rogers A. Effects of cholera toxin on ornithine decarboxylase activity in mouse skin.J Invest Dermatol. 1980; 75: 508-511Abstract Full Text PDF PubMed Scopus (14) Google Scholar). Epidermis was peeled from the rest of the skin and homogenized in a 50 mM KH2PO4 buffer using a Dounce homogenizer. The homogenate was sonicated three times for 10 s each while on ice. The samples were centrifuged at 10,000 × g for 15 min at 4°C. The supernatant protein was quantitated by Bradford assay and used for western blot analysis. Protein (5 μg) from mouse epidermis was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, blotted on to nitrocellulose, and probed with an anti-6-histidine tagged-human P-12LO antibody (Chen et al., 1994aChen X.-S. Sheller J.B. Johnson E.N. Funk C.D. Role of leukotrienes revealed by targeted disruption of the 5-lipoxygenase gene.Nature. 1994; 372: 179-182Crossref PubMed Scopus (340) Google Scholar). Detection was performed by enhanced chemiluminescence (Amersham, Bucks, UK). Tails and dorsal skin from both P-12LO–/– and P-12LO+/+ mice (6–10 wk old) were embedded in OCT and used in cryostat sectioning. Sections were fixed in cold acetone prior to incubation with a polyclonal antibody to a peptide sequence of P-12LO (KDDLPPNMRFHEEKRL; amino acids 157–172). The peptide was conjugated to 8-MAP prior to injection into rabbits (Research Genetics, Huntsville, AB). Antibody was purified from sera using the ImmunoPure A/G kit according to the manufacturer's instructions (Pierce, Rockford, IL). Specific staining in the wild-type sections with no staining of the P-12LO-deficient sections at a dilution of 1:5000, was observed. Sections were incubated with biotinylated secondary anti-sera and peroxidase-labeled tertiary anti-sera supplied with an ABC kit (Vector, Burlingame, CA) followed by visualization of immunoprecipitate with AEC chromogen (Biogenex, San Ramon, CA). Samples were counterstained in hematoxylin for 30 s prior to dehydration and mounting. Basal transepidermal water loss (TEWL) was assessed on dorsal skin of P-12LO–/– and P-12LO+/+ mice (6–10 wk old) using an evaporimeter (Servomed, Vällingby, Sweden). The temperature ranged from 21°C to 22°C and the relative humidity was maintained at 54%–55%. Measurements were recorded when TEWL readings were stabilized at approximately 45 s after the probe was placed on the skin. Data are shown as mean ± standard error. An unpaired Student's t-test was used to determine statistical significance. Permeability barrier recovery was evaluated as a function of the return of TEWL to basal levels. Acetone-soaked cotton balls were applied by gentle rolling to mouse skin for 2 min. This treatment resulted in barrier disruption manifested as increased TEWL measured at time zero as compared with basal levels measured previously. TEWL measurements were taken at time 2, 6, and 24 h after acetone treatment. The range of absolute TEWL values differed markedly upon acetone treatment; therefore, barrier recovery is expressed as a percentage with 100% recovery equal to basal TEWL. Mitotic activity was assayed after i.p. injection of 50 mg Brdu and 10 mg 5-fluoro-2′-deoxy (Sigma) uridine per kg of mouse body weight were injected intraperitoneally. Three hours later, 48 h after barrier disruption or no treatment, mice were killed and skin samples removed and fixed for paraffin embedding and staining. Paraffin sections were pretreated with 2 M HCl for 20 min followed by a 3 min trypsin digestion and overnight incubation with rat anti-Brdu (Accurate Chem & Scientific, Westbury, NY) at 4°C. Sections were incubated with biotinylated secondary anti-sera and peroxidase-labeled tertiary anti-sera supplied with an ABC kit (Vector) followed by visualization of immunoprecipitate with AEC chromogen (Biogenex, San Ramon, CA). For inflammation studies, arachidonic acid (2 mg in 20 μl) or vehicle (acetone) was applied topically to the inner surface of the left and right ear, respectively, of P-12LO-deficient and wild-type mice (6–10 wk old). After 1 h, punch biopsies (6 mm in diameter) were removed, fixed in paraformaldehyde, embedded in paraffin, and sectioned. The thickness of sections stained with hematoxylin was measured using an ocular ruler. Dorsal and tail skin from wild-type and P-12LO–/– mice were fixed for 24 h in 4% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 in accordance with our previous published methods (Morita et al., 1995Morita K. Hogan M.E. Nanney L.B. King L.K. Manabe M. Sun T.-T. Sundberg J.P. Cutaneous ultrastructural features of the Flaky Skin (fsn) mouse mutation.J Dermatol. 1995; 22: 385-395Crossref PubMed Scopus (24) Google Scholar). Samples were postfixed in 1% osmium tetroxide and stained en bloc with 2% uranyl acetate and dehydrated further in graded alcohols. Samples were embedded in Spurr resin and semithin sections were trimmed. Ultrathin sections were sectioned with a diamond knife, collected, and stained with uranyl acetate and lead citrate. Sections from both types of mice and both regions were compared and photographed with a Hitachi 800 electron microscope. Dorsal skin from shaved mice was placed epidermal side up on a block of dry ice. Epidermis was scraped with a scalpel, weighed, homogenized, and Folch extracted. Deuterated standards for palmitic, stearic and arachidonic acids were added just before extraction. Fatty acids were transesterified in acidic methanol and extracted into hexane. Samples were analyzed by gas chromatography–mass spectrometry in the electron impact mode. Here, we confirm that P-12LO–/– mice lack P-12LO expression in epidermis, and not just in platelets (Johnson et al., 1998Johnson E.N. Brass L.F. Funk C.D. Increased platelet sensitivity to ADP in mice lacking platelet-type 12-lipoxygenase.Proc Natl Acad Sci USA. 1998; 95: 3100-3105Crossref PubMed Scopus (100) Google Scholar), as determined by western blot analysis (Figure 1). Thus, a 75 kDa band, detected by a polyclonal anti-serum raised against the whole human protein, was present in the skin samples obtained from wild-type, but not in the samples from mice homozygous for the disrupted P-12LO allele. This antibody was shown to react well with mouse P-12LO by western blot analysis (Chen et al., 1994bChen X.-S. Kurre U. Jenkins N.A. Copeland N.G. Funk C.D. cDNA cloning, expression, mutagenesis of C-terminal isoleucine, genomic structure, and chromosomal localizations of murine 12-lipoxygenases.J Biol Chem. 1994; 269: 13979-13987Abstract Full Text PDF PubMed Google Scholar), but has not functioned well in immunohistochemical studies in mouse skin sections. A new antibody raised to a peptide sequence found exclusively in mouse and human P-12LO, but absent in all other cloned mammalian lipoxygenases, revealed epidermal expression of P-12LO in dorsal mouse skin (Figure 2). Because each epidermal layer of dorsal mouse epidermis is only one cell thick under normal conditions, immunohistochemistry of thick skin of the tail was used to localize P-12LO to the stratum granulosum of wild-type animals, whereas P-12LO-deficient samples exhibited no staining (Figure 2). In addition, blood vessels of wild-type animals were stained with the anti-P-12LO antibody presumably due to the presence of platelets. Because of the extensive history of the role of fatty acids in skin as well as our finding of P-12LO expression in the stratum granulosum, the permeability barrier of wild-type and P-12LO-deficient mice was examined as a function of TEWL. Basal TEWL measurements revealed that P-12LO-deficient animals exhibit greater water loss through the skin than wild-type control animals (Figure 3). Water loss may be the signal that results in increased DNA synthesis associated with barrier repair (Grubauer et al., 1989Grubauer G. Elias P.M. Feingold K.R. Transepidermal water loss: the signal for recovery of barrier structure and function.J Lipid Res. 1989; 30: 323-333Abstract Full Text PDF PubMed Google Scholar). Because P-12LO-deficient mice lose more water than wild-type mice under basal conditions, we examined the basal mitotic activity of epidermal cells via Brdu staining (Table 1). These studies illustrated that, although P-12LO-deficient mice exhibit an increase in TEWL, the basal mitotic activity is not altered, as there are comparable numbers of Brdu-stained cells.Table 1Mitotic activity of P-12LO-deficient and wild-type dorsal epidermis and tail skinaP-12LO-deficient and wild-type mice were treated with BrdU prior to skin sample isolation, paraffin embedding and sectioning. Values represent number of cells with stained nuclei in a 1648 μm2 field (n = 4).Wild-typeP-12LO deficientDorsal skin53.5 ± 6.041.0 ± 11.5Tail44.0 ± 8.028.0 ± 7.0a P-12LO-deficient and wild-type mice were treated with BrdU prior to skin sample isolation, paraffin embedding and sectioning. Values represent number of cells with stained nuclei in a 1648 μm2 field (n = 4). Open table in a new tab Disruption of the lipid barrier with acetone results in various signals of repair including the synthesis of lipid and DNA. To determine the requirement of P-12LO and its primary metabolite 12–HETE in barrier restoration, acetone was used to disrupt the lipid barrier of P-12LO-deficient and wild-type mice. Despite the differences observed in basal water loss, lack of P-12LO gene expression had no effect on barrier recovery as determined by the similar time course of return of transepidermal water loss to basal levels in P-12LO+/+ and P-12LO–/– mice (Figure 4). Because many eicosanoids [prostaglandins, leukotrienes, mono-hydroxylated derivatives of arachidonic acid (HETE)] have been associated with inflammation, we tested a standard inflammation assay in both P-12LO+/+ and P-12LO–/– mice (Opas et al., 1985Opas E.E. Bonney R.J. Humes J.L. Prostaglandin and leukotriene synthesis in mouse ears inflamed by arachidonic acid.J Invest Dermatol. 1985; 84: 253-256Abstract Full Text PDF PubMed Scopus (122) Google Scholar). Comparison of basal ear thickness as well as thickness changes evoked by arachidonic acid (measure of plasma leakage or edema) indicated no abnormality associated with P-12LO deficiency (Figure 5). Sections of dorsal skin from P-12LO-deficient and wild-type mice were analyzed for any distinction in ultrastructural properties or fatty acid content. Ultrastructural examination failed to detect an overt abnormality in structure (data not shown). No detectable difference in number or appearance of lamellar bodies was apparent. There was no obvious difference in the epidermal content of the major C16, C18, and C20 fatty acids (palmitic, stearic, oleic, linoleic, and arachidonic acids) as a result of P-12LO deficiency as judged by gas chromatographic–mass spectrometric analysis (n = 3; data not shown). In these studies, we have shown that mice generated by targeted disruption of the P-12LO gene fail to express P-12LO protein in the epidermis by two separate immunochemical methods using two distinct polyclonal antibodies (Figure 1 and Figure 2). This evidence compliments the previous data which indicate that P-12LO in platelets and in skin are products derived from the same gene (Takahashi et al., 1993Takahashi Y. Reddy G.R. Ueda N. Yamamoto S. Arase S. Arachidonate 12-lipoxygenase of platelet-type in human epidermal cells.J Biol Chem. 1993; 268: 16443-16448Abstract Full Text PDF PubMed Google Scholar;Chen et al., 1994bChen X.-S. Kurre U. Jenkins N.A. Copeland N.G. Funk C.D. cDNA cloning, expression, mutagenesis of C-terminal isoleucine, genomic structure, and chromosomal localizations of murine 12-lipoxygenases.J Biol Chem. 1994; 269: 13979-13987Abstract Full Text PDF PubMed Google Scholar). In contrast, immunohistochemical analysis of normal wild-type dorsal skin has localized P-12LO to epidermis; in particular, the thickened epidermis from the tail revealed expression in the stratum granulosum. Whereas previous reports have indicated basal keratinocyte expression in normal human skin (Hussain et al., 1994Hussain H. Shornick L.P. Shannon V.R. Wilson J.D. Funk C.D. Pentland A.P. Holtzman M.J. Epidermis contains platelet-type 12-lipoxygenase that is overexpressed in germinal layer keratinocytes in psoriasis.Am J Physiol. 1994; 266: C243-C253PubMed Google Scholar), this is new evidence to suggest an alternate P-12LO expression pattern. This discrepancy could be due to minimal accumulation of P-12LO protein below detection limits for the immunohistochemical analysis as well as variation between species. Localization of P-12LO to the granulosum rather than basal keratinocytes shifts functional implications away from cellular proliferation and migration to the water permeability barrier. The considerable body of literature examining fatty acids and the lipid barrier provided further incentive to examine the water barrier of P-12LO-deficient epidermis. Under basal conditions, P-12LO-deficient mice lose more water than control animals (Figure 3), indicating that the oxygenation of arachidonic acid to 12-H(P)ETE could aid in the function of the intact water barrier. The lack of increased basal mitotic activity in the epidermis of P-12LO–/– mice suggests that the role of P-12LO appears to be distinct from the proliferative signal of water loss in barrier function (Table 1). The normal recovery of the P-12LO–/– epidermal barrier after acetone disruption also suggests that P-12LO is not involved in cellular proliferation (Figure 4). These studies may indicate a possible separation of barrier repair, a process independent of P-12LO, with normal barrier function, in which P-12LO plays a part. Whereas water loss itself has been implicated as the signal which stimulates barrier repair processes (Grubauer et al., 1989Grubauer G. Elias P.M. Feingold K.R. Transepidermal water loss: the signal for recovery of barrier structure and function.J Lipid Res. 1989; 30: 323-333Abstract Full Text PDF PubMed Google Scholar), and P-12LO-deficient mice exhibit increased water loss without increased mitotic activity, the magnitude of increased water loss associated with P-12LO deficiency may not be sufficient to elicit cellular proliferation. It remains unclear whether 12-H(P)ETE acts as a signaling molecule in skin functions or simply as a lipid incorporated into the membrane. Nonetheless, these studies are the first to link P-12LO to a normal epidermal function in vivo. One possible explanation of the increased transepidermal water loss in P-12LO-deficient mice is that these mice suffer from a defective inflammatory state. A shunting of arachidonic acid to either the cyclooxygenase or the 5-lipoxygenase pathways may result in enhanced inflammation as both of these enzymes have been implicated in the inflammatory process (Chen et al., 1994aChen X.-S. Sheller J.B. Johnson E.N. Funk C.D. Role of leukotrienes revealed by targeted disruption of the 5-lipoxygenase gene.Nature. 1994; 372: 179-182Crossref PubMed Scopus (340) Google Scholar;Langenbach et al., 1995Langenbach R. Morham S. Tiano H. et al.Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and and indomethacin-induced gastic ulceration.Cell. 1995; 83: 483-492Abstract Full Text PDF PubMed Scopus (1002) Google Scholar). In order to address this issue, the inflammatory response of P-12LO-deficient and wild-type mice was assessed. Infiltration of neutrophils was not evident under unchallenged conditions nor was there any alteration in the edematous response upon arachidonic acid challenge in P-12LO-deficient mice (Figure 5). This finding would tend to rule out a generalized defect in inflammation causing the enhanced water loss phenotype. Examination of P-12LO-deficient skin sections using electron microscopy has indicated that P-12LO–/– epidermis exhibits no overt abnormality in structure. No detectable difference in number or appearance of lamellar bodies was apparent. This finding is not so surprising as the phenotype observed in these mice is subtle. It is entirely possible the P-12LO–/– mice have a biochemical alteration in their membrane coating granules that is not manifested as a morphologic change. Initial fatty acid analyses of P-12LO–/– epidermis have also not yielded clues to altered water loss in these mice. The content of major fatty acids was not different from wild-type mice. Future studies will need to focus on analysis of incorporation of 12-HETE or other P-12LO metabolites (epoxyhydroxy or trihydroxy derivatives from 12-HPETE) into specific membrane lipid classes with specific, high-sensitivity assays. Prior to the cloning and characterization of epidermal lipoxygenases,Nugteren and Kivits, 1987Nugteren D.H. Kivits G.A.A. Conversion of linoleic and arachidonic acid by skin epidermal lipoxygenases.Biochim Biophys Acta. 1987; 921: 135-141Crossref PubMed Scopus (55) Google Scholar, provided in vitro evidence for lipoxygenase metabolism in skin via two distinct lipoxygenases; a (n-9) lipoxygenase (equivalent to P-12LO here) that metabolizes arachidonic acid to 12-HETE and a (n-6) lipoxygenase that converts arachidonic acid to 15-HETE, linoleic acid to 13-hydroxy-octadecadienoic acid, and columbinic acid to 13-hydroxyoctadecatrienoic acid. They suggested that it was the latter lipoxygenase which serves a particular role in proper water barrier function through the oxygenation of linoleic acid contained in acyl ceramides but here we have some evidence for the former pathway. Interestingly, the EFA-deficient phenotype in mice has been characterized by various abnormalities including an increase in transepidermal water loss (Elias and Brown, 1978Elias P.M. Brown B.E. The mammalian cutaneous permeability barrier. Defective barrier function in essential fatty acid deficiency correlated with abnormal intercellular lipid deposition.Lab Invest. 1978; 39: 574-583PubMed Google Scholar). Some investigators have suggested that the rescue of the EFA-deficient phenotype by topical application of linoleic acid is due to the metabolism of linoleic acid by a lipoxygenase, not unlike the activity suggested byNugteren and Kivits, 1987Nugteren D.H. Kivits G.A.A. Conversion of linoleic and arachidonic acid by skin epidermal lipoxygenases.Biochim Biophys Acta. 1987; 921: 135-141Crossref PubMed Scopus (55) Google Scholar. Perhaps the increased TEWL of the EFA-deficient phenotype is due to a lack of arachidonic acid oxygenation to 12-H(P)ETE by P-12LO and to contribution by other lipoxygenases via direct linoleic acid oxygenation. At least four lipoxygenases are present in skin and a novel human 12(R)-lipoxygenase is capable of oxygenating C18 fatty acids (Sun et al., 1998Sun D. McDonnell M. Chen X.-S. et al.Human 12 (R) -lipoxygenase and the mouse ortholog. Molecular cloning, expression, and gene chromosomal assignment.J Biol Chem. 1998; 273: 33540-33547Crossref PubMed Scopus (63) Google Scholar). In conclusion, we have documented that the P-12LO pathway plays a part in maintaining the epidermal water barrier. Although under basal conditions the P-12LO-deficient phenotype is rather mild, perhaps under compromised conditions the lack of P-12LO would prove more severe. Whereas the molecular mechanism for the role of the P-12LO pathway is unclear at present, these data suggest that increased expression of P-12LO in disease states associated with heightened water loss such as psoriasis (Hussain et al., 1994Hussain H. Shornick L.P. Shannon V.R. Wilson J.D. Funk C.D. Pentland A.P. Holtzman M.J. Epidermis contains platelet-type 12-lipoxygenase that is overexpressed in germinal layer keratinocytes in psoriasis.Am J Physiol. 1994; 266: C243-C253PubMed Google Scholar) may be an epidermal coping mechanism designed to improve barrier function. We wish to express our gratitude to Dr. T. Stoudemeyer and Mr. I. Sadiq of S.K.I.N. Incorporated (Conshohocken, PA) for assistance with the water loss measurements. This work was supported in part by NIH grant HL53558 (C.D.F.) and by the Skin Disease Research Center (SDRC) grant 5P30 AR41943 from the NIH/NIAMS and GM40439 (L.B.N.) and by a Training Grant in Pharmacological Sciences GM07628 (E.N.J.).