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Cell Growth & Differentiation Vol. 11, 447-454, August 2000
© 2000 American Association for Cancer Research

8S-Lipoxygenase Products Activate Peroxisome Proliferator-activated Receptor {alpha} and Induce Differentiation in Murine Keratinocytes1

Stephanie J. Muga2, Philippe Thuillier2, Amy Pavone, Joyce E. Rundhaug, William E. Boeglin, Mitsuo Jisaka, Alan R. Brash and Susan M. Fischer3

University of Texas, M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957 [S. J. M., P. T., A. P., J. E. R., S. M. F.]; Division of Clinical Pharmacology, Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6602 [W. E. B., M. J., A. R. B.]


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
To determine the function and mechanism of action of the 8S-lipoxygenase (8-LOX) product of arachidonic acid, 8S-hydroxyeicosatetraenoic acid (8S-HETE), which is normally synthesized only after irritation of the epidermis, transgenic mice with 8-LOX targeted to keratinocytes through the use of a loricrin promoter were generated. Histological analyses showed that the skin, tongue, and stomach of transgenic mice are highly differentiated, and immunoblotting and immunohistochemistries of skin showed higher levels of keratin-1 expression compared with wild-type mice. The labeling index, however, of the transgenic epidermis was twice that of the wild-type epidermis. Furthermore, 8S-HETE treatment of wild-type primary keratinocytes induced keratin-1 expression. Peroxisome proliferator activated receptor {alpha} (PPAR{alpha}) was identified as a crucial component of keratin-1 induction through transient transfection with expression vectors for PPAR{alpha}, PPAR{gamma}, and a dominant-negative PPAR, as well as through the use of known PPAR agonists. From these studies, it is concluded that 8S-HETE plays an important role in keratinocyte differentiation and that at least some of its effects are mediated by PPAR{alpha}.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
8-LOX,4 one of several LOXs expressed in mouse epidermis, produces 8S-HETE from the metabolism of arachidonic acid and, to a much lesser extent, 9S-HODE from the metabolism of linoleic acid. Although 8-LOX enzymatic activity was found a decade ago (1) , the gene was only recently cloned, which has now permitted further characterization of its function (2) . Although 8-LOX expression is nearly undetectable in normal adult epidermis of most strains of mice, we and others have shown that 8-LOX mRNA and protein are highly inducible after topical application of the phorbol ester TPA (2-4) . This increase in expression is an early and sustained event such that high expression is observed by 4 h and persists for days (3) . It was suggested that 8-LOX may have a pathophysiological function, based on an observed correlation among strains of mice with differing sensitivities to TPA as an inflammatory and hyperplastic agent and the extent to which 8-LOX can be induced (4) . 8-LOX is also constitutively up-regulated in skin tumors (3) . Whether 8-LOX products play a role in the proliferative or differentiative aspects of the epidermis has been unclear. This enzyme is found in the upper or suprabasal, differentiated layer of the epidermis, the stratum granulosum, which suggests that it is associated with differentiation (2) . The mechanism or signaling pathways that are activated by 8-LOX metabolites have also not been determined. Whereas receptors have been reported for some of the other HETEs [e.g., 12S-HETE binds to and activates protein kinase C (5) ] many eicosanoids, including 8S-HETE and 9S-HODE, can activate PPARs (6-8) . These receptors belong to the steroid hormone superfamily of transcription factors and mediate changes in the expression of numerous genes associated with differentiation and fatty acid metabolism in several tissues (6-8) . The involvement of PPARs in differentiation processes and the localization of 8-LOX in the differentiated layers of the epidermis suggest that the two may be mechanistically linked. The 8S-HETE product of 8-LOX may act in either an autocrine or paracrine fashion, because it is readily released from keratinocytes. Thus, it is not known whether the target or responsive cells are the basal keratinocytes, which are undifferentiated and sit on the basement membrane, or the suprabasal keratinocytes, which are either in the process of differentiating or are fully differentiated.

To more completely determine the function and mechanism of action of 8-LOX and its 8S-HETE product in normal and neoplastic murine epidermis, targeted transgenic mice overexpressing the 8-LOX gene, under the control of a loricrin promoter, were generated. This keratinocyte-specific promoter was chosen because it is not expressed until shortly after birth, thus diminishing the possibility of embryonic lethality (9) . We report here that these mice have a hyperkeratotic epidermis and that 8S-HETE induces differentiation, as denoted by increased keratin-1 expression, through a mechanism involving PPAR{alpha}.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Generation of 8-LOX Transgenic Mice.
8-LOX was targeted to the epidermis of transgenic mice using a loricrin expression vector containing the loricrin promoter and polyadenylation site (9) . The expression construct containing the 8-LOX cDNA and the tyrosinase minigene which confers a dark coat color (10) were coinjected into the pronuclei of donor FVB(+/-) embryos. Use of the tyrosinase gene facilitated visual identification of transgenic animals. Southern analysis (Fig. 1ACitation ) shows the presence of the 8-LOX transgene (4.7 kb) in the transgenic mice. Four founders expressing various levels of the transgene were identified by Southern analysis and PCR. One mouse exhibited a high level of expression of the loricrin 8-LOX transgene, a second showed low expression, and the other two animals exhibited intermediate levels of transgene expression. The highest expressing mouse was used to generate the transgenic line described in these studies. The level of expression of 8-LOX in the transgenic mice was comparable with that occurring after phorbol-ester treatment of skin and in skin tumors (data not shown).



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  		Fig. 1. Transgene integration, expression and activity. A, Southern analysis of wild-type and transgenic mice. Genomic DNA was isolated from the tails of weanlings, digested with BamHI, fractionated on a 0.7% agarose/Tris-acetate/ethidium bromide gel, and hybridized with a radiolabeled 8-LOX cDNA probe. Lane 1 contains digested genomic DNA from wild-type mice and Lanes 2–7 contain digested genomic DNA from loricrin 8-LOX transgenic mice. The loricrin 8-LOX transgene has a length of 4.7 kb. B, Northern analysis of skin from wild-type and transgenic mice. Total RNA was extracted from the dorsal skin of wild-type and transgenic mice as described in "Materials and Methods." Ten µg of RNA was electrophoresed on a 1% agarose formaldehyde gel, blotted overnight onto a nylon membrane, and hybridized with a radiolabeled 8-LOX cDNA probe; stripped blots were probed with cDNA for GAPDH, as a control for gel loading. C, immunostaining of protein with 8-LOX antibody. Total protein was isolated from wild-type and loricrin 8-LOX transgenic skin as described under "Materials and Methods." Fifteen µg of total protein was fractionated on a 10% SDS polyacrylamide gel. The protein was transferred overnight to a polyvinylidene difluoride membrane and immunostained with the 8-LOX antibody as described previously (2) . Wild-type mice: M590, M591, M299; transgenic mice M351, M528, M179. Positive [Lox Std, 15-Lox-2 (protein)] and negative [12R Lox (protein)] controls are included for verification of antibody specificity (2) .

 
8-LOX Transgene Expression and Activity.
Northern analysis (Fig. 1BCitation ) shows a marked increase in expression of 8-LOX mRNA (3.2 kb) in transgenic mice as compared with the basal level seen in the wild-type mice. Protein from wild-type and transgenic mice was isolated and analyzed to assure that 8-LOX transgene message was translated. As shown in Fig. 1CCitation , 8-LOX protein is expressed in transgenic but not in wild-type epidermis. Specificity of the antibody was demonstrated through the use of negative and positive controls (2) . The enzymatic activity of 8-LOX in the epidermis of wild-type and transgenic mice was also analyzed to assess the functionality of the expressed transgene. Transgenic epidermal preparations produced a 42- to 180-fold increase in the level of 8S-HETE production, compared with wild-type preparations (Fig. 2Citation ). Rechromatography of the 8S-HETE fraction from normal phase HPLC on a chiral column showed that it was almost exclusively the S enantiomer (data not shown), as expected of an 8-LOX (11) .



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  		Fig. 2. Production of 8S-HETE by wild-type (A) and transgenic (B) keratinocytes. Epidermal homogenates were incubated with 100 µM [1-14C]arachidonic acid, and the metabolic products were separated by normal and reverse-phase HPLC as described in the text.

 
Histological Evaluation.
Visually there were no obvious differences in the appearance of the skin of transgenic and wild-type mice. Histological evaluation of the skin, however, revealed a thinned and stretched epidermis punctuated by a high degree of differentiation and keratinization (Fig. 3BCitation ) as compared with wild-type skin (Fig. 3ACitation ). Extensive keratinization was also observed in the tongue and forestomach of transgenic mice (data not shown). To determine whether this was associated with reduced cell proliferation, the labeling index in the interfollicular epidermis and in the follicles was measured (Fig. 4Citation ). The observed 2- to 3-fold increase in the labeling index of transgenic skin, as compared with wild-type skin, suggests that, although the transgenic epidermis is marked by hyperkeratosis, it also exhibits an increase in proliferation, perhaps to compensate for the rapid migration of epidermal keratinocytes from the basement membrane and the loss to terminal differentiation.



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  		Fig. 3. Histological analysis of wild-type and transgenic skin. Dorsal skin from wild-type (A) and transgenic mice (B) was removed, fixed in 4% paraformaldehyde, and processed for paraffin embedding. Tissue sections were stained with H&E and photographed at x400.

 


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  		Fig. 4. Labeling index of wild-type (WT) and transgenic (Tg) epidermis. Mice were given i.p. injections of BrdUrd 30 min prior to being killed, and skins were removed and processed for paraffin embedding, as described under "Materials and Methods." Immunohistochemistry was used to visualize BrdUrd-labeled cells. Labeling index was measured as the percentage of positive basal cells in the intrafollicular epidermis and in the hair follicles. Bars, mean ± SE.

 
Keratin-1 Induction by 8S-HETE.
Because the histological data revealed an increase in differentiation in transgenic epidermis, we also examined the protein levels of keratin-1, a marker for differentiation (12) . Immunostaining of a protein blot (Fig. 5ACitation ) shows that keratin-1 is produced to a greater extent in the transgenic epidermis than in wild-type epidermis. This suggests that expression of 8-LOX may contribute to keratinocyte differentiation through an increase in keratin-1. The double keratin-1 band seen in the transgenic sample is likely attributable to alterations in the state of phosphorylation (13) .



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  		Fig. 5. Keratin-1 expression. A, immunostaining of epidermal protein for keratin-1. Total protein was isolated from wild-type and loricrin 8-LOX transgenic skin as described under "Materials and Methods." Fifteen µg of total protein was fractionated on a 10% SDS polyacrylamide gel, blotted on a membrane, and immunostained with an antibody against keratin-1. The keratin-1 protein has a molecular mass of 65–69 kDa. B, immunostaining of wild-type and transgenic skin for keratin-1. Sections of skin from wild-type and transgenic mice were removed and processed for paraffin embedding. Keratin-1 expression was analyzed by immunostaining as described in the "Materials and Methods" section using hematoxylin as a counterstain. Sections were photographed at x400. Upper panel, wild-type skin; single arrow, a basal cell lacking keratin-1 staining. Lower panel, transgenic skin, double arrow, basal cell staining positively for keratin-1; single arrow, a basal cell lacking keratin-1 staining. C, 8S-HETE induction of keratin-1 mRNA in cultured wild-type keratinocytes. Primary cultures of keratinocytes grown in low-Ca2+ (0.05 mM) were treated with 1 µM of 8S-HETE for 24 h. Total RNA was isolated using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, Ohio), fractionated (5 µg) on a 1% agarose formaldehyde gel, and transferred to a nylon membrane. The immobilized RNA was hybridized with a keratin-1 cDNA probe. Keratin-1 mRNA has a length of 2.4 kb. Hybridization with GAPDH was used as a control for loading.

 
To assure that the differences in keratin-1 seen in Western blots was not attributable to an increase in the amount of stratum corneum, which is enriched in keratins, immunohistochemical staining of sections of skin of wild-type and transgenic animals was carried out. As shown in Fig. 5BCitation , keratin-1 staining in wild-type epidermis is primarily suprabasal, whereas staining of transgenic epidermis is not only more intense, it also occurs in many of the basal cells as well as in the suprabasal layers. Staining with antibodies against keratin-5 or keratin-14, which are expressed in basal cells, was the same in wild-type and transgenic mice with regard to both location and intensity (data not shown).

To demonstrate a more direct association between 8S-HETE and keratin-1 expression, primary cultures of keratinocytes were grown in a low-calcium media that normally precludes keratin-1 expression (12) and were treated with 1 µM 8S-HETE. Northern analysis (Fig. 5CCitation ) revealed a dramatic increase in the abundance of keratin-1 transcripts in the treated cultures, which indicated a regulatory relationship between 8S-HETE and the expression of a differentiation-related gene.

Role of PPAR{alpha} in Keratin-1 Induction.
On the basis of reports that PPAR{alpha} and PPAR{gamma} were induced on calcium-induced differentiation of human keratinocytes (1415) , we tested the possibility that the effects of 8S-HETE on differentiation occurs through a PPAR{alpha} mechanism. First, it was necessary to demonstrate that keratinocytes have functional PPARs. Wild-type keratinocytes were transfected with the PPAR response element reporter vector PPRE-Luc and treated exogenously with 8S-HETE or cotransfected with a dominant negative PPAR and treated with 8-HETE. As shown in Fig. 6ACitation , 8S-HETE caused an increase in reporter activity in a dose-responsive manner. Transfection with increasing amounts of the dominant negative construct reduced the ability of 8S-HETE to activate to below control levels. Although Western blots (data not shown) had indicated that keratinocytes contain all three PPAR isoforms, the data shown here indicate that one or more of these are active and respond directly or indirectly to 8S-HETE. To show specifically that PPAR{alpha} mediates 8S-HETE induction of keratin-1, keratinocytes were transiently transfected with expression vectors for PPAR{alpha}, PPAR{gamma}, and/or a dominant negative PPAR. As shown in Fig. 6BCitation , Lane 3, transfection with the PPAR{alpha} expression vector alone induced expression of keratin-1. This is likely to be attributable to endogenous PPAR activators because keratinocytes are known to produce high levels of both prostaglandins and LOX products. The ability of the dominant negative PPAR construct, which was reported to affect all of the three PPAR isoforms (16) , to prevent PPAR{alpha} from up-regulating keratin-1 expression (Lane 5) even in the in the presence of its high-affinity ligand, 8S-HETE (Lane 6) suggests that PPAR{alpha} can directly or indirectly regulate keratin-1 expression. This apparent involvement of PPAR{alpha} does not rule out a role for the other PPAR isoforms, particularly PPAR{gamma}, although 8S-HETE has been reported to preferentially activate PPAR{alpha} (6-8) .



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  		Fig. 6. Activation of PPAR{alpha} and its role in 8-HETE induction of keratin-1. A, activation of endogenous PPARs by 8S-HETE. Primary keratinocytes from wild-type or transgenic neonates were transfected with a luciferase reporter vector PPRE-Luc and treated with 1–10 µM 8S-HETE, or were transfected with increasing amounts of the dominant negative PPAR (dnPPAR) and treated with 10 µM 8S-HETE. All of the cultures were cotransfected with pCMV-ß-gal the activity of which was used to normalize promoter activity. B, effect of transient expression of PPAR{alpha} and a dominant negative PPAR on 8S-HETE induction of keratin-1. Total RNA was isolated from primary keratinocytes grown in low Ca2+ and transfected with or without 1-µg PPAR{alpha} expression vector in the presence or absence of 1 µg of dnPPAR, with or without addition of 1 µM 8S-HETE. Five µg of RNA was fractionated on an agarose gel and transferred to a nylon membrane. The immobilized RNA was hybridized with a keratin-1 cDNA probe (mRNA length, 2.4 kb). Equivalent gel loading was assessed by visualizing the 28S and 18S RNA.

 
A pharmacological approach was also used to further establish a role for PPAR{alpha} in the induction of keratin-1. Primary cultures of keratinocytes were grown in low-calcium (0.05 mM) medium, which does not promote the expression of keratin-1 (Fig. 7Citation , Lane 1). Differentiation can be induced by raising the calcium level to 0.12 mM (Lane 7) or treating with 8S-HETE, as shown in Lane 2 and in Fig. 5CCitation . Several unrelated natural and synthetic compounds previously documented as PPAR{alpha} agonists were also tested. The doses used were based on the EC50 values reported previously (617) . CLA is a naturally occurring fatty acid with a high degree of selectivity toward PPAR{alpha} (18) . As shown in Fig. 7Citation , Lane 4, 0.1 µM CLA caused a strong induction of keratin-1. Two members of the fibrate class of drugs, clofibrate and cibrofibrate, have been reported to be PPAR{alpha} activators (17) . ETYA is also a PPAR{alpha} activator (6) but for unknown reasons failed to activate keratin-1 to the same extent as the other agents. The observation that a variety of PPAR{alpha} agonists induce keratin-1 further suggests that this nuclear receptor plays a role in keratinocyte differentiation.



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  		Fig. 7. Induction of keratin-1 by several PPAR{alpha} agonists in murine keratinocytes. Primary cultures of keratinocytes were grown in low Ca2+ (0.05 mM) and were treated with either Wy14,643 (5 µM), CLA (0.1 µM), clofibrate (30 µM), ciprofibrate (30 µM), 8S-HETE (1 µM), or ETYA (1 µM) for 24 h. Total RNA was isolated using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, Ohio), fractionated (5 µg) on a 1% agarose formaldehyde gel, and transferred to a nylon membrane. The immobilized RNA was hybridized with a keratin-1 cDNA probe. Hybridization with GAPDH was used as a control for loading.

 
Activation of PPARs by Endogenous 8S-HETE.
To determine whether keratinocytes from transgenic mice produce sufficient endogenous 8S-HETE to activate endogenous PPARs, cultures from transgenic and wild-type mice were transfected with a PPAR response element reporter vector containing the luciferase gene and designated PPRE-Luc. As shown in Fig. 8Citation , the reporter activity in the transgenic cells was considerably higher than in the wild-type cells. When the cultures were cotransfected with the dominant negative PPAR construct, activity was reduced in both of the cell types. These data strongly suggest that 8S-HETE is an endogenous PPAR activator in keratinocytes.



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  		Fig. 8. PPAR response element reporter activity in wild-type (W) and transgenic (Tg) keratinocytes. Primary keratinocytes from wild-type or transgenic neonates were transfected with 1 µg of a luciferase reporter vector containing the PPAR response element, with or without cotransfection with 1 µg of dominant negative PPAR (dnPPAR) expression plasmid and with pCMV-ß-gal as a control for transfection efficiency. Promoter activity was normalized by ß-galactosidase activity. The data are representative of three independent experiments. Bars, means of triplicates ± SD.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Transgenic mice overexpressing 8-LOX under the direction of the loricrin promoter were generated to determine what role(s) this enzyme and its metabolites play in keratinocyte growth and differentiation. Several founder mice were obtained expressing various levels of the 8-LOX transgene. Expression of 8-LOX under the loricrin promoter produced a phenotype characterized by a thinning of the epidermis and marked by a high degree of differentiation and keratinization. A 2-fold increase in the labeling index in follicles and the interfollicular epidermis indicate a concomitant increase in proliferation, although this did not result in hyperplasia. The presence of keratin-1 in many but not all of the basal cells of the 8-LOX transgenic epidermis suggests that 8S-HETE induces keratin-1 only in those cells committed to terminal differentiation. It is likely that the proliferation observed in the basal cell population is driven by the accelerated differentiation of the suprabasal cells. This response may be a secondary, but important, effect of 8S-HETE stimulation of differentiation. This is reminiscent of the effect of TPA on the epidermis by which it causes the maturing amplifying cells to be displaced to the suprabasal layer and causes proliferation of the central proliferative unit basal cells (19) . An additional phenotype that was noted was the occurrence of sporadic, but mostly transient, dermatitis denoted by a rough inflamed appearance, which ranged from mild to severe. This observation is consistent with published reports of psoriatic skin exhibiting high levels of 8S-HETE (20) .

Recently several eicosanoids, including 8S-HETE, were shown to bind and activate PPARs (6-8) . Similar to other members of the nuclear steroid receptor family, which includes vitamin D3, retinoid, and thyroid hormone receptors, PPARs are ligand-activated transcription factors. An obligatory dimerization with the retinoid-X-receptor-{alpha} (RXR{alpha}) is also required for transcriptional activation (6) . PPARs control the expression of a large number of genes including those involved in adipocyte differentiation and fat metabolism in the liver (8) . Several PPAR isoforms exist, including PPAR{alpha}, PPAR{delta} (or PPARß; also called NUC-1 or FARR), and PPAR{gamma}2. Relative expression of each form is generally tissue-specific such that PPAR{alpha} is the predominant form in the liver, whereas PPAR{gamma}2 is the most prevalent form in adipocytes, spleen, and epithelial tissue (6-8) . We have recently shown that murine keratinocytes express all of the three isoforms, and that their levels of expression are elevated during carcinogenesis (data not shown). The involvement of PPARs in keratinocyte differentiation was previously suggested by studies in which the addition of the PPAR{alpha} ligands clofibrate or oleate to human keratinocytes that were cultured in low-calcium nondifferentiating media induced the expression of involucrin and transglutaminase, markers of late-stage differentiation (1415) .

In light of these observations, we hypothesized that 8S-HETE exerted its effects on keratin-1 expression through a mechanism involving PPARs. Several lines of evidence suggest that this was occurring: (a) treatment of keratinocyte cultures with 8S-HETE resulted in the induction of keratin-1; (b) the transfection of primary keratinocyte cultures with a PPAR{alpha} expression vector and subsequent treatment with 8S-HETE produced a dramatic increase in the mRNA levels of keratin-1; (c) this effect was inhibited by cotransfection with a dnPPAR construct; and (d) a similar trend was observed for other markers of fatty acid transport and metabolism including keratinocyte lipid-binding protein and acyl-CoA oxidase (Ref. 21 ; data not shown), which suggested that this was not a phenomenon unique to keratin-1. These in vitro observations are in agreement with the in vivo phenotype, in which high levels of 8-LOX expression produces a visible increase in differentiation and elevated keratin-1 expression in the epidermis. The observation that several unrelated agonists with high specificity toward PPAR{alpha} also induced keratin-1 provides strong supportive evidence that PPAR{alpha} can regulate keratinocyte differentiation and that this is not unique to 8S-HETE activation of PPAR{alpha}.

The final line of evidence that 8S-HETE is an endogenous activator of PPARs comes from experiments in which the activity from a PPAR response element was considerably higher in transgenic keratinocytes compared with wild-type keratinocytes in a manner that was inhibited by a dominant negative PPAR. This is the first demonstration of the activation of PPARs in vivo by endogenous factors.

The finding that 8S-HETE causes differentiation through a PPAR mechanism is intriguing for several reasons. This family of receptors have recently been implicated in colon and prostate tumor development, although the reports are in conflict with regard to whether PPAR activation inhibits or enhances tumor development (22-25) . Preliminary data (not shown) from an ongoing skin carcinogenesis study in the 8-LOX transgenic and wild-type mice indicates that elevated 8-LOX increases tumor incidence and accelerates the development of carcinomas. The mechanism(s) by which this occurs is currently unknown. Additional studies are needed to more clearly elucidate the functions of 8S-HETE and the extent to which its activation of PPARs contributes to tumor development or other pathologies. This is of particular importance to the human situation because the thiazolidinedione class of antidiabetic drugs are PPAR activators, as are some dietary fatty acids and their derivatives (6-8) .


   Materials and Methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Generation of Loricrin 8-LOX Transgenic Mice.
The vector (pML-B416) containing the loricrin promoter and poly(A)+ tail was a kind gift from Dennis Roop (Baylor College of Medicine, Houston, TX, Ref. 9 ). Because of incompatible restriction enzyme sites, the loricrin promoter and poly(A)+ region BamHI (Boehringer Mannheim) fragments were recloned into the pGEM11Z(+/-) vector (Promega) and subsequently renamed pGEM11 ML-B416. The 8-LOX cDNA was recloned into the pGEM3Z(+/-) vector (2) . The pGEM3Z.8S-LOX vector was digested and ligated into the pGEM-ML-B416 loricrin vector. The resulting plasmid pMLB416.8S-LOX was digested to create a 7.0-kb expression construct, which was isolated from a gel slice, purified by the Qiaex II (Qiagen) method, and subsequently used for injection into embryos. Donor embryos from superovulated female FVB(+/-) mice (National Cancer Institute, Research Facility, Frederick, MD) were isolated and injected with construct DNA and with DNA for the tyrosinase minigene, which confers a dark coat color (10) . Survivors were transferred to pseudopregnant FVB females. All of the mice were handled and housed in Association for Assessment and Accreditation of Laboratory Animal Care-accredited facilities and in accordance with USPHS and institutional guidelines.

Transgene Integration, Expression, and Activity.
Genomic DNA from wild-type and transgenic mice was screened for the integration of the transgene by Southern analysis. Genomic DNA isolated from the tails of weanlings (26) was digested with BamHI (Boehringer Mannheim), fractionated on a 0.7% agarose/TAE/ethidium bromide gel, and transferred to a nylon membrane. This digest generates a unique 4.7-kb DNA fragment that contains the loricrin promoter and the 8-LOX cDNA. A 32P-labeled 8-LOX cDNA probe was hybridized to the blot. The blot was washed twice for 15 min in 0.1% SDS/2x SSC at 42°C and once for 30 min with 0.1% SDS/0.1x SSC at 55°C and was exposed to X-ray film.

For transgene expression, frozen (-80°C) skins were pulverized in liquid nitrogen and solubilized in TriReagent (Molecular Research Center). Ten µg of total RNA was fractionated on a 1.1% agarose/6% formaldehyde/ethidium bromide-stained gel, blotted onto a nylon membrane, and hybridized with a radiolabeled 8-LOX cDNA probe. The washes were the same as used above. The blot was exposed to X-ray film, stripped, and reprobed with a radiolabeled probe homologous to GAPDH to verify equal loading of RNA. This cDNA was obtained by conventional reverse transcription-PCR methods using forward (GAPDH s756, 5'-ATG TGT CCG TCG TGG ATC TGA C-3') and reverse (GAPDH as1010, 5'-CCC TGT TGC TGT AGC CGT ATT C-3') oligonucleotides specific for GAPDH.

For protein expression, total protein was extracted, separated by SDS-PAGE, blotted onto a nitrocellulose membrane, and immunostained with an antibody specific for 8-LOX (2) . Proteins were detected by chemiluminescence using the Enhanced Chemiluminescence Western blotting kit (Amersham Life Sciences, Inc.). Wild-type mice were designated M590, M591, and M299 whereas transgenic mice are designated M351, M528, and M179. Positive (Lox standard, 15-LOX-2 protein) and negative (12R-LOX protein) controls were included for verification of antibody specificity (2) .

8-LOX activity was assessed using epidermal scrapings from transgenic and wild-type mice; the scrapings were homogenized and incubated with 100 µM [1-14C]arachidonic acid. The products were extracted with chloroform/methanol, and the extracts were analyzed by reverse phase and normal phase HPLC, as described previously to determine levels of 8S-HETE synthesis (2) .

Histological Analysis and Epidermal Cell Proliferation.
Dorsal skin from wild-type and transgenic mice was isolated, fixed in 4% paraformaldehyde, and processed for paraffin embedding. Tissue sections were stained with H&E and photographed at x400.

BrdUrd was injected i.p. (50 mg/kg body weight) into wild-type and transgenic mice 30 min before they were killed. Sections of dorsal skin were removed, fixed in 4% paraformaldehyde, and processed for paraffin embedding. Tissue sections were immunostained with anti-BrdUrd (Novocastra Laboratories, Inc.) and visualized with 3,3'-diaminobenzidene using a horseradish peroxidase-linked immunoglobulin. The number of BrdUrd positive cells in the interfollicular epidermis and in hair follicles was counted in several random skin sections from several mice at x400. The labeling index was calculated as the percentage of positive basal cells in the interfollicular epidermis and the percentage of positive cell in the follicles, and the mean percentage and SE were determined.

Immunostaining for keratin-1 used boiled paraffin-embedded sections of wild-type and transgenic skins incubated with rabbit polyclonal antimouse keratin-1 antibody at 1:500 dilution. After washing with 1% BSA in PBS, the sections were incubated with secondary antibody, goat antirabbit IgG, at 1:200 dilution. Finally, the sections were lightly counterstained with hematoxylin.

Transient Transfection.
Primary keratinocytes isolated from newborn mice were grown in low Ca2+ (0.05 mM) Eagle’s MEM media with 8% chelexed serum (Sigma). Cells were transiently transfected with an expression vector for PPAR{alpha} (1 µg) with and without a dominant-negative PPAR (dnPPAR, 1 µg) construct, using lipofectin (Life Technologies, Inc.). After 5 h, the medium was changed to conditioned media with or without the addition of 1 µM 8S-HETE, and the cells were incubated for an additional 24 h. Total RNA was isolated according to the Tri-Reagent protocol, fractionated on a 1% agarose/formaldehyde gel, and transferred to a nylon membrane (Amersham). The mRNA expression of keratin-1, a marker of differentiation, was determined using a radiolabeled cDNA probe for keratin-1 (the generous gift of Dennis Roop, Baylor College of Medicine) and GAPDH, as a gel-loading control. For experiments on PPAR response element activity, neonatal keratinocytes were plated in 35-mm dishes 40 h before transfection. Eight µg of tk-(PPRE)3 luciferase reporter vector (a gift from Ron Evans, The Salk Institute, La Jolla, CA) and 0.5 µg of pCMV-ß-gal vector (internal control) per dish were transfected when they were 80% confluent using 10 µl of lipofectin (Life Technologies). After 4 h of transfection, cells were washed with PBS twice and incubated in Eagle’s MEM for 24 h. Proteins were extracted according to the manufacturer’s protocol (Tropix Inc.). Luciferase and ß-galactosidase activities were measured by a luminometer (Tropix). ß-Galactosidase activity was used to normalize transfection efficiencies.


   Acknowledgments
 
We thank Debra Hollowell for doing the injections of expression cDNA and establishing the founder transgenic mice. We greatly appreciate the expertise of the Histology Core, University of Texas, M. D. Anderson Cancer Center, Science Park-Research Division, under the direction of Dr. Irma Gimenez-Conti for the histology work. We would also like to thank Aakosh Gaffar, Eric Dacus, and Amanda Walker for help with genotyping the transgenic mice.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants CA-34443, CA-83794, and CA-46886 (to S. M. F.) and National Institute of Environmental Health Sciences Center Grant P30 ES-07784. Back

2 Both of these authors contributed equally to this article. Back

3 To whom requests for reprints should be addressed, at University of Texas, M. D. Anderson Cancer Center, Science Park-Research Division, Department of Carcinogenesis, P. O. Box 389, Smithville, TX 78957. Phone: (512) 237-9482; Fax: (512) 237-9566; E-mail: sa83161{at}odin.mdacc.tmc.edu Back

4 The abbreviations used are: 8-LOX, 8S-LOX; LOX, lipoxygenase; CLA, conjugated linoleic acid; ETYA, eicosatetraynoic acid; HETE, hydroxyeicosatetraenoic acid; HODE, hydroxyoctadecadienoic acid; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; PPAR, peroxisome proliferator activated receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; BrdUrd, bromodeoxyuridine; HPLC, high-pressure liquid chromatography. Back

Received for publication 8/12/99. Revision received 4/18/00. Accepted for publication 7/ 3/00.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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