The role of lipoxygenases in epidermis.

Lipoxygenases (LOX) are key enzymes in the biosynthesis of a variety of highly active oxylipins which act as signaling molecules involved in the regulation of many biological processes. LOX are also able to oxidize complex lipids and modify membrane structures leading to structural changes that play a role in the maturation and terminal differentiation of various cell types. The mammalian skin represents a tissue with highly abundant and diverse LOX metabolism. Individual LOX isozymes are thought to play a role in the modulation of epithelial proliferation and/or differentiation as well as in inflammation, wound healing, inflammatory skin diseases and cancer. Emerging evidence indicates a structural function of a particular LOX pathway in the maintenance of skin permeability barrier. Loss-of-function mutations in the LOX genes ALOX12B and ALOXE3 have been found to represent the second most common cause of autosomal recessive congenital ichthyosis and targeted disruption of the corresponding LOX genes in mice resulted in neonatal death due to a severely impaired permeability barrier function. Recent data indicate that LOX action in barrier function can be traced back to the oxygenation of linoleate-containing ceramides which constitutes an important step in the formation of the corneocyte lipid envelope. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

Epidermis-type lipoxygenase 3 regulates adipocyte differentiation and peroxisome proliferator-activated receptor gamma activity.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) is essential for adipogenesis. Although several fatty acids and their derivatives are known to bind and activate PPAR gamma, the nature of the endogenous ligand(s) promoting the early stages of adipocyte differentiation has remained enigmatic. Previously, we showed that lipoxygenase (LOX) activity is involved in activation of PPAR gamma during the early stages of adipocyte differentiation. Of the seven known murine LOXs, only the unconventional LOX epidermis-type lipoxygenase 3 (eLOX3) is expressed in 3T3-L1 preadipocytes. Here, we show that forced expression of eLOX3 or addition of eLOX3 products stimulated adipogenesis under conditions that normally require an exogenous PPAR gamma ligand for differentiation. Hepoxilins, a group of oxidized arachidonic acid derivatives produced by eLOX3, bound to and activated PPAR gamma. Production of hepoxilins was increased transiently during the initial stages of adipogenesis. Furthermore, small interfering RNA-mediated or retroviral short hairpin RNA-mediated knockdown of eLOX3 expression abolished differentiation of 3T3-L1 preadipocytes. Finally, we demonstrate that xanthine oxidoreductase (XOR) and eLOX3 synergistically enhanced PPAR gamma-mediated transactivation. Collectively, our results indicate that hepoxilins produced by the concerted action of XOR and eLOX3 may function as PPAR gamma activators capable of promoting the early PPAR gamma-dependent steps in the conversion of preadipocytes into adipocytes.

The genotoxic air pollutant 3-nitrobenzanthrone and its reactive metabolite N-hydroxy-3-aminobenzanthrone lack initiating and complete carcinogenic activity in NMRI mouse skin.

3-Nitrobenzanthrone (3-NBA), a genotoxic mutagen found in diesel exhaust and ambient air pollution and its active metabolite N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) were tested for initiating and complete carcinogenic activity in the NMRI mouse skin carcinogenesis model. Both compounds were found to be inactive as either tumour initiators or complete carcinogens in mouse skin over a dose range of 25-400nmol. Topical application of 3-NBA and N-OH-3-ABA produced DNA adduct patterns in epidermis, detected by (32)P-postlabelling, similar to those found previously in other organs of rats and mice. 24h after a single treatment of 100nmol DNA adduct levels produced by 3-NBA (18+/-4 adducts/10(8) nucleotides) were 6 times lower than those by 7,12-dimethylbenz[a]anthracene (DMBA; 114+/-37 adducts/10(8) nucleotides). In contrast, identical treatment with N-OH-3-ABA resulted in adduct levels in the same range as with DMBA (136+/-25 adducts/10(8) nucleotides), indicating that initial DNA adduct levels do not parallel tumour initiating activity. When compounds were tested for tumour initiating activity by a single treatment followed by twice-weekly applications of TPA, DNA adducts formed by DMBA, but not by 3-NBA or N-OH-3-ABA, were still detectable 40weeks after treatment. When tested for activity as complete carcinogens by twice-weekly topical application, 3-NBA and N-OH-3-ABA produced identical DNA adduct profiles in mouse skin, with adducts still detectable after 40weeks. Only 3-NBA produced detectable adducts in other organs.

Development of an ichthyosiform phenotype in Alox12b-deficient mouse skin transplants.

12R-lipoxygenase (12R-LOX) represents a key enzyme of a recently identified eicosanoid pathway in the skin that plays an essential role in the establishment and/or maintenance of the epidermal barrier function. Genetic studies show that loss-of-function mutations in ALOX12B, encoding 12R-LOX, and in ALOXE3, encoding another closely related LOX involved in this pathway, are the second most common cause for autosomal recessive congenital ichthyosis (ARCI). To investigate the pathomechanism of ARCI and the function of 12R-LOX, we recently generated a 12R-LOX knockout model. 12R-LOX-deficient mice die rapidly after birth from severe barrier dysfunction without exhibiting an obvious cutaneous phenotype. Thus, we analyzed the adult phenotype of 12R-LOX(-/-) skin transplanted onto nude mice. 12R-LOX(-/-) skin develops an ichthyosiform appearance with thickening of the epidermis, hyperproliferation, hypergranulosis, focal parakeratosis, and severe hyperkeratosis. The adult mutant mouse skin phenotype closely reproduces the ichthyosis phenotype seen in patients with ALOX12B mutations. Western blot analysis revealed restoration of profilaggrin processing that used to be disturbed in neonatal mutant skin and overexpression of filaggrin, involucrin, and repetin. The results indicate that 12R-LOX knockout mice may represent a useful animal model for a detailed analysis of mechanisms involved in ARCI forms that are associated with impaired LOX metabolism.

Suprabasal BCL-2 expression does not sensitize to chemically-induced skin cancer in transgenic mice.

BACKGROUND: BCL-2 overexpression is frequently detected in nonmelanoma skin cancer. In normal skin, BCL-2 expression is restricted to the basal cell layer and the hair follicle bulge. Both contain stem cells targeted by carcinogens upon initiation of mouse skin carcinogenesis. It is unknown whether the anti-apoptotic activity of BCL-2 is involved in the susceptibility of this cell type to malignant transformation. If so, extending the pool of BCL-2-expressing cells to suprabasal skin layers should increase the likelihood of skin tumour formation. MATERIALS AND METHODS: To resolve this issue, we generated a novel transgenic mouse line overexpressing BCL-2 in suprabasal layers of the epidermis. The influence of suprabasal BCL-2 on tumour formation was then tested by chemically inducing skin cancer using the two-stage initiation-promotion protocol. RESULTS: Bcl-2 expression neither influenced the incidence nor the multiplicity of papillomas upon chemical tumour induction with 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), nor their progression to carcinomas. CONCLUSION: Suprabasal expression of BCL-2 in skin does not increase the formation of papillomas or their malignant progression to squamous cell carcinomas in two-stage mouse skin carcinogenesis.

RAGE signaling sustains inflammation and promotes tumor development.

A broad range of experimental and clinical evidence has highlighted the central role of chronic inflammation in promoting tumor development. However, the molecular mechanisms converting a transient inflammatory tissue reaction into a tumor-promoting microenvironment remain largely elusive. We show that mice deficient for the receptor for advanced glycation end-products (RAGE) are resistant to DMBA/TPA-induced skin carcinogenesis and exhibit a severe defect in sustaining inflammation during the promotion phase. Accordingly, RAGE is required for TPA-induced up-regulation of proinflammatory mediators, maintenance of immune cell infiltration, and epidermal hyperplasia. RAGE-dependent up-regulation of its potential ligands S100a8 and S100a9 supports the existence of an S100/RAGE-driven feed-forward loop in chronic inflammation and tumor promotion. Finally, bone marrow chimera experiments revealed that RAGE expression on immune cells, but not keratinocytes or endothelial cells, is essential for TPA-induced dermal infiltration and epidermal hyperplasia. We show that RAGE signaling drives the strength and maintenance of an inflammatory reaction during tumor promotion and provide direct genetic evidence for a novel role for RAGE in linking chronic inflammation and cancer.

15-lipoxygenase-1 production is lost in pancreatic cancer and overexpression of the gene inhibits tumor cell growth.

Pancreatic cancer patients have an abysmal prognosis because of late diagnosis and lack of therapeutic options. Pancreatic intraepithelial neoplasias (PanINs), the precursor lesions, are a potential target for chemoprevention. Targeting eicosanoid pathways is an obvious choice because 5-lipoxygenase (5-LOX) has been suggested as a tumor promoter in pancreatic carcinogenesis. Here we provide evidence that 15-lipoxygenase-1 (15-LOX-1) expression and activity may exert antitumorigenic effects in pancreatic cancer. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis showed absence or very weak expression of 15-LOX-1 in all pancreatic cancer cell lines tested. 15-LOX-1 was strongly stained in normal ductal cells, tubular complexes, and centroacinar cells, but no staining was seen in islets, cancer cells, PanIN lesions, or in tumor cells in lymph node metastases, indicating that 15-LOX-1 expression is lost during tumor development in human pancreas. Overexpression of 15-LOX-1 in pancreatic tumor cells or treatment with its arachidonic acid-derived metabolite resulted in decreased cell growth. These findings provide evidence that loss of 15-LOX-1 may play an important role in pancreatic carcinogenesis, possibly as a tumor suppressor gene. Thus, induction of 15-LOX-1 expression may be an attractive option for the prevention and treatment of pancreatic cancer.

The cyclooxygenase-2-mediated prostaglandin signaling is causally related to epithelial carcinogenesis.

Epidemiologic, pharmacologic, clinical, and experimental studies document the importance of prostaglandin (PG) signaling in cancer development, including non-melanoma skin cancer lesions in humans and mice. First of all, enzymes involved in PG biosynthesis, such as cyclooxygenase (COX)-2 and/or membrane prostaglandin E synthase (mPGES)-1, were found to be overexpressed in a wide range of premalignant and malignant epithelial tumors, including those of the skin, breast, esophagus, stomach, colorectum, pancreas, and bladder. On the other hand, 15-hydroxy-prostaglandin dehydrogenase (15-PGDH), which is involved in the degradation pathway of PG including PGE(2,) thus counteracting the activities of COX-2 and PGES, was found to be downregulated in human epithelial tumors, indicating a tumor suppressor activity of this enzyme. Most remarkably, genetic studies showed that mice, which are deficient in COX-2 and/or PGES are resistant to the development of cancer of skin, colon, and stomach. In contrast, the forced overexpression of COX-2 in proliferative compartments of simple or stratified epithelia such as skin epidermis, urinary bladder, mammary gland, and pancreas results in spontaneous hyperplasia and dysplasia in transgenic mice. In skin, the pathological changes are found to be due to an abnormal process of terminal differentiation, while in other tissues, hyperproliferation seems to be the main contributor to the pre-invasive neoplasms. Moreover, the COX-2 transgenic mouse lines are sensitized for cancer development.

The expression pattern of prostaglandin E synthase and EP receptor isoforms in normal mouse skin and preinvasive skin neoplasms.

Prostaglandin (PG) E(2), the predominant PG in skin, accumulates in experimentally produced mouse skin tumors. PGE(2) induces proliferation of mouse keratinocytes in vitro, epidermal hyperplasia and dysplasia, a promoted epidermis phenotype, and angiogenesis in keratin 5 promoter (K5) cyclooxygenase (COX)-2-transgenic NMRI mouse skin in vivo. PGE(2) is synthesized by COX-catalysed oxygenation of arachidonic acid to PGH(2) and its conversion to PGE(2) by prostaglandin E synthase (PGES) isoforms. PGE(2) signals via PGE(2) receptor isoforms EP1-EP4. Here, we investigated the expression profiles of PGES and EP receptors in wild type NMRI mouse skin constitutively expressing COX-1 when compared with the hyperplastic/dysplastic skin of homozygous K5 COX-2-transgenic mice and papillomas of both genotypes, which, in addition to COX-1, overexpress COX-2. The three PGES are constitutively expressed in normal and transgenic skin independent of the COX expression status. In papillomas, the increased PGE(2) levels correlate with an increased expression of mPGES-1 and cPGES. All four EP receptors were expressed in normal and transgenic skin. Only EP3 was slightly increased in transgenic skin. In papillomas of both genotypes, the expression levels of EP1 and EP4 were low when compared with those in wild type back skin. EP2 was the predominant receptor in papillomas of wild type and transgenic mice. In papillomas of wild type mice EP3 levels were slightly elevated when compared with transgenic tumors. EP1 and EP2 were localized in basal keratinocytes, sebaceous glands and CD31-positive vessels. Thus, normal and preinvasive mouse skin express the complete protein repertoire for PGE(2) biosynthesis and signalling.

12R-lipoxygenase deficiency disrupts epidermal barrier function.

12R-lipoxygenase (12R-LOX) and the epidermal LOX-3 (eLOX-3) constitute a novel LOX pathway involved in terminal differentiation in skin. This view is supported by recent studies showing that inactivating mutations in 12R-LOX and eLOX-3 are linked to the development of autosomal recessive congenital ichthyosis. We show that 12R-LOX deficiency in mice results in a severe impairment of skin barrier function. Loss of barrier function occurs without alterations in proliferation and stratified organization of the keratinocytes, but is associated with ultrastructural anomalies in the upper granular layer, suggesting perturbance of the assembly/extrusion of lamellar bodies. Cornified envelopes from skin of 12R-LOX-deficient mice show increased fragility. Lipid analysis demonstrates a disordered composition of ceramides, in particular a decrease of ester-bound ceramide species. Moreover, processing of profilaggrin to monomeric filaggrin is impaired. This study indicates that the 12R-LOX-eLOX-3 pathway plays a key role in the process of epidermal barrier acquisition by affecting lipid metabolism, as well as protein processing.

Tumor promotion as a target of cancer prevention.

Tumor promotion is an essential process in multistage cancer development providing the conditions for clonal expansion and genetic instability of preneoplastic and premalignant cells. It is caused by a continuous disturbance of cellular signal transduction that results in an overstimulation of metabolic pathways along which mediators of cell proliferation and inflammation as well as genotoxic by-products are generated. Among such pathways the oxidative metabolism of arachidonic acid has turned out to be of utmost importance in tumor promotion. The aberrant overexpression of cyclooxygenase-2, an inducible enzyme of prostanoid synthesis and lipid peroxidation, is a characteristic feature of more than two-thirds of all human neoplasias, and the specific inhibition of this enzyme has been found to have a substantial chemopreventive effect in both animal models and man. The prostaglandins produced by COX-2 promote tumor development by stimulating cell proliferation and angiogenesis and by suppressing programmed cell death and immune defense. In mice, a COX-2 transgene fused with the keratin 5 promoter, which is constitutively active in the basal (proliferative) compartment of stratified and simple epithelia, causes a preneoplastic and premalignant phenotype in several organs. Among these organs, skin, mammary gland, urinary bladder, and pancreas have been investigated in more detail. Histologically and biochemically, the COX-2-dependent alterations resemble an autopromoted state that--as shown for skin and urinary bladder--strongly sensitizes the tissue for carcinogenesis. In transgenic animals COX-2 expression is not restricted to keratin 5-positive cells but is seen also in adjacent keratin 5-negative cells. This spreading of the COX-2 signal indicates a paracrine mechanism of autoamplification. While cancer chemoprevention by COX-2 inhibition is a rapidly developing field, much less is known about other pathways of unsaturated fatty acid metabolism, although some of them may play a role in carcinogenesis rivaling that of prostaglandin formation. Here an urgent demand for systematic research exists.

Role of epidermis-type lipoxygenases for skin barrier function and adipocyte differentiation.

12R-lipoxygenase (12R-LOX) and epidermis-type LOX-3 (eLOX-3) are novel members of the multigene family of mammalian LOX. A considerable gap exists between the identification of these enzymes and their biologic function. Here, we present evidence that 12R-LOX and eLOX-3, acting in sequence, and eLOX-3 in combination with another, not yet identified LOX are critically involved in terminal differentiation of keratinocytes and adipocytes, respectively. Mutational inactivation of 12R-LOX and/or eLOX-3 has been found to be associated with development of an inherited ichthyosiform skin disorder in humans and genetic ablation of 12R-LOX causes a severe impairment of the epidermal lipid barrier in mice leading to post-natal death of the animals. In preadipocytes, a LOX-dependent PPARgamma activating ligand is released into the cell supernatant early upon induction of differentiation and available evidence indicates that this ligand is an eLOX-3-derived product. In accordance with this data is the observation that forced expression of eLOX-3 enhances adipocyte differentiation.

Inducible expression of 15-lipoxygenase-2 and 8-lipoxygenase inhibits cell growth via common signaling pathways.

Human 15-lipoxygenase (LOX)-2 and mouse 8-LOX represent orthologous members of the LOX family but display different positional specificities and tissue distribution. To study the functional role of 15-LOX-2 and 8-LOX in keratinocytes, an inducible Tet-On gene expression system was established in the premalignant mouse keratinocyte cell line 308. Doxycycline (dox)-induced expression of enzymatically active 15-LOX-2 and 8-LOX led to an inhibition of cell growth that was associated with an inhibition of DNA synthesis, as shown by a 15-46% reduction of 5-bromo-2-deoxy-uridine (BrdU) incorporation. The inhibitory effects were increased in the presence of exogenous arachidonic acid. In contrast, addition of linoleic acid or the LOX inhibitor baicalein reversed the growth-inhibitory effects. Treatment of the cells with 15-hydroxyeicosatetraenoic acid (HETE) or 8-HETE resulted in a similar inhibition of BrdU incorporation, whereas 13-hydroxyoctadecadienoic acid (HODE) and 9-HODE, in contrast, had no effects. Dox-induced keratinocytes showed increased levels of reactive oxygen species (ROS). The antioxidant N-acetyl-L-cysteine and a specific inhibitor of p38 mitogen-activated protein kinase, but not of extracellular signal-regulated kinase 1/2 or c-Jun N-terminal kinase/stress-activated kinases, completely abolished the LOX-induced growth inhibition, indicating a critical role of ROS and p38. Our data suggest that 15-LOX-2 and 8-LOX, although displaying different positional specificity, may use common signaling pathways to induce growth inhibition in premalignant epithelial cells.

Preinvasive duct-derived neoplasms in pancreas of keratin 5-promoter cyclooxygenase-2 transgenic mice.

BACKGROUND & AIMS: Basic research aimed at a better understanding of pancreatic carcinogenesis and improving the treatment of this disease is crucial because the majority of pancreatic cancers are highly aggressive and therapeutically nonaccessible. Cyclooxygenase (COX)-2, which is a key enzyme of prostaglandin (PG) biosynthesis, is overexpressed in around 75% of human carcinomas including those of the pancreas. METHODS: The pathologic changes of transgenic mouse pancreas with keratin 5-promoter-driven expression and activity of COX-2 were characterized. RESULTS: Aberrant expression of COX-2 in a few ductal cells and COX-2-mediated PG synthesis in the transgenic mice resulted in keratin 19- and mucin-positive intraductal papillary mucinous neoplasm- and pancreatic intraepithelial neoplasia-like structures, characterized by an increased proliferation index and serous cystadenomas. Moreover, Ras activation was enhanced and the HER-2/Neu receptor was overexpressed. Loss of acini, fibrosis, and inflammation were pronounced. Feeding a COX-2-selective inhibitor to the transgenic mice suppressed the accumulation of PG and the phenotype. The changes resemble the human disease in which COX-2 was overexpressed consistently. CONCLUSIONS: We present strong evidence for a causal relationship between aberrant COX-2 overexpression and COX-2-mediated PG synthesis and the development of serous cystadenoma, intraductal papillary mucinous, and pancreatic intraepithelial neoplasms. This model offers the unique possibility of identifying molecular pathways leading to the formation and malignant progression of the various types of preinvasive lesions of pancreatic adenocarcinomas that show different dismal outcomes.

A novel aspartic proteinase-like gene expressed in stratified epithelia and squamous cell carcinoma of the skin.

Homeostasis of stratified epithelia, such as the epidermis of the skin, is a sophisticated process that represents a tightly controlled balance between proliferation and differentiation. Alterations of this balance are associated with common human diseases including cancer. Here, we report the cloning of a novel cDNA sequence, from mouse back skin, that is induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and codes for a hitherto unknown aspartic proteinase-like protein (Taps). Taps represents a potential AP-1 target gene because TPA-induced expression in epidermal keratinocytes critically depends on c-Fos, and co-treatment with dexamethasone, a potent inhibitor of AP-1-mediated gene regulation, resulted in impaired activation of Taps expression. Taps mRNA and protein are restricted to stratified epithelia in mouse embryos and adult tissues, implicating a crucial role for this aspartic proteinase-like gene in differentiation and homeostasis of multilayered epithelia. During chemically induced carcinogenesis, transient elevation of Taps mRNA and protein levels was detected in benign skin tumors. However, its expression is negatively associated with dedifferentiation and malignant progression in squamous cell carcinomas of the skin. Similar expression was observed in squamous skin tumors of patients, suggesting that detection of Taps levels represents a novel strategy to discriminate the progression state of squamous skin cancers.

Sequence determinants for the reaction specificity of murine (12R)-lipoxygenase: targeted substrate modification and site-directed mutagenesis.

Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation. Site-directed mutagenesis identified sequence determinants for the positional specificity of these enzymes, and a critical amino acid for the stereoselectivity was recently discovered. To search for sequence determinants of murine (12R)-LOX, we carried out multiple amino acid sequence alignments and found that Phe(390), Gly(441), Ala(455), and Val(631) align with previously identified positional determinants of S-LOX isoforms. Multiple site-directed mutagenesis studies on Phe(390) and Ala(455) did not induce specific alterations in the reaction specificity, but yielded enzyme species with reduced specific activities and stereo random product patterns. Mutation of Gly(441) to Ala, which caused drastic alterations in the reaction specificity of other LOX isoforms, failed to induce major alterations in the positional specificity of mouse (12R)-LOX, but markedly modified the enantioselectivity of the enzyme. When Val(631), which aligns with the positional determinant Ile(593) of rabbit 15-LOX, was mutated to a less space-filling residue (Ala or Gly), we obtained an enzyme species with augmented catalytic activity and specifically altered reaction characteristics (major formation of chiral (11R)-hydroxyeicosatetraenoic acid methyl ester). The importance of Val(631) for the stereo control of murine (12R)-LOX was confirmed with other substrates such as methyl linoleate and 20-hydroxyeicosatetraenoic acid methyl ester. These data identify Val(631) as the major sequence determinant for the specificity of murine (12R)-LOX. Furthermore, we conclude that substrate fatty acids may adopt different catalytically productive arrangements at the active site of murine (12R)-LOX and that each of these arrangements may lead to the formation of chiral oxygenation products.

Mutation spectrum and functional analysis of epidermis-type lipoxygenases in patients with autosomal recessive congenital ichthyosis.

Autosomal-recessive congenital ichthyosis (ARCI) is a clinically and genetically heterogeneous group of severe hereditary keratinization disorders characterized by intense scaling of the whole integument, and differences in color and shape. It is often associated with erythema. To date, six loci for ARCI have been mapped. Mutations in ALOXE3 and ALOX12B on chromosome 17p13, which code for two different epidermal lipoxygenases, were recently found in patients with ichthyosiform erythroderma from Turkey, France, and North Africa. Here we describe molecular and clinical findings in 17 families with ARCI originating from Central Europe, Turkey, and the Indian subcontinent, with mutations in ALOXE3 or ALOX12B. We identified 11 novel point mutations in ALOX12B (one nonsense mutation and 10 missense mutations) and four different inactivating mutations in ALOXE3. The gene products of ALOX12B and ALOXE3, the epidermal lipoxygenases 12R-LOX and eLOX3, respectively, are preferentially synthesized in the skin. They act in sequence to convert arachidonic acid via 12(R)-HPETE to the corresponding epoxyalcohol, 8(R)-hydroxy-11(R),12(R)-epoxyeicosatrienoic acid. To assess the impairment of enzyme activity, we expressed the mutated genes in vitro and determined the activity of the recombinant proteins toward their genuine substrates. All but one of the recombinant mutants were enzymatically inactive. The characterization of disease-causing mutations in ALOXE3 and ALOX12B and the resulting ARCI phenotypes did not result in clear diagnostic criteria; however, we found a first correlation between the genetic findings and the clinical presentation of ichthyosis.

c-Fos-dependent induction of the small ras-related GTPase Rab11a in skin carcinogenesis.

Malignant transformation of mouse skin by tumor promoters and chemical carcinogens, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), is a multistage process leading to the formation of squamous cell carcinomas. It has been shown that mice lacking the AP-1 family member c-Fos exhibit an impaired transition from benign to malignant skin tumors. Here, we demonstrate enhanced expression of the small Ras-related GTPase Rab11a after short-term TPA treatment of mouse back skin. Expression of Rab11a in vivo and in vitro critically depended on c-Fos, because TPA application to the back skin of c-Fos-deficient mice and to mouse embryonic fibroblasts did not induce Rab11a mRNA or protein expression. Moreover, dexamethasone, which is a potent inhibitor of AP-1-mediated transactivation that exhibits anti-inflammatory and anti-tumor promoting activities, inhibited TPA-induced expression of Rab11a. Within the Rab11a gene promoter, we identified a functional AP-1 binding element that exhibited elevated c-Fos binding activity after TPA treatment of keratinocytes. Enhanced expression was not restricted to chemically induced mouse skin tumors but was also found in tumor specimens derived from patients with epithelial skin tumors. These data identify Rab11a as a novel, tumor-associated c-Fos/AP-1 target and may point to an as yet unrecognized function of Rab11a in the development of skin cancer.

Transitional cell hyperplasia and carcinomas in urinary bladders of transgenic mice with keratin 5 promoter-driven cyclooxygenase-2 overexpression.

The inducible form of cyclooxygenase (COX), COX-2, is up-regulated in many epithelial cancers and its prostaglandin products increase proliferation, enhance angiogenesis, and inhibit apoptosis in several tissues. Pharmacologic inhibition and genetic deletion studies showed a marked reduction of tumor development in colon and skin. COX-2 has also been strongly implicated in urinary bladder cancer primarily by studies with nonselective COX- and COX-2-selective inhibitors. We now show that forced expression of COX-2, under the control of a keratin 5 promoter, is sufficient to cause transitional cell hyperplasia (TCH) in 17% and 75% of the heterozygous and homozygous transgenic lines, respectively, in an age-dependent manner. TCH was strongly associated with inflammation, primarily nodules of B lymphocytes; some T cells and macrophage infiltration were also observed. Additionally, transitional cell carcinoma was observed in approximately 10% of the K5.COX-2 transgenic mice; no TCH or transitional cell carcinoma was observed in wild-type bladders. Immunohistochemistry for vascular proliferation and vascular endothelial growth factor showed significant increases above that in wild-type urinary bladders. Our results suggest that overexpression of COX-2 is sufficient to cause hyperplasia and carcinomas in the urinary bladder. Therefore, inhibition of COX-2 should continue to be pursued as a potential chemopreventive and therapeutic strategy.

Cystic duct dilatations and proliferative epithelial lesions in mouse mammary glands upon keratin 5 promoter-driven overexpression of cyclooxygenase-2.

Expression and pharmacological studies support a contribution of cyclooxygenase (COX)-2 to mammary gland tumorigenesis. In a recent transgenic study, mouse mammary tumor virus promoter-driven COX-2 expression in mouse mammary glands was shown to result in alveolar hyperplasia, dysplasia, and carcinomas after multiple rounds of pregnancy and lactation. In the study presented here, the effects of constitutive COX-2 overexpression in keratin 5-positive myoepithelial and luminal cells, driven by the keratin 5 promoter in a hormone-independent manner, was investigated. In nulliparous female mice, aberrant COX-2 overexpression correlated with increased prostaglandin (PG) E(2) levels and caused cystic duct dilatations, adenosis, and fibrosis whereas carcinomas developed rarely. This phenotype depended on COX-2-mediated PGE(2) synthesis and correlated with increased expression of proliferation-associated Ki67 in epithelial cells. No changes in the expression of apoptosis-related Bcl-2, caspase 3, or p53 were observed. Hyperproliferation of the mammary gland epithelial cells was associated with increased aromatase mRNA levels in this tissue. The spontaneous pathologies bear analogies to the human breast with fibrocystic changes. Intriguingly, strong COX-2 expression was observed in fibrocystic changes, as compared to low expression in normal breast epithelium. These results show for the first time that aberrant COX-2 expression contributes to the development of fibrocystic changes (FC), indicating that COX-2 and COX-2-mediated PG synthesis represent potential targets for the therapy of this most frequent benign disorder of the human breast.