A broad range of experimental and clinical evidence has highlighted the central role of chronic infl ammation in promoting tumor development. However, the molecular mechanisms converting a transient infl ammatory tissue reaction into a tumor-promoting microenvironment remain largely elusive. We show that mice defi cient for the receptor for advanced glycation end-products (RAGE) are resistant to DMBA/TPA-induced skin carcinogenesis and exhibit a severe defect in sustaining infl ammation during the promotion phase. Accordingly, RAGE is required for TPA-induced up-regulation of proinfl ammatory mediators, maintenance of immune cell infi ltration, 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 infl ammation 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 infi ltration and epidermal hyperplasia. We show that RAGE signaling drives the strength and maintenance of an infl ammatory reaction during tumor promotion and provide direct genetic evidence for a novel role for RAGE in linking chronic infl ammation and cancer.

Genetic and pharmacological evidence suggests that overexpression of cyclooxygenase-2 (COX-2) is critical for epithelial carcinogenesis and provides a major target for cancer chemoprevention by nonsteroidal antiinflammatory drugs. Transgenic mouse lines with keratin 5 promoter-driven COX-2 overexpression in basal epidermal cells exhibit a preneoplastic skin phenotype. As shown here, this phenotype depends on the level of COX-2 expression and COX-2-mediated prostaglandin accumulation. The transgenics did not develop skin tumors spontaneously but did so after a single application of an initiating dose of the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Long-term treatment with the tumor promoter phorbol 12-myristate 13-acetate, as required for tumorigenesis in wild-type mice, was not necessary for transgenics. The ratios of squamous cell carcinomas to papillomas and of sebaceous gland adenomas to papillomas plus squamous cell carcinomas were increased markedly in transgenic mice treated with DMBA alone compared with DMBA͞phorbol 12-myristate 13-acetate-treated transgenic and wild-type mice. Thus, COX-2 overexpression, which leads to high levels of epidermal prostaglandin E 2, prostaglandin F2␣, and 15-deoxy ⌬12,14 -PGJ2, is insufficient for tumor induction but transforms epidermis into an ''autopromoted'' state, i.e., dramatically sensitizes the tissue for genotoxic carcinogens.

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.

In prostanoid biosynthesis, the first two steps are catalyzed by cyclooxygenases (COX). In mice and humans, deregulated expression of COX-2, but not of COX-1, is characteristic of epithelial tumors, including squamous cell carcinomas of skin. To explore the function of COX-2 in epidermis, a keratin 5 promoter was used to direct COX-2 expression to the basal cells of interfollicular epidermis and the pilosebaceous appendage of transgenic mouse skin. COX-2 overexpression in the expected locations, resulting in increased prostaglandin levels in epidermis and plasma, correlated with a pronounced skin phenotype. Heterozygous transgenic mice exhibited a reduced hair follicle density. Moreover, postnatally hair follicle morphogenesis and thinning of interfollicular dorsal epidermis were delayed. Adult transgenics showed a body-sitedependent sparse coat of greasy hair, the latter caused by sebaceous gland hyperplasia and increased epicutaneous sebum levels. In tail skin, hyperplasia of scale epidermis reflecting an increased number of viable and cornified cell layers was observed. Hyperplasia was a result of a disturbed program of epidermal differentiation rather than an increased proliferation rate, as reflected by the strong suppression of keratin 10, involucrin, and loricrin expression in suprabasal cells. Further pathological signs were loss of cell polarity, mainly of basal keratinocytes, epidermal invaginations into the dermis, and formation of horn perls. Invaginating hyperplastic lobes were surrounded by CD31-positive vessels. These results demonstrate a causal relationship between transgenic COX-2 expression in basal keratinocytes and epidermal hyperplasia as well as dysplastic features at discrete body sites.

Adipocytes play a central role in whole-body energy homoeostasis. Complex regulatory transcriptional networks control adipogensis, with ligand-dependent activation of PPARγ (peroxisome proliferator-activated receptor γ) being a decisive factor. Yet the identity of endogenous ligands promoting adipocyte differentiation has not been established. Here we present a critical evaluation of the role of LOXs (lipoxygenases) during adipocyte differentiation of 3T3-L1 cells. We show that adipocyte differentiation of 3T3-L1 preadipocytes is inhibited by the general LOX inhibitor NDGA (nordihydroguaiaretic acid) and the 12/15-LOX selective inhibitor baicalein. Baicalein-mediated inhibition of adipocyte differentiation was rescued by administration of rosiglitazone. Treatment with baicalein during the first 4 days of the differentiation process prevented adipocyte differentiation; supplementation with rosiglitazone during the same period was sufficient to rescue adipogenesis. Accordingly, we demonstrate that adipogenic conversion of 3T3-L1 cells requires PPARγ ligands only during the first 4 days of the differentiation process. We show that the baicalein-sensitive synthesis of endogenous PPARγ ligand(s) increases rapidly upon induction of differentiation and reaches a maximum on days 3–4 of the adipocyte differentiation programme. The conventional platelet- and leucocyte-type 12(S)-LOXs and the novel eLOX-3 (epidermis-type LOX-3) are expressed in white and brown adipose tissue, whereas only eLOX-3 is clearly expressed in 3T3-L1 cells. We suggest that endogenous PPARγ ligand(s) promoting adipocyte differentiation are generated via a baicalein-sensitive pathway involving the novel eLOX-3.

Substantial evidence supports a functional role for cyclooxygenase- and lipoxygenase-catalyzed arachidonic and linoleic acid metabolism in cancer development. Genetic intervention studies firmly established cause-effect relations for cyclooxygenase-2, but cyclooxygenase-1 may also be involved. In addition, pharmacologic cyclooxygenase inhibition was found to suppress carcinogenesis in both experimental mouse models and several cancers in humans. Arachidonic acid-derived eicosanoid or linoleic acid-derived hydro[peroxy]fatty acid signaling are likely to be involved impacting fundamental biologic phenomena as diverse as cell growth, cell survival, angiogenesis, cell invasion, metastatic potential and immunomodulation. However, long chain unsaturated fatty acid oxidation reactions indicate antipodal functions of distinct lipoxygenase isoforms in carcinogenesis, i.e., the 5- and platelet-type 12-lipoxygenase exhibit procarcinogenic activities, while 15-lipoxygenase-1 and 15-lipoxygenase-2 may suppress carcinogenesis.

Transin RNA is a 1.9-kilobase RNA transcript induced by oncogenes in rat embryo fibroblast cell lines. We show that RNA species complementary to a cloned transin cDNA are present in mouse skin squamous cell carcinomas induced by a classical initiation-promotion protocol but not in premalignant, benign papillomas or in normal epidermis. A single application of a tumor promoting phorbol ester to normal epidermis elicits a transient increase in these RNA levels. Transin RNA encodes a secreted protease, an activity consistent with a functional role for enhanced expression of transin RNA in the progression of benign, encapsulated tumors to malignant, invasive carcinomas.During the process of either spontaneous or experimental carcinogenesis, the progression of normal cells through premalignancy to malignancy is accompanied by a variety of changes in morphological and biochemical properties. Some of these changes may result from alterations in cellular gene expression that are detectable as differences in the mRNA populations of transformed cells compared to their normal counterparts (1-7). For example, we have identified and sequenced a cloned cDNA corresponding to a 1.9-kilobase mRNA (transin RNA, formerly pTR1 RNA) that was present in significantly higher levels in rat cells transformed by polyoma virus, Rous sarcoma virus, and the activated cellular oncogene Ha-ras than in the normal parental cell lines (7). Transin RNA is also induced by epidermal growth factor in rat fibroblasts (7). A second RNA (transin-2 RNA) related to transin RNA and encoding a protein that is 71% homologous to transin is also induced by oncogenes in rat cells (L.M.M. and R.B., unpublished data). To investigate whether the expression of such transformation-specific transin RNAs occurs during the process of tumor progression in vivo, we have used the well-characterized mouse skin model of carcinogenesis (8-10), where it is known that invasive, malignant squamous cell carcinomas can be derived directly from benign, encapsulated, noninvasive papillomas (11) and that both tumor types are monoclonal in nature (12). We show that transin RNA can be detected in carcinomas and that transin is a secreted protease.MATERIALS AND METHODS RNA Analysis. Induction of tumors and preparation of RNA from papillomas, carcinomas, or skin samples was as described (6). RNA samples were subjected to gel electrophoresis, transferred to nitrocellulose, and hybridized to nick-translated pTR1 as described (7), except that the hybridization solution contained 40% (vol/vol) formamide and that filters were washed at 50°C in 2x SSC/0.1% NaDodSO4. (lx SSC = 0.015 M sodium citrate/0.15 M NaCl, pH 7.0.) Protein Analysis. Antibodies against a synthetic peptide were generated in rabbits by repeated injections of peptide in Freund's complete adjuvant. For immunoprecipitation studies, Rat-1 (± epidermal growth factor at 20 ng/ml), B-77, and Rat-1 1.2 cells (see ref. 7 for origins) in 60-mm culture dishes were placed in 2 ml of methionine-free Dulbecco's modified Eagle...

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.