Peter Krieg

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.

The simultaneous extraction of high-molecular-weight DNA and of RNA from solid tumors

A novel method for the isolation of both macromolecular DNA and RNA from solid tissues based upon the disruption by vibration of deep-frozen material in a mechanical device termed Mikro-dismembrator, is described. This technique reveals a yield of, on the average, 1 to 3 mg of either DNA or RNA per gram of tissue. The quality of the purified nucleic acids permits the detailed analysis of integrated tumor virus DNA sequences and their mRNA transcripts. Furthermore, the efficient isolation of papilloma virions from keratinized wart tissue is facilitated by the application of the Mikro-dismembrator.

Prostaglandin-H-synthase isozyme expression in normal and neoplastic human skin

Expression of prostaglandin‐H‐synthase (PGHS) isozymes was analyzed in 50 biopsies of normal human skin and of pre‐malignant and malignant skin lesions, by means of quantitative RT‐PCR, immunoprecipitation and Western blotting, as well as immunohistochemistry. Normal skin constitutively expressed PGHS‐1 in all cell layers of the epidermis, in endothelial cells of small blood vessels and in sweat‐gland epithelium. PGHS‐2 expression was very low and restricted to a few keratinocytes of the interfollicular and follicular epidermis. Steady‐state concentrations of PGHS‐1 and PGHS‐2 mRNA were similar in normal skin and in basal‐cell carcinomas, but PGHS‐1 mRNA was reduced and PGHS‐2 mRNA was elevated in actinic keratoses, squamous‐cell carcinomas and keratoacanthomas. PGHS‐1 protein was detected in all tumor biopsies, being occasionally increased in basal‐cell carcinomas. High amounts of PGHS‐2 protein were found in actinic keratoses, squamous‐cell carcinomas and keratoacanthomas, but not in basal‐cell carcinomas. Four malignant melanomas included in this study contained PGHS‐1 but no PGHS‐2 protein. Immunohistochemical analysis of the biopsies identified keratinocytes, in addition to cells of inflammatory infiltrates and of dendritic morphology, as the major PGHS‐expressing cell types. PGHS‐2‐specific signals were spread throughout the epidermal part of actinic keratoses and squamous‐cell carcinomas. These data suggest that constitutive up‐regulation of PGHS‐2 expression is a consistent pre‐malignant event in squamous‐cell cancer development in man, as it is in animal models of skin carcinogenesis. Thus, pre‐cancerous lesions such as actinic keratoses present a likely target for chemoprevention of skin cancer by selective PGHS‐2 inhibitors. Int. J. Cancer 82:648–656, 1999.

Molecular Analysis of 250 Patients with Autosomal Recessive Congenital Ichthyosis: Evidence for Mutation Hotspots in ALOXE3 and Allelic Heterogeneity in ALOX12B

In recent years several new genes for autosomal recessive congenital ichthyosis (ARCI) have been identified. However, little is known about the molecular epidemiology and pathophysiology of this genetically and clinically heterogeneous group of severe disorders of keratinization. ARCI is characterized by intense scaling of the whole integument often associated with erythema. We and others have shown that mutations in ALOX12B and ALOXE3, coding for the lipoxygenases 12R-LOX and eLOX-3 predominantly synthesized in the epidermis, can underlie this rare condition. Here we have surveyed a large group of 250 patients with ARCI for mutations in these two genes. We have identified 11 different previously unreported mutations in ALOX12B and ALOXE3 in 21 ARCI patients from 19 unrelated families and demonstrated that mutations in the two genes are the second most common cause for ARCI in this cohort of patients. Examination of the molecular data revealed allelic heterogeneity for ALOX12B and two mutational hotspots in ALOXE3. Functional analysis of all missense mutations and a splice site mutation demonstrated that complete loss of function of the enzymes underlies the phenotype. Our findings further establish the pivotal role of the 12-lipoxygenase pathway during epidermal differentiation.

Aloxe3 Knockout Mice Reveal a Function of Epidermal Lipoxygenase-3 as Hepoxilin Synthase and Its Pivotal Role in Barrier Formation

Loss-of-function mutations in the lipoxygenase (LOX) genes ALOX12B and ALOXE3 are the second most common cause of autosomal recessive congenital ichthyosis. The encoded proteins, 12R-LOX and epidermal LOX-3 (eLOX-3), act in sequence to convert fatty acid substrates via R-hydroperoxides to specific epoxyalcohol derivatives and have been proposed to operate in the same metabolic pathway during epidermal barrier formation. Here, we show that eLOX-3 deficiency in mice results in early postnatal death, associated with similar but somewhat less severe barrier defects and morphological changes than reported earlier for the 12R-LOX-knockout mice. Skin lipid analysis demonstrated that the severity of barrier failure is related to the loss of covalently bound ceramides in both 12R-LOX- and eLOX-3-null mice, confirming a proposed functional linkage of the LOX pathway to ceramide processing and formation of the corneocyte lipid envelope. Furthermore, analysis of free oxygenated fatty acid metabolites revealed strongly reduced levels of hepoxilin metabolites in eLOX-3-deficient epidermis, indicating an additional function of eLOX-3 in mammalian skin as a hepoxilin synthase linked to the 12S-LOX pathway.

cDNA cloning of a 8-lipoxygenase and a novel epidermis-type lipoxygenase from phorbol ester-treated mouse skin

Using a combination of PCR cloning and conventional screening procedures, we isolated from phorbol ester-treated mouse epidermis two full length cDNA clones encoding novel lipoxygenases. One of the cDNAs turned out to be identical to the recently cloned 8-lipoxygenase [Jisaka et al., J. Biol. Chem. 272 (1997) 24 410-24 416], the open reading frame of the second one corresponded to a protein of 701 amino acids with a calculated molecular mass of 80.6 kDa. The amino acid sequence showed 50.8% identity to human 15-lipoxygenase 2, approximately 40% to 5-lipoxygenase and 35% to 12- and 15-lipoxygenases. A unique structural feature is the insertion of 31 amino acid residues in the amino-terminal part of the molecule. Based on these data, we conclude that this epidermis-derived cDNA encodes a novel lipoxygenase isoform termed provisionally epidermis-type lipoxygenase 2 (e-LOX 2).

Cloning of SV40sv40 genomes from human brain tumors

From two human brain tumors SV40 genomes were isolated by recombinant DNA techniques. The SV40 genome cloned from a human meningioma DNA was shown to be indistinguishable from wild-type SV40. In contrast, the SV40 genome cloned from a human astrocytoma proved to be a nonviable deletion mutant with a truncated early region removing most of the large T-coding region. In addition, this mutant also carries a tandem duplication of an intact origin of replication.

Diversity of mouse lipoxygenases: Identification of a subfamily of epidermal isozymes exhibiting a differentiation-dependent mRNA expression pattern

By using reverse transcription-polymerase chain reaction technology (RT-PCR) and Northern blot analysis, the tissue-specific mRNA expression patterns of seven mouse lipoxygenases (LOX)—including 5S-, 8S-, three isoforms of 12S-, 12R-LOX, and a LOX of an as-of-yet unknown spacificity, epidermis-type LOX-3 (e-LOX-3)—were investigated in NMRI mice. Among the various tissues tested epidermis and forestomach were found to express the broadest spectrum of LOX. With the exception of 5S-and platelet-type 12S-LOX (p12S-LOX) the remaining LOx showed a preference to exclusive expression in stratifying epithelia of the mouse, in particular the integumental epidermis. The expression of the individual LOX in mouse epidermis was found to depend on the state of terminal differentiation of the keratinocytes. mRNA of epidermis-type 12S-LOX (e12S-LOX) was detected in all layers of neonatal and adult NMRI mouse skin, whereas experission of p12S-LOX, 12R-LOX, and e-LOX-3 was restricted to suprabasal epidermal layers of neonatal and adult mice. 8S-LOX mRNA showed a body-sitedependent expression in that it was detected in stratifying epithelia of footsole and forestomach but not in back skin epidermis. In the latter, 8S-LOX mRNA was strongly induced upon treatment with phorbol esters. With the exception of e12S-LOX and p12S-LOX, the isozymes that are preferentially expressed in stratifying epithelia are structurally related and may be grouped together into a distinct subgroup of epidermis-type LOX.

MURINE 12(R)-LIPOXYGENASE : FUNCTIONAL EXPRESSION, GENOMIC STRUCTURE AND CHROMOSOMAL LOCALIZATION

A cDNA, recently cloned (by Krieg et al. (1998)) from mouse skin, was shown to encode a 12(R)‐lipoxygenase. When expressed in HEK cells, the recombinant protein converted methyl arachidonate into the corresponding 12‐HETE ester which was shown to be the R‐enantiomer by chiral phase chromatography. Neither arachidonic acid nor linoleic acid were substrates for the recombinant protein. The structure of the 12(R)‐lipoxygenase gene is unique among all animal lipoxygenases in that it is divided into 15 exons and 14 introns spanning approximately 12.5 kb. By interspecific backcross analysis, the 12(R)‐lipoxygenase gene was localized to the central region of mouse chromosome 11.

Epidermis-Type Lipoxygenase 3 Regulates Adipocyte Differentiation and Peroxisome Proliferator-Activated Receptor γ Activity

ABSTRACT The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is essential for adipogenesis. Although several fatty acids and their derivatives are known to bind and activate PPARγ, 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 PPARγ 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 PPARγ ligand for differentiation. Hepoxilins, a group of oxidized arachidonic acid derivatives produced by eLOX3, bound to and activated PPARγ. 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 PPARγ-mediated transactivation. Collectively, our results indicate that hepoxilins produced by the concerted action of XOR and eLOX3 may function as PPARγ activators capable of promoting the early PPARγ-dependent steps in the conversion of preadipocytes into adipocytes.