Targeting NPM1 in irradiated cells inhibits NPM1 binding to RAD51, RAD51 foci formation and radiosensitizes NSCLC.

The ability of chemo-radiation therapy to control locally advanced stage III non-small cell lung cancer (NSCLC) is poor. While addition of consolidation immunotherapy has improved outcomes in subsets of patients there is still an urgent need for new therapeutic targets. Emerging research indicates that nucleophosmin1 (NPM1) is over-expressed in NSCLC, promotes tumor growth and that over-expression correlates with a lower survival probability. NPM1 is critical for APE1 base excision activity and for RAD51-mediated repair of DNA double strand breaks (DSBs). YTR107 is a small molecule radiation sensitizer that has been shown to bind to NPM1, suppressing pentamer formation. Here we show that in irradiated cells YTR107 inhibits SUMOylated NPM1 from associating with RAD51, RAD51 foci formation and repair of DSBs. YTR107 acts synergistically with the PARP1/2 inhibitor ABT 888 to increase replication stress and radiation-induced cell lethality. YTR107 was found to radiosensitize tumor initiating cells. Congruent with this knowledge, adding YTR107 to a fractionated irradiation regimen diminished NSCLC xenograft growth and increased overall survival. These data support the hypothesis that YTR107 represents a therapeutic target for control of NSCLC.

The importance of the lipoxygenase-hepoxilin pathway in the mammalian epidermal barrier.

This review covers the background to discovery of the two key lipoxygenases (LOX) involved in epidermal barrier function, 12R-LOX and eLOX3, and our current views on their functioning. In the outer epidermis, their consecutive actions oxidize linoleic acid esterified in ω-hydroxy-ceramide to a hepoxilin-related derivative. The relevant background to hepoxilin and trioxilin biochemistry is briefly reviewed. We outline the evidence that linoleate in the ceramide is the natural substrate of the two LOX enzymes and our proposal for its importance in construction of the epidermal water barrier. Our hypothesis is that the oxidation promotes hydrolysis of the oxidized linoleate moiety from the ceramide. The resulting free ω-hydroxyl of the ω-hydroxyceramide is covalently bound to proteins on the surface of the corneocytes to form the corneocyte lipid envelope, a key barrier component. Understanding the role of the LOX enzymes and their hepoxilin products should provide rational approaches to ameliorative therapy for a number of the congenital ichthyoses involving compromised barrier function. 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.

Gene regulation of CYP4F11 in human keratinocyte HaCaT cells.

Mechanisms regulating CYP4F genes remain under investigation, although characterization of CYP4F regulatory modalities would facilitate the discovery of new drug targets. This present study shows that all-trans- and 9-cis-retinoic acids can inhibit CYP4F11 expression in human keratinocyte-derived HaCaT cells. Transrepression of many genes by retinoic acids is mediated by interactions between retinoid receptors and the activator protein 1 (AP-1) complex. Proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta, which can activate the AP-1 complex, induce CYP4F11 transcription in HaCaT cells. The c-Jun N-terminal kinase (JNK)-specific inhibitor 1,9-pyrazoloanthrone (SP600125) blocked the induction of CYP4F11 by both cytokines, indicating involvement of the JNK pathway. Furthermore, TNF-alpha failed to induce CYP4F11 transcription when HaCaT cells were preincubated with retinoic acids. Retinoic acids are ligands for the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). The RXR agonist 6-(1(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl) nicotinic acid (LG268) greatly induced CYP4F11 transcription, whereas the RAR agonist 4-(2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid (TTNPB) markedly inhibited CYP4F11 transcription, indicating that down-regulation of CYP4F11 transcription by retinoic acid is mediated by RARs and may also be related to ligand competition for RXRs. Thus, the CYP4F11 gene is positively regulated by multiple signaling pathways in HaCaT keratinocytes, including RXR and JNK signaling pathways.

20-Hydroxylation is the CYP-dependent and retinoid-inducible leukotriene B4 inactivation pathway in human and mouse skin cells.

Metabolic inactivation of leukotriene B4 (LTB4) is an innate mechanism to resolve tissue inflammation. We studied the nine Cyp4f genes in the mouse genome, measuring cutaneous transcript levels by real-time polymerase chain reaction, and LTB4 metabolism in mouse and human skin. Transcripts arising from Cyp4f13 and 4f16 ranked most abundant, Cyp4f14, 4f17, and 4f37 ranked least abundant, and Cyp4f18 and 4f39 ranked intermediate. Those from Cyp4f15 and Cyp4f40 were highly variable or too low to measure in some animals. Retinoic acid exposure induced microsomal LTB4 hydroxylation activities in mouse and human skin cells. Two NADPH-dependent LTB4 metabolites eluted identically with 20-OH and 20-COOH LTB4 reference standards. Collision induced dissociation of the precursor ion m/z 351 confirmed that LTB4 products from CYP4F3A and human epidermal keratinocytes are identical structurally to 20-OH LTB4. We conclude 20-hydroxylation is the major CYP-dependent LTB4 inactivation pathway in skin; this retinoid-inducible metabolic pathway has capacity to modulate tissue levels of pro-inflammatory lipids.

Secreted Frizzled related protein-4 (sFRP4) promotes epidermal differentiation and apoptosis.

The skin provides vital protection from infection and dehydration. Maintenance of the skin is through a constant program of proliferation, differentiation and apoptosis of epidermal cells, whereby proliferating cells in the basal layer differentiating to form the keratinized, anucleated stratum corneum. The WNT signalling pathway is known to be important in the skin. WNT signalling has been shown to be important both in epidermal development and in the maintenance and cycling of hair follicles and epidermal stem cells. However, the precise role for this pathway in epidermal differentiation remains unknown. We investigated the role of the WNT signalling inhibitor sFRP4 in epidermal differentiation. sFRP4 is expressed in both normal skin and keratinocytes in culture. Expression of sFRP4 mRNA and protein increases with keratinocyte differentiation and apoptosis, whilst exposure of keratinocytes to exogenous sFRP4 promotes apoptosis and expression of the terminal differentiation marker Involucrin. These data suggest sFRP4 promotes epidermal differentiation.

Inflammation resolved by retinoid X receptor-mediated inactivation of leukotriene signaling pathways.

Leukotrienes are implicated in the pathogenesis of diverse, inflammation-driven diseases. Metabolic inactivation of leukotriene signaling is an innate response to resolve inflammation, yet little is known of mechanisms regulating disposition of leukotrienes in peripheral tissues afflicted in common inflammatory diseases. We studied leukotriene hydroxylases (CYP4F gene products) in human skin, a common target of inflammation and adverse drug reactions. Epidermal keratinocytes express at least six CYP4F enzymes; the most highly expressed and highly regulated is CYP4F3A-the main neutrophil leukotriene hydroxylase. Differentiation-specific factors and retinoids are positive CYP4F regulators in vitro, effecting increased leukotriene B4 hydroxylation (inactivation). CYP4F expression is up-regulated in situ in hyperproliferative dermatoses-an innate mechanism to repair and restore epidermal barrier competency-and after retinoid therapy. Enhanced CYP4F-mediated inactivation of leukotriene signaling is a previously unrecognized antiinflammatory property of therapeutic retinoids mediated by preferential interactions between retinoid X receptors and CYP4F promoter elements in epidermal cells.

Differentiation-specific factors modulate epidermal CYP1-4 gene expression in human skin in response to retinoic acid and classic aryl hydrocarbon receptor ligands.

Human epidermal keratinocytes express subsets of cytochromes P450 (P450) (CYP gene products) that are strongly up-regulated, not regulated, or down-regulated by differentiation-specific factors. We investigated how drug exposure affects epidermal expression of CYP1-4 genes, which encode many drug-metabolizing P450s. Real-time polymerase chain reaction (PCR) assays measured CYP1-4 mRNA levels in epidermal keratinocytes differentiated in vitro in the presence of drug or vehicle for 6 days. We confirmed the spinous phenotype at day 6 by changes in cellular morphology and upregulation of cytokeratin 10 and transglutaminase (TGM)1 mRNA in the differentiating keratinocytes. Effects of drug exposure depended on the influence of differentiation-specific factors in controlling epidermal CYP1-4 expression. CYP2C18, 2C19, 2C9, 2W1, 3A4, and 4B1 are up-regulated by cellular differentiation; mRNA levels for these CYP genes were inhibited in differentiating keratinocytes exposed to retinoic acid and aryl hydrocarbon receptor (AhR) ligands. These same drugs effected

A biosynthetic pathway generating 12-hydroxy-5,8,14-eicosatrienoic acid from arachidonic acid is active in mouse skin microsomes.

The epidermis expresses cyclooxygenases, lipoxygenases, and cytochromes P450, which utilize arachidonic acid to generate a diverse array of lipid mediators affecting epidermal cellular differentiation and functions. Recent studies show that mouse epidermis expresses CYP2B19, a keratinocyte-specific epoxygenase that generates 11,12- and 14,15-epoxyeicosatrienoic (EET) acids from arachidonate. We studied CYP2B19-dependent metabolism in mouse epidermal microsomes, reconstituted in the presence of [1-(14)C]arachidonic acid. The majority of the (14)C products formed independently of NADPH, indicative of robust epidermal cyclooxygenase and lipoxygenase activities. We studied two NADPH-dependent products generated in a highly reproducible manner from arachidonate. One of these (product I) coeluted with the CYP2B19 product 14,15-EET on a reversed-phase high-performance liquid chromatography (HPLC) system; there was no evidence for other regioisomeric EET products. Further analyses proved that product I was not an epoxy fatty acid, based on different retention times on a normal-phase HPLC system and failure of product I to undergo hydrolysis in acidic solution. We analyzed purified epidermal (14)C products by liquid chromatography negative electrospray ionization mass spectrometry. Structures of the NADPH-dependent products were confirmed to be 12-oxo-5,8,14-eicosatrienoic acid (I) and 12-hydroxy-5,8,14-eicosatrienoic acid (II). This was the first evidence for a 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway in mouse epidermis. Epidermal microsomes also generated 12-hydroperoxy, 12-hydroxy, and 12-oxo eicosatetraenoic acids from arachidonate, possible intermediates in the 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway. These results predict that hydroxyeicosatrienoic acids are synthesized from arachidonate in human epidermis. This would have important implications for human skin diseases given the known pro- and anti-inflammatory activities of stereo- and regioisomeric hydroxyeicosatrienoic acids.

Effects of the differentiated keratinocyte phenotype on expression levels of CYP1-4 family genes in human skin cells.

Epoxyeicosatrienoic acids produced by mouse CYP2B19 have been implicated in mechanisms regulating epidermal cornification (Ladd, P.A., Du, L., Capdevila, J.H., Mernaugh, R., Keeney, D.S., 2003. Epoxyeicosatrienoic acids activate transglutaminases in situ and induce cornification of epidermal keratinocytes. J. Biol. Chem. 278, 35184-35192). In this study, we aimed to identify CYPs that are up-regulated during keratinocyte differentiation and potentially responsible for epoxyeicosatrienoic acid formation in human skin. The cellular differentiation state of human epidermal cell cultures was manipulated to resemble the basal, spinous, and granular cell phenotypes in vivo. Changes in CYP mRNA levels were measured as a function of differentiation state for a panel of 15 CYPs that included known and putative arachidonate monooxygenases. Quantitative real-time PCR analyses showed that all of the CYPs were expressed in differentiating epidermal cell cultures and in human epidermis, with the exception of CYP2B6, which was poorly expressed in vitro. Six CYPs were strongly up-regulated at Day 6 and Day 8 of in vitro differentiation (CYP4B1, 2W1, 2C18, 3A4, 2C19, 2C9); the increase in mRNA levels ranged from 27- to 356-fold. Only CYP2U1 mRNA levels decreased (6-fold change) during cellular differentiation. Six CYPs showed little variation (<2-fold change) in mRNA levels during in vitro differentiation (CYP2S1, 2J2, 1B1, 1A1, 2E1, 2D6). No single CYP was identifiable as being a functional counterpart to CYP2B19 in mouse skin since none qualified as being mainly responsible for epidermal epoxyeicosatrienoic acid formation. Rather, the data suggest that epoxyeicosatrienoic acids in human skin are formed by several CYPs expressed in different cell layers of the epidermis. This would predict that CYP-derived eicosanoids have different functions in different epidermal cell layers.

Evidence that cytochrome P450 CYP2B19 is the major source of epoxyeicosatrienoic acids in mouse skin.

CYP2B19 is an arachidonic acid monooxygenase highly expressed in the outer, differentiated cell layers of mouse epidermis. We aimed to establish whether CYP2B19 is the source of epidermal epoxyeicosatrienoic acids (EETs), which are implicated in mechanisms regulating epidermal cornification. We show that primary cultures of mouse epidermal keratinocytes expressed native CYP2B19, as determined by mass spectrometry. Differentiation upregulated CYP2B19 mRNA levels ( approximately 39-fold) detected by real-time PCR, CYP2B19 immunoreactivity detected by Western blotting, and cellular levels of the CYP2B19 product 11,12-EET. Cellular 11,12-EET formed from endogenous arachidonic acid increased preferentially (4- to 12-fold) at Day 4 or 5 of differentiation, compared with undifferentiated (Day 0) keratinocyte cultures. Temporally, these results concur with the maximal levels of CYP2B19 mRNA measured at Day 2 and CYP2B19 immunoreactivity at Day 4. We conclude that while mouse epidermis likely expresses multiple cytochrome P450 enzymes, existing evidence supports native CYP2B19 as being the major source of epidermal EET formation.

Epidermal CYP2 family cytochromes P450.

Skin is the largest and most accessible drug-metabolizing organ. In mammals, it is the competent barrier that protects against exposure to harmful stimuli in the environment and in the systemic circulation. Skin expresses many cytochromes P450 that have critical roles in exogenous and endogenous substrate metabolism. Here, we review evidence for epidermal expression of genes from the large CYP2 gene family, many of which are expressed preferentially in extrahepatic tissues or specifically in epithelia at the environmental interface. At least 13 CYP2 genes (CYP2A6, 2A7, 2B6, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 2R1, 2S1, 2U1, and 2W1) are expressed in skin from at least some human individuals, and the majority of these genes are expressed in epidermis or cultured keratinocytes. Where epidermal expression has been localized in situ by hybridization or immunocytochemistry, CYP2 transcripts and proteins are most often expressed in differentiated keratinocytes comprising the outer (suprabasal) cell layers of the epidermis and skin appendages. The tissue-specific transcriptional regulation of CYP2 genes in the epidermis, and in other epithelia that interface with the environment, suggests important roles for at least some CYP2 gene products in the production and disposition of molecules affecting competency of the epidermal barrier.

Organization and evolution of the Cyp2 gene cluster on mouse chromosome 7, and comparison with the syntenic human cluster.

Genes from the cytochrome P450 (CYP) superfamily encode a diverse group of monooxygenases that play important roles in both endogenous processes and in the metabolism of exogenous compounds, including most drugs. A cluster of Cyp2 genes on mouse chromosome 7 was mapped and analyzed in detail and compared with the homologous cluster on human chromosome 19. The mouse cluster includes 22 loci from the same six CYP2 subfamilies--Cyp2a, Cyp2b, Cyp2f, Cyp2g, Cyp2s, Cyp2t--that are found in the human cluster. Twelve of these loci are functional genes, and 10 are pseudogenes. Parts of the mouse and human gene clusters are similarly arranged, but the data indicate that a significantly different series of duplication events created the modern gene organizations in the two species. The comparison of the mouse and human clusters provides new insights into the evolution of gene families, whereas the detailed analysis provides background information that should be informative for future studies on the expression, regulation, and function of specific mouse Cyp2 genes.

Epoxyeicosatrienoic acids activate transglutaminases in situ and induce cornification of epidermal keratinocytes.

The cytochrome P450 CYP2B19 is a keratinocyte-specific arachidonic acid epoxygenase expressed in the granular cell layer of mouse epidermis. In cultured keratinocytes, CYP2B19 mRNAs are up-regulated coordinately with those of profilaggrin, another granular cell-specific marker. We investigated effects of the CYP2B19 metabolites 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) on keratinocyte transglutaminase activities and cornified cell envelope formation. Keratinocytes were differentiated in vitro in the presence of biotinylated cadaverine. Transglutaminases cross-linked this substrate into endogenous proteins in situ; an enzyme-linked immunosorbent assay was used to quantify the biotinylated proteins. Exogenously added or endogenously formed 14,15-EET increased transglutaminase cross-linking activities in cultured human and mouse epidermal keratinocytes in a modified in situ assay. Transglutaminase activities increased approximately 8-fold (p < or = 0.02 versus mock control) in human keratinocytes transduced with adenovirus particles expressing a 14S,15R-EET epoxygenase (P450 BM3v). The physiological transglutaminase substrate involucrin was preferentially biotinylated in situ, determined by immunoblotting and mass spectrometry. P450 BM3v-induced transglutaminase activation was associated with increased 14,15-EET formation (p = 0.002) and spontaneous cell cornification (p < or = 0.001). Preferential involucrin biotinylation and the increased cornified cell envelope formation provided evidence that transglutaminases mediated the P450 BM3v-induced cross-linking activities. These results support a physiological role for 14,15-EET epoxygenases in regulating epidermal cornification, and they have important implications for epidermal barrier functions in vivo.