Retinoic Acid and Its 4-Oxo Metabolites Are Functionally Active in Human Skin Cells in Vitro

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Retinoic Acid and its 4-Oxo Metabolites are Functionally Active in Human Skin Cells In Vitro

Jens M. Baron,Ã,1 Ruth Heise,Ã,1 William S. Blaner,w Mark Neis,Ã Sylvia Joussen,Ã Alexandra Dreuw,z Yvonne Marquardt,Ã Jean-Hilaire Saurat,y Hans F. Merk,Ã David R. Bickers,z and Frank K. Jugertw ÃDepartment of Dermatology and Allergology, University Hospital of the RWTH, Aachen, Germany; wDepartment of Medicine, Columbia University, College of Physicians and Surgeons, New York, New York, USA; zInstitute of Biochemistry, University Hospital of the RWTH, Aachen, Germany; yDepartment of Dermatology, Hoˆ pital Cantonal University, Geneva, Switzerland; zDepartment of Dermatology, Columbia University, College of Physicians and Surgeons, New York, New York, USA

Retinoic acid exerts a variety of effects on gene transcription that regulate growth, differentiation, and inflammation in normal and neoplastic skin cells. Because there is a lack of information regarding the influence of metabolic transformation of retinoids on their pharmacologic effects in skin, we have analyzed the functional activity of all- trans-, 9-cis-, and 13-cis-retinoic acid and their 4-oxo-metabolites in normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts using gene and protein expression profiling techniques, including cDNA microarrays, two-dimensional gel electrophoresis, and MALDI-MS. It was previously thought that the 4-oxo-metabolites of RA are inert catabolic end-products but our results indicate instead that they display strong and isomer-specific transcriptional regulatory activity in both NHEKs and dermal fibroblasts. Microarray and proteomic analyses identified a number of novel genes/gene products that are influenced by RA treatment of NHEKs or fibroblasts, including genes for enzymes catalyzing biotransformation of retinoids, corticosteroids, and antioxidants and structural and transport proteins known to be essential for homeostasis. Our results expand current knowledge regarding retinoic acid action within skin cells and the target tissue/cell regulatory systems that are important for modulating the physiological and pharmacological effects of this important class of dermatological drugs. Key words: dermal fibroblasts/epidermal keratinocytes/functional proteomics/gene expression profile J Invest Dermatol 125:143 –153, 2005

Retinoids (vitamin A and its analogs) are crucial for main- eration and hyperkeratosis in the skin (Peck et al, 1949). taining the homeostasis of cells and tissues including pro- From studies of vitamin A-deficient animals and observation liferation and differentiation (Gudas et al, 1994). Aside from of vitamin A-deficient human infants, it was suggested that vision, the physiologic activity of retinoids is thought to be vitamin A deficiency gives rise to follicular keratoses re- mediated primarily by the all-trans- and 9-cis-retinoic acid sembling Darier’s disease or pityriasis rubra pilaris (Peck (RA) isomers that regulate transcription following binding and DiGiovanna, 1994). Initial attention focused on the to their cognate ligand-dependent transcription factors, naturally occurring all-trans- and 13-cis-RA isomers as the RA receptors (RAR), and retinoid X receptors (RXR) potentially effective for treating skin disease (Peck and (Chambon, 1994, 1996; Mangelsdorf et al, 1994, 1995; Ross DiGiovanna, 1994; Roos et al, 1998; Shroot et al, 1999; et al, 2000; Chawla et al, 2001; Clagett-Dame and DeLuca, Blaner, 2001; Zouboulis, 2001). More recently, a number of 2002). Several hundred genes may be regulated by all- synthetic RA analogs with enhanced therapeutic efficacy trans- and/or 9-cis-RA (Balmer and Blomhoff, 2002). have been developed (Peck and DiGiovanna, 1994; Roos The skin is a stratified squamous epithelium and a major et al, 1998; Shroot et al, 1999; Blaner, 2001; Zouboulis, site of RA action (Peck and DiGiovanna, 1994; Roos et al, 2001). It is important to emphasize that the skin has both a 1998; Blaner, 2001; Zouboulis, 2001). Vitamin A deficiency physiologic requirement for RA and responds to pharmaco- is known to result in squamous metaplasia of a variety of logic doses of natural or synthetic retinoids that are effective epithelia that is often associated with increased cell prolif- for treating patients with skin disease (Peck et al, 1949). Since human skin cells express both RAR and RXR (Peck and DiGiovanna, 1994; Roos et al, 1998; Shroot et al, 1999; Abbreviations: CYP, cytochrome P450; DMEM, Dulbecco’s mod- Zouboulis, 2001), it is reasonable to assume that retinoid ified Eagle’s medium; FCS, fetal calf serum; IL, interleukin; LRAT, effects in skin are mediated by retinoid-dependent tran- lecithin:retinol acyltransferase; MALDI-MS, matrix-assisted laser desorption ionization-mass spectrometry; MMP, matrix metal- scription factors. Interestingly, the binding affinity of 13-cis- loproteinase; NHEK, normal human epidermal keratinocyte; PBS, RA for the RAR and RXR and its activity in transactiva- phosphate-buffered saline; RA, retinoic acid; SDS-PAGE, sodium tion assays is substantially less than that for either all-trans- dodecyl sulfate-polyacrylamide gel electrophoresis; TBS, tris-buffered saline or 9-cis-RA (Mangelsdorf et al, 1992, 1994; Beard and 1Both authors contributed equally to this work. Chandraratna, 1999). Yet, it is well established that 13-cis-

Copyright r 2005 by The Society for Investigative Dermatology, Inc. 143 144 BARON ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

RA exerts pharmacological effects that cannot be recapit- binding protein A8 was upregulated by 13-cis- but down- ulated by either all-trans-or9-cis-RA, raising questions re- regulated by and all-trans- and 9-cis-RA. garding the molecular basis underlying the remarkable Interestingly, the 4-oxo-metabolites had very similar ef- efficacy of 13-cis-RA efficacy in acne vulgaris (Peck and fects on expression profiles as those observed for their DiGiovanna, 1994; Roos et al, 1998; Shroot et al, 1999; parent compounds (Table I and Table S1). The 4-oxo-met- Blaner, 2001; Zouboulis, 2001). One possibility is that the abolites of RA, especially 4-oxo-13-cis-RA, are generally superior clinical efficacy of 13-cis-RA in acne as compared thought to be inert catabolic end-products that are destined with all-trans-or9-cis-RA could relate to unique pharmaco- for elimination. As evidenced in Table I and Table S1, how- kinetic properties of this isomer (Peck and DiGiovanna, ever, these metabolites, which cannot be converted back to 1994; Blaner, 2001). In addition, the drug may act directly on RA, have substantial transcriptional activity. Incubation of proteins in skin cells by covalent and non-covalent binding. NHEK with these substances for 3 h revealed minor chang- There is a substantial literature indicating that RA can co- es in gene expression profiles (Table S2). valently bind to proteins, thereby altering cell signaling Incubation of dermal fibroblasts with the RA isomers for pathways (Takahashi et al, 1991a, b; Takahashi and Breit- 24 h revealed more substantial changes than those ob- man, 1992; Myhre et al, 1996; Hoyos et al, 2000; Ra- served in NHEK, including downregulation of p-450- dominska-Pandya et al, 2000; Imam et al, 2001; Korichneva dependent enzymes involved in xenobiotic/endogenous et al, 2003). Another possibility is that the effects of 13-cis- biotransformation, including cytochrome P450 (CYP)2D, RA in acne are indirect, acting for instance via competitive CYP3A7, CYP4A1, corticosteroid 11b-dehydrogenase, inhibition of enzymes involved in the bioactivation of an- prostaglandin-endoperoxide synthase 1, dual specificity drogenes (Karlsson et al, 2003). Alternatively, it is possible phosphatase 11, glutathione S transferase M1, and trans- that one or more metabolites formed from 13-cis-RA could port proteins like solute carrier family 16 (SLC16A1) (Table display transcriptional regulatory activity. To assess these S3). Similar effects were seen for most of the RA isomers possibilities, we have studied the metabolism and actions of and their 4-oxo-RA isomers. The RA and their 4-oxo met- 13-cis-RA in primary cultures of normal human skin cells. To abolites downregulated fibroblast Fas-activated serine/ conduct these studies, we used gene arrays and two-di- threonine kinase, growth factor receptor-bound protein 7, mensional (2D) sodium dodecyl sulfate-polyacrylamide gel epidermal growth factor receptor, collagen types VI and electrophoresis (SDS-PAGE) separations of proteins cou- XVII, as well as protease inhibitor 8 and 9 (4-oxo-) 13-cis-, pled with matrix-assisted laser desorption ionization-mass (4-oxo-) all-trans-, and (4-oxo-)-9-cis-RA. Expression of an- spectrometry (MALDI-MS) identification of the proteins to nexin A1, annexin A5, thymosin b4, ribosomal protein S13, explore various modes of action of all-trans-, 13-cis-, 9-cis- apolipoprotein D, and insulin-like growth factor-binding pro- RA, and their oxo-metabolites in these cells. tein 3 was upregulated in skin fibroblasts by 4-oxo-13-cis-, all-trans-, and 4-oxo-all-trans-RA but not by 9-cis-, 4-oxo- 9-cis-, and 13-cis-RA.

Results Real-time PCR (RT-PCR) confirmation of gene array data RT-PCR analysis in NHEK incubated with 106 M cDNA microarray analysis of the effects of RA and its retinoids using specific primers for the detection of cytok- oxo-metabolites on normal human epidermal keratin- eratins 1, 10, 13, and 19 confirmed strong upregulation of ocytes (NHEK) and dermal fibroblast gene expres- cytokeratins 13 and 19 by 13-cis-RA but a weaker upreg- sion To explore the mechanism whereby all-trans-, 13- ulation by all-trans-, 9-cis-, 4-oxo-13-cis-, 4-oxo-all-trans-, cis-, and 9-cis-RA isomers and their 4-oxo-metabolites and 4-oxo-9-cis-RA (Fig 2b and d). In accordance with the might differentially affect gene expression when added to cDNA microarray data, we also observed downregulation of cultured NHEK and fibroblasts, each of the RA isomers and cytokeratins 1 and 10 and MMP-3 by all-trans-, 9-cis-, and 4-oxo-RA species was added to primary cultures of NHEK 13-cis-RA and their 4-oxo-metabolites (Fig 2a, c, and e). or fibroblasts for 24 h, and analysis of differential gene ex- Since there were no available cDNA probes specific for the pression was performed using DNA-Chip technology (Fig 1). detection of lamin B1 on the microarrays used in this We analyzed the expression of sequence-validated cDNA project, our 2D-gel data were also directly confirmed by arrayed on PIQOR Skin Microarray using ImaGene software real-time PCR (Fig 3f). Only a weak upregulation of lamin B1 version 4.1 (Table I and Tables S1 and S2). There were mRNA expression in NHEK occurred after incubation with substantial differential effects of the RA isomers on gene each of the six different retinoids, especially after incubation expression. Incubation of NHEK with 106 M of each RA with 13-cis- or 4-oxo-13-cis-RA. Real-time PCR analyses of isomer for 24 h led to upregulation of keratins 7, 15, and 19 NHEK incubated with 107 M concentration of the retinoids as well as interleukin-1 (IL-1)b, tissue-type plasminogen revealed similar effects (Table S4). When cells were in- activator, and others (Table I and Table S1). We detected a cubated with these substances at a lower concentration significant downregulation of keratins 1, 10, 14, and 17 as (108 M), upregulation of keratins 13 and 19 and downreg- well as matrix metalloproteinase (MMP)-10, vascular end- ulation of MMP-3 were still detectable, whereas expression othelial growth factor (VEGF)-C, thrombospondin-1, plasm- of keratins 1, 10, and lamin B1 was not significantly reg- inogen activator inhibitor-2, cyclooxygenase-2, psoriasis- ulated (Table S4). associated fatty acid-binding protein (PA-FABP), and filaggrin and other genes by all-trans-, 13-cis-, and 9-cis- Immunofluorescence analysis of the effects of 13-cis- RA (Table I and Table S1). Expression of S100 calcium- and 4-oxo-13-cis-RA on NHEK expression of cytokera- 125 : 1 JULY 2005 4-OXO-RETINOIC ACIDS ARE ACTIVE IN SKIN CELLS 145

Figure 1 Analysis of differentially expressed genes in NHEK (a–d) and dermal fibroblasts (e–g) after treatment with 13-cis- and 4-oxo-13-cis-retinoic acid (RA) for 24 h using the DNA-Chip technology. Total RNA was isolated using peqGOLD RNA Pure. Two micrograms of aRNA from each probe was labeled by reverse transcription with Cy5 and Cy3 fluorescence, respectively, and subjected to a PIQOR skin microarray. Processing of microarrays was performed using a fully automated hybridization station. Image capture and signal quantification of hybridized cDNA arrays were carried out with the ScanArray Lite and ImaGene software version 4.1. Array images and Scatter blot analysis of keratinocytes induced by 10–6 M 13-cis-RA (a, c) and 4-oxo-13-cis-RA (b, d) compared with untreated control. For analysis of gene expression in dermal fibroblast, generation of 33P- labeled cDNA probe was carried out by reverse transcription of 10 mg total RNA with 33P dCTP for 90 min at 371C following the Mamalian GeneFilters (Research Genetics) protocol. Purified radiolabeled probe was applied to the array for hybridization at 421C overnight. Afterwards, filters were exposed to a Kodak X-OMAT AR-5 film at 701C with intensifying screens. Scatter blot analysis of fibroblasts induced by 10–5 M 13-cis-RA (e, f) and 4-oxo-13-cis-RA (e, g) compared with untreated control. tins 7 and 19 To determine whether the changes in mRNA analysis of integrated spot areas also indicated that both the levels detected following treatment with 10–6 M 13-cis-, all- cell treatments and the 2D electrophoresis contributed to the trans-, 9-cis-, 4-oxo-13-cis-, 4-oxo-all-trans-, or 4-oxo-9- measured variability. Most of the variability arose, however, cis-RA influence protein expression, we analyzed NHEK from the running of the 2D gels, the staining of the gels, and monolayers using specific antibodies and confirmed up- the subsequent densitometric measures carried out on the regulation of cytokeratins 7 (Fig 3a) and 19 (Fig 3b) expres- stained gels. From this analysis, it was possible to predict sion in response to all retinoids analyzed. As can be seen statistically which differences in spot intensities were because from this figure, both of these proteins are expressed at of treatment with all-trans-, 13-cis-RA, or both. We thus iden- higher levels in treated NHEK, indicating that protein ex- tified cellular proteins whose apparent concentrations within pression as well as mRNA levels for these two proteins are NHEK varied substantially (po0.01) after treatment with all- upregulated following addition of 13-cis-, all-trans-, 9-cis-, trans-and/or13-cis-RA from vehicle-treated cells. 4-oxo-13-cis-, 4-oxo-all-trans-, or 4-oxo-9-cis-RA. Overall, we identified 34 protein spots in NHEK whose intensities increased or decreased in response to treatment NHEK protein separation by 2D PAGE We further as- with either all-trans- and/or 13-cis-RA. The intensities of four sessed the effects of different RA isomers on protein ex- of the protein spots (i.e. the cellular concentration of the pro- pression patterns in these cells. We isolated proteins from tein) changed by more than 3-fold (three increased and one vehicle-, all-trans- and 13-cis-RA-treated NHEK and sepa- decreased). The magnitude of change in the concentrations rated them on 2D SDS-PAGE (2D PAGE) gels. Examples of for other protein spots was less substantial; nonetheless, for silver-stained 2D PAGE gels for all-trans-RA-, 13-cis-RA-, 34 of the remaining protein spots, statistically significant and vehicle-treated NHEK are shown in Figure S1. Since changes in spot intensities were observed following treat- each of the duplicate NHEK cell samples was analyzed by ment with all-trans- and/or 13-cis-RA. Ten NHEK protein two independent 2D PAGE runs, it was possible to assess concentrations responded solely to 13-cis-RA treatment and the variable intensity of the protein spots for each gel and not to all-trans-RA (five increased and five decreased), each treatment. For this analysis, the integrated areas (pro- whereas six NHEK protein concentrations responded solely tein spot percentages) for 371 protein spots on each gel for to all-trans-RA treatment and not to 13-cis-RA (all de- each of the thre treatments (vehicle, all-trans-, and 13-cis- creased). For 18 protein spots, NHEK protein concentrations RA) were included. Statistical analysis indicated that the responded similarly to both all-trans- and 13-cis-RA. cube root transformation of the data gave the best linear We conducted a second study using 26 h treatment with relationship between the magnitude of the mean spot per- all-trans-, 13-cis-RA, or vehicle to confirm these results. centage and the standard deviation of these measures. This Similar responses were documented, indicating that the 146 BARON ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table I. Differential gene expression in NHEK after incubation with 10–6 M 13-cis-, all-trans-, 9-cis-, 4-oxo-13-cis-, 4-oxo-all-trans-, or 4-oxo-9-cis-RA for 24 h using the PIQOR Skin microarray (complete data see Table S1)

4-oxo-13-cis- All-trans- 4-oxo-all- 4-oxo-9- Gene name 13-cis-RA RA RA trans-RA 9-cis-RA cis-RA Interleukin-1b 3.59 2.93 3.87 3.7 3.33 2.81 Tissue-type plasminogen 3.36 3.29 2.1 2.64 3.32 3.19 activator S100 calcium-binding protein 2.83 1.15 0.67 0.79 0.92 0.87 A8 S100 calcium- binding protein 5.08 2.49 1.17 1.6 1.99 2 A9 S100 calcium-binding protein 7.53 1.95 1.73 1.72 1.99 2.2 A7 (psoriasis)

Cytokeratin 15 3.55 4.09 3.46 3.53 3.58 4.07

Cytokeratin 19 2.15 3.2 2.2 2.14 2.64 2.81

Cytokeratin 7 3.26 3.96 2.57 3.9 4.43 4.54 Interleukin-1 receptor 0.54 0.66 0.4 0.47 0.59 0.45 Vascular endothelial growth 0.6 0.69 0.62 0.6 0.6 0.69 factor c Plasminogen activator inhibitor- 0.77 0.67 0.7 0.63 0.69 0.57 2 Stromelysin-2 (matrix 0.53 0.64 0.4 0.37 0.39 0.47 metalloproteinase-10) Thrombospondin1 0.85 0.78 0.94 0.88 0.57 0.69 Fatty acid-binding protein, 0.41 0.63 0.45 0.48 0.64 0.58 epidermal (psoriasis-associated fatty acid-binding protein homolog) Cyclooxygenase-2 0.18 0.21 0.19 0.17 0.17 0.15 Cytokeratin 1 0.54 0.48 0.33 0.35 0.33 0.49 Cytokeratin 10 0.47 0.37 0.29 0.3 0.39 0.31 Cytokeratin 14 0.56 0.78 0.55 0.68 0.66 0.83 Cytokeratin 17 0.55 0.72 0.43 0.6 0.6 0.61 Filaggrin (proﬁlaggrin) 0.88 0.39 0.3 0.28 0.28 0.27

NHEK, normal human epidermal keratinocytes; RA, retinoic acid. changes in protein spot intensity reflect changes in protein concentration after treatment with RA indicated by an in- concentrations within the NHEK that arise specifically in creased/decreased intensity of each silver-stained protein these cells as a result of exposure to all-trans- and/or 13- spots. The proteins can be divided into several groups cis-RA. based on their physiological roles. Several of the changes in For the 26 h study, changes in silver-stained protein spot protein expression such as those for cytokeratins 14, 17, 19 intensities reflect changes in protein concentrations within were predicted by our gene array studies and are known to the NHEK that arise specifically in these cells through ex- be RA responsive. Two of the proteins, DNAse I precursor posure to all-trans-RA and/or 13-cis-RA. Consequently, we protein and lamin B1, are localized in the nucleus and are excised protein spots from the dried silver-stained gels for known to participate in maintaining the structure and or- identification of their tryptic fragments by MALDI-MS. ganization of the cellular genome. As far as we are able to determine, neither DNAse I precursor protein nor Lamin B1 Identities of NHEK proteins whose concentrations have been previously reported to be RA responsive. One change following all-trans- and 13-cis-RA treat- of the proteins, lecithin:retinol acyltransferase (LRAT), is ments We were able to identify eight proteins by MALDI- thought to be crucial for retinoid homeostasis in the skin MS analysis, whose concentrations changed in a similar (Kurlandsky et al, 1996). LRAT expression in the liver and manner in response to treatments with all-trans- and 13-cis- lungs has previously been identified as being RA responsive RA. The identities of these eight proteins are shown in Table (Zolfaghari and Ross, 2000, 2002). Of 11 protein spots ex- S5. For our studies, we concentrated primarily on identifying cised from 2D gels for identification, we were unable to proteins that showed relatively large changes in protein identify three of these following trypsin digestion and MA- 125 : 1 JULY 2005 4-OXO-RETINOIC ACIDS ARE ACTIVE IN SKIN CELLS 147

Figure 2 Conﬁrmation of cDNA microarray and 2D-gel electrophoresis data using real-time PCR. (a–f) TaqMan real-time PCR analyses: normal human epidermal keratinocytes (NHEK) were stimulated with different RA isomers and metabolites at a concentration of 10–6 M for 24 h. Total RNA was isolated and untreated NHEK were used as control. The relative keratin 1, 10, 13, 19, matrix metalloproteinase-3, and lamin B1 RNA levels were normalized to 18S RNA.

LDI-MS analysis. Two of the spots contained insufficient S3). Each of the 4-oxo-metabolites showed similar expres- protein to provide an identifiable fragmentation pattern. The sion profiles but significantly different effects on gene reg- third consisted of a mixture of proteins for which unequiv- ulation (Figs 1–3, Table S1, Tables S1–S4). ocal identification was not possible. Our gene array data confirm and extend earlier work on the effects of RA on gene expression in the skin, especially regarding cytokeratin gene expression. It is known that Discussion cytokeratin 10 is a marker for epidermal hyperproliferation and differentiation (Mommers et al, 2000) and its expression Retinoids are among the most effective categories of ther- is known to be upregulated in psoriasis (Mommers et al, apeutic agents used in the treatment of dermatological dis- 2000) and downregulated in NHEK (Poumay et al, 1999). orders. Although extensive studies have been conducted to Similarly, cytokeratin 13 is thought to be a marker of em- define the pharmacological action of the retinoids in the bryonic differentiation and upregulation of cytokeratin 13 by skin, their mechanism of action remains ill defined. Using all-trans-RA and tazarotene has been shown by van Ros- cDNA-Chip technology, we showed that RA and their 4- sum et al (2000) and Virtanen et al (2000). Our data con- oxo-metabolites have differential effects on gene expres- firmed that the RA isomers and their 4-oxo-metabolites sion in NHEK and fibroblasts (Fig 1, Table I, and Tables S1– analyzed in this study exert similar effects on the expression S3). Interestingly, all of the 4-oxo-RA metabolites showed of cytokeratins 10 and 13 in NHEK (Fig 1, Table I, Tables S1 transcriptional regulatory activity including 4-oxo-13-cis- and S4). We showed, at both the mRNA and protein levels, RA, which has generally been thought to be an inactive that treatment of NHEK with each of the different RA iso- metabolite of 13-cis-RA (Figs 1–3, Table I, and Tables S1– mers and their 4-oxo-metabolites downregulates mRNA 148 BARON ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 3 Confirmation of cDNA microarray and 2D gel electrophoresis data using immunofluorescence. For immunofluorescent examination, normal human epidermal keratinocytes were seeded into chamber slides and induced with 10–6 M 13-cis-, all-trans-, 9-cis-, 4-oxo-13-cis-, 4-oxo-all-trans-, or 4-oxo-9-cis-RA for 24 h. The chamber slides were then incubated with specific primary antibodies against cytokeratin 7 (CK7) or cytokeratin 19 (CK19) for 60 min and subsequently with a secondary FITC-conjugated rabbit anti-mouse antibody for 30 min.

expression of keratin 14, a known basal cell layer keratin in It is well established that MMP are important for con- stratified squamous epithelium (Green et al, 2003). In ad- nective tissue structure and function. Furthermore, it has dition, our gene array data showed upregulation of mRNA been suggested that some of the beneficial actions of re- for keratins 7 and 19 that was confirmed by immunoflu- tinoids relate to effects on MMP gene expression. Our re- oresence studies of NHEK treated with the different results showed that RA treatment downregulated both MMP- tinoids (Fig 3, Table I, and Table S1). 3 (stromelysin-1, Fig 3) and MMP-10 (stromelysin-2, Table I, 125 : 1 JULY 2005 4-OXO-RETINOIC ACIDS ARE ACTIVE IN SKIN CELLS 149 and Table S1) in NHEK. Overexpression of MMP-3 is asso- tion of tissue-type plasminogen activator, especially after ciated with poor wound healing (Fray et al, 2003). Moreover, stimulation of NHEK with 13-cis- or 4-oxo-13-cis-RA and a MMP-3 is overexpressed in murine skin tumors and is as- downregulation of plasminogen activator inhibitor-2 by all sociated with a metastatic cell phenotype (Leite-Browning RA analyzed. This activation of the plasminogen activator et al, 2002). Increased expression of MMP-3 has also been system may be one mechanism whereby all-trans-RA ex- found in human malignant melanoma, especially in the exerts beneficial effects in cutaneous wound healing. tracellular matrix adjacent to blood vessels (Bodey et al, PA-FABP is a new member of a group of low-molecular- 2001). We propose that the downregulatory effect of each of weight proteins that are strongly upregulated in psoriatic the retinoids on MMP-3 expression could explain some of skin and that share a similarity with fatty acid-binding pro- the known anti-carcinogenic properties of the retinoids. A teins (Madsen et al, 1992). Ha et al (2004) recently showed similar downregulatory effect on the expression of other that expression of PA-FABP was elevated in senescent hu- MMP was described in vivo and in vitro after administration man dermal microvascular endothelial cells, suggesting that of all-trans-RA. Levels of latent and active MMP-9 and overexpression of FABP in cultured senescent human der- MMP-13 were reduced by 50%–75% (po0.05) by all-trans- mal microvascular endothelial cells is closely related to skin RA in organ cultures of skin from streptozotocin-diabetic aging. Our cDNA microarray in vitro study (Table I, Table S1) rats, and Fisher et al (1999) showed that pretreatment of revealed a 2-fold downregulation of PA-FABP, suggesting human skin with all-trans-RA inhibits UV induction of MMP, that retinoids can alter the fatty acid metabolism in human suggesting that this retinoid can reverse UV-induced dam- skin cells. age to connective tissue and may be able to limit or improve Kanekura et al (2000) showed that the constitutive ex- photoaging changes in the skin. MMP-10 (stromelysin-2) is pressions of cyclooxygenase (COX)-2 protein and pros- expressed in keratinocytes growing at the edge of healing taglandin E(2) (PGE(2)) biosynthesis were significantly skin wounds. Krampert et al (2004) showed that precise enhanced in human skin epidermal cancer cells and that regulation of stromelysin-2 is required for limited matrix cancer cell growth was effectively inhibited by the suppres- degradation at the wound site, thereby controlling keratin- sion of COX-2 expression by transfection with COX-2 an- ocyte migration. This correlates with our findings, revealing tisense oligonucleotide. Retinoids and antioxidants have a strong downregulatory effect of all analyzed retinoids on been used for the chemoprevention of cancers in several expression of MMP-10. tissues and treatment with 9-cis-RA suppressed COX-2 ex- In human epidermis, basal keratinocytes are the main pression and PGE(2) biosynthesis and cell growth in human source of IL-1, and IL-1a is the predominant isoform, skin squamous carcinoma cells. We have shown a 5-fold whereas IL-1b appears to be expressed predominantly in downregulation of COX-2 by all RA isomers and 4-oxo- cultured keratinocytes. Radioimmunoassay performed by metabolites analyzed (Table I, Table S1). These results fur- Gatto et al (1993) showed that after stimulation by all-trans- ther support the hypothesis that retinoids play a role in RA, intracellular IL-1b is predominantly increased, with no preventing skin cancer growth by influencing the expression significant modification of IL-1a expression. This is consist- of COX-2. ent with our findings, revealing a significant upregulation of Filaggrin is a basic protein normally present in the stra- IL-1b after stimulation with RA isomers and their 4-oxo- tum corneum. It derives from profillagrin, a high-molecular- metabolites both after 3 and 24 h of incubation (Table I, weight precursor synthesized in the stratum granulosum Tables S1 and S2). that is thought to be associated with the keratohyaline VEGF is overexpressed in hyperproliferative diseases, granules of granular cells. Asselineau et al (1990) showed such as psoriasis and cancers, in which there is increased that the accumulation of filaggrin in the outermost layers of angiogenesis. Using normal human keratinocytes in con- submerged cultured human keratinocytes is enhanced after ventional cultures and skin grafted onto nude mice in vivo, removing retinoids from the serum supplement and subse- Diaz et al (2000) showed that retinoids can inhibit TPA-me- quently inhibited when small concentrations of RA are re- diated VEGF gene induction at the transcriptional level. We added to the culture medium. This correlates with our cDNA obtained similar results in array experiments after incuba- array data revealing downregulaton of filaggrin after incu- tion with 10–6 M 13-cis-, all-trans-, 9-cis-, 4-oxo-13-cis-, 4- bation with RA isomers and especially their 4-oxo-met- oxo-all-trans-, or 4-oxo-9-cis-RA for 3 and 24 h. abolites (Table I, Table S1). Axel et al (2001) demonstrated downregulation of mRNA Each of the RA isomers and their 4-oxo-metabolites al- expression of the glycoprotein thrombospondin-1 in human tered gene expression in skin fibroblasts (Table S3). The arterial smooth muscle cells after all-trans-RA treatment. retinoids downregulated several CYP isoenzymes and Similar findings were detected in our in vitro studies using transporter proteins such as the monocarboxylate trans- NHEK (Table I, Tables S1 and S2). porter 1 (SLC16A1), which is responsible for the cellular The activation of the proteolytic plasminogen activator uptake of monocarboxylic acids, i.e., lactate, pyruvate, and system is important for the re-epithelialization of skin ketone bodies. (Garcia et al, 1994; Koehler-Stec et al, 1998; wounds. Keratinocytes synthesize and secrete the uroki- Daberkow et al, 2003). Regulation of the expression of these nase-type plasminogen activator, which binds to its specific transporters by RA (Table S3) has not been described pre- keratinocyte surface receptor. Receptor-bound urokinase- viously. Similarly, retinoid treatment downregulated the ex- type plasminogen activator efficiently activates cell surface- pression of collagen types VI and XVII in fibroblasts. Ogawa bound plasminogen. Braungart et al (2001) showed that all- et al (1998) showed that increasing concentrations of all- trans RA augments urokinase-type plasminogen activator. trans-RA decrease cell proliferation and synthesis of Our studies revealed similar effects, showing an upregula- collagen in human dermal fibroblasts. This observation 150 BARON ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY correlates well with our data and taken together, these strated that the 4-oxo-RA metabolites have transcriptional findings suggest that retinoids could have therapeutic ef- activity in skin cells and that the various isomers have dis- fects in hypertrophic scars and keloids (Daly and Weston, tinct transactivational profiles that are specific to NHEK and 1986; Ogawa et al, 1998). We also observed upregulation of dermal fibroblasts. In addition, we have identified several genes encoding annexin I and V in fibroblasts by all-trans- novel gene products, either as mRNA species or proteins and 13-cis-RA (Table S3). RA treatment of growth plate that are present in NHEK or fibroblasts and that were not chondrocytes is reported to cause an increase in cytosolic previously known to be RA responsive. Further studies are calcium concentration, followed by upregulation of annexin needed to clarify the mechanism whereby these very dis- gene expression, including annexins II, V, and VI gene ex- tinct actions are coordinately regulated and controlled in pression (Wang et al, 2003). Possibly, a similar mechanism maintaining skin homeostasis. is involved in the upregulation of annexins I and V in dermal fibroblasts. Through MALDI-MS analysis of 2D gels, we showed that Material and Methods the retinoids altered the expression of 8 proteins in NHEK. Three of these were cytokeratins (14, 17, and 19) for which Cell culture NHEK were obtained from healthy male donors un- changes in mRNA levels could account for an increase/de- dergoing circumcision. All studies were approved by the ethical crease in cellular protein level. In the case of lamin B1, we committee of the University Hospital, RWTH, Aachen. Participants gave their written informed consent, and the study was conducted were unable to detect changes in NHEK levels of mRNA according to the Declaration of Helsinki Principles. Skin specimens encoding the protein (Fig 3f). LRAT mRNA and activity levels were rinsed three times in sterile phosphate-buffered saline (PBS) in rodent liver and lungs are reported to be responsive to RA (PAA, Linz, Austria), containing antibiotics (penicillin/streptomycin, (Zolfaghari and Ross, 2000, 2002). LRAT has been pro- PAA), and amphothericin B (PAA) and cut into 2 2 mm small posed to be important in keratinocytes for maintaining re- pieces. These were placed in 2 U per mL dispase solution (Gib- tinoid availability to allow for the continued synthesis of RA coBRL, Paisley, Scotland, UK) overnight at 41C and additionally 1 in the cells (Kurlandsky et al, 1996; Zolfaghari and Ross, incubated at 37 C for 30 min. The epidermis was peeled from the dermis and incubated in trypsin/EDTA (Cambrex, Walkersville, 2000, 2002). It is thought that, when RA levels are sufficient Maryland) for 30 min at 371C. The enzymatic digestion was to meet cellular needs, LRAT level/activity is upregulated to stopped by Trypsin Neutralization Solution (Cambrex). The stratum allow for additional retinoid storage. Our finding that LRAT corneum was removed by filtering the digest through a 40 mm protein levels are upregulated in RA-treated NHEK is con- nylon strainer (Falcon, Franklin Lakes, New Jersey). The resulting sistent with this notion. This is a report of an increase in cell suspension was centrifuged for 6 min at 500 g (Heraeus LRAT protein levels in RA-treated cells that is not accom- Labofuge 400, Hanau, Germany), and the cell pellet was resus- panied by a change in LRAT mRNA levels as assessed by pended in fully supplemented keratinocyte growth medium (KGM, Cambrex, 0.015 mM calcium). The cells were cultured in a humid- Northern blot analysis. Our data do not provide an expla- ified atmosphere at 5% CO2 (MCBI incubator, Sanyo, Japan). Cells nation for this observation aside from suggesting that either used in our studies were always from the first and second passage the rate(s) of LRAT protein synthesis and/or degradation and in a state of late subconfluency. must be altered in NHEK treated with RA. Keratinocytes used in the 2D experiment were cultured in Lamin B1 proteins levels but not mRNA levels, were also Dulbecco’s modified Eagle’s medium (DMEM) (high glucose)/ elevated in RA-treated NHEK and this may be especially Ham’s F12 (3:1) Medium (GibcoBRL) supplemented with 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany), antibiotics interesting for understanding all-trans- and 13-cis-RA ac- (penicillin/streptomycin, PAA), amphothericin B (PAA), adenine tions within NHEK, suggesting for the first time that this (0.18 mM) (Sigma Chemical, St Louis, Missouri), cholera toxin protein is retinoid responsive. Lamin B1 is a major compo- (100 pM) (Sigma), epidermal growth factor (10 ng per mL) (Roche, nent of the nuclear lamina, a proteinaceous layer found at Mannheim, Germany), FCS (10%) (Biochrom), hydrocortisone the interface between chromatin and the inner nuclear (0.4 mg per mL) (Serva, Heidelberg, Germany), insulin (5 mg per membrane, and it is thought to interact with the basal tran- mL) (Roche), non-essential amino acids (PAA), transferrin (5 mg per scription machinery and to provide a scaffold for the as- mL) (Promo Cell, Heidelberg, Germany), and triiodothyronine (2 nM) (Sigma). sembly or stabilization of active transcription complexes Normal human fibroblasts were obtained from discarded fore- (Goldman et al, 2002; Holaska et al, 2002; Shumaker et al, skin specimens. After the removal of the epidermis, the dermis was 2003). Lamin assembly is proposed to be involved in trig- placed in a dry cell culture multi-well plate (Falcon) for 20 min, to gering apoptosis and is important for nuclear envelope for- allow adherence to the polystyrene surface of the culture plate. mation and integrity. Although lamin B1 is not currently DMEM (high glucose and containing L-glutamine; GibcoBRL) sup- known to play a role in the development of skin disease, it is plemented with 10% FCS (Biochrom), antibiotics (penicillin/strep- widely expressed in cells found in the skin (Broers et al, tomycin, PAA), and amphothericin B (PAA) was added. Fibroblast outgrowth was monitored by light microscopy (LEICA DMIL; Leitz, 1997; Tilli et al, 2003). Should the changes that we observed Wetzlar, Germany), and dermal remnants were removed after 5–7 in lamin B1 protein concentrations in NHEK not involve days. On reaching late subconfluency, cells were subcultivated transcriptional mechanisms, this might be very important for using the same media. Cells used in our experiments were from understanding non-genomic actions of all-trans- and/or 13- low passages (po5) and in a state of late subconfluency. cis-RA within skin cells. In summary, we have identified substantial metabolic Retinoid treatment of the cells Under dim red light, we added either RA isomers or their 4-oxo-metabolites (kindly provided by Dr differences between NHEK and human dermal fibroblasts in Ulf Wiegand, F. Hoffman La-Roche, Basel, Switzerland) freshly the processing of RA and their 4-oxo-metabolites. These prepared as stocks in dimethyl sulfoxide (Merck, Darmstadt, Ger- differences may help to explain the differential therapeutic many) to NHEK or dermal fibroblasts in culture. In all these exper- efficacy of RA isomers and their metabolites. We demon- iments, the final concentration of the solvent added to the culture 125 : 1 JULY 2005 4-OXO-RETINOIC ACIDS ARE ACTIVE IN SKIN CELLS 151 medium was less than 0.1% of the culture medium. Treated cells stimulated control of NHEK. Thus, results are referred to as tran- were incubated for 24 or 72 h at 5% CO2 and 371C in a humidified scription relative to the calibrator. atmosphere in the dark. Prior to expression studies, high-perform- ance liquid chromatography analyses of treated NHEK and fibro- 2PAGE Pelleted NHEK treated either with ethanol (vehicle), 2 mM blasts were conducted to verify the stability of RA isomers and all-trans-, or 2 mM 13-cis-RA were homogenized in buffer contain- their 4-oxo-metabolites in the presence of these cell types (data ing protease inhibitors and treated with nuclease to digest DNA. not shown). Cellular protein content was determined by the Coomassie dye- binding assay (Bio-Rad), and 50 mg total protein was loaded onto Microarray analysis (radiolabeled probes) Total RNA isolated an isoelectric focusing gel 2.0 mm inner diameter using 2% pH from dermal fibroblasts that had been incubated with the different range 3.5–10.0 ampholines (Amersham Pharmacia Biotech, Pi- RA isomers and their 4-oxo-metabolites was isolated using peq- scataway, New Jersey). Isoelectric focusing was carried out for GOLD RNA-Pure (peqlab, Biotechnologie GmbH, Erlangen, Ger- 20,000 Volt-h. Following isoelectric focusing, the proteins were many) according to protocols provided by the manufacturer. separated in the second dimension by SDS-PAGE on 10% gels. Generation of 33P-labeled cDNA probes was performed by re- The 2D electrophoresis separations were carried out according to verse transcription of 10 mg total RNA with 33P dCTP for 90 min at the method of O’Farrell (1975). SDS slab gel electrophoresis was 371C following the Mammalian GeneFilters (Research, Genetics, carried out for 4 h at 12.5 mA per gel. As molecular weight stand- Invitrogen, Karlsruhe Germany or Carlsbad, California) protocol. To ards, we used myosin (220,000), phosphorylase A (94,000), cat- remove unincorporated nucleotides, the probes were passed alase (60,000), actin (43,000), carbonic anhydrase (29,000), and through Bio-Spin 6 chromatography columns (Bio-Rad Labora- lysozyme (14,000) (Sigma Chemical). After electrophoresis, each tories GmbH, Mu¨ nchen, Germany). Purified radiolabeled probes 2D electrophoresis gel was silver stained (Bio-Rad) and scanned were applied to DermArray GeneFilters microarrays and allowed to by a laser densitometer (Molecular Dynamics, Sunnyvale, Califor- hybridize at 421C overnight. After hybridization, the filters were nia). Patterns of silver-stained proteins were analyzed by Progen- washed twice with 2 SSC, 0.1% SDS at 501C for 1 h and once esis software (Nonlinear Technology, Madison, Wisconsin), such with 0.5 SSC, 0.1% SDS for 30 min at 551C. Afterwards, filters that all major protein spots and all changing protein spots were were exposed to a Kodak X-OMAT AR-5 film at 701C in cassettes outlined, quantified, and matched for each duplicate gel for each of containing intensifying screens. We analyzed suitably exposed au- the three treatments. For each silver-stained 2D PAGE gel, the toradiograms using Pathway analysis software (Research Genet- intensity of each spot was calculated as a spot percentage that ics). equals the integrated density expressed as a percentage of the total density summed for all protein spots identified. Microarray analysis (fluorescent probes) Linear amplification of total RNA, labeling, and hybridization were performed according to Identification of NHEK proteins by MALDI-MS The identities of the manufacturer’s directions (Eberwine, 1996; Bosio et al, 2002). NHEK proteins present as silver-stained spots on the 2D PAGE Amplified RNA (aRNA) samples were quantified by spectropho- gels were determined through MALDI-MS analyses carried out by tometry, and the quality was verified by capillary electrophoresis the Protein Chemistry Core Facility of the Howard Hughes Medical (Bioanalyser 2001, Agilent Technologies GmbH, Boblingen, Ger- Institute at Columbia University. For this purpose, silver-stained many). Two micrograms of aRNA from each probe was labeled by protein spots were carefully dissected from the dried 2D PAGE gels reverse transcription with Cy5 and Cy3 fluorescent nucleotides, using a sharp pointed scalpel. The excised gel fragment was respectively, and subjected to a PIQOR Skin Microarray. Process- treated with trypsin to digest proteins in situ. The resulting tryptic ing of microarrays was performed using a fully automated hybrid- fragments were then subjected to MALDI-MS analysis, and the ization station according to the manufacturer’s guidelines (a-Hyb, identities of proteins present in the spots were determined through Memorec, Cologne, Germany). Image capture and signal quanti- search of the NCBI GenBank database. Detailed protocols for fication of hybridized PIQOR cDNA arrays were carried out with the these procedures are available online (http://cpmcnet.columbia. ScanArray Lite (Perkin Elmer, Life and Analytic Sciences, Boston, edu/dept/protein). Massachusetts) and ImaGene software version 4.1 (BioDiscovery, Los Angeles, California). Immunofluorescence For immunofluorescence examination of monolayers, cells were seeded into chamber slides (4-well Lab-Tek Quantitative real-time PCR Total RNA from NHEK was isolated II, Nunc (GmbH and Co.) Wiesbaden, Germany). NHEK were fixed using the High Pure RNA Isolation kit (Roche) according to the in ice-cold methanol for 10 min, rinsed in PBS, and washed in tris- manufacturer’s instructions with inclusion of the DNase step. Pu- buffered saline (TBS) (DAKO, Carpinteria, California) for 10 min. rified RNA was reverse transcribed with the TaqMan Reverse Tran- The chamber slides were incubated with primary monoclonal an- scription Reagents kit (Applied Biosystems, Weiterstadt, Germany) tibodies (anti-human cytokeratin 7 (Novocastra, Newcastle upon with random hexamers as primers. TaqMan experiments were car- Tyne, UK) diluted 1:50 or anti-human cytokeratin 19 (Research Di- ried out on an ABI PRISM 7000 Sequence Detection System (Ap- agnostics, Flanders, New Jersey) diluted 1:10). For each specific plied Biosystems) using ‘‘Assay on demand’’ primer/probe sets for antibody or the isotypic control (Sigma), incubations were for 60 human keratins 1, 10, 13, and 19, MMP-3, and lamin B1 (Applied min. Slides were washed three times for 10 min each with TBS. Biosystems). A pre-developed TaqMan Assay Reagent for human Subsequently, cells were incubated for 30 min with a secondary 18S-RNA (Applied Biosystems) was chosen as an endogenous FITC-conjugated rabbit anti-mouse antibody (DAKO), washed two control for normalization of the RNA load. times for 10 min with TBS, and mounted with Immu-Mount (Shan- don, Pittsburgh, Pennsylvania). Quantification of target gene expression The standard curve method was used for quantification of gene expression (Applied Biosystems, User Bulletin 2). Briefly, a cDNA sample of unstimu- These studies were supported by grants R01 DK052444 and R01 lated NHEK was 2-fold serial diluted to construct standard curves DK061310 from the National Institutes of Health. for human keratins 1, 10, 13, and 19, MMP-3, and lamin B1, and 18S RNA. The keratins, MMP-3, and lamin B1 messages in un- known samples were quantified, using the standard curves, to de- Supplementary Material termine a relative measure of the starting amount. Each sample The following material is available from http://www.blackwellpublish- was then normalized on the basis of 18S RNA expression. Finally, ing.com/products/journals/suppmat/JID/JID23791/JID23791.htm the normalized amount of the target gene is divided by the target Figure S1 Two-dimensional (2D) gel electrophoresis of total cellular gene quantity of the calibrator, with the calibrator being the un- proteins from NHEKs treated for 26 h with 2 mM 13-cis-RA. 152 BARON ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table S1 Differential gene expression in NHEKs after incubation with Fisher GJ, Talwar HS, Lin J, Voorhees JJ: Molecular mechanisms of photoaging 6 10 M 13-cis-RA, all-trans-RA, 9-cis-RA, 4-oxo-13-cis-RA, 4-oxo-all- in human skin in vivo and their prevention by all-trans retinoic acid. Pho- trans-RA or 4-oxo-9-cis-RA for 24 hours using the PIQOR Skin tochem Photobiol 69:154–157, 1999 microarray. Fray MJ, Dickinson RP, Huggins JP, Occleston NL: A potent, selective inhibitor of Table S2 Differential gene expression in NHEKs after incubation with matrix metalloproteinase-3 for the topical treatment of chronic dermal 106 M 13-cis-RA, all-trans-RA, 9-cis-RA, 4-oxo-13-cis-RA, 4-oxo-all- ulcers. J Med Chem 46:3514–3525, 2003 trans-RA or 4-oxo-9-cis-RA for 3 hours using the PIQOR Skin Garcia CK, Li X, Luna J, Francke U: cDNA cloning of the human monocarboxylate microarray. Table S3 Differential gene expression in dermal fibroblasts after transporter 1 and chromosomal localization of the SLC16A1 locus to incubation with the RA-isomers and 4-oxo metabolites for 24 hours 1p13.2–p12. Genomics 23:500–503, 1994 using the DermArray GeneFilters microarray (¼not regulated). Gatto H, Richard MH, Viac J, Charveron M, Schmitt D: Effects of retinoic acid Table S4 TaqMan real-time PCR analyses of NHEKs stimulated with on interleukin-1 alpha and -1 beta expression by normal human keratin- different RA isomers and metabolites at concentrations of 107and 108 ocytes cultured in defined medium. Skin Pharmacol 6:10–19, 1993 M for 24 hours. Goldman RD, Gruenbaum Y, Moir RD, Shumaker DK, Spann TP: Nuclear lamins: Table S5 Human keratinocyte proteins identified by MALDI-MS whose Building blocks of nuclear architecture. Genes Dev 16:533–547, 2002 levels change upon treatment with all-trans-retinoic acid or 13-cis- Green H, Easly K, Iuchi S: Marker succession during the development of ker- retinoic acid. atinocytes from cultured human embryonic stem cells. Proc Natl Acad Sci USA 100:15625–15630, 2003 DOI: 10.1111/j.0022-202X.2005.23791.x Gudas LJ, Sporn MB, Roberts AB: Cellular biology and biochemistry of the retinoids. In: Sporn MB, Roberts AB, Goodman DS (eds). The Retinoids: Manuscript received September 7, 2004; revised January 27, 2005; Biology, Chemistry, and Medicine, 2nd edn. New York: Raven Press, accepted for publication February 8, 2005 1994; p 443–520 Ha MK, Chung KY, Lee JH, Bang D, Park YK, Lee KH: Expression of psoriasis- Address correspondence to: PD Dr med Jens Malte Baron, Depart- associated fatty acid-binding protein in senescent human dermal micro- ment of Dermatology, University Hospital RWTH Aachen, Pauwels- vascular endothelial cells. Exp Dermatol 13:543–550, 2004 strae 30, D-52074 Aachen, Germany. Email: JensMalte.Baron@ Holaska JM, Wilson KL, Mansharamani M: The nuclear envelope, lamins and post.rwth-aachen.de nuclear assembly. Curr Opin Cell Biol 14:357–364, 2002 Hoyos B, Imam A, Chua R, et al: The cysteine-rich regions of the regulatory domains of Raf and protein kinase C as retinoid receptors. J Exp Med References 192:835–845, 2000 Imam A, Hoyos B, Swenson C, Levi E, Chua R, Viriya E, Hammerling U: Retinoids Asselineau D, Dale BA, Bernard BA: Filaggrin production by cultured human as ligands and coactivators of protein kinase C alpha. FASEB J 15:28–30, epidermal keratinocytes and its regulation by retinoic acid. Differentiation 2001 45:221–229, 1990 Kanekura T, Higashi Y, Kanzaki T: Inhibitory effects of 9-cis-retinoic acid and Axel DI, Frigge A, Dittmann J, et al: All-trans retinoic acid regulates proliferation, pyrrolidinedithiocarbamate on cyclooxygenase (COX)-2 expression migration, differentiation, and extracellular matrix turnover of human ar- and cell growth in human skin squamous carcinoma cells. Cancer Lett terial smooth muscle cells. Cardiovasc Res 49:851–862, 2001 161:177–183, 2000 Balmer JE, Blomhoff R: Gene expression regulation by retinoic acid. J Lipid Res Karlsson T, Vahlquist A, Kedishvili N, Torma H: 13-cis-retinoic acid competitively 43:1773–1808, 2002 inhibits 3 alpha-hydroxysteroid oxidation by retinol dehydrogenase Beard RL, Chandraratna RAS: RAR-selective ligands: Receptor subtype and RoDH-4: A mechanism for its anti-androgenic effects in sebaceous function selectivity. In: Nau H, Blaner WS (eds). The Handbook of Exper- glands? Biochem Biophys Res Commun 303:273–278, 2003 imental Pharmacology, The Retinoids. Heidelberg: Springer Verlag, 1999; Koehler-Stec EM, Simpson IA, Vannucci SJ, Landschulz KT, Landschulz WH: p 185–235 Monocarboxylate transporter expression in mouse brain. Am J Physiol Blaner WS: Cellular metabolism and actions of 13-cis-retinoic acid. J Am Acad 275:516–524, 1998 Dermatol 45:129–135, 2001 Korichneva I, Waka J, Hammerling U: Regulation of cardiac mitochondrial mem- Bodey B, Bodey B Jr, Siegel SE, Kaiser HE: Matrix metalloproteinase expression brane potential by retinoids. J Pharmacol Exp Ther 305:426–433, 2003 in malignant melanomas: Tumor–extracellular matrix interactions in inva- Krampert M, Bloch W, Sasaki T, et al: Activities of the matrix metalloproteinase sion and metastasis. In Vivo 15:57–64, 2001 stromelysin-2 (MMP-10) in matrix degradation and keratinocyte organi- Bosio A, Kno¨ rr C, Janssen U, Gebel S, Haussmann HJ, Mu¨ ller T: Kinetics of gene zation in wounded skin. Mol Biol Cell 15:5242–5254, 2004 expression profiling in Swiss 3T3 cells exposed to aqueous extracts of Kurlandsky SB, Duell EA, Kang S, Voorhees JJ, Fisher GJ: Auto-regulation of cigarette smoke. Carcinogenesis 23:741–748, 2002 retinoic acid biosynthesis through regulation of retinol esterification in Braungart E, Magdolen V, Degitz K: Retinoic acid upregulates the plasminogen human keratinocytes. J Biol Chem 271:15346–15352, 1996 activator system in human epidermal keratinocytes. J Invest Dermatol Leite-Browning ML, McCawley LJ, Choi OH, Matrisian LM, Ochieng J: Interac- 116:778–784, 2001 tions of alpha2-HS-glycoprotein (fetuin) with MMP-3 and murine Broers JLV, Machiels BM, Kuijpers HJH, Smedts F, van den Kieboom R, Raymond squamous cell carcinoma cells. Int J Oncol 21:965–971, 2002 Y, Ramaekers FCS: A- and B-type lamins are differentially expressed in Madsen P, Rasmussen HH, Leffers H, Honore B, Celis JE: Molecular cloning and normal human tissues histochem. Cell Biol 107:505–517, 1997 expression of a novel keratinocyte protein (psoriasis-associated fatty ac- Chambon P: The retinoid signaling pathway: Molecular and genetic analyses. id-binding protein [PA-FABP]) that is highly up-regulated in psoriatic skin Semin Cell Biol 5:115–125, 1994 and that shares similarity to fatty acid-binding proteins. J Invest Dermatol Chambon P: A decade of molecular biology of retinoic acid receptors. FASEB J 99:299–305, 1992 10:940–954, 1996 Mangelsdorf DJ, Borgmeyer U, Heyman RA, et al: Characterization of three RXR Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ: Nuclear receptors and lipid genes that mediate the action of 9-cis-retinoic acid. Genes Dev 6: physiology: Opening the X-files. Science 294:1866–1870, 2001 Clagett-Dame M, DeLuca HF: The role of vitamin A in mammalian reproduction 329–334, 1992 and embryonic development. Annu Rev Nutr 22:347–381, 2002 Mangelsdorf DJ, Thummel C, Beato M, et al: The nuclear receptor superfamily: Daberkow RL, White BR, Cederberg RA, Griffin JB, Zempleni J: Monocarboxylate The second decade. Cell 83:835–839, 1995 transporter 1 mediates biotin uptake in human peripheral blood mono- Mangelsdorf DJ, Umesono K, Evans RM: The retinoid receptors. In: Sporn MB, nuclear cells. J Nutr 133:2703–2706, 2003 Roberts AV, Goodman DS (eds). The Retinoids, Biology, Chemistry, and Daly TJ, Weston WL: Retinoid effects on fibroblast proliferation and collagen Medicine, 2nd edn. New York: Raven Press, 1994; p 319–350 synthesis in vitro and on fibrotic disease in vivo. J Am Acad Dermatol Mommers JM, van Rossum MM, van Erp PE, van De Kerkhof PC: Changes in 15:900–902, 1986 keratin 6 and keratin 10 (co-)expression in lesional and symptomless skin Diaz BV, Lenoir MC, Ladoux A, Frelin C, Demarchez M, Michel S: Regulation of of spreading psoriasis. Dermatology 201:15–20, 2000 vascular endothelial growth factor expression in human keratinocytes by Myhre AM, Takahashi N, Blomhoff R, Breitman TR, Norum KR: Retinoylation of retinoids. J Biol Chem 275:642–650, 2000 proteins in rat liver, kidney, and lung in vivo. J Lipid Res 37:1971–1977, Eberwine J: Amplification of mRNA populations using aRNA generated from 1996 immobilized oligo(dT)-T7 primed cDNA. Biotechniques 20:584–591, O’Farrell PH: High resolution two-dimensional electrophoresis of proteins. J Biol 1996 Chem 250:4007–4021, 1975 125 : 1 JULY 2005 4-OXO-RETINOIC ACIDS ARE ACTIVE IN SKIN CELLS 153

Ogawa A, Maeguchi M, Uchida Y, Yoshioka T, Kawashima M, Muraki T: Effect of Takahashi N, Jetten AM, Breitman TR: Retinoylation of cytokeratins in normal tazarotene, an acetylenic retinoid, on human dermal fibroblast. Jpn J human epidermal keratinocytes. Biochem Biophys Res Commun 180: Pharmacol 76:317–319, 1998 393–400, 1991a Peck GL, DiGiovanna JJ: Synthetic retinoids in dermatology. In: Sporn MB, Takahashi N, Liapi C, Anderson WB, Breitman TR: Retinoylation of the cAMP- Roberts AB, Goodman DS (eds). The Retinoids: Biology, Chemistry, and binding regulatory subunits of type II cAMP-dependent protein kinases in Medicine, 2nd Edition. New York: Raven Press Ltd., 1994; p 631–658 HL60 cells. Arch Biochem Biophys 290:293–302, 1991b Peck SM, Chargin L, Sobotka H: Keratosis follicularis (Darier’s disease)—a vi- Tilli CM, Ramaekers FC, Broers JL, Hutchison CJ, Neumann HA: Lamin expres- tamin A-deficiency disease. Arch Dermatol Syphilol 43:223, 1949 sion in normal human skin, actinic keratosis, squamous cell carcinoma Poumay Y, Herphelin F, Smits P, De Potter IY, Pittelkow MR: High-cell-density phorbol ester and retinoic acid upregulate involucrin and downregulate and basal cell carcinoma. Br J Dermatol 148:102–109, 2003 suprabasal keratin 10 in autocrine cultures of human epidermal keratin- van Rossum MM, Mommers JM, van de Kerkhof PC, van Erp PE: Coexpression ocytes. Mol Cell Biol Res Commun 2:138–144, 1999 of keratins 13 and 16 in human keratinocytes indicates association be- Radominska-Pandya A, Chen G, Czernik PJ, Little JM, Samokyszyn VM, Carter tween hyperproliferation-associated and retinoid-induced differentiation. CA, Nowak G: Direct interaction of all-trans-retinoic acid with protein Arch Dermatol Res 292:16–20, 2000 kinase C (PKC). J Biol Chem 275:22324–22330, 2000 Virtanen M, Torma H, Vahlquist A: Keratin 4 upregulation by retinoic acid in vivo:A Roos TC, Jugert FK, Merk HF, Bickers DR: Retinoid metabolism in the skin. sensitive marker for retinoid bioactivity in human epidermis. J Invest De- Pharmacol Rev 50:315–333, 1998 rmatol 114:487–493, 2000 Ross SA, McCaffery PJ, Drager UC, De Luca LM: Retinoids in embryonal de- Wang W, Xu J, Kirsch T: Annexin-mediated Ca2 þ influx regulates growth plate velopment. Physiol Rev 80:1021–1054, 2000 chondrocyte maturation and apoptosis. J Biol Chem 278:3762–3769, 2003 Shroot B, Gibson DFC, Lu XP: Retinoic receptor-selective agonists and their Zolfaghari R, Ross AC: Lecithin:retinol acyltransferase from mouse and rat liver: action in skin. In: Nau H, Blaner WS (eds). The Handbook of Experimental cDNA cloning and liver specific regulation by dietary vitamin A and re- Pharmacology, The Retinoids. Springer Verlag, Heidelberg: 1999; p 539–559 tinoic acid. J Lipid Res 41:2024–2034, 2000 Shumaker DK, Kuczmarski E, Goldman RD: The nucleoskeleton: Lamins and Zolfaghari R, Ross AC: Lecithin:retinol acyltransferase expression is regulated by actin are major players in essential nuclear functions. Curr Opin Cell Biol dietary vitamin A and exogenous retinoic acid in the lung of adult rats. J 15:358–366, 2003 Nutr 132:1160–1164, 2002 Takahashi N, Breitman TR: Covalent modification of proteins by ligands of steroid Zouboulis CC: Retinoids—which dermatological indications will benefit in the hormone receptors. Proc Natl Acad Sci USA 89:10807–10811, 1992 near future? Skin Pharmacol Appl Skin Physiol 14:303–315, 2001