Tài liệu Báo cáo khoa học: Role of ceramide kinase in peroxisome proliferatoractivated receptor beta-induced cell survival of mouse keratinocytes ppt

Role of ceramide kinase in peroxisome proliferatoractivated receptor beta-induced cell survival of mouse keratinocytes Kiyomi Tsuji1, Susumu Mitsutake2, Urara Yokose2, Masako Sugiura3, Takafumi Kohama4 and Yasuyuki Igarashi1,2 Laboratory of Biomembrane and Biofunctional Chemistry, Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan Laboratory of Biomembrane and Biofunctional Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Biological Research Laboratories II, Daiichi-Sankyo Co Ltd., Tokyo, Japan Exploratory Research Laboratories I, Daiichi-Sankyo Co Ltd., Tokyo, Japan Keywords cell survival; ceramide; ceramide 1-phosphate; CerK; PPARb Correspondence Y Igarashi, Laboratory of Biomembrane and Biofunctional Chemistry, Faculty of Pharmaceutical Sciences and Faculty of Advanced Life Sciences, Hokkaido University, Nishi 6, Kita 12, Kita-ku, Sapporo 060-0812, Japan Fax: +81 11 706 4986 Tel: +81 11 706 3970 E-mail: yigarash@pharm.hokudai.ac.jp Website: http://biomem.pharm.hokudai.ac.jp/ english/index.html (Received April 2008, revised 26 May 2008, accepted 29 May 2008) doi:10.1111/j.1742-4658.2008.06527.x Ceramide (Cer) is known to be a lipid mediator in apoptosis and to have an important role in cell fate, via control of intracellular Cer levels Recently, ceramide kinase (CerK) was identified as an enzyme that converts Cer to ceramide 1-phosphate (C1P) We examined potential functions of CerK in the regulation of keratinocyte survival, and the possible involvement of peroxisome proliferator-activated receptor beta (PPARb) PPARb is known to be a nuclear receptor acting as a ligand-inducible transcription factor and has been implicated in the control of keratinocyte survival In the mouse keratinocyte cell line SP1, serum starvation induced cell death and the accumulation of intracellular Cer, an apoptotic event However, apoptosis was inhibited by activation of PPARb Interestingly, activation of PPARb enhanced the mRNA expression of CerK and CerK activity Furthermore, the cell survival effect of PPARb was greatly diminished in keratinocytes isolated from CerK-null mice Chromatin immunoprecipitation revealed that, in vivo, PPARb binds to the CerK gene via a sequence located in the first intron Electrophoretic mobility-shift assays confirmed that PPARb associates with this sequence in vitro These findings indicated that CerK gene expression was directly regulated by PPARb In conclusion, our results demonstrate that PPARb-mediated upregulation of CerK gene expression is necessary for keratinocyte survival against serum starvation-induced apoptosis Ceramide (Cer) has been implicated in various cellular processes including proliferation, apoptosis and cell signaling [1] Intracellular Cer levels are strictly regulated by several enzymes, including ceramide kinase (CerK), which converts Cer to ceramide 1-phosphate (C1P) [2] Previous studies have suggested that CerK and C1P are involved in many cell functions, including membrane fusion, phagocytosis and degranulation in mast cells, among others [3] Recently, several studies have established a function for CerK in cell growth and apoptosis For example, in Arabidopsis plants, mutation of CerK was associated with an accumulation of Cer and enhanced symptoms during pathogen attack [4] In addition, in mammalian cells such as Abbreviations ABC, ATP-binding cassette; C1P, ceramide 1-phosphate; Cer, ceramide; CerK, ceramide kinase; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assays; LD, L-165,041; PI, propidium iodide; PPARb, peroxisome proliferator-activated receptor beta; PPRE, PPAR response element; RXR, retinoid X receptor; TEWL, transepidermal water loss FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS 3815 Role of CerK in PPARb-induced keratinocyte survival K Tsuji et al NIH 3T3 fibroblasts and A549 lung cancer cells, treatment with exogenous C1P at low concentrations enhanced cell survival, whereas high concentrations of C1P reduced cell survival and enhanced apoptosis induced by serum starvation [5] Although these previous reports suggest that CerK and C1P are involved in the regulation of cell survival or cell proliferation, the molecular mechanisms involved remain largely unknown Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily PPARs form heterodimers with the retinoid X receptor (RXR) in a ligand-dependent manner Together, these heterodimers regulate the expression of target genes by binding to their PPAR response elements (PPREs) [6,7] Three subtypes of PPARs have been identified, PPARa ⁄ NR1C1, PPARc ⁄ NR1C3 and PPARb ⁄ PPARd ⁄ NR1C2, each with reportedly unique tissue distribution and distinct cellular functions in lipid metabolism, diabetes, inflammation and tumor progression [8] PPARb is ubiquitously expressed and has been implicated by recent studies in tumor-promotion in various cell types [9] PPARb has also been suggested to have an important role in skin wound healing After a skin injury, PPARb expression is rapidly elevated in the epidermis at the wound edges; deletion of a single PPARb allele results in delayed wound healing [10] Furthermore, inflammation-induced apoptosis was found to be enhanced in keratinocytes isolated from PPARb-null mice [11] These studies suggest a role for PPARb in the regulation of keratinocyte survival and apoptosis Interestingly, the promoter activity of PPARb was reported to be increased by the treatment of exogenous Cer [11] However, any interactions between PPAR and sphingolipids remain to be further defined Skin epidermis contains abundant lipids, including cholesterol, fatty acids and Cer, each of which plays a critical role in water retention and epidermal permeability barrier functions [12] Recently, an abnormal sphingolipid distribution pattern was found in the keratinocytes of patients suffering from harlequin ichthyosis, a genetic disorder in which the keratinocytes carry abnormal lamellar granules [13] In harlequin ichthyosis, mutations have been identified in the ABCA12 gene, which encodes a member of the lipid transporter ATP-binding cassette (ABC) family that is also part of the ABCA subfamily ABCA12 is thought to function in keratinocytes as a glucosylceramide transporter to lamellar bodies [13] Interestingly, expression of ABCA12 mRNA has been shown to be induced by activation of PPARb or PPARc in cultured human keratinocytes [14] 3816 In this study, we examined the interaction between CerK and PPARb, and its role in regulating keratinocyte survival We report that in a mouse keratinocyte cell line upregulation of CERK expression by activation of PPARb results in a decrease in intracellular Cer levels and enhancement of cell survival Results Skin barrier disruption in hairless mice induces mRNA expressions for both PPARb and CerK PPARb plays an important role in keratinocyte survival during skin wound healing, and PPARb expression is elevated at injury sites [10,15] We investigated whether skin barrier disruption by tape-stripping would induce PPARb expression To quantify the skin barrier disruption, transepidermal water loss (TEWL), an indicator of skin barrier function [16], was measured in the dorsal skin of hairless mice treated with or without tape-stripping With normal skin barrier function, low levels of water loss from epidermal tissue and low TEWL rates were evident However, tapestripping the stratum corneum significantly increased the TEWL rate and resulted in skin barrier disruption (Fig 1A) PPARb mRNA expression was determined in keratinocytes isolated from the epidermal layer of these skins, and was found to be significantly increased in skin barrier disruption following tape-stripping (Fig 1Ba,b) Furthermore, CERK mRNA expression was also elevated following tape-stripping (Fig 1Ba,c) This is the first evidence that CERK expression increases in response to skin barrier disruption by tape-stripping, and that it is accompanied by increases in PPARb expression Stress-induced cell death is inhibited by the treatment of specific PPARb ligand: L-165,041 (LD) Cer has emerged as an apoptotic lipid mediator, and accumulation of Cer and apoptosis are known to be induced under various stimuli such as tumor necrosis factor-alpha, serum deprivation and c-radiation [17] Previous studies have provided evidence that control of intracellular Cer levels, through their modulation by enzymes active in sphingolipid metabolism, is important in drug resistance and cancer cell survival [18,19] Liu et al [18] reported that the activity of glucosylceramide synthase, the enzyme that converts Cer to glucosylceramide, and glucosylceramide levels were increased in adriamycin-resistant breast cancer cells Uchida et al [19] reported that in HL-60 ⁄ ADR cells FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS K Tsuji et al Role of CerK in PPARb-induced keratinocyte survival A TEWL (g·m–2·h–1) 100 P< 0.0001 80 60 40 20 NonTape stripped stripped b PPARβ CERK Mrpl27 Non- Tapestripped stripped c P < 0.0001 CERK mRNA (fold increase) a PPARβ mRNA (fold increase) B P < 0.0001 NonTape stripped stripped Non- Tape stripped stripped Fig Skin barrier disruption in hairless mice induces mRNA expression for both PPARb and CerK (A) Quantification of TEWL indicates skin barrier disruption The dorsal skin of each mouse was tape-stripped five to eight times to perturb the skin barrier function TEWL was measured in tape-stripped mice skin (tape-stripped) or control mice skin (not stripped) as described in Materials and methods Each column represents the mean ± SD of three animals for each group (B) Expression levels of PPARb and CERK mRNA in skin barrier disruption Total RNA was extracted from the epidermis of tape-stripped mice skin (tape-stripped) or control mice skin (not stripped), and the expression levels of PPARb, CERK and Mrpl27 mRNA were determined by RT-PCR as described in Materials and methods (a) Agarose gel electrophoresis of the products of PCR using specific primers for PPARb, CERK or Mrpl27 mRNA (b, c) Density of the mRNA expression of PPARb (b) or CERK (c) The results shown are normalized to the mRNA level of Mrpl27, and are relative to control mice skin (not stripped), and the mean ± SD of three animals for each group transcriptional upregulation of glucosylceramide synthase via doxorubicin-induced activation of Sp1 results in a decreased Cer level and obtained drug resistance Moreover, we previously reported that exogenous Cer is incorporated, hydrolyzed to sphingosine and then recycled into intracellular Cer itself, and that accumulation of Cer contributes to the induction of apoptosis [20] However, the signaling mechanism involved in Cer-mediated apoptosis remains to be further defined CerK is an enzyme that converts Cer to C1P Recently, CERK was cloned [2] C1P has been reported to be involved in the regulation of programed cell death in plants and in cell survival in mammalian cells [4] Arabidopsis, carrying a CerK mutation, exhibits a spontaneous cell-death phenotype and accumulates Cer late in development [4] In A549 human lung adenocarcinoma cells transfected with CERK siRNA, downregulation of CerK reduced cellular proliferation [5] We thought, therefore, that CerK could be involved in cell survival promoted by PPARb, and that a decrease in Cer content following activation of CerK would cause suppression of cell death In order to investigate this possibility, we examined whether CerK could be affected by activation of PPARb, using the mouse keratinocyte cell line SP1 Enhanced cell survival has previously been reported in the human keratinocyte cell line HaCat following treatment with the specific PPARb ligand L-165,041 (LD) [21] We confirmed that SP1 cell survival could be enhanced by PPARb As shown in Fig 2A, serum starvation stress induced limited cell growth and death in SP1 cells (Fig 2Ac), yet treatment with lm LD inhibited this cell death (Fig 2Ad) Furthermore, as determined using a cell proliferation assay (Fig 2B), the rate of cell survival in SP1 cells treated with serum starvation stress was reduced in a time-dependent manner However, treatment with lm LD inhibited the reduction of cell survival induced by serum starvation stress Flow cytometry analysis further demonstrated that treatment with LD (1 or 10 lm) suppressed the number of annexin V ⁄ propidium iodide (PI)-positive cells, representing late apoptotic or necrotic cells, which had increased upon serum starvation FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS 3817 Role of CerK in PPARb-induced keratinocyte survival A K Tsuji et al B LD ( + ) LD (−) 140 b % Cell survival a Stress (−) c d Stress (+ ) 120 LD (+ ) LD (−) * * 100 * 80 60 40 20 0 12 18 24 (h) Stress (+) C D Ceramide content (% control) Apoptotic cells (% control) 300 200 100 LD (μM) P < 0.001 250 P < 0.0001 400 10 Stress (− ) 10 Stress (+ ) P < 0.001 200 150 100 50 Stress LD − − − + + − + + Fig Activation of PPARb enhances cell survival and inhibits cell death induced by serum starvation stress (A) SP1 cells under serum starvation stress treated with LD SP1 cells were cultured for an additional 24 h in the presence [LD (+)] or absence [LD ())] of the specific PPARb ligand LD (1 lM), in medium lacking serum (serum-starvation conditions) or in medium containing fetal bovine serum (control conditions) [Stress (+) or Stress ()), respectively] for 24 h SP1 cells were observed under a phase-contrast microscope (B) Effect of LD on the cell survival rate of mouse keratinocyte cells SP1 cells were cultured in the presence [LD (+)] or absence [LD ())] of LD (1 lM), in serum starvation conditions [Stress (+)] After treatment for the indicated times, the cell survival rate was determined by Cell Counting Kit-8 as described in Materials and methods Significant difference from the corresponding LD ()) time point (*P < 0.05) (C) Inhibition by LD of serum starvation stress-induced cell death SP1 cells were cultured in serum starvation conditions [Stress (+)] or in control conditions [Stress ())], in the presence of LD (0, or 10 lM) for 24 h Cells were then stained with annexin V ⁄ fluorescein isothiocyanate and PI, and analyzed by flow cytometry The results shown are relative to untreated, unstressed cells the mean ± SD of three wells, and each experiment was repeated three times (D) Activation of PPARb inhibits the generation of cellular Cer induced by serum starvation stress SP1 cells were left untreated or treated with lM LD under control conditions [Stress ())] or serum starvation conditions [Stress (+)] for 24 h Total cellular lipids were extracted using the standard Bligh–Dyer protocol Total cellular Cer levels were measured by diacylglycerol kinase assay as described under Materials and methods and quantified using an Image Analyzer BAS2000 The results shown are relative to untreated, unstressed cells [Stress ()) ⁄ LD ())], and are the mean ± SD of three experiments stress (Fig 2C) These findings demonstrate that SP1 cells are sensitive to ligand activation of PPARb, which enhanced cell survival or inhibited cell death induced by serum starvation Next, we investigated the participation of Cer in cell survival promoted by PPARb, by examining intracellular Cer levels Total cellular lipids were extracted from serum-starved or non-starved SP1 cells cultured for 24 h in the presence or absence of lm LD, and the levels of endogenous Cer were determined by diacylglycerol kinase assay [22] Cer levels were significantly increased in cells stressed by serum starvation, compared with control cells (Fig 2D) However, similar to results observed in the survival studies, treatment with LD inhibited the increase in Cer levels induced by serum starvation stress These findings suggest that regulation of Cer levels is involved in enhanced cell survival resulting from activation of PPARb 3818 Activation of PPARb by LD induces CERK mRNA expression and increases CerK activity in SP1 cells To investigate the role of CerK and its regulation of Cer levels in the effect of PPARb activation on cell survival, the expression of CERK mRNA in SP1 cells treated with LD was determined using real-time PCR CERK mRNA expression was increased by LD treatment in a dose-dependent (Fig 3Aa) and timedependent (Fig 3Ab) manner, indicating that activation of PPARb is involved in the gene transcription of CERK Furthermore, an in vitro kinase assay determined that CerK activities in whole-cell lysates were significantly increased in SP1 cells treated for 24 h with lm LD, compared with untreated cells (Fig 3B) Although serum starvation stress did not affect CerK activity, the rate of increase in CerK activity in cells treated with LD was larger in cells FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS K Tsuji et al Role of CerK in PPARb-induced keratinocyte survival A a the effect of PPARb activation on cell survival in mouse keratinocytes CERK mRNA (% control) 300 250 200 * 150 100 50 b * * 0.1 0.5 1.0 5.0 LD (μ M) 10 CERK mRNA (% control) 300 200 * * 150 100 50 B * 250 12 24 Time (h) 48 400 CerK activity (% control) P < 0.001 300 P < 0.05 200 100 Stress LD − − − + + − + + Fig Activation of PPARb induces CERK mRNA expression and increased CerK activity in SP1 cells (A) CERK mRNA expression is induced by treatment with LD SP1 cells were treated with LD (0, 0.1, 0.5, 1, or 10 lM) under serum starvation conditions for 24 h (a), or with lM LD in serum-free medium for the indicated times (b) Total RNA was extracted from the cells, and the mRNA expression levels of CERK and Mrpl27 (a housekeeping gene) were determined by quantitative real-time PCR as described in Materials and methods The results shown are the mean ± SD of three wells, and each experiment was repeated three times *P < 0.05 compared with the controls at lM LD (a) or h (b) (B) CerK activity is enhanced in cells treated with LD SP1 cells were left untreated [LD ())] or treated [LD (+)] with lM LD in serum starvation conditions or control conditions [Stress (+) or Stress ()), respectively] After treatment for 24 h, cell lysates were collected and analyzed for in vitro CerK activities, as described in Materials and methods using C18-Ceras a substrate with [32P]ATP[cP] [32P]C1P was quantified using an Image Analyzer BAS2000 (Fuji Film) The results shown are relative to untreated, unstressed cells, are the mean ± SD of three experiments undergoing serum starvation stress than in unstressed cells [23] These findings suggest that CerK, through its regulation of Cer levels, plays an important role in PPARb binds to a putative PPRE in CERK and transactivates the CERK gene The results above suggest that PPARb can regulate expression of the CERK gene PPARs bind to response elements (PPREs) in target genes and regulate transcription Functional PPREs should reside in the regulatory region of the target gene Although the promoter region in the mouse CERK gene has not been identified, transcription start sequences at the 5¢-end of the gene reside  100 bp upstream of the protein-coding sequence, as identified by the Database of Transcriptional Start Sites (DBTSS) [24] and our experimental results (data not shown) Analysis of the region near the protein-coding sequence in the mouse CERK gene sequence (GenBank accession number NC_000081) was performed using nubiscan, an in silico tool for predicting nuclear receptor binding sites [25] This analysis revealed a putative PPRE, which we refer to as putative CERK-PPRE, in intron of the mouse CERK gene (Fig 4A) In order to determine whether PPARb binds to the mouse CERK gene in vivo, chromatin immunoprecipitation (ChIP) was carried out using SP1 cells, untreated [LD ())] or treated [LD (+)] with lm LD for 24 h Using immunoprecipitated chromatin, the CERK gene sequence containing putative CERK-PPRE was analyzed by PCR In the ChIP DNA obtained with the anti-PPARb IgG, the amount of DNA containing putative CERK-PPRE was significantly greater in the chromatin of cells treated with LD compared with untreated controls (Fig 4B,C) The results were comparable in the ChIP DNA obtained with acetylated histone H4 antibodies No PCR products with CERKnegative were obtained in ChIP DNA, indicating that the binding of putative CERK-PPRE to PPARb is specific Furthermore, the interaction between PPARb and CERK was confirmed by an EMSA using biotinlabeled putative CERK-PPRE and nuclear extract from SP1 cells As shown in Fig 4D, the binding of biotin-labeled putative CERK-PPRE to nuclear extract from SP1 cells was detected as shift bands The levels of these shift bands were reduced in the presence of competitors, including unlabeled putative CERKPPRE and PPRE-Wild, a known consensus sequence However, no reduction in shift band levels was observed in the presence of PPRE-Mutant, a mutation sequence of PPRE-Wild These results demonstrate specific binding of putative CERK-PPRE to PPARb in nuclear extracts of SP1 cells, and provide further FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS 3819 Role of CerK in PPARb-induced keratinocyte survival K Tsuji et al A CERK gene Exon-1 Exon-2 Intron -1 Putative CERK-PPRE: AGGCCAcAGGCCA ChIP DNA IgG anti-PPARβ anti-acH4 − + − + − + LD Input DNA − + C Putative CERK-PPRE (% control) B Putative CERK-PPRE CERK-negative P < 0.01 800 300 400 200 LD (−) LD (+ ) D R PP K- K- ER C iv e e tiv ta in in P- Lu c2 P- pu m -Δ c2 P Lu m Pin m E E − − + ve ct o − + − at + − − P- − − − ER − − − 500 r + + pu t + + c2 + + C + + P- Competitors (non-labeled) putative CERK-PPRE PPRE-Wild PPRE-Mutant − + Lu SP1 cell nuclear extracts Biotin-labeled putative CERK-PPRE 1000 R Free P < 0.0001 1500 PP Relative luciferase activity (% control) E Shift band Fig PPARb binds to putative CERK-PPRE and transactivates the CERK gene (A) Schematic representation of the CERK gene illustrating the position (bar) and sequence of the putative PPRE (putative CERK-PPRE) (B, C) ChIP demonstrates PPARb binding to putative CERK-PPRE in vivo SP1 cells were untreated or treated with lM LD in serum starvation medium for 24 h Nuclear extracts were collected and subjected to a ChIP assay, as described in Materials and methods, using antibodies against PPARb or acetylated histone H4, or IgG as a negative control ChIP DNA and aliquot of pre-immunoprecipitation samples of nuclear extracts (Input DNA) were analyzed by PCR with primers for putative CERK-PPRE or CER-negative as PCR negative control with primers for unrelated putative PPRE, as described in Materials and methods PCR products of ChIP DNA and Input DNA were analyzed with 1% agarose gel electrophoresis (B) The bands of PCR products corresponding to the binding of PPARb to putative CERK-PPRE were normalized to the bands of PCR product of Input DNA, and are relative to untreated sample [LD ())] (C) The results shown are the means ± SD of three experiments (D) EMSAs indicate that PPARb binds to putative CERK-PPRE in vitro SP1 cells were treated for 24 h with lM LD in serum starvation Nuclear extracts from the cells (5 lg) were incubated with biotin-labeled putative CERK-PPRE oligonucleotide (20 fmol) Competition assays were performed with non-biotinylated oligonucleotides (4 pmol) of putative CERK-PPRE, a specific DNA binding consensus sequence for PPARs (PPRE-Wild), or a mutant sequence of PPRE-Wild (PPRE-Mutant) Arrows indicate the labeled putative CERK-PPRE oligonucleotide in specific complex with SP1 nuclear extracts (Shift band) and unbound (Free) (E) Transfection assays indicate that PPARb transactivates the CERK gene SP1 cells were transiently cotransfected with pCMX–mPPARb, pCMX–mRXRa, the luciferase reporter constructs: minP-Luc2P-putative CERK-PPRE or minP-Luc2P-D putative CERK-PPRE, and pRL-SV40 control vector Transfected cells were treated for 24 h in Phenol Red-free Dulbecco’s modified Eagle’s medium containing with 10% charcoal stripped fetal bovine serum and lM LD Results were normalized with Renilla luciferase activity to correct for variability in transfection efficiency Values represent the means ± SD of three wells, and each experiment was repeated twice evidence that PPARb directly regulates expression of the CERK gene through PPRE Next, we assessed the function of putative CERKPPRE using the Dual-Luciferase Reporter Assay System In keeping with the genomic organization of the CERK gene, the 1807 bp fragment containing putative CERK-PPRE, or the 1007 bp fragmant not containing putative CERK-PPRE, in the CERK intron region was subcloned downstream of the 3820 minimal promoter and the Luc2P reporter gene in pGL4.27[luc2P ⁄ minP ⁄ Hygro], respectively named minP-Luc2P-putative CERK-PPRE or minP-Luc2P-D putative CERK-PPRE Reporter gene transfection studies showed that the region containing putative CERK-PPRE has the capacity to significantly influence transcriptional activity By contrast, constructs not containing putative CERK-PPRE were approximately equal to the control luciferase activity from cells FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS K Tsuji et al Role of CerK in PPARb-induced keratinocyte survival transfected with an empty vector (minP-Luc2P-vector) These findings suggest that putative CERK-PPRE is the functional PPRE in mouse CERK gene keratinocyte cultures, however, the inhibitory effect of LD was once again diminished (Fig 5D) These findings indicate that CerK is necessary for the enhanced cell survival associated with activation of PPARb Taken together, the data demonstrate that upregulation of CERK by PPARb suppresses Cer accumulation induced by serum starvation stress, which results in cell survival and inhibition of cell death in mouse keratinocytes Enhanced cell survival associated with activation of PPARb is diminished in keratinocytes from CerK-null mice The role of CerK in cell survival associated with PPARb activation was further examined using primary keratinocytes isolated from CerK-null mice [26] These mice lack exon of the mouse CERK gene, which encodes a region of the protein that is known to be essential for kinase activity and contains a diacylglycerol kinase-like catalytic domain [2] As shown in Fig 5A, cell death and limited cell growth following serum starvation stress for 24 h were apparent in primary keratinocytes isolated from wild-type mice In wild-type cells treated with lm LD under serum starvation conditions, cell death was inhibited, similar to results observed in SP1 cells (Fig 2A) However, in primary keratinocytes isolated from CerK-null mice, the inhibitory effect of LD on cell death induced by serum starvation stress was diminished (Fig 5B) To confirm this finding, serum deprivation-induced cell death was investigated by flow cytometry analysis with annexin V and PI staining A significant increase in the percentages of annexin V ⁄ PIpositive cells was observed in cultures of wild-type keratinocytes undergoing serum starvation stress for 24 h, but, as expected, treatment with lm LD suppressed this number (Fig 5C) In CERK-KO A Discussion The study reported here revealed that in mouse keratinocytes upregulation of CERK through activation of PPARb results in decreased intracellular Cer levels and increased cell survival with less cell death (Fig 6) There have been previous reports that PPARb activation improved skin wound healing by enhanced keratinocyte survival ⁄ anti-apoptosis [10,15] In this study, the effects on cell survival of PPARb were diminished in CERK-KO keratinocytes (Fig 5), suggesting the biological importance of CerK in PPARb functions This study provides the first evidence for the necessity of CerK in mouse keratinocyte survival associated with activation of PPARb Cer is known to have an important role in apoptosis and cell-cycle arrest induced by various stressors Intracellular Cer levels are adjusted by several sphingolipid production pathways, such as de novo ceramide synthesis by serine palmitoyltransferase and cleavage of sphingomyelin by sphingomyelinase, as well as by Cer metabolism pathways, including conversion to Wild-type keratinocytes LD (−) LD( +) a CERK-KO keratinocytes LD (−) LD( +) a b c b d Stress (−) Stress (−) c d Stress (+) Stress (+) C Wild-type keratinocytes 300 200 100 Stress LD D CERK-KO keratinocytes 300 P < 0.005 Apoptotic cells (% control) Apoptotic cells (% control) Fig Enhanced cell survival associated with activation of PPARb is diminished in keratinocytes from CerK-null mice (A–D) Mouse primary keratinocytes isolated from wild-type mice (A, C) or CerK-null mice (B, D) were grown to 80% confluence then grown for 24 h under serum starvation [Stress (+)] or control [Stress ())] conditions, in the presence [LD (+)] or absence [LD ())] of lM LD (A, B) Cells were observed under a phase-contrast microscope (C, D) Cells were stained with annexin V ⁄ fluorescein isothiocyanate and PI, and analyzed by flow cytometry Results shown are relative to untreated, unstressed cells, are the means ± SD of three wells, and each experiment was repeated three times B − − − + FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS + − + + P > 0.08 200 100 Stress LD − − − + + − + + 3821 Role of CerK in PPARb-induced keratinocyte survival K Tsuji et al Fig Schematic model illustrating the role of CerK in cell survival mediated by PPARb in mouse keratinocytes Under injury stress, endogenous ligands activate PPARb Subsequently, the activated PPARb promotes transcription of the CERK gene The conversion of Cer to C1P by the produced CerK results in decreased levels of intracellular Cer, and the inhibition of stress-induced cell death The interaction between CerK and PPARb may play an important role in regulating epidermal homeostasis in stress environments glucosylceramide by glucosylceramide synthase, degradation to sphingosine and conversion to C1P by CerK In some cancer cells, decreases in Cer levels by amplified activation of glucosylceramide synthase result in drug resistance [18,19] Mitra et al [5] reported that siRNA-induced downregulation of CerK reduced cell proliferation and promoted apoptosis Previous studies have suggested that metabolism of eicosanoids is regulated by feedback pathways through PPARs Recently, Xu et al [27] reported a positive feedback loop between PPARb and prostaglandin E2 through which PPARb promotes COX-2 expression and prostaglandin E2 synthesis, and subsequent activation of cystolic phospholipase A2a, which is responsible for arachidonic acid release, through mitogen-activated protein kinase and phosphatidylinositol 3-kinase-mediated phosphorylation This pathway results in cholangiocarcinoma cell growth CerK has also been reported to act as an upstream modulator of cystolic phospholipase A2a in the inflammatory response [28,29] CerK may also be implicated in the regulation of PPARb activation through inflammatory factors such as cystolic phospholipase A2a In this study, the skin of CerK-null mice appeared normal under specific pathogen-free conditions (data not shown), yet examinations of cultured CERK-KO keratinocytes showed a diminished effect on cell survival by activation of PPARb compared with that in wild-type keratinocytes (Fig 5) PPARb expression has been reported to be undetectable in the epidermal tissue of adult mice; however, it is apparently upregulated in various stress conditions, such as skin wound healing, that result in enhanced keratinocyte proliferation [10,15] Reportedly, CerK is also expressed highly at embryonic day but decreases rapidly thereafter [2] In the Arabidopsis plant, expression of CERK mRNA is induced after infection with a bacterial pathogen [4] Considering all this information, it appears that CerK 3822 function may be upregulated under stress conditions, such as those that induce PPARb expression Future studies will be required into the role of CerK using an animal model In conclusion, we have shown that PPARb-mediated upregulation of CerK gene expression is necessary for keratinocyte survival against serum starvation-induced apoptosis The interaction between CerK and PPARb may play an important role in regulating epidermal homeostasis in stress environments Materials and methods Materials Dispase was obtained from Godo Shusei (Tokyo, Japan) Keratinocyte serum-free medium, epidermal growth factor, bovine pituitary extract, Phenol Red-free Dulbecco’s modified Eagle’s medium, and charcoal stripped fetal bovine serum were obtained from GIBCO BRL (Gaithersburg, MD, USA) Minimum essential medium without calcium chloride was obtained from Cambrex (Walkersville, MD, USA) Penicillin–streptomycin, 0.125% trypsin–0.01% EDTA and LD were from Sigma (St Louis, MO, USA) Rabbit anti-PPARb IgG was from Santa Cruz Biotechnology Inc, (Santa Cruz, CA, USA) and the anti-acetylHistone H4 serum was from Upstate Biotechnology (Lake Placid, NY, USA) A MEBCYTO Apoptosis Kit was purchased from Medical and Biological Laboratories (Nagoya, Japan), and a Nuclear Extract Kit was from Active Motif (Carlsbad, CA, USA) The ChIP Assay Kit was also a product of Upstate Biotechnology Biotin 3¢-end DNA Labeling Kit and LightShift Chemiluminescent EMSA Kit were purchased from Pierce (Rockford, IL, USA) CulturPlate-96, White was purchased from Perkin-Elmer (Boston, MA, USA) and a lipofectamine 2000 reagent was from Invitrogen (Carlsbad, CA, USA) Dual-Luciferase reporter assay system was purchased from Promega (Madison, WI, USA) FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS K Tsuji et al Animals Hairless mice (HR-1), 4-week-old males, were purchased from Hoshino Experimental Animal Center (Saitama, Japan) C57BL ⁄ 6J mice were purchased from Clea Japan (Tokyo, Japan) All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (Hokkaido University Graduate School of Medicine, Japan) Animals were housed in plastic cages with metal lids at a temperature of 22 ± 3°C, with 50 ± 20% relative humidity, and were exposed daily to 12 h of light and 12 h of darkness Skin barrier disruption by tape-stripping In the dorsal skins of hairless mice, skin barrier disruption was performed by stripping with adhesive tape (P.P.S Nichiban, Tokyo, Japan: 2.5 · 3.0 cm) repeatedly, five to eight times An Evaporimeter AS-TW1 (Asahi Biomed, Co Ltd, Yokohama, Japan) was used to measure TEWL, in accordance with the ventilated chamber method [30] Measurements were carried out at a temperature of 22 ± 3°C, with 50 ± 20% humidity, and were performed in triplicate at each treatment skin spot Harvest and culture of keratinocytes Mouse keratinocytes were isolated from the epidermis of hairless mice or newborn of C57BL ⁄ 6J mice Briefly, the epidermis was separated from the dermis following an overnight incubation at 4°C in 2.5 mL)1 Dispase Keratinocytes isolated from the epidermis were harvested after treatment with 0.125% trypsin and 0.01% EDTA at 37 °C for Keratinocytes isolated from the epidermis of newborn mice were incubated in a humidified atmosphere of 5% CO2 in air at 37 °C with keratinocyte serum-free medium containing 0.02 mm Ca2+, ngỈmL)1 epidermal growth factor and 50 lgỈmL)1 bovine pituitary extract The isolated keratinocytes were used at a subconfluent state (80% confluency) The mouse keratinocyte cell line SP1 cells, a kind gift from S.H Yuspa (National Cancer Institute, Bethesda, MD, USA) [31], was cultured in minimum essential medium without calcium chloride supplemented with 0.02 mm Ca2+, 8% chelexed fetal bovine serum [32] and antibiotics (100 unitsỈmL)1 penicillin and 0.1 mgỈmL)1 streptomycin) The cultures were maintained in a humidified atmosphere of 5% CO2 in air at 37 °C Total RNA isolation The total RNA from each sample was isolated using an RNeasy Mini Kit (Qiagen, Chatsworth, CA, USA), according to protocols provided by the manufacturer To remove contaminating genomic DNA, the RNA samples were Role of CerK in PPARb-induced keratinocyte survival treated with RNase-free DNase I (Qiagen) at room temperature for 30 RT-PCR RT-PCR of each mRNA was performed with Omniscript RT Kit (Qiagen) following the manufacturer’s instructions using oligo(dT) primers and Taq DNA polymerase (Qiagen) with specific primers Sequences of the specific primers included, for PPARb forward 5¢-GCAGCCTCTTCCTCA ATGAC-3¢, for reverse 5¢-GTACTGGCTGTCAGGGTG GT-3¢; CERK forward 5¢-TCTGCAAGGACAGACCCT CT-3, reverse 5¢-CAAGTGCCATTTGCTGAGAA-3¢; and mitochondrial ribosomal protein L27 (Mrpl27) forward 5¢-GGGATAGTCCGCTACACGAA-3¢, reverse 5¢-ACCA TGTGGTTGTTGGGAA-3¢ The PCR condition of PPARb was as follows: 95 °C for 30 s, 60 °C for 30 s and 72 °C for 60 s, and 35 cycles were used The PCR condition of CERK was as follows: 95 °C for 30 s, 55 °C for 30 s and 72 °C for 60 s, and 35 cycles were used The PCR condition of Mrpl27 was as follows: 95 °C for 30 s, 60 °C for 30 s and 72 °C for 60 s, and 28 cycles were used PCR products were separated by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining The relative intensity of the gel bands was measured using nih image software, and results were normalized to the mRNA level of Mrpl27, a housekeeping enzyme [33] We performed these experiments using samples from three animal preparations Assessment of cell survival and cell death The rate of cell survival was determined using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan), following the manufacturer’s instructions Briefly, the cells were seeded onto a six-well plate (1.0 · 105 cellsỈmL)1), incubated at 37 °C for days, then, in the presence or absence of serum, cells were treated with or without LD (1 lm) After treatment, a solution of Cell Counting Kit-8, in ⁄ 10 volume of the culture medium, was added to each well, and the culture continued at 37 °C for h Each well was then assessed at D450 using an automatic enzyme-linked immunosorbent assay plate reader Cell death was quantified by flow cytometry analysis Cells were seeded onto six-well plates, grown to 80% confluence, then treated with or without varying concentrations of LD in the presence or absence of serum After a predetermined time, the cells were trypsinized and washed twice with NaCl ⁄ Pi The dead cells were stained with annexin V ⁄ and PI using a MEBCYTO Apoptosis kit according to the manufacturer’s instructions flow cytometry analysis was carried out on a FACSort cell sorter (Becton Dickinson, Mountain View, CA, USA) using cell quest software FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS 3823 Role of CerK in PPARb-induced keratinocyte survival K Tsuji et al solvent system Quantification of bands was carried out using the Imaging Analyzer BAS2000 Measurement of total intracellular Cer levels Total cellular Cer levels were measured by the diacylglycerol kinase method [22] Briefly, total cellular lipids were extracted using the Bligh–Dyer protocol as previously described [34] Extracts were suspended in micelle buffer containing 7.5% n-b-d-octyl glucopyranoside and 19.4 mgỈmL)1 a-dioleoylphosphatidylglycerol, then mixed with 0.1 unit of Escherichia coli diacylglycerol kinase and lCi [32P]ATP[cP], and incubated for h at 37 °C After the reaction, lipids were separated by a solvent system of chloroform ⁄ methanol ⁄ 15 mm CaCl2 (7.5 : 4.4 : 1, v ⁄ v ⁄ v) on Silica Gel 60 TLC plates (Merck, Darmstadt, Germany) Bands corresponding to C1P derived from intracellular Cer were quantified using an Imaging Analyzer BAS2000 (Fuji Film, Tokyo, Japan) Quantitative real-time PCR Quantification of CERK mRNA was performed using an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA) RNA samples were reverse transcribed to synthesize first-strand cDNA using the Omniscript RT kit (Qiagen), then analyzed by real-time PCR using specific primers and the double-stranded DNA dye SYBR Green I (Qiagen), according to protocols provided by the manufacturer Specific primer sequences used included for CERK forward 5¢-GAGTGGCAAGTGACA TGTGG-3¢ and for reverse 5¢-GCACTTCCGGATAAG GATGA-3¢; those for Mrpl27 were for forward 5¢-CTGC CCAAGGGTGCTGTGCTC-3¢ and for reverse 5¢-TTGTT CTCACCAGACCCTTGAC-3¢ All reactions were run with a hot-start preincubation step of 10 at 95 °C, followed by cycles of 15 s at 95 °C and at 60 °C The amount of template was quantified using the comparative cycle threshold method as outlined in the manufacturer¢s technical bulletin Quantified CERK mRNA levels were normalized to the Mrpl27 mRNA level for reporting Quantification of in vitro CerK activity CerK activity assays were performed as described by Bajjalieh et al [3] Briefly, cells were washed three times with ice-cold NaCl ⁄ Pi, then lysed in a buffer containing 10 mm Hepes, mm EGTA, mm dithiothreitol, 40 mm KCl and complete protease inhibitor mixture (Roche, Basel, Switzerland) The enzyme reactions were performed for 30 at 30 °C in a reaction mixture containing 20 mm Hepes, 80 mm KCl, mm CaCl2, mm cardiolipin, 1.5% b-octyl glucoside and 0.2 mm diethylenetriaminepentaacetic acid, with 40 mm Cer (C18:0, d18:1) as a substrate After the reaction, lipids were extracted and separated on Silica Gel 60 HPTLC plates (Merck, Darmstadt, Germany) in chloroform ⁄ acetone ⁄ methanol ⁄ acetic acid ⁄ water (10 : : : : 1, v ⁄ v ⁄ v ⁄ v ⁄ v) as the 3824 ChIP assays PPAR forms a heterodimer with RXR, and binds to PPRE sequences of the direct repeat-1 (DR-1) type (a repeat separated by one nucleotide) on DNA Using the nubiscan program [25], putative PPRE elements were identified within the first intron of the mouse CERK gene (putative CERK-PPRE) ChIP was performed using a ChIP Assay Kit (Upstate Biotechnology), according to protocols provided by the manufacturer, with some modifications Briefly, cells were seeded onto six-well plates, grown to 80% confluence, and then treated with or without LD (1 lm) in serum starvation medium for 24 h To cross-link the DNA, cells were fixed with 1% formaldehyde at 37 °C for 15 min, then sonicated to fragments ranging in size from 200 to 500 bp ChIP was carried out using PPARb antibodies and acetylated histone H4-specific antibodies, with normal rabbit IgG used as a negative control Reverse cross-linking of DNA fragments was achieved at 65 °C for h After phenol ⁄ chloroform treatment of purified DNA, the DNA was amplified by PCR using primers, for putative CERK-PPRE forward 5¢-GTAGGCATGAGAACGGGA AG-3 and for reverse 5¢-GGGGGTAAGAGGAGGAGA AA-3¢ and for CERK-negative forward 5¢-CCGCAAG AGGCTTTATTGTC-3 and reverse 5¢-TATGCCAAGGA CACGGAGAT-3¢, as a negative control PCR primer The condition for PCR amplification was as follows: 95 °C for 30 s, 60 °C for 30 s and 72 °C for 60 s; 32 cycles were used, depending on the abundance of DNA Electrophoretic mobility shift assays PPRE studies were performed using biotin-labeled oligonucleotides and nuclear extracts from SP1 cells The nucleotide sequences of putative CERK-PPRE, including the sense 5¢-CTCTCCAGGCCACAGGCCAGAGCGG-3¢ and anti-sense 5¢-GAGAGGTCCGGTGTCCGGTCTCGCC-3¢ sequences, were biotin-labeled using a Biotin 3¢-end DNA Labeling Kit (Pierce) Nuclear extracts from SP1 cells were prepared using a Nuclear Extract Kit (Active Motif) EMSA was performed using a LightShift Chemiluminescent EMSA Kit (Pierce), following the manufacturer’s protocols, with some modifications Briefly, nuclear extracts (5 lg) from SP1 cells and 20 fmol biotin-labeled putative CERK-PPRE, alone or with pmol unlabeled probe oligonucleotides (putative CERK-PPRE, PPRE-Wild, sense 5¢-CAAAACTAGGTCAAAGGTCA-3¢ and anti-sense 5¢-GTTTTGATCCAGTTTCCAGT-3¢; or PPRE-Mutant sense 5¢-CAAAACTAGCACAAAGCACA -3¢ and antisense 5¢-GTTTTGATCGTGTTTCGTGT-3¢) [35], were incubated in a reaction mixture at room temperature for 20 The mixtures were then separated by electrophoresis FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS K Tsuji et al on a 6% polyacrylamide gel at 4°C in 0.5· TBE at 100 V for 2–2.5 h The samples were subsequently transferred to Nylon Membranes, Positively Charged (Roche, Basel, Switzerland) and exposed to UV light (120 mJỈcm)2, min) to cross-link the DNA to the membrane In accordance with the manufacturer’s protocol, detection of Biotin-labeled DNA probe was performed Plasmid constructs The expression plasmids pCMX–mouse PPARb and pCMX were a kind gift from R M Evans (Salk Institute, San Diego, CA, USA) [36] The cDNA encoding mouse RXR a was subcloned into pCMX (pCMX–RXRa) The 1807 bp fragment containing putative CERK-PPRE (NC_000081:c86013544-86011738), or the 1007 bp fragmant non-containing putative CERK-PPRE (NC_000081:c86013544-86012538) in the CERK intron region were PCR amplified, and subcloned downstream of the minimal promoter and the Luc2P reporter gene in pGL4.27[luc2P ⁄ minP ⁄ Hygro] (Promega) The pRL-SV40 control vector was purchased from Promega Transfection and luciferase reporter assays SP1 cells (1.0 · 105 cellsỈmL)1) were trypsinized and replated into CulturPlate-96, White (Perkin-Elmer), and incubated for days The cells were cotransfected with 100 ng of either reporter plasmid: minP-Luc2P-vector, minPLuc2P-D putative CERK-PPRE or minP-Luc2P-D putative CERK-PPRE, 100 ng of pRL-SV40 control vector, 12.5 ng of pCMX–mRXRa and 12.5 ng of pCMX–mPPARb using lipofectamine 2000 reagent (Invitrogen) At 24 h after transfection, cells were rinsed with phosphate buffer and replaced with Phenol Red-free Dulbecco’s modified Eagle’s medium containing with 10% charcoal-stripped fetal bovine serum and lm LD After treatment for 24 h, luciferase activities were assayed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol The levels of Photinus pyralis (firefly) luciferase activity were normalized to the levels of Renilla luciferase activity of the cotransfected pRL-SV40 control plasmid Statistics Results are expressed as the mean ± SD for each sample Statistical analysis was performed using the unpaired Student’s t-test Statistical significance was defined as P < 0.05, 0.01 or 0.001 Acknowledgements This work was supported by the Sapporo Biocluster Bio-S, the Knowledge Cluster Initiative of the Ministry Role of CerK in PPARb-induced keratinocyte survival of Education, Sports, Science and Technology (MEXT), Japan References Hannun YA & Luberto C (2000) Ceramide in the eukaryotic stress response Trends Cell Biol 10, 73–80 Sugiura M, Kono K, Liu H, Shimizugawa T, Minekura H, Spiegel S & Kohama T (2002) Ceramide kinase, a novel lipid kinase Molecular cloning and functional characterization J Biol Chem 277, 23294–23300 Bajjalieh SM, Martin TF & Floor E (1989) Synaptic vesicle ceramide kinase A calcium-stimulated lipid kinase that co-purifies with brain synaptic vesicles J Biol Chem 264, 14354–14360 Liang H, Yao N, Song JT, Luo S, Lu H & Greenberg JT (2003) Ceramides modulate programmed cell death in plants Genes Dev 17, 2636–2641 Mitra P, Maceyka M, Payne SG, Lamour N, Milstien S, Chalfant CE & Spiegel S (2007) Ceramide kinase regulates growth and survival of A549 human lung adenocarcinoma cells FEBS Lett 581, 735–740 Delerive P, Fruchart JC & Staels B (2001) Peroxisome proliferator-activated receptors in inflammation control J Endocrinol 169, 453–459 Vanden Heuvel JP (1999) Peroxisome proliferator-activated receptors (PPARS) and carcinogenesis Toxicol Sci 47, 1–8 Issemann I & Green S (1990) Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators Nature 347, 645–650 Kliewer SA, Forman BM, Blumberg B, Ong ES, Borgmeyer U, Mangelsdorf DJ, Umesono K & Evans RM (1994) Differential expression and activation of a family of murine peroxisome proliferator-activated receptors Proc Natl Acad Sci USA 91, 7355–7359 10 Michalik L, Desvergne B, Basu-Modak S, Tan NS & Wahli W (2000) Nuclear hormone receptors and mouse skin homeostasis: implication of PPARbeta Hormone Res 54, 263–268 11 Tan NS, Michalik L, Noy N, Yasmin R, Pacot C, Heim M, Fluhmann B, Desvergne B & Wahli W (2001) Critical roles of PPAR beta ⁄ delta in keratinocyte response to inflammation Genes Dev 15, 3263–3277 12 Goldstein AM & Abramovits W (2003) Ceramides and the stratum corneum: structure, function, and new methods to promote repair Int J Dermatol 42, 256–259 13 Akiyama M, Sugiyama-Nakagiri Y, Sakai K, McMillan JR, Goto M, Arita K, Tsuji-Abe Y, Tabata N, Matsuoka K, Sasaki R et al (2005) Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer J Clin Invest 115, 1777–1784 14 Jiang YJ, Lu B, Kim P, Paragh G, Schmitz G, Elias PM & Feingold KR (2008) PPAR and LXR activators FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS 3825 Role of CerK in PPARb-induced keratinocyte survival 15 16 17 18 19 20 21 22 23 24 25 26 K Tsuji et al regulate ABCA12 expression in human keratinocytes J Invest Dermatol 128, 104–109 Michalik L, Desvergne B, Tan NS, Basu-Modak S, Escher P, Rieusset J, Peters JM, Kaya G, Gonzalez FJ, Zakany J et al (2001) Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice J Cell Biol 154, 799–814 Tsuji K, Mitsutake S, Ishikawa J, Takagi Y, Akiyama M, Shimizu H, Tomiyama T & Igarashi Y (2006) Dietary glucosylceramide improves skin barrier function in hairless mice J Dermatol Sci 44, 101–107 Dbaibo GS, Perry DK, Gamard CJ, Platt R, Poirier GG, Obeid LM & Hannun YA (1997) Cytokine response modifier A (CrmA) inhibits ceramide formation in response to tumor necrosis factor (TNF)-alpha: CrmA and Bcl-2 target distinct components in the apoptotic pathway J Exp Med 185, 481–490 Liu YY, Han TY, Giuliano AE, Hansen N & Cabot MC (2000) Uncoupling ceramide glycosylation by transfection of glucosylceramide synthase antisense reverses adriamycin resistance J Biol Chem 275, 7138–7143 Uchida Y, Itoh M, Taguchi Y, Yamaoka S, Umehara H, Ichikawa S, Hirabayashi Y, Holleran WM & Okazaki T (2004) Ceramide reduction and transcriptional up-regulation of glucosylceramide synthase through doxorubicin-activated Sp1 in drug-resistant HL-60 ⁄ ADR cells Cancer Res 64, 6271–6279 Takeda S, Mitsutake S, Tsuji K & Igarashi Y (2006) Apoptosis occurs via the ceramide recycling pathway in human HaCaT keratinocytes J Biochem (Tokyo) 139, 255–262 Di-Poi N, Tan NS, Michalik L, Wahli W & Desvergne B (2002) Antiapoptotic role of PPARbeta in keratinocytes via transcriptional control of the Akt1 signaling pathway Mol Cell 10, 721–733 Preiss J, Loomis CR, Bishop WR, Stein R, Niedel JE & Bell RM (1986) Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and rasand sis-transformed normal rat kidney cells J Biol Chem 261, 8597–8600 Di Nardo A, Wertz P, Giannetti A & Seidenari S (1998) Ceramide and cholesterol composition of the skin of patients with atopic dermatitis Acta Derm Venereol 78, 27–30 Suzuki Y, Yamashita R, Sugano S & Nakai K (2004) DBTSS, DataBase of Transcriptional Start Sites: progress report 2004 Nucleic Acids Res 32, D78–D81 Podvinec M, Kaufmann MR, Handschin C & Meyer UA (2002) NUBIScan, an in silico approach for prediction of nuclear receptor response elements Mol Endocrinol 16, 1269–1279 Mitsutake S, Yokose U, Kato M, Matsuoka I, Yoo JM, Kim TJ, Yoo HS, Fujimoto K, Ando Y, Sugiura M et al 3826 27 28 29 30 31 32 33 34 35 36 (2007) The generation and behavioral analysis of ceramide kinase-null mice, indicating a function in cerebellar Purkinje cells Biochem Biophys Res Commun 363, 519– 524 Xu L, Han C & Wu T (2006) A novel positive feedback loop between peroxisome proliferator-activated receptor-delta and prostaglandin E2 signaling pathways for human cholangiocarcinoma cell growth J Biol Chem 281, 33982–33996 Pettus BJ, Bielawska A, Spiegel S, Roddy P, Hannun YA & Chalfant CE (2003) Ceramide kinase mediates cytokine- and calcium ionophore-induced arachidonic acid release J Biol Chem 278, 38206–38213 Pettus BJ, Bielawska A, Subramanian P, Wijesinghe DS, Maceyka M, Leslie CC, Evans JH, Freiberg J, Roddy P, Hannun YA et al (2004) Ceramide 1-phosphate is a direct activator of cytosolic phospholipase A2 J Biol Chem 279, 11320–11326 Tomita Y, Akiyama M & Shimizu H (2005) Stratum corneum hydration and flexibility are useful parameters to indicate clinical severity of congenital ichthyosis Exp Dermatol 14, 619–624 Strickland JE, Greenhalgh DA, Koceva-Chyla A, Hennings H, Restrepo C, Balaschak M & Yuspa SH (1988) Development of murine epidermal cell lines which contain an activated rasHa oncogene and form papillomas in skin grafts on athymic nude mouse hosts Cancer Res 48, 165–169 Brennan JK, Mansky J, Roberts G & Lichtman MA (1975) Improved methods for reducing calcium and magnesium concentrations in tissue culture medium: application to studies of lymphoblast proliferation in vitro In Vitro 11, 354–360 Mohlke KL, Purkayastha AA, Westrick RJ & Ginsburg D (1998) Comparative mapping of distal murine chromosome 11 and human 17q21.3 in a region containing a modifying locus for murine plasma von Willebrand factor level Genomics 54, 19–30 Bligh EG & Dyer WJ (1959) A rapid method of total lipid extraction and purification Can J Biochem Physiol 37, 911–917 Juge-Aubry C, Pernin A, Favez T, Burger AG, Wahli W, Meier CA & Desvergne B (1997) DNA binding properties of peroxisome proliferator-activated receptor subtypes on various natural peroxisome proliferator response elements Importance of the 5¢-flanking region J Biol Chem 272, 25252–25259 Umesono K, Murakami KK, Thompson CC & Evans RM (1991) Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors Cell 65, 1255–1266 FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS ... 3821 Role of CerK in PPARb-induced keratinocyte survival K Tsuji et al Fig Schematic model illustrating the role of CerK in cell survival mediated by PPARb in mouse keratinocytes Under injury... accumulation induced by serum starvation stress, which results in cell survival and inhibition of cell death in mouse keratinocytes Enhanced cell survival associated with activation of PPARb is diminished... human keratinocytes [14] 3816 In this study, we examined the interaction between CerK and PPARb, and its role in regulating keratinocyte survival We report that in a mouse keratinocyte cell line