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• W2032992679 abstract "Psoriasis is a common skin disease characterized by hyperplastic regenerative epidermal growth and infiltration of immunocytes. The etiology of psoriasis is unknown, although several genetic and cellular factors have been elucidated. To find new psoriasis-related genes, we have cloned cDNAs that are differentially expressed between normal and psoriatic skins. Among these clones, we have identified a new gene that codes for a new member of the type IV cytosolic phospholipase A2 (cPLA2) family. We refer to this gene as cPLA2δ. It encodes a polypeptide of 818 amino acids that has significant homology with known cPLA2 proteins in the C2 and catalytic domains. The cPLA2δ gene was mapped to the 15q13-14 chromosomal locus, near to the locus of the cPLA2β gene, from which it is separated by a physical distance of about 220 kb. To identify the phospholipase A2 activity of cPLA2δ, we transfected COS-7 cells with His-tagged cPLA2δ. The cell lysate from these cells had calcium-dependent phospholipase A2 activity. Northern blot analysis revealed that a cPLA2δ transcript of about 4 kb is expressed in stratified squamous epithelia, such as those in skin and cervix, but not in other tissues. In situ hybridization and immunohistochemistry revealed that cPLA2δ is expressed strongly in the upper spinous layer of the psoriatic epidermis, expressed weakly and discontinuously in atopic dermatitis and mycosis fungoides, and not detected in the epidermis of normal skin; cPLA2α is not detected in either normal or psoriatic skin. These results suggest that cPLA2δ exhibits a unique distribution pattern compared with that of known cPLA2 subtypes, and it may play a critical role in inflammation in psoriatic lesions. Psoriasis is a common skin disease characterized by hyperplastic regenerative epidermal growth and infiltration of immunocytes. The etiology of psoriasis is unknown, although several genetic and cellular factors have been elucidated. To find new psoriasis-related genes, we have cloned cDNAs that are differentially expressed between normal and psoriatic skins. Among these clones, we have identified a new gene that codes for a new member of the type IV cytosolic phospholipase A2 (cPLA2) family. We refer to this gene as cPLA2δ. It encodes a polypeptide of 818 amino acids that has significant homology with known cPLA2 proteins in the C2 and catalytic domains. The cPLA2δ gene was mapped to the 15q13-14 chromosomal locus, near to the locus of the cPLA2β gene, from which it is separated by a physical distance of about 220 kb. To identify the phospholipase A2 activity of cPLA2δ, we transfected COS-7 cells with His-tagged cPLA2δ. The cell lysate from these cells had calcium-dependent phospholipase A2 activity. Northern blot analysis revealed that a cPLA2δ transcript of about 4 kb is expressed in stratified squamous epithelia, such as those in skin and cervix, but not in other tissues. In situ hybridization and immunohistochemistry revealed that cPLA2δ is expressed strongly in the upper spinous layer of the psoriatic epidermis, expressed weakly and discontinuously in atopic dermatitis and mycosis fungoides, and not detected in the epidermis of normal skin; cPLA2α is not detected in either normal or psoriatic skin. These results suggest that cPLA2δ exhibits a unique distribution pattern compared with that of known cPLA2 subtypes, and it may play a critical role in inflammation in psoriatic lesions. The phospholipase A2 (PLA2) 1The abbreviations used are: PLA2, phospholipase A2; cPLA2, cytosolic phospholipase A2; sPLA2IIA, secretory phospholipase A2 type IIA; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; iAFLP, introduced-amplified fragment length polymorphism; PC, phosphatidylcholine. 1The abbreviations used are: PLA2, phospholipase A2; cPLA2, cytosolic phospholipase A2; sPLA2IIA, secretory phospholipase A2 type IIA; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; iAFLP, introduced-amplified fragment length polymorphism; PC, phosphatidylcholine. family comprises a large number of structurally and mechanistically distinct enzymes that catalyze the hydrolysis of glycerophospholipids at the sn-2 position and then liberate free fatty acids and lysophospholipids (1Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (754) Google Scholar). One of the free fatty acids released by PLA2, arachidonic acid, is known to be the precursor of eicosanoids such as prostaglandins and leukotrienes, which are potent inflammatory mediators. Among various mammalian PLA2s, calcium-dependent 85-kDa cytosolic PLA2 (cPLA2α) is thought to have an important role in this process, because it is able to selectively release arachidonic acid (2Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1453) Google Scholar). cPLA2α, which encodes a polypeptide of 749 amino acids that contains an N-terminal calcium-dependent phospholipid binding domain (the C2 domain) and a catalytic domain (2Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1453) Google Scholar), is expressed ubiquitously in many different tissues (3Pickard R.T. Strifler B.A. Kramer R.M. Sharp J.D. J. Biol. Chem. 1999; 274: 8823-8831Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) and is regulated post-translationally. In response to a submicromolar concentration of intracellular free calcium ions, cPLA2α is translocated from the cytoplasm to the nuclear envelope and the endoplasmic reticulum (2Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1453) Google Scholar). This process is dependent on the N-terminal C2 domain (2Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1453) Google Scholar). In addition to the calcium-regulated mechanism, it is also known that MAP kinases can phosphorylate Ser-505 of cPLA2α and directly activate its intrinsic enzymatic activity (4Lin L.-L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1641) Google Scholar). Recently, two new members of the cPLA2 family (cPLA2β and cPLA2γ) have been identified (3Pickard R.T. Strifler B.A. Kramer R.M. Sharp J.D. J. Biol. Chem. 1999; 274: 8823-8831Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 5Underwood K.W. Song C. Kriz R.W. Chang X.J. Knopf J.L. Lin L.-L. J. Biol. Chem. 1998; 273: 21926-21931Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 6Song C. Chang X.J. Bean K.M. Proia M.S. Knopf J.L. Kriz R.W. J. Biol. Chem. 1999; 274: 17063-17067Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). cPLA2β encodes a polypeptide of 1012 amino acids that shares homology with both the C2 and catalytic domains of cPLA2α and is expressed ubiquitously (3Pickard R.T. Strifler B.A. Kramer R.M. Sharp J.D. J. Biol. Chem. 1999; 274: 8823-8831Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 6Song C. Chang X.J. Bean K.M. Proia M.S. Knopf J.L. Kriz R.W. J. Biol. Chem. 1999; 274: 17063-17067Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). On the other hand, cPLA2γ encodes a polypeptide of 541 amino acids that shares homology with only the catalytic domain of cPLA2α. cPLA2γ exhibits calcium-independent activity due to the loss of the C2 domain and also has a prenylation site at the C terminus. It is expressed in heart and skeletal muscle (5Underwood K.W. Song C. Kriz R.W. Chang X.J. Knopf J.L. Lin L.-L. J. Biol. Chem. 1998; 273: 21926-21931Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Further studies will be required to elucidate the function of these molecules in the arachidonic acid cascade and in inflammation. Psoriasis is a chronic inflammatory disease of the skin that is associated with both genetic and environmental risk factors (7Farber E.M. Nall L.M. Dermatologica. 1974; 148: 1-18Crossref PubMed Scopus (504) Google Scholar, 8Nickoloff B.J. J. Clin. Investig. 1999; 104: 1161-1164Crossref PubMed Scopus (109) Google Scholar). The psoriatic skin is characterized by the overgrowth and abnormal differentiation of epidermal keratinocytes, resulting in an impaired barrier function of the skin, and by the influx of activated immunocytes into both the epidermis and dermis, although the sequence of these events in the progression of psoriatic lesions is unclear (8Nickoloff B.J. J. Clin. Investig. 1999; 104: 1161-1164Crossref PubMed Scopus (109) Google Scholar). PLA2s are among the numerous molecules that reportedly participate in the progression of psoriatic lesions. They are thought to be involved in the pathogenesis of psoriasis by releasing arachidonic acid as the source of inflammatory eicosanoids, because free arachidonic acid and eicosanoids such as leukotriene B4 and 12-hydroxyeicosatetraenoic acid are known to accumulate in psoriatic skin (9Hammarstorm S. Hamberg M. Samuelsson B. Duell E.A. Stawiski M. Voorhees J.J. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 5130-5134Crossref PubMed Scopus (495) Google Scholar, 10Fogh K. Herlin T. Kragballe K. J. Investig. Dermatol. 1989; 92: 837-841Abstract Full Text PDF PubMed Scopus (63) Google Scholar). It has also been reported that transgenic mice overexpressing type IIA secretory phospholipase A2 (sPLA2IIA) exhibit epidermal hyperplasia and hyperkeratosis in the absence of inflammatory infiltrate (11Grass D.S. Felkner R.H. Chiang M.-Y. Wallace R.E. Nevalainen T.J. Bennett C.F. Swanson M.E. J. Clin. Investig. 1996; 97: 2233-2241Crossref PubMed Scopus (159) Google Scholar). In the present study, we have described a new member of the human cPLA2 family that is induced in the epidermis of psoriatic skin and shows an epithelial-specific tissue distribution. Samples and Materials—Skin samples were obtained from diseased and normal skin of psoriatic patients, from age-matched healthy controls, and from patients with atopic dermatitis and mycosis fungoides. Informed consent was obtained from all individuals who were subjected to skin biopsies. Restriction endonucleases and DNA-modifying enzymes were purchased from Takara Shuzo (Kyoto, Japan). COS-7 cells were obtained from Dainippon Pharmaceutical Co. (Osaka, Japan). Dulbecco's modified Eagle's medium and fetal calf serum were obtained from Invitrogen. [α-32P]dCTP and Hybond-N+ were from Amersham Biosciences. Affinity purified polyclonal anti-cPLA2α and anti-sPLA2IIA antibodies were obtained from Santa Cruz Biotechnology. Expression Profiling in Normal and Psoriatic Human Skin—Total RNA was isolated from all samples using an RNeasy kit (Qiagen) according to the manufacturer's instructions. A 3′-directed cDNA library was constructed from each total RNA sample derived from normal and psoriatic skin according to the methods of Okubo et al. (12Okubo K. Hori H. Matoba R. Niiyama T. Matsubara K. DNA Seq. 1991; 2: 137-144Crossref PubMed Scopus (76) Google Scholar). Random sequencing of 10,000 clones from each library was performed by the dideoxy chain termination method using an Applied Biosystems Model 373A automated sequencer and a dye terminator cycle sequencing kit (PE Biosystems). The frequency of appearance of each sequence was determined, and a search of sequence similarity against a DNA data bank was performed. Quantitative expression profiling of candidate genes in 7 normal skin samples, 10 psoriatic skin samples, 2 atopic dermatitis samples, and 3 mycosis fungoides samples was performed by a PCR-based method, introduced-amplified fragment length polymorphism (iAFLP) (13Kawamoto S. Ohnishi T. Kita H. Chisaka O. Okubo K. Genome Res. 1999; 9: 1305-1312Crossref PubMed Scopus (34) Google Scholar). Briefly, total RNA was annealed with a pUC19-based vector primer, and first and second cDNA syntheses were performed. cDNA was cleaved with MboI and ligated with an iAFLP adapter cassette with T4 ligase. Using this cDNA as template, a competitive PCR reaction was performed. An autosequencer 377XL and GeneScan program (PE Biosystems) were used for sizing and quantitation of dye-labeled fragments. For amplification of GS21015, the PCR reaction was performed using Tamra-labeled T7 primer (5′-ACGACTCACTATAGGGAGATTACTT-3′) and GS21015-specific antisense primer (5′-TACACGACAAAACCACAT GA-3′). Screening of the cDNA Library—The partial 3′-untranslated region fragment of cPLA2δ cDNA (about 1.2 kb) was amplified by PCR using sense (5′-GTCCAGCGCAGCCCCGCAGA-3′) and antisense (5′-AAATACACGACAAAACCACA-3′) primers. To obtain the full-length cPLA2δ cDNA, library screening was performed as reported previously (14Imai Y. Suzuki Y. Tohyama M. Wanaka A. Takagi T. Mol. Brain Res. 1994; 24: 313-319Crossref PubMed Scopus (24) Google Scholar) using the aforementioned cDNA fragment as a probe and a human keratinocyte cDNA library. A pBluescript II SK(-) plasmid containing an insert DNA was isolated from a positive plaque by an in vivo excision method (Stratagene), and the nucleotide sequence of the insert DNA was determined. Production of a Polyclonal Antibody to cPLA2δ—A fragment of cPLA2δ representing amino acids 1-224 (Fig. 1) was subcloned into a pGEX vector for expression of a glutathione S-transferase fusion protein in Escherichia coli. The expressed fusion protein, purified on a glutathione-Sepharose 4B column (Amersham Biosciences) according to the manufacturer's instructions, was used to raise polyclonal antibodies in rabbits. Expression of cPLA2s in COS-7 Cells and the PLA2 Assay—The open reading frame region of cPLA2α cDNA was PCR-amplified using the following primers: a 5′ sense primer with an associated EcoRV site (5′-GATCGATATCCATGTC-ATTTATAGATCCTTACCAGC-3′) and a 3′ antisense primer with an associated XhoI site (5′-GATCCTCGAGCTATGCTTTGGGTTTACTTAGAAAC-3′). The obtained cPLA2α cDNA and the open reading frame region of the cPLA2δ cDNA were each subcloned into a pcDNA4/HisMax vector (Invitrogen) and used to transfect COS-7 cells using a SuperFect transfection kit (Qiagen) according to the manufacturer's instructions. Cells harvested from a 10-cm dish were suspended in 500 μl of lysis buffer (10 mm HEPES, pH 7.5, 1 mm EDTA, 0.1 mm dithiothreitol, 0.34 m sucrose, and 1 mm phenylmethylsulfonyl fluoride) and lysed with sonication on ice. The lysate was centrifuged at 15,000 rpm for 10 min, and the supernatant was used as the lysate. The protein concentration was determined with a DC Protein Assay Kit (Bio-Rad). Anti-cPLA2δ polyclonal antibody and an Omni-probe (Santa Cruz Biotechnology) were used for Western blot analysis to detect proteins that were expressed in the lysate. PLA2 activity was determined in the presence of 10 mm CaCl2 or 5 mm EGTA as described previously (5Underwood K.W. Song C. Kriz R.W. Chang X.J. Knopf J.L. Lin L.-L. J. Biol. Chem. 1998; 273: 21926-21931Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) except for a minor change of substrate concentration (2 μm). 1-Palmitoyl-2-[1-14C]arachidonyl-phosphatidylcholine (PC), 1-palmitoyl-2-[1-14C]linoleoyl-PC, and 1-palmitoyl-2-[1-14C]-oleoyl-PC (PerkinElmer Life Science) were used as substrates in the assay. The released free arachidonic acid was measured as described previously (16Zhang Y.Y. Deems R.A. Dennis E.A. Methods Enzymol. 1991; 197: 456-468Crossref PubMed Scopus (20) Google Scholar). Northern Blot Analysis—A human multiple tissue Northern blot (Toyobo) was hybridized with a 32P-labeled cDNA probe as described for the library screening. Hybridization was performed by incubating the probe at 65 °C for 16 h in 6× SSC, 0.5% SDS, 5× Denhardt's solution, and 100 μg/ml salmon sperm DNA. Finally, the membrane was washed with 0.1× SSC and 0.5% SDS at 65 °C for 1 h and exposed to x-ray film at -70 °C for 2 days. In Situ Hybridization—A XbaI/AccI fragment of 720 bp in the 3′-untranslated region of cPLA2δ was subcloned into pBluescript II SK(+) and used for probe preparation. The cDNA fragments of the respective PLA2 subtypes were amplified by PCR and cloned into a pGEM-T easy vector (Promega) using the following primers: cPLA2α-5′ (5′-GGA-TCCTAATCAGGAAAATG-3′), cPLA2α-3′ (5′-GAATTCCTGATTCGTATAAT-3′), cPLA2β-5′ (5′-GAAGAGGCCATGGAGAAGGC-3′), cPLA2β-3′ (5′-CCTCCAGTT-GAACGTGCAAG-3′), cPLA2γ-5′ (5′-TCGAGGCTGACCTGAAACAT-3′), cPLA2γ-3′ (5′-CCAATTCTGAGCATCTCAG-3′), sPLA2IIA-5′ (5′-CCAT-GAAGACCCTCCTACTG-3′), sPLA2IIA-3′ (5′-GAGGGGACTCAGCAACGA-GG-3′). Digoxigenin-labeled sense and antisense cRNA probes were synthesized with T7, T3, and SP6 polymerase using a digoxigenin RNA labeling kit from Roche Diagnostics according to the manufacturer's instructions. In situ hybridization was performed on paraffin-embedded sections from normal and psoriatic skin as described previously (15Komminoth P. Merk F.B. Leav I. Wolfe H.J. Roth J. Histo-chemistry. 1992; 98: 217-228Google Scholar). Immunohistochemistry—Immunohistochemical observations were performed on paraffin-embedded sections from normal and psoriatic skin with an ABC kit (Vector Laboratory) using anti-cPLA2δ polyclonal antibody and affinity-purified anti-cPLA2α and anti-sPLA2IIA antibodies (Santa Cruz Biotechnology). Gene Expression Profiling in Normal and Psoriatic Human Skin—To monitor the difference in gene expression between normal and psoriatic skin, we constructed 3′-directed cDNA libraries from normal and psoriatic skin and determined the nucleotide sequence of 10,000 randomly selected clones from each of these libraries. Information was obtained for 3,156 independent 3′-expressed sequence tags (Gene Signature). The frequency information for the gene signature in each library provides a rough estimate of the mRNA composition of each source tissue. Some of the clones were identified by comparison with a DNA data bank. These included genes already known to be differentially expressed in psoriasis compared with normal skin, such as keratins 6, 16, and 17 (17Leigh I.M. Navsaria H. Purkis P.E. McKay I.A. Bowden P.E. Riddle P.N. Br. J. Dermatol. 1995; 133: 501-511Crossref PubMed Scopus (255) Google Scholar), psoriasin (18Madsen P. Rasmussen H.H. Leffers H. Honore B. Dejgaard K. Olsen E. Kiil J. Walbum E. Andersen A.H. Basse B. J. Investig. Dermatol. 1991; 97: 701-712Abstract Full Text PDF PubMed Scopus (350) Google Scholar), SKALP/elafin (19Schalkwijk J. de Roo C. de Jongh G.J. Biochim. Biophys. Acta. 1991; 1096: 148-154Crossref PubMed Scopus (67) Google Scholar), and loricrin (20Juhlin L. Magnoldo T. Darmon M. Acta Derm. Venereol. 1992; 72: 407-409PubMed Google Scholar). GS21015 Expression Profiling in Normal and Psoriatic Skin—We also obtained some undefined clones from the above procedure. To investigate the possible function of these genes, we searched their predicted cDNA sequences using exon the prediction software GenScan (21Burge C. Karlin S. J. Mol. Biol. 1997; 268: 78-94Crossref PubMed Scopus (2978) Google Scholar). Consequently, we obtained a new gene, referred to as GS21015. The predicted GS21015 cDNA consists of 25 exons and encodes a polypeptide of 1020 amino acids. The predicted amino acid sequence of GS21015 was highly homologous with cPLA2β, ∼50% within a region of 800 amino acids. The GS21015 sequence appeared three times in the 3705 clones from the psoriasis library. In contrast, it did not appear in the 4302 clones from the normal skin library. These results suggest that GS21015 mRNA expression is up-regulated in psoriatic skin. To confirm the up-regulation of GS21015 mRNA in psoriatic skin, we examined quantitative mRNA expression using the iAFLP method. Total RNA samples prepared from 7 normal and 10 psoriatic skin samples were used as templates for iAFLP. The relative data showed that the expression of GS21015 mRNA increased by about 9-fold (p < 0.001) in psoriatic skin compared with normal skin (Table I). Furthermore, to investigate whether GS21015 is up-regulated in other inflammatory skin diseases, two atopic dermatitis samples and three mycosis fungoides samples were analyzed in the same way. No significant difference was observed between normal skin and mycosis fungoides, but GS21015 was significantly increased (p < 0.001) in atopic dermatitis compared with normal skin.Table IComparison of cPLA2δ expression levels among different skin types using quantitative reverse transcription PCR (iAFLP)StateRelative peak area (no. of biopsies)Normal0.88 ± 0.38 (n = 7)Psoriasis7.89 ± 4.52 (n = 10)Atopic dermatitis4.35 ± 0.77 (n = 2)Mycosis fungoides2.11 ± 1.17 (n = 3) Open table in a new tab Cloning of the Full-length cPLA2δ cDNA (GS21015)—The full-length cDNA of GS21015 was isolated from a λ-phage human keratinocyte cDNA library by conventional hybridization screening. The cDNA contains an open reading frame of 2454 nucleotides that encodes a polypeptide of 818 amino acids. (The DNA sequence data are available from the DDBJ/EMBL/GenBank™ data bases under accession number AB090876.) A search of the GenBank™ Data Bank using the nucleotide and amino acid sequences revealed a significant homology with members of the cPLA2 family (Fig. 1). The full-length cDNA, designated as cPLA2δ, showed the highest homology with cPLA2β. The open reading frame sequence of cPLA2δ cDNA shares 44.5, 59.9, and 45.7% identity with cPLA2α, cPLA2β, and cPLA2γ, respectively. The amino acid sequence of cPLA2δ shares 29, 50, and 27% identity and 49, 66, and 42% similarity with cPLA2α, cPLA2β, and cPLA2γ, respectively. The genomic BAC clone RP11-35K2, which contains the cPLA2δ gene, maps to human chromosome 15q15, which is near the 15q11.2-q21.3 chromosomal locus of the cPLA2β gene. The cPLA2α and cPLA2γ genes are present at chromosomal locus 1q25 and 19q13.3, respectively. Alignment of the cPLA2δ cDNA sequence with the genomic sequence showed that cPLA2δ cDNA contained 20 exons, including the 7th-25th exons predicted by GenScan, and also showed that all of the intron/exon boundaries follow the GT-AG rule. Residues in cPLA2α that are considered necessary for catalysis, such as Arg-200, Ser-228, and Asp-549, were conserved in cPLA2δ at Arg-333, Ser-361, and Asp-647, respectively (Fig. 1, asterisks). The Ser-505 in cPLA2α, which is known to be phosphorylated by ERK1/2 MAP kinases, is absent in cPLA2δ, which has no MAP kinase phosphorylation site motif, similar to cPLA2β and cPLA2γ. Expression of cPLA2δ in COS-7 Cells and PLA2 Activity—To investigate the enzymatic activity of cPLA2δ, we transfected COS-7 cells with a pcDNA4/HisMax vector containing the full-length cDNA of cPLA2δ. The vector only was transfected as a control. cPLA2δ protein of about 90 kDa was detected in the cPLA2δ-transfected cell lysate by Western blot using an anti-cPLA2δ polyclonal antibody and an anti-express tag antibody (Omni-probe). cPLA2δ was detected as a doublet, probably because of post-translational modification (Fig. 2A). PLA2 activities were determined in cell lysates of the cPLA2δ-transfected cells and vector-transfected cells using 1-palmitoyl-2-[1-14C]arachidonyl-PC as a substrate. The amount of arachidonic acid released by the cPLA2δ-transfected cell lysate was 5-6 times higher than that released by the vector-transfected cell lysate (Fig. 2B). These results indicate that cPLA2δ is functional as a PLA2 enzyme. A similar level of PLA2 activity was obtained for a cPLA2α-transfected cell lysate (as a positive control) (Fig. 2B), although the quantity of expressed cPLA2δ protein estimated from the Western analysis was much higher than that of cPLA2α (Fig. 2A). This result suggests that the specific activity of cPLA2δ might be considerably lower than that of cPLA2α. The activity of cPLA2β has been reported to be about 3 orders of magnitude lower than that of cPLA2α (3Pickard R.T. Strifler B.A. Kramer R.M. Sharp J.D. J. Biol. Chem. 1999; 274: 8823-8831Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), and therefore the level of cPLA2δ activity may be similar to that of cPLA2β. When the PLA2 assay was performed in the absence of free calcium ions, no activity was detected in the lysate (Fig. 2C). This result indicates that cPLA2δ has a functional C2 domain. We next investigated the substrate specificity of cPLA2δ in the sn-2 position. PLA2 activities were determined in cell lysates of the cPLA2α-, cPLA2δ-, and vector-transfected cells using 14C-labeled substrates including oleic acid or linoleic acid instead of arachidonic acid, in the sn-2 position (Table II). cPLA2α lysate released arachidonic acid in the sn-2 position most efficiently, as described previously (2Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1453) Google Scholar). In contrast, cPLA2δ lysate released similar levels of oleic acid and arachidonic acid and released linoleic acid about 6 times more efficiently than arachidonic acid. This result suggests that the activity of cPLA2δ is not arachidonic acid-specific and that cPLA2δ has linoleic acid-specific activity.Table IISubstrate specificity of cPLA2δ at the sn-2 positionSubstrateVector controlcPLA2αcPLA2δ1-Pam-2-[14C]Ach-PtdCho2.49 ± 0.239.34 ± 0.819.23 ± 0.631-Pam-2-[14C]Lin-PtdCho−0.52 ± 0.686.13 ± 0.1058.56 ± 13.771-Pam-2-[14C]Ole-PtdCho−0.70 ± 0.400.27 ± 0.5110.99 ± 0.24 Open table in a new tab Epithelium-specific Expression of cPLA2δ—To determine whether the expression of the cPLA2δ transcript is specific to skin, Northern blot analysis was performed using human multiple tissue mRNA blots with the cPLA2δ cDNA fragment as a probe. The mRNA of cPLA2δ was detected as a band of about 4 kb in fetal skin, cervix, and, to a lesser extent, in prostate but not in other tissues (Fig. 3). Among the tissues examined, only skin and cervix contain stratified squamous epithelia, and both expressed cPLA2δ strongly. Other epithelial tissues such as lung, stomach and colon, which consist of simple or pseudostratified columnar epithelia, appeared not to express cPLA2δ. Therefore, this suggests that cPLA2δ is expressed specifically in stratified squamous epithelia. Increased Expression of cPLA2δ in the Epidermis of Psoriatic Skin—To confirm the increased expression of cPLA2δ in psoriatic skin compared with normal skin, and to identify the cells that express cPLA2δ in the skin, we performed an in situ hybridization analysis on paraffin-embedded tissue sections from samples of normal skin, psoriasis, atopic dermatitis, and mycosis fungoides using a cRNA probe derived from the 3′-cDNA fragment of cPLA2δ. The results showed that cPLA2δ mRNA was expressed strongly in the upper epidermis of psoriatic skin but not in the lower epidermis and dermis (Fig. 4B). In contrast, cPLA2δ was not detected in normal skin, even in the upper epidermis where normally differentiated keratinocytes were distributed (Fig. 4A). In atopic dermatitis, cPLA2δ expression was observed in the upper epidermis, but the signal was discontinuous, whereas a continuous signal was observed in psoriatic skin (Fig. 4C). In mycosis fungoides, a very weak, discontinuous signal was observed in the upper epidermis. No such signal was ever seen in normal skin. These results suggest that the expression of cPLA2δ is increased in inflammatory skin diseases, especially in psoriasis. To confirm the expression of other PLA2 subtypes in psoriatic skin, in situ hybridization was also performed using cPLA2α, cPLA2β, cPLA2γ, and sPLA2IIA cRNA probes. The results showed that none of the PLA2 subtypes could be detected in either normal or psoriatic skins (data not shown). Although the lack of detection may reflect the low sensitivity of this experiment, it is possible that over-expression of these subtypes does not occur in psoriatic skin compared with normal skin. To investigate the cPLA2δ protein expression further, we prepared a rabbit polyclonal antibody against the N-terminal 224 amino acids of cPLA2δ. An immunohistochemical analysis using the antibody showed that cPLA2δ was expressed in the upper epidermis of psoriatic skin but was not detected in normal skin (Fig. 5, A and C), a similar result to that found in in situ hybridization. A weak signal was seen in the basal layer of normal skin, but this was considered to be because of melanin pigmentation, as there is similar pigmentation in dewaxed-only sections with hematoxilin staining (Fig. 5B). Therefore it was concluded that this weak signal is not cPLA2δ-specific. We also investigated the expression of cPLA2α and sPLA2IIA in normal and psoriatic skin using commercially available antibodies. sPLA2IIA was detected in the upper epidermis of psoriatic skin but not in normal skin (Fig. 5, F and G), although no in situ hybridization signal was detected in either skin type. cPLA2α was not detected in either skin type (Fig. 5, D and E). In this study we isolated a novel cPLA2 subtype, which we designated as cPLA2δ. cPLA2δ has significant homology with the known cPLA2 subtypes, cPLA2α, cPLA2β, and cPLA2γ, in both the C2 and catalytic domains. Among the three known subtypes, cPLA2δ seems to be the most closely related to cPLA2β in terms of sequence similarity and chromosomal location. However, cPLA2δ is expressed specifically in stratified squamous epithelia, whereas cPLA2β is expressed widely in many tissues (3Pickard R.T. Strifler B.A. Kramer R.M. Sharp J.D. J. Biol. Chem. 1999; 274: 8823-8831Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). The expression of cPLA2δ is markedly increased in psoriatic skin, but cPLA2β is not detected in either psoriatic or normal skin. Although the possibility that cPLA2β mRNA expression is below the detection limit of in situ hybridization cannot be ruled out, these results suggest that cPLA2β and cPLA2δ are subject to different transcriptional regulatory mechanisms and may have distinct physiological roles in the human body. As expected, cPLA2δ has calcium-dependent PLA2 activity when expressed in COS-7 cells. Therefore, cPLA2δ activity might be regulated by intracellular calcium levels and by a variety of other stimuli, including inflammatory cytokines that may cause translocation of cPLA2δ as well as cPLA2α. cPLA2δ was detected as a doublet band by Western blotting, the cause of which is unclear. The activity of cPLA2α is regulated by phosphorylation (4Lin L.-L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1641) Google Scholar), and hence this raises the question of whether cPLA2δ is also activated enzymatically through phosphorylation. However, cPLA2δ contains no MAP kinase phosphorylation site motif, an issue that remains to be elucidated. There is strongly induced expression of cPLA2δ in the upper layer of the psoriatic epidermis, which consists of abnormally differentiated keratinocytes. These keratinocytes have been activated by inflammatory cytokines such as interferon-γ, which are released by infiltrated T cells in which diverse genes are activated transcriptionally or post-translationally. cPLA2δ could also be activated transcriptionally by such stimuli. In contrast, cPLA2α was not detected in the normal and psoriatic epidermis at both the mRNA and protein levels, and therefore it appears not to be increased in psoriatic skin compared with normal skin. cPLA2β and cPLA2γ mRNA were also not detected in normal or psoriatic skin. sPLA2IIA is the only PLA2 subtype that has previously been reported to be increased in psoriasis (22Andersen S. Sjursen W. Laegreid A. Volden G. Johansen B. Inflammation. 1994; 18: 1-12Crossref PubMed Scopus (69) Google Scholar); we also confirmed this result at the protein level although failing to detect the sPLA2IIA mRNA. A similar result has been reported in a different species. sPLA2IIA was detected in several rat tissues by immunohistochemistry and Northern blotting, but no in situ hybridization signal was detected (23Nyman K.M. Ojala P. Laine V.J. Nevalainen T.J. J. Histochem. Cytochem. 2000; 48: 1469-1478Crossref PubMed Scopus (25) Google Scholar). Therefore, in the case of this gene, mRNA expression may be below the detection limit of the current methods of tissue preservation and in situ hybridization. On the basis of the results of in situ hybridization and immunohistochemical analyses, cPLA2δ is expressed in psoriasis at a higher level than sPLA2IIA. It is thus likely that cPLA2δ is the most abundant PLA2 subtype in psoriatic skin. The result of the enzyme assay suggests that cPLA2δ preferentially releases linoleic acid in the sn-2 position and, to a lesser extent, arachidonic acid and oleic acid. In psoriatic skin, it has been reported that 13-hydroxyoctadecadienoic acids, which are linoleic acid metabolites, are the principal fatty acid derivatives (24Baer A.N. Costello P.B. Green F.A. J. Lipid Res. 1990; 31: 125-130Abstract Full Text PDF PubMed Google Scholar). Therefore it is possible that in psoriatic skin the release of linoleic acid from membrane phospholipids occurs efficiently along with arachidonic acid release due to cPLA2δ induction. It has been reported that PLA2 activity and the amount of free arachidonic acid were markedly increased in psoriatic skin compared with normal skin (9Hammarstorm S. Hamberg M. Samuelsson B. Duell E.A. Stawiski M. Voorhees J.J. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 5130-5134Crossref PubMed Scopus (495) Google Scholar) and that multiple topical applications of arachidonic acid to the skin of a mouse ear over several days induced acanthosis (epidermal hyperplasia), hyperkeratosis, parakeratosis, and intraepidermal neutrophil accumulation (25Doherty N.S. Beaver T.H. Rheins L.A. Nordlund J.J. J. Investig. Dermatol. 1988; 91: 298-302Abstract Full Text PDF PubMed Scopus (19) Google Scholar), which are the hallmarks of psoriatic skin. Therefore, it is suggested that the release of arachidonic acid in the epidermis is an important event in the onset of psoriasis, and cPLA2δ might have a central role in the production of free arachidonic acid in psoriatic skin. Hence, there is a possibility that the selective inhibition of cPLA2δ might result in the healing of psoriasis and would also have minimal side effects due to the restricted distribution of cPLA2δ. To investigate these possibilities, further studies, such as the construction of transgenic mice expressing cPLA2δ constitutively in the epidermis and a study of the effects of specific inhibitors on inflammatory skin models, will be required." @default.
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• W2032992679 title "Cloning of a Gene for a Novel Epithelium-specific Cytosolic Phospholipase A2, cPLA2δ, Induced in Psoriatic Skin" @default.
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