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Regulation of Vascular Endothelial Growth Factor Expression in Human Keratinocytes by Retinoids

2000; Elsevier BV; Volume: 275; Issue: 1 Linguagem: Inglês

10.1074/jbc.275.1.642

1083-351X

Bárbara Vega Diaz, Marie-Cécile Lenoir, Annie Ladoux, Christian Frelin, Michel Démarchez, Serge Michel,

melanin and skin pigmentation

Vascular endothelial growth factor (VEGF) is overexpressed in hyperproliferative diseases, such as psoriasis and cancers, which are characterized by increased angiogenesis. Experimentally, VEGF overexpression can be induced by the treatment of cell cultures and biological tissues with phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Using normal human keratinocytes in conventional cultures and skin grafted onto nude mice in vivo, we show that retinoids can inhibit TPA-mediated VEGF gene induction at the transcriptional level. Because retinoids are biologically active either by interacting with the nuclear retinoic acid receptors or by interfering with the activator protein 1 (AP1) transcription factor, we studied the effect of the retinoic acid derivative CD 2409, which exhibits strong anti-AP1 activity but does not bind to the known retinoic acid receptorsin vitro. The results demonstrate that the inhibition of VEGF expression by retinoids only depends on their anti-AP1 activity and does not require gene transactivation via retinoic acid response elements. Because the VEGF promoter contains four potential AP1 binding sites, we used different promoter constructs to identify the functional site responsible for TPA induction and retinoid inhibition. This site turned out to be localized at position −621 of the 5′ flanking region of the VEGF gene. Vascular endothelial growth factor (VEGF) is overexpressed in hyperproliferative diseases, such as psoriasis and cancers, which are characterized by increased angiogenesis. Experimentally, VEGF overexpression can be induced by the treatment of cell cultures and biological tissues with phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Using normal human keratinocytes in conventional cultures and skin grafted onto nude mice in vivo, we show that retinoids can inhibit TPA-mediated VEGF gene induction at the transcriptional level. Because retinoids are biologically active either by interacting with the nuclear retinoic acid receptors or by interfering with the activator protein 1 (AP1) transcription factor, we studied the effect of the retinoic acid derivative CD 2409, which exhibits strong anti-AP1 activity but does not bind to the known retinoic acid receptorsin vitro. The results demonstrate that the inhibition of VEGF expression by retinoids only depends on their anti-AP1 activity and does not require gene transactivation via retinoic acid response elements. Because the VEGF promoter contains four potential AP1 binding sites, we used different promoter constructs to identify the functional site responsible for TPA induction and retinoid inhibition. This site turned out to be localized at position −621 of the 5′ flanking region of the VEGF gene. vascular endothelial growth factor activator protein enzyme-linked immunosorbent assay normal human keratinocyte all-trans retinoic acid retinoic acid receptor retinoid X receptor retinoic acid response element 12-O-tetradecanoylphorbol-13-acetate glyceraldehyde-3-phosphate dehydrogenase polymerase chain reaction reverse transcription Vascular endothelial growth factor (VEGF)1 was first described as a tumor cell derived factor that induced vascular hyperpermeability to plasma proteins (1Senger D.R. Ledbetter S.R. Claffey K.P. Papadopoulos-Sergiou A. Peruzzi C.A. Detmar M. Am. J. Pathol. 1983; 149: 293-305Google Scholar). It was further characterized as an endothelial cell specific mitogen with the capacity to induce angiogenesis in a number of experimental in vivo models (2Leung D.W. Cachianes G. Kuang W.J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4532) Google Scholar, 3Keck P.J. Hauser S.D. Krivi G. Sanzo K Warren T. Feder J. Connoly D.T. Science. 1989; 246: 1309-1312Crossref PubMed Scopus (1849) Google Scholar, 4Ferrara N. Trends Cardiovasc. Med. 1993; 3: 244-250Crossref PubMed Scopus (172) Google Scholar). VEGF is a secreted homodimeric glycoprotein of 40–45 kDa that selectively binds to two high affinity tyrosine kinase receptors on endothelial cells (5De Vries C. Escobedo J.A. Veno H. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1903) Google Scholar,6Terman B.I. Dougher-Vermazen M. Carrion M.E. Dimitrov D. Armellino D.C. Gospodarowicz D. Böhlen P. Biochem. Biophys. Res. Commun. 1992; 187: 1579-1586Crossref PubMed Scopus (1414) Google Scholar). Four different human isoforms have been isolated to date, resulting from alternative splicing of VEGF mRNA (7Tischer E. Mitchell R. Hartman T. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. J. Biol. Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar). The two larger variants, VEGF 189 and VEGF 206, remain cell-associated, whereas the two smaller, forms VEGF121 and VEGF 165, are secreted (8Ferrara N. Houck K. Jakeman L. Leung D.W. Endocr. Rev. 1992; 13: 18-32Crossref PubMed Scopus (1568) Google Scholar). VEGF is the major angiogenic factor that regulates the growth of new capillaries from preexisting blood vessels, a process that involves the extravasion of plasma proteins, degradation of the extracellular matrix, and endothelial cell migration and proliferation, as well as capillary tube formation (9Detmar M. J. Invest. Dermatol. 1996; 106: 207-208Abstract Full Text PDF PubMed Scopus (80) Google Scholar). In normal human skin, VEGF is both expressed and secreted by epidermal keratinocytes. Neovascularisation, which occurs during wound healing, is associated with an enhanced expression of VEGF by migrating keratinocytes and with the up-regulation of VEGF receptors on dermal microvessels (10Brown L.F. Yeo K.T. Berse B. Yeo T.K. Senger D.R. Dvorak H.F. Van de Water L. J. Exp. Med. 1992; 176: 1375-1379Crossref PubMed Scopus (794) Google Scholar). VEGF expression is up-regulated in certain skin diseases involving vascular hyperproliferation, such as psoriasis (11Detmar M. Brown L.F. Claffey K.P. Yeo K.T. Kocher O. Jackman R.W. Berse B. Dvorak H.F. J. Exp. Med. 1994; 180: 1141-1146Crossref PubMed Scopus (657) Google Scholar), delayed-type skin hypersensitivity reactions, bullous diseases (12Brown L.F. Harrist T.J. Yeo K.T. Stahle-Backdahl M. Jackman R.W. Berse B. Tognazzi K. Dvorak H.F. Detmar M. J. Invest. Dermatol. 1995; 104: 744-749Abstract Full Text PDF PubMed Scopus (137) Google Scholar), and Kaposi's sarcoma (13Weindel K. Marme D. Weich H.A. Biochem. Biophys. Res. Commun. 1992; 183: 1167-1174Crossref PubMed Scopus (162) Google Scholar). In cultured human keratinocytes, the expression of VEGF is increased by serum, transforming growth factor-β1, tumor necrosis factor α, keratinocyte growth factor (14Frank S. Hubner G. Breier G. Longaker M.T. Greenhalgh D.G. Werner S. J. Biol. Chem. 1995; 270: 12607-12613Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar), UVB, oxidants such as H2O2 (15Brauchle M. Funk J.O. Kind P. Werner S. J. Biol. Chem. 1996; 271: 21793-21797Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), and hypoxia (16Detmar M. Brown L.F. Berse B. Jackman R.W. Elicker B.M. Dvorak H.F. Claffey K.P. J. Invest. Dermatol. 1997; 108: 263-268Abstract Full Text PDF PubMed Scopus (236) Google Scholar). Retinoids consist of both natural and synthetic vitamin A derivatives, which are potent agents for the treatment of different skin disorders (17Fritsch P.O. J. Am. Acad. Dermatol. 1992; 27: S8-S14Abstract Full Text PDF PubMed Scopus (72) Google Scholar). They exert their biological effects via two families of nuclear receptors, which belong to the superfamily of steroid/thyroid hormone nuclear receptors. They comprise the retinoic acid receptors (RAR α, β, and γ), which bind with both all-trans retinoic acid (RA) and 9-cis RA, and the retinoid X receptors (RXR α, β, and γ), which only bind with 9-cis RA. The two classes of receptors are ligand-dependent transactivating factors that regulate gene expression by interacting with the promoter of target genes in the form of RXR/RXR homodimers or RAR/RXR heterodimers (18Brand N. Petkovich M. Krust A. Chambon P. Marchio A. Tiollais P. Dejean A. Nature. 1988; 332: 850-853Crossref PubMed Scopus (867) Google Scholar, 19Mangelsdorf D.J. Borgmeyer U. Heyman R.A. Zhou J.Y. Ong E.S. Oro A.E. Kakizuka A. Evans R.M. Genes Dev. 1992; 6: 329-344Crossref PubMed Scopus (1085) Google Scholar, 20Petkovich M. Brand N.J. Krust A. Chambon P. Nature. 1987; 330: 444-450Crossref PubMed Scopus (1833) Google Scholar). They can also indirectly down-regulate the expression of certain genes, by antagonizing the effect of the AP1 transcription factor formed by heterodimers of proteins of the c-Jun and c-Fos family (21Schule R. Rangarajan P. Yang N. Kliewer S. Ransone L.J. Bolado J. Verma I.M. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6092-6096Crossref PubMed Scopus (519) Google Scholar). Because retinoids are used to treat cancers (22Triozzi P.L. Walker M.J. Pellegrini A.E. Dayton M.A. Cancer Invest. 1996; 14: 293-298Crossref PubMed Scopus (34) Google Scholar) and skin diseases such as psoriasis (17Fritsch P.O. J. Am. Acad. Dermatol. 1992; 27: S8-S14Abstract Full Text PDF PubMed Scopus (72) Google Scholar) in which an overexpression of VEGF is involved, we have studied their effect on the expression of VEGF at the mRNA and protein level in cultured keratinocytes and human skin grafted onto the nude mouse. There are preliminary data from our laboratory (23Vega B. Michel S. Ladoux A. World Patent EP 82636 A. 1997; Google Scholar) and others (24Weninger W. Rendl M. Mindler M. Tschachler E. J. Invest. Dermatol. 1998; 111: 907-911Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) indicating that natural and synthetic retinoids are able to down-regulate VEGF expression in cultured human keratinocytes; however, their mechanism of action has not been studied yet. In this paper we show, that it is the anti-AP1 activity of the retinoid molecules that is responsible for the inhibition of the VEGF expression, and the AP1 site in the human VEGF promoter responsible for this negative regulation has been identified. The retinoids used were as follows: RA, CD 367 (4-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-anthracen-2-yl)-benzoic acid), Am 580 (4-[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalene-2-carbonyl)-amino]-benzoic acid), CD 2019 (6-[4-methoxy-3-(1-methyl-cyclohexyl)-phenyl]-naphthalene-2-carboxylic acid), CD 437 (6-[3-(1-adamantyl-4-hydroxy-phenyl]-naphthalene-2-carboxylic acid), CD 271 (adapalene), CD 2665 (4-[6-methoxyethoxymethoxy-7-(1-adamantyl)2-naphthyl]benzoic acid), and CD 2409 (4-[1-hydroxy-3-(5,5, 8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-prop-2-ynyl]-benzoicacid). For references, see Table I.Table IBinding specificity for the different RAR subtypes and AP1 transrepression activity of retinoids used in this studyRetinoid (Ref.)Receptor binding affinity (K d)AP1 transrepression activity at 1 μm (Ref. 37Fanjul A. Dawson M. Hobbs P. Jong L. Cameron J. Harlev E. Graupner G. Lu X. Pfahl M. Nature. 1994; 372: 107-111Crossref PubMed Scopus (320) Google Scholar)SelectivityRARαRARβRARγnm% inhibitionRA (60Martin B. Bernardon J.M. Cavey M.T. Bernard B. Carlavan I. Charpentier B. Pilgrim W. Shroot B. Reichert U. Skin Pharmacol. 1992; 5: 57-65Crossref PubMed Scopus (83) Google Scholar)167370RARα, β, γCD367 (60Martin B. Bernardon J.M. Cavey M.T. Bernard B. Carlavan I. Charpentier B. Pilgrim W. Shroot B. Reichert U. Skin Pharmacol. 1992; 5: 57-65Crossref PubMed Scopus (83) Google Scholar)53260RARα, β, γAm580 (60Martin B. Bernardon J.M. Cavey M.T. Bernard B. Carlavan I. Charpentier B. Pilgrim W. Shroot B. Reichert U. Skin Pharmacol. 1992; 5: 57-65Crossref PubMed Scopus (83) Google Scholar)8 131 45080RARαCD 2019 (60Martin B. Bernardon J.M. Cavey M.T. Bernard B. Carlavan I. Charpentier B. Pilgrim W. Shroot B. Reichert U. Skin Pharmacol. 1992; 5: 57-65Crossref PubMed Scopus (83) Google Scholar)110034 160NDaND, not determined.RARβCD271 (61Shroot B. Michel S. J. Am. Acad. Dermatol. 1997; 36: S96-S103Abstract Full Text Full Text PDF PubMed Google Scholar)110034 13071RARβ, γCD 437 (60Martin B. Bernardon J.M. Cavey M.T. Bernard B. Carlavan I. Charpentier B. Pilgrim W. Shroot B. Reichert U. Skin Pharmacol. 1992; 5: 57-65Crossref PubMed Scopus (83) Google Scholar)650024807796RARγCD 240963563476353969Anti-AP1AntagonistCD 2665 (62Szondy Z. Reichert U. Bernardon J.M. Michel S. Toth R. Ancian P. Ajzner E. Fesus L. Mol. Pharmacol. 1997; 51: 972-982Crossref PubMed Scopus (83) Google Scholar)1544 4008121RARβ, γ (antagonist)a ND, not determined. Open table in a new tab Normal human keratinocyte (NHKs) were isolated from human skin obtained from plastic surgery. The cells were cultured by the method of Rheinwald and Green (25Rheinwald J.G. Green H. Cell. 1975; 6: 331-343Abstract Full Text PDF PubMed Scopus (4004) Google Scholar). They were propagated in serum-free keratinocyte basal medium (Clonetics, San Diego, CA) supplemented with 0.4% (v/v) bovine pituitary extract, 10 ng/ml epidermal growth factor, 5 μg/ml insulin, and 0.15 mm calcium. For all experiments, second passage keratinocytes were used. Subconfluent keratinocyte cultured in 60-mm dishes were incubated for 4 h in serum and growth factor-free keratinocyte basal medium either with or without retinoids. The latter were dissolved in Me2SO at the desired concentrations. In some experiments, the cells were preincubated with retinoids for 16 h before the addition of 100 nm12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma) for the last 8 h. Pathogen-free congenitally athymic nude mice, Swiss nu/nu (Iffa-Credo, Les Oncins, France), aged 5–7 weeks, were anesthetized with sodium pentobarbital (Nembutal). A graft site on the anterolateral back was prepared with 70% ethanol, after which a circular piece of skin (1 cm in diameter) was removed down to the panniculus carnosus. Human skin, obtained from plastic surgery after informed consent of the patients, was cut into 1-cm-diameter pieces and fitted into the prepared graft sites. To protect the human skin, grafts were first covered by a dermal equivalent and then protected by a surgical tape reinforced with an extensible bandage, which was changed twice a week over a 6-week period (26Démarchez M. Sengel P. Prunieras M. Dev. Biol. 1986; 113: 90-96Crossref PubMed Scopus (56) Google Scholar). Retinoids and TPA were simultaneously applied at the graft site for 6 h, and human skin was removed for RNA analysis. The assay was performed as described by Cavey et al. (27Cavey M.T. Martin B. Carlavan I. Shroot B. Anal. Biochem. 1990; 186: 19-23Crossref PubMed Scopus (35) Google Scholar). Briefly, COS-7 cells were transfected with the different pSG-derived expression vectors encoding for human RARs using the polybrene technique (28Farr A.J. McAteer A. Roman A. Cancer cells. 1987; 5: 171-177Google Scholar). Cells were lysed, and the nuclei were recovered by centrifugation. For competition binding assays, nuclear extracts were incubated with [3H]CD 367 (2 nm) as the radioligand and various concentrations of the retinoid to be tested. Separation of free and bound ligand was performed by high-performance size exclusion chromatography. The dissociation constant (K d value) for each retinoid was determined by nonlinear regression analysis using the Origin software (Microcalc Software Inc.). This assay was performed as described previously (29Bernard B. Bernardon J.M. Delescluse C. Martin B. Lenoir M.C. Maignan J. Charpentier B. Pilgrim W. Reichert U. Shroot B. Biochem. Biophys. Res. Commun. 1992; 186: 977-983Crossref PubMed Scopus (211) Google Scholar). Briefly, HeLa cells were cotransfected with 2 μg of expression vectors encoding for human RAR α, RAR β, or RAR γ and with 5 μg of the TRE3-tk-chloramphenicol acetyltransferase reporter plasmid, which responds equally well to RAR α, RAR β, and RAR γ. The cells were grown for 24 h in the presence of different concentrations of the various retinoids. Chloramphenicol acetyltransferase activity was determined in lysates by enzyme-linked immunosorbent assay (ELISA) (Roche Molecular Biochemicals). The retinoid concentrations that produced half maximal activation (AC50) were determined from dose response curves, using the Origin software (Microcalc Software Inc.) HeLa cells were transfected with a construct containing the collagenase promoter from position −73 to + 63 (30Angel P. Baumann I. Stein B. Delius H. Rahmsdorf H.J. Herlich P. Mol. Cell. Biol. 1987; 7: 2256-2266Crossref PubMed Scopus (627) Google Scholar) cloned upstream of the reporter gene encoding chloramphenicol acetyltransferase. Transfected cells were treated with retinoids at 1 μm for 5 h, and then 10 nm TPA was added for a further 16 h. The amount of chloramphenicol acetyltransferase in cell lysates was determined by ELISA. Total RNA was isolated from cultured keratinocytes or reconstructed epidermis using the Trizol method (Life Technologies, Inc.) according to the manufacturer's procedure and stored at −80 °C until use. Total RNA from epidermal skin grafts was isolated as described by Chomczynski and Sacchi (31Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (64501) Google Scholar). The oligonucleotide primers for PCR were synthesized by Life Technologies, Inc. The sequences were GAPDH sense (5′-AATCCCATCACCATCTTCCA-3′) and antisense (5′-GTCATCATATTTGGCAGGTT-3′) oligonucleotide and CRABPII sense (5′-GCCACCATGCCCAACTTCT-3′) and antisense (5′-GGCCACTCACTCTCGGACGTA-3′) oligonucleotide. The amplification products were predicted to be 558 base pairs for GAPDH and 427 base pairs for CRABPII. VEGF primers sequences were as follows: sense oligonucleotide, 5′-CCATGAACTTTCTGCTGTCTT-3′; antisense oligonucleotide, 5′-ATCGCATCAGGGGCACACAG-3′. The VEGF primers were chosen in exons 1 and 3, resulting in a 249-base pair PCR product irrespective of the splice form produced. RT-PCR was carried out using 5 μg of total RNA extracted from cultured cells and skin grafts. After denaturation in diethylpyrocarbonate-treated water for 10 min at 70 °C, RNA was reverse-transcribed into first strand cDNA using SuperScriptII RNase H-reverse transcriptase (10 units/reaction, Life Technologies, Inc.) and 0.5 μg of oligo(dT) as primer, at 42 °C for 50 min in a total volume of 20 μl in a buffer containing 20 mmTris-HCl, pH 8.4, 50 mm KCl, 1.5 mmMgCl2, 1 mm dNTP, 10 mmdithiothreitol, and 20 units RNasin. Reverse transcriptase was inactivated at 70 °C for 15 min, and the RNA template was digested by RNase H at 37 °C for 20 min. Each experiment included samples containing no reverse transcriptase (negative controls) to exclude amplification from contaminating genomic DNA. Semiquantitative RT-PCR amplification was performed with a PTC 225 thermal cycler (MJ Research), following a 1-min period of denaturation at 94 °C, under the following conditions: denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, for a total of 30 cycles. The assay mixture contained 20 mm Tris-HCl, pH 8.4, 50 mm KCl, 1.5 mm MgCl2, 0.1 μm of oligonucleotide primers, dNTPs (100 μm of dATP, dGTP, dTTP, 10 μm dCTP), 0.5 μCi of [32P]dCTP, 0.5 units of Taq DNA polymerase, and 5 μl of 100-fold diluted cDNA mixture. The final product was extended for 3 min at 72 °C. In each experiment, RT positive controls (templates containing cDNA encoding for VEGF) and negative control (without DNA) were included. The PCR products were then electrophoresed on 6% (w/v) acrylamide gels. Radioactivity in each band was quantified by the storage phosphorimaging technique. The screens were scanned using a Fuji BAS 2000. The signal was quantified in photostimulating luminescence units using the Tina image analysis software. Results were expressed for each sample as band intensity relative to that of GAPDH. An optimum number of PCR cycles was determined in the region of exponential amplification. 10-Fold logarithmic dilutions of the cDNA mixture were used to verify the linear correlation between the intensity of the radioactive signal and the initial amount of cDNA. 10 μg of total RNA were separated by denaturing electrophoresis on 1.2% agarose formaldehyde gels and transferred to Nytran membranes (Schleicher and Schuell) prior to hybridization with selected probes. The probe used for human VEGF was the coding sequence of VEGF165 subcloned into theBamHI site of the pBluescript SK(−) plasmid. The GAPDH probe was a gift of Dr. F. Moreau-Gachelin (Paris, France). cDNA probes were labeled using [32P]dCTP and the Prime-a-Gene labeling system (Promega). Radioactivity in each band was quantified according to the method described above for PCR products. The VEGF mRNA levels were normalized to GAPDH mRNA levels to compensate for loading errors. 96-well plates coated with anti-human VEGF monoclonal antibody were purchased from R&D Systems. Keratinocyte culture supernatants were added to the wells, and VEGF was bound by the immobilized antibody. After extensive washing, a peroxidase-linked polyclonal antibody recognizing VEGF121 and VEGF165 was added to the wells; after washing, a substrate solution was added, and the plates were incubated for 5 min at room temperature. Absorbance was measured at 620 nm with an ELISA plate reader (SLT Lab Instruments, 340 ATC). NHK cells were transiently transfected using the polybrene procedure (28Farr A.J. McAteer A. Roman A. Cancer cells. 1987; 5: 171-177Google Scholar) with 5 μg of different constructions containing VEGF promoter fragments cloned into the pGl2-basic luciferase reporter plasmid (32Ikeda E. Achen M.G. Breier G. Risau W. J. Biol. Chem. 1995; 270: 19761-19766Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar) kindly provided, with the permission of Dr J. Abraham (Scios Nova Inc., Sunnyvale, CA), by Drs. A. Damert and W. Risau (Max-Plank-Institut für Physiologische und Klinische Forschung, Bad Nauheim, Germany). After 6 h of incubation in the presence of polybrene (30 μg/ml) and plasmid DNA, the keratinocytes were shocked with 30% Me2SO for 5 min, washed twice with phosphate-buffered saline, and refed with culture medium. Cells were also transfected with 5 μg of a reporter plasmid containing three copies of the synthetic oligonucleotide (5′-GGCAAAGTGAGTGACCTGCTTT-3′) derived from position −614 to −635 of the VEGF promoter, cloned upstream of theHerpes virus thymidine kinase promoter in the TK-Luc+ (HSB) vector, a kind gift of Dr. P. Balaguer (Pathologie des Récepteurs Nucléaires, INSERM U 439, Montpellier, France). A 0.7 kb VEGF promoter fragment containing the putative −621 AP1 binding site was prepared from the full-length VEGF promoter using theNheI restriction enzyme. The fragment was subcloned into the corresponding restriction site of the pGl2 basic vector (Promega) and subjected to site-directed mutagenesis according to the manufacturer's procedure (QuickChangeTM site-directed mutagenesis kit, Stratagene). The −621 AP1 binding site displaying the nucleotide sequence TGAGTGA was mutated to give TTAGTTA, a sequence inactive for AP1 binding (33Risse G. Joos K. Neuberg M. Brüller H. Müller R. EMBO J. 1989; 8: 3825-3832Crossref PubMed Scopus (160) Google Scholar). After verification by sequencing, the 0.7-kilobase fragment was ligated in the correct orientation into theNheI-digested VEGF promoter. The mutated promoter thus obtained was subcloned into the pGl2-basic luciferase reporter plasmid. Transfected NHKs were treated with 100 nm CD 2409 or 100 nm dexamethasone for 16 h, than 100 nm TPA was added for an additional 8 h. Luciferase activity was determined using the Luclite kit (Packard) and the Microbeta Trilux (Wallac EG&G) luminescence counter. Nuclear extracts were prepared from NHK cells according to the method of Dignam et al. (34Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9586) Google Scholar). Cells were lysed in 10 mm Hepes, pH 7.9, containing 1.5 mm MgCl2, 10 mm KCl and 0.5 mm dithiothreitol. After centrifugation at 15,000 rpm for 15 min, the nuclear pellet was suspended in 20 mmHepes, pH 7.9, containing 25% (v/v) glycerol, 0.42 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, and 0.5 mm dithiothreitol. After 30 min of agitation, the nuclear suspension was centrifuged again, and the supernatant was dialyzed against 20 mm Hepes, pH 7.9, 20% (v/v) glycerol, 0.1m KCl, 0.2 mm EDTA, 0.5 mmdithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride. The nuclear extracts were incubated in loading buffer (20 mm Hepes, pH 7.9, 30 mm KCl, 20% glycerol, 0.1% Nonidet P-40, 0.2 mm EDTA, 4 mmMgCl2, 4 mm spermidine, 100 μg/ml each poly(dI-dC), and salmon sperm DNA) with the oligonucleotides that were previously labeled with 32P-ATP with T4polynucleotide kinase. The sequence of the AP1 consensus oligonucleotide derived from positions −621 of the human VEGF promoter was 5′-AGGGGCAAAGTGAGTGACCTGCTT-3′. The sequence of the AP1 consensus oligonucleotide derived from the collagenase promoter was 5′-CGCTTGATGAGTCAGCCGGAA-3′. The sequence of the AP2 consensus oligonucleotide was 5′-GATCGAACTGACCGCCCGCGGCCCGT-3′. The mixture was incubated for 30 min at 4 °C and subjected to 5% polyacrylamide gel electrophoresis. Following migration, the gel was analyzed by the storage phosphorimaging technique using a Fuji BAS 2000 screen. The results given in the form of histograms are the average (±S.E.) obtained from three independent experiments, each of which provided two to five samples for the same experimental condition. They were analyzed using the two-sided Student's t test. In a first series of experiments, the effect of different RAR subtype selective agonists and of an RAR antagonist on the basal expression of VEGF mRNA was determined in cultured human keratinocytes. The concentrations of retinoids were chosen according to their binding affinities for the different RARs (Table I). Time course experiments performed with RA showed that the down-regulation of VEGF mRNA is at its maximum after 4 h (Fig. 1 A). Fig. 1 B shows that the baseline level of VEGF mRNA is significantly reduced with the potent RAR pan-agonist CD 367, the selective RARα agonist Am 580, the selective RARβ,γ agonist CD 271, and the selective RAR γ agonist CD 437. The RARβ,γ antagonist CD 2665 had no effect on the VEGF mRNA transcription and did not inhibit the effect of the RARγ agonist CD 437. In a second series of experiments, the time course of VEGF expression during treatment of NHKs with 100 nm TPA was determined. The induction of VEGF mRNA was at a maximum after 8 h and then decreased slowly (Fig. 2 A). Subsequently, retinoids detailed in Table I were tested for their inhibitory effect on TPA-induced VEGF mRNA expression. All of the RAR agonists that displayed anti-AP1 activity (compare with Table I) inhibited VEGF mRNA induction regardless of their RAR subtype selectivity, as shown by Northen blot and RT-PCR analysis (Fig. 2 B). The RARβ,γ antagonist CD 2665 did not demonstrate any anti-AP1 activity, as it did not affect the level of VEGF mRNA. Because all molecules that diminished the VEGF mRNA level were active in the AP1 transrepression assay (Table I), we suggested that the VEGF inhibition by retinoids is related to their ability to antagonize the AP1 factor. In order to prove this hypothesis we used CD 2409, a selective anti-AP1 retinoid displaying no in vitro affinity for the three RAR subtypes (Table I) and a weak transcriptional activity via an RARE (Table II). As shown in Fig. 3 A, CD 2409 inhibited the binding of the AP1 nuclear protein complex to the AP1 consensus oligonucleotide sequence derived from the collagenase promoter. As shown in Fig. 3 B, CD 2409 did not inhibit the binding of the AP2 protein to the AP2 consensus oligonucleotide sequence.Table IIAC 50 values for the transactivation potential of CD 2409 and RARetinoidTransactivation potentialRARαRARβRARγAC 50 /nmCD 2409360360630RA2.13.62.5 Open table in a new tab CD 2409 inhibited the basal expression level of VEGF mRNA (Fig. 4 A) to a level similar to RA (see Fig. 1 A), and its effect was maximum after 4 h of treatment. CD 2409 also inhibited the TPA-induced VEGF mRNA level, as did dexamethasone, a well known anti-AP1 compound (35Jonat C. Rahmsdorf H.J. Park K.K. Cato A.B.C. Gebel S. Ponta H. Herrlich P. Cell. 1990; 62: 1189-1204Abstract Full Text PDF PubMed Scopus (1399) Google Scholar) (Fig. 4 B). This inhibition was dose-dependent and was maximum at 100 nm (result not shown). In addition, CD 2409 inhibited the basal and TPA-induced secretion of VEGF121and VEGF165 as determined by ELISA (Fig. 4 C). The discrepancy between the inhibition of basal VEGF expression at the mRNA (Fig. 4 B) and protein level (Fig. 4 C) can be explained by the fact that the inhibitory effect of retinoids on mRNA expression is maximum after 4 h and then diminishes (Fig. 1 B), whereas its manifestation at the protein level needs more time. In this particular experiment, the incubation time was 24 h. The effect of CD 2409 on VEGF mRNA was also evaluated in vivo in human skin grafted onto the nude mouse. Grafted human skin preserves most of its original characteristics for the life span of the graft (26Démarchez M. Sengel P. Prunieras M. Dev. Biol. 1986; 113: 90-96Crossref PubMed Scopus (56) Google Scholar). The VEGF mRNA level was markedly increased 6 h after topical treatment with 0.01 and 0.003% TPA, and it returned to control levels after 24 h (Fig. 5 A). In the subsequent experiments, TPA was used at 0.01%, and the VEGF mRNA levels were analyzed 6 h after treatment. CD 2409 alone displayed no significant effect on the basal level of VEGF mRNA at concentrations of either 0.01 or 0.1%. However, the TPA-induced VEGF mRNA expression was inhibited when skin grafts were simultaneously treated with 0.1% CD 2409 and TPA. Dexamethasone, th