The VCAM-1 Gene That Encodes the Vascular Cell Adhesion Molecule Is a Target of the Sry-related High Mobility Group Box Gene, Sox18
2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês
10.1074/jbc.m308512200
ISSN1083-351X
AutoresBrett Hosking, Shu-Ching Wang, Meredith Downes, Peter Koopman, George E.O. Muscat,
Tópico(s)Cell Adhesion Molecules Research
ResumoVCAM-1 (vascular cell adhesion molecule-1) and Sox18 are involved in vascular development. VCAM-1 is an important adhesion molecule that is expressed on endothelial cells and has a critical role in endothelial activation, inflammation, lymphatic pathophysiology, and atherogenesis. The Sry-related high mobility group box factor Sox18 has previously been implicated in endothelial pathologies. Mutations in human and mouse Sox18 leads to hypotrichosis and lymphedema. Furthermore, both Sox18 and VCAM-1 have very similar spatio-temporal patterns of expression, which is suggestive of cross-talk. We use biochemical techniques, cell culture systems, and the ragged opossum (RaOP) mouse model with a naturally occurring mutation in Sox18 to demonstrate that VCAM-1 is an important target of Sox18. Transfection, site-specific mutagenesis, and gel shift analyses demonstrated that Sox18 directly targeted and trans-activated VCAM-1 expression. Importantly, the naturally occurring Sox18 mutant attenuates the expression and activation of VCAM-1 in vitro. Furthermore, in vivo quantitation of VCAM-1 mRNA levels in wild type and RaOP mice demonstrates that RaOP animals show a dramatic and significant reduction in VCAM-1 mRNA expression in lung, skin, and skeletal muscle. Our observation that the VCAM-1 gene is an important target of SOX18 provides the first molecular insights into the vascular abnormalities in the mouse mutant ragged and the human hypotrichosislymphedema-telangiectasia disorder. VCAM-1 (vascular cell adhesion molecule-1) and Sox18 are involved in vascular development. VCAM-1 is an important adhesion molecule that is expressed on endothelial cells and has a critical role in endothelial activation, inflammation, lymphatic pathophysiology, and atherogenesis. The Sry-related high mobility group box factor Sox18 has previously been implicated in endothelial pathologies. Mutations in human and mouse Sox18 leads to hypotrichosis and lymphedema. Furthermore, both Sox18 and VCAM-1 have very similar spatio-temporal patterns of expression, which is suggestive of cross-talk. We use biochemical techniques, cell culture systems, and the ragged opossum (RaOP) mouse model with a naturally occurring mutation in Sox18 to demonstrate that VCAM-1 is an important target of Sox18. Transfection, site-specific mutagenesis, and gel shift analyses demonstrated that Sox18 directly targeted and trans-activated VCAM-1 expression. Importantly, the naturally occurring Sox18 mutant attenuates the expression and activation of VCAM-1 in vitro. Furthermore, in vivo quantitation of VCAM-1 mRNA levels in wild type and RaOP mice demonstrates that RaOP animals show a dramatic and significant reduction in VCAM-1 mRNA expression in lung, skin, and skeletal muscle. Our observation that the VCAM-1 gene is an important target of SOX18 provides the first molecular insights into the vascular abnormalities in the mouse mutant ragged and the human hypotrichosislymphedema-telangiectasia disorder. The formation of blood vessels occurs through two distinct mechanisms, vasculogenesis and angiogenesis (1Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4867) Google Scholar). Vasculogenesis leads to the vascularization of the endodermally derived organs such as lung, intestine, spleen, liver, and stomach, whereas angiogenesis involves the vascularization of the organs of mesodermal and ectodermal origin such as the limbs, kidney, and the brain. It also gives rise to the intersomitic and vertebral arteries (2Coffin J.D. Poole T.J. Anat. Rec. 1991; 231: 383-395Crossref PubMed Scopus (94) Google Scholar). We have recently implicated Sox18 in vascular development (10Pennisi D. Gardner J. Chambers D. Hosking B. Peters J. Muscat G. Abbott C. Koopman P. Nat. Genet. 2000; 24: 434-437Crossref PubMed Scopus (190) Google Scholar). Sox18 is a member of the Sry-related HMG 1The abbreviations used are: HMGhigh mobility groupSoxSryrelated HMG box-containing (protein)VCAM-1vascular cell adhesion molecule-1IRF-2interferon regulatory factor-2RaraggedRaJragged JacksonRaglragged-likeRaOPragged opossumRTreverse transcriptionEMSAelectrophoretic mobility shift assayGSTglutathione S-transferaseaaamino acids. box-containing (Sox) family of transcription factors. SOX proteins bind to DNA in a sequence-specific manner via the HMG domain, with all the proteins characterized to date binding to the heptameric motif (A/T)(A/T)CAA(A/T)G (11Wegner M. Nucleic Acids Res. 1999; 27: 1409-1420Crossref PubMed Scopus (755) Google Scholar). We have shown previously that Sox18 binds to the consensus sequence AACAAAG and trans-activates a heterologous promoter containing this element (12Hosking B.M. Muscat G.E. Koopman P.A. Dowhan D.H. Dunn T.L. Nucleic Acids Res. 1995; 23: 2626-2628Crossref PubMed Scopus (73) Google Scholar). high mobility group Sryrelated HMG box-containing (protein) vascular cell adhesion molecule-1 interferon regulatory factor-2 ragged ragged Jackson ragged-like ragged opossum reverse transcription electrophoretic mobility shift assay glutathione S-transferase amino acids. The Sox family displays both overlapping and distinct spatiotemporal expression patterns during embryogenesis and development. Aberrant Sox expression, mutation, or disruption leads to a number of diseases; for example, Sry and Sox9 are involved in sex reversal (13Koopman P. Gubbay J. Vivian N. Goodfellow P. Lovell-Badge R. Nature. 1991; 351: 117-121Crossref PubMed Scopus (1754) Google Scholar, 14Foster J.W. Graves J.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1927-1931Crossref PubMed Scopus (236) Google Scholar). The situation is similar for Sox18. In situ analysis of Sox18 has demonstrated expression in the mesenchyme underlying the developing hair follicle, in the presumptive heart, and in the developing vasculature (10Pennisi D. Gardner J. Chambers D. Hosking B. Peters J. Muscat G. Abbott C. Koopman P. Nat. Genet. 2000; 24: 434-437Crossref PubMed Scopus (190) Google Scholar). The naturally occurring mouse mutant ragged (Ra) of which there are four allelic variants, Ra, ragged Jackson (RaJ), ragged-like (Ragl) and RaOP, all contain mutations in Sox18 (15James K. Hosking B. Gardner J. Muscat G.E. Koopman P. Genesis. 2003; 36: 1-6Crossref PubMed Scopus (53) Google Scholar). All these mutants display defects in hair and skin development. However, most life threatening is the generalized edema suffered by these animals (16Slee J. J. Genet. 1957; 55: 570-584Crossref Scopus (18) Google Scholar), which is probably due to lymphatic aberrations (17Slee J. J. Genet. 1957; 55: 100-121Crossref Scopus (15) Google Scholar, 18Herbertson B.M. Wallace M.E. J. Med. Genet. 1964; 1: 10-23Crossref PubMed Scopus (9) Google Scholar, 19Wallace M.E. Heredity. 1979; 43: 9-18Crossref PubMed Scopus (8) Google Scholar). Recently, a report has been published describing the investigation of several mutations in SOX18 and the hypotrichosislymphedema-telangiectasia (HLT) disorder in humans (20Irrthum A. Devriendt K. Chitayat D. Matthijs G. Glade C. Steijlen P.M. Fryns J.P. Van Steensel M.A. Vikkula M. Am. J. Hum. Genet. 2003; 72: 1470-1478Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). Patients present with early onset alopecia of the scalp and lymphedema. The most severe lymphatic abnormality presented was non-immune hydrops fetalis (of unknown etiology). Telangiectasia was present in only in some of the patients studied, as were other anomalies such as thinness and transparency of the skin, hydrocele, and cutaneous papular vascular lesions. One of the major functions for the blood and lymphatic vascular system is to provide efficient access for leukocytes and other immune system molecules to all tissues of larger animals. The immune system responds to damage or illness via the accumulation of leukocytes, leading to a localized inflammation in the diseased area. This inflammatory response is necessarily tightly coordinated, as the lack of control can itself lead to various diseases, for example arthritis, psoriasis, multiple sclerosis, asthma, atherosclerosis, and allergy (21Steinman L. Zamvil S. Nat. Rev. Immunol. 2003; 3: 483-492Crossref PubMed Scopus (109) Google Scholar, 22Smolen J.S. Steiner G. Nat. Rev. Drug Discov. 2003; 2: 473-488Crossref PubMed Scopus (690) Google Scholar, 23Schieffer B. Drexler H. Am. J. Cardiol. 2003; 91: 12H-18HAbstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 24DeGraba T.J. Adv. Neurol. 2003; 92: 29-42PubMed Google Scholar). Of the multitude of proteins involved in the immune system response, the cell adhesion molecules play a major role in mediating immune function and inflammation (25Krieglstein C.F. Granger D.N. Am. J. Hypertens. 2001; 14: 44S-54SCrossref PubMed Google Scholar). Vascular cell adhesion molecule-1 (VCAM-1), a member of the immunoglobin gene superfamily of cell adhesion molecules, is expressed on the cell surface of activated endothelia, in the skin (26Groves R.W. Ross E.L. Barker J.N. MacDonald D.M. J. Am. Acad. Dermatol. 1993; 29: 67-72Abstract Full Text PDF PubMed Scopus (85) Google Scholar, 27Davies D. Larbi K. Allen A. Sanz M. Weg V.B. Haskard D.O. Lobb R.R. Nourshargh S. Immunology. 1999; 97: 150-158Crossref PubMed Scopus (9) Google Scholar), and in developing muscle and the lung (28Rosen G.D. Sanes J.R. LaChance R. Cunningham J.M. Roman J. Dean D.C. Cell. 1992; 69: 1107-1119Abstract Full Text PDF PubMed Scopus (316) Google Scholar, 29Stepp M.A. Urry L.A. Hynes R.O. Cell Adhes. Commun. 1994; 2: 359-375Crossref PubMed Scopus (54) Google Scholar, 30Beck-Schimmer B. Schimmer R.C. Madjdpour C. Bonvini J.M. Pasch T. Ward P.A. Am. J. Respir. Cell Mol. Biol. 2001; 25: 780-787Crossref PubMed Scopus (50) Google Scholar). VCAM-1 recognizes the integrin receptors α4β1 (31Elices M.J. Osborn L. Takada Y. Crouse C. Luhowskyj S. Hemler M.E. Lobb R.R. Cell. 1990; 60: 577-584Abstract Full Text PDF PubMed Scopus (1536) Google Scholar) and α4β7 (32Chan B.M. Elices M.J. Murphy E. Hemler M.E. J. Biol. Chem. 1992; 267: 8366-8370Abstract Full Text PDF PubMed Google Scholar, 33Postigo A.A. Sanchez-Mateos P. Lazarovits A.I. Sanchez-Madrid F. de L azuri M.O. J. Immunol. 1993; 151: 2471-2483PubMed Google Scholar) present on monocytes, eosinophils, and lymphocytes, whereas VCAM-1-deficient embryos die in utero due to abnormalities in chorio-allantoic fusion (34Gurtner G.C. Davis V. Li H. McCoy M.J. Sharpe A. Cybulsky M.I. Genes Dev. 1995; 9: 1-14Crossref PubMed Scopus (317) Google Scholar). In vivo animal studies demonstrate that attenuated VCAM-1 function in mice provided protection against atherosclerosis. For example, mice with reduced (not ablated) VCAM-1 expression and function crossed with low density lipoprotein receptor LDLR–/– mice prone to atherosclerosis produce animals resistant to atherogenesis (35Cybulsky M.I. Iiyama K. Li H. Zhu S. Chen M. Iiyama M. Davis V. Gutierrez-Ramos J.C. Connelly P.W. Milstone D.S. J. Clin. Investig. 2001; 107: 1255-1262Crossref PubMed Scopus (985) Google Scholar). Much of the previous research has focused on the control of VCAM-1 expression via extracellular signals. For example, lipopolysaccharide and cytokines such as interleukin-4 (IL-4), tumor necrosis factor-α (TNF-α) (36Barks J.L. McQuillan J.J. Iademarco M.F. J. Immunol. 1997; 159: 4532-4538PubMed Google Scholar, 37Briscoe D.M. Cotran R.S. Pober J.S. J. Immunol. 1992; 149: 2954-2960PubMed Google Scholar), interferon-γ (INF-γ), transforming growth factor-β1 (TGF-β1) (38Park S.K. Yang W.S. Lee S.K. Ahn H. Park J.S. Hwang O. Lee J.D. Nephrol. Dial. Transplant. 2000; 15: 596-604Crossref PubMed Scopus (51) Google Scholar), granulocytemacrophage colony-stimulating factor (GM-CSF) (39Henninger D.D. Panes J. Eppihimer M. Russell J. Gerritsen M. Anderson D.C. Granger D.N. J. Immunol. 1997; 158: 1825-1832PubMed Google Scholar), and vascular endothelial growth factor (VEGF) (40Kim I. Moon S.O. Kim S.H. Kim H.J. Koh Y.S. Koh G.Y. J. Biol. Chem. 2001; 276: 7614-7620Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar) cause an increase in the level of expression of VCAM-1 in endothelia as well as in other cell types, whereas angiopoietin 1 (ang1) can repress the activation of VCAM-1 by vascular endothelial growth factor (41Kim I. Moon S.O. Park S.K. Chae S.W. Koh G.Y. Circ. Res. 2001; 89: 477-479Crossref PubMed Scopus (301) Google Scholar). In contrast, transcriptional regulation of VCAM-1 is less well understood. Most notable is the role of interferon regulatory factor-2 (IRF-2) in the control of VCAM-1 expression in muscle (42Jesse T.L. LaChance R. Iademarco M.F. Dean D.C. J. Cell Biol. 1998; 140: 1265-1276Crossref PubMed Scopus (129) Google Scholar). Sox factors act as critical regulators of organ ontogeny via the modulation of expression of particular target genes. Surprisingly, relatively few target genes for this family of transcription factors have been described in the literature (11Wegner M. Nucleic Acids Res. 1999; 27: 1409-1420Crossref PubMed Scopus (755) Google Scholar). Therefore, to elucidate the function of Sox18 in the blood and lymphatic vascular system, the major site of expression and of abnormality in the ragged animal, it is important to find target genes in this organ. Our study demonstrates that native Sox18 (and not the mutant ragged form) is able to induce the activity of the VCAM-1 promoter. In biochemical assays we identified and characterized three Sox18 binding sites. However, we demonstrated that only the SoxB site at –715 is necessary for VCAM-1 trans-activation. In vivo validation of this data was obtained from the RaOP animals that have very significantly reduced levels (8–40-fold) of VCAM-1 expression (and not other adhesion molecules, including ICAM-1 and ICAM-2, JCAM, NCAM-1, and PECAM-1). RNA and cDNA Preparation—RNA for realtime PCR was isolated from organs of RaOP and wild-type adult male siblings as described previously (45Hosking B.M. Wyeth J.R. Pennisi D.J. Wang S.C. Koopman P. Muscat G.E. Gene. 2001; 262: 239-247Crossref PubMed Scopus (39) Google Scholar), with the exception that the lyophilized RNA was purified by processing ∼100 μg through a Qiagen RNeasy mini column. During this purification process the RNA was DNase-treated. Quantitation of the purified RNA was carried out as described previously (45Hosking B.M. Wyeth J.R. Pennisi D.J. Wang S.C. Koopman P. Muscat G.E. Gene. 2001; 262: 239-247Crossref PubMed Scopus (39) Google Scholar). First strand cDNA synthesis was carried out using 5 μg of purified RNA and primed with oligo(dT)18, using the Superscript III enzyme and the supplied manufacturer's protocol. Real Time PCR—Target cDNA levels were quantitated by real time RT-PCR using an ABI Prism™ 7700 Sequence Detector system utilizing SYBR Green I (Molecular Probes, Eugene OR; used at 0.8×) as a nonspecific PCR product fluorescence label. Quantitation was >45 cycles of 95 °C for 15 s and 60 °C for 1 min two-step thermal cycling preceded by an initial 95 °C for 2 min for activation of 0.75 units of Platinum® TaqDNA polymerase (Invitrogen). The 25-μl reaction also contained 20 mm Tris-HCl (pH 8.4), 50 mm KCl, 5 mm MgCl2, 200 μm each of dGTP, dATP, and dCTP, 400 μm dUTP, 0.5 units of uracil-N-glycosylase, 500 nm ROX reference dye (Invitrogen), and 200 nm each forward and reverse primers. Mus musculus primer sequences for VCAM-1 are as follows: forward, 5′-TGACAAGTCCCCATCGTTGA-3′; reverse, 5′-ACCTCGCGACGGCATAATT-3′. Promoter Construct Generation—The sequence of the murine VCAM-1 promoter has been published elsewhere (46Korenaga R. Ando J. Kosaki K. Isshiki M. Takada Y. Kamiya A. Am. J. Physiol. 1997; 273: C1506-C1515Crossref PubMed Google Scholar) and is available on NCBI (accession number U42327). Mouse genomic DNA (C57BL/6) was a gift from J. Rowland, and 10 ng was used in the outer PCR amplification to generate the VCAM-1 promoter. Nested PCR was necessary for amplification of the 1895-bp promoter that has been published previously. Outer primers were constructed at the published upstream limit of murine VCAM-1 promoter at –1895 bp and within the first exon at 19 bp. The outer PCR was performed with 100 ng of each primer (–1895, 5′-GCCGGTACCGATCTACATAGCCACGGAGAG-3′; and 19, 5′-CGACCATCTTCACAGGCATTT-3′), 1.25 units of Pfu®, and 0.2 mm dNTPs in a final volume of 50 μl containing the supplied buffer (Promega). Hot start PCR was performed at 95 °C for 5 min followed by 35 cycles of 95 °C for 1 min, 60 °C for 1 min, and 68 °C for 4 min. The inner primers were constructed such that the 5′-primer at –1889 contained the restriction enzyme site KpnI (–1887, 5′-CGGGGTACCATAGCCACGGAGAGTTCTT-3′), whereas, the 3′-primer had the restriction enzyme site XhoI (–1, 5′-GCCCTCGAGTTCAAGTCTCTGCTTCAAAGCC-3′). 1 μl of the outer PCR was combined with buffer B and 0.25 units of polymerase mix from the Fail-Safe PCR system (Epicenter) and 100 ng of each inner primer in a final volume of 10 μl. The VCAM-1 inner PCR profile was the same as that used for the outer PCR. The product was digested with KpnI and XhoI, inserted into pGL2-Basic (pGL2B-Promega), and sequenced completely via automated sequencing using the ABI system and reagents. This clone was then used as the parental plasmid to generate all other sub-clones. Deletion clones were generated either by restriction digests or PCR amplification. VC1219 was generated by restriction digest with PstI and KpnI, and the vector plus the remaining insert were then blunt-ended with Klenow (New England Biolabs) and religated. Both VC754 and 504 were generated via PCR amplification with the primers 5′-GACTTCCTGTCATCCAGCAATGGGTCAAA-3′ and 5′-CGGGGTACCTTTGTTGAAAGAG-3′, respectively. The 3′-primer was the inner primer –1 from the initial nested PCR. The PCR profile was essentially the same as that done for the nested PCR, with the exception that the annealing temperature was 55 °C. Site-directed mutagenesis of the VCAM-1 promoter was undertaken via the QuikChange® II kit from Stratagene and carried out according to the manufacturer's protocol. The primers used for the –1569 mutation are 5′-ATGACATGACATCATTGAGGTCCTCTAG-3′ and 5′-CTAGAGGACCTCAATGATGTCATGTCAT-3′. The primers used for the –715 mutation are 5′-GCTGGGGCATCATCAAACAAAA-3′ and 5′-TTTTGTTTGATGATGCCCCAG-3′. Cell Culture and Transient Transfections—COS-1 (simian fibroblast) and SVEC4-10 (mouse high venule endothelial) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in 6% CO2. C2C12 (mouse skeletal muscle) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum in 6% CO2. Cells for transfection were grown in 24-well dishes to 50–60% confluence before being transiently transfected with 2 μgofthe reporter plasmid and 1 μg of the expression plasmids in 0.5 ml of Dulbecco's modified Eagle's medium containing 10% fetal calf serum by a liposome-mediated procedure. Briefly, SVEC4-10 cells were transfected using N-[1-(2,3-dioleoyloxy)]-N,N,N-trimethylammonium propane methylsulfate (DOTAP) (Roche Molecular Biochemicals) in quantities 6-fold (v/w) the total amount of DNA, whereas COS-1 and C2C12 cells were transfected using 15 μl of DOTAP and 10 μl of 1,3-dioleoyloxy-2-(6-carboxyspermyl)-propylamid (DOSPER) (Roche Molecular Biochemicals). 24 h post-transfection the medium was replaced, and the cells were grown for a further 24–48 h. Each experiment represented at least two sets of independent quadruplicates to overcome the variability inherent in transfections. Cells were harvested and assayed for luciferase activity as described previously (47Chen S.L. Dowhan D.H. Hosking B.M. Muscat G.E. Genes Dev. 2000; 14: 1209-1228Crossref PubMed Google Scholar). Electrophoretic Mobility Shift Assay (EMSA)—We have described previously the cloning and protein expression of a murine Sox 18 fusion construct with glutathione-S-transferase (GST) using the pGEX-1 bacterial expression vector (12Hosking B.M. Muscat G.E. Koopman P.A. Dowhan D.H. Dunn T.L. Nucleic Acids Res. 1995; 23: 2626-2628Crossref PubMed Scopus (73) Google Scholar). Briefly, Escherichia coli DH5a cells containing this vector were induced for 1–2 h with 0.5 mm isopropyl thiogalactoside after the cells had grown to an A600 of 0.6. The pelleted cells were sonicated, and the cleared lysate containing the fusion protein was loaded onto glutathione-agarose columns in Dignam buffer C (containing protease inhibitors) (47Chen S.L. Dowhan D.H. Hosking B.M. Muscat G.E. Genes Dev. 2000; 14: 1209-1228Crossref PubMed Google Scholar). After extensive column washing, the fusion protein was eluted with Dignam buffer C supplemented with 5 mm reduced glutathione. Probes used in all EMSAs were annealed and then radiolabeled using γ-32P and T4 polynucleotide kinase (PNK). The oligonucleotide sequences used as probes are as follows: –1569, 5′-TTTTATGACATGACattgttGAGGTCCTC-3′ (top strand) and 5′-GAGGACCTCaacaatGTCATGTCATAAAA-3′ (bottom strand); –715, 5′-GGCTGGGGCattgtcAAACAAAAG-3′ (top strand) and 5′-CTTTTGTTTgacaatGCCCCAGCC-3′ (bottom strand); –491, 5′-GAAAGAGaacaatTTTTATTTTTTAAATTGCAAATGCATTTCTT-3′ (top strand) and 5′-AAGAAATGCATTTGCAATTTAAAAAATAAAAattgttCTCTTTC-3′ (bottom strand). The bases in lowercase letters represent the putative Sox binding sites. All EMSA experiments were carried out in a total of 20 μl in Dignam Buffer C containing 1–2 ng of T4 polynucleotide kinase-labeled probe and 2 μg of the purified, bacterially expressed GST-Sox 18. The assays were incubated at room temperature for 20 min and electrophoresed through a 6% (polyacrylamide/bisacrylamide; 20:1) gel in 80 mm Tris borate and 2 mm EDTA. Gels were briefly soaked in 10% acetic acid, dried, and autoradiographed. Competition EMSAs were carried out basically as above with the exception that unlabeled probe was added to the reaction in 20–80-fold molar excess as compared with the labeled probe. Both the probe and an unlabeled competitor were added to the reaction at the same time and then incubated and electrophoresed as before. For these experiments, the unlabeled competitor in each reaction was the same double-stranded oligonucleotide as the probe used in that reaction. Cell-specific Transcription of the Mouse VCAM-1 Is Regulated by the 5′-Upstream Flanking Sequences—The naturally occurring Sox18 mutations in mice and humans (15James K. Hosking B. Gardner J. Muscat G.E. Koopman P. Genesis. 2003; 36: 1-6Crossref PubMed Scopus (53) Google Scholar, 20Irrthum A. Devriendt K. Chitayat D. Matthijs G. Glade C. Steijlen P.M. Fryns J.P. Van Steensel M.A. Vikkula M. Am. J. Hum. Genet. 2003; 72: 1470-1478Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar) display defects in hair and skin development. However, the most life threatening is the generalized edema caused by lymphatic vascular dysfunction (17Slee J. J. Genet. 1957; 55: 100-121Crossref Scopus (15) Google Scholar, 18Herbertson B.M. Wallace M.E. J. Med. Genet. 1964; 1: 10-23Crossref PubMed Scopus (9) Google Scholar, 19Wallace M.E. Heredity. 1979; 43: 9-18Crossref PubMed Scopus (8) Google Scholar). Hence, we were particularly interested in the identification of Sox18 target genes that play a role in lymphatic function and thus may allow us to better understand the molecular basis of the ragged phenotype. We hypothesized that VCAM-1 was regulated by SOX18. Therefore, to test this supposition we examined the ability of VCAM-1 promoter sequences to direct the expression of the LUC gene in muscle, endothelial, and fibroblast cells. For this purpose, we designed primers to amplify the complete published murine VCAM-1 sequence (46Korenaga R. Ando J. Kosaki K. Isshiki M. Takada Y. Kamiya A. Am. J. Physiol. 1997; 273: C1506-C1515Crossref PubMed Google Scholar) and cloned the sequenced amplified product into the promoterless pGL2-Basic luciferase reporter vector. This plasmid was denoted as PGL2-VC1889 and encompasses 1889 bp immediately upstream of the murine VCAM-1 translation start codon (Fig. 1). To test if Sox18 could trans-activate the VCAM-1 promoter, we transfected it into fibroblasts and endothelial and skeletal muscle cell lines (Fig. 2). Transfection of pGL2-VC1889 into COS-1 fibroblasts and SVEC4-10 high venule endothelial cell lines demonstrated that the upstream promoter sequences of the VCAM-1 gene confer high level expression in a cell-specific manner. For example, pGL2-VC1889 activity in COS-1 fibroblasts is ∼ 5-fold greater than that of pGL2-Basic and 100-fold less than that of the constitutively active SV40 promoter. In contrast, in endothelial SVEC cells pGL2-VC1889 activity is ∼50-fold greater than that of pGL2-Basic and is similar in activity to the very efficient SV40 promoter. This is consistent with our previous study that reported a high level of Sox18 expression in the nuclei of SVEC4-10 (48Hosking B.M. Wang S.C. Chen S.L. Penning S. Koopman P. Muscat G.E. Biochem. Biophys. Res. Commun. 2001; 287: 493-500Crossref PubMed Scopus (53) Google Scholar). Similarly, when the VCAM promoter was transfected into proliferating C2C12 myoblasts, the VCAM-1 promoter activity was ∼50-fold greater than that of pGL2-Basic (data not shown), as has been reported previously (44Iademarco M.F. McQuillan J.J. Dean D.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3943-3947Crossref PubMed Scopus (74) Google Scholar). In conclusion, the efficient cell specific expression of VCAM-1 is consistent with the in vivo expression profile of VCAM-1.Fig. 2VCAM-1 promoter regulates transcription in a cell/tissue-specific manner. The activity of the full-length VCAM-1 promoter in COS-1 fibroblast (left) and SVEC4-10 endothelial (right) cells is depicted. pGL2 control (pGL2C), driven by the promiscuous SV40 enhancer and early promoter, was used as a control for transfection efficiency and as a comparison for the level of cell-specific activity of the VCAM-1 promoter. pGL2-Basic (pGL2) is the empty luciferase vector, whereas VC1889 refers to 1889 bp of the murine VCAM-1 promoter cloned into pGL2B. 2 μg of each plasmid was transfected in all cell lines using a liposome-mediated procedure. Results are expressed as mean ± S.D. of two sets of independent quadruplicates.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The VCAM-1 Gene Is a Target of the Sry-related HMG Box Gene Sox18 as Shown by the Identification of Three SOX18 Binding Sites—VCAM-1 expression can be stimulated via cytokines (e.g. tumor necrosis factor-α) (43Iademarco M.F. McQuillan J.J. Rosen G.D. Dean D.C. J. Biol. Chem. 1992; 267: 16323-16329Abstract Full Text PDF PubMed Google Scholar), mitogens (e.g. vascular endothelial growth factor) (49Klimiuk P.A. Sierakowski S. Latosiewicz R. Cylwik J.P. Cylwik B. Skowronski J. Chwiecko J. Ann. Rheum. Dis. 2002; 61: 804-809Crossref PubMed Scopus (108) Google Scholar), and transcription factors (e.g. IRF-2 and Oct-1) (42Jesse T.L. LaChance R. Iademarco M.F. Dean D.C. J. Cell Biol. 1998; 140: 1265-1276Crossref PubMed Scopus (129) Google Scholar, 43Iademarco M.F. McQuillan J.J. Rosen G.D. Dean D.C. J. Biol. Chem. 1992; 267: 16323-16329Abstract Full Text PDF PubMed Google Scholar). We investigated whether SOX18, could trans-activate the mouse VCAM-1 promoter in COS-1 fibroblasts. Co-transfection of a Sox18 expression construct with the pGL2-VC1889 promoter resulted in a moderate 5-fold activation (Fig. 3A). This activation is dose-dependent, as increasing amounts of Sox18 augmented the trans-activity of the VCAM-1 promoter (Fig. 3B). Thus, we demonstrate here for the first time that Sox18 trans-activates the promoter of VCAM-1. SOX and SRY proteins contain HMG domains that bind DNA in a sequence-specific manner. These proteins have been reported to bind in vitro and in vivo to sites with a core motif resembling (A/T)(A/T)CAA(A/T)G (for review, see Ref. 11Wegner M. Nucleic Acids Res. 1999; 27: 1409-1420Crossref PubMed Scopus (755) Google Scholar). We have reported previously that SOX18 can bind and trans-activate a core consensus motif of AACAAAG (12Hosking B.M. Muscat G.E. Koopman P.A. Dowhan D.H. Dunn T.L. Nucleic Acids Res. 1995; 23: 2626-2628Crossref PubMed Scopus (73) Google Scholar). As an initial step in the investigation to examine whether SOX18 was able to bind the murine VCAM-1 promoter, we searched the sequence of the promoter using MatInspector (50Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2427) Google Scholar) and found three potential Sox binding sites (Fig. 1). Of these, the two at –491 (SoxC) and –1569 (SoxA) contain a perfect core consensus site (AACAAT) for the binding of Sox/Sry transcription factors. The core sequence of the site at –715 (SoxB) is slightly different from the consensus, withaGat position 1 (GACAAT). Interestingly, it has been reported that SOX17 can bind this sequence from a pool of otherwise random oligonucleotides (51Kanai Y. Kanai-Azuma M. Noce T. Saido T.C. Shiroishi T. Hayashi Y. Yazaki K. J. Cell Biol. 1996; 133: 667-681Crossref PubMed Scopus (189) Google Scholar). To investigate whether GST-SOX18 can interact with the putative SoxA, SoxB, and SoxC binding sites, we performed an EMSA using 32P-labeled oligos that consist of the Sox binding site in addition to some surrounding sequence (see "Materials and Methods"). As seen in Fig. 4A, the SOX18 protein bound strongly to all three putative Sox sites. In contrast, GST protein alone could not bind to any of the three oligonucleotides used in this study. Thus, SOX18 can potentially interact with all three sites in the VCAM-1 promoter. To examine the sequence-specificity of the protein-DNA interaction, we attempted to compete the binding with cold/unlabeled double-stranded oligonucleotide with the same sequence as the
Referência(s)