Genomic Organization and Regulation of Expression of the Lectin-like Oxidized Low-density Lipoprotein Receptor (LOX-1) Gene

Abstract

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a recently identified receptor for oxidized low-density lipoprotein, one of the major atherogenic substances. Although LOX-1 was reported to be expressed abundantly in endothelial cells, including atheromatous lesions, the regulation of LOX-1 gene has not yet been clarified. In the present study, we isolated the rat LOX-1 gene and investigated the regulation of gene expression. The rat LOX-1 gene was encoded by a single copy gene spanning over 19 kilobases and consisted of eight exons. Exon boundaries correlated well with the functional domain boundaries of the receptor protein. The promoter region contained putative TATA and CAAT boxes and multiple cis-elements such as NF-κB, AP-1 and AP-2 sites, and a shear stress response element. Northern blot analysis revealed that LOX-1 gene expression was up-regulated 9-fold by shear stress, 21-fold by lipopolysaccharide, and 4-fold by tumor necrosis factor-α, in cultured vascular endothelial cells. LOX-1 was also expressed in macrophages but not in vascular smooth muscle cells. These data provide important information for elucidating the molecular mechanisms of LOX-1 gene regulation and suggest a role for LOX-1 in the pathophysiology of atherosclerotic cardiovascular disease. Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a recently identified receptor for oxidized low-density lipoprotein, one of the major atherogenic substances. Although LOX-1 was reported to be expressed abundantly in endothelial cells, including atheromatous lesions, the regulation of LOX-1 gene has not yet been clarified. In the present study, we isolated the rat LOX-1 gene and investigated the regulation of gene expression. The rat LOX-1 gene was encoded by a single copy gene spanning over 19 kilobases and consisted of eight exons. Exon boundaries correlated well with the functional domain boundaries of the receptor protein. The promoter region contained putative TATA and CAAT boxes and multiple cis-elements such as NF-κB, AP-1 and AP-2 sites, and a shear stress response element. Northern blot analysis revealed that LOX-1 gene expression was up-regulated 9-fold by shear stress, 21-fold by lipopolysaccharide, and 4-fold by tumor necrosis factor-α, in cultured vascular endothelial cells. LOX-1 was also expressed in macrophages but not in vascular smooth muscle cells. These data provide important information for elucidating the molecular mechanisms of LOX-1 gene regulation and suggest a role for LOX-1 in the pathophysiology of atherosclerotic cardiovascular disease. Oxidized low-density lipoprotein (OxLDL) 1The abbreviations used are: OxLDL, oxidized low-density lipoprotein; LOX-1, lectin-like OxLDL receptor; LPS, lipopolysaccharides; TNF, tumor necrosis factor; bp, base pair(s); PCR, polymerase chain reaction; EC(s), endothelial cell(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SSRE, shear stress response element; kb, kilobase(s). 1The abbreviations used are: OxLDL, oxidized low-density lipoprotein; LOX-1, lectin-like OxLDL receptor; LPS, lipopolysaccharides; TNF, tumor necrosis factor; bp, base pair(s); PCR, polymerase chain reaction; EC(s), endothelial cell(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SSRE, shear stress response element; kb, kilobase(s). is implicated in the pathogenesis of atherosclerotic cardiovascular disease (1Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. N. Engl. J. Med. 1989; 320: 915-924Crossref PubMed Google Scholar, 2Witztum J.L. Steinberg D. J. Clin. Invest. 1991; 88: 1785-1792Crossref PubMed Scopus (2455) Google Scholar). Previous studies indicate that OxLDL is present in atherosclerotic lesions (3Yla-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Butler S. Witztum J.L. Steinberg D. J. Clin. Invest. 1989; 84: 1086-1095Crossref PubMed Google Scholar) and that antioxidant drugs slow the progression of atherosclerosis (4Carew T.E. Schwenke D.C. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7725-7729Crossref PubMed Scopus (935) Google Scholar,5Kita T. Nagano Y. Yokode M. Ishii K. Kume N. Ooshima A. Yoshida H. Kawai C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5928-5931Crossref PubMed Scopus (791) Google Scholar). OxLDL possesses many atherogenic properties. First, OxLDL is thought to be taken up into macrophages via scavenger receptors, which promotes the deposition of lipid-laden foam cells in the vascular walls and leads to fatty streaks (1Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. N. Engl. J. Med. 1989; 320: 915-924Crossref PubMed Google Scholar). Second, recent data indicate that OxLDL alters various endothelial functions. OxLDL induces endothelial expression of several proteins, including adhesion molecules (6Khan B.V. Parthasarathy S.S. Alexander R.W. Medford R.M. J. Clin. Invest. 1995; 95: 1262-1270Crossref PubMed Scopus (406) Google Scholar, 7Kume N. Cybulsky M.I. Gimbrone J.M.A. J. Clin. Invest. 1992; 90: 1138-1144Crossref PubMed Scopus (718) Google Scholar), monocyte chemotactic protein-1 (8Cushing S.D. Berliner J.A. Valente A.J. Territo M.C. Navab M. Parhami F. Gerrity R. Schwartz C.J. Fogelman A.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5134-5138Crossref PubMed Scopus (957) Google Scholar, 9Liao F. Berliner J.A. Mehrabian M. Navab M. Demer L.L. Lusis A.J. Fogelman A.M. J. Clin. Invest. 1991; 87: 2253-2257Crossref PubMed Scopus (172) Google Scholar), smooth muscle growth factors (10Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9910) Google Scholar), and colony-stimulating factors (11Rajavashisth T.B. Andalibi A. Territo M.C. Berliner J.A. Navab M. Fogelman A.M. Lusis A.J. Nature. 1990; 344: 254-257Crossref PubMed Scopus (604) Google Scholar), some of which might be involved in endothelial cell-mediated recruitment of monocytes/macrophages into the intima. OxLDL also attenuates the endothelium-dependent vasodilatory response through reduced production of nitric oxide (12Kugiyama K. Kerns S.A. Morrisett J.D. Roberts R. Henry P.D. Nature. 1990; 344: 160-162Crossref PubMed Scopus (779) Google Scholar, 13Yokoyama M. Hirata K. Miyake R. Akita H. Ishikawa Y. Fukuzaki H. Biochem. Biophys. Res. Commun. 1990; 168: 301-308Crossref PubMed Scopus (169) Google Scholar). It has long been thought that there is a specific endothelial receptor for OxLDL. In 1997, Sawamura et al. (14Sawamura T. Kume N. Aoyama T. Moriwaki H. Hoshikawa H. Aiba Y. Tanaka T. Miwa S. Katsura Y. Kita T. Masaki T. Nature. 1997; 386: 73-77Crossref PubMed Scopus (1150) Google Scholar) identified a novel receptor for OxLDL (LOX-1) using expression cloning with bovine cultured endothelial cells. LOX-1 is a membrane protein abundantly expressed in endothelial cells. It binds, internalizes, and degrades OxLDL, but not native LDL or acetylated LDL. Its mRNA was shown to be expressed in human atheromatous lesions. This endothelial receptor might mediate some of the actions of OxLDL in the endothelium. The biologic roles of LOX-1, however, remain to be determined. In a previous study, we performed rat LOX-1 cDNA cloning and demonstrated that it encodes a single-transmembrane protein with its N terminus in the cytoplasm (15Nagase M. Hirose S. Sawamura T. Masaki T. Fujita T. Biochem. Biophys. Res. Commun. 1997; 237: 496-498Crossref PubMed Scopus (142) Google Scholar). The extracellular region consists of a spacer, 46-amino acid triple repeats, and C-type lectin-like domains. Quite unexpectedly, LOX-1 expression was markedly (>20-fold) up-regulated in the aorta of hypertensive rats (16Nagase M. Hirose S. Fujita T. Biochem. J. 1998; 330: 1417-1422Crossref PubMed Scopus (42) Google Scholar), which implies a pathophysiologic role for LOX-1 in hypertension or in hypertensive vascular remodeling. However, the genomic structure of LOX-1 or its regulation of expression in vitro has not yet been reported in any species. Here we report the genomic organization of rat LOX-1 and identified several consensus sequences in the 5′-flanking region. We demonstrated that the LOX-1 gene expression was markedly up-regulated by shear stress (9-fold), bacterial lipopolysaccharide (LPS) (21-fold), and tumor necrosis factor (TNF)-α (4-fold), in cultured vascular endothelial cells. We also examined its expression in cells other than endothelial cells, such as macrophages and vascular smooth muscle cells. A rat genomic DNA library (Sau3AI partial digest constructed in the λ phage, EMBL3 SP6/T7) (CLONTECH) was screened with a rat LOX-1 cDNA probe (432-bp XhoI/HindIII fragment,probe 1 in Fig. 1 A). Approximately 5 × 105 phages were plated at a density of 30,000 plaques/15-cm plate, and two replica nitrocellulose filters were prepared from each plate. High stringency screening was performed with hybridization in 50% formamide and final washes in 0.1 × SSC (1 × SSC is 150 mm NaCl, 15 mm sodium citrate, pH 7.0) containing 0.1% SDS at 60 °C. Plaques that produced positive signals on both replicas were selected and purified. The second and the third rounds of screening were carried out under the same conditions to isolate positive clones. Positive clones were digested withXhoI, EcoRI, SacI, HindIII, and ApaI. Each restriction fragment was subcloned into pBluescript II SK−. The 3′ portion was determined by PCR. Rat genomic DNA (100 ng) prepared from Wistar rat kidneys was amplified by PCR with an Expand Long Template PCR System (Boehringer Mannheim, Mannheim, Germany). Primer sets used were: 5′-AATCTGTGTCAGGACCATGGAGAACCCTCA-3′ (forward 1) and 5′-GCTTCCAGCTGAGGGTGTCTATCTGTTCCT-3′ (reverse 1) for RLG11, 5′-GAATCAGAATCTTCAAGAAGCCCTGCAGAG-3′ (forward 2) and 5′-GGGCCCATGGAAGAGGTAACAGTTTTCTTT-3′ (reverse 2) for RLG12, 5′-AAAGAAAACTGTTACCTCTTCCATGGGCCC-3′ (forward 3) and 5′-CAGCTGTCAAGGCCACATGTTTTACACCCA-3′ (reverse 3) for RLG13, and 5′-AGGAGTCCCAGAGAGAACTGAAGGAACAGA-3′ (forward 4) and 5′-CTGAAGAGTTTGCAGCTCTCTGCAGGGCTT-3′ (reverse 4) for RLG14. The amplification was carried out for 35 cycles of 94 °C for 10 s, 60 °C for 30 s, and 68 °C for 2 min. The products were subcloned into pBluescript II. Nucleotide sequences were determined on both strands using the dideoxynucleotide chain-termination method with a Sequi Therm Long-Read cycle sequencing kit (Epicentre Technologies, Madison, WI) and an Automated Laser Fluorescent DNA Sequencer (model 4000; LI-COR, Lincoln, NE). The sequence and consensus nucleotide motif analyses were performed using the GENETYX-MAC software (Software Development, Tokyo, Japan). Southern blot analysis was carried out according to Sambrook et al. (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 9.16-9.19Google Scholar). Rat genomic DNA (20 μg each) was digested with restriction enzymes (PstI,EcoRV, EcoRI, and BamHI), electrophoresed on a 0.7% agarose gel, and transferred onto a nylon membrane filter. An EcoRI/BamHI genomic DNA fragment (1.8 kb, probe 2 in Fig. 1 A) was used as a probe. Hybridization and wash were performed as described for the screening. Primer extension analysis was performed according to the manufacturer's recommendation (Promega, Madison, WI). In brief, a synthetic oligonucleotide, 5′-GCCACATGACTTCTGATCAGGCTGGCCATT-3′ (P1) (10 pmol), was end-labeled with [γ-32P]dATP using T4 polynucleotide kinase. Labeled primer (1 pmol) was annealed to poly(A)+ RNA (20 μg) from the rat cultured vascular endothelial cells (described below) at 58 °C for 1 h and then allowed to cool slowly to room temperature. The hybridized primer-RNA complex was extended using 1 unit of avian myeloblastosis virus reverse transcriptase at 42 °C for 1 h. Following ethanol precipitation, the extension products were separated on a 7 m urea/6% polyacrylamide sequencing gel and visualized with autoradiography. The mobility of the extended products was compared with that of a [γ-32P]dATP dideoxy sequence ladder obtained using the same primer. A 3.2-kb HindIII fragment of λRLG3, which contained the translation initiation site, was subcloned in the HindIII site of pBluescript II and used as a template. A single-stranded antisense DNA was synthesized with this template, end-labeled P1 primer, and T7 DNA polymerase. The products were digested with NheI, purified using alkaline gel electrophoresis, and used as a probe. The probe was hybridized to 20 μg of rat endothelial poly(A)+ RNA at 30 °C overnight. Nonannealed nucleic acids were digested with 1000 units/ml S1 nuclease. The nuclease-resistant products were ethanol-precipitated and electrophoresed in parallel with the primer extension product on a 7 m urea/6% polyacrylamide sequencing gel followed by autoradiography. Primary cultures of endothelial cells (ECs) were obtained from the rat aorta or the descending thoracic aorta of a bovine fetus by brief of the as described R. J. H. W. T. A. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). An cultured vascular smooth muscle were from the were in with bovine units/ml and in a of at °C. the were to shear stress bacterial or Wistar rats were from Laboratory Japan). The rats were with A was into the macrophages were by with 0.1% macrophages were by the with 0.1% were to a well using a parallel as described Y. H. M. M. A. Y. M. N. R. J. T. J. PubMed Scopus Google Scholar). In brief, the consisted of and which were separated by a were cultured on the (100 × to of the The of the were × × The was with A and Tokyo, were used to a The was in an in an of at °C. The of shear stress was as 2 is the at is the of the a and of the shear stress (20 was to the for h. were performed in a with cells from the same RNA was prepared using the acid method N. Biochem. 1987; PubMed Scopus Google Scholar). RNA was purified using an mRNA kit RNA was on a agarose gel and transferred to a nylon membrane filter. The probe was synthesized using reverse with RNA from and the and The amplification was carried out for 30 cycles of °C for 1 °C for 1 and °C for with The probe was with using the primer nylon were hybridized with cDNA probe (1 × in a containing 50% formamide at 42 °C for h. were then in × SSC containing 0.1% SDS at 60 °C. were to with an at °C. the Northern filters were to an and the of the was as with a (model Tokyo, Japan). The for LOX-1 was with that of glyceraldehyde-3-phosphate was rat In brief, the extracellular region of LOX-1 acid was subcloned into a of the was by the of The were then purified using a were with the purified protein μg) with an of the were with μg of the protein with an of 2 was performed 10 the third from of were separated by gel and transferred to a The membrane was with of the rat with containing the membrane was with of with alkaline (Boehringer for h. were visualized by and We isolated genomic clones rat LOX-1 using a of screening and PCR. Fig. 1 A the cloning genomic and partial restriction A rat genomic library was screened with a rat LOX-1 cDNA probe 1 in Fig. 1 A). genomic clones were isolated from × 105 The clones not the 3′ of the LOX-1 the portion was determined using PCR The nucleotide sequence was determined using the restriction of the clones λRLG3, of the genomic and cDNA sequences revealed that the region consisted of over 19 to 1 through through through through through through through and through Nucleotide sequences of were to sequences obtained from the rat 1 through 7 were determined to be and consensus sequences to the 1 Fig. 1 the organization in to the protein Exon 1 encodes the region and the acids in the Exon 2 encodes which is the Exon encodes acids and to the region and 1 in the extracellular and 5 to of 2 4) and which is the lectin-like Exon also encodes the region. Rat genomic DNA was digested with the restriction enzymes and and hybridized at stringency with probe 2 shown in Fig. 1 A. A single was in each that rat LOX-1 is encoded as a single copy gene the site, we performed primer extension analysis using a P1 to to to the The was end-labeled with hybridized to and extended by reverse shown in a single extension product of was obtained with endothelial RNA 1) but not with The sequence ladder revealed that the was of the translation site and to a this we performed an S1 using the same RNA and primer used for primer The from S1 was in with that from primer extension a fragment of was with endothelial RNA the of the translation initiation site was a site and as the nucleotide sequence of kilobases of the 5′-flanking region and the of rat The promoter region contained a putative TATA at to and a CAAT at to the other the promoter not analysis identified several factor AP-1 sites, AP-2 one site, and one shear stress response We examined the of shear stress, bacterial LPS, and on LOX-1 gene expression. In the of were to shear (20 were at h and for LOX-1 mRNA with Northern a cells were for h. shown in Fig. 5 we a for LOX-1 mRNA in of in a for h not any in LOX-1 gene expression. In shear stress a in LOX-1 The of LOX-1 mRNA to h of shear stress and a over at h. The mRNA to a over at h. were to (100 and (10 a in LOX-1 gene expression. The up to 21-fold at h and to over at h on the other LOX-1 expression to a The however, over h The LOX-1 regulation was also examined at the protein blot analysis with the rat LOX-1 a of in the from The of the h of 1 and We also the of the by of with not of LOX-1 gene we performed Northern blot analysis with RNA from and macrophages and cultured vascular smooth muscle cells as well as cultured LOX-1 was expressed in but not in cultured vascular smooth muscle cells LOX-1 expression was abundantly expressed in 1) and 2) macrophages in In the present study, we isolated and the rat LOX-1 The 5′-flanking region contained putative TATA and CAAT boxes and multiple such as NF-κB, AP-1 and AP-2 sites, and a LOX-1 gene expression was markedly up-regulated in response to bacterial and a role for LOX-1 in the pathogenesis of LOX-1 is a recently identified OxLDL receptor abundantly expressed in endothelial cells (14Sawamura T. Kume N. Aoyama T. Moriwaki H. Hoshikawa H. Aiba Y. Tanaka T. Miwa S. Katsura Y. Kita T. Masaki T. Nature. 1997; 386: 73-77Crossref PubMed Scopus (1150) Google Scholar). It is a II single-transmembrane protein with a N It was isolated in bovine and and identified the rat (16Nagase M. Hirose S. Fujita T. Biochem. J. 1998; 330: 1417-1422Crossref PubMed Scopus (42) Google Scholar). Previous data revealed that the LOX-1 protein binds, internalizes, and degrades OxLDL The protein not to to and not or or or The of this including the yet to be In the present study, we determined the genomic structure of the rat LOX-1 The LOX-1 gene consists of and each to the functional domain of the 2 to the domain and through to the lectin-like We reported that a 46-amino acid motif in the extracellular domain of the bovine and human LOX-1 was in the rat LOX-1 (16Nagase M. Hirose S. Fujita T. Biochem. J. 1998; 330: 1417-1422Crossref PubMed Scopus (42) Google Scholar). 2 and to of and The organization the of this motif as a functional The structure was also reported in the extracellular domain of the LDL receptor T. J.L. PubMed Scopus Google Scholar). and analyses of this unit to the In this study, we determined a major site of the rat LOX-1 gene in Primer extension and S1 analyses of RNA isolated from rat demonstrated that from the of The site was of the and of the the other there was might in one of the cDNA clones we isolated from a rat cDNA library region (16Nagase M. Hirose S. Fujita T. Biochem. J. 1998; 330: 1417-1422Crossref PubMed Scopus (42) Google Scholar). However, reverse with multiple primer sets that were from the site we identified in this study, at in cultured not of genomic clones up to of the promoter region of the rat LOX-1 gene revealed the of several such as NF-κB, AP-1 and AP-2 sites, and a Although studies to functional or the promoter structure several regulation of LOX-1 First, the of the site its regulation by and A. 1997; PubMed Google Scholar). Second, the of AP-1 and as well as the regulation by shear The was the identified for the of gene by shear stress N. T. W. C. Gimbrone M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar), which was in other factors NF-κB, and also been shown to be involved in the M.C. J. Y. M. S. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, N. Gimbrone M. T. J. Clin. Invest. 1995; PubMed Scopus Google Scholar, M.C. F. N. S. J. Clin. Invest. 1997; PubMed Scopus Google Scholar, Gimbrone M. N. T. 1997; PubMed Scopus (172) Google Scholar). LOX-1 expression was markedly up-regulated by We reported that the rat LOX-1 cDNA contained multiple in the region that be involved in the of mRNA (16Nagase M. Hirose S. Fujita T. Biochem. J. 1998; 330: 1417-1422Crossref PubMed Scopus (42) Google Scholar). However, we in the of shear stress the of LOX-1 mRNA not it is that the of LOX-1 mRNA in response to such as and shear stress was by not by of LOX-1 The biologic role of LOX-1 has not been determined It be involved in via the actions of OxLDL in the such as of adhesion molecules (6Khan B.V. Parthasarathy S.S. Alexander R.W. Medford R.M. J. Clin. Invest. 1995; 95: 1262-1270Crossref PubMed Scopus (406) Google Scholar, 7Kume N. Cybulsky M.I. Gimbrone J.M.A. J. Clin. Invest. 1992; 90: 1138-1144Crossref PubMed Scopus (718) Google Scholar) and growth factors (10Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9910) Google Scholar). The of LOX-1 by the such as LPS, and shear stress to be in this LOX-1 was expressed not in the but also in macrophages in LOX-1 mRNA was demonstrated to be expressed in such as the and (14Sawamura T. Kume N. Aoyama T. Moriwaki H. Hoshikawa H. Aiba Y. Tanaka T. Miwa S. Katsura Y. Kita T. Masaki T. Nature. 1997; 386: 73-77Crossref PubMed Scopus (1150) Google Scholar, M. Hirose S. Fujita T. Biochem. J. 1998; 330: 1417-1422Crossref PubMed Scopus (42) Google Scholar). the other the expression was in the and other It that LOX-1 is expressed in and which important roles in the atherosclerotic the expression of scavenger receptor OxLDL receptor abundantly expressed in was reported to be by and Fogelman A.M. J. 1992; Google Scholar). It is in to the of LOX-1 by receptor for OxLDL, was to be involved in atherosclerosis through R.M. 1998; PubMed Scopus Google Scholar, H. R.M. 1998; PubMed Scopus Google Scholar). et al. H. M. H. K. J. 1997; PubMed Scopus Google Scholar) identified a of endothelial scavenger receptor other scavenger receptors, it acetylated LDL with which was by OxLDL. it be that OxLDL and its on and macrophages a complex which leads to the atherosclerotic vascular We reported that LOX-1 gene expression is markedly in the aorta of hypertensive hypertensive and as compared with the in rats (15Nagase M. Hirose S. Sawamura T. Masaki T. Fujita T. Biochem. Biophys. Res. Commun. 1997; 237: 496-498Crossref PubMed Scopus (142) Google Scholar). This a role for LOX-1 in the pathophysiology of the other LOX-1 expression is not in hypertensive rats and rats (16Nagase M. Hirose S. Fujita T. Biochem. J. 1998; 330: 1417-1422Crossref PubMed Scopus (42) Google Scholar). the might be by or other factors to the hypertensive The present demonstrated in LOX-1 expression was markedly by it is that the factor has a on LOX-1 expression in hypertensive LOX-1 expression might the hypertensive vascular remodeling. In we determined the genomic structure of the rat LOX-1 The of LOX-1 gene expression by stress and as well as the of NF-κB, AP-1 and AP-2 sites, and suggest a role for LOX-1 in the pathophysiology of We to and for and