characterization of 5′-flanking region of human aggrecanase-1 (adamts4) gene

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Molecular Biology Reports 27: 167–173, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 167 Characterization of 5 0 -flanking region of human aggrecanase-1 (ADAMTS4) gene Yoshiharu Mizui, Kazuto Yamazaki, Yoshikazu Kuboi, Koji Sagane & Isao Tanaka Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3, Tokodai, Tsukuba, Ibaraki 300-2635, Japan; * Author for correspondence (Phone: +81-298-47-5809; Fax: +81-298-47-5367; E-mail: [email protected]) Received 30 August 2000; accepted 9 October 2000 Key words: aggrecanase-1, ADAMTS4, promoter, silencer, nuclear factor I Abstract Aggrecanase-1, also known as ADAMTS4 (a d isintegrin a nd m etalloproteinase with t hrombos pondin motifs 4), cleaves at the Glu 373 -Ala 374 site of aggrecan, thereby indicating aggrecan degradation. It is thought that ADAMTS4 plays a pivotal role in inflammatory joint diseases and cartilage degradation. To elucidate the mechanisms of regulation of ADAMTS4 gene expression, we cloned the 5 0 -flanking region of the human ADAMTS4 gene and characterized its promoter activity by means of reporter assay using porcine chondrocytes and NIH3T3 cells. Reporter gene analysis using deletion variants suggested that the region between -383 and +10 relative to the tentative transcription start site is necessary for full promoter activity; this region contains one Sp1 and three AP2 sites. In addition, the segment between -726 and -384 appears to contain silencer element(s). A complete deletion mutant of the nuclear factor I (NFI) binding site at -441 to -429 resulted in recovery of the promoter activity in chondrocytes, but not in NIH3T3 cells. Thus, the NFI site is involved in negative regulation of the human ADAMTS4 promoter activity in chondrocytes. Abbreviations: ADAMTS – a d isintegrin a nd m etalloproteinase with t hrombos podin motifs; bp – base pair(s); cDNA – DNA complementaryto RNA; DMEM – Dulbecco’s modified Eagle’s medium; FCS – fetal calf serum; kb – kilobase(s) or 1,000 bp; MMP – matrix metalloprotease; NFI – nuclear factor I; PCR – polymerase chain reaction; PLAP – placental alkaline phosphatase; RACE – rapid amplification of cDNA ends. Introduction Aggrecan is the major proteoglycan of cartilage and is responsible its mechanical properties, such as com- pressibility and stiffness. Aggrecan degradation is an important factor in the erosion of articular cartilage in arthritic diseases. Cleavage of aggrecan occurs at Asn 341 -Phe 342 and Glu 373 -Ala 374 . The cleavage at the Glu 373 -Ala 374 site is responsible for the aggrecan degradation in inflammatory joint diseases. Accumu- lation of a product of the Glu 373 -Ala 374 cleavage is observed in cartilage explants and chondrocyte culture treated with interleukin-1 and retinoic acid [1–3], and Sequence data presented in this article have been deposited with DDBJ/EMBL/GenBank Data Libraries under the following acces- sion number: AB039835 in the synovial fluid of patients with osteoarthritis and inflammatory joint disease [4, 5]. Several matrix metalloproteases (MMP-1, -2, -3, -7, -8, -9 and -13) have been shown to catalyze cleavage at the Asn 341 -Phe 342 site [6–9]. On the other hand, aggrecanases cleaving at the Glu 373 -Ala 374 site were recently discovered [10, 11]. These aggrecanases, ADAMTS4 (aggrecanase- 1) and ADAMTS11 (aggrecanase-2), belong to the novel a d isintegrin a nd m etalloproteinase with t hrombos pondin motifs (ADAMTS) family. Interest- ingly, both aggrecanases are expressed in articular car- tilage [11, 12], particularly highly in arthritic tissues [11]. Therefore, ADAMTS may play a pivotal role in inflammatory joint diseases and subsequent cartilage degradation. Little is yet known about the mechanisms

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Page 1: Characterization of 5′-flanking region of human aggrecanase-1 (ADAMTS4) gene

Molecular Biology Reports27: 167–173, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

167

Characterization of 5′-flanking region of human aggrecanase-1(ADAMTS4) gene

Yoshiharu Mizui, Kazuto Yamazaki, Yoshikazu Kuboi, Koji Sagane & Isao TanakaTsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3, Tokodai, Tsukuba, Ibaraki 300-2635, Japan;∗Author forcorrespondence (Phone:+81-298-47-5809; Fax:+81-298-47-5367; E-mail: [email protected])

Received 30 August 2000; accepted 9 October 2000

Key words:aggrecanase-1, ADAMTS4, promoter, silencer, nuclear factor I

Abstract

Aggrecanase-1, also known as ADAMTS4 (adisintegrin and metalloproteinase with thrombospondin motifs 4),cleaves at the Glu373-Ala374site of aggrecan, thereby indicating aggrecan degradation. It is thought that ADAMTS4plays a pivotal role in inflammatory joint diseases and cartilage degradation. To elucidate the mechanisms ofregulation of ADAMTS4 gene expression, we cloned the 5′-flanking region of the human ADAMTS4 gene andcharacterized its promoter activity by means of reporter assay using porcine chondrocytes and NIH3T3 cells.Reporter gene analysis using deletion variants suggested that the region between−383 and+10 relative to thetentative transcription start site is necessary for full promoter activity; this region contains one Sp1 and threeAP2 sites. In addition, the segment between−726 and−384 appears to contain silencer element(s). A completedeletion mutant of the nuclear factor I (NFI) binding site at−441 to−429 resulted in recovery of the promoteractivity in chondrocytes, but not in NIH3T3 cells. Thus, the NFI site is involved in negative regulation of the humanADAMTS4 promoter activity in chondrocytes.

Abbreviations:ADAMTS – a disintegrin and metalloproteinase with thrombospodin motifs; bp – base pair(s);cDNA – DNA complementary to RNA; DMEM – Dulbecco’s modified Eagle’s medium; FCS – fetal calf serum;kb – kilobase(s) or 1,000 bp; MMP – matrix metalloprotease; NFI – nuclear factor I; PCR – polymerase chainreaction; PLAP – placental alkaline phosphatase; RACE – rapid amplification of cDNA ends.

Introduction

Aggrecan is the major proteoglycan of cartilage andis responsible its mechanical properties, such as com-pressibility and stiffness. Aggrecan degradation is animportant factor in the erosion of articular cartilagein arthritic diseases. Cleavage of aggrecan occurs atAsn341-Phe342 and Glu373-Ala374. The cleavage atthe Glu373-Ala374 site is responsible for the aggrecandegradation in inflammatory joint diseases. Accumu-lation of a product of the Glu373-Ala374 cleavage isobserved in cartilage explants and chondrocyte culturetreated with interleukin-1 and retinoic acid [1–3], and

Sequence data presented in this article have been deposited withDDBJ/EMBL/GenBank Data Libraries under the following acces-sion number: AB039835

in the synovial fluid of patients with osteoarthritis andinflammatory joint disease [4, 5].

Several matrix metalloproteases (MMP-1, -2,-3, -7, -8, -9 and -13) have been shown tocatalyze cleavage at the Asn341-Phe342 site [6–9].On the other hand, aggrecanases cleaving at theGlu373-Ala374 site were recently discovered [10,11]. These aggrecanases, ADAMTS4 (aggrecanase-1) and ADAMTS11 (aggrecanase-2), belong tothe novel adisintegrin and metalloproteinase withthrombospondin motifs (ADAMTS) family. Interest-ingly, both aggrecanases are expressed in articular car-tilage [11, 12], particularly highly in arthritic tissues[11]. Therefore, ADAMTS may play a pivotal role ininflammatory joint diseases and subsequent cartilagedegradation. Little is yet known about the mechanisms

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Figure 1. Nucleotide sequence of the 5′-flanking region of the hu-man ADAMTS4 gene. The putative transcription start sites areshown by arrows. The site 406 bp upstream of the translation initia-tion codon is indicated by a bold arrow and tentatively designated as+1. Consensus sequences for binding of transcription factors, Sp1,AP-1, AP-2, PEA3, NF-II,6, a TATA box, NFI and a CCAAT box,are underlined.

of regulation of ADAMTS expression. We report herethe cloning of the 5′-flanking region of the humanADAMTS4 gene and partial characterization of itspromoter using porcine chondrocytes.

Materials and methods

Cloning of 5′-flanking region o human ADAMTS4gene

The 5′-flanking region of the human ADAMTS4 wasobtained by inverse PCR method as follows: humangenomic DNA purchased from Novagen (Novagen,Madison, WI) was digested overnight withBamHI,HindIII, EcoRI or EcoRV. Digested fragments wereself ligated with T4 DNA ligase (Takara, Kyoto,Japan), and purified with QIAprep Spin Miniprep kit(Qiagen, Chatsworth, CA). Purified DNA was used asa template in PCR. Primers used were hADAMTS4-1R (5′-GGAGAAAACTTAGTCCTTGGGC-3′, an-tisense, −180 to −159 numbered from the firstresidue of the ATG initiation codon of the humanADAMTS4 cDNA sequence, DDBJ/EMBL/GenBankaccession no. AF148213) and hADAMTS4-2 (5′-CAGCCCTTCTGAAAACTTTGCC-3′, sense,−140to −119). PCR conditions consisted of 45 cy-cles of 30 s at 98◦C and 10 min at 68◦C inMiniCycler (MJ Research, Watertown, MA). More-over, nested PCR was conducted with hADAMTS4-3R (5′-ATATGTGTCTGTGGGTCTCCCT-3′, anti-sense, −415 to −394) and hADAMTS4-4 (5′-AAAGCTAGGGTGTGGAGGACTT-3′, sense,−68to −47) under PCR conditions of 35 cycles of 30 sat 98◦C and 10 min at 68◦C. An approximate seven-kb fragment was produced and its nucleotide sequencewas determined directly with a Dye Terminator Cy-cle Sequencing kit (Applied Biosystems, Foster City,CA), using sequence-specific primers with an ABIPrism 377 DNA Sequencer (Applied Biosystems).PCR conditions for sequencing were as follows: 25cycles of 10 s at 96◦C, 5 s at 50◦C and 4 minat 40◦C in a GeneAmp PCR System 9600 (Perkin-Elmer, Palo Alto, CA). The computer analysis ofthe transcriptional factor binding sites was carried outusing GENETYX software (Software Development,Tokyo, Japan).

To identify tentative transcription initiation site(s),we conducted a 5′ RACE analysis [13] using a hu-man brain Marathon-Ready cDNA library (Clontech,Palo Alto, CA) according to the manual. Primers toamplify the 5′-fragment were: an adaptor primer 1 (5′-CCATCCTAATACGACTCACTATAGGGC-3′) andADAM-TS7 (5′-GCTTGGACTCCTGCCAAGGTC-3′, antisense,−115 to−95) in the first PCR, and anested adaptor primer 2 (5′-ACTCACTATAGGGCTCGAGCGGC-3′) and hADAMTS4-1R in the nested

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PCR. PCR conditions were 45 cycles of 30 s at 94◦C,30 s at 60◦C and 1 min at 72◦C with a final extensionfor 5 min at 72◦C in the first PCR, and 35 cyclesof 30 s at 94◦C, 30 s at 60◦C and 1 min at 72◦Cwith a final extension for 5 min at 72◦C in the nestedPCR. The nested PCR products were subcloned intopT7Blue(R)T vector (Novagen) and sequenced.

Plasmid constructs

For construction of reporter vectors, we de-signed the following primer: ADAM-TS11 (5′-GGTACCGGATCCGCCTTCTATAAAAGGAAAAGT-3′, where the underline indicates aKpnI site, sense)and ADAM-TS10 (5′-AAGCTTGGCACTGGTACTGCAGCTGGGAGGGAC-3′, where the underline in-dicates a HindIII site, antisense). We amplifiedthe 5′-flanking segment of the human ADAMTS4gene, using the primers and human genome un-der the same PCR conditions as in the inversePCR. The PCR product was cloned into theEcoRVsite with a T/A-hangover of the pT7Blue(R)T vec-tor (pT7-ADAMTS4-1) and sequenced. After theverification of its sequence, theKpnI/HindIII frag-ment was excised and inserted into theKpnI/HindIIIsites of the polylinker region of a placental alka-line phosphatase (PLAP) reporter plasmid [14] (p-1,132-PLAP vector,−726 to +406 relative to thetranscription start site). In order to construct dele-tion mutants, theKpnI/HindIII fragment from pT7-ADAMTS4-1 was inserted into theKpnI/HindIII sitesof the pT7Blue(R)T vector (pT7-ADAMTS4-2). TheBamHI/NcoI, BamHI/Bpu1102 I, and BamHI/NdeIfragments were deleted from the pT7-ADAMTS4-2by digestion and blunting-ligation (DNA blunting kit,Takara, Kyoto, Japan). Next, these vectors were di-gested withKpnI/HindIII and the resulting fragmentswere inserted into theKpnI/HindIII sites of the PLAPvector, generating p-789-PLAP (−383 to+406), p-506-PLAP (−100 to+406), and p-392-PLAP-vector(+11 to+406). These plasmids were transformed intoEscherichia coliDH5α and purified with an EndofreePlasmid Purification Kit (Qiagen), as indicated by themanufacturer.

Site-directed mutagenesis

Site-directed mutagenesis was performed accordingto the method of Deng and Nickoloff [15] us-ing a Transformer Site-Directed Mutagenesis kit(Clontech). The mutagenic oligonucleotide was 5′-AGTCCCCTCAGCCC∗CTAGGGCCAAAG-3′, from

which the NFI consensus motif, TGGAAACCAGCCA,has been completely deleted at∗.

Transfection and PLAP assay

Porcine chondrocytes were prepared as follows: ar-ticular cartilage was aseptically removed from youngmale porcine malleolar joints. Chondrocytes were iso-lated by sequential enzymatic digestion at 37◦C on arotator with 0.4% pronase (Calbiochem, San Diego,CA) in Ham F-12 without FCS for 1.5 h, and 0.0125%collagenase P (Boehringer Mannheim, Mannheim,Germany) in Ham F-12 without FCS for 15 h. Afterthe digestion, the cell suspension was passed througha nylon mesh in order to remove undigested fragments.Isolated cells were washed twice with Hank’s balancedsalt solution and resuspended in Dulbecco’s modifiedEagle’s medium (DMEM). Cell number and viabilitywere estimated with a hemocytometer after stainingwith trypan blue. The chondrocytes were grown inmixed medium of Ham F-12 and DMEM (1:1) with10% FCS. The transient PLAP assay was also carriedout with NIH3T3 cells. The cell line was purchasedfrom the American Type Culture Collection (ATCC,Manassas, VA).

Approximately 2× 105 chondrocytes were seededin a six-well plate and cultured in the medium asmentioned above. About 1× 105 NIH3T3 cells wereseeded in a 12-well plate and cultured in DMEM sup-plemented with 10% FCS. After 24 h, the culture waswashed to remove non-adherent cells and the mediumwas replaced, and then transfection was conductedwith FuGENE 6 (Boehringer Mannheim) accordingto the manual. A mixture of twoµg of a plasmidconsisting of the ADAMTS4 promoter region fusedto the PLAP reporter gene and oneµg of a pSV-β-galactosidase control vector (Promega, Madison, WI)was transfected into the cells. After two days, theculture supernatant was drawn from each well andthe PLAP activity was determined with the use ofa chemiluminescent substrate (CDP-Starready-to-usewith sapphire-II, Tropix, Bedford, MA). Samples wereheated at 65◦C for 20 min to inactivate non-specificalkaline phosphatase in the medium derived from FCS,and 10-µl aliquots of heat-treated samples were mixedwith 50µl of assay buffer (0.28 M Na2CO3-NaHCO3,pH 10.0, containing 8.0 mM MgSO4), followed byaddition of 50µl of chemiluminescent substrate. Themixtures were incubated at room temperature for60 min, and the steady-state chemiluminescence wasmeasured with a microplate luminometer (Wallac Eg

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Figure 2. Constructs of human ADAMTS4-PLAP fusion vectors. Numbers indicate the relative positions with respect to the tentative tran-scription start point. AP2, CCAAT box, an Sp1 and an NF-IL6 sites are indicated in the top scheme, with the restriction enzyme sites used inconstructing the deletion mutants.

Figure 3. The promoter activity of the human ADAMTS4-PLAP gene chimera transiently transfected into porcine chondrocytes. Values arethe means± standard deviations of three independent experiments. The relative promoter activity of 100% corresponds to the PLAP activity ofthe SV40 promoter. p-Basic-PLAP denotes the PLAP basic plasmid which contains no promoter.

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Figure 4. The location of the NFI binding site in the human ATAMTS4 promoter is shown in the upper panel (white box). Sequences ofthe wild or mutated oligonucleotides used in site-directed mutagenesis are indicated. The NFI site is represented in a box, and this site wascompletely deleted by the mutagenesis. Numbers are positions relative to the tentative transcription start site. A schematic representation of thestructures of wild-type reporter constructs and mutant variant is shown on the left in the lower panel. White and black boxes indicate wild andmutated sites, respectively. The promoter activity of the reporter genes, including an NFI-deleted variant, transiently transfected into porcinechondrocytes is shown at the bottom right. Values are the means and standard deviations of three independent experiments. The PLAP activityof the wild p-1,132-PLAP plasmid is set as 100%.

& G, Turku, Finland). Transfection with twoµg of aPLAP vector with the SV40 promoter (p-SV40-PLAP)was used as a positive control. The transfection ex-periments were repeated independently three or moretimes. Data were normalized by protein content andβ-galactosidase activity. The protein content andβ-galactosidase activity were measured with BCA pro-tein assay reagent (Pierce, Rockford, IL) and Galacto-Star (Tropix), respectively. Values are expressed asmeans and standard deviations.

Results and discussion

To identify potential transcriptional start point(s) ofthe human ADAMTS4 gene, we performed 5′ RACEusing a human brain Marathon-Ready cDNA library(Clontech). The 5′ RACE analysis suggests that tran-scription is initiated in a region at least 66 bp up-stream from the ATG. While the exact location of thetranscriptional start site remains to be established byprimer extension analysis, we have tentatively desig-nated nucleotide−66 upstream from the ATG as thebeginning of the promoter.

Using the genome walking technique, we cloned∼1.1-kb of the 5′-flanking region of the humanADAMTS4 gene. The nucleotide sequence of thecloned fragment is depicted in Figure 1. The com-puter sequence analysis failed to identify a classicalTATA box preceding the transcription start points atan appropriate distance, though an Sp1 site and a clas-sical CCAAT-box were located at (−28 to−20 and−76 to−72, respectively, referring to the major tran-scription start point. Several potential binding sitesfor transcription factors were also identified by thesequence analysis: multiple AP-2 transcription factorbinding sites were located at−319 to−312,−170 to−163, −165 to−158, +207 to+214 and+222 to+229. An NF-IL6 binding site was found at−144 to−136. In addition, PEA3 sites were present at−709 to−703,+32 to+37 and+167 to+172. The PEA3 siteis found in the promoters of several MMPs, such asMMP-1, -3, -7 and−13 [16, 17]. MMPs are known tobe associated with many disease conditions, includingarthritis and multiple sclerosis [18]. Since expressionlevel of several MMPs gene are increased in diseasetissue, ADAMTS4 gene transcription may work underregulatory conditions similar to those of MMPs. TwoNFI binding sites were also detected at−441 to−429

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Figure 5. The promoter activity of reporter genes, including an NFI-deleted variant, transiently transfected into NIH3T3 cells. Values are themeans and standard deviations of three independent experiments. The PLAP activity of the wild p-1,132-PLAP plasmid is set as 100%.

and−78 to−71, but the latter completely overlappedthe CCAAT box.

Various 5′-deletion mutants were constructed tocharacterize the 5′ flanking region of the ADAMTS4gene (Figure 2). The ability of each reporter constructto promote the transcription of the PLAP reporter genewas tested in porcine chondrocytes. Figure 3 showsthe PLAP activity of the human ADAMTS4-PLAP fu-sion plasmids containing various 5′-deletion mutants.The highest PLAP activity was detected in the p-789-PLAP vector; this corresponded to∼80% of that of thepositive control p-SV40PLAP vector, being∼13-foldhigher than that of the p-Basic-PLAP promoter-lessvector. The p-506-PLAP vector containing a CCAATbox and an Sp1 site had a PLAP activity only aboutfourfold higher than that of the p-Basic-PLAP vector.Therefore, the segment between−383 and−101 wasnecessary to maximize the promoter activity.

In regard to the p-1,132-PLAP vector, the PLAPactivity dropped to about a half of that of the p-789-PLAP vector. It is, thus, likely that silencer elementsexist within the region−726 to−384. As mentionedabove, we found an NFI binding site in this region.Several reports have shown negative regulation of NFIon certain genes, such as androgen receptor gene [19]and glutathione transferase P gene [20]. Therefore,it is possible that this NFI binding site is importantin negatively regulating the human ADAMTS4 gene.To address this possibility, we constructed a mutantvector in which the NFI binding site was completelydeleted, and compared the PLAP activity of the wildand mutated promoters, that is, p-1,132-PLAP and p-1,132(del/NFI)-PLAP vectors. The deletion of the NFIbinding site resulted in recovery of the promoter ac-tivity to a level almost corresponding to that of thep-789-PLAP vector (Figure 4), suggesting that this

NFI binding site may be a negative element in the re-gion−726 to+406 of the human ADAMTS4 gene inchondrocytes.

Figure 5 shows the PLAP activity of the reportergenes when transiently transfected into NIH3T3 cells.In contrast to chondrocytes, no difference of the PLAPactivities was observed between p-1,132-PLAP andp-789-PLAP vectors, or between p-1,132-PLAP andp-1,132del/NFI)PLAP vectors. Thus, different regu-latory mechanism(s) of the human ADAMTS4 genemay exist in chondrocytes and NIH3T3 cells.

In summary, we isolated the 5′-flanking region ofthe human ADAMTS4 gene. We also characterizedits promoter activity using porcine chondrocytes andNIH3T3 cells, and found that the region−383 to+10is necessary for basal promoter activity. Moreover,the NFI binding site at−441 to−429 was involvedin negative regulation of the human ADAMTS4 ex-pression. More detailed investigation will provide uswith further information on how the ADAMTS4 regu-lates the cleavage of aggrecan and what factors controlits transcription under physiological and pathologicalconditions.

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