From the Orthopaedic Research Laboratory, Department of Orthopaedic Surgery and the § Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032
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ABSTRACT |
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Identification and characterization of the regulatory elements of the human aggrecan gene are necessary first steps in addressing the molecular mechanisms through which the gene is regulated. Using luciferase reporter constructs driven by the human aggrecan promoter or the cytomegalovirus promoter, the 5'- and 3'-untranslated regions of the human aggrecan gene were found to regulate gene expression transcriptionally in a promoter- and/or cell type-specific manner. Independent of cell type, the 5'-untranslated region was inhibitory with respect to the cytomegalovirus promoter, but it was stimulatory to the human aggrecan promoter. The 5'-untranslated region inhibited the cytomegalovirus promoter by approximately 60% in both chondrocytes and NIH 3T3 cells, but it stimulated the activity of the human aggrecan promoter about 8-fold in chondrocytes and 40-fold in NIH 3T3 cells. In contrast, the 3'-untranslated region inhibited the activities of the human aggrecan promoter by 40-70% in both cell types, but it stimulated the cytomegalovirus promoter activities by 50-60% in NIH 3T3 cells and inhibited its activity by 70% in chondrocytes. The differential effects of the untranslated regions on the two types of promoters may be a reflection of differences in regulation of TATA-less promoters, such as the human aggrecan promoter, and TATA-containing promoters, such as the cytomegalovirus promoter.
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INTRODUCTION |
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Expression of aggrecan by chondrocytes is subject to modulation by
several factors, including cytokines, growth factors, and mechanical
stimuli. Interleukin-1 and tumor necrosis factor- both suppress
aggrecan gene expression and synthesis, whereas insulin-like growth
factor I and transforming growth factor-
stimulate aggrecan gene
expression and synthesis. Mechanical loading of cartilage has been
shown to modulate (increase and decrease) aggrecan gene expression and
synthesis depending on the type and extent of the load imposed
(1-7).1 Changes in aggrecan
synthesis have also been identified as important events in cartilage
pathology (8-10). Although aggrecan is expressed predominantly in
cartilage, it is also known to be expressed in other tissues such as
neural tissues (11-14) and neonatal lungs (15). In the embryonic
central nervous system, aggrecan is involved in regulating neural crest
migration (14, 16). Exposure of the lungs of neonatal rats to hyperoxia
up-regulates aggrecan expression (15). Mutations that preclude the
proper synthesis and deposition of aggrecan into the extracellular
matrix are often lethal. A point mutation identified in exon 12 of the
aggrecan core protein gene of the nanomelic chick introduces a
premature translation terminal codon that precludes synthesis of the
third globular domain (G3)2
(17), and a 7-bp deletional mutation in exon 5 of the cartilage matrix-deficiency (cmd) mouse aggrecan gene leads to a
premature translation termination codon in exon 6 (18). Despite these observations, the molecular mechanisms governing the regulation of
aggrecan gene expression and synthesis remain obscure.
The core protein of aggrecan consists of an amino-terminal globular domain (G1) that binds hyaluronan, an interglobular domain, a second globular domain (G2) whose function is yet unknown, an extended glycosaminoglycan attachment region, and G3, which is located at the carboxyl-terminal end of the molecule (19-21). Comparison of the aggrecan core protein structure and deduced amino acid sequences from various species (22-25) with the recently determined exon and intron organizations of the chick (26), human (27), mouse (25), and rat (28) aggrecan have revealed a highly modular structure of the gene. Various domains of the protein are coded by groups of exons or single exons, most of which must be expressed as modules of two or more exons because of restrictive exon splicing requirements (27). Alternative splicing of the epidermal growth factor-like (23, 29, 30) and complement-regulatory protein-like motifs (23) of the G3 domain in some species have, however, been reported.
The coding region of the aggrecan gene is flanked by exons 1 and 19 (27), from which the 5'- and 3'-untranslated regions (UTRs) are transcribed (23, 27). The UTRs of other genes, for example ferritin, have been shown to function as regulatory elements of the genes (31-35). We hypothesize that the UTRs of the aggrecan gene are involved similarly in regulating expression of the gene, potentially in concert with the promoter. Thus, in this study we have cloned and investigated the regulatory activities of the promoter and the 5'- and 3'-UTRs of the human aggrecan gene.
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EXPERIMENTAL PROCEDURES |
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Materials--
Dulbecco's modified Eagle's medium,
Ca2+/Mg2+-free Dulbecco's phosphate-buffered
saline, 50 × minimal essential amino acids, 100 × nonessential amino acids, 1 M Hepes, 0.75% sodium
bicarbonate, and antibiotic solution (5,000 IU/ml penicillin, 5,000 µg/ml streptomycin) were obtained from Mediatech, Inc. (Herndon, VA).
Fetal bovine serum and bovine serum albumin were purchased from Sigma.
The Wizard Midi-prep DNA isolation kit, Taq polymerase,
deoxynucleotide triphosphate mix (100 mM each), restriction
enzymes, the ProFection calcium phosphate mammalian transfection kit,
and the luciferase assay system were purchased from Promega Corporation
(Madison, WI). The pGEM-luc, pSV--galactosidase,
pGL3-basic, and pGL3-enhancer plasmids were also from Promega. The
pcDNA3 and pCR II vectors were purchased from Invitrogen
Corporation (Carlsbad, CA), and the Galacto-Light
-galactosidase
reporter gene assay system was from Tropix (Bedford, MA). The human
genomic PromoterFinder kit was from CLONTECH. The
Elongase Enzyme Mix was purchase from Life Technologies, Inc.
Gene-specific oligonucleotide primers were synthesized by Oligos, Etc.
(Wilsonville, OR).
5,6-Dichloro-1-
-D-ribofuranosylbenzimidazole (DRB) was
from Calbiochem-Novabiochem International. All other reagent-grade
chemicals were purchased from Sigma or Fisher Scientific.
Preparation of the Cytomegalovirus Promoter-driven 5'- and 3'-UTR
Constructs--
For investigation of the activities of the human
aggrecan 5'- and 3'-UTRs independent of the human aggrecan promoter,
luciferase constructs containing the 5'- and 3'-UTRs of the human
aggrecan mRNA (Fig. 1A)
were prepared in the cytomegalovirus (CMV) promoter-driven pcDNA3
eukaryotic expression vector. The luciferase gene cassette of the
pGEM-luc plasmid was first subcloned into the
BamHI and XhoI sites of pcDNA3 to generate
the control luciferase reporter plasmid pLUCneo. The previously cloned
5'-UTR of the human aggrecan mRNA (27) was amplified using the
primers 5'-ATC ACT AAG CTT GGC CCG ACC ACC TAC CTC-3' and
5'-TCA CGA GGA TCC AGA GTA AAG TGG TCA TAG TTC AC-3'. The
underlined sequences indicate restriction sites for HindIII
and BamHI, respectively. To preserve the Kozak sequence (36)
of the human aggrecan mRNA, TRP-2 was designed from nucleotides +78
to +54 of the published human aggrecan cDNA sequence (23). The
amplified product was digested with HindIII and
BamHI and cloned in the HindIII/BamHI
site immediately upstream of the luciferase reporter gene in pLUCneo to
generate the human aggrecan 5'-UTR construct pCMV/5UTR. Exon 19 (containing the 3'-UTR and two polyadenylation signals) of the human
aggrecan gene was amplified from the genomic cosmid cosHA-G3 (27),
using the primers 5'-TTT CGC TCG AGC CAC CAC CTA CAA ACG
CAG A-3' and 5'-TGC ACT GGG CCC TGG AAA GGC ACG ATG G-3',
and cloned into the XhoI/ApaI site immediately
downstream of the reporter gene in pLUCneo to generate the 3'-UTR
construct pCMV/3UTR. The construct pCMV/5_3UTR was generated by
cloning, respectively, the 5'-UTR and exon 19 in the
HindIII/BamHI and XhoI/ApaI
sites of pLUCneo. Constructs pCMV/5UTR-US and pCMV/3UTR-US, containing
the UTRs immediately upstream of the CMV enhancer/promoter, were
prepared by individually cloning the 5'- and 3'-UTRs upstream of the
CMV enhancer/promoter in the BglII/MluI site of
pLUCneo. The suffix -US denotes the upstream position of the UTRs
relative to the CMV promoter. All inserts were cloned, by design, in
the sense orientation. The -galactosidase (
-GAL) gene cassette of
the pSV-
-galactosidase expression vector was subcloned into the
HindIII/XhoI site of pcDNA3 for use as
transfection efficiency control.
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Preparation of the Human Aggrecan Promoter Constructs--
A
1.05-kb fragment (containing 0.7 kb of the proximal promoter and 0.35 kb of exon 1) of the upstream region of the human aggrecan gene was
amplified by PCR using the human genomic PromoterFinder kit and the
Elongase Enzyme Mix according to the suppliers' instructions. The PCR
product was cloned in the pCR II TA cloning vector and sequenced in
both directions. The promoter/exon 1 fragment was then digested with
MluI (located in the PromoterFinder adaptor) and
BsrBI (located at +25 of the human aggrecan exon 1 or
5'-UTR). The digested human aggrecan promoter (701 to +25) was
ligated to the 5'-UTR at the BsrBI site and subcloned, in
place of the CMV promoter, in pCMV/5UTR and pCMV/5_3UTR to generate the
chimeric luciferase constructs pAGC1(
701)/5UTR and
pAGC1(
701)/5_3UTR (Fig. 1B). Similarly, constructs
pAGC1(
701) and pAGC1(
701)/3UTR were prepared by cloning the
aggrecan promoter fragment into the MluI/BamHI
site immediately upstream of the luciferase gene in pLUCneo and
pCMV/3UTR. The aggrecan gene symbol AGC1 here denotes the human
aggrecan promoter.
Cell Culture and Transient Transfection--
Primary bovine
articular chondrocytes (1 × 106 cells/60-mm dish) or
NIH 3T3 cells (2 × 105 cells/60-mm dish) were
maintained in monolayer cultures at 37 °C for 24 h in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, amino acids (0.5 × minimal essential amino acids,
1 × nonessential amino acids), buffering agents (10 mM Hepes, 10 mM sodium bicarbonate, 10 mM TES, 10 mM BES), and antibiotics (100 units/ml penicillin, 100 µg/ml streptomycin). The cells were transiently transfected, using the ProFection calcium phosphate mammalian transfection kit, with 5 µg of each luciferase construct and 2-5 µg of -GAL cotransfection plasmid/60-mm dish for 48 h. Within experiments, all dishes were transfected with the same amount
of
-GAL cotransfection plasmid. At the time of harvesting, each dish
was rinsed twice with 5 ml of Ca2+/Mg2+-free
phosphate-buffered saline on ice, and the cells were lysed in 0.3 ml of
1 × Promega reporter lysis buffer. Luciferase activity in each
lysate was assayed using a LKB-Wallac liquid scintillation counter and
the Promega luciferase assay system.
-GAL activity was assayed using
the Galacto-Light
-GAL assay system. Luciferase activities were
normalized to
-GAL activities, and the data were subjected to
analysis of variance as well as the Student-Newman-Keuls multiple range
comparison test.
Measurement of Luciferase mRNA Half-life-- To assess the effects of the UTRs on stability of the luciferase mRNA, primary bovine articular chondrocytes were transfected with 10 µg each of the control luciferase plasmid pLUCneo and the pCMV/5UTR and pCMV/3UTR constructs. 45 h later (t = 0), the cells were treated with 65 µM DRB, an RNA synthesis inhibitor specific for RNA polymerase II (37, 38). Total RNA was subsequently isolated from the cells at 30-min intervals, starting at t = 0, and the luciferase mRNA levels were quantified by PCR (39).
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RESULTS |
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Effects of the Human Aggrecan 5'- and 3'-UTRs on Activities of the
CMV Promoter--
The human aggrecan mRNA contains a G/C-rich
382-bp leader sequence and a relatively short 3'-UTR (27). Computer
analysis predicts a complex secondary structure for the 5'-UTR which
could influence the rate of translation of the aggrecan gene (40). However, no information is available on how the aggrecan UTRs influence
expression of the aggrecan gene. Thus, to determine whether the UTRs of
the aggrecan gene have regulatory roles, the activities of the human
aggrecan 5'- and 3'-UTRs were first investigated, independent of the
aggrecan promoter, using chimeric luciferase constructs driven by the
CMV promoter/enhancer. In bovine articular chondrocytes, the activity
of the control pLUCneo luciferase plasmid was 1.84 × 104 ± 0.86 × 104 units of LUC/unit
-GAL. Relative to the activity of pLUCneo, the luciferase activity
of chondrocytes transfected with pCMV/5UTR was inhibited by 61%,
whereas that of the pCMV/3UTR-transfected chondrocytes was inhibited by
71% (p < 0.05, n = 18) (Fig.
2A). When the luciferase
reporter gene was flanked by both UTRs, the enzyme activity was reduced
by 92%. The results demonstrated that the human aggrecan UTRs contain
regulatory activities. Transfection of chondrocytes with pCMV/5UTR-US
and pCMV/3UTR-US, which contained the 5'-UTR and 3'-UTR immediately
upstream of the CMV promoter/enhancer, respectively, reduced the
expression of luciferase activities by 59 and 61% (Fig.
2A), demonstrating that transcription of the UTRs, and
therefore their physical presence in the luciferase transcript, is not
required for their regulatory activities. Assessment of the effects of
the UTRs on luciferase mRNA stability indicated that the presence
of the UTRs in the luciferase transcripts did not alter mRNA
stability significantly, as assessed by measurement of mRNA
half-lives (Fig. 3). The half-lives of
the luciferase transcripts ranged from 39 to 47 min, with or without
the UTRs.
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Sequence of the Human Aggrecan Promoter-- The proximal promoter (701 bp) and exon 1 (375 bp) of the human aggrecan gene have been cloned and sequenced in the forward and reverse directions. The current sequence of exon 1 differs from that obtained previously by cDNA cloning (27) at two nucleotide positions. At position +1, the current sequence reads as a C (Fig. 4), whereas it was determined as a G previously. Also, at position +7 the genomic sequence is a C compared with an A at the same position in the cDNA sequence. To verify the accuracy of these sequences, two separate clones containing the promoter and exon 1 were sequenced. The genomic sequence at position +7 is consistent with those reported for the rat and mouse aggrecan gene (25, 28).
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Effects of the Human Aggrecan 5'- and 3'-UTRs on Activities of the
Human Aggrecan Promoter--
To investigate the roles of the human
aggrecan promoter and the UTRs in expression of the aggrecan gene,
chimeric luciferase constructs containing the human aggrecan promoter
5'-UTR (exon 1) and/or 3'-UTR (exon 19) (Fig. 1B) were
prepared and used in transient transfection assays. The level of
expression induced by the promoter was 89-fold higher than that of the
promoter-less luciferase vector (data not shown), indicating that this
was a functional promoter. In the following experiments the activity of
pAGC1(701), containing the proximal promoter region (
701 to +25) of
the human aggrecan gene, served as base-line activity. The presence of
the 5'-UTR immediately downstream of the aggrecan promoter, construct
pAGC1(
701)/5UTR, significantly stimulated the expression of
luciferase activity 7.7-fold relative to the activity of pAGC1(
701)
(Fig. 5A). The expression of
luciferase activity was suppressed by 68% (p < 0.001, n = 6) when cells were transfected with
pAGC1(
701)/3UTR, the construct containing the human aggrecan exon 19 (3'-UTR) immediately downstream of the luciferase reporter gene. The
activity of pAGC1(
701)/5_3UTR, with the UTRs flanking the luciferase
gene, was 45% lower than that of pAGC1(
701) and 93% lower than the
activity of pAGC1(
701)/5UTR (Fig. 5A). In NIH 3T3
fibroblasts transfected with the aggrecan promoter/UTR constructs, a
pattern of luciferase expression similar to that in chondrocytes was
observed (Fig. 5B). Relative to the activity of the
pAGC1(
701) construct, expression of luciferase activity in
fibroblasts transfected with pAGC1(
701)/5UTR was 39.6-fold higher
(p < 0.001, n = 12). However, there
was no significant difference between the activities of
pAGC1(
701)/3UTR and pAGC1(
701)/5_3UTR compared with that of
pAGC1(
701).
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DISCUSSION |
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The structural gene for aggrecan is large and complex, giving rise
to an mRNA approximately 8 kb in size for the human gene. With
elucidation of the gene structure for aggrecan in several different
species (25-28), isolation and characterization of regulatory regions
of the gene can now be performed. We have cloned the human aggrecan
promoter and performed functional studies to determine regulatory
activities of the promoter and the 5'- and 3'-UTRs of the gene. The
human aggrecan promoter contains several putative SP-1/AP-2 binding
sites (-CCCGCC-), including a cluster of SP-1/AP-2 binding sites around
the transcriptional start site (Fig. 4). SP-1 plays a key role in
regulating transcription initiation of TATA-less promoters (44, 45).
The transcriptional start site of the human aggrecan promoter is
spanned by three overlapping SP-1/AP-2 binding sites. In the rat gene,
there is only one copy of the SP-1/AP-2 site (28) in a position
equivalent to that of the overlapping SP-1/AP-2 sites. Nonetheless, in
both genes, transcription is initiated 23-24 bp downstream of a
conserved TATG direct repeat (Fig. 4, reverse font),
suggesting that this sequence is important in transcriptional
initiation of the aggrecan gene. Search of the eukaryotic promoter data
base using the human aggrecan promoter sequence predicted a TATA box at
31, the position of the TATG repeat. Thus, this sequence may be the
equivalent of a TATA box in TATA-less promoters such as the aggrecan
promoter. Transcription initiation of the bovine aggrecan gene,
however, occurs at four different sites upstream of this TATG repeat
(25), suggesting other mechanisms of transcription initiation.
Expression of aggrecan can be modulated by mechanical forces,
cytokines, and other serum factors. The two NF-B sites in the promoter may confer responsiveness to cytokines. This is suggested by
the overlapping NF-
B and STAT binding sites at
375 to
362 (Fig.
4) and the fact that NF-
B is often activated by cytokines such as
tumor necrosis factor-
(46). The four SSREs (one in the promoter and
three in exon 1) are potential sites for regulating aggrecan gene
expression by shear stresses. The suppressive effects of interleukin-1
on aggrecan gene expression can be blocked or reversed by treatment
with platelet-derived growth factor (47). This stimulatory effect of
platelet-derived growth factor on aggrecan expression may be mediated
through the SIF response elements in the promoter (Fig. 4).
The 5'- and 3'-UTRs of the human aggrecan gene strongly modulated the activities of the human aggrecan promoter (Fig. 5) as well as those of the CMV promoter (Fig. 2). In chondrocytes and NIH 3T3 cells, the 5'-UTR stimulated the activities of the human aggrecan promoter greater than 7-fold (Fig. 5). In contrast, the 3'-UTR inhibited the activities of the human aggrecan promoter in both chondrocytes and NIH 3T3 cells. The stimulatory activity of the 5'-UTR was inhibited in the presence of the 3'-UTR. The mechanism by which the 3'-UTR suppresses aggrecan promoter activity is unclear, but it might be mediated through negative transcriptional elements including the Gfi-1 response elements found in both the promoter (Fig. 4) and the 3'-UTR (not shown). With respect to the CMV promoter, the 5'-UTR suppressed the expression of luciferase activity in chondrocytes or NIH 3T3 cells, but the 3'-UTR modulated the activity of CMV promoter in a cell type-specific manner. The 3'-UTR was inhibitory in chondrocytes (Fig. 2A) and stimulatory in NIH 3T3 cells (Fig. 2B). This cell type-specific effect of the 3'-UTR may be caused by the presence or absence of specific transcription factors in the transfected cells which bind the 3'-UTR and regulate transcription.
The effects of the 5'- and 3'-UTRs were mediated at the transcriptional level since 1) placement of the UTRs upstream of the CMV promoter did not alter their effects on expression of luciferase activities significantly (Fig. 2), and 2) their presence or absence from the luciferase transcript did not affect luciferase mRNA stability. The involvement of the 3'-UTR in modulating gene expression at the level of transcription is an unusual finding that has been demonstrated only for a few other genes. Typically, the 3'-UTR has been reported to control gene expression by modulating stability and translation of mRNAs (31-33). However, a role for the 3'-UTR in suppressing both basal and growth hormone-induced transcription has been reported in the rat serine protease inhibitor gene (35). Other examples of the transcriptional involvement of the 3'-UTR in regulating gene expression include the interaction between the tumor necrosis factor promoter and 3'-UTR in mediating the response of the gene to bacterial endotoxin (48) and the identification of an estrogen response element in the c-fos 3'-UTR which is capable of conferring estrogen responsiveness to a heterologous promoter such as the thymidine kinase promoter (34).
In the absence of inhibitory modulators, high level expression of the
human aggrecan gene is critically dependent on sequences present in
exon 1 (5'-UTR) (Fig. 6). This is indicated by the fact that sequential
deletions of the promoter from 701 to
52 have little effect on
promoter activity when the 5'-UTR is present. However, when the 5'-UTR
is removed, leaving a 5'-flanking sequence of
701 to +25 or
52 to
+25 (Fig. 6B), luciferase activity is reduced 85-100%. It
is possible that sequences within this region bind to general
transcription factors required during the assembly of the transcription
initiation complex. This region contains several putative SP-1 binding
sites, characteristic of TATA- and CAAT-less promoters, and may be
involved in recruiting the TFIID protein to the transcription
initiation complex.
Aggrecan is a member of a family of hyaluronan-binding proteoglycans that have been designated as hyalectans (49) or lecticans (50). Other members of this group include versican, neurocan, and brevican. The hyalectans share striking structural similarities, both at the protein and genomic levels (49). They differ primarily in the glycosaminoglycan attachment regions and also in the presence of the G2 domain in aggrecan. Promoter sequences for aggrecan (Fig. 3) (25, 28, 42), versican (51), neurocan (52), and brevican (53) have now been described. The versican (51) and neurocan (52) promoters both contain TATA boxes, whereas the aggrecan (Fig. 3) and brevican (53) promoters do not. Like the aggrecan promoter, the versican promoter contains multiple AP-2 sites (51). However, the 150-bp promoter region immediately upstream of the transcriptional start site of the versican gene lacks AP-2 or SP-1 sites, in contrast to that of the aggrecan promoter region (Fig. 3). This, along with the presence of a TATA box in the versican gene, suggests different mechanisms for transcriptional initiation of the aggrecan and versican genes.
Perlecan, a modular heparan-sulfate proteoglycan that is expressed
predominantly in vascular tissues (49, 54), is also expressed in
developing and adult cartilage (55, 56). The perlecan promoter, like
the aggrecan promoter (Fig. 3), lacks canonical TATA and CAAT boxes and
contains multiple SP-1/AP-2 binding sites in the promoter region and
exon 1 (57). Most of the perlecan SP-1/AP-2 binding sites are in the
reverse orientation compared with those of the aggrecan promoter.
Notwithstanding, the aggrecan (Fig. 6A) and perlecan (57)
proximal promoters exhibit very similar activities. Unidirectional
deletion of the promoters to 52 (for aggrecan) and
132 (for
perlecan) retained 80% and 40-50% of the wild-type promoter
activities, respectively. This is suggestive of the sufficiency of the
clusters of SP-1 and AP-2 sites near the transcriptional start sites,
in conjunction with sequences in exon 1, in mediating basal activities
of the aggrecan and perlecan promoters. The similarities in the
organizations and activities of the aggrecan and perlecan promoters may
provide clues to the mechanisms governing expression of the aggrecan
and perlecan genes, especially in relation to their expression in cartilage (55, 56).
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FOOTNOTES |
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* This work was supported in part by Grants AR43597 and AR42003 from the NIAMSD, National Institutes of Health, and Grant 95-024 from the Orthopaedic Research and Education Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF031586.
To whom correspondence should be addressed: Orthopaedic Research
Laboratory, Room BB-1412, Columbia-Presbyterian Medical Center, 630 West 168th St., New York, NY 10032. Tel.: 212-305-6446 or 3766; Fax:
212-305-2741; E-mail: valhmu{at}cuorma.orl.columbia.edu.
1 Valhmu, W. B., Stazzone, E. J., Bachrach, N. M., Saed-Nejad, F., Fischer, S. G., Mow, V. C., and Ratcliffe, A. (1998) Arch. Biochem. Biophys., in press.
2
The abbreviations used are: G1, G2, G3, globular
domains 1, 2, and 3, respectively; bp, base pair(s); UTR, untranslated
region; DRB,
5,6-dichloro-1--D-ribofuranosylbenzimidazole; CMV,
cytomegalovirus; LUC, luciferase;
-GAL,
-galactosidase; kb,
kilobase(s); PCR, polymerase chain reaction; TES,
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; BES,
N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic
acid; SSRE, shear stress response element; NF-
B, nuclear
factor-
B.
3 V. V. Solovyev and A. A. Salamov, manuscript in preparation.
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REFERENCES |
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