From the Departments of Medicine and of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110
Received for publication, April 11, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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Human matrix metalloproteinases-13 (HMMP13) shows
a wide substrate specificity, and its expression is limited to
pathological situations such as chronic inflammation and cancer. The
coding sequence for HMMP13 is 86% identical to rat matrix
metalloproteinases-13 (RMMP13); however, the regulation of HMMP13 and
RMMP13 protein synthesis in renal mesangial cells is strikingly
different. In human cells there is a discordance between HMMP13
mRNA levels and protein expression. Following IL-1 Matrix metalloproteinases
(MMPs)1 are proteolytic
enzymes that function in extracellular matrix (ECM) remodeling (1). It is also clear that MMPs influence many cellular functions, such as
migration, proliferation, apoptosis, and morphogenesis (2). Currently
20 distinct human MMPs have been described, of which 14 are secreted
extracellular proteinases and 6 are membrane-type MMPs. MMP-13,
together with MMP-1 and MMP-8, constitute the interstitial collagenase
subfamily (3). HMMP13 was initially identified in fibrosing breast
cancer (4). HMMP13 has a wide substrate specificity, and its expression
is limited to pathological situations in which rapid and effective
remodeling of collagenous ECM is required (5-7). High levels of
expression of HMMP13 have been documented in certain cancers that are
aggressive and invasive, and HMMP13 can serve as a prognostic marker of
tumor progression in a specific subset of cancers (8).
Numerous studies have documented the transcriptional regulation of
HMMP13 in several cell lines (9, 10). The HMMP13 gene can be
either up-regulated or down-regulated by a number of cytokines, growth
factors, and hormones. For example, HMMP13 expression was induced in
chondrocytes by IL-1 The 3'-UTR of certain genes can specifically control the nuclear
export, polyadenylation status, subcellular targeting, and rates of
translation or degradation of transcripts (16). The AREs found in the
3'-UTR of mRNA are one of the most prevalent and best studied
elements (17). They play important roles in the post-transcriptional
regulation of several genes involved in inflammation and tumorigenesis,
including COX-2 (18), iNOS (19), and TNF- Reagents--
Unless indicated, all reagents used for
biochemical methods were purchased from Sigma, VWR, or Fisher.
Restriction enzymes, DNA Polymerase I large fragment (Klenow), DNase I,
and alkaline phosphatases were all obtained from New England Biolabs
Inc. The pcDNA3 vector, cell culture medium, and fetal bovine serum
were from Invitrogen. Human recombinant IL-1 Cell Cultures--
Human mesangial cells were purchased from
Clonetics (CC-2599) and grown in supplied mesangial cell growth medium
(MsGM BulletKit, CC-3146) that contained 5% fetal bovine serum with 50 µg/ml gentamicin and 0.05 µg/ml amphotericin B. Rat primary
mesangial cell cultures were prepared from male Harlan Sprague-Dawley
rats as previously described (22). Cells were grown in RPMI 1640 medium
supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 0.6%
(v/v) insulin, 100 units/ml penicillin, 100 µg/ml streptomycin, 250 µg/ml amphotericin B, and 15 mM HEPES. Where indicated,
mesangial cells were stimulated with IL-1 RT-PCR and TaqMan Real-time PCR--
To test the expression of
MMP13 message, total RNA was isolated from cells using RNA STAT-60
reagent, DNase-treated, and reverse-transcribed by Omniscript reverse
transcriptase (Qiagen, Inc.) using Oligo (dT) 15 primers. The PCR
amplifications were carried out using human MMP13 amplification
primers, 5'-CCCCAACCCTAAACATCCAAAAAC-3' and
5'-CTTCCCCTACCCCG-CACTTCT-3', and rat MMP13 amplification primers,
5'-TGATGGGCCTTCTG-GTCTTCT-3' and 5'-AGGTCTCGGGATGGATGCT-3'. To
quantify HMMP13 mRNA levels, TaqMan real-time PCR was performed using gene-specific primers and the double-stranded DNA-binding dye,
SYBR green I. Fluorescence was detected with an ABI Prism 5700 sequence
detection system (PE Biosystems). HMMP13 amplification primers were
5'-TGAGCTGGACTCATTGTCGG-3' and 5'-GATTCCCGCGAGA-TTTGTAGG-3'. Amplification primers for glyceraldehyde-3-phosphate (GAPDH) were 5'-GAAGGTGAAGGTCGGAGTC-3' and 5'-GAAGATGGTGATGGGATTTC-3'. Primer pairs were tested to ensure a robust amplification signal of the expected size with no additional bands. The amount of HMMP13 message in
each RNA sample was quantified and normalized to GAPDH content. Relative amounts of HMMP13 cDNA were calculated by the comparative CT method.
Constructs--
MMP13 expression vectors were made by cloning
the RT-PCR-amplified human MMP13 coding region (HCR), human MMP13
3'-UTR (HUTR), and rat MMP13 3'-UTR (RUTR) into pcDNA3. Briefly,
after first-strand synthesis, cDNAs encoding HCR, HUTR, and RUTR
were generated by PCR using appropriate primers and then cloned into
pCR2.1-TOPO-TA cloning vector (Invitrogen). HCR forward primer was
5'-ATTCAAGATGCATCCAGGGGTC-3' and reverse primer was
5'-CACTTAAC-ACCACAAAATGGAATTTG-3'. HUTR primers were
5'-CAAATTCCATTTTGT-GGTGTTAAGTG-3' and
5'-TAAACAACATAAACACTTCCTAATACACTTTG-3'. RUTR primers were
5'-CGAATTCCTTATTGTGGTGTTAAACA-3' and 5'-GAAACAACATAAGCACAGTGTAACAC-3'. HCR was subcloned from pCR2.1 into pcDNA3 using restriction enzyme EcoRI to get HMMP13-HCR. The HUTR were excised from pCR2.1
using BstXI (filled in with Klenow) and EcoRV,
then ligated into HMMP13-HCR by using EcoRV and
NotI (filled in with Klenow) to generate HMMP13-HFL. The
RUTR were excised from pCR2.1 using BamHI (filled in with Klenow) and NotI and inserted into HMMP13-HCR by using
NotI and XbaI (filled in with Klenow) to obtain
HMMP13-HCRU. The FLAG-TIARa and FLAG-TIARb constructs were generated by
cloning the RT-PCR-amplified human TIAR-a (1185 bp) or
TIAR-b (1134 bp) gene into pcDNA3/FLAG expression vector
(Invitrogen). The forward primer was 5'-ACCATGATGGAAGACGACGG-3' and
reverse primer was 5'-GGCTCACTGT-GTTTGGTAACTTG-3'. The FLAG-TIAR and
F-TIA1 peptide constructs were generated by annealing two pairs of
complementary oligonucleotides,
(5'-GATCCATGCAACCCGATAGCAGAAGGGTCAACTCTTCTGTTGGATTTTCTGTTTTGCACTAAG-3' and
5'-ATTCTTAGTGCAAAACAGAAAATCAACAGAA-GAGTTGACCCTTC-TGCTATCGGGTTGCATG-3' for TIAR peptide or 5'-GATCCAGTAGTACCGTTGTCAGCACACAGCGTTCACAATAAG-3' and 5'-AATTCTTATTGTGAACGCTGTGTGCTGACAACGGTACTACTG-3' for TIA1 peptide) encoding the 17-amino acid stretch of TIAR-a or 11-amino acid
stretch of TIA1 flanked by BamHI and EcoRI
restriction sites. The oligos include a stop codon to terminate
translation. The annealed oligos were ligated in BamHI- and
EcoRI-cut pcDNA3/FLAG expression vector. The GFP-TIAR
and GFP-TIA1 peptides were made by annealing the above two pairs of
complementary oligonucleotides for TIAR peptide or TIA1 peptide and
then subcloning them into pcDNA3.1/NT-GFP vector (Invitrogen). All
constructs described above were sequenced to confirm sequence fidelity.
Transient Transfections--
Equimolar amounts of DNA were
transiently transfected into cells using SuperFect transfection reagent
(Qiagen Inc.) following the manufacturer's protocol.
Western Blot Analysis--
Culture medium from cells was
concentrated by Centriplus YM-30 centricons (Millipore Corp.), and
equal amounts of protein were assayed for MMP13 content by Western
blotting. Monoclonal anti-HMMP13 (Chemicon International Inc.) and
anti-RMMP13 (Oncogene) antibodies were used for Western blotting at a
1:500 dilution. Whole cell protein extracts were prepared from
confluent cells as described previously (22). Western blots of whole
cell extracts were probed with monoclonal anti-HuR (Santa Cruz
Biotechnology), monoclonal anti-FLAG (Sigma), polyclonal anti-GFP
(Clontech), or polyclonal anti-TIAR antibodies
(Santa Cruz Biotechnology) at a 1:1000 dilution. Signal detection was
carried out using enhanced chemiluminescence system (ECL, Amersham
Biosciences). The monoclonal anti-HMMP13 and anti-RMMP13
antibodies were specific for human and rat MMP13, respectively. The
monoclonal HuR and polyclonal TIAR antibodies reacted with both human
and rat proteins. Bands corresponding to the MMP13 were quantitated on
the Discovery Series densitometer, and the band intensities were
measured using Quantity One software, both from Bio-Rad. The
densitometer was calibrated against external standards and was linear
to 2 AUFs. We also calibrated it against different amounts of
expressed MMP-13 in serum-free cellular supernatants from
HCR-transfected HEK293 cells; the response was linear over 10-100 µl
of media, and we routinely used 50 µl for Western analysis.
Differential Regulation of MMP13 mRNA and Protein Expression in
Rat and Human Mesangial Cells--
Previous studies in our laboratory
have demonstrated that IL-1 The 3'-UTR of MMP13 Decreased HMMP13 Protein Expression in HEK293
and COS-7 cells but Not in Rat2 Cells--
To determine whether the
3'-UTR was responsible for the differential post-transcriptional
regulation of HMMP13 expression, we engineered a series of expression
plasmids, shown in Fig. 2. HMMP13-HCR
contains cDNA corresponding to the coding region of HMMP13, whereas
HMMP13-HFL contains the full-length cDNA (coding region plus the
3'-UTR). HMMP13-HCRU is the combination of the HMMP13 coding region and
the 3'-UTR of RMMP13. HEK293, COS-7, and Rat2 cells were transiently
transfected with equimolar amounts of these constructs. Human MMP13
protein expression was reduced by 70-80% in HEK293 cells transfected
with HMMP13-HFL or -HCRU when compared with cells transfected with
HMMP13-HCR (Fig. 3, A and
B). Similar results were also found in transiently
transfected COS-7 cells (Fig. 3, C and D). In
contrast, constructs expressed in Rat2 cells resulted in equivalent
amounts of MMP13 protein expression regardless of the 3'-UTR present in
the message (Fig. 3E). Interestingly, although only the
latent form of HMMP13 was detected in transfected HEK293 and COS-7
cells, both latent and active forms were expressed in transfected Rat2
cells. These data suggest that the suppression of HMMP13 expression
requires the 3'-UTR of MMP13, either human or rat, and occurs in human
and primate cell lines but not in Rat2 cells.
Translational Silencing of HMMP13 Gene Expression by an
Alternatively Spliced Form of RNA-binding Protein
TIAR--
Post-transcriptional regulation of certain genes has been
shown to be dependent on AREs present in the 3'-UTR (24). Several proteins, including AUF1/hnRNPD, HuR, TIA1, and TIAR, are reported to
bind AREs. Binding of these proteins to transcripts bearing AREs can
have either a positive or negative effect on gene expression (25). To
determine whether ARE binding proteins were differentially expressed,
we performed Western blot analysis on the cell lines of interest, using
anti-HuR and anti-TIAR antibodies. No significant differences in HuR
protein expression were observed in human and rat mesangial cells (Fig.
4A). However, TIAR protein
expression was different in human and primate cells compared with rat
cells (Fig. 4B). Human and primate cells expressed two
isoforms of TIAR, whereas rat cells expressed only one.
TIAR is a member of the RNA recognition motif (RRM) family of RNA
binding proteins that has been shown to act as a translational silencer
(26). This protein possesses three RRM domains that confer high
affinity binding to AREs. The TIAR gene encodes two isoforms, TIAR-a and TIAR-b, that arise from alternative splicing of a
common precursor transcript (27). TIAR-a differs from TIAR-b in that it
contains an additional 51 nucleotides in the middle of RRM1 resulting
in a 17-amino acid insertion (Fig. 4D), which has been
suggested to play a role in RNA binding specificity (28).
Interestingly, translational silencing occurred only in the human or
primate cell lines, the same cell lines that expressed TIAR-a. The
possibility arises, therefore, that species-dependent translational silencing of the HMMP13 gene is because of the
differential expression of TIAR-a in human and rat cell cultures. We
reasoned that if the 17-amino acid insert of TIAR-a serves as a binding site required for the translational repression of HMMP13, then expression of the peptide QPDSRRVNSSVGFSVLQ encoded by this
51-nucleotide insert may competitively inhibit this binding event and
reverse mRNA silencing observed in human and primate cell lines. To
test this possibility, we subcloned the 51-nucleotide cDNA into
FLAG-tagged pcDNA3 vector to generate the FLAG peptide. HEK293
cells transfected with HMMP13-HCR, HMMP13-HFL, or HMMP13-HCRU were
co-transfected with pcDNA3/FLAG (as control) and FLAG peptide.
Quantitative PCR demonstrated equivalent levels of HMMP13 mRNA for
all experimental conditions, confirming that co-transfection of the
plasmid encoding the 17-amino acid peptide had no effect on the steady
state level of the message (data not shown). The inclusion of the
3'-UTR of MMP13 resulted in a 60-70% decrease in protein expression,
and co-expression of the peptide blocked the reduction of HMMP13
protein expression because of the 3'-UTR, suggesting an interference of translational silencing conferred by TIAR-a (Fig.
5A).
TIA1 is another RNA-binding protein reported to be a translational
silencer similar to TIAR and has three RRMs that can bind to mRNAs
(29). TIA1 can be expressed as two alternatively spliced forms, the
difference due to an insert of a small (11-amino acid) peptide. In
contrast to the TIAR peptide insert that is located in the middle of
the first RRM, the TIA1 peptide insert (SSTVVSTQRSQ) is located between
RRM1 and RRM2 (30). We tested whether this peptide could also reverse
the translational silencing of HMMP13. Co-transfection of the FLAG-TIA1
peptide had no effect on HMMP13 protein expression because of the
3'-UTR (rat) and the minimal but not significant effect with human
3'-UTR of MMP13 (Fig. 5B). In all of these experiments there
was no difference between MMP13 expression when co-transfected with
pcDNA3, pcDNA3/FLAG, or pcDNA3/GFP vectors. The data were
normalized to the HCR data because of the variability in intensity of
Western blots between experiments. Within experiments all exposures
were carried out on the same blot and film. This result suggests that
interference of translational silencing of HMMP13 is specific for the
TIAR peptide. To confirm that the FLAG peptides were expressed, we
performed Western blot analysis for the FLAG protein. Unfortunately, we
were unable to detect FLAG peptide expression by routine Western
blotting. We believe this was because of technical problems generated
by the small molecular mass (less than 3 kDa) of the FLAG peptides.
To clearly document the expression of epitope-tagged peptide, we therefore engineered a GFP fusion protein (GFP peptide) whose expression was easily demonstrated by Western blotting (Fig.
6B). We repeated the transient
transfection experiments using the GFP peptides and confirmed that,
although the co-transfection of the GFP-TIAR peptide reversed the
translational block of HMMP13 protein expression because of the 3'-UTR,
the GFP-TIA1 peptide did not (Fig. 6A). Furthermore, the GFP
peptides were in fact expressed at equivalent levels (Fig.
6B).
Expression of TIAR-a but Not TIAR-b Produces Translational
Silencing on HMMP13 Protein Expression in Transfected Rat2
Cells--
Rat2 cells express only the TIAR-b isoform, and the
3'-UTR-dependent suppression of HMMP13 expression did not
occur in these cells (Fig. 4C). To determine whether TIAR-a
could cause translational silencing of HMMP13 in transfected Rat2
cells, we co-expressed human TIAR-a or TIAR-b (as control) in HCR-,
HFL-, or HCRU-transfected Rat2 cells and measured HMMP13 protein
levels. FLAG-tagged TIAR-a and TIAR-b were expressed at similar levels
in Rat2 cells (Fig. 7B).
Co-expression of TIAR-a resulted in a significant decrease in HMMP13
protein expression when the 3'-UTR of HMMP13 was present, whereas
TIAR-b had no significant effect (Fig. 7A). However, TIAR-b appeared to partially inhibit HMMP13 protein expression in Rat2 cells
co-transfected with the HCRU construct (about 25%). The results of
this experiment suggest that TIAR-a plays the major role in the
3'-UTR-dependent translational silencing in HMMP13 expression.
Thus we have shown, for the first time, species-dependent
MMP13 gene silencing in human and primate cell cultures. This
post-transcriptional regulation requires the participation of the
3'-UTR of the transcripts and is affected by the expression of a
peptide corresponding to the alternatively spliced form of TIAR, namely
TIAR-a. These results are consistent with the notion that TIAR-a binds
to the 3'-UTR of HMMP13 and inhibits translation. Expression of the
peptide competes for the RNA binding site or disrupts protein-protein interactions of TIAR-a. These experiments underscore the concept that
alternatively spliced transcripts can regulate the diversity of gene
expression. It may also provide a mechanism by which tumors derepress
MMP13 expression that may affect their metastasis potential.
or
TGF-
1 stimulation, HMMP13 mRNA levels increase
significantly, whereas the protein expression is absent. This
discordance is because of a species-dependent translational
repression. In addition to the 3'-untranslated region of the matrix
metalloproteinases-13 (MMP13) gene, the differential expression
of an alternatively spliced transcript of the RNA-binding protein TIAR
in human cell cultures is also critical for this post-transcriptional
regulation. Transient expression of the 17-amino acid insert of the
alternatively spliced form of TIAR reverses the HMMP13 mRNA
silencing observed in human and primate species. In addition,
co-transfection of the alternatively spliced form of TIAR and HMMP13
into Rat2 cells suppresses HMMP13 protein expression. Thus, we
report for the first time that a species-dependent
TIAR isoform plays a major role in the post-transcriptional silencing for HMMP13.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSIONS
REFERENCES
and in human gingival fibroblasts by TGF-
.
In contrast, IFN-
inhibited HMMP13 expression in
ras-transformed human keratinocytes (11). Recent work has
also shown that the RMMP13 transcript is stabilized by cortisol (12) or
alendronate (13) and destabilized by TGF-
in osteoblast cells (14).
The change in message stability was mediated by adenine- and
uridine-rich elements (AREs) in the 3'-untranslated region (3'-UTR) of
the RMMP13 mRNA. However, our understanding of the mechanisms for regulation of HMMP13 expression is far from complete (15).
(20). For example, in mice
with a targeted disruption of the AREs from TNF-
mRNA, the
animals developed chronic inflammatory arthritis and Crohn's-like
inflammatory bowel disease due to increased TNF-
synthesis (21). In
this study, we have shown that the HMMP13 gene undergoes a
species-dependent translational silencing in human and
primate cell cultures. This post-transcriptional regulation requires
the participation of the 3'-UTR of the transcripts and is affected by
the expression of a peptide corresponding to the alternatively spliced
form of TIAR, namely TIAR-a.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSIONS
REFERENCES
, TGF-
1,
and TNF-
were all purchased from Roche Molecular Biochemicals.
(100units/ml),
TGF-
1 (5 ng/ml), or TNF-
(20 ng/ml). All experiments
were performed with confluent cells and used at passages 3-8. HEK293
(CRL1573), COS-7 (CRL1651), and Rat2 (CRL1764) cells were all from
American Type Culture Collection (ATCC) and grown at 37 °C in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Transient transfections were performed using cells at 70-80% confluency.
RESULTS AND DISCUSSIONS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSIONS
REFERENCES
induces interstitial collagenase-3
(MMP13) gene expression and protein secretion in primary cultures of
rat mesangial cells (23). To further investigate the regulation of
MMP13 expression in renal mesangial cells, we used primary cultures of
mesangial cells from both human and rat. Cultured mesangial cells were
stimulated with IL-1
(100units/ml), TGF-
1 (5 ng/ml),
or TNF-
(20 ng/ml) for 24 or 48 h in serum-free medium. Total
RNA was extracted and analyzed for MMP13 message by RT-PCR, and cell
culture medium was concentrated and analyzed for MMP13 protein using
Western blots. Rat mesangial cells exposed to IL-1
for 24 h
showed increased MMP13 mRNA levels, whereas TGF-
1
and TNF-
had no effect (Fig. 1A). In human mesangial cells,
IL-1
also increased HMMP13 mRNA levels; however,
TGF-
1 was a more effective ligand and TNF-
had no
effect (Fig. 1B). Western blot analysis detected rat MMP13 (RMMP13) protein expression in media from IL-1
-treated rat mesangial cells (Fig. 1C). However, there was no HMMP13 protein
detected in human mesangial cell media after 48 h of stimulation
with IL-1
or TGF-
1 (Fig. 1D). We were also
unable to detect HMMP13 protein expression in whole cell lysates,
excluding the possibility that HMMP13 protein failed to be secreted
(data not shown). Thus, the discordance between the increase in
mRNA levels and the absence of HMMP13 protein expression following
IL-1
or TGF-
1 treatment suggests that the
HMMP13 gene is translationally silenced in human mesangial
cells. Furthermore, the differential regulation of MMP13 in human and
rat mesangial cells suggests a species-dependent mechanism
for post-transcriptional regulation.
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Fig. 1.
Differential regulation of MMP13 mRNA and
protein expression in rat and human mesangial cells. Cells were
treated with IL-1 (100units/ml), TGF-
1 (5 ng/ml), or
TNF-
(20 ng/ml) for 24 h. A, RT-PCR amplification of
a 500-bp region of RMMP13. IL-1
increased RMMP13 mRNA levels.
B, RT-PCR amplification of a 678-bp region of HMMP13.
IL-1
and TGF-
1 induced HMMP13 mRNA expression.
C, Western blot analysis of RMMP13 expression. IL-1
increased RMMP13 protein expression. p-aminophenylmercuric
acetate (APMA) treatment resulted in the cleavage of the
latent form (60 kDa) of MMP13 to active form (48 kDa). D,
Western blot analysis demonstrated that no HMMP13 protein was
expressed.
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Fig. 2.
DNA constructs utilized in this study.
HMMP13-HCR contained only the coding region of HMMP13. HMMP13-HFL
contained the full-length HMMP13 cDNA (coding region plus the
3'-UTR), and HMMP13-HCRU contained the coding region from HMMP13 plus
the 3'-UTR from RMMP13.
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Fig. 3.
The 3'-UTR of MMP13 decreased HMMP13 protein
expression in HEK293 and COS-7 cells but not in Rat2 cells. Cells
were transiently transfected with either empty vector
(Control), or HMMP13-HCR (HCR), HMMP13-HFL
(HFL), or HMMP13-HCRU (HCRU). HMMP13 protein
levels in transfected HEK293 (A, B), COS-7
(C, D), and Rat2 (E) cells were
detected by Western blot and quantitated by scanning densitometry.
Values with error bars are means ± S.E. for three
independent experiments.
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Fig. 4.
Differential expression of RNA binding
proteins in human and rat cell cultures. Western blot analysis of
HuR (A) and TIAR (B) protein expression in human
and rat mesangial cells that received no treatment (lane 1)
or were treated with IL-1 (100 units/ml, lane 2),
TGF-
1 (5 ng/ml, lane 3), or TNF-
(20 ng/ml, lane 4) for 24 h. C, Western blot
analysis of TIAR protein expression in HEK293, COS-7, and Rat2 cells
(in duplicate). D, schematic representation of TIAR
isoforms. TIAR-a differs from TIAR-b in that it contains an additional
17-amino acid peptide insertion in the middle of RRM1.
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Fig. 5.
Expression of the 17-amino acid insert of
TIAR-a, but not the 11-amino acid insert of TIA1, inhibited the
translational silencing of HMMP13 message. HEK293 cells
transfected with HMMP13-HCR, HMMP13-HFL (HFL) or
HMMP13-HCRU (HCRU) were co-transfected with either control
vector (pcDNA3/FLAG), FLAG-TIAR peptide (A), or
FLAG-TIA1 peptide (B) expression vector. The HMMP13 protein
levels were quantitated and normalized to that of HCR-transfected
cells. Data represent the mean ± S.E. from five independent
experiments (*, p < 0.05 versus
control).
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Fig. 6.
Expression of the GFP-TIAR peptide, but not
the GFP-TIA1 peptide, inhibited the translational silencing of HMMP13
message. A, HEK293 cells transfected with
HMMP13-HCR, HMMP13-HFL (HFL), or HMMP13-HCRU
(HCRU) were co-transfected with either GFP-TIAR peptide or
GFP-TIA1 peptide expression vector. The HMMP13 protein levels were
quantitated and normalized to that of HCR-transfected cells.
B, GFP protein expression was detected by Western blot
analysis using polyclonal anti-GFP antibody. Data represent the
mean ± S.E. from three independent experiments.
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Fig. 7.
Expression of TIAR-a translationally silenced
HMMP13 protein expression in Rat2 cells. Rat2 cells transfected
with HMMP13-HCR, HMMP13-HFL (HFL), or HMMP13-HCRU
(HCRU) were co-transfected with either pcDNA3/FLAG as
control or pcDNA3-FLAG-TIARa (FLAG-TIARa) or
pcDNA3-FLAG-TIARb (FLAG-TIARb) expression vector.
A, HMMP13 protein levels were quantitated and normalized to
that of HCR-transfected cells. B, FLAG protein expression
was detected by Western blot analysis using monoclonal anti-FLAG
antibody. Data represent the mean ± S.E. from four independent
experiments.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Award DK 09976 (to A. R. M).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.
Recipient of a research award from the National Kidney Foundation
of Eastern Missouri and Metro East, Inc.
§ To whom correspondence should be addressed: 822 Yalem Research Bldg., Barnes/Jewish Hospital, 216 S. Kings Hwy., St Louis, MO 63110. Tel.: 314-454-8495; Fax: 314-454-8430; E-mail: morrison@pcg.wustl.edu.
Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M203526200
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ABBREVIATIONS |
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The abbreviations used are: MMP, matrix metalloproteinase; IL, interleucine; TGF, transforming growth factor; IF, interferon; UTR, untranslated region; HUTR, human UTR; RUTR, rat UTR; RRM, RNA recognition motif; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; TIA1, T-cell-restricted intracellular antigen 1; TIAR, TIA1-related protein; HCR, human coding region; GFP, green fluorescence protein; ARE, adenine-rich element; AUFS, absorbance units full-scale.
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