Departments of Virology and Pathology, the Haartman Institute and
Biomedicum Helsinki, University of Helsinki and Helsinki University Hospital,
FIN-00014 Helsinki, Finland
*
Author for correspondence (e-mail:
katri.koli{at}helsinki.fi
Accepted April 23, 2001
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SUMMARY |
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Key words: LTBP, TGF-ß, 8-Cys repeat, Alternative splicing
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INTRODUCTION |
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LTBPs belong to the LTBP/fibrillin-family of extracellular matrix (ECM)
proteins (reviewed by Saharinen et al.,
1999;
Öklü and Hesketh,
2000
). The family includes
fibrillin-1 and - 2, in addition to four different LTBPs (Kanzaki et al.,
1990
;
Morén et al.,
1994
; Yin et al.,
1995
; Saharinen et al.,
1998
). The structures of these
large ECM glycoproteins are highly repetitive. They are mainly composed of
epidermal growth factor (EGF)-like repeats, eight cysteine (8-Cys) repeats
unique to this protein family, as well as flanking regions containing proline
rich areas, or in some cases glycine rich areas. LTBPs-1, -2 and -4 are known
to get assembled into the ECM (Gibson et al.,
1995
; Taipale et al.,
1996
; Saharinen et al.,
1998
;
Hyytiäinen et al.,
1998
). LTBP-1 and -2 localize
to microfibrillar structures in the ECM and associate with fibronectin rich
fibers (M. Hyytiäinen and J.K.-O., unpublished;
Gibson et al., 1995
; Taipale et
al., 1996
; Dallas et al.,
2000
). The N- and C-terminal
parts of LTBPs mediate matrix binding (Saharinen et al.,
1996
;
Unsöld et al.,
2001
), and the N-terminally
extended form of LTBP-1 associates more readily with the ECM (Olofsson et al.,
1995
). Only LTBPs-1, -3 and -4
can form large latent complexes with TGF-ß (Saharinen et al.,
1996
; Gleizes et al.,
1996
; Saharinen and Keski-Oja,
2000
) and mediate
extracellular matrix localization of these complexes. Several proteases
including plasmin can release the LTBPTGF-ß complexes from the ECM
(Saharinen et al., 1998
;
Taipale et al., 1992
; Taipale
et al., 1995
). Plasmin can
also cleave LTBP-2, which does not bind small latent TGF-ß The specific
cleavage sites have been located to the hinge region of LTBP-2, before a
proteolysis resistant core consisting mainly of EGF-like repeats
(Hyytiäinen et al.,
1998
).
LTBP molecules not containing small latent TGF-ß can also be detected
from the ECM (Taipale et al.,
1994), and it appears that
LTBPs are produced in molar excess compared with TGF-ßs. LTBPs are known
as structural components of the ECM, in addition to targeting TGF-ß.
Structural variation in LTBPs have been described and found to be caused by
various mechanisms, including proteolytic processing, alternative splicing and
the use of different promoters (Koski et al.,
1999
). Splice variants lacking
a specific domain or with additional repeats have been characterized from all
four LTBPs (reviewed by Saharinen et al.,
1999
). N-terminally extended
forms may associate with the ECM with differing affinity, while deletions of
part of the hinge region may provide protease resistance (Olofsson et al.,
1995
; Michel et al.,
1998
). The biological
functions of the numerous alternatively spliced forms are under keen
investigation at present.
The expression patterns of the four different LTBP isoforms have not been
well characterized so far. We analyzed here by RT-PCR the expression of LTBPs
in a panel of cultured cell lines including fibroblasts of different origin,
endothelial cells and immortalized keratinocytes. LTBPs were found to be
expressed in an overlapping manner. However, our semi-quantitative analyses
suggested differences in the expression levels of the different isoforms. A
novel LTBP-4 splice variant lacking the small latent TGF-ß binding domain
(LTBP-48-Cys3rd), was identified and found to be expressed
in the same tissues as intact LTBP-4. The targeting and activation of
TGF-ßs have numerous levels of regulation. LTBPs are necessary for the
correct folding and secretion of TGF-ß (Miyazono et al.,
1991
; Miyazono et al.,
1992
;
Eklöv et al.,
1993
). In the current work we
have identified a novel way to regulate this complex network, namely to
decrease the ECM-associated small latent complexes in certain cells expressing
LTBP-4. Regulation of the ratio of the two LTBP-4 variants may have an effect
on the cellular phenotype and be involved in the developmental control and in
the pathogenesis of diseases including cancers.
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MATERIALS AND METHODS |
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Total cellular RNA was isolated using RNeasy Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. RNA concentrations and purities were determined spectrophotometrically (Ultrospec 3000, Amersham Pharmacia Biotech, Uppsala, Sweden) as well as by agarose gel electrophoresis followed by ethidium bromide staining.
Antibodies
Rabbit polyclonal LTBP-4-specific antibodies #28-3 (against the
3rd 8-Cys repeat) and #33-4 (against the 4th 8-Cys
repeat) have been described previously (Saharinen et al.,
1998). The antibodies have
been affinity purified with the immunogenic peptide and are reactive in
immunoblotting assays under both reducing and nonreducing conditions. Mouse
monoclonal antihemagglutinin (
-HA) antibody 12CA5 was from BabCO
(Richmond, CA).
Primers
Primers were custom made by TAG Copenhagen A/S (Copenhagen, Denmark) except
for G3DPH primers, which were from Clontech (Palo Alto, CA). Primer sequences
and the sizes of expected PCR products are summarized in
Table 1.
|
cDNA constructs
Construct pFull codes for the 3rd 8-Cys repeat, the following
two EGF-like repeats and the 4th 8-Cys repeat of LTBP-4 and is
cloned in pSignal (Saharinen et al.,
1996), a eukaryotic secretory
expression vector derived from pcDNA3 (In Vitrogen, Carlsbad, CA). Constructs
p
8-Cys3rd and p
24 8-Cys are similar, but lack the
3rd 8-Cys repeat or the last 24 amino acids of the repeat,
respectively.
Reverse transcription and PCR
Reverse transcription was carried out with Oligo (dT)12-18
primer (Life Technologies) and Superscript II reverse transcriptase (Life
Technologies) using 5 µg of total RNA or 0.5 µg of poly A+
RNA (Clontech) according to manufacturer's instructions. The cDNAs were
amplified using AmpliTaq Gold (Perkin Elmer, Branschburg, NJ) in a 35 cycle
PCR reaction. For G3DPH (glyceraldehyde 3-phosphate dehydrogenase) only 25
cycles of amplification was needed to obtain quantifiable results. The number
of cycles was optimized so that the PCR reaction did not reach a plateau.
Semi-quantitation of the RT-PCR products was carried out after equalizing the
amounts of the specific products to the amounts of G3DPH in a particular cell
line.
The primers were designed to yield products spanning exon-intron boundaries so that a possible genomic contamination in total RNA preparation would result in a higher molecular weight product. Another test for genomic contamination was PCR performed directly from RNA samples that had not been reverse transcribed. In the 3rd negative control, the cDNA template was substituted with water. All these controls were negative.
Cloning and sequencing
PCR products were cloned into pGEM-T Easy Vector (Promega, Madison, WI) and
sequenced using an ABI 310 automatic DNA sequencer (Perkin-Elmer). Several
individual clones from separate PCR reactions were sequenced.
Ribonuclease protection assay
Ribonuclease (RNase) protection analysis was carried out following the
instructions of the commercial Direct Protect Kit (Ambion Inc., Austin, TX).
PCR products of size 331 (`RPA', nucleotides 3449-3779) or 439 bp (`RPA-2',
nucleotides 2943-3381) from LTBP-4 were cloned into pGEM-T Easy vector
(Promega), which contains the recognition site of T7 polymerase. The plasmids
were further linearized and antisense RNA probes were synthesized with T7
RNA-polymerase in the presence of [-32P]UTP (Amersham
Pharmacia Biotech) using a MAXIscript Kit (Ambion). Total RNA (5 µg) or 0.5
µg of polyA+ RNA (Clontech) were hybridized with the appropriate
antisense RNA probe. Following hybridization overnight, unpaired
single-stranded probes were digested with RNase A/Rnase T1 mix and the samples
were subsequently precipitated. Protected fragments were analyzed in a
denaturing 6% polyacrylamide gel containing 8 M urea, and their sizes were
determined by comparison with a Century Marker Template Plus (Ambion).
Northern hybridization analysis
For northern analysis 10 µg of total RNA was fractionated on 1.2%
agarose gels containing formaldehyde and transferred to Hybond-N nylon
membranes (Amersham Pharmacia Biotech) by capillary transfer.
Pre-hybridization and hybridization were performed at 68°C in ExpressHyb
hybridization solution (Clontech). PCR products from LTBP-4 spanning
nucleotides 3216-3554 (`3rd 8-Cys', which detects only LTBP-4 containing the
3rd 8-Cys repeat) and 3448-3779 (`RPA', which detects total LTBP-4,
whether or not it contains the 3rd 8-Cys repeat) were labeled with
[32P]-dCTP (>3000 Ci/mmol, Amersham Pharmacia Biotech) using a
Rediprime Labeling Kit (Amersham Pharmacia Biotech). Washes were carried out
under high stringency conditions, first with 2xSSC containing 0.05% SDS
and then with 0.1xSSC containing 0.1% SDS at 50°C for 40 minutes
each.
Multiple tissue expression array
Multiple tissue expression (MTE) arrays were carried out according to the
manufacturer's instructions (Clontech). The amounts of RNA in MTE array have
been equalized by the manufacturer by comparing the expression levels of eight
different housekeeping mRNAs. The blot was hybridized with cDNA probes as in
Northern blot. The MTE arrays have several controls including RNA and DNA from
yeast, E. coli and humans. The two probes used gave negative results
for all these controls and showed no nonspecific binding. The radioactivity
levels were quantified with a BAS-2500 bio-imaging analyzer (Fuji, Tokyo,
Japan).
Transfection of cell lines and immunoblotting
Approximately 7.5x105 293T cells were seeded per well in
six-well plates and transfected the following day with 2 µg of the plasmids
indicated using FuGENE6 liposome mediated transfection system (Roche Molecular
Biochemicals). Six hours after transfection the cells were washed, fed with
serum-free medium and the conditioned medium was collected after 48 hours.
Aliquots of medium samples were electrophoresed under reducing conditions in
4-15% gradient polyacrylamide gels in the presence of SDS and the proteins
were electrophoretically transferred to Protran nitrocellulose membranes
(Schleicher & Schuell, Dassel, Germany). Immunodetection using anti-HA
antibody or LTBP-4 specific antibodies was performed as described (Saharinen
et al., 1996).
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RESULTS |
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Two sets of primers (see Materials and Methods) were used in the detection
of LTBP isoforms. Because of the different splice variants described (see
Saharinen et al., 1999), we
selected two amplified areas; the first at the C-terminus and the second
around the 3rd 8-Cys repeat
(Fig. 1A). No alternatively
spliced LTBP isoforms have earlier been identified around these areas. Since
the exon-intron structures of human LTBP-2 (Bashir et al.,
1996
) and LTBP-3 (J.S. et al.,
unpublished) are known, we used this knowledge to design primers that would
probably amplify regions spanning exon-intron boundaries. Data obtained by
RT-PCR from these two different regions within each LTBP was very similar.
Since the amplification efficiency of the different primer pairs varied to a
certain extent, the levels of the different LTBP isoforms in each cell line
could be compared only in a semi-quantitative manner
(Fig. 1B). The two bands
detected with LTBP-4.2 primers are discussed below.
|
All cell lines assayed expressed at least some of each LTBP (summarized in
Fig. 1C). The expression levels
of the panel of cell lines were compared to those of CCL-137 cells. LTBP-1 was
abundant in embryonic lung fibroblasts and fibrosarcoma cells, while only low
levels of expression could be detected in transformed VA-13 cells. LTBP-2
expression was most prominent in skin fibroblasts and endothelial cells,
whereas only low levels were expressed in VA-13 cells. LTBP-3 expression was
more uniform with highest expression in HaCat keratinocytes, and lowest in
VA-13 cells. LTBP-4 was most prominent in embryonic lung fibroblasts and HaCat
keratinocytes and lowest levels of expression were detected in skin
fibroblasts and endothelial cells. Also, LTBP-4 was downregulated in VA-13
cells compared with the parental WI-38 cells, suggesting that malignant
transformation downregulates LTBP production in general (see also Taipale et
al., 1996; Koski et al.,
1999
).
Identification of a new LTBP-4 splice form
Amplification with LTBP-4 primers around the 3rd 8-Cys repeat
(primers LTBP-4.2) resulted in two products
(Fig. 2), the expected 486 bp
product and an additional 147 bp product. The small product was sequenced.
Nucleotides 3216-3554 encoding the 3rd 8-Cys repeat as well as
flanking regions were missing from this newly identified LTBP-4 splice form,
which we named LTBP-48-Cys3rd. The 147 bp fragment amplified
more readily and was always the prominent product seen in RT-PCR. All cell
lines studied expressed the LTBP-4
8-Cys3rd splice variant.
Since only the 3rd 8-Cys repeat is responsible for covalent binding
to small latent TGF-ß (Saharinen et al.,
1996
), the splice variant is
therefore unable to associate with small latent TGF-ß (Saharinen and
Keski-Oja, 2000
).
|
Besides LTBP-4, both LTBP-1 and -3 bind small latent TGF-ß readily
(Saharinen et al., 1996;
Gleizes et al., 1996
;
Saharinen and Keski-Oja,
2000
). No other splice forms
could be detected using the primers to amplify regions around the
3rd 8-Cys repeats in other LTBPs (see
Fig. 1). This suggests that
LTBP-4
8-Cys3rd is a unique splice form, although it cannot
be ruled out that for some reason the RT-PCR method would have failed in
amplifying a smaller product from the other LTBPs.
Genomic structure of LTBP-4 around the 3rd 8-Cys
repeat
To analyze the genomic structure of LTBP-4 around the 3rd 8-Cys
repeat, a PAC-clone containing LTBP-4 genomic sequences (Saharinen et al.,
1998) was used as a template
in PCR amplification. Primers LTBP-4.2 amplified a fragment of
4.5 kb
indicating the presence of
4 kb of intron sequences
(Fig. 3A). Primers (`3rd
8-Cys', see Materials and Methods) that amplify the 339 bp fragment absent
from LTBP-4
8-Cys3rd cDNA resulted in a
600 bp
amplification product from the PAC clone. This indicated that intron sequences
were also present between nucleotides 3216-3554 in LTBP-4 cDNA sequence.
|
The PCR products were cloned and partially sequenced. The positions of exon-intron boundaries as well as the flanking sequences are presented in Fig. 3B. The junctional sequences fit well with the consensus sequences at exon-intron boundaries in protein-coding genes. The 3rd 8-Cys repeat is encoded by two exons analogous to the exon-intron pattern in LTBP-2 and LTBP-3.
LTBP-4 mRNA expression analyses
RNase protection assays were used to analyze the expression of LTBP-4 mRNAs
in HT-1080, WI-38 and VA-13 cells. The probe was designed to result in
different sizes of protected fragments for mRNAs, whether or not they
contained the 3rd 8-Cys repeat (see Materials and Methods).
Fragment sizes of 331 bp and 225 bp corresponded to mRNAs containing or not
containing the 3rd 8-Cys repeat, respectively. Both mRNAs were
expressed in all three cell lines (Fig.
4A). In the transformed cell lines VA-13 and HT-1080, the
expression of both LTBP-4 forms was lower than in WI-38 cells. These results
are well in accordance with data obtained by RT-PCR.
|
Northern blotting of CCL-137 total RNA was performed with two different
probes to analyze the mRNA species detected by these probes for subsequent use
in Multiple Tissue Expression arrays (see below). `RPA' probe contains
sequences from the 3rd 8-Cys repeat as well as from the following
EGF-repeat and detects both LTBP-4 splice forms. The `3rd 8-Cys'
probe detects only mRNA species containing the 3rd 8-Cys repeat.
The sizes of the two mRNAs differ only by 339 bp and they migrate at the same
level in northern blots. Both probes detected the expected mRNA species of
5 kb (Fig. 4B).
Unexpectedly, a minor mRNA species of approximately 2 kb was also detected.
When the northern filters were washed under high stringency conditions it was
still detected, suggesting that it is specific and contains the 3rd
8-Cys repeat since both probes detected it. The identity of this minor mRNA
was not further investigated. Northern blotting analysis of WI-38 lung
fibroblasts gave similar results (data not shown).
Expression of LTBP-4 in different tissues
A multiple tissue expression array filter containing mRNAs from different
tissues, was probed with both `RPA' and `3rd 8-Cys' probes that detect total
LTBP-4 (including LTBP-48-Cys3rd), or specifically LTBP-4
containing the 3rd 8-Cys repeat, respectively. Radioactivity was
quantified using a phosphoimager. Each phosphoimager value obtained with one
probe, after subtracting the background, was divided by the average value from
all tissues. The expression pattern of LTBP-4 obtained using these two probes
correlated well with our previous results (Saharinen et al.,
1998
) revealing high
expression in aorta, heart ileum, jejunum, uterus and thyroid gland (data not
shown). The relative values obtained with the two different probes were very
similar in most tissues, indicating that the proportional expression of LTBP-4
forms were similar. However, in lymph node, bone marrow, peripheral blood
leukocyte, thymus, lung, kidney and liver the relative values differed
considerably, suggesting a different ratio of the two LTBP-4 isoforms compared
with most other tissues (data not shown). Those tissues were selected for
further analyses by RNase protection assay.
RNase protection assay from tissue mRNA
To analyze the expression of LTBP-48-Cys3rd and LTBP-4
containing the 8-Cys repeat in selected tissues, RNase protection assay was
first performed using the `RPA' probe as in
Fig. 4. From the commercial
tissue mRNAs, isolated from pooled tissue specimen, two additional protected
fragments of
240 bp and
90 bp were detected. This was probably due
to allelic variation at position 3685 leading to a mismatch with the probe and
degradation by RNase at this site (see below and
Fig. 6).
LTBP-4
8-Cys3rd was found to be expressed in all tissues
analyzed, but accurate quantification was not feasible (data not shown).
|
A new probe for RNase protection assay was designed (`RPA-2', see Materials
and Methods), and polyA+ RNAs from heart, lung, lymph node, bone
marrow, fetal liver, kidney and thymus were used in the assay. Three protected
fragments of different sizes were detected and found to correlate with full
length LTBP-4 (439 bp), LTBP-4 lacking the 13th EGF like repeat
(LTBP-4EGF-13, 313 bp) described previously (Saharinen et al.,
1998
), as well as
LTBP-4
8-Cys3rd (274 bp). A faint band corresponding to the
size of a splice form lacking both the EGF-like repeat and the 3rd
8-Cys repeat was consistently seen in lung mRNA samples, but quantification of
this minor fragment was not possible. Radioactivity of the protected fragments
was quantified using a phosphoimager (Fig.
5). LTBP-4
8-Cys3rd was found to be expressed in
all tissues analyzed, but the relative ratios of the full length LTBP-4 and
LTBP-4
8-Cys3rd were similar in tissues analyzed, not
supporting data obtained with multiple tissue expression arrays. RNase
protection assays from at least two different tissue mRNA preparations
indicated that LTBP-4 is very prominently expressed in the adult lung, which
was not clearly observed with the multiple tissue expression arrays. This
indicates that only a limited amount of information can be obtained from those
filters. More extended analyses of the expression of different splice variants
in various tissues will probably provide clues to the functions of the
different LTBP-4 forms.
|
LTBP-4 splice variant from HL-60 cells
HL-60 promyelocytic leukemia cells were included in the multiple tissue
expression arrays and originally showed a different relative ratio for the
LTBP-4 splice variants. It was therefore subjected to further analysis. RT-PCR
assay from HL-60 mRNA revealed another LTBP-4 splice variant, which lacks only
the second exon (nt 3483-3553) of the 3rd 8-Cys repeat
(Fig. 6). This exon codes for
24 amino acids, including the last cysteine residue of the 8-Cys repeat, and
its absence probably has an effect on the structure of the rest of the domain.
An LTBP-3 variant that lacks the first exon, coding the first seven cysteines,
of the fourth 8-Cys repeat has been described (Yin et al.,
1998). The variant identified
from HL-60 cells, named LTBP-4
24 8-Cys, was not observed from the
tissue mRNAs (data not shown), suggesting that it may be specific for cells of
haematopoietic origin or related to the malignant phenotype of HL-60 cells.
Sequencing of several RT-PCR products from separate experiments indicated that
in the heart or in the HL-60 cells there was cytosine or thymidine at position
3685, respectively. This, in addition to the sequence data from LTBP-4 cloning
papers (Saharinen et al.,
1998
; Giltay et al.,
1997
), suggests that there is
allelic variation at position 3685. The amino acid sequence does not differ
whether there is cytosine or thymidine at this position.
Computer search for human LTBP-4 sequences lacking the 3rd
8-Cys repeat
Full length LTBP-4 was used in a BLAST search (Altschul et al.,
1990) to obtain LTBP-4
sequences that lacked one or both of the exons coding for the 3rd
8-Cys repeat. Two sequences lacking the second exon were retrieved from
GenBank Human EST database (NCBI, NLM, NIH). They were cDNA sequences from
libraries made from bulk tissue of serous papillary carcinoma (GenBank
accession number: Aw118247) and normal germinal center B-cells (Aa832367).
This suggests that the splice form found in HL-60 cells is also present in
normal B-cells. cDNA sequences lacking the 3rd 8-Cys repeat were
more abundantly found and included clones from libraries made from total fetus
and adult brain (Ai039262, R87406, H46427, A1453016, H40747). Similar searches
with full length LTBP-1, -2 or -3 did not result in sequences lacking the
respective 8-Cys repeats, suggesting that this kind of splicing may be unique
to LTBP-4.
Expression of LTBP-4 fragments and immunoblotting from cell
conditioned medium
Since the expression of the full length recombinant LTBP-4 in cultured
cells is very inefficient (Saharinen et al.,
1998), we constructed
expression plasmids spanning the region from the 3rd 8-Cys repeat
to the end of the 4th 8-Cys repeat. pFull sequence is derived from
the published LTBP-4S sequence (Saharinen et al.,
1998
), whereas
p
8-Cys3rd and p
24 8-Cys plasmids lack sequences that
are missing from the newly identified splice variants,
LTBP-4
8-Cys3rd and LTBP-4
24 8-Cys, respectively. The
expression constructs were transfected into 293T human kidney epithelial cells
and aliquots of the conditioned media were subjected to immunoblotting
analyses as described in Materials and Methods. All constructs were expressed
and secreted to the conditioned media as detected by anti-HA antibodies
(Fig. 7). As expected, the
LTBP-4 antibody #28-3 made against the 3rd 8-Cys repeat did not
detect p
8-Cys3rd, but was able to detect p
24 8-Cys.
#33-4 antibody, which recognizes the 4th 8-Cys repeat was able to
react with all three constructs. These results suggest that cultured cells can
produce LTBP-4 fragments lacking parts of the 8-Cys repeat and secrete them
into the culture medium.
|
To demonstrate binding of full length LTBP-4 with TGFß1·LAP,
Saharinen et al. encountered problems with very low expression levels of the
LTBP-4 protein (Saharinen et al.,
1998). Also, smaller protein
fragments containing the TGF-ß binding domain showed lower binding
capacity to TGFß1·LAP than LTBP-1 fragments. We carried out
cotransfection experiments to demonstrate complex formation between the
mutated proteins and TGFß1·LAP as described previously (Saharinen
et al., 1998
). Our numerous
attempts proved to be constantly negative (data not shown; see also Saharinen
and Keski-Oja, 2000
).
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DISCUSSION |
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Latent TGF-ß complexes are integral parts of the extracellular
structures and, when activated, can regulate extracellular matrix composition
by increasing the synthesis of matrix components and decreasing protease
activity (Piek et al., 1999;
Taipale et al., 1998
). Matrix
association and activation of TGF-ß complexes form a finely tuned control
network for the maintenance of the organization of extracellular structures.
LTBPs function both as TGF-ß-binding proteins and as structural
components of the extracellular matrix (Taipale et al.,
1996
). The folding and
secretion of TGF-ß is dependent on simultaneously produced LTBP (Miyazono
et al., 1991
). LTBPs target
TGF-ß to extracellular structures for storage as well as for activation,
where LTBPs play a role as well (Flaumenhaft et al.,
1993
). A role for LTBPs as
components of the extracellular matrix 10 nm microfibrils as well as thicker
fibronectin rich fibers has been observed (M.
Hyytiäinen and J.K.-O., unpublished; Gibson et
al., 1995
; Taipale et al.,
1996
; Dallas et al.,
2000
). The existence of four
different LTBPs with only partially overlapping expression patterns suggests
important functions for LTBPs. LTBP-1 is mainly expressed in the heart,
placenta, lung, spleen, kidney and stomach, and LTBP-2 in lung, skeletal
muscle, liver and placenta (Kanzaki et al.,
1990
;
Morén et al.,
1994
; Tsuji et al.,
1990
). LTBP-3 (J.S. et al.,
unpublished) and -4 have similar expression patterns, predominantly found in
aorta, heart, small intestine and ovaries (Saharinen et al.,
1998
; Giltay et al.,
1997
). In fetal tissues, LTBP-1
and -2 are expressed more abundantly than LTBP-3 and -4 (reviewed by Saharinen
et al., 1999
).
We analyzed here the relative patterns of expression of LTBPs in cultured
fibroblasts of different origin as well as in umbilical vein endothelial cells
and HaCat keratinocytes by a semi-quantitative RT-PCR. We observed different
expression levels in cells from different tissues. This suggests differential
regulation of the LTBP/TGF-ß system in tissues, as well as possible
tissue-specific composition of the microfibrils. Fibrillins-1 and -2 are the
major components of the 10 nm microfibrils and show tissue specificity in
their expression levels (Sakai et al.,
1986; Zhang et al.,
1995
). Together with different
LTBPs and other components such as the microfibril associated glycoprotein
(MAGP, Gibson et al., 1998
),
the composition and function of microfibrils may be regulated in a tissue
specific manner.
In VA-13 cells (SV-40 virus-transformed WI-38 fibroblasts) the expression
of all four LTBPs was downregulated. This notion is in accordance with
previous reports indicating that malignant transformation can lead to
decreased LTBP levels (Koski et al.,
1999;
Eklöv et al.,
1993
; Mizoi et al.,
1993
). Our earlier results
indicate that VA-13 cells produce very little fibronectin matrix, which is
paralleled with a marked decrease in LTBP-1 fibers (Taipale et al.,
1996
). The current results
suggest that, in addition to LTBP-1, the other LTBPs are downregulated, and
that the regulation is at the level of transcription. Cancer cells have often
been found to produce aberrant amounts of TGF-ß. They also fail to
deposit TGF-ß complexes to their extracellular matrices, probably due to
decreased fibronectin matrix in cancer cells as well as decreased LTBP
production. This may be beneficial for tumor progression since the production
of soluble TGF-ß forms instead of tight LTBP-directed control may favor
various paracrine effects in tumors including stimulation of angiogenesis and
connective tissue formation.
LTBPs and fibrillins show structural variation. Proteins with different
N-terminal regions are commonly produced and they are thought to bind
extracellular matrix components with divergent affinities and possibly also
specificities. For example, the production of the two forms of LTBP-1 (the
longer protein having a 346 amino acid extension in the N-terminus) is
regulated by independent promoters (Koski et al.,
1999). Whether this is also
the case for the LTBP-4 isoforms with differing N-termini (Giltay et al.,
1997
; Saharinen et al.,
1998
) is still unclear.
Deletions or insertions of EGF-like domains are commonly found, and these
probably alter the structure and function of the produced proteins (see
Saharinen et al., 1999
; Sakai
et al., 1986
; Koli et al.,
2001
). EGF-like domains
participate in protein-protein interactions and via calcium binding also
provide stability to protein structures. Changes in the number of EGF-like
repeats alter the length of the molecule and possibly modulate its properties
in the association with microfibrils. Of the 34 known 8-Cys repeats found in
LTBPs and fibrillins only three are known to bind small latent TGF-ß
(Saharinen and Keski-Oja,
2000
). An LTBP-1 splice
variant that lacks a consensus heparin-binding domain has been suggested to be
less protease sensitive, changing the ability of this particular variant to be
released from the extracellular matrix (Michel et al.,
1998
;
Öklü et al.,
1998
).
We discovered here an LTBP-4 splice variant that lacks the TGF-ß
binding domain, namely the 3rd 8-Cys repeat
(LTBP-48-Cys3rd). This variant was produced by alternative
splicing over two exons. The exon-intron structure of LTBP-4 around this area
was similar to those of human LTBP-2 (Bashir et al.,
1996
) and LTBP-3 (J.S. et al.,
unpublished). In the mouse LTBP-3, a variant lacking most of the last 8-Cys
repeat has been described (Yin et al.,
1998
). Although no specific
function for the non-TGF-ß binding 8-Cys repeats have been suggested,
they are crucial to the structure of fibrillin-microfibrils (Lee et al.,
1991
). LTBP-1 and LTBP-3,
which also bind small latent TGF-ß via their respective 3rd
8-Cys repeats, were not found as similar splice variants in RT-PCR analyses.
The expression of LTBP-4 and LTBP-4
8-Cys3rd in cell lines
was analyzed by RNase protection assays and both forms were expressed, but
major differences in expression levels were not noted. Also,
LTBP-4
8-Cys3rd was expressed in a variety of normal human
tissues.
Regulation of the relative amounts of LTBP-48-Cys3rd and
LTBP-4 expressed by cells provides a novel level for the regulation of the
TGF-ß system. The existence of LTBP-4
8-Cys3rd, a
variant unable to bind small latent TGF-ß, is compatible with the idea of
LTBPs having functions not restricted to the TGF-ß system only. These
include the regulation of cell adhesion to fibronectin or chemotactic
properties, as described for LTBP-2 (M.
Hyytiäinen and J.K.-O., unpublished) and -1,
respectively (Kanzaki et al.,
1998
). Interestingly, the
coding sequence between the 3rd 8-Cys repeat and the previous
EGF-repeat in LTBP-4 is considerably longer than that in the other LTBPs and
contains proline-rich sequences. These regions are thought to provide
flexibility and protease sensitive sites to LTBP molecules. A proline-rich
region is also spliced out in the LTBP-4
8-Cys3rd, which may
lead to a more rigid structure and new functional properties.
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ACKNOWLEDGMENTS |
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