Departments of Virology and Pathology, Haartman Institute and Biomedicum Helsinki, University of Helsinki and Helsinki University Hospital, FIN-00014 Helsinki, Finland
* Author for correspondence (e-mail: jorma.keski-oja{at}helsinki.fi)
Accepted 10 June 2002
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Summary |
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Key words: LTBP, Latent TGF-ß, Alternative splicing
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Introduction |
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TGF-ßs are cytokine growth factors, which have various biological
functions, including regulation of cell growth and differentiation and
synthesis, degradation and remodeling of the ECM (reviewed in
Moses and Serra, 1996;
Roberts and Sporn, 1996
).
Small latent TGF-ß complexes contain the N-terminal pro-domain of
TGF-ß (illustrated in Fig.
2.). The prodomain, called the latency-associated propeptide
(LAP), is proteolytically cleaved from mature TGF-ß during secretion. It
is responsible for the latency of the mature growth factor by remaining
non-covalently associated with the complex. Most cell lines secrete
TGF-ßLAP in large latent complexes containing LTBPs. The small
latent TGF-ß complexes are attached by disulfide bonding of LAP-part to
the third 8-Cys repeat of LTBPs, forming large latent complexes
(Saharinen et al., 1996
;
Saharinen and Keski-Oja,
2000
). The third 8-Cys repeats of LTBP-1 and -3 are able to
associate efficiently with the propeptides of TGF-ß1, -2 and -3, whereas
LTBP-4 has weaker binding capacity and complex formation of LTBP-2 with any of
the TGF-ßs is negligible (Saharinen
and Keski-Oja, 2000
). Interestingly, a novel
non-TGF-ß-binding splice variant lacking the third 8-Cys repeat of LTBP-4
has been identified (Koli et al.,
2001a
).
|
After secretion, LTBP-1 targets TGF-ß1 to the extracellular matrix
(Taipale et al., 1994). Both
the N-terminal and C-terminal regions of LTBP-1 seem to mediate its
association with the ECM (Unsöld et
al., 2001
). LTBP-1L is an N-terminally extended form of LTBP-1,
which is capable of binding more efficiently to the constituents of the ECM
(Olofsson et al., 1995
). LTBPs
can be released from the matrix as truncated forms by various proteinases,
including plasmin and elastases (Taipale
et al., 1994
; Taipale et al.,
1995
; Hyytiäinen et al.,
1998
; Saharinen et al.,
1998
). The protease-sensitive sites of LTBP-1 and -2 have been
localized to the proline-rich `hinge' regions in their N-terminus. ECM is
possibly a storage place, from where latent TGF-ß can be released and
activated rapidly when needed. The importance of LTBPs in the regulation and
targeting of TGF-ß activity is emphasized by the fact that several
transformed cells show decreased production and secretion of LTBPs
(Taipale et al., 1996
;
Dallas et al., 1994
;
Koski et al., 1999
).
Immunohistochemical analysis of prostatic carcinoma indicated that TGF-ß1
is produced without associating with LTBP-1
(Eklöv et al., 1993
). The
lack of LTBP-1 expression in human gastrointestinal carcinomas seems to result
in the retention of TGF-ß1 inside the cells
(Mizoi et al., 1993
).
The purpose of this study was to analyze the expression and secretion of LTBP-3. For this reason we isolated the cDNA of human LTBP-3 and generated polyclonal antibodies against a large recombinant fragment of the protein. Human LTBP-3 is expressed in various human tissues, and its secretion is connected with the expression of TGF-ß1. Immunoblotting analysis indicated that overexpressed hLTBP-3 was secreted efficiently only if TGF-ß1 was simultaneously co-expressed. Moreover, in human osteosarcoma cell lines, the large latent complexes of hLTBP-3 and TGF-ß1LAP appeared to be the most prominent secreted forms of hLTBP-3.
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Materials and Methods |
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DNA cloning and sequence analysis
cDNA for human LTBP-3 was cloned from gt10 human heart
cDNA library (Clontech). The phage library was screened using
[
-32P]dCTP-labeled EST clone 49899 (GenBank accession number
H15208) as an initial probe. cDNA from positive, overlapping phage clones
(Fig. 1A) was extracted and
cloned into pBluescript II KS (+) cloning vector (Stratagene, La Jolla, CA)
and sequenced by ABI Prism 310 Genetic Analyzer (Perkin Elmer Instruments,
Shelton, CT). The 5' coding region surrounding the translation
initiation codon was obtained using the reverse transcriptase polymerase chain
reaction (RT-PCR) method. The oligo(dT)12-18 primer (Invitrogen,
Carlsbad, CA) was extended by Superscript II reverse transcriptase
(Invitrogen) according to the manufacturer's instructions. The cDNA was
further amplified by using a GC-RICH PCR System (Roche), which is designed
specially for amplification of GC-rich templates. The forward primer
5'-CCC CTC TAC TCC CTT CGG GCG-3' was designed using the genomic
sequence of human LTBP-3 (see below) as template, and the reverse
primer 5'-CTG CTT GGG CAG AGT GTC CTG AAA G-3' was obtained from
the cloned cDNA sequence of hLTBP-3. Finally, the contig was
assembled with Gap4 program in Staden software package
(Bonfield et al., 1995
).
|
The genomic clones containing human LTBP-3 gene were obtained by screening genomic PAC (Genome Systems, St Louis, MO) and lambda FIX II (Stratagene) libraries with PCR-derived probes corresponding to nucleotides 1951 to 1284 and from 385 to 539 of LTBP-3 cDNA, respectively. The exon-intron organization of hLTBP-3 gene was determined by cycle sequencing of the clones with primers specific for hLTBP-3. The sizes of the larger introns were determined by PCR and verified by sequencing.
To generate a phylogenetic tree, the amino-acid sequences of different LTBPs were derived from GenBank and aligned using the multiple alignment program ClustalX using Gonnet PAM 250 comparison matrix. The generated alignment was displayed by TREEVIEW (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).
cDNA expression constructs
A full-length human LTBP-3 cDNA expression construct named `phL3'
was constructed in a pcDNA3 eukaryotic expression vector (Invitrogen) from
overlapping cDNA clones CL44 and CL52 (Fig.
1A). The 5' coding region of hLTBP-3 was obtained
from the previously mentioned RT-PCR product (see DNA cloning and sequence
analysis). The cloning was accomplished in three individual steps using
internal recognition sites of ApaI, PshAI and XcmI
restriction endonucleases (Fig.
1A) and endonucleases that recognize the vector polylinkers.
The `phL3+ATG' cDNA expression construct contains an additional N-terminal extension of 42 bp compared with phL3 (Fig. 1B). It was constructed using the recognition site of AscI restriction endonuclease, which digests 34 bp upstream of the translation initiation site of phL3+ATG
For baculoviral expression, a cDNA fragment corresponding to nucleotides
2097-3459 of human LTBP-3 was amplified by PCR and cloned into a
eukaryotic secretory expression vector pSignal
(Saharinen et al., 1996). The
construct contained a HA-epitope tag of pSignal and an additional
six-histidine tag for purification and immunoblotting of the recombinant
protein, respectively. The cDNA construct was finally cloned from pSignal into
the pACGP67A baculovirus transfer vector (Pharmingen, San Diego, CA) as a
whole BamHI-XbaI restriction fragment and named
`phL3/699-1153'. The protein domains coded by phL3/699-1153 cDNA are shown in
Fig. 2A.
cDNA construct containing the full-length human pTGF-ß1 has been
described previously (Saharinen et al.,
1996).
Northern hybridization analysis
Total RNA was extracted from different cell lines by commercial RNeasy kit
(Qiagen, Hilden, Germany). Total RNA (7 µg) was electrophoresed on a 0.8%
formaldehyde-agarose gel and transferred to Hybond-N nylon filter (Amersham
Pharmacia Biotech). Commercial Human Multitissue Northern Blots were purchased
from Clontech. The BseRI-BsmI restriction fragment
corresponding to nucleotides from 2783 to 3103 or CL44 (see
Fig. 1A) was used as a cDNA
probe for hLTBP-3. The probe was labeled with
[-32P]dCTP by random priming. The hybridizations were
performed using Express Hyb hybridization solution (Clontech) according to the
manufacturer's instructions, and the filters were washed under high stringency
conditions followed by autoradiography. Loadings were controlled by
hybridizations with radioactively labeled ß-actin or
G3PDH cDNA probes.
Reverse transcription polymerase chain reaction (RT-PCR)
RT-PCR was carried out using as the templates eight normalized,
first-strand cDNA preparations from polyA+ RNA extracted from human
heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas (MTC
Panel, Clontech). The cDNAs were amplified using the GC-RICH PCR System and
the following primers: Fw 5'-CTA CCG CTG TGC CTG CAC-3' and Rev
5'-CAC ACT CGC AGC GGT AGG AG-3'. The primers were designed to
amplify a 154 bp cDNA product covering a part of the additional exon of the
splice variant and the 25th exon of hLTBP-3 in order to detect
possible genomic contamination in cDNA preparations. Primers specific for
abundantly expressed G3PDH were used to amplify a 983 bp product in
order to control the normalization of the cDNA panel. G3PDH was
amplified for only 22 cycles to obtain quantifiable results.
RT-PCR products were subjected to Southern hybridization analysis. An
oligonucleotide (5'-GAA GAG ATG GGA CGT GGA CGT GGA CGA GTG CCA
G-3') specific for the additional exon of the splice variant was labeled
by [-32P]ATP and hybridized with RT-PCR products, which were
immobilized to a Hybond-N nylon filter (Amersham Pharmacia Biotech).
Hybridizations were carried out using Express Hyb hybridization solution, and
the signals were detected by autoradiography.
Cell culture and transient transfection
WI-38 human embryonic lung fibroblasts (CCL-75, American Type Culture
Collection, Rockville, MD) and SV-40 virus-transformed WI-38/VA13 fibroblasts
(CCL-75.1, ATCC) were cultured in Minimal Essential Medium (MEM). Human MG-63
(CRL-1427, ATCC), G292 (CRL-1432, ATCC), Saos-2 (HTB-85, ATCC), and U-20S
(HTB-96, ATCC) osteosarcoma cells and human HaCaT keratinocytes
(Boukamp et al., 1988) were
maintained in Dulbeccos's Modified Eagle's Medium (D-MEM). All culture media
were supplemented with 10% heat-inactivated fetal calf serum (Gibco,
Invitrogen Corporation), 100 IU/ml penicillin and 50 µg/ml streptomycin.
Primary human osteoblasts, NHOst (CC-2538, Clonetics, Walkersville, MD) were
cultured in Osteoblast Growth Medium (Clonetics) and used within passages 7-9.
Spodoptera frugiperda (Sf-9) insect cells (Invitrogen) were grown in
TNM-FH insect medium (Pharmingen) and High Five insect cells (Invitrogen) in
Express Five SFM (Gibco, Invitrogen Corporation) supplemented with 100 IU/ml
penicillin and 50 µg/ml streptomycin.
Cells to be transfected were cultured in 35 mm diameter plates. Upon reaching 50-80% confluence, they were transfected with 1 µg of cDNA expression plasmid using FuGENE 6 transfection reagent (Roche). Co-transfections were carried out using a total of 2 µg of cDNAs. Six hours after transfection the cells were changed to fresh, serum-free culture medium.
Production of recombinant human LTBP-3 in High Five insect cells
Sf-9 insect cells were co-transfected with BaculoGold DNA (Pharmingen) and
recombinant phL3/699-1153 baculovirus transfer vector (see cDNA expression
constructs). The recombinant virus was amplified three times to obtain high
titer virus stocks. The virus stocks were subsequently used to infect High
Five insect cell cultures in order to produce large amounts of recombinant
hL3/699-1153 protein.
Purification of hL3/699-1153 recombinant protein
Serum-free conditioned medium from High Five insect cells was collected 3
days after infection and precipitated with 30%
(NH4)2SO4 at room temperature. The
precipitate was collected by centrifugation and redissolved in 50 mM Tris-HCl
buffer, pH 7.5, containing 0.3 M NaCl.
TALON Metal Affinity Resin (Clontech) was packed in column HR 5/2 (Amersham Pharmacia Biotech) for affinity purification of the recombinant protein by ÄKTAexplorer (Amersham Pharmacia Biotech) FPLC system. The bound proteins were eluted from the column by 50 mM Tris-HCl buffer, pH 7.5, containing 0.3 M NaCl and 0.1 M imidazole. The purified fractions were subjected to buffer change with a Fast Desalting Column HR10/10 (Amersham Pharmacia Biotech) equilibrated with phosphate-buffered saline (PBS, 0.14 M NaCl in 10 mM phosphate buffer, pH 7.4). The purity of hL3/699-1153 protein in different fractions was analyzed by Coomassie blue and silver staining. Total protein concentrations were finally quantified by Micro BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA).
Preparation of antibodies and immunoblotting
Polyclonal antibodies (Ab-hL3/2925) against recombinant hL3/699-1153
protein were raised in rabbits (Sigma-Genosys, Cambridge, UK). The rabbits
were immunized with 20 µg of purified
keyhole-limpet-hemocyanin-coupled hL3/699-1153 recombinant fragment in
Freund's complete adjuvant. Seven subsequent boosters in Freund's incomplete
adjuvant were given at 2 week intervals. Antibodies were purified by affinity
chromatography from whole serum. Recombinant hL3/699-1153 fragment was coupled
to HiTrap NHS-activated affinity columns (Amerham Pharmacia Biotech). The
antiserum was passed through the column for 1 hour and washed with PBS. Bound
proteins were eluted with 1 mM CH3COOH, 140 mM NaCl and neutralized
immediately with 1:10 volume of 1 M Tris-HCl buffer, pH 8.0. Affinity-purified
antibodies were finally subjected to buffer change with Fast Desalting Column
HR10/10 (Amersham Pharmacia Biotech) equilibrated with PBS. The resulting
antibody recognized LTBP-3 specifically, as determined by immunoblotting
assays using samples containing high amounts of LTBP-1, which Ab-hL3/2925 did
not recognize.
Proteins were separated by SDS-PAGE under nonreducing conditions using
commercial 4-15% gradient or 7.5% Tris-HCl gels (BioRad, Hercules, CA). When
necessary, conditioned cell culture medium was concentrated five-fold using
Microcon YM-30 centrifugal filter devices (Millipore, Bedford, MASS).
Electrophoretically separated proteins were transferred to nitrocellulose
membranes by semi-dry blotting, and subsequently treated with 5% nonfat milk
in PBS/Triton X-100 to saturate non-specific protein-binding sites. The
saturated membranes were incubated with affinity-purified Ab-hL3/2925 and
subsequently, after washing, with biotinylated anti-rabbit IgG antibodies
(DAKO A/S, Glostrup, Denmark) in TBS (Tris-buffered saline) buffer containing
0.05% Tween-20 and 5% bovine serum albumin (BSA). The bound antibodies were
finally detected using peroxidase-conjugated streptavidin and enhanced
chemiluminescence Western blotting detection system (Amersham Pharmacia
Biotech). Immunodetection using affinity-purified rabbit antihuman
TGF-ß1LAP (680) and anti-LTBP-1 (ab39) antibodies was carried out
as described previously (Taipale et al.,
1994).
Immunofluorescence analysis
For immunofluorescence analysis, the cells were plated on glass coverslips
and transfected the next day as described before (Cell culture and transient
transfection). After three days the cells were fixed with 3% paraformaldehyde
in PBS, permeabilized with 0.5% Nonidet P-40 in PBS and subsequently treated
with 3% BSA in PBS for 10 minutes. Pre-tested dilutions of affinity-purified
anti-hLTBP-3 (Ab-hL3/2925) were then applied to the cells in PBS containing 3%
BSA and incubated at room temperature for 1 hour. Binding of the primary
antibody was detected with biotinylated anti-rabbit IgG antibodies (DAKO A/S)
and lissamine rhodamine-conjugated streptavidin (Jackson Immuno Research
Laboratories, West Groove, PA). The coverslips were finally washed with PBS
and mounted on glass slides using Vectashield anti-fading reagent (Vector
Laboratories, Inc., Burlingame, CA).
Metabolic labeling and immunoprecipitation
For immunoprecipitation, the cells were seeded on 100 mm diameter dishes
and transiently transfected. The next day they were changed to serum-free
medium depleted of cysteine and methionine for 2 hours. Subsequently, 50
µCi of Easy Taq Express-[35S] protein labeling mix was added in
the same medium, and the cells were cultured for 24 hours.
After radiolabeling, the medium was collected, and the cells were lysed with 20 mM Tris-HCl buffer, pH 8.0, containing 120 mM NaCl, 0.5% Nonidet P-40 and protease inhibitors. The samples were incubated with anti-hLTBP-3 (Ab-hL3/2925) antibodies followed by precipitation of immune complexes with protein A sepharose beads (Sigma). Pre-adsorption with rabbit preimmune serum was used in order to reduce non-specific background. The immune complexes were separated by SDS-PAGE using 4-15% gradient gels under reducing conditions and detected by fluorography.
Proteinase digestion of hLTBP-3
COS-7 cells were transiently transfected with phL3 and pTGF-ß1
expression constructs (Cell culture and transient transfection). The
serum-free culture medium was collected and subjected to proteinase treatment
with plasmin (3, 10 and 30 µg/ml) or elastase (0.1, 0.5 and 1.0 µg/ml)
at 37°C for 1 hour. The samples were subsequently analyzed by
immunoblotting using the Ab-hL3/2955 antibody.
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Results |
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The genomic structure of the hLTBP-3 gene was determined by direct
sequencing of human genomic PAC and lambda FIXII clones and by PCR
amplification using gene-specific primers. The human LTBP-3 gene
spans a genomic region of about 19.8 kb and consists of 28 exons including an
exon that is alternatively spliced (Table
1). All the internal exon-intron junctions of hLTBP-3 are
defined by canonical 5' splice donor and 3' splice acceptor
sequences (Table 1).
Chromosomal localization of human LTBP-3 gene has been determined to
be 11q12 (Li et al.,
1995).
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Deduced primary protein structure of hLTBP-3
The translated open reading frame of hLTBP-3 encodes a polypeptide of 1256
amino-acid residues with a calculated molecular weight of 134 kDa and
estimated pI of 5.71. The signal peptidase cleavage site exists probably
between glycines at position 43-44, as was predicted by SignalP
(Nielsen et al., 1997). Human
LTBP-3 has 5 potential N-glycosylation sites. It consists structurally mainly
of two types of cysteine-rich motifs, EGF-like and 8-Cys repeats, which are
characteristic of members of the LTBP family
(Fig. 2A). Also another type of
8-Cys repeat, often known as a hybrid domain, is present in hLTBP-3. Most of
the EGF-like repeats of hLTBP-3 are of the calcium-binding type and contain a
characteristic consensus sequence
[D/N]X[D/N][E/Q]Xn[D/N]Xm[Y/F].
Comparison of the translated protein sequence with that of mouse Ltbp-3
confirmed that the cloned ORF codes for a human homologue of LTBP-3 protein.
It is 87% similar to mouse Ltbp-3, but only 47-32% similar to human LTBP-1,
LTBP-4 or LTBP-2 (Fig. 2B).
Expression of hLTBP-3 mRNA in different human tissues and
cell lines
Expression of hLTBP-3 was analyzed using northern blots containing
polyA+ and total RNA from various human tissues and cells. A
BseRI-BsmI restriction fragment or a cDNA fragment coding
for CL44 was used as a gene-specific probe. A single transcript species of
about 4.6 kb was detectable in various human tissues
(Fig. 3A). The transcript fits
quite well in size to the ORF of human LTBP-3. On the basis of the
hybridization results of the tissue northern blots, human LTBP-3 is
expressed prominently in heart, skeletal muscle, prostate and ovaries
(Fig. 3A). Significant
expression was detected also in testis and the small intestine. The result was
confirmed by another hybridization analysis using a similar Northern blot and
the same BseRI-BsmI restriction fragment (nt 2738-3103) as a
radioactively labeled probe.
Since the expression of mouse Ltbp-3 mRNA is induced during mouse
osteoblast differentiation (Yin et al.,
1995), the mRNA pattern of human LTBP-3 was determined
from some osteosarcoma cell lines and in osteoblasts. Of the analyzed cell
lines, human primary osteoblasts and G-292 and MG-63 human osteosarcoma cells
showed the highest expression of hLTBP-3 mRNA
(Fig. 3B). WI-38 human
embryonic fibroblasts were found to express hLTBP-3 mRNA at high
levels, whereas WI-38/VA13 fibroblasts, which are SV-40-virus-transformed
counterparts of normal WI-38 fibroblasts, showed very little expression
(Fig. 3B). Similar results of
the expression levels of hLTBP-3 in normal and transformed
fibroblasts have been obtained by semi-quantitative RT-PCR
(Koli et al., 2001a
). In
general, malignant transformation has been observed to result in
downregulation of the transcription, mRNA expression and protein levels of the
different LTBPs (Taipale et al.,
1996
; Koski et al.,
1999
; Koli et al.,
2001a
). Furthermore, an additional, slightly larger transcript was
detected in osteoblasts, MG-63 osteosarcoma cells and in WI-38 lung
fibroblasts. The size of this transcript is similar, but distinct from that of
LTBP-1S, and its general mRNA expression pattern in those cells is
clearly distinct from that of LTBP-1 (P. Vehviläinen, M.H. and
J.K-O., unpublished). For example, in WI-38 fibroblasts the expression of
LTBP-1 was very strong, whereas we detected only a trace of the large
hLTBP-3 transcript. By contrast, osteoblasts expressed slightly lower
levels of LTBP-1 than WI-38 fibroblasts but much higher levels of the
large hLTBP-3 transcript than WI-38 cells. In summary, the migration
of these two transcripts is different, and the differences in their expression
levels further indicate that they are distinct.
Northern blot hybridization analyses of LTBP-1 and -2
revealed the presence of two transcripts
(Moren et al., 1994;
Kanzaki et al., 1990
). The
different transcripts of LTBP-1 possess N-terminally distinct regions
(Olofsson et al., 1995
).
Molecular cloning of LTBP-4 also revealed an N-terminally extended
cDNA clone (Saharinen et al.,
1998
). The larger hLTBP-3 transcript could thus
correspond to an N-terminally extended transcript or some other alternative
splice variant of hLTBP-3.
Identification of an alternative splice variant of hLTBP-3
Screening of the cDNA library revealed a diverse cDNA clone CL21, which
contains an additional coding sequence compared with hLTBP-3. A
similar extension was detected in EST 49899 cDNA clone
(Fig. 1A). The variant splice
form named hL3+EGF contains an additional 141 bp exon, which codes
for an additional EGF-like repeat of the calcium-binding type
(Fig. 2A). hL3+EGF is
alternatively spliced between exons, which code for the 13th and 14th EGF-like
repeats of hLTBP-3 (Fig. 2A).
Inspection of the genomic sequence surrounding the alternative splice site
revealed the presence of characteristic splice donor and acceptor sites
(Table 1).
RT-PCR using cDNA prepared from human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas was performed to characterize the expression of hL3+EGF. Oligonucleotides for RT-PCR were designed to amplify the region encompassing the 13th EGF-like repeat and the novel EGF-like repeat of hL3+EGF. RT-PCR generated a 154 bp product, which was confirmed as hL3+EGF by Southern hybridization analysis using an internal oligonucleotide as a probe. hL3+EGF was found to be expressed in other studied tissues except skeletal muscle (Fig. 3C). Strong expression of the splice variant was detected in the pancreas and liver. In order to verify the normalization of the cDNA panel, the samples were amplified with oligonucleotides specific for abundantly expressed glyceraldehyde-3-phosphate dehydrogenase (G3-PDH; Fig. 3C). In order to determine whether this splice variant is expressed in other species as well, we carried out a Blast search (http://www.ncbi.nlm.nih.gov/BLAST/) on the mouse EST database against the mouse cDNA sequence coding for the 13th EGF-like repeat and the subsequent 8-Cys repeat. With this approach, we were able to identify two EST clones (Genbank accession numbers BI904087 and BI409427) containing sequences homologous to hL3+EGF, showing that the alternative splice variant is also expressed in other species.
Secretion of LTBP-3 requires co-expression of TGF-ß in COS-7
cells
To analyze the protein expression of hLTBP-3, rabbit polyclonal antibodies
were generated against human hL3/699-1153 recombinant protein coding for the
amino acids 699-1153 (Fig. 2A).
Specific antibodies were isolated from rabbit antiserum by affinity columns
containing the antigenic protein. The specificity of the isolated antibodies
was subsequently tested by immunofluorescence staining of transfected COS-7
cells and immunoblotting. The fluorescence signal of the translation product
of the transfected human LTBP-3 expression construct was detected
mainly inside the cells, probably to some extent in the Golgi
(Fig. 4A).
|
Immunoblot analysis was used to assess whether human LTBP-3 is released
into conditioned cell culture medium of COS-7 cells. Two expression vectors
containing hLTBP-3 cDNAs with the different translation initiation
codons (Fig. 1B) were
transiently transfected into COS-7 cells. A phL3/699-1153 cDNA construct in an
eukaryotic expression vector was used as a positive control. A recombinant
phL3/699-1153 cDNA construct was translated into a protein of 82 kDa.
Some higher molecular weight forms, which most probably correspond to
multimeric forms of it, were also detected
(Fig. 4B). Immunoblot analysis
did not reveal any specific signal for the full-length human LTBP-3 protein in
the conditioned medium of COS-7 cells unless an expression vector encoding for
human TGF-ß1 cDNA was co-transfected. Co-expression of hLTBP-3
and TGF-ß1 in COS-7 cells resulted in the secretion of high molecular
weight complexes of
240 kDa (Fig.
4B). No such complexes were detected if COS-7 cells were
transfected with pTGF-ß1 only (Fig.
4B) or if the cells overexpressing human LTBP-3 were treated with
5 ng/ml of TGF-ß1 (data not shown). In addition, co-transfection of
pTGF-ß1 cDNA increased the secretion of phL3/699-1153 protein as well as
its complexed forms in COS-7 cells. Both hLTBP-3 expression
constructs containing the different translation initiation codons were
translated into hLTBP-3 (Fig.
4B). Therefore, no definite proof of the functional translation
initiation site of human LTBP-3 was found.
The high molecular weight forms of the transfected COS-7 cells were
subsequently characterized by immunoblotting using an affinity-purified human
TGF-ß1LAP antibody, which recognizes the LAP part of TGF-ß1
(Taipale et al., 1995). An
immunoreactive signal corresponding to the
240 kDa band was detected in
the cell-conditioned medium of COS-7 cells co-expressing hLTBP-3 and
TGF-ß1 (Fig. 4C; arrow).
The high molecular weight forms were thus identified as large latent complexes
of human LTBP-3 and ß1LAP. Complex formation was also detected in
cells overexpressing the shorter phL3/699-1153 control plasmid and
pTGF-ß1 (Fig. 4C;
arrowhead).
Immunoprecipitation was used to confirm the composition of the large
240 kDa complex. COS-7 cells overexpressing hLTBP-3 and TGF-ß1 were
metabolically labeled, and the conditioned medium was collected for 24 hours.
The samples were immunoprecipitated with Ab-hL3/2925 antibody and analyzed by
4-15% gradient SDS-PAGE under reducing conditions. hLTBP-3 migrated at
180 kDa. Two lower molecular weight bands corresponding to
ß1LAP and unprocessed TGF-ß1LAP monomers were detected.
Their mobility was comparable to that observed by immunoblotting. The
unprocessed TGF-ß1LAP form results from overexpression, when the
furin cleavage required for converting TGF-ß1LAP precursor into a
mature growth factor does occur efficiently
(Dubois et al., 1995
).
Together, these findings indicate that overexpressed human LTBP-3
mRNA is translated into a protein, whose secretion seems to depend strictly on
the expression of and complex formation with TGF-ß1.
Secretion of hLTBP-3 from human osteosarcoma cells
As various human osteosarcoma cell lines express human LTBP-3 mRNA
(Fig. 3B), we assessed whether
hLTBP-3 protein is expressed and secreted by the cells at similar levels. The
conditioned medium of MG-63, U-2OS and Saos-2 human osteosarcoma cell lines
was subjected to immunoblotting analysis for hLTBP-3. Untransfected COS-7
cells and COS-7 cells overexpressing hLTBP-3 and TGF-ß1 were used as
negative and positive controls, respectively. A single, strongly
immunoreactive band of about 240 kDa was detected in the medium of MG-63 and
U-2OS human osteosarcoma cells, whereas Saos-2 osteosarcoma cells showed no
secretion of hLTBP-3 protein (Fig.
5A). The 240 kDa band co-migrated with the positive control. The
expression levels of hLTBP-3 protein corresponded well with its mRNA
expression pattern in the analyzed osteosarcoma cells
(Fig. 3B).
|
To determine whether the anti-ß1LAP antibody recognizes similar
endogenously expressed secreted complexes, we carried out an immunoblot
analysis of the serum-free conditioned medium from MG-63, U-2OS and Saos-2
osteosarcoma cells on the same, recycled filter. Secretion of two complexes of
slightly different molecular weights was detected in U-2OS osteosarcoma cells
(Fig. 5B; two arrows). The
slightly larger complex 260 kDa, which was more prominent in U-2OS cells,
was not detectable in the medium of MG-63 osteosarcoma cells. MG-63 cells
secreted only a complex of
240 kDa. Saos-2 cells did not secrete such
complexes. Instead, a smaller
90 kDa band corresponding to the migration
of ß1LAP dimer was detected with long exposure (data not
shown).
Since LTBP-1 associates with TGF-ß1LAP, we carried out a similar
immunoblotting analysis using an antibody against human LTBP-1 (Ab39).
Distinct forms of LTBP-1 were detected in U-2OS cells
(Fig. 5C). The smaller forms
most probably represent the free forms of LTBP-1
(Kanzaki et al., 1990),
whereas the larger form, which corresponds to the size of the larger
260
kDa band detected with anti-ß1LAP antibody
(Fig. 5B), represents the large
latent complex of LTBP-1 and TGF-ß1LAP. The molecular size of the
complex is similar to that secreted by human erythroleukemia cells
(Miyazono et al., 1991
). No
such complex was detected in MG-63 osteosarcoma cells. The secreted
TGF-ß1 seems to be exclusively complexed with LTBP-3 in MG-63
osteosarcoma cells, even though LTBP-1 is produced by these cells. Therefore,
the 240 kDa immunoreactive band detected in MG-63- and U-2OS-conditioned media
is likely to represent the large latent complex of human LTBP-3 and
TGF-ß1LAP, which seems to be the most prominent secreted form of
LTBP-3 in these osteosarcoma cells.
Proteolytic processing of hLTBP-3
LTBP-2 is proteolytically cleaved by plasmin and elastases from a
proline-rich, N-terminal hinge region located between the hybrid domain and
the long stretch of EGF-like repeats
(Hyytiäinen et al., 1998)
(Fig. 2A). LTBP-1 from
fibroblast-conditioned medium is processed into similar 120-140 kDa fragments
with serine proteases (Taipale et al.,
1994
; Taipale et al.,
1995
). Proteinase processing of LTBP-4 is also evident but it is
different from that of LTBP-1 and -2. LTBP-4 is processed to 230-220 kDa
fragments (Saharinen et al.,
1998
). To assess the susceptibility of hLTBP-3 for proteolytic
processing, the conditioned medium of COS-7 cells overexpressing the large
latent complex of hLTBP-3 and TGF-ß1LAP was subjected to digestion
with plasmin and leukocyte elastase. The molecular weight of the complex was
reduced from
240 kDa to
230-220 kDa when increasing concentrations
of plasmin were used (Fig. 6).
Leukocyte elastase was unexpectedly not able to cleave hLTBP-3
(Fig. 6). It therefore seems
that hLTBP-3 is not proteolytically cleaved at the proline-rich region
analogous to LTBP-1 and -2. The reduction of the molecular mass of the large
latent complex of hLTBP-3, instead, resembles that of LTBP-4.
|
![]() |
Discussion |
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---|
The 5' coding end of the human isoform has very high GC content, and
it contains two putative translation initiation codons separated by a 42 bp
region of GC-rich sequence. According to Kozak there are two positions around
the AUG initiator codon that are critical for function: 97% of vertebrate
mRNAs have a purine in position -3, and 46% have a G in position +4
(Kozak, 1991). The nucleotide
sequence CCTGAGAUGC surrounding the putative downstream initiator
codon of human LTBP-3 agrees with Kozak's GCCA/GCCAUGG
consensus sequence for initiation of translation, whereas the upstream
sequence CCCCGGAUGC does not. However, in most vertebrate mRNAs,
the first upstream AUG is commonly used as the ultimate initiation site.
Translation initiation efficiency of human LTBP-3 was studied in COS-7 cells
co-expressing hLTBP-3 and TGF-ß1. Both the upstream and downstream AUG
codons served as translation start codons when hLTBP-3 was overexpressed.
Therefore, either one of them may drive the initiation of the translation of
hLTBP-3 protein.
Structural variations have been described for all known LTBPs (reviewed by
Koli et al., 2001b). The
splice variants generally either contain additional or lack conserved protein
domains. Independent promoters regulate N-terminally distinct forms of LTBP-1,
which provides a means for their cell-type specific expression
(Koski et al., 1999
).
N-terminally extended forms may associate more efficiently with the ECM, as
has been shown for LTBP-1L (Olofsson et
al., 1995
). A splice variant lacking the third 8-Cys repeat is
unique to human LTBP-4 and may affect TGF-ß deposition into tissues
(Koli et al., 2001a
). Splice
variants of the hinge region may, in turn, provide protease resistance
(Gong et al., 1998
;
Michel et al., 1998
). We
identified here two cDNA clones, which contain an additional EGF-like repeat
in the C-terminus between the two C-terminal 8-Cys repeats of hLTBP-3. Tissue
distribution of hL3+EGF mRNA is different from that of
hLTBP-3, suggesting its functional specificity. In contrast to
hLTBP-3, hL3+EGF is not expressed in skeletal
muscle but is highly expressed in the pancreas. EGF-like repeats have been
found in many extracellular and cell-surface proteins, and they are considered
to be important in mediating protein-protein interactions (reviewed in
Davis, 1990
). The additional
EGF-like repeat of the hL3+EGF variant is located next to the homologous
region of LTBP-1S, which has recently been proposed to interact with the
extracellular matrix of human fibroblasts
(Unsöld et al., 2001
)
(Fig. 2A). Therefore, an
alternative hL3+EGF form may have altered affinity for the ECM.
LTBPs are essential for the efficient secretion and correct folding of
TGF-ßs (Miyazono et al.,
1991; Miyazono et al.,
1992
). In a recent study we found that overexpressed third 8-Cys
repeats of LTBP-1 and LTBP-3 associate covalently with all TGF-ßLAP
isoforms, whereas LTBP-4 has a binding capacity for TGF-ß1LAP only,
and LTBP-2 or fibrillins do not bind to any of the TGF-ßs
(Saharinen and Keski-Oja,
2000
). Different LTBPs may function and mediate the effects of
TGF-ßs in a tissue-specific manner (reviewed by
Koli et al., 2001b
). This
finding is supported by the fact that in all the osteosarcoma cells we
studied, the major fraction of human LTBP-3 was secreted into the large latent
complexes. Bone tissue is known to be a rich source of TGF-ßs, which are
involved in bone remodeling (reviewed in
Bonewald and Mundy, 1990
). A
recent report describes bone abnormalities in LTBP-3-null mice, suggesting
that hLTBP-3 has an essential role in regulating TGF-ß growth factor
deposition and availability in bone
(Dabovic et al., 2002
). Earlier
studies have, however, shown that certain bone cells and human glioblastoma
cells secrete TGF-ßs exclusively as free forms lacking LTBPs
(Olofsson et al., 1992
;
Dallas et al., 1994
). We found
that Saos-2 osteosarcoma cells secrete minor amounts of the growth factor in
free form without any LTBPs. Therefore, LTBPs may not be vital for efficient
secretion of TGF-ßs in all cell types. In this report we found that
efficient secretion of overexpressed hLTBP-3 in COS-7 cells required
co-expression of TGF-ß1. Overexpressed hLTBP-3 was not secreted unless
TGF-ß1 was co-expressed simultaneously. This finding demonstrates the
importance of hLTBP-3 as a binding protein for TGF-ß1. Dallas and
co-workers (Dallas et al.,
1994
) have reported earlier that MG-63 osteosarcoma cells secrete
TGF-ß1 in a 290 kDa complex containing LTBP-1. Our results favor the idea
that the large latent TGF-ß1 complex secreted by MG-63 cells contains
hLTBP-3 rather than LTBP-1. The major fraction of the secreted LTBP-1 was
found to be in free form in MG-63 cells. The discrepancy between the results
may be caused by dissimilar detection methods, which were not able to
distinguish slight size differences and to the fact that hLTBP-3 protein was
not known at that time.
LTBP-1, -2 and -4 associate with the extracellular matrix, from where they
can be released by treatment with serine proteases such as plasmin, elastases
and chymase (Taipale et al.,
1994; Hyytiäinen et al.,
1998
; Saharinen et al.,
1998
). We analyzed the cellular localization of hLTBP-3 by
immunofluorescence analysis. A fluorescence signal from the translation
product of the transfected hLTBP-3 cDNA was detected inside COS-7
cells, whereas no staining was observed in the extracellular matrix.
Co-transfection of hLTBP-3 and TGF-ß1 cDNA expression
vectors resulted in the secretion of high molecular weight complexes when
assessed by immunoblotting of the conditioned medium of COS-7 cells. By
immunofluorescence analysis, co-transfection appeared to have no effect in
possible matrix deposition of hLTBP-3 in COS-7 cells or in G292 osteosarcoma
cells, which produce high amounts of ECM (data not shown). Our preliminary
immunoblotting data from the extracellular matrices of CCL-137 embryonic lung
fibroblasts suggests, however, that a fraction of the large latent complexes
of hLTBP-3 and TGF-ß1 exists in SDS-soluble and plasmin-digested
matrices. Faint signals corresponding to the sizes of plasmin-digested
complexes of overexpressed hLTBP-3 and TGF-ß1
(Fig. 6) were detectable with
long exposures of the ECM immunoblots (data not shown), suggesting that
hLTBP-3 may be able to associate with the ECM to some extent.
Immunofluorescence analyses of transfected cells suggest that the faint signal
in matrix preparations is caused by inefficient matrix deposition of
hLTBP-3.
Characterization of the third human LTBP revealed that even though the
different LTBPs share structural and functional characteristics, they all are
unique. Their abilities to bind to latent TGF-ßs vary as do their
capacities for ECM association. For instance, analysis of hypomorphic
LTBP-4-/- mice indicated that LTBP-4 has several important
functions in the regulation of TGF-ß1 bioavailability in the heart, lung
and colon (Sterner-Kock et al.,
2002). Although LTBP-2 seems to function more as a structural
component of the microfibrillar network and resembles fibrillins, hLTBP-3
appears to be an essential binding protein for TGF-ß1. Novel functions
for LTBPs may include the regulation of cell adhesion as described for LTBP-2
(Hyytiäinen and Keski-Oja, unpublished data). The vital importance of
LTBP-2 for development has been observed in knockout mice, which are
embryonically lethal (Shipley et al.,
2000
). Mice lacking the alleles of LTBP-3 survive but
suffer from premature obliteration of synchondroses, osteosclerosis and
osteoarthritis (Dabovic et al.,
2002
). The finding that LTBP-3 does not get secreted from the
cells without co-expression of TGF-ß, and the fact that TGF-ß1 forms
complexes only with LTBP-3 in MG-63 cells, suggest very specific functions for
LTBP-3. These results indicate a clear and tight interaction between
TGF-ß1 and hLTBP-3 and support the observation that bone anomalies in
LTBP-3 knockout mice may result from deficient TGF-ß growth factor
availability. Further studies directed at determining the fate of secreted
large latent complexes of hLTBP-3 and TGF-ß1LAP will provide more
information on the role of hLTBP-3 in storage and activation of the latent
growth factor.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
After submission of this work we learned of similar, accordant results that demonstrated the need for TGF-ß and LTBP-3 co-expression for secretion of human LTBP-3 [Chen, Y., Dabovic, B., Annes, J. P. and Rifkin, D. B. (2002). FEBS Lett. 517, 277-280].
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