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From the
Proteolytic processing of the transforming growth factor
Transforming growth factor
TGF
The active 25-kDa TGF
Many
proteins including polypeptide hormones, viral proteins, growth
factors, and receptors are synthesized as large inactive precursor
proteins that must be proteolytically processed in order to release the
bioactive polypeptide
(21) . The most commonly occurring site of
proteolysis is at the carboxyl-terminal side of basic amino acids
residues found within the pro-protein
(21) . Recently, a family
of mammalian processing enzymes called SPCs (subtilisin-like
pro-protein convertases) has been characterized
(22, 23) . Up to six members of this family have been
identified to date. These are Ca
A series of cellular events take place during TGF
The
ubiquitous expression of both furin and TGF
The
evidence for furin as a relevant TGF
Interestingly, in vitro and in vivo experiments also revealed that the endoproteolytic cleavage
mediated by furin did not yield any intermediates which may have
occurred following processing at pairs of basic amino acids found
within the pro-TGF
We were quite surprised to observe an
important disparity between the efficiency of furin to cleave
pro-TGF
With the identification
and characterization of novel SPCs with similar substrate specificity
as furin
(51) , it has become important to delineate which
enzyme(s) in this class of proteases expresses TGF
Our findings may have broad biological implications.
TGF
In conclusion, our biochemical studies together with
coexpression studies provide evidence that furin is a good candidate as
a physiological endoprotease responsible for TGF
We thank Francine Grondin for skilled technical
assistance. We are greatly indebted to Dr. Nabil Seidah for his
contribution in the coinfection studies and helpful discussion and to
Dr. Claude Lazure for amino acid sequencing. We also thank Dr. Francis
W. Ruscetti for critical review and helpful discussion and Carole
Jacques for editorial assistance. Recombinant bovine TGF
Volume 270,
Number 18,
Issue of May 5, pp. 10618-10624, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
1 Precursor by Human Furin
Convertase (*)
ABSTRACT
INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
precursor (pro-TGF) is an essential step in the formation of the
biologically active TGF homodimeric protein (TGF). The
361-amino-acid precursor pro-TGF1 has within its primary structure
the R-H-R-R processing signal found in many constitutively secreted
precursor proteins and potentially recognized by members of the
mammalian convertase family of endoproteases. To determine whether
cleavage of pro-TGF1 can be achieved by the furin convertase
in vitro, purified precursor was incubated in the presence of
a truncated/secreted form of the enzyme. Immunoblots showed that the
55-kDa pro-TGF1 was converted into the 44 and 12.5 kDa bands
corresponding to the pro-region and the mature monomer, respectively.
Treatment of pro-TGF1 with furin resulted in a 5-fold increase in
the production of biologically active TGF1. Furthermore, when
expressed in the furin-deficient LoVo cells, no processing of
pro-TGF1 was observed. In contrast, efficient processing was
oberved when pro-TGF was coexpressed with the furin convertase.
Collectively, these results provide evidence that in our experimental
systems the TGF1 precursor is efficiently and correctly processed
by human furin thus permitting release of the biologically active
peptide.
(TGF)
()
is a 25-kDa homodimeric protein with potent effects on cell
growth and differentiation (for reviews, see Refs. 1-3). Three
different isoforms (TGF1, TGF2, and TGF3) with similar
bioactivities have been identified in mammalian tissues, TGF1
being the most extensively characterized isoform. Several proteins more
distantly related to TGFs such as activins, inhibins,
Müllerian inhibitory substance, bone morphogenic proteins,
products of the nodal gene in mice as well as products of the
decapentaplegic complex of Drosophila and the Vg1 gene from
Xenopus laevis appear to play an important role in cellular
differentiation
(4) .
isoforms are produced by a
wide variety of normal and malignant cells and have been isolated from
different tissues including blood platelets, bone, and placenta
(5, 6, 7, 8) . It has been shown that
most cell types secrete TGF1 in an inactive form
(9, 10) which does not interact with specific TGF cell surface
receptors thus failing to elicit TGF-induced biological responses.
Inactive TGF may take multiple forms: for example, the 25-kDa
mature TGF may be complexed with specific binding proteins such as
the TGF latency-associated peptide (NH
-terminal part
of the precursor sequence)
(3, 11) and the latent
TGF-binding protein and thus be unable to bind to its cognate
receptors; TGF may also be inefficiently processed from its
precursor
(12, 13) . The presence of TGF receptors
on most cell types
(14) and the ubiquity of the TGF
molecule itself
(15, 16) suggest that processing and
activation of TGF is an important step in the regulation of
TGF action.
1 molecule consists of
two identical disulfide-linked 12.5-kDa polypeptide chains
(17) . Cloning of the TGF1 precursor cDNA and determination
of its primary structure revealed that the mature 112-amino-acid chain
of TGF1 is derived from the COOH terminus of a 390-amino-acid
pre-pro-TGF1 by proteolytic cleavage
(18) . This processing
event is predicted to occur following an R-H-R-R sequence immediately
preceding the NH-terminal Ala 279 residue of the mature
growth factor. This suggests that processing of the growth factor
involves an endoprotease which shows cleavage specificity toward pairs
of basic amino acids
(18, 19, 20) .
-dependent serine
proteases that have been shown to cleave mostly at the R-R or K-R pairs
of basic amino acids. Furin, the first SPC member to be extensively
characterized, has been shown to process many proproteins including
pro-
-NGF
(24) , the insulin receptor
(25) , and the
HIV-1 glycoprotein gp160
(26) among others. Expression of the
fur gene, which encodes furin, appears to be ubiquitously
expressed in all tissues and cell types examined to date
(24, 27, 28) . Colocalization with TGN 38 and
failure to redistribute to the endoplasmic reticulum in the presence of
brefeldin A suggest that furin is mostly localized in the
trans-Golgi network
(24, 29) . Substrate
specificity studies have revealed that furin requires a
R- X-X-R motif for cleavage while the R- X-K/R-R
sequence provides an optimum processing site
(30) . Upon
inspection of the amino acid sequence of the TGF1 precursor, such
an optimum furin cleavage motif was revealed immediately upstream of
the amino acids constituting the NH-terminal of the mature
TGF. Colocalization of TGF precursor with mannosidase II and
its sensitivity to endoglycosidase H suggest that pro-TGF
processing occurs in the Golgi complex, the subcellular site of furin
location
(31) . Therefore, the nearly ubiquitous expression of
both furin and TGF, the correlation between their cellular
localization, and the nature of the TGF-processing site make furin
a good candidate for the physiological processing of TGF. Here we
provide evidence that furin can efficiently and correctly process
pro-TGF and that cleavage by this endoprotease occurs at the
carboxyl side of the consensus R-H-R-R cleavage motif.
Recombinant Vaccinia Viruses
Vaccinia virus (VV)
strain WR was used in this study. The VV wild type (VV:WT) and the VV
recombinant engineered to express a soluble COOH-terminal truncated
form of furin (VV:hFUR713t) or full-length furin (VV:FUR) have been
previously described
(30, 32) . The engineering of
VV:POMC has been previously described
(33) and VV:TGF was
prepared as described
(34) using full-length human TGF1
cDNA insert (obtained from the American Type Culture Collection
(ATTC)).
Production of Soluble Furin
For furin production,
BSC-40 cells were grown to near confluence on 100-mm plates in minimal
essential medium containing 10% fetal calf serum. Parallel plates were
infected with either VV:WT or VV:hFUR713t at an infection multiplicity
of 5. At 18-h post-infection, cells were washed twice with
phosphate-buffered saline and incubated at 37 °C for 4 h in 2 ml of
serum-free medium (MCDB 202; Life Technologies, Inc.) in the presence
of aprotinin (1 µg/ml), pepstatin (0.7 µg/ml), leupeptin (0.5
µg/ml) and phenylmethylsulfonyl fluoride (170 µg/ml). Media
were harvested, centrifuged (12,000 g, 10 min) at 4
°C, and the supernatants were concentrated by ultrafiltration using
Centricon-30 filtration units and kept on ice until used.
Coinfection Studies
LoVo cells (obtained from
ATCC) were grown to near confluence on 100-mm plates in Ham's F12
medium containing 10% fetal calf serum. Cultures were infected as
described previously
(35, 36) with a mixture of
VV:TGF and either VV:POMC or VV:FUR.
Enzymatic Assay
Fluorometric assay on the boc
RVRR-aminomethylcoumarin substrate (380-nm excitation, 460-nm emission)
was performed as described previously
(30) . One unit of
activity was defined as the amount of enzyme that can release 1 pmol of
aminomethylcoumarin from the substrate in 1 h.
Pro-TGF
TGF Purification1 precursor from
CHO cells transfected with simian TGF1 cDNA
(19) was
purified from a confluent roller bottle collected for 48 h containing
50 ml of serum-free media as described
(37) and then dialyzed
versus two changes of 0.2 M acetic acid and one
change of 4 mM HCl. The acidified TGF1 media was
lyophilized, redissolved in 2 ml of 40% acetonitrile-water, 0.1%
trifluoroacetic acid, and fractionated on an HPLC gel filtration
column, SEC-250 (Bio-Rad). The pro-TGF1 was collected
(37) and purified by HPLC chromatography on a C-18 column with a
gradient of water, 0.05% trifluoroacetic acid and propanol, 0.05%
trifluoroacetic acid, and the precursor appeared at 23% propanol, 0.05%
trifluoroacetic acid. In some cases, the monomeric pro-TGF1 was
purified on reducing SDS-PAGE gels.
Endoproteolytic Cleavage of Pro-TGF
Five µl of pro-TGF and Inhibitor
Assays1 purified as described above
was incubated in the presence of 27 µl of VV:WT-derived conditioned
medium or VV:hFUR713t-derived conditioned medium (3-5 units of
furin as defined above) in 8 µl of 5 reaction buffer (500
mM HEPES, 0.50% Triton X-100, and 5 mM
CaCl
, 5 mM 2-mercaptoethanol pH 7.5) at 30 °C
for 3 h. For inhibition experiments, EDTA was added to the reaction
mixture at a final concentration of 5 mM and the
decanoyl-arginine-valine-lysine-arginine-chloromethylketone
(decanoyl-RVKR-cmk) was dissolved in water at 1 mM and 0.2
µl (5 uM final) was added 30 min before the addition of
pro-TGF1. The reaction mixture was separated on 12% SDS-PAGE gels.
Separated proteins were then transferred onto nitrocellulose membrane,
blocked, and probed overnight with peptide antiserum that are specific
for NH-terminal (1:200 dilution) sequence, COOH-terminal
sequence (1:200 dilution) or the whole precursor polypeptide (1:800
dilution). The antibodies have been characterized previously
(19) . The membranes were then washed and incubated for 1 h with
either horseradish peroxydase-labeled anti-rabbit IgG (1:2500) and
immunoreactive bands revealed using the ECL detection system (Amersham)
or alkaline phosphatase-conjugated anti-rabbit IgG (1:5000) and
developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl
phosphate reagent (Bio-Rad).
Growth Inhibition Assay
Growth inhibition assays
were performed with Mv1Lu mink lung epithelial cells (CCL-64; ATCC)
essentially as originally described by Tucker et al.(38) . In brief, CCL64 cells were plated in 96-well flat
bottom plates at 2500 cells/well. After 24 h, the medium was removed
and serial dilutions of the samples to assay for TGF activity were
added. After 72 h incubation, the cells were pulsed with
[
H]thymidine. Cells were collected and
radioactivity counted in a liquid scintillation counter. One unit of
activity was defined as the amount of TGF required to give 50%
maximal response in the assay.
NH
The 55
kDa band corresponding to monomeric TGF-terminal Sequence Analysis1 precursor was excised
from preparative 7.5% SDS-PAGE gels accordingly to the position of
prestained molecular mass markers. The bands were sliced and eluted
from gels 4 h in 250 µl of enzymatic reaction buffer, concentrated
by ultrafiltration using Microcon-30 filtration units, and diluted in
enzymatic digestion buffer. Pro-TGF cleavage was achieved as
described above. The cleavage products were separated by 15% SDS-PAGE
gels, electrotransferred onto Immobilon P membranes, and stained with
Coomassie Brilliant Blue R-250. The 12.5 kDa band corresponding to the
mature form of TGF was excised and subjected to automated Edman
degradation in a PSQ-1 gas-phase sequencer.
In Vitro Cleavage of Purified TGF
To determine whether cleavage of the TGF1 Precursor by
Human Furin1
precursor can be mediated by furin, we initially used in vitro enzymatic digestions. As a source of pro-TGF, supernatants
from CHO cells expressing the simian pre-pro-TGF1 sequence were
used
(39) . Purification of these supernatants was performed as
described under ``Materials and Methods.'' The simian type 1
TGF cDNA encodes the entire growth factor precursor and displays a
high sequence homology to its human counterpart
(39, 40) . TGF-transfected CHO cells have been
shown to secrete two molecular forms of TGF1 proteins
(100-110 and 25 kDa) as determined by SDS-PAGE under non-reducing
conditions
(19) . Upon electrophoresis under reducing
conditions, followed by gel staining with Coomassie Brilliant Blue, the
corresponding monomers were found: a 55 kDa band (pro-TGF1), a 44
kDa band (pro-region), and a mature 12.5-kDa TGF1 monomer
(Fig. 1 B). Because of the glycosylation of the precursor
sequence
(41) , the 55 and 44 kDa bands appear as broad, stained
regions. In contrast, the mature TGF polypeptide that does not
contain glycosylation sites is detected as a sharp, discrete band.
Evidence that the 55, 44, and 12.5 kDa in Fig. 1 B represent the regions of the TGF precursor depicted in
Fig. 1A has been presented previously
(19) . Note
that as previously reported
(37) , current procedures used to
purify the intact 55-kDa pro-TGF to homogeneity also resulted in
the recovery of the 44- and 12.5-kDa species due to reassociation of
the pro-region with the mature polypeptide. Further attempts to
fractionate this TGF complex into separate components were
unsuccessful
(37) . Therefore, our precursor preparation
contains detectable amounts of the pro-region and the mature
polypeptide.
Figure 1:
A, schematic
representation of human pre-pro-TGF1 (27). The signal sequence
(residues 1-29, black box), the pro-peptide (residues
30-278, open box), and mature peptide (residues
279-390, hatched box) regions are indicated. Solid
lines above the molecule represent peptide sequences used for
antibody production; antibody designation is indicated above the lines.
The arrow indicates the cleavage site. B, Coomassie
Blue-staining pattern of proteins from stably transfected CHO cells
expressing TGF1. The CHO-conditioned media was purified as
described under ``Materials and Methods,'' and 2.5 µl was
fractionated on a 12% SDS-polyacrylamide gels under reducing conditions
followed by staining with Coomassie Brilliant Blue R-250. Arrows noted at the right of the figure correspond to TGF1 molecules
depicted in A.
As a source of human furin, supernatants from BSC-40
cells infected with recombinant VV containing cDNA encoding the soluble
secreted form of human furin (VV:hFUR713t) was used. Furin-containing
supernatants were compared in our assays with supernatants from VV:WT.
We were unable to detect furin-like activity from VV:WT supernatants
using the fluorogenic substrate boc RVRR-aminomethylcoumarin. Following
treatment with furin, the pro-TGF1 preparation was analyzed using
immunobloting techniques. Sequence-specific antibodies as indicated in
Fig. 1A were used to detect changes in immunoreactive
polypeptides after digestion with furin. As shown in Fig. 2,
lane 2, the overall intensity of the 55 kDa band was
diminished, and the 44 kDa band was intensified as detected with an
NH-terminal-specific antiserum. This shift was not observed
using supernatant from wild type VV-infected BSC-40 cells
(Fig. 2, lane 3). A similar immunoblot probed with the
COOH-terminal TGF1-specific antiserum indicated that after furin
treatment, a 12.5 kDa band was intensified (Fig. 2, lanes 2 and 1). This band has the same migration pattern as pure
recombinant TGF1 (data not shown). In a time course study, subtle
intensification of the 44 and 12.5 kDa bands was observed after a 1-h
digestion with a maximal effect seen at 3 h as revealed with antibodies
against the whole precursor (data not shown). Therefore, furin is able
to process the TGF1 precursor yielding the corresponding 44-kDa
pro-region and 12.5-kDa-TGF-related cleavage products as detected
under reducing conditions. Interestingly, prolonged digestion of
pro-TGF1 with furin or increasing concentrations of furin (data
not shown) did not yield any intermediates which may have occurred
following processing of pairs of basic amino acids found within the
pro-TGF 1 molecule
(39, 40) . This provides
evidence for the specificity of the in vitro cleavage of
pro-TGF1 by furin.
Figure 2:
In vitro processing of pro-TGF1 by
human furin. Immunoblot analysis of furin-treated CHO-derived TGF1
precursor. SDS-PAGE-separated proteins were transferred onto
nitrocellulose membranes and probed with antiserum no. 1125 directed
against the COOH-terminal region of the pro-region of the precursor or
antiserum no. 978 which is specific to the carboxyl-terminal region of
the mature sequence. Lane 1, purified pro-TGF; lane
2, pro-TGF incubated with supernatants from
VV:FUR713t(furin)-infected BSC-40 cells for 3 h; lane 3,
pro-TGF incubated with supernatants from VV:WT (control)-infected
cells for 3 h.
Peptidyl chloroalkylketones with peptide
moieties that mimic the furin cleavage site have been shown to be
specific inhibitors of furin enzymatic activity
(26) .
Fig. 3B shows the inhibitory properties of the
water-soluble decanoyl-RVKR-cmk on furin-mediated cleavage of TGF1
precursor. Densitometric measurement of the 44 and 12.5 kDa band
indicated that 5 µM of dec-RVKR-cmk blocked furin-mediated
conversion of the 55-kDa TGF precursor into the corresponding
12.5- and 44-kDa cleavage products. In fact, the intensity of the bands
corresponding to pro-TGF cleavage products (44- and 12.5-kDa
species) was similar to background level (due to 44 and 12.5-kDa
species found in the initial pro-TGF1 preparation used; see
Fig. 1B). Similar inhibition was observed using 5
mM EDTA showing the requirement of calcium for endoproteolytic
cleavage of pro-TGF by furin (Fig. 3 A).
Figure 3:
Inhibition of furin-mediated cleavage of
pro-TGF 1 by EDTA and decanoyl-RVKR-cmk. Pro-TGF was
incubated in the presence of VV:FUR713t (furin) for 3 h in the presence
or absence of EDTA (5 mM) ( A, lanes 2 and
3) or 5 µM of decanoyl-RVKR-cmk ( B,
lanes 2 and 3) and analyzed by immunoblotting as
described in the legend of Fig. 2.
NH
Proteolytic cleavage of TGF-terminal Sequencing of Cleavage
Product1 is predicted to occur
following a basic Arg-His-Arg-Arg sequence and immediately preceding
the NH-terminal Ala residue of the mature
growth factor
(37, 39, 40) . To specifically
localize the site of cleavage by furin, the purified 12.5-kDa
proteolytic fragment was subjected to NH
-terminal sequence
analysis. The resulting sequence Ala-Leu-Asp- X-Asn was
consistent with the predicted NH-terminal sequence of
mature simian TGF1 and indicates that cleavage by furin occurs
carboxyl to the consensus Arg-His-Arg-Arg furin cleavage motif.
Bioactivity of TGF
We next asked whether in vitro proteolytic
cleavage of pro-TGF1 Exposed in Vitro to
Furin1 by furin would release bioactive TGF1.
For this purpose, a rapid and sensitive growth inhibition assay using
CCL-64 mink lung epithelial cells was used
(38) . Pro-TGF1
incubated with supernatants from either VV:hFUR713t- or VV:WT-infected
BSC-40 cells was assayed for biological activity.
Fig. 4
demonstrates that treatment of pro-TGF1 with furin
results in a 5-fold increase in bioactive TGF with 213 µg/ml
of TGF detected compared to 42 µg/ml in control supernatant
(pro-TGF1 treated with supernatant from VV:WT-infected BSC-40
cells). These results indicate that processing of pro-TGF1 by
furin results in the production of biologically active TGF and
that furin processed pro-TGF at a physiologically relevant
cleavage site.
Figure 4:
Bioactivity of TGF1 exposed in
vitro to furin. Bioactive TGF was determined in a mink lung
cell growth inhibition assay. A representative experiment out of five
performed is shown.
In Vivo Cleavage of TGF
To assess
the ability of furin to cleave TGF1 by Furin precursor in cells, coinfection
experiments were performed, and the products of expression were
analyzed by electrophoresis of concentrated supernatants on reducing
SDS-PAGE gels followed by immunoblotting. For this experiment, we used
the furin-deficient LoVo cells
(42, 43) . As illustrated
in Fig. 5, lanes 1 and 2, the 55-kDa TGF1
precursor band was not observed in mock infected LoVo cells or cells
infected with control recombinant vaccinia (VV:POMC). LoVo cells
coinfected with TGF-expressing vaccinia recombinant (VV:TGF)
and a control vaccinia recombinant (VV:POMC) failed to cleave TGF1
precursor (Fig. 5, lane 3) as shown by the detection of
the 55 kDa precursor band only. Using the CCL-64 bioassay with a
sensitivity of 50 pg/ml, we did not detect any bioactive TGF from
these supernatants. This indicates that the 55-kDa pro-TGF has
very little or lacks bioactivity (). However, coinfection
with VV:TGF and VV:FuR resulted in a loss of immunoreactive 55 kDa
precursor band with the concomitant appearance of the 44-kDa pro-region
as detected with the NH-terminal-specific antibody
(Fig. 5, lane 4). Reprobing of the same blot with the
COOH-terminal-specific antisera indicate that the observed detection of
the 44-kDa species corroborated with the detection of the 12.5-kDa
polypeptide. No other species than the intact pro-region (44 kDa) and
the mature polypeptide (12.5 kDa) was observed even when the
immunoblots were overexposed (data not shown). Measure of the
biological activity of the (VV:TGF/VV:FUR) supernatants revealed
large amounts of bioactive TGF (20, 385 pg/ml; ).
Figure 5:
In vivo cleavage of pro-TGF1 by human
furin. LoVo cells in 10-cm dishes were coinfected with each virus added
at a multiplicity of infection of 3. 18-h post-infection, supernatants
were collected, concentrated, and resolved on 12% reducing SDS-PAGE
gels. Immunoblotting was performed with antiserum no. 1125 or antiserum
no. 978. Supernatants from mock-infected cells ( lane 1);
VV:POMC-infected cells ( lane 2); VV:TGF/VV:POMC-infected
cells ( lane 3); VV:TGF/VV:FUR-infected cells ( lane
4).
processing. Following synthesis of pre-pro-TGF, the signal peptide
is rapidly removed followed by N-glycosylation at predicted
sites
(41) . The TGF precursor is then translocated to the
Golgi network where other post-translational modifications occur
including the endoproteolytic processing at the carboxyl-terminal side
of a cleavage site consisting of basic residues
(13, 40) . No information is available to date
concerning the enzyme(s) involved in intracellular processing and
release of mature TGF1 from its larger precursor polypeptide.
Proteases such as plasmin, a relatively nonspecific serine protease, as
well as lysosomal cathepsin D have been shown to cleave and activate
pro-TGF isoforms
(13, 44, 45) . However,
these enzymes would unlikely be involved per se in TGF
processing events. In fact, multiple cleavage sites for these enzymes
are present in the sequence of TGF isoforms
(40, 46) , and it has been demonstrated that cleavage of
TGF1 precursor by plasmin results in degradation of the pro-region
and the mature TGF polypeptide; this later event results in a loss
of biological activity
(47) . This is inconsistent with unique
pro-TGF1 enzymatic processing site deduced by examination of the
amino acid sequence
(40) and further revealed in TGF1
overexpressing cells
(13) . In addition, plasmin and cathepsin D
enzymatic activities are found at the cell surface and in the
pericellular space (plasmin) or in the lysosomes (cathepsin D) which is
physically incompatible with the localization of TGF processing
events which has been demonstrated to take place during TGF
transit in the Golgi complex
(20, 31) .
1, the existing
correlation between their cellular localization, and the strategic
presence of an optimum furin consensus cleavage motif in the
pro-TGF1 amino acid sequence make furin a good candidate for the
physiological processing of this precursor. The data presented here
demonstrate that furin efficiently processes pro-TGF1 and that
cleavage by this endoprotease occurs at the physiologically relevant
R-H-R-R processing site. The identity of this cleavage site is
supported by studies in which the 12.5-kDa TGF1, secreted from
overexpressing CHO cells, was isolated and sequenced. The results
revealed an intact amino terminus sequence at Ala of the
mature growth factor, indicating that CHO cells properly processed
pro-TGF
1 at the R-H-R-R cleavage site
(37) . Since furin
mRNA was detected in these cells, it is possible that endogenous furin
is capable of producing the TGF1 secreted from these cells.
processing enzyme is
substantiated by data from coinfection studies in LoVo cells. These
cells are a human colon carcinoma cell line which was previously shown
to have a point mutation in the fur gene and was incapable of
processing the hepatocyte growth factor proreceptor
(42, 43) which bares a similar cleavage site (R-K-K-R) as
pro-TGF1. It was shown that this defect can be corrected by
genetic complementation of LoVo cells with mouse fur cDNA
(43) . In our study, analysis of supernatants from vaccinia virus-mediated overexpression of TGF1 precursor in LoVo cells
indicated that only the intact precursor form, which lacks biological
activity, was secreted whereas coexpression of TGF and furin
resulted in the release of significant amounts of immunoreactive and
bioactive TGF.
1 molecule
(39, 40) and provide
evidence for the selectivity of cleavage of pro-TGF1 by furin.
Indeed, under certain experimental conditions, furin has been found to
cleave (however with less efficiency) at pairs of basic amino acids
without the presence of Arg in the P4 position. For example, POMC was
partially converted by overexpressed furin, yielding physiologically
relevant peptides (-LPH) originating from cleavage at pairs of
basic amino acids only
(48, 49) . Additionally, analysis
of the enzymatic properties of furin on hemaglutinin of influenza virus
indicated that overexpression of the enzyme led to a broadening of its
specificity
(50) . Therefore, in our systems, the unique
cleavage site mediated by furin indicates that other basic moieties
found within the TGF pro-region and the mature polypeptide are
indeed not favored under our in vitro and in vivo experimental conditions.
in vivoversusin vitro. In
fact, we found a complete cleavage of TGF precursor in cells,
compared with the partial cleavage found in the in vitro enzymatic assays, even in the presence of higher levels of furin
enzymatic activity ( Fig. 5compared to Figs. 2 and 3). The exact
reason for this discrepancy is at the moment unknown. However, we can
speculate that some yet unidentified molecular chaperones, present in
cells, are needed to conduct proper folding and cleavage of TGF
precursor
(75) . We also cannot rule out the possibility that
the purification process somehow altered pro-TGF1, rendering it a
poor substrate for furin in vitro.
convertase
activity. Indeed, it has been recently demonstrated that furin and PACE
4 share similar substrate specificity
(52, 53) . Of the
six SPCs that have been characterized, PACE 4 and PC5/PC6, which show a
broad tissue distribution similar to TGF, might fulfill this role
while PC1, PC2, and PC4 which display restricted tissue specificity are
likely to be excluded from the list. As stated above, failure of LoVo
cells to process TGF precursor and the efficient processing
observed by overexpression of furin highlighted furin as a
TGF-processing enzyme. It should be stated that the LoVo cells, as
most of the constitutively secreting cell types, do not express PC1 or
PC2 mRNA
(54) . However, they express large amount of PACE 4
mRNA as well as PACE 4 enzymatic activity. Indeed, using the same
vaccinia-based overexpression system as used in this study,
LoVo cells were able to process pro-NGF at a R-S-K-R cleavage
motif, possibly implicating the endogenous PACE 4
convertase.()
In our study, failure of these
cells to process overexpressed TGF precursor suggests that
endogenous PACE 4 does not play a prime role in TGF processing
events. In addition, preliminary data from coinfection studies with
pro-TGF1 and PC1, PC2, PACE 4, PC5/PC6, or furin indicates that
furin is indeed a prominent TGF converting enzyme.()
has been shown to be a most potent natural inhibitor of many
inflammatory and immune pathways. TGF in vitro inhibits
the proliferation of several hematopoietic cells such as thymocytes
(55) , T and B cells
(56, 57, 58, 59) , and primitive bone
marrow progenitors
(60, 61, 62, 63) . In
addition, TGF suppresses IgM and IgG production by B cells
(56) , antagonizes the immunoregulatory effect of interleukin-1,
-2, and -3
(56, 57, 58, 59) ,
colony-stimulating factor
(62, 64) , and interferons
(65, 66) , reduces cell-surface receptor expression for
several growth factors
(67, 68) , and induces
interleukin-1 receptor antagonist
(69) . Therefore, the presence
of active TGF in biological fluids and tissues is likely to be of
prime biological importance. Supporting this, studies from several
teams have shown that targeted disruption of the murine TGF1 gene
resulted in uncontrolled inflammatory response that leads to premature
death
(70, 71, 72) . On the other hand,
increased production of activated TGF has been associated with
malignant progression
(73) . TGF has been shown to induce
angiogenesis, to act as an autocrine tumor growth factor, and to impair
tumor immune surveillance
(1, 2, 3, 74) . All these functions
would cooperate in the amplification of tumor progression. Since the
endoproteolytic cleavage of TGF precursor is likely the
cornerstone for full activity of the mature product, one can speculate
that a delicate balance of furin-like enzymatic activity is required
for normal physiological growth and differentiation of the cells and
that disturbance of this balance could lead to pathological
aberrations. In support of this, we have recently observed that
treatment of synovial cells and NIH-3T3 cells with TGF resulted in
a significant increase in fur mRNA levels suggesting that
TGF might auto-regulate its own converting enzyme.
()
1 processing. The
other mammalian TGF isoforms namely TGF2 and TGF3 are
also initially synthesized as larger precursor with similar furin
consensus motif at the junction of the pro-region and the mature
polypeptide. Efforts are underway to assess whereas these other
TGF isoforms are processed by furin and to confirm furin as
thebona fide pro-TGF convertase.
Table:
Measure of bioactive TGF in supernatants of
LoVo cells coinfected with recombinant vaccinia
,
transforming growth factor ; VV, Vaccinia virus; WT, wild type;
HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel
electrophoresis; FUR, furin; POMC, proopiomelanocortin; CHO, Chinese
hamster ovary.
1 was
generously provided by Oncogen/Bristol-Myers Company and the furin
inhibitor dec-RVKR-cmk was kindly provided by Drs. Herbert Angliker and
Elliott Shaw.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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