(Received for publication, August 26, 1994; and in revised form, November 29, 1994)
From the
Transforming growth factor- (TGF-
) is a potent growth
regulatory protein secreted by virtually all cells in a latent form. A
major mechanism of regulating TGF-
activity occurs through factors
that control the processing of the latent to the biologically active
form of the molecule. We have shown previously that thrombospondin 1
(TSP1), a platelet
-granule and extracellular matrix protein,
activates latent TGF-
via a protease- and cell-independent
mechanism and have localized the TGF-
binding/activation region to
the type 1 repeats of platelet TSP1. We now report that recombinant
human TSP1, but not recombinant mouse TSP2, activates latent TGF-
.
Activation was further localized to the unique sequence RFK found
between the first and the second type 1 repeats of TSP1 (amino acids
412-415) by the use of synthetic peptides. A peptide with the
corresponding sequence in TSP2, RIR, was inactive. In addition, a
hexapeptide GGWSHW, based on a sequence present in the type 1 repeats
of both TSP1 and TSP2, inhibited the activation of latent TGF-
by
TSP1. This peptide bound to
I-active TGF-
and
inhibited interactions of TSP1 with latent TGF-
. TSP2 also
inhibited activation of latent TGF-
by TSP1, presumably by
competitively binding to TGF-
through the WSHW sequence. These
studies show that activation of latent TGF-
is mediated by two
sequences present in the type 1 repeats of TSP1, a sequence (GGWSHW)
that binds active TGF-
and potentially orients the TSP molecule
and a second sequence (RFK) that activates latent TGF-
. Peptides
based on these sites have potential therapeutic applications for
modulation of TGF-
activation.
Transforming growth factor- (TGF-
) (
)is a
multifunctional growth regulatory protein that is involved in such
diverse biological activities as wound healing, growth and
differentiation, and
angiogenesis(1, 2, 3, 4) . TGF-
is secreted by virtually all cells in culture as a biologically
inactive molecule(1, 2, 3, 4) . An
essential means of regulating TGF-
activity occurs through factors
that control the processing of the latent to the active form of the
molecule(35) . Once activated, TGF-
can bind to high
affinity cellular receptors and elicit cellular responses.
Thrombospondin 1 (TSP1) activates cell-secreted latent TGF-
as
well as purified forms of small and large latent TGF-
in a
chemically defined system via binding
interactions(9, 10, 11) .
The
thrombospondins are a family of large multidomain glycoproteins
involved in modulating cell growth, adhesion, migration, and
angiogenesis(5, 6, 7, 8) . Our
laboratory recently determined that the TGF- binding/activation
site was localized to the type 1 (properdin-like) repeats of
TSP1(11) . Within the type 1 repeats of TSP1, there are two
well defined consensus sequences, CSVTCG and WSXW. Both
sequences are highly conserved within the TSP family members, TSP1 and
TSP2, that contain the properdin-like type 1 repeats. CSVTCG is an
attachment factor for melanoma and endothelial cells (12, 13, 14, 15) and is
anti-angiogenic in vivo(16) . The sequence WSHW binds
heparin and sulfated glycoconjugates with different affinities (17, 18) and inhibits the interaction of TSP with the
gelatin-binding domain of fibronectin(19) . Variations of this
motif are present in all of the type 1 repeats of both TSP1 and
TSP2(22) . The WSXW motif is also found within the
TGF-
and cytokine receptor
superfamilies(20, 21) . In order to further localize
the site within the TSP type 1 repeats that is involved in the
activation of latent TGF-
, we compared the activities of TSP1 and
TSP2 and tested the activities of synthetic peptides for the ability to
activate latent TGF-
.
In this work, we now report localization
of an activation site to a three-amino acid sequence between the first
and second type 1 repeats of TSP1. In addition, we have also identified
a second sequence in the type 1 repeats that binds active TGF-.
This second sequence, while unable to activate latent TGF-
,
inhibits activation of latent TGF-
by the trimeric TSP1 molecule.
Within the TSP family, only TSP1 and TSP2 contain the type 1
repeats and share 60% homology in the region(33) .
Therefore, in order to determine whether a sequence motif or tertiary
structure common to the type 1 repeats of both TSP1 and TSP2 is
involved in activation of latent TGF-
, human platelet TSP1,
recombinant TSP1, and recombinant TSP2 were tested for their ability to
activate latent TGF-
. A recombinant form of the full-length TSP2
trimer is inactive (Fig. 1). The lack of activity of the TSP2
molecule does not appear to be due to potential anomalies of the
expression system, since 0.1 nM of a recombinantly expressed
form of full-length TSP1 trimer (rTSP1) activates latent TGF-
with
a response identical to that observed for human platelet TSP1 (Fig. 1). Although TSP2 is unable to activate latent TGF-
,
it does inhibit activation of latent TGF-
by TSP1 (Table 1).
This suggests that in a system where TSP1 and TSP2 are both present,
there may be competition for binding to TGF-
. This is a reasonable
speculation given the ability of TSP2 to compete for the binding of
I-active TGF-
to TSP1 in a solid-phase binding assay
(data not shown). TSP1 and TSP2 have different regulatory elements
within their promoter regions, suggesting that their expression is
differentially controlled(33) . TSP1, in contrast to TSP2, is
an early response gene similar to jun and fos and is
up-regulated by serum. In addition, TSP1 and TSP2 have distinct
temporal and spatial patterns of expression. Thus, it is interesting to
speculate that elements that regulate the relative expression of TSP1
and TSP2 influence levels of TGF-
activity.
Figure 1:
Activation of latent TGF- is
specific for TSP1. Recombinant latent TGF-
(LTGF-
)
(2 nM) was incubated with increasing concentrations
(0.04-13 nM) of sTSP1, recombinant TSP1 (rTSP1), and recombinant TSP2 (rTSP2) for 1 h at 37
°C in a total volume of 0.5 ml of PBS. BSA (0.1%) was added to all
samples to decrease nonspecific binding to the tubes. Samples were
tested for TGF-
activity in the NRK colony-forming soft agar
assay. Results are expressed as the means of triplicate determinations
± S.D.
In order to
localize the region within the type 1 repeats of TSP1 that activates
latent TGF-, synthetic peptides corresponding to known sequence
motifs were obtained and tested for their ability to activate latent
TGF-
. Peptides correlating to the CSVTCG motif include Mal I
(amino acids 368-386, from the first type 1 repeat), Mal II
(amino acids 424-442, from the second type 1 repeat), Mal III
(amino acids 481-499, from the third type 1 repeat)(12) ,
and the peptide VTCGGGVQKRSRL (amino acids 488-500). In addition,
a peptide corresponding to amino acids 412-428, peptide 246
(KRFKQDGGWSHWSPWSS) from the second type 1 repeat of TSP1, was tested
to examine the WSHW motif for potential activity. Increasing
concentrations of the CSVTCG-containing synthetic peptides, ranging
from 11 nM to 11 µM, were incubated with
recombinant latent TGF-
and then tested for TGF-
activity in
the NRK soft agar assay. The CSVTCG-containing peptides were inactive
at all concentrations tested (Fig. 2a). In contrast, peptide
246 activated latent TGF-
as monitored by increased colony
formation (Fig. 2b). Maximal activation was observed using
0.03 nM peptide 246, which resulted in a 4-fold increase in
colony formation with activity remaining above base line at peptide
concentrations up to 11 µM (Fig. 2b and
data not shown). Dose-response curves show an estimated EC
of 0.02 nM for peptide 246, suggesting that this peptide
is slightly more effective at activating latent TGF-
than the TSP
trimer (EC
of
0.067 nM)(10) . These
data show that the activation site is within peptide 246
(KRFKQDGGWSHWSPWSS) and that the CSVTCG motif is not involved. The
amount of latent TGF-
(2 nM) used in these assays is in
significant molar excess to either the TSP or the peptides, suggesting
that activation by peptide 246 may be a catalytic event. However, the
effective molar concentration of the recombinant latent TGF-
is
significantly less as a result of misfolding of the recombinant small
latent molecule, with at most
10% of the protein mass being
activatable(36) ; thus the concentration of
``native'' or activatable latent TGF-
may actually be
closer to 0.02 nM. This suggests that TSP (and its peptides)
may at least be acting at a molar equivalency to latent TGF-
, if
not a molar excess.
Figure 2:
Peptides containing the sequence RFK
activate latent TGF- (LTGF-
). a,
recombinant latent TGF-
(2 nM) was incubated with 11
nM sTSP (bar) or increasing concentrations of
peptides for 1 h at 37 °C in a total volume of 0.5 ml of PBS.
Peptides include Mal I (residues 368-386, closedcircles, solidline), Mal II (residues
424-442, opencircles, dottedline), Mal III (residues 481-499, opendiamonds, dashedline), or
VTCGGGVQKRSRL (residues 488-500, opensquares, smalldashedline). Samples were then tested
for TGF-
activity using the NRK colony-forming soft agar assay.
Results are expressed as the means of triplicate determinations
± S.D. b, latent TGF-
(2 nM) was
incubated with increasing concentrations (0.01-60 nM) of
peptide 246 (residues 412-428, closedcircles, solidline), peptide 402 (KRFK, residues
412-415, opensquares, dottedline), or peptide 412 (RFK, residues 413-415, closedsquares, dashedline) for 1
h at 37 °C in a total volume of 0.5 ml of PBS. Samples were then
tested for TGF-
activity in the NRK soft agar assay. Results are
expressed as the means of triplicate determinations ±
S.D.
To further localize the activation site,
deletions were made to the C terminus of peptide 246. TSP1 (11
nM, based on TSP monomers) and equimolar concentrations of
peptide 246 or the truncated peptides were incubated with latent
TGF- and then tested for TGF-
activity in the NRK assay.
These experiments showed that the sequence KRFK, amino acids
412-415, is sufficient to activate latent TGF-
(Table 2). Although the peptides were used at 11 nM in
order to compare activation with that achieved using full-length TSP,
dose-response assays show that the EC
for these peptides
varied ( Table 2and Fig. 2b); peptides 246, 263
(KRFKQDGGWSHWSP), 352 (KRFKQDGGWSHW), and 402 (KRFK) have activities
with EC
values ranging from
0.02 to
0.06 nM (Table 2). However, in comparing the dose-response curves
directly, it appears that peptide 246 is significantly more active than
peptide 402 (KRFK) (Fig. 2b). These data show that
peptide 246 containing both the KRFK and WSHW motifs is the most
active, although the RFK sequence alone is sufficient to activate
latent TGF-
.
In order to confirm the results of our biological
assay, we also tested the peptides for their ability to activate latent
TGF- in an immunological assay using an ELISA that selectively
recognizes the active domain of TGF-
(10) . Consistent with
the results of the biological assay, incubation of latent TGF-
with sTSP1, peptide 246, or KRFK resulted in generation of
140 pg
of TGF-
activity (Table 3). The VTCGGGVQKRSRL peptide was
inactive in the ELISA. The results with the ELISA eliminate the
possibility that activation of latent TGF-
by TSP or the peptides
is due to increased protease activity or receptor binding in the
biological assays. Using the ELISA system, we observed that the
peptides activate latent TGF-
within 5 min of exposure to the
peptides, which is similar to the kinetics observed for intact
TSP(10) . In addition, activation of latent TGF-
by KRFK
also occurs in a complex cellular milieu, since KRFK activates bovine
aortic endothelial cell-secreted TGF-
in the presence of cells in
serum (20% fetal bovine serum)-containing medium as assayed by the NRK
soft agar biological assay (data not shown). This shows that the KRFK
peptide is active in an in vitro cellular system.
To determine if the sequence KRFK is functional in the full-length repeat, fusion proteins corresponding to the intron-exon boundaries of the first and second type 1 repeat of TSP1 were constructed and tested for activity(24) . The fusion protein containing KRF (11 nM) increased colony formation 2-fold above base line (Table 2), similar to the levels achieved using an equimolar concentration of sTSP (Table 2). The glutathione S-transferase vector control and the fusion protein lacking the N-terminal KRF (amino acids 412-414) had no activity (Table 2). These results suggest that the KRFK sequence is functional within the context of the second type 1 repeat.
To
determine which amino acids within the KRFK sequence are required for
activity, modifications of this sequence were examined for the ability
to activate latent TGF-. The corresponding sequence in TSP2, TRIR,
was completely inactive (Table 4). Although TSP2 lacks the KRFK
sequence between the first and second type 1 repeats, a similar
sequence, KKFK, is located within the procollagen-like domain. However,
the results with the full-length TSP2 trimer suggest this site is
potentially inaccessible within TSP2. Lys-412 is not required for
activity since it can be substituted with other amino acids (His or
Gln) or deleted without a loss of activity (Table 4). RFK has an
EC
of
0.06 nM, identical to that of the KRFK
sequence, further verifying that the first lysine is not required for
activity (Fig. 2b). In contrast, Phe-414 is essential
for activity, since it cannot be substituted with other aromatic
residues (Tyr or Trp) or with an Ala or Ile (Table 4). Phe-414
must be flanked by charged basic residues, since substituting either
Arg-413 or Lys-415 with Gln abolishes activity (Table 4). The
ability of a Lys residue to substitute for Arg-413 without a loss of
activity supports this finding (Table 4).
The minimal basic
sequence required for activation is BFB. A similar motif,
BBXB, has been shown to be a heparinbinding
motif(26) . However, not all BBXB motifs are sufficient for
activity, since a peptide from the heparin-binding domain of TSP1,
which contains this motif (ASLRQMKKTRGTLLALERKDHS,
residues 74-95)(27) , and the peptide
VTCGGGVQKRSRL are unable to activate latent TGF-
even at concentrations of 11 µM (data not shown). These
data further support our observation that Phe-414 is required for
activity. Peptide 246 has been shown to bind to heparin, albeit with
weak affinity(17, 18) . Therefore, we tested whether
heparin could inhibit activation of latent TGF-
by either peptide
246 or KRFK. Increasing concentrations of heparin, ranging from 0.01 to
100 µM, were preincubated with the peptides and then added
to latent TGF-
and tested for TGF-
activity. Heparin failed
to inhibit activation by the peptides (Fig. 3). In addition,
heparin had no effect on colony formation by active TGF-
(Fig. 3). Another preparation of heparin, the low molecular
weight form (3000 daltons), was also tested and found to be inactive
(data not shown). These data suggest that the peptides have a higher
affinity for latent TGF-
than for heparin and are capable of
activating latent TGF-
even when potentially bound to heparin (at
concentrations greater than 10 µM).
Figure 3:
Activation of latent TGF- by TSP1
peptides is not inhibited by heparin. Latent TGF-
(2 nM)
was incubated with 11 nM peptide 246 or KRFK (peptide 402) and
increasing concentrations of heparin (ranging from 0.01 to 100
µM) for 1 h at 37 °C in a total volume of 0.5 ml of
0.01 M phosphate buffer, pH 7.2, 0.05 M NaCl. Data
include heparin alone (closedcircles), heparin with
peptide 246 (closedsquares), heparin with KRFK (opensquares), or heparin with recombinant active
TGF-
(opencircles). Samples were then tested
for TGF-
activity using the NRK colony-forming soft agar assay.
Results are expressed as the means of triplicate determinations
± S.D.
The activation of
latent TGF- by KRFK-containing peptides is stable over the pH
range 5.4-8.0. Activation also occurred at NaCl concentrations
0.05-0.5 M (data not shown). This is consistent with our
previous observations that TSP
TGF-
remains complexed in the
presence of 0.55 M NaCl(23) . Although we were only
able to examine the effects of pH and salt over a limited range, these
data are suggestive that electrostatic interactions are not the primary
forces mediating interactions between the KRFK-containing peptides and
latent TGF-
. Consistent with this is the absolute requirement for
the hydrophobic phenyalanine residue. Moreover, preliminary data
suggest that KRFK may fit into a hydrophobic pocket within the
latency-associated peptide at the N terminus of the latent molecule. (
)
Activation of latent TGF- by KRFK is specific for
TSP1. Two common proteins that contain the RFK sequence, calcineurin (28, 29, 30, 31) and BSA (32) , fail to activate latent TGF-
at concentrations up
to 1 µM (data not shown), suggesting this sequence is
inaccessible in these proteins.
Although the sequence RFK is
sufficient for activation of latent TGF-, Trp-420 and Trp-423 in
the longer peptides 246 (amino acids 412-428) and 263 (amino
acids 412-425) are also important for activity ( Table 2and Fig. 4), since substitution of Trp-420 and Trp-423 with Ala
renders the peptides inactive at concentrations otherwise effective for
either TSP1 or the RFK peptide (Table 2). The EC
values of the peptides also show that substituting Trp-420 and
Trp-423 with Ala or changing the spacing of WSXW (peptide 262;
KRFKQDGGWWSP) results in a shift in the EC
from 0.02
nM for peptide 246 to 0.2 nM for peptide 262 and 200
nM for peptides 388 (amino acids 412-428 with Trp-420,
-423, and -426 substituted for Ala) and 266 (amino acids 412-425
with Trp-420 and Trp-423 substituted for Ala) ( Table 2and Fig. 4). In addition, peptides 263, 388, and 266 never reach the
same levels of activity as those achieved using peptide 246 (Fig. 4).
Figure 4:
Substitution of Trp-420 and Trp-423 with
Ala results in a loss of activity. Recombinant latent TGF- (2
nM) was incubated with increasing concentrations of peptide
246 (KRFKQDGGWSHWSPWSS, closedcircles), peptide 388
(KRFKQDGGASHASPASS, opencircles), or peptide 266
(KRFKQDGGASHASP, opensquares) for 1 h at 37 °C
in a total volume of 0.5 ml of PBS. Samples were then tested for
TGF-
activity using the NRK colony-forming soft agar assay.
Results are expressed as the means of triplicate determinations
± S.D.
In order to clarify the role of the WSXW
motif in modulation of TGF- activity, the peptide GGWSHW (amino
acids 418-423) was constructed and tested for activity. GGWSHW
(11 nM to 11 µM) was unable to activate latent
TGF-
(Fig. 5a). In contrast, incubation of 100-fold
molar excess (1.1 µM) GGWSHW with TSP completely inhibited
activation of latent TGF-
in the NRK assay (Fig. 5a).
In addition, GGWSHW (1 µM) partially (
60%) reversed
the TSP-mediated inhibition of bo-vine aortic endothelial cell
proliferation (data not shown). It does not appear likely that this
inhibitory activity is due to GGWSHW blocking active TGF-
from
binding to TGF-
receptors, since incubation of GGWSHW with active
TGF-
has no effect on TGF-
biological activity (data not
shown). The inhibitory activity of the GGWSHW sequence is specific
since related sequences, such as GGWSHY, GGWAHW, GGYSHW, and GGWSKW,
have no blocking effect (data not shown). In addition,
I-TGF-
specifically bound to immobilized GGWSHW (Fig. 5b). 100-fold molar excess soluble GGWSHW and
peptide 246 completely inhibit binding of
I-TGF-
to
the peptide. However, a 100-fold molar excess of peptide 402 (KRFK)
does not inhibit binding of
I-TGF-
to GGWSHW,
indicating that binding of active TGF-
to peptide 246 is mediated
through the GGWSHW sequence. Substituting the first Trp residue of
GGWSHW with a Tyr results in a loss in the ability to compete for
binding (Fig. 5b). Controls include the Hep I peptide
(ELTGAARKGSGRRLVKGPD, residues 17-35) (27) and BSA, both
of which are unable to inhibit the binding of
I-TGF-
to GGWSHW. Soluble GGWSHW also inhibits the binding of
I-TGF-
to immobilized TSP (data not shown). These
data suggest that GGWSHW-containing peptides inhibit activation of
latent TGF-
by TSP by blocking the ability of TSP to bind to the
latent TGF-
complex, and moreover this is the apparent mechanism
by which TSP2 inhibits activation of latent TGF-
by TSP1. Although
the peptide sequence used in these studies is from the second type 1
repeat of TSP1, the corresponding sequences from the first (DGWSPW) and
third (GGWGPW) type 1 repeats of both TSP1 and TSP2 also inhibit
TSP1-mediated activation of latent TGF-
(data not shown). This
implies that any of the three type 1 repeat WSXW motifs is
potentially capable of binding to the active TGF-
molecule.
Figure 5:
Peptide GGWSHW inhibits sTSP-mediated
activation of latent TGF-. a, latent TGF-
(2
nM) was incubated with 11 nM sTSP and increasing
concentrations of peptide GGWSHW for 1 h at 37 °C in a total volume
of 0.5 ml of PBS. BSA (0.1%) was added to all of the samples to reduce
nonspecific binding to the tubes. Samples were tested for TGF-
activity in the NRK colony-forming soft agar assay in the presence of
EGF. Results are expressed as the means of triplicate determinations
± S.D. b, peptide GGWSHW (0.1 µg) was immobilized
on a polyvinyl chloride-coated 96-well plate for 2 h at 37 °C in a
total volume of 100 µl of PBS. Nonspecific binding sites were
blocked with 1% BSA/PBS for 30 min at 37 °C.
I-TGF-
(0.125 ng) was added to the wells in the
presence of 100-fold molar excess unlabeled peptide GGWSHW or equimolar
amounts of peptide 246, KRFK, Hep I (ASLRQMKKTRGTLLALERKDHS), GGYSHW,
or BSA and incubated for 1.5 h at room temperature. Wells were then
washed four times, and bound radioactivity was counted in a
counter. Results are expressed as the total binding (cpm/well) and are
the means of triplicate determinations ± S.D.
These results show that the minimal sequence effective for
activation of latent TGF- is RFK. Within the type 1 repeats of
TSP1, RFK is followed by the conserved motif WSHW. We have shown that
peptides containing GGWSHW inhibit TSP1-mediated activation of latent
TGF-
, presumably by inhibiting the binding of TSP to TGF-
.
Based on the results of these studies, we propose that TSP1 utilizes a
two-step mechanism for the activation of latent TGF-
. The GGWSHW
sequence in TSP1 and TSP2 mediates TSP-TGF-
interactions,
potentially through the mature portion of TGF-
. Interactions
between GGWSHW (or potentially DGWSPW from the first type 1 repeat or
GGWGPW from the third type 1 repeat) and TGF-
may orient the TSP1
molecule so that the RFK sequence is in a favorable configuration for
interacting with latent TGF-
. In support of this theory is the
observation that peptide 246, which contains the entire WSXW
motif, is more active than the shorter peptides lacking the
WSXW motif. We are currently investigating the KRFK binding
site in the latent TGF-
complex. Initial data suggest that the
KRFK sequence may recognize a site in the latency-associated
peptide.
The interaction of the (K)RFK sequence with latent
TGF-
potentially induces a conformational change in the latent
complex, enabling the complex to recognize its cellular receptors. This
hypothesis is supported by data that show that TSP2, which has the
GGWSHW sequence but lacks the RFK activation sequence, binds
I-active TGF-
but does not activate latent
TGF-
. Furthermore, TSP2 inhibits activation of latent TGF-
by
TSP1, presumably by TSP2 competing with TSP1 for binding to the mature
portion of the latent TGF-
complex. An anti-peptide 246 polyclonal
antibody, which recognizes the GGWSHW sequence, also inhibits TSP1- and
peptide 246-mediated activation of latent TGF-
by
70% (data
not shown).
These studies are significant in that they confirm that
activation of latent TGF- by TSP results from TSP interactions
with multiple binding sites within the latent TGF-
complex.
Peptides containing RFK and GGWSHW sequences have potential therapeutic
uses in situations where TGF-
activity requires modulation, for
example during wound healing or in fibrotic scar formation.
Furthermore, these data suggest that regulation of the relative
expression levels of TSP1 and TSP2 is an important determinant
controlling TGF-
activity.