©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Transforming Growth Factor- Activation by Discrete Sequences of Thrombospondin 1 (*)

(Received for publication, August 26, 1994; and in revised form, November 29, 1994)

Stacey Schultz-Cherry (1) Hui Chen (2) Deane F. Mosher (2) Tina M. Misenheimer (2) Henry C. Krutzsch (3) David D. Roberts (3) Joanne E. Murphy-Ullrich (1)(§)

From the  (1)Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0019, the (2)Departments of Biomolecular Chemistry and Medicine, University of Wisconsin, Madison, Wisconsin 53706, and the (3)Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-1500

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transforming growth factor-beta (TGF-beta) is a potent growth regulatory protein secreted by virtually all cells in a latent form. A major mechanism of regulating TGF-beta 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 alpha-granule and extracellular matrix protein, activates latent TGF-beta via a protease- and cell-independent mechanism and have localized the TGF-beta 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-beta. 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-beta by TSP1. This peptide bound to I-active TGF-beta and inhibited interactions of TSP1 with latent TGF-beta. TSP2 also inhibited activation of latent TGF-beta by TSP1, presumably by competitively binding to TGF-beta through the WSHW sequence. These studies show that activation of latent TGF-beta is mediated by two sequences present in the type 1 repeats of TSP1, a sequence (GGWSHW) that binds active TGF-beta and potentially orients the TSP molecule and a second sequence (RFK) that activates latent TGF-beta. Peptides based on these sites have potential therapeutic applications for modulation of TGF-beta activation.


INTRODUCTION

Transforming growth factor-beta (TGF-beta) (^1)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-beta is secreted by virtually all cells in culture as a biologically inactive molecule(1, 2, 3, 4) . An essential means of regulating TGF-beta activity occurs through factors that control the processing of the latent to the active form of the molecule(35) . Once activated, TGF-beta can bind to high affinity cellular receptors and elicit cellular responses. Thrombospondin 1 (TSP1) activates cell-secreted latent TGF-beta as well as purified forms of small and large latent TGF-beta 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-beta 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-beta 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-beta, we compared the activities of TSP1 and TSP2 and tested the activities of synthetic peptides for the ability to activate latent TGF-beta.

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-beta. This second sequence, while unable to activate latent TGF-beta, inhibits activation of latent TGF-beta by the trimeric TSP1 molecule.


MATERIALS AND METHODS

Purification of Thrombospondin

TSP depleted of TGF-beta activity (stripped TSP (sTSP)) was purified as described, utilizing a gel permeation step in which the column is equilibrated at pH 11 in order to dissociate TGF-beta(9) . TSP purity was assessed by SDS-polyacrylamide gel electrophoresis using Coomassie Blue or silver staining. No contaminating TGF-beta activity was found associated with sTSP in NRK soft agar assays.

Peptide Synthesis

Peptides were synthesized as described (17, 18) . Briefly, peptides were synthesized corresponding to sequences of human TSP1 using standard Merrifield solid-phase synthesis protocols and t-butoxycarbonyl chemistry. Peptides were analyzed by reverse-phase high pressure liquid chromatography. The structure of the active peptides was verified by mass spectrometry.

Recombinant TSP1 and TSP2 Production in a Baculovirus System

The methods for baculovirally driven expression of mouse TSP2 (34) and human TSP1 (^2)in Spodoptera frugiperda cells are described in more detail elsewhere. Full-length cDNA clones were kind gifts from Vishva Dixit, University of Michigan. Convenient restriction sites were used to truncate the 5` ends so that the cDNAs could be cloned into baculoviral transfer vectors with a minimal sequence 5` to the translational start sites, 42 base pairs in the case of TSP2 and 29 in the case of TSP1. Cotransfection of transfer vectors was carried out with linearized mutant viral genome DNA so that there was positive selection of recombinant viruses. These viruses were plaque-purified prior to expansion. Recombinant TSPs were secreted into serum-free medium containing 0.2% BSA for 48-72 h after infection in spinner flasks. Purification was by affinity chromatography on heparin-agarose.

Cells

Bovine aortic endothelial cells were isolated from aortas obtained at a local abattoir and characterized by 1,1-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate-labeled acetylated low density lipoprotein uptake and staining for Factor VIII antigen. Stocks were maintained in Dulbecco's modified Eagle's medium (Cell-Gro, Mediatech, Herndon, VA) supplemented with 4.5 g/liter glucose, 2 mM glutamine, and 20% fetal bovine serum (Hyclone Laboratories, Logan, UT). NRK-49F cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD; CRL 1570), and stocks were maintained in Dulbecco's modified Eagle's medium supplemented with 4.5 g/liter glucose, 2 mM glutamine, and 10% calf serum (Hyclone Laboratories, Logan, UT) as described(25) . All cells tested negative for mycoplasma contamination.

NRK Colony Formation in Soft Agar

TGF-beta activity was assayed by determining the growth and colony formation of NRK cells in soft agar as described(23) . After 7 days of incubation, the number of colonies greater than 62 µm (8-10 cells) in diameter were counted. Experiments were performed in triplicate.

Activation of Recombinant Latent TGF-beta by sTSP

Equimolar amounts of sTSP or peptides (based on the molecular mass of TSP monomers) were incubated with 2 nM (200 ng/ml) recombinant latent TGF-beta in a final volume of 0.5 ml of PBS for 1 h at 37 °C. The concentration of 11 nM sTSP or peptides was used for most assays based on previous dose-response curves obtained with intact TSP(10) , which indicated that maximal activation of latent TGF-beta was obtained with this concentration of sTSP. 4 mM HCl was used as a positive control for activation. Samples were then tested for TGF-beta activity in the NRK colony-forming soft agar assay. Experiments were performed in triplicate.

ELISA for TGF-beta Activity

The ELISA assay was performed as described(10) . Briefly, 30 nM recombinant latent TGF-beta (1 µg/300 µl) was incubated with 56 nM sTSP (3 µg/300 µl) or equimolar amounts of peptides in a total volume of 300 µl of PBS for 5 min at 37 °C. Samples were then tested for increased TGF-beta activity using the TGF-beta Predicta kit (Genzyme Corp., Cambridge, MA) following the manufacturer's instructions. The ELISA utilizes monoclonal and polyclonal antibodies specific for an epitope within active TGF-beta. Increased TGF-beta activity was quantitated using a TGF-beta standard curve. Latent TGF-beta incubated with 1 N HCl for 1 h at 4 °C was used as a positive control for latent TGF-beta activation.

Peptide Binding Assays

Direct binding of I-TGF-beta to immobilized GGWSHW was determined as described previously(17, 18) . Briefly, the peptide was adsorbed on 96-well polyvinyl chloride microtiter plates (Falcon, Becton Dickinson, Oxnard, CA) for 2 h at 37 °C in a total volume of 100 µl of PBS. Nonspecific sites were blocked with 200 µl/well 1% BSA in PBS for 30 min at 37 °C. Wells were washed four times with wash buffer (PBS containing 1% BSA and 1% Triton X-100) and then incubated with 0.125 ng of I-TGF-beta (DuPont NEN) and test samples in 1% BSA/PBS in a total volume of 100 µl for 1.5 h at room temperature. The wells were washed three to four times, and the bound radioactivity was counted in the isolated wells using a counter. Experiments were performed in triplicate.

Additional Materials

Recombinant latent simian TGF-beta(1) was generously provided by Jane Ranchalis (Bristol-Myers Squibb, Seattle, WA). Fusion proteins corresponding to the intron-exon boundaries of the second TSP1 type 1 repeat were prepared by polymerase chain reaction amplification and expressed in Escherichia coli by glutathione S-transferase (24) . These proteins were generously provided by Dr. Jack Lawler, Harvard University. Heparin (disodium salt and low molecular weight forms) was purchased from Sigma.


RESULTS AND DISCUSSION

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-beta, human platelet TSP1, recombinant TSP1, and recombinant TSP2 were tested for their ability to activate latent TGF-beta. 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-beta with a response identical to that observed for human platelet TSP1 (Fig. 1). Although TSP2 is unable to activate latent TGF-beta, it does inhibit activation of latent TGF-beta 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-beta. This is a reasonable speculation given the ability of TSP2 to compete for the binding of I-active TGF-beta 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-beta activity.


Figure 1: Activation of latent TGF-beta is specific for TSP1. Recombinant latent TGF-beta (LTGF-beta) (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-beta 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-beta, synthetic peptides corresponding to known sequence motifs were obtained and tested for their ability to activate latent TGF-beta. 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-beta and then tested for TGF-beta 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-beta 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-beta 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-beta (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-beta 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-beta 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-beta, if not a molar excess.


Figure 2: Peptides containing the sequence RFK activate latent TGF-beta (LTGF-beta). a, recombinant latent TGF-beta (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-beta activity using the NRK colony-forming soft agar assay. Results are expressed as the means of triplicate determinations ± S.D. b, latent TGF-beta (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-beta 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-beta and then tested for TGF-beta activity in the NRK assay. These experiments showed that the sequence KRFK, amino acids 412-415, is sufficient to activate latent TGF-beta (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-beta.



In order to confirm the results of our biological assay, we also tested the peptides for their ability to activate latent TGF-beta in an immunological assay using an ELISA that selectively recognizes the active domain of TGF-beta(10) . Consistent with the results of the biological assay, incubation of latent TGF-beta with sTSP1, peptide 246, or KRFK resulted in generation of 140 pg of TGF-beta activity (Table 3). The VTCGGGVQKRSRL peptide was inactive in the ELISA. The results with the ELISA eliminate the possibility that activation of latent TGF-beta 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-beta 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-beta by KRFK also occurs in a complex cellular milieu, since KRFK activates bovine aortic endothelial cell-secreted TGF-beta 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-beta. 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-beta 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-beta 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-beta and tested for TGF-beta activity. Heparin failed to inhibit activation by the peptides (Fig. 3). In addition, heparin had no effect on colony formation by active TGF-beta (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-beta than for heparin and are capable of activating latent TGF-beta even when potentially bound to heparin (at concentrations greater than 10 µM).


Figure 3: Activation of latent TGF-beta by TSP1 peptides is not inhibited by heparin. Latent TGF-beta (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-beta (opencircles). Samples were then tested for TGF-beta 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-beta 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 TSPbulletTGF-beta 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-beta. 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. (^3)

Activation of latent TGF-beta 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-beta 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-beta, 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-beta (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-beta 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-beta 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-beta (Fig. 5a). In contrast, incubation of 100-fold molar excess (1.1 µM) GGWSHW with TSP completely inhibited activation of latent TGF-beta 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-beta from binding to TGF-beta receptors, since incubation of GGWSHW with active TGF-beta has no effect on TGF-beta 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-beta specifically bound to immobilized GGWSHW (Fig. 5b). 100-fold molar excess soluble GGWSHW and peptide 246 completely inhibit binding of I-TGF-beta to the peptide. However, a 100-fold molar excess of peptide 402 (KRFK) does not inhibit binding of I-TGF-beta to GGWSHW, indicating that binding of active TGF-beta 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-beta to GGWSHW. Soluble GGWSHW also inhibits the binding of I-TGF-beta to immobilized TSP (data not shown). These data suggest that GGWSHW-containing peptides inhibit activation of latent TGF-beta by TSP by blocking the ability of TSP to bind to the latent TGF-beta complex, and moreover this is the apparent mechanism by which TSP2 inhibits activation of latent TGF-beta 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-beta (data not shown). This implies that any of the three type 1 repeat WSXW motifs is potentially capable of binding to the active TGF-beta molecule.


Figure 5: Peptide GGWSHW inhibits sTSP-mediated activation of latent TGF-beta. a, latent TGF-beta (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-beta 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-beta (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-beta 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-beta, presumably by inhibiting the binding of TSP to TGF-beta. Based on the results of these studies, we propose that TSP1 utilizes a two-step mechanism for the activation of latent TGF-beta. The GGWSHW sequence in TSP1 and TSP2 mediates TSP-TGF-beta interactions, potentially through the mature portion of TGF-beta. Interactions between GGWSHW (or potentially DGWSPW from the first type 1 repeat or GGWGPW from the third type 1 repeat) and TGF-beta may orient the TSP1 molecule so that the RFK sequence is in a favorable configuration for interacting with latent TGF-beta. 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-beta complex. Initial data suggest that the KRFK sequence may recognize a site in the latency-associated peptide.^3 The interaction of the (K)RFK sequence with latent TGF-beta 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-beta but does not activate latent TGF-beta. Furthermore, TSP2 inhibits activation of latent TGF-beta by TSP1, presumably by TSP2 competing with TSP1 for binding to the mature portion of the latent TGF-beta complex. An anti-peptide 246 polyclonal antibody, which recognizes the GGWSHW sequence, also inhibits TSP1- and peptide 246-mediated activation of latent TGF-beta by 70% (data not shown).

These studies are significant in that they confirm that activation of latent TGF-beta by TSP results from TSP interactions with multiple binding sites within the latent TGF-beta complex. Peptides containing RFK and GGWSHW sequences have potential therapeutic uses in situations where TGF-beta 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-beta activity.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL50061 and American Cancer Society Grant CB-78 (to J. E. M.-U.), a predoctoral fellowship from the Department of Pathology (to S. S.-C.), Postdoctoral Fellowship HL08640 (to T. M. M.), and National Institutes of Health Grant HL49111 (to D. F. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, G038 Volker Hall, University of Alabama at Birmingham, Birmingham, AL 35294-0019. Tel.: 205-934-0415; Fax: 205-934-1775.

(^1)
The abbreviations used are: TGF-beta, transforming growth factor-beta; NRK, normal rat kidney; sTSP, thrombospondin free of TGF-beta activity; TSP, thrombospondin; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; EGF, epidermal growth factor.

(^2)
T. M. Misenheimer and D. F. Mosher, manuscript in preparation.

(^3)
S. Schultz-Cherry, S. M. F. Ribeiro, and J. E. Murphy-Ullrich, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Solange Ribeiro for many informative discussions and for critical review of the manuscript. We also thank Dr. Jack Lawler (Harvard University) for the polymerase chain reaction-based fusion constructs from the second type 1 repeat of TSP1; Dr. William Frazier (Washington University) for the Mal, Hep I, and Hep II peptides; and Jane Ranchalis (Bristol-Myers Squibb) for generously providing the latent TGF-beta.


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