Department of Cell Biology, Kaplan Cancer Center, and the Raymond and Beverly Sackler Foundation Laboratory, New York University Medical Center, New York 10016
Transforming growth factor- (TGF-
) is secreted by many cell types as part of a large latent complex composed of three subunits: TGF-
, the TGF-
propeptide, and the latent TGF-
binding protein (LTBP). To interact with its cell surface receptors,
TGF-
must be released from the latent complex by
disrupting noncovalent interactions between mature
TGF-
and its propeptide. Previously, we identified
LTBP-1 and transglutaminase, a cross-linking enzyme, as reactants involved in the formation of TGF-
. In this
study, we demonstrate that LTBP-1 and large latent
complex are substrates for transglutaminase. Furthermore, we show that the covalent association between
LTBP-1 and the extracellular matrix is transglutaminase dependent, as little LTBP-1 is recovered from matrix digests prepared from cultures treated with transglutaminase inhibitors. Three polyclonal antisera to
glutathione S-transferase fusion proteins containing
amino, middle, or carboxyl regions of LTBP-1S were used to identify domains of LTBP-1 involved in crosslinking and formation of TGF-
by transglutaminase.
Antibodies to the amino and carboxyl regions of
LTBP-1S abrogate TGF-
generation by vascular cell
cocultures or macrophages. However, only antibodies
to the amino-terminal region of LTBP-1 block transglutaminase-dependent cross-linking of large latent
complex or LTBP-1. To further identify transglutaminase-reactive domains within the amino-terminal region of LTBP-1S, mutants of LTBP-1S with deletions
of either the amino-terminal 293 (
N293) or 441 (
N441) amino acids were expressed transiently in
CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that
N293
LTBP-1S was matrix associated via a transglutaminasedependent reaction, whereas
N441 LTBP-1S was not.
This suggests that residues 294-441 are critical to the transglutaminase reactivity of LTBP-1S.
Most cell types secrete transforming growth factor- Interactions of the proteins involved in the activation of
large latent complex are not well understood. Plasmin can
release TGF- LTBP consists of a family of glycoproteins of ~120-210
kD that contain a central core of EGF-like repeats and
multiple unique eight-cysteine repeats (22, 28, 43, 46, 64).
LTBPs are structurally similar to the microfibrillar proteins fibrillin-1 and -2 (48, 50, 65). Defects in fibrillins are
responsible for the matrix fragility observed in patients
with Marfan syndrome and congenital contractural arachnodactyly (29, 47). The best characterized member of the
family is LTBP-1, which can exist as either short (LTBP1S) or long (LTBP-1L) forms (46). A number of cell types
secrete LTBP-1 as a higher order complex in which the
third eight-cysteine repeat in LTBP-1 is disulfide-linked to
the cysteine at position 33 of LAP (22a, 49). It is unknown whether the short and long forms of LTBP-1 are expressed
differentially. LTBP facilitates the secretion of small latent
complex, participates in the activation of large latent complex, targets large latent complex to the ECM of fetal rat
calvarial cells, fibroblasts, epithelial cells, and endothelial
cells, and contributes to the formation of fibrillar structures (13, 19, 41, 44, 59). LTBP-1S and -1L associate
differentially with the matrix, with LTBP-1L having a
greater affinity (46). Matrix association of LTBP-1 appears
to be covalent, as LTBP in the matrix is deoxycholate insoluble but is released upon proteolysis (Taipale, J., J. Saharinen, K. Hedman, and J. Keski-Oja. 1994. Mol. Biol.
Cell. 5[Suppl.]:311a). However, the mechanism of covalent
association between LTBP-1 and the ECM is unknown.
Transglutaminases are a family of structurally and functionally related calcium-dependent enzymes that catalyze
the formation of isopeptide bonds between Because LTBP-1 is structurally homologous to microfibrillar proteins that are stabilized by transglutaminasedependent cross-linking (10) and LTBP-2 has been localized to elastin-associated microfibrils (22), we explored the
possibility that the covalent association between LTBP-1
and ECM proteins is catalyzed by transglutaminase. Here
we show that LTBP-1 and large latent complex are substrates for transglutaminase both as isolated molecules
and in cell cultures. We demonstrate that matrix incorporation of LTBP-1 is transglutaminase-dependent and requires the amino-terminal region of LTBP-1 and that matrix association is an intermediate step in the activation
mechanism used by cells to generate TGF- Materials
Human plasmin (10 U/ml), isopropyl- Cell Culture
Human fibrosarcoma cells (HT 1080; American Type Culture Collection,
Rockville, MD) were grown in DME containing 10% (heat-inactivated) FCS, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, and 2 mM l-glutamine (P/S/Q). Rat osteosarcoma cells (UMR-106; American Type Culture Collection) were cultured in DME containing 10% (heat-inactivated) FCS and P/S/Q. Chinese hamster ovary cells (CHO K1) were cultured in
Primary BAE and BSM cells were isolated and cultured in Expression and Purification of Glutathione
S-Transferase-LTBP-1S Fusion Proteins
Three fusion proteins, each consisting of glutathione S-transferase (GST)
fused to a region of human fibroblast LTBP-1S, were generated by expressing pGEX1n constructs encoding LTBP-1S fragments in HB101 Escherichia coli followed by purification using glutathione-agarose (57). Fusion proteins GST-450, GST-1065, and GST-849 contain sequences from
the amino, middle, and carboxyl regions of human fibroblast LTBP-1S, respectively (Fig. 1) (28, 46).
pGEX-450 was constructed by cloning a 450-bp BanI fragment of the
LTBP-1S cDNA BP13 (nucleotides 419-869) (28) into a SmaI site of
pGEX1n. The pGEX-1065 construct was prepared by ligating a 1065-bp
BamHI-EcoRI fragment of the LTBP-1S cDNA BP13 (nucleotides 1087-
2152) and BamHI-EcoRI-digested pGEX1n. The pGEX-849 construct was generated by cloning an 849-bp StuI-SSpI fragment of the LTBP-1S cDNA BP13 (nucleotides 3779-4628) in the SmaI site of pGEX1n.
Once the insert orientation and sequence were verified for each construct, HB101 E. coli were transformed with the pGEX1n plasmid (control) or a pGEX-LTBP-1S expression construct by electroporation (Cell
Porator; GIBCO BRL, Gaithersburg, MD). Fusion proteins were purified
from IPTG-induced bacteria using glutathione-agarose according to the
manufacturer's instructions (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ). The integrity and apparent mol wts of the fusion proteins were
verified by reducing SDS-PAGE followed by Coomassie brilliant blue R
staining (26).
Preparation and Characterization of Antibodies to
GST-LTBP-1S Fusion Proteins
Polyclonal rabbit antisera raised against purified GST-450, GST-1065, or
GST-849 fusion proteins were prepared by Cocalico Biologicals, Inc.
(Reamstown, PA). Affinity-purified anti-450, anti-1065, and anti-849 IgG
were prepared by incubation of antisera with tosyl-activated agarose
(Pierce), to which fusion proteins had been coupled as recommended by
the manufacturer. Antibodies were characterized by Western blotting and
immunoprecipitation of recombinant large latent complex, recombinant
LTBP-1, or HT 1080 conditioned medium (CM).
Western blotting was performed using nonreducing or reducing conditions. Proteins separated by SDS-PAGE were transferred to Immobilon-P
membrane (Millipore Corp., Bedford, MA) (62). Membranes were blocked
with PBS containing 5% Carnation nonfat milk (Nestle Food Co., Glendale, CA), incubated with either Ab 39 serum or serum raised against
GST-LTBP fusion proteins in blocking buffer, washed with Tris-buffered
saline (150 mM NaCl, 50 mM Tris-HCl, pH 7.4) and 1% Triton X-100, incubated with either goat anti-rabbit IgG conjugated to alkaline phosphatase (Promega Corp., Madison, WI) or donkey anti-rabbit IgG conjugated to horseradish peroxidase (Amersham Corp., Arlington Heights,
IL), and washed as described above. Alkaline phosphate- and horseradish
peroxidase-containing immune complexes were revealed by incubating
the membrane with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium phosphatase substrate (Kirkegaard and Perry Laboratories, Inc.,
Gaithersburg, MD) and enhanced chemiluminescence detection reagents
(ECL; Amersham Corp.), respectively. The immunoreactivity of antisera
to GST-LTBP-1S fusion proteins was compared to that of Ab 39, a polyclonal antiserum raised against intact LTBP-1 purified from human platelets (40). The mol wts of large latent complex and LTBP-1 observed in our experiments are consistent with those reported by others (data not shown)
(28, 40).
Immunoprecipitations were performed using large latent complex (2-5
µg) or LTBP-1 (2-5 µg) iodinated using 250 µCi 125I-Na and 20 µg/ml
chloramine T as described by McConahey et al. (39). 35S-labeled HT 1080 CM was prepared by incubating confluent cultures overnight with DME
deficient in cysteine and methionine (Gibco Laboratories, Grand Island,
NY), 100 µCi/ml 35S-express translabel mix, and 1% normal DME culture
medium. Iodinated proteins and metabolically labeled CM were immunoprecipitated with Ab 39 serum or antiserum generated against one of the
three fusion proteins. Radiolabeled proteins were incubated with antiserum for 1-1.5 h at room temperature. Immune complexes were precipitated using protein A-agarose, washed with Tris-buffered saline and 1%
Triton X-100, and transferred to a 1.5-ml test tube followed by a water
wash. Protein A-precipitated complexes were solubilized in nonreducing
or reducing Laemmli sample buffer and separated by SDS-PAGE. Iodinated proteins were visualized by autoradiography, and metabolically labeled proteins were visualized by fluorography of gels treated with 1 M
sodium salicylate, dried, and exposed to film (X-OMAT; Eastman Kodak
Co., Rochester, NY) at Cross-Linking Reactions by Transglutaminase
Guinea pig liver transglutaminase was used at an enzyme/substrate molar
ratio of 1:5. Substrates for cross-linking reactions were iodinated using 250 µCi 125I-Na and 20 µg/ml chloramine T (39). Rabbit nonimmune IgG (1 mg/ml) was added as a carrier protein to all enzymatic assays, as IgG is not a
substrate for tissue transglutaminase (8). Human fibronectin (Collaborative Biomedical Products, Bedford, MA), recombinant large latent complex, and recombinant LTBP-1, all used at 167 nM, were incubated at
37°C for 1 h in 1.5-ml siliconized test tubes containing transglutaminase,
10 mM Tris-HCl, pH 8.0, 0.5 mM DTT, and 15 mM CaCl2. Specificity of
cross-linking reactions was verified by including either 100 µM MDC, a
competitive transglutaminase inhibitor, or 30 mM EDTA (33). Reactions
were stopped by addition of reducing SDS-PAGE Laemmli sample buffer.
Cross-linked proteins were separated by reducing SDS-PAGE, and gels
were examined by PhosphorImager scanning analysis (Molecular Dynamics, Sunnyvale, CA).
To identify regions of LTBP-1 that contain transglutaminase-reactive
sites, cross-linking reactions were performed as described above in the
presence of either affinity-purified antibodies generated against GST-
LTBP-1S fusion proteins or protein A-purified Ab 39, as well as 0.1 mg/
ml l-cystine, which was needed to minimize disulfide exchange within and
between proteins.
Immunoprecipitation of Matrix Digests Prepared from
Metabolically Labeled Cells
Subconfluent cultures were incubated in DME deficient in cysteine and
methionine for 1 h at 37°C. Cultures were treated with 0.06% DMSO
(control), 50 µM MDC, 100 µM cystamine, or affinity-purified antibodies
during the starvation and subsequent pulse-chase period. After the cells were
metabolically labeled for 3 h with 100 µCi/ml of l-[35S]cysteine in cysteine,
methionine-deficient DME supplemented with 2% Optimem (Gibco Laboratories), and 150 µg/ml of l-methionine, they were transferred to Optimem medium for an overnight incubation at 37°C in a 5% CO2 atmosphere. ECMs were prepared from metabolically labeled cells after lysis
with 0.5% sodium deoxycholate in Tris-buffered saline (10 mM Tris-HCl,
pH 8.0, and 150 mM NaCl) (59). Matrices were digested with 0.3 U/ml
plasmin in 0.1% n-octylglucoside, 3 mM MgCl2, 3 mM CaCl2, 10 mM TrisHCl, pH 8.0, and 150 mM NaCl for 1 h at 37°C. Plasmin was inhibited by
8 mM Pefabloc SC and 1 µg/ml aprotinin. Digests normalized to total
TCA-precipitable cpm as described by Harlow and Lane (26) were incubated with either nonimmune or Ab 39 serum, and immune complexes
were precipitated using protein A-agarose. Washed immunoprecipitates
were separated by reducing SDS-PAGE and visualized by fluorography.
Immunoprecipitation of Matrix Digests Prepared from
UMR-106 Matrices
35S-labeled CM were prepared using BAE or UMR-106 confluent cultures
that were metabolically labeled for 20 h with 100 µCi/ml of l-[35S]cysteine.
Metabolically labeled CM were concentrated fivefold using Centricon-30
ultrafilters (Amicon, Beverly, MA) and immediately added to UMR-106
ECMs prepared using 0.5% sodium deoxycholate in Tris-buffered saline
(59). Additions of antibodies or 50 µM MDC were done concurrently. To
catalyze the cross-linking of large latent complex present in radiolabeled
CM and UMR-106 matrix proteins, thrombin-activated Factor XIII (Factor XIIIa) was added (7). Each matrix incorporation reaction was done
using 1 ml of concentrated radiolabeled CM, 3 nmol of DTT-pretreated
Factor XIIIa, and 4 mM CaCl2 in Tris-buffered saline on 78.5 cm2 of
UMR-106 ECM for 1 h at 37°C. The LTBP-1 content of UMR-106 matrices was analyzed by immunoprecipitating plasmin digests of matrices with
Ab 39 followed by SDS-PAGE and fluorography of immunoprecipitates as described in this manuscript.
Transient Transfection of CHO Cells with LTBP-1S
cDNA Constructs
Three LTBP-1S cDNA constructs were generated using the pcDNA3 (InVitrogen, San Diego, CA) expression vector for transient expression of CHO
cells. The three constructs are pcDNA3-wild type (WT), pcDNA-
CHO cells were transiently transfected using lipofectamine (Gibco
Laboratories) according to the manufacturer's instructions. Briefly, cells
plated in 35-mm dishes received 1-4 µg of plasmid DNA premixed with lipofectamine in Optimem. After 24 h, media were harvested and replaced
with fresh Optimem. Transfected cultures were incubated for an additional 24 h, at which time media were collected and matrix digests were
prepared.
Western Blotting CM and Matrix Digests of Transfected
CHO Cultures
CM and matrix digests were prepared from transfected CHO cultures for
Western blotting analysis using Ab 39. The CM collected after the first
and second 24 h of transfection were pooled and concentrated 10-fold.
The concentrated CM and matrix digests were mixed with nonreducing
sample buffer for SDS-PAGE. Separated proteins were transferred electrophoretically to Immobilon-P for immunoblotting with Ab 39 serum, and
immune complexes were visualized using chemiluminescence reagents as
described earlier.
Preparation of CM from Cultures of BAE and BSM
Cells and Stimulated Macrophages
Serum-free CM were prepared from homo- and heterotypic cultures of
BAE and BSM cells and from LPS-stimulated thioglycollate-elicited macrophages as described in previous reports (44, 53, 54). CM collected after
14 h were immediately bioassayed for TGF- Mink Lung Epithelial Cells-Luciferase Assay
for TGF- This quantitative bioassay for TGF- Wound Migration Assay for TGF- To measure TGF- Transglutaminase Reactivity of LTBP and Large
Latent Complex
The apparent covalent interaction of LTBP-1 and ECM
(33, 36) as well as the requirement for tissue transglutaminase for activation of large latent complex described for
various culture systems suggested (30, 31, 44) that LTBP-1
and large latent complex might be substrates for transglutaminase. Therefore, iodinated recombinant LTBP-1
or large latent complex was incubated with guinea pig liver
transglutaminase and analyzed by reducing SDS-PAGE. Samples incubated with transglutaminase revealed high mol
wt complexes visible at the top of the 3% stacking polyacrylamide gel as well as intermediate-sized complexes at
the interface between the 3% stacking and 7% resolving
gels, indicating that both LTBP-1 and the large latent complex were polymerized by transglutaminase (Fig. 2). The
efficiency of cross-linking of either LTBP-1 or large latent
complex by transglutaminase varied among experiments as the relative amounts of polymerized and unreacted protein differed somewhat between reactions (data not shown).
This may reflect differences in the specific activity of different transglutaminase preparations. The observed polymerization of LTBP-1 and large latent complex implies
that LTBP-1 and the large latent complex each contain
both acyl donor and acyl acceptor sites necessary for formation of isopeptide bonds. Cross-linking LTBP-1 or large latent complex was Ca2+ dependent and transglutaminase
specific as generation of high mol wt complexes was attenuated by the addition of EDTA or MDC (Fig. 2) (33). MDC
acts as a competitive inhibitor of transglutaminase by competing for reactive glutamines (33). As large latent complex consists of small latent complex and LTBP-1, transglutaminase-dependent cross-linking of large latent complex to
itself could involve either LTBP-1, small latent complex,
or both. Cross-linking reactions using tissue transglutaminase and recombinant small latent complex also yielded
high mol wt complexes (data not shown), indicating it also
contains acyl donor and acceptor sites. However, this reaction is not responsible for the matrix incorporation of
large latent complex as shown by antibody inhibition studies (see below). Iodination of either LTBP-1 or large latent complex did not generate reactive sites absent in nonradiolabeled proteins because transglutaminase-catalyzed incorporation of MDC into nonradiolabeled LTBP-1 or
large latent complex was observed using black light to visualize MDC-containing proteins resolved by SDS-PAGE
(data not shown) (6).
Transglutaminase-dependent Cross-Linking of LTBP-1
and ECM by Cells
Both free LTBP-1 and large latent complex have been described to associate covalently with ECMs generated by fibroblasts, endothelial cells, and epithelial cells (59). The
results described above suggested the hypothesis that matrix incorporation of LTBP-1 and large latent complex occurs through transglutaminase-catalyzed cross-linking of
LTBP-1 and matrix proteins. To test this hypothesis, we
first confirmed that homotypic cultures of HT 1080 and
BAE cells as well as heterotypic cultures of BAE and BSM
cells generate matrix-associated LTBP-1. ECM-bound
LTBP-1 was analyzed by experiments in which matrix digests prepared from metabolically labeled cultures were
immunoprecipitated using Ab 39. Immunoprecipitated matrix digests prepared from HT 1080 and BAE cells, as well
as from cocultures of BAE and BSM cells, revealed fragments of 120-140 and 180-210 kD, respectively (Fig. 3).
These matrix fragments specifically reacted with Ab 39 because they were not observed in control immunoprecipitation experiments with nonimmune serum (data not shown).
The difference in mol wt of matrix fragments immunoprecipitated with Ab 39 obtained from HT 1080 and vascular
cells is consistent with a report that LTBP-1 recovered
from matrix digests prepared from different cell types varies in mol wt (61). The differences in size of the LTBP-1
fragments recovered from the ECM may reflect variations in matrix composition, in differential expression of LTBP-1
isoforms incorporated into the ECM (26), or in endogenous proteolytic activities in the cell culture.
Having confirmed that LTBP-1 was present in the ECM,
we next examined whether the incorporation of endogenous LTBP-1 into the ECM by cells was transglutaminase
dependent. Matrices were prepared from cultures metabolically labeled in the presence of the transglutaminase
competitive inhibitors MDC or cystamine (33), and the
LTBP-1 content of the ECM was examined by digesting
matrices with plasmin and immunoprecipitating the digests with Ab 39 followed by SDS-PAGE analysis. Significantly less LTBP-1 was recovered from matrices prepared
from cultures of HT 1080, BAE, and BSM cells treated
with MDC or cystamine than from control cultures (Fig. 4,
A and B). Matrix digests from cultures incubated with transglutaminase inhibitors contained 2-10-fold less LTBP-1
than respective control cultures as determined by laser
scanning densitometry. Cells treated with MDC incorporated less LTBP-1 into the matrix than did cystaminetreated cells (Fig. 4 A, second and fourth lanes), probably
because the concentration of cystamine used in cultures was below the saturating concentration needed to completely inhibit pericellular transglutaminase activity. Levels of MDC or cystamine greater than those used in our
experiments may have been necessary to completely inhibit matrix incorporation of LTBP-1. However, higher
levels of these transglutaminase inhibitors were toxic to
cells. To insure that results obtained using transglutaminase inhibitors did not result from effects on total protein
synthesis and secretion, all matrix digests were normalized
to total TCA precipitable cpms before immunoprecipitating with Ab 39 serum (11). In addition, the effect of MDC
on protein synthesis and secretion was monitored by immunoprecipitating fibronectin from metabolically labeled
CM harvested from untreated and MDC-treated HT 1080 cells. Fibronectin levels in treated and untreated HT 1080 CM were found to be equivalent (data not shown). Assuming that the transglutaminase inhibitors did not selectively affect LTBP-1 expression, the decreased amount of
LTBP-1 observed in matrix digests prepared from cultures
treated with inhibitors suggests that the matrix incorporation of LTBP-1 is transglutaminase dependent.
In similar experiments, twice as much LTBP-1 was recovered from BAE matrix digests than from BSM matrix
digests as determined by immunoprecipitating plasmin digests of metabolically labeled ECM using Ab 39 (Fig. 4 B).
This may reflect higher levels of tissue transglutaminase
expression by BAE cells compared to BSM cells rather
than differences in LTBP-1 expression, as BSM cells have
been reported to produce more LTBP-1 than BAE cells
(19, 31).
To establish whether the effect of transglutaminase inhibitors on the matrix incorporation of LTBP-1 as observed in culture was a consequence of altered matrix assembly and stabilization, matrix incorporation reactions
were done using matrices from untreated UMR-106 cells,
35S-labeled BAE CM as an exogenous source of large latent complex, and the plasma transglutaminase Factor XIIIa.
Matrices from UMR-106 cells were used because UMR106 cells produce latent TGF-
Identification of LTBP-1 Domains Involved in
Cross-Linking by Transglutaminase
Results presented in Figs. 2, 4, and 5 demonstrate that
LTBP-1 and large latent complex are substrates for transglutaminase and that matrix incorporation of large latent
complex is transglutaminase dependent. To determine
whether LTBP-1 is necessary for cross-linking of large latent complex, antibodies to intact LTBP-1 or different LTBP-1 sequences were included in cross-linking reactions of transglutaminase and iodinated large latent complex as described previously. Ab 39 inhibited transglutaminase-dependent cross-linking of large latent complex, as
measured by the absence of polymerized large latent complex present as a high mol wt band in the 3% stacking polyacrylamide SDS gel (Fig. 6 A). Ab 39 did not interfere
with cross-linking of small latent complex by transglutaminase (data not shown). This indicated that cross-linking of
the large latent complex probably requires lysine or glutamine residues present in LTBP-1. To identify what region
of LTBP-1 contains transglutaminase reactive residues, affinity-purified anti-450 and anti-849 IgG were added to
transglutaminase-dependent cross-linking reactions of iodinated LTBP-1 and large latent complex. Addition of
anti-450 IgG blocked cross-linking of large latent complex
and LTBP-1 to itself, as measured by the decreased formation of high mol wt polymers observed in the stacking gel,
whereas nonimmune, anti-849, and anti-1065 IgGs did not
(Fig. 6). These findings indicate that transglutaminasereactive sites are present in the amino-terminal region of
LTBP-1.
To establish whether the amino-terminal region also
contained transglutaminase reactive sites involved in the
matrix association of LTBP-1, affinity-purified antibodies
to the three GST-LTBP-1S fusion proteins were added to
cultures of HT 1080 and BAE cells throughout a pulsechase metabolic labeling period. Because HT 1080 cells
are proteolytically more active than BAE cells (18), we
added aprotinin, a serine protease inhibitor, to HT 1080 cultures to prevent antibodies from being degraded by endogenous proteases. The amount of LTBP-1 incorporated
in the matrix by antibody-treated HT 1080 and BAE cells
was assessed by immunoprecipitating matrix digests with
Ab 39 followed by SDS-PAGE. The immunoprecipitation experiments revealed that the amount of LTBP-1 recovered from matrices prepared from cultures treated with
anti-450 IgG was significantly decreased compared to that
recovered from cultures treated with nonimmune, anti1065, or anti-849 IgG (Fig. 7 A). Laser scanning densitometry of fluorographs revealed that digests prepared from
HT 1080 and BAE cultures receiving anti-450 IgG contained 5-10-fold and 70-fold less LTBP-1, respectively, than matrices prepared from cultures treated with the other antibodies. Addition of aprotinin did not affect the deposition of LTBP-1 into the matrix, as the cpms recovered by
immunoprecipitating matrix digests prepared from aprotinin-treated and untreated cultures with Ab 39 were similar
(data not shown). The weaker effect of anti-450 IgG on
HT 1080 versus BAE cells may reflect degradation of the
antibodies by metalloproteases; however, this was not verified. These results support findings reported by others
that incorporation of LTBP-1 into the matrix requires its
amino-terminal residues (46, 49).
To confirm the observation that addition of anti-450
IgG to cells producing large latent complex interferes with
the covalent incorporation of the complex to the ECM,
matrix incorporation reactions were done using UMR-106
matrices, 35S-labeled BAE CM, and Factor XIIIa, as described earlier, in the presence of antibodies to GST-
LTBP-1S fusion proteins. Western blotting analysis of matrix digests with Ab 39 indicated that addition of anti-450
IgG attenuated matrix deposition of large latent complex,
as less LTBP-1 was present in digests from matrix incorporation reactions containing anti-450 IgG compared to those receiving nonimmune, anti-1065, or anti-849 IgG (Fig. 7 B).
Results from these matrix incorporation reactions confirm
that cross-linking large latent complex to the ECM involves transglutaminase reactive residues at the amino terminus of LTBP-1.
Transglutaminase Reactivity of
Amino-truncated LTBP-1S
The antibody to the amino terminus of LTBP-1S (anti-450
IgG) identified the amino-terminal region of LTBP-1 as
containing transglutaminase-reactive sites. However, localization of reactive residues within the amino terminus is
not possible using antibodies, as their inhibitory effect may
be due to steric hindrance rather than binding to reactive
residues. To identify domains within the amino-terminal
region of LTBP-1S that contain the transglutaminase-reactive site(s), amino-terminal truncated mutants Effect of Antibodies to GST-LTBP-1S Fusion Proteins
on Latent TGF- Activation of latent TGF- Serum-free heterotypic cultures of BAE and BSM cells
were used as a cell system that generates mature TGF-
The results obtained using the MLEC-luciferase assay
to measure mature TGF- To determine whether the inhibitory effect of anti-450
or anti-849 IgG on latent TGF- The reactants involved in the activation of latent TGF- Matrix incorporation of LTBP-1 or large latent complex
(reported by others and reproduced by us) appears to occur through transglutaminase-dependent cross-linking of
LTBP-1 and matrix protein(s) (60). We observed that both
large latent complex and LTBP-1 are substrates for transglutaminase, the LTBP-1 of large latent complex contains
residues involved in cross-linking large latent complex, and
the inhibition of transglutaminase severely attenuates the
incorporation of LTBP-1 into the matrix. Transglutaminase-reactive sites of LTBP-1S appear to be located within
residues 294 and 441 of the amino terminus, as an LTBP-1S
mutant truncated at its amino terminus by 441 amino acids
was not cross-linked to the matrix, whereas a 293-amino
acid truncated LTBP-1S retained its matrix association.
Transglutaminase is more selective for glutamine residues than acyl acceptor sites (25). A consensus sequence
for glutamine residues that serve as amine acceptor sites in
transglutaminase-catalyzed cross-linking has not been described. However, LTBP-1S shares some structural features present in other transglutaminase substrates. Sequence analysis of reactive glutamine residues reveals that they are exposed in loop structures, as the glutamines tend
to be surrounded by positively or negatively charged amino
acids (4). In 60% of the substrates, glutamine residues are
located at the amino or carboxyl terminus. From sequence
alignment analysis of human LTBP-1S and LTBP-2, rat
LTBP-1 and murine LTBP-3 (28, 43, 45, 64), glutamine374 and a positively flanking amino acid within the second
cysteine-rich repeat of LTBP-1S is conserved among all
LTBPs, suggesting that it may be a transglutaminase reactive site. Glutamine-374 is present in The matrix protein(s) to which LTBP-1 is cross-linked
has not been identified. However, LTBP-1 has been described to colocalize with fibronectin (Taipale, J., J. Saharinen, K. Hedman, and J. Keski-Oja. 1994. Mol. Biol. Cell.
5[Suppl.]:311a) and with collagen-free fibrillar structures
generated by fetal rat calvarial cells (13). LTBP-2 appears
to closely associate with elastin-associated microfibrils (22).
Thus, fibronectin and proteins of elastic fibers, such as microfibril-associated glycoprotein, are candidate proteins, as they all contain residues susceptible to transglutaminase-catalyzed isopeptide bond formation (10, 37).
In addition to localizing the transglutaminase reactivity
of LTBP-1S to amino-terminal residues 294-441, LTBP-1S
domains participating in latent TGF- We have demonstrated that the carboxyl-terminal region of LTBP-1 is required for large latent complex activation, as antibodies to this region blocked TGF- Matrix incorporation of large latent complex may create
a concentrated pool of large latent complex. Others have
proposed that segregating pools of latent TGF- We propose that the activation of large latent complex,
to release, TGF-
This model suggests several testable questions. These include the potential significance of the putative plasmin
cleavage sites in LTBP-1S, the availability of the mannose
6-phosphate residues of LAP in the complex, and the location of plasmin-sensitive sites of LAP. It is also unknown
whether large latent complexes that differ in their LTBP
isoforms are activated by a similar mechanism. Answers to
these questions can be approached by altering specific residues in the components of large latent complex and monitoring rates of large latent complex activation.
1 (TGF-
)1 in a biologically inactive form
(42). Mature TGF-
is a homodimer composed
of two 12.5-kD polypeptides joined by a disulfide bond at
cysteine 77 (14). The monomeric subunits are produced by intercellular cleavage of a higher mol wt precursor at a
dibasic site immediately preceding Ala-279 (17, 21). However, after secretion the propeptides remain associated
with TGF-
through noncovalent interactions, rendering
TGF-
inactive (20). TGF-
with its propeptide, also known
as the latency associated peptide (LAP), is referred to as
the small latent complex. Both in vitro and in vivo, latent
TGF-
is secreted as part of a large latent complex in
which a second gene product, the latent TGF-
binding protein (LTBP), is disulfide-linked to LAP (42). The dissociation of TGF-
from LAP is required for TGF-
to
bind to its receptors and exert its effects on cell proliferation, extracellular matrix (ECM) deposition, cell migration, and differentiation (34, 38, 58). Latent TGF-
is activated by heat, acid or alkaline treatment, binding to
thrombospondin, deglycosylation, proteolysis, or irradiation (5, 9, 34, 35, 56). The most extensively studied process for activating large latent complex is a plasmin-dependent
mechanism observed in several tissue culture systems including bovine aortic endothelial (BAE) cells treated with
retinoids, cocultures of endothelial cells and either smooth
muscle cells or pericytes, and lipopolysaccharide (LPS)-
stimulated, thioglycollate-elicited peritoneal macrophages
(30, 31, 44, 54). Components of this activation mechanism
include the serine protease plasmin, the cross-linking enzyme transglutaminase, LTBP-1, and the mannose 6-phosphate/insulin-like growth factor type II receptor, which appears to bind to mannose 6-phosphate residues in LAP
(16, 19, 31, 54, 55).
from large latent complex under cell-free
conditions (36). The mannose 6-phosphate/insulin-like
growth factor type II receptor binds forms of latent TGF-
,
but the role of this interaction is not clear (32). The role of
LTBP-1 or tissue transglutaminase in large latent complex
activation is not known. As part of an effort to characterize the activation process of large latent complex, we have
initiated studies to examine the potential interactions of
LTBP-1 and tissue transglutaminase.
-carboxamide
groups of glutamine residues and
-amino groups of lysine
residues (24, 33). The family is comprised of five members:
plasma Factor XIII, keratinocyte transglutaminase, tissue/
endothelial transglutaminase, epidermal transglutaminase,
and prostate transglutaminase. Although transglutaminase does not have a signal sequence, the enzyme can be localized to cell surfaces as well as the ECM and reacts to stabilize interactions between extracellular substrates such as
plasminogen, fibronectin, nidogen, and vitronectin (3, 6,
37, 51, 63). Pericellular transglutaminase may play a role in
stabilizing cell and ECM interactions (24, 37). Transglutaminase is required for the conversion of large latent complex to TGF-
in several systems, including retinoid-treated
BAE cells, LPS-stimulated thioglycollate-elicited peritoneal
macrophages, and cocultures of BAE and bovine smooth muscle cells (BSM) (30, 31, 44).
.
Materials and Methods
-d-thiogalactopyranoside (IPTG),
1-o-n-octyl-
-d-glucopyranoside (n-octylglucoside), Pefabloc SC, and protein A-agarose were purchased from Boehringer Mannheim Corp. (Indianapolis, IN). Cystamine, glutathione-agarose, guinea pig liver transglutaminase, monodansylcadaverine (MDC), and nonimmune rabbit serum were
purchased from Sigma Chemical Co. (St. Louis, MO). Pyrogen-poor BSA
was purchased from Pierce (Rockford, IL). l-[35S]cysteine, 35S-labeled express translabel mix, and 125I-Na (pH 12-14, high concentration) were obtained from Du Pont Company Biotechnology Systems (Wilmington,
DE). Recombinant human TGF-
1 was a gift from Berlex Biosciences
(South San Francisco, CA). Pan-neutralizing monoclonal mouse anti-TGF-
IgG1 was either purchased from Genzyme (Cambridge, MA) or donated by
Celtrix (Santa Clara, CA) (15). Recombinant human LTBP-1S, human
large latent complex, and small latent complex were provided by Drs.
Hideya Ohashi and Haruhiko Tsumura (KIRIN Brewery Co., Ltd., Pharmaceutical Division, Gunma, Japan). Recombinant Factor XIII (166 kD)
was provided by Dr. P.D. Bishop (ZymoGenetics Inc., Seattle, WA) (7). The human fibroblast LTBP-1S cDNA (pSV7d-BP13) was a gift from Drs. K. Miyazono and C. Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden) (28, 46). Ab 39, a polyclonal rabbit anti-LTBP IgG, was
a gift from Dr. K. Miyazono (40). Ab 39 was generated against human
LTBP purified from human platelets and recognizes LTBP-1 but not
LTBP-2 (28, 43).
-MEM containing 10% (heat-inactivated) FCS and P/S/Q.
-MEM and
DME containing 10% (non-heat inactivated) calf serum plus P/S/Q, respectively (53). Stimulated peritoneal macrophages were harvested from
Swiss Webster mice, which had been injected i.p. with 4% thioglycollate
broth and activated by LPS in vitro as previously described (44).
Fig. 1.
Regions of LTBP-1S expressed in bacteria as GST-
LTBP-1S fusion proteins. Three GST-LTBP-1S fusion proteins
(GST-450, GST-1065, and GST-849) were generated by expressing regions of the human LTBP-1S cDNA using pGEX1n in E. coli.
Representation of LTBP-1S was adapted from Kanzaki et al. (28).
[View Larger Version of this Image (20K GIF file)]
80°C for 7-10 d. Anti-450 and anti-849 antisera
immunoprecipitated radiolabeled forms of LTBP-1 but were not as effective as Ab 39 (data not shown). Anti-1065 antisera failed to recognize any
of the radiolabeled preparations containing LTBP-1 (data not shown).
Anti-1065 antiserum was used as a negative control because its reactivity
with nondenatured LTBP-1 was negligible. These results indicated that
the antibodies to the amino- and carboxyl-terminal regions could be used
for immunoprecipitation of LTBP-1 and in experiments examining the role of the amino and carboxyl termini of LTBP-1 in the cross-linking and
activation of large latent complex.
N293,
and pcDNA-
N441 (see Fig. 8 A). To generate pcDNA3-WT LTBP-1S,
the fragment 68-4543 (DraI-DraI) of the human LTBP-1S cDNA BP13
(28) was subcloned into pcDNA3 downstream of the human cytomegalovirus immediate early gene promoter. pcDNA3-
N293 and pcDNA3
N441 were obtained by digesting BP13 with HpaI and DraI (nucleotides
1414-4543), and ScaI and DraI (nucleotides 970-4543), respectively; these
fragments were fused in frame with the baculovirus glycoprotein GP67
signal sequence as described elsewhere (22a) and subcloned into pcDNA3.
Fig. 8.
Expression and matrix incorporation of amino-truncated LTBP-1S mutants by CHO cells. (A) LTBP-1S cDNA constructs were generated by subcloning LTBP-1S cDNAs into
pcDNA3 for transient transfection of CHO cells. pcDNA3-WT
LTBP-1S encodes the entire coding sequence of LTBP-1S.
pcDNA3-N293 and -
N441 LTBP-1S encode LTBP-1S truncated at the amino terminus by 293 and 441 amino acids, respectively. (B) CM from untransfected (
) and LTBP-1S cDNA-
transfected CHO cells were immunoblotted using Ab 39 serum.
The LTBP-1S content of matrix digests from corresponding cultures was analyzed by immunoblotting digests using Ab 39 serum.
[View Larger Version of this Image (57K GIF file)]
activity (see below). TGF
-dependent activity was verified by adding neutralizing anti-TGF-
IgG
to parallel test samples before assay. Total TGF-
, which consists of cellactivated plus latent TGF-
, was quantitated after heating CM at 85°C for
12 min, a procedure known to convert latent TGF-
to TGF-
, before assay (9).
Activity
is based on the ability of TGF-
to
stimulate plasminogen activator inhibitor-1 (PAI-1) expression (1). Mink
lung epithelial cells (MLEC), stably transfected with an expression construct containing a truncated PAI-1 promoter fused to the firefly luciferase reporter gene, were incubated overnight with CM harvested from
either stimulated macrophages or cultures of BAE and BSM cells. Recombinant TGF-
1 in
-MEM or DME with 0.1% BSA was used to generate
a standard curve of TGF-
activity. Luciferase activity was quantitated using a luciferin substrate buffer in a ML3000 Microtiter Plate Luminometer
(Dynatech Laboratories Inc., Chantilly, VA) (1).
Activity
present in CM, wound migration assays were performed as described by Sato and Rifkin (52). This bioassay is based on the
ability of TGF-
to inhibit endothelial cell migration. Confluent monolayers of BAE cells plated in a 35-mm dish were wounded with a razor blade
and incubated with serum-free test samples for 18-20 h at 37°C in a 5%
CO2 atmosphere. Cells were fixed and stained with 0.5% crystal violet in
20% methanol (44). BAE cells that had migrated from the edge of the
wound were counted in successive 125-µm increments at 100× using a
light microscope with an ocular grid. The number of cells migrating beyond 125 µm from seven different fields was averaged. Data are presented as a percentage of control, where the control sample consists of homotypic
BAE and BSM media mixed 4:1.
Results
Fig. 2.
SDS-PAGE analysis of cross-linking large latent complex or LTBP-1 by transglutaminase. (A) Iodinated large latent
complex (LLC) was incubated with guinea pig liver transglutaminase (TGase) in the presence or absence of EDTA and CaCl2.
Proteins were separated by reducing SDS-PAGE using a 3%
stacking and 7% resolving gel and were revealed by PhosphorImager scanning. (B) Iodinated LTBP-1 was incubated with guinea
pig liver transglutaminase in the presence or absence of MDC.
Proteins were separated by reducing SDS-PAGE using a 3%
stacking and 12% resolving gel and were revealed by PhosphorImager scanning.
[View Larger Version of this Image (49K GIF file)]
Fig. 3.
Presence of LTBP-1
in matrix digests prepared
from HT 1080, BAE, and cocultures of BAE and BSM
cells. Subconfluent cultures of HT 1080 and BAE cells
and cocultures of BAE and
BSM cells were pulsed with
[35S]cysteine for 3 h and
chased overnight. Matrices
were prepared, followed by
digestion with 0.3 U/ml of
plasmin. Digests were immunoprecipitated using Ab 39 serum followed by protein
A-agarose. Immunoprecipitates were analyzed by SDSPAGE followed by fluorography. , 120-140-kD LTBP-1
matrix fragments.
, 180-
210-kD LTBP-1 matrix fragments.
[View Larger Version of this Image (47K GIF file)]
Fig. 4.
LTBP-1 content of matrix digests prepared from cultures treated with transglutaminase inhibitors. (A) Subconfluent
cultures of HT 1080 cells were either untreated or treated with
50 µM MDC, 0.06% DMSO, or 100 µM cystamine during the 3-h
pulse with [35S]cysteine and overnight chase. (B) Subconfluent
homotypic and heterotypic cultures of BAE and BSM cells were
either untreated or MDC-treated (50 µM) during the pulse-chase
period as described for HT 1080 cells. Matrix digests were prepared from all cultures and immunoprecipitated using Ab 39 serum. Immunoprecipitates were analyzed by SDS-PAGE followed
by fluorography.
[View Larger Version of this Image (37K GIF file)]
only as the small latent
complex (12). We confirm that large latent complex was
not generated by our UMR-106 cells, as no LTBP-1 was detected in Ab 39 immunoprecipitates of 35S-metabolically
labeled CM (Fig. 5 A, first lane). Metabolically labeled CM
from BAE cells was used as an exogenous source of large latent complex, as we have previously observed (19), and
confirmed that they produce LTBP-1 as large latent complex (220 kD; Fig. 5 A, second lane ). The LTBP-1 content
of UMR-106 matrices incubated with radiolabeled BAE
CM in the absence or presence of MDC was analyzed by
digesting matrices with plasmin and immunoprecipitating digests with Ab 39 (Fig. 5 B). Visualization of immunoprecipitates by fluorography revealed that matrices reacted
with transglutaminase and BAE CM contained crosslinked LTBP-1 (Fig. 5 B, first lane), whereas digests from a
parallel matrix reaction containing MDC did not (Fig. 5 B,
third lane). Control matrix incorporation reactions of
UMR-106 CM and UMR-106 matrices confirmed that the
ECM of UMR-106 cells does not contain endogenous
LTBP-1, as no LTBP-1 was recovered from matrix digests.
Results from matrix incorporation experiments indicate
that the decreased levels of matrix-associated LTBP-1 observed in cultures treated with MDC were due to the inhibition of cross-linking by transglutaminase rather than effects on matrix assembly and stability.
Fig. 5.
Cross-linking LTBP-1 in BAE CM to UMR-106 ECM
by Factor XIIIa. (A) UMR-106 and BAE cultures were metabolically labeled using [35S]cysteine and CM were immunoprecipitated using Ab 39 serum. LTBP-1 immunoprecipitates (IPs) from
CM were separated by nonreducing SDS-PAGE and visualized
by fluorography. (B) Matrix incorporation reactions were done
using metabolically labeled CM from BAE or UMR-106 cells and
UMR-106 ECMs. The cross-linking reaction was catalyzed by
Factor XIIIa in the absence () or presence (+) of 50 µM MDC.
Matrix digests were prepared by plasmin digestion and immunoprecipitated using Ab 39 serum. Immunoprecipitates were analyzed by reducing SDS-PAGE and fluorography.
[View Larger Version of this Image (48K GIF file)]
Fig. 6.
Effect of antibodies to LTBP-1 on
transglutaminase cross-linking of large latent
complex or LTBP-1. Ab 39 IgG (0.2 mg/ml) and
affinity-purified IgGs (0.8 mg/ml) generated
against GST-450, GST-1065, or GST-849 were
added to transglutaminase cross-linking reactions of iodinated large latent complex (LLC)
(A) or LTBP-1 (B). The presence of high mol wt
complexes was revealed by reducing SDS-PAGE
using a 3% stacking and 7% resolving gel followed by PhosphorImager scanning.
[View Larger Version of this Image (46K GIF file)]
Fig. 7.
Effect of antibodies to LTBP-1 on matrix incorporation
of LTBP-1. (A) Subconfluent cultures of HT 1080 and BAE cells
were treated during the 3-h pulse and overnight chase labeling
period with either protein A-purified nonimmune IgG or affinity-purified IgGs (20 µg/ml) generated against GST-450, GST1065, or GST-849. HT 1080 cultures also received 50 µg/ml aprotinin to prevent degradation of exogenously added antibodies.
Matrix digests were prepared and immunoprecipitated using Ab
39 followed by SDS-PAGE and fluorography. (B) Matrix incorporation reactions of 35S-labeled BAE CM and UMR-106 ECMs
were done in the absence () of additions or in the presence of
protein A-purified non-immune rabbit IgG or affinity-purified
IgGs (20 µg/ml) generated against GST-450, GST-1065, and
GST-849. Matrix digests were prepared as previously described
and their LTBP-1 content was analyzed by immunoblotting using
Ab 39 serum as described in Materials and Methods.
[View Larger Version of this Image (48K GIF file)]
N293 and
N441 LTBP-1S cDNAs (Fig. 8 A) were constructed for
transient transfection of CHO cells. Control cultures were
either untransfected or WT LTBP-1S cDNA-transfected
cells. CHO cells transfected with WT,
N293, or
N441
LTBP-1S cDNA expressed and secreted LTBP-1S protein
accordingly as determined by Western blotting CM using
Ab 39 (Fig. 8 B). Untransfected (Fig. 8 B) cells, as well as
mock (pcDNA3-cat) transfected cells (data not shown),
produce low levels of LTBP-1 compared to cells transfected with LTBP-1S cDNA constructs. To determine if
CHO cells incorporate LTBP-1S into ECM, matrix digests
were prepared by plasmin treating deoxycholate-extracted
matrices followed by immunoblotting using Ab 39. Matrix
digests prepared from cells transfected with WT and
N293 LTBP-1S cDNA contained LTBP-1S, whereas little to no LTBP-1S was detected in digests from untransfected and
N441 LTBP-1S cDNA-transfected cells (Fig.
8 B). To determine whether matrix association of WT and
N293 LTBP-1S was transglutaminase dependent, MDC
was added to untransfected and transfected CHO cultures.
Western blotting analysis of the LTBP-1S content of matrix digests generated from MDC-treated transfected CHO
cells revealed negligible levels of LTBP-1S present in digests from WT and
N293 LTBP-1S cDNA-transfected
cells, indicating that matrix association was transglutaminase dependent (data not shown). Results from the LTBP1S transfection experiments indicate that, unlike
N441
LTBP-1S,
N293 LTBP-1S was cross-linked to matrix suggesting that residues 294-441 are critical to the transglutaminase-dependent reactivity of LTBP-1S.
Activation
by cocultures of BAE and
BSM cells as well as by stimulated macrophages requires
transglutaminase and LTBP-1 (19, 31, 44). Transglutaminase-dependent cross-linking of LTBP-1 into the matrix
by cocultures is inhibited by anti-LTBP-1 antibodies (Fig.
4 B). Therefore, we examined whether antibodies to GST-
LTBP-1S fusion proteins affected activation of latent TGF-
using cocultures of BAE and BSM cells and cultures of
stimulated macrophages.
from endogenous large latent complex (14). Control cultures were serum-free homotypic cultures of BAE and
BSM cells. The cocultures of BAE and BSM cells received
either affinity-purified antibodies to one of the three
GST-LTBP-1S fusion proteins, protein A-purified Ab 39, or nonimmune IgG, as indicated in Fig. 9. After an overnight incubation, CM from these cultures were bioassayed
immediately for mature TGF-
using the MLEC-luciferase
assay. These experiments revealed that CM from cocultures treated with Ab 39, anti-450, or anti-849 IgG contained less mature TGF-
than CM from untreated cocultures or cocultures treated with nonimmune or anti-1065
IgG (Fig. 9 A). We previously observed that Ab 39 IgG
blocks latent TGF-
activation by cocultures of BAE and
BSM cells (19). To verify that the measured luciferase activity was TGF-
dependent, neutralizing anti-TGF-
IgG
was added to CM from untreated cocultures and found to
inhibit 86% of the luciferase response (Fig. 9 A), indicating that most of the measured luciferase activity was TGF-
dependent. Anti-450 and anti-849 IgG blocked TGF-
generation at ID50's of 20 and 1 µg/ml, respectively. CM
from cocultures treated with the anti-LTBP-1S IgGs contained levels of latent TGF-
similar to those from untreated cocultures, as determined by the MLEC-luciferase
assay (data not shown). Furthermore, addition of the anti-
LTBP-1S IgGs to CM from untreated cocultures did not
inhibit TGF-
activity (data not shown). Therefore, the effects of the antibodies did not result from changes in total
TGF-
levels.
Fig. 9.
Effect of antibodies to LTBP-1 on latent TGF- activation by cocultures of BAE and BSM cells, and stimulated macrophages. (A) The MLEC-luciferase assay was used to detect
TGF-
activity generated by cocultures of BAE and BSM cells.
Serum-free CM harvested from either untreated cultures or cultures treated with 10 µg/ml Ab 39 IgG, 50 µg/ml nonimmune
IgG, or 50 µg/ml of affinity-purified anti-450, anti-1065, or anti849 IgG were assayed using the MLEC-luciferase assay. To verify
that luciferase production was TGF-
-dependent, 20 µg/ml neutralizing anti-TGF-
IgG was added to CM from untreated cultures. Data presented are from one out of four experiments yielding similar results. (B) The wound migration assay was used to
detect TGF-
activity generated by cocultures of BAE and BSM cells. Serum-free CM harvested from untreated cocultures or cocultures treated with the same antibodies as in A were assayed
for TGF-
activity using the wound migration assay described in
Materials and Methods. Control (100%) is the migration observed in wounded BAE cultures treated with media from homotypic BAE and BSM cells mixed 4:1. These data are presented as
the percentage of migration observed in wounded cultures
treated with control media. Data are representative of four experiments yielding similar results. (C) The MLEC-luciferase assay was used to detect TGF-
activity generated by stimulated
macrophages. Serum-free CM from either untreated or antibodytreated cultures of LPS-stimulated thioglycollate-elicited peritoneal macrophages were harvested and assayed. Antibody treatments were as in A and B. Data presented are from one out of
three experiments yielding similar results.
[View Larger Version of this Image (22K GIF file)]
levels present in coculture CM
were confirmed using the wound migration assay, in which
the inhibitory effect of TGF-
on endothelial cell migration is quantitated. We observed that CM from cocultures
of BAE and BSM cells treated with Ab 39, anti-450, or
anti-849 IgG did not inhibit the migration of BAE cells,
nor did anti-TGF-
IgG-containing CM from untreated cocultures (Fig. 9 B). CM from cocultures treated with nonimmune or anti-1065 IgG inhibited the migration of endothelial cells by ~50% (Fig. 9 B). These results are similar
to those obtained using the MLEC-luciferase assay, indicating that less TGF-
is present in CM prepared from cocultures treated with Ab 39, anti-450, or anti-849 IgG.
activation was specific to
cocultures of BAE and BSM cells, we examined whether
these antibodies abrogated TGF-
formation by LPSstimulated thioglycollate-elicited peritoneal macrophages,
another cell system previously demonstrated to convert large latent complex to TGF-
(44). Serum-free CM from
stimulated macrophages was prepared and assayed for
TGF-
activity using the MLEC-luciferase assay. Levels of
mature TGF-
present in CM from cultures treated with
Ab 39, anti-450, or anti-849 IgG were similar to those
present in anti-TGF-
IgG containing CM harvested from
untreated cultures (Fig. 9 C). Like cocultures, stimulated macrophages were unaffected by anti-1065 IgG with respect to their ability to produce mature TGF-
(Fig. 9 C).
Assays of heat-activated CM from untreated or antibodytreated stimulated macrophages revealed that all cultures
generated similar levels of total TGF-
(data not shown).
Results obtained using the MLEC-luciferase assay could not be confirmed using the wound migration assay because the wound migration assay was unresponsive to
TGF-
present in CM from stimulated macrophages. Addition of either recombinant TGF-
1 or neutralizing anti-
TGF-
IgG to CM from stimulated macrophages did not
affect the migration of endothelial cells (data not shown).
This may be due to the presence of migration stimulatory
factors in macrophage CM (2). Nevertheless, anti-450 or
anti-849 IgG blocked TGF-
generation by stimulated
macrophages as determined by the MLEC-luciferase assay, suggesting that these antibody effects on large latent complex activation were not specific to cocultures of BAE
and BSM cells and that the role of LTBP-1 in activation
involves interactions with its amino- and carboxyl-terminal regions.
Discussion
by cocultures of endothelial and smooth muscle cells, retinoid-treated BAE cells, and stimulated macrophages include plasmin, cation-independent mannose 6-phosphate/
insulin-like growth factor type II receptor, tissue transglutaminase, and LTBP-1 (27). Interactions between the
reactants involved in the activation of latent TGF-
have not
been characterized in cell-free systems, with the exception that plasmin can cleave LAP, destabilizing noncovalent
interactions between LAP and TGF-
, and that recombinant small latent complex binds to the cell surface cationindependent mannose 6-phosphate/insulin-like growth factor type II receptor via mannose 6-phosphate residues present on LAP (16, 35, 36). In this article, we have identified and characterized interactions between two reactants,
LTBP-1 and tissue transglutaminase, that may be involved
in latent TGF-
activation by cocultures of BAE and BSM
cells as well as LPS-stimulated thioglycollate-elicited macrophages (31, 44, 54). These interactions include an enzyme-substrate relationship between transglutaminase and
LTBP-1 and transglutaminase-dependent anchoring of large
latent complex to the ECM. In addition, a second functional domain of LTBP-1 involved in latent TGF-
activation was identified.
N293 LTBP-1S,
which was incorporated into ECM but is absent in
N441
LTBP-1S, which appeared not to be cross-linked to ECM.
Identification of the transglutaminase-reactive glutamine(s)
is a current subject of investigation using biochemical and
molecular approaches.
activation were identified. Both the amino and carboxyl regions of LTBP-1S
appear to be involved in protein interactions required for
activation as antibodies to these two domains abrogated TGF-
generation by cocultures of BAE and BSM cells as
well as by LPS-stimulated thioglycollate-elicited macrophages. Previously, we reported that protein A-purified anti450 IgG (the antibody to the amino terminus of LTBP-1S)
did not affect activation of large latent complex by stimulated macrophages (44). This discrepancy results from the
fact that in the earlier work total IgG was used, whereas in
the experiments reported here affinity-purified IgG was
tested. Based upon the concentration of affinity-purified anti-450 IgG required to inhibit activation of large latent
complex, the amount of IgG used previously would have
been insufficient to observe an effect. Anti-450 IgG also
inhibited matrix association of LTBP-1 and large latent
complex, suggesting that the role of the amino terminus in
latent TGF-
activation may be to mediate the incorporation of large latent complex into the matrix.
generation (44). The carboxyl-terminal sequence of LTBP-1 does
not appear to contain sites involved in covalently attaching
LTBP-1 to the ECM, as addition of these antibodies to
cultures did not affect the matrix content of LTBP-1. This region does contain a putative protease-sensitive site at
residue 1257 located after the third cysteine-rich repeat
(28, 49, 60). Cleavage at this site might facilitate activation
of soluble forms of large latent complex.
may be
important in regulating the conversion of large latent complex to mature TGF-
at specific sites (23). Alternatively,
we speculate that soluble large latent complex may be resistant to plasmin activation. It has been demonstrated that activation of latent TGF-
in fibroblast CM by plasmin requires nonphysiological levels of plasmin (35). We
also find that recombinant large latent complex is not
readily activated by plasmin in solution (data not shown).
Therefore, cross-linking to the matrix may sequester large
latent complex that is not readily activated, and subsequent release from the matrix by proteolysis (60) could
generate a modified complex susceptible to plasmin activation.
occurs by the sequence of reactions illustrated in Fig. 10. Cross-linking large latent complex to
the ECM is an early step in activation of large latent complex. From our antibody inhibition studies on latent TGF-
activation, both the amino and carboxyl domains of
LTBP-1 are involved in protein interactions necessary for
activation. The role of the amino terminus of LTBP-1S appears to be in the cross-linking of large latent complex to the matrix, as we observed that the amino terminus contains transglutaminase reactive sites and transglutaminase
activity is required for activation (31). We propose that
matrix-bound large latent complex is released by cleaving
LTBP-1S at a potential tribasic protease site (arginine415) on the carboxyl side of the transglutaminase reactive
residue(s). This site has been proposed by Taipale et al. to
be plasmin sensitive (60). We hypothesize that the carboxyl domain of LTBP-1S is involved in forming noncovalent interactions, perhaps with matrix, as antibodies to this
domain did not interfere with the cross-linking to the matrix but did block TGF-
generation. An additional cleavage of LTBP-1S at the carboxyl terminus (residue 1257) as
proposed by Taipale et al. (60) would release a complex
with an LTBP-1 molecule containing only its core EGFlike repeats plus cysteine-rich repeats 3-4 bound to small
latent complex. This proteolytic processing of large latent
complex may be required for subsequent activation steps,
such as binding to cell surface mannose 6-phosphate/insulin-like growth factor type II receptors (16, 32). We speculate that cross-linking to the matrix occurs before targeting
to the cell surface mannose 6-phosphate/insulin-like factor
type II receptor, as the addition of excess mannose 6-phosphate does not interfere with the matrix association of
LTBP-1 or large latent complex (data not shown) but abrogates TGF-
generation (16). Once on the cell surface,
fragmented latent complex is susceptible to plasmin-dependent activation by proteolytic cleavage of LAP (27, 36).
The TGF-
released then binds to its receptor.
Fig. 10.
Model of plasmindependent activation of large
latent complex. A diagrammatic representation of large
latent complex is presented as covalently interacting with
the ECM by transglutaminase (TGase)-dependent cross-linking of LTBP-1 and
matrix protein(s). For activation of large latent complex
to proceed, large latent complex is solubilized by proteolysis of LTBP-1. It is proposed that the release of
large latent complex occurs
by cleavage occurring at the
amino terminus of LTBP-1
downstream of the TGase reactive site(s). In addition, there may be a second cleavage at the carboxyl terminus
of LTBP-1 resulting in matrix release and exposure of
mannose 6-phosphate (M6P)
residues of LAP involved in localizing latent TGF- to the cell surface. Proteolytically processed LTBP-1 is proposed to target to the
cell surface where cell-associated plasmin cleaves LAP to liberate mature TGF-
from the complex.
[View Larger Version of this Image (37K GIF file)]
Received for publication 12 April 1996 and in revised form 12 December 1996.
This research was supported by grants CA 2753 (D.B Rifkin), CA 34282 (D.B Rifkin), EY 06537 (I. Nunes), T32GM 07238 (I. Nunes), and CA 09161 (C.N. Metz) from the National Institutes of Health. P.-E. Gleizes was the recipient of a fellowship from the Association pour la Recherche Contre le Cancer.The authors thank Ms. Melinda Vassallo for her excellent technical assistance and Dr. John S. Munger for his critical review of the manuscript and stimulating discussions.
BAE, bovine aortic endothelial;
BSM, bovine smooth muscle;
CM, conditioned medium;
ECM, extracellular matrix;
GST, glutathione S-transferase;
LAP, latency associated peptide;
LPS, lipopolysaccharide;
LTBP-1, latent TGF-beta;
binding protein-1; MDC, monodansylcadaverine;
MLEC, mink lung epithelial cells;
PAI-1, plasminogen activator inhibitor-1;
TGF-, transforming growth factor-
;
WT, wild type.