©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Mast Cell Chymase and Leukocyte Elastase Release Latent Transforming Growth Factor-1 from the Extracellular Matrix of Cultured Human Epithelial and Endothelial Cells (*)

(Received for publication, October 3, 1994; and in revised form, November 17, 1994)

Jussi Taipale (1)(§) Jouko Lohi (1)(§) Juhani Saarinen (3) Petri T. Kovanen (3) Jorma Keski-Oja (1) (2)(¶)

From the  (1)Departments of Virology and (2)Dermatology and Venereology, University of Helsinki, FIN-00014 Helsinki, Finland and the (3)Wihuri Research Institute, Kalliolinnantie 4, FIN-00140 Helsinki, Finland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Monolayer cultures of human epithelial and endothelial cells were used to study the association of latent transforming growth factor-beta1 (TGF-beta1) to extracellular matrices and its release and activation during matrix degradation. Human umbilical vein endothelial cells and embryonic lung fibroblasts produced relatively high levels of TGF-beta1, its propeptide (beta1-latency-associated protein), and latent TGF-beta-binding protein and incorporated latent TGF-beta1 into their matrices as shown by immunoblotting. Amnion epithelial cells produced lower levels of these proteins. Confluent cultures of epithelial cells were exposed to matrix-degrading proteases and glycosidases. Mast cell chymase, leukocyte elastase, and plasmin efficiently released matrix-bound latent TGF-beta1 complexes, while chondroitinase ABC and heparitinases were ineffective. The ability of the proteases to activate recombinant latent TGF-beta1 was tested using growth inhibition assays and a novel sodium deoxycholate-polyacrylamide gel electrophoresis followed by immunoblotting. Sodium deoxycholate solubilized M(r) 25,000 TGF-beta1 but did not dissociate high M(r) latent TGF-beta1 complexes, allowing separation of these forms by polyacrylamide gel electrophoresis. Mast cell chymase and leukocyte elastase did not activate latent TGF-beta1, suggesting that its release from matrix and activation are controlled by different mechanisms. The release of TGF-beta from the matrix by leukocyte and mast cell enzymes may contribute to the accumulation of connective tissue in inflammation.


INTRODUCTION

Transforming growth factor-betas are a family of multifunctional polypeptide growth factors/growth inhibitors(1, 2, 3, 4, 5, 6, 7) . Three isoforms of TGF-beta (^1)have been found in mammals (TGF-beta1-TGF-beta3). TGF-betas are commonly secreted by cultured cells in a latent form(8) . Latency is caused by the amino-terminal pro-domain (LAP, latency-associated protein), which is cleaved from carboxyl-terminal active TGF-beta during secretion but remains non-covalently associated to the mature dimer(9) . The latent complex consisting of beta1-LAP and TGF-beta1 has been named small latent TGF-beta1(10) . A major fraction of latent TGF-beta from a variety of cell lines forms large latent TGF-beta complexes, containing additional high molecular mass proteins that associate with LAP(10, 11, 12, 13) . Best characterized of these is the latent TGF-beta-binding protein (LTBP), which associates to LAP by a disulfide bond(11, 14, 15, 16) . LTBP is important in the processing and secretion of latent TGF-beta(12) . We have recently observed that LTBP and large latent TGF-beta1 are extracellular matrix proteins forming insoluble high molecular mass complexes(13, 17) . These cross-linked forms can be solubilized by plasmin, which cleaves LTBP at specific sites, releasing both free LTBP and LTBP complexed to LAPbulletTGF-beta1 to the culture medium.

LTBP shows extensive homology in domain structure to fibrillins, a group of microfibrillar extracellular matrix proteins(14, 18, 19, 20) . LTBP localizes to the extracellular matrix of normal tissue and cancer stroma(20) . Staining of LTBP often parallels collagen fibers(20, 21, 22) . TGF-betas also often colocalize with interstitial extracellular matrix components and basement membranes(23, 24, 25) . In addition, soluble latent or active TGF-beta is commonly absent from the supernatant fluids of homogenized tissues(22) , unless extracted by acid/ethanol or denaturants(26, 27) .

Since most tissues and cultured cells express different isoforms of TGF-beta and its receptors(1, 6, 25) , the events that activate latent TGF-beta are probably the major mechanisms controlling TGF-beta activity in tissues. In vitro, latent forms of TGF-beta can be activated by extremes of pH, by heat treatment, and by certain glycosidases and the protease plasmin(8, 28, 29, 30, 31, 32, 33, 34) . Proteolysis (29, 30, 31, 32, 33, 34) , acidic cellular microenvironments(35) , and the extracellular matrix molecule thrombospondin (36) have been proposed to mediate TGF-beta activation under physiological conditions.

TGF-beta enhances the formation of connective tissue by stimulating the proliferation of matrix-producing mesenchymal cells such as smooth muscle cells and fibroblasts via an autocrine platelet-derived growth factor loop(37) , and by increasing the production of extracellular matrix components(1, 5, 38) . Connective tissue growth is further enhanced by the inhibition of epithelial and endothelial cell proliferation (39, 40) and decrease in matrix degradation by induction of proteinase inhibitors, such as plasminogen activator inhibitor-1 and tissue inhibitor of metalloproteinases-1(41, 42) . TGF-beta promotes tissue fibrosis and inflammatory cell infiltration by the induction of fibroblast, monocyte, and neutrophil chemotaxis(43, 44, 45) . On the other hand, TGF-beta is immunosuppressive, and mice lacking both TGF-beta1 alleles die shortly after weaning of excessive inflammatory response (46) .

These properties indicate that TGF-beta activity is important for the healing of wounds and damaged tissues(4, 47, 48) . Disturbances in the activation of TGF-beta may, in turn, contribute to fibrotic responses in a variety of diseases, such as glomerulonephritis(4) , lung fibrosis (49) , liver cirrhosis(50) , fibrosis of transplanted tissues during allograft rejection(22) , and in fibrosis of arteries after coronary angioplasty(51) , in carcinoid heart disease (21) and in arteriosclerosis(52) .

The present study was undertaken to understand the production and matrix deposition of latent TGF-beta1 complexes in two monolayer cell systems, namely cultured human epithelial and endothelial cells, and the roles of two important enzymes, human leukocyte elastase and mast cell chymase, on the TGF-beta1-containing extracellular matrices.


MATERIALS AND METHODS

Reagents

Plasmin (specific activity of 16.4 caseinolytic units/mg protein) was purchased from Chromogenix (Mölndal, Sweden). The proteinases porcine pancreatic elastase (specific activity 70 units/mg), bovine spleen cathepsin B (22 units/mg) and D (8.5 units/mg), human leukocyte (neutrophil) elastase (300 units/mg), human cathepsin G (66 units/mg), Clostridium histolyticum collagenase (2380 units/mg), Flavobacterium heparinum heparinases I (500 units/mg) and III (260 units/mg), and Proteus vulgaris chondroitinase ABC (0.55 units/mg) were from Sigma. Human urokinase (1 times 10^5 units/mg) was from Calbiochem. Human mast cell tryptase was a gift of Dr I. Harvima (Department of Dermatology, University of Kuopio, Finland; for details, see Refs. 53 and 54). Human mast cell chymase was purified to 99% homogeneity (specific activity 40 BTEE units/mg) from human skin as described(55) . Human stromelysin was a gift from Dr. Howard G. Welgus (Washington University School of Medicine, St. Louis, MO). Porcine intestinal mucosa heparin was from Sigma. Recombinant human small latent TGF-beta was a gift of Dr. P. Puolakkainen (Oncogen, Seattle, WA). Recombinant human large latent TGF-beta and LTBP were gifts from Dr. Hideya Ohashi (Kirin Brewery Co. Ltd., Gumma, Japan). The molecular mass of LTBP in these preparations is approx150 kDa, slightly larger than the ``platelet'' LTBP (120-140 kDa), but smaller than the full-length fibroblast LTBP (200 kDa). All other reagents were from commercial sources and of the highest purity available.

Antibodies

Affinity-purified peptide antibody 627 to amino acids 78-109 of mature TGF-beta1 was used in immunoblotting of TGF-beta1 (17) . Antibody Ab-39 to human platelet LTBP was a gift of Drs. K. Miyazono and C.-H. Heldin (Uppsala, Sweden), and has been described previously(11, 12) . Affinity-purified peptide antibody 680 against human beta1-LAP amino acids 111-134 was prepared essentially as described(17) . Antibody 680 is functional in immunoblotting, but has only weak reactivity in immunoprecipitation.

Cell Culture and Collection of Conditioned Medium

Human amniotic epithelial cells were extracted from amnions as described (56) and maintained in medium 199 with 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 50 µg/ml streptomycin. They were used as secondary cultures. Human umbilical vein endothelial (HUVE) cells were prepared according to Jaffe et al.(57) . The cells were cultured in medium 199 with 10% heat-inactivated fetal calf serum and 5% normal human serum on gelatin-coated culture plates and used after one or two passages. After two passages more than 80% of the cells stained positive for factor VIII (not shown). Human embryonic lung fibroblasts (CCL-137; ATCC, Rockville, MD) and Mv1Lu mink lung epithelial cells (CCL-64) were grown in Eagle's modification of minimal essential medium containing 10% fetal calf serum. All experiments were carried out under serum-free conditions. Before commencing experiments, the cells were washed twice and subsequently incubated in serum-free medium for 2-6 h to remove traces of serum proteins. For collection of conditioned medium, cells were subsequently incubated in serum-free medium (0.1 ml/cm^2) and culture supernatant was collected and clarified by centrifugation at the time points indicated.

Growth Inhibition Assay

The mink lung cell growth inhibition assay was performed essentially as described(31) . Briefly, subconfluent cultures of Mv1Lu cells were washed and incubated with 5 ng/ml epidermal growth factor and the protease digest indicated (in triplicate) under serum-free conditions for 16-24 h. The cells were subsequently labeled with 1 µCi/ml [6-^3H]thymidine for 2 h, followed by washing and methanol fixation. Radioactivity incorporated to DNA was solubilized by 0.2 M NaOH and determined by a liquid scintillation counter.

Extracellular Matrix Preparations

Extracellular matrices were prepared according to the sodium deoxycholate extraction procedure (58) . Where indicated, LTBP cross-linked to the matrix was unmasked and made soluble by digesting the sodium deoxycholate-insoluble residues by plasmin (0.3 caseinolytic unit/ml) in matrix digestion buffer (0.14 M NaCl, 1 mM CaCl, 1 mM MgCl and 0.1% n-octyl-D-beta-glucopyranoside in 10 mM sodium phosphate buffer, pH 7.4) at 37 °C for 1 h (see (13) for details). For immunoblotting, SDS (1% final concentration) was added to the digested matrices without removing the plasmin solution, and the plates were scraped with a rubber policeman. The samples were then transferred to microcentrifuge tubes and treated at 95 °C for 5 min before SDS-PAGE.

Polyacrylamide Gel Electrophoresis (PAGE) and Immunoblotting

Sodium deoxycholate-PAGE for the detection of different forms of latent and active TGF-beta1 was carried out as described(13) . Laemmli buffer system was used for gradient SDS-PAGE analysis. Immunoblotting was performed as described(13, 17) , with the exception that in order to enhance signal intensity, proteins from both sodium deoxycholate and sodium dodecyl sulfate polyacrylamide gels were transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) and the filters were treated at 80 °C under vacuum for 1 h to fix the transferred proteins.

Proteinase Digestion of Recombinant Latent TGF-beta1s

Large latent form of TGF-beta1 (10 µg/ml) was treated with the indicated concentrations of proteinases in phosphate-buffered saline containing 1 mM MgCl(2), 1 mM CaCl(2), 0.3 mg/ml octyl-D-beta-glucopyranoside, and 1 mg/ml bovine serum albumin at 37 °C for 4 h. Heparin (10 µg/ml) was added to some reactions. Proteinases were inhibited by the addition of aprotinin (200 IU/ml), soybean trypsin inhibitor (0.1 mg/ml), antipain (0.1 mg/ml), and leupeptin (0.1 mg/ml), and the incubations were continued for 20 min. TGF-beta1 activity was assayed immediately from aliquots of the reactions by Mv1Lu growth inhibition assay (in triplicate) and by sodium deoxycholate-PAGE followed by immunoblotting. The processing of LTBP and beta1-LAP was detected by SDS-PAGE and immunoblotting with specific antibodies.


RESULTS

Epithelial and Endothelial Cells Produce Large and Small Latent Forms of TGF-beta1 and Incorporate the Large Latent Form to their Matrices

We analyzed first the molecular forms of latent TGF-beta produced by epithelial and endothelial cells. The serum-free conditioned medium of human amniotic epithelial cells, umbilical vein endothelial cells, and control fibroblasts was collected after 4 days. The levels of LTBP, beta1-LAP, and TGF-beta were assayed by 4-15% gradient SDS-PAGE followed by immunoblotting. Endothelial cells and control fibroblasts secreted relatively high levels of LTBP, beta1-LAP, and TGF-beta1 (Fig. 1A). The majority of beta1-LAP comigrated as a complex with LTBP at 220 kDa, indicating that the majority of TGF-beta1 secreted by these cells was in the large latent complex. Approximately 10% and 5% of beta1-LAP from the conditioned medium of endothelial cells and fibroblasts, respectively, migrated at 80 kDa, indicating that it was in the small latent form. Epithelial cell conditioned medium contained very low levels of LTBP, beta1-LAP, and TGF-beta1 (Fig. 1A). The majority of beta1-LAP migrated at 80 kDa, indicating that the small latent complex is the major soluble form in epithelial cells.


Figure 1: Analysis of latent forms of TGF-beta1 from epithelial and endothelial cells by immunoblotting. After reaching confluence, cultures of human amniotic epithelial cells (Ep), umbilical vein endothelial cells (En), and embryonic lung fibroblasts (Fb) were washed and changed to serum-free medium, and conditioned medium was harvested at 96 h. A, secretion of LTBP, beta1-LAP, and TGF-beta1 to the culture medium. Samples of the conditioned medium (30 µl, equal to proteins secreted by 0.2 cm^2 of cells) were analyzed by 4-15% gradient SDS-PAGE (no stacking gel) under non-reducing conditions followed by immunoblotting with antibodies specific for LTBP, beta1-LAP, and TGF-beta1 as indicated. Lanes denoted LTBP and LL-TGFbeta1 contain 1 ng of recombinant human free form of LTBP and large latent TGF-beta1, respectively. Migration of the molecular mass markers (kDa) are shown on the left. B, analysis of TGF-beta1 complexes by sodium deoxycholate-PAGE. The panel shows the immunoblotting analysis for TGF-beta1 of the respective samples run in 4-15% gradient sodium deoxycholate-PAGE (40 µl of samples concentrated 20-fold by ultrafiltration; equal to proteins secreted by 5 cm^2 of cells). TGF-beta1 standard lanes L, S, and A contain 4 ng of large latent TGF-beta1 (L, 400 pg of TGF-beta equivalent), 10 ng of small latent TGF-beta (S, 2.5 ng of TGF-beta equivalent), and 700 pg of active TGF-beta1 (A), respectively. Activation control lanes contain fibroblast conditioned medium treated with medium only(-), acid (100 mM HCl), or alkali (Alk, 100 mM NaOH) prior to neutralization and sample loading. TGF-beta1 standard lanes are from a 10-fold shorter exposure than the first three lanes for clarity, see panel A for quantitative results. Arrow marks the position of the bottom of the sample wells (stacking gel was not used). C, analysis of the extracellular matrices. Extracellular matrices were prepared from confluent cultures of the respective cells. Immunoblotting analysis on 4-15% nonreducing gradient SDS-PAGE gels (no stacking gel) were carried out from samples of matrix (1 cm^2/lane) with antibodies specific for LTBP (upper panel), beta1-LAP (middle panel), and the TGF-beta1 (lower panel) after solubilization of the matrices by SDS(-) or limited plasmin digestion followed by SDS (+, see ``Materials and Methods''). Lane L contains 1 ng of large latent TGF-beta1 complex. The migration of molecular mass markers (kDa) is indicated on the right.



The molecular complexes of secreted latent TGF-beta were then characterized by sodium deoxycholate-PAGE. Sodium deoxycholate is less denaturing than SDS and does not dissociate TGF-beta1 from the small or large latent complexes(13) . In accordance with the results presented above, the majority of soluble fibroblast and endothelial cell-derived TGF-beta1 comigrated with recombinant large latent TGF-beta1 (LTBP:LAPbulletTGF-beta1). Approximately 10-20% of TGF-beta1 in endothelial cell and 5% in fibroblast conditioned medium comigrated with the small latent TGF-beta1 (LAPbulletTGF-beta1), respectively (Fig. 1B). Epithelial cells secreted significantly lower amounts of TGF-beta1 in two different TGF-beta1 complexes, the small latent complex (approx50%), and a novel large latent complex migrating above the LTBP:LAPbulletTGF-beta1 (approx50%).

Unlike active TGF-beta1 obtained by heat treatment, which aggregates on top of the gel(13) , acid-activated TGF-beta1 migrated as a low molecular mass band in sodium deoxycholate-PAGE (Fig. 1B). Immunoreactive band comigrating with active TGF-beta1 was not detectable in any cell type, confirming earlier observations that TGF-beta1 secreted by cultured cells is usually in the latent form.

Latent TGF-beta1 and LTBP associate with the extracellular matrices of cultured human fibroblasts and fibrosarcoma cells(13) . We therefore prepared extracellular matrices of confluent cultures of amnion epithelial cells, endothelial cells, and fibroblasts by sodium deoxycholate extraction (see ``Materials and Methods''). Since the major fraction of LTBP in fibroblast matrices is cross-linked to high molecular mass complexes that do not enter SDS-PAGE gels(13) , some matrix preparations were digested with plasmin (see ``Materials and Methods'') to unmask LTBP immunoreactivity. Immunoblotting of the plasmin-digested matrices revealed that matrices of all cell types contained comparable amounts of free LTBP (approximately 1-3 ng/cm^2; Fig. 1C, top panel). The matrices of endothelial cells and fibroblasts contained significant amounts of TGF-beta1 (30-70 pg/cm^2; Fig. 1C, bottompanel) and LTBP:LAP (200-600 pg/cm^2; Fig. 1C, middlepanel), whereas in epithelial cell matrices TGF-beta1 was barely detectable and LTBP:LAP was below detection limit (Fig. 1C, bottom and middlepanels). In matrices solubilized with SDS alone, multiple very high molecular mass LTBP immunoreactive bands were detected by immunoblotting, and some LTBP was aggregated on the top of the gel. beta1-LAP was not detectable without plasmin treatment. Endothelial cell matrices contained two nonspecific background bands (Fig. 1C; approx160 and 70 kDa). Small latent TGF-beta1 was not detectable in the matrix of any of the cells (Fig. 1C, middlepanel).

Amnion epithelial cells secreted very low levels of LTBP, while their matrices contained significant amounts of it. To test the possibility that these cells did not produce LTBP, but assembled exogenous LTBP from serum to their matrices, we metabolically labeled amnion epithelial cells with [S]cysteine. LTBP could be immunoprecipitated from plasmin solubilized matrix of these cells (see (13) for details), indicating that it was in part produced by the cells (data not shown). The efficient incorporation of LTBP to amnion epithelial cell matrices is probably related to the polar nature of this cell type.

Mast Cell Chymase and Leukocyte Elastase Release LTBP from the Matrix of Human Amnion Epithelial Cells

Since amnion epithelial cells were the most efficient in incorporating LTBP to their matrices (Fig. 1, A and C), we chose this cell type for analysis of matrix-degrading enzymes for the ability to release LTBP from the extracellular matrix.

For the analysis of matrix-degrading enzymes, confluent cultures of amnion epithelial cells were washed, changed to serum-free conditions for 4 h and treated with increasing concentrations of human mast cell chymase and tryptase, leukocyte elastase, plasmin, and cathepsin G, porcine pancreatic elastase, bovine spleen cathepsin B and D, C. histolyticum collagenase, F. heparinum heparinases I and III, and P. vulgaris chondroitinase ABC. Immunoblotting analysis of released material and matrices indicated that mast cell chymase, leukocyte and pancreatic elastases, and plasmin effectively released LTBP from the matrix of the cells ( Table 1and Fig. 2). Low activity was displayed by cathepsin G, mast cell tryptase, cathepsin B, and bacterial collagenase (in this order of potency), while cathepsin D and the glycosaminoglycan-degrading enzymes were negative ( Table 1and Fig. 2).




Figure 2: Release of LTBP from epithelial cell matrix by selected enzymes. Confluent cultures of human amnion epithelial cells were changed to serum-free conditions and incubated with matrix-degrading enzymes for 1 h. Medium was collected and extracellular matrices were prepared from the cells by sodium deoxycholate extraction followed by solubilization by limited plasmin digestion and SDS (see ``Materials and Methods'' for details). LTBP was assayed from the medium (Released) and solubilized extracellular matrix (Matrix) by non-reducing SDS-PAGE followed by immunoblotting (4-15% gradient gel). Chy, human mast cell chymase; PE, porcine pancreatic elastase; LE, human leukocyte elastase; Pla, plasmin; Try, mast cell tryptase; Col, bacterial collagenase; B, D, and G, respective cathepsins; HI and HIII, heparinases I and III; ABC, chondroitinase ABC; All, heparinases I, III, and chondroitinase ABC. Doses used are maximum tested doses of Table 1. The migration of molecular mass markers is indicated on the right.



Mast cell chymase, leukocyte and pancreatic elastases, and plasmin processed LTBP to similar molecular mass forms (90-130 kDa, Fig. 2) suggestive of protease-resistant ``core'' domain. Immunoreactive bands of lower molecular mass were not produced by any of the proteinases tested. The platelet-like LTBP is highly resistant to digestion with proteinases(12, 13, 59) . This was further emphasized by the observation that 100-fold higher concentrations of porcine pancreatic elastase that are sufficient to release all of matrix-bound LTBP could not further degrade the 90-kDa form of LTBP (Fig. 2).

Proteases Release LTBP and Large Latent TGF-beta1 from Endothelial Cell Matrix

The proteases having highest activity were tested for the release of LTBP and TGF-beta1 from endothelial cell matrices. Leukocyte elastase, mast cell chymase, thrombin, and plasmin released LTBP and TGF-beta1 from the matrix of confluent cultures of endothelial cells as shown by immunoblotting analysis (Fig. 3). The amounts of LTBP and TGF-beta1 remaining in the matrix were estimated from the corresponding matrix preparations after plasmin digestion to solubilize cross-linked LTBP complexes (see ``Materials and Methods''). Analysis of the immunoblots revealed a dose-dependent release of LTBP and TGF-beta1 from the matrix to the culture supernatant (Fig. 3). The amounts of LTBP and TGF-beta1 released correlated well to the remaining matrix-bound amounts, suggesting that the major fraction of cell layer latent TGF-beta1 is associated to the extracellular matrix and not to cell surface (cell membranes are solubilized by sodium deoxycholate). Mast cell chymase and leukocyte elastase released >90% of matrix-associated LTBP and TGF-beta1 at 10 nM. Plasmin was 90% effective at 200 nM, while 300 nM of thrombin released only 20% of LTBP (Fig. 3). These results indicate that mast cell chymase and leukocyte elastase are more effective than plasmin and thrombin, i.e. the proteinases originally reported to release TGF-beta1 from the matrix of fibroblasts and endothelial cells(17, 60, 61) .


Figure 3: Release of LTBP and TGF-beta1 from endothelial cell matrix by proteinases. Confluent cultures of human umbilical vein endothelial cells were changed to serum-free conditions and incubated for 1 h with increasing concentrations of human plasmin, mast cell chymase, leukocyte elastase, and thrombin. Release of LTBP and TGF-beta1 from the matrix (ECM) to culture supernatant (Released) was assayed by immunoblotting as described in caption to Fig. 2. In panelsLTBP, lower molecular mass band(s) represent free LTBP, and higher molecular mass band(s) LTBP complexed to beta-LAP.



Proteases in the Processing and Activation of Latent TGF-beta1

We have found that a high molecular mass form of LTBP is associated to the extracellular matrix, and that its release from the matrix is due to cleavage(s) from the high molecular mass ``fibroblast'' form (200 kDa) to a smaller ``platelet'' form (120-140 kDa; (13) ). We investigated therefore whether the ability of proteinases to release LTBP from the matrix correlates to the ability to process LTBP from fibroblast conditioned medium. Serum-free conditioned medium was collected from human fibroblasts after 3 days, and aliquots of the medium were incubated with porcine pancreatic elastase (10 nM), human plasmin (50 nM), mast cell chymase (10 nM), leukocyte elastase (10 nM) and cathepsin G (100 nM), and bovine cathepsins B (500 nM) and D (500 nM) at 37 °C for 1 h. Processing of LTBP was monitored by 4-20% gradient SDS-PAGE followed by immunoblotting. All enzymes that were able to release LTBP from the matrix (plasmin, elastases, and chymase) processed it to a fragment migrating at 85-140 kDa, while the enzymes that could not significantly release LTBP (cathepsins B, D, and G) were unable to process it (Fig. 4).


Figure 4: Processing of LTBP from fibroblast conditioned medium by selected proteinases. Fibroblast conditioned medium was harvested at 3 days and incubated with proteinases at 37 °C for 1 h. Reactions were terminated by the addition of SDS (2% final concentration), and the samples were analyzed by 4-20% gradient SDS-PAGE under nonreducing conditions followed by immunoblotting with LTBP-specific antibodies. Ct, untreated medium; Pla, plasmin (50 nM); Chy, chymase (10 nM); Leu, leukocyte elastase (10 nM); Pan, porcine pancreatic elastase (10 nM); G, cathepsin G (100 nM); B, cathepsin B (500 nM); D, cathepsin D (500 nM). The migration of molecular mass markers (kDa) is indicated on the right.



It is known that low concentrations of plasmin (10-100 nM) and thrombin release TGF-beta1 from the matrix of fibroblasts and endothelial cells in a latent form(17, 60, 61) . We studied next the bioactivity of TGF-beta released from the matrix of endothelial cells by mast cell chymase, leukocyte elastase, and cathepsin G. Confluent cultures of endothelial cells were treated with these proteinases for 2 h, and the activity of TGF-beta1 released to the culture medium was determined by Mv1Lu growth inhibition assay. The supernatant fluids were not growth-inhibitory to Mv1Lu cells, indicating that the samples contained less than 50 pg/ml active TGF-beta (Fig. 5). In addition, no TGF-beta1 activity was detected in the supernatants using an ELISA that is specific for active TGF-beta1 (R& Systems; detection limit approx 30 pg/ml; data not shown). Supernatant fluids of chymase, leukocyte elastase, and plasmin-treated cells were 95% growth inhibitory to Mv1Lu cells after heat treatment (80 °C, 5 min), indicating that these samples contained >1 ng/ml latent TGF-beta (Fig. 5). These results indicate that TGF-beta released by chymase, elastase, and plasmin was >95% latent, since >1 ng/ml latent TGF-beta and <0.05 ng/ml of active TGF-beta was released by these proteinases.


Figure 5: Analysis of the biological activity of released TGF-beta by Mv1Lu cell growth inhibition assay. Confluent cultures of human endothelial cells were changed to serum-free conditions and treated with mast cell chymase (30 nM), leukocyte elastase (3 nM), and cathepsin G (3 nM) for 2 h at 37 °C. Plasmin (30 nM) and human matrix metalloproteinase-3 (stromelysin; 100 nM) were used as positive and negative controls, respectively. Samples of medium representing 3 cm^2 of cell layer were diluted to 500 µl of minimal essential medium and analyzed by Mv1Lu cell growth inhibition assay in triplicate. Some samples were heat treated (80 °C, 5 min) prior to analysis to activate latent forms of TGF-beta. Growth inhibition was assessed after 18 h by [6-^3H]thymidine incorporation. Standards of active TGF-beta are also indicated.



We studied then the ability of the proteinases to activate recombinant human large latent TGF-beta1. Large latent TGF-beta1 (10 µg/ml) was incubated with mast cell chymase (500 nM), leukocyte cathepsin G (500 nM), or elastase (50 nM) for 4 h at 37 °C in the absence or presence of heparin (10 µg/ml). A sample treated with a high concentration of plasmin (1 µM) was included as a positive control of protease-mediated TGF-beta activation(29, 30) . The proteinases were inactivated by the addition of proteinase inhibitors and TGF-beta1 bioactivity was analyzed by Mv1Lu growth inhibition assay. Large latent TGF-beta1 (200 pM) treated with mast cell chymase or leukocyte elastase was not significantly growth inhibitory to Mv1Lu cells. Since the detection limit of the assay is approx2 pM, this indicates that less than 1% of large latent TGF-beta1 could be activated by these treatments. Approximately 1-2% of plasmin-treated large latent TGF-beta1 was activated, corresponding to 4 pM TGF-beta1 (see also (29) and (30) ). Cathepsin G treatment induced marginal activation of large latent TGF-beta1.

The samples were then analyzed by sodium deoxycholate-PAGE, followed by immunoblotting with TGF-beta1 specific antibodies. Treatment of the large latent TGF-beta1 with leukocyte elastase or mast cell chymase did not dissociate TGF-beta1 from the latent complex, while cathepsin G induced a very weak species comigrating with active TGF-beta1. In control samples treated with plasmin, a clear band comigrating with active TGF-beta1 was detected (representing approx1% of total TGF-beta1; for pH activation controls, see Fig. 1B). Similar levels of activation (approx1%) of large latent TGF-beta1 by plasmin was also detected by an ELISA that is specific for active TGF-beta1 (data not shown). Leukocyte elastase treatment converted approximately 50% of large latent TGF-beta1 to a band migrating between small and large latent complexes (Fig. 6, top panel). Analysis of the samples by SDS-PAGE followed by immunoblotting with LTBP specific antibodies indicated that the change in migration was caused by degradation of LTBP as indicated by the decreased LTBP immunoreactivity at 200 kDa (Fig. 6, bottompanel). This may represent an unphysiological effect, since LTBP released from the matrix was not completely degraded by leukocyte elastase (see Fig. 2and Fig. 3) and heparin, its physiological cofactor, totally inhibited the degradation of LTBP (Fig. 6, top and bottompanels). Other proteinases did not result in significant changes in the molecular mass of LTBP. Activation of TGF-beta occurs by proteolytic cleavage of LAP(29, 30) , while cleavage of LTBP results in the release of latent TGF-beta1 from the matrix(13) . We investigated therefore whether proteinase treatments resulted in processing of the LAP portion of the large latent TGF-betas. Analysis of the samples by 15% SDS-PAGE under non-reducing conditions indicated that degradation products of beta1-LAP were detectable only in samples treated with the activating proteinase plasmin (Fig. 6, middlepanel; approx100 kDa).


Figure 6: Activation of large latent TGF-beta1 by proteinases: analysis by sodium deoxycholate-PAGE and immunoblotting. Purified recombinant large latent TGF-beta1 (10 µg/ml) was incubated with human mast cell chymase (500 nM), leukocyte elastase (50 nM), and cathepsin G (500 nM) at 37 °C for 4 h. Treatment with plasmin (1 µM) was included as a positive control. Heparin (+) was included as a cofactor in some reactions (10 µg/ml). The reactions were terminated by the addition of proteinase inhibitors. The amount of 25-kDa active form of TGF-beta1 was assayed from the samples by 4-15% gradient sodium deoxycholate-PAGE followed by immunoblotting with antibodies specific for TGF-beta1 (toppanel; see ``Materials and Methods'' and caption to Fig. 1B for details). Activation of TGF-beta1 was also analyzed by Mv1Lu cell growth inhibition and an ELISA that is specific for active TGF-beta1 (R& Systems), and is expressed as + or -. Migratory positions of human recombinant large latent TGF-beta1 (Latent) and active TGF-beta1 (Active) are indicated on the right. The analyses of the respective samples by nonreducing 4-15% gradient SDS-PAGE followed by immunoblotting with antibodies specific for beta1-LAP (middlepanel) and LTBP (bottompanel) are also shown.




DISCUSSION

The present study was carried out to analyze the effects of two important proteases, mast cell chymase and leukocyte elastase, on matrix-associated latent TGF-beta complexes. Monolayer cultures of human umbilical vein endothelial cells and amnion epithelial cells were used as model system. Unlike fibroblastic cells, epithelial and endothelial cells grow in culture as sheets of polygonal monolayer cells and form a matrix resembling basement membrane(57, 62) .

Similar levels of LTBP and latent TGF-beta1 were produced by human umbilical vein endothelial cells and fibroblasts. Sodium deoxycholate-PAGE immunoblotting experiments indicated that the majority of the latent TGF-beta produced by endothelial cells was in a large latent complex, containing both LTBP and beta1-LAP. Significant proportion of total LTBP and large latent TGF-beta1 produced were also detected in the extracellular matrix of endothelial cells. Human amnion epithelial cells produced significantly less LTBP and very little TGF-beta1. However, higher proportion of the produced LTBP was deposited to the matrix. Small latent TGF-beta1 was not detected in the matrix of either cell type.

When a panel of proteinases and glycosaminoglycan-degrading enzymes were tested for their ability to release matrix-bound TGF-beta1, it was found that mast cell chymase and leukocyte elastase effectively released LTBP and TGF-beta1 from the matrix of epithelial and endothelial cells with ED (1 h, 37 °C) of 5 nM, as estimated from dose dependence studies. The enzymes were approximately 10-fold more efficient than plasmin (ED approx 50 nM). Concentrations of the above enzymes in this order of magnitude are likely to exist in the vicinity of cell surfaces following mast cell or neutrophil degranulation or plasminogen activation(63, 64, 65) . The kinetics of the cleavage reactions, as well as the fact that LTBP fragments produced by elastases, chymase, and plasmin differed in molecular mass, make it likely that these proteinases act directly and not via the activation of secondary proteinase(s). Significant levels of LTBP were not released from the matrix by glycosaminoglycan-degrading enzymes heparinase I and III and chondroitinase ABC, suggesting that LTBP is not bound to glycosaminoglycan structures. The ineffectiveness of bacterial collagenase to release LTBP suggests in turn that matrix-associated latent TGF-beta1 is not an integral part of collagen fibers (for in vivo distribution, see (20) ). However, our recent immunofluorescence studies reveal in fibroblasts a fibrillar staining of LTBP that colocalizes with fibronectin and collagen type IV (66) .

The proteinases releasing LTBP and latent TGF-beta1 from the matrix display significant differences in substrate specificity(67, 68, 69, 70, 71, 72) . The ability of enzymes with divergent P1 residue specificities to cleave LTBP and release it from the matrix supports the hypothesis (13) that there is a proteinase-sensitive hinge region in LTBP between extracellular matrix and latent TGF-beta binding domains. The presence of these two functional domains in LTBP is also supported by the fact that the ability of proteinases to release TGF-beta1 from the matrix correlated to the ability of the enzymes to cleave the 160-200-kDa fibroblast form of LTBP to a fragment whose size resembled platelet LTBP (90-140 kDa).

To study the activation of TGF-beta1, we developed a biochemical assay based on polyacrylamide electrophoresis in the presence of sodium deoxycholate. TGF-beta1 released from endothelial cell matrix by chymase and elastase was biologically latent (>95%). Recombinant large latent form of TGF-beta1 was not activated by chymase or elastase. Plasmin (1 µM) activated approximately 1-2% of the large latent complex, while the activation of TGF-beta1 by cathepsin G was marginal. Sodium deoxycholate-PAGE represents the first demonstration of a specific biochemical assay for TGF-beta activation, and can, in addition, distinguish between small and large latent TGF-beta complexes. Sodium deoxycholate-PAGE or analogous electrophoretic methods will facilitate studies on different latent TGF-beta complexes in tissues and cultured cells. They could also be used in the study of non-covalent protein-protein interactions in general.

Mast cells and neutrophils are particularly rich sources of proteolytic enzymes in peripheral tissues, and their granule proteinases participate in connective tissue degradation in inflammation. Mast cells often localize in the areas of uncontrolled tissue degradation, such as in the shoulder region of human coronary atheromas(73) , in sites of tumor invasion and metastasis(74, 75) , and in rheumatoid joints(76, 77) . Mast cell proteinases degrade the extracellular matrix directly(54, 72) , and via the activation of zymogens, such as procollagenase (55) and prostromelysin(78) . On the other hand, mast cells have been suggested to have a role in a variety of fibrotic conditions(79, 80) . LTBP and TGF-beta are present in the endothelial basement membrane(20, 60, 61) . During inflammatory processes, latent TGF-beta released from the subendothelial extracellular matrix by mast cell chymase and leukocyte elastase could specifically bind to and be activated at the surface of smooth muscle cells, as suggested by Sato et al. ((33) ; see also (28, 29, 30, 31, 32, 33, 34, 35, 36) ). Active TGF-beta could contribute to the recruitment of macrophages, resulting in inflammatory cell infiltration to the intima, and smooth muscle cell proliferation, which are associated with the development of atherosclerosis(81) . This hypothesis is strengthened by the finding that the expression of TGF-beta1 activity causes intimal and medial hyperplasia in porcine arteries transfected with constitutively active TGF-beta1 gene(52) . The excessive activity of proteinases and formation of feedback TGF-beta1 could lead to excessive remodeling (degradation and production) of extracellular matrix in chronic inflammation, resulting in the degradation of normal connective tissue and epithelia, and in the formation of granulation tissue(4, 47, 48) .

Low concentrations (10-100 nM) of plasmin release TGF-beta1 from the matrix in a latent form(17, 60) , while high concentrations (approx1 µM) activate a fraction of latent TGF-beta1(29, 30) . Recent genetic evidence suggests, however, that physiological mechanisms distinct from plasmin exist for the release of TGF-beta1 from the matrix and its activation. Mice lacking TGF-beta1 gene die shortly after weaning due to invasion of inflammatory cells to multiple organs (46) , whereas loss of plasminogen activator u-PA and t-PA function in mice does not severely affect the immune system(82) , indicating that u-PA- and t-PA-mediated plasminogen activation is not required for the expression of TGF-beta1 activity. Under physiological conditions plasmin is susceptible to inhibition by multiple serum protease inhibitors, such as alpha(1)-antiplasmin and alpha(2)-macroglobulin, and the conversion of plasminogen to plasmin can be controlled by plasminogen activator inhibitors. TGF-beta induces plasminogen activator inhibitor-1, which decreases the activation of plasminogen(41) . In contrast, mast cell chymase and leukocyte elastase are released from the respective cells as active, heparin-bound enzymes that are relatively resistant to inhibition(64, 83, 84, 85, 86) . These properties make mast cell chymase and leukocyte elastase attractive candidates for modulators of TGF-beta bioactivity during inflammatory reactions in vivo.

It is becoming increasingly clear that a major step in growth factor signaling is the modulation of growth factor and receptor activity by extracellular proteinases (see, for example, (3) and (87) -92). In the case of TGF-beta1, at least three specific proteolytic cleavages are likely to regulate TGF-beta1 activity in tissues, namely: 1) the release of TGF-beta1 from the matrix(13, 17) , 2) the activation of TGF-beta (29, 30, 31, 32, 33, 34) , and 3) negative regulation by the shedding of TGF-beta-binding protein betaglycan from the cell surface(92) . The present results indicate that a number of serine proteinases can release TGF-beta1 from the matrix of cultured human epithelial and endothelial cells, resulting in the formation of a soluble pool of latent TGF-beta1. Soluble latent TGF-beta1 could subsequently be activated at the surface of cells expressing binding sites for latent TGF-beta1(33) . Regulation of TGF-beta1 availability, activity, and cellular response by proteinases and the modulation of proteolytic activity by TGF-beta1 (41, 42) support a general model where proteinases and latent matrix-bound growth factors are components of an extracellular signal transduction machinery that directs tissue construction and remodeling and regulates the activity of infiltrating immune cells. Disturbances in these control circuits could operate in a variety of disease states affecting tissue morphology and function, such as arteriosclerosis, cancer, fibrotic diseases, and chronic inflammation.


FOOTNOTES

*
This work has been supported by the Sigrid Juselius Foundation, Novo Nordisk Foundation, Alfred Kordelin Foundation, Ida Montin Foundation, Oskar Öflund Foundation, Finnish Medical Association Duodecim, the Academy of Finland, and the University of Helsinki. 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.

§
Predoctoral fellow of the Academy of Finland.

To whom correspondence should be addressed: Dept. of Virology, University of Helsinki, P. O. Box 21 (Haartmaninkatu 3), University of Helsinki, FIN-00014 Helsinki, Finland. Tel.: 358-0-434-6476; Fax: 358-0-434-6491.

(^1)
The abbreviations used are: TGF-beta, transforming growth factor-beta; LAP, TGF-beta latency-associated protein; LTBP, latent TGF-beta-binding protein; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; u- and t-PA, urokinase and tissue type plasminogen activators.


ACKNOWLEDGEMENTS

We thank Dr. Ilkka Harvima for the gift of human mast cell tryptase, Dr. Pauli Puolakkainen for recombinant small latent TGF-beta1, Dr. Hideya Ohashi for recombinant LTBP and large latent TGF-beta1s, Drs. Kohei Miyazono and Carl-Henrik Heldin for the antibody Ab-39, and Dr. Howard G. Welgus for human stromelysin. We further thank Dr. Tapio Vartio for critical review of the manuscript and Sami Starast, Marja-Liisa Eskola, Raija Juhonen, Niina Saarela, and Anneli Virtanen for technical assistance.


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