©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Processing of Transforming Growth Factor 1 Precursor by Human Furin Convertase (*)

Claire M. Dubois (1)(§), Marie-Hélène Laprise (1) (2), Franois Blanchette (1), Larry E. Gentry (3)(¶), Richard Leduc (2)(**)

From the (1) Immunology Division, Department of Pediatrics, and (2) Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada and (3) Department of Biochemistry, Medical College of Ohio, Toledo, Ohio 43699

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Proteolytic processing of the transforming growth factor precursor (pro-TGF) is an essential step in the formation of the biologically active TGF homodimeric protein (TGF). The 361-amino-acid precursor pro-TGF1 has within its primary structure the R-H-R-R processing signal found in many constitutively secreted precursor proteins and potentially recognized by members of the mammalian convertase family of endoproteases. To determine whether cleavage of pro-TGF1 can be achieved by the furin convertase in vitro, purified precursor was incubated in the presence of a truncated/secreted form of the enzyme. Immunoblots showed that the 55-kDa pro-TGF1 was converted into the 44 and 12.5 kDa bands corresponding to the pro-region and the mature monomer, respectively. Treatment of pro-TGF1 with furin resulted in a 5-fold increase in the production of biologically active TGF1. Furthermore, when expressed in the furin-deficient LoVo cells, no processing of pro-TGF1 was observed. In contrast, efficient processing was oberved when pro-TGF was coexpressed with the furin convertase. Collectively, these results provide evidence that in our experimental systems the TGF1 precursor is efficiently and correctly processed by human furin thus permitting release of the biologically active peptide.


INTRODUCTION

Transforming growth factor (TGF)() is a 25-kDa homodimeric protein with potent effects on cell growth and differentiation (for reviews, see Refs. 1-3). Three different isoforms (TGF1, TGF2, and TGF3) with similar bioactivities have been identified in mammalian tissues, TGF1 being the most extensively characterized isoform. Several proteins more distantly related to TGFs such as activins, inhibins, Müllerian inhibitory substance, bone morphogenic proteins, products of the nodal gene in mice as well as products of the decapentaplegic complex of Drosophila and the Vg1 gene from Xenopus laevis appear to play an important role in cellular differentiation (4) .

TGF isoforms are produced by a wide variety of normal and malignant cells and have been isolated from different tissues including blood platelets, bone, and placenta (5, 6, 7, 8) . It has been shown that most cell types secrete TGF1 in an inactive form (9, 10) which does not interact with specific TGF cell surface receptors thus failing to elicit TGF-induced biological responses. Inactive TGF may take multiple forms: for example, the 25-kDa mature TGF may be complexed with specific binding proteins such as the TGF latency-associated peptide (NH-terminal part of the precursor sequence) (3, 11) and the latent TGF-binding protein and thus be unable to bind to its cognate receptors; TGF may also be inefficiently processed from its precursor (12, 13) . The presence of TGF receptors on most cell types (14) and the ubiquity of the TGF molecule itself (15, 16) suggest that processing and activation of TGF is an important step in the regulation of TGF action.

The active 25-kDa TGF1 molecule consists of two identical disulfide-linked 12.5-kDa polypeptide chains (17) . Cloning of the TGF1 precursor cDNA and determination of its primary structure revealed that the mature 112-amino-acid chain of TGF1 is derived from the COOH terminus of a 390-amino-acid pre-pro-TGF1 by proteolytic cleavage (18) . This processing event is predicted to occur following an R-H-R-R sequence immediately preceding the NH-terminal Ala 279 residue of the mature growth factor. This suggests that processing of the growth factor involves an endoprotease which shows cleavage specificity toward pairs of basic amino acids (18, 19, 20) .

Many proteins including polypeptide hormones, viral proteins, growth factors, and receptors are synthesized as large inactive precursor proteins that must be proteolytically processed in order to release the bioactive polypeptide (21) . The most commonly occurring site of proteolysis is at the carboxyl-terminal side of basic amino acids residues found within the pro-protein (21) . Recently, a family of mammalian processing enzymes called SPCs (subtilisin-like pro-protein convertases) has been characterized (22, 23) . Up to six members of this family have been identified to date. These are Ca-dependent serine proteases that have been shown to cleave mostly at the R-R or K-R pairs of basic amino acids. Furin, the first SPC member to be extensively characterized, has been shown to process many proproteins including pro--NGF (24) , the insulin receptor (25) , and the HIV-1 glycoprotein gp160 (26) among others. Expression of the fur gene, which encodes furin, appears to be ubiquitously expressed in all tissues and cell types examined to date (24, 27, 28) . Colocalization with TGN 38 and failure to redistribute to the endoplasmic reticulum in the presence of brefeldin A suggest that furin is mostly localized in the trans-Golgi network (24, 29) . Substrate specificity studies have revealed that furin requires a R- X-X-R motif for cleavage while the R- X-K/R-R sequence provides an optimum processing site (30) . Upon inspection of the amino acid sequence of the TGF1 precursor, such an optimum furin cleavage motif was revealed immediately upstream of the amino acids constituting the NH-terminal of the mature TGF. Colocalization of TGF precursor with mannosidase II and its sensitivity to endoglycosidase H suggest that pro-TGF processing occurs in the Golgi complex, the subcellular site of furin location (31) . Therefore, the nearly ubiquitous expression of both furin and TGF, the correlation between their cellular localization, and the nature of the TGF-processing site make furin a good candidate for the physiological processing of TGF. Here we provide evidence that furin can efficiently and correctly process pro-TGF and that cleavage by this endoprotease occurs at the carboxyl side of the consensus R-H-R-R cleavage motif.


MATERIALS AND METHODS

Recombinant Vaccinia Viruses

Vaccinia virus (VV) strain WR was used in this study. The VV wild type (VV:WT) and the VV recombinant engineered to express a soluble COOH-terminal truncated form of furin (VV:hFUR713t) or full-length furin (VV:FUR) have been previously described (30, 32) . The engineering of VV:POMC has been previously described (33) and VV:TGF was prepared as described (34) using full-length human TGF1 cDNA insert (obtained from the American Type Culture Collection (ATTC)).

Production of Soluble Furin

For furin production, BSC-40 cells were grown to near confluence on 100-mm plates in minimal essential medium containing 10% fetal calf serum. Parallel plates were infected with either VV:WT or VV:hFUR713t at an infection multiplicity of 5. At 18-h post-infection, cells were washed twice with phosphate-buffered saline and incubated at 37 °C for 4 h in 2 ml of serum-free medium (MCDB 202; Life Technologies, Inc.) in the presence of aprotinin (1 µg/ml), pepstatin (0.7 µg/ml), leupeptin (0.5 µg/ml) and phenylmethylsulfonyl fluoride (170 µg/ml). Media were harvested, centrifuged (12,000 g, 10 min) at 4 °C, and the supernatants were concentrated by ultrafiltration using Centricon-30 filtration units and kept on ice until used.

Coinfection Studies

LoVo cells (obtained from ATCC) were grown to near confluence on 100-mm plates in Ham's F12 medium containing 10% fetal calf serum. Cultures were infected as described previously (35, 36) with a mixture of VV:TGF and either VV:POMC or VV:FUR.

Enzymatic Assay

Fluorometric assay on the boc RVRR-aminomethylcoumarin substrate (380-nm excitation, 460-nm emission) was performed as described previously (30) . One unit of activity was defined as the amount of enzyme that can release 1 pmol of aminomethylcoumarin from the substrate in 1 h.

Pro-TGF Purification

TGF1 precursor from CHO cells transfected with simian TGF1 cDNA (19) was purified from a confluent roller bottle collected for 48 h containing 50 ml of serum-free media as described (37) and then dialyzed versus two changes of 0.2 M acetic acid and one change of 4 mM HCl. The acidified TGF1 media was lyophilized, redissolved in 2 ml of 40% acetonitrile-water, 0.1% trifluoroacetic acid, and fractionated on an HPLC gel filtration column, SEC-250 (Bio-Rad). The pro-TGF1 was collected (37) and purified by HPLC chromatography on a C-18 column with a gradient of water, 0.05% trifluoroacetic acid and propanol, 0.05% trifluoroacetic acid, and the precursor appeared at 23% propanol, 0.05% trifluoroacetic acid. In some cases, the monomeric pro-TGF1 was purified on reducing SDS-PAGE gels.

Endoproteolytic Cleavage of Pro-TGF and Inhibitor Assays

Five µl of pro-TGF1 purified as described above was incubated in the presence of 27 µl of VV:WT-derived conditioned medium or VV:hFUR713t-derived conditioned medium (3-5 units of furin as defined above) in 8 µl of 5 reaction buffer (500 mM HEPES, 0.50% Triton X-100, and 5 mM CaCl, 5 mM 2-mercaptoethanol pH 7.5) at 30 °C for 3 h. For inhibition experiments, EDTA was added to the reaction mixture at a final concentration of 5 mM and the decanoyl-arginine-valine-lysine-arginine-chloromethylketone (decanoyl-RVKR-cmk) was dissolved in water at 1 mM and 0.2 µl (5 uM final) was added 30 min before the addition of pro-TGF1. The reaction mixture was separated on 12% SDS-PAGE gels. Separated proteins were then transferred onto nitrocellulose membrane, blocked, and probed overnight with peptide antiserum that are specific for NH-terminal (1:200 dilution) sequence, COOH-terminal sequence (1:200 dilution) or the whole precursor polypeptide (1:800 dilution). The antibodies have been characterized previously (19) . The membranes were then washed and incubated for 1 h with either horseradish peroxydase-labeled anti-rabbit IgG (1:2500) and immunoreactive bands revealed using the ECL detection system (Amersham) or alkaline phosphatase-conjugated anti-rabbit IgG (1:5000) and developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent (Bio-Rad).

Growth Inhibition Assay

Growth inhibition assays were performed with Mv1Lu mink lung epithelial cells (CCL-64; ATCC) essentially as originally described by Tucker et al.(38) . In brief, CCL64 cells were plated in 96-well flat bottom plates at 2500 cells/well. After 24 h, the medium was removed and serial dilutions of the samples to assay for TGF activity were added. After 72 h incubation, the cells were pulsed with [H]thymidine. Cells were collected and radioactivity counted in a liquid scintillation counter. One unit of activity was defined as the amount of TGF required to give 50% maximal response in the assay.

NH-terminal Sequence Analysis

The 55 kDa band corresponding to monomeric TGF1 precursor was excised from preparative 7.5% SDS-PAGE gels accordingly to the position of prestained molecular mass markers. The bands were sliced and eluted from gels 4 h in 250 µl of enzymatic reaction buffer, concentrated by ultrafiltration using Microcon-30 filtration units, and diluted in enzymatic digestion buffer. Pro-TGF cleavage was achieved as described above. The cleavage products were separated by 15% SDS-PAGE gels, electrotransferred onto Immobilon P membranes, and stained with Coomassie Brilliant Blue R-250. The 12.5 kDa band corresponding to the mature form of TGF was excised and subjected to automated Edman degradation in a PSQ-1 gas-phase sequencer.


RESULTS

In Vitro Cleavage of Purified TGF1 Precursor by Human Furin

To determine whether cleavage of the TGF1 precursor can be mediated by furin, we initially used in vitro enzymatic digestions. As a source of pro-TGF, supernatants from CHO cells expressing the simian pre-pro-TGF1 sequence were used (39) . Purification of these supernatants was performed as described under ``Materials and Methods.'' The simian type 1 TGF cDNA encodes the entire growth factor precursor and displays a high sequence homology to its human counterpart (39, 40) . TGF-transfected CHO cells have been shown to secrete two molecular forms of TGF1 proteins (100-110 and 25 kDa) as determined by SDS-PAGE under non-reducing conditions (19) . Upon electrophoresis under reducing conditions, followed by gel staining with Coomassie Brilliant Blue, the corresponding monomers were found: a 55 kDa band (pro-TGF1), a 44 kDa band (pro-region), and a mature 12.5-kDa TGF1 monomer (Fig. 1 B). Because of the glycosylation of the precursor sequence (41) , the 55 and 44 kDa bands appear as broad, stained regions. In contrast, the mature TGF polypeptide that does not contain glycosylation sites is detected as a sharp, discrete band. Evidence that the 55, 44, and 12.5 kDa in Fig. 1 B represent the regions of the TGF precursor depicted in Fig. 1A has been presented previously (19) . Note that as previously reported (37) , current procedures used to purify the intact 55-kDa pro-TGF to homogeneity also resulted in the recovery of the 44- and 12.5-kDa species due to reassociation of the pro-region with the mature polypeptide. Further attempts to fractionate this TGF complex into separate components were unsuccessful (37) . Therefore, our precursor preparation contains detectable amounts of the pro-region and the mature polypeptide.


Figure 1: A, schematic representation of human pre-pro-TGF1 (27). The signal sequence (residues 1-29, black box), the pro-peptide (residues 30-278, open box), and mature peptide (residues 279-390, hatched box) regions are indicated. Solid lines above the molecule represent peptide sequences used for antibody production; antibody designation is indicated above the lines. The arrow indicates the cleavage site. B, Coomassie Blue-staining pattern of proteins from stably transfected CHO cells expressing TGF1. The CHO-conditioned media was purified as described under ``Materials and Methods,'' and 2.5 µl was fractionated on a 12% SDS-polyacrylamide gels under reducing conditions followed by staining with Coomassie Brilliant Blue R-250. Arrows noted at the right of the figure correspond to TGF1 molecules depicted in A.



As a source of human furin, supernatants from BSC-40 cells infected with recombinant VV containing cDNA encoding the soluble secreted form of human furin (VV:hFUR713t) was used. Furin-containing supernatants were compared in our assays with supernatants from VV:WT. We were unable to detect furin-like activity from VV:WT supernatants using the fluorogenic substrate boc RVRR-aminomethylcoumarin. Following treatment with furin, the pro-TGF1 preparation was analyzed using immunobloting techniques. Sequence-specific antibodies as indicated in Fig. 1A were used to detect changes in immunoreactive polypeptides after digestion with furin. As shown in Fig. 2, lane 2, the overall intensity of the 55 kDa band was diminished, and the 44 kDa band was intensified as detected with an NH-terminal-specific antiserum. This shift was not observed using supernatant from wild type VV-infected BSC-40 cells (Fig. 2, lane 3). A similar immunoblot probed with the COOH-terminal TGF1-specific antiserum indicated that after furin treatment, a 12.5 kDa band was intensified (Fig. 2, lanes 2 and 1). This band has the same migration pattern as pure recombinant TGF1 (data not shown). In a time course study, subtle intensification of the 44 and 12.5 kDa bands was observed after a 1-h digestion with a maximal effect seen at 3 h as revealed with antibodies against the whole precursor (data not shown). Therefore, furin is able to process the TGF1 precursor yielding the corresponding 44-kDa pro-region and 12.5-kDa-TGF-related cleavage products as detected under reducing conditions. Interestingly, prolonged digestion of pro-TGF1 with furin or increasing concentrations of furin (data not shown) did not yield any intermediates which may have occurred following processing of pairs of basic amino acids found within the pro-TGF 1 molecule (39, 40) . This provides evidence for the specificity of the in vitro cleavage of pro-TGF1 by furin.


Figure 2: In vitro processing of pro-TGF1 by human furin. Immunoblot analysis of furin-treated CHO-derived TGF1 precursor. SDS-PAGE-separated proteins were transferred onto nitrocellulose membranes and probed with antiserum no. 1125 directed against the COOH-terminal region of the pro-region of the precursor or antiserum no. 978 which is specific to the carboxyl-terminal region of the mature sequence. Lane 1, purified pro-TGF; lane 2, pro-TGF incubated with supernatants from VV:FUR713t(furin)-infected BSC-40 cells for 3 h; lane 3, pro-TGF incubated with supernatants from VV:WT (control)-infected cells for 3 h.



Peptidyl chloroalkylketones with peptide moieties that mimic the furin cleavage site have been shown to be specific inhibitors of furin enzymatic activity (26) . Fig. 3B shows the inhibitory properties of the water-soluble decanoyl-RVKR-cmk on furin-mediated cleavage of TGF1 precursor. Densitometric measurement of the 44 and 12.5 kDa band indicated that 5 µM of dec-RVKR-cmk blocked furin-mediated conversion of the 55-kDa TGF precursor into the corresponding 12.5- and 44-kDa cleavage products. In fact, the intensity of the bands corresponding to pro-TGF cleavage products (44- and 12.5-kDa species) was similar to background level (due to 44 and 12.5-kDa species found in the initial pro-TGF1 preparation used; see Fig. 1B). Similar inhibition was observed using 5 mM EDTA showing the requirement of calcium for endoproteolytic cleavage of pro-TGF by furin (Fig. 3 A).


Figure 3: Inhibition of furin-mediated cleavage of pro-TGF 1 by EDTA and decanoyl-RVKR-cmk. Pro-TGF was incubated in the presence of VV:FUR713t (furin) for 3 h in the presence or absence of EDTA (5 mM) ( A, lanes 2 and 3) or 5 µM of decanoyl-RVKR-cmk ( B, lanes 2 and 3) and analyzed by immunoblotting as described in the legend of Fig. 2.



NH-terminal Sequencing of Cleavage Product

Proteolytic cleavage of TGF1 is predicted to occur following a basic Arg-His-Arg-Arg sequence and immediately preceding the NH-terminal Ala residue of the mature growth factor (37, 39, 40) . To specifically localize the site of cleavage by furin, the purified 12.5-kDa proteolytic fragment was subjected to NH-terminal sequence analysis. The resulting sequence Ala-Leu-Asp- X-Asn was consistent with the predicted NH-terminal sequence of mature simian TGF1 and indicates that cleavage by furin occurs carboxyl to the consensus Arg-His-Arg-Arg furin cleavage motif.

Bioactivity of TGF1 Exposed in Vitro to Furin

We next asked whether in vitro proteolytic cleavage of pro-TGF1 by furin would release bioactive TGF1. For this purpose, a rapid and sensitive growth inhibition assay using CCL-64 mink lung epithelial cells was used (38) . Pro-TGF1 incubated with supernatants from either VV:hFUR713t- or VV:WT-infected BSC-40 cells was assayed for biological activity. Fig. 4 demonstrates that treatment of pro-TGF1 with furin results in a 5-fold increase in bioactive TGF with 213 µg/ml of TGF detected compared to 42 µg/ml in control supernatant (pro-TGF1 treated with supernatant from VV:WT-infected BSC-40 cells). These results indicate that processing of pro-TGF1 by furin results in the production of biologically active TGF and that furin processed pro-TGF at a physiologically relevant cleavage site.


Figure 4: Bioactivity of TGF1 exposed in vitro to furin. Bioactive TGF was determined in a mink lung cell growth inhibition assay. A representative experiment out of five performed is shown.



In Vivo Cleavage of TGF1 by Furin

To assess the ability of furin to cleave TGF precursor in cells, coinfection experiments were performed, and the products of expression were analyzed by electrophoresis of concentrated supernatants on reducing SDS-PAGE gels followed by immunoblotting. For this experiment, we used the furin-deficient LoVo cells (42, 43) . As illustrated in Fig. 5, lanes 1 and 2, the 55-kDa TGF1 precursor band was not observed in mock infected LoVo cells or cells infected with control recombinant vaccinia (VV:POMC). LoVo cells coinfected with TGF-expressing vaccinia recombinant (VV:TGF) and a control vaccinia recombinant (VV:POMC) failed to cleave TGF1 precursor (Fig. 5, lane 3) as shown by the detection of the 55 kDa precursor band only. Using the CCL-64 bioassay with a sensitivity of 50 pg/ml, we did not detect any bioactive TGF from these supernatants. This indicates that the 55-kDa pro-TGF has very little or lacks bioactivity (). However, coinfection with VV:TGF and VV:FuR resulted in a loss of immunoreactive 55 kDa precursor band with the concomitant appearance of the 44-kDa pro-region as detected with the NH-terminal-specific antibody (Fig. 5, lane 4). Reprobing of the same blot with the COOH-terminal-specific antisera indicate that the observed detection of the 44-kDa species corroborated with the detection of the 12.5-kDa polypeptide. No other species than the intact pro-region (44 kDa) and the mature polypeptide (12.5 kDa) was observed even when the immunoblots were overexposed (data not shown). Measure of the biological activity of the (VV:TGF/VV:FUR) supernatants revealed large amounts of bioactive TGF (20, 385 pg/ml; ).


Figure 5: In vivo cleavage of pro-TGF1 by human furin. LoVo cells in 10-cm dishes were coinfected with each virus added at a multiplicity of infection of 3. 18-h post-infection, supernatants were collected, concentrated, and resolved on 12% reducing SDS-PAGE gels. Immunoblotting was performed with antiserum no. 1125 or antiserum no. 978. Supernatants from mock-infected cells ( lane 1); VV:POMC-infected cells ( lane 2); VV:TGF/VV:POMC-infected cells ( lane 3); VV:TGF/VV:FUR-infected cells ( lane 4).




DISCUSSION

A series of cellular events take place during TGF processing. Following synthesis of pre-pro-TGF, the signal peptide is rapidly removed followed by N-glycosylation at predicted sites (41) . The TGF precursor is then translocated to the Golgi network where other post-translational modifications occur including the endoproteolytic processing at the carboxyl-terminal side of a cleavage site consisting of basic residues (13, 40) . No information is available to date concerning the enzyme(s) involved in intracellular processing and release of mature TGF1 from its larger precursor polypeptide. Proteases such as plasmin, a relatively nonspecific serine protease, as well as lysosomal cathepsin D have been shown to cleave and activate pro-TGF isoforms (13, 44, 45) . However, these enzymes would unlikely be involved per se in TGF processing events. In fact, multiple cleavage sites for these enzymes are present in the sequence of TGF isoforms (40, 46) , and it has been demonstrated that cleavage of TGF1 precursor by plasmin results in degradation of the pro-region and the mature TGF polypeptide; this later event results in a loss of biological activity (47) . This is inconsistent with unique pro-TGF1 enzymatic processing site deduced by examination of the amino acid sequence (40) and further revealed in TGF1 overexpressing cells (13) . In addition, plasmin and cathepsin D enzymatic activities are found at the cell surface and in the pericellular space (plasmin) or in the lysosomes (cathepsin D) which is physically incompatible with the localization of TGF processing events which has been demonstrated to take place during TGF transit in the Golgi complex (20, 31) .

The ubiquitous expression of both furin and TGF1, the existing correlation between their cellular localization, and the strategic presence of an optimum furin consensus cleavage motif in the pro-TGF1 amino acid sequence make furin a good candidate for the physiological processing of this precursor. The data presented here demonstrate that furin efficiently processes pro-TGF1 and that cleavage by this endoprotease occurs at the physiologically relevant R-H-R-R processing site. The identity of this cleavage site is supported by studies in which the 12.5-kDa TGF1, secreted from overexpressing CHO cells, was isolated and sequenced. The results revealed an intact amino terminus sequence at Ala of the mature growth factor, indicating that CHO cells properly processed pro-TGF1 at the R-H-R-R cleavage site (37) . Since furin mRNA was detected in these cells, it is possible that endogenous furin is capable of producing the TGF1 secreted from these cells.

The evidence for furin as a relevant TGF processing enzyme is substantiated by data from coinfection studies in LoVo cells. These cells are a human colon carcinoma cell line which was previously shown to have a point mutation in the fur gene and was incapable of processing the hepatocyte growth factor proreceptor (42, 43) which bares a similar cleavage site (R-K-K-R) as pro-TGF1. It was shown that this defect can be corrected by genetic complementation of LoVo cells with mouse fur cDNA (43) . In our study, analysis of supernatants from vaccinia virus-mediated overexpression of TGF1 precursor in LoVo cells indicated that only the intact precursor form, which lacks biological activity, was secreted whereas coexpression of TGF and furin resulted in the release of significant amounts of immunoreactive and bioactive TGF.

Interestingly, in vitro and in vivo experiments also revealed that the endoproteolytic cleavage mediated by furin did not yield any intermediates which may have occurred following processing at pairs of basic amino acids found within the pro-TGF 1 molecule (39, 40) and provide evidence for the selectivity of cleavage of pro-TGF1 by furin. Indeed, under certain experimental conditions, furin has been found to cleave (however with less efficiency) at pairs of basic amino acids without the presence of Arg in the P4 position. For example, POMC was partially converted by overexpressed furin, yielding physiologically relevant peptides (-LPH) originating from cleavage at pairs of basic amino acids only (48, 49) . Additionally, analysis of the enzymatic properties of furin on hemaglutinin of influenza virus indicated that overexpression of the enzyme led to a broadening of its specificity (50) . Therefore, in our systems, the unique cleavage site mediated by furin indicates that other basic moieties found within the TGF pro-region and the mature polypeptide are indeed not favored under our in vitro and in vivo experimental conditions.

We were quite surprised to observe an important disparity between the efficiency of furin to cleave pro-TGF in vivoversusin vitro. In fact, we found a complete cleavage of TGF precursor in cells, compared with the partial cleavage found in the in vitro enzymatic assays, even in the presence of higher levels of furin enzymatic activity ( Fig. 5compared to Figs. 2 and 3). The exact reason for this discrepancy is at the moment unknown. However, we can speculate that some yet unidentified molecular chaperones, present in cells, are needed to conduct proper folding and cleavage of TGF precursor (75) . We also cannot rule out the possibility that the purification process somehow altered pro-TGF1, rendering it a poor substrate for furin in vitro.

With the identification and characterization of novel SPCs with similar substrate specificity as furin (51) , it has become important to delineate which enzyme(s) in this class of proteases expresses TGF convertase activity. Indeed, it has been recently demonstrated that furin and PACE 4 share similar substrate specificity (52, 53) . Of the six SPCs that have been characterized, PACE 4 and PC5/PC6, which show a broad tissue distribution similar to TGF, might fulfill this role while PC1, PC2, and PC4 which display restricted tissue specificity are likely to be excluded from the list. As stated above, failure of LoVo cells to process TGF precursor and the efficient processing observed by overexpression of furin highlighted furin as a TGF-processing enzyme. It should be stated that the LoVo cells, as most of the constitutively secreting cell types, do not express PC1 or PC2 mRNA (54) . However, they express large amount of PACE 4 mRNA as well as PACE 4 enzymatic activity. Indeed, using the same vaccinia-based overexpression system as used in this study, LoVo cells were able to process pro-NGF at a R-S-K-R cleavage motif, possibly implicating the endogenous PACE 4 convertase.() In our study, failure of these cells to process overexpressed TGF precursor suggests that endogenous PACE 4 does not play a prime role in TGF processing events. In addition, preliminary data from coinfection studies with pro-TGF1 and PC1, PC2, PACE 4, PC5/PC6, or furin indicates that furin is indeed a prominent TGF converting enzyme.()

Our findings may have broad biological implications. TGF has been shown to be a most potent natural inhibitor of many inflammatory and immune pathways. TGF in vitro inhibits the proliferation of several hematopoietic cells such as thymocytes (55) , T and B cells (56, 57, 58, 59) , and primitive bone marrow progenitors (60, 61, 62, 63) . In addition, TGF suppresses IgM and IgG production by B cells (56) , antagonizes the immunoregulatory effect of interleukin-1, -2, and -3 (56, 57, 58, 59) , colony-stimulating factor (62, 64) , and interferons (65, 66) , reduces cell-surface receptor expression for several growth factors (67, 68) , and induces interleukin-1 receptor antagonist (69) . Therefore, the presence of active TGF in biological fluids and tissues is likely to be of prime biological importance. Supporting this, studies from several teams have shown that targeted disruption of the murine TGF1 gene resulted in uncontrolled inflammatory response that leads to premature death (70, 71, 72) . On the other hand, increased production of activated TGF has been associated with malignant progression (73) . TGF has been shown to induce angiogenesis, to act as an autocrine tumor growth factor, and to impair tumor immune surveillance (1, 2, 3, 74) . All these functions would cooperate in the amplification of tumor progression. Since the endoproteolytic cleavage of TGF precursor is likely the cornerstone for full activity of the mature product, one can speculate that a delicate balance of furin-like enzymatic activity is required for normal physiological growth and differentiation of the cells and that disturbance of this balance could lead to pathological aberrations. In support of this, we have recently observed that treatment of synovial cells and NIH-3T3 cells with TGF resulted in a significant increase in fur mRNA levels suggesting that TGF might auto-regulate its own converting enzyme.()

In conclusion, our biochemical studies together with coexpression studies provide evidence that furin is a good candidate as a physiological endoprotease responsible for TGF1 processing. The other mammalian TGF isoforms namely TGF2 and TGF3 are also initially synthesized as larger precursor with similar furin consensus motif at the junction of the pro-region and the mature polypeptide. Efforts are underway to assess whereas these other TGF isoforms are processed by furin and to confirm furin as thebona fide pro-TGF convertase.

  
Table: Measure of bioactive TGF in supernatants of LoVo cells coinfected with recombinant vaccinia



FOOTNOTES

*
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. This work was supported by the Arthritis Society of Canada.

§
Research Scholar of the Fonds de la Recherche en Santé du Québec. To whom reprint requests should be addressed: Immunology Division, Dept. of Pediatrics, Faculty of Medicine, Université de Sherbrooke, 3001 N. 12th Ave., Sherbrooke, Québec J1H 5N4, Canada. Tel.: 819-563-5555 (ext. 4851); Fax: 819-564-5215; E mail: cmdubois@courrier.usherb.ca.

Recipient of Grant A60848 from the National Institutes of Health.

**
Research Scholar of the Fonds de la Recherche en Santé du Québec.

The abbreviations used are: TGF, transforming growth factor ; VV, Vaccinia virus; WT, wild type; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; FUR, furin; POMC, proopiomelanocortin; CHO, Chinese hamster ovary.

N. Seidah, personal communication.

C. M. Dubois and N. Seidah,, manuscript in preparation.

Blanchette, F., Day, R., Laprise, M.-H. Grondin, F., and Dubois, C. M. (1995) FASEB J., in press.


ACKNOWLEDGEMENTS

We thank Francine Grondin for skilled technical assistance. We are greatly indebted to Dr. Nabil Seidah for his contribution in the coinfection studies and helpful discussion and to Dr. Claude Lazure for amino acid sequencing. We also thank Dr. Francis W. Ruscetti for critical review and helpful discussion and Carole Jacques for editorial assistance. Recombinant bovine TGF1 was generously provided by Oncogen/Bristol-Myers Company and the furin inhibitor dec-RVKR-cmk was kindly provided by Drs. Herbert Angliker and Elliott Shaw.


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