Evidence that Furin Is an Authentic Transforming Growth Factor-β1-Converting Enzyme
2001; Elsevier BV; Volume: 158; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)63970-3
ISSN1525-2191
AutoresClaire M. Dubois, François Blanchette, Marie‐Hélène Laprise, Richard Leduc, Francine Grondin, Nabil G. Seidah,
Tópico(s)Growth Hormone and Insulin-like Growth Factors
ResumoTransforming growth factor (TGF)-β1 plays an essential role in cell growth and differentiation. It is also considered as a gatekeeper of immune homeostasis with gene disruption leading to autoimmune and inflammatory diseases. TGF-β1 is produced as an inactive precursor polypeptide that can be efficiently secreted but correct proteolytic cleavage is an essential step for its activation. Assessment of the cleavage site has revealed a unique R-H-R-R sequence reminiscent of proprotein convertase (PC) recognition motifs and has previously demonstrated that this PC-like cleavage site is correctly cleaved by furin, a member of the PC family. Here we report that among PC members, furin more closely satisfies the requirements needed to fulfill the role of a genuine TGF-β1 convertase. Even though six members of the PC family have the ability to cleave TGF-β1, ectopic expression of α1-antitrypsin Portland (α1-AT-PDX), a potent furin inhibitor, blocked 80% of TGF-β1 processing mediated by endogenous enzymes as demonstrated in an in vitro digestion assay. Genetic complementation of a furin-deficient LoVo cell line with the wild-type gene restores the production of mature and bioactivable TGF-β1. Moreover, both furin and TGF-β are coordinately expressed and regulated in vitro and in vivo in the hematopoietic and immune system, an important tissue target. These results demonstrate for the first time that furin is an authentic and adaptive TGF-β1-converting enzyme whereas other members of the PC family might substitute or supplement furin activity. Our study advances our comprehension of the complexity of the TGF-β system and should facilitate the development of therapeutically useful TGF-β inhibitors. Transforming growth factor (TGF)-β1 plays an essential role in cell growth and differentiation. It is also considered as a gatekeeper of immune homeostasis with gene disruption leading to autoimmune and inflammatory diseases. TGF-β1 is produced as an inactive precursor polypeptide that can be efficiently secreted but correct proteolytic cleavage is an essential step for its activation. Assessment of the cleavage site has revealed a unique R-H-R-R sequence reminiscent of proprotein convertase (PC) recognition motifs and has previously demonstrated that this PC-like cleavage site is correctly cleaved by furin, a member of the PC family. Here we report that among PC members, furin more closely satisfies the requirements needed to fulfill the role of a genuine TGF-β1 convertase. Even though six members of the PC family have the ability to cleave TGF-β1, ectopic expression of α1-antitrypsin Portland (α1-AT-PDX), a potent furin inhibitor, blocked 80% of TGF-β1 processing mediated by endogenous enzymes as demonstrated in an in vitro digestion assay. Genetic complementation of a furin-deficient LoVo cell line with the wild-type gene restores the production of mature and bioactivable TGF-β1. Moreover, both furin and TGF-β are coordinately expressed and regulated in vitro and in vivo in the hematopoietic and immune system, an important tissue target. These results demonstrate for the first time that furin is an authentic and adaptive TGF-β1-converting enzyme whereas other members of the PC family might substitute or supplement furin activity. Our study advances our comprehension of the complexity of the TGF-β system and should facilitate the development of therapeutically useful TGF-β inhibitors. 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All three mammalian TGF-β isoforms are first synthesized as larger biologically inactive precursors which are proteolytically processed to yield a 25-kd homodimer.5Derynck R Jarrett JA Chen EY Eaton DH Bell JR Assoian RK Roberts AB Sporn MB Goeddel DV Human transforming growth factor-β complementary DNA sequence and expression in normal and transformed cells.Nature. 1985; 316: 701-705Crossref PubMed Scopus (1383) Google Scholar, 13Gentry LE Lioubin MN Purchio AF Marquardt H Molecular events in the processing of recombinant type I pre-pro-transforming growth factor beta to the mature polypeptide.Mol Cell Biol. 1988; 8: 4162-4168Crossref PubMed Scopus (210) Google Scholar The biosynthesis of the β1 isoform is the most extensively studied and generally regarded as the prototype of the TGF-β family. TGF-β1 is initially synthesized as a 390 amino-acid precursor molecule.5Derynck R Jarrett JA Chen EY Eaton DH Bell JR Assoian RK Roberts AB Sporn MB Goeddel DV Human transforming growth factor-β complementary DNA sequence and expression in normal and transformed cells.Nature. 1985; 316: 701-705Crossref PubMed Scopus (1383) Google Scholar Studies of the molecular events in the processing of this precursor dictate that important proteolytic sites are present and contribute in the formation of the final product. After synthesis, the signal peptide cleavage occurs at the Gly-29-Leu-30 peptide bond of the preproTGF-β1, yielding proTGF-β1 (amino acids 30 to 390). Proteolytic processing of the precursor to yield the mature TGF-β takes place at a cluster of basic amino acids (R-H-R-R) immediately preceding Ala-279.13Gentry LE Lioubin MN Purchio AF Marquardt H Molecular events in the processing of recombinant type I pre-pro-transforming growth factor beta to the mature polypeptide.Mol Cell Biol. 1988; 8: 4162-4168Crossref PubMed Scopus (210) Google Scholar Interestingly, this processing site is a consensus cleavage motif for the mammalian convertase furin and we have provided evidence that the TGF-β1 precursor is effectively processed in vitro and in vivo by human furin releasing a mature TGF-β1 moiety homologous to the natural product.14Dubois CM Laprise M-H Blanchette F Gentry LE Leduc R Processing of transforming growth factor β1 precursor by human furin convertase.J Biol Chem. 1995; 270: 10618-10624Crossref PubMed Scopus (335) Google Scholar Such observation shed new light on the process of TGF-β maturation events but further experiments were needed to verify if furin is a genuine TGF-β1-converting enzyme. 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The fur gene, which encodes furin, seems to be ubiquitously expressed in all tissues and cell types examined to date but in variable amounts among them.27Kiefer MC Tucker JE Joh R Landsberg KE Saltman D Barr JP Identification of a second human subtilisin-like protease gene in the fes/fps region of chromosome 15.DNA Cell Biol. 1991; 10: 757-769Crossref PubMed Scopus (248) Google Scholar, 28Hatsuzawa K Hosaka M Nakagawa T Nagase M Shoda A Murakami K Nakayama K Structure and expression of mouse furin, a yeast Kex2-related protease. 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According to their tissue distribution, the PCs can be classified into distinct subgroups where furin and PC7/PC8 are ubiquitously distributed, PACE-4, PC5/PC6A, and PC5/PC6B are expressed to varying degrees in many tissues and whereas the other convertases PC1, PC2, and PC4 are restricted to specific tissues such as neural and endocrine ones (PC1, PC2) and testicular spermatogenic cells (PC4). Unlike disruption of PC233Furuta M Yano H Zhou A Rouillé Y Holst J Carroll J Ravazzola M Orci L Furuta H Steiner DF Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2.Proc Natl Acad Sci USA. 1997; 94: 6646-6651Crossref PubMed Scopus (356) Google Scholar or PC4,34Mbikay M Tadros H Ishida N Lerner CP De Lamirande E Chen A El-Alfy M Clermont Y Seidah NG Chrétien M Gagnon C Simpson EM Impaired fertility in mice deficient for the testicular germ-cell protease PC4.Proc Natl Acad Sci USA. 1997; 94: 6842-6846Crossref PubMed Scopus (136) Google Scholar silencing of the expression of mouse furin results in embryonic lethality between days 10.5 and 11.5.35Roebroek AJ Umans L Pauli IG Robertson EJ Van Leuven F Van de Ven WJ Constam DB Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin.Development. 1998; 125: 4863-4876Crossref PubMed Google Scholar This is presumably because of hemodynamic insufficiency associated with several developmental defects including failure of the heart tube to fuse and undergo looping morphogenesis and failure of the embryo to undergo axial rotation. These findings are consistent with a role of furin in the maturation/activation of several members of the TGF-β family including TGF-β1, BMPs, nodal, dorsalin, and lefty-1 and -2 and suggest that they are candidate physiological furin substrates. Given the similarity of cleavage site specificity between all PCs and their overlapping expression in different tissues, it is often difficult to assign cleavage of a given precursor to a particular convertase. However, processing within the constitutive secretory pathway is probably achieved by either furin, PACE-4, PC5A and PC5B, or PC7. Because these proteases all have specificity toward multiple (clusters) of basic amino acids, it is possible one or more PCs are involved in the endoproteolytic cleavage of the TGF-β precursor. Among the processing competent PCs, an authentic protease responsible for TGF-β activation would have to fulfill several indispensable requirements. These include correct cleavage in vitro and in vivo at the naturally occurring cleavage site as well as coordinated expression and regulation of the precursor and the enzyme. Other essential proofs are provided from specific inhibition studies whereas inactivation or genetic alteration of the enzyme should invariably prevent processing of the precursor in the intact cell. This study was designed to define if furin meets the requirement of an authentic TGF-β-converting enzyme and if other members of the PC class of proteases also expresses TGF-β convertase activity. So far, our results indicate that furin fulfills the essential requirements needed for an authentic TGF-β1-converting enzyme whereas other members of the PC family might supplement or substitute in part furin activity. Possible involvement of these findings in embryogenesis as well as TGF-β-related biological and pathological conditions are discussed. The vaccinia virus wild type (VV:WT) and the VV recombinant engineered to express the proopiomelanocortin (POMC) polypeptide (VV:POMC) are used as control vaccinia virus and control recombinant vaccinia virus, respectively. Full-length hTGF-β1 cDNA (ATCC, Rockville, MD) was cloned into the vaccinia insertion plasmid pJM602 and homologous recombination performed according to established procedures.36Benjannet S Rondeau N Day R Chretien M Seidah NG PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues.Proc Natl Acad Sci USA. 1991; 88: 3564-3568Crossref PubMed Scopus (539) Google Scholar Recombinant vaccinia viruses expressing each PC (mPC1/PC3, mPC2, hPACE-4, mPC5A, mPC5B, hPC7, and hfurin) have been constructed and isolated as previously reported.37Munzer JS Basak A Zhong M Mamarbachi A Hamelin J Savaria D Lazure C Benjannet S Chretien M Seidah NG In vitro characterization of the novel proprotein convertase PC7.J Biol Chem. 1997; 272: 19672-19681Crossref PubMed Scopus (76) Google Scholar, 38Paquet L Bergeron F Boudreault A Seidah NG Chretien M Mbikay M Lazure C The neuroendocrine precursor 7B2 is a sulfated protein proteolytically processed by a ubiquitous furin-like convertase.J Biol Chem. 1994; 269: 19279-19285Abstract Full Text PDF PubMed Google Scholar, 39Benjannet S Savaria D Chretien M Seidah NG 7B2 is a specific intracellular binding protein of the prohormone convertase PC2.J Neurochem. 1995; 64: 2303-2311Crossref PubMed Scopus (91) Google Scholar, 40Vollenweider F Benjannet S Decroly E Savaria D Lazure C Thomas G Chretien M Seidah NG Comparative cellular processing of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp160 by the mammalian subtilisin/kexin-like convertases.Biochem J. 1996; 314: 521-532Crossref PubMed Scopus (82) Google Scholar VV:α1-AT was a generous gift from Dr. Gary Thomas (Vollum Institute, Oregon Health Science University, Portland, OR). BSC 40 or LoVo cells were infected with different recombinant vaccinia viruses according to previously published protocols36Benjannet S Rondeau N Day R Chretien M Seidah NG PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues.Proc Natl Acad Sci USA. 1991; 88: 3564-3568Crossref PubMed Scopus (539) Google Scholar with the exception that supernatants were collected 18 hours after infection. After co-infections, the supernatants were dialyzed overnight against 0.2 mol/L acetic acid, lyophilized, and resolved into reducing 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. In selected experiments, the cell pellets were lysed with 1% Nonidet P-40-containing lysis buffer supplemented with a cocktail of protease inhibitors (1 mmol/L phenylmethyl sulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). Separated proteins were then transferred onto nitrocellulose membranes, blocked, and probed overnight with affinity-purified goat anti-human LAP IgG (1:1,000; R&D Systems, Minneapolis, MN) or rabbit PAN-specific anti-TGF-β antibodies (1:1,000; R&D Systems), rabbit furin-specific antisera (1:10,000; Chiron Corp., Emeryville, CA) or rabbit α1-antitrypsin-specific antisera (1:2,000; DAKO Corp., Carpinteria, CA). The membranes were then washed and incubated 1 hour with horseradish peroxidase-labeled anti-rabbit IgG (1:5,000) or anti-goat IgG. (1:8,000) Immunoreactive bands were revealed using the enhanced chemiluminescence detection system (Amersham Canada Ltd., Oakville, Ontario, Canada). In vitro modulation of furin and TGF-β1 mRNA was performed as previously described.41Blanchette F Day R Dong W Laprise M-H Dubois CM TGFβ1 regulates gene expression of its own converting enzyme furin.J Clin Invest. 1997; 99: 1974-1983Crossref PubMed Scopus (119) Google Scholar For in vivo modulation, mice were injected intraperitoneally with 5 μg recombinant human TGF-β1, a generous gift from Dr. Antony F. Purchio (Oncogene Corp., Seattle, WA), or 1 μg lipopolysaccharide (Sigma, Oakville, Ontario, Canada). Tissues were collected 6 hours or 24 hours after mice injection, grinded in TriReagent (Molecular Research Center, Inc., Cincinnati, OH) solution, and mRNA extracted according to the TriReagent protocol. For the tissue expression or regulation of furin and TGF-β1, 5 μg of total RNA was used for Northern blot analysis. The rat cRNA furin, PC1, PACE-4, PC5, and PC7 riboprobes were generated as previously described.29Seidah NG Chretien M Day R The family of subtilisin/kexin like pro-protein and pro-hormone convertases: divergent or shared functions.Biochimie. 1994; 76: 197-209Crossref PubMed Scopus (383) Google Scholar, 42Seidah NG Hamelin J Mamarbachi M Dong W Tardos H Mbikay M Chretien M Day R cDNA structure, tissue distribution, and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases.Proc Natl Acad Sci USA. 1996; 93: 3388-3393Crossref PubMed Scopus (225) Google Scholar The rat cRNA TGF-β1 riboprobe was produced from a 985-nucleotide cDNA (ATCC) which corresponds to the coding region nucleotides 413 to 1582 of the published sequence.43Quian SW Kondaiah P Roberts AB Sporn MB cDNA cloning by PCR of rat transforming growth factor β-1.Nucleic Acids Res. 1990; 18: 3059-3065Crossref PubMed Scopus (265) Google Scholar Briefly, the cDNA insert was excised from pBluescript2KS+ by Hin dIII + Xba I digestion and transferred into pGEM-7Zf (Promega Corp., Nepean, Ontario, Canada). This new TGF-β1/pGEM vector was linearized with Xba I and the antisense riboprobe was transcribed with RNA polymerase SP6 as described for the other riboprobes. Radiolabeled riboprobes were prepared using [32P]UTP (800 Ci/mmol; Amersham Canada Ltd.) according to the Ambion MAXIscript in vitro transcription kit (Ambion Inc., Austin, TX). Briefly, transcription mixtures were constituted of 50 μCi of [32P]UTP, 10 mmol/L dithiothreitol, 0.5 mmol/L of ATP, CTP, and GTP, 1× transcription buffer, 12.5 U of RNase inhibitor, 1 μg of the appropriate linearized plasmid, and T7 or SP6 RNA polymerase in a total volume of 20 μl. The reactions were performed for 90 to 120 minutes at 37°C. One μl of RNase-free DNase1 was then added for 15 minutes at 37°C to remove the DNA template, and the riboprobes were purified over Sephadex G-50 (Pharmacia Fine Chemicals, Uppsala, Sweden) spin columns. As a control of RNA loading and integrity, blots were hybridized with a 1.0-kb Pst I cDNA probe of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; American Type Culture Collection, Rockville, MD) or a 3.45 kb cDNA probe of the mouse ribosomal 18S (ATCC). The GAPDH and 18S probe were labeled with a multiprime DNA-labeling system (Amersham) by using [32P]dCTP (specific activity >3,000 Ci/mmol; Amersham). Total RNA was extracted from cells according to the previously described Tri-Reagent protocol. Aliquots of 5 to 10 μg of total RNA were run on a horizontal gel apparatus in 1% agarose gel containing 1× MOPS and 6% formaldehyde submerged in 1× MOPS buffer (pH 7.0). The samples were transferred onto a nylon membrane Hybond N+ (Amersham) by overnight capillary action with 10× standard saline citrate (SSC). After blotting, the RNA was fixed with uv light, baked for 2 hours at 60°C, and the membranes were stained in 0.02% methylene blue in 0.3 mol/L sodium acetate (pH 5.5). The membranes were then prehybridized for 2 hours at 68°C with 1× hybridization buffer containing 120 mmol/L Tris (pH 7.4), 600 mmol/L NaCl, 8 mmol/L ethylenediaminetetraacetic acid (pH 8.0), 0.1% Na4PP, 0.2% SDS, 625 μg/ml heparin, and 10% dextran sulfate. Hybridization began with the addition of the [32P]UTP-labeled cRNA probe and performed overnight in one part 2× hybridization buffer and one part formamide. The membranes were sequentially washed in 2× SSC/1% SDS at room temperature, 2× SSC/1% SDS at 68°C, 0.1× SSC/0.2% SDS at 68°C, and 0.1× SSC/0.1% SDS at 68°C. For the cDNA GAPDH probe, prehybridization and hybridization were performed in the same prehybridization buffer used for the cRNA riboprobe. The membranes were prehybridized for 4 hours at 68°C and hybridization was performed overnight. The membranes were then washed once at room temperature for 20 minutes in 2× SSC, once with 0.1× SSC/0.5% SDS at 68°C for 60 minutes, and were rinsed off at room temperature in 0.1× SSC. The membranes were then exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) with intensifying screens at −80°C for times ranging from 2 hours to 3 days. Signal intensity was quantitated by densitometry with a Pharmacia LKB Ultrascan XL (Pharmacia Biotech, Uppsala, Sweden). Densitometric values are expressed as the ratio of convertase/GAPDH densitometric quantification with control values set at 1. Bioactive TGF-β was monitored using a growth inhibition assay with Mv1Lu mink lung epithelial cells (CCL-64; ATCC) essentially as originally described by Tucker et al.44Tucker RF Branum EL Shipley GD Ryan RJ Moses HL Specific binding to cultured cells of 125I-labeled type beta transforming growth factor from human platelets.Proc Natl Acad Sci USA. 1984; 81: 6757-6761Crossref PubMed Scopus (172) Google Scholar In brief, Mv1Lu cells were plated in 96-well flat-bottom plates at 2,500 cells/well. After 48 hours, the medium was removed and serial dilutions of the samples to assay for TGF-β activity were added. After 72 hours incubation, the cells were pulsed with [3H]thymidine for 24 hours. Cells were collected and radioactivity counted in a liquid scintillation counter. One unit of activity was defined as the amount of TGF-β required to give 50% maximal response in the assay. In selected experiments, samples were assayed for active TGF-β using a commercially available enzyme-linked immunosorbent assay kit specific for mature and bioactive TGF-β1 (R&D Systems). The limit of detection for this assay is 30 pg/ml TGF-β1. To delineate which enzyme(s) in the PC family of proteases expresses TGF-β1 convertase activity, we used vaccinia virus as an expression vector to produce both the substrate (proTGF-β1) and the enzymes in recombinant forms. In a first set of experiments, we infected LoVo cells, a furin-deficient cell line,45Takahashi S Nakagawa T Kasai K Banno T Duguay SJ Van de Ven WJ Murakami K Nakayama K A second mutant allele of furin in the processing-incompetent cell line, LoVo. Evidence for involvement of the homo B domain in autocatalytic activation.J Biol Chem. 1995; 270: 26565-26569Crossref PubMed Scopus (94) Google Scholar with both vaccinia recombinants for TGF-β1 precursor and each of the PCs. After co-infection, concentrated supernatants were analyzed for TGF-β processing by immunoblotting. As illustrated in Figure 1A (left), LoVo c
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