Sorting of Furin in Polarized Epithelial and Endothelial Cells : Expression Beyond the Golgi Apparatus
Département de Pathologie et Biologie Cellulaire (GM,MB) et Département de Biochimie (GB), Université de Montréal, Montréal, Québec, Canada
Correspondence to: Dr. Moïse Bendayan, Pathologie et Biologie Cellulaire, Université de Montréal, CP 6128 Succ Centre Ville, Montréal, Québec H3C 3J7, Canada. E-mail: moise.bendayan{at}umontreal.ca
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Summary |
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(J Histochem Cytochem 52:567579, 2004)
Key Words: furin cell surface endosomes electron microscopy
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Introduction |
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Furin (EC 3.4.21.85), the best-characterized member of the mammalian subtilisin/kexin-like convertases, is closely involved in the activation and function of several important proproteins in virtually all mammalian cells and tissues (Nakayama 1997). The type I transmembrane enzyme hydrolyzes the carboxy-terminal peptide bond of basic motifs, such as RXK/R
or RXXR
, contained in a number of substrates, including hormones, growth factors, cell surface receptors, extracellular matrix (ECM) proteins, matrix metalloproteinases (MMPs), and plasma proteins. Because of the types of protein it cleaves, furin has a broad role and is involved in vital mechanisms such as embryogenesis and general homeostasis (Molloy et al. 1999
). Furin may also participate in the proteolytic release of luminal or extracellular domains of transmembrane proteins in a process known as "shedding." This posttranslational modification modulates the biological activity of many proteins, such as receptors, growth factors, and enzymes. Even if proofs for such a role are still sparse and await further investigations, furin appears to be involved in the shedding of many proteins, including itself (Milhiet et al. 1995
; Chicheportiche et al. 1997
; Schneider et al. 1999
; Ghaddar et al. 2000
; Snellman et al. 2000
; Tasanen et al. 2000
; LopezFraga et al. 2001
; Wang and Pei 2001
; Williams and Wassarman 2001
; Balyasnikova et al. 2002
; Denault et al. 2002
; Banyard et al. 2003
).
Many studies using mutagenesis and overexpression in cells maintained in culture have shown that the localization of furin is tightly controlled by multiple sorting signals contained in the 56-amino-acid sequence of its cytoplasmic domain. The sorting signals include an acidic cluster (EECPSDSEEDE779) containing two serines that can be phosphorylated by the casein kinase 2 (CK2), a tyrosine motif (YKGL765), and two hydrophobic motifs (LI760, FI791). In addition, the cytoplasmic domain houses a sequence (VY753) that allows the interaction of furin with the actin-binding protein-280 (filamin; ABP-280) and constitutes a cell surface-tethering signal. The phosphorylation state of furin and the complex interactions among the different sorting signals with the cellular sorting machinery [mainly the adaptor proteins AP-1, AP-2, AP-4, the phosphofurin acidic cluster sorting protein-1 (PACS-1), and the ABP-280] are responsible for its presence in subcompartments of the late constitutive secretory pathway (Molloy et al. 1999; Simmen et al. 2002
).
Despite the major roles of furin in many physiological processes and the knowledge of its sorting signals, the exact subcellular sites of substrate processing and activation remain elusive. The TGN is the putative site at which furin cleaves a number of precursor proteins (Molloy and Thomas 2002). However, for many other proprotein substrates the site of processing is not clearly defined and may be positioned outside the Golgi apparatus, such as in endosomal subcompartments and/or at the cell surface (Sariola et al. 1995
; Band et al. 2001
; Chen et al. 2001a
; Thomas 2002
; Mayer et al. 2003
). The importance of this information is illustrated by the fact that furin is involved in severe diseases. Furin is upregulated in several types of cancer and acts upstream from the processes of cell transformation, acquisition of the tumorigenic phenotype, and metastasis formation (Bassi et al. 2001a
,b
). Mutation of the furin cleavage site present in profactor IX and in the insulin proreceptor results in a form of hemophilia B and insulin resistance, respectively (Nakayama 1997
). Furin is also closely involved in diseases such as rheumatoid arthritis and Alzheimer's. Finally, furin is required for the activation of several bacterial toxins and viruses such as those responsible for AIDS, anthrax, and Ebola fever (Thomas 2002
). Accordingly, there is a considerable stake in establishing the precise subcellular locations of furin in tissues and cells in situ.
In this study, furin antigenic sites were tracked in subcellular compartments of various tissues and corresponding cell lines by the use of specific anti-furin antibodies combined with high-resolution immunogold electron microscopy and Western blotting. In addition, plasmids containing the furin cDNA were transduced in animals and cultured cell lines and the expression, trafficking, and localization of furin were analyzed in these conditions of overexpression. In polarized epithelial cells, such as those lining the intestine and renal tubules, furin was found in the Golgi apparatus and, in appreciable amounts, in endosomes and at the apical and basolateral cell surfaces. The distribution of furin sites over the different types of blood capillary beds was also pinpointed. Strikingly, furin was localized over the luminal and abluminal cell surfaces of the continuous, fenestrated, and discontinuous endothelia. Even more remarkable was the labeling of furin around endothelial fenestrations and their diaphragms. These results were corroborated by those obtained with the furinHAcDNA-injected mouse tissues and with furin-transfected cell lines. The overexpressed enzyme has been found to travel to the Golgi apparatus and, further, to the plasma membrane and endosomes in transduced renal tubule epithelial cells, transfected MDCK cells, and transfected rat brain endothelial cells (RBE4 cell line). Our results show for the first time the widespread distribution of furin at the cell surface and endosomes of polarized epithelial and endothelial cells. Suggested roles such as physiological proprotein conversion and shedding of proteins at the cell surface are supported by our data.
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Materials and Methods |
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Cell Culture
All cell culture reagents were purchased from Gibco-BRL (Burlington, Canada). Cells were cultured as described previously (Azarani et al. 1998; Levy et al. 1999
; Bendayan et al. 2002
). Briefly, MadinDarby canine kidney (MDCK) cells were maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 0.25 µg/ml fungizone. The intestinal cell line Caco-2 was maintained in minimal essential medium supplemented with 1% non-essential amino acids, 10% fetal bovine serum, and 1% penicillin/streptomycin. The immortalized rat brain microvessel endothelial cells (RBE4) were grown on culture flasks precoated with type I rat tail collagen in a mixture of minimal essential medium:Ham's F10 (1:1) supplemented with 1 ng/ml basic fibroblast growth factor, 10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were maintained at 37C in a humidified atmosphere of 95% air and 5% CO2.
Furin cDNA-containing Plasmids
Plasmids pCDNA3/RSV/Furin and pCDNA3/RSV/Furin-hemagglutinin (HA) were generated by our group as described previously (Mayer et al. 2003). They contain the complete coding region of the furin cDNA, fused or not with the HA-tag reporter sequence, inserted into the mammalian expression vector pCDNA3/RSV. This vector also contains the aminoglycoside phosphotransferase 3' gene conferring resistance to the antibiotic Geneticin (G418) to cells that express it.
In Vivo Gene Delivery
Plasmid pCDNA3/RSV/Furin-HA was introduced and expressed in mouse tissues using the TransIT in vivo gene delivery system according to the manufacturer's instructions as previously described (Mayer et al. 2003; Mirus, Madison, WI). Briefly, the furin-HA vector was complexed with a cationic polymer generating stable non-aggregating DNA particles of less than 100 nm in diameter. The cationic furin-HA-encoding plasmid was then added to a 2-ml delivery solution and the entire volume was injected through a 27-gauge needle into the tail vein of an immunodeficient SCID mouse over 6 sec. The efficient expression of a transgene by this method is dependent on raising the intravascular hydrostatic pressure, which causes an extravasation of fluid that carries the plasmid DNA in contact with cells (Zhang et al. 1999
). The furin-HA vector was injected into seven mice. In control experiments, mice were injected with the plasmid pCDNA3/RSV without furin-HA cDNA. Twenty-four hours after gene delivery, animals were anesthetized and kidneys were sampled and processed for immunofluorescence as described below.
Cell Transfection
Cells were transfected as described previously (Azarani et al. 1998). Fifty to 60% confluent cells were transfected with 10 µg of plasmid pCDNA3/RSV/Furin by the calcium phosphateDNA co-precipitation method (Chen and Okayama 1987
). Stable transfected cells were selected by their ability to grow in the presence of G418 added to the culture medium. Surviving colonies were screened by Western blotting and immunofluorescence as described below. Control experiments included transfection with plasmid pCDNA3/RSV without the furin cDNA coding sequence.
Antibodies
In this study, four anti-furin antibodies were used. The rabbit polyclonal and the mouse monoclonal antibodies recognizing the N-terminal part of furin were from Alexis Biochemicals (San Diego, CA). The goat and rabbit antibodies recognizing the C-terminal part of furin were from Santa Cruz Biotechnology (Santa Cruz, CA) and Affinity Bioreagents (Golden, CO), respectively. The rabbit anti-HA tag and the FITC-conjugated anti-rabbit IgG antibodies were from Sigma (Oakville, Canada).
Immunofluorescence
Furin-transfected cells grown on microscope slides were washed in PBS (10 mM phosphate buffer, 150 mM NaCl, pH 7.2), fixed with 100% methanol for 5 min at 20C or, for cell surface labeling, fixed with 4% paraformaldehyde for 15 min at room temperature and washed again with PBS before proceeding to immunodetection as described below. Samples of furin-HA-transduced renal tissue were frozen in liquid nitrogen immediately after removal, embedded in cold Tissue-Tek OCT compound (VWR; Montreal, Canada), and frozen at 20C. Five-µm-thick cryosections were mounted on glass slides, fixed in acetone:ethanol (1:1) at 20C for 5 min, and rinsed with PBS.
Fixed cells were incubated with the rabbit anti-furin N-terminal antibody diluted 1:500 overnight at 4C. The fixed renal tissue cryosections were incubated with the rabbit anti-HA tag antibody diluted 1:50 overnight at 4C. The samples were then washed with PBS several times and incubated with the FITC-conjugated goat anti-rabbit IgG diluted 1:100 for 1 hr. The slides were then counterstained with 0.01% Evan's blue for 5 min and mounted in PBS containing 50% glycerol and 5% triethylenediamine (DABCO; Sigma). Cells and tissue sections were observed with a Leitz Orthoplan fluorescence microscope equipped with a Leitz Orthomat E camera (Leitz Microsystems; Montreal, Canada). Photographs were recorded on Kodak T-Max 400 film.
Immunogold Electron Microscopy
Confluent cells grown in 100-mm culture dishes were washed with 0.1 mol/liter cacodylate buffer (pH 7,2), fixed in 1% glutaraldehyde, washed again with the cacodylate buffer, scraped, and harvested in several 0.5- ml tubes. Small samples of various rat tissues were fixed by immersion in 1% glutaraldehyde for 2 hr at RT and then washed in 0.1 mol/liter phosphate buffer (pH 7.2). Fixed cells and tissues were then embedded in Lowicryl K4M at 20C (Canemco; Montreal, Canada) according to standard techniques (Bendayan 1995). One part of the glutaraldehyde-fixed MDCK cells was also postfixed with 1% osmium tetroxide for 1 hr and then embedded in Epon (Canemco) as described previously (Bendayan 1995
). Thin sections were mounted on Parlodion- and carbon-coated nickel grids. Sections of osmium-fixed MDCK cells were treated with sodium metaperiodate to unmask antigenic sites before the immunolabeling (Mayer and Bendayan 2001
).
For immunogold labeling, all grids were first floated on a drop of 0.15 mol/liter glycine in PBS, followed by 1% ovalbumin in PBS for 5 min each, and transferred to one of the four specific primary antibodies against furin overnight at 4C. After washing, the grids were incubated on a drop of the 1% ovalbumin for 5 min and transferred to a drop of protein A/10-nm gold (Bendayan 1995) or anti-mouse IgG/10-nm gold or anti-goat IgG/10-nm gold complex (Sigma) for 30 min. After several washes with PBS and distilled water, the sections were contrasted with uranyl acetate and observed with a Philips 410 electron microscope. Control experiments omitting the primary antibody, incubation with normal sera, and adsorption with corresponding antigens were performed systematically for each labeling protocol (Mayer et al. 2003
).
Cell and Tissue Lysis
To reveal furin biochemically, tissues of normal SpragueDawley rats were sampled, immersed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P40, 0.25% sodium deoxycholate, and a cocktail of protease inhibitors), cut into small pieces, and homogenized using a Tenbroeck tissue grinder with 15 strokes of a ground-glass pestle. Cells grown on a 100-mm plastic Petri dish were washed with PBS, scraped, collected in 15-ml conical tubes, and centrifuged for 10 min at 200 x g before addition of the lysis buffer. After incubation on ice for 1 hr, the homogenates were centrifuged for 20 min at 14,000 x g at 4C to remove insoluble material and the resulting supernatants were used for biochemical analysis. Protein concentrations of cell and tissue lysates were determined using the bicinchoninic acid protein assay kit (Pierce; Rockford, IL).
Immunoprecipitation
A portion of the lysates (2 mg) was cleared by incubation with 10 µl normal rabbit serum and 20 µl of a 50% protein A-Sepharose slurry (Sigma) for 1 hr at 4C. After centrifugation to remove the Sepharose beads, the cleared lysates were incubated with 5 µl of the rabbit anti-furin N-terminal antibody overnight at 4C under gentle agitation. Preimmune immunoglobulins were used for control conditions. Next, the antigen/antibody complexes were recovered by the addition of 20 µl of the 50% protein A-Sepharose slurry and incubation at 4C for 4 hr with end-over-end rotation. The immunoprecipitated proteins bound on Sepharose beads were collected by centrifugation, washed six times with ice-cold lysis buffer, and then resuspended in 40 µl of 1x electrophoresis sample buffer (Laemmli 1970) containing 5% ß-mercaptoethanol.
Western Blotting
Immunoprecipitated proteins were heated for 5 min at 95C, chilled, and separated by electrophoresis in slab gels of polyacrylamide (7.5% w/v) containing 1% sodium dodecyl sulfate (SDS-PAGE) using the Laemmli discontinuous buffer system and the Bio-Rad mini-protein III apparatus (Bio-Rad; Mississauga, Canada). After SDS-PAGE, proteins were electrophoretically transferred to 0.2-µm nitrocellulose membranes using the Bio-Rad mini Trans-Blot cell system. The membranes were blocked with 1% skimmed milk in 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl and 0.05% Tween-20, and incubated with the rabbit anti-furin N-terminal antibody diluted 1:1000 overnight at 4C. Bound antibodies were then revealed with the Lumi-Light Plus chemiluminescence detection kit (Roche Diagnostics; Montreal, Canada). The results were recorded by exposure of the membranes to Kodak X-Omat-AR film.
Enzymatic Deglycosylation
For glycosidase digestion experiments, the material resulting from the immunoprecipitation of furin-transfected MDCK cells was denatured and dissociated from the protein A-Sepharose by an incubation at 100C for 10 min in 0.5% SDS plus 1% mercaptoethanol. After centrifugation to remove the protein A-Sepharose, the samples were made up to a final concentration of either 50 mM sodium phosphate, 1% NP-40, pH 7.5, or 50 mM sodium citrate, pH 5.5, and incubated at 37C for 16 hr with 1000 U of peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H) (New England Biolabs; Mississauga, Canada), respectively. The reactions were stopped by the addition of 5x reducing SDS sample buffer and the resulting products were analyzed by SDS-PAGE and Western blotting as described above.
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Results |
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To further demonstrate the relevance of furin in endothelial cells, RBE4 cells, a rat brain endothelial cell line (Bendayan et al. 2002), were labeled for furin. Furin antigenic sites, revealed by immunogold, were located over membranes of the Golgi apparatus, of endosomes, and at the cell surfaces (not shown). Because of the relatively low signal obtained for endogenous furin, RBE4 cells were transfected with the plasmid pCDNA3/RSV/furin and labeled with anti-furin and FITC-conjugated secondary antibodies. Intracellular punctate and cell surface labelings were observed in addition to the strong perinuclear signal (Figure 2)
. The detection of furin in RBE4 cells by western Blotting (Figure 3A , Lane 2) demonstrates the expression of furin by endothelial cells in vitro.
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In light of these results, we decided to overexpress the enzyme in animals in vivo and to compare its localization in various organs with that of the normally expressed endogenous furin. For this purpose we used a gene delivery system that, after a very rapid (6-sec) tail vein injection of a high volume (2 ml) of a DNA/polymer complex solution in mouse, leads to transgene expression in multiple organs (Mayer et al. 2004). We have analyzed furin expression in the kidney of mice 24 hr after the injection of the plasmid pCDNA3/RSV/Furin-HA. The furin-HA encoding vector was used to discriminate and to reveal solely the overexpressed furin. The expression of the HA-tagged furin was clearly revealed by immunofluorescence, particularly in proximal tubules (Figure 6F). However, only a few cells expressed the protein, a result expected in tissues of tail vein plasmid DNA-injected mice (Budker et al. 1996
). When expressed, perinuclear, vesicular, and cell surface labeling was observed (Figure 6F). Both apical and basolateral membranes of proximal tubules were intensely labeled.
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Discussion |
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In this study we revealed subcellular sites of furin over several tissues and cell lines and compared them to those obtained by overexpression of the enzyme in the corresponding tissues and cells. At the EM level, we found that furin is positioned over the plasma membrane of endothelial cells of the continuous, fenestrated, and discontinuous capillaries. On overexpression of furin in RBE4 cells, an endothelial cell line, the enzyme was strongly expressed in the Golgi apparatus and, as found in vivo, in endosome-like structures and at the cell surface. The significance of the presence of the proprotein convertase furin over the endothelial cell surface could be multiple, a major feature being a role in endothelial permeability. Furin-processed proteins, such as cadherins, vascular endothelial growth factor, factor IX and von Willebrand factor, are known to be involved in vascular permeability (Wise et al. 1990; Wasley et al. 1993
; Gulino et al. 1998
; Eriksson et al. 2003
). The presence of furin over specialized areas of fenestrated endothelia, such as around the fenestrations and their diaphragms, provides strong support for a potential role in vascular permeability. Recent work also indicates that furin may participate in the shedding of the angiotensin-converting enzyme, thereby suggesting its implication in many vascular events (Gilbert et al. 2000
; Balyasnikova et al. 2002
). One can therefore envision that some of the above-mentioned proproteins are susceptible to processing by furin at the cell surface or in plasmalemmal vesicles of endothelial cells in vivo. Moreover, it cannot be excluded that some furin substrates are secreted as proproteins and activated at the cell surface in a paracrine fashion.
The localization of furin over the basal domain of endothelial cells also denotes a possible role in relation to the capillary basement membranes. Furin is involved in the cleavage of many ECM-related proteins, such as collagens and MMPs, as well as integrins and transforming growth factor-ß (Dubois et al. 1995; Snellman et al. 2000
; Tasanen et al. 2000
; Sternlicht and Werb 2001
). In a previous study, we have shown that furin is present as complexes with integrins and MMPs over the basal cell surface of specialized epithelial and endothelial cells in the renal glomerulus (Mayer et al. 2003
). Therefore, these results on the widespread distribution of furin over endothelial surfaces suggest important roles for furin in vascular permeability and in maintenance and turnover of basement membranes.
In polarized epithelial cells of the duodenum and kidney and in the corresponding Caco-2 and MDCK cells, respectively, furin was also found beyond the TGN. In addition to the Golgi, furin was located in endosomes and over apical and basolateral plasma membranes. These cells synthesize a great number of proproteins containing furin cleavage sites and, as in the case of endothelial cells, the proprotein-processing compartments are likely to be multiple. Maturation and activation of growth factors, hormones, and their receptors are important steps for cell fate. Survival, growth, differentiation, and cell death are in many cases controlled by the furin-catalyzed processing of the specific mediator (Thomas 2002). In addition to the cleavage of proproteins at sites delimiting the prodomain and the mature polypeptide, furin might be implicated in the proteolytic cleavage of the juxtamembrane stalk region of several transmembrane proteins. Besides the angiotensin-converting enzyme mentioned above, furin appears to be involved in the shedding of several other proteins (Milhiet et al. 1995
; Chicheportiche et al. 1997
; Schneider et al. 1999
; Ghaddar et al. 2000
; Snellman et al. 2000
; Tasanen et al. 2000
; LopezFraga et al. 2001
; Wang and Pei 2001
; Williams and Wassarman 2001
; Banyard et al. 2003
). Furin also acts upstream from the shedding process of other transmembrane proteins by participating in the maturation of bona fide secretases (Bennett et al. 2000
; Anders et al. 2001
; Peiretti et al. 2003
). Furthermore, aberrant furin cleavages leading to the release of protein ectodomains have been shown to occur in pathologies of familial amyloidosis of the Finnish type, anhydrotic ectodermal dysplasia, and Alzheimer's (Chen et al. 2001a
,b
; Elomaa et al. 2001
; Hashimoto et al. 2002
). Therefore, although furin is still widely considered as a standard "housekeeping" enzyme acting in the TGN, it is quite obvious that it is also involved in the mechanism of release of many transmembrane protein ectodomains.
Studies based on antibody uptake, toxin cleavage, and mutagenesis have led to a model for the complex trafficking itinerary of furin. It involves two local cycling loops, one between the TGN and endosomes and the other between the cell surface and early endosomes (Molloy et al. 1999). The phosphorylation state of the furin cytoplasmic domain is responsible for its transport between the loops. Our results introduce evidence that, under normal steady-state conditions, furin is present simultaneously in the exocytic and endocytic pathways of polarized cells. Hence, different pools of furin maybe caught in both cycling loops at the same time instead of being solely concentrated in the TGN/endosomes loop. In addition, using antibody uptake experiments and mutagenesis, it was shown that furin en route to the cell surface is targeted to the basolateral surface of polarized cells by interaction of a bipartite signal in its cytoplasmic domain with the sorting adaptor protein AP-4 (Simmen et al. 1999
,2002
). Our study shows that furin is not strictly confined to the basolateral domain of polarized cells but is also present at the apical cell surfaces.
Future developments of therapeutic agents are highly dependent on the establishment of precise subcellular localizations of proprotein maturation and transmembrane protein shedding sites. The widespread distribution found for furin at the cell surface and endosomes suggests its involvement in endothelial permeability, basement membrane turnover, and post-Golgi proprotein conversion, and also supports a role in the shedding of transmembrane proteins from the cell surface.
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Acknowledgments |
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We wish to thank Diane Gingras, Elizabeth Gervais, and Nadia Bonvouloir for their precious help with cell and molecular biology techniques.
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Footnotes |
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