1 Department of Pathology and Cell Biology, Université de
Montréal, Montreal, Quebec, H3C 3J7, Canada
2 Department of Biochemistry, Université de Montréal, Montreal,
Quebec, H3C 3J7, Canada
* Author for correspondence (e-mail: moise.bendayan{at}umontreal.ca)
Accepted 27 January 2003
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Summary |
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Key words: Furin, ProMT1-MMP, Integrin, Kidney glomerulus, Cell surface
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Introduction |
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Furin is a calcium-dependent serine protease of the subtilisin-like
proprotein convertase family. This type I transmembrane glycoprotein is
involved in pro-domain cleavage of many bioactive proteins traveling along the
constitutive secretory route. It cleaves proteins at the C-terminal side of
multibasic amino acid motifs such as R-X-K/R-R and R-X-X-R
(Molloy et al., 1999).
Knockout of furin gene is embryonic lethal (Roebroeck et al., 1998). This
underscores the importance of this protease in the maturation, function and
activation of several hormones, growth factors and cell surface receptors
(Molloy et al., 1999
). Furin
is also efficient at processing several adhesion-related proteins. Furin
processes and activates proproteins involved in cell-ECM and cell-cell
interactions such as pro-
-integrin subunits and pro-cadherins
(Lehmann et al., 1996
;
Posthaus et al., 1998
;
Lissitzky et al., 2000
). In
addition, furin cleaves protein substrates such as pro-membrane-type-1 matrix
metalloproteinases (proMT1-MMP) and pro-transforming-growth-factor ß,
which play major roles in the regulation of the ECM and basement membranes
(Dubois et al., 1995
;
Pei and Weiss, 1996
).
Incidentally, these adhesion and ECM-related proteins are important for the
structure and function of the glomerulus. Thus, by activating these proteins,
furin could play pivotal roles in pathophysiological processes. Although
significant information has been gathered using cell culture, little is known
about the trafficking and function of furin in situ. Moreover, the
physiological function of furin at the cell surface remains unknown.
The plasma-membrane-anchored matrix metalloproteinase (MMP) MT1-MMP is
involved in many important tissue-remodeling events because of its ability to
degrade ECM proteins either directly or indirectly by activating downstream
soluble MMPs such as the proMMP2
(Sternlicht and Werb, 2001).
MMP2, which is essential for glomerulogenesis
(Serluca et al., 2002
),
degrades the major constituent of basement membranes, type-IV collagen, and is
involved in various glomerular diseases
(Lenz et al., 2000
). In
addition, MT1-MMP and MMP2 can degrade several other components of the GBM
(Sternlicht and Werb, 2001
).
MT1-MMP is synthesized as a proenzyme with two potential furin cleavage sites
at the end of the propeptide domain. Yana and Weiss
(Yana and Weiss, 2000
) have
recently demonstrated that the removal of the pro-domain is a prerequisite for
MT1-MMP to acquire catalytic activity and that furin is probably its major
processing enzyme. Although it has been demonstrated that full-length
proMT1-MMP is the prominent form appearing at the plasma membrane of many cell
types, the subcellular site of activation and the identity of the activating
enzyme remain controversial (Sato et al.,
1994
; Okumura et al.,
1997
; Cao et al.,
1998
; Lehti et al.,
1998
; Sternlicht and Werb,
2001
). Nonetheless, a proprotein-convertase/MT1-MMP/MMP2 axis has
been proposed to conduct ECM remodeling
(Yana and Weiss, 2000
;
de Kleijn et al., 2001
).
In the present study, we report several novel findings that could shed some
light on a furin/MT1-MMP activation cascade operating at the renal glomeruli.
By using in vivo gene delivery, western blotting and immunogold electron
microscopy (EM), we provide evidence of significant pools of furin and
proMT1-MMP in glomeruli, concentrated along the basal surface of epithelial
and endothelial cells, lining the GBM. Furin was mainly found at the slit
diaphragm of podocytes and close to endothelial fenestrations, whereas
proMT1-MMP is concentrated at the slit diaphragm. Coimmunoprecipitation
experiments on isolated membrane fractions of glomerular cells indicate that
both enzymes are interacting and double immunogold labelings revealed that
furin and proMT1-MMP are colocalized on plasma membranes, particularly at the
points of insertion of the slit diaphragms. Moreover, we show that cell
surface furin interacts with the V integrin subunit, which participates
in the activation of proMMP2 (Deryugina et
al., 2001
). These results suggest that the interaction of furin
with these proteins could trigger GBM degradation either directly, by the
activation of cell surface proMT1-MMP, or indirectly, by promoting the
activation of proMMP2. Moreover, the very peculiar localization pattern of
furin and proMT1-MMP suggests that a mechanism of focalized pericellular
proteolysis is involved in GBM turnover and must play an important role in the
maintenance of the specialized structures of the glomerular wall.
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Materials and Methods |
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DNA manipulations and plasmid constructions
From the plasmid pSVLfur, the complete coding sequence of furin was
isolated, modified and amplified by PCR using chemically synthesized
oligonucleotides specific for furin (Life Technologies). Oligonucleotide
primers were designed to introduce restriction sites in the furin sequence to
subclone only the coding sequence (nucleotides 217-2601) of the gene into the
mammalian expression vector pCDNA3/RSV
(Jockers et al., 1996). The
forward primer
5'-AGCCACCTGTCCCCCAAGCTTACCATGGAGCTGAG-3'
allowed the introduction of a HindIII restriction site (italics)
upstream of the start codon (in bold). An adenine (underlined) was added to
complete an optimal sequence (ACCATGG) for initiation of translation by
eukaryotic ribosomes (Kozak,
1986
). The reverse primer
5'-GGTGGGCAGTGGGCTCATCTAGATCATGGCGCGCCGAGGGCGCTCTGGTCTTT-3'
allowed the introduction of an XbaI site (italics) immediately
downstream of the furin translational stop codon (in bold). An AscI
restriction site (underlined) was introduced upstream the stop codon to allow
the insertion of an HA-tag coding sequence. A thymine was also added
immediately after the AscI site to keep the good reading frame.
Following the PCR, the amplified 2.4 kb furin cDNA fragment was digested
with HindIII and XbaI restriction enzymes, and ligated into
the multiple cloning site of the vector pCDNA3/RSV resulting in
pCDNA3/RSV/Furin. The pCDNA3/RSV/Furin-HA vector was constructed by ligating
annealed oligonucleotides coding for the HA-tag peptide
(Lesage et al., 2000) into
AscI-digested pCDNA3/RSV/Furin vector. The resulting construct
encoded two copies of the HA epitope in the C-terminus tail of the furin
protein.
In vivo gene delivery
Furin and furin-HA-encoding vectors were introduced into mouse tissues
using the TransIT in vivo gene delivery system according to the manufacturer's
instructions (Mirus). Briefly, 15 µl of 1 mg ml-1
pCDNA3/RSV/Furin or pCDNA3/RSV/Furin-HA were mixed with 15 µl of a cationic
polymer solution and 170 µl sterile endotoxin-free water. The resulting
cationic DNA particles of less than 100 nm were added to a 2 ml delivery
solution and the entire volume was then injected through a 27-gauge needle
into the tail vein of an 18 g immunodeficient SCID mouse, in 6 seconds. For
each of the furin and furin-HA vectors, seven mice were injected. In control
experiments, mice were injected with the plasmid pCDNA3/RSV without furin
cDNA. 24 hours after gene delivery, animals were anesthetized and tissues were
sampled and processed for biochemical and immunocytochemical assays.
Tissue preparation
Kidneys from control 150 g male Sprague Dawley rats and from control and
plasmid-injected SCID mice were removed, divided into three parts and
processed as follows. One part was frozen in liquid nitrogen and embedded in
Tissue-Tek OCT compound. 5-µm-thick cryosections were mounted on glass
slides, fixed in acetone:ethanol (1:1) at -20°C and washed in PBS at room
temperature. A second part of the tissue was cut into 1 mm3 pieces,
fixed by immersion in 4% paraformaldehyde-lysine-periodate, embedded at low
temperature in Lowicryl K4M and cut into semi-thin (0.5 µm) and ultrathin
(100 nm) sections (Bendayan,
1995). Semi-thin sections were mounted on glass slides and the
ultrathin ones on Parlodion-carbon-coated nickel grids. The last part was
frozen in liquid nitrogen and kept at -80°C for biochemical assays.
Immunofluorescence
Fixed cryosections were incubated with the rabbit anti-furin-N-terminus
antibody or the rabbit anti-HA antibody overnight at 4°C followed by the
FITC-conjugated goat anti-rabbit IgG for 1 hour. Sections were examined with a
Leitz Orthoplan DM RB fluorescent microscope. Control experiments, omitting
the primary antibody, incubation with normal sera and adsorption with
corresponding antigens were performed for each labeling protocol.
Immunogold labeling experiments
The labeling procedure was carried out as previously described
(Bendayan, 1995). Ultrathin
sections were incubated overnight at 4°C with the appropriate primary
antibody followed by protein A-gold (5 nm or 10 nm; OD525=0.5) for
30 minutes. The tissue sections were observed with a Philips 410SL electron
microscope.
Double labeling experiments were conducted on both sides of ultrathin
sections mounted on uncoated nickel grids
(Bendayan, 1995). The labelings
were carried out essentially as described above using the anti-proMT1-MMP or
the anti-integrin
V antibodies on one side of the grids and the rabbit
anti-furin-N-terminus antibody on the other side of the grids. ProMT1-MMP and
integrin antigenic sites were detected with protein-A/5-nm-gold complex, and
furin with protein-A/10-nm-gold complex. Quantitative evaluations of the
double labelings were conducted as previously described
(Bendayan, 1995
).
Glomerulus isolation, fractionation and lysis
Glomeruli were isolated from freshly dissected rat renal cortex by
sequential sieving as previously described
(Regoli and Bendayan, 1999).
Intact isolated glomeruli were suspended in modified RIPA buffer (50 mM PBS,
pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, 200 µg
ml-1 PMSF and 1x protease inhibitor cocktail), homogenized
and centrifuged for 20 minutes at 14,000 g. The supernatants
were used for biochemical analysis.
Fractionation of isolated glomeruli was conducted in accordance to the
modified protocol of Kerjaschki et al.
(Kerjaschki et al., 1989).
Glomeruli were suspended in 200 mM Na2CO3, pH 11,
containing a protease inhibitor cocktail, homogenized and submitted to
sonication for 5 minutes in a Bransonic ultrasonic bath. The homogenate was
then separated into an insoluble pellet, containing mainly GBM and cell
debris, and a supernatant by centrifugation for 5 minutes at 2000
g. The supernatant was resolved into a total membrane pellet
and a soluble supernatant by centrifugation for 60 minutes at 100,000
g in a Beckman XL-70 ultracentrifuge using a SW-60Ti rotor.
The total membrane pellet was resuspended in ice-cold modified RIPA buffer,
homogenized and kept at 4°C for 60 minutes with end-over-end rotation. The
homogenate was centrifuged for 20 minutes at 14,000 g and the
resulting supernatant was stored at -80°C until needed for biochemical
assays.
Protein concentration was determined by the bicinchoninic acid method using bovine serum albumin as a standard.
Chemical crosslinking
In order to detect unstable protein-protein interactions, total glomerular
membrane fractions were crosslinked with the thiolcleavable reagent DSP
according to the protocol of Löster et al.
(Löster et al., 1995).
Briefly, the solubilized material (500 µl) was mixed with 12.5 µl of
DSP, from a freshly made 25 mM stock in dimethyl sulfoxide, and allowed to
react for 15 minutes at room temperature. The crosslinker was then quenched by
adding 50 µl of 100 mM Tris-HCl, pH 8.0, and the resulting fractions were
used for coimmunoprecipitation studies as described below.
Digestion of glomerular membranes by soluble recombinant furin
The experimental conditions for the assessment of proMT1-MMP cleavage by
purified soluble furin were adapted from a previous report
(Seger and Shaltiel, 2000).
Briefly, a fraction of solubilized glomerular membranes (50 µg) was
incubated with recombinant furin (5 U) in 25 mM Tris-HCl, pH 7.4, 0.5% Triton
X-100, 1 mM CaCl2 and 1 mM ß-mercaptoethanol for 4 hours at
37°C. Reducing sample buffer was then added and membrane proteins were
separated by SDS-PAGE and analyzed by western blotting.
Immunoprecipitation
Immunoprecipitations were conducted as previously described
(Arias et al., 2000). The
detergent lysates, precleared by incubations with preimmune rabbit serum and
protein-A/Sepharose, were incubated either with 5 µl of the rabbit
anti-proMT1-MMP or 5 µl of the rabbit anti-integrin-
V antibody for
overnight at 4°C under agitation. The immune complexes were recovered by
the addition of protein-A/Sepharose and washed with the modified RIPA buffer.
The immunoprecipitates were then analyzed by SDS-PAGE and immunoblotting.
Western blot analysis
Protein samples were boiled for 4 minutes in reducing SDS-sample buffer,
separated by SDS-PAGE on 7.5% or 10% Ready gels (Bio-Rad) or handmade
polyacrylamide gels, and electrophoretically transferred to nitrocellulose
membranes. Immunodetection of proteins on nitrocellulose membrane was
conducted as follows. Briefly, the membranes were blocked with TBS containing
0.05% Tween 20 and 1% skimmed milk, and incubated overnight at 4°C with
the appropriate antibodies. Bound antibodies were then revealed with the
Lumi-Light Plus chemiluminescence detection kit. The results were recorded by
exposition of the membranes to Kodak X-Omat-AR films.
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Results |
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In order to identify the precise subcellular location of endogenous furin in glomerular cells, immunogold was applied at the EM level. As expected, the Golgi apparatus of glomerular cells, particularly podocytes, was labeled (Fig. 1B). The endoplasmic reticulum was weakly labeled whereas very few gold particles were located over nuclei and mitochondria (Fig. 1B). The novel unexpected result concerned the labeling obtained at the surface of the glomerular cells. Indeed, furin immunolabeling, as revealed by gold particles, was present at both the lumenal and ablumenal plasma membranes of glomerular endothelial cells (Fig. 1C). Strikingly, the gold particles were very frequently associated with the endothelial fenestrations (Fig. 1C,D). Moreover, furin was associated to podocytes, particularly at the slit diaphragms and, to a lesser extent, at apical and basal membrane domains facing the urinary space and the GBM, respectively (Fig. 1C). Control experiments performed by adsorbing the antibody resulted in a major reduction of the labelings (Fig. 1E). The use of either one of the four anti-furin antibodies generated similar results.
The immunodetection of endogenous furin at the cell surface raises concern
about furin being an enzyme confined to the TGN as demonstrated in
overexpressing cell systems. Overexpression might lead to artefactual
distribution by saturating retention or retrieval mechanisms
(Wouters et al., 1998). In
order to compare the localization of the normally expressed endogenous and the
overexpressed furin in vivo, we used a gene delivery system that, upon
tail-vein injection of DNA-polymer complexes in mouse, leads to high levels of
transgene expression in multiple organs. We have analysed furin expression in
the kidney of mice 24 hours after the injection of the plasmid
pCDNA3/RSV/Furin or pCDNA3/RSV/Furin-HA. By immunofluorescence, cells
expressing high levels of furin were found in glomeruli
(Fig. 2A,B). In the
pCDNA3/RSV/Furin-injected mice, some renal cells showed normal staining,
whereas others displayed very intense signals, probably caused by
overexpression of furin (Fig.
2A). Staining of perinuclear regions, vesicular structures and
plasma membranes were similar, although of higher intensity, to those obtained
for endogenous normally expressed furin
(Fig. 1). Obviously, these
signals include those of endogenous as well as overexpressed furin
(Fig. 2A). In order to
discriminate and to reveal solely the overexpressed furin, we screened the
pCDNA3/RSV/Furin-HA injected mice. The expression of the HA-tagged furin in
glomerular cells was clearly revealed by immunofluorescence
(Fig. 2B). Only few cells
expressed furin-HA, a result expected in tissues of tail-vein
plasmid-DNA-injected mice (Budker et al.,
1996
). Perinuclear, vesicular and cell surface labelings similar
to those described above were obtained
(Fig. 2B).
|
The subcellular localization of overexpressed furin was carried out at the EM level. Furin- and furin-HA-transduced cells were identified on semi-thin sections and consecutive ultrathin sections were submitted to the immunogold labeling. For pCDNA3/RSV/Furin-transduced kidneys, furin was located at the cell surface of glomerular epithelial and endothelial cells (Fig. 2C). Moreover, as reported above, the signal was very frequently associated with endothelial fenestrations and podocyte slit diaphragms (Fig. 2C). Furin was also strongly expressed in the Golgi apparatus (not shown). Furin-HA immunolabeling distinguished the staining caused by endogenous furin. Furin-HA was found at the same locations as the untagged furin. A strong signal was located at the Golgi apparatus, particularly in what appears to be the trans side. Although of lower intensity, the labeling was over endothelial fenestrations and over podocyte slit diaphragms (Fig. 2D,E). Furthermore, the apical domain of foot processes and ablumenal membranes in contact with the GBM were also labeled. Nuclei, mitochondria, basement membranes and the urinary space were almost devoid of gold particles.
Cell surface furin colocalizes and associates with proMT1-MMP
The furin proprotein convertase is thus present in substantial amounts at
the surface of cells responsible for glomerular ultrafiltration. These cells
synthesize the proteins forming the glomerular basement membrane as well as
those regulating the state of this basement membrane. Precursor forms of many
regulatory proteins are potential substrates of furin. Among these, MT1-MMP
plays a major role in basement membranes turnover. Because furin could
intervene in the activation of proMT1-MMP at cell surfaces, we revealed the
full-length proMT1-MMP in rat renal glomeruli using an antibody specific to
the pro-domain of the enzyme. At the EM level, proMT1-MMP antigenic sites were
detected at the surface of glomerular mesangial (not shown), epithelial and
endothelial cells (Fig. 3). In
podocytes, the labeling was predominantly associated with the lateral membrane
domain of foot processes at the level of the slit diaphragms
(Fig. 3). In endothelial cells,
the labeling was mostly confined to the ablumenal side
(Fig. 3,
Fig. 4B-D). The gold particles
revealing the metalloproteinase antigenic sites were also present in
intracellular compartments including the rough endoplasmic reticulum, the
Golgi apparatus (Fig. 4A) and
podocytic vacuoles (Fig. 4B).
ProMT1-MMP immunoreactivity was also found over the GBM, which might reflect
the existence of the soluble, shed form of the proenzyme
(Kazes et al., 1998).
Biochemical results after SDS-PAGE of solubilized isolated glomeruli and
western blotting using the proMT1-MMP specific antibody, revealed a single
band displaying a molecular mass of
63 kDa. This is in agreement with the
molecular mass of the protein in its pro-form and also supports the high
specificity of the antibody (Fig.
1F) (Sato et al.,
1994
).
|
|
The detection of proMT1-MMP in the Golgi and at the cell surface in
glomerular cells prompted us to investigate its colocalization with furin.
When double immunogold labelings were performed on thin sections of rat
kidney, 5 nm and 10 nm gold particles (revealing proMT1-MMP and furin,
respectively) were often found within 15-25 nm distance suggesting close
association of the two antigenic sites. This is illustrated for the Golgi
apparatus (Fig. 4A) and
intracellular vacuoles of podocytes (Fig.
4B). Moreover, the furin/proMT1-MMP complexes were also present at
the slit diaphragm domain (Fig.
4B,C) at the ablumenal side of endothelial cells facing the GBM
and in the vicinity of endothelial fenestrations
(Fig. 4D). Not all gold
particles revealing these proteins were associated; a significant number
remained free. Morphometric analysis indicated that 40% of furin
immunogold particles are associated with those for proMT1-MMP at the surface
of glomerular cells facing the GBM.
Coimmunoprecipitation experiments were carried out to confirm the association between these enzymes. To overcome possible unstable association, a total glomerular membrane fraction was prepared and membrane proteins were chemically crosslinked with the thiol-cleavable homo-bifunctional reagent DSP. The crosslinked glomerular membranes were subjected to immunoprecipitation with the specific proMT1-MMP antibody and the immunoprecipitated material was analysed by SDS-PAGE, under reducing conditions, and western blotting with anti-furin antibodies. The experiment revealed a 98 kDa protein that coimmunoprecipitated with proMT1-MMP (Fig. 4E, lane 1). Furin was not, however, coimmunoprecipitated with proMT1-MMP in glomerular membranes not treated with DSP (Fig. 4E, lane 2) indicating that their normal association might be disrupted by the isolation and homogenization procedures or that furin has a low binding affinity towards MT1-MMP in the presence of detergents. Upon stripping the nitrocellulose membrane from the antibodies and reprobing it with the anti-proMT1-MMP antibodies (Fig. 4F), one band at 63 kDa was detected independently of chemical crosslinking (Fig. 4F, lanes 1,2). This supports the specificity of the coimmunoprecipitation experiment. Moreover, no protein was revealed using normal rabbit immunoglobulins (Fig. 4E,F, lane 3).
Additional results showing that furin could interact functionally with the proMT1-MMP in kidney glomeruli were obtained from the glomerular membrane digestion study using recombinant furin. Membranes were incubated with soluble furin and analyzed by western blotting to detect the proMT1-MMP. The signal revealing the proenzyme disappeared in the digested membranes indicating that the pro-domain is cleaved by the exogenous soluble furin (Fig. 5A). After stripping, the nitrocellulose membrane was reprobed with the anti-integrin-ß3 antibody and the resulting positive signal thus confirmed the integrity of the other membrane proteins (Fig. 5B).
|
Colocalization and interaction of furin with the V integrin
subunit
Furin has been involved in the endoproteolytic cleavage of the V
integrin subunit precursor (Lehmann et
al., 1996
; Lissitzky et al.,
2000
). This post-translational modification converts the
V
subunit into its mature form capable of signal transduction
(Berthet et al., 2000
).
Furthermore, furin has an integrin-binding motif (391RGD) and could
therefore bind to these ECM receptors at the cell surface as well as in the
extracellular milieu. However, this has never been demonstrated. The
vitronectin receptor
Vß3 is involved in the activation of MMP2,
colocalizes with MT1-MMP and allows the interaction of both metalloproteinases
(Puyraimond et al., 2001
;
Deryugina et al., 2001
). The
subcellular localization of the
V integrin subunit in the renal
glomerulus has been demonstrated previously
(Yoon et al., 2001
). Our
results confirm its presence at the surface of podocytes, particularly on
their basal side, around slit diaphragms as well as at the endothelial cell
surface (Fig. 6A). Because
V and furin share same locations, we assessed their colocalization. By
double immunogold labeling,
V (5 nm gold particles) and furin (10 nm
gold particles) were found associated at the podocyte/basement-membrane
interface, particularly near the filtration slit diaphragms
(Fig. 6B). The same association
was also encountered at the surface of endothelial cells but to a lesser
extent. Morphometrical analysis indicates that
30% of cell surface furin
is associated with the
V-integrin subunit. No labeling was detected
over mitochondria and nuclei and control experiments with adsorbed antibodies
as well as omitting the primary antibody yield negligible labeling.
|
Coimmunoprecipitation experiments were carried out to confirm the
morphological results. DSP-treated membranes were subjected to
coimmunoprecipitation with the specific anti-V-C-terminus antibody. A
98 kDa protein, corresponding to the molecular mass of furin,
coimmunoprecipitated with the
V-antigen-antibody complex
(Fig. 6C, lane 1). However,
furin was not coimmunoprecipitated with
V in membranes not treated with
DSP (Fig. 6C, lane 2),
indicating that the furin-
V complex displays weak affinity or that the
complex might have been disrupted during the homogenization procedures. When
the nitrocellulose membrane was stripped and reprobed with the anti-
V
antibody, one band with a molecular mass of
25 kDa (the molecular mass of
the C-terminus of the reduced
V subunit) was detected in the membrane
fractions, independently of the chemical crosslinking
(Fig. 6D, lane 1,2). This
supports the specificity of the coimmunoprecipitation experiment. No protein
was detected when immunoprecipitation was carried out with normal rabbit
immunoglobulins (Fig. 6C,D,
lane 3).
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Discussion |
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We have demonstrated that furin is expressed by the rat kidney, particularly by glomerular cells. At the EM level, we identified the precise locations of furin within the Golgi apparatus and at the plasma membrane. Additional evidence showing that furin is indeed gathered at the cell surface was provided by overexpressing the enzyme in glomerular cells using in vivo gene delivery. These results suggest that endogenous and overexpressed furin in intact organ cells are present in two cellular pools, concentrated in the Golgi and present in detectable amounts at the cell surface.
Furin has a short cytosolic domain containing well-defined sequence motifs
that direct its sorting to the TGN-endosomal system. The routing of furin
depends on its state of phosphorylation and on the interactions of its
trafficking motifs with the cellular sorting machinery
(Molloy et al., 1999).
Moreover, furin delivered to the plasma membrane can interact with the
underlying actin cytoskeleton by getting associated to the actin binding
protein ABP-280. This scaffolding protein, which organizes actin
microfilaments and serves as a docking site for various transmembrane cell
surface proteins and intracellular signal transduction proteins, tethers furin
to the cell surface and modulates its rate of internalization
(Liu et al., 1997
).
Interestingly, it has been proposed that anchoring furin at the plasma
membrane might provide a mechanism for concentrating the protease in discrete
regions where efficient extracellular processing could occur
(Liu et al., 1997
). We did
find evidence for such concentrations. Podocyte foot processes are rich in
F-actin and other cytoskeletal components such as myosin,
-actinin,
vinculin and talin. These cytoskeletal proteins are concentrated along the
basal domain of podocytes abutting the GBM and also surround the endothelial
fenestrations (Drenckhahn and Franke,
1988
). In light of our results on the localization of furin in the
glomerulus, it is tempting to speculate that ABP-280 crosslinks furin to
F-actin at the podocyte slit diaphragms and at the endothelial fenestrations
allowing the protease to act preferentially at these plasmalemmal domains. It
is important to realize that MT1-MMP and integrin
Vß3 also
interact with F-actin (Galvez et al.,
2001
; Tsuruta et al.,
2002
).
Involvement of furin in the activation and trafficking of proMT1-MMP
and integrin V
Conflicting results have been reported about the activation of proMT1-MMP.
Cao et al. have shown that the full-length proMT1-MMP could act as an active
enzyme, whereas others have shown that proMT1-MMP has to be processed to
activate MMP2 (Pei and Weiss,
1996; Yu et al.,
1997
; Cao et al.,
1998
; Yana and Weiss,
2000
). Recently, several laboratories have generated a wealth of
data showing strong correlations between furin activity and those of MT1-MMP
and MMP2 (Maquoi et al., 1998
;
Bassi et al., 2001
;
de Kleijn et al., 2001
).
Moreover, several groups have found proMT1-MMP at the plasma membrane of
different cell lines, adding weight to the proposition that the pro-domain of
MT1-MMP is required for the efficient trafficking of the enzyme to the cell
surface (Sato et al., 1994
;
Sternlicht and Werb, 2001
).
Still, the identity of the activating enzyme and the exact site of MT1-MMP
activation remain obscure. Our EM data demonstrate that proMT1-MMP is indeed
localized to the cell surface and our double immunogold labelings and
coimmunoprecipitation results indicate that furin associates with proMT1-MMP
on plasma membranes of glomerular cells. Moreover, the pro-domain of MT1-MMP
in glomerular membrane fractions was cleaved by a soluble form of furin under
cell-free conditions, indicating a functional interaction between both
enzymes. These results could reconcile the facts that the pro-domain is
necessary for MT1-MMP trafficking to the cell surface but that it might have
to be cleaved to allow activation. Hence, furin and proMT1-MMP associate at
defined plasmalemmal domains of the glomerular cells and could react together
to promote GBM turnover either directly through the activation of MT1-MMP or
indirectly through the downstream activation of proMMP2. However, our results
also show that furin and proMT1-MMP colocalize in the Golgi apparatus and in
intracellular post-Golgi/endosomal structures such as podocytic vacuoles.
Thus, in addition to the cleavage of MT1-MMP pro-domain, furin could be
involved in the trafficking of proMT1-MMP in the secretory pathway. It was
recently shown that furin colocalizes with proMT3-MMP and proadamalysin 19 in
MDCK cells independently of their enzyme-substrate relationship
(Kang et al., 2002a
;
Kang et al., 2002b
). These
results imply that pro-domain processing is not an obligatory step in a
furin-substrate complex and that furin could escort proproteins to the cell
surface.
In addition, we present evidence that furin interacts with the V
integrin subunit. Like other integrin
chains, the
V has a
tetrabasic furin recognition site. Furin also holds a
cell-adhesion/integrin-binding motif (RGD) in its lumenal domain. Until now,
furin has not been found to bind any integrin by virtue of this motif. We here
report the association of furin with the
V integrin subunit but cannot
attest to whether this is happening through the RGD motif. However, our data
also suggest that these proteins interact with low affinity. We can only
speculate about the fact that furin could escort MT1-MMP and the
V
integrin subunit to the cell surface by binding their pro-domain or other
domains with low affinity. It is worth emphasizing that another
TGN/cell-surface recycling protein, TGN38, has also been shown to be involved
in such trafficking of proteins (the ß1 integrin subunit)
(Wang and Howell, 2000
).
Involvement of furin in GBM turnover
Cell surface binding of furin with proMT1-MMP and integrin V
strongly indicates that it could be involved in an activation cascade leading
to GBM degradation. The activation of the soluble proMMP2 occurs on the plasma
membrane through a unique multistep pathway involving MT1-MMP, tissue
inhibitor of metalloproteinase TIMP-2 and integrin
Vß3
(Deryugina et al., 2001
). It
appears that MT1-MMP/TIMP-2 complexes and
Vß3 bind proMMP2 and
focalize its activation by other unbound, active, MT1-MMP and MMP2 molecules.
Furthermore, these docking systems restrict matrix proteolysis to a limited
microenvironment on plasma membranes. Accordingly, our results indicate that
the association of furin with proMT1-MMP and integrin
V is focused on
discrete microdomains at the surface of glomerular cells. This suggests that a
mechanism of focalized pericellular proteolysis could be implicated in GBM
turnover. Cytoskeletal interactions might be involved in recruiting these
complexes to the fenestrations and slit diaphragm area. Other mechanisms that
might be involved in the recruitment of furin/proMT1-MMP and furin/
V
complexes to specific plasmalemmal sites include their possible association
with lipid rafts. MT1-MMP, MMP2 and
Vß3 have all been shown to
colocalize with caveolin-1, a major component of caveolae, at the surface of
endothelial cells (Puyraimond et al.,
2001
). Podocyte slit diaphragms are specialized domains containing
lipid rafts and it has been shown that many of its components (such as
nephrin, podocin and CD2AP) interact with the raft marker caveolin-1
(Schwarz et al., 2001
).
Recently, Hofmann and colleagues have shown that overexpression of the
integrin
Vß3 in an
Vß3-negative cell line greatly
increased the processing and activation of proMT1-MMP and, by the same token,
that of proMMP2 (Hofmann et al.,
2000
). Furthermore, they found that MT1-MMP and
Vß3
interact at the cell membrane. This finding could explain our results on the
colocalization of furin with
V. In addition to clustering MMP2 near
active MT1-MMP, the integrin could also present furin to the MT1-MMP
precursor, thereby facilitating proMT1-MMP activation.
The significance of furin in the maturation of a wide array of proproteins in the secretory pathway has been well demonstrated. However, aside from the activation of opportunistic pathogenic entities, its physiological role at the cell surface has remained elusive. In view of recent results, some of its functions at the cell surface are on the verge of being deciphered. We have demonstrated for the first time the interaction of furin with two of its substrates at the cell surface, under physiological conditions. Our data also provide an explanation of some discrepancies in the mechanisms of proMT1-MMP trafficking and activation. We have also shown that, in renal glomeruli, the furin/MT1-MMP axis does exist and might be part of a focalized activation cascade leading to GBM turnover.
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