Experimental Cell Research 260, 292–303 (2000)
doi:10.1006/excr.2000.5031, available online at http://www.idealibrary.com on
Rac1-Induced Endocytosis Is Associated with Intracellular Proteolysis
during Migration through a Three-Dimensional Matrix
Mamoun Ahram,* ,† ,1 Mansoureh Sameni,* Rong-Guo Qiu,‡ ,2 Bruce Linebaugh,* David Kirn,‡
and Bonnie F. Sloane* ,† ,3
*Department of Pharmacology and †Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine,
Detroit, Michigan 48201; and ‡Onyx Pharmaceuticals, Richmond, California 94806
INTRODUCTION
Transfection of Rat1 fibroblasts with an activated
form of rac1 (V12rac1) stimulated cell migration in
vitro compared to transfection of Rat1 fibroblasts
with vector only or with dominant negative rac1
(N17rac1). To investigate the involvement of proteases in this migration, we used a novel confocal
assay to evaluate the ability of the Rat1 transfectants to degrade a quenched fluorescent protein substrate (DQ-green bovine serum albumin) embedded
in a three-dimensional gelatin matrix. Cleavage of
the substrate results in fluorescence, thus enabling
one to image extracellular and intracellular proteolysis by living cells. The Rat1 transfectants accumulated degraded substrate intracellularly. V12rac1 increased accumulation of the fluorescent product in
vesicles that also labeled with the lysosomal marker
LysoTracker. Treatment of the V12rac1-transfected
cells with membrane-permeable inhibitors of lysosomal cysteine proteases and a membrane-permeable
selective inhibitor of the cysteine protease cathepsin B significantly reduced intracellular accumulation of degraded substrate, indicating that degradation occurred intracellularly. V12rac1 stimulated
uptake of dextran 70 (a marker of macropinocytosis)
and polystyrene beads (markers of phagocytosis)
into vesicles that also labeled for cathepsin B. Thus,
stimulation of the endocytic pathways of macropinocytosis and phagocytosis by activated Rac1 may
be responsible for the increased internalization and
subsequent degradation of extracellular proteins.
© 2000 Academic Press
Key Words: Rac1; endocytosis; intracellular proteolysis; cysteine proteases; cathepsin B.
1
Present address: Pathogenetics Unit, Laboratory of Pathology,
National Cancer Institute, NIH, 9000 Rockville Pike, Building 10,
Room 2A33, Bethesda, MD 20892.
2
Present address: Department of Molecular Cell Biology, University of California at Berkeley, Berkeley, CA 94720.
3
To whom correspondence and reprint requests should be addressed. E-mail: [email protected].
0014-4827/00 $35.00
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.
The small GTP-binding protein Rac1 is involved in
several cellular functions including cell proliferation
and transformation, cell migration, and vesicular trafficking. Rac1 plays an essential role in the transformation of Rat1 and NIH-3T3 fibroblasts by oncogenic HRas [1, 2]. This includes H-Ras-induced invasiveness in
vitro and in vivo [3] since transfection with rac1 modulates fibroblast migration and invasion [1, 4]. Transfection of NIH-3T3 fibroblasts with oncogenic H-ras
increases the expression of multiple classes of proteases: the matrix metalloprotease (MMP) type IV collagenase [5], the serine protease urokinase-type plasminogen activator (uPA; [6]), and the cysteine
proteases cathepsins B and L [7]. Thus, induction of
migration and invasion by H-Ras may reflect an increased ability of the fibroblasts to degrade extracellular matrices. Rac1, like H-Ras, has been shown to
upregulate expression of proteases, in this case of collagenase-1 in rat synovial fibroblasts [8].
One of the cysteine proteases that is upregulated by
H-Ras is cathepsin B [7], a protease whose expression
parallels acquisition of malignancy in several human
tumors, including colon carcinomas and gliomas (for
review, see [9]). Increased immunostaining for cathepsin B correlates with loss of immunostaining for the
basement membrane proteins laminin, in invasive gastric and colorectal carcinomas [10, 11], and type IV
collagen in bladder tumors (Visscher and Sloane, unpublished observations). Cathepsin B can degrade
these extracellular matrix (ECM) proteins, either as
purified proteins or in situ in lens capsule [12–14], and
cysteine protease inhibitors can reduce focal proteolysis of laminin by U87 human glioblastoma cells [15].
Cathepsin B can also degrade ECM proteins indirectly
through a proteolytic cascade, as cathepsin B can activate latent proteases, such as pro-uPA [16, 17] and
prostromelysin-1 [18]. The ability of cysteine protease
inhibitors to reduce motility of melanoma cells [19] and
invasion of ovarian carcinoma cells [17, 20] suggests
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RAC1 AND INTRACELLULAR PROTEOLYSIS
that cysteine proteases like cathepsin B do participate
in tumor cell migration and invasion.
We have developed a novel confocal assay to examine
concomitantly proteolysis by living cells and their migration through a three-dimensional matrix (Sameni,
Moin, and Sloane, submitted for publication). Here, we
used this assay to analyze the ability of rac1-transfected Rat1 fibroblasts to migrate and invade through a
three-dimensional gelatin matrix using a quenched fluorescent derivative of bovine serum albumin (DQ-BSA)
as a marker for proteolysis. We demonstrated that
Rat1 fibroblasts accumulate degraded DQ-BSA intracellularly, that degradation of DQ-BSA occurred intracellularly, and that transfection of Rat1 fibroblasts
with activated rac1 (V12rac1) increased invasion and
intracellular degradation products for DQ-BSA. The
apparent increase in intracellular degradation induced
by Rac1 was due to increased phagocytosis and macropinocytosis of DQ-BSA, and degradation of DQ-BSA
was mediated intracellularly by the lysosomal cysteine
protease cathepsin B.
MATERIALS AND METHODS
Materials
All general chemicals were obtained from Sigma (St. Louis, MO).
CA074 was a generous gift from Dr. Nobuhiko Katunuma (Tokushima,
Japan). E64d and CA074Me were obtained from the Peptide Institute
(Louisville, KY). P35012 and P35047 were generously provided by Prototek-II (Dublin, CA) and BB3103 by British Biotech (Oxford, England).
The cathepsin B substrate carbobenzyloxy-argininyl-argininyl-7amino-4-methylcoumarin was obtained from Bachem Bioscience (King
of Prussia, PA). Human liver cathepsin B was purchased from Athens
Research and Technology (Athens, GA). DQ-green BSA, LysoTracker
red DND-99, fluorescein-dextran 70 (M r 70,000 kDa), fluorescein-conjugated transferrin, the Live/Dead Viability/Cytotoxicity Assay kit, and
the SlowFade antifade kit were purchased from Molecular Probes (Eugene, OR). Fluoresbrite plain yellow green 1.9-␮m polystyrene microsphere beads were from Polysciences (Warrington, PA). The primary
antibodies (polyclonal rabbit anti-human liver cathepsin B IgG [21] and
monoclonal mouse anti-Myc 9E10 antibody [2]) were isolated and characterized in our laboratories. Secondary horseradish peroxidase-conjugated antibodies for immunoblotting were obtained from Pierce Chemical Co. (Rockford, IL). Secondary donkey antibodies used for
immunofluorescence were from Jackson ImmunoResearch Laboratories (West Grove, PA). Fetal bovine serum, Geneticin (G418), puromycin, and Opti-MEM were purchased from Life Technologies (Grand
Island, NY), Matrigel from Collaborative Biomedical Products (Bedford,
MA), 24-well Transwells from Costar (Corning, NY), and the ECL
Western Blotting Detection Kit and Hyperfilm ECL from Amersham
Life Sciences (Arlington Heights, IL).
Cell Lines and Media
Rat1 fibroblasts were transfected with the pUHD10-3 vector (designated vector 5 or vector 8) or the same vector containing myctagged V12rac1 (V12Rac1-6a, V12Rac1-10a, V12Rac1-14a) or
N17rac1 (N17Rac1-7a, N17Rac1-10) as described previously [2].
Cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 4.5 g/liter D-(⫹)-glucose, and the
antibiotics 100 U penicillin, 100 ␮g/ml streptomycin, 400 ␮g/ml
293
G418, and 2 ␮g/ml puromycin. Cells were grown in a 95% O 2/5% CO 2
atmosphere at 37°C.
Invasion Assay
Transwells (24-well) were coated with 40 mg of Matrigel. Cells (5 ⫻
10 4) were resuspended in Opti-MEM and seeded on Matrigel. OptiMEM was added to the lower chamber in the presence or absence of
10 ng/ml platelet-derived growth factor (PDGF). Invasive cells were
stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium
bromide, thiazolyl blue solution and counted 72–96 h after seeding.
Experiments were performed in triplicate.
Measurement of Cathepsin B Activity
After incubation in serum-free medium for 18 h, cells at 60 –90%
confluency were scraped and resuspended in cold (4°C) buffer containing 250 mM sucrose, 25 mM 2-(morpholino)ethane sulfonic acid,
1 mM EDTA, and 0.1% Triton X-100, pH 6.5. Microcentrifuge tubes
were immersed in ice-cold water and sonicated twice for 10 s each.
The lysates were centrifuged (14,000g for 10 min at 4°C) and the
supernatants were divided into aliquots and stored at ⫺70°C until
assayed. Cathepsin B activity was measured against carbobenzyloxy-argininyl-argininyl-7-amino-4-methylcoumarin [21] and expressed as nmol of product/min/␮g DNA.
Immunoblotting
Cells were serum-starved for 18 h and lysed at 60 –90% confluency
in a buffer containing 50 mM Tris–HCl, pH 9.0, 2 mM EDTA, 100
mM NaCl, and 0.5% SDS. Lysates were boiled for 5 min and passed
through an 18-gauge syringe 10 times. Samples were separated by
12.5% SDS–PAGE and transferred to a nitrocellulose membrane.
The membrane was blocked with 5% nonfat milk in 0.05% Tween 20/
phosphate-buffered saline (PBS) overnight at 4°C and then immunoblotted for cathepsin B using anti-human liver cathepsin B IgG
followed by horseradish peroxidase-conjugated goat anti-rabbit IgG
[22]. The membrane was stripped at 65°C for 30 min with stripping
buffer (62.5 mM Tris, pH 6.8, 2% SDS, and 100 mM ␤-mercaptoethanol) and reprobed for Myc-tagged Rac1 using an anti-Myc monoclonal IgG [2], followed by horseradish peroxidase-conjugated goat antimouse IgG. Detection was performed using an ECL Western Blotting
Detection Kit and membranes were exposed to Hyperfilm ECL. Results are from four independent experiments.
In Situ Assay for Proteolysis of DQ-BSA in Three-Dimensional
Gelatin
Glass coverslips were coated with 2% gelatin containing 2% sucrose, 0.02% sodium azide, and 25 ␮g/ml DQ-BSA in PBS and placed
in 35- or 60-mm dishes. Cells (vector 5, V12Rac1-14a, or N17Rac110) were seeded on the coated coverslips and generation of fluorescence (i.e., degraded DQ-BSA) was examined over a period of 2– 6
days. To determine whether the intracellular compartment containing degraded DQ-BSA was the lysosomal compartment, cells were
incubated in the presence of 100 nM LysoTracker in medium for 2 h.
Cells were washed and chased for at least 2 h. They were then
examined on a Zeiss LSM 310 confocal microscope and digital images
recorded (0.4-␮m optical sections).
Alternatively, cells (V12Rac1-14a) were incubated in the presence
of DQ-BSA (50 ␮g/ml) for 48 h. Cells were then fixed in methanol
(⫺20°C) for 5 min and labeled for cathepsin B using polyclonal rabbit
anti-human liver cathepsin B IgG. After 2–3 h, cells were washed
and incubated with Texas red-conjugated donkey anti-rabbit IgG for
1 h. Cells were then fixed with methanol (⫺20°C) for 2 min, washed
with PBS, and mounted on slides with SlowFade antifade reagent
and observed by confocal microscopy as described above.
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AHRAM ET AL.
In Vitro Assay for in Vivo Proteolysis of DQ-BSA
V12rac1-transfected cells (vector 5, V12Rac1-14a, and N17Rac110) were grown in 60-mm dishes in the presence of DQ-BSA for 18 h.
Cells were washed with cold (4°C) PBS and scraped into 1 ml of cold
(4°C) PBS containing 0.1% Triton X-100. Cell lysates were sonicated
twice for 1 min each and centrifuged at 14,000g for 10 min at 4°C.
The amount of intracellular fluorescence in the supernatant was
measured with a Shimadzu RF-540 spectrofluorimeter (Columbia,
MD) at an excitation ␭ of 505 nm and an emission ␭ of 515 nm.
To determine which protease or protease class was involved in
degradation of DQ-BSA, V12rac1-transfected cells (V12Rac1-14a)
were incubated with protease inhibitors (10 ␮M E64d, CA074Me,
P35012, or pepstatin A or 1 ␮M aprotinin) for 7 h. DQ-BSA (25
␮g/ml) was then added to the cells along with the corresponding
inhibitor for 18 h. Intracellular fluorescence was analyzed as described above.
In Vitro Proteolysis of DQ-BSA
V12rac1-transfected cells grown in a 150-mm dish were scraped
into 4 ml of cold (4°C) PBS containing 0.1% TX-10, sonicated twice for
30 s each, and centrifuged at 14,000g for 10 min at 4°C in order to
remove cell debris that might interfere with fluorescence reading.
The supernatant was stored at ⫺20°C until assayed.
To assay, a 150-␮l aliquot of cell lysates was added to 150 ␮l of
citrate phosphate buffer (0.1 M citric acid and 0.2 M Na 2HPO 3), pH
5.2, 6.2, or 7.0. This was followed by addition of 300 ␮l of 10 mM DTT
and 4.8 mM EDTA, pH 5.2, reagent and samples were incubated at
37°C for 15 min. DQ-BSA (30 ␮g) was added to the lysates and the
samples were incubated at 37°C for 16 –18 h in the dark. Fluorescence was read using a Labsystems Fluoroscan II (Labsystems, Helsinki, Finland). In order to assess the effects of different protease
inhibitors, 10 ␮M E64, P35012, CA074, and pepstatin A were added
to the lysates prior to addition of DQ-BSA.
In order to determine whether cathepsin B degrades DQ-BSA, 2 ␮g
of purified human liver cathepsin B (150 ␮l) was added to 150 ␮l of
citrate phosphate buffer, pH 5.2, 6.2, or 7.0. Three hundred microliters of 10 mM DTT and 4.8 mM EDTA, pH 5.2, reagent was added
and samples were incubated for 15 min at 37°C to activate cathepsin
B. DQ-BSA (30 ␮g) was added and the reactions were incubated for
16 –18 h at 37°C in the dark. Fluorescence was read using a Labsystems Fluoroscan II.
Receptor-mediated endocytosis. Cells (vector 5, V12Rac1-14a,
and N17Rac1-10) grown on coverslips were serum-starved (⫹0.1%
BSA) for 18 h. Cells were washed with cold (4°C) PBS and incubated
on ice for 10 min. Cells were then incubated in the presence of 10
␮g/ml or 1 mg/ml fluorescein isothiocyanate–transferrin in PBS containing 0.1% BSA for 60 min at 4°C. Cells were washed three times
with cold (4°C) PBS, then washed with prewarmed (37°C) PBS and
incubated at 37°C for 30 min. Cells were washed with cold (4°C) PBS
and fixed with 4% formaldehyde for 45 min at 4°C. Cells were then
washed, mounted, and observed by confocal microscopy as described
above.
Phagocytosis. Cells (vector 5, V12Rac1-14a, and N17Rac1-10)
grown in 35-mm dishes were incubated with polystyrene microsphere beads (1.9 ␮m) at a ratio of 1 cell to 100 beads for 30 and 60
min. Cells were detached with 0.5 ml of 0.05% trypsin, neutralized in
1.5 ml of medium, and centrifuged at 200g for 5 min at 4°C. Cell
pellets were washed with cold (4°C) Ca 2⫹/Mg 2⫹-free PBS, centrifuged,
and resuspended in cold (4°C) PBS to approximately 1 ⫻ 10 6 cells/ml.
The cell suspension was kept on ice during the rest of the experimental analysis. The total number of phagocytic cells was measured
as previously described [23]. Briefly, cells (1 ⫻ 10 4) were sorted by
FACS Calibur and analyzed by Cell Quest version 3.1 (Becton–
Dickinson Immunocytometry System, San Jose, CA). Beads were
excited with an argon blue laser (wavelength 488 nm) and detected
with an FL1 detector (wavelength 530 ⫾ 30 nm).
For colocalization of cathepsin B and polystyrene beads, V12Rac114a cells were incubated for 90 min in the presence of beads at a ratio
of 1 cell to 100 beads. Cells were washed, chased for 120 min, and
then fixed in methanol (⫺20°C) for 5 min. Labeling for cathepsin B
was performed as described above. After incubation with secondary
antibody, cells were fixed with 3.7% formaldehyde for 5 min, washed,
mounted, and observed on a Zeiss LSM 310 as described above.
Inhibition of phagocytosis. V12Rac1-14a cells were cultured on
the gelatin-coated coverslips (⫹25 ␮g/ml DQ-BSA) in the presence or
absence of 5 ␮g/ml cytochalasin B. Degradation of DQ-BSA was
observed by confocal microscopy at 48 h postseeding as was described
above.
Statistical Analyses
Data are presented as means ⫾ SD. ANOVA (Tukey test) was used
for all statistical analyses and P values ⱕ0.05 were considered
significant.
Determination of Cell Viability
To determine cell viability, a Live/Dead Viability/Cytotoxicity Kit
was used. V12rac1-transfected cells (V12Rac1-14a) at 70% confluency were incubated with 4 ␮M calcein-AM and 2 ␮M ethidium
homodimer in PBS for 30 min at room temperature. This assay is
based on the permeability of calcein-AM, a marker that detects living
cells, and the impermeability of ethidium homodimer, a marker that
detects damaged and dead cells. As a control to confirm that we were
able to visualize damaged cells by this method, cells were incubated
in PBS containing 0.1% saponin for 10 min prior to addition of the
reagents. Cells were washed and observed immediately by confocal
microscopy as described above.
Analysis of Endocytosis
Pinocytosis. Cells (vector 5, V12Rac1-14a, and N17Rac1-10)
grown on glass coverslips were incubated for 2 and 24 h in the
presence of fluorescein-conjugated dextran 70 (1 mg/ml) to assess
macropinocytosis or Texas red-conjugated dextran 10 (1 mg/ml) to
assess micropinocytosis. Cells were washed and fixed in 3.7% formaldehyde for 10 min. To colocalize cathepsin B and dextran 70, cells
were fixed in 3.7% formaldehyde for 10 min followed by methanol
(⫺20°C) for 1 min. Cells were then labeled for cathepsin B and
observed by confocal microscopy as described above.
RESULTS
Activated Rac1 Increases Invasion in Vitro
Since Rac1 has been linked to fibroblast migration
and invasion [1, 4, 24], we analyzed the invasive ability
of rac1-transfected Rat1 fibroblasts: two clones transfected with vector (vector 5 and 8), three clones transfected with activated rac1 (V12rac1), and two clones
transfected with dominant negative rac1 (N17rac1).
The expression of Rac1 varied among the seven clones
(Table 1). Rat1 fibroblasts transfected with V12rac1
were spread and exhibited extensive lamellipodia and
membrane ruffles in which actin microfilaments were
concentrated (not shown), confirming the ability of
Rac1 to modulate the actin cytoskeleton of these fibroblasts [25]. Many of the V12rac1-transfected cells were
multinucleated, a phenomenon proposed to be due to a
defect in cytokinesis [26]. Transfection with V12rac1
increased invasion through Matrigel with invasiveness
295
RAC1 AND INTRACELLULAR PROTEOLYSIS
TABLE 1
Invasion of Rat1 Fibroblasts through Matrigel in Vitro
Invasive cells ⫻ 10 4 (⫾SD)
Pb
Cell line
Rac1 a
⫺PDGF
⫹PDGF
⫺PDGF
⫹PDGF
Pc
Vector 5
Vector 8
V12Rac1-10a
V12Rac1-6a
V12Rac1-14a
N17Rac1-7a
N17Rac1-10
⫺
⫺
⫹
⫹⫹
⫹⫹⫹⫹
⫹⫹
⫹⫹⫹
1.80 (0.31)
1.53 (0.45)
2.55 (0.97)
5.42 (1.40)
8.83 (1.47)
2.10 (0.36)
0.60 (0.24)
2.20 (0.76)
2.95 (1.43)
4.27 (1.06)
6.18 (0.76)
12.5 (2.24)
1.50 (0.54)
1.72 (0.61)
—
ns
ns
ⱕ0.001
ⱕ0.001
ns
ns
—
ns
ns
ⱕ0.001
ⱕ0.001
ns
ns
ns d
ⱕ0.05
ⱕ0.05
ns
ⱕ0.01
ⱕ0.05
ⱕ0.01
a
Levels of expression of Rac as determined by immunoblotting (see Materials and Methods).
Values were compared to those for vector 5 cells in the absence of PDGF (left column) and presence of PDGF (right column).
Values were compared for each cell line in the absence and presence of PDGF.
d
Not significant.
b
c
paralleling the expression of activated V12rac1 (Table
1). Invasion was reduced ⬃50% by the cysteine protease inhibitors P35012 and P35047 (data not shown),
thus implicating a cysteine protease in invasion of the
V12rac1-transfected cells. PDGF augmented invasion
of four of the seven transfectants, including one control
transfectant, two V12rac1-transfectants, and one
N17rac1-transfectant. Thus, PDGF appeared to increase cell invasion through a Rac1-independent pathway.
Activated Rac1 Induces Intracellular Accumulation of
Degraded DQ-BSA
Cell invasion is a multistep process that includes
proteolysis of the basement membrane [27]. In order to
evaluate proteolytic activity during invasion of Rat1
cells, we used a novel confocal assay in which vector-,
V12rac1-, and N17rac1-transfected Rat1 fibroblasts
were seeded onto a three-dimensional gelatin matrix
mixed with a quenched fluorescent substrate (DQgreen BSA), a substrate that fluoresces green when
cleaved by proteases. Over a 2- to 6-day incubation
period, two populations of cells could be observed: cells
that remained on top of the matrix (not illustrated) and
invasive cells that migrated through the matrix (Fig.
1). Fluorescent products resulting from cleavage of DQBSA were detected intracellularly, primarily in perinuclear vesicles. Fluorescent products were not detected
extracellularly. In general, the migratory or invasive
cells contained more intracellular fluorescence than
the cells that remained on top of the gelatin. The invasive V12rac1-transfected cells were characterized by
the presence of large vacuoles (compare Fig. 1B with
1A and 1C), yet were viable as determined with a
Live/Dead Viability/Cytotoxicity Kit (data not shown).
The invasive V12rac1-transfected fibroblasts contained more degraded DQ-BSA than either the inva-
sive vector-transfected cells or the invasive N17rac1transfected cells.
To quantitate the effects of Rac1 on the intracellular
accumulation of degraded DQ-BSA, transfectants were
grown in the presence of three concentrations of DQBSA (10, 25, and 50 ␮g/ml) for 18 h. Measurement of
intracellular fluorescence in vitro revealed that degraded DQ-BSA accumulated in a dose-dependent
manner (Fig. 2). The V12rac1-transfected cells contained more degraded DQ-BSA than did the other two
cell lines, thus confirming the above in situ microscopic
observations. Dominant negative Rac1 did not affect
the intracellular accumulation of degraded DQ-BSA.
Taken together, our results suggest that V12rac1 stim-
FIG. 2. V12rac1 increased intracellular accumulation of degraded DQ-BSA as assessed in an in vitro assay. More fluorescent
DQ-BSA was observed in V12rac1-transfected cells (‚) than in vector- (■) or N17rac1-transfected (F) cells. Cells were incubated with
DQ-BSA (10, 25, and 50 ␮g/ml) for 18 h. Intracellular fluorescence
was measured in cell lysates at an excitation ␭ of 505 nm and an
emission ␭ of 515 nm. The graph is representative of two independent experiments performed in triplicate. Data are presented as
means ⫾ SD. *P ⱕ 0.05, **P ⱕ 0.01 compared to vector-transfected
cells.
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AHRAM ET AL.
FIG. 1. V12rac1 increased intracellular accumulation of DQ-BSA as assessed in an in situ assay. Vector- (A), V12rac1- (B), and N17rac1(C) transfected cells were grown on a gelatin matrix containing DQ-BSA (25 ␮g/ml). More degraded DQ-BSA (green) accumulated in invasive
V12rac1-transfected cells than in invasive vector or invasive N17rac1-transfected cells. All images are of invasive cells day 4 postseeding and
are representative of three independent experiments. Bar, 25 ␮m.
FIG. 3. Colocalization in V12rac1-transfected cells of degraded DQ-BSA (A and B) with two lysosomal markers: LysoTracker (A) and the
cysteine protease cathepsin B (B). Colocalization of degraded DQ-BSA (green) with LysoTracker (red) and cathepsin B (red) is indicated by
the yellow fluorescence; however, degraded DQ-BSA and cathepsin B were also found in separate vesicles. Note in (B) that cathepsin B and
degraded DQ-BSA colocalized in large as well as small vesicles. Cathepsin B was labeled with rabbit anti-human liver cathepsin B IgG and
detected with donkey anti-rabbit IgG. The images are representative of two independent experiments performed in duplicate. Bar, 25 ␮m.
RAC1 AND INTRACELLULAR PROTEOLYSIS
297
ulated intracellular accumulation of degraded DQBSA.
Degraded DQ-BSA Colocalizes with Cathepsin B in
Lysosomes
To determine whether the perinuclear vesicles containing fluorescent (hence degraded) DQ-BSA were lysosomes, we grew V12rac1-transfected cells on DQBSA/gelatin in the presence of the lysosomal marker,
LysoTracker. There was extensive colocalization of degraded DQ-BSA and LysoTracker (Fig. 3A). These results suggest that lysosomes were the main site of
accumulation of degraded DQ-BSA and therefore that
lysosomal proteases such as the cysteine protease cathepsin B might be responsible for this degradation.
We confirmed that cathepsin B could degrade DQ-BSA
by incubating purified human liver cathepsin B with
DQ-BSA for 18 h at 37°C at pH 5.2, a pH comparable to
that of lysosomes and late endosomes (data not shown).
To demonstrate that cathepsin B and degraded DQBSA were colocalized in the same vesicles, we incubated the V12rac1-transfected cells with DQ-BSA and
then immunostained for cathepsin B. For this analysis,
we did not mix the DQ-BSA with gelatin because the
methanol fixation required for the immunostaining
damaged the three-dimensional gelatin matrix, thus
obscuring the fluorescent images. Considerable colocalization of cathepsin B and degraded DQ-BSA was observed (Fig. 3B), including colocalization in large vesicles (diameter of 2.6 ⫾ 0.7 ␮m, n ⫽ 37). The absence
of gelatin optimized diffusion of the anti-cathepsin B
antibodies so that we observed many vesicular compartments which labeled for cathepsin B (Fig. 3B, red
labeling), but not for DQ-BSA. The extensive staining
for cathepsin B in these cells may represent a redistribution of cathepsin B to endosomal compartments,
something we have previously observed in ras-transformed epithelial cells and in tumor cells [22, 28]. We
observed very few vesicles (Fig. 3B, green labeling)
which contained degraded DQ-BSA, but did not contain cathepsin B. Since degraded DQ-BSA present in
vesicles did not label for cathepsin B (Fig. 3B) and did
label with LysoTracker (Fig. 3A), we speculate that
lysosomal proteases other than cathepsin B may also
contribute to degradation of this substrate.
Inhibitors of Cysteine Proteases and Cathepsin B
Reduce Intracellular Accumulation of Degraded
DQ-BSA
The accumulation of degraded DQ-BSA intracellularly may be due to intracellular proteolysis or to extracellular proteolysis, followed by internalization of
degraded DQ-BSA by endocytosis and its delivery to
the lysosomes. To determine whether DQ-BSA was
degraded extracellularly or intracellularly and to de-
FIG. 4. Inhibitors of serine proteases, cysteine proteases, and
cathepsin B reduced intracellular accumulation of degraded DQBSA. Treatment of V12rac1-transfected cells with 10 ␮M membranepermeable cysteine protease inhibitors (E64d and P35012), 10 ␮M
membrane-permeable cathepsin B-selective inhibitor (CA074Me), or
1 ␮M serine protease inhibitor (aprotinin) reduced intracellular fluorescence. Pepstatin A (10 ␮M) did not significantly reduce intracellular accumulation of DQ-BSA. The figure is representative of three
independent experiments performed in triplicate. Data are represented as means ⫾ SD. *P ⱕ 0.05, **P ⱕ 0.001 compared to vehicle
(DMSO)-treated cells.
termine which protease(s) or protease class(es) was
responsible for degradation of DQ-BSA, we preincubated V12rac1-transfected cells with protease inhibitors for 7 h prior to adding DQ-BSA along with the
corresponding inhibitor. Inhibitors of cysteine proteases (E64d, P35012) and the highly selective inhibitor of intracellular cathepsin B (CA074Me) significantly reduced intracellular accumulation of degraded
DQ-BSA by living cells (Fig. 4). The comparable abilities of CA074Me, E64d, and P35012 to reduce accumulation of degraded DQ-BSA suggest that cathepsin B
was the cysteine protease responsible for DQ-BSA degradation. Aprotinin, an inhibitor of serine proteases,
also significantly reduced intracellular accumulation of
DQ-BSA, although to a lesser extent than the inhibitors of cysteine proteases and cathepsin B. Both pepstatin A (an inhibitor of aspartic proteases) and
BB3103 (an inhibitor of MMPs) were ineffective. Interestingly, a combination of E64d and pepstatin A, but
not of E64d and aprotinin, further reduced accumulation by 15–20% compared to E64d alone (data not
shown).
E64d and CA074Me are membrane-permeable forms
of E64 and CA074 that require activation by cytosolic
esterases [29, 30]. Thus, when incubated with cells,
E64d and CA074Me inhibit intracellular cysteine proteases and cathepsin B, respectively. P35012, the inhibitor that resulted in 50% reduction in cell invasion,
is membrane permeable and does not require activation, thus it can block the enzymatic activity of intracellular and extracellular cysteine proteases (M. Zimmerman, personal communication). The ability of these
298
AHRAM ET AL.
inhibitors to reduce intracellular accumulation of degraded DQ-BSA suggests that degradation occurred
intracellularly in lysosomes. To confirm that V12rac1transfected cells contain intracellular proteases that
can degrade DQ-BSA, we incubated cell lysates with
DQ-BSA in the absence and presence of inhibitors at
37°C and pH 5.2 for 18 h. P35012, E64, and CA074
reduced degradation of DQ-BSA by 32% (⫾ 1.7%). We
did not detect any difference in efficacy among the
cysteine protease inhibitors and the cathepsin B-selective inhibitor, indicating that cathepsin B was the primary cysteine protease in the cell lysates that was
capable of degrading DQ-BSA, thus supporting our
studies in vivo. Pepstatin A did not have any inhibitory
effect. Our results suggest that degradation of DQ-BSA
by Rat1 fibroblasts occurred intracellularly and that
cathepsin B was involved in this degradation.
Rac1 Does Not Alter Cathepsin B Levels
V12rac1-induced accumulation of degraded DQ-BSA
may reflect elevated levels of cathepsin B as has been
shown for collagenase-1 [8]. Therefore, we examined
the effect(s) of activated Rac1 on cathepsin B protein
and activity, both of which have been reported to increase in invasive cells [22, 30]. The primary form of
cathepsin B in the Rat1 transfectants was the mature
single-chain (31-kDa) enzyme. A smaller amount of the
mature double-chain (26-kDa heavy chain) enzyme
and a trace amount of procathepsin B (46 kDa) were
also observed. Levels of cathepsin B protein and activity (data not shown) did not correlate with expression
of activated or dominant negative Rac1 (Table 1). Thus,
Rac1 did not upregulate expression of cathepsin B and
thereby increase the intracellular accumulation of degraded DQ-BSA.
Activated Rac1 Induces Macropinocytosis and
Phagocytosis
Our observations of increased vacuolation in the
V12rac1-transfected cells and the specific accumulation of degraded DQ-BSA in those vacuolated cells
(Figs. 1B, 3A, and 3B) suggest that activated Rac1 may
have increased endocytosis. Thus, we analyzed the effect of Rac1 on macropinocytosis, micropinocytosis,
phagocytosis, and receptor-mediated endocytosis. Dextran 70 has been used previously to analyze stimulation of macropinocytosis by scatter factor [32] and v-Src
[33] and has a molecular weight (70 kDa) similar to
that of DQ-BSA (66 kDa). We found that dextran 70
accumulated in vacuolated V12rac1-transfected cells
within 1–2 h of incubation, whereas dextran 70 did not
accumulate in control transfectants and cells transfected with N17rac1 during the first 2 h of incubation
(data not shown). After longer incubation periods (i.e.,
18 and 24 h), the accumulation of dextran 70 in
V12rac1-transfected cells was substantially greater
than in vector- and N17rac1-transfected cells (Fig. 5).
The V12rac1-transfected cells were characterized by
vacuolation similar to that observed in cells containing
degraded DQ-BSA (see Fig. 1B).
Activated Rac1 did not affect micropinocytosis as
assessed by uptake of dextran 10. All three types of
Rat1 transfectants were capable of endocytosing dextran 10 during 2 or 24 h of incubation (data not shown).
Over a 24-h incubation period, vacuolated V12rac1transfected cells accumulated more dextran 10 than
did nonvacuolated cells (data not shown); however, this
may reflect stimulation of macropinocytosis by
V12rac1 (see above) as macropinocytosis can result in
internalization of smaller molecules such as dextran 10
as well as larger ones such as dextran 70 [34]. Transferrin, often used as a marker of receptor-mediated
endocytosis [35], was not internalized by the Rat1
transfectants. The transferrin receptor was expressed
in these cells as demonstrated by immunoblotting and
immunofluorescence techniques (data not shown).
Thus, by this criterion, receptor-mediated endocytosis
was not responsible for accumulation of degraded DQBSA by these cells.
Phagocytosis, in contrast to other types of endocytosis, can result in internalization of large proteins and
their transport to lysosomes for digestion. We used
1.9-␮m polystyrene beads to assess nonspecific phago-
FIG. 5. V12rac1 stimulated uptake of dextran 70 into macropinosomes, many of which stained for cathepsin B. Vacuolated V12rac1transfected cells (B) internalized more dextran 70 during a 24-h incubation than nonvacuolated V12rac1-transfected cells, cells transfected
with vector alone (A), or cells transfected with N17rac1 (C). (D) Colocalization of cathepsin B and dextran 70. V12rac1-transfected cells
incubated with dextran 70 (green) for 18 h were fixed and labeled for cathepsin B (red). Yellow fluorescence indicates colocalization of dextran
70 and cathepsin B. Note that colocalization occurs in large vesicles (2.2 ⫾ 0.7 ␮m, n ⫽ 41). Cathepsin B was labeled with rabbit anti-human
liver cathepsin B IgG and detected with donkey anti-rabbit IgG. The images are representative of two independent experiments performed
in duplicate. Bar, 25 ␮m.
FIG. 7. Colocalization in V12rac1-transfected cells of cathepsin B and microsphere beads. (A) Immunostaining for cathepsin B. (B)
Localization of microsphere beads. (C) Localization of microsphere beads superimposed on a phase-contrast image of the same field. Cells
incubated with the beads for 90 min and chased for 120 min were labeled for cathepsin B using rabbit anti-human liver cathepsin B IgG and
detected with donkey anti-rabbit IgG. In areas of colocalization, cathepsin B surrounded the beads forming a fluorescent ring structure
(compare insets in A and B). Some beads were not surrounded by a fluorescent ring (arrow, B), indicating the specificity of antibody staining.
The images are representative of duplicate experiments performed in duplicate. Bar, 25 ␮m.
RAC1 AND INTRACELLULAR PROTEOLYSIS
299
300
AHRAM ET AL.
cytosis as these beads cannot be internalized by macropinocytosis, but must be phagocytosed [23]. After 30
and 60 min of incubation with the polystyrene beads,
23 and 39% of V12rac1-transfected cells were found to
be phagocytic compared to 5 and 12% of vector control
transfectants and 6 and 15% of N17rac1-transfected
cells, respectively (Fig. 6). Statistical analyses revealed
that V12rac1 significantly stimulated phagocytosis.
We inhibited phagocytosis and macropinocytosis in the
V12rac1-transfected cells by incubating with cytochalasin B, a fungal derivative that prevents actin filaments from polymerizing [36]. Intracellular accumulation of degraded DQ-BSA was reduced by cytochalasin
B (not illustrated). This did not result in accumulation
of degraded DQ-BSA extracellularly, indicating that
degradation of DQ-BSA took place intracellularly.
Therefore, our studies indicate that V12rac1 increased
uptake of intact DQ-BSA and could presumably do so
for other protein substrates, primarily by increasing
phagocytosis and macropinocytosis.
Cathepsin B Colocalizes with Markers of
Macropinocytosis and Phagocytosis
The presence of cathepsin B in vesicles containing
dextran 70 and the phagocytosed beads might implicate cathepsin B in the degradation of DQ-BSA internalized by macropinocytosis or phagocytosis. Therefore, we immunostained V12rac1-transfected cells that
had been incubated with dextran 70 for cathepsin B
(Fig. 5D). Cathepsin B colocalized with dextran 70 in
small, perinuclear vesicles as well in large vesicles
(diameter of 2.2 ⫾ 0.7 ␮m, n ⫽ 41). When V12rac1transfected cells that had been incubated with polystyrene beads were stained for cathepsin B, the immunostaining for cathepsin B formed a fluorescent ring
surrounding the beads (Fig. 7A). Phagocytosed beads
were also observed in vesicles that did not stain for
cathepsin B, indicating that the antibodies did not bind
to the beads nonspecifically (Figs. 7A and 7B; arrows).
Furthermore, control antibodies (rabbit IgG) did not
bind to the beads (data not shown). The colocalization
of cathepsin B with macropinocytosed dextran 70 and
phagocytosed beads indicated that these vesicles must
have fused with lysosomes containing cathepsin B.
DISCUSSION
We have confirmed the reports of others that Rac1
plays a role in cell invasion [1, 4] by demonstrating
that activated Rac1 stimulated invasion of Rat1 fibroblasts through Matrigel (Table 1). The precise mechanism by which Rac1 induces cell migration is unknown,
although formation of lamellipodia at the leading edge
of motile cells seems to be involved. Two hypotheses
have been proposed to explain the role of Rac1 in shap-
FIG. 6. V12rac1 induced phagocytosis. Flow cytometric analysis
revealed an increased rate of phagocytosis by V12rac1-transfected
cells compared to vector- and N17rac1-transfected cells following
incubation with polystyrene beads for 30 (dotted bars) and 60 (shaded bars) min. The figure is representative of two independent experiments performed in triplicate. Data are presented as means ⫾ SD.
**P ⱕ 0.001 compared to vector-transfected cells.
ing the leading edge: (1) increased polymerization of
actin [37–39] and (2) redirection of exocytic vesicles to
the leading edge [40 – 42]. Both are thought to be responsible for forward movement and both were observed in the rac1-transfected Rat1 fibroblasts in this
study.
The novel confocal assay used here allowed us to
visualize degradation of a quenched fluorescent protein
substrate (DQ-BSA) by living cells as they migrated
through a three-dimensional gelatin matrix. The appearance of fluorescence signaled degradation. The
DQ-BSA substrate was available commercially at the
time we initiated this study; however, more relevant
ECM substrates are now available such as DQ-collagen
IV. We have demonstrated that human breast carcinoma cells (BT20, BT549) degrade DQ-BSA and DQcollagen IV comparably: the BT20 cells degrade the two
substrates extracellularly and the BT549 cells intracellularly (Sameni, Moin, and Sloane, submitted for
publication). In Rat1 cells transfected with activated
Rac1, we observed an increase in accumulation of degraded DQ-BSA within endocytic vesicles and resolved
that the mechanism for increased accumulation was
Rac1 stimulation of phagocytosis and macropinocytosis. Rac1 had previously been shown to be involved in
phagocytosis by professional phagocytes such as leukocytes and macrophages [43, 44]. Rac1 has been shown
to modulate the actin cytoskeleton in both cell types
[43– 45]. This might suggest that actin polymerization
is the mediator of Rac1-induced endocytosis, yet Rac1
is bound to phagosomes in macrophages, suggesting
that Rac1 may directly affect movement of phagocytic
vesicles [44]. Activated Rac1 has also been detected on
the cytoplasmic surface of macropinosomes [25, 46] and
has the ability to stimulate macropinocytosis [25;
present study]. A potential downstream mediator of
the Rac1 effect is protein-activated kinase-1, which
301
RAC1 AND INTRACELLULAR PROTEOLYSIS
binds to macropinocytic vesicles [47] and induces uptake of dextran 70 [48].
Increased phagocytosis by tumor cells parallels an
increase in invasiveness. Coopman et al. [49] reported
increased phagocytosis of ECM by invading breast carcinoma cells, and the invasion of glioma cells, is accompanied by phagocytosis and dissolution of normal brain
tissue [50]. A link between macropinocytosis and cell
invasion is less obvious. Nonetheless, macropinocytosis
has often been linked to membrane ruffling [25, 32, 51],
a phenomenon correlated with cell migration [32, 38].
Invasive fibrosarcoma cells and fibroblasts exhibit
zones of ECM clearing that are adjacent to membrane
ruffles and lamellipodia [52, 53]. Such observations
suggest that stimulation of phagocytosis and macropinocytosis may increase internalization of ECM proteins and thereby their degradation intracellularly.
The lysosomal proteases cathepsins B and D have
been reported to degrade ECM intracellularly. Cathepsin D colocalizes with internalized ECM in phagosomes
of breast carcinoma cells, where it is proposed to digest
the internalized ECM as a prerequisite to cell invasion
and metastasis [54, 55]. Intracellular degradation of
collagen I by cathepsin B has been demonstrated in
periosteal fibroblasts [56]. In the present study, by
using membrane-permeable inhibitors of cysteine proteases and of cathepsin B (Fig. 4), we verified that
cathepsin B could degrade an extracellular protein
substrate (DQ-BSA) that had been endocytosed.
Purified cathepsins B and D can degrade Bodipylabeled BSA in vitro and perhaps in situ as degradation
of BSA phagocytosed by macrophages can be reduced
by a protease inhibitor cocktail of pepstatin A, leupeptin (a cysteine protease inhibitor), and phenylmethylsulfonyl fluoride (a serine protease inhibitor) [57]. With
such a cocktail, the contribution of any individual aspartic, cysteine, and serine proteases to the degradation is not assessed. We were able to inhibit degradation of DQ-BSA by the rac1-transfected Rat1
fibroblasts with aprotinin, E64d, P35012, or CA074Me.
The reduction in intracellular degradation of DQ-BSA
by P35012 corresponded to the ability of this inhibitor
to compromise cell invasion. A combination of E64d
and pepstatin A (which was ineffective alone) was more
effective than E64d alone, perhaps indicating that cathepsin D and cysteine proteases participate in degradation of DQ-BSA in the Rat1 fibroblasts.
We were unable to completely inhibit DQ-BSA degradation by the rac1-transfected Rat1 fibroblasts with
inhibitors of serine proteases, cysteine proteases, or
cathepsin B, suggesting that additional proteases and
classes of proteases can degrade DQ-BSA. Surprisingly, the MMP inhibitor BB3103 was unable to reduce
DQ-BSA degradation. Nonetheless, MMPs may have
facilitated endocytosis of DQ-BSA by digesting the gelatin matrix in which it was embedded. As the gelatin
matrix was not labeled, degradation of gelatin would
not be observed under the conditions of the assay used
here. Consistent with a role for MMPs is that inhibitors of MMPs and serine proteases reduce gelatin uptake by breast carcinoma cells by ⬃50% [49]. Multiple
proteases and multiple sites of proteolysis are involved
in degradation of ECM proteins (elastin, glycoproteins,
collagens) by macrophages in which extracellular digestion is followed by intracellular digestion within
lysosomes [58, 59]. Similarly, degradation of collagen I
by periosteal fibroblasts involves MMPs extracellularly
and cathepsin B intracellularly within lysosomes [60].
The ability of aprotinin to inhibit accumulation of degraded DQ-BSA by the rac1-transfected fibroblasts
might suggest that extracellular cleavage of this substrate occurs, yet we did not observe extracellular accumulation of degraded DQ-BSA even in the presence
of cytochalasin B. We speculate that serine proteases
associated with caveoli on the surface of the fibroblasts,
i.e., tissue plasminogen activator and plasminogen
bound to the annexin II heterotetramer and urokinase
plasminogen activator bound to its receptor (for discussion, see [61]), may degrade DQ-BSA locally. The degraded DQ-BSA may not accumulate extracellularly,
but may be endocytosed [62], where it undergoes further degradation in the endolysosomal compartment.
Indeed, the combination of aprotinin and E64d was not
more effective than either inhibitor alone, suggesting
that serine proteases may act upstream of cysteine
proteases in a common pathway. The present study
shows that intracellular digestion of extracellular proteins occurred as living cells migrated through a threedimensional matrix. We therefore hypothesize that an
interaction between intracellular and extracellular
proteolytic pathways facilitates the migration and invasion of cells.
The authors thank Linda Mayernik for assistance in preparation
of figures. We also thank Dr. Michael McCabe and Kim Zukowski for
assistance with the flow cytometry studies. This work was supported
by NIH Grant 56586. The Microscopy and Imaging Resources Laboratory is supported, in part, by Center Grants P30ES06639 from the
National Institute of Environmental Health Sciences and
P30ES22453 from the National Cancer Institute. The Imaging and
Cytometry Facility Core of the EHS Center in Molecular and Cellular Toxicology with Human Applications is supported by Center
Grant P30ES06639 from the National Institutes of Environmental
Health Sciences.
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