Download Localization of Collagenase at the Basal Plasma Membrane of a

[CANCER RESEARCH 50, 6995-7002, November I. 1990]
Localization of Collagenase at the Basal Plasma Membrane of a Human Pancreatic
Carcinoma Cell Line1
Ute M. Moll, Bernard Lane, Stanley Zucker, Ko Suzuki, and Hideaki Nagase
Department of Pathology, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, New York 11794 [U. M. M., B. L.J; Department of
Medicine and Research, Veterans Administration Medical Center, Northport, New York 11786 [J. Z.]; and Department of Biochemistry, University of Kansas Medical
Center, Kansas City, Kansas 66103 fK. S., H. N.]
ABSTRACT
We have recently presented biochemical evidence for collagen and
gelatin degrading activities associated with plasma membranes of various
human cancer cell lines. In this report we describe the localization of
interstitial collagenase at the basal plasma membrane of the human
pancreatic cancer cell line RWP-I, using immunofluorescence and ultrastructural immunogold labeling techniques. Collagenase was expressed
on the extracellular face of the plasma membrane. Furthermore, the
immunogold labeling was concentrated on the long, finger-like microvillous projections typically seen on the basal cell surface, while the short,
brush-like projections characteristic of the apical cell surface were unlabeled. When the cytoplasmic face of the membrane was made accessi
ble, the number of reactive sites increased markedly, indicating a high
concentration of enzyme at the inner surface of the plasma membrane.
When plasma membrane fractions of RWP-I cells were prepared by
differential centrifugation, high salt washes virtually failed to extract
collagenase activity from the membrane, while detergent extraction with
n-octyl glucoside, a detergent used in the purification of integral mem
brane proteins, yielded soluble collagenase activity. When detergent
extracted membrane fractions were passed over an anticollagenase immunoaffmity column, collagenase was specifically bound, as demonstrated
by the TCAand K " degradation of type I collagen by the bound material.
Gelatinolytic activity did not bind to the column. Furthermore, immunoprecipitation of 125I-labeled detergent extracts of tumor membranes
yielded a single M, 55,000 band consistent with the zymogen form of the
connective tissue collagenase. These morphological and biochemical find
ings suggest that collagenase is a tightly associated component of the
basal plasma membrane, where it occupies a strategic location for direc
tional proteolysis during cell migration and invasion.
as an important mechanism in their invasiveness (10, 11).
Collagenase, synthesized by normal connective tissue cells in
culture, has been purified and its action on collagen types I, II,
III (12, 13) and X (14) has been characterized. The enzyme has
been shown to be a prototype of a secretory protein. It is
synthesized as preprocollagenase, processed to a proenzyme
within microsomal membranes and immediately secreted with
out intracellular storage (15).
In contrast, we described a metalloproteinase activity in the
total cell extract of highly metastatic mouse melanoma cells
that digests collagen type I and type IV and gelatin (16).
Furthermore, the collagenolytic activity of human small cell
carcinoma cells was highly enriched in the plasma membrane
fraction (17). These observations suggest the existence of cell
surface bound collagenase in human cancer cells and propose a
proteolytic function associated with the plasma membrane.
Their sanctuary status in the plasma membrane places them
optimally for the effective destruction of substratum in a con
trolled, directional way. This also allows the local concentration
of the enzyme to be maintained and may prevent it from binding
to proteinase inhibitors present in the extracellular milieu (18).
In this study we have used an ultrastructural and immunochemical approach to identify interstitial collagenase in RWP-I cells.
Tumor collagenase was predominantly localized at basal type
microvilli of plasma membrane where cells contact the substra
tum, suggesting that directional collagenolysis occurs at the
cell-stroma interface.
MATERIALS AND METHODS
INTRODUCTION
Cell Line
Dynamic interactions between cells and their extracellular
milieu play a crucial role in neoplastic processes of tumor
invasion and metastasis, as well as in normal phenomena such
as tissue remodeling during embryogenesis and inflammatory
responses. Basement membrane, extracellular matrix, and con
nective tissue fibrillary proteins present a natural barrier to the
migration of cancer cells. Much of our insight into the proteolytic degradation of the extracellular components during this
process comes from in vitro studies of malignant cells, which
serve as a readily available model for cell migration in general.
A spectrum of proteinases, such as interstitial collagenase (1,
2), type IV collagen degrading enzymes (3), gelatinase, serine
proteinases (4), cathepsin B (5), and plasminogen activator (6)
have been implicated in cancer invasion (7, 8).
Interstitial type collagen is the major structural protein of all
tissues. Tumor cells can penetrate dense stroma only if a
collagenase degrades the bundles of collagen (for review see
Ref. 9) and tumor cell derived collagenases have been proposed
The human RWP-I pancreatic cancer cell line, kindly provided by
Dr. D. L. Dexter (Department of Medicine, Roger Williams General
Hospital, Providence, RI), has previously been characterized (19). For
this study only cell cultures were used. Aliquots of these cells were
injected into athymic mice and gave rise to large s.c. tumors. RPMI
1640 (GIBCO, Grand Island, NY) and 5-10% fetal calf serum (Flow
Laboratories, Walkersville, MD) in 95% air and 5% CO2 at 37°Cwas
Received 10/13/89; accepted 7/16/90.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by Grant AR39189 from the NIH and by the Merit Review Grant
from the Veterans Administration.
2The abbreviations used are SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; anti-pColl, sheep anti-(human rheumatoid synovial
procollagenase) IgG; RAS. unconjugated rabbit anti-(sheep IgG) IgG; RWP-I,
human pancreatic cancer cell line; MMP-3, matrix metalloproteinase-3, synony
mous with stromelysin; APMA, p-aminophenylmercuric acetate; PBS, phosphate
buffered saline.
used.
Antisera
Human procollagenase from the culture medium of rheumatoid
synovial cells was affinity purified by exploiting cross-reactivity with
anti-rabbit collagenase (20). The anti-rabbit collagenase antibody was
directed against purified rabbit synovial fibroblast collagenase and has
been described previously (20). Its monospecificity was established by
double ininnili«
»lit
fusion, immunoprecipitation of '"I-labeled collagen
ase, and immunoblotting analysis. Cross-reactivity with human rheu
matoid synovial procollagenase has been shown (20). The human
antigen extracted on an anti-rabbit collagenase affinity column ran as
a doublet with molecular weights of 52,000 and 55,000 on SDS-PAGE.2
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COLLAGENASE
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The eluted antigen showed activity in a standard collagenase assay after
activation by 4-aminophenyl mercuric acetate. Antiserum was produced
by immunizing a sheep. The IgG fraction (referred to as anti-pColl)
was isolated at a final concentration of 18 mg/ml. Anti-pColl recognizes
interstitial type human collagenase and procollagenase, but not gelatinase and MMP-3/stromelysin as confirmed by immunoblot analysis.
The working dilution was 1:60 (300 Mg/ml).
Immunofluorescence Microscopy
Immunolocalization of collagenase at the light microscopic level was
carried out at room temperature. RWP-I cells were grown on coverslips
to semiconfluency. The adherent cells were washed in PBS and fixed
in 2% formaldehyde for 15 min. Others were washed in 150 ITIMNaCI,
50 mM Tris, 5 ITIMCaCl2, pH 7.5. No difference in staining was noted
between these two groups. Half of the coverslips were permeabilized in
0.25% Triton X-100/PBS for 5 min and the other half were kept in
PBS only. The specimens were then incubated with primary antiserum
(diluted 1:60 in PBS) for 60 min. After thorough washes in PBS,
fluorescein isothiocyanate-conjugated rabbit anti-|sheep IgG (H + L)]
IgG (Cappel, West Chester, PA) was applied (diluted 1:150 in PBS)
for 30 min. Preadsorbed anti-pColl as well as normal sheep serum were
used as controls. The preparations were mounted with Aquamount,
viewed with a Zeiss Axiomat microscope, and photographed with
Kodak TMAX film.
Distribution Analysis of Gold Grains on Plasma Membranes
The distribution of gold grains on the surface of 10 cells was assessed
by image analysis in order to determine whether collagenase sites on
the plasma membrane are concentrated on cell projections. The cells
were selected at random and photographed at X4000 which did not
allow an evaluation of the presence or location of gold particles. All
cells included a nuclear profile. All gold particles on the circumference
of each cell were counted and grouped into sites on cell projections
versus sites on the cell body. Small aggregates of gold grains were scored
as one site and grains had to be directly attached to the membrane to
be counted. Cell processes which were not in continuity with the cell
body were considered part of the cell if the profiles were no further
from the cell than the length of attached processes in the same plane
of section. To normalize the number of grains in each categories, the
perimeters were measured differentially with an electron image analysis
device (Zeiss ZIDAC). Scanning electron micrographs of similar cells
were taken to assess the 3-dimensional structure of the projections. For
this, cells were grown on coverslips, fixed in glutaraldehyde, critically
point dried, and examined with an AMC scanning electron microscope.
Preparation of Plasma Membrane Associated Collagenase
Postnuclear cell membranes were isolated from RWP-I cells using
nitrogen cavitation followed by differential centrifugation as previously
described in Ref. 16 and then subjected to sequential extraction. Ex
trinsic membrane proteins were extracted by 2 M KCI treatment (16).
Immunogold Labeling of Collagenase Sites at Ultrastructural Level
The remaining intrinsic membrane proteins were solubilized by treat
ment of the 100.000 x g pellet with 2.5% n-butyl alcohol (20 min on
Colloidal gold particles (10 nm) coupled to rabbit anti-[sheep IgG
ice) followed by a second 100,000 x g centrifugation. The supernatant
(H + L)] IgG at 40 ^g/ml protein concentration was purchased from
was divided into an upper half (lipid phase) and a lower half (protein
EY Laboratories, Inc., San Mateo, CA. The specificity of the reagent
phase). The pellet was treated with 1% n-octyl glucoside (Sigma Chem
was shown by incubating a nitrocellulose strip impregnated with 1 //)• ical Co., St. Louis, MO) overnight at 4°Cand subjected to a last
sheep IgG with 5 M' gold probe (20 Mg/ml); bovine serum albumin
100,000 x g centrifugation. The extracted proteins from each of the
served as control. After thorough washing, a dark red dot indicated
four supernatants were dialyzed against 25 mM cacodylate/5 mM CaCl2/
reaction.
0.05% Brij 35 (Sigma), pH 7.2. Samples undergoing immunoprecipiRWP-I cells were grown to confluence and harvested by scraping.
tation were concentrated by the addition of solid ammonium sulfate to
Cells were washed twice in culture medium and fixed in 0.1% glutaral60% saturation and dialyzed against the above buffer. Aliquots of pellets
dehyde/PBS for 40 min at room temperature. Aliquot portions (10 M') and supernatants were activated with trypsin, then inactivated with
of cells were packed into tubes, washed twice in PBS, and incubated in soybean trypsin inhibitor and assayed for 'H-collagenolysis using 2 Mg
of labeled substrate at 27'C for 18 h as described in Ref. 17. RWP-I
10% normal horse serum for 30 min at room temperature. Pellets were
then incubated with 70 i.l native or preabsorbed anti-pColl (300 Mg/ml) conditioned medium was collected after 48 h from serum-free cell
and rotated slowly for 18 h at 4°C.This gentle manipulation led to
cultures containing 1 x IO7cells/flask and spun at 770 x g for 10 min
partial detachment of the plasma membrane and exposure of the
followed by 50,000 x g for I h. The proteins in the supernatant were
cytoplasmic face in 30-60% of the cells. Nonspecifically bound IgG
precipitated with solid ammonium sulfate to 60% saturation, then
was removed by washing with PBS 6 times. The specimens were then
resuspended, and dialyzed against the above buffer. Aliquots were
incubated in 70 M'immunogold probe (3 Mg)for 20 h at 4°Cunder slow
activated with trypsin, inactivated with soybean trypsin inhibitor, and
rotation. For blocking controls, the immunogold step was preceded by assayed for collagen degradation, using 2 Mgof 'H-labeled collagen at
100 M' of affinity purified unconjugated rabbit anti-(sheep IgG) IgG
27°Cfor 18 h as described in Ref. 17. The protein concentration of
(2.9 Mg/ml) (referred to as RAS) (Chemicon, El Segundo, CA) for 18 h each extract and of conditioned medium was determined by the fluat 4°C(see controls). A final wash in 6 changes of PBS followed. Pellets
orescamine method (21).
of stained specimens appeared pink while unstained specimens ap
Immunoblotting
peared tan. The pellets were fixed in 3% glutaraldehyde/0.2 M sodium
cacodylate buffer and processed for electron microscopy. Ultrathin
Samples (100 M') of RWP-I whole cell homogenate and sequential
sections were cut, stained with lead citrate and uranyl acetate, and
membrane extracts in various PBS dilutions were applied to nitrocel
examined under a Zeiss EM 10 microscopy.
lulose filters in a slot blot apparatus (Bio-Rad Bio-Dot SF) and the
Controls
Preadsorption of Primary Antibody. Secreted procollagenase from
human rheumatoid synovial fibroblast cultures was purified to a final
concentration of 750 Mg/ml using an anti-(rabbit collagenase) immunoadsorbant column as described in Ref. 15. Anti-pColl (90 Mg) was
incubated with 53 Mgantigen in 70 n\ and diluted in PBS to a final
volume of 300 M' (i.e., anti-pColl 1:60). After overnight incubation at
4°C,the mixture was added to the cells.
membrane was washed several times with PBS; 100 M' nonimmune
rabbit IgG and PBS served as control. The nitrocellulose filters were
soaked in reconstituted 5% (w/v) nonfat dried milk for 40 min. They
they were incubated with anti-pColl diluted 1:125 in PBS overnight at
4°C.After extensive washing, the filters were reacted with donkey anti(sheep IgG) IgG-alkaline phosphatase conjugate (Sigma) 1:1000 for 1
h at 37°Cand the blot was developed in 0.1 M Tris-HCl, pH 9.5/0.1 M
NaCl/5 mM MgClj containing nitro blue tetrazolium (Sigma; 0.33 mg/
ml) and 5-bromo-4-chloro-3-indolyl phosphate (Sigma; 0.165 mg/ml).
The reaction was terminated by rinsing it in water.
Instead of Primary Antibody. Instead of primary antibody, cells were
incubated overnight in normal sheep serum diluted 1:10 in PBS.
Immunoadsorption of Collagenase
Blocking of Gold Probe. A 100-Ml portion of affinity purified RAS
RWP-I membrane extracts (260 M') containing 0.044 unit (1 unit
was added after incubation with the primary antibody and washed off
degrades 1 Mgof collagen/min) of total collagenase activity were applied
before the immunogold probe was applied.
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COLLAGENASE
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to a column of Affi-Gel 10 (Bio-Rad, Richmond, VA) coupled with
anti-pColl IgG (1.5 ml), which was equilibrated with 50 IÃŽIM
Tris-HCl/
0.15 M NaCl/10 mM Ca2V0.05% Brij 35/0.02% NaN3, pH 7.5. Spe
cifically bound procollagenase was eluted with the above buffer con
taining 6 M urea and fractions of 200 n\ were collected. The fractions
were then applied to spin columns to remove urea as described in Ref.
22. Each fraction was assayed for the degradation of 14C-acetylated type
I collagen and '4C-acetylated gelatin introducing 3200 cpm per tube
(22) in the presence of 1 mM APMA and 0.04 ng of MMP-3/stromelysin for 24 h at 24°Cand 37°C,respectively. MMP-3 is required for
the maximal activation of procollagenase (23, 24).
For the analysis of type I collagen degradation, 30 ¡Aof the peak
collagenase fraction were incubated with 15 /ig type I collagen in the
presence of 1 mM APMA/0.04 ng MMP-3 in a total volume of 50 n\
at 28°Cfor 48 h as described above and then analyzed on SDS-PAGE
(25).
Immunoprecipitation of Collagenase
One hundred /¿'
of concentrated n-octyl glucoside membrane extracts
(104 /¿g)were radioiodinated by using lodo-Gen ( 1,3,4,6-tetrachloro3a,6a-diphenylglycouril) (26) and free I25I ions were removed by spin
columns (22). The sample was then subjected to immunoprecipitation
by using 5 /¿I
of anti-pColl IgG in a total volume of 400 ß\according
to the method of Nagase et al. (15). Antigen-antibody complexes were
dissociated by boiling in SDS-PAGE sample buffer and the antigen was
analyzed by SDS-PAGE and subsequent autoradiography.
RESULTS
Immunolocalization of Membrane Bound Tumor Collagenase.
To characterize the extent and overall distribution of collagen
ase, RWP-I monolayer cultures were studied with an immunofluorescence technique by using anti-pColl. A discrete punctate
fluorescence was detected on approximately 50% of the cells,
indicating a moderate level of expression of collagenase on the
extracellular surface of the plasma membrane (Fig. \b). In
contrast, when cells were permeabilized with Triton X-100 prior
to incubation with the immunoprobe, the staining intensity
increased markedly (Fig. \c). The staining pattern was predom
inantly diffuse with some focal densities. This increase in stain
ing intensity was due to intracytoplasmic sites of enzyme as
well as sites on the cell surface. Preabsorption of the antibody
with the affinity purified immunogen (Fig. la) as well as re
placement of anti-pColl by normal sheep serum (data not
shown) completely abolished staining of permeabilized and
nonpermeabilized cells.
Polarized Localization of Collagenase in RWP-I Cells. At the
electron microscopic level, collagenase immunoreactivity was
detected at the cell surface in a nonrandom distribution. Al
though there was considerable variation between individual
cells, approximately 90% of all cell cross-sections were labeled
with gold grains. The short plump microvilli on the apical
surface were virtually devoid of gold labeling (Fig. 2A), while
the long, thin finger-like processes, which are typical for the
cell base, showed a moderate degree of collagenase reactivity
(Figs. 2a and 3a). The number of reactive sites increased greatly
when both the extracellular and the cytoplasmic face of the
plasma membrane became accessible to the immunoprobe (Fig.
3). The detached fragments of plasma membrane were heavily
labeled with gold grains in an irregular fashion. These fragments
also showed a distinct morphology. They usually formed long
convoluted strings with many small uniform ruffles decorating
the main thread (Fig. 36 and control Fig. 4). Labeling was
associated with these structures. To show that these peculiar
string configurations were plasma membrane rather than rough
endoplasmic reticulum or nuclear membrane, we have the fol
lowing evidence, (a) Some cells showed very early stages of
plasma membrane detachment, with loops peeling off (Fig. 4a).
Their morphology was identical to the detached membrane
fractions (Fig. 3b); (b) previously, we demonstrated that the
100,000 x g pellet of RWP-I cell homogenates contains a
membrane band which is highly enriched in collagenase activity
and exhibits the same ruffled profile as seen in this study (see
Ref. 27, Fig. 2, lower right). Other cell organelles such as
nuclei, mitochondria, and various vesicular structures showed
only occasional sparse labeling. No dense core granules which
might contain collagenase were noted.
Three types of controls demonstrated the specificity of the
immunolabeling at the electron microscopic level. Preabsorp
tion of anti-pColl with excess of purified human synovial pro
collagenase (Fig. 4a, b) and substitution of anti-pColl with
normal sheep serum (data not shown) completely abolished
immunogold labeling. Preincubation of the anti-pColl treated
specimens with unconjugated secondary antibody (RAS)
blocked the subsequent immunogold labeling virtually com
pletely (Fig. 4c). Immunolabeling of intact cells was abolished
as well (data not shown).
Quantitative Distribution Analysis of Collagenase Sites on
Intact Plasma Membrane. A comparison of the number of
particles per unit area of membrane on cell body versus projec
tions gave a measure of the differential distribution on the
Fig. 1. Plasma membrane associated colla
genase visualized by ¡mmunofluorescence. (a)
Preabsorption control of Triton X-100 per
meabilized RWP-I cells. (A) RWP-I cells with
out permeabilization. Collagenase is expressed
at the cell surface as demonstrated by a punc
tate staining pattern. (<•)
RWP-I cells after
Triton X-100 permeabilization. A marked in
crease in immunoreactive collagenase sites is
seen which is in part due to newly accessible
enzyme located at the cytoplasmic face of the
plasma membrane as demonstrated in Fig. 3h.
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COLLAGENASE
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Fig. 2. (a) Basal plasma membrane projections, which are long and finger-like, are decorated with anti-pColl immunogold complexes at the extracellular face, (hi
Apical projections, which are short and brush-like, are unlabeled; x 24.000.
Fig. 3. (a) Membrane on cell projections
(small arrow) and on the cell body (big arrow)
are immunolabeled with anti-pColl. (6) De
tached plasma membrane fragments are heav
ily labeled with anti-pColl immunogold com
plexes, indicating a much larger number of
enzyme sites at the cytoplasmic face. Note the
irregular distribution and the formation of ruf
fles (arrow) as shown in Fig. 4; x 24,000.
'•
**
*
intact cell surface (see Fig. 3a). We assume that a single gold
particle or an aggregate of gold particles attached to the mem
brane represents one collagenase site. Each pair of numbers in
Table 1 was generated from the same cross-section of a cell.
The total perimeter of cell body versus projecting membrane
profiles on each cell circumference was measured and the data
normalized. Table 1 shows that the plasma membrane of pro
jections exhibited twice as many collagenase sites than smooth
areas.
Extraction of Collagenolytic Activity from Detergent Treated
Plasma Membranes. Trypsin activated RWP-I whole cell homogenate, postnuclear preparations, and plasma membrane
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COLLAGENASE IN HUMAN PANCREATIC CARCINOMA
'
Fig. 4. RWP-I controls, (a and A) Preabsorption of the primary antibody with affinity purified immunogen. (a) Some cells show very early stages of plasma
membrane detachment with formation of the typical ruffled structures, (b) Large convoluted strands of plasma membrane show complete blockage of immunolabeling.
(c) Preabsorption of the antigen-antibody complex with unconjugated anti-(sheep IgG) prior to anti-(sheep lgG)-gold conjugate: x 24.000.
enriched fractions degraded 10, 40, and 1560 ng of type I [3H]
collagen/mg protein/ 18-h incubation, respectively. Treatment
with buffered 150 HIMNaCl, pH 7.5, followed by 2 M KC1 for
30 min resulted in no significant loss of membrane collagenolytic activity. In contrast, treatment with w-octyl glucoside re
sulted in extraction of 96% of the final collagenolytic activity
(1115 ng substrate degraded/mg protein/18 h). RWP-I condi
tioned medium also contained secreted collagenolytic activity;
48-h conditioned medium degraded 41 ng of [•'H]collagen/mg
protein/18-h incubation following trypsin activation. Ammo
nium sulfate precipitated (0-60% saturated) conditioned me
dium digested 244 ng of ['H]collagen/mg protein/18 h follow
ing trypsin activation.
Immunoblotting of Detergent Extracted Plasma Membrane
Fractions. The distribution of immunoreactive collagenase was
followed through each step of the membrane extraction se
quence. Aliquots of whole cell homogenate contained highly
concentrated collagenase-like material which could only be
extracted by the final w-octyl glucoside step (Fig. 5). However,
high salt extraction alone, which removes loosely associated
proteins, or butyl alcohol extraction, which removes certain
intrinsic membrane proteins yielded very little immunoreactive
material. These results suggest that collagenase is a tightly
associated membrane protein.
Selective Binding of Membrane Collagenase to an Immunoaffinity Column. Detergent extracted RWP-I membrane fractions,
which selectively bound to an anti-pColl affinity column, were
examined for collagenolytic and gelatinolytic activity (Fig. 6).
The degradation of 3H-type I collagen and pHjgelatin after
APMA and stromelysin activation at 27°Cand 37°C,respec
tively, across the column showed that collagenase activity was
eluted from the affinity column with 6 M urea, while gelatinase
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COLLAGENASE
IN HUMAN PANCREATIC CARCINOMA
did not bind to the column and was recovered in the flow
through fraction. The elution profile of collagenase seems to be
composed of two individual peaks, probably due to antibodypopulations of lower and higher affinity. When peak fractions
of the eluate were incubated with type I collagen at 27°Cand
then analyzed by SDS-PAGE, the TCA and the TCB products
were formed. No other degradation products were found (Fig.
6, inset). These products are considered specific for collagenase.
Conversely, the peak of the gelatinase fraction produced mul
tiple smaller fragments after incubation with gelatin at 37°C
(data not shown).
Immunoprecipitation of Collagenase. To identify the species
and determine the molecular weight of the membrane bound
collagenase, the detergent extract was radioiodinated and pre
cipitated with anti-pColl. As shown in Fig. 7, a single species
with a molecular weight of about 55,000 is recognized (Fig. 7,
Lane 2). The membrane bound collagenase required activation
cpm
1
2
3
500
oÃ- t
300
§ ñ
o o
O O
10
20
30
Fraction
50
No
60
' 0.2 ml/tube
I
Fig. 6. Purification of membrane collagenase by immunoaffinitv chromatography. Detergent extracted RWP-I membrane preparations were applied to an
anti-pColl immunoaffinity column and the bound collagenase was eluted as
described in "Materials and Methods." Each fraction was assayed for collagen
olytic activity in the presence of 1 mM APMA and 0.04 ng stromelysin at 27"C
for 24 h. Gelatinolytic activity was measured in the presence of 1 mM APMA at
37°Cfor 18 h; 3200 cpm/tuhe were introduced. The activities obtained were
Table 1 Differential distribution of gold grains on plasma membrane of cell
projections versus cell body in intact RH'P-¡cells
within the linear range of the assays. Inset: type I collagen degradation analyzed
by SDS-PAGE. Lane 1. 15 ^g collagen only; Lane 2, 15 ^g collagen incubated
with 0.018 unit of purified human synovial fibroblast collagenase at 27"C for 48
Cell
h (23); Lane 3, 15 /jg collagen incubated with 30 n\ of fraction 48. TCA and TC"
products are formed. Proteins were stained with Coumassie brilliant blue R-250.
ofgrains*54300161222Length'390383292222104143200266311225Projections"No.
ofgrains*8203211416425ISLength'24857318S520538561383692166742
A cluster of gold particles is counted as 1 grain. For definition see Fig. 3a.
bodyCell
no.12345678910No.
kDa
Total no. of grains
Total perimeter*
Grains x 10~3/relative
126
35
length unit
2536
13.8
4608
27.3
' Projection index = Grains on projected membrane/relative length unit
Grains on smooth membrane/relative length unit
* No. of grains on partial perimeter.
' Relative length unit.
wch
but
kcl
but
kcl
gl u
but
glu
but
glu
Fig. 7. Identification of membrane collagenase by immunoprecipitation. '"Ilabeled detergent extract of plasma membrane was immunoprecipitated with antipColl. Lane I, whole plasma membrane extract; Lane 2, a M, 55.000 collagenase
is the only immunoreactive membrane component. Molecular markers are phosphorylase a (M, 94.000). transferrin (M, 77,000), bovine serum albumin (M,
68,000), heavy chain of IgG (A/, 55,000), ovalbumin (A/, 43.000), and carbonic
anhydrase (M, 29.000). kDa. molecular weight in thousands.
Fig. 5. Immunoblot analysis of RVVP-I membrane collagenase. Aliquots of
whole cell homogcnate and of each extraction step were analyzed with anti-pColl.
Only detergent treatment was able to extract a significant amount of collagenase.
Left row. wch. whole cell homogenate. 79 ng; kcl. KCI extracts. 390 and 39 (¿g;
but. butyl alcohol extracts, lipid phase. 18 and 3 i>\i:right row. but. butyl alcohol
extracts, aqueous phase, 89 and 20 ng: glu. n-octyl glucoside extracts, 104. 21,
and 5 ng.
to produce collagenolysis, indicating that the enzyme is present
in a latent form (data not shown). The M, 55,000 species is
consistent in molecular weight with the procollagenase of con
nective tissue (15, 28). Other metalloproteinases, which have
partial homology with collagenase, are not recognized by our
antibody.
DISCUSSION
In the present study we have demonstrated tumor collagenase
at the plasma membrane. The enzyme seems to be primarily
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COLLAGENASE
IN HUMAN PANCREATIC CARCINOMA
present ¡nthe zymogen form and is activated with organomercurials and MMP-3 in vitro, ¡nvivo, other matrix proteinases,
such as the plasminogen activator/plasmin system could func
tion as activators as well as regulators of membrane associated
collagenase (29). Also, based on sequence homologies at the
site of activation between all members of the matrix metalloproteinase family known so far, autoactivation and mutai acti
vation have recently been shown to be important mechanisms
for these enzymes (23, 24, 30). The collagenase molecules are
concentrated at the cytoplasmic face of the membrane with
some degree of expression at the extracellular surface. To our
knowledge, this is the first ultrastructural immunolocalization
of a metalloproteinase. Our studies were possible because tumor
collagenase cross-reacts with an immunoprobe raised against
rheumatoid synovial fibroblast collagenase. Immunoprecipitation indicates that the tumor collagenase is a M, 55,000 mole
cule consistent with the molecular weight of the secreted con
nective tissue procollagenase. Furthermore, the immunological
cross-reactivity as well as the pattern of substrate degradation
suggest a high degree of conservation in the primary structures
between the two molecules, and even possible identity. The fact
that tumor collagenase was extractable from plasma membrane
with detergent, but not with high salt washes, is evidence that
the enzyme is tightly associated with the membrane and not
just loosely adsorbed. When the activity of membrane associ
ated collagenase is compared with secreted collagenase in con
ditioned medium, it appears that the cell membrane represents
a major portion of enzymatic activity in RWP-I cells. However,
differential half-life, activation status, and local inhibitor con
centration ultimately will determine the activity in vivo.
Our morphological data can reflect a number of possible
associations of enzyme and plasma membrane; e.g., (a) the
enzyme may be directly anchored within the lipid bilayer of the
membrane. Phosphatidyl inositol has been described as a covalently linked lipid anchor for a number of membrane associ
ated enzymes (31). (b) The enzyme may undergo transmembranous secretion but be partially or completely captured by a
high affinity membrane bound collagenase receptor. This type
of pathway has been shown to apply to the urokinase-type
plasminogen activator in normal and malignant cells (see Ref.
32 for a brief review). The failure to extract collagenase activity
from plasma membrane preparations with high salt solutions
makes this possibility unlikely but does not entirely exclude it.
(c) Collagenase may be transiently accumulated at the inner
surface of the plasma membrane and subsequently be released
as soluble enzyme via an exocytosis-like secretory pathway. The
ultrastructural image we observe could then be due to enzyme
in transit through the membrane, (d) The expression of colla
genase on the cell surface may be a transient phenomenon that
precedes the shedding of portions of the plasma membrane as
vesicles. Such a pathway has been described for cathepsin Blike cysteine proteinase (33). Also, electron microscopic studies
on invasive carcinoma in vivo suggested destruction of connec
tive tissue by membrane vesicles in the immediate vicinity of
the invading epithelial cells (34, 35). Although we favor the
possibility of a genuine collagenolytic activity of plasma mem
brane at points of contact between cell and substratum, our
data do not definitively distinguish among the above alterna
tives. A combination of mechanisms is also conceivable.
The pathway of collagenase in normal mesenchymal cells has
been well described and appears to be a simple secretory one,
but it is not known whether there is concentration at the cell
surface. In pulse chase experiments of human skin and rabbit
synovial fibroblast cultures, collagenase appears rapidly in the
culture medium within 35 min after synthesis (15, 28). Immunofluorescent collagenase sites are found abundantly within the
connective tissue matrix, whereas the cytoplasm is relatively
free of enzyme (36-38). These findings are consistent with the
secretion and diffusion of soluble enzyme into the matrix.
The notion of proteolytic activity at points of contact has
recently become more important. Urokinase-type plasminogen
activator has been localized to points of local contact in normal
and sarcoma cells (39). Fibronectin degradation by transformed
fibroblasts is confined to sites of contact involving the activation
of an integral membrane proteinase by pp60src which itself is
present at the inner surface of the plasma membrane (40). Our
findings of selective distribution of collagenase at basal type
microvillous projections suggests directional proteolysis via
contact sites at the cell-stroma interface. Electron microscopic
studies of reconstituted basement membranes have demon
strated that tumor cell invasion of extracellular matrix proceeds
by the formation of specialized pseudopodia that form adhesion
contacts and maximize local hydrolysis by membrane bound
proteinases at the interfacing surface area (39). Various human
breast cancer cell lines required direct contact for the degrada
tion of endothelial basement membrane (41). Clearing of sub
strate with release of degradation products of basement mem
brane type IV collagen, laminili and fibronectin occurs only
along the path of tumor cells and beneath cellular processes,
suggesting that it is due to cell surface proteinases rather than
secreted enzymes (42-44).
Collagenase localization studies in tumor cells have been
limited until now. Only light microscopic studies using immunofluorescence have been performed. In head and neck tumors
(45, 46) and in melanoma (38), the intercellular stroma exhibits
bright immunofluorescence when stained for collagenase. In
permeabilized cells, there is a sharp border between cytoplasm
and matrix, thus outlining the silhouette of the tumor cells,
while more than 90% of the tumor cells themselves are negative.
This is consistent with the existence of collagenase domains on
the cell surface, particularly in view of the limited resolution of
immunofluorescence. Our high resolution ultrastructural study
lends further support to this concept.
ACKNOWLEDGMENTS
We thank Edward Drummond and Rikako Suzuki for excellent
technical assistance, and Dr. James P. Quigley for his critical review.
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Localization of Collagenase at the Basal Plasma Membrane of a
Human Pancreatic Carcinoma Cell Line
Ute M. Moll, Bernard Lane, Stanley Zucker, et al.
Cancer Res 1990;50:6995-7002.
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