(Received for publication, May 12, 1995)
From the
Previous studies have demonstrated that the
Vitronectin is a conformationally labile plasma protein, which
is found in both plasma and the extracellular matrix. A variety of
functions have been described for vitronectin including mediating cell
adhesion, regulating the activity of both thrombin and plasminogen
activator, as well as modulating the membrane attack complex of
complement(3, 4) . The Arg-Gly-Asp (RGD) sequence in
vitronectin can interact with either the Vitronectin has been identified in at least two
conformations(15) . Native vitronectin is found in the plasma
in a monomeric form that binds weakly to
heparin(16, 17) . Treatment of vitronectin with
denaturants, such as urea, results in a multimeric form of vitronectin
with increased affinity for heparin and with an exposed epitope for the
8E6 monoclonal antibody(17, 18) . Such multimeric
forms of vitronectin have been identified in platelet
releasate(18) . Native vitronectin forms complexes with
components of the coagulation and complement cascades. The interaction
of vitronectin with thrombin-serpin complexes, as well as with the
terminal complement complex C5b-9, exposes both the heparin binding
domain and the 8E6 epitope in
vitronectin(15, 19, 20) . Thus, the
interaction of vitronectin with these complexes triggers the
conformational changes seen in the multimeric form of vitronectin. The binding of multimeric vitronectin as well as the ternary complex
of vitronectin-thrombin-antithrombin to the surface of vascular
endothelial cells has been shown to be dependent on the heparin binding
domain of vitronectin(21, 22) . The binding of
vitronectin to the endothelial cell surface is sensitive to
heparatinase and colocalizes with
proteoglycans(21, 22) , suggesting that
vitronectin-containing complexes bind to heparan sulfate proteoglycans
on the cell surface. In fibroblast cell layers, multimeric and native
vitronectin bind to the same site in the cell layer, and localize to
extracellular fibrils (23) . Fibroblasts internalize the
multimeric vitronectin and direct it to lysosomes for
degradation(1) . This endocytic process, but not initial
binding to the matrix, is inhibited by exogenous heparin and dependent
on the interaction of vitronectin with the The present studies
were conducted to determine the intracellular signaling pathways
involved in the endocytosis of vitronectin and to evaluate the relative
contribution of cell surface heparan sulfate proteoglycans and the
To
examine the role of integrins in the regulation of vitronectin
degradation, turnover of
Figure 1:
Effect of PMA and staurosporin on
vitronectin (Vn) degradation. Human fibroblasts were
pretreated with 500 nM PMA, Me
To
examine further the role of protein kinase C in vitronectin
degradation, confluent fibroblast monolayers were pretreated with
increasing doses of the protein kinase C inhibitors staurosporine or
calphostin C for 1 h. Degradation of
Figure 2:
Effect of calphostin C (A) and
staurosporine (B) on
Figure 3:
PMA overcomes the inhibition of
vitronectin degradation by heparin and heparinase (Hep'ase). Confluent fibroblasts were pretreated with 5
units/ml heparinase for 1 h prior to receiving PMA (500 nM) or
vehicle (Me
Figure 4:
Effect of PMA and calphostin C on
vitronectin internalization. Human fibroblasts were pretreated with
either 500 nM PMA or 1 µM calphostin for 1 h and
then incubated with vitronectin (12.5 µg/ml) for 5 h in F-12
containing 0.2% BSA in the presence or absence of 1 mg/ml heparin.
After the cells were fixed and permeabilized, vitronectin was
visualized by indirect immunofluorescence using the 8E6 monoclonal
antibody. A, control monolayers; B, monolayers
incubated with heparin; C, monolayers pretreated with
calphostin; D, monolayers pretreated with PMA and incubated
with heparin; E, monolayers pretreated with calphostin and PMA
and then incubated with heparin. Bar = 20
µM.
Figure 5:
PMA has no effect on inhibition of
vitronectin degradation by RGDS peptides or antibodies to the
Previously we have shown that conformationally altered
vitronectin was cleared from the extracellular matrix by
receptor-mediated endocytosis and subsequently degraded in
lysosomes(1) . The fact that degradation was blocked by
pretreating cell layers with heparinase or by antibodies against the
Binding of
vitronectin to cell surface proteoglycans has been demonstrated on
endothelial cells(21, 22) . Binding of vitronectin
multimers to proteoglycans on the surface of endothelial cells has been
proposed to mediate the internalization and transcytosis of plasma
vitronectin to the subendothelium(22) . Our studies suggest
that, in fibroblast cells, the actual internalization event may only
require the Degradation of vitronectin was sensitive to inhibitors of protein
kinase C. The doses of calphostin C used to block vitronectin
degradation were within the range of doses used in earlier studies on
fibroblasts, which have documented a role for protein kinase C in cell
spreading (31, 32) and matrix assembly(33) .
Indirect immunofluorescence showed that both calphostin C and
staurosporine blocked degradation by preventing internalization of
vitronectin. The initial binding of vitronectin to the cell layers was
not inhibited by either drug. PMA was able to increase base-line
degradation of vitronectin. PMA was also able to overcome the
inhibition of vitronectin degradation in the presence of heparin and
heparinase. PMA, staurosporine, and calphostin C appear to prevent
vitronectin degradation by regulating vitronectin internalization,
rather than by regulating the flow of vesicular traffic within the
cells. The absence of vitronectin-containing endocytic vesicles in the
presence of calphostin C and staurosporine and the return of endocytic
vesicles with PMA in heparin-blocked cells is consistent with the idea
that the protein kinase C signal regulates internalization. The
ability of PMA to activate the
Our study presents the first evidence that signaling pathways from
cell surface proteoglycans may regulate integrin function. The
inhibition of
integrin receptor functions in the
endocytosis and degradation of matrix-bound vitronectin by human skin
fibroblasts (Panetti, T. S., and McKeown-Longo, P. J.(1993) J.
Biol. Chem. 268, 11988-11993; Panetti, T. S., and
McKeown-Longo, P. J.(1993) J. Biol. Chem. 268,
11492-11495). These earlier studies demonstrated that vitronectin
degradation was inhibited by either antibodies to the
integrin or exogenous heparin, suggesting that both integrin
receptors and cell surface heparan sulfate proteoglycans are involved
in the endocytosis and degradation of vitronectin. The present study
was done to define intracellular signaling pathways involved in
endocytosis of vitronectin and to evaluate the relative contribution of
cell surface heparan sulfate proteoglycans and the
integrin in the activation of these
signaling pathways. The addition of the phorbol ester phorbol
12-myristate 13-acetate (PMA), a protein kinase C activator, to
monolayers of human skin fibroblasts, increased vitronectin
degradation. Staurosporine and calphostin C, inhibitors of protein
kinase C, blocked internalization and subsequent degradation of
vitronectin, while KT5720, an inhibitor of protein kinase A, had no
effect on the degradation of vitronectin. PMA was also able to reverse
the inhibition of vitronectin degradation seen when cells were
pretreated with heparinase or incubated with exogenous heparin. In
contrast, the inhibitory effect of either RGD peptides or
anti-
antibodies on vitronectin
degradation were not overcome by the addition of PMA. These data
suggest that the internalization of vitronectin from the matrix is
mediated by the
integrin following
activation of protein kinase C.
or the
integrin receptor on
the cell surface to mediate cell adhesion(5, 6) .
Vitronectin is also the primary binding site for plasminogen activator
inhibitor, Type I (PAI-1) (
)in the extracellular
matrix(7, 8, 9) . PAI-1 bound to vitronectin
can inhibit both plasminogen activator (10, 11) as
well as thrombin(12, 13, 14) , suggesting
that matrix vitronectin may be important in the regulation of
hemostasis.
integrin receptor(1, 2) , suggesting that
endocytosis of vitronectin requires the interaction of vitronectin with
both the
integrin and a species of
heparan sulfate proteoglycan. Binding to the extracellular matrix is a
prerequisite for internalization as multimeric vitronectin does not
appear to be degraded from the fluid phase(1, 23) .
Native vitronectin is not internalized and degraded but remains bound
in the native form (23) to the extracellular
matrix(1, 23) . Addition of
-thrombin, which is
known to alter the conformation of native vitronectin, triggers the
degradation of native vitronectin by the cells(1) . The fact
that degradation requires the exposure of vitronectin's heparin
binding domain and that degradation is inhibited by exogenous heparin
is consistent with the hypothesis that heparan sulfate proteoglycans
are involved in the endocytosis of vitronectin.
integrin in the activation of these
signaling pathways. Our data suggest that vitronectin internalization
is mediated by the
integrin
receptor, following a proteoglycan-dependent activation of protein
kinase C.
Cell Culture
Human foreskin fibroblasts were a
gift from Dr. Lynn Allen-Hoffmann (University of Wisconsin, Madison,
WI). The cells were cultured in Ham's F-12 nutrient medium (F-12)
(Life Technologies) supplemented with 10% fetal bovine serum (HyClone,
Logan, UT), penicillin (100 units/ml), and streptomycin (100
µg/ml). Fibroblasts were plated in T-75 flasks (Becton Dickinson
Laboratories, Lincoln Park, NJ) at 5 10
cells/flask
and reached confluence in 5-7 days. Experiments were done on
cells between passages 4 and 12. For experiments, fibroblasts were
grown to confluence in 12- or 24-well plates. After the cells achieved
confluence, fresh ascorbic acid (50 µg/ml) was added to the medium
daily for three to four days prior to use in experiments. The addition
of ascorbate was found to increase the binding of vitronectin to the
cell layer.
Purification and Iodination of
Vitronectin
Conformationally altered vitronectin was purified
from human plasma by heparin affinity chromatography according to the
method of Yatohgo et al.(24) as described
previously(1) . Conformationally altered vitronectin (400
µg) was labeled with NaI (DuPont NEN) using either
chloramine T or lactoperoxidase and glucose oxidase (Sigma) as
described previously(1, 2) . Iodinated vitronectin was
stabilized with 1% bovine serum albumin (BSA) and 0.1 mM
phenylmethylsulfonyl fluoride, dialyzed against phosphate-buffered
saline (PBS), and frozen at -80 °C until use. Integrity of
the labeled protein was assessed by gel electrophoresis and
autoradiography.
Binding of Vitronectin to Fibroblast
Monolayers
Monolayers of human skin fibroblasts were incubated
at 37 °C with F-12 containing 0.2% BSA and 620 ng/ml I-vitronectin. To measure bound vitronectin, medium
containing labeled protein was removed, cultures were rinsed three
times with PBS, and the monolayer was solubilized in 1 N NaOH.
Radioactivity was determined by
scintillation counting.
Degradation of Vitronectin by Fibroblasts
Cells
were incubated at 37 °C with F-12 containing 0.2% BSA and 620 ng/ml I-vitronectin. Degradation of
I-vitronectin
by the cultures was monitored by the appearance of radioactivity in the
medium that was soluble in 10% trichloroacetic acid. To estimate
background (i.e. cell-independent) degradation, blank culture
dishes were preincubated with complete medium (F-12, 10% fetal bovine
serum) for 1 h at 37 °C, rinsed, and subjected to the same
procedure as above. Vitronectin degradation in the absence of cells was
less than 1% over the time course of the experiment. Background
degradation was subtracted from total trichloroacetic acid-soluble
radioactivity. Radioactivity was determined in a
counter.
I-vitronectin by the cell layers
was measured in the presence of RGDS or RGES peptides (Sigma) or a
monoclonal antibody against the
integrin receptor, P1F6 (Life Technologies, Inc.). To determine
the role of glycosaminoglycans in vitronectin metabolism, degradation
of
I-vitronectin was examined in the presence of soluble
heparin (Sigma). In other experiments, heparan sulfate was removed from
the cell surface by pretreatment with 5 units/ml heparinase I
(heparinase) (Sigma), for 1 h. These enzyme amounts were similar to
those used earlier to demonstrate the binding of
vitronectin-thrombin-anti-thrombin III complexes to endothelial cell
surface proteoglycans(21) . Fresh enzyme was added with the
radiolabeled vitronectin and remained in the culture medium for the
duration of the experiment. The integrity of
I-vitronectin in the culture medium in the presence of
heparinase was verified by SDS-polyacrylamide gel electrophoresis and
autoradiography. All experiments were done at least three times. Data
presented are from one representative experiment done in triplicate.
Modulators of Protein Kinase
The protein kinase C
inhibitors calphostin C and staurosporine as well as the protein kinase
A inhibitor KT5720 (LC Laboratories, Woburn, MA) were solubilized in
dimethyl sulfoxide (MeSO). The stock solution of inhibitors
was diluted in F-12 with 0.2% BSA and incubated with the fibroblast
monolayers for 1 h before the addition of PMA or
I-vitronectin. Calphostin C was light-activated according
to the protocol of Bruns et al.(25) . The protein
kinase C activator, PMA, was solubilized in Me
SO, and
diluted to a working concentration of between 50 and 500 nM in
F-12 with 0.2% BSA.
Indirect Immunofluorescence
Vitronectin was
localized within cell monolayers by indirect immunofluorescence. Cell
layers were pretreated with PMA, calphostin C, or staurosporine 1 h
prior to addition of exogenous vitronectin (12.5 µg/ml). The
monolayers were rinsed with PBS and fixed with 3% paraformaldehyde.
Intracellular vitronectin was visualized by treating fixed cells with
-20 °C acetone for 10 min. Cell layers were incubated with
hybridoma medium (diluted 1:10 in PBS), which contained a monoclonal
antibody (8E6) directed against human vitronectin (generous gift from
Dr. Deane F. Mosher, University of Wisconsin, Madison, WI). The
secondary antibody was rhodamine isothiocyanate-conjugated goat
anti-mouse IgG (Cappel, Organon Teknika Corp., Durham, NC). Control
studies using no primary antibody were negative. Samples were viewed
using a Nikon Microphot microscope equipped with epifluorescence.
Vitronectin Degradation Is Modulated by Effectors of
Protein Kinase C
Previously we have demonstrated that
conformationally altered, multimeric vitronectin was internalized from
the extracellular matrix by receptor-mediated endocytosis and
subsequently degraded in lysosomes (1) . In these studies
soluble heparin, RGD peptides, and antibodies against the integrin were all shown to block the internalization and
degradation of vitronectin, without affecting the initial binding of
vitronectin to the extracellular matrix(1, 2) .
Because previous studies have shown that the
subunit
of the vitronectin receptor may be a substrate for protein kinase C (26, 27) , we asked whether protein kinase C may be
part of the intracellular signaling pathways involved in the regulation
of vitronectin endocytosis. As shown in Fig. 1, vitronectin
degradation was up-regulated 2.5-fold by a 1-h pretreatment of cells
with the activator of protein kinase C, PMA. PMA concentrations between
50 and 500 nm gave identical results (data not shown). Baseline levels
of vitronectin degradation were decreased about 60% in the presence of
5 mM staurosporin, an inhibitor of protein kinase C.
SO control (C), or 5 mM staurosporine (Staurosp'n) for 1 h. F-12 containing
I-vitronectin and 0.2% BSA was added to the cells for 5
h. Degradation of vitronectin was determined by measuring the
trichloroacetic acid-soluble radioactivity in the culture medium. Data
are expressed as mean ± S.E.
I-vitronectin was
measured over 5 h in the presence of increasing concentrations of the
drugs. Calphostin C inhibited vitronectin degradation in a
dose-dependent manner, with half-maximal inhibition occurring at 0.05
µM (Fig. 2A). Staurosporine also inhibited
vitronectin degradation (Fig. 2B). Neither
staurosporine nor calphostin C inhibited vitronectin binding to the
cell layer. The protein kinase A inhibitor, KT5720, had no effect on
vitronectin degradation (data not shown). These data demonstrate that
vitronectin degradation is protein kinase C-dependent.
I-vitronectin binding and
degradation. Confluent fibroblasts were incubated with increasing
concentrations of calphostin C, staurosporine, or vehicle
(Me
SO) for 1 h. F-12 containing
I-vitronectin, 0.2% BSA, and increasing concentrations of
protein kinase C inhibitors was added to the cells. After 5 h, the
trichloroacetic acid-soluble radioactivity in the culture medium was
determined as an index of degradation. To measure vitronectin binding,
the cell layers were rinsed and solubilized in 1 N NaOH.
Radioactivity was determined by
scintillation. Data are expressed
as percent of control, where binding and degradation in the absence of
drugs were set at 100%.
PMA Overcomes the Inhibition of Vitronectin Degradation
by Heparin and Heparinase
Our earlier studies have shown that
degradation of vitronectin is blocked by exogenous heparin, suggesting
a role for cell surface proteoglycans in the internalization of
vitronectin. These earlier observations, coupled with the ability of
PKC effectors to regulate vitronectin degradation, suggest that one
possible mechanism by which proteoglycans and
integrin could interact to mediate
internalization of vitronectin might be through a
proteoglycan-dependent activation of protein kinase C. We tested
whether the inhibition of vitronectin degradation by exogenous heparin
might be overcome with the addition of PMA. To block vitronectin
binding to cell surface proteoglycans, cell monolayers were either
incubated with exogenous heparin or glycosaminoglycans were digested
with heparinase as described under ``Materials and Methods.''
Heparinase-treated cell layers and non-digested cell layers were
pretreated with PMA for 1 h. The monolayers were then incubated with
I-vitronectin for 5 h in the presence of heparinase or
soluble heparin, and degradation was determined. As demonstrated
previously(1) , heparin inhibited the degradation of
vitronectin to trichloroacetic acid-soluble radioactivity (Fig. 3) but did not affect the binding of vitronectin to cell
layers (data not shown). Heparinase also blocked the degradation of
vitronectin (Fig. 3) but did not affect binding to the cell
layers (data not shown), confirming the role of proteoglycans in the
degradation of vitronectin. PMA was able to reverse the inhibition of
vitronectin degradation seen in the presence of heparin or in
heparinase-treated cells (Fig. 3). The PMA-dependent increase in
vitronectin degradation on heparinase-treated cells could be blocked
using staurosporine (data not shown). The ability of staurosporine to
block the PMA reversal of heparinase treatment demonstrates that a
protein kinase C signaling event generated by PMA triggers vitronectin
degradation.
SO). After a 1-h treatment with PMA, F-12
containing
I-vitronectin and 0.2% BSA was added to
monolayers in the presence of heparin (100 µg/ml) or heparinase (5
units/ml). After 5 h, the trichloroacetic acid-soluble radioactivity in
the culture medium was measured as an index of degradation. Data are
expressed as mean ± S.E.
Protein Kinase C Regulates Vitronectin
Internalization
Indirect immunofluorescence was used to localize
vitronectin in cell layers treated with various effectors of protein
kinase C. In control cells, vitronectin was localized to intracellular
vesicles (Fig. 4A), and localization within vesicles
was blocked by exogenous heparin (Fig. 4B). Cells
treated with calphostin C showed little vesicular staining (Fig. 4C), consistent with data showing that
staurosporine blocked vitronectin degradation (Fig. 2). The
addition of PMA to heparin-treated cells resulted in a reappearance of
vitronectin within vesicular structures (Fig. 4D),
indicating that the PMA-dependent increase in vitronectin degradation
seen in heparin-treated cells (Fig. 3) results from the ability
of PMA to trigger vitronectin internalization. The PMA-induced increase
in vitronectin internalization was blocked by calphostin C (Fig. 4E). Identical results were obtained when
staurosporine was used to inhibit protein kinase C (data not shown).
These data indicate that effectors of protein kinase C modulate
vitronectin degradation by regulating the internalization of
vitronectin. These studies demonstrate that under conditions where
protein kinase C activity is increased, vitronectin degradation does
not require interaction of vitronectin with cell surface heparan
sulfate proteoglycans.
PMA Has No Effect on Inhibition of Vitronectin
Degradation by RGDS Peptides or Antibodies to the
To demonstrate that vitronectin binding to the
Integrin
Receptor
vitronectin receptor was required
for vitronectin degradation, we examined the effect of PMA on
inhibition of vitronectin degradation by peptides and antibodies to the
vitronectin receptor. Confluent fibroblasts were pretreated with the
phorbol ester PMA for 1 h, and then vitronectin degradation was
examined in the presence of RGDS peptides or antibodies to the
integrin receptor. RGDS peptides and
antibodies to the
vitronectin
receptor inhibited vitronectin degradation by 80-85% (Fig. 5). The control peptide RGES had no effect on vitronectin
degradation (data not shown). PMA treatment had no effect on the
inhibition of vitronectin degradation by the antibodies or peptides.
The inability of PMA to overcome the inhibition of vitronectin
degradation by RGDS and anti-
antibodies suggests that the binding of vitronectin to the
integrin receptor is required for
internalization and degradation.
integrin receptor. Human fibroblasts
were pretreated with PMA (500 nM) or vehicle
(Me
SO) for 1 h. F-12 containing
I-vitronectin
and 0.2% BSA was added to the cells for 3 h in the presence of RGDS
peptides or antibodies to the
integrin receptor (P1F6). Degradation of vitronectin was
determined by measuring the trichloroacetic acid-soluble radioactivity
in the culture medium. Data are expressed as mean ±
S.E.
integrin indicated that the binding
of vitronectin to cell surface heparan sulfate proteoglycans as well as
the
integrin was required for
vitronectin's degradation. Localization of vitronectin by
indirect immunofluorescence indicated that soluble heparin blocked
vitronectin degradation by preventing the internalization of
vitronectin into intracellular vesicles. These data suggest that the
binding of vitronectin to both heparan sulfate proteoglycans as well as
the
integrin trigger
vitronectin's internalization by the cell.
integrin. The addition
of PMA to the cells could substitute for the proteoglycan binding
event, but not the integrin binding event. These findings are
consistent with a mechanism by which the proteoglycan acts as a
signaling molecule triggering the
-mediated internalization of
vitronectin. Recent studies have suggested that the
and
integrin receptors may function in the internalization of
adenovirus (28, 29) and fibrinogen(30) .
-dependent internalization of
vitronectin suggests that
function
can be regulated by protein kinase C. Whether PMA is triggering the
internalization of vitronectin bound to
or whether PMA is affecting the affinity of the
for vitronectin has not yet been
determined. Protein kinase C activation has been shown to increase the
rate of internalization of the transferrin receptor (34) and to
increase the formation of clathrin coated vesicles in Schizosaccharomyces pombe(35) , suggesting that
protein kinase C is involved in the up-regulation of the endocytic
process. Alternatively, PMA has been shown to alter the binding
properties of several integrins for their ligands, suggesting that
protein kinase C may be a component of the ``inside-out''
signaling pathways which affect the interaction of integrins with their
ligands (reviewed in (36) and (37) ). PMA-dependent
affinity modulation of the
(38) ,
(39) , and
(40) has been described. The
specific physiological mediators that activate protein kinase
C-dependent integrin modulation are not well understood. Thrombin (41) and ligation of T-cell receptor (42) can activate
integrin receptors on platelets and leukocytes. Activation of both the
and the
can also be stimulated by phorbol
esters(43, 44) , indicating that protein kinase C is
part of the signaling pathway between transmembrane signaling
receptors(45, 46) . Recent studies have suggested that
the
integrin receptor may regulate
the phagocytic function of the
receptor via a protein kinase C-dependent pathway(47) .
-dependent
internalization of vitronectin by both exogenous heparin as well as
heparinase treatment suggests that internalization requires the
interaction of vitronectin with heparan sulfate proteoglycans on the
cell surface. Previous studies have suggested that heparan sulfate
proteoglycans may transduce signals from the extracellular matrix that
promote the formation of focal contacts during cell adhesion to
fibronectin (48, 49, 50) . The fact that PMA
can subserve the vitronectin-proteoglycan binding suggests that the
interaction of vitronectin with heparan sulfate proteoglycans may
contribute to an increase in protein kinase C activity necessary to
trigger
-dependent endocytosis.
Regulation of vitronectin endocytosis may be important in the control
of hemostasis. Native vitronectin bound to the extracellular matrix in
a non-wound environment may mediate cell adhesion through interaction
with cell surface integrin receptors. During tissue injury, the
interaction of native vitronectin with PAI-1 and thrombin would be
expected to result in a ternary complex that is multivalent for both
integrin and proteoglycan binding. The interaction of such a
multivalent ligand with cell surface proteoglycans as well as the
integrin may result in the clearance
of vitronectin thrombin-serpin complexes from the pericellular space.
Further studies are needed to delineate the role of heparan sulfate
proteoglycans in receptor-mediated endocytosis of vitronectin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.