Department of Dermatology, School of Medicine, SUNY at Stony Brook, Stony Brook, New York 11794-8165
A three-dimensional collagen lattice can provide skin fibroblasts with a cell culture environment
that simulates normal dermis. Such a collagen matrix
environment regulates interstitial collagenase (type I
metalloproteinase [MMP-1], collagenase-1) and collagen receptor 2 subunit mRNA expression in both
unstimulated or platelet-derived growth factor-stimulated dermal fibroblasts (Xu, J., and R.A.F. Clark.
1996. J. Cell Biol. 132:239-249). Here we report that the collagen gel can signal protein kinase C (PKC)-
activation in human dermal fibroblasts. An in vitro kinase assay demonstrated that autophosphorylation of PKC-
immunoprecipitates was markedly increased by a collagen matrix. In contrast, no alteration in PKC-
protein levels or intracellular location was observed. DNA binding activity of nuclear factor
B (NF-
B), a downstream regulatory target of PKC-
, was also increased
by fibroblasts grown in collagen gel. The composition
of the NF-
B/Rel complexes that contained p50, was
not changed. The potential role of PKC-
in collagen gel-induced mRNA expression of collagen receptor
2
subunit and human fibroblast MMP-1 was assessed by
the following evidence. Increased levels of
2 and
MMP-1 mRNA in collagen gel-stimulated fibroblasts
were abrogated by bisindolylmaleimide GF 109203X
and calphostin C, chemical inhibitors for PKC, but retained when cells were depleted of 12-myristate 13-acetate (PMA)-inducible PKC isoforms by 24 h of pretreatment with phorbol PMA. Antisense oligonucleotides complementary to the 5
end of PKC-
mRNA sequences significantly reduced the collagen lattice-stimulated
2 and MMP-1 mRNA levels. Taken together,
these data indicate that PKC-
, a PKC isoform not inducible by PMA or diacylglycerol, is a component of
collagen matrix stimulatory pathway for
2 and MMP-1
mRNA expression. Thus, a three-dimensional collagen
lattice maintains the dermal fibroblast phenotype, in
part, through the activation of PKC-
.
The interactions of cells with extracellular matrix
(ECM)1 are essential for cell behavior such as morphology, growth, motility, differentiation, and gene
expression. In many biological and pathophysiological processes such as embryonic development, wound healing,
tumor invasion and metastasis, and fibrosis, ECM plays
this important role not only by its different components
but also by its tightly regulated spatial and temporal organizations (Hay, 1991 Much attention has been paid to the role of protein kinase C (PKC) in ECM-regulated cellular activities. A family of serine/threonine-specific protein kinases, PKC has
been linked to cell proliferation, differentiation, and regulation of gene expression. This enzyme family can be divided into three groups (for review see Nishizuka, 1995 The physiological activators of atypical PKC isoforms are
not known. Evidence in the past few years has suggested
that products of phosphoinositol 3-kinase, phosphoinositol
3,4-bisphosphate, and phosphoinositol 3,4,5-trisphosphate
can induce PKC- The regulatory role of PKC- Cell Culture
Human fibroblast cultures established by outgrowth from healthy human
skin biopsies were kindly provided by Marcia Simon (Department of Dermatology, SUNY at Stony Brook). The cells were maintained in DME
(GIBCO BRL, Gaithersburg, MD), supplemented with 10% FCS (Hyclone Labs, Logan, UT), 100 U/ml penicillin, 100 U/ml streptomycin (GIBCO BRL) and grown in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Cells between population doubling levels 15 and 20 (the
6th and 10th passage) were used for the experiments.
Antibodies and Inhibitors
Cycloheximide (CHX) was obtained from Sigma Chemical Co. (St. Louis,
MO). Bisindolylmaleimide GF 109203X (BIM) was purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA). Calphostin C and chelerythrine were purchased from LC Laboratories (Woburn, MA). Polyclonal
antibodies against human protein kinase C- Preparation of Collagen Lattices
Collagen gels were prepared according to a procedure previously described (Xu and Clark, 1996 After incubation at 37°C in 95% air, 5% CO2, and 100% humidity for
the indicated time, cultures were carefully washed twice in DME and processed for various analyses. In experiments where inhibitors were used,
the levels of lactate dehydrogenase activity released were measured (LD
Diagnostic kit, Sigma Chemical Co.) and found to be similar to cells cultured in the absence of inhibitors.
Coating of Petri Dishes
For monolayer collagen coating of plastic dishes, the collagen used for lattices was diluted to a final concentration of 50 µg/ml with PBS. This solution was added to plastic dishes at a final concentration of 6.4 µg/cm2 and
incubated overnight at 4°C. Coated dishes were blocked with 2% BSA for
2 h at room temperature and rinsed with PBS twice before use.
Incubation with Antibodies
Collagen gels minus fibroblasts and 1% FCS/DME were preincubated
with polyclonal antibodies against PDGF AB (100 µg/ml) and, as a control, cyclin A (100 µg/ml) for 1 h at 4°C. Fibroblasts starved in 1% FCS/
DME for 1 d were detached by trypsinization and then seeded into collagen gels containing relevant antibodies. For antibody blocking of PDGF
stimulation, the medium was replaced with fresh medium containing 30 ng/ml PDGF-BB preincubated with or without anti-PDGF for 1 h.
(PDGF-BB was generously provided by Charles Hart of ZymoGenetics, Seattle, WA.) All the control media were replaced accordingly, minus
PDGF-BB. Cells were further incubated for 18 h.
Northern Analysis of Total Cellular RNA
Total RNA was isolated from cell monolayers and collagen gel cultures
using a modification of guanidinium thiocyanate method (Chromczynski
and Sacchi, 1987 Preparation of Cell Extracts
Fibroblasts grown in collagen gels were released by digestion of gels with
collagenase D (Boehringer Mannheim Corp.). Cells grown on plastic
plates were scraped and subsequently processed as those released from
collagen gel. Cells from all culture conditions were then washed with icecold PBS twice and resuspended in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM EGTA, and proteinase
inhibitors). After incubation on ice for 30 min, cells were repeatedly
passed through a 26 1/2-gauge needle followed by centrifugation at 14,000 g
for 20 min. Protein content was determined using a bicinchonic acid assay
(Pierce, Rockford, IL).
Isolation of Nuclei
The nuclei were prepared according to a protocol previously described
with some modifications (Greenberg and Ziff, 1984 Western Immunoblotting
Proteins from cell and nuclear extracts were separated on 8% SDS-polyacrylmide gel and transferred to polyvinyl difluoride membranes (Milipore Corp., Bedford, MA). Samples containing 4-7 µg of total proteins
were loaded onto the gel. The membranes were incubated with a blocking
solution containing 2% BSA, 2% horse serum, 50 mM Tris, pH 7.5, 150 mM NaCl, and 0.05% Tween 20 for 1 h at room temperature and then incubated overnight at 4°C with various antibodies: monoclonal PKC- Immunoprecipitation and In Vitro PKC- Cell extracts prepared from 106 cells were incubated at 4°C overnight with a
rabbit polyclonal antibody against human PKC- Gel Mobility Shift Assay
Nuclear extracts were prepared by a modified miniextraction protocol
(Schreiber et al., 1989 Gel mobility shift assay was performed with nuclear extracts prepared
as described above. NF- Down-Regulation of PKC- The procedure was essentially as previously described (Xu et al., 1996 Inhibition of Collagen Gel-induced Previous studies have shown that collagen gel increases
The requirement of newly synthesized proteins and
posttranslational modifications was investigated next. Protein synthesis was required since both types of mRNA diminished in the presence of CHX, a protein synthesis inhibitor (Fig. 2 A). To understand what role PKC might play,
three specific PKC inhibitors, BIM, calphostin C (CalC),
and chelerythrine, were used in the study. Both BIM and
CalC blocked the induction of both mRNAs in a dose- dependent manner, indicating that protein phosphorylation by PKC is required for both
Since there are 12 PKC isoforms identified so far, nine
of which are phorbol 12-myristate 13-acetate (PMA)/DAGinducible, whereas three were not induced by PMA, the
identity of the PKC isoform(s) that mediated collagen-
induced The Expression of Atypical PKC Isoforms
To determine which atypical PKC may be required for the
collagen gel induction, protein levels of atypical PKC isoforms in human dermal fibroblasts were first examined.
We performed Western analysis of total cell extracts after
cells were grown in collagen gels for various time periods.
In these experiments, HeLa cell extracts were used as a
positive control (data not shown), as they contain all of the
PKC isoforms examined (Chun and Jacobson, 1996
Collagen Gel Induction of PKC- Since collagen gel induction of
Collagen Gel Induction of DNA Binding Activity of
Transcription Factor NF- PKC- Antisense Translational Inhibition of PKC- The results presented so far have suggested that collagen
gel was an activator of PKC-
Such cells were harvested for analysis of
The Cytoplasmic and Nuclear Localization of PKC- Several PKC isoforms have been reported to be present in
the nucleus, including PKC-
We have previously shown that a three-dimensional collagen lattice, either stressed or relaxed, can induce integrin
Although the nature of precise primary signals triggered
by a three-dimensional type I collagen construct is undefined, we report here that these collagen gels activate
PKC- Among known activators of PKC- The downstream targets of PKC- It is of great interest that collagen gel and PDGF (Xu et al.,
1996 It is well established that three-dimensional collagen
matrix sends integrated physical and chemical signals to
the interior of the cell. Nevertheless, detailed knowledge
of collagen matrix signal transduction is very limited. Results reported here connect a signal transduction protein,
PKC-; Lin and Bissell, 1993
; Grinnell, 1994
;
Clark, 1996
). Three-dimensional ECM culture systems
have been developed to simulate natural interactions between cells and ECM more closely than the traditional in
vitro monolayer culture (Grinnell, 1994
; Clark et al., 1995
;
Ronnov-Jessen et al., 1995
; Streuli et al., 1995
; Sankar et al.,
1996
). Among those systems, a relaxed collagen lattice
populated by fibroblasts is considered an in vitro system
representative of a normal fibrous stroma in vivo such as
the dermis (Grinnell, 1994
). When fibroblasts are embedded in the lattice consisting mainly of type I collagen, they
contract the initially loose network to a dense tissue-like structure. This process is accompanied by a fundamental
reprogramming of fibroblast morphology and metabolism.
This results in down-regulation of type I collagen synthesis
(Eckes et al., 1993
), attenuation of cellular response to
growth factors (Lin and Grinnell, 1993
; Clark et al., 1995
),
induction of collagenase (Unemori and Werb, 1986
) and
the collagen receptor
2 integrin subunit (Klein et al., 1991
),
and modulation of platelet-derived growth factor (PDGF) effects on integrin receptor expression (Xu and Clark,
1996
).
).
The classic group containing isoforms
,
I,
II, and
depends on Ca2+ and phorbol ester/diacylglycerol (DAG)
for activity. The novel group containing isoforms
,
,
,
,
and µ is phorbol ester/DAG-dependent but does not require Ca2+. An atypical group containing PKC-
,
, and
is not activated by phorbol ester/DAG. Cell adhesion has
been reported to signal PKC activation. For example, adhesion of HeLa cells to a collagen substratum induces
PKC activity (Chun and Jacobson, 1992
, 1993
). During
HeLa cell adhesion to a gelatin substratum, PKC-
is
translocated from cytosolic to membrane fractions (Chun
and Jacobson, 1996
). Integrin cell adhesion molecules
could be direct substrates for activated PKC since PKC
phosphorylates the cytoplasmic domain of
6A integrin
subunit in vitro (Gimond et al., 1995
). The PKC activity
has been shown to be required for the spreading of several cell types, such as macrophage on immunoglobulin-coated
surfaces (Li et al., 1996
), Hela cells on a collagen substratum (Chun and Jacobson, 1992
, 1993
), and CHO cells on
fibronectin (Vuori and Ruoslahti, 1993
). PKC is also involved in the formation of focal contacts by human
embryo fibroblasts on substrata composed of fibronectin
since its inhibitors reduced focal adhesion and stress fiber
formation (Woods and Couchman, 1992
).
autophosphorylation activity in a cellfree assay (Nakanishi et al., 1993
). Also, transducers of tumor necrosis factor (TNF)-
, such as ceramide, the product of sphingomyelin hydrolysis by acidic sphingomyelinase,
and arachidonic acid (AA), a product of cellular phospholipase A2, can activate PKC-
(Nakanishi and Exton, 1992
;
Muller et al., 1995
). The increased PKC-
activity by sphingomyelinase has also been observed (Lozano et al., 1994
).
In addition, PKC-
is directly associated with Ras (DiazMeco et al., 1994b).
in collagen-mediated cellular process has not been addressed. Nevertheless, studies
have shown that cell adhesion to collagen substratum increases AA release (Chun and Jacobson, 1992
; Auer and
Jacobson, 1995
) and Ras activity (Kapron-Bras et al.,
1993
), suggesting potential correlations between collagen
signaling processes and PKC-
activation. The aim of this
study is to understand how a three-dimensional collagen gel sends biochemical signals to induce cellular expression
of a collagen receptor integrin subunit,
2, and collagenase
type I metalloproteinase (MMP-1). In this report, we demonstrate that the collagen lattices activate fibroblast PKC-
.
The PKC-
activity appears to be a component of the nuclear signaling cascade that leads to integrin
2 and MMP-1
expression.
Materials and Methods
were purchased from
GIBCO BRL. Monoclonal antibodies against human protein kinase C-
,
-
and -
were purchased from Transduction Laboratories (Lexington,
KY). Polyclonal antibodies against PKC-µ, nuclear factor
B (NF-
B),
p65, and p50 were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Polyclonal antibodies against human PDGF AB and cyclin A were
purchased from Upstate Biotechnology (Lake Placid, NY).
). Pepsin-solubilized bovine dermal collagen
dissolved in 0.012 M HCl was 99.9% pure containing 95-98% type I collagen and 2-5% type III collagen (Vitrogen 100; Cetrix Laboratories, Palo
Alto, CA). Collagen for cultures was prepared by mixing 2.0 mg/ml of type I
collagen, 100 U/ml penicillin, 100 U/ml streptomycin, and 1% FCS in DME
at pH 7.0-7.4. Human dermal fibroblasts from subconfluent cultures
starved in 1% FBS for 24 h were mixed with 10 ml collagen solution for a
final concentration of 5 × 105 cells/ml. The collagen cell suspension (4 ml)
was immediately placed onto 2% BSA (Kankakee, IL)-coated 60-mm
petri dishes (Falcon; Becton-Dickinson Labware, Lincoln Park, NJ) and
incubated at 37°C for 2 h (or for the time periods described in figure legends) before the addition of 5 ml of 1% FCS/DME to each dish.
). After centrifugation at 14,000 g to remove culture medium, collagen gels were dissolved in 4 M guanidinium isothiocyanate and
repeatedly passed through a 20 1/2-gauge needle. For Northern blot hybridization, 3-5 µg of total RNA was treated with glyoxal/DMSO, separated by electrophoresis on an 1% agarose gel in 10 mM phosphate
buffer, pH 7.0, and transferred to Hybond+ nylon membranes (Amersham Corp., Arlington Heights, IL). Ethidium bromide (0.5 µg/ml) was included in the gel to monitor equal loading by the quantity of 18 S and 28 S
ribosomal RNA present. cDNA probes were labeled with [
-32P]dCTP by
the random primer procedure (Du Pont/NEN, Boston, MA). Oligonucleotide probes were end-labeled with [
-32P]ATP (Du Pont/NEN) and in
the presence of polynucleotide kinase (Boehringer Mannheim Corp.,
Indianapolis, IN). The filters were hybridized to the labeled probes in
QuickHyb solution (Stratagene, La Jolla, CA) for 3 h at 68°C and washed
according to manufacturer's protocol. The signals were detected by autoradiography (model X-Omat AR; Kodak Eastman, Rochester, NY) at
80°C for optimal exposure. All results shown are representative of at
least two independent experiments. Human
2 cDNA was a generous gift
from Dr. Yoshikazu Takada (Scripps Institute, La Jolla, CA) (Takada and
Hemler, 1989
). Human MMP-1 cDNA was purchased from American Type Culture Collection (Rockville, MD). Human
5 cDNA was purchased from GIBCO BRL. An oligonucleotide complementary to 28 S ribosomal RNA was purchased from Clontech (Palo Alto, CA).
). Cells released from
collagen gel or scraped from plastic plates were washed twice with ice-cold
PBS. Cells were precipitated by centrifugation at 500 g for 5 min at 4°C.
Cell pellet was washed in buffer A once (10 mM Tris, pH 7.4, 3 mM CaCl2, 2 mM MgCl2), resuspended in 1 ml lysis buffer B (0.5% NP-40 in buffer A)
and homogenized in a Dounce homogenizer with a "B" pestle. After examination under a microscope to monitor the release of nuclei, the homogenate was centrifuged at 500 g at 4°C. The pellet was designated as nuclei,
whereas the supernatant was designated as cytoplasmic fraction. The nuclear pellet was stored in buffer C (50 mM Tris, pH 8.0, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM PMSF, 1 µM leupeptin). The protein
content in cytoplasm and nuclei was determined using the bicinchonic acid
assay (Pierce).
, -
,
-
and
-tubulin, and polyclonal PKC-
and -µ. After incubation with
horseradish peroxidase-conjugated goat anti-rabbit antibody (1:1,000 dilution; Amersham Corp.) in 50 mM Tris, pH 7.5, 150 mM NaCl, and
0.05% Tween 20 for 1 h at room temperature, the blots were then visualized with enhanced chemiluminescence (Amersham Corp.).
Activity Assay
used in Western analysis.
The immune complexes were recovered by anti-rabbit IgG agarose beads
(Sigma Chemical Co.). The resulting immunoprecipitates were washed
three times with a cold buffer containing 20 mM Tris, pH 7.5, 0.5 mM
EDTA, 0.5 mM EGTA, 25 µg/ml leupeptin, and 25 µg/ml aprotinin before they were mixed in a final volume of 50 µl assay solution (35 mM
Tris, pH 7.5, 15 mM MgCl2, 1 mM MnCl2, 0.5 mM EGTA, 0.1 mM CaCl2,
1 mM sodium orthovanadate, and 100 µM [
-32P]ATP with or without 280 µg/ml phosphatidylserine) and incubated at 30°C for 10 min. After reactions were stopped by the addition of the equal volume of 2× gel loading
buffer, the samples were boiled for 3 min and precipitated. The supernatant was analyzed by SDS-PAGE followed by autoradiography.
). Cells were washed with ice-cold PBS twice and hypotonic buffer A once (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2,
0.5 mM PMSF, 0.5 mM DTT). Cells were lysed by incubation in lysis
buffer (0.2% NP-40 in buffer A) for 10 min on ice. After centrifugation at
500 g for 4 min at 4°C, the nuclear pellet was resuspended in extraction
buffer C (20 mM Tris, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 0.2 mM
EDTA, 20 mM KCl, 0.5 mM PMSF, 0.5 mM DTT) and D (20 mM Tris,
pH 7.9, 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 1.2 M KCl, 0.5 mM
PMSF, 0.5 mM DTT) at a 1:2 ratio followed by incubation on ice for 20 min. The supernatant was collected as nuclear extracts after centrifugation
at 14,000 g for 8 min. The nuclear protein was determined using the bicinchonic acid assay (Pierce).
B and Sp-1 enhancer element consensus sequences 5
-AGT TGA GGG GAC TTT CCC AGG C-3
and 5
-ATT CGA TCG GGG CGG GGC GAG C-3
, respectively, were purchased
(Promega Corp., Madison, WI). These oligonucleotides were labeled by
[
-32P]ATP. The nuclear extracts (3-5 µg) were incubated with 1 µg poly
(dI/dC) (Boehringer Mannheim Corp.) and 2 µg BSA (GIBCO BRL) in a
binding buffer (10 mM Tris, pH 7.9, 5 mM MgCl2, 50 mM KCl, 10% glycerol, and 1-5 × 104 cpm end-labeled oligonucleotides) for 20 min at room
temperature. The samples were separated on a 5% native polyacrylamide
gel in 0.5× TBE buffer (Tris-borate-EDTA). For supershift assays, the reaction mixture minus the probe was incubated with 32P-labeled oligonucleotides for 20 min followed by incubation with 2 µl antibodies for 30 min
at room temperature. The samples were separated on a 4% native polyacrylamide gel.
Protein by Antisense
Inhibition of Translation
)
with modifications to accommodate the collagen gel culture. Phosphorothioate DNA oligonucleotides with the sequences 5
-ATGCCCAGCAGGACC-3
(sense 1143), 5
-GGTCCTYGCTGGGCAT-3
(antisense 1142), and 5
-GGTCCTGCTGGGCATGCG AAAGC-3
(antisense 1144)
were synthesized by Promega. Subconfluent adult human dermal fibroblasts were treated with oligonucleotides in DME containing 20 µg/ml lipofectin (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride [DOTMA]; GIBCO BRL) for 6-8 h at 37°C in the presence of 5%
CO2. After this time, the medium containing lipofectin was replaced by
fresh medium containing appropriate oligonucleotides. After 48 h, cells
were subcultured into collagen gel containing freshly added oligonucleotides. After incubation for 18 h, total RNA or cell extracts were prepared
and analyzed with Northern blotting, Western blotting, or immunoprecipitation and kinase assay.
Results
2 and MMP-1
mRNA Levels
2
integrin mRNA steady-state levels in human foreskin
(Klein et al., 1991
) and adult dermal (Xu and Clark, 1996
)
fibroblasts. Relaxed collagen gels also induce fibroblast collagenase (Unemori and Werb, 1986
; Langholz et al., 1995
).
Here we demonstrate that the induction of
2 and MMP-1
mRNA occurred only when cells were cultured in a threedimensional collagen gel, not on collagen monolayer- coated surface (Fig. 1 A), in agreement with Langholz et al.
(1995)
. A time course showed that the mRNAs appeared
as early as 4 h after cells were placed in collagen lattices
(Fig. 1 B).
2 mRNA reached the maximum after 24 h,
whereas MMP-1 continued to increase up to 72 h (Fig. 1 B).
Since PDGF-BB also induces steady-state levels of
2
mRNA (Ahlen and Rubin, 1994
; Xu et al., 1996
), we asked whether the induction by collagen gel was caused by
PDGF present in collagen preparations. Antibody against
PDGF has been shown to neutralize PDGF biological effects (Ferns, 1991). Polyclonal antibody against PDGF AB
was therefore included in the collagen gel cultures and, as
control, in fibroblasts grown on conventional tissue culture
plates (TC) and in collagen gel (COL) before PDGF stimulation. As shown in Fig. 1 C, the antibodies did not affect the induction by COL (lanes 1 and 2) but drastically inhibited PDGF effects (lanes 3 and 4). The possibility that the
failure of anti-PDGF to inhibit COL induction may result
from PDGF-BB in collagen gel higher than 30 ng/ml, a
concentration used for PDGF effects on TC, was examined. Since the saturating concentration of PDGF-BB to induce
2 expression is between 10-15 ng/ml (Xu and Clark,
1996
), we rationalized that if high concentration of PDGF
in COL was responsible for
2 induction, the addition of PDGF cannot further increase
2 expression. Fibroblasts
grown in collagen gel were further stimulated by PDGF-BB
at 30 ng/ml, and integrin
2 mRNA level was greatly increased compared to COL or PDGF alone (Fig. 1 C, lanes
5 and 9), confirming our previous observation (Xu and
Clark, 1996
). This further induction was inhibited by antiPDGF in a dose-dependent manner (Fig. 1 C, lanes 10 and 11). A control antibody, cyclin A, did not have any effect
(Fig. 1 C, lane 12). Taken together, signals from three-
dimensional collagen lattices responsible for induction of
integrin
2 and MMP-1 do not emanate from PDGF contamination.
Fig. 1.
Northern analysis of integrin 2 subunit and MMP-1
mRNA induced by three-dimensional collagen lattices. Normal
human dermal fibroblasts were starved 1 d in 1% FCS/DME before subculture in test conditions. (A) Cells were subcultured on
tissue culture plates (TC), collagen monolayer coated surface (ML),
and in three-dimensional collagen lattices (COL). (B) Cells were
subcultured on tissue culture plates (TC) and in three-dimensional lattices (COL) for the time indicated. (C) Collagen gel mix
was preincubated with anti-PDGF at 100 ng/ml (lanes 2 and 6)
and at indicated concentrations (lanes 10 and 11), and with anticyclin A at 100 ng/ml (lane 12). Cells in collagen gel (lanes 5, 6,
and 10-12) or on TC (lanes 3, 4, and 8) were stimulated with 30 ng/ml PDGF-BB preincubated with (lanes 4, 6, 10, and 11) or
without anti-PDGF (lanes 3, 5, 8, and 9) or with anti-cyclin A
(lane 12) 4 h after subculture. Total RNA was probed with human
2 integrin or MMP-1 cDNAs as indicated. Equal loading
was monitored by UV light examination of ethidium bromide-
stained gel and confirmed by hybridization of the same blot with
[32P]labeled probe for 28 S ribosomal RNA. Results are representative of two experiments.
[View Larger Version of this Image (53K GIF file)]
2 and MMP-1 mRNA
expression (Fig. 2 B). Chelerythrine, however, did not
demonstrate the inhibitory effect on the induction (data
not shown).
Fig. 2.
Inhibition of collagen gel induction of 2 and MMP-1
mRNA levels. (A and B) Cells were preincubated with inhibitors
before subculture. (A) 10 µg/ml cycloheximide (CHX), a protein
synthesis inhibitor, for 15 min. (B) PKC inhibitors bisindolylmaleimide GF 109203X (BIM) and calphostin C (CalC) at concentrations indicated for 1 h and 30 min, respectively. The incubation
with inhibitors was continued for 18 h after subculture. (C) Quiescent cells either untreated or incubated for 24 h with PMA (300 ng/ml) were subcultured in collagen lattices or stimulated with
PMA (50 ng/ml) for 16 h. Total RNA was probed with human
2
integrin and MMP-1 cDNAs. Equal loading was monitored by
UV light examination of ethidium bromide-stained gel and confirmed by hybridization of the same blot with [32P]labeled probe
for 28S ribosomal RNA. Results are representative of two independent experiments. T, tissue culture plates; C, collagen lattices.
[View Larger Version of this Image (59K GIF file)]
2 and MMP-1 mRNA expression was sought. As
a first step, we investigated whether this PKC was PMAinducible or not. A widely used strategy is to deplete cellular PMA-inducible PKC levels by treating cell culture
chronically with PMA (Larrodera et al., 1990
). This approach depletes PKC isoforms sensitive to PMA/DAG activation. Thus, quiescent human fibroblast cultures were
exposed to PMA (300 ng/ml) for 24 h before they were
subcultured into collagen gel. PMA induction of cells cultured on tissue culture plates were performed in parallel as a control.
2 and MMP-1 mRNA steady-state levels were
then determined. As expected, PMA was unable to induce
either
2 or MMP-1 mRNA in cells chronically treated
with PMA (Fig. 2 C, right lane). In contrast, collagen gel
promoted a potent response in
2 mRNA expression in
PMA-pretreated cells, which was only slightly lower than
that in untreated cells (Fig. 2 C, middle two lanes). This result indicates the involvement of atypical PKC(s). However, MMP-1 mRNA response to collagen gel was reduced
from 10-fold to 6.3-fold increase in cells chronically treated
with PMA (Fig. 2 C, middle two lanes). These results suggest that collagen lattices activate an atypical PKC isoform
that, to a different extent, is required for
2 and MMP-1
mRNA expression.
). All
three known members of the atypical PKC subfamily,
,
,
and
, were detected in human dermal fibroblasts (Fig. 3). The antibody against the PKC-
isoform detected a high
level of immunoreactive proteins from the lysates. Collagen gel did not change its amount. PKC-
and -
were
detected at relatively low levels in both monolayer and
collagen gel cultures, and no obvious differences could be
discerned in the levels from the two culture conditions
(Fig. 3). When lysate amount, antibody concentration, or
exposure time was increased, the results became questionable because of the high background. A novel PKC isoform, PKC-µ, was also examined. Western blot detected
PKC-µ at a level comparable with PKC-
. In contrast to
atypical PKC isoforms that were not synthetically affected
by the collagen gel culturing, the cellular protein level of
PKC-µ decreased as cells were incubated in collagen gel
for 18 h (Fig. 3). These results demonstrate that collagen lattices, which were capable of altering some PKC isoform
protein levels, did not change the protein amount of atypical PKCs in human fibroblasts.
Fig. 3.
Western analysis of PKC isoforms in total cell lysates.
Normal human dermal fibroblasts were cultured 1 d in 1% FCS/
DME before subculture in test conditions. Cells were subcultured
on tissue culture plates (TC) or in three-dimensional collagen lattices (COL) for the time indicated. Total cellular proteins were
extracted, quantified with BCA assay, blotted, and detected with
antibodies against PKC-, -
, -
, and -µ.
[View Larger Version of this Image (29K GIF file)]
Kinase Activity
2 integrin and MMP-1
mRNA involved atypical PKC isoforms, collagen lattices
may induce atypical PKC enzyme activity. The kinase activity of PKC-
was investigated for the following reasons.
PKC-
is CalC (Larivee et al., 1994
) and BIM sensitive
(Xu et al., 1996
), chelerythrine insensitive (Thompson and
Fields, 1996
), and not down-regulated by PMA (Wooten, 1994), which is consistent with the results observed thus
far (Fig. 2, B and C). Furthermore, PKC-
was the only
member of atypical PKC subfamily detected in human fibroblasts in significant amounts (Fig. 3). To assess whether
collagen gel can induce PKC-
kinase activity, fibroblasts
grown in collagen gel for various time periods were assayed for PKC-
kinase activity. PKC-
present in the cell extracts was immunoprecipitated with a polyclonal antibody. PKC-
was autophosphorylated by kinase activities
associated with the immunoprecipitates. The kinase activity was induced 30 min after fibroblasts cultured in collagen gel (4.6-fold), reached the maximum at 4 h (9.2fold), and decreased after 4 h (Fig. 4, A and C). Cells grown on tissue culture plates, however, remained unstimulated during the entire time course (data not shown). The
presence of phosphatidylserine, a PKC activator, increased
the PKC-
activity impressively from unstimulated (sevenfold) and slightly from stimulated cells (11.5-fold, a 20%
increase). Therefore, collagen gel appears to be an activator for fibroblast PKC-
, as determined by kinase activity
associated with PKC-
immunoprecipitates.
Fig. 4.
Stimulation of PKC- and NF-
B DNA binding activity
by three-dimensional collagen lattices. Normal human dermal fibroblasts were cultured 1 d in 1% FCS/DME before subculture in
test conditions. Cells were subcultured on tissue culture plates
(TC) or in three-dimensional collagen lattices (COL) for the
times indicated. (A) PKC-
kinase activity assay. Total cellular
proteins were extracted, quantified with BCA assay, and immunoprecipitated with a polyclonal antibody against PKC-
in the
presence or absence of a synthetic peptide to which the antibody
was raised (Peptide). The immunoprecipitates were incubated in
a kinase assay buffer for 10 min at 30°C. Unless specified, the reactions were carried out in the absence of phosphatidylserine. The kinase activity was determined by autophosphorylation as
described in Materials and Methods. The results are representative of three independent experiments. (B) Gel mobility shift assay. Nuclear extracts were prepared and assayed for DNA binding activity of NF-
B and SP1 by gel mobility shift as described in
Materials and Methods. Arrows indicate specific bindings. The
results are representive of three independent experiments. (C)
Quantification of A and B. (D) Supershift assay. Nuclear extracts
prepared from cells stimulated with collagen gel (COL) for 24 h
and on tissue culture plates (TC) were incubated with labeled
NF-
B consensus sequences before futher incubation with antibodies against p65 and p50. I, the supershift caused by p65; II, the
supershift caused by p50; III, the specific NF-
B binding.
[View Larger Version of this Image (54K GIF file)]
B
has been reported as a positive regulator of the activity of a transcription factor, NF-
B (Diaz-Meco et al.,
1993
). Cells that display enhanced PKC-
phosphorylation
activity also increase NF-
B DNA binding activity in response to sphingomyelinase (Lozano et al., 1994
), ras p21
(Diaz-Meco et al., 1993
), and TNF-
(Muller et al., 1995
).
The induction of PKC-
activity by cells incubated in collagen gels prompted us to ask whether NF-
B DNA binding activity was also induced. Nuclear extracts were prepared from human fibroblasts grown in collagen gel from
30 min to 24 h. The NF-
B DNA binding activity present
in the nuclear extracts was detected by gel mobility shift
assay using a DNA probe encompassing the
B motif (see
Materials and Methods). As seen in Fig. 4 B, a 30-min incubation of human fibroblasts in collagen gel induced the
formation of specific
B DNA-protein complexes. The
NF-
B DNA binding activity remained similar from 30 min
to 4 h (Fig. 4 C, 2.9-3.8-fold increases) but increased modestly after 24 h of incubation in collagen gel (Fig. 4 C, 4.9fold increases). The quantification of the NF-
B DNA
binding activity revealed a kinetic pattern in concord with,
if not identical to, that of PKC-
kinase activity (Fig. 4 C).
Cells grown on tissue culture plates, on the contrary, did
not demonstrate any change in the NF-
B DNA binding
activity (data not shown). Competition experiments with an unlabeled
B consensus sequence confirmed the specificity of the binding complexes. As a control, the binding
activity to the Sp1, a transcription factor, consensus sequence was also examined (Fig. 4 B). The Sp1 DNA binding activity present in nuclear extracts was not altered by
cells cultured in collagen gel. Therefore, we conclude that
this three-dimensional cell culture system increased PKC-
activity in association with the activation of NF-
B DNA binding complexes. The composition of the NF-
B DNA
binding complexes was examined next. The supershift assay showed that p50 of NF-
B/Rel family proteins was one
component of the binding complex (Fig. 4 D, II) regardless of culture condition. Antibody to p65, however, only
shifted a very low amount of the binding complex (Fig.
4 D, I), suggesting that the binding complex may be composed of other member(s) of NF-
B/Rel family. Therefore, collagen gel induced the p50-containing complex binding to NF-
B site without changing protein composition.
activity and that collagen
gel-induced
2 and MMP-1 mRNA expression required
atypical PKC isoform activity. The potential role of PKC-
in collagen gel-induced
2 and MMP-1 mRNA expression
was then investigated. To establish a direct connection between these two events, we used a previously described
strategy to remove PKC-
protein through translation inhibition by antisense oligonucleotides (Xu et al., 1996
). Antisense phosphorothioate oligonucleotides 1142 and 1144 have sequences targeted at the begining of the open reading frame of PKC-
cDNA (Barbee et al., 1993
). This GCrich site is in a nonconserved variable region (V1) of PKC
family (Nishizuka, 1992
), which differs significantly among PKC isoforms. Antisense oligonucleotides 1142 and 1144 are essentially the same except that 1144 was designed to
have an additional looping secondary structure at its 3
end to reduce the possible degradation caused by 3
exonuclease (Tang et al., 1993
). Previously we have found that
the antisense oligonucleotides at concentrations higher than 2.5 µM can inhibit human fibroblast PKC-
protein
level by at least 70% (Xu et al., 1996
). Therefore, fibroblasts were lipid-transfected with antisense oligonucleotides 1142 or 1144 at 2.5 µM. As a control, some cells
were transfected with "sense" oligonucleotide 1143. The
transfected cells were then subcultured into collagen gels and further treated with oligonucleotides for 18 h. The depletion of PKC-
protein levels by antisense inhibition of
translation was monitored by Western blotting (Fig. 5 A)
and kinase activity of PKC-
immunoprecipitates (Fig. 5 B).
The results from Fig. 5 demonstrated a significant reduction of PKC-
protein and its associated kinase activity after treatment with both oligonucleotides, 1142 and 1144. The specificity of antisense inhibition was confirmed by
probing the membrane with an mAb against PKC-
. The
equal loading was confirmed by detection with an mAb
against
-tubulin (Fig. 5 A).
Fig. 5.
Specific down-regulation of protein levels and PKC-
activity by antisense inhibition of translation. Subconfluent normal dermal human fibroblasts were either untreated or treated
with sense (1143) or antisense oligonucleotides (1142 and 1144)
at 2.5 µM in DME containing 20 µg/ml DOTMA for 6-8 h. After
this time, the medium containing DOTMA was replaced by fresh
medium containing appropriate oligonucleotides. Cells were subcultured 48 h later on tissue culture plates (TC) or in three-
dimensional collagen lattices (COL) and continuously incubated
in appropriate oligonucleotides for 18 h. Total cellular proteins
were extracted, quantified with BCA assay, and assayed for
PKC-
in vitro kinase activity or protein level. (A) Western blot
detection of PKC-
, PKC-
, and
-tubulin. (B) In vitro kinase activity in immunoprecipitates of PKC-
.
[View Larger Version of this Image (25K GIF file)]
2 and MMP-1
mRNA expression. Antisense treatment inhibited collagen gel induction of
2 expression up to 60% (Fig. 6). Both
oligonucleotides 1142 and 1144 had similar effects. These
results are consistent with observations of cells treated
with the PKC inhibitors, BIM and CalC (Fig. 2 B), and
chronically with PMA (Fig. 2 C). MMP-1 expression was
also inhibited by antisense treatment, although less effectively with 1142 and more with 1144. The expression of a
fibronectin receptor integrin subunit,
5, was not affected
by either a collagen gel environment or antisense treatment, confirming the specificity of the antisense inhibition.
Therefore, PKC-
is involved in collagen gel induction of
the
2 integrin subunit and MMP-1 mRNA expression.
Fig. 6.
Antisense-mediated down-regulation of PKC- protein
inhibits collagen gel induction of integrin
2 and MMP-1 mRNA
expression. Total cellular RNA was extracted from normal human
dermal fibroblasts after incubation in the presence or absence of
sense (1143) or antisense (1142 and 1144) oligonucleotides complementary to the 5
-end of the PKC-
transcript at 2.5 µM. Before RNA harvest, cultures were incubated in collagen lattices for
18-24 h. Total RNA was probed with human
2 integrin, MMP-1
and
5 integrin cDNAs. Equal loading was monitored by UV light
examination of ethidium bromide-stained gel and confirmed by
hybridization of the same blot with [32P]labeled probe for 28S ribosomal RNA. Results are representative of two independent experiments.
[View Larger Version of this Image (31K GIF file)]
and PKC-
(Ventura et al.,
1995
). The nuclear localization of those PKC isoforms
seems directly correlated to its functional role in regulating cellular biosynthetic activities. For example, the phorbol ester-regulated expression of opioid peptide gene in
rat myocardial cells was reported as a possible target of nuclear PKC-
and -
through autocrine or paracrine mechanisms (Ventura et al., 1995
). The involvement of PKC-
in mRNA expression of
2 and MMP-1 prompted us to ask
whether PKC-
is a cytosolic or nuclear protein in human
dermal fibroblasts. Previously, PKC-
has been reported
present in cytoplasm and nucleus in species such as rat and
rabbit (Masmoudi et al., 1989
; Hagiwara et al., 1990
; Disatnik et al., 1994
; Rosenberger et al., 1995
). To assess this
possibility of human PKC-
, the distribution of PKC-
as
well as -µ in cytoplasm and nucleus was examined. Intact nuclei were isolated from fibroblasts grown as monolayer
or in three-dimensional collagen gel. Western analysis was
performed with cytoplasmic and nuclear fractions. While
PKC-µ was predominantly present in the cytoplasm, PKC-
was detected in both cytoplasm and nucleus (Fig. 7). Cells
grown in collagen gel did not change the distribution between the two subcellular compartments. Therefore, PKC-
,
the kinase required for collagen gel regulatory pathway leading to
2 and MMP-1 mRNA expression in human
dermal fibroblasts, is constitutively a nuclear as well as a
cytoplasmic protein.
Fig. 7.
Western analysis of PKC isoforms in cytoplasma and
nuclei. Normal human dermal fibroblasts were starved 1 d in 1%
FCS/DME before subculture in test conditions. Cells were subcultured on tissue culture plates (TC) or in three-dimensional collagen lattices (COL) for 18-24 h. Nuclear (Nuclei) and cytoplasmic (Cell Extracts) fractions were prepared, quantified with BCA
assay, blotted, and detected with antibodies against PKC- and -µ.
[View Larger Version of this Image (28K GIF file)]
Discussion
2 mRNA expression in human dermal fibroblasts (Xu and
Clark, 1996
). Interestingly, no induction of
2 or MMP-1
mRNA is observed when fibroblasts are plated on a collagen monolayer (Fig. 1 A) as previously reported by Langholz et al. (1995)
for MMP-1. The evidence presented in this report indicates that PKC-
is required for the full response of integrin
2 subunit and MMP-1 mRNA to collagen lattice (Fig. 6).
(Fig. 4 A) as a component of secondary transduction signals (second messenger pathway). How might collagen gel activate PKC-
? Previously, we showed that
PDGF can induce PKC-
activity (Xu et al., 1996
). Could
collagen gel use the same pathway as PDGF to activate
PKC-
? In fact, adhesion molecules such as CAMs (cell
adhesion molecules) and basic fibroblast growth factor
both activate FGF receptor to induce cell contact-dependent neurite outgrowth (Williams et al., 1994
). However,
several lines of evidence argue against the similar scheme
with collagen gel and PDGF. First, Lin and Grinnell (1993)
have presented evidence that PDGF, but not collagen gel, induces tyrosine phosphorylation of PDGF receptor in fibroblasts. Second, the same report also showed that collagen gel, especially relaxed collagen gel, actually reduces
PDGF-stimulated receptor autophosphorylation. Third,
we previously reported that collagen gel does not stimulate expression of integrin
3 and
5 subunits, two PDGFinducible genes (Xu and Clark, 1996
). Fourth, collagen gel
possesses both positive and negative impact on PDGF
stimulation of integrin subunit mRNA expression: positive
on
2, negative on
3 and
5 (Xu and Clark, 1996
). The
data taken together, in fact, indicate that collagen gel interferes with some PDGF pathways. Thus, collagen gel
and PDGF signaling pathways appear not to converge at
PDGF receptor site.
are AA generated
by phospholipase A2 and ceramide generated by phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC)/
sphingomyelinase. Adhesion of HeLa cells to collagen
(Chun and Jacobson, 1993
) or
1 integrin clustering (Auer
and Jacobson, 1995
) increased the release of AA. AA
could activate PKC-
either directly (Nakanishi and Exton, 1992
) or indirectly by triggering DAG release (Auer
and Jacobson, 1995
). DAG released from AA metabolism
may activate PKC-
by sphingomyelinase-ceramide pathway since it was able to increase sphigomyelin hydrolysis in some cell types (Kolesnick, 1987
). Alternatively, collagen gel may sequentially activate PC-PLC and PKC-
. In
our system, disruption of PC-PLC activity with its inhibitor D609 (Schutze et al., 1992
) blocked collagen gel induction of
2 mRNA expression (data not shown), implicating
the importance of this pathway. Another possibility for
PKC-
activation by collagen gel may be its phosphorylation by a second serine/threonine kinase. Several reports
have suggested that the phosphorylation may precede the PKC activation (Pears et al., 1992
; Cazaubon and Parker,
1993
). This phosphorylation requirement has been shown
with PKC-
and -
(Cazaubon et al., 1994
; Orr and Newton, 1994
). Potential phosphorylation sites are thought to
be present in several members of PKC family, including
(Tsutakawa et al., 1995
). In agreement with this, the clustering of
2 and
1, the subunits of collagen receptor integrin
2
1, which was reported to mediate collagen gel-induced MMP-1 expression (Langholz et al., 1995
), can induce
p21ras activation (Kapron-Bras et al., 1993
). Ras has been
shown to be coprecipitated with PKC-
and to induce PKC-
activity (Diaz-Meco et al., 1994b
). Indeed, our data showed
that collagen gel did not alter the cellular level of PKC-
(Fig. 3). Thus, posttranslational modification is a possible
mechanism by which PKC-
is regulated.
activity are yet to be
discovered. One consequence of PKC-
activation by extracellular stimuli such as TNF-
is the induction of NF-
B
activity (Diaz-Meco et al., 1994a
). Here we present evidence from gel mobility shift assays that collagen lattices induced NF-
B DNA binding activity in dermal fibroblasts
rapidly (
30 min) and persistently (
24 h) (Fig. 4 B). This
is in concordance with the observation that cells stimulated with TNF-
and p21Ras demonstrate both increased PKC-
and NF-
B activities (Diaz-Meco et al., 1994a
;
Muller et al., 1995
). In fact, TNF-
was also shown to induce NF-
B DNA binding rapidly and persistently (1/3-20 h)
(Johnson et al., 1996
; Roff et al., 1996
), similar to collagen
gel induction. One puzzling observation was that during a
24-h incubation period, PKC-
kinase activity peaked at 4 h,
whereas NF-
B DNA binding activity reached the maximum at 24 h with modest increase over the binding at 30 min (Fig. 4 C). While rapid increase of NF-
B activity is
predominantly caused by posttranslational modification
(for review see Siebenlist et al., 1994
), sustained nuclear
NF-
B activity over longer periods of stimulation may be
caused by either sustained reduction of NF-
B inhibitor B,
as demonstrated in vascular endothelial cells (Johnson et al.,
1996
), or induced expression of NF-
B subunits other than
RelA (p65), as reported for HL60 cells (Hohmann et al.,
1991
). Both c-Rel and RelB can be transcriptionally regulated through the
B element in their promoters (Hannink
and Temin, 1990
; Ryseck et al., 1992
), which p65 lacks
(Ueberla et al., 1993
). Stimulation of Jurkat cells over several hours results in increasing amounts of c-Rel relative
to p65 in the nucleus, possibly because of preferentially induced levels of c-Rel (Molitor et al., 1990
; Doerre et al.,
1993
). In support of this, results from supershift assay
showed very low levels of p65 in the
B-binding complex (Fig. 4 B). We speculate that the rapid binding observed
may be caused by phosphorylation of existing NF-
B/Rel
in cellular pool, whereas the persistent binding may result
from the newly synthesized NF-
B/Rel. Therefore, although
PKC-
reached the maximum at 4 h, NF-
B binding activity may not show identical kinetics and fold-increases because of regulatory transition from posttranscriptional modification of existing protein factors to synthesis and
modification of new proteins. In agreement with this, we
obtained evidence that collagen gel induction of
2 and
MMP-1 mRNA expression, a PKC-
-mediated event, requires both NF-
B (unpublished data) and protein synthesis (Fig. 2 A).
) both require PKC-
activity to induce integrin
2
mRNA expression. How is the induction further enhanced
when both signals are present (Fig. 1 C)? Although the
study is at a stage too early to provide answers, there are
several possibilities. First, PKC-
activity has been shown
to be regulated by protein-protein interactions with apoptosis gene par-4 product (Diaz-Meco et al., 1996b
),
-interacting protein (LIP) (Diaz-Meco et al., 1996a
), and Ras
(Diaz-Meco et al., 1994b
). Thus, collagen gel and PDGF
might induce different protein factors that in turn activate
PKC-
by protein-protein interaction. Copresence of both
stimuli could synergistically induce the kinase activity by
the interaction among multifactors. Second, there are multiple potential phosphorylation sites in PKC-
molecules
(Tsutakawa et al., 1995
). The fact that PKC-
is physically
interacting with and activated by Ras (Diaz-Meco et al.,
1994b
) suggests that phosphorylation is an important
means of regulating its activity. Thus, collagen gel and
PDGF may activate PKC-
by inducing phosphorylation
at different amino acid residue(s). A similar scheme was
suggested in the synergistic activation of NF-
B by calcium and Ras/Raf (for review see Baeuerle and Baltimore, 1996
). Third, PKC-
can induce transactivating activities
of different transcription factors such as NF-
B and AP-1
(Bjorkoy et al., 1995
). A differentially activated PKC-
could stimulate distinct downstream pathways. We have
observed that collagen gel stimulated DNA binding activity of NF-
B (Fig. 4 B) but not AP-1 (unpublished data).
In contrast, PDGF induced DNA binding of substantial
AP-1 but little NF-
B (unpublished data). It is tempting to
suggest that collagen gel/PKC-
and PDGF/PKC-
activate different transcription factors to induce integrin
2
and MMP-1 mRNA expression. The synergistic action then
would occur at the level of gene transcription. Investigations of these possibilities are ongoing in our laboratory.
, to the upstream initiator, the collagen lattice, and
to downstream outputs,
2 and MMP-1 mRNA expression.
Received for publication 30 August 1996 and in revised form 6 November 1996.
Funding for this work was provided by National Institutes of Health grant AG10114309 to R.A.F. Clark. J. Xu is supported by funding from the Dermatology Foundation and the School of Medicine, SUNY at Stony Brook.We thank Dr. Marcia Simon, Department of Dermatology, SUNY at
Stony Brook, NY, for adult human dermal fibroblasts, Dr. Yoshikazu Takada of The Scripps Institute, La Jolla, CA, for human 2 cDNA probe,
and Charles Hart of ZymoGenetics, Seattle, WA, for PDGF-BB.
AA, arachidonic acid;
BIM, bisindolylmaleimide GF 109203X;
CalC, calphostin C;
CAM, cell adhesion molecule;
CHX, cycloheximide;
COL, collagen gel;
DAG, diacylglycerol;
DOTMA, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride;
ECM, extracellular matrix;
MMP-1, type I metalloproteinase;
NF-B, nuclear factor
B;
PC-PLC, phosphatidylcholine-hydrolyzing phospholipase C;
PDGF, platelet-derived growth factor;
PKC, protein kinase C;
PMA, phorbol 12-myristate 13-acetate;
TC, tissue culture plates;
TNF, tumor necrosis factor.