From the
In addition to inhibiting the proteolytic activity of the matrix
metalloproteinases, tissue inhibitors of metalloproteinases (TIMPs)
promote the growth of cells in the absence of other exogenous growth
factors. TIMP-2 stimulates the proliferation of fibrosarcoma (HT-1080)
cells and normal dermal fibroblasts (Hs68) in a dose-dependent manner.
This response is evident as early as 2 h and persists up to 48 h after
treatment with recombinant TIMP-2 (rTIMP-2). The specificity of this
response is demonstrated by the ability of affinity-purified polyclonal
anti-TIMP-2 antibodies to ablate TIMP-2 mitogenesis and by the lack of
response to TIMP-1. This response is also blocked by the presence of an
adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (SQ22536).
Although SQ22536 did not affect untreated fibroblasts or fibrosarcoma
cells, this inhibitor completely abrogates the proliferative response
induced by rTIMP-2. Treatment of these cells with rTIMP-2 also
stimulates the production of cAMP in a time-dependent manner that
differs for the two cell lines. Moreover, treatment of purified cell
membranes with rTIMP-2 suppresses cholera toxin-mediated
ADP-ribosylation of the GTP-binding protein, Gs
The matrix metalloproteinases are a family of zinc-dependent
enzymes responsible for degrading the connective tissue matrix at
physiological pH
(1) . Members of this protease family possess
several conserved features including the consensus sequence for binding
zinc at the active site and an activation locus responsible for the
latency of the zymogen. The matrix metalloproteinase family is also
defined by the ability of tissue inhibitor of metalloproteinases
(TIMPs)
In
addition to their function as matrix metalloproteinase inhibitors, a
growing body of experimental evidence suggests that TIMPs behave as
cytokines and stimulate cellular proliferation in the absence of other
growth factors. In addition to their erythroid-potentiating activity
(EPA)
(7, 8) , TIMP-1, and TIMP-2 are mitogens for a wide
variety of cell types cultured in the absence of serum or exogenous
growth factors (9-11). Recent studies have shown that
SV40-transformed fibroblasts secrete TIMP-2 which is believed to
mediate the survival of nontransformed fibroblasts in the absence of
serum
(12) . TIMP-2 has also been shown to significantly increase
the survival of pituitary folliculo-stellate cells cultured in the
absence of serum
(13) .
These growth-promoting effects are
presumably mediated by specific TIMP binding sites on the cell
membrane, i.e. putative TIMP receptors. Recent studies have
demonstrated selective cell surface binding of TIMP-2 to HT-1080 cells
that is not competed by TIMP-1
(11, 14) . However,
neither the putative TIMP-1 or TIMP-2 receptor has been isolated or
characterized. Furthermore, the signal transduction mechanisms involved
in promoting growth by TIMPs have not been elucidated. Due to a growing
body of evidence for the growth-promoting activities of the TIMPs, we
have investigated the signal transduction mechanisms involved in
TIMP-2-mediated growth stimulation of normal dermal fibroblasts and
fibrosarcoma cells. We demonstrate that, in the absence of serum or
exogenous growth factors, rTIMP-2 mediates a mitogenic response in
normal dermal fibroblasts and fibrosarcoma cells by stimulating
adenylate cyclase to produce cAMP which, in turn, activates
cAMP-dependent protein kinase (PKA). This is the first report
demonstrating that TIMP-2 stimulates growth by activation of PKA.
PKA activity was determined by using the PKA assay
system from Life Technologies, Inc. with slight modification. Hs68 and
HT-1080 cells (1
rTIMP-2 stimulates the production of cAMP in HT-1080 and Hs68
cell lines in a time-dependent manner. Treatment of HT-1080 cells with
48 nM rTIMP-2 stimulated an approximately 50-fold increase in
the steady state levels of cAMP as early as 30 s compared to untreated
cells (Fig. 2,
Growth factors can stimulate cell proliferation through a
variety of different mechanisms including activation of tyrosine or
serine/threonine protein kinases
(39) . Although TIMP-2 has
previously been shown to possess growth-promoting
activity
(8, 11, 12, 13) , the mechanism
by which TIMP-2 transduces these growth signals has not been previously
elucidated. Identification of the signaling pathways utilized in growth
promotion is helpful not only in understanding the nature of the growth
factor response but also in characterization of the receptor. This
report gives the first description of the signal transduction events
that occur following treatment of normal dermal fibroblasts (Hs68) and
fibrosarcoma cells (HT-1080) with TIMP-2. We demonstrate in this study
that, in the absence of other exogenous growth factors, rTIMP-2 induces
a proliferative response in normal dermal fibroblasts and HT-1080
fibrosarcoma cells. This response to TIMP-2 treatment has also been
previously observed in Raji lymphoma cells, with the maximal effect in
these cells observed at sub-nanomolar concentrations
(11) . These
investigators also demonstrate that TIMP-2 can stimulate proliferation
of human Burkitt lymphoma cells following exposure in culture for
3-7 days. Moreover, the ability of TIMP-2 to increase thymidine
uptake in HSF4 fibroblasts has been shown to directly correlate with
the percent of cells in the S phase as determined by flow cytometric
DNA content analysis
(12) .
The differences in the maximal
effective concentrations are probably due to differences in cell line
responsiveness and/or inherent differences in the receptor affinities.
The observed stimulation of proliferation in Hs68 and HT-1080
fibrosarcoma cells is dependent on the concentration of rTIMP-2 and is
selectively blocked by affinity purified polyclonal anti-TIMP-2
antibodies and pretreatment of the cells with a specific inhibitor of
adenylate cyclase, SQ22536. HPLC purified TIMP-1 did not modulate cell
growth, thymidine incorporation, or cAMP production in these cell
lines. This selective growth modulating activity of TIMP-2 compared
with TIMP-1 has been observed previously in our laboratory
(17) .
A rapid increase in the production of cAMP is observed by treating
both cell lines with rTIMP-2. Two lines of experimental evidence
presented in this report suggest that TIMP-2 mediates production of
cAMP by activation of adenylate cyclase instead of suppressing
phosphodiesterase activity. These are the kinetics of cAMP production
in HT-1080 cells following rTIMP-2 treatment and the suppression of
mitogenesis by an adenylate cyclase inhibitor, SQ22536. Although both
cell lines respond to rTIMP-2 by initially increasing the production of
cAMP, the kinetics of the breakdown of cAMP differs dramatically
between these cells. HT-1080 cells treated with 48 nM rTIMP-2
results in an immediate increase in the steady state levels of cAMP as
soon as 30 s after treatment. The rapid decline of cAMP observed in
HT-1080 cells probably reflects the immediate activation of a
phosphodiesterase activity. The rapid spike in steady state levels of
cAMP (Fig. 2), followed by a return to basal levels at 10 min in
the HT-1080 cells, is consistent with the observations in the
8-azido-cAMP-labeling experiment (Fig. 4) and direct measurements
of PKA activity () in this same cell line. Treatment of
Hs68 cells with 48 nM rTIMP-2 also results in a 2.6-fold
production of cAMP as early as 2 min after treatment. However, the
production of cAMP in these cells remains elevated up to 10 min after
treatment with rTIMP-2. The persistence of elevated cAMP steady state
levels in these cells correlates with the 8-azido-cAMP labeling
experiment (not shown) and the percent active PKA ()
observed at 10 min following rTIMP-2 treatment. Moreover, the results
obtained from the 8-azido-cAMP labeling experiment demonstrate that
only one type of regulatory subunit is labeled in these cell lysates
and that this subunit binds to the cAMP produced in response to
treatment with rTIMP-2. Since binding of cAMP to the regulatory subunit
results in activation of PKA, we also demonstrate that the catalytic
activity of PKA is stimulated by treatment with TIMP-2.
The
differential responses of the Hs68 fibroblasts and HT-1080 fibrosarcoma
cells to TIMP-2 observed in several assays may be due to a number of
possibilities. These include differences in the cell cycle
synchronization following serum starvation as well as differences in
entry into S phase of the cell cycle. Alternative explanations for the
persistent difference in TIMP-2 responses between these cell lines
include the genetic backgrounds of these cells, differential expression
of the receptor for TIMP-2, differential regulation of
phosphodiesterase activity, and/or their inherent response to increases
in PKA activity. Stimulation of HT-1080 and Hs68 cells with rTIMP-2
results in the production of cAMP and enhanced catalytic activity of
PKA which may mediate a mitogenic response by phosphorylating
cytoplasmic and nuclear proteins targets
(40) . Previous reports
have shown that cAMP-mediated activation of PKA results in
translocation of the catalytic subunit of the PKA into the nuclei of
fibroblasts
(41) . The translocation of the PKA catalytic subunit
in NIH 3T3 fibroblasts has been previously reported to increase the
phosphorylation of transactivating proteins such as CREB, CREM, and
ATF-1 which facilitates their binding to cAMP responsive elements
(CREs:TGACGTCA) (42). Repressor molecules of the CRE such as
CREM-
Our findings demonstrate that
TIMP-2 mediates proliferation by binding a putative G-protein coupled
receptor which activates adenylate cyclase to produce cAMP and
ultimately activates PKA
(44) . It is postulated that when a
ligand binds a G-protein coupled receptor, GDP is exchanged for GTP on
the G
This
report is the first demonstration that rTIMP-2 induces proliferation in
normal dermal foreskin fibroblasts and fibrosarcoma cells by increasing
cAMP and the kinase activity of PKA. However, the overall significance
of these findings are presently not understood. Since TIMP-2 initiates
a cascade of events that result in the proliferation of neoplastic and
normal fibroblasts in the absence of exogenous growth factors, this
protein can be viewed as a growth factor for these cells. In the
absence of other exogenous growth factors, TIMP-2 could support the
survival and growth of normal fibroblasts, which may promote wound
healing
(45) . The ability of TIMP-2 to stimulate fibrosarcoma
cell proliferation suggests that TIMP-2 could also contribute to the
autocrine-stimulated neoplastic cell growth. However, recent studies
have demonstrated that increasing cAMP in HT-1080 cells decreases the
invasive potential of these cells when evaluated by in vitro invasion assays
(46) . Other studies have shown that TIMP-2
suppresses the in vitro invasive activity of HT-1080 cells
presumably through direct inhibition of the required matrix
metalloproteinase-2 activity
(47) . The present study suggests
that TIMP-2 may suppress the invasive phenotype not only by directly
inhibiting protease activity but also by stimulating the endogenous
production of cAMP.
Neither tumor cells nor fibroblasts in vivo are stimulated by a single growth factor, such as TIMP-2. Growth
of these cells is a result of the delicate integration of a number of
second messenger systems, not only from growth factors and their
receptors but also from extracellular matrix components and their
receptors. Thus, the ability of a cell to grow in response to TIMP-2
may depend on its sensitivity to cAMP at the time of TIMP-2
stimulation. The effects of TIMP-2 on cell growth under these in
situ conditions may be very different. In fact, we have recently
demonstrated that TIMP-2 selectively blocks the mitogenic effects of
basic fibroblast growth factor on human microvascular endothelial
cells
(17) . Thus, TIMP-2 may have distinctive effects on cell
growth depending on the specific cell type and presence of other growth
factors. We are currently investigating the interaction of signaling
pathways involved in the disparate effects of TIMP-2 on cell growth.
Cells
were treated with the maximally stimulating dose of TIMP-2 (48 nM for HT-1080 and 24 nM for Hs68). Cell lysates (20 µl)
from each treatment were tested on a Nunc 96-well plate in
quadruplicates and incubated at 30 °C for 10 min with either PKI or
cAMP or both as described under ``Experimental Procedures.''
Kemptide and
We wish to thank T. Clair for his assistance with the
8-azido-cAMP assays and Drs. E. Schiffmann and S. Aznavoorian for the
critical evaluation of this manuscript. We also thank Dr. Lance A.
Liotta for helpful suggestions and continued support.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit. These
results indicate that the
heterotrimer is dissociated
by treatment with rTIMP-2, which may facilitate the Gs
-mediated
activation of adenylate cyclase and subsequent production of cAMP.
Since cAMP binds to the regulatory subunit of cAMP-dependent protein
kinase and activates kinase activity, we evaluated how treatment with
rTIMP-2 affected both these parameters. We demonstrate in this report
that the cAMP produced in response to treatment with rTIMP-2 binds to
the type I regulatory subunit of cAMP-dependent protein kinase and
stimulates kinase activity. These results are the first demonstration
that TIMP-2 directly activates adenylate cyclase to pro-duce cAMP,
which increases cAMP-dependent protein kinase activity, resulting in
stimulation of fibroblast mitogenesis.
(
)
to inhibit proteolysis. Currently,
there are three well defined members of the TIMP family that share 40%
amino acid homology. These are TIMP-1 (2),
TIMP-2
(3, 4) , and TIMP-3
(5, 6) . The
TIMPs bind with high affinity in a 1:1 molar ratio to active matrix
metalloproteinases, resulting in loss of proteolytic activity.
Culture Conditions
Fibrosarcoma cells (HT-1080;
ATCC CLL 121) and normal human newborn foreskin fibroblasts (Hs68; ATCC
CRL 1635) were obtained from American Tissue Culture Collection
(Rockville, MD) at passage 19 and 14, respectively. The HT-1080 cells
obtained from ATCC are derived from a fibrosarcoma and have an
epithelial-like morphology with a karyotype of 2n = 46.
We used the HT-1080 cells between passages 19 and 30 and Hs68 cells
between passage 14 and 20. Cells were grown to 80% confluence in
Dulbecco's modified Eagle's media (DMEM; Life Technologies,
Inc.) containing 4500 mg/liter D-glucose,
D-glutamine, sodium pyruvate, 100 units/ml penicillin-G, 100
µg/ml streptomycin sulfate, and 10% heat inactivated fetal bovine
serum (FBS), unless otherwise indicated.
TIMP Proteins
The rTIMP-2 protein was expressed
using a vaccinia virus expression system and purified as described
previously (15). Affinity-purified rTIMP-2 was kindly provided by R.
Bird (Oncologix, Inc., Gaithersburg, MD). Native TIMP-2 was purified
from the human A2058 conditioned medium as described
previously
(3) . rTIMP-1 was isolated from the conditioned medium
of EPA-transfected Chinese hamster ovary cells (8/8 2G EPA2) (Genetics
Institute, Cambridge, MA) as described previously
(16) and then
purified by HPLC gel permeation chromatography using 50 mM
Tris-HCl, 150 mM NaCl, pH 7.5.
Thymidine Incorporation
HT-1080 and Hs68 cells
were plated at 5 10
cells/well of a 96-well Costar
plate for 18 h in DMEM with 10% FBS. The cells were then starved 18 h
in DMEM without serum. Fresh serum-free DMEM was added to the wells
prior to treatment with TIMP-2. After the indicated period of
incubation, [
H]thymidine (0.1 µCi/ml;
Amersham Corp.) was added and incubated for 2 h at 37 °C. The
percent of thymidine incorporated in a 2-h pulse correlated in a linear
fashion with the number of cells. The medium was subsequently
discarded, the wells were washed twice with phosphate-buffered saline
(PBS), and the cells were fixed in methanol:glacial acetic acid (3:1).
The incorporated [
H]thymidine was extracted as
described previously and quantitated by liquid scintillation
counting
(17) . The mean and standard deviation of triplicate
determinations were converted to percent control for graphic
representation. SQ22536 or 9-(tetrahydro-2-furyl)adenine
(Calbiochem/Novabiochem) was solubilized in sterile deionized water and
added to cells at a final concentration of 0.1
µM(18) . Affinity-purified polyclonal anti-TIMP-2
antibodies (5 µg/ml) were incubated with 24 nM rTIMP-2 for
1 h at 4 °C and 1 h with anti-rabbit affinity gel (Oraganon
Teknika, West Chester, PA). The supernatant from the immunoprecipitate
was added to the cells followed by thymidine.
cAMP Determination
Hs68 and HT-1080 cells were
plated at 1 10
cells/well of a 25-cm
Costar plate in 10% FBS for 18 h followed by an 18-h incubation
in serum-free DMEM. After the indicated period of incubation at 37
°C, the medium was discarded and the cells were washed once with
PBS. The cAMP was extracted by adding to the cells 200 µl of
Amersham's cAMP assay buffer containing 7% perchloric acid and 1
mM 3-isobutyl-1-methylxanthine (Sigma). After a 10-min
incubation at 4 °C, the cells were scraped, transferred to
siliconized Eppendorf tubes (PGC Scientific, Gaithersburg, MD) and the
precipitated protein pellet collected by a 10-min centrifugation in a
refrigerated bench-top microcentrifuge. The supernatants were
neutralized and the cAMP determined by
I-cAMP
radioimmunoassay (Amersham) according to the manufacturer's
instructions. The protein in the pellet was solubilized in 0.1
N sodium hydroxide, and the relative levels of protein were
determined by the Bradford method
(19) . The cAMP levels were
normalized to the protein concentration and expressed as fmol of cAMP
per µg of protein.
Cholera Toxin ADP-ribosylation
HT-1080 cells were
plated at 1 10
cells per 175-cm
Nunc
tissue culture flask and cultured in DMEM containing 10% FBS for 18 h
followed by 18 h in serum-free DMEM. The cells were washed in PBS,
scraped, and homogenized in 250 mM sucrose buffer containing
protease inhibitors (10 µg/ml 4-(2-aminoethyl)-benzenesulfonyl
fluoride hydrochloride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin). The homogenate was centrifuged for 5 min at 3000 rpm in a
bench-top centrifuge, and the pellet was discarded. The supernatant was
subjected to a 25-min high speed centrifugation (50,000 rpm) at 4
°C (TL100 Beckman ultracentrifuge). The pellet was resuspended in
100 µl of ribosylation buffer containing 100 mM Tris (pH
7.5), 1 mM MgCl
, 100 µM GTP, 1
mM ATP, 10 mM thymidine, and protease inhibitors
(same concentrations as above). rTIMP-2 (1 µg) was added to the
membranes as indicated in the figure legends and incubated at 30 °C
for 30 s prior to the addition of 1 µg of activated cholera toxin
(CT; List Biological Laboratories, CA) and 20 µCi of
[
P]NAD (Dupont NEN; 2 mCi/ml). The reaction was
then allowed to incubate for 20 min at 30 °C. The radioactive
membrane proteins were subsequently pelleted in a bench-top centrifuge,
washed twice in PBS, and resuspended in SDS-Laemmli sample buffer
containing 1%
-mercaptoethanol. The proteins were heated in a
boiling water bath for 2 min prior to loading and electrophoresis on a
10% Tris-glycine PAGE gel (Novex, San Diego, CA). Following
electrophoresis, the proteins were transferred onto a 0.2-µm
polyvinylidene difluoride (PVDF) membrane using 1
transfer
buffer (Novex) containing 0.001% SDS. If CT was omitted from the
reaction, the G-proteins were not labeled. The PVDF membrane was also
assayed for Gs
protein immunoreactivity by Western blot.
Nonspecific sites on the PVDF membrane were blocked using blocking
buffer containing 0.9 M NaCl, 90 mM sodium citrate
(pH 7.5), 1% non-fat dry milk, and 0.5% casein. The PVDF membrane was
then incubated with anti-Gs
antibody (Upstate Biotechnology,Inc.,
Lake Placid, NY) followed by donkey anti-rabbit horse radish peroxidase
antibody (Pierce), and the immunoreactive proteins were visualized by
chemiluminescence (Amersham) according to the manufacturer's
instructions. The extent of [
P]ADP-ribosylation
was quantitated by autoradiography of the Western blot using an Arcus
flatbed scanner (Agfa-Gevaert AG, Germany) and Image 1.49 program (NIH,
Bethesda, MD).
8-Azido PKA Labeling and Kinase Assay
Hs68 and
HT-1080 cells (1 10
) were plated on 175-cm
flasks in DMEM containing 10% FBS for 18 h followed by incubation
in serum-free DMEM for 18 h. Prior to rTIMP-2 treatment, the cells were
washed in serum-free DMEM. Cells were then incubated at 37 °C with
or without 48 nM rTIMP-2 (1 µg/ml) for 5 or 10 m. The
cells were then washed in PBS, scraped, and homogenized in buffer
containing 1% Triton X-100, 10 mM Tris (pH 7.5), 150
mM NaCl, 10% glycerol, 1 mM
3-isobutyl-1-methylxanthine, and proteinase inhibitors (described
above). 8-Azido-cAMP binding experiments utilized 2.5 µg of Hs68
and 4.5 µg of HT-1080 cell lysates. The cell lysates and 1 µg
of the positive controls were incubated with 20 µM
azido-[
P]cAMP (ICN, Costa Mesa, CA; 10
µCi/ml) for 30 min at 30 °C. The positive controls used were
bovine heart (contains 54- and 56-kDa type II regulatory subunits of
PKA), and rabbit skeletal muscle (contains the 49-kDa type I regulatory
subunit of PKA) (Sigma). Following incubation, the azido group was
activated by a 30-s irradiation at 254 nm UV light
(20) . If the
UV irradiation step is omitted, proteins were not labeled. The reaction
was stopped by adding SDS-Laemmli sample buffer containing 1%
-mercaptoethanol and heated in a boiling water bath for 2 min
prior to loading onto 8-16% Tris-glycine PAGE gel (Novex). After
electrophoresis, the proteins were Western blotted onto PVDF membrane
using 1
transfer buffer containing 0.001% SDS. The
P-labeled proteins were visualized by autoradiography of
the PVDF membrane and quantitated by densitometric analysis as
described above.
10
) were plated on 75-cm
flasks in DMEM containing 10% FBS for 18 h followed by 18 h in
serum-free DMEM. After treatment with 24 or 48 nM rTIMP-2 for
either 5 or 10 min, the cells were washed once with PBS, scraped, and
homogenized in 500 µl of extraction buffer (Life Technologies,
Inc.) containing 1 mM 3-isobutyl-1-methylxanthine and protease
inhibitors (described above). The cell lysates (20 µl) from the
samples were aliquoted in quadruplicate on a 96-well microtiter plate
(Nunc, Denmark) and incubated for 10 min with either PKA inhibitor
(PKI) or PKI plus cAMP (Life Technologies, Inc.).
Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide; Life Technologies, Inc.) and
[
-
P]ATP (20 µCi/ml; Amersham) were
added to the reaction and incubated for 10 min at 30 °C. Twenty
µl from each reaction were spotted onto phosphocellulose disks and
extracted with 1% phosphoric acid and water. The incorporated
radioactivity was quantitated by liquid scintillation. The percent
active PKA is derived from the picomoles of phosphate incorporated into
Kemptide per min as follows: (picomoles of phosphate/min inhibited by
PKI in the absence of cAMP) divided by (picomoles of phosphate/min
inhibited by PKI in the presence of cAMP) multiplied by 100 as per the
manufacturer's instructions.
Effect of TIMP-2 on Thymidine
Incorporation
Since TIMPs stimulate the growth of a variety of
cells, we determined whether TIMP-2 was mitogenic to normal human
foreskin fibroblasts (Hs68) and fibrosarcoma cells (HT-1080). Both
cells respond to rTIMP-2 exposure with a dose dependent increase in
[H]thymidine incorporation. Two hours after
exposure to rTIMP-2, the maximal stimulation in Hs68 cells is 2.5-fold
over basal rate of thymidine incorporation and occurs at a narrow range
of low nM concentration of TIMP-2 (24 nM or 500
ng/ml) (Fig. 1A,
). TIMP-2 concentrations 2-fold
greater than the maximally stimulating concentration also increases
thymidine incorporation, but to a slightly lesser degree. Further
increases in TIMP-2 concentrations above 48 nM markedly
diminished the mitogenic response in Hs68 fibroblasts. Increasing
concentrations of rTIMP-2 results in a dose dependent increase of
thymidine incorporation in HT-1080 cells similar to Hs68 fibroblasts
except that the maximal thymidine incorporation at 2 h was 1.5-fold
over the basal level of incorporation (Fig. 1A,
).
Incubating HT-1080 cells with 24 nM rTIMP-2 and native TIMP-2
(data not shown) for 24 h also stimulates a 2.5-fold increase in
mitogenesis (Fig. 1B). After a 48-h incubation with 48
nM rTIMP-2, a nearly 3-fold induction in thymidine
incorporation is observed in these cells (Fig. 1A,
). In Hs68 normal dermal fibroblasts, the response after a 2-h
treatment with rTIMP-2 is much more dramatic compared to that of
HT-1080 fibrosarcoma cells, but this difference is reduced by 24 h.
These differences probably reflect the degree of synchronization
induced by serum starvation, as well as inherent differences in the
cell cycle times for these cell lines.
Figure 1:
TIMP-2
mediated proliferation of normal dermal fibroblasts and fibrosarcoma
cells. Hs68 fibroblasts and HT-1080 fibrosarcoma cells were plated at 3
10
/well of a 96-well Costar plate in DMEM with 10%
FBS for 18 h and 18 h without serum prior to treatment with rTIMP-2.
[
H]Thymidine was added either 2 or 48 h after
treatment, and the percent thymidine incorporation was measured as
described under ``Experimental Procedures.'' Panel A represents the dose response of Hs68 (
) or HT-1080 cells
(
) after 2-h treatment with rTIMP-2 and HT-1080 cells (
)
following 48-h incubation with rTIMP-2. Panel B depicts the
ability of affinity-purified polyclonal anti-TIMP-2 antibodies (5
µg/ml) to block proliferation of HT-1080 cells following a 24-h
treatment with 24 nM rTIMP-2.
The specificity of TIMP-2
inducing this proliferative response in HT-1080
(Fig. 1B) and Hs68 cells (data not shown) is
demonstrated by the ability of affinity purified anti-TIMP-2 antibodies
to ablate this response and inability of TIMP-1 to stimulate
proliferation (Fig. 1B). Thus, the increase in thymidine
incorporation mediated by TIMP-2 is evident as soon as 2 h after
treatment and persists up to 24-48 h in HT-1080 cells. These
results demonstrate that TIMP-2 specifically mediates a proliferative
response that is not due to a contaminant in the TIMP-2 preparations
utilized for these experiments.
TIMP-2 Stimulates the Production of
cAMP
Previously, mitogenic responses have been observed by
treating fibroblasts with agents that either increase intracellular
cAMP
(21, 22) or suppress the cyclic nucleotide
phosphodiesterase activity
(21, 23) . The mitogenic
response observed by treating fibroblasts with agents such as
platelet-derived growth factor
(24) , interleukin-10
(25) ,
mastoparan (26), bombesin
(27, 28) , or vasoactive
intestinal peptide
(22) has been attributed to an increased
production of cAMP. In addition, reports have documented that ATP binds
to the A-adenosine receptor and stimulates proliferation in
fibroblasts by increasing cAMP levels and PKA
activity
(29, 30, 31) . The responses observed in
both the HT-1080 and Hs-68 cell lines following TIMP-2 treatment are
similar in magnitude to that observed if both cell lines are treated
with agents that are known to increase or mimic endogenous cAMP, such
as cholera toxin or low concentrations of dibutyryl cAMP. Hence, we
evaluated whether exposure to rTIMP-2 may stimulate the production of
cAMP.
). This rapid increase in the steady state
levels of cAMP quickly decreases to normal levels by 10 min. The
kinetics of cAMP production by Hs68 normal dermal fibroblasts in
response to rTIMP-2 treatment is different from that observed with
HT-1080 fibrosarcoma cells. Addition of 48 nM rTIMP-2
stimulates the production of cAMP as early as 30 s after treatment
(Fig. 2,
). The levels of cAMP in Hs68 cells stimulated by
a 2-min incubation with 48 nM rTIMP-2 are approximately
2.6-fold over basal cAMP levels. In these cells, the cAMP levels remain
elevated up to 10 min after addition of rTIMP-2. Clearly, the
production of cAMP in both cells is rapidly increased upon initial
exposure to low nanomolar concentrations of rTIMP-2. However, the rate
of cAMP breakdown following the initial stimulation differs
dramatically between these cells, presumably due to differential
regulation of phosphodiesterase activity. These kinetics may partially
explain the differences in the time course of the proliferative
responses of the two cell lines following rTIMP-2 treatment observed in
our initial [
H]thymidine incorporation
experiments (Fig. 1A). Nevertheless, these results
clearly demonstrate that TIMP-2 stimulates the production of cAMP which
may contribute to the mitogenic response in these cells. HPLC purified
rTIMP-1 did not stimulate cAMP production when measured in HT-1080
cells consistent with the lack of growth stimulatory response in both
cell lines to this inhibitor (Fig. 1B).
Figure 2:
TIMP-2 increases cAMP. HT-1080 ()
and Hs68 (
) cells were plated on Costar six-well plates (1
10
/well) in DMEM with 10% FBS for 18 h and in
serum-free DMEM for 18 h. At the indicated time points, 200 µl of
7% perchloric acid were added to the wells. The total cAMP in the
sample was determined by radioimmunoassay and normalized to the protein
concentration of the sample. The results are expressed as femtomoles of
cAMP/µg of protein as described under ``Experimental
Procedures.'' The left vertical axis represents the
response of HT-1080 fibrosarcoma cells, and the right vertical axis represents the response of Hs68 normal dermal
fibroblasts.
Inhibition of Adenylate Cyclase Blocks TIMP-2-mediated
Proliferation
To determine whether the increase in the steady
state production of cAMP directly contributed to the TIMP-2 mitogenic
responses, we evaluated whether blocking the production of cAMP with a
specific adenylate cyclase inhibitor, SQ22536, affected the stimulation
of proliferation by rTIMP-2. SQ22536 is a substituted adenine
derivative that binds to the P-site of adenylate cyclase and suppresses
prostaglandin-mediated proliferation. This analogue has been used in
biological systems to demonstrate that prostaglandins and
neurotransmitters
(31, 32) stimulate adenylate cyclase
activity. Treatment with SQ22536 (10M) was
not toxic to cells and did not affect basal levels of proliferation
(Fig. 3). However, pretreatment of Hs68 (Fig. 3A)
and HT-1080 (Fig. 3B) with SQ22536 prior to rTIMP-2
ablated the mitogenic response in both cell lines. These results
demonstrate that stimulation of proliferation by TIMP-2 is directly
dependent on activation of adenylate cyclase which increases the
production of cAMP in these cells.
Figure 3:
Effect of adenylate cyclase inhibitor on
TIMP-2-mediated proliferation. Hs68 (Panel A) and HT-1080
(Panel B) cells (3 10
/well) were plated on
96-well plates in DMEM with 10% FBS for 18 h and in serum-free DMEM for
18 h. Prior to stimulation, the cells were washed with DMEM without
FBS. 10
M SQ22536, rTIMP-2, and
[
H]thymidine were sequentially added to the cells
and incubated at 37 °C for 2 h. The percent of thymidine
incorporated was measured as described under ``Experimental
Procedures.'' Panel A, percent control of thymidine
incorporation in Hs68. Panel B, percent control of thymidine
incorporation in HT-1080.
TIMP-2 Stimulates Dissociation of Heterotrimeric Gs
Adenylate cyclase activity is stimulated by a
receptor-mediated activation of the GTP-binding protein,
Gs
Protein
(33) . ADP-ribosylating toxins such as pertussis toxin
(PT) and CT have been useful tools in determining the activation state
of G-proteins, because it has been previously demonstrated that
ADP-ribosylation of Gs
protein occurs only in the presence of the
subunit
(34, 35) . Previously, suppression of
PT ADP-ribosylation has been employed to characterize the receptor and
second messenger systems
(24) stimulated by thrombin
(36, 37) and vasopressin. Because ADP-ribosylating toxins, such as
CT and PT, more efficiently ADP-ribosylate the heterotrimeric
complex, the inactivation state of the
G-protein
(38) , we employed CT as a tool to evaluate whether
treatment with rTIMP-2 affected the activation state of the Gs
protein. Membranes from HT-1080 cells were isolated and pretreated with
rTIMP-2 prior to ADP-ribosylation with CT. The Gs
proteins in
these cells were identified based on susceptibility to CT-mediated
ADP-ribosylation, molecular weight, and immunoreactivity with specific
Gs
antibodies. Although two molecular mass species (46- and
52-kDa) were identified by Gs
antibody staining
(Fig. 4B), CT-mediated ADP-ribosylation favored the
46-kDa isoform (Fig. 4A). rTIMP-2 treatment
(Fig. 4A, lane 2) resulted in an approximately
40% decrease in CT-mediated [
P]ADP-ribosylation
of both the 46- and 52-kDa Gs
proteins when compared to untreated
cell membranes (Fig. 4A, lane 1). Western blot
analysis of this PVDF membrane (Fig. 4B) not only shows
that both Gs
proteins are expressed but also confirms that equal
amounts of the protein were tested in this assay. The decrease in the
radioactive labeling of these proteins (Fig. 4A)
suggests that treatment with rTIMP-2 (Fig. 4A, lane
2) results in the dissociation of the Gs
from the
subunits, a critical step in the subsequent activation of adenylate
cyclase.
Figure 4:
CT ADP-ribosylation is suppressed by
TIMP-2. HT-1080 cells (1 10
) were plated on 175
cm
flasks with FBS for 18 h and DMEM without FBS for 18 h.
The cells were washed in PBS, scraped in 250 mM sucrose
buffer, and homogenized. Equal amounts of membranes were resuspended in
100 µl of ribosylation buffer and incubated with (lane 2)
or without (lane 1) 1 µg of rTIMP-2 for 30 s prior to
addition of [
P]NAD and 1 µg of CT. The
reaction was allowed to incubate for 20 m at 30 °C. The membrane
proteins were subsequently washed in ribosylation buffer, resuspended
in Laemmli SDS sample buffer containing
-mercaptoethanol, and
boiled for 2 min. The proteins were separated on a 10% Tris-glycine
PAGE gel and transferred onto a PVDF membrane as described under
``Experimental Procedures.'' The relative quantity of the
radioactivity incorporated into the proteins was visualized by
autoradiography. Two proteins, 46 and 52 kDa, are ADP-ribosylated by CT
(Panel A) and recognized by the Gs
-specific antibody
(Panel B). The relative intensity of the
P-labeled proteins (Panel A) and immunoreactive
bands (Panel B) was quantitated by scanning the images on an
Arcus flatbed scanner, and the relative integrated intensity was
quantitated as described under ``Experimental
Procedures.''
TIMP-2 Treatment Results in cAMP Binding and Activation
of PKA
We have demonstrated that stimulation of cAMP production
by TIMP-2 is a critical step for the mitogenic stimulation in Hs68 and
HT-1080 cells. This signal transduction cascade can result in the
activation of the PKA
(39) . Hence, we evaluated if the cAMP,
produced in response to TIMP-2, bound to the regulatory subunit of PKA.
The photo-activable cAMP analog,
8-azido-[P]cAMP, was utilized for these studies
not only to radioactively label the type of regulatory subunit present
in these cells but also to test whether the endogenous cAMP produced by
rTIMP-2 treatment could interfere with the binding of this analog to
the sites on the regulatory subunit of PKA. Hs68 and HT-1080 cells were
untreated or treated with rTIMP-2, and cell lysates were generated from
these cells. Equal amounts of protein from these lysates were incubated
with 8-azido-[
P]cAMP and cross-linked to the
regulatory subunit by UV irradiation. The proteins were separated by
PAGE and transferred to PVDF as described under ``Experimental
Procedures.'' Autoradiography revealed that both Hs68 (data not
shown) and HT-1080 (Fig. 5, lane A) cell lysates
contained a single
-azido-[
P]cAMP-labeled
protein that co-migrated with the 49-kDa rabbit skeletal (Fig. 5,
lane D) type I regulatory subunit of PKA. Pretreatment of
HT-1080 cells for 30 s (data not shown) or 2 min (Fig. 5,
lane B) with 48 nM rTIMP-2 decreased the labeling of
this protein. However, 10 min after treatment with rTIMP-2
(Fig. 5, lane C), the labeling of this protein was back
to the levels observed without treatment (Fig. 5, lane
A). Addition of dibutyryl-cAMP to these lysates blocks the
P-labeling of these proteins by 8-azido-cAMP (data not
shown). These results show that the cAMP produced in response to
treatment with TIMP-2 binds to the type I regulatory subunit of PKA in
these cells. The kinetics of the cAMP response as determined by
competition for 8-azido-cAMP binding to the regulatory subunit of PKA
(Fig. 5) correlate with the results obtained by a direct
measurement of cAMP following rTIMP-2 treatment (Fig. 2).
Figure 5:
Binding of 8-azido-cAMP to type I
regulatory subunit of PKA. HT-1080 (1 10
) cells
were plated on 175-cm
Nunc flasks in DMEM with 10% FBS for
18 h and DMEM without FBS for 18 h. Prior to treatment with rTIMP-2,
the cells were washed in DMEM without FBS. Cells were either untreated
(lane A) or treated with 48 nM rTIMP-2 for 2 min
(lane B) or 10 min (lane C). 4.5 µg of the
HT-1080 cell lysates or 1 µg of positive controls were incubated
with 20 µM 8-azido-[
P]cAMP for 20
min at 30 °C. The samples were irradiated with 254 nm for 30 s, and
the reaction was stopped by adding Laemmli-SDS sample buffer containing
1%
-mercaptoethanol and boiling. The proteins were separated on a
10% Tris-glycine PAGE gel and transferred onto a PVDF membrane, and the
radioactive proteins were visualized by autoradiography as described
under ``Experimental Procedures.'' Lane D represents
the labeling of 1 µg of rabbit skeletal protein which contains the
49-kDa type I regulatory subunit of PKA. Lane E represents 1
µg of bovine heart protein which predominately contains the 54-kDa
type II regulatory subunit of PKA. The relative integrated density
units of the bands are as follows: lane A, 0.7; lane
B, 0.3; and lane C, 0.7.
Binding of cAMP to the regulatory subunit of PKA results in the
release of the catalytic subunit and increased protein kinase
activity
(39) . Therefore, we evaluated if rTIMP-2 increased the
kinase activity of PKA. In order to specifically analyze for PKA
activity, we utilized an assay system that measures the relative
phosphorylation of Kemptide in the presence and absence of a specific
PKA inhibitor, PKI. By using PKI in the presence and absence of cAMP,
the contribution of other kinases that may phosphorylate kemptide was
eliminated. Treatment of Hs68 normal dermal fibroblasts with 24
nM rTIMP-2 increased by 2- and 3-fold the kinase activity of
PKA at 5 and 10 min, respectively (). Stimulation of the
HT-1080 cells with 48 nM rTIMP-2 for 5 min also increases the
kinase activity 2-fold (). However, 10 min after treatment
with 48 nmol of rTIMP-2, the PKA activity in HT-1080 cells falls back
to normal levels. These results are consistent with the cAMP
determinations (Fig. 2) and the 8-azido-cAMP competition studies
(Fig. 5).
, -
, -
, ICER, and CREB-2 are also activated and
induced by cAMP and PKA. The activation of both activators and
repressors functions to fine tune and coordinate cellular responses to
cAMP production. Depending on the cell type and the concentration of
cAMP, these events may result in differentiation or
proliferation
(39, 43) .
subunit which results in its dissociation from G
.
The free G
subunit, in turn, activates effector functions such as
the adenylate cyclase activity
(33) . Toxins have been useful in
delineating the activation state of G-proteins because they selectively
recognize the inactive or heterotrimeric G-protein and transfer an
ADP-ribose moiety from NAD to the G
subunit
(34, 35) . The ability of ligand treatment to
suppress toxin ADP-ribosylation is believed to be due to dissociation
of the heterotrimeric complex or activation of G
subunit.
Therefore, the decrease in CT-mediated ADP-ribosylation of the Gs
by treatment with rTIMP-2 suggests that a receptor-ligand interaction
has induced the dissociation of Gs
from G
. The
dissociated Gs
may subsequently activate adenylate cyclase to
produce cAMP. These results imply that the putative TIMP-2 receptor may
be a G-protein-coupled receptor. Confirmation of this observation
awaits isolation and characterization of these receptors.
Table:
TIMP-2 increases PKA activity
-[
P]ATP was subsequently added
and incubated for 10 min at 30 °C. Aliquots (20 µl) from each
reaction solution were spotted on phosphocellulose disks and were
washed twice with 1% phosphoric acid and twice with water. The
picomoles phosphate incorporated into Kemptide/min by PKI-sensitive
kinase activity was determined by liquid scintillation. This assay
demonstrated a 1-6% error in the quadruplicate determinations for
phosphorylated Kemptide. The percent active PKA is the TIMP-2-activated
kinase activity (picomoles phosphate incorporated/min) divided by the
AMP-activated kinase activity (picomoles phosphate/min) multiplied by
100 as per the manufacturer's instructions. The relative PKA
activity column represents the kinase activity as a percentage of the
maximally stimulated activity obtained by adding cAMP to the cell
lysates.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.