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Reproductive Endocrinology Center (H.L., R.I.W.), University of California, San Francisco, San Francisco, California 94143; Laboratoire de Biologie Moleculaire et de Genie Genetique Universite de Liege (I.S., J.M.), Departamento De Fisiologia, Instituto de Investigaciones Biomedicas (C.C.), Universisdad Nacional Autonoma de Mexico, 04510 Mexico D.F., Mexico
Address all correspondence and requests for reprints to: Richard I. Weiner, Reproductive Endocrinology Center, University of California, San Francisco, San Francisco, California 94143. E-mail: richard_weiner{at}quickmail.ucsf.edu
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Abstract |
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Introduction |
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Both the 16 kDa N-terminal fragment of rat PRL (16K rPRL) (7) and human PRL (16K hPRL) are potent antiangiogenic factors in vivo in the chicken chorioallantoic membrane assay (10). The antiangiogenic action of 16K PRL appears to be mediated at multiple steps in the formation of new vessels. 16K PRL inhibits bFGF and VEGF-induced cell proliferation of cultured capillary endothelial cells from bovine and human (7, 10). Furthermore, 16K PRL inhibits the organization of BBEC cultured in type 1 collagen gels into capillary-like structures (10), and the migration of BBEC through the pores of collagen coated filters in Boyden chambers (data not shown). These observations led us to address the mechanisms by which 16K PRL activated cell invasiveness and migration.
Cellular invasiveness in various biological processes, including angiogenesis, requires the activation of proteases capable of degrading extracellular matrixes (11). Urokinase (urokinase type plasminogen activator, uPA) appears to be a key modulator in the neoplastic invasive process (12, 13). Urokinase converts widely distributed and inactive plasminogen into plasmin, a tryptic protease capable of degrading certain matrix components, and activating other matrix degrading enzymes like the metalloprotease, collagenase (14). Additionally, urokinase has also been demonstrated to be important in the migration of the capillary endothelial cells through the interstitial matrix (15, 16, 17).
Urokinase activity is regulated by the rate of its synthesis, conversion of the proenzyme to the active form of the enzyme, and the presence of the specific inhibitors of the enzyme activity (12). Plasminogen activator inhibitor-1 (PAI-1), a major endothelial cell derived component of the extracellular matrix, is believed to protect extracellular matrix proteins from excessive plasminogen activator catalyzed proteolysis (18, 19, 20, 21). PAI-1 irreversibly binds to and inactivates uPA (22). A balance between urokinase and PAI-1 levels has been proposed to be important in the regulation of angiogenesis. Consistent with an important role in the control of angiogenesis, PAI-1 gene expression is highly regulated, e.g. in vitro TGF-ß stimulates PAI-1 expression and is a potent antiangiogenic factor (23).
We hypothesized that the antiangiogenic actions of 16K PRL on cell invasiveness and migration could be mediated via the regulation of uPA activity. Data from in vitro experiments with BBEC showed that 16K PRL inhibits uPA activity apparently via stimulation of the expression of PAI-1. The stimulatory effect of 16K PRL on PAI-1 is at the transcriptional levels and is not dependent on the action of bFGF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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16K rPRL. Rat 16K PRL (16K rPRL)was generated by enzymatic cleavage of the intact, 23 kDa rat PRL (NIH, B-6), as previously described (7). Briefly, rPRL was cleaved by incubation with a particulate fraction from rat mammary gland homogenates, reduced in 2-mercaptoethanol, and the 16 kDa N-terminal fragment separated by gel filtration on Sephadex G-50. To prevent reformation of disulfide bonds, the 16K rPRL was carboidomethylated. 16K rPRL was reduced with DTT (20-fold molar excess over the disulfide bond molecular content) in the presence of 4 M guanidine HCl in 0.1 M NH4HCO3, pH 8.5, and subsequently alkylated with a 40 M excess of iodoacetamide over the DTT concentration. The sample was dialyzed, and its purity and concentration were assessed by SDS-PAGE and densitometry.
Cell culture
BBEC were isolated as previously described (25). The cells were
grown and serially passaged in low glucose DMEM supplemented with 10%
calf serum, 2 mM L-glutamine, and antibiotics
(100 U penicillin/streptomycin per ml and 2.5 mg of fungizone per ml).
Basic FGF (bFGF, Promega) was added (1 ng/ml) to the cultures every 2
days. Experiments were initiated with confluent cells between passages
5 and 13.
Cell stimulation and preparation of cell extracts
Confluent culture of BBEC were enzymatically dispersed and
plated at a density of 5 x 105 cells per well in
6-well plates in 1 ml of BBEC media. Twenty-four hours after plating,
cells were transferred to MEM containing low serum for 24 h (0.5%
calf serum). Cells were treated with bFGF or 16K PRL alone or together
for 16 h. The incubation was terminated by aspiration of the media
and addition of 200 µl of the lysis buffer (0.1 M Tris,
pH 8.1, and 0.5% Triton X-100) shake at 4 C for 20 min. The insoluble
fraction was removed by centrifugation at 4 C for 10 min at 14,000
x g, and the protein concentrations of the soluble fraction
were determined by BCA assay kit (Pierce, Rockford, IL).
Plasminogen activator substrate gel
Plasminogen activator (PA) substrate gels were performed
according to the method described by Blei et al. (26) with
minor modification. Briefly, aliquotes of conditioned medium and cell
extracts were separated under nonreducing conditions by SDS PAGE using
a 4% stacking and 10% resolving gel. The SDS gel was washed three
times (20 min each) in 2.5% Triton X-100 to remove the SDS and
overlaid on a fibrin-agar indicator gel as described above. The gel
complex was incubated overnight at 4 C to enable proteins in the
SDS-PAGE gel to diffuse into the substrate gel. Zones of lysis that
developed following incubation at 37 C (1–5 h) indicated PA activity.
The reaction was terminated by incubation with 10% acetic acid/40%
methanol for 20 min. The gel was stained in 1% amido black in 10%
acetic acid/40% methanol for 10 min and destained in 10% acetic
acid/40% methanol with several changes until the solution was clear.
The stained gels were dried and photographed on a light box. The bands
were scanned and quantified by the Scan Analysis Program.
RNA preparation
For the extraction of total RNA BBEC cultured on 10-mm dishes,
cells were lysed in guanidinium solution (4 M guanidinium
thiocyanate/25 mM sodium citrate, pH 7.0/0.5% sarcosyl/0.1
M 2-mercaptoethanol). The resulting lysates were layered on
5.7 M CsCl and centrifuged at 40,000 rpm for 12 h. The
RNA pellets were dissolved in DEPC treated H2O and
integrity of the RNA was assessed by electrophoresis on agarose gels.
The RNA concentrations were determined by spectrophotometry.
Northern blot analysis
For Northern blot analysis, RNA samples were electrophoresed in
1% agarose gels containing 2.2 M formaldehyde, 20
mM 3-(N-morpholino) propanesulfonic acid (MOPS,
Fisher Scientific, Pittsburgh, PA), 8 mM sodium
acetate, and 1 mM EDTA. RNA was transferred onto nylon
membranes (N-Hybond, Amersham) and covalently cross-linked
by irradiation with 120 mJ of UV light. 1535 bp of
EcoRI HinDIII fragment of Bovine urokinase cDNA (kindly
provided by Dr. W. D. Schleuning, Research Laboratory of Schering AG,
Berlin, Germany), 600 bp of PstI fragment of PAI-1
cDNA (kindly provided by Dr. M. J. Pepper, University Medical
Center, Geneva, Switzerland) and 110 bp of PstI
fragment of cyclophillin cDNA (kindly provided by Dr. M. Skinner) were
labeled by [32-P]dCTP with oligolabeling kit (Pharmacia,
Piscataway, NJ) and purified by Quick Spin columns (Boehringer
Mannheim, Indianapolis, IN). The hybridization reactions were
performed at 68 C for 1 h in Quickhyb solution (Stratagene, La
Jolla, CA). The hybridized blots were washed twice with 2
x SSC/0.1% SDS at RT for 15 min and once with 0.1 x SSC/0.1%
SDS at 65 C for 30 min. The resulting blots were subjected to
autoradiography. Quantification of the messenger RNA (mRNA) levels was
performed by densitometric scanning of the autoradiograms of
cyclophillin or methylene blue staining of 28S and 18S ribosomal
RNA as an internal control.
RNase protection assay for bovine urokinase
RNase protection assays were performed according to the
protocols of Werner et al. (27). The 445 bp
EcoRI-BglII fragment of the bovine urokinase cDNA
was transcribed with 32P uridine triphosphate using
T7 promoter to generate a radiolabeled riboprobe. Ten micrograms of the
RNA isolated from BBEC were hybridized with 2.5 x
105cpm of the riboprobes at 42 C for 12 h, digested
with RNase A and T1, and resolved on 8 M urea: 5%
polyacrylamide gels. Gels were dried and exposed to Kodak X-OMAT AR
film (Eastman Kodak, Rochester, NY) for 24–72 h with intensifying
screen to visualize the protected fragments.
Western blot analysis for bovine PAI-1
Cell homogenates or conditioned media from BBEC were resolved by
SDS/PAGE (4–10%) and transferred to nitrocellulose membrane using a
semidry transfer apparatus. The transfer blots were stained with
Ponceau Red for 1 min to visualize the even transfer of the proteins.
The blots were blocked with 5% milk in Tris-buffered saline containing
0.1% Tween 20 for 1 h and incubated with antibovine PAI-1 mouse
monoclonal antibody at a 1:2,000 dilution for 2 h (Gibco BRL, Life
Technologies, Gaithersburg, MD). The antigen-antibody complexes were
detected with horseradish peroxidase conjugated secondary antibody and
the enhanced chemiluminescence system (ECL, Amersham Life Science,
Arlington Heights, IL). The blots were exposed on reflection NEF
films (DuPont NEN, Boston, MA) to visualize the bands.
Immunoprecipitation of bovine PAI-1
Subconfluent cultures of BBEC grown on 60-mm Petri dishes were
incubated in MEM contain 0.5% calf serum for 24 h. Cells were
treated with bFGF (5ng/ml) or 16K PRL (10 nM) alone or
together for 16 h. The medium were removed, and the treated cells
were washed 3 times with methionine free MEM. To metabolically label
the protein pool, the washed cells were incubated in 500 µl of the
methionine free MEM and 50 µCi of the
L-[35S]methionine (Dupont NEN) was added to
the cells for 4 h. Conditioned media were collected. Cell extracts
were prepared by dissolving the cells in 1 ml of 0.1 M
Tris-HCl, pH 7.5 containing 0.5% Triton X-100, 0.1% SDS, 0.05% Tween
80, 0.15 M NaCl, and protease inhibitors (0.14 U aprotinin,
1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
20 µM leupeptin, final concentrations) at 4 C for 20 min
with shaking. The insoluble fraction was removed by centrifugation at 4
C for 20 min at 14,000 x g. Before
Immunoprecipitation, the samples were diluted 1:1 with 0.1
M Tris-HCl, pH 7.5, and precleared with 40 µl of 50%
(vol/vol) protein A Sepharose (Sigma) for 2 h at 4 C. The
resulting lysates were incubated with 5 µg of the antibovine PAI-1
(Gibco BRL, Life Technologies) or 5 µg normal mouse IgG at 4 C for
1 h. Fifty microliters of the 50% protein A Sepharose were added,
and the samples were incubated at 4 C for another hour. The
immunoprecipitates were washed 3 times with 0.1 M Tris-HCl,
pH 7.5/0.5%, Triton X-100, and finally dissolved in reducing Laemmli
sample buffer. The immunoprecipitated proteins were analyzed by
discontinuous 4–10% PAGE, and the gel dried and subjected to
autoradiography.
RNA half-life determination
BBEC were initially treated with 10 nM 16K hPRL for
6 h to stimulate PAI-1 mRNA expression. The treated BBEC were then
washed and fresh medium containing actinomycin D (3 µg/ml) added to
arrest transcription. Cells were harvested 0, 1, 4, and 8 h after
actinomycin D treatment. RNA samples were collected and studied by
Northern blot analysis.
Statistical analysis
Data are presented as the mean ± SE. Data were
statistically analyzed by one-way ANOVA followed by Fisher’s protected
least significant difference (StatView, Abacus Concepts, Berkeley, CA).
A P value of < 0.05 was considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This observation is
consistent with earlier reports that uPA is the predominant plasminogen
activator in bovine capillary endothelial cells (26, 28). No observable
lysis band was seen at 70 kDa, the expected size for tPA activity. Rat
16K PRL (40 nM) almost totally inhibited the bFGF-induced
increase in urokinase activity. At 1 nM concentration, 16K
hPRL inhibited both basal and bFGF-stimulated uPA activity (Fig. 1b).
These observations were repeated in three independent experiments with
16K rPRL and 3 times with 16K hPRL. Similar observations were made in
the secreted fraction (conditioned media) from treated cells (data not
shown). Importantly, 16K PRL inhibited basal urokinase activity to
almost undetectable levels. The increased potency of 16K hPRL compared
with 16K rPRL is consistent with differences seen on the inhibition of
mitogen-induced BBEC proliferation (10).
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), a–d. Treatment with 40
nM 16K rPRL had no effect on the stimulation of uPA mRNA
levels by bFGF nor on basal levels (Fig. 2, a–d). Similar results were
obtained in three independent Northern blot analyses and two RNase
protection assays. These findings suggested that the blockage in uPA
activity was not dependent on changes in the level of uPA mRNA.
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). This
observation was confirmed in three independent experiments. The
stimulation of cell associated and secreted PAI-1 by 16K PRL was first
observed at 1 h and was maximal by 8 h in the cell associated
fraction and by 24 h in the secreted fraction in time course
experiments (Fig. 4). The same pattern
was observed in two additional experiments. In the three experiments,
the maximum stimulation in the secretory fractions at 24 h varied
from 10- to 50-fold. The decrease in the cell associated PAI-1 at
24 h likely reflects the saturation of cell associated binding
sites and release into the media. Treatment with 10 ng/ml of bFGF
slightly stimulated cell associated PAI-1 protein levels (Fig. 3). The
stimulation by bFGF was additive with the effect of 16K PRL.
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, a and b). Treatment
with bFGF at 5 ng/ml had only a minor effect on the metabolic labeling
of PAI-1 in the cell associated fraction and no effect on PAI-1 in the
secreted fraction. The cotreatment of cells with bFGF and 16K hPRL
resulted in a decrease in the PAI-1 levels in the secreted fraction
compared with treatment with 16K hPRL alone. As will be discussed this
is likely due to the increased formation of PAI-1/uPA complexes
resulting from the stimulation of uPA by bFGF. These findings were
consistent with the increase in PAI-1 levels stimulated by 16K PRL
being associated with an increase in protein synthesis. The finding
that a larger proportion of the secreted PAI-1 was labeled suggests
that the newly synthesized PAI-1 is preferentially released. We then
asked if the increase in protein synthesis was associated with a change
in the level of PAI-1 mRNA.
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, a and b). Values were corrected for
loading relative to the intensity of the 28S and 18S bands. These
observations were confirmed in three independent experiments. Treatment
with 10 ng/ml of bFGF had no effect on PAI-1 mRNA levels, nor did it
affect the 16K PRL-induced stimulation.
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, 16K hPRL treatment did not
significantly affect the rate of disappearance of PAI-1 mRNA. This
observation is consistent with 16K PRL increasing the rate of
transcription of the PAI-1 gene rather than affecting mRNA
stability.
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Addition of 1 or 2 µg of recombinant PAI-1 to lysates of BBEC
resulted in the immunolocalization with an antibody to human PAI-1 of a
new band migrating at 99 kDa (Fig. 8a).
This is consistent with the expected size of the PAI-1/uPA complex. The
uPA activity of these lysates in the PA substrate gel assay was
partially inhibited by the addition of 1 µg of recombinant PAI-1, and
totally inhibited by the addition of 2 µg (Fig. 8b).
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. The more slowly migrating
90-kDa band was not seen in autoradiogram of the immunoprecipitates of
PAI-1 in Fig. 5 because the antibody against PAI-1 used did not
recognize the PAI-1/uPA complexes. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatment of BBEC with 16K PRL decreased uPA activity measured in substrate gels. The decrease in uPA activity did not appear to be due to a decrease in uPA gene expression as assessed by measurement of uPA mRNA levels by Northern analysis or an RNase protection assay. The direct measurement of uPA protein levels was not possible because no antibody to bovine uPA was available for Western blot or immunoprecipitation studies. In several systems, increases in uPA protein levels have been shown to be associated with increases in uPA mRNA levels (29, 30). Therefore, although we cannot exclude the possibility that 16K PRL decreases the levels of uPA, the lack of change in uPA mRNA levels is consistent with no substantial change in uPA levels.
An important component of the regulation of uPA activity is the level of production of the plasminogen inhibitor PAI-1. Treatment with 16K PRL results in a substantial increase in the level of PAI-1 protein in BBEC. The stimulation of PAI-1 by 16K PRL is independent of the action of bFGF. The 16K PRL-induced increases in PAI-1 levels were rapid and were first detected at 1 h and reached a maximum by 8 h. The increase in PAI-1 synthesis appeared to be associated with an increase in the level of expression of the PAI-1 gene. PAI-1 mRNA levels were increased following treatment with 16K PRL, and the increase in mRNA levels was not associated with any change in mRNA stability, findings consistent with the stimulation of the rate of transcription of the PAI-1 gene.
PAI-1 is known to form a complex with uPA that results in inactivation of the proteolytic activity of uPA, which can be measured in substrate gels. The formation of the complexes results in a band shift of uPA in Western blots to a higher molecular mass (90 kDa) (31). Addition of recombinant PAI-1 to BBEC lysates, caused a decrease in urokinase activity in substrate gels and formation of the 90 kDa uPA/PAI-1 complex in Western blots. Long-term exposure of the PAI-1 Western blots of lysates from 16K PRL treated BBEC, in which the levels of PAI-1 were elevated, also showed the presence of the 90-kDa complex. These results are consistent with the explanation that the decrease in urokinase activity in the 16K PRL-treated BBEC is due to the increase of PAI-1 production.
Because LPS has been shown to stimulate PAI-1 production in endothelial cells (32), it was essential to show that the effect of recombinant 16K hPRL was not due to bacterial contamination. However, a 100-fold excess of the level of LPS found in the recombinant 16K hPRL preparations used only mildly stimulated PAI-1 production in BBEC precluding this possibility (data not shown). 23KhPRL contained a similar amount of LPS but had no effect on PAI-1 production in BBEC. Furthermore, this was not an issue with the 16K rPRL used because it was made by enzyme cleavage and contained low levels of LPS.
It appears that stimulation of PAI-1 expression may be the common
action of several antiangiogenic factors in addition to 16K PRL
including: thrombospondin (33); TGFß (23); LIF (34); TNF-
(35, 36); and antiangiogenic steroids (26). In addition, the antiangiogenic
steroid, medroxyprogesterone acetate, inhibited urokinase activity by
stimulating PAI-1 expression (26).
Tissue remodeling associated with angiogenesis requires the delicate regulation of the proteolytic processes. Increased urokinase activity is necessary for degradation of the extracellular matrix and may also be involved in cell migration of capillary endothelial cells (15, 16, 17). Excess PAI-1 might result in endothelial cells being unable to penetrate through the basement membrane. Consistent with the hypothesis, addition of exogenous PAI-1 has been shown to inhibit endothelial cells migration (37). Antiangiogenic factors that have been shown to increase PAI-1 levels also inhibit capillary endothelial cell migration, i.e. 16K PRL, TGFß (38), thrombospondin (6), and LIF (34). These findings strongly support the conclusion that an important component of the antiangiogenic action of 16K PRL is mediated via the stimulation of PAI-1 production.
Received January 15, 1998.
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