From the
A hitherto unknown function of midkine (MK) was found in the
regulation of fibrinolytic activity of vascular endothelial cells.
Recombinant murine MK enhanced plasminogen activator (PA)/plasmin
levels in bovine aortic endothelial cells (BAECs) in a dose- and
time-dependent manner. After incubation with 10 ng/ml MK for 18 h, PA
and plasmin levels increased 6- and 4-fold, respectively. This effect
was attributed to a moderate up-regulation of urokinase-type PA
expression as well as to a significant down-regulation of PA
inhibitor-1 (PAI-1) expression. BAECs constitutively synthesized and
secreted MK and its production was enhanced 2-fold with 1
µ
M retinoic acid or 10 µ
M retinol. It was
found that MK served as a substrate for tissue transglutaminase. In the
culture medium, MK existed as a transglutaminase-mediated complex of 36
kDa. Addition of anti-MK antibody to BAEC cultures resulted in a
decrease of basal PA activity and an increase of basal PAI-l levels and
attenuated the ability of retinol to enhance PA activity 50% and
potentiated the ability to increase PAI-1 levels 4-fold. Furthermore,
MK and basic fibroblast growth factor (bFGF) acted more than additively
in enhancing PA levels. We conclude that in BAECs MK is a novel
autocrine factor sustaining the fibrinolytic property. MK functions as
a mediator of retinoid and cooperates with bFGF to enhance fibrinolytic
activity of BAECs.
Retinol (vitamin A) and its derivatives (retinoids) have
profound effects on the regulation of cell growth and differentiation
(1) . In an attempt to identify a gene responsible for the
control by retinoids, midkine (MK)
Vascular endothelial
cells play key roles in the maintenance of fibrinolysis. They produce
and secrete two immunologically distinct plasminogen activators (PAs),
tissue-type PA, and urokinase-type PA (uPA), in different ratios
depending upon the origin of the cells. Endothelial cells also produce
and secrete an inhibitor of PA, PA inhibitor-1 (PAI-1), that rapidly
inhibits the activity of both PAs
(15) . Hence, the balance
between the production of PAs and PAI-1 from endothelial cells is
important to maintain a normal fibrinolytic status. It has been
demonstrated that fibrinolysis reactions proceed on the surface of the
cells and/or extracellular matrix and that these surface reactions are
physiologically significant
(16, 17, 18) .
Although the primary physiological role of plasmin is to dissolve
fibrin clots, another important role of plasmin, especially
cell-associated plasmin, is to generate the active form of other
enzymes
(19) and cytokines
(20) . Furthermore, the
physiological relevance of cellular PA/plasmin in cell migration or in
metastasis has been pointed out
(21) .
Several functions of
endothelial cells are under the control of retinoids. An augmentation
in collagen production was first reported
(22) . Successively,
it has been demonstrated that retinoids enhance the PA/plasmin levels
of bovine endothelial cells through up-regulating both uPA and uPA
receptor expressions
(23, 24, 25, 26) .
Retinoids also enhance the synthesis of tissue type II transglutaminase
in bovine aortic endothelial cells (BAECs; Ref. 27). Because the
activation of latent transforming growth factor-
Since it has been
reported that MK, another retinoid-related cytokine, acts as a mediator
in retinoic acid-induced differentiation of P19 cells
(32) and
growth suppression of HL-60 cells
(33) , we have tested whether
MK affects endothelial cell functions using BAEC cultures. As the first
step, we have investigated the effect of MK on PA/plasmin levels in
BAECs. Here, we report that both exogenously added and endogenously
produced MK enhance PA/plasmin levels in BAEC cultures by stimulating
uPA expression and suppressing PAI-1 expression. Enhancement of
fibrinolytic activity is a hitherto unknown role of this cytokine and
may characterize MK as an autocrine factor involved in the regulation
of endothelial cell functions. In addition, we provide the evidence
that MK is a good substrate for tissue transglutaminase.
The present study demonstrates that MK enhances the
PA/plasmin activity of bovine vascular endothelial cells through
up-regulating uPA expression as well as down-regulating PAI-1
expression. This novel activity of MK is not due to a contaminate in
the MK preparation. Anti-MK antibody, which was affinity-purified using
a bacterial MK-fusion protein, abolished the enhancement activity of
MK. Chemically synthesized human MK showed an activity
indistinguishable from recombinant murine MK used herein
(44) .
We have further showed that MK is constitutively produced from BAECs
and that retinoids increase its production. BAECs expressed the same
molecular size of MK mRNA as reported previously
(3) , whereas
in the culture medium, a retinoid-sensitive MK-related band of 36 kDa
was detected with anti-MK antibody. The specificity of this antibody
has been guaranteed; it reacts so specifically with the C-terminal tail
of MK molecule
(45) that it does not react with any proteins,
even with PTN
(34) . We believed that this was not the result of
loose specificity of anti-MK antibody, but was a peculiar phenomenon
observed in BAECs, implying previously undetected modification of MK
molecules after synthesis from the cells. We investigated the
characteristics of these SDS- and
MK can be classified as a new member of
PA-inducible autocrine factors in endothelial cells, especially in the
case of retinoid-stimulated cells. Several cytokines such as
interleukin-1, tumor necrosis factor-
Retinoids regulate the
expression of certain genes and control behaviors of endothelial cells
(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 51) .
From the perspective of the regulation of the fibrinolytic status,
retinoids make endothelial cells more fibrinolytic and more
anti-thrombotic through the induction of PA/plasmin
(23, 24, 25, 26) and thrombomodulin
(51) . However, certain mechanism may be needed to suppress
excess of fibrinolytic activity. Therefore, retinoids may induce
TGF-
Finally, the present finding is important in considering MK function
in general. Because it has been reported that PA activity is associated
with neurite outgrowth and bone remodeling, the activities associated
with MK
(52, 53) , it is possible that some of the
previously reported activities of MK are mediated via the enhancement
of PA activity. PA is widely expressed in tumor cells and promotes
tumor cell invasion in vitro (21) . It will be of great
interest to examine whether MK, which is expressed in many human
carcinoma cells
(9) , also contributes to increase PA activity
in tumor cells.
We thank Dr. Daniel B. Rifkin for his critical
comments of this study and for correction of the English.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
was found as
a gene product whose expression was specifically induced by retinoic
acid in an embryonal carcinoma cell line
(2) . Among three
different MK cDNAs a 730-base pair cDNA produced major mRNA from which
15.5-kDa protein with a 2.5-kDa signal sequence was synthesized
(3) . The mature secreted form of MK has a molecular mass of 13
kDa and has a strong heparin binding activity
(4) . MK is
neurotrophic; it enhances neurite outgrowth and survival of various
embryonic neuron types
(5, 6, 7, 8) and
is mitogenic for certain fibroblastic cell lines
(5, 6) . Increased expression of MK was detected in many
human carcinoma cells
(9) . MK forms a family of structurally
related polypeptides with heparin-binding growth-associated molecule
(10) /pleiotrophin (PTN; Ref. 11) and with retinoic acid-induced
heparin-binding protein
(12) . Thus, MK is the first member of a
new protein family of developmentally regulated cytokines with diverse
biological activities
(13, 14) .
(TGF-
) is
performed by surface plasmin, elevation of surface plasmin and
transglutaminase levels by retinoids causes the formation of active
TGF-
(28, 29) , and TGF-
generated mediates
some of retinoid effects on endothelial cells such as suppression of
cell migration
(28) and enhancement of PAI-1 expression
(29) . Moreover, several other changes induced by retinoids in
endothelial cells are associated with the formation of TGF-
(30, 31) , indicating a strong linkage between retinoids
and TGF-
in endothelial cell biology.
Materials
Preparation of recombinant murine MK
and its neutralizing antibody were described previously
(5, 34) . The antibody was specific to MK and did not
cross-react with basic fibroblast growth factor (bFGF) nor with PTN
(34) . All- trans-retinol and retinoic acid were bought
from Sigma. Recombinant human bFGF, bovine serum albumin (BSA), human
urokinase, and guinea pig liver transglutaminase were from R&
(Minneapolis, MN), Miles (Kankakee, IL), Calbiochem, and Takara
Biochemicals (Ohtsu, Japan). Stock solutions of retinoids were prepared
in ethanol and were serially diluted into culture medium to yield a
final ethanol concentration of 0.5%
(28) .
Cellular PA Assay
BAECs were isolated and grown in
minimal essential medium (
MEM) containing 10% calf serum.
After cells were grown to confluence in a 96-well culture plate, the
cultures were rinsed with phosphate-buffered saline, pH 7.4, and
incubated in 100 µl of serum-free
MEM containing 0.1% BSA
(
MEM-BSA) plus various concentrations of MK. At the indicated
time, the medium was aspirated, the cultures washed with
phosphate-buffered saline, cellular PA extracted into 100 µl of
0.5% Triton X-100 in 0.1
M Tris-HCl, pH 8.1, and PA levels of
the extracts measured using the chromogenic substrate S-2251
(35) . PA activity was expressed as urokinase units/mg of
protein in the sample. Protein concentration was measured by BCA
(Pierce) assays using BSA as the standard.
Zymography and Reverse Zymography
Zymography and
reverse zymography were carried out according to the methods of
Loskutoff and Schleef
(36) . After proteins in the culture
medium were separated by SDS-polyacrylamide gel electrophoresis, the
gels were washed with 2.5% Triton X-100 and with phosphate-buffered
saline, applied onto fibrin-agar gels containing plasminogen without
(zymography) or with (reverse zymography) urokinase, and incubated at
37 °C. The uPA and PAI-1 visualized, respectively, as either a
lysis band in the zymography or a lysis-resistant band in the reverse
zymography.
Isolation of RNA and Northern Blot Analysis
Total
RNA was extracted from cells treated or untreated with MK or retinol
using acid guanidinium thiocyanate-phenol-chloroform extraction method
(37) . Each RNA (20 µg) was separated through either 1 or
1.6% agarose-formaldehyde-gel electrophoresis and transferred to BA85
nitrocellulose membranes (Schleicher & Schuell) according to the
published protocols
(38) . Membranes were hybridized with cDNA
for either bovine uPA, bovine uPA receptor (gifts from Dr. Wolf-Dieter
Schleuning, Research Laboratories of Schering AG, Berlin), human PAI-1
(gift from Dr. David J. Loskutoff, The Scripps Research Institute, La
Jolla, CA), or human MK
(9) and rehybridized with a probe for
glyceraldehyde-3-phosphate dehydrogenase. The cDNA probes were labeled
with [-
P]dCTP (DuPont) via random priming
using the kit supplied from Boehringer Mannheim
(39) .
Conditions for hybridizations and washings were described previously
(28) . Autoradiographyies were performed using Fujix BAS 2,000
Bio-imaging analyzer (Fuji Photo-Film, Tokyo). Each band was scanned,
and the signal intensity normalized to that obtained with
glyceraldehyde-3-phosphate dehydrogenase.
Fibrin Underlay Technique
Fibrin underlay
technique was carried out to observe fibrinolysis by endothelial cells
grown in the culture medium by the method described previously
(25) . The dissolution of a fibrin layer plated under the cells
was observed from the surroundings of each cell, reflecting cell
surface plasmin activity.
Assay of Cell-associated Plasmin Activity
Plasmin
bound to the cell surface was recovered with tranexamic acid and
assayed as described previously
(40) .
Western Blotting
Western blotting was performed
using affinity purified rabbit anti-MK antibody (final 5 µg/ml) and
goat anti-rabbit IgG antibody conjugated with peroxidase (Bio-Rad) by a
modification of the method described previously
(34) . The
signals were detected using Amersham (Buckinghamshire, United Kingdom)
ECL system.
Measurement of PAI-1 Levels in the Culture
Medium
The amount of PAI-1 antigen in the culture medium was
determined with specific enzyme-linked immunosorbent assay kit obtained
from Biopool (Umeå, Sweden). This assay uses the double-antibody
sandwich principle with peroxidase-conjugated murine anti-PAI-1
monoclonal antibody and the enzyme-linked colorimetric reaction with
ortho-phenylenediamine dihydrochloride
(41) .
Enzyme Immunoassay (EIA) for MK
EIA for MK was
performed using affinity-purified anti-MK antibody, the biotin-labeled
antibody, and streptavidin-labeled -galactosidase (Boehringer
Mannheim), adopting the procedure of EIA for nerve growth factor
(42) . The enzymatic activity was detected fluorometrically
using 4-methylumbelliferyl-
-
Dgalactoside (Sigma). Murine
MK in the range of 0.3-10 ng/ml can be measured. The details of
the EIA and the application will be described elsewhere.
(
)
Effect of MK on PA Levels in BAECs
In order to
know whether MK affects fibrinolytic property of endothelial cells, we
first determined the effect of addition of purified MK on cellular PA
levels in BAECs. After confluent BAEC cultures were incubated for 24 h
in serum-free medium containing the indicated amounts of recombinant
murine MK, cell lysates were prepared, and the levels of PA activity in
the lysate were measured (Fig. 1). MK increased PA activity
levels in a dose-dependent manner. Fig. 2 depicts the time course of MK
augmentation of PA activity with 10 ng/ml MK. Until 18 h PA activity
increased proportionally to the period of treatment of BAECs with MK.
About a 67-fold increase was achieved. Longer incubation, up to 48
h, decreased the PA activity. However, the cells still maintained
3-fold higher levels than the untreated control levels. A similar
enhancement of cellular PA activity by MK was observed using chemically
transformed BAECs. In this case, PA activity levels increased from 15
urokinase units/mg of protein to 42 urokinase units/mg protein after
incubation for 18 h with 10 ng/ml MK. MK itself did not express plasmin
activity nor directly activate plasminogen (data not shown). These data
indicate that MK enhances PA activity levels in BAECs.
Figure 1:
Dose-dependent enhancing effect of MK
on PA activity levels in BAECs. Confluent BAEC cultures were incubated
with the indicated amounts of MK in MEM-BSA for 24 h, and cellular
extracts were prepared as described under ``Experimental
Procedures.'' PA activity levels in each extract were measured
using the chromogenic substrate and expressed as urokinase
( UK) units/mg of protein of the
sample.
Changes in PA and PAI-1 Levels after Treatment with
MK
To analyze the ability of MK to potentiate cellular PA
activity, we measured the amounts of PA and PAI-1 at both protein and
mRNA levels, because the sum of PA and PAI-1 activities reflects total
PA activity. Fig. 3shows the results of zymography and reverse
zymography. After treatment of BAECs with increasing concentrations of
MK, the 55-kDa uPA band was moderately enlarged (upper bands), whereas
the 50-kDa PAI-1 band was markedly reduced (lower bands). The
quantitation using enzyme-linked immunosorbent assay showed that PAI-1
level in the culture medium prepared from a million BAECs was reduced
from 11.7 to 6.0 ng/ml after incubation with 50 ng/ml MK for 17 h.
Similar changes were detected in uPA and PAI-1 mRNA levels (Fig. 4). As
seen in column A, MK treatment of BAECs caused 1.5-fold increase in uPA
mRNA levels and 38% decrease in PAI-1 mRNA levels, whereas uPA Rc mRNA
levels unchanged ( lane 2). The similar results were observed
with transformed BAECs ( B). These results indicate that
moderate up-regulation of uPA expression and significant
down-regulation of PAI-1 expression cause the elevation of cellular PA
activity in MK-treated BAECs.
Figure 3:
MK-treated BAECs secrete increased amounts
of uPA and decreased amounts of PAI-1 into the culture medium. After
confluent BAEC cultures in 10-cm dishes were treated with the indicated
concentrations of MK for 17 h in 6 ml of serum-free MEM
supplemented with 0.1% gelatin, culture media were collected and
concentrated 50-fold on Centricon and Microcon concentrators (Amicon,
Danvers, MA). The amounts of PA and PAI in the concentrate were
measured by zymography ( upper black bands) or by reverse
zymography ( lower white bands), respectively, as described
under ``Experimental Procedures.''
Enhancement of Cell Surface Plasmin Levels by
MK
In the presence of a constant amount of plasminogen, changes
in PA activity are accompanied by the similar changes in cell surface
plasmin levels. Therefore, it was likely that MK-induced PA activity
provoked the elevation of cell surface plasmin levels in BAECs. To
confirm this, the fibrin underlay technique was employed
(Fig. 5). This assay system can detect the dissolution of a
fibrin layer, over which endothelial cells were grown in a culture
medium
(25) . Three and a half days after starting the
incubation, faint and small spots of dissolution were observed around
the center of the control dish ( A), reflecting the nominal
expression of surface PA/plasmin in BAEC cultures. Because the cells
are more dense in the center of the dish, a lysis zone first appeared
there. Larger and more obvious dissolution spots were seen with the
dish containing 100 ng/ml MK, reflecting the increased amounts of
surface plasmin. Indeed, membrane-associated plasmin levels were
increased from 10.5 to 38.5 ng/10cells after incubation
with 10 ng/ml MK for 18 h. These results indicate that MK enhances
surface plasmin activity of BAECs.
Figure 5:
Dissolution of fibrin layer by BAECs
treated with MK. BAECs were cultured in fibrin-coated dishes for 3.5
days with a serum-containing medium in the absence ( A) and the
presence of 100 ng/ml MK ( B). The medium was changed every
day.
Production of MK in BAECs
The presence of MK in
BAEC cultures and enhancement of the production by retinoids were
verified by Northern and Western blot analyses. As shown in
Fig. 6
, BAECs expressed a nominal level of a MK mRNA ( lane
1). Retinol enhanced the expression in a time-dependent manner
( lanes 2-6), and about a 2-fold increase was observed
after 16 h treatment ( lane 6). Fig. 7shows the result
of Western blotting. Due to its high contents of basic amino acids, the
recombinant murine MK migrated as a band of 16 kDa ( lane 1) as
reported in the previous paper
(34) . In culture media derived
from BAECs, a 36-kDa band was detected ( lanes 2-4).
Treatment of the cultures either with 10 µ
M retinol
( lane 3) or with 1 µ
M retinoic acid ( lane
4) for 17 h increased the amount of a 36-kDa band comparing with
the control ( lane 2). EIA showed that relative concentration
of MK in the culture medium increased from 2.5 to 5.8 ng/ml and 4.4
ng/ml with 10 µ
M retinol and with 1 µ
M
retinoic acid, respectively. The anti-MK antibody used in this
experiment has been shown to react specifically to MK and not to react
with any proteins other than 16-kDa MK itself and a 29-kDa MK-related
molecule
(34) ; the emergence of the 36-kDa band is a peculiar
phenomenon observed in BAECs. During the course of investigating the
characteristics of this 36-kDa band, we episodically found that MK
served as a good substrate for tissue transglutaminase. Incubation of
purified MK with purified tissue transglutaminase resulted in the
formation of high molecular weight multimers in a time-dependent manner
(Fig. 8). This reaction did not occur in the absence of
Ca(data not shown). The molecular masses of the
second (29 kDa) and the third (36 kDa) bands from the bottom matched
with those of MK-related bands detected in rat tissues
(34) and
in BAEC culture medium (Fig. 7), respectively. Therefore, we
included an inhibitor of transglutaminase, cystamine
(29) , into
BAEC cultures during the preparation of culture medium conditioned with
retinoic acid in order to examine if the 36-kDa band was a BAEC
transglutaminase-mediated MK-containing complex. As shown in lane 5 in Fig. 7, inclusion of 200 µ
M cystamine
attenuated the 36-kDa band and induced the emergence of the 16-kDa band
(MK monomer), suggesting that MK existed as a transglutaminase-mediated
complex in BAEC cultures. These results suggest that BAECs
constitutively produce and secrete MK, which may form a
transglutaminase-mediated complex in the culture medium, and that the
production is stimulated with retinoids.
Figure 6:
Enhancement of MK expression in BAECs by
retinol. Confluent BAECs were incubated in MEM-BSA for 16 h, and
10 µ
M retinol was included for the time indicated in the
figure. Thereafter, cell lysates were prepared, and the changes in MK
mRNA levels were analyzed by Northern blotting after fractionation of
RNA through 1.6% agarose-formaldehyde-gel electrophoresis as described
under ``Experimental Procedures'' ( A). The relative
changes after the normalization with the signal intensities of
glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) are plotted
versus treatment time ( B).
Figure 7:
Secretion of MK-related molecules from
BAECs. Confluent BAECs were incubated in serum- and BSA-free MEM
for 17 h in the absence and the presence of either 10 µ
M
retinol, 1 µ
M retinoic acid, or 1 µ
M retinoic
acid plus 200 µ
M cystamine. The culture media were
collected and passed through heparin-Sepharose column (Pharmacia).
Bound materials were eluted and applied onto the SDS-polyacrylamide gel
electrophoresis with 5-13% resolving gel under reducing
condition. The heparin-Sepharose column was operated as described
previously (34). The amounts of MK in each eluate were assessed by
Western blotting with affinity-purified anti-mouse MK antibodies as
described under ``Experimental Procedures.'' Lane 1,
recombinant murine MK (30 ng); lane 2, eluate derived from
control cell culture medium; lane 3, eluate derived from
retinol-treated cell culture medium; lane 4, eluate derived
from retinoic acid-treated cell culture medium; lane 5, eluate
derived from retinoic acid plus cystamine-treated cell culture
medium.
Figure 8:
Formation of MK multimers by tissue
transglutaminase. Purified MK (final: 2.9 µ
M) was
incubated at 37 °C with 1.25 µ
M tissue
transglutaminase isolated from guinea pig liver in a total volume of 24
µl in Hepes buffer containing 10 m
M Ca(29). After incubation for the indicated time, the reaction was
terminated by the addition of 50 m
M EDTA, and the formation of
MK multimers was assessed by Western blotting as
before.
Effect of Anti-MK Antibody on Retinol Effect
From
the above results and the fact that MK is primarily found as a product
of a retinoic acid-inducible gene
(2, 13, 14) ,
we speculated that MK might partially mediate the ability of retinol to
enhance PA levels in BAECs. This hypothesis was explored by the
inclusion of neutralizing antibodies to MK during the incubation of
BAECs with retinol, followed by the assay of PA activity
(Fig. 9 A) and PAI-1 levels (Fig. 9 B). As
illustrated in Fig. 9 A, sample 1, inclusion of 50
µg/ml anti-MK antibody into the control cultures suppressed the
basal level of PA activity about 36%, suggesting the secretion of
endogenous MK from the cells. The same concentration of the antibody
completely neutralized the effect of exogenously added MK at a final
concentration of 50 ng/ml ( sample 2). Simultaneous addition of
anti-MK antibody and retinol alleviated the enhancement of PA levels;
the 3.7-fold increase with 2 µ
M retinol was suppressed to
1.8-fold increase and the 5.6-fold increase with 10 µ
M
retinol suppressed to 3.2-fold. On the other hand, anti-MK antibody
increased PAI-1 levels in the control BAEC cultures (Fig. 9 B,
sample 1), and potentiated retinol augmentation of PAI-1 levels
( samples 3 and 4). These results suggest 1) BAECs
constitutively produce MK, 2) MK production is stimulated by retinol,
and 3) the augmented production of MK participates in the enhancement
of PA activity levels in retinol-stimulated cells.
Figure 9:
Effect of anti-MK antibody on retinol
up-regulation of PA activity and PAI-1 level. Confluent BAECs were
treated with 50 ng/ml MK or with 2 and 10 µ
M retinol for
17 h in the absence and the presence of 50 µg/ml non-immune
antibody ( NI IgG) or the same concentration of anti-MK
antibody ( anti-MK IgG). After the culture medium was
collected, cell lysates were prepared as before, PA activity levels in
the lysates determined with S-2251 ( A), and PAI-1 levels in
the culture medium measured with enzyme-linked immunosorbent assay
( B) as described under ``Experimental Procedures.''
The amount of PAI-1 was expressed as nanograms of PAI-1 secreted from
10cells.
Effect of Simultaneous Addition of MK and
bFGF
Finally, in order to know how MK affected the activity of
hitherto known PA-inducible factors, we selected bFGF
(43) and
examined the effect of a combination of these two cytokines on cellular
PA levels in BAECs. The result is depicted in Fig. 10. As shown
by the shift of curve A to curve B, a 6.8-fold
increase with 2050 ng/ml bFGF alone was potentiated to a 12.7-fold
increase in the presence of 2 ng/ml MK that alone increased the
activity only 2.6-fold, indicating that MK strengthened the ability of
bFGF to enhance PA levels more than additively. The similar phenomenon
was observed when a fixed amount of bFGF (2 ng/ml) was added to various
concentrations of MK ( curves C and D). These results
suggest that MK and bFGF exert a cooperativity in enhancing PA levels
of BAECs.
Figure 10:
Effect of simultaneous addition of MK and
bFGF on BAEC PA levels. Confluent BAECs were treated either with
various concentrations of bFGF in the absence ( curve A) and
the presence ( curve B) of 2 ng/ml MK or with various
concentrations of MK in the absence ( curve C) and the presence
( curve D) of 2 ng/ml bFGF for 15 h. Cell lysates were
prepared, and the PA activities were determined as
before.
-mercaptoethanol-resistant high
molecular mass bands and found that MK could be a substrate for
transglutaminase, which catalyzes the cross-linking among proteins
(27, 29) . Inhibitor experiment suggested that MK
existed as a transglutaminase-mediated complex in BAEC culture medium.
It remains unclear why MK exists the different molecular sizes of
transglutaminase-mediated complexes depending upon the tissues and/or
cell types. We are currently studying the physiological role of these
transglutaminase-mediated MK multimers and trying to determine the
amine acceptor(s) in the MK molecule. Finally, we have demonstrated
that endogenously produced MK functions to maintain BAEC fibrinolytic
levels and that the increased production of MK by retinol participates
in the ability of retinol to enhance cellular PA levels. Furthermore,
the result that MK and bFGF act cooperatively to enhance PA activity
levels may expand the possibility that the novel MK activity reported
here would be physiologically significant in the previously reported
bFGF-induced phenomena.
, and TGF-
are known to
up-regulate PAI-1 expression in endothelial cells
(46, 47) , whereas MK is the first cytokine that
down-regulates PAI-1 expression in the cells. The elevation of
PA/plasmin levels is often discussed in the context of cell migration
or invasion. Pepper et al. (48, 49) have
reported that migrating endothelial cells express high levels of both
uPA and uPA receptor. The increased levels of PA relate to the ability
of the cells to migrate
(21) . Accordingly, as the property of
bFGF to increase PA production categorizes bFGF as an angiogenic factor
(43) , the data presented here imply the possibility of MK also
being angiogenic. Recent work by Courty and co-workers
(50) has
demonstrated that PTN, a member of MK family, exerts angiogenic
activity toward BAECs, supporting this possibility. The hypothesis that
MK is a retinoid-inducible angiogenic factor is under investigation.
Furthermore, it will be of interest to examine whether up-regulation of
cellular PA/plasmin levels by MK causes the formation of TGF-
, the
multifunctional cytokine that regulates cell growth, differentiation,
and the production of matrix proteins as well as protease inhibitors,
including PAI-1
(20, 47) , because in BAECs the
activation of latent TGF-
is fully dependent upon cellular
PA/plasmin levels
(20, 28) .
formation
(28) and TGF-
-generated functions to
suppress PA levels via stimulation of PAI-1 expression
(29) .
Together with the present study, it is suggested that retinoids may
control homeostasis of endothelial cells, at least fibrinolytic levels,
positively and negatively (conversely, PAI-1 levels, negatively and
positively) via MK and TGF-
, two biologically opposing cytokines.
, transforming growth
factor-
; bFGF, basic fibroblast growth factor;
MEM,
minimal essential medium;
MEM-BSA,
MEM containing 0.1% BSA;
EIA, enzyme immunoassay.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.