©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Midkine Enhances Fibrinolytic Activity of Bovine Endothelial Cells (*)

Soichi Kojima (1)(§), Hisako Muramatsu (2), Hiroshi Amanuma (1), Takashi Muramatsu (2)

From the (1) Laboratory of Gene Technology and Safety, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Koyadai, Tsukuba, Ibaraki 305 and the (2) Department of Biochemistry, Nagoya University School of Medicine, Tsurumai-cho, Showa-ku, Nagoya, Aichi 466, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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)() 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) .

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- (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.

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.


EXPERIMENTAL PROCEDURES

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.()


RESULTS

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.




DISCUSSION

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 -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.

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-, 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) .

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- 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.

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.


FOOTNOTES

*
This study was partly supported by a special grant for promotion of research from RIKEN (to S. K.), by Grants-in-aid from the Japanese Ministry of Education, Science and Culture (06780597 (to S. K.) and 06454645 (to T. M.)), and by grants from the Ryoichi Naito Foundation for Medical Research (to S. K.) and the Uehara Memorial Foundation (to T. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Laboratory of Gene Technology and Safety, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Koyadai, Tsukuba, Ibaraki 305, Japan. Tel.: 81-298-36-3522; Fax: 81-298-36-9050.

The abbreviation used are: MK, midkine; PTN, pleiotrophin; PA, plasminogen activator; uPA, urokinase-type plasminogen activator; PAI-1, plasminogen activator inhibitor-1; BAECs, bovine aortic endothelial cells; TGF-, transforming growth factor-; bFGF, basic fibroblast growth factor; MEM, minimal essential medium; MEM-BSA, MEM containing 0.1% BSA; EIA, enzyme immunoassay.

H. Muramatsu and T. Muramatsu, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Daniel B. Rifkin for his critical comments of this study and for correction of the English.


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