JOURNAL OF BONE A N D MINERAL RESEARCH Volume 6, Number 10. 1991 Mary Ann Liebert, Inc.. Publishers

Tissue and Urokinase Plasminogen Activators in Bone Tissue and Their Regulation by Parathyroid Hormone GAETANE LELOUP,' CHANTAL PEETERS-JORIS,' JEAN-MARIE DELAISSE,' GHISLAIN OPDENAKKER,' and GILBERT VAES'

ABSTRACT The identification of the plasminogen activator (PA) types present in bone and the regulation of their activity by parathyroid hormone (PTH) were investigated in cultures of fetal mouse calvariae with the use of either a chromogenic substrate or a zymographic assay. PA was detected essentially in the tissue extracts of the explanted bones, with only 1-2'70 of the total activity released in the surrounding culture media. From their electrophoretic behavior compared to PAS of other mouse tissues and from their response to a specific antibody raised against the tissue type PA (tPA), two major molecular species, of 70 and 48 kD were identified as tPA and urokinase (uPA), respectively, a third minor species of 105 kD being likely to correspond to complexes between tPA and an inhibitor; the culture fluids, moreover, contained enzymatically active degradation products of uPA of 42 and 29 kD. The PA activity of the bone extracts was only minimally affected by the addition of fibrinogen fragments to the chromogenic assays. PTH induced bone resorption and stimulated in parallel the accumulation of PA in the tissue; other bone-resorbing agents, 1,25-dihydroxyvitamin D, and prostaglandin E,, had similar effects. Densitometric scanning of the zymograms of the bone extracts indicated that PTH stimulated only the production of tPA and had no effect on that of uPA. However, PTH also enhanced the release of uPA (both the 48 kD and the 29 kD forms) from the bones into the media. Although inhibiting bone resorption, calcitonin had no effect on the PTH-induced accumulation of PA in bone or on the release of tPA, but it prevented the PTH-induced accumulation of 29 kD uPA in the culture fluids. Thus these studies support the view that tPA and possibly also uPA may have a role in the physiology of bone; the nature of this role remains to be elucidated, however.

INTRODUCTION

I

NTEREST FOR THE POSSIBLE INVOLVEMENT of plasminogen activators (PA), either of the tissue (tPA) or of the urokinase (uPA) type (for a review, see Ref. l ) , in the physiology of bone arose from studies made on cultures of isolated osteoblast-like cell^(^-^' establishing that various bone-resorbing hormones increased the tPA activity expressed by the cells. These original observations led the a u t h o r ~ ' ~to. ~propose ' that the tPA-plasmin system may well be involved in the remodeling of the bone matrix, possibly in its resorption. It was suggested that the conversion

of plasminogen into plasmin by PA could be required to allow the action of collagenase during bone resorption. Indeed, according to the present views, bone resorption requires not only the action of osteoclast-secreted lysosomal cysteine-proteinases but also that of osteoblast-produced collagenase, the latter being possibly required to remove the nonmineralized collagen (osteoid) that prevents the access of osteoclasts to the bone surfaces (for a review, see Ref. 6 ) . Bone collagenase is produced as a latent zymogen,(') and our previous work has implicated plasmin as a possible physiologic activator of procollagenase.(8-'0' However, the studies on bone PAS have been done thus

'Laboratoire de Chimie Physiologique (Connective Tissue Group), Universitk de Louvain and International Institute of Cellular and Molecular Pathology, Bruxelles. Belgium. 'Rega Institute tor Medical Research, Universiteit van Leuven, Belgium.

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far on dispersed cells in monolayer culture, either cloned con 10). This concentration step and the subsequent assays osteogenic sarcoma cells or osteoblast-enriched prepara- were done, as a rule, on freshly harvested fluids or (when tions of dispersed calvarial cells. Considering the multiple indicated) after storage of the fluids at -20°C. Within interactions that may occur between the various cell types each experiment, the corresponding (controls, PTH, and present in bone tissue during its resorption, as well as those calcitonin + PTH) conditioned media were then pooled that may exist between the cells and their surrounding before the concentration step, as indicated in Results. extracellular matrix, it appeared important to investigate the production of PAS that may occur in organized bone Extraction of PA tissue in which resorption was induced by parathyroid hormone (PTH) or inhibited by calcitonin. The necessity to At the end of the cultures, the two calvariae were rinsed validate at the level of intact bone tissue the PA studies briefly with cold isotonic saline and immersed in a flat-botdone on cell cultures is further supported by a recent im- tomed polypropylene tube containing 1 ml extraction solumunohistochemical study done in sections of bone tis- tion to be extracted at 4°C under gentle agitation (12oscilsue'") that demonstrates tPA and uPA in osteoclasts (and lations per minute). In our first experiments, the bones uPA in macrophages) but not in osteoblasts or osteocytes. were extracted for 1 h following Jensen et al.(") with 2 M This validation constitutes the main object of the present KSCN, 1 mg/ml of Triton X-100,and 0.2 mg/ml of NaN3 work. (extraction procedure A). The extract was then dialyzed overnight against 0.1 M Tris-HCI, pH 8.1, 1 mM EDTA, 1 mg/ml of Triton X-100,and 0.2 mg/ml of NaN,. In subseMATERIALS AND METHODS quent experiments, the bones were extracted by the procedure developed for procollagenase in our earlier w ~ r k ( ' ~ . ' ~ ) Materials because we noticed that this method provided higher PA Human melanoma tPA and affinity-purified rabbit anti- activities than those obtained with the KSCN procedure human melanoma tPA antibody were prepared as previ- (for an illustration of the difference between the two exously described.(12'Rabbit antigoat IgG immunoglobulins, traction procedures, see Fig. 2A). Following this procedure purified on protein A-Sepharose, were kindly donated by (extraction procedure B), the calvariae were first extracted Dr. J.P. Vaerman [Experimental Medicine Unit, Univer- for 8 h in CNTN buffer (10 mM cacodylate-HCI, pH 6.0, 1 M ZnCI,, and sity of Louvain, Brussels). Human uPA was from Leo M NaCI, 0.1 mg/ml of Triton X-100, Laboratories, Ltd. (Aylesbury, England). Bovine parathy- 0.1 mg/ml of NaN,) to provide extracts 1. In some experiroid hormone-( 1-84) [PTH-(1-84); trichloroacetic acid ments, the extraction was repeated with the same buffer powder, 60 pg PTH per mg solid], calcitonin (synthetic sal- for three successive 24 h periods to provide extracts 2, 3, mon form), human plasminogen (6-9 units/mg protein), and 4. After each extraction, the extracts were dialyzed and HEPES were from Sigma (St. Louis, MO). Bovine overnight at 4°C against 100 vol of 10 mM HEPESPTH-( 1-34) and the chromogenic plasmin substrate H-D- NaOH, pH 8.3, containing 0.1 mg/ml of Triton X-100,0.1 Val-Leu-Lys-p-nitroanilide 2 HCI were from Bachem mg/ml of NaN,, and 1 mM EDTA. They were then used Feinchemikalien A.G. (Bubendorf, Switzerland); KSCN, for the PA assays either directly (for all the bone extracts) sodium dodecyl sulfate, and casein (Hammarsten quality) or after storage at 4°C (for extracts of other tissues). Unfrom Merck A.G. (Darmstadt, Germany); and cyanogen less otherwise indicated, all reported extracts of either bromide fragments of fibrinogen from Boehringer (Mann- bone or other mouse tissues (lung or kidney) were prepared heim, Germany). Other chemicals were from suppliers pre- following extraction procedure B. Conditioned media viously menti~ned."~' from mouse resident peritoneal macrophages were prepared as described(18'and stored frozen at -20°C before their use. Bone cultures and their evaluation Culture of fetal (19day) mouse calvariae and the evaluaPA assay tion of bone resorption were as previously des~ribed."~) PA was measured by monitoring spectrophotometricBriefly, two calvariae were cultured for 2 days in 2 ml medium 199 with or without PTH or calcitonin, with a daily ally, with a Titertek Multiskan enzyme-linked immunosorchange of medium; PTH-(1-84) was used for most experi- bent assay (ELISA) plate reader the activation of plasments, unless otherwise indicated. Bone resorption was minogen in the presence of the chromogenic plasmin subIncubations were evaluated by visual assessment of resorption lacunae using strate H-D-Val-Leu-Lys-p-nitroanilide. a scale from 0 to 5'") and by measuring the amounts of carried out at 37°C in microtiter plates in a total volume of calcium accumulated in the medium. In some experiments, 0.2 ml assay mixture containing 20 or 25 pl sample, 8 mM ~ ~ 'monitored in the cul- HEPES-NaOH, pH 8.3, 0.5 mg/ml of Triton X-100,0.2 the release of & g l u c u r ~ n i d a s e (was ture fluids and the accumulation of procollagenase was de- mg/ml of NaN,, 0.5 mM chromogenic substrate, and 25 termined in the bone t i s s ~ e ( ' ~at. ~ the ~ )end of the cultures. pg/ml of human plasminogen; care was exerted to keep the To allow the assay and the zymography of the PAS re- concentration of CI- anion, a potent inhibitor of the PA leased by the explants during their culture, conditioned reaction,(19)at noninterfering levels. In this amidolytic asthe rate of formation of p-nitroaniline, measured culture fluids were concentrated (15- to 25-fold, according say,(20.21) to the experiment) on microconcentrators (Amicon Centri- by its absorbance at 405 nm, is proportional to the concen-

PLASMINOGEN ACTIVATORS IN BONE TISSUE tration of plasmin, which itself results from the action of PA on plasminogen. Therefore, the acceleration of p-nitroaniline production is proportional to the initial rate of plasmin formation from plasminogen. At the early stage of activation, when the plasminogen concentration remains nearly constant, this acceleration is thus proportional to the concentration of PA. The acceleration can be evaluated from the slope of the straight line obtained by plotting the variation of absorbance at 405 nm versus the square of the incubation time: this slope corresponds to half the acceleration rate."') P A activity is calculated from the formula 2pN/t2, where pN is the amount (pmol) of p nitroaniline produced enzymatically over f minutes of incubation time. It is expressed in units, 1 unit corresponding to the amount of enzyme that generates 1 pmol plasmin per minute from plasminogen. Under our conditions of assay, a linear relationship was observed between 0.2 and 1.7 units of absorbance change when the amount of p-nitroaniline produced was related either to the time squared or to the concentration of bone extract.

Zymography of PA Samples were electrophoresed under nonreducing conditions in 9% sodium dodecyl sulfate (SDS)-polyacrylamide slab gels supplemented with plasminogen (100 pg/ml) and casein (0.5 mg/ml); the resolving gel was prerun at room temperature (around 20°C) for 2 h at a constant current of 25 mA."" After electrophoresis, the gels were washed three times for 20 minutes in 2.5% Triton X-100 and further incubated overnight at 35°C in 50 mM Tris-HCI pH 8.3. Finally, they were fixed and stained with 0.25% Coomassie blue. Controls were run without plasminogen. Individual casein digestion bands resolved from the samples were quantified by scanning densitometry of photographs of the gels using a Joyce-Loebl Chromoscan 3 densitometer.

Statistics

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Statistics in the tables and figures refer to mean standard deviation (SD). The significance p of the differences observed between groups was determined with the use of Student's f-test, analysis of variance, regression curve analysis, and Scheffe's F-test or binomial test, as most appropriate and as indicated with the results.

RESULTS PA activity in bone extracts and culture fluids P A activity was detected essentially in the extracts of the explanted calvariae. As with procollagenase,('b) lower P A activities were obtained in the bone extracts when the calvariae were homogenized before their extraction (not shown), possibly due to formation of enzyme-inhibitor complexes with inhibitors released from the cells (see Ref. 23). Using the chromogenic substrate assay, 76 11% of the recovered P A activity (mean f SD of 20 extractions) was found in the first 8 h extracts of the tissue (extracts I),

*

1083

22 f 11% in the second 24 h extracts (extracts 2), and the rest (about 2%) in the third and fourth 24 h extracts (extracts 3 and 4). Addition of trace amounts of plasmin to the chromogenic substrate assay (to activate possible proenzymes of PA; see Discussion) did not improve the PA activity recovered from the various extracts, although blank values for substrate degradation done in parallel in the absence of plasminogen indicated that plasmin was then active in the presence of the extracts (not shown). Addition of fibrinogen fragments, up to 50 polylysine, 125 pg/m1,(2b)or ovalbumin, 100 pg/m1,(27)to the assay mixtures or coating the wells of the microtitration plates used for the assay with fibrin(28)increased severalfold, as expected (see Discussion), the activity of a sample of purified human tPA on the chromogenic substrate but had a much lower effect on the P A activity measured in the bone extracts, which was either unchanged or only slightly enhanced (usually between 10 and 5OTo upon addition of 25-50 pg fibrinogen fragments per ml; not shown). Taken together, and as illustrated later in Fig. 1, these observations indicate that P A assays done with the chromogenic substrate on extracts 1 in the absence of added fibrinogen fragments provide valid estimations of the relative levels of P A activity present in the bone extracts. Only very low levels of activity were found in the culture fluids surrounding explanted bones. Their detection required both the concentration of the fluids ( I S - to 25-fold) and the addition of fibrinogen fragments (50 ,tg/ml) to the assays. Under these conditions, the P A activity of the culture fluids, assayed on the chromogenic substrate, amounted to only 1-2070 of the activity found in the corresponding bone extracts (not shown). Inhibitors of PA, assayed on purified human tPA, were not detected in the media (not shown).

Effect of PTH and calcitonin on bone PA activity and relation with bone resorption Higher P A activity was recovered from the explanted bones after 2 days of culture (14.0 6.0 pU per calvaria, mean SD of 23 cultures; extracts 1) than from noncultured controls (7.8 2.3 pU per calvaria, mean f SD of 13 determinations done each on two calvariae; p = 0.001 1 with Student's f-test). Addition of PTH to the cultures further stimulated the accumulation of PA in the bone explants in a dose-dependent manner, as illustrated in Fig. 1 for sequential bone extracts (1, 2, 3, and 4) assayed with or without the addition of fibrinogen fragments. Similar results were obtained and are shown in Fig. 2A for a series of experiments involving extracts 1. It was evident that the higher levels of P A in extracts 1 of PTH-treated bones could not result from a faster rate of extraction of PA from these bones compared to controls. Also, the addition of fibrinogen fragments (50 pg/ml) to extracts of either control (cultured or noncultured) or PTH-treated bones caused in all cases similar degrees of stimulation of their P A activity (Fig. I), indicating that the P A stimulation induced by PTH is unlikely to be due to the action of tPA stimulatory proteins extracted together with PA from the PTH-treated bones.

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LELOUP ET AL.

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PTH (xlO'M) FIG. 1. Stimulation of P A in explanted calvariae by PTH. Extraction of P A from tissue and the effects of fibrinogen fragments on its assay. Embryonic mouse calvariae were extracted following procedure B to provide the sequential extracts I , 2, 3, and 4 (see Materials and Methods). The calvariae belonged to six experimental groups each consisting of four sets of two calvariae, either noncultured (NC) or cultured with the indicated concentration of PTH. Each set of two calvariae was extracted independently, and the corresponding extracts 1-4 were pooled within each experimental group to be assayed for PA, either in the absence (-) or in the presence (+) of added cyanogen bromide fragments of fibrinogen (50 pg/ ml). The PA activities presented (in pU/calvaria) correspond to either extracts (El) or to the sum of the activities recovered in the four successive extracts (Esum); calv., calvaria.

4 a

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0.6

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50 40 30

20 The PTH-induced stimulation of P A occurred in parallel with the development of resorption lacunae and the loss of calcium from the explants (Fig. 2A and B), as well as with other known actions of PTH on bone: release of 0glucuronidase, a marker for the secretion of lysosomal enzymes,(lsl and accumulation of procollagenase in the tissue'13.161 (Fig. 2C). Other bone-resorbing agents, 1,25-diM) and PGE, M), hydroxycholecalciferol (2.6 x also stimulated the accumulation of P A in the calvariae in parallel with the development of resorption lacunae and the loss of calcium (not shown). The addition of indomethacin (1.4 x 10+ M) to the cultures did not prevent the stimulation of P A induced by PTH (lo-' M), indicating that the action of P T H was unlikely to be mediated by a production of prostaglandins (not shown). Although inhibiting bone resorption (calcium loss), calcitonin did not prevent the PTH-induced accumulation of PA (Table 1). The effects of PTH and calcitonin on the PAS released by the bone explants into their surrounding culture fluids were investigated in the three experiments presented in Table 1 (not shown). Within each experiment, the corresponding (controls, PTH, or P T H + calcitonin) conditioned media, collected after 24 and 48 h of culture, were pooled and concentrated to allow P A assays. Although slightly more P A activity was recovered from the media of PTH-treated bones (0.59 + 0.25 pU released per calvaria over 48 h culture; mean + S D for the three experiments) than of control bones (0.43 + 0.13 pU per calvaria per 48 h) when P A was assayed on the chromogenic substrate in the presence of fibrinogen fragments (50 pg/ml), this in-

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FIG. 2. Dose-response effect of PTH on the accumulation of P A in bone tissue in relation with the development of bone resorption. The open symbols in A, B, and C are the results (means f SD, the latter shown only if its magnitude exceeds that of the symbol used) of a single experiment in which embryonic mouse calvariae (calv.) were cultured for 2 days with increasing concentrations of PTH (three cultures of two calvariae each per concentration tested). Bone resorption and the related events were evaluated (B) by the extent of resorption lacunae (0)and the amounts of calcium released in the medium (A), as well as (C) by the release of 0-glucuronidase (V) and the accumulation of procollagenase in bone tissue (0). At the end of the cultures, P A was extracted from the calvariae following the KSCN extraction procedure A (see Materials and Methods) and assayed on the chromogenic substrate (A, 0).The closed symbols ( 0 ) in A report the P A activities assayed in extracts of calvariae obtained following the CNTN extraction procedure B (note the higher recovery of P A compared to procedure A). Each point is the mean f SD of a series of 23 cultures of two calvariae each, distributed over six experiments, except for the PTH concentrations of 0.15 and 1.5 x M, which involved four cultures each in a single experiment (p = lo-' by analysis of variance).

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PLASMINOGEN ACTIVATORS IN BONE TISSUE

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.O TABLE1 . EFFECTSOF CALCITONIN ON THE ACCUMULATION OF P A IN PTH-TREATED BONES~

Calcium loss PA (pmol per calvaria) (pU per calvaria) Control PTH PTH + calcitonin

0.36 f 0.17 0.51 f 0.18 0.19 f 0.16

19.1 71.4 66.6

* 8.58 * 39.4 f

F:

6 4

I

I

100 80 60

40.1

aFetal mouse calvariae were cultured for 2 days with or without PTH-(1-34), lo-' M, or calcitonin, 3 x 10.' M . Calcium released from the bones was measured in the culture medium, and PA was assayed in extracts of the bones done at the end of the cultures (extracts 1, extraction procedure B; see Materials and Methods). The results are the mean f SD for 12 cultures of two calvariae each, equally distributed over three experiments. Calcitonin significantly decreased the loss of calcium from PTH-treated bones (p < 0.01 by Scheffe's F test) but had no significant effect on their accumulation of PA.

0

0.5

1

1.5

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2.5

ANTIBODY (pgl FIG. 3. lmmunoinhibition of PA in bone extracts by antihuman tPA immunoglobulins. Human tPA (A), human uPA and extracts of embryonic mouse calvariae (extracts 1) that were cultured with PTH, 5 x M ((I), were preincubated for 30 minutes at 25°C with increasing amounts of either rabbit affinity-purified specific immuno0)or rabbit globulins raised against human tPA (A, 0, nonspecific (antigoat IgG) immunoglobulins (not shown). Controls were incubated in parallel with appropriate amounts of the immunoglobulins solvent buffer. The residual P A activity was then assayed and expressed as a percentage of the activities measured in the controls. Each point is the mean SD of five or six experiments.

(o),

crease (40%) was not significant; it was also not affected by the addition of calcitonin to PTH-treated cultures (0.63 0.22 pU per calvaria per 48 h).

*

tdentification of the types of PA present in bone extracts Affinity-purified specific immunoglobulins raised against human tPA inhibited most (about 90Vo) of the P A activity present in the extracts of PTH-treated bones; purified human uPA was only minimally affected (Fig. 3). Nonimmune gammaglobulins were not ihibitory (not shown). Analysis of the bone extracts by the zymography technique showed two and often three bands of proteolytic activity (Figs. 4 and 5 ) ; no band was visible when plasminogen was not included in the zymography gels (not shown). As seen in Fig. 4, the main activity comigrated with purified human tPA (approximately 70 kD) and with the PA activity of similar molecular mass found in mouse lung or kidney homogenates. This activity largely disappeared from the bone extracts after precipitation with the antihuman tPA antibody as it did also from the solutions of human tPA. It is thus likely to be tPA. A second band migrated at a faster rate and was not precipitated by the antibody. It migrated faster than purified human uPA (54 kD) but comigrated with the main P A activity found in mouse urine or in conditioned culture media of mouse macrophages (Fig. 4), none of which were precipitated by the anti-tPA antibody. It is thus likely to represent mouse uPA, whose molecular mass (48 kD) is known to be lower than that of human U P A . ( ~ ~ A - ~third, ') usually faint band of PA activity of higher molecular mass (about 105 kD) was sometimes visible. It was also precipitated by the antitPA antibody and is likely to correspond to complexes between tPA and an inhibitor that have been reactivated but not dissociated by the SDS-gel electrophoresis procedure used for the z y m ~ g r a r n s .32-341 '~~ As expected from the results of the chromogenic substrate assays (see Fig. l), the activity present in the zymograms of the successive bone extracts (1-4) decreased from extract to extract in both PTH-treated asnd control bones; this decrease was seen for each of the three bands of P A

activity (not shown). However, the extracts from PTHtreated bones always showed more activity on the zymograms than the corresponding extracts of control bones. Dose-response studies of the effect of PTH on the various components of P A activities visible on the zymograms of bone extracts (Fig. 5 ) showed that the accumulation of the 70 kD tPA was considerably stimulated by the hormone without significant changes in the accumulation of the 48 kD uPA. The minor 105 kD component was also stimulated by PTH, as expected if it represents mainly tPA bound to an inhibitor.

Effect of PTH and calcitonin on the PA types present in bone-conditioned culture fluids The concentrated, freshly harvested bone-conditioned culture fluids (collected separately for two successive 24 h culture periods) from the three experiments presented in Table 1 in which P A activity was assayed on the chromogenic substrate and that involved controls, PTH-treated, and P T H + calcitonin-treated cultures (see earlier), as well as the media of a fourth similar experiment that were stored at -20°C before their analysis, were analyzed by zymography. Moreover, zymograms of four pairs of similarly prepared concentrated pools of bone-conditioned culture media, which involved, however, only controls and PTH-treated cultures and were also stored at -2O"C, were considered as well for the analysis of the effects of PTH. The zymograms (Fig. 4C) showed the same three bands of P A activity of 105, 70, and 48 kD seen in zyrnograms of bone extracts; moreover, they showed two additional

LELOUP ET AL.

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FIG. 4. Identification of the types of PA present in bone extracts and culture fluids by zymographic analysis and the effects of PTH and calcitonin on the PA types released in the culture fluids. (A) SDS-polyacrylamide gels of PAS of mouse bone extracts compared to PAS of other sources: extracts 1 of PTH-treated calvariae (lanes 2 and 6); mouse macrophage-conditioned culture fluid (lane 3); human tPA (lane 4); mouse urine (lane 5); and extracts of mouse lung (lane 1) and kidney (lane 7). All these electrophoreses were run in gels containing plasminogen together with casein. In gels without plasminogen, no band of activity developed with bone extracts, mouse urine, or human tPA (not shown), but one or several bands of plasminogen-independent proteolytic activity remained in the zymograms of lung extract (lane l'), macrophage-conditioned culture fluid (lane 3'), or kidney extract (lane 7'). The molecular mass assignment of the bands was made by comparison with the migration of the plasminogen-dependent PAS observed in mouse macrophage culture fluid (48 kD uPA), mouse urine (mainly 48 kD uPA and its 29 kD degradation products, with some 70 kD tPA), and human tPA (70 kD).",29-3',52)(B) Absorption of PA activity bands from mouse bone extracts with antibodies to human melanoma tPA. Samples of PTH-treated mouse bone extracts (lanes 8 and 9), mouse macrophage-conditioned medium (lanes 10 and 1 l ) , mouse urine (lanes 12 and 13), human tPA (lanes 14 and 15), and human uPA (lanes 16 and 17) were preincubated for 30 minutes at 25°C in a total volume of 25 pI with 5 pg of either rabbit nonspecific (antigoat IgG) immunoglobulins (lanes 8, 10, 12, 14, and 16) or rabbit affinity-purified specific immunoglobulins against human melanoma tPA (lanes 9, 1 1 , 13, 15, and 17). Protein A-Sepharose was then added ( 5 pl sedimented gel), and 15 minutes later, the samples were centrifuged during 10 minutes at 13,000 x g. Zymograms were then obtained from the supernatants after their electrophoresis on SDS-polyacrylamide gels containing plasminogen and casein. (C) SDS-polyacrylamide gels of PAS of 15fold concentrated mouse calvariae-conditioned culture fluids from control (lane 18), PTH-treated (lane 19), or (calcitonin + PTH)-treated (lane 20) cultures. The cultures were done as indicated in Table 1 , and the media, collected between 24 and 48 h of culture, were concentrated and immediately analyzed by zymography. N o bands of activity developed when the gels were run without plasminogen (not shown).

bands (29 and 42 kD) corresponding (not shown) to plasminogen-dependent activity that was not precipitated by the anti-tPA antibody. These bands were sometimes also visible in extracts of mouse kidney (Fig. 4, lanes 7 and 7') or lung or in mouse urine (not shown), which were all stored for a few days at 4°C before being run in zymography and presumably correspond (see Discussion) to degradation products of the 48 kD mouse uPA; similar degradation products, although of slightly higher M,, can also be seen with the preparation of purified human urokinase (Fig. 4, lanes 16 and 17), itself of a higher M , (-54 kD) than mouse uPA. The ratio of uPA to tPA activity that could be seen in the zymograms was much higher in culture fluids than in bone extracts, suggesting either a preferential release of uPA from the bones or a greater stability of uPA in the fluids. Considering the large variability of the controls observed on the densitometric scanning of the zymograms in this series of experiments, the statistical significance of the differences observed for the various PA bands either between PTH-treated and controls or between calcitonin + PTHtreated and PTH-treated was analyzed by use of the binomial test. PTH appeared to increase significantly the 48 kD and the 29 kD uPA bands (both at p = 0.044), but its effects on the 42 kD band and on the two tPA bands (105 and 70 kD) were not significant. When added to PTH, cal-

citonin significantly decreased the 29 kD band (p = 0.016) without affecting the other PA bands. This suggested that PTH stimulates the release of uPA from the bones into the surrounding fluids and that it also increases the degradation of the 48 kD uPA into the 29 kD species. Calcitonin clearly inhibits the latter degradation, but the present experiments do not allow us to establish whether it does or does not affect the stimulatory action exerted by PTH on the release of uPA.

DISCUSSION PTH and other bone-resorbing agents are known to stimulate the production of tPA(2-5)as well as of latent proc011agenase'~~'~'' by osteoblast-type cells in monolayer culture. Considering that collagenase appears to be necessary for the process of bone r e ~ o r p t i o n ( ~and ~ . ~knowing ~) that plasmin is a potent activator of procollagenase,(8-10)it has been speculated that the activity of PA may be crucial for the initiation of bone resorption processes (for a review, see Ref. 6). To investigate the validity of this hypothesis, however, it is necessary to extend at the level of bone tissue the observations done thus far on cell monolayers. The present work represents a first attempt to study PAS in

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108M)

FIG. 5. Stimulation by PTH of the accumulation of tPA, but not uPA, in bone tissue. (A) Samples from the same extracts 1 that were assayed for PA on the chromogenic substrate in the experiment reported in Fig. 1 (see Fig. 1 for the abbreviations) were analyzed by zymography in the presence of plasminogen (no bands were visible in its absence). (B) Quantitative evaluation by scanning densitometry of the individual casein digestion bands, corresponding to the 48, 70, and 105 kD PA species, resolved in the zymogram presented in A. Because of technical problems encountered in the analysis of our zymograms (background variations due to the migration of casein in the gels), valid quantitative comparisons could be made only along horizontal axes, allowing us to compare the digestion bands of PA species of the same M,. The data are thus presented as percentages of the values obtained for each PA species in extracts of bones cultured without PTH. (C) Means f SD of the 48 and 70 kD PA species evaluated by scanning densitometry in a similar way as in B and in a total of five similar experiments. The PTH-induced increase in 70 kD tPA was significantly different by regression curve from that in controls (p = 2 x analysis). The differences observed for the 48 kD uPA were not significant (p = 0.32).

this tissue context in direct connection with the process of bone resorption. The evaluation of PAS within crude biologic media, such as tissue extracts or culture fluids, is rendered difficult by the complex molecular interactions that regulate the physiologic production of plasmin from its extracellular precursor, plasminogen (reviewed in Refs. I , 39, and 40). These media may indeed contain unknown proportions of different PA types, inhibitors and activators. Both tPA and uPA are produced as single-chain precursors that can be activated by plasmin into fully active two-chain Both PAS may be secreted in soluble form in the extracellular milieu or remain associated with the cell surfaces. Their activity is regulated by several specific inhibitors produced and secreted by a number of cell types, often concomitantly with the PAS, as well as by protein activators, most notably fibrin, which considerably stimulates plasminogen activation by tPA. Moreover, the plasmin generated by the initial PA reaction rapidly amplifies the reaction by producing the fully active two-chain form of the PAS; however, the activity of plasmin is itself regulated by specific inhibitors present in the extracellular fluids. In the present study, PA activities were determined by assay of their catalytic activities in both tissue extracts and conditioned culture media. Despite our exhaustive extraction procedure, the possibility remains that not all the enzyme was extracted, but it appears unlikely that much enzyme was left in the tissue considering that only about 2% of the total recovered PA activities were extracted in the last two extracts (3 and 4) of our extraction sequence. As is usually the case in such enzyme studies, however, part of the enzymes present in either tissues or media may have been irreversibly inactivated by either inhibitors or denaturation and thus escape detection. Our comparisons assume similar stabilities and recoveries of the various enzyme forms detected under our various experimental conditions, and we postulate that the amounts of enzyme detected are representative of the whole amounts present in either tissues or media. Although reasonable, the validity of this assumption may need to be verified by analyzing PA protein expression or mRNA levels in future work. To ensure maximal validity for our assays, the PA activities determined by the chromogenic substrate assay were validated in the present study by parallel evaluations done by zymography. Indeed, besides providing an estimation of the M , of the PA activities present, the SDS-gel electrophoresis procedure involved in this technique allows the reactivation and detection of the PAS present within PA-inhibitor complexes, although it does not usually dissociate these complexes.'3*-344 5 ) M oreover, by adding trace amounts of plasmin to the chromogenic assay, it was ascertained that there was no significant interference due to the presence of plasmin inhibitors in these assays. It is thus unlikely that zymogens or precursor forms of either uPA or tPA, known to be easily activated by plasmin and possibly present in the extracts, remained latent under the conditions of the chromogenic substrate assay due to the presence of plasmin inhibitors. Finally, addition of fibrinogen fragments [a substitute for fibrinlz42 5 ) ] to the assays (or several related

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procedures; see Results) had only minimal stimulatory effects on the P A activity found in the bone extracts. Considering that some bone extracts, devoid of P A activity, were found to stimulate considerably (up to 10-fold) the activity of purified human t P A (unpublished experiments), it appears likely that the mouse bone P A activity present in the extracts is almost fully stimulated, possibly by an endogenous component from the extracellular present in the extracts. A similar situation was observed by others for rat oocyte tPA.(28)This raises the interesting possibility(47)that fibrin, required for the full expression of t P A in fibrinolytic processes, could be replaced by other activating macromolecules for some specialized functions of tPA. With the use of these techniques, only minimal P A activity and low amounts of PA-inhibitor complexes were detected in the bone culture media. Active inhibitor of tPA was not detected either, possibly (but this possibility was not further investigated) because the most common of these inhibitors, P A inhibitor type I (PAI-1), produced under an active form by endothelial cells and several other cell types, including o s t e ~ b l a s t s , ( ~rapidly . ~ ) converts extracellularly to an inactive latent form.(*3)Most of the P A activity ( > 98%) was found in the bone extracts, however, indicating that it was mainly associated with the tissue, presumably the cells [possibly, however, also the matrix, because the presence of extracellular P A has been reported in mineralized The tissue-associated P A was distributed between three molecular species, corresponding to bands of approximately 105, 70, and 48 kD on the zymograms. From their electrophoretic behavior on the zymograms compared to that of PAS extracted from other mouse tissues, as well as from their response to a specific anti-tPA antibody, the 70 and 48 kD species were identified as tPA and uPA, respectively, and the third minor species of 105 kD appeared likely t o correspond to SDS-reactivated tPA-inhibitor complexes. Moreover, bone-conditioned culture fluids contained two P A molecular species (42 and 29 kD) that are likely to correspond to proteolytic degradation products of uPA. An active - 30 kD degraded form of uPA was indeed identified in several biologic products (reviewed in Ref. I), and the 42 kD P A band, which was clearly plasminogen dependent but was not precipitated by anti-tPA antibody, possibly represents an intermediary product in the conversion of 48 kD into 29 kD uPA, similar to the one that has sometimes been noted in samples of urine or of plasmin-activated urokinase. ( 3 1 . 4 9 ) Addition of P T H to the culture medium strongly stimulated the P A activity that could be recovered in the extracts of the bone explants. This was evident when P A was assayed with the chromogenic substrate as well as with the zymographic technique. Densitometric scanning of the zymograms allowed us t o establish that only tPA was involved in this stimulation, either in its main, free 70 kD form or in its minor, inhibitor-complexed 105 kD form; P T H had no effect on the accumulation of the 48 kD uPA within the bone tissue. The presence of increased amounts of the 105 kD tPA-inhibitor complex indicates that the accumulation of P A inhibitor, presumably PAI-1, was probably also stimulated by P T H within the bone tissue, an ob-

LELOUP ET AL. servation that contrasts with the apparent reduction of P A inhibitor observed by others in culture media conditioned by PTH-treated osteoblast-type cells(5);however, these authors did not assay P A inhibitors associated with either the cell surface or the extracellular matrix of their cultures, although more than 75% of the P A activity was located at that level. In our study, the stimulation of t P A was parallel with the extension of bone resorption in the explants, as evaluated by the development of resorption lacunae and the loss of calcium, as well as with other known effects of P T H on explanted bones: secretion of lysosomal enzymes, monitored by the release of /3-gl~curonidase,(’~) and accumulation of procollagenase in the tissue.(13,16)However, although calcitonin, a physiologic osteoclast inactivator as usual inhibited the effect of PTH on bone resorption, it did not prevent the PTH-induced accumulation of t P A [nor, as shown previousIy,(13.’6)of procollagenase], suggesting that these phenomena are unlikely to result from the direct activity of osteoclasts and that they are obviously not sufficient in themselves to determine bone resorption. Although the accumulation of uPA in bone tissue did not appear to be increased by PTH, zymograms of concentrated bone-conditioned culture fluids indicated that media from PTH-treated cultures contained more uPA, either the intact 48 kD form or the 29 kD degraded form of uPA, than nontreated controls. Careful analysis of the 48, 42, and 29 kD uPA bands of the zymograms did not allow us to establish whether calcitonin prevented the PTH-induced accumulation of uPA (i.e., the total amount of the three uPA species) in the media, but it was clear that calcitonin reduced the proportion of 29 kD uPA present. This suggests that P T H enhances the release of uPA by the bone explants into their surrounding media and, simultaneously, enhances the proteolytic conversion of 48 kD uPA into its 29 kD species, a process inhibited by calcitonin. The physiologic significance of the effects of P T H and calcitonin on the degradation of uPA is, however, unclear: they may well be related to the degradative action of lysosomal proteases whose secretion and accumulation in the culture fluids are stimulated by PTH(15,50) and inhibited by calcit ~ n i n ‘ ~in’ )parallel with the bone resorption events. The present observations have thus validated at the level of explanted bone tissue observations on the PTH induction of tPA done previously in culture systems of osteoblast-like cells. It is surprising that a recent immunohistochemical study‘’’’ did not identify tPA or uPA in osteoblasts and osteocytes but PAS were seen only in the phagolysosomal network of osteoclasts or macrophages. The presence of P A antigens at that level could perhaps result from an endocytosis-mediated catabolism of both PAS (see Refs. 53 and 54), and the possibility should be considered that it was only under conditions prevailing in these phagolysosomes that the antigens became “visible” to their respective antibodies. Our studies have shown, moreover, that PTH-induced t P A is not released in soluble form in the surrounding milieu but that it remains mainly associated with the tissue, presumably the cells, and that its accumulation occurs in parallel with other PTH-induced processes (secretion of lysosomal enzymes and accumulation

PLASMINOGEN ACTIVATORS IN BONE TISSUE of procollagenase) linked to bone resorption. Furthermore, they have indicated that P T H may also stimulate the release of uPA from bones. It remains for further work to establish the physiologic significance of these findings, however. tPA is often associated with fibrinolytic processes and uPA with tissue invasion and, possibly, connective tissue remodeling (see Refs. 1, 39, and 40). At the present stage, one can only speculate about possible roles of these two PAS in the bone-remodeling processes. Their functions might be linked either to the activation of procollagenase'8''o' and of the related metalloproteinase, stroby plasmin or to the remelysin (((prote~glycanase'')~~~) cruitment and migration of osteoclast precursor cells, two hypotheses that we are at present trying to evaluate. Others'"' have proposed that they may be involved in the local activation of transforming growth factor p, a potent regulator of bone metabolism, by plasmin.

ACKNOWLEDGMENTS This work was supported by the Fund for Medical Scientific Research (Belgium) and by the Belgian State Prime Minister's Office, Science Policy Programming (interuniversity attraction poles, Grant 7bis, and concerted actions, Grant 88/93-122). Leloup is a Research Assistant and Opdenakker is a Research Associate of the National Fund for Scientific Research (Belgium). We are grateful to Dr. A. Bouckaert for useful advice in the statistical treatment of the data and to Dr. J.P. Vaerman, for the gift of rabbit antigoat IgG immunoglobulins. The skillful secretarial assistance of Y. Marchand was greatly appreciated.

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Address reprint requests to: Gilbert Vaes Laboratoire de Chimie Physiologique UCL 75.39 Avenue Hippocrate, 75 B1200 Bruxelles. Belgium

Received for publication November 13, 1990; in revised form February 26, 1991;accepted March 9, 1991.

Tissue and urokinase plasminogen activators in bone tissue and their regulation by parathyroid hormone.

The identification of the plasminogen activator (PA) types present in bone and the regulation of their activity by parathyroid hormone (PTH) were inve...
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