On the mechanisms of bone resorption. The action of parathyroid hormone on the excretion and synthesis of lysosomal enzymes and on the extracellular release of acid by bone cells.

Bone resorption, characterized by the solubilization of both the mineral and the organic components of the osseous matrix, was obtained in tissue culture under the action of parathyroid hormone (PTH). It was accompanied by the excretion of six lysosomal acid hydrolases, which was in good correlation with the progress of the resorption evaluated by the release of phosphate, calcium 45 or hydroxyproline from the explants; there was no increased excretion of two nonlysosomal enzymes, alkaline phosphatase, and catalase. Balance studies and experiments with inhibitors of protein synthesis indicated that the intracellular stores of the acid hydrolases excreted were maintained by new synthesis. The release was not due to a direct disruption of the lysosomal membrane by PTH; it is presumed to result from an exocytosis of the whole lysosomal content and to involve mechanisms similar to those controlling the secretion of this content into digestive vacuoles. The resorbing explants acidified their culture fluids at a faster rate and released more lactate and citrate than the controls; this release was in good correlation, in the PTH-treated cultures, with the resorption of the bone mineral, but the amount of citrate released was considerably smaller than that of lactate. The acid released could account for the resorption of the mineral. It is proposed, as a working hypothesis, that the acid hydrolases of the lysosomes are active in the resorption of the organic matrix of bone and that acid, originating possibly from the stimulation of glycolysis, cares for the concomitant solubilization of bone mineral while also favoring the hydrolytic action of the lysosomal enzymes.

Influence and modelling of urban runoff on the peak flows in rivers.

Surface waters and urban drainage systems are usually studied separately. However there are important interactions between both systems. Urban drainage systems can have an important impact on the surface waters, mainly at combined sewer overflows. On the other hand during periods of high water levels in a river, the runoff from the urban drainage system can be significantly influenced by backwater, which increases the probability of flooding in is not obvious, because the modelling tools for both systems are often hard to combine properly. To properly assess the probability of flooding for this kind of integrated water systems, different submodels are needed for both subsystems. In practice often one single model is used to describe the runoff to rivers despite the presence of urban catchments. The main objective of this study is to show the limits of this simplified approach. Furthermore, it is necessary to use continuous long term simulations, because of the differences in runoff behaviour. Detailed hydrodynamic models do not really fit for this purpose because of long simulation times and high demands in memory and disk space. Therefore simplified conceptual models are more useful.

Science-policy interfacing in support of the Water Framework Directive implementation.

Many current water-related RTD projects have established operational links with practitioners, which allow the needs of policy makers to be taken into account. However, RTD results are not easily available to water policy implementers and research scientists may lack insight in the needs of policy makers and implementers (i.e. the European Commission and water managers). The SPI-Water project worked out a number of concrete actions to bridge these gaps in communication by developing and implementing a 'science-policy interface', enhancing the use of RTD results in the Water Framework Directive (WFD) implementation. This project is part of a wider EC perspective aiming to bridge the gap between science and policy, specifically with respect to the WFD implementation. As a first action, existing science-policy links are investigated. RTD and LIFE projects that are of direct relevance for the implementation of the WFD are identified and analysed. Secondly, an information system (Harmoni-CA's WISE RTD Web Portal) has been further developed to cater for an efficient and easy to use tool for dissemination as well as retrieval of RTD results. As third action, this science-policy interfacing of WFD related topics are extended to non-EU countries taking into account their specific needs.

Metastatic heterogeneity of cells from Lewis lung carcinoma.

To allow investigations of the role of tumor cell proteases in invasion and metastasis, an attempt was made to obtain well-defined homogeneous populations of Lewis Lung carcinoma cells differing widely in their metastatic potential. From a single Lewis lung carcinoma, a parental line of cells was established and subsequently cloned so as to provide 18 clonal tumor cell lines. These clones differed in their ability to produce spontaneous, macroscopically visible metastases in the lung after i.m. inoculation into syngeneic C57BL/6 mice. Several of them were less metastatic than the parental line. The parental line expressed a metastatic behavior close to that of the high-metastatic cell subpopulations that it contained. There was, within certain limits, a good correlation between the potential for spontaneous lung metastases arising from a primary tumor and that for "artificial" lung colonies obtained after i.v. injection of the Lewis lung carcinoma cells. Although positively correlated with the growth rate of the tumor cells, the metastatic ability of the clones could not be considered as a mere reflection of the proliferation rates of the cells constituting the primary tumors. Differences in metastatic behavior observed among clones persisted in several cases after the cells had been maintained in culture for prolonged periods. However, this stability of the clones in vitro was not absolute. Indeed, some subclones isolated from the low-metastatic clone H122 displayed metastatic abilities which were lower than that of the parent clone. Furthermore, a significant increase in metastatic potential was once observed after a prolonged culture period of that same clone, H122. Thus, new metastatic phenotypes can emerged under in vitro culture conditions. However, the relative rarity of this event suggests that some metastatic heterogeneity already preexisted in vivo among the tumor cells.

Collagen degradation by metastatic variants of Lewis lung carcinoma: cooperation between tumor cells and macrophages.

Interactions between cancer cells and host macrophages might have important regulatory roles in controlling the expression of the metastatic phenotype, particularly by regulating the production of proteases necessary for tissue invasion. To investigate that possibility, mouse macrophages and Lewis lung carcinoma (LLC) cells from four clonal subpopulations with either low or high metastatic ability were cultured on [14C]collagen (type l)-coated plates. They did not degrade collagen when they were cultured independently on that substrate, but they were induced to do so when macrophages and cancer cells were cultured together. An increased production of neutral collagenase and other neutral protease activities was observed simultaneously. The degree of stimulation of collagen degradation varied according to the cancer cell subpopulation present in the cocultures. For a given LLC cell subpopulation, similar degrees of stimulation of collagen degradation were achieved with either bone marrow-derived or resident peritoneal macrophages, either syngeneic (from C57BL/6 mice) or allogeneic; lower stimulations were obtained with thioglycolate-elicited peritoneal macrophages. Macrophage-conditioned culture media could be substituted for living macrophages to stimulate collagen degradation or collagenase secretion by LLC cells, but LLC cell-conditioned media did not stimulate collagen degradation by macrophages. This suggests that, in the cocultures, collagen degradation is achieved mainly by the cancer cells, not by the macrophages, and that it is induced by a soluble factor, a monokine, produced by the macrophages. That factor might be identical to a recently identified rabbit monokine that stimulates fibroblasts or synovial cells to degrade collagen and proteoglycan and to activate plasminogen, because rabbit macrophage-conditioned media containing that monokine also stimulated collagen degradation by LLC cells.

The enzymatic evaluation of procollagenase and collagenase inhibitors in crude biological media.

The validity of the enzymatic assay of procollagenase within crude biological media containing also the collagenase inhibitor TIMP (tissue inhibitor of metalloproteinases) as well as other (pro)metalloproteinases and sometimes, metalloproteinase-TIMP complexes, has been reevaluated. To be enzymatically assayed, procollagenase has to be activated. The standard activation procedures by either trypsin or 4-aminophenylmercuric acetate (APMA) both allow an optimal recovery of collagenase from procollagenase when the media do not contain free TIMP. However, they do not destroy TIMP nor do they reactivate the collagenase present in enzyme-inhibitor complexes. Therefore, the collagenase formed by the activation of procollagenase in the presence of free TIMP is immediately inactivated by binding to the inhibitor. As a result, both the bound collagenase and TIMP can no longer be assayed by enzymatic methods. An optimal recovery of collagenase can, however, be obtained if free TIMP is neutralized by the binding of other tissue metalloproteinases (such as those present in culture media of rabbit bone marrow-derived macrophages) prior to the activation and assay of procollagenase. Similarly, it is possible to recover under an active free form a large part of the TIMP present in collagenase- (or other metalloproteinase-)TIMP complexes by heating the complexes at acid pH under conditions which inactivate the collagenase.

Collagenase, procollagenase and bone resorption. Effects of heparin, parathyroid hormone and calcitonin.

1. The addition of heparin to the culture fluid of mouse tibiae or calvaria did not cause any significant resorption of bone collagen or mineral. However, heparin (or analogue sulfated polyanions), enhanced greatly the amount of latent, trypsin-activatable collagenase (i.e. procollagenase) released by the bones in the medium without influencing that of directly active collagenase which was always very low. Heparin appeared to act by increasing the production of the enzyme which is immediately excreted. Procollagenase and collagenase are not stored in bone tissue, even under conditions where it is in active resorption. 2. Parathyroid hormone induced in the explants a resorption of both mineral and collagen that was inhibited by calcitonin. These hormones, however, had no influence on the release of procollagenase or collagenase either in the presence or in the absence of heparin. 3. Once activated, bone collagenase digested the collagen of the bone explants, and more extensively after their demineralization. Thus the latent collagenase that accumulates around non-resorbing bones has to be considered as a precursor, (and not as a residue), of active enzyme. 4. Active collagenase added to incipient cultures of bones disappeared with a half-life of 24 h. The lost enzyme could, however, not be reactivated by trypsin and thus was not transformed into latent procollagenase.

Degradation of cartilage proteoglycan and collagen by synovial cells. Stimulation by macrophages under activation by phagocytosis, lymphocyte factors, bacterial products or other inflammatory stimuli.

When cultured together with dead 35S-labelled cartilage discs or at the surface of [3H]proteoglycan/[14C]collagen-coated plates, synovial cells from either arthritic or normal rabbit joints digested both the proteoglycan and the collagen of the substrates after a lag-period of 1-2 days. These digestions were inversely related to the age (number of subculture passages) of the synovial cells and they could be modulated by serum components that were either inhibitory or stimulatory. They were dependent on a protein synthesis by the cells and were paralleled, in young cultures, by the release of collagenase and of a proteoglycan-degrading neutral proteinase. The co-culture of synovial cells with macrophages or their culture with macrophage-conditioned culture media caused a more rapid and more extensive degradation of collagen and proteoglycan due to the stimulation of the synovial cells by a nondialysable macrophage factor. The production of this synovial cell-activating 'matrix regulatory monokine' by the macrophage was enhanced by several immunological or inflammatory stimuli such as lymphocyte factors, phagocytosis, asbestos fibres, endotoxin, adjuvant muramyl dipeptide or chemotactic formyl-methionyl peptide, as well as by other membrane-active agents (phorbol myristate acetate, concanavalin A). It is presumed that these interactions are of importance in the development of cartilage destruction in rheumatoid and other chronic inflammatory arthritis.

Production of gelatin-degrading matrix metalloproteinases ('type IV collagenases') and inhibitors by articular chondrocytes during their dedifferentiation by serial subcultures and under stimulation by interleukin-1 and tumor necrosis factor alpha.

The gelatin-degrading matrix metalloproteinase (MMP) activities and their inhibitors produced by rabbit articular chondrocytes have been characterized by gel substrate analysis ('zymography') after electrophoresis on non-reducing sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Differentiated chondrocytes in confluent primary culture produced constitutively only one gelatinase which presented the main characteristics of proMMP-2 ('72 kDa type IV procollagenase'). It had an apparent Mr of 66,000 (unreduced), which was partially or totally converted to 61,000 by, respectively, trypsin or APMA treatment; exogenous TIMP (tissue inhibitor or metalloproteinases) inhibited the conversion triggered by APMA but not that induced by trypsin. This proMMP-2 was also the predominant gelatinase found, together with its 61 kDa activation product, in extracts of articular cartilage. Differentiated chondrocytes simultaneously produced MMP inhibitors which on reverse zymograms were distributed over two bands with Mr of 27,500 and 20,400, resistant to both pH 2 and 100 degrees C, corresponding, respectively, presumably, to TIMP and TIMP-2. Interleukin-1 (IL1) and tumor necrosis factor alpha (TNF alpha) did not affect the production of the proMMP-2 nor of the two species of TIMP. However, IL1 induced the coordinated production of 91 and 55 kDa gelatinases. The 91 kDa activity is likely to correspond to proMMP-9. It could be converted to a 81 kDa gelatinase by trypsin or APMA treatment, in a process that was inhibited in both cases by exogenous TIMP. The 55 kDa gelatinolytic activity most probably represents the sum of the activities of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). It was sequentially converted to lower size forms (49 to 35 kDa) by either trypsin or APMA; that conversion was inhibited by TIMP, with the exception, however, of the first steps (from 55 to 49, then to 42 kDa) induced by trypsin. The 55 kDa and its conversion forms were all active on both gelatin and casein. TNF alpha did also stimulate the production of proMMP-9, although less efficiently than IL1, but it did not induce, or very poorly, that of the 55 kDa proMMP-1/proMMP-3 activity. Low levels of proMMP-9 and of its 81 kDa product of activation were also found in extracts of cartilage. With increasing passage number and cell dedifferentiation, confluent chondrocytes produced increasing amounts of proMMP-2 and of the two species of TIMP. A spontaneous low production of proMMP-9 and proMMP-1/proMMP-3 was only occasionally observed in cultures of dedifferentiated chondrocytes, accompanying a spontaneous low production of procollagenase.(ABSTRACT TRUNCATED AT 400 WORDS)

Partial characterization of the macrophage factor that stimulates fibroblasts to produce collagenase and to degrade collagen.

Rabbit bone marrow-derived macrophages in culture produce and release a soluble factor that activates collagenase secretion and collagen degradation by cultured skin fibroblasts from either rabbit, mouse or human origin. The factor is heat-labile and is inactivated by phenylglyoxal. After gel filtration, it is recovered in both an apparent high-Mr (67000-76000) and a low-Mr (9000-14000) form. Chromatography on cation exchangers suggests two molecular species with different charge properties. These characteristics are compatible with known properties of rabbit interleukin 1.

Production of collagens, collagenase and collagenase inhibitor during the dedifferentiation of articular chondrocytes by serial subcultures.

Rabbit articular chondrocytes were cultured in monolayer and the progressive loss of their differentiated phenotype was monitored from passage to passage. The cell densities achieved in confluent cultures decreased abruptly between the primoculture and the second or third subculture, and more slowly thereafter, reflecting parallel morphological changes. The synthesis of collagen (but not that of other proteins) decreased sharply, and a smaller proportion of collagen was incorporated into the matrix. Cells in primoculture synthesized mainly the cartilage-specific collagens, types II and XI, which were mostly deposited in the matrix, but no type I nor III collagen. With increasing passages, the synthesis of type II collagen decreased progressively while that of types I and III collagens increased, the latter being almost completely released in the culture medium. Simultaneously, the production of type XI collagen was apparently switched to that of type V. Fully differentiated confluent chondrocytes in primoculture produced the collagenase inhibitor TIMP (tissue inhibitor of metalloproteinases) but no detectable procollagenase; their production of procollagenase was, however, induced by interleukin 1. The production of TIMP increased from passage to passage. A spontaneous production of procollagenase was only occasionally observed in confluent cultures of dedifferentiated chondrocytes. However, interleukin 1 induced an always higher production of procollagenase from dedifferentiated chondrocytes than from cells in primoculture.

Modulation by interleukin 1 and tumor necrosis factor alpha of production of collagenase, tissue inhibitor of metalloproteinases and collagen types in differentiated and dedifferentiated articular chondrocytes.

The actions of interleukin 1 (IL1) and tumor necrosis factor alpha (TNF alpha) on several parameters of the collagen metabolism of rabbit articular chondrocytes were studied by comparing the responses of either differentiated chondrocytes in primoculture or dedifferentiated cells in late passage culture to human recombinant (hr) IL1 alpha, hr-TNF alpha and cytokine-enriched fractions of rabbit macrophage-conditioned media. In response to IL1 or TNF alpha, differentiated chondrocytes (i.e., producing the cartilage-specific collagens, types II and XI, but no type I), sharply reduced their synthesis of collagen, a reduction which involved both types II and XI collagens, without consistently changing their production of non-collagenous proteins; they also incorporated a smaller proportion of collagen into the matrix. Similar levels of response were obtained for hr-IL1 alpha at picomolar and for hr-TNF alpha at nanomolar concentrations. However, the action of TNF alpha, but not of IL1, was manifested only in the presence of serum. Simultaneously, IL1, but not TNF alpha, induced the chondrocyte production of procollagenase (a difference which contrasted with the similar levels of procollagenase induced by both cytokines in synovial and skin fibroblasts) but neither cytokine influenced the accumulation of the collagenase inhibitor TIMP. These effects were not affected by indomethacin and are thus unlikely to be prostaglandin-mediated. During their dedifferentiation in monolayer subcultures, chondrocytes became more sensitive to the procollagenase-inducing ability of IL1 and TNF alpha, but their response to TNF alpha was lower than to IL1. They also increased their production of TIMP, which remained unaffected by the cytokines. Simultaneously, they decreased their production of collagen and substituted progressively the synthesis of fibroblast-specific collagens, types I, III and V, for types II and XI. Acting on dedifferentiated cells, even in the presence of indomethacin, IL1 and TNF alpha further decreased the synthesis of collagen, reducing the production of both typical type I (i.e. [alpha 1(I)]2 x alpha 2(I) molecules) and type V collagens as well as their incorporation into the matrix, but increasing the synthesis of type III collagen. Therefore not only IL1, but also TNF alpha can exert profound influences on the collagen degradation and repair processes occurring in the pathology of articular cartilage.

Immunoreactive collagenase and bone resorption.

1. Active mouse bone collagenase is excluded from its inhibitory antibody by preincubation of that antibody with various forms of inactive enzyme, e.g. 'procollagenase', some collagenase-inhibitor complexes or partially denatured or degraded collagenase. This property allows the detection of several enzymatically inactive forms of collagenase. 2. The accumulation of immunoreactive collagenase in the culture fluid of mouse bones occurred only in the presence of heparin and was not correlated with bone resorption induced by parathyroid hormone. These experiments provide further (see Lenaers-Claeys, G. and Vaes, G., Biochim. Biophys. Acta (1979) 584, 375-388), more conclusive evidence that the critical role in the resorption of the organic matrix of these explants may be due to another enzyme system than collagenase.

Degradation of collagen and proteoglycan by macrophages and fibroblasts. Individual potentialities of each cell type and cooperative effects through the activation of fibroblasts by macrophages.

Fibroblasts and macrophages of various sources (peritoneal, alveolar or bone marrow-derived), from either rabbit or mouse, were cultured, independently or together, at the surface of [3H]proteoglycan/[14C]collagen-coated plates to evaluate their capacities for proteoglycan and collagen degradation. The various macrophage populations differed widely in their potentialities for proteoglycan and particularly, for collagen degradation, native collagen being significantly degraded, in this model only by rabbit alveolar macrophages. Fibroblasts were as active in proteoglycan degradation as the most active macrophage preparations, but their potential for collagen degradation appeared much higher than that of macrophages. Moreover, all types of macrophages secreted a factor, a monokine, that activated collagen and proteoglycan degradation by fibroblasts. Thus, fibroblasts might well be a major effector cell, active in connective tissue degradations occurring under chronic inflammatory situations.

Solids separation efficiency of combined sewer overflows.

The removal of sewer solids at combined sewer overflow locations depends on the flow patterns inside the overflow structure on the one hand and on the sediment characteristics on the other hand. Flow conditions can be described by the residence time distribution; sewer sediments can be characterised by their settling velocity distribution. The combination of both distributions leads to a dimensionless efficiency curve, which gives the removal efficiency as a function of the Hazen number. For field conditions this efficiency curve is mainly influenced by the settling velocity distribution of the sewer sediments and, as a consequence, nearly identical efficiency curves are found for different types of prototype CSO structure. For design purposes, a methodology using return frequency analysis is proposed.

Advection-dispersion modelling tools: what about numerical dispersion?

In general the transport of dissolved substances and fine suspended particles is governed by the one-dimensional advection-dispersion equation. In order to model the transport of dissolved substances and fine suspended particles, the advection-dispersion equation is incorporated into commonly used urban drainage modelling tools such as InfoWorks CS (Wallingford Software, United Kingdom) and MOUSE (DHI Software, Denmark). Two examples show the use of InfoWorks CS and MOUSE using standard model settings. Modelling results using tracer experiments show that numerical model parameters need to be altered in order to calibrate the model. Using tracer experiments as a model calibration tool, it is shown that a non-negligible amount of dispersion is generated by InfoWorks CS and MOUSE and that it is in fact the numerical dispersion that is calibrated.

Relationship of the plasminogen activator/plasmin cascade to osteoclast invasion and mineral resorption in explanted fetal metatarsal bones.

An attempt was made to establish whether the activation of plasminogen into plasmin is necessary either for the preparatory phases to bone resorption, involving the recruitment of osteoclast precursors, their migration toward mineralized surfaces, and their final differentiation, or for the subsequent osteoclastic resorption phase. 45Ca-labeled fetal (17 day) mouse metatarsals were cultured under conditions in which they pursue their modeling for a few days. In this model, the resorption phase, monitored by the release of 45Ca into the medium, is entirely dependent on the preparatory phases affecting osteoclast precursors. It was, as expected, stimulated by parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 and inhibited by calcitonin. PTH also enhanced the activity of tissue-type plasminogen activator (PA) in extracts of metatarsals but not that of urokinase (which is, however, the main PA present in the mouse fetal metatarsal culture model). The resorption processes were not dependent on the presence of plasminogen in the media, even when the rudiments were precultured with tranexamic acid to remove their endogenous plasminogen. Moreover, they were not influenced by inhibitors of plasmin, either the plasma inhibitors alpha 2-antiplasmin, alpha 2-macroglobulin, and alpha 1-antitrypsin, or aprotinin, which was tested under a variety of conditions. Aprotinin also did not influence the resorption (loss of calcium and hydroxyproline) of 19 day fetal mouse calvariae cultured with PTH in a medium devoid of plasminogen. It is concluded that the various steps implicated in the bone resorption processes that occur in the metatarsals and in the calvariae culture models are not dependent on the activity of plasmin. The function of PAs in bone, however, could be exerted through direct proteolysis of extracellular proteins other than plasminogen or be mediated by a molecular structural domain distinct from their catalytic domain.

Bone resorption and response to calcium-regulating hormones in the absence of tissue or urokinase plasminogen activator or of their type 1 inhibitor.

Plasminogen activators (PA) are implicated in cell migration and tissue remodeling, two components of the bone resorption processes. Using mice with inactivated tissue PA (tPA), urokinase PA (uPA), or type 1 PA inhibitor (PAI-1) genes, we evaluated whether these processes, or their stimulation by parathyroid hormone (PTH) or 1,25-dihydroxyvitamin (1,25[OH]2D3) are dependent on these genes. Two culture models were used, one involving 19-day fetal calvariae, to evaluate the direct resorptive activity of osteoclasis, and the other involving 45Ca-labeled 17-day fetal metatarsals, in which this activity depends on preliminary (pre)osteoclast migration. PTH similarly increased (about 10-fold) PA activity in calvariae from wild-type tPA+/+ and uPA+/+ or deficient uPA-/- and PAI-/- mice; it affected only tPA, not uPA. In tPA-/- bones, the low PA levels, due to uPA, were not influenced by PTH. Calcitonin did not affect PA responses to PTH. No differences were observed between tPA+/+, tPA-/-, uPA+/+, and uPA-/- calvariae for any parameter related to bone resorption (development of lacunae, release of calcium and lysosomal enzymes, accumulation of collagenase, loss of hydroxyproline), indicating similar responses to PTH or calcitonin. The progressive 45Ca release was largely similar in cultures of tPA+/+, tPA-/-, uPA+/+, uPA-/-, PAI+/+, or PAI-/- metatarsals and it was similarly enhanced by PTH or 1,25(OH)2D3. However, uPA-/- metatarsals released 45Ca at a slower rate at the beginning of the cultures, suggesting an impaired recruitment of the (pre)osteoclasts, which migrate at that time from the periosteum into the calcified cartilage. Thus, it appears that the direct resorptive activity of the osteoclasts does not necessitate the presence of either tPA or uPA, but uPA is likely to facilitate the migration of the (pre)osteoclasts toward the mineralized surfaces. Although considerably enhanced by PTH, tPA does not mediate the actions of PTH (nor of 1,25[OH]2D3) evaluated in these models.

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 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% 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 D3 and prostaglandin E2, 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.

Bone-resorbing agents affect the production and distribution of procollagenase as well as the activity of collagenase in bone tissue.

The participation of collagenase in bone resorption has been investigated by assaying the procollagenase extracted from fetal mouse calvaria cultured under a variety of conditions, and by evaluating its ability to degrade bone collagen. Procollagenase was found in two separate pools, one requiring demineralization for its extraction, the other not. Culturing the bones with PTH, 1,25-dihydroxyvitamin D3, prostaglandin E2, interleukin-1, tumor necrosis factor-alpha, catabolin, retinoic acid, or endotoxin (but not with heparin) induced resorption, enhanced lysosomal enzyme release, and markedly increased the procollagenase content of the second pool. The PTH-induced increase in procollagenase was dose dependent and paralleled the extent of calcium loss and lysosomal enzyme release. The increase in procollagenase was found in bone, periosteum, and sutures, where its distribution was similar to that of nonmineralized collagen. The increase in procollagenase was abolished by cycloheximide, but not by indomethacin, hydroxyurea, glucocorticoids, acetazolamide, bisphosphonates, or calcitonin. Calcitonin and bisphosphonates almost completely inhibited the PTH-induced Ca loss and lysosomal enzyme release, but only partially inhibited the PTH-induced loss of collagen. The latter was, however, completely prevented by the collagenase inhibitor, CI-1. CI-1 also partially inhibited the PTH-induced Ca loss. Moreover, collagen degradation occurred in PTH-precultured calvaria (but not in noncultured controls) when incubated in a buffer under nonviable and nondemineralizing conditions. This degradation was inhibited by collagenase inhibitors, either CI-1 or the natural tissue inhibitor of metalloproteinases. This work thus indicates that the resorption of fetal bone explants proceeds along with an accumulation of procollagenase, primarily within their nonmineralized matrix. Moreover the results suggest that collagenase is likely to participate in the degradation of the nonmineralized collagen of the bone explants. Whether it also participates in the degradation of the collagen of the mineralized matrix remains to be elucidated.