Comparing three novel endpoints for developmental osteotoxicity in the embryonic stem cell test.

Birth defects belong to the most serious side effects of pharmaceutical compounds or environmental chemicals. In vivo, teratogens most often affect the normal development of bones, causing growth retardation, limb defects or craniofacial malformations. The embryonic stem cell test (EST) is one of the most promising models that allow the in vitro prediction of embryotoxicity, with one of its endpoints being bone tissue development. The present study was designed to describe three novel inexpensive endpoints to assess developmental osteotoxicity using the model compounds penicillin G (non-teratogenic), 5-fluorouracil (strong teratogen) and all-trans retinoic acid (bone teratogen). These three endpoints were: quantification of matrix incorporated calcium by (1) morphometric analysis and (2) measurement of calcium levels as well as (3) activity of alkaline phosphatase, an enzyme involved in matrix calcification. To evaluate our data, we have compared the concentration curves and resulting ID(50)s of the new endpoints with mRNA expression for osteocalcin. Osteocalcin is an exclusive marker found only in mineralized tissues, is regulated upon compound treatment and reliably predicts the potential of a chemical entity acting as a bone teratogen. By comparing the new endpoints to quantitative expression of osteocalcin, which we previously identified as suitable to detect developmental osteotoxicity, we were ultimately able to illustrate IMAGE analysis and Ca(2+) deposition assays as two reliable novel endpoints for the EST. This is of particular importance for routine industrial assessment of novel compounds as these two new endpoints may substitute previously used molecular read-out methods, which are often costly and time-consuming.

Activation of the mitogen-activated protein kinase pathway by bone sialoprotein regulates osteoblast differentiation.

Bone sialoprotein (BSP) is an abundant protein in the extracellular matrix of bone that has been suggested to have several different physiological functions, including the nucleation of hydroxyapatite (HA), promotion of cell attachment and binding of collagen. Studies in our lab have demonstrated that increased expression of BSP in osteoblast cells can increase expression of the osteoblast-related genes Runx2 and Osx as well as alkaline phosphatase and osteocalcin and increase matrix mineralization. To determine the molecular mechanisms responsible for the BSP-mediated increase in osteoblastic differentiation, several functional domain mutants of BSP were expressed in primary rat bone osteoblastic cells, including the contiguous glutamic acid sequences (polyGlu) and the arginine-glycine-aspartic acid (RGD) motif. Markers of osteoblast differentiation, including matrix mineralization and alkaline phosphatase staining, were increased in cells expressing BSP mutants of the polyGlu sequences but not in cells expressing RGD-mutated BSP. We also determined the dependence on integrin-associated pathways in promoting BSP-mediated differentiation responses in osteoblasts by demonstrating the activation of focal adhesion kinase, MAP kinase-associated proteins ERK1/2, ribosomal s6 kinase 2 and the AP-1 protein cFos. Thus, the mechanism regulating osteoblast differentiation by BSP was determined to be dependent on integrin-mediated intracellular signaling pathways.

Metalloproteinases and their inhibitors-diagnostic and therapeutic opportunities in orthopedics.

Matrix metalloproteinases (MMPs) and related enzymes (ADAMs, ADAMTS) and their inhibitors control matrix turnover and function. Recent advances in our understanding of musculoskeletal conditions such as tendinopathy, arthritis, Dupuytren's disease, degenerative disc disease, and bone and soft tissue healing suggest that MMPs have prominant roles. Importantly, MMPs are amenable to inhibition by cheap, safe, and widely available drugs such as the tetracycline antibiotics and the bisphosphonates. This indicates that these MMP inhibitors, if proven effective for any novel indication, may be quickly brought into clinical practice.

[Effect of vitamin K on bone material properties].

Collagen cross-links are determinants of bone quality. Because vitamin K is thought to ameliorate bone quality, we summarized the literature regarding the effect of vitamin K such as menatetorenone (MK-4) on bone matrix property in the review. MK-4 seems to stimulate the osteoblastic activity. This results in the increase in collagen accumulation and lysyl oxidase controlled enzymatic cross-links in bone. Furthermore, vitamin K stimulates the secretion of collagen binding protein regulating proper fibrillogenesis such as leucine-rich repeat protein (tsukushi). This kinds of non-collagenous proteins induced by the treatment of vitamin K may also affect proper collagen cross-link formation and show the favorable effect on bone material quality.

Phosphatase actions at the site of appositional mineralization in bisphosphonate-affected bones of the rat.

Tissue-nonspecific alkaline phosphatase (TNSALP) and Ca-ATPase are known to play roles in bone mineralization, but how these enzymes contribute to appositional mineralization has been illusive. Here we examined the active sites of these enzymes in appositional mineralization using the bones of young rats being administered with 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) for 5 days. The doses of HEBP totally abolished mineralization of newly formed bone matrix except in matrix vesicles (MVs), and hence allowed precise localization of MVs and phosphatase reactions within non-mineralized extracellular matrix. Intense TNSALP and ATPase reactions were confirmed along the limited portions of osteoblast membranes where intimate cell-cell contacts were maintained. Diffuse reactions of these enzymes were throughout the osteoid implicating efflux of TNSALP and ATPase molecules into extracellular matrix from the osteoblast membranes. Phosphatase reactions associated with MVs varied both in intensity and location among the individual vesicles; newly formed MVs were almost free of reactions but appeared to gain those activities later in the osteoid. These data suggest that TNSALP and ATPase are released from the osteoblast membrane and later integrated into MVs within the osteoid. The osteoblasts may thus regulate appositional mineralization of bone from a distance at least in part by providing phosphatases via MVs.

Functional involvement of PHOSPHO1 in matrix vesicle-mediated skeletal mineralization.

UNLABELLED: PHOSPHO1 is a phosphatase highly expressed in bone. We studied its functional involvement in mineralization through the use of novel small molecule inhibitors. PHOSPHO1 expression was present within matrix vesicles, and inhibition of enzyme action caused a decrease in the ability of matrix vesicles to calcify. INTRODUCTION: The novel phosphatase, PHOSPHO1, belongs to the haloacid dehalogenase superfamily of hydrolases and is capable of cleaving phosphoethanolamine (PEA) and phosphocholine to generate inorganic phosphate. Our aims in this study were to examine the expression of PHOSPHO1 in murine mineralizing cells and matrix vesicles (MV) and to screen a series of small-molecule PHOSPHO1-specific inhibitors for their ability to pharmacologically inhibit the first step of MV-mediated mineralization. MATERIALS AND METHODS: q-PCR and immunohistochemistry were used to study the expression and localization profiles of PHOSPHO1. Inhibitors of PHOSPHO1's PEA hydrolase activity were discovered using high-throughput screening of commercially available chemical libraries. To asses the efficacy of these inhibitors to inhibit MV mineralization, MVs were isolated from TNAP-deficient (Akp2(-/-)) osteoblasts and induced to calcify in their presence. RESULTS: q-PCR revealed a 120-fold higher level of PHOSPHO1 expression in bone compared with a range of soft tissues. The enzyme was immunolocalized to the early hypertrophic chondrocytes of the growth plate and to osteoblasts of trabecular surfaces and infilling primary osteons of cortical bone. Isolated MVs also contained PHOSPHO1. PEA hydrolase activity was observed in sonicated MVs from Akp2(-/-) osteoblasts but not intact MVs. Inhibitors to PHOSPHO1 were identified and characterized. Lansoprazole and SCH202676 inhibited the mineralization of MVs from Akp2(-/-) osteoblasts by 56.8% and 70.7%, respectively. CONCLUSIONS: The results show that PHOSPHO1 localization is restricted to mineralizing regions of bone and growth plate and that the enzyme present within MVs is in an active state, inhibition of which decreases the capacity of MVs to mineralize. These data further support our hypothesis that PHOSPHO1 plays a role in the initiation of matrix mineralization.

Erk pathways negatively regulate matrix mineralization.

Skeletal mineralization is an important step regulating the mechanical properties of the calcified tissues, but molecular events underlying mineralization still remain elusive. We examined the role of extracellular signal-regulated kinase (Erk) pathways in matrix mineralization of osteogenic cells both in vitro and in vivo. Matrix mineralization by preosteocytic MLO-A5 cells and osteoblastic MC3T3-E1 cells was increased by either PD98059 Mek inhibitor treatment or adenovirus vector-mediated dominant negative Ras (Ras(DN)) expression and was suppressed by Erk activation by platelet-derived growth factor (PDGF) treatment or constitutively active Mek1 (Mek(CA)) expression. Administration of adenovirus vectors carrying Ras(DN) gene onto the calvaria of 1-day-old mice increased the mineralization of the tissues, while that of the Mek(CA) adenovirus suppressed it. These results suggest that the Erk pathway is a negative regulator of the matrix mineralization both in vitro and in vivo.

Proteolytic enzymes in skeletal development: histochemical methods adapted to the study of matrix lysis during the transformation of a "cartilage model" into bone.

The replacement of a "cartilage model" by definitive bone is characterized by a series of localized excavations of the cartilage which are eventually followed by bone deposition. Each excavation requires lysis of cartilage components (defined here as the breakdown of a peptide bond) and their eventual resorption (defined here as microscopical visible cartilage loss). More precisely we have proposed that the lysis is affected by proteases capable of breaking down the main proteoglycan "aggrecan" and the main fibril element, "type II collagen". Four approaches combining biochemical, immunologic and microscopic techniques have been adapted to test this hypothesis. Each is applied to the rat tibial head's "cartilage model" where proteases have been shown to be major contributors to secondary ossification center formation. The approaches have been found both effective and distinct as cartilage resorbing enzymes have not only been identified but also detected in situ before and after activation. Achieved overall is an understanding of when, where and how specified proteases contribute to tissue component lyses. While the focus resides on the in situ proteolysis of cartilage, three of the approaches could be translated without change to other tissues, whereas one may require tissue specific adjustments before use.

Nucleoside triphosphate pyrophosphatase of rabbit matrix vesicles, a mechanism for the generation of inorganic pyrophosphate in epiphyseal cartilage.

Inorganic pyrophosphate (PPi) may be important in the regulation of mineralisation but its origin in epiphyseal cartilage is ill-defined. Nucleoside triphosphate pyrophosphatase is one potential source, as this enzyme catalyses the formation of PPi from nucleoside triphosphates. This enzyme has been identified in matrix vesicles derived from rabbit epiphyseal cartilage and a method developed to measure the activity using ATP as substrate in intact matrix vesicles under relatively physiological conditions. The enzyme had a high affinity for ATP (Km less than 10 microM) and was also active towards GTP, CTP and UTP. Disruption of the matrix vesicle membrane by sonication failed to alter the activity. Treatment of sonicated matrix vesicles with Triton X-100 increased the activity which may indicate a direct effect of the detergent on the enzyme. Activity towards ATP was inhibited substantially by ADP and AMP and by another potential substrate beta,gamma-methyleneadenosine 5'-triphosphate. Dichloromethylene bisphosphonate, an analogue of the product PPi, inhibited the activity to a lesser extent. Two other potential substrates, NADP+ and thymidine 5'-monophosphate p-nitrophenyl ester were only weakly inhibitory as was 1-hydroxyethylidene 1,1-bisphosphonate. These results imply that nucleoside triphosphates are the substrates in vivo and the inhibitory effects of ADP and AMP suggest mechanisms whereby this activity could be regulated.

Evidence that ecto-nucleoside-triphosphate pyrophosphatase serves in the generation of extracellular inorganic pyrophosphate in human bone and articular cartilage.

Extracellular inorganic pyrophosphate (PPi) is important in the regulation of mineralisation of bone, and in the pathogenesis of chondrocalcinosis, an arthritic disease in which calcium pyrophosphate dihydrate crystals form in articular cartilage. Nucleoside-triphosphate pyrophosphatase, which catalyses the formation of PPi, was previously observed at the surface of human articular chondrocytes in culture. A similar enzyme has been identified in osteoblast-like human bone cells in culture, and is active towards purine and pyrimidine nucleoside triphosphates. The enzyme has high affinity for ATP and is located on the cell surface, and thus could serve in the generation of extracellular PPi. Moreover, no other mechanism for the catabolism of small amounts of exogenous ATP is present in human bone cells. Further evidence for ecto-nucleoside-triphosphate pyrophosphatase serving in the generation of extracellular PPi in articular cartilage and bone was obtained by studying the ability of alternative substrates (which do not yield PPi) to inhibit generation of PPi from ATP. In both articular chondrocytes and bone cells, the enzyme exhibited an apparent preference for ATP over dinucleotide and phosphodiester substrates. Some potential inhibitors of the enzyme activity were also studied in both cell types. ADP moderately inhibited the activity but two bisphosphonate drugs were only slightly inhibitory.

Allosteric modulation by ATP, calcium and magnesium ions of rat osseous plate alkaline phosphatase.

Alkaline phosphatase from rat osseous plate is allosterically modulated by ATP, calcium and magnesium at pH 7.5. At pH 9.4, the hydrolysis of ATP and PNPP follows Michaelis-Menten kinetics with K0.5 values of 154 microM and 42 microM, respectively. However, at pH 7.5 both substrates exhibit more complex saturation curves, while only ATP exhibited site-site interactions. Ca(2+)-ATP and Mg(2+)-ATP were effective substrates for the enzyme, while the specific activity of the enzyme for the hydrolysis of ATP at pH 7.5 was 800-900 U/mg and was independent of the ion species. ATP, but not PNPP, was hydrolyzed slowly in the absence of metal ions with a specific activity of 140 U/mg. These data demonstrate that in vitro and at pH 7.5 rat osseous plate alkaline phosphatase is an active calcium or magnesium-activated ATPase.

Bone matrix vesicle-bound alkaline phosphatase for the assessment of peripheral blood admixture to human bone marrow aspirates.

PURPOSE: Peripheral blood (PB) admixture should be minimized during numerical and functional, as well as cytokinetic analysis of bone marrow (BM) aspirates for research purposes. Therefore, purity assessment of the BM aspirate should be performed in advance. We investigated whether bone matrix vesicle (BMV)-bound bone alkaline phosphatase (ALP) could serve as a marker for the purity of BM aspirates. RESULTS: Total ALP activity was significantly higher in BM serum (97 (176-124)U/L, median (range)) compared to PB serum (63 (52-73)U/L, p < 0.001). Agarose gel electrophoresis showed a unique bone ALP fraction in BM, which was absent in PB. Native polyacrylamide gel electrophoresis revealed the high molecular weight of this fraction, corresponding with membrane-bound ALP from bone matrix vesicles (BMV), as evidenced by electron microscopy. A serial PB admixture experiment of bone cylinder supernatant samples, rich in BMV-bound ALP, confirmed the sensitivity of this proposed quality assessment method. Furthermore, a BMV ALP fraction of ≥ 15% is suggested as cut-off value for minimal BM quality. Moreover, the BM purity declines rapidly with larger aspirated BM volumes. CONCLUSION: The exclusive presence of BMV-bound ALP in BM could serve as a novel marker to assess purity of BM aspirates.

A histochemical study of acid phosphatases in medullary bone matrix and osteoclasts in laying Japanese quail.

Acid phosphatase activity in medullary bone matrix and osteoclasts of laying Japanese quail was examined histochemically. To avoid nonspecific staining, the reactivity of the enzyme was evaluated using both the azo dye method and the lead salt method and nonembedded thick sections and resin-embedded thin sections. The pH of the incubation medium was also varied from the acid range (pH 5.0 and 6.5) to the alkaline range (pH 8.5). Medullary bone osteoclasts contain both tartrate-resistant acid phosphatase (TRAP) activity and fluoride-resistant acid phosphatase (FRAP) activity, and no significant difference in intensity was detected between active and inactive osteoclasts. The entire matrix of medullary bone was positive for tartrate-resistant, fluoride-sensitive acid phosphatase activity. No reaction product was observed in sections incubated in substrate-free and pH 8.5 media. The results demonstrate the existence of FRAP in medullary bone osteoclasts and suggest that medullary bone matrix includes TRAP throughout the matrix.

New aspects of endochondral ossification in the chick: chondrocyte apoptosis, bone formation by former chondrocytes, and acid phosphatase activity in the endochondral bone matrix.

A detailed histological study of the growth plates from 9- to 20-day-old embryonic chick long bones was carried out with the aim of clarifying the long-debated question of the fate of the hypertrophic chondrocytes. Since resorption in chick bones does not occur synchronously across the plate as it does in mammals, specialized regions develop and the fate of the chondrocyte depends on its location within the growth plate. Where resorption took place, as at the sites of primary vascular invasion or at the main cartilage/marrow interface, chondrocytes underwent apoptosis before the lacunae were opened. In addition, spontaneous apoptosis of chondrocytes occurred at apparently random sites throughout all stages of chondrocyte differentiation. In older chick bones, a thick layer of endochondral bone matrix covered the cartilage edge. This consisted of type I collagen and the typical noncollagenous bone proteins but, in addition, contained tartrate-resistant acid phosphatase in the mineralized matrix. Where such matrix temporarily protected the subjacent cartilage from resorption, chondrocytes differentiated to bone-forming cells and deposited bone matrix inside their lacunae. At sites of first endochondral bone formation, some chondrocytes underwent an asymmetric cell division resulting in one daughter cell which underwent apoptosis, while the other cell remained viable and re-entered the cell cycle. This provided further support for the notion that chondrocytes as well as marrow stromal cells give rise to endochondral osteoblasts.

Osteosarcoma hybrids can preferentially target alkaline phosphatase activity to matrix vesicles: evidence for independent membrane biogenesis.

Alkaline phosphatase is the marker enzyme for matrix vesicles, extracellular organelles that play a major role in primary bone formation and calcification. Recently, we developed osteosarcoma x fibrosarcoma hybrids in which alkaline phosphatase expression was greatly reduced, a phenomenon known as extinction. In the present study, we used to cell hybrids, LTA-1 and LTA-5, constructed from a human osteoblast-like osteosarcoma. TE85, and a mouse fibrosarcoma, La-t-, to examine the differential distribution of alkaline phosphatase between matrix vesicles and the plasma membrane, postulated to be the parent membrane from which matrix vesicles are derived. While alkaline phosphatase in plasma membranes was extinguished, enzyme activity in matrix vesicles from LTA-1 hybrid cells was 34.2% of that present in matrix vesicles from the TE85 parent cells and 200 times that found in La-t- matrix vesicles. Matrix vesicles from LTA-5 had alkaline phosphatase levels similar to La-t-. When other membrane enzymes (phospholipase A2, 5'-nucleotidase, and Na+/K+ ATPase) were examined, hybrid matrix vesicle and plasma membrane levels were similar to those of TE85 and significantly higher than in La-t- membrane fractions. Northern analysis detected mRNA for alkaline phosphatase in TE85 cells, but not in the hybrids or La-t- cells. In contrast, reverse transcription-polymerase chain reaction (RT-PCR) revealed alkaline phosphatase mRNA in the hybrid cells, but at very low levels. Taken together, the data indicate that regulation of plasma membrane and matrix vesicle alkaline phosphatase is independent and suggest that matrix vesicle biogenesis is independent and distinct from that of plasma membrane biogenesis. Analysis of 1B- and 1L-type alkaline phosphatase mRNA by RT-PCR showed that alternate promoter usage of the alkaline phosphatase gene was not responsible for the differential localization of this enzyme in matrix vesicle. Thus, it is likely that matrix vesicle and plasma membrane alkaline phosphatase are regulated differently at a post-transcriptional level.

Cysteine proteinases and matrix metalloproteinases play distinct roles in the subosteoclastic resorption zone.

Digestion of calvarial bone by osteoclasts depends on the activity of cysteine proteinases and matrix metalloproteinases (MMPs). It is unknown, however, whether these enzymes act simultaneously or in a certain (time) sequence. In the present study, this was investigated by culturing mouse calvarial bone explants for various time intervals in the presence or absence of selective low molecular weight inhibitors of cysteine proteinases (E-64, Z-Phe-Tyr(O-t-Bu)CHN2 or CA074[Me]) and MMPs (CI-1, CT1166, or RP59794). The explants were morphometrically analyzed at the electron microscopic level. All proteinase inhibitors induced large areas of nondigested demineralized bone matrix adjacent to the ruffled border of actively resorbing osteoclasts. The appearance of these areas proved to be time dependent. In the presence of the cysteine proteinase inhibitors, a maximal surface area of demineralized bone was seen between 4 and 8 h of culturing, whereas the metalloproteinase inhibitors had their maximal effect at a later time interval (between 16 and 24 h). Because different inhibitors of each of the two classes of proteolytic enzymes had the same effects, our data strongly suggest that cysteine proteinases attack the bone matrix prior to digestion by MMPs. In line with the view that a sequence may exist were differences in the amount of proteoglycans (shown with the selective dye cuprolinic blue) in the subosteoclastic demineralized areas induced by the inhibitors. In the presence of the cysteine proteinase inhibitor, relatively high levels of cuprolinic blue precipitates were found, whereas this was less following inhibition of metalloproteinases. These data suggested that cysteine proteinases are important for digestion of noncollagenous proteins. We propose the following sequence in the digestion of calvarial bone by osteoclasts: after attachment of the cell to the mineralized surface an area with a low pH is created which results in dissolution of the mineral, then cysteine proteinases, active at such a low pH, digest part of the bone matrix, and finally, when the pH has increased somewhat, MMPs exert their activity.

The migration of purified osteoclasts through collagen is inhibited by matrix metalloproteinase inhibitors.

The most obvious proteolytic event controlled by the osteoclast is bone matrix removal in the resorption compartment. Here, however, we investigated whether matrix metalloproteinase (MMP) activity of the osteoclast might be involved in its migration to its future bone resorption site. We seeded either nonpurified or purified osteoclasts onto either uncoated or collagen-coated dentine slices and cultured them in the presence or absence of specific MMP inhibitors. When nonpurified osteoclasts were cultured on uncoated dentine, MMP inhibitors did not prevent pit formation, as previously reported. However, when collagen-coated dentine was used, pit formation was strongly inhibited by MMP inhibitors. The same results were obtained when performing these experiments with purified osteoclasts, thus demonstrating the ability of osteoclasts by themselves to migrate through collagen via an MMP-dependent pathway. This demonstration was confirmed by using collagen-coated invasion chambers. In addition, the invasions were not, or only slightly, inhibited by inhibitors of serine proteinases, cysteine proteinases, and carbonic anhydrase, though the latter two are well established bone resorption inhibitors that strongly inhibited pit formation. It is concluded that osteoclasts can migrate through collagen in the absence of other cells and that this migration relies on MMP activity, whereas other enzymes typically required for bone removal in the resorption compartment are not essential for migration. Some of the osteoclast MMPs might thus be relevant to the migratory/invasive activity of the osteoclast, rather than to its bone resorptive activity itself.

Development and cross-reactive properties of monoclonal antibodies to bovine matrix vesicle alkaline phosphatase.

Alkaline phosphatase (ALPase), concentrated in the membranes of matrix vesicles, is believed to play a role in initial calcification. To further purify, characterize, and identify this enzyme in tissue, a monoclonal antibody was developed against the ALPase of isolated fetal calf matrix vesicles. Splenic lymphocytes derived from mice immunized with Sepharose 6B-purified fetal calf matrix vesicle ALPase were fused with mouse plasmacytoma cells (line X63-Ag-8.653) using standard hybridoma technology. Hyperimmune sera and hybridoma culture supernatants were screened for the presence of specific antibody using a newly developed double-immunosorbent assay in which putative antibody is added to microtiter plate wells precoated with affinity-purified rabbit antimouse immunoglobulin. After incubation and washing, partially purified fetal calf matrix vesicle ALPase is added to each well. The enzyme adheres only to wells that contain specific anti-ALPase antibody. These wells are identified by adding the enzyme substrate p-nitrophenyl phosphate and reading the wells in a plate-reading spectrophotometer at 405 nm. A hybridoma-producing specific antibody was subsequently cloned and grown as ascities-producing tumors in pristane-primed mice. Ouchterlony analysis indicated that the cell line secretes an immunoglobulin of IgG1 class. This antibody reacts specifically with ALPase derived from calf matrix vesicles and cross-reacts with ALPase of bovine kidney, liver, and placental origin and human bone but does not cross-react with bovine intestinal ALPase or ALPase derived from matrix vesicles isolated from rachitic rat growth plate cartilage.

Isolation and characterization of a metalloprotease associated with chicken epiphyseal cartilage matrix vesicles.

A metalloprotease has been isolated from matrix vesicles of chicken epiphyseal cartilage and subsequently characterized. Matrix vesicles obtained by collagenase digestion and differential centrifugation were further purified by Sepharose CL2B gel filtration. The protease was solubilized from the vesicles by treatment with deoxycholate and freeze-thawing, and then isolated by Sephadex G150 gel filtration. Disc electrophoresis of the enzyme, which displayed protease activity toward azocasein substrate, gave a single protein band. Based on molecular weight (MW) determination, lack of immunocross reactivity, and differences in electrophoretic migration, there is little possibility of any contamination with external protease from the commercial collagenase used for vesicle preparation. The matrix vesicle protease had a MW of 33,000 and a pH optimum of 7.2 and was completely inhibited by 0.1 mM EDTA and 0.2 mM o-phenanthroline. alpha 2-Macroglobulin, ovalbumin, cysteine, penicillamine, ethane-1-hydroxy-1, 1-diphosphonate (EHDP) and pyrophosphate at higher concentrations were also inhibitory. The inhibition by omicron-phenanthroline was reversed by Co2+, Zn2+, Fe2+, and Cu2+. Protease activity was most abundant in the heavy fraction of matrix vesicles fractionated by discontinuous sucrose density gradient centrifugation. Release of this protease at the calcifying front could degrade noncollagenous protein moieties that inhibit precipitation of minerals in the extravesicular matrix and thus facilitate mineralization.

Procollagenase associated with the noncalcified matrix of bone and its regulation by parathyroid hormone.

Collagenolytic enzyme activity associated with the noncalcified pool of collagen was studied using calvarial matrices from which the periosteal cell envelope had been removed. Aminophenylmercuric acetate (APMA) stimulated degradation of about 5% of the noncalcified collagen in matrices prepared from freshly dissected bone. Significantly more activity was detected if intact calvaria were cultured 24 h before removal of the cells, in which case 20-30% of the noncalcified collagen was degraded following treatment with APMA. Trypsin elicited a similar response. The collagen being degraded was representative of the entire pool of noncalcified collagen and was not underhydroxylated. Treatment of intact calvaria with parathyroid hormone (PTH) before removal of the cells increased the level of both active collagenase and procollagenase activity associated with the matrix. Enhanced 3H release was noted for PTH treated intact bone in the prior 24 h. Inactivation of endogenous procollagenase by phenanthroline had no effect on the ability of isolated calvarial cells to resorb the bone upon treatment with resorptive agents. The data show that PTH-stimulated collagenolysis of noncalcified collagen involves increased deposition of procollagenase onto the noncalcified matrix in addition to activation of the enzyme.