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.     

Localisation of matrix metalloproteinases and TIMP-2 in resorbing mouse bone

There is strong evidence that matrix metalloproteinases (MMPs) play a crucial role during osteogenesis and bone remodelling. Their synthesis by osteoblasts has been demonstrated during osteoid degradation prior to resorption of mineralised matrix by osteoclasts and their activities are regulated by tissue inhibitors of metalloproteinases (TIMPs). For this study we developed and utilised specific polyclonal antibodies to assess the presence of collagenase (MMP13), stromelysin 1 (MMP3), gelatinase A (MMP2), gelatinase B (MMP9) and TIMP-2 in both freshly isolated neonatal mouse calvariae and tissues cultured with and without bone-resorbing agents. Monensin was added towards the end of the culture period in order to promote intracellular accumulation of proteins and facilitate antigen detection. In addition, bone sections were stained for the osteoclast marker, tartrate-resistant acid phosphatase (TRAP). In uncultured tissues the bone surfaces had isolated foci of collagenase staining, and cartilage matrix stained for gelatinase B (MMP9) and TIMP-2. Calvariae cultured for as little as 3 h with monensin revealed intracellular staining for MMPs and TIMP-2 in mesenchymal tissues, as well as in cells lining the bone plates. The addition of cytokines to stimulate bone resorption resulted in pronounced TRAP activity along bone surfaces, indicating active resorption. There was a marked upregulation of enzyme synthesis, with matrix staining for collagenase and gelatinase B observed in regions of eroded bone. Increased staining for TIMP-2 was also observed in association with increased synthesis of MMPs. The new antibodies to murine MMPs should prove valuable in future studies of matrix degradation.     

Mouse osteoblasts synthesize collagenase in response to bone resorbing agents

Bone cells isolated from mouse calvariae by a sequential digestion procedure have many osteoblast characteristics: they respond to PTH and prostaglandin E2 by activation of adenylate cyclase but not to calcitonin, they stain for alkaline phosphatase and they make only type I collagen. In confluent monolayer culture, they do not secrete collagenase in appreciable quantities, unless stimulated with resorptive substances such as PTH, prostaglandin E2, 1,25(OH)2 vitamin D-3 and monocyte-conditioned medium. This suggests they play a direct role in bone resorption.     

A new synthetic inhibitor of mammalian tissue collagenase inhibits bone resorption in culture

A specific and potent synthetic inhibitor of mammalian tissue collagenase and related metallo-proteinases inhibits the collagen matrix resorption induced by parathyroid hormone (PTH) in cultured embryonic mouse calvaria. The inhibition is reversible, dose-dependent and virtually complete at 50 microM inhibitor concentration whereas that due to a less potent stereoisomer is much weaker. The PTH-enhanced secretion of calvarial lysosomal enzymes and the small spontaneous leakage of lactate dehydrogenase are not affected by the inhibitor. These results suggest that collagenase plays a critical role in bone resorption. Its role is discussed in relation to that of cysteine-proteinases that have also been implicated in this process.     

Mammalian collagenase predisposes bone surfaces to osteoclastic resorption

Summary The cell-free endocranial surface of young adult rat parietal bones was used as a substrate for bone cell-derived mammalian collagenase. Incubation of parietal bones in a concentration of enzyme comparable to that secreted by osteoblastic cells in vitro caused destruction of surface osteoid, and resulted in exposure of mineral onto the bone surface. Bones so pre-treated were considerably more susceptible to osteoclastic resorption than bones preincubated in the absence of collagenase. These results are consistent with the view that the osteoid layer which covers bone surfaces acts as a barrier to osteoclastic contact with underlying, resorption — stimulating bone mineral; and that cells of the osteoblastic lineage induce osteoclastic resorption through collagenase secretion which, by digestion of the surface osteoid, exposes bone mineral to osteoclastic contact.     

Further studies on the activation of procollagenase, the latent precursor of bone collagenase. Effects of lysosomal cathepsin B, plasmin and kallikrein, and spontaneous activation.

1. Cathepsin B, a tissue (lysosomal) proteinase, and two humoral proteinases, plasmin and kallikrein, activate the latent collagenase ('procollagenase') which is released by mouse bone explants in culture. Other lysosomal proteinases (carboxypeptidase B, cathepsin C and D) and thrombin did not activate the procollagenase. Dialysis of the culture fluids against 3M-NaSCN at 4 degrees C and, for some culture fluids, prolonged preincubation at 25 degrees C also caused the activation of procollagenase. 2. In all these cases, activation of procollagenase involved at least two successive steps: the activation of an endogenous latent activator present in the culture fluids and the activation of procollagenase itself. 3. An assay method was developed for the endogenous activator. Human serum, bovine serum albumin, casein and cysteine inhibited the endogenous activator at concentrations that did not influence the collagenase activity. N-Ethylmaleimide and 4-hydroxy-mercuribenzoate stimulated the endogenous activator, but iodoacetate had no effect. 4. It is proposed that cathepsin B, kallikrein and plasmin may play a role in the physiological activation of latent collagenase and thus initiate degradation of collagen in vivo. This may occur whatever the molecular nature of procollagenase (zymogen or enzyme-inhibitor complex) might be.     

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.     

Effects of rat fetuin on stimulation of bone resorption in the presence of parathyroid hormone.

Rat fetuin, which is the rat counterpart of human alpha 2-HS glycoprotein and bovine fetuin, is only detectable in calcified tissues such as bone matrices and dentin, and bone cells such as osteoblasts and osteocytes immunohistochemically. The effect of this protein on bone resorption was examined to study its physiological role in bone metabolism. Rat fetuin increased bone resorption in the presence of low concentrations of parathyroid hormone (PTH), but it had no activity on bone resorption without PTH. The increase in bone resorption by PTH and PTH plus rat fetuin was inhibited by the addition of chymostatin, an inhibitor for cathepsin L. Moreover, we found that when type I collagen from rat was preincubated with rat fetuin, the digestion of rat type I collagen by cathepsin L was increased. These findings suggest that rat fetuin present in bone matrix is important in bone resorption.     

Parathyroid hormone temporal effects on bone formation and resorption

Parathyroid hormone (PTH) paradoxically causes net bone loss (resorption) when administered in a continuous fashion, and net bone formation (deposition) when administered intermittently. Currently no pharmacological formulations are available to promote bone formation, as needed for the treatment of osteoporosis. The paradoxical behavior of PTH confuses endocrinologists, thus, a model bone resorption or deposition dependent on the timing of PTH administration would de-mystify this behavior and provide the basis for logical drug formulation. We developed a mathematical model that accounts for net bone loss with continuous PTH administration and net bone formation with intermittent PTH administration, based on the differential effects of PTH on the osteoblastic and osteoclastic populations of cells. Bone, being a major reservoir of body calcium, is under the hormonal control of PTH. The overall effect of PTH is to raise plasma levels of calcium, partly through bone resorption. Osteoclasts resorb bone and liberate calcium, but they lack receptors for PTH. The preosteoblastic precursors and preosteoblasts possess receptors for PTH, upon which the hormone induces differentiation from the precursor to preosteoblast and from the preosteoblast to the osteoblast. The osteoblasts generate IL-6; IL-6 stimulates preosteoclasts to differentiate into osteoclasts. We developed a mathematical model for the differentiation of osteoblastic and osteoclastic populations in bone, using a delay time of 1 hour for differentiation of preosteoblastic precursors into preosteoblasts and 2 hours for the differentiation of preosteoblasts into osteoblasts. The ratio of the number of osteoblasts to osteoclasts indicates the net effect of PTH on bone resorption and deposition; the timing of events producing the maximum ratio would induce net bone deposition. When PTH is pulsed with a frequency of every hour, the preosteoblastic population rises and decreases in nearly a symmetric pattern, with 3.9 peaks every 24 hours, and 4.0 peaks every 24 hours when PTH is administered every 6 hours. Thus, the preosteoblast and osteoblast frequency depends more on the nearly constant value of the PTH, rather than on the frequency of the PTH pulsations. Increasing the time delay gradually increases the mean value for the number of osteoblasts. The osteoblastic population oscillates for all intermittent administrations of PTH and even when the PTH infusion is constant. The maximum ratio of osteoblasts to osteoclasts occurs when PTH is administered in pulses of every 6 hours. The delay features in the model bear most of the responsibility for the occurrence of these oscillations, because without the delay and in the presence of constant PTH infusions, no oscillations occur. However, with a delay, under constant PTH infusions, the model generates oscillations. The osteoblast oscillations express limit cycle behavior. Phase plane analysis show simple and complex attractors. Subsequent to a disturbance in the number of osteoblasts, the osteoblasts quickly regain their oscillatory behavior and cycle back to the original attractor, typical of limit cycle behavior. Further, because the model was constructed with dissipative and nonlinear features, one would expect ensuing oscillations to show limit cycle behavior. The results from our model, increased bone deposition with intermittent PTH administration and increased bone resorption with constant PTH administration, conforms with experimental observations and with an accepted explanation for osteoporosis.     

Paracrine interactions in bone-secreted products of osteoblasts permit osteoclasts to respond to parathyroid hormone

During bone remodeling, activation of resorption is followed by a cycle of formation and this ordered sequence of events has long suggested that local interactions between osteoclasts and osteoblasts are an important regulatory mechanism in bone metabolism. To study this phenomenon, we have prepared bone cells containing primarily osteoclasts by brief digestion of mice calvariae in collagenase, overnight attachment to polystyrene tissue culture flasks in serumless medium supplemented with OB (osteoblast) cell conditioned medium and subsequent growth in low serum. These OC (osteoclast) cells were found to be highly enriched in acid phosphatase activity and expressed cAMP responses to PTH (parathyroid hormone) and prostaglandin E2 but exhibited no PTH-stimulated hyaluronate synthesis in contrast to prostaglandin E2. PTH effects on hyaluronate, however, could be restored upon coculture of OC cells with OB cells (noncontact) or with OB cell conditioned medium, thereby suggesting that OB cells regulate OC cell PTH responsiveness and/or differentiation by soluble cell products secreted into the medium.     

Bisphosphonates and bone resorption: effects on collagenase and lysosomal enzyme excretion

When added to cultures of parathyroid hormone (PTH)- bones, dichloromethylenebisphosphonate (C12MBP) and 3-amino-1-hydroxypropydilene-1,1-bisphosphonate (AHPrBP) inhibit completely and in a parallel manner the development of resorption lacunae, the loss of calcium by the explants and their PTH-induced excretion of lysosomal hydrolases (β-glucuronidase and N-acetyl-β-glucosaminidase). The loss of collagen (hydroxyproline) by the bones is usually less inhibited than their loss of calcium and their heparin-induced excretion of collagenase is unaffected. To interpret these data, it is proposed that these bisphosphonates act more on the activity of osteoclasts, suppressing simultaneously their excretion of lysosomal enzymes and their erosion of mineralized bone matrix, than on that of other cell types (osteoblasts ?) responsible for collagenase production and the removal of uncalcified collagen.     

Monensin inhibits collagenase production in osteoblastic cell cultures and also inhibits both collagenase release and bone resorption in mouse calvaria cultures

Monensin, a monovalent cation ionophore, inhibited collagenase production in mouse osteoblast-rich bone cell and clonal osteogenic cell cultures. Inhibition of parathyroid hormone-stimulated bone resorption by monensin was also studied in calvaria cultures. Collagenase activity levels in the medium decreased concomitantly with the inhibition of bone resorption by monensin, indicating that monensin inhibited bone resorption by blocking collagenase secretion from osteoblasts in bone explants.     

Interaction of cyclosporine A and calcitonin on bone resorption in vitro

Cyclosporine A (CsA), which is a potent immunosuppressive agent, inhibits bone resorption in vitro. The inhibition of bone resorption by CsA is sustained, unlike the transient inhibition of bone resorption produced by calcitonin (CT). These different patterns of inhibition were studied by examining the interaction between CsA and CT on stimulated bone resorption in the neonatal mouse calvarial resorptive system. "Escape" from the CT inhibition of PTH stimulated bone resorption occurred after 24 hr of organ culture. Coincubation with CsA (1 micrograms/ml) delayed the "escape" response of CT + PTH treated bones, so that the full "escape" response did not occur until after 48 hr of organ culture. Likewise, a pretreatment of 24 hr with CsA (1 micrograms/ml) was sufficient to delay "escape" from CT inhibition of PTH stimulated bone resorption until after 48 hr of organ culture. A higher concentration of CsA (10 micrograms/ml) completely prevented the "escape" response. Our data could indicate an interaction between the CsA and CT inhibitory effects on resorption.     

Parathyroid hormone-induced bone resorption does not occur in the absence of osteopontin

Osteopontin is an RGDS-containing protein that acts as a ligand for the alpha(v)beta(3) integrin, which is abundantly expressed in osteoclasts, cells responsible for bone resorption in osteopenic diseases such as osteoporosis and hyperparathyroidism. However, the role of osteopontin in the process of bone resorption has not yet been fully understood. Therefore, we investigated the direct function of osteopontin in bone resorption using an organ culture system. The amount of (45)Ca released from the osteopontin-deficient bones was not significantly different from the basal release from wild type bones. However, in contrast to the parathyroid hormone (PTH) enhancement of the (45)Ca release from wild type bones, PTH had no effect on (45)Ca release from organ cultures of osteopontin-deficient bones. Because PTH is located upstream of receptor activator of NF-kappaB ligand (RANKL), that directly promotes bone resorption, we also examined the effect of RANKL. Soluble RANKL with macrophage-colony stimulating factor enhanced (45)Ca release from the bones of wild type fetal mice but not from the bones of osteopontin-deficient mice. To obtain insight into the cellular mechanism underlying the phenomena observed in osteopontin-deficient bone, we investigated the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bones subjected to PTH treatment in cultures. The number of TRAP-positive cells was increased significantly by PTH in wild type bone; however, no such PTH-induced increase in TRAP-positive cells was observed in osteopontin-deficient bones. These results indicate that the absence of osteopontin suppressed PTH-induced increase in bone resorption via preventing the increase in the number of osteoclasts in the local milieu of bone.     

Elevated bone collagenolytic activity and hyperplasia of parafollicular light cells of the thyroid gland in parathormone-treated grey-lethal mice

Summary By application of light and electron microscopy, histochemistry, tracer procedures and a collagenolytic assay procedure, it was established that the osteolytic response of grey-lethal mice to acute parathormone (PTH) therapy was decidedly more vigorous than that elicited from their normal littermates.Time and calcium dependency studies conducted on a cell-free extract obtained from PTH-treated grey-lethal mouse bone indicated that the collagenolytic factor present in the preparation was collagenase.The osteoclasts seen in osteopetrotic mouse bone eighteen hours after PTH injection were characteristically intensely basophilic and possessed secretory inclusions apparently derived from their nuclei. Karyorrhexis was of common occurrence in these cells.Histologic evidence indicated that osteocytes promote resorption of bone matrix in anticipation of becoming fused into osteoclasts.A large proportion of the epithelial cells in the thyroid glands of the PTH-treated grey-lethal mice was identified as parafollicular, light cells.Osteopetrosis may be considered a congenital endocrinopathy, the primary lesion of which is hyperplasia of the calcitonin-producing parafollicular cells of the thyroid gland.Aided by a grant from The National Foundation.     

Differential sensitivity of osteoclasts and osteoblasts suggests that prostaglandin E1 effects on bone may be mediated primarily through the osteoclasts

Prostaglandin E (PGE) stimulates resorption in bone. Since osteoblast-like osteosarcoma cells secrete PGE2, the possibility that osteoclasts were the major target for PGE was considered. To study this question, it was first established that in isolated bone cells enriched for either osteoclastic (OC) or osteoblastic (OB) characteristics, PGE1 can induce biochemical effects similar to those seen with bovine parathyroid hormone 1-84 (PTH), another potent stimulator of bone resorption. These changes include increased cAMP and hyaluronate synthesis in OC cells, and increased cAMP but decreased citrate decarboxylation in OB cells. By following these markers, it is demonstrated that PGE1 can activate OC cells at doses as low as 1 nM, whereas OB cells require 250 nM. Bone cell responses to various doses of PTH and PGE1 were also compared. In OC cells the lowest effective dose of PGE1 and PTH was similar (1 nM), but increasing response to PGE1 was seen up to 1000 nM in contrast to PTH response which peaked at 20 nM. In addition, the magnitude of PGE1-induced OC cell hyaluronate was two to four times greater than that of PTH at all doses tested. In OB cells, PTH induced significant decreases in citrate decarboxylation at 0.1 nM, compared to 250 nM for PGE1. Half-maximal inhibition of citrate decarboxylation (19% of control) by PTH occurred at 0.5 nM, whereas 500 nM of PGE1 was required for an equivalent effect. Thus, (i) OC cells responded to PGE1 doses that were approximately 200 times lower than the minimum required by OB cells, and (ii) OB cells responded to 100 times lower doses of PTH than PGE1.     

Comparison of inhibition of bone resorption and escape with calcitonin and dibutyryl 3',5' cyclic adenosine monophosphate

Both parathyroid hormone (PTH) and calcitonin (CT) can increase the concentration of cyclic 3',5' adenosine monophosphate (cAMP) in fetal rat bone in organ culture. Moreover, dibutyryl cAMP (dbcAMP) can both stimulate and inhibit 45Ca release from such bones depending on dose and experimental conditions. In this study we compared dbcAMP and CT for their effects on bones pretreated with PTH. Both compounds produced transient inhibition of bone resorption followed by escape. Escape from dbcAMP was independent of prostaglandin synthesis, since it occurred both in the presence and absence of indomethacin, a prostaglandin cyclo-oxygenase inhibitor.