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.     

Platelet factor 4 regulates osteoclastic bone resorption in vitro

Platelet factor 4, which inhibits collagenases derived from both human skin and granulocytic extracts, was found to block reversibly parathyroid hormone-stimulated ⁴⁵Ca release from fetal rat bone in vitro. The inhibitory property was equally effective in both actively resorbing bones and in bones stimulated to initiate resorption.     

Effects of parathyroid hormone on osteoclasts in vivo

Summary Young rats were treated with high doses of parathyroid hormone (PTH). Osteoclasts from these animals revealed characteristic alterations in comparison to control cells: a) The cytoplasm contained large vacuoles with phagocytosed cells, some of which resembled osteoblasts or osteocytes. The vacuoles were interpreted as lysosomes because the engulfed cells often appeared partly digested and the vacuoles contained acid phosphatase as demonstrated histochemically, b) lipid droplets were present in the cytoplasm and usually located close to the endoplasmic reticulum and/or in regions with many free ribosomes, c) the Golgi complex was more frequently separated from the nuclei than in control cells, d) small coated cytoplasmic bodies were numerous in the peripheral cytoplasm, e) the membranes of the endoplasmic reticulum were fused in some places, f) cytoplasmic regions with numerous free ribosomes were frequent, g) large ring-shaped granules occurred in some mitochondria. Energy dispersive X-ray analysis of these granules provided evidence that they contained calcium and probably phosphorus, h) in some osteoclasts the mitochondria were enlarged. — The findings are consistent with an increased activity of osteoclasts and in particular a stimulation of the lysosomal system in these cells.This research was supported by grants no. 512-819, 512-1545 and 512-1912 from the Danish Research Council. The observations were reported in part at the annual meeting of the Scandinavian Society for Electron Microscopy in Aarhus 1972 (Lucht, 1973). — The authors thank Mr. K. Ibe for cooperation in the energy dispersive X-ray analysis, which was carried out at the JEOL (Europe) S. A. Application Center, Paris.     

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.     

Potentiating effect of heparin in the activation of procollagenase by a low-Mr angiogenesis factor

A low-Mr freely dialysable endothelial cell-stimulating angiogenesis factor (ESAF) from conditioned medium of a mouse lymphoma cell line has previously been shown to activate latent skin fibroblast procollagenase. Activation comparable with the maximum that can be achieved with trypsin is obtained with chemically undetectable amounts of the factor. We now show that when even smaller amounts of ESAF are used heparin is able to potentiate its action in this system. The relationship between this activity and the mechanism of angiogenesis, which is itself potentiated by heparin, is discussed.     

Impact of parathyroid hormone on bone marrow-derived stem cell mobilization and migration

Parathyroid hormone (PTH) is well-known as the principal regulator of calcium homeostasis in the human body and controls bone metabolism via actions on the survival and activation of osteoblasts. The intermittent administration of PTH has been shown to stimulate bone production in mice and men and therefore PTH administration has been recently approved for the treatment of osteoporosis. Besides to its physiological role in bone remodelling PTH has been demonstrated to influence and expand the bone marrow stem cell niche where hematopoietic stem cells, capable of both self-renewal and differentiation, reside. Moreover, intermittent PTH treatment is capable to induce mobilization of progenitor cells from the bone marrow into the bloodstream. This novel function of PTH on modulating the activity of the stem cell niche in the bone marrow as well as on mobilization and regeneration of bone marrow-derived stem cells offers new therapeutic options in bone marrow and stem cell transplantation as well as in the field of ischemic disorders.     

3H-fucose incorporation into bone following exposure to parathyroid hormone,dibutyryl cyclic-AMP and colchicine

Summary Radioautographic and scintillation counting procedures were used to examine the effect of parathyroid hormone (PTH), dibutyryl cyclic-AMP (DB-cAMP), and colchicine on the incorporation of ³H-fucose into macromolecular material in organ cultures of bone. Radioautography demonstrated ³H-fucose incorporation into bone cells, with the heaviest uptake occurring in osteoclasts. A minimal incorporation occurred in pre-osteoblasts and osteoblasts of the osteogenic periosteum, and in fibroblasts of the fibrous periosteum. PTH appeared to produce a heavier label in association with osteoclasts while decreasing the limited labeling associated with cells of the osteogenic and fibrous periosteum. DB-cAMP and colchicine both markedly reduced the labeling associated with osteoclasts, while the minimal labeling of other bone cells remained. By contrast, scintillation counting results indicated that PTH had little or no effect on ³H-fucose incorporation, while DB-cAMP and colchicine considerably reduced the amount of labeled macromolecular material. The incorporation of ³H-fucose into glycoproteins and the role of glycoproteins are discussed.This investigation was aided by grants from the Orthopaedic Research and Education Foundation and the Minnesota Medical Foundation. The author gratefully acknowledges the excellent technical assistance of Karen Brintzenhofe and Cynthia Park     

Immunocytochemical evidence for endogenous calcitonin and parathyroid hormone in osteoblasts from the calvaria of neonatal mice

Summary Immunoreactivities to endogenous calcitonin, endogenous parathyroid hormone, endogenous estradiol and estradiol receptors were studied in osteoblasts from the calvaria of neonatal mice by immunocytochemistry with the use of ultrathin sections obtained by cryo-ultramicrotomy. Tissues were fixed in glutaraldehyde, postfixed in osmium tetroxide and frozen in liquid nitrogen. Estradiol and estradiol receptors could not be detected in osteoblasts, whereas calcitonin- and parathyroid hormone-like immunoreactivities were observed in this cell type. Calcitonin and parathyroid hormone had similar subcellular localizations: immunoreactivities were observed at the plasma-membrane level, in the cytoplasmic matrix, and in the nucleus. These results provide immunocytological evidence for: 1) the internalization of calcitonin and parathyroid hormone in osteoblasts; 2) a direct participation of calcitonin and parathyroid hormone in the regulation of osteoblasts; 3) the absence of estradiol receptors and estradiol in osteoblasts.     

The role of calcitonin and alpha-calcitonin gene-related peptide in bone formation

The Calca gene encodes two polypeptides, calcitonin (CT) and α-calcitonin gene-related peptide (α-CGRP), generated through alternative splicing. While CT, a hormone mainly produced by thyroidal C cells, has been described as a major regulator of bone resorption, α-CGRP, a neuropeptide expressed in the cells of the central and peripheral nervous system, is mostly known as a regulator of vascular tone. Surprisingly, the generation and skeletal analyses of two mouse deficiency models has recently uncovered a physiological function for both peptides in the regulation of bone formation. In the first model, where the replacement of exons 2-5 of the Calca gene resulted in the combined deficiency of CT and α-CGRP, an increased bone formation rate (BFR) was observed, whereas decreased BFR was found in the second model, where the introduction of a translational termination codon into exon 5 of the Calca gene resulted in the specific absence of α-CGRP.     

Effects of intermittent parathyroid hormone (PTH) administration on SOST mRNA and protein in rat bone

Summary Sclerostin, the secreted protein product of the SOST gene, which is mainly expressed by osteocytes, has recently been proposed as a negative regulator of bone osteoblastogenesis. Chronic elevation of PTH reduces SOST expression by osteocytes, while controversial results have been obtained by intermittent PTH administration. We have investigated the effects of intermittently administered PTH on SOST expression and sclerostin localization, comparing them with those of controls, as they appeared in three different bone segments of rat tibia: secondary trabecular metaphyseal and epiphyseal bone, and cortical diaphyseal bone. The histomorphometric results demonstrate that PTH enhances bone turnover through anabolic effects, as shown by the association of increased bone resorption variables with a significant rise in BV/TV, Tb.Th and Tb.N and a fall in Tb.Sp. PTH induces a SOST mRNA and protein fall in secondary metaphyseal trabeculae, diaphyseal bone and in epiphyseal trabeculae. Numbers of sclerostin immunopositive osteocytes/mm² show no change, compared with controls; there are fewer sclerostin-positive osteocytes in secondary metaphyseal trabeculae than in the other two bone areas, both in the control and PTH groups. The low numbers of sclerostin-positive osteocytes in the metaphyseal trabecular bone seem to be directly related to the fact that this area displays a high remodeling rate. The anabolic effects of PTH are in line with the fall of SOST mRNA and protein in all the three bone segments examined; the rise of bone turnover supports a negative role of SOST in bone formation.     

Rat enterocyte Na+ transport in vitro action of parathyroid hormone and calcitonin

Parathyroid hormone (PTH) and calcitonin exert well known effects on the renal tubule which are thought to involve specific hormone receptors and adenyl cyclase. In the intestine, it is not clear whether the action of PTH and calcitonin is only indirect or also direct, and their mechanisms of action are much less well established. In the present study, possibly direct effects of PTH and calcitonin on Na⁺ transport in isolated intestinal epithelial cells of rats were investigated. In the presence of bovine PTH (1.2 I.U./ml) in the incubation medium, the Na⁺ efflux rate constant (${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$) of isolated enterocytes was significantly reduced when compared to that in control experiments with the hormone vehicle only. The mean depression of ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$ induced by bovine PTH was 26% as compared to the control (100%) and to that induced by ouabain (4.0mM) which was 44%. No depressant effect of bovine PTH on ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$ was observed when the isolated enterocytes were incubated with ouabain (4.0 mM). Thus, bovine PTH appeared to inhibit the ouabain-sensitive Na⁺ pump. When incubating the isolated epithelial cells in an EGTA-containing Ca²⁺-free medium, bovine PTH lost its capacity to inhibit (${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$). Thus, the presence of extracellular Ca²⁺ appeared necessary for the inhibitory effect of bovine PTH. In contrast to its effect on ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$, bovine PTH induced no change in net Na⁺ uptake by isolated enterocytes. Moreover, no significant effect on enterocyte Na⁺ transport could be demostrated for salmon or porcine calcitonin at two different concentrations in the incubation medium. Neither bovine PTH nor salmon calcitonin induced significant changes in enterocyte cyclic AMP or cyclic GMP concentrations. It was concluded that bovine PTH, but not calcitonin, exerted a direct inhibitory effect on the ouabain-sensitive ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$ of isolated rat enterocytes. The effect of bovine PTH occured without measurable activation of the cyclic nucleotide system but needed the presence of Ca²⁺ in the incubation medium to be operative.     

Stimulation of bone resorption in vitro by a non-prostanoid factor released by human monocytes in culture

Cultured human monocytes produce a non-prostanoid factor which stimulates bone resorption in vitro. Production of the factor is not dependent on prostaglandin synthesis, but stimulation of resorption by the factor does involve prostaglandin production by bone. Salmon calcitonin inhibits the stimulatory activity by up to 80%. Production of the factor is not increased by the presence of lymphocytes in the monocyte cultures. Partial purification of the bone-resorbing activity by Ultrogel ACA 54 chromatography shows that it has an apparent molecular weight between 12 000 and 20 000. We suggest that the resorptive activity is due to an interleukin 1-like factor rather than an osteoclast activating factor.     

Identification of parathyroid hormone-regulated proteins in mouse bone marrow cells by proteomics

The ability of parathyroid hormone (PTH) to enhance bone formation has recently been exploited in the treatment of osteoporosis. Several studies have suggested that the activation of bone marrow stromal cells could be preceded to show the anabolic effect of PTH on bone formation, but little is known of PTH-regulated proteins in bone marrow cells. Therefore, protein profiling in the intermittent PTH-treated bone marrow cells was evaluated using proteomics. Daily treatment for 5 days consisting of subcutaneous injection of either 150 microg/kg per day of mouse PTH (1-84) or vehicle (0.9% normal saline) was performed on the ICR mouse. At the end of the treatment period, bone marrow cells were separated and used in proteomics. The expression levels of seven proteins including vimentin were decreased, but those of four proteins including calreticulin and thioredoxin domain containing 7 protein (Txnde7) were increased. Among these, the decrease of vimentin and the increase of both calreticulin Txnde7 in mRNA levels were confirmed by semi-quantitative RT-PCR. In PTH-treated mouse MC3T3-E1 osteoblast cells, mRNA expression levels were not totally consistent with the results observed in proteomics. In conclusion, the differentially expressed proteins in bone marrow cells depending on PTH could be highly linked to the differentiation of osteoprogenitor cells in the bone marrow into preosteoblast cells.     

A factor synthesized by rabbit periosteal fibroblasts stimulates bone resorption and collagenase production by connective tissue cells in vitro

Unstimulated monolayer cultures of confluent rabbit periosteal fibroblasts synthesize a factor that stimulates bone resorption in vitro. Furthermore it stimulates rabbit chondrocytes and mouse osteoblasts to synthesize collagenase. The factor has no effect on dead bone in culture, and its activity on live bone is mediated principally by osteoclasts, since it is 75% inhibited by salmon calcitonin. Characterization of the factor by gel filtration and isoelectric focusing indicates an M_r in the range 15 000–25 000 and a pI corresponding to approx. pH 4.7. These biological and physicochemical properties are similar to those reported for a factor released by peripheral blood monocytes. However, whereas human monocyte factor in both the crude and partially-purified state exhibits interleukin-1 activity, crude and fractionated periosteal fibroblast-conditioned medium does not. This is the first report of a conditioned medium containing a molecule like the monocyte-factor which appears to have no interleukin 1 activity. The factor may be synthesized by a wide range of cell types, and could have an important role in mediating connective tissue degradation during both physiological and pathological resorption.     

Bone matrix studies Influences of parathyroid extract,calcitonin, and cholecalciferol and of rickets and its treatment

Bones from young rats were incubated with radioactive glucosamine and proline. The concentrations and specific activities of matrix glycosaminoglycan fractions, prepared by a cetylpyridinium chloride method, and the specific activity of insoluble collagen hydroxyproline were determined. Acute parathyroid extract treatment increased labelling of hyaluronic acid and a glycopeptide fraction. These effects were partially blocked by calcitonin treatment which had no effect by itself. Parathyroid extract inhibited collagen synthesis and this effect was not blocked by calcitonin. Effects of these two hormones on labelling of chondroitin sulfate fractions were more variable. Vitamin D-3 caused an increase in labelling of all matrix fractions measured in bone from thyroparathyroidectomized rats, but its stimulating effect upon collagen synthesis was blocked by paratyroid extract.Bones from rats made rachitic on a phosphorus and vitamin D-deficient diet were incubated in vitro with radioactive glucosamine and proline. Over a three-week period rachitic bone exhibited a progressive fall in concentration and labelling of a glycopeptide-hyaluronic acid fraction, while pair-fed animals supplemented either with phosphorus alone or with phosphorus and vitamine D-3 not only remineralized their bones, but the bones showed a pronounced increment in concentration and labelling of this fraction. Both treatment regimens also enhanced chondroitin sulfate and collagen labelling.     

甲状旁腺素促进小鼠软骨细胞成软骨和抑制其终末期分化的实验研究

目的探讨甲状旁腺素(PTH)对小鼠软骨细胞成软骨性的促进作用和终末期分化的抑制作用。方法分离和培养新生小鼠胸骨软骨细胞,经PTH处理,倒置显微镜观察细胞形态的变化;Alcian蓝染色和碱性磷酸酶(ALP)染色方法检测软骨细胞蛋白多糖和ALP的分泌;RT-PCT法和Western blot方法检测细胞内成软骨因子和病理性肥大分化因子基因和蛋白的表达。结果新生小鼠胸骨软骨细胞具有自发成熟分化的特征,与对照组相比,经PTH处理的细胞更接近于软骨细胞形态;PTH明显提高软骨细胞Alcian蓝染色的强度,降低ALP染色的强度;PTH显著提高细胞内Sox9和Aggrecan基因和蛋白的表达,明显降低ALP和Runx2基因和蛋白的表达。结论 PTH具有促进小鼠软骨细胞成软骨和抑制其终末期分化的作用。     

Experimental study of changes in osteoblastic shape induced by calcitonin and parathyroid extract in an organ culture system

Summary Neonate rat endocranial osteoblasts were cultured on their bone surfaces in control medium (CC) or medium to which either parathyroid extract (PTE) or calcitonin (CT) had been added for 2, 4, 8 or 24 h. Some were cultured for 24 h in CC, then for 2, 4, 8 or 24 h in either CT or PTE medium; or for 24 h in PTE, then for 2, 4, 8 or 24 h in either CC or CT; or 24 h in CT and 2, 4, 8 or 24 h in CC. The dorsal ruffling of the cells in CC was found to be suppressed by later culturing with PTE and the disoriented cells reorganized to form arrays of parallel cells. The effects of PTE were also reversed by CC or CT: the osteoblasts in the second culture (CC) lost elongation and order, and proceeded through a proliferative phase before exhibiting the ruffling form similar to a single CC 24 h culture. PTE-cultured osteoblasts showed an increase in cell overlap and contact so that a more competent barrier was formed separating the bone from the medium. In control or CT medium, however, intercellular gaps were greater than in vivo.We are grateful for the expert technical assistance of Elaine Bailey, for laboratory facilities kindly provided by Dr. Martin Evans, and for financial support from the Medical Research Council     

Synthesis and characterization of novel biotinylated carboxyl-terminal parathyroid hormone peptides that specifically crosslink to the CPTH-receptor

Parathyroid hormone (PTH) regulates calcium, phosphorous and skeletal homeostasis via interaction with the G protein-coupled PTH/PTHrP receptor, which is fully activated by the amino-terminal 34 amino-acid portion of the hormone. Recent evidence points to the existence of another class of receptors for PTH that recognize the carboxyl (C)-terminal region of intact PTH (1–84) (CPTHRs) and are highly expressed by osteocytes. Here we report the synthesis and characterization of two novel bifunctional CPTH ligands that include benzoylphenylalanine (Bpa) substitutions near their amino-termini and carboxyl-terminal biotin moieties, as well as a tyrosine³⁴ substitution to enable radioiodination. These peptides are shown to bind to CPTHRs with affinity similar to that of PTH (1–84) and to be specifically and covalently crosslinked to CPTHRs upon exposure to ultraviolet light. Crosslinking to osteocytes or osteoblastic cells generates complexes of 80 and 220 kDa, of which the larger form represents an aggregate that can be resolved into the 80 kDa. The crosslinked products can be further purified using immunoaffinity and avidin-based affinity procedures. While the molecular structure of the CPTHR(s) remains undefined, these bifunctional ligands represent powerful new tools for use in isolating and characterizing CPTHR protein(s).     

Effects of calcitonin on osteoclasts in vivo

Summary Osteoclasts from the tibial metaphyses of young rats treated with porcine calcitonin were studied by electron microscopy. The animals were sacrificed 1 1/2, 4, 8 or 12 hours after injection of the hormone. In survey sections examined by light microscopy the osteoclasts appeared smaller than in control animals. At the ultrastructural level the osteoclasts showed the following alterations: 1) The typical ruffled border was absent. 2) Acid phosphatase was not present in the extracellular space between cell and bone. 3) The number of large vacuoles was decreased and there was no local accumulation of vacuoles in the cytoplasm. 4) The vacuoles did not contain bone crystals. 5) Vacuoles with cell organelles were increased in number. The majority of these vacuoles were identified as autolysosomes because they contained acid phosphatase and the enclosed cell organelles were partially digested. The above changes were present at all time intervals studied.The findings suggest that calcitonin decreases or inhibits bone resorption by osteoclasts. A decreased function of the osteoclasts may contribute to the hypocalcemic effect of the hormone. The increased number of autolysosomes is evidence of an enhanced autophagocytosis. Possible origins of the autolysosomes in osteoclasts are discussed.This research was supported by grants no. 512–819, 512–1545 and 512–1912 from the Danish Medical Research Council. The present observations were first reported at the annual meeting of the Scandinavian Society for Electron Microscopy in Umeå 1973 (Lucht, in press). I wish to thank Professor Arvid B. Maunsbach for valuable discussions and suggestions.