Endodontic status in older U.S. adults. Report of a survey.

BACKGROUND: Aging people experience a number of changes in the root canal systems of teeth that, while they are normal, have the potential to compromise the pulp's response to injury. METHODS: To better understand the dental perspective on these changes, the authors mailed a questionnaire to diplomates of the American Board of Endodontics. The questions dealt with the respondents' experiences, ages of their patient population and their perception of root canal changes in aging patients. The authors analyzed the data in terms of number of diplomates providing a response and stratified them on the basis of the respondents' number of years in practice. RESULTS: Respondents indicated that the number of patients aged 65 years and older in their practices is increasing. Virtually all of the diplomates agreed that the root canal gets smaller with age, but that this diminution does not contribute to the failure of treatment of affected teeth. Most respondents indicated that aging patients' teeth are in poorer condition than those of younger patients. CONCLUSIONS: As the U.S. population ages, clinicians need to have a better understanding of the physiological changes occurring in older patients' teeth that may influence the treatment required to help patients retain their natural dentition. Further studies are needed to determine the impact of aging on dental disease and treatment modalities. CLINICAL IMPLICATIONS: Recognition of changes in the dentition of aging patients will lead to more successful treatment, retention of functional natural dentition and better maintenance of general health.

The calpain-calpastatin system and cellular proliferation and differentiation in rodent osteoblastic cells.

The calpain-calpastatin system, which consists of calpains I and II (two ubiquitously distributed calcium-activated papain-like cysteine proteases), as well as calpastatin (the endogenous calpain inhibitor), plays an important role in cell proliferation and differentiation in many tissues. However, its contribution to the regulation of osteoprogenitor or pluripotent stem cell proliferation and differentiation into osteoblasts remains poorly defined. In these studies, rat pluripotent mesodermal cells (ROB-C26) and mouse MC3T3-E1 preosteoblasts were induced to differentiate into osteoblasts by long-term culture or in response to bone morphogenetic protein (BMP). The occurrence and distribution of calpain-calpastatin system proteins were determined by immunofluorescent microscopy, measurement of calcium-dependent proteolytic activity, and Western blotting. Treatment of intact MC3T3-E1 cells with an irreversible, membrane-permeable cysteine protease inhibitor attenuated proliferation and alkaline phosphatase upregulation under differentiation-enhancing conditions. Calpain II activity increased during differentiation of MC3T3-E1 cells in postconfluent culture. When ROB-C26 cells were maintained in long-term culture, neutral protease, calpain I, and calpain II activities increased 2- to 3-fold in the absence of BMP. In the presence of partially purified native BMP, neutral protease and calpain I activities also increased similarly, but calpain II activity increased by 10-fold in 3 days. The maximal increase in alkaline phosphatase occurred 4 to 11 days after the calpain II activity had peaked. Induction of differentiation in long-term MC3T3-E1 cultures was associated with higher calpain II and 70- and 110-kDa calpastatin protein levels and lower 17-kDa calpastatin degradation product levels. In conclusion, cysteine protease activity is essential for preosteoblastic proliferation and differentiation. The calpain-calpastatin system is regulated during osteoprogenitor proliferation and differentiation, as it is in other cells, and bone morphogenetic protein is a specific regulator of calpain II.

Vgr-1/BMP-6 induces osteoblastic differentiation of pluripotential mesenchymal cells.

The transforming growth factor-beta (TGF-beta) superfamily is a group of secreted growth factors that appears to play a central role in mesenchymal differentiation, including cartilage and bone formation. The present study examines the role of one member of this family, vgr-1, also called bone morphogenetic protein-6, in mesenchymal cell differentiation. This factor may be considered as a prototype for the largest subgroup of related factors within the TGF-beta superfamily, the function of which has as yet been poorly defined. vgr-1 has been localized previously to hypertrophic cartilage and has been shown to induce endochondral bone formation in vivo. To further characterize the role of vgr-1 in bone and cartilage differentiation, we stably transfected the pluripotent mesenchymal cell line ROB-C26 with a vector to overexpress vgr-1. Overexpression of this factor did not affect cell shape or morphology, but it enhanced osteoblastic differentiation in vitro and altered cellular responsiveness to retinoic acid. Furthermore, the extracellular matrix produced by these vgr-1-overexpressing cells induced ectopic bone formation in vivo and osteoblastic differentiation in vitro, similar to the matrix produced by C26 cells treated with retinoic acid. The osteoinductive effect of the matrix from vgr-1-overexpressing cells was blocked using a neutralizing vgr-1 antibody but not with a neutralizing TGF-beta 1 antibody, indicating that vgr-1 alone was required for this osteogenic effect. In contrast, the osteoinductive effect of matrix from retinoic acid-treated cells was blocked with both vgr-1 and TGF-beta 1 antibodies, suggesting that TGF-beta 1 may act prior to vgr-1 during osteoblastic differentiation. We further demonstrated that osteoinduction by vgr-1 was dependent on presentation of vgr-1 within the matrix, because the osteoinductive effect of matrix from vgr-1-overexpressing cells could not be mimicked with the addition of soluble vgr-1 to parental C26 cells. Finally, overexpression of MyoD within the C26 cells overexpressing vgr-1 converted the cells to myoblasts, indicating that vgr-1 had induced early osteoblastic.

Production and characterization of an antibody against the human bone GLA protein (BGP/osteocalcin) propeptide and its use in immunocytochemistry of bone cells.

We have generated and characterized an antibody that recognizes the C-terminal sequence of the propeptide of human bone GLA protein (BGP/osteocalcin)(amino acid -26 to -1, with +1 being the amino terminus of the mature protein). The range of sensitivity of the antibody, as determined by enzyme-linked immunosorbent assay (ELISA), was 0.5-250 ng/ml. The antibody effectively recognized pro-BGP in cell layer extracts of transformed cells (KT-005), but did not recognize mature, propeptide-less BGP in the medium from the same cultures. Strong labelling was obtained using this antibody in immunoperoxidase staining or immunofluorescence of both transformed and normal human bone cells in vitro. Monensin significantly altered the intracellular pattern of labelling in immunofluorescence studies, indicating that the recognized antigen was associated with the cellular secretory pathway. We also obtained a specific and strong staining of cells in tissue sections of human fetal bone. Antibodies against the mature protein strongly stained the mineralization front, but did not stain cells to any appreciable level. Newly embedded osteocytes were the predominant cell type stained in such material, suggesting that they may represent the major of BGP in the intact tissue. These observations indicate that BGP synthesis is a late event in osteoblastic development and that antibodies generated against the propeptide sequence are a potentially powerful tool in the analysis of bone tumors and evaluation of osteoblastic differentiation.

Interleukin-4 enhances murine bone marrow macrophage M-CSF receptor expression by a posttranscriptional mechanism.

Activated T-lymphocytes secrete interleukin-4 (IL-4), which has been shown to modulate a variety of monocyte activities requiring monocyte/macrophage colony-stimulating factor (M-CSF). To account for this interaction, we postulated that IL-4 acts on target cells by altering the expression of the M-CSF receptor (M-CSFr). To test this hypothesis, murine bone marrow macrophages were cultured under conditions that down-regulate M-CSFr and the effect of IL-4 on the reexpression of the receptor measured by binding of 125I-labeled M-CSF to the cells. The data show that incubation with IL-4 results in a dose-dependent, 2-3 x increase in M-CSFr with no change in binding affinity and a maximal effect on binding at about 12 h. This increase in M-CSFr is dependent upon new, specific protein synthesis as shown by the inhibitory action of cycloheximide, and gel analysis of radiolabeled, specific protein, immunoprecipitated with anti-M-CSFr antibody. Treatment with IL-4 does not stimulate M-CSFr mRNA expression but, consistent with enhanced receptor levels, does result in a heightened proliferative response to M-CSF. Thus, IL-4 affects M-CSF treated monocytic cells, at least in part, by altering the expression of M-CSFr.

Age-related bone loss in mice is associated with an increased osteoclast progenitor pool.

Age-associated osteopenia has been documented to occur in mice and, therefore, provides a model system whereby mechanisms of bone loss can be assessed in vivo and in vitro. One such mechanism, that could explain the increased resorptive activity seen in some forms of osteopenia, is an age-associated increase in the osteoclast precursor pool and osteoclastogenic formation. To test this hypothesis, we studied the bone marrow composition of aged (24 months) mice to determine if increased numbers of monocyte/macrophage/osteoclast precursor cells (MMOPC) were present when compared to young (4-6 months) animals. Our data show a moderate increase of 20-30% more hematopoietic cells obtained from the long bones of the aged animals. However, both liquid and semi-solid culture techniques demonstrate an approximately 2-3.5-fold increase in the numbers of plastic adherent macrophages or mononuclear colonies in bone marrow derived from the aged mice when stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF) or macrophage colony stimulating factor (GM-CSF), indicating a preferential increase in MMOPCs. In addition, cells derived from the aged mice show higher levels of cytokine stimulated incorporation of [3H]-thymidine and [3H]-leucine, with increased protein synthesis seen up to 7 days after cytokine stimulation, suggesting that these cells also have an enhanced sensitivity to cytokines.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies that recognize antigens in human osteosarcoma cells and normal fetal osteoblasts.

Four monoclonal antibodies (HOB-1-4) that react against human osteosarcoma cells (Saos-2) and human fetal osteoblasts in situ were developed by immunizing mice with Saos-2 cells. HOB-1 (IgG1, k) and HOB-2 (IgM, k) stain cytoplasmic antigens in Saos-2 cells, human authentic osteoblasts and occasional cells in liver, spleen and, in the instance of HOB-1, kidney and adrenal gland. On Western blotting of Saos-2 cell lysates, HOB-1 recognizes a single protein species of M(r) 59,000, while HOB-2 reacts with a different species of M(r) 57,000. HOB-3 (IgG1, k) stains a cell membrane associated antigen in Saos-2 cells and osteoblasts in situ. The reaction pattern of this antibody is virtually identical to that seen in alkaline phosphatase (ALP)-positive cells in all organs examined immunohistochemically, except for intestinal epithelium. Both immunoblotting and immunoprecipitation analyses confirm that the antigen detected by HOB-3 is ALP. HOB-4 (IgG1, k) reacts weakly against Saos-2 cells cultured under standard conditions, but strongly following the exposure of the cells to the secretory inhibitor monensin. On frozen section screening, this antibody reacts preferentially with the collagenous extracellular matrix not only of the periosteum but of other tissues and organs as well. The precise identity of the HOB-4 antigen remains to be established. We suggest that these four monoclonal reagents will be useful adjuncts in characterizing the osteoblastic phenotype.

Regulation of an H-ras-related transcript by parathyroid hormone in rat osteosarcoma cells.

The rat osteosarcoma cell line UMR 106-01 is a commonly used model system for the study of osteoblast function. However, it also expresses a phenotype characteristic of transformed cells. To test whether the latter could be accounted for by aberrant oncogene expression, we probed Northern blots of UMR and other osteoblastic cells with a panel of oncogene probes. These blots, when probed with a cDNA specific for v-H-ras, revealed a 7.0-kilobase (kb) H-ras-related transcript (designated HRRT) in UMR 106-01 cells that was not expressed in other osteoblastic cells. Osteoblast-enriched calvarial cells expressed the typical 1.1-kb H-ras mRNA, which was absent in UMR cells. Additionally, Western blots of lysates of UMR cells documented the presence of three proteins immunologically related to H-rasp21. To determine whether HRRT represented a recombinant retrovirus product, Northern blots were probed with a cDNA specific for the highly conserved gag-pol region of Moloney murine leukemia virus. These blots showed parallel cross-reactivity with an apparently identical transcript of 7.0 kb. The 7.0-kb transcripts detected by both v-H-ras and gag-pol probes declined to the same extent after treatment with concentrations of PTH known to inhibit proliferation of these cells. PTH regulated the abundance of HRRT in a time- and dose-dependent manner, with greatest repression of the transcript after 8 h of treatment with 10(-8) M PTH. The decrease in HRRT could not be completely accounted for by changes in transcriptional activity, as determined by nuclear run-on assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutral proteinase expression by human mononuclear phagocytes: a prominent role of cellular differentiation.

Human mononuclear phagocytes have the capacity to participate directly in extracellular matrix turnover via the secretion of neutral proteinases. These neutral proteinases include the serine proteinases, elastase and cathepsin G and the metalloproteinases, interstitial collagenase, 92 kD type IV collagenase, 72 kD type IV collagenase and stromelysin. Mononuclear phagocytes also produce the counter-regulatory metalloproteinase inhibitor, TIMP (tissue inhibitor of metalloproteinases). We have studied the capacity of normal human mononuclear phagocytes and of the human monocytic tumor line U937 to elaborate proteinases and inhibitors. The serine proteinases, elastase and cathepsin G, are present only at the earliest stages of mononuclear phagocyte differentiation (U937 cells in the basal state, freshly isolated peripheral blood monocytes) and are stored within intracellular granules. As human mononuclear phagocytes differentiate (U937 cells exposed to phorbol esters, human monocytes cultured in vitro), the cellular content of these serine proteinases declines rapidly. Accompanying the acquisition of a more differentiated state, the ability for regulated secretion of the neutral metalloproteinases is attained. This capacity is acquired in a sequential manner, with secretion of the 92 kD type IV collagenase observed at earlier states of differentiation while release of stromelysin requires a fully differentiated and LPS (lipopolysaccharide)-stimulated alveolar macrophage. Interstitial collagenase and 72 kD type IV collagenase are synthesized at intermediate stages of differentiation. In comparison to human fibroblasts, human mononuclear phagocytes produce approximately 10-30% of the interstitial collagenase, 10% of the stromelysin and 1-2% of the 72 kD type IV collagenase on a per cell basis. Synthesis of the 92 kD type IV collagenase is restricted to the inflammatory cell (but also occurs in neutrophils and keratinocytes).

Clonal osteogenic cell lines express myogenic and adipocytic developmental potential.

Clonal osteoblastic cell lines were isolated from neonatal rat calvariae and characterized with regard to a number of features associated with authentic osteoblasts. These included elevated alkaline phosphatase activity (relative to fibroblasts), PTH and PGE2-stimulated increases in cAMP, the predominant synthesis of type 1 collagen, and the production of a mineralized matrix in vitro. By these criteria, five clones with osteoblast-like phenotypes were identified (ROB-C8a, C11, C20, C23, and C26) which varied somewhat in shape, levels of alkaline phosphatase activity, and in responsiveness to PTH and PGE2. C11, C20, and C23 responded to both effector substances, whereas C8a only responded to PTH and C26 only responded strongly to PGE2. Upon further examination, two of the clones (C23 and C26) were also found to exhibit significant muscle myotube formation after reaching confluence, and three of the clones (C8a, C11, and C26) showed marked adipocyte differentiation after treatment with dexamethasone. Overall, these data add further supporting documentation to (1) the suspected ontogenetic relationships of osteoblasts to other connective tissue cells, and (2) the concept that osteoblastic cells associated with neonatal rat calvariae are in various stable stages of differentiation and developmental commitment.

Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro.

The in vitro effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on osteogenic and myogenic differentiation was examined in two clonal cell lines of rat osteoblast-like cells at different differentiation stages, ROB-C26 (C26) and ROB-C20 (C20). The C26 is a potential osteoblast precursor cell line that is also capable of differentiating into muscle cells and adipocytes; the C20 is a more differentiated osteoblastic cell line. Proliferation was stimulated by rhBMP-2 in C26 cells, but inhibited in C20 cells. rhBMP-2 greatly increased alkaline phosphate (ALP) activity in C26 cells, but not in C20 cells. The steady-state level of ALP mRNA was also increased by rhBMP-2 in C26 cells, but not in C20 cells. Production of 3',5'-cAMP in response to parathyroid hormone (PTH) was dose-dependently enhanced by adding rhBMP-2 in both C26 and C20 cells, though the stimulatory effect was much greater in the former. There was neither basal expression of osteocalcin mRNA nor its protein synthesis in C26 cells, but they were strikingly induced by rhBMP-2 in the presence of 1 alpha,25-dihydroxyvitamin D3. rhBMP-2 induced no appreciable changes in procollagen mRNA levels of type I and type III in the two cell lines. Differentiation of C26 cells into myotubes was greatly inhibited by adding rhBMP-2. The inhibitory effect of rhBMP-2 on myogenic differentiation was also observed in clonal rat skeletal myoblasts (L6). Like BMP-2, TGF-beta 1 inhibited myogenic differentiation. However, unlike BMP-2, TGF-beta 1 decreased ALP activity in both C26 and C20 cells. TGF-beta 1 induced neither PTH responsiveness nor osteocalcin production in C26 cells, but it increased PTH responsiveness in C20 cells. These results clearly indicate that rhBMP-2 is involved, at least in vitro, not only in inducing differentiation of osteoblast precursor cells into more mature osteoblast-like cells, but also in inhibiting myogenic differentiation.

Lysozyme synthesis in osteoclasts.

Osteoclasts may or may not be directly related to monocytes and macrophages, but it is well established that these cell types share a number of features in common. In the present study we sought to extend this comparison by assessing lysozyme synthesis in osteoclasts, an enzyme known to be produced and secreted in large amounts by monocytes and macrophages. Our data show that freshly isolated chicken osteoclasts and osteoclasts in situ contain an abundant amount of lysozyme and correspondingly high steady-state levels of the enzyme's messenger RNA. Marrow macrophages, at various stages of in vitro maturation, also possess lysozyme mRNA but in amounts approximately two to four times lower than osteoclasts. These observations reaffirm the monocyte-macrophage nature of the osteoclast but raise questions about the function of the lysozyme in this cell. At present, the role of the lysozyme in osteoclast activity remains unexplained.

Stimulation of collagenase production by rat osteosarcoma cells can occur in a subpopulation of cells.

Recent studies have indicated that neutral collagenase can be produced in bones of rats. In addition, it has been demonstrated by in vitro studies that the enzyme is likely secreted by osteoblasts. Cells of the osteoblastic tumor cell line UMR-106 can be stimulated to produce not only collagenase, but also collagenase inhibitor and plasminogen activator. However, it is conceivable that not all osteoblasts produce all of these proteins. In this study, in which UMR cells were maximally stimulated with PTH, only a subpopulation of cells was observed to produce enhanced levels of collagenase but all cells had the ability to synthesize plasminogen activator. Cells of the rat osteosarcoma line UMR-106-01 were stained for the presence of collagenase and tissue plasminogen activator using an immunohistochemical procedure. In many cases, the cells were exposed to monensin for the final 3 h of incubation as well as to the inducing agent PTH. Monensin prevented export of the enzymes, enabling them to be visualized within their cell or origin. Maximal stimulation of collagenase was demonstrated to occur 8 h after exposure to 10(-8) -10(-7) M PTH. Under these conditions, 14-17% of the cells appeared to synthesize elevated amounts of collagenase (as determined by intense staining). Without PTH stimulation, there was a low level of collagenase in all cells, but less than 1% of the cells stained heavily for the enzyme. In contrast, strong staining for plasminogen activator was observed in all cells with or without PTH treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial characterization of a parathyroid hormone-stimulated resorption factor(s) from osteoblast-like cells.

PTH stimulates osteoblast-like cells to produce a product(s) capable of increasing cellular bone resorption. We have investigated this phenomenon using primary cultures of osteoblasts and the clonal osteoblast-like cell line ROS 17/2. Conditioned medium from PTH-stimulated populations of either culture increases bone resorption compared to conditioned medium measured in three independent assay systems; the isolated osteoclast assay system, elicited macrophages, and the fetal bone rudiment system. Characterization of the factor(s) of PTH-stimulated osteoblast-like cell (ROS 17/2) suggests that the compound(s) is not prostaglandin (no inhibition by indomethacin; not extractable in diethylacetate). Rather, it is heat and protease sensitive. In addition, secretion of the product is sensitive to cycloheximide. These findings lead us to the conclusion that the factor(s) is protein. Further work demonstrates the necessity for a divalent cation for retention of factor activity. Finally, we have estimated the molecular radius of the factor as about 110,000 daltons and perhaps a second at about 70,000 daltons using a sizing column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of [35S]methionine-labeled PTH-stimulated ROS culture supernatants reveals relatively increased secretion of proteins with these approximate molecular radii.

Second messenger signaling in the regulation of collagenase production by osteogenic sarcoma cells.

Recent work indicates that PTH can stimulate osteoblastic cells to secrete neutral collagenase, an enzyme thought to be linked to bone matrix turnover. Since recent studies suggest that the calcium/protein kinase-C (PKC) message system is involved in signal transduction stimulated by PTH, we examined the role of these putative second messengers of PTH in the regulation of collagenase production by the osteoblastic tumor cell line UMR 106-01. Immunohistochemical staining of cells exposed to PTH (10(-7) M) revealed that about 20% of the entire population was positive for collagenase, compared to less than 3% staining positively in control untreated cells. Incubation with the cAMP analog 8-bromo-cAMP (8BrcAMP) increased the number of collagenase-staining cells in a dose-dependent manner (ED0.5 = 2.5 x 10(-4) M), but to a lower level than PTH, with the maximal effect producing about 15% positive cells. The calcium ionophore ionomycin (10(-7) M) was ineffective, whereas phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased collagenase-specific staining to about 5%, but only at high concentrations (10(-5) M). Incubation of UMR 106-01 cells with ionomycin and PMA did not change the effect of the latter. When the three agents were used in combination, an additive effect was observed, which fully reproduced that of PTH. Similarly, the amount of collagenase released into the medium by cells stimulated with maximal concentrations of 8BrcAMP (10(-3) M) was only 80% of that induced by maximal doses of PTH (10(-7) M). PMA (10(-5) M) was slightly stimulatory, and ionomycin was ineffective alone, but they were synergistic with submaximal doses of 8BrcAMP (10(-4) M). In agreement with the immunohistochemical results, the full hormonal effect was reproduced when the three second messenger analogs were used in combination. In conclusion, signal transduction from PTH receptor to collagenase production is mediated mainly by cAMP; the Ca2+/PKC system appears to have a contributory role necessary for the full expression of hormonal response. These results support the hypothesis of a dual pathway of target cell activation by PTH.

Parathyroid hormone inhibits collagen synthesis at both ribonucleic acid and protein levels in rat osteogenic sarcoma cells.

Cells of the clonal rat osteogenic sarcoma cell line, UMR 106-01, were used to investigate the regulation of collagen synthesis by PTH in osteoblastic cells. Monolayer cultures of cells were labeled with [3H] proline in order to determine both collagen type and rates of production. Analysis of labeled extracellular polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that UMR 106-01 cells synthesized predominantly type I collagen, accounting for 45.48 +/- 2.09% of the radioactivity incorporated into total protein. After 24-h treatment with bovine PTH (1-34, 10(-8) M), collagen synthesis (i.e. collagenase-digestible protein) was decreased to 29.45 +/- 1.39% of total protein production. This decrease was first observed 12 h after addition of hormone and greatest inhibition was achieved at 24 h. The effect of PTH was dose dependent, with half-maximal inhibition of collagen synthesis occurring at 5 x 10(-10) M after 24-h treatment. In contrast, when steady state levels of mRNA for type I collagen chains were examined by Northern blot analysis, the concentration of PTH that reduced collagen synthesis by 35-45% (10(-8) M), caused a net decrease of approximately 80-96% in the number of procollagen transcripts; a small reduction in beta-actin mRNA levels was also observed. The effect of the hormone on procollagen message level was dose dependent, with significant inhibition observed at 10(-10) M PTH and, as with collagen synthesis, maximal after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal regulation of the production of collagenase and a collagenase inhibitor activity by rat osteogenic sarcoma cells.

Collagenases that specifically cleave native collagen at neutral pH have been implicated in the maintenance and turnover of connective tissue. In bone, the origin of neutral collagenase has remained equivocal, although recent studies have indicated that it is synthesized by the osteoblast. In the present work, regulation of secretion of neutral collagenase and a collagenase inhibitory activity was investigated using the osteoblastic tumor cell line UMR 106-01 and a variety of bone-resorbing agents. Under basal conditions, UMR 106-01 cells produced very low levels of collagenase but substantial amounts of the inhibitory activity. Exposure to PTH and, to a lesser extent, 1,25-dihydroxyvitamin D3, prostaglandin E2, retinoic acid, and epidermal growth factor stimulated the release of collagenase, an effect not seen with interleukin-1 or heparin. The stimulation of collagenase by PTH was dose dependent, with a half-maximal response occurring at 10(-8) M. Inclusion of isobutylmethylxanthine decreased the concentration of PTH required to produce half-maximal stimulation to 2 X 10(-10) M, indicating action via cAMP. With respect to the inhibitory activity, PTH and epidermal growth factor were the only agents, among those tested, able to enhance its production. Both hormones caused a 50-100% increase over control levels 72 h after hormone administration. There were notable differences in the time courses of production of collagenase and the inhibitor. After treatment with PTH, the enzyme reached maximal concentrations between 12-48 h, but declined to undetectable levels by 96 h. In contrast, the inhibitory activity was secreted in a linear fashion, with the highest concentrations achieved around 72-96 h. These results suggest a complex pattern of regulation of collagenase and inhibitor secretion by the osteoblastic cell, with the steady accumulation of inhibitor perhaps being responsible for the ultimate curtailment of enzyme activity.

Thrombin chemotactic stimulation of HL-60 cells: studies on thrombin responsiveness as a function of differentiation.

Thrombin, a major procoagulant enzyme and growth factor, is also selectively chemotactic for monocytes and macrophages but not for neutrophils. This effect stands in contrast to other well-known chemotactic agents such as fMet-Leu-Phe, C5a fragments, and LTB4, which stimulate directed cell movement in both cell types, and have important physiological implications. The human leukemic cell line HL-60, which is capable of differentiating either along granulocytic or monocytic lineages, was therefore used to explore the development of this selective monocyte/macrophage chemotactic response to thrombin. Esterolytically inactive DIP-alpha-thrombin, as well as the thrombin-derived chemotactic peptide CB67-129, elicits a dose-dependent chemotactic response in HL-60 cells differentiated to monocytelike cells by treatment with 1,25(OH)2D3 (HL-60/mono), whereas no such response is evident in either undifferentiated HL-60 cells or in cells differentiated into granulocytes by treatment with DMSO (HL-60/gran). Similarly, early events which characterize stimulation of inflammatory cells by chemotactic agents are also evident, but only in monocyte-differentiated cells. In HL-60/mono, thrombin selectively stimulates rapid cytosolic Ca2+ elevation as well as rapid cytoskeletal association of cytosolic actin. Following thrombin stimulation, maximal actin association in these cells occurs within 30 sec (declining to basal levels at the end of 5 min), and maximal Ca2+ elevations are also evident within 15-20 sec, suggesting a temporal relationship between these two events. Thus, the events accompanying stimulation of HL-60/mono by thrombin are characteristic of those seen following stimulation of inflammatory cells by chemotaxins, with a major difference being the selectivity of thrombin as a chemotaxin for cells of macrophage/monocytic lineage. The selective chemotactic responsiveness of HL-60/mono to thrombin appears to relate to the development of specific receptors on these cells as part of monocytic differentiation: HL-60/mono (but HL-60/gran nor undifferentiated HL-60) are capable of significant specific 125-I-labeled alpha-thrombin-binding (ka approximately 20 nM), and possess an estimated 400,000 thrombin-binding sites per cell. Our findings further suggest that the thrombin response of HL-60 and particularly the expression of thrombin receptors on these cells may serve as a useful model system for exploring the biology of monocyte/macrophage differentiation.

Isolated osteoclasts resorb the organic and inorganic components of bone.

Osteoclasts are the principal resorptive cells of bone, yet their capacity to degrade collagen, the major organic component of bone matrix, remains unexplored. Accordingly, we have studied the bone resorptive activity of highly enriched populations of isolated chicken osteoclasts, using as substrate devitalized rat bone which had been labeled in vivo with L-[5-3H]proline or 45Ca, and bone-like matrix produced and mineralized in vitro by osteoblast-like rat osteosarcoma cells. When co-cultured with a radiolabeled substrate, osteoclast-mediated mineral mobilization reached a maximal rate within 2 h, whereas organic matrix degradation appeared more slowly, reaching maximal rate by 12-24 h. Thereafter, the rates of organic and inorganic matrix resorption were essentially linear and parallel for at least 6 d when excess substrate was available. Osteoclast-mediated degradation of bone collagen was confirmed by amino acid analysis. 39% of the solubilized tritium was recovered as trans-4-hydroxyproline, 47% as proline. 10,000 osteoclasts solubilized 70% of the total radioactivity and 65% of the [3H]-trans-4-hydroxyproline from 100 micrograms of 25-50 micron bone fragments within 5 d. Virtually all released tritium-labeled protein was of low molecular weight, 99% with Mr less than or equal to 10,000, and 65% with Mr less than or equal to 1,000. Moreover, when the 14% of resorbed [3H]proline-labeled peptides with Mr greater than or equal to 2,000 were examined for the presence of TCA and TCB, the characteristic initial products of mammalian collagenase activity, none was detected by SDS PAGE. In addition, osteoclast-conditioned medium had no collagenolytic activity, and exogenous TCA and TCB fragments were not degraded by osteoclasts. On the other hand, osteoclast lysates have collagenolytic enzyme activity in acidic but not in neutral buffer, with maximum activity at pH 4.0. These data indicate that osteoclasts have the capacity to resorb the organic phase of bone by a process localized to the osteoclast and its attachment site. This process appears to be independent of secretion of neutral collagenase and probably reflects acid protease activity.