Recruitment of osteoclast precursors by purified bone matrix constituents.

The osteoclast, the multinucleated giant cell of bone, is derived from circulating blood cells, most likely monocytes. Evidence has accrued that is consistent with the hypothesis that the recruitment of monocytes for osteoclast development occurs by chemotaxis. In the present study, we have examined the chemotactic response of human peripheral blood monocytes and related polymorphonuclear leucocytes to three constituents of bone matrix: peptides from Type I collagen, alpha 2-HS glycoprotein, and osteocalcin (bone gla protein). The latter two substances are among the major noncollagenous proteins of bone and are uniquely associated with calcified connective tissue. In chemotaxis assays using modified Boyden chambers, Type I collagen peptides, alpha 2HS glycoprotein, and osteocalcin evoke a dose-dependent chemotactic response in human monocytes. No chemotaxis is observed on PMNs despite their ontogenetic relationship to monocytes and their documented sensitivity to a broad range of other chemical substances. Our observations are consistent with the view that osteoclast precursors (monocytes) are mobilized by chemotaxis, and suggest that the chemoattractants responsible for this activity are derived from the bone matrix or, in the case of collagen and osteocalcin; directly from the osteoblasts which produce them.

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.

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.

Contact-mediated bone resorption by human monocytes in vitro.

Human circulating monocytes in tissue culture are capable of resorbing devitalized adult and fetal bone. An important component of this process is the adhesion of the cells to the mineralized substrate and the localized removal of matrix from beneath the attached cells. The process appears to involve both release of lysosomal enzymes onto the substrate and intracellular accumulation (transport) of resorbed matrix.

Defective binding of macrophages to bone in rodent osteomalacia and vitamin D deficiency. In vitro evidence for a cellular defect and altered saccharides in the bone matrix.

In the osteomalacic as well as normal skeleton, few osteoclasts are associated with osteoid-covered bone surfaces. The reason for this particular cellular deficit is not clear, but may relate to the inability of osteoclasts and/or osteoclast precursors (monocyte-macrophages) to attach to immature, unmineralized bone matrix, a step apparently essential for normal resorptive activity and osteoclast differentiation. In this study, we have examined cell-bone binding using macrophages (M phi) and bone isolated from vitamin D-deficient rats and hypophosphatemic, osteomalacic mice and from their normal counterparts. The data show that M phi-bone attachment is greatly reduced (P less than 0.001) in both vitamin D deficiency and hypophosphatemia, but that the mechanisms responsible for this reduction are apparently different in the two disorders. In hypophosphatemia, the reduction in binding appears solely attributable to the absence or inaccessibility of bone matrix oligosaccharides or glycoproteins essential to the attachment process. In vitamin D deficiency, on the other hand, not only is the bone matrix defective as a binding substrate, but the M phi, per se, is limited in its capacity to attach to normal, vitamin D-deficient, and hypophosphatemic bone.

Saccharides mediate the attachment of rat macrophages to bone in vitro.

Macrophages (M phi) are multipotential cells capable of giving rise to osteoclasts and of resorbing bone. Since both of these processes are ultimately dependent upon the attachment of cells to a mineralized bone surface, we have examined in this study the mechanism by which such attachment is achieved. The data show that elicited rat peritoneal M phi bind to bone in a temperature-dependent and -saturable manner with half-maximal attachment occurring within 10 min at 37 degrees C and reaching a plateau by approximately 60 min. The kinetics of binding are essentially the same whether devitalized bone particles or viable calvaria are used as a substrate. The attachment of M phi to bone is inhibited by some sugars (e.g., N-acetyl-galactosamine, thiogalactoside, beta-lactose), fetuin and asialofetuin, and by pretreating the bone with periodate. Binding is also significantly reduced when M phi are preincubated with tunicamycin and swainsonine at nontoxic concentrations sufficient to inhibit or alter glycosylation. On the other hand, exposing the cells to neuraminidase increases the capacity of M phi to bind to bone. Collectively, our observations indicate that the attachment of M phi to bone is a highly regulated process and is mediated, at least in part, by saccharides located on both the cell and the bone surface.

Differential action of the bisphosphonates (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD) and disodium dichloromethylidene bisphosphonate (Cl2MDP) on rat macrophage-mediated bone resorption in vitro.

The bisphosphonates (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD) and disodium dichloromethylidene bisphosphonate (Cl(2)MDP) effectively inhibit the accelerated bone resorption associated with some skeletal disorders, e.g., Paget's disease. However, it has not been established whether these compounds exert their inhibitory effect by rendering the bone mineral more resistant to degradation, by diminishing the activity of resorbing cells, or through some combination of both activities. In this study, we have tested these possibilities using an in vitro resorption assay system consisting of elicited rat peritoneal macrophages co-cultured with particles of (45)Ca-labeled, devitalized rat bone. This assay system permits the quantitative assessment of the action of APD and Cl(2)MDP on the two major phases of bone resorption (cell-substrate attachment and osteolysis) under circumstances where the drugs are present continuously or, most importantly for the issues in question, after the separate pretreatment of the particles or the resorbing cells. Our data indicate that (a) Both APD and Cl(2)MDP at concentrations >/=5 x 10(-6) M diminish macrophage-mediated (45)Ca release (i.e., bone resorption) in a log dose-dependent fashion. (b) A 10-min pretreatment of bone particles with either bisphosphonate (P-C-P) similarly inhibits resorptive activity, but is most pronounced with Cl(2)MDP. However, only APD is effective in reducing resorption when cells are preincubated (for 24 h) with P-C-P. (c) In cultures containing both labeled and unlabeled bone, significant inhibition occurs only when the labeled particles are coated with P-C-P (indicating that the action of P-C-P-treated bone is highly localized). (d) P-C-P does not diminish cell-bone particle attachment, an essential step in the resorptive process. On the other hand, delaying the addition of P-C-P until after cell-bone attachment is completed significantly reduces the resorption-inhibiting effect of these compounds. (e) Cl(2)MDP reduces culture DNA content in proportion to its inhibitory effect on resorption, and both the inhibitory and cytotoxic actions of this P-C-P are dependent upon the presence of bone. On the other hand, APD is cytotoxic only at very high concentrations (10(-4) M), acts independently of the presence of bone, and inhibits resorption without killing cells. We conclude that the mechanisms of action of APD and Cl(2)MDP are markedly different. Cl(2)MDP is a potent cytotoxin in the presence of bone and apparently exerts its inhibitory effect in this manner. APD is noncytotoxic at levels adequate to suppress resorption and, therefore, must inhibit macrophage activity by some other mechanism. Neither P-C-P appears to limit resorption by decreasing the solubility of mineralized bone matrix.

Interaction of diphenylhydantoin (phenytoin) and phenobarbital with hormonal mediation of fetal rat bone resorption in vitro.

Chronic administration of high doses of anticonvulsant drugs frequently produces classic osteomalacia with bone histologic changes characteristic of increased parathyroid hormone (PTH) effect in man. However, several reports have documented defects in calcified tissue metabolism suggestive of an end-organ resistance to PTH after chronic anticonvulsant drug therapy. To examine the direct action of anticonvulsant drugs on bone resorption, we investigated the effects of diphenylhydantoin (phenytoin) (DPH) (100-200 mug/ml) and phenobarbital (10-400 mug/ml) on basal and hormonally mediated resorption 5-day cultures of fetal rat forelimb rudiments. In this system both drugs significantly inhibited basal and PTH-stimulated (45)Ca and [(3)H]hydroxyproline release, as well as 1,25-dihydroxyvitamin D(3)-stimulated (45)Ca release. The effects of DPH and phenobarbital were additive, with DPH exhibiting a several-fold more potent inhibitory effect than phenobarbital. Whereas DPH exhibited a striking synergism with the inhibitory effects of human calcitonin (HCT) on PTH-induced resorption, the effect of phenobarbital was merely additive to that of HCT. PTH and PTH plus HCT-induced increases in bone cyclic AMP (cAMP) content were significantly inhibited by DPH but not by phenobarbital. However, in contrast to effects on (45)Ca release, DPH inhibition of cAMP generation was not accentuated in the presence of HCT. It is concluded that: (a) both DPH and phenobarbital can directly inhibit basal and hormonally stimulated bone resorption, with DPH being much more potent in this regard; (b) DPH appears to inhibit bone resorption via a cAMP-independent mechanism and has an additional suppressive effect on PTH-induced cAMP generation; and (c) the synergistic interaction of DPH and HCT in inhibiting (45)Ca release occurs at a site independent of cAMP generation.

Glucocorticoids modulate macrophage surface oligosaccharides and their bone binding activity.

The circumstantial evidence that indicates that glucocorticoids (GC) may stimulate osteoclastic resorption in vivo has recently found support in observations that demonstrate that these compounds effectively increase the activity of isolated resorptive cells (osteoclasts, macrophage polykaryons, and elicited macrophages [MO] ) in vitro. Data are presented here that indicate that this stimulation by GC is due to an enhancement of the initial stage of the resorption process, the attachment of cells to bone, and that this is caused by alterations of cell surface oligosaccharides. Specifically, dexamethasone and cortisol enhance by 80% the attachment of MO to bone surfaces in a dose dependent manner but do not alter or reduce the binding of these cells to other surfaces (plastic, collagen, and hydroxyapatite crystals). The effect of GC on cell-bone attachment is blocked by the glycosylation inhibitor, tunicamycin, and the glycosylation modifier, swainsonine; this demonstrates that asparagine-linked oligosaccharides are involved in the stimulatory process. Flow cytometric analysis of GC-treated cells using a panel of fluoresceinated lectins confirms this by indicating a selective, enhanced exposure of plasma membrane-associated N-acetylglucosamine and N-acetylgalactosamine residues, sugars we have previously shown to be pivotal in MO-bone binding. Finally, progesterone, a known GC antagonist, blocks GC-stimulated resorption, macrophage-bone binding, and membrane oligosaccharide modification, presumably by competing for the GC receptor. Progesterone alone alters none of these processes. Thus, GC stimulates the resorptive activity of macrophages by enhancing the initial events in the degradative process (cell-bone binding) and does so, apparently, via receptor-mediator alteration of cell surface glycoproteins.

Extracellular protons acidify osteoclasts, reduce cytosolic calcium, and promote expression of cell-matrix attachment structures.

Because metabolic acids stimulate bone resorption in vitro and in vivo, we focused on the cellular events produced by acidosis that might be associated with stimulation of bone remodeling. To this end, we exposed isolated chicken osteoclasts to a metabolic (butyric) acid and observed a fall in both intracellular pH and cytosolic calcium [( Ca2+]i). These phenomena were recapitulated when bone resorptive cells, alkalinized by HCO3 loading, were transferred to a bicarbonate-free environment. The acid-induced decline in osteoclast [Ca2+]i was blocked by either NaCN or Na3VO4, in a Na+-independent fashion, despite the failure of each inhibitor to alter stimulated intracellular acidification. Moreover, K+-induced membrane depolarization also reduced cytosolic calcium in a manner additive to the effect of protons. These findings suggest that osteoclasts adherent to bone lack functional voltage-operated Ca2+ channels, and they reduced [Ca2+]i in response to protons via a membrane residing Ca-ATPase. Most importantly, acidosis enhances formation of podosomes, the contact areas of the osteoclast clear zone, indicating increased adhesion to substrate, an early step in bone resorption. Thus, extracellular acidification of osteoclasts leads to decrements in intracellular pH and calcium, and appears to promote cell-matrix attachment.

Cytoplasmic pH regulation and chloride/bicarbonate exchange in avian osteoclasts.

Osteoclasts resorb bone by first attaching to the bone surface and then secreting protons into an isolated extracellular compartment formed at the cell-bone attachment site. This secretion of protons (local acidification) is required to solubilize bone hydroxyapatite crystals and for activity of bone collagen-degrading acid proteases. However, the large quantity of protons required, 2 mol/mol of calcium, would result in an equal accumulation of cytosolic base equivalents. This alkaline load must be corrected to maintain cytosolic pH within physiologic limits. In this study, we have measured cytoplasmic pH with pH-sensitive fluorescent compounds, while varying the extracellular ionic composition of the medium, to determine the nature of the compensatory mechanism used by osteoclasts during bone resorption. Our data show that osteoclasts possess a chloride/bicarbonate exchanger that enables them to maintain normal intracellular pH in the face of a significant proton efflux. This conclusion follows from the demonstration of a dramatic cytoplasmic acidification when osteoclasts that have been incubated in bicarbonate-containing medium are transferred into bicarbonate-free medium. This acidification is absolutely dependent on and proportional to medium [Cl-]. Furthermore, acidification is inhibited by the classic inhibitor of red cell anion exchange, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, and by diphenylamine-2-carboxylate, an inhibitor of chloride specific channels. However, the acidification process is neither energy nor sodium dependent. The physiologic importance of chloride/bicarbonate exchange is demonstrated by the chloride dependence of recovery from an endogenous or exogenous alkaline load in osteoclasts. We conclude that chloride/bicarbonate exchange is in large part responsible for cytoplasmic pH homeostasis of active osteoclasts, showing that these cells are similar to renal tubular epithelial cells in their regulation of intracellular pH.

Modulation of expression and cell surface binding of members of the transforming growth factor-beta superfamily during retinoic acid-induced osteoblastic differentiation of multipotential mesenchymal cells.

We have evaluated the effects of retinoic acid as a differentiating agent on two pluripotential mesenchymal stem cell lines, the mouse cell line C3H-10T1/2 (10T1/2), which has the capacity to differentiate in vitro into myoblasts, adipocytes, chondrocytes, and osteoblasts, and the rat cell line ROB-C26 (C26), which can, in culture, give rise to adipocytes, myoblasts, and osteoblasts. Retinoic acid (10(-6) M) reduces the incidence of myoblast and adipocyte formation and induces or increases alkaline phosphatase expression and responsiveness to PTH, two indicators of the osteoblastic phenotype. Because transforming growth factor-beta (TGF beta) superfamily members, including the different TGF beta isoforms and the bone morphogenetic proteins (BMPs), are thought to play a role in regulating bone and cartilage formation, and because exogenous TGF beta and BMP-2 have already been found to modulate osteoblastic differentiation of C26 and 10T1/2 cells, we evaluated the endogenous expression of these factors in both cell lines cultured in the presence or absence of retinoic acid. Our data show that C26 and 10T1/2 cells constitutively express a broad spectrum of TGF beta superfamily members. However, this pattern of expression is dramatically altered in response to retinoic acid. Specifically, expression of TGF beta 1 and especially TGF beta 2 is strongly increased, whereas TGF beta 3 expression is down-regulated. These changes are accompanied by a striking decline in TGF beta receptor expression levels at the cell surface. Furthermore, BMP-2 and -4 expression are decreased after treatment with retinoic acid, whereas vgr-1/BMP-6 expression is induced in C26 cells, but decreased in 10T1/2 cells. These results clearly show a dynamic changing pattern of TGF beta superfamily expression consequent to the induction of osteogenic differentiation and provide the first indication that TGF beta receptor down-regulation may be an essential part of this differentiation process. These data also establish the C26 and 10T1/2 cell lines as convenient in vitro model systems for exploring the autoregulation of osteogenic differentiation by members of the TGF beta superfamily.

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)

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)

Extracellular matrix synthesized by clonal osteogenic cells is osteoinductive in vivo and in vitro: role of transforming growth factor-beta 1 in osteoblast cell-matrix interaction.

ROB-C26 (C26) is a multipotential, clonal cell line known to express several members of the TGF-beta superfamily and to become more osteoblastic (e.g., express higher levels of alkaline phosphatase) upon treatment with 10(-6)M retinoic acid (RA). We hypothesize that the expression of this more osteoblastic phenotype subsequent to RA exposure is the result of the treated cell's extracellular matrix (ECM) becoming a repository and active source of putative osteoinductive growth factors including, specifically, select members of the TGF-beta superfamily. To test this hypothesis, we isolated the ECM from RA-treated and untreated C26 cells and assessed them for their ability to promote osteogenic differentiation in vivo and in vitro. We then explored whether the latter activities could be attributed specifically to TGF-beta 1. We found that the ECM of treated cells isolated by cell lysis and extensive washing induced endochondral bone formation in vivo when implanted into the thigh muscles of athymic nude mice and stimulated alkaline phosphatase (ALP) activity in vitro in freshly plated C26 cells. This latter stimulation was comparable to levels observed with direct RA treatment. This latter in vitro activity was only very partially mimicked by the ECM prepared from untreated cells and not duplicated at all by RA-treated collagen or the ECM from another RA-treated multipotential cell line. Moreover, the in vivo osteoinductive effect of the treated C26 cell ECM was not duplicated by comparable ECM prepared from untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of type I collagen in the regulation of the osteoblast phenotype.

Evidence from a variety of sources indicates that the extracellular matrix forms an important part of a feedback loop governing the migration, proliferation, and differentiation of the cells that produce it. In keeping with this, we showed previously that the extracellular matrix of a multipotential mesenchymal clonal cell line (ROB-C26) induced to differentiate into a more osteoblastic cell type by the addition of exogenous retinoic acid produces an extracellular matrix capable of osteoinductive activity in vivo and of stimulating alkaline phosphatase activity in vitro. Since type I collagen is the major structural component of this extracellular matrix, we sought to determine whether and to what extent this protein is responsible for the previously observed inductive/stimulatory activity. To this end, C26 cells are cultured on plastic, in the presence of retinoic acid, on a type I collagen film, or on an extracellular matrix from retinoic acid-treated C26 cells, and cell differentiation is assessed by measuring changes in the abundance of a number of osteoblast-related mRNAs. These determinations are made by RNAse protection assay after 3 or 6 days of incubation and include measurements of the RNAs for type I collagen, alkaline phosphatase, osteopontin, transforming growth factor alpha 1 and beta 2, and Vgr-1/BMP-6. In addition, C26 cells are incubated in the presence of retinoic acid and several established inhibitors of the synthesis or assembly of extracellular matrix components and the effects on induced alkaline phosphatase activity determined. Our data show that while the collagen substrate mimics some of the effects of retinoic acid and the extracellular matrix, it cannot reproduce all of them. Specifically, while the latter two culture conditions increase the abundance of all six mRNAs, type I collagen film increases the levels of only three of the six (collagen I, alkaline phosphatase, and osteopontin). Moreover, while type I collagen film produces an increase in alkaline phosphatase message, it falls to produce a similar change in alkaline phosphatase activity, an effect seen with both retinoic acid and extracellular matrix. However, interruption of collagen I synthesis by cis-4-hydroxy-L-proline blocks the increase in alkaline phosphatase activity associated with retinoic acid treatment. Thus, it appears likely that type I collagen is a necessary but, by itself, insufficient factor to elicit the comprehensive expression of the osteoblastic phenotype in immature mesenchymal cells.

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)

Age-related changes in osteogenic stem cells in mice.

Osteoblasts arise from partially differentiated osteogenic progenitor cells (OPCs) which in turn arise from undifferentiated marrow stromal mesenchymal stem cells (MSCs). It has been postulated that age-related defects in osteoblast number and function may be due to quantitative and qualitative stem cell defects. To examine this possibility, we compared osteogenic stem cell number and in vitro function in marrow cells from 4-month-old and 24-month-old male BALB/c mice. Histologic studies demonstrated that these mice undergo age-related bone loss resembling that seen in humans. In primary MSC cultures grown in media supplemented with 10 nM dexamethasone, cultures from older animals yielded an average of 41% fewer OPC colonies per given number of marrow cells plated (p < 0.001). This implies that for a given number of marrow cells there are fewer stem cells with osteogenic potential in older animals than there are in younger animals. The basal proliferative rate in cultures from older animals, as measured by 3H-thymidine uptake, was more than three times that observed in cultures from young animals (p < 0.005). However, the increase in proliferative response to serum stimulation was 10-fold in the younger cultures (p <0.001) and insignificant (p <0.4) in the older cultures. Colonies in both age groups became alkaline phosphatase positive at the same rate, and virtually all colonies were positive after 12 days of culture. Cultures from both age groups produced abundant type I collagen. These studies suggest that defects in the number and proliferative potential of MSCs may underlie age-related defects in osteoblast number and function.

Expression of helix-loop-helix regulatory genes during differentiation of mouse osteoblastic cells.

Although much is known about the hormonal regulation of osteoblastic cell differentiation, much less is known about the nuclear regulatory molecules that affect this process. We analyzed the expression of several regulatory molecules of the helix-loop-helix (H-L-H) group in primary mouse calvarial cells and in MC3T3-E1 mouse osteoblastic cells in situations representing different degrees of cellular differentiation. H-L-H class regulators are known to participate directly in directing cell fate and differentiation decisions in other mesodermal lineages. Two of the molecules that we studied, Id and E12, have well-established roles in this process. The other, mTwi, the murine homolog of the Drosophila twist gene, is a newly cloned mammalian H-L-H gene. Levels of E12 RNA remained unchanged during differentiation. On the other hand, in both primary osteoblastic cells and MC3T3-E1 cells, the abundance of Id and mTwi declined with cell maturation; mTwi less dramatically than Id. That Id expression is causally related to differentiation is suggested by the finding that MC3T3-E1 cells transfected with an Id-expression plasmid fail to undergo differentiation. We conclude that helix-loop-helix regulatory genes are expressed in mouse osteoblastic cells, where they are likely to participate in differentiation. The E12 gene product is likely to function as a positive modulating factor. In contrast, Id inhibits differentiation, probably by sequestering other H-L-H gene regulators, including E12, in inactive complexes. The precise role of mTwi is more speculative at this time, but the observed pattern of expression is consistent with a role in early and midmesodermal specification that is terminated as cells differentiate.