Onset and dynamics of osteosclerosis in mice induced by Reilly-Finkel-Biskis (RFB) murine leukemia virus. Increase in bone mass precedes lymphomagenesis.

Newborn NMRI strain mice were infected with Reilly-Finkel-Biskis (RFB) murine leukemia virus (MuLV), a murine leukemia virus that has been shown to induce lymphomas, osteosclerosis, and osteomas in susceptible strains of mice. Bone histomorphometry of the distal femoral metaphyses at 3-month intervals showed osteosclerosis 3 (100%), 6 (100%), and 9 (93%) months after infection. This was represented by significantly augmented cancellous bone mass and accompanied by distinct changes in bone architecture. High numbers of provirus copies were detected at 2-4 weeks in femora, humeri, and calvaria, and viral protein was highly expressed in trabecular and cortical bone cells, particularly in osteocytes. Infected mice showed enhanced bone formation and smaller numbers of osteoclasts relative to sex- and age-matched controls. Osteoclastic differentiation was significantly reduced in cocultures of spleen or bone marrow cells with RFB MuLV-infected osteoclastogenic, osteoblast-like cells. However, RFB MuLV did not impair the activity of mature osteoclasts. In infected mice lymphomas were only observed at 6 (22%) and 9 months (40%) of age. At 3 months, IgG gene and TCR-beta gene rearrangements were not detectable, and new proviruses showed a heterogeneous integration pattern, indicating the absence of lymphoma in early osteosclerotic mice. In contrast, lymphomas, which developed in 8- to 9-month-old infected mice, showed IgG rearrangements indicating development of B-cell lymphomas, together with mono- or oligoclonal expansion of distinct patterns of proviral integrations. These results indicate that RFB MuLV-induced osteosclerosis develops within 3 months after infection and precedes lymphomagenesis. It may therefore be considered an independent skeletal lesion in MuLV-infected mice.

Bisphosphonates induce osteoblasts to secrete an inhibitor of osteoclast-mediated resorption.

Current knowledge indicates that osteoblasts play an integral role in osteoclastic bone resorption through an osteoclast-stimulating activity produced by osteoblasts in response to resorption-promoting osteotropic factors. Previously, we have shown that the inhibitory action of the bisphosphonates on bone resorption in part is mediated by osteoblasts. The aim of the present study was to investigate whether the bisphosphonate-generated inhibition is due to these compounds decreasing the synthesis of the osteoclast-stimulating activity or is the result of osteoblasts synthesizing an osteoclast resorption inhibitor. Using the osteoblastic cell line CRP 10/30, which produces osteoclast- stimulating activity, constitutively and employing isolated rat osteoclasts cultured on ivory, evidence was obtained indicating that the bisphosphonates ibandronate and alendronate at a concentration of 10(-7) M induce osteoblasts to synthesize an osteoclast inhibitor that reduces pit formation by more than 50%. The inhibitor is heat and proteinase labile and has a molecular mass between 1-10 kDa. The reduction of resorption pits is paralleled by a decrease in tartrate-resistant acid phosphatase-positive mono- and multinucleated cells, whereas the mean area resorbed per pit was not changed, suggesting that the inhibitor affects osteoclast formation and/or survival and probably not the osteoclast resorption activity. Rat preosteoblastic cells and rat dermal fibroblasts were found not to produce the inhibitor. In conclusion, osteoblasts aside from their role of mediating osteoclastic resorption promoters are also involved in inhibiting bone resorption through the synthesis of an osteoclast resorption inhibitor.

Evidence for the synthesis of parathyroid hormone-related protein (PTHrP) by nontransformed clonal rat osteoblastic cells in vitro.

Parathyroid hormone-related protein (PTHrP) is synthesized by a variety of tumors and is thought to be the main cause of the clinical syndrome of humoral hypercalcemia of malignancy (HHM). In addition to its parathyroid hormone (PTH)-like actions, novel actions of PTHrP on placental calcium transport and inhibition of in vitro osteoclast activity have been demonstrated. The fact that osteoblasts act as mediators of osteoclastic bone resorption prompted us to investigate whether nontranformed, osteoblastlike cells produce PTHrP. PTHrP has been detected in developing human fetal bones and in rat long bones in culture. For this study, osteogenic cells, CRP 5/4 and CRP 10/30, were employed. Both cell types represent clonal bone cell populations established from 1-day-old rats. While CRP 10/30 cells express the osteoblastic phenotype, CRP 5/4 cells resemble cells with preosteoblastic properties. With a radioimmunoassay (RIA), utilizing antiserum directed against the amino-terminal PTHrP(1-40), it was found that both cell types synthesize PTHrP constitutively. CRP 10/30 cells produce about twice as much as CRP 5/4 cells. Transforming growth factor-beta (TGF-beta 1) was shown to increase the synthesis of PTHrP in CRP 5/4 cells by about 2.5-fold, while in CRP 10/30 cells it caused an approximate 50% reduction of PTHrP. Employing the reverse transcriptase polymerase chain reaction (RT-PCR) technique it was found that both bone cell types express mRNA for PTHrP and that the modulation of the PTHrP mRNA levels by TGF-beta 1 in CRP 5/4, and to a lesser degree in CRP 10/30 cells, was reflected in a change in the level of PTHrP protein in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of collagen type I and biglycan mRNA levels by hormones and growth factors in normal and immortalized osteoblastic cell lines.

Growth factors, such as transforming growth factor beta (TGF-beta) and insulin-like growth factors (IGF) I and II, have been shown to exert anabolic effects on bone cells in vitro. Hormones, such as PTH and probably insulin and growth hormone, were recently shown to stimulate bone formation in vivo as well. The aim of the present study was to assess by northern blots, which were quantitated by densitometry, the effects of these anabolic growth factors and hormones in two osteogenic cell populations: CRP 10/30 cells, a clonal cell population derived from primary rat calvarial cells, and IRC 10/30-myc cells, which were established from CRP 10/30 by immortalization. Transcripts for alpha 1(I) collagen, biglycan, osteonectin, osteopontin, and osteocalcin were detected in both cell populations, which is consistent with the phenotype expressed by mature osteoblasts. There were no difference in the basal expression of bone matrix mRNAs between the two cell populations. PTH increased alpha 1(I) collagen mRNA levels in both osteoblastic cells but had no effect on the biglycan transcripts. Neither insulin nor growth hormone affected mRNA levels of either matrix protein after 24 h exposure. All three growth factors, TGF-beta, IGF-I, and IGF-II, increased alpha 1(I) collagen transcripts in a time- and dose-dependent manner in both cell populations. Biglycan mRNA levels were enhanced in both osteoblastic lines only by IGF-I and IGF-II, but not TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Bisphosphonates act on rat bone resorption through the mediation of osteoblasts.

Bisphosphonates are generally considered to act on bone resorption by binding to bone mineral and subsequently inhibiting the activity of the osteoclasts which ingest them. This has been supported by the fact that bisphosphonates adsorbed on mineralized tissue inhibit the resorbing activity of isolated osteoclasts in vitro. However, the effectiveness of different bisphosphonates determined in this system does not reflect their relative potencies in vivo. Employing the well-described isolated osteoclast resorption pit assay, with ivory as the resorption substrate, we show here that this lack of correlation prevails only when the bisphosphonates are added to the mineral before addition of osteoclasts, but not when the cells are treated for a short time (5 min) before allowing them to adhere onto ivory. By using this approach with five different bisphosphonates, a stringent correlation of relative potencies was obtained with those found, both in the rat and in the human, in vivo. Furthermore, by using an osteoblastic cell line (CRP 10/30) which is a powerful promoter of osteoclastic resorption in vitro, we obtained evidence that the inhibitory effect of bisphosphonates was the result of an action on osteoblasts rather than on osteoclasts. Thus, in experiments in which the osteoblastic cells were pretreated for 5 min with bisphosphonates and then cocultured with osteoclasts, inhibition of osteoclastic resorbing activity was obtained. Moreover, it was found that this treatment resulted in a decrease of the stimulatory effect found in CRP 10/30-conditioned medium. In conclusion the present study shows that part of the osteoclast inhibiting action of the bisphosphonates is mediated through an action on osteoblasts.

Constitutive expression of osteoclast-stimulating activity by normal clonal osteoblast-like cells: effects of parathyroid hormone and 1,25-dihydroxyvitamin D3.

Osteoblasts are known to produce osteoclast-stimulating activity (OSA). The aim of the current study was to relate the expression of OSA to the osteoblastic phenotype and examine its regulation by calciotropic hormones. The study was performed with the normal osteoblastic cell clone CRP 10/30 and the preosteoblastic clone CRP 4/7. OSA was determined with the well described isolated osteoclast pit assay, using sperm whale dentine as substrate. In contrast to previous studies, the assay was carried out at pH 7.36, rather than at pH 6.4 or 6.9. The results indicate that over 24 h, CRP 10/30 cells produce constitutively OSA, which compared to controls corresponds to an about 7-fold increase in resorption pits. There was considerably less activity expressed by either CRP 4/7 cells or fibroblasts. OSA proved to be heat labile, and its mol wt was estimated to be over 10 kilodaltons. While PTH-(1-34) did not influence the synthesis of OSA, the number of pits formed by osteoclasts incubated with medium conditioned by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-treated CRP 10/30 cells was increased 3-fold above baseline values. A similar increase was obtained with 1.25-(OH)2D3 added directly to CRP 10/30-conditioned medium. These results could not be duplicated with 1,25-(OH)2D3 added to either control medium or medium conditioned by CRP 4/7 cells or fibroblasts. The present study shows that normal clonal bone cells synthesize constitutively OSA, which is not regulated by PTH or 1,25-(OH)2D3. Furthermore, the results suggest that the synthesis of bone cell-derived OSA is limited to cells expressing the mature osteoblastic phenotype. Finally, CRP 10/30-conditioned medium appears to permit 1,25-(OH)2D3 to function on osteoclasts.

The procalcitonin amino-terminal cleavage peptide (N-proCT) lacks biological activity on normal clonal rat osteoblastic and preosteoblastic cells in vitro.

The human-derived procalcitonin amino-terminal cleavage peptide, N-proCT has previously been shown to act mitogenically on isolated chicken and human osteoblast-like cells and on the human osteosarcoma cell line U-2 OS. We have examined the effect of N-proCT on growth and phenotype of cloned rat osteoblastic and preosteoblastic cells. Neither cell growth nor the phenotype (ALP, PTH response) of either cell type was significantly changed by the procalcitonin cleavage peptide.

Establishment and characterization of two immortalized cell lines of the osteoblastic lineage.

Osteoblastic cells were cloned by culturing rat calvariae cells in agarose in the presence of TGF-beta and EGF. Two bone cell lines were established by immortalizing such an osteoblastic clonal cell population by the introduction of the avian v-mycOK10 gene in the form of a mouse ecotropic retrovirus. Although originating from the same clonal cell population, the two lines exhibited somewhat differing properties. IRC10/30-myc1 expressed alkaline phosphatase (AP), showed PTH- and PGE2-induced cAMP production, synthesized mainly collagen type I and a minor fraction of type III, and produced mRNA for the bone-specific protein osteocalcin. IRC10/30-myc3 did not express AP, showed no PTH responsiveness, and synthesized only about one-third as much collagen as IRC10/30-myc1 (4 versus 12% of total protein synthesis). However, the cell line IRC10/30-myc3 was induced to synthesize cAMP by PGE2 and produced osteocalcin mRNA. When cultured in vivo in diffusion chambers, both lines proved to be osteogenic. Besides bone, both lines also formed cartilage and fibrous tissue. Thus, by immortalizing a clonal cell population of the osteoblastic phenotype, cell lines expressing varying properties can emerge. Furthermore, the expression of alkaline phosphatase and PTH-inducible adenylate cyclase are not prerequisites for a cell to form bone in vivo. Finally, cells expressing the phenotype of differentiated osteoblasts, including osteocalcin synthesis, still have a multipotential differentiation capacity and form bone and cartilage in vivo.

Production of granulocyte-macrophage (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) by rat clonal osteoblastic cell population CRP 10/30 and the immortalized cell line IRC10/30-myc1 stimulated by tumor necrosis factor alpha.

Previously, we have shown that primary cultures of murine calvarial cells produce both granulocyte macrophage (GM) and granulocyte (G) colony stimulating factor (CSF). Because of the heterogeneity of cell types in these cultures the osseous origin of these cytokines was not certain. Thus a non-transformed rat clonal osteoblastic cell population CRP 10/30 and the immortalized cell line IRC10/30-myc1 derived from it, which both express the osteoblastic phenotype, were now investigated. Both produced hemopoietic growth activity after treatment with recombinant murine tumor necrosis factor alpha. This activity eluted from diethylaminoethyl Sephacel at 0.2-0.3 M NaCl, and migrated on Sephacryl S-200 at a mol wt of around 30 k, as described for murine GM- and G-CSF. On a Phenyl Sepharose CL-4B column, it was separated into two peaks appearing at position where GM (peak I) and G-CSF (peak II) are expected to be eluted. Antisera against GM-CSF inhibited the activity of peak I. In the colony assay in semisolid medium, peak I induced colonies of the GM-type and peak II of the G-type. These data indicate that cloned osteoblasts produce GM- and G-CSF. Through CSF production, osteoblasts might regulate osteoclast formation, influence hemopoiesis and/or participate in local inflammatory reactions of bone.

Evidence for heterogeneity of the osteoblastic phenotype determined with clonal rat bone cells established from transforming growth factor-beta-induced cell colonies grown anchorage independently in semisolid medium.

Numerous reports have appeared in the literature indicating phenotypic heterogeneity among cells of the osteoblastic lineage. This diversity may be due to either certain stages of differentiation or a subspecialization of already terminally differentiated osteoblasts. To obtain answers to this question, we report on studies undertaken to clone bone cell populations from 1 day postnatal rat calvaria which express well defined differences in phenotype. To achieve this goal, we have used the soft agarose cloning technique which previously has almost exclusively been applied to clone cells of neoplastic origin. The reason for being able to employ this method is based on the fact that bone cells can be induced by transforming growth factor-beta to reversibly acquire the transformed phenotype, an event expressed by anchorage-dependent bone cells to form progressively growing colonies in soft agarose. Individual colonies, harvested from agarose, were expanded to clonal bone cell populations. Characterizing 48 cell clones by detection of osteoblastic cell markers such as alkaline phosphatase activity, PTH- and prostaglandin-E2-induced adenylate cyclase activity, osteocalcin mRNA synthesis, as well as collagen synthesis, 7 subsets of osteoblastic cell types were identified. Each subset was found to express a distinct phenotype, indicated by the absence or presence of osteoblastic cell markers. Some clones, previously found not to exhibit any osteoblastic traits, developed PTH responsiveness when treated with insulin-like growth factor-I/transforming growth factor-beta, suggesting that these clones may originate from the osteoprogenitor cell pool. While most clonal cell populations were characterized as fully functional osteoblastic cells, some clones expressed merely 1, 2, or 3 osteoblastic markers, which suggests that they may represent stages of differentiation along the osteogenic pathway. In addition, other subclones displayed the capacity to synthesize osteocalcin and showed PTH and prostaglandin-E2 responsiveness, but were found to be devoid of alkaline phosphatase activity. Others expressed all osteoblastic cell markers except PTH responsiveness. The phenotypic constellation of the latter suggests that these cell clones may represent mature osteoblast-like cells, which, perhaps due to environmental circumstances present at the time of isolation, have become altered in accordance with the physiological requirements of the tissue.

Effects of transforming growth factor type beta upon bone cell populations grown either in monolayer or semisolid medium.

Bone has been shown to store large amounts of transforming growth factor type beta (TGF beta) and this has recently been found to be synthesized by bone-forming cells. We report on studies undertaken to examine the effects of platelet-derived TGF beta on different bone cell populations, isolated from 1-day postnatal rat calvaria by sequential enzymatic digestion. In addition, we tried to determine which of these cell populations synthesize TGF beta. In this regard, evidence was collected to indicate that cell populations which were shown to be enriched with osteoblast-like cells synthesize TGF beta. Although the production of the factor appeared to be limited to a particular cell type, its action was found to be of a more general character, as all cell populations were found to respond to TGF beta. Contrary to earlier reports, TGF beta was shown to be inhibitory upon cell proliferation. In this context, growth of cells released during early digestions was reduced considerably more than growth of those released during late digestions. Studies on the effect upon protein synthesis revealed that TGF beta specifically inhibited collagen but not the synthesis of noncollagenous proteins. The synthesis of collagen was altered to a greater extent in cells isolated during late digestions than in cells of the early populations. Further information on the TGF beta-mediated effects on bone cell biology was provided by data showing that both alkaline phosphatase and cAMP production in response to PTH was greatly reduced by TGF beta. Finally, experiments performed to determine whether TGF beta induces any of the bone cell populations to acquire the transformed phenotype revealed that only populations previously shown to be enriched with osteoblast-like cells formed colonies in soft agarose.(ABSTRACT TRUNCATED AT 400 WORDS)

Fatty acid oxidation in bone tissue and bone cells in culture. Characterization and hormonal influences.

Fatty acid oxidation and its hormonal modulation were investigated in cultured rat calvaria and in cultivated cell populations. The latter were obtained from calvaria of newborn rats by sequential time-dependent digestion with collagenase, yielding eight cell populations: the early ones containing mainly fibroblasts, the middle ones being osteoblast-like, and late ones osteoblast-osteocyte-like. In calvaria, fatty acid oxidation was increased by adding 0.1 mM- and 1.0 mM-palmitate to the medium, containing 10% (v/v) fetal-calf serum. No effect was found after parathyrin addition in vitro or when injected in vivo. All cell populations obtained by sequential digestion were found to oxidize palmitate, whereby the osteoblast-like cells showed a lower oxidation rate than the other populations. Both parathyrin and calcitonin had no effect on fatty acid oxidation. 1,25-Dihydroxycholecalciferol at 1-100 nM and 24,25-dihydroxycholecalciferol at 100 nM increased oxidation primarily in the population enriched with osteoblast-like cells. Insulin at 1.6 microM diminished it in the cell populations enriched with osteoblast-like cells and in the late bone-cell fraction. However, glucagon had no effect. The energy provided by fatty acid oxidation in this system is approx. 40-80% of glucose metabolism, suggesting that this event may be of importance in the energy metabolism of bone.

Transforming growth factor-beta reduces the phenotypic expression of osteoblastic MC3T3-E1 cells in monolayer culture.

Transforming growth factor beta (TGF-beta) regulates cell growth and differentiation. Since it is abundant in bone, we have studied the effect of the polypeptide upon the growth and phenotypic expression of murine osteoblastic cells in monolayer culture. Its actions were compared to those of epidermal growth factor (EGF), another hormonally active polypeptide known to alter bone cell function. Picogram amounts of TGF-beta were found to inhibit the growth and phenotype (alkaline phosphatase and cAMP response to parathyroid hormone) of the clonal nontransformed MC3T3-E1 osteoblastic cell line. EGF also inhibited phenotypic expression, although at higher (nanogram) concentrations, but stimulated cell growth. The low concentration of TGF-beta required to inhibit growth and phenotype of osteoblastic cells together with its abundance in bone suggest that TGF-beta may be an important regulator of bone cell function.

Endothelial cells in culture synthesize a potent bone cell active mitogen.

Bovine aortic endothelial cells (BAEC) are known to synthesize in vitro multiple factors which act on a variety of cells in culture. The fact that vascularization appears to be required for endochondral and intramembranous ossification promoted us to examine whether BAEC produce a bone cell active mitogen. Conditioned media collected from exponential and confluent BAEC cultures were concentrated by ultrafiltration and assayed for growth-stimulating activity on bone cell populations isolated from 1-day-old rat calvaria by sequential enzymatic digestions. As assessed by the incorporation of [3H]thymidine into DNA, it was found that BAEC synthesized a potent bone cell active mitogen. Bovine and rat fibroblasts, as well as rabbit articular chondrocytes, were not affected by the factor which was produced by exponential and confluent cultures, regardless of whether BAEC were cultured in the presence or absence of fetal bovine serum. Employing gel filtration chromatography, Bio-Gel P60, the mitogenic activity eluted as a major peak. It was flanked on either side by a minor one. Apparent mol wt were calculated to be 43,000, 38,000, and 30,000, respectively. Upon heat treatment, 56 C for 30 min, the mitogenic activity was fully retained. No effect of the endothelial derived-growth factor on the synthesis of collagen was found. Our data suggest that endothelial cells, by virtue of their ability to synthesize bone cell active mitogen(s), may represent an important element in the genesis of bone formation.

The effect of pulsed electromagnetic fields on bone cell metabolism and calvaria resorption in vitro, and on calcium metabolism in the live rat.

The effects of three pulsed electromagnetic fields were investigated on bone cells and calvaria in culture, and on calcium metabolism in the live rat. No significant effect was seen on: 1) the proliferation of calvaria cells in culture; 2) alkaline phosphatase level, lactic acid release and collagen synthesis by confluent calvaria cells, with the exception of one pulse which produced a small increase in the latter when expressed as DNA; 3) resorption of calvaria in culture; 4) intestinal absorption, urinary excretion and net balance of calcium, bone formation and bone resorption in the live rat.

Differential response of bone cells isolated by sequential digestion to dichloromethylenebisphonate in culture.

Dichloromethylenebisphosphonate (Cl2-MBP), a compound structurally related to inorganic pyrophosphate but resistant to hydrolysis of endogenous phosphatase to yield inorganic phosphate, inhibits bone resorption and soft tissue mineralization in vivo. Previously, we have shown that bone cells isolated from rat calvaria respond profoundly to the exposure of Cl2MBP. To determine whether the cellular effects evoked by Cl2MBP are confined to a particular bone cell type, calvaria from 1 day postnatal rats were subjected to a sequential time-dependent enzyme digestion, yielding five bone cell populations marked by differences in PTH response, alkaline phosphatase activity and collagen, as well as hyaluronic acid synthesis. Culturing these bone cell populations with Cl2MBP revealed that previously observed results found with mixed bone cells (inhibition of cell proliferation, diminution of hyaluronic acid synthesis, and increase in alkaline phosphatase) were limited to cell populations which, according to the isolation scheme, stem from the outer tissue layer(s) of the calvaria. Collagen synthesis, however, was found to be equally increased regardless of cell type. These present results indicate that the action of Cl2MBP on bone may be cell specific.

Comparative study of deflazacort, a new synthetic corticosteroid, and dexamethasone on the synthesis of collagen in different rat bone cell populations and rabbit articular chondrocytes.

Deflazacort is a new synthetic glucocorticoid which is an oxazoline derivative of prednisolone. In previous studies, it was shown that deflazacort, depending on the test model used, not only showed considerably more antiinflammatory potency than prednisolone in animals but also caused less deleterious effects on bone mineral metabolism than equivalent amounts of other glucocorticoids in man. In this study, we have compared the effects of deflazacort with those of dexamethasone on the synthesis of collagen in various rat bone cell populations and chondrocytes. Three bone cell populations were prepared by sequential time-dependent collagenase treatment of 1-day-old rat calvaria. Each cell population was further purified on a Percoll gradient (10-90%) yielding three populations of which two are different in alkaline and acid phosphatase and response to parathyroid hormone. A 3-day treatment of bone cell populations with deflazacort and dexamethasone (10(-11)-10(-5) M) revealed that both glucocorticoids, although at different concentrations, inhibited collagen synthesis. 21-desacetyl-deflazacort (5 beta, 11 beta, 16 beta)-11,21-dihydroxy-2'-methyl-5-H-pregna-1-enol [17,16-d]oxazole-3,20-dione), the presumably active form of the steroid, which is formed in vivo after administration, produced nearly identical results as its precursor. Glucocorticoid concentrations at which inhibition was initially observed were 10(-9) M and 10(-7) M for dexamethasone and deflazacort respectively. Inhibition of collagen synthesis was significantly impaired only in cells isolated from bone during early tissue digestion, and not in those obtained during extensive collagenase treatment. Chondrocytes isolated from articular cartilage of 3-month-old rabbits and grown in primary cultures did not respond to either steroid.(ABSTRACT TRUNCATED AT 250 WORDS)

The subcellular distribution of [14C]dichloromethylenebisphosphonate and [14C]1-hydroxyethylidene-1,1-bisphosphonate in cultured calvaria cells.

Rat calvaria cells were cultured for 6 days in the presence or absence of [14C]dichloromethylenebisphosphonate [( 14C]Cl2MBP) or [14C]1-hydroxyethylidene-1, 1-bisphosphonate [( 14C]HEBP), after which cell organelles were separated by differential centrifugation. The distribution of protein, glutamate dehydrogenase, acid phosphatase, and 5'-nucleotidase was similar for cells treated or not treated with Cl2MBP. About 70-80% of the [14C]Cl2MBP and [14C]HEBP was found to be present in the supernatant. This was the only fraction that showed a ratio higher than 1 for the relative specific radioactivity, indicating that the bisphosphonates accumulated mainly in the cytosol. Rapid separation of particulate components and soluble cytoplasm of cells treated with [14C]Cl2MBP confirmed this finding, showing that it is unlikely that the result was due to leakage from the organelles. The uptake of [14C]Cl2MBP into cells was similar in different cell types. The binding of both bisphosphonates to macromolecules in the medium was 0.1-0.2% and 1-4% in the cells. This binding is not due to metabolic activity of the cells. About 15-20% of [14C]HEBP and [14C]Cl2MBP was modified by the living cells.

Rapid intracellular degradation of newly synthesized collagen by bone cells. Effect of dichloromethylenebisphosphonate.

Experiments were carried out to determine whether bone cells isolated from rat calvaria degrade newly synthesized collagen intracellularly prior to secretion and to assess the effect of dichloromethylenebisphosphonate, a compound shown to stimulate collagen synthesis during this event. The findings indicate that isolated bone cells grown in culture degraded a proportion (average 16%) of newly synthesized collagen prior to secretion. This process was markedly reduced by exposure to dichloromethylenebisphosphonate in a dose-related manner. Concomitantly with the observed decrease of degradation, an increase of collagen synthesis was detected as determined by the incorporation of [3H]proline into collagenase-digestible proteins or by the conversion of [3H]proline into [3H]hydroxyproline. No similar enhancement on total non-collagenous protein synthesis was evident. Dichloromethylenebisphosphonate did not influence the extracellular degradation of collagen. Although the reduction in intracellular degradation accounted only for part of the bisphosphonate mediated increase in net collagen synthesis, it is conceivable that the rate of collagen synthesis is regulated, at least in part, by mechanisms that modulate the level of intracellular degradation.