Biomedical subjects

C Löwik

Publications and source records attributed to C Löwik.

At least 19 recordsLinked to original sources

PubMed2001

Urokinase-receptor/integrin complexes are functionally involved in adhesion and progression of human breast cancer in vivo.

Interactions between specific cell-surface molecules, which include the urokinase receptor (uPAR) and integrins, are crucial to processes of tumor invasion and metastasis. Here we demonstrate that uPAR and beta1-integrins may cluster at distinct sites at the cell surface of metastatic MDA-MB-231 breast cancer cells and form functional complexes. Attachment assays performed in the presence of a synthetic peptide (p25), which interferes with the formation of uPAR-integrin complexes, reveal that uPAR is able to regulate the adhesive function of integrins in breast cancer cells. On dissociation of the uPAR-integrin complexes by p25, tumor cell attachment to the extracellular matrix was either decreased (vitronectin) or increased (fibronectin). Moreover, the tumor cells display remarkable morphological changes when cultured on fibronectin in the continuous presence of p25, leading to increased cell spreading and attachment. In marked contrast to control conditions, increased cellular adhesion to fibronectin after p25 treatment was entirely beta1-integrin-mediated. The role of uPAR-integrin complexes in tumor progression was studied in an in vivo bone xenograft model. Stably transfected MDA-MB-231 cells that overexpress p25 showed a significant reduction in tumor progression in bone (P < or = 0.0001 versus mock-control). In line with these observations, continuous administration of p25 (25 microg/mouse/day, osmotic minipumps) for 28 days resulted in significantly reduced tumor progression of MDA-MB-231 cells in bone (P < or = 0.005) when compared to scrambled control peptide. In conclusion, our data demonstrate that uPAR can act as an adhesion receptor in breast cancer and is capable of regulating integrin function. Our findings strongly suggest that adhesive and proteolytic events are tightly associated in metastatic breast cancer cells and that functional integrin-uPAR complexes are involved in tumor progression in vivo.

Bone Neoplasms↗

PubMed2001

Effect of angiogenic and antiangiogenic compounds on the outgrowth of capillary structures from fetal mouse bone explants.

Fetal mouse metatarsals are well-known models to study cartilage differentiation and osteoclastic resorption. We show here the outgrowth of PECAM-1 positive tubelike structures from the bone rudiments. This feature can be used to study angiogenesis in vitro. The area of outgrowth significantly increased with culture time, as shown by computerized image analysis of PECAM-1 positive tubelike structures. Treatment with recombinant human vascular endothelial growth factor (rhVEGF-A) stimulated the formation of tubelike structures. Treatment of explants with the angiogenesis inhibitor endostatin, the chemokine IP-10, and the thalidomide derivative phatolyl glutamic acid (PG-acid) resulted in an inhibition of the formation of PECAM-1 positive tubelike structures of 48.8% (+/- 4%), 50.2% (+/- 12%), and 80.8% (+/- 3%), respectively. Outgrowth of tubelike structures was partly dependent on endogenous VEGF-A because treatment with anti-mVEGF-A and truncated VEGF receptor 1 (soluble fms-like tyrosine kinase 1, sFIt1) strongly inhibited the formation of tubelike structures 74% (+/- 4%) and 38% (+/- 5%), respectively. Neither onset of tube formation nor total area of tubelike structures were changed when metatarsals were cultured on a fibrin gel or collagen type I gel. Tube formation required activation of matrix metalloproteinases because treatment of the bones with an inhibitor of matrix metalloproteinases completely inhibited migration and tube formation, whereas treatment with an inhibitor of plasmin had no effect. In conclusion, we describe a new in vitro model to study angiogenesis that can be used to test the angiogenic or antiangiogenic potential of novel test compounds that also combines the multicellularity of in vivo assays with the accessibility and flexibility of in vitro assays.

Animals↗

PubMed2001

Monitoring metastatic behavior of human tumor cells in mice with species-specific polymerase chain reaction: elevated expression of angiogenesis and bone resorption stimulators by breast cancer in bone metastases.

Tumor-stroma interactions are of primary importance in determining the pathogenesis of metastasis. Here, we describe the application of sensitive competitive polymerase chain reaction (PCR) techniques for detection and quantitation of human breast cancer cells (MDA-MB-231) in an in vivo mouse model of experimental metastasis. Human-specific oligonucleotide primers in competitive PCR reactions were used to quantify the amount of MDA-MB-231 cells per tissue per organ. Using this species-specific (semi)quantitative PCR approach, gene expression patterns of (human) tumor cells or (mouse) stromal cells in metastatic lesions in the skeleton or soft tissues were investigated and compared. In all metastatic lesions, MDA-MB-231 cells express angiogenic factors (vascular endothelial growth factors [VEGFs]; VEGF-A, -B, and -C) and bone-acting cytokines (parathyroid hormone-related protein [PTHrP] and macrophage colony-stimulating factor [M-CSF]). In these metastases, PECAM-1-positive blood vessels and stromal cells of mouse origin are detected. The latter express angiogenic factors and markers of sprouting vessels (VEGF receptors flt-1/flk - 1/flk-4 and CD31/PECAM-1). Strikingly, steady-state messenger RNA (mRNA) levels of VEGF-A and -B and the major bone resorption stimulators PTHrP and M-CSF by tumor cells were elevated significantly in bone versus soft tissues (p < or = 0.05, p < or = 0.0001, p < or = 0.001, and p < or = 0.05, respectively), indicating tissue-specific expression of these tumor progression factors. In conclusion, MDA-MB-231 breast cancer cells express a variety of factors in vivo that have been implicated in metastatic bone disease and that correlate with poor survival of patients with breast cancer. We hypothesize that the observed up-regulated expression of angiogenic and bone-resorbing factors by the breast cancer cells in the skeleton underlie the clinically observed osteotropism of breast cancer cells and pathogenesis of osteolytic bone metastases. The application of the species-specific competitive PCR-based assay in vivo can provide new information concerning the involvement of gene families in tumor progression and metastatic disease and greatly facilitates the study of tumor-stroma interactions in cancer invasion and metastasis.

Animals↗

PubMed2000

Tumour progression and angiogenesis in bone metastasis from breast cancer: new approaches to an old problem.

Breast cancer metastasizes frequently to the skeleton and causes considerable morbidity and deterioration of the quality of life. The clinical consequences of skeletal metastases are bone pain, pathological fractures, hypercalcaemia and nerve compression syndromes. From the moment breast cancer cells are located in the bone microenvironment, they may release factors which stimulate bone resorption and angiogenesis leading to growth of skeletal metastases and a subsequent selective increase in the attraction of new cancer cells to bone. In this review, emerging new concepts of breast cancer-bone interactions, in particular the involvement of angiogenesis, proteolysis and the role of cancer-induced bone resorption in skeletal metastasis are discussed. Better understanding of the processes involved in the metastasis of cancer cells to bone, local tumour growth and subsequent destruction of skeletal architecture can lead to optimal methods for the prevention and treatment of metastatic bone disease.

Animals↗

PubMed2000

The monoclonal antibodies 18d7/91f2 recognize a receptor regulatory protein on mouse bone marrow stromal cells.

Two monoclonal antibodies 18D7 and 91F2 were developed by immunizing rats with the mouse bone marrow-derived osteogenic cell line MN7. Hybridomas secreting rat antibodies against MN7 cell surface markers were selected by flow cytometry analysis. Both the monoclonal antibody 18D7 and the monoclonal antibody 91F2 are directed against the same cell surface antigen present on MN7 cells. Here, we report on the immunopurification of the 18D7/91F2 antigen and its identification as the prostaglandin F2 alpha receptor regulatory protein (FPRP). FPRP is expressed as a single messenger RNA (mRNA) of approximately 6 kilobases (kb) in MN7 cells and is differentially expressed in developing osteogenic cultures of bone marrow cells of the mouse. However, addition of the monoclonal antibodies 18D7 and 91F2 to these cultures did not inhibit bone formation in vitro. Both monoclonal antibodies reacted with mouse stromal cell lines established from bone marrow, thymus, spleen, and mandibular condyles. Immunohistochemical analysis of mature tibia of mice using the monoclonal antibody 18D7 revealed the presence of a distinct population of bone marrow cells close to trabecular and endosteal bone surfaces. In the central bone marrow, hardly any positive cells were found. In 17-day-old fetal mouse radius 18D7 immunoreactivity was restricted to cells in the periosteum in close vicinity to the bone collar. Mature osteoblasts, osteoclasts, osteocytes, growth plate chondrocytes, and mature macrophages were all negative. Taken together, these results suggest that FPRP plays a role in the osteogenic differentiation process.

Amino Acid Sequence↗

PubMed1999

Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates.

Bisphosphonates (Bps), inhibitors of osteoclastic bone resorption, are used in the treatment of skeletal disorders. Recent evidence indicated that farnesyl pyrophosphate (FPP) synthase and/or isopentenyl pyrophosphate (IPP) isomerase is the intracellular target(s) of bisphosphonate action. To examine which enzyme is specifically affected, we determined the effect of different Bps on incorporation of [(14)C]mevalonate (MVA), [(14)C]IPP, and [(14)C]dimethylallyl pyrophosphate (DMAPP) into polyisoprenyl pyrophosphates in a homogenate of bovine brain. HPLC analysis revealed that the three intermediates were incorporated into FPP and geranylgeranyl pyrophosphate (GGPP). In contrast to clodronate, the nitrogen-containing Bps (NBps), alendronate, risedronate, olpadronate, and ibandronate, completely blocked FPP and GGPP formation and induced in incubations with [(14)C]MVA a 3- to 5-fold increase in incorporation of label into IPP and/or DMAPP. Using a method that could distinguish DMAPP from IPP on basis of their difference in stability in acid, we found that none of the NBps affected the conversion of [(14)C]IPP into DMAPP, catalyzed by IPP isomerase, excluding this enzyme as target of NBp action. On the basis of these and our previous findings, we conclude that none of the enzymes up- or downstream of FPP synthase are affected by NBps, and FPP synthase is, therefore, the exclusive molecular target of NBp action.

Animals↗

PubMed1999

Nitrogen-containing bisphosphonates inhibit isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase activity with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo.

Bisphosphonates, synthetic compounds which suppress bone resorption, are used in the treatment of skeletal disorders. Their mode of action and intracellular targets have not yet been identified. Recent evidence suggested that enzymes of the mevalonate pathway are the potential targets. In this study, we examined the effect of four potent nitrogen (N)-containing bisphosphonates, clodronate and NH2-olpadronate, an inactive analogue of olpadronate, on isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase, geranylgeranyl pyrophosphate synthase, and protein geranylgeranyl transferase I activity. We found that all N-containing bisphosphonates inhibited isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase activity dose dependently with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo, whereas clodronate and NH2-olpadronate had no effect. Furthermore, none of the bisphosphonates tested affected geranylgeranyl pyrophosphate synthase or geranylgeranyl transferase I activity. Our study reveals for the first time the intracellular target of N-containing bisphosphonates and supports the view that all bisphosphonates do not share the same molecular mechanism of action.

Animals↗

PubMed1999

Inhibition of intravacuolar acidification by antisense RNA decreases osteoclast differentiation and bone resorption in vitro.

The role of proton transport and production in osteoclast differentiation was studied in vitro by inhibiting the transcription/translation of carbonic anhydrase II (CA II) and vacuolar H(+)-ATPase (V-ATPase) by antisense RNA molecules. Antisense RNAs targeted against CA II, or the 16 kDa or 60 kDa subunit of V-ATPase were used to block the expression of the specific proteins. A significant decrease in bone resorption rate and TRAP-positive osteoclast number was seen in rat bone marrow cultures and fetal mouse metacarpal cultures after antisense treatment. Intravacuolar acidification in rat bone marrow cells was also significantly decreased after antisense treatment. The CA II antisense RNA increased the number of TRAP-positive mononuclear cells, suggesting inhibition of osteoclast precursor fusion. Antisense molecules decreased the number of monocytes and macrophages, but increased the number of granulocytes in marrow cultures. GM-CSF, IL-3 and IL-6 were used to stimulate haematopoietic stem cell differentiation. The 16 kDa V-ATPase antisense RNA abolished the stimulatory effect of GM-CSF, IL-3 and IL-6 on TRAP-positive osteoclast formation, but did not affect the formation of monocytes and macrophages after IL-3 treatment, or the formation of granulocytes after IL-6 treatment. These results suggest that CA II and V-ATPase are needed, not only for the actual resorption, but also for osteoclast formation in vitro.

Acid Phosphatase↗

PubMed1999

The role of geranylgeranylation in bone resorption and its suppression by bisphosphonates in fetal bone explants in vitro: A clue to the mechanism of action of nitrogen-containing bisphosphonates.

Bisphosphonates, synthetic compounds used in the treatment of skeletal disorders, suppress osteoclast-mediated bone resorption by a yet unidentified mechanism. Previous studies showed that some bisphosphonates can inhibit enzymes of the mevalonate pathway, and nitrogen-containing bisphosphonates inhibit protein prenylation in mouse macrophages. In the present study, we examined the involvement of the mevalonate pathway in basal and bisphosphonate-inhibited osteoclastic resorption in fetal mouse long bone explants, an experimental model representative of the in vivo action of bisphosphonates. Mevastatin inhibited bone resorption at concentrations similar to those of the potent bisphosphonate ibandronate. This effect could be totally reversed by the addition of mevalnate and geranylgeraniol but not farnesol. The first two intermediates but not the latter could also stimulate basal bone resorption. The inhibitory effect of ibandronate on bone resorption could be totally reversed by the addition of geranylgeraniol and to a small extent only by mevalonate and farnesol, indicating that the bisphosphonate acts at a level of the mevalonate pathway different from that of mevastatin. Histologic sections of ibandronate-treated bone explants showed further rescue of functioning osteoclasts during concomitant treatment with geranylgeraniol. Finally, the reversibility of bisphosphonate inhibited osteoclastic resorption by geranylgeraniol was also demonstrated for the potent nitrogen-containing bisphosphonates alendronate, olpadronate, and risedronate but not for the non-nitrogen-containing bisphosphonates clodronate and etidronate. These studies demonstrate that protein geranylgeranylation but not farnesylation is important for osteoclast-mediated bone resorption and that nitrogen-containing bisphosphonates exert their antiresorptive action probably by affecting enzymes of the mevalonate pathway involved in the generation of geranylgeranyl pyrophosphate.

Animals↗

PubMed1997

Ovariectomy and orchidectomy induce a transient increase in the osteoclastogenic potential of bone marrow cells in the mouse.

Withdrawal of gender steroids in both women and men is associated with an increase in bone turnover with bone resorption exceeding bone formation leading to bone loss. To further investigate this process, the osteoclastogenic potential of mouse bone marrow cells was assessed at different timepoints after ovariectomy (ovx) or orchidectomy (orx). Cocultures of osteoclast-free fetal mouse long bones together with bone marrow from ovariectomized or orchidectomized mice indicated that the withdrawal of gender steroids in female and male mice induces a transient increase in osteoclastogenesis. The osteoclastogenic potential of the bone marrow cells was increased 7 days after ovx or orx. However, osteoclastic resorption was not increased at 3 days after surgery and had normalized 30 days after either ovx or orx. These results suggest that the withdrawal of gender steroids induces a transient increase in osteoclastogenesis in mice of both genders, which is associated with the early phase of rapid bone loss.

Animals↗

PubMed1996

Bisphosphonates inhibit the adhesion of breast cancer cells to bone matrices in vitro.

Bisphosphonates are used with increasing frequency in the management of skeletal complications in patients with breast cancer. In this paper, we have investigated whether bisphosphonates, besides their known beneficial effects on tumor-associated osteoclastic resorption, are capable of inhibiting breast cancer cell adhesion to bone matrix. For that we used two in vitro models for bone matrix (cortical bone slices and cryostat sections of trabecular bone from neonatal mouse tails). Four bone matrix-bound nitrogen-containing bisphosphonates (pamidronate, olpadronate, alendronate, and ibandronate) inhibited adhesion and spreading of breast cancer cells to bone dose-dependently, whereas etidronate and clodronate had little or no effect. Strikingly, the relative order of potency of the bisphosphonates in inhibiting the adhesion of cancer cells to cortical and trabecular bone corresponded to their relative antiresorptive potencies in vivo as well as their ranking in in vitro bone resorption assays with predictive value for their clinical efficacy. It appears that nitrogen-containing bisphosphonates alter selectively the adhesive properties of the extracellular bone matrix preventing the attachment of breast cancer cells to it. Besides the beneficial effects of bisphosphonates on tumor-induced osteoclastic resorption, the previously unrecognized effect presented in this paper makes these agents suitable for earlier pharmacologic intervention in patients with breast cancer at risk of developing bone metastases.

Animals↗

PubMed1996

Dissociation of binding and antiresorptive properties of hydroxybisphosphonates by substitution of the hydroxyl with an amino group.

The purpose of this study was to examine the role of the R1 moiety of bisphosphonates in binding to bone mineral and for antiresorptive action. For this, the R1 chain of three clinically useful hydroxybisphosphonates (etidronate, pamidronate, and olpadronate) was substituted with an amino group. The effects of the aminosubstituted bisphosphonates were compared with those of their hydroxy counterparts in a crystal growth assay and in fetal mouse long bone cultures which are representative of bisphosphonate actions in vivo. It was found that all three aminosubstituted compounds and their hydroxy analogs bound with similar affinity to bone mineral and inhibited the growth of calcium oxalate crystals to the same extent. Surprisingly, the antiresorptive effect of olpadronate was totally abolished by the amino substitution of the hydroxyl group while that of pamidronate was reduced by about six-fold and that of etidronate did not change. These studies demonstrate the involvement of the entire bisphosphonate molecule in the cellular mechanism of antiresorptive action. In addition, the aminosubstituted analog of olpadronate, which lacks any antiresorptive action but retains all other properties of olpadronate, provides an excellent tool for the study of specific cellular effects involved in bisphosphonate action.

Amines↗

PubMed1995

In vitro and ex vivo evidence that estrogens suppress increased bone resorption induced by ovariectomy or PTH stimulation through an effect on osteoclastogenesis.

The mechanism of bone loss following cessation of ovarian function is still unclear. Several studies have shown an increase in bone turnover following natural or surgical menopause which could be prevented by estrogen administration. However, a direct effect of estrogen on osteoclast-mediated bone resorption has been difficult to demonstrate in vitro. Recent evidence suggested that estrogen withdrawal stimulates the production of bone resorbing cytokines, (e.g., interleukin-6, IL-6), which regulate osteoclast formation in the bone marrow microenvironment. We studied the effects of 17beta-estradiol on osteoclastic resorption, measured as 45calcium release, in vitro using cultures of fetal mouse long bone explants in which different stages of osteoclast development and activity are represented. 17beta-estradiol (10(-12)-10(-8) M) had no effect on basal or parathyroid hormone (PTH)-stimulated resorption of bone explants in which mature osteoclasts (radii/ulnae) or osteoclast precursors/progenitors (metacarpals) are present. 17beta-estradiol, however, inhibited significantly the PTH-stimulated resorption of osteoclast-free metacarpals cultured together with mouse fetal liver as a source of early osteoclast progenitors; basal resorption was also not inhibited in this system. In ex vivo studies we further examined the effects of culturing bone marrow cells from ovariectomized (OVX) or sham-operated mice as an osteoclastic source together with osteoclast-free metacarpals on 45calcium release and bone histology. Cocultures of the bone marrow cells from OVX mice with osteoclast-free metacarpals increased significantly the osteoclast formation and subsequent osteoclastic resorption compared with control cocultures. This increase in resorption was prevented by either treatment of the OVX animals with estrogen for 1 week starting immediately after OVX or injection of the OVX animals with an IL-6 neutralizing antibody. We conclude that estrogens suppress the increased osteoclastic resorption induced by PTH or OVX through an effect on hematopoietic progenitor cells of the osteoclast lineage. Furthermore our data suggest that IL-6 is involved in the increase in osteoclastic resorption following OVX.

Animals↗

PubMed1995

Cytokine production by calvariae of osteopetrotic mice.

In the osteopetrotic op/op mouse, the absence of macrophage colony-stimulating factor (M-CSF) prevents the growth of macrophages and osteoclasts and, consequently, bone resorption. In the present study, we investigated whether this deficiency in M-CSF production alters the production of cytokines in op/op bones. Calvariae of phenotypically normal (+/?) and op/op mice were stimulated in vitro with lipopolysaccharide or Pasteurella multocida toxin to produce cytokines. Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) synthesis was the same both in calvaria from osteopetrotic and phenotypically normal animals. However, the production of granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha) was lower in calvaria from op/op animals than was the case in +/? calvaria. Thus, the lack of biologically active M-CSF causes defects which are not compensated by cells independent of M-CSF.

Animals↗

PubMed1995

Effects of experimental conditions on the release of 45calcium from prelabeled fetal mouse long bones.

Embryonic/neonatal bones in culture are commonly used for the study of osteoclastic resorption in vitro. For this purpose, the release of 45calcium (45Ca) from prelabeled bones is measured as an index of resorption. We studied 45Ca release from two types of long bone explants after different preparation methods: 17-day-old fetal mouse radii/ulnae with and without cartilage ends (intact radii/ulnae and shafts, respectively), and intact 18-day old metacarpals/metatarsals. In addition, we examined the effect of different culture conditions, such as cultures performed under the surface of the medium or at the interphase of medium and air, on 45Ca release and histology. When intact radii/ulnae were cultured under the surface of the medium, there was always a significant amount (10%) of net basal 45Ca release (corrected for physicochemical exchange) that was not due to osteoclastic resorption, as it could not be suppressed by inhibitors of resorption even at high concentrations. Moreover, histologically TRAcP-positive cells were almost absent after culture and the bone marrow/stromal cells in the center of the bone appeared necrotic, possibly due to a lack of oxygen. Under these culture conditions, osteoclasts could survive in shafts as well as in PTH-stimulated intact radii/ulnae, but a constant amount of 10% 45Ca, not due to resorption, was still released in the medium. When these explants were cultured at the interphase of medium and air, basal and stimulated 45Ca release originated from osteoclastic resorption. In contrast, in 18-day-old fetal mouse metacarpals/metatarsals, the experimental conditions applied did not affect 45Ca release, which was always due to resorption of the explants by osteoclasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗

PubMed1995

Hypercalcemia and cosecretion of interleukin-6 and parathyroid hormone related peptide by a human renal cell carcinoma implanted into nude mice.

Humoral hypercalcemia of malignancy is a paraneoplastic syndrome believed to be due to production by the tumor of substances that stimulate osteoclastic bone resorption primarily. The human renal cell carcinoma cell line RC-8, grown in nude mice, was investigated for factors involved in renal cancer-induced hypercalcemia. At a tumor load of 200 to 400 mm.3 the mice developed hypercalcemia and hypophosphatemia associated with a rise in serum 1,25-dihydroxyvitamin D concentration and cachexia. The tumor released 1) significant amounts of human interleukin-6 (IL-6) and 2) parathyroid hormone-related peptide (PTHrP) into the circulation. Cancer cells further expressed mRNA for both human IL-6 and PTHrP. No secretion of human tumor necrosis factor-alpha or interleukin-1 beta could be demonstrated in the circulation of the host. Antibodies to IL-6 caused a significant (p = 0.043) inhibition of tumor growth and decreased serum calcium concentrations compared with control animals. Our data suggest that IL-6 is involved, either directly or indirectly, in the development of hypercalcemia in renal cell carcinoma.

Animals↗

PubMed1994

Integrins and osteoclastic resorption in three bone organ cultures: differential sensitivity to synthetic Arg-Gly-Asp peptides during osteoclast formation.

We investigated possible inhibitory effects of five synthetic Arg-Gly-Asp (RGD)-containing peptides on osteoclastic resorption in three distinct in vitro resorption assays (17-day-old fetal mouse bone organ cultures) that differ in stages of osteoclast differentiation. RGD peptides, which can bind the adhesion receptors called integrins, inhibited osteoclastic resorption (45Ca release) in fetal mouse bone explants in which osteoclast precursors have yet to adhere to the mineralized matrix and develop into mature osteoclasts (metacarpals and coculture system). Treatment of metacarpals with RGD peptides inhibited the formation of multinucleated TRAP+ osteoclasts in the mineralized matrix because their mononuclear TRAP+ osteoclast precursors remained localized in the periosteum. In particular, echistatin, a viper venom protein with known affinity for alpha v beta 3 integrin, and GdRGDSP inhibited osteoclastic resorption dose dependently in these systems (ED50 10(-9) and 10(-4) M, respectively) but did not alter the activity of mature resorbing osteoclasts in radii. In addition, 45Ca release was significantly inhibited by the cyclic peptide GPenGRGDSPCA, which has a relatively higher affinity for the vitronectin than fibronectin receptor(s). In contrast, GRDGdSP, which has a much higher affinity for the fibronectin receptor (than the vitronectin receptors), had no effect on resorption at similar concentrations in any resorption system used. In summary, the data presented in this paper show that peptides with RGD motifs are capable of inhibiting osteoclastic resorption in bone organ cultures. Our studies not only support the hypothesis concerning the importance of alpha v beta 3 in osteoclastic resorption but also suggest an important role of integrin(s) in events preceding the actual resorption of calcified matrix by osteoclasts.

Amino Acid Sequence↗

PubMed1994

Structural requirements for bisphosphonate actions in vitro.

We investigated the structural requirements for the binding of bisphosphonates to bone mineral and the relation between their affinity for bone and their effects on bone resorption in vitro. For this we used fetal mouse long bones in culture and bisphosphonates with variable R1 and R2 structures. In addition, we studied the effect of structural differences in the incorporation of calcium into bone. We found that bisphosphonates containing a hydroxyl group in the R1 position have the highest affinity for bone mineral. This was related to their capacity to inhibit the incorporation of calcium into long bones but not to their antiresorptive potency. The latter was primarily determined by R2. Furthermore, the effect of bisphosphonates on calcification, but not on resorption of bone explants, was mainly determined by the mode of addition. The continuous presence of bisphosphonate during culture inhibited calcification even at very low concentrations, but short incubation of the bones with relatively high concentrations had no effect. This is probably a result of differences in the availability of the compound to the process of calcification. Because, in vivo, the more potent bisphosphonates inhibit resorption without adversely affecting mineralization of the skeleton and they disappear rapidly from the circulation after administration, we suggest that cultures of bone explants incubated with bisphosphonates for short times rather than cultures in which the drugs are continuously present provide more accurate information about the in vivo effect of these compounds on both resorption and calcification.

Animals↗