Biomedical subjects

C W Löwik

Publications and source records attributed to C W Löwik.

At least 19 recordsLinked to original sources

PubMed2001

Recombinant human extracellular matrix protein 1 inhibits alkaline phosphatase activity and mineralization of mouse embryonic metatarsals in vitro.

Two mRNAs are transcribed from the extracellular matrix protein 1 gene (Ecm1): Ecm1a and an alternatively spliced Ecm1b. We studied Ecm1 mRNA expression and localization during endochondral bone formation and investigated the effect of recombinant human (rh) Ecm1a protein on organ cultures of embryonic mouse metatarsals. Of the two transcripts, Ecm1a mRNA was predominantly expressed in fetal metacarpals from day 16 to 19 after gestation. Ecm1 expression was not found in 16- and 17-day-old metatarsals of which the perichondrium was removed. In situ hybridization and immunohistochemistry demonstrated Ecm1 expression in the connective tissues surrounding the developing bones, but not in the cartilage. Biological effects of rhEcm1a protein on fetal metatarsal cultures were biphasic: at low concentrations, Ecm1a stimulated alkaline phosphatase activity and had no effect on mineralization, whereas at higher concentrations, Ecm1a dose dependently inhibited alkaline phosphatase activity and mineralization. These results suggest that Ecm1a acts as a novel negative regulator of endochondral bone formation.

Alkaline Phosphatase↗

PubMed2001

Effect of interleukin-17 on nitric oxide production and osteoclastic bone resorption: is there dependency on nuclear factor-kappaB and receptor activator of nuclear factor kappaB (RANK)/RANK ligand signaling?

Interleukin-17 (IL-17) is a proinflammatory cytokine produced exclusively by activated memory T cells and has recently been found to stimulate osteoclastic resorption. Like other proinflammatory cytokines, IL-17 may affect osteoclastic bone resorption indirectly via osteoblasts, possibly by mechanisms previously reported for chondrocytes that respond in very similarly to osteoblasts. As in chondrocytes, but only in combination with tumor necrosis factor-alpha (TNF-alpha), IL-17 induced nitric oxide (NO) production in osteoblastic cells and fetal mouse metatarsals by a nuclear factor-kappaB (NF-kappaB)-dependent mechanism. This effect was associated with elevated mRNA levels of the NF-kappaB isoforms RelA and p50. In fetal mouse metatarsals, IL-17 stimulated osteoclastic bone resorption only in combination with TNF-alpha. The pathway by which the cytokine combination exerts this effect was examined using inhibitors of NO synthesis and NF-kappaB activation. Although both inhibitors used abolished NO production, they did not prevent the stimulatory effect of the cytokine combination on osteoclastic resorption. In contrast, the inhibitors slightly increased osteoclastic resorption, suggesting a suppressive rather than stimulatory effect of NO on cytokine-induced bone resorption. In addition, we showed that IL-17 + TNF-alpha stimulated osteoclastic resorption independent of NF-kappaB signaling. To further examine the pathway by which osteoclastic resorption was stimulated, we used osteoprotegerin, a specific inhibitor of the receptor activator of NF-kappaB (RANK)/receptor activator of the NF-kappaB ligand (RANKL) pathway. Osteoprotegerin partially inhibited IL-17 + TNF-alpha-stimulated osteoclastic resorption only at the high concentration of 1000 ng/mL, whereas it completely blocked parathyroid hormone-related peptide-stimulated resorption at 300 ng/mL. In conclusion, IL-17 stimulated NO production by an NF-kappaB-dependent pathway in osteoblastic cells and fetal mouse metatarsals only in combination with TNF-alpha. Neither NO production nor NF-kappaB signaling, and only partly the RANK/RANKL pathway, were involved in the stimulatory effect of the cytokine combination on osteoblastic bone resorption in these long bones, suggesting the existence of other pathways by which osteoclastic resorption can be stimulated.

3T3 Cells↗

PubMed2000

Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation.

Endochondral bone formation is regulated by systemically and locally acting growth factors. A role for vascular endothelial growth factor (VEGF) in this process has recently been proposed, because inactivation of VEGF inhibits endochondral bone formation via inhibition of angiogenesis. Despite the known effect of VEGF as specific endothelial growth factor, its effects on osteoblast differentiation have not been studied. We, therefore, examined the expression of VEGF-A, -B, -C, and -D and their receptors in a model of osteoblast differentiation using the mouse preosteoblast-like cell line KS483. Early in differentiation, KS483 cells express low levels VEGF-A, -B, and -D messenger RNA, whereas during mineralization, KS483 cells express high levels. In addition, expression of the VEGF receptors, VEGFR1, VEGFR2, and VEGF165R/neuropilin, coincided with expression of their ligands, being maximally expressed during mineralization. VEGF-A production during osteoblast differentiation was stimulated by insulin-like growth factor I that enhances osteoblast differentiation and was inhibited by PTH-related peptide that inhibits osteoblast differentiation. Furthermore, continuous treatment of KS483 cells with recombinant human VEGF-A stimulated nodule formation. Although treatment of KS483 cells with soluble FLT1, an agent that blocks binding of VEGF-A and -B to VEGFR1, did not inhibit nodule formation, this observation does not exclude involvement of VEGFR2 in the regulation of osteoblast differentiation. As it is known that VEGF-A, -C, and -D can act through activation of VEGFR2, other isoforms might compensate for VEGF-A loss. The expression pattern of VEGFs and their receptors shown here suggests that VEGFs play an important role in the regulation of bone remodeling by attracting endothelial cells and osteoclasts and by stimulating osteoblast differentiation.

Animals↗

PubMed2000

Expression of Indian hedgehog, parathyroid hormone-related protein, and their receptors in the postnatal growth plate of the rat: evidence for a locally acting growth restraining feedback loop after birth.

A locally acting growth restraining feedback loop has been identified in the murine embryonic growth plate in which the level of parathyroid hormone-related peptide (PTHrP) expression regulates the pace of chondrocyte differentiation. To date, it is largely unknown whether this feedback loop also regulates the pace of chondrocyte differentiation in the growth plate after birth. We therefore characterized the spatio-temporal expression of Indian hedgehog (IHH), PTHrP, and their receptors in the postnatal growth plate from female and male rats of 1, 4, 7, and 12 weeks of age. These stages are representative for early life and puberty in rats. Using semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) on growth plate tissue, IHH and components of its receptor complex, patched (PTC) and smoothened (SMO), PTHrP and the type I PTH/PTHrP receptor messenger RNA (mRNA) were shown at all ages studied irrespective of gender. Using in situ hybridization, IHH, PTHrP, and PTH/PTHrP receptor mRNA were detected in prehypertrophic and hypertrophic chondrocytes in both sexes during development. In addition, especially in the younger age groups, faint expression of PTH/PTHrP receptor mRNA also was shown in stem cells and proliferative chondrocytes. Immunohistochemistry confirmed the observations made with in situ hybridization, by showing the presence of IHH, PTC, PTHrP, and PTH/PTHrP receptor protein in prehypertrophic and hypertrophic chondrocytes. In addition, staining for hedgehog, PTC, and PTHrP also was observed in growth plate stem cells. No differences in staining patterns were observed between the sexes. Furthermore, no mRNA or protein expression of the mentioned factors was detected in the perichondrium. Our data suggest that in contrast to the proposed feedback loop in the early embryonic growth plate, which requires the presence of the perichondrium, a feedback loop in the postnatal growth plate can be confined to the growth plate itself. In fact, two loops might exist: (1) a loop confined to the transition zone and early hypertrophic chondrocytes, which might in part be autocrine and (2) a loop involving the growth plate stem cells.

Animals↗

PubMed1999

Immunohistochemical investigations on the differentiation marker protein E11 in rat calvaria, calvaria cell culture and the osteoblastic cell line ROS 17/2.8.

Until now, many extracellular matrix proteins, e.g. osteopontin and osteonectin, have been used to determine a cell's osteogenic maturation. The disadvantage in evaluation of these proteins is their relative wide-ranging appearance throughout the osteogenic differentiation process. Thus, the aim of this study was to establish an immunohistochemical setup using E11, a marker that binds selectively to cells of the late osteogenic cell lineage. In addition, the histochemical expression of the bone matrix proteins osteonectin, osteopontin and fibronectin was compared to that of E11 using monoclonal antibodies. For light microscopical detection of osteogenic markers in cultured cells we developed a simple paraffin technique using a fibrin glue as embedding medium. This allows the handling of cultured cells such as a tissue sample and includes the use of stored biological specimens for further immunohistochemical experiments. We used newborn rat calvariae for whole tissue preparations and for isolation and cultivation of bone cells. In addition, we included the rat osteosarcoma cell line ROS 17/2.8 in this study. For the first time, we have localised E11 in osteocytes of rat calvaria preparations at the electron microscopical level. E11 was detected at plasma membranes of osteocytes and their processes, but not at those of osteoblasts. Accompanying experiments with cultured newborn rat calvaria cells and ROS 17/2.8 cells revealed E11 reactivity on a subset of cells. The results obtained confirm the suitability of the differentiation marker E11 as a sensitive instrument for the characterisation of bone cell culture systems.

Animals↗

PubMed1999

Parathyroid hormone-related peptide (PTHrP) induces parietal endoderm formation exclusively via the type I PTH/PTHrP receptor.

A number of studies suggest a role for PTHrP and the classical PTH/PTHrP receptor (type I) in one of the first differentiation processes in mouse embryogenesis, i.e. the formation of parietal endoderm (PE). We previously reported that although in type I receptor (-/-) embryos PE formation seemed normal, the embryos were smaller from at least day 9.5 p.c. and 60% had died before day 12.5 p.c. Here we show that the observed growth defect commences even earlier, at day 8.5 p.c. Using two novel antibodies, we show that the expression of the type I receptor protein at this stage is confined to extraembryonic endoderm only. In addition, we show that large amounts of PTHrP protein are present in the adjacent trophoblast giant cells, suggesting a paracrine interaction of PTHrP and the type I PTH/PTHrP receptor in PE formation. The involvement in PE differentiation of other recently described receptors for PTHrP would explain a possible redundancy for the type I receptor in PE formation. However, deletion of the type I PTH/PTHrP receptor in ES cells by homologous recombination completely prevents PTHrP-induced PE differentiation. Based upon these observations, we propose that PTHrP and the type I PTH/PTHrP receptor, although not required for the initial formation of PE, are required for its proper differentiation and/or functioning.

Animals↗

PubMed1999

Inhibition of terminal chondrocyte differentiation by bone morphogenetic protein 7 (OP-1) in vitro depends on the periarticular region but is independent of parathyroid hormone-related peptide.

Bone morphogenetic protein-7, or BMP-7 (OP-1), is highly expressed in the perichondrium of embryonic long bones and is thought to play a role in endochondral ossification. Previously we have shown that BMP-7 inhibits terminal chondrocyte differentiation; that is, chondrocyte hypertrophy and mineralization in cultured explants of embryonic mouse metatarsals. However, the mechanism of this inhibition and the target cells of BMP-7 are still unknown. In this study we show that BMP-7 inhibits terminal chondrocyte differentiation indirectly, via an interaction with the periarticular region of the explants. This region also expresses parathyroid hormone-related peptide (PTHrP). PTHrP regulates terminal chondrocyte differentiation by inhibiting hypertrophic differentiation of prehypertrophic chondrocytes. The differentiating center in turn regulates PTHrP expression via a feedback loop involving Indian hedgehog (Ihh), which is expressed in the prehypertrophic chondrocytes. Ihh is thought to act on perichondrial cells, which in turn start to express an as yet unknown mediator that stimulates PTHrP expression in the periarticular region. It has been suggested that this factor belongs to the BMP-family. We investigated whether the inhibition of terminal chondrocyte differentiation by BMP-7 was due to upregulation of the PTHrP-Ihh feedback loop and whether BMP-7 was the unknown factor in the loop. Here we show that exogenous BMP-7 did not upregulate the mRNA expression of PTHrP, Ihh, or the PTH/PTHrP receptor in cultured wild-type embryonic metatarsals. Furthermore, BMP-7 could still inhibit terminal chondrocyte differentiation in the metatarsals of PTHrP-deficient (PTHrP-/-) mouse embryos. These data indicate that the BMP-7-mediated inhibition of terminal chondrocyte differentiation in vitro is independent of the PTHrP-Ihh feedback loop. We concluded that BMP-7 modulates terminal chondrocyte differentiation and cartilage mineralization of fetal bone explants in vitro via as yet unknown inhibitory factor(s) produced in the periarticular region.

Animals↗

PubMed1999

Degradation of extracellular matrix by metastatic follicular thyroid carcinoma cell lines: role of the plasmin activation system.

The plasmin activation system plays a key role in extracellular matrix degradation in many malignant tumors. Because no data are available on the involvement of the plasmin activation system in matrix degradation by thyroid carcinoma, the present study was performed using follicular thyroid carcinoma cell lines obtained from a primary tumor (FTC-133) and metastases (FTC-236 and FTC-238) of one patient. Matrix degradation by these cell lines was studied assessing the release of radioactivity from S35-methionine labeled extracellular matrix coated onto plastic. The involvement of constituents of the plasmin activation system as well as matrix metalloproteinases (MMPs), another class of proteolytic enzymes, which can be activated by plasmin, were assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and zymography. In the matrix degradation experiment, S35 release by FTC-133 was significantly higher than FTC-236 and FTC-238. S35 degradation could be inhibited by the plasmin inhibitor aprotinin and by anti-human urokinase-type plasminogen activator (uPA) antibody, indicating the involvement of the plasmin activation system. Matrix degradation could also be inhibited by the MMP inhibitor marimastat, thus demonstrating the involvement of MMPs in matrix degradation by these cell lines. Zymographic assays revealed activity of uPA in all cell lines. However, in contrast with FTC-236 and FTC-238, no plasminogen activator inhibitor (PAI) or PAI1 mRNA were found in FTC-133. Therefore, the differences in PAI activity as observed between the cell lines may originate from differences in PAI1 gene transcription. Differences in PAI1 expression did not affect the attachment of these cell lines to vitronectin. We conclude that the plasmin activation system is involved in extracellular matrix degradation by these metastatic follicular thyroid carcinoma cell lines. Differences in extracellular matrix degradation between the cell lines correspond with differences in PAI1 gene expression, indicating the significance of PAI1 in extracellular matrix degradation by metastatic follicular thyroid carcinoma.

Adenocarcinoma, Follicular↗

PubMed1999

A frame-shift mutation in the type I parathyroid hormone (PTH)/PTH-related peptide receptor causing Blomstrand lethal osteochondrodysplasia.

Blomstrand osteochondrodysplasia (BOCD) is a rare lethal skeletal dysplasia characterized by accelerated endochondral and intramembranous ossification. Comparison of the characteristics of BOCD with type I PTH/PTH-related peptide (PTHrP) receptor-ablated mice reveals striking similarities that are most prominent in the growth plate. In both cases, the growth plate is reduced in size due to a strongly diminished zone of resting cartilage and the near absence of columnar arrangement of proliferating chondrocytes. This overall similarity suggested that an inactivating mutation of the PTH/PTHrP receptor might be the underlying genetic defect causing BOCD. Indeed, inactivating mutations of the PTH/PTHrP receptor have been recently identified in two cases of BOCD. We describe here a novel inactivating mutation in the PTH/PTHrP receptor. Sequence analysis of all coding exons of the type I PTH/ PTHrP receptor gene and complementary DNA of a case with BOCD identified a homozygous point mutation in exon EL2 in which one nucleotide (G at position 1122) was absent. The mutation was inherited from both parents, supporting the autosomal recessive nature of the disease. The missense mutation resulted in a shift in the open reading frame, leading to a truncated protein that completely diverged from the wild-type sequence after amino acid 364. The mutant receptor, therefore, lacked transmembrane domains 5, 6, and 7; the connecting intra- and extracellular loops; and the cytoplasmic tail. Functional analysis of the mutant receptor in COS-7 cells and of dermal fibroblasts obtained from the case proved that the mutation was indeed inactivating. Neither the transiently transfected COS-7 cells nor the dermal fibroblasts responded to a challenge with PTH or PTHrP with a rise in intracellular cAMP levels, in sharp contrast to control cells. Our results provide further evidence that BOCD is caused by inactivating mutations of the type I PTH/PTHrP receptor and underscore the importance of this receptor in mammalian skeletal development.

Adult↗

PubMed1999

Identification of a retinoic acid-inducible element in the murine PTH/PTHrP (parathyroid hormone/parathyroid hormone-related peptide) receptor gene.

We have shown previously that the PTH/PTHrP (PTH-related peptide) receptor mRNA becomes expressed very early in murine embryogenesis, i.e. during the formation of extraembryonic endoderm. Retinoic Acid (RA) is a potent inducer of extraembryonic endoderm formation and PTH/PTHrP-receptor expression in embryonal carcinoma (EC) and embryonal stem (ES) cells. Using the P19 EC cell line, we have characterized promoter elements of the murine PTH/PTHrP-receptor gene that are involved in this RA-induced expression. The data show that RA-induced expression of the PTH/ PTHrP-receptor gene is mediated by the downstream P2 promoter. Analysis of promoter reporter constructs in transiently transfected P19 cells treated with RA identified an enhancer region between nucleotides -2714 and -2702 upstream of the P2 transcription start site that is involved in the RA effect. This region matches a consensus hormone response element consisting of a direct repeat with an interspacing of 1 bp (R-DR1). The R-DR1 efficiently binds retinoic acid receptor-alpha (RARalpha)-retinoid X receptor-alpha (RXRalpha) and chicken ovalbumin upstream promoter (COUP)-transcription factor I (TFI)-RXRalpha heterodimers and RXRalpha and COUP-TFI homodimers in a bandshift assay using extracts of transiently transfected COS-7 cells. RA differentiation of P19 EC cells strongly increases protein binding to the R-DR1 in a band-shift assay. This is caused by increased expression of RXR (alpha, beta, or gamma) and by the induction of expression of RARbeta and COUP TFI/TFII, which bind to the R-DR1 as shown by supershifting antibodies. The presence of RXR (alpha, beta, or gamma) in the complexes binding to the R-DR1 suggests that RXR homodimers are involved in RA-induced expression of the PTH/PTHrP-receptor gene. The importance of the R-DR1 for RA-induced expression of PTH/ PTHrP-receptor was shown by an inactivating mutation of the R-DR1, which severely impairs RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs. Since this mutation does not completely abolish RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs, sequences other than the R-DR1 might also be involved in the RA effect. Finally, we show that the RA-responsive promoter region is also able to induce expression of a reporter gene in extraembryonic endoderm of 7.5 day-old transgenic mouse embryos.

Animals↗

PubMed1999

Oestrogenic compounds modulate cytokine-induced nitric oxide production in mouse osteoblast-like cells.

Nitric oxide (NO) is a mediator of bone metabolism with effects on both bone resorption and formation. Its production by both the constitutive and inducible isoforms of nitric oxide synthase (NOS) is affected by oestrogen in several types of cell and in tissues other than bone cells. Recently, oestrogens were found to increase basal NO production by osteoblasts via enhanced activity or expression, or both, of NOS-3. Inflammatory cytokines, however, increase NO by increasing the expression of NOS-2. In this study we have examined whether cytokine-induced NO production by osteoblastic cells was affected by oestrogenic compounds by studying the effect of 17beta-oestradiol and the anti-oestrogens ICI164,384 and 4-hydroxytamoxifen on cytokine-induced NO production in oestrogen receptor positive MC3T3-E1 osteoblast-like cells. Combinations of the inflammatory cytokines interleukin-1beta, tumour necrosis factor-alpha, and interferon-gamma with lipopolysaccharide stimulated NO production up to 11-fold. This cytokine-induced NO production was further increased dose-dependently by the anti-oestrogens ICI164,384 and 4-hydroxytamoxifen (133.3 +/- 3.2% and 146.0 +/- 13.2%, respectively). 17Beta-oestradiol either had no effect on or slightly inhibited cytokine-induced NO production. It did, however, dose-dependently counteract the stimulatory effect of the anti-oestrogens. Concentrations of 17beta-oestradiol needed to prevent the stimulatory effect of 4-hydroxytamoxifen were ca tenfold that of ICI164,384. These findings show that, in addition to the stimulatory effect of oestrogen on basal NO production by NOS-3, cytokine-induced NO production is also affected by oestrogenic compounds in osteoblasts.

Animals↗

PubMed1999

Interleukin-17: A new bone acting cytokine in vitro.

Interleukin-17 (IL-17) is a recently cloned cytokine that is exclusively produced by activated T cells, but its receptor has been found on several cells and tissues. Like other proinflammatory cytokines produced by activated T cells, IL-17 may affect osteoclastic resorption and thereby mediate bone destruction accompanying some inflammatory diseases. In the present study, we investigated whether osteogenic cells possess the receptor for IL-17 (IL-17R) and whether IL-17 affects osteoclastic resorption. We found that IL-17R mRNA is expressed both in mouse MC3T3-E1 osteoblastic cells and fetal mouse long bones, suggesting that osteogenic cells may be responsive to IL-17. In fetal mouse long bones, IL-17 had no effect on basal and IL-1beta-stimulated osteoclastic bone resorption, but when given together with tumor necrosis factor-alpha (TNF-alpha) it increased bone resorption dose dependently in serum-free conditions. In addition, IL-17 increased TNF-alpha-induced IL-1alpha, IL-1beta, and IL-6 mRNA expression in fetal mouse metatarsals and IL-1alpha and IL-6 mRNA expression in MC3T3-E1 cells. In conclusion, IL-17R mRNA was expressed by mouse osteoblastic cells and fetal mouse long bones, and IL-17 in combination with TNF-alpha, but not IL-1beta, increased osteoclastic resorption in vitro. IL-17 may therefore affect bone metabolism in pathological conditions characterized by the presence of activated T cells and TNF-alpha production such as rheumatoid arthritis and loosening of bone implants.

Animals↗

PubMed1998

Binding and antiresorptive properties of heterocycle-containing bisphosphonate analogs: structure-activity relationships.

To define structure-activity relationships for bisphosphonate activity, we examined the bone binding and antiresorptive properties of heterocycle-containing analogs of risedronate, a pyridylbisphosphonate, in cultures of mouse fetal bone explants. Our studies indicated that hydroxybisphosphonates with the nitrogen molecule in the pyridyl ring were very potent inhibitors of osteoclastic resorption. Changing the place of the nitrogen in the ring structure of risedronate or its methylation did not significantly alter antiresorptive potency in relation to risedronate. Extension of the R2 chain, however, reduced efficacy. In binding experiments, we found that all heterocyclic bisphosphonates with a hydroxyl group in R1 had comparable affinity for bone mineral and inhibited calcium incorporation into bone explants to a similar extent. The affinity of a risedronate analog without R1 was markedly reduced. We also examined the properties of a risedronate analog (NE-10790) belonging to the group of phosphonocarboxylates in which one of the phosphonate groups is substituted by a carboxyl group. NE-10790 had strongly reduced binding affinity, but still retained some antiresorptive activity. Interestingly, the continuous presence of NE-10790 in cultures of fetal mouse metacarpal bones increased its antiresorptive efficacy by about 40-fold compared with 24 h preincubation, whereas, under the same conditions, the potency of high-affinity hydroxybisphosphonates did not change or only slightly increased. This may be explained by the differences in pharmacokinetic behavior between compounds of high and of low affinity for bone mineral. These data show that, as with alkylbisphosphonates, heterocycle-containing bisphosphonates with a nitrogen functionality in the R2 chain are potent antiresorptive agents and a hydroxyl substitution in the R1 chain confers high affinity for bone mineral, probably due to tridentate configuration. The group of phosphonocarboxylates, with strongly reduced bone affinity, provides an interesting therapeutic option.

Alendronate↗

PubMed1998

Role of integrins in the attachment of metastatic follicular thyroid carcinoma cell lines to bone.

One of the principle targets for metastasis of follicular thyroid carcinoma (FTC) is the skeleton. Because no data are available on the role of the integrin adhesion molecule family in the attachment of FTC to bone, we studied the attachment characteristics of three FTC cell lines to bone and the role of integrins. Three cell lines were used from the same patient, one (FTC-133) from the primary tumor and two (FTC-236 and FTC-238) from metastases. Attachment of FTC cell lines to bone was assessed on conditioned medium of an osteoblastic cell line, coated onto plastic, as an in vitro model of bone matrix. The synthetic RGD (Arg-Gly-Asp) peptide GRGDS impaired attachment of the FTC cell lines to bone matrix, demonstrating the role of integrins in the attachment of FTC to bone. Attachment of FTC-133 to bone matrix was blocked completely by GRGDS, whereas attachment of FTC-236 and FTC-238 could not be impaired completely. Semiquantitative polymerase chain reaction (PCR) of cDNA from the cell lines indicated stronger expression of alpha5 integrin mRNA in FTC-133 than in the other cell lines. In line with this, attachment of FTC-133 to bone matrix could be inhibited almost completely by anti alpha5 and beta1 integrin antibodies, indicating the importance of the fibronectin receptor in the attachment of FTC-133 to bone. Binding of FTC-236 and FTC-238 to bone matrix could not be inhibited completely by anti-integrin antibodies, suggesting an additional role of nonintegrin adhesion molecules in the attachment of FTC-236 and FTC-238 to bone. The synthetic bone sialoprotein cyclic peptide, CNB, revealed antiadhesive effects in the binding of FTC to bone. In conclusion, integrins play an important role in the attachment of metastatic FTC to bone. Differences in the functional involvement of integrins in the attachment to bone are observed between the three cell lines studied. From the present results, antiadhesive interventions with synthetic RGD peptides in FTC may be designed.

Adenocarcinoma, Follicular↗

PubMed1998

IL-1alpha, IL-1beta, IL-6, and TNF-alpha steady-state mRNA levels analyzed by reverse transcription-competitive PCR in bone marrow of gonadectomized mice.

Loss of gonadal function in both females and males is associated with increased rates of bone loss by a yet unidentified mechanism. There is ample evidence that cytokines that are produced in the bone microenvironment and stimulate the activity and/or formation of osteoclasts are involved. In the present study, we examined whether gonadectomy increases cytokine production via increased transcription in the bone marrow of mice. For this, the in vivo steady-state mRNA levels of multiple cytokines were determined in the central bone marrow compartment of mice at different time points following ovariectomy or orchidectomy by reverse transcription-competitive polymerase chain reaction. The limit of detectable differences in mRNA expression was approximately 2-fold. Bone marrow mRNA levels of the cytokines interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) were elevated up to 30-fold after treatment of mice with lipopolysaccharide. Following gonadectomy, there were no differences in the mRNA expression of these cytokines in bone marrow of female and male mice 4, 7, and 14 days after surgery. Gender steroid deficiency does not, therefore, increase steady-state mRNA levels of IL-1alpha, IL-1beta, IL-6, and TNF-alpha in cells of the central bone marrow compartment in mice. If changes have occurred these should have been less than 2-fold or in a small cell population. These results do not preclude an important role of these cytokines in the induction of bone loss after gonadectomy. For example, bone marrow cells situated close to the bone surface or bone cells may be responsible for increased cytokine synthesis. Alternatively, the loss of gender steroids may alter post-transcriptional events in cytokine synthesis and activity or may modify the responsiveness of target cells.

Animals↗

PubMed1998

Differential effects of aminosubstituted analogs of hydroxy bisphosphonates on the growth of Dictyostelium discoideum.

Replacing the hydroxyl group in the bone-binding site of three clinically useful bisphosphonates (etidronate, pamidronate, and olpadronate) by an amino group resulted in great differences in their antiresorptive potencies in vitro. In the present study, this is also shown in vivo in mice treated with the six bisphosphonates at doses of up to 16 microM/kg/day for 12 days. Because binding to bone mineral is nearly the same for all tested bisphosphonates, these findings suggest that the aminosubstitution affects the cellular action of the bisphosphonates. This was tested in the cellular slime mould Dictyostelium discoideum in which cellular effects of bisphosphonates can be examined independently of binding to bone mineral. Etidronate and its aminosubstituted analog were equipotent in inhibiting amebal growth, while pamidronate was somewhat more potent than its analog. Whereas olpadronate was a potent inhibitor of axenic growth of Dictyostelium amebae, the aminosubstitution reduced its potency drastically (IC50 12 microM and 700 microM, respectively). The similarities between the inhibitory effects of the bisphosphonates tested on bone resorption in vitro and in vivo and on the growth of Dictyostelium amebae confirm that the differences in antiresorptive potencies found reflect differences in cellular effects and suggest that bisphosphonates may bind to more than one intracellular target.

Amines↗

PubMed1998

Plasma nitrate+nitrite levels are regulated by ovarian steroids but do not correlate with trabecular bone mineral density in rats.

Nitric oxide (NO) is a mediator of bone metabolism and its production is under the control of gender hormones in several cell types or tissues. Changes in endogenous NO production, measured as plasma nitrate+nitrite levels, may therefore contribute to ovariectomy (OVX)-induced bone loss. We studied plasma nitrate+nitrite levels and trabecular bone mineral density (TBMD) 4 weeks after sham-operation or OVX in rats receiving various hormonal treatments. OVX decreased plasma nitrate+nitrite levels significantly and this was accompanied by a significant decrease in TBMD. Treatment with oral ethinyl oestradiol (EE) and subcutaneous 17beta-oestradiol dose-dependently prevented the decrease in plasma nitrate+nitrite levels after OVX, but treatment with oral 17beta-oestradiol did not. Oestrogen treatment, 17beta-oestradiol (s. c. or orally) or EE (orally), prevented the OVX-induced decrease in TBMD. Treatment of sham-operated rats with the anti-oestrogen ICI164, 384 induced a significant decrease in TBMD that corresponded to 54% of the decrease observed after OVX, but did not affect plasma nitrate+nitrite levels. Treatment of ovariectomized rats with Org 2058, a pure progestagen, did not prevent bone loss, but prevented the decrease in plasma nitrate+nitrite levels dose-dependently. Treatment with tibolone, a synthetic steroid with combined weak oestrogenic, progestagenic, and androgenic properties, or with progestagen in combination with EE completely prevented bone loss after OVX. These treatments, however, only partly prevented the OVX-induced decrease in plasma nitrate+nitrite levels. In conclusion, OVX decreased both TBMD and plasma nitrate+nitrite levels. Although plasma nitrate+nitrite levels were under the control of both oestrogen and progesterone, TBMD was affected by oestrogen only. Decreased systemic production of NO is, therefore, not involved in OVX-induced bone loss in rats.

Anabolic Agents↗

PubMed1998

Role of astrocyte-derived tissue-type plasminogen activator in the regulation of endotoxin-stimulated nitric oxide production by microglial cells.

In mixed glial cell cultures from cerebral cortices of newborn rats, endotoxin induces nitric oxide (NO) production in microglial cells. Earlier we demonstrated that endotoxin induced NO production by microglial cells is inhibited in the presence of astroglial cells by transforming growth factor beta (TGFbeta). Both microglial and astroglial cells produce TGFbeta in a biologically inactive form, which can be activated by plasmin generated by plasminogen activators (PA). In the present paper we describe studies on the mechanism by which glial cells may activate inactive TGFbeta and its potential inhibitory effect on NO production by microglial cells. Inhibition of plasmin increased NO production in endotoxin-treated mixed glial cell cultures. Subsequently, antibodies against tissue-type plasminogen activator (tPA) increased NO production in endotoxin-treated mixed glial cell cultures while amiloride, an inhibitor for urokinase (uPA), had no effect. We hereby concluded that tPA is the crucial PA involved in plasmin production resulting in inhibition of NO production in mixed glial cell cultures. Zymography and Northern blot analysis of purified astroglial, microglial, and mixed glial cell cultures demonstrated that astroglial cells produce tPA and a plasminogen activator inhibitor (PAI-1) and are thereby responsible for the production of plasmin which may activate the inactive TGF in these cultures. In conclusion, astroglial-derived tPA plays a major role in the inhibition of NO production by endotoxin-treated microglial cells through enhanced plasmin production and possible subsequent TGFbeta activation.

Amiloride↗