| 1. |
- Andersson, Martin K, et al.
(author)
-
Effects on osteoclast and osteoblast activities in cultured mouse calvarial bones by synovial fluids from patients with a loose joint prosthesis and from osteoarthritis patients.
- 2007
-
In: Arthritis research & therapy. - : Springer Science and Business Media LLC. - 1478-6362 .- 1478-6354. ; 9:1
-
Journal article (peer-reviewed)abstract
- Aseptic loosening of a joint prosthesis is associated with remodelling of bone tissue in the vicinity of the prosthesis. In the present study, we investigated the effects of synovial fluid (SF) from patients with a loose prosthetic component and periprosthetic osteolysis on osteoclast and osteoblast activities in vitro and made comparisons with the effects of SF from patients with osteoarthritis (OA). Bone resorption was assessed by the release of calcium 45 (45Ca) from cultured calvariae. The mRNA expression in calvarial bones of molecules known to be involved in osteoclast and osteoblast differentiation was assessed using semi-quantitative reverse transcription-polymerase chain reaction (PCR) and real-time PCR. SFs from patients with a loose joint prosthesis and patients with OA, but not SFs from healthy subjects, significantly enhanced 45Ca release, effects associated with increased mRNA expression of calcitonin receptor and tartrate-resistant acid phosphatase. The mRNA expression of receptor activator of nuclear factor-kappa-B ligand (rankl) and osteoprotegerin (opg) was enhanced by SFs from both patient categories. The mRNA expressions of nfat2 (nuclear factor of activated T cells 2) and oscar (osteoclast-associated receptor) were enhanced only by SFs from patients with OA, whereas the mRNA expressions of dap12 (DNAX-activating protein 12) and fcrgamma (Fc receptor common gamma subunit) were not affected by either of the two SF types. Bone resorption induced by SFs was inhibited by addition of OPG. Antibodies neutralising interleukin (IL)-1alpha, IL-1beta, soluble IL-6 receptor, IL-17, or tumour necrosis factor-alpha, when added to individual SFs, only occasionally decreased the bone-resorbing activity. The mRNA expression of alkaline phosphatase and osteocalcin was increased by SFs from patients with OA, whereas only osteocalcin mRNA was increased by SFs from patients with a loose prosthesis. Our findings demonstrate the presence of a factor (or factors) stimulating both osteoclast and osteoblast activities in SFs from patients with a loose joint prosthesis and periprosthetic osteolysis as well as in SFs from patients with OA. SF-induced bone resorption was dependent on activation of the RANKL/RANK/OPG pathway. The bone-resorbing activity could not be attributed solely to any of the known pro-inflammatory cytokines, well known to stimulate bone resorption, or to RANKL or prostaglandin E2 in SFs. The data indicate that SFs from patients with a loose prosthesis or with OA stimulate bone resorption and that SFs from patients with OA are more prone to enhance bone formation.
|
|
| 2. |
- Belibasakis, G N, et al.
(author)
-
Cytokine responses of human gingival fibroblasts to Actinobacillus actinomycetemcomitans cytolethal distending toxin.
- 2005
-
In: Cytokine. - : Elsevier BV. - 1043-4666 .- 1096-0023. ; 30:2, s. 56-63
-
Journal article (peer-reviewed)abstract
- Actinobacillus actinomycetemcomitans is implicated in the pathogenesis of localized aggressive periodontitis, and has the capacity to express a cytolethal distending toxin (Cdt). Gingival fibroblasts (GF) are resident cells of the periodontium, which can express several osteolytic cytokines. The aims of this study were a) to investigate the role of Cdt in A. actinomycetemcomitans-induced expression of osteolytic cytokines and their cognate receptors in GF and b) to determine if the previously demonstrated induction of receptor activator of NFkappaB ligand (RANKL) by A. actinomycetemcomitans is mediated by these pro-inflammatory cytokines or by prostaglandin E(2) (PGE(2)). A. actinomycetemcomitans clearly induced interleukin (IL)-6, IL-1beta, and to a minimal extent, tumor necrosis factor (TNF)-alpha mRNA expression. At the protein level, IL-6 but not IL-1beta or TNF-alpha expression was stimulated. The mRNA expression of the different receptor subtypes recognizing IL-6, IL-1beta and TNF-alpha was not affected. A cdt-knockout strain of A. actinomycetemcomitans had similar effects on cytokine and cytokine receptor mRNA expression, compared to its parental wild-type strain. Purified Cdt stimulated IL-6, but not IL-1beta or TNF-alpha protein biosynthesis. Antibodies neutralizing IL-6, IL-1 or TNF-alpha, and the PGE(2) synthesis inhibitor indomethacin, did not affect A. actinomycetemcomitans-induced RANKL expression. In conclusion, a) A. actinomycetemcomitans induces IL-6 production in GF by a mechanism largely independent of its Cdt and b) A. actinomycetemcomitans-induced RANKL expression in GF occurs independently of IL-1, IL-6, TNF-alpha, or PGE(2).
|
|
| 3. |
- Belibasakis, G N, et al.
(author)
-
The cytolethal distending toxin induces receptor activator of NF-kappaB ligand expression in human gingival fibroblasts and periodontal ligament cells.
- 2005
-
In: Infection and immunity. - 0019-9567 .- 1098-5522. ; 73:1, s. 342-51
-
Journal article (peer-reviewed)abstract
- Actinobacillus actinomycetemcomitans is associated with localized aggressive periodontitis, a disease characterized by rapid loss of the alveolar bone surrounding the teeth. Receptor activator of NF-kappaB Ligand (RANKL) and osteoprotegerin (OPG) are two molecules that regulate osteoclast formation and bone resorption. RANKL induces osteoclast differentiation and activation, whereas OPG blocks this process by acting as a decoy receptor for RANKL. The purpose of this study was to investigate the effect of A. actinomycetemcomitans on the expression of RANKL and OPG in human gingival fibroblasts and periodontal ligament cells. RANKL mRNA expression was induced in both cell types challenged by A. actinomycetemcomitans extract, whereas OPG mRNA expression remained unaffected. Cell surface RANKL protein was also induced by A. actinomycetemcomitans, whereas there was no change in OPG protein secretion. A cytolethal distending toxin (Cdt) gene-knockout strain of A. actinomycetemcomitans did not induce RANKL expression, in contrast to its wild-type strain. Purified Cdt from Haemophilus ducreyi alone, or in combination with extract from the A. actinomycetemcomitans cdt mutant strain, induced RANKL expression. Pretreatment of A. actinomycetemcomitans wild-type extract with Cdt antiserum abolished RANKL expression. In conclusion, A. actinomycetemcomitans induces RANKL expression in periodontal connective tissue cells. Cdt is crucial for this induction and may therefore be involved in the pathological bone resorption during the process of localized aggressive periodontitis.
|
|
| 4. |
- Bernhold Brechter, Anna, 1971-
(author)
-
Kinins : important regulators in inflammation induced bone resorption
- 2006
-
Doctoral thesis (other academic/artistic)abstract
- Inflammatory processes in, or in close vicinity of, the skeleton often lead to loss of bone tissue. Different cytokines have been shown to be involved as stimulators of inflammatory induced osteoclastic bone resorption. During inflammatory processes also the kallikrein-kinin system is activated, leading to production of kinins that can cause pain, vasodilation and increased permeability of vessels. Kinins can also induce bone resorption in vitro. All cytokines and kinins that stimulate bone resorption stimulate in parallell prostaglandin synthesis, and prostaglandins, per se, have also been shown to induce bone resorption. The aim of this project was to increase the knowledge about the mechanisms involved in the interactions between different inflammatory mediators (i.e. kinins, cytokines and prostaglandins) suggested to be involved in the pathogenesis of inflammatory bone resorbing diseases. Human osteoblasts (MG-63) are equipped with both kinin B1 and B2 receptors linked to prostaglandin release and the stimulation of prostaglandin release are likely mediated via separate molecular mechanisms (Paper I). Activation of B1 or B2 receptors causes synergistic stimulation of PGE2 synthesis induced by either interleukin-1b (IL-1b) or tumour necrosis factor-a (TNF-a) (Paper II). The molecular mechanism involves increased expression of cyclooxygenase-2 (COX-2) and results in synergistic potentiation of receptor activator of NF-kB ligand (RANKL) protein expression. The synergistic interaction is dependent on the activation of NF-kB and the mitogen-activated protein kinases (MAPK) p38 and JNK (Paper II). The synergistic increase in RANKL expression might be an explanation why kinins potentiate IL-1b induced bone resorption, a mechanism likely to be important in inflammation induced bone resorption in diseases such as periodontal disease and rheumatoid arthritis. The synergism between kinins and IL-1b or TNF-a might also be dependent on regulation of kinin receptors, since both IL-1b and TNF-a markedly upregulated B1 and B2 receptors, both at the mRNA level and protein level (Paper III). This upregulation is not further potentiated by the kinins, and different kinin receptor agonists do not regulate the receptors for IL-1b or TNF-a, in MG-63 cells. No other cytokines known to stimulate bone resorption regulates the expressions of B1 and B2 receptors. The IL-1b- or TNF-a-induced enhancements of B1 and B2 receptor expressions involve activation of NF-kB and MAPK. The enhancement of kinin receptors may also be an important mechanism in the synergistic interactions between the two pro-inflammatory cytokines and kinins (paper III). IL-4 and IL-13 are two cytokines that have been shown to inhibit bone resorption. We have shown that COX-2 and both B1 and B2 receptors are down-regulated by IL-4 and IL-13, via a ‘signal transducer and activator of transcription6’ (STAT6) dependent pathway, which might be an important regulatory mechanism in inflammation induced bone resorption (paper IV). In conclusion, the mechanisms behind the synergistic potentiation of prostaglandin formation and increased bone resorption caused by co-stimulation with kinins and IL-1b or TNF-a seem to involve both potentiation of COX-2 and subsequently increased levels of RANKL, as well as upregulation of B1 and B2 kinin receptors. Interestingly, IL-4 and IL-13 decreased the expressions of COX-2 and both B1 and B2 receptors. These events might be important in the regulation of inflammation induced bone resorption in diseases such as periodontitis and rheumatoid arthritis.
|
|
| 5. |
- Brage, M, et al.
(author)
-
Different cysteine proteinases involved in bone resorption and osteoclast formation.
- 2005
-
In: Calcified tissue international. - : Springer Science and Business Media LLC. - 0171-967X .- 1432-0827. ; 76:6, s. 439-47
-
Journal article (peer-reviewed)abstract
- Cysteine proteinases, especially cathepsin K, play an important role in osteoclastic degradation of bone matrix proteins and the process can, consequently, be significantly inhibited by cysteine proteinase inhibitors. We have recently reported that cystatin C and other cysteine proteinase inhibitors also reduce osteoclast formation. However, it is not known which cysteine proteinase(s) are involved in osteoclast differentiation. In the present study, we compared the relative potencies of cystatins C and D as inhibitors of bone resorption in cultured mouse calvariae, osteoclastogenesis in mouse bone marrow cultures, and cathepsin K activity. Inhibition of cathepsin K activity was assessed by determining equilibrium constants for inhibitor complexes in fluorogenic substrate assays. The data demonstrate that whereas human cystatins C and D are equipotent as inhibitors of bone resorption, cystatin D is 10-fold less potent as an inhibitor of osteoclastogenesis and 200-fold less potent as an inhibitor of cathepsin K activity. A recombinant human cystatin C variant with Gly substitutions for residues Arg8, Leu9, Val10, and Trp106 did not inhibit bone resorption, had 1,000-fold decreased inhibitory effect on cathepsin K activity compared to wildtype cystatin C, but was equipotent with wildtype cystatin C as an inhibitor of osteoclastogenesis. It is concluded that (i) different cysteine proteinases are likely to be involved in bone resorption and osteoclast formation, (ii) cathepsin K may not be an exclusive target enzyme in any of the two systems, and (iii) the enzyme(s) involved in osteoclastogenesis might not be a typical papain-like cysteine proteinase.
|
|
| 6. |
- Brechter, Anna Bernhold, et al.
(author)
-
Kinin B1 and B2 receptor expression in osteoblasts and fibroblasts is enhanced by interleukin-1 and tumour necrosis factor-alpha. Effects dependent on activation of NF-kappaB and MAP kinases.
- 2008
-
In: Bone. - : Elsevier BV. - 8756-3282 .- 1873-2763. ; 43:1, s. 72-83
-
Journal article (peer-reviewed)abstract
- Pro-inflammatory mediators formed by the kallikrein-kinin system can stimulate bone resorption and synergistically potentiate bone resorption induced by IL-1 and TNF-alpha. We have shown that the effect is associated with synergistically enhanced RANKL expression and enhanced prostaglandin biosynthesis, due to increased cyclooxygenase-2 expression. In the present study, the effects of osteotropic cytokines and different kinins on the expression of receptor subtypes for bradykinin (BK), des-Arg10-Lys-BK (DALBK), IL-1beta and TNF-alpha have been investigated. IL-1beta and TNF-alpha enhanced kinin B1 and B2 receptor binding in the human osteoblastic cell line MG-63 and the mRNA expression of B1 and B2 receptors in MG-63 cells, human gingival fibroblasts and intact mouse calvarial bones. Kinins did not affect mRNA expression of IL-1 or TNF receptors. EMSA showed that IL-1beta and TNF-alpha activated NF-kappaB and AP-1 in MG-63 cells. IL-1beta stimulated NF-kappaB via a non-canonical pathway (p52/p65) and TNF-alpha via the canonical pathway (p50/p65). Activation of AP-1 involved c-Jun in both IL-1beta and TNF-alpha stimulated cells, but c-Fos only in TNF-alpha stimulated cells. Phospho-ELISA and Western blots showed that IL-1beta activated JNK and p38, but not ERK 1/2 MAP kinase. Pharmacological inhibitors showed that NF-kappaB, p38 and JNK were important for IL-1beta induced stimulation of B1 receptors, and NF-kappaB and p38 for B2 receptors. p38 and JNK were important for TNF-alpha induced stimulation of B1 receptors, whereas NF-kappaB, p38 and JNK were involved in TNF-alpha induced expression of B2 receptors. These data show that IL-1beta and TNF-alpha upregulate B1 and B2 receptor expression by mechanisms involving activation of both NF-kappaB and MAP kinase pathways, but that signal transduction pathways are different for IL-1beta and TNF-alpha. The enhanced kinin receptor expression induced by the pro-inflammatory cytokines IL-1beta and TNF-alpha might be one important mechanism involved in the synergistic enhancement of prostaglandin formation caused by co-treatment with kinins and one of the two cytokines. These mechanisms might help to explain the enhanced bone resorption associated with inflammatory disorders, including periodontitis and rheumatoid arthritis.
|
|
| 7. |
- Brechter, Anna, et al.
(author)
-
Bradykinin potentiates cytokine-induced prostaglandin biosynthesis in osteoblasts by enhanced expression of cyclooxygenase 2, resulting in increased RANKL expression.
- 2007
-
In: Arthritis and rheumatism. - : Wiley. - 0004-3591 .- 1529-0131. ; 56:3, s. 910-23
-
Journal article (peer-reviewed)abstract
- OBJECTIVE: Bradykinin (BK) stimulates bone resorption in vitro and synergistically potentiates interleukin-1 (IL-1)-induced bone resorption and prostaglandin (PG) formation, suggesting that kinins are important in inflammation-induced bone loss. The present study was undertaken to study 1) the role of the kinin B1 and B2 receptors in the synergistic interaction with IL-1 and tumor necrosis factor alpha (TNFalpha), 2) the molecular mechanisms involved in synergistic enhancement of PG formation, and 3) the effects of kinins on cytokine-induced expression of RANKL, RANK, and osteoprotegerin (OPG) (the latter being crucial molecules in osteoclast differentiation). METHODS: Formation of PGs, expression of enzymes involved in arachidonic acid metabolism, and expression of RANKL, RANK, and OPG were assessed in the human osteoblastic cell line MG-63 and in mouse calvarial bones. The role of NF-kappaB and MAP kinases was studied using pharmacologic inhibitors. RESULTS: PGE(2) formation and cyclooxygenase 2 (COX-2) protein expression were induced by IL-1beta and potentiated by kinins with affinity for the B1 or B2 receptors, resulting in PGE(2)-dependent enhancement of RANKL. The enhancements of PGE(2) formation and COX-2 were markedly decreased by inhibition of p38 and JNK MAP kinases, whereas inhibition of NF-kappaB resulted in abolishment of the PGE(2) response with only slight inhibition of COX-2. CONCLUSION: Kinin B1 and B2 receptors synergistically potentiate IL-1- and TNFalpha-induced PG biosynthesis in osteoblasts by a mechanism involving increased levels of COX-2, resulting in increased RANKL. The synergistic stimulation is dependent on NF-kappaB and MAP kinases. These mechanisms might help to explain the enhanced bone resorption associated with inflammatory disorders, including that in rheumatoid arthritis.
|
|
| 8. |
- Conaway, H Herschel, et al.
(author)
-
Retinoids inhibit differentiation of hematopoietic osteoclast progenitors.
- 2009
-
In: The FASEB journal. - Bethesda, Md. : Wiley. - 1530-6860 .- 0892-6638. ; 23:10, s. 3526-38
-
Journal article (peer-reviewed)abstract
- Whether vitamin A promotes skeletal fragility, has no effect on fracture rate, or protects against bone loss is unclear. In the present study, effects of retinoids on osteoclast differentiation in cultured mouse bone marrow cells (BMCs), bone marrow macrophages (BMMs), spleen cells, and RAW264.7 cells were evaluated by analyzing osteoclast formation and expression of genes important in signal transduction and osteoclast function. All-trans-retinoic acid (ATRA) did not stimulate osteoclastogenesis in BMCs, but inhibited hormone and RANKL-induced gene expression and formation of osteoclasts. In BMMs, spleen cells, and RAW264.7 cells, osteoclast differentiation and formation stimulated by M-CSF/RANKL were inhibited (IC(50) = 0.3 nM) by ATRA. The effect was exerted at an early step of RANKL-induced differentiation. ATRA also abolished increases of the transcription factors c-Fos and NFAT2 stimulated by RANKL and suppressed down-regulation of the antiosteoclastogenic transcription factor MafB. By comparing effects of several compounds structurally related to ATRA, as well as by using receptor antagonists, evaluation pointed to inhibition being mediated by RARalpha, with no involvement of PPARbeta/delta. The results suggest that activation of RARalpha by retinoids in myeloid hematopoietic precursor cells decreases osteoclast formation by altering expression of the transcription factors c-Fos, NFAT2, and MafB.
|
|
| 9. |
- Granholm, Susanne, et al.
(author)
-
Calcitonin inhibits osteoclast formation in mouse haematopoetic cells independently of transcriptional regulation by receptor activator of NF-{kappa}B and c-Fms.
- 2007
-
In: The Journal of endocrinology. - 1479-6805. ; 195:3, s. 415-27
-
Journal article (peer-reviewed)abstract
- The effects of calcitonin (CT) on osteoclast formation and gene expression have been studied in cultured mouse spleen cells and mouse bone marrow macrophages (BMMs). CT inhibited the formation of multinucleated osteoclasts and resorption pits in spleen cell cultures and BMM as well as in CD115(+) CD3(-) CD45R(-)sorted BMM cultures, incubated in the presence of macrophage colony-stimulating factor and receptor activator of NF-kappaB ligand (RANKL). No effect on apoptosis by CT was observed. CT did not affect the mRNA expressions of RANK and c-Fms, or the mRNA expressions of a wide variety of transcription factors and genes important for osteoclast differentiation and activity. CT induced inhibition of tartrate-resistant acid phosphatase (TRAP), positive multinucleated osteoclast formation was not associated with any decrease of total TRAP activity, resulting in a large number of TRAP(+) mononucleated cells in CT-treated cultures. CT did not affect the mRNA expression of dendritic cell-specific transmembrane protein, d2 isoform of vacuolar (H(+)) ATPase v(o) domain, a disintegrin and metalloproteinase domain 8 (ADAM8), ADAM12, DNAX-activating protein or Fc receptor common gamma chain suggested to be involved in fusion of mononucleated osteoclast progenitor cells. The inhibitory effect by CT was mimicked not only by compounds activating cAMP and protein kinase A (PKA) but also by a cAMP analogue activating the exchange protein directly activated by cAMP (Epac) pathway. It is concluded that CT, through cAMP/PKA/Epac cascades, inhibits osteoclast formation and that this effect is not associated with decreased transcription of genes known to be important for osteoclast progenitor cell differentiation, fusion or function.
|
|
| 10. |
|
|
