RESUMEN
OBJECTIVE: Enamel matrix derivative (EMD) is widely used under the brand name Emdogain® to promote periodontal regeneration in surgical treatment of periodontitis and peri-implantitis. The molecular mechanisms are unclear, but it has been proposed that EMD has stimulatory effects on the root cementum and periodontal ligament cells. Since dental implants lack these structures, we hypothesized that EMD-induced bone gain involve interactions with osteoclast precursor cells, with consequent inhibitory effect on osteoclast formation and/or activity. The aim was to evaluate this hypothesis. MATERIAL AND METHODS: Primary mouse bone marrow macrophages (BMMs) and human peripheral blood monocytes were cultured in the presence of receptor activator nuclear factor-κB ligand (RANKL) to stimulate osteoclast formation. A purified Emdogain® fraction was added to the cell cultures and the effect on number and size of newly formed osteoclasts were evaluated. In cultures on natural bone slices, bioanalytical methods were used to assay osteoclast number and bone resorption. RESULTS: EMD had a negative effect on osteoclastogenesis in mouse cultures on plastic surface, whereas addition of EMD to osteoclast precursor cells on bone substrate did not affect osteoclast formation or bone resorption. CONCLUSIONS: The results on natural bone matrix contradict a direct effect of EMD on osteoclast precursor cells.
Asunto(s)
Resorción Ósea , Implantes Dentales , Animales , Médula Ósea/metabolismo , Diferenciación Celular , Humanos , Ligandos , Macrófagos/metabolismo , Ratones , Monocitos/metabolismo , FN-kappa B/metabolismo , FN-kappa B/farmacología , Osteoclastos , Plásticos/metabolismo , Plásticos/farmacología , Ligando RANK/metabolismoRESUMEN
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.
Asunto(s)
Hematopoyesis/efectos de los fármacos , Células Progenitoras Mieloides/efectos de los fármacos , Osteoclastos/citología , Tretinoina/farmacología , Animales , Línea Celular , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Ratones , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/fisiología , Ligando RANK/metabolismo , Receptores de Ácido Retinoico/agonistas , Receptores de Ácido Retinoico/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Glucocorticoid (GC) therapy is the greatest risk factor for secondary osteoporosis. Pathogenic mechanisms involve an initial increase in bone resorption followed by decreased bone formation. To gain a better understanding of the resorptive activity of GCs, we have used mouse bone marrow macrophages (BMM) to determine if GCs can directly modulate RANKL stimulated osteoclast formation and/or activity. In agreement with previous studies, experiments performed in plastic wells showed that GCs (dexamethasone, hydrocortisone, and prednisolone) inhibited osteoclast number and size during the initial phases of RANKL stimulated osteoclastogenesis; however, in prolonged cultures, decreased apoptosis was observed and escape from GC induced inhibition occurred with an enhanced number of osteoclasts formed, many with an increased area. When BMM cells were seeded on bone slices, GCs robustly enhanced RANKL stimulated formation of resorption pits and release of CTX without affecting the number or size of osteoclasts formed and with no effect on apoptosis. Stimulation of pit formation was not associated with increased life span of osteoclasts or an effect on mRNA expression of several osteoclastic or osteoclastogenic genes. The potentiation of RANKL induced CTX release by dexamethasone was significantly less in BMM cells from mice with conditional knockout of the osteoclastic glucocorticoid receptor and completely absent in cells from GRdim mice, which carry a point mutation in one dimerizing interface of the GC receptor. These data suggest that: 1. Plastic is a poor medium to use for studying direct effects of GCs on osteoclasts 2. GCs can enhance bone resorption without decreasing apoptosis, and 3. A direct enhancement of RANKL mediated resorption is stimulated by the dimeric GC-receptor.
Asunto(s)
Resorción Ósea/metabolismo , Resorción Ósea/patología , Diferenciación Celular , Osteoclastos/metabolismo , Multimerización de Proteína , Ligando RANK/farmacología , Receptores de Glucocorticoides/metabolismo , Células Madre/metabolismo , Animales , Biomarcadores/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células Gigantes/efectos de los fármacos , Células Gigantes/metabolismo , Células Gigantes/patología , Glucocorticoides/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Péptidos/metabolismo , Células Madre/efectos de los fármacosRESUMEN
The effects of cystatin C and other cysteine proteinase inhibitors on osteoclast formation and differentiation have been investigated. Cystatin C decreased osteoclast formation stimulated by parathyroid hormone (PTH), 1,25(OH)2-vitamin D3 or interleukin-6 (IL-6) (in the presence of its soluble receptor) as assessed by the number of tartrate-resistant acid phosphatase (TRAP+) multinucleated cells in mouse bone marrow cultures. The inhibitory effect was associated with decreased mRNA expression for the calcitonin receptor as well as decreased number of specific binding sites for 125I-calcitonin, and without any effect on the mRNA expression of receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL). Similarly, the cysteine proteinase inhibitors leupeptin, E-64 and benzyloxycarbonyl-Phe-Ala-diazomethane (Z-FA-CHN2) decreased PTH-stimulated formation of TRAP+ multinucleated cells and binding of 125I-calcitonin. A peptidyl derivative synthesized to mimic part of the proteinase-binding site of cystatin C (benzyloxycarbonyl-Arg-Leu-Val-Gly-diazomethane, or Z-RLVG-CHN2) also decreased PTH-stimulated osteoclast formation. In a 9-day culture, addition of cystatin C during the last 5 days was sufficient to cause substantial inhibition of osteoclast formation. Cystatin C-induced decrease of osteoclast formation was associated with enhanced number of F4/80-positive macrophages and increased mRNA expression of the macrophage receptor c-fms in the bone marrow culture. Osteoclast formation in mouse bone marrow cultures as well as in mouse spleen cell cultures, stimulated by macrophage colony-stimulating factor (M-CSF) and RANKL was also decreased by different cysteine proteinase inhibitors. In addition, cystatin C inhibited M-CSF/RANKL induction of calcitonin receptor mRNA in spleen cell cultures. The inhibitory effect by cystatin C in spleen cells was associated with decreased mRNA expression of RANK and the transcription factor NFAT2. It is concluded that cysteine proteinase inhibitors decrease formation of osteoclasts by interfering at a late stage of pre-osteoclast differentiation.
Asunto(s)
Inhibidores de Cisteína Proteinasa/farmacología , Osteoclastos/citología , Osteoclastos/enzimología , Animales , Bovinos , Células Cultivadas , Cistatina C , Cistatinas/farmacología , Cisteína Endopeptidasas/metabolismo , Relación Dosis-Respuesta a Droga , Glicoproteínas/biosíntesis , Humanos , Masculino , Ratones , Osteoclastos/efectos de los fármacos , Osteoprotegerina , Hormona Paratiroidea/farmacología , Receptores Citoplasmáticos y Nucleares/biosíntesis , Receptores del Factor de Necrosis TumoralRESUMEN
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.
Asunto(s)
Osteoartritis/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Falla de Prótesis , Cráneo/metabolismo , Líquido Sinovial/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Bovinos , Células Cultivadas , Humanos , Prótesis Articulares , Ratones , Osteoartritis/patología , Osteoblastos/citología , Osteoclastos/citología , Osteogénesis/fisiología , Cráneo/citología , Líquido Sinovial/citologíaRESUMEN
Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant > or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures stimulated by parathyroid hormone (PTH; 10 nM) or forskolin (FSK; 1 microM). Furthermore, CBX (100 microM) significantly inhibited prostaglandin E2 (PGE2; 10 microM) and 1,25(OH)2-vitamin D3 stimulated osteoclast differentiation in the mouse bone marrow cultures. Consequently, quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that CBX downregulated the expression of osteoclast phenotypic markers, but without having any significant effects on RANK, RANKL, and osteoprotegerin (OPG) mRNA expression. However, the results demonstrated that CBX significantly inhibits RANKL-stimulated (100 ng/ml) osteoclastogenesis in the mouse bone marrow cultures. Taken together, our results suggests that gap junctional diffusion of messenger molecules interacts with signaling pathways downstream RANKL in osteoclast differentiation. Further studies are required to define the precise mechanisms and molecular targets involved.
Asunto(s)
Uniones Comunicantes/metabolismo , Osteoclastos/metabolismo , Animales , Secuencia de Bases , Resorción Ósea/metabolismo , Calcitriol/farmacología , Carbenoxolona/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Comunicación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Colforsina/farmacología , Conexina 43/genética , Conexina 43/metabolismo , ADN Complementario/genética , Dinoprostona/farmacología , Uniones Comunicantes/efectos de los fármacos , Glicoproteínas/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoprotegerina , Hormona Paratiroidea/farmacología , Ligando RANK , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Activador del Factor Nuclear kappa-B , Receptores Citoplasmáticos y Nucleares/genética , Receptores del Factor de Necrosis Tumoral/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Interleukin (IL)-4 and IL-13 are cytokines that inhibit bone resorption. Data showing an inhibitory effect of IL-4 and IL-13 on RANK mRNA in mouse calvariae were first reported at the 22nd American Society for Bone and Mineral Research Meeting (Lerner, U.H., and Conaway, H. H. 2000) J. Bone Min. Res. 15, Suppl. 1, Abstr. SU 230). In the present study, release of 45Ca from cultured mouse calvarial bones stimulated by different cytokines, peptides, and steroid hormones was inhibited by IL-4 and IL-13. IL-4 and IL-13 decreased receptor activator of nuclear factor-kappaB ligand (RANKL) and RANK mRNA and increased osteoprotegerin (OPG) mRNA in calvariae. Additionally, the cytokines decreased RANKL protein and increased OPG protein in calvarial bones. In osteoblasts isolated from calvariae, both an increase in RANKL mRNA and a decrease in OPG mRNA and protein elicited by vitamin D3 were reversed by IL-4 and IL-13. IL-4 and IL-13 decreased the number of tartrate-resistant acid phosphatase positive multinucleated cells and the mRNA expression of calcitonin receptor, tartrate-resistant acid phosphatase, and cathepsin K in mouse spleen cells and bone marrow macrophages (BMM) treated with macrophage colony-stimulating factor and RANKL. Inhibition of mRNA for RANK and the transcription factor NFAT2 was also noted in spleen cell and BMM cultures treated with IL-4 and IL-13. In addition, RANK mRNA and RANK protein were decreased by IL-4 and IL-13 in RAW 264.7 cells. Osteoblasts, spleen cells, and BMM expressed mRNA for the four proteins making up the IL-4 and IL-13 receptors. No effects by IL-4 on bone resorption and osteoclast formation or on RANKL and RANK mRNA expression were seen in Stat6-/- mice. The data indicate that IL-4 and IL-13, via a STAT6-dependent pathway, inhibit osteoclast differentiation and bone resorption by activating receptors on osteoblasts and osteoclasts that affect the RANKL/RANK/OPG system.
Asunto(s)
Resorción Ósea/inducido químicamente , Proteínas Portadoras/genética , Glicoproteínas/genética , Interleucina-13/farmacología , Interleucina-4/farmacología , Glicoproteínas de Membrana/genética , Osteoclastos/citología , Receptores Citoplasmáticos y Nucleares/genética , Receptores del Factor de Necrosis Tumoral/genética , Factor de Transcripción STAT6/metabolismo , Animales , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Citocinas/farmacología , Glicoproteínas/análisis , Hormonas/farmacología , Ratones , Ratones Mutantes , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Osteoprotegerina , Ligando RANK , ARN Mensajero/análisis , Receptor Activador del Factor Nuclear kappa-B , Receptores Citoplasmáticos y Nucleares/análisis , Receptores del Factor de Necrosis Tumoral/análisis , CráneoRESUMEN
Several studies have demonstrated that connexin 43 (Cx43) mediates signals important for osteoblast function and osteogenesis. The role of gap junctional communication in bone resorption is less clear. We have investigated the expression of Cx43 mRNA in osteoclasts and bone resorption cultures and furthermore, the functional importance of gap junctional communication in bone resorption. RT-PCR analysis demonstrated Cx43 mRNA expression in mouse bone marrow cultures and in osteoclasts microisolated from the marrow cultures. Cx43 mRNA was also expressed in bone resorption cultures with osteoclasts and osteoblasts/stromal cells incubated for 48h on devitalized bone slices. An up-regulation of Cx43 mRNA was detected in parathyroid (PTH)-stimulated (0.1 nM) bone resorption. Two inhibitors of gap junction communication, 18alpha-glycyrrhetinic acid (30 microM) and oleamide (100 microM), significantly inhibited PTH- and 1,25-(OH)(2)D(3)-stimulated osteoclastic pit formation. In conclusion, our data indicate a functional role for gap junction communication in bone resorption.