RESUMEN
INTRODUCTION: Prostaglandins (PGs) can act on both hematopoietic and osteoblastic lineages to enhance osteoclast formation. METHODS: We examined PGE2 stimulated osteoclastogenesis in RAW 264.7 cells and the role of endogenous PGE2 in lipopolysaccharide (LPS) stimulated osteoclastogenesis. RESULTS: RANKL (1-100 ng/ml) increased formation of osteoclasts, defined as tartrate resistant acid phosphatase multinucleated cells, with peak effects at 30 ng/ml. Addition of PGE2 (0.01-1.0 microM) to RANKL (30 ng/ml) dose dependently increased osteoclast number 30-150%. Use of NS-398 (0.1 microM) or indomethacin (Indo, 1.0 micro M) to block endogenous PG synthesis had little effect on the response to RANKL alone but significantly decreased the response to PGE2. Addition of LPS (100 ng/ml) to RANKL increased osteoclast number 50%, and this response was significantly decreased by NS-398 and Indo. RANKL and PGE2 produced small, additive increases in COX-2 mRNA levels, while LPS produced a larger increase. PG release into the medium was not increased by RANKL and PGE2 but markedly increased by LPS. CONCLUSION: We conclude that RANKL stimulated osteoclastogenesis can be enhanced by PGE2 and LPS though direct effects on the hematopoietic cell lineage and that these effects may be mediated in part by induction of COX-2 and enhanced intracellular PG production.
Asunto(s)
Dinoprostona/farmacología , Lipopolisacáridos/farmacología , Osteoclastos/efectos de los fármacos , Animales , Línea Celular Tumoral , Ratones , Nitrobencenos/farmacología , Prostaglandinas F Sintéticas/farmacología , Ligando RANK/farmacología , Sulfonamidas/farmacologíaRESUMEN
We measured the effects of ovariectomy on the bone mass of mice that lacked type I interleukin-1 receptor (IL-I R1 -/- mice) in two genetic backgrounds (C57BL/6 x 129/Sv and C57BL/6) to investigate the role of interleukin-1 in the actions of estrogen on bone. At three weeks after surgery, ovariectomized wild-type mice decreased trabecular bone volume in the proximal humerus by 70% in a C57BL/6 x 129/Sv background and 48% in a C57BL/6 background compared to sham-operated controls. In contrast, there was no significant decrease in trabecular bone mass in IL-1 R1 -/- mice after ovariectomy. The estrogen status of all groups was confirmed by measurement of uterine wet weight. These results demonstrate that a functional IL-1 response pathway is required for mice to lose trabecular bone mass after ovariectomy in this model and they imply that IL-1 is an important mediator of the effects of ovariectomy on bone mass. Hence, therapeutic interventions that block the effects of IL-1 on bone may be beneficial for treating diseases of rapid bone loss such as post-menopausal osteoporosis.
Asunto(s)
Densidad Ósea , Ratones Noqueados/genética , Ovariectomía , Receptores de Interleucina-1/genética , Animales , Femenino , Húmero/metabolismo , Ratones , Tamaño de los Órganos , Periodo Posoperatorio , Valores de Referencia , Útero/anatomía & histologíaRESUMEN
Transforming growth factor-beta (TGFbeta) plays an important role in bone development and remodeling. TGFbeta stimulates PGE2 production, enhances interleukin-1-stimulated PGE2 production, and can stimulate PG-mediated bone resorption. We found that TGFbeta induced prostaglandin G/H synthase (PGHS-2) messenger RNA (mRNA) and PGE2 production in neonatal mouse calvarial cultures and in primary cells derived from these calvariae. We used MC3T3-E1 cells, an immortalized osteoblastic cell line derived from mouse calvariae, to examine the mechanism of PGHS-2 induction. PGHS-2 mRNA was rapidly induced by TGFbeta (10 ng/ml) in MC3T3-E1 cells; mRNA levels peaked at 4-8 h and were still elevated at 24 h. Induction of PGHS-2 protein and PGE2 production correlated with PGHS-2 mRNA levels. In contrast, TGFbeta had much less effect on PGHS-1 mRNA levels. Unlike the response to other agonists, PGHS-2 mRNA induction by TGFbeta was not enhanced by cycloheximide pretreatment, suggesting a requirement for new protein synthesis. To study transcriptional regulation, cells were stably transfected with a PGHS-2 promoter-luciferase reporter construct containing 371 bp of the 5'-flanking region and 70 bp of untranslated DNA from the PGHS-2 gene. TGFbeta-stimulated luciferase activity paralleled PGHS-2 mRNA induction. Stimulation of luciferase activity and PGHS-2 mRNA levels by other agonists, including interleukin-1, TGF alpha, forskolin, and phorbol 13-myristate 12-acetate, were enhanced by TGFbeta. A 90% drop in luciferase activity occurred with deletion of the region from -371 to -213 bp of the PGHS-2 promoter. The PG response to TGFbeta in MC3T3-E1 cells appears to be mediated primarily by transcriptional regulation of PGHS-2 expression through one or more cis-acting elements located between -371 and -213 bp in the 5'-flanking region of the PGHS-2 gene.
Asunto(s)
Isoenzimas/metabolismo , Osteoblastos/enzimología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Animales , Línea Celular , Mapeo Cromosómico , Cicloheximida/farmacología , Ciclooxigenasa 2 , Dinoprostona/biosíntesis , Amplificación de Genes , Humanos , Interleucina-1/farmacología , Isoenzimas/genética , Proteínas de la Membrana , Ratones , Ratones Endogámicos , Osteoblastos/fisiología , Fenotipo , Regiones Promotoras Genéticas/genética , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The production of prostaglandins by osteoblasts is an important mechanisms for the regulation of bone turnover. Bone cells contain both inducible and constitutive prostaglandin G/H synthase (PGHS-2 and PGHS-1) and these are differentially regulated. Nonsteroidal anti-inflammatory drugs (NSAIDs), which selectively inhibit one of these enzymes, would be useful in assessing their relative roles in bone metabolism. By Northern analysis, only PGHS-2 is expressed by the immortalized rat osteoblastic cell line, Py1a, while only PGHS-1 is expressed by the rat osteosarcoma cell line, ROS 17/2.8. We tested the relative inhibitory potency (IC50) of seven different NSAIDs on these two cell lines. A recently described selective inhibitor of PGHS-2, NS-398, was approximately 30 times more potent in inhibiting PGHS-2 than PGHS-1, and diclofenac was approximately 10 times more potent. Both had IC50's of approximately 3 nM for PGHS-2 in Py1a cells. Indomethacin, flurbiprofen, naproxen, and piroxicam were relatively nonselective with IC50's ranging from 30 nM to 1 microM, while 6-methoxy-2 naphthyl acetic acid, the active metabolite of nabumetone, was inhibitory only at concentrations greater than 1 microM. These results indicate that the presently available NSAIDs are unlikely to distinguish completely between effects mediated by PGHS-2 or PGHS-1. However, the cell systems employed could provide a model for the analysis of new compounds with greater selective activity.
Asunto(s)
Antiinflamatorios no Esteroideos/toxicidad , Huesos/efectos de los fármacos , Isoenzimas/biosíntesis , Osteoblastos/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Animales , Northern Blotting , Neoplasias Óseas/patología , Huesos/citología , Huesos/enzimología , Línea Celular , Células Cultivadas , Colágeno/biosíntesis , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inducción Enzimática/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Isoenzimas/genética , Proteínas de la Membrana , Osteoblastos/citología , Osteoblastos/patología , Osteosarcoma/patología , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandinas/biosíntesis , Prostaglandinas/fisiología , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , Ratas , Relación Estructura-Actividad , Células Tumorales CultivadasAsunto(s)
Huesos/enzimología , Dinoprostona/farmacología , Interleucina-1/farmacología , Isoenzimas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador alfa/farmacología , Factor de Crecimiento Transformador beta/farmacología , Células 3T3 , Animales , Ciclooxigenasa 1 , Ciclooxigenasa 2 , ADN Complementario , Dinoprostona/biosíntesis , Sinergismo Farmacológico , Inducción Enzimática , Cinética , Proteínas de la Membrana , Ratones , ARN Mensajero/biosíntesis , Transcripción Genética/inmunologíaRESUMEN
We have shown previously that prostaglandin (PG) production in 7-day-old neonatal mouse calvarial cultures is regulated largely by changes in prostaglandin G/H synthase-2 (PGHS-2) expression and to a lesser extent by changes in arachidonic acid (AA) release. In this study, we examined the effects of interleukin-4 (IL-4), and its interactions with other cytokines and with parathyroid hormone (PTH), on mRNA levels of PGHS-2, PGHS-1, and cytosolic phospholipase A2 (cPLA2) and on medium protaglandin E2 (PGE2) levels in calvarial cultures. IL-1 and tumor necrosis factor-alpha (TNF-alpha), both at 1-100 ng/ml, and PTH at 0.1-10 nM increased PGHS-2 and cPLA2 mRNA and medium PGE2 levels dose-dependently after 4 h of treatment. IL-6 and IL-11 at 1-100 ng/ml did not affect mRNA or PGE2 levels. IL-4 at 1-100 ng/ml decreased PGHS-2 and cPLA2 mRNA and PGE2 levels in control as well as IL-1, TNF-alpha, and PTH-stimulated cultures. The inhibition of PGHS-2 and cPLA2 mRNA expression by IL-4 (10 ng/ml) was present at 1 h, reached a maximum at 4 h, and persisted for 24 h. The effects were maintained in the presence of cycloheximide. IL-4 also decreased PGHS-2 protein levels in control and IL-1-stimulated cultures. PGHS-1 mRNA levels were not stimulated by any of the factors studied nor inhibited by IL-4. IL-4 partially inhibited control and PTH-stimulated 45Ca release from prelabeled mouse calvariae at 4 days. However, neither the inhibition of resorption by IL-4 nor the stimulation by IL-1 and PTH were altered by indomethacin (1 microM). We conclude that (1) IL-1, TNF-alpha, and PTH, but not IL-6 nor IL-11, can increase the expression of PGHS-2, cPLA2, and PGE2 production in cultured mouse calvariae; (2) IL-4 inhibits PGE2 production in both control and stimulated calvarial cultures by inhibiting PGHS-2 and cPLA2; and (3) IL-4 has an inhibitory effect on bone resorption which is independent of PG production.
Asunto(s)
Citosol/enzimología , Interleucina-4/farmacología , Hueso Parietal/enzimología , Fosfolipasas A/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Análisis de Varianza , Animales , Northern Blotting , Western Blotting , Resorción Ósea/metabolismo , Citosol/efectos de los fármacos , Dinoprostona/metabolismo , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Interleucina-1/farmacología , Interleucina-11/farmacología , Interleucina-6/farmacología , Ratones , Técnicas de Cultivo de Órganos , Hormona Paratiroidea/farmacología , Hueso Parietal/efectos de los fármacos , Fosfolipasas A/genética , Fosfolipasas A2 , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Prostaglandins (PGs) have been postulated to amplify their own production by stimulating cyclic adenosine monophosphate activity, which in turn stimulates PG production. We examined regulation of messenger RNA levels for the inducible and constitutive prostaglandin G/H synthases, PGHS-2 and PGHS-1, in murine osteoblastic MC3T3-E1 cells, which express both PGHS-1 and PGHS-2, and in rat osteoblastic Py1a cells, which express only PGHS-2. Prostaglandins E2, F2 alpha, and D2 induced PGHS-2 mRNA in both cell lines under serum-free conditions and stimulated small increases in PGHS-1 mRNA levels in MC3T3-E1 cells. PGE2 (1 microM) increased the transcription rate of PGHS-2 mRNA 9-fold at 2 h in serum-free cells and also induced PGHS-2 protein. In the presence of arachidonic acid or serum, PGs also increased medium PGE2. Both forskolin, a protein kinase A activator, and phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, have previously been shown to induce PGHS-2 mRNA in MC3T3-E1 cells, but in the present study only PMA induced PGHS-2 expression in Py1a cells. The induction of PGHS-2 mRNA in Py1a cells by PGs was inhibited by chelerythrine, a PKC inhibitor, and blocked by 24 h of pretreatment with PMA. The 2 h serum stimulation of PGHS-2 mRNA in MC3T3-E1 cells was inhibited 40-50% by three structurally unrelated nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting that endogenous PGs also amplify PG production through induction of PGHS-2.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Osteoblastos/enzimología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas/farmacología , Células 3T3/citología , Células 3T3/efectos de los fármacos , Análisis de Varianza , Animales , Antiinflamatorios no Esteroideos/farmacología , Northern Blotting , Western Blotting , Células Cultivadas , Colforsina/farmacología , ADN Complementario/genética , Dinoprost/farmacología , Dinoprostona/farmacología , Inducción Enzimática/efectos de los fármacos , Ratones , Hibridación de Ácido Nucleico , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Prostaglandina D2/farmacología , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/genética , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
A second prostaglandin G/H synthase (PGHS-2), encoded by a gene separate from that for the original PGHS (PGHS-1), has recently been identified. We have shown that PGHS-2 is expressed in cultured mouse calvariae and have compared regulation of PGHS-2 and PGHS-1 messenger RNA (mRNA) levels. PGHS-2 mRNA was not detectable in freshly isolated bones, but was induced during culture and further stimulated by interleukin-1 (IL-1) and PTH. Both factors also increased PGHS-2 protein levels. Changes in medium prostaglandin E2 (PGE2) production correlated with increases in PGHS-2 mRNA levels. However, with IL-1, PGE2 production was increased more than PGHS-2 mRNA levels (treated/control ratio, 3.4 and 1.5, respectively), whereas with PTH there was a closer correspondence (2.0 and 2.1). Cortisol reduced PTH-stimulated PGE2 production (treated/control ratio decreased from 3.1 to 0.2) more than PGHS-2 mRNA levels (2.8 to 0.8). In the presence of exogenous arachidonic acid, changes in PGHS-2 mRNA levels with IL-1, PTH, and cortisol correlated closely with changes in PGE2 production. PGE2 itself increased PGHS-2 mRNA, and nonsteroidal antiinflammatory drugs decreased PGHS-2 mRNA levels by 80%. In contrast, PGHS-1 mRNA was expressed constitutively and was not affected by IL-1, PTH, or cortisol when measured by competitive reverse transcriptase-polymerase chain reaction. We conclude that regulation of PGE2 production is predominantly through PGHS-2, rather than PGHS-1; that IL-1 and cortisol may also regulate arachidonic acid release; and that PGE2 may amplify its own production through stimulation of PGHS-2.
Asunto(s)
Dinoprostona/farmacología , Hidrocortisona/farmacología , Interleucina-1/farmacología , Hormona Paratiroidea/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Cráneo/enzimología , Animales , Animales Recién Nacidos/metabolismo , Secuencia de Bases , Western Blotting , Técnicas de Cultivo , Ratones , Ratones Endogámicos , Sondas Moleculares/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
Regulation of mRNA levels for the constitutive and inducible prostaglandin endoperoxide synthases, PGHS-1 and PGHS-2, was examined in murine osteoblastic MC3T3-E1 cells. Serum induction of PGHS-2 mRNA levels was rapid, transient, increased by cycloheximide, and inhibited 72% by cortisol. The cortisol inhibition was blocked by cycloheximide. Serum stimulation of PGHS-1 mRNA was slower, decreased by cycloheximide, and inhibited 28% by cortisol. Increased prostaglandin E2 (PGE2) production and induction of PGHS-2 immunoreactive protein paralleled changes in PGHS-2 mRNA. PGHS-2 mRNA was induced at 2 h in serum-free cells by transforming growth factor-beta (TGF-beta), phorbol 12-myristate 13-acetate, and, to a lesser extent, by forskolin. The combination of phorbol 12-myristate 13-acetate and forskolin was synergistic. TGF-beta induction was prolonged compared with serum, inhibited 67% by cortisol, and the inhibition was not blocked by cycloheximide. TGF-alpha had little effect on PGHS-2 mRNA at 2 h, but the combination of TGF-beta and TGF-alpha was synergistic for PGHS-1 and PGHS-2. PGE2 itself induced PGHS-2 mRNA, and inhibition of PGE2 production decreased the serum induction by 55%, suggesting an important role for autoamplification. The rapidity and amplitude of changes in PGHS-2 suggest that it may be involved in bone responses to acute stresses, such as mechanical strain, inflammation, and injury.
Asunto(s)
Osteoblastos/enzimología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , ARN Mensajero/metabolismo , Animales , Línea Celular , Colforsina/farmacología , Medios de Cultivo , Medio de Cultivo Libre de Suero , Cicloheximida/farmacología , ADN/metabolismo , Dactinomicina/farmacología , Dinoprostona/metabolismo , Inducción Enzimática , Hidrocortisona/farmacología , Cinética , Ratones , Osteoblastos/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/metabolismo , ARN Mensajero/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Parathyroid hormone-related peptide (PTHrP) has been shown to be the pathogenic agent in humoral hypercalcemia of malignancy (HHM), but the molecular forms that are secreted have not been fully characterized. PTHrP 1-34 has effects similar to parathyroid hormone (PTH), but C-terminal regions of the peptide, such as the 107-139 fragment found to inhibit resorption in a study by Fenton et al (1991), may have other biological activities not shared with PTH. We have compared the effects of the longer forms of recombinant human PTHrP (hPTHrP 1-84, 1-108, and 1-141) with hPTHrP 1-34 and synthetic bovine PTH (bPTH) 1-34 on bone resorption and formation in cultured neonatal mouse calvariae and fetal rat long bones. hPTHrP 1-84, 1-108, and 1-141 were qualitatively similar to hPTHrP 1-34 and PTH 1-34 in stimulating 45Ca release from both neonatal mouse calvariae and fetal rat long bones and in inhibiting the incorporation of [3H]-proline into collagenase digestible protein (CDP) and stimulating the incorporation of [3H]-thymidine (3H-TdR) in neonatal mouse calvariae. However, hPTHrP 1-108 and 1-141 were less potent at stimulating 45Ca release and inhibiting CDP labeling than hPTHrP 1-34, while hPTHrP 1-84 showed an intermediate potency. Since hPTHrP 1-108 and 1-141 were quite similar in potency, the difference cannot be attributed to an inhibitory effect of the 107-139 fragment. All the peptide lengths tested showed similar potency in stimulating [3H]-TdR incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Resorción Ósea/fisiopatología , Huesos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Proteína Relacionada con la Hormona Paratiroidea , Hormona Paratiroidea/farmacología , Animales , Colágeno/biosíntesis , ADN/biosíntesis , Ratones , Técnicas de Cultivo de Órganos , Fragmentos de Péptidos/farmacología , Biosíntesis de Proteínas , Proteínas/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacologíaRESUMEN
Potassium peroxydiphosphate (KPDP) is a slowly hydrolyzed pyrophosphate analog that can release hydrogen peroxide during hydrolysis. We tested its effects on the resorption of cultured fetal rat long bones as measured by the release of previously incorporated 45Ca, both by direct addition of KPDP to the medium and after preincubation of KPDP with large-molecular-weight resorbing factors followed by dialysis to reduce the KPDP concentration. With direct addition, KPDP at a concentration of 1 mM could inhibit the resortive response to bacterial lipopolysaccharide (LPS), parathyroid hormone (PTH), prostaglandin E2 (PGE2), and mouse recombinant interleukin-1 (mrIL-1). The response to LPS was partially inhibited at 0.3 mM KPDP. Control resorption in the absence of stimulators was also inhibited. Potassium pyrophosphate at 1 mM was less effective as an inhibitor of bone resorption. The inhibitory effects of KPDP did not appear to be due entirely to nonspecific toxicity since partial recovery occurred after it was removed. There was no significant decrease in [3H]thymidine or [3H]proline incorporation into bones incubated with KPDP at 1 mM for 5 days, but [3H]proline incorporation was decreased at 24 h, suggesting that KPDP may have a general inhibitory effect on bone cells. When media with and without stimulators of resorption were incubated overnight at 4 degrees C with KPDP at 5.8 mM and then dialyzed to bring the concentration to below 0.3 mM, the bone-resorbing activity of PTH, LPS, and mrIL-1 was completely lost. This may have been due to the slow release of hydrogen peroxide; however, preincubation with equimolar concentrations of H2O3 caused only partial inactivation of PTH and LPS. LPS.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Resorción Ósea , Calcio/metabolismo , Peróxidos/farmacología , Ácidos Fosfóricos/farmacología , Animales , Huesos/embriología , Huesos/metabolismo , Cricetinae , Medios de Cultivo , Técnicas de Cultivo , Dinoprostona/farmacología , Femenino , Peróxido de Hidrógeno/metabolismo , Hidrólisis , Interleucina-1/farmacología , Lipopolisacáridos , Peso Molecular , Hormona Paratiroidea/farmacología , Prolina/metabolismo , Ratas , Timidina/metabolismoRESUMEN
Inhibitory factors towards IL-1 have been identified in the urine and in the supernatants of human monocyte cultures and have been shown to act as receptor antagonists. We have investigated whether a natural inhibitor purified from human urine (uIL-1ra) and a recombinant molecule expressed using the gene for an IL-1 antagonist isolated from monocytes (rIL-1ra) can alter responses to human rIL-1 alpha in organ cultures of fetal rat long bones and neonatal mouse calvariae. The two preparations probably contained similar or identical molecules, because an antibody to rIL-1ra reacted with uIL-1ra by Western blot analysis. uIL-1ra and rIL-1ra specifically blocked stimulation of bone resorption by rIL-1 in both culture systems, as well as the increase in PGE2 production in cultured calvariae. Resorptive effects of parathyroid hormone and TNF-alpha were not blocked. The uIL-1ra preparation had some intrinsic resorbing activity, but on gel chromatography this appeared in fractions that eluted earlier than uIL-1ra. Concentration ratios of rIL-1ra to rIL-1 as low as 10 could block the resorptive response of fetal rat long bones, whereas concentration ratios of 100 to 1000 were required to block IL-1 action on neonatal mouse calvariae. The inhibitory effects appeared to be competitive, because increasing concentrations of IL-1 overcame the block of bone resorption in both systems and the inhibition of PGE2 production in calvariae.
Asunto(s)
Resorción Ósea , Dinoprostona/biosíntesis , Interleucina-1/antagonistas & inhibidores , Proteínas/farmacología , Receptores Inmunológicos/antagonistas & inhibidores , Sialoglicoproteínas/farmacología , Animales , Western Blotting , Calcio/metabolismo , Humanos , Técnicas In Vitro , Proteína Antagonista del Receptor de Interleucina 1 , Peso Molecular , Hormona Paratiroidea/antagonistas & inhibidores , Proteínas/genética , Proteínas/inmunología , Ratas , Receptores de Interleucina-1 , Proteínas Recombinantes/farmacología , Sialoglicoproteínas/genética , Sialoglicoproteínas/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Although most studies show that prostaglandin E2 (PGE2) is the most potent and effective of the prostanoids in bone, recent data in cell culture suggest that PGF2 alpha may have unique effects, particularly on cell replication. The present study was undertaken to compare the effects of PGF2 alpha and PGE2 in cultured neonatal mouse parietal bones by simultaneous measurement of bone resorption as release of previously incorporated 45Ca, bone formation as incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein, and DNA synthesis as incorporation of [3H]thymidine. PGF2 alpha was less effective than PGE2 as a stimulator of bone resorption, and its effects were partially inhibited by indomethacin and markedly inhibited by glucocorticoids. In contrast, the resorptive response to PGE2 was unaffected by indomethacin and only partially inhibited by cortisol. PGF2 alpha had little effect on bone formation, in contrast to the biphasic effect of PGE2, which inhibited labeling of CDP in the absence of cortisol and stimulated CDP labeling in the presence of cortisol. PGF2 alpha increased thymidine incorporation into DNA, but the effect was smaller than that of PGE2 and was inhibited by indomethacin. These observations suggested that PGF2 alpha might act in part by stimulating PGE2 production. By RIA, PGE2 concentrations were increased in the medium of bones treated with PGF2 alpha, and this increase was blocked by indomethacin. By HPLC, bones prelabeled with [3H]arachidonic acid showed an increase in labeled PGE2 release, and RIA showed an increase in PGE2 after PGF2 alpha treatment. These results indicate that PGF2 alpha is a relatively weak agonist in bone compared to PGE2 and that some of the effects of PGF2 alpha on bone resorption, formation, and cell replication may be mediated by an increase in endogenous PGE2 production.
Asunto(s)
Animales Recién Nacidos/metabolismo , Desarrollo Óseo/efectos de los fármacos , Resorción Ósea , Huesos/fisiología , Dinoprost/farmacología , Dinoprostona/biosíntesis , Animales , Huesos/efectos de los fármacos , Radioisótopos de Calcio/metabolismo , Cromatografía Líquida de Alta Presión , ADN/biosíntesis , Hidrocortisona/farmacología , Indometacina/farmacología , Cinética , Ratones , Técnicas de Cultivo de ÓrganosRESUMEN
To assess the possibility that diets rich in eicosapentaenoic acid (EPA) could have adverse effects on the skeleton, we examined the resorptive response to its major project, PGE3, and the effects and metabolism of EPA itself in cultured fetal rat long bones and neonatal rat calvaria. PGE3 stimulated bone resorption with a potency similar to that of PGE2. However, EPA was a much less effective precursor for PGE3 than was arachidonic acid (AA) for PGE2. In bones cultured with complement sufficient rabbit serum, which stimulates endogenous PGE release, addition of EPA had little effect on bone resorption while AA produced a substantial increase. Bones labeled with [3H]-AA and incubated with transforming growth factor-alpha (TGF-alpha), which stimulates endogenous PGE production, produced substantial amounts of PGE2, while bones labeled with [3H]-EPA and treated similarly produced less than 1/10th as much labeled PGE3. Thus, EPA appears to be a less effective precursor for the production of bone resorbing prostanoids than AA in cultured rat bone. However, since PGE3 is a potent stimulator of bone resorption, the possibility that dietary EPA can effect the production of bone resorbing prostanoids in man requires further study.
Asunto(s)
Alprostadil/análogos & derivados , Huesos/metabolismo , Ácido Eicosapentaenoico/farmacología , Prostaglandinas E/farmacología , Animales , Animales Recién Nacidos , Ácido Araquidónico , Ácidos Araquidónicos/farmacología , Sangre , Resorción Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Calcio/metabolismo , Dinoprostona/biosíntesis , Dinoprostona/farmacología , Ácido Eicosapentaenoico/metabolismo , Técnicas de Cultivo de Órganos , Ratas , Factores de Crecimiento Transformadores/farmacologíaRESUMEN
We compared the bone-resorbing activity in the conditioned medium from phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cell cultures with that of partially purified human monocyte-derived interleukin 1 (IL-1), human recombinant IL-1 alpha (pI 5) and IL-1 beta (pI 7), human recombinant tumor necrosis factor-alpha (TNF alpha), and PTH in fetal rat long bone cultures. An antiserum to the products of activated human mononuclear cells, including IL-1, completely blocked the bone-resorbing activity of all three forms of IL-1 and of unfractionated PHA-stimulated human peripheral blood mononuclear cell supernatants, but did not inhibit resorption stimulated by recombinant human TNF alpha. This antiserum also had no effect on the resorptive response to 3 nM PTH, but did decrease the response to 1 nM PTH slightly. These results imply that IL-1, but not TNF alpha, mediates the bone-resorbing activity found in the supernatants of PHA-stimulated human peripheral blood mononuclear cell cultures. It is not known whether the small inhibitory effect that the antiserum to IL-1 had on the response to 1 nM PTH resulted from a nonspecific action or an effect of PTH on local IL-1 synthesis in bone. Since cytokines are found in the circulation of normal individuals and are produced at local sites of pathology, these results suggest that they can influence both normal and abnormal skeletal metabolism.
Asunto(s)
Resorción Ósea/efectos de los fármacos , Glicoproteínas/farmacología , Interleucina-1/fisiología , Monocitos/fisiología , Hormona Paratiroidea/farmacología , Animales , Huesos/efectos de los fármacos , Huesos/metabolismo , Técnicas de Cultivo , Humanos , Sueros Inmunes/farmacología , Interleucina-1/inmunología , Fitohemaglutininas/farmacología , Prostaglandinas E/metabolismo , Ratas , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfaRESUMEN
Recent studies of rat and human calcitonin (CT) gene expression have uncovered a diversity of secretory peptides. Here we report the results of testing two such non-CT secretory peptides for CT-like action in live rats and in cultured fetal rat long bone. One peptide, the carboxyl-terminal CT-adjacent peptide that is cosynthesized with CT, has no hypocalcemic effect and no inhibitory action on bone resorption in vitro. The other peptide, CT gene-related peptide, lowers blood calcium and inhibits bone resorption. In vitro experiments are consistent with the idea that CT gene-related peptide is acting at CT receptors in bone.
Asunto(s)
Calcitonina/genética , Calcitonina/farmacología , Calcio/metabolismo , Proteínas del Tejido Nervioso/farmacología , Fragmentos de Péptidos/farmacología , Animales , Resorción Ósea/efectos de los fármacos , Huesos/embriología , Huesos/metabolismo , Péptido Relacionado con Gen de Calcitonina , Calcio/sangre , Radioisótopos de Calcio , Células Cultivadas , Femenino , Humanos , Hormona Paratiroidea/farmacología , Procesamiento Postranscripcional del ARN , Ratas , Ratas EndogámicasRESUMEN
We have compared the effects of thiophene 2-carboxylic acid (TCA) and a number of sulfur- and nitrogen-containing analogs for their ability to inhibit bone resorption in organ cultures of fetal rat long bones. Four compounds,--thionaphthene-2-carboxylic acid (TNCA), dibenzo-thiophene-4-carboxylic acid, indole-2-carboxylic acid and carbazole-1-carboxylic acid--caused a dose-related inhibition of PTH-stimulated bone resorption, although TCA was ineffective in this system. TNCA at 3 X 10(-4) M or 10(-4) M was the most potent inhibitor of PTH-stimulated bone resorption and was selected for further study. TNCA also inhibited stimulation of resorption by prostaglandin E2 and 1,25-dihydroxyvitamin D. Unlike calcitonin, the effect of TNCA was persistent and did not show escape. Moreover, TNCA could inhibit resorption in bones that had previously escaped from calcitonin. TNCA did not appear to be a nonspecific toxin, since it did not decrease incorporation of [3H]thymidine or [3H]proline into fetal rat long bones. The fact that resorption in unstimulated cultures was only decreased when the control rates were high also argues against nonspecific toxicity. Moreover, this suggests that TNCA will be most effective under conditions of accelerated bone resorption when an inhibiting effect is most desirable.
Asunto(s)
Resorción Ósea/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Tiofenos/farmacología , Animales , Calcitonina/farmacología , Calcio/metabolismo , Ácidos Carboxílicos , División Celular/efectos de los fármacos , Colecalciferol/farmacología , Técnicas de Cultivo de Órganos , Hormona Paratiroidea/farmacología , Prostaglandinas E/farmacología , RatasRESUMEN
We have compared two components of bacterial cell walls, muramyl dipeptide (MDP) and lipopolysaccharide (LPS), for their effects on bone resorption as measured by the release of previously incorporated 45Ca. MDP is the smallest active component of peptidoglycan, whereas LPS is the active component of endotoxin. Fetal rat long bones were cultured for 5 days in a chemically defined medium supplemented with bovine serum albumin (BSA) or serum. LPS increased 45Ca release at concentrations of 0.03-1.0 microgram/ml. LPS further purified by electrolytic dialysis (ED-LPS) was active at 0.01 microgram/ml. ED-LPS was ineffective at such low concentrations in the presence of serum. The response to MDP was more variable than that to LPS, but bone resorption was stimulated at concentrations of 10(-7)-10(-5) M. MDP was less effective or inactive in medium supplemented with serum. Stereoisomers of MDP that do not have adjuvant activity caused minimal stimulation of bone resorption, whereas 6-0-steroyl MDP stimulated resorption at 10(-8) M. The stimulation of bone resorption by LPS and MDP was not inhibited by indomethacin. Both LPS and MDP increased lysosomal enzyme release in proportion to their effects on 45Ca release. LPS also markedly increased collagenase activity in the medium, but MDP did not. These results indicate that chemically different products of bacterial cell walls can stimulate bone resorption in vitro. These products may be distinguished by differences in dose response curve, serum inhibition, and collagenase release.