RESUMO
High concentrations of prostaglandin E2 (PGE2) inhibit collagen synthesis and reduce alpha 1(I)procollagen messenger RNA (mRNA) levels in cultured fetal rat calvariae. To examine the mechanism of this effect, we used the immortalized rat osteoblastic clonal cell line, Py1a. PGE2 at 1 microM inhibited the incorporation of [3H]proline into collagenase-digestible protein (CDP) and increased incorporation into noncollagen protein, whereas 0.1 microM PGE2 increased both CDP and noncollagen protein labeling. Because insulin-like growth factor-I (IGF-I) is an anabolic hormone in bone and PGE2 can increase its production, we added exogenous IGF-I (10 nM) to Py1a cultures. In the presence of IGF-I, PGE2 from 10 nM to 1 microM had only an inhibitory effect on CDP labeling and alpha 1(I)procollagen mRNA levels. PGE2 at 1 microM decreased the rate of alpha 1(I)procollagen gene transcription in the presence or absence of IGF-I, determined by a nuclear run-on assay. Py1a cells were stably transfected with chimeric genes containing varying lengths of the 5'-upstream region of the rat alpha 1(I)procollagen promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene. In cells transfected with ColCAT 3.6, which contains 3520 base pairs of 5'-upstream DNA, CAT activity was inhibited by PGE2, but the inhibition was less than that observed for CDP labeling. With smaller 5'-upstream regions, there was no inhibitory effect of PGE2. These results demonstrate that PGE2 inhibits alpha 1(I)procollagen gene transcription and the activity of a region between -3.5 and -2.3 kilobases of the 5'-upstream collagen gene promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Dinoprostona/farmacologia , Osteoblastos/efeitos dos fármacos , Pró-Colágeno/biossíntese , Regiões Promotoras Genéticas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Animais , Linhagem Celular Transformada , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Osteoblastos/metabolismo , Pró-Colágeno/genética , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes de Fusão/biossínteseRESUMO
We previously showed that prostaglandin E2 (PGE2) can selectively inhibit collagen synthesis and gene transcription in the immortalized rat osteoblastic clonal cell line Py1a, particularly in the presence of insulin-like growth factor I (IGF-I). In the present study, we examined the structure-activity relations for this effect. PGF2 alpha was approximately 100 times more potent than PGE2. The prostaglandin F receptor (FP) selective agonist, fluprostenol, was the most potent agonist tested, significantly inhibiting incorporation of [3H]proline into both collagen and noncollagen protein at 10(-11) M, with more than 90% inhibition of collagen synthesis at 10(-8) M. The PGE2 analog, sulprostone, and PGD2 showed activity similar to that of PGE2. PGI2 and its stable analog, carbacyclin, were the least effective. Parathyroid hormone (PTH), forskolin, and isobutylmethylxanthine (IBMX) were ineffective. Phorbol myristate acetate (PMA) inhibited collagen synthesis in a manner similar to that of the prostanoids. The inhibitory effects of PGF2 alpha, fluprostenol, and PMA show a similar time course on alpha 1(I) procollagen mRNA levels. The inhibition appeared to be caused by a decrease in collagen gene transcription as measured by nuclear run-on analysis. Further evidence for a transcriptional effect was obtained with COL1A1 promoter-CAT reporter constructs, although these showed somewhat smaller effects of prostanoids on CAT activity than on mRNA levels or labeling.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Colágeno/biossíntese , Osteoblastos/metabolismo , Prostaglandinas/farmacologia , Animais , Linhagem Celular , Cloranfenicol O-Acetiltransferase/antagonistas & inibidores , Cloranfenicol O-Acetiltransferase/metabolismo , Osteoblastos/efeitos dos fármacos , Pró-Colágeno/antagonistas & inibidores , Pró-Colágeno/biossíntese , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Ratos , Acetato de Tetradecanoilforbol/farmacologia , Fatores de TempoRESUMO
The structure-activity relations and signal transduction pathways for the anabolic effects of prostaglandins were examined in cultured fetal rat calvariae. In the presence of cortisol prostaglandins of the E and F series (10(-9) to 10(-5) M) produced a dose-related increase in [3H]thymidine incorporation up to 4-fold at 24 h. Prostaglandin E2 (PGE2) was also effective in the absence of cortisol. Butaprost (10(-6) M), a selective EP-2 receptor agonist, produced partial stimulation. Prostaglandin D2, prostacyclin, sulprostone, an EP-1 and EP-3 receptor agonist, and fluprostenol, an FP receptor agonist, were ineffective. Forskolin (10(-4) M) increased [3H]thymidine incorporation 3-fold, while phorbol myristate acetate (PMA) (10(-6) M) produced a 1.8-fold increase. Isobutylmethylxanthine (IBMX) increased [3H]thymidine incorporation in control cultures, in the absence of cortisol, and increased the response to PGE2 in control and cortisol-treated cultures. [3H]proline incorporation into collagen and noncollagen protein was measured in the continuous presence of prostaglandins and cortisol for 72-96 h (continuous model) or when prostaglandins and cortisol were applied for 24 h, followed by culture for 48 h in control medium (on/off model). The effects on collagen were greater than on noncollagen proteins, so that the percent of collagen synthesis increased. The effects of prostaglandins and forskolin paralleled their mitogenic effects. PMA increased only noncollagen protein. Indomethacin did not diminish the anabolic response, while aphidicolin produced only partial inhibition. We conclude that the anabolic effects of prostaglandins on replication and differentiation of osteoblasts are likely to be mediated by an EP-2 receptor that stimulates adenylate cyclase.
Assuntos
Abortivos não Esteroides/farmacologia , Osteoblastos/efeitos dos fármacos , Ocitócicos/farmacologia , Prostaglandinas E Sintéticas/farmacologia , Transdução de Sinais/efeitos dos fármacos , 1-Metil-3-Isobutilxantina/farmacologia , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Colágeno/biossíntese , Dinoprostona/análogos & derivados , Dinoprostona/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Epoprostenol/farmacologia , Hidrocortisona/farmacologia , Marcação por Isótopo , Técnicas de Cultura de Órgãos , Osteoblastos/citologia , Inibidores de Fosfodiesterase/farmacologia , Prostaglandina D2/farmacologia , Prostaglandinas F Sintéticas/farmacologia , Ratos , Timidina/metabolismo , TrítioRESUMO
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.
Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Osso e Ossos/efeitos dos fármacos , Isoenzimas/biossíntese , Osteoblastos/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/biossíntese , Animais , Northern Blotting , Neoplasias Ósseas/patologia , Osso e Ossos/citologia , Osso e Ossos/enzimologia , Linhagem Celular , Células Cultivadas , Colágeno/biossíntese , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Indução Enzimática/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Isoenzimas/genética , Proteínas de Membrana , Osteoblastos/citologia , Osteoblastos/patologia , Osteossarcoma/patologia , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandinas/biossíntese , Prostaglandinas/fisiologia , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , Ratos , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
To test the hypothesis that insulin-like growth factors (IGFs) play a role in the response of bone to glucocorticoids, we determined the effects of cortisol on the incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP), the percent collagen synthesis, and the incorporation of [3H]thymidine into DNA of 21-day fetal rat calvariae cultured in the presence and absence of recombinant human insulin-like growth factor binding protein-2 (IGFBP-2). At 24 h, cortisol (100 nM) increased CDP labeling and the percent collagen synthesis, and these effects were blocked by IGFBP-2 (1000 nM). At 24 h, cortisol decreased the incorporation of [3H]thymidine into bone, which was not affected by the addition of IGFBP-2. At 48 h, cortisol (1000 nM) decreased CDP labeling, which was maintained in the presence of IGFBP-2. At 48 h, IGFBP-2 alone decreased basal levels of CDP and NCP labeling and the percent collagen synthesis. Our data suggest that endogenous IGFs maintain basal levels of collagen synthesis and mediate the early stimulatory effect of glucocorticoids on collagen synthesis in fetal rat calvariae. However, blocking endogenous IGFs does not abrogate the inhibitory effect of glucocorticoids on DNA synthesis and the later inhibition of collagen synthesis in calvariae.
Assuntos
Colágeno/biossíntese , Hidrocortisona/farmacologia , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/farmacologia , Crânio/efeitos dos fármacos , Crânio/metabolismo , Animais , DNA/biossíntese , Feto , Humanos , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Técnicas de Cultura de Órgãos , Ratos , Proteínas Recombinantes/farmacologiaRESUMO
Previous studies have shown that prostaglandin E2 (PGE2) has both inhibitory and stimulatory effects on the incorporation of proline into collagenase-digestible protein (CDP) in cultured fetal rat calvaria. The present studies were undertaken to analyze further these biphasic effects of PGE2. PGE2 increased [3H]thymidine incorporation at 24 h, and this effect was enhanced in the presence of cortisol (10(-8) and 10(-7) M). An inhibitory effect on CDP labeling was observed at 96 h with PGE2 (10(-6) M) in the absence or presence of indomethacin (10(-6) M), but not in the presence of cortisol (10(-8) or 10(-7) M). When the central osteoblast-rich bone and periosteum were analyzed separately, the inhibitory effect of PGE2, with or without indomethacin, was confined to the central bone. Addition of aphidicolin (30 microM), an inhibitor of cell replication, did not prevent the inhibitory effect of PGE2 on CDP labeling. Analysis of labeled collagen by polyacrylamide gel electrophoresis showed a decrease in labeling of type I collagen in central bone. Moreover, mRNA for alpha 1(I)procollagen was decreased, as measured by dot blot hybridization and Northern blot analysis. Cortisol (10(-8)-10(-6) M) decreased the labeling of CDP as well as noncollagen protein (NCP) at 96 h. In the presence of cortisol, PGE2 (10(-8)-10(-5) M) consistently stimulated labeling of CDP and NCP, with a greater increase in CDP, resulting in an increase in the percentage of collagen synthesized. In the presence of low concentrations of cortisol (10(-8) or 3 x 10(-8) M), PGE2 (10(-7) M) increased CDP labeling by 260-480%, and the absolute value was 145-160% of that in control cultures without any hormone addition. The stimulatory effect was seen in both central bone and periosteum, although absolute values for CDP and percentage of collagen synthesized were higher in central bone. PGE2 (10(-7) M) had similar effects on CDP at 24 and 96 h in the presence of cortisol, and the stimulation at 10(-7) M was the same in the presence and absence of aphidicolin, suggesting that it was not dependent on cell replication. Cortisol decreased labeling of type I collagen, determined by polyacrylamide gel electrophoresis, and alpha 1(I)procollagen mRNA levels, determined by both Northern and dot blot analysis. PGE2 reversed these effects, increasing both radiolabeled collagen type I chains and alpha 1(I)procollagen mRNA levels. These results indicate that PGE2 can regulate bone collagen synthesis at a pretranslational site.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Dinoprostona/farmacologia , Hidrocortisona/farmacologia , Osteogênese/efeitos dos fármacos , Crânio/embriologia , Animais , Colágeno/biossíntese , Técnicas de Cultura , Relação Dose-Resposta a Droga , Interações Medicamentosas , Feto/fisiologia , Colagenase Microbiana/farmacologia , Periósteo/embriologia , Periósteo/metabolismo , Prolina/metabolismo , Biossíntese de Proteínas , Ratos , Ratos Endogâmicos , Crânio/metabolismo , Timidina/metabolismoRESUMO
We have compared the effects of synthetic amino-terminal human PTH-(1-34)-related peptide (PTHrP) of malignancy with those of synthetic bovine PTH-(1-34) in cultures of half-calvariae from 21-day-old fetal rats and of parietal bones from 7-day neonatal mice. Incorporation of [3H] proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP), and percent collagen synthesis (PCS) were measured in both systems. Incorporation of [3H]thymidine and cAMP production were measured in fetal rat calvariae. Production of prostaglandin E2 and I2 and bone resorption, as assessed by release of previously incorporated 45Ca, were measured in mouse parietal bones. The effects of PTHrP and PTH were qualitatively similar. At 96 h CDP in rat calvariae was decreased by PTH at a concentration as low as 0.01 nM, while similar effects were seen with PTHrP at 0.1 nM. Effects on NCP were small, so PCS was reduced. At 24 h [3H]thymidine was not altered, but CDP and PCS were decreased by both PTH and PTHrP. cAMP production was increased in fetal rat calvariae at 30 min. Both PTH and PTHrP increased 45Ca release at low concentrations and prostaglandin production at high concentrations in mouse parietal bones. While PTH was about 10-fold more potent than PTHrP, there was no qualitative difference in the responses. These studies further suggest that PTHrP affects bone through the PTH receptor.
Assuntos
Doenças Ósseas Metabólicas/fisiopatologia , Proteínas de Neoplasias/farmacologia , Osteogênese/efeitos dos fármacos , Proteína Relacionada ao Hormônio Paratireóideo , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Proteínas , Animais , Animais Recém-Nascidos , Feto/fisiologia , Camundongos , Técnicas de Cultura de Órgãos , RatosRESUMO
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.
Assuntos
Animais Recém-Nascidos/metabolismo , Desenvolvimento Ósseo/efeitos dos fármacos , Reabsorção Óssea , Osso e Ossos/fisiologia , Dinoprosta/farmacologia , Dinoprostona/biossíntese , Animais , Osso e Ossos/efeitos dos fármacos , Radioisótopos de Cálcio/metabolismo , Cromatografia Líquida de Alta Pressão , DNA/biossíntese , Hidrocortisona/farmacologia , Indometacina/farmacologia , Cinética , Camundongos , Técnicas de Cultura de ÓrgãosRESUMO
We examined the effects of synthetic human atrial natriuretic factor (human ANF 99-126) on adenylate cyclase activity, cAMP and cyclic GMP (cGMP) levels, bone resorption, collagen and DNA synthesis, and prostaglandin E2 (PGE2) production in fetal rat bone organ cultures. ANF (100 nM) inhibited PTH- and PGE2-stimulated cAMP production but had no effect on basal cAMP production in 21-day fetal rat calvaria. ANF increased cGMP levels, and this was not affected by PTH. ANF (10 nM) partially inhibited bone resorption stimulated by PGE2 but had no effect on control or PTH-stimulated resorption in 19-day fetal rat long bones. ANF had no effect on collagen and DNA synthesis or PGE2 production and did not alter responses to PTH or PGE2 in the fetal rat calvaria. Thus, ANF has no major direct effect on bone resorption or formation, but it is possible that ANF modulates the local regulatory function of PGE2 in bone.
Assuntos
Fator Natriurético Atrial/farmacologia , Reabsorção Óssea , Osso e Ossos/metabolismo , Colágeno/biossíntese , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , DNA/biossíntese , Dinoprostona/biossíntese , Adenilil Ciclases/metabolismo , Animais , Osso e Ossos/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Dinoprostona/farmacologia , Feto , Hormônio Paratireóideo/farmacologia , Prolina/metabolismo , RatosRESUMO
Bovine PTH-(1-34) (PTH), human recombinant interleukin-1 alpha (IL-1), and cortisol were tested for their effects on bone resorption, prostaglandin (PG) production, and PG endoperoxide synthase (PGH synthase or cyclooxygenase) mRNA levels in cultured mouse parietal bones. Cultures were treated with PTH and IL-1 in the presence and absence of cortisol and arachidonic acid (AA). We found that both PTH and IL-1 stimulated the release of PGE2 and 6-keto-PGF1 alpha (the stable metabolite of PGI2). Stimulation of each metabolite by IL-1 at 0.6-60 pM was 2- to 118-fold, and that by PTH at 24 pM to 24 nM was 3- to 53-fold. Thus, IL-1 was 40-fold more potent than PTH in stimulating PG release. Moreover, IL-1 showed 2- to 3-fold greater efficacy than PTH in stimulating PGE2 release. However, IL-1 was only 4-fold more potent and no more effective than PTH in stimulating 45Ca release. IL-1 (60 pM) and PTH (2.4 nM) stimulation of PGE2 production showed a similar time course, with a lag phase of 0.75-1.5 h. Cortisol (1-100 nM) reduced basal PGE2 production and calcium release. The absolute amounts of PG produced in response to PTH and IL-1 were reduced in the presence of cortisol, but in the presence of AA the relative increases were still from 2.5- to 26-fold compared with levels in cultures treated with cortisol alone. Cortisol reduced the stimulation of 45Ca release by IL-1, but not by PTH. AA (10(-5) M) amplified PG production in response to PTH and IL-1, but not 45Ca release. In bones labeled with [3H]AA, IL-1 and PTH increased [3H]PGE2 and [3H]6-keto-PGF1 alpha release, as measured by HPLC and TLC. IL-1 slightly increased [3H]AA release, but PTH did not. Cortisol decreased [3H]AA release. To test for an effect on PG production at the level of PGH synthase, mRNA levels were measured. mRNA was increased by both PTH and IL-1 to a similar extent despite the greater effect of IL-1 on PGE2 production. Cortisol did not change PGH synthase mRNA levels and did not block the stimulation by PTH or IL-1. We conclude that IL-1 is a more potent stimulator of PG production and bone resorption than PTH. Stimulation of PG production by both PTH and IL-1 is mediated at least in part by increasing PGH synthase, but IL-1 may have an additional effect on AA release.
Assuntos
Hidrocortisona/farmacologia , Interleucina-1/farmacologia , Hormônio Paratireóideo/farmacologia , Osso Parietal/metabolismo , Prostaglandinas/biossíntese , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Ácidos Araquidônicos/farmacologia , Northern Blotting , Cálcio/metabolismo , Técnicas de Cultura , Relação Dose-Resposta a Droga , Indometacina/farmacologia , Camundongos , Camundongos Endogâmicos , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/metabolismoRESUMO
Prostaglandin E2 (PGE2) can stimulate collagen synthesis in bone at low concentrations or in the presence of cortisol. Moreover, cortisol inhibits and PGE2 stimulates the production of insulin-like growth factor (IGF-I) in cultured osteoblastic cells. Therefore, we examined the role of IGF-I in the response to PGE2. In 96-h fetal rat calvarial organ cultures, PGE2 increased, and cortisol and indomethacin decreased the medium IGF-I concentration, suggesting that both exogenous and endogenous PGs regulate IGF-I production. In the presence of cortisol, the stimulatory effects of PGE2 on medium IGF-I and incorporation of [3H] proline into collagenase-digestible protein were highly correlated (r = 0.95). When exogenous IGF-I (30 nM) was added, the stimulatory effect of PGE2 was abrogated in the absence, but not the presence, of cortisol. When we added IGF-binding proteins, which blocked the effects of IGF-I and IGF-II, collagenase-digestible protein labeling was decreased in control and cortisol-treated cultures, whereas the stimulatory effect of PGE2 was reduced, but not abrogated. We conclude that endogenous IGFs play a role in maintaining bone formation in cultured fetal rat calvariae and may mediate in part the anabolic response to PGE2. However, the PGE2 response probably involves additional IGF-independent pathways.
Assuntos
Dinoprostona/farmacologia , Feto/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Osteogênese/efeitos dos fármacos , Crânio/crescimento & desenvolvimento , Animais , Proteínas de Transporte/farmacologia , Células Cultivadas , Colagenases , Técnicas de Cultura , Feto/metabolismo , Inibidores do Crescimento/farmacologia , Hidrocortisona/farmacologia , Indometacina/farmacologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Crânio/embriologia , Timidina/farmacocinéticaRESUMO
There is evidence that estrogen decreases bone turnover in men as well as women. We therefore hypothesized that older men would show increased bone resorption in response to inhibition of the aromatase enzyme, which converts androgens to estrogen. Fifteen eugonadal men over 65 yr were treated for 9 weeks with 2.0 mg/day of anastrozole, an aromatase inhibitor. After 9 weeks of treatment, there were significant decreases in estradiol, estrone, and sex hormone-binding globulin levels by 29%, 73%, and 16%, respectively, and total testosterone increased significantly by 56%. Despite the limited decrease of estrogen and the increase in testosterone, C-telopeptide of type 1 collagen showed a progressive significant increase of 11%, 24%, and 33% (P for trend = 0.033) above baseline at 3, 6, and 9 weeks, respectively. N-telopeptide of type 1 collagen values were highly correlated with C-telopeptide of type 1 collagen, but the change in N-telopeptide of type 1 collagen was not statistically significant. Bone-specific alkaline phosphatase and N-terminal type I procollagen peptides showed significant decreases of 8% and 11% of baseline at 9 weeks. Osteocalcin decreased significantly by 30% at 18 weeks. We conclude that aromatase inhibition can reduce estrogen levels in older men, but this effect is limited, perhaps because of feedback stimulation of testosterone production, and that endogenous estrogen derived from aromatization of testosterone plays a role in bone metabolism of older men by limiting the rate of bone resorption.
Assuntos
Inibidores da Aromatase , Remodelação Óssea/fisiologia , Inibidores Enzimáticos/farmacologia , Hormônios Esteroides Gonadais/metabolismo , Gonadotropinas/metabolismo , Nitrilas/farmacologia , Triazóis/farmacologia , Idoso , Anastrozol , Biomarcadores/análise , Densidade Óssea , Humanos , Lipídeos/sangue , MasculinoRESUMO
To determine the mechanism for the anabolic effect of prostaglandins (PGs) on bone, [3H]thymidine(TdR) autoradiography was performed to localize the cells that replicate and to determine the fate of these labeled cells over time in cultured 20-day fetal rat calvariae. Treatment of bone organ cultures for 24 or 96 h with cortisol significantly decreased the number of [3H]TdR-labeled osteoblasts and periosteal cells. Treatment of bones with 0.1 or 1 microM PGE2 alone had no effect on [3H]TdR labeling of the osteoblast layer but doubled the number of labeled periosteal cells compared to control bones at 24 h. However, when both cortisol and PGE2 were added together, the number of labeled osteoblasts and periosteal cells increased. This effect was dependent on the concentration of PGE2. In bones treated with 0.1 microM PGE2 and 0.1 microM cortisol for 24 h, [3H]TdR labeling of periosteal cells was 5.5 times the labeling in bones treated with cortisol alone and osteoblast labeling increased 4.1-fold. Similar results in [3H]TdR labeling of osteoblasts and periosteal cells were found with 6-day neonatal mouse calvaria treated with PGE2 and cortisol. These data demonstrate that PGE2 in the presence of cortisol increases periosteal cell replication, which leads to an increased number of osteoblasts and increased bone formation.
Assuntos
Dinoprostona/farmacologia , Osteoblastos/citologia , Crânio/citologia , Animais , Animais Recém-Nascidos , Autorradiografia , Divisão Celular/efeitos dos fármacos , Hidrocortisona/farmacologia , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
We have studied the effects on bone of three structurally dissimilar non-steroidal anti-inflammatory drugs which inhibit prostaglandin cyclo-oxygenase activity (PGH synthase); indomethacin, flurbiprofen, and piroxicam. We used cultures of half calvaria from neonatal or fetal rats to measure effects on PGE2 production, measured by radioimmunoassay. In four day neonatal rat calvaria, indomethacin inhibited PGE2 release into the medium by 80% at 10(-8) M, while flurbiprofen and piroxicam produced similar inhibition at 10(-6) M. However, at 10(-10) M, treatment with all three compounds resulted in an increase in medium PGE2 concentration of 60 to 120%. To assess the mechanism of this effect, bones were labeled with [3H]-arachidonic acid, washed and cultured in the presence or absence of piroxicam. At 10(-6) M, piroxicam inhibited production of cyclo-oxygenase products and arachidonic acid release. However, at 10(-10) M, there was a substantial increase in labeled products, particularly PGE2, despite a further decrease in arachidonic acid release. In 21 day fetal rat cultures, flurbiprofen was found to increase PGE2 release both in control cultures and cultures which had been incubated with cortisol (10(-8) M) to reduce endogenous arachidonic acid release and supplied with exogenous arachidonic acid (10(-5) M) to provide a substrate. These results indicate that three potent inhibitors of PGH synthase can, paradoxically, increase prostaglandin production at low concentrations. The effect does not appear to be due to increased arachidonic acid release, and could be due to increased PGH synthase activity.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Osso e Ossos/metabolismo , Dinoprostona/biossíntese , Animais , Animais Recém-Nascidos , Osso e Ossos/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Inibidores de Ciclo-Oxigenase , Flurbiprofeno/farmacologia , Indometacina/farmacologia , Piroxicam/farmacologia , RatosRESUMO
Prostaglandins (PGs), particularly PGE2, are produced by bone and have powerful effects on bone metabolism. PGs have an initial, transient, direct inhibitory effect on osteoclast function. However, the major long-term effect in bone organ culture is to stimulate bone resorption by increasing the replication and differentiation of new osteoclasts. PGs also stimulate osteoclast formation in cell culture systems. Stimulation of osteoclastic bone resorption may be important in mediating bone loss in response to mechanical forces and inflammation. PGs have a biphasic effect on bone formation. At relatively low concentrations or in the presence of glucocorticoids, the replication and differentiation of osteoblasts is stimulated and bone formation is increased. This increase is associated with an increase in production of insulin-like growth factor-I (IGF-I). However, at high concentrations or in the presence of IGF-I, PGE2 inhibits collagen synthesis. In osteoblastic cell lines this inhibition can be shown to occur at the level of transcription of the collagen gene. The stimulatory effect on bone formation has been demonstrated when PGs are administered exogenously, but it is not clear how endogenous PG production affects bone formation in physiological or pathologic circumstances. The production of PGs in bone is highly regulated. The major source appears to be cells of the osteoblast lineage. A major site of regulation is at the level of the enzyme PG endoperoxide synthase (cyclooxygenase or PGH synthase). PGE2 production and PGH synthase mRNA are increased by PTH and interleukin-1 and decreased by estrogen. Glucocorticoids probably act by a different mechanism, decreasing either arachidonic acid or PGH synthase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Osso e Ossos/metabolismo , Osso e Ossos/fisiologia , Prostaglandinas/biossíntese , Prostaglandinas/fisiologia , Animais , Reabsorção Óssea/fisiopatologia , Humanos , Osteogênese/fisiologiaRESUMO
Biochemical markers of bone resorption have been used to characterize metabolic bone disease and assess therapeutic response. Most studies have used the urinary measurement of collagen crosslinks, but serum assays have recently been developed that may have less analytic and biologic variability. In the present study, we measured urine and serum N- and C-terminal crosslinked telopeptides of type I collagen (NTX and CTX) and serum bone sialoprotein (BSP) in postmenopausal women with or without hormone replacement therapy (HRT) and in men of similar age. In these populations, the variability of serum and urine markers was similar, except that serum NTX showed somewhat lower variability in postmenopausal women. Urine and serum assays correlated well with one another and were significantly lower in postmenopausal women on HRT compared with untreated women. The difference in women on HRT was similar for sNTX, uNTX and BSP (35-40%) and greater for sCTX and uCTX (52-53%). There was an inverse correlation between markers and bone mineral density, largely attributable to the high correlation in women not on HRT. Fractional excretion of NTX and CTX were estimated at 0.20+/-0.07 and 0.44+/-0.11, respectively. These values were independent of the concentration of the marker or of creatinine in the urine. We conclude that serum markers are useful measures of bone resorption in these populations, in whom the use of such markers is likely to be helpful in the management of osteoporosis.
Assuntos
Reabsorção Óssea/sangue , Reabsorção Óssea/urina , Colágeno/sangue , Colágeno/urina , Sialoglicoproteínas/sangue , Sialoglicoproteínas/urina , Adulto , Idoso , Densidade Óssea/fisiologia , Terapia de Reposição de Estrogênios , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Osteoporose Pós-Menopausa/sangue , Osteoporose Pós-Menopausa/tratamento farmacológico , Osteoporose Pós-Menopausa/urinaRESUMO
There is controversy concerning the effects of progestins on bone. Norethindrone acetate (NETA) is synthetic progesterone that also has estrogenic and androgenic effects. We tested its effects on hormone levels, lipids and biochemical markers of bone turnover in postmenopausal women who were on estrogen replacement therapy. Women were treated with NETA, 5 mg/d for 9 weeks. Estrogenic effects included a marked lowering of follicle stimulating hormone and luteinizing hormone. Androgenic effects included a decrease in sex hormone binding globulin and HDL cholesterol. Bone turnover showed inconsistent responses. Among markers of bone formation, bone specific alkaline phosphatase decreased significantly by 23% while procollagen peptides and osteocalcin showed a non-significant increase. The marker of bone resorption, N-telopeptide crosslinks of collagen, decreased by 19% at 6 weeks. These results indicate that NETA does not have a potent short-term anabolic effect on bone but does have effects that are likely to be mediated through the estrogen and androgen receptors.