RESUMEN
To determine the role of endogenous glucocorticoids in bone, we previously developed transgenic mice in which a 2.3 kb fragment of the Col1a1 promoter drives 11beta-hydroxysteroid dehydrogenase 2 expression in mature osteoblasts. This transgene should inactivate glucocorticoids upstream of all receptor signaling pathways. In the present study, we show that femoral cortical bone area and thickness were approximately 10-15% lower in transgenic mice than in wild-type littermates. Femur length was unchanged, indicating that bone elongation was not affected in this model. Expression of osteocalcin mRNA, pOBCol2.3-GFP (a green fluorescent protein marker of mature osteoblasts), and the formation of mineralized nodules were impaired in ex vivo transgenic primary calvarial cultures. The extent of crystal violet staining in bone marrow cultures, indicative of the number of adherent stromal cells, was also decreased. These data suggest that endogenous glucocorticoids are required for cortical bone acquisition and full osteoblast differentiation. It appears that blocking glucocorticoid signaling in vivo leads to a decrease in the commitment and/or expansion of progenitors entering the osteoblast lineage.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasas/genética , Glucocorticoides/metabolismo , Osteoblastos/citología , 11-beta-Hidroxiesteroide Deshidrogenasas/fisiología , Animales , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Linaje de la Célula , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Ratas , Transducción de SeñalRESUMEN
We identified quantitative trait loci (QTL) that determined the genetic variance in serum IGF-I through genome-wide scanning of mice derived from C57BL/6J(B6) x C3H/HeJ(C3H) intercrosses. One QTL (Igf1s2), on mouse chromosome 10 (Chr10), produces a 15% increase in serum IGF-I in B6C3 F2 mice carrying c3 alleles at that position. We constructed a congenic mouse, B6.C3H-10 (10T), by backcrossing c3 alleles from this 57-Mb region into B6 for 10 generations. 10T mice have higher serum and skeletal IGF-I, greater trabecular bone volume fraction, more trabeculae, and a higher number of osteoclasts at 16 wk, compared with B6 (P < 0.05). Nested congenic sublines generated from further backcrossing of 10T allowed for recombination and produced four smaller sublines with significantly increased serum IGF-I at 16 wk (i.e. 10-4, 10-7, 10-10, and 10-13), compared with B6 (P < 0.0003), and three smaller sublines that showed no differences in IGF-I vs. age- and gender-matched B6 mice. Like 10T, the 10-4 nested sublines at 16 wk had higher femoral mineral (P < 0.0001) and greater trabecular connectivity density with significantly more trabeculae than B6 (P < 0.01). Thus, by comprehensive phenotyping, we were able to narrow the QTL to an 18.3-Mb region containing approximately 148 genes, including Igf1 and Elk-3(ETS domain protein). Allelic differences in the Igf1s2 QTL produce a phenotype characterized by increased serum IGF-I and greater peak bone density. Congenic mice establish proof of concept of shared genetic determinants for both circulating IGF-I and bone acquisition.
Asunto(s)
Densidad Ósea/genética , Remodelación Ósea/genética , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Animales , Composición Corporal/genética , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Células Cultivadas , Mapeo Cromosómico , Cromosomas de los Mamíferos , Femenino , Fémur/anatomía & histología , Fémur/fisiología , Expresión Génica , Hígado/fisiología , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Fenotipo , Células del Estroma/citología , Células del Estroma/fisiologíaRESUMEN
CREM, the cyclic adenosine monophosphate (cAMP) responsive element modulator, belongs to a multigene family of cAMP-responsive transcription factors. CREM encodes a variety of different isoforms by utilizing four promoters and a complex pattern of alternative messenger ribonucleic acid (mRNA) splicing. We showed previously that parathyroid hormone induces the CREM P2 promoter products known as ICER (inducible cAMP early repressor) in osteoblasts. Herein we report that osteoblasts also express at least 15 CREM transcripts initiated from the P1 promoter, including 7 novel transcripts that result from alternative splicing. It is of interest that we found that CREM-X contains both exon theta1, previously identified only in P3 promoter products, and a new exon termed L, which is located upstream of exon theta1.
Asunto(s)
AMP Cíclico , Proteínas de Unión al ADN/metabolismo , Osteoblastos/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Células 3T3 , Empalme Alternativo/efectos de los fármacos , Animales , Animales Recién Nacidos , Secuencia de Bases , Clonación Molecular , Modulador del Elemento de Respuesta al AMP Cíclico , Proteínas de Unión al ADN/genética , Masculino , Ratones , Datos de Secuencia Molecular , Osteoblastos/efectos de los fármacos , Hormona Paratiroidea/farmacología , Isoformas de Proteínas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cráneo/efectos de los fármacos , Cráneo/metabolismo , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
We previously showed that parathyroid hormone (PTH) induces inducible cAMP early repressor (ICER) in osteoblastic cells and mouse calvariae. PTH signaling in osteoblastic cells is transduced by PTH receptor 1, which is coupled to cAMP-protein kinase A (PKA), protein kinase C (PKC), and calcium signaling pathways. In the present study, we examined the role of these pathways in mediating PTH-induced ICER mRNA and protein expression in osteoblastic MC3T3-E1 cells. Using RT-PCR, we found that PTH(1-34), forskolin (FSK), and 8-bromo-cAMP (8Br-cAMP) induced ICER expression, while phorbol myristate acetate (PMA), ionomycin, and PTH(3-34) did not. Similar results were found for the induction of ICER protein. PKA inhibition by H89 markedly reduced PTH- and FSK-induced ICER expression, while PKC depletion by PMA had little effect. We also tested ICER induction by other osteotropic signaling agonists. Other cAMP-PKA pathway activators, such as PTH-related protein (PTHrP), induced ICER expression, while agents that signal through other pathways did not. PTHrP maximally induced ICER mRNA at 2-4 h, which then returned to baseline by 10 h. Finally, PTH, FSK, and PTHrP induced ICER in cultured mouse calvariae and osteoblastic ROS 17/2.8, UMR-106, and Pyla cells. We conclude that ICER expression in osteoblasts requires activation of the cAMP-PKA signaling pathway.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Osteoblastos/fisiología , Proteínas Represoras , Transducción de Señal/fisiología , Células 3T3 , Animales , Colforsina/farmacología , Modulador del Elemento de Respuesta al AMP Cíclico , Inhibidores Enzimáticos/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Ratones , Ratones Endogámicos , Osteoblastos/citología , Hormona Paratiroidea/farmacología , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Embarazo , ARN Mensajero/análisis , Transducción de Señal/efectos de los fármacos , Cráneo/citologíaRESUMEN
The goals of this study were to examine the role of insulin-like growth factor I (IGF-I) on bone formation and to test the hypothesis that the inhibitory effects of glucocorticoids on bone formation are independent of the IGF-I pathway. In serum-free organ cultures of 18-day fetal mouse calvariae derived from Igf1 null mice (Igf1-/-) and their wild-type (Igf1+/+) and heterozygous (Igf1+/-) littermates, we measured the incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP), percent collagen synthesis (PCS), the incorporation of [3H]thymidine into DNA, and messenger RNA (mRNA) levels of osteoblast markers in the presence or absence of dexamethasone. After 24 h of culture, calvariae of all genotypes had similar levels of PCS. However, after 48-96 h of culture, PCS was significantly lower in Igf1-/- calvariae compared with Igf1+/+ calvariae. Treatment of calvariae with 100 nM of dexamethasone for 48-96 h decreased PCS in all genotypes. After 72 h of culture, [3H]thymidine incorporation was similar in all genotypes and 100 nM dexamethasone caused a significant reduction in [3H]thymidine incorporation in all genotypes. Dexamethasone at 100 nM decreased alpha1(I)-collagen (Colla1) mRNA and increased alkaline phosphatase, bone sialoprotein, and osteopontin mRNA in all genotypes after 72 h of culture. Type I IGF receptor mRNA levels were highest in Igf1-/- calvarial cultures. Dexamethasone at 100 nM increased Igf2 and type I IGF receptor mRNA levels in all genotypes. We conclude that one intact allele for Igf1 is sufficient to maintain normal rates of collagen synthesis in fetal mouse calvarial cultures. Moreover, the inhibitory effects of glucocorticoids on collagen synthesis and cell replication are at least partially independent of the IGF-I pathway in this model.
Asunto(s)
Colágeno/biosíntesis , Glucocorticoides/farmacología , Factor I del Crecimiento Similar a la Insulina/genética , Cráneo/efectos de los fármacos , Cráneo/embriología , Animales , Biomarcadores/análisis , Calcio/metabolismo , Colágeno/genética , ADN/biosíntesis , Dexametasona/farmacología , Desarrollo Embrionario y Fetal/efectos de los fármacos , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genotipo , Cinética , Ratones , Ratones Noqueados , Técnicas de Cultivo de Órganos , Tamaño de los Órganos/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Cráneo/metabolismoRESUMEN
We examined the effect of parathyroid hormone and various signaling molecules on collagen synthesis and chloramphenicol acetyltransferase activity in cultured transgenic mouse calvariae carrying fusion genes of the rat Col1a1 promoter and the chloramphenicol acetyltransferase reporter. After 48 h of culture, parathyroid hormone, forskolin, dibutyryl cAMP, 8-bromo cAMP, and phorbol myristate acetate inhibited transgene activity, while the calcium ionophore ionomycin had no effect. Pretreatment of calvariae with the phosphodiesterase inhibitor isobutylmethylxanthine potentiated the inhibitory effect of 1 nM parathyroid hormone on transgene activity and collagen synthesis. Parathyroid hormone further inhibited transgene activity and collagen synthesis in the presence of phorbol myristate acetate. Parathyroid hormone inhibition of transgene activity and collagen synthesis was not affected by indomethacin or interleukin-6. After 48 h of culture, parathyroid hormone inhibited chloramphenicol acetyltransferase activity by 50-85% in cultured calvariae carrying transgenes having progressive 5' upstream deletions of promoter DNA down to -1683 bp. These data show that the inhibitory effect of parathyroid hormone on Col1a1 expression in mouse calvariae is mediated mainly by the cAMP signaling pathway. Prostaglandins and IL-6 are not local mediators of the parathyroid hormone response in this model. Finally, regions of the Col1a1 promoter downstream of -1683 bp are sufficient for parathyroid hormone inhibition of the Col1a1 promoter.
Asunto(s)
Colágeno/biosíntesis , Colágeno/genética , Hormona Paratiroidea/farmacología , Cráneo/metabolismo , Animales , AMP Cíclico/metabolismo , Ratones , Ratones Transgénicos , Procolágeno/genética , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
We examined the effect of parathyroid hormone (PTH) on basic fibroblast growth factor-2 (FGF-2) and FGF receptor (FGFR) expression in osteoblastic MC3T3-E1 cells and in neonatal mouse calvariae. Treatment of MC3T3-E1 cells with PTH(1-34) (10-8M) or forskolin (FSK; 10-5M) transiently increased a 7 kb FGF-2 transcript with a peak at 2 h. The PTH increase in FGF-2 mRNA was maintained in the presence of cycloheximide. PTH also increased FGFR-1 mRNA at 2 h and transiently increased FGFR-2 mRNA at 1 h. FGFR-3 and FGFR-4 mRNA transcripts were not detected in MC3T3-E1 cells. In cells transiently transfected with an 1800-bp FGF-2 promoter-luciferase reporter, PTH and FSK increased luciferase activity at 2 h and 4 h. Immunohistochemistry showed that PTH and FSK increased FGF-2 protein labeling in the nuclei of MC3T3-E1 cells. PTH also increased FGF-2 mRNA, and FGFR-1 and FGFR-2 mRNA levels within 30 minutes in neonatal mouse calvarial organ cultures. We conclude that PTH and cAMP stimulate FGF-2 mRNA abundance in part through a transcriptional mechanism. PTH also regulated FGFR gene expression. We hypothesize that some effects of PTH on bone remodeling may be mediated by regulation of FGF-2 and FGFR expression in osteoblastic cells.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Osteoblastos/metabolismo , Hormona Paratiroidea/fisiología , Proteínas Tirosina Quinasas , ARN Mensajero/biosíntesis , Receptores de Factores de Crecimiento de Fibroblastos/genética , Animales , Células Cultivadas , Cicloheximida/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Tirosina Quinasas Receptoras/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos , Transcripción Genética , TransfecciónRESUMEN
Parathyroid hormone (PTH) regulates gene expression in skeletal osteoblasts mainly through the cAMP-protein kinase A (PKA) pathway. In neuroendocrine cells, activation of the cAMP-PKA signaling pathway leads to induction of the inducible cAMP early repressor (ICER), which is transcribed from an intronic promoter of the CREM gene and acts as a transcriptional repressor. To investigate whether PTH induces ICER expression in osteoblastic cells, RNA from MC3T3-E1 cells was subjected to reverse transcriptase-polymerase chain reaction using primers spanning the ICER sequence. Amplified products were subcloned, sequenced, and used as a probe for Northern blot analysis. In MC3T3-E1 cells, PTH induced ICER mRNA levels, which peaked at 2 h and declined to baseline by 8 h. Cycloheximide caused superinduction of ICER mRNA in response to PTH. In cultured mouse calvariae, PTH also induced ICER mRNA accumulation, which peaked at 2 h and returned almost to baseline by 10 h. Overexpression of ICER IIgamma decreased both baseline and PTH-stimulated prostaglandin G/H synthase 2 promoter activity in MC3T3-E1 cells. The induction of ICER represents a novel mechanism by which PTH regulates gene expression in osteoblastic cells.
Asunto(s)
AMP Cíclico/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Osteoblastos/metabolismo , Hormona Paratiroidea/farmacología , Proteínas Represoras/biosíntesis , Células 3T3 , Animales , Modulador del Elemento de Respuesta al AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cicloheximida/farmacología , Ciclooxigenasa 2 , Proteínas de Unión al ADN/genética , Femenino , Intrones , Isoenzimas/metabolismo , Ratones , Osteoblastos/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Represoras/genética , CráneoRESUMEN
The cytokine, interleukin-6 (IL-6), is produced by osteoblasts and may in part mediate parathyroid hormone (PTH)-stimulated bone resorption. The goals of the present study were: (1) to examine PTH induction of IL-6 expression in 7-day-old mouse calvarial organ cultures; (2) to assess the role of intracellular signaling pathways in this model; and (3) to determine whether PTH regulates IL-6 expression by a transcriptional mechanism. Northern blot analysis of calvarial RNA showed that PTH(1-34) at 0.1-100 nmol/L induced IL-6 mRNA at 0.5 h with a peak at 2 h. Forskolin at 10 micromol/L and 8-bromocyclic-AMP at 3 mmol/L also induced IL-6 mRNA with a peak at 2 h. Phorbol myristate acetate induced IL-6 expression, whereas ionomycin and PTH(3-34) amide, an N-terminal-truncated PTH analog that has reduced ability to activate the cAMP-PKA pathway, were much less effective. PMA pretreatment of calvariae greatly blocked IL-6 mRNA induction by a subsequent dose of PMA and decreased induction by PTH and forskolin to a much lesser extent. A reverse-transcriptase polymerase chain reaction (RT-PCR) assay was used to measure IL-6 heterogeneous nuclear RNA (hnRNA) and mRNA. A 5' primer spanning exons 1 and 2 and a 3' primer complementary to exon 5 of the murine IL-6 gene were used to detect IL-6 mRNA as a 638 bp product. A 5' primer corresponding to intron 4 of the murine IL-6 gene and the 3' primer were used to detect IL-6 hnRNA as a 370 bp product. RT-PCR of total calvarial RNA showed that the induction of IL-6 hnRNA by PTH and other agonists was similar to their induction of IL-6 mRNA. These data support the conclusion that PTH transcriptionally induces IL-6 gene expression in murine calvarial organ cultures mainly through the cAMP-PKA signaling pathway.
Asunto(s)
Interleucina-6/genética , Hormona Paratiroidea/farmacología , ARN Nuclear Heterogéneo/biosíntesis , ARN Mensajero/biosíntesis , Cráneo/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Animales Recién Nacidos , Núcleo Celular , Colforsina/farmacología , Cartilla de ADN/química , Interleucina-6/metabolismo , Ionomicina/farmacología , Ratones , Técnicas de Cultivo de Órganos , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Cráneo/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Transcripción GenéticaRESUMEN
Interleukin-1 (IL-1) inhibits collagen synthesis in osteoblastic cell lines and primary osteoblast-like cells. However, promoter elements regulating type I collagen A1 (COLIA1) expression in vivo and in organ culture may differ from those regulating expression in cell culture. We have examined the effects of IL-1 on reporter gene activity in neonatal transgenic mouse calvariae bearing COLIA1 promoter-chloramphenicol acetyltransferase (ColCAT) fusion genes. The parent construct, ColCAT 3.6, contains 3.5 kb of 5' flanking sequence and 115 bp of 5' untranslated region fused to the CAT reporter. In 48-h calvarial organ cultures, IL-1 repressed ColCAT 3.6 promoter activity and collagen synthesis in a dose-related manner, with a maximal inhibition of 40-65%. This repression was retained in 5' deletion constructs truncated to-1719 bp. The inhibition of transgene mRNA was blocked by cycloheximide, indicating a requirement for new protein synthesis. Pretreatment with indomethacin diminished the inhibitory effect of IL-1 on CAT activity and collagen synthesis, suggesting partial mediation by prostaglandins. Local in vivo injection of IL-1 (500 ng) decreased calvarial transgene mRNA after 8 h, an effect that was partially blocked by indomethacin. ColCAT transgenic mice represent a useful model for in vitro and in vivo assessment of COLIA promoter regulation by cytokines and other factors.
Asunto(s)
Cloranfenicol O-Acetiltransferasa/genética , Colágeno/genética , Interleucina-1/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Cráneo/efectos de los fármacos , Animales , Cloranfenicol O-Acetiltransferasa/antagonistas & inhibidores , Colágeno/antagonistas & inhibidores , Cicloheximida/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genes Reporteros/efectos de los fármacos , Indometacina/farmacología , Ratones , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/antagonistas & inhibidores , Cráneo/metabolismoRESUMEN
We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on organ cultures of transgenic mouse calvariae containing segments of the Col1a1 promoter extending to -3518, -2297, -1997, -1794, -1763, and -1719 bp upstream of the transcription start site fused to the chloramphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)2D3 had a dose-dependent inhibitory effect on the expression of the -3518 bp promoter construct (ColCAT3.6), with maximal inhibition of about 50% at 10 nM. This level of inhibition was consistent with the previously observed effect on the endogenous Col1a1 gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)2D3, suggesting that the sequences required for 1, 25(OH)2D3 inhibition are downstream of -1719 bp. The inhibitory effect of 1,25(OH)2D3 on transgene mRNA was maintained in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the inhibitory effect on Col1a1 gene transcription does not require de novo protein synthesis. We also examined the in vivo effect of 1,25(OH)2D3 treatment of transgenic mice on ColCAT activity, and found that 48 h treatment caused a dose-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25(OH)2D3 inhibits Col1A1 promoter activity in transgenic mouse calvariae, both in vivo and in vitro. The results indicate that there is a 1, 25(OH)2D3 responsive element downstream of -1719 bp. The inhibitory effect does not require new protein synthesis.
Asunto(s)
Calcitriol/farmacología , Colágeno/genética , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Cráneo/metabolismo , Animales , Animales Recién Nacidos , Cloranfenicol O-Acetiltransferasa/genética , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Genes Reporteros , Ratones , Ratones Transgénicos , Mutagénesis , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero , Ratas , Factores de TiempoRESUMEN
PTH increased PG synthase-2 transcription in osteoblastic MC3T3-E1 cells at 30 min, as assessed by nuclear run-on assays. To determine the signaling pathways used by PTH, the activity of a construct containing the PG synthase-2 gene between nucleotides -963 and +70 linked to a luciferase reporter was analyzed in stably transfected MC3T3-E1 cells. Agents that activate the cAMP-protein kinase A or protein kinase C pathways increased PG synthase-2 promoter activity. In contrast, the calcium ionophore ionomycin was ineffective. The protein kinase A inhibitor H89 blocked PTH stimulation of PG synthase-2 promoter activity, whereas an overnight pre-incubation with phorbol ester to down-regulate protein kinase C did not. PTH-(3-34), a peptide that has greatly reduced ability to activate the cAMP-protein kinase A pathway, did not increase PG synthase-2 transcription or promoter activity. PTH could induce PG synthase-2 messenger RNA accumulation and PG synthase-2 transcription in the presence of cycloheximide. In addition, PTH-stimulated PG synthase-2 transcription was maintained at a high level at 2 h in the presence of cycloheximide. We conclude that PTH rapidly increases PG synthase-2 transcription in MC3T3-E1 cells, mainly through a cAMP-protein kinase A-mediated pathway without the need for protein synthesis. In contrast, the attenuation of increased PG synthase-2 transcription by PTH requires de novo protein synthesis.
Asunto(s)
AMP Cíclico/metabolismo , Osteoblastos/enzimología , Hormona Paratiroidea/farmacología , Fragmentos de Péptidos/farmacología , Prostaglandina-Endoperóxido Sintasas/genética , Transcripción Genética/efectos de los fármacos , Animales , Calcio/metabolismo , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cicloheximida/farmacología , Inducción Enzimática/efectos de los fármacos , Ratones , Proteína Quinasa C/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Transducción de Señal , TransfecciónRESUMEN
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.
Asunto(s)
Colágeno/biosíntesis , Hidrocortisona/farmacología , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Cráneo/efectos de los fármacos , Cráneo/metabolismo , Animales , ADN/biosíntesis , Feto , Humanos , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Técnicas de Cultivo de Órganos , Ratas , Proteínas Recombinantes/farmacologíaRESUMEN
The objective of this study was to determine the acute effects of glucocorticoids on in vivo levels of bone collagen synthesis in neonatal mice. Mice were injected with vehicle or dexamethasone at the start of the experiment. At 22 h, mice were given a 10 microCi injection of [3H]proline. At 24 h, the mice were sacrificed and the incorporation of [3H]proline into collagenase-digestible CDP labeling) and noncollagen (NCP labeling) protein in calvariae were determined by digestion with bacterial collagenase. Calvarial RNA was analyzed for COL 1A1 and osteocalcin mRNA levels by Northern blotting. After 24 h, vehicle-treated mice showed a 9.8 +/- 1.0% weight gain while dexamethasone-treated mice (1 mg/kg) had a 7.4 +/- 0.8% weight loss. Dexamethasone (1 mg/kg) decreased CDP and NCP labeling in calvariae by 51 +/- 4% and 17 +/- 4%, respectively (13 experiments). The inhibitory effect on protein labeling was selective for collagen since dexamethasone decreased the percent collagen synthesis from 25.4 +/- 1.6% to 16.6 +/- 1.0% (13 experiments). Dexamethasone at 3 mg/kg also decreased CDP labeling and the percent collagen synthesis in calvariae. There was a 30% reduction in COL1A1 mRNA levels and a 67% decrease in osteocalcin mRNA levels. To determine the reversibility of the inhibition of collagen synthesis, mice were given a single injection of dexamethasone (1 mg/kg) and then injected with [3H]proline 2 h prior to sacrifice at 24, 48, or 72 h. The reduction in CDP labeling observed at 24 h was fully reversed by 48-72 h. Moreover, by 72 h, the-rate of weight gain by dexamethasone-treated mice was similar to vehicle-treated controls. These data show that administration of dexamethasone to neonatal mice leads to a selective decrease in bone collagen synthesis within 24 h that is accompanied by down-regulation of osteocalcin and COL1A1 mRNA levels. This model will be useful in determining mechanisms by which high dose glucocorticoids inhibit bone formation in vivo.
Asunto(s)
Colágeno/biosíntesis , Dexametasona/toxicidad , Glucocorticoides/toxicidad , Análisis de Varianza , Animales , Animales Recién Nacidos , Northern Blotting , Desarrollo Óseo/efectos de los fármacos , Colágeno/genética , Dexametasona/administración & dosificación , Relación Dosis-Respuesta a Droga , Marcaje Isotópico , Ratones , Osteocalcina/genética , Osteocalcina/metabolismo , Prolina/metabolismo , ARN Mensajero/metabolismo , Cráneo/citología , Cráneo/metabolismo , Transcripción Genética/efectos de los fármacos , Tritio/metabolismoRESUMEN
Prostaglandin G/H synthase (PGHS), a central enzyme for PG synthesis, is encoded by the constitutively expressed PGHS-1 and the inducible PGHS-2. The goal of this project was to study the regulation of PGHS-2 gene expression by PTH and its possible signaling pathways in osteoblastic MC3T3-E1 cultures. Bovine PTH-(1-34) at 0.01-10 nM increased PGHS-2, but not PGHS-1, messenger RNA (mRNA) levels. The effect of PTH was maximal at 1 h and decreased almost to control levels by 6 h. Phorbol myristate acetate (PMA), forskolin, and 8-bromo-cAMP increased PGHS-2 mRNA levels, whereas ionomycin had no effect. PTH, forskolin, and PMA increased the release of PGE2 into the culture medium. Pretreatment of cells with 0.1 microM PMA for 16 h blocked the induction of PGHS-2 mRNA levels by PMA, but did not alter the effects of PTH and forskolin. However, treatment of cells with 30 microM H-89, a protein kinase A inhibitor, significantly reduced the ability of PTH and forskolin to induce PGHS-2 mRNA levels. Moreover, PTH-(3-34) at 0.1-100 nM did not induce PGHS-2 mRNA levels. Our results show that PTH can rapidly and transiently induce PGHS-2 mRNA levels in osteoblastic MC3T3-E1 cells, primarily via the cAMP-protein kinase A signal transduction pathway. Induction of PGHS-2 may play a key role in mediating some actions of PTH on bone metabolism and gene expression.
Asunto(s)
AMP Cíclico/fisiología , Osteoblastos/enzimología , Hormona Paratiroidea/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Bovinos , Línea Celular , Ratones , Prostaglandina-Endoperóxido Sintasas/genética , Proteína Quinasa C/fisiología , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Our previous studies have shown that the 49-base pair region of promoter DNA between -1719 and -1670 base pairs is necessary for transcription of the rat COL1A1 gene in transgenic mouse calvariae. In this study, we further define this element to the 13-base pair region between -1683 and -1670. This element contains a TAAT motif that binds homeodomain-containing proteins. Site-directed mutagenesis of this element in the context of a COL1A1-chloramphenicol acetyltransferase construct extending to -3518 base pairs decreased the ratio of reporter gene activity in calvariae to tendon from 3:1 to 1:1, suggesting a preferential effect on activity in calvariae. Moreover, chloramphenicol acetyltransferase-specific immunofluorescence microscopy of transgenic calvariae showed that the mutation preferentially reduced levels of chloramphenicol acetyltransferase protein in differentiated osteoblasts. Gel mobility shift assays demonstrate that differentiated osteoblasts contain a nuclear factor that binds to this site. This binding activity is not present in undifferentiated osteoblasts. We show that Msx2, a homeodomain protein, binds to this motif; however, Northern blot analysis revealed that Msx2 mRNA is present in undifferentiated bone cells but not in fully differentiated osteoblasts. In addition, cotransfection studies in ROS 17/2.8 osteosarcoma cells using an Msx2 expression vector showed that Msx2 inhibits a COL1A1 promoter-chloramphenicol acetyltransferase construct. Our results suggest that high COL1A1 expression in bone is mediated by a protein that is induced during osteoblast differentiation. This protein may contain a homeodomain; however, it is distinct from homeodomain proteins reported previously to be present in bone.
Asunto(s)
Colágeno/biosíntesis , Colágeno/genética , Osteoblastos/metabolismo , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Sitios de Unión , Huesos/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cartilla de ADN , Proteínas de Unión al ADN/biosíntesis , Proteínas de Homeodominio/biosíntesis , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Osteoblastos/citología , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Ratas , Proteínas Recombinantes/biosíntesis , Cráneo/metabolismo , TransfecciónRESUMEN
Previous deletion studies using a series of COL1A1-CAT fusion genes have indicated that the 625 bp region of the COL1A1 upstream promoter between -2295 and -1670 bp is required for high levels of expression in bone, tendon, and skin of transgenic mice. To further define the important sequences within this region, a new series of deletion constructs extending to -1997, -1794, -1763, and -1719 bp has been analyzed in transgenic mice. Transgene activity, determined by measuring CAT activity in tissue extracts of 6- to 8-day-old transgenic mouse calvariae, remains high for all the new deletion constructs and drops to undetectable levels in calvariae containing the -1670 bp construct. These results indicate that the 49 bp region of the COL1A1 promoter between -1719 and -1670 bp is required for high COL1A1 expression in bone. Although deletion of the same region caused a substantial reduction of promoter activity in tail tendon, the construct extending to -1670 bp is still expressed in this tissue. However, further deletion of the promoter to -944 bp abolished activity in tendon. Gel mobility shift studies identified a protein in calvarial nuclear extracts that is not found in tendon nuclear extracts, which binds within this 49 bp region. Our study has delineated sequences in the COL1A1 promoter required for expression of the COL1A1 gene in high type I collagen-producing tissues, and suggests that different cis elements control expression of the COL1A1 gene in bone and tendon.
Asunto(s)
Colágeno/genética , Regulación de la Expresión Génica , Transgenes , Animales , Composición de Base , Secuencia de Bases , Colágeno/biosíntesis , Colágeno/aislamiento & purificación , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Eliminación de Secuencia , Piel/metabolismo , Cráneo/metabolismo , Tendones/metabolismoRESUMEN
The regulation of COL1A1 gene expression in bone was studied by measuring the activity of type I collagen promoter fusion genes (ColCAT) in permanently transfected osteoblastic cells and calvariae from transgenic animals. The basal activity of ColCAT fusion genes in transfected cells is mediated by DNA sequences between -3.5 to -2.3 kb while expression in vivo requires sequences between -2.3 and -1.7 kb. Parathyroid hormone, 1,25-dihydroxyvitamin D3 and interleukin-1 decrease the activity of ColCAT fusion genes in osteoblastic cells and transgenic calvariae. Because there may be differences between the expression of ColCAT fusion genes in cultured cells and intact bone, it will be important to compare data obtained from transfected cells with an in vivo model such as calvariae from transgenic mice.
Asunto(s)
Desarrollo Óseo/genética , Huesos/metabolismo , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/genética , Regulación del Desarrollo de la Expresión Génica/genética , Vitamina D/análogos & derivados , Animales , Fusión Artificial Génica/métodos , Desarrollo Óseo/efectos de los fármacos , Huesos/efectos de los fármacos , Células Cultivadas , Cadena alfa 1 del Colágeno Tipo I , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1/farmacología , Ratones , Ratones Transgénicos , Modelos Animales , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Hormona Paratiroidea/farmacología , Regiones Promotoras Genéticas/genética , Ratas , Cráneo/efectos de los fármacos , Cráneo/metabolismo , Vitamina D/farmacologíaRESUMEN
The synthesis of type I collagen in bone cells is inhibited by the calcium-regulating hormone 1,25-dihydroxyvitamin D3. Earlier work from our laboratories has indicated that vitamin D regulation is at the level of transcription, based on results from both nuclear run-off assays and functional promoter analysis of a hybrid gene consisting of a 3.6 kb COL1A1 promoter fragment fused to the chloramphenicol acetyltransferase reporter gene. In the present study, we investigated the molecular basis for vitamin D-mediated transcriptional repression of the COL1A1 gene and report the identification of a region within the COL1A1 upstream promoter (the HindIII-Pstl restriction fragment between nucleotides -2295 and -1670) which is necessary for 1,25-dihydroxyvitamin D3 responsiveness in osteoblastic cells. This hormone-mediated inhibitory effect on the marker gene parallels the inhibition of the endogenous collagen gene. A 41 bp fragment from this region (between nucleotides -2256 and -2216) contains a sequence which is very similar to vitamin D-responsive elements identified in the osteocalcin gene. Extracts from cultured cells which express a high level of vitamin D receptor contain a hormone:receptor complex that binds specifically to this 41 bp fragment, as demonstrated by bandshift analysis. However, deletion of this vitamin D receptor binding region from either a -3.5 kb or a -2.3 kb promoter fragment did not abolish vitamin D responsiveness. These results indicate that a vitamin D response element similar to that described for other vitamin D responsive genes (osteocalcin and osteopontin) does not alone mediate the repression of COL1A1 by 1,25-dihydroxyvitamin D3.
Asunto(s)
Calcitriol/farmacología , Colágeno/genética , Regulación de la Expresión Génica , Osteoblastos/metabolismo , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , Mapeo Cromosómico , Eliminación de Gen , Humanos , Ratones , Datos de Secuencia Molecular , Osteoblastos/citología , Regiones Promotoras Genéticas , Unión Proteica , Receptores de Calcitriol/metabolismo , Transcripción Genética/efectos de los fármacos , Vitamina D/metabolismo , Proteína de Unión a Vitamina D/metabolismoRESUMEN
The activity of fusion genes containing fragments of the COL1A1 promoter was measured in tissues from 6- to 8-day-old transgenic mice. ColCAT3.6 contains approximately 3.6 kb (-3521 to 115 bp) of the rat COL1A1 gene, the chloramphenicol acetyltransferase (CAT) reporter gene, and the SV40 splice and polyadenylation sequences. ColCAT2.3 and ColCAT1.7 are deletion constructs that contain 2296 and 1667 bp of COL1A1 upstream from the RNA start site, respectively. For each transgene, up to six lines of mice were characterized. Both ColCAT3.6 and ColCAT2.3 had similar activity in bone and tooth; ColCAT1.7 was inactive. In transgenic calvariae, levels of transgene mRNA paralleled levels of CAT activity. In tendon, the activity of ColCAT2.3 was 3- to 4-fold lower than that of ColCAT3.6, and the activity ColCAT1.7 was 16-fold lower than that of ColCAT2.3. There was little activity of the ColCAT constructs in liver and brain. These data show that DNA sequences between -2.3 and -1.7 kb are required for COL1A1 promoter expression in bone and tooth; sequences that control expression in tendon are distributed between -3.5 and -1.7 kb of the promoter, with sequences downstream of -1.7 kb still capable of directing expression to this tissue. The cis elements that govern basal expression of COL1A1 in transgenic calvariae appear to be different from those required for optimal expression of the COL1A1 promoter in stably transfected osteoblastic cells.