RESUMEN
Androgens play important endocrine roles in development and physiology. Here, we characterize activities of two "Andro" prohormones, androstenedione (A-dione) and 4-androsten-3beta,17beta-diol (A-diol) in MDA-MB-453 (MDA) and LNCaP cells. A-dione and A-diol, like cyproterone acetate, were partial agonists of transfected mouse mammary tumor virus (MMTV) and endogenous prostate-specific antigen (PSA) promoters. Different from bicalutamide but similar to CPA, both are inducers of LNCaP cell proliferation with only mild suppression of 5alpha-dihydrotestosterone (DHT)-enhanced cell growth. Like bicalutamide and cyproterone acetate, A-dione and A-diol significantly antagonized DHT/R1881-induced PSA expression by up to 30% in LNCaP cells. Meanwhile, in MDA cells, EC(50)s for the MMTV promoter were between 10 and 100nM. Co-factor studies showed GRIP1 as most active for endogenous androgen receptor (AR), increasing MMTV transcription by up to five-fold, without substantially altering EC(50)s of DHT, A-dione or A-diol. Consistent with their transcriptional activities, A-dione and A-diol bound full-length endogenous AR from MDA or LNCaP cells with affinities of 30-70nM, although binding to expressed ligand-binding domain (LBD) was >20-fold weaker. In contrast, DHT, R1881, and bicalutamide bound similarly to LBD or aporeceptor. Together, these data suggest that A-dione and A-diol are ligands for AR with partial agonist/antagonist activities in cell-based transcription assays. Binding affinities for both are most accurately assessed by AR aporeceptor complex. In addition to being testosterone precursors in vivo, either may impart its own transcriptional regulation of AR.
Asunto(s)
Androstenodiol/farmacología , Androstenodiona/farmacología , Neoplasias de la Mama/patología , Regiones Promotoras Genéticas/genética , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/patología , Proteínas Adaptadoras Transductoras de Señales , Antagonistas de Andrógenos/farmacología , Antagonistas de Receptores Androgénicos , Andrógenos/farmacología , Anilidas/farmacología , Animales , Neoplasias de la Mama/genética , Células COS , Proteínas Portadoras/metabolismo , División Celular/efectos de los fármacos , Chlorocebus aethiops , Acetato de Ciproterona/farmacología , Dihidrotestosterona/farmacología , Humanos , Ligandos , Macaca mulatta/genética , Masculino , Virus del Tumor Mamario del Ratón/genética , Ratones , Proteínas del Tejido Nervioso/metabolismo , Nitrilos , Neoplasias de la Próstata/genética , Receptores AMPA/metabolismo , Receptores Androgénicos/genética , Compuestos de Tosilo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
LXR and PPAR receptors belong to the nuclear receptor superfamily of transcriptional activating factors. Using ligand-dependent transcription assays, we found that 5-tetradecyloxy-2-furancarboxylic acid (TOFA) transactivates chimeric receptors composed of the glucocorticoid receptor DNA binding domain and the ligand binding regions of PPARalpha, PPARbeta (NUC-1) and LXRbeta (NER) receptors. In the same assays, ligands for PPARs (oleic acid, WY-14643 and L-631,033) and LXRs (hydroxycholesterols) maintain their respective receptor selectivity. TOFA and hydroxycholesterols also stimulate transcription from a minimal fibrinogen promoter that is under the control of AP-1 or NF-kappaB transcription factor binding sites. In addition to their effects on transcription, these LXRbeta activators induce neuronal differentiation in rat pheochromocytoma cells. TOFA and the natural LXR agonist, 22 (R)-hydroxycholesterol, stimulate neurite outgrowth in 55 and 28% of cells, respectively. No neurite outgrowth was induced by the related 22(S)-hydroxycholesterol, which does not activate the LXR family. These results suggest that the hydroxycholesterol signaling pathway has a complex effect on transcription that mediates the activity of TOFA and hydroxycholesterol on neuronal differentiation in pheochromocytoma cells.
Asunto(s)
Furanos/farmacología , Neuronas/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Transcripción Genética/efectos de los fármacos , Animales , Anticolesterolemiantes/farmacología , Células COS , Diferenciación Celular/efectos de los fármacos , Línea Celular , Colesterol/farmacología , Proteínas de Unión al ADN , Hipolipemiantes/farmacología , Receptores X del Hígado , Factores de Crecimiento Nervioso/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Ácido Oléico/farmacología , Receptores Nucleares Huérfanos , Proliferadores de Peroxisomas/farmacología , Pirimidinas/farmacología , Ratas , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/fisiología , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/fisiología , Activación Transcripcional/efectos de los fármacos , TransfecciónRESUMEN
The glucocorticoid receptor (GR) and peroxisome proliferator-activated receptors (PPARs) play important roles in the differentiation of mesenchymal cells. Glucocorticoids acting via the GR promote osteoblastic differentiation of bone marrow stromal cells, whereas PPAR ligands induce these cells to become adipocytes. To explore potential interactions between PPAR and GR pathways in osteoblasts, we studied the interaction between PPAR subtype-selective ligands and dexamethasone (DEX) in a murine calvaria-derived osteoblastic cell line (MB 1.8) that expresses endogenous GR and PPARs. In ligand-dependent transcription assays, the PPARgamma-selective ligand TZD [(5-(4-N-methyl-N(2-pyridyl)amino)ethoxy)benzyl)thiazolidine-2,4-dione], a thiazolidinedione antidiabetic, enhanced the effect of DEX to stimulate transcription of a glucocorticoid-inducible reporter gene (mouse mammary tumor virus-luciferase). No effect was seen with PPARalpha- or hNUC1/PPARdelta-selective ligands. The GR antagonist RU-486 inhibited the DEX and TZD responses, suggesting that the effects were mediated through endogenous GR. TZD also enhanced glucocorticoid-mediated transcription in SaOS-2/B10 human osteosarcomatous cells, but not in CV-1 cells, even though both cell lines were transfected with GR plasmid and expressed significant levels of endogenous PPARgamma messenger RNA. In MB 1.8 cells, TZD decreased alkaline phosphatase activity and the expression of osteoblast-associated genes while it up-regulated the adipocyte fatty acid-binding protein. DEX counteracted the effects of TZD on alkaline phosphatase enzyme activity and osteoblastic gene expression, but enhanced the actions of TZD on adipocyte fatty acid-binding protein. Interestingly, TZD inhibited in vitro bone nodule formation and mineralization, and DEX counteracted this effect. Thus, depending on the promoter context, TZD and DEX can oppose or enhance each other's actions on gene transcription. Collectively, these results point to a complex interaction between PPAR and GR signaling pathways that regulates the effects of TZD and DEX on osteoblastic differentiation. The mechanism of this interaction is still under investigation, but might involve PPAR -dependent and -independent pathways. As thiazolidinediones represent an important new class of drugs, our findings also raise the need for further studies in bone.
Asunto(s)
Osteoblastos/efectos de los fármacos , Osteoblastos/fisiología , Receptores de Glucocorticoides/fisiología , Tiazoles/farmacología , Tiazolidinedionas , Transcripción Genética/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Dexametasona/farmacología , Glucocorticoides/farmacología , Haplorrinos , Humanos , Riñón/citología , Riñón/fisiología , Ligandos , Ratones , Osteoblastos/citología , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Osteoblasts and adipocytes originate from common mesenchymal precursors. With aging, there is a decrease in osteoprogenitor cells that parallels an increase of adipocytes in bone marrow. We observed that rabbit serum (RS) induces adipocyte-like differentiation in human osteosarcoma SaOS-2/B10 and MG-63 cell lines, in rat ROS17/2.8 cells, and in mouse calvaria-derived osteoblastic MB1.8 cells, as evidenced by the accumulation of Oil Red O positive lipid vesicles and the decrease in alkaline phosphatase expression. Both SaOS-2/B10 and MG-63 cells, but not ROS17/2.8 nor MB1.8 cells, express significant levels of PPARgamma mRNA, a member of the peroxisome proliferator activated receptor (PPAR) family that has been implicated in the control of adipocyte differentiation. However, both ROS17/2.8 and MG-63 cells express significant levels of the adipocyte selective marker, aP2 fatty acid binding mRNA, which can be further increased by RS. These cell types express PPARdelta/NUC-1 but not PPARalpha, indicating that cells that do not express either PPARgamma or PPARalpha are capable of differentiating into adipocyte-like cells. Transfection experiments in COS cells showed that compared with fetal bovine serum (FBS), RS is rich in agents that stimulate PPAR-dependent transcription. The stimulatory activity was ethyl acetate extractable and was 35-fold more abundant in RS than in FBS. Purification and analysis revealed that the major components of this extract are free fatty acids. Furthermore, the same fatty acids, a mixture of palmitic, oleic, and linoleic acids, activate the PPARs and induce adipocyte-like differentiation of both ROS17/2.8 and SaOS-2/B10 cells. These findings suggest that fatty acids or their metabolites can initiate the switch from osteoblasts to adipocyte-like cells.
Asunto(s)
Adipocitos/citología , Ácidos Grasos/sangre , Osteoblastos/citología , Adipocitos/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Ácidos Grasos/aislamiento & purificación , Ácidos Grasos/fisiología , Sangre Fetal/fisiología , Humanos , Ratones , Proteínas Nucleares/fisiología , Osteoblastos/efectos de los fármacos , Conejos , Ratas , Receptores Citoplasmáticos y Nucleares/fisiología , Transactivadores/fisiología , Factores de Transcripción/fisiología , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Alendronate (4-amino-1-hydroxybutylidene-1,1-bisphosphonate) is a potent bisphosphonate that inhibits osteoclastic bone resorption and has proven effective for the treatment of osteoporosis. Its molecular mechanism of action, however, has not been defined precisely. Here we report that alendronate is a potent inhibitor of the protein-tyrosine-phosphatase-meg1 (PTPmeg1). Two substrates were employed in this study: fluorescein diphosphate and the phosphotyrosyl peptide src-pY527. With either substrate, alendronate was a slow binding inhibitor of PTPmeg1. Among the other bisphosphonates studied, alendronate was more potent and selective for PTPmeg1. The hydrolysis of fluorescein diphosphate by PTP epsilon and PTPmeg1 was sensitive to alendronate, with IC50 values of less than 1 microM; PTPsigma, however, under the same conditions, was inhibited by only 50% with 141 microM alendronate. Similarly, with the src-pY527 substrate, alendronate inhibition was also PTP dependent. Alendronate inhibited PTPmeg1 with an IC50 value of 23 microM, PTPsigma with an IC50 value of 2 microM, and did not inhibit PTP epsilon at concentrations up to 1 mM. The alendronate inhibition of these three PTPs and two substrates is consistent with the formation of a ternary complex comprised of enzyme, substrate, and inhibitor. PTP inhibition by hisphosphonates or vanadate was diminished by the metal chelating agent EDTA, or by the reducing agent dithiothreitol, suggesting that a metal ion and the oxidation of a cysteine residue are required for full inhibition. These observations show substrate- and enzyme-specific PTP inhibition by alendronate and support the possibility that a certain PTP(s) may be the molecular target for alendronate action.
Asunto(s)
Alendronato/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , ADN Complementario , Humanos , Proteína Tirosina Fosfatasa no Receptora Tipo 4 , Proteínas Tirosina Fosfatasas/metabolismo , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
Systemic administration of prostaglandins of the E series (PGEs) has an anabolic effect in bone. A large part of this osteogenic effect is due to recruitment of osteoblasts from their precursors. However, the immediate events initiated by the administration of an anabolic dose of PGEs or their target cells within bone tissue are not known. In this study we used Northern analysis to explore the induction of early-response genes in bone tissue following a single injection of an anabolic dose of PGE2 (6 mg/kg) and in situ hybridization to localize the responding cells. The mRNA levels of c-fos, c-jun, junB and early growth response gene-1 were markedly elevated in the tibial metaphysis as early as 15 min postinjection and returned to basal level by 180-300 min. The induction of c-fos was the earliest (significant at 15 min) and the greatest (sixfold at 60 min) and that of the other genes was smaller. Early-response gene expression was induced in the calvaria as well. Numerous cells in bone marrow (both in the tibia and calvaria) expressed high levels of c-fos in response to PGE2. In the tibia, these cells were localized in the secondary spongiosa and diaphysis and were absent from the primary spongiosa. Many, but not all, expressing cells were in relative proximity to cancellous or endosteal surfaces. In the calvaria, these cells were found in the marrow "windows" within the bony plate. Mature osteoblasts and osteoclasts were negative. Based on many reports of the stimulation of cancellous bone formation in tibiae of similar animals by PGE2 and the increased bone formation we found in the calvarial marrow spaces, the best candidate for these cells is a bone marrow-resident osteoblast precursor. The induction of early-response genes may thus be the first step in a chain of events which leads to the anabolic effect of PGE2 in vivo.
Asunto(s)
Dinoprostona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genes fos/efectos de los fármacos , Genes jun/efectos de los fármacos , Proteínas Inmediatas-Precoces , Oxitócicos/farmacología , Animales , Northern Blotting , Médula Ósea/efectos de los fármacos , Células de la Médula Ósea , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , División Celular/efectos de los fármacos , División Celular/genética , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Dinoprostona/administración & dosificación , Proteína 1 de la Respuesta de Crecimiento Precoz , Regulación de la Expresión Génica/genética , Genes fos/genética , Genes jun/genética , Hibridación in Situ , Inyecciones Subcutáneas , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Oxitócicos/administración & dosificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Tibia/citología , Tibia/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genética , Dedos de Zinc/efectos de los fármacos , Dedos de Zinc/genéticaRESUMEN
Non-steroidal antiandrogens have been employed in the management of prostate cancer, but the mechanism of action is unclear due to a lack of good tissue culture models. The growth of a hamster ductus deferens cell line (DDT1) is highly dependent upon the addition of 10 nM testosterone to synthetic serum-free media. We describe a non-steroidal compound N-(4-chlorophenyl)-(Z,Z)-2,3-bis(-cyclopropylmethylene) cyclopentanecarboxamide (L-245976) which antagonizes the action of testosterone on DDT1 cells at 10 microM but exhibits little or no effect on cell growth by itself. This compound also blocks the binding of 3H-dihydrotestosterone (DHT) to the human androgen receptor (AR) with an IC50 of approximately 28 microM. In addition, L-245976 was found to antagonize DHT-dependent transactivation of the AR via the probasin gene promoter at comparable doses with no agonist activity.
Asunto(s)
Amidas/farmacología , Antagonistas de Andrógenos/metabolismo , Antagonistas de Andrógenos/farmacología , Compuestos de Anilina/farmacología , Evaluación Preclínica de Medicamentos/métodos , Conducto Deferente/metabolismo , Antagonistas de Andrógenos/química , Proteína de Unión a Andrógenos/efectos de los fármacos , Proteína de Unión a Andrógenos/genética , Proteína de Unión a Andrógenos/metabolismo , Andrógenos/metabolismo , Andrógenos/farmacología , Animales , Células CHO/metabolismo , División Celular/efectos de los fármacos , Línea Celular , Colorimetría/métodos , Cricetinae , Dihidrotestosterona/metabolismo , Relación Dosis-Respuesta a Droga , Flutamida/análogos & derivados , Flutamida/metabolismo , Flutamida/farmacología , Formazáns/metabolismo , Humanos , Masculino , Receptores Androgénicos/efectos de los fármacos , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Testosterona/farmacología , Sales de Tetrazolio/análisis , Sales de Tetrazolio/metabolismo , Tiazoles/análisis , Tiazoles/metabolismo , Activación Transcripcional , Transfección , Conducto Deferente/citología , Conducto Deferente/efectos de los fármacosRESUMEN
The mammalian peroxisome proliferator-activated receptor (PPAR) family consists of three different subtypes, PPARalpha, hNUC1/PPARdelta and PPARgamma. Selective agonists have been identified for PPARalpha and PPARgamma but not for hNUC1, and consequently little is known about the genes that are controlled by this receptor. Using ligand-dependent transcription assays in COS-7 cells, we screened a variety of PPAR activating agents to identify a selective activator of hNUC1. We found that the potent peroxisome proliferator, Wy-14643, and the PPARgamma-selective thiazolidinedione, BRL 49653, were poor activators of hNUC1 (EC50s of > 100 microM). Short chain fatty acids (FAs) appeared more selective for PPARalpha than for hNUC1, whereas the very long chain FA, erucic acid (C22:1) was more selective for hNUC1. Using erucic acid as a probe, we conducted a topological similarity search of the Merck Chemical Collection and identified a fatty acid-like compound, L-631,033 4-(2-acetyl-6-hydroxyundecyl) cinnamic acid, that was a selective activator of hNUC1 (EC50 of 2 microM), but was much less selective for PPARalpha or PPARgamma (EC50s of > 100 microM). Structure-function analysis of PPAR activation by L-631,033 structural analogues showed that receptor selectivity depends on the position of the carboxyl group relative to the phenyl ring on the molecule. Transfection experiments in several cell types: an osteoblastic cell line (MB 1.8), a mouse liver cell line (ML-457), rat aortic smooth muscle cells (RSMCs) and COS-7 cells revealed differences in the activation profile of specific ligands. The most notable differences were observed in RSMCs, where transactivation by L-631,033 and Wy-14643, but not by BRL 49653, was markedly reduced, and in MB 1.8 cells, where oleic acid failed to activate PPARs. These findings identify certain structural features in PPAR-activating agents that modulate PPAR activation, and suggest that as with other nuclear receptors, activation is cell-type specific.
Asunto(s)
Receptores Citoplasmáticos y Nucleares/agonistas , Tiazolidinedionas , Factores de Transcripción/agonistas , Animales , Anticolesterolemiantes/química , Anticolesterolemiantes/farmacología , Células COS/efectos de los fármacos , Línea Celular , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Ligandos , Ratones , Especificidad de Órganos , Pirimidinas/química , Pirimidinas/farmacología , Ratas , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/metabolismo , Rosiglitazona , Relación Estructura-Actividad , Tiazoles/química , Tiazoles/farmacología , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
The nuclear hormone receptors NUC-1 (PPAR delta) and PPAR alpha are members of the peroxisome proliferator-activated receptor (PPAR) family. The members of this receptor family are activated by agents that stimulate peroxisome proliferation, free fatty acids, prostaglandin 12 metabolites, and agents considered for the therapy of insulin-independent diabetes mellitus. To identify putative physiological agents that activate NUC-1, we tested the ability of acetone extracts of various rat tissues to activate the transcription of an MMTV-luciferase reporter gene, via a GR/NUC-1 hybrid receptor. GR/NUC-1 contains the ligand binding region of the NUC-1 receptor and the DNA binding domain of the glucocorticoid receptor. Using this assay, we found stimulatory activity in the pancreas, which upon purification and characterization was identified as methyl-palmitate, known to be enriched in pancreatic lipids. In addition, we determined that ethyl esters of palmitic and oleic acids are also potent activators of this receptor. Thus, fatty acid ester formation may control the cellular concentrations of fatty acids, and acyl-ester formation may play a role in the control of metabolic pathways and the activation of the PPAR.
Asunto(s)
Ácidos Grasos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Ácidos Grasos/farmacología , Femenino , Genes Reporteros , Técnicas In Vitro , Luciferasas/genética , Palmitatos/metabolismo , Palmitatos/farmacología , Páncreas/metabolismo , Embarazo , Ratas , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacosRESUMEN
Two forms of the transmembrane human protein tyrosine phosphatase (PTP sigma), generated by alternative splicing, were identified by cDNA cloning and Northern hybridization with selective cDNA probes. The larger form of PTP sigma is expressed in various human tissues, human osteosarcoma, and rat tibia. The hPTP sigma cDNA codes for a protein of 1911 amino acid residues and is composed of a cytoplasmic region with two PTP domains and an extracellular region that can be organized into three tandem repeats of immunoglobulin-like domains and eight tandem repeats of fibronectin type III-like domains. In the brain, the major transcript of PTP sigma is an alternatively spliced mRNA, in which the coding region for the fibronectin type III-like domains number four to seven are spliced out, thus coding for a protein of 1502 amino acid residues similar to the rat PTP sigma and rat PTP-NE3. Using in situ hybridization, we assigned hPTP sigma to chromosome 6, arm 6q and band 6q15. The bacterial-expressed hPTP sigma exhibits PTPase activity that was inhibited by orthovanadate (IC50 = 0.02 microM) and by two bisphosphonates used for the treatment of bone diseases, alendronate (ALN) (IC50 = 0.5 microM) and etidronate (IC50 = 0.2 microM). In quiescent calvaria osteoblasts, micromolar concentrations of vanadate, ALN and etidronate stimulate cellular proliferation. These findings show tissue-specific alternative splicing of PTP sigma and suggest that PTPs are putative targets of bisphosphonate action.
Asunto(s)
Empalme Alternativo , Difosfonatos/toxicidad , Inhibidores Enzimáticos/efectos adversos , Proteínas Tirosina Fosfatasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Neoplasias Óseas/patología , Encéfalo/metabolismo , División Celular/efectos de los fármacos , División Celular/genética , Cromosomas Humanos Par 6/metabolismo , Clonación Molecular , Citoplasma/metabolismo , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Datos de Secuencia Molecular , Osteosarcoma/patología , Reacción en Cadena de la Polimerasa , Proteínas Tirosina Fosfatasas/efectos de los fármacos , Proteínas Tirosina Fosfatasas/metabolismo , ARN/genética , ARN/metabolismo , Células Tumorales Cultivadas , Vanadatos/toxicidadRESUMEN
Alendronate (ALN), an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its molecular target is still unknown. This study examines the effects of ALN on the activity of osteoclast protein-tyrosine phosphatase (PTP; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), called PTPepsilon. Using osteoclast-like cells generated by coculturing mouse bone marrow cells with mouse calvaria osteoblasts, we found by molecular cloning and RNA blot hybridization that PTPepsilon is highly expressed in osteoclastic cells. A purified fusion protein of PTPepsilon expressed in bacteria was inhibited by ALN with an IC50 of 2 microM. Other PTP inhibitors--orthovanadate and phenylarsine oxide (PAO)-inhibited PTPepsilon with IC50 values of 0.3 microM and 18 microM, respectively. ALN and another bisphosphonate, etidronate, also inhibited the activities of other bacterially expressed PTPs such as PTPsigma and CD45 (also called leukocyte common antigen). The PTP inhibitors ALN, orthovanadate, and PAO suppressed in vitro formation of multinucleated osteoclasts from osteoclast precursors and in vitro bone resorption by isolated rat osteoclasts (pit formation) with estimated IC50 values of 10 microM, 3 microM, and 0.05 microM, respectively. These findings suggest that tyrosine phosphatase activity plays an important role in osteoclast formation and function and is a putative molecular target of bisphosphonate action.
Asunto(s)
Difosfonatos/farmacología , Inhibidores Enzimáticos/farmacología , Expresión Génica , Osteoclastos/fisiología , Proteínas Tirosina Fosfatasas/metabolismo , Alendronato , Secuencia de Aminoácidos , Animales , Arsenicales/farmacología , Células de la Médula Ósea , Resorción Ósea , Clonación Molecular , Técnicas de Cocultivo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Isoenzimas/metabolismo , Cinética , Ratones , Datos de Secuencia Molecular , Osteoclastos/efectos de los fármacos , Osteoclastos/enzimología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/biosíntesis , Ratas , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Cráneo/citología , Vanadatos/farmacologíaRESUMEN
We recently defined an element (ACTAATTGG) within the rat osteocalcin (OC) promoter at -84 to -92 which provides approximately 70% of basal promoter activity in osteoblastic cell lines and binds a specific nuclear factor found in OC-producing ROS 17/2.8 osteosarcoma cells. Since this element closely resembles the recently described Msx-1 (Hox 7.1) homeodomain DNA binding cognate, we examined rodent osteoblastic cells lines for expression of Msx homeodomain-encoding messages. We have found and cloned a cDNA for rat Msx-2 (Hox 8.1) from a ROS 17/2.8 library and detect high levels of expression in various osteoblastic cell lines (ROS 17/2.8, RCT3, RCT1) as well as in culture passage 3 neonatal rat calvarial osteoblastic cells. Little to no expression was detected in phenotypically immature MC3T3E1 osteoblastic cells or in a variety of nonosteoblastic (ROS 25/1, C2C12, TRAB 11) mesenchymal cell lines. Dexamethasone (DEX) down-regulates Msx-2 message levels in both RCT3 and ROS 17/2.8 cells. Recombinant rat Msx-2 homeodomain expressed in Escherichia coli as a glutathione-S-transferase fusion protein binds to the rat OC promoter region -74 to -100 as determined by gel shift analysis. Recognition is dependent upon the intact ACTAATTGG motif at -84 to -92. In transient cotransfection assays using MC3T3E1 cells (which expresses very little or no endogenous Msx-2), Msx-2 suppresses the rat OC promoter 2- to 3-fold via the Msx-2 binding motif at -84 to -92. However, in ROS 17/2.8 cells, where a high level of endogenous Msx-2 mRNA is present, expression of exogenous Msx-2 does not suppress the rat OC promoter; surprisingly, Msx-2 further augments basal promoter activity by approximately 50-70%, again dependent upon the ACTAATTGG motif at -84 to -92. These data directly demonstrate that the Msx-2 homeodomain binds the rat OC promoter and that Msx-2 can act as a sequence-specific transcriptional regulator of the rat OC promoter in cultured osteoblastic cell lines. This activity is dependent upon the specific osteoblastic cellular context, similar to previous observations in nonosseous systems with other homeodomain transcription factors. These data suggest that Msx-2 may play a role in the transcriptional regulation of the osteoblast phenotype during development in the morphogenetic fields where it is expressed.
Asunto(s)
Proteínas de Unión al ADN/genética , Osteoblastos/metabolismo , Osteocalcina/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Animales , Secuencia de Bases , Células Cultivadas , ADN Complementario/química , ADN Complementario/genética , Proteínas de Unión al ADN/metabolismo , Dexametasona/farmacología , Regulación hacia Abajo , Factores de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Genes Homeobox , Proteínas de Homeodominio , Datos de Secuencia Molecular , Osteocalcina/biosíntesis , Osteocalcina/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/metabolismo , Factores de Crecimiento Transformadores/farmacología , Células Tumorales CultivadasRESUMEN
NER, a new member of the steroid hormone nuclear receptor (NR)-encoding gene family, was isolated from a human osteosarcoma SAOS/B10 cell line cDNA library. NER codes for a polypeptide of 461 amino acids which contains the conserved sequences of the DNA-binding and ligand-binding domains of typical steroid hormone NR. It has highest homology with the retinoic acid receptors: 55% at the DNA-binding domain and 38-40% at the ligand-binding domain. A single transcript of 2.3 kb was detected in all cells and tissues tested. Although no ligand was identified for NER-I, its wide distribution may indicate that this novel steroid hormone NR may play a basic role in cell function.
Asunto(s)
Receptores de Esteroides/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/genética , Clonación Molecular , ADN Complementario , Humanos , Receptores X del Hígado , Datos de Secuencia Molecular , Familia de Multigenes , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares , Receptores de Ácido Retinoico/genética , Células Tumorales CultivadasRESUMEN
We have identified a novel member of the steroid hormone receptor superfamily by cDNA cloning from a human osteosarcoma SAOS-2/B10 cell library. Sequence analysis predicts a protein of 441 amino acids, which includes the conserved amino acid residues characteristic of the DNA- and ligand-binding domains of nuclear receptors. Amino acid sequence alignment and transcriptional activation experiments revealed that the new protein is closely related to the mouse peroxisome proliferator activated receptor. The overall homology is 62%, and the highest similarity is seen in the DNA- and ligand-binding domains, 86% and 71%, respectively. Northern blot analysis showed that in mature rats, the receptor is highly expressed in heart, kidney, and lung as a transcript of approximately 3500 nucleotides. In human cells, the size of the mRNA is approximately 4000 nucleotides. Transcription assays using hybrid receptors consisting of the ligand-binding domain of the new protein and the DNA-binding domain of the glucocorticoid receptor showed weak stimulation by the peroxisome proliferator activator WY14643, suggesting a relationship to that receptor. Similar stimulation was observed with arachidonic and oleic acid (100-250 microM).
Asunto(s)
Ácido Araquidónico/farmacología , Núcleo Celular/metabolismo , Dexametasona/farmacología , Ácidos Oléicos/farmacología , Pirimidinas/farmacología , Receptores de Superficie Celular/genética , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides/metabolismo , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Biblioteca de Genes , Humanos , Cinética , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Ácido Oléico , Oligodesoxirribonucleótidos , Oligonucleótidos Antisentido , Osteosarcoma , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Esteroides/efectos de los fármacos , Receptores de Esteroides/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Osteocalcin is an abundant noncollagenous protein in bone, and its synthesis is stimulated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In this study, the rat osteocalcin gene was isolated, sequenced, and found to be a single-copy gene that is highly conserved between human and rat. Northern blot analysis of RNAs from a number of rat tissues revealed osteocalcin mRNA only in calvariae, consistent with bone-specific expression of osteocalcin. In order to investigate promoter activity and its modulation by 1,25(OH)2D3, plasmids containing the osteocalcin promoter region linked to the reporter enzyme bacterial chloramphenicol acetyltransferase (CAT) were used to transfect rat osteosarcoma ROS 17/2.8 cells, which express osteocalcin endogenously, and UMR 106 cells, which lack osteocalcin expression. Transfected ROS 17/2.8 cells exhibited a higher basal CAT activity than UMR 106 cells. Moreover, 1,25(OH)2D3 stimulated the CAT expression 5-10-fold only in ROS 17/2.8 cells and not in UMR 106 cells. By use of unidirectional deletion analysis, a domain strongly responsive to 1,25(OH)2D3 was identified between bases -1035 and -871 upstream from the site of transcription initiation, while a weakly responsive region was found further downstream.
Asunto(s)
Calcitriol/farmacología , Proteínas de Unión al Calcio/genética , Genes/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Huesos/metabolismo , Pollos , Humanos , Datos de Secuencia Molecular , Osteocalcina , ARN Mensajero/genética , Ratas , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
One cDNA clone encoding a truncated murine IL-1 beta (M IL-1 beta) sequence was isolated from a murine macrophage cDNA library. We reconstituted the coding sequence of the 152-residue mature protein and expressed it in Escherichia coli. rM IL-1 beta was purified to homogeneity and characterized by oligonucleotide and NH2-terminal sequence analysis. Purified rM IL-1 beta exhibited biologic activity equivalent to 7.8 x 10(7) units/mg in the murine thymocyte proliferation assay and 9.9 x 10(3) units/mg in the human gingival fibroblast PGE2 production assay, indicative of species specificity. The isoelectric point of rM IL-1 was found to be 8.85. The circular dichroism spectrum revealed that the secondary structure of M IL-1 is indistinguishable from that of the human protein. Receptor binding studies indicated the rM IL-1 bound to murine EL-4.1 thymoma cells in a specific and dose-dependent fashion with an affinity of 32 pM. Competition binding data suggested that murine and human IL-1 compete for a single class of receptor. Antisera were generated in rabbits against both murine and human IL-1. Results of ELISA binding and antisera neutralization assays indicated that there are common antigenic sites between the two IL-1 beta molecules. These domains are of functional importance because they are capable of mediating the neutralization of biologic activity.