RESUMEN

We compared the mechanical properties of bones from mice lacking either a functional cycloxygenase-1 (C57BL6/DBA COX-1-/-; n = 9) or COX-2 (C57BL6/DBA COX-2-/-; n = 9) gene and wild type mice (C57BL6/DBA; n = 10). Twenty-eight right femora from 3-month-old male mice were used to determine bulk structural and material properties of bone by three-point bending. Bone matrix properties were also measured by nanoindentation to access the changes in bulk mechanical properties due to changes in bone matrix or bone geometry. The bulk material properties (elastic modulus, P < 0.05; ultimate stress, P < 0.01) of COX-2-/- bones were lower than those of wild-type mice whereas the bulk structural properties (stiffness, P > 0.2; breaking force, P > 0.1) were similar to those of the wild-type mice. COX-2-/- mice had a longer moment of inertia but their cortical bones were thinner and contained many more intra-cortical pores compared with the bones of the other two groups. Finally, the bone matrix properties of COX-1-/- mice, COX-2-/- mice and their heterozygous littermates were similar to those of C57BL6/DBA wild-type mice.

Asunto(s)

Enfermedades Óseas , Matriz Ósea/fisiopatología , Fémur/fisiopatología , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintasas/genética , Animales , Fenómenos Biomecánicos , Enfermedades Óseas/congénito , Enfermedades Óseas/enzimología , Enfermedades Óseas/genética , Matriz Ósea/patología , Ciclooxigenasa 1 , Ciclooxigenasa 2 , ADN/análisis , Fémur/patología , Genotipo , Masculino , Ensayo de Materiales , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Nanotecnología , Reacción en Cadena de la Polimerasa

RESUMEN

The corpulent JCR:LA-cp rat (cp/cp) is a useful model for study of the metabolic consequences of obesity and hyperinsulinemia. To assess the effect of hyperinsulinemia on VLDL secretion in this model, we measured rates of secretion of VLDL in perfused livers derived from cp/cp rats and their lean littermates. Livers of cp/cp rats secreted significantly greater amounts of VLDL triglyceride and apolipoprotein, compared with lean littermates. The content of apoB, apoE, and apoCs in both perfusate and plasma VLDL was greater in the cp/cp rat, as was the apolipoprotein (apo)C, apoA-I, and apoA-IV content of plasma HDL. Triglyceride content was also greater in cp/cp livers, as was hepatic lipogenesis and expression of lipogenic enzymes and sterol regulatory element binding protein-1 (SREBP-1). Hepatic mRNAs for apoE, and apoA-I were higher in livers of cp/cp rats. In contrast, the steady state levels of apoC-II, apoC-III, and apoB mRNAs were unchanged. Thus, livers of obese hyperinsulinemic cp/cp JCR:LA-cp rats secrete a greater number of VLDL particles that are enriched in triglyceride, apoE, and apoC. Greater secretion of VLDL in the cp/cp rat in part results from higher endogenous fatty acid synthesis, which in turn may occur in response to increased expression of the lipogenic enzyme regulator SREBP-1c.

Asunto(s)

Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/metabolismo , Metabolismo de los Lípidos , Lípidos/biosíntesis , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Obesidad/metabolismo , Factores de Transcripción , Animales , Apolipoproteínas/genética , Glucemia/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas de Unión al ADN/genética , Hiperinsulinismo/metabolismo , Técnicas In Vitro , Insulina/sangre , Lípidos/análisis , Lipoproteínas VLDL/genética , Hígado/enzimología , Masculino , Obesidad/genética , Perfusión , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Mutantes , Ratas Sprague-Dawley , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Delgadez/genética , Delgadez/metabolismo , Triglicéridos/genética , Triglicéridos/metabolismo

RESUMEN

The nuclear factor-kappaB (NF-kappaB) family of transcription factors is involved in proliferation, differentiation, and apoptosis in a stage- and cell-dependent manner. Recent evidence has shown that NF-kappaB activity is necessary for both chicken and mouse limb development. We report here that the NF-kappaB family member c-rel and the homeodomain gene msx-1 have partially overlapping expression patterns in the developing chick limb. In addition, inhibition of NF-kappaB activity resulted in a decrease in msx-1 mRNA expression. Sequence analysis of the msx-1 promoter revealed three potential kappaB-binding sites similar to the interferon-gamma (IFN-gamma) kappaB-binding site. These sites bound to c-Rel, as shown by electrophoretic mobility shift assay (EMSA). Furthermore, inhibition of NF-kappaB activity significantly reduced transactivation of the msx-1 promoter in response to FGF-2/-4, known stimulators of msx-1 expression. These results suggest that NF-kappaB mediates the FGF-2/-4 signal regulation of msx-1 gene expression.

Asunto(s)

Extremidades/embriología , Factores de Crecimiento de Fibroblastos/farmacología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , FN-kappa B/fisiología , Factores de Transcripción , Animales , Sitios de Unión , Embrión de Pollo , Factor de Transcripción MSX1 , Proteínas Proto-Oncogénicas c-rel/genética , ARN Mensajero/análisis , Activación Transcripcional

RESUMEN

Members of the family of Sp transcription factors include Sp1, Sp3, and Sp4 and are important regulators of eukaryotic gene expression. We previously reported that Sp1 mediated stimulation of rat calmodulin I gene expression in response to insulin. To test whether other members of the Sp family are direct targets of insulin action, we compared the levels of Sp1 and Sp3 proteins from nuclear extracts obtained from both insulin-treated and untreated rat hepatoma (H-411E) cells. We demonstrated by Western blot analysis that levels of Sp1 and Sp3 proteins were increased more than 2-fold in the insulin-treated group. Additionally, the up-regulation of both Sp1 and Sp3 transcription factors by insulin was antagonized by tumor necrosis factor-alpha, a known inhibitor of insulin action. Immunohistochemical analysis demonstrated that H-411E cells treated with insulin (10,000 microU/ml) had a marked increase in demonstrable Sp1 in the nucleus compared with cells incubated in insulin-free medium. We extended these in vitro observations to in vivo studies in the streptozotocin-diabetic rat model. We demonstrated in rat liver tissue by both Western blot and immunohistochemical staining with anti-Sp1 antibody that 1) livers of fully diabetic streptozotocin rats have low levels of Sp1 transcription factor; and 2) insulin treatment of the diabetic rat rapidly reversed this process by markedly stimulating accumulation of Sp1 in rat liver. Studies of the signal transduction mechanisms involved in insulin's effect on Sp1 demonstrate a facilitating role for phosphoinositol 3-kinase and an inhibitory role for cyclic nucleotides. In summary, insulin stimulates Sp1 protein, a transcription factor that is shown to regulate calmodulin gene expression and most likely other, as yet untested, genes.

Asunto(s)

Diabetes Mellitus Experimental/metabolismo , Cetoacidosis Diabética/metabolismo , Insulina/fisiología , Neoplasias Hepáticas Experimentales/metabolismo , Factor de Transcripción Sp1/deficiencia , Animales , Western Blotting , Inmunohistoquímica , Masculino , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Factor de Transcripción Sp1/genética , Células Tumorales Cultivadas

RESUMEN

Msx1 promoter is known to be repressed by Msx1 protein [Shetty, Takahashi, Matsui, Iyengar and Raghow (1999) Biochem. J. 339, 751-758]. We show that in the transiently transfected C(2)C(12) myoblasts, co-expression of Msx3 also causes potent repression of Msx1 promoter that can be relieved by exogenous expression of cAMP-response-element-binding protein-binding protein (CBP) and p300 in a dose-dependent manner. Co-immunoprecipitation and Western blot analyses revealed that Msx3 interacts with CBP and p300 and this interaction significantly decreases the histone acetyltransferase (HAT) activity of both proteins. We also discovered that Msx3-mediated repression of Msx1 promoter is synergized by the exogenous co-expression of histone deacetylase 1 (HDAC1). Furthermore, the repression of Msx1 promoter by Msx3 could be relieved by treating transfected cells with trichostatin A, an inhibitor of HDAC(s). Finally, we show that Msx3 and HDAC1 can be co-immunoprecipitated in a complex that does not contain CBP and that Msx3 and HDAC1 proteins are co-localized in the nucleus. Taken together, our results strongly suggest that two distinct multiprotein complexes are present within the nuclei of C(2)C(12) cells: one containing Msx3 and HDAC(s) and another containing Msx3 and CBP and/or p300. On the basis of these results, we propose a dual mechanism of repression by Msx3 protein that involves the squelching of the HAT activity of co-activators, CBP and p300, and recruitment of HDAC(s).

Asunto(s)

Silenciador del Gen , Histona Desacetilasas , Histona Desacetilasas/metabolismo , Proteínas de Homeodominio/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae , Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/metabolismo , Animales , Proteína de Unión a CREB , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Proteína p300 Asociada a E1A , Técnica del Anticuerpo Fluorescente , Silenciador del Gen/efectos de los fármacos , Genes Reporteros , Histona Acetiltransferasas , Histona Desacetilasa 1 , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/genética , Proteínas de Homeodominio/genética , Ácidos Hidroxámicos/farmacología , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Transfección

RESUMEN

Murine myoblast cell lines stably transfected with expression vectors containing homeobox Msx1 cDNA in sense (F31-c) or antisense (F3R1) orientation have contrasting phenotypes. F3R1 cells readily differentiate in medium containing low serum whereas F31-c cells fail to differentiate under these conditions. The mechanism by which exogenous overexpression of Msx1 leads to the altered phenotype of F31-c cells and the downstream targets of Msx1 are unknown. Using the method of differential display, we have identified four cDNAs that represent transcripts up-regulated in F31-c. Two of these cDNAs are homologous to ribosomal proteins S23 and S24 while the third has homology to sequences in the murine Tcp-1 gene. A fourth cDNA does not have appreciable homology to cDNA sequences deposited in the NIH GenBank. Since withdrawal from the cell cycle and enhanced expression of MyoD commonly precede differentiation of myoblasts into myotubes, we also examined regulation of the major cell cycle proteins as well as MyoD by Western blot analysis. We show that the levels of Cdks 2, 4 and 6, cyclins A and D, and the Cdk inhibitor p27 in both proliferating and serum-starved F31-c cells were similar to those in F3R1. Finally, although MyoD protein levels increased in both cell types after 72 h incubation in serum depleted medium, the levels of MyoD in serum-starved F31-c cells were 2-4 fold lower. We postulate that the reduced amount of MyoD is sufficient to permit reversible withdrawal of F31-c cells from the cell cycle, but is inadequate to permit myogenesis.

Asunto(s)

Diferenciación Celular , Proteínas de Homeodominio/metabolismo , Músculos/citología , ARN Mensajero/biosíntesis , Factores de Transcripción , Animales , Northern Blotting , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Medio de Cultivo Libre de Suero , ADN Complementario/metabolismo , Perfilación de la Expresión Génica , Factor de Transcripción MSX1 , Ratones , Microscopía de Contraste de Fase , Desarrollo de Músculos , Músculos/metabolismo , Proteína MioD/metabolismo , Reacción en Cadena de la Polimerasa

RESUMEN

We have previously demonstrated that insulin positively regulates transcription of the rat calmodulin (CaM) I gene and that both basal and insulin stimulation of this gene are critically dependent on Sp1. Furthermore, a 392 bp CaM promoter was stimulated by insulin equal to the full promoter but lost activity with deletion of any of the three Sp1 sites (Solomon SS, Palazzolo MR, Takahashi T, Raghow R. Endocrinology 1997;138:5052-5054). Herein we document that Sp1 preferentially binds to the upstream sites Sp1(2) and Sp1(3) but not Sp1(1). Furthermore, gel-mobility super-shift assays demonstrate that both Sp1 and Sp3 protein are found in these complexes. When pPac-Spl, pPac-Sp3, pPac-USp3, and pPac-Sp4 were cotransfected with rCaM 1-392 promoter into Drosophila SL2 cells and challenged with 10,000 microU/mL insulin, we discovered that (1) Sp1 enhanced both basal and insulin-stimulated CaM I gene expression; (2) USp3, a "long" form of the Sp3 molecule, had a stimulatory effect on CaM I gene expression; (3) Sp1 or USp3 is involved in mediating insulin-stimulation of the CaM I gene in SL2 cells; and (4) Sp3, a "short" form of the Sp3 molecule, and Sp4 inhibited Spl-stimulated and insulin-stimulated Sp1-mediated CaM I gene expression. Together these data corroborate and extend our previous observations on Sp1 and elucidate that other members of the Sp family of transcription factors may also be involved in regulating the activity of the CaM promoter.

Asunto(s)

Calmodulina/genética , Regulación de la Expresión Génica , Factor de Transcripción Sp1/genética , Animales , Calmodulina/biosíntesis , Drosophila , Regulación de la Expresión Génica/efectos de los fármacos , Hipoglucemiantes/farmacología , Insulina/farmacología , Regiones Promotoras Genéticas , Ratas , Transfección , Células Tumorales Cultivadas

RESUMEN

We investigated the molecular mechanisms by which treatment of the human osteoblast-like cell line MG-63 with interleukin 1beta (IL-1) and/or fibroblast growth factor 1 (FGF-1) elicited prostaglandin biosynthesis. IL-1 induced a 5-fold increase in PGE(2) production compared to controls. While treatment with FGF-1 alone did not affect PGE(2) biosynthesis, it enhanced the formation of PGE(2) by IL-1 by an additional 3- to 5-fold. IL-1-induced PGE(2) biosynthesis accompanied increases in steady-state levels of mRNAs encoding cPLA(2) (10- to 15-fold) and PGHS-2 (>3-fold) and concomitant increases in cPLA(2) protein (>3-fold) and PGHS-2 protein (>1. 5-fold). FGF-1 treatment did not affect PGHS-2 gene expression, but enhanced the effect of IL-1 on PGHS-2 expression by an additional 2- to 3-fold. FGF-1 alone enhanced cPLA(2) expression (5-fold), and the combined effects of FGF-1 and IL-1 on cPLA(2) expression were additive. There was no measurable effect of either agonist on PGHS-1 expression. We also discovered that induction of PGE(2) biosynthesis in response to IL-1 or IL-1/FGF-1 was affected by the density of MG-63 cells in culture. Subconfluent cultures displayed a 3- to 10-fold greater response to IL-1 or IL-1/FGF-1 than confluent cultures. The decreased PGE(2) induction by IL-1 in confluent cultures was associated with reduced IL-1 receptor expression. We conclude that the signaling pathways resulting in PGE(2) biosynthesis in response to proinflammatory agents like IL-1 are subject to complex regulation by additional soluble mediators as well as cell-cell or cell-extracellular matrix interactions.

Asunto(s)

Dinoprostona/biosíntesis , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Interleucina-1/metabolismo , Animales , Bovinos , Recuento de Células , Células Cultivadas , Ciclooxigenasa 2 , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Factor 1 de Crecimiento de Fibroblastos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Interleucina-1/farmacología , Isoenzimas/biosíntesis , Isoenzimas/genética , Proteínas de la Membrana , Osteosarcoma , Fosfolipasas A/biosíntesis , Fosfolipasas A/genética , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero , Conejos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Receptores de Interleucina-1/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Células Tumorales Cultivadas

RESUMEN

Interleukin-1beta (IL-1) is a potent inducer of cyclooxygenase-2 (COX-2) and prostaglandin biosynthesis in many types of cells, yet little is known about the molecular mechanisms regulating IL-1 mediated prostanoid biosynthesis in the endothelium of the microvasculature. Therefore, we examined the cis- and trans-acting factors regulating IL-1-induced COX-2 expression in the human microvascular endothelial cell line, HMEC-1. IL-1 enhanced steady state levels of COX-2 protein and mRNA synthesis by approximately 2-fold which preceded a 2-fold increase in PGF(alpha) biosynthesis. Expression of a series of COX-2 promoter-luciferase constructs in IL-1 treated HMEC-1 cells revealed that the 'full length' (-1432/+59 bp) promoter was 10 times more active than the SV-40 promoter/enhancer and that it could be further activated by IL-1. Surprisingly however, all except for the shortest COX-2 promoter construct retained the ability to respond to IL-1 and luciferase activity driven by -191/+59 bp COX-2 promoter was as responsive to IL-1 as the full-length promoter. Moreover, site-directed promoter mutagenesis and electophoretic mobility shift assays (EMSA) indicate that the combinatorial actions of AP2, NF-IL6, and CRE elements are critical for both constitutive and IL-1-inducible COX-2 promoter activity. Understanding the mechanism(s) regulating COX-2 gene expression and prostaglandin biosynthesis in the microvasculature has important implications with regard to inflammation and angiogenesis in vivo.

Asunto(s)

Proteínas Potenciadoras de Unión a CCAAT , AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Endotelio Vascular/enzimología , Regulación Enzimológica de la Expresión Génica/genética , Isoenzimas/genética , Proteínas Nucleares/genética , Prostaglandina-Endoperóxido Sintasas/genética , Factores de Transcripción/genética , Transcripción Genética/genética , Secuencia de Bases , Proteína delta de Unión al Potenciador CCAAT , Línea Celular , Ciclooxigenasa 2 , Cartilla de ADN , Dinoprost/biosíntesis , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Proteínas de la Membrana , Mutación , Regiones Promotoras Genéticas , Factor de Transcripción AP-2

RESUMEN

The role of CD9 in cell adhesion and spreading on adhesive proteins was investigated using a transfected Chinese hamster ovary (CHO) cell system. CD9 cell surface expression resulted in reduced adhesion and increased spreading on fibronectin (Fn). Whereas mock-transfected (mock CHO) and naïve CHO cells assumed a typical fibroblast spindle shape morphology, CD9-transfected (CD9-CHO) cells were polygonal with many filipodial projections and exhibited a twofold greater surface area. The spread morphology of CD9-CHO cells, but not mock CHO cells, was inhibited by PB1 mAb blockade of alpha(5)beta(1), suggesting that the coexpression of alpha(5)beta(1) and CD9 influenced cell activity on Fn. The second extracellular loop of CD9 was implicated in regulation of adhesion since reduced CD9-CHO cell adhesion on Fn was reversed by either anti-CD9 antibody ligation to the second extracellular loop or with cells expressing a CD9 mutant lacking the second extracellular loop domain. Using cell adhesion assays and ELISA, we demonstrated CD9 binding to the HEP2/IIICS region of Fn. Finally, CD9 expression resulted in a twofold reduction in Fn-rich pericellular matrix assembly. Our observations show that CD9 dramatically influences CHO cell interactions with Fn and suggest that CD9 has an important role in modulating cell-extracellular matrix interactions.

Asunto(s)

Antígenos CD/metabolismo , Adhesión Celular/fisiología , Membrana Celular/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Animales , Antígenos CD/genética , Células CHO , Tamaño de la Célula , Cricetinae , Humanos , Integrinas/metabolismo , Fragmentos de Péptidos/metabolismo , Unión Proteica , Receptores de Fibronectina/metabolismo , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Relación Estructura-Actividad , Tetraspanina 29

RESUMEN

The TATA-less murine Msx1 promoter contains two Msx1-binding motifs, located at -568 to -573 and +25 to +30, and is subject to potent autorepression [Takahashi, Guron, Shetty, Matsui and Raghow (1997) J. Biol. Chem. 272, 22667-22678]. To investigate the molecular mechanism by which Msx1 represses the activity of its own promoter, we transfected C2C12 myoblasts with Msx1-promoter-luciferase constructs and assessed reporter gene activity, with and without the exogenous expression of Msx1. We demonstrate that Msx1-mediated autorepression remained unaffected, regardless of the presence or absence of the Msx1 recognition motifs on the promoter. Furthermore, graded exogenous expression of TATA-binding protein (TBP), Sp1 or cAMP-response-element-binding protein-binding protein (CBP/p300) could counteract the autoinhibitory activity of Msx1. Finally, we demonstrate that Msx1 protein can be immunoprecipitated in a multiprotein complex containing TBP, Sp1 and CBP/p300. We hypothesize that the interaction of Msx1 protein with one or more ubiquitous or tissue-restricted transcription factors mediates transcriptional autorepression of the Msx1 gene.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Proteínas de Homeodominio/genética , Proteínas Represoras/metabolismo , Factor de Transcripción Sp1/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Histona Acetiltransferasas , Proteínas de Homeodominio/metabolismo , Factor de Transcripción MSX1 , Ratones , Coactivador 3 de Receptor Nuclear , Pruebas de Precipitina , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Represoras/genética , Elementos de Respuesta/genética , Eliminación de Secuencia , Factor de Transcripción Sp1/genética , Proteína de Unión a TATA-Box , Transactivadores/genética , Factores de Transcripción/genética , Transcripción Genética/genética , Transfección , Células Tumorales Cultivadas

RESUMEN

We have previously shown that chronic insulin treatment of rat hepatocytes increases the fraction of edited apolipoprotein B (apoB) mRNA from approximately 50% to as much as 90%. We have now examined the effect of insulin on apobec-1 mRNA abundance and demonstrate that increased editing of apoB mRNA following insulin treatment is accompanied by elevated apobec-1 mRNA levels in primary rat hepatocytes. Time-course measurements of the effects of insulin on apoB mRNA editing and apobec-1 mRNA abundance showed that both were elevated almost maximally within 48 hours and sustained for at least 5 days of insulin treatment.

Asunto(s)

Apolipoproteínas B/genética , Citidina Desaminasa/biosíntesis , Citidina Desaminasa/genética , Insulina/farmacología , Hígado/metabolismo , Edición de ARN/efectos de los fármacos , ARN Mensajero/metabolismo , Desaminasas APOBEC-1 , Animales , Catálisis/efectos de los fármacos , Células Cultivadas , Citidina Desaminasa/efectos de los fármacos , Hígado/efectos de los fármacos , Masculino , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , Ratas , Ratas Sprague-Dawley

RESUMEN

The profound effects of transforming growth factor beta1 (TGF-beta1) on the immune system, cardiogenesis, in yolk sac hematopoeisis and in differentiation of endothelium have been demonstrated by detailed analyses of TGF-beta1 knockout mice during embryogenesis. We have systematically examined the autocrine and paracrine roles of TGF-beta1 in cell proliferation and in its ability to modulate the gene expression of selected components of extracellular matrix (ECM) using embryonic fibroblasts from TGF-beta1 null mice (TGF-beta-1(-/-)). The rates of cell proliferation of embryonic fibroblasts from normal mice (TGF-beta1(+/+)) and TGF-beta1 null mice were compared by cell counting, by 3H thymidine incorporation, and by measuring the fraction of cells in the G1, S, and G2/M phases of the cell cycle by fluorescent activated cell sorting (FACS). Concurrently, the expression of pro-alpha1(I) collagen, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) was also quantified by hybridization of total mRNA from TGF-beta1(+/+) and TGF-beta1(-/-) embryonic fibroblasts. We report that TGF-beta1(-/-) cells proliferated at about twice the rate of TGF-beta1(+/+) cells. Further, TGF-beta1 null fibroblasts accumulated and synthesized lower constitutive levels of pro-alpha1(I) collagen, fibronectin, and PAI-1 mRNA. The quantitative differences in the rates of cell proliferation and ECM gene expression between TGF-beta1(+/-) and TGF-beta1(-/-) cells could be eliminated by treatment of TGF-beta1(+/+) cells with a neutralizing antibody of TGF-beta1. Thus, our results are consistent with the hypothesis that TGF-beta1 acts as a negative autocrine regulator of growth and a positive autocrine regulator of ECM biosynthesis in embryonic fibroblasts.

Asunto(s)

División Celular/fisiología , Matriz Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Factor de Crecimiento Transformador beta/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Ciclo Celular/fisiología , Fibroblastos , Fibronectinas/genética , Citometría de Flujo , Eliminación de Gen , Ratones , Ratones Noqueados , Inhibidor 1 de Activador Plasminogénico/genética , Procolágeno/genética , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética

RESUMEN

Interleukin-1beta (IL-1) is a potent inducer of prostaglandin E2 (PGE2) synthesis. We previously showed that ceramide accumulates in fibroblasts treated with IL-1 and that it enhances IL-1-induced PGE2 production. The present study was undertaken to determine the mechanism(s) by which ceramide and IL-1 interact to enhance PGE2 production by examining their respective effects on the rate-limiting enzymes in PGE2 synthesis, cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A2 (cPLA2). IL-1-induced PGE2 synthesis required approximately 8 h even though COX-1 was constitutively expressed (both mRNA and protein) and enzymatically active in untreated cells. Conversely, COX-2 mRNA was barely detectable in untreated cells but within 2 h, ceramide or IL-1 alone induced a 5 and 20 fold increase in COX-2 mRNA, respectively. However, IL-1 induced COX-2 protein synthesis was only detectable 6-7 h after maximal COX-2 mRNA induction; COX-2 protein accumulation was not induced by ceramide alone. Ceramide however, reduced the length of time required for IL-1 to induce COX-2 protein accumulation and increased COX-2 protein accumulation. IL-1 induced a 15 fold increase in COX-1 mRNA including an alternatively spliced form of COX-1. IL-1, but not ceramide induced cPLA2 mRNA and protein expression which corresponded with the initiation of PGE2 synthesis. These observations indicate that, (1) while either ceramide or IL-1 rapidly induced COX-2 mRNA, COX-2 protein only accumulated in IL-1 treated cells after a delay of 6-7 h, (2) IL-1-induced PGE2 synthesis required both COX-2 and cPLA2 protein synthesis and, (3) ceramide enhanced (temporally and quantitatively) IL-1-induced COX-2 protein

Asunto(s)

Dinoprostona/biosíntesis , Interleucina-1/farmacología , Esfingosina/análogos & derivados , Empalme Alternativo , Ácido Araquidónico/farmacología , Células Cultivadas , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Fibroblastos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Proteínas de la Membrana , Fosfolipasas A/biosíntesis , Fosfolipasas A/genética , Fosfolipasas A2 , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , Esfingosina/farmacología

RESUMEN

Prostaglandin E2 (PGE2) production in immortalized, nontransformed cells derived from wild-type, cyclooxygenase 1-deficient (COX-1(-/-)) or cyclooxygenase 2-deficient (COX-2(-/-)) mice was examined after treatment with interleukin (IL)-1beta, tumor necrosis factor alpha, acidic fibroblast growth factor, and phorbol ester (phorbol myristate acetate). Compared with their wild-type counterparts, COX-1(-/-) or COX-2(-/-) cells exhibited substantially enhanced expression of the remaining functional COX gene. Furthermore, both basal and IL-1-induced expression of cytosolic phospholipase A2 (cPLA2), a key enzyme-regulating substrate mobilization for PGE2 biosynthesis, was also more pronounced in both COX-1(-/-) and COX-2(-/-) cells. Thus, COX-1(-/-) and COX-2(-/-) cells have the ability to coordinate the upregulation of the alternate COX isozyme as well as cPLA2 genes to overcome defects in prostaglandin biosynthetic machinery. The potential for cells to alter and thereby compensate for defects in the expression of specific genes such as COX has significant clinical implications given the central role of COX in a variety of disease processes and the widespread use of COX inhibitors as therapeutic agents.

Asunto(s)

Dinoprostona/biosíntesis , Isoenzimas/fisiología , Prostaglandina-Endoperóxido Sintasas/fisiología , Animales , Células Cultivadas , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inducción Enzimática , Interleucina-1/farmacología , Isoenzimas/biosíntesis , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Fosfolipasas A/biosíntesis , Fosfolipasas A2 , Prostaglandina-Endoperóxido Sintasas/biosíntesis

RESUMEN

Insulin positively regulates transcription of rat calmodulin (CaM) I gene and activates the low Km cyclic AMP (cAMP) phosphodiesterase (PDE). To elucidate the mechanism of transcriptional regulation, rat hepatoma (H-411E) cells were transfected with DNA constructs containing the putative CaM promoters coupled to a luciferase reporter and challenged with insulin. Activation of the full length 1835 bp rat CaM I promoter containing all three Sp1 sites or truncated promoters with combinations of one to three of the Sp1 sites was studied in Sp1 deficient Drosophilia SL2 cells and in SL2 cells co-transfected with an Sp1 expression vector and re-challenged with insulin. Our results demonstrate that Sp1 is obligatory for basal activation of the CaM promoter. The maximal insulin stimulation of CaM promoter is elicited only if it contains at least two Sp1 sites.

Asunto(s)

Calmodulina/genética , Insulina/farmacología , Factor de Transcripción Sp1/fisiología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiología , Animales , Drosophila/citología , Regiones Promotoras Genéticas/efectos de los fármacos , Ratas , Células Tumorales Cultivadas

RESUMEN

We have shown previously that insulin positively regulates transcription of the rat calmodulin (CaM) I gene. This activation occurs concomitantly with the activation of the low-Km adenosine 3':5'-cyclic phosphate phosphodiesterase (PDE), which appears to be coregulated with CaM. Rat hepatoma H-411E cells were transfected with plasmids containing various lengths of the putative CaM promoter coupled to a luciferease reporter and were challenged with insulin. We demonstrate that insulin-stimulated transcription of CaM I gene is mediated by a 392-bp 5'-flanking region of the CaM I gene, encompassing 185 bp downstream and 207 bp upstream of the start site of transcription. The CaM I promoter contains three potential Sp1 sites, located at -114 through -109 [(3), +], -77 through -72 [(2), -] and at +53 through +58 [(1), +]. The gel mobility shift assays demonstrated that nuclear protein(s) associate with all three sp1 sites. We present data demonstrating the relative importance of the three Sp1 sites for the insulin effect: prCaM I 1835, 3.8x, delta 1081; prCaM I 392, 5.3x, delta 1055; prCaM I 180, 3.7x, delta 462; prCaM I 237, 1.6x, delta 478; prCaM I 139, 2.6x, delta 182; prCaM I 130, 2.1x, delta 194; and prCaM I 1463, negligible activity. In summary, the maximal insulin stimulation of CaM gene expression is seen when the promoter region contains at least two Sp1 sites.

Asunto(s)

Calmodulina/biosíntesis , Regulación de la Expresión Génica , Insulina/farmacología , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas , Animales , Secuencia de Bases , Sitios de Unión , Calmodulina/genética , Genes Reporteros , Datos de Secuencia Molecular , Unión Proteica , Ratas , Eliminación de Secuencia , Transcripción Genética , Transfección , Células Tumorales Cultivadas

RESUMEN

To dissect the cis-regulatory elements of the murine Msx-1 promoter, which lacks a conventional TATA element, a putative Msx-1 promoter DNA fragment (from -1282 to +106 base pairs (bp)) or its congeners containing site-specific alterations were fused to luciferase reporter and introduced into NIH3T3 and C2C12 cells, and the expression of luciferase was assessed in transient expression assays. The functional consequences of the sequential 5' deletions of the promotor revealed that multiple positive and negative regulatory elements participate in regulating transcription of the Msx-1 gene. Surprisingly, however, the optimal expression of Msx-1 promoter in either NIH3T3 or C2C12 cells required only 165 bp of the upstream sequence to warrant detailed examination of its structure. Therefore, the functional consequences of site-specific deletions and point mutations of the cis-acting elements of the minimal Msx-1 promoter were systematically examined. Concomitantly, potential transcriptional factor(s) interacting with the cis-acting elements of the minimal promoter were also studied by gel electrophoretic mobility shift assays and DNase I footprinting. Combined analyses of the minimal promoter by DNase I footprinting, electrophoretic mobility shift assays, and super shift assays with specific antibodies revealed that 5'-flanking regions from -161 to -154 and from -26 to -13 of the Msx-1 promoter contains an authentic E box (proximal E box), capable of binding a protein immunologically related to the upstream stimulating factor 1 (USF-1) and a GC-rich sequence motif which can bind to Sp1 (proximal Sp1), respectively. Additionally, we observed that the promoter activation was seriously hampered if the proximal E box was removed or mutated, and the promoter activity was eliminated completely if the proximal Sp1 site was similarly altered. Absolute dependence of the Msx-1 minimal promoter on Sp1 could be demonstrated by transient expression assays in the Sp1-deficient Drosophila cell line cotransfected with Msx-1-luciferase and an Sp1 expression vector pPacSp1. The transgenic mice embryos containing -165/106-bp Msx-1 promoter-LacZ DNA in their genomes abundantly expressed beta-galactosidase in maxillae and mandibles and in the cellular primordia involved in the formation of the meninges and the bones of the skull. Thus, the truncated murine Msx-1 promoter can target expression of a heterologous gene in the craniofacial tissues of transgenic embryos known for high level of expression of the endogenous Msx-1 gene and found to be severely defective in the Msx-1 knock-out mice.

Asunto(s)

Proteínas de Homeodominio/genética , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción , Activación Transcripcional , Células 3T3 , Animales , Células Cultivadas , Medio de Cultivo Libre de Suero , Drosophila , Proteínas de Homeodominio/metabolismo , Factor de Transcripción MSX1 , Ratones , Ratones Transgénicos , Unión Proteica , Cráneo/metabolismo , Factor de Transcripción Sp1/metabolismo , beta-Galactosidasa/genética

RESUMEN

A construct containing human Proalpha1(I) collagen gene promoter/enhancer-driven chloramphenicol acetyltransferase (CAT), pCOL-KT, failed to be expressed significantly in Sp1-deficient Schneider Drosophila line 2 (SL2) cells. However, CAT expression was induced 200-fold in SL2 cells co-transfected with pCOL-KT and pPACSp1, an Sp1-expression vector driven by the Drosophila actin 5C promoter. Elimination of the four potential Sp1-binding sites from pCOL-KT (pCOL-KTDeltaI), by removal of the first intron, did not abrogate Sp1-mediated induction of CAT. Even more significantly, a minimal Proalpha1(I) collagen promoter (-100 to +117 bp), containing a TATA box (-28 to -25 bp) and one putative Sp1-binding site (-87 to -82 bp), elicited strong Sp1-induced transactivation. Furthermore, mutation of the Sp1 motif in the minimal Proalpha1(I) collagen promoter-CAT construct abolished Sp1-induced expression of the reporter gene. Purified Sp1 protein bound specifically to DNA fragments of the Proalpha1(I) minimal promoter encompassing the putative Sp1-binding site; Sp1 binding could be competed out by a double-stranded oligonucleotide containing the wild-type Sp1 sequence, while an oligonucleotide containing a mutated Sp1 site failed to compete. Based on these results, we postulate that Sp1 plays an obligatory role in the transcriptional activation of the human Proalpha1(I) collagen gene. Additionally, we propose that a bona fide Sp1 motif, located most proximal to the TATA box, is necessary and sufficient for Sp1-mediated activation of the minimal Proalpha1(I) collagen promoter.

Asunto(s)

Procolágeno/genética , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Transcripción Genética , Animales , Sitios de Unión , Drosophila , Humanos , Intrones , Mutagénesis Sitio-Dirigida , Eliminación de Secuencia , Relación Estructura-Actividad

RESUMEN

Regulation of myeloid cell proliferation and differentiation in the bone marrow is mediated, in part, by the interaction of integrins on early myeloid cells with the extracellular matrix proteins secreted by stromal cells. To further define adhesive protein receptors of early myeloid cells, we examined the expression of the integrin GPIIb-IIIa (alphaIIbbeta3) in leukemic cell lines KG-1a, KG-1, and HL-60, that represent early stages of myeloid differentiation. All three cell lines expressed surface GPIIb-IIIa as measured by flow cytometry and by binding of 125I-anti-GPIIb-IIIa monoclonal antibody. Preincubation of cells with human AB serum or platelet releasate increased GPIIb-IIIa surface expression. GPIIb transcripts were identified in all three cell lines by Northern blot analysis. Furthermore, we readily detected GPIIb transcripts in fluorescence activated cell sorted (FACS) myeloid cells from normal human bone marrow by RT-PCR. Cloning and sequencing of the PCR products established the identity of GPIIb transcripts in the leukemic cell lines and CD34+/CD33+ normal bone marrow cells. Since the normal myeloid cells also demonstrated markers corresponding to the maturational stage of KG-1a/KG-1 cells, we propose that GPIIb-IIIa may serve as a myeloid differentiation antigen and as a key integrin of myeloid precursors.

Asunto(s)

Antígenos CD34/biosíntesis , Antígenos CD/biosíntesis , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Células de la Médula Ósea/metabolismo , Leucemia Mieloide/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/biosíntesis , Secuencia de Bases , Sitios de Unión de Anticuerpos , Northern Blotting , Clonación Molecular , Citometría de Flujo , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Distribución Normal , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , ARN Mensajero/análisis , Alineación de Secuencia , Lectina 3 Similar a Ig de Unión al Ácido Siálico , Células Tumorales Cultivadas