RESUMEN
We have analyzed and compared the 5' promoter region, the intron structure and the exon-intron flanking sequences in the rat and human prohibitin-encoding genes (PHB). Comparative analysis of a 350-nt region immediately 5' to and including the first exon identifies eight highly conserved regions, four of which correspond to binding sites for known transcriptional control proteins (CCAAT box, 'SV40' site and two Sp1 sites). The promoter lacks a TATA box. Four transcription start points (tsp) clustered within a 35-bp region were identified by rapid amplification of cDNA ends (RACE). The exon-intron boundaries in rat and human are highly conserved, with identical positioning of splice junctions. PCR analysis with conserved exon primers was used to detect length variation between rat and human PHB, and length differences were observed in all of the introns.
Asunto(s)
Proteínas/genética , Proteínas Represoras , Animales , Secuencia de Bases , Evolución Biológica , División Celular/genética , Secuencia Conservada , Exones/genética , Genes Reguladores/genética , Humanos , Intrones/genética , Datos de Secuencia Molecular , Prohibitinas , Regiones Promotoras Genéticas/genética , Ratas , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
Prohibitin, a novel intracellular antiproliferative protein, blocks entry into the S phase of the cell division cycle when its mRNA is microinjected into normal fibroblasts or HeLa cells. To learn more about the interaction between prohibitin and the cell cycle, we studied the effect of microinjecting prohibitin mRNA at different points during the transition from G0 to S phase and analyzed prohibitin mRNA and protein levels in different parts of the cell cycle. The antiproliferative activity of microinjected prohibitin mRNA is high in G0/G1 and falls as cells approach S phase. Prohibitin mRNA and protein levels are high in G1, fall with S phase, rise again in G2, and fall in M. Together, these findings suggest that endogenous prohibitin contributes to the control of the G1 to S transition in cycling cells in a complex manner, which involves both a transcriptional and posttranslational mechanism.
Asunto(s)
Fibroblastos/citología , Proteínas/genética , ARN Mensajero/análisis , Proteínas Represoras , Animales , Western Blotting , Ciclo Celular/fisiología , División Celular/fisiología , Células Cultivadas , Fibroblastos/química , Fibroblastos/metabolismo , Humanos , Masculino , Microinyecciones , Prohibitinas , Proteínas/análisis , Proteínas/metabolismo , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , Ratas , Fase de Descanso del Ciclo Celular , Fase SRESUMEN
Inhibition of protein synthesis stabilizes a number of mRNAs, but little is known about the mechanism. To understand the relationship between protein synthesis and mRNA stability, we studied the degradation of calcitonin-induced urokinase-type plasminogen activator (uPA) mRNA in LLC-PK cells. uPA mRNA became highly stable by pretreatment with either cycloheximide or pactamycin, and the stabilizing effect of cycloheximide treatment was time dependent with the full effect exerted by 60 min. Stabilization was also observed with histone H4 mRNA but only partially with c-myc mRNA. To further analyze, we developed a cell-free decay reaction system based on post-mitochondrial supernatant (PMS). In this system, uPA mRNA was completely stable when fractions were obtained from cells pretreated with cycloheximide, but very unstable in control fractions, paralleling uPA mRNA stability in intact cells. However, in contrast to uPA mRNA and the in vivo observation, histone H4 mRNA was unstable whether or not the cells were pretreated with cycloheximide. These results suggest that inhibition of protein synthesis stabilizes mRNAs in at least two different ways in LLC-PK1 cells. When PMS from cycloheximide/calcitonin-treated cells was mixed with PMS from untreated cells, uPA mRNA was not destabilized. This suggests that a putative labile factor responsible for uPA mRNA degradation is not a soluble protein.
Asunto(s)
Cicloheximida/farmacología , Pactamicina/farmacología , ARN Mensajero/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Northern Blotting , Calcitonina/metabolismo , Línea Celular , Sistema Libre de Células , Técnicas In Vitro , Polirribosomas/metabolismo , Ribonucleasas/metabolismo , Porcinos , Factores de TiempoRESUMEN
In LLC-PK1 pig kidney cells, treatment with a cAMP-elevating peptide hormone, calcitonin, induces the accumulation of urokinase-type plasminogen activator (uPA) mRNA. When we used the method of differential hybridization to isolate uPA cDNA clones, we also obtained several calcitonin-inducible clones that were unrelated to uPA. Sequence analysis revealed 60% sequence homology between one of these clones and that of a Drosophila hsp70 gene. The uPA and the hsp70 cDNA clones were used as probes to compare the effects of various treatments on the accumulation of uPA mRNA and hsp70 mRNA in LLC-PK1 cells. Calcitonin or 8-bromo-cAMP treatment induced uPA mRNA accumulation, which was negligible in untreated cells. Heat treatment (42 degrees C) was ineffective. Calcitonin or heat treatment increased hsp70 mRNA accumulation, which was already high in untreated cells, but 8-bromo-cAMP was ineffective. Nuclear transcription of the hsp70 gene was increased by calcitonin but not by 8-bromo-cAMP treatment. These results suggest that calcitonin induces hsp70 mRNA accumulation in LLC-PK1 cells by a pathway apart from the activation of adenylate cyclase and through, at least partly, the activation of the gene transcription. Furthermore, induction of uPA mRNA accumulation by calcitonin or 8-bromo-cAMP treatment did not require protein synthesis. In contrast, induction of hsp70 mRNA accumulation by calcitonin or heat treatment did require protein synthesis. Other reports showed that protein synthesis is not required for heat induction of hsp70 mRNA in different cells, suggesting that the mechanism of induction of hsp70 mRNA accumulation in LLC-PK1 cells is not the same as in other cells.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Calcitonina/farmacología , Riñón/metabolismo , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Línea Celular , Drosophila/genética , Calor , Riñón/efectos de los fármacos , Datos de Secuencia Molecular , Porcinos , Transcripción Genética , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
In LLC-PK1 cells, a cyclic AMP (cAMP)-elevating peptide hormone, calcitonin, induces urokinase-type plasminogen activator (uPA) gene transcription without concomitant protein synthesis. To understand the molecular mechanism of the uPA gene regulation by cAMP, we developed a system which allows us to obtain mutant cells with modified regulatory proteins. A uPA-gpt hybrid gene was constructed, in which the regulatory region of the uPA gene was linked to a bacterial xanthine-guanine phosphoribosyltransferase gene (gpt), and it was transfected into LLC-PK1 cells. A stably transformed cell line, which expressed gpt only in the presence of calcitonin, was obtained, and then these cells were treated with a chemical mutagen, ethyl methanesulfonate. Cells were screened for constitutive gpt expression and, as mutations in regulatory proteins should affect the two genes at the same time, cells were further screened for an increased basal uPA mRNA level. Several such clones were obtained and none of them had modified cAMP-dependent protein kinase activity, suggesting that mutations were in the post-protein kinase step in the pathway of hormone action. Five clones were fused with the parent LLC-PK1 cells, and all of the fusion cells showed reduced basal uPA mRNA levels, indicating that they were recessive mutants. One clone was analyzed further for sensitivity to calcitonin in the induction of uPA mRNA, and it showed a significantly different dose-response pattern compared with parent cells. These results suggest that the uPA gene is regulated, at least partly, by a negatively regulating factor and that the action of cAMP is linked to this factor.
Asunto(s)
Calcitonina/farmacología , AMP Cíclico/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Activador de Plasminógeno de Tipo Uroquinasa/genética , Animales , Línea Celular , AMP Cíclico/farmacología , ADN Recombinante , Epitelio , Riñón , Mutación , Hibridación de Ácido Nucleico , Pentosiltransferasa/genética , Plásmidos , Proteínas Quinasas/metabolismo , ARN Mensajero/biosíntesis , Porcinos , Transcripción Genética/efectos de los fármacos , Transformación GenéticaRESUMEN
The peptide hormone calcitonin induces the accumulation of urokinase-type plasminogen activator (uPA) mRNA in pig kidney LLC-PK1 cells. By itself, inhibition of protein synthesis had a negligible effect on uPA mRNA accumulation. Inhibition of protein synthesis led to two superinductive effects: an increase in calcitonin-induced uPA mRNA accumulation over time, and a shift in the dose-response curve so that lower calcitonin doses became more potent. To explain these two superinductive effects of protein-synthesis inhibition on calcitonin treatment, we demonstrated that the inhibition of protein synthesis increased both calcitonin-induced uPA-gene transcription and uPA-mRNA stability. Different protein-synthesis inhibitors had similar actions, arguing against the possibility that the results were attributable to an anomalous action of a particular inhibitor. The superinductive effects of protein-synthesis inhibition could not be mimicked when a tumour promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), was used instead of calcitonin as an inducer. Calcitonin and TPA exert their effects through different pathways, suggesting a clue to the mechanism of superinduction. Although inhibition of protein synthesis has been reported to increase transcription and mRNA stability in a number of other systems, the one described here appeared unique in combining both effects in the context of hormonal regulation.
Asunto(s)
Calcitonina/farmacología , Activadores Plasminogénicos/genética , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Línea Celular , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Sustancias Macromoleculares , Porcinos , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
The peptide hormone, calcitonin, induces urokinase-type plasminogen activator (uPA) enzyme activity in cultured LLC-PK1 pig kidney cells. This induction occurs as a consequence of transcriptional activation of the uPA gene. Treatment with the synthetic glucocorticoid hormone, dexamethasone, was found to inhibit calcitonin induction of uPA enzyme activity by as much as 80%. The inhibitory effect of dexamethasone was attributed to at least two mechanisms: induction of an inhibitor of uPA enzyme activity, and reduction in uPA mRNA levels. Study on reduction of uPA mRNA levels showed that dexamethasone significantly reduced the transcription rate of the calcitonin-induced uPA gene, without affecting the half-life of uPA mRNA. Although dexamethasone has been reported to induce inhibitors of plasminogen activator enzyme activity and to inhibit transcription of various genes, the system described here appears novel in that both actions are coordinated.
Asunto(s)
Dexametasona/farmacología , Genes/efectos de los fármacos , Riñón/enzimología , Activador de Plasminógeno de Tipo Uroquinasa/genética , Animales , Calcitonina/farmacología , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Inducción Enzimática , Cinética , Porcinos , Transcripción Genética/efectos de los fármacos , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesisRESUMEN
We have isolated cDNA and genomic clones coding for porcine plasminogen activator (urokinase, uPA). The cDNA is 2375 nucleotides long: it consists of a 5'-non-coding region (104 nucleotides), an open reading frame of 1329 nucleotides, and 3'-non-coding region of 942 nucleotides apart from the poly A tail. The genomic segment corresponding to the transcribed sequence is 5.85 kb long; it is composed of 11 exons and 10 introns. The 5'-flanking genomic region contains a number of sequences of potential regulatory significance, including possible hormone receptor binding sites and a sequence which we tentatively propose may be involved in activation of transcription by cAMP. The full sequence of both cDNA and genomic clones, the latter including 1.3 kb of flanking region, is presented and discussed, and the deduced amino acid sequence compared with that of human uPA.