RESUMEN
The IgA1 protease secreted by the pathogenic Neisseriae cleaves Lamp1, a major integral membrane glycoprotein of lysosomes, and significantly reduces its steady-state levels in an infected cell. IgA1 protease hydrolysis of Lamp1 is inefficient at the low pH of lysosomes, strongly suggesting that the enzyme is unlikely to reduce Lamp1 levels within lysosomes to any appreciable extent. We therefore explored the possibility that the protease may reach Lamp1 through an alternative route. We demonstrate that Neisseria pili induce a transient increase in the levels of cytosolic free Ca2+ in A431 human epithelial cells, as demonstrated previously for ME180 cells. This Ca2+ flux triggers lysosome exocytosis, quickly altering the cellular distribution of Lamp1 and increasing surface Lamp1 levels. Finally, we demonstrate that surface Lamp1 is cleaved by IgA1 protease secreted by adherent bacteria. We conclude that the pilus-induced Ca2+ flux increases the amount of Lamp1 that is cleavable by the IgA1 protease.
Asunto(s)
Antígenos CD/metabolismo , Calcio/metabolismo , Exocitosis , Lisosomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Neisseria/enzimología , Serina Endopeptidasas/metabolismo , Antígenos CD/análisis , Línea Celular , Epitelio/microbiología , Exocitosis/fisiología , Fimbrias Bacterianas/metabolismo , Humanos , Proteína 1 de la Membrana Asociada a los Lisosomas , Proteínas de Membrana de los Lisosomas , Neisseria/crecimiento & desarrollo , Neisseria/patogenicidadRESUMEN
The renal cell line mIMCD3 exhibits markedly upregulated phosphorylation of the extracellular signal-regulated kinase (ERK) 1 and 2 in response to urea treatment (200 mM for 5 min). Previous data have suggested the involvement of a classical protein kinase C (cPKC)-dependent pathway in downstream events related to urea signaling. We now show that urea-inducible ERK activation requires extracellular calcium; unexpectedly, it occurs independently of activation of cPKC isoforms. Pharmacological inhibitors of known intracellular calcium release pathways and extracellular calcium entry pathways fail to inhibit ERK activation by urea. Fura 2 ratiometry was used to assess the effect of urea treatment on intracellular calcium mobilization. In single-cell analyses using subconfluent monolayers and in population-wide analyses using both confluent monolayers and cells in suspension, urea failed to increase intracellular calcium concentration. Taken together, these data indicate that urea-inducible ERK activation requires calcium action but not calcium entry. Although direct evidence is lacking, one possible explanation could include involvement of a calcium-dependent extracellular moiety of a cell surface-associated protein.
Asunto(s)
Calcio/metabolismo , Médula Renal/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal/fisiología , Urea/farmacología , Animales , Calcimicina/farmacología , Calcio/farmacología , Técnicas de Cultivo de Célula/métodos , Línea Celular , Activación Enzimática , Factor de Crecimiento Epidérmico/farmacología , Ionomicina/farmacología , Médula Renal/citología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Fosforilación , Proteína Quinasa C/metabolismo , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Tapsigargina/farmacologíaRESUMEN
Although ovarian surface epithelial (OSE) cells are the cell type responsible for malignant ovarian carcinoma, relatively little is known about either the extracellular stimuli or the intracellular signaling mechanisms responsible for regulating proliferation in these cells. We have demonstrated that OSE cells proliferate in response to elevation of extracellular calcium and that OSE cells express functional calcium-sensing receptors (CaR). Here we show that agonists of the CaR increase the kinase activity of Src and ERKs (extracellular signal-regulated kinases) in rat OSE cells and promote association between tyrosine-phosphorylated Shc and p120rasGAP. Expression of an interfering mutant CaR inhibited the proliferative response to elevated extracellular calcium, as well as CaR agonist-induced tyrosine phosphorylation and ERK activation. Transfection with dominant negative mutants of Ras, Raf, and MKK1 also inhibited the increase in ERK activity in response to calcium, as did treatment with herbimycin, a selective inhibitor for Src family kinases. These results indicate that the ability of OSE cells to proliferate in response to increases in extracellular calcium involves cross-talk between the G-protein-coupled CaR and the activation of a tyrosine kinase-dependent Ras-Raf-ERK signaling pathway.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/fisiología , Células Epiteliales/citología , Células Epiteliales/fisiología , Receptores de Superficie Celular/fisiología , Sustitución de Aminoácidos , Animales , Benzoquinonas , Calcio/farmacología , División Celular , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Espacio Extracelular/fisiología , Femenino , Flavonoides/farmacología , Riñón/metabolismo , Lactamas Macrocíclicas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutagénesis Sitio-Dirigida , Ovario/citología , Quinonas/farmacología , Ratas , Receptores Sensibles al Calcio , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rifabutina/análogos & derivados , Transfección , Familia-src Quinasas/metabolismoRESUMEN
The purpose of the present study was to determine whether human gastric mucous epithelial cells express a functional Ca2+-sensing receptor (CaR). Human gastric mucous epithelial cells were isolated from surgical tissues and cultured on glass coverslips, plastic dishes, or porous membrane filters. Cell growth was assessed by the MTT assay, CaR localization was detected by immunohistochemistry and confocal microscopy, CaR protein expression was assessed by Western immunoblotting, and intracellular Ca2+ concentration ([Ca2+]i) was determined by fura 2 spectrofluorometry. In paraffin sections of whole stomach, we found strong CaR immunohistochemical staining at the basolateral membrane, with weak CaR-staining at the apical membrane in mucous epithelial cells. Confocal microscopy of human gastric mucous epithelial cell cultures showed abundant CaR immunofluorescence at the basolateral membrane and little to no CaR immunoreactivity at the apical membrane. Western immunoblot detection of CaR protein in cell culture lysates showed two significant immunoreactive bands of 140 and 120 kDa. Addition of extracellular Ca2+ to preconfluent cultures of human gastric mucous epithelial cells produced a significant proliferative response. Changes in [Ca2+]i were also observed in response to graded doses of extracellular Ca2+ and Gd3+. The phospholipase C inhibitor U-73122 specifically inhibited Gd3+-induced changes in [Ca2+]i in the gastric mucous epithelial cell cultures. In conclusion, we have identified the localization of a functional CaR in human gastric mucous epithelial cells.
Asunto(s)
Mucosa Gástrica/metabolismo , Receptores de Superficie Celular/metabolismo , Western Blotting , Calcio/fisiología , División Celular/fisiología , Células Cultivadas , Espacio Extracelular/metabolismo , Gadolinio/metabolismo , Mucosa Gástrica/citología , Humanos , Inmunohistoquímica , Microscopía Confocal , Receptores Sensibles al Calcio , Valores de ReferenciaRESUMEN
Epidermal growth factor (EGF) is produced in the ovary and influences proliferation of the malignant ovarian surface epithelium (OSE); yet its role in malignancy or in regulating the normal surface epithelium is unclear. In human OSE cells derived from primary cultures of normal tissue transfected with SV40 large T antigen (IOSE cells), EGF promoted survival but not proliferation. This survival effect was reversed by acute treatment with the phorbol ester, 12-0-tetradecanoyl-13-phorbol acetate (TPA) which alone markedly inhibited IOSE proliferation. We tested whether the activities of the mitogen-activated protein kinases (ERK1/2 and JNK1) varied in response to EGF, TPA, or combinations of these agonists and if the same treatments altered patterns of immediate early gene expression. Alone, EGF activated ERK1/2, increased and sustained levels of c-jun mRNA, but had almost no effect on JNK1 activation. Conversely, PKC activation resulted in a rapid, but transient induction of c-fos RNA and of both kinases, JNK1 and ERK2. When combined, EGF and TPA further enhanced the phosphorylation of both enzymes despite inhibiting survival. Though JNKs and ERKs are thought to transduce opposing cellular responses, in IOSE cells, robust costimulation of the JNK and ERK pathways may redirect the survival message.
Asunto(s)
Apoptosis , Factor de Crecimiento Epidérmico/metabolismo , Ovario/citología , Proteína Quinasa C/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Activación Enzimática , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Genes Inmediatos-Precoces , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/genética , Acetato de Tetradecanoilforbol/farmacología , Factor de Crecimiento Transformador alfa/farmacologíaRESUMEN
Nerve endings of nociceptors (pain-sensing neurons) express an unusual subtype of ATP-gated ion channel, the P2X3 receptor, that rapidly desensitizes (<100 msec) and slowly recovers (>20 min). Here we show that Ca2+, or certain other polyvalent cations, binds to an extracellular site on rat sensory neurons and can increase current through P2X3 channels more than 10-fold. Importantly, Ca2+ facilitates P2X3 current to precisely the same level whether a transient Ca2+ change occurred just before or several minutes before activating the channels with ATP. This memory for past changes in Ca2+ is integrative in that a 90 sec Ca2+ stimulus delivered just before an ATP application has the same effect as an earlier series of three, separated 30 sec Ca2+ stimuli. These diverse phenomena are explained by a single mechanism: Ca2+ speeds recovery of P2X channels from desensitization. Recovery follows an exponential growth curve that depends on the duration, but not the timing, of changes in recovery rate. Modulation of desensitization underlies a well described short-term memory in bacteria, and it might be similarly used in the nervous system.
Asunto(s)
Calcio/farmacología , Activación del Canal Iónico/fisiología , Memoria/fisiología , Receptores Purinérgicos P2/fisiología , Adenosina Trifosfato/farmacología , Animales , Agonistas de los Canales de Calcio/farmacología , Células Cultivadas , Ganglios Espinales/química , Ganglios Espinales/fisiología , Humanos , Hiperalgesia/fisiopatología , Activación del Canal Iónico/efectos de los fármacos , Riñón/citología , Aprendizaje/fisiología , Mecanorreceptores/fisiología , Terminaciones Nerviosas/química , Terminaciones Nerviosas/fisiología , Neuronas Aferentes/química , Neuronas Aferentes/fisiología , Neuropéptidos/fisiología , Nociceptores/fisiología , Técnicas de Placa-Clamp , Ratas , Receptores Purinérgicos P2X3RESUMEN
OBJECTIVE: Our purpose was to determine whether human ovarian surface epithelial cells express calcium-sensing receptors and whether changes in extracellular calcium modulate the proliferation of these cells. STUDY DESIGN: Ovarian surface epithelial cells from normal patients and from ovarian tumors were tested for calcium-sensing receptor expression by Northern hybridization and immunoblot analysis. Functional responses to agonists of the calcium-sensing receptor were monitored by inositol triphosphate analysis and measurements of intracellular calcium release. The effect of extracellular calcium on proliferation was monitored by thymidine incorporation and growth curve analysis. RESULTS: Increasing extracellular calcium above 0.8 mmol/L produces a marked proliferative response in normal ovarian surface epithelial cells. Both normal and transformed cells express calcium-sensing receptor messenger ribonucleic acid and protein; some tumor lines overexpress calcium-sensing receptor messenger ribonucleic acid. The calcium-sensing receptors in normal ovarian surface epithelial cells respond to the agonist gadolinium with increases in inositol triphosphate production and calcium release. CONCLUSION: Human ovarian surface epithelial cells are growth regulated by extracellular calcium, and calcium-sensing receptors may mediate this response.
Asunto(s)
Expresión Génica , Ovario/química , Receptores de Superficie Celular/genética , Secuencia de Aminoácidos , Northern Blotting , Calcio/farmacología , División Celular/efectos de los fármacos , Células Epiteliales/química , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Gadolinio/farmacología , Humanos , Immunoblotting , Fosfatos de Inositol/metabolismo , Datos de Secuencia Molecular , Ovario/citología , Ovario/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ADN Polimerasa Dirigida por ARN , Receptores Sensibles al Calcio , Receptores de Superficie Celular/química , Receptores de Superficie Celular/fisiología , Alineación de SecuenciaRESUMEN
Changes in the concentration of extracellular calcium can affect the balance between proliferation and differentiation in several cell types, including keratinocytes, breast epithelial cells, and fibroblasts. This report demonstrates that elevation of extracellular calcium stimulates proliferation-associated signaling pathways in rat fibroblasts and implicates calcium-sensing receptors (CaR) as mediators of this response. Rat-1 fibroblasts express CaR mRNA and protein and respond to known agonists of the CaR with increased IP3 production and release of intracellular calcium. Agonists of the CaR can stimulate increased c-SRC kinase activity and increased extracellular signal-regulated kinase 1/mitogen-activated protein kinase activity. Both of the increases in SRC activity and mitogen-activated protein kinase activation are blocked in the presence of a nonfunctional mutant of the CaR, R796W. Proliferation of wild-type Rat-1 cells is sensitive to changes in extracellular calcium, but expression of the nonfunctional CaR mutant or inhibition of the calcium-dependent increase in SRC kinase activity block the proliferative response to calcium. These results provide evidence of a novel signal transduction pathway modulating the response of fibroblasts to extracellular calcium and imply that calcium-sensing receptors may play a role in regulating cell growth in response to extracellular calcium, in addition to their well known function in systemic calcium homeostasis.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calcio/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Receptores de Superficie Celular/metabolismo , Familia-src Quinasas/metabolismo , Animales , División Celular , Línea Celular , Clonación Molecular , Activación Enzimática , Espacio Extracelular/metabolismo , Fibroblastos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Mutagénesis , Fosforilación , Ratas , Receptores Sensibles al Calcio , Receptores de Superficie Celular/genética , Tirosina/metabolismoRESUMEN
Agents such as thapsigargin and endothelin elevate intracellular calcium levels by a combination of calcium release from intracellular stores and calcium influx across the plasma membrane; however, the relative contribution of influx vs. release in modulating calcium-dependent gene expression is not as well understood in nonexcitable cells as in excitable cells. In this report we have been able to separate thapsigargin-induced elevation of intracellular calcium into release and influx components, using carboxyamido-triazole (CAI), a known inhibitor of calcium influx with antiproliferative activity against a number of human carcinomas, to selectively inhibit influx without affecting release. The results of these experiments indicate that the ability of thapsigargin to induce calcium-dependent gene expression in nonexcitable cells is dependent on the induction of calcium influx, presumably through store-operated calcium channels. CAI treatment specifically inhibited thapsigargin- or endothelin-stimulated expression from the c-fos promoter in Rat-1 cells and in epithelial cell lines derived from ovary and breast. Use of the VL30 model system confirmed the ability of CAI to inhibit calcium-dependent gene expression and further demonstrated that the ability of elevated calcium to synergize with other signaling pathways required close temporal coupling. In addition to inhibiting endothelin-induced calcium influx, CAI treatment also resulted in a partial inhibition of IP3 production and calcium release. CAI treatment also blocked the increase in ERK1 kinase activity observed in response to either endothelin or thapsigargin, suggesting a role for calcium influx in the activation of mitogen-activated protein kinase pathways.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Calcio/fisiología , Endotelina-1/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genes fos/efectos de los fármacos , Tapsigargina/farmacología , Triazoles/farmacología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Endotelina-1/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , Genes Reporteros/efectos de los fármacos , Genes Reporteros/genética , Genes fos/genética , Imidazoles/farmacología , Ratas , Tapsigargina/antagonistas & inhibidoresRESUMEN
Induction of gene expression in response to calcium ionophores or thapsigargin, which inhibits the calcium-ATPase responsible for sequestering intracellular calcium, has frequently been attributed to direct stimulatory events subsequent to the elevation of intracellular free calcium. VL30 is a murine gene that is transcriptionally induced in response to a large array of mitogenic and transforming stimuli. We have shown previously that an enhancer element within the VL30 promoter region is dependent upon cotreatment with thapsigargin or calcium ionophore for a full-scale induction of gene expression. In this report, we demonstrate that both thapsigargin and calcium ionophores induce a transient inhibition of protein synthesis in Rat-1 cells transfected with a VL30 enhancer-driven reporter construct. Recovery of protein synthesis is facilitated by cotreatment with epidermal growth factor or phorbol esters. Furthermore, treatment with cycloheximide or DTT, which inhibit protein synthesis without altering intracellular calcium levels, can substitute for thapsigargin or ionophores in stimulating VL30 gene expression. These results suggest that the stimulatory effects of thapsigargin and calcium ionophores on VL30 expression may be mediated, at least in part, by the ability of these agents to initiate stress responses associated with inhibition of protein synthesis.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Genes Inmediatos-Precoces/fisiología , Inhibidores de la Síntesis de la Proteína/farmacología , Terpenos/farmacología , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Colina O-Acetiltransferasa/genética , Ionomicina/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , TapsigarginaRESUMEN
The VL30 family of defective retrovirus-like elements is abundantly transcribed in response to numerous transforming and proliferative stimuli. We have identified a novel enhancer element in the long terminal repeat of the transcriptionally active VL30 element RVL-3. The RVL-3 enhancer mediates a calcium-dependent induction of gene expression in response to treatment with either epidermal growth factor or the phorbol ester tumor promoter 12-O-tetradecanoylphorbol acetate. In this report we present in vivo and in vitro evidence indicating that the RVL-3 enhancer is also responsive to cAMP in the presence of elevated intracellular calcium. Proteins present in nuclear extracts obtained from Rat-1 fibroblasts bind specifically to a 20-basepair sequence within the RVL3 triple repeat. Competition binding studies and mutational analyses indicate that the cAMP responsiveness maps to the same region responsible for mediating the inductive response to epidermal growth factor and 12-O-tetradecanoylphorbol. The responsive sequence is different from previously described enhancer elements. This novel enhancer mediates transcription by multiple agonists and promotes a greater than additive increase in gene expression when more than one signal transduction pathway is stimulated simultaneously.
Asunto(s)
Calcio/farmacología , AMP Cíclico/farmacología , Elementos de Facilitación Genéticos , Expresión Génica/efectos de los fármacos , Secuencia de Bases , Unión Competitiva , Calcio/metabolismo , Línea Celular , Núcleo Celular/química , AMP Cíclico/análogos & derivados , AMP Cíclico/fisiología , ADN/química , ADN/metabolismo , Interacciones Farmacológicas , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Transducción de Señal , Terpenos/farmacología , Acetato de Tetradecanoilforbol/farmacología , TapsigarginaRESUMEN
Changes in intracellular Ca++ levels are observed as a second messenger in response to a number of cellular agonists, including epidermal growth factor, transforming growth factor beta 1, and endothelin-1. The role of elevated intracellular Ca++ in transducing the effects of these three agonists on gene expression has been studied using two target genes: transin/stromelysin-1 and the endogenous murine retrovirus VL30. Although the effects of EGF and TGF beta 1 on transin/stromelysin-1 mRNA expression appear to be independent of these agonists' effects on intracellular Ca++ levels, elevated Ca++ interacted synergistically with activators of pkC to induce transin expression, even though neither agent alone could induce transin/stromelysin-1 expression. In contrast, the integrated VL30 retrovirus could be induced by Ca++ ionophores alone, and induction of VL30 mRNA by other agonists was blocked if intracellular Ca++ levels were held below a threshold value of 165 nM with Ca++ chelators. Genetic analysis of the VL30 upstream regulatory region indicated that a triple-repeat element present in the VL30 long-terminal repeat could function as an inducible enhancer, but responsiveness to either EGF or pkC activation required the concomitant elevation of intracellular Ca++. Because EGF was capable of inducing expression even in pkC-depleted cells, providing Ca++ levels were elevated, these results indicate that elevated intracellular Ca++ is capable of interacting synergistically with multiple signaling pathways to stimulate increased gene expression.
Asunto(s)
Calcio/farmacología , Genes Virales/genética , Metaloendopeptidasas/genética , Animales , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Metaloproteinasa 3 de la Matriz , Transducción de SeñalRESUMEN
The VL30 family of defective murine retroviruses consists of 100 to 200 members, of which fewer than 5% appear to be transcriptionally active. A genomic clone of the transcriptionally active VL30 element RVL-3 was identified and sequenced. Genetic analysis indicated that a triple-repeat sequence within the RVL-3 long terminal repeat is capable of functioning as an inducible enhancer element responding to a variety of agonists. In Rat-1 fibroblasts, the ability of the RVL-3 enhancer to mediate induction of gene expression from a heterologous promoter in response to either epidermal growth factor (EGF) or phorbol ester treatment required coelevation of intracellular calcium. Two CArG boxes present in the triple-repeat sequence appeared to exert a negative effect on gene expression, as mutation of these sequences elevated the basal level of expression observed without altering the fold induction in response to either EGF or protein kinase C activation. In the presence of these CArG elements, mutation of AP-1-like sites adjacent to the CArG elements significantly inhibited the ability of either EGF or phorbol esters to induce gene expression. The effect of mutating these AP-1-like sites was relieved by simultaneous mutation of the CArG sites, indicating that interactions among these sites modulate RVL-3 expression. Mutational analysis and gel mobility shift experiments have identified a third sequence within the VL30 triple-repeat element that is required for the induction of gene expression and serves as a binding site for nuclear proteins. Sequence comparisons indicate that this enhancer element has not been described previously.
Asunto(s)
Calcio/fisiología , Elementos de Facilitación Genéticos , Factor de Crecimiento Epidérmico/farmacología , Regulación Viral de la Expresión Génica , Proteína Quinasa C/fisiología , Retroviridae/genética , Animales , Secuencia de Bases , Análisis Mutacional de ADN , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Virus Defectuosos/genética , Activación Enzimática , Regulación Viral de la Expresión Génica/efectos de los fármacos , Técnicas In Vitro , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Ratas , Secuencias Repetitivas de Ácidos Nucleicos , Acetato de Tetradecanoilforbol/farmacología , Transcripción GenéticaRESUMEN
Endothelin-1 (ET-1) has been shown to require Ca2+ influx for activation of vascular smooth muscle in vivo, but in vitro models show that ET-1 mobilizes intracellular Ca2+ and is independent of extracellular Ca2+. We present data that suggest ET-1 modulates cellular responses through a dual mechanism involving both phosphatidylinositol turnover and Ca2+ channel activation. Addition of low concentrations of ET-1 (less than 10(-9) M) to serum-deprived quiescent Rat-1 cells stimulated Ca2+ influx while having little effect on diacylglycerol (DG) release or intracellular Ca2+ levels. In contrast, higher concentrations of ET-1 (greater than 10(-9) M) stimulated intracellular Ca2+ transients and release of inositol trisphosphate (IP3) and DG but did not activate Ca2+ uptake. Stimulation of Ca2+ influx at low [ET-1] could not be accounted for by depletion of intracellular IP3-sensitive pools. Neither the stimulation of Ca2+ influx at low [ET-1] nor the inhibitory actions of high [ET-1] could be mimicked by the activation of protein kinase C. We tested the hypothesis that elevated intracellular Ca2+ was inhibitory for Ca2+ influx. When intracellular Ca2+ transients were maintained below approximately 165 nM by chelation with BAPTA or BAPTA derivatives with altered affinity for Ca2+, Ca2+ influx was stimulated over the entire range of ET-1 concentrations. In addition, experimentally elevating intracellular Ca2+ levels with the tumor promoter thapsigargin abolished ET-1-stimulated Ca2+ influx. These data suggest that the biological consequences of ET-1 release may be determined by local concentration differences. Thus in vascular smooth muscle cells ET-1 may act either to mobilize intracellular Ca2+ or to promote Ca2+ influx, depending on the distance from the endothelial cell source in the vascular wall. The activation of different processes by low and high ET-1 concentrations may determine the physiological response to ET-1 stimulation in vivo.
Asunto(s)
Calcio/metabolismo , Endotelinas/farmacología , Sistemas de Mensajero Secundario/efectos de los fármacos , Animales , Transporte Biológico/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Línea Celular , Quelantes/farmacología , Diglicéridos/metabolismo , Ácido Egtácico/farmacología , Retroalimentación , Inositol 1,4,5-Trifosfato/metabolismo , Cinética , RatasRESUMEN
In addition to its powerful vasoconstrictive activity, endothelin-1 (ET-1) is a potent agonist for stimulating a multitude of second messenger pathways. In the Rat-1 fibroblastic cell line, ET-1 induces a robust elevation of the intracellular levels of Ca2+, diacylglycerols (DGs), and inositol trisphosphate (IP3). Although low concentrations of ET-1 stimulate a significant increase in the rate of Ca2+ influx, this Ca2+ influx is not required for the observed increases in either IP3 or DG levels following ET-1 treatment, as both of these effects are observed even in the absence of extracellular Ca2+. The ability of ET-1 to stimulate Ca2+ influx shows a biphasic pattern, such that Ca2+ influx is stimulated at low ET-1 concentrations and inhibited at high concentrations. Investigations of the molecular mechanisms underlying this biphasic response indicate that elevated intracellular Ca2+ levels exert a negative feedback inhibition on Ca2+ influx, which can be relieved by the chelation of intracellular Ca2+. The ability of ET-1 to activate a number of distinct signal transduction pathways appears to have direct functional significance in determining the targeting of ET-1 activation. Short-term effects of ET-1 stimulation such as the induction of gene expression appear to be independent of ET-1's ability to activate protein kinase C (PKC) by elevating DG levels, as depletion of PKC activity has little or no effect on gene expression. In contrast, the ability of ET-1 to induce the rapid expression of the VL30 gene is totally dependent upon the ability of ET-1 to elevate intracellular Ca2+ levels above a specific threshold. Activation of PKC by ET-1, however, is essential for the long-term effects of ET-1 on cell proliferation and anchorage-independent growth, as the ability of ET-1 to promote DNA synthesis and to synergize with epidermal growth factor in augmenting anchorage-independent growth is significantly inhibited by prior PKC depletion. Thus, in fibroblasts, ET-1 appears to activate at least two bifurcating pathways: a Ca(2+)-sensitive pathway involved in the regulation of gene expression, and a PKC-dependent pathway required for the mitogenic effects of ET-1.
Asunto(s)
Calcio/metabolismo , Endotelinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , Células Cultivadas , ADN/análisis , ADN/biosíntesis , Diglicéridos/metabolismo , Ácido Egtácico/análogos & derivados , Fura-2 , Humanos , Fosfatos de Inositol/metabolismo , Proteína Quinasa C/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Sistemas de Mensajero Secundario/efectos de los fármacos , PorcinosRESUMEN
The transcriptionally active RVL3-VL30 element contains a triple repeat of TGACTCC, a sequence nearly identical to the AP-1 binding site. However, 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation was unable to elicit chloramphenicol acetyltransferase (CAT) expression from a construct containing these AP-1-like sequences upstream of the thymidine kinase promoter present in pTES. Endothelin, which activates protein kinase C (pkC) and elevates intracellular Ca2+ in Rat-1 cells, was effective in stimulating CAT expression from the VL30-pTES construct. We attempted to assess the relative importance of these second messenger systems by stimulating each pathway separately with exogenous agonists. We determined that neither stimulation of pkC by the tumor promoter TPA nor elevation of intracellular Ca2+ by the tumor promoter thapsigargin was sufficient to stimulate CAT expression from the VL30-pTES vector. When combined, the two tumor promoters induced a synergistic increase in CAT expression. Our data indicate that elevation of intracellular Ca2+ by thapsigargin was not required for full activation of pkC by TPA. First, TPA was able to stimulate expression of other genes in Rat-1 cells, indicating full activation of pkC. Second, thapsigargin synergized effectively with epidermal growth factor to stimulate CAT activity from the VL30-pTES construct in cells depleted of pkC activity by chronic TPA treatment. The permissive effects of thapsigargin on gene expression were also observed for an endogenous gene, transin/stromelysin. The permissive effects of elevated intracellular Ca2+ levels may represent a general mechanism for the stimulation of some genes by pkC-mediated pathways.
Asunto(s)
Carcinógenos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Terpenos/farmacología , Acetato de Tetradecanoilforbol/farmacología , Animales , Secuencia de Bases , Calcio/metabolismo , Línea Celular , Cloranfenicol O-Acetiltransferasa/biosíntesis , Proteínas de Unión al ADN/metabolismo , Interacciones Farmacológicas , Endotelinas/fisiología , Metaloproteinasa 3 de la Matriz , Metaloendopeptidasas/genética , Proteína Quinasa C/fisiología , Proteínas Proto-Oncogénicas c-jun , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos/genética , Secuencias Reguladoras de Ácidos Nucleicos/fisiología , Transducción de Señal/efectos de los fármacos , Tapsigargina , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
We investigated the role of intracellular [Ca2+] in mediating the independent signal transduction pathways leading to induction of VL30 RNA expression by multiple agonists in the Rat-1-derived RVL-3 cell line. This cell line contains a single integrated VL30 element, and displays a rapid transcriptional activation of VL30 following stimulation by epidermal growth factor, endothelin, or the phorbol ester tumor promoter 12-O-tetradecanoylphorbol acetate (Rodland, K. D., Brown, A. M. C., and Magun, B. E. (1987) M. Cell. Biol. 7, 2296-2298). Neither epidermal growth factor nor endothelin is dependent upon protein kinase C for activation of VL30 expression, as both of these agonists induce normal levels of VL30 RNA expression, even in cells which have been severely depleted of protein kinase C following chronic 12-O-tetradecanoylphorbol acetate exposure. Induction of VL30 RNA expression by either endothelin or 12-O-tetradecanoylphorbol acetate was blocked by concomitant exposure of RVL-3 cells to the intracellular Ca2(+)-chelating agent 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid at a concentration sufficient to buffer intracellular [Ca2+] below 200 nM, and VL30 RNA was induced by the application of the Ca2+ ionophore A23187 in the absence of agonist. Normal levels of VL30 expression in response to epidermal growth factor were observed at 165 nM [Ca2+], but were significantly inhibited at 115 nM [Ca2+]. Both the protein kinase C-dependent and protein kinase C-independent pathways leading to VL30 transcription were dependent upon the presence of an intracellular [Ca2+] exceeding 115 nM. The dependence upon intracellular Ca2+ transients for transcriptional induction by endothelin appears to be a characteristic of VL30 expression, as 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid treatment did not prevent the endothelin-induced transcription of the protooncogenes c-jun and c-fos.
Asunto(s)
Calcio/fisiología , Factor de Crecimiento Epidérmico/farmacología , Péptidos/farmacología , Proteína Quinasa C/metabolismo , ARN Viral/biosíntesis , Retroviridae/genética , Animales , Benzofuranos , Calcimicina/farmacología , Proteínas Portadoras/genética , Línea Celular , Ácido Egtácico/farmacología , Endotelinas , Endotelio Vascular , Colorantes Fluorescentes , Fura-2 , Fosfatos de Inositol/metabolismo , Isomerasa de Peptidilprolil , Ratas , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología , Transcripción GenéticaRESUMEN
Transforming growth factor beta (TGF-beta), initially identified in platelet extracts by virtue of its ability to confer anchorage-independent growth and a neoplastic phenotype on mesenchymal cells, has subsequently been identified as a potent inhibitor of proliferation in most cells of epithelial origin. Our laboratory has investigated the role of specific second messengers in mediating the transcriptional responses of fibroblasts following addition of TGF-beta 1. Our studies indicate that TGF-beta 1, alone and in conjunction with epidermal growth factor (EGF), is capable of stimulating increases in both phosphoinositide metabolism and calcium influx, leading to significant increases in intracellular levels of Ca++ and inositol trisphosphate (IP3). Our data indicated that Ca++ influx and inositol phosphate release are coupled in Rat-1 cells, and suggested that influx of Ca++ from the extracellular medium is required for the change in IP3 accumulation observed in response to both EGF and TGF-beta 1. Using nuclear run-on analysis of the transcription of rat transin, a secreted metalloproteinase homologous to human stromelysin, we have also demonstrated a significant inhibition of transin transcription within 10 min of TGF-beta 1 treatment. The ability of TGF-beta 1 to inhibit transin gene transcription was not related to the TGF-beta 1-induced influx of Ca++ or to an increase in intracellular inositol phosphates, since inhibiting production of these second messengers failed to inhibit repression of the transin gene.
Asunto(s)
Fibroblastos/metabolismo , Factores de Crecimiento Transformadores/fisiología , Animales , Calcio/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Espacio Extracelular/metabolismo , Regulación de la Expresión Génica , Fosfatos de Inositol/metabolismo , Membranas Intracelulares/metabolismo , Metaloproteinasa 3 de la Matriz , Metaloendopeptidasas/genética , Sistemas de Mensajero Secundario , Transducción de Señal , Factores de Crecimiento Transformadores/genética , Factores de Crecimiento Transformadores/inmunología , Cicatrización de HeridasRESUMEN
Stimulation of quiescent cultured fibroblasts with a variety of growth-promoting factors induces release of diacylglycerol (DG) and subsequent activation of protein kinase C (pkC), but the role of pkC in the induction of DNA synthesis and cell proliferation remains unclear. We have investigated the involvement of pkC in the response of Rat-1 fibroblasts to the newly described peptide endothelin-1 (Et-1), an agonist that is secreted by the vascular endothelium and that may play a role in the proliferative response of cells in the vessel wall. Addition of Et-1 to serum-deprived Rat-1 cells promoted DNA synthesis in the absence of additional factors and stimulated anchorage-independent growth in the presence of epidermal growth factor (EGF), indicating that Et-1 has many of the characteristics of a mitogen. The ability of Et-1 to stimulate both DNA synthesis and anchorage-independent growth was markedly reduced by the depletion of cellular pkC activity induced by prolonged exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, the ability of Et-1 to induce both second messenger production and transcription of c-fos and c-jun was largely independent of cellular pkC activity. Production of DG in response to Et-1 persisted for greater than 12 h and may account for the ability of Et-1 to augment the G1-S phase transition. Although these observations indicate that functional pkC is not an essential component of the proximal pathway leading to rapid changes in gene transcription and second messenger production in response to Et-1 treatment, the data suggest that activation of pkC is an essential component of the downstream events responsible for the stimulation of cell proliferation and anchorage-independent growth in Rat-1 cells exposed to Et-1.
Asunto(s)
Replicación del ADN/efectos de los fármacos , Endotelinas/farmacología , Fibroblastos/efectos de los fármacos , Proteína Quinasa C/fisiología , Animales , División Celular/efectos de los fármacos , Línea Celular , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Diglicéridos/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-fos , Proteínas Proto-Oncogénicas c-jun , Ratas , Sistemas de Mensajero Secundario/efectos de los fármacos , Estimulación Química , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacosRESUMEN
Various proteases have been found to be released by the growth cones of developing neurons in culture and have been hypothesized to play a role in the process of axon elongation. We report here that nerve growth factor (NGF) induced the gene encoding the metalloprotease transin in PC12 cells with a time course coincident with the initial appearance of neurites by these cells. Acidic and basic fibroblast growth factors also stimulated transin mRNA expression and neurite outgrowth, whereas various other agents had no effects on either of these phenomena. In contrast, dexamethasone was found to inhibit the induction of transin mRNA when added with, or following, NGF treatment. Finally, we show that sequences contained within 750 bp of the 5' untranscribed region of the transin gene confer responsiveness to NGF and dexamethasone.