RESUMEN
A retrospective clinicopathological analysis was performed to compare 35 proliferative verrucous leukoplakia (PVL), 40 leukoplakia without dysplasia (LK), 48 oral lichen planus (OLP)/oral lichenoid lesions (OLL), and 11 verrucous carcinoma (VC) (N = 134). The PVL group comprised 24 female and 11 male patients (mean age 66.5 years), with two to six sites involved (mean 3.1 sites) and multiple biopsies over time (mean 7.1/case). All PVL cases developed malignancy: 77.1% squamous cell and 40% verrucous carcinoma; 68.6% had multiple sites of malignancy. None showed local or distant metastatic spread. Five-year disease-specific survival was 88.6%. In LK and OLP/OLL, malignant transformation was significantly lower than in PVL (2.5% and 2.1%, respectively). Invasive squamous cell carcinoma was not reported in any conventional VC. Immunohistochemical histomorphometric analysis for p53, COX-2, and podoplanin showed no significant differences between the groups. PVL may overlap with LK, OLP/OLL, and VC, but has a persistent aggressive behaviour and high malignant transformation rate. The overlapping features may delay recognition as PVL. The results emphasize the need for a detailed clinicopathological definition of PVL, and long-term close monitoring to ensure progression to PVL and malignancy are recognized in time. The management of this persistent aggressive condition is challenging.
Asunto(s)
Carcinoma de Células Escamosas , Carcinoma Verrugoso , Neoplasias de la Boca , Anciano , Carcinoma de Células Escamosas/patología , Carcinoma Verrugoso/patología , Transformación Celular Neoplásica/patología , Femenino , Humanos , Leucoplasia Bucal , Masculino , Neoplasias de la Boca/patología , Estudios RetrospectivosRESUMEN
The single-copy gene for fibroblast growth factor-2 (FGF-2) encodes for multiple forms of the protein with molecular masses of 24, 22.5, 22, and 18 kDa. We reported previously that the 24-22-kDa FGF-2 forms inhibit the migration of endothelial and MCF-7 cells by 50% and 70%, respectively. Here we show that this inhibition of migration is mediated by the estrogen receptor (ER). We have found that depletion of the receptor in either cell line abrogates the inhibitory activity of 24-kDa FGF-2 while re-introduction of the ER into deficient cells once again promotes the inhibitory response. To determine whether exposure to 24-kDa FGF-2 resulted in the activation of the estrogen receptor, 3T3 cells were cotransfected with estrogen receptor cDNA and an estrogen regulatory element-luciferase gene reporter construct and treated with 24- and 18-kDa FGF-2. The high molecular weight form stimulated luciferase activity 5-fold while 18-kDa FGF-2 at the same concentration had no effect. Treatment of ER-positive MCF-7 cells transfected with the reporter construct only showed the same results. Inclusion of the pure estrogen antagonist ICI 182,780 blocked the increase in luciferase activity by 24-kDa FGF-2, further indicating that the response was estrogen receptor dependent. Expression of dominant negative FGF receptor 1 inhibited ER activation, indicating that this was the cell surface receptor mediating the effect. Although growth factor-dependent activation of the ER was reported to require mitogen-activated protein kinase-induced phosphorylation at Ser(118) in COS and HeLa cells, this mechanism is not involved with the activation by 24-kDa FGF-2. These results suggest that the addition of 55 amino acids to the amino-terminal end of 18-kDa FGF-2 by alternative translation alters FGF-2 function and allows for the activation of a second signaling pathway involving the estrogen receptor.
Asunto(s)
Movimiento Celular/fisiología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Receptores de Estrógenos/fisiología , Células 3T3 , Animales , Células COS , Células HeLa , Humanos , Ratones , Receptores de Estrógenos/química , Receptores de Estrógenos/metabolismo , Células Tumorales CultivadasRESUMEN
In previous studies of the role of tissue plasminogen activator (tPA) in the lung inflammatory response, we observed that tPA expression was present exclusively in the small arteries and arterioles within the lung and absent from the capillaries, veins, and large pulmonary arteries. To define more completely the expression pattern of tPA, we evaluated the distribution of this protein during prenatal and postnatal development. tPA was first observed in the rat fetus at day 13 in the large arteries of both the thoracic and cranial cavities, including the dorsal aortas and pulmonary arteries in the former and the internal carotid and middle cerebral arteries in the latter. By day 15, tPA was no longer detectable in the aortas but appeared throughout the pulmonary, subclavian, vertebral, and basilar arteries. At day 17, tPA had disappeared from the subclavian artery and the proximal portion of the vertebral artery but was found in the smaller arterial branches of these 2 large vessels. By the end of gestation, tPA had also disappeared from the main pulmonary arteries but remained in the branches at the hilus of the lung. At birth, tPA was concentrated in the endothelia of arteries within the pia mater, the basilar and superficial cerebral arteries, and the lung arterial system. As the animals reached maturity, tPA disappeared from the larger cerebral arteries and their cortical branches but continued to be expressed in the vessels of the pia mater and lung. This study indicates that tPA expression is a dynamic process that responds to a changing arterial environment during vascular development.
Asunto(s)
Arterias/embriología , Desarrollo Embrionario y Fetal , Activador de Tejido Plasminógeno/análisis , Animales , Aorta/química , Aorta/embriología , Arterias/química , Endotelio Vascular/química , Endotelio Vascular/embriología , Femenino , Edad Gestacional , Inmunohistoquímica , Pulmón/irrigación sanguínea , Pulmón/embriología , Piamadre/irrigación sanguínea , Piamadre/embriología , Embarazo , Arteria Pulmonar/química , Arteria Pulmonar/embriología , Ratas , Ratas Wistar , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Basic fibroblast growth factor (FGF2) is synthesized as four isoforms with molecular weights of 24, 22.5, 22, and 18 kDa, with each of the three higher molecular weight forms (hmwFGF2) produced by the initiation of translation at one of three upstream CUG codons. We have shown that bovine arterial endothelial cells export the high molecular weight forms of FGF2 (hmwFGF2) in a 17beta-estradiol-dependent manner (Piotrowicz et al., 1997, J Biol Chem 272:7042-7047). To determine whether the hmwFGF2 forms affected cell behavior after release, we evaluated the effect of recombinant hmwFGF2 on the growth and migration of endothelial cells and mammary carcinoma cells (MCF-7). Treatment with the recombinant protein resulted in the inhibition of endothelial cell migration by 45% and MCF-7 cell migration by 70%. HmwFGF2-dependent inhibition was observed when endothelial cell migration was stimulated by 18 kDa FGF2 or vascular endothelial growth, and MCF cell migration was stimulated with insulin-like growth factor. In each case, inclusion of an antibody against the 55 amino acid amino terminal end of 24 kDa FGF2 abrogated the inhibition of migration, while antibodies to the 18 kDa FGF2 domain had no effect. When endothelial cells were cultured under conditions which promoted export of hmwFGF2, a 40% decrease in motility was observed which was reversed by the antibodies to the 24 kDa FGF2. Thus, both recombinant and endogenously produced hmw-FGF2 are capable of inhibiting migration. In contrast to the ubiquitous effect on migration, hmwFGF2 had no effect on endothelial cell growth but stimulated MCF-7 growth equally as well as the 18 kDa FGF2 (threefold). Antibodies to the 18 kDa domain of 24 kDa FGF2 blocked the growth-promoting activity of hmwFGF2, but those to the amino terminal end were ineffective. These data suggest that hmwFGF2 has dual activities, an inhibitory effect on cell migration and a growth-stimulating effect. The two activities can be localized to different parts of hmwFGF2: inhibitory activity to the amino terminal 55 amino acids (which are absent from the 18 kDa FGF2) and growth-promoting activity to the 18 kDa domain. Therefore, the ratio of hmwFGF2 and 18 kDa FGF2 in the extracellular space may provide a mechanism of control for angiogenesis and mammary tumor development.
Asunto(s)
División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Animales , Células COS , Bovinos , División Celular/fisiología , Línea Celular , Movimiento Celular/fisiología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/lesiones , Femenino , Factor 2 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/fisiología , Humanos , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/fisiologíaRESUMEN
Plasma levels of tissue plasminogen activator (tPA) increase rapidly in response to specific vasoactive agents, trauma, and neural stimulation. This response has been attributed to acute release of tPA from stored pools within the vascular endothelium and from catecholamine storage vesicles of chromaffin cells. We have tested directly whether tPA can be sorted into the regulated secretory pathway using the murine pituitary-derived neuroendocrine cell line AtT-20 transfected with tPA cDNA. Clones of AtT-20 cells expressing tPA were isolated, and targeting of tPA into the regulated secretory pathway was demonstrated by (1) stimulation of tPA secretion with 8-bromo-cAMP, the secretagogue which promotes the release of dense granule contents; (2) colocalization with ACTH, an endogenous protein that is stored in dense core granules; and (3) retention of newly synthesized tPA in the cell for prolonged periods of time. Laser scanning confocal microscopy analysis of cells immunostained with antibodies to tPA and ACTH showed colocalization at the tips of the neuritic processes under the cytoplasmic membrane, a region where dense granules are known to migrate after maturation. Treatment of the cells with 5 mM 8-bromo-cAMP for 30 min resulted in a 2.41+/-0.36-fold increase in tPA secretion. Both the magnitude of the stimulatory effect and the fraction of the intracellular tPA released were the same regardless of the tPA expression level in the various clones. Pulse-chase experiments showed that a portion of newly synthesized tPA is retained in the cell for at least 4 h and is released into the culture medium in response to 8-bromo-cAMP. These studies indicate that tPA, under the appropriate conditions, can be targeted into the regulated secretory pathway and can be stored for later release by cellular stimuli.
Asunto(s)
Adenohipófisis/metabolismo , Señales de Clasificación de Proteína/fisiología , Activador de Tejido Plasminógeno/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Hormona Adrenocorticotrópica/metabolismo , Animales , Células CHO , Línea Celular , Células Clonales , Cricetinae , Espacio Extracelular/metabolismo , Inmunohistoquímica , Líquido Intracelular/metabolismo , Ratones , Factores de Tiempo , Activador de Tejido Plasminógeno/genética , TransfecciónRESUMEN
The effects of enhanced HSP27 expression or expression of a nonphosphorylatable form of HSP27 on the migration of bovine arterial endothelial cells was assessed. Expression of the wild-type protein enhanced migration by twofold compared to control transfectants, whereas expression of the mutant protein retarded migration by 40%. Since homologs of the small heat shock protein inhibit F-actin polymerization in vitro and may alter basolateral F-actin content in vivo, it was postulated that the 27 kDa heat shock protein affects microfilament extension essential for cell motility. Expression of the wild-type protein promoted the generation of long cellular extensions, whereas expression of the dominant negative mutant protein resulted in a marked reduction of lamellipodia and generated aberrant microfilament morphology at the wound edge. Immunofluorescence combined with phalloidin staining demonstrated the colocalization of the HSP27 gene products with lamellipodial microfilament structures. These data suggest that the 27 kDa heat shock protein regulates migration by affecting the generation lamellipodia microfilaments.
Asunto(s)
Movimiento Celular , Endotelio Vascular/citología , Proteínas de Choque Térmico/metabolismo , Actinas/metabolismo , Animales , Bovinos , Células Cultivadas , Microscopía Fluorescente , FosforilaciónRESUMEN
A clinical diagnostic scale of infravesical obstruction (IVO) in patients with benign prostatic hyperplasia is proposed which provides the diagnosis of IVO in BPH patients with probability up to 89% basing only on clinical evidence obtained at a detailed urological examination (size of the gland, size and index of the prostatic transitional zone, residual urine, micturition urine, maximal micturition rate).
Asunto(s)
Hiperplasia Prostática/diagnóstico , Obstrucción del Cuello de la Vejiga Urinaria/diagnóstico , Urodinámica , Humanos , Masculino , Persona de Mediana Edad , Hiperplasia Prostática/diagnóstico por imagen , Hiperplasia Prostática/fisiopatología , Programas Informáticos , Ultrasonografía , Obstrucción del Cuello de la Vejiga Urinaria/diagnóstico por imagen , Obstrucción del Cuello de la Vejiga Urinaria/fisiopatologíaRESUMEN
The in vivo activity of the 27-kDa heat shock protein, a barbed-end microfilament capping protein, may be localized to the plasma membrane. To investigate this putative association, bovine endothelial cells expressing the human wild type or a mutant nonphosphorylatable 27-kDa heat shock protein were subjected to subcellular fractionation and immunoblot analysis. The 25-kDa endogenous bovine homolog and both exogenous gene products partitioned with cytosolic or plasma membrane components, indicating that phosphorylation is not required for membrane association. Phorbol ester treatment resulted in phosphorylation of only membrane-associated 25-kDa and wild type 27-kDa heat shock protein and did not induce redistribution. In a second fractionation protocol, streptavidin-agarose precipitation of extracts prepared from cells biotinylated at either the apical or basal surface localized membrane 25- and 27-kDa heat shock protein exclusively to the basolateral surface. Stimulation of transfectants expressing the wild type 27-kDa heat shock protein resulted in its phosphorylation and a doubling in the amount of membrane-associated F-actin precipitated, whereas the mutant protein decreased the amount of F-actin precipitated. These data suggest that membrane-associated 25- and 27-kDa heat shock proteins inhibit the generation of basolateral microfilaments and that phosphorylation releases this inhibition.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas de Choque Térmico , Polímeros/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Membrana Basal/enzimología , Bovinos , Endotelio Vascular/metabolismo , Activación Enzimática , Proteínas de Choque Térmico HSP27 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Chaperonas Moleculares , Proteínas de Neoplasias/metabolismo , Fosforilación , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Basic fibroblast growth factor is an important mitogenic and angiogenic factor that stimulates endothelial cell growth and migration. This hormone is not secreted via the classical vesicular pathway, and the identification of intracellular proteins that facilitate its release remains lacking. Transfection and expression of the 27-kDa human heat shock protein in bovine arterial endothelial cells doubles the rate of estrogen-induced basic fibroblast growth factor secretion, preferentially inducing the release of high molecular weight forms of the hormone. The secreted basic fibroblast growth factor is mitogenic to breast adenocarcinoma cells cultured in the conditioned medium obtained from the transfected endothelial cells. In contrast, decreasing the level of the endogenous heat shock protein homolog with an antisense vector markedly decreases basic fibroblast growth factor release. Anti-heat shock protein or anti-basic fibroblast growth factor antibodies co-precipitate both proteins from endothelial cell extracts, demonstrating a direct association between the two proteins. This interaction is likely to be an important step in the mechanism of basic fibroblast growth factor secretion.
Asunto(s)
Endotelio Vascular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteínas de Choque Térmico/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Bovinos , Células Cultivadas , Expresión Génica , Técnicas de Transferencia de Gen , Proteínas de Choque Térmico/genética , Humanos , Receptores de Estrógenos/genéticaRESUMEN
Plasma tissue plasminogen activator (tPA) has long been considered to be the product of the endothelial cells that line the various parts of the vascular system regardless of vessel size or location. To determine whether this was truly the case in vivo, the distribution of tPA in the endothelium of the mouse lung and other tissues was evaluated. Immunohistochemical analysis of normal lung tissue showed positive staining limited to the endothelial cells of the bronchial arteries regardless of size with few cells of the pulmonary circulation associated with tPA. The pulmonary vessels that did contain endothelial cell-derived tPA were consistently between 7 and 30 microns in diameter. No capillary or large vessel pulmonary endothelium ever stained positive. These results were also observed in primate lung tissue where the bronchial endothelium of all vessels, even down to capillary size, contained tPA while none of the pulmonary endothelium did. Prolonged exposure of mice to hyperoxic conditions promotes acute lung injury and associated inflammation. Using this model, the effect of inflammation on endothelial cell tPA expression was evaluated. A 4.5-fold increase in the number of pulmonary vessels staining positive for tPA was observed after 66 hours with all of these vessels having a diameter between 7 and 30 microns. Again, none of the endothelium of large arteries or veins nor the capillaries had tPA. Whole tissue tPA mRNA increased dramatically with hyperoxia and in situ hybridization analysis showed tPA mRNA in the endothelium of the same types of vessels as antigen. The tPA localized to both the bronchial and pulmonary endothelium was active with neither tPA-PAI-1 complexes nor urokinase found in perfused lung tissue. These results indicate that endothelial cell tPA expression, either constitutive or induced by a pathologic event, is a function of a highly select group of endothelial cells which are defined by their association with vessels of discrete size and/or anatomic location. Thus, the widely held concept that the steady state level of plasma tPA is maintained through its constitutive production by all endothelial cells of the vascular system is invalid. Also suggested is the possibility that endothelial cell tPA might play a broader role than simply maintaining vessel patency as a component of the fibrinolytic pathway and contribute to complex dynamic processes such as inflammation.
Asunto(s)
Activadores Plasminogénicos/metabolismo , Animales , Antígenos/metabolismo , Endotelio , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Oxígeno/metabolismo , Activadores Plasminogénicos/genética , Primates/metabolismo , ARN Mensajero/análisis , Conejos , RatasRESUMEN
The small molecular mass heat shock protein of 27 kDa (HSP27) has been shown to influence actin filament dynamics and endothelial cell behavior in ways similar to those observed during laminar flow. We have employed human umbilical vein endothelial cells to determine whether fluid shear stress affects HSP27 expression or phosphorylation. After a shear stress of 16 dyn/cm2, HSP27 became more highly phosphorylated, with maximum increase in phosphorylation levels (3-fold) attained by 30 min and sustained for at least 20 h. HSP27 antigen levels did not change; however, HSP27 mRNA levels decreased by 20% after 16 h. In bovine aortic endothelial cells stably transfected with the wild-type human HSP27 gene, shear stress induced the phosphorylation of both the exogenous human HSP27 and the endogenous bovine HSP25. The product of a transfected mutant HSP27 gene in which the putative phosphorylation sites Ser-15, Ser-78, and Ser-82 had been replaced with Gly was not phosphorylated. Thus the modulation of HSP27 and its activity by shear stress is mediated through a posttranslational mechanism and differs from the shear stress induction of immediate early genes at the level of transcription.
Asunto(s)
Endotelio Vascular/metabolismo , Proteínas de Choque Térmico/metabolismo , Animales , Bovinos , Células Cultivadas , Endotelio Vascular/patología , Humanos , Fosforilación , ARN Mensajero/biosíntesis , Estrés MecánicoRESUMEN
Bovine arterial endothelial cells were stably transfected with the human wild-type (wt) HSP27 or a mutant gene (mu) encoding a nonphosphorylatable form of the protein. At early passage both cultural and cellular morphology were similar, although the vacuole content in wtHSP27 was much higher than muHSP27 cells. As the cultures aged, wtHSP27 cells became large, polymorphic, highly vacuolated, and reached senescence before muHSP27 transfected cultures, which remained small and polygonal with few detectable vacuoles. Vector control cells showed an intermediate phenotype. Tritiated thymidine incorporation studies were performed with multiple wtHSP27 and muHSP27 clones and the results compared with 11 vector control clones. The results showed an average increase in growth rate for the wtHSP27 cells of 3.0 +/- 0.6 times. The growth rate of eight muHSP27 clones showed a slight decrease. Estradiol treatment of endothelial cells resulted in an increase in both bovine and human HSP27, with peak expression at 100 nM. Treatment of the vector-transfected cells with 100 nM estradiol resulted in a 1.44 +/- 0.18 fold increase in growth rate, which was blocked by expression of muHSP27. These data demonstrate a role for HSP27 in controlling the growth rate of endothelial cells in an estrogen-responsive manner.
Asunto(s)
Arterias/crecimiento & desarrollo , Endotelio Vascular/crecimiento & desarrollo , Proteínas de Choque Térmico/biosíntesis , Actinas/análisis , Animales , Arterias/citología , Arterias/efectos de los fármacos , Células CHO , Bovinos , Células Cultivadas , Senescencia Celular , Clonación Molecular , Cricetinae , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Estradiol/farmacología , Proteínas de Choque Térmico/genética , Humanos , Immunoblotting , Focalización Isoeléctrica , Pinocitosis , Proteínas Recombinantes/biosíntesis , Selección Genética , TransfecciónRESUMEN
Lp(a) competes with plasminogen for binding to cells but it is not known whether this competition is due to the ability of Lp(a) to interact directly with plasminogen receptors. In the present study, we demonstrate that Lp(a) can interact directly with plasminogen binding sites on monocytoid U937 cells and endothelial cells. The interaction of Lp(a) with these sites was time dependent, specific, saturable, divalent ion independent and temperature sensitive, characteristics of plasminogen binding to these sites. The affinity of plasminogen and Lp(a) for these sites also was similar (Kd = 1-3 microM), but Lp(a) bound to fewer sites (approximately 10-fold less). Both gangliosides and cell surface proteins with carboxy-terminal lysyl residues, including enolase, a candidate plasminogen receptor, inhibited Lp(a) binding to U937 cells. Additionally, Lp(a) interacted with low affinity lipoprotein binding sites on these cells which also recognized LDL and HDL. The ability of Lp(a) to interact with sites on cells that recognize plasminogen may contribute to the pathogenetic consequences of high levels of circulating Lp(a).
Asunto(s)
Lipoproteína(a)/metabolismo , Receptores de Superficie Celular/metabolismo , Sitios de Unión , Endotelio Vascular/citología , Gangliósidos/farmacología , Gangliósidos/fisiología , Humanos , Linfoma de Células B Grandes Difuso/patología , Glicoproteínas de Membrana/fisiología , Monocitos/metabolismo , Proteínas de Neoplasias/metabolismo , Unión Proteica/efectos de los fármacos , Receptores de Lipoproteína/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Células Tumorales Cultivadas , Venas UmbilicalesRESUMEN
The immunolocalization of tissue plasminogen activator (tPA) was assessed in vessels of various sizes from baboons. Femoral artery and vein, carotid artery, aorta, and sections from basal ganglia and cerebral cortex were stained for tPA and CD31, an endothelial cell-specific surface antigen. In each case, the endothelium of the large vessel stained positively for anti-CD31 but not for tPA. However, vascular structures in the adventitia corresponding to the vasa vasorum were found to be associated with tPA antigen. In situ hybridization of femoral artery with 35S-labeled cRNA probes detected tPA mRNA in the vasa vasorum but not the large vessel endothelium. Analysis of the microvasculature of the basal ganglia and cerebral cortex showed limited immunohistochemical staining for tPA; only 3% of the vessels measuring 4 to 100 mu were positive. Even so, tPA was mostly distributed within a narrow range of vessel size; 90% of the positive vessels were classified as precapillary arterioles and postcapillary venules (7.5 to 30.0 mu), whereas only 3% of the capillaries were positive, despite accounting for 40% of all vessels. Thus, tPA-containing endothelium are distributed mainly in smaller vessels, excluding the capillaries.
Asunto(s)
Endotelio Vascular/química , Activador de Tejido Plasminógeno/análisis , Vasa Vasorum/química , Animales , Arteriolas/química , Ganglios Basales/irrigación sanguínea , Arteria Femoral/química , Masculino , Papio , Vénulas/químicaRESUMEN
We studied the effect of lipoprotein(a) [Lp(a)], low-density lipoprotein (LDL), and high-density lipoprotein (HDL) on tissue plasminogen activator (TPA) secretion from human endothelial cells. At 1 mumol/L, Lp(a) inhibited constitutive TPA secretion by 50% and phorbol myristate acetate- and histamine-enhanced TPA secretion by 40%. LDL and HDL also depressed TPA secretion by 45% and 35% (constitutive) and 40% to 60% (stimulated). TPA mRNA levels were also examined and found to change in parallel with antigen secretion. In contrast to TPA, plasminogen activator inhibitor type-1 secretion and mRNA levels were not affected by any of the three lipoproteins. These results suggest that the interaction of lipoproteins with certain cell-surface binding sites may interfere with the proper production and/or secretion of TPA.
Asunto(s)
Endotelio Vascular/metabolismo , Lipoproteínas/farmacología , Activador de Tejido Plasminógeno/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Humanos , Lipoproteína(a)/farmacología , Lipoproteínas HDL/farmacología , Lipoproteínas LDL/farmacología , Inhibidor 1 de Activador Plasminogénico/sangre , ARN Mensajero/análisis , Activador de Tejido Plasminógeno/genéticaRESUMEN
Placement of endothelial cells under hypoosmotic or hyperosmotic conditions results in the reduction and increase, respectively, in the phosphorylation of a M(r) = 16,500 protein (P17). The changes were dose-dependent with a 3.3 +/- 0.3-fold increase occurring at 485 mosm/kg H2O and negligible phosphorylation observed at 202 mosm/kg H2O. The phosphorylation and dephosphorylation were rapid and prolonged; modified phosphorylation levels were maintained as long as the anisotonic conditions were present. However, return to isotonic medium reversed the phosphorylation back to normal within 1 h. Cellular fractionation studies showed that P17 was associated only with the nuclear compartment under isotonic, hypertonic, or hypotonic conditions. Two forms of P17 with pI values of 9.2 and 9.6 were resolved by isoelectric focusing; both forms showed enhanced phosphorylation by hyperosmotic treatment. Phosphorylation occurred on serines exclusively. These studies demonstrate that a nuclear protein with characteristics similar to histones is affected by cell shrinkage or swelling through changes in its phosphorylation state.
Asunto(s)
Histonas/metabolismo , Células Cultivadas , Cromatografía en Capa Delgada , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Cinética , Concentración Osmolar , FosforilaciónRESUMEN
Shear, stretch, and the generation of oxygen radicals stimulate increases in tissue plasminogen activator (t-PA) mRNA levels and antigen production, suggesting that environmental stress may regulate t-PA gene expression. We have examined whether t-PA production is also responsive to a hyperosmotic environment. Endothelial and HeLa cells were treated with hyperosmotic medium, and t-PA mRNA and antigen secretion were measured. Endothelial cells incubated in hyperosmotic medium showed a dose-dependent decrease in cell volume and a 1.9 +/- 0.3- and 3.7 +/- 0.9-fold increase in t-PA secretion at 425 and 485 mosmol/kgH2O, respectively. HeLa cells showed a 3.3 +/- 0.6- and 5.1 +/- 1.2-fold increase at the same osmolalities. Increased secretion began between 8 and 16 h and continued through 24 h. Cultures returned to isosmotic medium after 8 h of treatment continued to release 98.1 +/- 7% of the maximum levels of t-PA for the next 16 h, despite the reversal of other responses to hyperosmotic environment. t-PA mRNA levels also increased between 8 and 16 h to five times control levels but returned to baseline by 24 h. No change in intracellular Ca2+ concentration, inositol 1,4,5-trisphosphate, or diacylglycerol content was detected, suggesting that a different intracellular signal pathway may be involved in the response to hyperosmolar stimulus. Thus environmental stress may be a general stimulatory signal through which t-PA production can be induced.