RESUMEN
Cumulative exposure of hippocampal neurons to stress-like levels of corticosterone produces a negative spectrum of cellular alterations from ultrastructural changes to disruption of dendritic morphology and eventual degeneration. An experimental system which adapts itself to characterization of corticosteroid-induced proteins which mediate such effects is the hippocampal slice incubated in the presence of a radiolabeled amino acid following treatment of rats with corticosterone. The most consistently observed response to elevated corticosterone levels produced by exogenous injection is synthesis of a hippocampal cytosolic protein which has characteristics of glycerol phosphate dehydrogenase. Because synthesis of this protein is enhanced with a short latency as serum corticosterone levels are increased and terminated quickly upon re-establishment of basal conditions, it serves as a valid biological marker of the response of the hippocampus to short-term stress. In contrast, alterations in synthesis of other proteins following corticosterone treatment only become apparent under defined conditions or after chronic treatment. For example, steroid-inhibited synthesis of a hippocampal protein with an approximate molecular weight of 25,000 is only observed when slices are incubated at an elevated temperature. Such negative changes may represent loss of adaptive responses that protect the neuron from damage by cellular insults.
Asunto(s)
Corticosterona/farmacología , Proteínas de Choque Térmico/biosíntesis , Hipocampo/fisiopatología , Proteínas del Tejido Nervioso/biosíntesis , Neuronas/fisiología , Estrés Fisiológico/fisiopatología , Animales , Proteínas de Choque Térmico/aislamiento & purificación , Hipocampo/efectos de los fármacos , Hipocampo/patología , Calor , Técnicas In Vitro , Peso Molecular , Neuronas/efectos de los fármacos , Neuronas/patología , Ratas , Estrés Fisiológico/patologíaRESUMEN
To investigate the mechanism of glucocorticoid-induced lymphocytolysis, we used two-dimensional gel electrophoresis to analyze the effect of dexamethasone on the synthesis of individual proteins in S49 mouse lymphoma cells. We found that synthesis of a 78-Kd protein is preferentially maintained following dexamethasone treatment, at a time when the synthesis of most other cellular proteins is decreased. Synthesis of this protein could also be induced by tunicamycin, suggesting that it might be the 78-Kd glucose-regulated protein (GRP78). The identity of the 78-Kd protein with GRP78 was confirmed by limited chymotryptic mapping and immunoprecipitation analysis. Preferential synthesis of GRP78 in dexamethasone-treated cells was not secondary to alterations in the glycosylation of cellular proteins. Significantly, steady-state levels of GRP78 mRNA were unchanged following dexamethasone treatment. Preferential synthesis of GRP78 in glucocorticoid-treated S49 cells may reflect the unique property of GRP78 mRNA to be translated under conditions that interfere with the translation of most other cellular mRNAs. GRP78 is a highly conserved protein that is essential for cell viability. Preferential synthesis of GRP78 may be a protective response to metabolic events that interfere with normal mRNA translation in glucocorticoid-treated mouse lymphoma cells.
Asunto(s)
Proteínas Portadoras/biosíntesis , Dexametasona/farmacología , Proteínas de Choque Térmico , Linfoma/metabolismo , Chaperonas Moleculares , Animales , Autorradiografía , Proteínas Portadoras/genética , Quimotripsina , Electroforesis en Gel Bidimensional , Chaperón BiP del Retículo Endoplásmico , Glicosilación , Cadenas Pesadas de Inmunoglobulina , Ratones , Ratones Endogámicos BALB C , Mapeo Peptídico , ARN Mensajero/metabolismo , Células Tumorales Cultivadas , Tunicamicina/farmacologíaRESUMEN
The rat glucocorticoid receptor is a 795-amino acid protein with the hormone binding domain located in the C-terminal portion of the molecule. In the absence of hormone, this domain displays a protein inactivation activity that represses the nuclear localization, DNA binding, and transcriptional regulatory activities of the receptor. This inactivation activity, which appears to be mediated by the 90-kilodalton heat shock protein (HSP90), is stronger in the glucocorticoid receptor than the corresponding activity of the estrogen receptor hormone binding domain. In order to analyze these differences, we have directly compared in vitro translated glucocorticoid and estrogen receptors in terms of their interaction with HSP90 by a coimmunoprecipitation assay employing two monoclonal antibodies, AC88 and 8D3, which react with different regions of the HSP90 molecule. Intact forms of both the glucocorticoid receptor and the estrogen receptor coimmunoprecipitated with endogenous HSP90 in reticulocyte lysates, indicating that both receptors were capable of binding to HSP90 when translated in vitro. By assaying a series of receptor deletion mutants, we found that the sequences required for HSP90 binding mapped to a similar region within the hormone binding domain of both receptors. While the hormone binding domain was found to be the only structural requirement for HSP90 binding to the glucocorticoid receptor, additional sequences N-terminal to the hormone binding domain were shown to be required for HSP90 binding to the estrogen receptor. These results are consistent with a postulate that differences in the protein inactivation activities of the glucocorticoid and estrogen receptor hormone binding domains may be secondary to differences in the interactions of these domains with HSP90.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Glucocorticoides/metabolismo , Animales , Técnicas In Vitro , Mutación/genética , Pruebas de Precipitina , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , Ratas , Receptores de Estrógenos/genética , Receptores de Glucocorticoides/genéticaRESUMEN
Increased synthesis of a rat hippocampal protein with an apparent molecular weight (Mr) of 35,000 Da occurs in response to elevation of serum corticosterone levels. Subcellular fractionation has localized this protein in the cytosol. Two-dimensional gel electrophoresis indicated that this protein has an isoelectric point (IEP) of 6.6. A similar protein in liver has a slightly higher Mr and an IEP of 6.8. Increased synthesis of one additional hippocampal protein with an Mr of 46,000 Da and an IEP of 6.2 and of two other liver proteins, one with an Mr of 53,000 Da and an IEP of 6.2 and the other with an Mr of 45,000 Da and a range of IEPs from 8.7 to 7.8, was also seen after injection of corticosterone into rats. One possible identity of the 35,000 Da protein is glycerol 3-phosphate dehydrogenase (GPDH), based upon the reported Mr and IEP of this enzyme. The 35,000 Da hippocampal protein co-eluted from a gel filtration column with GPDH activity. No alteration of hippocampal GPDH activity was seen in intact rats 4 or 24 h after injection of either corticosterone or the type II receptor-specific agonist RU 28362. However, daily administration of corticosterone to rats beginning 10 days after adrenalectomy returned hippocampal GPDH activity to normal values after 2-3 days. In contrast, synthesis of the 35,000 Da protein was maximally increased 4 h after a single injection of steroid and not elevated at later times.
Asunto(s)
Corticosterona/farmacología , Hipocampo/química , Proteínas del Tejido Nervioso/análisis , Glándulas Suprarrenales/fisiología , Animales , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Glicerolfosfato Deshidrogenasa/análisis , L-Lactato Deshidrogenasa/análisis , Masculino , Ratas , Ratas Endogámicas , Fracciones Subcelulares/químicaRESUMEN
Following treatment of rats with a subcutaneous injection of 5 mg of corticosterone, hippocampal slices in vitro show increased labeling from 35S-methionine of a protein with an apparent molecular weight (Mr) of 35,000. Increased protein labeling is seen in response to corticosterone, dexamethasone, and aldosterone, steroids that associate with glucocorticoid receptors. Little or no response occurs after administration of progesterone or estradiol. Because the injected dose of steroids is high and responses to an injection of this magnitude may be pharmacological, several experiments have been done to determine whether stimuli that increase endogenous levels of corticosterone have the same effect on labeling of the 35,000 Mr protein. One hour after various stresses (immobilization, cold, ether, and sham-injection), when plasma levels of corticosterone are elevated, labeling of the 35,000 Mr protein is increased. Injection of ACTH also stimulates the synthesis of this protein in intact animals in a manner analogous to that seen with corticosterone injections. In addition, a dose-response curve for corticosterone with adrenalectomized rats shows that synthesis of the protein is maximally increased when the injected dosage causes serum levels of corticosterone to increase to the levels seen during stress. The increase in labeling of the 35,000 Mr protein in adrenalectomized animals is only half as great as that observed in intact animals. Injections of the type II glucocorticoid (GR) receptor agonist, RU 28362, into adrenalectomized rats differentially stimulates the synthesis of the 35,000 Mr protein compared with the mineralocorticoid aldosterone, which has a higher affinity for the type I (CR) receptor.(ABSTRACT TRUNCATED AT 250 WORDS)