v-mos oncoproteins affect the nuclear retention and reutilization of glucocorticoid receptors

Expression of the p85gag-mos oncoprotein in temperature sensitive transformed 6m2 cells results in desensitization of glucocorticoid induction of metallothionein-1 mRNA. Indirect immunofluorescence analyses demonstrate that hormone insensitivity in v-mos transformed cells is associated with inefficient nuclear retention of glucocorticoid receptor (GR) protein. Desensitized receptors that accumulate in the cytoplasm of transformed 6m2 cells do not regain the capacity for hormone-dependent nuclear translocation after turnover of the thermo-labile p85gag-mos oncoprotein. Although ligand induced down-regulation of immunoreactive GR protein occurs in transformed 6m2 cells, desensitized receptors appear to retain some capacity to bind hormone in vivo. Thus alterations in the intracellular partitioning of GR protein in v-mos-transformed cells result in the generation of a novel desensitized receptor that is apparently trapped in the cytoplasm and incapable of being reutilized.     

Steroid-induced nuclear binding of glucocorticoid receptors in intact hepatoma cells

Some of the early steps of steroid hormone action have been studied in cultured hepatoma cells, in which glucocorticoids induce tyrosine aminotransferase. The hypothesis that inducer steroids promote the binding of specific cytoplasmic receptors to the cell nucleus has been examined in intact cells.Binding of steroids such as dexamethasone and cortisol results in a loss of most of the receptor sites from the cytoplasm. This coincides with the binding of an equivalent number of steroid molecules in the nucleus. Both processes occur concomitantly, even when their kinetics are altered by reducing the temperature. When the inducer is removed from the culture, steroid dissociates from the nucleus while the level of cytoplasmic receptor returns to normal, even if protein or RNA synthesis is inhibited. These results suggest that nuclear binding of glucocorticoids is due to the association with the nucleus of the cytoplasmic receptor-steroid complex itself and make it unlikely that the receptor acts as a mere carrier for the intracellular transfer of the steroid.Steroids that differ in their effects on tyrosine aminotransferase induction were also studied. In contrast to those bound with inducer steroids, receptors complexed with the anti-inducer progesterone did not leave the cytosol. Further, a suboptimal inducer (deoxycorticosterone) produced an intermediate level of depletion. Thus, the biological effect of different classes of steroids can be related to their capacity to promote nuclear binding of the receptor. These data support a model proposed earlier, according to which the receptor is an allosteric regulatory protein directly involved in the hormone action, under the control of specific steroid ligands. They further suggest that the conformational state influenced by the inducer is such that a nuclear binding site on the receptor is exposed.Evidence is also presented that a distinct reaction takes place between the binding of the steroid to the receptor and the association of the complex with the nucleus. At 0 °C, this change is rate-limiting. It could correspond to the “activation” of receptor-steroid complexes known to be required for binding of the complexes by isolated nuclei, and thus represent an additional step in hormone action.     

Conversion of transformed androgen and glucocorticoid receptors to higher molecular forms

The 4 S transformed androgen receptor from rat prostate and thymus converted to a higher molecular form (5-7 S) on low-salt conditions. The converted receptor retained the DNA-binding capacity as well as the 4 S transformed receptor. This conversion was also demonstrated on glucocorticoid receptor from rat liver and thymus. The sedimentation coefficients of both the converted receptors were affected by sodium molybdate, i.e., the receptors converted to a relatively smaller molecule in the presence of molybdate than in the absence of the reagent. These observations suggest that molybdate directly binds to the transformed receptors and prevents the excessive association of a factor(s) to the transformed receptors.     

The effects of ras gene expression on glucocorticoid receptors in mouse fibroblasts

Analysis of induction of glutamine synthetase activity by dexamethasone showed a 2-fold increase in NIH3T3 but no change in NIH3T3 ras (EJ-ras) cells. The observed increase could be abolished by the antagonist RU486. The lack of response in ras transformed cells might reflect oncoprotein effects on the glucocorticoid receptor (GR). Several GR parameters were studied in order to clarify this point. Total GR level was the same for both cells; cytoplasmic receptor level however, was 3 times lower in NIH3T3 ras than in NIH3T3 cells. Hormone-receptor binding affinity, specificity, thermostability, sedimentation coefficient, molecular weight as well as the cytoplasmic GR transformation ratio were similar for the two cell lines. On the other hand, the fraction of the total receptor pool involved with the recycling process was approximately 20% lower in NIH3T3 ras than in NIH3T3 cells. After 24 h of dexamethasone treatment, no GR down regulation was observed in NIH3T3 ras cells, whereas normal NIH3T3 cells exhibited a decrease of GR binding capacity around 80%. Further studies are necessary to define the mechanisms underlying the association between glucocorticoid insensitivity, and modifications in the GR nuclear/cytoplasmic ratio, in the recycling GR fraction and in the down-regulation process observed in ras transformed cells.     

Effects of desensitization of capsaicin-sensitive afferent neurons on gastric microcirculation in rats depend on the blood glucocorticoid hormone level

The effects of desensitization of capsaicin-sensitive afferent neurons on gastric microcirculation were investigated before and after administration of indomethacin at ulcerogenic dose in adrenalectomized rats with or without corticosterone replacement and in sham-operated animals. We estimated the blood flow velocity in submucosal microvessels; the diameters and permeability of mucosal venous microvessels as parameters of gastric microcirculation. Desensitization of capsaicin-sensitive neurons was performed with capsaicin at the dose 100 mg/kg two weeks before the experiment. Adrenalectomy was created one week before experiment. In vivo microscopy technique for the direct visualization of gastric microcirculation and the analysis of the blood flow was employed. Indomethacin at ulcerogenic dose decreased the blood flow velocity in submucosal microvessels, caused dilatation of superficial mucosal microvessels and increased their permeability. Desensitization of capsaicin-sensitive afferent neurons potentiated indomethacin-induced microvascular disturbances in gastric submucosa-mucosa. These potentiated effects of the desensitization are obviously promoted by concomitant glucocorticoid deficiency. Thus, glucocorticoid hormones have a beneficial effect on gastric microcirculation in rats with desensitization of capsaicin-sensitive afferent neurons.     

转衣藻染色质烟草细胞核的变化及衣藻核基因的表达

应用反复冻融法以衣藻原生质体为材料制备衣藻染色质,应用显微操作技术准确将衣藻染色质转移到烟草叶片外植体中,进行连续培养并镜检观察。结果表明,经染色质转移处理的烟草叶片外植体、衣藻细胞核与烟草细胞核均发生形态上的变化。同时烟草叶片外植体正常发芽并获得再生苗,经RT-PCR检测发现,有衣藻核基因(rbcs2)的表达。     

Energy-dependent conversion of transformed cytosolic glucocorticoid receptors from soluble to particulate-bound form.

We have recently published that soluble cytosolic glucocorticoid receptors are converted to a particulate form when they are incubated at 37 degrees C in a tubulin-polymerizing buffer [Pratt, W. B., Sanchez, E. R., Bresnick, E. H., Meshinchi, S., Scherrer, L. C., Dalman, F. C., & Welsh, M. J. (1989) Cancer Res. (Suppl.) 49, 2222s-2229s]. In this work, we further define this phenomenon and demonstrate that the L-cell glucocorticoid receptors are binding to a protein particulate composed largely of cytoskeletal proteins. Incubation of L-cell cytosol with glutamate at 37 degrees C converts the glucorticoid receptor to a form that pellets when cytosol is centrifuged at 150000g. The particulate material formed in a temperature-dependent and glutamate-dependent manner contains a large amount of tubulin, actin, and vimentin, but it is not the product of a cold-labile, colchicine-sensitive polymerization process. Very few cytosolic proteins are present in this complex, but the glucocorticoid receptor is tightly bound to it. Binding of the receptor to the cytoskeletal complex occurs after receptor transformation and is at least partially energy-dependent. Examination of the behavior of beta-galactosidase receptor fusion proteins and the nti glucocorticoid receptor demonstrates that residues 445 to the COOH-terminus of the receptor (DNA-binding and hormone-binding domains) contain the features required for binding to the cytoskeletal complex. Although it is the transformed receptor that associates tightly with the complex, DNA-binding activity is not required for association with the cytoskeletal particulate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in integrin receptors on oncogenically transformed cells

Oncogenically transformed cells show reduced assembly of fibronectin-rich extracellular matrixes and diminished ability to adhere to fibronectin. The molecular bases of these phenotypic alteration are not fully understood. We report here alterations in the spectrum of integrins, including two fibronectin receptors, on oncogenic transformation of rodent cells. Transformation of rat1, NRK, and Nil8 cells by Rous sarcoma virus or by murine sarcoma viruses encoding ras oncogenes leads to reductions in the level of integrin alpha 5 beta 1, which is a well-defined fibronectin receptor, and of two other integrin receptors. In contrast, another receptor, alpha 3 beta 1, which is a polyspecific receptor for fibronectin, laminin, and collagen, is retained by transformed cells. These results provide explanations for earlier results concerning the interactions of extracellular matrix proteins with the surfaces of tumor cells and offer leads to further understanding of the altered adhesive and migratory behavior of malignant cells.     

db—cAMP对转化细胞钙调素基因表达与细胞骨架的影响

We have demonstrated that the distribution of microtubules (MT), microfilaments (MF) and fibronectin (FN) were diminished, while the gene expression of the calmodulin and c-fos enhanced in the transformed C3 H10 T1/2 cells. After treatment with 1 mM db-cAMP for 1 hr. and 2 hrs., there was an early and rapidly reduced in gene expression of calmodulin and c-fos respectively. After db-cAMP treatment for 4-5 days, the number of Capping cells of ConA binding decreased significantly and the cell surface microvilli decreased also. The growth of treated cells was inhibited markedly. By using 4F1 cDNA probe, which is preferentially expressed in G1 phase, we have found that the db-cAMP treated cells were accumulated at G1 phase. Of particular interest is the fact that the distribution of microtubules, microfilaments and fibronectin were recovered after treatment with 1 mM db-cAMP for 6 days. It is suggested that the inhibition of proliferation, alteration of phenotype and recovery of cytoskeleton in transformed cells after treatment with db-cAMP are related to the inhibition of gene expression of calmodulin.     

Polymorphisms in the glucocorticoid receptor gene that modulate glucocorticoid sensitivity are associated with rheumatoid arthritis

Introduction  

The glucocorticoid receptor (GR) plays an important regulatory role in the immune system. Four polymorphisms in the GR gene are associated with differences in glucocorticoid (GC) sensitivity; the minor alleles of the polymorphisms N363 S and BclI are associated with relative hypersensitivity to GCs, while those of the polymorphisms ER22/23EK and 9β are associated with relative GC resistance. Because differences in GC sensitivity may influence immune effector functions, we examined whether these polymorphisms are associated with the susceptibility to develop Rheumatoid Arthritis (RA) and RA disease severity.     

Nicotine-induced up-regulation and desensitization of alpha4beta2 neuronal nicotinic receptors depend on subunit ratio

Desensitization induced by chronic nicotine exposure has been hypothesized to trigger the up-regulation of the alpha4beta2 neuronal nicotinic acetylcholine receptor (nAChR) in the central nervous system. We studied the effect of acute and chronic nicotine exposure on the desensitization and up-regulation of different alpha4beta2 subunit ratios (1alpha:4beta, 2alpha:3beta, and 4alpha:1beta) expressed in Xenopus oocytes. The presence of alpha4 subunit in the oocyte plasmatic membrane increased linearly with the amount of alpha4 mRNA injected. nAChR function and expression were assessed during acute and after chronic nicotine exposure using a two-electrode voltage clamp and whole-mount immunofluorescence assay along with confocal imaging for the detection of the alpha4 subunit. The 2alpha4:3beta2 subunit ratio displayed the highest ACh sensitivity. Nicotine dose-response curves for the 1alpha4:4beta2 and 2alpha4:3beta2 subunit ratios displayed a biphasic behavior at concentrations ranging from 0.1 to 300 microm. A biphasic curve for 4alpha4:1beta2 was obtained at nicotine concentrations higher than 300 microm. The 1alpha4:4beta2 subunit ratio exhibited the lowest ACh- and nicotine-induced macroscopic current, whereas 4alpha4:1beta2 presented the largest currents at all agonist concentrations tested. Desensitization by acute nicotine exposure was more evident as the ratio of beta2:alpha4 subunits increased. All three alpha4beta2 subunit ratios displayed a reduced state of activation after chronic nicotine exposure. Chronic nicotine-induced up-regulation was obvious only for the 2alpha4: 3beta2 subunit ratio. Our data suggest that the subunit ratio of alpha4beta2 determines the functional state of activation, desensitization, and up-regulation of this neuronal nAChR. We propose that independent structural sites regulate alpha4beta2 receptor activation and desensitization.