Ceramide regulation of nuclear protein import

Nucleocytoplasmic trafficking is an essential and responsive cellular mechanism that directly affects cell growth and proliferation, and its potential to address metabolic challenge is incompletely defined. Ceramide is an antiproliferative sphingolipid found within vascular smooth muscle cells in atherosclerotic plaques, but its mechanism of action remains unclear. The hypothesis that ceramide inhibits cell growth through nuclear transport regulation was tested. In smooth muscle cells, exogenously supplemented ceramide inhibited classical nuclear protein import that involved the activation of cytosolic p38 mitogen-activated protein kinase (MAPK). After application of SB 202190, a specific and potent pharmacological antagonist of p38 MAPK, sphingolipid impingement on nuclear transport was corrected. Distribution pattern assessments of two essential nuclear transport proteins, importin-alpha and Cellular Apoptosis Susceptibility, revealed ceramide-mediated relocalization that was reversed upon the addition of SB 202190. Furthermore, cell counts, nuclear cyclin A, and proliferating cell nuclear antigen expression, markers of cellular proliferation, were diminished after ceramide treatment and effectively rescued by the addition of inhibitor. Together, these data demonstrate, for the first time, the sphingolipid regulation of nuclear import that defines and expands the adaptive capacity of the nucleocytoplasmic transport machinery.     

Importin 7 and importin alpha/importin beta are nuclear import receptors for the glucocorticoid receptor

The vertebrate glucocorticoid receptor (GR) is cytoplasmic without hormone and localizes to the nucleus after hormone binding. GR has two nuclear localization signals (NLS): NL1 is similar in sequence to the SV40 NLS; NL2 is poorly defined, residing in the ligand-binding domain. We found that GR displayed similar hormone-regulated compartmentalization in Saccharomyces cerevisiae and required the Sxm1 nuclear import receptor for NL2-mediated import. Two metazoan homologues of Sxm1, importin 7 and importin 8, bound both NL1 and NL2, whereas importin alpha selectively bound NL1. In an in vitro nuclear import assay, both importin 7 and the importin alpha-importin beta heterodimer could import a GR NL1 fragment. Under these conditions, full-length GR localized to nuclei in the presence but not absence of an unidentified component in cell extracts. Interestingly, importin 7, importin 8, and importin alpha bound GR even in the absence of hormone; thus, hormonal control of localization is exerted at a step downstream of import receptor binding.     

Tissue-specific regulation of glucocorticoid receptor mRNA by dexamethasone

The effect of glucocorticoids on tissue-specific regulation of glucocorticoid receptor mRNA was studied in intact and adrenalectomized rats. Glucocorticoid receptor mRNA was examined by Northern blot hybridization and quantitated by slot blot hybridization using a glucocorticoid cRNA probe. Glucocorticoid receptor mRNA was greatest in the lung with the relative levels in other tissues as follows: spleen, 70%; brain, 55%; liver, 50%; kidney, 43%; heart, 35%; adrenal, 13%; and testis only 8%. A tissue-specific difference in glucocorticoid receptor mRNA accumulation was found after adrenalectomy. There was little change in glucocorticoid receptor mRNA levels in liver and lung, but the brain and kidney demonstrated a 40 and 80% increase in mRNA, respectively. In contrast, dexamethasone treatment resulted in a consistent decrease of 40-60% in the accumulation of glucocorticoid receptor mRNA in all tissues studied. These results provide in vivo evidence for the autoregulation of the glucocorticoid receptor by its homologous ligand and demonstrate the existence of tissue-specific regulation of the glucocorticoid receptor mRNA levels in states of glucocorticoid excess and depletion.     

Cell cycle regulation of glucocorticoid receptor function.

Glucocorticoid receptor (GR) nuclear translocation, transactivation and phosphorylation were examined during the cell cycle in mouse L cell fibroblasts. Glucocorticoid-dependent transactivation of the mouse mammary tumor virus promoter was observed in G0 and S phase synchronized L cells, but not in G2 synchronized cells. G2 effects were selective on the glucocorticoid hormone signal transduction pathway, since glucocorticoid but not heavy metal induction of the endogenous Metallothionein-1 gene was also impaired in G2 synchronized cells. GRs that translocate to the nucleus of G2 synchronized cells in response to dexamethasone treatment were not efficiently retained there and redistributed to the cytoplasmic compartment. In contrast, GRs bound by the glucocorticoid antagonist RU486 were efficiently retained within nuclei of G2 synchronized cells. Inefficient nuclear retention was observed for both dexamethasone- and RU486-bound GRs in L cells that actively progress through G2 following release from an S phase arrest. Finally, site-specific alterations in GR phosphorylation were observed in G2 synchronized cells suggesting that cell cycle regulation of specific protein kinases and phosphatases could influence nuclear retention, recycling and transactivation activity of the GR.     

Postnatal regulation of glucocorticoid receptor in the liver of chicken

The specific binding of [3H]-dexamethasone to glucocoticoid receptor (GR) and activation of hormone-receptor (H-R) complexes from the liver of chicken at day 0, 5, 10, 30, 60 and 90 were investigated to find out GR regulation during postnatal development. Results showed that GR level (fmol/mg protein) reached a peak by day 5 of postnatal age and was significantly higher (+ 42%) than observed at day-0 (day of hatching), as evidenced also by protein blot experiments and Scatchard analysis of binding data. The GR concentration declined gradually up to day 30, and thereafter, no significant change was observed at day 60 and 90 of postnatal ages. The temperature and salt-dependent activation of GR showed no significant differences in 0 and 30-day old chicken, as determined by DNA-cellulose binding assay. However, nuclear binding of temperature and salt-activated GR complexes was significantly higher in 0-day old chicken. Cross-mixing experiments (wherein nuclei of day-0 were incubated with the H-R complexes of day-30 and vice-versa) revealed the role of nuclear specificity in higher binding of temperature and salt-activated H-R complexes at day-0 of postnatal age. DNase I extraction of nuclei bound to activated H-R complexes showed higher extractability at day-0 (70%), compared to day-30 (44%). Above findings suggested that changes in GR concentration as well as chromatin organization might play an important role in glucocorticoid-mediated responses during postnatal development of chicken.     

A photoregulated ligand for the nuclear import receptor karyopherin alpha.

The ability to orchestrate the transport of proteins between nucleus and cytoplasm provides cells with a powerful regulatory mechanism. Selective translocation between these compartments is often used to propagate cellular signals, and it is an intimate part of the processes that control cell division, viral replication, and other cellular events. Therefore, precise experimental control over protein localization, through the agency of light, would provide a powerful tool for the study and manipulation of these events. To this end, a prototype photoregulated nuclear localization signal (NLS) was derived from a native NLS. A library of 30 mutants of the bipartite NLS from Xenopus laevis nucleoplasmin containing a novel, photoisomerizable amino acid was prepared by parallel, solid-phase synthesis and screened in vitro for binding to the nuclear import receptor karyopherin alpha, which mediates the nuclear import of cellular proteins. A single peptide was identified in which the cis and trans photoisomers bind the receptor differentially. The strategy used to obtain this peptide is systematic and empirical; therefore, it is potentially applicable to any peptide-receptor system.     

Isolation and characterization of rat liver nuclear glucocorticoid receptor

The rat liver nuclear glucocorticoid receptor has a molecular weight of 90 000. Using antibody bound to the stationary matrix, the cytosol and nuclear glucocorticoid receptors from rat liver were purified. The translocation of glucocorticoid receptor from rat liver cytosol into the nucleus was studied using immunoaffinity chromatography. Immediately after the intraperitoneal injection of rats with the hormone, the receptor translocation started and was complete within 10 min. The 90 000 dalton nuclear receptor component is identical to the 90 000 dalton cytosol component. They have identical molecular weights in the same gel electrophoresis system and produce identical peptide fragments after digestion with Staphyolococcal aureus V8 protease. The receptor component enriched by immunoaffinity chromatography from cytosol of adrenalectomised rats contained mainly a 45 000 dalton component.     

Transcriptional regulation by the nuclear receptor superfamily

In the past year, additional experimental data have expanded our understanding of the molecular mechanisms that underlie nuclear receptor control of regulatory programs. It is increasingly clear -that steroid members (e.g. glucocorticoid and estrogen) and non-steroid members (e.g. retinoic acid, thyroid hormone, and vitamin D) of the nuclear receptor superfamily may utilize distinct strategies in achieving their complex control of gene regulation.     

Mutations in squirrel monkey glucocorticoid receptor impair nuclear translocation

To identify the determinants of impaired glucocorticoid receptor (GR) signaling in a model of glucocorticoid resistance, cloned GR from Guyanese squirrel monkeys (gsmGR) was tagged with enhanced green fluorescent protein, and nuclear translocation was examined in transfected COS1 cells. In keeping with evidence that gsmGR transactivational competence is impaired, we found that nuclear translocation is likewise diminished in gsmGR relative to human GR (hGR). Experiments with GR chimeras revealed that replacement of the gsmGR ligand binding domain (LBD) with that from hGR increased translocation. Truncated gsmGR constructs lacking the LDB after amino acid 552 also showed increased translocation even in the absence of cortisol. Three back-mutations of gsmGR to hGR (Thr551Ser, Ala616Ser, and Ser618Ala) in the LBD confirmed that these amino acids play a role in diminished translocation.     

Differential roles of heat shock protein 70 in the in vitro nuclear import of glucocorticoid receptor and simian virus 40 large tumor antigen.

Nuclear import of glucocorticoid receptors (GRs) was analyzed in vitro with digitonin-permeabilized cells (S. A. Adam, R. Sterne-Marr, and L. Gerace, J. Cell Biol. 111:807-816, 1990). Indirect immunofluorescence methods were used to monitor the transport of GRs from rat hepatoma and fibroblast cell cytosol into HeLa nuclei. In vitro nuclear import of GRs was shown to be hormone dependent and to require ATP and incubation at ambient temperatures (i.e., 30 degrees C). Hormone-dependent dissociation of GR-bound proteins, such as the 90-kDa heat shock protein, hsp90, is part of an activation process that is obligatory for the expression of the receptor's DNA-binding activity. Inhibition of in vitro GR activation by Na2MoO4 blocked hormone-dependent nuclear import, demonstrating that receptor activation is required for nuclear import. The addition to GR-containing cytosol of antiserum directed against the cytosolic 70-kDa heat shock protein, hsp70, while effective in blocking the nuclear import of simian virus 40 large tumor antigen (SV40 TAg), did not affect hormone-dependent nuclear import of endogenous, full-length GRs or an exogenously added truncated GR protein (i.e., XGR556) that lacks a hormone-binding domain but possesses a constitutively active nuclear localization signal sequence (NLS). Depletion of hsp70 from HeLa cell cytosol did not affect the nuclear import of exogenously added XGR556 but led to inhibition of SV40 TAg nuclear import. Thus, two closely related NLSs, one contained within GRs and the other contained within SV40 TAg, are distinguished by their differential requirements for hsp70 in vitro.