Inhibition of glucocorticoid receptor nucleocytoplasmic shuttling by okadaic acid requires intact cytoskeleton.

It has been shown previously that glucocorticoid receptors (GRs) that have undergone hormone-dependent translocation to the nucleus and have subsequently exited the nucleus upon hormone withdrawal are unable to recycle into the nucleus if cells are treated during hormone withdrawal with okadaic acid, a cell-permeable inhibitor of certain serine/threonine protein phosphatases. Using a green fluorescent protein (GFP) GR chimera (GFP-GR), we report here that okadaic acid inhibition of steroid-dependent receptor recycling to the nucleus is abrogated in cells treated for 1 h with colcemid to eliminate microtubule networks prior to steroid addition. After withdrawal of colcemid, normal cytoskeletal architecture is restored and okadaic acid inhibition of steroid-dependent GFP-GR nuclear recycling is restored. When okadaic acid is present during hormone withdrawal, GR that is recycled to the cytoplasm becomes complexed with hsp90 and binds steroid, but it does not undergo the normal agonist-dependent dissociation from hsp90 upon retreatment with steroid. However, when the cytoskeleton is disrupted by colcemid, the GR in okadaic acid-treated cells recycles from the cytoplasm to the nucleus in an agonist-dependent manner without dissociating from hsp90. This suggests that under physiological conditions where the cytoskeleton is intact, a dephosphorylation event is required for loss of high affinity binding to hsp90 that is required for receptor translocation through the cytoplasm to the nucleus along cytoskeletal tracts.     

A pH-sensitive polymer that enhances cationic lipid-mediated gene transfer.

The efficient release of nonviral gene carriers from endosomes is an important step for the successful delivery of DNA into the cell nucleus. A synthetic pH-sensitive anionic polymer, poly(propylacrylic acid) (PPAA), was designed to aid in endosomal escape of nonviral vectors and improve the transfection efficiencies with these vectors. Transfection of NIH3T3 fibroblasts with ternary physical mixtures of the cationic lipid DOTAP, pCMVbeta plasmid DNA, and PPAA showed marked enhancement of both gene expression levels and fraction of cells transfected compared to binary control mixtures of DOTAP and DNA. PPAA also significantly improved the serum-stability of DOTAP/DNA vectors. The DOTAP/DNA/PPAA vectors maintained high levels of transfection in media containing up to 50% serum. The striking enhancement of transfection efficiency with cationic lipid/DNA/PPAA mixtures, along with the enhanced serum-stability, suggests that PPAA may provide significant improvements for the in vivo intracellular delivery of drugs such as DNA, oligonucleotides, proteins, and peptides.     

Functional abnormality of glucocorticoid receptor in Shionogi carcinoma 115 cells as evidenced by gene transfer experiments.

The assay systems for steroid receptor functions in steroid-sensitive cells (SC-3 cells) were developed in which hormone-responsive element linked to a reporter gene [chloramphenicol acetyl transferase (CAT) gene] was transfected by the electroporation technique. Stimulation with androgen of SC-3 cells transfected with mouse mammary tumor virus promoter-CAT gene (MMTV-CAT) resulted in clear enhancement of CAT activity, whereas glucocorticoid required abnormally high concentrations to obtain significant stimulation. The simultaneous addition of glucocorticoid surprisingly inhibited androgen-induced CAT activity in SC-3 cells, whereas glucocorticoid and androgen acted together synergistically to activate CAT activity in T 47D cells. When SC-3 cells were cotransfected with the expression vector of human glucocorticoid receptor (GR) gene, inhibition with glucocorticoid of androgen-enhanced CAT activity was abolished. These results would suggest that SC-3 cells contain functionally abnormal GR.     

Influence of estradiol on NADPH diaphorase/neuronal nitric oxide synthase activity and colocalization with progesterone or type II glucocorticoid receptors in ovine hypothalamus

Nitric oxide (NO) has been shown to play an important role in both the neuroendocrine reproductive and stress axes, which are closely linked. Because progesterone (P4) receptors (PRs) and glucocorticoid receptors (GRs) are not found in GnRH neurons and the NOergic system has been implicated in the control of GnRH secretion, this study aimed to ascertain whether steroids altered the NOergic system. Our first objective was to map the distribution of NO synthase (NOS) cells in the ovine preoptic area (POA) and hypothalamus and to determine whether NOS activity is enhanced by estradiol (E2) treatment. Using NADPH diaphorase (NADPHd) histochemistry, we found that NADPHd-positive neurons were spread throughout the ovine POA and hypothalamus, and that all NADPHd cells were immunoreactive for NOS. In response to estradiol, a significant increase in the number of NADPHd cells was noted only in the ventrolateral region of the ventromedial nucleus (VMNvl), with no significant difference in the POA or arcuate nucleus. Progesterone and glucocorticoid receptors were colocalized with NADPHd reactive neurons in the POA, arcuate nucleus, and VMNvl of ewes in both treatment groups. In ewes receiving estradiol, the number of NADPHd-positive cells containing steroid receptors in the POA (PR, 81%; GR, 79%) and arcuate nucleus (PR, 89%; GR, 84%) was similar, but in the VMNvl, fewer NADPHd-positive cells contained GR (PR, 88%, GR, 31%). These data show that estradiol up-regulates NOS activity in a site-specific manner and that the influence and possible interaction of progesterone and corticosteroids on NO producing cells may differ according to the neural location.     

A quantitative comparison of dual control of a hormone response element by progestins and glucocorticoids in the same cell line

Progesterone receptor-containing T47D human breast cancer cells are responsive to progestins but fail to respond to other steroid hormones, in particular dexamethasone, because they have no measurable levels of receptors for estrogens, androgens, or glucocorticoids. To quantitatively study dual responsiveness of the mouse mammary tumor virus (MMTV) promoter to progestins and glucocorticoids, we have stably transfected T47D cells with a glucocorticoid receptor (GR) expression vector. A cloned derivative (A1-2) was isolated that expresses a normal, full length GR, as assessed by steroid binding and Western immunoblot with a monoclonal anti-GR antibody. Moreover, GR is expressed at levels (80,000-100,000 molecules per cell) comparable to the high levels of endogenous progesterone receptor (200,000 molecules per cell). In A1-2 cells transiently transfected with an MMTV-chloramphenicol acetyl transferase reporter gene, induction by glucocorticoid was substantially greater (5-fold) than induction mediated by progestins. These results suggest that glucocorticoids may be the primary regulator of MMTV.     

Recycling and desensitization of glucocorticoid receptors in v-mos transformed cells depend on the ability of nuclear receptors to modulate gene expression

In v-mos transformed cells, glucocorticoid receptor (GR) proteins that bind hormone agonist are not efficiently retained within nuclei and redistribute to the cytoplasmic compartment. These cytoplasmic desensitized receptors cannot be reutilized and may represent trapped intermediates derived from GR recycling. We have used the glucocorticoid antagonist RU486 to examine whether v-mos effects can be exerted on any ligand-bound GR. In the rat 6m2 cell line that expresses a temperature-sensitive p85gag-mos oncoprotein, RU486 is a complete antagonist and suppresses dexamethasone induction of metallothionein-1 mRNA at equimolar concentrations. Using indirect immunofluorescence, we observe efficient nuclear translocation of GR in response to RU486 treatment in either the presence or absence of v-mos oncoproteins. However, in contrast to the redistribution of agonist-bound nuclear receptors to the cytoplasm of v-mos-transformed cells, RU486-bound GRs are efficiently retained within nuclei. Interestingly, withdrawal of RU486 does not lead to efficient depletion of nuclear GR in either nontransformed or v-mos transformed cells. It is only after the addition of hormone agonist to RU486 withdrawn v-mos-transformed cells that GRs are depleted from nuclei and subsequently redistributed to the cytoplasm. Thus, only nuclear GRs that are agonist-bound and capable of modulating gene activity can be subsequently processed and recycled into the cytoplasm.     

Long duration electroporation for achieving high level expression of glucocorticoid receptors in mammalian cell lines

A method is presented that utilizes long duration electroporation (LDE) to more efficiently introduce DNA into mammalian cell lines than standard electroporation techniques. With SV40-based vectors, more than 550,000 glucocorticoid receptors (GRs) per cell could be obtained in COS-7 cells with good cell survival. In experiments with a CMV-driven vector expressing an enhanced Green Fluorescent Protein (EGFP), 54% of the cells were transfected, and 77% of EGFP positive cells expressed EGFP at moderate to high levels. In cell lines not containing the large T antigen, a CMV-driven vector for the GR was superior to the SV40-based vector. In EDR3, DG44, and CV-1 cell lines approximately 220,000, 190,000 and 150,000 GRs/cell were obtained, respectively. Transfection efficiency of the EGFP vector ranged from 44 to 55% for the three cell lines. Cortisol treatment of COS-7 and DG44 cultures cotransfected with vectors expressing the GR and a GRE driven luciferase gene produced 4 to 12 times more enzyme activity per plate with LDE than conventional electroporation protocols. LDE allows transient overexpression of proteins in COS-7 cells at the high levels generally achieved by mammalian overexpression systems only in stable cell lines.     

Application of an HIV gp41-derived peptide for enhanced intracellular trafficking of synthetic gene and siRNA delivery vehicles

Endosomal release is an efficiency-limiting step for many nonviral gene delivery vehicles. In this work, nonviral gene delivery vehicles were modified with a membrane-lytic peptide taken from the endodomain of HIV gp41. Peptide was covalently linked to polyethylenimine (PEI) and the peptide-modified polymer was complexed with DNA. The resulting nanoparticles were shown to have similar physicochemical properties as complexes formed with unmodified PEI. The gp41-derived peptide demonstrated significant lytic activity both as free peptide and when conjugated to PEI. Significant increases in transgene expression were achieved in HeLa cells when compared to unmodified polyplexes at low polymer to DNA ratios. Additionally, peptide-modified polyplexes mediated significantly enhanced siRNA delivery compared to unmodified polyplexes. Despite increases in transgene expression and siRNA knockdown, there was no increase in internalization or binding of modified carriers as determined by flow cytometry. The hypothesis that the gp41-derived peptide increases the endosomal escape of vehicles is supported by confocal microscopy imaging of DNA distributions in transfected cells. This work demonstrates the use of a lytic peptide for improved trafficking of nonviral gene delivery vehicles.     

Effects of acetylation, polymerase phosphorylation, and DNA unwinding in glucocorticoid receptor transactivation

Varying the concentration of selected factors alters the induction properties of steroid receptors by changing the position of the dose-response curve (or the value for half-maximal induction=EC(50)) and the amount of partial agonist activity of antisteroids. We now describe a rudimentary mathematical model that predicts a simple Michaelis-Menten curve for the multi-step process of steroid-regulated gene induction. This model suggests that steps far downstream from receptor binding to steroid can influence the EC(50) of agonist-complexes and partial agonist activity of antagonist-complexes. We therefore asked whether inhibitors of three possible downstream steps can reverse the effects of increased concentrations of two factors: glucocorticoid receptors (GRs) and Ubc9. The downstream steps (with inhibitors in parentheses) are protein deacetylation (TSA and VPA), DNA unwinding (CPT), and CTD phosphorylation of RNA polymerase II (DRB and H8). None of the inhibitors mimic or prevent the effects of added GRs. However, inhibitors of DNA unwinding and CTD phosphorylation do reverse the effects of Ubc9 with high GR concentrations. These results support our earlier conclusion that different rate-limiting steps operate at low and high GR concentrations versus high GR with Ubc9. The present data also suggest that downstream steps can modulate the EC(50) of GR-mediated induction, thus both supporting the utility of our mathematical model and widening the field of biochemical processes that can modify the EC(50).