Proinflammatory cytokines regulate human glucocorticoid receptor gene expression and lead to the accumulation of the dominant negative beta isoform: a mechanism for the generation of glucocorticoid resistance.

Inflammatory responses in many cell types are coordinately regulated by the opposing actions of NF-kappaB and the glucocorticoid receptor (GR). The human glucocorticoid receptor (hGR) gene encodes two protein isoforms: a cytoplasmic alpha form (GRalpha), which binds hormone, translocates to the nucleus, and regulates gene transcription, and a nuclear localized beta isoform (GRbeta), which does not bind known ligands and attenuates GRalpha action. We report here the identification of a tumor necrosis factor (TNF)-responsive NF-kappaB DNA binding site 5' to the hGR promoter that leads to a 1.5-fold increase in GRalpha mRNA and a 2.0-fold increase in GRbeta mRNA in HeLaS3 cells, which endogenously express both GR isoforms. However, TNF-alpha treatment disproportionately increased the steady-state levels of the GRbeta protein isoform over GRalpha, making GRbeta the predominant endogenous receptor isoform. Similar results were observed following treatment of human CEMC7 lymphoid cells with TNF-alpha or IL-1. The increase in GRbeta protein expression correlated with the development of glucocorticoid resistance.

The activation and inhibition of human nicotinic acetylcholine receptor by RJR-2403 indicate a selectivity for the alpha4beta2 receptor subtype.

Human nicotinic acetylcholine (ACh) receptor subtypes expressed in Xenopus oocytes were characterized in terms of their activation by the experimental agonist RJR-2403. Responses to RJR-2403 were compared with those evoked by ACh and nicotine. These agonists were also characterized in terms of whether application of the drugs had the effect of producing a residual inhibition that was manifest as a decrease in subsequent control responses to ACh measured 5 min after the washout of the drug. For the activation of alpha4beta2 receptors, RJR-2403 had an efficacy equivalent to that of ACh and was more potent than ACh. RJR-2403 was less efficacious than ACh for other human receptor subtypes, suggesting that it is a partial agonist for all these receptors. Nicotine activated peak currents in human alpha4beta2 and alpha3beta2 receptors that were 85 and 50% of the respective ACh maximum responses. Nicotine was an efficacious activator of human alpha7 receptors, with a potency similar to ACh, whereas RJR-2403 had very low potency and efficacy for these receptors. At concentrations of <1 mM, RJR-2403 did not produce any residual inhibition of subsequent ACh responses for any receptor subtype. In contrast, nicotine produced profound residual inhibition of human alpha4beta2, alpha3beta2, and alpha7 receptors with IC(50) values of 150, 200, and 150 microM, respectively. Co-expression of the human alpha5 subunit with alpha3 and beta2 subunits had the effect of producing protracted responses to ACh and increasing residual inhibition by ACh and nicotine but not RJR-2403. In conclusion, our results, presented in the context of the complex pharmacology of nicotine for both activating and inhibiting neuronal nicotinic receptor subtypes, suggest that RJR-2403 will be a potent and relatively selective activator of human alpha4beta2 receptors.

Immunocytochemical analysis of the glucocorticoid receptor alpha isoform (GRalpha) using GRalpha-specific antibody.

The alpha isoform of the glucocorticoid receptor (GRalpha) binds glucocorticoids and functions as a ligand-dependent transcription factor. Although GRalpha is expressed in almost all tissues and cells, its subcellular distribution is controversial. Many studies have reported that GRalpha translocates from the cytoplasm to the nucleus in a hormone-dependent manner whereas others have concluded that GRalpha is constitutively located in the nucleus. These conflicting data may result from the use of antibodies that do not discriminate GRalpha from a splice variant of the GR gene termed GRbeta. Using a GRbeta-specific antibody, we have recently demonstrated that GRbeta resides in the nucleus of cells independent of glucocorticoid treatment. In the following study we have generated a novel GRalpha-specific antibody (AShGR) in order to assess, unambiguously, the subcellular distribution of GRalpha. AShGR recognizes recombinant GRalpha on Western blots and in immunoprecipitation experiments but does not cross-react with recombinant GRbeta. Endogenous GRalpha is detected by AShGR in a variety of human cell lines including HeLa S3, CEM-C7, HEK-293, MCF-7, Hep G2, and secondary lung epithelial cells. In addition, AShGR detects endogenous rat and mouse GRalpha. Immunocytochemistry was performed with AShGR on COS-I cells transfected with human GRalpha and on HTC rat hepatoma cells expressing endogenous GRalpha. In both systems, GRalpha was found in the cytoplasm of cells in the absence of hormone and in the nucleus after hormone treatment. These studies mark the first time a GRalpha-specific antibody has been employed to examine the expression and subcellular distribution of endogenous GRalpha.

Antagonist activities of mecamylamine and nicotine show reciprocal dependence on beta subunit sequence in the second transmembrane domain.

We show that a portion of the TM2 domain regulates the sensitivity of beta subunit-containing rat neuronal nicotinic AChR to the ganglionic blocker mecamylamine, such that the substitution of 4 amino acids of the muscle beta subunit sequence into the neuronal beta4 sequence decreases the potency of mecamylamine by a factor of 200 and eliminates any long-term effects of this drug on receptor function. The same exchange of sequence that decreases inhibition by mecamylamine produces a comparable potentiation of long-term inhibition by nicotine. Inhibition by mecamylamine is voltage-dependent, suggesting a direct interaction of mecamylamine with sequence elements within the membrane field. We have previously shown that sensitivity to TMP (tetramethylpiperidine) inhibitors is controlled by the same sequence elements that determine mecamylamine sensitivity. However, inhibition by bis-TMP compounds is independent of voltage. Our experiments did not show any influence of voltage on the inhibition of chimeric receptors by nicotine, suggesting that the inhibitory effects of nicotine are mediated by binding to a site outside the membrane's electric field. An analysis of point mutations indicates that the residues at the 6' position within the beta subunit TM2 domain may be important for determining the effects of both mecamylamine and nicotine in a reciprocal manner. Single mutations at the 10' position are not sufficient to produce effects, but 6' 10' double mutants show more effect than do the 6' single mutants.

The novel progesterone receptor antagonists RTI 3021-012 and RTI 3021-022 exhibit complex glucocorticoid receptor antagonist activities: implications for the development of dissociated antiprogestins.

We have identified two novel compounds (RTI 3021-012 and RTI 3021-022) that demonstrate similar affinities for human progesterone receptor (PR) and display equivalent antiprogestenic activity. As with most antiprogestins, such as RU486, RTI 3021-012, and RTI 3021-022 also bind to the glucocorticoid receptor (GR) with high affinity. Unexpectedly, when compared with RU486, the RTI antagonists manifest significantly less GR antagonist activity. This finding indicates that, with respect to antiglucocorticoid function, receptor binding affinity is not a good predictor of biological activity. We have determined that the lack of a clear correlation between the GR binding affinity of the RTI compounds and their antagonist activity reflects the unique manner in which they modulate GR signaling. Previously, we proposed a two step "active inhibition" model to explain steroid receptor antagonism: 1) competitive inhibition of agonist binding; and 2) competition of the antagonist bound receptor with that activated by agonists for DNA response elements within target gene promoters. Accordingly, we observed that RU486, RTI 3021-012, and RTI 3021-022, when assayed for PR antagonist activity, accomplished both of these steps. Thus, all three compounds are "active antagonists" of PR function. When assayed on GR, however, RU486 alone functioned as an active antagonist. RTI 3021-012 and RTI 3021-022, on the other hand, functioned solely as "competitive antagonists" since they were capable of high affinity GR binding, but the resulting ligand receptor complex was unable to bind DNA. These results have important pharmaceutical implications supporting the use of mechanism based approaches to identify nuclear receptor modulators. Of equal importance, RTI 3021-012 and RTI 3021-022 are two new antiprogestins that may have clinical utility and are likely to be useful as research reagents with which to separate the effects of antiprogestins and antiglucocorticoids in physiological systems.

Glucocorticoid receptor phosphorylation: overview, function and cell cycle-dependence.

All steroid hormone receptors are phosphorylated and undergo hormone-induced hyperphosphorylation. Most phosphorylated residues identified so far are serines in the N-terminal domain. Other residues and domains may also be phosphorylated, e.g. the estrogen receptor is phosphorylated on tyrosine in the hormone-binding domain. Many sites lie in consensus sequences for proline-directed, cell cycle-associated kinases. In some receptors hyperphosphorylation is induced by hormone antagonists as well as agonists, and leads to new phosphorylated sites. With glucocorticoid receptors, hyperphosphorylation is specific for glucocorticoid agonists, follows receptor activation and produces no new sites. Rate studies suggest that hyperphosphorylation is due to accelerated phosphorylation rather than delayed dephosphorylation. Evidence to date indicates that steroid hormone receptor phosphorylation serves not as an on-off switch but modulates function more subtly. Mutations of phosphorylated sites to alanine have been found to decrease activity by 0 to 90%, depending on mutated site, cell type, reporter gene and hormone concentration. With glucocorticoid receptors, some alanine mutants are up to 75% less active in hormone-induced transactivation of certain reporter genes. They are also inactive in hormone-induced repression of transcription of their own gene and down regulation of the receptor protein. Furthermore, they are much less sensitive to degradation. Both basal phosphorylation and hormone-dependent hyperphosphorylation of these receptors are cell cycle-dependent, basal phosphorylation being low in S phase and high in G2/M and hyperphosphorylation the reverse, suggesting a causal relation to the cell cycle-dependence of glucocorticoid activity reported with several cell lines. Hyperphosphorylation appears to be regulated by basal phosphorylation through negative charge in the N-terminal domain, which in S phase is relatively low and permits hyperphosphorylation, but in G2/M is relatively high and blocks hyperphosphorylation.

Expression and subcellular distribution of the beta-isoform of the human glucocorticoid receptor.

Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two highly homologous protein isoforms, termed hGR alpha and hGRbeta, that differ at their carboxy-termini. In contrast to the well characterized hGR alpha isoform, which modulates gene expression in a hormone-dependent fashion, the biological significance of hGRbeta has only recently begun to emerge. We and others have shown that the hGRbeta messenger RNA transcript is widely expressed in human tissues and that the hGRbeta protein functions as a dominant negative inhibitor of hGR alpha in transfected cells. Unfortunately, these initial studies did not determine whether the hGRbeta protein was made in vivo. Such analyses are hindered because available anti-hGR antibodies cannot discriminate between the similarly sized hGR alpha and hGRbeta proteins. Therefore, to investigate the expression of the hGRbeta protein, we have produced an antipeptide, hGRbeta-specific antibody termed BShGR. This antibody was made against the unique 15-amino acid peptide at the carboxy-terminus of hGRbeta and recognizes both the native and denatured conformations of hGRbeta, but does not cross-react with hGR alpha. Using BShGR on Western blots and in immunoprecipitation experiments, we detected the hGRbeta protein in a variety of human cell lines and tissues. Immunocytochemistry was then performed with BShGR on HeLa S3 and CEM-C7 cells and on tissue sections prepared from lung, thymus, and liver to assess the cellular and subcellular distribution of hGRbeta. In all immunopositive cells, hGRbeta was found in the nucleus independent of glucocorticoid treatment. Within tissues, the hGRbeta protein was expressed most abundantly in the epithelial cells lining the terminal bronchiole of the lung, forming the outer layer of Hassall's corpuscle in the thymus, and lining the bile duct in the liver. As a potential in vivo inhibitor of hGR alpha activity, expression of hGRbeta may be an important factor regulating target cell responsiveness to glucocorticoids.

Mouse glucocorticoid receptor phosphorylation status influences multiple functions of the receptor protein.

Although studies have shown that the mouse glucocorticoid receptor (mGR) contains eight phosphorylation sites (Bodwell, J. E., Ortí, E. , Coull, J. M., Pappin, D. J. C., Smith, L. I., and Swift, F. (1991) J. Biol. Chem. 266, 7549-7555), the effect of phosphorylation on receptor function is unclear. We have examined the consequences of single or multiple phosphorylation site mutations on several properties of mGR including receptor expression, ligand-dependent nuclear translocation, hormone-mediated transactivation, ligand-dependent down-regulation of mGR, and receptor protein half-life. Mutations had little effect on receptor expression, subcellular distribution, ligand-dependent nuclear translocation, or on the ability to activate hormone-mediated transcription from a complex (murine mammary tumor virus) promoter. In contrast, the phosphorylation status of the mGR had a profound effect on the ability to transactivate a minimal promoter containing simple glucocorticoid response elements after hormone administration. Similarly, ligand-dependent down-regulation by glucocorticoids of both receptor mRNA and protein was abrogated in mutants containing three or more phosphorylation site alterations. Finally, we show that the phosphorylation status of mGR has a profound effect on the stability of the glucocorticoid receptor protein. Receptors containing seven or eight mutated sites have a markedly extended half-life and do not show the ligand-dependent destabilization seen with wild type receptor. These data show that receptor phosphorylation may play a crucial role in regulating receptor levels and hence control receptor functions.

Immunocytochemical analysis of hormone mediated nuclear translocation of wild type and mutant glucocorticoid receptors.

We have analyzed structural and functional features of the human glucocorticoid receptor (hGR) for their effects on receptor subcellular distribution. COS 1 cells transiently transfected with wild type and mutant hGR cDNAs were assessed immunocytochemically using well-characterized antipeptide antibodies to the hGR. The effect of administration of steroid hormones (and the antiglucocorticoid RU486) on receptor localization was evaluated. Unliganded wild type receptors expressed in COS 1 cells were predominately cytoplasmic. Addition of glucocorticoids or the glucocorticoid receptor antagonist, RU486, resulted in complete translocation of these receptors into the nucleus whereas non-glucocorticoid steroids or dibutyryl cAMP were not effective in promoting nuclear translocation. Thus, nuclear translocation was specific for steroids capable of high affinity binding to the hGR. To elucidate the potential role of receptor domains in receptor localization, COS 1 cells transiently transfected with various receptor cDNA mutants were analyzed in a similar manner. Translocation of an hGR deletion mutant lacking the majority of the amino terminus (deletion of amino acids 77-262) was identical to the wild type receptor despite the absence of a transactivation domain. Receptors in which the DNA binding domain was either partially or totally deleted showed an impaired capacity to undergo hormone-inducible nuclear translocation. Deletion of the hinge region of the hGR (which also contains part of the nuclear localization signal, NL1) resulted in receptor localization in the cytoplasm. Mutants in the ligand binding domain exhibited two localization phenotypes, exclusively nuclear or cytoplasmic. Receptor mutants truncated after amino acid 550 were found in the nucleus in the presence and absence of hormone consistent with the existence of nuclear localization inhibitory sequences in the ligand binding domain of the receptor. However, a linker insertion mutant (at amino acid 582) which results in a receptor deficient in ligand binding did not undergo nuclear translocation indicating that nuclear localization inhibitory sequences were intact in this mutant. The role of receptor phosphorylation on hormone induced nuclear translocation was also examined. Mouse glucocorticoid receptors which contained mutations of certain hormone inducible phosphorylation sites exhibited translocation properties similar to wild type mGR indicating that these phosphorylation sites on the receptor do not play a major role in hormone inducible nuclear translocation.

The 5'-flanking region of the ovine follicle-stimulating hormone-beta gene contains six progesterone response elements: three proximal elements are sufficient to increase transcription in the presence of progesterone.

Progesterone (P4) can alter the synthesis and secretion of FSH from pituitary gonadotropes of sheep. In this study, the 5'-flanking region (4.7 kilobases) of the ovine FSH beta gene was tested for binding by human progesterone receptors (hPR), using an immunoprecipitation technique. Three fragments were bound by hPR. Competition experiments using homologous and heterologous DNA fragments revealed this binding to be specific and of high affinity (Kd = 1.2-47 nM). The fragment sequences were screened for potential P4 response elements (PREs). Six PRE-like elements were found among the three immunoprecipitated fragments. Band shift experiments discerned that each of these PRE-like sequences could be bound by hPR. In functional studies, each of the PRE-like elements could enhance the expression of a reporter gene driven by a heterologous promoter in a hormone-dependent manner. The 5'-flanking region of the ovine FSH beta gene was tested for P4 responsiveness using a luciferase reporter. In the presence of P4, there was a 2- to 3-fold increase in luciferase activity when the entire 4.7 kilobases of the 5'-flanking sequence were present, whereas no increase was seen in a construct that contained only 84 basepairs 5' to the transcription start site. This effect on transcription was dose dependent for P4. Deletion studies revealed that the three PRE-like elements closest to the transcription start site (-250 to -137) were sufficient to create the hormone-dependent enhancement. These results indicate that the 5'-flanking sequence of the ovine FSH beta gene contains sequences capable of being bound by hPR and may be responsible for the effects of P4 on FSH beta synthesis and secretion. This study is the first to show binding and function of PR for a gonadotropin gene.

Effect of hearing loss of cochlear origin on the auditory brain stem response.

Auditory brain stem response (ABR) testing is widely used to detect lesions of the auditory neural pathways. The ABR waves depend not only on the integrity of the neural pathways, but also on the condition of the cochlea. To properly interpret the ABR response, it is necessary to understand the effects of cochlear hearing loss on the ABR wave latencies. We studied two populations of subjects with cochlear hearing loss: one with varying degrees of high-frequency hearing loss and the other with varying degrees of flat configuration hearing loss. The degree of cochlear hearing loss was quantified in several different ways and subjected to one linear and three nonlinear regression analyses to test for accuracy in predicting ABR wave latencies and interpeak intervals (waves I, III, V, I-V, I-III, and III-V) for three click intensities. Hearing loss levels from 2 to 6 kHz, in particular 4 kHz, were superior to other audiometric test frequencies as predictors of ABR wave latencies for the group with the high-frequency losses. No particular characterization was found to be superior for the flat hearing loss configurations. From these results, modeled predictions of wave latencies as a function of degree and configuration of hearing loss were made. The modeled predictions are then used to suggest guidelines for interpretations of ABR results where hearing impaired patients are involved.

Inhibin increases and progesterone decreases receptors for gonadotropin-releasing hormone in ovine pituitary culture.

Treatments (48 h) with highly purified bovine or porcine inhibins (10 ng/ml) induced ovine pituitary cells to increase their binding for des-Gly10-[D-Ala6]LHRH-ethylamide by 3.6- and 5-fold, respectively. Studies with less pure inhibin from porcine follicles showed that increased binding could reach 7-fold within 48 h and was due to higher numbers of receptors for GnRH. The 48-h increase in GnRH receptors was linear with time and was rapidly reversible, since removal of inhibin at 24 h decreased GnRH binding below control levels at 48 h. Inhibin (bovine or porcine) also increased GnRH-stimulated secretion of LH by 2-fold. The ED50 for both inhibin actions noted above was in the range of 0.5-2.0 ng/ml (in terms of highly purified bovine inhibin). Progesterone (P) totally counteracted inhibin induction of GnRH binding and GnRH-stimulated LH secretion at 48 h. In the absence of inhibin, P decreased GnRH binding below control levels by as much as 80% within 48 h, but did not affect GnRH-stimulated LH secretion at 48 h. The ED50 for P action was near 1 nM, which is within the physiological range for P during the luteal phase of the sheep estrous cycle. The data suggest that P may act during the luteal phase to decrease receptors for GnRH. The rapid decrease in P during the 48 h before the preovulatory LH surge should permit GnRH receptors to rise under the influence of inhibin (and estradiol) to boost gonadotroph responsiveness to GnRH so the LH surge may occur to its fullest.

Estradiol alters the effectiveness of gonadotropin-releasing hormone (GnRH) in ovine pituitary cultures: GnRH receptors versus responsiveness to GnRH.

17 beta-Estradiol (E) rapidly (5-12 h) induced ovine pituitary cultures to increase their binding of des-Gly10-[D-Ala6] GnRH ethylamide by 2.5- to 4.5-fold. The ED50 for E was near 0.1 nM. Scatchard analysis indicated that E increased binding by increasing receptors for GnRH. GnRH-stimulated release of LH increased 2-fold along with the increase in GnRH receptors, but heightened responsiveness to GnRH disappeared after 27 h of E treatment even though GnRH receptors remained elevated. These data are consistent with the concepts that 1) E increased GnRH receptors, which initially enhanced gonadotroph responsiveness to GnRH, and 2) E subsequently nullified the increased response to GnRH by blocking a mechanism of LH secretion not associated with GnRH receptor number. Further support for this hypothesis came from studies with porcine follicular inhibin. Inhibin in ovine pituitary culture concomitantly increased GnRH receptors by 5-fold and GnRH-stimulated LH secretion by 2-fold. Inhibin maintained both effects long term (48 h). When E was added along with inhibin, the increase in GnRH receptor number became greater than with either hormone alone (7-fold increase at 48 h), but the initial increase in GnRH-stimulated LH secretion was fully inhibited by E at 48 h. The increase in GnRH receptors caused by E may be important physiologically to help create the preovulatory LH surge; the delayed inhibitory action of E on GnRH-stimulated LH secretion may limit the LH surge.

Treatment of behavioral problems with pindolol.

A study was conducted on a group of chronically hospitalized, brain-damaged male patients to assess the effectiveness of treatment with pindolol on behavioral problems. The study was conducted in two parts. The first was a double-blind, placebo-controlled analysis of the effect of pindolol on assaultive behavior, both verbal and physical. The second part was open and sought to determine whether pindolol would diminish such behaviors as resistance to care, sexual preoccupation, or provocation of others, which were sufficient to preclude placement at a lower level of care. These target behaviors and nursing interventions were monitored and clinical global assessments of improvement in behavior and of suitability for lower levels of care were developed. Eight of 13 patients were considered improved. Those with significant premorbid personality disorders showed little benefit. Pindolol appears to ameliorate some management problems and, by inference, improve the quality of life in many patients with behavioral pathology due to organic brain disease.