The neuroregenerative mechanism mediated by the Hsp90-binding immunophilin FKBP52 resembles the early steps of neuronal differentiation.

BACKGROUND AND PURPOSE The immunosuppressive macrolide FK506 (tacrolimus) shows neuroregenerative action by a mechanism that appears to involve the Hsp90-binding immunophilin FKBP52. This study analyses some aspects of the early steps of neuronal differentiation and neuroregeneration. EXPERIMENTAL APPROACH Undifferentiated murine neuroblastoma cells and hippocampal neurones isolated from embryonic day-17 rat embryos were induced to differentiate with FK506. Subcellular relocalization of FKBP52, Hsp90 and its co-chaperone p23 was analysed by indirect immunofluorescence confocal microscopy and by Western blots of axonal fractions isolated from cells grown on a porous transwell cell culture chamber. Neuroregeneration was evaluated using a scratch-wound assay. KEY RESULTS In undifferentiated cells, FKBP52, Hsp90 and p23 are located in the cell nucleus, forming an annular structure that disassembles when the differentiation process is triggered by FK506. This was observed in the N2a cell line and in hippocampal neurones. More importantly, the annular structure of chaperones is reassembled after damaging the neurones, whereas FK506 prompts their rapid regeneration, a process linked to the subcellular redistribution of the heterocomplex. CONCLUSIONS AND IMPLICATIONS There is a direct relationship between the disassembly of the chaperone complex and the progression of neuronal differentiation upon stimulation with the immunophilin ligand FK506. Both neuronal differentiation and neuroregeneration appear to be mechanistically linked, so the elucidation of one mechanism may lead to unravel the properties of the other. This study also implies that the discovery of FK506 derivatives, devoid of immunosuppressive action, would be therapeutically significant for neurotrophic use.

The p160 nuclear receptor co-activator RAC3 exerts an anti-apoptotic role through a cytoplasmatic action.

The p160 nuclear receptor co-activators represent a family of molecules, which are recruited by steroid nuclear receptors as well as other transcription factors that are overexpressed in several tumors. We investigated the role of one member of this family on the sensitivity of cells to apoptosis. We observed that overexpression of the RAC3 (receptor-associated co-activator-3) p160 co-activator inhibits hydrogen peroxide-induced cell death in human embryonic kidney 293 (HEK293) cells. The mechanism involves the activation of anti-apoptotic pathways mediated through enhanced nuclear factor kappa B (NF-kappaB) activity, inhibition of caspase-9 activation, diminished apoptotic-inducing factor (AIF) nuclear localization and a change in the activation pattern of several kinases, including an increase in both AKT and p38 kinase activities, and inhibition of ERK2. Moreover, RAC3 has been found associated with a protein complex containing AIF, Hsp90 and dynein, suggesting a role for the co-activator in the cytoplasmatic nuclear transport of these proteins associated with cytoskeleton. These results demonstrate that there are several molecular pathways that could be affected by their overexpression, including those not restricted to steroid regulation or the nuclear action of co-activators, which results in diminished sensitivity to apoptosis. Furthermore, this could represent one mechanism by which co-activators contribute to tumor development.

Progestin-induced caveolin-1 expression mediates breast cancer cell proliferation.

Progestin regulation of gene expression was assessed in the progestin-dependent murine tumor line C4HD which requires MPA, a synthetic progestin, for in vivo growth and expresses high levels of progesterone receptor (PR). By using suppressive subtractive hybridization, caveolin-1 was identified as a gene whose expression was increased with in vivo MPA treatment. By Northern and Western blot analysis, we further confirmed that caveolin-1 mRNA and protein expression increased in MPA-treated tumors as compared with untreated tumors. When primary cultures of C4HD cells were treated in vitro with MPA, caveolin-1 levels also increased, effect that was abolished by pre-treatment with progestin antagonist RU486. In addition, MPA promoted strong caveolin-1 promoter transcriptional activation both in mouse and human breast cancer cells. We also showed that MPA regulation of caveolin-1 expression involved in activation of two signaling pathways: MAPK and PI-3K. Short-term MPA treatment of C4HD cells led to tyrosine phosphorylation of caveolin-1 protein, where Src was the kinase involved. Additionally, we showed that MPA-induced association of caveolin-1 and PR, which was detected by coimmunoprecipitation and by confocal microscopy. Finally, we proved that MPA-induced proliferation of C4HD cells was inhibited by suppression of caveolin-1 expression with antisense oligodeoxynucleotides to caveolin-1 mRNA. Furthermore, we observed that inhibition of caveolin-1 expression abrogated PR capacity to induced luciferase activity from a progesterone response element-driven reporter plasmid. Comprehensively, our results demonstrated for the first time that caveolin-1 expression is upregulated by progestin in breast cancer. We also demonstrated that caveolin-1 is a downstream effector of MPA that is partially responsible for the stimulation of growth of breast cancer cells.

Molecular mechanism of activation and nuclear translocation of the mineralocorticoid receptor upon binding of pregnanesteroids.

The mineralocorticoid receptor (MR) is primarily localized in the cytoplasm of the cell in the absence of ligand. The first step in the genomic-dependent mechanism of action of mineralocorticoids is the binding of steroid to the MR, which in turn triggers MR nuclear translocation. The regulation of hormone-binding to MR is complex and involves a multifactorial mechanism, making it difficult to determine the optimal structure of a steroid for activating the MR and promoting its nuclear translocation. Here we review the structure-activity relationship for several pregnanesteroids that possess various functional groups, and suggest that a flat conformation of the ligand rather than the presence of particular chemical groups is a critical parameter for the final biological effect in vivo. We also discuss how the MR undergoes differential conformational changes according to the nature of the bound ligand, which in turn affects the dynein-dependent retrograde rate of movement for the steroid/receptor complex.

Differential responsivity of the hypothalamic-pituitary-adrenal axis to glucocorticoid negative-feedback and corticotropin releasing hormone in rats undergoing morphine withdrawal: possible mechanisms involved in facilitated and attenuated stress responses.

Chronic morphine treatment produces profound and long-lasting changes in the pituitary-adrenal responses to stressful stimuli. The purpose of the present study was to explore the mechanisms involved in these altered stress responses. Chronic morphine administration increased basal plasma concentrations of corticosterone and adrenocorticotropic hormone (ACTH), which peaked at 36 h after the final morphine injection and returned to normal levels within 84-h. Whole brain glucocorticoid receptor protein expression was reduced (approximately 70%) in morphine-treated rats 4-h after the final morphine injection and these levels recovered within 16-h. Twelve hours following morphine withdrawal, rats displayed normal ACTH, but potentiated and prolonged corticosterone responses to restraint stress. Both the ACTH and corticosterone responses to restraint in acutely withdrawn rats were insensitive to dexamethasone. Furthermore, acutely withdrawn rats displayed reduced ACTH but prolonged corticosterone responses to peripheral corticotropin releasing hormone (CRH) administration. These findings suggest that the normal ACTH and enhanced corticosterone responses to stress in acutely withdrawn rats involved decreased sensitivity of negative-feedback systems to glucocorticoids, reduced pituitary responsivity to CRH, and enhanced sensitivity of the adrenals to ACTH. Eight days following morphine withdrawal, rats displayed dramatically reduced ACTH, but normal corticosterone responses to restraint stress. These rats displayed enhanced sensitivity to dexamethasone and normal pituitary-adrenal responses to CRH. These data suggest that the reduced ACTH responses to stress in 8-day withdrawal rats involved increased sensitivity of negative-feedback systems to glucocorticoids as well as reduced CRH and/or AVP function in response to stress. Taken together, the results of this study illustrate some of the mechanisms mediating altered stress responsivity in rats that have received chronic morphine treatment.

Stoichiometry, abundance, and functional significance of the hsp90/hsp70-based multiprotein chaperone machinery in reticulocyte lysate.

Rabbit reticulocyte lysate contains a multiprotein chaperone system that assembles the glucocorticoid receptor (GR) into a complex with hsp90 and converts the hormone binding domain of the receptor to its high affinity steroid binding state. This system has been resolved into five proteins, with hsp90 and hsp70 being essential and Hop, hsp40, and p23 acting as co-chaperones that optimize assembly. Hop binds independently to hsp70 and hsp90 to form an hsp90.Hop.hsp70 complex that acts as a machinery to open up the GR steroid binding site. Because purified hsp90 and hsp70 are sufficient for some activation of GR steroid binding activity, some investigators have rejected any role for Hop in GR.hsp90 heterocomplex assembly. Here, we counter that impression by showing that all of the Hop in reticulocyte lysate is present in an hsp90.Hop.hsp70 complex with a stoichiometry of 2:1:1. The complex accounts for approximately 30% of the hsp90 and approximately 9% of the hsp70 in lysate, and upon Sephacryl S-300 chromatography the GR.hsp90 assembly activity resides in the peak containing Hop-bound hsp90. Consistent with the notion that the two essential chaperones cooperate with each other to open up the steroid binding site, we also show that purified hsp90 and hsp70 interact directly with each other to form weak hsp90.hsp70 complexes with a stoichiometry of 2:1.

Evidence that the peptidylprolyl isomerase domain of the hsp90-binding immunophilin FKBP52 is involved in both dynein interaction and glucocorticoid receptor movement to the nucleus.

We have previously shown that immunoadsorption of the FKBP52 immunophilin component of steroid receptor.hsp90 heterocomplexes is accompanied by coadsorption of cytoplasmic dynein, a motor protein involved in retrograde transport of vesicles toward the nucleus. Coimmunoadsorption of dynein is competed by an expressed fragment of FKBP52 comprising its peptidylprolyl isomerase (PPIase) domain (Silverstein, A. M., Galigniana, M. D., Kanelakis, K. C., Radanyi, C., Renoir, J.-M., and Pratt, W. B. (1999) J. Biol. Chem. 52, 36980-36986). Here we show that cotransfection of 3T3 cells with the FKBP52 PPIase domain and a green fluorescent protein (GFP) glucocorticoid receptor (GR) chimera inhibits dexamethasone-dependent movement of the GFP-GR from the cytoplasm to the nucleus. Cotransfection with FKBP12 does not affect GFP-GR movement. Inhibition of movement by the FKBP52 PPIase domain is abrogated in cells treated with colcemid to eliminate microtubules prior to steroid addition. After withdrawal of colcemid, microtubules reform, and PPIase inhibition of GFP-GR movement is restored. These observations are consistent with the notion that FKBP52 targets retrograde movement of the GFP-GR along microtubules by linking the receptor to the dynein motor. Here, we also show that native GR.hsp90 heterocomplexes immunoadsorbed from L cell cytosol contain dynein and that GR.hsp90 heterocomplexes assembled in reticulocyte lysate contain cytoplasmic dynein in a manner that is competed by the PPIase domain of FKBP52.

hsp70 interacting protein Hip does not affect glucocorticoid receptor folding by the hsp90-based chaperone machinery except to oppose the effect of BAG-1.

Reticulocyte lysate contains a chaperone system that assembles glucocorticoid receptor (GR).hsp90 heterocomplexes. Using purified proteins, we have prepared a five-protein heterocomplex assembly system consisting of two proteins essential for heterocomplex assembly-hsp90 and hsp70-and three proteins that act as co-chaperones to enhance assembly-Hop, hsp40, p23 [Morishima, Y., Kanelakis, K. C., Silverstein, A. M., Dittmar, K. D., Estrada, L., and Pratt, W. B. (2000) J. Biol. Chem. 275, 6894-6900]. The hsp70 co-chaperone Hip has been recovered in receptor.hsp90 heterocomplexes at an intermediate stage of assembly in reticulocyte lysate, and Hip is also thought to be an intrinsic component of the assembly machinery. Here we show that immunodepletion of Hip from reticulocyte lysate or addition of high levels of Hip to the purified five-protein system does not affect GR.hsp90 heterocomplex assembly or the activation of steroid binding activity that occurs with assembly. Despite the fact that Hip does not affect assembly, it is recovered in GR.hsp90 heterocomplexes assembled by both systems. In the five-protein system, Hip prevents inhibition of assembly by the hsp70 co-chaperone BAG-1, and cotransfection of Hip with BAG-1 opposes BAG-1 reduction of steroid binding activity in COS cells. We conclude that Hip is not a component of the assembly machinery but that it could play a regulatory role in opposition to BAG-1.

Mechanism of action of the potent sodium-retaining steroid 11, 19-oxidoprogesterone.

We have demonstrated previously that a planar conformation of the molecular frame is required for steroids to acquire optimal sodium-retaining activity and binding properties to the mineralocorticoid receptor (MR). One of the most active sodium-retaining compounds tested in those studies was 11, 19-oxidoprogesterone. Despite its biological potency, the relative affinity of 11,19-oxidoprogesterone for the MR is 5-fold lower than that of 21-deoxycorticosterone and 10-fold lower than aldosterone. Such a discrepancy may be assigned to uncommon biopharmacological properties of this synthetic steroid or an unusual molecular mechanism of action. In this work, we studied the biopharmacological and mechanistic features of 11,19-oxidoprogesterone. We show that both the pharmacokinetic properties of 11,19-oxidoprogesterone and its ability to transform and translocate the MR into the nucleus are undistinguishable from aldosterone. However, the capability of the serine/threonine phosphatase inhibitor tautomycin to impair nuclear translocation of the aldosterone-MR complex is not observed for the 11,19-oxidoprogesterone-MR complex. In addition, the binding properties of both steroids are differentially affected by modification of crucial lysyl residues of the MR. Kinetic studies performed on the aldosterone-MR complex in the presence of low concentrations of oxidopregnane suggest that 11,19-oxidoprogesterone may bind to the MR in a different binding site from the aldosterone binding pocket. Consistent with this postulate, a biologically inactive dose of 0.6 ng of oxidopregnane is able to potentiate the mineralocorticoid effect of a suboptimal dose of aldosterone.

Oxidative stress induced by L-buthionine-(S,R)-sulfoximine, a selective inhibitor of glutathione metabolism, abrogates mouse kidney mineralocorticoid receptor function.

In vitro studies have demonstrated that cysteine groups present in most of the steroid receptors play an essential role in the steroid binding process as well as in the ability of this superfamily of signaling proteins to function as transcription factors. However, there is poor experimental evidence, if any, which demonstrates that under conditions of oxidative stress the steroid receptors in general, and the mineralocorticoid receptor in particular, are affected in vivo in a similar fashion as has been described for cell-free systems or cells in culture. In the present work we report that when mice are exposed to oxidative stress by treatment with L-buthionine-(S,R)-sulfoximine (L-(S,R)-BSO), a glutathione depleting agent, the aldosterone-dependent mineralocorticoid biological response (measured as sodium retention and potassium elimination) was diminished in a directly proportional manner with respect to the depletion of renal glutathione. Accordingly, the steroid binding capacity of the mineralocorticoid receptor was also abrogated, whereas the receptor protein level remained unchanged. The harmful effects observed in mice after glutathione depletion were efficiently prevented by co-treatment with glutathione monoethyl ester. Similar inhibition in the steroid binding capacity was also generated in vitro by receptor alkylation and receptor oxidation, an effect which was prevented in the presence of reducing agents. Since the glutathione deficit generated in vivo by treatment with L-(S,R)-BSO did not significantly affect other renal proteins which are known to be required for the mineralocorticoid mechanism of action, we suggest that in renal cells a low redox potential exerts drastic and uncompensated inhibition of the receptor-mediated mineralocorticoid biological response. This effect was ascribed to the loss of steroid binding capacity of oxidized receptor, most likely by modification of essential cysteines as supported by experiments where a decreased number of reactive thiols and reduced covalent binding of thiol-reactive ligand were evidenced on immunopurified receptor after in vivo treatment with L-(S,R)-BSO.

Different regions of the immunophilin FKBP52 determine its association with the glucocorticoid receptor, hsp90, and cytoplasmic dynein.

FKBP52 is a high molecular mass immunophilin possessing peptidylprolyl isomerase (PPIase) activity that is inhibited by the immunosuppressant drug FK506. FKBP52 is a component of steroid receptor.hsp90 heterocomplexes, and it binds to hsp90 via a region containing three tetratricopeptide repeats (TPRs). Here we demonstrate by cross-linking of the purified proteins that there is one binding site for FKBP52/dimer of hsp90. This accounts for the common heterotetrameric structure of native receptor heterocomplexes being 1 molecule of receptor, 2 molecules of hsp90, and 1 molecule of a TPR domain protein. Immunoadsorption of FKBP52 from reticulocyte lysate also yields co-immunoadsorption of cytoplasmic dynein, and we show that co-immunoadsorption of dynein is competed by a fragment of FKBP52 containing its PPIase domain, but not by a TPR domain fragment that blocks FKBP52 binding to hsp90. Using purified proteins, we also show that FKBP52 binds directly to the hsp90-free glucocorticoid receptor. Because neither the PPIase fragment nor the TPR fragment affects the binding of FKBP52 to the glucocorticoid receptor under conditions in which they block FKBP52 binding to dynein or hsp90, respectively, different regions of FKBP52 must determine its association with these three proteins.

Differential effects of the hsp70-binding protein BAG-1 on glucocorticoid receptor folding by the hsp90-based chaperone machinery.

The heat shock protein hsp70/hsc70 is a required component of a five-protein (hsp90, hsp70, Hop, hsp40, and p23) minimal chaperone system reconstituted from reticulocyte lysate that forms glucocorticoid receptor (GR).hsp90 heterocomplexes. BAG-1 is a cofactor that binds to the ATPase domain of hsp70/hsc70 and that modulates its chaperone activity. Inasmuch as BAG-1 has been found in association with several members of the steroid receptor family, we have examined the effect of BAG-1 on GR folding and GR.hsp90 heterocomplex assembly. BAG-1 was present in reticulocyte lysate at a BAG-1:hsp70/hsc70 molar ratio of approximately 0.03, and its elimination by immunoadsorption did not affect GR folding and GR. hsp90 heterocomplex assembly. At low BAG-1:hsp70/hsc70 ratios, BAG-1 promoted the release of Hop from the hsp90-based chaperone system without inhibiting GR.hsp90 heterocomplex assembly. However, at molar ratios approaching stoichiometry with hsp70, BAG-1 produced a concentration-dependent inhibition of GR folding to the steroid-binding form with corresponding inhibition of GR.hsp90 heterocomplex assembly by the minimal five-protein chaperone system. Also, there was decreased steroid-binding activity in cells that were transiently or stably transfected with BAG-1. These observations suggest that, at physiological concentrations, BAG-1 modulates assembly by promoting Hop release from the assembly complex; but, at concentrations closer to those in transfected cells and some transformed cell lines, hsp70 is continuously bound by BAG-1, and heterocomplex assembly is blocked.

Comparative inhibition by hard and soft metal ions of steroid-binding capacity of renal mineralocorticoid receptor cross-linked to the 90-kDa heat-shock protein heterocomplex.

We analysed the inhibitory effects in vitro and in vivo of several metal ions on aldosterone binding to the rat kidney mineralocorticoid receptor with the purpose of assessing possible toxic effects of those ions on sodium retention, as well as to obtain information on receptor structural requirements for ligand binding. For the assays in vitro, the inhibitory effects of 20 metal ions were analysed on steroid-binding capacity for renal receptor cross-linked to 90-kDa heat-shock protein (hsp90) by pretreatment with dimethyl pimelimidate. Cross-linking prevented the artifactual dissociation of hsp90 (and, consequently, the loss of steroid binding) from the mineralocorticoid receptor due to the presence of high concentrations of salt in the incubation medium. Cross-linked heterocomplex showed no difference in ligand specificity and affinity with respect to native receptor, but increased stability upon thermal- or ionic-strength-induced destabilization was observed. Treatments in vitro with metal ions in the range 10(-8)-10(-1) M resulted in a differential inhibitory effect for each particular ion on aldosterone binding. Using the negative logarithm of metal concentration for 50% inhibition, the ions could be correlated with their Klopman hardness constants. The analysis of this relationship led us to postulate three types of reaction: with thiol, imidazole and carboxyl groups. The essential role played by these residues in steroid binding was confirmed by chemical modification of cysteines with dithionitrobenzoic acid, histidines with diethyl pyrocarbonate and acidic amino acids with Woodward's reagent (N-ethyl-5-phenylisoxazolium-3'-sulphonate). Importantly, the toxic effects of some metal ions were also observed by treatments in vivo of adrenalectomized rats on both steroid-binding capacity and aldosterone-dependent sodium-retaining properties. We suggest that those amino acid residues are involved in the activation process of the mineralocorticoid receptor upon steroid binding. Thus toxic effects observed with these metal ions may be a consequence of modifications of those essential groups. Our results support the notion that toxicity of metals on renal mineralocorticoid function may be predicted according to their chemical hardness.

The glucocorticoid properties of the synthetic steroid pregna-1,4-diene-11beta-ol-3,20-dione (deltaHOP) are not entirely correlated with the steroid binding to the glucocorticoid receptor.

The natural steroid 11beta-hydroxyprogesterone is not only a modulator of 11beta-hydroxy-steroid dehydrogenase activity, but also an efficient inducer of tyrosine aminotransferase activity in hepatocytes. In contrast with the low affinity for the mineralocorticoid receptor. 11beta-hydroxyprogesterone binds well to both the glucocorticoid receptor and the carrier protein transcortin. It is accepted that the introduction of a 1:ene double bond into 3-keto 4:ene steroids increases the glucocorticoid potency, so that 3-keto-1,4:diene steroids show improved chemical stability and are more potent glucocorticoids than their respective 4:ene analogs. The steroid pregna-1,4-diene-11beta-ol-3,20-dione (deltaHOP) had previously been described as an anti-inflamatory compound and an inhibitor of macromolecular biosynthesis in thymocytes and lymphocytes. In such studies, deltaHOP also exhibited some particular glucocorticoid properties which made it attractive as a tool for the study of the mechanism of action of glucocorticoids. In the present paper we show that deltaHOP possesses some classical biological actions of glucocorticoids such as deposition of glycogen in rat liver, induction of TAT activity in hepatocytes, and inhibition of the uptake of leucine and thymidine by thymocytes. It also exhibits minimal sodium-retaining properties. Consistent with these biological effects, deltaHOP shows a 70 times lower relative binding affinity for the mineralocortioid receptor than aldosterone, but a reasonable affinity for the glucocorticoid receptor, and is as efficient as dexamethasone in dissociating the 90 kDa heat shock protein from the glucocorticoid receptor heterocomplex. However, the inhibition of the uptake of amino acids and nucleotides observed in the presence of deltaHOP is not efficiently blocked when thymocytes are coincubated in the presence of steroids with known antiglucocorticoid activity. deltaHOP is similarly inefficient in inducing chloramphenicol-acetyl transferase activity in cells transfected with a plasmid that possesses two canonical glucocorticoid-responsive elements. Unlike most glucocorticoids, deltaHOP does not induce the fragmentation of DNA in a regular pattern characteristic of apoptosis and it does not reduce thymus weight. This unusual dissociation of glucocorticoid parameters makes deltaHOP a useful tool to discriminate between mechanisms of action by which steroids can exert their biological effects.

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.

Inhibition of glucocorticoid receptor binding by nitric oxide.

Septic shock is a dangerous condition with high mortality rates. In sepsis, the inducible form of nitric oxide (NO) synthase is induced, releasing high amounts of NO. Glucocorticoids have potent anti-inflammatory properties and are very effective in inhibiting the induction of this enzyme if administered before the shock onset. It is known that glucocorticoid receptor (GR) has critical cysteine residues for steroid binding in its hormone-binding and DNA-binding domains. It has also been reported that NO reacts with ---SH groups, forming S-nitrosothiols. Therefore, we examined the potential effect of NO on the ligand-binding ability of GR. NO donors (S-nitroso-acetyl-DL-penicillamine, S-nitroso-DL-penicillamine, or S-nitroso-glutathione) decreased, in a time- and dose-dependent manner, the binding of [3H]triamcinolone to immunoprecipitated GR from mouse L929 fibroblasts. The nonnitrosylated parent molecules, N-acetyl-DL-penicillamine, and reduced gluthatione were without effect. Scatchard plots revealed that the number of ligand binding sites and Kd were reduced (50%) by NO donors. Western blot analysis ruled out the possibility that dissociation of GR/heat shock protein 90 heterocomplex or decrease in GR protein would account for the inhibitory effect of NO. Decreased ligand binding to GR was found when NO donors were incubated with intact fibroblasts. Incubation with NO donors also decreased the steroid-induced reduction in [3H]uridine incorporation into RNA. All of these NO effects were inhibited by the thiol-protecting agent dithiothreitol. Therefore, S-nitrosylation of critical ---SH groups in GR by NO with consequent decreases in binding and affinity may be the mechanisms which explain the failure of glucocorticoids to exert their anti-inflammatory effects in septic shock.

A model for the cytoplasmic trafficking of signalling proteins involving the hsp90-binding immunophilins and p50cdc37.

A number of transcription factors and protein kinases involved in signal transduction exist in heterocomplexes with the ubiquitous and essential protein chaperone hsp90. These signalling protein x hsp90 heterocomplexes are assembled by a multiprotein chaperone system comprising hsp90, hsp70, Hop, hsp40, and p23. In the case of transcription factors, the heterocomplexes with hsp90 also contain a high molecular weight immunophilin with tetratricopeptide repeat (TPR) motifs, such as FKBP52 or CyP-40. In the case of the protein kinases, the heterocomplexes contain p50cdc37. The immunophilins bind to a single TPR acceptor site on hsp90, and p50cdc37 binds to an adjacent site so that binding is exclusive for p50cdc37 or an immunophilin. Direct interaction of immunophilins with the transcription factors or p50cdc37 with the protein kinases leads to selection of different heterocomplexes after their assembly by a common mechanism. Studies with the glucocorticoid receptor, for which translocation from the cytoplasm to the nucleus is under hormonal control, suggest that dynamic assembly of the heterocomplexes is required for rapid movement of the receptor through the cytoplasm along cytoskeletal tracts. As for the similar short-range trafficking of vesicles along microtubules, there must be a mechanism for linking the signalling protein solutes to the molecular motors involved in movement. We present here a model in which the immunophilins and p50cdc37 target, respectively, the retrograde or anterograde direction of signalling protein movement by functioning as connectors that link the signalling proteins to the movement machinery.

Tautomycin inhibits phosphatase-dependent transformation of the rat kidney mineralocorticoid receptor.

The binding of aldosterone (ALDO) to the mineralocorticoid receptor (MR) induces a conformational change of the protein referred to as 'transformation'. This feature can be evidenced in vivo by the capacity of the MR to interact with chromatin, and in vitro by the ability of the MR to bind to DNA strands or to shift the sedimentation coefficient (S) to lower values. The transformation process allows MR to work as a transcription factor after interacting with specific sequences of DNA. The signal transduction pathway for the MR transformation remains unknown. As a first step towards elucidating the mechanism of steroid-dependent MR transformation, we asked if the MR-signaling pathway is affected by the phosphorylation status of the MR-heterocomplex, and how that pathway may be regulated. Incubation of preformed [3H]ALDO-MR complex with bovine intestinal alkaline phosphatase led to an increase in the rate of MR-transformation (measured as 9.4-5.4S shift). This alkaline phosphatase-dependent MR transformation was inhibited by the specific alkaline phosphatase-type inhibitor levamisole, and was not evident in incubations performed with acid phosphatases. A direct correlation between the DNA-cellulose binding capacity of the [3H]ALDO-MR complex and the percentage of transformed 5.4S MR form was also observed. When rat kidney cytosol was incubated in the absence of both exogenous phosphatase and stabilizing agents (such as molybdate or vanadate), MR transformation also took place, in a time- and temperature-dependent process. In contrast with the inhibitory effect observed upon alkaline phosphatase-promoted transformation, levamisole was unable to inhibit the endogenous transforming activity of MR, suggesting that an endogenous phosphatase other than those which belong to the alkaline-type may be responsible for that transformation. Tautomycin, a polyketide produced by the soil bacteria Streptomyces which inhibits serine/threonine phosphatases of the PP1/PP2A subgroup, was able to inhibit the endogenous phosphatase activity in a concentration-dependent form (Ki(app)=7.35 nM). These results support the idea that the endogenous renal activity involved in the regulation of rat kidney MR transformation may be a protein phosphatase which belongs to the PP1/PP2A subgroup.

Heat shock protein 90-dependent (geldanamycin-inhibited) movement of the glucocorticoid receptor through the cytoplasm to the nucleus requires intact cytoskeleton.

We use here a chimera of the green fluorescent protein (GFP) and the glucocorticoid receptor (GR) to test the notion that the protein chaperone heat shock protein-90 (hsp90) is required for steroid-dependent translocation of the receptor through the cytoplasm along cytoskeletal tracks. The GFP-GR fusion protein undergoes steroid-mediated translocation from the cytoplasm to the nucleus, where it is transcriptionally active. Treatment of 3T3 cells containing steroid-bound GFP-GR with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, inhibits dexamethasone-dependent translocation from the cytoplasm to the nucleus. The t1/2 for translocation in the absence of geldanamycin is approximately 5 min, and the t1/2 in the presence of geldanamycin is approximately 45 min. In cells treated for 1 h with the cytoskeletal disrupting agents colcemid, cytochalasin D, and beta,beta'-iminodipropionitrile to completely disrupt the microtubule, microfilament, and intermediate filament networks, respectively, the GFP-GR still translocates rapidly to the nucleus in a strictly dexamethasone-dependent manner but translocation is no longer affected by geldanamycin. After withdrawal of the cytoskeletal disrupting agents for 3 h, normal cytoskeletal architecture is restored, and geldanamycin inhibition of dexamethasone-dependent GFP-GR translocation is restored. We suggest that in cells without an intact cytoskeletal system, the GFP-GR moves through the cytoplasm by diffusion. However, under physiological conditions in which the cytoskeleton is intact, diffusion is limited, and the GFP-GR utilizes a movement machinery that is dependent upon hsp90 chaperone activity. In contrast to the GR, GFP-STAT5B, a signaling protein that is not complexed with hsp90, undergoes GH-dependent translocation to the nucleus in a manner that is not dependent upon hsp90 chaperone activity.

Influence of calf serum on glucocorticoid-responses of certain progesterone derivatives.

UNLABELLED: The following in vitro glucocorticoid (GC) parameters of progesterone (P), 1-ene progesterone (deltaP), 11beta-hydroxyprogesterone (HOP), 11beta-1-ene progesterone (deltaHOP) and dexamethasone (Dexa) were assayed in the presence or absence of bovine calf serum (BCS): binding to thymus cytosol, dissociation of the glucocorticoid receptor (GR)-heat shock protein 90 (hsp90) complex (diss.), tyrosine aminotransferase (TAT) induction in hepatocytes and the inhibition of 3H-uridine and 35S-methionine uptake by thymocytes. Without BCS, steroids were in most cases active in this general order: Dex > deltaHOP > HOP > deltaP > P. BCS abolished all activities in P and deltaP, but left them unaltered in all other steroids, except diss. in HOP, which diminished intermediately. Binding of P, deltaP, HOP and deltaHOP to GR and CBG paralleled their in vivo activating effects on glycogen deposition. CONCLUSIONS: in this steroid series, BCS, but not CBG, inhibits GC responses of P and deltaP. 11-Beta hydroxylation frees those molecules from the inhibitory effects of BCS.