Generation of corticosteroid binder IB from binder II by a sulfhydryl dependent renal cytosolic factor

It has long been debated whether binder IB represents a unique form of the glucocorticoid receptor or is derived from the larger molecular weight form, binder II, by limited proteolysis. Transformed glucocorticoid receptors in kidney, liver and mixed kidney/liver cytosols were examined using anion exchange and gel filtration chromatography. The transformed receptor in liver cytosols chromatographs as binder II on DEAE-Sephadex A-50 anion exchange columns and has a Stokes radius of approx 6.0 nm. The transformed receptor in kidney cytosols chromatographs as binder IB on DEAE-Sephadex A-50 anion exchange columns and has a Stokes radius of 3.0-4.0 nm (3.2 nm on agarose; 3.0-4.0 nm on Sephadex G-100). Using cytosols prepared from mixed homogenates (2 g kidney plus 8 g liver tissue), our experiments show that binder II is converted to a lower molecular weight form (Rs = 3.2 nm on agarose; Rx = 3.9 nm on Sephadex G-100) that is identical to binder IB in its elution position from DEAE-Sephadex anion exchange resin. Identical results are obtained using kidney/liver/cytosols mixed in vitro in which only the hepatic receptor, binder II, is labelled with [3H]TA. These results support the hypothesis that the renal receptor, binder IB, is a proteolytic fragment of binder II and does not represent a polymorphic form of the glucocorticoid receptor. The renal converting activity is dependent on free-SH for full activity but is insensitive to the protease inhibitors leupeptin, antipain, and PMSF. The conversion of hepatic binder II to binder IB in in vitro mixing experiments can be prevented if kidney cytosol is gel filtered on Sephadex G-25 and the eluted macromolecular fraction is adjusted to 10 mM EGTA (or EDTA) prior to mixing with the [3H]TA labelled hepatic cytosol.     

Immunochemical characterization of the rat kidney glucocorticoid receptor. The role of proteolysis in the formation of corticosteroid binder IB

Glucocorticoid receptors of rat kidney and liver were compared by physicochemical and immunochemical methods to investigate the role of proteolysis in the formation of corticosteroid binder IB. Kidney cytosol prepared in the presence of sodium molybdate contained receptor forms comparable to rat liver glucocorticoid receptor; [3H]triamcinolone acetonide-labeled receptors eluted from Sephacryl S-300 as a multimeric 6.1 nm component in the presence of molybdate and as a monomeric 5.7 nm component in the absence of molybdate. Both forms were recognized by the monoclonal antibody BUGR-1 which was raised against rat liver glucocorticoid receptor. When kidney cytosol was prepared in the absence of molybdate, labeled receptor complexes eluted from Sephacryl S-300 as a 5.8 nm component in the presence of molybdate. However, in the absence of molybdate, the receptor eluted as a smaller 3.4 nm component which was identical with the size of activated kidney glucocorticoid receptor chromatographed in either the presence or absence of molybdate. The 3.4 nm activated kidney glucocorticoid receptor did not bind to DEAE-cellulose under conditions where activated liver receptor was retained. These properties of the activated kidney receptor are characteristic of corticosteroid binder IB. Incubation of the activated kidney receptor complex with BUGR-1 resulted in a shift in apparent Stokes radius from 3.4 nm to 5.4 nm, indicating immunochemical similarity with rat liver receptor. Identification of the immunoreactive receptor subunit by Western blotting demonstrated that kidney cytosol prepared in the presence of molybdate contained a major 94-kDa immunoreactive component which co-migrated with rat liver glucocorticoid receptor, while cytosol prepared in the absence of molybdate contained principally a 44-kDa immunoreactive species. These results suggest that corticosteroid binder IB can be generated by in vitro proteolysis and does not represent a polymorphic form of the glucocorticoid receptor.     

Glucocorticoid-receptor activation in hypertrophied skeletal muscle

This investigation was undertaken 1) to determine whether the increased glucocorticoid-receptor binding activities, observed in hypertrophied plantaris muscles, are associated with a reduced ability to undergo receptor activation and 2) to examine whether glucocorticoid-receptor complexes in hypertrophied muscles undergo a shift in the relative distribution of the two thermally activated receptor forms (termed binder II and corticosteroid binder IB) to a distribution that is found in slow-twitch or heart muscle types. Plantaris muscles of female adrenalectomized rats, enlarged by surgical removal of synergists, were 60% heavier and had higher glucocorticoid cytosol binding (125 +/- 14 vs. 79 +/- 8 fmol/mg protein) than these muscles of controls. Activation, which was quantitated by the ability of the steroid-receptor complex to bind to DNA, was similar in overloaded and control muscles (57 +/- 2 vs. 62 +/- 4%). Diethylaminoethyl-cellulose chromatography of activated receptors showed approximately 16% of the radioactivity appearing as binder II and 38% as binder IB in both hypertrophied and control muscles. These results show that although enlarged plantaris muscles are undergoing certain fast- to slow-twitch biochemical transformations, the activated glucocorticoid-receptor distribution does not shift to that observed in slow fibers.     

Heat Stress Modulates Both Anabolic and Catabolic Signaling Pathways Preventing Dexamethasone‐Induced Muscle Atrophy In Vitro

It is generally recognized that synthetic glucocorticoids induce skeletal muscle weakness, and endogenous glucocorticoid levels increase in patients with muscle atrophy. It is reported that heat stress attenuates glucocorticoid‐induced muscle atrophy; however, the mechanisms involved are unknown. Therefore, we examined the mechanisms underlying the effects of heat stress against glucocorticoid‐induced muscle atrophy using C2C12 myotubes in vitro, focusing on expression of key molecules and signaling pathways involved in regulating protein synthesis and degradation. The synthetic glucocorticoid dexamethasone decreased myotube diameter and protein content, and heat stress prevented the morphological and biochemical glucocorticoid effects. Heat stress also attenuated increases in mRNAs of regulated in development and DNA damage responses 1 (REDD1) and Kruppel‐like factor 15 (KLF15). Heat stress recovered the dexamethasone‐induced inhibition of PI3K/Akt signaling. These data suggest that changes in anabolic and catabolic signals are involved in heat stress‐induced protection against glucocorticoid‐induced muscle atrophy. These results have a potentially broad clinical impact because elevated glucocorticoid levels are implicated in a wide range of diseases associated with muscle wasting. J. Cell. Physiol. 232: 650–664, 2017. © 2016 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.     

Murine glucocorticoid receptors: new evidence for a discrete receptor influenced by H-2

The glucocorticoid receptor contents in the lungs of females of two congenic strains of mice, B10.A (H-2a) and B10 (H-2b), differing only in the H-2 histocompatibility region of chromosome 17, have been measured by the dextran-charcoal method and by our previously described methods of molecular sieving and ion exchange chromatography [M. Katsumata, C. Gupta, and A. S. Goldman (1985) Arch. Biochem. Biophys. 243, 385-395]. As reported, two receptors, II and IB, are demonstrable by each column chromatographic method, and 5,5-diphenylhydantoin binds to receptor IB but not to receptor II. Receptor IB cannot be detected unless molybdate is added in cytosols prepared with hypotonic buffer [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 10 mM dithiothreitol, pH 7.35) according to S. L. Liu, J. F. Grippo, R. P. Erickson, and W. B. Pratt (1984) J. Steroid Biochem. 21, 633-637], a method which has been reported to give maximal receptor levels. Using hypotonic buffer containing 10 mM molybdate we observed a small but significantly higher content of receptor IB in B10.A mice than that in B10 mice, but no significant difference in receptor II or total receptor content. On the other hand, cytosols prepared with isotonic buffer (50 mM Tris-HCl, 120 mM NaCl, 1 mM EDTA, 10 mM dithiothreitol, and 10 mM molybdate, a modification of the buffer used in our previous report) contained significantly higher levels of receptor IB and of total binding in pulmonary cytosols of B10.A as compared to those of B10. There was no difference in receptor II content. Molybdate stabilizes receptor IB in both buffers. These results explain the apparent contradiction between our results and those of Liu et al. by showing that the hypotonic buffer used by them allows for determination of maximal levels of receptor II, but permits selective destruction of receptor IB. However, the use of isotonic buffer gives maximal values of both receptors II and IB. With isotonic buffer, it is demonstrated that only the level of receptor IB is influenced by H-2-linked genes.     

Muscle Atrophy in Response to Cytotoxic Chemotherapy Is Dependent on Intact Glucocorticoid Signaling in Skeletal Muscle

Cancer cachexia is a syndrome of weight loss that results from the selective depletion of skeletal muscle mass and contributes significantly to cancer morbidity and mortality. The driver of skeletal muscle atrophy in cancer cachexia is systemic inflammation arising from both the cancer and cancer treatment. While the importance of tumor derived inflammation is well described, the mechanism by which cytotoxic chemotherapy contributes to cancer cachexia is relatively unexplored. We found that the administration of chemotherapy to mice produces a rapid inflammatory response. This drives activation of the hypothalamic-pituitary-adrenal axis, which increases the circulating level of corticosterone, the predominant endogenous glucocorticoid in rodents. Additionally, chemotherapy administration results in a significant loss of skeletal muscle mass 18 hours after administration with a concurrent induction of genes involved with the ubiquitin proteasome and autophagy lysosome systems. However, in mice lacking glucocorticoid receptor expression in skeletal muscle, chemotherapy-induced muscle atrophy is completely blocked. This demonstrates that cytotoxic chemotherapy elicits significant muscle atrophy driven by the production of endogenous glucocorticoids. Further, it argues that pharmacotherapy targeting the glucocorticoid receptor, given in concert with chemotherapy, is a viable therapeutic strategy in the treatment of cancer cachexia.     

Dosing schedule-dependent attenuation of dexamethasone-induced muscle atrophy in mice

Many inflammatory and autoimmune diseases are treated using synthetic glucocorticoids. However, excessive glucocorticoid can often cause unpredictable effects including muscle atrophy. Endogenous glucocorticoid levels robustly fluctuate in a circadian manner and peak just before the onset of the active phase in both humans and nocturnal rodents. The present study determines whether muscle atrophy induced by exogenous glucocorticoid can be avoided by optimizing dosing times. We administered single daily doses of the glucocorticoid analog dexamethasone (Dex) to mice for 10 days at the times of day corresponding to peak (early night) or trough (early morning) endogenous glucocorticoid levels. Administration at the acrophase of endogenous glucocorticoids significantly attenuated Dex-induced wasting of the gastrocnemius (Ga) and tibialis anterior (TA) muscles that comprise mostly fast-twitch muscle fibers. Real-time RT-PCR revealed that the Dex-induced mRNA expression of genes encoding the atrophy-related ubiquitin ligases Muscle Atrophy F-box (Fbxo32, also known as MAFbx/Atrogin-1) and Muscle RING finger 1 (Trim63, also known as MuRF1) in the Ga and TA muscles was significantly attenuated by Dex when administered during the early night. Dex negligibly affected the weight of the soleus (So) muscle that mostly comprises slow-twitch muscle fibers, but significantly and similarly decreased the weight of the spleen at both dosing times. These results suggest that glucocorticoid-induced muscle atrophy can be attenuated by optimizing the dosing schedule.     

Glucocorticoid receptors in purified subpopulations of human peripheral blood lymphocytes.

On the premise that the differential effects of glucocorticoids on various aspects of the immune response may be mediated by differences in the glucocorticoid receptors in the effector cells, subpopulations of human peripheral blood lymphocytes were examined for these receptors as well as for glucocorticoid responsiveness. Purified T and non-T lymphocytes, when studied by a sensitive whole cell assay technique, contained equivalent amounts of specific glucocorticoid receptor, which, by binding affinity and specificity measurements, were indistinguishable from each other. Furthermore, under in vitro incubation conditions, macromolecular synthesis in both of these cell populations was inhibited by glucocorticoid at concentrations which saturated the receptor sites. It is concluded that the putative differential effects of glucocorticoids on T and non-T lymphocyte-associated functions are probably not mediated by differences in the glucocorticoid receptors in these cell populations.     

Receptor properties of corticosteroid binder IB

Corticosteroid binder IB, present in liver and kidney, is pronounced in liver cytosol after injection of [³H]triamcinolone acetonide. Following injection of the radioactive ligand, livers homogenized in the presence of 20 mm molybdate, 2 mm leupeptin hemisulfate, 2 mm antipain, or 2 mm phenylmethylsufonyl fluoride produce cytosols with Chromatographie profiles of binders II and IB identical to controls, as determined by DEAE-Sephadex chromatography, suggesting that IB is a cellular constituent rather than a product of protease action (sensitive to the above inhibitors) after cell breakage. Generation of IB in kidney cytosols in vitro appears to be unrelated to protease activity. Liver binder IB has an S value of 5–6 and a Stokes radius of about 26 Å producing a calculated range of molecular weight from 40,000 to 50,000 with frictional coefficient and axial ratio close to spherical values. As expected of a steroid receptor, IB, like II, binds to DNA and to liver cell nuclei but IB binds more tightly as evidenced by the fact that KCl is more effective in eluting II than IB from nuclei. Because recovery of bound radioactivity from acceptors is sometimes difficult to achieve, indirect experiments have been used frequently to determine the binding. Pyridoxal phosphate extracts liver IB and II equally from nuclei but spermidine is ineffective. While IB and II can be extracted partially from nuclei by pancreatic DNase I, more binder II is extracted by this method than IB. Micrococcal nuclease is poorly effective in either case. Binder II is extracted to a greater degree from DNA-cellulose than is IB by spermidine, MgCl₂, pyridoxal phosphate, and NaCl. IB binds more extensively to homodeoxypolymers than II. The extent of binding of liver IB to homodeoxypolymers is in the order: poly(dC) ≥ poly(dG) > poly(dA) ? poly(dT), whereas the order for liver binder II is: poly(dG) ≥ poly(dT) > poly(dC) ? poly(dA). Binders IB and II may be separate gene products or IB may arise in the cell from post-translational action. In the latter case, the activity of a protease cannot be ruled out.     

Glial cells express both mineralocorticoid and glucocorticoid receptors.

In the brain, the action of glucocorticoid steroids is mediated via two intracellular receptors, the mineralocorticoid (MR), or type I receptor, and the glucocorticoid (GR), or type II receptor. These receptors are expressed in many types of neurons and are co-expressed in some neurons such as the hippocampal pyramidal cells. Although glucocorticoids are known to affect gliogenesis and glial cell differentiation, the expression of the GR in different types of glial cells throughout the brain has not been thoroughly studied and the expression of the MR in glia not previously reported. Here we review studies suggesting that both receptors are expressed in astrocytes and oligodendrocytes.     

Dependence of dexamethasone-induced Akt/FOXO1 signaling,upregulation of MAFbx,and protein catabolism upon the glucocorticoid receptor

The muscle ubiquitin ligases MAFbx and MuRF1 are upregulated in and promote muscle atrophy. Upregulation of MAFbx and MuRF1 by glucocorticoids has been linked to activation of FOXO1 and FOXO3A resulting from reduced Akt activity. We determined the requirements for the glucocorticoid receptor (GR) in these biological responses in C2C12 cells in which GR expression was knocked down by stable expression of an shRNA. Loss of GR prevented dexamethasone-induced increases in protein catabolism. Loss of GR, or inhibition of ligand binding to GR with RU486, prevented upregulation of MAFbx and MuRF1 by dexamethasone. Loss of GR also prevented dexamethasone-induced decreases in Akt phosphorylation, and increases in the fraction of FOXO1 that was unphosphorylated. The findings establish a requirement for the GR in activating molecular signals that promote muscle protein catabolism.     

Testosterone protects against dexamethasone-induced muscle atrophy, protein degradation and MAFbx upregulation

Administration of glucocorticoids in pharmacological amounts results in muscle atrophy due, in part, to accelerated degradation of muscle proteins by the ubiquitin-proteasome pathway. The ubiquitin ligase MAFbx is upregulated during muscle loss including that caused by glucocorticoids and has been implicated in accelerated muscle protein catabolism during such loss. Testosterone has been found to reverse glucocorticoid-induced muscle loss due to prolonged glucocorticoid administration. Here, we tested the possibility that testosterone would block muscle loss, upregulation of MAFbx, and protein catabolism when begun at the time of glucocorticoid administration. Coadministration of testosterone to male rats blocked dexamethasone-induced reduction in gastrocnemius muscle mass and upregulation of MAFbx mRNA levels. Administration of testosterone together with dexamethasone also prevented glucocorticoid-induced upregulation of MAFbx mRNA levels and protein catabolism in C2C12 myotube expressing the androgen receptor. Half-life of MAFbx was not altered by testosterone, dexamethasone or the combination. Testosterone blocked dexamethasone-induced increases in activity of the human MAFbx promotor. The findings indicate that administration testosterone prevents glucocorticoid-induced muscle atrophy and suggest that this results, in part at least, from reductions in muscle protein catabolism and expression of MAFbx.     

Selective regulation of bone cell apoptosis by translational isoforms of the glucocorticoid receptor

Glucocorticoids are widely used in the treatment of inflammatory and other diseases. However, high-dose or chronic administration often triggers troublesome side effects such as metabolic syndrome and osteoporosis. We recently described that one glucocorticoid receptor gene produces eight translational glucocorticoid receptor isoforms that have distinct gene-regulatory abilities. We show here that specific, but not all, glucocorticoid receptor isoforms induced apoptosis in human osteosarcoma U-2 OS bone cells. Whole human genome microarray analysis revealed that the majority of the glucocorticoid target genes were selectively regulated by specific glucocorticoid receptor isoforms. Real-time PCR experiments confirmed that proapoptotic enzymes necessary for cell death, granzyme A and caspase-6, were induced by specific glucocorticoid receptor isoforms. Chromatin immunoprecipitation assays further suggested that glucocorticoid receptor isoform-dependent induction of proapoptotic genes was likely due to selective coregulator recruitment and chromatin modification. Interestingly, the capabilities to transrepress proinflammatory genes were similar among glucocorticoid receptor isoforms. Together, these findings provide new evidence that translational glucocorticoid receptor isoforms can elicit distinct glucocorticoid responses and may be useful for the development of safe glucocorticoids with reduced side effects.     

Glucocorticoids mediate differential anti-apoptotic effects in human fibroblasts and keratinocytes via sphingosine-1-phosphate formation

Glucocorticoids are potent anti-inflammatory and immunomodulatory drugs which also induce growth inhibition in a variety of cell types. For this reason long-term treatment of inflammatory skin diseases may result in irreversible skin atrophy. To elucidate whether the antiproliferative action of glucocorticoids in fibroblasts is accompanied by induction of apoptosis we investigated the influence of dexamethasone (DEX) on both parameters. Interestingly, we revealed that growth inhibitory concentrations of this glucocorticoid did not induce fibroblast apoptosis. Moreover, DEX protected these cells from apoptosis induced by tumor necrosis factor alpha (TNFalpha)/actinomycin, UV-irradiation, and cell permeable ceramides. These findings are in contrast to the lack of anti-apoptotic effects detected in keratinocytes. Although DEX inhibited TNFalpha mediated nuclear factor-kappa (NF-kappaB) activity in fibroblasts, this mechanism was not involved in its cytoprotection as it was verified by specific NF-kappaB inhibitors. Therefore, we looked for alternative intracellular mediators. Coincubation of fibroblasts with the sphingosine kinase inhibitor N,N-dimethylsphingosine, which blocks formation of the sphingolipid degradation product sphingosine-1-phosphate (S1P), abrogated the protective glucocorticoid effect almost completely. As preincubation with S1P reduced the number of apoptotic cells after stimulation with TNFalpha/actinomycin and moreover DEX increased the intracellular S1P content a role of this sphingolipid in the cytoprotection by DEX is suggested.