Localization Of Glucocorticoid Receptor Research Articles

Immunocytochemical assessment of FKBP51 and Glucocorticoid receptor localization in asthmatic patients

Asthma is a chronic inflammatory disease affecting 5% of the world population. FKBP51 is an important immunophilin modular protein of the glucocorticoid receptor (GC).
 The aim of the present study was to evaluate the levels and immunocytochemical distribution of FKBP51 and GR in lymphocyte cells of asthmatic patients, by use of immunocytochemistry method, and to assesslevels ofstress hormones (cortisol and ACTH) by radioimmuniassay (RIA).
 The results showed significantly increased nuclear localization and decreasedcytoplasmic distribution of FKBP51, while they showed a significant increase in nuclear localization and a non-significant decrease in cytoplasmic distribution of GRin asthmatic patients(P<0.05).
 Cortisol and ACTH levels were also measured and showedinsignificant increases(P<0.05)in steroid treated (338.85 ±139.5 mMol/L, 35.05±3.77 ng/ml, respectively)and non steroid treated asthmatics(280.5 ± 74.6 mMol/L, 32.0±6.43 ng/ml, respectively)as compared with the control group (234.33±29.13 mMol/L, 29.0±7.02 ng/ml, respectively).

Elevated utero/placental GR/NR3C1 is not required for the induction of parturition in the dog.

The endocrine mechanisms that lead to initiation of parturition in dogs are still not fully understood. The prepartum luteolysis is associated with increased prostaglandin (PG) F2α secretion; however, there is no pregnancy- or parturition-related increase in estrogens. Moreover, unlike in other mammalian species, in the dog, increased peripartum levels of cortisol measured sporadically in maternal peripheral blood are not mandatory for normal parturition. Nevertheless, auto/paracrine effects of cortisol at the placental feto-maternal level cannot be excluded. Therefore, the aim of this study was to investigate the expression and localization of glucocorticoid receptor (GR/NR3C1) in canine utero/placental (Ut/Pl) units and uterine interplacental sites at selected time points during pregnancy (pre-implantation, post-implantation and mid-gestation), and at normal and antigestagen-induced parturition. The Ut/Pl expression of GR/NR3C1 did not change significantly from pre-implantation until mid-gestation; however, it was strongly induced during the prepartum luteolysis. Within the interplacental samples, expression of GR/NR3C1-mRNA was greater post-implantation than pre-implantation and did not change afterward, i.e. toward mid-gestation. Compartmentalization studies within the Ut/Pl units, involving placenta, endometrium and myometrium separately, performed at the prepartum luteolysis revealed the highest GR/NR3C1-mRNA levels in placenta compared with endometrium and myometrium. Interestingly, in antigestagen-treated mid-pregnancy dogs, Ut/Pl and interplacental GR/NR3C1-mRNA expression remained unaffected. At the cellular level, placental GR/NR3C1 was clearly detectable in placenta fetalis, i.e. in trophoblast cells. In conclusion, increased expression of GR/NR3C1 during normal parturition, but not following antigestagen-treatment, suggest that it is not required for initiating the signaling cascade of PG synthesis leading to the induction of parturition in the dog.

Localization and Dynamics of Glucocorticoid Receptor at the Plasma Membrane of Activated Mast Cells

In addition to their actions in the cell nucleus, glucocorticoids exhibit rapid non-nuclear responses that are mechanistically not well understood. To explain these effects, the localization of a glucocorticoid receptor (GR) expressed in mast cells as a GFP fusion was analyzed after activation of the cells on allergenic lipid arrays. These arrays were produced on glass slides by dip-pen nanolithography (DPN) and total internal reflection (TIRF) microscopy was used to visualize the GR. A rapid glucocorticoid-independent and -dependent recruitment of the GR-GFP to the plasma cell membrane was observed following contact of the cells with the allergenic array. In addition, the mobility of the GR at the membrane was monitored by fluorescence recovery after photobleaching (FRAP) and shown to follow binding kinetics demonstrating interactions of the receptor with membrane-bound factors. Furthermore the recruitment of the GR to the cell membrane was shown to result in a glucocorticoid-mediated increase in Erk phosphorylation. This is evidenced by findings that destruction of the membrane composition of the mast cells by cholesterol depletion impairs the membrane localization of the GR and subsequent glucocorticoid-mediated enhancement of Erk phosphorylation. These results demonstrate a membrane localization and function of the GR in mast cell signaling.

Glucocorticoid receptor isoforms in human hepatocarcinoma HepG2 and SaOS-2 osteosarcoma cells: Presence of glucocorticoid receptor alpha in mitochondria and of glucocorticoid receptor beta in nucleoli

In the context of a possible direct action of glucocorticosteroids on mitochondrial transcription and/or apoptosis by way of cognate mitochondrial receptors, the possible localization of glucocorticoid receptors α and β (GRα and GRβ) in mitochondria was explored in human hepatocarcinoma HepG2 and osteosarcoma SaOS-2 cells, in which glucocorticoids exert an anabolic and apoptotic effect, respectively. In both cell types, GRα was detected in mitochondria, in nuclei and in cytosol by immunofluorescence labeling and confocal scanning microscopy, by immunogold electron microscopy and by Western blotting. GRβ was shown to be almost exclusively restricted to the nucleus of the two cell types, being particularly concentrated in nucleoli, pointing to a solely nuclear role of this receptor isoform and to a possible function in nucleoli related processes. Computer analysis identified a putative internal mitochondrial targeting sequence within the glucocorticoid receptor. The demonstration of mitochondrially localized GRα in HepG2 and SaOS-2 cells corroborates previous findings in other cell types and further supports a direct role of this receptor in mitochondrial functions.

Localization of glucocorticoid receptors at postsynaptic membranes in the lateral amygdala

Glucocorticoids, released in high concentrations from the adrenal cortex during stressful experiences, bind to glucocorticoid receptors in nuclear and peri-nuclear sites in neuronal somata. Their classically known mode of action is to induce gene promoter receptors to alter gene transcription. Nuclear glucocorticoid receptors are particularly dense in brain regions crucial for memory, including memory of stressful experiences, such as the hippocampus and amygdala. While it has been proposed that glucocorticoids may also act via membrane bound receptors, the existence of the latter remains controversial. Using electron microscopy, we found glucocorticoid receptors localized to non-genomic sites in rat lateral amygdala, glia processes, presynaptic terminals, neuronal dendrites, and dendritic spines including spine organelles and postsynaptic membrane densities. The lateral nucleus of the amygdala is a region specifically implicated in the formation of memories for stressful experiences. These newly observed glucocorticoid receptor immunoreactive sites were in addition to glucocorticoid receptor immunoreactive signals observed using electron and confocal microscopy in lateral amygdala principal neuron and GABA neuron soma and nuclei, cellular domains traditionally associated with glucocorticoid immunoreactivity. In lateral amygdala, glucocorticoid receptors are thus also localized to non-nuclear-membrane translocation sites, particularly dendritic spines, where they show an affinity for postsynaptic membrane densities, and may have a specialized role in modulating synaptic transmission plasticity related to fear and emotional memory.

Immunophilin Directs Bcl Proteins to the Mitochondria

The antiapoptotic proteins Bcl-2 and Bcl-x L block the mitochondrial route to apoptosis by localizing to the mitochondria to prevent the release of proapoptotic proteins that drive apopotosome assembly and caspase activation. But just how Bcl-2 and Bcl-x L are targeted to the mitochondria has not been clear. Shirane and Nakayama show that both Bcl-2 and Bcl-x L associate with a member of the FKBP family of immunophilins called FKBP38. The interaction is required for the mitochondrial localization of both Bcl proteins and, hence, their anti-apoptotic activity. The endogenous proteins also colocalize at the mitochondria. Unlike other immunophilins, FKBP38 does not require association with the immunosuppressant molecule FK506 to inhibit calcineurin, a phosphatase implicated in cell death regulation. Overexpression of FKB38 in mammalian cells promoted mitochondrial localization of Bcl-2 and Bcl-x L and reduced apoptosis in response to various proapoptotic stimuli. Reduction of FKBP38 expression by RNA interference altered the localization of Bcl-2 and Bcl-x L and increased cell sensitivity to apoptosis. Because other immunophilins regulate localization of glucocorticoid receptors to the nucleus, it possible that immunophilins in general mediate intracellular protein transport. M. Shirane, K.I. Nakayama, Inherent calcineurin inhibitor FKBP38 targets Bcl-2 to mitochondria and inhibits apoptosis. Nature Cell Biol. 5 , 1-10 (2003). [Online Journal]

Localization of glucocorticoid receptors in the murine inner ear.

We performed an immunohistochemical investigation of the distribution of glucocorticoid receptors (GRs) in the murine inner ear and found that GRs were expressed extensively, but with various degrees of immunoreactivity in different regions. We observed the strongest GR expression in the type III fibrocytes of the spiral ligament. Although the immunoreactivity of the cochlear hair cells and of the vestibular sensory epithelia was weak, the neighboring cochlear supporting cells and the subepithelial regions of the vestibular sensory epithelia were immunostained. Staining for GRs was also positive in the spiral ganglia and vestibular ganglia, as well as in the endolymphatic sac. The role of GRs in the inner ear is discussed.

Selective apoptosis of luteal endothelial cells in dexamethasone-treated rats leads to ischemic necrosis of luteal tissue.

In infertile cycles in rats, the corpus luteum (CL) ceases producing progesterone in about 2 days and is eliminated by structural luteolysis. Glucocorticoids disrupt the ovarian cycle and interfere with structural luteolysis. We studied the effects of the glucocorticoid dexamethasone (DEX) on rat luteolysis. Cycling rats were treated during 3 days (from estrus to diestrus) with different doses (0.025, 0.1, 0.4, and 1 mg/rat) of DEX or vehicle. DEX-treated rats showed a necrotic pattern of cell death, affecting exclusively the last generation of regressing CLs. In these animals, selective apoptosis of luteal endothelial cells, detected by both morphological characteristics and TUNEL assay, was observed on the morning of proestrus and was followed by necrosis of the luteal tissue. These effects were dose related. With the lowest DEX doses (0.025 and 0.1 mg), only some of the animals were affected and showed smaller necrotic areas in CLs. The deleterious effects of DEX on endothelial cells were in keeping with the immunohistochemical localization of glucocorticoid receptors in the endothelial cells of the last CL generation. The results of this study strongly suggest that DEX-induced selective apoptosis of endothelial cells leads to ischemic necrosis of the luteal tissue and raises the possibility that actions on endothelial cells may be underlying glucocorticoid-induced effects on the ovary.

Heat shock preconditioning and pretreatment with glucocorticoid antagonist RU 486 protect rat myogenic cells H9c2 against glutamate-induced cell death.

We have observed that the treatment of rat-heart derived H9c2 myoblasts for 20 h with the excitatory amino acid glutamate resulted in cell death in a dose dependent manner as determined by LDH release. The optimum cardiotoxicity was seen at 25 mM glutamate. Preconditioning with either sublethal heat shock (42 degrees C for 30 min) or pretreatment with 500 nM of the glucocorticoid antagonist RU 486 for 24 h almost completely protected H9c2 cells against subsequent 20 h treatment with 25 mM lethal glutamate. In addition, we have observed that glutamate treatment resulted in intense nuclear localization of glucocorticoid receptors (GR) in H9c2 cells as judged by the confocal immunofluorescence microscopy. Furthermore, pretreatment with either heat shock or RU 486 followed by glutamate treatment resulted in dramatic decrease in GR nuclear localization which was almost comparable to that observed with control untreated cells. In conclusion, we have shown for the first time using H9c2 cells that (i) protection from glutamate cardiotoxicity occurs with prior treatment with sub lethal heat shock or RU 486 and (ii) these measures down regulate the intense nuclear localization of GR induced by glutamate. The block to GR nuclear localization is likely to be involved in cardioprotective effects offered against glutamate toxicity by pretreatment with heat shock or RU 486.

Immunohistochemical localization of glucocorticoid receptors in the forebrain of the rainbow trout (Oncorhynchus mykiss)

Immunohistochemical localization of glucocorticoid receptors in the forebrain of the rainbow trout (Oncorhynchus mykiss)

Dynamic changes in subcellular localization of glucocorticoid receptor in living cells visualized with a green fluorescent protein

Dynamic changes in subcellular localization of glucocorticoid receptor in living cells visualized with a green fluorescent protein

モルモットか牛におけるグルココルチコイド受容体の局在

モルモットか牛におけるグルココルチコイド受容体の局在

Distribution of glucocorticoid receptor immunoreactivity and mRNA in the rat brain: an immunohistochemical and in situ hybridization study

The localization of glucocorticoid receptor (GR) immunoreactivity and mRNA in the adult rat brain was examined by light microscopic and electron microscopic immunohistochemistries, and in situ hybridization. For the purpose of detailed investigation of the distribution and comparison of GR immunoreactivities and mRNAs, specific polyclonal antibodies against a part of the transcription modulation (TR) domain of rat GR were used in the immunohistochemistry, whereas fluorescein-labeled RNA probes, complementary to the TR domain in the GR cDNA were used in the in situ hybridization. In the rat brain, GR immunoreactivity was predominantly localized in the cell nucleus, and the expression of GR mRNA was detected in the cytoplasm. GR-immunoreactive and GR mRNA-containing cells were widely distributed from the olfactory bulb of the forebrain to the gracile-cuneate nuclei of the medulla oblongata. The highest densities of GR-immunoreactive and mRNA-containing cells were observed in the subfields of cerebral cortex, olfactory cortex, hippocampal formation, amygdala, septal region, dorsal thalamus, hypothalamus, trapezoid body, cerebellar cortex, locus coeruleus and dorsal nucleus raphe. The distributional pattern of GR immunoreactivity in many regions was well-correlated with that of GR mRNA, but in the CA3 and CA4 pyramidal layers of the hippocampus, different localization was noted. The present study provides the groundwork for elucidating the role of GRs in brain function.

Glucocorticoid receptor localization in human epidermal cells

Glucocorticoid receptor localization in human epidermal cells

Immunocytochemical localization of glucocorticoid receptor in rat skin

The aim of the present study was to map immunohistochemically the distribution of the glucocorticoid receptor (GR) in rat skin. Nuclear GR-like immunoreactivity (LI) was found in both epidermis and dermis. In the epidermis, the basal cell layer showed an intense immunoreaction; the lower part of the spinous layer was also labelled. In the dermis, the fibroblasts as well as the sweat glands, sebocytes and adipocytes were GR-immunoreactive (IR). In the root sheath of the hair follicle the staining was most intensive in the outer layer. The endothelial cells comprising the smooth muscle cells of the blood vessels, as well as the arrector pili muscle, showed GR-LI. In the peripheral nerves, the immunoreaction was localized to the nuclei of the Schwann cells and in the perineurial fibroblasts. Mast cells did not show nuclear GR-LI. Based on our immunocytochemical findings that several cell types of the skin are GR-IR, the variable physiological and pharmacological effects of glucocorticoids are easier to understand.

Localization of glucocorticoid receptors and glucocorticoid receptor mRNAs in the rat cochlea.

The distribution of glucocorticoid (GR) receptor messenger RNAs (mRNAs) and GR receptors was studied by in situ hybridization histochemistry and immunocytochemistry, respectively. In situ hybridization histochemistry was performed with a biotin-labeled riboprobe complementary to rat GR receptor mRNA. GR receptor mRNAs were demonstrated in spiral ligament cells, spiral limbus cells, and spiral ganglion cells. GR receptor mRNAs were demonstrated neither in cells of the stria vascularis nor in cells of the organ of Corti region. With the use of a monoclonal and a polyclonal antibody, GR receptors were observed in the spiral ligament cells, stria vascularis cells, spiral limbus cells, and spiral ganglion cells by immunocytochemistry. Binding of anti-GR-receptor antibodies to a lesser extent was observed in the organ of Corti region; however, cellular distribution of the GR receptors could not be resolved with the applied techniques. These results suggest that the GR receptor is expressed differently in the heterogeneous cochlear tissues.

Immunolocalization of gluco- and mineralocorticoid receptors in rabbit kidney

The localization of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) was determined in the rabbit kidney by immunohistochemistry with the use of a monoclonal, anti-GR antibody and a monoclonal, anti-idiotypic, anti-MR antibody. Immunostaining was performed on serial histological sections from normal and adrenalectomized rabbits. The specificity of immunostaining was assessed for MR by in situ competition studies with steroids and for GR by presaturation of the antibody with GR preparation. Immunostaining by both the anti-MR and the anti-GR antibodies was present in all parts of the distal nephron (beyond proximal tubule) and absent in the glomerulus and proximal tubule. The absence of staining by the anti-GR antibody in the proximal tubule suggests that the effects of glucocorticoids in this structure involve either a GR different from that of distal structures or a non-receptor mediated mechanism of action. MR immunostaining predominates in the distal and all along the collecting tubule in its cortical, medullary, and papillary portions. GR immunostaining was most abundant in the medullary ascending limb and distal tubule. Immunostaining by both antibodies was present in papillary interstitial cells and cells of the epithelium lining the papilla. Fifteen to twenty percent of the cells of the cortical collecting tubule, presumably intercalated cells, were devoid of MR and GR immunostaining. Immunostaining was present in both nuclear and cytoplasmic cell compartments. No clear difference was observed between normal and adrenalectomized rabbits. This study is the first report on renal immunolocalization of GR compared with MR. In addition, we show evidence for new targets for corticosteroid hormones such as papillary interstitial cells and papillary epithelium.

Localization of glucocorticoid receptor in neuropeptide Y-containing neurons in the arcuate nucleus of the rat hypothalamus

Glucocorticoid receptor (GR) was localized immunohistochemically in nuclei of neurons in the arcuate nucleus of the rat hypothalamus. The double immunostaining method further revealed that about half of the GR-positive neurons in the arcuate nucleus were also immunoreactive for neuropeptide Y (NPY).