Receptor Signal Transduction Protocols: Third Edition

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We used a 35 S-labelled anti-sense riboprobe for the TSH receptor as described previously Herwig et al. Following hybridisation, sections were apposed to film for 7 days. Adenylate cyclase activation by TSH was performed in triplicate and in two independent experiments.

Each treatment was performed in duplicate or triplicate, and the assay was performed twice. Protein detection was accomplished using a standard western blot protocol. Brain explants were dissected as described in the previous section for primary cell cultures. The resulting PCR amplification products were separated through 2. Total RNA was extracted and reverse transcribed as described in the previous paragraph.

Each PCR procedure included a negative control reaction without template, and each sample was run in triplicate with the experiment being performed twice. In situ hybridisation confirmed that TSH receptor expression in the day-old rat brains was localised in the hypothalamus exclusively to the ependymal cell layer lining the wall of the third ventricle Fig. A In situ hybridisation for TSH receptors on a brain section of a old-rat shows a high level of expression in the ventral region of the rat hypothalamus originating from cells that constitute mainly tanycytes arrowheads.

Signal Transduction Protocols, 3rd Edition

B and C Immunohistochemistry for vimentin B and GFAP C on formaldehyde-fixed tissue at a ventral location of the hypothalamus in the region of the arcuate nucleus. Citation: Journal of Molecular Endocrinology 54, 3; Immunocytochemical staining of the day-old rat brain hypothalamic sections with anti-vimentin antibody showed that this type 3 intermediate filament was localised to cell soma in the ependymal wall and to processes that extended into the surrounding neuropil Fig.

Similarly, staining with an anti-GFAP antibody was observed in tanycytes cells of the sub-ependymal regions with a morphological appearance characteristic of astrocytes Fig. In primary cell cultures derived from the micro-dissected ependymal wall, all of the cells were found to express vimentin Fig. In comparison, cultures derived from the brain cortex extensively expressed both vimentin and GFAP markers Fig.

Vimentin immunocytochemistry on primary ependymal cell cultures A or primary cortical cultures D. Primary cell cultures were assessed and compared to tissue explants for the expression of genes known to be mostly localised to tanycytes: Dio2 , Gpr50 , Vimentin , Darpp and the TSH receptor Fig. Transcripts for all of these genes were detected in both primary cell cultures and explants. Figure 3 Download Figure Download figure as PowerPoint slide Expression of tanycyte markers in primary ependymal cell cultures.

Glyceraldehydephosphate dehydrogenase G3pdh was used as a housekeeping gene.

Receptor Signal Transduction Protocols - Third Edition | Gary B. Willars | Springer

RNA isolated from the pars distalis PD was used as a positive control. NS, not significant. Shown is one representative experiment of two independent experiments for each assay with treatments in duplicate or triplicate. Treatments were performed in triplicate in two independent experiments. To investigate the signal transduction mechanism of the TSH receptor in the hypothalamic ependymal layer, we chose to utilise primary cell cultures of these cells from day-old rat brains, as described by Prevot et al. First, as found in other species, Tsh receptor mRNA expression was confirmed by in situ hybridisation in the hypothalamus of day-old rats, confined to the cells adjacent to the third ventricle Hanon et al.

Using primary ependymal layer cell cultures prepared from day-old rats, we investigated the signal transduction mechanism used by TSH receptors to transduce hormone binding in these cells. The cultures showed characteristics of tanycyte cells with the expression of mRNAs that are mostly restricted to tanycytes of the third ventricle, such as Gpr50 , Darpp , Dio2 and Tshr Ma et al. Stimulation of the tanycyte primary cell cultures with TSH produced a dose-dependent increase in secreted cAMP levels.

The functionality of the TSH receptor signalling was also evident, with a 2. However, the functional significance of this potential promiscuity of G protein coupling is not understood, and the outcome of TSH receptor activation is likely to be dependent on the cell type expressing the receptor and the repertoire of available G proteins.

The relevance of a bifurcation of TSH receptor signalling in tanycytes is unknown. Furthermore, whether all tanycytes have a dual signalling pathway or whether there may be regionalisation in this capacity based on tanycyte subtype distribution in the third ventricle Rodriguez et al.

A specific mineralocorticoid response element has not been identified. Receptor specificity is also demonstrated by specific agonists and antagonists. The synthetic GR agonist Dex, which with prolonged use can cause a steroid-induced cataract, 36 binds the MR with high affinity, but it does not activate the MR with the same effectiveness as it does the GR. Our results Fig. The hypophosphorylated GR undergoes hyperphosphorylation at multiple serine residues on hormone binding. Seven phosphorylated residues have been identified, mostly serines, clustered in the N-terminal domain.

The hormone-dependent phosphorylation of the GR at S and S in hLECs is similar to what is seen in other cell types, suggesting that the lens GR is functionally activated on hormone treatment. Posterior subcapsular cataracts are detected in patients after many months to years of steroid treatment.

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No changes in hLEC proliferation were observed during the same period Fig. Although we could not identify changes in these cellular functions, we were able to identify a transcriptional response as early as 2 to 4 hours, demonstrating an acute GC response in hLECs, despite the lack of detectable physiological changes. Although genes were modulated after an acute 4—16 hours Dex treatment, they could not be coordinately related to a change in lens physiology, consistent with our findings that Dex did not induce LEC proliferation or apoptosis.

Instead, changes in signaling were suggested. The pathway analyzer Pathway Assist; Ariadne Genomics 31 was used to analyze previously reported microarray data from 4 and 16 hour GC treatment to understand the resultant biological effect of Dex-induced changes in LEC gene expression.

The predictions are not absolutely precise, but they do identify trends in gene expression and allow for the study of signaling networks as opposed to individual genes. We chose to start the analysis with DSIP because microarray studies revealed that its expression was more than fivefold expression at 4 hours 14 and more than twofold expression as late as 48 hours. The pathway analyzer Pathway Assist; Ariadne Genomics revealed that nearly 30 genes identified by microarray were connected to regulation of the MAPK pathway.

Predictions were confirmed by real-time PCR and Western blot analysis. Primary cultures were used to examine protein expression because lens organ cultures did not yield sufficient protein amounts and we were limited by the number of donor specimens. The absence of an effect on p38 at 16 hours may be due to the ethanol vehicle inhibiting the expression of phospho-p38 in all samples. Ethanol has been reported to inhibit the activation of p38 in human mononuclear cells, but the mechanism is not understood.

Dex did not inhibit phospho-JNK expression Fig. Dex has been reported to activate or inhibit the MAPKs differentially, depending on the cell type and conditions. The finding of nucleated epithelial cells in the posterior region of the lens suggests that GCs play a role in LEC proliferation, differentiation, apoptosis, survival, or migration. It has been suggested that these signals are not transmitted in a linear fashion from one point to the next, but through a combination of signals that converge on a final target or cellular function.

Instead, the inhibition of both of these pathways may be a redundant effect to inhibit a final cellular target or physiological function, such as inhibition of cell survival. We have now identified a GC signaling pathway in LECs but have not been able to identify a concomitant physiological response. Although we have identified an acute transcriptional GC response, researchers have been able to identify cellular changes in hLECs only after prolonged or chronic high-dosage GC treatment.

The final physiological response may be due to the background of other effectors, hormones, and growth factors present in the environment after prolonged treatment. It is interesting to note that while activated ERK and AKT play roles in cell survival, activated p38 plays a role in apoptosis. Decreased phospho-p38 expression may be a mechanism to compensate for the inhibition of cell survival. This demonstrates the complexity of the lens GC response and may account for the inability to identify an acute physiological GC response. We predict that long-term GC treatment would lead to more dramatic changes in gene expression, which then can lead to prolonged modulation of these important signaling pathways leading to increased proliferation, decreased differentiation, increased migration, decreased apoptosis, or increased cell survival, all of which can account for the finding of nucleated epithelial cells in the posterior region of the lens.

Animal models have not been useful thus far in understanding the formation of a steroid cataract in humans. The chick is a common model used to study a lens GC response, but prolonged GC treatment results in a nuclear cataract, which differs from the posterior subcapsular cataract found in humans. In , a posterior subcapsular cataract was observed in rabbits treated with GC for 72 hours, but since that time, it has not been repeated. Ideally, these results would have been repeated in human posterior subcapsular cataract specimens, but available specimens are limited.

The current studies are being repeated in primary hLEC cultures, and preliminary studies have demonstrated a decrease in phospho-RAF, but not in RAF, expression with Dex treatment, similar to that observed in the B-3 cells. This suggests that the B-3 cells can serve as a model to examine before repeating the results in primary cultures. Although a direct link to posterior subcapsular cataract remains elusive, this is the first demonstration of a lens GC signaling pathway.

In relationship to a mechanism of cataractogenesis it is interesting to note that prolonged high-dosage Dex treatment of rat lenses resulted in migration of epithelial cells to the posterior region of the lens and decreased E-cadherin expression. This novel insight is an essential first step in understanding the mechanism of a lens GC response and cataractogenesis, and the foundation for a rational approach to the treatment and prevention of a steroid-induced cataract. Submitted for publication July 31, ; revised November 20, ; accepted January 25, Disclosure: V. Gupta , None; N.

Awasthi , None; B. Wagner , None. The publication costs of this article were defrayed in part by page charge payment. F igure 1. View Original Download Slide. Dex bound to and activated the GR, but not the MR, to induce transcription. Error bars, SE. A HLE B-3 cells. B Primary cultures. Two experiments were performed on cultured cells from two donor pairs. F igure 2. F igure 3. Dex had no effect on HLE B-3 cell apoptosis or proliferation.

A flow cytometry of vehicle- and Dex-treated cells. Error bars, SD. F igure 4. Pathway analysis of DSIP. Pathway analysis was performed with Pathway Assist 2. Pathway Assist generated a pathway around DSIP and its interactions with other gene products from the microarray and the current literature database.

Proteins are depicted as pink ovals , whereas proteins identified by microarray are depicted as pink ovals with blue outlines. Gray lines : interaction through regulation. F igure 5. Experiment was repeated with a second preparation of RNA and yielded similar results. Vehicle- 32 hours and Dex-treated samples were electrophoresed on agarose gels.

F igure 6. Each time point was examined two to three times, each in triplicate, with similar results.

F igure 7. Each time point was examined three times in triplicate. Dex-mediated decreases were inhibited by GR antagonist, RU D There was no change in phospho-JNK expression with treatment. F igure 8. Ocular complications of topical, peri-ocular, and systemic corticosteroids. Curr Opin Ophthalmol. The association of cataract formation and systemic corticosteroid therapy.

Int Ophthalmol Clin. Charge heterogeneity in wildtype and variant glucocorticoid receptors. Mol Cell Endocrinol. Autoregulation of glucocorticoid receptor gene expression. Glucocorticoid receptor interactions with glucocorticoids: evaluation by molecular modeling and functional analysis of glucocorticoid receptor mutants.

The structure of the human glucocorticoid receptor and its gene. J Steroid Biochem. Glucocorticoid receptors. Monogr Endocrinol. Invest Ophthalmol Vis Sci. Mol Cell Endocr. Receptors for glucocorticoids in the lens epithelium of the calf. Is there a glucocorticoid receptor in the bovine lens?.


Exp Eye Res. GuptaV, WagnerBJ. Expression of the functional glucocorticoid receptor in mouse and human lens epithelial cells. Global gene profiling reveals novel glucocorticoid induced changes in gene expression of human lens epithelial cells. Mol Vis. The effects of steroids on the human lens epithelium. What causes steroid cataracts? Clin Exp Optom. Mechanism of action of glucocorticoids. Aldosterone: its receptor, target genes, and actions. Vitam Horm. The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response.

Transactivation and synergistic properties of the mineralocorticoid receptor: relationship to the glucocorticoid receptor. Mol Endocrinol. PI3K-Akt pathway: its functions and alterations in human cancer. The role of phosphatidylinositol 3-kinase in the regulation of cell response to steroid hormones.


Biochim Biophys Acta. RouxPP, BlenisJ. Microbiol Mol Biol Rev. ClarkAR, LasaM. Crosstalk between glucocorticoids and mitogen-activated protein kinase signalling pathways. Curr Opin Pharmacol. Propagation and immortalization of human lens epithelial cells in culture. Glucocorticoids induce rapid up-regulation of mitogen-activated protein kinase phosphatase-1 and dephosphorylation of extracellular signal-regulated kinase and impair proliferation in human and mouse osteoblast cell lines. AwasthiN, WagnerBJ.

Differences in the determinants of eplerenone, spironolactone and aldosterone binding to the mineralocorticoid receptor. Clin Exp Pharmacol Physiol. Inhibition of glucocorticosteroid action in cultured L mouse fibroblasts by RU , a new anti-glucocorticosteroid of high affinity for the glucocorticosteroid receptor. Exp Cell Res. Bioinformatics: from genes to pathways. Nat Methods. Glucocorticoid-induced changes in the global gene expression of lens epithelial cells.

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J Ocul Pharmacol Ther. J Biol Chem. Glucocorticoid-induced leucine zipper inhibits the Raf-extracellular signal-regulated kinase pathway by binding to Raf Mol Cell Biol. Glucocorticoid receptor interaction with 14—3—3 and Raf-1, a proposed mechanism for cross-talk of two signal transduction pathways. MohanR, MuralidharanAR. Steroid induced glaucoma and cataract.