PMID: 9176246;Abstract:
Glucocorticoids are an effective anti-inflammatory therapy for the treatment of asthma. The anti-inflammatory effects of glucocorticoids may be due to the inhibition of transcription factors that regulate cytokine synthesis. Because of the potential role of the bronchial epithelium in asthmatic inflammation and the possibility that this cell may be the main target of inhaled glucocorticoids, we have characterized glucocorticoid receptors (GR) and GR signaling in the human bronchial epithelial cell line BEAS-2B. Western blot analysis and radioligand binding studies demonstrated that BEAS-2B cells have functional GR that bind to dexamethasone (Dex) (dissociation constant = 5.6 nM and maximal density of binding sites = 228 ± 3.3 fmol/mg protein). GR were activated by Dex as assessed using a glucocorticoid-responsive reporter plasmid. Transfection of BEAS-2B cells with an activator protein-1 (AP-1) reporter construct followed by 12-O- tetradecanoylphorbol-13-acetate (TPA) treatment resulted in a fivefold induction of reporter gene activity. Transfection with a nuclear factor (NF)- κB reporter construct followed by tumor necrosis factor-α (TNF-α) treatment resulted in a 10-fold induction of reporter gene activity. Dex (10-7 M) markedly repressed both the induced AP-1 and NF-κB activity. The GR antagonist RU-486 inhibited the repressive effect of Dex on TNF-α- induced NF-κB activity by 81% but only counteracted the repressive effect of Dex on TPA-induced AP-1 activity by 43%. These studies demonstrate that cross-signaling between AP-1 and NF-κB with GR may explain the anti- inflammatory properties of glucocorticoids in airway epithelial cells.
PMID: 17693064;PMCID: PMC2683348;Abstract:
Glucocorticoid-induced gene-1 (Gig1) was identified in a yeast one-hybrid screen for factors that interact with the MyoD core enhancer. The Gig1 gene encodes a novel C2H2 zinc finger protein that shares a high degree of sequence similarity with two known DNA binding proteins in humans, Glut4 enhancer factor and papillomavirus binding factor (PBF). The mouse ortholog of PBF was also isolated in the screen. The DNA binding domain of Gig1, which contains TCF-E-tail CR1 and CR2 motifs shown to mediate promoter specificity of TCF-E-tail isoforms, was mapped to a C-terminal domain that is highly conserved in Glut4 enhancer factor and PBF. In mouse embryos, in situ hybridization revealed a restricted pattern of expression of Gig1 that overlaps with MyoD expression. A nuclear-localized lacZ knockin null allele of Gig1 was produced to study Gig1 expression with greater resolution and to assess Gig1 functions. X-gal staining of Gig1nlacZ heterozygous embryos revealed Gig1 expression in myotomal myocytes, skeletal muscle precursors in the limb, and in nascent muscle fibers of the body wall, head and neck, and limbs through E14.5 (latest stage examined). Gig1 was also expressed in a subset of Scleraxis-positive tendon precursors/rudiments of the limbs, but not in the earliest tendon precursors of the somite (syndetome) defined by Scleraxis expression. Additional regions of Gig1 expression included the apical ectodermal ridge, neural tube roof plate and floor plate, apparent motor neurons in the ventral neural tube, otic vesicles, notochord, and several other tissues representing all three germ layers. Gig1 expression was particularly well represented in epithelial tissues and in a number of cells/tissues of neural crest origin. Expression of both the endogenous MyoD gene and a reporter gene driven by MyoD regulatory elements was similar in wild-type and homozygous null Gig1nlacZ embryos, and mutant mice were viable and fertile, indicating that the functions of Gig1 are redundant with other factors. © 2007 Elsevier Ireland Ltd. All rights reserved.
PMID: 18708143;PMCID: PMC2570706;Abstract:
Blood meal digestion in mosquitoes occurs in two phases, an early phase that is translationally regulated, and a late phase that is transcriptionally regulated. Early trypsin is a well-characterized serine endoprotease that is representative of other early phase proteases in the midgut that are only synthesized after feeding. Since the kinase Target of Rapamycin (TOR) has been implicated as a nutrient sensor in other systems, including the mosquito fat body, we tested if TOR signaling is involved in early trypsin protein synthesis in the mosquito midgut in response to feeding. We found that ingestion of an amino acid meal by female mosquitoes induces early trypsin protein synthesis, coincident with phosphorylation of two known TOR target proteins, p70S6 kinase (S6K) and the translational repressor 4E-Binding Protein (4E-BP). Moreover, in vitro culturing of midguts from unfed mosquitoes led to amino acid-dependent phosphorylation of S6K and 4E-BP which could be blocked by treatment with rapamycin, a TOR-specific inhibitor. Lastly, by injecting mosquitoes with TOR double stranded RNA (dsRNA) or rapamycin, we demonstrated that TOR signaling was required in vivo for both phosphorylation of S6K and 4E-BP in the midgut, and for translation of early trypsin mRNA in response to amino acid feeding. It may be possible to target the TOR signaling pathway in the midgut to inhibit blood meal digestion, and thereby, decrease fecundity and the spread of mosquito borne diseases. © 2008 Elsevier Ltd. All rights reserved.
PMID: 3343225;Abstract:
The effect of glucocorticoids on the regulation of glucocorticoid receptor mRNA was studied in two different cell lines, human IM-9 lymphocytes and rat pancreatic acinar AR42J cells. Using a glucocorticoid receptor cDNA probe, glucocorticoid receptor mRNA was examined by Northern blot hybridization and quantitated by slot-blot hybridization. In IM-9 and AR42J cells, dexamethasone decreased steady-state glucocorticoid receptor mRNA levels to approximately 50% of control. This decrease occurred with a one-half time of 3 h for IM-9 cells and 6 h for AR42J cells. Dexamethasone was the most potent steroid tested with a one-half maximal effect occurring at 10 nM and a maximal effect occurring at 100 nM. Glucocorticoid receptor mRNA half-life and gene transcription were then studied to determine the mechanism of decreased mRNA levels. The glucocorticoid mRNA half-life was approximately 120 min in IM-9 cells and 240 min in AR42J cells; these rates were not affected by dexamethasone treatment. In contrast, the rate of glucocorticoid gene transcription as measured by run-on assays in IM-9 cells was decreased to 50 ± 6% of control by dexamethasone. These results indicate therefore that glucocorticoids regulate glucocorticoid receptor mRNA levels by influencing gene transcription.
PMID: 2651007;Abstract:
This review has highlighted several topics in the study of steroid hormone action. The unanswered questions regarding the mechanism of ligand-controlled LRF activity, the extent of evolutionary conservation and specificity of DNA binding, and the validity of various models of transcriptional regulation mediated through gene networks point to the future direction of research in this field. Steroid hormones are used extensively in clinical treatments, especially glucocorticoids. Our laboratory is attempting to determine which gene networks are responsible for some of these clinical phenotypes. Figure 5 points out that the study of glucocorticoid action holds a unique position because it spans both the basic sciences and the field of applied molecular biology. Now that we have a fundamental knowledge of the necessary elements required for steroid-dependent regulation of gene expression, we can better investigate the clinical responses to steroid therapy (which include devastating side effects) by isolating and characterizing the important target gene(s). In this author's opinion, future directions in the study of steroid responsiveness will have to include a systematic approach toward deciphering a variety of these LRF-regulated gene networks in experimentally feasible systems. Hopefully, work in this area may be revealing and perhaps beneficial to ongoing clinical studies. In addition, the study of mechanisms of transcriptional induction and repression, using the model system of LRFs, could be applicable to many gene regulatory systems which are controlled by such processes as development and differentiation.
