Regulated expression of microinjected DNA in adult Aedes aegypti mosquitoes.

We have developed a novel molecular genetic approach to investigating gene regulation in adult mosquitoes called whole body transfection (WBT). This DNA microinjection method allows for both constitutive and regulated expression of plasmid vectors in the fat body and midgut of adult mosquitoes within 24 h of injection. Using a luciferase reporter gene containing the Aedes aegypti heat shock protein 70 (Hsp70) promoter, we optimized the WBT protocol at various times post-injection and used these parameters to measure the expression of a vitellogenin-luciferase reporter gene in response to blood meal feeding. These studies showed that a 843 bp fragment of the Ae. aegypti vitellogenin-C (VgC) promoter caused a greater than 200-fold induction of luciferase activity in a strict tissue-specific manner, and only in response to feeding. Functional mapping of the VgC promoter by WBT identified essential upstream regulatory elements in the region spanning -780 to -182 bp from the transcriptional start site. We also constructed a lipopolysaccharide-regulated expression vector using a 1096 bp genomic fragment of the Ae. aegypti cecropin B (CecB) promoter. Our data show that four days after WBT injection, the CecB-luciferase reporter gene could be induced more than 100-fold in the fat body following lipopolysaccharide injection. Moreover, we found that lipopolysaccharide-induction of the CecB reporter gene occurred up to 28 days post-WBT injection. These data suggest that WBT could provide a novel strategy to express recombinant proteins and RNAi constructs in adult mosquitoes using conventional microinjection methods.

Differential control of eosinophil survival by glucocorticoids.

Glucocorticoids are effective drugs for eosinophil-related disorders, such as asthma and allergy. Previous studies have demonstrated that glucocorticoids increase eosinophil apoptosis and block the survival effect of submaximal concentrations of interleukin-5 (IL-5). We investigated the effect of glucocorticoids on eosinophil survival in the presence of a higher concentration of IL-5 (1 ng/ml), comparable to IL-5 levels in bronchoalveolar lavage and sputum specimens from patients with asthma. In contrast to incubation in the presence of submaximal concentrations of IL-5, the addition of dexamethasone (DEX) to media containing 1 ng/ml IL-5 led to a significant increase in eosinophil cell viability from 58 +/- 6.9% to 87 +/- 2.4% ( p < 0.005) after 72 hours in culture. We found that RU486 blocked the DEX effect on cell viability confirming that glucocorticoid receptor functions are required. We investigated the possibility that the glucocorticoid enhancement of eosinophil survival may be due to an effect on IL-5 receptor expression. Our results show that the IL-5 associated decrease in IL-5 receptor alpha-subunit expression was blocked significantly after 24 hrs in culture with media containing IL-5 plus DEX compared to IL-5 alone. It is tempting to speculate that the observed glucocorticoid enhancement of eosinophil survival in the presence of elevated concentrations of IL-5 could be a mechanism that contributes to glucocorticoid resistance in asthma.

Analysis of glucocorticoid and androgen receptor gene fusions delineates domains required for transcriptional specificity.

Androgen receptor (AR) and glucocorticoid receptor (GR) influence distinct physiologic responses in steroid-responsive cells despite their shared ability to selectively bind in vitro to the same canonical DNA sequence (TGTTCT). While the DNA-binding domains (DBDs) of these receptors are highly conserved, the amino N-terminal domain (NTD) and hormone-binding domain (HBD) are evolutionarily divergent. To determine the relative contribution of these functional domains to steroid-specific effects in vivo, we constructed a panel of AR/GR gene fusions by interchanging the NTD, DBD, and HBD regions of each receptor and measured transcriptional regulatory activities in transfected kidney and prostate cell lines. We found that GR was approximately 10-fold more active than AR when tested with the mouse mammary tumor virus promoter, and that this difference in activity was primarily owing to sequence divergence in the NTDs. We also tested transcriptional activation of the androgen-dependent rat probasin promoter, and in this case, AR was at least twofold more active than GR. Analysis of the chimeric receptors revealed that this difference mapped to the DBD region of the two receptors. Transcriptional repression functions of the wild-type and chimeric receptors were measured using an activator protein 1 (AP-1) transrepression assay and identified the GR HBD as a more potent transrepressor of AP-1 transcriptional activation than the AR HBD. Taken together, our analyses reveal that evolutionary sequence divergence between AR and GR functional domains results in unique promoter-specific activities within biologic systems in which both AR and GR are normally expressed.

Characterization of Apt- cell lines exhibiting cross-resistance to glucocorticoid- and Fas-mediated apoptosis.

Apoptosis induction by staurosporine, ceramide, and Fas stimulation was investigated in the mouse thymoma cell line W7.2 and a panel of dexamethasone (dex)-resistant W7.2 mutant cell lines, Apt3.8, Apt4.8 and Apt5.8, and a Bcl-2 transfected W7.2 cell line (Wbcl2). While W7. 2 cells were found to be sensitive to these apoptosis inducers, the Apt- mutants and Wbcl2 cells were shown to be resistant to some or all of the treatments. Specifically, all three Apt- mutants and Wbcl2 cells were found to be resistant to ceramide and Fas-mediated apoptosis, whereas, Apt4.8 and Apt5.8 were sensitive to staurosporine-induced apoptosis under conditions in which Apt3.8 and Wbcl2 cells were resistant. Measurements of caspase activity and cytochrome c release in cytosolic extracts of dex and staurosporine-treated cells indicated that the recessive Apt- mutations effect steps upstream of mitochondrial dysfunction. Steady-state RNA levels of apoptosis-associated gene transcripts showed that the observed differential resistance of the Apt- cell lines could not be explained by altered expression of numerous Bcl-2 or Fas related genes. Transient transfection of human Fas gene coding sequences into the Apt- mutants and Wbcl2 cells did not induce apoptosis, even though these same cell lines were sensitive to ectopic expression of the FADD and caspase 8 genes. Taken together, these data provide genetic evidence for the existence of shared components in the dex- and Fas-mediated apoptotic pathways in W7.2 cells.

Glucocorticoid regulation of GM-CSF: evidence for transcriptional mechanisms in airway epithelial cells.

Inflammation plays a central role in the pathogenesis of asthma. Glucocorticoids are first-line anti-inflammatory therapy in the treatment of asthma and are effective inhibitors of inflammatory cytokines. Clinical data demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF) production by airway epithelial cells may be an important target of inhaled glucocorticoid therapy. We examined the regulatory mechanisms of GM-CSF expression by interleukin-1beta (IL-1beta) and the synthetic glucocorticoid dexamethasone in the BEAS-2B human bronchial epithelial cell line. IL-1beta stimulation resulted in a 15-fold induction of GM-CSF protein, which was associated with a corresponding 47-fold maximal induction of GM-CSF mRNA levels. Treatment with the transcriptional inhibitor actinomycin D before IL-1beta stimulation completely abolished induction of GM-CSF mRNA, whereas incubation with cycloheximide had no effect. Taken together, these data demonstrate that IL-1beta induction of GM-CSF is mediated through transcriptional mechanisms. Dexamethasone treatment of BEAS-2B cells produced an 80% inhibition of IL-1beta-induced GM-CSF protein and a 51% inhibition of GM-CSF mRNA. GM-CSF mRNA was rapidly degraded in these cells, and dexamethasone treatment did not significantly affect this decay rate. We conclude that, in the BEAS-2B bronchial epithelial cell line, IL-1beta induction and dexamethasone repression of GM-CSF expression are mediated predominantly through transcriptional mechanisms.

Glucocorticoid receptor signaling in a bronchial epithelial cell line.

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)-kappa B reporter construct followed by tumor necrosis factor-alpha (TNF-alpha) treatment resulted in a 10-fold induction of reporter gene activity. Dex (10(-7) M) markedly repressed both the induced AP-1 and NF-kappa B activity. The GR antagonist RU-486 inhibited the repressive effect of Dex on TNF-alpha-induced NF-kappa B 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-kappa B with GR may explain the anti-inflammatory properties of glucocorticoids in airway epithelial cells.

Crosstalk during Ca2+-, cAMP-, and glucocorticoid-induced gene expression in lymphocytes.

In the WEHI7.2 thymoma cell line, cAMP, glucocorticoids, or increases in cytosolic Ca2+ concentration lead to cell death by apoptosis. In the present study, we examined the effects of these compounds on cAMP response element (CRE)-mediated gene expression. Thapsigargin and A23187 were employed to increase cytosolic Ca2+ levels and induce apoptosis. Both compounds enhanced transcription from a CRE preceding apoptotic death. Moreover, the transcriptional response to the combination of forskolin and either thapsigargin or A23187 was synergistic mirroring the effect on cell death. Importantly, dexamethasone treatment, which causes an efflux of Ca2+ from the ER, induced transcription from a CRE alone or in synergy with forskolin. The increase in CRE-controlled gene expression correlated with a decrease in cell viability. Following treatment with forskolin, thapsigargin, or dexamethasone, the CRE binding protein (CREB) was phosphorylated at levels correlating with the level of induced gene expression. These data suggest that transcriptional crosstalk between independent signaling pathways occurs in lymphocytes, and CREB may play a central role in the mediation of CRE-dependent transcription by these diverse set of apoptotic agents.

Delineation of two distinct type 1 activation functions in the androgen receptor amino-terminal domain.

Based on the finding that some transcription factors contain multiple transcriptional regulatory activities, we constructed a panel of rat androgen receptor (AR) mutants containing small internal deletions and point mutations within the amino-terminal region of the receptor. Trans-activation assays in CV-1 cells using AR-responsive reporter genes were performed and led to the identification of two noncontiguous trans-activation regions in the AR amino terminus. One of these regions, termed activator function 1a (AF-1a) is a highly-conserved 14-amino acid segment that is predicted to form a beta-turn followed by an acidic amphipathic alpha-helix. Point mutagenesis within AF-1a revealed that two adjacent hydrophobic residues were required for full AR trans-activation function, as arginine substitutions resulted in a 60% reduction in transcriptional activity. A second amino-terminal region was also identified and has been designated AF-1b. Deletion of the 65-amino acid AF-1b segment, which contains numerous glutamate and aspartate residues, caused a 55% decrease in trans-activation function. An AF-1a/AF-1b double mutant retains less than 10% trans-activation function compared with wild-type AR, suggesting that AF-1a and AF-1b may each contribute separately to maximal AR activity. To determine whether AF-1a and AF-1b play a role in AR-mediated trans-repression of AP-1 function, we tested single and double AF-1a/AF-1b mutants in a transient trans-repression assay. Our results showed that neither AF-1a nor AF-1b was required for AP-1 trans-repression, demonstrating that AR-mediated trans-repression and trans-activation are discrete functions.

Transcriptional control of steroid-regulated apoptosis in murine thymoma cells.

Early studies in murine T cell lines indicated that transcriptional transactivation functions encoded in the glucocorticoid receptor (GR) N-terminal domain are required for glucocorticoid-mediated apoptosis. However, more recent studies in human T cell lines have suggested that the N-terminal domain is not necessary for steroid-regulated apoptosis and that GR-mediated transrepression may be the more critical mechanism. To better understand the contribution of the GR N-terminal transactivation domain in mediating murine thymocyte apoptosis, we stably transfected GR, GR variants, and the androgen receptor (AR) into receptor-negative S49 murine thymoma cells. GR expression levels were shown to be rate-limiting for initiating the apoptotic pathway, and a positive correlation between steroid sensitivity and GR-mediated induction of an integrated mouse mammary tumor virus (MMTV) LTR reporter gene was observed. Analysis of GR chimeric receptors containing the potent VP16 and E1A viral transactivation domains in place of the GR N terminus revealed that even low level expression of these receptors resulted in both enhanced steroid sensitivity and MMTV induction, thus supporting a role for transactivation in apoptosis. In contrast, we found that AR can initiate apoptosis in S49 cells after treatment with 5 alpha-dihydrotestosterone, despite its relative inability to induce high level expression of MMTV. To investigate this further, we examined the steroid-regulated expression of an endogenous thymocyte-specific gene called GIG18. We found that GIG18 was rapidly induced to comparable levels by both AR and GR, demonstrating that AR can indeed function as a transcriptional activator in S49 cells and, moreover, that GIG18 induction may be a marker of early apoptotic events in steroid-treated cells. Taken together, these results support our conclusion that transcriptional transactivation is a necessary signaling component of S49 cell apoptosis, although an additional role for GR-mediated transrepression cannot be excluded.

A cell-specific and selective effect on transactivation by the androgen receptor.

The androgen (AR) and glucocorticoid receptors (GR) are related ligand-activated transcriptional regulators which bind the same cis-acting element and are coexpressed in a variety of cell types. Despite a shared DNA binding site, these receptors mediate diverse cellular responses. To explain this paradox, the existence of cell-specific factors that interact with, and modulate the function of, distinct receptors has been proposed. Prostate epithelial cell growth is sensitive to androgens, but is not affected by glucocorticoids, even though both AR and GR are expressed in these cells. We have recently isolated a unique panel of prostate epithelial cell lines from normal rats and have used these cell lines to examine cell-specific steroid responses. In this study, we compared the abilities of AR and GR to enhance transcription of several different reporter genes regulated by simple (i.e., noncompsite) hormone response elements (HREs) in prostate and nonprostate cell lines. The cell-specific effect occurred independently of the AR hormone binding domain and could be observed with a GAL4 fusion protein containing only the AR N-terminal regulatory domain. Gel shift analyses showed that the relative DNA binding affinity of AR for a probe containing a simple HRE was similar in prostate and nonprostate cell extracts. Presently, the only factors known to mediate steroid receptor-specific gene regulation are cJun and cFos, but there were no cell-specific differences in the functional levels of these proteins which could account for a preferential effect on AR-dependent transcription. Taken together, these results suggest that cell-specific activities exist which can preferentially modulate transcriptional transactivation by AR.