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.
PMID: 7723369;Abstract:
A putative explanation of the effect of sulindac on adenomatous colon and duodenal polyps from clinical observations and related in vitro experiments is presented. In cells with mutant APC genes, persistent high prostaglandin content of polyps leads to desensitization, downregulation of adenylate cyclase, uncoupling of cAMP synthesis from prostaglandin, and inactivation of protein kinase A (PKA). It is suggested that in normal cells, (APC) protein binds to catenins and microtubules to maintain structure and contribute to cell-cell communication, adherence, and the dephosphorylated state, a necessary condition for such functions. Cells with mutant APC product become isolated, deprived of communication and adhesion to other epithelial cells, overphosphorylated, and without corrective capability. The latter is largely due tn downregulation of cAMP synthesis and protein kinase A activity secondary tn high prostaglandin. Three main biochemical defects ensue: (1) the restrictive influence of PKA catalyzed phosphorylation of Raf-1 kinase and resultant effects on the MAP kinase cascade and transcription is lost, (2) the transcription of immediate early genes, including cyclooxygenase is stimulated, and (3) the stimulation of protein tyrosine phosphatase (PTPase) by PKA is in abeyance. These putative abnormalities are reversed by inhibition of cyclooxygenase-1 by sulindac. cAMP synthesis and PKA activity return to normal. PKA catalyzed phosphorylations block Raf-1 kinase at the confluence of the Ras and protein kinase C pathways. The MAP kinase cascade is inhibited as is transcription of immediate early genes. At the same time PKA stimulates PTPase, which dephosphorylates the cytoskeleton and restores cell-cell communication, adherence, and structure. The transformed phenotype is circumvented by adjustment of the phosphorylation state and mutant cells rejoin the epithelial community. The redox state of cytoplasm in mutant cells may be shifted toward reduction.
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.
